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Direct oral anticoagulants (DOACs) are increasingly used in the treatment and prophylaxis of thromboembolism because of several advantages over vitamin K antagonists, including no need for laboratory monitoring. However, it has become increasingly important in certain clinical scenarios to know either actual DOAC concentration (quantitative) or presence of DOAC (qualitative). These clinical conditions include patients presenting with major bleeding or requiring urgent surgery who may need a reversal or hemostatic agent, extremes of body weight, failed therapy, etc. Prothrombin time and activated partial thromboplastin time are variably affected by factor Xa inhibitors (FXaIs) and direct thrombin inhibitor (DTI), respectively, depending on reagents’ sensitivity, and hence, they cannot be relied on confidently. Thrombin time is highly sensitive to very low amounts of DTI; thus, normal value rules out a clinically significant amount. Liquid chromatography mass spectrometry accurately measures DOAC levels but is clinically impractical. Dilute thrombin time and ecarin-based assays using appropriate calibrators/controls provide an accurate DTI level. Anti-Xa assay using corresponding FXaI calibrators/controls provides accurate drug levels. However, these assays are not readily available in the United States compared with some other parts of the world. Heparin assays using anti-Xa activity often have a linear relationship with calibrated FXaI assays, especially at the lower end of on-therapy levels, and they may provide rapid assessment of drug activity for clinical decision making. Currently, there is very limited knowledge of DOAC effect on viscoelastic measurements. Although there is uniformity in expression of DOAC concentrations in nanograms per milliliter, a universal FXaI DOAC assay is urgently needed.

Introduction: In patients with SARS-CoV-2 COVID-19 infection elevated D-dimers are associated with a poor prognosis. Although suggestive of DIC, the majority of patients do not fulfil ISTH criteria for DIC. Studies have demonstrated that when compared to D-dimer, fibrin monomers are more sensitive and specific when differentiating overt DIC from non-overt DIC. Our special coagulation laboratory (SCL) routinely measures soluble fibrin monomer complex (SFMC) as part of our DIC profile in samples with elevated D-dimers. Here we aimed to investigate the percentage of patients with COVID-19 infection with elevated SFMC as performed as part of the DIC profile. Methods: Between April 1st 2020 and July 26, 2020 inpatients at the Mayo Clinic Rochester, MN campus with COVID-19 infection who underwent DIC testing in SCL were identified through SCL database. Patients were excluded if SARS-CoV2 PCR results were not available in the Mayo Clinic electronic medical record (EMR). DIC profile components include prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen and D-dimer. Reflex SFMC is performed if D-dimer is elevated. Data abstracted from EMR included results of laboratory tests, date of COVID-19 symptom onset, date of SARS-CoV2 PCR positivity, and death within 30 days of hospital admission. The ISTH DIC score was calculated and patients were labeled as overt DIC or non-overt DIC based off of ISTH DIC score ≥5. Results: 35 patients (19 female), median age 59 years (28-91) met our study criteria DIC profiles were obtained 6.5 (1-37) days after COVID-19 symptom onset (n=33) with 28 obtained within 14 days of symptom onset. Time to DIC profile from SARS-CoV2 PCR testing was 4 (0-37) days (n=35) with 31 having testing completed within 14 days of PCR positivity. 8 patients were intubated during hospitalization and of the remaining 27, 67% required supplemental oxygen. 21/35 (60%) patients had an elevated fibrinogen (526.5 mg/dL (201-800)) while 19/35 (54%) had a prolonged PT (12.8 seconds (10.1-99.6) and 5/35 (14%) had a prolonged APTT (31 seconds (24-68)). 17 patients with prolonged PT and 3 patients with prolonged APTT had factor activity assays performed (Figure 1A). 9/35 (25%) had thrombocytopenia (230 x 109/L (12-709) and 27/35 (77%) patients had an elevated D-dimer (895 ng/ml FEU (220- >100,000). D-dimer was higher in males than in females (p: 0.049). Of those with elevated D-dimer only 5/27 (18.5%) had an elevated SFMC (5 mcg/ml (5- >1100) (Figure 1B). 4/35 (11%) patients had ISTH DIC scores ≥ 5 and all had score of 5. All of these patients had D-dimer > 2000 ng/ml while only 2 had elevated SFMC however SFMC in both of these patients was < 15 mcg/ml. Of these, one patient with SFMC of 12 mcg/ml had known cirrhosis and the other with an SFMC of 11 mcg/ml had acute decompensation and associated shock liver. This patient also had venous thrombosis 3 days prior to DIC profile and died from complications related to COVID-19 infection. Of the two other patients with high ISTH score and normal SFMC, one had previous chronic thrombocytopenia of < 50 x109/L while the other had concomitant admission for acute and recurrent pancreatitis in the setting of alcoholism with acute intoxication. Six patients died within 30 days of admission and in 2/5 performed SFMC was elevated (Figure1C). Conclusion: Compared to 77% of patients with elevated D-dimers, of those tested only 18.5 % of patients had elevated SFMC. It has been hypothesized that the elevated D-dimer noted in COVID-19 pulmonary infection is a direct consequence of acute lung injury and not overt DIC. Although preliminary, the small percentage of patients with overt DIC by ISTH criteria and normal SFMC in the majority of the current cohort support this hypothesis; however studies in a larger more controlled cohort are needed to confirm these findings. Disclosures Sridharan: Alexion: Honoraria. Pruthi:Merck: Honoraria; Instrumentation Laboratory: Honoraria; HEMA Biologics: Honoraria; Bayer Healthcare: Honoraria; Genentech Inc.: Honoraria; CSL Behring: Honoraria.

Tigecycline, a first representative of the new class of glycylcycline antibacterial drugs, has been approved by FDA for using in the treatment of complicated skin and skin structure infections and community-acquired pneumonia in adults in 2005. It has been widely used in hospitals due to its broad-spectrum activity against pathogens. Pre-clinical studies showed that tigecycline has a significant anti-bacterial effect with low and mild adverse drug reactions (ADRs), including nausea, vomit or some other ADRs. Accompany with the huge increase of tigecycline usage in China, more and more side effects began to arouse people's attention. Here, we found that tigecycline can cause coagulation abnormalities in cancer patients, especially with hematological malignance, through clinical observation and retrospective analysis. To analyze the effect and risk factors of tigecycline on coagulation index in severely infected patients with hematologic cancer, we screened the cases admitted to department of hematology from three hospitals treated with tigecycline from Nov, 2015 to Oct, 2019. Then we compared the differences among the prothrombin time (PT), prothrombin time activity (PT %), the international standardization ratio (INR), part of the activation of prothrombin time (APTT), fibrinogen (FBG), thrombin time (TT), D-Dimer, fibrin degradation products (FDP), platelet (PLT), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CRE), and c-reactive protein (CRP) before and after the tigecycline treatment. Meanwhile, we tend to establish the relationship between coagulation function and the time of tigecycline usage. In addition, for exploring the potential cause of abnormal coagulation induced by tigecycline, we evaluated the underlying biological mechanisms via Chinese hamster ovary (CHO) cells treated by tigecycline in vitro and subsequent RNA-sequencing analysis. A total of 129 qualified cases were included for analysis, including 71 males (55%) and 58 females (45%), with average age of 49.5±15.9 years old. After the treatment with tigecycline, PT, APTT and TT were significantly prolonged (P < 0.05). The INR, D-Dimer and FDP levels also increased (P < 0.05), but the FBG concentration was significantly reduced (P < 0.05) which indicated the possibility of hypofibrinogenaemia. Moreover, the coagulation parameters were tremendously changed in the early stage after intravenous infusion of tigecycline for anti-infection. There was no significant change in PLT count, but the levels of ALT, AST, CRE and CRP in the patients were statistically significant declined (P < 0.05). Most of all, the liver and kidney function of the patients was recovered after the infection was controlled by tigecycline treatment. In addition, the adhesion and stretching function of CHO cells were significantly suppressed after tigecycline treatmentin vitro. Lastly, bulk RNA-sequencing assay showed significant abnormalities in gene sets associated with platelet-activation and coagulation, such as PKFP, PLA2g7, PDGFRA, PDGFRB, PAFAH1b3, PDGFA, PECAM1, PDGFC and MPIG6b. Furthermore, adhesion regulating molecules RPN13, CHL1, ICAM5, TLCN were also downregulated by tigecycline treatment. What's more, these key genes which showed above need to be further validated in blood samples of AML patients when treated with tigecycline. Taken together, tigecycline can cause coagulation disorders in blood cancer patients. As we known, blood cancer could disturb the normal hematopoietic reconstitution in bone marrow, which resulted in abnormal numbers and functions of the peripheral blood lineages. Hence, these ADRs can accelerate the disease progression, cause severe bleeding and increase the risk of mortality in these patients. The biological mechanism study of this abnormality showed that tigecycline could inhibit platelet activation and the expression of adhesion regulators, which may increase the risk of bleeding and threaten the safety of patients. Therefore, the monitoring of coagulation index during tigecycline treatment should be adopted. Disclosures No relevant conflicts of interest to declare.

Although the direct oral anticoagulants (DOACs) do not require routine monitoring and reduce bleeding compared with warfarin, there are special circumstances in which laboratory measurement or reversal of their anticoagulant effect may be indicated. The dilute thrombin time and ecarin-based assays are able to quantify dabigatran across a broad range of concentrations, but are not widely available. A normal thrombin time excludes clinically relevant levels and a normal activated partial thromboplastin time probably excludes excess levels of dabigatran. Factor Xa inhibitors may be quantified with an anti-Xa assay calibrated with drug-specific standards. A normal prothrombin time probably excludes excess levels of rivaroxaban and edoxaban, but not apixaban. Patients with minor and moderate DOAC-associated bleeding can be treated with supportive care and general hemostatic measures. Nonspecific reversal agents (eg, prothrombin complex concentrate, activated prothrombin complex concentrate) are of unproven benefit, carry a risk of thrombosis, and should be reserved for severe bleeding. Specific reversal agents, such as idarucizumab (a monoclonal antibody fragment that binds dabigatran) and andexanet alfa (a recombinant factor Xa variant that binds factor Xa inhibitors but lacks coagulant activity), are in clinical development.

Background: The concept of developmental hemostasis has been universally accepted. Plasma concentrations of many coagulation factors in childhood are significantly different from adults for both mean values and ranges of normal. Thus, an understanding of developmental hemostasis and the development of appropriate age-dependent reference ranges are pivotal for prevention, diagnosis, and treatment of hemostatic problems during childhood. However, no data of developmental hemostasis are available in healthy Chinese children. Methods: Coagulation data from children aged 0-18 years old receiving minor elective surgery in Chengdu Women and Children's Central Hospital, from Sep. 2017 to Feb. 2019 was collected, and patient medical records were reviewed. Ethical approval was obtained from the parents of all children and the study was approved by the Ethics Review Committee at Chengdu Women and Children's Central Hospital. To qualify for the study, enrolled subjects had to meet the following criteria: (1) Children aged 0-18 years receiving minor elective surgery.(2) No history of bleeding problems.(3)No family history of bleeding nor thrombosis .(4) No history of heart, lung, liver or kidney diseases, normal physical examination. (5) Normal blood routine test. Normal liver function and kidney function, (6)No history of medication use for at least two weeks prior to specimen collection.(7) The operation gone successfully, and postoperative recovery was well. Blood samples (3 mL) were obtained by venipuncture into plastic tube containing 3.2% (109 mmol/L) buffered sodium citrate (1 part citrate:9 parts blood). The specimens were centrifuged immediately at 2500 g for 15 min at room temperature to prepare platelet-poor plasma (PPP) for the preoperative coagulation screening tests, consisting of activated partial thromboplastin time (APTT), prothrombin time (PT)), international normalized ratio (INR), thrombin time (TT) and fibrinogen. Activity of coagulation factors VIII, IX, XI, XII,Ⅱ,Ⅴ,Ⅶ,Ⅹ were determined using one-stage clotting methods with respective factor-deficient plasma. Results: A total of 82 samples were collected, while only 67 met the enrollment criteria. Specimens with inappropriate ratio of blood to anticoagulant, hemolysis or abnormal values of coagulation tests (APTT, PT, TT, fibrinogen) were excluded from analysis. The total of 67 children (40 Male 27 female), with a median age of 2.0 years (range: 1month -14 years) were divided into three groups according to the age: 6y group 7cases. (1) The values of APTT, PT, TT, and fibrinogen were (36.1±5.2) seconds, (11.1±0.85) seconds, (20.3±1.6) seconds, (2.4±0.89) g/L respectively. No significant differences were found between groups. (2) In all children ,the activities of factor VIII、IX、XI、XII、vWF、II、V、VII、X were(123.6±48.3)%、(75.9±16.9)%、(95.9±24.3)%、(43.7±16.3)%、(111.3±50.4)%、(90.2±14.0)%、(104.7±21.1)%、(81.6±19.1)%、(93.0±21.8)% respectively. Factor VIII and vWF activities were significantly higher than other factors, while factor XII activities were significantly lower than others. (3)Mean values of FII:C, FVIII:C, FIX:C, FXI:C were significantly lower in children below Conclusions: Coagulation test is just a simple and easy screening test. Coagulation factor activities changed dynamically with age during childhood, especially the FII:C, FVIII:C, FIX:C and FXI:C. Physiological reference ranges for coagulation factor activities in Chinese children of different ages should be established in order to evaluate the children with congenital or acquired bleeding diseases correctively. Table Disclosures No relevant conflicts of interest to declare.

Introduction: Andexanet Alpha (Coagulation factor Xa recombinant, inactivated Zh-zo; AA, Portola Pharmaceuticals) is a recombinant factor Xa decoy protein which is designed to reverse the effects of apixaban and rivaroxaban and is approved for the control of bleeding complications associated with their use. The molecular modification in this recombinant protein involves the substitution of serine active site by alanine and the removal of the gamma-carboxyglutamic acid (GLA) domain to restrict its assemblage into prothrombinase complex. Beside the reversal of the effects of anti-Xa agents AA is also reported to neutralize the biologic effects of heparin and related drugs. Assay dependent variations in the neutralization profile of various factor Xa inhibitors by andexanet has been recently reported https://doi.org/10.1177/1076029619847524. Since heparin and related drugs also mediate their biologic actions by inhibiting factor Xa via AT complexation, it is hypothesized that AA may also inhibit their biologic effects as measured in various laboratory assays. It is the purpose of this study is to compare the relative neutralization profile of heparin (UFH), a low molecular weight heparin, enoxaparin (E) and a chemically synthetic pentasaccharide, Fondaparinux (F) by AA. Materials and Methods: API versions of UFH, E and F were commercially obtained in powdered forms and dissolved in saline at a working dilution of 1mg/ml. AA was dissolved in saline to obtain a 10mg/ml working solution. The anticoagulant profile of UFH, E and F was studied using the activated partial thromboplastin time (APTT) and thrombin time (TT) in a concentration range of 0 - 10 ug/ml in pooled human plasma. The anti-Xa and anti-IIa studies were carried out in amidolytic assays in the same concentration range. The thrombin generation inhibition was studied using calibrated automated thrombin generation systems (CAT, Diagnostica Stago). The effect of AA on the reversal of the anticoagulant and anti-protease and thrombin generation effects of each of these agents were studied by supplementing this agent at 100 ug/ml. The results are compared to determine the difference between pre and post AA neutralization settings. Results: All agents produce a concentration dependent effect in the anticoagulant and anti-protease assays with the exception of F which showed mild anticoagulant effects, and very weak anti-IIa actions and strong anti-Xa activity. In the anti-Xa assay the IC-50 for UFH was 2.1ug/ml (0.13 um), E 4.3 ug/ml (0.95 um) and F 0.7 ug/ml (0.41 um) upon supplementation of AA the IC50s for UFH was increased to 5 ug/ml (0.31 um) and for E 5 ug/ml (1.11 um). However, there was no neutralization of the anti-Xa effects of the F by AA and the IC50 remained the same for both pre and post andexxa studies. The anticoagulant effects of UFH as measured by aPTT and TT was strongly neutralized whereas E was only partially neutralized in the aPTT assay and almost completely neutralized in the thrombin time assay. At concentrations of up to 10 ug/ml F did not produced any significant anticoagulant effects, both in the presence and absence of AA. In the thrombin generation inhibition assays, UFH produced a complete inhibition of thrombin generation which was completely reversed by AA. Although both E and F produced strong inhibition of thrombin generation, AA did not completely neutralize these effects. The results are tabulated on table 1 for the studies carried out at 10 ug/ml of UFH, E and F. Conclusion: These results indicate that AA is capable of differentially neutralizing anticoagulant and anti-protease effects of UFH in an assay dependent manner. However, AA is incapable of neutralizing the anti-Xa effects of E and F. This may be due to the relatively differential affinities of enoxaparin and fondaparinux AT complex to factor Xa rendering it inhibited in the presence of AA. These studies also demonstrate that the primary surrogate marker anti-Xa activity for measuring the activities of anti-Xa agents is not proportional to the anticoagulant and thrombin generation inhibitory effects of these agents. A global clotting assay may be a better indication of the biologic effects of these agents and their reversal by AA. Disclosures Tafur: Recovery Force: Consultancy; Janssen: Other: Educational Grants, Research Funding; BMS: Research Funding; Idorsia: Research Funding; Daichi Sanyo: Research Funding; Stago: Research Funding; Doasense: Research Funding.

Whole genome sequencing of an individual completely devoid of plasma- and platelet-derived factor V (FV) identified 167 variants in his F5 gene including previously identified and damaging missense mutations at rs6027 and Leu90Ser. Because the administration of fresh frozen plasma (FFP) prevents gastrointestinal bleeding in this individual, its effects on his plasma- and platelet-derived FV concentrations were assessed. The patient's plasma FV levels peaked by 2 hours following FFP administration and were undetectable 96 hours later. In contrast, increased platelet-derived FV/Va concentrations were observed within 6 hours, peaked at 24 hours, decreased slowly over 7 days, and originated from megakaryocyte endocytosis and intracellular processing of plasma FV. Ten days after transfusion, no thrombin was generated in a tissue factor-initiated whole blood clotting assay unless exogenous FV was added, consistent with the complete absence of plasma FV. In marked contrast, release of the patient's platelet-derived FV/Va (7% of normal) following platelet activation resulted in robust thrombin generation, similar to that in an individual with normal plasma- and platelet-derived FV concentrations. Thus, total FV deficiency can be corrected by plasma administration, which partially repletes and sustains the platelet cofactor pool, thereby highlighting the critical role of platelet-derived FV/Va in ensuring hemostatic competence.

Fibrinogen Milano XII was detected in an asymptomatic Italian woman, whose routine coagulation test results revealed a prolonged thrombin time. Fibrinogen levels in functional assays were considerably lower than levels in immunologic assays. Polymerization of purified fibrinogen was strongly impaired in the presence of calcium or ethylenediaminetetraacetic acid (EDTA). Two heterozygous structural defects were detected by DNA analysis: Aalpha R16C and gamma G165R. As seen previously with other heterozygous Aalpha R16C variants, thrombin-catalyzed release of fibrinopeptide A was 50% of normal. Additionally, the release of fibrinopeptide B was delayed. Immunoblotting analysis with antibodies to human serum albumin indicated that albumin is bound to Aalpha 16 C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of plasmin digests of fibrinogen Milano XII in the presence of calcium or EDTA showed both normal and novel D1 and D3 fragments. Further digestion of abnormal D3 fragments by chymotrypsin resulted in degradation products of the same size as the fragments derived from normal fibrinogen. SDS-PAGE analysis under reducing conditions showed no difference between normal fibrinogen and fibrinogen Milano XII or between their plasmic fragments. Circular dichroism analysis revealed a shift in the mean residual ellipticity and a significant reduction of the alpha-helix content in the variant D3 fragment. It is concluded that the Aalpha-chain substitution is mainly responsible for the coagulation abnormalities, whereas the substitution in the gamma-chain induced a conformational change in the D3 fragment.

Background: The incidence of venous thromboembolism (VTE) in children is increasing. The current standard of care is unfractionated heparin (UFH), low-molecular-weight heparin (LMWH) and Vitamin K antagonists (VKAs), all of which have limitations. Dabigatran, which is orally administered as the prodrug, dabigatran etexilate (DE), is effective for the treatment of VTE in adults and may overcome some of the limitations associated with standard care. Objective: The objectives of this phase IIa study were to obtain pharmacokinetic (PK) and pharmacodynamic (PD) data and to evaluate the tolerability and safety of an oral liquid formulation (OLF) of DE in patients aged 1 to < 12 years. Methods: The open-label, multinational, multicenter, non-randomized, single-arm study was conducted in patients aged 1 to < 2 years and 2 to < 12 years with a diagnosis of VTE, who had completed planned treatment with LMWH or oral anticoagulants (VKAs) for VTE. DE was administered as an OLF at a weight and age-adjusted dose (calculated using a nomogram) equivalent to 150 mg twice daily (bid) in adults. Originally, all patients were to receive multiple dosing (bid for 3 days). However, this was replaced with single dosing (SD) in the course of the study. A 30-day follow-up was included. Here we report data from SD patients only. The primary PK endpoints were plasma concentration of total dabigatran, free dabigatran, unchanged DE and intermediate metabolites. To compare the exposure to DE in pediatrics with that in adults with VTE, gMean concentrations at 10 hours after SD of DE were extrapolated to steady-state trough concentration. An accumulation ratio was calculated based on the terminal half-life (t1/2) and the dosing interval (for a bid dosing regimen) to project the steady-state dabigatran plasma concentration. Primary PD endpoints were activated partial thromboplastin time (aPTT), ecarin clotting time (ECT), and diluted thrombin time (dTT). Primary safety endpoints were incidence of all bleeding events and incidence of all adverse events (AEs). Results: Six patients aged 1 to < 2 years and 9 patients aged 2 to < 12 years received SD DE; 3 patients aged 2 to < 12 received multiple doses (bid for 3 days). In the SD groups, plasma concentration of total dabigatran increased at 2 hours (peak) after dosing then decreased until 10 hours after dosing. The projected steady-state dabigatran trough concentrations of both age groups of children studied were largely comparable to those observed in adults with VTE, i.e. the projected results for children fell between the first and the third quartile of adult trough concentrations (Table). Data from the SD groups indicated that there was a linear PK/PD relationship for ECT and dTT (see Figure) and a non-linear PK/PD relationship for aPTT. The PK/PD relationships were similar to those seen in adults (for all coagulation parameters analyzed) and adolescents (dTT and ECT) with VTE. During the on-treatment period there were no study drug related AEs; one patient had AEs (leukopenia and dizziness), which were mild in intensity and unrelated to the study drug. No AEs were reported during the post-treatment or post-study periods. No bleeding events were reported. Conclusion: The OLF of DE was well tolerated in pediatrics, with no drug-related AEs or bleeding events. Projected steady-state dabigatran trough concentrations were largely comparable to those seen in adults with VTE. The observed PK/PD relationship was similar to that seen in adults and adolescents with VTE. Disclosures Halton: Boehringer Ingelheim: Other: Pediatric Expert Working Group for Boehringer Ingelheim. Albisetti:Boehringer Ingelheim: Other: Pediatric Expert Working Group. Luciani:Boehringer Ingelheim: Other: Member of Pediatric Expert Working Group for Boehringer Ingelheim. Huang:Boehringer Ingelheim: Employment. Biss:Boehringer Ingelheim: Employment. Brueckmann:Boehringer Ingelheim: Employment. Gropper:Boehringer Ingelheim: Employment. Maas:Boehringer Ingelheim: Employment. Tartakovsky:Boehringer Ingelheim: Employment. Mitchell:Boehringer Ingelheim: Consultancy; Pfizer: Consultancy; Bristol Myers Squibb: Consultancy.

Introduction: In a recent study, we performed autologous serum infusions to evaluate the elimination kinetics of hemostasis-related biomarkers in healthy human subjects. In order to monitor a serum-induced activation of coagulation, we measured free thrombin in the infused serum and in plasma samples taken during and after infusion, but did not detect any de novo thrombin formation [PLoS One. 2015; 10(12): e0145012]. To study if the low levels of free thrombin in the infused serum induce generation of activated protein C (APC) we additionally measured APC in samples drawn after autologous serum infusion and in vitro in a purified system. Methods: Autologous serum was infused (50 mL/30 min) into 19 healthy volunteers. Four of them were simultaneously receiving infusions of the thrombin inhibitor argatroban (1 µg/kg/min), initiated 1 h before and ceased 1 h after starting the infusion of serum. Thrombin and APC were measured in serum and in plasma samples drawn before and in 15-min intervals during the infusion of serum, using a highly-sensitive oligonucleotide-based enzyme capture assay (OECA) platform. In in vitro experiments, APC formation was induced by addition of purified thrombin or serum to buffer containing protein C and thrombomodulin in excess, and CaCl2 at physiological concentrations. The formation of APC was subsequently measured by OECA. Results: In the autologous serum median (interquartile range) concentrations of thrombin and APC were 6.68 (4.63 - 8.73) ng/mL and 9.17 (7.63 - 13.91) ng/mL, thus doses of 0.12 (0.07 - 0.15) ng/mL of thrombin and 0.16 (0.14 - 0.22) ng/mL of APC were infused per mL of the subjects' plasma volume. In the plasma of probands, that did not receive argatroban, peak thrombin levels of 0.04 (0.00 - 0.08) ng/mL were measured, indicating a rapid inactivation of thrombin by endogenous inhibitors present in the plasma. However, with 1.41 (0.76 - 2.97) ng/mL peak APC levels exceeded the infused APC doses by a multiple. This was also true for the plasma samples from the probands that received argatroban, in which peak levels of APC of 0.94 (0.79 - 1.22) ng/mL were measured despite thrombin inhibition indicated by prolongation of the aPTT of 42.9 (40.1 - 44.4) s and thrombin time of 78.3 (69.3 - 87.2) s. In the in vitro experiments addition of argatroban at the concentrations achieved in the probands completely abolished APC generation up to a thrombin concentration of 5 ng/ml. Addition of human serum as a source for thrombin in the same purified system consistently induced generation of greater amounts of APC than expected on the basis of the amount of thrombin present in the serum samples. Conclusions: The data obtained provide evidence for a thrombin-independent mechanism of APC formation. Further in vitro studies with endothelial cells are required to identify the components that are involved in this alternative way of APC generation. Disclosures Rühl: CSL Behring: Research Funding; Bayer: Consultancy, Honoraria. Müller:Novo Nordisk: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Introduction: Dabigatran etexilate is a pro-drug which is used to prevent embolic stroke in patients with atrial fibrillation. This oral anticoagulant is also approved for other indications in Europe. As with all anticoagulants, there is a potential for serious hemorrhage with dabigatran usage which may require antidotes to control bleeding. Idarucizumab is an anti-dabigatran Fab fragment (Boehringer-Ingelheim) that binds to the benzamidine group on dabigatran and inhibits its anti-thrombin activity. Idarucizumab has recently been approved by the US FDA for the control of bleeding associated with dabigatran. Materials and Methods: Such antithrombin agents as argatroban, melagatran, hirudin, and bivalirudin, human antithrombin, thrombomodulin, heparin cofactor II, and heparin-AT complex were commercially obtained. Anti-factor Xa agents (rivaroxaban, apixaban and DX-9065a were also obtained from various sources To test the specificity of the inhibitory effects of idarucizumab, each of these agents were supplemented to whole blood and citrated plasma at concentrations ranging from 0.1 to 100 µg/mL. Idarucizumab was added to each mixture at a concentration of 1 mg/mL and anticoagulant activities were assessed using PT, aPTT, thrombin time and chromogenic anti-IIa/Xa and flurometric thrombin generation assays. Results: Idarucizumab itself did not produce any anticoagulant effects on whole blood or plasma clotting profile. However it showed a slight procoagulant effect in the whole blood and plasma based assays. It produced a strong concentration dependant inhibition of both dabigatran and melagatran. The antibody showed strong specificity for the inhibition of dabigatran amd melagatran and did not affect the anticoagulant and other effects of the other synthetic and natural thrombin and FXa inhibitors. The prolongation of the PT, APTT and thrombin time by melagatran was completely inhibited by idarucizumab. Idarucizumab more effectively inhibited the prolongation of thrombin time by dabigatran than the prolongation induced by melagatran. Discussion: The cross-reactivity of idarucizumab with melagatran may result from the presence of a common benzamidine pharmacophore which is present in both of these anticoagulant agents. Since the benzamidine pharmacophore is present in a number of serine protease inhibitors as well as drugs such as pentamidine, propamidine and dibromopropamidine. These observations suggest that simultaneous administration of idarucizumab may compromise the pharmacodynamics profile of benzamidine derived drugs such as the anti-malarials, anti-psychotic, anti-fungal and other compounds. Thus there is a need for a systemic screening of idarucizumab for its potential interactions with drugs containing benzamidine based therapeutic agents. Disclosures No relevant conflicts of interest to declare.

Introduction: The currently used unfractionated heparin (UFH) and low molecular weight heparins (LMWH) are mostly derived from Porcine mucosal tissue. Since the technology to manufacture heparin has advanced and the quality assurance practices are in place, improved products with high potency and purity are now available. Considering these factors the resourcing of heparins utilizing bovine (cow) and ovine (sheep) tissues is discussed at regulatory and pharmaceutical levels. The purpose of this study is to compare multiple individual batches of UFH obtained from Bovine, Ovine, and Porcine origin and their depolymerized product obtained by benzylation followed by alkaline hydrolysis representing enoxaparins. Materials and Methods: The molecular profile of the heparins and enoxaparins from various sources were determined using the size exclusion chromographic method. The anticoagulant potency was measured using clot based methods such as aPTT and Thrombin Time. The AT mediated anti-Xa and anti-IIa activities were also measured in defined biochemical sysytems. A comercially obtained chromogenic substrate based methods were used to determine the USP potency in terms of anti-Xa and anti-IIa activities (Hyphen Biomedical, Ohio, USA). The relative interaction of the heparins and enoxaparins with heparin induced thrombocytopenia (HIT) antibody induced aggregation of platelets were investigated using serum pool obtained from clinically confirmed HIT cases using aggregometry. Results: The molecular profile of multiple batches of the Bovine, ovine, and porcine heparins and enoxaparin were almost comparable and ranged from 15-18 kDa. The global anticoagulant and amidolytic protease assays for the bovine heparin were consistently lower than porcine and ovine samples. In the purified system the Porcine and Ovine preparations consistently showed lower IC50 values for both the thrombin and Xa inhibition in contrast to the bovine heparin. Similar trends were observed in the anti IIa assays. The USP potency of 28 batches of porcine ranged from 170-210 U/mg, whereas the 21 batches of ovine heparins exhibited comparable potencies in the range of 160-210 U/mL. In contrast the USP potency of 30 batches of bovine mucosal heparin was much lower and ranged from 110-140 U/mg. The anti-Xa - IIa ratio for the porcine and ovine heparins were comparable. However, the anti-Xa - IIa ratio of bovine heparin was somewhat lower. The ovine and porcine enoxaparin exhibited comparable potencies which ranged from 94-110 U/mg whereas bovine enoxaparin was slightly lower, ranging from 80-87 U/mg. However the antiXa and anti-IIa ratios of the enoxaparins derived from various species were comparable. In the HIT screening, there was no difference between the HIT responses in the heparins from different species. Similar results were obtained amongst enoxaparins of different origins. Conclusions: These studies show that heparins from bovine, ovine, and porcine origin exhibit comparable molecular profiles. While the porcine and ovine heparins exhibit similar biological potencies, the bovine heparin was found to be weaker. The enoxaparins derived from these species also exhibit similar molecular profiles, however, in functional assays, they exhibited similar trends where the bovine derived products were weaker. In the HIT screening profile, the heparins from all of these species produced stronger effects in comparison to their enoxaparin counterparts. These results suggest that heparin and enoxaparin derived from ovine mucosal tissue exhibit comparable biosimilar profiles. However, the bovine heparins and enoxaparin derived from bovine mucosal tissues are somewhat weaker. Disclosures Yao: Ronnsi Pharma: Employment.

Congenital homozygous dysfibrinogenemia was diagnosed in a man with a history of 2 thrombotic strokes before age 30. His hemostatic profile was characterized by a dramatically prolonged plasma thrombin clotting time, and no clotting was observed with reptilase. Complete clotting of the abnormal fibrinogen occurred after a prolonged incubation of plasma with thrombin. The release of fibrinopeptides A and B by thrombin and of fibrinopeptide A by reptilase were both normal. Thrombin-induced fibrin polymerization was impaired, and no polymerization occurred with reptilase. The polymerization defect was characterized by a defective site “a,” resulting in an absence of interaction between sites A and a, indicated by the lack of fragment D1 (or fibrinogen) binding to normal fibrin monomers depleted in fibrinopeptide A only (Des-AA fm). By SDS-PAGE, the defect was detected on the γ-chain and in its fragment D1. The molecular defect determined by analysis of genomic DNA showed a single base change (A→T) in exon VIII of the γ-chain. The resulting change in the amino acid structure is γ 330 aspartic acid (GAT) → valine (GTT). It is concluded that the residue γ-Asp330 is essential for the normal functioning of the polymerization site a on the fibrinogen γ-chain.

A novel BβAsn-160 (TAA) to Ser (TGA) substitution has been identified in fibrinogen Niigata derived from a 64-year-old asymptomatic woman, who is heterozygotic for this abnormality. The mutation creates an Asn-X-Ser–type glycosylation sequence, and a partially sialylated biantennary oligosaccharide was linked to the BβAsn-158 residue. The functional abnormality was attributed to delayed lateral association of normally formed double-stranded protofibrils based on normal cross-linking of fibrin γ-chains and tissue-type plasminogen activator-catalyzed plasmin generation by polymerizing fibrin monomers. Enzymatic removal of all the N-linked oligosaccharides from fibrinogen Niigata accelerated fibrin monomer polymerization that reached the level of untreated normal fibrin monomers, but the thrombin time was prolonged from 18.2 seconds to 113 seconds (normal: 11.2 seconds to 8.9 seconds). By scanning electron micrographic analysis, Niigata fibrin fibers were found to be more curvilinear than normal fibrin fibers. After deglycosylation, Niigata fibers became straight being similar to untreated normal fibrin fibers, whereas normal deglycosylated fibrin appeared to be less-branched than untreated normal or deglycosylated Niigata fibrin. Although normal and Niigata fibrins were similar to each other in permeation and compaction studies, deglycosylated normal and Niigata fibrins had much higher permeability and compaction values, indicating that deglycosylation had brought about the formation of more porous networks. The enzymatic deglycosylation necessitates an Asn to Asp change at position Bβ-158 that is responsible for reducing the fiber thickness because of either local repulsive forces or steric hindrance in the coiled-coil region.

Vitronectin (VN) binds to plasminogen activator inhibitor-1 (PAI-1) and integrins and may play an important role in the vascular response to injury by regulating fibrinolysis and cell migration. However, the role of VN in the earliest response to vascular injury, thrombosis, is not well characterized. The purpose of this study was to test the hypothesis that variation in vitronectin expression alters the thrombotic response to arterial injury in mice. Ferric chloride (FeCl3) injury was used to induce platelet-rich thrombi in mouse carotid arteries. Wild-type (VN +/+, n = 14) and VN-deficient (VN −/−, n = 15) mice, matched for age and gender, were studied. Time to occlusion after FeCl3 injury was determined by application of a Doppler flowprobe to the carotid artery. Occlusion times of VN −/− mice were significantly shorter than those of VN +/+ mice (6.0 ± 1.2 minutesv 17.8 ± 2.3 minutes, respectively, P < .001). Histologic analysis of injured arterial segments showed that thrombi from VN +/+ and VN −/− mice consisted of dense platelet aggregates. In vitro studies of murine VN +/+ andVN −/− platelets showed no significant differences in ADP-induced aggregation, but a trend towards increased thrombin-induced aggregation in VN −/− platelets. Purified, denatured VN inhibited thrombin-induced platelet aggregation, whereas native VN did not. Thrombin times of plasma from VN −/− mice (20.5 ± 2.1 seconds, n = 4) were significantly shorter than those ofVN +/+ mice (34.2 ± 6.7 seconds, n = 4, P < .01), and the addition of purified VN to VN −/− plasma prolonged the thrombin time into the normal range, suggesting that VN inhibits thrombin-fibrinogen interactions. PAI-1-deficient mice (n = 6) did not demonstrate significantly enhanced arterial thrombosis compared with wild-type mice (n = 6), excluding a potential indirect antithrombin function of VN mediated by interactions with PAI-1 as an explanation for the accelerated thrombosis observed in VN−/− mice. These results suggest that vitronectin plays a previously unappreciated antithrombotic role at sites of arterial injury and that this activity may be mediated, at least in part, by inhibiting platelet-platelet interactions and/or thrombin procoagulant activity.

Systemic administration of ISIS 2302, a 20-mer antisense phosphorothioate oligonucleotide targeting human intercellular adhesion molecule-1 mRNA, causes prolongation of plasma clotting times in both monkey and human studies. The anticoagulant effects of ISIS 2302 were investigated with both in vitro coagulation assays in human plasma and purified enzyme systems. At high oligonucleotide plasma concentrations (>100 μg/mL), prolongation of the prothrombin and thrombin times was observed. In a thrombin time assay using purified components, high concentrations of ISIS 2302 inhibited thrombin clotting activity both by stimulating inhibition by heparin cofactor II and directly competing with fibrinogen for binding to anion binding exosite I. In contrast, low concentrations of ISIS 2302 (© 1998 by The American Society of Hematology.

Introduction: The currently used unfractionated heparin (UFH) and low molecular weight heparins (LMWH) are mostly derived from Porcine mucosal tissue. Since the technology to manufacture heparin has advanced and the quality assurance practices are in place, improved products with high potency and purity are now available. At the same time the demand for heparins has increased requiring alternate sources to obtain heparin and LMWH. Considering these factors the resourcing of heparins utilizing bovine (cow) and ovine (sheep) tissues is discussed at regulatory and pharmaceutical levels. The pharmaceutical industry has already initiated programs to manufacture Bovine, Ovine, and Porcine heparins and depolymerized enoxaparin from these products some of which are currently in various phases of development. The purpose of this study is to compare 5 individual batches of UFH obtained from Bovine, Ovine, and Porcine origin and their depolymerized product obtained by benzylation followed by alkaline hydrolysis representing enoxaparins. Methods: The molecular profile of the heparins and enoxaparins from various sources were determined using the size exclusion method. A narrow range calibration method was used for comparing the molecular weight of heparin, whereas the EP method was used to cross-reference the molecular weight of enoxaparins. The anticoagulant potency was measured use clot based methods such as aPTT and Thrombin Time. Chromogenic substrate based methods were used to determine the USP potency in terms of anti-Xa and anti-IIa activities (Hyphen Biomedical, Ohio, USA). The interaction between AT and heparins and enoxaparin were investigated in a purified biochemical system, using AT supplemented buffered assay. Thrombin Generation inhibition studies were carried out using a flourometric method (Technoclone, Vienna, Austria). The relative interaction of the heparins and enoxaparins with heparin induced thrombocytopenia (HIT) antibody induced aggregation of platelets were investigated using serum pool obtained from clinically confirmed HIT cases using aggregometry. Results: The molecular profile of the Bovine, ovine, and porcine heparins and enoxaparin were almost identical. In the clot based assays, such as PT and PTT, Porcine and Ovine heparin produce consistently comparable anticoagulant effects, which were stronger in comparison to the bovine derived heparins. In contrast, the enoxaparins derived from these three sources showed minimal differences. In the amidolytic anti Xa and IIa assays both ovine and porcine heparins produced similar inhibitory effects, whereas the bovine heparin exhibited lower activity. In the purified system the Porcine and Ovine preparations consistently showed lower IC50 values for both the thrombin and Xa inhibition in contrast to the bovine heparin. Similar trends were observed in the anti IIa assays. The USP potency of the Porcine and Ovine heparins ranged from 180 to 190u/mg, whereas the Bovine was found to be 130-140 u/mg. The anti-Xa - IIa ratio for the heparin were comparable. The ovine and porcine enoxaparin exhibited comparable potencies which ranged 94-110 u/mg whereas bovine enoxaparin was slightly lower 80-87 u/mg. However the antiXa and anti-IIa ratios were comparable. The AT mediated inhibition of factor Xa and anti-IIa was stronger with heparins in comparison to the enoxaparins. Similarly heparins produced stronger inhibition of thrombin generation in comparison to the enoxaparin. In the HIT screening there was no difference between the HIT responses in the heparins from different species. Similar results were obtained with enoxaparins. Conclusions: These studies show that while bovine, ovine and porcine heparins and enoxaparins exhibit comparable molecular profiles however in some of the functional assays bovine heparin and enoxaparin exhibited somewhat lesser potencies especially in the pharmacopeial assays. No differences were noted in the HIT antibody interactions among heparins and enoxaparins from different species. These studies demonstrate that ovine and porcine heparins are biosimilar and can be developed as such for clinical purposes. The bovine derived heparins exhibit slightly weaker potencies in functional assays despite comparable molecular profile. Potency adjustment for in vivo usage may be required to obtain comparable anticoagulant responses for the bovine heparin and enoxaparin. Disclosures No relevant conflicts of interest to declare.

CONTEXT: Triton X-100 is a non-ionic surfactant that has been proposed as a virus inactivator in a laboratory setting for it causes cell lysis through lipid membrane disruption without denaturing proteins. Very few studies have tried to evaluate its effect on laboratory tests, especially on coagulation tests that require a phospholipid subtract. METHOD: Plasma samples were collected. Triton X-100 was spiked at a concentration of 0,25% and specimens were incubated for an hour. We performed international normalized ratio (INR), activated partial thromboplastin time (aPTT), thrombin time (TT), fibrinogen dosing and D-Dimer dosing, comparing the standard plasma with the triton X-100 exposed plasma. We also dosed coagulation factors, and performed dilution and lupus anticoagulant studies. Finally, we compared prothrombin time and aPTT using reagents from various manufacturers. RESULTS: We observed an important difference for the INR and the aPTT with addition of triton x-100. A slight decrease of an average of 6.6 to 16.4% was also found in factor assays. Differences in the INR and the aPTT were dependent respectively on the ISI and the phospholipids content of the reagent. CONCLUSION: The addition of triton x-100 significantly modifies INR and aPTT results with every reagent tested. The impact on TT and fibrinogen is not clinically relevant, and the D- Dimer dosage is not affected. The slight decrease in all coagulation factors does not explain those results. The interference could result from an interaction with phospholipids, calcium or some enzymatic reactions caused by the presence of triton x-100 in the analyzed specimen. Table. Test Mean Mean Mean of difference p-value (paired t-test) without Triton with Triton INR 1,59 2,87 68,80% p Disclosures No relevant conflicts of interest to declare.

Background: A proportion of trauma patients presenting with coagulopathy also receive anticoagulant therapy, which can exacerbate coagulopathy and increase bleeding. Emergency reversal of anticoagulation therapy may require therapy with specific reversal agents, as with idarucizumab for dabigatran or non-specific reversal agents, such as prothrombin complex concentrates (PCC). Methods: Using a lethal polytrauma model under dabigatran anticoagulation, we investigated the mechanism and procoagulant properties of a four-factor PCC or idarucizumab in dabigatran-anticoagulated pigs following multiple injuries. Blood markers of coagulation associated with procoagulant effects, platelet activation and hyperfibrinolysis were determined over time and related to blood loss. After ethical approval, dabigatran etexilate was given orally for 3 days and iv on day 4 to 18 pigs after anesthesia. Animals were randomized to receive idarucizumab (60 mg/kg), PCC (50 IU/kg) or saline (control), administered 15 minutes after bilateral femur fracture and blunt liver injury. Plasma dabigatran was determined using diluted thrombin time. Thrombin-antithrombin (TAT) complexes and fibrinopeptide A (FPA) were quantified using ELISA and D-Dimer with a clotting assay. Thrombin generation (ETP) in plasma was measured using Calibrated Automated Thrombogram. Platelet function was evaluated as aggregation using platelet-rich plasma in response to tissue factor, collagen, or adenosine diphosphate (ADP) agonism using light transmission aggregometry. Results: Mean dabigatran levels were 543±186 ng/ml prior to injury. Blood loss was 4056±537 mL after saline treatment (control), 1822±222 mL after PCC (P Conclusion: Although four-factor PCC (50 IU/kg) is effective in reducing blood loss, its efficacy is less than therapy with idarucizumab (60 mg/kg). Most importantly the activation of coagulation is explained by different mechanisms. Supernormal activation of coagulation (e.g. TAT, FPA, ETP) was evident after PCC administration, whereas idarucizumab normalized ETP to baseline values. Since thrombin is a potent activator of platelets via PAR, restoration of active thrombin after PCC also partially restored aggregation. In contrast, idarucizumab binds dabigatran and forms an irreversible complex, inactivating dabigatran in plasma, thereby restoring hemostatic function. This difference may influence the choice of therapy when idarucizumab is clinically available. Disclosures van Ryn: Boehringer Ingelheim: Employment. Grottke:NovoNordisk: Research Funding; Portola Pharmaceuticals: Consultancy; CSL Behring: Honoraria, Research Funding; Boehringer Ingelheim: Consultancy, Honoraria, Research Funding; Biotest: Research Funding.

Introduction Hemostasis in childhood differs from that of adults; and the hemostatic system is still developing during childhood. These differences offer a protective advantage to children with hemorrhagic and thrombotic complications. Plasma levels of coagulation factors (except for fibrinogen, factor V and factor VIII), as well as plasma levels of protein C, protein S and antithrombin are reduced. Hereditary dysfibrinogenemia is a rare disorder wherein an inherited abnormality in fibrinogen structure may result in defective fibrin function and/or structure. Clinical symptoms may vary from asymptomatic to life-threatening bleeding complications. Venous or arterial thrombotic complications occur extremely rarely during childhood. Fibrinogen, a key component in hemostasis, is a 340-kDa glycoprotein. The molecule consists of three different pairs of polypeptide chains (Aa, Bb, and g) each encoded by a distinct gene (FGA, FGB, and FGG). N-terminal parts of the Aa chain - fibrinopeptides A and the Bb chain - fibrinopeptides B are situated in the central part of the molecule and block polymerization of the molecules. Conversion of fibrinogen to fibrin occurs after the cleavage of N-terminal fibrinopeptides by the serine protease thrombin. Correct conformation of fibrinopeptides is important for the cleavage by thrombin. Methods Routine coagulation tests were performed with citrated plasma samples on a STA-R coagulation analyzer. The functional fibrinogen level was measured by the Clauss method. Total fibrinogen level was determined by an immunoturbidimetric assay performed on a UV-2401PC spectrophotometer. Fibrin polymerization induced by either thrombin or reptilase and fibrinolysis experiments were obtained by the turbidimetrical method. Fibrinopeptide release was measured as a function of time; and the fibrinopeptides were determined by the reversed-phase, high-performance liquid chromatography (RP-HPLC) method. The purified genomic DNA was amplified by polymerase chain reaction, using specific primers; and dideoxysequencing was performed with Dye Terminator Cycle Sequencing with a Quick Start kit and a CEQ 8000 genetic analysis system). Results We have examined four unrelated children suspected dysfibrinogenemia. The first patient was a 5-yr old boy with prolonged thrombin time and low functional fibrinogen level. He presented with easy bruising and epistaxis. Genetic analysis revealed a heterozygous substitution Aalpha R16H. The second patient was a 13-yr old girl with prolonged thrombin and reptilase times and low functional fibrinogen level. She presented with menorrhagia. Genetic analysis revealed a heterozygous substitution Aalpha G17V. The third patient was a 12-yr old asymptomatic boy. Genetic analysis revealed a heterozygous substitution Aalpha R16H. Kinetics of fibrinopeptide release was impaired in all these cases. The fourth patient was a 3-yr old girl with low functional fibrinogen level. She presented with easy bruising. Genetic analysis revealed a heterozygous substitution Aalpha K448N. All patients presented with impaired fibrin polymerization. Conclusion All patients were found to be heterozygous for point mutations in FGA gene. Three mutations were found in the site of fibrinopeptide cleavage and one in the alphaC-domain. Mutations had different clinical manifestations from asymptomatic to bleeding. Mutations Aalpha Arg16His and Aalpha Gly17Val are among the most common fibrinogen mutations and both decelerate fibrinopeptide A cleavage from mutant fibrinogens. We describe here a novel previously unreported mutation Aalpha Lys448Asn affecting fibrin formation. Acknowledgment This work was supported by the project of the Ministry of Health of the Czech Republic for conceptual development of the research organization 00023736, by Grant from the Academy of Sciences, Czech Republic (P205/12/G118), and by ERDF OPPK CZ.2.16/3.1.00/28007. Disclosures No relevant conflicts of interest to declare.

In patients with acute promyelocytic leukemia (APL), in spite of recent advances, early treatment related mortality (TRM) still remains a challenge and is largely related to the associated coagulopathy. It is well recognized that conventional coagulation parameters used in the clinic do not accurately predict the risk of adverse outcomes like major bleeding or thrombosis. There is limited data on the clinical utility of whole blood viscoelastic assays and extended coagulation parameters for evaluation of coagulopathy in patients with APL. Consecutive patients with PML-RARA positive APL were enrolled. Blood samples were collected prior to infusion of blood products on day 1 and again on day 4 for thromboelastometry done on whole blood and activated with recombinant tissue factor at a final dilution of 1:50,000 (ROTEM analyzer, Tem Innovations GmbH, Basel) and an extended panel of coagulation parameters done by immunoassays (TAT complex, protein C, ATIII, PIC complex, tPAIC, d-dimer and thrombomodulin). Platelet count and conventional coagulation parameters, which were prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen and thrombin time (TT), were measured daily for the first 14 days (more frequently as clinically indicated). Patients received platelet transfusions to target a platelet count of 30x109/L (in the absence of bleeding). Fresh frozen plasma and cryoprecipitate transfusions were done to target a normal PT and aPTT and maintain a fibrinogen level above 140 mg%. Major bleeding, thrombosis and death during induction therapy were the outcome variables. Of the 61 patients enrolled, 11 were excluded (3 did not continue treatment at our hospital, 2 died within 48 hours of admission, and 6 took discharge against medical advice before completing induction). Of the remaining 50 cases that were evaluated, 40 were newly diagnosed and 10 were relapsed. The median age was 34.5 years (range 9-68). 25 were males (50 %). The WBC count was above 10 x 109/L in 20 patients (40%). The median platelet count was 28 x 109/L (range 4-339). There were 13 (26%) deaths in induction. Six patients had a major bleeding event. Five patients had a thrombotic event. Of the evaluated conventional coagulation parameters, extended coagulation parameters, platelet counts and ROTEM parameters at diagnosis (ROTEM data not available at diagnosis in 2 cases) only the ROTEM parameter of maximum clot firmness (MCF) as a continuous variable was significantly associated with death (p=0.012). On univariate cox regression analysis with death as the outcome, MCF≤ 30mm was a poor prognostic marker (hazard ratio of 13.24, 95%CI 1.71-102.19; p =0.013). On multivariate cox regression analysis including high WBC count (>10 x 109/L), low platelet count (< 40 x 109/L) and history of relapse, MCF ≤30mm was an independent poor prognostic variable (HR: 11.89; 95% CI 1.43 - 98.75; p 0.022). Four of the 6 major bleeding events, four of five thrombotic events and all deaths related to coagulopathy occurred in patients with MCF ≤30mm. The descriptive statistics of patients with MCF >30mm (n=21) vs those with MCF ≤ 30mm (n=27) are shown in table 1. Figure 1 illustrates the type of discordance one can see between conventional coagulation parameters and ROTEM values. This study suggests that the MCF value from a ROTEM assay at diagnosis is probably a better measure of the hemostatic defect at diagnosis in APL. Patients with APL with low MCF on ROTEM at diagnosis are at an increased risk of death during induction therapy despite an adequate blood product replacement strategy which was based on conventional coagulation parameters. In these patients, there is a need to develop novel and possibly a more intensive replacement strategy. Table 1. Comparison of baseline characteristics and clinical outcomes. Variables MCF ≤30 (n = 27)N (%) /Median (range) / Mean±SD MCF > 30 (n=21)N (%) /Median (range) / Mean±SD p value Age (years) 40(12-68) 27(9-48) 0.006 WBC >10 x x109/L 17(62.96) 2(9.52) 0.003 Platelet count (x109/L) 17(4-89) 45(10-339) 0.001 PT in seconds 14.5(11-87.5) 11.8(10.1-14.8) 0.004 aPTT in seconds 28(23.2-77.9) 30.2(22-53.6) 0.104 Fibrinogen in mg% 134.8(24.7-393) 172.2(14.7-549.5) 0.066 Major bleeding 4(14.8) 1(4.8) 0.368 Thrombosis 4(14.8) 1(4.8) 0.369 Death 12(44.4) 1(4.8) 0.003 Disclosures No relevant conflicts of interest to declare.

The ability of heparin to dramatically enhance the inactivation of thrombin (IIa) by antithrombin III (ATIII) in buffer is negated through formation of a IIa-fibrin-heparin ternary complex (Hogg and Jackson, Proc Natl Acad Sci USA 86:3619, 1989; Hogg and Jackson, J Biol Chem 265:241, 1990). IIa, in this ternary complex, is protected from inactivation by ATIII. Our aim was to determine whether fibrin also compromises heparin efficacy in plasma. We found that soluble fibrin ablated the heparin-mediated prolongation of the thrombin time with half-maximal effect at 60 nmol/L fibrin. The heparin-mediated prolongation of the activated partial thromboplastin time (APTT) was also reduced by fibrin with half-maximal effects at 140 nmol/L fibrin using 0.12 U/mL heparin and 500 nmol/L fibrin using 0.25 U/mL heparin. The mechanism of inhibition of heparin activity by fibrin in plasma was determined by measuring IIa-ATIII complexes by enzyme-linked immunosorbent assay (ELISA). Fibrin was found to inhibit the heparin- catalyzed inactivation of IIa by ATIII with half-maximal effect at 97 +/- 19 nmol/L fibrin. Fibrin had no effect on the heparin-catalyzed inactivation of factor Xa by ATIII in plasma, using either standard heparin, a heparinoid preparation (Orgaran; Organon, Lane Cove, Sydney, Australia), or low-molecular weight heparin. These findings imply that fibrin is a potent modulator of heparin activity in vivo by inhibiting heparin-catalyzed IIa-ATIII complex formation through formation of ternary IIa-fibrin-heparin complexes.

A 68-year-old man on chronic hemodialysis for 6 years, presented with a spontaneous psoas muscle hemorrhage. Investigations showed intermittently elevated activated partial-thromboplastin time and thrombin time. Preliminary investigations suggested a heparin-like inhibitor in the patient's plasma, but no anti-Xa activity could be detected. Investigation of the ability of patient plasma to inhibit exogenous thrombin showed that most thrombin was inhibited by heparin cofactor II, in contrast to normal plasma in which most thrombin was inhibited by antithrombin III. Treatment of plasma with glycosaminoglycan-degrading enzymes suggested the presence of dermatan sulfate (DS) in patient plasma. This was confirmed in a heparin cofactor II-dependent antithrombin assay for DS that showed anticoagulant equivalent to 2.2 +/- 0.3 micrograms/mL (mean +/- SD) of porcine mucosal DS. Of this activity, approximately 90% was sensitive to enzymes that degrade DS. The glycosaminoglycan containing fraction of plasma was isolated and subjected to gel chromatography. Anticoagulant activity eluted from Sephadex G-100 (Pharmacia, Montreal, Quebec, Canada) as two peaks with Kav of 0.10 and 0.45. After treatment with base, the Kav of the higher molecular weight species was increased to 0.55. This activity was completely sensitive to enzymes that degrade DS. Thus, the active DS was present as a proteoglycan. The lower molecular weight material was not sensitive to enzymes that degrade DS or heparan sulfate and it was active in the heparin cofactor II- dependent antithrombin assay but not in an antithrombin III-dependent antithrombin assay. This activity was not degraded by heating. Subsequently, measurement of DS activity was performed in plasmas obtained from eight other patients on hemodialysis before administration of heparin that showed that all patients had DS activity present that varied from 0.05 to 0.4 microgram/mL. No enzyme-resistant activity could be shown in these patients. In summary, a circulating anticoagulant with properties of DS is present in patients requiring hemodialysis.

A fibrinogen fragment obtained by limited-plasmin proteolysis has been isolated and purified to apparent homogeneity by gel filtrations. This fragment, denoted as 24-Kd fragment, has an apparent M(r) approximately 24,000 and contains an N-terminal sequence of met-glu-leu-glu-arg-pro- gly-gly-asn-glu-ile. The fragment contains a large number of acidic amino acid residues, and its amino acid composition is similar to several fibrinogen A alpha chains degradation fragments isolated previously. It corresponds to a peptide of the fibrinogen A alpha chains, the N-terminal of which starts at alpha Met-240. This peptide delays thrombin plasma clotting time. It does not bind calcium ions and does not inhibit thrombin's amidolytic activity. It binds to immobilized fibrin but not fibrinogen. It also inhibits the polymerization of desAA and desAABB fibrin monomers by simultaneously decreasing the maximum rate and the maximum level of the polymerization reaction. However, the initial lag period of this reaction is not affected by the fragment.

To explore the effect(s) of growth hormone signaling on thrombosis, we studied signal transduction and transcription factor 5 (STAT5)–deficient mice and found markedly reduced survival in an in vivo thrombosis model. These findings were not explained by a compensatory increase in growth hormone secretion. There was a modest increase in the activity of several procoagulant factors, but there was no difference in the rate or magnitude of thrombin generation in STAT5-deficient mice relative to control. However, thrombin-triggered clot times were markedly shorter, and fibrin polymerization occurred more rapidly in plasma from STAT5-deficient mice. Fibrinogen depletion and mixing studies indicated that the effect on fibrin polymerization was not due to intrinsic changes in fibrinogen, but resulted from changes in the concentration of a circulating plasma inhibitor. While thrombin-triggered clot times were significantly shorter in STAT5-deficient animals, reptilase-triggered clot times were unchanged. Accordingly, while the rate of thrombin-catalyzed release of fibrinopeptide A was similar, the release of fibrinopeptide B was accelerated in STAT5-deficient plasma versus control. Taken together, these studies demonstrated that the loss of STAT5 resulted in a decrease in the concentration of a plasma inhibitor affecting thrombin-triggered cleavage of fibrinopeptide B. This ultimately resulted in accelerated fibrin polymerization and greater thrombosis susceptibility in STAT5-deficient animals.

Patients with primary systemic amyloidosis (AL) often experience bleeding, and we report a newly recognized coagulation abnormality in AL. Of 103 patients with primary systemic AL studied over 2 years, 41 had prolongation of the thrombin time (range, 25 to 46 seconds; normal, less than 22 seconds) and reptilase time (range, 17 to 39 seconds; normal, 14 to 16 seconds). The fibrinogen from the plasma of 36 patients was precipitated by beta-alanine and diluted to a concentration of approximately 200 mg/dL. The thrombin times of the precipitated fibrinogens were normal in 34 patients, implying that an inhibitor was responsible for the abnormal tests. The addition of patient fibrinogen-free plasma to normal plasma prolonged the thrombin times, and this result confirmed the presence of an inhibitor. The inhibitor is more likely to be present in patients with nephrotic syndrome (20 of our patients) and congestive heart failure (six). A circulating monoclonal protein (24 patients), the presence of amyloid liver involvement (eight), and the presence of amyloid neuropathy (nine) were not predisposing factors. Only one patient had deficiency of factor X. We conclude that inhibition of fibrinogen conversion to a fibrin clot rather than dysfibrinogenemia is the cause of the prolonged thrombin time in primary systemic AL.

A new case of heterozygous dysfibrinogenemia characterized by the replacement of NH2-terminal amino acid of fibrin beta-chain was found in a 50-year-old man. Despite a prolonged thrombin time, the propositus' fibrinogen had a normal reptilase time with the normal release of fibrinopeptide A. Release of fibrinopeptide B by thrombin was strongly affected, but a very high concentration of thrombin almost completely released fibrinopeptide B with a normal elution pattern on reversed-phase high performance liquid chromatography (HPLC). Lysylendopeptidase-cleavage of purified B beta-chains analyzed on HPLC showed the decrease of one peptide compared with the normal and the appearance of an abnormal peptide peak. These peptides were treated with thrombin and further separated on HPLC. Amino acid sequence analysis of the abnormal peptide demonstrated that B beta glycine-15, NH2-terminus of the fibrin beta-chain, was replaced by cysteine. These findings will be of particular importance because they strongly support the hypothesis that the NH2-terminal portion of the fibrin beta-chain is involved in the polymerization reaction by thrombin. The propositus' daughter and two sisters had the same abnormal fibrinogen. This unique inherited abnormal fibrinogen was designated as fibrinogen Ise. During these studies, we found that a very high concentration of thrombin cleaves not only the A alpha Arg19-Val20 bond but also the COOH- terminal region of alpha-chains, which results in the generation of further degraded alpha-chains with apparent molecular weights of 44,000 or less.

A 68-year-old man, following mitral valve replacement, presented with a low-grade chronic consumptive coagulopathy. Laboratory analysis showed mild fibrinolysis, minimal effect of coumadin therapy, and a prolonged thrombin time (greater than 150 seconds using bovine IIa). When purified human IIa was used the thrombin time normalized to within 17 seconds of controls, suggesting a possible inhibitor of bovine IIa. An anti-IIa antibody was isolated by protein A-Sepharose (Sigma, St Louis, MO) chromatography followed by affinity chromatography using a bovine IIa-Sepharose column. The effects of this purified anti-IIa antibody on both bovine and human IIa procoagulant and anticoagulant functions were studied. The isolated immunoglobulin G (IgG) was observed to inhibit bovine IIa in all assays tested. This IgG was also able to slightly prolong fibrinogen clotting by human IIa. Using an enzyme-linked immunosorbent assay it was observed that the IgG bound to bovine IIa, bovine II, human IIa, but not to human II. Further, binding was detectable at approximately 50-fold lower concentrations to bovine IIa (1 nmol/L IgG concentration) than to human IIa (50 nmol/L IgG concentration). The effect of the antibody on the reaction between IIa and AT III/heparin was investigated. Human IIa was found to be protected from AT III/heparin neutralization in the presence of this antibody. These results suggest that this patient developed an antibody that strongly binds to and inhibits the bovine IIa in all assays tested. However, the antibody only significantly affects human IIa neutralization by AT III/heparin, and has little effect on the human IIa procoagulant activity. These data suggest that the decreased effect of AT III/heparin on this patient's IIa may have been a contributing factor in his coagulopathy. The exact cause of this antibody development is unclear, but the role of bovine topical thrombin used during cardiac valve replacement surgery is suspect.

Antithrombotic effect of recombinant human thrombomodulin in mice, both in vitro and in vivo, was studied. The soluble recombinant human thrombomodulin was expressed in Chinese hamster ovary cells and purified from the conditioned medium by a modification of the conventional method. Recombinant thrombomodulin prolonged thrombin clotting time for mouse plasma in a dose-dependent manner. Thrombin was injected into the lateral tail vein of mice and caused acute thromboembolism. All mice injected with thrombin died of thromboembolism; however, preinjection with recombinant human thrombomodulin neutralized the lethal effect of thrombin in a concentration-dependent manner. Histologic examination showed that fibrin deposits were found in all large and small arteries in the lung from mice injected with thrombin; however, fibrin deposits were not detected in any large arteries from the mouse preinjected with thrombomodulin.

A synthetic, tyrosine-sulfated, dodecapeptide (BG8865) modeled on residues 53–64 of hirudin was found to elevate the activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) of human plasma in a dose-dependent manner. The most sensitive assay was the TT, which was prolonged 2 and 3 times control values at 2.2 and 4.1 micrograms/mL hirudin peptide, respectively. The sulfated dodecapeptide exhibited no dependency on antithrombin III as monitored by the APTT in the presence of sheep anti-human antithrombin III antibodies, and its activity was not neutralized by platelet releasates or platelet factor 4. In studies of thrombin-induced platelet activation, the hirudin peptide was found to block aggregation, serotonin release and thromboxane A2 generation. At thrombin concentrations of 0.25 U/mL, the IC50 (concentration resulting in 50% inhibition) for inhibition of platelet aggregation was 0.72 micrograms/mL peptide. Inhibition of TXA2 generation and serotonin release correlated closely with inhibition of aggregation. Using platelets from patients with clinically documented heparin-induced thrombocytopenia anticoagulant doses of heparin were found to induce platelet aggregation and thromboxane A2 generation. In sharp contrast, anticoagulant-equivalent doses of hirudin peptide had no effect on patient platelets, as evidenced by a lack of platelet aggregation and thromboxane A2 generation. These data provide compelling in vitro evidence that the hirudin peptide has several potential advantages over heparin, namely effective inhibition of thrombin-induced platelet activities, co-factor independence, insensitivity to endogenous heparin- neutralizing factors, and an apparent lack of direct or immune-mediated platelet stimulating properties.

Introduction: MST-188 (purified poloxamer 188) is a tri-block co-polymer with high affinity to hydrophobic cellular surfaces that inhibits hydrophobic adhesive interactions in the circulation. It also facilitates blood flow by reducing blood viscosity and reportedly exhibits anti-adhesion and anti-inflammatory properties. Currently, this agent is under study in with patients experiencingsickle cell crisis and in patients with acute limb ischemia. Since MST-188 may be administered with other anti-coagulant agents such as heparin its potential interaction to modulate the anti-coagulant effects of this drug require experimental validation. These studies are designed to investigate the potential interactions between heparin and MST-188 in a rat model of tail transection bleeding and jugular vein clamping induced thrombosis model. Materials and Methods: The in vitro interactions between MST-188 were investigated by supplementing this agent to normal rat plasma (NRP) and heparinized rat plasma at a fixed concentrations of 1.25 and 2.50 mg/mL. The concentration of heparin was kept at 1.25 and 2.50 μg/mL. In the in vivo studies individual groups of rats (n=6-8) were administered with saline as a control, heparin in the dosage range of 125-500 ug/kg intravenously and MST-188 at 25 mg/kg IV followed by heparin at the 125-500 ug/kg dosages. Rat tail resection time was measured 5 minutes after administration of heparin and clot occlusion index was measured as number of jugular vein clamping required to occlude the blood vessel. After the completion of the procedure blood samples were obtained through cardiac puncture and used for ex vivo analysis of PT, aPTT, heptest and thrombin time. Results: In the in vitro studies heparin produced a concentration dependent prolongation of the aPTT, heptest and thrombin time. MST-188 did not produce any effects on the aPTT and heptest time, however it decreased the thrombin time. MST-188 at a higher concentration of 2.5 and 5.0 mg/ml produced a shorting of heparins anticoagulant responses, as measured by aPTT and thrombin time. Heparin produced a dose dependent increase in both the bleeding time (p Conclusion: These studies suggest a differential response of MST-188. While in vitro it exhibits a prohemostatic response as evident by shortening of thrombin time, in the in vivo setting it enhances the anticoagulant effects of heparin as evident by increased bleeding time and increased number of jugular vein clamps required to occlude the vessel. Thus, both of these mechanisms are involved in the mediation of the beneficial effects observed with this agent in vaso-occlusive and thrombotic processes. Disclosures Emanuele: Mast Therapeutics: Employment.

Introduction Hemostasis in childhood differs from that of adults; and the hemostatic system is still developing during childhood. These differences offer a protective advantage to children with hemorrhagic and thrombotic complications. Plasma levels of coagulation factors (except for fibrinogen, factor V and factor VIII), as well as plasma levels of protein C, protein S and antithrombin are reduced. Hereditary dysfibrinogenemia is a rare disorder wherein an inherited abnormality in fibrinogen structure may result in defective fibrin function and/or structure. Clinical symptoms may vary from asymptomatic to life-threatening bleeding complications. Venous or arterial thrombotic complications occur extremely rarely during childhood. Fibrinogen, a key component in hemostasis, is a 340-kDa glycoprotein. The molecule consists of three different pairs of polypeptide chains (Aα, Bβ, and γ) each encoded by a distinct gene (FGA, FGB, and FGG). N-terminal parts of the Aα chain - fibrinopeptides A and the Bβ chain - fibrinopeptides B are situated in the central part of the molecule and block polymerization of the molecules. Conversion of fibrinogen to fibrin occurs after the cleavage of N-terminal fibrinopeptides by the serine protease thrombin. Correct conformation of fibrinopeptides is important for the cleavage by thrombin. Methods Routine coagulation tests were performed with citrated plasma samples on a STA-R coagulation analyzer. The functional fibrinogen level was measured by the Clauss method. Total fibrinogen level was determined by an immunoturbidimetric assay performed on a UV-2401PC spectrophotometer. Fibrin polymerization induced by either thrombin or reptilase and fibrinolysis experiments were obtained by the turbidimetrical method. Fibrinopeptide release was measured as a function of time; and the fibrinopeptides were determined by the reversed-phase, high-performance liquid chromatography (RP-HPLC) method. The purified genomic DNA was amplified by polymerase chain reaction, using specific primers; and dideoxysequencing was performed with Dye Terminator Cycle Sequencing with a Quick Start kit and a CEQ 8000 genetic analysis system). Results We have examined two unrelated boys aged 4 (boy 1) and 14 (boy 2) for susp. dysfibrinogenemia. Routine coagulation tests revealed prolonged thrombin and reptilase time in both boys and also showed decreased functional fibrinogen levels in both kids (Tab. 1). Boy 1 presented with bleeding manifestation – easy bruising, epistaxis, and bleeding after tooth extraction. His 26-year-old mother presented with similar coagulation findings and with mild bleeding complications. Boy 2 was asymptomatic. Fibrin polymerization experiments carried out on plasma samples showed prolonged lag time and significantly reduced final turbidity in both cases. Measurement of kinetics of fibrinopeptide release showed a decreased amount of the released fibrinopeptide A in both patients. DNA sequencing of boy 1 revealed a point mutation in exon 2 of the FGA gene at the position 3456 G/A, which causes the substitution of Aα 16 Arg to His (fibrinogen Praha V). The boy was found to be heterozygous for the mutation as well as his mother. DNA analysis of boy 2 revealed the same point mutation in the FGAgene, causing the same substitution of Aα 16 Arg to His (fibrinogen Kralupy nad Vltavou). Conclusion In this study we report two cases of congenital defects in the fibrinogen Aα Arg16-Gly17 bond found during childhood. It has been described earlier that the replacement of Aα 16 arginine by histidine decelerates thrombin catalyzed fibrinopeptide A release. Although both kids presented with the same genetic defect and with similar coagulation results, they have different clinical manifestation of the disease. Acknowledgment This work was supported by the project of the Ministry of Health of the Czech Republic for conceptual development of the research organization 00023736, by Grant from the Academy of Sciences, Czech Republic (P205/12/G118), and by ERDF OPPK CZ.2.16/3.1.00/28007. Table 1: Routine coagulation test results Boy 1 Boy 1's mother Boy 2 Normal APTT 40.1 s 34.6 s 35.3 s 27.5 - 36.1 s Prothrombin time 17.5 s 14.4 s 16.9 s 11.7 - 15.1 s Thrombin time 40.9 s 36.2 s 44.8 s 17.6 - 21.6 s Reptilase time 58.3 s 51.6 s 55.7 s 16.0 - 20.0 s Fibrinogen (Clauss) 0.51 g/l 1.12 g/l 0.58 g/l 2.00 - 4.20 g/l Fibrinogen (Immuno) 2.47 g/l 2.68 g/l 2.31 g/l 2.00 - 4.20 g/l Age 4 26 14 - Disclosures No relevant conflicts of interest to declare.

Background: The most frequent side effect associated with anticoagulant therapy is bleeding. Testing reversal strategies in patients is difficult due to the unpredictability, rarity and heterogeneity of these events. Currently methods to reverse this bleeding are being tested in a variety of clinical settings in volunteers by using reversal of anticoagulation as a marker of reversal of bleeding in patients. However, the limitation of volunteer studies is the lack of understanding between the assay being used to measure anticoagulation and if its reversal has any relevance for bleeding reversal in patients. A marker of bleeding that was safe to use in volunteers and also relevant for predicting reversal of bleeding in a patient would be advantageous. Objective: The feasibility of two markers of bleeding - fibrin formation (fibrinopeptide A, FPA) in blood as it exits a wound and blood loss measured via a washed blood method, both at a superficial wound site - were tested in pigs under high dabigatran anticoagulation. Reversal of dabigatran anticoagulation and bleeding following trauma injury were evaluated with idarucizumab and outcomes compared to the markers of bleeding from superficial wound. Methods: After ethical approval dabigatran etexilate (30 mg/kg p.o. twice daily, n=24) or placebo (n=6, no dabigatran anticoagulation) was administered to male pigs for 3 days. On Day 4, the pigs were anesthetized and given a 90 min infusion of active dabigatran. A standardized blunt liver injury (0 min) was performed and animals underwent hemorrhagic shock, 15 min later were randomized to receive idarucizumab (30, 60 or 120 mg/kg) or vehicle. Blood loss was measured at defined time points over 4 hrs post injury. At the same time points a standardized cut was made on the inner side of the ear using an adult Surgicutt® device, one cut for each method. Shed blood was collected from the wound site as it emerged over 4 min, placed in protease inhibitor solution and processed for FPA measurement by ELISA. For washed blood method, blood as it exited the wound in 10 sec intervals was diluted in saline into 96 wells and measured photometrically. AUC over 15 min was recorded as blood loss. Blood samples were collected over time to measure active dabigatran by diluted thrombin time (dTT). Data shown as mean ± SE. Results: Cumulative blood loss was 630 ± 56 mL in the placebo group and 2977 ± 129 mL in the dabigatran-treated group. There was a dose-dependent reduction in blood loss by 47% (1586 ± 253 mL), 64% (1064 ± 40 mL) and 62% (1140 ± 44 mL) following treatment with 30, 60 and 120 mg/kg idarucizumab (p FPA in blood from the wound in dabigatran-treated groups was decreased ~74% vs non-anticoagulated group (211.2 ± 86 vs 53.7 ± 13 ng/mL). FPA increased by 2.6-fold (136.3 ± 96 ng/mL), 1.4-fold (103.9 ± 59 ng/mL) and 6.2-fold (251.9 ± 139 ng/mL) over baseline following 30, 60 and 120 mg/kg idarucizumab, respectively. At 240 min, FPA levels were 12%, 25% and 52% of non-anticoagulated animals with increasing idarucizumab doses and correlated with levels of dabigatran at 240 min. Washed blood loss (AUC) in dabigatran-treated animals was increased ~7-fold prior to injury vs non-anticoagulated groups (2110 ± 420 vs 309.1 ± 11.2 OD*sec). There was a dose-dependent reduction in washed blood loss by 2.5% (1682 ± 688 OD*sec), 48% (889 ± 304 OD*sec), and 79% (370 ± 97 OD*sec) 30 min following 30, 60 and 120 mg/kg idarucizumab vs dabigatran-treatment (1724 ± 298 OD*sec). Washed blood loss was comparable to the non-anticoagulated group (186 ± 59 vs 281 ± 201 OD*sec) with 120 mg/kg idarucizumab after 240 min, also correlating with lack of dabigatran anticoagulant activity. Conclusions: This study shows that bleeding and its reversal with a dabigatran-specific antidote can be evaluated using alternative methods that measure FPA levels in shed blood and washed blood loss at non-trauma superficial wound sites. The reversal of blood loss and FPA generation correlated with reversal of bleeding from a trauma wound. These methods may serve as potential markers in experimental bleeding and trauma models to sensitively assess blood loss and to also monitor reversal agents. Disclosures Grottke: Boehringer Ingelheim : Consultancy, Research Funding; CSL Behring: Research Funding. Honickel:Boehringer Ingelheim : Travel support Other. Schurer:Boehringer Ingelheim Pharma GmbH & Co KG: Employment. van Ryn:Boehringer Ingelheim Pharma: Employment.

BACKGROUND Dabigatran etexilate has received its market authorization for various indications worldwide. It was developed to be used in fixed dose regimens without the need of regular monitoring. However, the perioperative management of dabigatran could require an assessment of the drug plasma levels to ensure a safe use of the product, especially in absence of specific antidotes. The EMA stated that dabigatran concentrations under 48 ng/mL should be reached before invasive intervention. The GIHP put the threshold at 30 ng/mL. Therefore, a specific laboratory assay, accurate in the low concentration range, easily available and performable 24h/7 is requested but, until now, all coagulation tests dedicated to the measurement of plasma dabigatran concentrations showed a lower limit of quantitation from 30 to 50 ng/mL. This limits their perioperative utility and the thrombin time (TT) is currently presented as an alternative due to its very high sensitivity to dabigatran. This interesting approach has limitations because TT is affected by several analytical and biological variables that could lead to misinterpretations and expose the patient at riskier hemostatic conditions. In this study, we propose to investigate the performance of two coagulation tests (the Hemoclot Thrombin Inhibitors LOW (HTI LOW) (Hyphen BioMed) and the Ecarin Chromogenic Assay II (ECA-II) (Diagnostica Stago)) specifically developed to measure low plasma dabigatran concentrations and to compare their results with a reference LC-MS/MS. We also assessed the performance of the standard procedure of HTI and TT. MATERIALS AND METHODS Thirty-three plasma samples of patients treated with dabigatran etexilate for stroke prevention in non-valvular atrial fibrillation, were included in the study. Plasma samples were taken randomly and included after a first screening using the HTI to select plasma concentrations For HTI, tested plasma was diluted 1:8 in Owren-Koller® buffer. Fifty μl of tested plasma were mixed with 100 μl of normal pooled plasma and were incubated during 240 sec. One hundred μl of highly purified human thrombin pre-incubated at 37°C was then added to start the reaction. For HTI LOW, the dilution of the sample was reduced to 1:2 and specific calibrators at lower concentrations were used. For ECA-II, tested plasma was diluted 1:5 in Owren-Koller® buffer. Fifty μl of tested plasma were mixed with 140 μl of prothrombin and then 70 µl of chromogenic substrate were added and incubated during 240 sec. Seventy μl of ecarin pre-incubated at 37°C were then added to start the reaction. Thrombin time was performed using Thrombin Time® reagent 1.5NIH (Diagnostica Stago) and the limit of measurement was extended to 300 sec. All of these procedures were performed on a STA-R Evolution® coagulometer (Diagnostica Stago). RESULTS AND DISCUSSION The plasma concentrations ranged from 0 to 200 ng/mL as provided by LC-MS/MS measurements. Among these samples, 17 were between 0 and 50 ng/mL. Linear correlation, Spearman correlation and Bland-Altman analyses versus LC-MS/MS are provided in Figure 1, for assays that express results in ng/mL, i.e. HTI, HTI LOW and ECA-II. For TT, the relation is described by linear correlation. Our results show that HTI LOW performs better than HTI and ECA-II on the whole concentration range providing closer correlation and a lower systematic difference compared to LC-MS/MS. For concentrations below 50 ng/mL HTI LOW and ECA-II reveal similar systematic difference with higher 95% CI for ECA-II and perform better than HTI to estimate plasma concentrations. Thrombin time is less useful than dedicated assays to provide an estimation of the pharmacodynamics of dabigatran. For concentrations above 50 ng/mL it often exceeds the limit of measurement, i.e. 120 sec as defined by the manufacturer, and is influenced by the level of fibrinogen. However, it gives an acceptable correlation for concentration below 50 ng/mL. This may help the biologist to choose which test to use to avoid unnecessary costs and ensure the use of the more accurate dedicated assay to estimate the plasma concentrations. When TT is lower than 120 sec, HTI LOW or ECA-II should be preferred to HTI. CONCLUSION We recommend the use of specific coagulation assays to assess the dabigatran plasma concentrations before invasive procedure. Thrombin time may guide the biologist on the dedicated coagulation test to perform if he uses the HTI platform. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction: It is well know that most of the coagulation factor increase during pregnancy in healthy women. Nevertheless the uninterrupted course of coagulation parameters from the beginning until the end of a pregnancy in healthy women has not been described yet. Only reference ranges for the third month and the sixth month of pregnancy are evaluated. There aren't any data available for the course of coagulation parameters during pregnancy in women with known coagulation disorders. In 2012 we started a study to investigate reference ranges during pregnancy for all coagulation factors, anticoagulants and activation markers of coagulation in 100 healthy pregnant women and 100 pregnant women with a previously known mild bleeding disorders. The study has been approved by the Ethics Committee Nordrhein. Samples and Methods: We analysed samples of pregnant women by conducting the following tests: Blood count, VWF:RCo, VWF:Ag, VWF:CB, Fibrinogen (Clauss), activities of FII, FV, FVII, FVIII (clotting and chromogenic), FIX, FX, FXI, FXII, FXIII. d-dimer, prothrombin fragment 1.2, Quick, partial thromboplastin time, plasma thrombin time , CRP, proteine S, proteine C, antithrombin, Lupus antigoaculant, ACA, ß2-GP in week 10, 16, 22, 28, 34, 40 and 6 weeks post partum (max. +/- 1) Interim Results: Currently 21 pregnant women were included in our study. 16 obviously healthy women were used to calculate the reference ranges for pregnancy. Because of strict inclusion and exclusion criteria (no previous spontaneous abortion, no previous placenta haematoma, no previous pre-eclampsia and only natural pregnancies) most of the women are in the group of the no known coagulation disorder so far. Discussion/Conclusion: There are signs that defects in the coagulation system can be associated to complications during pregnancy like child loss, intrauterine haematoma and genital bleeding. The evaluation of reference ranges helps to detect and to value coagulation disorders during pregnancy. It might be possible to explain the higher abortion rate in women with mild bleeding disorders by determination of reference values of all pro- and anticoagulants during pregnancy. If a treatment with coagulation factor concentrates can help to prevent miscarriage is still subject of ongoing studies. Determination of reference ranges for coagulation factors in the third trimenon might help to determine the peripartum bleeding risk of women with mild coagulation disorders and to help to decide whether a women needs coagulation factor concentrate during labour. Limitation: Based on the strict inclusion and exclusion criteria the number of patients is small. Disclosures Halimeh: Biotest: The authors declare that they receive research grant from Biotest AG Other. Rott:Biotest: The authors declare that they receive research grant from Biotest AG Other. Osseiran:Biotest: The authors declare that they receive research grant from Biotest AG Other. Kappert:Biotest: The authors declare that they receive research grant from Biotest AG Other. Siebert:Biotest: The authors declare that they receive research grant from Biotest AG Other.

One of the most popular features utilized to promote direct oral anticoagulants diffusion in stroke prevention in non-valvular atrial fibrillation is that these drugs do not require monitoring of their anticoagulant activity or plasma level measurements. We believe that there are clinical scenarios in which monitoring can help guide clinical management. Here we describe a clinical case in which close monitoring of thrombin inhibitor dabigatran peak plasma levels helped in maintaining the patient drug administration schedule, despite the interference of mirabegron, a beta-3 adrenergic agonist, utilized for the symptomatic treatment overactive bladder (OAB) syndrome, and of amiodarone, an effective and commonly prescribed antiarrhythmic drug. These P-glycoprotein (P-gp) inhibitors drugs may increase the blood levels and effects of dabigatran. Combining these medications may increase the risk of bleeding complications and anemia, especially when associated to mild to moderate renal insufficiency. Dabigatran etexilate is a pro-drug that has a low (3%-7%) bioavailability. Once dabigatran etexilate is absorbed, the pro-drug is hydrolyzed to the active drug, dabigatran, by carboxylesterases in the bloodstream. Dabigatran etexilate is a substrate of the efflux transporter P-gp, but dabigatran, the active drug, is not. P-gp is present on the luminal side of absorptive cells in the small intestine and takes drug molecules from the cell cytoplasm and transports them back into the intestinal lumen for excretion. An inhibitor of P-gp, as mirabegron or amiodarone, will increase bioavailability of a P-gp substrate by reducing drug efflux. Our patients suffers from persistent non-valvular atrial fibrillation, complicated by ischemic stroke. He has been assigned a dabigatran 150 mg b.i.d. treatment in may 2014. His creatinine clearance was 75 mL/min. One month later, due to OAB syndrome, he started mirabegron, a drug which might potentially increase the risk of bleeding and which generally causes to stop dabigatran administration. We decided instead to set up a monitoring plan, utilizing plasma-diluted thrombin time to measure dabigatran concentrations. Calibration material consisted of pooled normal plasma with known quantities of dabigatran. By using this calibration material, we constructed a dose-response curve ranging from 0 to 500 ng/mL of dabigatran. An examination of the published literature of the pharmacology of dabigatran indicated that the expected peak steady-state concentration of dabigatran in patients with atrial fibrillation (150 mg, 2 times daily) was approximately 180 ng/mL with a trough of approximately 90 ng/mL (12 hours after last dose). After 4 weeks of mirabegron administration, dabigatran plasma trough level of our patient was 126.4 ng/mL. We therefore decided not to modify the 150 mg b.i.d. treatment. Three weeks later, patient suffered from acute cardiac failure associated to a creatinine clearance reduction to 41 mL/min. As a consequence, mirabegron treatment was stopped. At this point, dabigatran plasma trough level was 148.6 ng/mL. Again, we decided not to modify the 150 mg b.i.d. treatment. Six weeks later, due to an elevated cardiac rate (140/min) a treatment with 300 mg o.d. of amiodarone was started. A week later dabigatran plasma trough level was 219.6 ng/mL, with a creatinine clearance of 58mL/min. We decided then to reduce the administration of dabigatran to 110 mg twice daily. After two weeks, dabigatran plasma trough level lowered to 120.2 ng/mL. Our patient is still on dabigatran treatment which has so far never been stopped. At this time, studies addressing the correlation of dabigatran concentration with risk of bleeding (overdose) or breakthrough thrombosis (underdosing) are unavailable. The lack of such studies currently limits the predictive power of the plasma-diluted thrombin time to determine hemorrhage/thrombosis risk. However, we believe that in situations like our patient’s the adoption of this assay would be a valuable tool to aid adjusting dabigatran treatment. With access to appropriate pharmacodynamic and pharmacokinetic data and relevant calibration material, the plasma–diluted thrombin time assay can easily be applied for use in the monitoring dabigatran etexilate. Disclosures No relevant conflicts of interest to declare.

The direct thrombin inhibitor dabigatran etexilate is a novel oral anticoagulant agent. The drug is used for the prevention of stroke and systemic embolism in nonvalvular atrial fibrillation. There is no specific antidote for the reversal of the anticoagulant effects of dabigatran etexilate. Therefore, the management of the clinical bleeding due to dabigatran etexilate represents a great challenge for the clinician. We would like to share our experience regarding the massive life-threatening hemorrhages due to toxic oral intake of 3750 mg dabigatran etexilate as a suicide attemp and the clinical management. A 83-year-old man with a medical history of atrial fibrillation, coronary artery disease, hypertension and myelodisplastic syndrome presented to the emergency room with massive hematuria. Because of atrial fibrilation, he was using dabigatran etexilate as a prophylaxis of stroke or systemic embolism. He had received about 50 capsules of dabigatran etexilate (3750 mg) with the purpose of suicide, 30 hours before transfer to hospital. On the admission, physical assessment showed hypotension, massive hematuria and spontaneous pethechial areas and purpuras on the skin. The thrombin clotting time (TT) was 130 s (normal range 20- 30 s), activated partial thromboplastine time (aPTT) was 59 s (normal 22-35 s), prothrombin time - international normalized ratio (INR) was 1,4. Complete blood count showed that hemoglobin was 7,4 g/dL and platelets were 30000 /µL. The patient received 1000 U prothrombin complex concentrates (PCC), for two days. And 2 units of erytrocyte suspensions were administered. The bleeding ceased within the first day and the thrombin time normalized 3 days later. Dabigatran etexilate is a safer drug than the other anticoagulants. When compared to the warfarin and enoxaparin, dabigatran etexilate has been shown to reduce the rates of bleeding. However, the patients receving dabigatran etexilate have still a risk of bleeding. Because of the predictable pharmacokinetic and pharmacodynamic profile of dabigatran etexilate, there is no need for routine therapeutic coagulation monitoring while using the drug. In case of active bleeding, a possible overdose, or the need for an invasive procedure, the monitoring gains importance. TT and aPTT are the most sensitive laboratory assays to determine the dabigatran etexilate levels. Since the prothrombin time is not affected by dabigatran etexilate, it is not sensitive. There is a strong correlation between the TT measurements and dabigatran etexilate plasma levels. So in monitoring effectivity of therapy or determining the dabigatran etexilate levels in plasma, thrombin clotting time is very useful. However, at dabigatran etexilate concentrations above 600 ng/mL, the maximum measurement of the test is exceeded, correlating to a TT of greater than 120 seconds. Our patient on admission had an elevated TT of >120 seconds indicating accumulation of dabigatran etexilate with levels >600 ng/mL and massive hematuria. There were a bleeding associated dabigatran etexilate and overdose of drug. In order to stop bleeding we need to reverse anticoagulant effects of dabigatran etexilate. There is no specific antidote for dabigatran etexilate and there is limited guidance for treatment in these situations. Fortunately, dabigatran etexilate has short half-life which provides a relatively quick decline in plasma concentrations after discontinuance of the drug in a patient with normal renal function In case of overdose, it is suggested that discontinuing dabigatran etexilate therapy, initiating supportive care, the potential use of hemodialysis, and investigating the source of the bleed. In patients who require more urgent reversal of their anticoagulation, the use of prothrombin complex concentrates (PCCs) may be helpful. PCCs contain vitamin K–dependent coagulation factors II, VII, IX, and X. We started the patient PCCs at the dose of 1000 IU/d and supportive care. The bleeding was ceased in the first day and there was no need to hemodialysis. Thus, the advantages of using dabigatran etexilate instead of warfarin will lead to widespread use. But it must be kept in mind that dabigatran etexilate is not completely innocent drug and the patients using dabigatran etexilate still have the significant risk of bleeding. And acute reversal of the effects of dabigatran etexilate in the actively bleeding patient is a significant challenge. Disclosures No relevant conflicts of interest to declare.

Introduction Anti-thrombin antibodies are a rare cause of acquired bleeding disorder. We report a case of a patient with a monoclonal gammapathy of unknown significance (MGUS) referred to us for an acquired bleeding tendency. We found that he had an isolated anti-thrombin antibody and showed that this antibody inhibit platelet accumulation and fibrin generation in vivo in a mouse model of arterial thrombus formation. Patient A 40-year-old man with no personal or familial history of bleeding was referred to us for multiple bleeding episodes (rectal bleeding, hematoma) for 18 years. He was diagnosed 12 years previously with an IgG Kappa MGUS which was stable. Complete blood counts were normal. Activated partial thromboplastin time (APTT) and thrombin time (TT) were prolonged at 76 seconds (control 35) and > 120 seconds (control 18) respectively. The prolonged TT was observed using human and bovine thrombin. A TT mixing study was performed by measuring the TT of serial dilutions of patient’s plasma in normal pooled plasma. This study confirmed an acquired anti-thrombin activity of patient’s plasma, as TT of the mix was prolonged from dilutions 1:1 to 1:16 (>120 to 25 seconds). Other coagulation factors were normal. No other auto-antibodies were detected except an isolated lupus anticoagulant. Thrombin generation in patient’s platelet rich plasma measured using the Calibrated Automated Thrombogram (CAT) showed a prolonged lag time (15.5 versus 4.7 minutes) associated with a decreased endogenous thrombin potential (ETP, 834 versus 1730 nmol/L min). The diagnosis of an acquired anti-thrombin antibody associated with a MGUS was proposed. The patient received multiple treatments, including immunosuppressive agents, anti-CD20 monoclonal antibody therapy and bortezomib, in order to eradicate the antibody with no success. Methods Anti-thrombin IgG antibodies were affinity purified from the patient’s plasma using activated agarose beads covalently coupled to human alpha thrombin (Aminolink, ThermoScientific). A mouse model of thrombosis was used to test the effect of the infusion of the purified anti-thrombin IgG on thrombus formation in vivo. Platelet accumulation and fibrin generation at the site of injury was imaged after laser-induced injury of cremaster arterioles using intravital microscopy in wild type mice. Platelet and fibrin labeling were performed using anti-CD42 antibody conjugated to DyLight 488 (Emfret) and an anti-fibrin antibody conjugated with AlexaFluor 647 (Sekisui). Results Immunoblotting using human and bovine alpha-thrombin thrombin confirmed the purified IgG from the patient’s plasma (yield 86 µg/ml) to be anti-thrombin antibodies. At a concentration of 2.45 mg/mL, these anti-thrombin IgG were able to prolong APTT and TT (59 versus 33 seconds and >120 versus 19 seconds, respectively). Control IgG (2.5 mg/mL) purified from healthy individuals had no effect on APTT and TT. Using the mouse model of laser-induced arteriolar injury, we found that patient-derived anti-thrombin IgG (690 microg) caused a significant inhibition of platelet accumulation and fibrin generation of more than 70% at the site of vessel wall injury. Conclusion We report a case of severe acquired bleeding tendency secondary to an anti-thrombin antibody in a patient with an IgG Kappa MGUS. Anti-thrombin IgG antibodies purified from the patient’s plasma retain the anti-thrombin activity in vitro. Additionally, infusion of purified anti-thrombin IgG from the patient induced a dramatic decrease in platelet thrombus accumulation and fibrin generation at the site of injury in a mouse model of arterial injury, proving the etiology of patient’s bleeding tendency. This is the first description of the anti-thrombotic effect of anti-thrombin antibodies directly in vivo. Disclosures: Turhan: BMS, Novartis: Honoraria, Research Funding.

Introduction Possibilities to monitor the intensity of dabigatran etexilate (DE) treatment may be valuable before urgent intervention. The Working Group on Perioperative Haemostasis proposed that the drug plasma concentration ([]) should be less or equal to 30ng/mL. However, plasma levels where it is safe to carry out an invasive procedure or surgery have not been confirmed prospectively. In addition, no biological test has been correlated with bleeding risk. Literature showed heterogeneous data regarding the arrest of DE depending on the renal function and the haemorrhagic risk. In addition, physicians request guidance for patients excluded from the clinical trials. Consequently, there is still a need for a rapid and widely available biological test. Some authors make proposals based on activated Partial Thromboplastin Time (aPTT), Hemoclot Thrombin Inhibitor® (HTI) or Thrombin Time (TT). TT displayed several advantages over aPTT. However, TT is affected by numerous analytic variables. Therefore, the objectives of the present study were: 1. To determine the optimal [thrombin] with a variety of instruments and reagents 2. To assess the repeatability of TT at optimized conditions 3. To compare the sensitivity and linearity of TT at residual [dabigatran] (DA) with those of aPTT and HTI 4. To validate the fibrinogen reagent (with heparin inhibitor) for TT experiments Methods DA was spiked at increasing [] in pooled citrated normal human platelet-poor plasma (NPP). The following [DA] were prepared: 0, 5, 10, 20, 30, 40 and 50ng/ml. 1. Optimal [thrombin] determination. The optimal [thrombin] was defined with 2 reagents: bovine thrombin (HemosIL® TT) and human thrombin (STA®-Thrombin) on 4 instruments: STA-R Evolution®, ACLTOP® , CS2000i® and KC10®. TT higher than Tmax is not informative for the physician. Thus the optimized [thrombin] was defined as the maximum [] giving, in a reproducible way, a TT at 50ng/ml lower than Tmax and a TT at 0ng/ml higher than the minimum TT. The Tmax was set arbitrarily at 120 sec. 2. Repeatability The repeatability of optimized TT was assessed by running aliquots of NPP spiked with all [DA] on 10 consecutive days 3. TT (1.5 NIH/ml, STA®-Thrombin), aPTT with SynthasIL® and STA®-C.K.Prest, and HTI were also determined within 1 hour on STA-R® on replicates of all NPP solutions. 4. STA®- Fibrinogen-5 was diluted to the optimized [thrombin] (1.5 NIH/ml) on STA-R® and used as a thrombin reagent. Results 1. The thrombin origin is a more important variable in comparison to the type of coagulometer. At [DA] of 30ng/ml and [thrombin] of 1.5 NIH/ml, the STA®-TT ranges from 56 sec to 74 sec according to the instrument, whereas on a same instrument, the TT varied of minimum 43 sec, depending on the thrombin origin. The optimized [thrombin] are the following : a) STA-R®, STA®-Thrombin : 1.5 NIH/ml, b) STA-R®, HemosIL® TT : 3.8 NIH/ml, c) ACLTOP®, STA®-Thrombin 3.8 NIH/ml, d) ACLTOP®, HemosIL® TT 5.0 NIH/ml, e) KC10®, STA®-Thrombin 1.5 NIH/ml, f) KC10®, HemosIL® TT 5.0 NIH/ml, g) CS2000i®, STA®-Thrombin 1.4 NIH/ml, h) CS2000i®, HemosIL® TT 3.8 NIH/ml. Except for STA®-Thrombin on STA-R®, the [optimized] is not the one recommended by the manufacturer. Figure 1 shows the results of optimized TT according to thrombin origin and coagulometer. 2. Repeatability experiments showed that variability increased with the [DA] (i.e. STA®-Thrombin (STA-R®): coefficient of variation: 8.0% and 28.9% for 0ng/ml and 50ng/ml, respectively) and that the variability depends on the coagulometer and the reagent. 3. Comparison of sensitivity and linearity of TT, aPTT and HTI are presented in Table 1. APTT is not sensitive enough in low [DA], whatever the reagent, whereas HTI is not suitable in [DA] lower than 50ng/ml. 3. TT performed with fibrinogen reagent shows longer times in comparison to thrombin reagent (>120 sec vs 109 sec at 30ng/ml DA). Conclusions TT may be more informative than aPTT and HTI to provide with guidance to carry out an urgent procedure or surgery for patients receiving DE. However, TT is affected by a lot of analytic variables that should be understood by laboratories. Each laboratory should optimize its TT procedure according to its combination coagulometer-reagent. Disclosures: No relevant conflicts of interest to declare.

Introduction The concentration of clottable fibrinogen in plasma decreases rapidly after severe traumatic injury and resuscitation, a reduction associated with both greater transfusion requirements and mortality. Fibrinogen is very sensitive to oxidative modification by oxidants generated during ischemia and inflammation, including hypochlorous acid and peroxynitrite, which both can oxidize methionine to methionine sulfoxide. Here, we investigated the potential of this mechanism to contribute to the coagulopathy of trauma. Methods We obtained plasma, clinical data, and outcomes from a biorepository of trauma patients presenting to the Emergency Department of a local Level I trauma center. Subjects with plasma INR ≥1.3 were defined as being coagulopathic while those with INR Results Trauma patients who were coagulopathic (N=31) required more blood products in the first 24 hours of care compared to non-coagulopathic subjects (N=30) (mean PRBC: 3.9 vs. 0.5 units, p < 0.001; mean plasma transfused: 3.2 vs. 0.2 units, p < 0.001; and mean platelets transfused: 0.8 vs. 0.2 units, p=0.008) and were more likely to die (29% vs. 10% mortality, ChiSqr Likelihood Ratio p=0.056). Methionine sulfoxide was selectively increased in coagulopathic trauma patients at position 476 in the alpha C domain compared to controls [mean(SEM) 1.8(0.1)% vs. 2.5(0.1)%, p=0.003] and this low-level of oxidation was confirmed to be associated with an increasing reptilase time in vitro. Mean thrombin time (16 vs. 17.8 sec, p = 0.01) and reptilase times (18.2 vs. 23.7 sec, p Conclusions The level of oxidation of methionine residues in fibrinogen was increased in coagulopathic trauma patients presenting to the Emergency Department. Even at low levels, this modification was associated with altered fibrin polymerization suggesting that it may contribute mechanistically to coagulopathy by altering fibrin alpha C domain dimerization during the lateral aggregation of fibrin monomer. Disclosures: White: Stasys Medical Corp: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Vidacare Corp: Honoraria; NIH: Research Funding; Coulter Foundation: Research Funding; Washington State Life Sciences Discovery Fund: Research Funding.

Background Children with acute, severe cardiopulmonary compromise may require support with ECMO. Adequate anticoagulation without inducing life-threatening hemorrhage plays a critical role in the management of these patients. Recent data from adult studies suggest that patients undergoing cardiac bypass for coronary artery bypass grafting (CABG) often have reduced Factor XIII levels (FXIII). This relative FXIII deficiency may be predictive of hemorrhage or severe blood loss. Such studies have never been conducted in the pediatric population. Our primary objective will be to determine if a relative deficiency in FXIII occurs in children undergoing ECMO and to determine any time dependency of these changes. Our secondary objective will be to determine if blood loss and/or transfusion requirements during ECMO correlates with FXIII levels. Methods Single center ongoing study utilizing a prospective design involving pediatric patients (ages 0-18) undergoing ECMO. Samples for FXIII, D-Dimers, Fibrinogen and Thrombin Time are being drawn at designated time points as follows: prior to or during ECMO cannulation, 2 hours, 24 hours, 3-5 days and 14-21 days after initiation of ECMO and 1-3 days after discontinuation of ECMO. Mixed effects analysis of covariance (ANCOVA) will be used for data analysis. Results To date, fifteen patients have been enrolled and analyses have been conducted for n=7 patients with a total of 36 time points of data. Figure 1a and 1b show the mean and standard deviation (SD) of FXIII and Fibrinogen, respectively. Figure 1c shows the mean ratio of FXIII to Fibrinogen. Error bars represent 1 SD around the mean (central point). Conclusion and Significance This is the first pediatric study evaluating FXIII activity in children undergoing ECMO. Firm conclusions cannot be drawn with the interim analyses conducted to date. If variations in FXIII are found, our results will provide important information towards determining if children undergoing ECMO could potentially benefit from FXIII replacement, in turn minimizing bleeding complications and/or transfusion requirements often encountered with this treatment modality. Disclosures: No relevant conflicts of interest to declare.

A 88 year-old woman was referred to our center for 3 weeks of gastrointestinal bleeding and a spontaneous right rectus femoris hematoma. The prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT) and reptilase time (RT) were markedly prolonged (Table 1). Assays for coagulation factors V, VIII, IX and X were normal. A fibrinogen concentration of < 50 mg/dl was measured by Clauss method while fibrinogen concentration measured by radial immunodiffusion was 180 mg/dl. Following infusions of cryoprecipitate there was accelerated clearance of fibrinogen as measured by the Clauss method. An inhibitor of fibrinogen function was suspected.Table 1TestTime (sec)1:1 mix (sec)Normal Range (sec)PT>902212-15aPTT70.73923-36TT>120>12015-19Reptilase Time>120>12014.8-20.4 When the patient’s plasma was tested towards immobilized fibrinogen in ELISA plates, a concentration dependent specific binding of IgG was seen while under similar conditions control IgG had no significant binding (Figure 1). The IgG antibody was polyclonal comprising of both kappa and lambda subtypes. The IgG fraction was purified on a Protein A column and the isolated IgG fraction prolonged the thrombin time of normal plasma in a concentration dependent manner. The effect of the isolated IgG fraction on fibrin polymerization was measured by monitoring the absorbance at λ390 in a spectrophotometer. The IgG inhibited fibrin polymerization in a concentration dependent manner. Control IgGs had no significant effect on polymerization under these conditions (Figure 2). The immunologic specificity of antibody was tested in immunoblots of fibrinogen subjected to SDS-PAGE. The patients IgG reacted with fibrinogen in unreduced buffers. Under reducing conditions, the IgG reacted with Bβ and γ chains of fibrinogen while the control IgG did not (Figure 3). The patient was treated with rituximab and steroids. After four, weekly doses of rituximab, fibrinogen levels by Clauss method normalized (450 mg/dl). There was no detectable antibody to fibrinogen and no further bleeding was noted. Conclusion This is a description of a rare case of a polyclonal autoantibody to Bβ and γ chains of fibrinogen that inhibited fibrin polymerization leading to a severe bleeding diathesis. Disclosures: No relevant conflicts of interest to declare.

Abstract 2272 Background and Aim: Dialysis based approaches can provide rapid removal of dabigatran in cases of emergency due to its low protein binding of ∼35%. However the in vitro properties of these filtration devices have not yet been characterized in detail. This study in the porcine system (both in vitro and in vivo) was performed to evaluate dabigatran elimination by hemodialysis and activated charcoal perfusion as compared to normal renal elimination. Methods: Porcine blood (5L) was supplemented with 1000 ng/mL dabigatran and circulated through an circuit of tubing allowing attached to an activated charcoal filter (Absorba 300 C, Gambro). Further supplementation of dabigatran allowed the determination of maximum binding capacity of the filter. A similar set up was used to also test dialysis (Polyflux 140H, Gambro) and determine the dependence of the flow rate on dabigatran removal. Dialysate flow rates were increased up to 500 ml/min. Anesthetized pigs (Domestic swine, female, ∼60 kg) were attached to an activated charcoal column or a High-Flux hemodialysis filter with a blood flow rate of 200 ml/min. Animals were given an initial i.v. infusion of dabigatran (7.5 mg in 15 min) and then reduced to 5 mg/hr to achieve steady state dabigatran over 1hr. Infusion was then stopped and elimination methods were applied over 4 hrs. An observation time of 1 hr followed. Dabigatran plasma levels were quantitated with diluted thrombin time. Preliminary settings/flow rates were obtained in vitro using 5L citrated porcine whole blood exposed to different AC or HD conditions. Results: Activated charcoal completely removed dabigatran within 1 hr from the 5L whole blood supplemented with 1000 ng/mL dabigatran, with a clearance rate of 100%. By repeatedly reapplying dabigatran, it was shown the active charcoal filter had a maximum binding capacity of ∼30 mg drug. Upon saturation there was no further clearance of dabigatran. Hemodialysis removed dabigatran with increasing clearance rates depending on dialysate flow rates (100 ml/min-35%, 200 ml/min-60%, 300 ml/min-65%) reaching a plateau of ∼65%. Further increases of dialysate flow to 500 ml/min had no further effect on drug clearance. Initial plasma levels of dabigatran ranged between 200–450 ng/mL after 60 min infusion in pigs. Exposure to activated charcoal or hemodialysis (dialysate flow 300 ml/min) resulted in 75–80% reduction in circulating dabigatran after 1 hr as compared to ∼25% reduction untreated controls after 1 hr. After 2 hrs dabigatran levels were below the detection limit using both elimination methods. Conclusions: Dabigatran can effectively be removed from the circulation in this in vivo porcine model using dialysis based approaches, which results in a restoration of blood coagulation. Active charcoal perfusion was fast and effectively removed dabigatran, but may be saturated if dabigatran plasma levels are too high (human body load for 150 mg dose in steady state is ∼14g). Stationary hemodialysis with sufficiently high dialysate flows achieves similar results in this model without saturation limitations; however, the set up for dialysis is much more specialized than the simpler approach of activated charcoal filtration. Disclosures: Formella: Boehringer Ingelheim: Employment. Clemens:Boehringer Ingelheim (Anticoagulant Therapy): Employment. van Ryn:Boehringer Ingelheim: Employment. Schenk:Boehringer Ingelheim: Research Funding.

Abstract 22 Background: The objective of this study is to determine the pharmacokinetics (PK) and pharmacodynamics (PD) of dabigatran (a small molecule thrombin inhibitor) and its antidote (a humanized Fab against dabigatran) in the monkey and to develop a combined mechanistic mathematical model to describe the data. Methods: There were three groups: control, antidote alone and dabigatran etexilate (DE) + antidote. Rhesus monkeys (n = 2/group) received either 12 mg/kg/day of DE or vehicle orally on Days 1–4, 15–18 and 29–32 with a single IV dose of the antidote administered 90 minutes after DE on Days 4, 18 and 32. Doses of the antidote were 30, 90 or 175 mg/kg, respectively. PK parameters of the antidote and sum dabigatran (dabigatran plus its glucuronides) were determined after measurements of plasma concentrations. Coagulation activity was measured using a diluted thrombin time assay to determine the activity of the unbound sum dabigatran. Results: The PK of the antidote were not affected by dabigatran. Clearance of the antidote was low (0.87 mL/min/kg) and steady-state volume of distribution was small (0.06 L/kg), indicating that the antidote was mostly restricted to plasma. The plasma profile of the antidote was bi-phasic with a short initial phase t1/2 of 0.4 hour (h) and a terminal phase t1/2 of 4.3 h. Immediately after antidote dosing, plasma concentrations of sum dabigatran increased, a consequence of the rapid redistribution of dabigatran and its glucuronides from tissue to plasma due to binding to the antidote. Complete reversal of dabigatran's anticoagulant activity was observed immediately after antidote dosing at all three dose levels, as measured by the diluted thrombin time assay, which indicates that all dabigatran was bound to the antidote. The degree to which this reversal effect was maintained over an extended period (24 h) was dose-dependent. A mechanistic ordinary differential equation model, based on the mass action kinetics for describing the distribution, binding and elimination of dabigatran and its antidote, was developed by combining the PK models for dabigatran and the antidote and adding the binding interaction (1:1 stoichiometry) between the two compounds. The distribution and elimination parameters of the dabigatran-antidote complex were assumed to be the same as those of the antidote, based on similar measured PK parameters of the antidote with and without dabigatran in the monkey. The combined PK/PD model of dabigatran and antidote was able to describe the in vivo PK/PD data observed in monkeys. Conclusion: The dabigatran-specific antidote successfully reversed the anticoagulant activity of dabigatran in the monkey in a dose-dependent manner, and our combined mathematical model accurately describes monkey PK/PD data of sum dabigatran and its antidote. Insights gained from this model will be used to guide model development for clinical trials. Disclosures: Toth: Boehringer Ingelheim: Employment. Gan:Boehringer Ingelheim: Employment. van Ryn:Boehringer Ingelheim: Employment. Dursema:Boehringer Ingelheim: Employment. Isler:Boehringer Ingelheim: Employment. Coble:Boehringer Ingelheim: Employment. Burke:Boehringer Ingelheim: Employment. Lalovic:Boehringer Ingelheim: Employment. Olson:Boehringer Ingelheim: Employment.

Abstract 3424 Introduction: Dabigatran etexilate is a new orally direct thrombin inhibitor and rivaroxaban is a new oral direct Factor Xa (FXa) inhibitor. The increasing use of the new oral anticoagulants administered for prevention of VTE in patients after orthopedic surgery creates the need of their measurement in the clinical routine. A modified thrombin time based assay can be used for measurement of thrombin inhibitors like dabigatran and the direct Xa inhibitor rivaroxaban can be measured with a chromogenic anti-Xa assay. The aim of this study was to evaluate the performance of the two assays on coagulation analyzers using lyophilized calibrators and controls. Method: The assay for dabigatran measurement is a clotting assay based on the inhibition of a constant and defined concentration of thrombin. Clotting times measured are directly related to thrombin inhibitor concentrations. Calibration of the assay is performed using the lyophilized calibrators with assigned dabigatran values. Lyophilized controls in two ranges with assigned values for dabigatran are measured to calculate recovery and precision. A one stage chromogenic assay for the determination of Xa inhibitor activity in human citrated plasma is used for rivaroxaban measurement. The assay is based on the inhibition of activated factor × (FXa) by the direct Xa inhibitor rivaroxaban as measured by a chromogenic FXa substrate. Lyophilzed calibrators with assigned rivaroxaban values are used for assay calibration. Controls in 3 ranges are measured to calculate recovery and precision. Both assays are performed automated using the coagulation analyzers Ceveron alpha, Coasys Plus C and STA Compact with adapted pipetting schemes for the two anticoagulants on each analyzer. Results: For dabigatran measurement calibration curves in the range of 0–500ng/mL were made on each analyzer and curves with R2=1.0±0.1 were obtained on all analyzers. The recovery of control was ± 10% of target value for all controls on each analyzer. Precission data of the assay are summarized below: For rivaroxaban calibration curves in low range 0–150ng/mL and high range 0–500ng/mL were made using two adjusted analyzer settings. Calibration curves with R2=1.0±0.1 were obtained for rivaroxaban in the range of 0–150ng/mL, as well as for the range of 0–500ng/mL on all analyzers. The recovery of control was ± 10% of target value on all analyzers and precision was very good with intra-assay variations between 0.9%-4.2% and inter-assay variations between 1.8–4.9%. The detection limit for rivaroxaban was 10ng/mL using low range calibration curve and 25ng/mL using high range calibration curve. Conclusions: Our data demonstrate that the diluted thrombin time assay is suitable for mmeasurement of dabigatran and the chromogenic anti-Xa assay is suitable for measurement of rivaroxaban if lyophilized calibrators are used for assay calibration. Optimised pipetting schemes have to be validated for each of the analyzers used in order to obtain results with good precision. Disclosures: No relevant conflicts of interest to declare.

Abstract 23 Background: To date, all published studies assessing the effects of the new oral anticoagulants dabigatran (dabi) and rivaroxaban (riva) on coagulation assays have used normal pooled plasma spiked with known concentrations of anticoagulant and therefore do not reflect the variability expected from patients taking these medications. Methods: We collected citrated venous blood samples from patients taking either dabi (n=43) or riva (n=10) for management of either atrial fibrillation or acute venous thrombosis. All subjects were required to have taken at least 5 doses of drug in order to reflect steady-state conditions and informed consent was obtained. The interval between last dose and phlebotomy ranged from 1 – 18 hours. Platelet-poor plasma was prepared and using the STA-R.. coagulometer (Diagnostica Stago), the following assays were performed: PT/INR using Neoplastin Plus.. (NP) and Innovin.. (IN), aPTT (Actin.. FS), thrombin time (TT), clot-based protein S (PS) activity, free PS antigen, chromogenic protein C (PC) activity, clot-based antithrombin activity, Clauss fibrinogen, dilute Russell's viper venom time (DRVVT), and factor VIII (FVIII) activity at several dilutions (1:10 to 1:160). Hemoclot.. dilute thrombin time and STA.. -Rotachrom.. anti-Xa assays were performed for quantitation of dabi and riva drug levels, respectively, using Aniara commercial plasma calibrators. Results: Median INR levels for patients on dabi were 1.1 (IN) and 1.2 (NP) and for riva were 1.15 (IN) and 1.3 (NP). The proportion of subjects with INR levels above 1.3 for dabi were 15% (IN) and 28% (NP) and for riva were 0% (IN) and 40% (NP). Median aPTT levels were 42s dabi and 32.5s riva. Although 73% of patients on dabi had an elevated aPTT (>38s), 60% of these were only mildly elevated (39–45s). Only one subject on riva had an elevated aPTT. The TT was extremely sensitive to the presence of dabi, as 100% of treated subjects had an elevated TT (>20s) and 73% were above the linearity cutoff (>100s). All subjects on riva had normal TT. As shown in Figure 1A, the clot-based PS assay was affected markedly by dabi, with patients showing artificially high PS activity with increasing drug levels, but no such effect was observed for riva (Figure 1B). For both drugs, the PS free antigen and chromogenic PC assays were unaffected. Figure 1C shows dabi also caused false elevations in antithrombin activity, though it did not affect the Clauss fibrinogen assay, which uses a much higher concentration of thrombin. Riva had no discernable effect on the antithrombin and fibrinogen assays (data not shown). Figure 1D shows that increasing levels of both anticoagulants caused artefactual decreases in FVIII activity. Increasing dilutions of patient plasma caused progressive elevation of FVIII activity, with median activity levels nearly doubling between 1:10 and 1:160 dilutions for both dabi (0.65 to 1.28) and riva (0.81 to 1.41). Finally, the DRVVT assay was prolonged (>20% elevated versus normal plasma) for 86% of dabi and 60% of riva patient plasmas. No differences were noted for any of these results between dabi 110 mg (n=14) and 150 mg (n=29) dosing. Conclusions: For the assay platforms assessed in this study, the majority of patients taking dabi and riva had near-normal INR and aPTT levels. This is discrepant with results from studies using spiked plasma samples. Clinicians should be cautioned that normal INR and aPTT results do not rule out the presence of therapeutic anticoagulant levels and the results should not be used to guide clinical decisions. Clotting-based thrombophilia testing should be avoided for patients taking these medications due to assay interferences, particularly with dabigatran. Disclosures: Teal: Bayer: Consultancy, Honoraria; Boehringer-Ingelheim: Consultancy, Honoraria. Lee:Bayer: Honoraria; Boehringer-Ingelheim: Honoraria.

Abstract 1169 INTRODUCTION: New oral anticoagulants (NOACs) have been recently approved by the European Medicine Agency (EMA) and the Food and Drug Administration (FDA) for several indications. New oral anticoagulants include anti-IIa agent (dabigatran etexilate) and anti-Xa agents (rivaroxaban, apixaban and edoxaban). NOACs do not require monitoring nor frequent dose adjustment. However, searching for the optimal dose in the individual patient may be useful in some situations. Recent studies have shown that aPTT, HTI and ECT could be used to monitor dabigatran whereas PT and anti-Xa chromogenic assays could be used to monitor anti-Xa agents, while standardizing the time between the last intake of rivaroxaban and the sampling is mandatory. These tests only measure the initiation phase of the coagulation cascade. Thrombin generation assay (TGA) which measures the entire thrombin generation process could be used to better discriminate the inhibitory profile of the NOACs in patients. OBJECTIVES: To study the impact of dabigatran and rivaroxaban in patients by thrombin generation assay compared to other traditional coagulometric and chromogenic assays. MATERIALS AND METHODS: Five patients under dabigatran and 5 patients under rivaroxaban for atrial fibrillation were included in this study. Blood samples were taken at different intervals: at Ctrough, 2h and 3h after drug administration. The following tests were performed at each timepoint Rivaroxaban: Prothrombin Time (PT) using the following reagents: Triniclot PT Excel S.. and Innovin.., Thrombin Generation Assay (TGA) using PPP-Reagent and PPP-Reagent High, Biophen Direct Factor-Xa Inhibitor.. (DiXaI). Dabigatran: Activated Partial Thromboplastin Time (aPTT) using CK-Prest.. and Synthasil.., Hemoclot Thrombin Inhibitor.. (HTI) and Thrombin Generation Assay (TGA) using PPP-Reagent and PPP-Reagent Low. All the tests were calibrated by spiking rivaroxaban or dabigatran at increasing concentrations in pooled citrated normal human platelet poor plasma (PPP). RESULTS AND DISCUSSIONS: In vitro Rivaroxaban The Peak and mVRI were the most sensitive CAT parameters with a high sensitivity (Peak IC50 was 3ng/mL with PPP-Reagent Low and PPP-Reagent and 14ng/mL with PPP-Reagent High; mVRI IC50 was 1ng/mL with PPP-Reagent Low and PPP-Reagent and 3ng/mL with PPP-Reagent High) and both reagent showed a low variability (CV The PPP-reagent Low does not allow resolving TGA profiles with different rivaroxaban concentrations. Consequently, PPP-Reagent and PPP-Reagent High were only tested ex vivo. The low sensitivity of the lag time explains the lack of sensitivity of PT, even with the more sensitive reagent (i.e. Triniclot PT Excel S..). Dabigatran Dabigatran mainly delayed the initiation phase with a strong dose-dependent increase of lag time and Tmax and a slight dose-dependent decrease of Cmax and ETP. The concentration in dabigatran needed to double the lag time was 66 ng/mL and 70ng/mL with the PPP-Reagent and the PPP-Reagent Low respectively. The PPP-reagent High is less sensitive in comparison to PPP-Reagent and PPP-Reagent Low. Ex-Vivo Similar concentrations of rivarovaban or dabigatran in different patients provided with Biophen DiXaI.. and HTI are associated with differences in TGA profile (Figure 1 & 2 showing the dispersion of the different measurement). Therefore, anti-Xa tests and dilute thrombin time are not the most accurate tests to use to propose cut-off associated with a bleeding or thrombosis risk. This illustrates the interest to have tests able to evaluate the entire thrombin generation process to propose cut-off in one or more parameters associated with a bleeding or thrombosis risk. CONCLUSION Thrombin generation assay could be superior to traditional coagulometric and chromogenic assays to monitor new oral anticoagulants in terms of prediction of bleeding or thrombosis risk. Disclosures: No relevant conflicts of interest to declare.

Abstract 4366 Introduction: Dabigatran is the first oral anticoagulant since warfarin to be approved by the FDA for prevention of stroke in patients with non-valvular atrial fibrillation. Unlike warfarin, dabigatran monitoring has not yet been recommended. However, dabigatran accounted for 3781 serious adverse events (including 542 deaths) as reported in QuarterWatch by the Institute for Safe Medicine Practices in 2011. This has prompted clinicians to consider situations in which drug level measurements may provide clinically useful information to guide dosage. Aside from managing the acutely bleeding patient, drug level monitoring may be useful in the outpatient setting. For example, establishing a baseline drug level may be relevant given the direct correlation with bleeding risk, especially in patients with decreased creatinine clearance. It can also assess patient compliance given the short half-life of the drug. We performed a retrospective analysis of patient samples that were tested for dabigatranlevels at our facility to determine whether drug levels were within the therapeutic range. Method: Dabigatran levels were assayed for 172 frozen, citrated plasma samples from 127 patients (25 patients had multiple samples). The assay is a laboratory-developed, dilute thrombin clotting time test (CV ≤7.3%) that was validated by LC-MS/MS and is performed using drug-specific calibrators (Clin Chem 2012 abstract, in press). Specimens should be collected 90 to 120 minutes after the last dose and after ≥3 doses. The therapeutic range (64–443 ng/mL) was derived from the PETRO trial (van Ryn et al Throm Haemost2010). Patient demographics were correlated with drug levels, but clinical history was not available. Results: Dabigatran levels were within the therapeutic range for 76 samples (44.2%), in the subtherapeutic range for 82 samples (47.7%), and in the supratherapeutic range for 14 (8.1%) samples. The median dabigatran level for the samples within the therapeutic range was 152 ng/mL. These samples also had a skewed distribution such that 44 (57.9%) were within the lowest quartile and 62 (81.6%) were within the lower half. Table 1 shows the distribution of dabigatranlevels by age and gender. Women ≥65 years of age were the most likely to have levels in the therapeutic range. Independent of gender, people aged Of the 25 patients with multiple samples (2–6 samples/patient), 17 patients were consistently in or out of range: 8 therapeutic, 7 subtherapeutic, and 2 supratherapeutic. The remaining 8 patients were variable (Table 2). Conclusions: (1) A surprisingly high percentage of patients taking dabigatran were subtherapeutic. (2) The cause of the subtherapeutic levels is not readily discernable given the lack of clinical history, but may be explained by poor compliance, improper collection time, drug interactions, or decreased absorption (anecdotal evidence). Disclosures: No relevant conflicts of interest to declare.

Abstract 2273 Introduction Reversal of pharmacologic anticoagulation is an issue that arises when an anticoagulated patient has a major bleeding or when an emergency surgery needs immediate correction of coagulation. However, the new oral anticoagulants (NOAC) Rivaroxaban (anti-FXa) and Dabigatran (anti-FIIa) lack specific antidotes and only limited data is available regarding the antidotal effect of non-specific haemostatic agents. Therefore, in this ex vivo study reversal of anticoagulant activity after the administration of either 20mg Rivaroxaban or 150mg Dabigatran was tested in vitro using two different PCC (Beriplex®, Cofact®), aPCC (FEIBA®; factor eight inhibitor bypassing activity) or rFVIIa (NovoSeven®) at various concentrations. Patients and Method Subjects 10 healthy caucasian subjects (female: n=5; male: n=5) were first randomized to receive either 20 mg Rivaroxaban or 150 mg Dabigatran in one oral dose. The patients had to abide a 7-day wash-out period before administration of the respective second drug. Methods Citrated venous blood was taken right before (T0) and 2 hours after administration (T2) of either Dabigatran or Rivaroxaban. The potential of 4 commercially available haemostatic agents to reverse the anticoagulant effect of the NOACs was evaluated. • Beriplex: 0.25; 0.5 (corresp. to 25 U/kg); 1; 2 U/mL • Cofact: 0.25; 0.5 (corresp. to 25 U/kg); 1; 2 U/mL • FEIBA:0.25; 0.5; 1 (corresp. to 80 U/kg); 2 U/mL • NovoSeven: 1.25; 2.5; 5; 10 μg/mL (a dose of 90–100 μg/kg corresponds to a plasma level of about 2 μg/mL) Thrombin generation using Calibration Automated Thrombinography (CAT) was the primarily applied assay, along with the parameters aPTT, PT, thrombin time, Rivaroxaban- and Dabigatran levels and Ecarin time. Parameters of interest concerning the thrombin generation assay (TGA) were the endogenous thrombin potential (ETP), Peak (maximum reaction velocity) and Lag time (LT; length of the latent phase). Thrombin generation in platelet poor plasma was initiated by adding 1 pM tissue factor and 4 μM phospholipids. Results 2 hours after administration (T2), Rivaroxaban showed remarkable inhibitory effects on the investigated TGA parameters ETP, Peak and Lag time, with a more pronounced inhibitory effect on the Peak. In contrast, Dabigatran at T2 only showed a slight effect on ETP and no effect on the Peak whereas LT was significantly prolonged (fourfold). Rivaroxaban-induced inhibition of ETP and Peak were reversed by FEIBA in a concentration dependent manner with an over-correction for the two highest concentrations (1 and 2 U/mL). Cofact and rFVIIa restored ETP dose-dependently and both reached baseline T0 at their highest concentrations. Compared to FEIBA, rFVIIa and Cofact only had a slight but dose-dependent effect on the Peak. Interestingly, the other PCC, Beriplex, did not show any reversal effects on ETP and Peak. Regarding Rivaroxaban-prolonged LT all concentrations of rFVIIa and FEIBA were responsible for a significant LT-reduction close to baseline, whereas both PCCs did not correct prolonged LT. Regarding Dabigatran, all doses of rFVIIa, Cofact and FEIBA reduced the LT, with a more pronounced and dose-dependent effect of Cofact and FEIBA. For Beriplex only a slight reduction in LT was observed. Conclusion FEIBA and rFVIIa showed significant reversal of anticoagulant activity already at low therapeutic concentrations for both anticoagulants in TGA. Surprisingly, regarding both investigated PCCs, only Cofact showed an antidotal effect on TGA parameters, especially in higher therapeutic concentrations (1 and 2 U/mL). Considering that the main difference between the two PCCs is that Beriplex contains small amounts of heparin and Cofact does not, it is conceivable that this may explain the observed differences in TGA. Further clinical validation is needed. Disclosures: No relevant conflicts of interest to declare.

Abstract 2271 Introduction: Dabigatran and Rivaroxaban have both been approved in Canada for the prevention and treatment of thrombosis. Although they have different mechanisms of action; they both share the same advantage over warfarin that is their predictable anticoagulation effect, which precludes the need for routine monitoring. However, there are special situations (emergent surgery, bleeding, therapeutic failure) where dosing would be clinically relevant. A major concern is that patients treated with these drugs may present themselves in non-tertiary centers where the availability of specialized coagulation assays is usually limited, especially in an emergency setting. So, although specific monitoring assays are under development for dabigatran and rivaroxaban, we sought to investigate the widely available coagulation assays prothrombin time (PT), international normalized ratio (INR), activated partial thromboplastin time (aPTT) and thrombin time (TT) as means to estimate plasma levels of these new anticoagulants. Study design: Our study aimed at emulating real life situations. We purposely recruited academic (15) and non-academic centers (26), to reflect the diversity of the laboratories in the province of Quebec, Canada. Method: Solutions containing increasing concentrations of dabigatran (from 0,01 μmol/mL to 0,48 μmol/mL) and rivaroxaban (from 0,012 μmol/mL to 0,490 μmol/mL) were prepared. Frozen aliquots of each solution were shipped to each of the 41 participating center (blinded for concentration). Each laboratory then performed automated assays for PT, aPTT and TT, according to each local protocol. Results: Dabigatran The TT was exquisitely sensitive to dabigatran but was not available in every hospital (available in 25 out of 41). All values were three times above the normal value even for low concentrations (> 0,03μg/mL). With regards to aPTT, we were able to demonstrate a difference in sensitivity of different cephalins for low concentrations of dabigatran. The value was abnormal in 74% centers for a concentration of 0,01 μg/mL. Whereas no abnormal value was obtained with APTT-SP, all values were above normal for CK-PREST. As for high concentrations, normal values were obtained in 5% of centers for a concentration of 0,06 μg/mL. An aPTT value over 80 was obtained for 25% of centers testing the 0,26 μg/mL concentration and of 68% centers with the 0,48 μg/mL samples. We thus have shown that a normal aPTT could be obtained, although rarely, with concentrations that are within or above the therapeutic range. Accordingly with what has already been published, our study demonstrated that INR was less sensitive to dabigatran than aPTT. Rivaroxaban The TT was not significantly prolonged with therapeutic concentrations of rivaroxaban (at the highest concentrations we observed a slight prolongation with a ratio on normal value < 1,5). INR prolongation with rivaroxaban was linear and we observed a variation in sensitivity with the 5 studied thromboplastins. Neoplastin C1 and Neoplastin C1 plus showed the greatest sensitivity while Thromborel S showed the least. We were able to demonstrate a statically significant difference between high ISI (>1,15) and low ISI ( Conclusion: Dabigatran and Rivaroxaban represent a new challenge for hemostasis laboratories. Our study reflects the diversity of coagulation assays used throughout the province of Quebec. The variation in sensitivity that was observed between laboratories makes it difficult to provide broad recommendations on the use of common coagulation assays as a dosing method for dabigatran or rivaroxaban. We suggest that each center provide recommandations based on a calibrator generated dose response curve before using these assays in a clinical setting. Disclosures: No relevant conflicts of interest to declare.