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Vitamin K-dependent matrix Gla protein (MGP) has been suggested to play a role in the inhibition of soft-tissue calcification. Here we report the expression of recombinant prokaryotic MGP as part of a fusion protein and the preparation of two antibodies that specifically recognize MGP. Monoclonal antibodies were raised against synthetic peptides homologous to the sequences 3-15 and 63-75 of human MGP. Both antibodies recognize recombinant and synthetic human MGP. Immunohistochemical analysis showed that MGP was associated with the extracellular matrix of noncalcified bone and with chondrocytes in cartilage. In the healthy human arterial vessel wall, MGP antigen was demonstrated in association with smooth muscle cells and elastic laminae of the tunica media and with the extracellular matrix of the adventitia. Colocalization with the elastic laminae was lost at sites of medial calcification; in both human and rat arteries, high amounts of MGP were found in the extracellular matrix at borders of intimal and medial calcification. Our data demonstrate the close association between MGP and calcification. It is suggested that undercarboxylated MGP is biologically inactive and that poor vascular vitamin K status may form a risk factor for vascular calcification.

Matrix gamma-carboxyglutamic acid protein (MGP) is a mineral-binding extracellular matrix protein synthesized by vascular smooth muscle cells (VSMCs) and chondrocytes that is thought to be a key regulator of tissue calcification. In this study, we identified four polymorphisms in the promoter region of the human MGP gene. Transfection studies showed that the G-7A and T-138C polymorphisms have an important impact on in vitro promoter activity when transiently transfected into VSMCs. We found that one of these polymorphisms (T-138C) is significantly correlated with serum MGP levels in human subjects. Promoter deletion analysis showed that this polymorphism lies in a region of the promoter critical for transcription in VSMCs. This region contains a potential activating protein-1 (AP-1) binding element located between -142 and -136. We have demonstrated that the T-138C polymorphism results in altered binding of an AP-1 complex to this region. The -138T allelic variant binds AP-1 complexes consisting primarily of c-Jun, JunB and its partners Fra-1 and Fra-2 in rat VSMC. Furthermore, the -138T variant form of the promoter was induced following phorbol 12-myristate 13-acetate treatment, while the -138C variant was refractive to phorbol 12-myristate 13-acetate treatment, confirming that AP-1 factors preferentially bind to the -138T variant. This study therefore suggests that a common polymorphism of the MGP promoter influences binding of the AP-1 complex, which may lead to altered transcription and serum levels. This could have important implications for diseases such as atherosclerosis and aortic valve stenosis, since it strongly suggests a genetic basis for regulation of tissue calcification.

Objectives To provide a rational basis for recommended daily allowances (RDA) of dietary phylloquinone (vitamin K1) and menaquinone (vitamin K2) intake that adequately supply extrahepatic (notably vascular) tissue requirements. Background Vitamin K has a key function in the synthesis of at least two proteins involved in calcium and bone metabolism, namely osteocalcin and matrix Gla-protein (MGP). MGP was shown to be a strong inhibitor of vascular calcification. Present RDA values for vitamin K are based on the hepatic phylloquinone requirement for coagulation factor synthesis. Accumulating data suggest that extrahepatic tissues such as bone and vessel wall require higher dietary intakes and have a preference for menaquinone rather than for phylloquinone. Methods Tissue-specific vitamin K consumption under controlled intake was determined in warfarin-treated rats using the vitamin K-quinone/epoxide ratio as a measure for vitamin K consumption. Immunohistochemical analysis of human vascular material was performed using a monoclonal antibody against MGP. The same antibody was used for quantification of MGP levels in serum. Results At least some extrahepatic tissues including the arterial vessel wall have a high preference for accumulating and using menaquinone rather than phylloquinone. Both intima and media sclerosis are associated with high tissue concentrations of MGP, with the most prominent accumulation at the interface between vascular tissue and calcified material. This was consistent with increased concentrations of circulating MGP in subjects with atherosclerosis and diabetes mellitus. Conclusions This is the first report demonstrating the association between MGP and vascular calcification. The hypothesis is put forward that undercarboxylation of MGP is a risk factor for vascular calcification and that the present RDA values are too low to ensure full carboxylation of MGP.

Prothrombin complex concentrate (PCC) has been suggested as a measure to terminate trauma and dabigatran-induced bleeding. Owing to the conflicting data concerning such therapy, we investigated the impact of a four-factor PCC to terminate massive bleeding following the infliction of multiple trauma in dabigatran anticoagulated pigs.

Introduction Acute myocardial infarction (AMI) is still an important cause of morbidity and mortality. One of the contributing mechanisms in AMI is plasma hypercoagulability, with recent studies suggesting activation of factor (F) XI to play a role in the onset of the thrombotic event. By serving as an amplification loop that enhances thrombin generation (TG), FXIa is thought to be involved in thrombotic diseases. To quantify FXIa levels in plasma, we developed a new TG based assay and hypothesized that in AMI patients FXIa plasma levels are increased at the time of the thrombotic event, as compared to 6 months after and compared to healthy controls. Methods A prospective cohort study was performed including patients with a first AMI. Citrated blood samples were collected upon admission (before administration of heparin) and after 6 months. Citrated blood samples from apparently healthy individuals served as a control population. FXIa TG was measured by means of the Calibrated Automated Thrombogram. All plasma samples were diluted at a 1:5 ratio in FXI deficient plasma and triggering was by addition of 4 µM phospholipids (without tissue factor). Active site-inhibited factor VII (30 nM) was added to inhibit the extrinsic coagulation pathway. FXIa plasma concentrations were established using a FXIa reference curve, which was obtained by addition of varying concentrations of FXIa (0-100 pM) to a corn trypsin inhibitor (CTI) pool-FXI deficient plasma mixture. The established FXIa concentrations were related to the 1-year outcome: cardiovascular death, recurrent MI, a second coronary intervention and ischemic stroke. Results A total of 56 consecutive AMI patients (71% men), with a mean age on admission of 60 (SD ±12) years, were included. The healthy control population consisted of 30 subjects (67% men) with a mean age of 50 (SD ±6) years. FXIa plasma concentrations were significantly increased in AMI patients at day 0 compared to 6 months after the event (4.8 pM [3.6-6.9] vs. 3.9 pM [2.4-5.7], median ± IQR; P=0.0015), and also compared to the FXIa levels in apparently healthy subjects (4.8 pM [3.6-6.9] vs. 3.8 pM [2.4-4.9], median ± IQR; P=0.0075). No significant difference in FXIa concentrations was demonstrated between AMI patients at 6 months follow-up and apparently healthy controls. In the 12-month follow-up period, 11 patients reached an endpoint. However, FXIa concentrations in the highest quartile were not significantly associated with the development of a recurrent thrombotic cardiovascular event (OR 3.2 [95%CI 0.4-28.2], P=0.29). Conclusions This study presents the first application of a new TG based FXIa assay. Results showed that in AMI patients at the time of the thrombotic event there is a significantly increased FXIa plasma concentration, compared to 6 months after the event and compared to healthy controls. However, no significant association could be established between the FXIa concentration and the risk of recurrent ischemic cardiovascular complications. Acknowledgment This research was supported by the Center for Translational Molecular Medicine and the Dutch Heart Foundation (INCOAG). Disclosures: No relevant conflicts of interest to declare.

Introduction New oral anticoagulants are effective alternatives to warfarin; however, no specific reversal agents are currently available for life-threatening bleeding or emergency surgery. This study measured the effects of prothrombin complex concentrate (PCC), activated PCC (aPCC) and recombinant factor VIIa (rFVIIa) and compared this to reversal with the specific antibody fragment for dabigatran after high-dose dabigatran etexilate (DE) treatment in a porcine model of trauma, to assess reversal of anticoagulation. Methods Studies were performed in 5 male pigs after ethical approval. DE was given orally for 3 days (30 mg/kg bid) and, on the 4th day, dabigatran was infused at 0.77mg/kg/h for 30 min followed by 0.52 mg/kg/min for 60 min. The infusion was administered immediately before trauma (blunt liver insult). Blood samples were taken before dabigatran infusion and 60 min post-injury. Two doses of PCC (30 and 60 IU/kg), aPCC (30 and 60 IU/kg) and rVIIa (90 and 180 μg/kg) and two doses anti-Dabi Fab (30 and 60 mg/kg) were added to blood samples ex vivo. Coagulation was assessed by thromboelastometry (ExTEM assay) and PT and dabigatran levels with diluted TT. Data were analyzed by ANOVA (± SEM). Results Oral DE prolonged clot formation (CT: 389 ± 128 sec; CFT: 159 ± 39 sec) and PT (27 ± 9 sec), but coagulopathy was more severe after achieving high dabigatran plasma levels (943 ± 147 ng/ml) and inducing trauma (CT: 2378 ± 480 sec; CFT: 3374 ± 625 sec; PT: 167 ± 40 sec). At this time point, 30 IU/kg PCC and 30 IU/kg aPCC significantly accelerated clot formation (PCC: CT 416 ± 70 sec, CFT 250 ± 91 sec; aPCC: CT 525 ± 126 sec, CFT 309 ± 60 sec; p Conclusion The ex vivo addition of either PCC or aPCC is effective in reducing coagulopathy in a porcine model of high-dose dabigatran therapy and trauma. However the specific aDabi-Fab is the most promising agent to reverse the anticoagulant effects of dabigatran. The potential for reducing blood loss remains to be investigated. Disclosures: van Ryn: Boehringer Ingelheim Pharma GmbH & Co. KG: Employment. Spronk:Boehringer Ingelheim Pharma GmbH & Co. KG: Research Funding. Rossaint:Boehringer Ingelheim: Research Funding. Grottke:Boehringer Ingelheim: Research Funding; CSL Behring: Consultancy.

Introduction A specific antibody fragment (Fab) is currently in development to reverse the effects of dabigatran in cases of life-threatening bleeding or emergency surgery. In trauma, different volume expanders are used for resuscitation to compensate for blood loss and hemorrhagic shock. The influence of volume expanders on binding of dabigatran to the Fab, however, is not known. Therefore, in this study we investigated whether frequently used infusion solutions affect binding of dabigatran to the Fab antidote and the resulting effects on coagulation assays. Methods All studies were performed after ethical approval. Dabigatran etexilate was given to pigs for 3 days (30 mg/kg bid) and on day 4 a 90 min infusion of dabigatran (30 min:0.77mg/kg/h; 60 min:0.26 mg/kg/min) was performed in anesthetized pigs. Then ∼50% (100 mL/min) of total blood volume was removed and animals were randomized to (n=5/group): balanced Ringer’s solution, 6% HES 130/0.4, 6% HES 200/0.5, 4% gelatin, retransfusion of washed red blood cells (RBCs) or control (no haemodilution). Resuscitation consisted of, 1:1 to blood loss for crystalloids, 25 mL/kg for colloids, and 12 mL/kg for RBC. Antidote was then given (30 mg/kg iv) and serial blood samples were taken for up to 24 hrs to measure active dabigatran as diluted TT (Hemoclot) and total dabigatran (LC-MS/MS). Coagulation was assessed by means of aPTT, diluted PT (dPT, Neoplastin R, 1:100), i-Stat ACT celite (ACT), and ROTEM, ExTem and InTem reagents. Data expressed as mean ± SE and analyzed with ANOVA. Results Hemodilution resulted in a substantial reduction of hemostatic parameters (Hemoglobin, platelets and leukocytes) with all agents, except after RBC transfusion vs control. Mean plasma dabigatran levels measured at dTT were 640 ± 60 ng/mL after infusion and 625 ± 123 ng/mL after hemodilution without significant differences between groups. Administration of 30 mg/kg iv Fab resulted in a comparable reduction of active dabigatran in all groups, with a calculated half-life of 3.2 ± 0.1 hrs. The best correlation between dTT and other coagulation assays was obtained for the dPT (r2=0.6321), the ACT-celite (r2=0.6605) and clotting time from ExTEM and InTEM ROTEM (r2=0.5169 and 0.7303, respectively). The correlation between aPTT and dTT was moderate: r2=0.2321. Overall, coagulation was prolonged upon dabigatran treatment as indicated by the dPT (from 32.3±10.6 to 162.7±18.1 sec), aPTT (fom 44.6±6.2 to 67.9±10.3 sec), ACT (from 97±6 to 297±25 sec), CT ExTem (from 36.5±3.7 to 1205.4±598.3 sec) and CT InTem (from 122.0±11.9 to 989.3±218.9 sec). Fab treatment restored coagulation by on average 73% (between 58 and 95%, depending on the applied assay) at 5 minutes following administration. The dPT shortened to 43.8±24.1 sec, the aPTT to 30.6±5.5 sec, the ACT to 126±25 sec, the CT ExTem to 124.3±83.4 sec, and the CT InTem to 300.7±168.7 sec, with no significant differences in coagulation parameters between the various volume expanders. However, resuscitation with 4% gelatin and Fab treatment resulted in a significant lesser reduction of the CT InTem compared to the other volume expanders: 631.6±360.4 sec for 4% gelatin and 203.8±22.4, 251.7±157.2, and 280.8±235.3 sec for 6% HES 200/0.5, 6% HES 130/0.4, and Ringers, respectively. All coagulation parameters were comparable between the various volumes expanders from 60 minutes onward. Twenty four hours after Fab administration all coagulation parameters were almost back to baseline values with 26.9±62.8 ng/mL dabigatran, 50.9±12.6 sec for the dPT, 48.6±13.5 sec for the aPTT, 121±6 sec for ACT, 50.0±3.4 sec for CT ExTem, and 189.7±24.5 sec for CT InTem. Conclusions Clinically used infusion solutions for volume resuscitation do not interfere with binding of dabigatran to its specific antidote. Inhibition of dabigatran with its specific Fab antidote at this particular dose, restored coagulation by almost 70% This effect was independent of the volume applied for resuscitation. Disclosures: Spronk: Boehringer Ingelheim Pharma GmbH & Co. KG: Research Funding. van Ryn:Boehringer Ingelheim Pharma GmbH & Co. KG: Employment. Grottke:Boehringer Ingelheim Pharma GmbH & Co. KG: Research Funding.

Background Exposure to ambient particulate matter (PM) has been associated with adverse cardiovascular effects in epidemiological studies. Current knowledge of independent effects of individual PM characteristics remains limited. Methods Using a semi-experimental design we investigated which PM characteristics were consistently associated with blood biomarkers believed to be predictive of the risk of cardiovascular events. We exposed healthy adult volunteers at 5 different locations chosen to provide PM exposure contrasts with reduced correlations among PM characteristics. Each of the 31 volunteers was exposed for 5 h, exercising intermittently, 3–7 times at different sites from March to October 2009. Extensive on-site exposure characterization included measurements of PM mass and number concentration, elemental- (EC) and organic carbon (OC), trace metals, sulfate, nitrate, and PM oxidative potential (OP). Before and 2 h and 18 h after exposure we measured acute vascular blood biomarkers - C-reactive protein, fibrinogen, platelet counts, von Willebrand Factor, and tissue plasminogen activator/plasminogen activator inhibitor-1 complex. We used two-pollutant models to assess which PM characteristics were most consistently associated with the measured biomarkers. Results and Conclusion We found OC, nitrate and sulfate to be most consistently associated with different biomarkers of acute cardiovascular risk. Associations with PM mass concentrations and OP were less consistent, whereas other measured components of the air pollution mixture, including PNC, EC, trace metals and NO2, were not associated with the biomarkers after adjusting for other pollutants.

Abstract 103 Background: Besides its well-known role in initiation and propagation of thrombus formation, thrombin may have multiple pro-atherogenic actions. Hypothesis: Pharmacologic thrombin inhibition attenuates the onset and progression of atherosclerosis and favors plaque stability in hypercoagulable mice prone to aggressive atherosclerosis development. Methods and Results: Hypercoagulability in TMPro/Pro:ApoE−/− mice resulted in severe atherosclerotic burden formation compared to ApoE−/− mice (389.1*103 μm2 vs. 187.0*103 μm2, p 80%, suppressed leukocyte recruitment, enhanced plaque stability, whereas numerous pro-inflammatory cytokines were diminished to levels comparable to non-atherosclerotic mice. Conclusion: These data establish a pivotal role for thrombin generation in controlling the level of inflammation related to atherosclerosis development in ApoE−/− mice. The potential clinical impact of long term administration of novel classes of anticoagulants on atherosclerosis merits further study. Disclosures: Weiler: BloodCenter of Wisconsin: Patents & Royalties. van Ryn:Boehringer Ingelheim Pharma GmbH & Co KG: Employment.

Abstract 1207 Background: Although the recombinant factor VIIa (rFVIIa) has been registered for use in hemophilia patients with inhibitors, there is still no method to monitor the effects of rFVIIa in restoring the coagulation balance in plasma. Hence, information is lacking about the individual optimal dose needed to normalize thrombin generation. Methods: The calibrated automated thrombogram (CAT) method was modified to increase sensitivity for rFVIIa addition to plasma at concentrations of 0, 2.5, 5, 10, 20, 40 and 80 nM, which covers the expected plasma concentration of 26 nM reached after standard administration. Thrombin generation was triggered using combinations of TF concentrations between 0 and 4 pM, and phospholipids concentrations between 0 and 4 μM. Endogenous thrombin potential (ETP), peak height, and velocity index were calculated in platelet poor plasmas (PPP) of different donors. All blood was collected in citrated tubes containing corn trypsin inhibitor (CTI) to minimize any contact activation. Results: The optimal conditions for discriminating rFVIIa (0–80 nM) in the CAT assay were determined in PPP: 0 or 0.25 pM TF with 4 μM of phospholipids. Also at higher TF concentrations, the CAT method was able to detect varying rFVIIa concentrations. The optimal concentration of phospholipids was 4 μM for all TF concentrations. In plasma of 6 healthy volunteers, thrombin generation triggered with 0.25 pM TF dose dependently increased using varying rFVIIa concentrations between 0 and 80 nM ( Figure 1 , left panel). The mean values for ETP, peak height and velocity index are depicted in Table 1 . On average, addition of 2.5, 5, 10, 20, 40 or 80 nM of rFVIIa resulted in a 146, 156, 161, 174, 206, and 285 % of the peak height compared to 0 nM rFVIIa, which was set at 100 %. At 4 pM TF the maximum ETP, peak height, and velocity index were reached at concentrations less than 20 nM rFVIIa for all donors. The mean values are depicted in Table 2 . Surprisingly, at 80 nM rFVIIa, thrombin generation was decreased compared to lower rFVIIa concentrations ( Figure 1 , right panel). Addition of 2.5, 5, 10, 20, 40 or 80 nM of rFVIIa resulted in 107, 109, 107, 103, 100, or 94 % of the peak height without addition of rFVIIa (0 nM set at 100 %). In FVIII deficient patient plasma (PPP), this effect was also present and even more pronounced. Here, a dose dependent effect of rFVIIa addition was visible at low (0 or 0.25 pM) TF trigger, whereas at 4 pM TF trigger ETP, peak height and velocity index were maximal in the presence of 10 nM rFVIIa. Overall, the peak height was 136, 142, 126, 102, 94, and 81 % upon addition of 5, 10, 20, 30, 40, or 80 nM rFVIIa respectively compared to 0 nM rFVIIa (set to 100 %). Download : Download high-res image (20KB) Download : Download full-size image Figure 1: . The concentration of rFVIIa is plotted against the % of ETP (ETP values obtained without rFVIIa addition are set at 100%). In the left panel, results measured at 0.25 pM of TF are depicted, whereas the right panel shows the % ETP at 4 pM TF addition. Each number represents a different donor. Table 1: . Thrombin generation at 0.25 pM of TF. All values are means ± SD of 6 volunteers. 0.25 pM TF 0 nM rFVIIa 80 nM rFVIIa ETP (nM.min) 333 ± 213 751 ± 137 Peak Height (nM) 26 ± 19 74 ± 14 Velocity Index (nM/min) 4.1 ± 3.5 14.1 ± 2.1 Table 2: . Thrombin generation at 4 pM of TF. All values are means ± SD of 6 volunteers. 4 pM TF 0 nM rFVIIa 20 nM rFVIIa 80 nM rFVIIa ETP (nM.min) 1191 ± 232 1222 ± 207 1179 ± 194 Peak Height (nM) 250 ± 71 257 ± 54 235 ± 66 Velocity Index (nM/min) 107.5 ± 62.0 104.5 ± 46.0 91.3 ± 50.9 Discussion: In hemophilic as well as normal plasma, the addition of rFVIIa dose dependently altered thrombin generation triggered with a low TF trigger (0 or 0.25 pM). At a higher trigger of 4 pM TF, maximal thrombin generation was obtained at rFVIIa concentrations of less then 20 nM. Remarkably, thrombin generation was attenuated in the presence of 80 nM rFVIIa. This paradox may be explained by assuming that the endogenously activated VIIa is more active than the rVIIa that was added. At higher rVIIa dosages the fraction of TF occupied by endogenous VIIa will decrease resulting in less active TF:VIIa complexes. This effect will be more pronounced when FXa formation is dependent on TF:FVIIa alone without the involvement of the tenase complex, which shows from our analysis in hemophilic plasma. Overall, these data suggest that the assay is most sensitive to added rVIIa when the contribution to Xa formation of TF:VIIa complex is small compared to that of rVIIa alone, i.e., in conditions where there is no or very little TF present. Disclosures: Giesen: Thrombinoscope bv: Employment.

Several epidemiological studies associated exposure to increased levels of particulate matter in Augsburg, Germany with cardiovascular mortality and morbidity. To elucidate the mechanisms of cardiovascular impairments we investigated the cardiopulmonary responses in spontaneously hypertensive rats (SHR), a model for human cardiovascular diseases, following intratracheal instillation of dust samples from Augsburg.250 μg, 500 μg and 1000 μg of fine ambient particles (aerodynamic diameter2.5 μm, PM₂(.)₅-AB) collected from an urban background site in Augsburg during September and October 2006 (PM₂(.)₅ 18.2 μg/m³, 10,802 particles/cm3) were instilled in 12 months old SHRs to assess the inflammatory response in bronchoalveolar lavage fluid (BALF), blood, lung and heart tissues 1 and 3 days post instillation. Radio-telemetric analysis was performed to investigate the cardiovascular responses following instillation of particles at the highest dosage based on the inflammatory response observed.Exposure to 1000 μg of PM₂(.)₅-AB was associated with a delayed increase in delta mean blood pressure (ΔmBP) during 2(nd)-4(th) day after instillation (10.0 ± 4.0 vs. -3.9 ± 2.6 mmHg) and reduced heart rate (HR) on the 3rd day post instillation (325.1 ± 8.8 vs. 348.9 ± 12.5 bpm). BALF cell differential and inflammatory markers (osteopontin, interleukin-6, C-reactive protein, and macrophage inflammatory protein-2) from pulmonary and systemic level were significantly induced, mostly in a dose-dependent way. Protein analysis of various markers indicate that PM₂(.)₅-AB instillation results in an activation of endothelin system (endothelin1), renin-angiotensin system (angiotensin converting enzyme) and also coagulation system (tissue factor, plasminogen activator inhibitor-1) in pulmonary and cardiac tissues during the same time period when alternation in ΔmBP and HR have been detected.Our data suggests that high concentrations of PM₂(.)₅-AB exposure triggers low grade PM mediated inflammatory effects in the lungs but disturbs vascular homeostasis in pulmonary tissues and on a systemic level by affecting the renin angiotensin system, the endothelin system and the coagulation cascade. These findings are indicative for promotion of endothelial dysfunction, atherosclerotic lesions, and thrombogeneis and, thus, provide plausible evidence that susceptible-predisposed individuals may develop acute cardiac events like myocardial infarction when repeatedly exposed to high pollution episodes as observed in epidemiological studies in Augsburg, Germany.

Abstract 2127 Poster Board II-102 Background: The revised model of coagulation proposes that factor XI (FXI) can be activated by thrombin, which is generated upon activation of the tissue factor (TF) pathway. This concept, however, has not been tested in vivo. A recent study questioned the existence of this feedback loop and suggested that factor XII (FXII) is the sole activator of FXI. Here, we analyze the feedback activation of FXI in plasma and in genetically altered mice. Methods and results: Fluorescence-based assays indicated that particle-bound thrombin caused thrombin generation in plasma both in the absence of TF and in the presence of active site inhibited factor VIIa. Thrombin failed to activate FXII and thrombin generation was almost completely abolished by an anti-FXIa antibody and in FXI-deficient plasma. Surface bound thrombin induced complex formation of FXI, with its major inhibitor C1 inhibitor, even in FXII-deficient plasma in a time and dose dependent manner. To determine if thrombin-driven FXI activation is important for hemostasis in vivo we used TF deficient mice (low TF), which have severely reduced thrombin formation. Low TF mice were crossed with mice deficient in one of the intrinsic pathway proteases FXII, FXI, or FIX. Double deficiency in TF and either FIX or FXI resulted in the intrauterine death of embryos due to hemorrhage. In contrast low TF/FXII-null mice were viable and the bleeding phenotype was unchanged from low TF animals. Conclusions: Surface-bound thrombin, a model for fibrin clot-protected thrombin, generates thrombin in a FXI dependent manner, independently from FXII. In addition to corroborating an amplifying role of FXI in thrombin generation, we provide the first evidence that at low TF levels FXI is essential in generating a sufficient ambient level of thrombin to permit embryonic development. Disclosures: No relevant conflicts of interest to declare.

The classic contact activation coagulation system emerges as a potentially important mechanism in arterial thrombosis. Recent studies not only indicate that coagulation factor (F) XII is associated with atherothrombotic coronary disease, but also that it contributes to clot formation via activation of downstream FXI. The time course of contact activation after an acute myocardial infarction (AMI), has not been studied. In addition, its relevance for arterial thrombosis has not been addressed in prospective studies. We measured the plasma concentrations of activated FXII (FXIIa), FXIa and Kallikrein in complex with their natural inhibitors in 135 consecutive patients with a documented first AMI (chest pain between 30 minutes and 24 hours accompanied by ST-segment elevation on ECG and/or elevated plasma levels of creatine kinase and troponin T). FXIIa-C1 inhibitor, kallikrein-C1 inhibitor, FXIa-C1 inhibitor and FXIa-antitrypsin complexes were measured in established homemade ELISAs (Govers-Riemslag et al. J Thromb Haemost2007; 5: 1896–1903). Mean FXIIa-C1 inhibitor complexes were highest on admission (0.081 U/mL) as compared to day 4 (0.065 U/ml), when most patients were still hospitalized and treated with low molecular weight heparin (LMWH) and dual platelet inhibition (aspirin and clopidogrel). At 90 and 180 days after admission mean FXIIa-C1 inhibitor values were 0.064 U/mL, (P

The principles of Virchov's triad appear to be operational in atherothrombosis or arterial thrombosis: local flow changes and particularly vacular wall damage are the main pathophysiological elements. Furthermore, alterations in arterial blood composition are also involved although the specific role and importance of blood coagulation is an ongoing matter of debate. In this review we provide support for the hypothesis that activated blood coagulation is an essential determinant of the risk of atherothrombotic complications. We distinguish two phases in atherosclerosis: In the first phase, atherosclerosis develops under influence of "classical" risk factors, i.e. both genetic and acquired forces. While fibrinogen/fibrin molecules participate in early plaque lesions, increased activity of systemic coagulation is of no major influence on the risk of arterial thrombosis, except in rare cases where a number of specific procoagulant forces collide. Despite the presence of tissue factor – factor VII complex it is unlikely that all fibrin in the atherosclerotic plaque is the direct result from local clotting activity. The dominant effect of coagulation in this phase is anticoagulant, i.e. thrombin enhances protein C activation through its binding to endothelial thrombomodulin. The second phase is characterized by advancing atherosclerosis, with greater impact of inflammation as indicated by an elevated level of plasma C-reactive protein, the result of increased production influenced by interleukin-6. Inflammation overwhelms protective anticoagulant forces, which in itself may have become less efficient due to down regulation of thrombomodulin and endothelial cell protein C receptor (EPCR) expression. In this phase, the inflammatory drive leads to recurrent induction of tissue factor and assembly of catalytic complexes on aggregated cells and on microparticles, maintaining a certain level of thrombin production and fibrin formation. In advanced atherosclerosis systemic and vascular wall driven coagulation becomes more important and elevated levels of D-dimer fragments should be interpreted as markers of this hypercoagulability.

Background Carotid atherosclerosis is an important cause of stroke. Intra-plaque haemorrhage (IPH) on magnetic resonance imaging (MRI) increases stroke risk. Development of IPH is only partly understood. Thrombin is an essential enzyme in haemostasis. Experimental animal studies have shown conflicting results on the relation between thrombin and plaque vulnerability. We hypothesize that decreased thrombin generation (TG) is associated with IPH and plaque vulnerability. Objective This article investigates whether TG is associated with IPH and other features of plaque vulnerability in stroke patients. Methods Recently symptomatic stroke patients underwent carotid MRI and blood sampling. MRI plaque features include plaque burden, presence of IPH, amount of lipid-rich necrotic core (LRNC), calcified tissue and fibrous tissue (% of total wall volume). TG was assessed in platelet-poor plasma and expressed as: peak height (PH) and endogenous thrombin potential (ETP). MR images could be analysed in 224 patients. Blood samples were available in 161 of 224 patients. Binary multivariate logistic and linear regression were used to investigate the association between TG and MRI plaque features. Results IPH and LRNC were present in 65 (40%) and 102 (63%) of plaques. There were no significant associations between TG and IPH; PH odds ratio (OR) = 1, 95% confidence interval (CI): 0.76 to 1.45 and ETP OR = 1, 95% CI: 0.73 to 1.37. After correction for age, sex and hypercholesterolaemia, the association was weak but non-significant; PH: OR = 0.76, 95% CI: 0.52 to 1.10 and ETP: OR = 0.73, 95% CI: 0.53 to 1.37. Conclusion Features of carotid plaque on MRI show no significant association with TG in stroke patients. Systemic TG does not seem to be an important factor in IPH development.

The reversal of the anticoagulant effects of the new oral anticoagulants in severely bleeding patients requires new therapeutic strategies. This study investigated the effectiveness of a specific antidote for idarucizumab to reverse bleeding in a dabigatran anticoagulated pig trauma model.

This study investigated the effectiveness of a specific antidote for dabigatran (aDabi-Fab) to reverse bleeding in a dabigatran anticoagulated pig trauma model. After ethical approval, male pigs (n=24) were given dabigatran etexilate for 3 days (30 mg/kg bid PO), the sham group (n=6) received

Background Increase in tissue factor (TF) and loss in thrombomodulin (TM) antigen levels has been described in various inflammatory disorders. The functional consequences of such changes in antigen concentrations in the coagulation balance are, however, not known. This study was designed to assess the consequences of inflammation-driven organ specific functional properties of the procoagulant response. Methods Tissue specific procoagulant activity was assessed by adding tissue homogenate to normal human pool plasma and recording of the thrombin generation curve. The new technique was subsequently applied on two inflammation driven animal models: 1) mouse lipopolysaccharide (LPS) induced endotoxemia and 2) spontaneously hypertensive rats exposed to environmental air pollution (particulate matter (PM). Results Addition of lung tissue from untreated animals to human plasma suppressed the endogenous thrombin potential (ETP) (175 ± 61 vs. 1437 ± 112 nM.min for control). This inhibitory effect was due to TM, because a) it was absent in protein C deficient plasma and b) lungs from TMpro/pro mice allowed full thrombin generation (ETP: 1686 ± 209 nM.min). The inhibitory effect of TM was lost after LPS administration to mice, which induced TF activity in lungs of C57Bl/6 mice as well as increased the ETP (941 ± 523 vs. 194 ± 159 nM.min for control). Another pro-inflammatory stimulus, PM dose-dependently increased TF in the lungs of spontaneously hypertensive rats at 4 and 48 hours after PM exposure. The ETP increased up to 48 hours at the highest concentration of PM (1441 ± 289 nM.min vs. saline: 164 ± 64 nM.min, p < 0.0001), suggesting a concentration- and time dependent reduction in TM activity. Conclusion Inflammation associated procoagulant effects in tissues are dependent on variations in activity of the TF-TM balance. The application of these novel organ specific functional assays is a useful tool to monitor inflammation-driven shifts in the coagulation balance within animal or human tissues.