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Background: Andexanet alfa is a Gla-domainless FXa (GDXa) analog used as an antidote to FXa inhibitors. Despite its clinical use, laboratory monitoring for anti-Xa reversal and the effect of andexanet on fibrinolysis has not been explored. We used a GDXa with a serine-to-alanine mutation at position 195 (chymotrypsin numbering) to model the interaction between andexanet and apixaban., Methods: Six batches of pooled plasma, and whole blood from healthy volunteers were treated with increasing concentrations of apixaban with/without GDXa. Thrombin generation and plasmin generation (TG and PG) were tested in plasma, and whole blood thrombus formation was tested using thromboelastometry or a flow-chamber system. FXa was also tested in isolation for its ability to support plasmin activation with/without apixaban and GDXa., Results: Apixaban (250-800 nM) concentration-dependently decreased the velocity and peak of TG in plasma. Apixaban prolonged the onset of thrombus formation in thromboelastometry and flow-chamber tests. GDXa normalized apixaban-induced delays in TG and whole blood thrombus formation. However, GDXa minimally affected the low PG velocity and peak caused by apixaban at higher concentrations (500-800 nM). FXa promoted plasmin generation independent of fibrin that was inhibited by apixaban at supratherapeutic concentrations., Conclusions: This study demonstrated the feasibility of assessing coagulation lag time recovery in plasma and whole blood following in vitro apixaban reversal using GDXa, a biosimilar to andexanet. In contrast, GDXa-induced changes in plasmin generation and fibrinolysis were limited in PG and tPA-added ROTEM assays, supporting the endogenous profibrinolytic activity of FXa and its inhibition at elevated apixaban concentrations., Competing Interests: Declaration of competing interest JHL is a member of the advisory boards or steering committees for Merck, Octapharma, Takeda, and Werfen; ALB and KAT have received research support from Cellphire Therapeutics, Hikari DX and Octapharma., (Copyright © 2023. Published by Elsevier Ltd.)

Introduction: The methods of viscoelastic coagulation testing (VCT) have evolved since the original invention of thrombelastography over 60 years ago, and new generations of devices are clinically used to guide hemostatic interventions at bedside. The utility of VCTs has been demonstrated in several clinical trials, but diagnostic performance of VCT may vary between devices, various transfusion algorithms, and patient populations., Areas Covered: Working principles and currently available data on the evolving VCTs for coagulation monitoring in acute care settings are reviewed. The PubMed database was used to search pertinent retrospective, prospective, and meta-analysis data on VCTs., Expert Opinion: Point-of-care VCTs provide clinically useful information on platelet count, fibrin polymerization, and other procoagulant factor activities in acute bleeding due to trauma or major surgery, and antithrombotic therapy. The addition of fibrin-specific VCT channel has brought renewed attention to early correction of fibrinogen deficiency using fibrinogen-rich component therapy, and stabilization of fibrin with antifibrinolytic agents. Normal reference ranges vary between devices, and diagnostic and treatment algorithms should be specifically established for each device and indication. There is interest in utilizing VCTs in complex coagulation management relating to hemophilia with inhibitors, but the standardized protocol has not been established.

Background: Acute traumatic coagulopathy is common in trauma patients. Prompt diagnosis of hypofibrinogenemia allows for early treatment with cryoprecipitate or fibrinogen concentrate. At present, optimal cutoffs for diagnosing hypofibrinogenemia with kaolin thrombelastography (TEG) have not been established. We hypothesized that kaolin kaolin-TEG parameters, such as kinetic time (K-time), α-angle, and maximum amplitude (MA), would accurately diagnose hypofibrinogenemia (fibrinogen <200 mg/dL) and severe hypofibrinogenemia (fibrinogen <100 mg/dL)., Methods: Adult trauma patients (injury severity score >15) presenting to our trauma center between October 2015 and October 2017 were identified retrospectively. All patients had a traditional plasma fibrinogen measurement and kaolin-TEG performed within 15 minutes of each other and within 1 hour of admission. Some patients had additional measurements after. Receiver operating characteristic (ROC) curve analysis was performed to evaluate whether K-time, α-angle, and MA could diagnose hypofibrinogenemia and severe hypofibrinogenemia. Area under the ROC curve (AUROC) was calculated for each TEG parameter with a bootstrapped 99% confidence interval (CI). Further, ROC analysis was used to estimate ideal cutoffs for diagnosing hypofibrinogenemia and severe hypofibrinogenemia by maximizing sensitivity and specificity. In addition, likelihood ratios were also calculated for different TEG variable cutoffs to diagnose hypofibrinogenemia and severe hypofibrinogenemia., Results: Seven hundred twenty-two pairs of TEGs and traditional plasma fibrinogen measurements were performed in 623 patients with 99 patients having additional pairs of tests after the first hour. MA (AUROC = 0.84) and K-time (AUROC = 0.83) better diagnosed hypofibrinogenemia than α-angle (AUROC = 0.8; P = .03 and P < .001 for AUROC comparisons, respectively). AUROCs statistically improved for each parameter when severe hypofibrinogenemia was modeled as the outcome (P < .001). No differences were found between parameters for diagnosing severe hypofibrinogenemia (P > .05 for all comparisons). The estimated optimal cutoffs for diagnosing hypofibrinogenemia were 1.5 minutes for K-time (95% CI, 1.4-1.6), 70.0° for α-angle (95% CI, 69.8-71.0), and 60.9 mm for MA (95% CI, 59.2-61.8). The estimated optimal cutoffs for diagnosing severe hypofibrinogenemia were 2.4 minutes for K-time (95% CI, 1.7-2.8), 60.6° for α-angle (95% CI, 57.2-67.3), and 51.2 mm for MA (95% CI, 49.0-56.2). Currently recommended K-time and α-angle cutoffs from the American College of Surgeons had low sensitivity for diagnosing hypofibrinogenemia (3%-29%), but sensitivity improved to 74% when using optimal cutoffs., Conclusions: Kaolin-TEG parameters can accurately diagnose hypofibrinogenemia and severe hypofibrinogenemia in trauma patients. Currently recommended cutoffs for the treatment of hypofibrinogenemia are skewed toward high specificity and low sensitivity. Many patients are likely to be undertreated for hypofibrinogenemia using current national guidelines.

Background: Recombinant activated factor VII (rFVIIa) concentrate reduces allogeneic blood transfusions, but it may increase thromboembolic complications in complex cardiac surgery. The mixture of activated factor VII (FVIIa) and factor X (FX) (FVIIa/FX) (FVIIa:FX = 1:10) is a novel bypassing agent for hemophilia patients. We hypothesized that the combination of FX and FVIIa could improve thrombin generation (TG) in acquired multifactorial coagulation defects such as seen in cardiac surgery and conducted in vitro evaluation of FVIIa/FX in parallel with other coagulation factor concentrates using in vitro and in vivo diluted plasma samples., Methods: Plasma samples were collected from 9 healthy volunteers and 12 cardiac surgical patients. We measured TG (Thrombinoscope) using in vitro 50% dilution plasma and in vivo dilution plasma after cardiopulmonary bypass, in parallel with thromboelastometry (ROTEM) and standard coagulation assays. In vitro additions of FVIIa/FX (0.35, 0.7, and 1.4 μg/mL, based on the FVIIa level), rFVIIa (1.4, 2.8, and 6.4 μg/mL), prothrombin complex concentrate (0.3 international unit), and 20% plasma replacement were evaluated., Results: In diluted plasma, the addition of either FVIIa/FX or rFVIIa shortened the lag time and increased the peak TG, but the effect in lag time of FVIIa/FX at 0.35 μg/mL was more extensive than rFVIIa at 6.4 μg/mL. Prothrombin complex concentrate increased peak TG by increasing the prothrombin level but failed to shorten the lag time. No improvement in any of the TG variables was observed after 20% volume replacement with plasma. The addition of factor concentrates normalized prothrombin time/international normalized ratio but not with plasma replacement. In cardiac patients, similar patterns were observed on TG in post-cardiopulmonary bypass samples. FVIIa/FX shortened clotting time (CT) in a concentration-dependent manner on CT on thromboelastometry. Plasma replacement did not improve CT, but a combination of plasma and FVIIa/FX (0.35 μg/mL) more effectively shortened CT than FVIIa/FX alone., Conclusions: The combination of FVIIa and FX improved TG more efficiently than rFVIIa alone or plasma in dilutional coagulopathy models. The required FVIIa dose in FVIIa/FX was considerably lower than those reported during bypassing therapy in hemophilia patients (1.4-2.8 μg/mL). The combination of plasma could restore coagulation more efficiently compared to FVIIa/FX alone. Lesser FVIIa requirement to exert procoagulant activity may be favorable in terms of reducing systemic thromboembolic complications.

Perioperative management of cardiovascular surgical procedures requiring cardiopulmonary bypass (CPB) in patients with hemophilia A poses a clinical challenge in coagulation management. Use of CPB requires the administration of an anticoagulant, usually unfractionated heparin, and also causes dilutional coagulopathy, platelet dysfunction or platelet consumption coagulopathy. Hypothermia and activation of the inflammatory cascade also affect coagulation. The effects of CPB on circulating levels of factor VIII have not been clearly defined. In this review, the effects of CPB and hemodilution on FVIII are shown in a case presentation, and perioperative laboratory testing in patients with hemophilia A having cardiac surgery is discussed along with perioperative and postoperative coagulation management.

Prolonged prothrombin time (PT) and its ratio are routinely used for the assessment of candidates for liver transplantation (LT), but intraoperative coagulation management of transfusion is hindered by its long turnaround time. Abnormal reaction time (R time) on thromboelastography (TEG) or clotting time (CT) of rotational thromboelastometry (ROTEM) are presumably an alternative, but there is a paucity of clinical data on abnormal R time/CT values compared to PT during LT. After receiving institutional review board approval and informed consent, we obtained blood samples from 36 LT patients for international normalized ratio (INR), factor (F) X level, and viscoelastic tests (EXTEM/INTEM and kaolin/rapid TEG) at baseline and 30 minutes after graft reperfusion. Receiver operating characteristic (ROC) curves were calculated for INR > 1.5 and viscoelastic R time/CT thresholds to assess the ability to diagnose FX deficiency at the moderate (<50%) or severe (<35%) level. The FX deficiency data were calculated using cutoff values of INR (>1.5) and abnormal R time/CT for TEG and ROTEM. Tissue factor (TF)-activated INR and EXTEM-CT performed well in diagnosing FX below 50%, but rapid TEG with combined TF and kaolin activators failed. Improved performance of INTEM-CT in diagnosing FX below 35% underlies multifactorial deficiency involving both intrinsic and common pathways. In conclusion, the differences among different viscoelastic tests and clinical situations should be carefully considered when they are used to guide transfusion during LT., (© 2015 American Association for the Study of Liver Diseases.)

Unlike kaolin thrombelastography (k-TEG), the clinical utility of rapid thrombelastography (r-TEG) and functional fibrinogen thrombelastography (FF-TEG) has not been tested in liver transplantation (LT). These thrombelastography techniques were simultaneously performed at the time of the skin incision (the baseline) and 30 minutes after graft reperfusion (III + 30) for 27 consecutive adult LT patients. k-TEG and r-TEG parameters [alpha angle (α) and maximum amplitude of the clot (MA)] were compared in addition to the assay time. Estimated FF-TEG fibrinogen levels were compared with plasma fibrinogen measurements. At the baseline, the values of Spearman's correlation coefficient (r) between k-TEG and r-TEG were moderate for α (r = 0.40, P = 0.06) and strong for MA (r = 0.90, P < 0.01). At III + 30, r was 0.46 (P < 0.05) for α and 0.80 (P < 0.01) for MA. The average time required to measure MA via r-TEG was decreased in comparison with k-TEG [from 29.7 to 21.6 minutes at the baseline (a 22% reduction) and from 29.6 to 22.9 minutes at III + 30 (a 23% reduction)]. FF-TEG correlated strongly with the plasma fibrinogen level at the baseline (r = 0.90, P < 0.01); however, FF-TEG overestimated the fibrinogen level at III + 30 (r = 0.58, P = 0.01). In conclusion, in adult LT, r-TEG correlates with k-TEG strongly for MA but only moderately for α. FF-TEG estimates the plasma fibrinogen level well at the baseline; however, it must be interpreted with caution because of its overestimation after graft reperfusion when the plasma fibrinogen level often decreases to less than 100 mg/dL., (© 2014 American Association for the Study of Liver Diseases.)

Introduction: Prasugrel is a thienopyridyl P2Y12 antagonist with potent antiplatelet effects. At present, little is known about its effects on thrombin generation or what strategies may emergently reverse its anticoagulant effects. In the current study we evaluated whether recombinant activated factor VII may reverse prasugrel induced effects and increase thrombin generation in an in vitro model., Methods: The effect of prasugrel active metabolite, PAM (R-138727), was evaluated on platelet aggregation, thrombin generation, and rotational thromboelastometry parameters using blood from 20 healthy volunteers. Additionally, we evaluated the effects of adenosine diphosphate (ADP) and recombinant activated factor VII on restoring these parameters towards baseline values., Results: PAM reduced maximum platelet aggregation and led to platelet disaggregation. It also decreased peak thrombin, increased lag time, and increased time to peak thrombin. Treatment with recombinant activated factor VII restored all three parameters of thrombin generation towards baseline. ADP decreased lag time and time to peak thrombin, but had no effect on peak thrombin. When recombinant activated factor VII and ADP were combined they had a greater effect on thrombin parameters than either drug alone. PAM also increased thromboelastometric clotting time and clot formation time, but had no effect on maximum clot firmness. Treatment with either recombinant activated factor VII or ADP restored these values towards baseline., Conclusions: Recombinant activated factor VII restores thrombin generation in the presence of PAM. In patients taking prasugrel with life-threatening refractory bleeding it has the potential to be a useful therapeutic approach. Additional clinical studies are needed to validate our findings., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: The activated clotting time (ACT) is widely used for monitoring heparin anticoagulation during cardiac surgery. Celite-based ACT values are prolonged when aprotinin is administered. MDCO-2010, a novel serine protease inhibitor, is currently being evaluated as a possible alternative to aprotinin. Therefore, we evaluated the in vitro effects of this novel agent on ACT values using 3 different point-of-care instruments with kaolin or celite as an activator., Methods: The study was performed in 2 parts. In the first part, blood samples were obtained from 15 healthy volunteers. Samples were pipetted into small Eppendorf tubes and 2 concentrations of the MDCO-2010 (100 and 500 nM, final concentration) alone or with heparin (1.2 or 2.4 U/mL) were added. ACTs were measured using Helena (celite), Hemochron (kaolin), and Medtronic (kaolin) devices. In the second part of the study, blood samples were obtained intraoperatively, at 5 time points, from 15 patients undergoing cardiopulmonary bypass. MDCO-2010 at a final concentration of 100 or 500 nM was added and ACT testing was performed as before. Additional coagulation tests included prothrombin time, activated partial thromboplastin time, fibrinogen, antithrombin, prothrombin, and anti-Xa levels., Results: Addition of MDCO-2010 concentration-dependently prolonged ACTs in volunteers' and patients' blood samples regardless of the ACT activator or device used. In volunteer samples (no heparin) and in patient samples (baseline and intensive care unit) percent changes in ACTs due to MDCO-2010 were on average 3.1 ± 1.8 times higher (95% confidence interval 2.6-3.6; P < 0.001) for the celite-based Helena device compared with either Hemochron or Medtronic devices., Conclusion: MDCO-2010 causes less ACT prolongation with kaolin than with celite activation.

Background: Rotational thromboelastometry (ROTEM®)-based FIBTEM is used perioperatively to assess the extent of fibrin polymerization in whole blood. In FIBTEM, cytochalasin D eliminates the contribution of platelets to whole blood clotting, but changing levels in fibrin(ogen) and erythrocytes may differently affect clot formation. Because dynamic changes of hematocrit are not reflected in plasma fibrinogen measurements, we hypothesized that the lack of erythrocytes in isolated plasma measurements would affect the relationship between the Clauss method and whole blood-based FIBTEM during cardiac surgery. Therefore, in the current study we investigated the influence of perioperative hematocrit changes on FIBTEM and fibrinogen measurements., Methods: Blood samples were collected from 6 consenting healthy volunteers. FIBTEM tests were run before and after serial in vitro dilutions of whole blood with saline or autologous plasma (5:1, 2:1, and 1:1 v/v). We then evaluated the relationship between FIBTEM-maximal clot firmness (MCF) and the Clauss fibrinogen method in relation to hematocrit values before and after cardiac surgery. Pearson correlation coefficients were determined between laboratory test results and ROTEM variables., Results: Upon in vitro hematocrit reduction, FIBTEM-MCF was progressively decreased depending on the extent of saline dilution, but it was increased by 31% after 1:1 volume replacement with autologous plasma (P < 0.05). In samples from cardiac patients (150 measurements in 50 patients), the overall correlation coefficient between FIBTEM-MCF and plasma fibrinogen was 0.80 (P < 0.001). In hemodiluted blood samples (during surgery or at intensive care unit), FIBTEM-MCF 10 mm corresponded to plasma fibrinogen levels of 200 mg/dL. In the subgroup analysis (n = 50 each), according to hematocrit levels (<25%, ≥25% to 30%, ≥30%), plasma fibrinogen levels of 200 mg/dL corresponded to 11 mm, 10 mm, and 8 mm of FIBTEM-MCF, respectively. The correlation between FIBTEM-MCF and plasma fibrinogen was higher at lower hematocrit (<25%) than at higher hematocrit (>30%) (r = 0.88 and 0.67, respectively)., Conclusions: Perioperative changes in hematocrit affect the correlation between plasma fibrinogen levels and FIBTEM-MCF values. The higher correlation between FIBTEM-MCF and plasma fibrinogen with lower hematocrit (<25%) indicates that FIBTEM is a practical method to determine the need for fibrinogen replacement in bleeding patients who typically develop perioperative anemia.

Background: Coagulopathy after cardiopulmonary bypass (CPB) is caused by multiple perturbations in cellular and humoral elements of coagulation. A timely and comprehensive method to evaluate hemostasis would be helpful in the management of bleeding patients after CPB. The assessment of whole blood coagulation using rotation thromboelastometry (ROTEM) was compared to coagulation tests routinely performed during cardiac surgery., Study Design and Methods: Blood was obtained from 26 patients undergoing CPB surgery at baseline, at 60 minutes on CPB, at the end of CPB, and on admission to intensive care unit. ROTEM tests (extrinsically activated [EXTEM], intrinsically activated [INTEM], specific clot formation [FIBTEM]), prothrombin time, activated partial thromboplastin time, platelet (PLT) count, fibrinogen, prothrombin level, antithrombin level, and thrombin generation (TG) measurement were performed., Results: We observed strong correlations between FIBTEM-amplitude at 10 minutes (A10) and fibrinogen level (r=0.87; p<0.001) and between EXTEM/ INTEM-A10 variables and PLT count (r=0.72 and 0.67, respectively; p<0.001). Receiver operating characteristic analysis demonstrated that EXTEM-A10 and INTEM-A10 are predictive of thrombocytopenia below 80×10(9)/L (area under the curve [AUC], 0.83 and 0.82, respectively), and FIBTEM-A10 was highly predictive of fibrinogen level below 200 mg/dL (AUC, 0.96). There were only weak correlations found between TG peak and clot formation time of EXTEM or INTEM (r=0.30 and 0.29, respectively; p<0.05)., Conclusion: ROTEM variables demonstrated clinically relevant correlations with PLT counts and fibrinogen levels. In particular, decreasing levels of fibrinogen can be quickly determined (<15-20 min) using FIBTEM., (© 2011 American Association of Blood Banks.)

In the recent years, thromboelastography has become a popular monitoring device for hemostasis and transfusion management in major surgery, trauma, and hemophilia. Thromboelastography is performed in whole blood and assesses the viscoelastic property of clot formation under low shear condition. Thromboelastography can be performed with a variety of activator and inhibitors at different concentrations representing the most important factors for different intervals and clot formation variables reported in multiple studies and algorithms. Furthermore, fibrinogen levels and platelet counts have a major influence on thromboelastographic variables. In addition, differences in patient populations, devices, and preanalytical conditions contribute to some conflicting findings in different studies., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Objective: The authors hypothesized that various hemostatic products may differently affect viscoelastic clot formation depending on their respective procoagulant activity and fibrinogen content., Design: In vitro coagulopathy modeling using warfarin-treated plasma (international normalized ratio, 2.8-3.8) and fibrinogen-deficient plasma evaluated by rotational thromboelastometry (ROTEM; Pentapharm, Munich, Germany)., Setting: A university laboratory., Intervention: Different volumes of cryoprecipitate, fresh frozen plasma (FFP), fibrinogen concentrate, and platelet concentrate were mixed with each abnormal plasma to simulate the in vivo transfusions of 250 mL to 1,000 mL. Three thromboelastometric variables that reflect the rate and extent of clot growth were measured: (1) coagulation time (CT), (2) angle, and (3) maximal clot firmness (MCF)., Measurements and Main Results: In warfarin-treated plasma, the addition of FFP, cryoprecipitate, and platelets led to a dose-dependent improvement of CT and angle, whereas MCF increased with cryoprecipitate or platelets only. The addition of fibrinogen concentrate improved MCF and angle but not CT. In fibrinogen-deficient plasma, the addition of cryoprecipitate, platelets, and fibrinogen concentrate led to a dose-dependent improvement of ROTEM variables, whereas the addition of FFP resulted in significantly longer CT and lower MCF values compared with other hemostatic products. The addition of platelets in the presence of cytochalasin D (a platelet inhibitor) resulted in improvements of ROTEM variables that were similar to when FFP was added to warfarin-treated and fibrinogen-deficient plasma., Conclusions: Cryoprecipitate supports clot formation on ROTEM more efficiently than FFP because of the high fibrinogen content. Improved ROTEM variables after platelet addition are presumably caused by increased interaction among thrombin-activated platelets and fibrinogen., (Copyright 2010 Elsevier Inc. All rights reserved.)

Perioperative bleeding is a major challenge particularly because of increasing clinical use of potent antithrombotic drugs. Understanding current concepts of coagulation is important in determining the preoperative bleeding risk of patients, and in managing hemostatic therapy perioperatively. The serine protease thrombin plays pivotal roles in the activation of additional serine protease zymogens (inactive enzymatic precursors), cofactors, and cell-surface receptors. Thrombin generation is closely regulated to locally achieve rapid hemostasis after injury without causing uncontrolled systemic thrombosis. During surgery, there are major disturbances in coagulation and inflammatory systems because of hemorrhage/hemodilution, blood transfusion, and surgical stresses. Postoperative bleeding often requires allogeneic blood transfusions, which support thrombin generation and hemostasis. However, procoagulant activity and inflammation are increased postoperatively; thus, antithrombotic therapy may be required to prevent perioperative thrombotic complications. There have been significant advances in the management of perioperative hemostasis and thrombosis because of the introduction of novel hemostatic and antithrombotic drugs. However, a limitation of current treatment is that conventional clotting tests do not reflect the entire physiological processes of coagulation making optimal pharmacologic therapy difficult. Understanding the in vivo regulatory mechanisms and pharmacologic modulation of thrombin generation may help control bleeding without potentially increasing prothrombotic risks. In this review, we focus on the regulatory mechanisms of hemostasis and thrombin generation using multiple, simplified models of coagulation.

Despite improvements in surgical techniques, the risk for perioperative bleeding remains significant. Transfusion of allogeneic red blood cells, platelets, and hemostatic factors remains the mainstay of current therapy strategy for management of perioperative bleeding. Transfusions significantly contribute to perioperative adverse events. Pharmacologic strategies to prevent or decrease perioperative bleeding are also important therapeutic approaches to reduce the need for allogeneic transfusions. This article discusses systemic pharmacologic prohemostatic agents (aprotinin, lysine analogues, protamine, desmopressin, and recombinant factor VIIa).

Background: Antithrombin (AT) levels decrease during cardiopulmonary bypass (CPB), particularly when combined with deep hypothermic circulatory arrest (DHCA). Low AT levels might lead to imbalance of pro- and anticoagulant factors promoting systemic thrombotic events. We hypothesized that low levels of AT might lead to increased in vitro thrombin generation when procoagulant factors are added to the patient's plasma after CPB., Methods: Blood samples were obtained before heparinization and after separation from CPB from five patients undergoing cardiac surgery with DHCA. AT levels were determined by chromogenic assay and expressed as a percent of normal activity. The balance between procoagulant and anticoagulant elements was manipulated in the patients' plasma by adding normal donor plasma, AT-deficient plasma, or purified AT. The Thrombinoscope system was used to evaluate thrombin generation with and without AT supplementation., Results: AT levels (median, range) were 82.0% (71.0, 109) and 37.0% (34.0, 41.0) of normal before and after separation from CPB, respectively (P < 0.05). Peak thrombin generation (median, range) was 56.6 nM (42.1, 61.0) in plasma after CPB, and it remained at 61.1 nM (54.9, 64.5) when a donor plasma with normal AT (105%) was added. When AT-deficient plasma was added to the patient's plasma, peak thrombin generation (median, range) was increased from 56.6 nM (42.0, 61.0) to 117 nM (95.0, 188) (P < 0.05 versus control). After the addition of purified AT, the peak thrombin generation was reduced to 12.2 nM (9.0, 29.3) (P < 0.05 versus control)., Conclusion: Plasma AT activity is severely decreased after CPB with DHCA. Our data suggest that the administration of coagulation factor components without AT repletion may lead to excessive thrombin generation, which clinically, may potentially lead to a hypercoagulable state.

Purpose: The authors analyzed the coagulation data of patients who underwent on-pump coronary artery bypass graft (CABG) or off-pump coronary artery bypass surgery (OPCAB) in a randomized prospective trial., Methods: CABG and OPCAB patients received heparin anticoagulation at 400 U x kg(-1), and 180 U x kg(-1) plus 3000 U every 30 min, respectively. In addition, OPCAB patients received a rectal aspirin, 650 mg, during the procedure. Perioperative coagulation test results (platelet count, fibrinogen, prothrombin time, partial thromboplastin time [PTT], activated clotting time [ACT], and thromboelastography [TEG; Haemoscope] were collected from CABG (n = 99) and OPCAB (n = 98) patients. Residual heparin activity after protamine was measured, using an anti-activated factor X (Xa) assay, in 10 patients from each group., Results: Our study showed that the current anticoagulation regimen in the OPCAB patients achieved a peak ACT of 445 +/- 73 s, and it preserved platelet counts and fibrinogen levels. A residual heparin effect was detected, with residual anti-Xa heparin activity of 0.2 U x ml(-1) up to 2 h after surgery in the OPCAB group. Despite the residual anticoagulation, the OPCAB group had a similar TEG index of native blood, postoperative chest tube drainage, and non-erythrocyte transfusion rate as compared with the CABG group., Conclusion: We have shown that the heparin anticoagulation regimen in OPCAB patients does not lead to an immediate hypercoagulable state. Total doses of heparin and protamine were lower in the OPCAB group compared with the CABG group, and there was a residual heparin effect on TEG and PTT in the early postoperative period in the OPCAB group.

Heparin is the current mainstay drug for anticoagulation during cardiac surgery, but it requires normal levels of antithrombin (AT) for optimal anticoagulation. Heparin anticoagulation may be less effective in cardiac surgical patients because of decreased AT levels due to the prolonged heparin therapy. Therefore, other anticoagulants that would work well in AT deficient patients may be more desirable. One such agent currently being evaluated is Intimatan, which catalyzes heparin cofactor II (HCII) dependent inhibition of thrombin. In the current in vitro study we examined the effects of Intimatan (20 microg/ml), heparin (0.25 U/ml), or both drugs in combination on thrombin generation in plasma with decreasing levels of AT, HCII or both cofactors, using a novel method based on the continuous measurement of thrombin generation. For the study, we collected blood samples from healthy volunteers, isolated platelet poor plasma by centrifugation and mixed it with AT, HCII, or AT-HCII deficient plasma samples to achieve different levels of AT, HCII and AT-HCII. Thrombin generation was inhibited equally well with heparin or Intimatan when the level of their respective cofactors was within the normal range. With decreasing levels of AT or HCII, heparin and Intimatan became less effective in thrombin inhibition, respectively. With the absence of both cofactors, neither agent alone or in combination had any effect on thrombin generation. We conclude that Intimatan may be an effective adjunct to heparin therapy under low AT conditions.

In this study, we characterized clot morphology with a scanning electron microscope (SEM) at time points corresponding to the commonly used thrombelastography (TEG) variables, illustrating the correlation of the physical clot formation with TEG(R) tracings. The first channel of the TEG analyzer was used to obtain the tracings of clot formation, while the sub-samples for the SEM were obtained from the second TEG channel. Different types of samples were examined, including whole blood, abciximab-treated whole blood, platelet-rich plasma (PRP), and abciximab-treated PRP. The SEM images were obtained at reaction time, different amplitudes (5-30 mm), maximum amplitude (MA), and at amplitude 60 min after MA. In the whole blood, coarse fibrin and activated platelets were observed at reaction time and fibrin strands progressively became more solid and intertwined at amplitude 10 mm and thereafter. Red blood cells were surrounded with fibrin strands at amplitude 30 mm and were tightly packed by fibrin strands at MA. In abciximab-treated whole blood, red blood cell shape was maintained at MA. The process of fibrin formation and platelet activation was also examined in PRP. Abciximab did not block platelet shape change, although the blockage of fibrin binding to platelets was shown on the TEG analyzer. In summary, we have shown structural changes of the forming clot in relation to TEG variables.

Introduction: Our study aim was to explore how different protamine-heparin ratios impacted enzymatic coagulation and acellular fibrin clot growth in plasma using an in vitro model. We hypothesized that a low protamine-heparin ratio would be associated with superior fibrin clot growth dynamics. Methods: We performed an in vitro study using 15 plasma samples from a commercial supplier. Different protamine-heparin ratios were added to each donor plasma sample: low ratio (0.7–1), traditional ratio (1–1), and high ratio (1.3–1) and clot formation dynamics were evaluated using a Thrombodynamics analyzer. Study outcomes were initial clot growth velocity and clot size at 30 min. Results: Plasma samples treated with a one-to-one protamine-heparin ratio had significantly lower mean initial clot growth velocity compared to samples treated with a low protamine-heparin ratio; mean difference −2.3 μm/min (95% CI = −4.0 to −0.7, p =.004). Plasma samples treated with a one-to-one protamine-heparin ratio also had significantly smaller mean clot size at 30 min compared to samples treated with a low protamine-heparin ratio; mean difference −54.0 μm (95% CI = −107.6 to −0.4, p =.048). There were no significant differences in mean initial clot growth velocity or clot size at 30 min between plasma samples treated with a high protamine-heparin ratio and those treated with a one-to-one or low protamine-heparin ratio (all p >.05). Conclusions: Plasma samples treated with a low protamine-heparin ratio had superior clot growth velocity and larger clot size at 30 min compared to a one-to-one ratio, supporting the notion that a low protamine-heparin ratio may optimize enzymatic coagulation after cardiopulmonary bypass. [ABSTRACT FROM AUTHOR]

This study in vitro comprehensively assessed reversal of the anticoagulant effects of rivaroxaban, an oral factor Xa inhibitor, using andexanet alfa and various prothrombin complex concentrate (PCC) products in a battery of tests. In static coagulation assays, andexanet alpha outperformed PCCs except for activated PCC being more effective in standard coagulation times. However, in a flow chamber model mimicking arterial shear, both andexanet alpha and high-concentration PCC restored fibrin formation, but not platelet adhesion. In the Russell's viper venom test and anti-Xa assay, only andexanet alpha could be tested for efficacy. The antidote effects of andexanet alpha and PCCs in restoring coagulation can be qualitatively or selectively demonstrated using in vitro coagulation tests. [ABSTRACT FROM AUTHOR]

Background: The purpose of this study was to survey liver transplant centers in the United States to assess baseline practices in blood utilization and identify opportunities for standardization to optimize blood use in these complex cases. Study Design and Methods: Two surveys, one for transfusion medicine physicians and the other for anesthesiologists, were distributed to high‐volume liver transplant centers. Results: The response rate was 52% for both surveys. The majority of respondents (90%) indicated they issue a standardized number of blood products to start surgeries. The most common number of products issued before the start of cases were 10 red blood cells (RBC) and 10 plasma units with no platelets or cryoprecipitate. On average, fewer RBC (7.5) and plasma (7) units were transfused than issued. Decisions to transfuse RhD+ RBCs to RhD‐ patients and use antigen untested units in alloimmunized patients were mainly handled on a case‐by‐case basis. Many centers reported utilizing viscoelastic testing (97%) and cell salvage (97%). Most centers reported standardized, laboratory‐based intraoperative transfusion goals for RBCs (65%) and fibrinogen replacement (52%) but lacked a standardized approach for plasma (55%) and platelets (58%). Discussion: More blood products are issued during surgery than are transfused. Responses from anesthesiology providers suggest a broad consensus on practice. Almost all respondents use viscoelastic testing in the management of intraoperative coagulopathy, either alone or in combination with classical coagulation tests. The majority of programs do not transfuse clotting factor concentrates, including fibrinogen concentrate, prothrombin complex concentrates, and recombinant activated FVII, and do not use antifibrinolytics prophylactically. [ABSTRACT FROM AUTHOR]

This article discusses a case study involving a patient undergoing kidney transplantation who exhibited highly elevated functional fibrinogen on a thromboelastography (TEG) test. The authors successfully treated the condition using desmopressin, but no changes were observed in the repeat TEG test. The authors express concerns about inferring hemostatic function solely based on functional fibrinogen and highlight the need for additional laboratory coagulation data. They also discuss the potential impact of chronic kidney disease (CKD) on coagulation abnormalities and anemia, emphasizing the need for further research in this area. [Extracted from the article]

Background: Heparin management in hemophilia A (HA) patients with a factor VIII (FVIII) inhibitor can be challenging due to severe activated clotting time (ACT) prolongations. It is important to better understand the impact of emicizumab, a FVIII mimetic on ACT, and tissue factor (TF)‐based coagulation assays. Methods: Whole blood from 18 patients undergoing cardiopulmonary bypass (CPB) were mixed in vitro with pooled normal plasma, FVIII‐deficient or FVIII‐inhibitor plasma to affect functional FVIII levels. ACTs and heparin concentration by protamine titration were measured in whole blood mixture with/without emicizumab (50‐100 μg/ml). Thrombin generation and plasmin generation were measured in the patient's plasma mixed with normal plasma or FVIII‐inhibitor plasma to assess the impact of emicizumab under low TF activation. Results: FVIII inhibitors prolonged ACTs by 2.2‐fold compared to those in normal plasma mixture at baseline. During CPB, ACTs in normal plasma mixture, and FVIII‐deficient mixture were in 400s, but ACTs reached 900s in FVIII‐inhibitor mixture. Emicizumab shortened ACTs by up to 100s in normal plasma mixture, and FVIII‐deficient mixtures. ACTs remained over 600s in FVIII‐inhibitor mixture, despite adding emicizumab at 100 μg/ml. Heparin concentration measured by TF‐based protamine titration was unaffected. Emicizumab enhanced thrombin peak in the presence of FVIII inhibitors, whereas plasmin generation was mainly affected by thrombin generation, and systemic use of ɛ‐aminocaproic acid. Conclusions: FVIII inhibitors extensively prolong ACTs in heparinized whole blood, and clinical levels of emicizumab partially reverse ACT values. Protamine titration should be considered for optimal heparin monitoring in emicizumab‐treated patients with FVIII inhibitors. [ABSTRACT FROM AUTHOR]

Bleeding complications after cardiac surgery are common and are associated with increased morbidity and mortality. Their etiology is multifactorial, and treatment decisions are time sensitive. Point-of-care (POC) testing has an advantage over standard laboratory tests for faster turn-around times, and timely decision on coagulation intervention(s). The most common POC coagulation testing is the activated clotting time (ACT), used to monitor heparin therapy while on cardiopulmonary bypass. Viscoelastic coagulation tests including thromboelastometry (ROTEM) and thromboelastography (TEG) have been recommended for the treatment of postoperative bleeding after cardiac surgery because the ROTEM/TEG-guided treatment algorithms reduced the use of blood products. Other POC tests are commercially available, but there is sparse evidence for their routine use in cardiac surgery. These devices include heparin management systems, POC prothrombin time and activated partial thromboplastin time, POC fibrinogen assay, and whole blood platelet function tests. There are multiple confounding elements and conditions associated with cardiac surgery, which can significantly alter test results. Anemia and thrombocytopenia are regularly associated with deviations in many POC devices. In summary, POC coagulation testing allows for rapid clinical decisions in hematological interventions, and, when used in conjunction with a proper transfusion algorithm, may reduce blood product usage, and potentially complications associated with blood transfusion. [ABSTRACT FROM AUTHOR]

Background: Thrombin generation (TG) is a pivotal process in achieving hemostasis. Coagulation profiles during pregnancy and early neonatal period are different from that of normal (non-pregnant) adults. In this ex vivo study, the differences in TG in maternal and cord plasma relative to normal adult plasma were studied. Methods: Twenty consented pregnant women and ten consented healthy adults were included in the study. Maternal and cord blood samples were collected at the time of delivery. Platelet-poor plasma was isolated for the measurement of TG. In some samples, anti-FIXa aptamer, RB006, or a TFPI inhibitor, BAX499 were added to elucidate the contribution of intrinsic and extrinsic pathway to TG. Additionally, procoagulant and inhibitor levels were measured in maternal and cord plasma, and these values were used to mathematically simulate TG. Results: Peak TG was increased in maternal plasma (393.6±57.9 nM) compared to adult and cord samples (323.2±38.9 nM and 209.9±29.5 nM, respectively). Inhibitory effects of RB006 on TG were less robust in maternal or cord plasma (52% vs. 12% respectively) than in adult plasma (81%). Likewise the effectiveness of BAX499 as represented by the increase in peak TG was much greater in adult (21%) than in maternal (10%) or cord plasma (12%). Further, BAX499 was more effective in reversing RB006 in adult plasma than in maternal or cord plasma. Ex vivo data were reproducible with the results of the mathematical simulation of TG. Conclusion: Normal parturient plasma shows a large intrinsic pathway reserve for TG compared to adult and cord plasma, while TG in cord plasma is sustained by extrinsic pathway, and low levels of TFPI and AT. [ABSTRACT FROM AUTHOR]

ntifibrinolytic agents have been prophylactically administered to patients undergoing cardiopulmonary bypass (CPB) to reduce postoperative bleeding due to plasmin-mediated coagulation disturbances. After the recent market withdrawal of aprotinin, a potent bovine-derived plasmin inhibitor, two lysine analogs, ε-aminocaproic acid and tranexamic acid are currently available for clinical use. Although the use of aprotinin recently raised major concerns about postoperative thrombosis and organ dysfunctions, there is a paucity of information on the potential complications related to lysine analogs. Using the available preclinical and clinical data, we present current perspectives on the hemostatic mechanism and potential harms of antifbirnolytic therapy related to cardiac surgery. Fibrin formation is the critical step for hemostasis at the site of vascular injury, and localized fibrinolytic activity counterbalances excess fibrin formation which might result in vascular occlusion. Inhibition of the endogenous fibrinolytic system may be associated with thrombotic complications in susceptible organs. It is thus important to understand CPB-related changes in endogenous fibrinolytic proteins (e.g., tissue plasminogen activator (tPA), plasminogen) and antifibrinolytic proteins (e.g., α-antiplasmin). [ABSTRACT FROM AUTHOR]

The Sonoclot Analyzer provides a functional test of whole blood coagulation by measuring the viscous property of the blood sample. In this study, we used a modified Sonoclot assay, using cuvettes with a glass bead activator containing heparinase, and compared the Sonoclot data before and after cardiopulmonary bypass (CPB) to assess the usefulness in predicting postoperative hemorrhage. In 41 cardiac surgery patients, Sonoclot data were obtained immediately after heparin administration (pre-bypass) and just before protamine administration (post-bypass). Excessive bleeding was defined as chest tube drainage greater than 2 ml·kg−1·h−1 in 1 h during the first 4 h after surgery. There were no significant differences in Sonoclot values before and after CPB in patients with acceptable bleeding ( n = 29). In patients with excessive bleeding ( n = 12), Sonoclot variables reflecting fibrin formation (activated clotting time [ACT], rate of fibrin formation [clot rate], and peak clot signal) were preserved after CPB; however, the variables reflecting platelet-fibrin interaction (time to peak, peak angle, and clot retraction rate) were significantly different from their respective pre-bypass values. Sonoclot analysis showed impairment of clot maturation after CPB in patients with excessive postoperative bleeding. Our results suggest that abnormal postoperative hemorrhage can be predicted by Sonoclot analysis with a new glass bead-activated heparinase test performed after CPB. [ABSTRACT FROM AUTHOR]

Thrombin plays a central role in coagulation and haemostasis. Binding of thrombin to thrombomodulin generates activated protein C (APC), which exerts a negative feedback on thrombin formation. Aprotinin, a natural proteinase inhibitor is used extensively during cardiac surgery because this procedure is often associated with profound activation of coagulation and inflammatory pathways. Some in vitro evidences suggest that aprotinin inhibits APC, but the clinical relevance is unclear. The recombinant human soluble thrombomodulin (rhsTM)-modified thrombin generation (TG) assay was used to investigate the effects of aprotinin on APC in plasma samples obtained from healthy volunteers, aprotinin-treated cardiac surgical patients and in protein C (PC)-depleted plasma. Based on the results of in vitro TG assay, addition of rhsTM (0·75–3·0 μg/ml) to volunteer or patient platelet-poor plasma significantly reduced (70·8 ± 21·9 and 95·3% ± 4·6%, respectively) thrombin formation when compared with PC-depleted plasma (8·3% ± 5·2%). Aprotinin (100–200 KIU) caused a small, statistically insignificant decrease in the peak thrombin formation in normal and PC-deficient plasma (12·0 ± 6·1%). In cardiac surgical patients, levels of functional PC, factor II, antithrombin and platelet significantly decreased after cardiopulmonary bypass (CPB). Soluble thrombomodulin concentrations were increased after CPB (3·5 ± 2·2 to 5·0 ± 2·2 ng/ml), but they were still within the normal range for human plasma. Our results showed that, even though endogenous PC level is decreased after CPB, it retains its activity in the presence of thrombomodulin, and aprotinin has limited inhibitory effect on APC generation. [ABSTRACT FROM AUTHOR]

Background: DX-88 is a potent kallikrein inhibitor that is being studied for the treatment of hereditary angioedema (HAE) and represents a potential alternative to aprotinin in cardiac surgical patients. The current study was designed to evaluate in vitro effects of DX-88 on coagulation in comparison with aprotinin. Methods: Blood samples were obtained from consented 12 healthy volunteers. DX-88 or aprotinin was added to blood at 200 and 800 kallikrein inhibitory units (KIU) per milliliter for aprotinin, and at 1.1, 2.2, or 8.8 μg/ml for DX-88. Thromboelastography (TEG®) was performed using celite, kaolin, or tissue factor (TF) activation. Kaolin-based activated clotting times (ACTs) were measured at different heparin levels. The whole blood prothrombin time (PT)/PTT values were also measured. The endogenous thrombin generation was assessed with a fluorogenic assay using platelet-poor plasma (PPP). Results: With celite and kaolin activation of TEG, the reaction time was prolonged with DX-88 and aprotinin. With tissue factor activation, TEG parameters were not affected. DX-88 caused dose-dependent kaolin-ACT prolongation that was augmented by increasing doses of heparin. DX-88 or aprotinin had no significant effects on the PT values, but PTT values were dose-dependently prolonged. Both agents delayed the onset of thrombin generation when PTT reagent was used as a trigger, whereas no change was observed when tissue factor was used. Conclusion; We found that DX-88 delayed contact activator induced coagulation without affecting tissue factor mediated coagulation. For evaluation of coagulation during DX-88 therapy, the use of PT or tissue factor-activated TEG may be preferable. [Copyright &y& Elsevier]

Anticoagulant therapy is challenging to modern surgical practice because it complicates risks of bleeding and the need for allogeneic blood transfusions. In an aging population, there is extensive use of antiplatelet agents, and patients present for operations receiving these agents. Hemostatic inhibitors are reviewed here, including anticoagulants, platelet inhibitors (clopidogrel), low-molecular-weight heparins, pentasaccharide (fondaparinux), and other factor Xa inhibitors. Agents used to manage bleeding, including aprotinin, lysine analogs, desmopressin, and recombinant factor VIIa, are discussed. [Copyright &y& Elsevier]