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Competing Interests: Conflicts of Interest: See Disclosures at the end of the article.

Background: Plasma transfusion remains the mainstay hemostatic therapy during liver transplantation (LT) in most countries. However, a large volume is required for plasma to achieve clinically relevant factor increases. Prothrombin complex concentrate (PCC) is a low-volume alternative to plasma in warfarin reversal, but its efficacy has not been well studied in LT., Methods: Blood samples were collected from 28 LT patients at baseline (T0) and 30 minutes after graft reperfusion (T1). Factor X and antithrombin levels were measured. Ex vivo effects of PCC (0.2 and 0.4 IU/mL) and 10% volume replacement with normal plasma were compared in LT and warfarin plasma by measuring lag time, thrombin peak, and endogenous thrombin potential (ETP) using thrombin generation (TG) assay., Results: Coagulation status was worsened at T1 as international normalized ratio increased from 1.7 to 3.0, and factor X was decreased from 49% to 28%. TG measurements showed normal lag time and ETP at T0 and T1, but low-normal peak at T0, and below-normal peak at T1. Both doses of PCC increased peak and ETP, while 10% volume plasma had minimal effects on TG. Thrombin inhibition appears to be very slow after adding 0.4 IU/mL of PCC in LT plasma due to low antithrombin. The same doses of PCC and plasma were insufficient for warfarin reversal., Conclusions: Reduced TG in LT can be more effectively restored by using PCC rather than plasma. The required doses of PCC for LT patients seem to be lower than warfarin reversal due to slow thrombin inhibition.

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.

Background: Bleeding is a serious complication after pediatric cardiopulmonary bypass (CPB) that is associated with an increase in perioperative morbidity and mortality. Four-factor prothrombin complex concentrates (4F-PCCs) have been used off-label to supplement transfusion protocols for bleeding after CPB in adults; however, data on their use in neonates are limited. In this study, we hypothesized that 4F-PCCs administered ex vivo to neonatal plasma after CPB will increase thrombin generation., Methods: Fifteen neonates undergoing complex cardiac repairs requiring CPB were enrolled in this prospective study. Arterial blood was obtained after anesthesia induction but before CPB (baseline), after CPB following heparin reversal, and after our standardized transfusion of a quarter of a platelet apheresis unit (approximately 20 mL·kg) and 3 units of cryoprecipitate. Kcentra (CSL Behring), a 4F-PCC with nonactivated factor VII (FVII), and factor 8 inhibitor bypassing activity (FEIBA; Baxter Healthcare Corporation), a 4F-PCC with activated FVII, were added ex vivo to plasma obtained after CPB to yield concentrations of 0.1 and 0.3 IU·mL. Calibrated automated thrombography was used to determine thrombin generation for each sample., Results: The addition of Kcentra to plasma obtained after CPB resulted in a dose-dependent increase in the median (99% confidence interval) peak amount of thrombin generation (42.0 [28.7-50.7] nM for Kcentra 0.1 IU·mL and 113.9 [99.0-142.1] nM for Kcentra 0.3 IU·mL). The rate of thrombin generation was also increased (15.4 [6.5-24.6] nM·min for Kcentra 0.1 IU·mL and 48.6 [29.9-66.6] nM·min for Kcentra 0.3 IU·mL). The same was true for FEIBA (increase in peak: 39.8 [27.5-49.2] nM for FEIBA 0.1 IU·mL and 104.6 [92.7-124.4] nM for FEIBA 0.3 IU·mL; increase in rate: 17.4 [7.4-28.8] nM·min FEIBA 0.1 IU·mL and 50.5 [26.7- 63.1] nM·min FEIBA 0.3 IU·mL). In the posttransfusion samples, there was a significant increase with Kcentra in the median (99% confidence interval) peak amount (41.1 [21.0-59.7] nM for Kcentra 0.1 IU·mL and 126.8 [106.6- 137.9] nM for Kcentra 0.3 IU·mL) and rate (18.1 [-6.2 to 29.2] nM·min for Kcentra 0.1 IU·mL and 53.2 [28.2-83.1] nM·min for Kcentra 0.3 IU·mL) of thrombin generation. Again, the results were similar for FEIBA (increase in peak: 43.0 [36.4-56.7] nM for FEIBA 0.1 IU·mL and 109.2 [90.3-136.1] nM for FEIBA 0.3 IU·mL; increase in rate: 25.0 [9.1-32.6] nM·min for FEIBA 0.1 IU·mL and 59.7 [38.5-68.7] nM·min for FEIBA 0.3 IU·mL). However, FEIBA produced in a greater median reduction in lag time of thrombin generation versus Kcentra in samples obtained after CPB (P = 0.003 and P = 0.0002 for FEIBA versus Kcentra at 0.1 and 0.3 IU·mL, respectively) and in samples obtained after transfusion (P < 0.0001 for FEIBA versus Kcentra at 0.1 and 0.3 IU·mL)., Conclusions: After CPB, thrombin generation in neonatal plasma was augmented by the addition of 4F-PCCs. The peak amount and rate of thrombin generation were enhanced in all conditions, whereas the lag time was shortened more with FEIBA. Our findings suggest that the use of 4F-PCCs containing activated FVII may be an effective adjunct to the initial transfusion of platelets and cryoprecipitate to augment coagulation and control bleeding in neonates after CPB.

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: Increased levels of factor VIII occur as a response to vascular injury and/or inflammation, and may increase thrombotic risks. In contrast, factor VIII deficiency poses a major hemostatic challenge. The role of factor VIII in modulating hemostasis/thrombosis was investigated in plasma models of hypocoagulable and hypercoagulable state using thrombin generation (TG) assay., Methods: TG was performed in undiluted/diluted control, FVIII-deficient, FVIII-deficient with low antithrombin (AT activity, ~59%), and factor XI-deficient plasma samples using relipidated tissue factor (TF, 2 pM) or dilute Actin as activators. The impact of elevated FVIII on TG was simulated by adding Humate-P (0 to 3 U/ml) to the above plasma samples. In fondaparinux (1 μg/ml) treated plasma with normal or lower AT activity effects of Humate-P vs. 60 nM of recombinant activated factor VII (rFVIIa) were also evaluated., Results: Humate-P increased TG concentration dependently in undiluted and diluted control plasma with TF activation. With Actin activation, only the concentration dependent shortening of lag time, but no change in peak thrombin was observed. In FVIII-deficient, FVIII-deficient with low AT, and FXI-deficient samples, 3 U/ml of Humate-P increased TG, and decreased its onset with either activator. The reduced peak thrombin due to fondaparinux was reversed with Humate-P (3 U/ml) more than with rFVIIa. Elevated FVIII levels seem to favor intrinsic tenase formation and antagonize fondaparinux because anti-FIXa aptamer added to fondaparinux effectively attenuated TG., Conclusion: Elevated FVIII supports the propagation of TG via intrinsic tenase formation under low TF condition, factor XI deficiency or in the presence of fondaparinux., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Decreased levels of factor VIII (FVIII) limit the amount of thrombin generated at the site of injury, but not the rate that thrombin is neutralised by antithrombin (AT). We hypothesised that FVIII-deficient mice with heterozygous AT deficiency will demonstrate increased thrombin generation and therefore less in vivo bleeding compared to FVIII-deficient mice with normal AT levels. Therefore, we performed tail bleeding experiments in wild-type (WT), heterozygous AT deficient (AT(+/-)) mice, FVIII-deficient (FVIII(-/-)) mice, and FVIII-deficient mice with heterozygous AT deficiency (FVIII(-/-)/AT(+/-)). Amount of bleeding was assessed by measuring absorbance of haemoglobin released from lysed red blood cells collected after tail transection. In addition, we measured thrombin generation, activated partial thromboplastin time (aPTT), and AT activity in plasma from the different mice groups. Tail bleeding was significantly reduced in FVIII(-/-)/AT(+/-) mice compared to FVIII(-/-) mice. On the other hand, there was no difference in tail bleeding between AT(+/-) and wild-type mice. Thrombin generation was dependent on the mice genotype, and increased in the following order: FVIII(-/-) < FVIII(-/-)/AT(+/-) < WT < AT(+/-). The aPTT was not influenced by reduced AT activity (i.e. AT(+/-) genotype), but was significantly prolonged in FVIII(-/-) and FVIII(-/-)/AT(+/-) mice. Using FVIII-deficient mice as an in vivo murine model of reduced thrombin generation, we demonstrated that moderately reduced AT levels increase thrombin generation and decrease bleeding after traumatic tail vessel injury. In agreement with congenital thrombotic conditions, our data elucidate that bleeding phenotypes can be modulated by the balance between procoagulant and anticoagulant proteins.

Background: Warfarin is routinely used in the prevention and treatment of prothrombotic events. During initiation of warfarin therapy levels of factor (F) VII and protein C decrease rapidly but prothrombin, FIX and FX decline much slower. Therefore, propagation of thrombin generation (TG) remains unaffected much longer, increasing the risk of inadequate anticoagulation. Recently, a novel agent, anti-IXa aptamer, RB006, has been developed. Therefore, we have evaluated the in vitro effects of this agent in warfarin plasma., Methods: The investigation consisted of two parts. First, a computer simulated time course of TG with warfarin alone and in combination with FIXa inhibition was evaluated and, second, normal volunteer, protein C deficient, FVII deficient and commercial warfarin plasmas (INR 2.1 and 3.1) were spiked with increasing concentrations of aptamer (0-24 microg/ml) and its anticoagulant effects were evaluated using prothrombin time (PT), activated partial thromboplastin time (aPTT) and TG with tissue factor and Actin as activators. Direct effects of aptamer on protein C were also evaluated., Results: Simulation of coagulation during warfarin induction showed that TG can be significantly delayed and decreased by inhibiting FIXa (i.e., with anti-FIXa aptamer). The anti-FIXa aptamer inhibited TG in all tested plasmas, but was most efficacious in warfarin and FVII deficient plasma. The aptamer itself did not inhibit protein C and had no effect on PT, but concentration-dependently increased aPTT., Conclusion: The anti-FIXa aptamer potentiates the inhibitory effects of warfarin on TG, and may fill the need as an adjuvant agent during initiation of warfarin therapy., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

The REG1 system consists of factor IXa inhibitor, RB006, an aptamer-based anticoagulant and its antidote, RB007. The optimal use of RB006 can be facilitated by understanding its effect on the formation of thrombin and fibrin, and other standard tests of coagulation. Blood from consented volunteers was drawn into 3.2% citrate (9:1 v/v) and either used immediately or centrifuged to obtain platelet-poor plasma. Increasing concentrations of aptamer (6-24 microg/ml) alone or in combination with heparin (0.1 U/ml) or lepirudin (0.2 microg/ml) were added to blood and plasma samples. Activated clotting times (ACT+, low range-ACT), thrombelastometry (ROTEM) or thrombelastography (TEG) were performed in recalcified whole blood samples. Thrombin generation, prothrombin time (PT) and activated partial thromboplastin time (aPTT) were performed in plasma samples. To some samples the antidote RB007 was added to neutralise the anticoagulation activity of RB006. In all experiment the ratio of RB006 to RB007 was kept 1:2. RB006 dose-dependently prolonged aPTT and low range-ACT, but, as expected, had no effect on PT. RB006 prolonged the lag time and decreased the peak of Actin-triggered thrombin generation. Thrombin-activated TEG demonstrated that RB006 decreases the rate of clot formation. These effects were potentiated when RB006 was combined with heparin or lepirudin. In all experiments RB007 reversed the effects of RB006 back to baseline. In conclusion, RB006 inhibits thrombin generation and clot formation in a concentration-dependent manner. It is feasible to monitor RB006 and its reversal with RB007 using aPTT, low range-ACT, and thrombin-activated TEG.

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.

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.

Background: Thromboembolic complications can occur with recombinant activated factor VII (rFVIIa) treatment in trauma and surgical patients but they are infrequent in hemophiliacs. Bleeding diathesis in these conditions is often attributed to reduced thrombin generation, which may be improved with rFVIIa. Normally, thrombin that diffuses from local vascular injury sites is quickly inactivated by antithrombin (AT). Evaluating the influence of AT levels on thrombin generation in hypocoagulable FVIII-deficient plasma would be a simple approach to better understand how procoagulant stimuli, such as rFVIIa, might result in postoperative thrombotic complications. We hypothesize that reduced AT concentrations would increase the procoagulant effects of rFVIIa in FVIII-deficient plasma., Methods: Thrombin generation was evaluated in vitro in FVIII-deficient and AT/FVIII-deficient plasma using thrombelastography and a thrombin generation assay (Thrombinoscope). The effect of added rFVIIa on these variables was evaluated., Results: Delayed thrombus formation based on thrombelastography in FVIII-deficient plasma was predictably reversed by rFVIIa. Improved thrombus formation and responses to rFVIIa were observed when AT levels were 20%-50% of normal. Thrombin generation in FVIII-deficient plasma increased in an inverse relationship to AT levels. Supplemental rFVIIa decreased the lag time of thrombin generation but not the amount of thrombin generated., Conclusions: Using FVIII-deficient plasma as a model of reduced thrombin generation, we demonstrate that low AT levels enhance in vitro hemostatic responses to rFVIIa. Reduced AT levels in trauma and surgical patients with normal or increased FVIII levels may be considered potentially prothrombotic. Monitoring of AT levels during rFVIIa therapy may thus reduce thrombotic complications in nonhemophiliacs.

Thrombin is a key hemostatic enzyme, which propagates its own generation by activating factors V, VIII, and XI. Sustained thrombin generation also activates thrombin-activatable fibrinolysis inhibitor (TAFI), which stabilizes fibrin clot against fibrinolysis. Recombinant activated factor VII (rFVIIa) is considered a novel hemostatic intervention for refractory bleeding, but rebleeding episodes related to fibrinolysis still occur. The present study aimed to investigate the antifibrinolytic effects of rFVIIa in relation to thrombin generation. Using thrombelastography, the effects of rFVIIa on thrombin-activated fibrin formation and on fibrinolysis induced by tissue plasminogen activator were evaluated in various factor-deficient plasma samples. A Thrombinoscope was used to quantitate thrombin generation. Thrombin increased antifibrinolytic activity in a concentration-dependent manner as demonstrated by a longer clot lysis time. In plasma deficient in factors V, VIII, IX, X, or XI, clot lysis occurred early (< 20 min), and rFVIIa addition had minimal effect, except for improved antifibrinolytic effect in factor-XI-deficient plasma. A normal clot lysis time was observed in factor-XIII-deficient or dual antithrombin/factor-VIII-deficient plasma. Inhibition of TAFI increased the rate of fibrinolysis. Thrombin generation was delayed or decreased in single factor-deficient plasma except for factor XIII deficiency. After rFVIIa addition, the peak thrombin generation reached over 100 nmol/l in factor-XI-deficient plasma, but not in plasma deficient in factors V, VIII, IX, or X. Thrombin generation and subsequent activation of TAFI were important for clot stability. We conclude that rFVIIa therapy does not compensate for increased susceptibility to fibrinolysis due to lack of factor(s) necessary for the formation of tenase and prothrombinase.

For intravenous anticoagulation, heparin has been the mainstay drug, but its use may be contraindicated in heparin-induced thrombocytopenia and thrombosis. Heparin alternatives including direct thrombin inhibitors are available, but clotting assays (e.g. partial thromboplastin time) measure the time required to form fibrin gel when only a small amount of thrombin is generated. It was hypothesized that the extent of thrombin inhibition varies among inhibitors, and thrombin-activated thrombelastography would provide useful data on therapeutic responses to thrombin inhibitors. Thrombin was added (0-100 nmol/l final concentration) to nonrecalcified whole blood to evaluate clot formation on thrombelastography. Effects of direct thrombin inhibitors (argatroban 3.75 microg/ml, bivalirudin 15 microg/ml, and lepirudin 3.0 microg/ml), and heparin cofactor II activator and dermatan disulfate (20 microg/ml) were evaluated in the presence of 100 nmol/l thrombin. The interactions of thrombin and respective inhibitors were also compared by fluorogenic thrombin substrate cleavage. Increasing concentrations of thrombin progressively shortened the lag time and increased viscoelasticity on thrombelastography. Only 20 nmol/l thrombin caused instantaneous clotting, but maximal viscoelastic force was obtained at 50-100 nmol/l thrombin. All thrombin inhibitors prolonged the lag time (lepirudin > bivalirudin > argatroban = dermatan disulfate), but full recovery of thrombelastography viscoelasticity was observed with argatroban and bivalirudin. Lepirudin abrogated clotting, and dermatan disulfate suppressed clot development on thrombelastography. Thrombin substrate cleavage was observed only for bivalirudin, and heparin cofactor II without dermatan disulfate. The modified thrombelastography technique using nonrecalcified whole blood may be useful in evaluating the extent and reversibility of thrombin blockade with direct or indirect thrombin inhibitors.

Background: Coagulation tests, such as activated partial thromboplastin time and activated clotting time, are used to monitor the effects of unfractionated heparin and the direct thrombin inhibitor, bivalirudin. These tests reflect only the initial phase of blood clotting, when <5% of thrombin has been formed. In this study, we sought to determine if similar increases in activated partial thromboplastin time or activated clotting time due to heparin or bivalirudin would reflect the same degree of inhibition of thrombin formation., Methods: Thrombin formation was evaluated in platelet-poor plasma activated in the presence of heparin (0-5 U/mL) or bivalirudin (0-30 microg/mL) using a thrombin generation assay (Thrombinoscope). Prothrombin activation was measured by prothrombin fragment 1.2 (F1.2) formation. Thrombus formation was further evaluated in kaolin-activated whole blood samples containing heparin (1.5 or 2.5 U/mL) or bivalirudin (12.5 or 25 microg/mL) using Sonoclot and thromboelastography., Results: Based on the Thrombinoscope results, increasing concentrations of bivalirudin and heparin progressively delayed the onset of thrombin formation, but only heparin dose-dependently decreased the amount of thrombin generated. Heparin and bivalirudin delayed the onset of F1.2 formation, but there was no difference in peak F1.2 levels between bivalirudin and non-anticoagulated samples (206 +/- 28.2 vs 182 +/- 23.9 nmol/L, P = 0.09). In heparinized samples, F1.2 levels were significantly lower (75.7 +/- 29.8 nmol/L, P < 0.05) than controls. Heparin and bivalirudin prolonged the onset of clotting on viscoelastic monitors, but only heparin decreased the rate of thrombus formation., Conclusion: Thrombus formation kinetics differs between heparin and bivalirudin despite similar prolongation of clotting test values.

Thrombin is an essential serine protease for survival. Since the discovery of heparin in the early twentieth century, significant advances have been made in the understanding of thrombin structure and function in coagulation system. Endogenous anticoagulant proteins in blood tightly regulate thrombin generation, but additional anticoagulant agents may be necessary to suppress excessive thrombin formation or defective anticoagulant proteins. Despite the availability of an array of anticoagulant agents based on chemical and biological engineering technologies, anticoagulation therapy remains a challenge for clinicians in terms of balancing bleeding and thrombosis. The aim of this article is to review endogenous serine protease inhibitors and novel antithrombotic agents in relation to pharmacologic regulation of thrombin.

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 microg/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.

We evaluated argatroban, a direct thrombin inhibitor, as a heparin adjunct for anticoagulation. Platelet-poor plasma (PPP) was isolated from blood collected from 12 volunteers. Thrombin generation measurements were performed in donor PPP that was mixed with antithrombin (AT)-poor plasma to yield AT levels of 0%, 20%, 60%, and 100%. Effects of argatroban (0-1.0 microg/mL), heparin (0.25 U/mL), or the combination of argatroban (0.5 microg/mL) and heparin were also studied. The addition of increasing concentrations of argatroban, heparin, or both to donor PPP (AT level approximately 100%) caused progressive decreases in the lag time and peak formation of thrombin generation. Heparin (0.25 U/mL) at small AT concentrations had a minimal effect on lag time or peak thrombin formation; its effectiveness of inhibiting thrombin was directly correlated with the concentration of AT. Argatroban at 0.5 microg/mL was effective in decreasing thrombin formation at both low and normal AT levels, but it was most effective when combined with heparin. Additionally, blood samples were obtained from 47 cardiac surgical patients, and the interaction of heparin (>1.5 U/mL) and AT or argatroban on clot formation was evaluated with kaolin activated clotting times (ACTs). Significant increases of ACTs at all heparin levels were observed with the addition of argatroban (0.125 and 0.25 microg/mL). The addition of AT (0.2 U/mL) to heparinized blood samples further prolonged ACTs. In summary, we showed that argatroban, unlike heparin, could effectively reduce thrombin generation regardless of AT levels and could prolong ACTs in vitro at clinically used concentrations.

Abstract: Introduction: Warfarin and its derivatives are widely used for prevention of thrombotic incidents. Prothrombin complex concentrate (PCC) and recombinant activated factor VII (rFVIIa) have been used clinically for the acute reversal of this agent but there is a paucity of data on comparative efficacies of these hemostatic interventions. Materials and methods: Using in vivo rat and in vitro human models of anticoagulation, we compared PCC and rFVIIa on the recovery of endogenous thrombin generation. For in vivo reversal of anticoagulation, saline (control), PCC 50 U ml−1, or rFVIIa100 μg ml−1 was given to rats which received phenprocoumon (2.5 mg kg−1) orally. For in vitro model, plasma samples from warfarin-treated individuals with INR values of 2.1–6.7 were spiked with PCC (0.2, 0.4, or 0.72 U ml−1) or rFVIIa (3.0 μg/ml). The treatments were evaluated using prothrombin time (PT) and thrombin generation (Thrombinoscope™). Results: Administration of phenprocoumon to rats prolonged PT (14.7±0.5 to 50.43±0.7 s) and decreased peak thrombin generation by 89±2.3%. Administration of PCC dose dependently reversed the anticoagulation effects both in warfarin-treated human plasma and in phenprocoumon-treated rats by shortening PT and increasing peak thrombin levels. However, rFVIIa only reversed PT, but had minimal effects on peak thrombin levels. Conclusion: Both PCC and rFVIIa reverse warfarin anticoagulation based on PT, but only PCC restores overall thrombin generation. [Copyright &y& Elsevier]

Platelet activation is involved in physiological hemostasis and in a variety of thromboembolic events leading to stroke, myocardial ischemia, and other organ dysfunction. Apart from congenital platelet dysfunction, acquired platelet defects are increasingly common in patients due to the widespread use of antiplatelet agents. Newer antiplatelet agents targeting platelet glycoprotein IIb-IIIa and adenosine-5'-diphosphate receptors inhibit platelet activation, and therefore assessing platelet inhibition may be important to prevent major bleeding, especially before a surgical intervention. In the past decade, the knowledge from genotypic analyses and prospective outcome studies suggested inter-patient variability in the therapeutic response to antiplatelet therapies, which further stresses the need for individual platelet testing. In managing bleeding patients, platelet transfusion may be better guided by the use of platelet function tests because transfusion therapy is costly and potentially harmful if administered inappropriately. Basic understanding of the hemostatic roles of platelets, etiologies of platelet defects, and characteristics of current platelet assessment methods should help physicians who manage complex coagulation issues in critically ill patients. [ABSTRACT FROM AUTHOR]

We studied whether there was a relationship between the anticoagulant effects of recombinant human soluble thrombomodulin (rhsTM) and activation of protein C in a primate model of acute vascular graft thrombosis in 11 baboons ( Papio species). Baboons were pretreated with 0·1, 1 and 5 mg/kg of rhsTM, with or without co-injection of a neutralising monoclonal antibody to protein C (HPC4) in the 1 mg/kg rhsTM group. Subsequently, thrombogenic polyester grafts were deployed for 3 h into chronic exteriorised arteriovenous shunts. Thrombus growth in the graft, plasma-activated protein C (APC) levels, coagulation and thrombosis markers were determined. In untreated baboons, baseline circulating APC levels more than doubled and graft thrombi propagated until reaching equilibrium in about 1 h. Treatment with rhsTM reduced thrombus propagation rates, prolonged the clotting and bleeding times, decreased thrombin–antithrombin complex, β-thromboglobulin and fibrinopeptide A levels, and, surprisingly, also decreased systemic APC levels, in a dose-dependent manner. In the presence of HPC4 antibody to inhibit APC generation, the acute antithrombotic activity of rhsTM on graft thromboses was not attenuated for up to 80 min, but sustained thrombus accumulation was observed over a 180-min period. These findings suggest that, in contrast to the prevailing hypotheses, the primary antithrombotic activity of rhsTM is independent of protein C, at least in this primate model. Direct inhibition of thrombin's prothrombotic activity upon complex formation with rhsTM might explain the molecular mechanism of the observed antithrombotic effect. [ABSTRACT FROM AUTHOR]

Andexanet alfa is a Gla-domainless mutant (S195A) factor Xa (GDXa) approved for acute reversal of oral factor Xa inhibitors. Cardiac surgery patients exposed to andexanet before cardiopulmonary bypass often exhibit severe heparin resistance. There is a paucity of data on the effectiveness and optimal dosage of antithrombin use in this setting. The objective of this study was to evaluate the in vitro effect of increased heparin with antithrombin levels on attenuating heparin resistance induced by GDXa. Heparinised normal pooled plasma and cardiopulmonary bypass plasma were spiked with GDXa 4 μM. Tissue factor-activated thrombin generation was used to assess heparin reversal effects of GDXa and restoration of anticoagulation with additional heparin with and without antithrombin. Serum thrombin–antithrombin complex, antithrombin activity, and tissue factor pathway inhibitor were also measured in tissue factor-activated, recalcified cardiopulmonary bypass plasma spiked with GDXa. In normal pooled plasma, GDXa-induced heparin reversal was mitigated by maintaining a high heparin concentration (12 U ml−1) and supplementing antithrombin (1.5–4.5 μM) based on peak and velocity of thrombin generation. Heparin reversal by GDXa was also demonstrated in cardiopulmonary bypass plasma, but supplementing both heparin (8 U ml−1) and antithrombin (3 μM) attenuated GDXa-induced changes in peak and velocity of thrombin generation by 72.5% and 72.2%, respectively. High heparin and antithrombin levels attenuated thrombin–antithrombin complex formation in tissue factor-activated, GDXa-spiked cardiopulmonary bypass plasma by 85.7%, but tissue factor pathway inhibitor remained depleted compared with control cardiopulmonary bypass plasma. Simultaneous supplementation of heparin and antithrombin mitigate GDXa-induced heparin resistance by compensating for the loss of tissue factor pathway inhibitor. [ABSTRACT FROM AUTHOR]

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. 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. 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. 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. Schemata of putative mechanisms of factor Xa modulation of plasmin generation. A trace amount of fibrin initiates the formation of plasmin (Pln), which proteolyzes phospholipid-bound factor Xaα into Xaβ. The latter is further proteolyzed by plasmin into a heterodimer of 33 kDa and 13 kDa fragments (Xa33/13). Both Xaβ and Xa33/13 express carboxy-terminal lysine residues (⊕ in yellow) to which plasminogen (Plg) can be bound and subsequently activated by tissue plasminogen activator (tPA), facilitating the initiation of fibrinolysis. After binding of apixaban to factor Xaα, thrombin (IIa) generation is slowed down (|| in red), and Xa33/13 formation is blocked (× in red), resulting in reduced velocities of plasmin generation. [Display omitted] • Gla-domainless factor Xa S195A (GDXa) is useful in modeling a factor Xa inhibitor reversal with andexanet alpha in vitro • Profibrinolytic activity of factor Xa is inhibited by apixaban. • Despite the recovery of thrombin generation, GDXa had minimal effect on the changes in plasmin generation and fibrinolysis at elevated apixaban concentrations. [ABSTRACT FROM AUTHOR]

The article provides information on a study which examined the effects of hemodilution on thrombin generation, fibrin polymerization and fibrinolysis, in relation to the mechanisms of dilutional coagulopathy. It states that anticoagulant factor levels are decreased proportional to the extent of hemodilution, while fibrinolytic and antifibrinolytic activities are also affected in massive hemorrhage. In addition, amplified generation of thrombin and thrombin-activated FXIII produced the cleavage of fibrinogen bound to platelet glycoprotein receptors.

Abstract: Introduction: Thrombin-mediated activation of human platelets involves the G-protein-coupled protease-activated receptors PAR1 and PAR4. Inhibition of PAR1 and/or PAR4 is thought to modulate platelet activation and subsequent procoagulant reactions. However, the antithrombotic effects of PAR1 and PAR4 antagonism have not been fully elucidated, particularly under flow conditions. Materials and Methods: A microchip-based flow chamber system was used to evaluate the influence of SCH79797 (PAR1 antagonist) and YD-3 (PAR4 antagonist) on thrombus formation mediated by collagen and tissue thromboplastin at shear rates simulating those experienced in small- to medium-sized arteries (600s-1) and large arteries and small veins (240s-1). Results: At a shear rate of 600s-1, SCH79797 (10μM) efficiently reduced fibrin-rich platelet thrombi and significantly delayed occlusion of the flow chamber capillary (1.44 fold of control; P <0.001). The inhibitory activity of SCH79797 was diminished at 240s-1. YD-3 (20μM) had no significant effect at either shear rate. The antithrombotic effects of SCH79797 were significantly augmented when combined with aspirin and AR-C66096 (P2Y12 antagonist), but not with YD-3. In contrast, no significant inhibition of tissue factor-induced clot formation under static conditions was observed in blood treated with SCH79797 and YD-3, although thrombin generation in platelet-rich plasma was weakly delayed by these antagonists. Conclusions: Our results suggest that the antithrombotic activities of PAR1 and/or PAR4 antagonism is influenced by shear conditions as well as by combined platelet inhibition with aspirin and a P2Y12-antagonist. [Copyright &y& Elsevier]