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Although thrombin is a key enzyme in the coagulation cascade and is required for both normal hemostasis and pathologic thrombogenesis, it also participates in its own negative feedback via activation of protein C, which downregulates thrombin generation by enzymatically inactivating factors Va and VIIIa. Our group and others have previously shown that thrombin's procoagulant and anticoagulant activities can be effectively disassociated to varying extents through site-directed mutagenesis. The thrombin mutant W215A/E217A (WE thrombin) has been one of the best characterized constructs with selective activity toward protein C. Although animal studies have demonstrated that WE thrombin acts as an anticoagulant through activated protein C (APC) generation, the observed limited systemic anticoagulation does not fully explain the antithrombotic potency of this or other thrombin mutants. AB002 (E-WE thrombin) is an investigational protein C activator thrombin analog in phase 2 clinical development (clinicaltrials.gov NCT03963895). Here, we demonstrate that this molecule is a potent enzyme that is able to rapidly interrupt arterial-type thrombus propagation at exceedingly low doses (<2 µg/kg, IV), yet without substantial systemic anticoagulation in baboons. We demonstrate that AB002 produces APC on platelet aggregates and competitively inhibits thrombin-activatable fibrinolysis inhibitor (carboxypeptidase B2) activation in vitro, which may contribute to the observed in vivo efficacy. We also describe its safety and activity in a phase 1 first-in-human clinical trial. Together, these results support further clinical evaluation of AB002 as a potentially safe and effective new approach for treating or preventing acute thrombotic and thromboembolic conditions. This trial was registered at www.clinicaltrials.gov as #NCT03453060., (© 2020 by The American Society of Hematology.)

Background: It is well known that warfarin inhibits the synthesis of vitamin K-dependent anticoagulants, including thrombin, protein C and S, and factor Xa, leading, paradoxically, to an initial hypercoagulable state. Edoxaban, a direct inhibitor of activated factor X is widely used for the treatment of acute venous thromboembolism (VTE). However, the effect of edoxaban on circulating coagulation factors, in patients with acute VTE, remains unknown., Methods and Results: We enrolled 57 patients with acute VTE with/without pulmonary embolism treated with edoxaban (n=37) or warfarin (n=20) in a clinical setting. Before treatment and 2 weeks after treatment, we evaluated thrombotic burden using ultrasound or computed tomography angiography. We also evaluated thrombin generation, represented by prothrombin fragment F1+2; thrombus degradation, represented by D-dimer; and levels of anticoagulants, including protein C, protein S, and antithrombin III. Both edoxaban and warfarin treatment improved thrombotic burden and decreased prothrombin fragment F1+2, and D-dimer. Edoxaban treatment preserved protein C and protein S levels. In contrast, warfarin decreased protein C and protein S levels. Neither treatment affected antithrombin III., Conclusions: Edoxaban improves VTE while preserving protein C and protein S levels, thereby indicating that edoxaban improves thrombotic burden while maintaining levels of anticoagulants., (Copyright © 2017 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.)

Background: Protein C is a vitamin K dependent plasma zymogen. It inhibits clotting by inhibiting clotting by inactivating factor V and factor VIII. Protein C activation pathway involves three steps: (i) Activation of protein C; (ii) Inhibition of coagulation through inactivating factor V and VIII by activated protein C and (iii) Inhibition of activated protein C by plasma protease inhibitors specific for this enzyme. Proteinases converting the zymogen protein C (PC) of vertebrates into activated PC have been detected in several snake venoms. Most PC activators have been purified from venom of snake species belonging to the genera of the Agkistrodon complex. Unlike the physiological thrombin-catalyzed PC activation reaction which requires thrombomodulin as a cofactor, most snake venom activators directly convert the zymogen PC into the catalytically active form which can easily be determined by means of coagulation or chromogenic substrate techniques., Conclusion: The fast-acting PC activator Protac® from Agkistrodon contortrix contortrix (Southern copperhead snake) venom has found a broad application in diagnostic practice for the determination of disorders in the PC pathway. Recently, screening assays for the PC pathway have been introduced, based on the observation that the PC pathway is probably the most important physiological barrier against thrombosis., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Objectives: The risk of venous thromboembolism (VTE) is highest during the initial months of oral contraceptive (OC) use. We sought to evaluate the extent of hemostatic variable changes during the initial OC cycle and if such changes are related to systemic ethinyl estradiol (EE2) exposure., Study Design: Participants provided multiple blood samples during a 21-day OC cycle (30mcg EE2; 150mcg levonorgestrel) and after a single dose following a washout period. Analytes included D-dimer, factor VIII activity, protein C total antigen and the hepatic proteins corticosteroid-binding globulin (CBG) and sex-hormone-binding globulin (SHBG). EE2 pharmacokinetic analyses related to the 24h after the first OC tablet (OC1) and at steady state (OC21)., Results: Seventeen women completed the study. D-dimer more than doubled by OC6 (p=.013) and remained elevated at OC21 (p=.012). D-dimer levels within women varied widely from day to day. Factor VIII increased 27% by OC2 (p<.001) but declined to a 9% increase by OC21. Protein C increased only 6%. EE2 steady-state area-under-the-curve ranged from 488 to 1103pg∙h/mL; higher levels were not correlated with greater increases in clotting variables. CBG and SHBG increased significantly but were not significantly correlated with levels of EE2 or with the hemostatic variables., Conclusions: D-dimer increases during the first OC cycle were at least as great as increases seen with longer OC use. These results provide support for the increased VTE risk during initial OC use. The extreme variability in D-dimer levels may be an important component of this risk., Implications: This study showed that increases in D-dimer are clearly evident in the first cycle of OC use and may be larger than are seen after a longer duration of use and thus provide biological support for the increased VTE risk during initial OC use found in epidemiological studies., Competing Interests: None of the other authors have any conflicts to report., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose of Investigation: To compare the effects of desogestrel (DSG) 150 mcg/ethinyl estradiol (EE) 20 mcg for 21 days followed by either seven days of EE ten mcg (21/7-active) or no treatment (DSG/EE+no Tx) on hemostatic markers., Materials and Methods: This was a randomized, multicenter, open-label study that enrolled healthy premenopausal women. Non-inferiority of 21/7-active to DSG/EE+no Tx was determined if the upper limit of the two-sided 95% CI of the mean treatment difference in prothrombin fragment 1+2 (F1+2) over 24 weeks between groups was < 130 pmol/L., Results: 246 subjects (n = 125, 21/7-active; n = 121, DSG/EE+no Tx) comprised the primary analysis. Mean F1+2 levels increased in both 21/7-active and DSG/EE+no Tx regimens (least square [LS] mean changes +45 pmol/L and +56.8 pmol/L, respectively). LS mean treatment difference was -11.8 pmol/L (95% CI: -54.8, 31.2)., Conclusion: The effect of adding EE ten mcg to the seven-day hormone-free interval of DSG/EE on F1+2 levels was non-inferior to traditional DSG/EE.

Objective: To investigate the influence of hydroxyethyl starch solution on exogenous coagulation and active protein C ( APC ) in the patients with septic shock., Methods: A single-center prospective study was conducted. Eighty-four consecutive patients with septic shock admitted to intensive care unit ( ICU ) of Peking University People's Hospital from November 2009 to October 2014 were enrolled. The patients were randomized into two study groups by random digits table: Ringer lactate solution group ( RL group, n = 40 ) and hydroxyethyl starch group ( HES group, n = 44 ), and Ringer lactate solution or hydroxyethl starch 130/0.4 was used for resuscitation respectively. Peripheral blood was collected at four time points: before resuscitation, 6, 12, and 24 hours after resuscitation. The prothrombin time ( PT ), tissue factor ( TF ), tissue factor pathway inhibitor ( TFPI ) and APC were determined, and the length of ICU stay and the mortality were recorded., Results: There were no significant differences in PT, TF, TFPI, and APC before and after resuscitation in RL group. No change in PT was found after resuscitation in HES group, and no significant difference was found as compared with RL group. TF after resuscitation in HES group was decreased gradually, and the level at the 24 hours after resuscitation was significantly lower than that before resuscitation ( U/L: 15.80±7.32 vs. 31.40±2.75, P<0.05 ); but there was no significant difference at all time points when compared with that of RL group ( all P>0.05 ). TFPI at 12 hours and 24 hours after resuscitation in HES group was increased when compared with before resuscitation ( μg/L: 1.32±0.22, 1.14±0.09 vs. 0.63±0.54 ). TFPI in HES group was significantly higher than that in RL group ( μg/L: 0.84±0.69, 0.95±0.30 ), but there was no significant differences between two groups ( both P>0.05 ). APC after resuscitation in HES group was decreased gradually, which was significantly lower than that in RL group at 6, 12, 24 hours after resuscitation ( mg/L: 3.38±3.00 vs. 5.98±4.12, 3.31±1.94 vs. 5.33±3.71, 3.42±2.64 vs. 7.53±4.67, P<0.05 or P<0.01 ). The length of ICU stay in HES group was significantly shorter than that in RL group ( days: 12.50±8.83 vs. 17.10±16.60, t = 9.037, P<0.001 ), but there was no significant difference in mortality between HES group and RL group [ 40.9% ( 18/44 ) vs. 60.0% ( 24/40 ), χ (2) = 2.339, P = 0.126 ]., Conclusions: Both RL and hydroxyethyl starch fluid resuscitation did not affect the PT of the patients. The use of hydroxyethyl starch probably inhibits excessive activation of the exogenous coagulation and hyper-coagulation in the early stage of sepsis, and inhibits activation of protein C as well.

The protein C pathway provides multiple important functions to maintain a regulated balance between hemostasis and host defense systems in response to vascular and inflammatory injury. The anticoagulant protein C pathway is designed to regulate coagulation, maintain the fluidity of blood within the vasculature, and prevent thrombosis, whereas the cytoprotective protein C pathway prevents vascular damage and stress. The cytoprotective activities of activated protein C (APC) include anti-apoptotic activity, anti-inflammatory activity, beneficial alterations of gene expression profiles, and endothelial barrier stabilization. These cytoprotective activities of APC, which require the endothelial protein C receptor (EPCR) and protease-activated receptor-1 (PAR1), have been a major research focus. Recent insights, such as non-canonical activation of PAR1 at Arg46 by APC and biased PAR1 signaling, provided better understanding of the molecular mechanisms by which APC elicits cytoprotective signaling through cleavage of PAR1. The discovery and development of anticoagulant-selective and cytoprotective-selective APC mutants provided unique opportunities for preclinical research that has been and may continue to be translated to clinical research. New mechanisms for the regulation of EPCR functionality, such as modulation of EPCR-bound lipids that affect APC's cytoprotective activities, may provide new research directions to improve the efficacy of APC to convey its cytoprotective activities to cells. Moreover, emerging novel functions for EPCR expand the roles of EPCR beyond mediating protein C activation and APC-induced PAR1 cleavage. These discoveries increasingly develop our understanding of the protein C pathway, which will conceivably expand its physiological implications to many areas in the future., (© 2013 International Society on Thrombosis and Haemostasis.)

Protein C in its activated form (APC) limits thrombin generation. Protein C inhibitor (PCI) readily neutralizes APC. Heparin accelerates this reaction, which may complicate anticoagulant treatment in patients with varying APC generation potential. A potent anticoagulant conjugate of antithrombin and heparin (ATH) was prepared, and its effect on APC+PCI reactions was tested. Second order rate constants for APC+PCI reactions were measured by discontinuous rate experiments in the presence of heparin or ATH. Similarly, low molecular weight fractions of heparin (LMWH) and ATH (LMWATH) were tested, as was high molecular weight ATH (HMWATH). Mechanisms of heparin or ATH binding to APC or PCI were assessed using electrophoresis. While heparin gave a higher maximal APC inhibition rate compared to ATH, peak inhibition rate was achieved at comparatively lower ATH concentrations. Since LMWH was ineffective at enhancing APC inhibition by PCI, unfractionated heparin likely acts by bridging APC and PCI. Unlike heparin, ATH may conformationally activate either APC or PCI since LMWATH significantly catalyses APC inhibition. Binding studies showed that ATH readily associates with APC. Thus, although a small fraction of ATH efficiently catalyses APC inhibition by PCI, complete ATH preparations induce a decreased maximal rate of APC-PCI formation compared to unfractionated heparin.

Valproic acid is one of the most frequently prescribed antiepileptic drugs for the therapy of generalized and focal epilepsies. Valproate induces a variety of hemostatic disorders such as thrombocytopenia, abnormal platelet function, hypofibrinogenemia, and decreased concentrations of von Willebrand factor, and it rarely causes serious bleeding complications. It may also lead to atherosclerosis and thrombosis. However, there is still lack of knowledge about the incidence and occurrence of these particular side effects. In this prospective systematic study, we assessed the early effects of sodium valproate on both pro- and anticoagulatory factors, homocysteine, and lipoprotein (a) in 24 newly diagnosed epileptic children treated with valproate. Valproate causes decreased factor VII levels, platelet count, factor VIII, Protein C, fibrinogen, and increased lipoprotein (a) levels. To the best of our knowledge, our report is the first in the medical literature, which describes that valproate significantly reduces factor VII levels even during short-term therapy.

Background: A recently halted clinical trial showed that intensive treatment of type 2 diabetes mellitus was associated with increased mortality. Given the phenotypic heterogeneity of diabetes, therapy targeted at insulin status may maximize benefits and minimize harm., Methods: In this longitudinal cohort study, we followed 503 patients with type 2 diabetes who were free of cardiovascular disease from 1996 until data on mortality and cardiovascular outcomes were censored in 2005. Phenotype-targeted therapy was defined as use of insulin therapy in patients with a fasting plasma C peptide level of 0.2 nmol/L or less and no insulin therapy in patients with higher C peptide levels., Results: The mean age of the cohort was 54.4 (standard deviation 13.1) years, and 56% were women. The mean duration of diabetes was 4.6 years (range 0-35.9 years). Of the 503 patients, 110 (21.9%) had a low C peptide level and 111 (22.1%) were given insulin. Based on their C peptide status, 338 patients (67.2%) received phenotype-targeted therapy (non-insulin-treated, high C peptide level [n = 310] or insulin-treated, low C peptide level [n = 28]), and 165 patients (32.8%) received non-phenotype-targeted therapy (non-insulin-treated, low C peptide level [n = 82] or insulin-treated, high C peptide level [n = 83]). Compared with the insulin-treated, low-C-peptide referent group, the insulin-treated, high-C-peptide group was at a significantly higher risk of cardiovascular events (hazard ratio [HR] 2.85, p = 0.049) and death (HR 3.43, p = 0.043); the risk was not significantly higher in the other 2 groups. These differences were no longer significant after adjusting for age, sex and diabetes duration., Interpretation: Patients with low C peptide levels who received insulin had the best clinical outcomes. Patients with normal to high C peptide levels who received insulin had the worst clinical outcomes. The results suggest that phenotype-targeted insulin therapy may be important in treating diabetes.

Tadalafil is a phosphodiesterase 5 (PDE5) inhibitor that is used to treat male erectile dysfunction. It is currently undergoing clinical trials for the treatment of pulmonary hypertension. Although at present there is no evidence of hypercoagulation states being associated with the use of PDE5 inhibitors, several studies suggest that PDE5 inhibitors may, in rare cases, cause arterial and venous thrombotic or embolic events in the heart, eye, brain, and ear. We report a 54-year-old male patient with reduced plasma levels of protein C who developed pulmonary embolism (PE) just after he started taking tadalafil. The Naranjo probability scale indicates tadalafil as the possible cause of a PE in our patient. To the best of our knowledge, this is the first case report of tadalafil-induced PE in the literature.

Objective: Various lung diseases are associated with local activation of coagulation and concurrent inhibition of fibrinolysis. Although salmeterol, a beta2-adrenoceptor agonist with profound bronchodilatory properties, has been studied extensively, the effects of this compound on the pulmonary hemostatic balance are not elucidated., Design: A single-blinded, placebo-controlled study., Setting: University hospital and laboratory., Subjects: A total of 32 human volunteers., Interventions: Subjects inhaled 100 microg of salmeterol or placebo (t = -30 mins) followed by 100 microg of lipopolysaccharide (LPS) or normal saline (t = 0 mins; n = 8 per group)., Measurements and Main Results: Measurements were performed in bronchoalveolar lavage fluid obtained 6 hrs postchallenge. Inhalation of LPS enhanced pulmonary coagulation as determined by an increase in the concentrations of thrombin-antithrombin complexes, factor VIIa, and soluble tissue factor in bronchoalveolar lavage fluid (all p < .05 vs. saline). LPS concurrently inhibited pulmonary fibrinolysis, as reflected by a decrease in bronchoalveolar lavage fluid plasminogen activator activity together with an increase in plasminogen activator inhibitor type 1 (both p < .05 vs. saline). Moreover, LPS inhalation was associated with a suppression of the anticoagulant protein C pathway, as indicated by an increase in soluble thrombomodulin and decreases in protein C and activated protein C levels in bronchoalveolar lavage fluid (all p < .05 vs. saline). Salmeterol, either with or without LPS inhalation, enhanced fibrinolysis (plasminogen activator activity and tissue-type and urokinase-type plasminogen activator levels) but did not influence LPS-induced changes in coagulation or the protein C pathway., Conclusions: Salmeterol has profibrinolytic properties in the normal lung and when applied in a model of sterile pulmonary inflammation.

Phosphatidylserine is known to significantly accelerate the blood coagulation reaction. In a previous communication submitted for publication, we demonstrated that phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine showed effects on the blood coagulation reaction using the factor Xa-prothrombin reaction system, and discuss a new function of membrane phospholipids. The present study examined the role of phospholipids in the blood coagulation regulatory reaction (anticoagulation system), by studying the effects of phospholipids on the protein C/protein S reaction. We have established quantitative methods for measuring activated protein C activity and protein S activity, and used them to measure their activity after the addition of liposomes with different phospholipid compositions. We found that phosphatidylcholine inhibited activated protein C and protein S activities in a dose-dependent manner, as in the factor Xa-prothrombin reaction system. On the other hand, phosphatidylethanolamine and lysophosphatidylcholine showed no effect on activated protein C activity. Phosphatidylethanolamine inhibited and lysophosphatidylcholine accelerated coagulation activity in the factor Xa-prothrombin system, but such effects were not observed in the protein C/protein S reaction system. The coagulation and anticoagulation reactions are exquisitely balanced by thrombin, with a role both as a procoagulant and anticoagulant. Therefore, it is understandable that phosphatidylethanolamine and lysophosphatidylcholine show different effects in the factor Xa-prothrombin and protein C/protein S reaction systems. It appears that coagulation and anticoagulation reactions are co-ordinated and controlled by changes in phospholipid composition of the cellular membrane where the coagulation reaction takes place.

Activated protein C (APC) anticoagulant activity and the ability to be inhibited by auto-antibodies associated with thrombosis are strongly augmented by the presence of phosphatidylethanolamine (PE) and phospholipid oxidation. beta(2)-glycoprotein I (beta(2)-GPI) is a major antigen for antiphospholipid antibodies present in patients with the antiphospholipid syndrome. We therefore investigated whether anti-beta(2)-GPI monoclonal antibodies (mAbs) could inhibit APC with similar membrane specificity. Five mouse mAbs that reacted with different epitopes on beta(2)-GPI were examined. Each inhibited the PE-, phospholipid oxidation-dependent enhancement of APC anticoagulant activity and required antibody divalency. A chimeric APC that retains anticoagulant activity but is relatively unaffected by protein S, PE, or oxidation was not inhibited by the antibodies. In purified systems, anti-beta(2)-GPI mAb inhibition of factor Va inactivation was greater in the presence of protein S and required beta(2)-GPI. Surprisingly, although the mAbs did increase beta(2)-GPI affinity for membranes, PE and oxidation had little influence on the affinity of the beta(2)-GPI antibody complex for the membrane vesicles. We conclude that antibodies to beta(2)-GPI inhibit APC function specifically and contribute to a hypercoaguable state by disrupting specific protein-protein interactions induced by oxidation of PE-containing membranes.

Background and Aims: N-acetylcysteine is used to treat paracetamol overdose but depresses the activity of plasma coagulation factors II, VII, and X, which are often used to assess liver injury. The aim of this study was to investigate the effect of N-acetylcysteine on haemostasis in normal volunteers., Methods: Haemostatic parameters in 10 healthy subjects were analysed before and following intravenous infusion of therapeutic doses of N-acetylcysteine, as well as in vitro., Results: N-acetylcysteine induced significant decreases in plasma levels of vitamin K dependent haemostatic proteins in vivo, being maximal at one hour following the start of infusion, with maximal decreases from 1.00 to 0.73 (0.67-0.79) (mean (95% confidence interval)), 0.66 (0.58-0.73), 0.81 (0.73-0.90), 0.64 (0.57-0.70), 0.74 (0.65-0.82), and 0.61 (0.54-0.67) for factor II, VII, IX, and X activities, protein C activity, and free protein S reactivity, respectively. These data suggest that N-acetylcysteine induces protein modifications affecting activity. Five subjects developed an adverse reaction to infusion of N-acetylcysteine and these were associated with a rapid increase in levels of factor VIII and its carrier protein von Willebrand factor (vWf) from 1.0 to 1.85 (1.08-2.62) and 1.77 (0.83-2.71), respectively, which suggests that the allergic reaction induced release of vWf from endothelial cells. N-acetylcysteine did not affect factor VIII or vWf in subjects without adverse reactions, and nor did it affect factor V or antithrombin in any of the subjects., Conclusion: Therapeutic doses of N-acetylcysteine cause abnormal haemostatic activity, and this should be taken into account when using haemostatic function tests as an indicator of hepatic injury.

Objective: To investigate the protein C-activating property and experimental conditions that may affect the activities of protein C activator (PCA-SV)., Methods: PCA-SV's functional characteristic, possible anticoagulant and amidase activities were measured by method of APTT and that of specific chromogenic substrate respectively in various experimental conditions., Results: PCA-SV can specifically act on protein C. The pH-optimum and temperature-optimum for the action of PCA-SV were 6.0 - 7.0 and 37 - 40 degrees C respectively. The anticoagulants (heparin, sodium citrate) had no effect on the activities., Conclusion: PCA-SV is a new protein C-activating reagent that has high potency and specificity.

Objective: Sepsis is frequently associated with coagulatory activation, which may contribute to deteriorated organ function. Antithrombin is one important endogenous coagulation inhibitor that is therapeutically applied during sepsis. This study investigates the effect of 14-day antithrombin application on coagulatory variables., Design: Prospective study., Setting: Surgical intensive care unit of a university hospital., Patients: Forty patients with severe sepsis., Interventions: Patients with severe sepsis were randomly assigned to receive either conventional intensive care treatment (n = 20, controls) or antithrombin substitution that aimed at a plasma antithrombin activity > or =120% during a long-term (14-day) study period (n = 20, antithrombin). To allow comparative analysis of laboratory variables over time, all patients who did not survive the 14-day-period (five controls and six antithrombin patients) were prospectively excluded from the final evaluation. Their data were included in an intent-to-treat analysis., Measurements and Main Results: Antithrombin supplementation normalized global coagulation tests and increased prothrombin activity as well as fibrinogen concentration, reflecting less coagulation factor consumption (percent change from baseline in prothrombin activity, p <.01 vs. controls at days 9, 11-14 of antithrombin vs. controls [unpaired Student's t-test]; fibrinogen concentration, p <.01 vs. controls at days 10, 11, 13, and 14 of antithrombin). Simultaneously, antithrombin reduced contact system activation as indicated by increasing prekallikrein activities over time (% change, p <.01 vs. controls at days 6, 9-14) and increased protein C activities when compared with controls (% change, p <.01 vs. controls at days 10-14). Most changes occurred from day 7 to day 14 of antithrombin supplementation. Antithrombin did not influence C1 esterase inhibitor, plasminogen, alpha2 antiplasmin, or platelet counts (p >.01)., Conclusion: In this first study on long-term antithrombin therapy, antithrombin significantly reduced septic coagulatory response in patients with severe sepsis when given over 14 days.

Background: There is evidence for the activation of the coagulation system and a hypercoagulable state following cardioversion. The aim of the study was to determine whether electrical cardioversion in patients with chronic atrial fibrillation induced a prothrombotic state despite optimal anticoagulation. We studied the effects of electrical cardioversion on plasma levels of fibrinogen, antithrombin III, protein C and D-dimers., Methods: We studied 24 patients with chronic atrial fibrillation who were on optimal anticoagulation and were referred for electrical cardioversion. Samples of venous blood were taken 2 h pre and post cardioversion and 1 month later., Results: Plasma median concentrations of fibrinogen decreased significantly from 3.8 g/l (interquartile range 3.1-4.2 g/l) before cardioversion to 3.5 g/l (interquartile range 2.9-3.9 g/l) 2 h after cardioversion levels (P=0.004). The fibrinogen levels at 1 month post cardioversion (3.45 g/l, interquartile range 3.1-3.9 g/l) were also significantly lower than baseline (P=0.02). Plasma median levels of antithrombin III fell from 93.5 U/dl (interquartile range 89.3-97.0 U/dl) pre cardioversion to 89.5 U/dl (interquartile range 83.0-93.0 U/dl) 2 h after cardioversion (P=0.001) and returned to normal by 1 month (94.0 U/dl; interquartile range 89.3-98.5 U/dl; P=0.0001). There were no significant changes in plasma median D-dimer or protein C levels at any time., Conclusions: We have demonstrated a significant fall in the plasma fibrinogen and antithrombin III levels in patients with chronic atrial fibrillation early after electrical cardioversion, indicating thrombin generation. This study suggests that there are haemostatic changes of thrombogenesis induced by cardioversion despite optimal anticoagulation with warfarin.

A plausible mechanism to explain thrombotic risk differences associated with the use of second- and third-generation oral contraceptives (OCs), particularly in carriers of factor V(Leiden), is still lacking. In a double-blind trial, 51 women without and 35 women with factor V(Leiden) were randomized to either a second- (30 microg ethinylestradiol/150 microg levonorgestrel) or third- (30 microg ethinylestradiol/150 microg desogestrel) generation OC. After 2 cycles of use and a wash-out of 2 cycles, the participants continued with the corresponding progestagen-only preparation. Hemostatic variables that probe the activity of the anticoagulant protein C system were determined. Compared with levonorgestrel, desogestrel-containing OCs significantly decreased protein S and increased activated protein C (APC) resistance in both groups. OCs with desogestrel had the most pronounced effects in carriers of factor V(Leiden). Progestagen-only preparations caused changes of anticoagulant parameters opposite to those of combined OCs, which in a number of cases were more pronounced with levonorgestrel. Our data show that progestagens in combined OCs counteract the thrombotic effect of the estrogen component. The higher thrombotic risk associated with third-generation OCs compared with second-generation OCs may be explained by the fact that desogestrel appeared less antithrombotic than levonorgestrel, especially in women with factor V(Leiden).

Introduction: Clinical trials evaluating direct thrombin inhibitors in unstable coronary artery disease (CAD) have been disappointing. The hypothesis tested in the present study was that these agents may inhibit the anticoagulant effect of thrombin to a further extent than the procoagulant effect of thrombin., Materials and Methods: We studied both reversible and irreversible thrombin inhibitors and compared the effects of each inhibitor on activated protein C (APC) generation vs. the effect on fibrinopeptide A (FPA) generation. A mixture of protein C, thrombin inhibitor, fibrinogen, fibrin polymerisation blocker and thrombin was incubated with thrombomodulin (TM)-expressing human saphenous vein endothelial cells (HSVECs). The inhibitors investigated were melagatran, inogatran, hirudin, hirugen, D-Phe-D-Pro-D-arginyl chloromethyl ketone (PPACK), and antithrombin (AT) alone or in combination with unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH)., Results: All agents, except hirugen, inhibited APC and FPA generation in a dose-dependent manner. FPA inhibition/APC inhibition ratios, based on IC50 for inogatran, melagatran, hirudin, PPACK, AT, AT-UFH and AT-LMWH were 1.73, 0.85, 0.55, 2.1, 0.5, 0.65 and 3.1 respectively., Conclusions: All agents, except hirugen, inhibited APC and FPA generation approximately to a similar extent. Thus, it can be inferred that the poor efficacy of thrombin inhibitors in recent clinical trials in patients with unstable CAD is unlikely to be a consequence of their effects on the protein C system.

Background: Raloxifene therapy is associated with a three-fold increase in the risk for venous thromboembolism; however, its effects on the hemostatic system in postmenopausal women have not been well defined., Objective: To determine the effects of raloxifene therapy on the levels of natural anticoagulant proteins in postmenopausal women., Methods: Sixteen healthy postmenopausal women were enrolled in this prospective longitudinal study. The patients were treated with raloxifene hydrochloride (60 mg/day) for a period of 6 months. Antithrombin and protein C activities and protein S antigen levels were measured in all users at baseline, and after 1, 3 and 6 months of treatment. Statistical analysis included one-way analysis of variance (ANOVA) and the Bonferroni test for multiple comparisons among the study periods., Results: Statistically significant 5.1% and 6.5% reductions of plasma antithrombin activity were observed at 3 and 6 months of therapy, respectively (p < 0.05). Compared with baseline, raloxifene did not significantly affect protein C activity or protein S level., Conclusions: The results of this prospective study show for the first time that raloxifene use is associated with a significant reduction in plasma antithrombin activity. This effect may contribute to a procoagulant state and partly explain the increased risk of venous thromboembolism in raloxifene users.

Solvent/detergent (S/D) plasma is being increasingly widely used in clinical practice, as it carries significantly lower risk of lipid-enveloped viral transmission than standard fresh frozen plasma (FFP). However, previous reports have suggested that S/D processing might influence the coagulation factor content of plasma. We have investigated this question by measuring procoagulant factors (fibrinogen, factor V and factor VIII), anticoagulant factors (protein C and protein S) and routine coagulation screening tests (prothrombin time and activated partial thromboplastin time) in 48 single-donor units of FFP, and in 16 units of S/D plasma (Octaplas). All routine coagulation screening tests, factor VII and protein C levels were within the normal reference range for both S/D plasma and FFP. However, we found significant reductions in factor V (31%), factor VIII (28%) and protein S (50%) in S/D plasma. The observed quantitative differences in coagulation factor levels may be further exacerbated by the lower volume of solvent detergent plasma units (200 ml) compared with units of standard fresh frozen plasma (250 ml). These findings are of potential clinical significance, particularly in those patients with liver disease, constitutional factor V deficiency and congenital or acquired protein S deficiency.

The use of oral contraceptives (OCs) has been known for many years to affect significantly almost all hemostatic parameters, but the challenge to relate these changes in a meaningful way to OC-induced increased venous thrombotic risk has not been met. New insights indicate that at least part of the answer can be found in the net effect of OC use on the efficacy with which the protein C pathway down-regulates thrombin formation. During OC use the (blood) plasma of a woman becomes resistant to the anticoagulant action of activated protein C (APC). The extent of this so-called acquired APC resistance as determined in a thrombin generation-based assay correlates remarkably well with the risk increases observed in clinical studies. Recent evidence indicates that the prothrombotic effect of the estrogen component ethinylestradiol in combined OC is counteracted by the progestagen component present in these preparations and that third-generation progestagens such as desogestrel or gestodene are less efficient with respect to this than the second-generation progestagen levonorgestrel.

Here we report the in vitro selection of novel small peptide motifs that bind to human alpha-thrombin. We have applied mRNA display to select for thrombin binding peptides from an unbiased library of 1.2 x 10(11) different 35-mer peptides, each containing a random sequence of 15 amino acids. Two clones showed binding affinities ranging from 166 to 520 nM. A conserved motif of four amino acids, DPGR, was identified. Clot formation of human plasma is inhibited by the selected clones, and they downregulate the thrombin-mediated activation of protein C. The identified peptide motifs do not share primary sequence similarities to any of the known natural thrombin binding motifs. As new inhibitors for human thrombin open interesting possibilities in thrombosis research, our newly identified peptides may provide further insights into this field of investigation and may be possible candidates for the development of new anti-thrombotic agents.

Oral anticoagulant therapy, which is used for prophylaxis and management of thrombotic disorders, causes similar reductions in plasma levels of vitamin K-dependent procoagulant and anticoagulant clotting factor zymogens. When we measured levels of circulating activated protein C, a physiologically important anticoagulant and anti-inflammatory agent, in patients on oral anticoagulant therapy, the results unexpectedly showed that such therapy decreases levels of activated protein C substantially less than levels of protein C, prothrombin, and factor X, especially at lower levels of prothrombin and factor X. Thus, we suggest that oral anticoagulant therapy results in a relatively increased expression of the protein C pathway compared with procoagulant pathways not only because there is less prothrombin to inhibit activated protein C anticoagulant activity, but also because there is a disproportionately higher level of circulating activated protein C.

Antiphospholipid antibodies may interfere with the PC pathway, displaying a resistance to the activated PC (resistant phenotype). This effect was evaluated by the APCR and the ProCG systems in 36 lupus anticoagulant samples, yielding abnormal results in 47% of APCR(original), 17% of APCR(modified), and 22% of ProCG test. ProCG values correlated with APCR(original) but not with APCR(modified). Most of lupus anticoagulants affecting the PC pathway showed abnormal APCR(original) results but not abnormal ProCG values. The different behavior between both systems may be due to the heterogeneity of the antibodies or could be attributed to the fact that, in the ProCG, a PC activator is added, while the APCR employs already activated exogenous PC., (Copyright 2002 Wiley-Liss, Inc.)

Although the use of oral contraceptives has been frequently associated with an increased risk of thromboembolic events, definitive prothrombotic mechanisms have not so far been fully elucidated. The aim of our investigation was the evaluation of the activities of antithrombin, protein C, protein S and the resistance to activated protein C in 137 healthy users of third-generation oral contraceptives and in 170 healthy women who were not consuming oral contraceptives. Women on oral contraceptives showed a marked prothrombotic pattern, characterized by reduced activities of antithrombin and protein S, and increased resistance to activated protein C. Nearby 50% of oral contraceptive users displayed activities of protein S below the lower value of the reference range (controls, 10%; P < 0.001). No significant differences were observed between two progestagens (desogestrel or gestodene) on the coagulation parameters tested. We believe that, due to the adverse effect on haemostasis, the administration of third-generation oral contraceptives should be carefully considered in women carrying prothrombotic abnormalities.

Coagulation factor V has been at the centre of investigation for several years. In addition to factor V Leiden, various other polymorphisms are becoming the object of interest. Different results have been published about the association of the HR2 haplotype with decreased factor V levels and with reduced response to activated protein C (APC). Due to the central position of factor V in the clotting process, its activity can be determined in both thromboplastin-based and activated partial thromboplastin time (aPTT)-based assays. A multitude of assays are known for the determination of APC response. The aim of our study was to investigate whether different methods disclose genotype-dependent differences in factor V activity as well as APC response. Three wild-type carriers, three carriers homozygous for the R2 allele (4070G), and three carriers homozygous for the G allele (2391G, 2663G, 2684G, 2863G) were investigated. For each individual plasma sample, the factor V activity was determined using 12 different reagent combinations of three different thromboplastins, three different aPTT reagents, and two different factor V deficient plasma sources. The determination of factor V activity in the thromboplastin system revealed differences between the genotypes. These differences were independent of the thromboplastin reagent and the factor V-deficient plasma. The aPTT system exhibited a dependency on the aPTT reagent and the factor V-deficient plasma. Analysis of APC response disclosed genomic differences in specific test systems only. One type of assay could be more appropriate than other types in dependence of the position of genomic variations. Therefore, the applied assay is an important influential factor in investigations of functional consequences of genomic variations.

It has been suggested that aprotinin results in significantly increased risk for perioperative thrombotic complications in patients with Factor V(LEIDEN) (F5L) due to its ability to competitively inhibit activated protein C (APC) function in vitro. No clinical studies have been performed to assess the effect of aprotinin on APC function of F5L in vivo. We developed an ex vivo model to mimic the effects of cardiopulmonary bypass with the exclusion of the patient in order to assess APC function. Blood from normal (n = 2) and F5L heterozygous donors (n = 2) was treated with aprotinin or placebo (saline). The blood was heparinized, added to the prime and circulated at 2 l/min through a modified cardiopulmonary bypass circuit. After 60 min of circulation, the heparin was neutralized with protamine sulfate. Blood samples, drawn at specific time points, were analysed for APC ratio. Results showed a decrease in APC ratio for both F5L and normal bloods with the addition of aprotinin (18% and 40%, respectively). APC ratios also decreased with the commencement of extracorporeal circulation for all bloods, resulting in an APC ratio of 1.35 in normal placebo-treated blood and 0.67 in F5L placebo-treated blood. The combined effect of aprotinin and extracorporeal circulation resulted in APC ratios of 0.90 for normal blood and 0.63 for F5L blood, corresponding to a severe dysfunction of APC intraoperatively (reference range 1.9-4.0). The data from this model predict an increased risk of perioperative thrombosis due to inhibition of APC function in cardiac surgical patients heterozygous for the F5L mutation. Aprotinin further compounds the severity of APC dysfunction, though the effect is more severe in normal blood. The ex vivo model employed was an effective tool for the investigation of the haemostatic effect of aprotinin. This model may be exploited for other applications such as the investigation of novel or emerging haemostatic agents prior to clinical trial.

Objective: In the pathogenesis of disseminated intravascular coagulation, dysfunctional natural anticoagulant pathways appear to play a pivotal role. In this article, we will address the mechanisms that contribute to this defect in the regulation of coagulation activation. Furthermore, we will explore the experimental and clinical evidence that restoration of these anticoagulant pathways results in clinical improvement., Data Sources: We have searched and reviewed published articles on experimental studies of disseminated intravascular coagulation models in animals and clinical studies in patients with disseminated intravascular coagulation., Data Synthesis: All three major anticoagulant pathways, that is, the antithrombin pathway, the protein C system, and tissue factor pathway inhibitor, are defective in sepsis and disseminated intravascular coagulation. Several mechanisms contribute to this defect. Restoration of these pathways, in principle, by administration of coagulation inhibitor concentrates or recombinant anticoagulant factors, appears to ameliorate the coagulation disorder and, more important, result in improvement of clinically relevant outcomes, such as a reduction of organ failure and mortality., Conclusions: Restoration of disrupted physiologic anticoagulant pathways in disseminated intravascular coagulation is not only a logical point of impact in patients with sepsis and an activated coagulation system, but also is associated with an improved outcome in experimental and (initial) clinical studies.

Endothelial cell protein C receptor (EPCR) augments protein C activation by the thrombin-thrombomodulin complex about 5-fold in vitro. Augmentation is EPCR concentration dependent even when the EPCR concentration is in excess of the thrombomodulin. EPCR is expressed preferentially on large blood vessel endothelium, raising questions about the importance of protein C-EPCR interaction for augmenting systemic protein C activation. In these studies, this question was addressed directly by infusing thrombin into baboons in the presence or absence of a monoclonal antibody to EPCR that blocks protein C binding. Activated protein C levels were then measured directly by capturing the enzyme on a monoclonal antibody and assaying with chromogenic substrate. Blocking protein C-EPCR interaction resulted in about an 88% decrease in circulating activated protein C levels generated in response to thrombin infusion. Leukocyte changes, fibrinogen consumption, fibrin degradation products, and vital signs were similar between the animals infused with thrombin alone and those infused with thrombin and the anti-EPCR antibody. The results indicate that EPCR plays a major role in protein C activation and suggest that defects in the EPCR gene might contribute to increased risk of thrombosis.

Induction of procoagulant factors in malignant cells is considered to be the major cause of coagulation disorders in cancer. Thrombomodulin (TM), a negative regulator of coagulation was also found to be expressed in cancer cells. We report here evidence for another anticoagulant, the endothelial cell protein C receptor (EPCR), in cancer cells. EPCR was detected in several cell lines derived from various types of cancer. Significant levels of protein C (PC) activation were detected only with cell lines expressed both EPCR and TM. Anti-EPCR monoclonal antibodies (mAbs) specifically inhibited the activation. Thus, EPCR function appears to be important for PC activation by cancer cells. In addition, we detected EPCR expression in tumor cells from breast cancer patients, with an extremely high frequency. EPCR function may contribute to progression or pathogenesis of some types of cancer, and may explain the complexity of coagulopathy in cancer patients.

The dysfunctional mutant R352W-protein C was found in two patients with venous thrombosis. The mutant R352A-protein C was constructed to define the contribution of charge/size of the residue at 352 on protein C (chymotrypsin numbering 187). Compared with wild type-protein C, R352W-protein C showed no difference in activation by thrombin-thrombomodulin or alpha-thrombin. However, R352W-activated protein C (APC) anticoagulant activity (aPTT assay) was reduced to approximately 65%. Although the catalytic efficiency of R352W-APC towards the oligopeptide substrate S-2366 was unperturbed, factor Va and R506Q-factor Va were not efficiently inactivated by R352W-APC compared with wild type-APC. R352A-APC showed reduced anticoagulant activity and reduced efficiency in factor Va inactivation and in factor VIIIa-inactivation in the presence of protein S. These observations suggest that the dysfunction of R352W-APC in factor Va inactivation may be one of the mechanisms leading to venous thrombosis in affected patients and that R352 plays an important role in the physiological functioning of APC.

Warfarin, an antagonist of vitamin K, causes diminution of vitamin K-dependent coagulation factors in the circulation. Although all vitamin K-dependent factors have Gla domains, the warfarin-induced decrease in their plasma concentration differs among factors. In warfarin-treated HepG2 cells, we found modest and severe intracellular degradation of prothrombin and protein C, respectively. To investigate the structural features of these proteins that contribute to their warfarin sensitivity, chimeric prothrombin containing the prepropeptide and Gla domain of protein C was expressed in baby hamster kidney (BHK) cells. This chimera showed similar secretion kinetics and warfarin sensitivity to those of wild-type prothrombin, demonstrating that the Gla domain cannot solely explain the warfarin sensitivity of protein C. In contrast, two chimeric protein Cs containing either the Gla domain alone or the prepropeptide and Gla domain of prothrombin showed impaired secretion. Even though gamma-carboxylation proceeded normally, both chimeras were degraded intracellularly by the proteasome. From these results, we conclude that not only the folding of the Gla domain, but the entire structure and conformation of protein C and prothrombin, contribute to their quality control and susceptibility to warfarin-induced ER (endoplasmic reticulum)-associated degradation.

The zinc endopeptidases mutalysin I (100 kDa) and mutalysin II (22.5 kDa) have been previously isolated from bushmaster (Lachesis muta muta) snake venom. Hemorrhagic activity was observed with as little as 0.5 microg (2000 units/mg) and 17.8 microg (56.2 units/mg) for mutalysin I and II, respectively. Additionally, the proteases hydrolyse the Aalpha>Bbeta chain of fibrinogen without clot formation. The specific fibrinogenolytic activity was estimated as 5. 25 and 16.3 micromol fibrinogen/min/micromol protein for mutalysin I and II, respectively. In vitro, the enzymes act directly on fibrin and are not inhibited by serine proteinase inhibitors (SERPINS). Analysis by SDS-PAGE of fibrin hydrolysis by both enzymes showed that mutalysin II (0.22 microM) completely digested the alpha- and gamma-gamma chains and partially the beta-chain (in 120 min incubation). In contrast, mutalysin I (three fold higher concentration than mutalysin II) hydrolyzed selectively the alpha-chain of fibrin leaving the beta and gamma-gamma chains unaffected. Unlike with the plasminogen activator-based thrombolytic agents (e.g., streptokinase), mutalysins do not activate plasminogen. Neither enzyme had an effect on protein C activation. Mutalysin II does not inhibit platelet aggregation in human PRP induced by collagen or ADP. However, mutalysin I showed a selective inhibitory effect on collagen-induced aggregation of human PRP; it did not affect platelet aggregation with ADP as the agonist. The present investigation demonstrates that both native and EDTA-inactivated mutalysin I dose dependently blocked aggregation of human PRP elicited by 10 microg/mL of collagen with an IC(50) of 180 and 580 nM, respectively. These studies suggest that, in addition to the metalloprotease region of mutalysin I, the disintegrin-like domain also participates in the inhibitory effect. The proteolytic activity of mutalysin II against dimethylcasein and fibrin was completely abolished by alpha2-macroglobulin (alpha2-M). The stoichiometry of inhibition was 1.0 mol of enzyme per mol of alpha2-M. In contrast, the proteolytic effect of mutalysin I against the same substrates was not significantly inhibited by alpha2-M. Therefore, the data explain why mutalysin I contributes significantly not only to local but also to systemic bleeding associated with the observed pathological effects of the venom.

In order to study the effect of different intensities of anti-vitamin K treatment on coagulation parameters, 23 patients with venous thromboembolism were given, after the initial treatment period, warfarin at doses giving an International Normalised Ratio of 1.3-2.0 for 4 weeks, and of 1.1-1.3 for another 4 weeks. Blood samples were taken at the end of each of these periods and 4 weeks after the end of warfarin treatment. The vitamin K-dependent coagulation factors VII, IX, and X, as well as the inhibitor protein C and its cofactor protein S, all showed a highly significant correlation with treatment intensity. This was to some extent also true for the coagulation activation markers, prothrombin fragment 1+2 and thrombin-antithrombin complex. Ratios of pro- and anticoagulant factors in some instances showed a decrease at therapeutical (International Normalised Ratio) levels, and also sometimes with reduced warfarin treatment intensity. Taken together, our results encourage further research addressing issues of varying treatment intensity with warfarin and alternative methods for monitoring of anti-vitamin K treatment.

A method for detecting activated protein C (APC)-resistant factor V, especially factor V Leiden, is described, which uses reagents containing two unfractionated snake venoms. The procedure can be used for testing plasma samples from patients receiving oral anticoagulant therapy, heparin therapy and patients with lupus anticoagulant, and does not require the use of factor-V-deficient plasma. The sample plasma is first incubated with dilute venom from Agkistrodon contortrix contortrix (Southern Copperhead) which activates the endogenous protein C and then a dilute Russell's viper venom time test is performed. In individuals with APC-resistant factor V, especially factor V Leiden, a marginal prolongation of dilute Russell's viper venom time was noted [1.14 +/- 0.14 s (n = 16)]. Non-carriers were easily discriminated in each patient group, with a prolongation of 2.69 +/- 0.30 s for normal blood donors (n = 127), 2.61 +/- 0.38 s for patients taking oral anticoagulants (n = 102), 2.41 +/- 0.45 s for patients taking heparin (n = 96), and 2.38 +/- 0.41 s for patients with lupus anticoagulant (n = 22). Patients taking oral anticoagulants with moderate prolongation (between 1.5- and 2.0-fold) may have low levels of functional protein C and this might additionally indicate a subgroup of such patients at higher than normal thrombotic risk.

In the treatment of unstable coronary artery disease, direct thrombin inhibitors have shown no or only limited benefit as compared with heparin, despite theoretical advantages. One explanation may be that the direct thrombin inhibitors to a greater extent than heparin have an inhibiting effect on the generation and activity of activated protein C. In the present study, this hypothesis was tested in an in vitro, "purified" system, where human protein C underwent activation to activated protein C by the thrombin-thrombomodulin complex. Direct thrombin inhibitors, inogatran and hirudin, unfractionated heparin+antithrombin, or dalteparin+antithrombin, were added to the system before activation to evaluate their inhibitory effect on the generation of activated protein C. At inhibitor concentrations well below the achieved plasma levels in major clinical trials, the thrombin-thrombomodulin-mediated activation of protein C was inhibited by all the studied inhibitors in a dose-dependent manner, but, contrary to our hypothesis, to a greater extent by unfractionated heparin+antithrombin and dalteparin+antithrombin than by the direct thrombin inhibitors, hirudin and inogatran. Despite difficulties to draw conclusions for the in vivo situation, the in vitro inhibition, by all studied inhibitors, of the generation of activated protein C, found in this study may be a possible explanation for ongoing cardiovascular events despite adequate treatment with thrombin inhibitors, in patients with unstable coronary artery disease. This inhibition of the generation of activated protein C may also contribute to the rebound in cardiovascular events after withdrawal of effective antithrombotic treatment.

The mechanism of thrombosis in patients with antiphospholipid syndrome is not clear. To investigate it, we examined the effect of monoclonal anticardiolipin (aCL) antibodies and beta2-glycoprotein I (beta2-GPI), which is required for formation of the aCL epitopes, on activated protein C (APC) and on fibrinolytic activity. First, APC activities were measured in the presence and absence of beta2-GPI or gamma M immunoglobulin (IgM) monoclonal aCLs (EY1C8 and EY2C9), or both, established from peripheral blood lymphocytes obtained from a patient with aCL. beta2-GPI exhibited a procoagulant activity by inhibiting APC activity as well as an anticoagulant activity by inhibiting thrombin generation. Any further inhibition of APC activity was caused by monoclonal aCL, and then only in the presence of beta2-GPI. The remaining tissue plasminogen activator (t-PA) of the sample consisting of beta2-GPI, two-chain recombinant t-PA, and plasminogen activator inhibitor (PAI)-1 was measured by a chromogenic assay using the synthetic substrate S-2251, Glu-plasminogen, and soluble fibrin monomer. beta2-GPI protected t-PA activity from inhibition by PAI-1. However, monoclonal aCLs (EY1C8 and EY2C9) inhibited the effect of beta2-GPI on fibrinolytic activity; that is, monoclonal aCLs inhibited fibrinolytic activity by elevating PAI-1 activity. Thrombosis in patients with aCL can be explained in part by both the inhibition of APC anticoagulant activity and the impairment of fibrinolytic activity by aCL.

Given the central role of thrombin in arterial thrombogenesis, most treatment strategies for acute coronary syndromes are aimed at inhibiting its generation or blocking its activity. Although heparin has been widely used, it has limitations in the setting of arterial thrombosis. These limitations reflect the inability of heparin to inactivate thrombin bound to fibrin, a major stimulus for thrombus growth. In addition, the anticoagulant response to heparin varies from patient to patient, and heparin is neutralized by platelet Factor IV, large quantities of which are released from platelets activated at sites of plaque rupture. Consequently, heparin requires careful laboratory monitoring to ensure an adequate anticoagulant effect. Direct thrombin inhibitors, such as hirudin and bivalirudin, overcome the limitations of heparin. These agents inhibit fibrin-bound thrombin, as well as fluid-phase thrombin, and produce a predictable anticoagulant response. Bivalirudin has both safety and potential efficacy advantages over hirudin. Bivalirudin appears to have a wider therapeutic window than hirudin, possibly because bivalirudin only transiently inhibits the active site of thrombin. The better safety profile of bivalirudin permits administration of higher doses, which may give it an efficacy advantage. Hirudin prevents thrombin from activating protein C, thereby suppressing this natural anticoagulant pathway. In contrast, bivalirudin may promote protein C activation by transiently inhibiting thrombin until it can be bound by thrombomodulin. Differences between bivalirudin and hirudin, as well as other direct thrombin inhibitors, highlight the pitfalls of considering all direct thrombin inhibitors to have equivalent risk-benefit profiles.

In some studies third-generation oral contraceptives have been reported to be associated with a higher risk of venous thromboembolism than are second-generation oral contraceptives, whereas recent, more refined studies have not confirmed this. The reasons for the alleged differences are under discussion, and differential effects on hemostasis have been proposed. Eighteen studies comparing second- and third-generation oral contraceptives with respect to their effects on hemostasis were analyzed. Significant changes from baseline were reported for many variables with both second- and third-generation oral contraceptives without significant between-group differences. Also, in a combined analysis of nonsignificant changes, no consistent pattern of change emerged for any marker, with the exception of higher factor VII levels associated with third-generation oral contraceptives. However, factor VII is not related to venous thromboembolism risk. In addition, 1 cross-sectional study with an unvalidated assay reported a higher ratio of activated protein C sensitivity with third-generation oral contraceptives. Only 2 components of the hemostatic system (factor VII and activated protein C sensitivity ratio) emerged as potentially differentially affected by second- and third-generation oral contraceptives; the association with venous thromboembolism risk is questionable in the former case and unknown in the latter.