Procoagulant activities of skeletal and cardiac muscle myosin depend on contaminating phospholipid.

Recent reports indicate that suspended skeletal and cardiac myosin, such as might be released during injury, can act as procoagulants by providing membrane-like support for factors Xa and Va in the prothrombinase complex. Further, skeletal myosin provides membrane-like support for activated protein C. This raises the question of whether purified muscle myosins retain procoagulant phospholipid through purification. We found that lactadherin, a phosphatidyl-l-serine-binding protein, blocked >99% of prothrombinase activity supported by rabbit skeletal and by bovine cardiac myosin. Similarly, annexin A5 and phospholipase A2 blocked >95% of myosin-supported activity, confirming that contaminating phospholipid is required to support myosin-related prothrombinase activity. We asked whether contaminating phospholipid in myosin preparations may also contain tissue factor (TF). Skeletal myosin supported factor VIIa cleavage of factor X equivalent to contamination by ∼1:100 000 TF/myosin, whereas cardiac myosin had TF-like activity >10-fold higher. TF pathway inhibitor inhibited the TF-like activity similar to control TF. These results indicate that purified skeletal muscle and cardiac myosins support the prothrombinase complex indirectly through contaminating phospholipid and also support factor X activation through TF-like activity. Our findings suggest a previously unstudied affinity of skeletal and cardiac myosin for phospholipid membranes.

Hippocampal Gene Expression Is Highly Responsive to Estradiol Replacement in Middle-Aged Female Rats.

In the hippocampus, estrogens are powerful modulators of neurotransmission, synaptic plasticity and neurogenesis. In women, menopause is associated with increased risk of memory disturbances, which can be attenuated by timely estrogen therapy. In animal models of menopause, 17ß-estradiol (E2) replacement improves hippocampus-dependent spatial memory. Here, we explored the effect of E2 replacement on hippocampal gene expression in a rat menopause model. Middle-aged ovariectomized female rats were treated continuously for 29 days with E2, and then, the hippocampal transcriptome was investigated with Affymetrix expression arrays. Microarray data were analyzed by Bioconductor packages and web-based softwares, and verified with quantitative PCR. At standard fold change selection criterion, 156 genes responded to E2. All alterations but 4 were transcriptional activation. Robust activation (fold change > 10) occurred in the case of transthyretin, klotho, claudin 2, prolactin receptor, ectodin, coagulation factor V, Igf2, Igfbp2, and sodium/sulfate symporter. Classification of the 156 genes revealed major groups, including signaling (35 genes), metabolism (31 genes), extracellular matrix (17 genes), and transcription (16 genes). We selected 33 genes for further studies, and all changes were confirmed by real-time PCR. The results suggest that E2 promotes retinoid, growth factor, homeoprotein, neurohormone, and neurotransmitter signaling, changes metabolism, extracellular matrix composition, and transcription, and induces protective mechanisms via genomic effects. We propose that these mechanisms contribute to effects of E2 on neurogenesis, neural plasticity, and memory functions. Our findings provide further support for the rationale to develop safe estrogen receptor ligands for the maintenance of cognitive performance in postmenopausal women.

Decrease in factor V activity in patients treated with azathioprine or 6-mercaptopurine.

BACKGROUND AND OBJECTIVE: A decrease in factor V activity has been reported in some patients treated with azathioprine or 6-mercaptopurine. This may lead to unnecessary treatment discontinuation in otherwise asymptomatic patients. Our aim was to review spontaneously reported cases of decreased factor V activity associated with both drugs and to identify the possible impact on patient care. METHODS: Cases of decrease in prothrombin (PT) or factor V activity involving purine analogs were extracted from the French pharmacovigilance database. Reports with evidence of disseminated intravascular coagulation, signs of acute hepatocellular failure, liver cirrhosis or concomitant vitamin K antagonist treatment were excluded. RESULTS: Twenty-four cases (azathioprine: 13 and 6-mercaptopurine: 11) were retained. Therapeutic indications were inflammatory bowel diseases in 11 patients, acute leukemia in eight, and other autoimmune diseases in five. PT activity before treatment was normal in all nine tested patients. The decrease in PT or factor V activity occurs after a median of 10 weeks of treatment and all patients were asymptomatic. The median PT and factor V activities values were 51.5% and 36.4%, respectively. Other coagulation factors were inconsistently decreased. Full recovery was observed within 3-60 days following purine analogs discontinuation. In four patients, drug rechallenge was associated with recurrence of the coagulation disorders. CONCLUSIONS: Although the mechanism remains unknown, this series that includes cases with positive drug reintroduction strongly suggests the causative role of these drugs. As all patients remained asymptomatic, treatment discontinuation should be carefully considered in patients who clearly benefits from this treatment.

Selection of contraceptive therapy for patients with thrombophilia: a review of the evidence.

BACKGROUND: Awareness of the contributions of thrombophilia to thrombosis-related morbidity and mortality has been growing in the last few decades. Thrombophilia is especially concerning in females seeking contraception because some types of hormonal contraception have been associated with venous thromboembolism (VTE). Clinicians face a growing need for awareness of evidence-based contraception selection for this population. METHODS: PubMed literature searches were conducted to provide a review of the literature describing contraceptive use in patients with thrombophilia. This review also describes contraceptive selection and counseling for this population. RESULTS: Studies of combined hormonal contraceptive (CHC) use demonstrate a 2- to 50-fold increase in VTE in individuals with thrombophilia, depending on the type of thrombophilia and the reference group identified. Two small studies describing VTE incidence in progesterone-only contraceptive (POC) users with thrombophilia were identified but they did not provide conclusive information regarding VTE risk in this population. CONCLUSIONS: POC may be recommended for contraception in patients with most thrombophilias, but studies should be undertaken to further define the safety of POC use in this population.

Antiplatelet agents can promote two-peaked thrombin generation in platelet rich plasma: mechanism and possible applications.

BACKGROUND: Thrombin generation assay is a convenient and widely used method for analysis of the blood coagulation system status. Thrombin generation curve (TGC) is usually bell-shaped with a single peak, but there are exceptions. In particular, TGC in platelet-rich plasma (PRP) can sometimes have two peaks. OBJECTIVE: We sought to understand the mechanism underlying the occurrence of two peaks in the PRP thrombin generation curve. METHODS: Tissue factor-induced thrombin generation in PRP and platelet-poor plasma (PPP) was monitored using continuous measurement of the hydrolysis rate of the thrombin-specific fluorogenic substrate Z-Gly-Gly-Arg-AMC. Expression of phosphatidylserine (PS) and CD62P on the surface of activated platelets was measured by flow cytometry using corresponding fluorescently labeled markers. RESULTS: The addition of the P(2)Y(12) receptor antagonist MeS-AMP (160 µM), 83 nM prostaglandin E(1) (PGE(1)), or 1.6% DMSO to PRP caused the appearance of two peaks in the TGC. The PS exposure after thrombin activation on washed platelets in a suspension supplemented with DMSO, PGE(1) or MeS-AMP was delayed, which could indicate mechanism of the second peak formation. Supplementation of PRP with 1.6% DMSO plus 830 nM PGE(1) mediated the disappearance of the second peak and decreased the amplitude of the first peak. Increasing the platelet concentration in the PRP promoted the consolidation of the two peaks into one. CONCLUSIONS: Procoagulant tenase and prothrombinase complexes in PRP assemble on phospholipid surfaces containing PS of two types--plasma lipoproteins and the surface of activated platelets. Thrombin generation in the PRP can be two-peaked. The second peak appears in the presence of platelet antagonists as a result of delayed PS expression on platelets, which leads to delayed assembly of the membrane-dependent procoagulant complexes and a second wave of thrombin generation.

Rivaroxaban delivery and reversal at a venous flow rate.

OBJECTIVE: Rivaroxaban is an oral anticoagulant that directly targets both free factor Xa and factor Xa in complex with its protein cofactor, factor Va, in the prothrombinase complex. It is approved in the United States for the prophylaxis of deep vein thrombosis and stroke in patients with atrial fibrillation; however, it also carries a black box warning regarding the risk of thrombosis after discontinuation of treatment. The purpose of this study was to determine the degree to which rivaroxaban, over a range of physiologically relevant free plasma concentrations, inhibits preassembled prothrombinase at a typical venous shear rate (100 s(-1)) and to determine the dynamics of rivaroxaban washout. METHODS AND RESULTS: Prothrombinase was assembled on phospholipid-coated glass capillaries. Its activity was characterized with respect to the activation of prothrombin (mean plasma concentration, 1.4 µmol/L) in the absence and presence of rivaroxaban (2, 5, and 10 nmol/L). The degree of inactivation of preassembled prothrombinase is sensitive to the solution-phase rivaroxaban concentration; however, prothrombinase unmasking upon removal of rivaroxaban is concentration independent. CONCLUSIONS: The model system presented suggests that when rivaroxaban plasma concentrations decrease after cessation of therapy, there will be an unmasking of thrombus-associated prothrombinase that may be related to the reported rebound phenomena.

Tranexamic acid: less bleeding and less thrombosis?

The early administration of tranexamic acid (TXA) to bleeding trauma patients reduces all-cause mortality without increasing the risk of vascular occlusive events. Indeed, the risk of arterial thrombosis appears to be reduced with TXA. In this commentary we hypothesize that TXA has an antithrombotic effect and explore potential mechanisms. These include inhibition of the inflammatory effects of plasmin, effects on platelets and effects on factors V and VIII. If proven, these antithrombotic effects would have major implications for the systemic use of TXA in surgical patients, where TXA has been clearly shown to reduce bleeding.

Residual iliofemoral thrombosis in a 31-year-old woman on oral contraceptives possessing heterozygous factor V Leiden, factor XIII Val34Leu allele and alpha-fibrinogen Thr312Ala allele: case report.

We present the case of a 31-year-old woman on oral contraceptives with a 3-year history of iliofemoral thrombosis resistant to recanalization despite satisfactory anticoagulation therapy and absence of concomitant diseases. Thrombophilia screening revealed heterozygous factor V Leiden mutation. We also detected the presence of factor XIII (FXIII) Leu34 allele and alpha-chain fibrinogen 312Ala allele, which are known to adversely affect fibrin clot structure and lysis. It might be speculated that the presence of 3 polymorphisms in this patient could contribute to proximal thrombosis resistant to treatment. We postulate that determination of FXIII and alpha-fibrinogen polymorphisms can be useful in the evaluation of some young patients with deep vein thrombosis.

Effect of dietary omega-3 and omega-6 fatty acids on clotting activities of Factor V, VII and X in fatty liver haemorrhagic syndrome-susceptible laying hens.

1. The relationship between concentrations of omega-3 and omega-6 fatty acids in plasma and Factor V, VII and X clotting activities was determined using a crossover feeding trial with diets supplemented with either soy oil or flax oil. 2. Laying hens on the soy diet, which is high in omega-6 fatty acids, had substantially higher clotting activity for all three factors compared to laying hens on the flax diet that was high in omega-3 fatty acids. 3. Positive associations were seen between liver haemorrhage score and the percentage of liver weight and between the percentage of liver weight and the severity of haemorrhagic and fatty changes seen on histology. 4. These results support the hypothesis that concentrations of omega-6 and omega-3 fatty acids in plasma affect clotting activity; however, there was no relationship between the extent of liver haemorrhages and the composition of plasma fatty acids.

Successful treatment with rituximab in a patient with an acquired factor V inhibitor.

Rituximab has already been successfully used to treat immune-mediated bleeding disorders such as acquired factor VIII inhibitor. We report here a case of severe acquired factor V (FV) inhibitor deficiency due to FV inhibitor which has been dramatically improved after rituximab.

Effect of phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine on the activated factor X-prothrombin system.

Membrane phospholipids are essential in blood coagulation reactions. The importance of negatively changed phosphatidylserine has been shown. The roles of other phospholipids in the blood coagulation system, however, are not clear. This study examined the effects of phosphatidylcholine on the blood coagulation system using liposomes containing varying concentrations of phosphatidylcholine in the presence of phosphatidylserine at a constant concentration. In addition, with phosphatidylserine and phosphatidylcholine at constant concentrations, the effects of phosphatidylethanolamine and lysophosphatidylcholine on the blood coagulation system were examined. Using an in vitro reconstructed system of the activated factor X-prothrombin system, blood coagulation was measured by the rate of thrombin formation after the addition of liposome preparations. The results showed suppression of the system by phosphatidylcholine and phosphatidylethanolamine and acceleration by lysophosphatidylcholine. The results of the present study suggest that the cell membrane, the 'location' of blood coagulation, is one of the regulatory factors, and that changes in phosphatidylcholine content and phospholipid composition of the cell membrane regulate the coagulation reaction.

Effect of intravenous N-acetylcysteine infusion on haemostatic parameters in healthy subjects.

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.

Factor Xa is highly protected from antithrombin-fondaparinux and antithrombin-enoxaparin when incorporated into the prothrombinase complex.

Antithrombin and its cofactor, heparin, target both the product of prothrombin activation by prothrombinase, thrombin, as well as the enzyme responsible for the reaction, factor (F)Xa. These studies were carried out to quantify the effects of each of the prothrombinase components on the half-life of FXa in the presence of antithrombin and the low-molecular-weight heparins (enoxaparin, Aventis, Laval, Quebec, Canada) or the heparin pentasaccharide (fondaparinux, Organon Sanofi-Synthelabo, Cypress, TX, USA). Experiments were carried out using a recombinant form of prothrombin in which the active site serine has been mutated to cysteine and subsequently labeled with fluorescein. This mutant allowed calculation of the second order rate constant for inhibition of FXa by antithrombin in such a way that competition for antithrombin by thrombin is eliminated and competition for FXa by prothrombin is accounted for. Intrinsic rate constants for the inhibition of FXa by antithrombin-enoxaparin and antithrombin-fondaparinux, in the presence of the various prothrombinase components, were calculated. Addition of phospholipid had no significant effect on the second order rate constant for inhibition of FXa by antithrombin, while addition of FVa appeared to be mildly protective. Further addition of prothrombin however, caused profound protection of FXa, increasing its half-life from 1.1 to 353 s in the case of fondaparinux, and from 0.4 to 42 s in the case of enoxaparin. Similar results were reported for unfractionated heparin previously [1]. Therefore, in the presence of unfractionated heparin, fondaparinux, or enoxaparin, prothrombinase is profoundly protected from antithrombin.

Effect of hyperhomocysteinemia on protein C activation and activity.

Hyperhomocysteinemia has been proposed to inhibit the protein C anticoagulant system through 2 mechanisms: decreased generation of activated protein C (APC) by thrombin, and resistance to APC caused by decreased inactivation of factor Va (FVa). We tested the hypotheses that generation of APC by thrombin is impaired in hyperhomocysteinemia in monkeys and that hyperhomocysteinemia produces resistance to APC in monkeys, mice, and humans. In a randomized crossover study, cynomolgus monkeys were fed either a control diet or a hyperhomocysteinemic diet for 4 weeks. Plasma total homocysteine (tHcy) was approximately 2-fold higher when monkeys were on the hyperhomocysteinemic diet than when they were on the control diet (9.8 +/- 2.0 microM versus 5.6 +/- 1.0 microM; P <.05). After infusion of human thrombin (25 microg/kg of body weight), the peak level of plasma APC was 136 +/- 16 U/mL in monkeys fed the control diet and 127 +/- 13 U/mL in monkeys fed the hyperhomocysteinemic diet (P >.05). The activated partial thromboplastin time was prolonged to a similar extent by infusion of thrombin in monkeys fed the control diet and in those fed the hyperhomocysteinemic diet. The sensitivity of plasma FV to human APC was identical in monkeys on control diet and those on hyperhomocysteinemic diet. We also did not detect resistance of plasma FV to APC in hyperhomocysteinemic mice deficient in cystathionine beta-synthase (plasma tHcy, 93 +/- 16 microM) or in human volunteers with acute hyperhomocysteinemia (plasma tHcy, 45 +/- 6 microM). Our findings indicate that activation of protein C by thrombin and inactivation of plasma FVa by APC are not impaired during moderate hyperhomocysteinemia in vivo.

Functional characterization of recombinant FV Hong Kong and FV Cambridge.

In factor V (FV) Cambridge (Arg306Thr) and Hong Kong (Arg306Gly), a cleavage site for anticoagulant activated protein C (APC), which is crucial for the inactivation of FVa, is lost. Although patients carrying FV Hong Kong have a normal APC response, those with FV Cambridge were reported to be APC resistant. To elucidate the molecular characteristics of the 2 FV mutants, we recreated them in a recombinant system and evaluated their functional properties. The 2 FV variants yielded identical APC resistance patterns, with APC responses being intermediate to those of wild-type FV and FV Leiden (Arg506Gln), which is known to be associated with the APC resistance phenotype. In the absence of protein S, APC mediated FVa inactivation curves obtained with the 2 variants were identical, resulting in partial FVa inactivation. In the presence of protein S, both FVa variants were almost completely inactivated because of protein S stimulation of the cleavage at Arg679. In a FVIIIa degradation system, both FV variants demonstrated slightly impaired APC cofactor activity. The ability of APC to cleave at Arg506 and at Arg679 in FVa Cambridge and Hong Kong and the slight decrease in APC cofactor activity of the 2 FV variants may explain the low thrombotic risk associated with these Arg306 mutations. In conclusion, we demonstrate that recombinant FV Cambridge and Hong Kong behave identically in in vitro assays and provide a mechanism for the low thrombotic risk associated with these FV mutations.

Venous thromboembolism in young women; role of thrombophilic mutations and oral contraceptive use.

AIMS: The interaction between the R506Q mutation of factor V and the G20210A mutation of prothrombin with oral contraceptives on venous thromboembolism was evaluated. METHODS AND RESULTS: Three hundred and one women of reproductive age who had venous thromboembolism (140 while using oral contraceptives) and 650 healthy women (173 on oral contraceptives at presentation) were examined. Of the patients, 19.3% were carriers of R506Q (two homozygotes) and 9.6% were heterozygous carriers of G20210A; eight patients (2.7%) were heterozygous for both mutations. Among controls, 2.9% were carriers of R506Q, 3.1% of G20210A, while one case was a heterozygous carrier of both mutations. The relative risk (odds ratio) associated with carriership of R506Q or G20210A mutations was 10.3 and 4.7, respectively; it was 45.6 in carriers of both mutations. The odds ratio of using oral contraceptives in the absence of both mutations was 2.4. The odds ratios according to oral contraceptives use and the presence of R506Q or G20210A or both mutations were 41.0, 58.6 and 86.5, respectively. While the odds ratio for R506Q remains elevated (8.9) in non-oral contraceptive users, the odds ratio for G20210A was 2.0 and did not reach statistical significance. CONCLUSIONS: Our data showed a strong interaction between oral contraceptive use and the presence of either R506Q or G20210A mutations. In non-oral contraceptive users the risk of venous thromboembolism was significantly increased in carriers of R506Q but not in those with the G20210A mutation.

Anti-c5a ameliorates coagulation/fibrinolytic protein changes in a rat model of sepsis.

Sepsis and trauma are the two most common causes of disseminated intravascular coagulation and multiple organ dysfunction syndrome. Both disseminated intravascular coagulation and the systemic inflammatory response syndrome often lead to multiple organ dysfunction syndrome. The current studies have evaluated the relationship between the anaphylatoxin, C5a, and changes in the coagulation/fibrinolytic systems during the cecal ligation and puncture (CLP) model of sepsis in rats. CLP animals treated with anti-C5a had a much improved number of survivors (63%) compared to rats treated with pre-immune IgG (31%). In CLP rats treated with pre-immune IgG there was clearly increased procoagulant activity with prolongation of the activated partial thromboplastin time and prothrombin time, reduced platelet counts, and increased levels of plasma fibrinogen. Evidence for thrombin formation was indicated by early consumption of factor VII:C, subsequent consumption of factors XI:C and IX:C and anti-thrombin and increased levels of the thrombin-anti-thrombin complex and D-dimer. Limited activation of fibrinolysis was indicated by reduced plasma levels of plasminogen and increased levels of tissue plasminogen activator and plasminogen activator inhibitor. Most of these parameters were reversed in CLP rats that had been treated with anti-C5a. Production of C5a during sepsis may directly or indirectly cause hemostatic defects that can be reduced by blockade of C5a.

Prothrombin protects factor Xa in the prothrombinase complex from inhibition by the heparin-antithrombin complex.

Heparin is a commonly used anticoagulant drug. It functions primarily by accelerating the antithrombin inhibition of coagulation proteinases, among which factor Xa and thrombin are believed to be the most important targets. There are conflicting results as to whether anticoagulant heparins can catalyze the antithrombin inhibition of factor Xa in the prothrombinase complex (factor Va, negatively charged membrane surfaces, and calcium ion), which is the physiologically relevant form of the proteinase responsible for the activation of prothrombin to thrombin during the blood coagulation process. In this study, a novel assay system was developed to compare the catalytic effect of different molecular-weight heparins in the antithrombin inhibition of factor Xa, either in free form or assembled into the prothrombinase complex during the process of prothrombin activation. This assay takes advantage of the unique property of a recombinant mutant antithrombin, which, similar to the wild-type antithrombin, rapidly inhibits factor Xa, but not thrombin, in the presence of heparin. A direct prothrombinase inhibition assay, monitoring thrombin generation under near physiological concentrations of prothrombin and antithrombin in the presence of therapeutic doses of low- and high-molecular-weight heparins, indicates that factor Xa in the prothrombinase complex is protected from inhibition by antithrombin more than 1000 times, independent of the molecular size of heparin.