Factor V Leiden paradox in a middle-aged Swedish population: A prospective study.

Few prospective studies have examined the factor V paradox: factor V Leiden (FVL) is a stronger risk factor for deep venous thrombosis (DVT) than for pulmonary embolism (PE). The present study, to the best of our knowledge, is the first population-based study aimed to examine the relationship between FVL and incidence of venous thromboembolism (VTE), DVT and PE in a prospective cohort study of middle-aged Swedish individuals. FVL was determined in 4890 subjects (aged 46-68 years, 57% women) from the general population without previous VTE or cancer, who participated in the Malmö Diet and Cancer study between 1991 and 1994. Incident cases of VTE were identified from the Swedish patient register during a mean follow-up of 15.6 years. Of 4890 subjects with determination of FVL (10.2% carriers), 220 had VTE during follow-up (113 DVT, 78 PE, 29 both). Incidence of VTE was significantly higher in subjects with heterozygous and homozygous FVL: adjusted hazard ratios (HR) were 1.8 (95% CI 1.3-2.6, p=0.001) and 6.5 (2.1-21, p=0.001), respectively. The population attributable fraction was 8.7% for FVL. Adjusted HRs for DVT were 2.2 (1.4-3.3, p<0.001) for heterozygotes and 3.3 (0.5-24, p=0.233) for homozygotes. Adjusted HRs for PE were 1.2 (0.65-2.2, p=0.582) for heterozygotes and 8.7 (2.1-36, p=0.003) for homozygotes. The FVL paradox was confirmed for heterozygotes for FVL. However, homozygotes for FVL had a high risk for PE, suggesting that the FVL paradox is related to the carriership of one wild type and one mutated factor V allele.

Protease-activated receptor (PAR) 1 and PAR4 differentially regulate factor V expression from human platelets.

With the recent interest of protease-activated receptors (PAR) 1 and PAR4 as possible targets for the treatment of thrombotic disorders, we compared the efficacy of protease-activated receptor (PAR)1 and PAR4 in the generation of procoagulant phenotypes on platelet membranes. PAR4-activating peptide (AP)-stimulated platelets promoted thrombin generation in plasma up to 5 minutes earlier than PAR1-AP-stimulated platelets. PAR4-AP-mediated factor V (FV) association with the platelet surface was 1.6-fold greater than for PAR1-AP. Moreover, PAR4 stimulation resulted in a 3-fold greater release of microparticles, compared with PAR1 stimulation. More robust FV secretion and microparticle generation with PAR4-AP was attributable to stronger and more sustained phosphorylation of myosin light chain at serine 19 and threonine 18. Inhibition of Rho-kinase reduced PAR4-AP-mediated FV secretion and microparticle generation to PAR1-AP-mediated levels. Thrombin generation assays measuring prothrombinase complex activity demonstrated 1.5-fold higher peak thrombin levels on PAR4-AP-stimulated platelets, compared with PAR1-AP-stimulated platelets. Rho-kinase inhibition reduced PAR4-AP-mediated peak thrombin generation by 25% but had no significant effect on PAR1-AP-mediated thrombin generation. In conclusion, stimulation of PAR4 on platelets leads to faster and more robust thrombin generation, compared with PAR1 stimulation. The greater procoagulant potential is related to more efficient FV release from intracellular stores and microparticle production driven by stronger and more sustained myosin light chain phosphorylation. These data have implications about the role of PAR4 during hemostasis and are clinically relevant in light of recent efforts to develop PAR antagonists to treat thrombotic disorders.

Evaluation of factor V mRNA to define the residual factor V expression levels in severe factor V deficiency.

We evaluated FV mRNA in severe factor V deficiency caused by the -12T/A IVS18 mutation, activating a cryptic splice site and leading to premature translation termination. Quantitative evaluation of factor V cDNA from homozygous and heterozygous subjects, and correction for nonsense mediated decay, suggested the presence of 0.1% of normal factor V mRNA.

Biosynthesis of coagulation Factor V by a human hepatocellular carcinoma cell line.

A human hepatocellular carcinoma line, HepG2, was found to secrete coagulation Factor V. Factor V activity in HepG2 culture fluid increased nearly linearly during a 20-h time course (5 ng Factor Va/h per 10(6) cells). Thrombin treatment increased Factor V activity in HepG2 culture medium six- to ninefold, indicating that the medium accumulates a mixture of Factors V and Va. To demonstrate de novo synthesis of Factor V, HepG2 cells were incubated in culture medium containing [35S]methionine. Labeled Factor V was immunoprecipitated from the medium and was shown to co-migrate with purified plasma Factor V upon sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography. When medium was treated with thrombin before immunoprecipitation and fluorography, the 330,000-Mr [35S]methionine-labeled Factor V was converted to Factor Va. Factor Va coagulant activities from HepG2 cells and human plasma were inhibited in parallel by anti-Factor V antibody, indicating that HepG2 and plasma Factor Va have the same intrinsic activity. If normal hepatocytes synthesize Factor V at the same rate as HepG2 cells, then hepatocyte secretion can account for the total Factor V present in plasma. The production of Factor V by cultured human umbilical vein endothelial cells was also examined. Spent culture medium from endothelial cells contained only Factor Va and the amount was less than 1% of the activity found in medium from HepG2 cells under comparable conditions. The amount of Factor V activity in endothelial cell culture fluid did not change with time in culture.

Activation of coagulation factor V by a platelet protease.

Factor V must be converted to Factor V(a) in order to bind to a high affinity platelet surface site and participate in prothrombin activation. Osterud et al. (10) presented data that suggested that human platelets contain an activated form of Factor V and a Factor V activator. We find that the Factor V released when platelets are disrupted by freezing and thawing or sonication is activated 3- to 10-fold by thrombin as determined by coagulation assay and is therefore stored as the relatively inactive procofactor rather than in the active form Factor V(a). We incubated purified Factor V, which had a specific activity of 140+/-30 U/mg, with Factor V-deficient frozen and thawed platelets (10(9) platelets/ml) obtained from a patient with Factor V deficiency. The specific activity of the Factor V increased to a maximum of 740+/-240 U/mg (mean+/-SD of three experiments). When this partially activated Factor V was incubated with thrombin its specific activity increased further to 1,440+/-280 U/mg, which is similar to the activity of Factor V activated with thrombin alone (1,540+/-60 U/mg). The platelet Factor V activator is not inhibited by dansyl arginine-4-ethylpiperidine amide, 93 muM, indicating that it is not thrombin. When thrombin-stimulated platelets, to which dansyl arginine-4-ethylpiperidine amide had been added to inhibit the further action of thrombin, were incubated with (125)-labeled Factor V, there was no detectable proteolysis of the Factor V molecule. Our failure to detect activation of Factor V under these conditions suggests that <4% of the platelet protease is released by thrombin. Subcellular fractionation of platelets indicates that the platelet protease that activates Factor V is in the soluble fraction. When Factor V(a) formed by the action of platelet protease is incubated with platelets, peptides with M(r) = 105,000, 87,000, and 78,000 bind to the platelet surface. All three radiolabeled peptides are displaced from platelets by unlabeled Factor V(a) formed by the action of thrombin. The stoichiometry of binding suggests that the 105,000-dalton peptide is associated with either an 87,000- or a 78,000-dalton peptide. The 78,000-dalton peptide binds with greater affinity and probably accounts for the bulk of the activity of Factor V(a) in coagulation assays. Whether or not the platelet protease serves to activate Factor V before thrombin formation during normal hemostasis remains to be determined.

Biosynthesis of factor V in isolated guinea pig megakaryocytes.

Although platelets contain Factor V, localized primarily in the alpha-granules, the origin of this coagulation cofactor in these cells is not known. We therefore explored whether isolated megakaryocytes could biosynthesize Factor V. Guinea pig plasma Factor V coagulant activity was demonstrated to be neutralized by human monoclonal and rabbit polyclonal antibodies directed monospecifically against human Factor V. These antibodies had been used earlier to purify human Factor V. These antibodies had been used earlier to purify human Factor V and to quantify Factor V antigen concentration, respectively (1983. Chiu, H. C., E. Whitaker, and R. W. Colman. J. Clin. Invest. 72:493-503). As determined by a competitive enzyme-linked immunosorbent assay with guinea pig plasma as a standard, Factor V solubilized from guinea pig megakaryocytes was present at 0.098 +/- 0.018 micrograms/10(5) cells. Each megakaryocyte contained about 500 times as much Factor V as is in a platelet (0.234 +/- 0.180 micrograms/10(8) platelets). The content of Factor V antigen in guinea pig plasma was greater (27.0 +/- 3.0 micrograms/ml) than that of Factor V antigen in human plasma (11.1 +/- 0.4 micrograms/ml). In contrast, human platelets contain ninefold more Factor V antigen (2.01 +/- 1.09 micrograms/10(8) platelets) than do guinea pig were 2.85 +/- 0.30 U/ml plasma, 0.022 +/- 0.012 U/10(8) platelets, and 0.032 +/- 0.03 U/10(5) megakaryocytes, compared with human values of 0.98 +/- 0.02 U/ml plasma and 0.124 +/- 0.064 U/10(8) platelets. Isolated megakaryocytes were found to contain Factor V by cytoimmunofluorescence. The megakaryocytes were incubated with [35S]methionine, and radiolabeled intracellular proteins purified were on a human anti-Factor V immunoaffinity column. The purified protein exhibited Factor V coagulant activity and neutralized the inhibitory activity of a rabbit antihuman Factor V antibody, which suggests that megakaryocyte Factor V is functionally and antigenically intact. These results indicate that Factor V is synthesized by guinea pig megakaryocytes. Nonetheless, megakaryocyte Factor V was more slowly activated by thrombin and in the absence of calcium was more stable after activation than was plasma Factor Va. Electrophoresis in sodium dodecyl sulfate and autoradiography of the purified molecule showed a major band of Mr 380,000 and a minor band of Mr 350,000, as compared with guinea pig and human plasma Factor V, where the protein had an Mr of 350,000. Both forms of Factor V were substrates for thrombin. Possible explanations for the higher molecular weight and different thrombin sensitivity and stability observed are that a precursor of Factor V was isolated or that the megakaryocyte Factor V had not been fully processed before isolation.

Human thrombomodulin is not an efficient inhibitor of the procoagulant activity of thrombin.

The effect of human thrombomodulin isolated from placenta on the procoagulant activity of thrombin was studied and compared to that of rabbit thrombomodulin. The isolated protein was proved to be thrombomodulin because a rabbit antibody against the isolated protein blocked protein C activation by thrombomodulin in solution and also blocked the protein-C-activating cofactor activity of human umbilical vein endothelial cells. The affinity of human thrombomodulin for human thrombin in the presence of fibrinogen is 30 times less than that of rabbit thrombomodulin. This value is based on the measurements of the clotting time of human fibrinogen and thrombin in the presence of increasing amounts of thrombomodulin. Human thrombomodulin was also much less effective compared with rabbit thrombomodulin in inhibiting thrombin-induced human coagulation factor V activation. The ability to inhibit release of [3H]serotonin from washed human platelets was at least 10 times less using human thrombomodulin compared with rabbit thrombomodulin. A partially purified preparation of human lung thrombomodulin was also relatively ineffective in inhibiting thrombin-induced serotonin release from platelets, indicating that the difference between rabbit and human thrombomodulin is one of species rather than of tissue. Thus, while human thrombomodulin is a potent cofactor in protein C activation, it is not an efficient inhibitor of the procoagulant actions of thrombin.

Blood coagulation at the site of microvascular injury in healthy and coumadin-treated subjects heterogenous for factor V Leiden mutation.

There is little knowledge regarding the in-vivo course of coagulation reactions in subjects with factor (F)V Leiden (G1691A, R506Q). The aim of the current study was to evaluate the effect of FV Leiden on coagulant reactions triggered by vascular injury. At the site of microvascular injury, prothrombin activation and FVa formation and inactivation have been evaluated in 16 apparently healthy subjects and 16 patients on long-term anticoagulation with acenocoumarol, with eight heterogenous carriers of FV Leiden in the healthy and in the anticoagulated group. Thrombin formation, measured by levels of thrombin-antithrombin complexes (TAT) and thrombin B-chain, proceeded at similar rates in the bleeding-time blood in carriers and non-carriers of FV Leiden. The onset and rate of FV activation did not differ significantly among the four groups. When healthy carriers of FV Leiden were compared to healthy non-carriers, the onset of FVa inactivation by activated protein C (APC) was delayed by 60 to 90 seconds (p=0.01). Anticoagulated individuals also showed the same pattern of FV Leiden associated differences in the time of occurrence of the 30 kD FVa heavy-chain fragment (p=0.021). Our results indicate that when blood clotting is triggered by microvascular injury, the heterozygous expression of FV Leiden has no effect on thrombin generation in healthy or coumadin-treated subjects. Inactivation of FVa by the APC mechanism is attenuated significantly in carriers of FV Leiden but the magnitude of this effect is smaller than that observed in most purified systems.

Vascular smooth muscle cells synthesize, secrete and express coagulation factor V.

Expression of cellular procoagulant activity may be one of the more important responses to vascular injury. Because factor V, a coagulation cofactor in the prothrombinase complex, catalyzes the conversion of prothrombin to thrombin, it may be a key to understanding this response. Therefore, we have investigated the synthesis, secretion and expression of factor V by vascular smooth muscle cells, which proliferate at sites of vascular injury. Cultured aortic vascular smooth muscle cells constitutively secreted Factor V activity, as determined by a functional assay. Labeled factor V was immunoprecipitated from conditioned medium of [35S]methionine-labeled cells, indicating that the secreted factor V was synthesized by vascular smooth muscle cells. Treatment of vascular smooth muscle cells with tunicamycin prevented secretion of factor V, suggesting that its secretion was dependent on the presence of N-linked carbohydrate. Factor V activity was also expressed on the vascular smooth muscle cell surface, as indicated by the ability of cultured cells to promote factor Xa-catalyzed prothrombin activation. These data suggest that the proliferation of smooth muscle cells in response to vascular injury may be one mechanism that links vascular disease with thrombosis.

Increased prevalence of factor V Leiden in patients with retinal vein occlusion and under 60 years of age.

Retinal vein occlusion (RVO) is a multifactorial disease involving vessel damage, stasis, viscosity and thrombosis. Conflicting findings on hereditary thrombophilic risk factors have been reported and their impact on RVO features remains to be defined. The aim of the present study was to evaluate the prevalence of hereditary thrombophilic risk factors (HTRF) and characteristics of RVO in patients with or without HTRF. The design of the study was a prospective, observational case series. Two hundred and thirty-four patients with RVO were included consecutively. A French healthy population of the same region was studied as control group. The HTRF studied were protein C (PC), protein S (PS) and antithrombin (AT) deficiencies, factor V Leiden (FVL) and factor II 20210A polymorphisms. Chi-Square was used for comparison with the healthy subjects and between RVO patient with and without HTRF according to localisation (branch vs. central), type of RVO (ischemic or non-ischemic), recurrence, age at first event and classical vascular risk factors. Twenty-two patients had HTRF (12 FV Leiden heterozygotes, 9 FII 20210A heterozygotes and 1 PS deficiency). No AT or PC deficiency was detected. Frequencies of PS deficiency, FVL and FII 20210A allele were similar to the reference population as well as to published data in the general caucasian population. Eighty-six patients experienced their first episode before the age of 60 years. Systemic hypertension, glaucoma and angina were significantly less frequent in patients with RVO before 60 years. Fourteen of the 22 patients with one HTRF (64%) experienced their first episode of RVO before the age of 60 years compared to 72 of 212 without HTRF (34%) (p = 0.006). Heterozygote status for FV Leiden was significantly more frequent in patients who had experienced their first episode of RVO before 60 years (p = 0.027). In conclusion, this study suggests a role of FV Leiden in the occurrence of RVO in patients younger than 60 years who exhibit fewer acquired vascular risk factors than in older patients.

Lipoprotein (a) and other prothrombotic risk factors in Caucasian women with unexplained recurrent miscarriage. Results of a multicentre case-control study.

From 1998 to 2003, 133 Caucasian women aged 17-40 years (median 29 years) suffering from unexplained recurrent miscarriage (uRM) were consecutively enrolled. In patients and 133 age-matched healthy controls prothrombotic risk factors (factor V (FV) G1691A, factor II (FII) G20210A, MTHFR T677T, 4G/5G plasminogen activator inhibitor (PAI)-1, lipoprotein (Lp) (a), protein C (PC), protein S (PS), antithrombin (AT), antiphospholipid/anticardiolipin (APA/ACA) antibodies) as well as associated environmental conditions (smoking and obesity) were investigated. 70 (52.6%) of the patients had at least one prothrombotic risk factor compared with 26 control women (19.5%; p<0.0001). Body mass index (BMI; p=0.78) and smoking habits (p=0.44) did not differ significantly between the groups investigated. Upon univariate analysis the heterozygous FV mutation, Lp(a) > 30 mg/dL, increased APA/ACA and BMI > 25 kg/m(2) in combination with a prothrombotic risk factor were found to be significantly associated with uRM. In multivariate analysis, increased Lp(a) (odds ratio (OR): 4.7/95% confidence interval (CI): 2.0-10.7), the FV mutation (OR:3.8/CI:1.4-10.7), and increased APA/ACA (OR: 4.5/CI: 1.1-17.7) had independent associations with uRM.

Assembly and regulation of prothrombinase complex on B16F10 melanoma cells.

A number of studies indicate that coagulation proteases play significant roles in cancer biology. Melanoma is a highly metastatic cancer, and there is evidence that thrombin contributes to this aggressive pattern. However, few studies correlate this type of cancer with formation of the prothrombinase complex, which is responsible for conversion of prothrombin into thrombin in the coagulation system. The aim of this study was to investigate the assembly and regulation of prothrombinase complex on the murine melanoma cell line, B16F10. B16F10 cells were unable to activate prothrombin except when previously incubated with factor Xa. This effect was dependent on factor Xa binding to cell membranes, since no activation was detected with Gla-domainless factor Xa. The thrombin formation by B16F10-bound factor Xa was enhanced approximately 10 fold in the presence of factor Va, indicating the assembly of prothrombinase complex. Differently from platelets, B16F10-assembled prothrombinase complex was inhibited by prothrombin fragment 1 but not by fragment 2. In addition, bothrojaracin, a specific ligand of proexosite I on prothrombin, caused a significant decrease in the zymogen activation. Our data demonstrate that B16F10 melanoma cells generate thrombin by promoting assembly of the prothrombinase complex. This ability might be correlated with the increased metastatic potential of this cell line. Moreover, B16F10-assembled prothrombinase complex seems to be modulated in a different way from that found for the physiological complex assembled on platelets.

Factor V and thrombotic disease: description of a janus-faced protein.

The generation of thrombin by the prothrombinase complex constitutes an essential step in hemostasis, with thrombin being crucial for the amplification of blood coagulation, fibrin formation, and platelet activation. In the prothrombinase complex, the activated form of coagulation factor V (FVa) is an essential cofactor to the enzyme-activated factor X (FXa), FXa being virtually ineffective in the absence of its cofactor. Besides its procoagulant potential, intact factor V (FV) has an anticoagulant cofactor capacity functioning in synergy with protein S and activated protein C (APC) in APC-catalyzed inactivation of the activated form of factor VIII. The expression of anticoagulant cofactor function of FV is dependent on APC-mediated proteolysis of intact FV. Thus, FV has the potential to function in procoagulant and anticoagulant pathways, with its functional properties being modulated by proteolysis exerted by procoagulant and anticoagulant enzymes. The procoagulant enzymes factor Xa and thrombin are both able to activate circulating FV to FVa. The activity of FVa is, in turn, regulated by APC together with its cofactor protein S. In fact, the regulation of thrombin formation proceeds primarily through the upregulation and downregulation of FVa cofactor activity, and failure to control FVa activity may result in either bleeding or thrombotic complications. A prime example is APC resistance, which is the most common genetic risk factor for thrombosis. It is caused by a single point mutation in the FV gene (factor V(Leiden)) that not only renders FVa less susceptible to the proteolytic inactivation by APC but also impairs the anticoagulant properties of FV. This review gives a description of the dualistic character of FV and describes the gene-gene and gene-environment interactions that are important for the involvement of FV in the etiology of venous thromboembolism.

[Impact of inherited thrombophilia on the development of some pregnancy complications].

AIM: To assess the clinical significance of inherited thrombophilia [IT] for the development of some pregnancy complications. MATERIAL AND METHODS: The incidence of the following factors was studied in 97 pregnant women with pregnancy complications and in 103 controls: R506Q mutation encoding Factor V Leiden [FVL] synthesis, Prothrombin G20210A mutation, T677 methylenetetrahydropholate reductase mutation [MTHFR], 4G/4G polymorphism of the plasminogen activator inhibitor [PAI 4G/4G]. Among 97 patients in the group studied 39 had early onset severe preeclampsia [PE], 14--placental abruption [AP] without PE, 18--intrauterine growth restriction [IUGR] without PE, 12--stillbirth [SB] without PE, 14--habitual spontaneous abortions [HSA]. The control group included 103 clinically healthy pregnant women with at least one previous uneventful pregnancy, without history of thromboembolic disorders. In addition, patients with severe PE with and without IT were compared regarding g. a. and birthweight at delivery and intrauterine fetal loss rate. DNA analysis was performed according to internationally accepted standards. Pregnancy outcomes were ascertained from hospital records. Statistical significance (p < 0.05) was assessed by means of Student's t-test. RESULTS: FVL mutation was found in 23.7% (23/97) of the patients from the studied group and in 5.8% (6/103) of the controls. Prothrombin G20210A carriers were 11% (11/97) of the studied and 3.8% (4/103) of the controls, while with PAI 4G/4G polymorphism they were 30.9% (30/97) and 14.5% (15/103) respectively. MTHFR T677 was not more frequent in the studied group (8.2%) compared to the control one (29%). Eight of the patients (9.6%) were carriers of more than one mutation. In 22 cases with early onset severe PE and IT gestational age and birthweight at delivery were lower than in the cases with severe PE without IT while intrauterine fetal loss rate did not differ significantly between the two groups. CONCLUSIONS: Inherited thrombophilia is found more frequently in women with pregnancy complications like PE, IUGR, AP, SB, HSA. The incidence of homozygous MTHFR T677 is not higher in these cases. IT worsens the prognosis of severe PE. The diagnosis of IT is important since anithrombotic therapy has to be considered to protect the mother and the fetus.

Assembly of prothrombinase complex.

The approaches described in this article have resulted in an increased understanding of the reaction steps involved in the stabilization and assembly of the prothrombinase complex. Because prothrombinase is considered an archetype for some of the other coagulation complexes, the quantitative information derived from these studies (Table I) provides the framework for future studies of prothrombinase and suggests experimental approaches for studies of the other analogous coagulation reactions.

Successful extracorporeal porcine liver perfusion for 72 hr.

BACKGROUND: Improvements in extracorporeal perfusion technology and the production of transgenic pigs resistant to hyperacute rejection have stimulated several groups to re-explore the possibility of supporting patients in hepatic failure with extracorporeal porcine livers. The success of organ transplantation has also stimulated interest in using extracorporeal perfusion as a means of organ preservation and resuscitation of organs from marginal donors. The present study describes a method by which livers can be maintained in a viable condition for a minimum of 72 hr of normothermic, extracorporeal perfusion. METHODS: Five extracorporeal porcine liver perfusions were performed, each with a duration of 72 hr. Hepatectomy was performed, followed by cold preservation, cannulation of vessels, and initiation of perfusion with normothermic, oxygenated porcine blood. Organ viability was assessed by metabolic, synthetic, hemodynamic, and histologic parameters. RESULTS: After 72 hr of normothermic, extracorporeal perfusion, the isolated livers demonstrated maintenance of normal physiological levels of pH and electrolytes. Continued hepatic protein synthesis (complement and factor V) was maintained throughout the perfusion. Hemodynamic parameters remained within normal physiological range. Histology demonstrated good preservation of the liver with no overall architectural change. CONCLUSION: It is possible to maintain a liver in a viable condition for a minimum of 72 hr of extracorporeal perfusion. This technique has been developed primarily as a preclinical model of extracorporeal liver support with the intention of proceeding to a clinical trial in patients with fulminant liver failure. However, it also has potential applications in organ preservation or resuscitation before transplantation and in the experimental study of isolated liver physiology.

Thrombin-induced platelet factor Va formation in patients with a gray platelet syndrome.

The present study was initiated to establish the functional factor V concentration in platelets of patients with a mild bleeding disorder ascribed to a gray platelet syndrome. This inherited platelet disorder has been characterized by a specific deficiency of alpha-granules and subsequent deficiencies in the alpha-granule proteins. We found that the concentration of plasma factor V was slightly decreased (70% of normal values). In contrast, platelet factor Va formation was severely impaired. Besides a much lower factor V content than in control platelets (10-20% of normal), the dependency of platelet factor Va formation on thrombin concentration was altered. Increasing the thrombin concentration 4-fold compared to the concentration that results in maximal factor Va generation from normal platelets did not result in a maximal factor Va formation from gray platelets. When a suspension of washed gray platelets was incubated with a prostacyclin analogue prior to the stimulation with thrombin, a 10-fold lower factor Va activity was measured. Thus, thrombin-induced factor Va formation in a suspension of gray platelets is the result of a release reaction, followed by the thrombin-catalyzed activation of released factor V. Whereas the kinetics of the former reaction are apparently impaired, the kinetics of the latter one were found to be identical to those observed for normal platelet and plasma factor V activation.

Human platelets contain forms of factor V in disulfide-linkage with multimerin.

Factor V is an essential cofactor for blood coagulation that circulates in platelets and plasma. Unlike plasma factor V, platelet factor V is stored complexed with the polymeric alpha-granule protein multimerin. In analyses of human platelet factor V on nonreduced denaturing multimer gels, we identified that approximately 25% was variable in size and migrated larger than single chain factor V, the largest form in plasma. Upon reduction, the unusually large, variably-sized forms of platelet factor V liberated components that comigrated with other forms of platelet factor V, indicating that they contained factor V in interchain disulfide-linkages. With thrombin cleavage, factor Va heavy and light chain domains, but not B-domains,were liberated from the components linked by interchain disulfide bonds, indicating that the single cysteine in the B-domain at position 1085 was the site of disulfide linkage. Since unusually large factor V had a variable size and included forms larger than factor V dimers, the data suggested disulfide-linkage with another platelet protein, possibly multimerin. Immunoprecipitation experiments confirmed that unusually large factor V was associated with multimerin and it remained associated in 0.5 M salt. Moreover, platelets contained a subpopulation of multimerin polymers that resisted dissociation from factor V by denaturing detergent and comigrated with unusually large platelet factor V, before and after thrombin cleavage. The disulfide-linked complexes of multimerin and factor V in platelets, which are cleaved by thrombin to liberate factor Va, could be important for modulating the function of platelet factor V and its delivery onto activated platelets. Factor Va generation and function from unusually large platelet factor V is only speculative at this time.

Abnormal formation of the prothrombinase complex: factor V deficiency and related disorders.

A membrane-bound, Ca2-dependent complex of the cofactor factor Va and the enzyme factor Xa comprises the prothrombinase coagulation complex, which catalyzes the proteolytic conversion of prothrombin to thrombin. In normal hemostasis, the platelet is presumed to supply the surface membrane and thus constitutes the site at which an enzymatically functional complex assembles and thrombin generation occurs. Factor Va, the two subunit protein produced by thrombin activation of factor V, is an essential, nonenzymatic cofactor of the prothrombinase complex. Factor Va performs its cofactor role in part by binding to the platelet membrane and functioning as the membrane receptor for factor Xa in a 1:1 stoichiometric complex of high affinity (Kd = 10(-10) M). Factor Va also appears to participate in the binding of prothrombin to the enzymatic complex. Because deletion of factor Va from the prothrombinase complex decreases the rate of thrombin generation by four orders of magnitude, the essential role it plays is easily understood. Therefore, in the evaluation of factor Va function in the prothrombinase complex, the ability of factor Va to support various binding interactions with the platelet, factor Xa, and prothrombin must be considered. Factor Va can be made available from two potential blood compartments: the plasma and platelets. Approximately 80 per cent of the total blood factor V circulates in plasma whereas the remaining 20 per cent is contained within platelet granules. The relative contribution of plasma versus platelet factor V to factor Va binding interactions in the prothrombinase complex are not clearly defined. However, data from our laboratory and several others suggest that factor V stored and released from platelets is of utmost importance in maintaining normal hemostasis. A discussion of these data relative to congenital and acquired deficiencies of both plasma and platelet factor V is the subject of this report.