Severe factor V (FV) deficiency is associated with mild to severe bleeding diathesis, but many patients with FV levels lower than 1% bleed less than anticipated. We used calibrated automated thrombography to screen patients with severe FV deficiency for protective procoagulant defects. Thrombin generation in FV-deficient plasma was only measurable at high tissue factor concentrations. Upon reconstitution of FV-deficient plasma with purified FV, thrombin generation increased steeply with FV concentration, reaching a plateau at approximately 10% FV. FV-deficient plasma reconstituted with 100% FV generated severalfold more thrombin than normal plasma, especially at low tissue factor concentrations (1.36 pM) or in the presence of activated protein C, suggesting reduced tissue factor pathway inhibitor (TFPI) levels in FV-deficient plasma. Plasma TFPI antigen and activity levels were indeed lower (P < .001) in FV-deficient patients (n ‫؍‬ 11; 4.0 ؎ 1.0 ng/mL free TFPI) than in controls (n ‫؍‬ 20; 11.5 ؎ 4.8 ng/mL), while persons with partial FV deficiency had intermediate levels (n ‫؍‬ 16; 7.9 ؎ 2.5 ng/mL). FV immunodepletion experiments in normal plasma and surface plasmon resonance analysis provided evidence for the existence of a FV/TFPI complex, possibly affecting TFPI stability/clearance in vivo. Low TFPI levels decreased the FV requirement for minimal thrombin generation in FV-deficient plasma to less than 1% and might therefore protect FV-deficient patients from severe bleeding. (Blood. 2008;112:3615-3623) IntroductionCoagulation factor V (FV) is a large multidomain glycoprotein structurally and functionally homologous to factor VIII (FVIII). 1 After biosynthesis in the liver, FV is released in the bloodstream, where it is found in both plasma (80%; concentration of 21-25 nM) and platelets (20%). The activated form of FV (FVa) acts as an essential cofactor of activated factor X (FXa) in prothrombin (PT) activation, thereby enhancing thrombin formation by several orders of magnitude. 2 The generation of thrombin is physiologically down-regulated by several anticoagulant mechanisms, including the protein C pathway 3 and the tissue factor pathway inhibitor (TFPI) system. 4 Activated protein C (APC) is a vitamin K-dependent serine protease which, in concert with its nonenzymatic cofactor protein S, inactivates FVa and FVIIIa by limited proteolysis. A poor anticoagulant response of plasma to exogenous APC (APC resistance 5 ) is the most common risk factor for venous thrombosis. Conversely, TFPI is a Kunitz-type protease inhibitor that binds and inhibits both FXa and the tissue factor (TF)/FVIIa complex in a 2-step reaction, 6 the first step being stimulated by protein S. 7,8 TFPI is synthesized primarily by the vascular endothelium, and most of it (approximately 80%) is associated with the endothelial surface as a full-length protein, the form that most effectively inhibits FXa. 9 Another 2% of all TFPI is stored in platelets. 10,11 The remainder circulates in plasma at a concentration of 2.0 to 2.5 nM, of which app...

Coagulation factor V (FV), present in plasma and platelets, is indispensable to thrombin formation, yet patients with undetectable plasma FV seldom experience major bleeding. We used thrombin generation assays to explore the role of platelet

SummaryCoagulation factor V (FV), present in plasma and platelets, is an indispensable clotting factor, as demonstrated by the uniform lethality of FV knock-out mice. Surprisingly, however, severe FV deficiency is rarely fatal in humans. In fact, although several cases of life-threatening intracranial haemorrhage have been reported in FV-deficient newborns, many patients with undetectable FV levels experience only mild to moderate bleeding and do not require routine prophylaxis. While the reasons for this variable phenotypic expression are largely unknown, several observations from different laboratories indicate platelets as crucial players in FV deficiency. Moreover, we have recently shown that plasma levels of tissue factor pathway inhibitor are considerably reduced in FV-deficient plasma, which results in enhanced thrombin generation especially at very low FV levels (<2%). The present review discusses and integrates these findings in the context of the biology of FV and the clinical features of FV deficiency.

To cite this article: Castoldi E, Duckers C, Radu C, Spiezia L, Rossetto V, Tagariello G, Rosing J, Simioni P. Homozygous F5 deep-intronic splicing mutation resulting in severe factor V deficiency and undetectable thrombin generation in platelet-rich plasma. J Thromb Haemost 2011; 9: 959-68. Summary. Background: Coagulation factor (F) V deficiency is associated with a bleeding tendency of variable severity, but phenotype determinants are largely unknown. Recently, we have shown that three patients with undetectable plasma FV and mild bleeding symptoms had sufficient residual platelet FV to support thrombin generation in platelet-rich plasma (PRP). Therefore, we hypothesized that FV-deficient patients with severe bleeding manifestations may lack platelet FV. Objectives: To characterize a FV-deficient patient with a severe bleeding diathesis. Patients/Methods: We performed FV mutation screening and functional studies in a 31-year-old male (FV:C < 1%) with umbilical bleeding at birth, recurrent hemarthrosis and muscle hematomas, and a recent intracranial hemorrhage. Results: The proband was homozygous for a deep-intronic mutation (F5 IVS8 +268A fi G) causing the inclusion of a pseudo-exon with an in-frame stop codon in the mature F5 mRNA. Although platelet FV antigen was detectable by immunoprecipitation followed by Western blotting, no FV activity could be demonstrated in the probandÕs plasma or platelets with a prothrombinase-based assay. Moreover, no thrombin generation was observed in PRP triggered with 1-50 pM tissue factor (even in the presence of platelet agonists), whereas an acquired FV inhibitor was excluded. Clot formation in the probandÕs whole blood, as assessed by thromboelastom-etry, was markedly delayed but not abolished. Conclusions: This is the first report of a pathogenic deep-intronic mutation in the F5 gene. Our findings indicate that the minimal FV requirement for viability is extremely low and suggest that thrombin generation in PRP may predict bleeding tendency in patients with undetectable plasma FV.

Summary. Background: The tissue factor pathway inhibitor (TFPI)/protein S anticoagulant system is a potent inhibitor of blood coagulation. TFPI and protein S are major determinants of thrombin generation (TG) tests determined at low tissue factor (TF) and at high TF concentrations in the presence of activated protein C (APC). Both TFPI and protein S protect against venous thrombosis, but the importance of the TFPI/protein S system in arterial thrombosis remains unclear. Objectives: To investigate the influence of the TFPI/protein S anticoagulant system on the risk of myocardial infarction (MI) in young women. Methods: The RATIO study is a case-control study in women under 50 years of age, including 205 patients and 638 controls. TFPI and protein S were quantified using ELISA. The TFPI/ protein S activity (nTFPIr) and the APC sensitivity ratio (nAPCsr) were determined using TG tests. Odds ratios (ORs) adjusted for putative confounders and corresponding 95% confidence intervals (95% CI) were determined. Results: Women with MI had higher TFPI levels than controls (135.9 ± 40% vs. 124.2 ± 41%), resulting in increased TFPI/protein S activities and increased APC sensitivity. Furthermore, an increased TFPI activity was associated with MI [nTFPIr: adjusted OR Q1 vs. Q4 = 2.1 (95%CI 1.1-4.1)]. Additionally, an increased APC sensitivity was associated with MI [nAPCsr: adjusted OR Q1 vs. Q4 = 1.7 (95% CI 0.9-3.2)] Conclusion: Women with MI had increased TFPI levels compared with controls. Consequently, the TFPI/protein S activity and APC sensitivity are increased in women with MI. Whether this increase in TFPI activity acts as a compensating mechanism for an increased procoagulant state or is a marker of endothelial damage remains to be investigated.