The lag time, ETP and peak height of thrombin generation depend on the levels of multiple coagulation factors and inhibitors. The specific assay determinants vary with the experimental conditions.

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...

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SummaryThree novel polymorphisms were found in the repeated region of the large exon 13 of factor V gene, one giving rise to a codon dimorphism (Serl240) and two causing aminoacid substitutions (Hisl299Arg, Leul257Ile). An increasing frequency of the Argl299 (R2 allele) correlated with a decreasing mean plasma factor V activity in the groups of subjects under study, which included 26 unrelated subjects with partial factor V deficiency. Family studies supported the co-inheritance both of low factor V activity and of R2 allele. The reduction of factor V activity associated with the R2 allele was not clinically symptomatic even in the homozygous condition and was characterized by a parallel reduction of antigen in plasma, in which abnormal molecules were not detected. Data suggest that the R2 allele represents a marker in linkage with an unknown defect rather than a functional polymorphism.These studies provide the first evidence of a genetic component in determining factor V levels in plasma and of a genetic linkage between the factor V gene and factor V deficiency. They also define specific haplotypes which are associated with factor V deficiency or with APC resistance (Arg506Gln) and are valuable fools for the study of factor V defects.

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