We report a case of juvenile thrombophilia associated with a substitution of leucine for arginine at position 338 (R338L) in the factor IX gene (factor IX-R338L). The level of the mutant factor IX protein in plasma was normal, but the clotting activity of factor IX from the proband was approximately eight times the normal level. In vitro, recombinant factor IX-R338L had a specific activity that was 5 to 10 times as high as that in the recombinant wild-type factor IX. The R338 substitution causes a gain-of-function mutation, resulting in factor IX that is hyperfunctional.
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
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