Low plasma levels of tissue factor pathway inhibitor in patients with congenital factor V deficiency

Abstract

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...

Factor V‐short and protein S as synergistic tissue factor pathway inhibitor (TFPIα) cofactors

“…It is interesting to note that FV810 recently was reported to have no protein S‐dependent TFPIα‐cofactor activity despite having the binding site for TFPIα,20, 21 suggesting that FV‐Short (deletion of 756‐1458) contains structural elements important for the protein S interaction which FV810 is lacking. FV is also known to bind TFPIα but with much lower affinity (Kd estimated with Biacore to be 13 nmol L −1 ),10 which is compatible with the concept that the acidic region in intact FV is engaged in an interaction with the positively charged cluster in the B domain.…”

“…It has been shown that FV‐deficiency is associated with low plasma levels of tissue factor pathway inhibitor (TFPIα), which regulates the initiation step of the extrinsic tissue factor (TF) pathway 10. Immune depletion of FV results in depletion of TFPIα, which demonstrates that TFPIα is circulating in complex with FV 10, 11…”

Platelets Contain Tissue Factor Pathway Inhibitor-2 Derived from Megakaryocytes and Inhibits Fibrinolysis

“…In previous studies, pdFV bound to TFPI-1 with ϳ15 nM K d (46). 6 Because pdFV and FV-1033 both contain all essential features for binding to TFPI-1 (57), the moderate variation in K d previously observed with pdFV (46) and currently obtained with FV-1033 could reflect slight differences in the experimental conditions used in the two laboratories. Importantly, pulldown and Western blot analysis data presented in Fig.…”

“…Binding of TFPI-2 and TFPI-1 to FV-1033 and rFVa Using SPR-TFPI-1 has been shown previously to bind to pdFV with ϳ15 nM K d using SPR (46). Here, we employed SPR to study the binding of FV-1033 and rFVa to immobilized TFPI-2 or TFPI-1.…”

“…TFPI-2 is abundantly made by syncytiotrophoblasts during pregnancy (45); its role could be to supplement inhibition of fibrinolysis to control bleeding. Noticeably, TFPI-1 is associated with FV in normal plasma (46,47) and binds to purified FV (46) and FVa (47). Currently, association of TFPI-2 with purified FV or FV in plasma of pregnant women is not known; further, contribution of plasma TFPI-2 toward the inhibition of intrinsic coagulation and/or fibrinolysis has not been determined.…”