Unique in Vivo Modifications of Coagulation Factor V Produce a Physically and Functionally Distinct Platelet-derived Cofactor

Abstract

Platelet-and plasma-derived factor Va (FVa) serve essential cofactor roles in prothrombinase-catalyzed thrombin generation. Platelet-derived FV/Va, purified from Triton X-100 platelet lysates was composed of a mixture of polypeptides ranging from ϳ40 to 330 kDa, mimicking those visualized by Western blotting of platelet lysates and releasates with anti-FV antibodies. The purified, platelet-derived protein expressed significant cofactor activity such that thrombin activation led to only a 2-3-fold increase in cofactor activity yet expression of a specific activity identical to that of purified, plasma-derived FVa. Physical and functional differences between the two cofactors were identified. Purified, platelet-derived FVa was 2-3-fold more resistant to activated protein C-catalyzed inactivation than purified plasma-derived FVa on the thrombin-activated platelet surface. The heavy chain subunit of purified, plateletderived FVa contained only a fraction (ϳ10 -15%) of the intrinsic phosphoserine present in the plasma-derived FVa heavy chain and was resistant to phosphorylation at Ser 692 catalyzed by either casein kinase II or thrombin-activated platelets. MALDI-TOF mass spectrometric analyses of tryptic digests of platelet-derived FV peptides detected an intact heavy chain uniquely modified on Thr 402 with an N-acetylglucosamine or N-acetylgalactosamine, whereas Ser 692 remained unmodified. N-terminal sequencing and MALDI-TOF analyses of plateletderived FV/Va peptides identified the presence of a fulllength heavy chain subunit, as well as a light chain subunit formed by cleavage at Tyr 1543 rather than Arg 1545 accounting for the intrinsic levels of cofactor activity exhibited by native platelet-derived FVa. These collective data are the first to demonstrate physical differences between the two FV cofactor pools and support the hypothesis that, subsequent to its endocytosis by megakaryocytes, FV is modified to yield a platelet-derived cofactor distinct from its plasma counterpart.Factor Va (FVa), 1 a heterodimeric protein composed of heavy chain (105 kDa) and light chain (74 kDa) subunits, is formed by limited proteolysis of factor V (FV) (1). In normal hemostasis, FVa functions as a non-enzymatic cofactor of the prothrombinase complex, which consists of a 1:1 stoichiometric and Ca 2ϩ -dependent complex of the serine protease factor Xa and FVa, bound to the membrane of appropriately activated platelets, and catalyzes the proteolytic conversion of prothrombin to thrombin (2). When incorporated into the prothrombinase complex, the catalytic activity of factor Xa is increased by approximately 5 orders of magnitude, and FVa contributes substantially to this increase (3). Removal of FVa from the prothrombinase complex results in a 10,000-fold decrease in the rate of thrombin generation (3), the physiologic effect of which is demonstrated in the bleeding diatheses expressed by FV-deficient individuals (4 -8)Factor V circulates in two pools in whole blood. The majority (75-80%) is found in the plasma as an inactive, singl...

Polyphosphate modulates blood coagulation and fibrinolysis

“…Platelet factor V is more active in supporting clotting than is plasma factor V, and in particular, platelet factor V is activated by factor Xa at an accelerated rate relative to plasma factor V (9). Platelet factor V is structurally modified compared to the plasma protein, which may help explain its enhanced activity (8,10). We propose that an additional factor enhancing the procoagulant function of platelet factor V is the concomitant secretion of polyP upon platelet activation, and that polyP serves as a cofactor to enhance the rate of factor V activation by both thrombin and factor Xa.…”

Platelets and platelet-derived factor Va confer hemostatic competence in complete factor V deficiency

“…3 This platelet-derived FV pool originates solely from megakaryocyte endocytosis of the plasma procofactor through a process that results in the formation of a partially proteolytically activated cofactor (FV/Va) 4 and phenotypically alters it to a more procoagulant phenotype. [4][5][6][7][8][9][10] The most common treatment of individuals with symptomatic FV deficiency is administration of fresh frozen plasma (FFP) to temporarily maintain plasma FV at minimally hemostatic levels (20% to 30%). 11 Its effect on platelet-derived FV/Va concentrations is unknown.…”

Acquired Factor V Inhibitors: A Review of Literature

“…Given the success rate with steroids alone this is a reasonable first line treatment. In some cases, where patients have recurrence of the inhibitor, rituximab has been used as maintenance treatment with good results [14]. 60% of asymptomatic patients in this cohort had remission of the inhibitor.…”

“…20% of our factor V is stored in platelets and this FV seems to have different properties that allows it to escape FV inhibitors [12,13]. There is also evidence to suggest functionally distinct features of platelet and plasma FV [14,15]. One small study showed that patients with severe congenital FV with undetectable plasma FV had functional FV in their platelets [6].…”