Human platelets contain forms of factor V in disulfide-linkage with multimerin

Abstract

Factor V is an essential cofactor for blood coagulation that circulates in platelets and plasma. Unlike plasma factor V, platelet factor V is stored complexed with the polymeric alpha-granule protein multimerin. In analyses of human platelet factor V on nonreduced denaturing multimer gels, we identified that approximately 25% was variable in size and migrated larger than single chain factor V, the largest form in plasma. Upon reduction, the unusually large, variably-sized forms of platelet factor V liberated components that comigrated with other forms of platelet factor V, indicating that they contained factor V in interchain disulfide-linkages. With thrombin cleavage, factor Va heavy and light chain domains, but not B-domains,were liberated from the components linked by interchain disulfide bonds, indicating that the single cysteine in the B-domain at position 1085 was the site of disulfide linkage. Since unusually large factor V had a variable size and included forms larger than factor V dimers, the data suggested disulfide-linkage with another platelet protein, possibly multimerin. Immunoprecipitation experiments confirmed that unusually large factor V was associated with multimerin and it remained associated in 0.5 M salt. Moreover, platelets contained a subpopulation of multimerin polymers that resisted dissociation from factor V by denaturing detergent and comigrated with unusually large platelet factor V, before and after thrombin cleavage. The disulfide-linked complexes of multimerin and factor V in platelets, which are cleaved by thrombin to liberate factor Va, could be important for modulating the function of platelet factor V and its delivery onto activated platelets. Factor Va generation and function from unusually large platelet factor V is only speculative at this time.

An acquired factor V inhibitor associated with defective factor V function, storage and binding to multimerin 1

“…Plasma was tested for IgG autoantibodies against FV, as previously described [8]. Plasma and platelet lysates were tested by: FV ELISA (using detecting antibodies against heavychain, light-chain, and B-domain epitopes) [12]; western blotting (using polyclonal antisera after reduced sodium dodecylsulfate polyacrylamide gel electrophoresis) [13]; and MMRN1 and FV-MMRN1 complex ELISA [12]. CAT was used to test FVa-dependent thrombin generation by plateletpoor plasma (PPP), platelet-rich plasma (PRP), and washed platelets [14], resuspended in control, patient or FV-deficient plasma (final in assay: 135 · 10 9 platelets L -1…”

Identification of the MMRN1 Binding Region within the C2 Domain of Human Factor V

“…The recombinant MMRN1 contained 18.5 g of MMRN1 per unit of antigen (amount contained in 10 9 pooled normal platelets) (10). Because of variability in MMRN1 polymer size, molar concentrations of recombinant MMRN1 were calculated based on the amounts of MMRN1 subunits (average subunit size was 186 kDa).…”

“…Third, after conversion of factor V to Va, there was a considerable (84%) reduction in binding of liberated B domain fragments to MMRN1. Recently, we identified that human platelets contain forms of factor V that are linked to MMRN1 by a disulfide bridge involving cysteine 1085 in the factor V B domain (10). Although it is possible that the B domain enhances factor V-MMRN1 binding indirectly by altering conformation of the factor V light chain, the covalent linkages between the factor V B domain and MMRN1 in platelets suggests that the B domain directly contributes to the MMRN1 binding site of factor V.…”

“…In blood, much of the procoagulant factor V is stored in platelets as a complex with the ␣-granule protein multimerin 1 (MMRN1) for activation-induced release during clot formation (10,11). Several differences have been noted in the functional properties of platelet and plasma factor V (reviewed in Ref.…”

Location of the multimerin 1 binding site in coagulation factor V: An update

“…In blood, approximately 25% of the factor V (FV) is retained in platelet α-granules, where FV is stored as a partially activated cofactor complexed to the polymeric protein multimerin 1 (MMRN1) [2,3]. In humans, MMRN1-FV binding is predominantly noncovalent, although 25% of platelet FV is disulfide linked to MMRN1 [3]. Noncovalent FV-MMRN1 binding involves an extended region of the FV light chain C2 domain, that overlaps the FVa membrane binding site [4].…”