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 α-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. Because 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 all unusually large factor V in platelets was associated with multimerin and it remained associated in 0.5 M salt. Multimerin immunodepletion of the normal pooled platelet lysate removed 100 ± 0% of multimerin and 47.0 ± 2.4% of total factor V antigen, whereas sham immunodepletion removed 12.0 ± 3.0 % of multimerin and 4.0 ± 4.0% of factor V antigen (means ± 1 S.D. for 3 experiments). Analyses of serial factor V immunopurified samples indicated that 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 suggestion that only a subpopulation of multimerin was covalently linked to factor V was consistent with the estimated 17 fold molar excess of multimerin subunits to factor V molecules in platelets. The disulfide-linked complexes of multimerin and factor V in platelets, that 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. Multimerin could function to hold about half of the platelet pool of factor V in covalent and noncovalent linkages, until granule release occurs and thrombin cleavages liberate factor Va for prothrombinase assembly on the platelet surface, akin to the way supporting scaffolds hold pieces of plastic models in a unit until their removal for model assembly is desired.

Platelet adhesion to multimerin 1 in vitro: influences of platelet membrane receptors, von Willebrand factor and shear

“…Proteins for adhesion and binding assays were >95% pure using silver staining (verified as described [19]). MMRN1 and bovine serum albumin (BSA) were labeled with biotin, as described [24] and tested after biotinylated MMRN1 was confirmed to support platelet adhesion.…”

“…Sources of reagents and supplies included: thrombin receptor‐activating peptide (TRAP) SFLLRN (Bachem Bioscience Inc., King of Prussia, PA, USA); calcein acetoxymethylester (calcein‐AM; Invitrogen Canada Inc., Burlington, ON, Canada); unfractionated heparin (Leo Pharma Inc., Ajax, ON, Canada); O‐sialoglycoprotein endopeptidase (O‐SGP) from Cedarlane Labs (Hornby, ON, Canada); Vitrocom hollow rectangle glass microcapillary tubes (100‐mm length, 0.2 × 2‐mm cross‐section; Fiber Optic Center, New Bedford, MA, USA); normal mouse IgG (nmIgG; BD Biosciences, Pharmingen, Franklin Lakes, NJ, USA); monoclonal αvβ3 antibody MAB1976 (Chemicon International, Temecula, CA, USA); monoclonal GPIbα antibodies SZ2 [16] (Beckman Coulter Canada Inc., Mississauga, ON, Canada), AN51 [16] (Gene Tex Inc., San Antonio, TX, USA), AK2 [16] (AbD Serotec, Oxford, UK); monoclonal antibodies 7E3 (anti‐αIIbβ3/αvβ3) and 10E5 (anti‐αIIbβ3) (gifts from Dr Barry S. Coller, Rockefeller University, New York, NY, USA); rabbit anti‐VWF (Dako Canada Inc, Mississauga, ON, Canada); Alexa‐488‐conjugated goat anti‐rabbit IgG (Invitrogen‐Molecular Probes, Burlington, ON, Canada); peroxidase‐conjugated goat anti‐human VWF (Affinity Biologicals Inc, Ancaster, ON, Canada); peroxidase‐conjugated goat anti‐rabbit IgG (Jackson Immunoresearch, West Grove, PA, USA); rabbit and monoclonal anti‐human MMRN1 (sources described [17]); platelet, recombinant wild type (WT) and RGE‐MMRN1 (sources described [3,18,19]); purified human VWF (gift from Dr C. Mazurier, Laboratoire Français du Fractionnement et des Biotechnologies LFB, Lille, France); Horm (equine) collagen (95% and 5%, types I and III respectively; Nycomed Linz, Austria); human collagen type I (recombinant) (BD Biosciences, Bedford, MA, USA); EZ‐Link Sulfo‐NHS‐LC Biotin (spacer arm length 22.4 Å) (Pierce, Rockford, IL, USA); ristocetin (American Biochemical and Pharmaceutical Company (Marlton, NJ, USA); adenosine 5′‐diphosphate (ADP) and other reagents (Sigma‐Aldrich Canada, Oakville, ON, Canada).…”

Factor Va, bound to microparticles released during platelet storage, is resistant to inactivation by activated protein C

“…This might leave the PLT FVa associated with the multimerin complex via its C2 domain, 32 possibly preventing cleavage by APC. 10,12 A more hypothetical explanation could be the following: the homology between FVIII and FV might point to a role for a low-density lipoprotein receptor, for example, apolipoprotein ER2 (LRP-8) 33 present on the PLT and/or MP surface that acts as a "receptor" binding FVa and rendering it less susceptible to cleavage by APC. Another explanation for the impaired inactivation of FVa on the MP and PLT surface could be that FVa resides in lipid rafts and therefore might be less accessible for cleavage by APC.…”

Understanding the Clotting Cascade, Regulators, and Clinical Modulators of Coagulation

“…FV is a single polypeptide encoded by chromosome 1 and primarily synthesized in the liver and some evidences show that FV is also produced by megakaryocytes [68][69][70]. The activated FV acts as a cofactor for FXa, to form a prothrombinase complex and it also serves as a target for APC-PS complex in inhibiting the coagulation cascade [71].…”