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.
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.
The Quebec Platelet Disorder (QPD) is an inherited disorder associated with delayed bleeding, increased expression and storage of active urokinase-type plasminogen activator (u-PA) in platelets, normal to increased u-PA in plasma, consumption of platelet plasminogen activator inhibitor-1 (PAI-1), and increased plasmin generation in platelets with proteolysis of stored α-granule proteins, including factor V. Although accelerated fibrinolysis has been proposed to contribute to QPD bleeding, the effects of QPD blood on the lysis of forming and preformed fibrin have not been evaluated. In this study, we modeled fibrinolysis in vitro using thromboelastography, biochemical evaluations of whole blood clots formed at low shear, and perfusion of blood over preformed fibrin. Thromboelastography (TEG) indicated that the clot formation and lysis phases were normal in QPD whole blood clots generated with tissue factor and observed for 180 min, which is the maximum time allowed by the TEG® software. However, when the TEG® were done with limiting amounts of tissue-type plasminogen activator (t-PA), QPD clots showed a shortened lysis phase. The addition of QPD platelets to normal blood hastened t-PA mediated lysis. In QPD whole blood clots, generated at low shear with exogenous thrombin (without t-PA), there was abnormal plasmin generation, with increased fibrinolysis unless fibrinolytic inhibitors were used to inhibit plasmin. These data excluded the defect in platelet factor V as the cause of abnormal lysis. Perfusion studies indicated that QPD platelets adhered to fibrin, although the amount of adhesion was reduced compared to control samples, likely because of the reduced platelet count in QPD blood. When QPD blood was perfused over labeled, preformed fibrin, without a stimulus for thrombin generation, there was accelerated loss of fibrin, and a corresponding increased generation of labeled fibrin degradation products. These results indicate that the QPD is associated with a “gain of function” abnormality that increases the lysis of forming or preformed clots, independent of thrombin generation. Our findings suggest accelerated fibrinolysis is an important contributor to QPD bleeding and support the clinical observation that transfusion of normal platelets does not correct the defect in this bleeding disorder.
Multimerin is a large soluble protein, with an uncertain function, found in platelets, megakaryocytes, endothelium and extracellular matrix fibers but not in plasma. The observation that multimerin contains structural features of an adhesive protein, including an Arg-Gly-Asp (RGD) sequence, led us to investigate its ability to support adhesion of platelets, megakaryocytes, endothelial cells and other cell types. Multimerin had the ability to support the adhesion of both platelets and megakaryocytes and this required cellular activation and the multimerin RGD site. Studies of normal and Glanzmann platelets indicated that multimerin interacted with the major platelet integrin receptor, αIIbβ3 and radioimmunoprecipitation analyses confirmed that multimerin bound to αIIbβ3. Multimerin also supported adhesion of endothelial cells, neutrophils and other cells including smooth muscle cells, fibroblast cells, human embryonic kidney (HEK293) and epithelial cells. Unlike platelets, these cells do not express αIIbβ3; this indicated that other integrin or non-integrin receptors could be involved in cellular adhesion to multimerin. Comparisons of cell adhesion to wild-type and RGE-multimerin indicated that unlike platelets and megakaryocytes, some other cell types (e.g. endothelial cells, smooth muscle cells and neutrophils) were capable of adhering to RGE-multimerin. This suggested that cellular adhesion to multimerin occurs by both RGD and non-RGD dependent mechanisms. Finally, unlike platelets, megakaryocytes and neutrophils, adhesion of other cell types to multimerin did not require cellular activation. In conclusion, our data indicate multimerin has fairly broad proadhesive properties, involving RGD and non-RGD dependent mechanisms, and that cellular receptors including αIIbβ3 interact with multimerin to mediate its binding to activated platelets, endothelial cells and potentially other cell types.
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