Investigation of the interaction of multimerin 1 with components of prothrombinase, in vitro

Abstract

<p>Prothrombinase is an enzymatic complex that accelerates the conversion of prothrombin to thrombin for efficient blood clot formation at sites of vessel injury. Prothrombinase consists of the enzyme factor Xa and its cofactor factor Va, assembled on a phosphatidyl serine-containing membrane, in the presence of calcium. Multimerin 1 (MMRN1) is a polymeric, factor V/Va-, prothrombin-, and phosphatidyl serine-binding protein that is stored in platelet and endothelial cell secretion granules. When released, MMRN1 binds to their cell surface and to the extracellular matrix. Unlike plasma factor Va, platelet factor Va is stored complexed to MMRN1 in platelet α-granules, and is resistant to inactivation by activated protein C (APC). Previous studies revealed that exogenous MMRN1 inhibits thrombin generation in plasma. My thesis investigated the interaction of MMRN1 with components of prothrombinase in order to elucidate the molecular mechanisms by which MMRN1 modulates coagulation. ELISA binding assays that used prothrombin derivatives revealed that the prothrombin gamma-carboxyglutamic acid and kringle domains have potential MMRN1 binding sites. Thrombin generation assays that used purified proteins and/or phospholipid vesicles revealed that MMRN1 inhibits thrombin generation in the presence and absence of factor Va, but not in the absence of phospholipid vesicles. These findings suggest that MMRN1 inhibits phospholipid-dependent thrombin generation through its interactions with factor Va and prothrombin. MMRN1 did not affect APC-mediated loss of factor Va, which suggest other post-translational modifications in platelet factor Va account for its increased APC resistance. In thrombin generation assays with plasma, immobilized MMRN1 captured factor V/Va of human and mouse origin for subsequent thrombin generation, consistent with preservation of function that could be important for localizing factor V/Va for prothrombinase assembly. Collectively, these findings suggest that MMRN1 has a dual role in coagulation: as an inhibitor in fluid phase, and as a promoter when immobilized.</p>