| Factor V is an essential coagulation cofactor, with pro- and anticoagulant functions that regulate blood coagulation. Activated factor V (factor Va) accelerates thrombin generation by the enzyme complex prothrombinase. Approximately 25% of the factor V in blood is stored in platelets in complex with multimerin 1 (MMRN1), a disulfide-linked, homopolymeric platelet and endothelial cell protein, for activation induced release during clot formation. The focus of my thesis was to identify the structural basis and functional consequence(s) of factor V and Va binding to MMRN1. Modified enzyme-linked immunosorbent assays (ELISA) with mutated factor V constructs were used to identify MMRN1 binding sites in the light chain C1 and C2 domains of factor V/Va, with a key role of the C2 domain membrane binding residues of factor V/Va (W2063, and W2064) in MMRN1 binding. Accordingly, anti C2 domain autoantibodies (that impaired factor V membrane binding and Va procoagulant function) also inhibited factor V-MMRN1 binding. Surface plasmon resonance analyses of factor V/Va-MMRN1 binding indicated that factor V and Va bound to MMRN1 with high affinities (1<.0 values of approximately 2 and 7 nM, respectively). Compared to factor Va, factor V dissociated more slowly from MMRN1, possibly because factor V-MMRN1 complexes underwent stabilizing conformational changes that were detectable by circular dichroism spectroscopy. The functional consequences of high affinity factor V/Va-MMRN1 binding were evaluated by calibrated automated thrombinography. The addition of MMRN1 delayed and reduced thrombin generation in plasma, in the presence and absence of platelets. These inhibitory effects were at least partly based on delayed factor V activation by thrombin and factor Xa in the presence of MMRN1. MMRN1 also reduced thrombin generation by preformed factor Va, possibly by interfering with factor Va-membrane interactions. Collectively, these findings offer insights into the molecular mechanism of factor V and Va binding to MMRN1, provide an explanation for factor V-MMRN1 complex formation in platelets, and suggest that MMRN1 released from platelets and endothelial cells downregulates factor Va generation and procoagulant functions. Now that factor V/Va-MMRN1 interactions have been examined in vitro, it is appropriate to consider assessing their structure-function relationships by animal models.;... |
