PROTHROMBIN KRINGLE-1 DOMAIN INTERACTS WITH FACTOR VA DURING THE ASSEMBLY OF PROTHROMBINASE COMPLEX

The kringle 2 domain of prothrombin has been shown to interact with fa ctor Va during the activation of prothrombin by the prothrombinase com plex composed of factor Xa, factor Va, negatively charged phospholipid s and Ca2+ ions. However, contradictory results have been reported abo ut the role of the kringle 1 domain of prothrombin during the assembly of the prothrombinase complex. In an attempt to clarify the role of t he kringle 1 domain of prothrombin, its effect on the activation of pr othrombin by the prothrombinase complex and its direct binding to huma n factor Va were assessed. Comparative evaluation with the effects cau sed by other prothrombin structural components [a fragment 1 (gamma-ca rboxyglutamic acid and kringle 1 domains), a kringle 2 domain and a ca talytic protease domain] was also performed. In the presence of factor Va, each kringle 1 and kringle 2 fragment significantly inhibited the factor Xa-catalysed prothrombin activation in the absence of phosphol ipids. However, in the absence of both factor Va and phospholipids, kr ingle 2 fragment, but not kringle 1 fragment, inhibited prothrombin ac tivation. Evaluation of the molecular interaction of the kringle domai ns with factor Va in assays with solid-phase phospholipid vesicles sho wed that each kringle 1 and kringle 2 fragment inhibited the prothromb inase complex activity. Assessment of the direct binding of prothrombi n and each kringle domain of prothrombin with factor Va by fluorescenc e polarization showed that prothrombin, kringle 1 and kringle 2 fragme nts bind directly to factor Va with dissociation constants of 1.9 +/- 0.1, 2.3 +/- 0.1 and 2.0 +/- 0.4 mu M (means +/- S.D.) respectively. T hese findings suggest that both kringle 1 and 2 domains of prothrombin interact with factor Va during the assembly of the prothrombinase com plex.