The kringle 2 domain of prothrombin has been shown to interact with factor Va during the activation of prothrombin by the prothrombinase complex composed of factor Xa, factor Va, negatively charged phospholipids and Ca2+ ions. However, contradictory results have been reported about the role of the kringle 1 domain of prothrombin during the assembly of the prothrombinase complex. In an attempt to clarify the role of the kringle 1 domain of prothrombin, its effect on the activation of prothrombin by the prothrombinase complex and its direct binding to human factor Va were assessed. Comparative evaluation with the effects caused by other prothrombin structural components [a fragment 1 (γ-carboxyglutamic acid and kringle 1 domains), a kringle 2 domain and a catalytic 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 phospholipids. However, in the absence of both factor Va and phospholipids, kringle 2 fragment, but not kringle 1 fragment, inhibited prothrombin activation. Evaluation of the molecular interaction of the kringle domains with factor Va in assays with solid-phase phospholipid vesicles showed that each kringle 1 and kringle 2 fragment inhibited the prothrombinase complex activity. Assessment of the direct binding of prothrombin and each kringle domain of prothrombin with factor Va by fluorescence polarization showed that prothrombin, kringle 1 and kringle 2 fragments bind directly to factor Va with dissociation constants of 1.9ŷ0.1, 2.3ŷ0.1 and 2.0ŷ0.4 ƁM (meansŷS.D.) respectively. These findings suggest that both kringle 1 and 2 domains of prothrombin interact with factor Va during the assembly of the prothrombinase complex.
Prothrombin kringle 1 domain interacts with factor Va during the assembly of prothrombinase complex
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Hiroshi DEGUCHI, Hiroyuki TAKEYA, Esteban C. GABAZZA, Junji NISHIOKA, Koji SUZUKI; Prothrombin kringle 1 domain interacts with factor Va during the assembly of prothrombinase complex. Biochem J 1 February 1997; 321 (3): 729–735. doi: https://doi.org/10.1042/bj3210729
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