The Gla domain of human prothrombin has a binding site for factor Va

The role of the Gla domain of human prothrombin in interaction with the pro thrombinase complex was studied using a peptide with the sequence of the fi rst 46 residues of human prothrombin, PT-(1-46). Intrinsic fluorescence mea surements showed that PT-(1-46) undergoes a conformational alteration upon binding calcium; this conclusion is supported by one-dimensional H-1 NMR sp ectroscopy, which identifies a change in the chemical environment of trypto phan 41. PT-(1-46) binds phospholipid membranes in a calcium dependent mann er with a K-d Of 0.5 muM and inhibits thrombin generation by the prothrombi nase complex with a K-i of 0.8 muM. In the absence of phospholipid membrane s, PT-(1-46) inhibits thrombin generation by factor Xa in the presence but not absence of factor Va, suggesting that PT-(1-46) inhibits prothrombin-fa ctor Va binding. The addition of factor Va to PT-(1-46) labeled with the fl uorophore sulfosuccinimidyl-7-amino-4-methylcoumarin-3-acetic acid (PT-(1-4 6)AMCA) caused a concentration-dependent quenching of AMCA fluorescence, pr oviding direct evidence of a PT-(1-46)-factor Va interaction. The K-d for t his interaction was 1.3 muM. These results indicate that the N-terminal Gla domain of human prothrombin is a functional unit that has a binding site f or factor Va. The prothrombin Gla domain is important for interaction of th e substrate with the prothrombinase complex.