Modulation of human prothrombin activation on phospholipid vesicles and platelets using monoclonal antibodies to prothrombin fragment 2

Prothrombin contains two kringle domains that are removed during activation to the blood clotting enzyme alpha-thrombin. By analogy with other kringle-containing proteins the prothrombin kringles may play a role in the protein-protein interactions necessary for prothrombin activation. Four monoclonal antibodies to prothrombin kringle 2 have been produced against human prothrombin, and a fifth monoclonal antibody was produced against a synthetic peptide consisting of amino acid residues 216-231 of kringle 2. Each antibody was tested for its ability to block prothrombin activation by factor Xa. In the presence of phosphatidylcholine/phosphatidylserine vesicles and factor Va, two of the antibodies, alpha HII-3 and alpha HII-4, inhibited prothrombin activation at a 90 and 50% level, respectively. Two other monoclonal antibodies (alpha HII-6 and alpha HII-7) and the antipeptide antibody (alpha HII-5) had no effect on prothrombin activation. When factor Xa was the catalyst alone, antibody alpha HII-3 lost the ability to inhibit prothrombin activation whereas antibody alpha HII-4 again partially inhibited the reaction. When human platelets were the reaction surface, the patterns of inhibition by the anti-fragment 2 antibodies were identical to that observed with phospholipid vesicles. These data suggest a role for prothrombin fragment 2 in activation, possibly by mediating the interaction of substrate prothrombin with factor Xa or factor Va on the phospholipid surface.