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.
Church
W R
WR
Department of Biochemistry, University of Vermont College of Medicine, Burlington 05405.
Ouellette
L A
LA
Messier
T L
TL
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001792:Blood Platelets / Q000378:metabolism*; D004789:Enzyme Activation; D015943:Factor Va / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D010713:Phosphatidylcholines / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D011516:Prothrombin / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D010743:Phospholipids; D015943:Factor Va; D011516:Prothrombin; D015951:Factor Xa