Identification of human platelet membrane fibrinogen receptors by immunochemical techniques.

The fibrinogen receptors of platelets were investigated with the use of three types of anti-platelet membrane antibodies and three types of platelets. We found that antisera raised in rabbits against membranes prepared from human intact, chymotrypsin- or pronase-treated platelets inhibited the fibrinogen-induced aggregations of ADP-stimulated intact platelets, chymotrypsin-treated platelets and pronase-treated platelets. These antisera also blocked the binding of 125I-fibrinogen to ADP-stimulated intact, chymotrypsin-treated, and pronase-treated platelets. These results suggest that all three antisera blocked the interaction of fibrinogen with its receptor on the surface of the three types of platelets studied. Fibrin clot retraction by intact platelets was also inhibited by these three antibodies indicating an important role of platelet membrane proteins in clot retraction. As demonstrated by techniques using 125I-surface labeling, Staphylococcus aureus immunoprecipitation, SDS-polyacrylamide gel electrophoresis and autoradiography, anti-intact platelet membrane antibody immunoprecipitated the membrane glycoproteins GPIIb, GPIII and a protein with an apparent molecular weight of 66 000 from detergent solubilized surface 125I-iodinated chymotrypsin-treated platelets. Anti-chymotrypsin and anti-pronase-treated platelet membrane antisera immunoprecipitated mostly GPIII and the 66 000 molecular weight protein from detergent solubilized, surface 125I-iodinated chymotrypsin-treated platelets. The 66 000 Mr protein was not found on the surface of intact (unstimulated) platelets which do not bind 125I-fibrinogen and are not aggregated by fibrinogen without the prior addition of ADP. The ability of anti-platelet membrane antibodies to block fibrinogen-induced platelet aggregation and fibrinogen binding to platelets correlated with their ability to immunoprecipitate a 66 000 Mr protein from the platelet surface. It is proposed that the 66 000 Mr protein may be the fibrinogen binding domain of GPIII which becomes permanently exposed on the surface of chymotrypsin and pronase-treated platelets following proteolysis and which becomes exposed upon stimulation of intact platelets by agents such as ADP.