Localization of a thrombin-binding site on human platelet membrane glycoprotein Ib determined by a monoclonal antibody.
To determine a thrombin-binding site on GPIb alpha on platelet membrane, we have examined the binding activities of tryptic or chymotryptic fragments of purified GPIb alpha to a monoclonal antibody against GPIb (TM60) and thrombin using (immuno)affinity chromatography. When purified GPIb alpha was digested with trypsin, two fragments (94-kDa, and 43-kDa) were obtained. The 43-kDa fragment was shown to bind to both affinity columns of TM60- and thrombin-Affi-Gel, while the 94-kDa fragment did not bind to either Affi-Gel columns. When trypsin fragments were incubated with TM60 and then applied to the column of thrombin-Affi-Gel, neither fragments were bound to the column. When the same experiment was performed using chymotrypsin, three fragments (94-kDa, 45-kDa and 39-kDa) were observed. On TM60- and thrombin-Affi-Gel columns, the smaller fragments (45-kDa and 39-kDa) were bound to the column. After incubation of these fragments with TM60, neither bound to the thrombin column. These results indicate (i) that the epitope for TM60 is located near, or on the thrombin-binding site of GPIb alpha, and (ii) that the thrombin-binding site is located on the tail portion of GPIb alpha, especially on a chymotrypsin cleavage site.
D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D001345:Autoradiography; D001665:Binding Sites; D002846:Chromatography, Affinity; D002918:Chymotrypsin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis; D006023:Glycoproteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D008565:Membrane Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D010980:Platelet Membrane Glycoproteins; D013917:Thrombin / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D006023:Glycoproteins; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D010980:Platelet Membrane Glycoproteins; D002918:Chymotrypsin; D014357:Trypsin; D013917:Thrombin