Monoclonal antibodies against human platelet membrane glycoprotein IIIa and collagen-induced platelet activation.

Abstract

Two monoclonal antibodies against human platelet membrane glycoprotein IIIa (GPIIIa) were obatined. One monoclonal antibody, designated as 1B1, was found to inhibit both collagen-induced platelet aggregation and release eractions. This antibody also inhibited the binding of ¹²⁵I-labeled collagen to human platelets. On the other hand, the other antibody, designated as B10, had no effect on platelet activation induced by a number of physiological stimulants including collagen. Direct binding studies involving ¹²⁵I-labeled 1B1 or B10 demonstrated that the binding sites for these antibodies on unstimulated platelets have dissociation constants of 4.2 and 14.0 nM, respectively. The binding of ¹²⁵I-labeled 1B1 or B10 to platelets was not inhibited by the other antibody. Purified 1B1 and B10 were covalently coupled to Affi-Gel and then proteolytic fragments of GPIIIa were applied to the Affi-Gel immunoadsorbent columns. Of the several proteolytic fragments, the 56 kilodaltons (kDa) fragment obtained on digestion with V8 protease bound to both of the columns. The 69 and 55 kDa fragments obtained with BrCN bound to only the 1B1 Affi-Gel column, while the 63 kDa fragment obtained with chymotrypsin only bound to the B10-Affi-Gel column. Based on the partial amino acid sequences of these fragments and the amino acid sequence of GPIIIa (C. A. Fitzgerald, B. Steiner, S. C. Rall, Jr., S. Lo and D. R. Phillips, J. Biol. Chem., 262, 3936(1987)), the epitopes for 1B1 and B10 were concluded to be located at amino acids 335 to 582 and 206 to 335, respectively. These results indicate that a certain epitope of GPIIIa or the GPIIb/IIIa complex on the platelet cell surface constitutes a receptor for collagen, and that the epitope for 1B1 is located near or in the collagen-binding site of GPIIIa. Thus, the collagen binding site is located near the transmembrane portion of GPIIIIa, namely, amino acid residues 335 to 582. Furthermore, the 1B1 antibody was found to induce the aggregation of washed platelets, suggesting that GPIIIa is directly involved in the activation of platelets.