Identification of two distinct regions within the binding sites for fibrinogen and fibronectin on the IIb–IIIa human platelet membrane glycoprotein complex by monoclonal antibodies P2 and P4

Abstract

The effect of two monoclonal antibodies P2 (LyP 2) or P4 (LyP 4), specific for the platelet membrane glycoprotein I1 bj1Il a complex, on binding of '251-labelled fibrinogen or '251-labelled fibronectin to thrombinstimulated platelets was studied. These monoclonal antibodies are directed against different determinants on the I1 b-IIIa complex and react only with the complex and not with the individual glycoproteins. Fibrinogen binding to thrombin-stimulated platelets was significantly inhibited by P2 but not by P4. Fibronectin binding to thrombinstimulated platelets was significantly inhibited by P4 but only poorly by P2. These results indicate the presence of specific regions on the glycoprotein I1 b-I11 a complex which act as binding sites for fibrinogen or fibronectin. glycoprotein IIb or IIIa had no effect on platelet functionsRecently a number of monoclonal antibodies have been used to investigate the role of glycoproteins IIb and IIIa in platelet function [S]. Monoclonal antibodies, directed against glycoproteins I1 b and/or Ill a, inhibit platelet aggregation and binding of '251-labelled fibrinogen either completely, partially or have no effect [9-171. The degree of inhibition caused by these monoclonal antibodies appears to be linked to the position of their determinants in relation to the fibrinogenbinding site in the Ilb-IIIa complex 19-171. Recent reports suggest that the I1 b-111 a complex contains receptor sites not only for fibrinogen but also for fibronectin, thrombospondin and von Willebrand factor (vWf) [18-211. Very little is known about the regions of the 11 b-IIIa complex that serve as binding sites for these four adhesive proteins (fibrinogen, fibronectin, thrombospondin and vWf). A tetrapeptide, Arg-Gly-Asp-Ser (RGDS), was shown to inhibit the binding of three of these proteins to stimulated platelets [22, 231. It was, therefore. suggested that fibrinogen, fibronectin and vWf interact with a common site on thrombinactivated platelets. To test this hypothesis this study has examined the binding of 1251-labelled fibrinogen or '251-labelled fibronectin to thrombin-stimulated platelets in the presence or absence ofmonoclonal antibodies P2 (LyP 2) or P4 (LyP 4). These monoclonal antibodies are directed against different [71.

Platelet dysfunction during Bothrops jararaca snake envenomation in rabbits

“…The epitope recognized by P2 is close to the fibrinogenbinding site of GPIIb-IIIa (92,93). The results presented herein showed a significant reduction of P2 binding to platelets of envenomed rabbits between 1 and 2 h. Since no alteration of GPIIb-IIIa expression was observed by polyclonal antibodies, the diminution of P2 binding probably reflected the occupation of P2-binding site in GPIIb-IIIa.…”

Separation of important new platelet glycoproteins (GPIa, GPIc, GPIc*, GPIIa and GMP-140) by f.p.l.c. Characterization by monoclonal antibodies and gas-phase sequencing

“…Glycoproteins eluted from the Mono Q column were further isolated and identified by immunoaffinity chromatography, using MAbs LYP22 (specific for GPIIa [13]), or LYP4 (specific for GPIIb/IIIa [14]) and by Western blotting using an anti-GMP-140 MAb (LYP20) [15].…”

Effects of dietary linoleic acid and gamma linolenic acid on platelets of patients with multiple sclerosis

“…to get platelet-rich-plasma (PRP). Platelets were washed according to the Mustard technique (17) with slight modifications as previously described (18).…”

“…The fibrinogen was iodinated at a specific activity of (6 x lO5cpm/pg) and stored at -70°C for no more than a week. The fibrinogen binding studies were carried out as previously described (18). Briefly, platelets (final concentration 108/ml) were stimulated with 0.8U/ml human alpha-thrombin for 5 min at 25°C.…”