Independent modulation of von Willebrand factor and fibrinogen binding to the platelet membrane glycoprotein IIb/IIIa complex as demonstrated by monoclonal antibody.

Abstract

In this study we have used two new monoclonal antibodies, designated LIP5 and LJP9, as well as a previously described one, AP2, all specific for the platelet membrane glycoprotein (GP)IIb/ IIIa complex. None of them reacted with dissociated GPIIb or GPIIIa. The monovalent Fab fragment of both LJP5 and LJP9 bound to unstimulated platelets in a saturable manner, but binding was markedly decreased after platelets had been incubated at 370C in the absence of added extracellular calcium. The binding of LJP9 was not affected by AP2, but was blocked by excess UP5. On the contrary, the binding of LJP5 was blocked in the presence of both AP2 and LJP9. Thus, these antibodies bound to distinct epitopes of GPIIb/IIIa. At saturation, the binding to unstimulated platelets was between 2.41 and 10.9 X 104 molecules/platelet for LJP5 and between 3.47 and 9.1 X 104 molecules/platelet for LJP9 (range of 11 and 10 experiments, respectively). Binding increased up to 50% after thrombin stimulation. The estimated association constant, K., was 2.7 X 107 M-1 for LJP5 and 3.85 X 107 M-1 for LJP9. Both UP5 and 1JP9 partially inhibited the association of 45Ca2W with the surface of unstimulated platelets. Moreover, both antibodies blocked the binding of von Willebrand factor (vWF) to stimulated platelets, whereas only UJP9, but not UP5, blocked fibrinogen binding. LJP9 was also a potent inhibitor of platelet aggregation, whereas UP5 was without effect in this regard. The results of the present study demonstrate that independent modulation of vWF and fibrinogen binding to stimulated platelets can be attained with monoclonal antibodies directed against distinct epitopes of GPIIb/IIIa.

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

“…The inhibition by these peptides also suggests that these adhesive proteins share a common binding site on the activated I1 bIIIa complex. However, data mentioned in this study and those recently reported by Trapani Lombardo et al [47] show that monoclonal antibodies can specifically inhibit fibronectin or vWf binding to stimulated platelets without affecting fibrinogen binding or platelet aggregation. Plow et al 1451 have recently suggested that high-affinity interactions of any of these four adhesive proteins with platelets may call for common as well as unique binding sites.…”

von Willebrand factor interaction with the glycoprotein IIb/IIa complex. Its role in platelet function as demonstrated in patients with congenital afibrinogenemia.

“…Accordingly, this antibody inhibited 1251I-vWF binding to normal or afibrinogenemic platelets stimulated by ADP plus adrenaline (Table I). In previous studies we have shown that antibody UPS has no effect on the binding of fibrinogen to GPIIb/IIIa (see reference 24 for full details). Moreover, antibody UP5 has no effect on the aggregation of normal platelet-rich plasma (Fig.…”

“…belong to three different kindreds and have been previously described in detail (19-2 1 Monoclonal antibody. The anti-GPIIb/IIIa monoclonal antibody UP5 has been recently described in detail (24). It blocks vWF binding to stimulated platelets without affecting fibrinogen binding.…”

“…It blocks vWF binding to stimulated platelets without affecting fibrinogen binding. Monovalent Fab fragment of this antibody was prepared as described (24). It was stored in 0.02 M Tris * HCI-0.…”

“…The method for preparation ofwashed platelet suspensions using the albumin density gradient technique has also been reported (24,27).…”