Synergistic inhibition of platelet aggregation by fibrinogen-related peptides.

We have evaluated the ability of the fibrinogen-related peptides Gly-Arg-Gly-Asp-Ser (GRGDS), Gly-Gln-Gln-His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (gamma-chain peptide), and Gly-Pro-Arg-Pro (GPRP) to inhibit platelet aggregation in platelet-rich plasma individually and in combination. When used alone, GRGDS totally inhibited ADP-induced aggregation of human platelets in platelet-rich plasma; however, the maximum inhibitory effect of the other peptides was less than 80%. The concentrations necessary to inhibit platelet aggregation in plasma by 50% were 100 mumols/l and 1 and 3.2 mmol/l for GRGDS, gamma-chain peptide, and GPRP, respectively. When evaluating the effect of peptide mixtures, we discovered that the combination GPRP + GRGDS worked together synergistically (p less than 0.001, analysis by surface response methodology), whereas GPRP + gamma-chain peptide did not. For example, our analysis indicated that a mixture of 50 mumols/l GRGDS plus 180 mumols/l GPRP would produce 50% inhibition of platelet aggregation. This is an effect twofold greater than that produced by 50 mumols/l GRGDS alone, and one that would require an 18-fold greater concentration of GPRP if used alone. These data indicate that the combination GPRP + GRGDS inhibited platelet aggregation in plasma in a synergistic fashion and suggest the potential value of their combined use in antithrombotic therapy.

Plasminogen interactions with platelets in plasma.

In this report we used a fluorescent flow cytometry-based assay to examine plasminogen binding to platelets in plasma. Our data indicate that platelets activated in platelet-rich plasma (PRP) by adenosine-5'-diphosphate (ADP) or thrombin bind plasminogen to their surface. Fab fragments of the monoclonal antibody LJ-CP8 that are directed against the fibrinogen binding site on the glycoprotein (GP) IIb-IIIa complex inhibit both plasminogen and fibrinogen binding to ADP-stimulated platelets as does 5 mmol/L EDTA. Platelet aggregation and plasminogen and fibrinogen binding are also concurrently inhibited by the Gly-Arg-Asp (RGD) analogue Gly-Arg-Gly-Asp-Ser (GRGDS) when it is added to PRP before ADP stimulation. The scrambled peptide analogue SDGRG has no effect. The monoclonal antibody 6D1, directed against the von Willebrand factor binding site on GPIb, has no effect on plasminogen-platelet binding, nor does antithrombospondin antibody. epsilon-Aminocaproic acid (EACA), however, inhibits plasminogen binding to ADP-activated platelets. These data indicate that plasminogen binds to platelets activated in plasma, that binding occurs on platelet GPIIb/IIIa, and that binding may be mediated via plasminogen association with fibrinogen via lysine binding domains. Finally, we found both plasminogen and fibrinogen on resting platelets in PRP and demonstrated that they are equally displaced by EDTA, LJ-CP8, and 10E5 (an additional anti-GPIIb/IIIa monoclonal antibody). Plasminogen is also equally displaced by EACA. These data suggest that plasminogen is also bound to GPIIb/IIIa on resting platelets, possibly also via interaction with fibrinogen.