LOCALIZATION OF THE DOMAINS OF FIBRIN INVOLVED IN BINDING TO PLATELETS
PLATELET GYCOPROTEINS IIb-IIIa
The molecular basis of platelet-fibrin interactions has been investigated by using synthetic peptides as potential inhibitors of binding fibrin protofibrils and fibrinogen to ADP-stimulated platelets, adhesion of fibrin fibers to the platelet surface, and platelet-mediated clot retraction. Synthetic peptides RGDS and HHLGGAKQAGDV, corresponding to regions of the fibrinogen α and γ chains previously identified as platelet recognition sites, inhibited the binding of radiolabelled soluble fibrin
... ed soluble fibrin oligomers to ADP-stimulated platelets with IC50 values of 12 and 40 μM, respectively. The IC50 values obtained with fibrinogen as the ligand were 3-fold higher. Synthetic GPRP and GHRP, corresponding to the N-terminal sequences of the fibrin α and β chains, were minimally effective in blocking soluble fibrin oligomer binding to ADP-stimulated platelets. The extent of fibrin:platelet adhesion was determined with a microfluorimetric technique which measures the quantity of fluorescein-labelled fibrin attached to the surface of platelets. The signal obtained from the brightly fluorescent platelet:fibrin adducts was time- and concentration-dependent, and was fully inhibited by a monoclonal antibody directed against the glycoprotein II:IIIa complex (HP1-1D, kindly provided by Dr. W. Nichols). Inhibition of fibrin:platelet adhesion by RGDS, HHGGAKQAGDV, and GHRP all exhibited a similar, linear dependence on the peptide concentration, reaching 1/2 maximum at about 200 μM, suggesting nonspecific effects. GPRP inhibited fibrin assembly but did not appear to have specific effects on fibrin:platelet adhesion. The time course of clot retraction was followed by right angle light scattering intensity measurements. Only RGDS affected clot retraction, causing a 4-fold decrease in rate at 230 μM. These results indicate that fibrinogen and fibrin protofibrils, which are obligatory intermediates in the fibrin assembly pathway, share a set of common platelet recognition sites located at specific regions of the α and γ chains of the multinodular fibrin(ogen) molecules. The RGDS site is also involved in mediating interactions between the three dimensional fibrin network and ADP-stimulated platelets.