Fibrinogen binding to human blood platelets: Effect of ? chain carboxyterminal structure and length

Abstract

Recent evidence suggests that fibrinogen binding to platelets is mediated by the 12 carboxyterminal amino acid residues of the ? chain. Because human plasma fibrinogen ? chains differ in mol wt and carboxyterminal amino acid sequence, we examined the effect of such ? chain heterogeneity on platelet-fibrinogen interactions, using two fibrinogens of distinct composition, separated by ion exchange chromatography. One fibrinogen possessed only ? chains of mol wt 50,000 (F(?50)), the predominant ? chain species found in plasma. The other fibrinogen possessed equal amounts of ? chains with mol wt 50,000 and 57,500 (F(?50,57.5)), with the longer ? chain (?57.5) possessing an amino acid extension at the carboxyterminal end. The latter fibrinogen was 50% less effective than F(?50) in supporting ADP-induced platelet aggregation at concentrations of .01 to 2 mg/mL. Scatchard analysis revealed no difference in the binding affinities of the two fibrinogens to ADP-treated platelets, but the amount of F(?50,57.5) that was bound to platelets at saturation was only 50% that of F(?50). Fibrinogen receptors that remained unoccupied in the presence of saturating concentrations of F(?50,57.5), however, could be occupied by fresh F(?50). Excess unlabeled F(?50) displaced both radiolabeled fibrinogens from activated platelets, and both fibrinogens bound to the same platelet receptor, as judged by the inhibition of binding to stimulated platelets by a monoclonal antibody directed against the glycoprotein (GP) IIb/IIIa complex. Furthermore, an intact GPIIb/IIIa complex was required for these reactions, since platelets incubated with EDTA at 37 °C at alkaline pH failed to aggregate and bound neither fibrinogen in response to ADP following recalcification. Approximately 50% of each fibrinogen bound irreversibly to platelets after one hour and failed to dissociate in the presence of 10 mmol/L of EDTA or excess unlabeled F(?50). The data demonstrate that heterodimeric F(?50,57.5) binds less well to platelets and supports platelet aggregation only half as well as homodimeric F(?50). These results support prior conclusions that the carboxyterminal portion of the ? chain is important in platelet-fibrinogen interactions, and suggest that the 20 amino acid, hydrophobic ? chain carboxyterminal extension of F(?50,57.5) may sterically hinder the interaction of this fibrinogen with platelet receptors.