A model of platelet aggregation involving multiple interactions of thrombospondin-1, fibrinogen, and GPIIbIIIa receptor

Thrombospondin-l (TSP) may, after secretion from platelet Lu granules, part icipate in platelet aggregation, but its mode of action is poorly understoo d. We evaluated the capacity of TSP to form inter-platelet crossbridges thr ough its interaction with fibrinogen (Fg), using either Fg-coated beads or Fg bound to the activated GPIIbIIIa integrin (GPIIbIIIa*) immobilized on be ads or on activated fixed platelets (AFP), i.e. in a system free of platele t signaling and secretion mechanisms. Aggregation at physiological shear ra tes (100-2000 s(-1)) was studied in a microcouette device and monitored by flow cytometry. Soluble TSP bound to and induced aggregation of Fg-coated b eads dose-dependently, which could be blocked by the amino-terminal heparin -binding domain of TSP, TSP18. Soluble TSP did not bind to GPIIbIIIa*- coat ed beads or AFP, unless they were preincubated with Fg. The interaction of soluble TSP with Fg-GPIIbIIIa'''-coated beads or Fg-AFP resulted in the for mation of aggregates via Fg-TSP-Fg cross-bridges, as demonstrated in a syst em where direct cross-bridges mediated by GPIIbIIIa*'-Fg on one particle an d free GPIIbIIIa* on a second particle were blocked by the RGD mimetic Ro 4 4-9885. Soluble TSP increased the efficiency of Fg-mediated aggregation of AFP by 30-110% over all shear rates and GPIIbIIIa* occupancies evaluated. S urprisingly, TSP binding to Fg already bound to its GPIIbIIIa* receptor app ears to block the ability of this occupied Fg to recognize another GPIIbIII a* receptor, but this TSP can indeed cross-bridge to another Fg molecule on a second platelet. Finally, TSP-coated beads could directly coaggregate at shear rates from 100 to 2000 s(-1). Our studies provide a model for the co ntribution of secreted TSP in reinforcing inter-platelet interactions in fl owing blood, through direct Fg-TSP-Fg and TSP-TSP cross-bridges.