A Model of Platelet Aggregation Involving Multiple Interactions of Thrombospondin-1, Fibrinogen, and GPIIbIIIa Receptor

Abstract

Thrombospondin-1 (TSP) may, after secretion from platelet ␣ granules, participate in platelet aggregation, but its mode of action is poorly understood. We evaluated the capacity of TSP to form inter-platelet crossbridges through its interaction with fibrinogen (Fg), using either Fg-coated beads or Fg bound to the activated GPIIbIIIa integrin (GPIIbIIIa*) immobilized on beads or on activated fixed platelets (AFP), i.e. in a system free of platelet signaling and secretion mechanisms. Aggregation at physiological shear rates (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 beads dose-dependently, which could be blocked by the amino-terminal heparin-binding domain of TSP, TSP18. Soluble TSP did not bind to GPIIbIIIa*-coated 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 formation of aggregates via Fg-TSP-Fg cross-bridges, as demonstrated in a system where direct cross-bridges mediated by GPIIbIIIa*-Fg on one particle and free GPIIbIIIa* on a second particle were blocked by the RGD mimetic Ro 44 -9883. Soluble TSP increased the efficiency of Fg-mediated aggregation of AFP by 30 -110% over all shear rates and GPIIbIIIa* occupancies evaluated. Surprisingly, TSP binding to Fg already bound to its GPIIbIIIa* receptor appears to block the ability of this occupied Fg to recognize another GPIIbIIIa* 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 contribution of secreted TSP in reinforcing inter-platelet interactions in flowing blood, through direct Fg-TSP-Fg and TSP-TSP cross-bridges.

Molecular and Functional Differences Induced in Thrombospondin-1 by the Single Nucleotide Polymorphism Associated with the Risk of Premature, Familial Myocardial Infarction

“…3a). This finding supports previous data showing that fibrinogen bound to integrin ␣ IIb ␤ 3 provides a primary pathway for TSP-1 binding to platelets (33), a reflection of the high affinity of TSP-1 for fibrinogen. In the presence of fibrinogen (300 nM, ϳK d for ␣ IIb ␤ 3 ), the amount of Asn-700 TSP-1 bound to platelets increased by 8-fold, whereas the amount of bound Ser-700 TSP-1 increased by 16-fold.…”

“…3b). It has been proposed that TSP-1 forms additional bridges between aggregating platelets and, thereby, increases the extent of platelet aggregation (33). Thus, our data showing that Ser-700 TSP-1 has increased binding to fibrinogen provides a potential explanation as to why this variant supports enhanced platelet aggregation and increases thrombus burden.…”

Plasma fibronectin depletion enhances platelet aggregation and thrombus formation in mice lacking fibrinogen and von Willebrand factor

“…However, it is currently unknown whether EDA domain-positive cellular Fn 36 (a megakaryocyte synthesized form of Fn present in platelets in a small proportion; 10%-20%) is necessary for this process. It is probably that the cellular Fn and other adhesion ligands (such as thrombospondin-1, vitronectin, and CD40L or combinations thereof) 21,34,[37][38][39][40][41][42] synergistically contribute to this Fg/VWF/pFn-independent platelet aggregation.…”

Thrombospondins: from structure to therapeutics

“…The integrins α3β1 and αvβ3, CD36, and CD47 similarly mediate distinct responses to TSP1 in vascular smooth muscle cells (VSMC) [18][19][20]. In platelets, CD36 and CD47 appear to be the major direct receptors for TSP1, although TSP1 binding to fibrinogen may also allow indirect binding to the major platelet integrin αIIbβ3 [21,22].…”