Thrombospondin-1 stimulates platelet aggregation by blocking the antithrombotic activity of nitric oxide/cGMP signaling

Abstract

Platelet alpha-granules constitute the major rapidly releasable reservoir of thrombospondin-1 in higher animals. Although some fragments and peptides derived from thrombospondin-1 stimulate or inhibit platelet aggregation, its physiologic function in platelets has remained elusive. We now show that endogenous thrombospondin-1 is necessary for platelet aggregation in vitro in the presence of physiologic levels of nitric oxide (NO). Exogenous NO or elevation of cGMP delays thrombin-induced platelet aggregation under high shear and static conditions, and exogenous thrombospondin-1 reverses this delay. Thrombospondin-1-null murine platelets fail to aggregate in response to thrombin in the presence of exogenous NO or 8Br-cGMP. At physiologic concentrations of the NO synthase substrate arginine, thrombospondin-1-null platelets have elevated basal cGMP. Ligation of CD36 or CD47 is sufficient to block NO-induced cGMP accumulation and mimic the effect of thrombospondin-1 on aggregation. Exogenous thrombospondin-1 also reverses the suppression by NO of alphaIIb/beta3 integrin-mediated platelet adhesion on immobilized fibrinogen, mediated in part by increased GTP loading of Rap1. Thrombospondin-1 also inhibits cGMP-mediated activation of cGMP-dependent protein kinase and thereby prevents phosphorylation of VASP. Thus, release of thrombospondin-1 from alpha-granules during activation provides positive feedback to promote efficient platelet aggregation and adhesion by overcoming the antithrombotic activity of physiologic NO.

Thrombospondin-1 Inhibits VEGF Receptor-2 Signaling by Disrupting Its Association with CD47

“…At nanomolar TSP1 concentrations, this inhibition can be mediated by CD36 (14), but at physiological circulating plasma TSP1 concentrations (100 -200 pM), CD47 is the necessary TSP1 receptor for inhibiting activation of soluble guanylate cyclase (20) or cGMP-dependent protein kinase (21). CD47-null and TSP1-null mice show similarly enhanced survival of full thickness skin grafts and increased angiogenic responses in the wound bed (22).…”

Regulation of Cellular Redox Signaling by Matricellular Proteins in Vascular Biology, Immunology, and Cancer

“…The NOinsensitive state of sGC persisted in cell-free lysates and was prevented by the protein kinase inhibitor staurosporine, which suggests that TSP1/CD47 signaling induces an inhibitory phosphorylation of sGC. Inhibition of sGC by TSP1 was first reported in endothelial cells, but subsequent studies have confirmed this result in VSMCs (96), platelets (95,162), T cells (202), and macrophages (206) of human and nonhuman origin, broadly implicating TSP1 as limiter of sGC activity.…”

“…-/-and Cd47 -/-platelets showed clear defects in thrombin-mediated activation (95). Correspondingly, cGMP levels were elevated in Thbs1 -/-platelets but restored to normal levels by addition of exogenous TSP1 or a CD47-binding peptide.…”

“…TSP1 also inhibited endothelial cell adhesion stimulated by 8-Br-cGMP, indicating that TSP1 has a second inhibitory target downstream of sGC (93). TSP1 limited the antithrombotic activity of NO and the cell-permeable cyclic analogue 8-Br-cGMP in WT platelets (95) (Fig. 5).…”

Cyclic nucleotide‐dependent inhibitory signaling interweaves with activating pathways to determine platelet responses

“…Interestingly, low NO concentrations have been seen to slightly potentiate thrombin‐induced Ca 2+ ‐elevations thus potentially contributing to platelet activation 61, 62. Platelet activators like thrombospondin‐1 (TSP1) have been shown to cross‐inhibit sGC through some unknown mechanism 63. Further studies are required to clarify interactions between different receptors, NO, sGC, cGMP, and Ca 2+ keeping in mind the methodological issues that might affect experiments on NO and cGMP pathways 64, 65…”