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A novel mechanism regulating human platelet activation by MMP-2–mediated PAR1 biased signaling

Manuela Sebastiano, Stefania Momi, Emanuela Falcinelli, Loredana Bury, Marc F. Hoylaerts and Paolo Gresele

Key Points

  • Active MMP-2 enhances platelet activation by cleaving PAR1 at an extracellular site different from the thrombin cleavage site.

  • The novel PAR1-tethered ligand exposed by MMP-2 selectively stimulates PAR1-dependent Gq and G12/13 pathway activation.

Abstract

Platelets contain and release several matrix metalloproteinases (MMPs). Among these, active MMP-2 enhances platelet aggregation by favoring the activation of phosphatidylinositol 3- kinase (PI3K) and contributes to arterial thrombosis. The platelet surface target of MMP-2 and the mechanism through which it primes platelets to respond to subsequent stimuli are still unknown. We show that active MMP-2 enhances platelet activation induced by weak stimuli by cleaving PAR1 at a noncanonical extracellular site different from the thrombin-cleavage site and thus initiates biased receptor signaling, triggering only some of the signaling pathways normally activated by full PAR1 agonism. The novel PAR1-tethered ligand exposed by MMP-2 stimulates PAR1-dependent Gq and G12/13 pathway activation, triggering p38-MAPK phosphorylation, Ca+2 fluxes, and PI3K activation, but not Gi signaling; this is insufficient to cause platelet aggregation, but it is enough to predispose platelets to fully respond to Gi-activating stimuli. Integrin αIIbβ3 is a necessary cofactor for PAR1 cleavage by MMP-2 by binding the MMP-2 hemopexin domain, thus favoring the interaction of the enzyme with PAR1. Our studies unravel a novel mechanism regulating platelet activation that involves the binding of MMP-2 to integrin αIIbβ3 and the subsequent cleavage of PAR1 by active MMP-2 at a noncanonical site, exposing a previously undescribed tethered ligand that triggers biased G-protein agonism and thus predisposes platelets to full activation by other stimuli. These results identify the MMP-2-αIIbβ3-PAR1 interaction as a potential target for the prevention of arterial thrombosis.

  • Submitted June 27, 2016.
  • Accepted December 14, 2016.
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