The PAR-1-Stimulated Platelet Subpopulation That Binds Factor Xa Also Expresses Matrixmetalloproteinase and Calpain Activities Resulting in Population-Specific GP1bα Shedding and Platelet Vesiculation.

We previously showed that only a subpopulation of platelets responds to stimulation by strong agonists with exposure of coagulation factor binding sites. The subpopulation of SFLLRN-amide or collagen-stimulated platelets that bound coagulation factor Xa showed 50% decreased surface glycoprotein 1bα (GP1bα) and 60% reduced forward scatter relative to unstimulated platelets. While collagen-stimulated Xa-negative platelets lost no GP1b/IX/V surface density, the PAR-1-stimulated Xa-negative population lost 25% of both GP1bα and GPIX surface density signifying decreased GP1b/IX/V complex following PAR-1 stimulation. The PAR-1-stimulated Xa-positive platelets showed a discrepancy between the surface densities of GPIX and GP1bα that suggested a procoagulant subpopulation-specific shedding of GP1bα. While Xa-positive PAR-1-stimulated platelets pretreated with 10μM GM6001, a nonspecific inhibitor of matrixmetalloproteinases, recovered 30% of the lost surface GP1bα, bringing the GP1bα /GPIX ratio to ~1, collagen-stimulated loss of GP1bα on Xa-positive platelets was little affected by GM6001 pretreatment, and GP1bα on Xa-negative platelets was unaffected by GM6001 pretreatment. While inhibition of calpains by pretreatment of platelets with 100μM calpeptin had little effect on agonist-stimulated GP1bα shedding, calpeptin pretreatment resulted in 50% decreased loss of GP1bα/GPIX from PAR-1-stimulated Xa-negative platelets, in a 35–45% loss of the Xa-positive subpopulation resulting from PAR-1 stimulation, in 25% increased forward scatter from all agonist-induced Xa-positive populations, and in a 40–50% decrease in positive events occurring outside the defined platelet region. On the contrary, GM6001 inhibition of GP1bα shedding had no effect on either the size of the Xa-positive subpopulation, on the density of binding sites or on forward scatter. Platelet vesiculation would produce smaller platelets with decreased forward scatter, and microparticles. Calpain, previously implicated in platelet vesiculation, appears to mediate specific vesiculation of Xa-positive platelets. In summary, PAR-1 agonists and collagen recruit a platelet subpopulation to undergo specific membrane changes exposing binding sites for coagulation enzymes factors IXa and Xa. This procoagulant subpopulation recruited by PAR-1 signaling is also notable for specific matrixmetalloprotease activities leading to procoagulant population-specific GP1bα shedding. Calpain activity mediates membrane vesiculation of procoagulant platelets resulting from both collagen- and PAR-1-stimulation leading to smaller procoagulant platelets and to platelet microparticles.