Abstract
Glycoprotein V (GPV) is a subunit of the GPIb-IX-V receptor for von Willebrand factor and thrombin and has been shown to modulate platelet responses to the two strongest physiological agonists, thrombin and collagen. Thrombin directly cleaves GPV from the platelet surface, yielding a 69-kDa fragment GPV f1 of unknown function. We show here that a approximately 82-kDa fragment of GPV is shed from the platelet surface upon cellular activation with phorbol 12-myristate 13-acetate or the collagen-related peptide. This shedding was inhibited by the broad range metalloproteinase inhibitor GM6001, the two potent ADAM17 inhibitors GW280264X and TAPI-2, and was absent in mice lacking functional ADAM17 (ADAM17 lacking Zn-binding domain; ADAM17(DeltaZn/DeltaZn)). Furthermore, we show that recombinant ADAM17 ectodomain efficiently releases GPV from the platelet surface. GPV is known to be associated with the intracellular regulatory protein calmodulin, which has previously been shown to be involved in ADAM17-mediated shedding of l-selectin from the surface of leukocytes. As in these reports, inhibition of calmodulin led to rapid GPV shedding from the platelet surface, a process that was again blocked by GM6001 or ADAM17 inhibitors and that was absent in ADAM17(DeltaZn/DeltaZn) mice. Inhibition of outside-in signaling through GPIIb/IIIa did not significantly affect GPV shedding, excluding an essential role of this pathway for the regulation of ADAM17 activity. These results demonstrate that GPV is cleaved upon agonist-induced platelet activation and show that ADAM17 is the major enzyme mediating this process.
Highlights
Platelet adhesion and aggregation at sites of vascular injury is crucial to limit post-traumatic blood loss but may harm tissue by occluding diseased vessels
We show here that a ϳ82-kDa fragment of Glycoprotein V (GPV) is shed from the platelet surface upon cellular activation with phorbol 12-myristate 13-acetate or the collagen-related peptide
The receptor complex is associated with the regulatory cytoplasmic protein calmodulin, which is involved in the free Ca2ϩ uptake upon platelet activation [3], but its exact role in GPIb-IX-V function is unclear
Summary
Platelet adhesion and aggregation at sites of vascular injury is crucial to limit post-traumatic blood loss but may harm tissue by occluding diseased vessels. We show that agonist-induced platelet activation or inhibition of calmodulin results in metalloproteinase-dependent shedding of an 82-kDa fragment of the receptor from the platelet surface. GPIb surface levels were only slightly decreased or unchanged in PMA- or CRP-stimulated platelets, respectively, indicating that down-regulation of GPV was rather the effect of shedding than internalization (Fig. 1B).
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