Evidence for a Role of ADAM17 (TACE) in the Regulation of Platelet Glycoprotein V

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 ϳ82-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 ⌬Zn/⌬Zn ). 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 ⌬Zn/⌬Zn 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.Platelet adhesion and aggregation at sites of vascular injury is crucial to limit post-traumatic blood loss but may also harm tissue by occluding diseased vessels. The membrane glycoprotein (GP) 1 Ib-IX-V complex plays a central role in these events in that it mediates the initial platelet tethering to the damaged vessel wall under conditions of elevated shear by interacting with collagen-bound von Willebrand factor (1). The receptor complex consists of four distinct polypeptides, GPIb␣ (M r ϭ 143,000), Ib ␤ (M r ϭ 22,000), V (M r ϭ 83,000), and IX (M r ϭ 20,000), in 1:1:0.5:1 stoichiometry with ϳ25,000 copies per platelet. GPs Ib, V, and IX are structurally related and belong to the family of leucine-rich glycoproteins (2). The receptor complex is associated with the regulatory cytoplasmic protein calmodulin, which is involved in the free Ca 2ϩ uptake upon platelet activation (3), but its exact role in GPIb-IX-V function is unclear.The importance of the GPIb-IX-V complex is emphasized by the study of the Bernard-Soulier syndrome, an inherited bleeding disorder in which the complex is congenitally missing or dysfunctional because of mutations in the genes encoding GPIb or GPIX (2). Likewise, targeted deletion of the GPIb␣ (4) or GPIb␤ (5) genes in mice reproduces the Bernard-Soulier phenotype, as characterized by thrombocytopenia, giant platelets, and massively prolonged bleeding times. Although the essential role of GPIb-IX for normal platelet funct...

Regulated surface expression and shedding support a dual role for semaphorin 4D in platelet responses to vascular injury

“…Immunoblots with an antibody against the ADAM17 catalytic domain show the zymogen (or immature) form of ADAM17 is present in both resting and activated platelets (Fig. 3C), consistent with an earlier report (22). A band corresponding to the mature (active) form of ADAM17 is also detectable but was less abundant than the immature form.…”

“…Recent reports have shown that the metalloprotease ADAM17, or TACE, is required for cleavage of two platelet membrane proteins, GP Ib␣ (21) and GP V (22). ADAM17 is a Zn-dependent protease that is synthesized as an inactive precursor.…”

Improved platelet survival after cold storage by prevention of glycoprotein Ib clustering in lipid rafts

“…Shedding of GPV is regulated by ADAM17 and inhibited by the broad-spectrum matrix metalloproteinase inhibitor GM6001. 22 As expected, the inhibitor prevented GPV loss during cold storage (Online Supplementary Figure S3C), but had no inhibitory effect on [GPIbα-GM1] and [GPIbα-GPIbα] associations (Online Supplementary Figure S3D).…”

Pathologic shear triggers shedding of vascular receptors: a novel mechanism for down-regulation of platelet glycoprotein VI in stenosed coronary vessels

“…10,12,32,33 As platelets are sensitive to changes in shear forces, we determined whether shear-mediated platelet activation could also activate GPVI shedding in vitro. Citrated PRP from 19 healthy persons were either untreated or subjected to 10 000-second Ϫ1 shear rate for 5 minutes PRP from a single donor was subjected to a shear rate of 7500 seconds Ϫ1 for up to 10 minutes in the presence of an inhibitor of ␣IIb␤3, or treated with 5mM NEM for 15 minutes.…”