Differentially regulated GPVI ectodomain shedding by multiple platelet–expressed proteinases

Abstract

Glycoprotein VI (GPVI) mediates platelet activation on exposed subendothelial collagens at sites of vascular injury and thereby contributes to normal hemostasis, but also to the occlusion of diseased vessels in the setting of myocardial infarction or stroke. GPVI is an attractive target for antithrombotic therapy, particularly because previous studies have shown that anti-GPVI antibodies induce irreversible down-regulation of the receptor in circulating platelets by internalization and/or ectodomain shedding. Metalloproteinases of the a disintegrin and metalloproteinase (ADAM) family have been proposed to mediate this ectodomain shedding, but direct evidence for this is lacking. Here, we studied GPVI shedding in vitro and in vivo in newly generated mice with a megakaryocyte-specific ADAM10 deficiency and in Adam17 ex/ex mice, which lack functional ADAM17. We demonstrate that GPVI cleavage in vitro can occur independently through either ADAM10 or ADAM17 in response to distinct stimuli. In contrast, antibody (JAQ1)-induced GPVI shedding in vivo occurred in mice lacking both ADAM10/ADAM17 in their platelets, suggesting the existence of a third GPVI cleaving platelet enzyme. This was supported by in vitro studies on ADAM10/ADAM17 double-deficient platelets. These results reveal that ectodomain shedding of GPVI can be mediated through multiple differentially regulated platelet-expressed proteinases with obvious therapeutic implications. (Blood. 2010;116(17):3347-3355) IntroductionAt sites of vascular injury, the subendothelial extracellular matrix (ECM) becomes exposed to the flowing blood and triggers platelet adhesion and aggregation. Under high shear flow conditions, the initial contact is mediated by the interaction between collagenbound von Willebrand factor (VWF) and the glycoprotein (GP)Ib-V-IX complex on the platelet surface. This interaction is transient and reduces the velocity of the cells and thereby enables interactions between the low-affinity platelet collagen receptor, glycoprotein VI (GPVI) and collagen, leading to platelet activation and subsequent firm adhesion and aggregation. 1 GPVI is a type I transmembrane protein of approximately 68 kDa that is noncovalently associated with the FcR␥-chain, 2-4 which represents the signaling subunit of the receptor complex. 3,5 Ligand binding of GPVI results in phosphorylation of the immunoreceptor tyrosinebased activation motif (ITAM) of the FcR␥-chain, followed by a cascade of tyrosine phosphorylation steps involving the adapter linker of activated T cells (LAT) and leading to the activation of various effector molecules, including phospholipase (PL)C␥2. 6 GPVI-deficient patients 7-9 and mice 3,10,11 display defective platelet responses to collagen, but only a moderate bleeding tendency, making GPVI a promising antithrombotic target. We have previously shown that in vivo administration of monoclonal anti-GPVI antibodies (JAQ1, 2, 3) leads to down-regulation of the GPVI receptor in circulating murine platelets 12,13 and long-term protection in models of arteri...

Megakaryocyte-specific deletion of the protein-tyrosine phosphatases Shp1 and Shp2 causes abnormal megakaryocyte development, platelet production, and function

“…ADAM10 is an established sheddase of GPVI and GPIba in platelets. 38 Collectively, these findings demonstrate differences in platelet counts, volumes, and surface glycoprotein expression in MP-Shp1 and MP-Shp2 KO mice that were severely exacerbated in DKO mice, suggesting Shp1 and Shp2 regulate platelet production.…”

The TspanC8 Subgroup of Tetraspanins Interacts with A Disintegrin and Metalloprotease 10 (ADAM10) and Regulates Its Maturation and Cell Surface Expression

“…The metalloprotease ADAM10 cleaves over 40 transmembrane targets, including the platelet-activating collagen receptor glycoprotein VI (7,8). Because ADAM10 is compartmentalized into tetraspanin membranes on various cell types (23,24), including platelets (Fig.…”

“…Indeed, transgenic overexpression of ADAM10 prevents amyloid plaque deposition in a mouse model of Alzheimer disease, suggesting that ADAM10 is a promising target for disease treatment in humans (6). ADAM10 targets in the vasculature include the platelet-activating collagen receptor glycoprotein VI (7,8) and endothelial proteins with roles in angiogenesis and inflammation, such as vascular endothelial growth factor receptor 2 (9), the junctional adhesion molecule VE-cadherin (10), and transmembrane chemokines CX3CL1 and CXCL16 (11). Despite the importance of ADAM10, the regulation of its trafficking, activation, and localization to targets is poorly understood.…”

Sandwich Immunoassay for Soluble Glycoprotein VI in Patients with Symptomatic Coronary Artery Disease

“…On the other hand, platelet activation induces matrix metalloproteinase-dependent pGPVI cleavage, which is followed by an ectodomain shedding of sGPVI (21,22 ). This shedding is regulated by at least 3 different types of plateletexpressed proteinases/sheddases (23 ). Because pGPVI is the key to both types of responses, platelet aggregation and phosphatidylserine expression, loss of pGPVImediated phosphatidylserine exposure may result in reduced prothrombinase activity (24 ).…”