Tumor Necrosis Factor-α–Converting Enzyme (ADAM17) Mediates GPIbα Shedding From Platelets In Vitro and In Vivo

Abstract

Abstract-Interaction of the platelet receptor glycoprotein (GP) Ib-V-IX with von Willebrand factor exposed at a site of vascular injury is an essential step in the initiation of a hemostatic plug. Proteolytic cleavage (shedding) of the GPIb␣ subunit was first described Ͼ25 years ago, the protease mediating this event as well as its physiological function, however, have not been elucidated. We reported recently that shedding of GPIb␣ induced by platelet storage or mitochondrial injury involves a platelet-derived metalloproteinase(s). Here we show that GPIb␣ shedding in response to mitochondrial injury or physiological activation is inhibited in platelets obtained from chimeric mice, which express inactive tumor necrosis factor-␣ converting enzyme (TACE ⌬Zn/⌬Zn ) in blood cells only. Shedding was also inhibited in mouse and human platelets in the presence of 2 potent TACE inhibitors: TAP1 and TMI-1. Our data further suggest that TACE is important in the regulation of GPIb␣ expression in vivo because we observed an Ϸ90% reduction in soluble GPIb␣ (glycocalicin) in plasma of TACE ⌬Zn/⌬Zn chimeras as well as significantly increased levels of GPIb␣ on circulating platelets. In contrast, shedding of P-selectin from activated platelets was not affected by the mutation in TACE. Damaged TACE ⌬Zn/⌬Zn platelets were further characterized by a markedly improved post-transfusion recovery and hemostatic function in mice. In conclusion, our data demonstrate that TACE is expressed in platelets and that it is the key enzyme mediating shedding of GPIb␣. Key Words: platelets Ⅲ TACE Ⅲ GPIb␣, shedding C ellular activation often results in the rapid proteolysis of a wide range of transmembrane proteins such as growth factors/cytokines, growth factor/cytokine receptors, and adhesion molecules. This process, known as ectodomain shedding, regulates many cellular functions and has been implicated in pathologies such as Alzheimer's disease and cancer. 1 The majority of these shedding events are mediated by zinc-dependent proteinases, most importantly members of the matrix metalloproteinase (MMP) 2 and a disintegrin and metalloproteinase (ADAM) 3 family of proteinases. Initially identified as a sheddase for tumor necrosis factor-␣ (TNF-␣), TNF-␣-converting enzyme (TACE; ADAM17) was also shown to mediate the release of many other cell surface transmembrane proteins, including adhesion molecules such as L-selectin and vascular cell adhesion molecule 1 (VCAM-1). 4 -6 Inactivation of the metalloproteinase activity of TACE by targeted deletion of the Zn 2ϩ -binding domain in mice (TACE ⌬Zn/⌬Zn ) results in perinatal lethality, demonstrating the importance of ectodomain shedding in vivo. 4 In platelets, proteolytic cleavage has been identified as a key mechanism to regulate the surface expression of a variety of adhesion receptors, including P-selectin, CD40 ligand, and the glycoprotein (GP) V and GPIb␣ subunits of the von Willebrand factor receptor complex, GPIb-V-IX. 7-9 GPIb-V-IX plays an important role in the adhesion of circulating platelet...

Dietary α-linolenic acid increases the platelet count in ApoE−/− mice by reducing clearance

“…This is an important negative feedback mechanism by which platelets are rendered progressively less capable of adhering to vWF and of aggregating, thus avoiding excessive platelet activation. 27,28 Since we found a reduction of plasma glycocalicin following a high-ALA diet (Figure 2A), we hypothesized that GPIba cleavage by TACE following platelet stimulation would be reduced. The results were in line with our previous finding: although GPIba levels on resting platelets were not different in the 2 groups (data not shown), GPIba shedding was significantly reduced after platelet activation with both thrombin concentrations used ( Figure 2C).…”

FlnA-null megakaryocytes prematurely release large and fragile platelets that circulate poorly

“…Because ADAM17 cleaves GPIb␣ in platelets 20 and FlnA absence leads to MMP9 overexpression in human melanoma cells, 21 the expression of these 2 metalloproteinases was investigated in platelets and MKs. Expression of both ADAM17 and MMP9 was increased in FlnA-null platelets, compared with controls ( Figure 6C), but normal in FlnA-null MKs ( Figure 6D).…”

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

“…9 This activation required active Src family kinases, Syk and PI3K, and resulted in rapid tyrosine phosphorylation of Syk, indicating that engagement of GPIb␣ by the A1 domain of VWF could generate signaling events comparable with events downstream of GPVI engagement by collagen. 9 GPVI, GPIb␣, and GPV are shed on engagement of GPVI by physiologic ligands in humans [10][11][12] and in mice [13][14][15][16] in a metalloproteinase-mediated process that requires activation of Src family kinases, Syk and PI3K, and yields a soluble approximately 55-kDa ectodomain fragment of GPVI (sGPVI) in plasma and approximately 10-kDa GPVI, approximately 16-kDa GPIb␣, or approximately 5-kDa GPV cytoplasmic tail fragments that remain associated with platelets. 12 Shedding of GPVI in response to ligands is probably mediated by members of the A Disintegrin And Metalloproteinase (ADAM) family of proteases.…”