Metalloprotease disintegrins are a family of membrane-anchored glycoproteins that are known to function in fertilization, myoblast fusion, neurogenesis, and ectodomain shedding of tumor necrosis factor (TNF)-␣. Here we report the analysis of the intracellular maturation and catalytic activity of the widely expressed metalloprotease disintegrin MDC9. Our results suggest that the pro-domain of MDC9 is removed by a furin-type pro-protein convertase in the secretory pathway before the protein emerges on the cell surface. The soluble metalloprotease domain of MDC9 cleaves the insulin B-chain, a generic protease substrate, providing the first evidence that MDC9 is catalytically active. Soluble MDC9 appears to have distinct specificities for cleaving candidate substrate peptides compared with the TNF-␣ convertase (TACE/ADAM17). The catalytic activity of MDC9 can be inhibited by hydroxamic acid-type metalloprotease inhibitors in the low nanomolar range, in one case with up to 50-fold selectivity for MDC9 versus TACE. Peptides mimicking the predicted cysteineswitch region of MDC9 or TACE inhibit both enzymes in the low micromolar range, providing experimental evidence for regulation of metalloprotease disintegrins via a cysteine-switch mechanism. Finally, MDC9 is shown to become phosphorylated when cells are treated with the phorbol ester phorbol 12-myristate 13-acetate, a known inducer of protein ectodomain shedding. This work implies that removal of the inhibitory pro-domain of MDC9 by a furin-type pro-protein convertase in the secretory pathway is a prerequisite for protease activity. After pro-domain removal, additional steps, such as protein kinase C-dependent phosphorylation, may be involved in regulating the catalytic activity of MDC9, which is likely to target different substrates than the related TNF-␣-convertase.Metalloprotease-disintegrin proteins (also known as MDC 1 proteins, metalloprotease/disintegrin/cysteine-rich proteins; ADAMs, a disintegrin and metalloprotease(1)) are a family of membrane-anchored glycoproteins that play a role in spermegg binding and fusion (2-10), muscle cell fusion (11), neurogenesis, and modulation of the Notch receptor signaling pathway in Drosophila melanogaster and in Caenorhabditis elegans (12-16) and processing of the pro-inflammatory cytokine TNF-␣ (17-19) (for recent reviews see Refs. 1 and 20). Metalloprotease disintegrins are usually comprised of several different protein modules as follows: an N-terminal signal sequence is followed by a pro-domain, metalloprotease domain, disintegrin domain, cysteine-rich domain, epidermal growth factor repeat, transmembrane domain, and cytoplasmic tail (see Fig. 2A). About half of the currently known metalloprotease disintegrins are predicted to be active metalloproteases due to a catalytic site consensus sequence (HEXXH) in their metalloprotease domain. The family members that lack a catalytic site are not predicted to be active metalloproteases. Both catalytically active and inactive metalloprotease disintegrins may play a role in ce...

BackgroundPeroxisome proliferator-activated receptor-γ (PPARγ) is expressed in human platelets although in the absence of genomic regulation in these cells, its functions are unclear.ObjectiveIn the present study, we aimed to demonstrate the ability of PPARγ ligands to modulate collagen-stimulated platelet function and suppress activation of the glycoprotein VI (GPVI) signaling pathway.MethodsWashed platelets were stimulated with PPARγ ligands in the presence and absence of PPARγ antagonist GW9662 and collagen-induced aggregation was measured using optical aggregometry. Calcium levels were measured by spectrofluorimetry in Fura-2AM-loaded platelets and tyrosine phosphorylation levels of receptor-proximal components of the GPVI signaling pathway were measured using immunoblot analysis. The role of PPARγ agonists in thrombus formation was assessed using an in vitro model of thrombus formation under arterial flow conditions.ResultsPPARγ ligands inhibited collagen-stimulated platelet aggregation that was accompanied by a reduction in intracellular calcium mobilization and P-selectin exposure. PPARγ ligands inhibited thrombus formation under arterial flow conditions. The incorporation of GW9662 reversed the inhibitory actions of PPARγ agonists, implicating PPARγ in the effects observed. Furthermore, PPARγ ligands were found to inhibit tyrosine phosphorylation levels of multiple components of the GPVI signaling pathway. PPARγ was found to associate with Syk and LAT after platelet activation. This association was prevented by PPARγ agonists, indicating a potential mechanism for PPARγ function in collagen-stimulated platelet activation. Conclusions: PPARγ agonists inhibit the activation of collagen-stimulation of platelet function through modulation of early GPVI signalling.

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Thrombocytopenia is a condition of multiple etiologies affecting the megakaryocyte lineage. To perturb this lineage in transgenic mice, the tsA58 mutation of the simian virus 40 large tumor antigen was targeted to megakaryocytes using the platelet factor 4 promoter. Ten of 17 transgenic lines generated exhibited low platelet levels, each line displaying a distinct, heritable level of thrombocytopenia. Within a line, the degree of the platelet reduction correlated directly with transgene zygosity. The platelet level could be further reduced by the inactivation of one copy of the endogenous retinoblastoma gene. Western blot analysis detected large tumor antigen protein in the most severely affected lines; less affected lines were below the level of detection. Platelets and megakaryocytes from thrombocytopenic mice exhibited morphological abnormalities. Mice with either normal or reduced platelet levels developed megakaryocytic malignancies with a mean age of onset of about 8 months. There was no correlation between severity of thrombocytopenia and onset of malignancy. These mice provide a dermed genetic model for thrombocytopenia, and for megakaryocytic neoplasia, and implicate the retinoblastoma protein in the process of megakaryocyte differentiation.