Background: ADAM10 is a transmembrane metalloprotease that regulates development, inflammation, cancer, and Alzheimer disease. Results: The TspanC8 subgroup of tetraspanin membrane proteins interacts with and promotes ADAM10 maturation and cell surface localization. Conclusion: This study defines the TspanC8 tetraspanins as essential regulators of ADAM10. Significance: Focusing on specific TspanC8-ADAM10 complexes may allow ADAM10 therapeutic targeting in a cell typeand/or substrate-specific manner.

It has become increasingly appreciated that receptors coupled to G␣ i family members can stimulate platelet aggregation, but the mechanism for this has remained unclear. One possible mediator is the small GTPase, Rap1, which has been shown to contribute to integrin activation in several cell lines and to be activated by a calcium-dependent mechanism in platelets. Here, we demonstrate that Rap1 is also activated by G␣ i family members in platelets. First, we show that platelets from mice lacking the G␣ i family member G␣ z (which couples to the ␣ 2A adrenergic receptor) are deficient in epinephrine-stimulated Rap1 activation. We also show that platelets from mice lacking G␣ i2 , which couples to the ADP receptor, P2Y12, exhibit reduced Rap1 activation in response to ADP. In contrast, platelets from mice that lack G␣ q show no decrease in the ability to activate Rap1 in response to epinephrine but show a partial reduction in ADP-stimulated Rap1 activation. This result, combined with studies of human platelets treated with ADP receptor-selective inhibitors, indicates that ADPstimulated Rap1 activation in human platelets is dependent on both the G␣ i -coupled P2Y12 receptor and the G␣ q -coupled P2Y1 receptor. G␣ i -dependent activation of Rap1 in platelets does not appear to be mediated by enhanced intracellular calcium release because no increase in intracellular calcium concentration was detected in response to epinephrine and because the calcium response to ADP was not diminished in platelets from the G␣ i2 ؊/؊ mouse. Finally, using human platelets treated with selective inhibitors of phosphatidylinositol 3-kinase (PI3K) and mouse platelets selectively lacking the G␤␥-activated form of his enzyme (PI3K␥), we show that G i -mediated Rap1 activation is PI3K-dependent. In summary, activation of Rap1 can be stimulated by G␣ iand PI3K-dependent mechanisms in platelets and by G qand Ca 2؉ -dependent mechanisms, both of which may play a role in promoting platelet activation.

Heterotrimeric G proteins mediate the earliest step in cell responses to external events by linking cell surface receptors to intracellular signaling pathways. Gz is a member of the Gi family of G proteins that is prominently expressed in platelets and brain. Here, we show that deletion of the ␣ subunit of Gz in mice: (i) impairs platelet aggregation by preventing the inhibition of cAMP formation normally seen at physiologic concentrations of epinephrine, and (ii) causes the mice to be more resistant to fatal thromboembolism. Loss of Gz␣ also results in greatly exaggerated responses to cocaine, reduces the analgesic effects of morphine, and abolishes the effects of widely used antidepressant drugs that act as catecholamine reuptake inhibitors. These changes occur despite the presence of other Gi␣ family members in the same cells and are not accompanied by detectable compensatory changes in the level of expression of other G protein subunits. Therefore, these results provide insights into receptor selectivity among G proteins and a model for understanding platelet function and the effects of psychoactive drugs.H eterotrimeric guanine nucleotide binding proteins (G proteins) comprised of ␣, ␤, and ␥ subunits mediate diverse cellular responses by linking receptors on the cell surface to intracellular signaling pathways. At least 20 human genes are known to encode GTP-binding ␣ subunits. Half of these are members of the G i␣ family, including the ubiquitously expressed and nearly identical G i1␣ , G i2␣ , and G i3␣ , as well as several with restricted expression, such as G z␣ , G o␣ , and transducin. The best described effector for most G i family members is adenylyl cyclase. However, others exist as well, including cGMP phosphodiesterase, ion channels, phospholipase C, and Rap1GAP (1-5). One unresolved issue is the need for such a multiplicity of G i␣ family members, many of which are commonly expressed within the same cell. One approach to addressing this question has been the development of mice that lack individual family members. Of the three broadly expressed members of the family, mice lacking G i2␣ have the most striking phenotype with abnormalities of T cell function and thymocyte maturation, as well as an increased susceptibility to develop inflammatory bowel disease (6-8). Deletion of G i1␣ or G i3␣ , on the other hand, has not been reported to produce an obvious effect. Clearly, however, if there are functional differences among these and the other G i family members, then receptor selection among them is potentially a major determinant for cellular responses.Of the 10 known members of the G i␣ family, the sequence of G z␣ bears the least similarity to the others. G z␣ has a limited distribution in humans with prominent expression in blood platelets and selected areas of the brain. Like other members of the family, G z␣ has been shown to inhibit cAMP formation by adenylyl cyclase when over-expressed (9), but it is not known whether this is part of its role in vivo. Similarly, in over-expression systems, ...

Antiplatelet agents are proven efficacious treatments for cardiovascular and cerebrovascular diseases. However, the existing drugs are compromised by unwanted and sometimes life-threatening bleeding that limits drug usage or dosage. There is a substantial unmet medical need for an antiplatelet drug with strong efficacy and low bleeding risk. Thrombin is a potent platelet agonist that directly induces platelet activation via the G protein (heterotrimeric guanine nucleotide-binding protein)-coupled protease-activated receptors PAR1 and PAR4. A PAR1 antagonist is approved for clinical use, but its use is limited by a substantial bleeding risk. Conversely, the potential of PAR4 as an antiplatelet target has not been well characterized. Using anti-PAR4 antibodies, we demonstrated a low bleeding risk and an effective antithrombotic profile with PAR4 inhibition in guinea pigs. Subsequently, high-throughput screening and an extensive medicinal chemistry effort resulted in the discovery of BMS-986120, an orally active, selective, and reversible PAR4 antagonist. In a cynomolgus monkey arterial thrombosis model, BMS-986120 demonstrated potent and highly efficacious antithrombotic activity. BMS-986120 also exhibited a low bleeding liability and a markedly wider therapeutic window compared to the standard antiplatelet agent clopidogrel tested in the same nonhuman primate model. These preclinical findings define the biological role of PAR4 in mediating platelet aggregation. In addition, they indicate that targeting PAR4 is an attractive antiplatelet strategy with the potential to treat patients at a high risk of atherothrombosis with superior safety compared with the current standard of care.

A disintegrin and metalloprotease 10 (ADAM10) is a ubiquitously expressed transmembrane metalloprotease that cleaves the extracellular regions from its transmembrane substrates. ADAM10 is essential for embryonic development and is implicated in cancer, Alzheimer, and inflammatory diseases. The tetraspanins are a superfamily of 33 four-transmembrane proteins in mammals, of which the TspanC8 subgroup (Tspan5, 10, 14, 15, 17, and 33) promote ADAM10 intracellular trafficking and enzymatic maturation. However, the interaction between TspanC8s and ADAM10 has only been demonstrated in overexpression systems and the interaction mechanism remains undefined. To address these issues, an antibody was developed to Tspan14, which was used to show co-immunoprecipitation of Tspan14 with ADAM10 in primary human cells. Chimeric Tspan14 constructs demonstrated that the large extracellular loop of Tspan14 mediated its co-immunoprecipitation with ADAM10, and promoted ADAM10 maturation and trafficking to the cell surface. Chimeric ADAM10 constructs showed that membrane-proximal stalk, cysteine-rich, and disintegrin domains of ADAM10 mediated its co-immunoprecipitation with Tspan14 and other TspanC8s. This TspanC8-interacting region was required for ADAM10 exit from the endoplasmic reticulum. Truncated ADAM10 constructs revealed differential TspanC8 binding requirements for the stalk, cysteine-rich, and disintegrin domains. Moreover, Tspan15was the only TspanC8 to promote cleavage of the ADAM10 substrate N-cadherin, whereas Tspan14 was unique in reducing cleavage of the platelet collagen receptor GPVI. These findings suggest that ADAM10 may adopt distinct conformations in complex with different TspanC8s, which could impact on substrate selectivity. Furthermore, this study identifies regions of TspanC8s and ADAM10 for potential interaction-disrupting therapeutic targeting.

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Objective-Recently, mice made deficient in growth arrest-specific gene 6 product (Gas6) or in which Gas6 gene expression was inhibited were shown to have platelet dysfunction and to be less susceptible to thrombosis. The aim of this study was to define and characterize the relevant Gas6 receptor or receptors involved in platelet function. Methods and Results-Using RT-PCR and Western blot analysis we found that mer was the predominantly expressed subtype in mouse and human platelets, whereas axl and rse were not detected. We generated mer-deficient mice by targeted disruption of the mer receptor gene. Platelets derived from mer-deficient mice had decreased platelet aggregation in responses to low concentrations of collagen, U46619, and PAR4 thrombin receptor agonist peptide in vitro. However, the response to ADP was not different from wild-type platelets. Knockout of the mer gene protected mice from collagen/epinephrine-induced pulmonary thromoembolism and inhibited ferric chloride-induced thrombosis in vivo. Tail bleeding times, coagulation parameters, and peripheral blood cell counts in mer-deficient mice were similar to wild-type mice. Key Words: receptor tyrosine kinase Ⅲ Gas6 Ⅲ mer Ⅲ platelet Ⅲ thrombosis M er (c-mer, Nyk, Eyk) 1,2 tyrosine kinase belongs to a family of transmembrane receptors that also includes axl (Ark, Ufo, Tyro7) 3 and rse (Tyro3, Dtk, Etk, Brt, Tif). 4,5 This family of proteins has been implicated in reversible cell growth arrest, 6 survival, 7 proliferation, 7-9 and cell adhesion. 10 -12 Recently, the product of the growth arrest-specific gene 6 (Gas6) was identified as a heterophilic ligand for axl, rse, and mer, whereas protein S was identified as the ligand for the rse receptor. [13][14][15][16] Both Gas6 and protein S belong to the vitamin K-dependent protein family. Proteins in this family, which also includes prothrombin; coagulation factors VII, IX, and X; protein C; and protein Z, are characterized by posttranslational ␥-carboxylation of certain glutamic acid residues by carboxylase, using vitamin K as a cofactor. 6,17 Gas6 is structurally similar to protein S with 48% amino acid identity. Protein S is a cofactor for activated protein C that inactivates the coagulation factors Va and VIIIa 18 and is thus involved in the anticoagulation cascade. Genetic deficiency of protein S in humans is one of the most severe inherited risk factors for thrombosis. 19 Gas6, originally isolated as a growth arrest-specific gene from quiescent fibroblasts, 6 has recently been shown to participate in the regulation of platelet activation and aggregation. 20 Gas6 amplified platelet aggregation and secretion in response to known platelet agonists including ADP, collagen, and the thromboxane A2 analogue U46619. 20 Inactivation of the Gas6 gene prevented venous and arterial thrombosis in mice, and protected against fatal collagen/epinephrine-induced thromboembolism. 20 In addition, Gas6 antibodies inhibited platelet aggregation in vitro and protected mice against fatal thromboembolism in vivo. 20 Con...