Activated platelets shed surface proteins, potentially modifying platelet function as well as providing a source of bioactive fragments. Previous studies have identified several constituents of the platelet sheddome, but the full extent of shedding is unknown. Here we have taken a global approach, analyzing protein fragments in the supernate of activated platelets using mass spectroscopy and looking for proteins originating from platelet membranes. After removing plasma proteins and microparticles, 1048 proteins were identified, including 69 membrane proteins. Nearly all of the membrane proteins had been detected previously, but only 10 had been shown to be shed in platelets. The remaining 59 are candidates subject to confirmation. Based on spectral counts, protein representation in the sheddome varies considerably. As proof of principle, we validated one of the less frequently detected proteins, semaphorin 7A, which had not previously been identified in platelets. Surface expression, cleavage, and shedding of semaphorin 7A were demonstrated, as was its association with ␣-granules. Finally, cleavage of semaphorin 7A and 12 other proteins was substantially reduced by an inhibitor of ADAM17, a known sheddase. These results define a subset of membrane proteins as sheddome candidates, forming the basis for further studies examining the impact of ectodomain shedding on platelet function. (Blood. 2011;117(1): e15-e26)

Objective-In dyslipidemic states, platelets become hyperreactive, secreting molecules that promote atherosclerosis. We have shown that the semaphorin family member, sema4D (CD100), is expressed on the surface of platelets and proposed that its role includes promoting thrombus growth by binding to nearby platelets and endothelial cells, both of which express sema4D receptors. Here we tested the hypothesis that deleting sema4D will attenuate the adverse consequences of dyslipidemia on platelets and the vessel wall. Methods and Results-Platelet function and atherosclerotic lesion formation were measured in LDLR(Ϫ/Ϫ) and sema4D(Ϫ/Ϫ)LDLR(Ϫ/Ϫ) mice after 6 months on a high-fat diet. All of the mice developed the dyslipidemia expected on this diet in the absence of functional LDL receptors. However, when compared to LDLR(Ϫ/Ϫ) mice, sema4D(Ϫ/Ϫ) LDLR(Ϫ/Ϫ) mice had reduced lipid deposition in the descending aorta, a 6-fold decrease in the frequency of arterial occlusion and a reduction to near wild-type levels in the accumulation of platelets after injury. These differences were retained ex vivo, with a marked decrease in platelet accumulation on collagen under flow and in platelet aggregation. Conclusions-These results show that loss of sema4D expression reduces the platelet hyperactivity otherwise found in dyslipidemia, and confers protection against the development of atherosclerosis. (Arterioscler Thromb Vasc Biol.

We recently reported that Semaphorin 4D (Sema4D) and its receptors are expressed on the platelet surface and showed that Sema4D (