Deciphering the human platelet sheddome

Abstract

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)

Calpain inhibition stabilizes the platelet proteome and reactivity in diabetes

“…It also fits well with a recent report that identified calpain in the platelet secretome. 17 We also observed that the in vitro incubation of CCL5 with calpain resulted in the modification of CCL5 and the generation of a truncated variant (CCL5 3-21) that was more chemotactic for monocytes than the full-length protein.More than 19 variants of CCL5 are known to exist, 32 and proteases such as dipeptidyl peptidase IV and cathepsin G can generate 3-68 and 4-68 variants of CCL5. 33,34 Although the clinical significance of these variants is unclear, higher plasma levels of CCL5 (3-68) have been reported in patients with type 2 diabetes.…”

“…It also fits well with a recent report that identified calpain in the platelet secretome. 17 We also observed that the in vitro incubation of CCL5 with calpain resulted in the modification of CCL5 and the generation of a truncated variant (CCL5 3-21) that was more chemotactic for monocytes than the full-length protein.…”

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

“…12 Shedding of GPVI in response to ligands is probably mediated by members of the A Disintegrin And Metalloproteinase (ADAM) family of proteases. 17,18 Whether exposure of platelets to elevated shear rates is sufficient to down-regulate GPVI expression and/or increase GPVI shedding has not yet been addressed; however, it may be of clinical relevance in thrombotic diseases where disturbed blood flow is important, for example, in aortic valve stenosis 19 or in patients on coronary bypass. 20 In this regard, elevated surface levels of GPVI on circulating platelets correlate with increased incidence of stroke and acute coronary syndrome, 21 and elevated plasma levels of sGPVI have been reported in a stable angina pectoris (SAP) cohort.…”

A Short Overview of the Components in Mass Spectrometry Instrumentation for Proteomics Analyses

“…Tryptic peptides originated from abundant proteins predominate, no matter what kind of analysis is performed in the mass spectrometer. Highly abundant proteins such as albumin in blood samples, cytoskeleton proteins in pelleted cells, among other examples, need to be removed to allow detection of less abundant ones, and require separate analyses [7]. The same situation happens in plant derived materials, in which the amount of RuBisCO in leaves and of storage proteins in seeds and seedlings mask the non abundant proteins in the samples.…”

“…Besides different strategies used to deal with this situation (exclusion lists, dynamic exclusion, affinity columns), it is still challenging and will not be focused in this chapter. For further reading, please refer to selected references cited ahead [7][8][9][10]. The last step is to combine the peptide identification results into a list of proteins that are most likely present in the sample [56,58].…”