title: Deubiquitinases Modulate Platelet Proteome Ubiquitination, Aggregation, and Thrombosis.
abstract: OBJECTIVE
  Platelets express a functional ubiquitin-proteasome system. Mass spectrometry shows that platelets contain several deubiquitinases, but whether these are functional, modulate the proteome, or affect platelet reactivity are unknown.
  
  
  RESULTS
  Platelet lysates contained ubiquitin-protein deubiquitinase activity hydrolyzing both Lys48 and Lys63 polyubiquitin conjugates that was suppressed by the chemically unrelated deubiquitinase inhibitors PYR41 and PR619. These inhibitors acutely and markedly increased monoubiquitination and polyubiquitination of the proteome of resting platelets. PYR41 (intravenous, 15 minutes) significantly impaired occlusive thrombosis in FeCl3-damaged carotid arteries, and deubiquitinase inhibition reduced platelet adhesion and retention during high shear flow of whole blood through microfluidic chambers coated with collagen. Total internal reflection microscopy showed that adhesion and spreading in the absence of flow were strongly curtailed by these inhibitors with failure of stable process extension and reduced the retraction of formed clots. Deubiquitinase inhibition also sharply reduced homotypic platelet aggregation in response to not only the incomplete agonists ADP and collagen acting through glycoprotein VI but also to the complete agonist thrombin. Suppressed aggregation was accompanied by curtailed procaspase activating compound-1 binding to activated IIb/IIIa and inhibition of P-selectin translocation to the platelet surface. Deubiquitinase inhibition abolished the agonist-induced spike in intracellular calcium, suppressed Akt phosphorylation, and reduced agonist-stimulated phosphatase and tensin homolog phosphatase phosphorylation. Platelets express the proteasome-associated deubiquitinases USP14 and UCHL5, and selective inhibition of these enzymes by b-AP15 reproduced the inhibitory effect of the general deubiquitinase inhibitors on ex vivo platelet function.
  
  
  CONCLUSIONS
  Remodeling of the ubiquitinated platelet proteome by deubiquitinases promotes agonist-stimulated intracellular signal transduction and platelet responsiveness.
authors.affiliation: From the Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland, OH; and Department of Molecular Medicine, Case Western Reserve University, Cleveland, OH.
authors.forename: Nilaksh
authors.initials: N
authors.lastname: Gupta
authors.affiliation: From the Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland, OH; and Department of Molecular Medicine, Case Western Reserve University, Cleveland, OH.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.affiliation: From the Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland, OH; and Department of Molecular Medicine, Case Western Reserve University, Cleveland, OH. mcintyt@ccf.org.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: McIntyre
mesh_terms: D000631:Aminopyridines / Q000494:pharmacology; D000818:Animals; D001565:Benzoates / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D002712:Chlorides; D004195:Disease Models, Animal; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005290:Ferric Compounds; D005663:Furans; D006801:Humans; D008810:Mice, Inbred C57BL; D046210:Microfluidic Analytical Techniques; D008857:Microscopy, Interference; D010881:Piperidones / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects; D010979:Platelet Function Tests; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000097:blood*; D011720:Pyrazoles / Q000494:pharmacology; D038981:Receptors, Collagen / Q000097:blood; D018179:Receptors, Thrombin / Q000097:blood; D015398:Signal Transduction; D013861:Thiocyanates / Q000494:pharmacology; D013927:Thrombosis / Q000097:blood / Q000139:chemically induced / Q000201:enzymology* / Q000517:prevention & control; D043222:Ubiquitin Thiolesterase / Q000037:antagonists & inhibitors / Q000097:blood; D064570:Ubiquitin-Specific Proteases / Q000037:antagonists & inhibitors / Q000097:blood*; D054875:Ubiquitination
chemical_list: C570894:2,6-diaminopyridine-3,5-bis(thiocyanate); C569633:3,5-bis((4-nitrophenyl)methylidene)-1-prop-2-enoylpiperidin-4-one; C523934:4(4-(5-nitro-furan-2-ylmethylene)-3,5-dioxo-pyrazolidin-1-yl)-benzoic acid ethyl ester; D000631:Aminopyridines; D001565:Benzoates; D002712:Chlorides; D004791:Enzyme Inhibitors; D005290:Ferric Compounds; D005663:Furans; D010881:Piperidones; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011720:Pyrazoles; D038981:Receptors, Collagen; D018179:Receptors, Thrombin; D013861:Thiocyanates; C509299:USP14 protein, human; C421245:UCHL5 protein, human; D043222:Ubiquitin Thiolesterase; D064570:Ubiquitin-Specific Proteases; D046988:Proteasome Endopeptidase Complex; C024555:ferric chloride
keywords: blood platelets; proteasome endopeptidase complex; thrombosis; ubiquitin; ubiquitin-specific protease