title: Targeted gene inactivation reveals a functional role of calpain-1 in platelet spreading.
abstract: BACKGROUND
  Calpains are implicated in a wide range of cellular functions including the maintenance of hemostasis via the regulation of cytoskeletal modifications in platelets.
  
  
  OBJECTIVE
  Determine the functional role of calpain isoforms in platelet spreading.
  
  
  RESULTS
  Platelets from calpain-1(-/-) mice show enhanced spreading on collagen- and fibrinogen-coated surfaces as revealed by immunofluorescence, differential interference contrast (DIC) and scanning electron microscopy. The treatment of mouse platelets with MDL, a cell permeable inhibitor of calpains 1/2, resulted in increased spreading. The PTP1B-mediated enhanced tyrosine dephosphorylation in calpain-1(-/-) platelets did not fully account for the enhanced spreading as platelets from the double knockout mice lacking calpain-1 and PTP1B showed only a partial rescue of the spreading phenotype. In non-adherent platelets, proteolysis and GTPase activity of RhoA and Rac1 were indistinguishable between the wild-type (WT) and calpain-1(-/-) platelets. In contrast, the ECM-adherent calpain-1(-/-) platelets showed higher Rac1 activity at the beginning of spreading, whereas RhoA was more active at later time points. The ECM-adherent calpain-1(-/-) platelets showed an elevated level of RhoA protein but not Rac1 and Cdc42. Proteolysis of recombinant RhoA, but not Rac1 and Cdc42, indicates that RhoA is a calpain-1 substrate in vitro.
  
  
  CONCLUSIONS
  Potentiation of the platelet spreading phenotype in calpain-1(-/-) mice suggests a novel role of calpain-1 in hemostasis, and may explain the normal bleeding time observed in the calpain-1(-/-) mice.
authors.affiliation: Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL, USA.
authors.forename: S M
authors.initials: SM
authors.lastname: Kuchay
authors.forename: A J
authors.initials: AJ
authors.lastname: Wieschhaus
authors.forename: M
authors.initials: M
authors.lastname: Marinkovic
authors.forename: I M
authors.initials: IM
authors.lastname: Herman
authors.forename: A H
authors.initials: AH
authors.lastname: Chishti
mesh_terms: D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D002154:Calpain / Q000037:antagonists & inhibitors / Q000172:deficiency* / Q000235:genetics; D002448:Cell Adhesion; D048430:Cell Shape / Q000187:drug effects; D003094:Collagen / Q000378:metabolism; D004789:Enzyme Activation; D005340:Fibrinogen / Q000378:metabolism; D020868:Gene Silencing; D005838:Genotype; D006487:Hemostasis; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D008857:Microscopy, Interference; D010641:Phenotype; D010766:Phosphorylation; D011480:Protease Inhibitors / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1 / Q000172:deficiency / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors; D014443:Tyrosine; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D020742:rhoA GTP-Binding Protein
chemical_list: D011480:Protease Inhibitors; D047428:Protein Kinase Inhibitors; C107953:RAC1 protein, human; D011994:Recombinant Proteins; D014443:Tyrosine; D005340:Fibrinogen; D003094:Collagen; D054460:rho-Associated Kinases; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; C516609:Ptpn1 protein, mouse; D002154:Calpain; C112682:Capn1 protein, mouse; C526380:RhoA protein, mouse; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein