Targeted gene inactivation reveals a functional role of calpain-1 in platelet spreading

Authors

  • S. M. KUCHAY,

    1. Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL
    2. Howard Hughes Medical Institute, New York University School of Medicine, New York, NY
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  • A. J. WIESCHHAUS,

    1. Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL
    2. Department of Molecular Physiology and Pharmacology, Sackler School Programs in Physiology, Pharmacology, and Microbiology, Tufts University School of Medicine, Boston, MA, USA
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  • M. MARINKOVIC,

    1. Department of Molecular Physiology and Pharmacology, Sackler School Programs in Physiology, Pharmacology, and Microbiology, Tufts University School of Medicine, Boston, MA, USA
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  • I. M. HERMAN,

    1. Department of Molecular Physiology and Pharmacology, Sackler School Programs in Physiology, Pharmacology, and Microbiology, Tufts University School of Medicine, Boston, MA, USA
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  • A. H. CHISHTI

    1. Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL
    2. Department of Molecular Physiology and Pharmacology, Sackler School Programs in Physiology, Pharmacology, and Microbiology, Tufts University School of Medicine, Boston, MA, USA
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  • 1These authors contributed equally to this work.

Athar Chishti, Department of Molecular Physiology and Pharmacology, Tufts University School of Medicine, 136 Harrison Avenue, Room 701A, Boston, MA 02111, USA.
Tel.: +1 312 355 1293; fax: +1 312 355 1297.
E-mail: athar.chishti@tufts.edu
Shafi Kuchay, Howard Hughes Medical Institute, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA.
Tel.: +1 212 263 9267; fax: +1 212 263 5107.
E-mail: shafi.kuchay@nyumc.org

Abstract

Summary.  Background:  Calpains are implicated in a wide range of cellular functions including the maintenance of hemostasis via the regulation of cytoskeletal modifications in platelets.

Objectives:  Determine the functional role of calpain isoforms in platelet spreading.

Methods and 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.

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