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Molecular and Cellular Biology, March 2001, p. 2213-2220, Vol. 21, No. 6
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.6.2213-2220.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Disruption of the Mouse µ-Calpain Gene Reveals an Essential Role in Platelet Function

Mohammad Azam,1 Shaida S. Andrabi,1 Kenneth E. Sahr,1 Lakshmi Kamath,2 Athan Kuliopulos,2 and Athar H. Chishti1,*

Section of Hematology-Oncology Research, Departments of Medicine, Anatomy, and Cellular Biology, St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02135,1 and Division of Hematology-Oncology, New England Medical Center, Departments of Medicine and Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 021112

Received 15 September 2000/Returned for modification 8 November 2000/Accepted 27 December 2000

Conventional calpains are ubiquitous calcium-regulated cysteine proteases that have been implicated in cytoskeletal organization, cell proliferation, apoptosis, cell motility, and hemostasis. There are two forms of conventional calpains: the µ-calpain, or calpain I, which requires micromolar calcium for half-maximal activation, and the m-calpain, or calpain II, which functions at millimolar calcium concentrations. We evaluated the functional role of the 80-kDa catalytic subunit of µ-calpain by genetic inactivation using homologous recombination in embryonic stem cells. The µ-calpain-deficient mice are viable and fertile. The complete deficiency of µ-calpain causes significant reduction in platelet aggregation and clot retraction but surprisingly the mutant mice display normal bleeding times. No detectable differences were observed in the cleavage pattern and kinetics of calpain substrates such as the beta 3 subunit of alpha IIbbeta 3 integrin, talin, and ABP-280 (filamin). However, µ-calpain null platelets exhibit impaired tyrosine phosphorylation of several proteins including the beta 3 subunit of alpha IIbbeta 3 integrin, correlating with the agonist-induced reduction in platelet aggregation. These results provide the first direct evidence that µ-calpain is essential for normal platelet function, not by affecting the cleavage of cytoskeletal proteins but by potentially regulating the state of tyrosine phosphorylation of the platelet proteins.

* Corresponding author. Mailing address: Biomedical Research, CBR 404, St. Elizabeth's Medical Center, 736 Cambridge St., Boston, MA 02135. Phone: (617) 789-3118. Fax: (617) 789-3111. E-mail: Athar_Chishti{at}cchcs.org.

Molecular and Cellular Biology, March 2001, p. 2213-2220, Vol. 21, No. 6
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.6.2213-2220.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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