Inhibition of Calpain Blocks Platelet Secretion, Aggregation, and Spreading<sup><a href="#FN1" rid="FN1" class=" fn">*</a></sup>

K Croce

R Flaumenhaft

M Rivers

B Furie

I Herman

D Potter

^{Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111}

^{Center for Hemostasis and Thrombosis Research, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215}

^{Howard Hughes Medical Institute, Tufts University School of Medicine, Boston, Massachusetts 02111}

^{Division of Hematology/Oncology and Tupper Research Institute, Department of Medicine, New England Medical Center, Boston, Massachusetts 02111}

^{Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111}

^{To whom all correspondence should be addressed: Dept. of Medicine, New England Medical Center 245, 750 Washington St., Boston, MA 02111. Tel.: 617-636-8499; Fax: 617-636-5649; E-mail:}ude.stfut.lapo@chm_rettopd

Abstract

Previous studies have indicated that the Ca^{-dependent protease, calpain, is activated in platelets within 30–60 s of thrombin stimulation, but specific roles of calpain in platelets remain to be identified. To directly test the functions of calpain during platelet activation, a novel strategy was developed for introducing calpain’s specific biological inhibitor, calpastatin, into platelets prior to activation. This method involves treatment of platelets with a fusion peptide, calpastat, consisting of the cell-penetrating signal sequence from Kaposi’s fibroblast growth factor connected to a calpain-inhibiting consensus sequence derived from calpastatin. Calpastat specifically inhibits thrombin peptide (SFLLR)-induced α-granule secretion (IC}₅₀ = 20 µm) during the first 30 s of activation, thrombin-induced platelet aggregation (IC₅₀ = 50 µm), and platelet spreading on glass surfaces (IC₅₀ = 34 µm). Calpastat-Ala, a mutant peptide in which alanine is substituted at conserved calpastatin residues, lacks calpain inhibitory activity and fails to inhibit secretion, aggregation, or spreading. The peptidyl calpain inhibitors calpeptin, MDL 28,170 (MDL) and E64d also inhibit secretion, aggregation and spreading, but require 3–10-fold higher concentrations than calpastat for biological activity. Together, these findings demonstrate that calpain regulates platelet secretion, aggregation, and spreading and indicate that calpain plays an earlier role in platelet activation following thrombin receptor stimulation than had been previously detected.

Abstract

Reorganization of the actin cytoskeleton is required for a number of platelet activation processes, including shape change, aggregation and spreading. In rapid response to platelet agonists, intracellular Ca^{transients regulate the early platelet activation events of shape change (}1, 2) and α-granule secretion (3–5), occurring within seconds of platelet stimulation, as well as aggregation (3, 6) and spreading, occurring within minutes. Since the discovery of the Ca^{-activated protease, calpain, in platelets (}7), there has been considerable interest in determining whether the Ca^{transients occurring early in platelet activation function, in part, through calpain-mediated cytoskeletal remodeling. Calpain has been found to modulate the late event of platelet-mediated fibrin clot retraction (}8–10). We have recently found that calpain regulates actin remodeling events of endothelial wound healing and fibroblast spreading, including lamellipodial and filopodial protrusion formation (11, 12). Calpain has also been implicated in cell motility through its effects on release of rear integrin contacts (13). Calpain translocates to the plasma membrane within seconds of platelet activation, where it is activated and may cleave cytoskeletal proteins (14–16). These findings together suggest a regulatory role for calpain in early cytoskeletal remodeling, including secretion, aggregation, and spreading.

There has been growing interest in the role of calpain in the site-specific cleavage of actin-associated proteins such as talin, actin-binding protein 280 (ABP 280¹ or filamin) (7, 17), and the cytoplasmic domain of β₃ integrin (18). Regulatory kinases and phosphatases, including protein kinase C (PKC) (19), pp60⁽20), inositol polyphosphate 4-phosphatase (4-phosphatase) (21), focal adhesion kinase (22), and protein-tyrosine phosphatase 1-B (23) are also cleaved by calpain during platelet activation. Nonetheless, despite the identification of cytoskeletal and kinase/phosphatase targets for calpain in platelets, a functional role for calpain in aggregation and spreading has not been demonstrated.

A major obstacle to determining the physiological functions of calpain in platelets is lack of specific cell-permeant inhibitors. Peptidyl calpain inhibitors inhibit other cysteine proteases, including cathepsins (24). In addition, peptidyl calpain inhibitors require high micromolar concentrations to inhibit calpain in platelets (25), in contrast to fibroblasts (12). The lack of efficacy of peptidyl calpain inhibitors in platelets may be due to the high calpain content of platelets, where calpain represents 2% of platelet protein (26). While calpain’s reversible biological inhibitor, calpastatin, is the only known specific inhibitor of calpain (24, 27–32) simple strategies for increasing calpastatin in platelets have not been available. Calpastatin is cell-impermeant, and thus unsuitable for conventional studies of platelet activation (24). To solve this problem, we developed a cell-permeant specific inhibitor of calpain by joining the minimal inhibitory sequence of calpastatin to the cell-penetrating signal sequence of k-FGF, which confers cellular peptide importation (33–35). Using this specific cell-penetrating inhibitor and a set of peptidyl calpain inhibitors, with appropriate specificity controls, we demonstrate that calpain regulates platelet degranulation, aggregation, and spreading.

Footnotes

^{This work was supported by National Institutes of Health Grants K08-CA 1562 and P30 DK34928 (GRASP Digestive Disease Research Center grant to D. A. P. and I. M. H.), GM 55110, EY09033 and P30 DK34928 (GRASP Imaging Core grant) (to I. M. H.), and HL42443 and HL51926 (to B. F. and B. C. F.), and by a New England Medical Center grant (to D. A. P.).}

^{The abbreviations used are: ABP 280, actin-binding protein 280; AMC, aminomethylcoumarin; k-FGF, Kaposi’s fibroblast growth factor; MDL, MDL28,170; MIC, minimal inhibitory concentration; SFLLR, thrombin agonist peptide; suc-LLVY-AMC, succinyl-leucyl-leucyl-valyltyrosyl-7-amino-4-methylcoumarin; ZLLYCHN}₂, benzyloxycarbonylleucyl-leucyl-tyrosyl-diazomethane; PI, phosphatidylinositol; PKC, protein kinase C; PAGE, polyacrylamide gel electrophoresis; PIPES, 1,4-piperazinediethanesulfonic acid.

Footnotes

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