Inhibition of calpain blocks platelet secretion, aggregation, and spreading

Abstract

Previous studies have indicated that the Ca(2+)-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 alpha-granule secretion (IC(50) = 20 microM) during the first 30 s of activation, thrombin-induced platelet aggregation (IC(50) = 50 microM), and platelet spreading on glass surfaces (IC(50) = 34 microM). 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.

FIG. 1. Amino acid sequence of calpastat and related peptides

Calpastat, 40 amino acids in length, consists of amino acids 7–22 of human k-FGF at the amino terminus, followed by the 24-amino acid calpastatin consensus sequence of Croall and McGrody (42). Calpastat-Ala is a control peptide, in which the most highly conserved amino acids of the calpastatin consensus sequence are replaced with alanine. The calpastatin 24-mer is the calpastatin consensus sequence (42).

FIG. 2. Calpastat inhibits calpain in vitro

A, the dose-response curve of calpastat for inhibition of μ-calpain cleavage of the substrate suc-LLVY-AMC, assayed as described (see “Experimental Procedures”). The IC₅₀ of calpastat for inhibition of μ-calpain is 70 nm. The calpastat-Ala mutant peptide has no inhibitory activity in this assay. B, the cell-penetrating calpastatin peptide inhibits calpain in vivo. Platelets were preincubated with ZLLYCHN₂ (Z), Me₂SO vehicle (D), calpastat (C), or HEPES vehicle for calpastat (B), and treated with the ionophore A23187 for 0, 5, 8, or 30 min. Platelet proteins were analyzed by SDS-PAGE, as described (see “Experimental Procedures”). Arrowheads designate the 135- and 93-kDa calpain breakdown products of ABP280 and the 190-kDa calpain breakdown product of talin. The 49-kDa calpain breakdown product of talin is not visualized in this gel system. Free calpastat is visualized at the bottom of lanes corresponding to conditions where the peptide was added.

FIG. 3. P-selectin surface expression is calpain-dependent

A, calpastat, but not calpastat-Ala nor the calpastatin 24-mer, inhibits P-selectin surface expression on gel-filtered platelets. The extent of P-selectin surface expression from SFLLR-treated platelets was determined from the geometric mean of P-selectin fluorescence, measured by flow cytometry (see “Experimental Procedures”). The SFLLR concentration utilized, 50 µm, results in maximal P-selectin expression. B, the dose response curve for inhibition of platelet P-selectin surface expression by calpastat. The IC₅₀ for calpastat inhibition of SFLLR-induced P-selectin presentation on gel-filtered platelets is 20 µm. Measurement of P-selectin surface expression was performed as in A.

FIG. 4. Aggregation of thrombin-stimulated platelets is calpaindependent

Calpastat (50 µm) but not calpastat-Ala (50 µm) inhibits thrombin-induced (1.0 unit/ml) platelet aggregation. Aggregation of stirred platelets was initiated by thrombin treatment, as described (see “Experimental Procedures”). The abscissa, indicating time, is marked by broken lines in intervals of 15 s. The ordinate, indicating percentage of light transmission, is marked by broken lines in intervals of 10%. 100% and 0% light transmission were defined by buffer and unaggregated platelets in buffer, respectively. The time of thrombin addition is indicated with an arrow. Gel-filtered platelets were preincubated with calpastat or calpastat-Ala for 20 min before recalcification and subsequent addition of thrombin.

FIG. 5. Spreading of platelets on glass is calpain-dependent

A, calpastat inhibits platelet spreading, dependent upon the calpastatin consensus sequence, and requires a pre-incubation period of between 30 and 60 min. Platelets were preincubated with calpastat-Ala (black bar) or calpastat (white bar) peptide (100 µm) for 30, 60, or 90 min and then spread for 20 min on glass coverslips. The coverslips were fixed, stained with Oregon Green-phalloidin, and photographed by fluorescence microscopy. The cell areas of the spread platelets were measured by computerized image analysis (NIH Image 1.61). One area unit (AU) is 0.625 µm². Although calpastat-Ala demonstrated a 20% nonspecific inhibition of spreading relative to a vehicle control at 90 min of preincubation (data not shown), this inhibition was 4-fold less than the 85% inhibition observed for calpastat, compared with the vehicle control. Unspread platelets (gray bar) were obtained by treating platelets with 3% Me₂SO, which completely blocks spreading, and allowing them to adhere to glass coverslips for 20 min as above. B, calpastat inhibits platelet actin remodeling during spreading, dependent upon the calpastatin consensus sequence. Platelets were incubated with peptide for 90 min and then spread on glass for 20 min. The coverslips were processed, stained with Oregon Green-phalloidin, and photographed by fluorescence microscopy. The size bar is 10 µm. C, similar to calpastat, MDL and calpeptin inhibit platelet actin remodeling during spreading. Calpastat (100 µm), MDL (400 µm), and calpeptin (300 µm) were used at their respective IC₆₀ concentrations for spreading inhibition. Preincubation times were 90 min for calpastat and HEPES vehicle control and 10 min for MDL, calpeptin, and the Me₂SO vehicle control. NH₄Cl (10 µm) treatment for 10 min had no detectable effect on spreading (data not shown). The coverslips were processed, stained with Oregon Green-phalloidin, and photographed by fluorescence microscopy. The size bar is 10 µm. The IC₆₀ concentrations of inhibitors were chosen to demonstrate the morphologies of the actin cytoskeleton for calpastat and peptidyl calpain inhibitor-treated platelets, under conditions of partial and equal spreading.