Although calpain is activated in agonist-stimulated platelets, the precise roles of calpain in platelet activation are unknown. The present study demonstrates the Ca2+ and calpain dependence of platelet secretion, aggregation, and spreading. A cell-permeant calpastatin motif, calpastat, and three distinct peptidyl calpain inhibitors, calpeptin, MDL, and E64d, block platelet secretion, aggregation, and spreading. These inhibitors block the expression of P-selectin on the platelet surface, remodeling of the actin cytoskeleton during filopodial and lamellipodial protrusion formation, and cleavage of the membrane-associated proteins ABP 280 and talin during platelet activation events. These results implicate calpain in Ca2+-associated secretion events occurring during the first 30 s of platelet activation by the thrombin receptor agonist peptide, SFLLR. This suggests a novel and direct connection between thrombin receptor signaling and early activation of calpain. Blocking of platelet aggregation and spreading by calpain inhibition also indicates that calpain regulates later platelet activation events involving integrin function, as well as actin remodeling. These results suggest that calpain plays a critical, rate-limiting role in the activation of platelets by thrombin, promoting remodeling of actin networks and the platelet membrane cytoskeleton.
Prior studies of thrombin-induced platelet aggregation have demonstrated that an early sign of calpain activation, autolysis, occurs within 30–60 s of agonist stimulation, but not before (15
). Calpain also rapidly translocates to the plasma membrane within 10 s of thrombin stimulation, but it has been proposed that this event alone is not sufficient for calpain activation (15
). Activation of calpain was reported to be dependent upon both engagement of integrin IIb/IIIa (15
) and stirring (16
). Nonetheless, α-granule secretion due to thrombin receptor stimulation is independent of integrin IIb/IIIa function (50
). The results presented here indicate that calpain is functionally active in thrombin peptide-stimulated α-granule secretion during the first 30 s of activation, suggesting that rapid translocation of calpain to the platelet membrane may be of functional significance earlier in platelet activation than had been previously anticipated. Of interest, activation of calpain, measured by autolysis, is greatly reduced in Glanzmann’s thrombasthenia platelets (15
), despite the fact that α-granule secretion is not defective (50
). It is possible that secretion may require activation of only a small fraction of the calpain pool, not detectable by Western blotting. We hypothesize that there may be a direct connection between thrombin receptor engagement and initial calpain activation that may be sufficient to activate secretion and precede integrin IIb/IIIa mechanisms. Further studies will address this possibility.
transients regulate secretory processes in platelets and other cells, its precise role in platelet degranulation has yet to be elucidated. Our investigations have demonstrated that inhibition of calpain by calpastat or by peptidyl inhibitors resulted in 50–80% inhibition of P-selectin surface expression and α-granule secretion. Consistent with these findings are reports that calpain regulates secretion in type II alveolar cells (52
). At present it is unknown whether resting platelet α-granules exist both pre-docked at the plasma membrane and free in the cytoplasm. The ability of the calpain inhibitors to block 50–80%, but not all α-granule secretion may result from a specific role for calpain in initial events enabling the priming of undocked rather than pre-docked granule stores.
Recently, it was demonstrated that calpain cleaves spectrin, α-SNAP, syntaxin I, and synaptobrevin II during activationdirected degranulation of type II alveolar cells (52
). Our recent finding that SNARE family members are involved in α-granule secretion raises the possibility that calpain proteolysis facilitates granule fusion via cleavage of α-granule associated SNARE family members (5
). The role of proteolysis in vesicle fusion is nonetheless unknown, and future investigations will examine the cleavage of these proteins during platelet activation. At present, our experiments also have not eliminated the possibility that calpain is involved in upstream signaling events required for α-granule secretion.
Platelet aggregation draws on coordinated regulation of numerous signaling, cytoskeletal and adhesive processes. Calpain may be involved in the proteolytic cleavage of signaling kinases and phosphatases to active or inactive forms. Calpain cleavage of protein kinase C generates a constitutively active isoform, protein kinase M (PKM), which no longer requires diacyl glycerol for activity (24
). Calpain cleavage of phosphatidylinositol 4-phosphatase results in inactivation of this enzyme, which may facilitate platelet activation by elevating levels of phosphatidylinositol 3,4-bisphosphate (21
). Alternatively, calpain may facilitate the activation of phosphatidylinositol 3-phosphate 4-kinase, through mechanisms that remain to be fully elucidated (57
). It is also possible that calpain regulates the clustering and ligand engagement of platelet integrins, which are involved both in signaling process and in adhesive events. Thrombin-induced cleavage of the cytoplasmic domain of β3
integrin is dependent on calpain (18
), and calpain is known to regulate integrin clustering in other cell types (8
In the present study, inhibition of calpain with calpastat or the peptidyl calpain inhibitors blocks cell spreading. This finding is consistent with our previous report that calpain regulates spreading in fibroblasts (12
). Notably, the MICs of calpeptin, MDL, and E64d for spreading are comparable to the MICs of these inhibitors for spreading of fibroblasts (12
). When calpain is inhibited in platelets, filopodial and lamellipodial protrusion formation is significantly attenuated, demonstrating a role for calpain in remodeling of the cortical actin cytoskeleton in platelets. Together, the platelet and fibroblast results suggest that modulation of cell spreading by calpain may be a general mechanism used by a variety of different cell types.
There are at least three mechanisms by which calpain could modulate platelet spreading. First, calpain cleavage of actin filament capping complexes may expose barbed ends for polymerization and/or untethering the filamentous actin cytoskeleton from the plasma membrane. Although there is evidence to support a role for calpain in the uncapping of β-actin filaments through cleavage of the cytoskeleton-membrane linking protein, ezrin (11
), ezrin is not abundant in platelets (59
). The dominant member of the ezrin/radixin/moesin family found in platelets is moesin, which is not a calpain substrate (60
). Recently, adducin, a calpain substrate, has been identified as an actin capping protein in a variety of cell types (61
). It is possible that adducin may be a calpain target during actin remodeling events in platelets. Calpain might also facilitate platelet spreading by raising intracellular levels of the 4-phosphate phosphatidylinositols (PIs) PI (4,5)P and PI (3,4)P, which can directly uncap β-actin filaments (56
). Finally, calpain could facilitate platelet spreading by activating PKC (19
), which is functionally active during platelet activation and is known to facilitate cell spreading by an undetermined mechanism (67
Regulated proteolysis plays an important role in the regulation of intracellular signal transduction and cytoskeletal remodeling events. Our data provide strong evidence that calpain is a critical Ca2+ triggered molecular switch that regulates a series of complex processes during platelet activation. Identification of the critical physiologic substrates of calpain during platelet activation will add considerably to our understanding of calpain’s specific role in cell activation.