Calpain cleavage of focal adhesion proteins regulates the cytoskeletal attachment of integrin alphaIIbbeta3 (platelet glycoprotein IIb/IIIa) and the cellular retraction of fibrin clots.

The intracellular thiol protease calpain catalyzes the limited proteolysis of various focal adhesion structural proteins and signaling enzymes in adherent cells. In human platelets, calpain activation is dependent on fibrinogen binding to integrin αIIbβ3 and subsequent platelet aggregation, suggesting a potential role for this protease in the regulation of postaggregation responses. In this study, we have examined the effects of calpain activation on several postaggregation events in human platelets, including the cytoskeletal attachment of integrin αIIbβ3, the tyrosine phosphorylation of cytoskeletal proteins, and the cellular retraction of fibrin clots. We demonstrate that calpain activation in either washed platelets or platelet-rich plasma is associated with a marked reduction in platelet-mediated fibrin clot retraction. This relaxation of clot retraction was observed in both thrombin and ionophore A23187-stimulated platelets. Calcium dose-response studies (extracellular calcium concentrations between 0.1 μM and 1 M) revealed a strong correlation between calpain activation and relaxed clot retraction. Furthermore, pretreating platelets with the calpain inhibitors calpeptin and calpain inhibitor I prevented the calpain-mediated reduction in clot retraction. Relaxed fibrin clot retraction was associated with the cleavage of several platelet focal adhesion structural proteins and signaling enzymes, resulting in the dissociation of talin, pp60c-src, and integrin αIIbβ3 from the contractile cytoskeleton and the tyrosine dephosphorylation of multiple cytoskeletal proteins. These studies suggest an important role for calpain in the regulation of multiple postaggregation events in human platelets. The ability of calpain to inhibit clot retraction is likely to be due to the cleavage of both structural and signaling proteins involved in modulating integrin-cytoskeletal interactions.

Calpains are a family of calcium-dependent cysteine proteinases widely expressed in mammalian cells (1)(2)(3). Activation of these enzymes occurs in response to a wide range of physiological stimuli and is associated with limited proteolysis of several key cellular proteins, including the c-Fos and c-Jun transcription factors (4), the cytoskeletal proteins talin and actin-binding protein (filamin) (5), and multiple signaling enzymes, in-cluding protein kinase C, pp60 c-src , and the tyrosine phosphatase PTP-1B 1 (6 -8). Although calpain-mediated proteolysis has been implicated in a broad range of pathophysiological processes, including postischemic tissue damage and degenerative diseases (3), the precise role of these enzymes in cell function has not been established.
Calpains have been localized to points of attachment between cells and the extracellular matrix (focal adhesions) and in the cytoskeletal fraction of thrombin-stimulated platelets (9,10). The recruitment of calpain to these sites is thought to promote its activation by membrane phospholipids and calcium and to co-localize it with target substrates (11). A growing number of these substrates have been identified in focal adhesions, in which they may participate in the assembly of cytoskeletal signaling complexes and in anchoring integrin adhesion receptors to the contractile cytoskeleton. Once anchored, integrins form a stable transmembrane linkage between extracellular matrix proteins and cytoskeletal elements, thereby allowing the extracellular transmission of cytoskeletal contractile forces necessary for fibrin clot retraction, wound healing, and tissue morphogenesis.
Studies in thrombin-stimulated platelets have demonstrated calpain-catalyzed proteolysis of multiple focal adhesion structural proteins and signaling enzymes (7,12). Many of these proteins translocate to the cytoskeleton in an aggregation-dependent manner and participate in the formation of integrinrich cytoskeletal signaling complexes. The formation of these complexes is thought to be critical for the tyrosine phosphorylation of multiple cytoskeletal proteins and for the stable incorporation of integrin ␣ IIb ␤ 3 into the contractile cytoskeleton. In this report, we have investigated the effects of calpain activation on a number of postaggregation events in human platelets. Our studies demonstrate that the calpain-catalyzed proteolysis of focal adhesion structural proteins and signaling enzymes leads to a selective defect in the ability of platelets to retract fibrin clots. This calpain-mediated relaxation of clot retraction was associated with the detachment of integrin ␣ IIb ␤ 3 from the contractile cytoskeleton and the dephosphorylation of multiple cytoskeletal proteins on tyrosine residues.

EXPERIMENTAL PROCEDURES
Materials-Calpeptin was obtained from Biomol Research Laboratories (Plymouth Meeting, PA). Ionophore A23187 and calpain inhibitor I were from Calbiochem. All other materials were from sources we have described previously (13,14).
Antibodies-Anti-phosphotyrosine MAbs PY20 and 4G10 were sup-* This work was funded by a grant from the National Health and Medical Research Council of Australia. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Preparation of Washed Platelets and Platelet Aggregation Studies-Human platelets were obtained from healthy volunteers who had not taken antiplatelet medication in the preceding 2 weeks and washed as described previously (14). Washed platelets were finally resuspended in modified Tyrode's buffer (10 mM Hepes, 12 mM NaHCO 3 , pH 7.5, 137 mM NaCl, 2.7 mM KCl, and 5 mM glucose), and preincubated with either calpeptin (2-100 g/ml), calpain inhibitor I (2-10 M), or vehicle (0.3% Me 2 SO) for 30 min at room temperature. Washed platelets (3 ϫ 10 8 /ml) were stimulated with 0.01-1 M ionophore A23187 and/or thrombin (1 unit/ml) in the presence of the indicated concentration of CaCl 2 . Fibrinogen (0.3 mg/ml) was included in the platelet suspensions when ionophore A23187 was used as a single agonist. All aggregations were initiated by stirring the platelet suspensions at 950 rpm for 10 min at 37°C in a four-channel automated platelet analyzer (Kyoto Daiichi, Japan). The extent of platelet aggregation was defined arbitrarily as the percentage of change in optical density as measured by the automated platelet analyzer.
Antiphosphotyrosine, Calpain, pp60 c-src , GP IIb, and Talin Immunoblots-Equal quantities of platelet extracts were separated by 7.5% SDS-PAGE, under reducing conditions, then transferred to PVDF membranes. Western blots were performed as described by Towbin et al. (16), using specific primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies. Blots were developed using ECL according to the manufacturer's instructions (Amersham International).
Clot Retraction-Clot retraction studies using platelet-rich plasma (PRP) or washed platelets were performed as described (13), with some minor modifications. Platelet-rich plasma (3 ϫ 10 8 platelets/ml) was anticoagulated with 2 mM EGTA and 2 mM MgCl 2 and activated with thrombin (5 units/ml) at room temperature in the presence or absence of 10 mM calcium. Reaction mixtures were either left unstirred or were stirred for a maximum period of 45-60 s to induce platelet aggregation. Washed platelets were pretreated with calpeptin (2-100 g/ml), calpain inhibitor I (2-10 M), or vehicle (0.3% Me 2 SO) for 30 min prior to activation with thrombin (1 unit/ml) and/or ionophore A23187 (0.01-1 M). For studies with ionophore A23187 alone, atroxin (0.1 g/ml) was added to induce clot formation. In all studies with ionophore A23187, the platelets were left unstirred during the assay. When thrombin was used as the agonist, platelets were stirred for a maximum period of 45-60 s, unless otherwise indicated. Reproducible clot retraction results in thrombin-aggregated washed platelets were dependent on not stirring the platelets excessively, as the formation of large aggregates resulted in their sedimentation within the tube and ineffective clot retraction. For calcium dose-response studies, the free ionized calcium concentration was maintained using Ca 2ϩ and EGTA buffers (17). In both the PRP and washed platelet assays, clot retraction was assessed after 60 min of platelet activation. The extent of clot retraction (percentage of clot retraction) was quantitated by measuring the residual volume of serum (after removal of the fibrin clot) in each reaction mixture and expressing this as a percentage of the total reaction volume.
Miscellaneous Methods-SDS-PAGE was performed according to the method of Laemmli (18). Fibrinogen was purified from fresh frozen plasma as described previously by Jakobsen and Koerulf (19). Protein concentrations were measured using the Bio-Rad protein assay with bovine serum albumin as a standard.

RESULTS
Studies in human platelets have demonstrated that several cytoskeletal-associated signaling enzymes are substrates for activated calpain. These enzymes include the Src family of nonreceptor tyrosine kinases and the nonreceptor tyrosine phosphatase PTP-1B (7,20,21). As these enzymes are highly expressed in platelets and translocate to the cytoskeletal fraction of aggregated platelets, they are likely to play a major role in regulating the tyrosine phosphorylation status of cytoskeletal proteins. Although the precise role of these phosphorylation events has yet to be clearly established, studies with tyrosine kinase inhibitors have suggested a potentially important role for these enzymes in regulating the cytoskeletal attachment of integrin ␣ IIb ␤ 3 (13). We therefore aimed to investigate the effects of calpain activation on the level of tyrosine phosphorylation of cytoskeletal proteins and to correlate these effects with specific platelet postaggregation events, including the cytoskeletal attachment of integrin ␣ IIb ␤ 3 and fibrin clot retraction.
Calpain-mediated pp60 c-src Cleavage is a Postaggregation Event in Human Platelets-pp60 c-src is the major protein tyrosine kinase identified in platelets, constituting 0.2-0.4% of total platelet protein (22). In our initial studies, we examined the time course for pp60 c-src cleavage in ionophore A23187-and thrombin-stimulated platelets and correlated this with platelet aggregation. As demonstrated in Fig. 1, pp60 c-src cleavage occurred well after the initiation of platelet aggregation by ionophore A23187 (1 M), with no cleavage detected at 1 min, 20% proteolysis by 2 min, and complete proteolysis after 5 min of platelet stimulation. Consistent with a role for platelet aggregation in calpain activation and pp60 c-src cleavage (5, 6, 12, 23), we found that not stirring ionophore A23187-stimulated plate- Platelet aggregation was examined in a four-channel platelet aggregometer, and the extent of aggregation was determined, as described under "Experimental Procedures." Results represent the mean Ϯ S.E. (bars) of three separate experiments. Platelet aliquots were removed at the indicated time points and lysed in Laemmli reducing buffer, and total lysates were examined for pp60 c-src proteolysis by immunoblot analysis, as described under "Experimental Procedures." The extent of proteolysis was assessed by performing densitometry on the pp60 c-src immunoblots. These results are from one experiment, representative of three. lets prevented platelet aggregation and significantly delayed pp60 c-src cleavage. In contrast, in thrombin-stimulated platelets, pp60 c-src cleavage was completely dependent on integrin ␣ IIb ␤ 3 -mediated platelet aggregation (data not shown) (6). Inhibition of calpain-catalyzed cleavage of pp60 c-src by calpeptin had no effect on platelet aggregation induced by ionophore A23187 (Fig. 1) or thrombin (data not shown). These observations are consistent with the hypothesis that calpain-mediated proteolysis of pp60 c-src and other target substrates is likely to regulate platelet responses that occur after platelet aggregation.
Proteolysis of pp60 c-src Abolishes its Cytoskeletal Localization in Aggregated Platelets-We examined the possibility that calpain-mediated proteolysis of pp60 c-src may regulate the cytoskeletal association of this kinase. Studies were performed in ionophore A23187-stimulated platelets, as this agonist has the unique ability to stimulate platelet activation in the absence of calpain activation when used at low concentrations (0.01-0.1 M), whereas at higher concentrations (1 M) it induces both platelet and calpain activation. The results from these experiments are presented in Fig. 2, A and B, and demonstrate that low concentrations of ionophore A23187 (0.01-0.1 M) stimulate the redistribution of pp60 c-src from the Triton X-100-soluble ( Fig. 2A, I) to the cytoskeletal ( Fig. 2A, II) fraction of aggregated platelets. With higher concentrations of ionophore A23187 (1 M), pp60 c-src was completely proteolyzed to its 52and 47-kDa forms ( Fig. 2A, I) and was no longer associated with the cytoskeleton ( Fig. 2A, II). Time course studies in ionophore A23187-stimulated platelets (1 M) revealed an initial increase in the cytoskeletal content of pp60 c-src , which was followed by its proteolysis and dissociation from the cytoskeleton (Fig. 2B, I). This time-dependent cytoskeletal dissociation of pp60 c-src correlated with the appearance of the 52-and 47-kDa proteolytic fragments in the Triton X-100-soluble fraction (data not shown). An important role for calpain in regulating the cytoskeletal association of pp60 c-src was confirmed by the ability of calpeptin to prevent pp60 c-src cleavage and its subsequent dissociation from the cytoskeleton (Fig. 2B, II).
Recent studies in thrombin-stimulated platelets have suggested that the calpain-mediated cleavage of nonreceptor tyrosine kinases and phosphatases in human platelets leads to a substantial reduction in protein tyrosine phosphorylation (24). Many of these proteins are phosphorylated in an aggregationdependent manner and are specifically localized within the cytoskeletal fraction of aggregated platelets (25,26). We therefore performed antiphosphotyrosine immunoblot analysis on the cytoskeletal extracts of ionophore A23187-stimulated platelets to examine for time-dependent changes in cytoskeletal protein phosphorylation. As demonstrated in Fig. 2B, tyrosinephosphorylated proteins of 138, 110 -90, 80, 71, 60 -50, and 38 kDa were consistently identified within the cytoskeletal fraction of aggregated platelets. Consistent with previous reports (24), we found that the tyrosine phosphorylation of cytoskeletal proteins was a transient phenomenon, with dephosphorylation occurring within 2 min of platelet stimulation (Fig. 2B, I). The pretreatment of platelets with either calpeptin (Fig. 2B, II) or calpain inhibitor I (data not shown) completely abolished calpain-mediated cleavage of pp60 c-src and the tyrosine phosphatase PTP-1B (data not shown) and dramatically reduced these dephosphorylation events. These observations are consistent with the hypothesis that the cleavage of cytoskeletal-associated nonreceptor tyrosine kinases and phosphatases regulates the tyrosine phosphorylation status of multiple cytoskeletal proteins.
Regulation of Platelet-mediated Fibrin Clot Retraction by Activated Calpain-Our previous studies have suggested that the phosphorylation of cytoskeletal proteins on tyrosine residues by nonreceptor tyrosine kinases may be important for regulating the cytoskeletal attachment of integrin ␣ IIb ␤ 3 and the cellular retraction of fibrin clots (13). The ability of calpain to regulate the phosphorylation status of cytoskeletal proteins FIG. 2. Effect of calpain activation on the cytoskeletal association of pp60 c-src and the tyrosine phosphorylation of cytoskeletal proteins. Washed platelets (3 ϫ 10 8 /ml) were pretreated with 0.3% Me 2 SO or calpeptin (100 g/ml) for 30 min at room temperature in the presence of CaCl 2 (1 mM). Platelets were stimulated with the indicated concentrations of ionophore A23187 while stirring. Platelets were lysed at the indicated time points and fractionated into Triton X-100soluble and cytoskeletal extracts. The platelet extracts were separated by 7.5% SDS-PAGE, transferred to PVDF membranes, and examined for pp60 c-src content and protein tyrosine phosphorylation by immunoblot analysis. A, effect of calpain cleavage on the subcellular localization of pp60 c-src . I, Triton X-100-soluble fraction; II, cytoskeletal fraction. B, correlation between cytoskeletal localization of pp60 c-src and tyrosine phosphorylation of cytoskeletal proteins in the absence (I) and presence (II) of calpeptin. and to cleave focal adhesion structural proteins involved in anchoring integrins to the cytoskeleton has suggested a potential role for this protease in the regulation of clot retraction. We therefore performed a series of experiments in PRP and washed platelets to correlate calpain activation with changes in clot retraction. Previous studies examining the role of activated calpain in the regulation of clot retraction have reported no differences in the rate and extent of clot retraction in the presence or absence of calpeptin (23). However, it is unlikely that calpain was substantially activated in these studies, as the platelets were not aggregated during the clot retraction assay. In preliminary studies, we examined clot retraction in a PRP assay system, as PRP is the normal physiological medium used for platelet aggregation and clot retraction studies. The stimulation of platelets with thrombin (5 units/ml) alone (Fig. 3A,I, tube 1) or thrombin (5 units/ml) in the presence of calcium (10 mM), without stirring (i.e. no aggregation; Fig. 3A, I, tube 3), resulted in 88 Ϯ 2% (Fig. 3A, II, lane 1) and 84 Ϯ 6% (Fig. 3A, II, lane 3) (n ϭ 5) retraction of fibrin clots, respectively. However, when platelets were stimulated with thrombin (5 units/ ml) and calcium (10 mM) while stirring for 45 s (i.e. conditions that promote platelet aggregation and calpain activation; Fig.  3A, I, tube 4), the extent of clot retraction was markedly reduced to 32 Ϯ 11% (n ϭ 5) (Fig. 3A, II, lane 4). It was unlikely that this defect in clot retraction was purely a technical artifact related to the formation of large platelet aggregates, as an 83 Ϯ 5% (n ϭ 5) (Fig. 3A, II, lane 2) retraction of fibrin clots was observed in thrombin-aggregated platelets when calcium was omitted from the reaction mixture (Fig. 3A, I, tube 2).
To confirm that these inhibitory effects on platelet-mediated clot retraction were due to calpain activation, we examined clot retraction in a washed platelet assay system. With this assay it was much easier to examine the effects of pharmacological inhibitors of calpain, as these platelet suspensions contained no plasma components, such as albumin or plasma lipoproteins, which may bind these lipophilic compounds and sequester them from platelets. Furthermore, to exclude the possibility that the reduction in clot retraction observed in aggregated platelets was a technical artifact due to uneven platelet dispersion throughout the fibrin clot, we activated calpain in the absence of platelet stirring and aggregation by stimulating platelets with ionophore A23187 (1 M). This agonist was particularly useful in these studies, as it has the unique ability to activate calpain in the absence of platelet aggregation (27). As demonstrated in Fig. 3B, platelet stimulation by thrombin (1 unit/ml) alone (Fig. 3b, lane 1) or thrombin with calpeptin (100 g/ml) (Fig. 3B, lane 2) resulted in 90 Ϯ 7% and 90 Ϯ 9% (n ϭ 5) retraction of fibrin clots, respectively. However, in the presence of ionophore A23187, thrombin-stimulated clot retraction was reduced to 49 Ϯ 9% (n ϭ 5) (Fig. 3B, lane 3). This reduction in clot retraction was prevented by pretreating platelets with 100 g/ml calpeptin (83 Ϯ 5%) (Fig. 3B, lane 4).
To further strengthen our hypothesis that calpain is the responsible protease mediating relaxed fibrin clot retraction, we performed ionophore A23187 dose-response studies. We monitored calpain activation in these studies using antibodies against the inactive large subunit of calpain (80 kDa) and the autoproteolytic activated form (76 kDa). Previous studies in human platelets have demonstrated calcium-dependent autoproteolytic conversion of the 80-kDa subunit of calpain to its 76-kDa active form (3). The antibodies used in these studies have been raised against synthetic peptides corresponding to the N-terminal sequence of the large subunit of both forms of calpain (28). Immunoblot analysis of whole cell lysates with these antibodies represents a sensitive, specific, and direct means of monitoring calpain activation within the cell (29). As shown in Fig. 4, concentrations of ionophore A23187 that activate platelets without activating calpain (0.01 and 0.05 M), as monitored by calpain autolysis and pp60 c-src cleavage, resulted in 72.7 Ϯ 3.5 and 70 Ϯ 5.4% retraction of fibrin clots, respectively. Higher concentrations of ionophore A23187 (0.25 and 1 M) resulted in calpain activation and pp60 c-src cleavage and were associated with a substantial reduction in clot retraction (46 Ϯ 2.8 and 37.4 Ϯ 5.2%, respectively). The chelation of extracellular calcium by the addition of 1 mM EGTA and 2 mM MgCl 2 to the platelet reaction mixtures abolished calpain activation by 1 M ionophore A23187 and restored effective clot retraction (72.3 Ϯ 2.9%).
Further evidence supporting a role for calpain in the regulation of clot retraction stemmed from calcium dose-response studies. Previous reports have demonstrated an absolute requirement for extracellular calcium for calpain activation induced by both pharmacological and physiological agonists (2). As demonstrated in Fig. 5, A and B, calpain activation in both ionophore A23187 and thrombin-stimulated platelets required the presence of low micromolar concentrations of extracellular calcium. Higher concentrations of extracellular calcium were associated with a progressive increase in calpain activation, as monitored by either calpain autolysis or calpain substrate proteolysis, with maximal activation observed with 1.0 mM calcium (Fig. 5, A and B). In each of these experiments, there was a strong correlation between the extent of calpain activation and the reduction in clot retraction. Furthermore, in all of the studies reported here the inhibitory effects of calpain were limited to the clot retraction process, with normal platelet aggregation and [ 14 C]serotonin release in response to thrombin or ionophore A23187 (data not shown).

Calpain-mediated Cleavage of Talin and the Dissociation of Talin and Integrin ␣ IIb ␤ 3 from the Contractile Cytoskeleton-
The ability of cells to transmit cytoskeletal contractile forces to extracellular matrices is dependent on the anchorage of integrins to the cytoskeleton. The stable association of integrins with actin filaments requires a number of intermediary proteins, including talin, ␣-actinin, and vinculin (30). We investigated whether the calpain-catalyzed cleavage of talin was associated with the dissociation of either talin or integrin ␣ IIb ␤ 3 from the contractile cytoskeleton. Immunoblot analysis of total cell lysates, with an antibody that recognizes the native 230-kDa form of talin and the largest 190-kDa talin fragment, revealed rapid proteolysis of talin in ionophore A23187-stimulated platelets (Fig. 6A). Cleavage of talin from its 230-kDa native form to the 190-kDa fragment was observed within 15 s of platelet stimulation and was complete by 3 min. In agreement with previous studies (23), we observed that calpeptin (100 g/ml) pretreatment of platelets dramatically slowed the rate of talin cleavage but did not consistently prevent cleavage altogether. The effect of this cleavage on the cytoskeletal association of talin was investigated by fractionating platelets into Triton X-100-soluble and -insoluble (cytoskeletal) extracts. Ionophore A23187 stimulation of platelets was associated with a progressive increase in the cytoskeletal content of talin throughout the first 60 s of platelet activation (Fig. 6B). The association of both the 230-and 190-kDa forms of talin with the cytoskeleton was transient, however, with complete dissociation of the 190-kDa fragment observed after 4 min of platelet stimulation. This cytoskeletal dissociation was not due to further proteolysis of the fragment, as the total cell levels of the 190-kDa fragment remained constant throughout the period of examination (Fig. 6A). An important role for calpain in regulating the cytoskeletal attachment of talin was confirmed in calpeptin-treated platelets, in which reduced proteolysis of talin prevented its dissociation from the cytoskeleton (Fig. 6B).
We correlated the effect of talin cleavage on the cytoskeletal attachment of integrin ␣ IIb ␤ 3 . Time course experiments revealed a close correlation between the cytoskeletal dissociation of talin with that of integrin ␣ IIb ␤ 3 (GP IIb) (Fig. 6, B-D). As with talin, pretreating platelets with calpeptin resulted in both a higher and more sustained level of integrin ␣ IIb ␤ 3 incorporation into the cytoskeleton. Previous reports have suggested that the dissociation of integrin ␣ IIb ␤ 3 from the membrane cytoskeleton does not correlate with talin cleavage (23). Our studies are consistent with these findings, as we observed extensive proteolysis of talin within the first 30 s of platelet stimulation, yet the bulk of integrin ␣ IIb ␤ 3 (GP IIb) did not dissociate from the cytoskeleton until 2-3 min after platelet activation (Fig. 6, compare A and B with C). To exclude the possibility that the dramatic reduction in the cytoskeletal content of talin and integrin ␣ IIb ␤ 3 was due to a global reduction in total cytoskeletal protein, the talin and integrin ␣ IIb ␤ 3 immunoblots were stained with Coomassie Brilliant Blue. The total amount of filamentous actin (Fig. 6D) and a range of other cytoskeletal proteins (not shown) increased approximately 2-3fold following ionophore A23187 stimulation of platelets and was largely maintained throughout the period of examination. Hence it is unlikely that the calpain-mediated reduction in the cytoskeletal content of integrin ␣ IIb ␤ 3 and talin is attributable to gross changes in the total amount of cytoskeletal protein.
Relationship Between Calpain Activation, the Cytoskeletal Attachment of Integrin ␣ IIb ␤ 3 , and Clot Retraction-To examine in more detail the relationship between calpain activation and the cytoskeletal attachment of integrin ␣ IIb ␤ 3 , we per- formed experiments on ionophore A23187-stimulated platelets that had been resuspended in either calcium-free or calciumcontaining buffers. Platelet stimulation with 1 M ionophore A23187 for 5 min in the presence of 1 mM CaCl 2 resulted in complete conversion of inactive calpain to its activated form. Under these assay conditions, integrin ␣ IIb ␤ 3 was no longer detectable within the cytoskeleton by immunoblot analysis (Fig. 7A). In contrast, the resuspension of platelets in buffers containing 1 mM EGTA and 2 mM MgCl 2 , prior to ionophore A23187 stimulation, prevented calpain activation and restored the association of integrin ␣ IIb ␤ 3 with the contractile cytoskel- Washed platelets (3 ϫ 10 8 /ml) were pretreated with 0.3% Me 2 SO or calpeptin (100 g/ml) for 10 min at room temperature and then stimulated with ionophore A23187 (1 M) for the indicated time points while stirring. Platelets were lysed and fractionated into Triton X-100soluble or cytoskeletal extracts, as described under "Experimental Procedures." Whole cell lysates or cytoskeletal extracts were subjected to immunoblot analysis using monoclonal antibodies against talin or GP IIb. Cytoskeletal actin was quantitated by densitometry after staining the PVDF membranes with Coomassie Brilliant Blue. Results are from one experiment, representative of three. A, talin proteolysis in whole cell lysates; B, association of talin with the cytoskeleton; C, association of GP IIb with the cytoskeleton; D, comparative time course for cytoskeletal dissociation of GP IIb and talin.
(1 mg/ml) and the indicated concentrations of CaCl 2 . The platelets were stirred for 45-60 s, then left unstirred for 60 min. The extent of clot retraction (Histogram) was quantitated as described in Fig. 2 eton. Under each of these experimental conditions, the ability of integrin ␣ IIb ␤ 3 to associate with the cytoskeleton correlated well with the ability of platelets to retract fibrin clots. Further evidence supporting a role for calpain in regulating the cytoskeletal attachment of integrin ␣ IIb ␤ 3 stemmed from calpeptin dose-response studies. As demonstrated in Fig. 7A, pretreating platelets with doses of calpeptin greater than 2 g/ml inhibited calpain activation in a dose-dependent manner. In these dose-response experiments there was an excellent correlation between the extent of calpain activation, the amount of integrin ␣ IIb ␤ 3 incorporated into the cytoskeleton, and the extent of clot retraction. These results were not unique to calpeptin-treated platelets, as calpain inhibitor I also restored clot retraction by ionophore A23187-stimulated platelets in a dosedependent manner (data not shown).
In further studies, we examined the ability of calpeptin to restore clot retraction by thrombin-aggregated platelets. Reproducible clot retraction results in these experiments were dependent on limiting the duration of platelet stirring for a maximum of 45-60 s after the addition of thrombin. Stirring the platelet reaction mixtures for longer periods resulted in the formation of large platelet aggregates, which tended to sediment in the tube and retract fibrin clots poorly. Consistent with our studies in platelet-rich plasma, the stirring of thrombinstimulated washed platelets for 45-60 s dramatically reduced the extent of clot retraction (Fig. 7B). The pretreatment of these cells with calpeptin, prior to the addition of thrombin, restored the ability of these aggregated platelets to retract fibrin clots. As with ionophore A23187-stimulated platelets, the effect of calpeptin on the clot retraction process was dose-dependent (Fig. 7B) and correlated with its ability to inhibit calpain activation (data not shown).

DISCUSSION
The studies presented in this article define an important role for calpain in the regulation of multiple postaggregation events in human platelets. We have demonstrated under a variety of different experimental conditions that the calpain-catalyzed cleavage of several focal adhesion structural proteins and signaling enzymes in human platelets leads to a selective defect in the ability of platelets to retract fibrin clots. This reduction in clot retraction was associated with reduced incorporation of integrin ␣ IIb ␤ 3 into the contractile cytoskeleton and the dephosphorylation of multiple cytoskeletal proteins on tyrosine residues.
Although there are a number of cysteine and serine proteases in human cells, we have provided several lines of evidence suggesting that calpain is likely to be the responsible platelet protease regulating fibrin clot retraction. First, relaxed fibrin clot retraction was only observed under experimental conditions that promoted calpain activation. These conditions include the requirement for extracellular calcium in both thrombin-and ionophore A23187-stimulated platelets and the necessity for platelet aggregation when thrombin was used as a single agonist. Second, dose-response studies in ionophore A23187-stimulated platelets revealed a close correlation between calpain activation and relaxed clot retraction. Third, pretreatment of platelets with two different inhibitors of calpain restored clot retraction in a dose-dependent manner. Fourth, an excellent correlation was observed between the extent of clot retraction and the degree of calpain activation under all experimental conditions examined. Finally, the ability of calpain to proteolyze cytoskeletal proteins involved in regulating cellular contractile processes is consistent with a role for this protease in the regulation of clot retraction.
Although previous studies have indicated an important role for calpain in the regulation of postaggregation responses, such as the release of procoagulant-rich microparticles from the platelet membrane, a role for calpain in the regulation of fibrin clot retraction has not been established (23,31). One of the major technical obstacles in examining the role of calpain in clot retraction is the need to induce platelet aggregation to activate the protease. The formation of platelet aggregates may lead to uneven platelet dispersion throughout the fibrin clot, resulting in an artifactual decrease in clot retraction. To overcome this technical problem we have used washed platelets treated with the pharmacological agonist ionophore A23187. This agonist has well characterized effects on platelet function and has the advantage of activating calpain in the absence of platelet aggregation (27). Several lines of evidence suggest that the results we have obtained in ionophore A23187-stimulated platelets are not unique to this agonist and are likely to be physiologically relevant. First, the inhibitory effects of calpain activation on clot retraction were not limited to ionophore A23187-stimulated platelets, as we observed a similar functional defect in thrombin-stimulated platelets under assay conditions that favored calpain activation. Second, calpain-mediated proteolysis of Src family kinases, PTP-1B and talin is also observed in platelets activated by physiological agonists, such as thrombin and collagen, and is associated with the dissociation of integrin ␣ IIb ␤ 3 from the membrane cytoskeleton (31). Third, the ability of calpain to regulate the cytoskeletal attachment and signaling function of pp60 c-src and PTP-1B is a feature of both ionophore A23187-and thrombin-stimulated platelets (6,7,21). Finally, the calpain-induced dephosphorylation of multiple platelet proteins that we have observed in ionophore A23187-stimulated platelets has recently been reported in thrombin-aggregated platelets (24).
Previous studies in thrombin-and collagen-stimulated platelets have suggested that calpain-mediated cleavage of cytoskeletal proteins is responsible for the dissociation of as much as 16% of total platelet integrin ␣ IIb ␤ 3 from the membrane cytoskeleton of aggregated platelets (31). Although talin is considered to play a critical role in anchoring integrins to the contractile cytoskeleton, these studies have highlighted that the calpain-mediated cleavage of talin does not appear to correlate with the release of integrin ␣ IIb ␤ 3 from the cell surface. Our time course experiments in ionophore A23187-stimulated platelets are consistent with this possibility, as they clearly demonstrate that the cytoskeletal dissociation of integrin ␣ IIb ␤ 3 lags well behind the initial cleavage of talin. These observations suggest a role for additional calpain-mediated proteolytic events in dissociating integrin-cytoskeletal contacts. A recent report has demonstrated that the cytoplasmic domain of ␤ 3 -integrins is cleaved at multiple sites by calpain in vivo, raising the distinct possibility that direct proteolysis of integrins can regulate their association with the cytoskeleton (32).
In previous studies we have demonstrated that tyrosine phosphorylation events in human platelets play a key role in regulating the cytoskeletal attachment of integrin ␣ IIb ␤ 3 (13). The studies reported in this article are consistent with these findings, as they demonstrate a close correlation between cytoskeletal protein dephosphorylation and the cytoskeletal dissociation of integrin ␣ IIb ␤ 3 . Furthermore, studies in human fibroblasts have demonstrated that the tyrosine phosphorylation of cytoskeletal proteins, following the ligation and aggregation of integrins on the cell surface, is essential for stable interaction between filamentous actin and integrins (33). Although these observations highlight the importance of tyrosine phosphorylation events in regulating cytoskeletal-integrin contacts, they provide limited insight into the molecular events modulating this interaction. For example, the level of tyrosine phosphorylation of talin, ␤-integrin, and vinculin is low in normal adherent cells, suggesting that direct phosphorylation of these proteins is an unlikely mechanism by which integrins become anchored to the cytoskeleton. In contrast, the vinculinbinding proteins paxillin and tensin are prominent tyrosinephosphorylated proteins in focal adhesions (34). The phosphorylation of paxillin may also be important for regulating its association with other cytoskeletal proteins, such as vinculin and talin. An attractive hypothesis is that the phosphorylated forms of paxillin and/or tensin bind to vinculin and unmask its talin and actin binding sites. This "active conformation" of vinculin may in turn stabilize the interaction between integrins and the underlying cytoskeleton (35). It is likely that the calpain-mediated cleavage of tyrosine kinases and phosphatases leads to a reduction in the level of phosphorylation of cytoskeletal proteins, such as paxillin and tensin. The dephosphorylation of these proteins may undermine the stable association of vinculin with actin filaments and talin, ultimately leading to the disassembly of integrin-cytoskeletal contacts.
The studies reported here on human platelets clearly have important implications for adhesion processes in other cells. Calpain is a ubiquitous protease, which has been localized to focal adhesions in a variety of adherent cells (9). The ability of calpain to cleave a growing number of focal adhesion proteins suggests a potentially important role for this enzyme in the regulation of these cellular structures. Our studies indicate that one of the functions of this protease is to regulate the transmission of cytoskeletal contractile forces to extracellular matrices. Furthermore, our studies suggest that calpain may also have an important signal-terminating role within the cell. The ability of integrins to promote calpain activation, leading to the proteolysis and down-regulation of cytoskeletal-associated signaling enzymes, suggests a potentially novel means by which these adhesion receptors can limit their own signaling function. Whether these proteolytic events have flow-on effects to other signaling pathways linked to cell adhesion will be an important area for future investigation.