Calpain Cleavage of Focal Adhesion Proteins Regulates the Cytoskeletal Attachment of Integrin αIIbβ3 (Platelet Glycoprotein IIb/IIIa) and the Cellular Retraction of Fibrin Clots

Abstract

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 ␣ IIb ␤ 3 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 ␣ IIb ␤ 3 , 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 M 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, pp60 c-src , and integrin ␣ IIb ␤ 3 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 (1-3). 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, including 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 numb...

A novel form of integrin dysfunction involving β1, β2, and β3 integrins

“…Calpain is also activated following FMLP stimulation of neutrophils (38). However, in platelets, calpain activation is reported to lie downstream of αIIbβ3 ligation, because calpain inhibitors have no effect on platelet aggregation but do affect fibrin clot retraction (39). Because the patient in this study has no platelet aggregation, it is unlikely that the defect affects calpain.…”

Platelet Factor XIII and Calpain Negatively Regulate Integrin αIIbβ3 Adhesive Function and Thrombus Growth

“…An alternative mechanism relates to calpain-mediated cleavage of various signaling enzymes (FAK, Src, and PTP-1B) and cytoskeletal proteins (talin, filamin-1, and cortactin) (33)(34)(35)(36)(37). Several of these proteins have been implicated in the regulation of integrin ␣ IIb ␤ 3 adhesive function (38,39) and cleavage and disassembly of cytoskeletal signaling complexes may undermine the sustained activation of integrin ␣ IIb ␤ 3 and/or its cytoskeletal anchorage (16). These mechanisms are not necessarily mutually exclusive and it remains possible that each may contribute in varying degrees to the down-regulation of integrin ␣ IIb ␤ 3 adhesive function.…”

A revised model of platelet aggregation

“…For scanning electron microscopy (SEM) studies, anticoagulated whole blood was perfused over spread platelet monolayers at 600 s -1 for 2 minutes. Adherent platelets were prepared for SEM as described previously (14). For the imaging of thrombus formation in real time (see Figure 3c), platelet-collagen interactions and subsequent thrombus growth was viewed using fluorescence confocal microscopy (×63; Leica TCS SP; Leica, Heidelberg, Germany) and monitored at 1-minute intervals over a 5-minute time period.…”