Clinicaltrials.gov registry number: NCT00296829.
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...
Focal adhesion kinase (125 kDa form; pp125FAK) is a widely expressed non-receptor tyrosine kinase that is implicated in integrin-mediated signal transduction. We have identified a novel means of pp 125FAK regulation in human platelets, in which this kinase undergoes sequential proteolytic modification from the native 125 kDa form to 90, 45 and 40 kDa fragments in thrombin-, collagen- and ionophore A23187-stimulated platelets. The proteolysis of pp125FAK was prevented by pretreating platelets with the calpain inhibitors calpeptin or calpain inhibitor-1, and was reproduced in vitro by incubating immunoprecipitated pp125FAK with purified calpain. Proteolysis of pp125FAK resulted in a dramatic reduction in its autokinase activity and led to its dissociation from the cytoskeletal fraction of platelets. These studies define a novel signal-terminating role for calpain, wherein proteolytic modification of pp125FAK attenuates its autokinase activity and induces its subcellular relocation within the cell.
Platelet adhesion to sites of vascular injury is initiated by the binding of the platelet glycoprotein (GP) Ib-V-IX complex to matrix-bound von Willebrand factor (vWf). This receptor-ligand interaction is characterizedby a rapid on-off rate that enables efficient platelet tethering and rolling under conditions of rapid blood flow. We demonstrate here that platelets adhering to immobilized vWf under flow conditions undergo rapid morphological conversion from flat discs to spiny spheres during surface translocation. Studies of Glanzmann thrombasthenic platelets (lacking integrin ␣ IIb  3 ) and Chinese hamster ovary (CHO) cells transfected with GPIb/IX (CHO-Ib/IX) confirmed that vWf binding to GPIb/IX was sufficient to induce actin polymerization and cytoskeletal reorganization independent of integrin ␣ IIb  3 . vWf-induced cytoskeletal reorganization occurred independently of several well characterized signaling processes linked to platelet activation, including calcium influx, prostaglandin metabolism, protein tyrosine phosphorylation, activation of protein kinase C or phosphatidylinositol 3-kinase but was critically dependent on the mobilization of intracellular calcium. Studies of Oregon Green 488 1,2-bis(o-amino-5-fluorophenoxy)ethane-N,N,N,N-tetraacetic acid tetraacetoxymethyl ester-loaded platelets and CHO-Ib/IX cells demonstrated that these cells mobilize intracellular calcium in a shear-dependent manner during surface translocation on vWf. Taken together, these studies suggest that the vWf-GPIb interaction stimulates actin polymerization and cytoskeletal reorganization in rolling platelets via a shear-sensitive signaling pathway linked to intracellular calcium mobilization.The ability of platelets to adhere to subendothelial matrix proteins and to other activated platelets at sites of vascular injury is essential for the arrest of bleeding and for subsequent vascular repair. The first step in the hemostatic process involves the binding of the platelet adhesion receptor, GPIb/V/IX, to the vascular adhesive protein, vWf.1 Under conditions of rapid blood flow this receptor-ligand interaction is indispensable for tethering platelets to the injured vessel wall as a prerequisite step for integrin-mediated cell arrest (1, 2). This multi-step adhesion mechanism is remarkably similar to that utilized by leukocytes to adhere to post-capillary venules in vivo. Rolling of leukocytes is mediated by one or more selectin family members, whereas irreversible cell adhesion requires activation of  2 integrins (3, 4).vWf is a unique adhesive ligand in that it has the ability to support both the initial transient phase of platelet adhesion as well as integrin ␣ IIb  3 -mediated cell arrest. The A1 domain of vWf contains the binding site for GPIb␣, whereas the C1 domain peptide sequence Arg-Gly-Asp (RGD) binds integrin ␣ IIb  3 (platelet GPIIb/IIIa) (5). Bond formation between vWf and GPIb is rapid, reversible, and inherently resistant to detachment by high shear forces. This latter property of the vWf-GPIb interaction is...
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