Activation of calpain I and hydrolysis of calpain substrates (actin-binding protein, glycoprotein Ib, and talin) are not a function of thrombin-induced platelet aggregation.

Abstract

Calcium-activated neutral proteinase (calpain) has been shown to cleave proteins involved in the maintenance of cell structure. In human platelets, substrates of calpain include glycoprotein Ib (GPIb), actin-binding protein (ABP), and talin. GPIb-ABP complexes can be isolated in detergent extracts and are thought to represent membrane-cytoskeleton attachment sites. It has been hypothesized that the hydrolysis of GPIb-ABP by calpain is regulated by the extent of binding of this proteinase to the plasma membrane-cytoskeleton interface with platelet activation. Recently, another calpain substrate (talin) has been shown to redistribute from the cytoplasm to the plasma membrane-cytoskeleton interface as the result of thrombin stimulation. To investigate the intracellular distribution of calpain I, we employed the monoclonal antibody B27D8, specific for the heavy chain (catalytic subunit) of calpain I. Indirect immunofluorescent staining of resting human platelets revealed undetectable surface antigen. Permeabilization with Triton X-100, however, revealed a diffuse intracellular antigen consistent with a cytosolic distribution. To determine whether this antigen distribution reflected the proenzyme or the activated form of calpain I and to assess the degree of hydrolysis of ABP, GPIb, and talin, we employed B27D8 and murine monoclonal antibodies against ABP (1B3 and 3D1), GPIb (LJIblO), and rabbit polyclonal antibodies against talin (A2 and Bll) in a quantitative immunotransblot assay. Examination of resting platelets revealed that calpain I existed as the 85 -kd proenzyme form and that ABP, GPIb, and talin existed in their native intact forms. When platelets were aggregated with thrombin, autoproteolysis of calpain I occurred within the 30 seconds required to completely solubilize platelet aggregates in sodium dodecyl sulfate-containing buffer and not as a direct result of thrombin-induced activation. When EDTA was added to platelet samples before the addition of sodium dodecyl sulfate lysis buffer, negligible proteolysis of calpain I, ABP, or talin occurred. When platelets were lysed with sodium dodecyl sulfate in the absence of EDTA, autoproteolysis of calpain I and cleavage of ABP and talin occurred. GPIb is not proteolyzed under either condition. In parallel immunofluorescent studies, examination of thrombin-stimulated platelets demonstrated minimal surface staining. Permeabilization revealed a redistribution of the calpain I epitope to an intracellular circumferential ring pattern. We conclude that with thrombin-induced platelet activation, calpain I redistributes intracellularly in the absence of activation of the 85-kd proenzyme form or significant hydrolysis of three important calpain substrates, ABP, GPIb, and talin. Failure to chelate divalent cations at the time of platelet lysis in sodium dodecyl sulfate enables the redistributed calpain I in thrombin-activated platelets to efficiently proteolyze ABP and talin. This proteolysis is initiated at the time of solubilization and is unrelated to thrombin aggrega...

Calpain is a signal transducer and activator of transcription (STAT) 3 and STAT5 protease

“…Methods were the same as those described in the legend for Figure 1C except that STAT3 was recognized by the anti-STAT3 MoAb as in Figure 2A. 19,20 a significant loss of STAT5 immunoreactivity was observed ( Figure 1D), resulting in appearance of a major cleaved band (as in Figure 1A). The band was not recognized by anti-STAT5 antibodies reactive with the carboxy-terminal domain of STAT5 (data not shown).…”

Effect of cold preservation on intracellular calcium concentration and calpain activity in rat sinusoidal endothelial cells

“…A number of studies in different models ranging from the lens of the eye 13 to spinal cord neurons 14 have shown that calpains are important in actin remodeling, and this study extends those observations to cold preserved SEC. Yoshida 13 reported a direct effect of calpain on actin, but calpain is also active against actin-associated proteins such as talin 33 and actin-binding protein 34,35 and localizes in some models to the site on the cell membrane where these proteins are present. 34 Therefore, the effect of calpain on actin disassembly may be partly or entirely indirect, i.e., actin disassembly may be induced by degradation of actin-related proteins.…”

Identification, purification, and characterization of a zyxin-related protein that binds the focal adhesion and microfilament protein VASP (vasodilator-stimulated phosphoprotein).

“…2A by using [32P]VASP overlays as a detection tool. Detection of the VASP-binding protein p83 was strongly dependent op the presence of a specific fibrinogen receptor (integrin a1nb33) antagonist or on high concentrations of a Ca2+ chelator during platelet preparation or platelet lysis, respectively (not shown), suggesting that p83 is susceptible to platelet aggregationdependent proteolysis by calpain (21,22). In a first chromatographic step VASP-binding activity was recovered at neutral pH in the 70-220 mM NaCl eluate of a cation exchanger (S-Sepharose FF) and was nearly quantitatively precipitated by the addition of ammonium sulfate (30% saturation).…”