Calpain I remains intact and intracellular during platelet activation. Immunochemical measurements with monoclonal and polyclonal antibodies

Samis, John A.; Zboril, G; Elce, John S.

doi:10.1042/bj2460481

Cited by 52 publications

(43 citation statements)

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“…The monoclonal antibody to hnRNP C (4F4) has been described previously (Choi and Dreyfuss, 1984) and was a kind gift from Dr. Gideon Dreyfuss (Philadelphia, USA). The antibody to human calpain-I has been previously described (Samis et al, 1987). Monoclonal antibody to Fas (clone CH11) was from Immunex (Seattle, Washington, USA.).…”

Section: Reagentsmentioning

confidence: 99%

Calpain activation is upstream of caspases in radiation-induced apoptosis

et al. 1998

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The molecular events involved in apoptosis induced by ionizing radiation remain unresolved. In this paper we show that the cleavage of fodrin to a 150 kDa fragment is an early proteolytic event in radiation-induced apoptosis in the Burkitts' Lymphoma cell line BL30A and requires 100 mM zVAD-fmk for inhibition. Caspases-1, -3, -6 and -7 were shown to cleave fodrin to the 150 kDa fragment in vitro and all were inhibited by 10 mM zVAD-fmk. We also show that the in vitro cleavage of fodrin by calpain is inhibited by 100 mM zVAD-fmk as was the calpain-mediated hydrolysis of casein. We demonstrate that calpain is activated within 15 min after radiation exposure, concomitant with the cleavage of fodrin to the 150 kDa fragment whereas caspase-3 is activated at 2 h correlating with the cleavage of fodrin to the 120 kDa fragment. These results support a role for calpain in the early phases of the radiation-induced apoptosis pathway, upstream of the caspases.

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Section: Reagentsmentioning

confidence: 99%

Calpain activation is upstream of caspases in radiation-induced apoptosis

et al. 1998

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“…Polyclonal antibodies to brain spectrin (Ab992) and to calpain II and a monoclonal antibody to calpastatin were purchased from Chemicon. The monoclonal antibody against a-actin (clone B-5-l-2) was from Boehringer Mannheim and the monoclonal antibody against the 80-kDa subunit of calpain I (CIAb) was a generous gift of Dr. J. Elce (Samis et al, 1987). Horseradish peroxidase-conjugated secondary antibodies were from BioRad.…”

Section: Antibodies and Chemicalsmentioning

confidence: 99%

Ceramide Selectively Decreases Tau Levels in Differentiated PC12 Cells Through Modulation of Calpain I

Xie

Johnson

1997

Journal of Neurochemistry

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Ceramide has been recently proposed to be a signal mediator in several important physiological processes including apoptosis, cellular growth, and differentiation. Because the microtubule-associated protein tau plays an important role in the establishment and maintenance of neuronal morphology, the effects of ceramide on tau were examined. Treatment of differentiated P012 cells with the cell-permeable ceramide derivative N-acetylsphingosine (C2) resulted in a significant reduction in tau levels. Significant decreases in tau levels were also observed when the cells were treated with another ceramide derivative, N-hexanoylsphingosine (06). In addition, C2 treatment increased the levels of a calpain-derived spectrin breakdown product but did not alter the levels of two cytoskeletal proteins, a-actin and a-tubulin. Because both tau and spectrin are proteolyzed in vitro by the calcium-activated cysteine protease calpain, the effects of ceramide analogues on the activity of this protease were examined. Treatment of P012 cells with C2 enhanced calcium-stimulated proteolytic activity significantly, as revealed by monitoring the hydrolysis of the membrane-permeable calpain-selective fluorescence probe N-succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine-7-amido-4-methylcoumarin. This activity increase was not due to a direct effect of 02 on calpains, because 02 did not alter the activities of purified calpain I or II. In addition, 02 treatment of PC12 cells resulted in a significant increase in the levels of calpain I and, to a lesser extent, the levels of calpastatin (an endogenous calpain inhibitor protein), whereas the levels of calpain II were not changed. Moreover, treatment of the cells with the synthetic calpain-specific inhibitor N-carbobenzoxy-L-leucyl-L-leucyl-L-tyrosine diazomethyl ketone blocked the 02-induced decreases in tau levels. These results indicate that tau levels are regulated in response to a physiological factor and, thus, have implications for ceramidemediated changes in normal and pathological neuronal processes.

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“…13 The specificity of B27D8 was confirmed by quantitative immunotransblot of a platelet cytosolic fraction as described. 16 Although B27D8 also reacts with calpain II, the cross reactivity is extremely low and negligible in these experiments.…”

Section: Antibodiesmentioning

confidence: 99%

“…16 Although B27D8 also reacts with calpain II, the cross reactivity is extremely low and negligible in these experiments. 13 The cell line B27D8 sc E l l was used to produce ascites fluid in BALB/c BYJ mice (Jackson Labs, Bar Harbor, Me. ), and IgG was purified by combining and modifying 17 …”

Section: Antibodiesmentioning

confidence: 99%

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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.

Wencel-Drake

Okita

Annis

et al. 1991

Arterioscler Thromb

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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...

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Calpain I remains intact and intracellular during platelet activation. Immunochemical measurements with monoclonal and polyclonal antibodies

Cited by 52 publications

References 44 publications

Calpain activation is upstream of caspases in radiation-induced apoptosis

Calpain activation is upstream of caspases in radiation-induced apoptosis

Ceramide Selectively Decreases Tau Levels in Differentiated PC12 Cells Through Modulation of Calpain I

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.

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