Eleanor B. McGowan

Inhibition of calpain in intact platelets by the thiol protease inhibitor E-64d

E-64d, a membrane permeant derivative of E-64c, a thiol protease inhibitor (Tamai et al. (1986) J. Pharmacobio-Dyn. 9, 672-677), was tested for ability to inhibit calpain activity in intact platelets. Calpain activity was measured by proteolysis of actin-binding protein and talin, two known substrates of calpain. Incubation of platelets with E-64c (not permeant) or E-64d before lysis prevented proteolysis after lysis. When the platelets were incubated with E-64c or E-64d and then washed to remove the drugs before lysis, only E-64d inhibited proteolysis. When platelets were incubated with E-64c or E-64d and then activated with A23187 plus calcium, a treatment that activates intraplatelet calpain, only E-64d inhibited proteolysis. These results indicate that E-64d can enter the intact cell and inhibit calpain.

Delayed degeneration of dystrophic and normal muscle cell cultures treated with pepstatin, leupeptin, and antipain.

Abstract The effect of the proteinase inhibitors, pepstatin, leupeptin, and antipain, on dystrophic and normal embryonic chicken muscle cells growing in tissue culture was determined. The three inhibitors are effective against lysosomal cathepsins as well as other proteinases. The inhibitors appeared to delay atrophy and degeneration of dystrophic muscle fibers markedly; the effect on the normal muscle fibers was less striking. Catheptic activity and acidic autolysis are known to increase in the dystrophic chicken. These results support the suggestion that lysosomal proteases are involved, by an unknown mechanism, in the degradative process in dystrophic tissue. Delay in the process of degradation of muscle tissue suggests that these low molecular weight, nontoxic inhibitors offer some prospects as therapeutic agents for treatment of muscular dystrophy and other degenerative muscle diseases.

The action of calcium-dependent protease on platelet surface glycoproteins

The action of exogenous calcium-dependent protease (CDP) on tritium-labeled surface glycoproteins was analyzed by incubation of labeled, washed human platelets with CDP partially purified from human platelets. Labeled glycoproteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fluorography. Incubation of the labeled platelets with the protease led to a loss (calcium-dependent) from the platelets of glycoproteins Ib and V and concomitant appearance in the supernatant solution of glycocalicin (a proteolytic fragment of glycoprotein Ib), glycoprotein V, and other, unidentified glycoproteins. These changes in surface label were accompanied by alterations in three parameters of platelet function. Compared to control platelets, the CDP-treated platelets were activated by thrombin more slowly and showed less saturable and nonsaturable binding of thrombin. The CDP-treated platelets, but not the controls, aggregated on addition of fibrinogen, indicating that treatment with CDP had exposed fibrinogen receptors. The alterations in surface glycoproteins and functional parameters were compared over a 1000-fold range of CDP treatment. The decreased binding of thrombin and the exposure of fibrinogen receptors were correlated with the release of surface glycoproteins to the supernatant solution, but the slow activation by thrombin was observed under conditions where no release of labeled glycoproteins was detected (i.e., brief incubations with low concentrations of CDP). Activation of the endogenous CDP with 2.5 mM calcium chloride plus the ionophore A23187 was accompanied by hydrolysis of actin-binding protein, a known substrate, and release to the supernatant solution of labeled glycocalicin and glycoprotein V plus a faster-migrating glycoprotein not released by exogenous protease. This effect was observed in the presence of leupeptin, which completely inhibited action of exogenous protease, suggesting that platelet calcium-dependent protease may modify the platelet surface in ways that can cause alterations of platelet function.

In vivo effect of protease inhibitors in denervation atrophy

The protease inhibitors leupeptin and pepstatin were used in vivo to examine their effect on denervation-induced atrophy in chicken pectoralis muscle. Comparison of muscle weights, fiber diameters, and light meromyosin paracrystals in treated versus untreated animals indicated that these inhibitors had a significant effect in delaying denervation atrophy. The studies suggest that protease inhibitors may have potential therapeutic capability in a variety of neuromuscular degenerative disorders.

Succinate potentiates the action of platelet agonists.

Succinate potentiated the actions of ADP, epinephrine, and the endoperoxide analog U46619 on platelets in plasma. Potentiation was observed at concentrations as low as 400 microM. Potentiation occurred immediately and did not change during incubations as long as five min. It was also observed with maleate and malonate but not with fumarate, oxalate, glutarate or succinate analogs with one or both carboxyls blocked. Succinate was able to reverse the inhibition of platelet activation due to elevated cAMP, and succinate and maleate were comparable to ADP in their ability to inhibit PGI2-induced synthesis of cAMP.

Characterization of the thrombin-induced desensitization of platelet activation by thrombin

Brief exposure of platelets to thrombin makes them less sensitive to subsequent activation by thrombin, a phenomenon demonstrated by Shuman, Botney, and Fenton [J. Clin. Invest., 63, 1211-1218, 1979] by incubating prostacyclin-inhibited platelets with thrombin; after removal of thrombin and prostacyclin, the platelets were selectively desensitized to subsequent activation by thrombin. The conditions for this desensitization have been further defined. Inhibition of thrombin-induced platelet activation by prostacyclin was not absolute, it was only temporary, it could be overcome with higher thrombin concentrations, and it varied with platelet concentration and temperature. With low enough thrombin concentrations, high enough prostacyclin concentrations and short enough times of exposure, platelets could be pretreated with thrombin with no evidence of activation. After addition of hirudin to inhibit thrombin, the platelets were washed and tested for thrombin-induced secretion of ATP. Desensitization to thrombin depended on the concentration of thrombin during pretreatment and on the length of pretreatment, consistent with a catalytic modification of a receptor. A less extensive desensitization was observed when platelets without inhibitor were incubated with a sub-threshold level of thrombin before addition of an activating concentration of thrombin. This desensitization also varied with the time of pretreatment and the concentration of sub-threshold thrombin.

INHIBITION OF MUSCLE DEGENERATION USING PROTEASE INHIBITORS

Publisher Summary This chapter discusses the inhibition of muscle degeneration using protease inhibitors. Although, the primary lesion responsible for muscle degeneration in muscular dystrophy is unknown, both the human and animal disease are characterized by an extensive loss of sarcoplasmic and contractile protein and their replacement by fat and connective tissue. There is little doubt that proteases are somehow involved, be they lysosomal or cytoplasmic, when one considers the large quantity of contractile protein that must be catabolized. In dystrophic untreated animals (DU), there was a progressive increase of fatty tissue with considerable fiber degeneration. In contrast, dystrophic treated muscle (DT) showed little fatty infiltration and the general architecture of the muscle was well preserved. It is possible that dystrophic muscle is embryonic in nature and contains contractile proteins in their embryonic form, which are more susceptible to proteolysis.