Background:

E-64d, a membrane permeant derivative of E-64c, a thiol protease inhibitor¹, was tested for ability to inhibit calpain activity in intact platelets.

E-64d inhibits severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 surface glycoprotein incorporation into pseudotyped vesicular stomatitis virus (VSV) particles in Vero cells, an effect that is reduced by expression of the serine protease TMPRSS2.

E-64c or E-64d also inhibited (lanes 3-8), demonstrating their effect on calpain. When the platelets were incubated with these inhibitors for I0 min and were then washed to remove extracellular inhibitor before lysis, neither E-64c nor leupeptin inhibited proteolysis, but E-64d did inhibit. E-64d was able to penetrate the platelet and was thus not removed by washing.E-64c failed to inhibit proteolysis in intact platelets, but E-64d, the permeant inhibitor, did? inhibit intracellular proteolysis.E-64c and E-64d were each able to inhibit the protease activity in lysed platelets. This protease activity has been attributed to calpain by its absolute dependence on Ca²⁺and by inhibition by known inhibitors of calpain. E-64d is able to enter the? intact platelet: i) after washing to remove extracellular inhibitor, there was? no protease activity detected after platelet lysis, and ii) activation of? platelets preincubated with E-64d, but not E-64c, resulted in inhibition of? proteolysis by calpain activated in intact platelets by A23187 plus calcium.

Reference:
1. M. Tamai, K. Matsumoto, S. Omura, I. Koyama, Y. Ozawa, K. Hanada J. Pharmacobio-Dyn., 9 (1986), pp. 672–677
2. E. B. McGowan, E. Becker, and T. C. Detwiler, INHIBITION OF CALPAIN IN INTACT PLATELETS BY THE THIOL PROTEASE INHIBITOR E-64d. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS , Vol. 158, No. 2, 1989
3. Carmen JC, Sinai AP. The Differential Effect of Toxoplasma Gondii Infection on the Stability of BCL2-Family Members Involves Multiple Activities. Front Microbiol. 2011 Jan 24;2:1.