E 64d,88321-09-9,IC-0209408

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 Ca 2+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.