Evidence that agonist-induced activation of calpain causes the shedding of procoagulant-containing microvesicles from the membrane of aggregating platelets.
One of the responses of platelets to stimulation is activation of intracellular calpain (the Ca(2+)-dependent protease). Previously, we have shown that activation of calpain in platelets is involved in the generation of platelet procoagulant activity. Because procoagulant activity is present on the microvesicles that are shed from activated platelets, in this study we examined whether calpain is involved in the shedding of microvesicles. Platelets were incubated with the physiological agonists collagen or thrombin. The extent of activation of calpain correlated positively with the amount of procoagulant-containing microvesicles that formed, and the shedding of procoagulant-containing microvesicles was inhibited by calpeptin, MDL, and EST (E-64-d), three membrane-penetrating inhibitors of calpain. The protein composition of the microvesicles shed from aggregating platelets was similar to that of microvesicles shed by platelets in which the association of the membrane skeleton with the plasma membrane had been disrupted by incubation of platelets with dibucaine or ionophore A23187. Furthermore, like microvesicles shed from dibucaine- or ionophore A23187-treated platelets, those shed from the aggregating platelets possessed procoagulant activity. These results are consistent with the possibility that activation of calpain in aggregating platelets causes the shedding of procoagulant-containing microvesicles. We suggest that the shedding of microvesicles results from the calpain-induced hydrolysis of the platelet membrane skeleton.
Fox
J E
JE
Gladstone Foundation Laboratories for Cardiovascular Disease, Department of Pathology, University of California, San Francisco 94140-0608.
Austin
C D
CD
Reynolds
C C
CC
Steffen
P K
PK
D001779:Blood Coagulation Factors / Q000378:metabolism*; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D000001:Calcimycin / Q000494:pharmacology; D002154:Calpain / Q000097:blood*; D002462:Cell Membrane / Q000502:physiology; D003094:Collagen / Q000494:pharmacology; D003992:Dibucaine / Q000494:pharmacology; D004789:Enzyme Activation; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D015539:Platelet Activation; D010974:Platelet Aggregation; D010980:Platelet Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology
D001779:Blood Coagulation Factors; D010980:Platelet Membrane Glycoproteins; C049426:platelet procoagulant factor; D000001:Calcimycin; D003094:Collagen; D013917:Thrombin; D002154:Calpain; D003992:Dibucaine