Calpain I remains intact and intracellular during platelet activation. Immunochemical measurements with monoclonal and polyclonal antibodies.
As a step towards understanding the physiological function of calpain (Ca2+-activated neutral proteinase, EC 3.4.22.17) in blood platelets, and in view of some suggestions that calpain is transferred to the platelet external surface during platelet activation, the enzyme was studied with immunochemical methods in resting and thrombin-activated cells. (1) A mouse IgG1 monoclonal antibody was prepared which binds strongly only to the denatured large subunit of human calpain I, and weakly to that of human calpain II. A polyclonal antibody raised against rat calpain II was available which, apart from binding strongly to rat calpain II, binds to the large subunits of human calpain I and II about equally. (2) With these antibodies, it was found that calpain could be detected in fixed platelets in suspension only after permeabilization with 0.1% saponin, and could not be detected on the exterior surface of resting or of activated platelets, or in the supernatant media of these platelets. It was concluded that calpain is not significantly externalized during platelet activation. (3) Immunoblotting showed that conversion of the larger calpain I subunit from 80 kDa into 76-78 kDa occurred only when thrombin-activated platelets were stirred to permit aggregation, and did not occur during unstirred thrombin activation. Although an action of calpain in the 80 kDa form on possible platelet substrates such as cytoskeletal proteins cannot be excluded, calpain is certainly not present as the 76-78 kDa form, which is assumed to be its active form, until aggregation is initiated.
Samis
J A
JA
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Zboril
G
G
Elce
J S
JS
D000906:Antibodies; D001792:Blood Platelets / Q000201:enzymology*; D002154:Calpain / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007122:Immunoelectrophoresis; D010974:Platelet Aggregation
D000906:Antibodies; D002154:Calpain