Membrane expression of platelet calpain

Abstract

Platelet calpain has many platelet substrates, including external membrane proteins. We thus investigated whether platelet calpain II was associated with platelet membranes in unstimulated and thrombin- activated platelets. A monospecific, goat polyclonal antibody was reared to purified platelet calpain II. Sixteen whole platelet lysates were found to contain 4.5 +/- 0.7 micrograms calpain antigen II per 10(8) platelets (mean +/- SEM) as determined by a competitive enzyme- linked immunosorbent assay. Using the dipeptide fluorogenic substrate, Suc-Leu-Tyr-MCA, 17 human platelet lysates contained 3.6 +/- 0.4 micrograms calpain activity per 10(8) platelets. Platelet calpain II was associated with the Triton X-100 insoluble platelet cytoskeletons from both unstimulated and thrombin-activated platelets. When compared with the total cell content of platelet calpain II, calpain antigen (10% to 13%) and calpain activity (24% to 28%) was associated with platelet cytoskeletons in unstimulated and thrombin-activated platelets, respectively. On immunoblot, the heavy chain (80 Kd) of calpain II was detected in platelet cytoskeletons. Subcellular fractionation studies on both unstimulated and thrombin-activated platelets, revealed that half of the total platelet calpain II antigen was associated with cytosol, and the other half was associated with the membrane fraction. Platelet calpain II was not seen on the surface of unstimulated, paraformaldehyde fixed platelets by immunofluorescence. However, on thrombin-activated platelets, rim immunofluorescence was seen, indicating that activated platelets externalize their calpain. This observation was confirmed by the finding that about 2,000 molecules per platelet of an 125I-anti-calpain II Fab' specifically bound to thrombin-activated but not unstimulated platelets. Both dibucaine (1 mmol/L) and platelet activating factor (1.86 mumol/L) in the absence of external Ca++, but not collagen (5 micrograms/mL) or ionophore A23187 (2.5 mumol/L) in the absence of external Ca++, were also able to externalize platelet calpain II antigen, as indicated by a similar level of specific 125I-anti-calpain II Fab'-platelet binding. These combined studies indicate that platelet calpain II is a major protein, comprising 2% of total platelet protein, a substantial portion of which is membrane-associated. When platelets are activated by thrombin and platelet activating factor, calpain II antigen also becomes present on the external platelet surface.

Calcium‐activated neutral proteinase (CANP; calpain) activity in Schwann cells: Immunofluorescence localization and compartmentation of μ‐ and mCANP

“…The finding of mCANP in the tSc particulate fraction is comparable to the situation with human neutrophils (Pontremoli et al, 1986), mCANP localization in myelin (Banik et al, 1987a,b;Yanagisawa et al, 1988;DeRosbo et al, 1986;Kolehmainen and Kaisto, 1989), and the recent demonstration of CANP in platelet membrane (Schmaier et al, 1990). The finding of mCANP in the Schwann cell membrane fraction is consistent with the interpretation that mCANP is in PNS myelin (Badalamente et al, 1987;Kamakura et al, 1983;Chakrabarti et al, 1989).…”

“…In this context, it is noteworthy that CANP immunoreactivity has recently been demonstrated in the basal lamina of Schwann cells (Badalamente et al, 1987). Externalization of mCANP has also been demonstrated in thrombin-activated platelet membrane (Schmaier et al, 1990).…”

“…In non-neural tissue, the CANP is mainly in the cytosol and a membrane association has not often been suspected (Kleese et a]., 1987;Yoshimura et al, 1986). However, in platelets, mCANP localization in activated platelet plasmalemma, extracellular space (synovial fluid), and in sarcolemma has been implicated (Barth and Elce, 1987;Dayton and Schollmeyer, 1981;Schmaier et al, 1990;Fukui et al, 1989;Suzuki et al, 1990). Recent immunoelectron-microscopic studies have indicated localization of CANP in sarcoplasmic reticulum, sarcolemma, and other membranous organelles (e.g., mitochondria) of canine myocardium .…”

Calcium‐activated neutral proteinase (calpain) activity in C6 cell line: Compartmentation of μ and m calpain

“…The extraction of the major band (80 kD calpain) in the supernatant is also evident by Coomassie blue staining of the gel. Virtually complete solubilization or release of mcalpain from mylein, synaptosomes, and other membranes by Triton X-100, has been found in many laboratories (Banik et al, 1987a;Zalewska et al , 1988;Schmaier et al, 1990). This was confirmed as well by immunoprecipitation of mcalpain from myelin only in the presence of Triton X-100 and not in its absence (Yanagisawa et al, 1988) indicating that calpain is tightly bound to the membrane.…”

“…We examined distribution of calpain in the particulate (membrane) and cytosol fractions following centrifugation of a homogenate of C6 cells. mCalpain is associated with myelin and other cell membranes (Banik et al, 1987a;Yanagisawa et al, 1988;Schmaier et al, 1990). The true total activity can be determined only after extraction of membranes with detergent (Banik et al, 1987a) and removal of the endogenous inhibitor.…”