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Membrane expression of platelet calpain
AH Schmaier, HN Bradford, D Lundberg, A Farber and RW Colman
Hematology/Oncology Section, Temple University School of Medicine,
Philadelphia, PA 19140.
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.
Volume 75,
Issue 6,
pp. 1273-1281,
03/15/1990
Copyright © 1990 by The American Society of Hematology
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