Summary
Calponin, a basic smooth-muscle protein capable of binding to F-actin, tropomyosin
and calmodulin in vitro, was tested for its expression and subcellular localization
in resting and stimulated human platelets. Using immunoblotting techniques calponin
was revealed as a single protein band with a molecular weight of 34 kDa. Although
calponin has been shown to be proteolytically degraded by calpain, in the presence
of the calpain inhibitor E-64 and EGTA a significant hydrolysis of calponin could
not be detected. Upon stimulation with 10 μM arachidonic acid calponin became increasingly
incorporated into Triton X-100 insoluble cytoskeletal fractions reaching a plateau
after 15 s. The accumulation of calponin in the cytoskeletons of stimulated platelets
paralleled the polymerization of actin into newly formed microfilaments. Immunofluorescence
microscopy revealed a sub-membranous co-localization of calponin and actin in aggregated
platelets. Since isolated calponin is phosphorylated by protein kinase C and Ca2+/calmodulin-dependent protein kinase II thereby losing its inhibitory effect on the
actomyosin MgATPase activity, we examined whether changes in cell shape due to platelet
stimulation are accompanied by a phosphorylation of calponin. By performing immunoblotting
analysis on either resting or stimulated platelets phosphorylation of calponin on
tyrosine, serine or threonine residues could not be demonstrated. In line, [32P]radiolabeling experiments were unable to detect phosphate incorporation into calponin.
These observations support the hypothesis that calponin plays a physiological role
in regulating contraction and secretion of human platelets even in the absence of
its phosphorylation.
