Interaction of calmodulin with the cytoplasmic domain of the platelet membrane glycoprotein Ib-IX-V complex

Engagement of platelet membrane glycoprotein (GIP) Ib-IX-V by von Willebrand factor triggers Ca++-dependent activation of alpha IIb beta3, resulting in (patho)physiological thrombus formation. It is demonstrated here that the cytoplasmic domain of GPIb-IX-V associates with cytosolic calmodulin. First, an anti-GPIb alpha antibody coimmunoprecipitated GPIb-IX and calmodulin from platelet lysates. Following platelet stimulation, calmodulin dissociated from GPIb-IX and, like the GPIb-IX-associated proteins 14-3-3 zeta and p85, redistributed to the activated cytoskeleton. Second, a synthetic peptide based on the cytoplasmic sequence of GPIb beta, R149-L167 (single-letter amino acid codes), affinity-isolated calmodulin from platelet cytosol in the presence of Call as confirmed by comigration with bovine calmodulin on sodium dodecyl sulfate-polyacrylamide gels, by sequence analysis, and by immunoreactivity with the use of an anticalmodulin antibody. The membrane-proximal GPIb beta sequence was analogous to a previously reported calmodulin-binding sequence in the leukocyte adhesion receptor, L-selectin. In addition, the cytoplasmic sequence of GPV, K529-G544, was analogous to a calmodulin-binding IQ motif within the alpha 1c subunit of L-type Ca++ channels. Calmodulin coimmunoprecipitated with GPV from resting platelet lysates, but was dissociated in stimulated platelets. A GPV-related synthetic peptide also bound calmodulin and induced a Ca++-dependent shift on nondenaturing gels. Together, these results suggest separate regions of GPIb-IX-V can directly bind calmodulin, and this novel interaction potentially regulates aspects of GPIb-IX-V-dependent platelet activation. (C) 2001 by The American Society of Hematology.