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

Abstract

Engagement of platelet membrane glycoprotein (GP) Ib-IX-V by von Willebrand factor triggers Ca⁺⁺-dependent activation of αIIbβ3, 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-GPIbα antibody coimmunoprecipitated GPIb-IX and calmodulin from platelet lysates. Following platelet stimulation, calmodulin dissociated from GPIb-IX and, like the GPIb-IX–associated proteins 14-3-3ζ and p85, redistributed to the activated cytoskeleton. Second, a synthetic peptide based on the cytoplasmic sequence of GPIbβ, R149–L167 (single-letter amino acid codes), affinity-isolated calmodulin from platelet cytosol in the presence of Ca⁺⁺ 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 GPIbβ 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 α1c 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.