HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Interaction of calmodulin with the cytoplasmic domain of the platelet membrane glycoprotein Ib-IX-V complex

Engagement of platelet membrane glycoprotein (GP) Ib-IX-V by von Willebrand factor triggers Ca11-dependent activation of aIIbb3, 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-GPIba antibody coimmunoprecipitated GPIb-IX and calmodulin from platelet lysates. Following platelet stimulation, calmodulin dissociated from GPIb-IX and, like the GPIb-IX– associated proteins 14-3-3z and p85, redistributed to the activated cytoskeleton. Second, a synthetic peptide based on the cytoplasmic sequence of GPIbb, R149– L167 (single-letter amino acid codes), affinity-isolated calmodulin from platelet cytosol in the presence of Ca11 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 membraneproximal GPIbb sequence was analogous to a previously reported calmodulinbinding sequence in the leukocyte adhesion receptor, L-selectin. In addition, the cytoplasmic sequence of GPV, K529– G544, was analogous to a calmodulinbinding IQ motif within the a1c subunit of L-type Ca11 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 Ca11-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. (Blood. 2001;98:681-687)