OC 58.4 - CIB1 Binding to Cytoplasmic Tail of αIIb Is Required for αIIbβ3 Activation in Platelets

Background: It is well-established that platelet integrin αIIbβ3 activation occurs when talin interacts with the β3-subunit, but the role of the αIIb-subunit and its binding partner calcium- and integrin-binding protein 1 (CIB1) in modulating αIIbβ3 activation is unknown.

Aims: To investigate the consequence of CIB1 and αIIb-cytoplasmic tail interaction in αIIbβ3 activation.

Methods: Using a high throughput screening assay a small molecule compound, CI306, was identified to specifically inhibit the interaction of CIB1 and IIb-cytoplasmic tail. Platelet aggregation and JON/A binding were performed in WT and Cib1-/- murine platelets in the presence or absence of CI306. Platelet functional assays such as aggregation, FITC-PAC-1 binding, P-selectin surface expression, fibrinogen binding, thrombin-induced Ca+2 rise and Rap1-activation were assessed in the presence of CI306 in human platelets. To study the interaction of CIB1 and αIIbβ3, immunoprecipitation and blue native PAGE was employed.

Results: Preincubation of platelets with CI306 (100-500nM) dose-dependently attenuated thrombin-induced (0.1U) WT mouse platelet aggregation and JON/A binding reducing it to the level seen in vehicle treated Cib1-/- platelets, but CI306 didn’t affect aggregation or JON/A binding in Cib1-/- platelets confirming the specificity of CI306. Thrombin-stimulated platelet aggregation, and αIIbβ3 activation were abrogated upon pre-treatment with CI306 (500nM). Interestingly, thrombin (0.1U) induced Ca2+ rise an early step and Rap1 activation a late step of inside-out signaling were unaffected upon preincubation with CI306 (500nM) suggesting no effect on inside-out signaling. Platelet accumulation over a collagen surface under arterial shear stress were significantly (P < 0.0001) attenuated by pretreatment with CI306 (500nM) suggesting defective αIIbβ3 activation. We found that translocation of αIIbβ3 and CIB1 to cytoskeleton were significantly inhibited (P < 0.0001) in the presence of CI306, however, interaction between β3 and talin were not affected.

Conclusion(s): In addition to talin binding to β3 tail, CIB1 binding to αIIb tail is necessary for αIIbβ3 activation.