Colocalization of endogenous CIB1 and αIIbβ3 in nonstimulated and agonist-stimulated megakaryocytes. (A) Nonstimulated, PAR4P, or PAR4P + fibrinogen (Fg)–treated megakaryocytes were adhered to poly-l-lysine, fixed, and stained with antibodies against CIB1 and αIIb. CIB1 is shown in green, αIIb in red, and colocalization in yellow. Boxed areas are enlarged to show membrane distribution of CIB1 and αIIb. Bars, 20 μm. (B) Histogram depicts relative colocalization R values calculated as described in Materials and methods. *, P < 0.01, as compared with agonist-stimulated conditions. Data represent means ± SEM (n = 4) for each condition.

Model of CIB1 regulation of αIIbβ3 activation. (A) In the resting state, a limited amount of CIB1 may be associated with a portion of resting αIIbβ3. (B) Upon agonist stimulation, most αIIbβ3 molecules are predicted to become associated with talin, which converts αIIbβ3 to an activated state such that the integrin can bind fibrinogen (Fg). (C) Agonist stimulation may induce a redistribution of additional CIB1 to the plasma membrane to facilitate further association of CIB1 with a portion of αIIbβ3 molecules via binding to the αIIb tail. CIB1-associated αIIbβ3 is unable to bind talin in a fully functional manner and therefore remains in a resting or intermediate state unable to bind fibrinogen.

CIB1 inhibits talin binding to the αIIb cytoplasmic tail and αIIbβ3. (A and B) Solid-phase binding studies using recombinant CIB1 and THD. Increasing concentrations of CIB1 (A) or THD (B) were added to wells coated with αIIb cytoplasmic tail peptide. CIB1 or THD binding was detected using an anti-CIB1 or anti-talin antibody, respectively. (C) Increasing concentrations of αIIb cytoplasmic tail peptide were incubated with a constant concentration of 10 nM CIB1 or THD before addition to αIIb peptide-coated wells. Binding of CIB1 or THD was detected as in A and B, respectively. (D) Competitive inhibition of THD binding to immobilized αIIb cytoplasmic tail peptide by CIB1. 10 nM of soluble THD was added to wells in the presence of increasing concentrations of soluble CIB1. THD binding was detected using an anti-THD rabbit pAb. (E) Competitive inhibition of 10 nM CIB1 binding to immobilized αIIb cytoplasmic tail peptide by increasing concentrations of THD. CIB1 binding was detected using a chicken anti-CIB1 antibody. (F) CIB1 partially inhibits αIIbβ3 binding to immobilized THD. Soluble, activated RGD affinity–purified αIIbβ3 (Frelinger et al., 1990) was incubated with increasing concentrations of CIB1 or CIB1 F173A before addition to immobilized THD. Integrin αIIbβ3 binding was detected using an anti-αIIb mAb. Data represent means ± SEM (≥3).

CIB1 coimmunoprecipitation and inhibition of agonist-induced fibrinogen binding to megakaryocytes. (A) αIIbβ3 coimmunoprecipitates with CIB1 in washed human platelets. CIB1 was immunoprecipitated from lysates of resting or thrombin receptor activating peptide (TRAP)–stimulated human platelets using either a control IgY or anti-CIB1 chicken IgY antibody. The membrane was probed with an anti-αIIb antibody and an anti-CIB1 chicken antibody. Whole cell lysates (WCL) indicate the position of αIIb. Blot represents three separate experiments. (B) Untransduced, EGFP-, CIB1-EGFP–, or CIB1 F173A-EGFP–expressing megakaryocytes were tested for agonist-induced increases in fibrinogen binding upon stimulation with 3 mM PAR4P. Data are percent increases in mean fluorescence over basal binding (i.e., total minus basal binding). *, P < 0.001, as compared with all other groups. The inset shows expression of CIB1-EGFP and CIB1 F173A-EGFP fusion proteins and endogenous CIB1 in transduced megakaryocytes, quantified by densitometry. Data are presented as fold increase over endogenous CIB1 expression. (C) Expression of endogenous CIB1 in control and CIB1 siRNA–transfected megakaryocytes. The membrane was also probed for PAK1 as a loading and siRNA-specificity control. (D) Percent increase in fibrinogen binding to siRNA-treated megakaryocytes stimulated with 1 and 6 mM PAR4P. The P value of murine (m) CIB1 siRNA–treated megakaryocytes was compared with untransfected (untr) or human CIB1 siRNA control cells (ctrl siRNA) at 1 and 6 mM PAR4P. All data represent means ± SEM (≥3). *, P < 0.02; **, P < 0.04.