The mechanism by which platelets regulate the function of integrin ␣ IIb ␤ 3 (or GPIIb/IIIa), the platelet fibrinogen receptor, is unknown but may involve the binding of proteins or other factors to integrin cytoplasmic domains. To identify candidate cytoplasmic domain binding proteins, we screened a human fetal liver cDNA library in the yeast two-hybrid system, using the ␣ IIb cytoplasmic domain as "bait," and isolated a novel 855-base pair clone. The open reading frame encodes a novel 191-amino acid polypeptide (termed CIB for calciumand integrin-binding protein) that appears to be specific for the cytoplasmic domain of ␣ IIb , since it does not interact with the ␣ v , ␣ 2 , ␣ 5 , ␤ 1 , or ␤ 3 integrin cytoplasmic domains in the yeast two-hybrid system. This protein has sequence homology to two known Ca 2؉ -binding regulatory proteins, calcineurin B (58% similarity) and calmodulin (56% similarity), and has two EF-hand motifs corresponding to the two C-terminal Ca 2؉ binding domains of these proteins. Moreover, recombinant CIB specifically binds 45 Ca 2؉ in blot overlay assays. Using reverse transcriptase-polymerase chain reaction and Western blot analysis, we detected CIB mRNA and protein (ϳ25 kDa), respectively, in human platelets. An enzyme-linked immunosorbent assay performed using either immobilized recombinant CIB or monoclonal antibody-captured ␣ IIb ␤ 3 indicates a specific interaction between CIB and intact ␣ IIb ␤ 3 . These results suggest that CIB is a candidate regulatory molecule for integrin ␣ IIb ␤ 3 .Integrin-mediated adherence of cells to extracellular matrix is important for a variety of physiological processes, such as hemostasis, angiogenesis, and cell differentiation (1). Integrin ␣ IIb ␤ 3 , the platelet fibrinogen receptor, is specific to the megakaryocytic lineage. In unstimulated platelets the majority of ␣ IIb ␤ 3 is in an inactive conformation or low affinity state and is unable to bind soluble ligands. Platelet agonists through their respective receptors transduce a cascade of intracellular signals ultimately converting ␣ IIb ␤ 3 to an active, high affinity state, capable of binding soluble ligands. This activation process is termed inside-out signaling (2).The role of the ␣ IIb and ␤ 3 cytoplasmic domains in inside-out signaling has been indicated using mutants of ␣ IIb ␤ 3 transfected into Chinese hamster ovary cells (3). When wild type ␣ IIb ␤ 3 is expressed in Chinese hamster ovary cells, it is unable to bind soluble fibrinogen. However, when an ␣ IIb ␤ 3 construct lacking the ␣ IIb cytoplasmic domain is expressed, the integrin becomes constitutively active and binds soluble fibrinogen with high affinity (4). Moreover, deletion of the conserved GFFKR motif of the ␣ IIb cytoplasmic domain makes the integrin constitutively active. These data suggest that the ␣ IIb cytoplasmic domain helps to maintain a default low affinity state of ␣ IIb ␤ 3 . Interestingly, a naturally occurring point mutation, Ser-752 3 Pro in the ␤ 3 cytoplasmic domain, disrupts inside-out signaling (5), indica...

Following platelet stimulation by agonists, integrin-alpha IIb beta 3 (or glycoprotein IIb-IIIa) is converted to an activated state that can bind soluble fibrinogen and mediate platelet aggregation. However, little is known about modulation of alpha IIb beta 3 in cell lines. In the present study, we show that agonist stimulation modulates alpha IIb beta 3-dependent adhesive properties of a human erythroleukemic (HEL) cell line. Brief treatment with phorbol 12-myristate 13-acetate (PMA) caused a significant increase in HEL cell adhesion to monoclonal antibodies (MoAbs) specific for activated alpha IIb beta 3 (PAC1 or pl- 55). This adhesion was inhibited by blocking MoAbs or peptides specific for alpha IIb beta 3, but not by anti-Fc gamma receptor-specific MoAb. Similarly, PMA enhanced HEL cell adhesion to immobilized fibrinogen by 10-fold. However, the activation-dependent ligands in solution (ie, PAC1, pl-55, or fibrinogen) did not inhibit the enhanced HEL cell adhesion to immobilized MoAbs PAC1 or pl-55 after PMA treatment. Thus, PMA may increase alpha IIb beta 3-dependent adhesion to immobilized activation-dependent antibodies and fibrinogen by increasing the local concentration of alpha IIb beta 3 to participate in low-affinity interactions, resulting in an increased avidity, changing the affinity state of alpha IIb beta 3, or both.

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