ICAM-1 is involved in both adhesion and extravasation of leukocytes to endothelium during inflammation. It has been shown that the ICAM-1 cytoplasmic domain is important for transendothelial migration of leukocytes but the precise molecular mechanisms involving the intracytoplasmic portion of ICAM-1 is not known. To characterize precisely the molecular scaffolding associated with ICAM-1, we have used the yeast two-hybrid system, and we have identified six different proteins interacting with the ICAM-1 cytoplasmic domain. In this study, we report that the two forms of nonmuscle α-actinin (i.e., α-actinin 1 and α-actinin 4) associate with ICAM-1, and that these interactions are essential for leukocyte extravasation. These interactions were further confirmed by coimmunoprecipitation and immunofluorescence in endothelial cells and in ICAM-1-transfected Chinese hamster ovary cells. The function of these interactions was analyzed by point mutation of charged amino acids located on ICAM-1 cytoplasmic domain. We have identified three charged amino acids (arginine 480, lysine 481, and arginine 486) which are essential in the binding of α-actinins to the ICAM-1 cytoplasmic tail. Mutation of these amino acids completely inhibited ICAM-1-mediated diapedesis. Experiments with siRNA inhibiting specifically α-actinin 1 or α-actinin 4 on endothelial cells indicated that α-actinin 4 had a major role in this phenomenon. Thus, our data demonstrate that ICAM-1 directly interacts with cytoplasmic α-actinin 1 and 4 and that this interaction is required for leukocyte extravasation.

Abstract. The cytoadhesins represent a group of RGD receptors that belongs to the integrin superfamily of adhesion molecules. Members of this cytoadhesin family include the platelet GPIIb-IIIa and the vitronectin receptors. These glycoproteins share the same/~-subunit, which is associated with different ot subunits to form an a/[3 heterodimer. In the present study, we have analyzed the fine recognition specificity of the cytoadhesins from platelets and endothelial cells for the adhesive protein, fibrinogen. Two sets of synthetic peptides, RGDX peptides and peptides corresponding to the COOH terminus of the fibrinogen 3' chain, were compared for their structure-function relationships in the two cellular systems. The results indicate that: (a) both RGDX and 3'-chain peptides inhibit the binding of fibrinogen to platelets and endothelial cells; (b) a marked influence of the residue at the COOH-and NH2-terminal positions of each peptide set can be demonstrated on the two cell types; and (c) RGDX and "t peptides have differential effects on platelets and endothelial cells with respect to fine structural requirements. These results clearly indicate that while the platelet and endothelial cytoadhesins may interact with similar peptidic sequences, they express a different fine structural recognition.

Participation of fibrinogen in platelet aggregation is contingent upon the capacity of various stimuli to induce specific receptors for the molecule on the surface of the cell. The interaction of fibrinogen with this receptor results directly in platelet aggregation, and dissociation of fibrinogen is associated with disaggregation. While the role of exogenous fibrinogen in this process has been fully documented, the mechanisms which control the surface exposure of platelet fibrinogen are less understood. In the present study Fab fragments of antibodies monospecific for fibrinogen have been used to examine the surface expression of intracellular fibrinogen and its involvement in platelet aggregation. Radiolabelled Fab fragments did not interact with non-stimulated platelets but significant binding was observed when the cells were stimulated by ADP, thrombin, collagen and Ca ionophore A23187. Binding was specific for fibrinogen, was not observed with thrombasthenic platelets and was dependent upon the presence of extracellular calcium. With all stimuli tested, the binding of the Fab probe to platelets correlated with platelet secretion. At the following concentrations of stimuli: 30 pM ADP, 4 pg/ml collagen, 3 pM A23187 and 0.05 U/ml thrombin, the immune Fab fragments inhibited platelet aggregation. A monoclonal antibody to glycoprotein IIb/IIIa complex and a synthetic peptide y400 -41 1, that inhibited the interaction of plasma fibrinogen with platelets, did not inhibit the binding of 12'I-FAB fragments. Taken together these results support the hypothesis that endogenous fibrinogen becomes surface-expressed during stimulation of the cell and can support platelet aggregation, particularly that induced by low concentrations of stimuli. The mechanism for the surface expression of platelet fibrinogen may be distinct from that for the binding of plasma fibrinogen.

The effect of buflomedil (Fonzylane®; Laboratoire Lafon, Maisons-Alfort, France) on platelet function, a drug used clinically for the treatment of peripheral vascular diseases, was investigated in vitro. The compound significantly inhibits epinephrine-induced aggregation at the micromolar level. At higher doses (approximately 1 mM), a weak inhibition of ADP- and collagen-induced aggregation was observed; at these concentrations, buflomedil inhibits granular secretion and the interaction of fibrinogen with its receptor on platelet. Further investigations indicate that the drug affects calcium uptake at the membrane level and inhibits the binding of [³H]-yohimbine to the same extent as observed with phentolamine. The IC₅₀ determined from competition binding assays was 1 ± 0.5 μM. This value was consistent with the affinity constant approximated for the binding of [³H]-buflomedil to non-stimulated platelets. Taken together, these results indicate that the vasoactive compound buflomedil is a weak antiaggregating agent which exhibits α₂-adrenergic antagonistic properties.

Two mouse monoclonal antibodies have been characterized. Both antibodies specifically recognized the amino acid sequences which are responsible for the presence of the HPA-la and HPA-lb alloantigens at the surface of human platelet. The monoclonality allied with an exquisite specificity of these antibodies allow a perfect typing of platelet HPA-1 status. A fast and reliable Elisa assay which fulfills all the needs and requirements of hematology laboratories has been set up and is described here. The Elisa assay has been tested on a population of 689 blood donors. The results agreed with the known phenotypic frequencies of the HPA-1 alloantigens.