Journal: The Journal of Experimental Medicine

Article Title: The Glycoprotein Ib-IX-V Complex Is a Platelet Counterreceptor for P-Selectin

doi:

Figure Lengend Snippet: (A) Adhesion of CHO-P cells to glycocalicin. Glycocalicin, the soluble extracellular portion of GP Ibα, was immobilized on the wells of plastic microtiter plates. 51 Cr-labeled CHO-P cells or untransfected CHO cells were then incubated in the wells under static conditions. After the wells were washed, the residual radioactivity was quantitated. (B) Inhibition of CHO-P adhesion to glycocalicin. The experiment was performed as in A, except that the relevant inhibitor or control was either preincubated with the bound glycocalicin (the anti-GP Ibα antibodies) or incubated with the cells during the adhesion assay. The adhesion is displayed as a percentage of the adhesion under control conditions after subtracting the background adhesion of untransfected CHO cells. Data are segregated into three sets. In the first set, the calcium dependence of the interaction was assessed by performing the assay either in the presence or absence of 5 mM EDTA; the second set depicts the effects of GP Ibα mAbs with an irrelevant antibody as a control; and the third set depicts the effect of an anti–P-selectin antibody, with nonimmune rabbit IgG serving as the control. The antibodies used are 2C9, an anti–von Willebrand factor antibody (control), and SZ2, WM23, and AK3, which are all directed against GP Ibα. SZ2 maps to the anionic sulfated region bounded by amino acid residues 276–282; WM23 and AK3 both bind within the mucin-like macroglycopeptide region that lies between the anionic sulfated region and the plasma membrane. The anti-PS antibody is an affinity-purified rabbit polyclonal antibody prepared against P-selectin purified from platelets. The experiment was performed three times in quadruplicate; data shown here are derived from one representative experiment and are presented as mean ± SE of the mean for that experiment. (C) Proteoglycan inhibition of CHO-P adhesion to glycocalicin. Different proteoglycans were tested for their ability to inhibit the adhesion of CHO-P to glycocalicin. The experimental conditions were as in A, except that the CHO-P cells were preincubated with the indicated proteoglycan at the following concentrations: 0.4 mg/ml fucoidin and chondroitin sulfate and 5 mg/ml heparin. As in B, the results are displayed as a percent of the adhesion of the control, untreated CHO-P cells, after subtracting the background adhesion of untransfected CHO cells. Experiments were performed twice in quadruplicate; the results of one representative experiment are shown.

Article Snippet: Anti-GP Ibα mAbs used were: WM23 and AK3, both of which bind within the mucin-like macroglycopeptide ; SZ2, which recognizes an epitope within the anionic/sulfated tyrosine region of GP Ibα bounded by residues Tyr276 and Glu282 ; and AK2 (RDI Research Diagnostics), which binds within the GP Ibα NH 2 terminus and inhibits ristocetin- and botrocetin-induced von Willebrand factor binding .

Techniques: Labeling, Incubation, Radioactivity, Inhibition, Control, Cell Adhesion Assay, Clinical Proteomics, Membrane, Affinity Purification, Purification, Derivative Assay