The firm adhesion and transplatelet migration of leukocytes on vascular thrombus are both dependent on the interaction of the leukocyte integrin, Mac-1, and a heretofore unknown platelet counterreceptor. Here, we identify the platelet counterreceptor as glycoprotein (GP) Ibα, a component of the GP Ib-IX-V complex, the platelet von Willebrand factor (vWf) receptor. THP-1 monocytic cells and transfected cells that express Mac-1 adhered to GP Ibα–coated wells. Inhibition studies with monoclonal antibodies or receptor ligands showed that the interaction involves the Mac-1 I domain (homologous to the vWf A1 domain), and the GP Ibα leucine-rich repeat and COOH-terminal flanking regions. The specificity of the interaction was confirmed by the finding that neutrophils from wild-type mice, but not from Mac-1–deficient mice, bound to purified GP Ibα and to adherent platelets, the latter adhesion being inhibited by pretreatment of the platelets with mocarhagin, a protease that specifically cleaves GP Ibα. Finally, immobilized GP Ibα supported the rolling and firm adhesion of THP-1 cells under conditions of flow. These observations provide a molecular target for disrupting leukocyte–platelet complexes that promote vascular inflammation in thrombosis, atherosclerosis, and angioplasty-related restenosis.

We have identified platelet glycoprotein (GP) Ibα as a counterreceptor for P-selectin. GP Ibα is a component of the GP Ib-IX-V complex, which mediates platelet adhesion to subendothelium at sites of injury. Cells expressing P-selectin adhered to immobilized GP Ibα, and GP Ibα–expressing cells adhered to and rolled on P-selectin and on histamine-stimulated endothelium in a P-selectin–dependent manner. In like manner, platelets rolled on activated endothelium, a phenomenon inhibited by antibodies to both P-selectin and GP Ibα. Unlike the P-selectin interaction with its leukocyte ligand, PSGL-1 (P-selectin glycoprotein ligand 1), the interaction with GP Ibα required neither calcium nor carbohydrate core-2 branching or α(1,3)-fucosylation. The interaction was inhibited by sulfated proteoglycans and by antibodies against GP Ibα, including one directed at a tyrosine-sulfated region of the polypeptide. Thus, the GP Ib-IX-V complex mediates platelet attachment to both subendothelium and activated endothelium.

Here, we present evidence that glycoprotein (GP) Ib alpha, one of three polypeptides that make up the GP Ib--IX complex--the receptor for von Willebrand factor (vWf) on the surface of unactivated platelets--is modified by sulfation of tyrosine residues. Only GP Ib alpha was found to incorporate 35S when the GP Ib--IX complex was immunoprecipitated from [35S]sulfate metabolically labeled L and CHO cells that express the recombinant complex. The occurrence of sulfation on tyrosine residues of the polypeptide backbone was determined by removing O- and N-linked oligosaccharides. Limited proteolytic digestion of metabolically labeled GP Ib alpha revealed that sulfated tyrosine residues are located in the 45-kDa globular region containing the vWf binding site. By mutating potentially sulfated tyrosine residues to phenylalanine and comparing the stoichiometry of sulfate incorporation of these mutants to the incorporation in wild-type GP Ib alpha, three clustered tyrosine residues--Tyr-276, Tyr-278, and Tyr-279-were identified that undergo the modification. Culturing cells in sulfate-depleted medium containing sodium chlorate and guaiacol completely inhibited GP Ib alpha sulfation but did not decrease GP Ib-IX expression on the cell surface. Similarly, transiently transfected CHO cells expressed the mutant GP Ib alpha polypeptide on their surfaces at the same levels as they expressed wild-type GP Ib alpha. These results suggest that tyrosine sulfation of GP Ib alpha has little or no effect on the synthesis, assembly, and surface expression of the GP Ib-IX complex. Nevertheless, inhibiting sulfation of GP Ib alpha reduced the binding of 125I-labeled vWf in the presence of ristocetin by up to 37%.(ABSTRACT TRUNCATED AT 250 WORDS)

Factor XI binds to high affinity sites on the surface of stimulated platelets where it is efficiently activated by thrombin. Here, we provide evidence that the factor XI binding site on platelets is in the glycoprotein (GP) Ib␣ subunit of the GP Ib-IX-V complex as follows. 1) Bernard-Soulier platelets, lacking the complex, are deficient in factor XI binding; 2) two GP Ib␣ ligands, SZ-2 (a monoclonal antibody) and bovine von Willebrand factor, inhibit factor XI binding to platelets; 3) by surface plasmon resonance, factor XI bound specifically to glycocalicin (the extracellular domain of GP Ib␣) in Zn 2؉ -dependent fashion (K d app ϳ 52 nM). We then investigated whether glycocalicin could promote factor XI activation by thrombin, another GP Ib␣ ligand. In the presence of high molecular weight kininogen (45 nM), Zn 2؉ and Ca 2؉ions, thrombin activated factor XI in the presence of glycocalicin at rates comparable with those seen in the presence of dextran sulfate (1 g/ml). With higher high molecular weight kininogen concentrations (360 nM), the rate of thrombin-catalyzed factor XI activation in the presence of glycocalicin was comparable with that on activated platelets. Thus, factor XI binds to the GP Ib-IX-V complex, promoting its activation by thrombin.Coagulation factor XI (FXI) 1 is a disulfide-linked homodimer that can be bound to activated platelets and subsequently activated by thrombin, FXIIa or FXIa (1-10). In plasma, FXI circulates as a complex with high molecular weight kininogen (HK) (1-7). In the presence of HK or prothrombin, FXI binds specifically and reversibly to high affinity sites on the surface of activated human platelets in the presence of zinc and calcium ions (8,9). We have reported that activated platelets promote FXI activation by thrombin in the presence of HK or prothrombin at optimal rates, thereby initiating intrinsic coagulation independent of contact proteins (8).The primary structure of FXI reveals the presence of four repeated Apple domains (A1-A4) within the heavy chain followed by a light chain containing the serine protease catalytic domain (11). Our laboratory has demonstrated that the A3 domain of FXI is essential for the binding of FXI to platelets as both A3 domain peptides and a recombinant A3 domain bind specifically and saturably to the platelet surface (10,12). However, the platelet receptor(s) responsible for this interaction has not been identified.One potential receptor was suggested by our earlier work (13) with platelets from patients with the congenital bleeding disorder Bernard-Soulier syndrome (14,15). This disorder results from a deficiency or functional defects of the platelet glycoprotein (GP) Ib-IX-V complex, a large plasma membrane complex comprising four polypeptide chains: GP Ib␣, GP Ib␤, GP IX, and GP V, arranged in a stoichiometry of 2:2:2:1, respectively (16 -18). Approximately 25,000 copies of the first three peptides reside in the platelet surface along with half as many copies of GP V. The GP Ib-IX-V complex is the receptor responsible for adhering...

The glycoprotein (GP) Ib-IX complex is a platelet surface receptor that binds thrombin as one of its ligands, although the biological significance of thrombin interaction remains unclear. In this study we have used several approaches to investigate the GPIb␣-thrombin interaction in more detail and to study its effect on the thrombin-induced elaboration of fibrin. We found that both glycocalicin and the aminoterminal fragment of GPIb␣ reduced the release of fibrinopeptide A from fibrinogen by about 50% by a noncompetitive allosteric mechanism. Similarly, GPIb␣ caused in thrombin an allosteric reduction in the rate of turnover of the small peptide substrate D-Phe-Pro-Arg-pNA. The K d for the glycocalicin-thrombin interaction was 1 M at physiological ionic strength but was highly salt-dependent, decreasing to 0.19 M at 100 mM NaCl (⌫ salt ‫؍‬ ؊4.2). The salt dependence was characteristic of other thrombin ligands that bind to exosite II of this enzyme, and we confirmed this as the GPIb␣-binding site on thrombin by using thrombin mutants and by competition binding studies. R68E or R70E mutations in exosite I of thrombin had little effect on its interaction with GPIb␣. Both the allosteric inhibition of fibrinogen turnover caused by GPIb␣ binding to these mutants, and the K d values for their interactions with GPIb␣ were similar to those of wild-type thrombin. In contrast, R89E and K248E mutations in exosite II of thrombin markedly increased the K d values for the interactions of these thrombin mutants with GPIb␣ by 10-and 25-fold, respectively. Finally, we demonstrated that low molecular weight heparin (which binds to thrombin exosite II) but not hirugen (residues 54-65 of hirudin, which binds to exosite I of thrombin) inhibited thrombin binding to GPIb␣. These data demonstrate that GPIb␣ binds to thrombin exosite II and in so doing causes a conformational change in the active site of thrombin by an allosteric mechanism that alters the accessibility of both its natural substrate, fibrinogen, and the small peptidyl substrate D-Phe-Pro-Arg-pNA.