We have identified platelet glycoprotein (GP) Ibalpha as a counterreceptor for P-selectin. GP Ibalpha 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 Ibalpha, and GP Ibalpha-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 Ibalpha. Unlike the P-selectin interaction with its leukocyte ligand, PSGL-1 (P-selectin glycoprotein ligand 1), the interaction with GP Ibalpha required neither calcium nor carbohydrate core-2 branching or alpha(1,3)-fucosylation. The interaction was inhibited by sulfated proteoglycans and by antibodies against GP Ibalpha, 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.
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) Ibα 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 Ibα 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 Ibα) in Zn2+-dependent fashion (Kdapp ∼ 52 nm). We then investigated whether glycocalicin could promote factor XI activation by thrombin, another GP Ibα ligand. In the presence of high molecular weight kininogen (45 nm), Zn2+and Ca2+ ions, thrombin activated factor XI in the presence of glycocalicin at rates comparable with those seen in the presence of dextran sulfate (1 μg/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.
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 35Ssulfate 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%.
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.
To study the role of the glycoprotein (GP) Ibα cytoplasmic domain in the mobility of the GP Ib−IX complex within the plasma membrane and in its ability to bind vWf, we established eight cell lines ...expressing GP Ib−IX complexes (these complexes lack GP V but function normally as receptors for vWf) that contain either wild-type GP Ibα or one of a series of GP Ibα truncation mutants missing different lengths of the cytoplasmic domain. To test the mobility of these complexes within the plasma membrane, we used the technique of fluorescence recovery after photobleaching after labeling them with a fluorescein-conjugated anti-GP Ibα monoclonal antibody. Fluorescence recovery within a bleached area on the cell surface was evaluated by scanning the cell surface with a low-intensity laser for 3 min after bleaching and then extrapolating the recovery values to infinite time. Fluorescence recovery in cells expressing wild-type GP Ibα was negligible. However, when only six amino acids were removed from the GP Ibα carboxyl terminus (t604 mutant, polypeptide length of 604 vs 610 residues for wild-type GP Ibα), complex mobility increased greatly, as judged by a more rapid recovery of fluorescence in the bleached area (48% recovery). The mobility increased further in the t594 mutant and remained approximately the same through the t534 mutant (55−67% recovery). A further increase in mobility was observed with the t518 mutant (>80% recovery), which lacks almost all of the GP Ibα cytoplasmic domain. The ristocetin-dependent binding of the mutant cell lines was also evaluated. Binding of vWf to cells expressing any of the mutant complexes was markedly lower than that to cells expressing the wild-type complex. These studies demonstrate that the cytoplasmic domain of GP Ibα fixes the position of the GP Ib−IX complex on the platelet surface and that this orientation is an important determinant of the complex's ability to bind vWf.
Despite the known importance of the sequences surrounding ATG start codons (Kozak sequences) for efficient translation of proteins, few reports have appeared that describe the natural variations in ...these sequences. Here, we report a human polymorphism in the Kozak sequence of the platelet adhesion receptor, glycoprotein (GP) Ibα, a component of the GP Ib-IX-V complex, which mediates the initial adhesion of platelets to the blood vessel wall following injury. The polymorphism is based on the presence of either thymine (T) or cytosine (C) at position −5 from the initiator ATG in the GP Ibα gene. The less common allele, −5C, represented 8% to 17% of the alleles in four ethnic populations surveyed. This allele more closely resembles the sequence considered optimal for efficient initiation of protein translation and is associated with increased expression of the receptor on the cell membrane, both in transfected cells and in the platelets of individuals carrying the allele. In vitro transcription/translation studies indicate that the increased expression results from more efficient translation of the −5C form of the GP Ibα mRNA. Other mutations made to approximate more closely the consensus sequence described by Kozak did not increase expression of the receptor. This is the first known description of Kozak sequence polymorphism as a determinant of the surface levels of a cell adhesion receptor. This polymorphism may influence an individual's susceptibility for the development of cardiovascular disease.
The polypeptides of the platelet von Willebrand factor (vWf) receptor, the GP Ib-IX-V complex, each contain tandem repeats of a sequence that assigns them to the leucine-rich repeat protein family. ...Here, we studied the role of conserved Asn residues in the leucine-rich repeats of GP Ibα, the ligand-binding subunit of the complex. We replaced the Asn residue in the sixth position of the first or sixth leucine-rich repeat (of seven) either with a bulky, charged Lys residue or with a Ser residue (sometimes found in the same position of other leucine-rich repeats) and studied the effect of the mutations on complex expression, modulator-dependent vWf binding, and interactions with immobilized vWf under fluid shear stress. As predicted, the Lys substitutions yielded more severe phenotypes, producing proteins that either were rapidly degraded within the cell (mutant N158K) or failed to bind vWf in the presence of ristocetin or roll on immobilized vWf under fluid shear stress (mutant N41K). The binding of function-blocking GP Ibα antibodies to the N41K mutant was either significantly reduced (AK2 and SZ2) or abolished (AN51 and CLB-MB45). Ser mutations were tolerated much better, although both mutants demonstrated subtle defects in vWf binding. These results suggest a vital role for the conserved asparagine residues in the leucine-rich repeats of GP Ibα for the structure and functions of this polypeptide. The finding that mutations in the first leucine-rich repeat had a much more profound effect on vWf binding indicates that the more N-terminal repeats may be directly involved in this interaction.
Despite the known importance of the sequences surrounding ATG start codons (Kozak sequences) for efficient translation of proteins, few reports have appeared that describe the natural variations in ...these sequences. Here, we report a human polymorphism in the Kozak sequence of the platelet adhesion receptor, glycoprotein (GP) Ib, a component of the GP Ib-IX-V complex, which mediates the initial adhesion of platelets to the blood vessel wall following injury. The polymorphism is based on the presence of either thymine (T) or cytosine (C) at position −5 from the initiator ATG in the GP Ib gene. The less common allele, −5C, represented 8% to 17% of the alleles in four ethnic populations surveyed. This allele more closely resembles the sequence considered optimal for efficient initiation of protein translation and is associated with increased expression of the receptor on the cell membrane, both in transfected cells and in the platelets of individuals carrying the allele. In vitro transcription/translation studies indicate that the increased expression results from more efficient translation of the −5C form of the GP Ib mRNA. Other mutations made to approximate more closely the consensus sequence described by Kozak did not increase expression of the receptor. This is the first known description of Kozak sequence polymorphism as a determinant of the surface levels of a cell adhesion receptor. This polymorphism may influence an individual’s susceptibility for the development of cardiovascular disease.
We investigated the crucial hemostatic interaction between von Willebrand factor (VWF) and platelet glycoprotein (GP) Ib alpha . Recombinant VWF A1 domain (residues Glu super(497)-Pro super(705) of ...VWF) bound stoichiometrically to a GPIb alpha calmodulin fusion protein (residues His super(1)-Val super(289) of GPIb alpha ; GPIb alpha CaM) immobilized on W-7-agarose with a K sub(d) of 3.3 mu M. The variant VWF A1(R545A) bound to GPIb alpha CaM 20-fold more tightly, mainly because the association rate constant k sub(on) increased from 1,100 to 8,800 M super(-1) s super(-1). The GPIb alpha mutations G233V and M239V cause platelet-type pseudo-von Willebrand disease, and VWF A1 bound to GPIb alpha (G233V)-CaM and GPIb alpha (M239V)-CaM with a K sub(d) of 1.0 and 0.63 mu M, respectively. The increased affinity of VWF A1 for GPIb alpha (M239V)-CaM was explained by an increase in k sub(on) to 4,500 M super(-1) s super(-1). GPIb approximately aCaM bound with similar affinity to recombinant VWF A1, to multimeric plasma VWF, and to a fragment of dispase-digested plasma VWF (residues Leu super(480)/Val super(481)-Gly super(718)). VWF A1 and A1(R545A) bound to platelets with affinities and rate constants similar to those for binding to GPIb alpha CaM, and botrocetin had the expected positively cooperative effect on the binding of VWF A1 to GPIb alpha CaM. Therefore, allosteric regulation by botrocetin of VWF A1 binding to GPIb alpha , and the increased binding affinity caused by mutations in VWF or GPIb alpha , are reproduced by isolated structural domains. The substantial increase in k sub(on) caused by mutations in either A1 or GPIb alpha suggests that productive interaction requires rate-limiting conformational changes in both binding sites. The exceptionally slow k sub(on) and k sub(off) provide important new constraints on models for rapid platelet tethering at high wall shear rates.
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