Specific heteromeric association of four transmembrane peptides derived from platelet glycoprotein Ib-IX complex

As the receptor on the platelet surface for von Willebrand factor, glycoprotein (GP) Ib-IX complex is critically involved in hemostasis and thrombosis. How the complex is assembled from GP Ibα, GP Ibβ and GP IX subunits, all of which are type I transmembrane proteins, is not entirely clear. Genetic and mutational analyses have identified the transmembrane (TM) domains of these subunits as active participants in assembly of the complex. In this study, peptides containing the transmembrane domain of each subunit have been produced and their interaction with one another characterized. Only the Ibβ TM sequence, but not the Ibα and IX counterparts, can form homo-oligomers in SDS-PAGE and TOXCAT assays. Following up on our earlier observation that a Ibβ-Ibα-Ibβ peptide complex (αβ₂) linked through native juxtamembrane disulfide bonds could be produced from isolated Ibα and Ibβ TM peptides in detergent micelles, we show here that addition of the IX TM peptide facilitates formation of the native αβ₂ complex, reproducing the same effect by the IX subunit in cells expressing the GP Ib-IX complex. Specific fluorescence resonance energy transfer was observed between donor-labeled αβ₂ peptide complex and acceptor-conjugated IX TM peptide in micelles. Finally, the mutation D135K in the IX TM peptide could hamper both the formation of the αβ₂ complex and the energy transfer, consistent with its reported effect in the full-length complex. Overall, our results have demonstrated directly the native-like heteromeric interaction among the isolated Ibα, Ibβ and IX TM peptides, which provides support for the four-helix bundle model of the TM domains in the GP Ib-IX complex and paves the way for further structural analysis. The methods developed in this study may be applicable to other studies of heteromeric interaction among multiple TM helices.     

Glycoprotein Ib and glycoprotein IX in human platelets are acylated with palmitic acid through thioester linkages

The glycoprotein (GP) Ib-IX complex is a major component of the platelet membrane which mediates adhesion of platelets to exposed subendothelium. GP Ib is a heterodimer with a large alpha chain (Mr = 135,000-145,000) and small beta chain (Mr = 22,000-27,000) linked by a disulfide bond(s). GP Ib is bound in a noncovalent 1:1 complex with GP IX (Mr = 17,000-22,000). We labeled isolated human platelets with [3H] palmitate or surface-labeled platelet membrane glycoproteins with sodium periodate-[3H]sodium borohydride and immunoprecipitated the GP Ib-IX complex from radiolabeled platelet lysates using a mouse monoclonal antibody (SZ.1) which recognizes the intact complex. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitates from [3H]palmitate-labeled platelets revealed two radiolabeled bands under reducing conditions at 24 and 19 kDa and two bands under nonreducing conditions at 170 and 19 kDa. As demonstrated by the parallel analysis of immunoprecipitates from periodate-[3H]sodium borohydride-labeled platelets, the [3H]palmitate-labeled bands obtained under reducing conditions corresponded to GP Ib beta and GP IX and the ones obtained under nonreducing conditions to intact GP Ib and GP IX, respectively. Using alkaline methanolysis followed by high pressure liquid chromatography analysis of the methanolysis products, we demonstrated that the radioactivity associated with the GP Ib-IX complex from [3H]palmitate-labeled platelets was, in fact, covalently bound [3H]palmitate in ester linkage to protein. The protein-fatty acid linkage was also disrupted by hydroxylamine at neutral pH. Thus, this study demonstrates that GP Ib beta and GP IX in human platelets are both fatty acid-acylated with palmitate through thioester linkages.     

The dimerization interface of the glycoprotein Ibβ transmembrane domain corresponds to polar residues within a leucine zipper motif

Experiments with the transmembrane (TM) domains of the glycoprotein (GP) Ib-IX complex have indicated that the associations between the TM domains of these subunits play an important role in the proper assembly of the complex. As a first step toward understanding these associations, we previously found that the Ibβ TM domain dimerized strongly in Escherichia coli cell membranes and led to Ibβ TM-CYTO (cytoplasmic domain) dimerization in the SDS-PAGE assay, while neither Ibα nor IX TM-CYTO was able to dimerize. In this study, we used the TOXCAT assay to probe the Ibβ TM domain dimerization interface by Ala- and Leu-scanning mutagenesis. Our results show that this interface is based on a leucine zipper-like heptad repeat pattern of amino acids. Mutating either one of polar residues Gln129 or His139 to Leu or Ala disrupted Ibβ TM dimerization dramatically, indicating that polar residues might form part of the leucine zipper-based dimerization interface. Furthermore, these specific mutational effects in the TOXCAT assay were confirmed in the thiol-disulfide exchange and SDS-PAGE assays. The computational modeling studies further revealed that the most likely leucine zipper interface involves hydrogen bonding of Gln129 and electrostatic interaction of the His139 side chain. Correlation of computer modeling results with experimental mutagenesis studies on the Ibβ TM domain may provide insights for understanding the role of the association of TM domains on the assembly of GP Ib-IX complex.     

Identification of a region in the cytoplasmic domain of the platelet membrane glycoprotein Ib-IX complex that binds to purified actin-binding protein.

The platelet membrane glycoprotein (GP) Ib-IX complex is a major site of attachment of the platelet membrane skeleton to the plasma membrane. This association is mediated by the interaction of actin-binding protein with the GP Ib-IX complex. The aim of the present work was to identify domains on the GP Ib-IX complex that interact with actin-binding protein. Synthetic peptides corresponding to sequences of the GP Ib alpha-chain and beta-chain cytoplasmic domains were analyzed for their ability to bind to purified actin-binding protein. Two overlapping peptides encompassing a sequence (Thr-536-Phe-568) from the central region of the cytoplasmic domain of GP Ib alpha were the most effective in binding 125I-actin-binding protein, as assessed by a microtiter well approach and peptide affinity chromatography. One of the active peptides (Thr-536-Leu-554) was chosen to evaluate the likelihood that the central region of the cytoplasmic domain of GP Ib alpha is involved in binding of the intact complex to actin-binding protein. This peptide could be specifically cross-linked to purified actin-binding protein in solution. Rabbit polyclonal antibody against this peptide inhibited the binding of purified actin-binding protein to the purified GP Ib-IX complex. Finally, as in intact platelets, the calpain-induced hydrolytic fragments of purified actin-binding protein (M(r) = 200,000 and M(r) = 91,000) showed little binding to the GP Ib alpha peptide. Taken together, these results provided evidence that a region between Thr-536 and Phe-568 of the cytoplasmic domain of GP Ib alpha participates in the interaction of the GP Ib-IX complex with actin-binding protein.     

Interaction of purified actin-binding protein with the platelet membrane glycoprotein Ib-IX complex

The interaction of platelet membrane glycoprotein (GP) Ib-IX complex with the cytoplasmic membrane skeleton is potentially of major importance in regulating platelet function. Indirect evidence suggested that this interaction is mediated by actin-binding protein, but it is not known whether GP Ib-IX and actin-binding protein associate directly. To examine more closely the nature of this association, purified GP Ib-IX complex was specifically bound and oriented on the surface of impermeable polymer beads via a monoclonal antibody, AK 2, directed against the extracytoplasmic domain of GP Ib alpha (glycocalicin). Binding was specific since 1) it was abolished by excess unlabeled actin-binding protein; 2) there was no detectable specific binding of radiolabeled actin-binding protein to beads coated with glycocalicin, the major extracytoplasmic proteolytic fragment of GP Ib alpha; and 3) unlike actin-binding protein, there was no specific binding of bovine serum albumin or human platelet vinculin to the GP Ib-IX complex-coated beads. Binding of actin-binding protein to the GP Ib-IX complex-coated beads, but not to the glycocalicin-coated beads, was saturable and reversible (apparent Kd = 1 x 10(-7) M). These experiments provide direct evidence that actin-binding protein can bind to the cytoplasmic domain of a membrane glycoprotein. Because actin-binding protein is found submembranously in cells other than the platelet, it is possible that this protein may link actin filaments to the plasma membrane in those cells.     

Interaction between platelet glycoprotein Ibalpha and filamin-1 is essential for glycoprotein Ib/IX receptor anchorage at high shear.

The interaction of the glycoprotein (GP) Ib-V-IX receptor complex with the membrane skeleton of platelets is dependent on a specific interaction between the cytoplasmic tail of GPIbalpha and filamin-1. This interaction has been proposed to regulate key aspects of platelet function, including the ligand binding of GPIb-V-IX and the ability of the cells to sustain adhesion to von Willebrand factor (vWf) under high shear. In this study we have examined sequences in the GPIbalpha intracellular domain necessary for interaction of the receptor with filamin-1. We have identified two adjacent sequences involving amino acids 557-568 and 569-579 of the GPIbalpha cytoplasmic domain that are critical for normal association between the receptor complex and filamin-1. Under flow conditions, Chinese hamster ovary (CHO) cells expressing these two mutant receptors exhibited an increase in translocation velocity that was associated with increased cell detachment from the vWf matrix at high shear. The shear-dependent acceleration in velocity of mutant Delta557-568 and Delta569-579 CHO cells was associated with a critical defect in receptor anchorage, evident from significant extraction of GPIb-IX from the CHO cell membrane at high shear. These studies define a critical role for amino acids within the 557-579 sequence of GPIbalpha for interaction with filamin-1.     

Necessity of conserved asparagine residues in the leucine-rich repeats of platelet glycoprotein Ib alpha for the proper conformation and function of the ligand-binding region

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 Ib alpha, 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 Ib alpha 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 Ib alpha 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.     

Efficient plasma membrane expression of a functional platelet glycoprotein Ib-IX complex requires the presence of its three subunits.

The glycoprotein (GP) Ib-IX complex of the platelet plasma membrane mediates the adhesion of platelets to damaged blood vessel wall. The complex is composed of three membrane-spanning polypeptides, GP Ib alpha, GP Ib beta, and GP IX, all of which are absent from the platelets of patients with the hereditary bleeding disorder Bernard-Soulier syndrome. In this study we report stable expression of the recombinant receptor in three cell lines and demonstrate that the three subunits of the complex are necessary for its efficient expression on the plasma membrane. The expressed complex associates with the cytoskeleton of the transfected cells through an interaction with actin-binding protein and binds its ligand, von Willebrand factor. These data suggest that the lack of plasma membrane GP Ib-IX complex in the Bernard-Soulier syndrome could potentially arise from mutations affecting any one of its three subunits.     

Purification and preliminary characterization of the glycoprotein Ib complex in the human platelet membrane

Human platelet glycoprotein Ib (GP Ib) is a major integral membrane protein that has been identified as the platelet-binding site mediating the factor VIII/von Willebrand-factor-dependent adhesion of platelets to vascular subendothelium. Recent evidence suggests that GP Ib is normally complexed with another platelet membrane protein, GP IX. In this study, human platelet plasma membranes were selectively solubilized with a buffer containing 0.1% (v/v) Triton X-100. The GP Ib complex (GP Ib plus GP IX) was purified to homogeneity in approximately 30% yield by immunoaffinity chromatography of the membrane extract using the anti-(glycoprotein Ib complex) murine monoclonal antibody, WM 23, coupled to agarose. GP Ib and GP IX were subsequently isolated as purified components by immunoaffinity chromatography of the GP Ib complex using a second anti-(glycoprotein Ib complex) monoclonal antibody, FMC 25, coupled to agarose. As assessed by dodecyl sulphate/polyacrylamide gel electrophoresis, purified GP Ib was identical to the molecule on intact platelets and had an apparent relative molecular mass of 170 000 under nonreducing conditions and 135 000 (alpha subunit) and 25 000 (beta subunit) under reducing conditions. GP IX had an apparent Mr of 22 000 under both nonreducing and reducing conditions. Purified Gb Ib complex and GP Ib inhibited the ristocetin-mediated, human factor VIII/von Willebrand-factor-dependent and bovine factor VIII/von Willebrand-factor-dependent agglutination of washed human platelets suggesting the proteins had been isolated in functionally active form. GP Ib alpha had a similar amino acid composition to that previously reported for its proteolytic degradation product, glycocalicin. The amino acid compositions of GP Ib beta and GP IX were similar but showed marked differences in the levels of glutamic acid, alanine, histidine and arginine. The N-termini of GP Ib alpha and GP IX were blocked; GP Ib beta had the N-terminal sequence, Ile-Pro-Ala-Pro-. On crossed immunoelectrophoresis, both GP Ib and GP IX were found to occur in the same immunoprecipitin arc(s) whether the platelets had been solubilized in the absence or presence of the calcium-dependent protease inhibitor, leupeptin. Binding studies in platelet-rich plasma indicated a similar number of binding sites (means +/- SD) for three anti-(glycoprotein Ib complex) monoclonal antibodies: AN 51, epitope on GP Ib alpha (22 000 +/- 2700, n = 3), WM 23, epitope on GP Ib alpha (21 000 +/- 3400, n = 3), FMC 25, epitope on GP IX (20 100 +/- 2700, n = 3), and FMC 25 (Fab')2 (27 100 +/- 800, n = 2).(ABSTRACT TRUNCATED AT 400 WORDS)     

Cross-linking of a monomeric 39/34-kDa dispase fragment of von Willebrand factor (Leu-480/Val-481-Gly-718) to the N-terminal region of the alpha-chain of membrane glycoprotein Ib on intact platelets with bis(sulfosuccinimidyl) suberate

A 39/34-kilodalton (kDa) monomeric dispase fragment of von Willebrand factor (vWF) has been purified by heparin affinity chromatography. Detailed structural analysis of the individual 39- and 34-kDa fragments indicated that they had identical amino acid sequences extending from Leu-480/Val-481 to Gly-718 with an intramolecular disulfide bond between Cys-509 and Cys-695. In addition to the binding site for heparin, the 39/34-kDa fragment also contained binding sites for collagen and for platelet membrane glycoprotein (GP) Ib. Unlike native vWF, the 39/34-kDa fragment bound to GP Ib without the requirement for a modulator but showed increased binding in the presence of botrocetin. The 39/34-kDa vWF fragment was cross-linked to intact human platelets by using the membrane-impermeable, homobifunctional cross-linking reagent bis(sulfosuccinimidyl) suberate. Two distinct cross-linked species of similar molecular weight (220/200 kDa, nonreduced; 190/175 kDa, reduced) were identified by SDS-polyacrylamide gel electrophoresis and autoradiography, consistent with the cross-linking of the 125I-labeled 39/34-kDa vWF fragment to GP Ib. The formation of these cross-linked species was enhanced 1.5-2.5-fold in the presence of the modulator botrocetin. The platelet membrane protein involved in cross-linking was shown unequivocally to be GP Ib since (i) neither cross-linked species was formed with Bernard-Soulier syndrome platelets, which genetically lack the GP Ib-IX complex, (ii) both cross-linked species were specifically immunoprecipitated by anti-GP Ib polyclonal and monoclonal antibodies, and (iii) the formation of the cross-linked species was completely inhibited only by those anti-GP Ib-IX complex monoclonal antibodies that inhibited vWF-GP Ib-IX complex interaction. Proteolysis of cross-linked platelets with endoproteinase Lys-C, which preferentially cleaves off the N-terminal peptide domain on the alpha-chain of GP Ib, indicated that the 39/34-kDa vWF fragment was cross-linked exclusively to this region of the GP Ib-IX complex.     

The Transmembrane Domains of β and IX Subunits Mediate the Localization of the Platelet Glycoprotein Ib-IX Complex to the Glycosphingolipid-enriched Membrane Domain

We have previously reported that the structural elements of the GP Ib-IX complex required for its localization to glycosphingolipid-enriched membranes (GEMs) reside in the Ibβ and IX subunits. To identify them, we generated a series of cell lines expressing mutant GP Ibβ and GP IX where 1) the cytoplasmic tails (CTs) of either or both GP Ibβ and IX are truncated, and 2) the transmembrane domains (TMDs) of GP Ibβ and GP IX were swapped with the TMD of a non-GEMs associating molecule, human transferrin receptor. Sucrose density fractionation analysis showed that the removal of either or both of the CTs from GP Ibβ and GP IX does not alter GP Ibα-GEMs association when compared with the wild type. In contrast, swapping of the TMDs of either GP Ibβ or GP IX with that of transferrin receptor results in a significant loss (∼50%) of GP Ibα from the low density GEMs fractions, with the largest effect seen in the dual TMD-replaced cells (>80% loss) when compared with the wild type cells (100% of GP Ibα present in the GEMs fractions). Under high shear flow, the TMD-swapped cells adhere poorly to a von Willebrand factor-immobilized surface to a much lesser extent than the previously reported disulfide linkage dysfunctional GP Ibα-expressing cells. Thus, our data demonstrate that the bundle of GP Ibβ and GP IX TMDs instead of their individual CTs is the structural element that mediates the β/IX complex localization to the membrane GEMs, which through the α/β disulfide linkage brings GP Ibα into the GEMs.     

The transmembrane domain of glycoprotein Ibbeta is critical to efficient expression of glycoprotein Ib-IX complex in the plasma membrane

Lack of expression of glycoprotein (GP) Ib-IX-V complex in platelets often results from mutations in its three subunits: GP Ibalpha, GP Ibbeta, or GP IX. The requirement of all three subunits in the efficient surface expression of the receptor complex has been reproduced in Chinese hamster ovary cells. Here, we probed the role of the transmembrane domains in expression of the GP Ib-IX complex and potential interactions between these domains. Replacing the transmembrane domains of either GP Ibalpha or GP Ibbeta, but not that of GP IX, with unrelated sequences markedly diminished surface expression of the GP Ib-IX complex in transiently transfected Chinese hamster ovary cells. Replacement of the Ibbeta transmembrane domain produced the largest effect. Furthermore, several single-site mutations in the Ibbeta transmembrane domain were found to significantly decrease overall expression as well as surface expression of GP Ibalpha, probably by perturbing the interaction between the Ibalpha and Ibbeta transmembrane domains and in turn reducing the stability of GP Ibalpha in the cell. Mutations S503V and S503L in the Ibalpha transmembrane domain partly reversed the expression-decreasing effect of mutation H139L, but not the others, in the Ibbeta transmembrane domain, suggesting a specific interaction between these two polar residues. Together, our results have demonstrated the importance of the Ibbeta transmembrane domain, through its interaction with the Ibalpha counterpart, to the proper assembly and efficient surface expression of the GP Ib-IX complex.     

Purification of botrocetin from Bothrops jararaca venom. Analysis of the botrocetin-mediated interaction between von Willebrand factor and the human platelet membrane glycoprotein Ib-IX complex

Interaction of von Willebrand factor (vWF) with its platelet receptor only occurs in vitro in the presence of a modulator such as ristocetin. We have recently confirmed that the human platelet membrane glycoprotein (GP) Ib-IX complex is the receptor involved in the ristocetin-dependent binding of vWF by reconstitution with the purified components [Berndt, M.C., Du, X., & Booth, W.J. (1988) Biochemistry 27, 633-640]. We have now developed a similar solid-phase reconstitution assay using an alternate modulator, botrocetin, for the competitive analysis of functional domains in both vWF and the GP Ib-IX complex. Botrocetin was purified from Bothrops jararaca venom by ammonium sulfate fractionation and subsequent DEAE-cellulose and hydroxylapatite chromatography. The purified protein was a 25-kilodalton (kDa) disulfide-linked dimer with apparent subunit molecular weights of 14,000 and 14,500. Binding studies with immobilized botrocetin demonstrated that botrocetin bound to vWF and to a 52/48-kDa region of vWF that contains the GP Ib binding domain, but not to glycocalicin, a proteolytic fragment of GP Ib that contains the vWF binding site. Binding of 125I-labeled vWF to GP Ib-IX complex coated beads and to platelets was strictly botrocetin-dependent with half-maximal binding at a botrocetin concentration of congruent to 0.27 microM. Botrocetin-dependent binding of vWF was specific, saturable, and comparable to that observed with ristocetin. An anti-vWF monoclonal antibody, 3F8, inhibited ristocetin- but not botrocetin-dependent binding of vWF, suggesting the presence of distinct ristocetin and botrocetin modulator sites on vWF. The botrocetin reconstitution assay was at least an order of magnitude more sensitive than the corresponding ristocetin assay for the competitive analysis of functional domains on both vWF and the GP Ib-IX complex and has confirmed the localization of the vWF-binding domain to the 45-kDa N-terminal region of GP Ib.     

Ristocetin-dependent reconstitution of binding of von Willebrand factor to purified human platelet membrane glycoprotein Ib-IX complex

Whether the human platelet membrane glycoprotein (GP) Ib-IX complex is the receptor for ristocetin-dependent binding of von Willebrand factor (vWF) has been examined by reconstitution with the purified components using a solid-phase bead assay. Purified GP Ib-IX complex was bound and orientated on the beads via a monoclonal antibody, FMC 25, directed against the membrane-associated region of the complex. Specific binding of 125I-labeled vWF to the GP Ib-IX complex coated beads was strictly ristocetin dependent with maximal binding occurring at ristocetin concentrations greater than or equal to 1 mg/mL. Ristocetin-dependent specific binding of 125I-labeled vWF was saturable. The observed binding was specific to the interaction between vWF and the GP Ib-IX complex since there was no ristocetin-dependent specific binding of vWF if the physicochemically related platelet membrane glycoprotein, GP IIb, was substituted for the GP Ib-IX complex in a corresponding bead assay. Further, neither bovine serum albumin nor other adhesive glycoproteins, such as fibrinogen or fibronectin, specifically bound to the GP Ib-IX complex in the presence of ristocetin. Ristocetin-dependent binding of vWF to platelets and to GP Ib-IX complex coated beads was inhibited by monoclonal antibodies against a 45,000 molecular weight N-terminal region of GP Ib but not by monoclonal antibodies directed against other regions of the GP Ib-IX complex. Similar correspondence between platelets and purified GP Ib-IX complex with respect to the ristocetin-dependent binding of vWF was obtained with anti-vWF monoclonal antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of the glycoprotein Ib.IX complex from platelet membranes

The glycoprotein Ib.IX complex is a major component of the platelet membrane. It mediates the adhesion of platelets to exposed subendothelium and provides an attachment site for the membrane skeleton on the plasma membrane. The present study was designed to characterize the structure of the glycoprotein Ib.IX complex. Electron microscopy of purified glycoprotein Ib.IX complex in detergent showed that each complex existed as a flexible rod with a globular domain on either end. The overall length of the complex was approximately 59.5 nm. The smaller globular domain had a diameter of approximately 8.9 nm; the larger, a diameter of approximately 15.9 nm. In the absence of detergent, the glycoprotein Ib.IX complexes tended to self-associate through the larger globular domain, suggesting that this domain contained the hydrophobic region that inserts into the membrane. Proteases known to cleave glycoprotein Ib alpha close to its membrane-insertion site released the larger globular domain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that this domain was composed of glycoprotein Ib beta, glycoprotein IX, and a Mr = 25,000 fragment of glycoprotein Ib alpha. Proteolysis at the external end of glycoprotein Ib alpha reduced the size of the smaller globular domain. This study shows that the glycoprotein Ib.IX complex has an elongated shape, with a globular domain on the end that inserts into the membrane and a smaller globular domain on the end of glycoprotein Ib alpha that is oriented external to the plasma membrane.     

Identification of a site in the alpha chain of platelet glycoprotein Ib that participates in von Willebrand factor binding

The binding of von Willebrand factor (vWF) to the platelet receptor glycoprotein (GP) Ib-IX complex is a key event in hemostasis and may participate in the development of thrombotic vascular occlusion. We present here evidence that residues Ser251-Tyr279 in the GP Ib alpha-chain participate in this function. Initial studies suggested that the modality of vWF interaction with GP Ib depended on the conditions used for induction of binding, either in the presence of ristocetin, or botrocetin, or with asialo-vWF. In fact, only the 45-kDa amino-terminal fragment of GP Ib alpha inhibited the vWF-GP Ib interaction under all conditions tested, while the 84-kDa macroglycopeptide was significantly effective only in the presence of ristocetin. Moreover, the 45-kDa fragment with reduced disulfide bonds still inhibited ristocetin-induced binding but had no effect, at the concentrations tested, on botrocetin-mediated or direct asialo-vWF binding. In order to localize in more detail the functional site, the entire sequence of the 45-kDa fragment was reproduced in 27 overlapping synthetic peptides that were then used in inhibition of binding assays. This led to the identification of a linear GP Ib alpha sequence (residues Ser251-Tyr279) that effectively inhibited platelet interaction with vWF mediated by ristocetin and, at higher concentration, also by botrocetin. A shorter peptide overlapping with the longer one (residues Gly271-Glu285) was the second most active inhibitory species. This region of the molecule contains several residues with a high surface probability index, as expected for a site involved in ligand binding. Thus, while native conformation of GP Ib alpha appears to be important for optimal interaction with vWF, the results obtained with short synthetic peptides may help in defining the amino acid residues participating in this essential function.     

The von Willebrand factor-glycoprotein Ib/V/IX interaction induces actin polymerization and cytoskeletal reorganization in rolling platelets and glycoprotein Ib/V/IX-transfected cells

Platelet adhesion to sites of vascular injury is initiated by the binding of the platelet glycoprotein (GP) Ib-V-IX complex to matrix-bound von Willebrand factor (vWf). This receptor-ligand interaction is characterized by a rapid on-off rate that enables efficient platelet tethering and rolling under conditions of rapid blood flow. We demonstrate here that platelets adhering to immobilized vWf under flow conditions undergo rapid morphological conversion from flat discs to spiny spheres during surface translocation. Studies of Glanzmann thrombasthenic platelets (lacking integrin alpha(IIb)beta(3)) and Chinese hamster ovary (CHO) cells transfected with GPIb/IX (CHO-Ib/IX) confirmed that vWf binding to GPIb/IX was sufficient to induce actin polymerization and cytoskeletal reorganization independent of integrin alpha(IIb)beta(3). vWf-induced cytoskeletal reorganization occurred independently of several well characterized signaling processes linked to platelet activation, including calcium influx, prostaglandin metabolism, protein tyrosine phosphorylation, activation of protein kinase C or phosphatidylinositol 3-kinase but was critically dependent on the mobilization of intracellular calcium. Studies of Oregon Green 488 1, 2-bis(o-amino-5-fluorophenoxy)ethane-N,N,N',N-tetraacetic acid tetraacetoxymethyl ester-loaded platelets and CHO-Ib/IX cells demonstrated that these cells mobilize intracellular calcium in a shear-dependent manner during surface translocation on vWf. Taken together, these studies suggest that the vWf-GPIb interaction stimulates actin polymerization and cytoskeletal reorganization in rolling platelets via a shear-sensitive signaling pathway linked to intracellular calcium mobilization.     

Functional modulation of the isolated glycoprotein Ib binding domain of von Willebrand factor expressed in Escherichia coli

We have expressed in Escherichia coli the domain of von Willebrand factor (vWF) containing the binding site for platelet glycoprotein (GP) Ib and used it to study the regulation of vWF-platelet interaction. The recombinant fragment, comprising residues 445-733 of the mature vWF subunit and designated rvWF445-733, did not have the native conformation of the corresponding domain in the intact molecule because, in order to prevent formation of random aggregates, the seven cysteine residues in the sequence were reduced and alkylated. Unlike native vWF, rvWF445-733 bound to GP Ib in the absence of any modulator, suggesting that the lack of disulfide bonds and/or carbohydrate side chains within this domain may expose platelet interaction sites. In the presence of two modulators, the glycopeptide ristocetin and the snake protein botrocetin, rvWF445-733 inhibited native vWF binding to GP Ib as well as platelet aggregation mediated by vWF, suggesting that both the fragment and the native molecule interact with the same site on platelets. This conclusion was also supported by the observation that the recombinant fragment competed with the binding to platelets of an anti-GP Ib monoclonal antibody known to inhibit vWF binding. Botrocetin formed a complex with rvWF445-733, but the affinity of this interaction was approximately 25-fold lower than with native vWF. However, the complexes of botrocetin with either rvWF445-733 or multimeric native vWF bound to GP Ib with similar dissociation constant. Therefore, conformational attributes of vWF regulate its affinity for botrocetin, but once the complex is formed, interaction with GP Ib is independent of native vWF conformation. These findings provide insights into the regulation of vWF-platelet interaction.     

Platelet glycoprotein Ib beta is phosphorylated on serine 166 by cyclic AMP-dependent protein kinase

Platelet responses are inhibited by agents such as prostaglandin E1 that increase the cytoplasmic concentration of cyclic AMP. Inhibition is thought to result from phosphorylation of specific proteins. One protein that becomes phosphorylated is glycoprotein (GP) Ib beta, a component of the GP Ib.IX complex. We have suggested that phosphorylation of GP Ib beta inhibits the collagen-induced polymerization of actin. The aim of the present study was to identify the amino acid(s) in GP Ib beta that is phosphorylated. Purified GP Ib.IX complex was phosphorylated by the catalytic subunit of purified bovine cyclic AMP-dependent protein kinase in the presence of [gamma-32P]ATP. Phosphoamino acid analysis showed that in GP Ib beta, [32P]phosphate was incorporated only into serine and was in a single tryptic peptide. Amino acid sequencing showed that this peptide was from the cytoplasmic domain of GP Ib beta and encompassed residues 161-175. A single serine residue, serine 166, contained the radiolabel. To determine whether the same residue was phosphorylated in intact platelets, GP Ib beta was isolated from 32P-labeled platelets before or after their exposure to prostaglandin E1. In both cases, radiolabel was present in phosphoserine and was in a single tryptic peptide. This peptide was the same as that which was phosphorylated in the purified GP Ib.IX complex, as shown by its identical mobility on two-dimensional tryptic maps, the presence of a positively charged residue in the fourth position, and the presence of the radiolabel in the sixth position of the peptide. This study shows that when cyclic AMP concentrations rise in platelets, the cytoplasmic domain of GP Ib beta is phosphorylated on serine 166, probably by cyclic AMP-dependent protein kinase. We suggest that phosphorylation of this residue may contribute to the inhibitory actions of cyclic AMP by inhibiting collagen-induced polymerization of actin.     

Cyclic AMP-dependent phosphorylation of glycoprotein Ib inhibits collagen-induced polymerization of actin in platelets

Platelet function is inhibited by agents such as prostaglandin E1 (PGE1) that elevate the cytoplasmic concentration of cyclic AMP. Inhibition presumably results from the cyclic AMP-stimulated phosphorylation of intracellular proteins. Polypeptides that become phosphorylated are actin-binding protein, P51 (Mr = 51,000), P36 (Mr = 36,000), P24 (Mr = 24,000), and P22 (Mr = 22,000). Recently, we identified P24 as the beta-chain of glycoprotein (GP) Ib, a component of the plasma membrane GP Ib.IX complex. The existence of Bernard-Soulier syndrome, a hereditary disorder in which platelets selectively lack the GP Ib.IX complex, enabled us to examine whether the phosphorylation of GP Ib beta (P24) is responsible for any of the inhibitory effects of elevated cyclic AMP on platelet function. Exposure of control platelets to PGE1 increased phosphorylation of actin-binding protein, P51, P36, GP Ib beta, and P22. Prostaglandin E1 induced the same phosphorylation reactions in Bernard-Soulier platelets, except that of GP Ib beta, which is absent. In control platelets, PGE1 inhibited collagen-induced phosphorylation of myosin light chain, phosphorylation of P47 (an unidentified Mr 47,000 cytoplasmic protein that is phosphorylated by protein kinase C in stimulated platelets), aggregation, and the secretion of granule contents. Despite the absence of GP Ib beta, PGE1 also inhibited these collagen-induced responses in Bernard-Soulier platelets. However, while PGE1 inhibited collagen-induced polymerization of actin in control platelets, it did not inhibit actin polymerization in Bernard-Soulier platelets. These results suggest that cyclic AMP-induced phosphorylation of GP Ib inhibits collagen-induced actin polymerization in platelets. Because actin polymerization is required for at least some of the functional responses of platelets to an agonist, phosphorylation of Gp Ib beta may be one way in which cyclic AMP inhibits platelet function.