GPIb Beta Research Articles

The highly specific platelet glycoprotein (GP) VI agonist trowaglerix impaired collagen-induced platelet aggregation ex vivo through matrix metalloproteinase-dependent GPVI shedding.

C-type lectin proteins (CLPs) have diverse targets including platelet GPIb, GPVI and integrin alpha(2)beta(1), and affect platelet function in a various way. In this study, we characterized a huge, heterodimeric venom protein, trowaglerix, which belongs to the CLP family. We purified a potent platelet-aggregation inducer, trowaglerix, from the crude venom of Tropidolaemus wagleri. Biotinylated trowaglerix was used for binding assays, and immunoblotting was used to investigate the signal transduction involved. Two distinct subunits of trowaglerix with similar masses of around 16 kDa were eluted by high-performance liquid chromatography after reduction and alkylation. Trowaglerix induced platelet aggregation of washed human platelets and platelet-rich plasma (PRP) in a concentration-dependent manner. Biotinylated trowaglerix specifically bound to platelet membrane GPVI, but not to GPIb or alpha(2) integrin. Treatment with trowaglerix induced GPVI loss in human platelets in vitro and impaired the platelet aggregation of mouse PRP ex vivo in response to collagen but not in response to adenosine diphosphate (ADP). However, GM6001, a matrix metalloproteinase (MMP) inhibitor, inhibited trowaglerix-induced GPVI cleavage and restored the platelet responsiveness of PRP to collagen. Trowaglerix activates platelets through specific binding to GPVI, leading to kinases-dependent exposure of functional alpha(IIb)beta(3) and platelet aggregation, and also induces MMP-dependent GPVI shedding from platelets.

Two Mutations (1717 T〉C, GP IX;124del145, GPIb beta) Occur Frequently among Patients with Bernard Soulier Syndrome in India.

Bernard Soulier syndrome (BSS) is a rare (1:1000000) hereditary bleeding disorder of platelet adhesion, caused by a qualitative or quantitative defect of glycoprotein (GP)Ib/IX/V complex. We report here the molecular basis of BSS in eight unrelated patients from India. The diagnosis was based on low platelet count, presence of giant platelets and aggregometry studies (absence of response to ristocetin) in eight unrelated patients who presented with mucocutaneous bleeding at 17.9 years of age (range:9 months–60yrs). Flow cytometry to assess surface GPIb/IX/V complex using anti CD42b antibodies showed reduced (7.7%–21.1%, n=6) expression. Genomic DNA was screened for mutations in the GPIb alpha, GPIb beta, GP IX genes by a novel PCR and conformation sensitive gel electrophoresis method. Mutations were identified in all the 8 patients. Six different mutations, including missense (n=4) mutations, a deletion and a duplication were identified of which 5 are novel. Three novel missense mutations were identified in a patient (CD42b:21.1%) of which, Tyr492His in GPIb alpha gene was in heterozygosity and the Glu129His, Leu132Pro mutations in GPIb beta was detected in a double homozygous condition. All these substitutions occur at a highly conserved codon in the transmembrane region of GPIb alpha and beta chains, a region critical for anchoring the glycoprotein subunits into the platelet membrane. A novel homozygous deletion of 22 bp in exon 2 of GPIb beta (124del145, p.Arg17CysfsX14) gene was identified in 3 different patients (CD42b: range, 10.6–15.9%). This deletion occurs at a sequence flanking the leucine rich repeat at the NH2-side of GPIb beta protein results in a premature termination of translation (PTC) and a truncated mutant with 30 amino acids. A novel homozygous duplication of 38 nucleotides (2119_2120 dup38) in the transmembrane region resulted in a frameshift (142AlafsX76) in one of the patient. This frameshift predicts termination after the addition of 75 amino acids. A previously reported homozygous transition (1717 T >C) in the GP IX gene resulting in a Cys8Arg substitution was identified in the remaining 3 patients (CD42b:10.02%, 7.7%). The frequent presence of GP IX 1717 T>C and GPIb beta 124del145 in 6 out of 8 (75%) patients studied, suggests that this could be due to a founder effect. This data adds significantly to the mutation database of this condition and will be useful for genetic diagnosis of this condition in India.

Shear Stress Induced Release of Von Willebrand Factor and Thrombospondin-1 in Uvec Extracellular Matrix Enhances Breast Tumour Cell Adhesion

Endothelial cells in vivo are exposed to blood shear forces and flow perturbations induce their activation. Such modifications of hemodynamic can be observed in patients with cancer. We have submitted endothelial cells (HUVEC) to shear stress (13 dynes/cm(2)) and isolated their extracellular matrix (ECM) prior perfusion with breast adenocarcinoma cells (MDA-MB-231) in whole blood at a shear rate of 1500 s(-1). Exposure of HUVEC to 13 dynes/cm(2) (tau(13)) for 2 h enhanced the secretion of von Willebrand factor (vWF) and thrombospondin-1 (TSP-1) in the ECM. Moreover, MDA-MB-231 cell adhesion was enhanced to such treated-ECM. This over-adhesion was inhibited by pre-incubating the ECM with anti-vWF or anti-TSP-1 antibodies, or by blocking tumour cell alpha(v)beta(3) integrin. Although blood platelets were involved in tumour cell adhesion to ECM, blockade of platelet GPIb or alpha(IIb)beta(3) receptors did not specifically inhibit the enhanced tumour cell adhesion observed on tau(13). ECM. These findings indicate that shear stress can modulate the expression of adhesive proteins in ECM, which favours direct tumour cell adhesion via alpha(v)beta(3) and other receptors.

A palmitylated peptide derived from the glycoprotein Ibβ cytoplasmic tail inhibits platelet activation

The platelet receptor GPIb/IX/V mediates a crucial role in hemostasis, yet the signaling mechanisms involved are incompletely understood. The complex consists of four polypeptides GPIb alpha, GPIb beta, GPIX and GPV. We identified an amino acid sequence in the cytoplasmic tail of the GPIb beta subunit between residues R151 and A161 that is highly conserved across species and hypothesized that it has functional importance. To target this motif, we synthesized a corresponding cell-permeable palmitylated peptide (Pal-RRLRARARARA) and investigated its effect on platelet function. Pal-RRLRARARARA completely inhibited low dose thrombin- and ristocetin-induced aggregation in washed platelets but only partially inhibited collagen- and U46619-induced aggregation. Thromboxane production in platelets stimulated with thrombin was significantly reduced by Pal-RRLRARARARA compared with collagen. Activation of the integrin alpha IIb beta 3 in response to thrombin was significantly reduced when platelets were preincubated with Pal-RRLRARARARA. The adhesion of washed platelets to von Willebrand factor (VWF) under static conditions was significantly reduced by Pal-RRLRARARARA. Under conditions of high shear, the velocity of platelets rolling on VWF was significantly increased when platelets are preincubated with Pal-RRLRARARARA. This study defines a novel function for the RRLRARARARA motif of GPIb beta in platelet activation.

Role of the intracellular domains of GPIb in controlling the adhesive properties of the platelet GPIb/V/IX complex

Glycoprotein (GP) Ib/V/IX complex-dependent platelet adhesion to von Willebrand factor (VWF) is supported by the 45-kd N-terminal extracellular domain of the GPIb alpha subunit. Recent results with an adhesion blocking antibody (RAM.1) against GPIb beta, which is disulfide linked to GPIb alpha, have suggested a novel function of this subunit in regulating VWF-mediated platelet adhesion, possibly involving its intracellular face. A putative cooperation between the GPIb alpha and GPIb beta cytoplasmic domains was investigated by measuring the adhesion under flow to immobilized VWF of K562 and Chinese hamster ovary (CHO) cells transfected with GPIb/(V)/IX containing mutations in this region. Adhesion of cells carrying a glycine substitution of the GPIb beta Ser166 phosphorylation site was 50% lower than normal and became insensitive to inhibition by RAM.1. In contrast, forskolin or PGE(1) treatment increased both the phosphorylation of GPIb beta and adhesion of control cells, both effects being reversed by RAM.1, but had no influence on cells expressing the Ser166Gly mutation. A role of the GPIb alpha intracellular domain was also apparent as the VWF-dependent adhesion of cells containing deletions of the entire (Delta 518-610) or portions (Delta 535-568, Delta 569-610) of the GPIb alpha cytoplasmic tail was insensitive to RAM.1 inhibition. Cells carrying progressive 11 amino acid deletions spanning the GPIb alpha 535-590 region were equally unresponsive to RAM.1, with the exception of those containing GPIb alpha Delta 569-579, which behaved like control cells. These findings support a role of the GPIb beta intracellular domain in controlling the adhesive properties of the GPIb/V/IX complex through phosphorylation of GPIb beta Ser166 and point to the existence of cross-talk between the GPIb beta and GPIb alpha intracellular domains.

Distinct Glycoprotein Ib/V/IX and Integrin αIIbβ3-dependent Calcium Signals Cooperatively Regulate Platelet Adhesion under Flow

We have investigated the calcium signaling relationship between the two major platelet adhesion receptors, glycoprotein Ib/V/IX (GPIb/V/IX) and integrin alpha(IIb)beta(3), involved in regulating platelet adhesion on von Willebrand factor (vWf) under flow. Our studies demonstrate that GPIb engagement of immobilized vWf elicits a transient calcium spike that may function to promote reversible arrest of translocating platelets. Subsequent integrin alpha(IIb)beta(3) engagement of vWf promotes sustained calcium oscillations that are essential for the maintenance of irreversible adhesion. GPIb-induced calcium spikes appear distinct from those initiated by integrin alpha(IIb)beta(3), in that the former are exclusively mediated through release of intracellular calcium stores via a signaling mechanism independent of PI 3-kinase. In contrast, integrin alpha(IIb)beta(3)-dependent calcium flux involves a PI 3-kinase-dependent signaling mechanism linked to intracellular calcium mobilization and subsequent transmembrane calcium influx. Studies employing the caged calcium chelator (o-nitrophenyl-EGTA) demonstrate that transient calcium spikes initiate a transient phase of platelet arrest that is converted to irreversible adhesion with the development of sustained oscillatory calcium flux. These studies demonstrate the existence of a dual step calcium signaling mechanism utilized by GPIb and integrin alpha(IIb)beta(3) that serves to regulate the dynamics of platelet adhesion under flow.

Genetic polymorphism in human platelet glycoprotein GP Ib/IX/V complex is enriched in GP V (CD42d).

Platelet glycoproteins Ib beta (CD42c), IX (CD42a), and V (CD42d), together with GP Ib alpha (CD42b), form a receptor whose interaction with the von Willebrand factor is essential in the initial stages of haemostasis. Genetic variation in these proteins can cause alloimmunization leading to neonatal alloimmune thrombocytopenia and platelet transfusion refractoriness. Defective mutations cause a rare bleeding disorder, Bernard-Soulier syndrome. Only two antigenic polymorphisms have thus far been established in these proteins: the HPA-2 in GP Ib alpha and the rare Iy variant in GP Ib beta. Recently, we reported that only a limited degree of polymorphism can be found in the GP Ib alpha gene; the level of variation in the other components is not known. We therefore systematically screened polymorphism in the GP Ib beta, GP IX, and GP V genes in 50 unrelated Finnish blood donors. Nine polymorphic sites were found in the GP V gene, of which four changed the amino acid code and five were silent. The gene frequencies for substitutions Asp114Tyr, Met273Ile, Gly341Arg, and Leu397Arg were 1%, 1%, 2%, and 1% respectively. The five silent polymorphisms also had low frequencies, 1-4%. No polymorphism was found in the GP Ib beta gene and only one mutation was found in the 3' untranslated region of the GP IX gene. Our results indicate that genetic variation in the GP Ib/IX/V complex is mostly tolerated in the GP V protein--whose function in the complex is not clear whereas the other components have only very limited genetic polymorphism.

Characterization of cDNA encoding full-length mouse platelet glycoprotein IX.

Glycoprotein (GP) IX is a platelet membrane 20 kD protein, which is associated with GPIbalpha, GPIb beta, and GPV to form GPIb/IX/V complex. GPIb/IX/V complex is a major receptor for von Willebrand factor, which mediates platelet adhesion and aggregation under high shear stress conditions. The relevance of this receptor for hemostasis has been implicated by a congenital bleeding disorder lacking the receptor, Bernard-Soulier syndrome. All subunits for the human receptor have been cloned and characterized. However, the function of GPIX is still elusive. To obtain further information of GPIX, we have determined a cDNA sequence of mouse GPIX (811 bp). The deduced amino-acid sequence (177aa) was 71% identical to the human GPIX protein. All cysteine residues in extracytoplasmic domain and putative N-linked glycosylation site (Asn44) were conserved. Mouse GPIX contained a leucine-rich glycoprotein sequence composed of 24 amino acids, as did human GPIX.

Distinct sequences of the glycoprotein Ib-binding domain of von Willebrand factor involved in shear induced platelet aggregation

Shear-induced platelet aggregation (SIPA) requires von Willebrand factor (vWF) binding to the platelet receptors GPIb and alpha beta. In order to determine the vW F sequences involved in SIPA at 4000/s, we studied the llb 3 effect of three monoclonal antibodies (mabs) 724, 713 and 328 to the A1 domain of vWF. We found that mab 724 induced an enhanced SIPA via a Fc gamma -receptor independent mechanism. In contrast, mab 713 and mab 328 could inhibit SIPA by 52 and 91%, respectively. Based on distinct effects on SIPA, we can propose the following working model for the interaction between vWF and GPIb: mabs 713 and 328, which block SIPA, may recognize an epitope that is involved in binding to GPIb, whereas mab 724, which increases SIPA in the presence of vWF, may mimic the effect of botrocetin when binding to vWF, by inducing an active conformation of vWF, which may be more sensitive to high shear rate.

Platelet glycoprotein (Gp) IX associates with GpIb alpha in the platelet membrane GpIb complex

The platelet membrane glycoprotein (Gp) Ib complex consists of four polypeptides: the disulfide-linked GpIb alpha and GpIb beta subunits; GpIX, tightly, but noncovalently associated with GpIb alpha-beta; and the more weakly associated GpV. It is not certain whether the association of GpIX to Gplb alpha-beta is via GpIB alpha, GpIb beta, or both subunits, although recently published evidence implicates an interaction with GpIb beta. We have investigated the interaction of GpIX with GpIb alpha-beta using polyclonal rabbit antibodies to GpIb alpha and GpIb beta raised by immunization with purified glycocalicin and with synthetic peptide sequences from GpIb beta, respectively, as well as monoclonal antibodies directed against GpIX (FMC-25) and against GpIb alpha (AP-1). We performed two types of experiments, using either purified GpIb complex or platelets. (1) When wells were coated with nonreduced GpIb complex, the binding of FMC-25 was inhibited 73% by GpIb alpha antibody, but only 30% by the GpIb beta antibody; when wells were coated with reduced complex, FMC-25 binding was inhibited by the same two antibodies by 86% and 13%, respectively. When wells were coated with polyclonal GpIb alpha or GpIb beta antibodies and then incubated with reduced GpIb complex, only wells coated with GpIb alpha antibodies captured GpIX reactivity. When wells were coated with FMC-25 and then incubated with nonreduced GpIb complex, both the GpIb alpha and GpIb beta polyclonal antibodies reacted strongly; in contrast, only GpIb alpha reactivity was retained when wells coated with FMC-25 were incubated with reduced GpIb complex. In the reciprocal experiment, AP-1-coated wells incubated with either nonreduced or reduced GpIb complex bound radiolabeled FMC-25. (2) The ability of polyclonal GpIb alpha and GpIb beta antibodies to inhibit binding of FMC-25 to platelets was studied by ELISA and by flow cytometry. In both systems, FMC-25 binding was inhibited by the GpIb alpha antibody, but not significantly by the GpIb beta antibody. We conclude that GpIX is strongly associated with GpIb alpha in the purified platelet GpIb complex and in the platelet membrane.

Biosynthetic Defect in Platelet Glycoprotein IX Mutants Associated With Bernard-Soulier Syndrome

To evaluate the biosynthetic basis for decreased glycoprotein (GP) Ib-IX expression resulting from GP IX mutations described in three siblings with Bernard-Soulier syndrome, we introduced each mutation into the cDNA for GP IX by site-directed mutagenesis (GP IX Asp21 --> Gly and GP IX Asn45 --> Ser) and examined the associations of the mutants with the two other subunits of the GP Ib-IX complex in transfected cells. Unlike wild-type GP IX, neither of the mutants was able to increase GP Ib expression on the cell surface, either when transfected into Chinese hamster ovary (CHO) alpha beta cells or when cotransfected with GP Ib alpha and GP Ib beta into wild-type CHO cells. We also evaluated whether cotransfecting wild-type or mutant GP IX with GP Ib beta would result in the appearance of GP IX on the surface of the transfected cells; the wild-type protein was detected on the surface of the cells, whereas neither mutant reached the cell surface in appreciable quantities. Immunofluorescence microscopy of permeabilized cells revealed that the failure to express mutant GP IX on the cell surface did not result from failure to synthesize the polypeptide. Both mutants were detected in intracellular compartments, albeit at lower levels than the wild-type polypeptide (the fluorescence of cells expressing the GP IX Asp21 --> Gly was consistently the lowest). Direct evidence that the mutants associate poorly with Gp Ib beta was obtained of 35S-labeled cells transiently expressing GP Ib beta and wild-type or mutant GP IX. The amount of GP IX coprecipitated with GP Ib beta was greatly diminished in cells expressing either mutant. These findings suggest an important role for the conserved leucine-rich motif of GP IX in the association of this polypeptide with GP Ib beta and provide further evidence for the importance of GP IX in the stability of the GP-Ib-IX complex.

The platelet glycoprotein Ib-V-IX system: regulation of gene expression.

Platelet glycoproteins (GPs) Ib-V-IX form the surface receptor for von Willebrand factor, and this receptor-ligand interaction mediates the shear-dependent adhesion of platelets to damaged arterial vessel walls. The receptor is a multicomponent structure consisting of four distinct polypeptides (heterodimeric GPIb: Ib alpha-Mr143k and Ib beta-Mr22k; GPV-Mr83k; GPIX-Mr20k), and each of the four cDNAs and genes has been cloned and characterized. The genes appear to have evolved from a common progenitor genomic sequence related to that encoding GPIX. They share simple structures with few introns and possess common consensus regulatory sequences (GATA, ets, Sp-1) in their 5' flanks. Both the GPIb alpha and the GPIX promoters have been analyzed by transfection of reporter constructs into hematopoietic and nonhematopoietic cells. The promoters function in a tissue-specific fashion, and gel shift and mutational analyses indicate that GATA and ets sequences regulate activity. In the case of the GPIX promoter, footprints confirm the role of the ets-related consensus region. Recent studies of GPIb beta transcriptional regulation suggest that an aberrant polyadenylation signal, located in the 3' end of the gene immediately upstream of the GPIb beta gene, allows in vitro expression of a rare extended fusion transcript encoding both the upstream protein and GPIb beta. Little detailed information is available in regard to expression of the GPV gene. In summary, the genes of the GPIb-V-IX system display features of other megakaryocyte/platelet genes, but the unique regulatory events that direct the selective expression of these genes in megakaryocytes remain to be defined.

Functional Expression of Single Chain Glycoprotein lb Alpha on the Surface of COS Cells and BHK Cells

The GpIb-IX complex constitutes the major receptor for von Willebrand factor (vWF) on the surface of blood platelets. The vWF-binding site has been mapped to one of the three constituent chains, GpIb alpha. Surface expression of GpIb alpha depends on the correct intracellular assembly with the GpIb beta and GpIX chains. We have now grafted a portion of the extracellular domain of GpIb alpha containing the vWF binding site onto the transmembrane/intracellular domain of the single chain surface molecule ICAM-1. Transient transfection of this chimeric protein in COS cells resulted in surface expression as assessed by immunostaining of live cells. Similar results were obtained after stable transfection into BHK cells. Purified vWF bound to the surface of transfected cells in the presence of ristocetin and botrocetin with a Kd of 52 ng/ml, comparable to the Kd for fixed platelets (65.5 ng/ml). This study indicates that functional expression of the vWF-binding domain of GpIb alpha on the surface of mammalian cells can be obtained in the absence of GpIb beta and GpIX. Furthermore, this model system simplifies existing methods for the assessment of the functional consequences of mutations in GpIb alpha as found in pseudo-von Willebrand disease and Bernard-Soulier syndrome.

Platelet GPIb-V-IX complex. Structure, function, physiology, and pathology.

In the early phase of primary hemostasis, platelets adhere to damaged vessel wall by binding via the platelet glycoprotein (GP) Ib-V-IX complex to von Willebrand factor (vWf) exposed on the subendothelium. The complex is composed of four glycoprotein subunits, GPIb alpha, GPIb beta, GPIX and GPV, each with a variable number of leucine-rich repeats. GPIb alpha and GPIb beta are linked by a disulphide bridge while GPIX and GPV associate noncovalently with the complex. The study of defects in the expression of the GPIb-V-IX complex at the platelet surface leading to pathological disorders, like Bernard-Soulier syndrome (BSS), or in the affinity of platelets for vWf, like pseudo-von Willebrand disease, has helped to delineate the binding site for vWf on GPIb alpha. However, the mechanism by which the complex binds to vWf has not yet been elucidated but it must involve changes in the conformation of the molecules as no interaction between platelets and vWf occurs in the plasma. The GPIb-V-IX complex has a binding site for thrombin on GPIb alpha which participates in the platelet activation by that agonist. GPV is also cleaved by thrombin but the function of this proteolysis is not clear. The platelet response to thrombin is slower and weaker when the thrombin binding site on GPIb alpha is blocked or cleaved or when the GPIb-V-IX complex is not expressed on the platelet surface as in classic BSS. At low doses of thrombin, the rapid activation of the platelets via the seven-transmembrane thrombin receptor is dependent on the presence of the GPIb-V-IX complex.

Bernard-Soulier syndrome Kagoshima: Ser 444-->stop mutation of glycoprotein (GP) Ib alpha resulting in circulating truncated GPIb alpha and surface expression of GPIb beta and GPIX [see comments

The platelet membrane glycoprotein (GP)Ib/IX complex is composed of three polypeptides, GPIb alpha, GPIb beta, and GPIX, and functions as a platelet receptor for von Willebrand factor. All three subunits are reported to be requisite for efficient surface expression of the complex. The absence of the GPIb/IX complex on platelet membrane is the hallmark of a congenital qualitative platelet disorder, termed the Bernard-Soulier syndrome (BSS). We describe here the molecular basis of a novel variant phenotype of BSS in a female patient, designated as BSS Kagoshima. Her platelets completely lacked the surface expression of GPIb alpha, but expressed a residual amount of GPIb beta and GPIX. Unexpectedly, her platelets and plasma contained a truncated GPIb alpha polypeptide with an apparent molecular weight of 116 kD (under nonreducing conditions). The amounts of truncated protein were 23% and 60% of the normal values in platelets and plasma, respectively. The abnormal protein contained a normal amount of sialic acid as demonstrated by digestion with neuraminidase. DNA sequencing analysis showed a homozygous single nucleotide substitution from the serine codon (TCA) to a nonsense codon (TAA) at residue 444 in the GPIb alpha gene. The mutant gene generated a truncated GPIb alpha molecule lacking the transmembrane region and cytoplasmic tail. Her parents were heterozygotes for the mutation. These findings suggest that this type of truncated GPIb alpha was produced, normally glycosylated, and subsequently secreted into the plasma. Furthermore, the truncated GPIb alpha might be associated with the process of the surface expression of incomplete GPIb/IX complex, GPIb beta and GPIX.

Bernard-Soulier Syndrome Kagoshima: Ser 444 → Stop Mutation of Glycoprotein (GP) Iba Resulting in Circulating Truncated GPIbα and Surface Expression of GPIbβ and GPIX

The platelet membrane glycoprotein (GP)Ib/IX complex is composed of three polypeptides, GPIb alpha, GPIb beta, and GPIX, and functions as a platelet receptor for von Willebrand factor. All three subunits are reported to be requisite for efficient surface expression of the complex. The absence of the GPIb/IX complex on platelet membrane is the hallmark of a congenital qualitative platelet disorder, termed the Bernard-Soulier syndrome (BSS). We describe here the molecular basis of a novel variant phenotype of BSS in a female patient, designated as BSS Kagoshima. Her platelets completely lacked the surface expression of GPIb alpha, but expressed a residual amount of GPIb beta and GPIX. Unexpectedly, her platelets and plasma contained a truncated GPIb alpha polypeptide with an apparent molecular weight of 116 kD (under nonreducing conditions). The amounts of truncated protein were 23% and 60% of the normal values in platelets and plasma, respectively. The abnormal protein contained a normal amount of sialic acid as demonstrated by digestion with neuraminidase. DNA sequencing analysis showed a homozygous single nucleotide substitution from the serine codon (TCA) to a nonsense codon (TAA) at residue 444 in the GPIb alpha gene. The mutant gene generated a truncated GPIb alpha molecule lacking the transmembrane region and cytoplasmic tail. Her parents were heterozygotes for the mutation. These findings suggest that this type of truncated GPIb alpha was produced, normally glycosylated, and subsequently secreted into the plasma. Furthermore, the truncated GPIb alpha might be associated with the process of the surface expression of incomplete GPIb/IX complex, GPIb beta and GPIX.

Neutrophil proteinase cathepsin G is proteolytically active on the human platelet glycoprotein Ib-IX receptor: characterization of the cleavage sites within the glycoprotein Ib alpha subunit.

The proteolytic activity of the neutrophil serine-proteinase cathepsin G (CG) on platelet adherence receptors, the glycoprotein (GP) Ib-IX complex and the integrin alpha IIb beta 3, has been investigated. In the range 50 to 200 nmol/l, CG is a potent platelet agonist which induces shape change, granule exocytosis and aggregation. Investigation of the proteolysis of the receptors' subunits during the course of platelet activation by CG was performed by immunoblot analysis of platelet proteins using a panel of specific antibodies. Exposure of platelets for 3 min at 37 degrees C to CG at a concentration that induces full cell activation resulted in an extensive cleavage of the N-terminal region of the extracellular domain of GPIb alpha, the largest (relative molecular mass, M(r), 143,000) of the three subunits constituting the GPIb-IX complex. In contrast, no detectable proteolytic modification of the two other subunits, GPIb beta and GPIX, was detected. Similarly, we observed that neither of the two subunits of the alpha IIb beta 3 receptor were proteolytically modified by CG. Cleavage of GPIb alpha by CG leaves a remnant of the polypeptide chain with M(r) approx. 106,000 in the plasma membrane, while releasing into the extracellular milieu the N-terminal domain with M(r) in the range 40,000 to 46,000. N-terminal sequencing of the CG-derived fragments of GPIb alpha indicated that the Leu275-Tyr276 peptide bond was the primary cleavage site for this proteinase. Proteolysis of GPIb alpha was already detectable at concentrations of CG as low as 25 nmol/l, while with 200 nmol/l the cleavage was detected as soon as 10 s after exposure of platelets to the proteinase. Comparison of the kinetics and concentration dependency for the proteolysis of GPIb alpha and for the activation of platelets by CG showed that cleavage of the GPIb-IX receptor is an early event that accompanies exocytosis and aggregation. Quantitative evaluation of the conversion of GPIb alpha into its membrane fragment indicated that, under optimal conditions, a maximum of approx. 50% of the total GPIb alpha can be affected by proteolysis. However, this proteolysis was > 90% complete when platelets were in the presence of the potent antagonist prostacyclin, suggesting that cellular redistribution of the GPIb-IX receptor may also occur during activation by CG. These results thus indicate that the very early phase of platelet activation by CG is accompanied by extensive modifications in the structure and expression of the GPIb-IX receptor, an effect that might be of functional significance for the interaction of platelets with the vessel wall.

Structural characterization and chromosomal location of the gene encoding human platelet glycoprotein Ib beta

Human platelet glycoprotein Ib beta (GPIb beta) (M(r) 22,000) is part of the GPIb-V-IX system that constitutes the receptor for von Willebrand factor and mediates platelet adhesion in the arterial circulation. The four members of the receptor (GPs Ib alpha, Ib beta, V, and IX) share structural and functional features. Individually, GPIb beta contributes to surface expression of the receptor and participates in transmembrane signaling through phosphorylation of its intracellular domain. To define the structure of the GPIb beta gene, a cosmid clone from a human genomic library was analyzed. The transcriptional start site was located by both primer extension and the "anchored" polymerase chain reaction. Similar to the genes for Ib alpha, V, and IX, the Ib beta gene is compact with a single 274-base intron inserted into the 5' end of the open reading frame. The 5'-flanking region of the gene contains both GATA and ets sites that are also found in the 5' promoter regions of other described megakaryocyte/platelet genes. The GPIb beta gene was localized to chromosome 22q11.2 by fluorescence in situ hybridization. The GPIb beta gene has a simple structure, similar to that of other described megakaryocyte/platelet genes, including those of the GPIb-V-IX system.

Complementary DNA cloning of the alternatively expressed endothelial cell glycoprotein Ib beta (GPIb beta) and localization of the GPIb beta gene to chromosome 22.

Glycoprotein Ib beta (GPIb beta) exists in platelets disulfide-linked to glycoprotein Ib alpha (GPIb alpha), a major receptor for von Willebrand factor. Both GPIb alpha and GPIb beta are expressed in endothelial cells (EC). While the GPIb alpha mRNA and protein appear similar in platelets and EC, EC GPIb beta mRNA is larger than platelet GPIb beta and encodes a larger protein. We have cloned and sequenced EC GPIb beta cDNA and report a 2793-nucleotide sequence which contains a 411-amino acid open reading frame. The EC sequence contains all of the platelet cDNA sequence and all but three amino acids of the primary translation product. Like the genes encoding GPIb alpha, GPIX, and GPV, the GPIb beta gene appears simple in structure. Using human hamster hybrids, we have localized the GPIb beta gene to chromosome 22pter-->22q11.2. When we examined poly (A)+ RNA from several human tissues for GPIb beta mRNA expression, we found that GPIb beta mRNA was expressed in a variety of tissues but was most abundant in heart and brain, while GPIb alpha and GPIX mRNA expression was found only in lung and placenta at very low levels. The broad distribution of GPIb beta mRNA suggests that it may be playing a role different than or additional to its function in platelets.

Double heterozygosity for mutations in the platelet glycoprotein IX gene in three siblings with Bernard-Soulier syndrome

Bernard-Soulier syndrome (BSS) giant platelets have defective and/or deficient glycoprotein (GP) Ib/IX complexes, causing absent ristocetin- induced aggregation, defective interaction with von Willebrand factor, morphologic abnormality, and a clinical bleeding tendency. Recently several mutations have been described in the platelet GPIb alpha gene in individuals exhibiting the BSS phenotype. We have studied a family with classical BSS, and have excluded lesions at the GPIb alpha locus by restriction fragment length polymorphism linkage analysis. Analysis of the genes for two other components of the platelet GPIb:IX complex, namely GPIb beta and GPIX, showed two different missense mutations in the coding region of the GPIX gene: an A-->G transition in codon 21 results in conversion of an aspartic acid to glycine and an A-->G change in codon 45 converts an asparagine residue to serine. Three affected individuals are doubly heterozygous for these mutations, which alter conserved residues in or flanking the GPIX leucine-rich glycoprotein motif. Both mutations create new recognition sites for the enzyme Fnu 4H1; therefore, this enzyme was used to screen 60 normal subjects (120 alleles). Neither mutation was detected in any subject other than direct relatives of the affected individuals. Although low levels of GPIb were demonstrable by both flow cytometry and immunoblot analysis in an affected individual's platelets, there was no evidence of GPIX immunoreactivity. We propose that expression of abnormal GPIX prevents stable assembly of the GPIb/IX complex, causing BSS in the doubly heterozygous individuals in this family.