Genetic and structural characterization of an amino acid dimorphism in glycoprotein Ib alpha involved in platelet transfusion refractoriness.

The platelet-specific alloantigen, Siba, located within the alpha-subunit of the glycoprotein (GP) Ib-IX membrane receptor, has been found to be involved in the pathogenesis of platelet transfusion refractoriness. We have identified the existence of a naturally occurring threonine/methionine dimorphism at position 145 of the GPIb alpha sequence, and determined that the Siba antigen corresponds to the molecule containing methionine145. The diallelic codons can be detected by restriction enzyme analysis of amplified genomic DNA fragments from the GPIb alpha gene. Evaluation of 61 healthy blood donors showed that the allele frequencies are 89% and 11% for the threonine145 and methionine145 codons, respectively. A positive correlation exists between platelet reactivity with the anti-Siba antibody and the presence of a methionine145-encoding allele. Moreover, recombinant expression of two soluble GPIb alpha fragments differing only at residue 145, provided definitive evidence that the human anti-Siba antibody reacts only with the molecule containing methionine145. These results explain the structural basis of the Siba human alloantigen system and define screening methodologies useful in transfusion medicine to match donor and recipient platelets accordingly.     

Flow induces loop-to-beta-hairpin transition on the beta-switch of platelet glycoprotein Ib alpha

Interaction of glycoprotein Ibα (GPIbα) with von Willebrand factor (VWF) initiates platelet adhesion to injured vascular wall to stop bleeding. A major contact between GPIbα and VWF involves the β-switch region, which is a loop in the unliganded GPIbα but switches to a β-hairpin in the complex structure. Paradoxically, flow enhances rather than impedes GPIbα-VWF binding. Gain-of-function mutations (e.g., M239V) in the β-switch reduce the flow requirement for VWF binding, whereas loss-of-function mutations (e.g., A238V) increase the flow requirement. These phenomena cannot be explained by crystal structures or energy calculations. Herein we demonstrate that the β-hairpin is unstable without contacting VWF, in that it switches to a loop in free molecular dynamics simulations. Simulations with a novel flow molecular dynamics algorithm show that the loop conformation is unstable in the presence of flow, as it switches to β-hairpin even without contacting VWF. Compared with the wild-type, it is easier for the M239V mutant but harder for the A238V mutant to switch to β-hairpin in the presence of flow. These results elucidate the structural basis for the two mutants and suggest a regulatory mechanism by which flow activates GPIbα via inducing a loop-to-β-hairpin conformational transition on the β-switch, thereby promoting VWF binding.     

Genetic polymorphism of platelet glycoprotein Ib

Platelet glycoprotein (GP) Ib from 131 healthy Japanese was analyzed using SDS-polyacrylamide gel electrophoresis and specific staining with peroxidase-coupled wheat germ agglutinin after it was transferred to nitrocellulose membranes. Four slightly different species of GPIb were observed and designated as A, B, C, and D for glycoproteins with molecular weights of 168,000, 162,000, 159,000, and 153,000 daltons, respectively. The respective gene frequencies were calculated to be .073, .011, .561, and .355 for A-, B-, C-, D-type GPIb. Portions from each type of GPIb molecule (alpha-chain and glycocalicin) showed heterogeneity with the same molecular weight difference, indicating that the variance would be derived from the polypeptide portion that is exposed to the outer medium. The different types of GPIb were the same with respect to their accessibility to lactoperoxidase, reactivity to lectins, and affinity to TLCK-thrombin. Although Bolin et al reported patients with a bleeding tendency whose platelets have double GPIb bands, here we found that platelets with different GPIb phenotypes showed no significant differences in aggregating activity and platelet retention. Analysis of GPIb phenotype should be important for structural and physiologic studies on GPIb and glycocalicin.     

Plasmin-induced redistribution of platelet glycoprotein Ib

Platelet membrane glycoprotein Ib (GPIb), a receptor for von Willebrand factor and thrombin, is present on the platelet surface membrane, in intraplatelet stores, and in plasma (as the proteolytic fragment glycocalicin). We examined the hypothesis that after plasmin-mediated cleavage of platelet surface GPIb, platelets can replenish their surface GPIb pool. Incubation of washed platelets with plasmin (1 hour, 22 degrees C) resulted in loss of platelet surface GPIb, but further incubation (3 hours, 37 degrees C) in autologous plasma resulted in restoration of platelet surface GPIb, as determined by ristocetin- induced platelet agglutination and a flow cytometric assay of platelet binding of three GPIb-specific monoclonal antibodies. Despite the restoration of platelet surface GPIb after the 3-hour incubation of plasmin-treated platelets in autologous plasma, the whole platelet GPIb content (measured by enzyme-linked immunosorbent assay [ELISA], sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and flow cytometry) remained reduced, quantitatively corresponding to an increase in plasma glycocalicin concentration (measured by ELISA). The loss and restoration of platelet surface GPIb occurred on all platelets and, as evidenced by lack of inhibition by prostaglandin E1, EDTA, and cytochalasins, was not mediated by cyclic AMP, extracellular Ca2+, or the platelet microfilament system. In summary, this study shows that after plasmin-mediated cleavage of platelet surface GPIb, platelets can replenish their surface GPIb pool by recruitment of GPIb molecules from the intraplatelet pool (or from a sequestered surface site).     

Reduction of platelet glycoprotein Ib in uraemia

Patients with uraemia have abnormal platelet function that may be partially corrected by haemodialysis, cryoprecipitate or 1-desamino-8-d-arginine vasopressin (DDAVP). We studied the platelet von Willebrand factor receptor. glycoprotein Ib (GPIb). and plasma von Willebrand factor (vWF) in uraemic patients undergoing chronic haemodialysis. Using the slope of agglutination of formalin-fixed platelets as an index of response to ristocetin (with a constant amount of normal plasma as a source of vWF), we found the response of platelets from uraemic patients, both before (2·7± 1·5. n = 40) and after dialysis (1·2 ± 1·2, n = 40) to be significantly less than that for normal controls (14·1 ± 10·2. n = 20: P < 0·001). In addition, the agglutination response of platelets obtained after dialysis was less than that of platelets obtained before dialysis ( P < 0·001). Immunoblotting demonstrated decreased or absent staining of glycocalicin, a subunit of GPIb, in platelet lysates from 25 patients. All platelet samples with reduced glycocalicin also had decreased responses to ristocetin. Tritium-labelled platelets from seven patients showed decreased labelling of a protein with an electrophoretic mobility equivalent to that of GPIb (140000 daltons). In addition, platelets with the lowest levels of surface GPIb, as demonstrated by flow cytometry. also had decreased ristocetin agglutination and decreased staining on immunoblot. Levels of von Willebrand factor antigen and ristocetin cofactor in plasma from 10 patients were generally within the normal range, although post-dialysis levels tended to be higher than pre-dialysis levels. The pre- and post-dialysis plasma vWF multimeric patterns were normal.     

High-affinity alpha-thrombin binding to platelet glycoprotein Ib alpha: identification of two binding domains.

alpha-Thrombin binding to and activation of platelets are of major importance in the initiation of physiologic thrombi and in the genesis of arterial thrombus formation. We have studied the site(s) and affinity of thrombin binding to human platelets. Our studies of the peptide inhibition of thrombin binding indicate that the glycoprotein Ib alpha binding site is of high affinity, Kd approximately 10(-10) M, while the seven-transmembrane-domain site is a moderate-affinity thrombin binding site, Kd approximately 10(-8) M. Further studies to modulate the high- or moderate-affinity thrombin binding can be directed to a specific class of sites. This would allow partial or total inhibition of specific thrombin-platelet interaction(s) in different clinical settings.     

Functional analysis of the C-terminal flanking sequence of platelet glycoprotein Ib alpha using canine-human chimeras.

Platelet glycoprotein Ib-IX-V (GPIb-IX-V) mediates adhesion to von Willebrand factor (vWF) in (patho)physiological thrombus formation. vWF binds the N-terminal 282 residues of GPIb alpha, consisting of an N-terminal flank (His1-Ile35), 7 leucine-rich repeats (Leu36-Ala200), a C-terminal flank (Phe201-Gly268), and a sulfated tyrosine sequence (Asp269-Glu282). By expressing canine-human chimeras of GPIb alpha on Chinese hamster ovary cells, binding sites for functional anti-GPIb alpha antibodies to individual domains were previously mapped, and it was shown that leucine-rich repeats 2 to 4 were required for optimal vWF recognition under static or flow conditions. Using novel canine-human chimeras dissecting the C-terminal flank, it is now demonstrated that (1) Phe201-Glu225 contains the epitope for AP1, an anti-GPIb alpha monoclonal antibody that inhibits both ristocetin- and botrocetin-dependent vWF binding; (2) VM16d, an antibody that preferentially inhibits botrocetin-dependent vWF binding, recognizes the sequence Val226-Gly268, surrounding Cys248, which forms a disulfide-bond with Cys209; (3) vWF binding to chimeric GPIb alpha is comparable to wild-type in 2 chimeras in which the sixth leucine-rich repeat was of the same species as the first disulfide loop (Phe201-Cys248) of the C-terminal flank, suggesting an interaction between these domains may be important for optimal vWF binding; and (4) replacing the C-terminal flank second disulfide loop (Asp249-Gly268) in human GPIb alpha with the corresponding canine sequence enhanced vWF binding under static and flow conditions, providing the first evidence for a gain-of-function phenotype associated with the second loop of the C-terminal flank.     

Human anti-PlEl antibody recognizes epitopes associated with the alpha subunit of platelet glycoprotein Ib

Together, a platelet-reactive antibody in the serum of a polytransfused patient (proband) and a platelet-reactive antibody in the serum of a mother of an infant with neonatal thrombocytopenia have served to establish the diallelic, platelet-specific alloantigen system, PlE. We now provide evidence that the platelet-specific antibody in the serum of the proband, anti-PlE1, recognizes epitopes associated with the alpha subunit of glycoprotein (GP) Ib. By 51Cr release, platelets from two of three patients with the Bernard-Soulier syndrome (BSS) responded sub-normally to anti-PlE1, and the apparently normal response of platelets from the last BSS patient was attributable to anti-HLA-A2 antibodies in the proband serum. These results suggested that the PlE1 antigen is associated with the GPIb complex (glycoproteins Ib kX) known to be absent from BSS platelets. This possibility was confirmed by ELISA using the purified GPIb complex or glycocalicin, the N-terminal fragment of GPIb alpha produced by proteolysis with endogenous platelet calpain, as solid-phase antigen. Anti-PlE1 antibody bound specifically to both the GPIb complex and glycocalicin. 3H-labelled platelet membrane glycoproteins with apparent molecular weights of 130k, 25k, and 21k (under reduced conditions) corresponding to GPIb alpha, GPIb beta, and GPIX were immunoprecipitated by anti-PlE1 plasma. Finally, at a titre of 1:16, anti-PlEl completely inhibited ristocetin-induced platelet agglutination, a property of platelets mediated by GPIb.     

Mutation in the gene encoding the alpha chain of platelet glycoprotein Ib in platelet-type von Willebrand disease.

Platelet-type von Willebrand disease (PT-vWD) is an autosomal dominant bleeding disorder characterized by abnormally enhanced binding of von Willebrand factor (vWF) by patient platelets. Although the platelet glycoprotein (GP) Ib/IX complex is known to constitute the platelet's ristocetin-dependent receptor for vWF, a unique structural abnormality within this complex has not previously been identified in PT-vWD. Using the polymerase chain reaction to amplify genomic DNA coding for the alpha chain of GP Ib (GP Ib alpha) and then sequencing the amplified DNA following cloning into M13mp18 and M13mp19 phage vectors, we have found a single point mutation in the GP Ib alpha coding region of PT-vWD DNA resulting in the substitution of valine for glycine at residue 233. This substitution within the vWF-binding region of GP Ib alpha is likely to exert a significant influence on the conformation of the resulting protein. Competitive oligonucleotide primer assay for this mutation showed a homozygous wild-type pattern in genomic DNA from the 161 normal volunteers studied and from 6 phenotypically normal members of a PT-vWD family. All 7 affected members of this family studied were heterozygous for the mutant allele. Platelet GP Ib alpha mRNA reverse-transcribed and studied by competitive oligonucleotide primer assay showed similar expression of the mutant and wild-type alleles in the affected PT-vWD patients. Absence in the normal population, tight linkage with phenotypic expression of disease, and absence of any additional abnormality of GP Ib alpha in these patients identify the glycine-to-valine substitution as a point mutation underlying functional abnormality of the vWF receptor in PT-vWD.     

Polymorphisms of platelet membrane glycoprotein Ib alpha and plasma von Willebrand factor antigen in coronary artery disease.

To examine the relationships of two polymorphisms of platelet glycoprotein (GP) Ib alpha and coronary artery diseases (CAD) in Japanese patients, we conducted a case-control study with 158 Japanese patients and 169 control subjects. The frequencies of HPA-2 polymorphism and the variable number of tandem repeat (VNTR) polymorphisms in the macroglycopeptide region did not significantly differ between CAD patients and control subjects. The polymorphisms of GPIb alpha were not associated with the number of affected vessels in CAD patients. When patients with acute coronary syndrome only were analyzed, the frequencies of the two polymorphisms of GPIb alpha showed no significant difference. Although plasma von Willebrand antigen (vWF:Ag) levels in patients were significantly higher than in controls, no association between vWF concentration and GPIb genotypes was observed. In patient groups with higher or lower vWF:Ag concentrations, no increase in the frequencies of Met145 or larger VNTR polymorphisms was seen in either group. Our findings indicate that no association exists between the frequencies of the two polymorphisms of GPIb alpha and CAD.     

Reversibility of thrombin-induced decrease in platelet glycoprotein Ib function

Summary. Thrombin induces a redistribution of glycoprotein (GP) Ib/GP IX complex from the platelet surface into the surface connected canalicular system (SCCS). This redistribution results in a reduced interaction of platelet GP Ib with von Willebrand factor (vWF) bound to subendothelium leading to impaired platelet adhesion. In this study we show that the platelet aggregation and degranulation require concentrations of thrombin above 0.05 U/ml, while the decrease in GP Ib function (about 50% of control value), as determined by ristocetin induced platelet agglutination, can be induced by lower concentrations (0.01–0.04 U/ml). Moreover, we show that when adding thrombin inhibitors to the platelets preincubated with <0.04 U/ml thrombin for 5 min, their agglutinability by ristocetin was gradually recovered within 30 min. indicating that in these conditions the decrease in platelet adhesiveness is reversible. Immuno-electromicroscopic study showed that this restoration of platelet GP Ib function was associated with a reversed translocation of GP Ib from the SCCS to the plasma membrane. The data obtained from counting gold particles showed that the ratio of GP Ib immunolabelling on the external membrane versus that on the SCCS was 3.31±0.90 for resting platelets, down-regulated to 0.84±0.13 ( P >0.05 versus resting platelets) for the platelets treated with 0.04 U/ml thrombin and returned to 2.63±2.21 ( P >0.05 versus resting platelets) after incubation for 30 min with hirudin. However, the translocation of GP Ib was poorly reversed by thrombin inhibitors when higher concentrations of thrombin were used which induced platelet aggregation and large extent of degranulation. We conclude that thrombin affects platelets in a dose dependent manner, and that at low concentrations the decrease in platelet GP Ib related function is a reversible phenomenon.     

Effect of phosphopyridoxylation on thrombin interaction with platelet glycoprotein Ib.

The purpose of this study was to determine the effect of chemical modification of lysyl residues on thrombin interaction with platelet membrane proteins. Modification of lysyl residues by pyridoxal-5'-phosphate affected two different sites on thrombin and resulted in a greatly decreased binding to platelets. Using a crosslinking bifunctional reagent [bis(sulphosuccinimidyl) suberate (BS3)], we show that modified thrombin retained the ability to form high molecular mass (greater than or equal to 400 kDa) complexes with yet unidentified platelet proteins and to bind to platelet protease nexin I, but had lost the ability to bind to platelet glycoprotein Ib (GPIb). As previously reported by others, heparin protected one of the two sites from phosphopyridoxylation. In contrast modified thrombin, heparin-protected modified thrombin retained the ability to bind to GPIb, indicating that the lysyl residue(s) protected by heparin from the modification are essential for GPIb binding. While unprotected modified thrombin failed to bind hirudin, heparin-protected modified thrombin retained its ability to bind the carboxy-terminal hirudin peptide H54-65. Tritium-labelling of the modified lysyl residues and degradation of modified thrombins by CNBr or trypsin confirmed that the lysyl residue(s) protected by heparin and essential for GPIb binding are located in the thrombin binding domain for the carboxyl-terminal tail of hirudin, within the sequence 18-73 of the thrombin B chain.     

Cytoskeletal regulation of the platelet glycoprotein Ib/V/IX-von willebrand factor interaction

Shear-induced binding of von Willebrand factor (vWf) to the platelet glycoprotein (GP) Ib/V/IX complex plays a key role in initiating platelet adhesion and aggregation at sites of vascular injury. This study demonstrated that pretreating human platelets with inhibitors of actin polymerization, cytochalasin D or latrunculin B, dramatically enhances platelet aggregation induced by vWf. The effects of these inhibitors were specific to the vWf-GPIbalpha interaction because they enhanced vWf-induced aggregation of Glanzmann thrombasthenic platelets and Chinese hamster ovary (CHO) cells transfected with GPIb/V/IX. Moreover, cytochalasin D enhanced the extent of platelet aggregation induced by high shear stress (5000 s(-1)) and also lowered the shear threshold required to induce aggregation from 3000 s(-1) to as low as 500 s(-1). Studies of CHO cells expressing GPIbalpha cytoplasmic tail truncation mutants that failed to bind actin-binding protein-280 (deletion of residues 569-610 or 535-568) demonstrated that the linkage between GPIb and actin-binding protein-280 was not required for vWf-induced actin polymerization, but was critical for the enhancing effects of cytochalasin D on vWf-induced cell aggregation. Taken together, these studies suggest a fundamentally important role for the cytoskeleton in regulating the adhesive function of GPIb/V/IX.     

Evaluation of platelet glycoprotein Ib by fluorescence flow cytometry

Platelet glycoprotein Ib (GpIb), a receptor for von Willebrand's factor (vWF), was studied by way of fluorescence flow cytometry. Using a sandwich staining technique, GpIb was identified by a monoclonal antibody (6D1) directed against an epitope close to the vWF binding site. Platelets from normal individuals were symmetrically distributed with respect to GpIb content. Treatment of washed platelets with plasmin resulted in progressive loss of GpIb as measured by fluorescence flow cytometry and by loss of agglutination response when combined with ristocetin in the presence of vWF. In mixing experiments with GpIb-deficient and normal platelets, it was possible to detect a subpopulation of deficient cells comprising 2% of the total population. Streptokinase treatment of platelet-rich plasma caused loss of the agglutination response to ristocetin and the emergence of a population of GpIb-deficient platelets. Fluorescence flow cytometry appears to be an important new technique by which to study platelet surface receptors.     

Cloning of the alpha chain of human platelet glycoprotein Ib: a transmembrane protein with homology to leucine-rich alpha 2-glycoprotein.

Glycoprotein Ib is a surface membrane glycoprotein of platelets that functions as a receptor for von Willebrand factor. It is a heterodimer composed of an alpha and a beta chain linked by a disulfide bond(s). A phage lambda gt11 cDNA expression library prepared from mRNA from a human erythroleukemia cell line, HEL, was screened using an affinity-purified antibody to the glycocalicin portion of the alpha chain of glycoprotein Ib. Eleven positive clones were isolated and plaque-purified. The largest cDNA insert was 2420 nucleotides in length and coded for a leader sequence of 16 amino acids, a mature protein of 610 amino acids, and a stop codon. It also contained 42 nucleotides of 5' noncoding sequence and 497 nucleotides of 3' noncoding sequence, including a poly(A) tail. The amino acid sequence of the alpha chain of GPIb predicted from the cDNA agreed completely with the sequence of 156 amino acids that was determined by Edman degradation of peptides isolated from human platelet glycocalicin after digestion with trypsin or Staphylococcus aureus V8 protease. The extracytoplasmic domain of the alpha subunit of GPIb contains several noteworthy structural features, including a region of seven tandem repeats of 24 amino acids that are homologous with those present in leucine-rich alpha 2-glycoprotein. The extracytoplasmic domain also contains two hydrophilic regions, one rich in charged amino acids and a second rich in serine and threonine residues. The region rich in serine and threonine includes five repeats of nine amino acids as well as the majority of the O-linked carbohydrate sites present in the molecule. The extracytoplasmic domain is followed by a potential transmembrane segment of approximately 29 amino acids and a potential intracellular domain of approximately 100 amino acids located at the carboxyl end of the molecule.     

Variation in surface platelet glycoprotein Ib expression in Bernard-Soulier syndrome

Seven patients with Bernard-Soulier syndrome (BSS) and 15 presumed heterozygotes of BSS from four families are presented. Evaluation of their platelet membranes was performed by an enzyme-linked radioimmunosorbent assay (ELISA) technique including monoclonal antibodies specific for glycoprotein (GP) Ib and GPIIb/IIIa. Analyses of platelets from the patients revealed 6-33% of the normal GPIb concentration; siblings showed nearly equal amounts of this component. One of the relatives had 44% of the normal level, while 85-98% (mean 92%) of the normal GPIb content was observed in the remaining relatives. Normal or slightly elevated levels of GPIIb/IIIa were detected in the patients as well as in the relatives. All relatives had normal platelet count, size distribution, bleeding time, and ristocetin-induced platelet aggregation. The patients and their relatives may account for a biological variation in GPIb expression or may represent a variant type of BSS.     

Platelet glycoprotein Ib alpha supports experimental lung metastasis

The platelet paradigm in hemostasis and thrombosis involves an initiation step that depends on platelet membrane receptors binding to ligands on a damaged or inflamed vascular surface. Once bound to the surface, platelets provide a unique microenvironment supporting the accumulation of more platelets and the elaboration of a fibrin-rich network produced by coagulation factors. The platelet-specific receptor glycoprotein (GP) Ib-IX, is critical in this process and initiates the formation of a platelet-rich thrombus by tethering the platelet to a thrombogenic surface. A role for platelets beyond the hemostasis/thrombosis paradigm is emerging with significant platelet contributions in both tumorigenesis and inflammation. We have established congenic (N10) mouse colonies (C57BL/6J) with dysfunctional GP Ib-IX receptors in our laboratory that allow us an opportunity to examine the relevance of platelet GP Ib-IX in syngeneic mouse models of experimental metastasis. Our results demonstrate platelet GP Ib-IX contributes to experimental metastasis because a functional absence of GP Ib-IX correlates with a 15-fold reduction in the number of lung metastatic foci using B16F10.1 melanoma cells. The results demonstrate that the extracellular domain of the alpha-subunit of GP Ib is the structurally relevant component of the GP Ib-IX complex contributing to metastasis. Our results support the hypothesis that platelet GP Ib-IX functions that support normal hemostasis or pathologic thrombosis also contribute to tumor malignancy.     

A role for glycoprotein Ib in Streptococcus sanguis-induced platelet aggregation

Numerous studies have implicated bacteria in cardiovascular disease, but there is a paucity of information on the mechanism involved. In this study we show how the common oral bacterium Streptococcus sanguis can directly interact with platelets, resulting in activation and aggregate formation. Platelet aggregation was dependent on glycoprotein IIb/IIIa (GPIIb/IIIa) and thromboxane. Platelets could also directly bind to S sanguis, but this interaction was not inhibited by GPIIb/IIIa antagonists. Antibodies to GPIb could inhibit both platelet aggregation and platelet adhesion to bacteria. This suggested a direct interaction between GPIb and S sanguis; however, this interaction did not require von Willebrand factor, the normal ligand for GPIb. By use of a range of monoclonal antibodies to GPIb and the enzyme mocharagin, which cleaves GPIb at amino acid 282, the interaction was localized to a region within the N-terminal 1-225 portion of GPIbalpha. Furthermore S sanguis failed to induce aggregation of platelets from a patient with Bernard-Soulier disease, the organism bound to Chinese hamster ovary cells transfected with the GPIbalpha gene but did not bind to mock-transfected cells and biotin-labeled S sanguis cells bound to purified GPIb in ligand blots. It is suggested that the interaction between S sanguis and GPIb is important in the pathogenesis of infective endocarditis and may also play a contributory role in some cases of myocardial infarction.