A novel isoform of platelet glycoprotein Ibα is prevalent in African Americans

The heavy chain of platelet glycoprotein Ib (GPIb) contains two prevalent sequence polymorphisms. The first, Thr/Met¹⁴⁵ is responsible for the human platelet alloantigen system, human platelet antigen (HPA)-2. The second is a tandem repeat polymorphism that consists of four variants, A, B, C, and D. Previous linkage studies in Caucasian and Eastern Asian populations have demonstrated that HPA-2a (Thr¹⁴⁵) is associated with variants C and D, while HPA-2b (Met¹⁴⁵) is associated with variants A and B. We have determined HPA-2 and variable number of tandem repeats (VNTR) genotypes in three different North American ethnic groups. The gene frequency of HPA-2b in the North American Indians was intermediate between African Americans and Caucasians, and similar to the frequency previously reported in Japanese. Furthermore, the VNTR-A allele, which previously has been reported only in Eastern Asian populations, was present in two of 101 North American Indian individuals. These data are consistent with the hypothesis that the first Native Americans migrated to North America from Eastern Asia. Analysis of HPA-2 and VNTR haplotypes demonstrated an unexpected linkage pattern in the African American population. A rare GPIbα isoform, HPA-2b/VNTR-C, was present in 2.2% of African American haplotypes. Furthermore, a novel GPIbα isoform, HPA-2a/VNTR-B, was present in 6.5% of African American haplotypes. These data suggest a more complex evolutionary pattern of GPIbα isoforms than previously proposed. Am. J. Hematol. 60:77–79, 1999. © 1999 Wiley-Liss, Inc.     

New alleles of the platelet glycoprotein Ibα gene

Platelet membrane glycoprotein Ibα (GP Ibα) bears two molecular polymorphisms which are in linkage disequilibrium: the C/T dimorphism at codon 145 (HPA-2) and the variable number of tandem repeats (VNTR) polymorphism in the macroglycopeptide region. The frequencies of these two polymorphisms, and of another three recently described silent polymorphisms, were investigated by genotypic identification in 729 Caucasian individuals from the south of Spain. Eight different alleles of this gene, including the longest VNTR A allele of the GP Ibα gene, were found in this population. Moreover, we detected an unexpected linkage between the B and A variants of the VNTR polymorphism and the HPA-2a allele in 5.9% of this population. These results suggest a new evolutionary model of GP Ibα, in which homologous recombination could account for the genetic diversity of the GP Ibα.     

Endothelial nitric oxide synthase gene polymorphism and type 2 diabetic retinopathy among Asian Indians

Endothelial nitric oxide synthase (eNOS) has been shown to play an essential role in retinal vascular function, and disequilibrium in its production can lead to diabetic retinopathy (DR). Genetic polymorphisms of eNOS gene have been suggested to play a role in nitric oxide (NO) abnormalities which may contribute to the development and progression of DR. In view of the variable results that have been reported for the association between eNOS gene polymorphisms and DR, the present study was designed to study the association and interaction between eNOS gene polymorphisms and the development and progression of DR in Asian Indian type 2 diabetes mellitus patients (T2DM). We screened 1,720 T2DM patients, belonging to two independently ascertained cohorts out of which 1,446 were genotyped for three polymorphisms of eNOS (two SNPs: T-786C, G894T and one 27-bp repeat polymorphism in intron 4 (27VNTR)) using validated PCR?CRFLP assays. In both the cohorts, consistently lower prevalence and decreased risk of DR was observed in patients with ba, aa and ba?+?aa genotype of 27VNTR (a/b), C-a-G and C-a-T haplotype (allele of T-786C, 27VNTR a/b and G894T) carrying ??C?? allele of T-786C and ??a?? allele of 27VNTR (a/b). Also, mean NO levels in T2DM subjects carrying ba?+?aa genotype were higher as compared to bb genotype. Our results suggest that eNOS genotypes 27VNTR carrying ??aa?? genotype is an independent protective factor for DR and is associated with low risk of DR.     

The effect of human platelet alloantigen polymorphisms on the in vitro responsiveness to adrenaline and collagen

A number of clinical studies have suggested that carriage of the low frequency allele (b) of the human platelet antigen 1 (HPA-1) system is a risk factor for coronary thrombosis. We have examined the effect of a series of HPA biallelic polymorphisms (systems -1, -2, -3 and -5) on the in vitro platelet aggregation in response to adrenaline and collagen in 30 healthy volunteers. There was a significantly higher prevalence (10 out of 18) of carriers of the HPA-1b polymorphism among subjects showing a > 50% aggregation response to adrenaline ('responders') than the prevalence (1/12) in 'non-responders' (P < 0.05). Platelets heterozygous for the HPA-1b polymorphism showed a significantly higher rate (slope) and greater extent (%) of adrenaline-induced aggregation than platelets not carrying the HPA-1b allele (P < 0.05). A greater extent of collagen-induced aggregation was also demonstrated in HPA-1ab platelets (P < 0.05). Inhibition of adrenaline-induced aggregation following incubation with aspirin was greater (P < 0.01) in HPA-1ab than in HPA-1aa platelets. Collagen-induced aggregation was slower in carriers of the HPA-5b allele than in HPA-5aa subjects (P < 0.05). Polymorphisms of the HPA-2 and HPA-3 systems were not associated with different aggregation responses to either adrenaline or collagen. These results support the clinical observation that polymorphism HPA-1b may predispose to increased platelet thrombogenicity and suggest that the presence of polymorphism HPA-5b might render the platelet less reactive to collagen.     

The largest variant of platelet glycoprotein Ib alpha has four tandem repeats of 13 amino acids in the macroglycopeptide region and a genetic linkage with methionine145

Platelet membrane glycoprotein Ib alpha (GPIb alpha) bears the human platelet alloantigen (HPA)-2 and molecular weight (MW) polymorphisms on sodium dodecyl sulfate-polyacrylamide gels. HPA-2 arises from a threonine/methionine dimorphism at residue 145 of the GPIb alpha sequence, whereas different numbers of tandem repeats of a 39-bp sequence encoding 13-amino acids corresponding to a region between serine399 and threonine411 of the GPIb alpha account for the latter. To identify the genetic basis of the MW polymorphism among Japanese, we counted the tandem repeats in 103 individuals. In addition to the reported three variants with one, two, or three tandem repeats, we identified a new variant with four perfect tandem repeats of the 39-bp sequence that corresponded to the largest phenotype. Phenotypic analysis of the MW polymorphism on 12 individuals including all four phenotypes completely accorded in the genotype. We also determined the genotype of HPA-2 and found that methionine145 was in complete linkage disequilibrium, with the larger variants containing three or four tandem repeats. These results imply a model of evolutionary steps in the gene encoding GPIb alpha.     

多巴胺转运体基因可变数目串联重复多态在帕金森病易感性中的作用

目的　探讨多巴胺转运体 (DopamineTransporter ,DAT)基因可变数目串联重复多态(variablenumberoftandemrepeats,VNTR)在帕金森病的疾病易感性中的作用。　方法　采用扩增片段长度多态法 (Amplifiedfragmentlengthpolymorphism ,Amp FLP) ,在上海汉族人群中选择 14 4例帕金森病患者和 184例健康人 ,进行DAT基因VNTR多态与帕金森病的遗传关联研究。　结果　(1)上海地区汉族人群中 ,DAT基因VNTR多态以 4 80bp重复片段为主 ,其基因频率为 93% ;(2 )帕金森病患者与健康人群间无DAT基因VNTR多态分布的差异 ,该多态与帕金森病亦无关联 (P >0 0 5 )。按发病年龄中位数为界将帕金森病组分层后 ,无论是 <6 0岁组人群 ,还是≥ 6 0岁组人群 ,都不存在该多态性与帕金森病的相关。　结论　上海地区汉族人群中DAT基因多态性与帕金森病无关。     

Glycoprotein Ib polymorphisms influence platelet plug formation under high shear rates

Platelet polymorphisms (Kozak, VNTR and HPA-2) within glycoprotein (GP)Ib alpha may be associated with an increased risk of arterial thrombosis. However, the functional role of these polymorphisms has not been clarified. Their influence on platelet plug formation under high shear rates was, therefore, examined in 233 healthy individuals. Collagen-adrenaline-induced closure time was shorter in carriers of the C/D versus C/C VNTR allele and in homozygotes with the (-5)T/T versus (-5)C/T Kozak genotype as determined by novel polymerase chain reaction methods. The HPA-2 genotype had no effects, and the density of GPIb alpha molecules was not influenced by GPIb alpha genotypes.     

Platelet functional implications of glycoprotein Ibalpha polymorphisms in African Americans

The platelet glycoprotein Ibalpha is crucial in the binding of platelets to Von Willebrand Factor within areas of high stress. A single nucleotide polymorphism of GP Ibalpha gives rise to the Ko(a) (HPA-2b) and the -5C Kozak polymorphism. The presence of these polymorphisms has been associated with an increased risk for atherothrombotic disease. The Ko(a) polymorphism has been shown to have a higher prevalence in African Americans compared to American Caucasians. However, very little is known regarding any functional consequences of these platelet polymorphisms in African Americans. We assessed the prevalence of the Ko and -5C Kozak polymorphisms in a population of both African American and American Caucasian patients with and without CAD and determined whether there were platelet functional consequences in both groups. We studied 99 patients of which 22 were African American and 77 were American Caucasian. Aggregations were performed and shear induced platelet plug formation was tested using a platelet function analyzer. The HPA-2b allele was significantly higher in African Americans when compared to Caucasians (P = 0.001). Genotype frequencies of the -5C Kozak polymorphism were not significantly different between the two groups. We found no differences in platelet aggregation in African Americans who were either heterozygous or homozygous for the HPA-2b allele or the -5C Kozak allele when compared to American Caucasians of the same category. We found no significant differences in PFA-100 testing. We conclude from our study that these polymorphisms do not lead to altered platelet function in African Americans.     

Polymorphism in the 5'-leader cistron of the beta2-adrenergic receptor gene associated with obesity and type 2 diabetes.

We screened the 5'-untranslated region of the beta2-adrenergic receptor gene from 40 obese subjects by the PCR-direct sequencing technique. Two polymorphic sites were identified; a T-->C substitution at -47 and a T-->C substitution at -20. We further analyzed the association of the polymorphisms with obesity in 574 subjects by PCR and restriction digestion. The substitution at -47 was in tight linkage disequilibrium with that at -20. The polymorphisms were also in linkage disequilibrium with codon 16 and codon 27 polymorphisms. Subjects carrying the -47C/-20C allele had greater body mass index (25.5+/-4.5 vs. 24.4+/-4.1 kg/m2, p=0.007) and higher serum triglyceride levels (166+/-160 vs. 139+/-95 mg/dl, p=0.015) than -47T/-20T homozygotes. The variant allele frequency was significantly higher in obese subjects than in non-obese subjects (0.18 vs. 0.11, p=0.0026). Furthermore, an increased frequency of the variant allele was shown in diabetic patients compared with non-diabetic subjects (0.19 vs. 0.11, p=0.0005). The association may be attributable to the greater proportion of diabetic patients in the obese group. The exchange at -47 may alter the expression level of the beta2-adrenergic receptor gene, because the nucleotide substitution at -47 results in a Cys-->Arg exchange at the C terminal of the leader peptide. The -47C/-20C allele may be associated with genetic predisposition to obesity and obesity-related metabolic disorders.     

Platelet glycoprotein Ibalpha Kozak polymorphism is associated with an increased risk of ischemic stroke.

Platelets are pivotal to the process of arterial thrombosis resulting in ischemic stroke. Occlusive thrombosis is initiated by the interaction of von Willebrand factor (vWf) and platelet glycoprotein (GP) Ibalpha. Three polymorphisms have been described in GP Ibalpha (Kozak T/C polymorphism, variable number of tandem repeats [VNTR], and the human platelet antigen 2a [HPA-2a] [Thr] or HPA-2b [Met] at position 145), each of which may enhance the vWf and GP Ibalpha interaction. This study investigated whether these polymorphisms are candidate genes for first-ever ischemic stroke. A hospital-based case-control study was conducted of 219 cases of first-ever ischemic stroke and 205 community controls randomly selected from the electoral roll and stratified by age, sex, and postal code. The subtypes of stroke were classified, the prevalence of conventional risk factors was recorded, and blood was collected to perform genotyping analysis for Kozak C or T alleles, VNTR, and HPA-2a/b. It was found that the Kozak T/C genotype was over-represented in the stroke group (32.2%) compared with controls (22.8%) (odds ratio [OR], 1.6; 95% confidence interval [CI], 1.03-2.54; P <.03), and the association was still present even after adjusting for conventional risk factors. There was a trend in the increased prevalence of HPA-2a/b in stroke patients (15%) compared with controls (9.9%) (adjusted OR, 1.8; 95% CI, 0.94-3.4; P =.07). No associations were seen with the VNTR polymorphism or with any of the polymorphisms with stroke subtype. It was concluded that the Kozak T/C polymorphism, which is associated with an increase in platelet GP Ibalpha surface expression, is an independent risk factor for first-ever ischemic stroke.     

Allelic and genotypic frequencies of platelet glycoprotein polymorphisms in a Portuguese population

Introduction and objectivesWe studied genotypic and allelic frequencies of polymorphisms that can affect platelet function, namely the Kozak, VNTR and HPA-2 polymorphisms of glycoprotein Ibα, the Pl^A polymorphism of glycoprotein IIIa and the C807T polymorphism of glycoprotein Ia, in a Portuguese population composed of 227 donors.MethodsPCR-RFLP was used to assess the Kozak, HPA-2, Pl^A and C807T polymorphisms. The VNTR polymorphism was discriminated by different weight bands on electrophoresis.ResultsAll genotypic frequencies were in Hardy-Weinberg equilibrium and do not differ from other Caucasian populations. Genotypic frequencies were 68.3%, 26.9% and 4.8% for Pl^A1/A1, Pl^A1/A2 and Pl^A2/A2 genotypes of the Pl^A polymorphism, 79.3%, 20.3% and 0.4% for TT, TC and CC genotypes of the Kozak polymorphism, 81.1%, 18.9% and 0.0% for aa, ab and bb genotypes of the HPA-2 polymorphism, 15.4%, 0.9%, 70.5%, 11.5%, 1.3% and 0.4% for BC, BD, CC, CD, DD and CE genotypes of the VNTR polymorphism, and 39.7%, 50.2% and 10.1% for CC, CT and TT genotypes of the C807T polymorphism.ConclusionsThe Portuguese population has now been characterized in terms of major platelet glycoprotein polymorphisms, which will be an important tool for further studies to assess the role of platelet glycoproteins in individual predisposition to prothrombotic conditions and response to antithrombotic therapy.     

From the Cover: Evidence of positive selection acting at the human dopamine receptor D4 gene locus

Associations have been reported of the seven-repeat (7R) allele of the human dopamine receptor D4 (DRD4) gene with both attention-deficit/hyperactivity disorder and the personality trait of novelty seeking. This polymorphism occurs in a 48-bp tandem repeat in the coding region of DRD4, with the most common allele containing four repeats (4R) and rarer variants containing 2-11. Here we show by DNA resequencing/haplotyping of 600 DRD4 alleles, representing a worldwide population sample, that the origin of 2R-6R alleles can be explained by simple one-step recombination/mutation events. In contrast, the 7R allele is not simply related to the other common alleles, differing by greater than six recombinations/mutations. Strong linkage disequilibrium was found between the 7R allele and surrounding DRD4 polymorphisms, suggesting that this allele is at least 5-10-fold "younger" than the common 4R allele. Based on an observed bias toward nonsynonymous amino acid changes, the unusual DNA sequence organization, and the strong linkage disequilibrium surrounding the DRD4 7R allele, we propose that this allele originated as a rare mutational event that nevertheless increased to high frequency in human populations by positive selection.     

The apolipoprotein(a) gene: linkage disequilibria at three loci differs in African Americans and Caucasians

Lipoprotein(a) (Lp(a)) is an independent, genetically regulated cardiovascular risk factor. Lp(a) plasma levels are largely determined by the apolipoprotein(a) (apo(a)) component, and differ across ethnicity. Although a number of polymorphisms in the apo(a) gene have been identified, apo(a) genetic regulation is not fully understood. To study the relation between apo(a) gene variants, we constructed haplotypes and assessed linkage equilibrium in African Americans and Caucasians for three widely studied apo(a) gene polymorphisms (apo(a) size, +93 C/T and pentanucleotide repeat region (PNR)). Apo(a) size allele frequency distributions were different across ethnicity (p<0.01). For African Americans, PNR frequencies were similar across apo(a) sizes, suggesting linkage equilibrium. For Caucasians, the PNR and the PNR-C/T haplotype frequencies differed for large and small apo(a), with the T and PNR 9 alleles associated with large apo(a) size (p<0.0002); also, the PNR 9 allele was more common on a T allele, while PNR 8 was more common on a C allele. On a C allele background, small PNR alleles were more common and the PNR 10 allele less common among African Americans than Caucasians (p<0.001). The ethnic difference in apo(a) size distribution remained controlling for C/T and PNR alleles (p=0.023). In conclusion, allele and haplotype frequencies and the nature of the linkage disequilibrium differed between African Americans and Caucasians at three apo(a) gene polymorphisms.     

Polymorphisms in the endothelial nitric oxide synthase gene are associated with Behçet's disease

OBJECTIVE: Reduced plasma nitric oxide (NO) levels in Beh?et's disease (BD) patients have been implicated in the development of the endothelial abnormalities and thrombotic complications occurring in these patients. This study investigated the association of the endothelial NO Synthase (eNOS) gene polymorphisms with BD. METHODS: A case-control study was carried out using 193 unrelated Turkish BD patients and 106 healthy controls. All individuals were genotyped by PCR for two single-nucleotide polymorphisms (SNPs): -786 T-->C in the promoter region and 894 G-->T in exon 7 (Glu298Asp). A variable number of tandem repeats (VNTR) polymorphism in intron 4 was also investigated. RESULTS: The VNTR polymorphism was associated with BD, detected by an increased frequency of the b allele (odds ratio = 1.9, P = 0.0069) and b/b genotype (odds ratio = 2.2, P = 0.002) in patients. After the stratification of cases according to the family history, a significant difference between familial cases and controls in the -786 SNP was observed, with an increase in the frequency of the T allele (odds ratio = 2.5, P = 0.0016) and T/T genotype (odds ratio = 2.5, P = 0.0085), and the association of the VNTR polymorphism with BD became stronger. The -786*T and VNTR*b alleles were in linkage disequilibrium (D' = 0.65, P <0.0001), and the number of individuals homozygous for the -786*T/VNTR*b haplotype was significantly increased in the patients. CONCLUSIONS: eNOS gene polymorphisms are associated with BD, which might contribute to the reduced NO activity observed in BD patients.     

Rapid typing of apolipoprotein B DNA polymorphisms by DNA amplification. Association between Ag epitopes of human apolipoprotein B-100, a signal peptide insertion/deletion polymorphism, and a 3'flanking DNA variable number of tandem repeats polymorphism of the apolipoprotein B gene.

We present here rapid and efficient methods for the analysis of multiple variable apolipoprotein (apo) B loci using polymerase chain reaction based techniques. For illustrative purposes, we have applied these methods to establish an association between these polymorphisms and the apo B Ag immunological epitopes. The 5 DNA polymorphisms include 3 restriction endonuclease sites (for XbaI, EcoRI and MspI), a variable number of tandem repeat (VNTR) locus at the 3' end of the apo B gene, and an insertion/deletion polymorphism involving the signal peptide region of apo B. The latter two newly described polymorphisms are directly detectable following amplification and may have physiological effects on apo B expression because of their critical locations. All of these sites were typed using flanking oligonucleotides and the newly developed polymerase chain reaction. Amplification products were typed either directly (3' VNTR and signal peptide insertion/deletion alleles), or following specific enzyme digestion (for the restriction sites), or by allele specific oligonucleotides. The detailed methods presented will prove generally useful for rapidly typing DNA variation in the apo B gene. Using these techniques, we found a significant linkage disequilibrium between the Ag(t/z) locus and the 3' VNTR, and the Ag(c/g) locus and the signal peptide length polymorphism. Future association studies using these DNA polymorphisms should take into consideration that observed effects may be related to its linkage disequilibrium with the Ag loci and vice versa.     

洛阳地区献血员人类血小板抗原1～10频率分布调查

目的:研究洛阳地区献血员人类血小板抗原(HPA)1~10基因多态性,为患者提供HPA相配合的血小板。方法:随机收集250例无偿献血员样本,应用聚合酶链式反应-序列特异引物(PCR-SSP)技术检测HPA-1~10基因型及等位基因频率。结果:HPA-1aa、-1ab所占百分比分别为98.80%和1.20%,HPA-2aa、-2ab所占百分比分别为90.80%和9.20%,HPA-4aa、-4ab所占百分比分别为98.00%和2.00%,HPA-5aa、-5ab所占百分比分别为96.40%和3.60%,HPA-aa、-ab、-bb三种基因型在HPA-3和HPA-6中的比例分别为29.20%、46.00%、24.80%和91.60%、6.80%、1.60%,在HPA-7、-8、-9、-10中仅检测到HPA-aa纯合子。结论:洛阳地区HPA-1~HPA-10基因分布与文献报道相似,HPA-3特异性抗体是引起本地区HPA同种免疫性疾病的首要原因。     

Identification of the Rare,Four Repeat Allele of IL-4 Intron-3 VNTR Polymorphism in Indian Populations

Background: Cytokines are cell signaling molecules which upon release by cells facilitate the recruitment of immune-modulatory cells towards the sites of inflammation. Genetic variations in cytokine genes are shown to regulate their production and affect the risk of infectious as well as autoimmune diseases. Intron-3 of interleukin-4 gene (IL-4) harbors 70-bp variable number of tandem repeats (VNTR) that may alter the expression level of IL-4 gene. Objective: To determine the distribution of IL-4 70-bp VNTR polymorphism in seven genetically heterogeneous populations of Chhattisgarh, India and their comparison with the finding of other Indian and world populations. Methods: A total of 371 healthy unrelated individuals from 5 caste and 2 tribal populations were included in the present study. The IL-4 70-bp VNTR genotyping was carried out using PCR and electrophoresis. Results: Overall, 3 alleles of IL-4 70-bp VNTR (a2, a3 and a4) were detected. The results demonstrated the variability of the IL-4 70-bp VNTR polymorphism in Chhattisgarh populations. Allele a3 was the most common allele at the 70-bp VNTR locus in all populations followed by a2 allele. This study reports the presence four repeat allele a4 at a low frequency in the majority of the Chhattisgarh populations studied. Further, the frequency of the minor allele (a2) in Chhattisgarh populations showed similarity with the frequencies of European populations but not with the East Asian populations where the a2 allele is a major allele. Conclusions: Our study provides a baseline for future research into the role of the IL-4 locus in diseases linked to inflammation in Indian populations.     

Role of 4 platelet membrane glycoprotein polymorphisms on experimental arterial thrombus formation in men.

This study investigates whether the polymorphisms of 3 important platelet receptors affected experimental thrombus formation in men. Forty healthy male volunteers randomly recruited were genotyped for the variable number of tandem repeat (VNTR) of GPIbalpha, the -5T/C polymorphism in the Kozak sequence of GPIbalpha, the 807C/T polymorphism of GPIa, and the PI(A1)/PI(A2) polymorphism of GPIIb/IIIa. Platelet thrombus formation was induced ex vivo by exposing a collagen-coated coverslip in a parallel plate perfusion chamber to native blood for 4 minutes. The shear rates at the collagen surface were 650 and 2600 x s(-1). At 2600 x s(-1) platelet thrombus formation was significantly related only to the 807C/T polymorphism. In contrast, at 650 x s(-1) thrombus formation was significantly altered only by the Kozak sequence polymorphism. The VNTR and the PI(A1)/PI(A2) polymorphisms did not influence thrombus formation. Thus, platelet thrombus formation is significantly influenced by genetic variations of the GPIbalpha and GPIa receptors. The effect of these polymorphisms was dependent on the blood flow rate.     

Genetic linkage of Kozak sequence polymorphism of the platelet glycoprotein Ib alpha with human platelet antigen-2 and variable number of tandem repeats polymorphism, and its relationship with coronary artery disease

The -5 C/T polymorphism of platelet glycoprotein (GP) Ib alpha is a major determinant of the level of GP Ib/V/IX complex surface expression. We investigated the frequency of this polymorphism among Asian populations. The gene frequencies of cytosine (C) in this polymorphism were 0.283 and 0.219 in Japanese and Korean populations respectively. The C allele is linked with human platelet antigen (HPA)-2a and smaller types of variable number of tandem repeats (VNTR). A novel allele, C-HPA-2a-D of VNTR, was found. No association was observed between these alleles and coronary artery disease in this case-control study. The clinical relevance of this polymorphism in the thrombotic status remains undetermined.     

Threonine-145/methionine-145 variants of baculovirus produced recombinant ligand binding domain of GPIbalpha express HPA-2 epitopes and show equal binding of von Willebrand factor

Glycoprotein (GP) Ibalpha is the functionally dominant subunit of the platelet GPIb-IX-V receptor complex, with the von Willebrand factor (vWF) binding site residing on the amino-terminus. A threonine for methionine-145 replacement of GPIbalpha is associated with the human platelet antigen (HPA)-2 system. To study the structural and functional consequences of this mutation, both forms of GPIbalpha were expressed as calmodulin fusion proteins in insect cells. Both recombinant proteins were recognized by their respective alloantibodies, independent of glycosylation or intactness of disulfide bonds, and gave similar results to platelet-derived GPIbalpha in antibody detection assays. Resonant mirror studies showed that vWF binding was not affected by the HPA-2 mutation; however, vWF binding was partially inhibited by IgG HPA-2 antibodies. Our data are compatible with an involvement of the leucine-rich repeat domain of GPIbalpha in vWF binding and indicate that recombinant GPIbalpha may be used to detect HPA-2 antibodies. (Blood. 2000;95:205-211)