Association of the –92C/G and 807C/T Polymorphisms of the 2 Subunit Gene With Human Platelets 2?1 Receptor Density
http://www.100md.com
动脉硬化血栓血管生物学 2005年第8期
From the Departments of Hematology (N.A., P.D., M.-C.G.), Anesthesiology (C.B., I.P.), and Biochemistry (B.G.), the Clinical Investigation Center (V.H.), Biostatistics (P.V.), Hopital Bichat, Assistance Publique-Hopitaux de Paris, and INSERM U698 (N.A., C.B., M.-C.G., J.B.), Hopital Bichat, University Paris, France.
Correspondence to Dr N. Ajzenberg, Service d’Hématologie et Immunologie, H?pital Bichat, 46 rue Henri Huchard, 75018, Paris, France. E-mail nadine.ajzenberg@bch.ap-hop-paris.fr
Abstract
Objective— Platelet adhesion to the subendothelial tissue via the collagen receptor 2?1 is a crucial event in vascular biology. Although evidence has been provided that the number of platelets 2?1 copies is genetically determined, the molecular change primary responsible has not been yet elucidated. The aim of our present study was to investigate the effect of combined polymorphisms within both regulatory (–52C/T and –92C/G) and coding regions (807C/T and 1648A/G) of the 2 subunit gene on human platelets 2?1 receptor density and/or susceptibility to coronary artery disease (CAD).
Methods and Results— Among 254 cardiac surgery patients, no evidence was found for an association between the 2 subunit gene polymorphisms and CAD. In contrast, in a subgroup of 113 patients, we observed a significant association between all polymorphisms except –52C/T and 2?1 receptor level. Furthermore, when 3 groups of patients were defined according to the tertiles of platelets 2?1 copies, the –92C/807T haplotype was more frequent in the group of patients with high 2?1 receptor level.
Conclusion— These results suggest that an individual effect of each polymorphism located either in the coding or promoter sequence of the 2 gene may act in combination to modulate variations in platelets 2?1 receptor density.
An important combined effect of the –92C and 807T polymorphisms of the 2 gene in increasing the expression of human platelet 2?1 receptors has been observed, suggesting that this haplotype could modulate variations in 2?1 receptor density.
Key Words: –92C/G ? 807C/T polymorphism ? 2?1 density
Introduction
Vessel wall injury triggers platelet activation and platelet plug formation, followed by the formation of fibrin-containing thrombi that occlude the site of injury. The interaction of platelet receptors with subendothelial components such as collagen is central to these events, which both limit blood loss at sites of tissue trauma but may also obstruct diseased vessels, leading to ischemia and infarction of vital organs. The direct platelet–collagen interaction is mediated by 2 receptors, the integrin 2?1 and the nonintegrin receptor glycoprotein VI (GPVI), although there are likely to be others.1 It is now considered that GPVI provides the primary collagen signal that activates and recruits the integrin receptor 2?1 to further amplify collagen signals and fully activate platelets through a common intracellular signaling pathway.2
Integrin 2?1 is a heterodimer composed of 2 noncovalently associated subunits (2 and ?1) that are encoded by separate genes. By virtue of its expression on platelets and vascular cells,3–5 the integrin 2?1 may play a significant role in vascular pathobiology. Among healthy individuals, platelet 2?1 density is highly variable and correlates with the rate and extent of platelet adhesion to collagen type I or type III under static conditions.4,6 The differences in platelet 2?1 density also correlate with the inheritance of certain allelic combinations, defined by linked polymorphisms within the coding sequence of the 2 gene.7–10 In addition, 2 single-base substitutions at positions –52 and –92 have been identified11 within the "core" region defined by Zutter et al12 in the proximal 5'-regulatory region of the gene. In vitro, the –52C/T and –92C/G dimorphisms have been shown to influence the rate of the 2 gene transcription in transfected human megakaryocytic cell lines.11 It has also been suggested that 1 of the 2 promoter dimorphisms (–52C/T) could correlate with platelet 2?1 density in healthy individuals.13
A potentially important role for the 2?1 integrin is suggested by recent epidemiologic data.7,14,15 Several studies suggest a direct correlation between the genetically determined number of copies of 2?1 at the platelet surface and the risk of thrombotic events. The T allele of 807C/T polymorphism, which is associated with high-level expression of 2?1 on platelets, has been reported as an independent risk factor for myocardial infarction in selected groups of patients16–18 for the development of diabetic retinopathy in patients with type 2 diabetes mellitus19 and for stroke in the young,20 although other studies failed to find such a correlation.21–24 However, no data are yet available for the clinical impact of the dimorphisms present in the core region of the promoter that may modulate the expression of 2?1 at the cell surfaces.
The aim of the present study was to examine the distribution of the –92C/G, –52C/T, 807C/T, and 1648A/G dimorphisms in a population of patients undergoing cardiac surgery and to analyze the influence of particular haplotypes on platelets 2?1 receptor density. We hypothesized that the profile of an individual with combined polymorphisms in coding and regulatory regions of the 2 subunit gene may constitute a hereditary background for the susceptibility to the development of coronary artery disease (CAD).
Patients, Materials, and Methods
Patient Selection
The protocol was approved by the local ethics committee, and all patients gave their written informed consent to participate in the study. The total study population comprised 254 consecutive white patients (179 men and 75 women; 59±14 years of age) scheduled for either coronary artery bypass grafting or valve surgery. By means of coronary angiography, the study population was divided into patients with CAD (n=171) for subjects referred to the hospital for coronary artery bypass grafting in whom coronary artery stenosis was >70% (50% for left main artery) and non-CAD patients (n=83) for subjects referred to the hospital for valve surgery and without any angiographic signs of CAD (no atherosclerotic coronary plaque). We excluded patients with acute coronary syndrome who required emergency coronary artery bypass.
Blood Sampling
Blood sample was collected before coronary artery bypass or valve surgery. Blood was drawn in evacuated container tubes (Vacutainer; Becton Dickinson) containing EDTA for DNA analysis or 0.129 mol/L trisodium citrate for platelet 2?1 density determination.
DNA Genotyping
We obtained genomic DNA from blood samples after extraction from blood leukocytes by a standard procedure with the QIAmp DNA blood Midi kit (Qiagen GmbH) according to manufacturer instructions.
We studied 2 nucleotide polymorphisms located at 807C/T bp and 1648A/G bp within the coding region of the 2 gene25,26 and single-base substitutions at 2 positions, –92C/G and –52C/T, within the proximal 5'-regulatory region of the 2 gene.13 Genotyping of these 4 polymorphisms was conducted using an adapted method of DNA amplification by polymerase chain reaction (PCR) procedure with specific primers. PCR products were digested by specific restriction enzymes and separated by appropriate electrophoresis.
Quantitation of Platelet 2?1
We quantified the platelet 2?1 level in a subgroup of 113 patients (CAD n=49; non-CAD n=64). Determination of platelet 2?1 density was performed on citrated whole blood by flow cytometry using a kit "platelet Gp Screen test" (Biocytex). Briefly, whole blood was incubated with mouse monoclonal antibody against 2?1 integrin (CD 69b) and followed by an incubation with a polyclonal antibody anti-mouse IgG coupled to fluorescein isothiocyanate.27 Cytometric analysis was performed on a FACScalibur (Becton Dickinson). The number of platelet receptors was determined by converting the fluorescence intensity into corresponding number of sites per platelet on the basis of a calibrated bead standard curve using beads varying from 260 to 80 000 sites per platelet.
Statistical Analysis
Genotype distributions in different groups were compared by testing the hypothesis of homogeneity using 2 test and SAS software (SAS Institute; V8.0). Estimation of haplotype frequencies and linkage disequilibrium (D and D') were computed using the Arlequin software V2.000. Data were expressed as mean±SD and analyzed within genotype groups using ANOVA and Tukey–Kramer test for post hoc comparisons. For categorical variables and tertile comparisons, 2 test was performed. Differences were considered to be significant when P<0.05.
Results
Distribution of the 2 Gene Polymorphisms
Table 1 summarizes the genotype distribution of the 807C/T, 1648 A/G, –92 C/G, and –52 C/T polymorphisms of the 2 gene in the CAD and non-CAD groups. We reported similar genotype distribution in both groups. The genotype distribution is in agreement with the frequencies predicted by Hardy–Weinberg equilibrium (P=0.76, P=0.48, P=0.54, and P=0.38, respectively.)
TABLE 1. Genotype Distribution in CAD and Non-CAD Patients
Measure of Linkage Disequilibrium Between 2 Gene Polymorphic Sites
In the total population of 254 patients, we investigated the linkage disequilibrium between the 4 polymorphic sites of 2 gene (Table 2). A significant linkage disequilibrium was observed between the 807C/T and 1648A/G polymorphisms and between the –52C/T and –92C/G polymorphisms within the proximal 5' regulatory region of the 2 gene: all patients carrying the –92C allele were found to carry the –52T allele.
TABLE 2. Measure of Linkage Disequilibrium (D and D') Between 2 Polymorphic Sites in the Total Population
In contrast, we observed a moderate linkage disequilibrium between the 807C/T and the promoter sequence –92C/G and –52C/T (Table 2).
Relationship Between 2 Genotypes and Differences in Platelet 2?1 Levels
A subgroup of 113 patients (CAD n=49; non-CAD n=64) was available for the measurement of platelet 2?1 levels in whole blood by flow cytometry. No difference in 2?1 density was observed between CAD and non-CAD patients (3960±1098 and 3881±1084 receptors per platelet, respectively). In consequence, the 2 groups were pooled to examine the relationship between genotypes and 2?1 platelet density. In Figure 1, the number of platelet 2?1 copies per platelet is plotted according to the 2 genotypes within the coding sequence of each individual. As originally reported by Kunicki et al28 and confirmed by others,21,29 in healthy subjects, we observed (Figure 1A) an association between 2?1 densities and 807 polymorphism (ANOVA P<0.0001). Lowest 2?1 densities (3200±906 receptors per platelet) were observed in 807CC patients; and conversely, highest receptor density in 807TT patients (4782±1216 receptors per platelet; P<0.05). In the same way, the platelet 2?1 levels measured in 1648GG patients were lower than in heterozygous 1648AG patients (ANOVA P=0.0082), reaching 3774±1090 and 4435±958 receptors per platelet, respectively (P<0.05). Unfortunately, no homozygous 1648AA patient was available for quantification of platelet 2?1 copies.
Figure 1. Relationship between platelet 2?1 density and polymorphisms within the 2 coding region: 807C/T (A) and 1648 G/A (B) in a population of 113 patients. Box and whisker plots show median value (horizontal lines), 25th and 75th percentiles (boxes), and 10th and 90th percentiles (error bars). *P<0.05.
Interestingly, we also found a significant correlation between the –92 C/G polymorphism and the platelet 2?1 level (ANOVA P=0.0056; Figure 2). The –92CC homozygous patients exhibited higher levels of 2?1 receptors than the heterozygous –92CG patients (4066±1122 and 3384±744 receptors per platelet, respectively; P<0.05). The 4 homozygous patients for the –92 G allele had low density of 2?1 (2810±646 receptors per platelet), but the difference with the –92CC homozygous did not reach significance. In contrast, the platelet 2?1 receptor level was not found to be associated with the –52 C/T polymorphism (ANOVA P=0.565) because density of 2?1 varies between 3792±1115 receptors per platelet for –52CC and 3829±911 receptors per platelet for –52TT.
Figure 2. Relationship between platelet 2?1 density and polymorphisms within the 5' regulatory region: –92 C/G in a population of 113 patients. Box and whisker plots show median value (horizontal lines), 25th and 75th percentiles (boxes), and 10th and 90th percentiles (error bars). *P<0.05.
We looked for a possible influence of combined polymorphisms within regulatory and coding regions of the 2 subunit gene on human platelet 2?1 receptor density. Because of the strong linkage disequilibrium between the 807C/T and 1648G/A polymorphisms on one hand, and the absence of association between the –52C/T polymorphism and the 2?1 receptor density on the other hand, we determined the frequencies of haplotypes defined by the –92C/G and 807C/T polymorphisms in a panel of 113 patients in which the platelet 2?1 receptor level has been quantified (Table 3). Patients were studied according to tertiles of platelet 2?1 receptor level: low (<3348 copies per platelet), medium (ranging from 3348 to 4191), and high (>4191) platelet 2?1 receptor level. We observed that haplotype distribution was significantly different among the 3 groups of patients (ANOVA P<0.001). The most striking observation was that the –92C/807T haplotype is more frequent in the high-platelet 2?1 receptor level group (0.60 versus 0.12 in the first tertile), whereas the –92G/807C haplotype is more frequent in the low-platelet 2?1 receptor level group (0.16 versus 0.02 in the third tertile). Moreover, to determine whether this haplotype frequency was related to the linkage disequilibrium of polymorphisms or to the effect of each polymorphism, we compared the 2?1 density according to the 807 genotype in the subgroup of 88 patients homozygous for –92C: significant differences in 2?1 receptor level between 807TT, 807CT, and 807CC were observed, suggesting an individual effect of each polymorphism (ANOVA P<0.0001) and reaching 4872±1290, 4251±917, and 3316±912 receptors per platelet, respectively).
TABLE 3. Haplotype Frequency in the 3 Groups of Patients Defined by Tertiles of Platelet 2?1 Receptors per Platelet
Discussion
Platelet adhesion to the subendothelial tissue via the collagen receptor 2?1 is a crucial event in vascular biology and interindividual variations in 2?1 expression levels could have a significant impact on vascular pathology and risk of arterial thrombosis. Although evidence has been provided that the number of platelets 2?1 copies is genetically determined,7–10 the molecular change primarily responsible has not been yet elucidated. The aim of our present study was to investigate the effect of combined polymorphisms within regulatory and coding regions of the 2 subunit gene on human platelet 2?1 receptor density or susceptibility to CAD.
Among a total population of 254 patients, we report new data about the –92C/G, –52C/T, 807C/T, and 1648G/A polymorphic sites of the 2 gene. The linkage disequilibrium between the 807C/T and 1648A/G nucleotide polymorphisms on one hand, and between the –92C/G and –52C/T single-base substitutions on the other hand, is confirmed, as reported previously by other groups.26,28 The platelet 2?1 receptor quantification performed in freshly drawn whole blood also confirms that the level of collagen receptor 2?1 can vary up to 4-fold among individuals.6 As originally reported by Kunicki et al28 in healthy subjects and confirmed by others,10,29 we observed an obvious association between the 807C allele and low 2?1 level. However, homozygous patients for the 1648G allele exhibited the lowest 2?1 density, in agreement with previous data of Corral et al.10 No association was observed between the –52C/T dimorphism and platelet receptor density. Interestingly, we found that the platelet 2?1 receptor level was significantly associated with the –92 C/G promoter nucleotide dimorphism. This finding is supported by the previous work of Jacquelin et al,13 demonstrating that in vitro, in transfected human megakaryocytic cell lines, the –92G promoter sequence has a negative regulatory effect on the 2 gene transcription. Altogether, these findings emphasize the potential importance of the –92C promoter substitution on human platelet 2?1 receptor expression.
When 3 groups of patients were defined according to the tertiles of the number of platelets 2?1 copies, it appeared that haplotype distribution was different: the –92G/807C haplotype was more frequent in the group of patients with low 2?1 receptor levels, whereas the –92C/807T haplotype was in a large majority found in the group of patients with high 2?1 receptor levels. Moreover, in the subgroup of patients, –92CC significant differences of 2?1 density were observed according to 807C/T polymorphism. Because of moderate linkage disequilibrium between the –92C/G and 807C/T polymorphisms, the present results confirm evidence for more than a single genetic factor involved in the number of 2?1 molecules per platelet. Our findings indicate that an individual effect of each polymorphism in either the coding or promoter sequence may act in combination to modulate variation in receptor density. Although the –92C/G promoter substitution may have a direct impact on expression levels of 2 gene, the silent 807C/T polymorphism within the coding sequence that does not modify the deduced amino acid sequence of the translated protein may be linked to other polymorphisms within the 2 gene, which remain to be determined.
Looking for a clinical relevance of the 2 alleles, we investigated the relationship between the platelet 2?1 receptor density and nucleotide polymorphisms of the 2 subunit gene and CAD. No significant difference was observed in the surface expression of platelet 2?1 in CAD versus non-CAD patients. It is likely that the 2?1 receptor number alone is not sufficient to compromise platelet function and an individual’s susceptibility to arterial diseases, which are probably under the dependence of an other major collagen receptor, GPVI,30 or of other platelet receptors such as GPIb or 2b?3. Abnormalities of the vessel wall (as atherosclerosis) could also modulate platelet adhesion. Genotype distributions and allele frequencies were not significantly different in CAD versus non-CAD patients. With regard to nucleotide polymorphisms located within the coding region of the gene, several previous reports have extensively studied 807C/T and 1648A/G dimorphisms. In agreement with us, some authors failed to show an association between 807C/T polymorphism and CAD,21,22,24,31 whereas conflicting studies showed a significant correlation between 807T allele and myocardial infarction.16,17 Furthermore, Kroll et al found in a large population a significant association between the 1648A/G polymorphism and CAD.32 Possible explanations for these apparent discrepancies may be the differences of population size and the fact that numerous other factors are involved in the progression of atherosclerosis. Furthermore, our negative results may be attributable to the choice of an anatomic (angiographic) criteria rather than a more clinically relevant one such as evolutivity of coronary disease (occurrence of acute coronary syndrome).
In summary, we showed evidence for a possible influence of the inheritance of particular haplotypes of 2 gene on differences in human platelet 2?1 receptor density. Our results suggest an important combined effect of the –92C and 807T nucleotide sequences in increasing the expression of human platelet 2?1 receptors. These results must be confirmed in a larger study to precisely determine the involvement of the 2 gene allelic combinations in coronary disease through the platelet 2?1 receptor expression. Further studies are also required in a larger population to test whether or not the –92C/G and 807C/T polymorphisms have a functional effect on gene expression, and, if not, to identify the causal single-nucleotide polymorphisms in linkage disequilibrium with –92C/G and 807C/T polymorphisms. A more generalized haplotype strategy on the basis of polymorphisms described in single-nucleotide polymorphism databases would probably be most pertinent.
References
Moog S, Mangin P, Lenain N, Strassel C, Ravanat C, Schuhler S, Freund M, Santer M, Kahn M, Nieswandt B, Gachet C, Cazenave JP, Lanza F. Platelet glycoprotein V binds to collagen and participates in platelet adhesion and aggregation. Blood. 2001; 98: 1038–1046.
Chen H, Kahn ML. Reciprocal signaling by integrin and nonintegrin receptors during collagen activation of platelets. Mol Cell Biol. 2003; 23: 4764–4777.
Santoro SA, Rajpara SM, Staatz WD, Woods VL Jr. Isolation and characterization of a platelet surface collagen binding complex related to VLA-2. Biochem Biophys Res Commun. 1988; 153: 217–223.
Kunicki TJ, Nugent DJ, Staats SJ, Orchekowski RP, Wayner EA, Carter WG. The human fibroblast class II extracellular matrix receptor mediates platelet adhesion to collagen and is identical to the platelet glycoprotein Ia-IIa complex. J Biol Chem. 1988; 263: 4516–4519.
Santoro SA, Zutter MM. The alpha 2 beta 1 integrin: a collagen receptor on platelets and other cells. Thromb Haemost. 1995; 74: 813–821.
Kunicki TJ, Orchekowski R, Annis D, Honda Y. Variability of integrin alpha 2 beta 1 activity on human platelets. Blood. 1993; 82: 2693–2703.
Santoro SA. Platelet surface collagen receptor polymorphisms: variable receptor expression and thrombotic/hemorrhagic risk. Blood. 1999; 93: 3575–3577.
Santoso S, Kalb R, Walka M, Kiefel V, Mueller-Eckhardt C, Newman PJ. The human platelet alloantigens Br(a) and Brb are associated with a single amino acid polymorphism on glycoprotein Ia (integrin subunit alpha 2). J Clin Invest. 1993; 92: 2427–2432.
Kritzik M, Savage B, Nugent DJ, Santoso S, Ruggeri ZM, Kunicki TJ. Nucleotide polymorphisms in the alpha2 gene define multiple alleles that are associated with differences in platelet alpha2 beta1 density. Blood. 1998; 92: 2382–2388.
Corral J, Rivera J, Gonzalez-Conejero R, Vicente V. The number of platelet glycoprotein Ia molecules is associated with the genetically linked 807C/T and HPA-5 polymorphisms. Transfusion. 1999; 39: 372–378.
Jacquelin B, Rozenshteyn D, Kanaji S, Koziol JA, Nurden AT, Kunicki TJ. Characterization of inherited differences in transcription of the human integrin alpha 2 gene. J Biol Chem. 2001; 276: 23518–23524.
Zutter MM, Santoro SA, Painter AS, Tsung YL, Gafford A. The human alpha 2 integrin gene promoter. Identification of positive and negative regulatory elements important for cell-type and developmentally restricted gene expression. J Biol Chem. 1994; 269: 463–469.
Jacquelin B, Tarantino MD, Kritzik M, Rozenshteyn D, Koziol JA, Nurden AT, Kunicki TJ. Allele-dependent transcriptional regulation of the human integrin alpha2 gene. Blood. 2001; 97: 1721–1726.
Kunicki TJ. The role of platelet collagen receptor (glycoprotein Ia/IIa; integrin alpha2 beta1) polymorphisms in thrombotic disease. Curr Opin Hematol. 2001; 8: 277–285.
Kunicki TJ, Ruggeri ZM. Platelet collagen receptors and risk prediction in stroke and coronary artery disease. Circulation. 2001; 104: 1451–1453.
Moshfegh K, Wuillemin WA, Redondo M, Lammle B, Beer JH, Liechti-Gallati S, Meyer BJ. Association of two silent polymorphisms of platelet glycoprotein Ia/IIa receptor with risk of myocardial infarction: a case-control study. Lancet. 1999; 353: 351–354.
Santoso S, Kunicki TJ, Kroll H, Haberbosch W, Gardemann A. Association of the platelet glycoprotein Ia C807T gene polymorphism with nonfatal myocardial infarction in younger patients. Blood. 1999; 93: 2449–2453.
Roest M, Banga JD, Grobbee DE, de Groot PG, Sixma JJ, Tempelman MJ, van der Schouw YT. Homozygosity for 807T polymorphism in alpha(2) subunit of platelet alpha(2)beta(1) is associated with increased risk of cardiovascular mortality in high-risk women. Circulation. 2000; 102: 1645–1650.
Petrovic M, Hawlina M, Peterlin B, Petrovic D. BglII gene polymorphisms of the alpha2beta1 gene is a risk factor for diabetic retinopathy in Caucasians with type 2 diabetes. J Hum Genet. 2003; 48: 457–460.
Carlsson LE, Santoso S, Spitzer C, Kessler C, Greinacher A. The alpha2 gene coding sequence T807/A873 of the platelet collagen receptor integrin alpha2beta1 might be a genetic risk factor for the development of stroke in younger patients. Blood. 1999; 93: 3583–3586.
Corral J, Gonzalez-Conejero R, Rivera J, Ortuno F, Aparicio P, Vicente V. Role of the 807C/T polymorphism of the alpha2 gene in platelet GP Ia collagen receptor expression and function–effect in thromboembolic diseases. Thromb Haemost. 1999; 81: 951–956.
Croft SA, Hampton KK, Sorrell JA, Steeds RP, Channer KS, Samani NJ, Daly ME. The GPIa C807T dimorphism associated with platelet collagen receptor density is not a risk factor for myocardial infarction. Br J Haematol. 1999; 106: 771–776.
Cole VJ, Staton JM, Eikelboom JW, Hankey GJ, Yi Q, Shen Y, Berndt MC, Baker RI. Collagen platelet receptor polymorphisms integrin alpha2beta1 C807T and GPVI Q317L and risk of ischemic stroke. J Thromb Haemost. 2003; 1: 963–970.
Morita H, Kurihara H, Imai Y, Sugiyama T, Hamada C, Sakai E, Mori M, Nagai R. Lack of association between the platelet glycoprotein Ia C807T gene polymorphism and myocardial infarction in Japanese. An approach entailing melting curve analysis with specific fluorescent hybridization probes. Thromb Haemost. 2001; 85: 226–230.
Kalb R, Santoso S, Unkelbach K, Kiefel V, Mueller-Eckhardt C. Localization of the Br polymorphism on a 144 bp exon of the GPIa gene and its application in platelet DNA typing. Thromb Haemost. 1994; 71: 651–654.
Reiner AP, Aramaki KM, Teramura G, Gaur L. Analysis of platelet glycoprotein Ia (alpha2 integrin) allele frequencies in three North Am populations reveals genetic association between nucleotide 807C/T and amino acid 505 Glu/Lys (HPA-5) dimorphisms. Thromb Haemost. 1998; 80: 449–456.
Schmitz G, Rothe G, Ruf A, Barlage S, Tschope D, Clemetson KJ, Goodall AH, Michelson AD, Nurden AT, Shankey TV. European Working Group on Clinical Cell Analysis: Consensus protocol for the flow cytometric characterisation of platelet function. Thromb Haemost. 1998; 79: 885–896.
Kunicki TJ, Kritzik M, Annis DS, Nugent DJ. Hereditary variation in platelet integrin alpha 2 beta 1 density is associated with two silent polymorphisms in the alpha 2 gene coding sequence. Blood. 1997; 89: 1939–1943.
Roest M, Sixma JJ, Wu YP, Ijsseldijk MJ, Tempelman M, Slootweg PJ, de Groot PG, van Zanten GH. Platelet adhesion to collagen in healthy volunteers is influenced by variation of both alpha(2)beta(1) density and von Willebrand factor. Blood. 2000; 96: 1433–1437.
Nieswandt B, Brakebusch C, Bergmeier W, Schulte V, Bouvard D, Mokhtari-Nejad R, Lindhout T, Heemskerk JW, Zirngibl H, Fassler R. Glycoprotein VI but not alpha2beta1 integrin is essential for platelet interaction with collagen. EMBO J. 2001; 20: 2120–2130.
Mikkelsson J, Perola M, Penttila A, Karhunen PJ. Platelet collagen receptor GPIa (C807T/HPA-5) haplotype is not associated with an increased risk of fatal coronary events in middle-aged men. Atherosclerosis. 2002; 165: 111–118.
Kroll H, Gardemann A, Fechter A, Haberbosch W, Santoso S. The impact of the glycoprotein Ia collagen receptor subunit A1648G gene polymorphism on coronary artery disease and acute myocardial infarction. Thromb Haemost. 2000; 83: 392–396(Nadine Ajzenberg; Clariss)
Correspondence to Dr N. Ajzenberg, Service d’Hématologie et Immunologie, H?pital Bichat, 46 rue Henri Huchard, 75018, Paris, France. E-mail nadine.ajzenberg@bch.ap-hop-paris.fr
Abstract
Objective— Platelet adhesion to the subendothelial tissue via the collagen receptor 2?1 is a crucial event in vascular biology. Although evidence has been provided that the number of platelets 2?1 copies is genetically determined, the molecular change primary responsible has not been yet elucidated. The aim of our present study was to investigate the effect of combined polymorphisms within both regulatory (–52C/T and –92C/G) and coding regions (807C/T and 1648A/G) of the 2 subunit gene on human platelets 2?1 receptor density and/or susceptibility to coronary artery disease (CAD).
Methods and Results— Among 254 cardiac surgery patients, no evidence was found for an association between the 2 subunit gene polymorphisms and CAD. In contrast, in a subgroup of 113 patients, we observed a significant association between all polymorphisms except –52C/T and 2?1 receptor level. Furthermore, when 3 groups of patients were defined according to the tertiles of platelets 2?1 copies, the –92C/807T haplotype was more frequent in the group of patients with high 2?1 receptor level.
Conclusion— These results suggest that an individual effect of each polymorphism located either in the coding or promoter sequence of the 2 gene may act in combination to modulate variations in platelets 2?1 receptor density.
An important combined effect of the –92C and 807T polymorphisms of the 2 gene in increasing the expression of human platelet 2?1 receptors has been observed, suggesting that this haplotype could modulate variations in 2?1 receptor density.
Key Words: –92C/G ? 807C/T polymorphism ? 2?1 density
Introduction
Vessel wall injury triggers platelet activation and platelet plug formation, followed by the formation of fibrin-containing thrombi that occlude the site of injury. The interaction of platelet receptors with subendothelial components such as collagen is central to these events, which both limit blood loss at sites of tissue trauma but may also obstruct diseased vessels, leading to ischemia and infarction of vital organs. The direct platelet–collagen interaction is mediated by 2 receptors, the integrin 2?1 and the nonintegrin receptor glycoprotein VI (GPVI), although there are likely to be others.1 It is now considered that GPVI provides the primary collagen signal that activates and recruits the integrin receptor 2?1 to further amplify collagen signals and fully activate platelets through a common intracellular signaling pathway.2
Integrin 2?1 is a heterodimer composed of 2 noncovalently associated subunits (2 and ?1) that are encoded by separate genes. By virtue of its expression on platelets and vascular cells,3–5 the integrin 2?1 may play a significant role in vascular pathobiology. Among healthy individuals, platelet 2?1 density is highly variable and correlates with the rate and extent of platelet adhesion to collagen type I or type III under static conditions.4,6 The differences in platelet 2?1 density also correlate with the inheritance of certain allelic combinations, defined by linked polymorphisms within the coding sequence of the 2 gene.7–10 In addition, 2 single-base substitutions at positions –52 and –92 have been identified11 within the "core" region defined by Zutter et al12 in the proximal 5'-regulatory region of the gene. In vitro, the –52C/T and –92C/G dimorphisms have been shown to influence the rate of the 2 gene transcription in transfected human megakaryocytic cell lines.11 It has also been suggested that 1 of the 2 promoter dimorphisms (–52C/T) could correlate with platelet 2?1 density in healthy individuals.13
A potentially important role for the 2?1 integrin is suggested by recent epidemiologic data.7,14,15 Several studies suggest a direct correlation between the genetically determined number of copies of 2?1 at the platelet surface and the risk of thrombotic events. The T allele of 807C/T polymorphism, which is associated with high-level expression of 2?1 on platelets, has been reported as an independent risk factor for myocardial infarction in selected groups of patients16–18 for the development of diabetic retinopathy in patients with type 2 diabetes mellitus19 and for stroke in the young,20 although other studies failed to find such a correlation.21–24 However, no data are yet available for the clinical impact of the dimorphisms present in the core region of the promoter that may modulate the expression of 2?1 at the cell surfaces.
The aim of the present study was to examine the distribution of the –92C/G, –52C/T, 807C/T, and 1648A/G dimorphisms in a population of patients undergoing cardiac surgery and to analyze the influence of particular haplotypes on platelets 2?1 receptor density. We hypothesized that the profile of an individual with combined polymorphisms in coding and regulatory regions of the 2 subunit gene may constitute a hereditary background for the susceptibility to the development of coronary artery disease (CAD).
Patients, Materials, and Methods
Patient Selection
The protocol was approved by the local ethics committee, and all patients gave their written informed consent to participate in the study. The total study population comprised 254 consecutive white patients (179 men and 75 women; 59±14 years of age) scheduled for either coronary artery bypass grafting or valve surgery. By means of coronary angiography, the study population was divided into patients with CAD (n=171) for subjects referred to the hospital for coronary artery bypass grafting in whom coronary artery stenosis was >70% (50% for left main artery) and non-CAD patients (n=83) for subjects referred to the hospital for valve surgery and without any angiographic signs of CAD (no atherosclerotic coronary plaque). We excluded patients with acute coronary syndrome who required emergency coronary artery bypass.
Blood Sampling
Blood sample was collected before coronary artery bypass or valve surgery. Blood was drawn in evacuated container tubes (Vacutainer; Becton Dickinson) containing EDTA for DNA analysis or 0.129 mol/L trisodium citrate for platelet 2?1 density determination.
DNA Genotyping
We obtained genomic DNA from blood samples after extraction from blood leukocytes by a standard procedure with the QIAmp DNA blood Midi kit (Qiagen GmbH) according to manufacturer instructions.
We studied 2 nucleotide polymorphisms located at 807C/T bp and 1648A/G bp within the coding region of the 2 gene25,26 and single-base substitutions at 2 positions, –92C/G and –52C/T, within the proximal 5'-regulatory region of the 2 gene.13 Genotyping of these 4 polymorphisms was conducted using an adapted method of DNA amplification by polymerase chain reaction (PCR) procedure with specific primers. PCR products were digested by specific restriction enzymes and separated by appropriate electrophoresis.
Quantitation of Platelet 2?1
We quantified the platelet 2?1 level in a subgroup of 113 patients (CAD n=49; non-CAD n=64). Determination of platelet 2?1 density was performed on citrated whole blood by flow cytometry using a kit "platelet Gp Screen test" (Biocytex). Briefly, whole blood was incubated with mouse monoclonal antibody against 2?1 integrin (CD 69b) and followed by an incubation with a polyclonal antibody anti-mouse IgG coupled to fluorescein isothiocyanate.27 Cytometric analysis was performed on a FACScalibur (Becton Dickinson). The number of platelet receptors was determined by converting the fluorescence intensity into corresponding number of sites per platelet on the basis of a calibrated bead standard curve using beads varying from 260 to 80 000 sites per platelet.
Statistical Analysis
Genotype distributions in different groups were compared by testing the hypothesis of homogeneity using 2 test and SAS software (SAS Institute; V8.0). Estimation of haplotype frequencies and linkage disequilibrium (D and D') were computed using the Arlequin software V2.000. Data were expressed as mean±SD and analyzed within genotype groups using ANOVA and Tukey–Kramer test for post hoc comparisons. For categorical variables and tertile comparisons, 2 test was performed. Differences were considered to be significant when P<0.05.
Results
Distribution of the 2 Gene Polymorphisms
Table 1 summarizes the genotype distribution of the 807C/T, 1648 A/G, –92 C/G, and –52 C/T polymorphisms of the 2 gene in the CAD and non-CAD groups. We reported similar genotype distribution in both groups. The genotype distribution is in agreement with the frequencies predicted by Hardy–Weinberg equilibrium (P=0.76, P=0.48, P=0.54, and P=0.38, respectively.)
TABLE 1. Genotype Distribution in CAD and Non-CAD Patients
Measure of Linkage Disequilibrium Between 2 Gene Polymorphic Sites
In the total population of 254 patients, we investigated the linkage disequilibrium between the 4 polymorphic sites of 2 gene (Table 2). A significant linkage disequilibrium was observed between the 807C/T and 1648A/G polymorphisms and between the –52C/T and –92C/G polymorphisms within the proximal 5' regulatory region of the 2 gene: all patients carrying the –92C allele were found to carry the –52T allele.
TABLE 2. Measure of Linkage Disequilibrium (D and D') Between 2 Polymorphic Sites in the Total Population
In contrast, we observed a moderate linkage disequilibrium between the 807C/T and the promoter sequence –92C/G and –52C/T (Table 2).
Relationship Between 2 Genotypes and Differences in Platelet 2?1 Levels
A subgroup of 113 patients (CAD n=49; non-CAD n=64) was available for the measurement of platelet 2?1 levels in whole blood by flow cytometry. No difference in 2?1 density was observed between CAD and non-CAD patients (3960±1098 and 3881±1084 receptors per platelet, respectively). In consequence, the 2 groups were pooled to examine the relationship between genotypes and 2?1 platelet density. In Figure 1, the number of platelet 2?1 copies per platelet is plotted according to the 2 genotypes within the coding sequence of each individual. As originally reported by Kunicki et al28 and confirmed by others,21,29 in healthy subjects, we observed (Figure 1A) an association between 2?1 densities and 807 polymorphism (ANOVA P<0.0001). Lowest 2?1 densities (3200±906 receptors per platelet) were observed in 807CC patients; and conversely, highest receptor density in 807TT patients (4782±1216 receptors per platelet; P<0.05). In the same way, the platelet 2?1 levels measured in 1648GG patients were lower than in heterozygous 1648AG patients (ANOVA P=0.0082), reaching 3774±1090 and 4435±958 receptors per platelet, respectively (P<0.05). Unfortunately, no homozygous 1648AA patient was available for quantification of platelet 2?1 copies.
Figure 1. Relationship between platelet 2?1 density and polymorphisms within the 2 coding region: 807C/T (A) and 1648 G/A (B) in a population of 113 patients. Box and whisker plots show median value (horizontal lines), 25th and 75th percentiles (boxes), and 10th and 90th percentiles (error bars). *P<0.05.
Interestingly, we also found a significant correlation between the –92 C/G polymorphism and the platelet 2?1 level (ANOVA P=0.0056; Figure 2). The –92CC homozygous patients exhibited higher levels of 2?1 receptors than the heterozygous –92CG patients (4066±1122 and 3384±744 receptors per platelet, respectively; P<0.05). The 4 homozygous patients for the –92 G allele had low density of 2?1 (2810±646 receptors per platelet), but the difference with the –92CC homozygous did not reach significance. In contrast, the platelet 2?1 receptor level was not found to be associated with the –52 C/T polymorphism (ANOVA P=0.565) because density of 2?1 varies between 3792±1115 receptors per platelet for –52CC and 3829±911 receptors per platelet for –52TT.
Figure 2. Relationship between platelet 2?1 density and polymorphisms within the 5' regulatory region: –92 C/G in a population of 113 patients. Box and whisker plots show median value (horizontal lines), 25th and 75th percentiles (boxes), and 10th and 90th percentiles (error bars). *P<0.05.
We looked for a possible influence of combined polymorphisms within regulatory and coding regions of the 2 subunit gene on human platelet 2?1 receptor density. Because of the strong linkage disequilibrium between the 807C/T and 1648G/A polymorphisms on one hand, and the absence of association between the –52C/T polymorphism and the 2?1 receptor density on the other hand, we determined the frequencies of haplotypes defined by the –92C/G and 807C/T polymorphisms in a panel of 113 patients in which the platelet 2?1 receptor level has been quantified (Table 3). Patients were studied according to tertiles of platelet 2?1 receptor level: low (<3348 copies per platelet), medium (ranging from 3348 to 4191), and high (>4191) platelet 2?1 receptor level. We observed that haplotype distribution was significantly different among the 3 groups of patients (ANOVA P<0.001). The most striking observation was that the –92C/807T haplotype is more frequent in the high-platelet 2?1 receptor level group (0.60 versus 0.12 in the first tertile), whereas the –92G/807C haplotype is more frequent in the low-platelet 2?1 receptor level group (0.16 versus 0.02 in the third tertile). Moreover, to determine whether this haplotype frequency was related to the linkage disequilibrium of polymorphisms or to the effect of each polymorphism, we compared the 2?1 density according to the 807 genotype in the subgroup of 88 patients homozygous for –92C: significant differences in 2?1 receptor level between 807TT, 807CT, and 807CC were observed, suggesting an individual effect of each polymorphism (ANOVA P<0.0001) and reaching 4872±1290, 4251±917, and 3316±912 receptors per platelet, respectively).
TABLE 3. Haplotype Frequency in the 3 Groups of Patients Defined by Tertiles of Platelet 2?1 Receptors per Platelet
Discussion
Platelet adhesion to the subendothelial tissue via the collagen receptor 2?1 is a crucial event in vascular biology and interindividual variations in 2?1 expression levels could have a significant impact on vascular pathology and risk of arterial thrombosis. Although evidence has been provided that the number of platelets 2?1 copies is genetically determined,7–10 the molecular change primarily responsible has not been yet elucidated. The aim of our present study was to investigate the effect of combined polymorphisms within regulatory and coding regions of the 2 subunit gene on human platelet 2?1 receptor density or susceptibility to CAD.
Among a total population of 254 patients, we report new data about the –92C/G, –52C/T, 807C/T, and 1648G/A polymorphic sites of the 2 gene. The linkage disequilibrium between the 807C/T and 1648A/G nucleotide polymorphisms on one hand, and between the –92C/G and –52C/T single-base substitutions on the other hand, is confirmed, as reported previously by other groups.26,28 The platelet 2?1 receptor quantification performed in freshly drawn whole blood also confirms that the level of collagen receptor 2?1 can vary up to 4-fold among individuals.6 As originally reported by Kunicki et al28 in healthy subjects and confirmed by others,10,29 we observed an obvious association between the 807C allele and low 2?1 level. However, homozygous patients for the 1648G allele exhibited the lowest 2?1 density, in agreement with previous data of Corral et al.10 No association was observed between the –52C/T dimorphism and platelet receptor density. Interestingly, we found that the platelet 2?1 receptor level was significantly associated with the –92 C/G promoter nucleotide dimorphism. This finding is supported by the previous work of Jacquelin et al,13 demonstrating that in vitro, in transfected human megakaryocytic cell lines, the –92G promoter sequence has a negative regulatory effect on the 2 gene transcription. Altogether, these findings emphasize the potential importance of the –92C promoter substitution on human platelet 2?1 receptor expression.
When 3 groups of patients were defined according to the tertiles of the number of platelets 2?1 copies, it appeared that haplotype distribution was different: the –92G/807C haplotype was more frequent in the group of patients with low 2?1 receptor levels, whereas the –92C/807T haplotype was in a large majority found in the group of patients with high 2?1 receptor levels. Moreover, in the subgroup of patients, –92CC significant differences of 2?1 density were observed according to 807C/T polymorphism. Because of moderate linkage disequilibrium between the –92C/G and 807C/T polymorphisms, the present results confirm evidence for more than a single genetic factor involved in the number of 2?1 molecules per platelet. Our findings indicate that an individual effect of each polymorphism in either the coding or promoter sequence may act in combination to modulate variation in receptor density. Although the –92C/G promoter substitution may have a direct impact on expression levels of 2 gene, the silent 807C/T polymorphism within the coding sequence that does not modify the deduced amino acid sequence of the translated protein may be linked to other polymorphisms within the 2 gene, which remain to be determined.
Looking for a clinical relevance of the 2 alleles, we investigated the relationship between the platelet 2?1 receptor density and nucleotide polymorphisms of the 2 subunit gene and CAD. No significant difference was observed in the surface expression of platelet 2?1 in CAD versus non-CAD patients. It is likely that the 2?1 receptor number alone is not sufficient to compromise platelet function and an individual’s susceptibility to arterial diseases, which are probably under the dependence of an other major collagen receptor, GPVI,30 or of other platelet receptors such as GPIb or 2b?3. Abnormalities of the vessel wall (as atherosclerosis) could also modulate platelet adhesion. Genotype distributions and allele frequencies were not significantly different in CAD versus non-CAD patients. With regard to nucleotide polymorphisms located within the coding region of the gene, several previous reports have extensively studied 807C/T and 1648A/G dimorphisms. In agreement with us, some authors failed to show an association between 807C/T polymorphism and CAD,21,22,24,31 whereas conflicting studies showed a significant correlation between 807T allele and myocardial infarction.16,17 Furthermore, Kroll et al found in a large population a significant association between the 1648A/G polymorphism and CAD.32 Possible explanations for these apparent discrepancies may be the differences of population size and the fact that numerous other factors are involved in the progression of atherosclerosis. Furthermore, our negative results may be attributable to the choice of an anatomic (angiographic) criteria rather than a more clinically relevant one such as evolutivity of coronary disease (occurrence of acute coronary syndrome).
In summary, we showed evidence for a possible influence of the inheritance of particular haplotypes of 2 gene on differences in human platelet 2?1 receptor density. Our results suggest an important combined effect of the –92C and 807T nucleotide sequences in increasing the expression of human platelet 2?1 receptors. These results must be confirmed in a larger study to precisely determine the involvement of the 2 gene allelic combinations in coronary disease through the platelet 2?1 receptor expression. Further studies are also required in a larger population to test whether or not the –92C/G and 807C/T polymorphisms have a functional effect on gene expression, and, if not, to identify the causal single-nucleotide polymorphisms in linkage disequilibrium with –92C/G and 807C/T polymorphisms. A more generalized haplotype strategy on the basis of polymorphisms described in single-nucleotide polymorphism databases would probably be most pertinent.
References
Moog S, Mangin P, Lenain N, Strassel C, Ravanat C, Schuhler S, Freund M, Santer M, Kahn M, Nieswandt B, Gachet C, Cazenave JP, Lanza F. Platelet glycoprotein V binds to collagen and participates in platelet adhesion and aggregation. Blood. 2001; 98: 1038–1046.
Chen H, Kahn ML. Reciprocal signaling by integrin and nonintegrin receptors during collagen activation of platelets. Mol Cell Biol. 2003; 23: 4764–4777.
Santoro SA, Rajpara SM, Staatz WD, Woods VL Jr. Isolation and characterization of a platelet surface collagen binding complex related to VLA-2. Biochem Biophys Res Commun. 1988; 153: 217–223.
Kunicki TJ, Nugent DJ, Staats SJ, Orchekowski RP, Wayner EA, Carter WG. The human fibroblast class II extracellular matrix receptor mediates platelet adhesion to collagen and is identical to the platelet glycoprotein Ia-IIa complex. J Biol Chem. 1988; 263: 4516–4519.
Santoro SA, Zutter MM. The alpha 2 beta 1 integrin: a collagen receptor on platelets and other cells. Thromb Haemost. 1995; 74: 813–821.
Kunicki TJ, Orchekowski R, Annis D, Honda Y. Variability of integrin alpha 2 beta 1 activity on human platelets. Blood. 1993; 82: 2693–2703.
Santoro SA. Platelet surface collagen receptor polymorphisms: variable receptor expression and thrombotic/hemorrhagic risk. Blood. 1999; 93: 3575–3577.
Santoso S, Kalb R, Walka M, Kiefel V, Mueller-Eckhardt C, Newman PJ. The human platelet alloantigens Br(a) and Brb are associated with a single amino acid polymorphism on glycoprotein Ia (integrin subunit alpha 2). J Clin Invest. 1993; 92: 2427–2432.
Kritzik M, Savage B, Nugent DJ, Santoso S, Ruggeri ZM, Kunicki TJ. Nucleotide polymorphisms in the alpha2 gene define multiple alleles that are associated with differences in platelet alpha2 beta1 density. Blood. 1998; 92: 2382–2388.
Corral J, Rivera J, Gonzalez-Conejero R, Vicente V. The number of platelet glycoprotein Ia molecules is associated with the genetically linked 807C/T and HPA-5 polymorphisms. Transfusion. 1999; 39: 372–378.
Jacquelin B, Rozenshteyn D, Kanaji S, Koziol JA, Nurden AT, Kunicki TJ. Characterization of inherited differences in transcription of the human integrin alpha 2 gene. J Biol Chem. 2001; 276: 23518–23524.
Zutter MM, Santoro SA, Painter AS, Tsung YL, Gafford A. The human alpha 2 integrin gene promoter. Identification of positive and negative regulatory elements important for cell-type and developmentally restricted gene expression. J Biol Chem. 1994; 269: 463–469.
Jacquelin B, Tarantino MD, Kritzik M, Rozenshteyn D, Koziol JA, Nurden AT, Kunicki TJ. Allele-dependent transcriptional regulation of the human integrin alpha2 gene. Blood. 2001; 97: 1721–1726.
Kunicki TJ. The role of platelet collagen receptor (glycoprotein Ia/IIa; integrin alpha2 beta1) polymorphisms in thrombotic disease. Curr Opin Hematol. 2001; 8: 277–285.
Kunicki TJ, Ruggeri ZM. Platelet collagen receptors and risk prediction in stroke and coronary artery disease. Circulation. 2001; 104: 1451–1453.
Moshfegh K, Wuillemin WA, Redondo M, Lammle B, Beer JH, Liechti-Gallati S, Meyer BJ. Association of two silent polymorphisms of platelet glycoprotein Ia/IIa receptor with risk of myocardial infarction: a case-control study. Lancet. 1999; 353: 351–354.
Santoso S, Kunicki TJ, Kroll H, Haberbosch W, Gardemann A. Association of the platelet glycoprotein Ia C807T gene polymorphism with nonfatal myocardial infarction in younger patients. Blood. 1999; 93: 2449–2453.
Roest M, Banga JD, Grobbee DE, de Groot PG, Sixma JJ, Tempelman MJ, van der Schouw YT. Homozygosity for 807T polymorphism in alpha(2) subunit of platelet alpha(2)beta(1) is associated with increased risk of cardiovascular mortality in high-risk women. Circulation. 2000; 102: 1645–1650.
Petrovic M, Hawlina M, Peterlin B, Petrovic D. BglII gene polymorphisms of the alpha2beta1 gene is a risk factor for diabetic retinopathy in Caucasians with type 2 diabetes. J Hum Genet. 2003; 48: 457–460.
Carlsson LE, Santoso S, Spitzer C, Kessler C, Greinacher A. The alpha2 gene coding sequence T807/A873 of the platelet collagen receptor integrin alpha2beta1 might be a genetic risk factor for the development of stroke in younger patients. Blood. 1999; 93: 3583–3586.
Corral J, Gonzalez-Conejero R, Rivera J, Ortuno F, Aparicio P, Vicente V. Role of the 807C/T polymorphism of the alpha2 gene in platelet GP Ia collagen receptor expression and function–effect in thromboembolic diseases. Thromb Haemost. 1999; 81: 951–956.
Croft SA, Hampton KK, Sorrell JA, Steeds RP, Channer KS, Samani NJ, Daly ME. The GPIa C807T dimorphism associated with platelet collagen receptor density is not a risk factor for myocardial infarction. Br J Haematol. 1999; 106: 771–776.
Cole VJ, Staton JM, Eikelboom JW, Hankey GJ, Yi Q, Shen Y, Berndt MC, Baker RI. Collagen platelet receptor polymorphisms integrin alpha2beta1 C807T and GPVI Q317L and risk of ischemic stroke. J Thromb Haemost. 2003; 1: 963–970.
Morita H, Kurihara H, Imai Y, Sugiyama T, Hamada C, Sakai E, Mori M, Nagai R. Lack of association between the platelet glycoprotein Ia C807T gene polymorphism and myocardial infarction in Japanese. An approach entailing melting curve analysis with specific fluorescent hybridization probes. Thromb Haemost. 2001; 85: 226–230.
Kalb R, Santoso S, Unkelbach K, Kiefel V, Mueller-Eckhardt C. Localization of the Br polymorphism on a 144 bp exon of the GPIa gene and its application in platelet DNA typing. Thromb Haemost. 1994; 71: 651–654.
Reiner AP, Aramaki KM, Teramura G, Gaur L. Analysis of platelet glycoprotein Ia (alpha2 integrin) allele frequencies in three North Am populations reveals genetic association between nucleotide 807C/T and amino acid 505 Glu/Lys (HPA-5) dimorphisms. Thromb Haemost. 1998; 80: 449–456.
Schmitz G, Rothe G, Ruf A, Barlage S, Tschope D, Clemetson KJ, Goodall AH, Michelson AD, Nurden AT, Shankey TV. European Working Group on Clinical Cell Analysis: Consensus protocol for the flow cytometric characterisation of platelet function. Thromb Haemost. 1998; 79: 885–896.
Kunicki TJ, Kritzik M, Annis DS, Nugent DJ. Hereditary variation in platelet integrin alpha 2 beta 1 density is associated with two silent polymorphisms in the alpha 2 gene coding sequence. Blood. 1997; 89: 1939–1943.
Roest M, Sixma JJ, Wu YP, Ijsseldijk MJ, Tempelman M, Slootweg PJ, de Groot PG, van Zanten GH. Platelet adhesion to collagen in healthy volunteers is influenced by variation of both alpha(2)beta(1) density and von Willebrand factor. Blood. 2000; 96: 1433–1437.
Nieswandt B, Brakebusch C, Bergmeier W, Schulte V, Bouvard D, Mokhtari-Nejad R, Lindhout T, Heemskerk JW, Zirngibl H, Fassler R. Glycoprotein VI but not alpha2beta1 integrin is essential for platelet interaction with collagen. EMBO J. 2001; 20: 2120–2130.
Mikkelsson J, Perola M, Penttila A, Karhunen PJ. Platelet collagen receptor GPIa (C807T/HPA-5) haplotype is not associated with an increased risk of fatal coronary events in middle-aged men. Atherosclerosis. 2002; 165: 111–118.
Kroll H, Gardemann A, Fechter A, Haberbosch W, Santoso S. The impact of the glycoprotein Ia collagen receptor subunit A1648G gene polymorphism on coronary artery disease and acute myocardial infarction. Thromb Haemost. 2000; 83: 392–396(Nadine Ajzenberg; Clariss)