Polymorphisms of the human platelet alloantigens HPA-1, HPA-2, HPA-3, and HPA-4 in ischemic stroke Sarra Saidi,1 Touhami Mahjoub,1 Lamia B. Slamia,2 Sofyan B. Ammou,2 Abeer M. Al-Subaie,3 and Wassim Y. Almawi3* Polymorphism in human platelet antigen (HPA)-1 and HPA-3 (GPIIb/IIIa), HPA-2 (GPIb/IX), HPA-4 (GPIIIa), and HPA-5 (GPIa/IIa) was investigated in 329 stroke patients and 444 matched control subjects. HPA genotyping was done by PCR-SSP method. Lower HPA-1a (P < 0.001) and higher HPA-1b (P < 0.001) allele frequencies were seen in patients than control subjects, and homozygosity for HPA-1b (P < 0.001) alleles was more prevalent in stroke cases than in controls. The allele and genotype distributions of the other HPA polymorphic variants were similar between cases and controls. Select HPA combined genotypes comprising the 2121 (Pc 5 0.008) and 2221 (Pc 5 0.018) genotypes, which were positively associated, and the 1111 (Pc < 0.001), which was negatively associated with stroke, thereby conferred a disease susceptibility and protective nature to these genotype combinations. Multivariate analysis confirmed the negative association of the 1111 (P < 0.001) and the positive association of the 2121 (P 5 0.017) combined genotypes with stroke, after adjustment for a number of covariates. This is the first evidence demonstrating differential association of the common 4 HPA gene variants and specific HPA genotype combinations with stroke. Am. J. Hematol. C 2008 Wiley-Liss, Inc. 83:570–573, 2008. V
Introduction Platelets regulate several aspects of primary hemostasis by maintaining vascular integrity, wound healing and repair at sites of injury, and by responding to endothelial damage [1]. Altered platelet activation and platelet-dependent thromboembolism was associated with the pathogenesis of cerebrovascular diseases [2], including stroke [3]. Platelet adhesion and activation is mediated by human platelet alloantigens (HPA), a complex of platelet membrane glycoprotein (Gp) with other cell-bound factors [4,5]. In excess, 19 HPAs were identified [6,7] with HPA-1, -2, -3, -4, and -5 being the most clinically relevant [8]. By altering platelet receptor sensitivity, polymorphism in platelet Gp directly impacts platelet susceptibility in activating stimuli and was linked with heightened risk of atherothrombotic events [9], including acute myocardial infarction [10]. Eleven of the 19 HPAs are on integrin aIIbb3 or GPIIb/IIIa, while of the remaining eight, three are on GPIb/IX/V, two on the integrin a2b1, and one each on GPIV, GPV, and CD109 [6,7]. All HPAs identified are bi-allelic and map to specific membrane proteins. HPA polymorphisms are due to single base-pair substitutions, resulting in single amino acid replacement. HPA-1 (T196C, Leu33Pro), HPA-3 (T2622G, Ile843Ser), and HPA-4 (G526A; Arg143Glu) are present on the GPIIb/IIIa receptor complex [11], while HPA-2 (T524C, Met145Thr) is found on the von Willebrand Factor (vWF) receptor GPIb/IX [12], and HPA-5 (G1648A, Glu505Lys) is carried on the collagen receptor GPIa/IIa [13]. The distribution of these gene variants is geographically and ethnically restricted, evidenced by the high prevalence of HPA-5b and HPA-2b among Africans [14], their low frequencies among Asians [15– 17], and the high frequency of HPA-1b among Caucasians [18] and Berbers [19] compared to Africans and Asians. Insofar as they modulate platelet receptor density and function, platelet structure, and/or expression levels of adhesion proteins [7], HPA polymorphic variants were linked with thrombotic events [9]. Their contribution to stroke was examined but with inconsistent results. This was due to differences in the selection of study subjects, and also due to differences in study design, since the impact of single
genetic factors on the risk of stroke may be higher when analyzed in the context of other inherited or acquired risk factors. Here, we examined the association of four common HPA polymorphisms with stroke in a group of 329 consecutive stroke patients, compared with 444 age- and gendermatched healthy controls, and we assessed the interactions between these HPA polymorphic variants and other traditional risk factors of stroke. Results HPA distribution The distribution of HPA-2 (P 5 0.23) and HPA (P 5 0.43), but not HPA-1 (P 5 0.002), or HPA-3 (P 5 0.003) genotypes was in Hardy-Weinberg equilibrium among controls. The allelic distribution of the 4 HPA polymorphisms among stroke cases and control subjects is shown in Table II. The allele frequency of HPA-1a (0.46 vs. 0.59; P < 0.001) was lower, while the frequency of HPA-1b (0.54 vs. 0.41; P < 0.001) was higher in patients than in control subjects, respectively. The frequencies of the other alleles were comparable between patients and controls. Homozygosity for the HPA-1b (22.2% vs. 10.4%; P < 0.001) alleles was more prevalent in stroke cases than in controls 1 Research Unit of Hematological and Autoimmune Diseases, Faculty of Pharmacy, University of Monastir, Monastir, Tunisia; 2Department of Neurology, CHU Sahloul, Sousse, Tunisia; 3Department of Medical Biochemistry, College of Medicine and Medical Sciences, Arabian Gulf University, Manama, Bahrain
Contract grant sponsors: NIH; Contract grant number: PHS R01 DK63088, PHS RR-08084, and PHS U54 HL070871. *Correspondence to: Wassim Y. Almawi, Department of Medical Biochemistry, College of Medicine and Medical Sciences, Arabian Gulf University, PO Box 22979, Manama, Bahrain. E-mail:
[email protected] Received for publication 12 September 2007; Revised 29 January 2008; Accepted 30 January 2008 Am. J. Hematol. 83:570–573, 2008. Published online 13 February 2008 in Wiley InterScience (www.interscience. wiley.com). DOI: 10.1002/ajh.21171
C 2008 Wiley-Liss, Inc. V
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http://www3.interscience.wiley.com/cgi-bin/jhome/35105
TABLE I. Characteristics of Study Participants Cases (329) Mean age ± SD (years) Gender (M:F) Smokers (%) Systolic BP (mmHg) Diastolic BP (mmHg) Hypertension (%) BMI (kg/m2) Glucose (mmol/L) CPK (U/L) Cholesterol (mmol/L) HDL (mmol/L) LDL (mmol/L) Triglycerides (mmol/L) a b
61.8 ± 12.2 180:149 130 (39.8) 160.1 ± 30.0 88.7 ± 14.7 208 (63.2) 26.4 ± 1.9 7.6 ± 3.3 59.9 ± 31.5 5.2 ± 1.1 1.2 ± 0.7 3.6 ± 1.0 1.5 ± 0.6
Controls (444) 60.3 ± 12.8 245:199 131 (34.6) 122.7 ± 15.6 79.1 ± 11.1 98 (22.1) 26.3 ± 2.8 6.0 ± 2.0 64.9 ± 19.9 4.8 ± 1.1 1.0 ± 0.2 2.9 ± 0.5 1.2 ± 0.6
TABLE III. HPA Combined Genotypes in Patients and Controls P a
0.104 0.942b 0.160b
