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Enhancing effect of the 145Met-allele of GPIb alpha on platelet sensitivity to aspirin under high-shear conditions
Thrombosis Research (2008) 123, 331–335
www.elsevier.com/locate/thromres
BRIEF COMMUNICATION
Enhancing effect of the 145 Met-allele of GPIb alpha on platelet sensitivity to aspirin under high-shear conditions Yumiko Matsubara a,⁎, Mitsuru Murata b , Gentaro Watanabe c , Yasuo Ikeda a a b c
Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan, Department of Laboratory Medicine, School of Medicine, Keio University, Tokyo, Japan, Medical Center, Mitsui-Sumitomo Bank, Tokyo, Japan
Received 26 July 2007; received in revised form 24 January 2008; accepted 6 February 2008 Available online 16 April 2008
Introduction Aspirin (ASA) is widely used as an antiplatelet drug, and a large number of clinical trials with ASA have demonstrated significant efficacies for the prevention and treatment of atherothrombosis [1,2]. Recent accumulating evidence, however, indicates that there are individuals with unexpected platelet reactivity to ASA [3]. This subpopulation, called ASA resistant, has lower sensitivity to ASA in ex vivo and in vitro platelet function tests and has poor clinical outcomes. The mechanism underlying the variability in ASA sensitivity is largely unknown. The glycoprotein (GP) Ib/IX/V complex is a receptor for von Willebrand factor (VWF) that mediates sheardependent platelet function. GPIbα, the largest subunit of this complex, contains the VWF binding site [4]. Binding of VWF to GPIbα has critical roles in thrombus formation at the site of vascular injury, especially under high shear conditions. The GPIbα/ ⁎ Corresponding author. Department of Internal Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo, Japan, 160-8582. Tel.: +81 3 3353 1211; fax: +81 3 3353 3515. E-mail address: [email protected] (Y. Matsubara).
VWF interaction in vitro occurs under high shear conditions or in the presence of nonphysiologic inducers such as ristocetin or botrocetin. ASA seems to affect GPIb/IX/V function: ASA causes shedding (proteolytic ectodomain cleavage) of GPIbα and GPV in vitro and in vivo via a disintegrin and metalloprotease 17 (ADAM17), a membrane-bound metalloprotease, although the mechanism of the ASA-induced shedding remains unclear [5]. Shedding of GPIbα might be associated with suppression of platelet function [5]. Also, ristocetin-induced platelet activation, but not primary agglutination, was reduced by ASA [6]. Several polymorphisms have been reported in GPIbα gene, including − 5T/C, 145Thr/Met, and variable number tandem repeat (VNTR) polymorphisms [7]. The 145Thr/Met and VNTR [1–4 repeats (1R to 4R)] of the 13-amino acid sequence are in linkage disequilibrium. The 145Met-allele is tightly linked to the 3R- or 4R-allele, and there is strong linkage between the 145Thr-allele and the 1R- or 2R-allele. There is a race difference in the genotype distribution of the VNTR polymorphism. These polymorphisms are reportedly associated with risk of atherothrombotic disorders and receptor function. However, only a few published studies have addressed the effects of the
0049-3848/$ - see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2008.02.008
332
Y. Matsubara et al.
− 5T/C
[8–10], 145Thr/Met [10], and VNTR [9] polymorphisms on variability in the platelet response to ASA, and study subjects of these previous investigations were patients with atherothrombosis who might have various clinical backgrounds. Therefore, in the present study, we examined the association between platelet reactivity to ASA and the − 5T/C and 145The/ Met polymorphisms among healthy (i.e., no apparent hematologic or vascular disorders) subjects.
this study. According to previous reports for the determination of platelet response to ASA, study subjects were divided into two groups based on the CEPI-CT of ASA (+) samples [11,12]: a good responder group was defined as having a CEPI-CT N250s and a poor responder group as having a CEPI-CT ≤250s.
VWF antigen level
Materials and methods
After centrifugation of citrated blood samples at 2500 rpm for 10 min, the plasma samples were stored at −80 degree until assay. Plasma VWF antigen level was measured using a microlatex particlemediated immunoassay (LIA) (Roch Diagnostics, Mannheim, Germany).
Study subjects
Statistics
The protocol was approved by the ethics committee of the School of Medicine, Keio University. Written informed consent was obtained from all study subjects (n = 176), who were genetically unrelated Japanese males at their regular checkups. Ninety-five subjects included in the present study were the same as those in their study [11]. The mean age was 47.0 ± 5.1 years. The study subjects had no apparent hematologic or vascular disorders and were not taking any medications that affect platelet function.
Genetic analysis Genomic DNA was extracted from peripheral blood. Genotypes of the − 5T/C and 145The/Met polymorphisms were determined using the MegaBACE1000 (Amersham Biosciences, Piscataway, NJ) according to the manufacturer's protocol for the single nucleotide primer extension-based method.
Assessment of platelet sensitivity to ASA For studies of platelet sensitivity to ASA, citrated (0.129 M) whole blood was incubated with ASA (final concentration 10 μM, which roughly represents a peak plasma concentration when low dose ASA was orally administrated) [ASA (+)] or vehicle [ASA (−)] for 30 min at room temperature, and then each blood sample was analyzed with a Platelet Function Analyzer-100® (PFA-100®, Dade Behring, Deerfield, IN). This PFA-100® system using the collagen/epinephrine (CEPI) or collagen/ADP (CADP) cartridge is a relatively accurate device for assessing platelet function, as assessed by closure time (CT), under high shear flow conditions (5000–6000/s). The maximum measurement of CT is 300s, and a longer CT indicates lower platelet function. The manufacturer's instruction for PFA-100® shows that the CEPI-CT is sensitive to the effects of ASA, and thus a good responder to ASA has a longer CEPI-CT. We measured the CEPI-CT for ASA (+) samples, and the CEPI-CTand CADP-CT for ASA (−) samples in
Table 1
Paired t-test was used to compare the CEPI-CT value between the ASA (+) and ASA (−) samples. A chi-square test was performed to compare two proportions between the poor and good responders. Multiple logistic regression analysis was performed to evaluate the relationship between the poor and good responders (categorical variable) and other variables. Independent variables included in the analysis were 145Thr/Met genotype (categorical variable), VWF level (quantitative value), platelet count (quantitative value), and hematocrit value (quantitative value), because the manufacturer's instructions for the PFA-100® indicate the possibility that the CT value is affected by VWF level, platelet count, and hematocrit value. To examine whether base line platelet function [i.e. the CEPI-CT in ASA (−)] affects the platelet sensitivity to ASA, we analyzed another multiple logistic regression model with the CEPICT in ASA (−) as independent variable. Because multiple regression analysis revealed a significant contribution of VWF level to the CEPI-CT in ASA (−), VWF level as independent variable was excluded from this model. Thus, in the another model, the dependent variable was the poor and good responders (categorical variable), and independent variables were 145Thr/Met genotype (categorical variable), the CEPI-CT in ASA (−) (quantitative value), platelet count (quantitative value), and hematocrit value (quantitative value). Also, the relationship between the GPIb α polymorphisms and the continuous variable of CEPI-CT or CADP-CT was examined using analysis of covariance (ANCOVA) with VWF level as covariate. Statistical analyses were performed using StatView (ver 5.0, for Macintosh, SAS, Cary, NC). A p value less than 0.05 was considered to be statistically significant.
Results We observed the inhibitory effect of ASA on platelet function; the CEPI-CT in ASA (+) samples was significantly longer than that in
GPIbα polymorphisms and platelet reactivity to aspirin −5
Good responders⁎ Poor responders⁎
TT, n (%)
TC+CC, n (%)
p value
72 (53.3) 17 (41.5)
63 (46.7) 24 (58.5)
145
− 145
p value
98 (72.6) 38 (92.7)
37 (27.4) 3 (7.3)
0.0072
ThrThr, n (%)
Good responders⁎ Poor responders⁎
−5
ThrMet+MetMet, n (%)
0.1831
⁎A good responder group was defined as having a CEPI-CT N 250s, and a poor responder group as having a CEPI-CT ≤ 250s.
Effect of the
145
Met-allele of GPIb alpha on platelet sensitivity to aspirin under high-shear conditions 333
ASA (−) samples (p b 0.0001). Study subjects were divided into two groups based on the CEPI-CT of ASA (+) samples as described in materials and methods section: a good responder group (n = 135) and a poor responder group (n = 41). The frequency of poor responders was 23.3%, which was consistent with previous reports [11,13]. To examine whether the− 5T/C and/or 145Thr/ Met polymorphisms affect ASA efficacy in platelet function, we analyzed these genotype distributions between the good and poor responders. As shown in Table 1, there was no significant difference in the − 5T/C genotype distribution between the good and poor responders (p = 0.1831). On the other hand, the genotype distribution of the 145Thr/Met polymorphism was significantly different between the two groups: the frequency of 145Thr/Met and 145Met/Met genotypes in the poor responders was significantly lower than that in the good responders (p = 0.0072). A multiple logistic regression analysis was performed with the dependent variable, good responder vs poor responder, as assessed by CEPI-CT in ASA (+) samples, and the independent variables including 145Thr/Met genotype, plasma VWF antigen level, platelet count, and hematocrit value. As a result, the 145Thr/Met genotype (p = 0.0247) and VWF level (p = 0.0133) were shown to be independent predictors for CEPICT in ASA (+) samples (Table 2A). Additionally, we examined whether base line platelet function [i.e. the CEPI-CT in ASA (−)] affects the platelet sensitivity to ASA, and another multiple logistic regression model with the CEPI-CT in ASA (−) as independent variable was analyzed. Because multiple regression analysis revealed a significant contribution of VWF level to the CEPI-CT in ASA (−), VWF level as independent variable was excluded from this model. Results demonstrated that the 145Thr/ Met genotype (p = 0.0424) and the CEPI-CT in ASA (−) (p = 0.0002) were independent predictors for CEPI-CT in ASA (+) samples (Table 2B). These results for two models suggest that the 145Thr/ Met genotype is independently associated with the CEPI-CT in ASA (+). For ASA (−) samples, we examined whether the − 5T/C or 145Thr/ Met polymorphism affects CEPI-CT or CADP-CT. The association between the − 5T/C or 145Thr/Met polymorphism and CEPI-CT or CADP-CTwas analyzed by ANCOVA for statistical adjustment of VWF levels, because multiple regression analysis with independent variables (plasma VWF level, platelet count, and hematocrit value) revealed a significant contribution of VWF level to both CEPI-CT (p b 0.0001) and CADP-CT (p b 0.0001). As a result, the − 5T/C polymorphism was not associated with the CEPI-CT (136.6 ± 35.9 for −5 TT, 147 ± 38.9 for − 5CC+− 5TC, p = 0.8601) or CADP-CT (96.0 ± 15.6 for − 5TT, 96.7±16.2 for − 5CC+− 5TC, p = 0.7904). There was no association between the 145Thr/Met polymorphism and the CEPI-CT (138.6 ± 37.5 for 145Thr/Thr, 152.7±36.1 for 145Thr/Met+145Met/
Table 2
Multiple logistic regression analyses
Independent variables
Odds ratio
95% confidence interval
p value
(A) VWF level 145 Thr/Met genotype Platelet count Hematocrit value
1.009 4.179 1.018 0.943
1.002–1.017 1.200–14.553 0.937–1.105 0.809–1.099
0.0133 0.0247 0.6776 0.4528
(B) CEPI-CT in ASA (−) 145 Thr/Met genotype Platelet count Hematocrit value
0.972 3.741 1.000 0.882
0.958–0.987 1.046–13.378 0.919–1.087 0.749–1.039
0.0002 0.0424 0.9921 0.1320
Met, p = 0.1132) or CADP-CT (94.5± 15.0 for 145Thr/Thr, 102.5 ± 17.2 for 145Thr/Met+145Met/Met, p = 0.0583).
Discussion The efficacy of ASA to inhibit platelet function is mainly due to irreversible acetylation of a specific serine residue on the platelet cyclooxygenase-1 enzyme. The inhibition of platelet functions by ASA also occurs via a cyclooxygenase-1-independent pathway. Although the mechanism of platelet reactivity to ASA has been widely studied, the mechanism underlying the inter-individual variations in platelet response to ASA is not fully understood. Recently, the association between the variability in platelet response to ASA and the polymorphisms of platelet membrane receptors was highlighted [7,14]. Among the receptors, the GPIIIa PlA1/A2 polymorphism is the most studied polymorphism. The PlA2-allele, however, is extremely rare in the Japanese population. With regard to the association of GPIbα polymorphisms with platelet reactivity to ASA resistance status assessed by the CEPI-CT, Macchi et al reported that there was no relation between the − 5T/C polymorphism and the ASA resistance status among ASA-treated patients [8], which is compatible with the present observation. There are more than 10 publications of study on the effect of the 145 Thr/Met polymorphism on atherothrombotic disorders and receptor functions [7,15]. However, Williams et al recently reported an association between this polymorphism and variability in platelet response to ASA, showing no relationship between the − 5TC and 145 Thr/Met polymorphisms and ASA resistance status among patients taking ASA of 81–325 mg within 24 hours prior to enrollment [10]. In their study, ASAtreated patients were divided into two groups based on the CEPI-CT (N167s or ≤167s), and this cut-off value was different from our study (250s) in ASA (+) samples. Also, in our study, samples were from healthy subjects, because we intended to exclude individual pharmacokinetic variations and to reduce bias of study subjects. The difference in background of study subjects is likely to cause conflicting views. Our findings suggest that the inhibitory effect of ASA on platelet function is more efficient in subjects with the 145Met-allele. This 145Met-allele is suggested to be a risk factor for atherothrombotic disorders and to have higher interaction with immobilized VWF under high shear conditions, although these findings are somewhat controversial [7,15–17]. The mechanism of the present finding that the 145 Met-allele is associated with increased sensitivity to ASA in vitro is largely unknown. Recently, Aktas et al reported that ASA induces ADAM17-mediated shedding of GPIbα and GPV in vivo and in vitro,
334 which is associated with the downregulation of thrombus formation [5], although the mechanism of the ASA-induced shedding, as well as physiologic relevance of GPIbα shedding, is not fully understood. More recently, Gardiner et al showed that the synthetic peptides based on the region of GPIbα (450 Lys-483 Phe) were cleaved by ADAM17 at 464Gly/ 465 Val [18]. This cleaving site lies adjacent to the sequence for VNTR polymorphism which is closely linked to 145 Thr/Met polymorphism. We speculate as follows: the GPIbα/ADAM 17 interaction is likely to be enhanced by ASA, and platelets with the 145 Met/VNTR 3R or 4R-allele have a structure that allows easier access for ADAM17. Thus, enhancing effect of the 145 Met/VNTR 3R or 4R-allele on the GPIbα shedding by ADAM 17 might become evident in the presence of ASA, as compared with basal condition. The present study did not provide the evidence that the GPIbα shedding is associated with the difference in platelet sensitivity to ASA between the genotypes of GPIbα polymorphisms. Further studies are needed to elucidate the molecular mechanism of the difference in the ASA inhibitory effect on platelet reactivity between the 145Thr/Met genotypes. Also, it is important to learn the stage at which the ASA-induced shedding occurs. For ASA(−) samples, we observed no association between the − 5T/C polymorphism and CEPI-CT or CADP-CT. Also, there was no effect of the 145Thr/Met polymorphism on CEPI-CTand CADP-CT. We previously reported that the 145Met/VNTR 4R-expressing cell lines had a higher interaction with immobilized VWF under high shear conditions [16]. It is likely that the CT in PFA 100® system is affected by various stimulations, such as collagen and epinephrine or ADP. Thus, it is difficult to examine the difference in GPIb/VWFmediated platelet function between GPIbα polymorphisms, under an experimental condition with no stimulation, using samples from healthy (i.e., no evidence of bleeding or atherothrombosis) subjects. We performed a single measurement of the CT value. Because only small amounts of blood from samples taken during regular checkups were available for this study, we examined the validity of the values obtained in the PFA-100® system in a pilot study. Reproducibility and reliability of the assay were confirmed by repeated measurements of the CT value. Also, samples in the pilot study were evaluated by the light transmission method, and the CEPI-CT significantly correlated with the maximum aggregation of collagen (1 ug/ml)-, ADP (5 uM)-, or epinephrine (10 uM)-induced platelet aggregation. We did not observe these correlations with the CADP-CT in the pilot study. Because PFA-100® assay system requires only small amount of whole blood (0.8 ml/assay), this
Y. Matsubara et al. method is suitable for point of care testing and is convenient at site of blood collection in regular checkup. The CT value of PFA-100® system has cutoff value of 300s. In this study, of the 176 subjects, 133 had a CEPI-CT N300s in ASA (+) samples. Consequently, we did not analyze actual value of the CEPI-CT in ASA (+) samples but analyzed the proportion of poor responder categorized by the CEPI-CT. Also, we could not define low responders by relative extension of the CEPI-CT between the ASA (+) and ASA (−) samples. It is necessary, however, to examine platelet sensitivity to ASA, using other measurement tools as well. Additionally, we observed that the mean CTof CEPI or CADP in healthy Japanese subjects differed from that described in PFA-100® manufacturer's instructions. This may suggest the importance of PFA-100® data analysis in different ethnic groups. In summary, we examined the effect of GPIbα − 5T/C and 145Thr/Met polymorphisms on platelet sensitivity to ASA among healthy subjects. The results suggested that the 145Thr/Met polymorphism, but not − 5T/C polymorphism, affects the platelet sensitivity to ASA. The present study is the first to show the enhancing effect of the 145Met-allele on platelet sensitivity to ASA upon CEPI-CTamong healthy subjects. This finding could have a potential clinical impact on the design of pharmacogenetic antiplatelet therapies.
Acknowledgment This study is supported by a grant from the Japanese Ministry of Health and Welfare for the Scientific Research Project (H16-Genome-002).
References [1] Antithrombotic Trialists' Collaboration. Collaborative metaanalysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. BMJ 2002;324:71–86. [2] Patrono C, Garcia Rodriguez LA, Landolfi R, Baigent C. Lowdose aspirin for the prevention of atherothrombosis. N Engl J Med 2005;353:2373–83. [3] Szczeklik A, Musial J, Undas A, Sanak M. Aspirin resistance. J Thromb Haemost 2005;3:1655–62. [4] Andrews RK, Gardiner EE, Shen Y, Whisstock JC, Berndt MC. Glycoprotein Ib-IX-V. Int J Biochem Cell Biol 2003;35:1170–4. [5] Aktas B, Pozgajova M, Bergmeier W, Sunnarborg S, Offermanns S, Lee D, et al. Aspirin induces platelet receptor shedding via ADAM17 (TACE). J Biol Chem 2005;280: 39716–22. [6] Garcia A, Quinton TM, Dorsam RT, Kunapuli SP. Src family kinase-mediated and Erk-mediated thromboxane A2 generation are essential for VWF/GPIb-induced fibrinogen receptor activation in human platelets. Blood 2005;106:3410–4. [7] Yee DL, Bray PF. Clinical and functional consequences of platelet membrane glycoprotein polymorphisms. Semin Thromb Hemost 2004;30:591–600.
Effect of the
145
Met-allele of GPIb alpha on platelet sensitivity to aspirin under high-shear conditions 335
[8] Macchi L, Christiaens L, Brabant S, Sorel N, Ragot S, Allal J, et al. Resistance in vitro to low-dose aspirin is associated with platelet PlA1 (GP IIIa) polymorphism but not with C807T(GP Ia/IIa) and C-5T Kozak (GP Ibalpha) polymorphisms. J Am Coll Cardiol 2003;42:1115–9. [9] Douglas H, Davies GJ, Michaelides K, Gorog DA, Timlin H, Ahmed N, et al. Detection of functional differences between different platelet membrane glycoprotein Ibalpha variable number tandem repeat and Kozak genotypes as shown by the PFA-100 system. Heart 2006;92:676–8. [10] Williams MS, Ng'alla LS, Vaidya D. Platelet functional implications of glycoprotein Ibalpha polymorphisms in African Americans. Am J Hematol 2007;82:15–22. [11] Takahashi S, Ushida M, Komine R, Shimizu A, Uchida T, Ishihara H, et al. Increased basal platelet activity, plasma adiponectin levels, and diabetes mellitus are associated with poor platelet responsiveness to in vitro effect of aspirin. Thromb Res 2007;119:517–24. [12] Favaloro EJ. Clinical application of the PFA-100®. Curr Opin hematol 2002;9:407–15. [13] Fontana P, Nolli S, Reber G, de Moerloose P. Biological effects of aspirin and clopidogrel in a randomized cross-over study in 96 healthy volunteers. J Thromb Haemost 2006;4:813–9.
[14] Rozalski M, Boncler M, Luzak B, Watala C. Genetic factors underlying differential blood platelet sensitivity to inhibitors. Pharmacol Rep 2005;57:1–13. [15] Murata M, Matsubara Y, Kawano K, Zama T, Aoki N, Yoshino H, et al. Coronary artery disease and polymorphisms in a receptor mediating shear stress-dependent platelet activation. Circulation 1997;96:3281–6. [16] Ulrichts H, Vanhoorelbeke K, Cauwenberghs S, Vauterin S, Kroll H, Santoso S, et al. von Willebrand factor but not alphathrombin binding to platelet glycoprotein Ibalpha is influenced by the HPA-2 polymorphism. Arterioscler Thromb Vasc Biol 2003;23:1302–7. [17] Matsubara Y, Murata M, Hayashi T, Suzuki K, Okamura Y, Handa M, et al. Platelet glycoprotein Ib alpha polymorphisms affect the interaction with von Willebrand factor under flow conditions. Br J Haematol 2005;128:533–9. [18] Gardiner EE, Karunakaran D, Shen Y, Arthur JF, Andrews RK, Berndt MC. Controlled shedding of platelet glycoprotein (GP)VI and GPIb-IX-V by ADAM family metalloproteinases. J Thromb Haemost 2007;5:1530–7.
www.elsevier.com/locate/thromres
BRIEF COMMUNICATION
Enhancing effect of the 145 Met-allele of GPIb alpha on platelet sensitivity to aspirin under high-shear conditions Yumiko Matsubara a,⁎, Mitsuru Murata b , Gentaro Watanabe c , Yasuo Ikeda a a b c
Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan, Department of Laboratory Medicine, School of Medicine, Keio University, Tokyo, Japan, Medical Center, Mitsui-Sumitomo Bank, Tokyo, Japan
Received 26 July 2007; received in revised form 24 January 2008; accepted 6 February 2008 Available online 16 April 2008
Introduction Aspirin (ASA) is widely used as an antiplatelet drug, and a large number of clinical trials with ASA have demonstrated significant efficacies for the prevention and treatment of atherothrombosis [1,2]. Recent accumulating evidence, however, indicates that there are individuals with unexpected platelet reactivity to ASA [3]. This subpopulation, called ASA resistant, has lower sensitivity to ASA in ex vivo and in vitro platelet function tests and has poor clinical outcomes. The mechanism underlying the variability in ASA sensitivity is largely unknown. The glycoprotein (GP) Ib/IX/V complex is a receptor for von Willebrand factor (VWF) that mediates sheardependent platelet function. GPIbα, the largest subunit of this complex, contains the VWF binding site [4]. Binding of VWF to GPIbα has critical roles in thrombus formation at the site of vascular injury, especially under high shear conditions. The GPIbα/ ⁎ Corresponding author. Department of Internal Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo, Japan, 160-8582. Tel.: +81 3 3353 1211; fax: +81 3 3353 3515. E-mail address: [email protected] (Y. Matsubara).
VWF interaction in vitro occurs under high shear conditions or in the presence of nonphysiologic inducers such as ristocetin or botrocetin. ASA seems to affect GPIb/IX/V function: ASA causes shedding (proteolytic ectodomain cleavage) of GPIbα and GPV in vitro and in vivo via a disintegrin and metalloprotease 17 (ADAM17), a membrane-bound metalloprotease, although the mechanism of the ASA-induced shedding remains unclear [5]. Shedding of GPIbα might be associated with suppression of platelet function [5]. Also, ristocetin-induced platelet activation, but not primary agglutination, was reduced by ASA [6]. Several polymorphisms have been reported in GPIbα gene, including − 5T/C, 145Thr/Met, and variable number tandem repeat (VNTR) polymorphisms [7]. The 145Thr/Met and VNTR [1–4 repeats (1R to 4R)] of the 13-amino acid sequence are in linkage disequilibrium. The 145Met-allele is tightly linked to the 3R- or 4R-allele, and there is strong linkage between the 145Thr-allele and the 1R- or 2R-allele. There is a race difference in the genotype distribution of the VNTR polymorphism. These polymorphisms are reportedly associated with risk of atherothrombotic disorders and receptor function. However, only a few published studies have addressed the effects of the
0049-3848/$ - see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2008.02.008
332
Y. Matsubara et al.
− 5T/C
[8–10], 145Thr/Met [10], and VNTR [9] polymorphisms on variability in the platelet response to ASA, and study subjects of these previous investigations were patients with atherothrombosis who might have various clinical backgrounds. Therefore, in the present study, we examined the association between platelet reactivity to ASA and the − 5T/C and 145The/ Met polymorphisms among healthy (i.e., no apparent hematologic or vascular disorders) subjects.
this study. According to previous reports for the determination of platelet response to ASA, study subjects were divided into two groups based on the CEPI-CT of ASA (+) samples [11,12]: a good responder group was defined as having a CEPI-CT N250s and a poor responder group as having a CEPI-CT ≤250s.
VWF antigen level
Materials and methods
After centrifugation of citrated blood samples at 2500 rpm for 10 min, the plasma samples were stored at −80 degree until assay. Plasma VWF antigen level was measured using a microlatex particlemediated immunoassay (LIA) (Roch Diagnostics, Mannheim, Germany).
Study subjects
Statistics
The protocol was approved by the ethics committee of the School of Medicine, Keio University. Written informed consent was obtained from all study subjects (n = 176), who were genetically unrelated Japanese males at their regular checkups. Ninety-five subjects included in the present study were the same as those in their study [11]. The mean age was 47.0 ± 5.1 years. The study subjects had no apparent hematologic or vascular disorders and were not taking any medications that affect platelet function.
Genetic analysis Genomic DNA was extracted from peripheral blood. Genotypes of the − 5T/C and 145The/Met polymorphisms were determined using the MegaBACE1000 (Amersham Biosciences, Piscataway, NJ) according to the manufacturer's protocol for the single nucleotide primer extension-based method.
Assessment of platelet sensitivity to ASA For studies of platelet sensitivity to ASA, citrated (0.129 M) whole blood was incubated with ASA (final concentration 10 μM, which roughly represents a peak plasma concentration when low dose ASA was orally administrated) [ASA (+)] or vehicle [ASA (−)] for 30 min at room temperature, and then each blood sample was analyzed with a Platelet Function Analyzer-100® (PFA-100®, Dade Behring, Deerfield, IN). This PFA-100® system using the collagen/epinephrine (CEPI) or collagen/ADP (CADP) cartridge is a relatively accurate device for assessing platelet function, as assessed by closure time (CT), under high shear flow conditions (5000–6000/s). The maximum measurement of CT is 300s, and a longer CT indicates lower platelet function. The manufacturer's instruction for PFA-100® shows that the CEPI-CT is sensitive to the effects of ASA, and thus a good responder to ASA has a longer CEPI-CT. We measured the CEPI-CT for ASA (+) samples, and the CEPI-CTand CADP-CT for ASA (−) samples in
Table 1
Paired t-test was used to compare the CEPI-CT value between the ASA (+) and ASA (−) samples. A chi-square test was performed to compare two proportions between the poor and good responders. Multiple logistic regression analysis was performed to evaluate the relationship between the poor and good responders (categorical variable) and other variables. Independent variables included in the analysis were 145Thr/Met genotype (categorical variable), VWF level (quantitative value), platelet count (quantitative value), and hematocrit value (quantitative value), because the manufacturer's instructions for the PFA-100® indicate the possibility that the CT value is affected by VWF level, platelet count, and hematocrit value. To examine whether base line platelet function [i.e. the CEPI-CT in ASA (−)] affects the platelet sensitivity to ASA, we analyzed another multiple logistic regression model with the CEPICT in ASA (−) as independent variable. Because multiple regression analysis revealed a significant contribution of VWF level to the CEPI-CT in ASA (−), VWF level as independent variable was excluded from this model. Thus, in the another model, the dependent variable was the poor and good responders (categorical variable), and independent variables were 145Thr/Met genotype (categorical variable), the CEPI-CT in ASA (−) (quantitative value), platelet count (quantitative value), and hematocrit value (quantitative value). Also, the relationship between the GPIb α polymorphisms and the continuous variable of CEPI-CT or CADP-CT was examined using analysis of covariance (ANCOVA) with VWF level as covariate. Statistical analyses were performed using StatView (ver 5.0, for Macintosh, SAS, Cary, NC). A p value less than 0.05 was considered to be statistically significant.
Results We observed the inhibitory effect of ASA on platelet function; the CEPI-CT in ASA (+) samples was significantly longer than that in
GPIbα polymorphisms and platelet reactivity to aspirin −5
Good responders⁎ Poor responders⁎
TT, n (%)
TC+CC, n (%)
p value
72 (53.3) 17 (41.5)
63 (46.7) 24 (58.5)
145
− 145
p value
98 (72.6) 38 (92.7)
37 (27.4) 3 (7.3)
0.0072
ThrThr, n (%)
Good responders⁎ Poor responders⁎
−5
ThrMet+MetMet, n (%)
0.1831
⁎A good responder group was defined as having a CEPI-CT N 250s, and a poor responder group as having a CEPI-CT ≤ 250s.
Effect of the
145
Met-allele of GPIb alpha on platelet sensitivity to aspirin under high-shear conditions 333
ASA (−) samples (p b 0.0001). Study subjects were divided into two groups based on the CEPI-CT of ASA (+) samples as described in materials and methods section: a good responder group (n = 135) and a poor responder group (n = 41). The frequency of poor responders was 23.3%, which was consistent with previous reports [11,13]. To examine whether the− 5T/C and/or 145Thr/ Met polymorphisms affect ASA efficacy in platelet function, we analyzed these genotype distributions between the good and poor responders. As shown in Table 1, there was no significant difference in the − 5T/C genotype distribution between the good and poor responders (p = 0.1831). On the other hand, the genotype distribution of the 145Thr/Met polymorphism was significantly different between the two groups: the frequency of 145Thr/Met and 145Met/Met genotypes in the poor responders was significantly lower than that in the good responders (p = 0.0072). A multiple logistic regression analysis was performed with the dependent variable, good responder vs poor responder, as assessed by CEPI-CT in ASA (+) samples, and the independent variables including 145Thr/Met genotype, plasma VWF antigen level, platelet count, and hematocrit value. As a result, the 145Thr/Met genotype (p = 0.0247) and VWF level (p = 0.0133) were shown to be independent predictors for CEPICT in ASA (+) samples (Table 2A). Additionally, we examined whether base line platelet function [i.e. the CEPI-CT in ASA (−)] affects the platelet sensitivity to ASA, and another multiple logistic regression model with the CEPI-CT in ASA (−) as independent variable was analyzed. Because multiple regression analysis revealed a significant contribution of VWF level to the CEPI-CT in ASA (−), VWF level as independent variable was excluded from this model. Results demonstrated that the 145Thr/ Met genotype (p = 0.0424) and the CEPI-CT in ASA (−) (p = 0.0002) were independent predictors for CEPI-CT in ASA (+) samples (Table 2B). These results for two models suggest that the 145Thr/ Met genotype is independently associated with the CEPI-CT in ASA (+). For ASA (−) samples, we examined whether the − 5T/C or 145Thr/ Met polymorphism affects CEPI-CT or CADP-CT. The association between the − 5T/C or 145Thr/Met polymorphism and CEPI-CT or CADP-CTwas analyzed by ANCOVA for statistical adjustment of VWF levels, because multiple regression analysis with independent variables (plasma VWF level, platelet count, and hematocrit value) revealed a significant contribution of VWF level to both CEPI-CT (p b 0.0001) and CADP-CT (p b 0.0001). As a result, the − 5T/C polymorphism was not associated with the CEPI-CT (136.6 ± 35.9 for −5 TT, 147 ± 38.9 for − 5CC+− 5TC, p = 0.8601) or CADP-CT (96.0 ± 15.6 for − 5TT, 96.7±16.2 for − 5CC+− 5TC, p = 0.7904). There was no association between the 145Thr/Met polymorphism and the CEPI-CT (138.6 ± 37.5 for 145Thr/Thr, 152.7±36.1 for 145Thr/Met+145Met/
Table 2
Multiple logistic regression analyses
Independent variables
Odds ratio
95% confidence interval
p value
(A) VWF level 145 Thr/Met genotype Platelet count Hematocrit value
1.009 4.179 1.018 0.943
1.002–1.017 1.200–14.553 0.937–1.105 0.809–1.099
0.0133 0.0247 0.6776 0.4528
(B) CEPI-CT in ASA (−) 145 Thr/Met genotype Platelet count Hematocrit value
0.972 3.741 1.000 0.882
0.958–0.987 1.046–13.378 0.919–1.087 0.749–1.039
0.0002 0.0424 0.9921 0.1320
Met, p = 0.1132) or CADP-CT (94.5± 15.0 for 145Thr/Thr, 102.5 ± 17.2 for 145Thr/Met+145Met/Met, p = 0.0583).
Discussion The efficacy of ASA to inhibit platelet function is mainly due to irreversible acetylation of a specific serine residue on the platelet cyclooxygenase-1 enzyme. The inhibition of platelet functions by ASA also occurs via a cyclooxygenase-1-independent pathway. Although the mechanism of platelet reactivity to ASA has been widely studied, the mechanism underlying the inter-individual variations in platelet response to ASA is not fully understood. Recently, the association between the variability in platelet response to ASA and the polymorphisms of platelet membrane receptors was highlighted [7,14]. Among the receptors, the GPIIIa PlA1/A2 polymorphism is the most studied polymorphism. The PlA2-allele, however, is extremely rare in the Japanese population. With regard to the association of GPIbα polymorphisms with platelet reactivity to ASA resistance status assessed by the CEPI-CT, Macchi et al reported that there was no relation between the − 5T/C polymorphism and the ASA resistance status among ASA-treated patients [8], which is compatible with the present observation. There are more than 10 publications of study on the effect of the 145 Thr/Met polymorphism on atherothrombotic disorders and receptor functions [7,15]. However, Williams et al recently reported an association between this polymorphism and variability in platelet response to ASA, showing no relationship between the − 5TC and 145 Thr/Met polymorphisms and ASA resistance status among patients taking ASA of 81–325 mg within 24 hours prior to enrollment [10]. In their study, ASAtreated patients were divided into two groups based on the CEPI-CT (N167s or ≤167s), and this cut-off value was different from our study (250s) in ASA (+) samples. Also, in our study, samples were from healthy subjects, because we intended to exclude individual pharmacokinetic variations and to reduce bias of study subjects. The difference in background of study subjects is likely to cause conflicting views. Our findings suggest that the inhibitory effect of ASA on platelet function is more efficient in subjects with the 145Met-allele. This 145Met-allele is suggested to be a risk factor for atherothrombotic disorders and to have higher interaction with immobilized VWF under high shear conditions, although these findings are somewhat controversial [7,15–17]. The mechanism of the present finding that the 145 Met-allele is associated with increased sensitivity to ASA in vitro is largely unknown. Recently, Aktas et al reported that ASA induces ADAM17-mediated shedding of GPIbα and GPV in vivo and in vitro,
334 which is associated with the downregulation of thrombus formation [5], although the mechanism of the ASA-induced shedding, as well as physiologic relevance of GPIbα shedding, is not fully understood. More recently, Gardiner et al showed that the synthetic peptides based on the region of GPIbα (450 Lys-483 Phe) were cleaved by ADAM17 at 464Gly/ 465 Val [18]. This cleaving site lies adjacent to the sequence for VNTR polymorphism which is closely linked to 145 Thr/Met polymorphism. We speculate as follows: the GPIbα/ADAM 17 interaction is likely to be enhanced by ASA, and platelets with the 145 Met/VNTR 3R or 4R-allele have a structure that allows easier access for ADAM17. Thus, enhancing effect of the 145 Met/VNTR 3R or 4R-allele on the GPIbα shedding by ADAM 17 might become evident in the presence of ASA, as compared with basal condition. The present study did not provide the evidence that the GPIbα shedding is associated with the difference in platelet sensitivity to ASA between the genotypes of GPIbα polymorphisms. Further studies are needed to elucidate the molecular mechanism of the difference in the ASA inhibitory effect on platelet reactivity between the 145Thr/Met genotypes. Also, it is important to learn the stage at which the ASA-induced shedding occurs. For ASA(−) samples, we observed no association between the − 5T/C polymorphism and CEPI-CT or CADP-CT. Also, there was no effect of the 145Thr/Met polymorphism on CEPI-CTand CADP-CT. We previously reported that the 145Met/VNTR 4R-expressing cell lines had a higher interaction with immobilized VWF under high shear conditions [16]. It is likely that the CT in PFA 100® system is affected by various stimulations, such as collagen and epinephrine or ADP. Thus, it is difficult to examine the difference in GPIb/VWFmediated platelet function between GPIbα polymorphisms, under an experimental condition with no stimulation, using samples from healthy (i.e., no evidence of bleeding or atherothrombosis) subjects. We performed a single measurement of the CT value. Because only small amounts of blood from samples taken during regular checkups were available for this study, we examined the validity of the values obtained in the PFA-100® system in a pilot study. Reproducibility and reliability of the assay were confirmed by repeated measurements of the CT value. Also, samples in the pilot study were evaluated by the light transmission method, and the CEPI-CT significantly correlated with the maximum aggregation of collagen (1 ug/ml)-, ADP (5 uM)-, or epinephrine (10 uM)-induced platelet aggregation. We did not observe these correlations with the CADP-CT in the pilot study. Because PFA-100® assay system requires only small amount of whole blood (0.8 ml/assay), this
Y. Matsubara et al. method is suitable for point of care testing and is convenient at site of blood collection in regular checkup. The CT value of PFA-100® system has cutoff value of 300s. In this study, of the 176 subjects, 133 had a CEPI-CT N300s in ASA (+) samples. Consequently, we did not analyze actual value of the CEPI-CT in ASA (+) samples but analyzed the proportion of poor responder categorized by the CEPI-CT. Also, we could not define low responders by relative extension of the CEPI-CT between the ASA (+) and ASA (−) samples. It is necessary, however, to examine platelet sensitivity to ASA, using other measurement tools as well. Additionally, we observed that the mean CTof CEPI or CADP in healthy Japanese subjects differed from that described in PFA-100® manufacturer's instructions. This may suggest the importance of PFA-100® data analysis in different ethnic groups. In summary, we examined the effect of GPIbα − 5T/C and 145Thr/Met polymorphisms on platelet sensitivity to ASA among healthy subjects. The results suggested that the 145Thr/Met polymorphism, but not − 5T/C polymorphism, affects the platelet sensitivity to ASA. The present study is the first to show the enhancing effect of the 145Met-allele on platelet sensitivity to ASA upon CEPI-CTamong healthy subjects. This finding could have a potential clinical impact on the design of pharmacogenetic antiplatelet therapies.
Acknowledgment This study is supported by a grant from the Japanese Ministry of Health and Welfare for the Scientific Research Project (H16-Genome-002).
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