Study Of The Risk Factors And Possible Mechanisms Of High On Treatment Platelet Reactivity With Clopidogrel Therapy

Objective:We aimed at investigating the incidence and related risk factors of high on treatment platelet reactivity (HTPR) with clopidogrel therapy, primarily analysing the correlation between genetic polymorphisms and HTPR. We also purposed to explore the possible mechanism how diabetes mellitus increasing HTPR with clopidogrel.Methods:The clinical study:578 patients with coronary heart disease undergoing PCI treatment were enrolled. They all received dual antiplatelet therapy with aspirin (300mg) plus clopidogrel (300mg) over 24h, or aspirin (100mg/day) and clopidogrel (75mg/day) over three days. Patients were divided into two groups according the result of ADP-inhibition rate (ADP-IR%). The single nucleotide polymorphism (SNP) were detected by MassArray genotyping system. The follow-up lasted at least 12 months and adverse endpoints events were recorded. The experimental study:Fifty male SD rats were randomly divided into four groups:BLA, DM, BLA+CLO and DM+CLO groups. BLA+CLO group and DM+CLO group were given clopidogrel. CD62P and intracytoplasmic calcium concentration ([Ca2+]) were detected by flow cytometry. The changes of homocysteine (Hcy), high sensitive c-reactive protein (hs-CRP), superoxide dismutase (SOD), malonaldehyde (MDA), et al. were assessed by Elisa. Expressions of CYP450, P2Y12 and PKC were asssessed by realtime-PCR and Western-blot.Results:The clinical study:① Total incidence of HTPR was 15.74%, and the female’s was significantly higher than male’s (24.29% vs 13.01%, P<0.01). ② Female (OR = 2.023,95% CI:1.146-3.571, P = 0.015), diabetes mellitus (OR = 2.815, 95% CI:1.737-4.561, P = 0.000), Hcy (OR = 2.479,95% CI:1.092-5.629, P= 0.030) and hs-CRP (OR= 8.476,95%CI:1.588-45.240, P=0.012) were positively correlated with the occurrence of HTPR.③ We found four positive SNPs polymorphism: rs1057910, rs2242480, rs2246709 and rs776746. ④ The polymorphism of rs1057910 was positively correlated with HTPR (OR = 2.90,95% CI:1.44-5.68, P = 0.003), while the polymorphism of rs2246709 (OR = 0.69,95% CI:0.49-0.98, P = 0.039) and rs776746 (OR = 0.66.95% CI:0.45-0.97, P = 0.034) were negatively correlated with HTPR. Female was positively correlated with rs 1057910 mutation (OR = 3.24.95% CI:1.46-7.18, P = 0.004), and negatively correlated with rs776746 mutation(OR= 0.57.95% CI:0.35-0.93, P= 0.025).⑤Compared with patients who had single SNP polymorphism. There were no significant difference of HTPR incidence in those had coexisting SNPs polymorphism (14.6% vs 14.8%, P>0.05). ⑥The endpoints events was significantly higher in HTPR group than those in non-HTPR group. HTPR was correlated with recurrent angina (OR= 1.486,95% CI:1.104-1.999, P = 0.009) and restenosis (OR = 1.86,95% CI stent:1.079-3.207, P=0.026). ⑦ The ADP-IR% in patients with ticagrelor therapy was sgnificantly higher than those with clopidogrel therapy. The incidence of endpoints events were significantly lower than those with clopidogrel therapy. The experimental study:① ADP-IR% in DM+CLO group was significantly lower than that in BLA+CLO group; The levels of CD62P and [Ca2+] in DM+CLO group were also significantly higher; ② Levels of GLU, GSP, TC, MDA, TXA2, TF, hs-CRP and Hcy in DM and DM+CLO groups were both significantly higher than the other two groups, while levels of HDL-C, PGI2, SOD and NO were significantly lower than the other two groups. ③ Compared with BLA+CLO groups, the expression levels of PKC and P2Y12 in DM and DM+CLO groups were significantly higher, while CYP450 protein level was significantly lower.④ ADP-IR% was negatively correlated with levels of SOD, TXA2 and CD62P.Conclusions:1) The total incidence of HTPR was 15.74%. Female, diabetes mellitus, high levles of Hey and hs-CRP were risk factors for HTPR; 2) There were four positive SNPs polymorphism:rs 1057910, rs2242480, rs2246709 and rs776746; rs1057910 polymorphism was a risk factor of HTPR, while rs2246709 and rs776746 polymorphisms were protective factors; Female was a risk factor of rs1057910 mutation, and was a protective factor of rs776746 mutation; 3) Coexisting SNPs polymorphism didn’t increase the incidence of HTPR; 4) HTPR and endpoint events were significantly correlated; 5) Ticagrelor can significantly decrease the aggregation of platelets, and reduce the cardiovascular endpoint events.6) There were three possible mechanisms that diabetes mellitus resulted in clopidogrel HTPR: ① The increasing of inflammation and oxidative stress, endothelial dysfunction, high levels of PKC and CD62P expression, combined with the increased cytoplasmic [Ca2+] that caused platelets hyperfunction; ② The upregulation of P2Y12 signalling increased the combination of ADP and P2Y12 receptor, so that clopidogrel can not completely inhibited the activation of platelets; ③ The reduction of CYP450 enzymes decreased the activation product of clopidogrel, which may diminished the clopidogrel effect.