| Acute myocardial infarction(AMI) is one of the major diseases that seriously threatened human health and life. It has become a major public health problem in china. Platelet aggregation and activation play important roles in the plaque formation and thrombosis of AMI. Recently, percutaneous coronary interventional(PCI) and dual antiplatelet therapy(aspirin and clopidogrel) have become the main therapy for AMI, which not only relieve clinical symptoms, but also improve the prognosis for AMI patients. However, 4-30% individuals don’t respond to clopidogrel, which is called clopidogrel resistance(CR). The risk for cardiovascular events in CR patients is obviously increased. Increasing the dose of clopidogrel, using the triple antiplatelet drugs(aspirin, clopidogrel plus cilostazol) or using the new antiplatelet drug ticagrelor could effectively reduce the risk for cardiovascular events in CR patients. However, the thrombus events can’t be completely eliminated, indicating that a certain degree of platelet aggregation and activation may still exist in CR patients. Therefore, to search for the key molecules and signaling pathway regulating platelet functions could provide the basis and strategies for AMI and other cardiovascular diseases.C-C motif ligand(CCL2), also named monocyte chemotactic protein 1(MCP-1), is currently the most widely studied CC chemokine. CCL2 play important roles in the atherosclerosis, angiogenesis, heart damage and heart repair. CCL2 expression in the plasma of patients with acute coronary syndrome(ACS) is significantly higher than healthy controls, and its expression is positively correlated with cardiovascular events and clinical outcome. Besides, the expressions of CCL2 and CCR2 in human atherosclerotic plaque tissues are higher than that in normal arterial tissues. Subsequent studies indicate CCL2 and CCR2 play important roles in the formation of atherosclerotic plaque in animal models. The above results show that CCL2 and CCR2 play important roles in ACS and atherosclerosis. However, whether CCL2 could participate in the development of AMI by affecting platelet functions, there is still unknown.To clarify the roles of CCL2 in platelet functions, CCL2 expressions in the plasma and coronary thrombus tissues of ST-elevation myocardial infarction(STEMI) patients were firstly examined. Next, the effects of CCL2 on platelet aggregation, activation, granule secretion and adhesion were examined using by light transmission aggregometry, flow cytometry and ELISA assay, and the roles of PKCα-P38 MAPK signal pathway in this process were studied. This study may provide the basis for diagnosis and therapy for AMI and other cardiovascular diseases.The major results were presented below: 1. CCL2 expressions in the plasma and coronary thrombus tissues of STEMI patients were increased.(1) Forty STEMI patients and 40 age-gender matched healthy controls were collected in our study. The proportion of smoking, hypertension history and the glucose level in STEMI patients were significantly higher than healthy controls(p=0.014, p=0.004 and p=0.003, respectively).The expressions of CCL2 in the plasma of STEMI patients and healthy controls were examined using by ELISA assay. We found that CCL2 expressions in STEMI patients were significantly higher than controls(299.17 ± 78.73 pg/ml vs. 177.00 ± 30.92 pg/ml, n=40, p<0.01).(2) The coronary thrombus tissues from 10 STEMI patients were obtained by aspiration catheter. The expressions and locations of CCL2 and CCR2 in the coronary thrombus tissues of STEMI patients were examined using by real time quantitative PCR, Western Blot, immunohistochemistry and immunofluorescence staining. Compared with coronary blood clots, the expressions of CCL2 and CCR2 in the coronary thrombus tissues of STEMI patients were significantly increased at m RNA and protein levels(n=10,p<0.01). Besides, CCL2 and CCR2 were co-located with platelet activation marker CD62 p in the coronary thrombus tissues.The above results showed that CCL2 expressions in the plasma and coronary thrombus tissues of STEMI patients were higher than controls, indicating that CCL2 might play an important role in the thrombus formation of STEMI patients.2. CCL2 increased platelet aggregation, activation, granule secretion and adhesion(1) Twenty healthy volunteers were included. After obtaining the purified platelets using CD45+ magnetic beads, the expressions of CCR2 in human platelets were examined using by RT-PCR, Western Blot and immunofluorescence staining. Results showed that CCR2 was obviously expressed in human platelets.(2) Effects of different concentrations of CCL2(0, 50, 100, 200, 500 and 1000 ng/ml) on platelet aggregation were examined using by light transmission aggregometry. When the concentration of CCL2 was 100 ng/ml, platelet aggregation was significantly increased compared with baseline(15.03 ± 2.54% vs. 6.92 ± 2.12%, n=20, p<0.01). With the increase of CCL2 concentration, platelet aggregation was gradually increased. Therefore, 100 ng/ml was selected as the final concentration in vitro experiments.Next, the effects of CCL2 on the expressions of platelet activation markers PAC-1 and CD62 p were detected using by flow cytometry. Compared with baseline, CCL2 could significantly increase the expressions of platelet PAC-1(20.54 ± 3.18% vs. 10.23 ± 2.58%, n=20, p<0.01) and CD62p(7.95 ± 2.19% vs. 5.12 ± 0.69%, n=20, p<0.01).Besides, the effects of CCL2 on the expressions of platelet granule secretion markers CD40 L and TXB2 were examined using by ELISA assay. Compared with baseline, CCL2 significantly increased the expressions of platelet CD40L(160.09 ± 19.66 pg/ml vs. 73.58 ± 6.48 pg/ml, n=20, p<0.01) and TXB2(23.44 ± 7.38 ng/ml vs. 9.62 ± 5.55 ng/ml, n=20, p<0.01). Meanwhile, CCL2 also increased the adhesion between platelets and HUVECs(54.25 ± 8.42 vs. 18 ± 2.94, n=20, p<0.01).(3) Effects of CCL2 neutralizing antibody and CCR2 antagonist(RS102895) on platelet functions were examined. Compared with CCL2 group, CCL2 neutralizing antibody significantly inhibited platelet aggregation(9.33 ± 1.72%, n=20, p<0.01), the expressions of platelet activation markers PAC-1 and CD62p(PAC-1: 11.58 ± 1.87%; CD62p: 5.35 ± 0.53%, n=20, p<0.01), the expressions of platelet CD40 L and TXB2(CD40L: 87.88 ± 12.01 pg/ml; TXB2: 11.77 ± 2.77 ng/ml, n=20, p<0.01), and adhesion(27.25 ± 8.66, n=20, p<0.01).Meanwhile, RS102895 also inhibited platelet aggregation(10.58 ± 2.07%, n=20, p<0.01), the expressions of platelet activation marker PAC-1 and CD62p(PAC-1: 12.55 ± 1.62%; CD62p: 5.48 ± 0.83%, n=20, p<0.01), the expressions of platelet CD40 L and TXB2(CD40L: 85.69 ± 14.68 pg/ml; TXB2: 12.02 ± 3.11 ng/ml, n=20, p<0.01), and adhesion(30 ± 6.27, n=20, p<0.01).(4) The effects of CCL2 combined with different platelet inductors(ADP or Collagen) on platelet functions were examined. Compared with ADP(5 μM), CCL2 combined with ADP could significantly increase platelet aggregation(60.2 ± 6.06%, n=20, p<0.01), platelet activation marker PAC-1 and CD62p(PAC-1: 53.85 ± 7.64%; CD62p: 34.77 ± 2.79%, n=20, p<0.05), platelet CD40 L and TXB2(CD40L: 425.37 ± 64.58 pg/ml; TXB2: 41.88 ± 5.09 ng/ml, n=20, p<0.01).Meanwhile, compared with Collagen(10 μg/ml), CCL2 combined with Collagen also increased platelet aggregation(69.53 ± 4.65%, n=20, p<0.01), the expression of platelet PAC-1(57.14 ± 10.72%, n=20, p<0.05), platelet α granule CD40L(449.03 ± 47.88 pg/ml, n=20, p<0.01) and dense granule TXB2(51.53 ± 8.72 ng/ml, n=20, p<0.05).These above results showed that CCL2 could significantly increase platelet aggregation, activation, granule secretion and adhesion, which were inhibited by CCL2 neutralizing antibody and RS102895.3. PKCα-P38 MAPK pathway mediated the effects of CCL2 on platelet functions(1) In view of the important roles of PKCs, MAPKs and PI3K/AKT pathways in the regulation of platelet activation and granule secretion, the phosphorylation expressions of platelet PKCs, MAPKs and PI3K/AKT were firstly examined using by Western Blot. CCL2 significantly increased the phosphorylation levels of PKCα and P38 MAPK in human platelets(n=5, p<0.01), however, the phosphorylation levels of PKCβII, ERK1/2, JNK1/2, PI3 K and AKT were not changed. The total protein levels of PKCα, PKCβII, P38 MAPK, ERK1/2, JNK1/2, PI3 K and AKT were also unchanged.CCL2 neutralizing antibody and RS102895 could inhibit the phosphorylation levels of PKCα and P38 MAPK, which were induced by CCL2(n=5, p<0.01). The above results indicated that PKCα-P38 MAPK signal pathway in human platelets was activated after CCL2 stimulation.(2) PKCα inhibitor(RO318220) or P38 MAPK inhibitor(SB203580) was used to pre-treat human platelets, and the phosphorylation levels of PKCα and P38 MAPK were examined by Western Blot. RO318220 significantly inhibited the phosphorylation levels of PKCα and P38 MAPK. However, SB203580 only inhibited the phosphorylation level of P38 MAPK.Next, the effects of RO318220 and SB203580 on CCL2-induced platelet aggregation, activation, granule secretion and adhesion were examined. Compared with CCL2 group, RO318220 significantly inhibited platelet aggregation(8.67 ± 1.06%, n=20, p<0.01), the expressions of platelet PAC-1 and CD62p(PAC-1: 11.08 ± 2.36%; CD62p: 4.93 ± 1.92%, n=20, p<0.01), the expressions of platelet CD40 L and TXB2(CD40L: 85.61 ± 7.91 pg/ml; TXB2: 11.82 ± 2.69 ng/ml, n=20, p<0.01), and adhesion(24.5 ± 8.39%, n=20, p<0.01).Meanwhile, SB203580 also significantly inhibited platelet aggregation(7.1 ± 1.51%, n=20, p<0.01), platelet PAC-1 and CD62p(PAC-1:10.77 ± 1.95%; CD62p: 4.5 ± 1.05%, n=20, p<0.01), platelet CD40 L and TXB2(CD40L: 87.03 ± 5.51 pg/ml; TXB2: 9.49 ± 1.92 ng/ml, n=20, p<0.01), and adhesion(23.75 ± 9.64%, n=20, p<0.01).These above results indicated that CCL2 could promote platelet aggregation, activation, granule secretion and adhesion through inducing PKCα-P38 MAPK pathway activation.In summary, CCL2 expressions in the plasma and coronary thrombus tissues of STEMI patients were significantly higher than controls. CCL2 could increase platelet aggregation, activation, granule secretion and adhesion in virto experiments, and PKCα-P38 MAPK pathway was activated during the above processes. RO318220 and SB203580 could significantly inhibit platelet aggregation, activation, granule secretion and adhesion, which were induced by CCL2. This study revealed the important roles of CCL2 and PKCα-P38 MAPK signal pathway in platelet functions, which not only could elucidate the roles of CCL2 in the development of AMI, but also provide new therapeutic targets for searching new antiplatelet drugs. |
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