The Diagnostic Value Of Soluble C-type Lectin Like Receptor 2 In Coronary Heart Disease And The Related Mechanism In The Level Change

Cardiovascular diseases (CVDs) are the leading cause of death all over the world. Understanding of mechanism underlying CVD has become a priority task for early diagnosis and effective treatment. Consequently, the last decades have seen an astonishing amount of studies in this field. Importantly, the obligatory roles of platelets in CVD is hallmarked by their involvement in the thrombus formation after rupture of atherosclerotic plaques. Being a member of C-type lectin receptor family encoded by the gene CLEC1B located on human chromosome 12, CLEC-2 is highly expressed in megakaryocytes and platelets. A growing body of evidence has demonstrated the important roles of CLEC-2 in a variety of physiological processes including platelet activation and lymphatic development. We previously showed two isoforms of mouse CLEC-2 due to differential cleavage. Full length CLEC-2 can be hydrolyzed into a soluble form, which binds to the ligands of CLEC-2 and inhibits membrane-bound CLEC-2 activation. However, whether CLEC-2 plays a role in coronary atherosclerotic heart disease (CHD) remains largely unknown.Part I Diagnostic value of plasma soluble CLEC-2 in CHDObjective:To evaluate the plasma level and of soluble CLEC-2 in CHD patients and explore the clinical significance of CLEC-2 in CHD.Methods:One hundred and forty-six patients with CHD, including 14 cases of non-ACS and 132 cases of ACS, hospitalized in the first affiliated hospital of Soochow University and first affiliated hospital to Chinese Medical University from May 2011 through October 2016 were enrolled in the study. The control group contains 31 healthy subjects. All patients and control subjects underwent venous blood collection to detect plasma sCLEC-2 and sGPVI levels using the enzyme-linked immunosorbent assay (ELISA). Plasma levels of sCLEC-2 were compared between patients with CHD and healthy controls. Subgroup analysis was performed to compare serum sCLEC-2 levels among ACS patients, non-ACS patients and healthy controls. Comparison of sCLEC-2 level was also carried out among ACS patients with different numbers of affected coronary branches. GRACE risk score was calculated for patients with ACS and sCLEC-2 levels in patients with different risk were compared. Logistic regression was used to evaluate plasma sCLEC-2 as an independent risk factor for ACS. Receiver operating curve (ROC) was constructed to analyze the diagnostic value of plasma sCLEC-2 in CHD. The relation of plasma sCLEC-2 and plasma sGPVI as a classical indicator of platelet activation was evaluated using Pearson's linear regression.Results:Patients with CHD had a significantly higher plasma sCLEC-2 level (181.86 ±146.21 pg/mL vs.121.68 ± 56.24 pg/mL, P<0.001) compared to healthy controls. Subgroup analysis showed that ACS patients had had significantly increased plasma sCLEC-2 levels compared to healthy controls (187.74±151.42 pg/mL vs 121.68±56.24 pg/mL, P<0.001) and non-ACS patients (187.74±151.42 pg/mL vs 126.47±60.98 pg/mL, P<0.05). No significant difference of plasma sCLEC-2 was displayed between non-ACS patients and healthy controls (P>0.05). Plasma sCLEC-2 levels in CAD patients with single, double and triple coronary branch lesions were 196.98±161.44pg/mL,151.73 ±118.79 pg/mL, and 190.29±145.87 pg/mL, without any observed statistical difference (P>0.05). Patients with ACS were stratified as low risk, medium and high risk according to their GRACE scores on admission. Plasma levels of sCLEC2 (Low risk 205.46±185.98 pg/mL, Medium risk 177.83±137.53 pg/mL, High risk 190.37±149.48 pg/mL) were not significantly different among these groups (P>0.05). Logistic regression showed that high plasma sCLEC-2 level (OR= 1.006,95% CI:1.000-1.012, P=0.035) is an independent risk factor for ACS. Receiver operating curve demonstrated that high level of plasma sCLEC-2 can be an effective diagnostic index for CHD, with an AUC of 0.627,95% CI of 0.525?0.728, threshold value of 193.65 pg/mL, sensitivity of 25.3% and specificity of 96.8%(P<0.05). Pearson's linear regression indicated that plasma sCLEC2 level is positively correlated with plasma sGPVI that is an established marker for platelet activation (r=0.474, P<0.001).Conclusions:Plasma level of sCLEC-2 is significantly increased in patients with CAD. Patients with ACS have higher plasma sCLEC-2 levels compared to non-ACS patients and controls. High plasma sCLEC-2 level is an independent risk factor of ACS. Elevated plasma sCLEC-2 level is an effective diagnostic index for CHD. Plasma level of sCLEC-2 is positively correlated with plasma sGPVI level, rendering it a novel indicator of platelet activation. Part ? The regulatory mechanism of elevated level plasma soluble CLEC-2Objective:To investigate the causes resulting in the increased plasma level of CLEC-2 in CHD and explore the related mechanisms.Methods:The surface expression of CLEC-2 was determined by flow cytometry analysis. Washed platelets were isolated from the healthy volunteers and stimulated with 50 ?g/mL of ox-LDL to detect the mean fluorescence intensity to confirm CLEC-2 shedding. Moreover, the related signaling pathway in platelet CLEC-2 shedding induced by ox-LDL tested in the presence of kinase inhibitor PP2, PRT-060318 and Ro-31-8820. CLEC-2 levels in the supernatants were measure by ELISA when washed platelets were stimulated by the combination of thrombin, oxLDL and 8-pCPT-cGMP. to stimulated the washed platelets and the level of CLEC-2 secretion from platelets was determined by ELISA. The pathways involved in platelet CLEC-2 secretion were determined by modulation of different signals by MnTMPyP, PP2 or Ro-31-8220 when platelet secretion magnification signaling and integrin outside-in signaling were blocked.Results:High expression of CLEC-2 was found in platelet surface rather than dendritic cells, B cells, T cells, monocytes and neutrophils. Ox-LDL stimulation led to CLEC-2 shedding from platelet surface, which was mediated by Src family kinase-Syk-PKC signaling. PKG-cGMP signaling was found to be involved in CLEC-2 secretion from platelets, as well as upstream ROS, Src family kinase and PKC.Conclusions:The elevated plasma level of soluble CLEC-2 in was achieved in 2 ways, including shedding from platelet surface and secretion from platelet granules. These mechanisms are mediate through the Src family kinase-Syk-PKC pathway and Src-PKC-ROS-cGMP signaling, respectively.