Platelet P-selectin(CD62P) and soluble TREM-like transcript-1(sTLT-1) are associated with coronary artery disease-A case control study

Background/Aims: Platelet activation plays a crucial role in the pathogenesis of coronary artery disease(CAD) (cid:0) platelet P-selectin(CD62P )is a platelet classic activation indicator in platelet surface and soluble TREM-like transcript-1 (sTLT-1) is a new one,the relation between these two makers and acute coronary syndromes(ACS) has not been elucidated. This study aimed to investigate the expression of CD62P in platelet surface and sTLT-1 on serum and their relationship with CAD. Methods: We measured the levels of CD62P and sTLT-1 in 83 patients undergoing CAD compared to 49 controls.The associations with age, blood pressure,lipid proles, body mass index and liver injury marker levels were also examined. Results: A stepwise increase in CD62P concentration was found based on the number of CAD patients (P <0.01), especially in AMI (P <0.01) . Serum sTLT-1 concentration of AMI and UAP were more higher than the NC group (P <0.01). Conclusions: The consistent of sTLT-1 and CD62P expression levels in CAD,indicating that sTLT-1 level may be a new maker of platelet activating and positively related with CAD.


Introduction
With an increasing global burden of coronary artery disease (CAD), early detection and timely management of risk factors are crucial to reduce morbidity and mortality in such patients. Platelet activation plays a crucial role in the pathogenesis of CAD[1], Circulating-activated platelets are thought to trigger ischaemic complications after angiography, angioplasty and vascular surgery.The onset of CAD, especially in acute coronary syndromes ACS),is closely associated with the enhanced platelet adhesion and aggregation,Therefore early detection and antiplatelet therapy are extremely important for the treatment of coronary heart disease. P-selectin, also called CD62p, is a kind of glycoprotein stored in Weibel-Palade bodies of vascular endothelial cells and a-granules of platelets. Activated CD62P is a transmembrane protein expressed on the surface of platelets[2] During platelet activation, the expression of CD62P on the surface of the cell membrane was dramatically increased, accompanied by the simultaneous increase of the expression level of plasma soluble CD62, which played an important role in the initiation, formation and expansion of thrombus. Therefore, it is agreed that the detection of the expression level of CD62P on the platelet membrane is one of the gold standards re ecting platelet activation.
TREM-like transcript-1 (TLT-1) , coexistence of CD62P in platelet alpha particles, is a new platelet activation secretory expression in platelet membrane surface [3,4]at the same time, releasing a soluble TLT -1 (sTLT -1) in the plasma, enhance the aggregation of platelet[2,5],therefore we hypothesised that detecting potentially physiologically active substances sTLT -1 expression level can re ect the expression of platelet membrane TLT-1[6],and indirectly re ect the platelet activation status.Despite of the proof of the biological function of CD62P on platelets and sTLT-1 on serum, there has been little information about its role either in patients with with CAD.Therefore,our study was done to elucidate the association between CD62P sTLT-1 and CAD.Based on the potential link between CD62p,sTLT-1 and platelet activation, we systematically analyzed their correlation with CAD.

Clinical samples
This study was planned to enroll consecutive patients with CAD whose maximal coronary stenosis < 50% con rmed by coronary angiograms were included in the control group, of whom 29 had acute myacardial imfraction (AMI), 26 had unstable angina pectoris (UAP), and 28 had stable angina pectoris (SAP).
Patients Compared with 49 patients without CAD, patients with any evidence of tumors or systemic disease were excluded, had no recent history of acute disease, injury or surgery. They agreed to participate in this study and signed the informed consent.A detailed clinical history was collected from all patients, including clinical characteristics. This study was approved by Medical Ethics Committee of Xiangya Hospital of Central South University.

Information Collection
Clinical data on general condition (age, gender, height, and weight), medical history (hypertension, diabetes or stroke), personal history (smoking or drinking), medication history, family history and laboratory tests were obtained from all patients.

Measurement of plasma
Blood venous samples were collected directly into plastic tubes after an overnight fast, collected blood samples of about 5ml, placed in a vacuum blood collection vessel, centrifuged for 3min (1500r/min) to separate the upper platelet-rich plasma and placed in a sterile EP tube. The expression level of CD62P on the platelet surface was detected by ow cytometry(BD FACSCalibur,America).The concentration of sTLT -1 in serum was measured by enzyme-linked immunosorbent assay according to the manufacturer's instructions.

Statistical analysis
The statistical analysis was performed by IBM SPSS version 22.0 software.Quantitative data were presented as the mean±SD deviation.Any differences among ≥3 groups were evaluated by ANOVA with the Scheffe's test for parametric variablesand by the Kruskal-Wallis test with the Steel-Dwass test for nonparametric variables,Any differences between 2 groups were evaluated by the unpaired t-test for parametric variables. Correlations between sTLT-1 CD62P and the severity of coronary atherosclerosis were evaluated by the Pearman's rank correlation test. P-value levels of <0.05 was considered statistically signi cance.

Clinical population clinical characteristics of participants
Of the 132 study patients the details of demographic, clinical and laboratory data are listed in Table 1. The results showed no signi cant differences were found with respect to age, sex, BMI, systolic blood pressure, DBP, and platelet count between the CAD and non-CAD groups. LDL and TG in ACS group were higher than those in NC group (△P<0.05). A stepwise decrease in HDL levels was found d levels in AMI and UAP groups than those in control group (△P<0.05).BS of AMI group was higher than that of NC group ( P = 0.03).

Levels of Plasma sTLT-1 platelet CD62p in Patients with CAD and Healthy Controls
We found that the expression level of CD62P in platelet membrane in CAD group was signi cantly higher than that in NC group, and the difference was statistically signi cant ( gure a-e and table1).The expression level of platelet CD62P in AMI group was signi cantly higher than that in SAP group (P = 0.011), and the difference was statistically signi cant (P<0.01).
Given that elevated plasma levels of sTLT-1 are implicated in the activation of platelet, we measured plasma sTLT-1 in patients diagnosed with CAD. Soluble TLT-1 levels were signi cantly increased in patients with AMI UAP compared to matched, healthy control participants signi cant correlation was observed between patients with AMI versus SAP P<0.01) 2.3 Correlation analysis of serum sTLT, platelet membrane CD62P and other risk factors of coronary heart disease Pearman regression was used to analyze the correlation between platelet membrane CD62P and serum sTLT-1 and other risk factors of coronary heart disease in ACS patients, including HDL, LDL and TG(Table3 Table4 . The results showed that serum sTLT-1 level was positively correlated with TG (r

Discussion
The world health organization (WHO) divides coronary heart disease into ve clinical types: asymptomatic, angina pectoris, myocardial infarction, ischemic cardiomyopathy and sudden death. ACS(Acute Coronary Syndrome) refers to as Coronary atherosclerosis plaque rupture and thrombosis or vasospasm and clinical Syndrome, Acute or subacute myocardial ischemia including unstable angina pectoris (UAP) and Acute myocardial infarction (AMI), and its typical performance for myocardial ischemia caused by chest pain, its development is associated with platelet activation. The adhesion molecule P-selectin (CD62P) plays a dominant role in modulating interactions between platelets and the endothelium, and involved in acute cardiovascular events[7],Studies agree that CD62P is one of the classic indicators of platelet activation.The activation of platelets is one of the targets of antiplatelet therapy.
The results of our study showed that the platelet CD62P level in AMI, UAP and SAP group was signi cantly higher than that in the normal control group, and the platelet CD62P level in AMI group was signi cantly higher than that in UAP and SAP group, which was consistent with previous research results.The results of this experiment showed that there was no signi cant difference between the UAP group and the SAP group, which may be related to the small amount of specimens collected in this experiment.
TLT-1 is a type-1 transmembrane protein [8,9,5]that is highly and exclusively expressed in megakaryocytes and platelets[10],stored in a-granules and possibly in another platelet compartment, and it is rapidly upregulated to the surface of activated platelets,with the most obvious increase in expression being CD62P[11] Upon platelet activation, TLT-1 transports to the membrane where it enhances Ca2+ in uence and promotes platelet aggregation,TLT-1 is a new player in platelet aggregation.
It has been proved that the expression level of sTLT-1 in patients with chest pain is signi cantly higher than that in the control group[12,13]and inAcute Respiratory Distress Syndrome The results showed that the expression level was closely related to coronary heart disease and was also one of the indexes of platelet activation. Morales-Ort´ız et al show in a murine model of acute lung injury (ALI) that TLT-1 mediates brinogen deposition in the lungs and facilitates platelet-neutrophil release during transmigration [14].In mice, inhibition of TLT-1 has been shown to reduce thrombosis in carotid artery thrombosis models and to protect mice from excessive bleeding during pulmonary embolism [12]. It has been proved that the serum level of sTLT-1 is correlated with the incidence of acute coronary syndrome and has certain predictive value[15] The speci c mechanism needs to be further studied.
Combined with our experimental results, the serum sTLT-1 level in AMI group and UAP group was signi cantly higher than that in NC group and SAP group.The expression level of sTLT-1 in AMI group was signi cantly higher than that in SAP group.Therefore, it is speculated that in the early stage of rapid platelet activation, TLT-1 may also have an obvious increase in expression as CD62P, which is also one of the indicators of platelet activation.The level of serum sTLT-1 in ACS patients can provide some reference for the diagnosis of coronary heart disease.Antiplatelet therapy is the cornerstone of the treatment of thrombotic diseases especially acute coronary syndrome.To better characterize sTLT-1 involvement in CAD, plasma levels of sTLT-1 should be evaluated in a larger cohort of patients and should places TLT-1 as an important factor to be considered to better unbe correlated with cytokines levels, disease severity, and survival rate. This study undoubtedly derstand platelet's function in CAD.TLT-1 is a newly discovered indicator of platelet activation, and further study and understanding of this indicator may provide new directions and targets for diagnosis of thrombotic related diseases and antiplatelet therapy.