| PART ? Evaluation of the Diagnostic Value of Platelet CEACAM5 in Acute Coronary SyndromeObjectives:Acute coronary syndrome(ACS),the initial presentation of complete or incomplete coronary occlusion,is the umbrella term of clinical manifestations induced by acute myocardial ischemia.ACS could lead to severe adverse cardiovascular events,such as cardiogenic shock and sudden cardiac death.Rapid and accurate diagnosis of ACS is critical for effective clinical decision-making and timely revascularization,which could effectively reduce mortality.However,cardiac troponins(cTns)assays lack sensitivity within first few hours from the onset of symptoms,and cannot discern the mechanisms of myocardial injury.Therefore,studies on novel biomarkers with higher sensitivity and specificity are essential for the diagnosis of ACS.The purpose of this study was to explore the expression of carcinoembryonic antigen-associated adhesion molecule 5(CEACAM5)on human platelet and its value in diagnosis of ACS,and to examine the effect of rhCEACAM5 on platelet aggregation,release,and adhesion.Methods:We observed the expression of CEACAM5 on platelet by Western Blot and two-color flow cytometry,respectively.Then we observed the expression of CEACAM5 on platelet of ACS by two-color whole blood flow cytometry,and the expression of CEACAM5 and CD62P on platelet of ACS by three-color whole blood flow cytometry.The expression level of CEACAM5 in serum of ACS was detected by a custom-made Luminex multiplex assay.We examined the effect of rhCEACAM5 on platelet aggregation induced by thrombin,collagen,AA with aggregometer.We observed the effect of rhCEACAM5 on platelet P-selectin release after stimulated by thrombin and collagen with flow cytometry.The effect of rhCEACAM5 on platelet adhesion to collagen was observed under a microscope.We examined the proteolysis of the extracellular domain of CEACAM5 by MMP-2?-7?-9?-12 with in vitro digest assays.Results:1.Expression of CEACAM5 on human platelet1)CEACAM5 was expressed on human platelet surface;2)Platelet CEACAM5 expression significantly enhanced when stimulated with different doses of thrombin(P<0.01).2.The expression level of platelet CEACAM5 in ACS and its correlation with serum CEACAM5,markers of myocardial necrosis,and BNP1)The expression level of platelet CEACAM5 increased significantly in ACS compared with healthy control(P<0.01);2)No statistically significant correlation was found between the expression of platelet CEACAM5 and serum CEACAM5 in ACS(P>0.05);3)No statistically significant correlation was found between the expression of platelet CEACAM5 and cTnI,CKMB,and MYO in ACS(P>0.05);4)The expression of platelet CEACAM5 was positively correlated with BNP in ACS(P<0.05).3.Predicted and diagnostic value of platelet CEACAM5 expression in ACS1)Platelet CEACAM5 expression correlated with ACS independent of sex,age,CVF,and cTnI,was an independent risk factor for ACS(P<0.01);2)Platelet CEACAM5 expression was similar to cTnI in diagonsis of ACS with high sensitivity(88.7%)and specificity(75%),and the sensitivity improved significantly(60%to 93%),but the specificity reduced(95%to 75%)than cTnI test alone when combined with cTnI;3)Platelet CEACAM5 expression positively correlated with platelet CD62P expression level in ACS(P<0.01).4.Effect of rhCEACAM5 on platelet activation4.1 Effect of rhCEACAM5 on platelet aggregation induced by different agonists1)rhCEACAM5 dose-dependently inhibited platelet aggregation induced by low-dose thrombin(P<0.01),and the inhibition rate of 5 ?g/ml and 10 ?g/ml rhCEACAM5 is 70%and 94%,respectively;2)rhCEACAM5 dose-dependently inhibited platelet aggregation induced by low-dose collagen(P<0.01),and the inhibition rate of 10 ?g/ml rhCEACAM5 is 78%;3)2-10 p.g/ml rhCEACAM5 had no significant effect on platelet aggregation induced by high-dose thrombin,high-dose collagen,and AA(P>0.05).4.2 Effect of rhCEACAM5 on platelet P-selectin release and platelet adhesion to collagen1)rhCEACAM5 dose-dependently inhibited platelet P-selectin release induced by thrombin(P<0.01),the inhibition rate of 2,5,and 10 ?g/ml rhCEACAM5 is 28%,40%and 53%,respectively;2)rhCEACAM5 had no significant effect on platelet P-selectin release induced by collagen(P>0.05);3)There was no significant difference in the numbers of platelet adhesion to collagen after incubated with 2-10?g/ml rhCEACAM5(P>0.05).5.Digestion of the extracellular domain of CEACAM5 by MMP-12,-7,-2,and-9There was no significant attenuation of rhCEACAM5 bands,and no fragments were found after incubated by rhMMP-12,2,7,and 9 at 3 7?.Conclusions:1.CEACAM5 was expressed on human platelet surface,and increased when stimulated by thrombin;2.The expression level of platelet CEACAM5 could diagose ACS with high sensitivity and specificity,and when combined with cTnI the sensitivity improved significantly while the specificity reduced compared with cTnI test alone;3.Platelet CEACAM5 expression correlated with ACS independent of sex,age,CVF,and cTnI,might be an independent risk factor for ACS;4.Platelet CEACAM5 expression correlated with BNP significantly,and might predict cardiac function;5.Platelet CEACAM5 expression correlated with P-selectin significantly,and might predict platelet activation;6.rhCEACAM5 inhibited platelet aggregation and P-selectin release induced by low-dose thrombin,and inhibited platelet aggregation induced by low-dose collagen;7.MMP-12,-2,-7,and-9 could not cleave the extracellular domain of CEACAM5.PART ? Study of Peptide KM 17 on Platelet ActivationObjectives:CEACAM1,highly homologous to CEACAM5,was expressed on human platelet surface.We found MMP-12,-2,-7,and-9 could not cleave the extracellular domain of CEACAM5 in Part ?,but We have found multiple active fragments were generated after platelet CEACAM1 digested by MMP-12.Many of these fragments could affect collagen-platelet interactions,and all of which were derived from IgC2 or IgV-like domains.The effect of peptides at the junction of the two domains on platelet activation is unknown.This part aims to explore the effect of KM17,containing the amino acid sequences of CEACAM1 IgV and IgC2 domains,on platelet aggregation,release,and adhesion.Methods:We examined the effect of KM 17 on platelet aggregation induced by different agonists(ADP,thrombin,collagen,and AA)with aggregometer.We observed the effect of KM 17 on platelet P-selectin release after stimulated by ADP with FCM.The effect of KM 17 on platelet adhesion to collagen was observed under a microscope.Results:1.Effect of KM17 on platelet aggregation induced by different agonists1)KM 17 dose-dependently promoted ADP-induced platelet aggregation,the promotion rate of 50 and 100 ?M KM 17 is 31.4%and 66%,respectively;2)KM 17 had no significant effect on platelet aggregation induced by AA,collagen,and thrombin(P>0.05).2.Effect of KM17 on platelet P-selectin release and platelet adhesion to collagen1)KM 17 had no significant effect on platelet P-selectin release induced by ADP(P>005);2)There was no significant difference in the numbers of platelet adhesion to collagen after incubated with 10-100 ?M KM 17(P>0.05).Conclusions:1.KM 17 dose-dependently promoted platelet aggregation induced by ADP;2.KM 17 had no significant effect on platelet P-selectin release induced by ADP,and on platelet adhesion to collagen.PART ? Study on Site 84 in Detection of Enzymatic activity of MMP-2,-7,-9,-12Objectives:We have found that multiple active fragments and restriction sites generated after the extracellular domain of CEACAM1 cleavaged by MMP-12.Synthetic peptides containing restriction sites are expected to be used for the detection of enzymatic activity of MMPs.This part aims to explore the use of Site 84,a synthetic CEACAM1-derived peptide fluorescent substrate,in detection of enzymatic activity of MMP-2,-7,-9,-12,and to explore the digestion of the extracellular domain of CEACAM1 by MMP-7.Methods:We observed the role of Site 84 in detection of enzymatic activity of MMP-2,-7,-9,and-12 by fluorescence resonance energy transfer(FRET).We examined the proteolysis of the extracellular domain of CEACAM1 by MMP-7 with in vitro digest assays.Results:1.Site 84 in detection of enzymatic activity of MMP-2,-7,-9,and-121)The enzymic kinetics curves of Site 84 with MMP-12,-7,-2,but not with MMP-9,were obtained;2)Site 84 could detect enzymatic activity of MMP-2 from gelatinase specifically,and the kinetics of the enzymic reaction were Km=315 ?M,Kcat/Km=2565 M-1·S-1.2.Digestion of the extracellular domain of CEACAM1 by MMP-7MMP-7 cleavaged the extracellular domain of CEACAM1 dose-dependently,and fragments around 10kDa were generated.Conclusions:1.The enzymic kinetics curves of Site 84 with MMP-12,-7,-2 were obtained;2.Site 84 could detect the enzymatic activity of MMP-2 from gelatinases specifically;3.MMP-7 could cleave the extracellular domain of CEACAM1 as same as MMP-12. |
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