Basic And Clinical Research Of MMP12 Facilitated Platelet Activation By Shedding CEACAM1

[Objective]Coronary arterial thrombosis secondary to plaque rupture or erosion is a key step in ST-segment elevation myocardial infarction(STEMI).Platelets express carcinoembryonic antigen-binding molecules 1(CEACAM1)and a variety of matrix metalloproteinases(MMPs),which can regulate platelet activation and thereby affect thrombosis.But the mechanism is currently unclear.Recent studies have reported that CEACAM1 has a negative regulation of collagen-platelet interactions and thrombosis,and ceacaml-/-mouse platelets are more susceptible to collagen activation than wild-type mouse platelets and are prone to large and stable thrombosis.Our group previous reported that:platelets can express MMP12;synthetic MMP12 can cleave CEACAM1 protein in vitro.Therefore,we propose the hypothesis that an agonist(such as collagen)can stimulate platelet release of MMP12,MMP12 cleaves platelet membrane protein CEACAM1,up regulate platelet activation and promotes thrombosis.Therefore we conducted a basic and clinical studies.The aim of this study was to investigate the mechanism of shedding of CEACAM1-mediated platelet activation by MMP12.Clinical study was designed to investigate the expression of CEACAM1 fragment in coronary thrombosis and serum in patients with STEMI[Method]Part ?(basic research).The platelets were collected by filtration with Sepharose 2B glue.In order to clarify the agonist that stimulate platelet express MMP12,this study measure the concentration and activity of MMP12 in the supernatant of platelet after stimulated by different types of platelet stimulants(thrombin,adenosine diphosphate,arachidonic acid,type ? collagen),by enzyme-linked immunosorbent assay(ELISA)and fluorescence Resonance energy transfer(FRET).To elucidate the experimental evidence of shedding of CEACAM byMMP12 on the platelet level,the platelets were incubated with MMP12 at 37 ? for 120 minutes and the CEACAM1 fragment was detected by immunoblotting(WB).Meanwhile,the platelets were labeled with fluorescently labeled CD61,and the extracellular domain of CEACAM1 was labeled with fluorescently labeled antibody.The fluorescence density of CEACAM1 was detected by flow cytometry.In order to clarify the effect of MMP 12 on platelet activation,this study used different types of platelet agonists to stimulate the isolated platelets after plateletpheresis,and then through the platelet aggregation test,static adhesion test and P selectin release test,to evaluate the effect of MMP 12 on platelet aggregation,adhesion and release function.In addition,according to the results of this team preliminary study,we synthesized a short peptide-WYKG from the shedding of CEACAM mediated by MMP12.The isolated platelets pre-incubated with WYKG were measured the aggregation and P selectin release.Part ?(clinical study).In this study,128 cases were included in the study group:STEMI group(n = 46),SAP group(n = 52)and control group(n = 30).The STEMI group was divided into large thrombotic burden subgroup(LTB,n = 26)and small thrombotic burden subgroup(STB,n = 20)according to the thrombotic load rating.In LTB,the intracoronary thrombosis was aspirated and the CEACAM1 fragment was detected by WB,immunohistochemistry and immunofluorescence.Meanwhile,three groups of peripheral blood were collected and the concentration of CEACAM1 was detected by ELISA.[Results]Part ?(basic research).Type ? collagen and thrombin can stimulate platelet to express and secrete MMP 12,but the effect of type ? collagen was significantly stronger than thrombin.MMP 12 can cleave the platelet membrane protein CEACAMI and produces a small molecule peptide with a molecular weight of less than 10 kd by immunoblot(WB)and a decrease in CEACAM1 fluorescence density in platelet surface by a flow cytometer(FCM).Platelets pre-incubated with MMP12 had more and faster platelet aggregation(51.3±2.1%VS.711±2.7%,*P<0.05;n=3),higher static adhesion(17.35±0.12 VS.11.48±0.36,*P<0.05;n=3),more release of P-selectin(43.15±2.46 VS.26.10±3.05,*P<0.05;n=3)and increased PLCy2 tyrosine phosphorylation responses to type ? collagen.While ADP,AA and thrombin did not have this effect.Similarly,Platelets pre-incubated with the synthetic CEACAM1 short peptide WYKG and then stimulated with type ? collagen showed more release of P-selectin(40.57±3.56 VS.23.05±5.03,*P<0.05;n=3).Part ?(clinical study).Baseline information between the study groups is basically the same.CEACAM1 was detected in human coronary thrombosis(MW = 58kd),and there were some small molecule fragments(MW<36kd).In immunohistochemistry and immunofluorescence,we used antibodies that identify long or short fragments of CECAM1.As a result,the antibody that identify short fragment of CECAM1 was positive but not the antibody that identify long fragment of CECAM1.The concentration of CEACAM1 in serum was significantly higher than that in SAP group and control group[(84.51 ± 16.81)ng/ml vs(61.25 ± 15.96)ng/ml vs(39.0 ± 10.16)in the STEMI group,0.05)],but there was no significant difference in the concentration of CEACAM1 between the high and low thrombosis subgroups[84.82± 19.05)vs(84.1 ± 14.38);p = 0.877].[Conclusion]1.Type ? collagen can stimulate platelet expression and secrete MMP12.2.MMP12 can cleave platelet membrane protein CEACAM1.3.MMP12 can facilitate type ? collagen induced platelet aggregation,adhesion and release function and the signal pathways might involve in PLC?2 of the lysine phosphorylation.Similarly,one short peptide,WYKG,which is a fragment of CEACAM1 facilitated type ? collagen induced platelet alpha granule secretion.4.CEACAM1 protein is present in the coronary thrombus of patients with STEMI and may exist in small fragments.5.Serum CEACAM1 concentration is related to STEMI...