The Antiplatelet Effects Of Human CEACAM1 Derived Peptides

Objectives:Recent studies indicated that CEACAM1 acts as an important negative regulator of collagen receptor GP?.CEACAM1-L protein has two ITIMs that can inhibit the ITAMs signaling pathway of the GPVI-FcRy chain complex.Until now,little is known about the role of the extracellular domain of CEACAM1 on regulating platelet function.Previous studies have suggested that Matrix Metalloproteinase-12(MMP-1 2),which stimulates platelet activation,can enzymatically cut the extracellular domain of platelet membrane protein CEACAM 1.And the negative regulation role of CEACAM 1 may be impaired after cleavage,which leads to enhanced platelet activation in response to collagen.However,the role and underlying mechanism of the soluble cleavage fragments in modulating platelets' function remains unexplored.Therefore,this project intends to explore the functions and mechanisms of extracellular domain of CEACAM 1 on platelet activation.This might provide new theoretical basis to decipher mechanisms of CEACAM1's role in platelet function and to develop novel reagents.Methods:Part I:This part aims to explore the effects of recombinant human CEACAM 1 extracellular domain proteins(rhCEACAM 1 s)and soluble cleavage fragments of CEACAM 1 on platelet function.We examined the effect of purified rhCEACAM1s and synthetic enzymatic peptide fragments on platelet aggregation,which was induced by various platelet agonists(collagen,arachidonic acid,thrombin and ADP).The effects of rhCEACAM1s and peptides on platelets' static adhesion to collagen coated slides were observed under the microscope.We also use His-pull down to explore the target protein of peptides on platelet.Part II:This part aims to study the mechanism of the effect of peptides on platelet function.First,we investigated the effects of peptides on platelet activation mediated by collagen receptor GPVI.A platelet aggregation analyzer was used to observe the effect of peptides on GPVI selectively agonist convulxin-induced platelet aggregation.Flow cytometry and platelet aggregation analyzer were used to examine the effects of peptides on convulxin-induced secretion of ATP and dense granules.The effects of peptides on protein phosphorylation in platelet was studied by Western Blot.Secondly,we investigated the effects of peptides on platelet "inside-out" and "outside-in'activation mediated by platelet integrin receptor ??b?3.Using fluorescence resonance energy transfer(FRET)and flow cytometry,we explored the effects of peptides on platelet Ca2+release and PAC-1 binding rate.The effects of peptides on platelets spreading on fibrinogen coated slides were observed under the microscope.We also inspected the effects of peptides on platelet clot retraction.Results:Part ?.? rhCEACAMs inhibited low dose collagen-induced platelet aggregation.?CEACAMI IgC2 domain-derived peptides QDTT and QLSN significantly inhibited collagen-induced platelet aggregation,QDTT and QLSN also inhibited arachidonic acid,thrombin and ADP-induced platelet aggregation.? QDTT and QLSN inhibited platelet static adhesion to collagen.Part ?.?QDTT and QLSN attenuated GPVI selectively agonist convulxin-induced platelet aggregation and release.? When QDTT and QLSN were used together with PI3K broad-spectrum inhibitor Wortmannin or SFKs broad-spectrum inhibitor PP2,the effects of QDTT and QLSN on inhibiting collagen-induced platelet aggregation were enhanced.QDTT and QLSN inhibited the global phosphorylation of platelet proteins mediated by GPVI and phosphorylation of the ITAMs-mediated platelet activation signaling molecules Src,Syk,PLC y 2 and Akt.?QDTT and QLSN inhibit platelet integrin ??b?3-mediated platelet "inside-out" activation.?QDTT inhibits platelet integrin ??b?3-mediated platelet "outside-in" activation.Conclusions:The inhibition effects of QDTT and QLSN on collagen-induced platelet activation were mainly dependent on GPVI mediated platelet aggregation and release,and were involved in ITAMs-mediated phosphorylation of Src,Syk,PLCy2 and Akt.The CEACAM1-derived peptide QDTT also inhibited integrin ??b?3-medicated platelet "inside-out" and "outside-in" activation.