Effects Of Anti-inflammatory And Anticoagulant Fusion Protein TAP-SSL5on Platelets Function

Background and Objective:Atherosclerosis (AS) is a chronic inflammatory response, which usually occurs onthe basis of intimal injury leaded by oxLDL (oxidized low density lipoprotein), variousinfection or other risk factors. The activation of both inflammation and coagulationsystem contributes key roles to atherosclerosis. The recombinant fusion proteinTAP-SSL5is composed of SSL5(staphylococcus aureus superantigen-like protein-5) andtick anticoagulant peptide (TAP). SSL5can bind to P-selectin glycoproteinligand-1(PSGL-1) on the leukocytes and inhibit leukocyte adhesion on P-selectin coatedsurface or activated endothelial cells, meanwhile TAP can inhibit the activation ofcoagulation factor Xa (FXa). We have demonstrated previously that SSL5could bind toGPIbα and activate platelets. In this study, we further observed the effects of TAP-SSL5on platelet. OS-1as antagonistic peptides of GPIbα can inhibit the binding of GPIbα withvWF-A1. In this study, the anti-thrombotic and platelet activation effects of OS-1weredetected as well.Research Methods1. The construction of TAP-SSL5expression vector. The expression andpurification of fusion protein TAP-SSL5were according to the methods in our lab.2. in vitro experiments: Blood was collected by venipuncture from healthyvolunteers. All subjects were definitely free of drugs for14days. Blood was anti-coagulated with one-tenth volume of citrate solution, platelet-rich plasma (PRP) wasobtained by centrifugation at200g for10minutes, and gel-filtered platelets (GFPs) wereprepared from PRP using a sepharose CL-2B column and modified Tyrode’s buffer foreluting. Samples were analyzed using flow cytometry, the binding of TAP-SSL5toplatelets was detected. The binding of HIP1(mouse anti-human GPIbα monoclonal antibody) to platelets competitively inhibited by TAP-SSL5or OS-1was observed. BothPAC-1–FITC and anti-CD62P-PE were used to detect platelet activation. TAP-SSL5induced platelets aggregation in whole blood and PRP was measured using electricalaggregometry (Chrono-Log Corp). The static adhesion of platelets on fibrinogen-coatedsurface were assayed. The effect of OS-1on mitochondrial membrane potential inplatelets was detected by JC-1kit.3. Animal experiments: Mouse tail bleeding time was assayed after bolus injectionof TAP-SSL5to evaluate its bleeding risk. Ferric chloride-induced rat vena cavathrombus formation model was used to study the antithrombotic effect of OS-1.Results:1. TAP-SSL5can bind to platelets and competitively inhibited the HIP1binding toplatelets, which indicates that TAP-SSL5may bind to GPIbα on platelets and inhibit the“GPIbα-vWF” interaction. Meanwhile TAP-SSL5at high concentrations could activateplatelets, when platelets were stimulated with30mg/L TAP-SSL5, the positive rate ofP-selectin and PAC-1was90.4%and66.3%respectively； TAP-SSL5at highconcentrations (10mg/L and30mg/L) promoted the platelets aggregation as well.30mg/LTAP-SSL5increased platelets adhesion on immobilized fibrinogen in the static condition.OS-1could inhibit the HIP1binding to platelets competitively. OS-1at highconcentrations could activate platelets. OS-1didn’t affect the mitochondrial membranepotential in platelets.2. A bolus injection of10mg/kg TAP-SSL5into mice could prolong tail bleedingtime obviously,753.6±127.3s vs647.1±33.7s in control group (p<0.01). There were nosignificant difference between3mg/kg TAP-SSL5group and control group in tailbleeding time (p＞0.05). Bolus injection of1.22mg/kg or3.66mg/kg OS-1into SpragueDawley rat could significantly reduce the thrombus weight in ferric chloride-induced ratvena cava thrombus formation model compared with control group, the thrombus weightwas as following:17.3±5.6mg in the vehicle control (DMSO),10.5±3.5mg in1.22mg/kgOS-1group (p<0.05vs control),8.2±4.1mg in3.66mg/kg OS-1group (p<0.01vscontrol). Conclusion: TAP-SSL5can bind to GPIbα on platelets, indicates that TAP-SSL5remains the GPIbα binding function of SSL5as reported previously, which may improvethe anti-thrombosis function of TAP-SSL5by inhibiting the GPIbα-vWF interaction. Butit’s noticeable that TAP-SSL5at high concentration may prolong the bleeding time andactivate platelets. OS-1can reduce ferric chloride-induced venous thrombus formationsignificantly and activate platelets at high concentration as well.