Effect Of Fusion Protein TAP-SSL5on Platelet-leukocyte Interactions

Background and ObjectiveAtherosclerosis（AS） is an important pathological basis of cardiovascular andcerebrovascular diseases. P-selectin glycoprotein ligand-1(PSGL-1) is expressed widely onthe surface of leukocytes, including monocytes, neutrophils and lymphocytes. PSGL-1canbind to P-/E-selectin which expressed on the surface of activated platelets or endothelialcells. The interaction of P-/E-selectin and PSGL-1mediates the leukocytes adhesion to theinjured vascular endothelium, and the leukocytes will be activated subsequently.Staphylococcal superantigen-like protein-5(SSL5) can bind to PSGL-1and inhibitleukocyte’s adhesion on endothelial cells, it can also bind to GPCRs and inhibit leukocyteactivation induced by chemokines (CCL2/MCP-1, CXCL8/IL-8) and anaphylatoxins (C3a,C5a). Tick antico-agulant peptide (TAP) is an inhibitor of factor Xa (FXa). In our previousstudy, a novel fusion protein TAP-SSL5was constructed which can inhibit FXa activity andleukocyte adhesion to P-selectin-coated surface. In this study, the effects of TAP-SSL5onthe formation of platelet-leukocyte aggregates, platelet-induced IL-8protein expression inTHP-1cells and MCP-1-induced THP-1cells migration were evaluated. The purpose of thisstudy was to further explore the possible anti-inflammatory mechanisms of TAP-SSL5.Methods1. Cell viability was assessed by colorimetric Cell Counting Kit-8（CCK-8）. Flowcytometry （FACS） was applied for the inhibition effect of TAP-SSL5on KPL-1(a mouseanti-human CD162monoclonal antibody) binding to leukocytes. Platelet-rich plasma (PRP)from healthy adult was offered from blood bank in Southwest hospital. Platelets werewashed with CGS buffer by centrifugation, then finally resuspended in modified Tyrode’sbuffer; THP-1cells or neutrophils were incubated with TAP-SSL5or SSL5for20minuteson ice and washed twice, the platelets activated by20μmol/L ADP was added at the ratio of50:1to THP-1cells or neutrophils, and were further incubated for5min. P-selectionexpression on activated platelets was assayed by PE-labeled anti-CD62P monoclonalantibody, the binding rate of activated platelets to THP-1cells or neutrophils was assayedby FACS and Wright-Giemsa staining respectively.2. THP-1cells were treated with TAP-SSL5or SSL5. After washing, THP-1cells wereincubated with patelets (1:100) which were activated by20mg/L TRAP-6(thrombinreceptor activator peptide6) for18hours. IL-8protein expression in THP-1cells wasassayed by ELISA. The effect of TAP-SSL5on THP-1cells migration induced by MCP-1was evaluated by transwell culture chamber.Results1. TAP-SSL5under30mg/L didn’t affect the viability of THP-1cells or humanneutrophils. PSGL-1is highly expressed in THP-1cells, the binding rate of KPL-1toTHP-1cells was approximately90%.10mg/L TAP-SSL5competitively inhibited KPL-1binding to THP-1cells significantly. The binding rates of activated platelets（by20μmol/LADP）to THP-1cells or neutrophils were (31.3±8.4)%and (35.6±5.9)%，and decreased to(13.4±6.7)%and (10.4±6.4)%respectively (p <0.01) in the groups of10mg/L TAP-SSL5preincubated THP-1cells and neutrophils respectively. rThe number of binding platelets per100neutrophils by Wright-Giemsa staining were (364.3±79.1) in the control group, andreduced to (189.3±53.9) in the groups of10mg/L TAP-SSL5or SSL5(p<0.05).2. The IL-8protein levels in the supernatant of THP-1cells stimulated by TRAP-6activated platelets were as following,(1068.3±410.7) pg/mL in the control group,(737.2±167.4) pg/mL in the TAP-SSL5group, which indicated that TAP-SSL5could significantlyinhibit platelet-induced IL-8protein expression in THP-1cells (p<0.05). As comparing tonegative control group, the number of migrated cells in the other groups was calculated bymultiple relationship as following,(14.1±1.9) times in MCP-1group (positive control),(6.2±0.2) times in TAP-SSL5group,（6.1±0.7）times in SSL5group, which indicates that bothTAP-SSL5and SSL5can obviously inhibit THP-1cell migration induced byMCP-1(p<0.001).ConclusionsFusion protein TAP-SSL5can not only inhibit the formation of platelet-leukocyte aggregates and IL-8expression in THP-1cells induced by activated platelets, but alsoinhibit THP-1cell migration induced by MCP-1, which are the possible anti-inflammatorymechanisms of TAP-SSL5.