Platelet Hem Itam Signaling Mediated Cancer-associated Venous Thrombosis

Background and objective: Venous thromboembolism(VTE)is a common complication of malignant tumors.There is a lack of specific indicators to accurately determine the prethrombotic state of the patients or effective therapeutic drugs which do not increase the risk of bleeding while treating venous thrombosis in the clinic.Recent studies have found that platelet activation plays an important role in cancer-associated VTE.Podoplanin(PDPN)and C-type lectin-like receptor 2(CLEC-2)mediate platelet activation through a hem-immunoreceptor tyrosine-based activation motif(hemITAM).The purpose of this study is to determine the clinical relevance of platelet hem ITAM signaling in the development of VTE in patients with cancer and to investigate a potential novel therapeutic strategy for cancer patients via reducing thrombosis.Materials and methods: Two human malignant cell lines(NCI-H226 and C8161)expressing high levels of PDPN were screened by FCM,CHO-PNPN was used as a positive control.We conducted to investigate the interaction between tumor cells with high PDPN expression and platelets in vitro and to explore its effects on platelet aggregation and activation,using the PDPN monoclonal antibody SZ168 or Syk inhibitor R406 line inhibition experiment developed by our laboratory.Further,we studied the role of hemITAM tyrosine kinase signaling pathway in the activation of platelets.And then established a nude mouse model of tumor thrombosis,observed tumor growth and venous thrombosis in nude mice to evaluate the hem ITAM pathway in tumors associated VTE in vivo.Immunohistochemical staining using anti-PDPN antibody was also performed in the pulmonary squamous cell carcinoma specimens of 166 patients.R software was used to calculate the cumulative incidence of VTE in the presence of competitive death.The survival curve was plotted to analyze the correlation between PDPN expression and prognosis.Results: Both NCI-H226 and C8161 cells expressing high PDPN triggered platelet activation via CLEC-2 in vitro,which was abrogated by SZ168 in a concentration-dependent manner or R406.In the cell-induced platelet activation assay,the release concentration of platelet factor 4(PF4)after tumor cells were co-incubated with PRP was detected by ELISA.NCI-H226 and C8161 cells significantly increased PF4 secretion in PRP,and tumor cells co-incubated with SZ168 in advance did not differ significantly from control PBS.To further confirm that SZ168 can inhibit platelet activation,SZ168 were incubated with tumor cells and the expression of P-selectin on the platelet surface was detected by flow cytometry.The results showed that CHO-PDPN,NCI-H226 and C8161 induced platelet activation,and the expression of P-selectin on platelet surface increased significantly.Co-incubation with SZ168 significantly reduced the expression of P-selectin on the platelet surface in a concentration-dependent manner.C8161 cells were pre-incubated with Mouse IgG and SZ168,subcutaneously injected into the right shoulder of nude mice.After 25 days,the inferior vena cava stenosis was performed.The nude mice were dissected 48 h after operation,tumors and thrombus were removed.Tumors,thrombi size and platelet counts in Mouse Ig G group were significantly larger than that in SZ168 group.Consistent with the observation in the C8161 tumor thrombosis model,CHO-PDPN cells were injected into the nude mice via the tail vein.The thrombi size,thrombus rate and platelet level of the Mouse IgG group were significantly higher than those of the SZ168 group.PDPN-mediated platelet activation was also intervened using the Syk tyrosine kinase inhibitor R406 in the hemITAM pathway.Flow cytometry analysis showed that R406 partially blocked PDPN-induced platelet activation in vitro.Western blot results showed that p-Syk,which is a Syk activated form,was significantly inhibited in the presence of R406.The platelet counts and thrombi size of tumor-bearing mice treated with R406 and SZ168 were significantly reduced.Of the 166 lung cancer specimens,105 were positive for PDPN staining(63.2%).During a 5-year follow-up,a total of 20(12.05%)patients developed VTE.In the competitive risk model analysis,patients with different PDPN expression had significant differences in the 1-year cumulative thrombosis rate of VTE.The risk of PDPN-positive patients was higher than that of PDPN-negative patients.Survival analysis showed that the median time to death in PDPN-negative patients was approximately 18.5 months,compared with 9.8 months in PDPN-positive patients.Conclusion: PDPN expression in tumors induced platelet activation and was related to a high risk of VTE via platelet hemITAM signaling.SZ168 inhibited PDPN-induced platelet activation in vitro and decreased the incidence of VTE in mice.PDPN expressed by tumor cells and platelet receptor CLEC-2 are expected to become new targets for the prevention and treatment of cancer-associated venous thrombosis.Antiplatelet therapy may be a new strategy for the treatment of cancer-associated venous thrombosis.