Study on the mechanisms of dengue virus nonstructural protein 1-induced platelet activation

Abstract

Dengue the most common mosquito-transmitted viral infection can cause a range of diseases from self-limiting dengue fever to severe dengue One of the major characters of severe dengue is bleeding disorder resulting from thrombocytopenia and vascular leakage Dengue virus (DENV) nonstructural protein 1 (NS1) the only nonstructural protein that could be secreted by DENV-infected cells has been considered as the last puzzle of the pathogenesis of dengue during the decade Several studies have demonstrated that NS1 plays a crucial role in vascular leak during DENV infection However limited studies have been addressed whether DENV NS1 contributes to thrombocytopenia in dengue Platelets small anucleate cells present in the circulation play a key role in regulating coagulation and hemostasis Recent studies have identified platelets as major contributors to inflammation in viral infections especially dengue Compare to platelets from healthy donors isolated platelets from dengue patients present increased P-selectin a platelet activation marker and exposed phosphatidylserine (PS) on their surface Besides a previous study has indicated that the circulation NS1 negatively correlates to platelet count in dengue patients Based on these observations we hypothesized that DENV NS1 could induce platelet activation and vascular leak thereby leading to the bleeding disorder We used an established DENV-induced hemorrhagic mice model and found DENV NS1 plays a critical role in DENV-induced hemorrhage bleeding time prolong and thrombocytopenia Indeed direct injection of DENV NS1 recombinant protein could trigger glycocalyx degradation vascular leak and decrease of platelet count in mice These results indicated that DENV NS1 could directly induce vascular leak and thrombocytopenia and then causes the bleeding disorder Next we investigated whether DENV NS1 could directly trigger platelet activation and if so what is the underlying mechanism At first we demonstrated that DENV but not Zika virus supernatant could induce P-selectin expression and PS exposure in human washed platelets both of which were abolished when NS1 was depleted from the DENV supernatant In addition recombinant DENV NS1 but not ZIKV NS1 could induce P-selectin expression and PS exposure in platelets in a dose- and time-dependent manner Moreover DENV NS1 could also trigger platelet aggregation in both washed platelet system and platelet-rich plasma The activation of platelets triggered by DENV NS1 enhanced platelet adhesion to endothelial cells and phagocytosis by monocytes and macrophages All of the effects induced by DENV NS1 could be blocked in the presence of anti-DENV NS1 F(ab’)2 Next we investigated whether DENV NS1 activates platelets via binding to Toll-like receptor 4 (TLR4) Our results showed that TLR4 inhibition could significantly diminish DENV NS1-induced platelet activation in vitro as well as DENV-induced hemorrhage bleeding time prolong and thrombocytopenia in vivo However the binding of DENV NS1 to platelets and DENV NS1-induced platelet aggregation could only be partially inhibited by TLR4 blocking Therefore it is possible that there is not only one receptor for DENV NS1 in platelets To test this possibility we used the washed platelet aggregation system We demonstrated that DENV NS1-induced platelet activation is completely abolished by R406 a tyrosine kinase Syk (spleen tyrosine kinase Syk) specific inhibitor In addition DENV NS1-induced platelet aggregation was dependent on secondary mediators such as secreted ADP and thromboxane A2 which is similar to platelet activation by collagen and CLEC-2 agonists Furthermore treatment of platelets with a CLEC-2 antagonist AAWAP could fully inhibit the DENV NS1-induced platelet aggregation indicating that DENV NS1 mediates platelet activation via CLEC-2 Finally we tested the therapeutic potential of dasatinib a FDA-approved Src (Proto-oncogene tyrosine-protein kinase) inhibitor Dasatinib could only partially rescue DENV-induced skin hemorrhage and bleeding time prolong but significantly rescued the DENV-induced thrombocytopenia In conclusion our results suggested that DENV NS1 activates platelets not only through TLR4 but a Src family kinase (SFK)-dependent signaling pathway mediated by CLEC-2 receptors

Date of Award	2021
Original language	English
Supervisor	Trai-Ming Yeh (Supervisor)