Background: Inhibition of PAR1 has been shown to ameliorate TLR3-dependent procoagulant and inflammatory responses in mouse models of viral infection, potentially via thrombin-PAR1 signaling on innate immune cells. However, procoagulant responses to TLR3 stimulation have been shown in human endothelium but failed in human myeloid cells.

Aims: To characterize the role of thrombin-PAR signaling for TLR3-mediated responses in EC.

Methods: In vivo responses to the TLR3 agonist poly(I:C) and PAR1/2 inhibitors were analyzed by TAT-complex in C57BL/6J mice. The genome-wide effect of thrombin and PAR1/2 modulation of the poly(I:C) response was analyzed by transcriptional profiling of EC. PAR1/2 effects on endothelial procoagulant and cytokine responses to poly(I:C) were studied in human EA.Hy926 and HUVEC, and mouse bEND3 cells with cleavage resistant antibodies and pharmacological inhibitors. TM-thrombin mediated PAR activation was studied using CRISPR/cas9-mediated TM^-/- EC.

Results: Pharmacological inhibition of PAR1/2 altered the in vivo systemic TAT-complex, indicating that PAR1/2 modifies the PCA response to poly(I:C). Transcriptional profiling in human EC revealed that a subset of the poly(I:C) response, including TF, IL8, IL6, and adhesive molecules, was augmented by PAR1/2 or thrombin co-stimulation, indicating an overlapping target spectrum for both receptors. Thrombin, but not the ternary (TF/VIIa/Xa) complex, replicated the potentiating effect of PAR1/2 agonist peptides on the poly(I:C)-response. PAR1/2 cleavage resistant antibodies (WEDE15/ATAP2; SAM-II) revealed that thrombin cleaves PAR1, but not PAR2. However, both PAR1/2 inhibitors blocked thrombin-mediated potentiation of the poly(I:C) response, indicating PAR1-PAR2 crosstalk/heterodimer. Blocking of VCAM-1/E-SELE reduced monocytes adhesion on EC surface, confirming leukocytes-EC interaction. TM-null EC did not alter the thrombin-mediated augmentation responses, suggesting that thrombin does not required TM to activate PAR1/2.

Conclusions: Our data demonstrate that a thrombin-PAR1/2 positive feedback loop amplifies TLR3-mediated endothelial cell procoagulant, proinflammatory, proadhesion responses to poly(I:C) which is independent of TM-thrombin complex. Both PAR1 and PAR2 is required for thrombin-mediated augmentation of poly(I:C)-induced responses.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/thrombin-par1-2-signaling-axis-modulates-tlr3-mediated-procoagulant-proinflammatory-and-proadhesive-responses-in-vascular-endothelial-cells/