Thrombin-PAR1/2 Signaling Axis Modulates TLR3-Mediated Procoagulant, Proinflammatory, and Proadhesive Responses in Vascular Endothelial Cells

Activation of the blood coagulation mechanism is an integral part of the innate immune response to infection by viral and bacterial pathogens.  Pattern-recognition receptors (PRRs) on cells recognize pathogen-associated molecular patterns (PAMPs) produced by the pathogen, such as lipopolysaccharide (LPS), double-stranded (ds) or single-stranded (ss) RNA, or viral DNA.  Activation of these PRRs leads to the expression of the type I interferons (IFNs), chemokines, and cytokines. Toll-like receptor 3 (TLR3) recognizes dsRNA, an intermediate generated during most viral infections. It was therefore widely accepted that TLR3 acted as a sentinel against viruses.  Vascular endothelial cells (ECs) stripe the luminal surface of blood vessels, providing a critical barrier between the vasculature and organ systems. ECs represent an important target for infection of most human viruses, including beta- and gamma-herpesviruses, adenovirus, parainfluenzavirus, poliovirus, echovirus, measles, mumps, CMV, human T-cell leukemia virus type 1, and HIV. Recent pandemic SARS-CoV2 also targets ECs. The innate immune system also senses infection-driven proteolytic enzymes through a family of protease-activated receptors (PARs), nonclassical PRRs. Extracellular proteinases are tightly regulated by several mechanisms, including the regulation of protease expression and their negative control by protease inhibitors, in physiological conditions. The PARs, 7-transmembrane G protein–coupled receptors (7-TM GPCRs), detect serine proteinases derived from pathogens and the host during infection. Given that TLR3 and PARs are concurrently present on ECs, Saravanan Subramaniam, Ph.D., of the Versiti-Blood Research Institute in Milwaukee, WI, stated on Monday that he and others sought out to characterize the role of thrombin-PAR signaling for TLR3-mediated responses in ECs.

Here, Subramaniam and colleagues reported that, using synthetic and endogenous PAR-specific ligands, the thrombin-PAR1/2 signaling axis distinctly regulates poly(I:C)-induced gene expression. Transcriptional profiling in human ECs revealed that a subset of the poly(I:C) response, including TF, IL-8, IL-6, and adhesive molecules, was augmented by PAR1/2 or thrombin co-stimulation, indicating an overlapping target spectrum for both receptors. Thrombin, but not the binary (TF:VIIa, TF:Xa), 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 and PAR2 inhibitors blocked thrombin-mediated potentiation of the poly(I:C) response, indicating PAR1–PAR2 crosstalk/heterodimer. Blocking of vascular cell adhesion molecule 1/E-selectin (VCAM-1/E-SELE) reduced monocyte adhesion on the EC surface, confirming leukocyte-EC interaction and leukocyte infiltration. Besides, TM-null ECs did not alter the thrombin-mediated augmentation responses, suggesting that thrombin does not require TM to activate PAR1/2. Based on these findings, Subramaniam proposed that PAR1 and PAR2 activation by the endogenous ligand, thrombin, worsens infection severity by augmenting the inflammatory cytokine, chemokine, and TF expressions in ECs that were reversed with PAR1/2 inhibitors. In addition, these observations support that PAR1/2 activation plays a significant role in viral sepsis pathology. For in vivo validation, Subramaniam has been actively working on mouse models with a real viral infection.

Read the full abstract here.