Thrombomodulin Regulates Platelet and Extracellular Vesicle Mediated Sterile Inflammation in the Placenta

Scientific Research Question: Thrombomodulin (TM) deficiency causes tissue-factor initiated thrombin generation, maternal platelet-activation and embryonic lethality in mice. However, the mechanism remains poorly understood. We have recently shown that procoagulant extracellular vesicles (EVs) activate maternal platelets, which results in purinergic receptor mediated inflammasome activation in the trophoblast and subsequently preeclampsia (PE). We therefore investigated whether this EV-mediated thrombo-inflammatory pathway in placenta is controlled by thrombomodulin and whether this mechanism contributes to the lethality of TM-null embryos.

Methodology: Procoagulant EVs were injected into C57/Bl6 pregnant mice. Placental-TM expression was studied using immunoblotting and immunofluorescence. EV-injected pregnant mice were treated with soluble TM (solulin) to establish causality. Genetic and pharmaceutical inflammasome inhibition was conducted to establish mechanistic relevance in TM-null placenta. Differentiated mouse trophoblast stem cells were treated with EV or IL-1β to study the inflammasome associated loss of TM. Trophoblast proliferation and cell death was studied using Ki-67 and TUNEL staining respectively. Translational relevance was corroborated by analysis of human PE placentae and trophoblast cells.

Findings: EV caused PE and accumulation of activated platelets within the placenta. EV treatment resulted in a reduced TM expression and trophoblast proliferation in mouse placenta in-vivo and EV-treated mouse or human trophoblast cells in-vitro. Trophoblast cell death was increased. Solulin treatment ameliorated the PE-like phenotype in mice and prevented thrombo-inflammation and cell death. Furthermore, TM expression was negatively correlated with IL-1β expression and platelet counts in human PE placentae. However, genetic (NLRP3^-/-) or pharmaceutical (Anakinra, Apyrase) inflammasome inhibition did not rescue the TM-null embryos from lethality. Treatment of mouse or human trophoblast cells with exogenous IL-1β resulted in reduced TM expression indicating that loss of TM is the consequence, and not cause of inflammasome activation. Mechanistically, inflammasome induced TM loss was associated with NF-κB activation. Over-expression of TM in the trophoblast restored the reduced proliferation due to EV treatment establishing causality.

Conclusions: These results demonstrate that EV-mediated platelet-activation, thrombo-inflammation and PE are associated with loss of placental TM and support a patho-physiological association of thrombophilia associated pregnancy complications and sterile inflammation. While loss of TM seems to be a consequence rather than cause of inflammasome activation, treatment with solulin efficiently prevented the EV-induced thrombo-inflammatory response and PE-like phenotype, presumably by restricting coagulation and platelet activation.

No conflict of interest has been declared by the author(s).