Thrombin weakens the amnion extracellular matrix (ECM) directly rather than through protease activated receptors

Introduction: Preterm premature rupture of fetal membranes (pPROM) is a major cause of preterm birth. Abruption associated thrombin production, and infection-inflammation associated cytokine production reportedly play major roles in pPROM. Utilizing an in vitro model-system we have confirmed that both thrombin and inflammatory cytokines remodel and biomechanically weaken amnion, the load-bearing component of FM. Also, we have shown thrombin directly weakens isolated amnion but cytokines weaken amnion only indirectly by initially interacting with choriodecidua and releasing unidentified soluble activator(s). This study's purpose was to determine whether thrombin weakens the isolated amnion through thrombin receptor-protease activated receptors (PARs 1,2,3,4), activation of previously secreted extracellular matrix (ECM) enzymes, or by direct action on the ECM. Methods: Primary amnion cells and isolated amnion were tested for PARs by immunohistochemistry, Western Blot and rtPCR. Cell-free amnion ECM was produced by devitalizing isolated amnion by exposure to UV light and subsequent freeze thaw cycles. Devitalized amnion membrane explants were incubated with thrombin and biomechanically tested. Results: PARS were not found in amnion or amnion cells. Thrombin induced dose-dependent weakening of devitalized amnion explants. Preincubation with the thrombin inhibitor hirudin prevented thrombin-induced weakening. Thrombin converted pro-MMP2 embedded in the devitalized amnion ECM to multiple active forms. Thrombin also directly digested gelatin gels in zymograms suggesting the possibility of direct degradation of amnion ECM components. Discussion: Thrombin appears to directly weaken the amnion ECM and additionally activates stored pro-MMP2 to active forms that may further enhance amnion ECM degradation. Conclusion: Thrombin weakens amnion directly rather than through PARs. (C) 2013 Elsevier Ltd. All rights reserved.