Human thrombomodulin regulates complement activation as well as the coagulation cascade in xeno‐immune response

Abstract

Background: With the introduction of the a1, 3-galactosyltransferase gene-knockout (GT-KO) pig and its pivotal role in preventing hyperacute rejection (HAR), coagulation remains a considerable obstacle yet to be overcome in order to provide long-term xenograft survival. Thrombomodulin (TBM) plays a critical anticoagulant and anti-inflammatory role in its part of the protein C pathway. Many studies have demonstrated the strong anticoagulant effects of TBM in xenotransplantation, but its complement regulatory effects have not been appropriately examined. Here, we investigate whether TBM can regulate complement activation as well as coagulation in response to xenogeneic stimuli. Methods: We transfected porcine endothelial cells (MPN-3) with adenovirus vectors containing the human TBM gene (ad-hTBM), or a control gene containing GFP (ad-GFP). The expression level of ad-hTBM was measured by flow cytometry. To confirm the anticoagulant effect of TBM, coagulation time was measured after treatment with recalcified human plasma in adhTBM-transfected MPN-3, and a thrombin activity assay was performed after treatment with 50% human serum in ad-hTBM-infected MPN-3. Results: Thrombin generation was significantly decreased in a dosedependent manner in ad-TBM group, and coagulation time was increased in the ad-hTBM group when compared to the ad-GFP group. Complement-dependent serum toxicity assays were performed after treatment with 20% human serum or heat-inactivated human serum by LDH assay. Complement-dependent toxicity was significantly attenuated in the ad-hTBM group, but complement-independent toxicity was not attenuated in the ad-hTBM group. These results suggest that human thrombomodulin (hTBM) has complement regulatory effects as well as anticoagulant effects. To further investigate the mechanisms of complement regulation by hTBM, we deleted the EGF5, 6 domains that are involved in thrombin generation or the lectin-like domain involved in inflammation of TBM and functional tests were performed using these modified forms. We showed that the EGF5, 6 domain of TBM principally inhibits complement activation rather than the lectin domain. Conclusion: The EGF5, 6 domains of TBM appear to be the major domains for down-regulating the complement system rather than the lectin-like domain during xenogenic stimuli. The role of EGF5, 6 domains of hTBM may be due to inhibition of thrombin as thrombin can cleave C3a and C5a directly and hTBM may also be involved in complement regulation. Clearly then human TBM has complement regulatory effects as well as anticoagulant effects in xeno-immune response, and it is a promising target for attenuating xenograft rejection.

Expression of human thrombomodulin on porcine endothelial cells can reduce platelet aggregation but did not reduce activation of complement or endothelium – an experimental study

“…Only in combination with GTKO and hCD46, activation of coagulation was inhibited as both complement and endothelial cell activation were reduced (Figs ). The interference of hTBM with thrombin generation is in line with the data from other studies . In regard to the aspects of anti‐inflammatory and complement regulatory properties and in contrast to other studies , we found no protective effect of the mono‐transgenic PAEC expressing hTBM.…”

“…The interference of hTBM with thrombin generation is in line with the data from other studies . In regard to the aspects of anti‐inflammatory and complement regulatory properties and in contrast to other studies , we found no protective effect of the mono‐transgenic PAEC expressing hTBM. The complement regulatory effect of hTBM observed in other studies was described as based on thrombin inhibition, since thrombin can activate the complement system via cleavage of C3 and C5 to C3a and C5a .…”

“…The effect of hTBM has been studied extensively in static cell culture assays . Studies in dynamic assays are less common and rarely focus on hTBM specifically .…”

“…Therefore, it is difficult to rule out the effect of hTBM alone in this setting. There are only few studies focusing on the effect of hTBM alone . Two major effects of hTBM in this context have been described: first, regulation of the procoagulant activity by prevention of thrombin generation and second, anti‐inflammatory and complement regulatory properties .…”

The Vitamin E Analog Gamma-Tocotrienol (GT3) and Statins Synergistically Up-Regulate Endothelial Thrombomodulin (TM)

“…TM is structurally divided into five domains (i.e., N-terminal lectin-like domain, epidermal growth factor (EGF)-like repeats domain, serine-threonine rich domain, transmembrane domain, and cytoplasmic tail), each of which has unique functional properties [3]. Studies by various groups have shown that TM alone or the TM-thrombin complex suppress inflammation, coagulation, oxidative stress, complement activation, sepsis, adhesion of immune cells to the endothelium, and radiation damage [4,5,6,7,8]. Moreover, the TM-thrombin complex transforms protein C to activated protein C (APC), which is known to have anti-inflammatory, anti-coagulant, and cytoprotective properties [4].…”