Pig Thrombomodulin Binds Human Thrombin but Is a Poor Cofactor for Activation of Human Protein C and TAFI

Abstract

Incompatibility between pig thrombomodulin (TM) and primate thrombin is thought to be an important factor in the development of microvascular thrombosis in rejecting pig-to-primate xenografts. To examine this interaction at the molecular level, we cloned pig TM and measured its ability to bind human thrombin and act as a cofactor for the activation of human protein C and TAFI. The 579-residue pig TM protein showed approximately 69% sequence identity to human TM. Within the EGF domains necessary for binding of thrombin (EGF56), protein C (EGF4) and TAFI (EGF3), all of the amino acids previously identified as critical for the function of human TM, with the exception of Glu-408 in EGF5, were conserved in pig TM. Comparison of transfected cells expressing pig or human TM demonstrated that both proteins bound human thrombin and inhibited its procoagulant activity. However, pig TM was a poor cofactor for the activation of human protein C and TAFI, with domain swapping showing that EGF5 was the most important determinant of compatibility. Thus, while pig TM may be capable of binding thrombin generated in the vicinity of xenograft endothelium, its failure to promote the activation of human protein C remains a significant problem.

Pig-to-Baboon Liver Xenoperfusion Utilizing GalTKO.hCD46 Pigs and Glycoprotein Ib Blockade.

“…Coagulation cascade activation is exacerbated in the xenotransplantation setting because porcine VWF, in contrast to human VWF, has been shown to constitutively activate human platelets, resulting in uncontrolled platelet aggregation [6]. Furthermore, negative feedback provided by porcine thrombomodulin is inefficient, and activated protein C levels are not maintained effectively [7][8][9]. It is also unclear whether porcine tissue factor pathway inhibitor is able to limit initiation of the clotting cascade [10,11].…”

Sequence alignment analysis of proteins involved in platelet-endothelial cell interaction identifies molecular incompatibilities between Homo sapiens and Sus scrofa

“…For example, porcine thrombomodulin has about 78% sequence similarity with human thrombomodulin, and porcine thrombomodulin is able to recognize and bind human thrombin, but the resulting complex is a weak activator of both human protein C and thrombin-activatable fibrinolysis Inhibitor. [38] Thus, from our analysis, the function of candidate targets needs to be confirmed experimentally. Furthermore, it is worthy of note that the availability, annotation, integrity of the Sus scrofa genome provide some obstacles that may ultimately affect the accuracy and integrity of our results.…”

“…Although porcine thrombomodulin is able to recognize and bind human thrombin, the resulting complex is a weak activator of both human protein C and thrombin-activatable fibrinolysis Inhibitor. [38] In contrast, porcine von Willebrand factor has been reported to be a strong agonist for human platelet GPIb receptors, resulting in robust human platelet activation. [39] In order to control the biological effects of these incompatibilities, the generation of genetically-modified pigs has been suggested as an additional strategy to prolong xenograft survival.…”

Progress and prospects: genetic engineering in xenotransplantation

“…Furthermore, incompatibility between pig thrombomodulin (TM) and primate thrombin could be involved in microvascular thrombosis during xenograft rejection. Roussel et al 25 recently showed in vitro that pig TM bound to human thrombin inhibits its procoagulant activity but is a poor cofactor for human protein C activation (APC) and thrombin-activated fibrinolysis inhibitor, which interacts with various cofactors to shut down coagulation.…”