Pig BMSCs Transfected with Human TFPI Combat Species Incompatibility and Regulate the Human TF Pathway in Vitro and in a Rodent Model

Abstract

Background: The activation of tissue factor (TF) is one of the major reasons for coagulation dysregulation after pig-to-primate xenotransplantation. Tissue factor pathway inhibitor (TFPI) is the most important inhibitor of TF. Studies have demonstrated species incompatibility between pig TFPI and human TF. Methods: A pig-to-macaque heterotopic auxiliary liver transplantation model was established to determine the origin of activated TF. Chimeric proteins of human and pig TFPI were constructed to assess the role of Kunitz domains in species incompatibility. Immortalised pig bone marrow mesenchymal stem cells transfected with human TFPI were tested for their ability to inhibit clotting in vitro. Results: TF from recipient was activated early after liver xenotransplantation. Pig TFPI Kunitz domain 2 bound human FXa, but Kunitz domain 1 did not effectively inhibit human TF/FVIIa. Immortalised pig bone marrow mesenchymal cells (BMSCs) transfected with human TFPI showed a prolonged recalcification time in vitro and in a rodent model. Conclusion: Recipient TF is relevant to dysregulated coagulation after xenotransplantation. Kunitz domain 1 plays the most important role in species incompatibility between pig TFPI and human TF, and clotting can be inhibited by human TFPI-transfected pig BMSCs. Our study shows a possible way to resolve the incompatibility of pig TFPI.

A review of pig liver xenotransplantation: Current problems and recent progress

“…20 Red blood cell and platelet debris were found in the hepatic sinusoidal endothelial cells (LSECs) and hepatocytes. 20 Two years later, our group 14 Confusingly, recipients in the studies by Yeh's group developed severe thrombocytopenia (compared to our group) that was even lower than that following conventional orthotopic liver transplantation (possibly associated with different NHP species, surgical procedures, and therapeutic strategies). Notably, relatively stable blood counts, without any clinical signs of bleeding, were recorded.…”

Pig Liver Xenotransplantation

“…( A is reproduced with permission from Ekser et al Transplantation 2010;90:483–493; B is reproduced with permission from Ji H, et al 28 …”

“…At necropsy some days later, some livers showed macroscopic changes consistent with cholestasis, except for some patchy dark areas which microscopically showed hemorrhagic necrosis, platelet-fibrin thrombi, monocyte/macrophage margination, and vascular endothelial cell hypertrophy (Figure 2C). 12,28,36–38 No cell infiltration was seen. Confocal microscopy has confirmed tissue factor expression on platelets, and platelet and platelet/leukocyte aggregates in liver sinusoids.…”

“…28 Their innovative surgical technique is illustrated in Figure 4. Although it requires native splenectomy, the Xi’an technique has the advantage that none of the native liver needs to be excised as the graft fits comfortably into the splenic fossa.…”

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

“…In addition, molecular incompatibilities of some proteins such as TFPI or h-DAF are still controversial. [43][44][45] In the present study, we systematically analyzed the amino acid sequence of proteins involved in platelet-EC interaction between humans (Homo sapiens) and pigs (Sus scrofa) by sequence alignment, aiming to explore the potential target proteins responsible for dysregulated platelet activity.…”