The long-term survival of hearts and kidneys from a1,3-galactosyltransferase gene knockout (GT-KO) pigs transplanted into nonhuman primates is currently limited by a thrombotic microangiopathy (TM), even when the T cell and elicited antibody responses are largely suppressed. We shall explore the role of dysregulated coagulation in the pathogenesis of TM.
Aim 1 : To investigate in vitro the mechanisms underlying the development of the TM in pig-to-baboon studies. Several hypotheses will be tested using GT-KO pig endothelial cells (ECs) exposed to primate blood constituents, with expression of tissue factor (TF) defining the activated phenotype: -(I) exposure of porcine EC to fresh primate sera of blood type AB leads to the development of a procoagulant phenotype by the EC; (2) exposure of porcine EC by primate platelets, monocytes, or both, in close proximity is sufficient for the development of a procoagulant phenotype by the EC; (3) removal of anti-nonGal antibody and interruption of both thrombin and platelet interactions are necessary and sufficient to prevent activation of GT-KO ECs; and (4) in vitro, expression of hTFPI on porcine EC will inhibit coagulation initiated by TF expressed on either activated EC, monocytes, or platelets, and facilitate protection from antibody and platelet adhesive interactions.
Aim 2 : To investigate the effects of pharmacologic agents that inhibit thrombin formation or platelet aggregation on the development of TM after GT-KO pig-to-baboon heart and kidney Tx. We shall carry out GT-KO pig-to-baboon kidney or heart Tx, using a regimen that has allowed survival of GT-KO pig hearts for 58 to 179 days, but in which TM developed. We shall add agents that are known to inhibit (1) thrombin formation (antithrombin, melagatran), or (2) platelet aggregation (aspirin, eptifibatide), to assess their effect on the development of TM. Pathway-specific parameters of hemostasis and coagulation will be measured at regular intervals.
Aim 3 : To assess the effect of the Tx of organs from GT-KO pigs transgenic for human tissue factor pathway inhibitor (hTFPI) on the development of TM. A recent study in rodents suggests that fibrin deposition, platelet aggregation, and related dysregulated coagulation may not be the secondary response hitherto believed, but that prevention of the development of local fibrin deposition, platelet aggregation, and coagulation in the xenograft may play a primary role in preventing acute vascular rejection/TM. This hypothesis will be examined in the pig-to-baboon model using GT-KO pigs that are also transgenic for hTFPI. If TM is not completely prevented, agents demonstrated in Aim 2 to inhibit its development will be added to the regimen. It is anticipated that these approaches will abrogate the development of TM and allow long-term survival of pig organs in baboons.
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