Mechanisms of thrombotic microangiopathy following xenogeneic hematopoietic progenitor cell transplantation

Introduction. Attempts to induce tolerance though mixed hematopoietic chime rism in the discordant pig-to-baboon xenotransplantation model are sometime s complicated by a potentially fatal thrombotic microangiopathy in the reci pient baboons. This state develops immediately after the infusion of porcin e mobilized peripheral blood leukocytes, containing progenitor cells (PBPC) , In our study, we examined the interaction of infused porcine PBPC with re cipient platelets in vivo in baboons and investigated the underlying mechan isms using an in vitro model. Methods. Two naive baboons and six baboons preconditioned with irradiation and immunosuppression that received porcine PBPC were evaluated in vivo. Th e interaction of porcine and baboon PBPC with baboon platelets was investig ated by an in vitro platelet aggregation assay. Fresh and cryopreserved PBP C were evaluated as well as PBPC obtained from growth-factor mobilized and unmobilized pigs. Furthermore, cellular subsets of PBPC were assessed for p otential to induce platelet aggregation. Immunohistochemical staining was p erformed on platelet-leukocyte aggregates and potential inhibition of aggre gation with anti-P-selectin and anti-CD154 mAbs, or eptifibatide (a GPIIb/I IIa receptor antagonist), was tested. Results. All baboons that received porcine PBPC rapidly developed marked th rombocytopenia (<20,000/<mu>L), elevated serum lactate dehydrogenase (>1,50 0U/Liter), schistocytosis, and platelet aggregates on blood smear. Three ba boons died (two untreated and one preconditioned), and substantive platelet aggregates containing porcine leukocytes were observed in the microvascula ture of lungs and kidneys, In vitro, porcine, but not baboon, PBPC induced aggregation of baboon platelets in a dose-dependent manner. Immunohistologi cal examination of these aggregates confirmed the incorporation of porcine leukocytes, Cryopreserved PBPC caused less aggregation than fresh PBPC, and growth-factor-mobilized PBPC induced less aggregation than unmobilized PBP C, Aggregation was fully abrogated by the addition of eptifibatide, and mod ulated by anti-P-selectin and anti-CD154 monoclonal antibodies that recogni ze adhesion receptors on activated platelets. Purified fractions (granulocy tes, CD2+, and CD- cells) of porcine PBPC did not initiate aggregation, whe reas addition of exogenous porcine PBPC membranes (erythrocytes, dead cells , and/or platelets) to the purified fractions exacerbated the aggregation r esponse, Conclusions, These data indicate that porcine PBPC mediate aggregation of b aboon platelets, This process likely contributes to the thrombotic microang iopathy observed after PBPC transplantation in the pig-to-baboon model. Ept ifibatide can fully abrogate platelet aggregation induced by porcine PBPC i n vitro. Purification of the progenitor cells from porcine PBPC and/or trea tment of baboons with eptifibatide may be beneficial.