Ex Vivo Perfusion And In Vivo Xenotransplantation Of Pig Lungs With Humanized Von Willebrand Factor Demonstrate Reduced Platelet Sequestration

PurposeIn normal hemostasis, activated von Willebrand factor (vWF) stimulates platelet adhesion and aggregation, but porcine vWF (pvWF) is “always active” when exposed to human blood. We hypothesized that humanized vWF (h*pvWF) would reduce this nonphysiologic platelet adhesion in lung xenotransplantation models.MethodsGTKO.hCD46.h*pvWF pig lungs were perfused with human blood ex vivo and compared to reference GTKO.hCD46 lungs. Anti-GPIb Fab, 1-BIA (thromboxane synthase inhibitor), and antihistamines were used in all perfusions. One lung from each pair (n=5 h*pvWF, n=5 ref) also received integrin (H52/IB4) and selectin (GMI-1271, PSGL-1) blockade. Lungs surviving 8 hrs of perfusion were terminated electively. Platelets were measured by flow cytometry. h*pvWF lungs were also transplanted into baboon recipients (n=3 h*pvWF, n=10 ref), with platelets measured by CBC.ResultsAll ex vivo groups had similar survival, with most electively terminated. Pulmonary vascular resistance was very low and constant in all groups. Platelet sequestration was significantly reduced and delayed in h*pvWF lungs compared to pvWF (p<0.01), not further modulated by selectin and integrin blockade (Fig.1). No significant differences were seen in BTG (platelet activation) levels, F1+2 (thrombosis) levels, or neutrophil counts. h*pvWF lung baboon recipients trended toward longer survival (p=0.07) with attenuated thrombocytopenia compared to reference lungs (p<0.01)(Fig.2).ConclusionWhen perfused with human blood, h*pvWF lungs had reduced sequestration of platelets compared to pvWF lungs, with similar reductions in lung xenotransplant recipients. “Humanization” of porcine vWF attenuates the nonphysiologic platelet adhesion and aggregation associated with xenograft rejection. Further study will focus on Fc-receptors, adenosine and coagulation pathway dysregulation, and activated neutrophils through additional drugs and genetic modifications. In normal hemostasis, activated von Willebrand factor (vWF) stimulates platelet adhesion and aggregation, but porcine vWF (pvWF) is “always active” when exposed to human blood. We hypothesized that humanized vWF (h*pvWF) would reduce this nonphysiologic platelet adhesion in lung xenotransplantation models. GTKO.hCD46.h*pvWF pig lungs were perfused with human blood ex vivo and compared to reference GTKO.hCD46 lungs. Anti-GPIb Fab, 1-BIA (thromboxane synthase inhibitor), and antihistamines were used in all perfusions. One lung from each pair (n=5 h*pvWF, n=5 ref) also received integrin (H52/IB4) and selectin (GMI-1271, PSGL-1) blockade. Lungs surviving 8 hrs of perfusion were terminated electively. Platelets were measured by flow cytometry. h*pvWF lungs were also transplanted into baboon recipients (n=3 h*pvWF, n=10 ref), with platelets measured by CBC. All ex vivo groups had similar survival, with most electively terminated. Pulmonary vascular resistance was very low and constant in all groups. Platelet sequestration was significantly reduced and delayed in h*pvWF lungs compared to pvWF (p<0.01), not further modulated by selectin and integrin blockade (Fig.1). No significant differences were seen in BTG (platelet activation) levels, F1+2 (thrombosis) levels, or neutrophil counts. h*pvWF lung baboon recipients trended toward longer survival (p=0.07) with attenuated thrombocytopenia compared to reference lungs (p<0.01)(Fig.2). When perfused with human blood, h*pvWF lungs had reduced sequestration of platelets compared to pvWF lungs, with similar reductions in lung xenotransplant recipients. “Humanization” of porcine vWF attenuates the nonphysiologic platelet adhesion and aggregation associated with xenograft rejection. Further study will focus on Fc-receptors, adenosine and coagulation pathway dysregulation, and activated neutrophils through additional drugs and genetic modifications.