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Role of recombinant hirudin in a pig-to-human lung transplantation model
Role of Recombinant Hirudin in a Pig-to-Human Lung Transplantation Model H. Schelzig, A. Vogel, C. Krischer, F. Simon, and D. Abendroth
X
ENOTRANSPLANTATION may be an alternative to allogeneic organ transplantation, particularly for patients requiring lung transplantation, an area where the organ shortage is a major problem. Despite the progress in genetic engineering of nonprimate species as future source for solid organ transplantation, the mechanisms of severe acute graft dysfunction are not fully understood. The major mechanisms responsible for hyperacute rejection (HAR) include binding of antispecies antibodies, complement activation and antibody-independent activation of endothelial cell/leucocyte interactions.1– 4 Although some of the major factors mediating antibody- or complement-induced acute lung damage have been nearly eliminated, clear signs of HAR occur.5,6 One experimental approach to understand the cellular and subcellular mechanisms of HAR is isolated ex vivo hemoperfusion of whole organs. Our group developed a system for isolated ex vivo hemoperfusion of kidneys to detect early changes in genetically modified organs perfused with human whole blood.7 Based on this model a device was developed for ventilated ex vivo lung-perfusion.8 Since this system contains artificial surfaces—for example, platinum coated tubes and deoxygenator—anticoagulation with heparin is required as has been true in nearly every system used for this purpose to date. Jurd reported that porcine endothelial cells were activated by prothrombin in absence of xenoreactive antibodies and complement and that Hirudin, a potent inhibitor of thrombin, blocked this activation in vitro.9 The original source of Hirudin is the saliva of Hirudo medicinalis (leech). Since 1986 hirudin has been available as a recombinant molecule. It inhibits fibrinogen, factor V, factor VIII, and factor XIII of the coagulation cascade, leading to attenuated platelet aggregation, fibroblast growth and smooth muscle cell activation. The threshold for the AT III-independent anticoagulatory effect is 5–10 g hirudin per mL human blood. No allergic reactions to this preparation have been reported in the literature. Since the end-point of HAR and vascular rejection is blood coagulation, xenotransplantation research needs to investigate these mechanisms and their regulation. In this study we investigated the influence of recombinant hirudin (Lepuridin, Refludan, Hoechst Marion Roussel, Germany) in a pig-to-human lung-transplant model. 0041-1345/02/$–see front matter PII S0041-1345(02)03288-8 2384
MATERIAL AND METHODS The lung perfusion system consists of a heated (37 °C) organ chamber in which the excised heart-lung block is mounted through cannulation of pulmonary artery and the left atrium. The right main stem bronchus, pulmonary artery and pulmonary veins are ligated. The lungs are ventilated (Servo B, Siemens, Sweden) with room air. Blood deoxygenation was achieved by N2 and CO2. Blood flow which was maintained by rollerpumps (IPS, Ismatec SA, Zurich, Switzerland) contained an hematocrit of the perfusate diluted by PBE to 25%. The pig lungs were harvested and cooled with Celsior preservation solution to 4 °C. Perfusion was performed using fresh heparinized human blood (n ⫽ 6) or human blood supplemented with lepuridin (n ⫽ 6). Blood gas analyses (BGA) and pulmonary arterial pressure (PAP) were monitored. sP-selectin and thromboxane B2 were measured by ELISA (Elisa MedSystems Diagnostics, Austria). Tissue samples were taken to evaluated microscopic changes.
RESULTS
When pig-lungs were perfused with heparinized human blood they were destroyed within the first hour of perfusion [pO2 after 1 minute 61 (⫾7) mm Hg; 5 minutes, 85 (⫾4) mm Hg; 10 minutes 96 (⫾7) mm Hg; 15 minutes 100 (⫾8) mm Hg; 30 minutes, 103 (⫾6) mm Hg; 45 minutes 70 (⫾9) mm Hg; 60 minutes, 56 (⫾6) mm Hg]. When 5 g lepuridin per mL was added to human blood, effective gas exchange was stabilized [pO2 after 1 minute, 59.6 (⫾7.5) mm Hg; 5 minutes, 79 (⫾8.4) mm Hg; 10 minutes, 134.6 (⫾10) mm Hg; 15 minutes, 133.3 (⫾9.3) mm Hg; 30 minutes, 110.3 (⫾13) mm Hg; 45 minutes, 102.3 (⫾9) mm Hg; 60 minutes, 108.6 (⫾12) mm Hg; 120 minutes, 107.6 (⫾6.5) mm Hg; 180 minutes 86 (⫾13) mm Hg (P ⬍ .05 human blood plus heparin vs human blood plus lepuridin after 60 minutes)]. Increased pulmonary arterial pressure (PAP) associated with HAR was observed after 45 minutes of perfusion of pig-lungs with heparinized human blood. [PAP after 15 minutes, 30 (⫾9) mm Hg; 30 minutes, 33 (⫾7) mm Hg; 45 minutes, 50 (⫾13) mm Hg; 60 minutes 110 (⫾16) mm Hg]. From the Department of Thoracic and Vascular Surgery, University of Ulm, Ulm, Germany. Address reprint requests to Dr. H. Schelzig, Dpt. of Thoracic and Vascular Surgery, University of Ulm, Steinho¨velstrasse 9, 89075 Ulm, Germany. © 2002 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 34, 2384 –2386 (2002)
ROLE OF RECOMBINANT HIRUDIN
2385
Fig 1. Heparin vs lepuridin: Pulmonalarterial pressure (PAP) over time, general thrombosis of the vascular bed in the heparin group.
Lepuridin attenuated the effects of HAR [PAP after 15 minutes, 33 (⫾9) mm Hg; 30 minutes, 35 (⫾5) mm Hg; 45 minutes, 37 (⫾4) mm Hg; 60 minutes, 36 (⫾5) mm Hg; 120 minutes, 35 (⫾5) mm Hg; 180 minutes, 36 (⫾3) mm Hg] (P ⬍ .05 human blood plus heparin vs human blood plus lepuridin after 60 min). P-selectin showed a constant increase in the heparin group [1 minute after perfusion, 97 (⫾19) ng/mL; 5 minutes, 110 (⫾14) ng/mL; 10 minutes, 134 (⫾17) ng/mL; 15 minutes, 127 (⫾21) ng/mL; 30 minutes, 144 (⫾22) ng/mL; 45 minutes, 136 (⫾9) ng/mL] but was stable in the lepuridin-group [1 minute after perfusion, 101 (⫾9) ng/mL; 5 minutes, 85.9 (⫾15) ng/mL; 10 minutes, 86.2 (⫾13) ng/mL; 15 minutes, 71.2 (⫾23) ng/mL; 30 minutes, 72.9 (⫾17) ng/mL; 45 minutes, 81.5 (⫾13) ng/mL; 60 minutes, 89.4 (⫾22) ng/mL; 120 minutes, 78.4 (⫾14) ng/mL; 180 minutes, 78 (⫾17) ng/mL (P ⬍ .05 human blood plus heparin vs human blood plus lepuridin after 45 minutes)]. Thromboxane B2 showed a consistent increase in the lepuridin-group [1 minute after perfusion, 528 (⫾117) pg/mL; 5 minutes, 591.3 (⫾185.5) pg/mL; 10 minutes, 633.2 (⫾39.5) ng/mL; 15 minutes, 941.13 (⫾75.6) ng/mL; 30 minutes, 973.8 (⫾89.8) ng/mL; 45 minutes, 1011.9 (⫾130.9) ng/mL]. Unfortunately investigation was not performed in the heparin-group due to lack of material. While HE stainings of lungs perfused with heparinized human blood showed destruction of the blood-air barrier and marked signs of hemorrhage and edema, the lepuridin group revealed intact architecture of parenchyma. Neutrophil infiltration was observed, indicating early reactions of the human cellular system to the organ.
Immunohistological stainings indicated widespread human IgG and IgM deposition on pig-lung endothelium as well as activation of the classical pathway complement (C4). In addition the non-treated group showed common-pathway activation and deposition of membrane attack complex (C3a and C5b-9). Although activation was observed, there was markedly less destruction among grafts in the lepuridin group. DISCUSSION
A potential solution to the shortage of human organs for transplantation lies in the use of animal organs; the pig is the most likely donor. Within minutes to hours after perfusion of porcine organs with human blood, HAR occurs as characterized by intravascular thrombosis. The pathogenesis of the reaction has been attributed to xenoreactive natural antibodies that bind to epitopes on porcine endothelium thereby activating complement with subsequent activation of haemostasis. To investigate the pathophysiology of HAR in pig-lung grafts, we established an ex-vivo-hemoperfusion system.8 Perfusion of the pig lung with heparinized whole human blood produced HAR with thrombosis and loss of function. Jurd found that human prothrombin was activated by porcine but not human endothelium in the absence of xenoreactive antibodies and complement, suggesting that this phenomenon may pose a nonimmunological obstacle to pig-to-human xenotransplantation.9 Thrombin is involved in the inflammatory process by inducing neutrophil infiltration, endothelial P-selectin expression and release of platelet activating factor. Thrombin also functions as a long
2386
acting proinflammatory agent leading to endothelial cell synthesis of the mediators required for neutrophil activation and extravasation during inflammation.10 When human blood was anticoagulated with recombinant hirudin, thereby blocking thrombin, the signs of HAR were moderate and there was prolonged gas-exchange. Platelet activation is detected by the expression of Pselectin on platelet surfaces.11 As thrombotic vascular occlusion occurs within the first hour of reperfusion using heparinized human blood, platelet activation as shown by P-selectin, increases consistently during the first 45 minutes. Reduced activation of P-selectin was demonstrated among the hirudin-group. The expression of vasoactive components like thromboxan B2 may be activated during organ preservation, explantation and cooling,12 ie, the ischemia/reperfusioninjury. We found that inhibition of thrombin by hirudin does not have any effect on the increase in thromboxane B2. In summary, the inert, selective thrombin-inhibitor and hirudin-analog lepuridin reduces activation of host-platelets by pig-endothelium thereby improving the initial function of transplant. There was no influence on the liberation of thromboxane B2, a nonspecific mediator of inflamma-
SCHELZIG, VOGEL, KRISCHER ET AL
tion. We conclude that hirudin could serve as future adjunct for xenogeneic lung transplantation. REFERENCES 1. Hammer C: Transplant Proc 19:4443, 1987 2. Marks R, Todd R, Ward P: Nature 339:314, 1989 3. Baldwin WM, Pruitt SK, Brauer RB, et al: Transplantation 59:797, 1995 4. Sheikh S, Parhar R, Kwaasi A, et al: Transplantation 70:917, 2000 5. Pierson RN, Kasper-Ko ¨nig W, Tew DN, et al: Transplantation 63:594, 1997 6. Kaplon RJ, Platt JL, Kwiatkowski PA: Transplantation 59: 410, 1995 7. Storck M, Abendroth D, Prestel R, et al: Transplantation 63:304, 1997 8. Schelzig H, Simon F, Brem W, et al: Europ Surg Res 31:162, 1999 9. Jurd KM, Gibbs RV, Hunt BJ: Blood-Coagul-Fibrinolysis 7:336, 1996 10. Kaplanski G, Fabrigoule M, Boulay V, et al: J Immunol 158:5435, 1997 11. Schneider DJ, Tracy PB, Mann KG, et al: Circulation 96:2877, 1997 12. Shachleton CR, Ettinger SL, Mc Longlein MG, et al: Transplantation 49:641, 1990
X
ENOTRANSPLANTATION may be an alternative to allogeneic organ transplantation, particularly for patients requiring lung transplantation, an area where the organ shortage is a major problem. Despite the progress in genetic engineering of nonprimate species as future source for solid organ transplantation, the mechanisms of severe acute graft dysfunction are not fully understood. The major mechanisms responsible for hyperacute rejection (HAR) include binding of antispecies antibodies, complement activation and antibody-independent activation of endothelial cell/leucocyte interactions.1– 4 Although some of the major factors mediating antibody- or complement-induced acute lung damage have been nearly eliminated, clear signs of HAR occur.5,6 One experimental approach to understand the cellular and subcellular mechanisms of HAR is isolated ex vivo hemoperfusion of whole organs. Our group developed a system for isolated ex vivo hemoperfusion of kidneys to detect early changes in genetically modified organs perfused with human whole blood.7 Based on this model a device was developed for ventilated ex vivo lung-perfusion.8 Since this system contains artificial surfaces—for example, platinum coated tubes and deoxygenator—anticoagulation with heparin is required as has been true in nearly every system used for this purpose to date. Jurd reported that porcine endothelial cells were activated by prothrombin in absence of xenoreactive antibodies and complement and that Hirudin, a potent inhibitor of thrombin, blocked this activation in vitro.9 The original source of Hirudin is the saliva of Hirudo medicinalis (leech). Since 1986 hirudin has been available as a recombinant molecule. It inhibits fibrinogen, factor V, factor VIII, and factor XIII of the coagulation cascade, leading to attenuated platelet aggregation, fibroblast growth and smooth muscle cell activation. The threshold for the AT III-independent anticoagulatory effect is 5–10 g hirudin per mL human blood. No allergic reactions to this preparation have been reported in the literature. Since the end-point of HAR and vascular rejection is blood coagulation, xenotransplantation research needs to investigate these mechanisms and their regulation. In this study we investigated the influence of recombinant hirudin (Lepuridin, Refludan, Hoechst Marion Roussel, Germany) in a pig-to-human lung-transplant model. 0041-1345/02/$–see front matter PII S0041-1345(02)03288-8 2384
MATERIAL AND METHODS The lung perfusion system consists of a heated (37 °C) organ chamber in which the excised heart-lung block is mounted through cannulation of pulmonary artery and the left atrium. The right main stem bronchus, pulmonary artery and pulmonary veins are ligated. The lungs are ventilated (Servo B, Siemens, Sweden) with room air. Blood deoxygenation was achieved by N2 and CO2. Blood flow which was maintained by rollerpumps (IPS, Ismatec SA, Zurich, Switzerland) contained an hematocrit of the perfusate diluted by PBE to 25%. The pig lungs were harvested and cooled with Celsior preservation solution to 4 °C. Perfusion was performed using fresh heparinized human blood (n ⫽ 6) or human blood supplemented with lepuridin (n ⫽ 6). Blood gas analyses (BGA) and pulmonary arterial pressure (PAP) were monitored. sP-selectin and thromboxane B2 were measured by ELISA (Elisa MedSystems Diagnostics, Austria). Tissue samples were taken to evaluated microscopic changes.
RESULTS
When pig-lungs were perfused with heparinized human blood they were destroyed within the first hour of perfusion [pO2 after 1 minute 61 (⫾7) mm Hg; 5 minutes, 85 (⫾4) mm Hg; 10 minutes 96 (⫾7) mm Hg; 15 minutes 100 (⫾8) mm Hg; 30 minutes, 103 (⫾6) mm Hg; 45 minutes 70 (⫾9) mm Hg; 60 minutes, 56 (⫾6) mm Hg]. When 5 g lepuridin per mL was added to human blood, effective gas exchange was stabilized [pO2 after 1 minute, 59.6 (⫾7.5) mm Hg; 5 minutes, 79 (⫾8.4) mm Hg; 10 minutes, 134.6 (⫾10) mm Hg; 15 minutes, 133.3 (⫾9.3) mm Hg; 30 minutes, 110.3 (⫾13) mm Hg; 45 minutes, 102.3 (⫾9) mm Hg; 60 minutes, 108.6 (⫾12) mm Hg; 120 minutes, 107.6 (⫾6.5) mm Hg; 180 minutes 86 (⫾13) mm Hg (P ⬍ .05 human blood plus heparin vs human blood plus lepuridin after 60 minutes)]. Increased pulmonary arterial pressure (PAP) associated with HAR was observed after 45 minutes of perfusion of pig-lungs with heparinized human blood. [PAP after 15 minutes, 30 (⫾9) mm Hg; 30 minutes, 33 (⫾7) mm Hg; 45 minutes, 50 (⫾13) mm Hg; 60 minutes 110 (⫾16) mm Hg]. From the Department of Thoracic and Vascular Surgery, University of Ulm, Ulm, Germany. Address reprint requests to Dr. H. Schelzig, Dpt. of Thoracic and Vascular Surgery, University of Ulm, Steinho¨velstrasse 9, 89075 Ulm, Germany. © 2002 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 34, 2384 –2386 (2002)
ROLE OF RECOMBINANT HIRUDIN
2385
Fig 1. Heparin vs lepuridin: Pulmonalarterial pressure (PAP) over time, general thrombosis of the vascular bed in the heparin group.
Lepuridin attenuated the effects of HAR [PAP after 15 minutes, 33 (⫾9) mm Hg; 30 minutes, 35 (⫾5) mm Hg; 45 minutes, 37 (⫾4) mm Hg; 60 minutes, 36 (⫾5) mm Hg; 120 minutes, 35 (⫾5) mm Hg; 180 minutes, 36 (⫾3) mm Hg] (P ⬍ .05 human blood plus heparin vs human blood plus lepuridin after 60 min). P-selectin showed a constant increase in the heparin group [1 minute after perfusion, 97 (⫾19) ng/mL; 5 minutes, 110 (⫾14) ng/mL; 10 minutes, 134 (⫾17) ng/mL; 15 minutes, 127 (⫾21) ng/mL; 30 minutes, 144 (⫾22) ng/mL; 45 minutes, 136 (⫾9) ng/mL] but was stable in the lepuridin-group [1 minute after perfusion, 101 (⫾9) ng/mL; 5 minutes, 85.9 (⫾15) ng/mL; 10 minutes, 86.2 (⫾13) ng/mL; 15 minutes, 71.2 (⫾23) ng/mL; 30 minutes, 72.9 (⫾17) ng/mL; 45 minutes, 81.5 (⫾13) ng/mL; 60 minutes, 89.4 (⫾22) ng/mL; 120 minutes, 78.4 (⫾14) ng/mL; 180 minutes, 78 (⫾17) ng/mL (P ⬍ .05 human blood plus heparin vs human blood plus lepuridin after 45 minutes)]. Thromboxane B2 showed a consistent increase in the lepuridin-group [1 minute after perfusion, 528 (⫾117) pg/mL; 5 minutes, 591.3 (⫾185.5) pg/mL; 10 minutes, 633.2 (⫾39.5) ng/mL; 15 minutes, 941.13 (⫾75.6) ng/mL; 30 minutes, 973.8 (⫾89.8) ng/mL; 45 minutes, 1011.9 (⫾130.9) ng/mL]. Unfortunately investigation was not performed in the heparin-group due to lack of material. While HE stainings of lungs perfused with heparinized human blood showed destruction of the blood-air barrier and marked signs of hemorrhage and edema, the lepuridin group revealed intact architecture of parenchyma. Neutrophil infiltration was observed, indicating early reactions of the human cellular system to the organ.
Immunohistological stainings indicated widespread human IgG and IgM deposition on pig-lung endothelium as well as activation of the classical pathway complement (C4). In addition the non-treated group showed common-pathway activation and deposition of membrane attack complex (C3a and C5b-9). Although activation was observed, there was markedly less destruction among grafts in the lepuridin group. DISCUSSION
A potential solution to the shortage of human organs for transplantation lies in the use of animal organs; the pig is the most likely donor. Within minutes to hours after perfusion of porcine organs with human blood, HAR occurs as characterized by intravascular thrombosis. The pathogenesis of the reaction has been attributed to xenoreactive natural antibodies that bind to epitopes on porcine endothelium thereby activating complement with subsequent activation of haemostasis. To investigate the pathophysiology of HAR in pig-lung grafts, we established an ex-vivo-hemoperfusion system.8 Perfusion of the pig lung with heparinized whole human blood produced HAR with thrombosis and loss of function. Jurd found that human prothrombin was activated by porcine but not human endothelium in the absence of xenoreactive antibodies and complement, suggesting that this phenomenon may pose a nonimmunological obstacle to pig-to-human xenotransplantation.9 Thrombin is involved in the inflammatory process by inducing neutrophil infiltration, endothelial P-selectin expression and release of platelet activating factor. Thrombin also functions as a long
2386
acting proinflammatory agent leading to endothelial cell synthesis of the mediators required for neutrophil activation and extravasation during inflammation.10 When human blood was anticoagulated with recombinant hirudin, thereby blocking thrombin, the signs of HAR were moderate and there was prolonged gas-exchange. Platelet activation is detected by the expression of Pselectin on platelet surfaces.11 As thrombotic vascular occlusion occurs within the first hour of reperfusion using heparinized human blood, platelet activation as shown by P-selectin, increases consistently during the first 45 minutes. Reduced activation of P-selectin was demonstrated among the hirudin-group. The expression of vasoactive components like thromboxan B2 may be activated during organ preservation, explantation and cooling,12 ie, the ischemia/reperfusioninjury. We found that inhibition of thrombin by hirudin does not have any effect on the increase in thromboxane B2. In summary, the inert, selective thrombin-inhibitor and hirudin-analog lepuridin reduces activation of host-platelets by pig-endothelium thereby improving the initial function of transplant. There was no influence on the liberation of thromboxane B2, a nonspecific mediator of inflamma-
SCHELZIG, VOGEL, KRISCHER ET AL
tion. We conclude that hirudin could serve as future adjunct for xenogeneic lung transplantation. REFERENCES 1. Hammer C: Transplant Proc 19:4443, 1987 2. Marks R, Todd R, Ward P: Nature 339:314, 1989 3. Baldwin WM, Pruitt SK, Brauer RB, et al: Transplantation 59:797, 1995 4. Sheikh S, Parhar R, Kwaasi A, et al: Transplantation 70:917, 2000 5. Pierson RN, Kasper-Ko ¨nig W, Tew DN, et al: Transplantation 63:594, 1997 6. Kaplon RJ, Platt JL, Kwiatkowski PA: Transplantation 59: 410, 1995 7. Storck M, Abendroth D, Prestel R, et al: Transplantation 63:304, 1997 8. Schelzig H, Simon F, Brem W, et al: Europ Surg Res 31:162, 1999 9. Jurd KM, Gibbs RV, Hunt BJ: Blood-Coagul-Fibrinolysis 7:336, 1996 10. Kaplanski G, Fabrigoule M, Boulay V, et al: J Immunol 158:5435, 1997 11. Schneider DJ, Tracy PB, Mann KG, et al: Circulation 96:2877, 1997 12. Shachleton CR, Ettinger SL, Mc Longlein MG, et al: Transplantation 49:641, 1990