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Progress towards overcoming coagulopathy and hemostatic dysfunction associated with xenotransplantation
International Journal of Surgery 23 (2015) 296e300
Contents lists available at ScienceDirect
International Journal of Surgery journal homepage: www.journal-surgery.net
Review
Progress towards overcoming coagulopathy and hemostatic dysfunction associated with xenotransplantation Peter J. Cowan a, b, *, Simon C. Robson c a
Immunology Research Centre, St Vincent's Hospital Melbourne, Melbourne, Victoria, Australia Department of Medicine, University of Melbourne, Melbourne, Victoria, Australia c Transplant Institute and Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA b
h i g h l i g h t s Dysregulated coagulation is a major barrier to successful xenotransplantation. Microvascular thrombosis is frequently observed in rejected xenografts. Consumptive coagulopathy can develop in recipients. Likely causes include antibodies and cross-species molecular incompatibilities. Genetic modification of the donor pig may provide the solution to this problem.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 July 2015 Received in revised form 16 July 2015 Accepted 17 July 2015 Available online 26 July 2015
Dysregulation of coagulation and disordered hemostasis are frequent complications in the pig-to-nonhuman primate preclinical xenotransplantation model. The most extreme manifestations are the systemic development of a life-threatening consumptive coagulopathy, characterized by thrombocytopenia and bleeding, which is balanced at the opposite extreme by local complications of graft loss due to thrombotic microangiopathy. The contributing mechanisms include inflammation, vascular injury, heightened innate, humoral and cellular immune responses, and molecular incompatibilities affecting the regulation of coagulation. There also appear to be organ-specific factors that have been linked to vascular heterogeneity. As examples, liver xenografts rapidly induce thrombocytopenia by sequestering human/primate platelets; renal xenografts cause a broader coagulopathy, linked in some cases to reactivation of porcine CMV, whereas cardiac xenografts often succumb to microvascular thrombosis without associated systemic coagulopathy but with local perturbations in fibrinolysis. Overcoming coagulation dysfunction will require a combination of genetic and pharmacological strategies. Deletion of the xenoantigen aGal, transgenic expression of human complement regulatory proteins, and refinement of immunosuppression to blunt the antibody response have all had some impact, without providing a complete solution. More recently, the addition of approaches specifically targeted at coagulation have produced promising results. As an example, heterotopic cardiac xenografts from donors expressing human thrombomodulin have survived for more than a year in immunosuppressed baboons, with no evidence of thrombotic microangiopathy or coagulopathy. © 2015 IJS Publishing Group Limited. Published by Elsevier Ltd. All rights reserved.
Keywords: Xenotransplantation Coagulation Inflammation Coagulopathy Thrombocytopenia
Contents 1. 2.
Factors underlying dysregulated coagulation in xenograft recipients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297 Strategies to prevent dysregulation of coagulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298 2.1. Genetic modification of the donor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298 2.2. Treatment of the recipient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298
* Corresponding author. Immunology Research Centre, St Vincent's Hospital Melbourne, PO Box 2900, Fitzroy 3065, Victoria, Australia. E-mail address: [email protected] (P.J. Cowan). http://dx.doi.org/10.1016/j.ijsu.2015.07.682 1743-9191/© 2015 IJS Publishing Group Limited. Published by Elsevier Ltd. All rights reserved.
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3.
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Recent insights from preclinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298 3.1. Heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298 3.2. Kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298 3.3. Liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 3.4. Lung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 Ethical approval . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 Author contribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 Conflicts of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 Guarantor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
1. Factors underlying dysregulated coagulation in xenograft recipients Coagulation is a tightly controlled physiological process designed to preserve the integrity of the vascular system [1]. Endothelial injury results in exposure of active tissue factor (TF) to the blood, triggering a molecular cascade that results in the formation of a fibrin-enmeshed platelet plug. Finely tuned regulatory systems, comprising cell surface and circulating components, cooperate to ensure that clotting is localized, proportionate to injury, and resolved at the appropriate time. The key endothelial anticoagulant/antithrombotic proteins include thrombomodulin (TBM), tissue factor pathway inhibitor
(TFPI), endothelial protein C receptor (EPCR) and CD39. Because coagulation and complement activation are interconnected, the complement regulatory proteins (CRPs) CD46, CD55 and CD59 also play a role. Porcine xenografts face a series of challenges that tip the balance towards coagulation and inflammation (Fig. 1). First, human and nonhuman primate (NHP) sera contain significant levels of preexisting anti-pig antibodies [2,3]. The target antigens are predominantly carbohydrate structures on glycoproteins and glycolipids, indicating that these ‘natural’ antibodies arose as a consequence of cross-species differences in glycosylation. While the key specificity is galactose-a1,3-galactose (aGal), antibodies to N-glycolylneuraminic acid (Neu5Gc) are also likely to be important in the pig-to-
Fig. 1. Pathways to dysregulated coagulation and inflammation in solid organ xenotransplantation. Poor control of TF and thrombin by TFPI and TBM, respectively, will potentially drive a pro-inflammatory, procoagulant positive-feedback loop. Genetic strategies designed to disrupt this process are shown in green.
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human setting, because humans (unlike pigs and Old World primates) do not express Neu5Gc. Second, the adaptive immune response to xenografts contains a strong humoral component that is difficult to control. Elicited antibodies promote coagulation by activating porcine endothelial cells and platelets, but may also target protective endothelial surface proteins including EPCR and CRPs [4]. In addition, the development of antibodies to clotting factor VIII in xenograft recipients [5] has potential implications for coagulopathy. Third, xenografts can induce a systemic inflammatory response that may indicate precedent adaptive immunity, which may promote coagulation. Baboons receiving porcine hearts or kidneys exhibit steady rises in levels of the inflammatory marker C-reactive protein before developing consumptive coagulopathy [6]. Some proinflammatory cytokines (notably IL-6) are also increased in this context, even when the xenograft was limited to a small artery patch [6]. This inflammatory milieu is conducive to systemic upregulation or recruitment of recipient TF on platelets and monocytes [7]. Finally, several in vitro studies have identified molecular incompatibilities affecting the capacity of porcine endothelial anticoagulants to regulate human/primate coagulation factors. Pig TBM binds human thrombin but is a poor co-factor for subsequent activation of human protein C [8,9]. Similarly, recombinant pig TFPI binds and neutralizes human factor Xa but is a less efficient inhibitor of human TF/factor VIIa than human TFPI [10], although the latter incompatibility is not observed in all model systems [11]. Furthermore, the tendency for coagulation and thrombosis may be heightened by an abnormal interaction between porcine von Willebrand Factor (vWF) and human glycoprotein Ib (GPIb) [12], which causes the induction of active TF on human platelets incubated with porcine aortic endothelial cells [13]. Thus the exquisite balance between coagulation and thromboregulatory mechanisms is intrinsically disturbed in xenotransplantation: the endogenous defenses of the xenograft, already partly compromised by cross-species differences, are gradually overwhelmed by ongoing activating stimuli and loss of protective factors. 2. Strategies to prevent dysregulation of coagulation 2.1. Genetic modification of the donor Several complementary genetic strategies that have been developed to protect xenografts are pertinent to the regulation of coagulation. The xenograft can be made less ‘visible’ to the recipient immune system by eliminating xenoantigens such as aGal (GGTA1 KO or GTKO) and Neu5Gc (CMAH KO). The capacity of the xenograft to regulate complement activation and inflammation can be increased by expressing protective molecules such as human CRPs, hemeoxygenase-1 and A20. Anticoagulant defenses can be bolstered, with concomitant correction of molecular incompatibilities, by expressing human TBM, TFPI, EPCR and CD39. Expression of procoagulants such as TF can be reduced by siRNAmediated knockdown [14]. For liver xenografts, the aberrant phagocytosis of human platelets can be addressed by deleting the porcine asialoglycoprotein receptor (ASGR) [15]. Where data are available, the impact of these existing modifications on coagulation dysfunction will be discussed organ-byorgan later in this article. Advances in genome editing technology hold the promise of even more precise modifications, such as correction of the incompatibility between pig vWF and human GPIb. 2.2. Treatment of the recipient
in
Many anticoagulants and anti-inflammatories have been tested xenotransplantation models, including heparin, aspirin,
warfarin, clopidogrel, statins, adenosine agonists, apyrases, antithrombin, and activated protein C (reviewed in [16,17]). These agents have been used in combination with immunosuppression and/or genetically modified donors, making it difficult to assess their impact individually, although it does appear that increasing anticoagulant treatment does not fully correct dysregulated coagulation [18,19]. Rather, judicious pharmacological anticoagulation is seen as an adjunct to the other strategies [20]. Anti-inflammatories such as etanercept (soluble TNF receptor), anakinra (IL-1 receptor antagonist) and tocilizumab (anti-IL-6 receptor) hold promise, but have not been widely tested in xenotransplantation to date. 3. Recent insights from preclinical studies Progress in pig-to-NHP xenotransplantation models from 1998 to the end of 2013 has been extensively reviewed [21]. We will therefore restrict our discussion to some of the more recent studies. 3.1. Heart The intractable nature of coagulation and fibrinolytic dysfunction was illustrated several years ago by a pig-to-baboon heterotopic cardiac xenograft study using inbred GTKO donors, a comprehensive immunosuppressive protocol (antithymocyte globulin (ATG), anti-CD154, cobra venom factor, mycophenolate mofetil (MMF), steroids), and anticoagulation with continuous heparin, antithrombin and/or aspirin as an antiplatelet drug [22]. Even this combination was not sufficient to prevent the eventual development of thrombotic microangiopathy, restricting maximum survival to 179 days [22]. Using a similar regimen with outbred GTKO/hCD46 donors and the addition of anti-CD20, maximum survival was extended to 236 days, but microvascular thrombosis was still evident in long-surviving grafts [23]. In contrast, survival exceeding 600 days was achieved by using GTKO/hCD46/hTBM hearts and substituting high-dose anti-CD40 for anti-CD154, and biopsies at 1 year showed minor fibrosis but no thrombotic microangiopathy [24]. The experimental design did not allow definitive conclusions to be drawn on the relative contributions of the genetic modifications and the treatment protocol, which included continuous heparin infusion to maintain activated clotting time at twice baseline. Nevertheless, the results suggested that expression of hTBM was important for controlling coagulation and promoting long-term cardiac xenograft survival. In a separate study, GTKO/hCD46/hTBM hearts were transplanted into two baboons treated with ATG, anti-CD40, belatacept, steroids, low molecular weight heparin, and rapamycin or tacrolimus [25]. The xenografts did not survive as long (maximum survival 130 days) as in the earlier study, possibly due to the absence of anti-CD20. However, the recipients showed no features of dysregulated coagulation, and importantly did not require continuous heparinization. This further suggests the potential benefit of hTBM in cardiac xenotransplantation. 3.2. Kidney Kidney xenografts show a greater tendency to induce consumptive coagulopathy than hearts, which may reflect differential regulation of coagulation and fibrinolysis in renal and cardiac vasculature [26]. Baboons transplanted with GTKO/hCD46 kidneys and treated with ATG, anti-CD154, MMF and heparin all developed coagulopathy by day 16 [7]. This was preceded by the progressive expression of TF on platelets and monocytes. In contrast, GTKO thymokidney xenografts survived up to 83 days in baboons treated with a variety of costimulation blockade-based immunosuppressive
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regimens, with minimal thrombotic microangiopathy and coagulopathy [27]. The many differences between these studies preclude drawing any definitive conclusions about the reason for the disparate outcomes. Disappointingly, mean published survival of GTKO thymokidneys has decreased from 53.2 days in studies prior to mid-2008 to 13.7 days subsequently [28]. The likely cause of this fall has been ascribed to be inadvertent introduction of porcine CMV into the donor herd, leading to viral activation of xenograft endothelial cells and vascular injury [28,29]. Porcine CMV replication was also observed in some, but not all, baboon recipients of GTKO/hCD55/ hCD59/hCD39 renal xenografts [30]. Treatment was with tacrolimus, MMF, steroids, human C1 inhibitor, and cyclophosphamide or bortezomib, with or without plasma exchange. The xenografts were lost by day 15 to acute humoral rejection, with developing signs of consumptive coagulopathy, highlighting the importance of controlling the antibody response. No conclusions could be drawn about the efficacy of hCD39, which was expressed at modest levels in the kidney [30]. A recent small study reported the longest survival of pig-to-NHP kidney xenografts to date [31]. GTKO/hCD55 kidneys were transplanted into rhesus macaques treated with anti-CD4, anti-CD8, MMF, steroids, and anti-CD154 or belatacept. In the anti-CD154 group, xenograft survival was only 6 days in a recipient with very high titers of pre-existing anti-pig IgM and IgG, accompanied by profound thrombocytopenia. Strikingly, however, survival was prolonged to >125 and >133 days in two low-titer animals, both of which exhibited stable platelet counts and no clinical or histological evidence of rejection over that time [31]. This was achieved even in the absence of specific anticoagulation and the presence of CMV in the donor pigs. Even more recently, a single case study has reported renal xenograft survival of 136 days in a baboon recipient [32]. The donor pig was GTKO/hCD46/hCD55/hTBM/hEPCR/hCD39, and the recipient was treated with ATG, anti-CD20, anti-CD40, rapamycin, steroids, etanercept, tocilizumab and low molecular weight heparin. Although these remarkable results remain to be confirmed, it suggests that it is possible to delay or even prevent coagulation dysfunction in renal xenograft recipients. 3.3. Liver Realistically, liver xenografts are unlikely to take the place of allografts, but could provide time to allow native liver recovery/ regeneration or bridging to allotransplantation. However, while genetically modified (GTKO ± hCD46) liver xenografts can support life for at least 9 days in immunosuppressed baboons, recipient survival is cut short by the rapid development of severe thrombocytopenia, coagulopathy, and bleeding resulting in death [33,34]. This occurs even in the apparent absence of rejection, and is believed to stem largely from molecular incompatibility between pig asialoglycoprotein receptor (ASGR) and its human/NHP ligand [35], resulting in platelet sequestration by pig liver sinusoidal endothelial cells and Kupffer cells. In a promising recent development, pigs lacking ASGR have been generated, and their livers showed significantly reduced uptake of human platelets in an ex vivo perfusion model [15]. In a GTKO pig-to-baboon model in which the native liver was retained, thrombocytopenia was still observed, but bleeding complications were reduced (presumably due to the continued production of baboon clotting factors) and one liver xenograft survived for 15 days [36]. Interestingly, the cause of graft failure in this setting appears to have been thrombotic microangiopathy, suggesting that a balance in the coagulation system must be achieved to prevent rejection whilst retaining adequate hemostasis.
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3.4. Lung The lung appears to be the vascularized xenograft most difficult to obtain acceptable results, with maximum survival of less than 5 days in the pig-to-baboon model [37,38]. Inflammation and dysregulated coagulation are key factors in lung xenograft failure. Although the data are limited, it is clear that genetic modifications that have been shown to significantly prolong cardiac and renal xenograft survival (i.e. GTKO ± hCRP expression) have only a marginal impact in pulmonary xenotransplantation [39,40]. This suggests that the lung is more sensitive to xenogeneic injury and/or that additional mechanisms, perhaps involving pulmonary intravascular macrophages [37,39] and platelet sequestration, play a role. A recent paper from the Pierson group analyzed a large body of data obtained using their ex vivo model in which pig lungs are perfused with human blood [41]. An extensive range of genetic modifications has been tested in this model. Although the data did not allow definitive conclusions on the benefit of each individual modification, the authors identified several strong trends: (i) lung xenograft ‘survival’ improved incrementally with each additional transgene; (ii) GTKO, hCD46, hCD55, hEPCR, and hemeoxygenase-1 were associated with protection; and (iii) hTBM and hHLA-E had no apparent effect. While the impact of hCD39 appeared inconclusive, a study using a similar model and different transgenic pig lines has shown that hCD39 expression is protective on the GTKO/hCRP background [42]. The ex vivo model has also provided evidence suggesting that long-term survival of genetically modified lung xenografts may require specific anticoagulant/anti-inflammatory treatment, including targeting of GPIb, GPIIb/IIIa, thromboxane synthesis and histamine signaling (reviewed in [38]). 4. Conclusion Coagulation dysregulation remains a major problem in xenotransplantation, with each organ type presenting a different, albeit overlapping, set of challenges. Any solution must take into account the tight integration of the processes that govern and regulate coagulation and inflammation. Recent progress has been significant, with a limited number of cases indicating the potential for long-term survival of heart (>600 days [24]) and kidney (>126 days [31]) xenografts in the absence of major disturbances to coagulation. Further advances are anticipated with the development of more efficient techniques for precise editing of the porcine genome [43]. We conclude that the right combination of pre-transplant antibody screening, donor genetics, and immunosuppressive and antiinflammatory therapy may be sufficient to prevent coagulation dysfunction in xenotransplantation. Ethical approval Not applicable. Author contribution Peter Cowan wrote the paper with input from Simon Robson. Conflicts of interest None. Funding The National Health and Medical Research Council of Australia.
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The Juvenile Diabetes Research Foundation. The National Institutes of Health. [22]
Guarantor Not applicable.
[23]
Acknowledgments [24]
The work of the authors described in this review was supported by funding from the National Health and Medical Research Council of Australia, the Juvenile Diabetes Research Foundation, and the National Institutes of Health NHLBI and NIDDK.
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Surg. 148 (3) (2014 Sep) 1106e1113. H. Iwase, B. Ekser, V. Satyananda, J. Bhama, H. Hara, M. Ezzelarab, et al., Pig-tobaboon heterotopic heart transplantation - exploratory preliminary experience with pigs transgenic for human thrombomodulin and comparison of three costimulation blockade-based regimens, Xenotransplantation 22 (3) (2015 May) 211e220. C. Knosalla, K. Yazawa, A. Behdad, N. Bodyak, H. Shang, L. Buhler, et al., Renal and cardiac endothelial heterogeneity impact acute vascular rejection in pigto-baboon xenotransplantation, Am. J. Transpl. 9 (5) (2009 May) 1006e1016. A.D. Griesemer, A. Hirakata, A. Shimizu, S. Moran, A. Tena, H. Iwaki, et al., Results of gal-knockout porcine thymokidney xenografts, Am. J. Transpl. 9 (12) (2009 Dec) 2669e2678. K. Yamada, M. Tasaki, M. Sekijima, R.A. Wilkinson, V. Villani, S.G. Moran, et al., Porcine cytomegalovirus infection is associated with early rejection of kidney grafts in a pig to baboon xenotransplantation model, Transplantation 98 (4) (2014 Aug) 411e418. B. Gollackner, N.J. Mueller, S. Houser, I. Qawi, D. Soizic, C. Knosalla, et al., Porcine cytomegalovirus and coagulopathy in pig-to-primate xenotransplantation, Transplantation 75 (11) (2003 Jun) 1841e1847. S. Le Bas-Bernardet, X. Tillou, J. Branchereau, N. Dilek, N. Poirier, M. Chatelais, et al., Bortezomib, C1-inhibitor and plasma exchange do not prolong the survival of multi-transgenic GalT-KO pig kidney xenografts in baboons, Am. J. Transpl. 15 (2) (2015 Feb) 358e370. L. Higginbotham, D. Mathews, C.A. Breeden, M. Song, A.B. Farris 3rd, C.P. Larsen, et al., Pre-transplant antibody screening and anti-CD154 costimulation blockade promote long-term xenograft survival in a pig-toprimate kidney transplant model, Xenotransplantation 22 (3) (2015 May) 221e230. H. Iwase, H. Liu, M. Wijkstrom, H. Zhou, J. Singh, H. Hara, et al., Pig kidney graft survival in a baboon for 136 days: longest life-supporting organ graft survival to date, Xenotransplantation (2015 Jun 29), http://dx.doi.org/10.1111/ xen.12174 [Epub ahead of print]. B. Ekser, G.J. Echeverri, A.C. Hassett, M.H. Yazer, C. Long, M. Meyer, et al., Hepatic function after genetically engineered pig liver transplantation in baboons, Transplantation 90 (5) (2010 Sep) 483e493. K. Kim, C. Schuetz, N. Elias, G.R. Veillette, I. Wamala, M. Varma, et al., Up to 9day survival and control of thrombocytopenia following alpha1,3-galactosyl transferase knockout swine liver xenotransplantation in baboons, Xenotransplantation 19 (4) (2012 JuleAug) 256e264. C. Burlak, L.L. Paris, R.K. Chihara, R.A. Sidner, L.M. Reyes, S.M. Downey, et al., The fate of human platelets perfused through the pig liver: implications for xenotransplantation, Xenotransplantation 17 (5) (2010 SepeOct) 350e361. H. Yeh, Z. Machaidze, I. Wamala, J.W. Fraser, N. Navarro-Alvarez, K. Kim, et al., Increased transfusion-free survival following auxiliary pig liver xenotransplantation, Xenotransplantation 21 (5) (2014 SepeOct) 454e464. E. Cantu, K.R. Balsara, B. Li, C. Lau, S. Gibson, A. Wyse, et al., Prolonged function of macrophage, von Willebrand factor-deficient porcine pulmonary xenografts, Am. J. Transpl. 7 (1) (2007 Jan) 66e75. D.G. Harris, K.J. Quinn, S. Dahi, L. Burdorf, A.M. Azimzadeh, R.N. Pierson 3rd, Lung xenotransplantation: recent progress and current status, Xenotransplantation 21 (6) (2014 NoveDec) 496e506. E.L. Bush, A.S. Barbas, Z.E. Holzknecht, G.W. Byrne, C.G. McGregor, W. Parker, et al., Coagulopathy in alpha-galactosyl transferase knockout pulmonary xenotransplants, Xenotransplantation 18 (1) (2011 JaneFeb) 6e13. B.N. Nguyen, A.M. Azimzadeh, T. Zhang, G. Wu, H.J. Schuurman, D.H. Sachs, et al., Life-supporting function of genetically modified swine lungs in baboons, J. Thorac. Cardiovasc. Surg. 133 (5) (2007 May) 1354e1363. D.G. Harris, K.J. Quinn, B.M. French, E. Schwartz, E. Kang, S. Dahi, et al., Metaanalysis of the independent and cumulative effects of multiple genetic modifications on pig lung xenograft performance during ex vivo perfusion with human blood, Xenotransplantation 22 (2) (2015 MareApr) 102e111. G.P. Westall, B.J. Levvey, E. Salvaris, J. Gooi, S. Marasco, F. Rosenfeldt, et al., Sustained function of genetically modified porcine lungs in an ex vivo model of pulmonary xenotransplantation, J. Heart Lung Transpl. 32 (11) (2013 Nov) 1123e1130. P. Li, J.L. Estrada, C. Burlak, J. Montgomery, J.R. Butler, R.M. Santos, et al., Efficient generation of genetically distinct pigs in a single pregnancy using multiplexed single-guide RNA and carbohydrate selection, Xenotransplantation 22 (1) (2015 JaneFeb) 20e31.
Contents lists available at ScienceDirect
International Journal of Surgery journal homepage: www.journal-surgery.net
Review
Progress towards overcoming coagulopathy and hemostatic dysfunction associated with xenotransplantation Peter J. Cowan a, b, *, Simon C. Robson c a
Immunology Research Centre, St Vincent's Hospital Melbourne, Melbourne, Victoria, Australia Department of Medicine, University of Melbourne, Melbourne, Victoria, Australia c Transplant Institute and Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA b
h i g h l i g h t s Dysregulated coagulation is a major barrier to successful xenotransplantation. Microvascular thrombosis is frequently observed in rejected xenografts. Consumptive coagulopathy can develop in recipients. Likely causes include antibodies and cross-species molecular incompatibilities. Genetic modification of the donor pig may provide the solution to this problem.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 July 2015 Received in revised form 16 July 2015 Accepted 17 July 2015 Available online 26 July 2015
Dysregulation of coagulation and disordered hemostasis are frequent complications in the pig-to-nonhuman primate preclinical xenotransplantation model. The most extreme manifestations are the systemic development of a life-threatening consumptive coagulopathy, characterized by thrombocytopenia and bleeding, which is balanced at the opposite extreme by local complications of graft loss due to thrombotic microangiopathy. The contributing mechanisms include inflammation, vascular injury, heightened innate, humoral and cellular immune responses, and molecular incompatibilities affecting the regulation of coagulation. There also appear to be organ-specific factors that have been linked to vascular heterogeneity. As examples, liver xenografts rapidly induce thrombocytopenia by sequestering human/primate platelets; renal xenografts cause a broader coagulopathy, linked in some cases to reactivation of porcine CMV, whereas cardiac xenografts often succumb to microvascular thrombosis without associated systemic coagulopathy but with local perturbations in fibrinolysis. Overcoming coagulation dysfunction will require a combination of genetic and pharmacological strategies. Deletion of the xenoantigen aGal, transgenic expression of human complement regulatory proteins, and refinement of immunosuppression to blunt the antibody response have all had some impact, without providing a complete solution. More recently, the addition of approaches specifically targeted at coagulation have produced promising results. As an example, heterotopic cardiac xenografts from donors expressing human thrombomodulin have survived for more than a year in immunosuppressed baboons, with no evidence of thrombotic microangiopathy or coagulopathy. © 2015 IJS Publishing Group Limited. Published by Elsevier Ltd. All rights reserved.
Keywords: Xenotransplantation Coagulation Inflammation Coagulopathy Thrombocytopenia
Contents 1. 2.
Factors underlying dysregulated coagulation in xenograft recipients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297 Strategies to prevent dysregulation of coagulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298 2.1. Genetic modification of the donor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298 2.2. Treatment of the recipient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298
* Corresponding author. Immunology Research Centre, St Vincent's Hospital Melbourne, PO Box 2900, Fitzroy 3065, Victoria, Australia. E-mail address: [email protected] (P.J. Cowan). http://dx.doi.org/10.1016/j.ijsu.2015.07.682 1743-9191/© 2015 IJS Publishing Group Limited. Published by Elsevier Ltd. All rights reserved.
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3.
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Recent insights from preclinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298 3.1. Heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298 3.2. Kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298 3.3. Liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 3.4. Lung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 Ethical approval . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 Author contribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 Conflicts of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 Guarantor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
1. Factors underlying dysregulated coagulation in xenograft recipients Coagulation is a tightly controlled physiological process designed to preserve the integrity of the vascular system [1]. Endothelial injury results in exposure of active tissue factor (TF) to the blood, triggering a molecular cascade that results in the formation of a fibrin-enmeshed platelet plug. Finely tuned regulatory systems, comprising cell surface and circulating components, cooperate to ensure that clotting is localized, proportionate to injury, and resolved at the appropriate time. The key endothelial anticoagulant/antithrombotic proteins include thrombomodulin (TBM), tissue factor pathway inhibitor
(TFPI), endothelial protein C receptor (EPCR) and CD39. Because coagulation and complement activation are interconnected, the complement regulatory proteins (CRPs) CD46, CD55 and CD59 also play a role. Porcine xenografts face a series of challenges that tip the balance towards coagulation and inflammation (Fig. 1). First, human and nonhuman primate (NHP) sera contain significant levels of preexisting anti-pig antibodies [2,3]. The target antigens are predominantly carbohydrate structures on glycoproteins and glycolipids, indicating that these ‘natural’ antibodies arose as a consequence of cross-species differences in glycosylation. While the key specificity is galactose-a1,3-galactose (aGal), antibodies to N-glycolylneuraminic acid (Neu5Gc) are also likely to be important in the pig-to-
Fig. 1. Pathways to dysregulated coagulation and inflammation in solid organ xenotransplantation. Poor control of TF and thrombin by TFPI and TBM, respectively, will potentially drive a pro-inflammatory, procoagulant positive-feedback loop. Genetic strategies designed to disrupt this process are shown in green.
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human setting, because humans (unlike pigs and Old World primates) do not express Neu5Gc. Second, the adaptive immune response to xenografts contains a strong humoral component that is difficult to control. Elicited antibodies promote coagulation by activating porcine endothelial cells and platelets, but may also target protective endothelial surface proteins including EPCR and CRPs [4]. In addition, the development of antibodies to clotting factor VIII in xenograft recipients [5] has potential implications for coagulopathy. Third, xenografts can induce a systemic inflammatory response that may indicate precedent adaptive immunity, which may promote coagulation. Baboons receiving porcine hearts or kidneys exhibit steady rises in levels of the inflammatory marker C-reactive protein before developing consumptive coagulopathy [6]. Some proinflammatory cytokines (notably IL-6) are also increased in this context, even when the xenograft was limited to a small artery patch [6]. This inflammatory milieu is conducive to systemic upregulation or recruitment of recipient TF on platelets and monocytes [7]. Finally, several in vitro studies have identified molecular incompatibilities affecting the capacity of porcine endothelial anticoagulants to regulate human/primate coagulation factors. Pig TBM binds human thrombin but is a poor co-factor for subsequent activation of human protein C [8,9]. Similarly, recombinant pig TFPI binds and neutralizes human factor Xa but is a less efficient inhibitor of human TF/factor VIIa than human TFPI [10], although the latter incompatibility is not observed in all model systems [11]. Furthermore, the tendency for coagulation and thrombosis may be heightened by an abnormal interaction between porcine von Willebrand Factor (vWF) and human glycoprotein Ib (GPIb) [12], which causes the induction of active TF on human platelets incubated with porcine aortic endothelial cells [13]. Thus the exquisite balance between coagulation and thromboregulatory mechanisms is intrinsically disturbed in xenotransplantation: the endogenous defenses of the xenograft, already partly compromised by cross-species differences, are gradually overwhelmed by ongoing activating stimuli and loss of protective factors. 2. Strategies to prevent dysregulation of coagulation 2.1. Genetic modification of the donor Several complementary genetic strategies that have been developed to protect xenografts are pertinent to the regulation of coagulation. The xenograft can be made less ‘visible’ to the recipient immune system by eliminating xenoantigens such as aGal (GGTA1 KO or GTKO) and Neu5Gc (CMAH KO). The capacity of the xenograft to regulate complement activation and inflammation can be increased by expressing protective molecules such as human CRPs, hemeoxygenase-1 and A20. Anticoagulant defenses can be bolstered, with concomitant correction of molecular incompatibilities, by expressing human TBM, TFPI, EPCR and CD39. Expression of procoagulants such as TF can be reduced by siRNAmediated knockdown [14]. For liver xenografts, the aberrant phagocytosis of human platelets can be addressed by deleting the porcine asialoglycoprotein receptor (ASGR) [15]. Where data are available, the impact of these existing modifications on coagulation dysfunction will be discussed organ-byorgan later in this article. Advances in genome editing technology hold the promise of even more precise modifications, such as correction of the incompatibility between pig vWF and human GPIb. 2.2. Treatment of the recipient
in
Many anticoagulants and anti-inflammatories have been tested xenotransplantation models, including heparin, aspirin,
warfarin, clopidogrel, statins, adenosine agonists, apyrases, antithrombin, and activated protein C (reviewed in [16,17]). These agents have been used in combination with immunosuppression and/or genetically modified donors, making it difficult to assess their impact individually, although it does appear that increasing anticoagulant treatment does not fully correct dysregulated coagulation [18,19]. Rather, judicious pharmacological anticoagulation is seen as an adjunct to the other strategies [20]. Anti-inflammatories such as etanercept (soluble TNF receptor), anakinra (IL-1 receptor antagonist) and tocilizumab (anti-IL-6 receptor) hold promise, but have not been widely tested in xenotransplantation to date. 3. Recent insights from preclinical studies Progress in pig-to-NHP xenotransplantation models from 1998 to the end of 2013 has been extensively reviewed [21]. We will therefore restrict our discussion to some of the more recent studies. 3.1. Heart The intractable nature of coagulation and fibrinolytic dysfunction was illustrated several years ago by a pig-to-baboon heterotopic cardiac xenograft study using inbred GTKO donors, a comprehensive immunosuppressive protocol (antithymocyte globulin (ATG), anti-CD154, cobra venom factor, mycophenolate mofetil (MMF), steroids), and anticoagulation with continuous heparin, antithrombin and/or aspirin as an antiplatelet drug [22]. Even this combination was not sufficient to prevent the eventual development of thrombotic microangiopathy, restricting maximum survival to 179 days [22]. Using a similar regimen with outbred GTKO/hCD46 donors and the addition of anti-CD20, maximum survival was extended to 236 days, but microvascular thrombosis was still evident in long-surviving grafts [23]. In contrast, survival exceeding 600 days was achieved by using GTKO/hCD46/hTBM hearts and substituting high-dose anti-CD40 for anti-CD154, and biopsies at 1 year showed minor fibrosis but no thrombotic microangiopathy [24]. The experimental design did not allow definitive conclusions to be drawn on the relative contributions of the genetic modifications and the treatment protocol, which included continuous heparin infusion to maintain activated clotting time at twice baseline. Nevertheless, the results suggested that expression of hTBM was important for controlling coagulation and promoting long-term cardiac xenograft survival. In a separate study, GTKO/hCD46/hTBM hearts were transplanted into two baboons treated with ATG, anti-CD40, belatacept, steroids, low molecular weight heparin, and rapamycin or tacrolimus [25]. The xenografts did not survive as long (maximum survival 130 days) as in the earlier study, possibly due to the absence of anti-CD20. However, the recipients showed no features of dysregulated coagulation, and importantly did not require continuous heparinization. This further suggests the potential benefit of hTBM in cardiac xenotransplantation. 3.2. Kidney Kidney xenografts show a greater tendency to induce consumptive coagulopathy than hearts, which may reflect differential regulation of coagulation and fibrinolysis in renal and cardiac vasculature [26]. Baboons transplanted with GTKO/hCD46 kidneys and treated with ATG, anti-CD154, MMF and heparin all developed coagulopathy by day 16 [7]. This was preceded by the progressive expression of TF on platelets and monocytes. In contrast, GTKO thymokidney xenografts survived up to 83 days in baboons treated with a variety of costimulation blockade-based immunosuppressive
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regimens, with minimal thrombotic microangiopathy and coagulopathy [27]. The many differences between these studies preclude drawing any definitive conclusions about the reason for the disparate outcomes. Disappointingly, mean published survival of GTKO thymokidneys has decreased from 53.2 days in studies prior to mid-2008 to 13.7 days subsequently [28]. The likely cause of this fall has been ascribed to be inadvertent introduction of porcine CMV into the donor herd, leading to viral activation of xenograft endothelial cells and vascular injury [28,29]. Porcine CMV replication was also observed in some, but not all, baboon recipients of GTKO/hCD55/ hCD59/hCD39 renal xenografts [30]. Treatment was with tacrolimus, MMF, steroids, human C1 inhibitor, and cyclophosphamide or bortezomib, with or without plasma exchange. The xenografts were lost by day 15 to acute humoral rejection, with developing signs of consumptive coagulopathy, highlighting the importance of controlling the antibody response. No conclusions could be drawn about the efficacy of hCD39, which was expressed at modest levels in the kidney [30]. A recent small study reported the longest survival of pig-to-NHP kidney xenografts to date [31]. GTKO/hCD55 kidneys were transplanted into rhesus macaques treated with anti-CD4, anti-CD8, MMF, steroids, and anti-CD154 or belatacept. In the anti-CD154 group, xenograft survival was only 6 days in a recipient with very high titers of pre-existing anti-pig IgM and IgG, accompanied by profound thrombocytopenia. Strikingly, however, survival was prolonged to >125 and >133 days in two low-titer animals, both of which exhibited stable platelet counts and no clinical or histological evidence of rejection over that time [31]. This was achieved even in the absence of specific anticoagulation and the presence of CMV in the donor pigs. Even more recently, a single case study has reported renal xenograft survival of 136 days in a baboon recipient [32]. The donor pig was GTKO/hCD46/hCD55/hTBM/hEPCR/hCD39, and the recipient was treated with ATG, anti-CD20, anti-CD40, rapamycin, steroids, etanercept, tocilizumab and low molecular weight heparin. Although these remarkable results remain to be confirmed, it suggests that it is possible to delay or even prevent coagulation dysfunction in renal xenograft recipients. 3.3. Liver Realistically, liver xenografts are unlikely to take the place of allografts, but could provide time to allow native liver recovery/ regeneration or bridging to allotransplantation. However, while genetically modified (GTKO ± hCD46) liver xenografts can support life for at least 9 days in immunosuppressed baboons, recipient survival is cut short by the rapid development of severe thrombocytopenia, coagulopathy, and bleeding resulting in death [33,34]. This occurs even in the apparent absence of rejection, and is believed to stem largely from molecular incompatibility between pig asialoglycoprotein receptor (ASGR) and its human/NHP ligand [35], resulting in platelet sequestration by pig liver sinusoidal endothelial cells and Kupffer cells. In a promising recent development, pigs lacking ASGR have been generated, and their livers showed significantly reduced uptake of human platelets in an ex vivo perfusion model [15]. In a GTKO pig-to-baboon model in which the native liver was retained, thrombocytopenia was still observed, but bleeding complications were reduced (presumably due to the continued production of baboon clotting factors) and one liver xenograft survived for 15 days [36]. Interestingly, the cause of graft failure in this setting appears to have been thrombotic microangiopathy, suggesting that a balance in the coagulation system must be achieved to prevent rejection whilst retaining adequate hemostasis.
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3.4. Lung The lung appears to be the vascularized xenograft most difficult to obtain acceptable results, with maximum survival of less than 5 days in the pig-to-baboon model [37,38]. Inflammation and dysregulated coagulation are key factors in lung xenograft failure. Although the data are limited, it is clear that genetic modifications that have been shown to significantly prolong cardiac and renal xenograft survival (i.e. GTKO ± hCRP expression) have only a marginal impact in pulmonary xenotransplantation [39,40]. This suggests that the lung is more sensitive to xenogeneic injury and/or that additional mechanisms, perhaps involving pulmonary intravascular macrophages [37,39] and platelet sequestration, play a role. A recent paper from the Pierson group analyzed a large body of data obtained using their ex vivo model in which pig lungs are perfused with human blood [41]. An extensive range of genetic modifications has been tested in this model. Although the data did not allow definitive conclusions on the benefit of each individual modification, the authors identified several strong trends: (i) lung xenograft ‘survival’ improved incrementally with each additional transgene; (ii) GTKO, hCD46, hCD55, hEPCR, and hemeoxygenase-1 were associated with protection; and (iii) hTBM and hHLA-E had no apparent effect. While the impact of hCD39 appeared inconclusive, a study using a similar model and different transgenic pig lines has shown that hCD39 expression is protective on the GTKO/hCRP background [42]. The ex vivo model has also provided evidence suggesting that long-term survival of genetically modified lung xenografts may require specific anticoagulant/anti-inflammatory treatment, including targeting of GPIb, GPIIb/IIIa, thromboxane synthesis and histamine signaling (reviewed in [38]). 4. Conclusion Coagulation dysregulation remains a major problem in xenotransplantation, with each organ type presenting a different, albeit overlapping, set of challenges. Any solution must take into account the tight integration of the processes that govern and regulate coagulation and inflammation. Recent progress has been significant, with a limited number of cases indicating the potential for long-term survival of heart (>600 days [24]) and kidney (>126 days [31]) xenografts in the absence of major disturbances to coagulation. Further advances are anticipated with the development of more efficient techniques for precise editing of the porcine genome [43]. We conclude that the right combination of pre-transplant antibody screening, donor genetics, and immunosuppressive and antiinflammatory therapy may be sufficient to prevent coagulation dysfunction in xenotransplantation. Ethical approval Not applicable. Author contribution Peter Cowan wrote the paper with input from Simon Robson. Conflicts of interest None. Funding The National Health and Medical Research Council of Australia.
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The Juvenile Diabetes Research Foundation. The National Institutes of Health. [22]
Guarantor Not applicable.
[23]
Acknowledgments [24]
The work of the authors described in this review was supported by funding from the National Health and Medical Research Council of Australia, the Juvenile Diabetes Research Foundation, and the National Institutes of Health NHLBI and NIDDK.
[25]
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