Thrombotic microangiopathy (TM) is a serious complication of bone marrow transplantation (BMT) that resembles thrombotic thrombocytopenic purpura (TTP). In attempting to achieve hematopoietic cell chimerism in the pig-to-baboon model, we have observed TM following infusion of high doses (>1010 cells/kg) of porcine peripheral blood mobilized progenitor cells (PBPC) into baboons. We performed investigations to analyze the pathobiology of this TM and to test therapeutic interventions to ameliorate it. PBPC were obtained by leukapheresis of cytokine-stimulated swine. The initial observations were made in two baboons that underwent a non-myeloablative regimen (NMR) prior to PBPC transplantation (TX) (group 1). We then studied three experimental groups. Group 2 (n = 2) received NMR without PBPC TX. Group 3 (n = 2) received PBPC TX alone. Group 4 (n = 6) received NMR + PBPC TX combined with prostacyclin, low-dose heparin, methylprednisolone, and cyclosporine was replaced by anti-CD40L mAb in five cases. Baboons in groups 1 and 3 developed severe thrombocytopenia (<10 000/mm3), intravascular hemolysis with schistocytosis (>10/high powered field (hpf)), increase in plasma lactate dehydrogenase (LDH) (2500–9000 U/l), transient neurologic changes, renal insufficiency, and purpura. Autopsy on two baboons confirmed extensive platelet thrombi in the microcirculation, and, similar to clinical BMT-associated TM/TTP, no unusually large vWF multimers or changes in vWF protease activity were observed in the plasma of baboons with TM. In group 2, self-limited thrombocytopenia occurred for 10–15 days following NMR. Group 4 baboons developed thrombocytopenia (<20 000/mm3) rarely requiring platelet transfusion, minimal schistocytosis (<3/hpf), minor increase in LDH (<1000 U/l), with no clinical sequelae. We conclude that high-dose porcine PBPC infusion into baboons induces a microangiopathic state with vWF biochemical parameters resembling clinical BMT-associated TM/TTP and that administration of antithrombotic and anti-inflammatory agents can ameliorate this complication. Bone Marrow Transplantation (2001) 27, 1227–1236.
Thrombotic microangiopathy (TM) is a serious complication of clinical bone marrow transplantation (BMT) with an incidence of 14% and 7% with allo- and autografts, respectively.1 The clinical picture in BMT-associated TM has comparable features to ‘classical TTP’ (acute idiopathic and familial forms), with thrombocytopenia, microangiopathic hemolytic anemia, fever, neurological symptoms and renal impairment. However, there are distinct differences in that plasma exchange therapy is beneficial only in classical TTP.2 Decreased activity of the von Willebrand factor (vWF) multimer-cleaving metalloprotease has been demonstrated only in classical TTP.3,4 This deficiency leads to accumulation of unusually large multimers of vWF,5 that result in spontaneous platelet aggregation and microthrombus formation. However in BMT-associated TM, normal metalloprotease activity has recently been demonstrated,6 suggesting a different mechanism. No animal model of BMT-associated TM has been reported to date, and the etiological basis for this disease remains unclear.
To achieve specific immunologic tolerance to allografts and concordant xenografts, our laboratory has developed models in which mixed hematopoietic chimerism has been established in rodents7,8 and nonhuman primates.9,10 In view of the extreme shortage of suitable organs for clinical allografting, we have attempted to extend this approach to the discordant xenograft pig-to-baboon combination. Initially, we infused pig peripheral blood mobilized progenitor cells (PBPC) at doses of 2–30 × 108/kg without complication, although chimerism was undetectable.11,12 To attempt to achieve more durable engraftment, we have recently infused higher doses of porcine PBPC (2–4 × 1010 cells/kg) in baboons undergoing non-myeloablative conditioning regimens. The first two baboon recipients of PBPC developed a microangiopathic hemolytic anemia with features comparable to BMT-associated TM. From an ethical perspective with regard to the health of the experimental animal, we immediately investigated this complication and explored therapy to prevent it in further animals. We describe here the experimental observations and pathobiology of xenotransplantation-associated TM, and a therapeutic approach that has allowed a significant amelioration in the clinical manifestations and microangiopathy.
Materials and methods
Baboons (Papio anubis, n = 12) of known ABO blood group and body weight 10–15 kg (Biological Resources, Houston, TX, USA) were used as recipients. Massachusetts General Hospital MHC-inbred miniature swine (n = 7) of blood group O, 2–4 months old, 18–40 kg body weight (Charles River Laboratories, Wilmington, MA, USA) and Landrace pigs transgenic for human decay-accelerating factor (hDAF) (n = 3) (the generous gift of Imutran, Ltd, Cambridge, UK) of blood group O or A, 1.5 to 2.5 months old, weighing 20 to 25 kg, served as donors of PBPC.
Care of animals was in accordance with the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health. Protocols were approved by the Massachusetts General Hospital Subcommittee on Research Animal Care.
Mobilization and collection of leukocytes (PBPC) in pigs
Pigs were treated with recombinant hematopoietic growth factors (cytokines)14,15 before and during the period of leukapheresis. Porcine interleukin-3 (BioTransplant, Inc., Charlestown, MA, USA) 100 μg/kg/day by subcutaneous (s.c.) injection and porcine stem cell factor (BioTransplant) 100 μg/kg/day s.c. were administered daily on days 0–15, and human granulocyte colony-stimulating factor (Amgen, Thousand Oaks, CA, USA) 10 μg/kg/day s.c. was given on days 11–15. Leukapheresis was performed on days 5–9 and 12–16 using a Cobe Spectra apheresis machine (Cobe, Lakewood, CO, USA). After collection, the pig leukocytes (designated peripheral blood progenitor cells, PBPC) were washed and, after red blood cell lysis, were frozen with 5% DMSO. Details of the collection procedure and preparation of the leukocyte product have been described elsewhere.16
Extracorporeal immunoadsorption in baboons
Natural anti-α1–3 galactose (αGal) antibody was depleted from the baboon's circulation by the perfusion of plasma through immunoadsorption columns containing synthetic Galα1–3Galβ1–4Glc-X-Y (αGal type VI trisaccharide, Alberta Research Council, Edmonton, Alberta, Canada), as reported previously.11,12,17,18
Conditioning regimen in baboons
All recipient baboons underwent splenectomy on day −8. In groups 1, 2 and 4, the baboons underwent a nonmyeloablative regimen consisting of whole body irradiation in two fractions (150 cGy each) on days −6 and −5 (total dose 300 cGy), thymic irradiation (700 cGy) on day −1, horse anti-human thymocyte globulin (ATGAM; Upjohn, Kalamazoo, MI, USA) 50 mg/kg/day i.v. on days −3, −2 and −1, extracoporeal immunoadsorption on days −2, −1 and 0.12 This was followed by porcine PBPC TX on day 0 and further PBPC TX on days 1 and 2. The baboons also received mycophenolate mofetil (MMF) by continuous i.v. infusion (at approximately 80 mg/kg/day, administered with an Abbott Omniflow 4000 infusion device, Abbott Laboratories, Mississauga, Ontario, Canada) from days −8 to 28 to maintain whole blood levels of 3–6 μg/ml.19
Other therapeutic components in some experiments (Table 1) included cobra venom factor i.v. at approximately 100 units/kg/day on days −1 to 14 or 28 to deplete complement levels so that CH50 is approximately equal to 0%.20 Groups 1, 2 and one group 4 animal also received cyclosporine (Novartis, Basel, Switzerland) (at approximately 15 mg/kg/day) by continuous i.v. infusion from days −8 to 28 to maintain a whole blood level of 1200–1400 ng/ml. In five of six group 4 animals, cyclosporine was replaced by a course of murine anti-human CD40L mAb (5C8; ATCC, Rockville, MD, USA) (20 mg/kg/i.v.) administered on alternate days from days 0 to 14 (total of eight doses).
Porcine PBPC transplantation in baboons
After rapidly thawing in a water bath at 37°C, the stored PBPC were washed twice with calcium/magnesium-free Hank's balanced salt solution and immediately infused through a central systemic (n = 7) or intraportal (n = 3) venous catheter. The total number of PBPCs administered to each baboon is indicated in Table 1. Group 4 baboons received therapy aimed at preventing platelet and endothelial activation, consisting of prostacyclin (PGI2; 20 ng/kg/min by continuous i.v. infusion), heparin (10 U/kg/h by continuous i.v. infusion), and methylprednisolone (2 mg/kg × 2 daily i.v. for 7 days, followed by tapering and discontinuation over the next 7 or 21 days). This therapy (designated PHM therapy) commenced immediately before the first PBPC infusion and continued for a minimum of 14 days.
Hematopoietic growth factor (cytokine) therapy in baboons
Recombinant porcine interleukin-3 (100–200 μg/kg/day s.c. or i.v.) and porcine stem cell factor (100–1000 μg/kg/day s.c. or i.v.) were adminstered to baboons from days 0 to 14 in group 1, and 1–28 in group 4.
Blood cell count, blood smear, chemistry including serum lactate dehydrogenase (LDH), plasma prothrombin time, partial thromboplastin time, fibrinogen and levels of immunosuppressive drugs were determined by routine methods. Washed irradiated red blood cells from ABO-matched baboon donors were administered to maintain the hematocrit >20%. Erythropoietin was administered to some baboons at a dose of 100 units/kg s.c. × 3 weekly. Thrombocytopenia of <10 000 platelets/mm3 was corrected by the transfusion of fresh washed irradiated baboon platelets. No pig platelets were ever administered. A persistent white blood cell count of <1000 cells/mm3 was on occasions treated with a course of recombinant human granulocyte colony-stimulating factor (10 μg/kg s.c.). All pigs and baboons received daily cefazolin sodium (500 mg/day i.v.) throughout the periods of leukapheresis and therapy, respectively. Temperature was not monitored, as such measurements require extensive handling and, frequently, sedation of the animals.
Phosphate-buffered saline Tween 20 (PBST) (100 μl) was added to a 96-well plate precoated with polyclonal goat anti-D-dimer antibody (American Diagnostica, Inc., Greenwich, CT, USA). The test sample or a D-dimer standard (25 μl) (2000, 1000, 500, 250, 125, 62.5, 32, 0 ng/ml) was added to the 96-well plate and incubated for 1 h on a shaker at room temperature. After washing the wells four times with PBST, 50 μl of anti-D-dimer antibody was added and incubated for 1 h at room temperature on a shaker. After further washing, 100 μl of substrate solution was added. The enzymatic reaction was stopped after 15 min by adding 50 μl of stopping reagent. Absorbances were read on a microplate reader (Biotek, Wihooski, VT, USA) at a wavelength of 405 nm. D-dimer was not measured in the group 1 baboons.
Measurement of vWF antigen by ELISA
Baboon plasma samples were diluted 1:10 in PBST and 3% bovine serum albumin. Diluted test sample or vWF standard (100 μl) (10, 5, 2, 1 and 0.5 mU/ml) was added to a 96-well plate precoated with polyclonal goat anti-vWF antibody (American Diagnostica) and incubated for 1 h on a shaker at room temperature. After washing the wells four times with PBST, 100 μl of horseradish peroxidase-conjugated vWF antibody was added and incubated for 1 h at room temperature on a shaker. After further washing, 100 μl of substrate solution was added. The enzymatic reaction was stopped after 20 min by adding 50 μl of 0.5 M sulfuric acid. Absorbances were read on a micro-test plate reader (Molecular Devices, Sunnyvale, CA, USA) at a wavelength of 450 nm. The vWF concentration was determined by reference to standard curve using calibrated human antigen.
SDS-agarose gel electrophoresis
Baboon citrated plasma samples were diluted 1:10 in PBS. Prediluted plasma samples were then diluted 1:1.5 in Fairbanks denaturing buffer (final concentration 20 mM Tris, 2% SDS, 0.1 mM disodium EDTA, pH 8.0 and 0.01% bromophenol blue as a tracking dye) and then incubated at 60°C for 15 min. Aliquots of 25 μl were loaded on a 1% SDS-agarose gel (ultra pure HGT agarose; FMC, Rockland, ME, USA). SDS-agarose gel electrophoresis was performed in Fairbanks running buffer (final concentration 37.5 mM Tris, 0.1% SDS, 2 mM disodium EDTA, pH 7.4) until the dye front was near the anode. Transfer was performed semi-dry in transfer buffer (39 mM Tris, 48 mM glycine, 0.0375% SDS and 20% methanol) on Millipore PVDF (polyvinylidene difluoride nitrocellulose) membrane (Millipore, Bedford, MA, USA). Multimeric patterns were visualized by immunochemical detection using anti-vWF antibody-horseradish peroxidase conjugate at 1:500 dilution (Dako, Glostrup, Denmark).
vWF-cleaving protease assay
Baboon citrated plasma samples were diluted 1:3 to 1:8 in a Tris-buffer (50 mM sodium chloride, 2.5 mM calcium chloride, 50 mM Tris pH 8.0 and 1 mM PMSF (phenylmethyl sulphonyl fluoride)) in the absence or presence of 5 mM EDTA. Either cryoprecipitate (obtained from healthy human donors) at a dilution of 1:50 or purified vWF obtained from plasma and treated with 1.5 M guanidine hydrocholoride at a dilution of 1:10 was added to the test samples and incubated at 37°C for 1 h. The enzymatic reaction was terminated by the addition of a Tris-buffer (0.125 mM Tris, 5 mM EDTA, 2% SDS, 5% glycerol) at a 1:5 dilution. The extent of vWF cleavage was then determined by 4–10% SDS-PAGE, immunoblotting and autoradiography as previously described.3 Group 1 baboons were not studied for vWF parameters.
Histopathology and immunohistopathological studies
Tissues taken from baboons were fixed in 10% formalin, paraffin-embedded, and stained with hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) for light microscopy. For analysis of the nature of microthrombi, immunohistochemical staining was performed. To detect platelet aggregates, an indirect immunoperoxidase technique was applied with a murine monoclonal antibody directed to human CD62P (P-selectin) (Becton Dickinson, San Jose, CA, USA), crossreactive with baboon platelets. For vWF detection, a rabbit polyclonal antibody to human vWF crossreactive with baboon antigen was used (Dako). For the detection of pig cells, a monoclonal mouse anti-pig leukocyte antibody (TBRC 1030H-1–19) was used. For the detection of fibrin deposition, frozen tissue sections were stained using direct immunofluorescence with FITC-conjugated rabbit polyclonal antibody to human fibrin (Dako), crossreactive with baboon antigen. Controls consisted of tissue staining with an irrelevant murine monoclonal antibody or rabbit-anti-human albumin. The pathologist graded the positivity of the immunohistochemistry and immunofluorescence studies as mild (corresponding to <25% of capillaries involved), moderate (<50% capillaries involved) or strong (>50% capillaries involved).
Experimental groups (Table 1)
The baboons in which the clinical observation was first made (group 1 (n = 2)) received the non-myeloablative conditioning regimen combined with cyclosporine therapy. Both baboons received PBPC harvested from miniature swine.
The three experimental groups subsequently studied were: group 2 (n = 2) received the non-myeloablative regimen alone without PBPC transplantation. Group 3 (n = 2) received no preparative therapy. Both baboons received PBPC harvested from miniature swine. Group 4 (n = 6) received the same nonmyeloablative regimen as in groups 1 and 2, but all received the PHM therapy. Cyclosporine was omitted and replaced by a 14-day (eight dose) course of anti-CD40L mAb in five of six baboons. Three baboons received PBPC from miniature swine and three from human decay accelerating factor (hDAF) transgenic pigs; one baboon (B57–9) receiving hDAF pig PBPC did not receive cobra venom factor (Table 1).
PBPC mobilization and leukapheresis
The total yield of cells collected per day was between 2–10 × 1010 cells. These products typically consist of 70–90% mononuclear cells. The content of progenitor cells assayed as GEMM (granulocyte-erythroid-monocyte-megakaryocyte) colony-forming units was 0.1% of mononuclear cells. The viability after freezing, thawing and washing was between 50 and 90%. The platelet content in each case was <10 000/mm3.
Clinical outcome of PBPC transplantation
Group 1 (n = 2):
Within 1–2 days, baboons developed severe thrombocytopenia (Figure 1), with schistocytosis (>10/high powered field (hpf)) and a marked increase of serum LDH (peak of 5900 U/l; normal values in baboons <300) (Figure 1). There was also evidence of renal insufficiency (peak serum creatinine of 3.7 mg/dl). Neurological signs during the pig cell infusions were manifested by lethargy or transient loss of consciousness. One animal died 1 day after the last infusion of pig cells, and autopsy showed hemorrhage and infarcts in multiple organs and lymph nodes.
Group 2 (n = 2):
Baboons receiving the conditioning regimen alone, without PBPC TX, developed less pronounced thrombocytopenia within 10 to 15 days (Figure 1). The thrombocytopenia recovered spontaneously and did not require platelet transfusion. Blood smears showed infrequent schistocytes (1/hpf). There were no increases in LDH levels (Figure 1) and no neurological signs or renal insufficiency developed.
Group 3 (n = 2):
Baboons that received PBPC TX without the non-myeloablative regimen developed complications comparable to the group 1 animals, ie thrombocytopenia (Figure 1), marked schistocytosis, and increase in LDH (peak of 9000 U/l) (Figure 1). Neurological signs were observed during the PBPC infusions, and severe renal insufficiency developed in one baboon (peak creatinine of 4 mg/dl). One animal died 4 days after the last PBPC infusion and, at autopsy, massive hemorrhage in multiple organs was observed macroscopically.
Group 4 (n = 6):
Baboons that received PBPC TX with prostacyclin/heparin/methylprednisolone (PHM) therapy +/− anti-CD40L mAb had attenuated features of TM. Lesser degrees of thrombocytopenia (requiring only occasional platelet transfusions) (Figure 1), mild schistocytosis (<3/hpf), minor increases in LDH (<775 U/l) (Figure 1) and no renal or neurological sequelae were observed. No significant differences were noted in the features of TM following the transplantation of either hDAF or miniature swine PBPC. No mortality was observed in this group.
These parameters were comparable in all four groups and only minimal changes were observed. PT, PTT and fibrinogen showed only minor self-limiting fluctuations during the pre-treatment phase (associated with extracorporeal immunoadsorption) and after the pig cell infusions (data not shown).
All baboons that received PBPC (groups 1, 3 and 4) showed platelet counts that reached a nadir within 12–24 h following PBPC infusion. This rapid decrease was in contrast with the group 2 baboons that received induction therapy without PBPC infusion, and showed a progressive fall of platelets over 10–12 days. This progressive fall in platelets was attributed to decreased production, whereas the rapid fall in the baboons receiving PBPC was attributed to platelet consumption. All baboons that received platelet transfusions showed an immediate but transient increase in platelet count. We did not assay for anti-platelet Ab, but serum obtained from baboons in groups 1 and 3 did not produce thrombocytopenia when injected into normal baboon recipients (data not shown).
D-dimer was not measured in the preliminary group 1 baboons in which the TM was initially observed. Baboons in group 2 had increases in D-dimer level observed during the conditioning regimen to 900 ng/ml. Baboons in group 3 showed an increase in D-dimer levels to peak levels around 4000 ng/ml on day 3 after PBPC infusion, with an observed decrease during the following days. Baboons in group 4 showed transitory increases of D-dimer levels at the time of the conditioning regimen with a second peak after the PBPC infusion to 1800 ng/ml.
Group 1 baboons were not studied for vWF parameters. Baboons in group 2 showed minor fluctuations in vWF plasma levels ranging between 90 and 115 mU/ml vWF (Figure 2). Group 3 showed acute fluctuations in vWF plasma levels after infusion of PBPC with an increase from the 50 to 90 mU/ml range of vWF values on days 0 to 2 (Figure 2). Baboons in group 4 showed minor fluctuations in vWF plasma levels between 80 and 100 mU/ml vWF (Figure 2). The vWF multimeric pattern in groups 2 and 4 showed only minor changes and no unusually large vWF (ULvWF) multimers were observed. Baboons in group 3 showed minor transient shifts in vWF multimeric patterns (Figure 3). Following treatment with EDTA, the calcium-dependent cleaving protease activity was inhibited in all groups studied (data not shown).
Pathology and histopathology
Autopsies were performed in the two baboons of groups 1 and 3 that died after PBPC TX (B17–5 and B75–34). In both baboons, the macroscopic features were hemorrhage and infarcts in multiple organs (heart, lungs, kidneys, adrenals) and, in one case, the mesenteric lymph nodes. Microscopically, thrombi were detected in the microcirculation of several major organs (heart, lungs, kidneys) in both baboons. Immunohistological staining was strongly positive for a platelet marker (CD62P) and vWF, but only moderately positive for fibrin (Figure 4a and b), in keeping with a microangiopathic deposition of platelet aggregates with minimal activation of coagulation pathways. Porcine leukocytes were also detected in these lesions (Figure 4c).
There is a critical shortage of donor organs for allotransplantation that prevents many suitable candidates from benefiting from this form of therapy and results in a significant mortality in patients awaiting transplantation.21 A potential solution would be the use of organs from a suitable animal, such as the pig.21,22 The strengths of the humoral and cellular rejection responses in primates to a transplanted pig organ are known to be strong, and current opinion by several groups, including our own, is that these responses may only be overcome if a state of immunological tolerance can be induced in the recipient.23 One way of achieving this would be through the induction of mixed hematopoietic cell chimerism, an approach that has been shown to be successful in primate allograft and concordant xenograft models.9,10
Early studies involving relatively low-dose (2–30 × 108 cells/kg) pig hematopoietic cell infusions into nonhuman primates (undergoing a very similar conditioning regimen to that in the present studies) were not accompanied by thrombotic complications, but were unsuccessful in achieving chimerism.11,12 With these lower numbers of cells infused, the pig hematopoietic cells were undetectable in the circulation within minutes, which was believed to result from clearance of circulating cells by the reticuloendothelial system (Buhler L et al, manuscript submitted), and no thrombocytopenia was observed in the recipient baboons. Our subsequent studies using higher doses of pig PBPC, reported herein, were unexpectedly complicated by a thrombotic condition that could prove fatal. These comparative observations strongly suggest that the development of TM is related to the number of pig hematopoietic cells infused.
This thrombotic condition consisted of consumptive thrombocytopenia, hemolytic anemia with profound peripheral blood schistocytosis, marked elevations of LDH, and, in some cases, neurologic changes, renal failure and death. Notably, examination of baboon tissues at autopsy revealed microvascular angiopathic lesions predominantly in arterioles consisting of platelet thrombi with minimal deposition of fibrin. This thrombotic, microangiopathic condition only developed in baboons receiving PBPC (groups 1, 3 and 4). Although cyclosporine has been implicated in causing TM and may induce vWF secretion from endothelial cells,24,25 this disorder occurred independently of cyclosporine administration. Furthermore, it was not a result of the conditioning regimen since it occurred in animals that underwent PBPC TX without receiving any pretransplant conditioning (group 3). The severity of the microangiopathic disorder was attenuated by treatment with antithrombotic/anti-inflammatory agents such as heparin, methylprednisolone and prostacyclin, and by the administration of anti-CD40L monoclonal antibody (group 4). In all baboons that survived and exhibited restoration of their platelet counts, resolution was accompanied by normalization of serum LDH levels and by a decrease in peripheral blood schistocytosis.
Several features of this thrombotic microangiopathic condition resemble those of TTP, a clinical illness which was first described by Moschcowitz in 1924.26 Classical TTP represents a clinical syndrome typified by a pentad of signs: microangiopathic hemolytic anemia, thrombocytopenia, fever, neurologic changes and renal failure. Due to the inherent technical difficulties in handling the animals for routine temperature measurement, we did not obtain information on fever, but all other elements of the pentad were observed in the baboons receiving porcine PBPC. In TTP, thrombocytopenia is secondary to consumption of platelets caused by inappropriate clumping in the microcirculation. These platelet aggregates within the microvasculature result in both traumatic hemolysis of red cells (evidenced by schistocytes on blood smears) and in tissue ischemia/infarction, resulting in release of LDH from both red cells and tissues (such as the liver, skeletal muscle and heart27). The ischemia induced by platelet aggregates in the cerebral and renal microcirculation results in impairment of central nervous and renal function, respectively.
In a variant presentation of TM known as the hemolytic-uremic syndrome (HUS), the platelet aggregates are confined primarily to the kidney vasculature, and other organs are relatively spared. Both TTP and HUS are characterized histopathologically by platelet thrombi predominantly in arterioles. In contrast to disseminated intravascular coagulation (DIC), the venules are conspicuously uninvolved and there is minimal fibrin deposition and less prominent vascular injury in TTP.28
In both platelets and endothelial cells, vWF is stored as large multimers which, following release into the circulation, are cleaved by a metalloprotease into smaller forms.3,4 The highly multimeric forms of vWF are extremely efficient mediators of platelet aggregation. In ‘classical’ TTP, such as acute idiopathic TTP and familial (chronic relapsing) TTP, unusually large vWF multimers may circulate in the plasma. In both of these conditions, the vWF-cleaving protease activity is diminished. In familial TTP this is due to congenital deficiency of the protease, whereas in idiopathic TTP protease activity is blunted due to the presence of a circulating IgG protease inhibitor.3,4,29,30 In both conditions, TTP is treatable by plasma infusion. In the case of idiopathic TTP, plasma exchange (to remove the inhibitor) is particularly efficacious.31
In addition to a classical form, a variant form of TTP characterized by TM occurs secondary to administration of certain pharmacologic agents (including cyclosporine24,25), and as a consequence of BMT, both autologous and allogeneic.1,32 The incidence of TM in BMT recipients may be as high as 14% in allogeneic recipients and as high as 7% in autologous recipients,1 indicating that this complication is not uniquely associated with graft-versus-host disease or with the use of cyclosporine as prophylaxis or treatment of GVHD. Moreover, in contrast to classical TTP, TM secondary to BMT is refractory to treatment by plasma exchange,33,34 and is frequently irreversible.
Important observations within the past year have clarified the differences between classical TTP and BMT-associated TM.6 In classical TTP, the absence of the vWF-cleaving serum metalloprotease activity may result in the circulation of extraordinarily large vWF multimers, and their presence was initially proposed as a means of differentiating between TTP and HUS.5,35 However, in BMT-associated TTP, there is no deficiency of vWF-cleaving activity.6 Subtle changes in vWF structure, however, cannot be excluded by these data. These findings suggest that the pathophysiology of the microvascular platelet aggregation differs between these various conditions and that disruption of vWF-multimeric structure may occur secondary to shear stress in the injured microvasculature in TM. Though animal (dog, pig and rat) models of TTP exist, these models have utilized injection of botrocetin, a snake venom factor which initiates platelet agglutination only in the presence of vWF.36,37 To date, no primate model of TTP has been established, and no model of BMT-associated TTP/TM has been described in any animal.
Athough the TM observed in baboons following xenogeneic PBPC TX differs from clinical BMT-associated TM both in timing (developing almost immediately after PBPC infusion, as compared to the typical evolution at >4 weeks post-BMT32) and in the marked suddenness and severity of the thrombocytopenia and hemolysis, it is similar to clinical BMT-associated TM in that it occurs without significant changes in the vWF multimer patterns or in vWF-cleaving protease activity. Though group 1 and 3 animals showed some acute fluctuations in vWF plasma levels determined by ELISA, these were self-limiting with only minor shifts in the vWF multimeric pattern. In addition, there was no evidence of unusually large vWF multimers. Group 4 animals showed minor, if any, fluctuations in vWF plasma levels without shifts in vWF multimeric patterns, and group 2 animals showed no changes in vWF levels or multimer patterns. No changes in vWF-cleaving protease activity were observed in any of the groups. These data indicate that aberrant vWF-protease activity does not play a crucial role in the evolution of TM in these animals, analogous to the findings in BMT-associated TM in humans.6
The baboon platelet consumption observed in the animals following PBPC TX could result, in addition to TM, from immune-mediated destruction and/or from DIC. The thrombocytopenia observed did not appear secondary to immune-mediated consumption, as all animals were splenectomized prior to PBPC TX, and the platelet count rose immediately after each transfusion of baboon platelets. Furthermore, serum from affected baboons did not induce thrombocytopenia when injected into healthy baboons. Activation of the complement cascade is unlikely to have played a role in the development of TM as all baboons had received cobra venom factor to deplete complement 1 day prior to the infusion of PBPC, and depletion of complement was maintained throughout the experiment by daily cobra venom therapy. We have found cobra venom therapy to maintain the CH50 at zero, and to be without side-effects.20
Though minor increases in D-dimer were observed, an important distinguishing feature supporting a central role of TTP was the absence of significant changes in plasma fibrinogen levels. Moreover, the pathology was most consistent with TTP or TM; the thrombotic lesions were predominantly arteriolar in distribution, platelet antigens were observed in association with vWF and fibrin deposition was minimal. The presence of porcine leukocytes in these lesions further suggests the formation of baboon platelet-xenogeneic leukocyte aggregates in vivo. The finding of transiently elevated D-dimers suggests that fibrinolytic mechanisms were activated as a consequence of the microangiopathic condition, a common finding in advanced TTP.25,31
Despite undergoing pre-transplant splenectomy, an intervention which can ameliorate TTP,38 all animals receiving PBPC TX developed TM. The rationale for the protective PHM therapy used in this study was to combine agents that have anti-thrombotic and anti-inflammatory effects. Heparin has antithrombin activity and also inhibits thrombin-mediated platelet activation.39 Steroids stabilize cellular membranes by inhibiting phospholipases that would release lipid metabolites with stimulating effects on endothelial cells40 and prostacyclin is a potent anti-platelet aggregatory and vasodilator agent,41 which has been used clinically to treat TTP.42 The rationale to use anti-CD40L mAb was primarily to block costimulation of recipient T cells and to facilitate donor stem cell engraftment. However, CD40L is also on activated platelets.43 It has not yet been established if anti-CD40L mAb can modulate aggregation of activated platelets in vivo. Only one baboon in group 4 was treated with the combination therapy PHM and without anti-CD40L mAb. All other animals in this group received PHM therapy in combination with anti-CD40L mAb. The protective effect was similar in all group 4 baboons, with minimal schistocytosis and a mild increase of LDH during the PBPC infusion.
We are currently investigating the mechanisms whereby PBPC activate primate endothelial cells, leukocytes and/or platelets. Of note, we do not observe TM following allogeneic high-dose peripheral blood mobilized progenitor cell TX in baboons utilizing the same regimen as described here. This suggests that there may be a role for structural disparities in relevant molecules that mediate or regulate inflammatory processes between pig cells and baboon platelets, leukocytes and/or endothelium.
Many of the natural anticoagulants and complement regulators are expressed by endothelial cells and monocyte–macrophages. These anticoagulant proteins are largely ineffective across discordant xenogeneic species barriers44 and these molecular incompatibilities could predispose to the TM seen in our studies.
In conclusion, we describe here a TM condition arising as a complication of the TX of xenogeneic porcine PBPC into baboons. We recognized this complication early in our studies, and we immediately tested an antithrombotic and anti-inflammatory regimen to ameliorate this potentially lethal condition. The pharmacologic approach we have described here has now allowed us to successfully prevent morbidity and mortality in 13 subsequent porcine PBPC transplants into baboons, and it is now an accepted and critical component of the treatment plan in our model of xenotransplantation. Of note, anti-platelet agents, such as prostacyclin, heparin and steroids, have each been used to treat TTP with only modest efficacy, but these three agents have not previously been combined as in the present study. Our results, therefore, raise the possibility that this combination of agents may have efficacy in ameliorating BMT- or PBPC TX-associated TM. From an ethical perspective with regard to the health of these animals, we are continuing to examine therapeutic approaches to prevent or treat this TM. Beyond the impact of this information on the future and practical applicability of clinical xenotransplantation, our observations of TM may help facilitate the development of therapeutic agents to treat this serious complication of clinical hematopoietic stem cell transplantation.
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We thank Drs J Fishman and T Spitzer for their considerable advice throughout this study, Drs C Ferran and T Spitzer for their review of the manuscript, A Watts, S Treter and K Nash for excellent technical assistance and Mrs Lisa Bernardo for help in manuscript preparation. We also wish to record our gratitude to Ms Stephanie Spaide of Abbott Laboratories for making available additional Omniflow 4000 infusion pumps on several occasions, and to the following companies for generous gifts of their products: Abbot Laboratories (Hetastarch 6%), Baxter Healthcare (Albumin 5%), Novartis Pharmaceuticals (Sandimmune iv), Glaxo-Wellcome (Zantac iv), and Roche Laboratories, Inc. (CellCept iv). This work was supported in part by National Institutes of Health grant No. 5P01 AI39755 and by a Sponsored Research Agreement between the Massachusetts General Hospital and BioTransplant, Inc.
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Bühler, L., Goepfert, C., Kitamura, H. et al. Porcine hematopoietic cell xenotransplantation in nonhuman primates is complicated by thrombotic microangiopathy. Bone Marrow Transplant 27, 1227–1236 (2001). https://doi.org/10.1038/sj.bmt.1703067
- thrombotic thrombocytopenic purpura
- bone marrow transplantation
- von Willebrand factor
- mixed chimerism
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