Transgenic Expression of Human CD46 on Porcine Endothelium: Effect on Coagulation and Fibrinolytic Cascades During Ex Vivo Human-to-Pig Limb Xenoperfusions : Transplantation

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Original Basic Science—General

Transgenic Expression of Human CD46 on Porcine Endothelium

Effect on Coagulation and Fibrinolytic Cascades During Ex Vivo Human-to-Pig Limb Xenoperfusions

Bongoni, Anjan K. PhD; Kiermeir, David MD; Schnider, Jonas MD; Jenni, Hansjörg; Garimella, Pavan BSc; Bähr, Andrea DVM; Klymiuk, Nikolai PhD; Wolf, Eckhard DVM; Ayares, David PhD; Voegelin, Esther MD; Constantinescu, Mihai A. MD; Seebach, Jörg D MD; Rieben, Robert PhD

Author Information
Transplantation 99(10):p 2061-2069, October 2015. | DOI: 10.1097/TP.0000000000000746

Complement activation and dysregulation of the coagulation system are pivotal issues in pig-to-human xenotransplantation. Indeed, thrombotic microangiopathy and consumptive coagulopathy appear to play a more important role than in allotransplantation, and this has increasingly been recognized as a major barrier to the success of xenotransplantation (reviewed in Satyananda et al and Schmelzle et al1,2). The ability to generate genetically modified pigs with α 1,3-galactosyltransferase-gene knockout (GalTKO) was a major step forward to overcome hyperacute rejection mediated by binding of natural antipig antibodies to the important xenoantigen galactose-α1,3-galactose (Gal).3-6 However, the remaining barriers are by no means insignificant, for instance, the occurrence of non-Gal anti-pig antibody mediating complement activation and dysregulation of coagulation. To overcome these barriers human regulators of complement activation, including CD46,7-9 CD55,10 and CD59,11 have been expressed in transgenic pigs on the GalTKO background.12,13 Because there are molecular interactions between the complement and the coagulation systems,14 regulation of complement activation in xenotransplantation might have beneficial effects beyond the complement system, including a reduction of endothelial cell (EC) activation and coagulation.15

Activation of porcine EC by recipient antibodies and complement and/or by direct exposure to recipient platelets, immune cells, and cytokines changes the normally anticoagulant phenotype of the endothelium to a procoagulant one.16 In addition, there are a number of molecular incompatibilities between the pig and human coagulation systems that boost the activation of coagulation.17,18 Tissue factor is exposed and accumulated by activated EC induced by both immune-dependent and -independent mechanisms.19,20 Activated factor VII binds to tissue factor and initiates the generation of thrombin. In vitro studies have demonstrated that activated porcine EC express another procoagulant factor, fibrinogen-like protein-2 (FGL2), which directly generates thrombin from human prothrombin without the involvement of the regular coagulation cascade.21

The formation of thrombin on activated EC has important physiological consequences. It generates fibrin from fibrinogen22 and also activates platelets through protease activator receptors 1 and 4 to provide a platform for the assembly of the coagulation complexes.23 In addition, thrombin can directly cleave C5 in the absence of classical C3-dependent complement C5 convertase14,24,25 and by this opens a new route for the formation of C5b-9 (the membrane attack complex) that is not controlled by CD46 or CD55. Shedding of membrane-bound heparan sulfate and antithrombin, which is bound to it, contribute to the loss of the natural anticoagulant properties of the endothelium.18,26 Finally, endothelial expression of several other antithrombotic factors, such as tissue factor pathway inhibitor, thrombomodulin, and endothelial protein C receptor, is downregulated (reviewed in Cowan et al18).

Physiological hemostasis depends on a finely tuned process balancing between coagulation (clot formation) and fibrinolysis that leads to the removal of fibrin deposits (clot resolution). The key component of the fibrinolytic system is plasminogen and its active form, plasmin, which is generated by tissue plasminogen activator (tPA) and urokinase plasminogen activator.27 Endothelial cells play a key role in the regulation of fibrinolysis by secreting tPA and its inhibitor, plasminogen activator inhibitor (PAI)-1. Upon exposure to human xenoreactive antibodies and complement, vascular EC in the graft are activated and express PAI-1, which blocks the activity of tPA and changes the endothelium to an antifibrinolytic state.28 Unbalanced production of these components has been implicated in, or associated with, acute vascular rejection.29,30 However, the role of the fibrinolytic system has not been investigated in detail in pig-to-human xenotransplantation.

In addition to antibodies and the plasma cascade systems, human natural killer (NK) cells participate in the innate immune attack against porcine xenografts. The NK cells are able to infiltrate pig organs perfused with human blood and mediate lysis of porcine cells in vitro, either directly or via antibody-dependent cellular cytotoxicity.31-34 One major reason or direct xenogeneic NK cell-mediated cytotoxicity is the failure of porcine Major Histocompatibility Complex class I molecules to be recognized by human NK cell inhibitory receptors.35,36 Consequently, transgenic expression of HLA-E, a nonclassical human Major Histocompatibility Complex class I molecule, on porcine cells has been demonstrated to provide partial protection against human-antipig NK cytotoxicity.37-39

To study the protective effects of transgenic expression of human CD46 and HLA-E on porcine xenografts, forelimbs of wild-type and GalTKO-heterozygous, hCD46/HLA-E double transgenic pigs were ex vivo xenoperfused with whole, heparinized human blood for 12 hours as previously reported. This model represents a useful tool to study immunological, coagulatory, and fibrinolytic-antifibrinolytic responses associated with pig-to-human vascularized xenograft transplantation in the absence of hyperacute rejection. We have already shown that the transgenic expression of hCD46 provided protection against complement-mediated responses.9,40 Our hypothesis for the current study was that the expression of CD46 would also attenuate activation of the coagulation system and prevent the induction of an anti-fibrinolytic state of the endothelium during pig-to-human xenoperfusion.

MATERIALS AND METHODS

Ex Vivo Perfusion Model

Animal experiments in this study were performed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and Swiss National Guidelines. The local animal experimentation committee of the Canton Bern approved this study (permission no. BE45/11). Wild-type (n = 8) and GalTKO-heterozygous, hCD46/HLA-E double transgenic (n = 10) forelimbs of large white pigs (30-40 kg) of both sexes were used to perform ex vivo xenoperfusion with heparinized whole human blood as described previously.9,40 Briefly, 500 mL of whole blood were withdrawn from individual human donors into standard transfusion bags containing 10,000 IU of heparin (Liquemin). The amputated porcine forelimbs were connected to extracorporeal circuits and xenogeneic perfusions were performed for 12 hours. The flow rate was maintained at 100 to 150 mL/min (MEDOS DataStream blood pump, model DP2), temperature was kept at 32°C (Heater-Cooler Unit HCU30), and O2 at 21% by membrane oxygenator (MEDOS Hilite 800 LT).

Tissue and Blood Sampling

Biopsies of skeletal muscle as well as serum and ethylene diamine tetra acetate (EDTA) plasma samples were obtained at baseline and after 12 hours (end) of perfusion. Snap-frozen tissue samples were used for analysis by immunofluorescence staining. Blood samples were centrifuged at 3000 rpm for 7 minutes at 4°C so separate plasma from cells, aliquoted and stored at −80°C for further analysis.

Immunofluorescence

Snap-frozen biopsy samples were cut into 5-μm-thick sections, air dried, and either processed immediately or stored at −80°C until further analysis. After fixation with acetone and hydration, the sections were stained using either 1-step direct or 2-step indirect immunofluorescence techniques. The following reagents were used: BS-I-B4 lectin (fluorescein isothiocyanate [FITC]–labeled, Sigma), goat antihuman IgM-FITC (Sigma), goat antihuman IgG-FITC (Sigma), mouse antihuman C5b-9 (Diatec), rabbit antihuman FGL2 (Aviva Systems Biology), sheep antihuman tissue factor (Affinity Biologicals), goat antihuman fibrinogen (Santa Cruz Biotechnologies), mouse antihuman PAI-1 (Hycult Biotech), rabbit antihuman tPA (Abcam). Cross-reactivity with the respective porcine antigens was verified. Secondary antibodies were goat antimouse IgG-Alexa488, donkey antigoat IgG-Alexa488, donkey antisheep IgG-Alexa488 (all from Molecular Probes), and sheep antirabbit IgG-Cy3 (Sigma). Nuclear staining was done by using 4′,6-diamidino-2-phenylindole (DAPI, Boehringer). The slides were analyzed using a fluorescence microscope (DMI4000B, Leica). Quantification of fluorescence intensity as raw integrated density was performed using Image J software, version 10.2 (National Institutes of Health) on unmanipulated TIFF images.

Markers for Thrombin Generation and Fibrinolysis

Plasma levels of hemostatic markers, including D-dimer (RayBiotech) and prothrombin fragment F1 + 2 (US Biologicals), were measured using commercially available enzyme-linked immunosorbent assay kits according to the manufacturer's instructions.

Cleavage of Human Plasminogen by Human or Porcine tPA

Cleavage of human plasminogen by human or porcine tPA was analyzed in vitro by plasmin-specific chromogenic substrate activity assay. Briefly, purified human plasminogen, as full-length protein (Abcam), was incubated at a concentration of 15 and 30 μg/mL, respectively, with either active human tPA (Abcam) or porcine tPA (Molecular Innovations) at various concentrations (0, 10, and 20 μg/mL). Plasminogen conversion to plasmin was measured as plasmin activity in the reaction mixtures using 3 mmol/L chromogenic S-2251 substrate (Chromogenix).

Inhibition of tPA-Mediated Human Plasminogen Cleavage by Porcine PAI-1

To study the inhibitory effect of porcine PAI-1 on human/porcine tPA activity, 15 and 30 μg/mL of purified human plasminogen was incubated with various concentrations of human or porcine tPA (0, 10, and 20 μg/mL) either in the presence or absence of 20 μg/mL of purified porcine active form of PAI-1 (Innovative Research) for 30 minutes at 37°C. The reaction mixtures were then used for conversion of the plasmin-specific chromogenic S-2251 substrate (3 mmol/L).

PAI-1/tPA Complex Formation

The PAI-1/tPA complexes were measured in EDTA plasma samples from ex vivo xenoperfusions by an inhouse developed Bio-Plex assay using xMAP technology, as described previously.41 Briefly, mouse anti–PAI-1 antibody was coupled to carboxylated polystyrene beads (Luminex) using a Bio-Plex amine coupling kit (Bio-Rad). The coupled beads were then incubated with samples and bound PAI-1/tPA complexes detected using biotinylated rabbit anti-tPA antibody (Molecular Innovations) and streptavidin-PE (Qiagen). Measurement and data analysis were performed with a Bio-Plex 100 array reader and the Bio-Plex Manager software version 6.1.

Statistical Analysis

Data are shown as mean ± standard deviation. They were analyzed using GraphPad Prism 6 (GraphPad Software). Significance was tested using one-way analysis of variance with Bonferroni correction (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001).

RESULTS

Expression of the Gal Epitope and Deposition of Human IgM, IgG, and C5b-9 on Xenoperfused Tissue

The expression of the Gal epitope was analyzed by immunofluorescence on freshly frozen muscle biopsies obtained from pig forelimbs after xenoperfusion with human blood for 12 hours. Staining with the BS-I-B4 lectin showed no difference in Gal epitope expression between wild-type and GalTKO-heterozygous/hCD46/HLA-E double transgenic pig muscle tissues (Figure 1A). Furthermore, deposition of human IgM, IgG, and C5b-9 on EC was tested at baseline and 12-hour xenoperfused tissue samples. Immunofluorescence staining showed significantly higher human IgM and IgG deposition on both wild-type (IgG: P = 0.030, IgM: P < 0.0001) and transgenic (IgG: P = 0.020, IgM: P = 0.031) xenoperfusion samples as compared to their respective baseline samples, with no significant differences between wild-type and transgenic limbs (Figure 1B-C). Also, C5b-9 deposition was present after both wild-type (P < 0.0001) and transgenic limb (P = 0.002) xenoperfusion, but it was significantly (P < 0.0001) lower in transgenic limbs (Figure 1D).

F1-13
FIGURE 1:
Expression of the Gal epitope and deposition of human IgM, IgG and C5b-9 on xenoperfused tissue. A, Gal epitope expression on wild-type and hCD4/HLA-E transgenic pig forelimb muscle samples was tested using BS-I-B4 lectin (from Bandeiraea simplicifolia) by immunofluorescence staining. All tissue samples stained positive for Gal expression with no difference between groups. B-D, Effect of transgenic expression of hCD46 and HLA-E on immunoglobulin and complement deposition. Freshly frozen tissue samples from ex vivo xenoperfused pig limbs were analyzed for deposition of human IgM and IgG as well as C5b-9 using 1-step direct or 2-step indirect immunofluorescence staining. Representative images of (B) IgM, (C) IgG and (D) C5b-9 deposition on 12 hours perfused tissue samples. Scale bars: 75 μm. Wild-type and transgenic xenogeneic perfusion samples showed increased deposition of IgM, IgG, and C5b-9 over baseline, whereas transgenic hCD46 expression resulted in significantly reduced C5b-9 deposition. Quantitative estimation of IgM, IgG, and C5b-9 deposition was performed using Image J software, and statistical analysis was carried out by one-way ANOVA testing with Bonferroni correction. Data are mean ± SD, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001; n = 8 for wild-type and n = 10 for hCD46/HLA-E transgenic pigs. ANOVA indicates analysis of variance.

In vitro, supplementary control experiments were performed using hCD46-only and hCD46/HLA-E double transgenic porcine aortic EC (PAEC). Wild-type, hCD46-only and hCD46/HLA-E transgenic PAEC were treated with normal human serum (NHS, 1:10) for 4 hours and then assessed for complement deposition as well as endothelial activation by measuring CD62E expression and complement-mediated cytotoxicity. The same cells were also grown on microcarrier beads and exposed to whole, nonanticoagulated human blood to assess clotting time. Transgenic expression of hCD46 alone or in combination with HLA-E on PAEC both resulted in significantly reduced C5b-9 deposition (P < 0.0001), CD62E expression (P < 0.0001), PAEC cytotoxicity (P < 0.0001), and prolonged clotting time (P < 0.002) as compared to wild-type PAEC (Figure S1, SDC, https://links.lww.com/TP/B158). In none of these assays did we observe a significant difference between hCD46-single and hCD46/HLA-E double transgenic PAEC.

Reduction of Endothelial Induction of FGL2 by hCD46/HLA-E

Induction of the procoagulant protein FGL2 on the vascular endothelium constitutes a direct mechanism for the production of thrombin from prothrombin. Ex vivo xenoperfusion samples showed a significantly increased FGL2 expression in wild-type (P = 0.0002) and transgenic samples (P = 0.036) as compared to their respective baseline samples. The expression of FGL2 was significantly (P = 0.028) lower in the presence of hCD46/HLA-E as compared to wild-type limb ex vivo xenoperfusions (Figure 2A).

F2-13
FIGURE 2:
Reduction of endothelial expression of tissue factor, deposition of fibrin, and of plasma levels of D-dimers and prothrombin fragment F1 + 2 by hCD46/HLA-E expression on xenoperfused tissue. Xenoperfusion-induced endothelial procoagulant activity was evaluated by immunofluorescence staining on freshly frozen ex vivo xenoperfusion tissue samples for the expression of FGL2, tissue factor and deposition of fibrin as coagulation activation markers. Representative images of IF staining for (A) FGL2, (B) tissue factor and (C) fibrin on sections of muscle biopsies at baseline and after 12 hours of perfusion are shown. Expression of FGL2 and tissue factor as well as deposition of fibrin was significantly lower in samples from hCD46/HLA-E transgenic xenoperfusions as compared to wild-type xenoperfusion limbs. Immunofluorescence staining intensities of FGL2, tissue factor and fibrin were quantitatively measured using Image J software. Measurement of (D) D-dimers and (E) prothrombin fragment F1 + 2 were done in citrate plasma collected at baseline and 12 hours of xenoperfusion as soluble coagulation markers using commercial ELISA kits. After 12 hours of xenoperfusion, these markers were significantly upregulated in both wild-type and transgenic samples, but significantly lower in transgenic as compared to wild-type limb xenoperfusions. Column graphs are mean ± SD analyzed by one-way ANOVA with Bonferroni correction. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001; n = 8 for wild-type and n = 10 for hCD46/HLA-E transgenic pigs. ELISA indicates enzyme linked immunosorbent assay; BL, baseline.

Reduction of Endothelial Expression of Tissue Factor and Deposition of Fibrin by hCD46/HLA-E

To test whether human antipig humoral responses induce a procoagulant phenotype of the pig endothelium, expression of tissue factor and deposition of fibrin were investigated by immunofluorescence staining on ex vivo xenoperfused tissue samples. Quantitative analysis using Image J software revealed a significant increase in tissue factor expression and fibrin deposition on both wild-type (tissue factor: P < 0.0001; fibrin: P = 0.0002) and transgenic (tissue factor: P = 0.004; fibrin: P = 0.003) xenoperfusion samples as compared to respective baseline samples. However, tissue factor expression (P = 0.005) and fibrin deposition (P = 0.009) were significantly reduced in transgenic xenoperfusion samples as compared to wild-type xenoperfusions (Figure 2B-C).

Reduction of Plasma Levels of D-Dimers and Prothrombin Fragment F1 + 2 by hCD46/HLA-E

The direct measurement of thrombin is unreliable because active thrombin is rapidly neutralized by antithrombin. However, quantification of prothrombin fragment F1 + 2 allows for monitoring of the activation of the coagulation cascade, culminating in the formation of thrombin.42 Therefore, citrate plasma samples were collected to quantify prothrombin fragment F1 + 2 and D-dimers (a marker for coagulation/thrombolysis) before and after 12 hours of xenoperfusion. D-dimer levels were markedly increased after perfusion in both wild-type (249.0 ± 40.4 at baseline to 858.9 ± 78.0 ng/mL after 12 hours, P < 0.0001) and transgenic (205.4 ± 12.8 to 738.3 ± 60.5 ng/mL, P < 0.0001) limbs. However, the presence of hCD46/HLA-E transgenes resulted in significantly (P = 0.044) reduced D-dimer formation in transgenic as compared to wild-type limb xenoperfusions (Figure 2D). Also, the prothrombin fragment F1 + 2 was significantly elevated over baseline levels after 12 hours of wild-type (27.5 ± 0.2 to 87.0 ± 8.6 ng/mL, P = 0.0001) and transgenic (27.1 ± 0.3 to 65.9 ± 14.1 ng/mL, P = 0.005) limb xenoperfusions. Comparison of wild-type versus transgenic limb xenoperfusions revealed significantly reduced prothrombin fragment F1 + 2 levels in transgenic perfusions (P = 0.031) (Figure 2E).

Cross-Species Cleavage of Human Plasminogen by Porcine tPA and Inhibition of Human tPA by Porcine PAI-1

Tissue plasminogen activator is a key molecule in the initiation of the fibrinolytic cascade. Potentially, fibrinolysis could be compromised in pig-to-primate xenotransplantation due to a molecular incompatibility between tPA, produced by the porcine endothelium, and plasminogen in the primate blood. Therefore, we investigated the conversion of human plasminogen to plasmin by porcine tPA in vitro. As shown in Figure 3, human plasminogen was cleaved by porcine tPA in a dose-dependent manner with no difference between human and porcine tPA activity when tested on human plasminogen. In addition, incubation of human plasminogen with either human or porcine tPA in the presence of porcine PAI-1 revealed no plasminogen conversion to plasmin. Porcine PAI-1 thus inhibits the cleavage of human plasminogen by both human and porcine tPA (Figure 3B).

F3-13
FIGURE 3:
Cross-species cleavage of human plasminogen by porcine tPA and inhibition of human tPA by porcine PAI-1. A, Conversion of human plasminogen by human or porcine tPA was investigated using purified proteins in vitro and plasmin-specific chromogenic S-2251 substrate. Incubation of human plasminogen full length protein (15 and 30 μg/mL) with 0, 10 and 20 μg/mL of active human or porcine tPA protein resulted in a dose-dependent conversion of plasminogen to plasmin. No significant difference between human and porcine tPA activity was observed. B, Inhibition of human and porcine tPA activity by porcine PAI-1. Incubation of human plasminogen with human as well as porcine tPA in the presence of porcine PAI-1 showed significantly reduced plasmin activity, suggesting that porcine PAI-1 efficiently inhibited both human and porcine tPA activity on human plasminogen. Column graphs are mean ± SD analyzed by one-way ANOVA with Bonferroni correction. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, n ≥ 3 separately performed assays.

Improved Fibrinolysis by Transgenic hCD46/HLA-E Expression

Fibrinolysis was analyzed during ex vivo human-to-pig xenoperfusion by measuring tPA and PAI-1 expression. Baseline tissue samples showed strong tPA staining, mainly on vascular endothelium, which is characteristic for the healthy, profibrinolytic state of the endothelium. Activation of the endothelium is characterized by downregulation of tPA and upregulation of PAI-1 expression, leading to an antifibrinolytic endothelial phenotype. Indeed, after 12 hours of xenoperfusion, a significant decrease of tPA expression was observed in wild-type (P = 0.003 vs baseline), but not in transgenic limbs (P = 0.152 vs baseline). After 12 hours of xenoperfusion, expression of porcine tPA in wild-type pig limbs was significantly (P = 0.009) lower than that in transgenic limbs (Figure 4A). In contrast, no PAI-1 expression was observed at baseline, neither in wild-type nor in transgenic limbs. Moreover, the expression of PAI-1 was significantly induced in both the vascular endothelium of wild-type and transgenic xenoperfused limbs (both P < 0.0001 vs baseline). The final PAI-1 expression was significantly higher in wild-type as compared to transgenic limbs (P = 0.022) at the end of the 12 hour xenoperfusion period (Figure 4B). To determine the changes in the fibrinolytic activity also in the fluid phase, the formation of PAI-1/tPA complexes was measured in EDTA plasma samples obtained from ex vivo human-to-pig limb xenoperfusions. Baseline values of PAI-1/tPA complexes were minimal and not significantly different between wild-type and transgenic xenoperfused limbs. Formation of PAI-1/tPA complexes significantly increased after 12 hours of xenoperfusion in both wild-type (P = 0.0007 vs baseline) and transgenic limbs (P = 0.007 vs baseline). However, the presence of hCD46/HLA-E resulted in a significantly reduced PAI-1/tPA complex formation (P = 0.015; Figure 4C).

F4-13
FIGURE 4:
Improved fibrinolysis by transgenic hCD46/HLA-E expression on xenoperfused tissue. A-B, Tissue samples obtained from baseline and 12 hours xenoperfused porcine limbs were stained by immunofluorescence for the expression of tPA and PAI-1. A, Expression of tPA was significantly reduced in 12 hours xenoperfused wild-type tissue samples, but not in transgenic limbs, as compared to respective baselines and xenoperfused transgenic limbs. In transgenic limbs, expression of tPA was significantly higher after 12 hours xenoperfusion than in wild-type limbs. B, PAI-1 expression was significantly upregulated after 12 hours xenoperfusion in wild-type and transgenic limbs as compared to respective baselines. However, expression of PAI-1 was significantly lower in transgenic than in wild-type limbs after 12 hours of xenoperfusion. Immunofluorescence staining intensities of tPA and PAI-1 were quantitatively measured using Image J software. C, Formation of PAI-1/tPA complexes was analyzed as fluid-phase antifibrinolytic marker in EDTA plasma samples. Significantly increased PAI-1/tPA complex formation over baseline was observed in both, transgenic and wild-type limbs after 12 hours perfusion, but the levels were significantly lower in transgenic as compared to wild-type limbs. Statistical analysis was done by one-way ANOVA testing with Bonferroni correction. Data are mean ± SD, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001; n = 8 for wild-type and n = 10 for hCD46/HLA-E transgenic pigs.

DISCUSSION

Because clot formation in the vasculature is detrimental for the affected tissue or organ, coagulation is tightly regulated on several different levels. To achieve efficient clot formation, the production of tissue factor needs to be complemented by dysfunctional or inhibited anticoagulant pathways along with suppression of fibrinolysis.43,44 In the current study, we demonstrate using a human-to-pig ex vivo limb perfusion model that expression of hCD46 attenuates the activation of the coagulation system as well as that it supports the establishment of a procoagulant, antifibrinolytic phenotype of the porcine endothelium. We used heparinized human blood to perfuse porcine extremities, which is known to have an effect on both the complement and the coagulation system.45-47 The observed prothrombotic and antifibrinolytic effects induced by the perfusion with human blood may therefore have been attenuated. However, despite of the heparinization, fibrin deposition, thrombin generation as well as presence of several markers for the activation of the complement system and inhibition of fibrinolysis were clearly detectable. This underscores the strength of the procoagulant and antifibrinolytic reaction occurring in this xenoperfusion model. Heparinization was equally used in perfusions of GalTKO-heterozygous/hCD46/HLA-E double transgenic and wild-type limbs, and the observed effects of the transgenes are therefore independent of the heparin used in the perfusate.

Several factors contribute to hyperacute rejection of xenografts, but the key events are binding of xenoreactive natural anti-Gal antibodies to graft endothelium via Gal epitopes and the subsequent activation of the complement system.48 In addition, anti–non-Gal antibodies also mediate humoral responses against xenografts, but only after days or weeks.49,50 We measured the expression of the Gal epitope as well as binding of human IgM and IgG in tissue of porcine limbs xenoperfused with human blood, without finding differences between GalTKO-heterozygous/hCD46/HLA-E transgenic and wild-type pig limbs. In contrast, a significantly reduced C5b-9 deposition in transgenic tissue was shown, confirming our previous data on reduction of soluble C5b-9 by transgenic expression of hCD46/HLA-E during xenoperfusion.9 These data corroborate that the expression of hCD46 indeed inhibits the terminal pathway of complement activation by blocking the central complement proteins C3b and C4b. The pig limbs, which we used for perfusion with human blood, also expressed HLA-E to inhibit NK cell activation. We did not have hCD46-only transgenic pigs available as control for the effect of HLA-E in our experiments. However, we performed in vitro experiments in which we measured complement activation, EC activation based on E-selectin expression, cytotoxicity, and clotting time with whole human blood. These experiments were performed with both, hCD46 single and hCD46/HLA-E double transgenic PAEC and they revealed no difference between single and double transgenic cells (Figure S1, SDC, https://links.lww.com/TP/B158). We assume, therefore, that the HLA-E transgene only played a minor role, if any, in the present study. Data on the effect of HLA-E on NK cell activation in our perfusion model will be reported elsewhere by Puga Yung et al.

Local expression of procoagulant proteins on the vascular endothelium is thought to play an important role in thrombus formation. FGL2, for example, is expressed and differentially regulated in many cell types, including EC.51 Membrane-associated FGL2 has an inducible prothrombinase activity that directly converts prothrombin into thrombin, resulting in fibrin production independent of the classical coagulation cascade. In addition, it participates in cell adhesion and migration as well as regulation of transcription factors.52,53 The secreted form of FGL2 also has an immunomodulatory activity.21,54 Here, we show that FGL2 was significantly upregulated in ex vivo xenoperfused wild-type limbs, indicating an important role in the pathophysiology of xenograft associated coagulation dysregulation. In contrast, hCD46/HLA-E transgenic xenoperfused limbs expressed significantly less FGL2. Furthermore, the generation of the prothrombin fragment F1 + 2, an activation peptide released from prothrombin during thrombin formation,42 and D-dimers was reduced by transgenic hCD46/HLA-E expression on xenoperfused tissue. These data, together with the reduction of tissue factor expression and the reduced deposition of fibrin, provide evidence for a protective anti-coagulant effect of the hCD46/HLA-E transgenes. However, prevention of EC activation by human membrane-bound complement regulators was shown to be insufficient to avoid thrombosis formation in pig-to-primate xenotransplantation.18 This may in part be due to the molecular incompatibilities between human thrombin and porcine thrombomodulin, preventing the activation of human protein C and leading to a procoagulant state.55 Additional strategies to prevent coagulation, such as expression of human anticoagulant proteins, are therefore needed in pig-to-primate xenotransplantation (reviewed in18). Transgenic expression of human thrombomodulin has indeed been shown to be a promising approach.56

Little is known on the fibrinolytic system in xenotransplantation models. In a very early report, Jørgensen et al30 showed activation of fibrinolysis in a model of rabbit kidney xenoperfusion. However, the short duration of these perfusions, only 60 minutes, did most probably not allow for detection of the antifibrinolytic effect of PAI-1, which is only produced by EC several hours after endothelial activation.57 In fact, Kalady et al29 described a progressive loss of the fibrinolytic capacity of porcine endothelium in actual pig-to-baboon xenotransplantation. We therefore explored whether molecular incompatibilities between human and pig also exist in the fibrinolytic cascade. In our experiments, this was not the case. We found that human plasminogen was cleaved into active plasmin by porcine tPA as effectively as with human tPA and that the activity of both human and porcine tPA was inhibited by porcine PAI-1. The PAI-1 expressed by activated porcine EC will therefore bind both human and porcine tPA and inhibit their plasminogen-cleaving activities, thereby preventing the degradation of emerging fibrin clots. Indeed, in wild-type xenoperfusions, we found an increased expression of PAI-1, whereas the expression of tPA was decreased, indicating the induction of an antifibrinolytic state of the endothelium. This was further supported by the formation of PAI-1/tPA complexes, which are known as reliable antifibrinolytic markers.58,59 Transgenic expression of hCD46/HLA-E resulted in significantly reduced PAI-1 expression as well as PAI-1/tPA complex formation during xenoperfusion.

For pig-to-nonhuman primate transplantation, it is currently accepted standard to use homozygous GalTKO pigs as the “platform” on which additional genetic modifications can be assembled, such as one or more human regulators of complement activation and possibly other human proteins like thrombomodulin, TFPI, or CD39.5,60 Gal is the major target for human and nonhuman primate antiporcine antibodies, which initiate complement activation and subsequently hyperacute rejection. Deletion of Gal epitopes by the generation of GalTKO pigs has greatly reduced the incidence of hyperacute rejection of porcine grafts in nonhuman primates.61-63 It is therefore a limitation of our study that we did not have homozygous GalTKO pigs available for our experiments and instead used heterozygous GalTKO animals which express Gal at approximately the same level as wild-type pigs. However, the combination of GalTKO and hCD46 does not prevent rejection in actual pig-to-nonhuman primate transplantations. Instead, the occurrence of thrombotic microangiopathy has been reported, despite the absence of the Gal antigen.64 We assume, therefore, that the observed effect of hCD46/HLA-E expression on coagulation and fibrinolysis, will also be of importance in a more clinically relevant, homozygous GalTKO setting.

In conclusion, transgenic hCD46/HLA-E expression on pig vascular endothelium resulted not only in a significant reduction of complement activation but also in prevention of the establishment of a procoagulant and antifibrinolytic state of the porcine endothelium during human-to-pig limb xenoperfusion with human blood.

ACKNOWLEDGMENTS

We thank Dr. Daniel Mettler, Mrs. Olgica Beslac and Mr. Daniel Zalokar from the Experimental Surgery Unit, as well as Alain Despont, and Julie Denoyelle, Department of Clinical Research, University of Bern, for expert technical support. Images were acquired on equipment and service supported by the Microscopy Imaging Center of the University of Bern.

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