Expression of human thrombomodulin by GalTKO.hCD46 pigs modulates coagulation cascade activation by endothelial cells and during ex vivo lung perfusion with human blood

Abstract

Thrombomodulin is important for the production of activated protein C (APC), a molecule with significant regulatory roles in coagulation and inflammation. To address known molecular incompatibilities between pig thrombomodulin and human thrombin that affect the conversion of protein C into APC, GalTKO.hCD46 pigs have been genetically modified to express human thrombomodulin (hTBM). The aim of this study was to evaluate the impact of transgenic hTBM expression on the coagulation dysregulation that is observed in association with lung xenograft injury in an established lung perfusion model, with and without additional blockade of nonphysiologic interactions between pig vWF and human GPIb axis. Expression of hTBM was variable between pigs at the transcriptional and protein level. hTBM increased the activation of human protein C and inhibited thrombosis in an in vitro flow perfusion assay, confirming that the expressed protein was functional. Decreased platelet activation was observed during ex vivo perfusion of GalTKO.hCD46 lungs expressing hTBM and, in conjunction with transgenic hTBM, blockade of the platelet GPIb receptor further inhibited platelets and increased survival time. Altogether, our data indicate that expression of transgenic hTBM partially addresses coagulation pathway dysregulation associated with pig lung xenograft injury and, in combination with vWF-GP1b-directed strategies, is a promising approach to improve the outcomes of lung xenotransplantation.

Keywords: activated protein C; coagulation; ex vivo perfusion; lung; thrombomodulin; xenotransplantation.

Figure 1

Relative hTBM expression and function in GalKO.hCD46.hTBM PAEC. Porcine aortic endothelial cells (PAEC) isolated from pICAM-2 (n=13) and pTBM (n=5) promoter groups were analyzed along with GalTKO.hCD46 (#504-7) PAEC and HUVEC as negative and positive controls respectively. (A) hTBM mRNA levels were measured by RT-qPCR. Values represent mean ± SEM of at least two independent experiments. (B) PAEC cell surface hTBM protein expression was analyzed by flow cytometry as shown in SI Figure 1. Results are expressed as the proportion of endothelial cells (CD31+) expressing hTBM (CD141+) compared to the isotype control. Values represent mean ± SEM of at least three independent experiments. (C) Analysis of APC generation by GalTKO.hCD46.hTBM as described in Methods. APC results were normalized to a HUVEC reference of 1.0. APC assay revealed that human protein C was effectively converted to APC in the presence of each transgenic PAEC line studied here. Values represent mean ± SEM of at least three independent experiments.

Figure 2

In-vitro flow chamber perfusion of human blood over confluent porcine endothelia. (A, C) Representative images showing thrombosis formation stimulated by GalTKO.hCD46 and GalTKO.hCD46.hTBM endothelia. (B, D) Qualitatively confirmed results by 3-dimensional surface representations visually depicting thrombus volume. (E) Summary of results from replicate in-vitro xenoperfusions of porcine wild-type (WT), GalTKO.hCD46 and GalTKO.hCD46.hTBM endothelia with human blood. a.u., arbitrary units; TV, thrombus volume; Δ, relative change; SA, percent surface area coverage; FR, fluorescence ratio; WT, wild type. For Δ & P values, GalTKO.hCD46 are compared to WT, and GalTKO.hCD46.hTBM are compared to GalTKO.hCD46. Values are expressed as mean ± SEM.

Figure 3

Survival of porcine lungs perfused ex vivo with human blood, by experimental group. Survival was defined by hemodynamic (PVR) or physiologic (gas exchange, loss of barrier function) lung failure endpoint criteria, or attainment of an arbitrary 4 hour interval with preserved graft function. Experiments using hTBM-expressing lungs that received αGPIb-treatment all ‘survived’ to the elective termination at four hours. This survival was significantly longer than in any other group (* p=0.031 vs. GalTKO.hCD46.hTBM; p=0.027 vs. GalTKO.hCD46 + αGPIb; p=0.001 vs. GalTKO.hCD46).

Figure 4

Physiologic perturbations during ex vivo lung perfusion. (A) Pulmonary vascular resistance during ex vivo lung perfusion. PVR is expressed as a function of perfusion time, by experimental group. Time 0 represents measurements obtained during the first minute of lung perfusion. Organs in all experimental groups showed a rise in PVR during the first 30min of perfusion with GalTKO.hCD46 + αGPIb lungs showing the lowest values between 1 and 4 hours of perfusion (e.g. at 1h: 140±14 vs. 229±42 mmHg*min/L, p=0.012). (B) Complement cascade activation. Plasma levels of complement activation byproduct C3a, expressed as the amount of complement fragments produced above the pre-perfusion baseline. Reference GalTKO.hCD46 +/− αGPIb experiments showed less C3a elaboration than experiments using lungs with hTBM.

Figure 5

Platelet counts and platelet activation during ex vivo lung perfusion. (A) Platelet sequestration, expressed as the percentage of platelets remaining in the perfusate, was similar in GalTKO.hCD46 and GalTKO.hCD46.hTBM groups during the first 2 hours of perfusion but was significantly delayed with both genetics during the first 60 minutes when αGPIb be was given (e.g. ⁰ at 30min: 60±9 vs. 40±5; p=0.036). (B) Total activation of platelets, as plasma levels of βTG, was significantly reduced in hTBM lungs when compared to GalTKO.hCD46 lungs (e.g. * at 4h: 545±125 vs. 1134±124; p=0.007). αGPIb-treatment further reduced βTG elaboration. (C) Activation of circulating platelets, as CD41+ platelets expressing CD62P, was significantly reduced in association with hTBM lungs when compared to GalTKO.hCD46 (e.g. # at 4h: 8.0± 3.6 vs. 23.2±4.6; p=0.045) and nearly completely prevented when hTBM lungs were treated with αGPIb (e.g. + at 4h: 1.7±1.6; p=0.001).

Figure 6

Physiologic perturbations during ex vivo lung perfusion. (A) Thromboxane elaboration: Plasma thromboxane B2 levels were similar for all groups at the 15min time point. Both hTBM groups showed higher TXB2 values between 1 and 4 hours of perfusion. (B) Thrombin generation: Activation of the coagulation cascade was detected by the formation of thrombin, measured in plasma as level of prothrombin fragment F1+2. No significant differences were observed between groups. (C) Blood neutrophil count: Neutrophil sequestration from the blood perfusate was not significantly different between groups. Data is expressed as change from the baseline and shown as the mean ± SEM of surviving experiments.

Figure 7

hTBM expression by IHC in lung tissue prior to ex vivo lung perfusion. (A) Control WT lung (747C) does not show hTBM expression. (B) shows a representative staining of a pTBM promotor lung (A111-5); (C) and (D) show hTBM stainings in lungs with ICAM-2 promoter driving hTBM expression (558-3 and 558-1). HTBM expression in the endothelium was overall strong and more ubiquitously distributed in ICAM-2 promoter lungs when compared to pTBM promoter lungs. Expression analyses are summarized in SI Table 1.

Figure 8

Lung hTBM expression during perfusion with human blood. (A) Pig lung tissue was stained by two-color immunofluorescence for von Willebrand (green), identifying endothelium, and TBM (red) as described in Methods. Samples were randomly selected from 3 lungs in each group. Representative pictures from GalTKO.hCD46 and GalTKO.hCD46.hTBM from the ICAM-2 and TBM promoter groups at various time-points during lung perfusion with human blood. There was no expression of hTBM seen in GalTKO.hCD46 control lungs (fourth row). Lungs from both the ICAM-2 (left) and pTBM (right) promoter-derived GalTKO.hCD46.hTBM transgenic pigs showed high levels of vascular hTBM expression. (B) Slides were evaluated in a blinded fashion with respect to transgene, promoter, and time point, for the expression of hTBM on a semi-quantitative scale of 0-3. Expression of hTBM was found to decrease slightly at later time points. Each colored line represents an individual animal scored for hTBM expression. Red points indicate that hTBM was not detected in the GalTKO.hCD46 control pig lungs perfused with human blood. (C) Soluble thrombomodulin levels: While reference experiments only show a slight increase in soluble TBM levels in blood plasma, experiments using hTBM lungs show a 3-4 times higher rise, measured between 2 and 4 hours of perfusion (Final value (120-240), presumably reflecting thrombomodulin shed from the lung xenograft.