Can regulatory T cells improve outcomes of sensitised patients after HLA-Ab incompatible renal transplantation: study protocol for the Phase IIa GAMECHANgER-1 trial.

BACKGROUND: Kidney transplantation is the gold-standard treatment for patients with kidney failure. However, one-third of patients awaiting a kidney transplant are highly sensitized to human leukocyte antigens (HLA), resulting in an increased waiting time for a suitable kidney, more acute and chronic rejection, and a shorter graft survival compared to non-highly sensitised patients. Current standard immunosuppression protocols do not adequately suppress memory responses, and so alternative strategies are needed. Autologous polyclonally expanded regulatory T cells (Tregs) have been demonstrated to be safe in transplant settings and could be a potential alternative to modulate memory immune alloresponses. METHODS: The aim of this trial is to determine whether adoptive transfer of autologous Tregs into HLA sensitised patients can suppress memory T and B cell responses against specific HLA antigens. This is a two-part, multi-centre, prospective clinical trial, comprising an observational phase (Part 1) aiming to identify patients with unregulated cellular memory responses to HLA (Pure HLA Proteins) followed by an interventional phase (Part 2). The first 9 patients identified as being eligible in Part 1 will undergo baseline immune monitoring for 2 months to inform statistical analysis of the primary endpoint. Part 2 is an adaptive, open labelled trial based on Simon's two-stage design, with 21 patients receiving Good Manufacturing Practice (GMP)-grade polyclonally expanded Tregs to a dose of 5-10 × 106 cells/kg body weight. The primary EP is suppression of in vitro memory responses for 2 months post-infusion. 12 patients will receive treatment in stage 1 of Part 2, and 9 patients will receive treatment in stage 2 of Part 2 if ≥ 50% patients pass the primary EP in stage 1. DISCUSSION: This is a prospective study aiming to identify patients with unregulated cellular memory responses to Pure HLA Proteins and determine baseline variation in these patterns of response. Part 2 will be an adaptive phase IIa clinical trial with 21 patients receiving a single infusion of GMP-grade polyclonally expanded Tregs in two stages. It remains to be demonstrated that modulating memory alloresponses clinically using Treg therapy is achievable. TRIAL REGISTRATION: EudraCT Number: 2021-001,664-23. REC Number: 21/SC/0253. Trial registration number ISRCTN14582152.

Thrombalexins: Cell-Localized Inhibition of Thrombin and Its Effects in a Model of High-Risk Renal Transplantation.

Allograft transplantation into sensitized recipients with antidonor antibodies results in accelerated antibody-mediated rejection (AMR), complement activation, and graft thrombosis. We have developed a membrane-localizing technology of wide applicability that enables therapeutic agents, including anticoagulants, to bind to cell surfaces and protect the donor endothelium. We describe here how this technology has been applied to thrombin inhibitors to generate a novel class of drugs termed thrombalexins (TLNs). Using a rat model of hyperacute rejection, we investigated the potential of one such inhibitor (thrombalexin-1 [TLN-1]) to prevent acute antibody-mediated thrombosis in the donor organ. TLN-1 alone was able to reduce intragraft thrombosis and significantly delay rejection. The results confirm a pivotal role for thrombin in AMR in vivo. This approach targets donor organs rather than the recipient and is intended to be directly translatable to clinical use.

Thrombalexin: Use of a Cytotopic Anticoagulant to Reduce Thrombotic Microangiopathy in a Highly Sensitized Model of Kidney Transplantation.

Early activation of coagulation is an important factor in the initiation of innate immunity, as characterized by thrombotic microangiopathy (TMA). In transplantation, systemic anticoagulation is difficult due to bleeding. A novel "cytotopic" agent, thrombalexin (TLN), combines a cell-membrane-bound (myristoyl tail) anti-thrombin (hirudin-like peptide [HLL]), which can be perfused directly to the donor organ or cells. Thromboelastography was used to measure time to clot formation (r-time) in both rhesus and human blood, comparing TLN versus HLL (without cytotopic tail) versus negative control. Both TLN- and HLL-treated rhesus or human whole blood result in significantly prolonged r-time compared to kaolin controls. Only TLN-treated human endothelial cells and neonatal porcine islets prolonged time to clot formation. Detection of membrane-bound TLN was confirmed by immunohistochemistry and fluorescence activated cell sorter. In vivo, perfusion of a nonhuman primate kidney TLN-supplemented preservation solution in a sensitized model of transplantation demonstrated no evidence of TLN systemically. Histologically, TLN was shown to be present up to 4 days after transplantation. There was no platelet deposition, and TMA severity, as well as microvascular injury scores (glomerulitis + peritubular capillaritis), were less in the TLN-treated animals. Despite promising evidence of localized efficacy, no survival benefit was demonstrated.

Highly sensitised individuals present a distinct Treg signature compared to unsensitised individuals on haemodialysis.

Introduction: Highly sensitised (HS) patients represent up to 30% of patients on the kidney transplant waiting list. When they are transplanted, they have a high risk of acute/chronic rejection and long-term allograft loss. Regulatory T cells (Tregs) (CD4+CD25hiCD127lo) are T cells involved in the suppression of immune alloresponses. A particular subset, called T follicular regulatory T cells (Tfr, CXCR5+Bcl-6+), is involved in regulating interactions between T effectors and B cells within the germinal centre and can be found in peripheral blood. Therefore, we wanted to identify specific subsets of Tregs in the peripheral blood of HS individuals. Methods: We recruited prospectively healthy volunteers (HV) (n = 9), non-sensitised patients on haemodialysis (HD) (n = 9) and HS individuals, all of whom were on haemodialysis (n = 15). Results: We compared the Treg phenotypes of HV, HD and HS. HS patients had more CD161+ Tregs (p = 0.02) and more CD45RA-CCR7- T effectors (Teffs) (p = 0.04, memory Teffs able to home to the germinal centre) compared to HVs. HS patients had more Bcl-6+ Tregs (p < 0.05), fewer Th1-like Tregs, more Th2-like Tregs (p < 0.001) and more CD161+ (p < 0.05) Tregs compared to HD patients. This population has been described to be highly suppressive. HD had a deficiency in a Th17-like CD161+ effector Treg cluster (cluster iii., CCR6+CCR4+CXCR3- CD39+CD15s+ICOS-CCR7-CD161+) (p < 0.05). Discussion: This is the first study presenting a deep Treg phenotype in HS patients. We confirmed that HS patients had more of a Th17-like CD161+ effector Treg from population III (CD4+CD25hiCD127loCD45RA-) compared to non-sensitised patients on HD. The clinical relevance of this highly suppressive Tregs population remains to be determined in the context of transplantation.

Transplant accommodation--are the lessons learned from xenotransplantation pertinent for clinical allotransplantation?

"Accommodation" refers to a vascularized transplant that has acquired resistance to antibody-mediated rejection (AMR). The term was coined in 1990, but the phenomenon was first described after clinical ABO-incompatible (ABOi) renal transplantation in the 1980s and is recognized as a common outcome in this context today. Because of the absence, until recently of reliable animal models of allograft accommodation, it has been studied extensively by investigators in the xenotransplantation field. With recent advances in the ability to recognize and diagnose AMR in human organs, the growth of desensitization programmes for transplantation into sensitized recipients and the availability of therapies that have the potential to promote accommodation, it is timely to review the literature in this area, identifying lessons that may inform preclinical and clinical studies in the future.

Renal allograft recipients fail to increase interferon-γ during invasive fungal diseases.

Invasive fungal diseases are a major cause of death in renal allograft recipients. We previously reported that adjunctive recombinant human interferon-γ therapy has clinical utility for invasive fungal diseases after renal transplantation. We have now developed a rapid peripheral blood-based quantitative real-time PCR assay that enables accurate profiling of cytokine imbalances. Our preliminary studies in renal transplant patients with invasive fungal diseases suggest that they fail to mount an adequate interferon-γ response to the fungal infection. In addition, they have reduced IL-10 and increased TNF-α when compared to stable renal transplant patients. These preliminary cytokine profiling-based observations provide a possible explanation for the therapeutic benefit of adjunctive human interferon-γ therapy in renal allograft recipients with invasive fungal diseases.

Outcome of patients with preformed donor-specific antibodies following alemtuzumab induction and tacrolimus monotherapy.

It has been shown that low-level preformed donor-specific antibodies (DSAbs) detected by luminex beads in the setting of a negative CDC and flow cytometry crossmatch (CDC/FCXM) are associated with inferior allograft outcomes. The relevance of preformed DSAbs in patients receiving alemtuzumab induction and tacrolimus monotherapy has not been studied. Four hundred and eighty renal transplant recipients with a negative CDC/FCXM had their pretransplant sera retrospectively screened for DSAbs. 45/480 (9.4%) of patients were found to have preformed DSAbs. Females and patients receiving regrafts were more likely to have a DSAb (p = 0.008 and p < 0.0001, respectively). Patients with DSAbs had inferior allograft survival (p = 0.047), increased incidence of antibody-mediated rejection (p < 0.0001) and inferior allograft function at 6 months posttransplant (p = 0.017). Patients with HLA class I DSAb (alone or in combination with a Class II DSAb) with high mean fluorescence intensities (MFIs) were at highest risk. We conclude that patients with preformed DSAb are at high risk of adverse outcomes when receiving a minimal immunosuppressive regime incorporating alemtuzumab induction. Patients found to have a preformed DSAb despite a negative crossmatch might benefit from augmented immunosuppression.

Major histocompatibility complex class II-expressing endothelial cells induce allospecific nonresponsiveness in naive T cells.

The role of endothelial cells (EC) in initiating a primary T cell response is of importance in clinical transplantation and autoimmunity since EC are the first allogeneic target encountered by the recipient's immune system and may display tissue-specific autoantigens in the context of an inflammatory response. In this study, we have investigated the antigen-presenting cell function of human umbilical vein-derived EC (HUVEC), depleted of constitutively major histocompatibility complex class II+ cells and induced to express class II molecules by interferon-gamma. The results show that HUVEC do not express B7 but can support proliferation by antigen-specific T cell clones. In contrast, they were unable to initiate a primary alloresponse using three independent HUVEC cultures and MHC class II-mismatched CD4+ T cells from eight donors. The response to HUVEC was reconstituted by trans-costimulation provided by DAP.3 transfectants expressing human B7.1. Coculture of peripheral blood T cells with EC expressing allogeneic DR molecules had markedly different effects on CD45RO+ and RA+ subsets. Subsequent reactivity of the RO+ T cells was unaffected by exposure to EC, indicating a neutral encounter. In contrast, culture with DR+ EC induced allospecific nonresponsiveness in RA+ T cells.

Exogenous interferon-gamma immunotherapy for invasive fungal infections in kidney transplant patients.

The incidence of invasive fungal infections (IFIs) in nonneutropenic solid organ transplant patients is increasing. We report our clinical experience with the use of interferon-gamma (IFN-gamma) immunotherapy in seven renal transplant patients who developed life threatening, disseminated IFIs refractory to conventional antifungal drug therapy. The infections were all microbiologically and histologically proven. The rapid cure of these disseminated infections with exogenous IFN-gamma injections was not associated with impaired kidney allograft function despite the use of liposomal amphotericin B in all cases. No clinical toxicity from the IFN-gamma immunotherapy was seen and no IFI relapsed during long-term follow-up. Our experience is both uncontrolled and in patients with unpredictable fungal infection-related outcomes. However, compared to standard approaches, the accelerated cure of life threatening, disseminated IFIs with 6 weeks of combination antifungal drug therapy and IFN-gamma immunotherapy saved lives, retained allograft function and led to substantial cost savings in this small patient group.

Recipient tissue factor expression is associated with consumptive coagulopathy in pig-to-primate kidney xenotransplantation.

Consumptive coagulopathy (CC) remains a challenge in pig-to-primate organ xenotransplantation (Tx). This study investigated the role of tissue factor (TF) expression on circulating platelets and peripheral blood mononuclear cells (PBMCs). Baboons (n = 9) received a kidney graft from pigs that were either wild-type (n = 2), alpha1,3-galactosyltransferase gene-knockout (GT-KO; n = 1) or GT-KO and transgenic for the complement-regulatory protein, CD46 (GT-KO/CD46, n = 6). In the baboon where the graft developed hyperacute rejection (n = 1), the platelets and PBMCs expressed TF within 4 h of Tx. In the remaining baboons, TF was detected on platelets on post-Tx day 1. Subsequently, platelet-leukocyte aggregation developed with formation of thrombin. In the six baboons with CC, TF was not detected on baboon PBMCs until CC was beginning to develop. Graft histopathology showed fibrin deposition and platelet aggregation (n = 6), but with only minor or no features indicating a humoral immune response (n = 3), and no macrophage, B or T cell infiltration (n = 6). Activation of platelets to express TF was associated with the initiation of CC, whereas TF expression on PBMCs was concomitant with the onset of CC, often in the relative absence of features of acute humoral xenograft rejection. Prevention of recipient platelet activation may be crucial for successful pig-to-primate kidney Tx.

Prospects for xenografting.

New insights into the mechanisms of hyperacute rejection, endothelial cell activation and accommodation are clarifying the processes that determine the rejection of discordant xenografts. Recent advances include the identification of endothelial cell antigens targeted by human anti-pig antibodies and an increasing understanding of the mechanisms underlying endothelial cell activation. Novel ways to prevent hyperacute rejection, including new therapeutic agents and the use of organs from transgenic animals, are promising to significantly improve the early survival of pig organs transplanted into man. Clinical xenotransplantation has become a realizable goal.

Postinjury vascular intimal hyperplasia in mice is completely inhibited by CD34+ bone marrow-derived progenitor cells expressing membrane-tethered anticoagulant fusion proteins.

BACKGROUND: Coagulation proteins promote neointimal hyperplasia and vascular remodelling after vessel injury, but the precise mechanisms by which they act in vivo remain undetermined. OBJECTIVES: This study, using an injury model in which the neointima is derived from bone marrow (BM)-derived cells, compared inhibition of tissue factor or thrombin on either BM-derived or existing vascular smooth muscle cells. METHODS: Two transgenic (Tg) mouse strains expressing membrane-tethered tissue factor pathway inhibitor (TFPI) or hirudin (Hir) fusion proteins driven by an alpha smooth muscle actin (SMA) promoter were generated (alpha-TFPI-Tg and alpha-Hir-Tg) and the phenotype after wire-induced endovascular injury was compared with that in wild-type (WT) controls. RESULTS: WT mice developed progressive neointimal expansion, whereas injury in either Tg was followed by repair back to a preinjured state. This was also seen when WT mice were reconstituted with BM from Tg mice but not when Tgs were reconstituted with WT BM, in which injury was followed by slowly progressive neointimal expansion. Injection of CD34+ cells from Tg mice into injured WT mice resulted in the accumulation of fusion protein-expressing cells from day 3 onwards and an absence of neointimal hyperplasia in those areas. CONCLUSIONS: Neointimal development after wire-induced endovascular injury in mice was completely inhibited when BM-derived cells infiltrating the damaged artery expressed membrane tethered anticoagulant fusion proteins under an alpha-SMA promoter. These findings enhance our understanding of the pathological role that coagulation proteins play in vascular inflammation.

Factor VII promotes hepatocellular carcinoma progression through ERK-TSC signaling.

We previously demonstrated PAR2 starts upstreamed with tissue factor (TF) and factor VII (FVII), inhibited autophagy via mTOR signaling in HCC. However, the mechanism underlying for merging functions of PAR2 with the coagulation system in HCC progression remained unclear. The present study aimed to investigate the role of TF, FVII and PAR2 in tumor progression of HCC. The expressions of TF, FVII and PAR2 from HCC specimens were evaluated by immunohistochemical stains and western blotting. We found that the expression of FVII, but not TF and PAR2, directly related to the vascular invasion and the clinical staging. Importantly, a lower level of FVII expression was significantly associated with the longer disease-free survival. The addition of FVII but not TF induced the expression of PAR2 and phosphorylation of ERK1/2, whereas knockdown of FVII decreased PAR2 expression and ERK1/2 phosphorylation in HCC cell lines. Furthermore, levels of phosphor-TSC2 (Ser664) were increased after treatment with FVII and PAR2 agonist whereas these were significantly abolished in the presence of a potent and specific MEK/ERK inhibitor U0126. Moreover, mTOR knockdown highly reduced Hep3B migration, which could be reverted by FVII but not TF and PAR2. These results indicated that FVII/PAR2 signaling through MEK/ERK and TSC2 axis for mTOR activation has potent effects on the migration of HCC cells. In addition, FVII/PAR2 signaling elicits an mTOR-independent signaling, which promotes hepatoma cell migration in consistent with the clinical observations. Our study indicates that levels of FVII, but not TF, are associated with tumor migration and invasiveness in HCC, and provides clues that evaluation of FVII expression in HCC may be useful as a prognostic indicator in patients with HCC and may form an alternative target for further therapy.

"Accomodated" pig endothelial cells promote nitric oxide-dependent Th-2 cytokine responses from human T cells.

BACKGROUND: Cardiac and renal allo- and xenografts can become naturally resistant to vascular rejection. Understanding this process of "accommodation" would enhance our understanding of vascular inflammatory responses and have implications for immune manipulation and tolerance induction. A feature of these grafts is infiltration by leukocytes secreting a Th-2 pattern of cytokines. METHODS: HLA-DR-1-transfected, immortalized porcine endothelial cells (IPEC) were incubated with polyclonal human immunoglobulin G (IgG) for 6 days before incubation with purified human CD4+ T cells. RESULTS: IgG-incubated IPEC stimulated a normal proliferative response from alloreactive T cells. However, interferon (IFN)-gamma levels were significantly reduced, whereas interleukin (IL)-5 and IL-10 were maintained at levels equivalent to those stimulated by control IPEC. Cognate interaction between T cells and IPEC was not required for this effect, because IgG-incubated, MHC-class II-negative IPEC caused reduced IFN-gamma secretion during a response to human Epstein-Barr virus-transformed B cells. Experiments with the nitric oxide (NO) donor, (z)-1-2-[2-Aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO), and the NO synthase inhibitor, NG-monomethyl-L-arginine.monoacetate (L-NMMA), showed that NO released by the IgG-incubated IPEC was actively involved in the development of this phenotype. CONCLUSIONS: These data suggest a novel, IgG-mediated, NO-dependent mechanism by which endothelial cells (EC) influence T cell responsiveness and that the Th-2 cytokine skewing seen in "accommodated" grafts may be a secondary phenomenon, resulting from the T-EC interactions.

Nitric oxide-mediated expression of Bcl-2 and Bcl-xl and protection from tumor necrosis factor-alpha-mediated apoptosis in porcine endothelial cells after exposure to low concentrations of xenoreactive natural antibody.

BACKGROUND: Cardiac and renal allo- and xenografts can acquire a natural resistance to vascular rejection. This "accommodation" involves endothelial cell (EC) expression of "survival genes" such as Bcl family members and hemoxygenase 1. Understanding what initiates this protective process would have profound implications; our hypothesis is that low concentrations of antigraft antibodies may mediate these changes. METHODS: In vitro cultured primary and immortalized porcine EC were incubated with polyclonal human IgG for 6 days and then examined for phenotype changes. RESULTS: The cells acquired resistance to tumor necrosis factor-alpha-mediated apoptosis (50-100% reduction at 6 hr) and up-regulated expression of Bcl-2 and Bcl-xl; sustained expression was accompanied by inducible nitric oxide (NO) synthase expression and by enhanced production of NO by EC. Two observations suggested that NO was actively involved in the process of Bcl-2 and Bcl-xl induction. First, (z)-1-2-[2-aminoethyl)-N- (2-ammonioethyl)amino]diazen-1-ium-1,2-diolate, an NO donor, was able to induce similar changes in porcine EC to those induced by anti-pig antibodies. Second, an NO synthase inhibitor NG-monomethyl-L-arginine.monoacetate was able to specifically inhibit the anti-pig antibody-mediated expression of Bcl-2 or Bcl-xl. CONCLUSIONS: These data strongly support the hypothesis that Bcl-2 and Bcl-xl expression and protection from apoptosis in EC may result from antibody-mediated NO production through the neoexpression of inducible NO synthase.

In vitro accommodation of immortalized porcine endothelial cells: resistance to complement mediated lysis and down-regulation of VCAM expression induced by low concentrations of polyclonal human IgG antipig antibodies.

The capacity of vascularized xenografts to survive in the face of normal levels of circulating antigraft antibodies and complement has been ascribed to a phenomenon referred to as "endothelial cell accommodation." The mechanisms whereby accommodation might occur have remained obscure. We have investigated this phenomenon in an in vitro system. A preparation of polyclonal immunoglobulin, human normal globulin (HNG), induced a change in the phenotype of immortalized porcine endothelial cells (IPEC) suggestive of accommodation; the cells became resistant to complement mediated lysis and displayed a reduced expression of surface VCAM and MHC class I. The accommodated phenotype only manifested after 72 hr incubation with HNG and was optimal after 120 hr. In an analysis of all the experiments performed, the development of resistance to complement mediated lysis appeared independent of the inducing dose of HNG. However, down-regulation of VCAM was only manifest when subsaturating doses were used. Our results suggest that IgG xenoreactive antibodies can mediate changes in porcine endothelial cell phenotype consistent with accommodation. The dependence on both time and dose of antibody applied might explain why accommodation has been difficult to achieve consistently in in vivo models of discordant xenotransplantation. By demonstrating a functional interaction between human VLA-4 and porcine VCAM, we speculate that the down-regulation in expression of VCAM on accommodated endothelium may have an important regulatory effect on traffic of inflammatory cells into xenografts. Our results have important implications for the development of strategies to promote accommodation of xenografts.

Cloning of porcine intercellular adhesion molecule-1 and characterization of its induction on endothelial cells by cytokines.

BACKGROUND: The transplantation of pig organs into humans requires a detailed knowledge of similarities and differences between the two species in the molecular physiology of host defense mechanisms. We therefore set out to identify porcine intercellular adhesion molecule (ICAM)-1 and to characterize its expression by endothelial cells. METHODS: Porcine ICAM-1 cDNA was isolated from an endothelial cell cDNA library. An anti-pig ICAM-1 monoclonal antibody was generated and used to investigate the regulation by cytokines of ICAM-1 expression by porcine aortic endothelial cells (PAEC), using flow cytometry. RESULTS: We found that porcine ICAM-1 was similar in primary structure to human ICAM-1, with five Ig-like domains. COS-7 cells transfected with porcine ICAM-1 supported beta2 but not alpha4 integrin-dependent adhesion of human T lymphoblasts. There was a low-level surface expression of ICAM-1 on unstimulated PAEC and increased expression after stimulation with tumor necrosis factor (TNF)-alpha. However expression of ICAM-1 seemed to be significantly lower than that of vascular cell adhesion molecule-1, both on unstimulated and TNF-alpha-activated PAEC. Recombinant porcine interferon-gamma weakly stimulated ICAM-1 expression when incubated alone with PAEC but had an inhibitory effect on the increase in ICAM-1 due to TNF-alpha, both at 8 and 24 hr. CONCLUSIONS: Our observations confirm the existence of ICAM-1 in the pig and provide novel insights into how porcine and human endothelial cells differ in terms of adhesion molecule expression and cytokine responsiveness. Such differences are potentially important in interpreting models of inflammation in the pig and also in understanding the process of rejection of porcine xenografts.

Regulated inhibition of coagulation by porcine endothelial cells expressing P-selectin-tagged hirudin and tissue factor pathway inhibitor fusion proteins.

BACKGROUND: Thrombotic vascular occlusion resulting in infarction occurs during hyperacute rejection of allografts transplanted into sensitized patients and remains a major problem in experimental xenotransplantation. A similar process is also found in disorders of diverse etiology including atherosclerosis, vasculitis, and disseminated intravascular coagulation. METHODS: We have previously constructed two membrane-tethered anticoagulant fusion proteins based on human tissue factor pathway inhibitor and the leech anticoagulant hirudin and demonstrated their functional efficacy in vitro. These constructs have now been modified by the addition of a P-selectin sequence to the cytoplasmic tail to localize them in Weibel-Palade bodies. They have been transfected into Weibel-Palade body-positive endothelial cells isolated from the inferior vena cava of normal pigs. RESULTS: In resting endothelial cells, fusion protein expression colocalized with P-selectin and was confined to Weibel-Palade bodies. These cells had a procoagulant phenotype in recalcified human plasma. However, after activation with phorbol ester the anticoagulant proteins were rapidly relocated to the cell surface where they specifically inhibited the clotting of human plasma. CONCLUSIONS: Novel anticoagulant molecules may prove useful therapeutic agents for gene therapy in thrombotic disease and postangioplasty or for transgenic expression in animals whose organs may be used for clinical xenotransplantation. Expression in vascular endothelial cells may be regulated by inclusion of P-selectin cytoplasmic sequence, to restrict cell surface expression to activated endothelium.

Inhibition of tissue factor-dependent and -independent coagulation by cell surface expression of novel anticoagulant fusion proteins.

BACKGROUND: Thrombotic vascular occlusion occurs in disorders of diverse etiology, including atherosclerosis, vasculitis, and disseminated intravascular coagulation. The same process results in hyperacute rejection of renal allografts transplanted into sensitized patients and remains a major problem in experimental xenotransplantation. METHODS: We have previously described the design and expression of several genetic constructs encoding novel fusion proteins with anticoagulant properties. They are based on two naturally occurring soluble anticoagulant proteins, human tissue factor pathway inhibitor (hTFPI) and the leech protein hirudin, which act early and late in the clotting cascade, respectively. We report the expression of human hTFPI-CD4 on the surface of immortalized porcine endothelial cells (IPEC), and show that it functions across the species divide as evidenced by the binding of membrane-expressed porcine tissue factor (pTF)-human factor VIIa complexes. RESULTS: Using a human plasma recalcification clotting assay, we distinguished between pTF-dependent and pTF-independent fibrin generation, and we have demonstrated that expression of hTFPI-CD4 on IPEC effectively prevented pTF-dependent clotting. Moreover, we show that when hTFPI-CD4 was co-expressed with the hirudin construct, the procoagulant properties of in vitro cultured, activated IPEC were almost completely abolished. CONCLUSIONS: These results suggest that these novel anticoagulant molecules may prove useful therapeutic agents for gene therapy or for transgenic expression in animals whose organs may be used for cliniCal xenotransplantation.