Randomized, placebo-controlled study on efficacy, safety and tolerability of drug-induced defibrinogenation for sudden sensorineural hearing loss: the lessons learned.

PURPOSE: Disturbance of cochlear microcirculation is discussed as final common pathway of various inner ear diseases. Hyperfibrinogenemia causing increased plasma viscosity is a possible factor for a critical reduction of cochlear blood flow that might lead to sudden sensorineural hearing loss (SSHL). The aim was to determine the efficacy and safety of drug-induced defibrinogenation by ancrod for SSHL. METHODS: Double-blind, randomized, placebo-controlled, multicenter, parallel group, phase II (proof-of-concept) study (planned enrollment: 99 patients). Patients received an infusion of ancrod or placebo (day 1) followed by subcutaneous administrations (day 2, 4, 6). Primary outcome was the change in pure tone audiogram air conduction average until day 8. RESULTS: The study was terminated early due to slow recruiting (31 enrolled patients: 22 ancrod, 9 placebo). A significant improvement of hearing loss was registered in both groups (ancrod: - 14.3 dB ± 20.4 dB, - 39.9% ± 50.4%; placebo: - 22.3 dB ± 13.7 dB, - 59.1% ± 38.0%). A statistically significant group-difference was not detected (p = 0.374). Placebo response of 33.3% complete and 85.7% at least partial recovery was observed. Plasma fibrinogen levels were reduced significantly by ancrod (baseline: 325.2 mg/dL, day 2: 107.2 mg/dL). Ancrod was tolerated well, no adverse drug reaction was of severe intensity, no serious adverse events occurred. CONCLUSION: Ancrod reduced fibrinogen levels that support its mechanism of action. The safety profile can be rated positively. Since the planned number of patients could not be enrolled, no efficacy conclusion can be drawn. The high rate of placebo response challenges clinical trials for SSHL and needs to be considered in future investigations. Trial registrations This study was registered in the EU Clinical Trials Register, EudraCT-No. 2012-000066-37 at 2012-07-02.

Defibrinogenation Ameliorates Retinal Microgliosis and Inflammation in A CX3CR1-Independent Manner.

SUMMARY STATEMENT: Diabetic human and murine retinas revealed pronounced microglial morphological activation and vascular abnormalities associated with inflammation. Pharmacological fibrinogen depletion using ancrod dampened microglial morphology alterations, resolved fibrinogen accumulation, rescued axonal integrity, and reduced inflammation in the diabetic murine retina.

Drug-induced Defibrinogenation as New Treatment Approach of Acute Hearing Loss in an Animal Model for Inner Ear Vascular Impairment.

OBJECTIVE: Disturbance of cochlear microcirculation is considered to be the final common pathway of various inner ear diseases. Hyperfibrinogenemia causing increased plasma viscosity is a known risk factor for sudden sensorineural hearing loss and may lead to a critical reduction of cochlear blood flow. The aim of this study was to evaluate the effect of a substantial reduction of plasma fibrinogen levels by drug-induced defibrinogenation for the treatment of acute hearing loss in vivo. METHODS: Acute hearing loss was induced by hyperfibrinogenemia (i.v. injection of 330 mg/kg BW fibrinogen), using a guinea pig animal model. Parameters of cochlear microcirculation and hearing thresholds were quantified by intravital microscopy and evoked response audiometry. After obtaining baseline values, the course of hearing loss and disturbances of microcirculation were investigated under influence of intravenous defibrinogenation therapy (ancrod), corticosteroid, or placebo treatment, using 5 animals/group. RESULTS: Acute hyperfibrinogenemia caused hearing loss from 10 ±â€Š7 to 26 ±â€Š10 dB SPL at baseline. Drug-induced reduction of fibrinogen levels showed a significant increase of cochlear microcirculation (1.6-fold) and recovered hearing threshold (11 ±â€Š6 dB SPL). Placebo or corticosteroid treatment had no effect on hearing loss (35 ±â€Š7 dB SPL and 32 ±â€Š18 dB SPL, respectively). CONCLUSION: Acute hyperfibrinogenemia resulted in hearing loss. Drug-induced reduction of elevated fibrinogen levels caused an increase in cochlear blood flow and a decrease in hearing thresholds. Placebo or corticosteroid treatment had no effect. Reduction of plasma fibrinogen levels could serve as a clinical treatment option for acute hearing loss.

Carbon monoxide releasing molecule-2 inhibition of snake venom thrombin-like activity: novel biochemical "brake"?

A complication of defibrinogenation therapy with snake venom enzymes such as ancrod is hypofibrinogenemia associated bleeding secondary to no human-derived inhibitor being available to inactivate or diminish the activity of such enzymes. Of interest, ancrod contains a critical histidine residue without which enzymatic activity is inhibited, and carbon monoxide has been demonstrated to inhibit biomolecular function by interacting with histidine moieties in ion channels. We tested the hypothesis that exposure of three different snake venoms containing serine proteases with thrombin-like activity (which included ancrod) to carbon monoxide derived from carbon monoxide releasing molecule-2 would diminish their effects on plasmatic coagulation as assessed by thrombelastography. In the case of the Malayan pit viper and Eastern diamondback rattlesnake venoms, carbon monoxide diminished the effects of thrombin-like activity. In contrast, timber rattlesnake venom demonstrated enhancement of "thrombin-generating" activity with simultaneous loss of thrombin-like activity in response to carbon monoxide exposure. These findings may serve as the rational basis for not just continuing to investigate the potential of snake venom enzymes as clinical defibrinogenating agents, but to also to assess the potential to stop such agents from becoming a catalytic "runaway train" by judicious application of a biochemical "brake" such as carbon monoxide.

Ancrod revisited: viscoelastic analyses of the effects of Calloselasma rhodostoma venom on plasma coagulation and fibrinolysis.

Fibrinogen depletion via catalysis by snake venom enzymes as a therapeutic strategy to prevent or treat thrombotic disorders was utilized for over four decades, with ancrod being the quintessential agent. However, ancrod eventually was found to not be of clinical utility in large scale stroke trial, resulting in the eventual discontinuation of the administration of the drug for any indication. It was hypothesized that ancrod, possessing thrombin-like activity, may have unappreciated robust coagulation kinetics. Using thrombelastographic methods, a comparison of equivalent tissue factor initiated thrombin generation and Calloselasma rhodostoma venom (rich in ancrod activity) on plasmatic coagulation kinetics was performed. The venom resulted in thrombi that formed nearly twice as fast compared to thrombin formed clots, and there was no difference in fibrinolytic kinetics initiated by tissue-type plasminogen activator. In plasma containing iron and carbon monoxide modified fibrinogen, which may be found in patients at risk of stroke, the coagulation kinetic differences observed with venom was still more vigorous than that seen with thrombin. These phenomena may provide insight into the clinical failure of ancrod, and may serve as an impetus to revisit the concept of fibrinogen depletion via fibrinogenolytic enzymes, not those with thrombin-like activity.

Pharmacological and genetic depletion of fibrinogen protects from kidney fibrosis.

Fibrinogen (Fg) has been implicated in the pathogenesis of several fibrotic disorders by acting as a profibrotic ligand for a variety of cellular surface receptors and by modulating the provisional fibrin matrix formed after injury. We demonstrated increased renal Fg expression after unilateral ureteral obstruction and folic acid (FA) nephropathy in mice, respectively. Urinary Fg excretion was also increased in FA nephropathy. Using in vitro and in vivo approaches, our results suggested that IL-6 mediates STAT3 activation in kidney fibrosis and that phosphorylated (p)STAT3 binds to Fgα, Fgß, and Fgγ promoters in the kidney to regulate their transcription. Genetically modified Fg heterozygous mice (∼75% of normal plasma Fg levels) exhibited only 3% kidney interstitial fibrosis and tubular atrophy after FA nephropathy compared with 24% for wild-type mice. Fibrinogenolysis through Ancrod administration after FA reduced interstitial fibrosis more than threefold compared with vehicle-treated control mice. Mechanistically, we show that Fg acts synergistically with transforming growth factor (TGF)-ß1 to induce fibroblast proliferation and activates TGF-ß1/pSMAD2 signaling. This study offers increased understanding of Fg expression and molecular interactions with TGF-ß1 in the progression to kidney fibrosis and, importantly, indicates that fibrinogenolytics like Ancrod present a treatment opportunity for a yet intractable disease.

Fibrinogen depleting agents for acute ischaemic stroke.

BACKGROUND: Fibrinogen depleting agents reduce fibrinogen in blood plasma, reduce blood viscosity and hence increase blood flow. This may help remove the blood clot blocking the artery and re-establish blood flow to the affected area of the brain after an ischaemic stroke. The risk of haemorrhage may be less than with thrombolytic agents. This is an update of a Cochrane review first published in 1997 and last updated in 2003. OBJECTIVES: To assess the effect of fibrinogen depleting agents in patients with acute ischaemic stroke. SEARCH METHODS: We searched the Cochrane Stroke Group Trials Register (July 2011), the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library 2011, Issue 7), the Chinese Stroke Trials Register (September 2011), MEDLINE (1950 to July 2011), EMBASE (1980 to July 2011) and Web of Science Conference Proceedings (1990 to July 2011). In addition, we searched six Chinese databases, four ongoing trials registers (July 2011) and relevant reference lists. For previous versions of the review, we handsearched journals and contacted researchers in China and Japan and relevant drug companies. SELECTION CRITERIA: Randomised trials of fibrinogen depleting agents started within 14 days of stroke onset, compared with control in patients with definite or possible ischaemic stroke. DATA COLLECTION AND ANALYSIS: Two review authors independently selected trials, assessed trial quality and extracted the data. We resolved disagreement by discussion. MAIN RESULTS: We included eight trials involving 5701 patients. Six trials tested ancrod and two trials tested defibrase (patients were treated for less than three hours to less than 48 hours). Allocation concealment was adequate in seven trials. Fibrinogen depleting agents marginally reduced the proportion of patients who were dead or disabled at the end of follow-up (risk ratio (RR) 0.95, 95% confidence Interval (CI) 0.90 to 0.99, 2P = 0.02). There was no statistically significant difference in death from all causes during the scheduled treatment or follow-up period. There were fewer stroke recurrences in the treatment group than in the control group (RR 0.67, 95% CI 0.49 to 0.92, 2P = 0.01). However, symptomatic intracranial haemorrhage was about twice as common in the treatment group compared with the control group (RR 2.42, 95% CI 1.65 to 3.56, 2P < 0.00001). AUTHORS' CONCLUSIONS: The current evidence is promising but not yet sufficiently robust to support the routine use of fibrinogen depleting agents for the treatment of acute ischaemic stroke. Further trials are needed to determine whether there is worthwhile benefit, and if so, which categories of patients are most likely to benefit.

Ancrod and fibrin formation: perspectives on mechanisms of action.

BACKGROUND AND PURPOSE: Ancrod, derived from Malayan pit viper venom, has been tested as ischemic stroke treatment in clinical trials with inconsistent results. We studied the actions of ancrod on fibrinolysis pathways in patient plasma samples and endothelial cell culture systems. METHODS: We analyzed fibrinogen levels during the first 6 hours of ancrod infusion in patients entered in the Stroke Treatment with Ancrod Trial. For the in vitro study, human brain microvascular endothelial cells incubated with plasminogen or with human brain microvascular endothelial cell-conditioned medium were co-incubated with ancrod and fibrinogen under normal or oxygen-glucose deprivation conditions over 6 hours. RESULTS: Fibrinogen levels decreased both in vivo and in vitro. Ancrod generated fibrinopeptide A, caused visible clot formation, and reduced levels of tissue-type plasminogen activator antigen in the human brain microvascular endothelial cell system and in a cell-free system with conditioned media. CONCLUSIONS: The in vitro results indicate that ancrod causes local fibrin formation and secondary depletion of tissue-type plasminogen activator by binding to fibrin clot. Ancrod-induced fibrin formation could result in cerebral microvascular occlusion and may explain the suboptimal clinical effects of ancrod in human stroke trials.

Co-overexpression of PpPDI enhances secretion of ancrod in Pichia pastoris.

Ancrod, a serine protease purified from the venom of Agkistrodon rhodostoma, is highly specific for fibrinogen. It causes anticoagulation by defibrinogenation and has been used as a therapeutic anticoagulant for the treatment of moderate to severe forms of peripheral arterial circulatory disorders in a variety of countries. The DNA of ancrod was amplified by recursive PCR with a yeast bias codon and cloned into the pGEM-T Easy vector. In order to achieve a high level secretion and a full activity expression of ancrod in Pichia pastoris (P. pastoris), the P. pastoris protein disulfide bond isomerase (PpPDI) was co-overexpressed in the strain. The secretion characteristics of ancrod with and without PpPDI were examined. With co-overexpression of PpPDI, the production of recombinant ancrod (rAncrod) was increased to 315 mg/L in the culture medium, which is twofold higher than the control strain carrying only the ancrod gene. Through purified by Ni²âº affinity chromatography and phenyl Sepharose column, the purity of rAncrod was found to be as high as 95.2%. The fibrinogenolytic and zymographic activities of the rAncrod were determined and found to be similar to that of the native protein. This improved expression system can facilitate further studies and the industrial production of ancrod.

Ancrod in acute ischemic stroke: results of 500 subjects beginning treatment within 6 hours of stroke onset in the ancrod stroke program.

BACKGROUND AND PURPOSE: Previous studies of multiple-day dosing with the defibrinogenating agent, ancrod, in acute ischemic stroke yielded conflicting results but suggested that a brief dosing regimen might improve efficacy and safety. The Ancrod Stroke Program was designed to test this concept in subjects beginning ancrod or placebo within 6 hours of the onset of acute ischemic stroke. METHODS: Five hundred subjects with acute ischemic stroke who could begin receiving study material within 6 hours of symptom onset were infused intravenously with either ancrod (0.167 IU/kg per hour) or placebo over 2 or 3 hours. The primary efficacy outcome was a dichotomized, modified Rankin score at 90 days with less stringent cut-points for higher prestroke modified Rankin score and pretreatment NIHSS total score ("responder analysis"). Safety variables included mortality, major bleeding, and intracranial hemorrhage. RESULTS: Although the desired changes in fibrinogen level were seen in >90% of ancrod subjects, interim analysis for futility led to the study being halted for lack of efficacy. Positive responder status in the interim dataset was seen in 39.6% of ancrod subjects and 37.2% of placebo subjects (P=0.47). Ninety-day mortality did not differ between the 2 groups (ancrod, 15.6%; placebo, 14.1%; P=0.32), and the incidence of symptomatic intracranial hemorrhage within the first 72 hours, although not significantly different in ancrod compared to placebo subjects (P=0.19), was approximately twice as high (3.9% vs 2.0%; P=0.19). CONCLUSIONS: These results demonstrate that intravenous ancrod starting within 6 hours after symptom onset in a broad selection of subjects with ischemic stroke did not improve their outcome and revealed a trend to increased bleeding despite successful efforts to achieve rapid initial defibrinogenation and avoid prolonged hypofibrinogenemia.

Microstructure and mechanics of collagen-fibrin matrices polymerized using ancrod snake venom enzyme.

The relationship between microstructural features and macroscopic mechanical properties of engineered tissues was investigated in pure and mixed composite scaffolds consisting of collagen Type I and fibrin proteins containing embedded smooth muscle cells. In order to vary the matrix microstructure, fibrin polymerization in mixed constructs was initiated using either the blood-derived enzyme thrombin or the snake venom-derived enzyme ancrod, each at low and high concentrations. Microstructural features of the matrix were quantified by analysis of high resolution scanning electron micrographs. Mechanical properties of the scaffolds were assessed by uniaxial tensile testing as well as creep testing. Viscoelastic parameters were determined by fitting creep data to Burger's four-parameter model. Oscillatory dynamic mechanical testing was used to determine the storage modulus, loss modulus, and phase shift of each matrix type. Mixed composite scaffolds exhibited improved tensile stiffness and strength, relative to pure collagen matrices, as well as decreased deformation and slower relaxation in creep tests. Storage and loss moduli were increased in mixed composites compared with pure collagen, while phase shift was reduced. A correlation analysis showed that the number of fiber bundles per unit volume was positively correlated with matrix modulus, strength, and dynamic moduli, though this parameter was negatively correlated with phase shift. Fiber diameter also was negatively correlated with scaffold strength. This study demonstrates how microstructural features can be related to the mechanical function of protein matrices and provides insight into structure-function relationships in such materials. This information can be used to identify and promote desirable microstructural features when designing biomaterials and engineered tissues.

Ancrod for acute ischemic stroke: a new dosing regimen derived from analysis of prior ancrod stroke studies.

BACKGROUND: Ancrod, a fibrinogen-reducing agent, has been evaluated as treatment beginning within 3 or 6 hours of onset of acute ischemic stroke with inconsistent results. The data sets from these studies provide an opportunity to determine whether ancrod-related variables are associated with efficacy and safety. OBJECTIVE: This post hoc analysis of data from the Stroke Treatment with Ancrod Trial (STAT) analyzed ancrod-related variables as potential determinants of efficacy or safety. The resulting hypotheses were then tested in the European STAT (ESTAT) database. METHODS: The relationships between ancrod-related variables and the outcomes of efficacy and symptomatic intracranial hemorrhage (ICH) were analyzed using a 3-stage multivariate process. RESULTS: Good clinical outcome at 3 months based on the Barthel Index occurred almost twice as often in rapid defibrinogenators (>or=30 mg/dL/h) (52%) as in slow defibrinogenators (26%), with no increase in mortality or symptomatic ICH. Compared with a 20.7% incidence of symptomatic ICH in patients with mean post-9-hour fibrinogen levels less than or equal to 60 mg/dL, symptomatic ICH incidence was 0.8% in those with mean levels greater than 60 mg/dL (with no loss of efficacy). There were no symptomatic ICHs among 220 North American patients with mean levels greater than 70 mg/dL. It was hypothesized that an initial controlled rapid ancrod infusion with mean post-9-hour fibrinogen levels greater than 70 mg/dL would yield superior efficacy and safety. Such ESTAT patients had statistically significant efficacy versus placebo and a marked reduction in the incidence of symptomatic ICH versus patients taking ancrod with lower maintenance fibrinogen levels. CONCLUSION: Modifications to ancrod dosing may substantially improve efficacy while reducing the rate of symptomatic ICH.

A leaky blood-brain barrier, fibrinogen infiltration and microglial reactivity in inflamed Alzheimer's disease brain.

This study has used immunohistochemical examination of tissue obtained from Alzheimer's disease (AD) brains and rat hippocampus injected with Abeta(1-42) peptide to determine effects of induced inflammatory reactivity on integrity of blood-brain barrier (BBB) and viability of neurons. Tissue from AD, but not non-demented, brains exhibited a diffuse pattern of staining for fibrinogen and immunoglobulin (IgG) indicative of BBB leakiness with considerable fibrinogen immunoreactivity (ir) appearing in association with Abeta deposits. Immunostaining for the endothelial cell specific glycoprotein, von Willebrand factor, showed morphological evidence for altered blood vessels in AD tissue. AD brains also demonstrated extensive areas of fibrinogen ir in association with microglial reactivity. In vivo, intra-hippocampal injection of Abeta(1-42) caused time-dependent (1-7 days after injection) increases in double staining of fibrinogen with areas of microgliosis. Two independent pharmacological strategies were employed to examine how Abeta(1-42) stimulation (7 days injection) may be linked to neurodegeneration. The defibrinogenating compound, ancrod, reduced inflammatory reactivity, levels of parenchymal fibrinogen and IgG, and was neuroprotective. These results prompted use of Abeta(1-42) plus fibrinogen as a novel in vivo inflammatory stimulus and this combination significantly enhanced inflammatory reactivity, vascular perturbations and neuronal damage compared to Abeta(1-42) alone. A second approach, using anti-Mac-1 (antibody for antigen CD11b) to block activation of microglia, was highly effective in attenuating effects of Abeta(1-42) plus fibrinogen amplification of inflammatory and vascular responses and conferred significant neuroprotection. The overall findings from study of AD tissue and in vivo in Abeta(1-42) and Abeta(1-42) plus fibrinogen stimulated rat hippocampus suggest microglial responses to promote increased extravasation of blood protein as a critical component in amplifying inflammatory reactivity and causing neuronal damage in inflamed AD brain.

Expression and purification of ancrod, an anticoagulant drug, in Pichia pastoris.

Ancrod is known as a thrombin-like enzyme from the venom of Calloselasma rhodostoma. The cDNA encoding ancrod was synthesized with a yeast bias codon and inserted into the eukaryotic expression vector pPIC9 and was subsequently expressed in the yeast Pichia pastoris. Recombinant ancrod was produced in 5-L bioreactor using a sorbitol-methanol feeding strategy and recovered from the fermentation broth by hydrophobic, affinity, and ion exchange chromatography. SDS-PAGE analysis revealed that ancrod was heterogeneously glycosylated and running at the expected molecular weight of 43-48 kDa which decreased to about 29 kDa after deglycosylation with N-glycosidase F. The fibrinogenolytic and zymographic activity of the recombinant ancrod were determined and were found to be similar to that of the native protein.

A novel nuclear interactor of ARF and MDM2 (NIAM) that maintains chromosomal stability.

The ARF tumor suppressor signals through p53 and other poorly defined anti-proliferative pathways to block carcinogenesis. In a search for new regulators of ARF signaling, we discovered a novel nuclear protein that we named NIAM (nuclear interactor of ARF and MDM2) for its ability to bind both ARF and the p53 antagonist MDM2. NIAM protein is normally expressed at low to undetectable levels in cells because of, at least in part, MDM2-mediated ubiquitination and proteasomal degradation. When reintroduced into cells, NIAM activated p53, caused a G1 phase cell cycle arrest, and collaborated with ARF in an additive fashion to suppress proliferation. Notably, NIAM retains growth inhibitory activity in cells lacking ARF and/or p53, and knockdown experiments revealed that it is not essential for ARF-mediated growth inhibition. Thus, NIAM and ARF act in separate anti-proliferative pathways that intersect mechanistically and suppress growth more effectively when jointly activated. Intriguingly, silencing of NIAM accelerated chromosomal instability, and microarray analyses showed reduced NIAM mRNA expression in numerous primary human tumors. This study identifies a novel protein with tumor suppressor-like behaviors and functional links to ARF-MDM2-p53 signaling.

Intravenous ancrod for acute ischaemic stroke in the European Stroke Treatment with Ancrod Trial: a randomised controlled trial.

BACKGROUND: Intravenous tissue plasminogen activator is the only approved specific treatment for acute ischaemic stroke. Ancrod, a natural defibrinogenating agent from snake venom, has proved to have a favourable effect when given within 3 h after an acute ischaemic stroke. The European Stroke Treatment with Ancrod Trial was undertaken to assess the effects of ancrod when given within 6 h. METHODS: 1222 patients with an acute ischaemic stroke were included in this randomised double-blind placebo-controlled trial. Brain CT scans were done to exclude intracranial haemorrhages and large evolving ischaemic infarctions. Patients were randomly assigned ancrod (n=604) or placebo (n=618). The primary outcome was functional success at 3 months (survival, Barthel Index of 95 or 100, or return to prestroke level). The analysis was by intention-to-treat. This trial is registered with ClinicalTrials.gov, trial number NCT00343174. FINDINGS: Functional success at 3 months did not differ between patients given ancrod (42%) and those given placebo (42%) (p=0.94, OR=0.99, 95% CI, 0.76-1.29). INTERPRETATION: On the basis of our findings, ancrod should not be recommended for use in acute ischaemic stroke beyond 3 h.

A coagulation factor VII deficiency protects against acute inflammatory responses in mice.

Upregulation of the activated Factor VII (FVIIa)/Tissue Factor complex, downregulation of natural anticoagulation pathways, and inhibition of fibrinolysis, are major contributors to coagulopathies associated with acute inflammation. Provision of FVIIa, and consequent downstream coagulation-related proteases, also stimulates further inflammatory changes, which can result in disseminated intravascular coagulation. Thus, the potential protective effects in vivo of a genetic-based reduction in FVII levels have been investigated in a murine model of acute inflammation, namely lipopolysaccharide (LPS)-induced lethal endotoxaemia. Mice with a total FVII deficiency do not survive the neonatal period. Therefore mice expressing low levels of FVII (FVII(tTA/tTA)), producing sufficient amounts of FVII for survival (approximately 5% of wild-type (WT) FVII), were employed to investigate in vivo pathways involved in the crosstalk between coagulation, inflammation, and survival, consequent to administration of a lethal dose of LPS. The FVII(tTA/tTA) mice presented with reduced mortality, coagulation, and inflammatory responses in comparison with similarly treated WT mice after administration of LPS. The attenuated inflammatory responses in FVII(tTA/tTA) mice were associated with downregulation of Egr-1 signalling. Administration, in vivo, of specific inhibitors of FXa and thrombin demonstrated that the inflammatory responses were unaltered in WT mice, but further reduced in FVII(tTA/tTA) mice. Therefore, a FVII deficiency enhances survival from lethal endotoxaemia both through attenuation of inflammatory responses that result directly from reduced FVIIa levels, and, indirectly, from downregulation of coagulation proteases downstream of the FVII-dependent cascade.

NK-cell-dependent acute xenograft rejection in the mouse heart-to-rat model.

BACKGROUND: Acute humoral xenograft rejection is characterized by widespread intravascular thrombosis with a significant NK-cell and macrophage infiltrate. Although in vitro and ex vivo data have shown that NK cells are capable of killing xenogeneic tissue, the precise role they play in vivo is still not certain. Consequently, there are few tested strategies for dealing with NK-cell-mediated rejection, should this prove to be a problem. One reason for this has been the lack of a relevant rodent model in which rejection by these cells can be easily studied. METHODS: Prior to transplantation of mouse hearts, we depleted rat recipients of fibrinogen using a snake venom, ANCROD, from the Malayan pit viper. Graft survival was examined by manual palpation and the rejected hearts were examined by histology. Levels of circulating interferon gamma (IFN-gamma), used as a surrogate marker for NK-cell activation, were determined by an enzyme-linked immunosorbent assay. RESULTS: Depletion of fibrinogen to approximately 5% of normal allowed surgery without a significant increase in the technical failure rates and prolonged graft survival compared with that seen in unmanipulated rats. Rejected hearts showed no evidence of intravascular thrombosis but did show significant antibody and complement deposition. There was little T-cell infiltration and cyclosporin had no influence on survival. Instead, hearts were infiltrated with NK cells and macrophages and rejection was associated with significant IFN-gamma production. Depletion of NK cells with anti-asialo-GM-1 from ANCROD-treated recipients led to a further significant prolongation of graft survival. CONCLUSIONS: Inhibition of intravascular thrombosis by fibrinogen depletion, in the absence of any other manipulation, unmasks NK-cell-dependent acute xenograft rejection in the mouse-to-rat heart transplantation model. This relatively simple model is expected to be useful to investigate the mechanisms of NK-cell-mediated rejection and to provide insight into the types of graft manipulation that could modify this process.