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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-γ), 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-γ 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.

Ancrod is a purified fraction of venom from the Malayan pit viper Calloselasma rhodostoma, containing a serine protease that cleaves fibrinopeptides A from fibrinogen. We report on a study that involved intravenous and subcutaneous application of ancrod in healthy subjects in which it was shown that ancrod induces the formation of desAA-fibrin complexes that are partially crosslinked by factor XIII proenzyme, and act as cofactor in tPA induced plasminogen activation. The plasmin generated degrades fibrin, as well as fibrinogen, leading to the appearance of large amounts of fibrinogen and fibrin degradation products in the circulation, including fragment D-dimer. At low concentrations of ancrod, formation of desAA-fibrin is preceded by production of desA-profibrin, lacking only one fibrinopeptide A.

The thrombin-like serine protease ancrod from the Malayan pit viper Agkistrodon rhodostoma was expressed in mouse epithelial cells (C127). Oligosaccharide constituents were liberated from tryptic glycopeptides by treatment with peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F. Neutral oligosaccharide alditols obtained after reduction and enzymic desialylation were separated by two-dimensional HPLC and characterized by methylation analysis, liquid secondary-ion mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and sequential degradation with exoglycosidases. In contrast to natural ancrod, the recombinant glycoprotein carries exclusively diantennary, triantennary and tetraantennary N-glycans with Gal beta 4 GlcNAc beta (type-2) antennae which were, in part, further substituted by host-cell-specific structural elements such as Gal alpha 3 residues or N-acetyllactosamine repeats. As a characteristic feature, a substantial proportion of the oligosaccharides bears a GalNAc beta 4Glc-NAc antenna. Studies at the level of individual N-glycosylation sites demonstrated that glycans with N, N'-diacetyllactosediamine units are not specifically attached but occur at all sites in varying amounts. Hence, the putative recognition signal (Pro70-Lys-Lys) for glycoprotein hormone N-acetylgalactosaminyltransferase, present in this glycoprotein in close proximity to Asn79, does not convey site-specific transfer of GalNAc residues in these cells.

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 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 is a purified coagulant venom which renders blood incoagulable by cleaving fibrinopeptide A (FPA) from fibrinogen, but the mechanism involved in the clearance of fibrin from the circulation is unknown. To investigate the fibrinolytic response to ancrod, and to increase understanding of clearance mechanisms, six patients with peripheral vascular disease causing claudication were infused with ancrod at 2 u/kg over 6 h followed by 2 u/kg at 12 h intervals for 38 h. Venous blood samples were taken at time 0, 3, 6, 25 and 49 h for assay of fibrinogen (Fbg), fibrinopeptide A (FPA), total fibrin(ogen) degradation products (TDP), fibrin degradation products (FbDP), fibrinogen degradation products (FgDP), cross-linked fibrin degradation products (XL-FDP), tissue plasminogen activator (tPA), urinary type plasminogen activator (u-PA), plasminogen, alpha 2 antiplasmin (alpha 2 AP) and plasminogen activator inhibitor-1 (PAI-1). Fibrinogen (median and range) was 2.3 (1.4-3.90) g/l at time 0 and thereafter was undetectable. FPA rose from 2.5 (1.8-3.6) to 600 and 188 pmol/l at 3 h and 6 h and remained elevated. TDP, FbDP and FgDP increased greatly following ancrod while there was no evidence of XL-FDP. The surprising increase in FgDP during defibrination suggests either that fibrinogen is digested following its incorporation into circulating fibrin protofibrils or that some of the fibrin subunits in the photofibril retain one of the two fibrinopeptide A's. tPA and uPA remained unchanged. Plasminogen fell from 125 (100-155)% to 79 (40-118)% at 49 h and alpha 2 AP fell from 91 (75-107)% to 24 (10-35)% at 49 h. The level of PAI-1 was depressed during defibrination, with the exception of the 6 h data. The results demonstrate that ancrod removes FPA from fibrinogen to produce non-cross-linked (soluble) fibrin. This is cleared from the circulation without evidence of an increase in the circulating activities of the plasminogen activators, tPA or UK, but with evidence of plasminogen activation and consumption.

Effects of heparin, urokinase, and ancrod were concurrently compared on intraglomerular coagulation using a model of progressive Masugi nephritis. Evaluations were performed with light and immunofluorescent microscopy. Treatment significantly reduced crescent formation, more markedly with ancrod. In contrast, endocapillary proliferation was enhanced in the groups treated with urokinase and, especially, ancrod. Ancrod appeared to be most effective in decreasing the glomerular deposition of fibrinogen-related materials as judged from the immunofluorescence, and a relatively high dose was required for urokinase to be comparable with ancrod. However, in some glomeruli from two of 13 animals given ancrod, mesangiolytic lesion developed. Thus, although promising, ancrod has many problems to be solved before using it to control endocapillary proliferation and mesangiolysis.

Measurements of stress relaxation in uniaxial extension have been made on fibrin film prepared from fine bovine fibrin clots (i.e., clots in which there is minimal lateral aggregation of protofibrils), both ligated and unligated, and polymerized with both thrombin and ancrod, plasticized with either aqueous buffer or glycerol. The stress 100 s after imposition of strain was approximately proportional to In λ, where λ is the stretch ratio. Ligated thrombin films showed comparatively little relaxation over a period of one day and almost complete recovery after release of stress. In unligated thrombin films, there was substantial relaxation in two stages, as previously observed for coarse films, and substantial irrecoverable deformation. The extent of relaxation and the proportion of strain that was irrecoverable increased with the magnitude of the strain. In ancrod films (unligated), there was much more relaxation (stress decaying by as much as a factor of 10) and much more irrecoverable deformation (about 70% of the initial deformation); these results did not depend on the magnitude of the strain. When an ancrod film was released after relaxation and submitted to a second stretch, the extent of the second relaxation was much less. These observations are discussed in relation to the structure of fine films and possible mechanisms for relaxation and irrecoverable deformation.

Summary. The fibrin digestion products which appear in serum of patients having treatment with ancrod have been compared with derivatives of human fibrinogen produced by plasmin by means of immunoelectrophoresis, immunodiffusion and gel filtration techniques. In ancrod-treated patients the large molecular weight derivatives X and Y, as well as fragments D and E were seen 4 hr after the start of therapy. By 24 hr the main serum component was fragment D with low concentrations of large molecular weight derivatives still present, but fragment E had been cleared from the circulation. The identity of these fragments to the known products of fibrinogen proteolysis by plasmin has been confirmed. It is suggested that the initial degradation of fibrin micro-clots produced in the circulation during ancrod infusion is mediated by the fibrinolytic enzyme system.

A patient with a functionally defective fibrinogen (fibrinogen Chapel Hill) has been investigated. Fibrinogen Chapel Hill is characterized by hypofibrinogenemia, with a plasma concentration about one third of normal, as measured both functionally and immunochemically. Fibrinogen survival is normal; so also is fibrinopeptide release. A polymerization defect in this fibrinogen results in the delay of fibrin fibrils in solution to form a normal three-dimensional gel. This defect is not associated with end-to-end aggregation or with lateral associations in solution. Delayed gelation results from an abnormality in a tertiary contact site involved in network branching, which is associated with the hydrophilic, carboxy-terminal segment of the alpha chain. Fibrinogen Chapel Hill exhibits two additional abnormal responses, which are also associated with the same region. The early plasmin cleavages of fibrinogen and fragment X are delayed, and there is a concomitant delay in the appearance of fragments Y, D, and E. This fibrinogen also has an unusual sensitivity to Ancrod proteolysis, whereby Ancrod cleaves a large carboxy-terminal segment of the alpha chain more rapidly than in normal fibrinogen. The abnormalities in fibrinogen Chapel Hill can be explained by a structural abnormality which is functionally related to an alpha chain associated polymerization domain.

Summary. The serum FDP produced in response to defibrination with ancrod and to thrombolytic therapy with intermittent streptokinase/plasminogen infusion have been characterized using a method of solid phase immuno-precipitation followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Using this method it is possible to distinguish between the plasmin degradation products of fibrinogen and factor XIIIa induced crosslinked fibrin. During ancrod administration, fragments of similar mobility to fibrinogen fragments X, Y, D and E are present in serum samples taken 24 h after the initiation of treatment, while subsequent samples contain mainly fragments with the same mobility as fibrinogen fiagment D. The intermittent nature of the streptokinase/plasminogen infusions produces fibrinogen fragments X, Y, D and E in the serum 1 h after each daily streptokinase/plasminogen administration. An early clearance of fragment E from the circulation of patients receiving either ancrod or streptokinase/plasminogen infusions accords with the results of other investigations. The presence of the D dimer fragment, which is produced only by plasmin lysis of factor XIIIa crosslinked fibrin, could not be conclusively confirmed in either group of patients.

Summary. The effect of the coagulant fraction from Agkistrodon rhodostoma venom (Arvin = ancrod) on the generation of microthrombi was studied in rabbits. Ancrod infusion caused hypofibrinogenaemia and induced the formation of circulating fibrin monomer complexes, but microthrombi were not detected in the glomerular capillaries. If, however, endotoxin was injected into ancrod-infused rabbits, 73% of the animals showed microclots in the glomerular capillaries typical of the generalized Shwartzman reaction. These microclots exhibited antigenic characteristics of fibrinogen derivatives. Injection of endotoxin only did not produce glomerular capillary thrombosis and the mean fibrinogen levels did not decrease significantly, although fibrin monomer complexes could be detected in all the rabbits. Generation of microthrombosis by ancrod infusion and endotoxin injection could not be prevented by treating the animals with heparin. Thus endotoxin mediated the generation of microclots in ancrod-infused rabbits by a mechanism different from its procoagulant activity. Heparin treatment interfered neither with the decrease in fibrinogen levels caused by ancrod infusion nor with the fall in the platelet counts caused by endotoxin injection.

Summary The results of the use of 1251 fibrinogen in 51 studies performed on 45 patients after renal transplantation are reported. The control patients did not show increased transplant radioactivity and there was no significant difference between the clearance slopes of the heart and transplant sites. All 24 studies of acute rejection episodes within the first 50 days after transplantation showed increased transplant radioactivity with a significant difference between 1251 fibrinogen clearance slopes of the heart and transplant sites. Rejection episodes were predicted in 12 patients before clinical and biochemical evidence of rejection. There were no positive results with rejection episodes occurring more than 50 days after transplantation. False positive results were obtained with haematomata, urinary leak, malakoplakia and during ancrod therapy, although in some cases these can be recognised by examination of the clearance slopes. The technique is a useful aid in the diagnosis of acute rejection episodes in transplants within 50 days of the operation but not in chronic rejection.

Summary. Plasma fibrinogen and its degradation products (FDP) were examined in mice given single injections of Ancrod, the purified coagulative fraction of Malayan pit viper venom previously called Arvin. Clottable fibrinogen had disappeared completely 5 min after the injection and slowly reappeared 12 hr later. At least 2 FDP appeared in the plasma 30 min after the injections while 5 hr later only one FDP was present which corresponded to human fragment E. This was in contrast to in vitro lysis of mouse fibrinogen which yielded two late FDP corresponding to human fragments D and E. The fibrinolytic system also was examined in the plasma of these animals. 30 min after the injection, plasminogen was decreased in amount but increased in susceptibility to activation by urokinase. Transient, modest increases in fibrinolytic activity were evident in diluted plasma samples 1 hr and again 8 hr after the injections. Inhibitors of fibrinolysis did not differ from those in normal mice. These observations are correlated with our previous immunofluorescent and histochemical studies on these and other animals. The present data support our earlier conclusion that fibrinolysis within small vessels of the tissues is of major importance in the reaction to Ancrod.

1 Ancrod is inactivated by diisopropyl [32P]phosphofluoridate when one g-atom of 32P is irreversibly bound to 34600 g enzyme. 2 Acid hydrolysis of the radioactive and inactive enzyme confirms that the label is attached to a serine residue in l-serine-O-[32P]phosphate. 3 Ancrod is inactivated by the substrate analogue α-N-(nitrobenzyloxycarbonyl)-l-arginyl chloromethyl ketone. Kinetic analysis shows that the irreversible inactivation is preceded by a reversible binding of inactivator to the enzyme. This inactivation is prevented by the addition of α-N-benzoyl-l-arginine ethyl ester. 4 The rate of inactivation of ancrod by the active-site-directed reagent depends on the ionisation of a group with pK(app) of 7.4. This group is probably the histidine residue which is lost when the enzyme is inactivated by this compound. 5 A modified figure of approximately 38000 is suggested for the molecular weight of the enzyme which contains one active site.

A method for measuring progressive antithrombin activity is described. Higher levels of antithrombin activity were found in plasma defibrinated with ancrod than in plasma defibrinated by heating to 56-degrees C or by thrombin. Because ancrod alone has no antithrombin activity it is recommended as the best agent for defibrinating plasma prior to antithrombin estimation. The relationship between plasma and serum antithrombin concentration was studied in 8 patients during oral contraceptive therapy. There was a fall in both plasma and serum antithrombin during therapy but the fall in serum antithrombin was significantly greater than that seen in plasma. This reflects the fact that the serum antithrombin concentration is dependent on the degree of thrombin generation which has taken place during clotting.(AUTHORS MODIFIED)

Summary. Coagulation of plasma fibrinogen in the presence of fibrin degradation products was studied in rabbits treated with ancrod (Arvin), Two techniques were employed: the first, based on trace-labelling of plasma samples with labelled fibrinogen in vitro, was to measure the degree to which solubilized fibrin inhibited coagulation; in the second, two differently labelled fibrinogens were used, one injected intravenously and the other added in vitro, by which estimation of fibrinogen in serum relative to the amount of circulating breakdown products was attempted. The first technique is considered to provide reliable information about the extent of defective coagulation, and hence the proportion of masked fibrinogen present in serum. Because of the difficulty in obtaining resolution between fibrinogen and its breakdown products in clots, the second technique is thought to be more suitable for comparative studies rather than for measurements in absolute terms. High doses of hydrocortisone administered to rabbits for several days prior to ancrod significantly reduced the speed of the dissolution of microclots. The data obtained are consistent with the hypothesis that the hormone interfered with fibrinolysis at the plasminogen activator level.