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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 ± 7 to 26 ± 10 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 ± 6 dB SPL). Placebo or corticosteroid treatment had no effect on hearing loss (35 ± 7 dB SPL and 32 ± 18 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.

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.

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.

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.

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.

Group A streptococci, a common human pathogen, secrete streptokinase, which activates the host's blood clot-dissolving protein, plasminogen. Streptokinase is highly specific for human plasminogen, exhibiting little or no activity against other mammalian species, including mouse. Here, a transgene expressing human plasminogen markedly increased mortality in mice infected with streptococci, and this susceptibility was dependent on bacterial streptokinase expression. Thus, streptokinase is a key pathogenicity factor and the primary determinant of host species specificity for group A streptococcal infection. In addition, local fibrin clot formation may be implicated in host defense against microbial pathogens.

Background: Tissue factor initiated glomerular fibrin deposition is an important mediator of injury in crescentic glomerulonephritis. Recent data have suggested noncoagulant roles for tissue factor in inflammation., Methods: To test the hypothesis that in addition to its effects in initiating coagulation, tissue factor has proinflammatory effects in glomerulonephritis, rabbits given crescentic anti-glomerular basement membrane (GBM) antibody-induced glomerulonephritis were defibrinogenated with ancrod. One group of defibrinogenated rabbits was also given anti-tissue factor antibodies. Comparisons were made between these groups, as well as a third group that was neither defibrinogenated with ancrod nor given anti-tissue factor antibodies., Results: Defibrinogenation alone abolished glomerular fibrin deposition, reduced crescent formation, and limited renal impairment (ancrod-treated, serum creatinine 274 +/- 37 micromol/L; untreated 415 +/- 51 micromol/L; P < 0.01). Tissue factor inhibition in defibrinogenated rabbits resulted in further protection of renal function (creatinine 140 +/- 19 micromol/L, P < 0.01) and reduced proteinuria (0.4 +/- 0.2g/day, untreated 2.6 +/- 0.4 g/day, P <0.01), which was significantly increased by defibrinogenation alone (ancrod-treated, 5.6 +/- 1.2 g/day). Anti-tissue factor antibodies (but not defibrinogenation alone) attenuated glomerular T-cell and macrophage recruitment, and major histocompatibility complex (MHC) class II expression., Conclusion: These results demonstrate important proinflammatory effects of tissue factor in crescentic glomerulonephritis that are fibrin independent and provide in vivo evidence for tissue factor's proinflammatory effects on MHC class II expression and leukocyte accumulation.

The role of active thrombosis in the pathophysiology of pulmonary embolism is unclear. We tested the hypothesis that venous thrombi significantly increase their thrombotic activity once they embolize into the high-flow circulation of the pulmonary arteries. Thrombotic activity was measured using an immunoassay that measures both fibrinopeptide B (FPB) as well as its most abundant metabolite des-arginine FPB. Thrombi were formed in the femoral veins of adult dogs. In one group, the thrombi were embolized without anticoagulation. In the second group, heparin (300 U/kg bolus, then 90 U x kg(-1) x h(-1) infusion) was administered before embolization to prevent subsequent thrombotic activity. Plasma FPB concentrations were significantly suppressed in the heparinized group relative to the nonheparinized group for 1 h postembolization (P = 0.038). We conclude that pulmonary embolization itself causes preexisting venous thrombi to greatly intensify their thrombotic activity and that embolization-associated thrombus propagation can be prevented by heparin.

Background: Infective endocarditis (IE) caused by Staphylococcus aureus is associated with significant morbidity and mortality rates. Platelets play a dual role as adhesive cells forming associates with bacteria as well as specialized inflammatory cells. The specific role of the various factors involved in bacteria-platelet association has not yet been fully elucidated., Methods and Results: We observed a dramatic increase in the capability to bind S aureus when platelets were activated with thrombin (from 5% to 30%, P<0.001). To pinpoint platelet-binding sites involved in the interaction, platelets from knockout mice and from patients with selective inherited deficiency of membrane proteins or of granules were used. CD36, GPIIb/IIIa, and P-selectin were excluded as receptors for S aureus. Platelets from patients with alpha-delta-storage pool disease and Gray platelet syndrome indicate the requirement of alpha-granule contents. Platelet activation by ADP did not promote platelet-S aureus associate formation, although these platelets were covered with bound fibrinogen. Only small numbers of associates between fibrinogen-covered bacteria and ADP-activated platelets were observed. Formation of fibrin alone was also not sufficient to induce association. Only when fibrin formation and platelet activation occurred together were large numbers of associates formed (P<0.001). A potential receptor for fibrin on S aureus is clumping factor A. Addition of thrombospondin-1 to control platelets increased the number of associates (P=0.02)., Conclusions: Soluble fibrin but not fibrinogen is the main mediator of platelet-S aureus association. In addition, platelet activation and the release of alpha-granule contents, particularly thrombospondin-1, is a requirement for platelet-S aureus association.

Snakes' venom is a mixture of biologically active substances, containing proteins and peptides. A number of these proteins interact with haemostasis system components. Activators and inhibitors affecting blood coagulation and fibrinolysis systems are of special interest. Venom components can be classified into three main groups, such as procoagulants, anticoagulants and fibrinolytic enzymes according to their action. This review is focused on enzymes from Agkistrodon halys halys venom. They are thrombine-like enzyme, named Ancystron-H, flbrinogenolytic enzyme, protein C activator and platelet aggregation inhibitor. Ancystron-H is used for determination of fibrinogen level in blood plasma of patients undergoing heparin treatment and blood coagulation inhibitors accumulation. The fibrinogenolytic enzyme can be used as the instrument for protein-protein interactions in fibrinogen-fibrin system. The protein C activator is used for protein C level determination in blood plasma with different pathologies. Functions of the platelet aggregation inhibitor, belonging to disintegrins group, can be used for development of antithrombotic preparations. Information about the use of snake venoms in science and medicine is presented.

Reperfusion of the ischemic heart is necessary to prevent irreversible injury of the myocardium, which leads to permanent organ dysfunction. However, reperfusion in itself leads to myocardial ischemia/reperfusion (I/R) injury, which is characterized by an acute inflammatory response mediated by activated inflammatory cells, chemokines, cytokines, and adhesion molecules. The molecular mechanisms of myocardial I/R injury are not completely known. Tissue factor (TF) and thrombin, two potent procoagulant and proinflammatory mediators, are recognized to play significant roles in myocardial I/R injury. To investigate the role of TF and thrombin in myocardial I/R injury, we used rabbit and murine in situ coronary artery ligation models. Increased TF mRNA, antigen, and activity were found in ischemic cardiomyocytes. Administration of an inhibitory antirabbit TF monoclonal antibody before or during the onset of ischemia resulted in a significant reduction in infarct size. Functional inhibition of thrombin with hirudin also reduced the infarct size. However, defibrinogenating rabbits with ancrod had no effect on infarct size, suggesting a requirement of thrombin generation but not fibrin deposition in myocardial I/R injury.

Ancrod is a purified fraction of venom from the Malayan pit viper, Calloselasma rhodostoma, currently under investigation for treatment of ischemic stroke. The therapeutic effect is ascribed to a lowering of plasma fibrinogen. Thirty-two healthy volunteers received subcutaneous ancrod at doses of 1.0, 1.5 and 2.0 IU/kg body weight or placebo. Blood samples were drawn before the injection and at various time points until 96 h after the injection. Ancrod leads to the formation of desAA-fibrin, which serves as cofactor in tissue plasminogen activator activity (tPA)-induced plasminogen activation. Unchanged concentrations of prothrombin fragment F1.2 and thrombin-antithrombin complex (TAT) indicate that fibrin formation occurs independent of thrombin. Plasmin generation is independent of an increase in tPA activity or changes in plasminogen activator inhibitor-1 (PAI-1) concentration in plasma. Subcutaneous injection of ancrod leads to a generalized fibrino(geno)lytic response caused solely by providing tPA with soluble fibrin as its cofactor in plasminogen activation. Maximal plasmin activity is present 12 h after subcutaneous injection.

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.

Ancrod is a purified fraction of venom from the Malayan pit viper, Calloselasma rhodostoma, currently under investigation for treatment of acute ischemic stroke. Treatment with ancrod leads to fibrinogen depletion. The present study investigated the mechanisms leading to the reduction of plasma fibrinogen concentration. Twelve healthy volunteers received an intravenous infusion of 0.17 U/kg body weight of ancrod for 6 hours. Blood samples were drawn and analyzed before and at various time points until 72 hours after start of infusion. Ancrod releases fibrinopeptide A from fibrinogen, leading to the formation of desAA-fibrin monomer. In addition, a considerable proportion of desA-profibrin is formed. Production of desA-profibrin is highest at low concentrations of ancrod, whereas desA-profibrin is rapidly converted to desAA-fibrin at higher concentrations of ancrod. Both desA-profibrin and desAA-fibrin monomers form fibrin complexes. A certain proportion of complexes carries exposed fibrin polymerization sites E(A), indicating that the terminal component of the protofibril is a desAA-fibrin monomer unit. Soluble fibrin complexes potentiate tissue-type plasminogen activator-induced plasminogen activation. Significant amounts of plasmin are formed when soluble fibrin in plasma reaches a threshold concentration, leading to the proteolytic degradation of fibrinogen and fibrin. In the present setting, high concentrations of soluble fibrin are detected after 1 hour of ancrod infusion, whereas a rise in fibrinogen and fibrin degradation products, and plasmin-alpha(2)-plasmin inhibitor complex levels is first detected after 2 hours of ancrod infusion. Ancrod treatment also results in the appearance of cross-inked fibrin degradation product D-dimer in plasma. (Blood. 2000;96:2793-2802)

We analyzed the polymerization of BbetaA68T fibrinogen, the recombinant counterpart of fibrinogen Naples, a variant known to have decreased thrombin binding. When polymerized with equal thrombin concentrations, BbetaA68T fibrinogen had a longer lag time and lower rate of lateral aggregation, V(max), than normal recombinant fibrinogen, but a similar final turbidity. At thrombin concentrations that equalized the rates of fibrinopeptide A release, BbetaA68T fibrinogen polymerized with a lag time and V(max) similar to normal, but reached a significantly lower final turbidity. Similar results were produced when BbetaA68T was polymerized with Ancrod, which cleaves fibrinopeptide A at the same rate from either fibrinogen, and when BbetaA68T desA monomers were polymerized. The polymerization of desAB fibrin monomers, which circumvents fibrinopeptide release, was the same for both fibrinogens. We confirmed that turbidity was indicative of fiber thickness by scanning electron microscopy of fibrin clots. Here, we present the first experimental evidence of fibrin polymerization with a normal period of protofibril formation and rate of lateral aggregation, but with a significantly decreased extent of lateral aggregation. We conclude that the decreased lateral aggregation seen in BbetaA68T fibrinogen is due to an altered step in the enzymatic phase of its polymerization process. We propose that during normal polymerization a subtle conformational change in the E domain occurs, between the release of FpA and FpB, and that this change modulates the mechanism of lateral aggregation. Without this change, the lateral aggregation of BbetaA68T fibrinogen is impaired such that variant clots have thinner fibers than normal clots.

Reptilase, defibrase and ancrod are thrombin-like venom enzymes that cleave fibrinogen to release fibrinopeptide-A and generate fibrin monomers. Although these enzymes decrease fibrinogen levels in vivo, presumably by enhancing fibrinolytic activity, the mechanism has not been identified. In the present study, we analyzed their effects on the inhibitors of fibrinolysis. Plasminogen activator inhibitor-1 (PAI-1) was cleaved at its C-terminus by reptilase and lost its specific activity. Alpha2-antiplasmin (alpha2-AP) was cleaved both at the Pro19-Leu20 peptide bond and at its C-terminus by reptilase, and also lost its specific activity. The apparent second-order rate constants (mol/l per min per Batroxobin unit) were 0.22 for the cleavage of PAI-1 (3.2 micromol/l) and 0.19 for that of alpha2AP (6.4 micromol/l), which were approximately 200-fold lower than that (47.0) for the cleavage of fibrinogen (1.1 micromol/l). Neither defibrase nor ancrod cleaved and inactivated these inhibitors. Only reptilase enhanced euglobulin clot lysis in vitro at high concentration, due probably to PAI-1 inactivation. Since all these three enzymes enhance fibrinolysis similarly during defibrination therapy, the neutralization or inactivation of the inhibitors of fibrinolysis appeared not to represent the main mechanism for the enhancement.

Heparin-induced thrombocytopenia (HIT) may be complicated by severe thrombotic complications and death. Currently no specific laboratory test is available to make the diagnosis. When HIT is clinically suspected, heparin should be discontinued immediately. While no specific therapy for HIT exists, there is increasing evidence that acute antithrombin therapy may significantly reduce morbidity and mortality. Among several agents, the direct antithrombins, such as r-hirudin and argatroban, look the most promising for acute treatment.

Fibrinogen and platelets rapidly saturate the exposed subendothelium of a freshly deendothelialized aorta in vivo. As thrombin generated within the site of injury is largely responsible for fibrin(ogen) deposition, we questioned whether various anticoagulant treatments would inhibit uptake of both fibrinogen and platelets in vivo. Rabbits were anticoagulated by pretreatment with either Warfarin, Ancrod, or recombinant hirudin. Each anesthetized, anticoagulated (or saline-injected control) rabbit was injected i.v. with rabbit 51Cr-platelets and 125I-fibrinogen before a balloon-catheter deendothelializing (or sham) injury of the thoracic aorta. At 10 minutes after injury, the rabbit was exsanguinated and the aorta excised. Platelet adsorption by the deendothelialized aorta surface was substantially reduced in anticoagulated rabbits (controls, 2.2x10(5)/mm2; Warfarin-treated, 1.2x10(5)/mm2; Ancrod-treated, 5.3x10(4)/mm2; r-hirudin-treated [5 mg/kg], 5.3x10(4)/mm2), and a significant reduction of fibrinogen associated with the platelet layer (from 5.3 to 1 to 2 pmol/cm2) and within the underlying intima-media layer (from 16.9 to 5 to 6 pmol/cm2) was observed in the r-hirudin-and Warfarin-treated rabbits. The pattern of aorta-deposited 51Cr-platelets and 125I-fibrin in the anticoagulated rabbits corresponded well with an assessment by transmission electron microscopy of aortic tissue samples. We conclude that approximately 70% of fibrinogen uptake is thrombin dependent and that approximately 80% of platelet adsorption depends on codeposited fibrin(ogen) during the 10-minute interval after balloon injury. Pretreatment with an agent that interferes with either thrombin or fibrin production will inhibit the immediate interaction of fibrinogen and platelets with the freshly exposed subendothelium.

The venoms from 3 snakes have been shown to induce defibrinogenation: ancrod from the venom of Calloselasma rhodostoma (formerly known as Agkistrodon rhodostoma), batroxobin from the venom of Bothrops atrox moojeni, and crotalase from the venom of Crotalus adamanteus. The purified fractions of ancrod, batroxobin, and crotalase possess coagulant, proteolytic and esterolytic properties, although their primary mechanism of action is a proteolytic effect on circulating fibrinogen. Ancrod cleaves only the A-fibrinopeptides, but not the B-fibrinopeptides, from fibrinogen; this contrasts with thrombin, batroxobin and crotalase, which cleave both fibrinopeptides A and B. Within minutes of administration of ancrod or batroxobin, there is a significant reduction in plasma fibrinogen levels, and these remain exceedingly low with repeated administration (once or twice daily). The rapid fall in plasma fibrinogen levels is accompanied by a slightly delayed but marked rise in the level of fibrinogen-fibrin degradation products. Plasminogen levels are decreased and blood viscosity is reduced, but formed elements in the circulating blood remain unaltered. Ancrod and batroxobin have been investigated in patients with stroke, deep-vein thrombosis, myocardial infarction, peripheral arterial thrombosis, priapism, and sickle-cell crisis; crotalase has not been administered to humans. However, results have been difficult to interpret, and additional well designed trials are needed to better define the optimum role of ancrod and batroxobin in the management of these conditions. Overall, treatment is well tolerated and serious adverse events are infrequent. In the coagulation laboratory, ancrod, batroxobin and crotalase may be used as reagents to perform coagulation studies on specimens of blood that contain heparin. These venom fractions can be substituted for thrombin in performing the thrombin time and in removing fibrinogen from plasma for accurate determination of fibrinogen-fibrin degradation products.

Heparin-induced thrombocytopenia and thrombosis, also known as heparin-associated thrombocytopenia and thrombosis, was diagnosed in a 73-year-old man who had sustained a pelvic fracture, which was complicated by a left, deep-vein thrombosis. Heparin was administered and thrombocytopenia and arterial thrombosis of his left foot developed, which required amputation of three lateral toes. Four years later, the patient experienced a heart attack, and subsequently postinfarction angina developed, which was refractory to treatment with aspirin, nitrates, and beta-blockers. He was referred to a large, 750-bed teaching hospital for cardiac catheterization and possible coronary artery bypass grafting. An alternative treatment was needed for rapid anticoagulation. Ancrod, snake venom from the Malayan pit viper, was used to lower plasma fibrinogen levels, which allowed successful cardiac catheterization and coronary artery bypass grafting. We present a case study of the successful treatment of this patient with use of ancrod, and the nursing management for patients with heparin-induced or heparin-associated thrombocytopenia and thrombosis receiving this drug.

Previous results demonstrated that rats given Escherichia coli lipopolysaccharide (LPS; 4 mg/kg, i.v.) experience hepatocellular necrosis that begins within 4 hr and that prior treatment with anticoagulants (e.g., heparin) which target thrombin prevents the liver injury. In this study, hepatocellular injury, as marked by increased plasma alanine aminotransferase (ALT) activity and histologic changes, was prevented when heparin or hirudin was administered to rats shortly before the onset of injury. These results suggest that thrombin is a critical mediator that acts distally in the series of inflammatory events that culminates in hepatocellular damage. To explore further this hypothesis, livers isolated from rats 2 hr after LPS administration were perfused with various media. Perfusion of livers with medium comprising diluted blood from heparin-treated donors resulted in no release of ALT activity. By contrast, perfusion with similar medium anticoagulated with ancrod, which prevents clotting by depleting fibrinogen but does not inhibit thrombin, resulted in hepatocellular injury evidenced as a time-dependent appearance of ALT activity in the medium. Moreover, when livers from rats treated 2 hr previously with LPS were perfused with buffer to which thrombin had been added, injury resulted. No injury resulted when thrombin was omitted from the buffer or when livers from saline-treated rats were used. These results indicate that thrombin is a critical and distal mediator of LPS-induced liver damage and contributes to hepatocellular injury through a mechanism that is independent of clot formation. Furthermore, inflammatory events triggered by LPS exposure are a prerequisite for thrombin-induced injury.

Ancrod-generated fibrin has been shown to stimulate prostacyclin synthesis of human umbilical vein endothelial cells (Chang et al., 1994, Biochem. Biophys. Res. Commun. 203, 1920). We further investigated its mechanism of action. The increment of 6-keto prostaglandin F1 alpha stimulated by ancrod-generated fibrin was almost completely inhibited when endothelial cells were either pretreated with 50 microM 8-(N,N'-diethylamino)octyl-3,4,5- trimethoxybenzoate (TMB-8) or preloaded with 15 microM 1,2-bis(2- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). 6-Keto prostaglandin F1 alpha production during 2 and 10 h incubation period was also inhibited by 1.2 mM ethyleneglycol-bis-(beta-aminoethylether)-N,N,N',N'-tetraace tic acid (EGTA) (41 +/- 12 and 53 +/- 17% inhibition, respectively). Further, ancrod-generated fibrin caused a rapid-onset increase in [3h]inositol monophosphate (IP1) formation in endothelial cells. This increase in IP1 was significantly inhibited by 1 mM Gly-Pro-Arg-Pro, 1 mM neomycin or 100 ng/ml pertussis toxin. At the same time, neomycin and pertussis toxin also significantly inhibited 6-keto prostaglandin F1 alpha synthesis of endothelial cells stimulated by ancrod-generated fibrin. Additionally, the increment of IP1 production as well as prostacyclin production were significantly inhibited by monoclonal antibodies directed against alpha v beta 3. These results suggest that intra- and extra-cellular Ca2+ participate in prostacyclin synthesis stimulated by ancrod-generated fibrin. Ancrod-generated fibrin stimulates pertussis toxin-sensitive G-protein regulated phosphoinositide breakdown, which is responsible for prostacyclin synthesis. This augmentation in prostacyclin synthesis and phosphoinositide breakdown caused by ancrod-generated fibrin area, at least in part, mediated by fibrin binding to integrin alpha v beta 3 on endothelial cells.

The objective of this investigation was to evaluate the effect of ancrod, a fibrinogenolytic protease from Malayan pit viper venom, locally delivered through a photopolymerized biodegradable hydrogel in preventing postoperative adhesions. The experimental model involved ischemic and serosal injury to the uterine horns of rats with measurement of adhesions 7 days after injury. Ancrod was delivered intravenously for 5 days preoperatively through 3 days postoperatively, intraperitoneally for 5 days preoperatively, intraperitoneally for 3 days postoperatively, and locally via the hydrogel formed upon the uterine horns by photopolymerization of an aqueous precursor solution. Systemic defibrinogenation by intravenous administration pre-through postoperatively reduced the extent of adhesions by 63% without dose sensitivity from 5 to 20 units/kg/day. Preoperative defibrinogenation by intraperitoneal administration reduced adhesion extent by up to 57%, while postoperative administration was more effective, reducing adhesions by up to 84% with a dose-dependent response from 5 to 20 units/kg/day. Administration of ancrod by local release from a tissue-adherent hydrogel was more effective than either the hydrogel alone or the same amount of ancrod administered by postoperative intraperitoneal injection. Adhesions were reduced by 82% at a local dose of 10 units/kg, compared to a reduction of 68% due to the barrier properties of the gel alone (P < 0.01) and of 19% due to the same amount of drug given at the time of surgery (P < 0.001). Local delivery of ancrod from a tissue-adherent hydrogel barrier thus provided an efficacious prevention to postoperative adhesions while permitting administration of a low total dose of the protease.

Ancrod, a thrombin-like enzyme purified from the venom of Calloselasma rhodostoma, was administered to rabbits intravenously, and blood samples were obtained at 1, 3, 6, 10, and 24 hours after infusion. Ancrod caused a rapid and sustained defibrinogenation within the first 6 hours, with production of fibrinogen degradation products (FDPs) peaking at 1 hour and declining to background level at 6 hours. No significant changes in platelet count, white cell count, or hematocrit was observed. Citrated PRP prepared 1, 3, and 6 hours after ancrod infusion showed diminished aggregation, adenosine triphosphate (ATP) release, and thromboxane B2 formation on the addition of collagen. Although platelet suspension prepared from defibrinogenated platelet-rich plasma (PRP) at 3 hours showed no significant change in aggregation and ATP-releasing activity, the latent period of platelet aggregation was prolonged. When the remaining platelet-poor plasma obtained from defibrinogenated PRP at 3 hours was used to suspend the normal washed platelets prepared from PRP before ancrod infusion, the platelets showed a similar defect in aggregation and release action. Addition of fibrinogen (200 micrograms/ml to 2 mg/ml) to the above preparation partially restored aggregation but not capacity for secretion and thromboxane formation. When normal washed platelets were suspended with the defibrinogenated plasma, prepared by mixing ancrod with normal plasma in vitro and removing the formed fibrin, the platelet suspension showed impaired platelet aggregability, and the aggregability could be restored to the normal level by the addition of exogenous fibrinogen to this preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence suggests that components of the coagulation system contribute to the pathogenesis of liver injury after exposure to lipopolysaccharide (LPS) from gram-negative bacteria. Although the mechanism by which the coagulation system mediates liver injury remains unknown, it has been proposed that the conversion of fibrinogen to insoluble fibrin and consequent deposition in liver microvasculature may contribute to the development of liver injury. The purpose of this study was to test the hypothesis that the coagulation system contributes to LPS hepatotoxicity by a mechanism which is dependent on circulating fibrinogen. A marked reduction in plasma fibrinogen concentration occurred in rats after LPS exposure. The decrease in circulating fibrinogen, which marked activation of the coagulation cascade: 1) occurred at doses of LPS that caused liver injury; 2) was temporally associated with the onset of liver injury; and 3) was attenuated by pretreatment with heparin or warfarin under conditions which afforded protection against liver injury. Pretreatment with either pentoxifylline or antiserum to tumor necrosis factor-alpha, both of which protect against LPS hepatotoxicity, also attenuated the LPS-induced decrease in circulating fibrinogen. Polymorphonuclear leukocyte (neutrophil) depletion protected against liver injury after administration of either a small (2 mg/kg) or a large (8 mg/kg) dose of LPS and attenuated the decrease in circulating fibrinogen albeit to a lesser degree after the larger LPS dose. Depletion of circulating fibrinogen with ancrod did not afford protection against LPS hepatotoxicity. These results suggest that the coagulation system contributes to the pathogenesis of LPS-induced liver injury, but it does so by a mechanism which is independent of circulating fibrinogen.

Isometric contraction to direct supramaximal tetanic stimulation of the anterior tibialis (AT) muscle was measured in 50 New Zealand White rabbits after ischemia and reperfusion. Ischemia was produced unilaterally by collateral ligation and temporary inflow control until AT muscle function decreased to < 5% of contralateral (control) AT muscle and the ischemic interval was recorded. Reperfusion was carried out in one of the following ways: group I (n = 20), release of vascular clamps (blood reperfusion [BR]); group II (n = 10), release of vascular clamps and simultaneous intraarterial administration of 50,000 units of urokinase (urokinase reperfusion [UR]); group III (n = 10), release of vascular clamps and simultaneous administration of 50,000 units of urokinase and 28 mg (5 units) of purified rabbit plasminogen (urokinase plasminogen reperfusion [UPR]); and group IV (n = 10), animals defibrinated to < 50 mg/dl with ancrod prior to ischemia and received BR (ancrod blood reperfusion [ABR]). During reperfusion, function was recorded every 60 min for 2 hr. Recovery of experimental muscle function is expressed as the percentage of contralateral control limb function. The mean ischemic interval (mean +/- SEM), to achieve < 5% of contralateral control limb function, was 206.7 +/- 9.9, 209.5 +/- 16.6, 221.7 +/- 12.5, and 272.0 +/- 14.2 min for animals in groups I-IV, respectively. The mean experimental muscle function (mean +/- SEM) following the ischemic interval was 3.2 +/- 0.8, 4.5 +/- 1.4, 4.4 +/- 1.2, and 3.3 +/- 1.0 for groups I-IV, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Ancrod caused defibrinogenation and exhibited ex vivo antiplatelet activity in experimental rabbits. In this study, platelet thrombus was induced by irradiation of the mesenteric microvessels with filtered light in mice pretreated with fluorescein sodium intravenously. Ancrod (0.5-2 U/kg) dose-dependently, significantly prolonged the time lapse of inducing platelet plug formation in mesenteric venules when it was intravenously infused. At these doses, ancrod depleted plasma fibrinogen and displayed ex vivo antiplatelet aggregation induced by collagen. Ancrod (1 U/kg) prolonged the occlusion time about 2.1 folds (from control 103.2 +/- 17.0 to 211.2 +/- 26.3 seconds) with a duration longer than 60 min. On the other hand, PGI2 briefly prolonged the occlusion time about 1.5 folds when it was given by continuous infusion (250-500 ng/kg/min). Heparin (100-250 U/kg) had no significant effect in this model. Therefore, ancrod may be used as a therapeutic agent not only in treatment of venous thrombosis and possibly in prevention of arterial thrombosis.

The role of defective fibrinolysis caused by elevated activity of plasminogen activator inhibitor-1 (PAI-1) in promoting fibrin deposition in vivo has not been well established. The present study compared the efficacy of thrombin or ancrod, a venom-derived enzyme that clots fibrinogen, to induce fibrin formation in rabbits with elevated PAI-1 levels. One set of male New Zealand rabbits received intravenous endotoxin to increase endogenous PAI-1 activity followed by a 1-hour infusion of ancrod or thrombin; another set of normal rabbits received intravenous human recombinant PAI-1 (rPAI-1) during an infusion of ancrod or thrombin. Thirty minutes after the end of the infusion, renal fibrin deposition was assessed by histopathology. Animals receiving endotoxin, rPAI-1, ancrod, or thrombin alone did not develop renal thrombi. All endotoxin-treated rabbits developed fibrin deposition when infused with ancrod (n = 4) or thrombin (n = 6). Fibrin deposition occurred in 7 of 7 rabbits receiving both rPAI-1 and ancrod and in only 1 of 6 receiving rPAI-1 and thrombin (P < .01). In vitro, thrombin but not ancrod was inactivated by normal rabbit plasma and by purified antithrombin III or thrombomodulin. The data indicate that elevated levels of PAI-1 promote fibrin deposition in rabbits infused with ancrod but not with thrombin. In endotoxin-treated rabbits, fibrin deposition that occurs with thrombin infusion may be caused by decreased inhibition of procoagulant activity and not increased PAI-1 activity.

Although "biocompatible" polymeric elastomers are generally nontoxic, nonimmunogenic, and chemically inert, implants made of these materials may trigger acute and chronic inflammatory responses. Early interactions between implants and inflammatory cells are probably mediated by a layer of host proteins on the material surface. To evaluate the importance of this protein layer, we studied acute inflammatory responses of mice to samples of polyester terephthalate film (PET) that were implanted intraperitoneally for short periods. Material preincubated with albumin is "passivated," accumulating very few adherent neutrophils or macrophages, whereas uncoated or plasma-coated PET attracts large numbers of phagocytes. Neither IgG adsorption nor surface complement activation is necessary for this acute inflammation; phagocyte accumulation on uncoated implants is normal in hypogammaglobulinemic mice and in severely hypocomplementemic mice. Rather, spontaneous adsorption of fibrinogen appears to be critical: (a) PET coated with serum or hypofibrinogenemic plasma attracts as few phagocytes as does albumin-coated material; (b) in contrast, PET preincubated with serum or hypofibrinogenemic plasma containing physiologic amounts of fibrinogen elicits "normal" phagocyte recruitment; (c) most importantly, hypofibrinogenemic mice do not mount an inflammatory response to implanted PET unless the material is coated with fibrinogen or the animals are injected with fibrinogen before implantation. Thus, spontaneous adsorption of fibrinogen appears to initiate the acute inflammatory response to an implanted polymer, suggesting an interesting nexus between two major iatrogenic effects of biomaterials: clotting and inflammation.

Kistomin, a metalloprotease purified from venom of Calloselasma rhodostoma, dose- and time-dependently prolonged the latent period of aggregation and inhibited ATP secretion of human washed platelets stimulated by thrombin. It inhibited aggregation induced by low concentrations of thrombin (< or = 0.2 U/ml) whereas it had only slight effect on aggregation induced by high concentrations of thrombin (> or = 0.5 U/ml). Meanwhile it also inhibited ristocetin-induced platelet aggregation in a dose- and time-dependent manner. It significantly inhibited cytosolic calcium rise of Quin 2--loaded platelets, completely blocked thromboxane B2 formation, and blocked [3H]inositol phosphates formation of [3H]myoinositol loaded platelets stimulated by 0.1 U/ml of thrombin. Kistomin inhibited significantly thromboxane but not [3H]inositol phosphates formation of platelets stimulated by a high concentration of thrombin (1 U/ml). Incubation of platelets with kistomin resulted in a selective cleavage of platelet membrane glycoprotein Ib as revealed by SDS/PAGE stained by periodic acid/Schiff reagent. These results suggested that thrombin activates platelets at least through two receptors/or effectors-mediated events. In addition to glycoprotein Ib, other surface membrane component(s) (e.g., the seven transmembrane domain thrombin receptor) may also be important in regulating the biochemical events of human platelets in response to thrombin. However, the extent and rate of platelet aggregation stimulated by low concentrations of thrombin ( < or = 0.2 U/ml) are closely related with the intactness of glycoprotein Ib.

By means of CM-Sephadex C-50 column chromatography, gel-filtration on sephadex G-75 and Sephacryl S-200 columns, a purified fibrinogenase, kistomin, was obtained from venom of Agkistrodon rhodostoma. It was a single peptide-chain with a molecular mass of about 21,800 Da containing about 202 amino-acid residues as revealed by amino acid analysis. Kistomin preferentially cleaved A alpha- and subsequently the gamma-chain of fibrinogen, leaving the B beta-chain unaffected. Its fibrinogenolytic activity was estimated to be 36.6 +/- 4.5 mg/min per mg protein and was inhibited by the pretreatment of EDTA, suggesting that it is a metalloproteinase. Its fibrinogenolytic activity in platelet-poor plasma is much less potent as compared to that in purified fibrinogen solution. It inhibited ristocetin-induced aggregation of human platelets in a dose-dependent manner in the presence of von Willebrand factor.

Neutrophils (polymorphonuclear neutrophils [PMNs]) have been implicated as mediators of reperfusion injury. Heparin, urokinase, and ancrod are agents used routinely to prevent and treat thrombosis, yet their effects on PMN function are unknown. Therefore human PMNs were obtained and incubated for 30 minutes with either saline solution or one of the following pharmacologic agents, each tested at three different concentrations: group 1, saline solution (control, n = 14); groups 2 through 4, heparin (5000 units/ml, n = 8; 2500 units/ml, n = 6; and 1250 units/ml, n = 6, respectively); groups 5 through 7, urokinase (50,000 units/ml, n = 8; 25,000 units/ml, n = 6; and 12,500 units/ml, n = 6, respectively), and groups 8 through 10, ancrod (70 units/ml, n = 8; 35 units/ml, n = 6; and 17.5 units/ml, n = 6). Superoxide anion production was measured by the reduction of cytochrome c in a spectrophotometric assay. Chemotaxis was evaluated by the number of PMNs migrating across a filter with a Neuro Probe chamber (Neuro Probe, Cabin John, Md.). Phagocytosis was determined by the ingestion of opsonized zymosan particles by PMNs. Serum obtained from each PMN donor was used both to opsonize the zymosan and as a chemoattractant in the chemotaxis assay. Statistical comparison was evaluated by analysis of variance, and post hoc comparisons for each agent with control were performed with the unpaired Student t test. No agent, at any dose, significantly changed superoxide anion production compared with control cells. All three agents significantly inhibited PMN chemotaxis (p < 0.01). In the control group the number of PMNs counted was 27.6 +/- 4.9.(ABSTRACT TRUNCATED AT 250 WORDS)

We have previously reported that heparin can enhance the removal of antigen from the glomeruli of rats with chronic serum sickness glomerulonephritis. We have performed two studies to investigate the relevance of anticoagulation to this effect. The first experiment was essentially a repeat of those described before, but instead of using heparin, anticoagulation was achieved using Ancrod, which causes depletion of fibrinogen. The amount of antigen in the glomeruli of these rats at the end of the experiment did not differ from controls. In the second experiment, both kidneys were excised from rats with glomerulonephritis induced by a radiolabelled cationic antigen. Of each pair of kidneys, one was perfused for 30 min at 5 ml/min with Hank's balanced salt solution (HBSS) at 37 degrees C. The other was perfused with HBSS to which a total of 4500 units of heparin had been added. The antigen content of isolated glomeruli was subsequently measured. In every case, the kidney perfused with heparin had less antigen per glomerulus than the control contralateral kidney. The ability of heparin to enhance removal of glomerular immune complex deposits is therefore not mediated by anticoagulation.

High baseline levels of plasma fibrinogen have been correlated with increased risk for cardiovascular disease. Since fibrinogen plays a central role in both coagulation and platelet aggregation and is a primary component of thrombi, this study was designed to assess the relationship of circulating fibrinogen concentration and vein anastomotic patency. Subcutaneously injected turpentine was used to increase, and intravenous ancrod (snake venom) to decrease, plasma fibrinogen levels. Rat femoral vein anastomoses were performed, and patency was assessed after 120 min of flow. Rat tail bleeding times were obtained, and blood samples were drawn to determine fibrinogen and plasma protein concentrations, Lee-White clotting times, and activated partial thromboplastin times (APTT). Increased patency was found in the ancrod group (88%) (P < 0.05 vs. controls); turpentine-treated and control groups were not significantly different (71% and 63%, respectively). The ancrod group also showed significantly prolonged tail bleeding times and APTT. Fibrinogen levels were significantly decreased in the ancrod group (1.73 mg/ml) and elevated in the turpentine group (4.91 mg/ml) vs. controls (2.34 mg/ml; P < 0.005). These results indicate that elevated fibrinogen levels, in particular when triggered by an acute-phase response, do not appear to predispose small vessel repairs toward thrombosis. Furthermore, this study supports the use of ancrod as an anticoagulant for microvascular surgery.

Deposition of fibrin within the peritoneal cavity is an integral host response to local infection. To directly assess the role of fibrin deposition in the pathogenesis of intraabdominal abscess formation, the ability to induce abscesses in fibrinogen-depleted mice was examined. We hypothesized that systemic defibrinogenation with ancrod would limit the availability of fibrinogen for deposition within the peritoneal cavity and would therefore impair intraabdominal abscess formation. A gelatin capsule containing 50% sterile feces plus Bacteroides fragilis 1 x 10(9) CFU was inserted IP into control or defibrinogenated mice. System defibrinogenation resulted in alteration of the character of abscess formation, as manifested by reduced abscess size and degree of purulence. Abscesses were significantly smaller (0.18 +/- 0.02 gm [n = 29] vs. 0.09 +/- 0.02 gm [n = 11], p less than 0.01) and less purulent (p less than 0.001) in the ancrod-treated mice than in control animals, despite equal numbers of bacteria in the abscesses recovered from both groups. The effect of ancrod was specific for defibrinogenation, because IP repletion with fibrinogen reversed the ancrod effect on abscess size. In addition to its local effects, systemic fibrinogen depletion resulted in a significant elevation in mortality following IP infection (1 of 30 control animals vs. 10 of 23 ancrod-treated animals, p less than 0.01). However, this was not due to an increase in the magnitude of the B. fragilis bacteremia. These studies demonstrate that fibrin deposition contributes to the pathogenesis of purulent abscess formation and that systemic depletion of fibrinogen may alter host susceptibility to the consequences of infection.

Recently evidence was provided for a pathway whereby circulating fibrinogen enters megakaryocyte granules by an endocytic mechanism. Synthesis of fibrinogen by megakaryocytes has been reported. To determine the relationship between plasma fibrinogen and alpha-granule fibrinogen in megakaryocytes and platelets, the fibrinogen content of these cells was studied in rats defibrinated by use of Ancrod, a thrombinlike enzyme purified from the venom of Agkistrodon rhodostoma. Unlike thrombin, Ancrod does not induce platelet secretion. Rats were injected with Ancrod (50 units/kilogram body weight) at 8-hour intervals for 5 days. There were no significant changes in platelet counts. Blood from the treated rats failed to clot, and plasma fibrinogen levels were less than 15 mg/dl. Bone marrow from defibrinated rats and untreated control rats was stained immunohistochemically for fibrinogen and two other alpha-granule proteins, albumin and platelet factor 4 (PF4), in plastic-embedded sections. The presence of these three proteins in platelets was detected by Western blots. Only trace amounts of fibrinogen were detected in megakaryocytes and platelets from defibrinated rats, but fibrinogen in control megakaryocytes and platelets was readily demonstrated. However defibrinated and control rats did not differ in albumin and PF4 content in megakaryocytes and platelets. It is concluded that a major portion of rat platelet fibrinogen is derived from plasma by endocytosis by megakaryocytes.

Reduction of blood viscosity by venesection may improve peripheral blood flow but similar haemorheological changes have not been conclusively demonstrated following reduction of plasma viscosity by defibrinogenation. Ancrod is a defibrinogenating enzyme from the venom of the Malayan pit viper. We studied six male patients with severe intermittent claudication before, during and after treatment with ancrod for 48 h. Ancrod was given by initial infusion of 2 U/kg over 6 h and four subsequent bolus i.v. injections of 2 U/kg. Skin blood flow in feet and toes were measured repeatedly by laser Doppler velocimetry and calf blood flow by electronic plethysmography both before and after 2 min arterial occlusion. Plasma fibrinogen [median (range)] fell from 2.3(1.4-3.9) to 0.1(0.1-0.3) g/l and plasma viscosity from 1.81(1.61-1.90) to 1.49(1.45-1.72) cp. Dorsal foot and toe skin blood (resting or post-ischaemic) flows were not changed significantly by ancrod. Resting calf blood flow fell significantly after 49 h treatment with ancrod (P less than 0.04). Brachial and ankle blood pressures remained unchanged and haematocrit was not changed. Thus, treatment with ancrod for 48 h did not improve peripheral blood flow in patients with peripheral vascular disease. The fall observed in calf blood flow could be related to increased circulating fibrin/fibrinogen high-molecular-weight complexes and degradation products.

Ten dogs were subjected to defibrinogenation with an intravenous perfusion of ancrod (1 unit/kg) (Arvin, Knoll AG, Ludwigshafen, Federal Republic of Germany) over a 2 1/2 hour period. Six of them were subjected to extracorporeal elimination of carbon dioxide with a polypropylene membrane lung by means of veno-venous bypass. The remaining four dogs did not undergo extracorporeal circulation and served as control subjects. In both groups, ancrod administration itself resulted in a marked drop in alpha 2-antiplasmin (33% and 67%, respectively, of the baseline values) and in slight but significant decreases in factor II and plasminogen activities of 25% and 20%, respectively (p less than 0.05), in the group subjected to carbon dioxide removal. There were no significant changes in platelet number or factor V and antithrombin III activities. During the 6-hour bypass period, platelet count and antithrombin III and factor II and V levels decreased significantly. No bleeding was observed. Histologic examination of lung biopsy tissue showed no pathologic features. Analysis of the membrane of the artificial lungs revealed no fibrin deposits. In the control group, except for a drop in alpha 2-antiplasmin levels (54%), no significant changes in hemostatic parameters occurred during the corresponding 6 hours. We conclude that, despite the drop in coagulation factors and in alpha 2-antiplasmin activity during bypass, ancrod can be considered as a valuable alternative anticoagulant for extracorporeal carbon dioxide removal.

In vitro platelet aggregation was studied in dogs following defibrination with Ancrod. There was marked inhibition of aggregation immediately after defibrination. A gradual return toward normal aggregation was observed over the 96 hours study period, but aggregation was still abnormal at 96 hours after defibrination. There was an inverse and highly significant correlation between aggregation and fibrin degradation product level. Fibrinogen was not measurable after defibrination for the entire study period. Aggregation was thus independent of fibrinogen concentration in this study.

Earlier studies suggest that adverse reactions to injected radiographic contrast media are idiosyncratic. In an attempt to gain a better understanding of pathophysiologic events underlying these reactions, rabbit models injected with lethal dose ranges of a cholangiographic contrast material were studied. These animals showed activation of both the complement and coagulation systems. Externally applied heat potentiated complement consumption and increased mortality. Depleting complement components C3-C9 by cobra venom factor did not prevent activation of coagulation or diminish mortality. However, depleting fibrinogen diminished complement activation and markedly diminished mortality. Heparin, administered at several hourly intervals after contrast challenge, also diminished mortality. These studies suggest that the adverse effects of contrast media in this model system are mediated chiefly by the coagulation system, and that complement, if it participates deleteriously, must involve components up to, but not including, C3. A logical role for the inhibitor of C1 esterase in adverse contrast reactions is considered.

Fifteen patients with severe intermittent claudication were treated by therapeutic defibrination with subcutaneous injections of ancrod for 5 weeks. Mean plasma-fibrinogen was maintained below 50% of the initial value throughout the treatment period. This reduction in plasma-fibrinogen was accompanied by a parallel fall in whole-blood viscosity and a pronounced clinical improvement. Objective measurements showed maximum benefit on the 21st day of treatment, when the mean resting ankle/arm pressure index had increased by 37%, the post-exercise pressure index had increased by 50%, and the time taken for the pressure index to return to a resting value after a constant exercise had decreased by 33%. (The claudication-count had increased by 59%).

Despite considerable uncontrolled clinical data implying a beneficial effect of defibrinogenation in peripheral vascular disease, this is not supported by double blind placebo controlled trials. A hypothetical explanation for this discrepancy is developed from experimental studies suggesting that in the absence of adequate shear forces defibrinogenation might induce occlusion of the impaired microcirculation and therefore can lead to an aggravation of tissue hypoxia.

Plasma fibronectin (FN) binds fibrin in vitro by both noncovalent and covalent bonds and is decreased in DIC. In rabbits, conventionally purified 125I-FN had a complex blood clearance with a late t1/2 of 71 hr. A large portion was apparently altered, as evinced by rapid clearance and an intravascular/total body ratio (C1) of 0.28-0.51. 3H-labeled FN, made in vivo by injection of 3H amino acids, had a t1/2 of 73 hr. Crosstransfusion of 131I-FN and 3H-FN into a second set of animals gave similar t1/2s and C1s of 0.74-0.82, indicating the altered 125I-FN was biologically screened in the first animals. Other animals were given 125I-fibrinogen and "screened" 131I-FN. Intravenous thrombin (50-60 U/kg/1 hr) caused a 25%-50% decrease in both 125I-fibrinogen and 131I-FN. Ancrod injection reduced fibrinogen by greater than 90% but had no effect on 131I-FN. 131I-FN levels did not change when thrombin was given after ancrod. No cross-linked FN-fibrinogen alpha-chain was found in the plasma, nor was the thrombin-induced fall in FN affected by spermidine blockade. These experiments demonstrate that FN and fibrin bind in vivo during defibrination and are rapidly cleared from the blood. The abnormal fibrin resulting from ancrod either does not bind FN in vivo or does so reversibly.