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Background Thrombus is composed of two main substances i.e. red blood cells and aggregated platelets which make a web of inter-connected fibrin proteins. During injury it prevents bleeding, so it is very useful but it can be very dangerous if it is produced in healthy blood vessels and block the blood flow through it. Mural thrombi attaches with the blood vessels but in most cases do not block it completely. Venoms are an incredible source of peptides having amazing bioactivities with varying number of amino acid residues. Anticoagulant venom peptides however inhibit the enzyme taking part in coagulation like factor Xa and thrombin. The anticoagulant potential of venom peptides have also been reported by the degradation of the fibrin or fibrinogen related to serine or metalloproteases. Designing and development of numerous therapeutic agents or lead molecules mostly for cardiovascular diseases have been motivated from toxins/proteins from snake venoms. For example, disintegrins, a large family of platelet aggregation inhibitors found in viperid and crotalid snake venoms were the basis for designing of platelet aggregation inhibitors such as eptifibatide and tirofiban. Conclusion Ancrod isolated from Malayan pit viper venom can cause reduction in level of blood fibrinogen and has been effectively tried in various ischemic conditions, including stroke. In order to search for novel lead molecules, the emphasis should be on isolation and characterization of pharmacologically active snake venoms proteins affecting blood coagulation and platelet aggregation. In this review an attempt has been made to recapitulates and discuss venoms of different animals and arthropod having anticoagulant peptides for their potential use in therapeutics and diagnostics.

Microglia-mediated inflammation plays a significant role in neuronal and vascular damage in diabetic retinopathy (DR), but the mechanism linking inflammation, neurodegeneration, and impaired vascular integrity is still unclear. Previous studies from diabetic mouse models showed accumulation of fibrinogen at vessel lesions surrounded by perivascular microglial clusters. The purpose of this study was to evaluate whether the pathological hallmarks of gliosis and vascular aberrations characterized in diabetic animal models are consistent with those in diabetic human retinas, and to assess the effects of the defibrinogenating agent ancrod in retinal pathology and visual acuity in a two-hit inflammatory diabetic mouse model. Post-mortem human eyes were assessed for retinal and inflammatory gene expression by quantitative PCR. Immunohistochemical analyses in human and murine retinas were performed using markers of gliosis, vascular integrity, and fibrinogen deposition. An inflammatory microenvironment, with microgliosis and microaneurysms, was found in the diabetic human eye. Microglial activation, fibrinogen deposition, and axonal loss were also observed in the diabetic murine retina. Ancrod treatment correlated with reduced microgliosis, less fibrinogen deposition, and reduced pro-inflammatory cytokine levels in diseased retinal tissues. Together, these data suggest that fibrinogen contributes to microglia-mediated inflammation in the diabetic retina. Since retinal microgliosis, vascular pathology, and vision deficits manifest in diabetic mice irrespective of CX3CR1 genotype, our results indicate that defibrinogenation can dampen systemic neuroinflammation and vascular insults, thereby improving vision at early stages of diabetes. 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.

While snake venom derived enzymes, such as the thrombin-like activity possessing ancrod, have been used to treat thrombotic disease by defibrinogenating patients, the therapeutic potential of fibrinogenolytic snake venom enzymes, such as those derived from Crotalus atrox, have not been fully explored. However, one of the potential risks of administering fibrinogenolytic enzymes to effect defibrinogenation is hemorrhage secondary to hypofibrinogenemia. The present investigation sought to determine if human fibrinogen modified with carbon monoxide (CO) and iron (Fe) could resist degradation by C. atrox venom as has been seen in vitro in a recently developed rabbit model of envenomation. Compared with unmodified human fibrinogen, CO/Fe modified fibrinogen administered prior to envenomation had significantly shorter onset of coagulation and greater strength; however, when administered after envenomation, there was no differences between the two types of fibrinogen. Of interest, when administered after envenomation, both types of fibrinogen delayed the onset of coagulation while increasing plasma clot strength, a mixed effect likely secondary to formation of fibrinogen degradation products. Further preclinical investigations are needed to further define the benefits and risks of the use of fibrinogenolytic enzymes as defibrinogenating agents, as well as the risks of the "biochemical brakes" used to modulate the activity or substrate of the fibrinogenolytic enzyme.

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.

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.

Heparin anticoagulation followed by protamine reversal is commonly used in cardiopulmonary bypass (CPB) cardiac procedures, but this strategy has some limitations. The primary objective of this study was to determine the reliable alternatives for anticoagulation during CPB for cardiac surgery. For each drug proposed, the secondary objectives were to outline the main advantages and disadvantages, to propose a therapeutic protocol, and to provide a cost-benefit analysis. A systematic review of the literature was performed between September 2012 and December 2013. It was based on the protocol established by the “Cochrane collaboration Handbook”. Twenty articles were analyzed. The Theriaque database from the University Hospital of Grenoble made the economic analysis possible. Seven alternative anticoagulation strategies were considered: danaparoid sodium, lepirudin, argatroban, bivalirudin, ancrod, idraparinux, and EP217609. Danaparoid sodium has issues with individual variability. Several studies (EVOLUTION-ON, CHOOSE-ON) proposed a reliable therapeutic protocol for bivalirudin. Ancrod resulted in an increase in the transfusion of blood products. Direct thrombin inhibitors offer a promising alternative. EP217609 is a synthetic anticoagulant currently undergoing Phase IIa clinical trials. It is an indirect inhibitor of factor Xa, a direct inhibitor of free and bound thrombin, and can be neutralized by avidin. The ideal anticoagulation strategy for cardiac surgery with CPB does not exist. Heparin and protamine remain the gold standard for anticoagulation therapy. To date, bivalirudin is the most promising molecule despite its high cost and lack of a readily available antagonist.

© 2017 Elsevier Inc. Blood-brain barrier (BBB) disruption alters the composition of the brain microenvironment by allowing blood proteins into the CNS. However, whether blood-derived molecules serve as extrinsic inhibitors of remyelination is unknown. Here we show that the coagulation factor fibrinogen activates the bone morphogenetic protein (BMP) signaling pathway in oligodendrocyte progenitor cells (OPCs) and suppresses remyelination. Fibrinogen induces phosphorylation of Smad 1/5/8 and inhibits OPC differentiation into myelinating oligodendrocytes (OLs) while promoting an astrocytic fate in vitro. Fibrinogen effects are rescued by BMP type I receptor inhibition using dorsomorphin homolog 1 (DMH1) or CRISPR/Cas9 activin A receptor type I (ACVR1) knockout in OPCs. Fibrinogen and the BMP target Id2 are increased in demyelinated multiple sclerosis (MS) lesions. Therapeutic depletion of fibrinogen decreases BMP signaling and enhances remyelination in vivo. Targeting fibrinogen may be an upstream therapeutic strategy to promote the regenerative potential of CNS progenitors in diseases with remyelination failure. Extrinsic inhibitors contribute to remyelination failure in neurological diseases. Petersen et al. identify the blood coagulation factor fibrinogen as an activator of BMP receptor signaling in oligodendrocyte progenitor cells that may be targeted therapeutically to promote remyelination.

Urgent reperfusion of the ischaemic brain is the aim of stroke treatment and there has been ongoing research to find a drug that can promote vessel recanalisation more completely and with less side effects. In this review article, the major studies which have validated the use and safety of tPA are discussed. The safety and efficacy of other thrombolytic and anticoagulative agents such as tenecteplase, desmoteplase, ancrod, tirofiban, abciximab, eptifibatide, and argatroban are also reviewed. Tenecteplase and desmoteplase are both plasminogen activators with higher fibrin affinity and longer half-life compared to alteplase. They have shown greater reperfusion rates and improved functional outcomes in preliminary studies. Argatroban is a direct thrombin inhibitor used as an adjunct to intravenous tPA and showed higher rates of complete recanalisation in the ARTTS study with further studies which are now ongoing. Adjuvant thrombolysis techniques using transcranial ultrasound are also being investigated and have shown higher rates of complete recanalisation, for example, in the CLOTBUST study. Overall, development in medical therapies for stroke is important due to the ease of administration compared to endovascular treatments, and the new treatments such as tenecteplase, desmoteplase, and adjuvant sonothrombolysis are showing promising results and await further large-scale clinical trials.

The venom of Malayan pit viper (Calloselasma rhodostoma) is highly toxic but also valuable in drug discovery. However, a comprehensive proteome of the venom that details its toxin composition and abundance is lacking. This study aimed to unravel the venom complexity through a multi-step venomic approach. At least 96 distinct proteins (29 basic, 67 acidic) in 11 families were identified from the venom. The venom consists of mainly snake venom metalloproteinases (SVMP, 41.17% of total venom proteins), within which the P-I (kistomin, 20.4%) and P-II (rhodostoxin, 19.8%) classes predominate. This is followed by C-type lectins (snaclec, 26.3%), snake venom serine protease (SVSP, 14.9%), L-amino acid oxidase (7.0%), phospholipase A2 (4.4%), cysteine-rich secretory protein (2.5%), and five minor toxins (nerve growth factor, neurotrophin, phospholipase B, 5′ nucleotidase and phosphodiesterase, totaling 2.6%) not reported in the proteome hitherto. Importantly, all principal hemotoxins unveiled correlate with the syndrome: SVSP ancrod causes venom-induced consumptive coagulopathy, aggravated by thrombocytopenia caused by snaclec rhodocytin, a platelet aggregation inducer, while P-II rhodostoxin mediates hemorrhage, exacerbated by P-I kistomin and snaclec rhodocetin that inhibit platelet plug formation. These toxins exist in multiple isoforms and/or complex subunits, deserving further characterization for the development of an effective, polyspecific regional antivenom. Biological significance Advents in proteomics and bioinformatics have vigorously propelled the scientific discoveries of toxins from various lineages of venomous snakes. The Malayan pit viper, Calloselasma rhodostoma, is a medically important species in Southeast Asia as its bite can cause envenomation, while the venom is also a source of bioactive compounds for drug discovery. Detailed profiling of the venom, however, is inadequate possibly due to the complex nature of the venom and technical limitation in separating the constituents into details. Integrating a multi-step fractionation method, this study successfully revealed a comprehensive and quantitative profile of the composition of the venom of this medically important venomous snake. The relative abundance of the various venom proteins is determined in a global profile, providing useful information for understanding the pathogenic roles of the different toxins in C. rhodostoma envenomation. Notably, the principal hemotoxins were identified in great details, including the variety of toxin subunits and isoforms. The findings indicate that these toxins are the principal targets for effective antivenom neutralization, and should be addressed in the production of a pan-regional polyspecific antivenom. In addition, minor toxin components not reported previously in the venom were also detected in this study, enriching the current toxin database for the venomous snakes.

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.

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 Ni²⁺ 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.

See related article, pages 1687–1691. In this issue of Stroke , del Zoppo et al1 report new data on the association between hyperfibrinogenemia and functional prognosis after ischemic stroke using the placebo data from 2 well-known clinical interventional trials on the use of defibrinogenating agent ancrod in acute ischemic stroke.2,3 Briefly, the authors explore the relationship of fibrinogen with the ischemic stroke outcome showing that patients with lower initial fibrinogen levels (

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 (≥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.

Of the various prophylactic agents evaluated, four have been found to be effective. These are oral anticoagulants, low-dose heparin, mechanical devices which increase venous blood-flow in the leg, and Dextran. Oral anticoagulants have been shown to be effective in patients having abdominal, thoracic, or hip surgery, when treatment was started either before surgery or in the immediate postoperative period. They have also been shown to be effective in medical patients. The evidence derives from studies which showed that treatment can reduce total mortality, prevents venous thromboembolism detected clinically or at autopsy, and prevents thrombosis diagnosed with venography. On the other hand, the incidence of thrombosis diagnosed by 125I-fibrinogen scanning was not reduced when oral anticoagulants were started just before or just after surgery. This suggests that oral anticoagulant treatment starting in the immediate postoperative period may not prevent formation of the initial thrombotic nidus, but is clinically effective because it prevents extension of the nidus to form a significant thrombus. Bleeding has been a significant complication in almost all studies of surgical patients, and this is the major factor which has prevented widespread use of oral anticoagulant prophylaxis. In addition, the need for careful laboratory monitoring makes this approach inconvenient and adds to its expense. Low-dose heparin has been shown to be effective in general surgical and medical patients, but results have been inconclusive in patients having elective hip surgery, and this approach is probably ineffective in patients with hip fracture. In general surgical patients, low-dose heparin prophylaxis has been shown to prevent pulmonary embolism diagnosed at autopsy examination or with lung scanning, and calf and thigh vein thrombosis diagnosed with 125I-fibrinogen leg-scanning. A slight, but statistically significant, increase in the frequency of wound hematoma and a greater postoperative hematocrit fall have been reported when heparin was given three times daily, but not with the twice daily heparin injection regimen. In these studies, low-dose heparin was given without laboratory control of its anticoagulant effect, so that this prophylactic approach is simple, but the need for subcutaneous injections is a disadvantage of this approach. Results with methods which increase venous blood-flow in the leg have varied, depending on the technic used. Active measures, such as intermittent pneumatic calf compression or peroperative electrical calf muscle stimulation, have been shown to prevent thrombosis detected with 125I-fibrinogen leg-scanning. However, while the evidence suggests that both methods are effective in relatively low risk patients, they may have limited value in the high risk patient who is confined to bed for a long time. These methods are free of side effects and relatively inexpensive, but intermittent calf compression, in particular, is slightly cumbersome...

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.

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.

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.

The effect of molecular “aging” of fibrinogen stimulated by preincubation in solution on the fibrin three-dimensional architecture, its ability to crosslink fibrin-stabilizing factor, and the sensitivity of fibringel to plasmin hydrolysis have been studied. The method of elastic light scattering was used to demonstrate that fibrin generated from “defective” fibrinogen had a coarser structure with a higher mean mass-length ratio of polymeric fibers compared to native fibrinogen (2.24 × 109 and 1.46 × 109 g/(mol cm), respectively). Crosslinking had no effect on the architecture of both control and experimental fibrin samples. Spectrophotometric and electrophoretic analysis has shown a higher sensitivity of coarse fibrin gels to plasmin. A close correlation between spontaneous local conformational reconstructions in fibrinogen molecule and its functional activity is concluded.

Cerebrovascular dysfunction contributes to the pathology and progression of Alzheimer's disease (AD), but the mechanisms are not completely understood. Using transgenic mouse models of AD (TgCRND8, PDAPP, and Tg2576), we evaluated blood–brain barrier damage and the role of fibrin and fibrinolysis in the progression of amyloid-β pathology. These mouse models showed age-dependent fibrin deposition coincident with areas of blood–brain barrier permeability as demonstrated by Evans blue extravasation. Three lines of evidence suggest that fibrin contributes to the pathology. First, AD mice with only one functional plasminogen gene, and therefore with reduced fibrinolysis, have increased neurovascular damage relative to AD mice. Conversely, AD mice with only one functional fibrinogen gene have decreased blood–brain barrier damage. Second, treatment of AD mice with the plasmin inhibitor tranexamic acid aggravated pathology, whereas removal of fibrinogen from the circulation of AD mice with ancrod treatment attenuated measures of neuroinflammation and vascular pathology. Third, pretreatment with ancrod reduced the increased pathology from plasmin inhibition. These results suggest that fibrin is a mediator of inflammation and may impede the reparative process for neurovascular damage in AD. Fibrin and the mechanisms involved in its accumulation and clearance may present novel therapeutic targets in slowing the progression of AD.

A n ONTROLLED ANTICOAGULATION is essential for the safe performance of cardiac surgery. Unfractionated heprin (UFH) is the standard anticoagulant used in patients unergoing both onand off-bypass cardiac procedures. Advanages of UFH include low cost, rapid onset of effect, ease of itration and monitoring, and rapid reversal with protamine. lthough UFH has a long record of safety in the cardiac urgical patient population, heparin can induce thrombocytoenia via immune and nonimmune mechanisms. Non–immuneediated thrombocytopenia (heparin-induced thrombocytopeia type I [HIT type I]) is caused by direct platelet activation by eparin. The reduction in platelet count with HIT type I is mild nd self-limiting and requires no treatment. Heparin-induced hrombocytopenia type II is an immune-mediated disorder that ccurs when antibodies (predominantly IgG) are produced gainst platelet factor 4 (PF4)-heparin complexes. The binding f heparin to PF4 results in the expression of several antigenic eoepitopes, which trigger the production of IgG.1 The macroolecular complex of heparin, PF4, and IgG results in platelet ctivation, platelet destruction, and the release of prothromotic microparticles from platelets.2 The paradoxic prothromotic state (low platelet count, arterial and venous thrombosis) bserved in patients with HIT type II is because of platelet ctivation and the generation of thrombin by the systemic elease of microparticles. Further discussion of HIT refers to IT type II. HIT is a clinicopathologic syndrome. Patients should only be iagnosed with HIT when thrombocytopenia (with or without hrombosis) occurs during or after heparin treatment and labratory tests confirm the presence of heparin-dependent antiodies.3 Most typically, HIT presents as an unexpected fall in he platelet count occurring within 5 to 10 days of heparin dministration (the time period required to generate HIT antiodies). On occasion, HIT may present as an abrupt fall in latelet count within hours of the administration of heparin if he patient had received heparin in the previous 100 days rapid-onset HIT, which is likely caused by the presence of esidual HIT antibodies induced by the previous heparin expoure).4 Typically, platelet counts drop by approximately 50% in he first 2 to 3 days after cardiac surgery because of hemodiution and platelet consumption during cardiopulmonary byass (CPB). This thrombocytopenia, occurring within 4 days of urgery, should not be attributed to HIT. Platelet counts begin o rise, often above baseline values, on postoperative day 3. A p

Iron-bound fibrinogen has been noted to accelerate plasmatic coagulation in patients with divergent conditions involving upregulation of heme oxygenase activity, including hemodialysis, Alzheimer's disease, sickle cell anemia, and chronic migraine. Our goal was to determine if a site of iron-fibrinogen interaction was on the alpha chain. Using thrombelastography, we compared the coagulation kinetic profiles of plasma exposed to 0-10 µM ferric chloride after activation of coagulation with thrombin generated by contact activation of plasma with the plastic sample cup or by exposure to 1 µg/ml of Calloselasma rhodostoma venom (rich in ancrod activity), which causes coagulation via polymerization of alpha chain monomers. Venom mediated coagulation always occurred before thrombin activated thrombus formation, and ferric chloride always diminished the time of onset of coagulation and increased the velocity of clot growth. Iron enhances plasmatic coagulation kinetics by modulating the alpha chain of fibrinogen.

Summary 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.

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.

A novel metalloproteinase, recombinant fibrinogenase IV (rFIVa), was expressed and purified from Agkistrodon acutus venom. It is a single-chain protein with an apparent molecular weight of 27 kDa. Western blot showed that it had a good immunological reaction against anti-FIVa rabbit serum. The kinetic parameters Km and Kcat of rFIVa on the substrate T6140 were 7.471 x 10(-4) mol/l and 5.103 x 10(-5) s(-1). RFIVa cleaved preferentially the alpha-chain, and the beta- and gamma-chains of fibrinogen were also cleaved when the incubation time was prolonged. The administration of rFIVa (1.8 and 5.4 mg/kg) to animals with acute blood-stasis model produced a decrease in fibrinogen to control values. To our knowledge, this is the first report of the expression, purification, and evaluation of recombinant fibrinogenase IV, which belongs to class P-I metalloproteinase from A. acutus venom.

The direct effect of ancrod is defibrinogenation. This results in several secondary effects that differentiate ancrod from typical thrombolytics such as rt-PA. The combined actions of indirect fibrinolysis, reduced fibrin deposition, and decreased blood viscosity represent a constellation of actions that should be beneficial in a variety of thrombotic conditions. The clinical condition explored most thoroughly in recent years with ancrod has been its acute application to ischemic stroke. Two clinical studies (one of them with a 6 h time-to-treatment window) have demonstrated significant benefit in increasing the proportion of patients alive and disability-free at 3 months, but higher doses of the drug appear to increase the risk of symptomatic intracranial hemorrhage and reduce efficacy. Although the evidence is not conclusive, it suggests that, with modified dosing, ancrod could yield a favorable risk–benefit ratio in stroke by limiting the risk of symptomatic intracranial hemorrhage.

A novel model to induce occlusive thrombus formation was developed in mice in vivo. Mice were simultaneously treated with ligation and cuff placement at the left carotid artery. At 7 days after the treatment, occlusive thrombus was observed at the intracuff region, but not in the distal and proximal regions of the cuff, and not induced by a single treatment of ligation or cuff placement. The plasma levels of von Willebrand factor (vWF), which represent the endothelial status, were significantly increased in combined treatment of ligation and cuff placement 1 day after the operation. Whereas no significant changes in plasma vWF were observed in either single treatment of ligation or cuff placement. The expression of vWF, considered to be the endothelial marker, was detected on the luminal surface distal and proximal to the cuff and the carotid artery in the single treatment groups treated with either ligation or cuff placement, but was not detected in the intracuff region. Furthermore, the binding of Griffolia Simplicifolia Lectin-I (GSL-I) and endothelial nitric oxide synthase (eNOS) expression indicating the endothelial integrity was not detected in the intracuff region. Intermittent injections of ancrod, which decreases the plasma fibrinogen, inhibited occlusive thrombus formation in the intracuff region. The expression of eNOS was detected at the distal and proximal but not the intracuff region of the carotid artery treated with ancrod. Daily administration of aspirin significantly suppressed the thrombus formation in this model. These results indicate that occlusive thrombus formation accompanied by endothelial damage or dysfunction is induced by the combined application of ligation and cuff placement at the carotid artery, and suggest that this endothelial damage or dysfunction may be one pathogenesis of thrombogenesis in this model.

Fibrin independent proinflammatory effects of tissue factor in experimental crescentic glomerulonephritis.BackgroundTissue 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.MethodsTo 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.ResultsDefibrinogenation alone abolished glomerular fibrin deposition, reduced crescent formation, and limited renal impairment (ancrod-treated, serum creatinine 274 ± 37 μmol/L; untreated 415 ± 51 μmol/L; P < 0.01). Tissue factor inhibition in defibrinogenated rabbits resulted in further protection of renal function (creatinine 140 ± 19 μmol/L, P < 0.01) and reduced proteinuria (0.4 ± 0.2g/day, untreated 2.6 ± 0.4 g/day, P

A safe and effective alternative is needed for patients in whom unfractionated heparin (UFH) or protamine is contraindicated (e.g., those with heparin-induced thrombocytopenia or allergy to protamine). Furthermore, choice of anticoagulant may influence graft patency in coronary surgery and may therefore be important even when there is no contraindication to UFH. Direct thrombin inhibitors have several potential advantages over UFH, demonstrated in acute coronary syndromes. However, there are also potential difficulties with their use related to lack of reversal agents and paucity of clinical experience in monitoring their anticoagulant activity at the levels required for cardiac surgery with cardiopulmonary bypass (CPB). In the first prospective randomized trial of an alternative to heparin in cardiac surgery, we compared bivalirudin (a short-acting direct thrombin inhibitor) with UFH in 100 patients undergoing off-pump coronary artery bypass (OPCAB) surgery. Blood loss for the 12 hours following study drug initiation in the bivalirudin group was not significantly greater than in the heparin group. Median graft flow was significantly higher in the bivalirudin group. We concluded that anticoagulation for OPCAB surgery with bivalirudin was feasible without a clinically important increase in perioperative blood loss. A larger study is needed to investigate the impact of improved graft patency on other clinical outcomes after cardiac surgery.