hirudin and Cerebral-Hemorrhage

This review describes some natural proteins, which can be employed, either as factor concentrates derived from human plasma or as recombinant drug, to modulate the coagulation system. I will address some biochemical characteristics and the physiological role of von Willebrand factor, the coagulation factors of the extrinsic and intrinsic pathways, and the physiological anticoagulant protein C. In addition, I will detail the pharmacological compounds, which are available for influencing or substituting the coagulation proteins: desmopressin (DDAVP), single coagulation factor concentrates, prothrombin complex concentrates, and protein C concentrate. In particular, I will address some treatment topics of general medical interest, such as the treatment of massive bleeding, the correction of the coagulopathy induced by vitamin K-antagonists in patients with cerebral haemorrhage, and of the coagulopathy of meningococcemia. Finally, I will describe some properties and practical clinical applications of the recombinant anticoagulans lepirudin and bivalirudin, which are derived from hirudin, the natural anticoagulant of the medical leech.

Topics: Anticoagulants; Blood Coagulation Disorders; Blood Coagulation Factors; Blood Proteins; Cerebral Hemorrhage; Hemorrhage; Hirudins; Humans; Peptide Fragments; Protein C; Recombinant Proteins; Vitamin K; von Willebrand Factor

In the case of acute coronary syndrome with prolonged ST elevation on ECG showing an acute coronary obstruction, the urgent institution of fibrinolysis is a widely validated treatment. Since the first placebo controlled studies with streptokinase until the development of bolus administration rt-PA varieties, fibrinolytic agents have lowered mortality. Associated anti-thrombotic drugs are multiplying in parallel. Their association is recognised as necessary in order to avoid early reocclusions which worsen the prognosis of infarction, the fibrinolysis triggering a harmful prothrombotic effect, notably due to the clot thrombin re-exposed during thrombolysis. Aspirin has an essential place formally demonstrated in ISIS 2. Non-fractionated heparin has more complex effects and its administration protocol in association with fibrinolysis has recently been reviewed with a reduction in dosage because prolonged clotting times during fibrinolysis have provoked a distinct increase in the risk of intracranial haemorrhage. The low molecular weight heparins seem to have become the adjuvant treatment of choice following publication of the ASSENT-3 trial. Pentasaccharide seems attractive. The place of hirudine and its derivatives in the acute phase of MI appear limited after the results of the HERO-2 trial, associating hirulog and streptokinase, with the earlier studies also having been disappointing. The GPIIbIIIa blockers in association with a half dose of fibrinolysis do not aggravate the intracerebral haemorrhagic risk before 75 years old and clearly reduce hospital morbidity in infarction, at the price however of an increase in transfusions.

Topics: Aspirin; Cerebral Hemorrhage; Drug Costs; Electrocardiography; Fibrinolytic Agents; Hirudin Therapy; Hirudins; Myocardial Infarction; Myocardial Reperfusion; Randomized Controlled Trials as Topic; Risk Factors; Streptokinase

Topics: Cerebral Hemorrhage; Clinical Trials, Phase III as Topic; Dose-Response Relationship, Drug; Double-Blind Method; Hirudin Therapy; Hirudins; Humans; Infusions, Intravenous; Myocardial Infarction; Randomized Controlled Trials as Topic; Risk Assessment; Thrombosis

Topics: Anticoagulants; Blood Coagulation; Blood Platelets; Cerebral Hemorrhage; Clinical Trials as Topic; Fibrinolysis; Heparin; Hirudin Therapy; Hirudins; Humans; Multicenter Studies as Topic

Worldwide, streptokinase continues to be used widely in the treatment of myocardial infarction because it is inexpensive and causes fewer intracranial hemorrhages than other thrombolytic regimens. However, in the Global Use of Strategies to Open Occluded Coronary Arteries (GUSTO-I) trial, the 90-minute angiographic Thrombolysis in Myocardial Infarction (TIMI) trial grade 3 flow rate with streptokinase was 43% lower than that with accelerated tissue plasminogen activator, and there was a higher incidence of death or disabling stroke with streptokinase (7.8% vs 6.9%, p <0.01). In the first Hirulog and Early Reperfusion/Occlusion (HERO-1) trial, 48% of patients given the direct thrombin inhibitor bivalirudin (formerly Hirulog, The Medicines Company) after streptokinase had TIMI 3 patency at 90 minutes, compared with 35% of patients given intravenous heparin (p <0.05). Other angiographic and clinical studies and animal research have shown that early infarct artery blood flow may be increased markedly if a direct thrombin inhibitor is administered before the thrombolytic agent. In the HERO-2 trial, 17,000 patients presenting within 6 hours after the onset of acute myocardial infarction will be given aspirin and randomized to receive either intravenous heparin or bivalirudin before streptokinase is administered. The primary endpoint will be 30-day mortality, and secondary endpoints will include death or myocardial infarction within 30 days, and death or nonfatal disabling stroke. If the thrombin hypothesis is supported by improved clinical outcomes with bivalirudin in the HERO-2 trial, large-scale clinical trials will be needed to evaluate the administration of direct thrombin inhibitors before other thrombolytic regimens.

Topics: Antithrombins; Cerebral Hemorrhage; Cerebrovascular Disorders; Coronary Angiography; Drug Therapy, Combination; Fibrinolytic Agents; Heparin; Hirudin Therapy; Hirudins; Humans; Myocardial Infarction; Peptide Fragments; Recombinant Proteins; Research Design; Streptokinase

Although intravenous heparin is routinely used in the treatment of patients with acute coronary syndromes, this anticoagulant requires antithrombin III as a cofactor, has no affinity to clot-bound thrombin, and is bound or inactivated by several plasma proteins and platelet factor 4. Recombinant hirudin, the prototypic direct thrombin inhibitor, has been demonstrated in pilot studies to yield improved angiographic and clinical outcomes compared with heparin. We compared these two antithrombins in a large-scale randomized trial.. At 275 participating hospitals in 12 countries, patients within 12 hours from the onset of ischemic chest discomfort with an abnormal ECG were randomly assigned to receive a 72- to 120-hour infusion of heparin (5000-U bolus and 1000- to 1300-U/h infusion, adjusted to activated partial thromboplastin time (APTT) of 60 to 90 seconds or hirudin (0.6-mg/kg bolus and 0.2-mg/kg per hour infusion without aPTT adjustment) on a double-blind basis. Although recruitment of 12,000 patients was planned, the trial was stopped earlier because of an excess of intracerebral hemorrhagic events after 2564 patients were enrolled. The overall incidence of hemorrhagic stroke tended to be higher for patients receiving hirudin (1.3%) compared with heparin (0.7%), P = .11, but the incidence was significantly higher in patients receiving thrombolytic therapy (1264 patients, 1.8%) compared with those who did not (1168 patients, 0.3%), P < .001. The hemorrhagic stroke rate varied by the thrombolytic and antithrombin combination: tissue-type plasminogen activator and heparin, 0.9%; with hirudin, 1.7%; streptokinase with heparin, 2.7%; with hirudin, 3.2%. All these rates are higher than the overall incidence of hemorrhagic stroke in the patients receiving thrombolytic therapy and intravenous heparin in the GUSTO I trial (30,892 patients with rate of 0.7%, 95% CI of 0.6 to 0.8%). Among the 26 patients who had intracerebral hemorrhages, the aPTT was significantly elevated compared with the event-free patients (110 +/- 46 versus 87 +/- 36 seconds at 12 hours of therapy, respectively), P = .03.. At the dose of hirudin tested, there was a trend of an excess of hemorrhagic stroke compared with heparin. Heparin, at a slightly higher dose than previously used in a large-scale trial (approximately 20% increase) was accompanied by a twofold risk of hemorrhagic stroke in patients receiving thrombolytic therapy. With both thrombin inhibitors, the aPTT appears to be a useful index for predicting risk of hemorrhagic stroke in patients receiving thrombolytic therapy.

Topics: Acute Disease; Aged; Cerebral Hemorrhage; Cerebrovascular Disorders; Coronary Disease; Double-Blind Method; Female; Heparin; Hirudin Therapy; Hirudins; Humans; Injections, Intravenous; Male; Middle Aged; Recombinant Proteins; Syndrome; Thrombolytic Therapy

Thrombin is a unique factor that triggers post-intracerebral hemorrhage (ICH) angiogenesis by increasing hypoxia-inducible factor-1α (HIF-1α) at the protein level. However, HIF-1α mRNA remains unchanged. MicroRNAs (miRNAs) mediate posttranscriptional regulation by suppressing protein translation from mRNAs. This study aimed to determine if miRNAs might be involved in thrombin-induced angiogenesis after ICH by targeting HIF-1α or its upstream prolyl hydroxylase domains (PHDs).. The study was divided into two parts. In part 1, rats received an injection of thrombin into the right globus pallidus. An miRNA array combined with miRNA target prediction, luciferase activity assay, and miRNA mimic/inhibitor transfection were used to identify candidate miRNAs and target genes. Part 2 included experiments 1 and 2. In experiment 1, rats were randomly divided into the sham group, ICH group, and ICH+hirudin-treated (thrombin inhibitor) group. In experiment 2, the rats were randomly divided into the sham group, ICH group, ICH+antagomir group, ICH+antagomir-control group, and ICH+vehicle group. Western blotting and quantitative real-time polymerase chain reaction were used to determine the expression of protein and miRNA, respectively. The coexpression of miR-24-1-5p (abbreviated to miR-24) and von Willebrand factor was detected by in situ hybridization and immunohistochemical analysis. The angiogenesis was evaluated by double-labeling immunofluorescence. Neurological function was evaluated by body weight, modified Neurological Severity Scores, and corner turn and foot-fault tests.. In part 1, it was shown that miR-24, which is predicted to target PHD1, was upregulated (fold-change of 1.83) after thrombin infusion, and that the miR-24 mimic transfection decreased luciferase activity and downregulated PHD1 expression (p < 0.05). miR-24 inhibitor transfection increased PHD1 expression (p < 0.05). In part 2, it was shown that miR-24 was expressed in endothelial cells. The HIF-1α protein level and proliferating cell nuclear antigen-positive (PCNA+) nuclei in vessels were increased, while the PHD1 protein level was decreased after ICH, and these effects were reversed by hirudin (p < 0.05). The antagomiR-24-treated rats exhibited a markedly lower body weight and significantly poorer recovery from neurological deficit compared with those in ICH groups (p < 0.05). AntagomiR-24 intervention also led to lower miR-24 expression, a higher PHD1 protein level, and fewer PCNA+ nuclei in vessels compared with those in ICH groups (p < 0.05).. The present study suggests that thrombin reduces HIF-1α degradation and initiates angiogenesis by increasing miR-24, which targets PHD1 after ICH.

Topics: Animals; Antagomirs; Cerebral Hemorrhage; Gene Expression Regulation; Genes, Reporter; Globus Pallidus; Hirudins; Hypoxia-Inducible Factor 1, alpha Subunit; MicroRNAs; Neovascularization, Physiologic; Prolyl Hydroxylases; Random Allocation; Rats; Rats, Sprague-Dawley; Thrombin; Up-Regulation; von Willebrand Factor

Pericyte coverage on the endothelial tubes leads to the formation of a mature and stable microvessel system, which is critical for brain repair after intracerebral hemorrhage (ICH). We report herein that thrombin promotes pericyte coverage by activating Tie2 and the downstream signaling pathway PI3K/Akt in a rat model of ICH. ICH was induced by injection of autologous blood with or without thrombin inhibitor hirudin. Rats were treated with thrombin alone or in combination with a Tie2 inhibitor. The expression of total- and phospho-Tie2, PI3K and phospho-Akt, blood perfusion, pericyte coverage, IgG extravasation, neuron survival and neurological deficits were evaluated by western blot, fluorescein-5-isothiocyanate-dextran staining, immunohistochemistry, Nissl staining and modified neurological severity scores respectively. Induction of ICH resulted in increased phosphorylation of Tie2 on endothelial cells and pericyte coverage, better formation of integral and functional microvessels, more surviving neurons and accelerated motor function recovery, all of which were significantly attenuated by hirudin at 7 and 14 days after ICH induction. Furthermore, thrombin increased phosphorylation of Tie2 and Akt, expression of PI3K, and pericyte coverage, which were however reversed by pharmacological inhibition of Tie2. Our results demonstrated that thrombin promotes pericyte coverage on microvessels following ICH by enhancing activation of Tie2, in which the downstream PI3K/Akt signaling pathway might be involved.

Topics: Animals; Brain; Cerebral Hemorrhage; Disease Models, Animal; Endothelial Cells; Hirudins; Male; Microvessels; Pericytes; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Receptor, TIE-2; Recovery of Function; Signal Transduction; Thrombin

Intracerebral hemorrhage (ICH) is a severe type of stroke without effective treatment. The coagulation cascade is activated after blood flows into the brain parenchyma. The conversion of fibrinogen to fibrin is an essential step of coagulation processes, but its influences on neuroinflammation and long-term outcome after ICH have not been adequately studied. Hirudin binds to thrombin and inhibits the conversion of fibrinogen to fibrin. We therefore investigated the impact of hirudin treatment on brain inflammation and long-term outcome of ICH in mice.. Fibrinogen levels were measured in plasma samples from patients with ICH. In mice subjected to collagenase injection, fibrinogen levels were measured in the plasma and brain. The impact of hirudin on neuroinflammation and long-term neurological outcome was determined in ICH mice.. Circulating fibrinogen level was increased in patients with ICH at day 1 and day 4 after onset. In ICH mice, fibrinogen levels in the blood and brain were increased at day 7. Delayed daily administration of hirudin from day 7 to day 28 significantly improved long-term outcome in ICH mice. Hirudin treatment reduced leukocyte accumulation in the brain and shifted microglia toward an anti-inflammatory phenotype. In addition, depletion of microglia in ICH mice diminished the benefit of hirudin in ICH mice.. These results suggest that inhibition of fibrin formation alleviates brain inflammation and improves long-term outcome after ICH.

Topics: Animals; Brain; Cerebral Hemorrhage; Encephalitis; Female; Fibrin; Fibrinogen; Hirudin Therapy; Hirudins; Humans; Male; Mice, Inbred C57BL

To investigate the effects of microglia/macrophages activation induced by intrastriatal thrombin injection on dentate gyrus neurogenesis and spatial memory ability in mice.. The male C57BL/6 mice were divided into 4 groups of 10: sham, intracerebral hemorrhage (ICH), ICH + hirudin (thrombin inhibitor), and ICH + indometacin (Indo, an anti-inflammation drug). ICH model was created by intrastriatal thrombin (1U) injection. BrdU (50 mg/kg) was administrated on the same day after surgery for 6 consecutive days. Motor functions were evaluated with rotarod and beam walking tests. The spatial memory deficit was measured with Morris water maze (MWM). Cell quantification was performed for doublecortin (DCX, immature neuron), BrdU (S-phase proliferating cell population) and CD68 (activated microglia/macrophage) immune-reactive cells.. Microglia/macrophages activation induced by intrastriatal thrombin injection reduced hippocampal neurogenesis and impaired spatial memory ability, but did not affect the motor function at 3 and 5 days post-injury. Both hirudin and indometacin reduced microglia/macrophages activation, enhanced hippocampal neurogenesis, and improved spatial memory ability in mice.. Microglia/macrophages activation induced by intrastriatal thrombin injection might be responsible for the spatial memory deficit. Targeting both thrombin and inflammation systems in acute phase of ICH might be important in alleviating the significant spatial memory deficits.

Topics: Animals; Cell Proliferation; Cerebral Hemorrhage; Corpus Striatum; Dentate Gyrus; Doublecortin Protein; Hirudins; Indomethacin; Male; Maze Learning; Mice; Microglia; Microinjections; Motor Skills; Neurogenesis; Spatial Memory; Thrombin

This study aimed to investigate the effects of thrombin released in hematoma after intracerebral hemorrhage (ICH) on the cerebral injury of perihematomal tissues and to evaluate the protection effect of hirudin on the cerebral injury after ICH. We used the autologous uncoagulated blood injection method to prepare the ICH rat model, and all rats were randomly divided into a normal group, an ICH group, or a hirudin group. At different time points, rat heads were cut to harvest brain sections. Immunohistochemical staining, histochemical staining, and hematoxylin and eosin staining were conducted for CD34, microglia, and neutrocytes. CD34-positive microvessels were most abundant in brain tissues of the sham-operation group. At 12 h after ICH, CD34 expression reduced and reached the minimum level at 72 h (P<0.01). At 6 h after ICH, microglia expression was visible and reached a peak at 48 h (P<0.01). At 12 h after ICH, neutrocyte infiltration was visible and the number was greatest at 48 h (P<0.01). The early application of hirudin after ICH could significantly reduce microglia and neutrocyte expression and could significantly slow down the CD34 decrease trend (P<0.01). However, hirudin application in the edematization stage after ICH did not significantly increase CD34- positive microvessel abundance (P>0.05). A thrombin-mediated inflammatory reaction is involved in the cerebral injury after ICH, and the early application of hirudin has a protective effect.

Topics: Animals; Antigens, CD34; Brain Edema; Cerebral Hemorrhage; Disease Models, Animal; Hirudins; Leukocytes; Male; Microglia; Microvessels; Rats; Rats, Sprague-Dawley; Thrombin

Spontaneous intracerebral hemorrhage (ICH) is one of the most severe types of stroke. Thrombin has been reported to participate in brain repair following ICH and play an important role in angiogenesis. Our previous studies have shown that ICH induces angiogenesis in damaged rat brain, accompanied by upregulation of expression of thrombospondin (TSP)-1 and TSP-2. The aim of the present study was to investigate whether the expression of TSP-1 and TSP-2 was regulated by thrombin in rat brain following ICH. A rat model of ICH was induced by injection of autologous blood into the right globus pallidus (GP). Hirudin, a thrombin specific inhibitor, or thrombin was injected into the GP. Immunohistochemistry, quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) and western blot assays were applied. Results showed that ICH induced an increase in the expression of TSP-1 mRNA and TSP-2 mRNA after ICH, whereas hirudin significantly inhibited the expression of TSPs mRNA after ICH (P<0.05). In contrast, sole thrombin treatment in normal rats induced strong expression of TSP-1 or TSP-2 in the blood vessels around the damaged brain region when compared with those without thrombin treatment. Western blot analysis data confirmed that the protein levels of TSPs were significantly increased when compared with those in the sham control group (P<0.01). These findings support that thrombin positively regulates the expression of TSP-1 and TSP-2 after ICH, which may be involved in modulating angiogenesis in injured brains following ICH.

Topics: Animals; Antithrombins; Blood Vessels; Cerebral Hemorrhage; Gene Expression Regulation; Hirudins; Male; Neovascularization, Physiologic; Random Allocation; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; RNA, Messenger; Thrombin; Thrombospondin 1; Thrombospondins; Up-Regulation

Edema formation has been linked to thrombin toxicity induced by blood clot at the acute stage of intracerebral hemorrhage. Thrombin induces cell toxicity in neuron, microglia and astrocyte. Aquaporin (AQP) 4 and 9 are proteins expressed on astrocyte in rat brain and involved in the brain water accumulation in brain edema. Recombinant hirudin (r-Hirudin) is a direct inhibitor of thrombin that can block the toxicitic effect of thrombin. In this study, we demonstrated that autologous whole blood infusion in caudate nucleus up-regulates the expression of AQP4 and AQP9 mRNAs and proteins. AQP4 and AQP9 mRNAs expression peaked at about 6 h after blood infusion. The AQP4 protein peaked at about 48 h while AQP9 at about 24 h after blood infusion. Thrombin induced up-regulation of AQP4 and AQP9 were inhibited by r-Hirudin administration and significantly decreased the expression of both AQPs. We further investigated the relationship between edema formation and expression of AQP4 and AQP9. The data presented here may be helpful in optimizing r-Hirudin as an anti-thrombin drug in the treatment of edema at the acute stage of ICH.

Topics: Animals; Aquaporin 4; Aquaporins; Brain Edema; Cerebral Hemorrhage; Gene Expression Regulation; Hirudins; Magnetic Resonance Imaging; Rats; Rats, Wistar; Recombinant Proteins; RNA, Messenger

To reduce bleeding and damage to central nervous system tissue in intracerebral hemorrhage, the coagulant effect of thrombin is essential. However, thrombin itself can kill neurons in intracerebral hemorrhage as can the matrix metalloproteinases (MMPs), which are also elevated in this condition, in part due to thrombin-mediated activation of MMPs. It is thus important to understand and block the neurotoxic effects of thrombin without inhibiting its therapeutic outcomes. In this study, we have investigated the relative roles of proteinase activated receptor-1, a thrombin receptor, and MMPs in brain injury induced by thrombin or blood.. Mice were subjected to stereotactic intracerebral injections of saline, thrombin, and autologous blood, with or without hirudin, a thrombin inhibitor, or GM6001, an MMP inhibitor. Twenty-four hours later, tissue sections were obtained to evaluate the area of brain damage and extent of dying neurons. Data from wild-type mice were compared with results obtained with proteinase activated receptor-1 null mice.. In blood-induced damage to the brain parenchyma, both hirudin and GM6001 significantly reduced injury to a comparable extent (>40%) implicating both thrombin and MMPs in neurotoxicity. In proteinase activated receptor-1 null mice, blood-induced brain damage was reduced by 22.6% relative to wild-type animals; by comparison, the blood-induced brain damage was reduced by 48.3% using GM6001.. The neurotoxicity of blood in intracerebral hemorrhage involves both proteinase activated receptor-1 and MMP activation, with the latter appearing more prominent in causing death.

Topics: Animals; Antithrombins; Blood Transfusion, Autologous; Brain; Cell Death; Cerebral Hemorrhage; Dipeptides; Female; Gelatin; Hirudins; Male; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Mice; Mice, Inbred C57BL; Mice, Knockout; Neurotoxicity Syndromes; Protease Inhibitors; Receptor, PAR-1; Thrombin

Topics: Anticoagulants; Antiphospholipid Syndrome; Cerebral Hemorrhage; Drug Administration Schedule; Half-Life; Heparin; Hirudins; Humans; Lupus Erythematosus, Systemic; Male; Middle Aged; Recombinant Proteins; Renal Insufficiency; Thrombocytopenia; Treatment Outcome

Neurogenesis in intracerebral hemorrhage (ICH) has not been investigated. Thrombin formation causes acute brain injury after ICH, but thrombin also can stimulate cell proliferation. The present study examined whether neurogenesis takes place in ICH and the role of thrombin in ICH-related neurogenesis.. This study was divided into four parts. (1) Rats received either an ICH or a needle insertion (sham). The rats were killed for doublecortin (DCX) Western blot analysis and immunohistochemistry. (2) Rats had an ICH or a sham operation, and then received intraperitoneal injections of 5-bromo-2'-deoxyuridine (BrdU) at day-7 and day-9 later. Brains were perfused to identify BrdU-positive cells. (3) Rats had an intracaudate injection of thrombin (1 U) and brains were sampled for Western blots. (4) Rats had an ICH with or without a thrombin inhibitor, hirudin. The brains were sampled for DCX quantitation.. DCX levels in the ipsilateral basal ganglia started to increase as early as 7 days after ICH, peaked at 14 days, and then gradually decreased at 1 month. Immunohistochemistry also demonstrated that DCX immunoreactivity was increased in the ipsilateral subventricular zone and basal ganglia at 2 weeks after ICH. Some DCX-positive cells were BrdU-positive. One unit thrombin, which does not cause marked brain injury, was injected into the caudate. Thrombin increased DCX levels in the ipsilateral basal ganglia and hirudin blocked ICH-induced upregulation of DCX.. Our results demonstrated that neurogenesis occurs in the brain after ICH and that thrombin may play a role in ICH-induced neurogenesis.

Topics: Animals; Brain; Bromodeoxyuridine; Cell Proliferation; Cerebral Hemorrhage; Doublecortin Domain Proteins; Doublecortin Protein; Hirudins; Immunohistochemistry; Male; Microtubule-Associated Proteins; Models, Biological; Neurons; Neuropeptides; Rats; Rats, Sprague-Dawley; Thrombin; Time Factors

To explore the protective effects of hirudin on acute experimental intracerebral hemorrhage (ICH) by observing the changes of histologic pathology and brain water content as well as GFAP-positive cells in the perihematomal brain regions.. The models of rat ICH were made with infusion of autologous blood into the right neucleus caudatus. The rats were divided randomly into control group, intracerebral hemorrhage group and treating group with hirudin. Brain water content was measured, and pathological and GFAP changes were observed.. The pathological impairation after ICH were gradually deteriorated and peaked at the third day. Brain water content after ICH was gradually increased and obviously after one day(P < 0.05) and peaked at the third day. GFAP-positive cells were gradually increased and peaked at the seventh day after ICH. In the treating groups, the pathological impairation and brain water content as well as the GFAP-positive cells were decreased as compared to those in the intracerebral hemorrhage group and the control group. And the positive correlation between GFAP-positive cell numbers and brain water content were shown by linear regression.. The local administration of hirudin, a special inhibitor of thrombin, has protective effects within the first week after ICH.

Topics: Acute Disease; Animals; Brain; Cerebral Hemorrhage; Female; Glial Fibrillary Acidic Protein; Hirudins; Male; Neuroprotective Agents; Random Allocation; Rats; Rats, Wistar

Neonatal periventricular hemorrhage (PVH) is a devastating complication of prematurity in the human infant. Based upon observations made primarily in adult rodents and the fact that the immature brain uses proteolytic systems for cell migration and growth, we hypothesized that thrombin and plasmin enzyme activities contribute to the brain damage after PVH. The viability of mixed brain cells derived from newborn rat periventricular region was suppressed by whole blood and thrombin, but not plasmin. Following injection of autologous blood into the periventricular region of newborn rat brain, proteolytic activity was detected in a halo around the hematoma using membrane overlays impregnated with thrombin and plasmin fluorogenic substrates. Two-day old rats received periventricular injection of blood, thrombin, and plasminogen. After 2 days, thrombin and blood were associated with significantly greater damage than saline or plasminogen. Two-day old mice received intracerebral injections of blood in combination with saline or the proteolytic inhibitors hirudin, alpha2macroglobulin, or plasminogen activator inhibitor-1. After 2 days, hirudin significantly reduced brain cell death and inflammation. Two-day-old mice then received low and high doses of hirudin mixed with blood after which behavioral testing was conducted repeatedly. At 10 weeks there was no statistically significant evidence for behavioral or structural brain protection. These results indicate that thrombin likely plays a role in neonatal periventricular brain damage following PVH. However, additional factors are likely important in the recovery from this result.

Topics: Animals; Brain; Cells, Cultured; Cerebral Hemorrhage; Fibrinolytic Agents; Hirudins; Injections, Intraventricular; Male; Mice; Rats; Rats, Sprague-Dawley; Serine Endopeptidases; Thrombin

In order to explore the PAR-1 mRNA and protein expression around hemotoma following intracerebral hemorrhage and the relation between the PAR-1 expression and thrombin, collagenase VII was stereotaxically injected into right caudate nucleus in rats. The PAR-1 mRNA expression was detected by RT-PCR method and the PAR-1 protein expression by immunohistochemical method respectively. It was found that the PAR-1 mRNA and protein expression around hemotoma was increased at 6 h after intracerebral hemorrhage (P<0.05), peaked at 2 days (P<0.01), and then declined. The change pattern of the PAR-1 mRNA and protein expression was similar to that of intracerebral hemorrhage after thrombin intracerebral injection. The PAR-1 mRNA and protein expression in hirudin group showed no significant difference with control group. These results indicated that the PAR-1 mRNA and protein expression were markedly increased after intracerebral hemorrhage, which may be closely related to thrombin. Upregulation of the PAR-1 expression may involve in neurotoxic injury induced by thrombin.

Topics: Animals; Cerebral Hemorrhage; Female; Fibrinolytic Agents; Hematoma; Hirudins; Male; Random Allocation; Rats; Rats, Sprague-Dawley; Receptor, PAR-1; RNA, Messenger; Thrombin

In humans, intracerebral hemorrhage (ICH) causes marked perihematomal edema formation and neurological deficits. A rat ICH model, involving infusion of autologous blood into the caudate, has been used extensively to study mechanisms of edema formation, but an examination of behavioral outcome would improve its preclinical utility and provide a more rigorous assessment of the pathological cascade of events over time. The purpose of this study was to use a battery of sensorimotor function tests to examine the neurological effects of ICH in the rat and to examine which components of the hematoma are involved in generating those effects.. The behavioral tests used were forelimb placing, preference for forelimb use for weight shifts during vertical exploration of a cylindrical enclosure, and a corner turn test. Rats were tested from day 1 to day 28 after injection of autologous whole blood; injection of blood plus hirudin (thrombin inhibitor), packed red blood cells, thrombin, or saline; or needle placement only.. The battery of tests indicated that there were marked neurological deficits by day 1 after ICH, with progressive recovery of function over 4 weeks. The forelimb placing score paralleled changes in edema. Injection of thrombin caused and injection of hirudin reduced the ICH-induced neurological deficits. Injection of packed red blood cells, which causes delayed edema formation, induced delayed neurological deficits. These tests allow continuous monitoring of neurological deficits after rat ICH and assessment of therapeutic interventions. The time course of the neurological deficit closely matched the time course of cerebral edema for both ICH and injection of blood components. There was marked recovery of function after ICH, which may be amenable to therapeutic manipulation.

Topics: Animals; Behavior, Animal; Brain; Brain Chemistry; Brain Edema; Cerebral Hemorrhage; Diagnostic Techniques, Neurological; Disease Models, Animal; Disease Progression; Drug Administration Routes; Fibrinolytic Agents; Forelimb; Hirudins; Male; Rats; Rats, Sprague-Dawley; Recovery of Function; Thrombin; Water

Serine proteases, such as thrombin and tissue-type plasminogen activator, play an important role in brain injury after intracerebral hemorrhage and other neurologic disorders. Plasminogen activator inhibitor-1 is one of the serine protease inhibitors, or serpins. The balance between serine proteases and serpins may affect the outcome of intracerebral hemorrhage. The purpose of this study was to determine whether plasminogen activator inhibitor-1 and tissue-type plasminogen activator are upregulated after intracerebral hemorrhage and the role that thrombin plays in that induction. Plasminogen activator inhibitor-1 protein levels were upregulated after intracerebral hemorrhage. Brain plasminogen activator inhibitor-1 content also increased after thrombin infusion in a dose-dependent manner. Hirudin, a specific thrombin inhibitor, blocked the upregulation of plasminogen activator inhibitor-1 after intracerebral hemorrhage. Time courses showed that plasminogen activator inhibitor-1 levels around the hematoma peaked at the first day. Plasminogen activator inhibitor-1-positive cells were detected in the perihematomal area and the ipsilateral basal ganglia after thrombin infusion, but not in the contralateral hemisphere. Plasminogen activator inhibitor-1 messenger RNA levels were increased at 24 hours after intracerebral hemorrhage and after thrombin infusion. However, tissue-type plasminogen activator protein levels were the same in the control, whole-blood, and thrombin-infusion groups. In conclusion, intracerebral hemorrhage and thrombin infusion stimulate plasminogen activator inhibitor-1 but not tissue-type plasminogen activator production in the brain. The upregulation of plasminogen activator inhibitor-1 may be neuroprotective by limiting thrombin or other serine protease-induced toxicity.

Topics: Animals; Blotting, Western; Brain; Cerebral Hemorrhage; Enzyme Induction; Enzyme-Linked Immunosorbent Assay; Fibrinolytic Agents; Hirudins; Immunohistochemistry; Male; Plasminogen Activator Inhibitor 1; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Serine Proteinase Inhibitors; Thrombin; Tissue Plasminogen Activator; Up-Regulation

We conducted a study using diffusion-weighted (DWI) and perfusion-weighted (PWI) magnetic resonance imaging (MRI) to evaluate the efficacy of thrombolysis in an embolic stroke model with recombinant tissue plasminogen activator (rt-PA) and hirulog, a novel direct-acting antithrombin. DWI can identify areas of ischemia minutes from stroke onset, while PWI identifies regions of impaired blood flow. Right internal carotid arteries of 36 rabbits were embolized using aged heterologous thrombi. Baseline DWI and PWI scans were obtained to confirm successful embolization. Four animals with no observable DWI lesion on the initial scan were excluded; therefore, a total of 32 animals were randomized to one of three treatment groups: rt-PA (n = 11), rt-PA plus hirulog (n = 11), or placebo (n = 10). Treatment was begun 1 h after stroke induction. Intravenous doses were as follows: rt-PA, 5 mg/kg over 0.5 h with 20% of the total dose given as a bolus; hirulog, 1 mg/kg bolus followed by 5 mg/kg over 1 h. MRI was performed at 2, 3, and 5 h following embolization. Six hours after embolization, brains were harvested, examined for hemorrhage, then prepared for histologic analysis. The rt-PA decreased fibrinogen levels by 73%, and hirulog prolonged the aPTT to four times the control value. Posttreatment areas of diffusion abnormality and perfusion delay were expressed as a ratio of baseline values. Significantly improved perfusion was seen in the rt-PA plus hirulog group compared with placebo (normalized ratios of the perfusion delay areas were as follows: placebo, 1.58, 0.47-3.59; rt-PA, 1.12, 0.04-3.95; rt-PA and hirulog, 0.40, 0.02-1.08; p < 0.05). Comparison of diffusion abnormality ratios measured at 5 h showed trends favoring reduced lesion size in both groups given rt-PA (normalized ratios of diffusion abnormality areas were as follows: placebo, 3.69, 0.39-15.71; rt-PA, 2.57, 0.74-5.00; rt-PA and hirulog, 1.95, 0.33-6.80; p = 0.32). Significant cerebral hemorrhage was observed in one placebo, two rt-PA, and three rt-PA plus hirulog treated animals. One fatal systemic hemorrhage was observed in each of the rt-PA groups. We conclude that rt-PA plus hirulog improves cerebral perfusion but does not necessarily reduce cerebral injury. DWI and PWI are useful methods for monitoring thrombolysis.

Topics: Animals; Anticoagulants; Blood Coagulation; Brain; Cerebral Hemorrhage; Cerebrovascular Circulation; Cerebrovascular Disorders; Hirudins; Intracranial Embolism and Thrombosis; Male; Peptide Fragments; Plasminogen Activators; Rabbits; Recombinant Proteins; Tissue Plasminogen Activator

Multiple clinical trials have proven that thrombolytic therapy is an effective treatment for acute myocardial infarction. Spontaneous intracranial hemorrhage (ICH) occurs in a small percentage of patients as a result of the treatment. The etiology of the ICH is unknown and there is currently no established experimental model for this side effect. A model of ICH during thrombolytic therapy has been developed using spontaneously hypertensive rats (SHR). The SHR were made susceptible to ICH during thrombolytic therapy by bilateral ligation of the external jugular veins. This procedure produced asymptomatic hemorrhagic lesions in the brains of the animals in the hours preceding the administration of t-PA/heparin. The incidence of ICH following the administration of test substances was assessed by histological examination and by measuring the red blood cell count in a sample of cerebrospinal fluid taken from the atlanto-occipital space. t-PA administration produced a low frequency of ICH in this model. The incidence and severity of ICH were dramatically increased, and significant mortality at 24h was observed, by combining heparin were administered sequentially rather than simultaneously. Furthermore, ICHs were observed whether the t-PA dose was administered over 4 h, 1 h, or as a double bolus 30 min apart. The potentiation of ICH by heparin was dose dependent and proportional to the prolongation of the aPTT. Although the precise mechanism of ICH during thrombolytic therapy is unknown, many similarities exist between the observations made in this model and in the human clinical experience.

Topics: Animals; Antithrombin III; Cerebral Hemorrhage; Disease Models, Animal; Drug Interactions; Fibrinolytic Agents; Heparin; Hirudins; Humans; Hypertension; Male; Rats; Rats, Inbred SHR; Streptokinase; Tissue Plasminogen Activator

Topics: Cerebral Hemorrhage; Clinical Trials as Topic; Fibrinolytic Agents; Heparin; Hirudins; Humans; Myocardial Infarction; Risk Factors

Topics: Anticoagulants; Cerebral Hemorrhage; Drug Therapy, Combination; Fibrinolytic Agents; Heparin; Hirudins; Humans; Multicenter Studies as Topic; Myocardial Infarction; Randomized Controlled Trials as Topic; Risk Factors; Thrombolytic Therapy