hirudin and Disease-Models--Animal

Limitations and contraindications of heparins and oral vitamin K antagonists have led to the development of new anticoagulant drugs over the last few years. Argatroban is an intravenous direct thrombin inhibitor currently indicated for the prophylaxis and treatment of thrombosis associated with heparin-induced thrombocytopenia (HIT) and for patients at risk of HIT undergoing percutaneous coronary intervention (PCI). The role of argatroban for the treatment of acute coronary syndrome (ACS) is under evaluation.. This article reviews the potential use of argatroban for the treatment of ACS and presents the pharmacokinetic data currently available. The authors also present the pharmacodynamic literature of agratroban in addition to highlighting the safety and tolerability of the drug.. Theoretically, argatroban's pharmacokinetics makes it an attractive alternative to heparin. Pharmacological advantages of argatroban over heparin include a more-predictable anticoagulant response and the absence of a risk of HIT. Furthermore, argatroban has a fast and predictable dose-dependent anticoagulant effect with low inter-individual variability. It is non-immugenic, not susceptible to degradation by proteases and it is cleared via the liver. These characteristics confer argotroban a different profile from other anticoagulants. Agatroban is an effective alternative for patients when heparin, lepirudin and bivalirudin cannot be used. Its utility in ACS and PCI in non-HIT patients has been evaluated but further studies are warranted to define its role in this context.

Topics: Acute Coronary Syndrome; Animals; Antithrombins; Arginine; Disease Models, Animal; Drug Evaluation; Heparin; Hirudins; Humans; Peptide Fragments; Percutaneous Coronary Intervention; Pipecolic Acids; Randomized Controlled Trials as Topic; Recombinant Proteins; Sulfonamides; Thrombocytopenia; Thrombosis

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.

Topics: Ancrod; Animals; Antibodies, Monoclonal; Disease Models, Animal; Fibrinolytic Agents; Hirudins; Mice; Mice, Knockout; Myocardial Reperfusion Injury; Myocardium; Rabbits; Receptor, PAR-1; Receptors, Thrombin; RNA, Messenger; Thrombin; Thromboplastin

Heparin-induced thrombocytopenia (HIT), a drug-induced immunohaematological adverse reaction, is a rare but potentially very severe condition. The main problem for this complex syndrome is its recognition and management, which should be as early as possible to avoid the development of life-threatening complications. Most studies have reported heterogeneous populations of patients with other diseases that potentially induce thrombocytopenia. There is no gold standard diagnostic criteria, and we have established a score with anamnestic criteria that allows us to evaluate the likelihood of HIT. In clinical practice, the diagnosis is based on the analysis of clinical features and laboratory tests. Platelet aggregation test (PAT) and an ELISA test (heparin platelet-induced antibodies) are generally performed by expert laboratories to confirm the occurrence of HIT. In our experience, both tests are concordant in the majority of patients. PAT seems to correlate better with the clinical features while ELISA appears more specific. Regarding their limits, both are complementary in the determination of HIT diagnosis coupled to the clinical score system. The treatment often requires a multidisciplinary approach. Danaparoid (Orgaran) or lepirudin (Refludan) are the two alternative treatments for HIT patients with marketing approval. To avoid further exposure to heparin, every HIT patient should carry a written document that confirms the immunoallergy.

Topics: Animals; Anticoagulants; Chondroitin Sulfates; Dermatan Sulfate; Disease Models, Animal; Drug Combinations; Fibrinolytic Agents; Heparin; Heparitin Sulfate; Hirudin Therapy; Hirudins; Humans; Immunoassay; Platelet Activation; Recombinant Proteins; Thrombocytopenia

Topics: Animals; Disease Models, Animal; Fibrinolytic Agents; Heparin; Hirudins; Humans; Thrombin; Thrombosis

Damage to podocytes is an important determinant of renal pathology. The puromycin aminonucleoside (PAN) mice nephropathy model is commonly used in the study of renal disease with podocyte injury. Hirudin has a broad nephroprotective effect and has been shown to treat renal interstitial fibrosis in previous studies. Mice were given PAN by gavage to prepare animal models, and MPC5 cells were incubated with PAN in vitro. Twenty-four hours urine was collected for analysis of urinary protein levels. Renal pathological changes were observed by hematoxylin and eosin staining. Immunofluorescence detection of nephrin in kidney tissues and cells. Apoptosis was analyzed with over TUNEL. Cytoskeleton, endoplasmic reticulum stress (ERS), p38 MAPK signaling, and apoptosis-related proteins were assessed by western blot analysis. The data suggested that hirudin attenuated reduced renal injury and increased urine protein in PAN mice. Hirudin also attenuated cytoskeletal protein (synaptopodin, nephrin, and podocin) disruption, ERS activation, and apoptosis in PAN mice and PAN-induced podocytes. In addition, hirudin inhibited the expression of p38 MAPK signaling key proteins upregulated by PAN, thereby suppressing ERS. The p38 MAPK agonist was able to partially antagonize the inhibition of p38 MAPK signaling by hirudin in PAN-induced podocytes, thereby reactivating the ERS inhibited by hirudin, promoting cytoskeletal protein degradation and increasing the level of apoptosis. In conclusion, hirudin could decrease podocyte injury by inhibiting p38 MAPK signaling-mediated ERS, resulting in the protection of the kidney from PAN damage. These findings may provide an experimental basis for hirudin treatment of podocyte injury diseases.

Topics: Animals; Cytoskeletal Proteins; Disease Models, Animal; Endoplasmic Reticulum Stress; Hirudins; Kidney Diseases; Mice; p38 Mitogen-Activated Protein Kinases; Podocytes; Puromycin Aminonucleoside

Topics: Animals; Antithrombins; Blood Vessel Prosthesis; Carotid Arteries; Carotid Artery Injuries; Cell Proliferation; Coated Materials, Biocompatible; Disease Models, Animal; Endothelial Cells; Hirudins; Indoles; Male; Peptide Fragments; Polymers; Polytetrafluoroethylene; Recombinant Proteins; Swine; Swine, Miniature; Thrombosis; Wettability

Renal interstitial fibrosis (RIF) often occurs in many chronic kidney diseases (CKD). Hirudin now is applied to treat fibrosis in some organs. In this study, we verified the treatment effects of hirudin on RIF in vivo and in vitro with the underlying mechanism. The RIF in vivo was the unilateral ureteral obstruction (UUO) model and RIF in vitro was the renal tubular epithelial cells induced by TGF-β. The renal pathological changes and renal fibrosis were observed by hematoxylin and eosin (H&E) staining and Masson staining. The α-SMA in renal tissues was detected by immunohistochemistry. The inflammatory factors were analyzed by the ELISA assay. The cell apoptosis was observed by TUNEL assay. The related proteins of fibrosis, epithelial-mesenchymal transition (EMT) and apoptosis were assessed by western blot analysis. The experimental data demonstrated that hirudin decreased fibrosis, EMT, inflammation and cell apoptosis in renal tissues of UUO rats and TGF-β-induced renal tubular epithelial cells. Furthermore, hirudin also reduced the expression of collgen-I, FN, α-SMA, N-cad, slug, E-cad, IL-1β, IL-6 and TNF-α in mice serum and TGF-β-induced renal tubular epithelial cells. The apoptosis related proteins (pro-caspase3, pro-caspase9, bcl2 and bax) expression was also down-regulated in renal tissues of UUO rats. In conclusion, hirudin depressed the fibrosis in renal tissues and renal tubular epithelial cells by inhibiting the inflammation, regulating the related proteins of fibrosis and ETM and decreasing the apoptosis of renal tubular epithelial cells. These findings may offer an effective treatment method for RIF.

Topics: Animals; Anti-Inflammatory Agents; Apoptosis; Cells, Cultured; Disease Models, Animal; Fibrosis; Hirudin Therapy; Hirudins; Humans; Inflammation; Kidney Tubules; Male; Medicine, Chinese Traditional; Mice; Mice, Inbred BALB C; Rats; Renal Insufficiency, Chronic; Ureteral Obstruction

To investigate protective effects of hirudin on oxidative stress and apoptosis of spinal dorsal root ganglion cells in high-glucose rats at the cellular and molecular level.. Dorsal root ganglion neurons (DRGn) were harvested from embryonic day in 15 SD rats, purified and identificated after primary culture. They were divided into the normal control group, high-glucose (HG) group, positive control (alpha-lipoic acid, ALA) group, low-dose hirudin group (H1), medium-dose hirudin group (H2) and high-dose hirudin group (H3). The control group was cultured by neuron specific culture medium, while the HG group was cultured by neuron specific culture medium and 20 mmol/L glucose (HG medium). The hirudin groups were cultured by HG medium+0.25 IU/mL hirudin (H1), HG medium+0.5 IU/mL hirudin (H2) and HG medium+1 IU/mL hirudin (H3). The ALA group was cultured by HG medium+100 µ mol/L ALA. 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenylt etrazolium bromide (MTT) assay was used to explore the optimum concentration and intervention time. Flow cytometry assay was used to detect the level of reactive oxygen series (ROS). Western blot and quantificational realtime polymerase chain reaction (qRT-PCR) were used to detect the expression of protein and mRNA of nuclear factor erythroid 2-related factor 2 (Nrf-2), hemeoxygence-1 (HO-1), nuclear factor-κ B (NF-κ B) and Caspase-3. TUNEL assay was used to test the apoptosis rate of different groups.. After 24 h of culture, the cell activity of hirudin and ALA groups were higher than that of HG group, and there was a statistical difference between the H1 group and HG group (P<0.05). In hirudin groups, the apoptosis rate of cells, the expression of activated Caspase-3 protein and Caspase-3 mRNA were lower than those of HG group (P<0.01), higher than those of ALA group (P<0.01 or P<0.05). The ROS level of hirudin groups was higher than that of ALA group (P<0.01), lower than that of HG group (P<0.01 or P<0.05). The expression of NF-κ B (P65) protein in H3 group were lower than those of HG group (P<0.05). The expression of Nrf-2 protein in hirudin groups was higher than that of HG group (P<0.01), lower than that of ALA group (P<0.01 or P<0.05). The expression of HO-1 protein in hirudin groups was lower than that of ALA group (P<0.01 or P<0.05), higher than that of HG group (P<0.01 or P<0.05).. The activity of DRGn cells can be promoted by hirudin under HG conditions. The effects of hirudin on the inhibition of HG on DRGn cells damage mainly include scavenging ROS, up-regulating Nrf-2/HO-1 pathway, inhibiting activation of NF-κ B pathway, down-regulating the expression of and Caspase-3 and reducing DRGn cell apoptosis.

Topics: Animals; Apoptosis; Caspase 3; China; Disease Models, Animal; Ganglia, Spinal; Heme Oxygenase (Decyclizing); Hirudins; NF-E2-Related Factor 2; Oxidative Stress; Rats; Rats, Sprague-Dawley; Transcription Factor RelA

BACKGROUND This study aimed to investigate the effects of hirudin on the production of extracellular matrix (ECM) factors by renal tubular epithelial cells in a rat model of diabetic kidney disease (DKD) and HK-2 human renal tubule epithelial cells. MATERIAL AND METHODS Sprague-Dawley rats were divided into the normal control group (n=10), the normal control+hirudin group (n=10), the DKD model group (n=12) and the DKD+hirudin group (n=12). At the end of the study, renal histopathology was undertaken, and the expression of type IV collagen, fibronectin, hypoxia-inducible factor-1alpha (HIF-1alpha), and vascular endothelial growth factor (VEGF) were evaluated using immunohistochemistry, Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR). HK-2 cells were cultured in glucose and treated with hirudin. Protein and mRNA expression of fibronectin, type IV collagen, HIF-1alpha, and VEGF were evaluated following knockdown or overexpression of HIF-1alpha. RESULTS Hirudin significantly improved renal function in the rat model of DKD (P<0.01), and significantly down-regulated the expression of fibronectin, type IV collagen, HIF-1alpha, and VEGF proteins (P<0.05). The expression of ECM associated proteins was increased in HK-2 cells treated with high glucose and reduced in the high glucose+shRNA HIF-1alpha group (P<0.05). Compared with the control group, the expression of ECM associated proteins was increased in the HIF-1alpha over-expressed group, and decreased following treatment with hirudin (P<0.05). CONCLUSIONS Hirudin reduced the expression of markers of ECM by inhibiting the HIF-1alpha/VEGF signaling pathway in DKD renal tubular epithelial cells.

Topics: Animals; Biomarkers; Cells, Cultured; China; Diabetic Nephropathies; Disease Models, Animal; Epithelial Cells; Extracellular Matrix; Hirudins; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney Tubules; Male; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Inflammation-induced podocyte apoptosis plays an important role in kidney injury during diabetic nephropathy (DN). Hirudin (HIR), a natural compound extracted from leeches, can inhibit inflammation. However, whether HIR can protect the kidneys against inflammation during DN is unknown. In the present study, we aimed to study the effects of HIR on kidney damage in a DN rat model and explore its anti-inflammatory properties.. A streptozotocin-induced DN rat model was generated, and HIR was administered subcutaneously. Immortal podocytes and primary peritoneal macrophages were used for vitro studies. Hematoxylin and eosin staining was used to evaluate renal pathological changes; quantitative polymerase chain reaction and immunoblotting were used to detect gene expression; and TUNEL staining was used to detect apoptotic cells.. Our results showed that HIR protected against renal injury, as indicated by kidney weight/body weight, serum creatinine, renal pathological changes, blood urea nitrogen, and detection of urine proteins. Notably, HIR treatment reduced macrophage infiltration, pro-inflammatory cytokine expression, and podocyte apoptosis in the kidney tissues of DN rats. In vitro, high glucose (HG) induced the activation of M1 macrophages, which was accompanied by increased podocyte apoptosis. HIR could decrease HG-induced podocyte apoptosis and suppress pro-inflammatory cytokine expression in podocytes in vitro. This was achieved via inhibition of p38 MAPK/NF-κB activation in renal tissues and podocytes.. HIR could inhibit inflammation via the p38 MAPK/NF-κB pathway, prevent podocyte apoptosis, and protect against kidney damage in a DN rat model.

Topics: Animals; Apoptosis; Diabetic Nephropathies; Disease Models, Animal; Hirudins; Inflammation; Injections, Subcutaneous; Kidney; Leeches; Macrophages; Male; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Podocytes; Protective Agents; Rats; Rats, Sprague-Dawley; Streptozocin

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

Bone morphogenetic and activin membrane-bound inhibitor (BAMBI) is a transmembrane protein related to the type I transforming growth factor- β (TGF-β) receptor family that is present on both platelets and endothelial cells (ECs). Bambi-deficient mice exhibit reduced hemostatic function and thrombus stability characterized by an increased embolization.. We aimed to delineate how BAMBI influences endothelial function and thrombus stability.. Bambi-deficient mice were subjected to the laser-induced thrombosis model where platelet and fibrin accumulation was evaluated. Expression of thrombomodulin and tissue factor pathway inhibitor (TFPI) was also assessed in these mice.. Thrombus instability in Bambi. We demonstrate that endothelial BAMBI influences fibrin generation and thrombus stability by modulating thrombomodulin and TFPI anticoagulant function of the endothelium; we also highlight the importance of these anticoagulant proteins in the laser-induced thrombosis model.

Topics: Animals; Anticoagulants; Blood Coagulation; Cells, Cultured; Disease Models, Animal; Endothelial Cells; Female; Fibrin; Hirudins; Lipoproteins; Lung; Male; Membrane Proteins; Mice, Inbred C57BL; Mice, Knockout; Thrombomodulin; Thrombosis

Topics: Animals; Antithrombins; Cell Survival; Cells, Cultured; Disease Models, Animal; Gene Expression; Hirudins; Humans; Immunophenotyping; Kidney Glomerulus; Nephrosis; Podocytes; Proteinuria; Rats; Receptor, PAR-1; Receptors, Thrombin; Signal Transduction; Thrombin

Topics: Animals; Anticholesteremic Agents; Biological Therapy; Disease Models, Animal; Estrogens; Hirudins; Hyperlipidemias; Liver; Metalloendopeptidases; Mice; Non-alcoholic Fatty Liver Disease; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Quail; Rats; Recombinant Fusion Proteins; Treatment Outcome

Coagulation factor V (FV) is distributed in plasma and platelet pools, which are distinguished by physical and functional differences. FV has been extensively studied for its roles in coagulation. The roles of FV in other physiologic pathways remain understudied.. Hind limb ischemia was produced in transgenic mice by femoral artery ligation, with different levels of FV gene expression restricted to the plasma or platelets.. Hind limb blood flow perfusion in mice with higher platelet FV was significantly increased. The expression of major angiogenesis-related factors was correlated with the level of FV during ischemia. Furthermore, a platelet depletion and transfusion procedure showed that the transfusion of platelets with higher levels of FV into transgenic mice with undetectable platelet FV significantly rescued the ischemia-mediated impairments in blood flow perfusion. Immunohistochemistry analysis also indicated markedly increased capillary formation in the ischemic muscle of mice with higher platelet FV. Moreover, thrombin activity was significantly higher in the mice with higher platelet FV. Platelets expressing higher levels of FV stimulated increased endothelial cell migration. Hind limb blood flow perfusion was significantly blocked by thrombin inhibitor.. These findings suggest that platelet-derived FV contributes to the control of angiogenesis and is likely associated with thrombin generation.

Topics: Angiogenic Proteins; Animals; Antithrombins; Blood Flow Velocity; Blood Platelets; Cell Movement; Cells, Cultured; Disease Models, Animal; Endothelial Cells; Factor V; Genotype; Hindlimb; Hirudins; Ischemia; Mice, Inbred C57BL; Mice, Transgenic; Muscle, Skeletal; Neovascularization, Physiologic; Phenotype; Platelet Endothelial Cell Adhesion Molecule-1; Platelet Transfusion; Regional Blood Flow; Thrombin; Time Factors

OBJECTIVE Painful neuropathic injuries induce blood-spinal cord barrier (BSCB) breakdown, allowing pro-inflammatory serum molecules to cross the BSCB, which contributes to nociception. The goal of these studies was to determine whether the blood-borne serine protease thrombin also crosses a permeable BSCB, contributing to nociception through its activation of protease-activated receptor-1 (PAR1). METHODS A 15-minute C-7 nerve root compression, which induces BSCB breakdown and painful behaviors by Day 1, was administered in the rat (n = 10); sham operation (n = 11) and a 3-minute compression (n = 10) that does not induce sensitivity were administered as controls. At Day 1 after root compression, spinal cord tissue was co-immunolabeled for fibrin/fibrinogen, the enzymatic product of thrombin, and IgG, a serum protein, to determine whether thrombin acts in areas of BSCB breakdown. To determine whether spinal thrombin and PAR1 contribute to hyperalgesia after compression, the thrombin inhibitor hirudin and the PAR1 antagonist SCH79797, were separately administered intrathecally before compression injuries (n = 5-7 per group). Rat thrombin was also administered intrathecally with and without SCH79797 (n = 6 per group) to determine whether spinal thrombin induces hypersensitivity in naïve rats through PAR1. RESULTS Spinal fibrin(ogen) was elevated at Day 1 after root compression in regions localized to BSCB breakdown and decreased in those regions by Day 7. Blocking either spinal thrombin or PAR1 completely prevented compression-induced hyperalgesia for 7 days. Intrathecal thrombin induced transient pain that was prevented by blocking spinal PAR1 before its injection. CONCLUSIONS The findings of this study suggest a potent role for spinal thrombin and its activation of PAR1 in pain onset following neuropathic injury.

Topics: Animals; Antithrombins; Capillary Permeability; Central Nervous System Agents; Cervical Vertebrae; Disease Models, Animal; Fibrin; Hirudins; Hyperalgesia; Injections, Spinal; Male; Pain; Pain Measurement; Peripheral Nervous System Diseases; Pyrroles; Quinazolines; Radiculopathy; Rats, Sprague-Dawley; Receptor, PAR-1; Spinal Cord

The effects of antithrombotic drugs on random and free flap survival have been investigated in the past, but the experimental and clinical results are not in agreement. A perforator-based critical ischaemia model was used to evaluate the effects of different perioperatively administered pharmaceutical agents on tissue ischaemia and to assess the potential additional haemorheological or vasodilative effects of antithrombotics on flap microcirculation. Combined laser Doppler flowmetry and remission spectroscopy revealed an increase in certain microcirculation parameters in most groups in comparison with saline controls, and these changes correlated with flap survival. Clopidogrel and hirudin significantly improved the amount of viable flap tissue in comparison with controls, while unfractioned heparin had a negative effect on flap survival. Low molecular weight heparin, aspirin, pentoxifylline, and hydroxyethyl starch had no impact on the amount of viable flap tissue. A higher complication rate was observed in all experimental groups, but only clopidogrel had a negative impact on the flap viability. Our results add to the body of evidence supporting the conclusion that perioperative antithrombotic treatment improves flap survival. Clopidogrel and hirudin are effective pharmacological agents that significantly increased the viability of perforator-based skin flaps in rats, but at a higher risk of postoperative bleeding.

Topics: Animals; Aspirin; Clopidogrel; Disease Models, Animal; Fibrinolytic Agents; Graft Survival; Heparin; Hirudins; Ischemia; Laser-Doppler Flowmetry; Male; Microcirculation; Pentoxifylline; Postoperative Complications; Rats; Rats, Wistar; Skin Diseases; Skin Transplantation; Surgical Flaps; Ticlopidine

The leech protein hirudin is a potent natural thrombin inhibitor. Its potential as an antithrombotic agent is limited by its promotion of bleeding. We attempted to modify this profile by positioning albumin and a plasmin cleavage site on its N-terminus, in recombinant protein HSACHV3 [comprising hirudin variant 3 (HV3) fused to the C-terminus of human serum albumin (HSA) via a plasmin cleavage site (C)], Previously we showed that HSACHV3 inhibited thrombin in a plasmin-dependent manner, and that, unlike HV3, it did not increase bleeding in vivo when administered to mice. Here we tested HSACHV3 for the ability to reduce thrombosis and assist enzymatic thrombolysis in animal models. Intravenous administration of HSACHV3, but not a control protein lacking the plasmin cleavage site (HSAHV3), reduced thrombus weight by 2.1-fold in the ferric chloride-injured mouse vena cava. Similarly, thrombi formed in a rabbit jugular vein stasis model were 1.7-fold lighter in animals treated with HSACHV3 compared to those receiving HSAHV3. Administration of 60 mg/kg body weight HSACHV3 prolonged the time to occlusion in the ferric chloride-injured mouse carotid artery by threefold compared to vehicle controls, while equimolar HSAHV3 had no effect. HSACHV3 had no ability to restore flow to the murine carotid arteries occluded by ferric chloride treatment, but combining HSACHV3 (60 mg/kg) with recombinant mutant tissue plasminogen activator (TNKase) significantly reduced the time to restore patency to the artery compared to TNKase alone. Unlike unfused HV3, HSACHV3 did not increase bleeding in a mouse liver laceration model. Our results show that HSACHV3 acts as an antithrombotic agent that does not promote bleeding and which speeds the time to flow restoration when used as an adjunct to pharmacological thrombolysis in animal models.

Topics: Animals; Chlorides; Disease Models, Animal; Ferric Compounds; Hirudins; Humans; Mice; Rabbits; Recombinant Proteins; Thrombolytic Therapy; Thrombosis

Platelets are known to facilitate tumor metastasis and thrombocytosis has been associated with an adverse prognosis in ovarian cancer. However, the role of platelets in primary tumour growth remains to be elucidated. The present study demonstrated that the expression levels of various markers in platelets, endothelial adherence and angiogenesis, including, platelet glycoprotein IIb (CD41), platelet endothelial cell adhesion molecule 1 (CD31), vascular endothelial growth factor (VEGF), lysyl oxidase, focal adhesion kinase and breast cancer anti‑estrogen resistance 1, were expressed at higher levels in patients with malignant carcinoma, compared with those with borderline cystadenoma and cystadenoma. In addition, the endothelial markers CD31 and VEGF were found to colocalize with the platelet marker CD41 in the malignant samples. Since mice transplanted with human ovarian cancer cells (SKOV3) demonstrated elevated tumor size and decreased survival rate when treated with thrombin or thrombopoietin (TPO), the platelets appeared to promote primary tumor growth. Depleting platelets using antibodies or by pretreating the cancer cells with hirudin significantly attenuated the transplanted tumor growth. The platelets contributed to late, but not early stages of tumor proliferation, as mice treated with platelet‑depleting antibody 1 day prior to and 11 days after tumor transplantation had the same tumor volumes. By contrast, tumor size in the early TPO‑injected group was increased significantly compared with the late TPO‑injected group. These findings suggested that the interplay between platelets and angiogenesis may contribute to ovarian cancer growth. Therefore, platelets and their associated signaling and adhesive molecules may represent potential therapeutic targets for ovarian cancer.

Topics: Animals; Antithrombins; Blood Coagulation; Blood Platelets; Cell Line, Tumor; Disease Models, Animal; Female; Heterografts; Hirudins; Humans; Mice; Neoplasm Staging; Neovascularization, Pathologic; Ovarian Neoplasms; Prognosis; Thrombophilia; Thrombosis; Tumor Burden

Although it is recognized that thrombin plays a key role in airway remodeling during chronic asthma. In a previous study, we have proved that thrombin promotes airway remodeling via PAR-1 in OVA-allergic rats, but little is known about intracellular signaling pathway involved in the event.. In this study, we intend to explore the impact of pERK1/2 signaling pathway on the process of thrombin-induced airway remodeling in OVA-allergic rats.. A rat model of chronic asthma was set up by systemic sensitization and repeated challenge to OVA. The doses of thrombin, recombinant hirudin, PAR-1 inhibitor ER-112780-06, and pERK1/2 inhibitor PD98059 varied for different groups. The expression of pERK1/2 was analyzed by western blot and RT-PCR. Secretion of TGF-β1 and IL-6 was detected by ELISA.. The expression of pERK1/2 was higher in the airway of asthmatic rats than those of normal rats, and was significantly increased by thrombin treatment but decreased by thrombin-inhibitor treatment. Airway remodeling was enhanced by thrombin but weakened by pERK1/2 inhibitor. Expression of growth factors and IL-6 in asthmatic rats was significantly increased by thrombin treatment and decreased by thrombin-inhibitor treatment and pERK1/2 inhibitor treatment.. These results suggest that ERK1/2 signaling pathway may play an important role in the process of thrombin-promoting airway remodeling in OVA-allergic rats, and pERK1/2 inhibitor effectively inhibits the process.

Topics: Administration, Inhalation; Airway Remodeling; Animals; Antithrombins; Asthma; Disease Models, Animal; Female; Hirudins; Interleukin-6; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Ovalbumin; Rats, Wistar; Receptor, PAR-1; Thrombin; Transforming Growth Factor beta1

The risk of thrombotic complications such as deep vein thrombosis (DVT) during tumor development is well known. Tumors release into the circulation procoagulant microparticles (MPs) that can participate in thrombus formation following vessel injury. The importance of this MP tissue factor (TF) in the initiation of cancer-associated DVT remains uncertain.. To investigate how pancreatic cancer MPs promote DVT in vivo.. We combined a DVT mouse model in which thrombosis is induced by flow restriction in the inferior vena cava with one of subcutaneous pancreatic cancer in C57BL/6J mice. We infused high-TF and low-TF tumor MPs to determine the importance of TF in experimental cancer-associated DVT.. Both tumor-bearing mice and mice infused with tumor MPs subjected to 3 h of partial flow restriction developed an occlusive thrombus; fewer than one-third of the control mice did. We observed that MPs adhered to neutrophil extracellular traps (NETs), which are functionally important players during DVT, whereas neither P-selectin nor glycoprotein Ib were required for MP recruitment in DVT. The thrombotic phenotype induced by MP infusion was suppressed by hirudin, suggesting the importance of thrombin generation. TF carried by tumor MPs was essential to promote DVT, as mice infused with low-TF tumor MPs had less thrombosis than mice infused with high-TF tumor MPs.. TF expressed on tumor MPs contributes to the increased incidence of cancer-associated venous thrombosis in mice in vivo. These MPs may adhere to NETs formed at the site of thrombosis.

Topics: Animals; Antithrombins; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell-Derived Microparticles; Disease Models, Animal; Extracellular Traps; Hirudins; Ligation; Male; Mice, Inbred C57BL; Mice, Knockout; P-Selectin; Pancreatic Neoplasms; Platelet Glycoprotein GPIb-IX Complex; Regional Blood Flow; Thromboplastin; Vena Cava, Inferior; Venous Thrombosis

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

Hirulog-like peptide (HLP) and low-molecular-weight heparin (LMWH) are thrombin inhibitor peptides. Our previous study demonstrated that HLP could reduce vascular neointimal formation or restenosis in animals undergoing balloon catheter injury in the carotid artery. However, the function of HLP during ischemic stroke is largely unknown. The present study investigated the effect of HLP on brain injury, which was induced by suture of middle cerebral artery occlusion in mice. Mice were divided into four groups, which included a sham group and three treatment groups. Ischemia was induced by transient suture insertion into the middle cerebral artery for 90 min, and mice were either treated with saline, HLP or LMWH. Infarct volume, neurologic deficits and apoptotic factors were measured following 1-14 days of ischemia. We demonstrated that HLP intravenous injection alleviated brain infarct volume and improved neurologic outcomes (p<0.05). HLP decreased levels of protease-activated receptor-1 (PAR-1), caspase-3, malondialdehyde (MDA) and Bcl-2-associated X protein (Bax), increased the activities of catalase and B cell lymphoma-2 (Bcl-2), and improved the ratio of Bcl-2/Bax compared with the control (p<0.05). This study indicates that HLP and LMWH reduced infarct volume and improved neurobehavioral outcomes induced by transient middle cerebral artery occlusion (tMCAO). In addition, HLP had a beneficial effect on the regulation of the thrombin receptor and key apoptosis regulators in the mouse brain. These results suggest that HLP may be a potential alternative therapy for arterial occlusion-induced cerebral ischemia.

Topics: Animals; Antithrombins; Apoptosis; Blotting, Western; Brain; Disease Models, Animal; Down-Regulation; Heparin, Low-Molecular-Weight; Hirudins; Immunohistochemistry; Infarction, Middle Cerebral Artery; Male; Malondialdehyde; Mice, Inbred ICR; Motor Activity; Neuroprotective Agents; Peptide Fragments; Receptors, Thrombin; Rotarod Performance Test; Severity of Illness Index

Aptamers are nucleic acid based molecular recognition elements with a high potential for the theranostics. Some of the aptamers are under development for therapeutic applications as promising antithrombotic agents; and G-quadruplex DNA aptamers, which directly inhibit the thrombin activity, are among them. RA-36, the 31-meric DNA aptamer, consists of two thrombin binding pharmacophores joined with the thymine linker. It has been shown earlier that RA-36 directly inhibits thrombin in the reaction of fibrinogen hydrolysis, and also it inhibits plasma and blood coagulation. Studies of both inhibitory and anticoagulation effects had indicated rather high species specificity of the aptamer. Further R&D of RA-36 requires exploring its efficiency in vivo. Therefore the development of a robust and adequate animal model for effective physiological studies of aptamers is in high current demand. This work is devoted to in vivo study of the antithrombotic effect of RA-36 aptamer. A murine model of thrombosis has been applied to reveal a lag and even prevention of thrombus formation when RA-36 was intravenous bolus injected in high doses of 1.4-7.1 µmol/kg (14-70 mg/kg). A comparative study of RA-36 aptamer and bivalirudin reveals that both direct thrombin inhibitors have similar antithrombotic effects for the murine model of thrombosis; though in vitro bivalirudin has anticoagulation activity several times higher compared to RA-36. The results indicate that both RA-36 aptamer and bivalirudin are direct thrombin inhibitors of different potency, but possible interactions of the thrombin-inhibitor complex with other components of blood coagulation cascade level the physiological effects for both inhibitors.

Topics: Animals; Anticoagulants; Antithrombins; Aptamers, Nucleotide; Blood Coagulation; Disease Models, Animal; Drug Evaluation, Preclinical; Fibrinogen; Fibrinolytic Agents; G-Quadruplexes; Hirudins; Male; Mice; Mice, Inbred C57BL; Peptide Fragments; Recombinant Proteins; Thrombin; Thrombosis

Edoxaban is a novel, potent and orally active direct Factor Xa (FXa) inhibitor under development for prophylaxis and treatment of thromboembolic diseases. Properties of dose response and margin of safety of anticoagulants are the key factors for a positive risk/benefit of novel oral anticoagulants.. To compare the dose response of antithrombotic effect and margin of safety between antithrombotic and hemorrhagic effects of edoxaban with conventional anticoagulants, unfractionated heparin (UFH), dalteparin (low molecular weight heparin), lepirudin, and warfarin in rat models of thrombosis and hemorrhage.. Rats were treated with edoxaban, UFH, dalteparin, and lepirudin by continuous intravenous (iv) infusion, or with oral warfarin for 4 days before inducing thrombosis or bleeding. Thrombosis was induced by inserting a platinum wire into the inferior vena cava for 60 minutes. Tail template bleeding time was measured after making an incision on the tail.. In rats, iv infusion of edoxaban inhibited venous thrombosis in a dose-dependent manner. The other anticoagulants also exerted dose-dependent antithrombotic effects. The slopes of the dose-response curves of edoxaban were significantly shallower than the slopes of UFH, dalteparin, and warfarin. At supratherapeutic doses, edoxaban prolonged bleeding time in a rat tail bleeding model. To determine bleeding risk, the margins between antithrombotic and bleeding-time prolongation were compared. The margins of safety of edoxaban were wider than those of UFH, dalteparin, lepirudin, and warfarin.. These results suggest that edoxaban may be more easily controlled and has the potential for a more positive risk/benefit ratio compared to conventional anticoagulants.

Topics: Animals; Anticoagulants; Disease Models, Animal; Dose-Response Relationship, Drug; Factor Xa Inhibitors; Heparin; Hirudins; Male; Pyridines; Rats; Rats, Wistar; Recombinant Proteins; Thiazoles; Thromboembolism; Warfarin

Reocclusion is one of the major root causes for secondary complications that arise during thrombolytic therapy. A multifunctional staphylokinase variant SRH (staphylokinase (SAK) linked with tripeptide RGD and didecapeptide Hirulog) with antiplatelet and antithrombin activities in addition to clot specific thrombolytic function, was developed to address the reocclusion problem. We preferred to use Escherichia coli GJ1158 as the host in this study for economic production of SRH by osmotic (0.3 mol/L sodium chloride) induction, to overcome the problems associated with the yeast expression system. The therapeutic potential of SRH was evaluated in the murine model of vascular thrombosis. The SAK protein (1 mg/kg body mass) and SRH protein (1 mg/kg and 2 mg/kg) were administered intravenously to the different treatment groups. The results have shown a dose-dependent antithrombotic effect in carrageenan-induced mouse tail thrombosis. The thrombin time, activated partial thromboplastin time, and prothrombin time were significantly prolonged (p < 0.05) in the SRH-infused groups. Moreover, SRH inhibited platelet aggregation in a dose-dependent manner (p < 0.05), while the bleeding time was significantly (p < 0.05) prolonged. All of these results inferred that the osmotically produced multifunctional fusion protein SRH (SAK-RGD-Hirulog) is a promising thrombolytic agent, and one which sustained its multifunctionality in the animal models.

Topics: Animals; Antithrombins; Blood Coagulation; Carrageenan; Disease Models, Animal; Dose-Response Relationship, Drug; Escherichia coli; Hirudins; Male; Metalloendopeptidases; Mice; Mice, Inbred BALB C; Oligopeptides; Partial Thromboplastin Time; Peptide Fragments; Platelet Aggregation; Platelet Aggregation Inhibitors; Prothrombin Time; Rats; Rats, Sprague-Dawley; Recombinant Fusion Proteins; Recombinant Proteins; Sodium Chloride; Thrombin Time; Thrombosis; Time Factors

The aim of this study was to observe the effect of hirudin on the expression of lung tissue protease activated receptor-1 (PAR-1) and the correlation between inflammation factors and the expression of PAR-1 after hirudin pre-treatment and to provide the theoretical basis for the treatment of lung injury by hirudin. Wistar rats of the model group were intraperitoneally administered endotoxin by injection (LPS 10 mg/kg) to copy acute lung injury (ALI) animal models, while the rats of the control group were injected with an equal amount of physiological saline. The rats of the hirudin groups were injected with hirudin and endotoxin intraperitoneally at the same time. The lung tissue was stained by HE dye to detect tumor necrosis factor α (TNF-α) and matrix metallo-proteinase 12 (MMP12) content. RT-PCR was applied to test PAR-1 mRNA expression. The results showed that the expression of PAR-1 mRNA of lung tissue increased significantly, but declined with the increased doses of hirudin when lung injury due to endotoxin occurred. The content of TNF-α and MMP12 was significantly lower compared to that of the endotoxin group. The difference was statistically significant (p<0.05). Hirudin reduced the release of TNF-α and MMP12 in mice by inhibiting the production of PAR-1 and reduced the content of TNF-α and MMP12. Thus, we deduced that hirudin inhibits the inflammation and fibrosis caused by lung injury and plays a role in lung protection as an anti-inflammatory mediator.

Topics: Acute Lung Injury; Animals; Disease Models, Animal; Fibrinolytic Agents; Gene Expression Regulation; Hirudin Therapy; Hirudins; Lipopolysaccharides; Matrix Metalloproteinase 12; Rats; Rats, Wistar; Receptor, PAR-1; Tumor Necrosis Factor-alpha

Thrombin induces CD40 ligand (CD40L) and matrix metalloproteinases (MMPs) under inflammatory/prothrombotic conditions. Thrombin and CD40L could modulate endothelial MMP-10 expression in vitro and in vivo.. Human endothelial cells were stimulated with thrombin (0.1-10 U/mL), CD40L (0.25-1 μg/mL), or their combination (thrombin/CD40L) to assess MMP-10 expression and microparticle generation. Thrombin/CD40L elicited higher MMP-10 mRNA (5-fold; P<0.001) and protein levels (4.5-fold; P<0.001) than either stimulus alone. This effect was mimicked by a protease-activated receptor-1 agonist and antagonized by hirudin, a-protease-activated receptor-1, α-CD40L, and α-CD40 antibodies. The synergistic effect was dependent on p38 mitogen-activated protein kinase and c-Jun N-terminal kinase-1 pathways. Thrombin also upregulated the expression of CD40 in endothelial cell surface increasing its availability, thereby favoring its synergistic effects with CD40L. In mice, thrombin/CD40L further increased the aortic MMP-10 expression. Septic patients with systemic inflammation and enhanced thrombin generation (n=60) exhibited increased MMP-10 and soluble CD40L levels associated with adverse clinical outcome. Endothelial and systemic activation by thrombin/CD40L and lipopolysaccharide also increased microparticles harboring MMP-10 and CD40L.. Thrombin/CD40L elicited a strong synergistic effect on endothelial MMP-10 expression and microparticles containing MMP-10 in vitro and in vivo, which may represent a new link between inflammation/thrombosis with prognostic implications.

Topics: Adult; Aged; Aged, 80 and over; Animals; Antibodies; Blood Coagulation; Case-Control Studies; CD40 Antigens; CD40 Ligand; Cell-Derived Microparticles; Cells, Cultured; Disease Models, Animal; Disseminated Intravascular Coagulation; Endothelial Cells; Endotoxemia; Female; Gene Expression Regulation, Enzymologic; Hirudins; Human Umbilical Vein Endothelial Cells; Humans; Lipopolysaccharides; Male; Matrix Metalloproteinase 10; Mice; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Mitogen-Activated Protein Kinase 8; Multivariate Analysis; p38 Mitogen-Activated Protein Kinases; Peptides; Protein Kinase Inhibitors; Receptor, PAR-1; Risk Assessment; Risk Factors; RNA, Messenger; Sepsis; Signal Transduction; Spain; Survival Analysis; Thrombin; Time Factors; Up-Regulation

Cell therapy is a promising option for the treatment of acute or chronic myocardial ischemia. The intracoronary infusion of cells imposes the potential risk of cell clotting, which may be prevented by the addition of anticoagulants. However, a comprehensive analysis of the effects of anticoagulants on the function of the cells is missing.. Here, we investigated the effects of heparin and the thrombin inhibitor bivalirudin on bone marrow-derived mononuclear cell (BMC) functional activity and homing capacity.. Heparin, but not bivalirudin profoundly and dose-dependently inhibited basal and stromal cell-derived factor 1 (SDF-1)-induced BMC migration. Incubation of BMCs with 20 U/mL heparin for 30 minutes abrogated SDF-1-induced BMC invasion (16±8% of control; P<0.01), whereas no effects on apoptosis or colony formation were observed (80±33% and 100±44% of control, respectively). Pretreatment of BMCs with heparin significantly reduced the homing of the injected cells in a mouse ear-wound model (69±10% of control; P<0.05). In contrast, bivalirudin did not inhibit in vivo homing of BMCs. Mechanistically, heparin binds to both, the chemoattractant SDF-1 and its receptor, chemokine receptor 4 (CXCR4), blocking CXCR4 internalization as well as SDF-1/CXCR4 signaling after SDF-1 stimulation.. Heparin blocks SDF-1/CXCR4 signaling by binding to the ligand as well as the receptor, thereby interfering with migration and homing of BMCs. In contrast, the thrombin inhibitor bivalirudin did not interfere with BMC homing or SDF-1/CXCR4 signaling. These findings suggest that bivalirudin but not heparin might be recommended as an anticoagulant for intracoronary infusion of BMCs for cell therapy after cardiac ischemia.

Topics: Animals; Anticoagulants; Antithrombins; Bone Marrow Cells; Cell Movement; Cell- and Tissue-Based Therapy; Cells, Cultured; Chemokine CXCL12; Disease Models, Animal; Female; Heparin; Hirudins; Humans; In Vitro Techniques; Leukocytes, Mononuclear; Mice; Mice, Inbred Strains; Myocardial Infarction; Peptide Fragments; Receptors, CXCR4; Recombinant Proteins; Signal Transduction

SAK-RGD-K2-Hir and SAK-RGD-K2-Hirul are recombinant proteins that are derivatives of r-SAK (recombinant staphylokinase). They are characterized by their fibrin-specific plasminogen activation properties and their antithrombin and antiplatelet activities. The difference between these proteins is the presence of the antithrombotic fragment (hirudin or hirulog) in the C-terminal portion of the r-SAK. The aim of the present study was to examine the thrombolytic potentials of SAK-RGD-K2-Hir and SAK-RGD-K2-Hirul in an electrically induced carotid artery thrombosis model in rats and to compare the potentials to that of r-SAK. We determined that a bolus injection of SAK-RGD-K2-Hirul was more effective than one of r-SAK in the improvement and maintenance of carotid patency and in arterial thrombus weight reduction; however, it had the same potency as SAK-RGD-K2-Hir. The bleeding time, prothrombin time and activated partial thromboplastin time were significantly prolonged in the animals that were treated with either dose (1.5 or 3.0 mg/kg) of SAK-RGD-K2-Hir or SAK-RGD-K2-Hirul, whereas no changes were observed in the plasma fibrinogen concentration or the α2 plasmin inhibitor level. r-SAK alone did not change the bleeding time or coagulation parameters. In conclusion, our findings demonstrate the thrombolytic activity of intravenous bolus injection of the novel thrombolytic agent SAK-RGD-K2-Hirul in rats. Although this protein compares favorably with r-SAK, we were unable to show the presence of any beneficial effects of SAK-RGD-K2-Hirul over those of SAK-RGD-K2-Hir. Furthermore, our results suggest that high doses of SAK-RGD-K2-Hirul bear the risk of bleeding.

Topics: Animals; Bleeding Time; Blood Coagulation; Carotid Artery Thrombosis; Disease Models, Animal; Dose-Response Relationship, Drug; Fibrinolytic Agents; Hirudins; Injections, Intravenous; Male; Metalloendopeptidases; Rats; Rats, Wistar; Recombinant Fusion Proteins

We previously described a model of laser-induced thrombosis in mesenteric arterioles with superficial and deep levels of injury producing a transient thrombus resolving within 2 min and a larger almost occlusive thrombus, respectively. Both types of lesion were sensitive to platelet GPIIb-IIIa and P2Y(12) inhibition, whereas only deep injuries were sensitive to thrombin blockade.. The aim of the present study was to use histologic methods and electron and intravital microscopy to characterize the lesions and thrombi and to extend our knowledge of the sensitivity of this model to genetic and pharmacologic inhibition.. A superficial injury was found to detach the endothelial cells and expose a collagen III- and IV-rich subendothelium where platelets could adhere. Tissue factor and fibrin were not detected. Deeper penetration of the external elastic lamina occurred in deep injuries, with exposure of collagen I, III and IV. Here the thrombus was composed of platelets exhibiting a decreasing gradient of degranulation from the deepest lesion area to the surface. Fibrin was found close to the most activated platelets. Consistently, glycoprotein VI (GPVI)-collagen and GPIb-von Willebrand factor (VWF) interactions were found to be critical in superficial injuries. After deep lesion, thrombus formation was modestly reduced in GPVI-immunodepleted mice and still strongly inhibited in VWF(-/-) mice. Combined hirudin infusion and GPVI depletion further inhibited thrombosis after deep injury.. This study confirms the feasibility of inducing arterial thrombosis with distinct levels of severity and establishes the central roles of collagen and VWF in thrombus formation after superficial injury. Collagen, VWF and thrombin all appear to contribute to thrombosis after deep arterial lesion.

Topics: Animals; Blood Platelets; Collagen Type I; Collagen Type III; Collagen Type IV; Disease Models, Animal; Endothelium, Vascular; Feasibility Studies; Fibrin; Fibrinolytic Agents; Hirudins; Injections, Subcutaneous; Lasers, Gas; Male; Mesenteric Arteries; Mesenteric Vascular Occlusion; Mice; Mice, Knockout; Platelet Adhesiveness; Platelet Membrane Glycoproteins; Severity of Illness Index; Thrombosis; Time Factors; von Willebrand Factor

To understand the role of epidermal growth factor receptor (EGFR) transactivation in G protein-coupled receptor (GPCR) agonist-induced signaling events, we have studied the capacity of thrombin in the activation of Gab1-SHP2 in vascular smooth muscle cells (VSMCs). Thrombin activated both Gab1 and SHP2 in EGFR-dependent manner. Similarly, thrombin induced Rac1 and Cdc42 activation, and these responses were suppressed when either Gab1 or SHP2 stimulation is blocked. Thrombin also induced PAK1 activation in a time- and EGFR-Gab1-SHP2-Rac1/Cdc42-dependent manner. Inhibition of activation of EGFR, Gab1, SHP2, Rac1, Cdc42, or PAK1 by pharmacological or genetic approaches attenuated thrombin-induced VSMC stress fiber formation and motility. Thrombin activated RhoA in a time-dependent manner in VSMCs. LARG, a RhoA-specific GEF (guanine nucleotide exchange factor), was found to be associated with Gab1 and siRNA-mediated depletion of its levels suppressed RhoA, Rac1 and PAK1 activation. Dominant negative mutant-mediated interference of RhoA activation inhibited thrombin-induced Rac1 and PAK1 stimulation in VSMCs and their stress fiber formation and migration. Balloon injury induced PAK1 activity and interference with its activation led to attenuation of SMC migration from media to intima, resulting in reduced neointima formation and increased lumen size. Inhibition of thrombin signaling by recombinant hirudin also blocked balloon injury-induced EGFR tyrosine phosphorylation and PAK1 activity. These results show that thrombin-mediated PAK1 activation plays a crucial role in vascular wall remodeling and it could be a potential target for drug development against these vascular lesions.

Topics: Angioplasty, Balloon; Animals; Carotid Artery Diseases; Carotid Stenosis; cdc42 GTP-Binding Protein; Cell Movement; Cells, Cultured; Disease Models, Animal; ErbB Receptors; Fibrinolytic Agents; Gene Transfer Techniques; Genetic Therapy; Guanine Nucleotide Exchange Factors; Hirudins; Humans; Muscle, Smooth, Vascular; Mutation; p21-Activated Kinases; Phosphoproteins; Phosphorylation; Protein Kinase Inhibitors; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Quinazolines; rac1 GTP-Binding Protein; Rats; Rho Guanine Nucleotide Exchange Factors; rhoA GTP-Binding Protein; RNA Interference; RNA, Small Interfering; Stress Fibers; Thrombin; Time Factors; Transfection; Tyrphostins

To resolve the therapeutic dilemma between efficacy of thrombolysis and bleeding risk associated with the use of a combination of thrombolytic and anticoagulant treatments, we created a fusion protein. Staphylokinase was fused to the N-terminus of hirudin using thrombin recognition sequence as linker peptide, resulting in a fusion protein STH. We hypothesised that STH would be cleaved by thrombin at the thrombus site, releasing staphylokinase and hirudin to perform bifunctionally, and attenuating bleeding risk. SDS-PAGE and Western blot analyses indicated that the linker peptide could be specially recognised and cleaved by thrombin. Amidolytic and thromboelastogram assays showed that the N-terminus of hirudin in STH was blocked by staphylokinase and linker peptide, impeding hirudin's anticoagulant activity. Once cleaved, STH displayed 35.7% of the anticoagulant activity of equimolar hirudin and exhibited anticoagulant effects in the fibrin clot lysis assay. Thrombin-binding and fibrin clot lysis assays showed that the C-terminus of hirudin retained its high affinity for thrombin. Moreover, STH showed improved thrombolytic effects and a lower bleeding risk in animals. Thus, STH may have the capacity to perform bifunctionally and release anticoagulant activity in a thrombus-targeted manner in vivo, which may reduce the bleeding risk that often accompanies high thrombolytic efficacy in the treatment of thromboembolic diseases.

Topics: Animals; Base Sequence; Cattle; Disease Models, Animal; DNA Primers; Fibrinolytic Agents; Hemorrhage; Hirudins; Humans; In Vitro Techniques; Metalloendopeptidases; Mice; Plasminogen; Rats; Recombinant Fusion Proteins; Thrombelastography; Thrombin; Thromboembolism; Thrombolytic Therapy; Vena Cava, Inferior

The purpose of this study was to design and evaluate hirudin (HIR) derivatives with low bleeding risk. In these derivatives, the factor (F) XIa, FXa, and thrombin recognition peptides (EPR, GVYAR, and LGPR, respectively) were linked to the N-terminus of HIR. The intact derivatives have no anticoagulant activity because of the extension of the N-terminus of HIR. After cleavage by the corresponding coagulation factor that occurs on the activation of the coagulation system and in the presence of the thrombus, its activity is released. This limited the anticoagulant activity of these derivatives to the vicinity of the thrombus, and as a result, systemic bleeding complications were avoided. The definite antithrombotic effect and low bleeding parameters of these derivatives were investigated in rat carotid artery and inferior vena cava thrombosis models. In both models, the three derivatives showed significant antithrombotic effects, indicating that anticoagulant activity could be successfully released in vivo. Moreover, the bleeding parameters of these derivatives were lower than that of HIR as indicated by the values of activated partial thromboplastin time (APTT) and thrombin time (TT). To further assess the safety of these derivatives, bleeding time was measured in a mouse tail-cut model. Although the derivatives had obvious effects on bleeding at a dose of 6 mg/kg, the effect of these derivatives on bleeding was significantly weaker than that of HIR at a dose of 1.5 mg/kg. Thus, the benefit-to-risk profiles of the derivatives were superior to that of HIR.

Topics: Animals; Anticoagulants; Bleeding Time; Blood Coagulation; Cloning, Molecular; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Fibrinolytic Agents; Hemorrhage; Hirudins; Male; Mice; Partial Thromboplastin Time; Prodrugs; Rats; Rats, Wistar; Recombinant Fusion Proteins; Risk Assessment; Thrombin Time; Thrombosis; Venous Thrombosis

In an effort to evaluate pharmacologic agents for optimal anticoagulant prophylaxis in patients undergoing free tissue transfer, we evaluated the efficacy of desirudin (Canyon Pharmaceuticals, Hunt Valley, MD), a recombinant hirudin that acts as a direct thrombin inhibitor, using a rat model of microvenous thrombosis.. Randomized, blinded study using an in vivo rat model of microvenous failure.. Thirty-two rats received either desirudin or saline in a randomized, blinded fashion 30 minutes prior to performance of a standardized thrombogenic procedure on rat femoral veins. Bleeding time, vessel patency, and presence of clot within the anastomosis were subsequently assessed. Appropriate statistical analyses were then performed.. There was a significant increase in vessel patency in rats treated preoperatively with desirudin compared to controls receiving saline (96.9% vs. 53.1%, P < .001). In evaluating patent vessels for non-occluding clot, 41.2% of control rats had non-obstructive clot at the site of anastomosis, versus 3.2% of rats treated with desirudin (P = .002). Bleeding times were longer in desirudin-treated rats than those that received saline (7.17 +/- 3 minutes vs. 5.15 +/- 1.2 minutes, P = .027).. The use of preoperative desirudin increases the rate of microvascular anastomotic patency, decreases the occurrence of non-occluding clot, and increases bleeding time in an in vivo rat model, indicating potential efficacy in patients undergoing microvascular free tissue transfer.

Topics: Anastomosis, Surgical; Animals; Disease Models, Animal; Fibrinolytic Agents; Graft Survival; Hirudins; Male; Microcirculation; Microsurgery; Preoperative Care; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Surgical Flaps; Thrombosis

There are continuing needs for new antithrombotic agents and procedures. We hypothesized that the slowly cleared recombinant fusion proteins barbourin--albumin (BLAH6) and hirudin--albumin (HLAH6) would be effective in limiting fibrin(ogen) and/or platelet deposition in a rabbit model of arterial injury.. Recombinant fusion proteins were expressed in Pichia pastoris fermenter cultures and purified by nickel-chelate affinity chromatography. They were injected intravenously into rabbits prior to blood sampling and platelet aggregometry, assessment of deposition of 125I-fibrin(ogen) and 51Cr-platelet onto the balloon-injured thoracic aorta, electron microscopy (EM) and immunohistochemistry of aortic sections, and determination of bleeding time following a standardized ear incision.. BLAH6 administration elicited a dose- and time-dependent inhibition of platelet aggregation in post-injection whole blood samples, and reduced both fibrin(ogen) and platelet deposition on the injured aorta, although the former effect was both more durable and more significant than the latter. In contrast, HLAH6 injection reduced fibrin(ogen) but not platelet deposition. Doses of the two proteins ineffective in preventing fibrin(ogen) deposition when given alone were effective when combined, suggesting at least additive effects. Immunohistochemistry and EM supported the radioactive deposition studies, while bleeding times were decreased with combined BLAH6 and HLAH6 administration compared to HLAH6 alone in a rabbit ear bleeding model. The data show that these fusion proteins exert an antithrombotic effect in vivo and may indicate that combined low-dose administration of antiplatelet and antithrombin agents could offer safety advantages in the treatment of thrombosis.

Topics: Albumins; Animals; Aorta; Blood Coagulation Tests; Catheterization; Crotalid Venoms; Disease Models, Animal; Drug Therapy, Combination; Fibrin; Fibrinolytic Agents; Hirudins; Immunohistochemistry; Microscopy, Electron; Platelet Adhesiveness; Rabbits; Recombinant Fusion Proteins; Thrombosis

Transient left-ventricular dysfunction because of myocardial reperfusion injury is a significant problem after cardiac surgery, but the underlying complex pathophysiology is still poorly understood. The authors studied early functional recovery of the postischemic myocardium and explored potential effects of thrombin inhibition on procoagulatory, proinflammatory, and proapoptotic features of myocardial ischemia-reperfusion injury.. A randomized, blinded study.. University research laboratory.. Porcine model.. Twenty pigs undergoing 60 minutes of aortic clamping and 75 minutes of normothermic cardiopulmonary bypass (CPB) received an intravenous bolus of r-hirudin (10 mg, 0.4mg/kg, n = 10) or placebo (n = 10) 15 minutes before aortic declamping, followed by a 135-minute intravenous infusion of r-hirudin (3.75 mg, 0.15 mg/kg/h) or placebo.. Hemodynamic parameters were measured before CPB, after weaning from CPB, and at 30, 60, 90, and 120 minutes after aortic declamping. Blood was sampled, and myocardial biopsies were taken before CPB, just before aortic declamping, during reperfusion, and after 120 minutes of reperfusion to measure thrombin antithrombin complexes and to quantitate leukocyte infiltration (myeloperoxidase activity) for histologic evaluation and detection of apoptosis with caspase-3 and the TUNEL method. The r-hirudin group showed significantly higher stroke volume and cardiac output than the control group at 60 minutes and at 90 minutes after aortic declamping (p < 0.05). Microthrombosis was not observed in either group, indicating sufficient anticoagulation and excluding intravascular clots as explanations for LV dysfunction in the current experiment. Instead, ample myocardial activation of inflammation was present, but only a trend of r-hirudin-associated anti-inflammatory effect was observed. Compared with the controls, TUNEL-positive myocytes were detected significantly less frequently in the r-hirudin group (0.05 +/- 0.06 v 0.13 +/- 0.07 TUNEL-positive nuclei %, p = 0.042).. The improved cardiac recovery in the r-hirudin group during reperfusion after cardioplegia-induced cardiac arrest was associated with significant differences in cardiomyocyte apoptosis and anti-inflammatory effects. Thus, in clinical cardiac surgery, inhibition of reperfusion- induced thrombin may offer beneficial effects by mechanisms other than direct anticoagulation.

Topics: Analysis of Variance; Animals; Apoptosis; Blood Pressure; Cardiac Output; Cardiopulmonary Bypass; Disease Models, Animal; DNA Fragmentation; Enzyme-Linked Immunosorbent Assay; Female; Fibrinolytic Agents; Heart Rate; Hirudins; In Situ Nick-End Labeling; Male; Myocardial Reperfusion Injury; Myocytes, Cardiac; Random Allocation; Swine; Thrombin; Time Factors; Ventricular Dysfunction, Left; Ventricular Pressure; Whole Blood Coagulation Time

Myocardial ischemia/reperfusion (I/R) injury is partly mediated by thrombin. In support, the functional inhibition of thrombin has been shown to decrease infarct size after I/R. Several cellular responses to thrombin are mediated by a G-protein coupled protease-activated receptor 1 (PAR1).However, the role of PAR1 in myocardial I/R injury has not been well characterized. Therefore, we hypothesized that PAR1 inhibition will reduce the amount of myocardial I/R injury. After we detected the presence of PAR1 mRNA and protein in the rat heart by RT-PCR and immunoblot analysis,we assessed the potential protective role of SCH 79797, a selective PAR1 antagonist, in two rat models of myocardial I/R injury. SCH 79797 treatment immediately before or during ischemia reduced myocardial necrosis following I/R in the intact rat heart. This response was dose-dependent with the optimal dose being 25 microg/kg IV. Likewise, SCH 79797 treatment before ischemia in the isolated heart model reduced infarct size and increased ventricular recovery following I/R in the isolated heart model with an optimal concentration of 1 microM. This reduction was abolished by a PAR1 selective agonist. SCH 79797-induced resistance to myocardial ischemia was abolished by wortmannin, an inhibitor of PI3 kinase; L-NMA, a NOS inhibitor; and glibenclamide, a nonselective K(ATP) channel blocker. PAR1 activating peptide,wortmannin, L-NMA and glibenclamide alone had no effect on functional recovery or infarct size. A single treatment of SCH 79797 administered prior to or during ischemia confers immediate cardioprotection suggesting a potential therapeutic role of PAR1 antagonist in the treatment of injury resulting from myocardial ischemia and reperfusion.

Topics: Androstadienes; Animals; Cardiotonic Agents; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glyburide; Hirudins; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Nitric Oxide; Nitric Oxide Synthase; Oligopeptides; omega-N-Methylarginine; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Potassium Channel Blockers; Potassium Channels; Proto-Oncogene Proteins c-akt; Pyrroles; Quinazolines; Rats; Rats, Sprague-Dawley; Receptor, PAR-1; Recombinant Proteins; Research Design; RNA, Messenger; Signal Transduction; Thrombin; Time Factors; Ventricular Function, Left; Wortmannin

Despite the fact that lytic therapy of thromboembolic disorder has been achieved, reocclusion of the damaged vessels and bleeding complication frequently reduce the therapeutic effect. In order to prevent the vessel reocclusion and enhance the therapeutic effect, combining the anticoagulant with the thrombolytic was assumed. Herein, we propose that restraining but locally releasing anticoagulant activity in the vicinity of thrombus is a way to alleviate the bleeding risk. A bifunctional fusion protein, termed as SFH (Staphylokinase (SAK) linked by FXa recognition peptide at N-terminus of Hirudin (HV)), was designed. SFH retained thrombolytic activity but no anticoagulant activity in thrombus-free blood due to the extension of the N-terminus of HV. However, it could locally liberate intact HV and exhibit anticoagulant activity when FXa or fresh thrombus was present. At equimolar dose, both improved antithrombotic and thrombolytic effects of SFH were observed in kappa-carrageenin inducing mouse-tail thrombosis model and rat inferior vena cava thrombosis model, respectively. Moreover, we observed significantly lower bleeding risk in mice and rats treated with SFH than with the mixture of SAK and HV with monitoring TT (P < 0.01), aPTT (P < 0.05) and PT (P < 0.05), and bleeding time (P < 0.05). In conclusion, SFH is a promising bifunctional therapeutic candidate with lower bleeding risk.

Topics: Animals; Anticoagulants; Blood Coagulation Tests; Disease Models, Animal; Fibrinolytic Agents; Hirudins; Male; Metalloendopeptidases; Mice; Rats; Recombinant Fusion Proteins; Recombinant Proteins; Thrombolytic Therapy; Venous Thrombosis

The recombinant protein SAK-RGD-K2-Hir is characterized by its fibrin-specific properties of plasminogen activation combined with antithrombin and antiplatelet activities. It was previously shown in our in-vitro studies to be a more potent and faster-acting thrombolytic agent compared with standard r-SAK. In order to document the effects of the thrombolytic potential of SAK-RGD-K2-Hir we examined this protein in an electrically induced carotid artery thrombosis model and stasis-induced venous model in rats. In the arterial thrombosis model, a bolus injection of SAK-RGD-K2-Hir was less effective than rt-PA and r-SAK. However, the most effective in the improvement and maintenance of carotid patency and in arterial thrombus mass reduction was SAK-RGD-K2. In contrast, all r-SAK derivatives reduced venous thrombus weight significantly in comparison to r-SAK and r-Hir. However, the most observable decrease in thrombus weight was obtained after application of recombinant proteins containing the r-Hir. The bleeding time was significantly prolonged in the animals treated with proteins containing r-Hir at a dose of 1.0 mg/kg. There were no observable changes in plasma fibrinogen concentration. In conclusion, our findings show thrombolytic activity in intravenous bolus injection of the novel thrombolytic agent SAK-RGD-K2-Hir in rats. Although this protein compares favourably with r-SAK in rat venous thrombolysis, we were unable to confirm the beneficial effects of SAK-RGD-K2-Hir over r-SAK and rt-PA in the carotid artery thrombolysis model. Furthermore, our results also suggest that SAK-RGD-K2-Hir bears a risk of bleeding, but this may be true for higher doses.

Topics: Animals; Bleeding Time; Blood Coagulation; Carotid Artery Thrombosis; Disease Models, Animal; Dose-Response Relationship, Drug; Electric Stimulation; Fibrin; Fibrinolytic Agents; Hemorrhage; Hirudins; Ligation; Male; Metalloendopeptidases; Partial Thromboplastin Time; Rats; Rats, Wistar; Recombinant Fusion Proteins; Thrombin Time; Time Factors; Tissue Plasminogen Activator; Vascular Patency; Venae Cavae; Venous Thrombosis

Fibrillar collagens are among the most potent activators of platelets and play an important role in the initiation of thrombosis. The glycoprotein VI (GPVI)/FcRgamma-chain complex is a central collagen receptor and inhibitors of GPVI produce a major defect in arterial thrombogenesis. In this study we have examined arterial thrombus formation in mice lacking the GPVI/FcRgamma-chain complex (FcRgamma(-/-)). Using 3 distinct arterial thrombosis models involving deep vascular injury, we demonstrate that deficiency of GPVI/FcRgamma is not associated with a major defect in arterial thrombus formation. In contrast, with milder vascular injury deficiency of GPVI/FcRgamma was associated with a 30% reduction in thrombus growth. Analysis of FcRgamma(-/-) platelets in vitro, using thrombin-dependent and -independent thrombosis models, demonstrated a major role for thrombin in overcoming the thrombosis defect associated with GPVI/FcRgamma deficiency. Inhibition of thrombin in vivo produced a much greater defect in thrombus formation in mice lacking GPVI/FcRgamma compared with normal controls. Similarly, thrombin inhibition produced a marked prolongation in bleeding time in FcRgamma(-/-) mice relative to wild-type mice. Our studies define an important role for thrombin in overcoming the hemostatic and thrombotic defect associated with GPVI/FcRgamma deficiency. Moreover, they raise the interesting possibility that the full antithrombotic potential of GPVI receptor antagonists may only be realized through the concurrent administration of anticoagulant agents.

Topics: Animals; Arterial Occlusive Diseases; Blood Platelets; Blood Vessels; Disease Models, Animal; Hirudins; Mice; Mice, Knockout; Platelet Activation; Platelet Membrane Glycoproteins; Receptors, IgG; Thrombin; Thrombosis

Thrombin plays a role in cerebral ischemia as rats subjected to focal cerebral ischemia were protected by the intracerebral injection of hirudin, a selective thrombin inhibitor. To separate the roles of thrombin in cell death and in coagulation, we have used an in vitro approach to test the effect of hirudin and of protease nexin-1 (PN-1), a cerebral thrombin inhibitor, on neuronal ischemia. Rat organotypic hippocampal slice cultures were subjected to oxygen (5%) and glucose (1 mmol/L) deprivation (OGD) during 30 min. Hirudin or PN-1 administered after OGD significantly prevented neuronal death in the CA1 region. After 24 h, there was a marked increase in thrombin immunoreactivity on Western blots. Thrombin therefore contributes to ischemic damage in neural tissue in vitro.

Topics: Animals; Animals, Newborn; Blotting, Western; Cell Death; Disease Models, Animal; Dose-Response Relationship, Drug; Gene Expression Regulation; Glucose; Hippocampus; Hirudin Therapy; Hirudins; Hypoxia; In Vitro Techniques; Ischemia; Neurons; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Thrombin

Thrombin is the most potent agonist of platelets and plays a critical role in the development of arterial thrombosis. Human platelets express dual thrombin receptors, protease-activated receptor (PAR) 1 and PAR4; however, there are no therapeutic strategies that effectively target both receptors.. Platelet aggregation studies demonstrated that PAR4 activity is markedly enhanced by thrombin-PAR1 interactions. A combination of bivalirudin (hirulog) plus a novel PAR4 pepducin antagonist, P4pal-i1, effectively inhibited aggregation of human platelets to even high concentrations of thrombin and prevented occlusion of carotid arteries in guinea pigs. Likewise, combined inhibition of PAR1 and PAR4 with small-molecule antagonists and pepducins was effective against carotid artery occlusion. Coimmunoprecipitation and fluorescence resonance energy transfer studies revealed that PAR1 and PAR4 associate as a heterodimeric complex in human platelets and fibroblasts. PAR1-PAR4 cofactoring was shown by acceleration of thrombin cleavage and signaling of PAR4 on coexpression with PAR1.. We show that PAR1 and PAR4 form a stable heterodimer that enables thrombin to act as a bivalent functional agonist. These studies suggest that targeting the PAR1-PAR4 complex may present a novel therapeutic opportunity to prevent arterial thrombosis.

Topics: Animals; Blood Platelets; Cell Line; Chemotaxis; Dimerization; Disease Models, Animal; Drug Therapy, Combination; Guinea Pigs; Hirudins; Humans; Peptide Fragments; Platelet Aggregation; Protein Binding; Receptor, PAR-1; Receptors, Thrombin; Recombinant Proteins; Thrombin; Thrombosis; Transfection

Emerging findings have demonstrated the critical role of blood clotting factors in the formation and stabilization of embryonic blood vessels. Whether a similar role is true during post-natal angiogenesis remains to be determined. Here we sought to determine whether the suppression of thrombin generation with anticoagulant drugs at doses routinely used for therapeutic purposes would affect the angiogenesis pattern following hindlimb ischemia in rats. Animals were treated with r-hirudin or enoxaparin within six hours post induction of hindlimb ischemia, whereas two other groups received oral anticoagulation warfarin beginning at day 3 post-ischemia or saline (as control). The revascularization anatomical and functional responses were evaluated 30 days following tissue ischemia. Chronic administration of the drugs resulted in stable anticoagulation in all animals throughout the experiment. Animals that received drugs with fast anticoagulation effects (i.e. r-hirudin and enoxaparin) presented a significant decrease in capillary density and capillary-to-myocyte ratio compared to control animals. These effects were not associated with changes in relative perfusion of the hindlimb at steady state. These anti-angiogenic effects occur in a time-dependent manner, since delayed inhibition of coagulation (>72 hours) presents no adverse effect on the angiogenic response. We conclude that the use of anticoagulant drugs immediately after tissue ischemia induction hampers in vivo angiogenic response in a rodent hindlimb ischemia model.

Topics: Animals; Anticoagulants; Blood Coagulation Factors; Disease Models, Animal; Enoxaparin; Hindlimb; Hirudins; Ischemia; Kinetics; Neovascularization, Physiologic; Rats; Rats, Inbred Lew; Time Factors; Warfarin

A recently published post-hoc analysis of a trial using high-dose antithrombin (AT) in septic patients (KyberSept) revealed significant reduction of lethality when no concomitant heparin was administered, whereas patients with the combination of heparin and AT did not benefit in terms of survival. Therefore, it seems feasible to study the capability of AT in prevention of microvascular thrombus formation to avoid concomitant application of heparin and AT. Using fluorescence microscopy and a light/dye-injury mouse ear model, the kinetics of thrombus formation were analyzed quantitatively in vivo upon single iv bolus of saline (control), heparin (100 IU/kg), hirudin (1 mg/kg) or AT (25, 50, 100 or 250 IU/kg) (N = 7 animals per group each). In controls, light/dye-injury induced complete thrombotic occlusion in all arterioles and venules studied. Heparin and hirudin prevented thrombotic vessel occlusion in 62% and 43% of arterioles and 11% and 28% of venules. AT-250 was found to be more effective than heparin and hirudin, because thrombus formation was completely banned in all arterioles and venules. AT-100 and AT-50 were also capable of significantly blocking thrombus formation in both arterioles and venules. In blood vessels, which finally clogged, the time for development of complete vessel occlusion was delayed after heparin, hirudin and AT-25, but in particular after AT-50 and AT-100. In conclusion, AT-mediated antithrombotic activity has been characterized in a model of phototoxicity-induced microvascular thrombosis formation, demonstrating that AT delays and prevents thrombus formation in arterioles and venules at least comparably effective as heparin and hirudin.

Topics: Animals; Antithrombins; Disease Models, Animal; Female; Fibrinolytic Agents; Heparin; Hirudins; Homozygote; Kinetics; Male; Mice; Microcirculation; Microscopy, Fluorescence; Thrombosis; Treatment Outcome

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.

Topics: Ancrod; Animals; Anticoagulants; Antithrombin III; Biomarkers; Blood Coagulation; Disease Models, Animal; Down-Regulation; Early Growth Response Protein 1; Endotoxemia; Factor VII Deficiency; Factor Xa; Fibrinogen; Fondaparinux; Hirudins; Lipopolysaccharides; Male; Mice; Mice, Inbred C57BL; Neutrophils; Peptide Hydrolases; Polysaccharides; Recombinant Proteins; Signal Transduction; Thrombin

Little is known with regard to efficacy of heparin as an adjunct to fibrinolytics under conditions of severe vascular damage. In this study, we compared the effects of unfractionated heparin (UH), low-molecular weight heparin (LMWH), and recombinant desulfohirudin (HIR) in combination with streptokinase (SK) in such settings.. We used an established rabbit model, in which thrombosis, critical stenosis, and vascular wall damage were introduced to a segment of the abdominal aorta and the effects of the respective therapies were assessed by time to patency (TTP in minutes), cumulative patency (CP (%)), lysis of original clot (CL (%)), and net clot accretion (NCA (%)). Treatments were administered over 90 min at the following doses: SK: 33,000 U/kg, UH: 125-250 U/kg, LMWH: 1.25-2.5 mg/kg, HIR: 0.25-0.55 mg/kg.. Unexpectedly, UH and LMWH had a paradoxical and detrimental effect on SK-mediated recanalization as measured by both TTP and CP. Thus, administration of SK vs. SK+UH or SK+LMWH resulted in TTP values of 43+/-8 min vs. 70+/-11 min (p<0.05) and 67+/-12 min. (p<0.08), respectively. For CP, the corresponding values were 21+/-7%, 0.5+/-0.3% and 9+/-8%. This delay in vessel recanalization occurred despite excessive systemic anticoagulation (activated partial thromboplastin time (aPTT) and thrombin clotting time (TCT) ratios >6 and >34, respectively). Of interest, both heparinoids completely inhibited SK-induced fibrinogen consumption (FC). In contrast, recombinant desulfohirudin (HIR) shortened SK-induced TTP (4.97+/-0.81 min) without preserving fibrinogen.. Our findings suggest that caution needs to be exercised, when using the combination of SK and heparinoids for the treatment of arterial thrombosis under conditions of severe vascular damage and stenosis.

Topics: Animals; Aorta, Abdominal; Aortic Valve Stenosis; Disease Models, Animal; Drug Antagonism; Drug Therapy, Combination; Fibrinolysis; Heparin; Heparin, Low-Molecular-Weight; Hirudins; Male; Rabbits; Recombinant Proteins; Streptokinase; Thrombosis; Time Factors; Vascular Patency

Recently, it has been reported that both thrombin-sensitive protease-activated receptor 1 (PAR-1) and platelet-derived growth factor (PDGF) are present not only in platelets, but also in the CNS, which indicates that they have various physiological functions. In this study, we evaluated whether PAR-1/PDGF in the spinal cord could contribute to the development of a neuropathic pain-like state in mice. Thermal hyperalgesia and tactile allodynia induced by sciatic nerve ligation were significantly suppressed by repeated intrathecal injection of hirudin, which is characterized as a specific and potent thrombin inhibitor. Furthermore, a single intrathecal injection of thrombin produced long-lasting hyperalgesia and allodynia, and these effects were also inhibited by hirudin in normal mice. In nerveligated mice, the increase in the binding of [35S]GTPgammaS to membranes of the spinal cord induced by thrombin and PAR-1-like immunoreactivity (IR) in the spinal cord were each greater than those in sham-operated mice. Thermal hyperalgesia and tactile allodynia induced by sciatic nerve ligation were also suppressed by repeated intrathecal injection of either the PDGF alpha receptor (PDGFRalpha)/Fc chimera protein or the PDGFR-dependent tyrosine kinase inhibitor AG17 [(3,5-di-tert-butyl-4-hydroxybenzylidene)-malononitrile]. Moreover, thermal hyperalgesia and tactile allodynia induced by thrombin in normal mice were virtually eliminated by intrathecal pretreatment with PDGFRalpha/Fc. In immunohistochemical studies, PAR-1-like IR-positive cells in the spinal dorsal horn were mostly colocated on PDGF-like IR-positive neuronal cells. These data provide novel evidence that PAR-1 and PDGF-A-mediated signaling pathway within spinal cord neurons may be directly implicated in neuropathic pain after nerve injury in mice.

Topics: Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Functional Laterality; Hirudin Therapy; Hirudins; Immunohistochemistry; Male; Mice; Mitogen-Activated Protein Kinase Kinases; Neuralgia; Neurons; Pain Measurement; Physical Stimulation; Platelet-Derived Growth Factor; Receptor, PAR-1; Sciatic Neuropathy; Spinal Cord; Thrombin; Time Factors

Myointimal hyperplasia is the condition usually responsible for recurrent stenosis (restenosis) after endarterectomy, bypass grafting and angioplasty. Its cause is still not known. The present study examined whether inhibition of thrombin by tissue plasminogen activator (r-TPA) or polyethylene glycol recombinant hirudin (PEG-hirudin) could reduce restenosis in an animal model. Restenosis was induced in 20 cholesterol-fed rabbits. The right carotid artery underwent a double-balloon injury while left carotid artery acted as a control. Recombinant tissue plasminogen activator (1 mg kg(-1) s.c.) and PEG-hirudin (0.7 mg kg(-1) s.c.) were given subcutaneously with normal saline acting as a control. Blood levels of PEG-hirudin were measured by both ELISA and an Ecarin (activity) assay. Vessel dimensions were measured in histological sections, obtained from perfusion-fixed tissue, using computerised planimetry. The model reproduced many of the histological changes found in human restenosis, such as intramural thrombus, rupture of the elastic lamina, macrophage infiltration and smooth muscle migration. Reinjury caused an almost three-fold reduction in the area of the lumen (median 0.25 mm(2)) compared with uninjured vessels (median 0.72 mm(2)). The mean plasma levels of PEG-hirudin and r-tPA achieved were 291 ng/ml (S.E.M. 28 ng/ml) and 34 IU/ml (S.E.M. 12 IU/ml), respectively. PEG-hirudin significantly inhibited the effect of balloon injury on luminal area compared with saline-treated controls (0.21 versus 0.44 mm(2), respectively, P<0.05). Recombinant tPA also had a similar inhibitory affect, but this did not reach statistical significance (0.16 versus 0.44 mm(2), respectively, P>0.05). The magnitude of luminal narrowing was significantly reduced by subcutaneous injection of PEG-hirudin. Further studies are required to determine whether this effect can be enhanced by other antithrombins or improved methods of delivery.

Topics: Animals; Anticoagulants; Carotid Arteries; Carotid Artery Diseases; Catheterization; Constriction, Pathologic; Disease Models, Animal; Hirudins; Rabbits; Recombinant Proteins; Recurrence; Tissue Plasminogen Activator

We have generated transgenic mice expressing the leech anticoagulant hirudin and human tissue factor pathway inhibitor tethered to the cell surface by fusion with fragments of human CD4 and P-selectin. Expression of the transgenes is under the control of the CD31 (platelet endothelial cell adhesion molecule [PECAM]) promoter, limiting expression to endothelial cells, monocytes, and platelets. In addition, the P-selectin sequence directs expression to secretory granules. Functional cell surface expression only occurs when the cells are activated. In a mouse model of systemic lipopolysaccharide (LPS)-induced endotoxemia, we show that expression of either anticoagulant on activated endothelium inhibits the widespread intravascular thrombosis, thrombocytopenia, and consumptive coagulopathy associated with endotoxemia. Importantly, non- LPS-treated transgenic mice had normal baseline bleeding times. We speculate that targeted delivery of anticoagulants to the endothelium may be a strategy worth pursuing in clinical sepsis to improve efficacy of systemic anticoagulation while minimizing potential hemorrhagic side effects.

Topics: Animals; Bone Marrow; Disease Models, Animal; Endotoxemia; Genetic Therapy; Hirudins; Humans; Leeches; Lipoproteins; Mice; Mice, Transgenic; Recombinant Fusion Proteins; Thrombosis

Many functions of the coagulation system have nonthrombotic effects. The indirect thrombin inhibitor heparin has been previously shown to be effective in limiting intimal hyperplasia (IH). We sought to study the effect of thrombin on IH by using two direct thrombin inhibitors (DTIs), argatroban and lepirudin. Sprague-Dawley rats underwent interposition vein grafting to the carotid artery. Vein grafts were treated with either saline (n = 6) or one of the two DTIs (n = 6 for both). At 30 days, the rats were sacrificed and vessels were perfusion fixed. Sections of the proximal carotid artery, graft, and both anastomoses were stained with both hematoxlyin/eosin and von Gieson's elastin stain. Sections were examined and compared for luminal area and intima-to-media (IM) ratio. The vessels treated with DTIs had less (p < 0.05) IH (IM ratio for proximal anastomosis: control 1.036 +/- 0.857, lepirudin 0.373 +/- 0.21, argatroban 0.182 +/- 0.118) and better lumen preservation than the control vessels (lumen area of proximal anastomosis: control 1.69 +/- 0.9, lepirudin 2.45 +/- 0.74, argatroban 2.81 +/- 0.78). There were no thromboses in the DTI-treated vessels. Dilatation of the graft segment was noted in the argatroban group. Thus, DTIs are effective at reducing IH in a small-animal model, suggesting that inhibition of thrombin has a protective role in IH. In addition, a difference of action between DTIs is suggested by the dilatation seen only in the argatroban-treated graft sections.

Topics: Anastomosis, Surgical; Animals; Carotid Artery, Common; Disease Models, Animal; Fibrinolytic Agents; Hirudins; Hyperplasia; Male; Models, Cardiovascular; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Thrombin; Thrombosis; Tunica Intima; Veins

Topics: Animals; Clinical Trials as Topic; Disease Models, Animal; Endothelium, Vascular; Escherichia coli; Hirudins; Humans; Recombinant Proteins; Shiga Toxin; Thrombin

Cardiopulmonary bypass and surgical stress are accompanied by a systemic inflammatory response and activation of coagulation. Thrombin forms fibrin and activates platelets and neutrophils. Consequently, disseminated microthrombosis might increase capillary vascular resistance and thus impair reperfusion. We hypothesized that recombinant hirudin, a direct inhibitor of thrombin, could attenuate coagulation and enhance microvascular flow during reperfusion.. Twenty pigs undergoing 60 minutes of aortic clamping and 75 minutes of normothermic perfusion were randomized in a blinded setting to receive an intravenous bolus of recombinant hirudin (10 mg, 0.4 mg/kg; n = 10) or placebo (n = 10) 15 minutes before aortic declamping and then continued with an intravenous 135-minute infusion of recombinant hirudin (3.75 mg/h, 0.15 mg/kg) or placebo. Thrombin-antithrombin complexes, activated clotting times, and several hemodynamic parameters were measured before cardiopulmonary bypass, after weaning from cardiopulmonary bypass, and at 30, 60, 90, and 120 minutes after aortic declamping. Intramucosal pH and Pco(2) were measured from the luminal surface of ileum simultaneously with arterial gas analysis at 30-minute intervals.. Recombinant hirudin inhibited thrombin formation after aortic declamping; at 120 minutes, thrombin-antithrombin complexes levels (microg/L, mean +/- SD) were 75 +/- 21 and 29 +/- 44 (P <.001) for placebo and pigs receiving recombinant hirudin, respectively. When compared with the placebo group, pigs receiving recombinant hirudin showed significantly higher stroke volume, cardiac output, and lower systemic vascular resistance at 60 and 90 minutes after aortic declamping (P <.05). Based on arteriomucosal Pco(2) and pH differences, progressive worsening of intestinal microcirculatory perfusion occurred in the placebo group but not in the recombinant hirudin group.. Infusion of thrombin inhibitor recombinant hirudin during reperfusion was associated with attenuated postischemia left ventricular dysfunction and decreased vascular resistance. Consequently microvascular flow was improved during ischemia-reperfusion injury. Control of thrombin formation during reperfusion may be a feasible approach to improve oxygen delivery to reperfused vascular beds.

Topics: Animals; Anticoagulants; Blood Gas Analysis; Cardiopulmonary Bypass; Disease Models, Animal; Female; Hemodynamics; Hirudins; Intestines; Male; Manometry; Postoperative Complications; Random Allocation; Recombinant Proteins; Swine; Vascular Resistance

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

We previously reported that plasminogen activator inhibitor 1 (PAI-1), in the presence of vitronectin (VN), inhibits thrombin activity in vitro. Furthermore, we demonstrated in human atherosclerotic plaques the colocalization of thrombin, PAI-1, and VN, as well as activity of thrombin and PAI-1. Here, we show that PAI-1 is a local thrombin inhibitor in vivo.. We used the murine carotid artery ligation model to assess the role of PAI-1 and VN in stenosis by using PAI-1-deficient (PAI-1(-/-)) and VN(-/-) mice. Ligation resulted in a smooth muscle cell (SMC)-rich intima without infiltrating cells. We show that PAI-1(-/-) and VN(-/-) mice generate a larger intima than wild-type mice as the result of more extensive SMC proliferation, as evidenced by cell counting and staining for proliferating cell-nuclear antigen.. In PAI-1(-/-) mice, excessive intima formation is prevented by the thrombin-specific inhibitor hirudin. Finally, immunohistochemical analysis revealed PAI-1, VN, and (pro)thrombin antigen in intimal lesions. Our observations are compatible with inhibition of thrombin-mediated SMC proliferation by PAI-1/VN complexes.

Topics: Animals; Carotid Arteries; Carotid Stenosis; Cell Division; Connective Tissue; Disease Models, Animal; Hirudins; Immunohistochemistry; Ligation; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Smooth, Vascular; Plasminogen Activator Inhibitor 1; Prothrombin; Tunica Intima; Tunica Media; Vitronectin

The occurrence of peritoneal adhesions in surgical patients is positively correlated with tumor necrosis factor (TNF) levels. In a model of septic peritonitis-cecal ligation and puncture-TNF neutralization prevented formation of peritoneal adhesions and increased mortality, most likely because localization of the septic focus was prevented. To discriminate between the coagulation-independent protective TNF effect and a potential protective procoagulant TNF effect, formation of peritoneal adhesions after CLP was inhibited with heparin, hirudin, or urokinase. Each treatment increased mortality and increased the number of bacteria in the peritoneal lavage fluid, kidney, and liver to various degrees. Under these experimental conditions, antibiotics prevented death. In coagulation-compromised mice, lethality was further enhanced by additional TNF neutralization. These findings demonstrate that peritoneal adhesions early in septic peritonitis are an important mechanism of innate immunity that prevents increased spread of bacteria and reduces mortality.

Topics: Animals; Cecum; Colony Count, Microbial; Disease Models, Animal; Heparin; Hirudins; Immunity, Innate; Ligation; Male; Mice; Peritonitis; Sepsis; Tissue Adhesions; Tumor Necrosis Factor-alpha; Urokinase-Type Plasminogen Activator

In an effort to reduce the risks of a possible iatrogenic transmission of bovine spongiform encephalitis (BSE) through the use of bovine-derived medicinal products, we patented in the USA in 1999 a polysaccharide from brown algae, endowed with interesting pharmacological activities: (a) concentration-dependent inhibition of thromboplastin or cephalin-kaolin-induced thrombin generation from platelets, (b) concentration-dependent inhibition of thrombin-induced platelet aggregation, (c) thrombin has hypotensive effect, which was blunted and zeroed by our fucansulfate in a dose-dependent way, (d) when aortae are stimulated with thrombin, they become stickier for polymorphonucleated leukocytes (PMNs); our fucansulfate decreased concentration-dependently, PMNs sticking to autologous rabbit aortae, (e) dose-dependent inhibition of thrombin-induced thrombosis. All the above data suggest that our fucansulfate could be a heparin substitute endowed with antithrombotic and anti-inflammatory activities, devoid or the problems caused to heparin by its animal origin, i.e., possible prion protein contamination.

Topics: Animals; Antithrombins; Aorta; Cell Adhesion; Disease Models, Animal; Dose-Response Relationship, Drug; Fibrinolytic Agents; Heparin; Hirudins; Male; Neutrophils; Phaeophyceae; Platelet Aggregation; Polysaccharides; Rabbits; Thrombin; Thrombosis

The clinical complications of Extracorporeal Circulation (ECC) have been linked to disturbances in the microcirculation. In order to prevent these deleterious effects, a biodegradeable agent to coat the extracorporeal circuit was tested.. Intravital fluorescence microscopy was used on the hamster skinfold chamber model in permanently instrumented, awake animals. ECC was introduced via a micro-roller-pump and a silicon tube shunted between the carotid artery and the jugular vein. The ECC-tube system was coated with PEG-Hirudin-Iloprost, two additional groups received either Iloprost i.v. (0.8 mg/kg/h) or Hirudin i.v. (1 mg/kg b.w.).. ECC for 20 minutes resulted in an increase in rolling and adherent leukocytes in postcapillary venules (Roller 9 to 36 [%]; Sticker 24 to 330 [n/mm2]). Use of the coated tube system reduced L/E cell interaction (Roller 9 to 24* [%], Sticker 28 to 194* [n/mm2]; *p<0.05), whereas Hirudin i.v. nearly abolished it.. The protective effects of the coating and of Hirudin i.v are probably a result of an attenuated activation of the coagulation-fibrinolytic system.

Topics: Animals; Antithrombins; Arterioles; Cell Adhesion; Cell Communication; Circadian Rhythm; Cricetinae; Disease Models, Animal; Electrocardiography; Endothelium, Vascular; Extracorporeal Circulation; Hemodynamics; Hirudins; Leukocytes; Mesocricetus; Microcirculation; Muscle, Smooth, Vascular; Time Factors; Venules

The development of new antithrombotic drugs to inhibit the blood coagulation system and to block receptors of platelets represents an important area of medical research. The results of the efficacy in animal models are of critical importance for the further development of these compounds for human use. The transferability of such data from animal to human species still remains controversial. The antithrombotic effects of direct thrombin inhibitors and a glycoprotein IIb/IIIa (GPIIb/IIIa) receptor antagonist were investigated in an experimental thrombosis model using human blood in order to analyze the feasibility of such a model.

Topics: Abciximab; Animals; Antibodies, Monoclonal; Antithrombin III; Antithrombins; Blood Coagulation Tests; Disease Models, Animal; Dose-Response Relationship, Drug; Fibrinolytic Agents; Heparin; Heparin, Low-Molecular-Weight; Heparinoids; Hirudins; Humans; Immunoglobulin Fab Fragments; Peptide Hydrolases; Platelet Aggregation Inhibitors; Platelet Factor 4; Platelet Glycoprotein GPIIb-IIIa Complex; Reproducibility of Results; Thrombin; Thrombosis

Recombinant hirudin, a potent and direct inhibitor of thrombin, effectively inhibits platelet-dependent thrombosis. Our aim was to establish the plasma concentration at which r-hirudin expresses its optimal antithrombotic effect. We measured the extent of inhibition of (111)In-labeled platelet deposition onto 0.6 cm(2) segments of Dacron vascular grafts. These grafts were incorporated as extension segments into exteriorized permanent femoral arteriovenous shunts in baboons. In six control studies a mean of 1.99 +/- 0.26 x 10(9) platelets were deposited at the end of 120 min. In the treatment studies, a thrombus was allowed to form for 10 min in six animals. Treatment for 30 min with r-hirudin at dosages of 140, 70, and 35 microgram/kg/min, but not 14 microgram/kg/min, dose dependently interrupted platelet deposition. The relationship between the percent inhibition of platelet deposition caused by r-hirudin and the plasma concentration of hirudin was exponential (i.e., % Inhibition = 95(1-e(0.23 x [r-hirudin])) (R(2) = 0.76). From this, we estimated that 50% inhibition of platelet deposition will occur at a plasma concentration of approximately 3.3 microgram r-hirudin/mL and 80% at 8.1 microgram/mL. The relationship between the inhibition of platelet deposition and the plasma concentration of hirudin makes it possible to estimate the dose of hirudin that will result in a given level of inhibition of platelet deposition.

Topics: Animals; Antithrombins; Blood Platelets; Disease Models, Animal; Half-Life; Hirudin Therapy; Hirudins; Male; Papio; Recombinant Proteins; Thrombosis

Detailed studies of tumor cell-associated procoagulants and fibrinolytic factors have implied that local thrombin generation and fibrin deposition and dissolution may be important in tumor growth and dissemination. To directly determine whether fibrin(ogen) or plasmin(ogen) are determinants of the metastatic potential of circulating tumor cells, this study examined the impact of genetic deficits in each of these key hemostatic factors on the hematogenous pulmonary metastasis of 2 established murine tumors, Lewis lung carcinoma and the B16-BL6 melanoma. In both tumor models, fibrinogen deficiency strongly diminished, but did not prevent, the development of lung metastasis. The quantitative reduction in metastasis in fibrinogen-deficient mice was not due to any appreciable difference in tumor stroma formation or tumor growth. Rather, tumor cell fate studies indicated an important role for fibrin(ogen) in sustained adhesion and survival of tumor cells within the lung. The specific thrombin inhibitor, hirudin, further diminished the metastatic potential of circulating tumor cells in fibrinogen-deficient mice, although the inhibitor had no apparent effect on tumor cell proliferation in vitro. The absence of plasminogen and plasmin-mediated fibrinolysis had no significant impact on hematogenous metastasis. The authors concluded that fibrin(ogen) is a critical determinant of the metastatic potential of circulating tumor cells. Furthermore, thrombin appears to facilitate tumor dissemination through at least one fibrin(ogen)-independent mechanism. These findings suggest that therapeutic strategies focusing on multiple distinct hemostatic factors might be beneficial in the containment of tumor metastasis.

Topics: Animals; Carcinoma, Lewis Lung; Cell Adhesion; Disease Models, Animal; Fibrinogen; Fibrinolysin; Fibrinolysis; Fibrinolytic Agents; Hemostasis; Hirudins; Histocytochemistry; Lung Neoplasms; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasm Metastasis; Neoplastic Cells, Circulating; Neovascularization, Pathologic; Thrombin

Current antithrombotic compounds have several limitations in clinical practice. The present study was designed to investigate a novel orally available direct thrombin inhibitor, BSF 208791. Intravenous administration of BSF 208791 showed superior antithrombotic properties as compared with Polyethylenglycol-Hirudin (PEG-Hirudin) and low molecular weight heparin (LMWH) in a model of venous thrombosis in rabbits. The thrombus growth was 22%, 30%, 37% and 50% after BSF 208791, PEG-Hirudin. LMWH, and saline administration, respectively. Moreover, bleeding time was less affected after administration of BSF 208791 as compared with PEG-Hirudin. The oral administration of BSF 208791 resulted in adequate bioavailability and significantly reduced venous thrombus growth to 36% as compared with 60% in the saline treated rabbits. The antithrombotic effect of BSF 208791 appears to be superior to PEG-Hiridin and LMWH without affecting the bleeding time. BSF 208791 is an orally available agent that might be a promising candidate for future antithrombotic therapy.

Topics: Administration, Oral; Animals; Anticoagulants; Antithrombins; Disease Models, Animal; Heparin, Low-Molecular-Weight; Hirudins; Oligopeptides; Rabbits; Thrombosis

Topics: Animals; Clopidogrel; Disease Models, Animal; Female; Fibrinolytic Agents; Heparin; Hirudins; Rats; Rats, Wistar; Thrombosis; Ticlopidine

CX-397, a recombinant hirudin analog, is a potent and specific inhibitor of human alpha-thrombin. We conducted a comparative study of CX-397 and heparin in a canine model of left circumflex (LCX) coronary artery thrombosis to evaluate the anti-thrombotic efficacy of CX-397. Administration of drugs (i.v.; bolus + infusion) was commenced 10 min prior to the initiation of LCX coronary artery electrolytic injury (100 microA for 300 min). All saline-treated control animals (7/7) developed thrombotic occlusion during the experimental period, leaving a residual thrombus mass of 15.4 +/- 3.8 mg. Treatment with CX-397 at three incremental dose levels reduced the incidence of occlusion (4/7, 2/5 and 0/7) and decreased thrombus weight (12.6 +/- 2.5 mg, 6.3 +/- 3.0 mg and 2.1 +/- 1.3 mg, respectively) in a dose-dependent manner. At the intermediate dose (15,000 ATU/kg + 15,000 ATU/kg/h) or higher, CX-397 showed significant anti-thrombotic effects (p <0.05 and p <0.01) and suppressed increases in thrombin-antithrombin III complex (TAT) levels (p <0.01 and p <0.001). In the heparin (80 U/kg + 60 U/kg/h)-treated group, the incidence of occlusion (5/7) and thrombus weight (14.1 +/- 6.2 mg) did not differ significantly from the control group. Plasma TAT levels in the heparin group decreased compared with the control group (p <0.01), but was less potent than the intermediate dose CX-397 (p <0.01). The anti-coagulation (activated partial thromboplastin time and activated clotting time) and template bleeding time prolongation effects of heparin were more potent than those of the intermediate dose CX-397 which showed significant anti-thrombotic effects. In conclusion, CX-397 dose-dependently suppressed thrombus formation by inhibition of thrombin activity in a canine coronary artery injury model. The anti-thrombotic efficacy of CX-397 was more potent than that of heparin at equivalent anti-coagulation dosage.

Topics: Animals; Anticoagulants; Blood Coagulation; Coronary Thrombosis; Disease Models, Animal; Dogs; Heparin; Hirudins; Humans; Infusions, Intravenous; Injections, Intravenous; Recombinant Proteins

The importance of thrombin in arterial and venous thrombosis renders thrombin inhibition an important therapeutic target. Identification of novel inhibitors requires an appropriate animal model. We modified a previously reported rat arterial thrombosis model to allow simultaneous assessment of the arterial and venous antithrombotic efficacies of heparin, hirudin, hirulog, a novel thrombin inhibitor H-(N-Me-D-Phe)-Pro-L-trans-4-aminocyclohexyl-Gly-[CO-CO]-NHCH3+ ++ (L-370,518) and the factor Xa inhibitor tick anticoagulant peptide in rabbits. Thrombosis was induced through application of 70% ferric chloride to the femoral artery and jugular vein. Incidence of occlusion, thrombus weight, aPTT and plasma inhibitor concentrations were determined. Heparin was efficacious in preventing arterial and venous occlusive thrombosis but at a dose that profoundly elevated aPTT. On a molar dosing basis, the approximate order of potency of the thrombin and factor Xa inhibitors was similar in artery and vein: hirudin>tick anticoagulant peptide>hirulog> or =L-370,518. Data suggested that compounds tended to be more potent in preventing venous thrombosis than arterial. This thrombin-dependent model is an economical and efficient approach to arterial and venous antithrombotic efficacy screening that eliminates variabilities encountered when multiple model/multiple animal strategies are employed.

Topics: Animals; Antithrombins; Arteries; Arthropod Proteins; Disease Models, Animal; Hirudins; Intercellular Signaling Peptides and Proteins; Peptide Fragments; Peptides; Rabbits; Rats; Recombinant Proteins; Thrombin; Thrombosis; Veins

Current therapeutic use of heparin as an adjunct to thrombolytic therapy for myocardial infarction is suboptimal with respect to efficacy and bleeding risk. In a rat carotid arterial thrombolysis model (FeCl3-induced injury) we evaluated the combined effect of tPA (2.0 mg/kg/30 min) with our potent injectable direct thrombin inhibitor, BCH-2763 (Ki 0.11 nM; MW 1.5 kDa), which, unlike heparin, inhibits bound and free thrombin; comparisons were with standard heparin (SH), other direct thrombin inhibitors, r-hirudin (MW 6.5 kDa) and hirulog (MW 2.3 kDa), or tPA alone. Time to lysis (TL), patency time (PT), aPTT (fold increase) and bleeding time (BT) were determined. ED100 (100% of rats reperfused) for BCH-2763, hirulog or r-hirudin was 1, 3 or 2 mg/kg/60 min, respectively; 67% of rats reperfused with SH at the highest dose tested (220 U/kg/60 min) and 43% with tPA alone. At these doses, TL (min) was shorter (p < 0.01) with BCH-2763 (0.5 +/- 0.1), hirulog (3.3 +/- 2.3) or r-hirudin (2.3 +/- 1.0) than SH (66.3 +/- 30.8) or tPA alone (93.4 +/- 21.4). The aPTT fold increase after 15 min infusion was markedly greater (p < 0.001) for SH (32.0 +/- 0.8) than BCH-2763 (3.7 +/- 0.5), hirulog (5.2 +/- 0.3) or r-hirudin (4.5 +/- 0.8) in combination with tPA or tPA alone (1.1 +/- 0.1). In addition, the BT (min) for BCH-2763 (3.0 +/- 0.4) was similar to tPA alone (1.6 +/- 0.3), but prolonged (p < 0.05) for hirulog (7.5 +/- 2.7), r-hirudin (6.6 +/- 0.8) or SH (7.3 +/- 1.8). Comparisons at same aPTT fold increase revealed that in combination with tPA, BCH-2763 required a lower anticoagulant level to shorten the TL and prolong the PT than hirulog, r-hirudin or SH. Thus, in this rat arterial thrombolysis model direct thrombin inhibitors are more effective than SH as antithrombotic adjuncts to tPA. BCH-2763 is effective at a lower gravimetric dose and more modest aPTT fold increase than hirulog or r-hirudin with less alteration in haemostasis, which may confer an improved safety index.

Topics: Amino Acid Sequence; Animals; Anticoagulants; Carotid Arteries; Disease Models, Animal; Fibrinolysis; Fibrinolytic Agents; Heparin; Hirudin Therapy; Hirudins; Injections; Male; Molecular Sequence Data; Oligopeptides; Peptide Fragments; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Thrombin; Tissue Plasminogen Activator

The endothelial vasoconstrictor endothelin (ET) can induce acute renal failure when fibrinolysis and vasodilatory prostanoids (PGs) are inhibited. This study compares therapeutic agents preventing ET-induced acute renal failure in anesthetized female pigs. We investigated the effect of four ET boli (1.5 microg/kg, i.v.) after pretreatment with indomethacin (2 mg/kg) and epsilon-aminocaproicacid (100 + 50 mg/kg) alone (controls, group 1) or during additional nifedipine (10 microg/kg/h; group 2), hirudin (0.5 mg/kg; group 3), or enalapril (2 x 0.15 mg; group 4) on coagulation, PGs, and renal function. The ET-induced blood pressure increase was lower in groups 2 to 4 (lowest in group 3, P < 0.05). PG synthesis was blocked in all groups. The initial hypercoagulability (controls) resulted in disseminated intravascular coagulation that was prevented by hirudin and was attenuated in groups 2 and 4. At the end of the experiment, creatinine clearance was significantly (P < 0.05) decreased. The recovery of renal function two hours after the last ET bolus was most pronounced in the hirudin group. All therapeutic drugs attenuated ET-induced impairment of renal function. Hirudin seems to be the most potent protective drug. Prevention of further ET release evoked by ET-mediated secretion of thrombin might explain this. These results suggest three important pathways for ET's hemodynamic and renal effects.

Topics: Acute Kidney Injury; Angiotensin-Converting Enzyme Inhibitors; Animals; Anti-Inflammatory Agents, Non-Steroidal; Antithrombins; Blood Pressure; Calcium Channel Blockers; Creatinine; Disease Models, Animal; Enalapril; Endothelins; Female; Hirudins; Indomethacin; Nifedipine; Renal Circulation; Swine; Vasoconstrictor Agents

Systemic anticoagulation is required during cardiopulmonary bypass (CPB) to inhibit the activation of platelets, the coagulation system and ultimately thrombus formation. Unfractionated heparin is most commonly used, but it is neither entirely safe nor completely effective. The use of protamine sulphate to reverse the anticoagulant effect further complicates the use of heparin. The clinical need for a heparin substitute is therefore obvious. We evaluated the efficacy of r-Hirudin, a potent and specific inhibitor of thrombin, as anticoagulant in a baboon model of cardiopulmonary bypass.. Ten baboons, divided into two groups of five each, were used. The one group received 0.7 mg/kg r-Hirudin as a bolus before CPB was started, followed by a constant infusion of 1.4 mg/kg/hr for the 90 min of CPB. The other group received a bolus of 2.5 mg/kg heparin before the start of CPB, followed by maintenance dosages to maintain the activated clotting time (ACT) >400 sec.. Adequate anticoagulation was obtained with both anticoagulants. Haemodilution due to priming the extracorporeal system with Ringer's lactate and appropriately anticoagulated donor blood, was equivalent in both groups. During CPB with heparin, but not with hirudin, there was a significant increase in the number of circulating platelet aggregates, thrombin-antithrombin (TAT) complexes and 111In-labelled platelet accumulation in the oxygenator. After the initial decrease in platelet count due to haemodilution, it further decreased significantly during CPB with heparin but remained relatively constant when r-Hirudin was used.. Our results strongly suggest that r-Hirudin is superior to heparin especially with respect to its inhibitory effect on platelet dependent thrombogenesis caused by the biomembranes of the oxygenator.

Topics: Animals; Anticoagulants; Antithrombin III; Antithrombins; Cardiopulmonary Bypass; Disease Models, Animal; Hemodilution; Heparin; Hirudin Therapy; Hirudins; Infusions, Intravenous; Papio; Peptide Hydrolases; Platelet Activation; Platelet Count; Recombinant Proteins; Safety; Thrombosis; Treatment Outcome

Rabbit being a common animal model to evaluate the antithrombotic effect of heparins, our purpose was to apply the heparin Standard Independent Unit (SIU) approach to rabbit plasma. To take into account the specificities of the enzymes we have measured the decay constants of factor Xa and of thrombin from autologous and heterologous origins, in presence and in absence of heparin. Different heparins or heparin fractions with a mean molecular weight from 1.7 to 10.5 kDa were used. We found that: a) the decay constants varied strongly between species and between enzymes; b) the decay constants of thrombin were always higher than those of factor Xa; c) the specific anti-factor Xa activity of heparins increased with the molecular weight and was 1.33 times higher when determined with bovine factor Xa than with rabbit factor Xa; d) the specific antithrombin activity of heparins also increased with the molecular weight but was similar when determined with rabbit and human thrombin; e) the specific anti-factor Xa activity was always lower than the specific antithrombin activity; f) the calibration of the heparins and heparin fractions against the 4th International Standard of Heparin expressed in International Units (IU) lead to a systematic overestimation of the anti-factor Xa activity and to an under-estimation of the antithrombin activity. These observations indicate that it may be very important to use the SIU approach and to know the accurate activities to better understand the mechanism of the antithrombotic activity of heparins in experimental models.

Topics: Animals; Anticoagulants; Antithrombins; Cattle; Disease Models, Animal; Factor Xa; Factor Xa Inhibitors; False Negative Reactions; False Positive Reactions; Heparin; Heparin, Low-Molecular-Weight; Hirudins; Humans; Kinetics; Male; Rabbits; Recombinant Proteins; Species Specificity

NF-6505, a bi-O-Tyr-sulfated decapeptide, which specifically interacts with the anion-binding exosite of the thrombin molecule, was chemically synthesized and assessed for its antithrombotic effects in vitro and in vivo. The IC50 value of this peptide on fibrin-clot formation in vitro was about 0.05 microgram/ml, which indicated a potency similar to that of a recombinant hirudin. NF-6505 caused a 2-fold prolongation of activated partial thromboplastin time when intravenously administered at 1 mg/kg in rats. In a rat venous thrombosis model, a bolus intravenous administration of this peptide dose-dependently inhibited the thrombus formation with an ED50 value of 0.03 mg/kg, a value smaller than that of recombinant hirudin (ED50 = 0.1 mg/kg) or of argatroban (ED50 = 0.2 mg/kg). These results suggest that NF-6505 is a highly potent and safe agent for the clinical treatment of venous thrombosis diseases.

Topics: Amino Acid Sequence; Animals; Anions; Binding Sites; Blood Coagulation; Disease Models, Animal; Fibrin; Hirudins; Humans; In Vitro Techniques; Injections, Intravenous; Male; Oligopeptides; Partial Thromboplastin Time; Rats; Rats, Sprague-Dawley; Thrombin; Thrombophlebitis

Air desiccation endothelial injury followed by cholesterol feeding is known to induce focal femoral atherosclerosis in rabbits. We previously demonstrated the effectiveness of hirudin in limiting restenosis after balloon angioplasty (BA) in this double instrumentation injury (DI) model. In the present study, we sought to determine whether BA without prior air desiccation endothelial injury (single instrumentation injury (SI)) would lead to similar femoral lesions, and whether the response to this injury might also be limited by hirudin. Accordingly, 38 femoral arteries of cholesterol-fed rabbits underwent BA with (n = 18, DI group) or without (n = 20, SI group) prior air desiccation endothelial injury. Animals were killed 24 hours or 28 days after BA. Twenty-four hours after BA, the SI group (n = 10) had a significantly smaller percentage of cross-sectional area narrowing by plaque than the DI group (n = 8) (0% versus 42% +/- 9%, p = 0.008). However, 28 days after BA, the percentages of cross-sectional area narrowing by plaque in the SI (n = 10) and DI (n = 10) groups were similar (59% +/- 6% versus 68% +/- 1%, p = NS). The percentages of intima (16% +/- 3% versus 16% +/- 3%, p = NS) and media occupied by foam cells were also similar in the two groups. To test whether hirudin administration would limit arterial narrowing after injury in the SI model, we randomly assigned cholesterol-fed rabbits that had not undergone air desiccation injury to either bolus hirudin followed by repeat dosing 24 hours after BA or bolus heparin (150 U/kg) at the time of BA. The hirudin-treated group showed significantly less angiographic and histologic restenosis 28 days after BA, despite no difference in early (0 to 72 hours) cumulative cellular proliferation between the two groups. Thus, in the cholesterol-fed rabbit, plaque formation and foam cell accumulation are similar after BA of a non-air-desiccated (SI) or focally atherosclerotic (DI) artery. Thrombin inhibition with hirudin limits arterial narrowing after SI, further emphasizing the role of thrombin in neointimal growth after injury.

Topics: Angioplasty, Balloon; Animals; Cell Division; Cholesterol, Dietary; Coronary Disease; Disease Models, Animal; Endothelium, Vascular; Fibrinolytic Agents; Hirudins; Male; Models, Biological; Rabbits

Tissue factor pathway inhibitor is a naturally occurring protein inhibitor of factor X and the tissue factor-factor VII complex of the extrinsic pathway of coagulation. The potential of tissue factor pathway inhibitor as a topical antithrombotic agent was evaluated in a rabbit model of thrombosis that combined intimal injury, anastomosis, and a twisted pedicle. In 207 rabbit ears, a near-complete amputation was performed, preserving the central ear artery and vein. The central ear artery was transected, the intima was removed mechanically over a 1-cm length, the artery was anastomosed, and the ear was twisted 360 degrees, wrapping the intact vein around the artery. Before recirculation, the lumen was irrigated on a blinded, randomized basis with either hirudin (100 or 500 units/ml), heparin (50 or 100 units/ml), tissue factor pathway inhibitor (10, 40, 125, or 250 microgram/ml), heparin and tissue factor pathway inhibitor together, or vehicle (control). Upon arterial reflow, the ears were observed for 7 days. Patency rates after 7 days were as follows: hirudin, 30 and 55 percent; heparin, 43 and 50 percent; tissue factor pathway inhibitor, 75 and 90 percent; heparin and tissue factor pathway inhibitor, 75 percent; and vehicle, 6 percent. The higher concentrations of tissue factor pathway inhibitor led to significantly higher patency rates than heparin, hirudin, or control solutions. Electron microscopic evaluation of specimens irrigated with gold- labeled tissue factor pathway inhibitor revealed the inhibitor bound to the injured intimal surface for at least 3 days postoperatively. Coagulation studies showed no change in the clotting profile upon intravascular infusion with tissue factor pathway inhibitor even at the highest dose used topically. We conclude that tissue factor pathway inhibitor is a more effective topical antithrombotic agent than either heparin or hirudin.

Topics: Administration, Topical; Animals; Anticoagulants; Antithrombins; Blood Coagulation; Disease Models, Animal; Drug Evaluation, Preclinical; Heparin; Hirudins; Lipoproteins; Microcirculation; Rabbits; Recombinant Proteins; Thrombosis; Vascular Patency

Aprotinin has recently been approved for clinical use in cardiopulmonary bypass. Although unfractionated heparin has been the only anticoagulant widely used for cardiopulmonary bypass, disadvantages involving heparin have led to ongoing investigations of alternative anticoagulant agents.. The objective of this study was to evaluate the efficacy of aprotinin in combination with other anticoagulant agents, specifically low molecular weight heparin and recombinant hirudin, using a dog model of cardiopulmonary bypass.. The blood conservation resulting from the use of aprotinin was observed only with unfractionated heparin. Efficacy of anticoagulation as measured by protein deposits in the bypass circuit filter revealed an unexpected reduction in the quantity of deposits when aprotinin was used in combination with low molecular weight heparin.. As alternative anticoagulant agents are sought, the potential benefits of aprotinin in the reduction of operative blood loss must be evaluated independently for each anticoagulant agent.

Topics: Animals; Anticoagulants; Antithrombins; Aprotinin; Blood Coagulation; Blood Loss, Surgical; Cardiopulmonary Bypass; Disease Models, Animal; Dogs; Drug Combinations; Filtration; Hemostatics; Heparin; Heparin, Low-Molecular-Weight; Hirudin Therapy; Hirudins; Male; Partial Thromboplastin Time; Recombinant Proteins

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

1. We compared the direct thrombin inhibitor, desulfatohirudin (REVASC) and the indirect thrombin inhibitor, heparin, as adjuncts to thrombolytic therapy with reteplase in a canine model of coronary artery thrombosis. 2. Reteplase (BM 06.022) is a recombinant unglycosylated variant of human tissue-type plasminogen activator. Thrombus formation in anaesthetized open chest dogs was induced by electrical injury. Left circumflex coronary artery blood flow was monitored for 210 min with an electromagnetic flow probe. Twenty eight dogs were randomized to receive i.v. heparin (120 iu kg-1 bolus plus 80 iu kg-1 per h) or i.v. hirudin (2.0 mg kg-1 bolus plus 2.0 mg kg-1 per h) 10 min before thrombolysis preceded by i.v. acetylsalicyclic acid (20 mg kg-1) 5 min prior to anticoagulation. Every dog received an i.v. double bolus injection of 0.14 + 0.14 u kg-1 ( = 0.24 + 0.24 mg kg-1) reteplase, 30 min apart, 1 h after thrombus formation. 3. At comparable reperfusion rates (12 out of 12 vs. 15 out of 16 dogs), hirudin enhanced time to reperfusion (14.3 +/- 1.4 vs. 23.2 +/- 3.4 min; P < 0.05) and completely prevented reocclusion after reperfusion in contrast to heparin (0 out of 11 vs. 7 out of 11 dogs; P < 0.05). Coronary blood flow quality was improved by hirudin as shown by a higher maximum blood flow after reperfusion (130 +/- 14.3 vs. 83 +/- 9.3% of baseline; P < 0.05), a higher blood flow level at 20, 30, 40, and 50 min after onset of thrombolysis (P < 0.05) and a longer cumulative patency time (195 +/- 1.7 vs. 166 +/- 12 min; P < 0.05). Activated partial thromboplastin time and buccal mucosa bleeding time were prolonged (P < 0.05) by either anticoagulant, but did not differ significantly between groups. 4. The direct thrombin inhibitor, desulfatohirudin, enhanced thrombolysis, prevented reocclusion and increased blood flow as compared with the indirect thrombin inhibitor, heparin, when investigated at one dose level each and used in conjunction with reteplase.

Topics: Animals; Coronary Circulation; Coronary Thrombosis; Disease Models, Animal; Dogs; Drug Therapy, Combination; Female; Fibrinolytic Agents; Half-Life; Heparin; Hirudin Therapy; Hirudins; Humans; Male; Plasminogen Activators; Recombinant Proteins; Thrombolytic Therapy; Tissue Plasminogen Activator

The administration of gram-negative bacterial lipopolysaccharide (LPS) to rats results in hepatic parenchymal cell injury within 6 hr. The coagulation system is critical to the pathogenesis, but previously reported results suggested that its critical role is independent of insoluble clot formation and that thrombin may be a key mediator of liver injury. To test the hypothesis that thrombin is involved in LPS-induced liver injury, animals were treated with the selective thrombin inhibitor, hirudin. The hirudin treatment regimen effectively inhibited thrombin, as evidenced by prolonged activated partial thromboplastin time and by maintenance of plasma fibrinogen concentrations in LPS-treated rats. Treatment with hirudin prevented LPS-induced liver injury, assessed by plasma alanine aminotransferase activity and histological evidence of hepatocellular necrosis. Previous studies have shown that LPS exposure results in the accumulation of neutrophils and platelets within the liver and that both of these cell types are critical for the development of LPS-induced liver injury. Hirudin attenuated in part the decrease in blood platelet concentration that accompanied LPS administration, but did not alter hepatic platelet or neutrophil accumulation. These results support the hypothesis that thrombin is required for hepatic injury from LPS exposure, but that it does not act by promoting the accumulation of platelets or neutrophils within the liver.

Topics: Animals; Blood Platelets; Disease Models, Animal; Female; Hirudins; Lipopolysaccharides; Liver; Rats; Rats, Sprague-Dawley

Proliferation of medial smooth muscle cells (SMC) plays a major role in restenosis after coronary angioplasty and can be inhibited by heparin. Platelets stimulate SMC proliferation, and their aggregation after angioplasty can be reduced by the direct thrombin inhibitor hirudin. In a porcine coronary stent-angioplasty model (16 animals, 4 per group), we studied the effect of 3- and 14-day treatment with a novel long-lasting polyethyleneglycol-conjugated (PEG-hirudin, LU 57291), at a dose of 1 mg/kg intravenously (i.v.) and then subcutaneously once daily as compared with chronic low-molecular-weight heparin (LMWH) clivarine at a dose of 150 IU/kg as an intravenous bolus, followed by 10 IU/kg/h i.v. for 24 h, followed by 75 IU/kg twice daily, as compared with acute unfractionated heparin (100 IU/kg) as a control. Sixteen animals were randomly assigned to the four treatment groups. Four weeks after angioplasty, hearts were perfusion-fixed and six slices per angioplasty segment were analyzed for neointimal thickness and neointimal area (% of total vessel cross-sectional area). Maximal neointimal thickness was 1.10 +/- 0.2 mm in the control group (mean +/- SD, n = 9 arteries) and was significantly lower in both PEG-hirudin (0.62 +/- 0.22 and 0.86 +/- 0.18 mm, 11 and 10 arteries) and in clivarine-treated animals (0.75 +/- 0.33 mm, 11 arteries, p < 0.01). Similarly, neointimal area was smaller in PEG-hirudin groups (20 +/- 12 and 21 +/- 12%) and in the clivarine group (24.8 +/- 13.2%) as compared with the control group (41 +/- 17%, p < 0.02). We conclude that PEG-hirudin and clivarine reduce neointimal proliferation in a coronary stent-angioplasty model. Prolonged PEG-hirudin has no better effect than therapy limited to 3 days.

Topics: Angioplasty, Balloon, Coronary; Animals; Anticoagulants; Antithrombins; Cell Division; Coronary Vessels; Disease Models, Animal; Heparin; Heparin, Low-Molecular-Weight; Hirudins; Muscle, Smooth, Vascular; Swine; Swine, Miniature

Thrombin, a potent stimulator of smooth muscle cell proliferation and inhibitor of endothelial cell growth, has been implicated as an important mediator of restenosis after angioplasty. Acute administration of the thrombin inhibitor r-hirudin reduced restenosis in animal models of angioplasty, possibly by inhibiting smooth muscle cell proliferation. Because thrombin-induced proliferation requires prolonged exposure to the agonist, it was hypothesized that a greater reduction in lesion size could be achieved by chronic administration of r-hirudin.. To determine whether prolonged treatment with r-hirudin would reduce lesion size and improve vascular function in a rabbit model of neointimal proliferation.. Male New Zealand white rabbits were fed a high-cholesterol diet for 4 weeks, after which they were subjected to balloon injury of the left subclavian artery. The rabbits were assigned to one of three groups: control (no drug); acute r-hirudin treatment (0.33 mg/kg intravenously plus 0.48 mg/kg per h subcutaneously for 24 h); or chronic r-hirudin treatment (0.33 mg/kg intravenously plus 0.6 mg/kg per h subcutaneously for 28 days). After surgery the rabbits were fed a normal diet and killed 30 days later. Left (angioplastied) and right (control) subclavian arteries were removed for morphological and functional analysis.. Angioplasty in control, untreated rabbits produced large neointimal lesions [15.0 +/- 1.8% of the area within the external elastic lamina (EEL)], comprised mainly of smooth muscle cells (34 +/- 16 cells/section) and lipid-rich macrophage or foam cells (118 +/- 51 cells/section). Acute r-hirudin treatment neither inhibited smooth muscle cell proliferation (35 +/- 12 cells/section) nor reduced neointimal lesion size (23.5 +/- 4.6% of the area within the EEL). Chronic r-hirudin treatment significantly increased the number of proliferating cells (55 +/- 15 cells/section, P < 0.05) and the size of the lesions (28.5 +/- 5.6% of the area within the EEL, P < 0.05). Further more, treatment with r-hirudin appeared to exacerbate, rather than improve, angioplasty-induced functional alterations.. Prolonged treatment with r-hirudin neither inhibits vascular smooth muscle cell proliferation in rabbits after angioplasty of the left subclavian artery nor reduces the size of neointimal lesions. Furthermore, treatment with r-hirudin might impair endothelial cell function after angioplasty. This suggests that prolonged thrombin inhibition using this r-hirudin regimen is not suitable as an antirestenotic intervention.

Topics: Angioplasty; Animals; Antithrombins; Cholesterol, Dietary; Disease Models, Animal; Endothelium, Vascular; Hirudins; Immunohistochemistry; Injections, Intravenous; Injections, Subcutaneous; Male; Rabbits; Reference Values; Subclavian Artery

The effects of heparin boluses comparable with those commonly used during angioplasty procedures were compared with equal gravimetric doses of recombinant desulfatohirudin (CGP 39393) in a rabbit model of microarterial thrombosis. Seven-millimeter segments of the central arteries of the ears were isolated between microvascular clamps and subjected to arteriotomy and deep vessel wall trauma. Arteriotomies were closed with continuous 10-0 sutures. Five minutes before vascular reperfusion (opening of vascular clamps), boluses of heparin (1.00 mg/kg), hirudin (1.00 mg/kg), hirudin (0.25 mg/kg), or vehicle (saline) were administered to groups of 10 rabbits in a blind, random fashion. Neither agent prolonged arteriotomy bleeding times relative to vehicle. All active agents significantly increased patency rates versus vehicle at 30 minutes after reperfusion, though reduced vessel patency was noted in a substantial portion of vessels in the groups receiving 0.25 mg hirudin or 1.00 mg/kg heparin. Patency rates at 120 minutes were only improved by the 1.00 mg/kg hirudin dose, and in accord, thrombus weights were significantly reduced only by the 1.00 mg/kg dose of hirudin. In contrast, the anticoagulant response (measured as the activated partial thromboplastin time and anti-IIa and anti-Xa activities) was considerably more pronounced and of longer duration after administration of heparin and hirudin. This is consistent with the hypothesis that the inactivation of clot-bound thrombin that is produced by specific thrombin inhibition (hirudin) but not by cofactor-mediated thrombin inhibition (heparin) is of pivotal importance in achieving a profound and sustained antithrombotic effect following vascular trauma. While heparin boluses seem to be of doubtful value, hirudin seems a promising strategy in context with angioplasty procedures.

Topics: Animals; Disease Models, Animal; Female; Heparin; Hirudin Therapy; Hirudins; Male; Rabbits; Random Allocation; Recombinant Proteins; Thrombin; Thrombosis

Hirulog, a thrombin-specific inhibitor, has shown efficacy in reducing arterial thrombosis in patients treated with aspirin who require angioplasty or have unstable angina. In this study, the effect of hirulog on reducing deposition of indium 111-labeled platelets was assessed in a surgical model of aspirin-treated rats undergoing carotid endarterectomy.. Animals were randomly assigned to one of five groups: control (no aspirin or hirulog); aspirin alone (10 mg/kg); aspirin plus low-dose hirulog (0.2 mg/kg bolus followed by 0.5 mg/kg/hr); aspirin plus medium-dose hirulog (0.4 mg/kg bolus followed by 1.0 mg/kg/hr); or aspirin plus high-dose hirulog (0.6 mg/kg bolus followed by 1.5 mg/kg/hr). Hirulog was infused before surgery and continued until termination of the experiment 30 minutes after endarterectomy.. Platelet deposition in rats receiving aspirin alone was reduced by 19% +/- 23% SE (p = 0.26) compared with controls. Deposition in aspirin-treated groups receiving low-, medium-, and high-dose hirulog decreased in a dose-dependent manner by 37% +/- 20% (p = 0.048), 44% +/- 19% (p = 0.061), and 56% +/- 13% (p = 0.022), respectively. As the dose of hirulog was increased, the plasma hirulog levels and activated partial thromboplastin time ratios (final:initial) also increased in a dose-dependent manner. The mean plasma hirulog levels ranged from 0.74 +/- 0.08 micrograms/ml in the low-dose hirulog group to 2.55 +/- 0.08 micrograms/ml in the high-dose hirulog group, and the corresponding activated partial thromboplastin time ratios were 1.5 +/- 0.12 (p = 0.001) and 3.3 +/- 0.63 (p = 0.001). Bleeding was easily controlled by local hemostatic measures for all experimental groups.. Hirulog causes significant decrease in 111In-labeled platelet deposition in aspirin-treated rats subjected to microsurgical endarterectomy at doses that allow surgical hemostasis to be easily established.

Topics: Animals; Aspirin; Blood Platelets; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Therapy, Combination; Endarterectomy, Carotid; Hirudin Therapy; Hirudins; Male; Peptide Fragments; Postoperative Complications; Random Allocation; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Thrombin; Thrombosis

A rat thrombosis model was developed to assess the efficacity of antithrombotic drugs. It had the following characteristics: controlled hemodynamic and rheological conditions corresponding to arterial flow, a collagen coated surface as a relevant thrombogenic stimulus, a method of measurement allowing dynamic monitoring of thrombus formation and the possibility to assess the thrombus structure. A shunt composed of polyethylene and silicone catheters, including in the middle of the shunt a collagen coated glass capillary, was inserted between the two primitive carotids of the rat. The duration of patency of the shunt was recorded using a thermic probe fixed on its central part. In this model, the patency of the shunt was 539 +/- 55 s. Platelet and fibrinogen-fibrin accumulation in successive one centimeter segments along the shunt were measured using 111In labeled platelets and 125I labeled fibrinogen. Platelet accumulation occurred on the collagen coated surface and at the junctions between the different components of the shunt, where flow was disturbed. The effects of four antithrombotic agents were measured: aspirin, clopidogrel, heparin and r-hirudin. Clopidogrel, heparin and hirudin significantly prolonged patency duration of the shunt, whereas aspirin was inactive. Aspirin did not reduce platelet or fibrinogen-fibrin accumulation on the collagen coated surface. Platelet accumulation on the collagen surface was significantly lower in the clopidogrel group (50 mg/kg) than in the group treated with heparin (500 U/kg), demonstrating the direct antiplatelet effect of clopidogrel. Hirudin at doses giving similar values of APTT as heparin (500 U/kg) prolonged the occlusion time to over 2 h while the heparin occlusion time was only 20 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Anticoagulants; Arterio-Arterial Fistula; Aspirin; Carotid Artery Thrombosis; Clopidogrel; Collagen; Disease Models, Animal; Drug Evaluation, Preclinical; Fibrin; Fibrinogen; Fibrinolytic Agents; Glass; Heparin; Hirudin Therapy; Hirudins; Male; Platelet Adhesiveness; Platelet Aggregation Inhibitors; Polyethylenes; Rats; Rats, Wistar; Recombinant Proteins; Regional Blood Flow; Silicones; Thrombin; Ticlopidine

A model for thrombolysis in rats was developed. Repeated, focal external heating was applied to the carotid artery which leads to the development of a cyclic blood flow with slow, steady decreases followed by abrupt increases. When this cyclic blood flow stops spontaneously, the entire arterial segment (approximately 10 mm) can be demarcated with snares to create an arterial thrombus of fixed size, with a platelet-rich head and an erythrocyte-rich tail. The usefulness of the model was tested by evaluating the thrombolysis induced by a low dose of recombinant tissue-type plasminogen activator (rt-PA) alone and rt-PA in combination with standard heparin and recombinant hirudin. Re-canalization of the artery was measured as blood flow and as the residual 125I-radioactivity in the artery at the end of the experiment, resulting from 125I-fibrinogen incorporated during the formation of the thrombus. Both blood flow and 125I-activity measurements show that hirudin, but not heparin in combination with rt-PA, significantly improves thrombolysis, which is in accordance with previous experimental findings. It is concluded that the model, with a thrombus resembling the thrombus found in man after coronary occlusion, enables complicated experiments with thrombolysis frequently performed only in large animals to be performed in rats.

Topics: Animals; Blood Platelets; Carotid Arteries; Disease Models, Animal; Drug Combinations; Erythrocytes; Hirudins; Hot Temperature; Iodine Radioisotopes; Male; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Thrombosis; Tissue Plasminogen Activator

Venous thrombosis was induced by ligature of the inferior vena cava in rats whose blood was made hypercoagulable by intravenous (IV) administration of tissue thromboplastin. From a dose-response showing that the administration of increasing doses of tissue thromboplastin resulted in a subsequent progressive increase of thrombus weight, two concentrations of tissue thromboplastin were chosen: a high dose (550 microL/kg, IV) where thrombus formation was optimal and a concentration (7 microL/kg, IV) where tissue thromboplastin-hypercoagulability was intermediate. In both experimental conditions, leukopenia provoked by a myelotoxic drug did not influence the development of venous thrombosis. However, after thrombocytopenia induced by an antiplatelet antiserum, a dramatic decrease in thrombus formation was observed in animals that had been pre-challenged with the lower dose of tissue thromboplastin, whereas decrease in platelet count did not affect venous thrombosis under high thrombogenic challenge. When administered orally 2 hours before thrombosis induction, the ticlopidine analogue clopidogrel showed dose-dependent inhibition of thrombus formation in animals that were pre-challenged with a low dose of tissue thromboplastin (ED50 = 7.9 +/- 1.5 mg/kg, orally) but remained ineffective against high tissue thromboplastin-induced venous thrombosis. We further determined the effect of heparin and hirudin, and showed that both of these drugs exhibited a more potent antithrombotic activity after injection of the lower dose of tissue thromboplastin than after injection of a high dose of tissue thromboplastin. Therefore, using our model of stasis and hypercoagulability, platelet activation played a major role in the development of venous thrombosis when the thrombogenitic stimulus was mild.

Topics: Animals; Blood Platelets; Clopidogrel; Disease Models, Animal; Heparin; Hirudins; Leukopenia; Male; Mechlorethamine; Partial Thromboplastin Time; Platelet Aggregation Inhibitors; Rabbits; Rats; Rats, Sprague-Dawley; Thrombocytopenia; Thrombophlebitis; Thromboplastin; Ticlopidine

The primary bleeding time is prolonged when tested during the infusion of both plasminogen activators and anticoagulants, and such sites frequently exhibit rebleeding after initial hemostatic control. This study describes an animal (rabbit) model which distinguishes fibrinolytic from anticoagulant hemorrhage and further applies the model to the study of hemostatic plugs of increasing age. In this model, rebleeding occurred from hemostatically-stable ear puncture sites induced prior to infusion of streptokinase (SK) or recombinant tissue-plasminogen activator (rt-PA), but not of heparin or hirudin. This distinction was apparent even for lesions induced only 15 minutes prior to the infusion and fibrinolytic bleeding was observed in such lesions induced up to 24 hours earlier. Post-infusion sites bled more quickly than did pre-infusion sites, and there was a gradual decrease in susceptibility of such prior trauma sites for rebleeding, evidenced not only by a lower proportion of sites that rebled, but also by a longer lag time after starting SK or rt-PA before such rebleeding occurred. At the dosages tested, SK showed a trend (not statistically significant) toward more sites that rebled, while rt-PA showed a trend towards a longer duration of rebleeding. Thus, this animal model of rebleeding appears to be unique for fibrinolytic agents and allows for more detailed study of the physiological mechanisms of such bleeding and for a multifaceted comparison of the bleeding potential of plasminogen activators.

Topics: Animals; Anticoagulants; Bleeding Time; Disease Models, Animal; Female; Fibrinolysis; Fibrinolytic Agents; Hemorrhage; Hemostasis; Heparin; Hirudins; Male; Rabbits; Recombinant Proteins; Streptokinase; Tissue Plasminogen Activator

The in vitro anticoagulant activity of recombinant desulphated hirudin (HBW 023) and its antithrombotic activity in a rabbit venous stasis model were assessed in comparison to unfractionated heparin (UH). The specific activity of r-hirudin in rabbit plasma is similar to that of unfractionated heparin on a weight basis when using the whole blood clotting time or APTT, while it was five times more potent according to the thrombin clotting time (TCT). Forty-eight (6x8) anaesthetized New Zealand male rabbits were randomized to receive HBW 023 (12.5, 25, 50, 100, 200, 400 micrograms.kg-1), standard heparin (90 micrograms.kg-1) or placebo. Five minutes after administration of the drug, the experimental thrombosis was induced by an injection of glass activated overnight human serum into the marginal vein of the ear and ligation of the jugular vein (Wessler model). The jugular vein was removed after 10 min stasis and examined by a researcher unaware of the treatment administered. In the Wessler stasis model the fresh thrombus weight and a score as well as the circulating level of r-hirudin using a chromogenic substrate assay were used to determine the inhibitory effect of the drug. Highly significant inverse correlations were found between fresh thrombus weight and the injected doses as well as r-hirudin plasma levels. The ID50 which was the dose of the drug that induced a complete inhibition of thrombosis in 50% of the dose group tests was about 200 micrograms.kg-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Constriction; Disease Models, Animal; Hemostasis; Heparin; Hirudins; In Vitro Techniques; Jugular Veins; Male; Rabbits; Random Allocation; Recombinant Proteins

An existing arterio-venous shunt thrombosis model in the rat has been modified to increase its usefulness for the testing of anti-thrombotic agents and characterised using morphological and radiometric techniques. The thrombus formed on the cotton thread held in the shunt was found to be composed of platelet aggregates surrounded by red thrombus. After 30 min of blood flow there was a 15-fold increase in the platelet content of the thrombus and a 4-fold increase in the fibrin(ogen) content compared with an equivalent weight of whole blood. Use of the anticoagulants recombinant desulphatohirudin (CGP 39393, 4 mg/kg, s.c.) and unfractionated heparin (800 IU/kg, s.c.) showed that approx. 90% inhibition of thrombus weight and approx. 80% inhibition of fibrin(ogen) content could be achieved without significant effect on the platelet content. Conversely, using the platelet inhibitor Iloprost (1 microgram kg-1 min-1), a reduction in thrombus weight of 50% was associated with 75% inhibition of platelet content and only 20% inhibition of fibrin(ogen). These observations suggest that the growth of this type of thrombus is largely the result of continued fibrin formation rather than continued platelet recruitment and activation.

Topics: Animals; Arteriovenous Shunt, Surgical; Disease Models, Animal; Fibrinolytic Agents; Hematologic Tests; Heparin; Hirudin Therapy; Hirudins; Iloprost; Male; Radiometry; Rats; Rats, Inbred Strains; Recombinant Proteins; Thrombosis

Various reactions of disseminated intravascular coagulation (DIC) were experimentally induced by infusion of thrombokinase in rats, by administration of endotoxin in rabbits and pigs and by infusion of adrenaline and thrombin in dogs. Low plasma concentrations of recombinant hirudin (r-hirudin; 20-200 ng/ml) were sufficient for the inhibition of the different triggering mechanisms. The studies on the pharmacological profile of r-hirudin in DIC therapy confirm the efficacy of this specific tight-binding thrombin inhibitor.

Topics: Animals; Disease Models, Animal; Disseminated Intravascular Coagulation; Dogs; Endotoxins; Epinephrine; Factor Xa; Factor Xa Inhibitors; Female; Fibrinolytic Agents; Hemodynamics; Hirudin Therapy; Hirudins; Lipid A; Male; Platelet Count; Rabbits; Rats; Rats, Inbred Strains; Recombinant Proteins; Species Specificity; Swine; Thrombin; Thrombolytic Therapy; Thrombosis; Viscera

The objective of the study was to evaluate the ability of heparin to enhance the thrombolytic effect of recombinant tissue type plasminogen activator (rt-PA) and to prevent thrombus growth during and after thrombolysis with rt-PA. In the thrombolysis studies, three groups of rabbits were infused with rt-PA at a dose of 0.5 mg, 1 mg, or 2.5 mg over 3 hours, respectively. Rabbits in each group were randomized to receive, in addition to rt-PA, heparin, 20 or 60 antifactor Xa U/kg/h, or saline over 6 hours. The three doses of rt-PA produced the same extent of thrombolysis both in the two groups treated with heparin (34% +/- 6%, 52% +/- 7%, and 79% +/- 8% in the lower dose group; 39% +/- 6%, 49% +/- 4%, and 81% +/- 6% in the higher dose group) and in the group treated with saline (37% +/- 4%, 47% +/- 5%, and 84% +/- 7%). In the thrombus growth inhibition studies 0.5 mg of rt-PA was infused over 3 hours in each rabbit. In addition, the rt-PA-treated rabbits were randomized to receive heparin, 20 or 60 antifactor Xa U/kg/h over 6 hours, or saline. At the end of infusion, no statistically significant differences in thrombus growth were found in three groups of rabbits (54.8 +/- 7.4 micrograms and 52.4 +/- 12.1 micrograms in the low and high dose of heparin groups, respectively, and 59.4 +/- 10.4 micrograms in the saline group). In different experiments rabbits were randomized to receive heparin, 60 antifactor Xa U/kg/h, or saline at the end of the rt-PA infusion. In these experiments heparin inhibited thrombus growth more efficiently than saline (41.1 +/- 6.5 micrograms and 58.7 +/- 12.9 micrograms, respectively, P less than .05). In vitro experiments confirmed that heparin is unable to prevent fibrin accretion on the clots during lysis with rt-PA while both D-Phe-Pro-Arg-CH2-Cl (PPACK) and hirudin are able to prevent the accretion of fibrin. We conclude that the data obtained in these animal models do not support the concomitant use of heparin and rt-PA. However, heparin could be used successfully after rt-PA to inhibit thrombus growth.

Topics: Amino Acid Chloromethyl Ketones; Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Factor Xa; Fibrin; Heparin; Hirudins; Rabbits; Recombinant Proteins; Thrombolytic Therapy; Thrombosis; Tissue Plasminogen Activator

The antithrombotic effects of three thrombin inhibitors (hirudin, NAPAP and argidipine) were investigated in an experimental thrombosis model using laser lesions of rat mesenteric venules. Furthermore, their in vitro anticoagulant activity (partial thromboplastin time, thrombin time, Heptest, inhibition of factor IIa and factor Xa) in human platelet poor plasma (PPP) and their in vitro and ex vivo activities were studied in rat plasma. All three thrombin inhibitors showed significant and dose-dependent antithrombotic effects after intravenous injection, if venules were damaged, which lasted for more than 4 but less than 8 h. The anticoagulant effect observed in vitro did not differ much between human and rat PPP. The two synthetic thrombin inhibitors NAPAP and argidipine were about as effective as hirudin in vitro; however, the ex vivo effect after intravenous injection of hirudin in rats was more pronounced than that observed with the two synthetic thrombin inhibitors. The antithrombotic effect of all three thrombin inhibitors in the laser model lasted much longer than the anticoagulant activity. This fact needs further investigations in the future.

Topics: Animals; Antithrombins; Arginine; Disease Models, Animal; Hirudins; Humans; Injections, Intravenous; Lasers; Light Coagulation; Pipecolic Acids; Rats; Rats, Inbred Strains; Sulfonamides; Thrombosis

The antithrombotic activity of hirudin was studied in a rat coronary thrombosis model. The thrombus formation was induced by external application of silver nitrate solution onto the left anterior descending coronary artery. Following subcutaneous injection, hirudin in doses of 0.25, 0.5 and 1.0 mg/kg reduced the development of coronary thrombosis in a dose-dependent manner. The most pronounced antithrombotic effect of hirudin in the described model was related with plasma concentrations between 0.20 and 0.35 microgram hirudin/ml.

Topics: Animals; Coronary Disease; Coronary Thrombosis; Disease Models, Animal; Female; Fibrinolytic Agents; Hirudins; Injections, Subcutaneous; Male; Rats; Rats, Inbred Strains