Anticoagulants ; Antithrombins/therapeutic use* ; Coronary Disease ; Heparin ; Hirudins ; Humans ; Peptide Fragments ; Percutaneous Coronary Intervention ; Recombinant Proteins ; Treatment Outcome

Objective: To compare the safety and efficacy of bivalirudin versus unfractionated heparin during perioperative period of percutaneous coronary intervention(PCI) in real-world. Methods: A total of 13 097 serial patients who underwent PCI from January 2016 to November 2018 in the Northern Theater Command were enrolled in the present study. Patients were stratified as the bivalirudin group or the heparin group according to antithrombotic therapy during PCI. The primary efficiency endpoint was 30-day net adverse clinical event(NACE), defined as all-cause death, re-infarction, urgent target lesion revascularization (uTLR), stroke or any bleeding. The second efficiency endpoint was 30-day major cardiac and cerebral events (MACCE), defined as all-cause death, re-infarction, uTLR and stroke. Additional end points included the rates of stent thrombosis at 30 days. Propensity scores included clinical and demographic variables, with 1∶2 matching. Compared the incidence of events above between the two groups before and after matching. Results: Among the 13 097 included patients(age was (61±10) years old), 3 421 (26.1%) were female. And 2 734 patients were divided into the bivalirudin group, and 10 363 patients to the heparin group(5 468 after matching). Before propensity score matching, patients in bivalirudin group were older and received higher levels of CRUSADE score than heparin group. These patients were more likely to have hypertension and more with ST-segment elevation acute coronary syndromes（all P＜0.05）. After propensity score matching, the incidence of 30-day NACE(3.8%(103/2 734) vs.5.0%(271/5 468), P=0.015) and any bleeding (2.0%(54/2 734) vs. 2.8%(151/5 468), P=0.032) in the bivalirudin group were lower than that in the heparin group, but the incidence of MACCE (1.9%(51/2 734) vs. 2.3%(127/5 468), P=0.180) and stent thrombosis (0.1%(2/2 734) vs. 0.1%(3/5 468), P=1.000) were comparable between the two groups. Conclusion: The risk of bleeding and the incidence of NACE are significantly lower for patients using bivalirudin during perioperative period of PCI compared to heparin, without significant differences in ischemic events.
Aged ; Anticoagulants/therapeutic use* ; Female ; Heparin/therapeutic use* ; Hirudins ; Humans ; Middle Aged ; Peptide Fragments ; Percutaneous Coronary Intervention ; Perioperative Period ; Recombinant Proteins ; Treatment Outcome

OBJECTIVE: To investigate the antithrombotic effects of recombinant hirudin and its mechanism. METHODS: Sixty male Kunming mice were randomly divided into 6 group (=10):control group, model group, aspirin (25 mg/kg) group, recombinant hirudinlow, middle and high dose (0.05, 0.1, 0.2 mg/kg) groups.Except mice in control group, 2.5 mg/kg carrageenan was injected intraperitoneallyto mice in the other groups to produce thrombosis on the mice tail. The mice in aspirin group were administrated intraperitoneally 25 mg/kg aspirin, the mice in recombinant hirudinlow, middle and high dose groups were administrated intraperitoneally 0.05, 0.1, 0.2 mg/kg combinanthirudin, the mice in control group and model group were administrated intraperitoneallynormal saline at the same volume respectively at 24 h, 0.5 h before injecting carrageenan and 24 h after injecting carrageenan. The black tail length of mice and the incidence of black tail were observed at 48h after injection of carrageenan; prothrombin time (PT), activated partial thromboplastin time (APTT), tissue plasminogen activator (t-PA), type-1 plasminogen activator inhibitor (PAI-1), 6-keto-PGF1α, and thromboxane B2 (TXB2) level in mice plasma were determined. RESULTS: As compared with control group, the mice in model group presented tail thrombosis; PT level in plasma was significantly shortened (<0.01), PAI-1 and TXB2levels in plasma were significantly increased (<0.01), while the t-PA and 6-keto-PGF1α levels in plasma in model group were significantly decreased (<0.01). As compared with model group, the thrombus length in the tail was significantly shortened (<0.05, <0.01), PT level was obviously prolonged (<0.01), and the plasma levels of PAI-1 and TXB2 were significantly decreased (<0.01), while the plasma levels of t-PA and 6-keto-PGF1α were significantly increased (<0.01)in the mice of recombinant hirudin low dose, middle dose, high dose groups and aspirin group. As compared with aspirin group, the thrombus length in the tail was significantly increased (<0.05), PT level was obviously shortened (<0.01), and the plasma levels of PAI-1 and TXB2 were significantly increased (<0.01)in the mice of recombinant hirudin low dose group; the plasma level of 6-keto-PGF1α was significantly decreased (<0.01, <0.05) in the mice of recombinant hirudin low dose and middle dose groups; the plasma levels of PAI-1 and TXB2 were significantly increased (<0.01, <0.05)in the mice of recombinant hirudin middle dose group. CONCLUSIONS The recombinant hirudin can fight against thrombosis, its antithrombotic mechanisms may be related to its influence on the exogenous coagulation system and the promotion of fibrinolysis function.
Animals ; Blood Coagulation ; Fibrinolytic Agents ; Hirudins ; pharmacology ; Male ; Mice ; Recombinant Proteins ; Thromboxane B2 ; Tissue Plasminogen Activator

OBJECTIVEThis study aimed to investigate the effects of hirudin on the expression of transforming growth factor (TGF-β1) and basic fibroblast growth factor (bFGF) in human gingival fibroblasts (HGFs) in vitro, as well to explore its func- tion in the mechanism of gingival remodeling.

METHODSAfter culturing was performed with classic tissue-explant method, HGFs were derived from normal gingival and gingival hyperplasia tissues followed by orthodontic treatments with different concentrations of hirudin. The mRNA and protein expression levels of TGF-β1 and bFGF were respectively detected by real time quantity polymerase chain reaction and immunocytochemistry.

RESULTSCompared with normal HGFs, TGF-β1 expression promoted collagen synthesis of fibroblasts, whereas bFGF collagen synthesis was decreased in hyperplasia HGFs without hirudin (P < 0.05). Hirudin significantly upregulated the expression levels of bFGF but downregulated TGF-β1 in hyperplasia HGFs (P < 0.05).

CONCLUSIONOrthodontic force may influence the balance of collagen synthesis and degradation in HGFs. Hirudin may modulate the balance of HGF collagen metabolism, thereby promoting gingival remodeling.

Fibroblast Growth Factor 2 ; Fibroblasts ; Gingiva ; Hirudins ; Humans ; RNA, Messenger ; Transforming Growth Factor beta ; Transforming Growth Factor beta1

OBJECTIVETo explore the effect and mechanism of hirudin on atherosclerotic plaques in apolipoprotein E knockout (ApoE(-/-)) mice.

METHODSTotally 24 ApoE(-/-) mice, 7-8 weeks old were fed with high fat diets. They were randomly divided into the recombinant hirudin treatment group (drug group) and the model group according to body weight and different dens, 12 in each group. Twelve C57BL/6J mice, 7-8 weeks old fed with high fat diet were recruited as the normal control group. Recombinant hirudin (0.25 mg/kg) was intraperitoneally injected to mice in the drug group from the 10th week old once every other day for five successive weeks. Equal volume of normal saline was injected to mice in the model group. Mice in the normal control group received no treatment. All mice were sacrificed after fed with high fat diet until they were 20 weeks old. Serum levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), high-sensitive C-reactive protein (hs-CRP), E-selectin, interleukin-6 (IL-6), and stromal metalloproteinase-2 (MMP-2) were detected. The plaque/lumen area and extracellular lipid composition/ plaque area were analyzed by HE staining and morphometry. Changes of signaling molecules in store-operated calcium channels, including stromal interacting molecule 1 (STIM1), Orail protein, and transient receptor potential channel 1 (TRPC1) were determined by Western blot. Results Lipid plaque formed in the aorta vessel wall of 20-week old mice in the model group. Compared with the normal control group, serum levels of TC, TG and LDL increased (P<0.01), hs-CRP, E-selction, IL-6, and MMP-2 obviously increased (P<0.01, P<0.05) in the model group; expression levels of STIM1, TRPC1, and Orail significantly increased (P<0.01). Compared with the model group, the plaque/lumen area and the extracellular lipid composition/plaque area significantly decreased in the drug group (P<0.05, P<0.01); serum levels of TC and LDL, hs-CRP, E-selction, IL-6, and MMP-2 obviously decreased (P<0.05, P<0.01); expression levels of STIM1, TRPC1, and Orail were significantly down-regulated (P<0.05, P<0.01).

CONCLUSIONHirudin could significantly improve lipids and endothelial functions of ApoE(-/-) mice, down-regulate expression levels of STIM1, Orai1, and TRPC1, and thus delaying the occurrence and development of atherosclerosis.

Animals ; Aorta ; Apolipoproteins E ; metabolism ; Atherosclerosis ; C-Reactive Protein ; Cholesterol ; Diet, High-Fat ; Drugs, Chinese Herbal ; E-Selectin ; Hirudins ; metabolism ; Interleukin-6 ; Lipids ; Lipoproteins, HDL ; Lipoproteins, LDL ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Plaque, Atherosclerotic ; metabolism ; Recombinant Proteins ; metabolism ; Triglycerides

OBJECTIVE: Over-expression of thrombin in ovarian cancer cells is associated with poor prognosis. In this study, we investigated the role of thrombin in inducing epithelial-mesenchymal transition (EMT) in SKOV3 epithelial ovarian cancer cells. METHODS: After thrombin treatment SKOV3 cells were subjected to western blots, reverse-transcription PCR, and enzyme-linked immunosorbent assay to quantify EMT-related proteins, mRNA expression of SMAD2, DKK1, and sFRP1, and the secretion of matrix metalloproteinases (MMPs) and cytokines. Meanwhile, invasion ability was evaluated using transwell assays. RESULTS: The results indicated a dose- and time-dependent down-regulation of E-cadherin and upregulation of N-cadherin and vimentin in thrombin-treated SKOV3 cells, compared with the thrombin-free control group (p<0.05). There was a dose- and time-dependent increase in the levels of SMAD2 and DKK1 mRNAs and a decrease in the levels of sFRP1 mRNA in thrombin-treated SKOV3 cells compared to control cells (p<0.05). Thrombin-treated SKOV3 cells exhibited increased secretion of MMP-9, MMP-2, interleukin (IL)-8, and IL-6 and increased invasion compared to untreated cells (p<0.05). Thrombin altered the morphology of SKOV3 cells to a spindle-like phenotype. Addition of hirudin to thrombin-treated cells reversed the effects of thrombin. CONCLUSION: Thrombin induced EMT and promoted the invasion of SKOV3 cells, possibly via distinct signaling pathways. Hirudin inhibited the effects of thrombin, suggesting that anticoagulant therapy could be a novel therapeutic strategy for ovarian carcinoma.
Blotting, Western ; Cadherins ; Cytokines ; Down-Regulation ; Enzyme-Linked Immunosorbent Assay ; Epithelial-Mesenchymal Transition ; Hirudins ; Interleukin-6 ; Interleukins ; Matrix Metalloproteinases ; Neoplasms, Glandular and Epithelial ; Ovarian Neoplasms ; Phenotype ; Polymerase Chain Reaction ; Prognosis ; Proteins ; RNA, Messenger ; Thrombin ; Up-Regulation ; Vimentin

BACKGROUNDBivalirudin was widely used as an anticoagulant during coronary interventional procedure in western countries. However, it was not available in China before this clinical trial was designed. This randomized, single-blind and multicenter clinical trial aimed to evaluate the efficacy and the safety of domestic bivalirudin during percutaneous coronary intervention (PCI).

METHODSA randomized, single-blind, multicenter trial was designed. Elective PCI candidates in five centers were randomized into a bivalirudin group and a heparin group, which were treated with domestic bivalirudin and non-fractional heparin during the PCI procedure. The efficacy was evaluated by comparing the activated coagulation time (ACT), the procedural success rate (residual stenosis < 20% in target lesions without any coronary artery related adverse events within 24 hours after PCI), and the survival rate without major adverse cardiac events at 30 days after PCI between the two groups. Safety was evaluated by the major/minor bleeding rate.

RESULTSA total of 218 elective PCI patients were randomized into a bivalirudin group (n = 110) and heparin group (n = 108). Except for two patients needing additional dosing in the heparin group, the ACT values of all other patients in both groups were longer than 225 seconds at 5 minutes after the first intravenous bolus. Procedural success rates were respectively 100.0% and 98.2% in the bivalirudin group and heparin group (P > 0.05). Survival rates without major adverse cardiac events at 30 days after PCI were 100.0% in the bivalirudin group and 98.2% in the heparin group (P > 0.05). Mild bleeding rates were 0.9% and 6.9% (P < 0.05) at 24 hours, and 1.9% and 8.8% (P < 0.05) at 30 days after PCI in the bivalirudin group and heparin group respectively. There was one severe gastrointestinal bleeding case in the heparin group.

CONCLUSIONSDomestic bivalirudin is an effective and safe anticoagulant during elective PCI procedures. The efficacy is not inferior to heparin, but the safety is superior to heparin.

Aged ; Antithrombins ; adverse effects ; therapeutic use ; Female ; Heparin ; therapeutic use ; Hirudins ; adverse effects ; Humans ; Male ; Middle Aged ; Peptide Fragments ; adverse effects ; therapeutic use ; Percutaneous Coronary Intervention ; Recombinant Proteins ; adverse effects ; therapeutic use ; Single-Blind Method ; Survival Rate ; Whole Blood Coagulation Time

BACKGROUND: The Multiplate analyzer (Dynabyte GmbH) has been recently introduced as a platelet function test for patients taking antiplatelet drugs. The study aimed at providing basic data for determining the reference interval of parameters produced by Multiplate in Koreans and to study the factors that influence those parameters. METHODS: Blood was collected from 35 healthy volunteers (female 18, male 17) into tubes containing hirudin or 3.2% sodium citrate. Whole blood platelet aggregations triggered by adenosine-5'-diphosphate (ADP), ADP-high sensitive (ADP+PGE1 only in hirudin samples), arachidonic acid (AA), collagen or thrombin receptor activator peptide (TRAP) were investigated using Multiplate according to the manufacturer's instructions. Data from healthy volunteers for the area under the curve (AUC) were determined from the central 95th percentile of the results. RESULTS: The values of AUC in hirudin samples for all agonists were significantly higher than those in sodium citrate samples. The AUC values in hirudin (sodium citrate) samples were as follows: ADP 38-107 (18-119) U; ADP+PGE1 16-91 U; AA 64-156 (32-117) U; collagen 53-112 (26-108) U; and TRAP 81-163 (49-149) U. The parameters from Multiplate were significantly correlated with leukocyte counts, but not with hematocrit levels. CONCLUSIONS: Although our data were derived from only 35 subjects, the results are expected to be helpful in determining the reference interval at a single institute and may serve as basic data for future cumulative data of reference intervals from multiple institutes in Korea.
Academies and Institutes ; Adenosine Diphosphate ; Arachidonic Acid ; Area Under Curve ; Blood Platelets ; Citrates ; Citric Acid ; Collagen ; Hematocrit ; Hirudins ; Humans ; Korea ; Leukocyte Count ; Male ; Platelet Aggregation Inhibitors ; Platelet Function Tests ; Receptors, Thrombin ; Sodium

OBJECTIVETo study the extraction system of hirudin emulsion liquid membrane with the Poecilobdella manillensis as raw material, di-(2-ethylhexyl) phosphate (D2EHPA) as carrier, Span 80 as emulsifier, octane and D2EHPA mixed to constitute membrane solution, diluted HCl solutions as internal aqueous phase.

METHODUsing the orthogonal experiment to optimize the extraction conditions of hirudin reference substance such as membrane phase, internal aqueous phase volume ratio (MIPVR), external aqueous phase pH, internal aqueous phase pH, mobile carrier concentration and so on, and then using hirudin crude extracts to do purifying experiment, and gaining experimental samples.

RESULTThe optimal conditions of hirudin extraction were as follows: MIPVR 10: 3, internal aqueous phase pH 2.6, external aqueous phase pH 3.4, the mass fraction of carrier D2EHPA 2%. In the optimal extraction conditions, when the initial concentration of hirudin was one anti-thrombin activity units (ATU) x mL(-1), ATU recovery rate of the reference substance was 83.06%. In the purifying experiment of crude extracts, ATU recovery rate was 82.99%, and the specific activity of sample was 3 289.48 the ATU x mg(-1). Discontinuous polyacrylamide gel electrophoresis and spectral scanning, the results showed that the purity and reference substance were considerable.

CONCLUSIONThe method of preparation hirudin was relatively simple, the purity of the experimental samples and ATU recovery were both high.

Animals ; Emulsions ; chemistry ; Hirudins ; analysis ; isolation & purification ; Leeches ; chemistry ; Membranes, Artificial ; Solid Phase Extraction ; instrumentation ; methods

OBJECTIVETo observe the effect of local injection of recombinant hirudin on survival of skin flaps with venous congestion in a rabbit model.

METHODSEighteen healthy rabbits were enrolled and divided into heparin-treatment (HT), recombinant hirudin treatment (RHT) and control (C) groups according to the random number table, with 6 rabbits in each group. After intravenous anesthesia with 20 g/L pentobarbital sodium, model of skin flaps with venous congestion in the size of 6 cm × 3 cm was reproduced in the dorsal side of left ear of each rabbit, in which central artery of ear served as the only blood supply, and a pedicle of 1 cm in width including central vessel of ear and its accompanying nerves as the only venous return pathway. Each flap in RHT, HT, C groups was respectively given 1 mL recombinant hirudin (1 U), low-molecular-weight heparin (625 U), and isotonic saline via multi-point and homogenous injection, then they were sutured in site. Appearance and survival rate of the flaps were observed after operation. Specimens of the distal part of flaps were harvested for determination of thromboxane B2 (TXB2) on post operation day (POD) 1, 3, 5, 7. Data were processed with one-way analysis of variance and t test.

RESULTSRabbit model of skin flaps with venous congestion was reproduced successfully. Obvious hair loss was observed in completely necrotic parts of flap in each group. Obvious edema was observed in all flaps with venous congestion at distal site. The color of flaps in HT and RHT groups were lighter as compared with that in C group, and apparent hematoma of flap was observed in 1 rabbit of RHT group, 2 rabbits of HT group, 4 rabbits of C group on POD 1. The survival rate of flap in HT and RHT groups was respectively (92.3 ± 1.7)% and (94.8 ± 1.9)%, both higher than that in C group [(77.9 ± 1.2)%, F = 191.29, P < 0.05]. There was no statistical difference in survival rate of flap between HT group and RHT group (t = 2.75,P > 0.05). The content of TXB2 in HT and RHT groups on POD 3, 5 was respectively lower than that in C group (with t value from 6.68 to 30.55, P values all below 0.01), but there was no statistical difference between HT and RHT groups (with t value respectively 1.22, 6.44, P values all above 0.05).

CONCLUSIONSLocal injection of low-molecular-weight heparin or recombinant hirudin can significantly ameliorate venous congestion of skin flap in rabbit ear, and improve its survival rate.

Animals ; Ear ; blood supply ; Graft Survival ; drug effects ; Hirudins ; pharmacology ; Hyperemia ; Rabbits ; Recombinant Proteins ; pharmacology ; Skin ; blood supply ; Surgical Flaps ; blood supply