BACKGROUND AND OBJECTIVES: Thrombin and its interaction with platelets play a pivotal role in arterial thrombus formation. Hirudin, an anticoagulant agent derived from medicinal leeches(Hirudo medicinalis), is a unique and specific thrombin inhibitor with no effect on other serine protease. We investigated the inhibitory effect of hirudin on platelet deposition in a rabbit carotid artery eversion model of acute arterial thrombosis. MATERIALS AND METHODS: The everted arterial segments were perfused with 111 Indium-labeled human platelets only(control, n=8), and a mixed solution of 111 Indium-labeled human platelets and hirudin(30, 45, 60, 90 microgram/ml, n=3, respectively). Platelet deposition was calculated by a gamma-counter and confirmed by scanning electron microscopy. RESULTS: 1) Indium-111 labeling efficiency of platelets was 87.0+/-6.6%, and the aggregation of platelets was not changed after labeling. The number of platelets perfused through each arterial segment was 4.3 +/-0.2x10(8) platelets/ml. 2) The control group showed a platelet deposition rate of 23.9+/-7.0 % and a number of platelet deposition of 9.8+/-2.5x10(8) platelets/cm2 . 3) Platelet deposition of arteries perfused with hirudin(60 microgram/ml) was significantly decreased compared with that of the control group(2.9+/-0.6 vs 9.8+/-2.5x10(8)/cm2 , p<0.05). 4) The number of deposited platelets in hirudin-perfused arteries was dose-dependently decreased(30 microgram/ml:6.7+/-1.4x10(8) /cm2 , 45 microgram/ml: 4.8+/-1.7x10(8) /cm2 , 60 microgram/ml: 2.9+/-1.8x10(8)/cm2, 90 microgram/ml:2.9+/-1.4x10(8)/cm2: p<0.05 vs. control, respectively). 5) Scanning electron microscopic examination revealed significantly reduced platelet deposition in hirudin-perfused groups compared with control group. CONCLUSION: Hirudin inhibits effectively platelet deposition and arterial thrombus formation in a rabbit carotid artery eversion model. The antiplatelet effect of hirudin in this model suggests that hirudin may be an useful antithrombotic agent therapeutically useful in the prevention of acute arterial thrombus formation.
Arteries ; Blood Platelets ; Carotid Arteries* ; Hirudins* ; Humans ; Microscopy, Electron, Scanning ; Serine Proteases ; Thrombin ; Thrombosis

A 45 year old man was admitted for aggravated dyspnea, abdominal distension and poor oral intake.On Echocardiogram,mitral stenosis(severe),tricuspid regurgitaion(IV),and LA thrombus were diagnosed.We used heparin with continuous infusion for prevention of systemic thrombo embolism. On the 11 th day of admissin, the patient showed thrombocytopenia and we suspected Heparin-induced thrombocytopenia.Hirudin was used in this case as alternative anticoagulant during cardiopulmonary bypass to prevent serious complication of heparin.The patient was recovered without any complication as postoperative bleeding or systemic thromboembolism.
Cardiopulmonary Bypass ; Dyspnea ; Embolism ; Heart* ; Hemorrhage ; Heparin ; Hirudins ; Humans ; Middle Aged ; Thoracic Surgery* ; Thrombocytopenia* ; Thromboembolism ; Thrombosis

Heparin-induced thrombocytopenia (HIT) is a heparin-dependent antibody-mediated platelet activating syndrome frequently accompanying thrombocytopenia, thromboembolism. We experienced a case of cardiopulmonary bypass using hirudin, a direct thrombin inhibitor, in a patient with HIT. The patient who showed thrombocytopenia and thrombosis after heparin re-exposure was highly suspected of having HIT. Hirudin was used in this case as an anticoagulating agent during cardiopulmonary bypass (CPB) to prevent serious complications of heparin. Hirudin 0.3 mg/kg was mixed with a priming solution of CPB and a 0.2 mg/kg IV bolus followed by a continuous infusion of hirudin 0.15 mg/kg given for anticoagulation. After CPB, forced diuresis and platelet transfusion was performed and the patient was recovered without complication.
Blood Platelets ; Cardiopulmonary Bypass* ; Diuresis ; Heparin ; Hirudins* ; Humans ; Platelet Transfusion ; Thrombin ; Thrombocytopenia* ; Thromboembolism ; Thrombosis

Four different methanol feeding modes were evaluated in the hirudin production in high-density fermentation by Pichia pastoris. It was difficult to avoid methanol excessive in the broth with the feeding strategy only based on DO level. On the other hand, the fluctuation in methanol concentration was observed with methanol feeding strategy by off-line gas chromatography. However, the stable methanol concentration was perfectly achieved by the on-line monitoring with methanol sensor. The supply of energy was improved by feeding glycerol at a limited rate as well as methanol in the induction phase. Therefore, the high cell dry weight (162 g/L) and high hirudin activity (2.4 x 10(4) ATU/mL or 1.7 g/L) was obtained in the fed-batch fermentation of recombinant Pichia pastoris by methanol-glycerol mixed feeding.
Fermentation ; Glycerol ; pharmacology ; Hirudins ; biosynthesis ; Methanol ; pharmacology ; Pichia ; genetics ; metabolism ; Recombination, Genetic

Chemostat culture was performed to characterize the growth, substrate consumption and the hirudin production, and to disclose their interrelations in the fermentation of recombinant Pichia pastoris. The Andrew substrate-inhibited growth model is more suitable than Monod model to simulate the growth of Pichia pastoris on methanol. Therefore, two stationary states can be obtained in the continuous culture at a certain dilution rate because of the substrate inhibition on cell growth. The stationary state could be obtained if only the dilution rate not more than 0.048 h(-1) in the continuous fermentation. The concentrations of cell, methanol and hirudin were constant after 50 h continuous culture with dilution rate at 0.04 h(-1). However, it could not be obtained when the dilution rate more than 0.048 h(-1) because the other stationary point at S > 0.048 h(-1) is unstable. Therefore, it was found that the cell concentration declined and the methanol concentration increased from 2.9 g/L to 18.1 g/L within 18h at dilution rate 0.06 h(-1). Thus, the fed-batch culture with a constant specific growth rate was carried out to disclose the fermentation behavior at high and constant methanol concentration in aid of a methanol sensor. The theoretical maximum specific growth rate, microm = 0.0464 h(-1), was found under critical methanol concentration, Scrit = 3.1 g/L. The growth of P. pastoris was typically methanol-limited at the methanol concentration S < Scrit. It was, however, inhibited at S > Scrit. The maximum specific Hir65 production rate qp was obtained at 0.2 mg/(g x h) when methanol concentration and mu were 0.5 g/L and 0.02 h(-1), respectively. The specific Hir65 production rate qp increased with the increase of mu and S at mu < 0.02 h(-1), and decreased at mu > 0.02 h(-1). The specific methanol consumption rate increased with the increase of S when S < 5 g/L, but decreased when S > 5 g/L. At last, the high Hir65 production rate 0.2 mg/(g x h) was obtained in the fermentation conducted under methanol-limited concentration and mu controlled at 0.5 g/L and 0.02 h(-1), respectively, while the specific methanol consumption rate is low only at 0.04 g/(g x h), showing the potential for the strategy of getting high Hir65 production rate at the low consumption of methanol.
Fermentation ; physiology ; Hirudins ; metabolism ; Methanol ; metabolism ; Pichia ; growth & development ; metabolism ; Recombination, Genetic

OBJECTIVETo determine the contents of hirudin's hydrolysates in processed leeches, set up a new evaluating method of dot blotting to evaluate the qualities of those processed Chinese medicines as leeches.

METHODContents of hirudin's hydrolysates in processed leeches were determined by dot blotting with rat antibody of anti-hirudin as the first antibody. Blotting signal was analyzed by software of Quantity One.

RESULTContents of hirudin's hydrolysates in four batches of processed leeches were 296.51, 165.47, 95.58, and 298.05 microg g(-1), respectively.

CONCLUSIONDifference among four batches of processed leeches was significant in the content of hirudin's hydrolysates. Dot blotting, as a convenient and accurate method can be broadly used for evaluating processed products of Chinese crude drugs similar to leeches.

Animals ; Drugs, Chinese Herbal ; chemistry ; Hirudins ; immunology ; metabolism ; Immunoblotting ; methods ; Leeches ; chemistry ; Reproducibility of Results

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

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

OBJECTIVETo study the effect of hirudin on the function of human hyperplastic scar fibroblasts (HSFBs).

METHODSHSFBs were cultured in vitro. Hirudin solution in the concentration of 1, 10, and 50 kU/L was respectively added into DMEM culture medium to form 1, 10, and 50 kU/L hirudin groups, with 9 wells in each group. HSFBs cultured without hirudin were set up as control group. Cell inhibition rate, secretion level of TGF-beta1 from cells, and expression levels of mRNA of type I and III precollagen were determined at 24, 48, and 72 h after culture.

RESULTSInhibition rates of HSFBs growth was respectively (29.3 +/- 0.9)%, (30.1 +/- 0.3)%, and (45.2 +/- 1.9)% when cultured with 10 kU/L hirudin for 24, 48, and 72 hs, which were higher than those in control group [(0.0 +/- 0.0)%, P < 0.05]. There was statistically significant difference between control group and 1 and 50 kU/L hirudin groups in the inhibition rates of HSFBs at some time points (P < 0.05). Secretion level of TGF-beta1 of HSFBs in 1, 10, 50 kU/L hirudin groups was respectively (228.5 +/- 1.8), (210.5 +/- 11.1), and (168.5 +/- 14.1) pg/mL when cultured for 48 hs, of which the last 2 figures were significantly lower than that of control group [(265.0 +/- 1.5) pg/mL, P < 0.05]. Hirudin in the concentration of 10 and 50 kU/L could inhibit the expression of mRNA of type I and III precollagen in HSFBs.

CONCLUSIONSHirudin solution in the concentration of 10 and 50 kU/L can inhibit the proliferation of HSFBs and secretion of TGF-beta1 and collagen in certain degree.

Cells, Cultured ; Cicatrix, Hypertrophic ; pathology ; Fibroblasts ; cytology ; drug effects ; secretion ; Hirudins ; pharmacology ; Humans ; Transforming Growth Factor beta1 ; metabolism

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