CN1502693A - Zymoplasmoid of venom of Deinagkistrodon acutus with hemostatic activity - Google Patents
Zymoplasmoid of venom of Deinagkistrodon acutus with hemostatic activity Download PDFInfo
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- CN1502693A CN1502693A CNA021386307A CN02138630A CN1502693A CN 1502693 A CN1502693 A CN 1502693A CN A021386307 A CNA021386307 A CN A021386307A CN 02138630 A CN02138630 A CN 02138630A CN 1502693 A CN1502693 A CN 1502693A
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- 239000002435 venom Substances 0.000 title 1
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- 238000000746 purification Methods 0.000 claims abstract description 6
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- 206010053476 Traumatic haemorrhage Diseases 0.000 claims abstract description 3
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 10
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invented Dernagkistrodon acutus snake venom thrombase relates to the field of biological medicine and zymology. In particular, it relates to a structural feature of enzyme white is extracted from Dernagkistrodon acutus snake venom and has hemostatic activity, its purification and application in medicine field. The total molecular weight of said enzyme is 32 KD (SDS-PAGE method), and is formed from similar two subunits of 17KD and 15KD. HPLC is single peak, the optimum temp. of said enzyme is 35 deg.C, optimum pH is 7.5 and isoelectric point is 5.9. Said enzyme is composed of 18 or 20 amino acids, and its plasma coagulation test is 60 plus minus 20 sec. and simultaneous fibre source coagulation test is 230 plus or minus 50 sec. Said invention can be used for preventing clinical operative hemorrhage and curing traumatic hemorrhage, etc.
Description
The present invention relates to biological medicine and protein zymetology field.Concretely, it relates to a kind of a kind of constitutional features with Thrombin-like enzyme of styptic activity of extracting, the purposes of purification process and field of medicaments aspect from agkistrodon acutus (Agkisdroton acutus) snake venom.The Thrombin-like enzyme of this hemostatic effect is strengthened blood coagulation by the activation fiber proteinogen, simultaneously because of not activating the XIII factor, can not cause thrombosis.In preclinical study, drawn efficient utmost point hypotoxicity effect.Agkistrodon acutus claims Agkistrodon or long-nosed pit viper again, is distributed in the each province, south China.Arginine ester enzymic activity in its snake venom is lower, impassivity poison and kallikrein activity.In its component, there are several compositions can make people or fine former the condensing of ox, but cause that in animal body various physiological effect is as falling fine anti-freezing, platelet aggregation and hemostasis etc. external.F.Kornalic will be that the snake venom enzyme of substrate is divided into three classes by its mode of action difference with the Fibrinogen: a class is: Thrombin-like enzyme (Thrombin-like enzyme) or blood coagulating protein enzyme (Thrombic protease), they only decompose the N end peptide chain of fibrinogenic α chain and/or β chain, discharge A peptide (α chain) and/or B peptide (β chain), make blood coagulation.Two classes are: Fibrinogen lyase (Fibrinogenolytic enzyme), directly from the C end hydrolysis of three peptide chains of Fibrinogen (α, β and γ), with the former fragment that resolves into of fibre, blood is not solidified for they.Three classes are: Profibrinolysin activating enzyme (Enzyme activating plasminogen), and it is Fibrinolytic indirect activation agent, it produces plasmin by plasminogen activation, comes cracking scleroproein or Fibrinogen.The invention belongs to the first kind. the research of sTLE is Cheng in 1967 the earliest, H.C. and Ouyang, C. etc., they isolated two Thrombin-like enzyme components in succession in 1971 and 1972, and through the experimentation on animals checking, they all have anticoagulation in various degree.Xiao Changhua etc. from 1988 with Guan Jinhua etc. from 1992, repeatedly repay examination and from the agkistrodon acutus snake venom, separate the component that the animal clotting time is shortened, but all because of purifying, can't be accurately qualitative and quantitative to its structure, successfully do not abandon.External similar product has partial purification from the Bothropsjararaca snake venom such as Klobusitzky and Konig to go out a local and non-stomach and intestine hemostatic agent safe in utilization, be named Hemocoagulase, by Milan Ravizzo company extracting section from the Bothrops jararaca snake venom, extract from the Bothropsatrox snake venom with trade(brand)name Bothropase with by Basle Pentapharm company, on sale with the preparation of trade(brand)name Reptilase the America and Europe.The kind that wherein only has commodity to be called reptilase (Reptilase) is built the agency and sales of peace pharmaceuticals by Shenzhen at home, the reptilase determined curative effect, the effect of paying is little, and clinical indication is wide, be better than characteristics such as other hemostatic drug, and entered the China national essential drugs in 1996.
The Thrombin-like enzyme mode of action of the present invention is identical with Reptilase, but its molecular structure is different from reptilase (reptilase) fully.This Thrombin-like enzyme excises acid A peptide in the fine original molecule by enzyme, and human plasma and bovine fibrinogen are solidified external, because of not activating the XIII factor, causes producing non-crosslinked fibrin monomer mixture.In the wound, this monomer complex and other thrombin interact and produce hemostatic effect, and in complete blood vessel, this monomer transformation period is shorter, can be degraded by plasmin rapidly.This Thrombin-like enzyme makes the Citrated clotting of plasma external, can use the clotting time in vivo significantly to shorten, and does not cause thrombosis again simultaneously, reaches the hemostasis of wound location or prevents hemorrhage purpose.
It is hemorrhage to the purpose of this invention is to provide the prevention clinical operation, and the agkistrodon acutus Thrombin-like enzyme of treatment traumatic hemorrhage and whole body and local hemostasis aspect is determined its physicochemical property and constitutional features accurately.
The purpose of this invention is to provide this enzyme molecule solidifying of blood is not subjected to the influence of heparin, be used for measuring clinically going out the clotting time of some use heparin patient so can make diagnostic reagent.
The present invention also aims to provide the comprehensive layout and the chromatography condition of method, the especially purifying process of the agkistrodon acutus Thrombin-like enzyme that purifying has hemostatic effect.
A kind of have a hemorrhage snake venom thrombin-like enzyme of control, and recording its full molecular weight with the SDS-PAGE method is 32KD, form by close two subunits, and molecular weight subunit 17KD and 15KD, this enzyme optimum temperuture is 35 ℃, makes PH 7.5 most, iso-electric point is 5.9.Confirm that according to document and our test-results this Thrombin-like enzyme structure is different fully with reptilase.
Survey this Thrombin-like enzyme amino acid with liquid phase chromatography and consist of (%): Asp6.2, Thrl.43, Ser4.16, Glu8.74, Gly2.14, Ala2.15, Val2.88, Met1.33, Ile2.35, Leu2.28, Tyr2.45, Phe4.16, Lys4.19, His1.58, Arg1.83, Pro1.8, Cys4.34, Trp4.93, NH
32.68.
The purification process of the snake venom thrombin-like enzyme of this hemostatic effect comprises the dissolving of agkistrodon acutus snake venom, Sephedex G75 gel-filtration, and DEAE-Sepharose Fast Flow ion-exchange, dialysis and Sephedex G-25 desalination constitute.It is characterized in that: this snake venom thrombin-like enzyme adopts FPLC DEAE-Sepharose Fast Flow chromatograph packing material to carry out purifying, flow velocity 40ml/min. mobile phase A: 10mmol/L phosphate buffered saline buffer PH8.0, B liquid are the A liquid that contains 0.35mol/L sodium-chlor. gradient is the sodium-chlor of linear 0～0.35mol/L.
The method of pure this enzyme of concrete preparation is: get the agkistrodon acutus snake venom of having verified, and with damping fluid dissolving back centrifugal.Get Sephdaex G-75 chromatography column on the supernatant liquor, DEAE-Sepharose Fast Flow chromatography column on the active peak, DEAE-Sepharose Fast Flow chromatography column is gone up again in active peak after dialysis, collect active peak, is pure product.
Thrombin-like enzyme behind the purifying, the full molecular weight position 32KD with the SDS-PAGE method is surveyed is made up of 17KD and two polypeptide chains of 15KD. and positive HPLC is single band for simple spike .PAGE, at the external Freshman blood plasma that can condense.
Pharmacodynamics checking in the body: the agkistrodon acutus Thrombin-like enzyme behind the purifying is through New Zealand white rabbit ear vein and buttocks intramuscular injection injection, three dosage groups [0.01 are adopted in experiment, 0.025,0.05 (U/kg)], the result all makes whole blood clotting time (table 1) obviously shorten, and makes drug effect keep 6 hours more than the reptilase.Each test dose group intravenous injection makes fibrinogen content (table 2) and blood viscosity (table 7) is constant or minimizing.To rabbit activated partial thromboplastin time (table 3), platelet counts (table 4) does not have obviously influence.To euglobulin lysis time (table 5), but its shortening during heavy dose, the middle low dose of obviously influence of not having.Onset in this product ear vein injection 10 minutes reached the peak in 30 minutes.Reptilase positive controls and physiological saline negative control group are set up in test.No matter intramuscular injection or quiet notes, this product group drug effect of same dose is close with the reptilase positive controls, but is better than physiological saline group control group.Experiment mice tail vein is cut off hemorrhage experiment (table 6), these product (0.5U/Kg, 1.0U/Kg, 2.0U/Kg) abdominal injection, each dosage group mouse tail vein bleeding time is compared with the reptilase group with the physiological saline group, shortening is in various degree all arranged, and wherein 0.5u/kg dosage group and control group relatively have significant difference (P＜0.01).
Hemostasis Thrombin-like enzyme of the present invention, its feature are that also this enzyme molecule can pass through hemato encephalic barrier, and the treatment cranium is hemorrhage.
The approach that is suitable for commonly used has: intramuscular injection, intravenous injection; Can the part or whole body use etc.
Table 1. agkistrodon acutus Thrombin-like enzyme (sTLE) is to the influence of new zealand rabbit whole blood clotting time (X ± SD)
The influence of table 2. agkistrodon acutus Thrombin-like enzyme (sTLE) New Zealand white rabbit fibrinogen content (X ± SD)
Table 3. agkistrodon acutus Thrombin-like enzyme (sTLE) is to the influence of New Zealand white rabbit APTT (X ± SD)
Table 4. agkistrodon acutus Thrombin-like enzyme (sTLE) is to the influence of platelet counts
Table 5. agkistrodon acutus Thrombin-like enzyme (sTLE) is to the influence of rabbit euglobulin lysis time.
Table 6. agkistrodon acutus Thrombin-like enzyme (sTLE) is cut the influence in tail bleeding time to mouse
Table 7. agkistrodon acutus Thrombin-like enzyme (sTLE) is to the influence of new zealand rabbit whole blood viscosity (mPaS)
Real row one: get agkistrodon acutus snake venom 1 gram of having checked, after adding phosphate buffered saline buffer dissolving snake venom, 6000 rev/mins of centrifugal precipitations of abandoning, get Sephdaex G-75 chromatography column (4*80cm) on the supernatant liquor, use the phosphate buffered saline buffer wash-out, collect DEAE-SepharoseFast Flow chromatography column (2*15cm) on the active peak, with the phosphate buffered saline buffer gradient elution that comprises 0.35M sodium-chlor, collect active peak and after 8～10KD holds back the semi-permeable membranes dialysis, go up DEAE-Sepharose Fast Flow chromatography column (2*15cm) again, with the phosphate buffered saline buffer gradient elution that comprises with 0.35M sodium-chlor, collect active peak, promptly receive pure product 10.6mg.
Thrombin-like enzyme behind the purifying, the full molecular weight position 31849D that surveys with the SDS-PAGE method, two molecular weight subunit 16465D and 15384D, molecular sieve HPLC is a simple spike, purity 100%.PAGE is single band, at the external Freshman blood plasma that can condense.Albumen is than 15.38U/mg alive (reptilase unit).Iso-electric point is 5.9.Consist of (%): Asp6.2, Thr1.43, Ser4.16, Glu8.74 with the Thrombin-like enzyme amino acid behind the purifying of liquid phase chromatography survey, Gly2.14, Ala2.15, Val2.88, Met1.33, Ile2.35, Leu2.28, Tyr2.45, Phe4.16, Lys4.19, His1.58, Arg1.83, Pro1.8, Cys4.34, Trp4.93, NH
32.68.
Pharmacodynamics sees Table 1～7.
Real row two: get agkistrodon acutus snake venom 20 grams of having checked, after adding 150ml phosphate buffered saline buffer dissolving snake venom, 6000 rev/mins of centrifugal precipitations of abandoning, get supernatant liquor and divide upward Sephdaex G-75 chromatography column (7*100cm) five times, use the phosphate buffered saline buffer wash-out, collect DEAE-Sepharose Fast Flow chromatography column (4.5*30cm) on the active peak, with the phosphate buffered saline buffer gradient elution that comprises with the 0.35M sodium chloride, collect active peak and after 8～10KD holds back the semi-permeable membranes dialysis, go up DEAE-Sepharose Fast Flow chromatography column (4.5*30cm) again, with the phosphate buffered saline buffer gradient elution that comprises 0.35M sodium-chlor, collect active peak, promptly get pure product 189.9mg.
Thrombin-like enzyme behind the purifying, the full molecular weight position 32540D that surveys with the SDS-PAGE method, two molecular weight subunit 17036D and 15504D, molecular sieve HPLC is a simple spike, purity 97.5%.PAGE is single band, at the external Freshman blood plasma that can condense.Enzyme is than 17.5U/mg alive.
Pharmacodynamics sees Table 1～7.
Table 1. agkistrodon acutus Thrombin-like enzyme (sTLE) is to the influence of new zealand rabbit whole blood clotting time (X ± SD)
| Group dosage (u/kg) n 0min 10min 30min 1hour 3hour 6hour | |
| ????sTLE | ??0.05?????????6????535.7±257.9???381.9±83.1??????294.1±203.2?????368.3±120 *??????361.9±133.3??????286.2±163 *??0.025????????6????581.8±199.6???345.7±143. *????342.1±103 *?????286.9±133.5 *????411±104.3 *?????329.4±91.1 *??0.01?????????6????592.7±189.2???376.9±113.3 *???412±244?????????424.5±150 *??????292.6±249.2 *???345.1±214.4 |
| Reptilase | ??0.025????????6????811.4±148.7???423.4±230.4?????364.6±174.5 *???385.6±134.5 *????546.3±161.6??????554.69±162. * |
*P＜0.05?by?paired?t?test
The influence of table 2. agkistrodon acutus Thrombin-like enzyme (sTLE) New Zealand white rabbit fibrinogen content (X ± SD)
| Group dosage (u/kg) n 0 10min 30min 1hour 3hour 6hour | |
| ?sTLE | ?0.05????????6????156.0±32.1????188.0±21.6????186.2±20.9?????174.3±27.2?????178.1±23.7?????209.9±19.9 ?025?????????6????222.1±47.4????210.7±44.4????204.3±60.2?????206.6±53.9?????209.0±53.1?????219.0±58.6 ?0.01????????6????254.0±56.1????247.3±55.7????218.7±107??????249.0±57.7?????218.2±42.9 *???221.8±83.7 |
| Reptilase | ?025?????????6????265.3±34.9????219.8±31.3 *??216.5±25.9 *??220.7±24.4 *???221.5±38.5 *???212.5±24.7 ** |
*P＜0.05，
**P＜0.01?by?paired?t?test.
Table 3. agkistrodon acutus Thrombin-like enzyme (sTLE) is to the influence of New Zealand white rabbit APTT (X ± SD)
| Group dosage (u/kg) n 0 10min 30min 1hour 3hour 6hour | |
| ?sTLE | ?0.05????????6????66.10±17.89????77.40±21.74????76.57±25.85??????67.58±17.93??????81.98±15.67?????60.43±8.62 ?025?????????6????84.52±14.67????77.48±14.89????72.28±16.78??????72.33±15.56 **???73.45±11.86 **??83.92±12.43 ?0.01????????6????75.35±15.17????76.20±16.35????78.70±19.06??????72.68±13.36??????56.03±7.02 *????56.95±14.03 * |
| Reptilase | ?025?????????6????80.38±18.07????74.98±13.23????69.15±14.43 *????70.63±13.36??????70.60±10.91?????63.75±12.37 * |
*P＜0.05，
**P＜0.01?by?paired?t?test.
Table 4. agkistrodon acutus Thrombin-like enzyme (sTLE) is to the influence of platelet counts
Group number of animals platelet counts (10
9/ L)
10min??????????????1h????????????????????3h
Physiological saline group 7 319.4 ± 72 317.7 ± 79.6 237.1 ± 90.3
The sTLE group
0.025u/kg?????????7????????????288.0±79.1????????255.0±59.6???????????233.0±12.7
0.05u/kg??????????7????????????272.0±77.9????????226.1±46.4
*?????????272.2±29
0.1u/kg???????????7????????????339.0±88.1????????330.9±88.7???????????238.3±70.3
Reptilase 0.05u/kg 7 267.0 ± 73.9 306.2 ± 81.2 269.4 ± 62.1
Compare with the physiological saline group
*P＜0.05
Table 5. agkistrodon acutus Thrombin-like enzyme (sTLE) is to the influence of rabbit euglobulin lysis time
Group number of animals euglobulin lysis time
(n)??????10min?????????????60min?????????????180min
Physiological saline 7 246.0 ± 7.7 225.1 ± 76.4 191.4 ± 3.8
sTLE?0.025u/kg????7????????133.8±7.4
**?????141.7±34.7
*?????235.0±7.6
***
0.05u/kg?????8????????169.0±96.6???????156.3±3.5????????151.4±3.8
***
0.1u/kg??????8????????85.0±28.3
***?????75.0±28.3
***????136.3±7.4
***
Reptilase 0.05u/kg 8 211.4 ± 85.5 138.8 ± 86.3 286.3 ± 90.4
*
Compare with the physiological saline group
*P＜0.05
*P＜0.01
* *P＜0.001
Table 6. agkistrodon acutus Thrombin-like enzyme (sTLE) is cut the influence in tail bleeding time to mouse
Group dosage (u/kg) the route of administration number of animals bleeding time
Physiological saline---Ip 10 34.1 ± 11.1
sTLE????????????0.5?????????????Ip???????????10????????????19.7±7.5
**
sTLE????????????1.0?????????????Ip???????????11????????????26.9±12
sTLE????????????2.0?????????????Ip???????????12????????????26±18.0
Reptilase 0.5 Ip 10 28.7 ± 17.1
*Compare P＜0.01 with the physiological saline group
Table 7. agkistrodon acutus Thrombin-like enzyme (sTLE) is to the influence of new zealand rabbit whole blood viscosity (mPaS)
Group number of animals shear rate 38.4s
-1
10min???????????1h?????????????3h
Physiological saline group 7 8.22 ± 3.46 7.01 ± 1.20 6.89 ± 2.70
The sTLE group:
0.025u/kg?????????7?????????6.89±1.00??????6.09±0.80?????6.29±1.32
0.05u/kg??????????7?????????7.21±1.40??????6.08±1.65?????5.74±0.90
0.1u/kg???????????7?????????7.28±0.95??????7.48±2.02?????6.39±1.85
Reptilase 0.05u/kg 7 7.43 ± 1.88 6.02 ± 0.86 6.46 ± 1.56
Claims (8)
1. prevent and treat hemorrhage sTLE for one kind. it is characterized in that: its full molecular weight 32KD, form iso-electric point 5.9 by 17KD and two polypeptide chains of 15KD.
2. hemostasis Thrombin-like enzyme according to claim 1 is characterized in that: amino acid consists of (%): Asp6.2, Thr1.43, Ser4.16, Glu8.74, Gly2.14, Ala2.15, Val2.88, Met1.33, Ile2.35, Leu2.28, Tyr2.45, Phe4.16, Lys4.19, His1.58, Arg1.83, Pro1.8, Cys4.34, Trp4.93, NH
32.68.
3. hemostasis Thrombin-like enzyme according to claim 1 is characterized in that: external, promote the acidifying clotting of plasma of people's citron and bovine fibrinogen is solidified, the clotting time is shortened.
4. the purification process of the hemorrhage sTLE of control: comprise and get the agkistrodon acutus snake venom of having verified, centrifugal with damping fluid dissolving back, get Sephdaex G-75 chromatography column on the supernatant liquor, DEAE-Sepharose Fast Flow chromatography column on the active peak, DEAE-Sepharose FastFlow chromatography column is gone up again in active peak after dialysis, collect active peak, be pure product.
5. the purification process that comprises described this Thrombin-like enzyme of claim 4, it is characterized in that: this snake venom thrombin-like enzyme adopts FPLC DEAE-Sepharose Fast Flow chromatograph packing material to carry out purifying, flow velocity 40ml/min. mobile phase A: 10mmol/L phosphate buffered saline buffer PH8.0, B liquid are the A liquid that contains 0.35mol/L sodium-chlor. gradient is the sodium-chlor of linear 0～0.35mol/L.
6. hemostasis Thrombin-like enzyme according to claim 1 is characterized in that preventing clinical operation hemorrhage, treatment traumatic hemorrhage and whole body and local hemostasis.
7. hemostasis Thrombin-like enzyme according to claim 6, but it is characterized in that not only intramuscular injection, but also intravenous injection and intravenous drip.Not only whole body also can locally use.
8. according to claim 1 and 6 described hemostasis Thrombin-like enzymes, it is characterized in that this enzyme molecule is not subjected to the influence of heparin to solidifying of blood, be used for measuring clinically going out the clotting time of some use heparin patient so can make diagnostic reagent.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010034176A1 (en) * | 2008-09-27 | 2010-04-01 | 康辰医药股份有限公司 | A throbin-like enzyme of agkistrodon acutus |
| CN101812436B (en) * | 2009-12-30 | 2011-12-28 | 唐松山 | Agkistrodon acutus venom thrombin-like enzyme, preparation method and application thereof |
| CN101684151B (en) * | 2008-09-26 | 2012-06-20 | 江苏正大天晴药业股份有限公司 | Protein matter with coagulation activity |
-
2002
- 2002-11-22 CN CNA021386307A patent/CN1502693A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101684151B (en) * | 2008-09-26 | 2012-06-20 | 江苏正大天晴药业股份有限公司 | Protein matter with coagulation activity |
| WO2010034176A1 (en) * | 2008-09-27 | 2010-04-01 | 康辰医药股份有限公司 | A throbin-like enzyme of agkistrodon acutus |
| US8476054B2 (en) | 2008-09-27 | 2013-07-02 | Konruns Pharmaceutical Co., Ltd. | Thrombin-like enzyme of Agkistrodon acutus |
| CN101812436B (en) * | 2009-12-30 | 2011-12-28 | 唐松山 | Agkistrodon acutus venom thrombin-like enzyme, preparation method and application thereof |
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