British Journal of Haematology, 1971, 21, 43. Reptilase@'-R-A New Reagent in Blmd

C. FUNK,J. GMUR,R. HEROLDAND P. W. STRAUB Department of Medicine, Kantonsspital, University of Zurich, Switzerland

(Received 8 September 1970; accepted for piiblication 29 September 1970)

SUMMARY.Reptilase@-R, a purified -like snake-venom enzyme from Bothrops atrox, has been tested for its usefulness as a substitute for thrombin in two coagulation assay systems. Reptilase was found to be more stable than thrombin and not to be inhibited by and . Performance of the allows the rapid exclusion of heparin con- tamination in plasma samples with prolonged thrombin times. In the presence of fibrinogenlfibrin split products, the Reptilase time is less prolonged than the , whereas in patients with prolonged thrombin times because of congenital dysfibrinogenaemia the inverse is found. Thus, performance of both Reptilase time and thrombin time allows the rapid identification of three major causes for a prolongation of the thrombin time. In patients on heparin therapy because of a defibrination syndrome, the Reptilase time may replace the thrombin time as a guide for the degree of coagulation inhibition by FDP, although our re- sults show that it is somewhat less sensitive. Reptilase can also be substituted for thrombin in the rapid chronometric fibrino- gen assay. Unfortunately the advantages of reagent stability and insensitivity to heparin are balanced by a lesser reproducibility, especially in the presence of FDP.

While thrombin liberates the fibrinopeptides of both the a- and p-chains of , the thrombin-like snake-venom enzyme Reptilase exclusively acts on the a-chain, thus leading to coagulation by liberating only fibrinopeptide A (Blombick et al, 1957; Stocker & Straub, 1970). Since Reptilase, in contrast to thrombin, is stable, and because we expected that normal human plasma would be devoid of any inhibitor directed against this exogenous substance, we have tested the usefulness of Reptilase as a substitute for thrombin in various assay systems.

METHODS

For the preparation of plasma nine parts of blood were collected directly into one part of 0.1 M sodium- in siliconized glass tubes or into one volume of a three: two mixture of 0.166 M sodium oxalate with Trasylol 5000 u/inl. For some experiments nine parts of blood were collected into one part of 3.8% (w/v) sodium citrate. Dilutions were done in veronal-acetate buffer, pH 7.39 (Michaelis). Routine methods were used for the assay of clotting factors (Duckert, 1958). Fibrinogen (factor I) was determined with a biuret method on a washed clot, with the h.eat

D 43 44 C. Funk et a1 precipitation method of Schulz (1955) and with the thrombin-clotting time method of Clauss (1957). In the latter method the clotting time of 0.1 ml of I in 10 diluted plasma is recorded after addition of 0.1 ml of a thrombin dilution. When Reptilase (34 mg/ml) was substituted for thrombin in this assay, one volume of the test plasma was diluted with four volumes of ammonium acetate buffer, pH 7.1 (Shainoff & Page, 1962). Calibration curves were estab- lished using normal plasmas with a known (biuret method) fibrinogen content and dilutions thereof. Thrombin time and Reptilase time were recorded after addition of 0.1 ml of a thrombin dilution or Reptilase to 0.2 ml of undiluted plasma. The dilution of thrombin was chosen so as to give the same normal range of 13-18 s as undiluted Reptilase. was assayed using a method (von Kaulla & Schultz, 1958) and the fibrin plate technique (Astrup & Miillertz, 1952). Plasminogen was determined according to Alkjaersig et a1 (1959). Fibrinogenlfibrin degradation products (FDP) were detected qualitatively using immunoelectrophoresis (Straub et aE, 1966) and quantitatively using the tanned red cell haemagglutination inhibition immunoassay (TRCHII) of Merskey et a1 (1966). Fibrinogen degradation products (FDP) were produced by incubation of purified fibrinogen with Urokinase or plasmin for various periods. The fibrinolytic activity was blocked by addition of Trasylol400 u/ml.

Materials Purified fibrinogen ‘Kabi’, 95% clottable, was obtained from Globopharm AG, Zurich. Bovine thrombin ‘Roche’ was acquired from Hoffmann-La Roche, Basel, one vial containing 5000 NIH-U = IOO mg of the dry powder. Hirudin, Grade 111, was obtained from Sigma Co., St Louis, U.S.A. Reptilase (reagent-grade) was obtained from Pentapharm Ltd, Basel, one vial containing 34 mg of the dry powder, which is reconstituted to I ml with distilled water. The solution is already buffered with glycine and stabilized with partially hydrolysed gelatin. It is stable for 5 days at 4°C and for 24 hr at room temperature. Trasylol ‘Bayer’ ampoules of 5 ml (5000 u/ml) was used. Urokinase was obtained through the courtesy of Dr F. Duckert, Burgerspital, Basel.

RESULTS I. REPTILASE TIME VERSUS THROMBIN TIME For the comparison of Reptilase times with thrombin times Reptilase was used undiluted (34 mg/ml) and thrombin was diluted so as to produce identical clotting times in normal plasma as Reptilase.

Injuence of Heparin and Hirudin As shown in Table I, the Reptilase time is not influenced by increasing amounts of heparin in vitro. Similarly the Reptilase time is found normal in heparinized patients (Table 11).

Injluence of FibrinogenlFibrin Split Products and of Congenital Fibrinogen Variants Fig I shows Reptilase and thrombin times obtained in mixtures of normal plasma with Reptilase and Blood Coagulation 45 ncreasing amounts of FDP, as obtained by digestion of fibrinogen ‘Kabi’ to a thrombin time of 60 s. The Reptilase time is obviously less sensitive to interference by FDP than the thrombin time. This was also observed during progressive digestion of normal plasma with Urokinase, as shown in the lower part of Fig 2. Thus, early FDP had the same effect as later products. Fig I also demonstrates that admixture of dysfibrinogenaemia plasma produces the opposit

TABLEI. Influence of heparin and hirudin addition to normal plasmas in vitro on thrombin- and Reptilase- times

Clottirig time (3) of normal plasma containing increasing amounts of heparin and hirudin or buffer

Heparin (NIH-U/ml) Hirudin I rng/nil I 2

Thrombin 4 NIH-U/ml Plasma I co Plasma 2 co Plasma 3 co Reptilase 34 mg/ml Plasma I I4 Plasma 2 16 Plasma 3 I3

Fibrinogen values Thrombiti time Reptilase time

Heat precipit. Clam method (mg%) 4 NIH-U/ml 16 NIH-U/ml 34 wlml (v%) (4 (4 (3) Thrombin Reptilase 33 ulml 34 mginil

Group I (u = 10) ISO-330 120-380 115-360 > 60 9-> 60 13-19 Group I1 (n = 6) 100-350 75-210 125-390 > 60 > 60 12-16

Normal values 150-soo 150-500 150-500 13-IS* 5-9 12-IS*

relation between Reptilase and thrombin times. Identical results were obtained when citrate instead of oxalate was used as an . As shown in Fig 3 and Table 111, the Reptilase time was consistently shorter than the throm- bin time in 18 patients with elevated serum FDP and a prolonged thrombin time not nor- malized by BaSO, adsorption and thus not due to heparin, while it was longer than the 46 C. Funk et a1 thrombin time in all I I tested patients with congenital dysfibrinogenaemia. Details of the latter cases have been reported earlier (von Felten et af, 1966; Funk & Straub, 1970).

loo/ (0) I I I I *O I 1 I

40

20 p--" L - I I I

Normal plasma in mixture (%) FIG I. Comparison of thrombin times (0)and Reptilase times (0)in mixtures of normal plasma with increasing amounts of plasma of a patient (a) with Fibrinogen Zurich, (b) with a fibrinogen digest obtained with urokinase and (c) with buffer.

11. RAPID CHRONOMETRIC FIBRINOGEN DETERMINATIONS USING REPTILASE INSTEAD OF THROMBIN Normal lndivicltials Fig 4 shows typical calibration curves for the Clauss method using Reptilase and thrombin, and Fig 5 shows the correlation between fibrinogen values as determined with Reptilase and with thrombin in 27 normal subjects. Reptilase values are obviously less reliable than values obtained with thrombin, especially in the low and high range. Reptilase and Blood Coagulation 47 Iv$uence of Heparin and Himdin The fibrinogen values as obtained with Reptilase were compared with the values obtained with two different dilutions of thrombin in plasmas before and after in vitro addition of various amounts of heparin or hirudin. The Reptilase assay is insensitive to these inhibitors. Table I1 shows the fibrinogen values in two groups of heparinized patients: The first group

Urokinase

:' 0.. \ '.\ \ 50 - -., \ ...)A-- - - -6- 0.. - --- ', -. a------*. +/

Reptilase

Is, ...G '0 20-

II I I I 0 10 35 60 90 '-0 Incubation time (min)

FIG 2. Effect of urokinaseinduced fibrinolysis on normal plasma. Upper graph: fibrinogen as deter- mined by heat precipitation, .;biuret determination of clottable protein, 0;chronometric method of Clauss using thrombin, A ; and chronometric method using Reptilase, 0. had conventional heparin therapy, the second group consists of six patients who had under- gone cardiac surgery using the extracorporeal circulation immediately before the assay was done. The fibrinogen assay using Reptilase gives accurate values even in highly heparinized patients.

Itlflztence of Fibrinogen Split Products Fig z shows the influence of fibrinogen split products, as produced by incubation of normal plasma with Urokinase, on the results of the different fibrinogen assays. The Reptilase assay is even more affected by split products than the assay with thrombin. It is noteworthy that 48 C. Funk et a1 because of difficulties in endpoint detection the Reptilase values were not well reproducible and therefore the variation was considerable. The values obtained with the heat precipitation method remained virtually unchanged because this method measures fibrinogen plus some FDP.

I2C

I00

- 8C -m ._E c

;60 4 D ._-c a d

40 OA

A

2c

20 4041 60 60 I00 Thrombin time (5)

FIG 3. Reptilase time versus thrombin time in 11 patients with congenital dysfibrinogenaemia(e), and in patients with elevation of serum FDP due to various disease (0)or due to thrombolytic therapy (A).

DISCUSSION We have found that determination of the Reptilase time, when performed in conjunction with the thrombin time and with reagent concentrations to give identical normal values, is a very valuable screening test for several coagulation disorders. Similar results were obtained with oxalate- or citrate-plasma. When the thrombin time is prolonged and the Reptilase time is normal, heparin is likely to be present in the plasma. By this comparison heparin contamination may thus rapidly be excluded. The presence of heparin in a sample can subsequently be confirmed by BaSO, adsorption, which normalizes the prolonged thrombin time of oxalated plasma when it is due to heparin (Gmur et al, 1970). A prolonged thrombin time associated with a prolonged Reptilase time points to an abnormality in the fibrinogen-fibrin conversion: (a) a Reptilase time longer than the throm- bin time should lead to a search for fibrinogen variants. Such abnormalities are probably Reptihe and Blood Co'gulution 49

TABLE111. Results of coagulation and fibrinolysis assays in patients with elevation of serum FDP due to various diseases and to thrombolytic therapy with urokinase and in patients with dysfibrinogenaemia

Euglob. Quick Thrombin time (s) Reptilase lysis Immuno- Case Diagnosis (%) beJore aj?er time time rlectro- adsorption with BaSO, (s) (hr) phoresis -- I Cirrhosis of liver 37 210 25 24 IS >4 neg. 2 2 Cirrhosis of liver 47 200 15s 22 23 IS >4 neg. 4 3 Foetal death in utero 30 140 76 27 25 IS - neg. 8 4 Chronic hepatitis 43 240 190 23 23 I7 - neg. 16 5 Budd Chiari syndrome 38 300 235 23 24 16 - neg. 2 6 Cirrhosis of liver 53 c 500 660 20 I9 IS - neg. 7 Cl. perfringens septicaemia 32 530 190 32 27 18 >4 pos. 128 8 Budd Chiari syndrome 35 190 140 28 27 18 >4 neg. 8 9 Acute leukaemia 52 I00 70 26 - 18 - pos. 8 10 Carcinoma of prostate 18 SO I4 60 - 40 ? pos. 32

47 150 I00 30 60 zoo 80 30 pos. 64 49 pos. 32 26 neg. 4 84 270 210 20 neg. 2 80 270 180 26 neg. 2 7 58 210 80 30 neg. 16 8 88 240 175 I7 neg. 2

I 7s 22 23 30 >4 neg. neg. 66 30 31 4s >4 neg. neg. 56 25 24 64 >4 neg. neg. 49 48 48 68 >4 neg. neg. - 70 30 4s 4 neg. 66 24 24 3s >4 neg. neg. 78 23 24 30 >4 neg. neg. 8 50 40 39 I20 14 neg. neg. 9 64 27 26 45 >4 neg. neg. I0 70 30 28 36 >4 neg. neg. -- I- Normal values 170-IwI 13-18 13-18 12-18 I >4 neg. 50 C. Funk et a1 much more common than previously expected, as is shown by the rapid succession of per- tinent reports in the literature. (b) When the Reptilase time was less markedly prolonged than the thrombin time, this discrepancy was so far regularly due to the presence of fibrinogen/ fibrin split products. As to the mechanism responsible for the difference between thrombin- and Reptilase time in plasmas with FDP, it may be speculated that both clotting rimes are prolonged by inter- ference of FDP with fibrin monomer aggregation, but that the thrombin time is more pro- longed because of the specific action (Latallo et d, 1964) of FDP.

50

20

51 + I111111 I I I ,0111 20 50 100 200 500 1000 mg % FIG 4. Calibration curves for the chronometric fibrinogen determination with: (a) thrombin, 33 NIH-U/ml; plasma dilution I :IO in Michaelis buffer, pH 7.4; and (b) Reptilase, 34 mg/ml; plasma dilution 1:s in ammonium acetate 0.15 M.

When the above results were presented (Funk et al, 1g70), Britten et a2 (1970)reported on the use of the Reptilase time as a monitor of progress in disseminated intravascular coagula- tion. In patients with the defibrination syndrome treated with heparin these authors found the Reptilase time analogous to the thrombin time. From our own results it appears that in Reptilase and Blood Coagirlation SI heparin-treated patients with the defibrination syndrome and circulating FDP, the Reptilase time may indeed replace the thrombin time as a useful indicator of the degree of coagulation inhibition by FDP. However, it must be stressed that the Reptilase time is less sensitive to FDP than the thrombin time. UnfortunateIy we have not yet had the opportunity to study patients with prolonged thrombin times due to paraproteins. Reptilase can also be substituted for thrombin in the chronometric fibrinogen assay of Clauss. However, the results are less accurate than those obtained with thrombin, particu- larly for high and low fibrinogen concentrations. The test plasma should not be diluted more than I in 5. When higher dilutions are used, the variation becomes too great. However, this difficulty to properly dilute the test plasma is of no importance since, in contrast to chrono- metric assays using thrombin, no natural inhibitors of the enzyme have to be expected. The

r-0.07

0 60 120 180 240 300 360 420 Thrombin (mg %) FIG5. Correlation between fibrinogen values as determined chronometrically with Reptilase and with thrombin in 30 normal subjects.

rapid fibrinogen assay with Reptilase may be definitely useful in plasmas of heparinized patients or plasmas where heparin contamination is suspected. On the other hand, our results demonstrate that the assay is not useful in the presence of fibrinogenlfibrin split products, because of an insufficient reproducibility and because it gives even lower values than the chronometric assay with thrombin. Thus, in our laboratory, Reptilase has not proved to be superior to thrombin in the rapid chronometric fibrinogen assay. Still, because of the stability of Reptilase, the method might be useful in non-specialized laboratories with only occasional fibrinogen determinations, where the Clauss method is impractical due to the instability of thrombin.

ACKNO WLBD GMENT This work has been supported by grant Nr 4963 from the Swiss National Fund for Scientific Research. 52 C.Funk et a1

REFERENCES ALKJAERSIG,N., FLETCHER,A.P. & SHERRY,S. (1959) Zurich 11. (Abstract). 3. Annual Meeting European The mechanism of clot dissolution by plasmin. Society for Clinical Investigation, Scheveningen, 1970. Journal of Clinical Investigafion, 38, 1086. GMUR,J., VON FELTEN,A. & STRAUEI,P.W. (1970) ASTRUP,T. & MULLERTZ,S. (1952) The fibrin plate Gerinnungsuntersuchungen an Nabelvenenblut : method for estimating fibrinolytic activity. Archives fetales Fibrinogen?. Schweizerisclie Medizinishe ofBiochemistry, 40, 346. Wochenschrij, 100, 299. BLOMBACK,B., BLOMBACK,M. & NILSSON,I.M. VON KAULLA,K.N. & SCHULTZ,R.L. (1958) Methods (1957) Coagulation studies on ‘Reptilase’, an extract for the evaluation of human fibrinolysis: studies of the venom from Bothrops jararaca. Thrombosis et with two combined technics. American Journal of Diathesis Haemorrhagica, I, 76. Clinical , 29, 104. BRITTEN,A.F.H., CARD,R.T. & ~~XELKE,C.H., JR LATALLO,Z.S., BUDZY~~SKI,A.Z., LIPI~SKI,B. & (1970) Reptilase time-useful monitor of progress in KOWALSKI,E. (1964) Inhibition of thrombin and of disseminated intravascular coagulation. XIIZ Inter- fibrin polymerization, two activities derived from national Congress of Haematology, Munich, 2-8 plasmin-digested fibrinogen. Nature, 203, I 184. August 1970, Abstract volume, p 109. MERSKEY,C., &EWER, G.J. &JOHNSON,A.J. (1966) CLAUSS,A. (1957) Gerinnungsphysiologische Schnell- Quantitative estimation of split products of fibrino- methode zur Bestimmung des . Acta gen in human serum, relation to diagnosis and Haematologica, 17, 237. treatment. Blood, d), I. DUCKERT,F. (1958) Le diagnostic des coagulopathies. SCHULZ,F.H. (1955) Eine einfache Bewertung von 3. Congr& International de Biologie Clinique, Bruxelles, Leberparenchymschiden. Volumetrische Fibrin- 1957, p 635. Presse Acadtmique Europeenne, bestimmung. Acta Hepatologica, 7, 306. Bruxelles. SHAINOFF,J.R. & PAGE,I.H. (1962) Significance of VON FELTEN,A., DUCKET,F. & FRICK,P.G. (1966) cryoprofibrin in fibrinogen-fibrin conversion. Familial disturbance of fibrin monomer aggregation. Journal ofExperimenta1 Medicine 116, 687. BritishJournal of Haematology, 12, 667. STOCKER,K. & S~UB,P.W. (1970) Rapid detection of FUNK,C., STOCKER,K. & STRAUB,PW. (1970) fibrinopeptides by bidimensional paper electro- Reptilase in diagnosis and evaluation of dysfibrino- phoresis. Thrombosis et Diathesis Haemorrhagica, genemias, with observations in a new fibrinogen 24, 248. variant. XIIIth International Congress of Haematology, STRAUB,P.W., VON FELTBN,A. & FRICK,P.G. (1966) Munich, 2-8 August 1970, Abstract volume, p 13. Recurrent intravascular coagulation with renal FUNK,C. & STRAUB,P.W. (1970) Hereditary abnor- cortical necrosis and recovery. Annals of Internal malities of fibrin monomer aggregation. Fibrinogen Medicine, 64, 643. 本文献由“学霸图书馆-文献云下载”收集自网络，仅供学习交流使用。

学霸图书馆（www.xuebalib.com）是一个“整合众多图书馆数据库资源，

提供一站式文献检索和下载服务”的24 小时在线不限IP 图书馆。 图书馆致力于便利、促进学习与科研，提供最强文献下载服务。

图书馆导航：

图书馆首页 文献云下载 图书馆入口 外文数据库大全 疑难文献辅助工具