Scand. J . Haemat. (1973) 11, 360-366

The Influence of Fibrinogen Degradation Products

on the Reptilase-Time of Plasma

HARALD ARNESEN, M.D., PETER KIERULF, M.D. 8~ HANS CHR. GODAL, M.D.

Haematological Research Laboratory (Chief, H . C . Godal), and Department I X (Chief, K . Aas), Ullevdl Hospital, University Clinic, Oslo, Norway

The Reptilase and thrombin clotting times of plasma containing increasing amounts of various FDP, and from patients on urokinase therapy, were equally prolonged. At the point of visible gelation, equal amounts of fibrin were formed with Reptilase and thrombin, as indicated by generation of N-terminal glycine. Dialysis of the Reptilase reagent markedly increased the effect of FDP on the Rep- tilase time. The presence of glycine in the Reptilase reagent is suggested to counteract the influence of polymerization inhibitors, by lowering pH in the clotting mixture and by decreasing the solubility of fibridfibrinogen.

Reptilase, a proteolytic enzyme from Bo- throps atrox, like thrombin, clots fibrino- gen. The clotting time of plasma with Rep- tilase is not influenced by thrombin in- hibitors such as heparin (Blomback et a1 1957, Funk et al 1971, Latallo et al 1971). In the presence of fibrinogen degradation products (FDP), mainly acting as poly- merization inhibitors (Alkjaersig et al 1962, Arnesen & Godal 1973), some authors have found the Reptilase-time to be less affected than the thrombin time (Funk et a1 1971), while others have failed to demonstrate this discrepancy (Britten et al 1970, Latallo et a1 197 1).

The commercial Reptilase reagent (Penta- pharm, Basle) contains considerable amounts of glycine. Due to its buffer capacity, the presence of this compound may affect the pH of the clotting mixture. Being a pre- cipitant of fibrinogen (Blomback & Blom-

back 1956), glycine might also contribute to the aggregation of fibrin and fibrinogen.

In the present study, Reptilase- and thrombin clotting times were compared in plasma from patients on urokinase therapy, and in mixtures of plasma and purified FDP. To compare the amount of fibrin formed at visible gelation with Reptilase and thrombin, N-terminal amino acid analyses were carried out at intervals during their incubation with purified fibrinogen. Finally, the effect of glycine on the clotting times with Reptilase and thrombin was studied.

MATERIALS AND METHODS

Patients on urokinase treatment. 10 consecutive urokinase-treated patients were part of the Euro- pean urokinase trial in myocardial infarction (1972). Samples were taken at regular intervals 4 times during the 18 h treatment in all patients.

Citrated plasma. 9 vol of blood were collected

REPTILASE-TIME AND FDP 361

into precooled (+ 4O C) plastic tubes containing 1 vol of 0.1 M sodium citrate. Plasma was pro- cessed by centrifugating 2000 g for 20 min at + 4 O C and used fresh or after stored deep-frozen for less than 3 months.

Fibrinogen. Purified human fibrinogen, more than 90 % clottable (Jacobson 1955), and con- taminated with plasminogen, was obtained from Kabi, Stockholm, Sweden. Solutions containing 12 mg/ml were dialysed against 200 vol of (9 parts of 0.3 M NaCl and 1 part of veronal buffer pH 7.4) and stored at - 2 O O C . Before use, the solutions were thawed and diluted to desired concentrations with distilled water and veronal buffer, ionic strength and pH being adjusted to 0.15 and 7.4. (For N-terminal analysis, the pH of one fibrinogen solution was adjusted to 8.7).

Urokinase. Kindly supplied by Hoffmann-La Roche, Basle, Switzerland. Vials containing 20,000 CTA-U were dissolved in phophate buffer pH 7.6 to a concentration of 2000 CTA-U/ml, and stored at - 2 O O C .

Thrombin. Bovine thrombin, Topostasine, Hoff- mann-LaRoche, Basle, Switzerland, was dissolved in 0.15 M NaCl to a final concentration of 30 NIH-U/ml, and stored in lusteroid tubes at -2OO C. Further dilutions were made with 0.15 M NaCl (or as specially stated), and kept at + 4O C during the experiments.

Reptilase reagent was obtained from Penta- pharm Ltd., Basle, Switzerland. The vials were dissolved in 1 ml distilled water as recommended by the manufacturer, immediately before use. Dialysed Reptilase was obtained by dialysis against 200 vol of 0.15 M NaCl for 40 h in the cold.

Soybean trypsin inhibitor (STI), type I-S from Sigma, St. Louis, Mo., USA, was dissolved in phosphate buffer pH 8.0 to a concentration oE 10 mg/ml, and stored at -20° C. Final concentra- tion in all experiments was 0.1-0.4 mg/ml.

Heparin from ‘AL‘, Oslo, Norway, 5000 IE/ml was used. Dilutions were made with 0.15 M NaCI.

Glycine. Glycocoll analytical grade, mol w 75.07 from Merck, Darmstadt, Western Germany, was dissolved in 0.15 M NaCl.

Rabbit antihuman antisera against products D and E from Behringwerke AG, Marburg Lahn, Western Germany, were used.

Verona1 buffer pH 7.4 containing 0.74 % NaCI, ionic strength 0.15 (Owren 1947), was used.

DEAE-cellulose from Serva Entwicklungslabor, Heidelberg, Western Germany, was used. Chroma- tography was performed at room temperature in a 2.0 x 40 cm column equilibrated with phos- phate buffer 0.01 M, pH 8.6 (buffer I). Elution was performed with 300 ml buffer I, followed by a gradient of 375 ml buffer I and 300 ml sodium dihydrogen phosphate buffer 0.3 M, pH 4.3 (buffer 11). The sample size was 25 ml (stage 111 FDP from about 0.25 g fibrinogen), the flow rate about 30 ml per h, and eluate aliquots 7-15 ml.

Progressive fibrinogenolysis. Fibrinogen (1 vol, 3 mg/ml or 1.5 mg/ml) was incubated with uro- kinase (1/5 vol, final conc. 400 CTA-U/ml) at 37O C. At intervals, aliquots were withdrawn, and STI (1/50 vol) added before testing. Maximal anticoagulant effect was obtained after 25 min incubation (FDP-25’).

Purification of products D and E. Fibrinogen was incubated with urokinase at 37O C for 3 h to produce stage 111 FDP (according to Marder et a1 1969). Purification was performed on DEAE- cellulose (vide supra), where the typical pattern of products D and E (as described by Nussen- zweig et a1 1961) was obtained. The peak frac- tions were concentrated, dialysed to obtain a pH 7.4 and ionic strength 0.15, and stored at -20° C. Dilution to desired concentrations was made with saline. Purity of the separated products was checked by double immunodiffusion in 1 % agarose gel (Ouchterlony 1958) with rabbit anti- human antisera against products D and E. Pro- tein concentration of products D and E was de- termined by absorption at 280 nm using extinc- tion coefficient of 20 and 10, respectively (Marder et a1 1969, Nilkhn 1967).

N-terminal analysis was performed according to Edman (1950), modified by Blomback & Yamashina (1958), as described by Kierulf & Abildgaard (1971).

Fibrinogen (42 mg with pH 7.4 or 32 mg with pH 8.7) was incubated at 37OC. Thrombin (1/10 vol, final conc. about 0.025 NIH-U/ml) or Rep- tilase (conc. adjusted with saline to give equal clotting time as with thrombin) was added. The reaction was stopped by initiating N-terminal analysis after 0, 10, 45 and 90 min. The results were expressed as pmol glycine per pmol fibrino- gen using 340,000 as mo1.w. for fibrinogen (Cas- pary & Kekwick 1954).

3 62 H. ARNESEN, P. KIERULF & H. C. GODAL

TABLE I Reptilase and thrombin clotting times in plasma

from patients on urokinase therapy for I8 h

No of patients 10 No of samples 40

% prolongation of Reptilase time 79.9 pre-treatment value Thrombin time 76.2

Test system: 0.2 ml citrated plasma was incubated at 37O C for 60 s before the addition of 0.1 ml of Reptilase or thrombin. The time of visible gela- tion was recorded. All tests in duplicate. Control values in normal plasma were 24-30 s. The results have been expressed as % prolongation of pre- treatment values. Lowest fibrinogen value with the method of Jacobson (1955): 75 mg%.

INCUBATION (MIN.)

THROMBIN CLOTTING TIME (-1 AND REPTILASE-TIME (-1

Figure 1. Reptilase and thrombin clotting times during progressive fibrinogenolysis. FDP/F indi- cates the concentration of FDP, expressed as fibrinogen originally present in the incubation mixture (cfr. Methods), and of fibrinogen as mea- sured in the normal plasma used in the test sys- tem. - Test system: 0.2 ml normal citrated plasma incubated with 0.2 ml FDP (or saline as control) at 37O C for 60 s before addition of 0.2 ml of Reptilase or thrombin. Results recorded and ex- pressed as in Table I.

RESULTS

Patients on urokinuse therapy. As shown in Table I, the Reptilase and thrombin clotting times were equally prolonged in patient plasmas during treatment with urokinase for 18 h.

Progressive fibrimgenolysis. During pro- gressive degradation of purified fibrinogen, the Reptilase and thrombin clotting times were equally prolonged when FDP were present in moderate amounts. With very high concentrations of FDP, a discrepancy

*O t L I

I I

100 200 300 0’

DIE (mgxl

-x D - Reptilase time - A D - Thrombin time - 0 D + Heparin -Reptilase time

----- x E - Reptilase time ----- A E -Thrombin time

0 E +Heparin-Reptilase time

Figure 2. Reptilase and thrombin clotting times in the presence of purified products D or E. Test system as in Figure 1. In addition 0.1 ml of heparin (final conc. 1 1Wml) (or saline as control) was added to the incubation mixture. The re- corded clotting times are directly given in the ordinate. - Thrombin clotting times with heparin were all unmeasurably prolonged.

REPTILASE-TIME AND FDP 363

became evident, the Reptilase time being still measurable in spite of thrombin inco- agulability (Figure 1).

Similar results were encountered when purified fibrinogen was used instead of plasma in the test system, excluding the progressive anti-thrombin I11 of plasma as responsible for the discrepancy.

Purified products D or E . While purified product E has been shown to exert anti- thrombin activity (Larrieu et a1 1972, A r e - sen 1973), no discrepancy between the two clotting times was encountered in the pres- ence of purified products D or E (Figure 2).

The presence of heparin (final conc. 1 IE/ ml) did not influence the Reptilase times, while the thrombin times were unmeasurably prolonged.

N-rerminal analysis. At pH 7.4, equal amounts of N-terminal glycine ( a b u t 0.5 pmollpmol fibrinogen) were generated at the point of visible gelation with Reptilase and with thrombin. At pH 8.7, more N- terminal glycine (about 1.2 pmollpmol fibrinogen) was generated before visible

1.4 1 L

- Rept.pH 8,7 - - Rept. pH 7,4 T pH 8,7

-- T pH 7,4

1 I U,I

2

20 40 60 90 lncu ba t ion- t i m e ( min.)

Figure 3. Generation of N-terminal glycine during incubation of purified fibrinogen with Reptilase or thrombin at pH 7.4 or 8.7 (cfr. Methods). Arrows indicate point of visible gelation.

gelation occurred, the values with thrombin being slightly higher than those with Rep- tilase (Figure 3).

The influence of glycine present in the Reptilase reagent. The results are shown in Figure 4. The Reptilase reagent obtained from the manufacturer contained glycine (about 22.5 mg/ml when dissolved as re- commended). In the standard test system (0.2 ml citrated plasma, 0.2 ml FDP, 0.2 ml Reptilase/thrombin), the final pH with Reptilase was 7.45 as compared to 7.8 with thrombin. Dialysis of the Reptilase reagent against 0.15 M NaCl markedly increased the sensitivity of Reptilase times to the presence of FDP.

Dilution of the thrombin stock solution with veronal buffer pH 7.4 similarly reduced

FDP-25'fmg%)

X-I dialysed Reptilase,final pH 7.8 A-A .. - 7.45 P O Reptilase(22.5mghl glycine)-- x----x thrombin in saline,final pH 7.8

0 -- 9- ,.-glycine(22,5mg/ml)-..- A---.A--- buffer-..-7.45

Figure 4. The influence of pH and glycine on Reptilase and thrombin clotting times in the pres- ence of various amounts of 'FDP-25" (cfr. Meth- ods). Test system as in Figure 2.

364 H. ARNESEN, P. KIERULF & H. C. GODAL

TABLE I1 Reptilase-time, with or without the presetice of

FDP-25' (final conc. about 0.4 mg/ml), us influenced by various concentrations of glycine

Without FDP

0 62 30.5 5 42

10 40 28 20 38 30 35 25 40 31.5 50 28.5 22.5

Test system as in Figure 1.

the sensitivity of thrombin times to the pres- ence of FDP.

A shortening of both clotting times was found in the presence of glycine (final conc. as with Reptilase reagent), as compared with clotting mixtures without glycine, but of equal pH.

The influence of various concentrations of glycine. As shown in Table 11, the clotting times with dialysed Reptilase were highly dependent on the concentration of glycine, especially in the presence of FDP.

DISCUSSION

Thrombin leads to the formation of fibrin by splitting off fibrinopeptides A and B from fibrinogen, whereas Reptilase does so by splitting off fibrinopeptide A only (Blom- back 1958, Stocker & Straub 1970). During clotting of human fibrinogen with thrombin at pH 7.4, only small amounts of fibrino- peptide B are released at visible gelation (Abildgaard 1965). Thus, the present N- terminal amino1 acid data at pH 7.4 indicate that fibrinopeptide A is released to the same extent at visible gelation with Reptilase as with thrombin. At pH 8.7, polymerization of fibrin monomers is impaired (Scheraga & Ehrenpreis 1958). This would be in agree-

ment with the present finding of more N- terminal glycine being generated at this pH, reflecting a higher degree of fibrinopeptide release at the point of visible gelation. The slightly higher value obtained with thrombin than with Reptilase may reflect the addi- tional release of fibrinopeptide B by throm- bin.

The action of Reptilase is suggested to lead to only end-twend polymerization of fibrin, whereas thrombin-produced monoh mers also polymerize sidetocside (Laurent & Blomback 1958). If this were correct, the considerable prololngation of the Reptilase- time in the presence of FDP might imply that their anticoagulant effect is mainly due to inhibition of end-to-end polymerization of fibrin monolmers. Furthermore, Reptilase- produced fibrin would be s u p p e d to poly- merize more slowly than fibrin produced by thrombin in the presence of FDP. As shown above, this was apparently not the case. In fact, increasing amounts of FDP produced thrombin incoagulability when the clotting time with Reptilase was still measurable. This is most likely explained by the pres- ence of glycine in the Reptilase reagent. The effect of glycine may be twefold. Being a buffer, the pH of citrated plasma will be lowered, whereby polymerization is en- hanced (Scheraga & Ehrenpreis 1958). Further, glycine decreases fibrinogen solu- bility (Blomback & Blomback 1956), and will thus tend to shorten the clotting time. As pointed out by Latallo et al (1971), the presence of FDP was found to render the clotting times highly sensitive to changes in pH. Furthennore, the sensitivity of the clot- ting times to FDP markedly increased if glycine was removed from the Reptilase preparation by dialysis. Finally, when the buffering capacity of glycine was simulated by adding small amounts of 0.1 N HCl to

KEPTILASE-TIME AND FDP 365

plasma, the clotting times with dialysed Reptilase were still somewhat longer than with undialysed Reptilase in normal plasma, probably due to an additional procoagulant effect of glycine per se. Similar results were obtained with thrombin clotting times when glycine was added, or when pH of the clot- ting mixture was otherwise lowered.

The present findings indicate that the coagulation of fibrinogen with Reptilase is more sensitive to polymerization inhibitors like FDP than is coagulation with thrombin. This sensitivity, however, is counteracted by the presence of glycine in the Reptilase reagent.

As shown above, equal prolongations of the clotting times of plasma with Reptilase (containing glycine) and thrombin were found in the presence of various FDP with- in a wide range of concentrations. The same was found in plasma from patients on uro- kinase therapy. This is in agreement with the observations of Britten et al (1970) and Latallo et al (1971). On the other hand, Funk et al (1971) found the Reptilase-time to be less prolonged than the thrombin time by FDP. According to the present results, differences in the glycine content of the Reptilase preparations may probably explain this discrepancy.

Product E has been shown to exert anti- thrombin effect (Larrieu et al 1972, Arne- sen 1973). The equal prolongation of the Reptilase and thrombin clotting times found in the presence of product E, suggests that this product inhibits the action of thrombin and Reptilase on fibrinogen in a similar way. If the anti-thrombin effect of product E is progressive, however, the present clotting time systems may not have been sensitive enough to detect this. The observed pro- longation, of the clotting times in the pres- ence of product E would then need further

explanation, as for instance polymerization inhibition in addition.

ACKNOWLEDGEMENTS

The skilled technical assistance of Renate Ruyter, Lise-Mette Aamodt and Karl Gravem is gratefully acknowledged.

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Accepted for publication July 25, 1973.

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Correspondence to

Dr. Harald Arnesen Haematological Research Laboratory Department IX, Ullevil Hospital Oslo, Norway