Scand. J . Haemat. (1973) 10,232-240 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the authods)

Studies on the Thrombin Clotting Time I. The Influence of Fibrinogen Degradation Products

HARALD ARNESEN, M.D. & HANS CHR. GODAL, M.D.

Haematological Research Laboratory (Chief, H. C . Godal), Department IX (Chief, K . Aas), Ullevdl Hospital, University Clinic,

Oslo, Norway

Mixtures of fibrinogen degradation products (FDP) from purified fibrinogen, as well as purified products from defined stages of fibrinogen degradation have been tested as to their influence on the thrombin clotting time. The prolongation of the thrombin clotting time exerted by various FDP was found to depend on the relative concentrations of FDP and fibrinogen in the test system. In addition, decreasing concentrations of fibrinogen increased the relative inhibitory influence of FDP. N-terminal amino acid analysis revealed that the strong effect exerted by purified stage I1 FDP was mainly due to polymerization inhibition, while the moderate effect exerted by products (D + E) was due to combined thrombin and polymerization inhibition,

The influence of different fibrinogen degra- dation products (FDP) on the thrombin clotting time has been extensively studied since the first report by Niewiarowski & Kowalski in 1957 (Fletcher et al 1962, Gormsen I& Laursen 1967, Larrieu et al 1966, Marder & Shulman 1969, Nilthn 1967a, Triantaphyllopoulos 1959). All au- thors agree that early degradation products prolong the thrombin clotting time to a con- siderable degree, while the effect of late de- gradation products is small. It is still not clear, however, whether the anticoagulent effect of FDP is mainly due to inhibition of fibrin polymerization (Alkjaersig et al 1962, Godal & Helle 1963), or whether an anti- thrombin effect is of significant importance (Latallo et al 1964, NilBhn 1967a, Trianta- phyllopoulos & Triantaphyllopoulos 1966). .

In the present study, various FDP (defined according to Marder et al 1969) from pun- fied fibrinogen were tested as to their in- fluence on the thrombin clotting time. Fur- ther, the influence of the relative amcen- trations of fibrinogen and FDP on the throm- bin clotting time, was studied.

The transformation of fibrinogen to fibrin leads to the appearance of glycine as N- terminal amino acid in fibrin. During in- cubation of fibrinogen and thrombin, the in- crease in N-terminal glycine gives direct in- formation on the amount of fibrin formed. Thus, thrombin inhibition may be studied. To obtain more information as to the mecba- nism of action of FDP on the last stage of coagulation, therefore, N-terminal amino acid analyses were included.

THROMBIN CLOTTING TIME AND FDP 233

MATERIALS AND METHODS

Fibrinogen. Purified human fibrinogen, more than 90 % clottable (Jacobson 1955), and contaminated with plasminogen, was obtained from Kabi, Stock- holm, Sweden. Solutions containing about 12 mp/ ml were dialysed against 0.3 M NaCl and stored at -20° 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 respectively.

Citrated plasma. 9 vol. of blood were collected into precooled (+ 4 O C) plastic tubes containing 1 vol. of 0.1 M sodium citrate. Plasma was pro- cessed by centrifugating 2000 g for 20 min at +4O C and used fresh or after stored deep-frozen for less than 3 months.

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 - 20° C. Further dilutions were made with 0.15 M NaCl and kept at + 4 O C during the experi- ments.

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

Soybean trypsin inhibitor (STZ), type I-S from Sigma, St. Louis, Mo., USA,, was dissolved in phosphate buffer pH 8.0 to a concentration of 1 mg/ml, and stored at -20° C. The final concen- tration in all experiments was 0.02 mg/ml.

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

Saline phosphate buffer pH 7.6. 4.00 g NaCl, 7.15 g Na,HPO,, 1.30 g KH,PO, dissolved in 900 ml distilled water. pH was adjusted to 7.6 with 1 N NaOH and the volume to lo00 ml with distilled water.

Sephadex G-200, from Pharmacia, Uppsala, Sweden, was used. Gel filtration was performed at room temperature in a 5 x 55 cm column with 1.0 M NaCc 0.05 M Tris-hydrochloric acid buffer at pH 8.3. The flow rate was 30 ml per h, sample size 20 ml (about 10 mglml), and eluate aliquots about 5 ml. STI was added to the elution solution to prevent further proteolysis.

DEAE-cellulose, from Serva Entwicklungs- labor, Heidelberg, Western Germany, was used.

Chromatography was performed at room temper- ature in a 2.0 x 40 cm column equilibrated with phosphate buffer 0.01 M, pH 8.6 (buffer I). Elu- tion was done 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 (buf- fer 11). The flow rate was about 30 ml per h, sample size 25 ml (stage I11 FDP from about 0.25 g fibrinogen), and eluate aliquots 7-15 ml.

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

EXPERIMENTALS

Mixtures o f degradation products from purified fibrinogen Fibrinogen (1.5 mg/ml) was incubated with uro- kinase (115 vol., final conc. 400 CTA-U/ml) at 37O C. At intervals, aliquots were withdrawn and STI added.

According to the nomenclature of Marder et a1 (1969), stage I FDP (mainly consisting of product X, mol. w. 240,000), lasts until loss of clottability, and stage I1 FDP (mainly consisting of praduct Y, mo1.w. 155,000) until no further proteolysis with plasmin is observed, that is when stage I11 FDP (consisting of product D, mol. w. 83,000 and product E, mo1.w. 50,000) is reached (Figure 1).

Purification of FDP Fibrinogen was incubated with urokinase to pro- duce FDP corresponding to stage I, I1 and III. Stage I FDP from just before loss of clottability, and stage I1 FDP giving maximal prolongation of the thrombin clotting time (cfr. Figure l), were chosen for purification. Plasmin activity was in- hibited with STI, and purification was performed on Sephadex G-200 (cfr. ‘Materials and Methods’). The peak fractions were subjected to concentra- tion, 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.

Separation of products D and E was performed from stage I11 FDP on DEAE-cellulose by gra- dient elution (cfr. ‘Materials and Methods’). The peak fractions were subjected to concentration, 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

234 HARALD ARNESEN & HANS CHR. GODAL

Stage I FDP

immunodifision in agarose gel (Ouchterlony 1958) with rabbit antihuman antisera against pro- ducts D and E (cfr. ‘Materials and Methods’). Protein concentration of products D and E was determined by adsorption at 280 nm using ex- tinction coefficients of 20 and 10, respectively (Marder et a1 1969, Nil6hn 1967b).

Stage II FDP

Dialysable fraction from late FDP Stage I11 FDP from purified fibrinogen were dialysed against distilled water in a volume ratio 1/30 (Stachurska et a1 1970). The dialysate was vacuum concentrated and reconstituted to desired concentration assuring pH 7.4 and ionic strength 0.15.

N-terminal analysis was performed according to Edman (1950), modified by Blomback & Yama- shina (1958), as described by Kierulf & Abild- gaard (1971). To study the influence of stage I1 FDP on the thrombin-fibrinogen reaction, 27 mg fibrinogen, 27 mg purified stage I1 FDP, or a mixture of 27 mg fibrinogen and 9 mg purified stage I1 FDP, were incubated at 37O C. Thrombin (1110 vol., final conc. about 0.07 NIH-U/ml) was added, and N-terminal analysis was performed after 0, 10, 30 and 75 min. To study the influence of stage In FDP on the thrombin-fibrinogen re- action, the same procedure was followed with 20 mg fibrinogen, 33 mg products (D + E) (purified on Sephadex G-200), or a mixture of 20 mg fibri- nogen and 33 mg products (D + E).

6 10

c + 60

50

40

30

The following test system for thrombin clotting times was used:

0.2 ml citrated plasma + 0.2 ml test solution/buffer

were incubated at 37O C for 60 s. 0.2 ml thrombin, 3 NIH-U/ml was added.

The time of visible gelation was recorded. All tests were run in duplicate. Control values with veronal buffer pH 7.4 were 24-26 s.

RESULTS

Thrombin clotting time in the presence of mixtures of FDP

Maximal prolongation of the thrombin clot- ting time was found after 15-20 min incu- bation with urokinase (Figure 1). At this time the incubation mixture was incoagul- able with thrombin. Upon further incubation with urokinase, the effect on the thrombin clotting time decreased and finally levelled off, corresponding to stage 111 FDP.

Thrombin clotting time in the presence of purified FDP

It is seen (Table I) that the greatest inhibitory effect was obtained with purified stage I1

Stage 111 FDP

20

0 10 20 30 120 Incubation time (min.)

Figure 1. The influence on the thrombin clotting time of mix- tures of FDP from the different fibrinogenolytic stages (according to Marder et a1 1969) (cfr. ‘Ex- perimentals’).

THROMBIN CLOTTING TIME AND FDP 235

~ 1 0 0

.gl 75

Q) In J

.- +.'

c a 50 E e

25

TABLE I The influence of isolated products of fibrinogen

degradation on the thrombin clotting time The protein concentrations are

equal, and adjusted to give measurable results in the standard test system

Conc. of fibrinogen in citrated plasma 2 mg/ml, conc. of FDP in testsamples 1.5 mg/ml

Product

Purified stage I Purified stage I1 Products (D + E) D E Dialysable fraction

from late FDP Control

Thrombin 1 clotti;! time

76 92 32 30 36

25 25

FDP. Purified stage I FDP was less effective, whereas only slight prolongation was ob- tained with products @+E). The effect of purified product E was found to be some- what stronger than that of purified product D. Dialysable fraction from late FDP did not affect the thrombin clotting time.

Figure 2. The influence on the thrombin clotting time of various concentrations of fibrinogen in relation to a fixed concentration of early stage I1 FDP (final conc. 40 mg/100 ml). Tested with two different thrombin concentrations (final conc. 1 and 3 NIH-U/ml respectively). Control: The lower curve repre- sents the dilution curve for fi-

l l 12

\ \ \ \ \

The importance of the fibrinogenJFDP ratio in the test system

The influence of the different types of FDP on the thrombin clotting time depended on the fibrinogedFDP ratio in the test system (Figures 2 and 3). In addition, the absolute concentration of fibrinogen was important (Table 11), the inhibitory effect of FDP being accentuated upon dilution with heat defibrin- ated plasma.

N-terminal d y s i s

During incubation of 27 mg of purified stage I1 FDP with thrombin, the generation of N- terminal glycine was small as compared with what was generated from an equal amount of fibrinogen (Figure 4). Addition of 9 mg of FDP to fibrinogen did not influence sig- nificantly the thrombin-induced generation of N-terminal glycine. In the presence of this amount of FDP, however, visible gelation was delayed from 15 to 24 min, and thus

\ a *--- ass

I 25 50 75 100 Fibrinogen (final conc. mg o/O)

brinogen without the presence of degradation products (final conc. 1 NIH-U/ml).

236 HARALD ARNESEN & HANS CHR. GODAL

occurred at a much higher level of N-terminal glycine (fibrin), indicating that fibrin poly- merization was markedly impaired.

Figure 5 gives the results from a similar experiment with products (D+E). Due to the weak anticoagulant effect of these FDP, much higher concentrations were needed to give a similar delay of visible gelation. It is seen that no increase in N-terminal glycine

2 4 0

2 2 0

2 0 0

180 - u 2 160

E

- 0

.- 140

2 120

- c .-

0 L

c' 100

80

6 0

4 0

2c

S t a d i u m I[ - F D P

was obtained upon incubation of products @+E) with thrombin. The appearance of N-terminal glycine from fibrinogen occurred more slowly in the presence of products (D-tE), indicating thrombin inhibition. In addition, visible gelation occurred at a some- what higher level of N-terminal glycine than with fibrinogen alone, suggesting that even polymerization was affected.

Figure 3. The influence on the thrombin clotting time of various concentrations of different FDP in relation to a fixed concentra-

1 0 0 2 0 0 4 0 0 tion of fibrinogen (170 mg/100 ml) .

2 0 5 0

log conc. F D P ( r n g , )

THROMBIN CLOTTING TIME AND FDP

F (mg/100 ml) I Tz F (mg/100 ml)

237

I T5 FDP (mg/100 ml)

TABLE I1 The effect of dilution on the clotting time of plasmas containing fibrinogen (F)

or fibrinogen/fibrinogen degradation products (FDP) respectively Dilution with the same plasma heatdefibrinated

Titration of the thrombin concentration so as to give the same clotting time in the two plasmas undiluted

DISCUSSION

The observed effects on the thrombin clot- ting time of various mixtures of degradation products from purified fibrinogen are in ac- cordance with previous studies (Latallo et al 1964, Marder & Shulman 1969). Thus,

maximal anticoagulant effect was obtained with early stage I1 FDP, whereas stage I11 FDP exerted only a small effect. Similar re- sults were obtained with purified FDP. In agreement with Stachurska et al (1970), the dialysable fraction obtained from late FDP was inactive.

2.4

2 2

2.0

1.6 Y.

0 - : 1.6

2 0 b

.c N 1.4 - 6 : o . 1.2 L

U c - 1.0

0.0

0.0

Figure 4. The increase in N-terminal 0.4

glycine during incubation of 27 mg fibrinogen, 27 mg purified stage I1 FDP, or a mixture of 27 mg fibrinogen and 9 mg purified stage I1 FDP, with throrn- bin.

0.2

I f 10 30 75

In c uba t i on - t i me ( mlnJ

23 8 HARALD ARNESEN & HANS CHR. GODAL

In accordance with previous reports (Al- kjaersig et al 1962, Godal & Helle 1963, Triantaphyllopoulos & Triantaphyllopoulos 1959), the degree of prolongation of the thrombin clotting time depended on the con- centration ratio between fibrinogen and the various FDP in the mixture. In addition, the absolute concentration of fibrinogen proved important. These findings may possibly pro- vide a rational basis for the therapeutic bene- fit of fibrinogen administration in cases with bleeding tendency caused by FDP.

N-terminal analysis accurately reflects the action of thrombin on fibrinogen (Blomback 1958). By this technique thrombin inhibition may be studied separately. In the present study, the thrombin-induced generation of N-terminal glycine in purified fibrinogen was practically unaffected by the presence of purified stage I1 FDP, although the time of visible gelation was considerably delayed. The interpretation of these findings is com-

2 .o

1.6

plicated by the fact that purified stage I1 FDP are susceptible to thrombin, giving a minor increase in N-terminal glycine during incubation with thrombin. If it is assumed that the fibrinogen-thrombin reaction is part- ly zero and partly first order with respect to substrate (Blomback 1967, Kierulf 1972), the present results possibly indicate slight thrombin inhibition by stage I1 FDP. How- ever, much more N-terminal glycine, or fibrin, had been formed at the time of visible gelation, indicating that polymerization in- hibition is the dominating mechanism by which stage I1 FDP prolong the thrombin clotting time.

No N-terminal glycine was generated dur- ing incubation of products @+E) with thrombin (Figure 5) . In spite of this, pro- ducts (D+E) inhibited the action of throm- bin on fibrinogen. The mechanism of inhibi- tion is obscure, but it may be noted that the inhibitory effect was moderate, in spite of

F - 4 1.4, /

/

a

Figure 5. The increase in N-terminal (DIE) glycine during incubation of 20 mg

a mixture of 20 mg fibrinogen and 33 mg products (D + E), with thrombin.

- 6 . fibrinogen, 33 mg products (D + E), or

10 30 7 5

I n c u b a t i o n - t i m a ( m i n.)

THROMBIN CLOTTING TIME AND FDP 239

great molecular excess of products (D+E). As more fibrin had been formed at the time of visible gelation in the presence of products (D+E), fibrin polymerization was also af- fected. As compared with stage I1 FDP, this effect appeared to be very weak.

After separation on DEAE-cellulose, pro- duct E was found to prolong the thrombin clotting time somewhat more than product D. So far, contradictory results have been re- ported on the effect on the thrombin clotting time of isolated products D and E (Marder & Shulman 1969, NilBhn 1967a). These dis- crepancies may partly be due to the use of different test systems. In order to obtain further information, the influence of sepa- rated products D and E on the thrombin- induced generation of N-terminal glycine from fibrinogen is now being studied.

ACKNOWLEDGEMENTS

The skilled technical assistance of Mrs. LiseMette Aamodt and Mrs. Renate Ruyter is gratefully ac- knowledged. During performance of the N-termi- nal analyses, the advices of Dr. Peter Kierulf and the excellent technical assistance of Karl Gravem have been of great help.

The author is research fellow of Nasjonalfor- eningen for Folkehelsen, Hjerte-Karrfidet.

REFERENCES

Alkjaersig, N., Fletcher, A. P. & Sherry, S. (1962) Pathogenesis of the coagulation defect devel- oping during pathological plasma proteolytic (‘fibrinolytic’) states. 11. The significance, mech- anism and consequences of defective fibrin polymerization. J. clin. Invest. 41, 917-34.

Blomback, B. (1958) Studies on the action of thrombic enzymes on bovine fibrinogen as measured by N-terminal analysis. Arkiv fiir Kemi 12, 321-35.

Blomback, B. (1967) Fibrinogen to fibrin transfor- mation. In W. H. Seegers (ed) Blood clotting enzymology, p 176. Acad. Press, New York and London.

Blomback,B. & Yamashina,I. (1958) On the N- terminal amino acids in fibrinogen and fibrin. Arkiv for Kemi 12, 299-318.

Edman, P. (1950) Preparation of phenyl thio- hydantoins from some natural amino acids. Acta chem. scand. 4, 277-82.

Fletcher, A. F., Alkjaersig, N. & Sherry, S. (1962) Pathogenesis of the coagulation defect devel- oping during pathological plasma proteolytic (‘fibrinolytic’) states. I. The significance of fibri- nogen proteolysis and circulating fibrinogen breakdown products. J. clin. Invest. 41, 896- 916.

Godal, H. C. & Helle, I. (1963) In vitro studies on fibrinogenolysis induced by streptokinase. Scand. J. clin. Invest. 15, 303-10.

Gormsen, J. & Laursen, B. (1967) Fibrinogen breakdown products and clotting parameters. Thrombos. Diathes. haemorrh. (Stuttg.) 17, 467- 82.

Jacobsson, K. (1955) Studies on the determination of fibrinogen in human blood plasma. Scand. J . clin. Lab. Invest. 7, Suppl. 14, 1-54.

Kierulf, P. & Abildgaard, U. (1971) Studies on soluble fibrin in plasma. I. N-terminal analysis of a modified fraction I (Cohn) from normal and thrombin-incubated plasma. Scand. J. clin. Lab. Invest. 28, 231-40.

Larrieu, M. J., Marder, V. J. & Luceman, S. (1966) Effects of fibrinogen degradation products on platelets and coagulation. Thrombos. Diathes. haemorrh. (Stuttg.), Suppl. 20, 215-26.

Latallo, Z. S., Budzynski, A. Z., Lipinski, B. & Ko- walski,E. (1964) Inhibition of thrombin and of fibrin polymerization, two activities derived from plasmin-digested fibrinogen. Nature (Lond.) 203, 1184-85.

Marder, V. J., Shulman, N. R. & Carroll, W. R. (1969) High molecular weight derivatives of human fibrinogen produced by plasmin. I. Physiochemical and immunochemical charac- terization. J. biol. Chem. 244, 2111-19.

Marder,V. J. & Shulman, R. (1969) High mo- lecular weight derivatives of human fibrinogen produced by plasmin. 11. Mechanism of their anticoagulant activity. J . biol. Chem. 244, 212& 24.

Niewiarowski, S. & Kowalski, E. (1957) Anti- thrombin formation during proteolysis of fibri- nogen. Trans. 6th Congr. Europ. SOC. Haemat., Copenhagen, p 560.

240 HARALD ARNESEN & HANS CHR. GODAL

NilChn, J.-E. (1967a) Influence of split products of fibrinogen on results of blood coagulation tests and platelet adhesiveness. Scand. I . Haemat. 4, 1-11.

Nilbhn, J.-E. (1967b) Split products of fibrinogen after prolonged interaction with plasmin. Thrombos. Diathes. haemorrh. (Stuttg.) 18, 89- 100.

Ouchterlony, 0. (1958) Difision-in-gel methods for immunological analysis. Progr. Allergy 5, 1.

Owren, P. A. (1947) The coagulation of blood. In- vestigations on a new clotting factor. Acta med. scand. 128, Suppl. 194.

Accepted for publication December 20, 1972.

Stachurska, J., Latallo, Z. & Kopec, M. (1970) Inhibition of platelet aggregation by dialysable fibrinogen degradation products (FDP). Throm- bos. Diathes. haemorrh. (Stuttg.) 23, 91-98.

Triantaphyllopoulos, D. C. (1959) Nature of the thrombin inhibiting effect of incubated fibrino- gen. Amer. J. Physiol. 191, 515-79.

Triantaphyllopoulos, D. C. & Triantaphyllopoulos, E. (1966) Evidence of antithrornbic activity of the anticoagulant fraction of incubated fibrino- gen. Brit. J. Haemat. 12, 145-51.

Correspondence to

Harald Amesen, M.D. Haematological Research Laboratory Department IX, Ullev%l Hospital Oslo, Noyay

Books received

Rosemary Biggs: Human Blood Coagulation, Haemostasis and Thrombosis. Blackwell Scientific Publications, Oxford, England, 1972; p 697. 7.50 Engl. Pounds.

P. L. Mollison: Blood Transfusion in Clinical Medicine. Fifth ed. Blackwell Scientific Publications, Oxford, England, 1972; p 830. 8.00 Engl. Pounds.

Stewart Sell: Immunology, Immunopathology and Immunity. First ed. Harper & Row Publishers, Hagmtown, Maryland, USA; p 277. 12.95 US Dollars.

Center National de la Recherche Scientifique: Colloques No. 203: Buses Mole- culaires de la Pathologie. Gif-Sur-Yvette 11-13 Sept. 1971. Masson et Cie, Editeurs, Paris; p 806. 74.90 FF.

F. Ciscar & P. Farreras: Diugnostico hematobgico Laboratorio y Clinica. Tom0 1-11; p 1962. Editorial Jims, Barcelona.