Scand. J . Haemat. (1973) 10,291-297

Studies on the Thrombin Clotting Time 11. The Influence of Fibrin Degradation Products

HARALD -SEN, M.D.

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

Oslo, Norway

The influence on the thrombin clotting time of degradation products obtained from soluble or polymerized fibrin was studied. Plasmin degradation of soluble fibrin gave products which markedly prolonged the thrombin clotting time, whereas degradation products from polymerized fibrin (extra clot lysis) were almost inactive. The effect on the thrombin clotting time of degrada- tion products obtained by intra clot lysis varied considerably, dependent on the relative concentrations of thrombin and plasmin used. The present observations stress the importance of a strict definition of fibrin as a source of degradation products, and are also related to the clinical interpretation of the thrombin clotting time.

During plasmin proteolysis of fibrinogen, fibrinogen degradation products (FDP) are formed. Early FDP (X and Y) markedly prolong the thrombin clotting time, whereas late FDP (D and E) are much less effective (Latallo et al 1964, Marder & Shulman 1969). The main effect of FDP on the clot- ting of fibrinogen by thrombin is on the polymerization phase (Alkjaersig et al 1962, Latallo et al 1962, Godal & Helle 1963a, Arnesen 1973).

The degradation products from fibrin (fdp) have been less studied. Godal & Helle (1963b), using extra clot lysis, found a much smaller prolongation of the thrombin clotting time with fdp than with FDP. This has later been confirmed by Allington (1967), Mitchel & Beller (1970), and Arnesen (1971). In

contrast, Marder et a1 (1971) and Niewia- rowski & Nandi (1971) found that intra clot lysis of fibrin gave rise to high molecular weight products similar to X and Y, pro- ducing considerably prolonged thrombin clotting time.

The reason for this discrepancy is obscure, but different experimental conditions might lead to the formation of diflerent degrada- tion products. Blomback (1958) and Abiid- gaard (1965) have shown by N-terminal analysis that considerable amounts of the fibrinogen molecules may be converted to des-A-fibrin during incubation with thrombin before visible gelation takes place. Thus, the term fibrin comprises many transitional forms from soluble fibrin monomer to fully polymerized fibrin. Consequently, the term fdp is ambigous.

292 HARALD ARNESEN

For correct interpretation of the thrombin clotting time it is of importance to know the individual influence of the various fibrinogen/ fibrin degradation products.

In the present report, the influence on the thrombin clotting time of fdp obtained from various sources of fibrin (i.e. from soluble fibrin and from polymerized fibrin) was studied, and the results compared with those obtained with different FDP.

MATERIALS AND METHODS

Fibrinogen. Purified human fibrinogen, found to be more than 90 % clottable (Jacobsson 1955), and contaminated with plasminogen and fibrin stabilizing factor, was obtained from Kabi, Stock- holm, Sweden. Solutions containing about 12 mg/ ml were dialyzed against 0.3 M NaCl and kept at - 2 O O C. Before use the solutions were thawed and diluted to desired concentrations with distilled water and veronalbuffer, 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 centrifugation 2000 g at + 4 O C for 20 min and used fresh, or deep-frozen within 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 - 2 O O C. Further dilutions werc made with 0.15 M NaCl and kept at + 4O 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 - 2 O O C.

Soybean trypsin inhibitor (STI). Type I-S, Sig- ma, St. Louis, Mo., USA. Dissolved in phosphate buffer pH 8.0 to a concentration of 1 mg/ml, and stored at -20° C. The final concentration in all experiments was 0.02 mg/ml.

Antithrombin 111 (AT-III). Highly purified AT- 111 was kindly prepared by Abildgaard (1968). The activity was expressed in per cent of that in nor-

mal pooled plasma. A stock solution containing 520 % was used. The final concentration in all experiments was 52 %.

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,HP04, 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 1000 ml with distilled water.

EXPERIMENTALS

Soluble fibrin (‘des-A-fibrin’). Soluble fibrin was produced by incubating fibrinogen (1.5 mg/ml) with minute amounts of thrombin (final conc. about 0.005 NIH-U/ml) at 37O C, so as to give visible gelation in about 60 min. Thrombin clotting time was performed on a subsample from this mixture of soluble fibrin and fibrinogen just before visible gelation (Blomback 1958, Abild- gaard 1965).

f d p from soluble fibrin. Soluble fibrin was pre- pared as described above. Just before visible gela- tion, urokinase (115 vol.) was added (final conc. 400 CTA-U/ml). This mixture remained incoag- ulable. During further incubation at 37O C, aliquots were withdrawn at intervals, STI added, and the samples tested. In order to compare with FDP, equal amounts of fibrinogen were incubated with 0.15 M NaCl and urokinase as above.

fdp obtained by extra clot lysis (d+e) . Fibrino- gen (3 mg/ml) was clotted with thrombin (1110 vol., final conc. 1 NIH-U/ml) at 37O C for 2 h. Clot lysis was achieved by adding urokinase (1/5 vol., final conc. 1000 CTA-U/ml). At the time of clot lysis STI was added. Residual thrombin ac- tivity was neutralized with AT-I11 before testing (vide infra).

Prolonged incubation of (d+e) with urokinase after clot lysis did not change their influence on the thrombin clotting time.

fdp obtained by intra clot lysis. Aliquots of fibrinogen (3 mg/ml) were clotted in two series with thrombin (1/10 vol., final conc. 0.5 and 15 NIH-U/ml respectively) a t 37O C. Just prior to the addition of thrombin, urokinase (1/5 vol.) was added so as to give a clot lysis time of about 14 min. At intervals before total clot lysis, clots were thoroughly syneresed by winding on to a glass rod, and removed. The residual fluid was

THROMBIN CLOTTING TIME AND fdp 293

added STI and AT-111, and analysed spectro- photometrically for protein content (expressed as fibrinogen, using an extinction coefficient of 16, Blomblck & Blomback 1956), before testing. The same procedure was followed after clot lysis. To test that thrombin was totally neutralized by AT-111, a subsample of the mixture was added to fibrinogen (1.5 mg/ml) at 37O C. No visible gela- tion took place.

Plasmin resistant FDP ( D + E). Fibrinogen (3 mg/ml) was incubated with urokinase (1/5 vol., final conc. 400 CTA-U/ml) at 37O C for 2 h (Arnesen 1973), before the addition of STI.

Secondary incubation of ( D + E) with thrombin. (D+E) was prepared as described above. The mix- ture was incubated with thrombin (1/10 vol., final conc. 3 NIH-U/ml) at 37OC for 1 h, and AT-111 added. This mixture was tested for surplus throrn- bin activity as described above.

Secondary incubation of early FDP with throm- bin. Fibrinogen (1.5 mg/ml) was incubated with urokinase (1/5 vol., final conc. 400 CTA-U/ml) at 37O C until maximal prolongation of the standard thrombin clotting time occurred. A subsample of the mixture was then incoagulable with thrombin. STI was added, and the mixture incubated with thrombin (1/10 vol., final conc. 3 NIH-U/ml) at 37OC for 1 h, when AT-I11 was added. Testing for surplus thrombin activity was performed as described above.

The following test system was used:

+ 0.2 ml test solutiodcontrol 0.2 ml standard citrated plasma

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. Normal control was 24-26 s.

RESULTS

The influence of soZubZe fibrin. Soluble fibrin invariably gave shorter thrombin clotting times than did fibrinogen alone (Table I).

The influence of degradation products from soluble fibrin. Incubation with urokin- ase for about 20 min led to a marked pro- longation of the thrombin ’ clotting time, even more pronounced than with correspond-

TABLE I The influence of soluble fibrin

(cfr. ‘Experirnentals’) on the thrombin clotting time

The results are the mean of 12 different readings on 3 different days

Thrombin I clotti;; time

Soluble fibrin 21

Control (fibrinogedor buffer) 24

ing FDP (Figure 1). Upon further incubation with urokinase, the thrombin clotting times gradually decreased, reaching a plateau after about 60 min. At this time moderate and equal effects were obtained with fdp and FDP (Figure 1).

The influence of fdp obtained by extra clot lysis. fdp obtained by extra clot lysis exterted only a slight effect on the thrombin clotting time, even less than identical con- centrations of plasmin resistant FDP @+E). (Table 11). Incubation of @+E) with thrombin did not reduce their effect on the thrombin clotting time (Table 11).

The influence of fdp obtained by intra clot lysis. The degradation products obtained

soluble fibrin + fibrinogen

-

I 0 30 69 90

Incubation with urokinase (rnin.)

Figure 1. The influence on the thrombin clotting time of fdp from soluble fibrin (cfr. ‘Experimen- tals’) and corresponding FDP,

294 HARALD ARNESEN

TABLE I1 The influence of fibrin degradation products

obtained by extra clot lysis (d + e) on the trombin clotting time, as compared to

that of plasmin resistant FDP (D + E) Relative concentrations of (D + E) or (d + e) to

fibrinogen in the test system, 1.5 : 1

Thrombin clotting time

(D + E) 42 (D + E) + thrombin 42 (d + e) 33 Control 25

when a low thrombin concentration was used, caused a marked prolongation of the thrombin clotting time, maximum effect be- ing obtained at the time of clot lysis. In con- trast, equal concentrations of degradation products obtained when a high thrombin concentration was used, were almost inactive. (Figure 2).

6C

The influence of early degradation pro- ducts from fibrinogen after secondary incu- bation with thrombin. The results are given in Table 111. It is seen that the effect of early FDP on the thrombin clotting time was moderately reduced upon secondary incuba- tion with thrombin. The effect was however, still much more pronounced than with cor- responding concentrations of fdp obtained by extra clot lysis.

DISCUSSION

Early degradation products from fibrinogen (FDP) markedly interfere with the poly- merization of fibrin (Arnesen 1973). Prob- ably, soluble complexes between fibrin mo- nomer, fibrinogen and various degradation products are formed (Lipinski et a1 1967), whereby gelation is postponed.

The present study has shown that early

C - J- - n 200’; .A .-

-I 0

100

h 0 10 10 30 60

Before clot lvsis After clot I v i h -~ Incubation time (min.)

Figure 2. The influence on the thrombin clotting time of fdp obtained during intra clot lysis after initial clotting with two different concentrations of thrombin (cfr. ‘Experi- mentals’). Protein concentration in test samples (expressed as mg/100 ml fibrinogen, given on the right vertical axis) is indicated by columns. Black columns refer to samples with the highest initial thrombin concentration.

THROMBIN CLOl'TING TIME AND fdp 295

TABLE I11 The influence on the thrombin clotting time of

early FDP after secondary incubation with thrombin

Relative concentrations of FDP or (d + e) to fibrinogen in the test system, 0.5 : 1

Thrombin clotting time

Early FDP 58 Early FDP + thrombin 50 d + e 26 Control 24

degradation products obtained from soluble fibrin prolonged the thrombin clotting time even more than did early products from fibrinogen alone. The observation of Marder & Shulman (1969) that fibrin monomer is more active in complexing FDP than is fibrinogen, might be relevant to the present finding. On the other hand, Blomback & Laurent (1958) have shown that during in- cubation of fibrinogen with thrombin, soluble aggregates with increasing molecular weights are formed. Plasmin proteolysis of such ag- gregates might give rise to products which strongly interfere with fibrin polymerization. A third explanation would be that degrada- tion products from fibrin monomer differ from those obtained from fibrinogen. The initial action of thrombin on fibrinogen might change the conditions for plasmin proteolysis.

In contrast to our findings with fdp from soluble fibrin, fdp obtained by extra clot lysis (d+e, Dudek et a1 1970) exerted only a slight effect on the thrombin clotting time. This is in accordance with Allington (1967) and Mitchell & Beller (1970). As shown (Table 11), these products were even less effective than were (D+E), and this difference was not eliminated after incubation of @+E) with thrombin. These findings indicate that the polymerization process involves condi- tions of importance for plasmin proteolysis

other than the release of fibrinopeptides. Ac- cordingly, recent investigations (Dudek et al. 1970, Pizzo et al 1972, Plow & Edginton 1972, Catanzaro 1972) support the sugges- tion of dissimilarity between @+E) and (d+e). In addition, the greater inhibitory effect of non-polymerized derivatives may in- dicate that their cross-linking sites are of importance for the inhibition.

The results obtained with the model of intra clot lysis (Figure 2) probably link the results obtained with fdp from different sources together. Simultaneous addition of plasmin and thrombin gives rise to proteo- lysis products with different anticoagulant properties depending on the relative concen- trations of thrombin and plasmin used. Thus, with high concentrations of thrombin the de- gradation products obtained mainly originate from polymerized fibrin, and accordingly exert only a slight effect on the thrombin clotting time. With low concentrations of thrombin, appreciable amounts of degrada- tion products from fibrinogen and even from soluble fibrin are formed, giving considerable prolongations of the thrombin clotting time. The present findings may also be in accor- dance with those of Marder et al (1971) and of Niewiarowski & Nandi (1971), who found that high molecular weight fdp, giving con- siderable prolongations of the thrombin clot- ting time, were formed.

It is of interest to note that the influence on the thrombin clotting time of proteolytic products from fibrinogen obtained by incu- bation with plasmin and thrombin depended on the sequence of enzyme addition. Thus, initial incubation with thrombin gave higher maximal prolongation of the thrombin clot- ting time upon secondary plasmin proteolysis than did plasmin proteolysis alone (Figure 1). On the other hand, if a mixture of early FDP was secondary incubated with thrombin

HARALD ARNESEN 296

(Table of the tered.

The

111), a slightly reduced prolongation thrombin clotting time was encoun-

last finding indicates maintained thrombin susceptibility in a mixture of early FDP. This is in accordance with the sug- gestion of WallCn (1971) that the N-terminal part of the a (A)-chain of fibrinogen is un- changed during the first stages of plasmin proteolysis, and also with our finding that N-terminal glycine increases during incuba- tion of early FDP with thrombin (Arnesen 1973). As shown in Table 11, a similar effect was not recorded when late FDP was sec- ondary incubated with thrombin. This is in agreement with our recent finding that in- cubation of products (D+E) with thrombin does not produce any increase in N-terminal glycine (Arnesen 1973), and with Pizzo et a1 (1972) that D and E were lacking fibrino- peptides.

For the clinical interpretation of the thrombin clotting time, it is of importance that lysis of polymerized fibrin or fibrin de- posits will not give rise to any considerable prolongation whereas plasminolytic products from soluble fibrin may exert a strong effect. Degradation products from soluble fibrin may probably form during intravascular coagulation accompanied by fibrinolysis, and thus contribute to the prolongation of the thrombin clotting time seen in such cases.

ACKNOWLEDGEMENTS

The skilled technical assistance of Mrs. Lise-Mette Aamodt and Mrs. Renate Ruyter is gratefully ac- knowledged.

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

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THROMBIN CLOTTING TIME AND fdp 297

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Correspondence to

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