PART G

FIBRINOGEN DEGRADATION PRODUCTS AND FIBRINOGEN-TO-FIBRIN CONVERSION

Chairman : G.P. McNicol

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3 1 . FIBRINOGEN DEGRADATION PRODUCTS AND FIBRINOGEN TO FIBRIN CONVERSION

Harald Arnesen

Haemtological Research Imboratoory (Chief: H,C W a l ) Ulleval Hospital, Depr.lX ( ch ie f : K . Aas)

Oslo, N o ~ y

1 will mainly report on the influence of different fibrinogen degradation products on the thrombin clotting time, and only sporadi- cally discuss the influence of FDP on the polymerization step in contrast to the enzyma- tic step. (Alkjaersig et al. 1962 ; Latallo et al. 1964 ; Miller and Sanchez-Avalos 1968 ; Nilehn 1967). The standard test system has contained :

0.2 ml citrated plasma added 0.2 ml test solution or control this being incubated for 60” at 37°C before the addition of

0.2 ml thrombin in a final concentration

All experiments have been done at pH 7,4 and ionic strength 0.15, usually achieved by dialysis. All results are the mean of at least two readings, all done by the same technician.

Fibrinogen degradation products from pun- fied human fibrinogen have been prepared at 37°C with urokinase, and fibrinogenolysis was stopped with soybean trypsin inhibitor (figure

As is well known (Latallo et al. 1964, Marder and Shulman 1969), a marked but largely transient prolongation of the thrombin time is obtained, the maximum prolongation in my system being found after 15 minutes incubat ion.

If the Corresponding incubation experiment

of 1 NlH-u/ml.

1).

is done in plasma the results vary widely between plasmas from different persons. Some plasmas will never be incoagulable with the chosen concentrations of urokinase, while others will be so after even short incubation. The results from a plasma with a high degree of fibrinbgenolysis are shown in figure 2.

A high degree of prolongation is reached after 5 minutes incubation, the maximum effect not being reached until 2 hours incuba- tion. But in contrast to purified fibrinogea the prolongation is not greatly reduced on pro- longed incubation.

Purified products D and E, have been prepared from 24 hours digest of human fibrinogen with urokinase by DEAE-cellulose- chromatography with gradient elution (Nilehn 1967, Nussenzweig et al. 1961).

The product D gave a slight prolongation of the thrombin clotting time, a little less than that of late fibrinogen degradation products in corresponding concentration.

The product E also gave a slj&t prolonga- tion, a little less than that of product D in corresponding concentration.

Dialysable fraction from late fibrinogen de- gradation products has been prepared by dia- lysis of 24 hours digest of human fibrinogen with urokinase, against distilled water in a volume of 1 : 30. The dialysate being vacuum- concentrated and reconstituted to a final con-

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control 5 10 15 20 25 30 1 9 120

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Test-sampie diluted to give measurable results.

centration of 45 mg %, expressed as fibrinogen, assuring a pH of 7,4 and ionic strength 0.15. That is according to Stachurska et al. (1970). This dialysable fraction was not found to give any prolongation of the thrombin clotting time.

Fibrinolytic degradation products have been prepared by intra clot lysis or extra clot lysis.

In intra clot lysis human fibrinogen or citrated plasma was allowed to coagulate in the presence of urokinase in a final concentration so as to give a lysis time of about 10 minutes. (200 and 100 CTA-u/ml respectively). Clotting was brought about by thrombin in different concentrations, added directly after urokinase. At different times before clot lysis the clots were thoroughly squeesed and removed. To the residual fluid was added soybean trypsin in-

hibitor, and in the case of purified fibrinogen also purified antithrombin I11 (Abildgaard 1968).

The results obtained with purified fibrinogen (figure 3) varied with the initial thrombin con- centration used, but the pattern was similar in all experiments. Around clot lysis time the thrombin clotting time was moderately pro- longed. Further incubation gave a slightly les- sened prolongation. The smallest variations, that is the flatest curve, was obtained with the highest initial thrombin concentration used.

All changes were very slight as compared to those of fibrinogenolytic degradation products.

The results with intra clot lysis of citrated plasma (figure 4) were very similar. With low initial thrombin concentration a biphasic curve

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control 5 15 30 120 180 1080' Incubation time (min.)

Figure 2

The plasma thrombin time as influenced by fibrinogenolytic degradation products from plasma

was obtained. With high initial thrombin con- centrations the fluctuations were almost omit- ted. The maximum prolongation was a little less than with purified fibrinogen and negligible in relation to that obtained with fibrinogenolytic degradation products.

In extra clot lysis fibrinogen or citrated plasma was incubated with 10 NIH-u/ml of thrombin for 2 hours, synerized and incubated with urokinase. Fibrinolysis was stopped with soybean trypsin inhibitor and remaining throm- bin inhibited with purified antithrombin 111.

The products obtained by extra clot lysis did only prolong the thrombin clotting time 10% or less.

Fraction D prepared by DEAE-cellulose- chromatography from eextra clot lysis had a similar effect on the thrombin clotting time as fraction D from lysed fibrinogen.

These experiments show that fibrinolytic degradation products give a very slight pro- longation of the thrombin clotting time as

compared to that of early fibrinogenolytic degradation products, which in most plasmas tested seem to be the dominating products. The prolongation achieved by fibrinolytic degra- dation products is also less than that of late degradation products of fibrinogen (Allington 1967 ; Coda1 and Helle 1963).

On this background it was of interest to see if the influence of fibrinogenolytic degradation products could be reduced by secondary incu- bation with thrombin.

Early degradation products from purified fibrinogen or citrated plasma was incubated with thrombin for 15 to 60 minutes. Remaining thrombin activity was inhibited' with purified antithrombin Ill. In the same way, secondary incubation with thrombin was performed with late degradation products of fibrinogen, frac- tion D from fibrinogenolysis and fibrinolytic degradation products after extra clot lysis.

(table I) that secondary in- cubation with thrombin of early degradation

It was found

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t

9 9 I9

3 6 9 10 30 120 after clot lysis Incubation time (min.)

> before clot lysis

Figure 3

The plasma thrombin time as influenced by fibrinolytic degradation products from purified fibrinogen. Two different concentrations of thrombin used for the initial coagulation.

products slightly reduced the prolonging effect on the thrombin clotting time, but the pro- longation was still much more pronounced than by fibrinolytic degradation products in corres- ponding concentrations.

The prolonging effect of late degradation products, fraction D and fibrinolytic degra- dation products was not reduced by secondary incubation with thrombin.

Therefore it can also be stated that large fibrinogenolytic degradation products cannot be reduced to fibrinolytic degradation products by secondary proteolysis with thrombin (Coda1 and Helle 1963).

These results seem to indicate that a marked prolongation of the thrombin clotting time as observed in fibrinolytic states must be ex-

plained by fibrinogenolysis rather than by fibrinolysis, this being valid for fibrinolytic degradation products after primary polymeri- zation of the fibrin monomers.

However, we have prepared soluble fibrin from purified fibrinogen by a limited action of thrombin, the so-called des-A monomer (25-40% of the mixture according to Abild- gaard (1 965)).

By degradation of this soluble fibrin with urokinase for 30 minutes the products had an effect on the thrombin clotting time which was almost as great as that of early degradation products from fibrinogen.

Therefore the conclusion seems to be valid that during fibrinolytic states the early degra- dation products of fibrinogen and soluble fibrin

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before clot lysis after clot lysis lncubationtime (min.)

Figure 4

The plasma thrombin time as influenced by degradation products from plasma. Two different concentrations of thrombin used for the initial coagulation.

are of main importance for the prolongation of the thrombin clotting time, while late degra- dation products of fibrinogen and fibrinolytic degradation products after polymerization play a minor role in this respect.

I would also like to say some words on the influence of different concentrations of fibrino- gen and degradation products (figure 5) .

Citrated plasma was diluted with the same plasma heat-defibrinated. In the absence of degradation products, that is the lower curve with’ 1 NIH-u/ml as final thrombin concen- tration, the thrombin clotting time is moderate- ly prolonged until very low fibrinogen concen- trations.

With early degradation products in a fiied concentration in the mixture, a quickly in-

creased prolongation of the thrombin clotting time was found, even with only slight dilution of the plasma fibrinogen. At a final concen- tration of fibrinogen corresponding to the chosen concentration of degradation products the thrombin clotting time was unmeasurably prolonged. This was not overcome by increasing concentrations of thrombin, and was found to be valid for concentrations down to 50 mg 7%.

In a similar experiment (figure 6) with a fixed concentration of plasma fibrinogen and increasing concentrations of early degradation products identical results were obtained, the plasma being incoagulable when the concen- trations of fibrinogen and degradation products were equal.

These experiments show that the relative

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Figure 5

The plasma thrombin time as influenced by different concentrations of fibrinogen in relation to a fixed concentration of early degradation products. (40 mg $% final concentration), tested with two different thrombin concentrations. Control : The dilution curve for fibrinogen without the presence of degradation products.

concentrations of fibrinogen and degradation products beside the type of degradation pro- duct are of main importance for the prolonga- tion of the thrombin clotting time.

REFERENCES

Abildgaard, U. (1965) “N-terminal analysis during coagulation of purified human fibrinogen, fraction I, and plasma”. Scand. J. Clin. Lab. Invest. I 7,

Abildgaard, U. (1968) “Highly purified antithrombin 111 with heparin cofactor activity prepared by disc electrophoresis”. Scand.J.Clin.Lab.Invest. 21,

Alkjaersig, N., Fletcher, A.P. and Sherry S. (1962) “Pathogenesis of the coagulation defect developing

529-536.

89-9 1.

during pathological plasma proteolytic (fibrino- lytic) states. 11. The significance, mechanism and consequences of defective fibrin polymerization.” J. Clin. In vest. 4 I , 9 1 7-9 3 4.

Allington, M.J. (1967) “Fibrinogen and fibrin degra- dation products and the clumping of staphylococci by serum”. Brit.J.Haernat. 13. 550-567.

Godal, H.C. and Helle, 1. (1963) “The influence of fibrinogenolytic and fibrinolytic split products on the last stage of coagulation”. Scand.J.Clin. Lab. Invest. 15,’327-330.

Latallo, Z.S., Budzynski, A.Z., Lipinski, 8. and -KO- walski, E. (1964) “Inhibition of thrombin and fibrin polymerization, two activities derived from p l a s m i n d i g e s t ed fibrinogen”. Nature 203, 1184-1185.

Marder, V.J. and Shulman, N.R. (1969) “High mole- cular weight derivatives of human fibrinogen pro- duced by plasmin”. J.Biol.Chern 244, 212Cb2124.

25 1

Figure 6

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25 50 75 100 125 ’ Early FDP (final conc. mg%)

The plasma thrombin time as influenced by different concentrations of early degradation products in relation to a fixed concentration of fibrinogen (105 mg %I final concentration). Tested with two diffcrent thrombin conccnt rat ions.

Table I

Purified Plasma fibrinogen

Fibrinogen split products 5 8” 12”

idem + thrombin

45” 5 1”

Fibrin split products 3 2” 30”

Control 28” 28”

Table I

The plasma thrombin time as influenced by early degradation products from fibrinogen after secondary incubation with thrombin (final concentration 8.3 NIH-u/ml). Comparison with fibrin degradation products in cor- responding concentrations. Test system :

0,2 ml citrated plasma 0,1 ml test s o l u t i o n / c p r o l 60” incubation at 37 C + 0.1 ml thrombin.

Miller, S.P. and Sanchez-Avalos, J. (1968) “Degra- dation of fibrinogen by proteolytic enzymes. 11. Ef- fect of the products on coagulation”. Z’hrombos Dhtheshaemorrh. 20, 15-30.

Nilehn, J.-E. (1967) “Split products of fibrinogen after prolonged interaction with plasmin”. Thromb.Dhth.haemorrh. IS, 89- 100.

Nilehn, J.E. (1967) “Influence of split products on results of blood coagulation tests and platelet

adhesiveness”. Scand..J.Haemat. 4, 1-1 1. Nussenzweig, V., Seligmann, M., Pelmont, J. and

Grabar, P. (1961) “Les produits de degradation du fibrinogene humain par la plasmine”. Ann.lnst.Pas- teur 100, 317-389.

Stachurska, 3. (1970) “Inhibition of platelet aggrega- tion by dialysable fibrinogen degradation products (FDP)”. Thrombos,Diathes.haemorrh. 23, 91-98.