Temperature dependent dissociation of soluble fibrin monomer complexes demonstrated by agarose gel filtration

0049-3848/80/210325-09$02.00/O THROMBOSIS RESEARCH20; 325-333,1980 Printedin the USA. All rightsreserved.Copyright(c) 1980PergamonPressLtd

TEMPERATURE DEPENDENT DISSOCIATION OF SOLUBLE FIBRIN MONOMER COMPLEXES DEMONSTRATED BY AGAROSE GEL FILTRATION

R. Hafter, R. von Hugo, M. Baumgartner, F.K. Hiller, H. Graeff I. Frauenklinik der Universitat Maistrasse 11, 8000 Mtinchen2, FRG

(Received 7.8.1980;in revisedform 13.10.1980. Acceptedby EditorA. Henschen) ABSTRACT The temperature dependent dissociation behaviour of soluble fibrin monomer complexes (SFMC) in plasma of post surgical patients was studied using gel filtration chromatography of B-Ala precipitated plasma samples. A temperagure depgndency in the investigated range between 12 and 37 C was demonstrated with a linear regression y = 5.56 - 0.07 x and a correlation coefficient r = 0.83. Additional experiments using 125 I-desA fibrin injected into heparinized rabbits revealeQ that after gel filtration of whole plasma samples at 20 C 68 % of the activity was eluted in front of the fibrinogen @ution volume and 32 % with the fibrinogen volume. At 37 C 24 % of the activity was eluted in front of fibrinogen, while 76 % eluted with the fibrinogen peak. The experiments indicate that SFMC are subject to a temperature dependent dissociation behaviour which is also influenced by the type of the complexes.

INTRODUCTION A so-called state of hypercoagulability is observed in patients with predisposing conditions for thromboembolic disease such as pregnancy and the early puerperium (1,2), the postoperative course (3), or advanced stages of cancer (4). This hypercoagulability is reflected by the occurrence of thrombin mediated catabolic products of fibrinogen, which form soluble fibrin monomer complexes (SFMC) with fibrinogen in plasma. These complexes seem Rey words: Soluble fibrin dissociation.

complexes, temperature dependent 325

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be arranged predominantly in a one to one molecular ratio (5,6,7). They can be fractionated from plasma together with the non-complexed fibrinogen by precipitation with f3-alanine (8). The separation of the complexes from the bulk of fibrinogen and contaminating serum proteins can then be accomplished grossly by gel filtration chromatography. The complexes are stable at pH 7.4 but dissociate at pH 5.3 into the fibrinogen and fibrin part as shown by Sasaki et al (9) in sedimentation studies. The same authors also found that the complexes dissociate at neutral pH by warming up and that the rate of dissociation is dependent from the concentration of the accompanying fibrinogen. It was the aim of this study to investigate whether the temperature dependent dissociation of SFMC as observed in vitro by gel filtration might also be demonstrable in plasma samples of patients with signs of hypercoagulability. Further, to supplement this study, plasma samples of rabbits in which I25 I-des-A fibrin complex formation with fibrinogen was induced, were investigated. to

MATERIALS AND METHODS Human plasma samples: A plasma pool from atients with gynecologlcal operations (2 days p.o.) (n = 20P was used (fibrinogen content: j; = 331 mg per 100 ml plasma). The plesma samples were pooled in 2 ml portions and kept frozen at -25 C until use. All determinations were done within 2 weeks. Preparation of plasma samples: Blood (9 ml) was withdrawn into plastic syringes containing 1 ml of anticoagulant: 0.129 M trisodium citrate, 0.06 M N-tris (hydroxymethyl) methyl-2aminoethanesulfonic acid (TES), 1000 KIU/ml Trasylol (Bayer), pH 7.5. The hematocrit was determined in microhematocrit tubes by standard technique. Platelet-poor plasma was prepared by sequential centrifugation of the blood in a refrigerated centrifuge for 15 min at 1.400 x g and 10 min at 3.600 x g. Animals: Rabbits (n = 5) weighing 2 - 2,5 kg were used. They were fed rabbit pellet food and water ad libitum containing NaI (1 g/liter) starting 3 days before the experiment. Two hours before injection of labelled des-A fibrin (0.5 mg/kg), the animals received 1000 u/kg/24 h heparin (Liquemin, Roche, Grenzach, Germany) in a continuous intravenous infusion into the marginal ear vein. Blood was withdrawn 15 min after the injection of I25 I-des-A fibrin in a one way syringe containing anticoagulant (1:lO). Des-A fibrin: Rabbit fibrinogen was repared from plasma by man01 precipitation (IOP The clottability of fdcG"reEEra&on was 93 96.Rabbit fibrinogen was labelled by the iodine monochloride method of McFarlane (11). Fibrinopeptide A release from fibrinogen was achieved by incubating 10 mg fibrinogen with 5 NIH U thrombin in 5 ml buffered 2 M urga (0.05 M Iris; 0.0025 M EDTA; 1000 KIU/ml aprotinin) at 37 C (12). After 2 hours the reaction was stopped with 5 AT U

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hirudin (Plantorgan, Bad Zwischenahn, Germany). Fibrinogen determination: Assays were done in duplicate by the d momback (13) in a slightly modified th d f Bl b‘-k %syon'as d~~c~~be~elsewhere (8). rose gel filtration and calculation of percentage of soluble rln monomer complexes: Flbrlnogen and its derlvatlves were precipitated from 2 ml plasma samples in plastic tubes by slow addition of 2 ml 4.2 M solution of R-alanine. The precipitate was spun down at 3.600 x g for 5 min in a refrigerated centrifuge. The pellet was washed with 3 ml glycine solution (2.1 M), centrifuged again and dissolved in 2 ml TES buffer, pH 7.5 (0.06 M TES, 0.081 M NaCl, 0.0129 M trisodium citrate) containing 200 KIU aprotinin/ml. Four percent agarose gel filtration was performed at different temperatures on Biogel A-15 m, 100-200 mesh (Bio-Rad Lab.). Two ml of the redissolved precipitate (approx. 6 mg protein) or 0.5 ml rabbit plasma containing I25 I-des-A fibrin were applied to a waterjacket column (1.6 x 90 cm) equilibrated and eluted with Tris-NaCl-citrate-buffer, pH 7.6 (0.05 M tris, 0.116 M NaCl, 0.0129 M trisodium citrate, 0.025 M E-aminocaproic acid and 0.025 $ sodium azide). The temperature in the column was maintained constant by thermostated water circuit. The flow rate of the effluent was kept at approx. 13 ml/h and the fractionation rate was 3.6 ml. The optical density was monitored at 280 nm. Percentage of soluble fibrin monomer complexes (SFMC) was calculated by planimetric evaluation of the elution pattern as described elsewhere (8). The I25 I-activity was measured in the eluted fractions with a scintillation counter (Gammascint BF 5300, Fa. Berthold). Percentage of complexed and of non-complexed fibrin was calculated as above from plotted elution profiles (counts vs. elution volume). Recovery rates of the gel filtrated protein were calculated from the concentrations of protein (O.D. or counts) before and after gel filtration. Statistical evaluation: The hypothesis of a linear relation between percentage of SFMC and temperature had to be checked. For this purpose regression analysis was applied to estimate the relationship between the dependent parameter, i.e. the percentage of SFMC, and the independent parameter, i.e. the temperature (14). RESULTS With the plasma pool of postoperative patients 14 gel filtrations were run at 4 different temperatures in the range between 12 and 37'C on the same column. At 20°C a mean of 4.2 + 0.5 (n = 4) percent SFMC coubd be demonstrated. Lowering thg filtration temperature to 12 C resulted in higher value8 (4.7 + 0.4; n = 4) and rise of temperature to 30 and 37 C in lower-values (3.8 + 0.5; n = 3 and 3.0 + 0.4; n = 3, respectively). The mean Recovery rate of the gel filtrated protein was 98 percent and not influenced by the temperature. The corre-

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6

0

2

y = 5.56 -0.07x r = - 0.86

1

I

I

I

12

20

30 Temperature

I

37 OC

FIG. 1 Correlation of SFMC- in - percent of total _._ . fibrinogen in a pool of postoperative patients after gel filtration at various temperatures.

lation between percent SFMC and temperature (Fig. 1) can be described by y = 5.56 - 0.07 x, (p = 0.05). The correlation coefficient was r = 0.83 and the standard deviation Sy . x = 0.46. Linearity of the regression was proved by the linearity test by which the F-value is estimated in respect to the level of si ificance p = 0.05. The calculation of F-value resulted in F Vn 2; 10; 0.05) = 0.403. For the uppermost tabulated critical value (14) with 2 and 10 degrees of freedom we received in the F-distribution F' (2; 10; 0.05) = 4.10. Since F' was considerably higher than F, the hypothesis was proved that the percentage of SFMC stands in a linear relation to the temperature (Fig. I). After application of I25 I-des-A fibrin to Bnticoagulated rabbits gel filtration chromatography at 20 C resulted in the occurrence of 68 + 6.8 percent of the activity as high molecular weight fibriii,while 32 + 6.8 percept of the activity cochromatographed with fibrinogen. At 37 C only 24 + 6 percent of the activity could be demonstrated as high molecclar weight fibrin while 76 2 percent cochromatographed with fibrinogen.

6

cts/min ~10~

5

20% --37c

4

Des A- Fibrin

3

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20-c ,FdymerI Monomer x-681x -32 26.8

2 1

I

50

I

100

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FIG. 2 4 percent agarose ge& filtration pattern of plasma at 20 and 3'7C: 125 I-des-A fibrin in a representative plasma sample of an anticoagulated rabbit. Fibrinogen is eluted at 95 ml. DISCUSSION A distinct risk of thromboembolism is given in gynecological patients following major operations (15). Elevated levels of thrombin mediated soluble fibrin monomer complexes could be demonstrated in plasma of these patients 2 hours postoperatively (16). There is some indication that the occurrence of SFMC in plasma coincides with the so-called state of hypercoagulability. A correlation of thrombosis detected by I25 I-fibrinogen leg scanning and elevated levels of SFMC detected b gel filtration was established by Fletcher and coworkers (17s . We were able to confirm these data in patients with gynecological cancer under radiotherapy (18). A rise in fibrinopeptide A levels in postsurgical patients was observed and sug ested to originate from fibrin I (desA fibrin) formation ?19). The delayed release of fibrinopeptide B in vitro together with the presence of antithrombin in blood also suggests that des-A fibrin mainly re resents the type of fibrin generated in circulating blood (20P .

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The data presented in this paper are concerned with thermodissociation during gel filtration of fibrin monomer complexes originating from plasma of postsurgical patients. Following major gynecological operatigns a mean of 4.2 percent of SFMC could be demonstrated at 20 C. This finding was in agreement with earlier obsgrvations (16). Lowering the temperature of gel filtration to 12 C regulted in 4.7 percent, elevation of temperature to 30 and 37 C in a decline of SFMC to 3.8 and 3.0 percent, respectively. Considering that the estimation of SFMC by gel filtration has its limitations especially in plasma with low SFMC content (8), an auxiliary study was undertaken with I25 I labelled des-A fibrin monomer injegted into rabbits. Following gel filtration of plasma at 20 C, 68 percent of the activity was eluted as SFMC with a molecular weight higher than fibrinogen. 32 percent cochromatographed with fibrinogen indicating that to somg extent des-A fibrin can exist in dissociated monomer form at 20 C. Similar data were reported by Brosstad (21) from in vitro studies in which 26 percent of des-A fibrin coeluted with fibrinogen at gel filtration. In our study 76 percent of I25 I-des-A fibrin eluteg in dissociated monomer form together with fibrinogen at 37 C. However, 24 percent could still be demonstrated as high molecular weight SFMC, substantiating the assumption thBt in vivo generated SFMC are not completely dissociated at 37 C. Thermodissociation of SFMC from des-A fibrin and fibrinogen in the experimental animal seems similar to the one of SFMC from postsurgical patients. This is conceivable under the assumption that des-A fibrin is also the main fibrin part in plasma of patients. It was reported that fibrin is precipitated onto the matrix of columns to some extent during the filtration procedure (22,23). In our experiments with patients' plasma the recovery rate of the chromatographed protein was close to 100 percent (24). From this it is unlikely that fibrin was retained in the column. In experiments using I25 I-des-AB fibrin prepared in urea we observed however a loss of about 50 % after gel filtration of respective plasma samphes (252. There was no difference in recovery rate between 20 and 37 C. Similar recoveries were observed in this study with plasma containing I25 I-des-A fibrin. The loss might be related to the labelling process and the preparation in urea. In addition, fibrin of SFMC observed during hypercoagulability is usually slightly plasmin degraded (26) by which solubility ameliorates. Reversible complex formation of fibrinogen and fibrin monomer is demonstrated by various methods during the induction period that precedes polymerization. Cleavage of fibrinopeptide A seems to be the prerequisite for this type of complex formation by which the thrombin induced activation of a domain (A) located in the N-DSK portion of fibrinogen interacts with a C-terminal a) in the D portion of fibrinogen that needs no acti12). In this context it should be mentioned that also calcium ions play an important role in the nonenzymatic reversible stage of fibrin polymerization. They accelerate the polymerization step and shorten subsequent gel formation (27).

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Dissociation of the complexes at 37'C became evident, thus confirming observations of other investigators (22,23,28). It can be assumed that aff&nity between-fibrinogen and.des-A fibrin is reduced at 37 C. However, In accor$ance with observations of Shainoff and Dardik (29) even at 37 C 24 percent of the deB-A fibrin was eluted as complexed fibrin. Our findings at 37 C may support the assumption that complete dissociation is suppressed by an excess of fibrinogen (29). On the other hand, it can be discussed that different rates of dissociation and reassembly of the fibrinogen fibgin complexes occur at different temperatures. This favours at 37 C the dissociation of fibrin from fibrinogen and the permeation of fibrin into the gel, thus resulting in an elution behaviour of fibrin similar to the one of fibrinogen. However, this gel related behaviour pattern certainly does not exclude the existence of soluble complexes in vivo. ACKNOWLEDGEMENTS This study was supported_by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, FRG (Sonderforschungsbereich 51, bEnchen, grant no. B/6). The technical assistance of Miss B. von Brunn is gratefully acknowledged. REFERENCES 1.

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2.

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3.

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4.

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8.

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11.

MCFARLANE, A.S. Efficient trace-labelling of proteins with iodine. Nature 182, 535 1958.

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14.

SACHS, L. Statistische Auswertungsmethoden. HeidelbergNew York: Springer, 1972.

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SCHORR, D.M. and GRUBER, U. Prophylaxe thromboembolischer Komplikationen in der operativen Gynakologie. Geburtsh. u. Frauenheilk. 37, 291-296, 1977.

16.

von HUGO, R., DREISER, M., HAFTER, R. and GRAEFF, H. Postoperative Thromboseprophylaxe in der Gynakologie. In: Klinische und ambulante Anwendung klassiscber Antikoagulantien, R. Marx and H.A. Thies (ads.) Stuttgart-New York: Schattauer, 1977, p. 225-228.

17.

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18.

von HUGO, R., HILLER, K.F., RAFTER, R., LOCHMULLER, H. and GRAEFF, H. Hypercoagulability and thrombosis in patients with gynecological cancer under radiotherapy. Thrombos. Haemostas. 5 341, 1979.

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21.

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23.

KRELL, W., MAHN, I. and IfiLLER-BERGHAUS,Gb Gel filkration of 125-I fibrin and 131-I fibrinogen at 20 C and 37 C. Thrombosis Res. 2, 299-310, 1979.

24.

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25.

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26.

GOLLWITZER, R., HAFTER, R., TIMPL, R. and GRAEFF, H. Immunological assay for a carboxy terminal peptide of the fibrinogen A&chain in normal and pathological human sera. Thrombosis Res. l_l,859-868, 1977.

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29.

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