Impaired clot lysis in the presence of human neutrophil elastase

Thrombosis Research, Vol. 80, No. 2, pp. 1X+-159,1995 Cowieht 0 1995 Elsevier Science Ltd .< Printed-in the USA. All rights reserved 0049-3848/95 $9.50 + 00

Pergamon 0049-3848(9S)W161-1

IMPAIRED

CLOT LYSIS IN THE PRESENCE OF HUMAN NEUTROPHIL ELASTASE

Edvin T. Bach-Gansmol, Sigrun Halvorsen 1,2, Hans Christian Godal and Ole H. Skjpmsbergl. ‘Department of Pulmonary Medicine and 2Hematological Research Laboratory, Ullev&l Hospital, University of Oslo, Oslo, Norway.

(Received 7 February 1995 by Editor t? Kierulf; revisetiaccepted

7 August 7995)

Neutrophil granulocytes contain human neutrophil elastase (HNE), a se&e protease with fibrino(geno)lytic capacity. Leukocytes accumulate within thrombi and also migrate to sites of fibrin deposition in the inflammatory response (1). Neutrophil granulocytes are hence found in intimate association with fibrin in several pathological processes. Initial release of HNE from the neutrophil granulocytes has been found to occur concomitantly or slightly after clotting of whole blood (2). HNE has been shown to degrade fibrin (3). However, we have recently shown that this degradation of fibrin by HNE has an inhibitory effect on the plasminogen/plasmin mediated fibrinolysis, by reducing the stimulating effect of fibrin on tissue plasminogen activator (t-PA) mediated plasminogen activation (3). Toinvestigate the net effect of these two opposing actions, we studied the lysis of whole blood clots in the presence and absence of HNE. In addition to visual evaluation of clot lysis, we measured the release of erythrocytes and D-dimers into the clot supernatant at different intervals, since they have been found to correspond to other parameters of fibrinolysis, and reflect fibrinolytic capacity in a whole blood sample (4,5). MATERIALS

AND METHODS

Alphal-proteinase inhibitor (Prolastit-0, Miles Inc., Cutter Biological, Elkhart IN USA). Enzyme immunoassay of D-Dimer (AsserachromQ D-Di, Diagnostic0 Stago, Asnieres-sur-Seine, France). N-Sue-Ala-Ala-Ala-pNA (Elastin Products Company, Owensville, MO, USA). Phosphate buffered saline (PBS)(O.OlM K@P0~3H~O, O.OlM NaH,POaH,O, 0.14M NaCl, pH 7.4) Tris NaCl buffer (0.1 M Tris, 0.5 M NaCl, 1.5 mM NaN3, pH 7.5) Key words: Elastase, fibrin, fibrinolysis, whole blood clot, D-dimer. Corresponding author: Edvin Bach-Gansmo, Medical Department, Ulleviil Hospital, 0407 Oslo, Norway.

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Purified human neutrophil elastase isolated from human sputum (Elastin Products Company Owensville, MO, USA). Bovine thrombin (Thrombostat@)(Parke-Davis, USA), dissolved in supplied diluent to 1000 II-l/ml, subsequently diluted to 100 IU/ml (final concentration) in Owrens buffer (6) (a modified Verona1 buffer, containing sodium chloride 0.74 %, pH 7.38, ionic strength 0.15) and used immediately. Preparation of HNE solution Purified human neutrophil elastase was dissolved in Tris NaCl buffer, and aliquots containing 0.5 mg/ml were stored at -70 oC. The HNE activity was found to be 183 Units/ml, based on its capacity to hydrolyse the synthetic substrate N-Sue-Ala-Ala-Ala-pNA (as described by the manufacturer). Venous occlusion The venous occlusion test was carried out according to Robertson et al. (7). Samples of blood were taken From 4 healthy volunteers. After an overnight fast, and at least 10 minutes rest in the supine position, a blood pressure cuff was inflated to a point halhay between systolic and diastolic blood pressure for 20 minutes to induce t-PA release. Blood samples were drawn immediately before deflating the cuff. Sample preparation The blood (4 ml) was immediately mixed with PBS (4 ml) containing HNE in various concentrations (0, 17, 34 and 51 ug/ml)(final concentration) and thrombin 0.2 III/ml (final concentration). Thrombin was added to make the clotting time sufficiently short in order to avoid extensive degradation of fibrinogen at the highest concentrations of HNE. Visual determination of clot formation The time before clot formation following mixing of blood and PBS containing HNE and thrombin was recorded for each sample. Visual determination of clot lysis The time before complete clot lysis was recorded for each sample. Determination of whole blood clot lysis by release of erythrocytes This was performed essentially as described by Kahn et al (4). At different intervals samples were withdrawn from the mixtures of blood and PBS containing HNE and thrombin. The concentrations of erythrocytes in the supernatants were measured by using an automated haematology analyser (SysmexB K- 1000 from TOA Medical Electronics Co Ltd, Kobe, Japan). Determination of whole blood clot lysis by release of D-dimer This was performed essentially according to Haaland et al. (5), with the exception that in our experiments the whole blood was diluted in PBS. In a pilot study, corresponding results were obtained when using undiluted whole blood. Samples to be used for D-dimer estimation were mixed with alphal-proteinase inhibitor (5.0 mg/ml) (final concentration), to inhibit HIVE activity, and stored at -70° C until analysed. The samples were subsequently analysed by an ELISA-technique as described by the manufacturer. The values were corrected for the dilution by alphal-proteinase inhibitor.

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iao1603140'0 5 1208 z-100-

.g ao-5

60s

8

40I I

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34 microgramiml

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concentration

FIG.1 The clotting time of diluted whole blood in the presence of increasing amounts of HNE (0, 17, 34 and 51 p g/ml final concentration).Resultsaregiven asmedianandrange(n=4).

45

hrs

30

hrs 400 350 1

I

I 0 microgram/ml

I 17 microgram/ml

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FIG.2 The time before lysis of clots formed of diluted whole blood in the presence of increasing amounts of HNE (0, 17, 34 and 51 pg/ml final concentration).Resultsxe given asmedianandrange(n=4).

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L----I I

0 min

50 min

75 min 100 min Incubation time

+

0 microgram/ml

+

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51 microgram/ml

125 min 150 min

FIG.3 The concentration of Rbc‘s in the supernatant as a measure of clot lysis in the presence of increasing amounts of HNE (0,17,34 and 51 pg/mi final concentration). The samples were incubated for various time intervals. Results are given as median values (n=4).

I

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FIG.4 concentration of D-dimer in. the supernatant as a measure of clot lysis in the presence of increasing amounts of HNE (0,17,34 and 51 pg/ml final concentration). The samples were incubated for various time intervals. Results are given as median values (n=4).

The

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RESULTS Visuaf determination of clot formation In Figure 1 the results of the visual determination of clot formation time are shown. As can be seen, there was only a modest increase in clotting time, occurring in the presence of the highest concentrations of HNE. Visual determination of clot lysis In Figure 2 the results of the visual determination of clot lysis time are shown. In all experiments, the time before clot lysis increased with increasing HNE concentration. Whole blood clot Eysis estimated by release of erythrocytes The results of the release of erythrocytes are shown in Figure 3. With increasing HNE concentration, the amount of erythrocytes released into the supernatant successively decreased. whole blood clot lysis estimated by release of D-dimer The results of the D-dimer measurements are shown in Figure 4. The pattern of D-dimer release into the supernatant was similar to the pattern observed when measuring the release of erythrocytes. With increasing HNE concentration, the release of D-dimer into the supernatant successively decreased. DISCUSSION The present study has shown that when whole blood, obtained after venous occlusion to stimulate t-PA release, was clotted in the presence of increasing amounts of HNE, a delay of clot lysis, as determined by visual evaluation and impaired release of erythrocytes and D-dimers, was observed. The influence of HNE on the formation and degradation of fibrin is complex. Thus, impaired polymerisation of degraded fibrinogen as well as direct degradation of fibrin by HNE have been demonstrated (3,s). In addition, HNE may convert plasminogen to miniplasminogen (des-kringle 1-4 plasminogen), which is more sensitive than plasminogen to activation by t-PA. Also, HNE disables the inhibitor system of fibrinolysis (PAL1 and cL2-antiplasmin)(9-12). All these factors would indicate a more rapid removal of a clot when HNE is present. However, we have recently shown that HNE degradation of purified fibrin is associated with reduced t-PA mediated activation of plasminogen. The present observations indicate that this effect is dominating in the concerted action of HNE and t-PA mediated activation of plasminogen in whole blood. Whether the observed inhibitory effect of HNE on clot lysis is of clinical relevance remains to be shown. Obviously, high concentrations of HNE would be required to interfere with clots produced in whole blood due to the presence of plasma HNE inhibitors. On the other hand, the finding of free HNE activity in plasma obtained from patients with septicaemia indicate that release of HNE in such situations is sufficient to overwhelm available inhrbitors in circulating blood (13). Moreover, HNE may be released in close proximity to its substrates, or in protected extracellular compartments (14-17). Finally, once HIVE is bound to the surface of clot fibrin, it has been found to escape inhibition (18). Consequently, it can not be excluded that the inhibitory effect of HNE on clot lysis observed above, may affect the lysis of a thrombus or the resolution of excess fibrin in vivo.

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Acknowledgments The authors wish to thank Karl Gravem and Anne Vaeret for expert technical assistance, and Forskningsforum, Ulleviil sykehus (FUS) and Glaxo Norway, for financial support. REFERENCES 1. PLOW, E.F. and EDGINGTON, T.S. An alternative pathway for fibrinolysis. I.The cleavage of fibrinogen by leukocyte proteases at physiological pH. J. Clin. Invest. 56, 30-38, 1975. 2. PLOW, E.F. Leukocyte elastase release during blood coagulation. A potential mechanism for activation of the alternative fibrinolytic pathway. J.Clin. Invest. 69, 564-572, 1982. 3. BACH-GANSMO, E.T., HALVORSEN, S., GODAL, H.C. and SKJPINSBERG, O.H, Degradation of the cc-chain of fibrin by human neutrophil elastase reduces the stimulating effect of fibrin on plasminogen activation. Thromb. Res. 75, 307-3 17, 1994. 4. KAHN, M.B., PALMER S., MARLAR, R.A. and FINK, L. A modified quantitative whole blood clot lysis method for general laboratory analysis of fibrinolysis. Thromb. Res. 59, 17 1- 18 1, 1990. 5. HAALAND, A.K., SKJ(ZINSBERG, O.H., VBRET, A., RUYTERR and GODAL, H.C. A novel principle for assessment of stimulated fibrinolysis. Thromb. Res. 62, 725-735, 1991. 6. OWREN, P. A. The coagulation of blood. Acta Med. Stand. 128, suppl, 194, 1947. 7. ROBERTSON, B.R., PANDOLFI, M. and NILSSON, I.M. “Fibrinolytic capacity” in healthy volunteers as estimated from effect of venous occlusion of arms. Acta Chir. Stand. 138, 429-436, 1972. 8. BACH-GANSMO, E.T., HALVORSEN, S., GODAL, H.C. and SKJBNSBERG, O.H. Impaired coagulation of fibrinogen due to digestion of the C-terminal end of the Acr-chain by human neutrophil elastase. Thromb. Res. 73,61-68, 1994. 9. MOROZ, L.A. Mini-Plasminogen: A mechanism for leukocyte modulation of plasminogen activation by urokinase. Blood 58, 97- 104, 198 1. 10. MACHOWICH, R., and OWEN, W.G. An elastase-dependent pathway of plasminogen activation. Biochemistry 28,45 17-4522, 1989. 11. LEVIN, E.G. and SANTELL, L. Association of a plasminogen activator inhibitor @‘AI-l) with the growth substratum and membrane of human endothelial cells. J. Cell Biol. 105, 25432549, 1987. 12. BROWER, MS. and HARPEL, P.C. Proteolytic cleavage and inactivation of olz-plasmin inhibitor and Cl inactivator by human polymorphonuclear leukocyte elastase. J. Biol. Chem. 257, 9849-9854, 1982. 13. COHEN, J.R., SARFATI, I., BIRNBAUM, E., BENACQUISTA T. and WISE, L. The inactivation of antithrombin III by serum elastase in patients with surgical infections. The American Surgeon 56,665-667, 1990. 14. JANOFF, A. Elastase in tissue injury. Ann. Rev. Med. 36,207-216, 1985. 15. CAMPBELL, E.J., SENIOR R.M., MCDONALD, J.A. and COX, D.L. Proteolysis by neutrophils. Relative importance of cell-substrate contact and oxidative inactivation of proteinase inhibitors in vitro. J. Clin. Invest. 70, 845-852, 1982. 16. WEITZ, J.I., HUANG, A.J., LANDMAN, S.L., NICHOLSON, S.C. and SILVERSTEIN, S.C. Elastase-mediated fibrinogenolysis by chemoattractant-stimulated neutrophils occurs in the presence of physiologic concentrations of antiproteinases. J. Exp. Med. 166, 1836-1850, 1987.

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17. LOIKE, J.D., SILVERSTEIN, R., WRIGHT, S.D., WEITZ, J.I., I-WANG, A.J. and SILVERSTEIN, S.C. The role of protected extracellular compartments in interactions between leukocytes, and platelets, and fibrin/fibrinogen matrices. Ann. N.Y. Acad. Sci. 667, 163-172, 1992. 18. KOLEV, K., L&ANT, I., TENEKEJIBV, K. and MACHOVICH, R. Regulation of fibrinolytic activity of neutrophil leukocyte elastase, plasmin and miniplasmin by plasma protease inhibitors. J. Biol. Chem. 269,25, 17030-17034, 1994.