Influence of granulocyte elastase-like proteinase (ELP) on platelet functions

THROMBOSIS RESEARCH 41; 837-846, 1986 0049-3848/86 $3.00 t .OO Printed in the USA. Copyright (c) 1986 Pergamon Press Ltd. All rights reserved.

INFLUENCE OF GRANULOCYTE ELASTASE-LIKE PROTEINASE ON PLATELET FUNCTIONS

(ELP)

* M. Taki, T. Miura, M.+Inagaki, Y. Tomita, N. $$ito, Y. Okad:, Y. Tsuda, T. Megurg*and K. Yamada Department of Pediatrics, Keio University, School of Medicine, Tokyo, Department of Medical Chemistry, Faculty of Pharmaceutical Sciences, Kobe-GakuinUniversity,Kobe*and Department of Pediatrics,St.Marianna University, School of Medicine, Kawasaki:* Japan

(Received 17.5.1985;

Accepted in revised form 12.12.1985 by Editor U. Okamoto)

ABSTRACT The influence of granulocyte elastase-like proteinase (ELP) on platelet functions was investigated. ELP inhibited the platelet aggregations induced by a wide variety of agonists. The inhibition was marked in the case of receptor-mediated agonists such as thrombin, ristocetin, etc. It was moderate with the pervading agonist, arachidonic acid, and mild with the bypassing agonist, Ca'+ionophore A23187. ELP inhibited the release of thromboxane A2 from platelets On the in the case of the platelet aggregation induced by thrombin. other hand, ELP did not inhibit the release of thromboxane A2 from platelets in the platelet aggregation induced by arachidonic acid or Ca'+ionophore A23187. ELP suppressed the release of serotonin from platelets induced by thrombin, while it did not markedly suppress Treatment the release of serotonin induced by Ca2+ionophore A23187. of platelets with ELP resulted in a slight increase of intraplatelet CAMP levels. These results suggest that ELP acts on receptors and inhibits platelet functions. As a results, ELP markedly inhibits the platelet functions such as aggregation or release of serotonin or thromboxane A2 stimulated by receptor-mediated agonists. ELP slightly elevates the CAMP level in the platelets, resulting in the mild inhibition of the platelet functions stimulated by the pervading agonist, arachidonic acid, or the bypassing agonist, Ca2+ionophore A23187.

INTRODUCTION Release of granulocyte elastase-like proteinase (ELP) is observed in situation such as inflammation (1) or during blood coagulation (2). ELP was found to destroy the activity of coagulation factors II, v, VII (3,4),VIII (3, 5,6),

Key words: elastase-like proteinase reaction, CAMP, receptor

(ELP), platelet aggregation,

837

release

ELP ON PLATELET FUNCTIONS

838

Vol. 41, No. 6

XII (3, 4) and XIII (3, 7). The enzyme degrades fibrinogen (8, 9), von Willebrand factor (5, 6) and fibronectin (8,10,11). The enzyme inactivates plasma inhibitors in coagulation and fibrinolysis system such as antithrombin III (12), Cl-inhibitor (12) and a2-plasmin inhibitor (12, 13). Several studies have suggested a potential role for granulocytes in the modulation of thrombosis and hemostasis (14-20). The present report describes studies on the mechanism of influence of ELP on platelet functions. The findings obtained suggested that treatment of platelets with ELP resulted in a change of receptors, and slight increase of the intraplatelet CAMP levels. Consequently, ELP markedly inhibits platelet functions such as aggregation or release ofserotonin or thromboxane A2 stimulated by receptor mediated-agonists, and mildly inhibits platelet functions stimulated by the pervading agonist, arachidonic acid, or the bypassing agonist, Ca'+ionophore A23187.

METHODS Purification

of ELP

ELP was purified from healthy human granulocytes by affinity chromatography using a specific synthetic inhibitor for ELP according to the method of Okada et al. (21-23). The affinity Sepharose containing specific inhibitor for ELP, Sue-L-Tyr-D-Leu-D-Val-pNA-Sepharose, was synthesized in our laboratories (21, 24). The amidolytic activity was measured using Suc-L-Ala-L-Tyr-LVal-pNA which was synthesized in our laboratories for the assay of ELP (25). The level of ELP activity was estimated from the level ofpNA (410 nm) released from the substrate (U=pmol/min, tentatively). SDS polyacrylamide

gel electrophoresis

7% SDS polyacrylamide gel electrophoresis method of Weber and Osborn (26). Preparation

was performed according to the

of washed platelet suspensions

Citrated whole blood was obtained from healthy adults by venepuncture. Platelet rich plasma (PRP) was prepared from the blood. In the studies on platelet aggregation, t$romboxane A2 release or intraplatelet cAMPlevel,platelet suspensions (3 X 10 /ml) were prepared from the PRP by washing with 0.3% bovine serum albumin in Ca2+ -free Hepes-Tyrode buffer (pH 7.4). Calcium chloride was added to give final concentration of 0.5 mM just before the addition of agonists. In the study on serotonin release, platelet suspensions were prepared from the PRP by washing with 10 mM Tris-HCl, 1 mM EDTA, 150 mM NaCl (pH 7.3). Measurement

of platelet aggregation

in washed platelet suspensions

Platelet aggregation was observed with an NKK Aggregometer or Chrono-Log The various kinds of agonists for induction of plateCo. Lumi Aggregometer. let aggregation were used in following final concentrations: ADP (2 PM) with fibrinogen (0.1 mg/ml), epinephrine (50 PM) with fibrinogen (O.lmg/ml), thrombin (0.1 U/ml), collagen (5 ug/ml), ristocetin (1.2 mg/ml) with von Willebrand factor (0.1 U/ml), arachidonic acid (50 PM), and Ca'+ionophore A23187 (5 FM). In order to assess the influence of ELP on platelet aggregation, platelets were incubated with various concentrations of ELP for 10 min, 30 min,etc. at 37OC, before the addition of agonists.

Vol. 41 No. 6

ELP ON PLATELET FUNCTIONS

839

Assay of thromboxane A2 release from platelets The release of thromboxane A2 from platelets in the platelet aggregation induced by agonists such as thrombin (0.1 U/ml), arachidonic acid (50 PM) or Ca*+ionophore A23187 (5 PM) was measured by radioimmunoassay of the thromboxane B2 at 5 min after the addition of agonists. In order to examine the influence of ELP on the release of thromboxane A2 from platelets in the platelet aggregation, platelets were incubated with various concentrations of ELP for 10 min or 30 min at 37'C before the addition of agonists. Assay of serotonin release from platelets 14 C-serotonin from prelabeled platelets stimulated by agoThe release of nists such as thrombin (0.1 U/ml) or Ca*+ionophore A23187 (1 PM) was measured according to the method of Mills et al. (27). In order to examine the influence of ELP on the release of serotonin from platelets, platelets were incubated with various concentrations of ELP for 30 min at 37OC before the addition of agonists. Assay of intraplatelet CAMP The intraplatelet CAMP was estimated by radioimmunoassay. In order to examine the influence of ELP on the level ofcAMPinthe platelets, platelets were incubated with various concentrations of ELP for 10 min or 30 min at 37OC.

RESULTS ELP The purified ELP fraction was found to be homogeneous on 7% SDS polyacrylamide gel electrophoresis (Fig. 1). The molecular weight was approximetely 30 kilo-daltons. Mw

FIG. 1 57200 42900 e

28600

a)

b)

ELP

SDS polyacrylamide gel electrophoresis of ELP: a) calibration protein, b) ELP. The electrophoresis was performed at a constant current of 8 mA per gel for 4 hr using 7% gel.

840

ELP ON PLATELET FUNCTIONS

Vol. 41, No. 6

Effects of ELP on platelet aggregation Preliminary examinations demonstrated that platelet aggregation was not observed when platelets were incubated with ELP. The platelet aggregation induced by a wide variety of agonists was inhibited by ELP, depending on the concentration of ELP and the incubation time with ELP (Fig. 2). The inhibition was marked with thrombin, collagen, epinephrine or ristocetin. It was moderate with arachidonic acid or ADP, and mild with Ca'+ionophore A23187 (Fig. 3).

o-

o-

s

IO-

2

10

z zo-

g

20-

p :

30

z

30-

f

40-

f

40-

$

50-

:

50-

2

60-

;

60-

=

70-

3

no-

= 0 1

no-

?

goIOO-

70-

goIOOb)

FIG. 2 Inhibition of thrombin (0.1 U/ml)-induced platelet aggregation in ELP-treated platelets. a)

Platelets were incubated with 0.016 U/ml of ELP for 10, 30, 45 or 60 min at 37'C. b) Platelets were incubated with various concentrations of ELP for 30 min at 37'C.

Effects of ELP on thromboxane A2 release from platelets The release of thromboxane A2 from platelets which were treated with ELP was suppressed in the case of the platelet aggregation induced by thrombin in a dose- and time-dependent manner, but was not suppressed in the case of the platelet aggregation induced by arachidonic acid or Ca*+ionophore A23187 (Fig. 4). Effects of ELP on serotonin release from olatelets Preliminary examinations demonstrated that release of serotonin was not observed when platelets were incubated with various concentrations of ELP for 30 min at 37OC. The release of serotonin from platelets which were treated with ELP was markedly suppressed in the stimulation with thrombin, while it was not so greatly suppressed in the stimulation with Ca*+ionophore A23187 (Fig. 5).

Vol. 41 No. 6

841

ELP ON PLATELET FUNCTIONS

a)

e)

ADP+fg

ristocetin

+vWF

b) epinephrine+fg

L

8) A23187

control

t

control

FIG. 3 Inhibition of platelet aggregation

in ELP-treated

platelets.

Platelets were incubated with ELP (0.016 U/ml) for 30 min at 37OC before the fg: fibrinogen (0.1 mg/ml), vWF: von Willebrand factor addition of agonists. (0.1 U/ml), ADP (2 PM), epinephrine (50 PM), thrombin (0.1 U/ml), collagen (5 pg/ml, ristocetin (1.2 mg/ml), AA: arachidonic acid (50 PM), A23187 (5 PM).

842

ELP ON PLATELET FUNCTIONS

41, No. 6

Vol.

thrombin

(0.1 U/ml)

30min .. .’ arachidonic acidt50NI

..” ,. .,

30

so time (set) A23187 ( 1 PM)

0

o.olo

0.005

o.Ol5

0

ELP ( u/ml I

30

60

180

time (set)

FIG. 5

FIG. 4 Thromboxane A2 release from platelets induced by thrombin (0.1 U/ml), arachidonic acid (50 PM) or Ca*+ionophore A23187 (5 PM). Platelets were incubated with various concentrations of ELP for 10 min or 30 min at 37'C before the addition of agonists.

Effects of ELP on intraplatelet

0

Serotonin release from platelets stimulated by thrombin (0.1 U/ml) or Ca*+ionophore A23187 (1 PM). Platelets were incubated with various concentrations of ELP for 30 min at 37'C before the addition of agonists.

CAMP

Treatment of platelets with ELP resulted in a slight elevation of the intraplatelet CAMP levels (Table 1).

843

ELP ON PLATELET FUNCTIONS

Vol. 41 No. 6

TABLE 1 Effects of ELP on intraplatelet CAMP intraplaselet CAMP (pmol/lO platelets) 10

incubation time (min) buffer

+ platelets

ELP (0.04 U/ml) + platelets ELP (0.08.U/ml) + platelets

8.3 + 0.4 15.6 + 0.4* 17.0 + 0.8*

30 7.6 + 0.0 16.4 + 1.6** 16.3 + 0.6*

Mean + SD, n=3. +p
DISCUSSION The present series of experiments revealed that the platelet aggregation induced by a wide variety of agonists was inhibited by ELP. The inhibition was marked in the case of receptor-mediated agonists such as thrombin, ristocetin, etc. It was moderate with a pervading agonist such as arachidonic acid, and mild with a bypassing agonist such as Ca*+ionophore A23187. Furthermore, release of thromboxane A2 from platelets which were treated with ELP in the case of the platelet aggregation induced by thrombin was suppressed. On the other hand, the release was not suppressed in the case of the platelet aggregation induced by arachidonic acid or Ca*+ionophore A23187. Release of serotonin from platelets which were treated with ELP was markedly suppressed in the stimulation with thrombin, while it was not so greatly suppressed in the stimulation with Ca*+ionophore A23187. These results suggest that ELP acts on receptors and inhibits platelet functions, and that ELP does not inhibit the arachidonic acid metabolism after liberation of free arachidonic acid. ELP was recently reported to show selective proteolysis of platelet membrane glycoprotein Ib (28). This finding is consistent with the fact that ELP can inhibit platelet functions stimulated by thrombin or ristocetin. ELP also inhibited platelet aggregation induced by ADP, epinephrineorcollagen. Therefore, it was suggested that treatment of platelets with ELP resulted in a change of receptors including membrane glycoprotein Ib. The role of CAMP in the regulation of platelet aggregation is well documented. The precise mechanism.of action is not yet clearly understood, although an elevation of the intraplatelet CAMP level results in an inhibition of aggregation (29). Treatment of platelets with ELP resulted in a slight increase of the intraplatelet CAMP levels. Therefore, ELP also mildly inhibited the platelet functions such as aggregation or release reactions stimulated by the pervading agonist, arachidonic acid, or the bypassing agonist, Ca*+ionophore A23187. In summary, the present findings suggest that ELP acts on receptors and

844

ELP ON PLATELET FUNCTIONS

Vol. 41, No. 6

inhibits platelet functions. The treatment of platelets with ELP results in a slight elevation of the intraplatelet CAMP levels, so that ELP mildly inhibits the platelet functions stimulated by the pervading agonist, arachidonic acid, or the bypassing agonist, Ca*+ionophore A23187.

ACKNOWLEDGEMENTS We wish to thank Drs. Y. Ando, K. Watanabe and Y. Ikeda, Keio University, School of Medicine, for their helpful discussions.

REFERENCES 1.

OHLSSON, K., OLSSON, I. The extracellular release of granulocyte collagenase and elastase during phagocytosis and inflammatory processes. Stand. J. Haematol. -19, 145-152, 1977.

2.

PLOW, E.F. Leukocyte elastase release during blood coagulation: A potential mechanism for activation of the alternative fibrinolytic pathway. J. Clin. Invest. E, 564-572, 1982.

3.

SCHMIDT, W., EGBRING, R., HAVEMANN, K. Effect of elastase-like and chymotrypsin-like neutral proteases from human granulocytes on isolated clottThromb. Res. 6, 315-326, 1975. ing factors.

4.

EGBRING, R., SCHMIDT, W., FUCHS, G., HAVEMANN, K. Demonstration of granulocytic proteases in plasma of patients with acute leukemia and septiBlood 49, 219-231, 1977. cemia with coagulation defects. --

5.

KOPEC, M., BYKOWSKA, K., LOPACIUK, S., JELENSKA, M., KACZANOWSKA, J., SOPATA, I., WOJTECKA, E. Effects of neutral proteases from human leukocytes on structure and biological properties of human factor VIII. Thromb. Haemostas. 43, 211-217, 1980.

6.

VARADI, K., MAROSSY, K., ASBOTH, G., ELijDI, P., ELijDI, S. Inactivation Thromb. Haemostas. 43, of human factor VIII by granulocyte proteases. 45-48, 1980.

7.

HENRIKSSON, P., NILSSON, I.M., OHLSON, K., STENBERG, P. Granulocyte elastase activation and degradation of factor XIII. Thromb. Res. 18, 343-351. 1980.

8.

KOPEC, M., KACZANOWSKA, J., STACHURSKA, J., TOMCZAK, Z., WOJTECKA-LUKASIK, E ., BYKOWSKA, K., LOPACIUK, S. Effect of granulocytic neutral proteases In: Progress in Fibrinolysis. Davidson, J.F., on some clotting factors. Nilsson, I.M., ftstedt, B. (Eds.). Churchill Livingstone, Edinburgh, London, Melbourne, New York, 2, 75-77, 1981.

9.

GRAMSE, M., BINGENHEIMER, C., SCHMIDT, W., EGBRING, R., HAVEMANN, K. Degradation products of fibrinogen by elastase-like neutral protease from J. Clin. Invest. g, 1027-1033, 1978. human granulocytes.

10.

MCDONALD, J.A., KELLEY, D.G. Degradation of fibronectin by human leukoJ. Biol. Chem. 255, 8848-8858, 1980. cytic elastase.

Vol. 41 No. 6

ELP ON PLATELET FUNCTIONS

845

11.

VARTIO, C. Characterization of binding domains in the fragments cleaved by cathepsin G from human plasma fibronectin. Eur. J. Biochem. E,223233, 1982.

12.

BROWER, M.S, HARPEL, P.C. Proteolytic cleavage and inactivation of e2plasmin inhibitor and Cl inactivator by human polymorphonuclear leukocyte elastase. J. Biol. Chem. 257, 9849-9854, 1982.

13.

KLINGEMAN, H.C., EGBRING, R., HOLST, F., GRAMSA, M.,HAVEMANN, K. Digestion of a2-plasmin inhibitor by neutral proteases from human leukocytes. Thromb. Res. 2, 479-483, 1981.

14.

HENRY, R.L. 46, 1965.

15.

WESTER, J., SIXMA, J.J., GEUZE, J.J., HEIJNEN, H. Morphology of the hemostatic plug in human skin wounds: Transformation of the plug. Lab. Invest. 4l_,182-192, 1979.

16.

MARCUS, A.J., BROEKMAN, M.J., SAFIER, L.B., ULLMAN, H.L., ISLAM, N., SERHAN, C.N., RUTHERFORD, L.E., KORCHAK, H.M., WEISSMAN, G. Formation of leukotrienes and other hydroxy acids during platelet-neutrophil interactions in vivo. Biochem. Biophys. Res. Commun. 109, 130-137, 1982.

17.

MACLOUF, J., FRUTEAU DE LACLOS, B., BORGEAT, P. Stimulation of leukotriene biosynthesis in human blood leukocytes by platelet derived 12hydroperoxy-icosatetraenoicacid. Proc. Natl. Acad. Sci. USA, 3, 60426046, 1982.

18.

SERHAN, C.N., RADIN, A., SOMOLEN, J.E., KORCHAK, H., SAMUELSSON, B., WEISSMAN, G. Leukotriene B4 is a complete secretagogue in human neutrophils: A kinetic analysis. Biochem. Biophys. Res. Commun. 107, 10061012, 1982.

19.

LONKY, S.A., MARSH, J., WOHL, H. Stimulation of human granulocyte elastase by platelet factor 4 and heparin. Biochem. Biophys. Res. Commun. 85, 1113-1118, 1978.

20.

LONKY, S.A., WOHL, H. Stimulation of human leukocyte elastase by platelet factor 4: Physiologic, morphology, and biochemical effects on hamster lungs in vitro. J. Clin. Invest. 67, 817-826, 1981.

21.

OKADA, Y., TSUDA, Y., NAGAMATSU, Y., OKAMOTO, U. Convenient purification of human spleen fibinolytic proteinase (SFP) and human leucocyte elastase by affinity chromatography. Chem. Pharm. Bull. 30, 152811530, 1982.

22.

OKAMOTO, U., NAGAMATSU, Y., OKADA, Y., TSUDA, Y. Novel synthetic selective peptide inhibitors of human spleen fibrinolytic proteinase (SFP) and leukocyte elastase like proteinase (ELP): with special reference to the use of inhibitors for the purification of the enzymes. In: Progress in Fibrinolysis. Davidson, J.F., Nilsson, I.M., Astedt, B. (Eds.). Churchill Livingstone, Edinburgh, London, Melbourne, New York, 5, 391393, 1981.

23.

NAGAMATSU, Y., OKAMOTO, U., TSUDA, Y., OKADA, Y. Human leukocyte elastase-like proteinase purified by affinity chromatography with Suc-L-TyrD-Leu-D-Val-pNA, and its identification with human spleen fibrinolytic

Leukocytes and thrombosis. Thromb. Diath. Haemorrh. 13, 35-

846

ELP ON PLATELET FUNCTIONS

proteinase.

Vol. 41, No. 6

Thromb. Haemostas. 51, 243-247, 1984.

24.

OKADA, Y., TSUDA, Y., NAGAMATSU, Y., OKAMOTO, U. Synthesis of stereoisomeric Sue-Tyr-Leu-Val-pNA and their properties as substrate and inhibitor for human spleen fibrinolytic proteinase (SFP). Chem. Pharm. Bull. 29, 1483-1486, 1981.

25.

OKADA, Y., TSUDA, Y., HIRATA, A., NAGAMATSU, Y., OKAMOTO, U. Synthesis of chromogenic substrates specific for human spleen fibrinolytic proteinase (SFP) and human leukocyte elastase (LE). Chem. Pharm. Bull. 30, 4060-4068, 1982.

26.

WEBER, K., OSBORN, M. The reliability of molecular weight determination J. Biol. Chem. by dodecyl sulfate-polyarylamide gel electrophoresis. 244, 4406-4412, 1969.

27.

The release of nucleotides, MILLS, D.C.B., ROBB, I.A., ROBERTS, G.C.K. 5-hydroxytryptamine and enzymes from human blood platelets during aggre195, 715-729, 1968. gation. J. Physiol. -

28.

BROWER, M.S., LEVIN, R.I., GARRY, K. Human neutrophil elastase modulates platelet function by limited proteolysis of membrane glycoproteins. J. Clin. Invest. 2, 657-666, 1985.

29.

WHITTLE, B.J.R., MONCADA, S. Pharmacological interactions between prosBr. Med. Bull. 2, 232-238, 1983. tacyclin and thromboxanes.