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protein kinase C inhibitors
THROMBOSIS RESEARCH 49; 205-214, 1988 0049-3848/88 $3.00 + .OO Printed in the USA. Copyright (c) 1988 Pergamon Journals Ltd. All
rights
reserved.
AUGMENTATION OF PROCOAGULANT ACTIVITY IN MONOKINE STIMULATED HUMAN ENDOTHELIAL CELLS BY CALMODULIN/PROTEIN KINASE C INHIBITORS
Steven H. Zuckerman and Yvonne M. Surprenant Department of Immunology, Lilly Research Labs, Indianapolis, IN 46285, (Received
31.8.1987;
Accepted in revised by Editor N.U. Bang)
USA
form 28.10.1987
ABSTRACT The incubation of human umbilical cord endothelial cell cultures with inflammatory mediators results in the induction of procoagulant activity. As many of these mediators activate protein kinase C, the effect of calmodulin and protein kinase C inhibitors on IL-l, TNF, phorbol ester and LPS stimulated procoagulant activity was determined. Incubation of endothelial cell cultures with these inflammatory agents in the presence of phenothiazine derivatives or other classes of calmodulin and protein kinase C antagonists resulted in a 2-4 fold increase in procoagulant activity compared to parallel stimulated cultures in the absence of antagonists. The augmented response of IL-1 stimulated endothelial cells to these antagonists was actinomycin D sensitive.
The vascular endothelium plays a central role in separating blood elements from extravascular tissue. Stimulation of primary endothelial cell cultures with inflammatory agents including lipopolysacchande, LPS, phorbol esters, or the monokines Interleukin-1 , IL-l, and Tumor Necrosis Factor, TNF, results in significant changes in membrane components and in endothelial-endothelial and endothelialleukocyte cellular interactions. Specifically, increased adherence of peripheral blood monocytes, neutrophils and the human leukemic cell lines HL-60 and U937 to IL-l, LPS, or phorbol ester stimulated endothelial cells has been repotted and correlates with the induction of a leukocyte adherence molecule H 4118 on the endothelial cell surface (l-4). The increased secretion of platelet activating factor, GranulocyteMacrophage Colony Stimulating Factor, prostacyclins, plasminogen activator inhibitor, Key words: cells
calmodulin, protein kinase C, monokine, procoagulant activity, endothelial
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and IL-l has also been detected in stimulated endothelial cell culture supernatants (5 9). Endothelial cells can also be induced to express procoagulant activity (tissue factor, thromboplastin, factor Ill) by inflammatory mediators thus resulting in the activation of the coagulation cascade by the extrinsic pathway (10-14). The activation of procoagulant activity could contribute to localized or disseminated intravascular coagulation observed during inflammation or endotoxemia respectively. While the induction of procoagulant activity in human endothelial cell cultures by inflammatory mediators is well documented, little is known concerning the intracellular processes which modulate the level of this activity. Many of the mediators which induce procoagulant activity on endothelial cells have been demonstrated to activate protein kinase C (15). The diversity of biologic processes regulated by calcium or phospholipid dependent protein kinases through calmodulin or protein kinase C would suggest that antagonists of these calcium binding proteins might affect procoagulant expression. The present study was undertaken to investigate the effect of calmodulin and protein kinase C inhibitors on the induction of endothelial cell procoagulant activity by inflammatory mediators. We report that protein kinase C and calmodulin antagonists actually increase the amount of procoagulant activity detected on LPS, phorbol ester, IL-l, or TNF stimulated primary human endothelial cell cultures.
MAFRlAl SAND METHODS Compounds. Phenothiazine derivatives, and the tricyclic antidepressants, imipramine, chloroimipramine, and amitriptyline were provided by Drs. Robert Archer and Ray Fuller (Lilly Research Labs, Indianapolis, IN). Staurosporine was provided by Dr. Jeff Howbert (Lilly Research Labs). Compounds W-7 (N-(6-aminohexyl)S-chloro-lnapthalenesulfonamide), 48/80, calmidazolium, actinomycin D, phorbol 12-myristate 13-acetate, as well as calmodulin and mellitin were obtained from Sigma Chemical (St. Louis, MO). Endothelial Cell Cultures. Primary human umbilical cord endothelial cells were established as primary cultures, and provided at passage 1 by Dr. Francoise Booyse (University of Alabama, Birmingham, AL). Endothelial cell cultures were grown in medium 199, M199, (GIBCO Labs, Grand Island, NY) containing 10% fetal calf sera (Hyclone, Logan, UT), 1% glutamine (GIBCO), 2 units/ml of heparin (Lilly Res. Labs, Indianapolis, IN) and 60 ug/ml of Endothelial Cell Growth Supplement, ECGS, (Collaborative Research, Bedford, MA) on fibronectin (Collaborative Research) coated (1 ug/cm*) tissue culture flasks. Procoagulant Assay. Human umbilical cord endothelial cell cultures between passages 2-20 were trypsinized, plated at 3 x 10s cells/well on fibronectin coated 96 well plates (Costar, Cambridge, MA) and allowed to recover for 24 hours prior to assay. Procoagulant activity was assayed by a modified two stage amidolyltic assay using the chromogenic substrate S-2222 (13). Briefly, microtiter plate cultures in triplicate were induced with 2 ng/ml of IL-1 (human recombinant IL-1 j3 provided by Dr. J.L. Bobbitt, Lilly Research Labs, Indianapolis, IN), 1 unit/ml of TNF (Amgen Biologicals, Thousand Oaks, CA) 20
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ng/ml of LPS (E coli 05585, Difco Labs, Detroit, MI) or 300 pg/ml of phorbol 12-myristate 13-acetate, PMA, for 4 hours in the presence or absence of calmodulin or protein kinase C inhibitors. All inhibitors were used at 10 ug/ml, approximately 2.5 x 10-s M unless otherwise indicated. Following incubation, plates were washed twice with phosphate buffered saline and cells were then incubated for 2 hrs at 37 degrees C with 100 uliwell of Ml99 without phenol red (GIBCO Labs) containing 1.3 factor VII units/ml of a coagulation factor concentrate consisting of factors II, VII, IX, and X (Proplex T, Travenol Labs, Inc., Glendale, CA) and 500 ug/ml of S2222 (Helena Labs, Beaumont, TX).’ The change in optical density at 410 nm was quantitated using a Dynatech MR 600 microplate reader (Dynatech Laboratories Inc., Alexandria, VA).
The demonstration that protein kinase C activators were capable of inducing procoagulant activity in human endothelial cells (10-14) suggested that compounds which block calcium dependent processes could modulate procoagulant induction. Several phenothiazine derivatives have been demonstrated to inhibit both calmodulin and protein kinase C dependent processes (16-20) and these and other phenothiazines were utilized in the present study (figure 1).
Trivlrl
Name
phenothiazine 2-CF3phenothiezine 2aetylphenothiazine chlorpromazine triflupromazine acetylpromazine promelhazine trimeprazine
prochlorperazine trifluoperazine perphenazine
X
H H H CH+H2CH2N(Me)2 CH&H2CH2N(Me)2 CH,CH,CH,N(Me),
x CF3 COCH3 Cl CFa COCHI
CHzCH(Me)N(Meh CHzCH(Me)CHzN(Meh
H H
CHa CHa CH&HzOH
CF, Cl
Cl
FIG. 1. Structure of Phenothiazine Derivatives. These phenothiazine derivatives differ in their IC50 for calmodulin-induced of phosphodiesterase and in their medicinal application as tranquilizers,
activation
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chlorpromazine, triflupromazine, acetylpromazine, prochlorperazine, perphenazine, as an antihistaminic, promethazine, as an antipruritic, as an antihelminthic, phenothiazine (20). Incubation of endothelial cells with IL-1 and the phenothiazine ug/ml resulted in a 2-4 fold increase in procoagulant activity over that alone (Fig. 2).
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trifluoperazine, trimeprazine, and derivatives at 10 observed for IL-l
Perphenazine Trifluoperazine Prochlorperazine Trimeprazine Promethazine Acetylpromazine Triflupromazine Chlorpromazine 2-Acetylphenothiazine 2-CF3-Phenothiazine Phenothiazine IL-1 Control
Optical Density FIG. 2. Effect of Phenothiazine Derivatives on IL-1 induced Procoagulant Activity. Phenothiazine derivatives at 10 ug/ml (2.5 x 1O-5M) were incubated for 4 hours with endothelial cell cultures in the presence of IL-l and procoagulant activity was determined. Endothelial cells without IL-l, control, had no detectable procoagulant activity. Representative experiment of four separate experiments. Brackets indicate the standard error of the mean.
This was evident with all the phenothiazine derivatives except those simply consisting of the basic ring structure, phenothiazine, 2-CF3-phenothiazine, and 2acetlyphenothiazine. The augmentation in procoagulant activity observed in endothelial cells induced with IL-1 plus phenothiazine derivatives such as chlorpromazine or prochlorperazine was dependent upon &I~QYQ transcription as the addition of actinomycin D at 1 ug/ml completely inhibited the induction of procoagulant activity (data not shown). Furthermore, in the absence of IL-l, the phenothiazines alone did not result in an increase in procoagulant activity. In a representative experiment, an optical density of .039 was observed for endothelial cells plus prochlorperazine, .362 for cells plus IL-l, and .707 for cells plus prochlorperazine plus IL-l. The addition of phenothiazines at the substrate stage of the procoagulant assay did not atter the amount of substrate conversion observed (data not shown). Augmentation of procoagulant activity by phenothiazine derivatives was also seen when a suboptimal concentration (300 pg/ml) of phorbal myristate acetate was used as the inducing agent. As demonstrated in table 1, both chlorpromazine and prochlorperazine augmented procoagulant activity in endothelial cell cultures by 175 and 200 percent respectively.
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Table 1 Effect of Phenothiazine
Derivatives on Phorbol Ester Induced Procoagulant Activity
cal Density
fUt.U
.007
Control PMA PMA + Chlorpromazine PMA + Prochlorperazine
.168 .295 .359
(.OOl) (.012) (.026) (.031)
Mean value of triplicate wells is presented. Brackets indicate the standard error of the mean. The effect of non-phenothiazine related calmodulin, protein kinase C inhibitors on the induction of procoagulant activity was determined to exclude the possibility that the augmentation observed was unique to the phenothiazine class of compounds. As seen in figure 3, other classes of calmodulin and protein kinase C inhibitors also resulted in a 2-3 fold increase in procoagulant activity over that observed for IL-l.
Stau rosporine Calm idazolium 48180 Amitriptyline Imipramine Chloroimipramine w-7 Mellitin Proc‘hlorperazine IL-I I’ 0
1
100
’
I
200
I
’
3do
41
Percent Increase FIG. 3. Augmentation of Procoagulant Activity by different classes of calmodulin/protein kinase C inhibitors. Compounds were incubated with endothelial cells in the presence of IL-l. The increase in procoagulant activity induced by IL-1 was expressed as lOO%, and all values are expressed relative to IL-1 stimulated cells. Representative experiment of six. Standard errors are indicated by brackets.
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This included the tricyclic antidepressants imipramine, chloroimipramine, and amiyltryptline, compounds W-7, 48/80, and calmidazolium, staurosporine and mellitin (19-24). All compounds were used at a final concentration of 10 ug/ml except for calmidazolium at 1 ug/ml, mellitin at 0.1 ug/ml, and staurosporlne at 1 pg/ml. The augmented response of IL-1 stimulated endothelial cells was no longer apparent when these compounds were diluted 10 or 100 fold (staurosporine). Therefore, phenothiazine and non-phenothiazine related protein kinase C and calmodulin inhibitors augmented the induction of procoagulant activity by IL-l. The increase in procoagulant activity observed by these calmodulin, protein kinase C inhibitors was not restricted to IL-1 or PMA mediated induction of procoagulant activity. A similar augmentation was also observed with these compounds at comparable concentrations when endothelial cells were stimulated with LPS’or TNF (Fig. 4 a,b).
Calmidazolium 48180 Chlorpromazine Staurosporine LPS
Calmidazolium 48180 Chlorpromazine Staurosporine TNF 200 100 Percent Increase
FIG. 4. Augmentation of LPS or TNF induced procoagulant activity by calmodulin/protein kinase C antagonists. Endothelial cells were incubated with compounds in the presence of LPS (a) or TNF (b). Procoagulant activity induced by LPS or TNF alone was expressed as 100 percent and all other values were expressed relative to the control stimulated cultures. Representative experiment of four, standard errors are indicated by brackets. Increases between 150-500 percent were observed relative to cultures stimulated with LPS or TNF alone. Therefore, calmodulin and protein kinase C inhibitors augment procoagulant activity induced by a variety of inflammatory mediators.
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Changes in the endothelial cell surface during inflammation can contribute to the severity of the inflammatory response by activating the coagulation cascade and by promoting leukocyte influx into perivascular regions. The induction of procoagulant activity in human endothelial cell cultures by inflammatory molecules such as monokines, LPS, and phorbol esters serves as a model for the identification of compounds which modulate endothelial cell surface changes. In the present study, protein kinase C and calmodulin antagonists have been demonstrated to augment the induction of procoagulant activity mediated by IL-l, TNF, PMA, or LPS. This enhancing effect was seen with distinct series of inhibitors including phenothiazine derivatives, tricyclic antidepressants, a substituted napthalene sulfonamide (W-7), the antibiotic staurosporine as well as with other classes of compounds. Whether this effect on procoagulant induction is related to calmodulin or protein kinase C dependent processes cannot be definitively determined as most of these inhibitors act on both pathways (19-24). Furthermore, many of these compounds are antagonists in other systems. Phenothiazines have been reported to block dopamine receptors (25), 5-HT2 receptors (26), and calcium channels (27). The increase in procoagulant activity induced by IL-1 and chlorpromazine or prochlorperazine however, was not reversible by excess dopamine and could not be demonstrated with the 5-HT2 antagonist Ketanserin or with the calcium channel blocker Nifedipine (data not shown). The increased procoagulant activity observed with calmodulin, protein kinase C inhibitors was apparent when inhibitors were present in the induction phase of the assay, not during the substrate phase and was dependent upon de novo transcription. The observation that calmodulin/protein kinase C antagonists augment the level of procoagulant activity observed following induction with known protein kinase C activators would appear a priori to be paradoxical. The phorbol ester induced increase in transferrin endocytosis by rabbit reticulocytes (28) and the phorbol ester induced expression of the human monocyte surface antigen Mo3e on human monocytic cell lines (29) for example, have been reported to be inhibited by calmodulin/protein kinase C antagonists. Protein kinase C activators however, have also been shown to down-regulate TNF receptors on human T cells (30) transferrin receptors on HL60 (31), epidermal growth factor receptors (32), and gamma interferon receptors on the murine T cell line EL-4 (33). A Protein kinase C antagonist, H7 (1-(5isoquinolinesulfonyl)-2-methylpiperazine), has been reported to partially block the down-regulation of TNF receptors on T cells (30). In view of these observations, the augmentation in procoagulant induction observed in the presence of protein kinase C/calmodulin antagonists may be explained by the following hypothesis. First is that the induction of procoagulant by IL-l, TNF, PMA, and LPS is occurring through a nonprotein kinase C dependent mechanism. The activation of protein kinase C does, however, result in the down-regulation of TNF receptors and presumably IL-1 and LPS binding components as well thus rendering the endothelial cells refractory to subsequent stimulation by inflammatory mediators. The protein kinase C/calmodulin antagonists prevent receptor down regulation and therefore, these cells can be further stimulated by the continued presence of inflammatory mediators. While other explanations are possible the proposed model represents a working hypothesis amenable to further investigation. The in vivo significance of these findings that clinically relevant phenothiazine derivatives augment human endothelial cell procoagulant activity in response to an inflammatory stimulus cannot be determined from this study. The in vivQ concentration
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and metabolism of these compounds as well as the complexities of endothelial cell interaction with both peripheral blood and extravascular elements must be considered before & yivcr extrapolation of these observations can be considered. Clearly, protein kinase C, calmodulin antagonists alter the dynamics of the endothelial cell response to inflammatory mediators. Whether other parameters of endothelial cell function, such as changes in their secretory capacity, are also altered remains to be determined.
We wish to thank Drs. Robert Archer, Marlene Cohen, Jeff Howbert, and Winston S. Marshall for providing us with the necessary antagonists and for their helpful discussions and Dr. Francoise Booyse for providing us with early passage human endothelial cell cultures.
1. BEVILACQUA, M.P., POBER, J.S., MAJEAU, G.R., COTRAN, R.S., and GIMBRONE JR., M.A. lnterleukin 1 (IL-l) induces biosynthesis and cell surface expression of procoagulant activity in human vascular endothelial cells. J. Exp. Med., 160, 618-623, 1984. 2. SCHLEIMER, R.P. and RUTLEDGE, B.K. Cultured human vascular endothelial cells acquire adhesiveness for neutrophils after stimulation with interleukin 1, endotoxin, and tumor-promoting phorbol diesters. J. Immunol., 136, 649-654, 1986. 3. BEVILACQUA, M.P., POBER, J.S., WHEELER, M.E., COTRAN, R.S., and GIMBRONE JR., M.A. Interleukin-1 activation of vascular endothelium. Effects on procoagulant activity and leukocyte adhesion. Am. J. Pathol. 121, 393-403, 1985 4. POBER, J.S., BEVILACQUA, M.P., MENDRICK, D.L., LAPIERRE, L.A., FIERS, W., and GIMBRONE JR., M.A. Two distinct monokines, interleukin 1 and tumor necrosis factor, each independently induce biosynthesis and transient expression of the same antigen on the surface of cultured human vascular endothelial cells. J. lmmunol., 136, 1680, 1986. 5. NAWROTH, P.P., BANK, I., HANDLER, D., CASSIMERIS, J., CHESS, L., and STERN, D. Tumor necrosis factor/cachectin interacts with endothelial cell receptors to induce release of interleukin 1. J. Exp. Med., 163, 1363-1375, 1986. 6. KAWAKAMI, M., ISHIBASHI, S., OGAWA, H., MURASE, T., TAKAKU, F., and SHIBATA, S. Cachectin/TNF as well as interleukin-1 induces prostacyclin synthesis in cultured vascular endothelial cells. Biochem. Biophys. Res. Comm., 141, 482-487, 1986. 7. SIEFF, C.A., TSAI, S., and FALLER, D.V. Interleukin 1 induces cultured human endothelial cell production of Granulocyte-Macrophage Colony-stimulating factor. J. Clin. Invest., 79, 48-51, 1987.
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8. MANTOVANI, A., and DEJANA, E. Modulation of endothelial function by interleukin-1 . Biochem. Pharm., 36, 301-305, 1987. 9. GROSS, J.L, MOSCATELLI, D., JAFFE, E.A., and RIFKIN, D.B. Plasminogen activator and collagenase production by cultured capillary endothelial cells. J. Cell Biol., 95, 974-981, 1982. 10. NAWROTH, P.P., and STERN, D.M. Modulation of endothelial cell hemostatic properties by tumor necrosis factor. J. Exp. Med., 163, 740-745, 1986. 11. BROX, J.H., OSTERUD, B., BJORKLID, E., and FENTON, J.W. Production and availability of thromboplastin in endothelial cells: the effects of thrombin, endotoxin and platelets. Brit. J. Haematol., 57, 239-246, 1984. 12. GALDAL, K.S. Thromboplastin 378-385, 1984.
synthesis in endothelial cells. Haemostasis, 14,
13. COLUCCI, M., BALCONI, G., LORENZET, R., PIETRA, A., LOCATI, D., DONATI, M.B., and SEMERARO, N. Cultured human endothelial cells generate tissue factor in response to endotoxin. J. Clin. Invest., 71, 1893-1896, 1983. 14. JOHNSEN, U.L.H., LYBERG, T., GALDAL, K.S., and PRYDZ, H. Platelets stimulate thromboplastin synthesis in human endothelial cells. Thromb. Haemostas., 49, 69-72, 1983. 15. NISHIZUKA, Y. Studies and perspectives of protein kinase C. Science, 233, 3b5312, 1986. 16. SCHATZMAN, R.C., WISE, B.C., and KUO, J.F. Phospholipid-sensitive calciumdependent protein kinase: Inhibition by anti-psychotic drugs. Biochem. Biophys. Res. Comm., 98, 669-676, 1981. 17. MORI, T., TAKAI, Y., MINAKUCHI, R., YU, B., and NISHIZUKA, Y. Inhibitory action of chlorpromazine, dibucaine, and other phospholipid-interacting drugs on calciumactivated, phospholipid-dependent protein kinase. J. Biol. Chem., 255, 8378-8380, 1980. 18. WISE, B.C.,GLASS, D.B., CHOU, C.H.J., RAYNOR, R.L., KATOH, N., SCHATZMAN, R-C., TURNER, R.S., KIBLER, R.F., and KUO, J.F. Phospholipidsensitive Ca2+ dependent protein kinase from heart. II. Substrate specificity and inhibition by various agents. J. Biol. Chem., 257, 8489-8495, 1982. 19. WEISS, B., PROZIALECK, W.C., and WALLACE, T.L. interaction of drugs with calmodulin. Biochemical, pharmacological and clinical implications. Biochem. Pharmacol., 31, 2217-2226, 1982. 20. PROZIALECK, W.C., and WEISS, B. Inhibition of calmodulin by phenothiazines and related drugs: structure-activity relationships. J. Pharm. Exp. Therapeut., 222, 509-516, 1982.
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21. BARNETTE, M.S., DALY, FL, and WEISS, B. Inhibition of calmodulin activity by
insect venom peptides.
Biochem. Pharm., 32, 2929-2933, 1983.
22. GIETZEN, K. Comparison of the calmodulin antagonists compound 48/80 and calmidazolium. Biochem. J., 216, 61 l-61 6, 1983. 23. MAZZEI, G.J., SCHATZMAN, R.C., TURNER, R.S., VOGLER, W.R., and KUO, J.F.
Phospholipid-sensitive Ca 2+ dependent protein kinase inhibition by R-24571, a calmodulin antagonist. Biochem. Pharm., 33, 125-l 30, 1984. 24. TAMAOKI, T., NOMOTO, H., TAKAHASHI, I., KATO, Y., MORIMOTO, M., and TOMITA, F. Staurosporine, a potent inhibitor of phospholipid/Ca++ dependent protein kinase. Biochem. Biophys. Res. Comm.,l35, 397-402, 1986. 25. GIETZEN, K., WUTHRICH, A., and BADER, H. R 24571: A new powerful inhibitor of red blood cell Ca++ transport ATPase and of calmodulin-regulated functions. Biochem. Biophys. Res. Comm., 101, 418-425, 1981. 26. COHEN, M.L., CARPENTER, R., SCHENCK, K., WITTENAUER, L., and MASON, N. Effect of nitrendipine, diltiazem, trifluoperazine and pimozide on serotoninn (5-HT2) receptor activation in the rat uterus and jugular vein. J. Pharm. and Exp. Therapeut., 238, 860-867, 1986. 27. GOULD, R.J., MURPHY, M.M., REYNOLDS, I.J., and SNYDER, S.H. Antischizophrenic drugs of the diphenylbutylpiperidine type act as calcium channel antagonists. Proc. Natl. Acad. Sci. USA., 80, 5122-5125, 1983. 28. HEBBERT, D., and MORGAN, E.H. Calmodulin antagonists inhibit and phorbol esters endhance transferrin endocytosis and iron uptake by immature erythroid cells. Blood, 65, 758-763, 1985. 29. TODD III, R.F., BURY, M.J., ALVAREZ, P.A., BROTH, D.A., and LIU, D.Y. Regulation of human monocyte surface antigen expression. I. Up-modulation of Mo3e antigen expression on U-937 and HL-60 cells stimulated by pharmacologic activators of protein kinase C. Blood, 68, 1154-l 161, 1986. 30. SCHEURICH, P., UNGLAUB, R., MAXEINER, B., THOMA, B., ZUGMAIER, G., and PFIZENMAIER, K. Rapid modulation of tumor necrosis factor membrane receptors by activators of protein kinase C. Biochem. Biophys. Res. Comm., 141, 855-860, 1986. 31. MAY, W.S., JACOBS, S., and CUATRECASAS, P. Association of phorbol esterinduced hyperphosphorylation and reversible regulation of transferrin membrane receptors in HL60 cells. Proc. Natl. Acad. Sci. USA., 81, 2016-2020, 1984. 32. DOWNWARD, J., WATERFIELD, M.D., and PARKER, P.J. Autophosphorylation and protein kinase C phosphorylation of the epidermal growth factor receptor. Effect on tyrosine kinase activity and ligand binding affinity. J. Biol. Chem., 260, 1453814546, 1985. 33. FASSIO, A., COFANO, F., CAVALLO, G., and LANDOLFO. S. Activation of Protein Kinase-C down regulates IFN-Gamma receptors. B&hem. Biophys. Res. Comm., 144, 337-344, 1987.
rights
reserved.
AUGMENTATION OF PROCOAGULANT ACTIVITY IN MONOKINE STIMULATED HUMAN ENDOTHELIAL CELLS BY CALMODULIN/PROTEIN KINASE C INHIBITORS
Steven H. Zuckerman and Yvonne M. Surprenant Department of Immunology, Lilly Research Labs, Indianapolis, IN 46285, (Received
31.8.1987;
Accepted in revised by Editor N.U. Bang)
USA
form 28.10.1987
ABSTRACT The incubation of human umbilical cord endothelial cell cultures with inflammatory mediators results in the induction of procoagulant activity. As many of these mediators activate protein kinase C, the effect of calmodulin and protein kinase C inhibitors on IL-l, TNF, phorbol ester and LPS stimulated procoagulant activity was determined. Incubation of endothelial cell cultures with these inflammatory agents in the presence of phenothiazine derivatives or other classes of calmodulin and protein kinase C antagonists resulted in a 2-4 fold increase in procoagulant activity compared to parallel stimulated cultures in the absence of antagonists. The augmented response of IL-1 stimulated endothelial cells to these antagonists was actinomycin D sensitive.
The vascular endothelium plays a central role in separating blood elements from extravascular tissue. Stimulation of primary endothelial cell cultures with inflammatory agents including lipopolysacchande, LPS, phorbol esters, or the monokines Interleukin-1 , IL-l, and Tumor Necrosis Factor, TNF, results in significant changes in membrane components and in endothelial-endothelial and endothelialleukocyte cellular interactions. Specifically, increased adherence of peripheral blood monocytes, neutrophils and the human leukemic cell lines HL-60 and U937 to IL-l, LPS, or phorbol ester stimulated endothelial cells has been repotted and correlates with the induction of a leukocyte adherence molecule H 4118 on the endothelial cell surface (l-4). The increased secretion of platelet activating factor, GranulocyteMacrophage Colony Stimulating Factor, prostacyclins, plasminogen activator inhibitor, Key words: cells
calmodulin, protein kinase C, monokine, procoagulant activity, endothelial
205
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and IL-l has also been detected in stimulated endothelial cell culture supernatants (5 9). Endothelial cells can also be induced to express procoagulant activity (tissue factor, thromboplastin, factor Ill) by inflammatory mediators thus resulting in the activation of the coagulation cascade by the extrinsic pathway (10-14). The activation of procoagulant activity could contribute to localized or disseminated intravascular coagulation observed during inflammation or endotoxemia respectively. While the induction of procoagulant activity in human endothelial cell cultures by inflammatory mediators is well documented, little is known concerning the intracellular processes which modulate the level of this activity. Many of the mediators which induce procoagulant activity on endothelial cells have been demonstrated to activate protein kinase C (15). The diversity of biologic processes regulated by calcium or phospholipid dependent protein kinases through calmodulin or protein kinase C would suggest that antagonists of these calcium binding proteins might affect procoagulant expression. The present study was undertaken to investigate the effect of calmodulin and protein kinase C inhibitors on the induction of endothelial cell procoagulant activity by inflammatory mediators. We report that protein kinase C and calmodulin antagonists actually increase the amount of procoagulant activity detected on LPS, phorbol ester, IL-l, or TNF stimulated primary human endothelial cell cultures.
MAFRlAl SAND METHODS Compounds. Phenothiazine derivatives, and the tricyclic antidepressants, imipramine, chloroimipramine, and amitriptyline were provided by Drs. Robert Archer and Ray Fuller (Lilly Research Labs, Indianapolis, IN). Staurosporine was provided by Dr. Jeff Howbert (Lilly Research Labs). Compounds W-7 (N-(6-aminohexyl)S-chloro-lnapthalenesulfonamide), 48/80, calmidazolium, actinomycin D, phorbol 12-myristate 13-acetate, as well as calmodulin and mellitin were obtained from Sigma Chemical (St. Louis, MO). Endothelial Cell Cultures. Primary human umbilical cord endothelial cells were established as primary cultures, and provided at passage 1 by Dr. Francoise Booyse (University of Alabama, Birmingham, AL). Endothelial cell cultures were grown in medium 199, M199, (GIBCO Labs, Grand Island, NY) containing 10% fetal calf sera (Hyclone, Logan, UT), 1% glutamine (GIBCO), 2 units/ml of heparin (Lilly Res. Labs, Indianapolis, IN) and 60 ug/ml of Endothelial Cell Growth Supplement, ECGS, (Collaborative Research, Bedford, MA) on fibronectin (Collaborative Research) coated (1 ug/cm*) tissue culture flasks. Procoagulant Assay. Human umbilical cord endothelial cell cultures between passages 2-20 were trypsinized, plated at 3 x 10s cells/well on fibronectin coated 96 well plates (Costar, Cambridge, MA) and allowed to recover for 24 hours prior to assay. Procoagulant activity was assayed by a modified two stage amidolyltic assay using the chromogenic substrate S-2222 (13). Briefly, microtiter plate cultures in triplicate were induced with 2 ng/ml of IL-1 (human recombinant IL-1 j3 provided by Dr. J.L. Bobbitt, Lilly Research Labs, Indianapolis, IN), 1 unit/ml of TNF (Amgen Biologicals, Thousand Oaks, CA) 20
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ng/ml of LPS (E coli 05585, Difco Labs, Detroit, MI) or 300 pg/ml of phorbol 12-myristate 13-acetate, PMA, for 4 hours in the presence or absence of calmodulin or protein kinase C inhibitors. All inhibitors were used at 10 ug/ml, approximately 2.5 x 10-s M unless otherwise indicated. Following incubation, plates were washed twice with phosphate buffered saline and cells were then incubated for 2 hrs at 37 degrees C with 100 uliwell of Ml99 without phenol red (GIBCO Labs) containing 1.3 factor VII units/ml of a coagulation factor concentrate consisting of factors II, VII, IX, and X (Proplex T, Travenol Labs, Inc., Glendale, CA) and 500 ug/ml of S2222 (Helena Labs, Beaumont, TX).’ The change in optical density at 410 nm was quantitated using a Dynatech MR 600 microplate reader (Dynatech Laboratories Inc., Alexandria, VA).
The demonstration that protein kinase C activators were capable of inducing procoagulant activity in human endothelial cells (10-14) suggested that compounds which block calcium dependent processes could modulate procoagulant induction. Several phenothiazine derivatives have been demonstrated to inhibit both calmodulin and protein kinase C dependent processes (16-20) and these and other phenothiazines were utilized in the present study (figure 1).
Trivlrl
Name
phenothiazine 2-CF3phenothiezine 2aetylphenothiazine chlorpromazine triflupromazine acetylpromazine promelhazine trimeprazine
prochlorperazine trifluoperazine perphenazine
X
H H H CH+H2CH2N(Me)2 CH&H2CH2N(Me)2 CH,CH,CH,N(Me),
x CF3 COCH3 Cl CFa COCHI
CHzCH(Me)N(Meh CHzCH(Me)CHzN(Meh
H H
CHa CHa CH&HzOH
CF, Cl
Cl
FIG. 1. Structure of Phenothiazine Derivatives. These phenothiazine derivatives differ in their IC50 for calmodulin-induced of phosphodiesterase and in their medicinal application as tranquilizers,
activation
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chlorpromazine, triflupromazine, acetylpromazine, prochlorperazine, perphenazine, as an antihistaminic, promethazine, as an antipruritic, as an antihelminthic, phenothiazine (20). Incubation of endothelial cells with IL-1 and the phenothiazine ug/ml resulted in a 2-4 fold increase in procoagulant activity over that alone (Fig. 2).
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trifluoperazine, trimeprazine, and derivatives at 10 observed for IL-l
Perphenazine Trifluoperazine Prochlorperazine Trimeprazine Promethazine Acetylpromazine Triflupromazine Chlorpromazine 2-Acetylphenothiazine 2-CF3-Phenothiazine Phenothiazine IL-1 Control
Optical Density FIG. 2. Effect of Phenothiazine Derivatives on IL-1 induced Procoagulant Activity. Phenothiazine derivatives at 10 ug/ml (2.5 x 1O-5M) were incubated for 4 hours with endothelial cell cultures in the presence of IL-l and procoagulant activity was determined. Endothelial cells without IL-l, control, had no detectable procoagulant activity. Representative experiment of four separate experiments. Brackets indicate the standard error of the mean.
This was evident with all the phenothiazine derivatives except those simply consisting of the basic ring structure, phenothiazine, 2-CF3-phenothiazine, and 2acetlyphenothiazine. The augmentation in procoagulant activity observed in endothelial cells induced with IL-1 plus phenothiazine derivatives such as chlorpromazine or prochlorperazine was dependent upon &I~QYQ transcription as the addition of actinomycin D at 1 ug/ml completely inhibited the induction of procoagulant activity (data not shown). Furthermore, in the absence of IL-l, the phenothiazines alone did not result in an increase in procoagulant activity. In a representative experiment, an optical density of .039 was observed for endothelial cells plus prochlorperazine, .362 for cells plus IL-l, and .707 for cells plus prochlorperazine plus IL-l. The addition of phenothiazines at the substrate stage of the procoagulant assay did not atter the amount of substrate conversion observed (data not shown). Augmentation of procoagulant activity by phenothiazine derivatives was also seen when a suboptimal concentration (300 pg/ml) of phorbal myristate acetate was used as the inducing agent. As demonstrated in table 1, both chlorpromazine and prochlorperazine augmented procoagulant activity in endothelial cell cultures by 175 and 200 percent respectively.
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209
Table 1 Effect of Phenothiazine
Derivatives on Phorbol Ester Induced Procoagulant Activity
cal Density
fUt.U
.007
Control PMA PMA + Chlorpromazine PMA + Prochlorperazine
.168 .295 .359
(.OOl) (.012) (.026) (.031)
Mean value of triplicate wells is presented. Brackets indicate the standard error of the mean. The effect of non-phenothiazine related calmodulin, protein kinase C inhibitors on the induction of procoagulant activity was determined to exclude the possibility that the augmentation observed was unique to the phenothiazine class of compounds. As seen in figure 3, other classes of calmodulin and protein kinase C inhibitors also resulted in a 2-3 fold increase in procoagulant activity over that observed for IL-l.
Stau rosporine Calm idazolium 48180 Amitriptyline Imipramine Chloroimipramine w-7 Mellitin Proc‘hlorperazine IL-I I’ 0
1
100
’
I
200
I
’
3do
41
Percent Increase FIG. 3. Augmentation of Procoagulant Activity by different classes of calmodulin/protein kinase C inhibitors. Compounds were incubated with endothelial cells in the presence of IL-l. The increase in procoagulant activity induced by IL-1 was expressed as lOO%, and all values are expressed relative to IL-1 stimulated cells. Representative experiment of six. Standard errors are indicated by brackets.
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This included the tricyclic antidepressants imipramine, chloroimipramine, and amiyltryptline, compounds W-7, 48/80, and calmidazolium, staurosporine and mellitin (19-24). All compounds were used at a final concentration of 10 ug/ml except for calmidazolium at 1 ug/ml, mellitin at 0.1 ug/ml, and staurosporlne at 1 pg/ml. The augmented response of IL-1 stimulated endothelial cells was no longer apparent when these compounds were diluted 10 or 100 fold (staurosporine). Therefore, phenothiazine and non-phenothiazine related protein kinase C and calmodulin inhibitors augmented the induction of procoagulant activity by IL-l. The increase in procoagulant activity observed by these calmodulin, protein kinase C inhibitors was not restricted to IL-1 or PMA mediated induction of procoagulant activity. A similar augmentation was also observed with these compounds at comparable concentrations when endothelial cells were stimulated with LPS’or TNF (Fig. 4 a,b).
Calmidazolium 48180 Chlorpromazine Staurosporine LPS
Calmidazolium 48180 Chlorpromazine Staurosporine TNF 200 100 Percent Increase
FIG. 4. Augmentation of LPS or TNF induced procoagulant activity by calmodulin/protein kinase C antagonists. Endothelial cells were incubated with compounds in the presence of LPS (a) or TNF (b). Procoagulant activity induced by LPS or TNF alone was expressed as 100 percent and all other values were expressed relative to the control stimulated cultures. Representative experiment of four, standard errors are indicated by brackets. Increases between 150-500 percent were observed relative to cultures stimulated with LPS or TNF alone. Therefore, calmodulin and protein kinase C inhibitors augment procoagulant activity induced by a variety of inflammatory mediators.
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211
Changes in the endothelial cell surface during inflammation can contribute to the severity of the inflammatory response by activating the coagulation cascade and by promoting leukocyte influx into perivascular regions. The induction of procoagulant activity in human endothelial cell cultures by inflammatory molecules such as monokines, LPS, and phorbol esters serves as a model for the identification of compounds which modulate endothelial cell surface changes. In the present study, protein kinase C and calmodulin antagonists have been demonstrated to augment the induction of procoagulant activity mediated by IL-l, TNF, PMA, or LPS. This enhancing effect was seen with distinct series of inhibitors including phenothiazine derivatives, tricyclic antidepressants, a substituted napthalene sulfonamide (W-7), the antibiotic staurosporine as well as with other classes of compounds. Whether this effect on procoagulant induction is related to calmodulin or protein kinase C dependent processes cannot be definitively determined as most of these inhibitors act on both pathways (19-24). Furthermore, many of these compounds are antagonists in other systems. Phenothiazines have been reported to block dopamine receptors (25), 5-HT2 receptors (26), and calcium channels (27). The increase in procoagulant activity induced by IL-1 and chlorpromazine or prochlorperazine however, was not reversible by excess dopamine and could not be demonstrated with the 5-HT2 antagonist Ketanserin or with the calcium channel blocker Nifedipine (data not shown). The increased procoagulant activity observed with calmodulin, protein kinase C inhibitors was apparent when inhibitors were present in the induction phase of the assay, not during the substrate phase and was dependent upon de novo transcription. The observation that calmodulin/protein kinase C antagonists augment the level of procoagulant activity observed following induction with known protein kinase C activators would appear a priori to be paradoxical. The phorbol ester induced increase in transferrin endocytosis by rabbit reticulocytes (28) and the phorbol ester induced expression of the human monocyte surface antigen Mo3e on human monocytic cell lines (29) for example, have been reported to be inhibited by calmodulin/protein kinase C antagonists. Protein kinase C activators however, have also been shown to down-regulate TNF receptors on human T cells (30) transferrin receptors on HL60 (31), epidermal growth factor receptors (32), and gamma interferon receptors on the murine T cell line EL-4 (33). A Protein kinase C antagonist, H7 (1-(5isoquinolinesulfonyl)-2-methylpiperazine), has been reported to partially block the down-regulation of TNF receptors on T cells (30). In view of these observations, the augmentation in procoagulant induction observed in the presence of protein kinase C/calmodulin antagonists may be explained by the following hypothesis. First is that the induction of procoagulant by IL-l, TNF, PMA, and LPS is occurring through a nonprotein kinase C dependent mechanism. The activation of protein kinase C does, however, result in the down-regulation of TNF receptors and presumably IL-1 and LPS binding components as well thus rendering the endothelial cells refractory to subsequent stimulation by inflammatory mediators. The protein kinase C/calmodulin antagonists prevent receptor down regulation and therefore, these cells can be further stimulated by the continued presence of inflammatory mediators. While other explanations are possible the proposed model represents a working hypothesis amenable to further investigation. The in vivo significance of these findings that clinically relevant phenothiazine derivatives augment human endothelial cell procoagulant activity in response to an inflammatory stimulus cannot be determined from this study. The in vivQ concentration
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and metabolism of these compounds as well as the complexities of endothelial cell interaction with both peripheral blood and extravascular elements must be considered before & yivcr extrapolation of these observations can be considered. Clearly, protein kinase C, calmodulin antagonists alter the dynamics of the endothelial cell response to inflammatory mediators. Whether other parameters of endothelial cell function, such as changes in their secretory capacity, are also altered remains to be determined.
We wish to thank Drs. Robert Archer, Marlene Cohen, Jeff Howbert, and Winston S. Marshall for providing us with the necessary antagonists and for their helpful discussions and Dr. Francoise Booyse for providing us with early passage human endothelial cell cultures.
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21. BARNETTE, M.S., DALY, FL, and WEISS, B. Inhibition of calmodulin activity by
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