Regulation of endothelial cell protein c activation and fibrinolysis by procoagulant albumin

THROMBOSIS RESEARCH 70; 459-469,1993 00493848193 $6.00 + .OO Printed in the USA. Copyright (c) 1993 Pergamon Press Ltd. All rights reserved.

REGULATION

OF ENDOTHELIAL CELL PROTEIN C ACTIVATION FIBRINOLYSIS BY PROCOAALBUMIN

AND

David

B. Gubler', Brent D. Wilson+, Charles J. Parker+', and George M. Rodgers'"* Departments of Medicine' and Pathology*, University of Utah Medical Center, and Veterans Affairs Medical Center' Salt Lake City, UT (Received

Abstract

8.1 .1993; accepted

in revised form 12.3.1993

by Editor H.C. Pirkle)

Endothelial cell regulation of protein C activation and fibrinolysis are important components of the hemostatic response to vascular injury or perturbation. Procoagulant of normal human plasma has albumin (P-Al), a constituent been purified and identified as an inducer of endothelial The purpose of the studies cell tissue factor activity. reported herein was to investigate the effects of P-Al on cell protein C activation and endothelial human P-Al suppressed protein C activation, fibrinolysis. enhanced release of plasminogen activator inhibitor-l, effect on tissue-plasminogen activator but had no Plasminogen activator inhibitor-l released by release. P-Al was functional as evidenced by the capacity to form a covalent complex with 1251-urokinase. Inactive albumin (isolated during the same purification procedure as P-Al, but without tissue factor-inducing activity) did not suppress protein C activation or increase plasminogen activator inhibitor-l release. These results indicate of human plasma, that P-Al, a component can modulate multiple vascular hemostatic properties, and support the hypothesis that P-Al is involved in normal or pathologic hemostasis.

Key words: Endothelium, protein C, fibrinolysis, albumin Correspondence to: Dr. George M. Rodgers, Division of HematologyUniversity of Utah Medical Center, Salt Lake City, UT Oncology, 84132, USA

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Compelling evidence indicates that modulation of endothelial cell hemostatic properties contributes to the regulation of blood coagulation (1). Activation of protein C, mediated by the thrombin-thrombomodulin complex, appears to be an important component of this process, since certain patients deficient in components of the protein C pathway experience recurrent thrombosis The fibrinolytic mechanism that is responsible for lysis of (2). the fibrin clot is initiated by generation of plasmin from the plasminogen by tissue-plasminogen zymogen activator (t-PA). Suppression of vascular fibrinolytic activity occurs by interaction of t-PA with its physiological inhibitor, plasminogen activator inhibitor (PAI)-1. PAIalso inhibits the plasminogen activator, PAIinhibition of these plasminogen activators occurs urokinase. by formation of a stable covalent protease-protease inhibitor Deficient fibrinolysis,resulting from decreased complex (3,4). plasminogen activator release or increased PAIsecretion, may be an important risk factor in thrombosis (5,6). our laboratory identified a constituent of normal human Recently, plasma that induces tissue factor activity in human endothelial cells and monocytes (7). This constituent was identified as an anionic form of albumin that we termed procoagulant albumin (P-Al). The purpose of this study was to determine if, in addition to inducing tissue factor expression, P-Al modulates other hemostatic properties of endothelial cells. Accordingly, the effects of P-Al on endothelial cell protein C activation and fibrinolysis were investigated. MATERIALS

AND

METHODS

obtained from Falcon Plastics culture plasticware was Tissue ammonium sulfate, rabbit brain Trizma base, (Oxnard, CA). an endotoxin affinity column (E-Toxate), buffers, thromboplastin, iodoacetic acid, polymyxin B, Triton X-100, and gel electrophoresis supplies were purchased from Sigma Chemical Co. (St. Louis, MO). Medium 199 (M199) and other tissue culture reagents were obtained from the University of California, San Francisco Cell Culture by Baxter-Hyland Proplex-T concentrate was supplied Facility. The chromogenic substrates, S-2222 and S-2238 were (Glendale, CA). purchased from Kabi Vitrum (Franklin, OH). "'1 was obtained from Iodogen was supplied by Pierce Amersham Corp. (Clearbrook, IL). Enzyme-linked immunosorbent assays (Rockford,IL). Chemical Co. (ELISA) for t-PA and PAIwere provided by American Diagnostica A silver stain kit for identifying proteins in (Greenwich, CT). Purified human gels was obtained from Bio-Rad (Richmond, CA). protein C and activated protein C were provided by Enzyme Research Laboratories (South Bend, IN). Elution of activity that suppressed protein C activation from a non-denaturing gel (8) was accomplished by electrophoresing 20 pg of purified P-Al (5 pg/lane using 4 gel lanes), and cutting the Individual gel slices were lanes into horizontal slices of 5 mm. eluted electrophoretically with Tris-buffered saline (TBS) using a The eluates were dialyzed against TBS, then Centrilutor apparatus. endothelial cell protein C assayed for the capacity to suppress

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A P-Al standard was run simultaneously in an adjacent activation. gel lane and silver-stained to correlate biologic activity with gel protein mobility. Human umbilical vein endothelial cells (HUVEC) were prepared as described (9) and were used during the first passage. Assays were performed on cells plated in 24-well trays. Endothelial cell protein C activation was measured using a chromogenic substrate assay (10). Protein concentrations of endothelial cell lysates were determined using a protein-dye binding method (11). Data is expressed as activated protein C (ng/ml/min)per pg cell protein (12). To determine whether P-Al directly inhibited amidolytic activity of activated protein C, P-Al (100 pg/ml) was incubated with activated protein C (2 pg/ml) in the absence of cells for 4 hr, then the To amidolytic assay for activated protein C was performed. determine whether P-Al induced a HUVEC inhibitor of activated protein C, P-Al (100 pg/ml) was incubated with HUVEC and activated activity of activated protein C (2 pg/ml) for 4 hr; amidolytic protein C was then measured. To determine whether the free sulfhydryl group of P-Al mediated the suppressive effect on protein C activation, P-Al (100 pg/ml) was treated with iodoacetic acid (0.1 mM, 30 min), then dialyzed against TBS prior to assay. t-PA and PAIwere using Immunologic assays of conducted conditioned media from cells treated with serum-free media alone or containing was media P-Al. Conditioned serum-free media centrifuged (10,000 g x 10 min), and the supernate was recovered. Next, Triton X-100 was added to the supernate so that the final concentration was 0.01%. Samples were stored at -7OoC until assayed by ELISA according to the manufacturer's instructions. PAIData is expressed as (ng/ml) per pg cell protein. protein Endothelial cell concentrations were determined as described above for the protein C experiments (12). The ability of P-Al to release functional PAIwas monitored using 1251-urokinase (UK, recombinant single-chain form provided by Dr. Jack Henkin, Abbott Laboratories, Abbott Park, IL). UK was iodinated using the Iodogen technique (13) in which 0.5-l mg of protein was incubated at 4°C with 1 mCi oflz51 in an Iodogen-coated tube. After 20 min, Eppendorf the radiolabeled plasminogen activator was from separated free I251 by filtration gel chromatography. The specific activity of the radiolabeled plasminogen activator ranged between 20-40~10~ cpm/pg. Conditioned media from control and P-Al-treated cells that had been incubated for 16 hr was obtained, centrifuged at 10,000 g x 10 min, and 0.1 ml of the supernate was incubated with lo6 cpm of1251-DK for 30 min at 370C. Next, each sample was then treated with nonreduced gel buffer, boiled and analyzed by 10% SDS-PAGE. Following electrophoresis, the gel was dried and an autoradiograph was prepared using Kodak XAK film.

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RESULTS Effects of P-Al on protein C activation activation in a P-Al suppressed HUVEC protein C time-and concentration-dependent manner. Time-course studies showed that maximal reduction in protein C activation occurred after 4 hr of incubation of HUVEC with P-Al (data not shown). Figure 1 P-Al illustrates concentration-response relationships of the suppression of protein C activation after a 4 hr incubation. At a concentration of 25 pg/ml, P-Al reduced protein C activation by approximately 20%; higher concentrations (150 pg/ml) reduced protein C activation by approximately 50%. Similar experiments in which inactive albumin (albumin isolated from the same purification protocol as P-Al, but without tissue factor-inducing activity) was tested showed no reduction in protein C activation. These results demonstrate that modulation of protein C activity is specific for P-Al.

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Albumin(pg/ml) FIG.

1.

Concentration-response relationships of P-Al suppression HUVEC were of endothelial cell protein C activation. incubated with varying concentrations of P-Al (lo-200 pg/ml). After 4 hr, the cells were washed with serum-free media and protein C activation was quantitated using a chromogenic substrate assay (10). Each value represents the mean + SEM of triplicate experiments. Solid symbols indicate the effect of inactive albumin on protein C activation.

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Alternative explanations for these results are that P-Al directly inhibits activated protein C (APC) activity or P-Al induces an endothelial cell inhibitor to APC. To test these possibilities, PAl (100 pg/ml) was incubated with purified APC (2 pg/ml) with or without HDVEC for 4 hr in serum-free media. The chromogenic substrate assay for APC activity was then performed. There was no diminution of APC activity by P-Al when incubations were done with or without endothelial cells. In experiments designed to exclude endotoxin as a contaminant inhibitor of protein C activation in the P-Al samples, P-Al was chromatographed on an endotoxin affinity column (E-Toxate) . This column did not remove the inhibitory activity from the P-Al sample. did not prevent Incubation of P-Al with polymyxin B (5 pg/ml) suppression of protein C activation. Additionally, endotoxin assay of active and inactive samples of albumin using a limulus lysate method indicated equivalent levels of endotoxin in both samples (~40 ng/ml) . These data indicate that endotoxin contamination does not account for the ability of P-Al to suppress protein C activation. A gel-slice elution experiment was performed to demonstrate that Figure biologic activity of the sample was associated with P-Al. 2 indicates that functional activity could be eluted from a nondenatured gel at a position that correlated with the mobility of PAl. We previously identified a free sulfhydryl group on P-Al (data not indicated that agents shown), and a report by other investigators with intact sulfhydryl groups may inhibit protein C activation by the reducing critical disulfide bonds within thrombomodulin To determine whether the free sulfhydryl of P-Al molecule (14). mediated the suppressive effect on protein C activation, P-Al (100 pg/ml) was treated with iodoacetic acid (O.lmM, 30 min). Next, the dialyzed and subsequently assayed sample was for suppressive activity. Treatment of P-Al with iodoacetic acid did not prevent reduction in protein C activation compared to the buffer-treated control (data not shown). However, this treatment blocked the free sulfhydryl group of P-Al as determined by using Ellman's reagent and a spectrophotometric assay (15). Induction of tissue factor activity by P-Al appears to be mediated by a scavenger-like receptor present on IWVEC, because this effect of P-Al is inhibited by agents such as heparin sulfate and dextran sulfate, but not by ferritin and ovalbumin (7, 16). Accordingly, we examined whether the suppressive effects of P-Al on HUVEC protein C activation could also be prevented by these agents. Heparin sulfate (100 pg/ml), ovalbumin (400 pg/ml) and ferritin individually had no effect on basal protein c (700 pg/ml) activation, and none of these 3 reagents inhibited the suppressive effect of P-Al (100 pg/ml, 4 hr) on protein C activation. Dextran sulfate, by itself (lo-100 pg/ml), totally inhibited basal protein C activation (data not shown). These results suggest that the effect of P-Al on endothelial cell protein C activation is mediated by a differentmechanismthan the effect on tissue factor induction.

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FIG. 2. that suppresses HUVEC protein C Elution of activity activation from a nondenaturing gel. Purified P-Al (20 pg) was electrophoresed in a 10 % nondenaturing gel, the gel was sliced, and protein eluted from individual segments. Following dialysis against TBS, the capacity of each eluate to inhibit protein C activation was measured using a chromogenic substrate assay. Inhibitory activity was eluted at a gel slice position that corresponded to the result is This protein. mobility of the purified representative of two experiments.

Effects of P-Al on fibrinolysis Levels of t-PA and PAI- were measured by ELISA in control and PBasal secretion of t-PA was Al-treated HUVEC conditioned media. not substantially affected by P-Al; t-PA levels after 19 hrs incubation with 200 pg/ml P-Al were 0.87 ng/ml, compared to 0.90 ng/ml in untreated media. In contrast to these results with t-PA secretion, P-Al significantly affected PAIsecretion. Timecourse studies indicated peak P-Al-induced PAI- secretion occurred after 18-24 hr incubation. A concentration-response curve of PAIaccumulation

over

a 19 hr incubation

period

is shown

in Figure

3.

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Albumin(pLg/ml) FIG. 3. Concentration-response relationships of P-Al induced secretion of PAI- into HWEC conditioned media. Confluent HUVEC grown in 24-well trays were washed with serum-free media, then incubated in 0.4 ml serum-free media with varying concentrations of P-Al (O-200 pg/ml) for 19 hr. PAI1 levels were then measured as described in Methods. Each value represents the mean + SEM of triplicate experiments. Solid symbols indicate the effect of inactive albumin on PAI- secretion.

in this study was 45 ng/ml/pg. Basal accumulation of PAIMaximally-treated cells (P-Al concentration 2 50 pg/ml) secreted PAI3-fold increased amount of nearly a (112 ng/ml/pg). Incubation of "inactive" albumin with HUVEC did not result in enhanced secretion of PAIcompared to untreated cells (Fig. 3). Experiments were performed to exclude a role for endotoxin in these Heating P-Al (EOOC, 15 min) abolished the experimental results. ability of P-Al to enhance PAIsecretion; these conditions do not Additionally, inactivate endotoxin (17). co-incubation of P-Al with polymyxin B did not prevent enhanced PAI(50 jq/ml)

secretion. Figure 4 indicates that PAIsecreted in response to P-Al is functional in that complex formation with 1251-UE occurs. A covalent complex (M,=106,000) is seen in the sample with P-Al

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conditioned media (lane 3), but not in the sample with control conditioned media or 1251-UK alone. These results are consistent with stable complex formation between UK (~=54,000) and PAIWhen "'I-UK was incubated with purified P-Al in serum(M,=52,000). free media not exposed to HUVEC, complex formation did not occur.

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FIG. 4. Demonstration of complex formation between PAI- secreted by P-Al-treated HUVEC and 125I-UK. UK was radiolabeled as described in Methods, then incubated either with serumfree media (lane l), media from untreated endothelial cells (lane 2), or media from P-Al-treated cells (200 pg/ml, 16 hr), (lane 3). Media-plasminogen activator incubations were conducted for 30 min. Samples were then boiled in nonreduced gel buffer and analyzed by 10 % SDS-PAGE. An autoradiogram of the dried gel is shown; complex formation between PAI- and x251-UK is indicated by the arrow.

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DISCUSSION These data indicate that P-Al can regulate endothelial cell protein C activation and PAIsecretion, but not t-PA secretion. Together describing P-Al-induced tissue factor with our previous report activity in endothelial cells and monocytes (7), these data suggest that P-Al significantly alters vascular hemostatic function. Previous studies indicated that agents that stimulate endothelial cell tissue factor activity also reduce protein C activation and PAIsecretion. These agents include endotoxin, enhance and tumor necrosis factor (1). Stimulation of interleukin-1, endothelial cell tissue factor activity, suppression of protein C secretion of PAIenhanced all occur at activation, and concentrations of P-Al ranging from 50-100 pg/ml, and would result in a vascular surface that promotes thrombosis. In contrast to the effects of P-Al on induction of endothelial cell tissue factor activity that appear to involve a scavenger-like suppression of protein C activation receptor (161, P-Al-mediated may occur by a different mechanism. Studies by others indicate that protein C activation can be inhibited by reducing agents (14), turnover (endocytosis) of thrombomodulin enhanced (181, or decreased expression of thrombomodulin mRNA (19). The observation that treatment with iodoacetic acid had no effect on the capacity of P-Al to inhibit protein C activation suggests that P-Al does not inhibit protein C activation by reducing intrachain disulfide bonds that are critical for the structural/functional integrity of thrombomodulin. Whether P-Al enhances thrombomodulin endocytosis expression of thrombomodulin nRNA remains or reduces to be determined. With regard to the mechanism for enhanced PAIsecretion into HUVEC conditioned media, a recent study suggests that agonists promoting PAIsecretion do so by stimulation of PAImRNA accumulation (20). Identifying the exact mechanism by which P-Al enhances PAIsecretion will be the aim of future studies. Acknowledgments The authors acknowledge Teri McCluskey and Li-Hua Liu for excellent technical assistance. This work was supported by a grant from the University of Utah Undergraduate Research Opportunities Program (DBG and BDW) and by the Utah affiliate, American Heart Association (GMR), and by the Research Service of the Department of Veterans Affairs (GMR and CJP), and by PHS grant ROl DK 35830 awarded to CJP. CJP is the recipient of a Shannon Award and a Research Career Development Award from the NIH. REFERENCES 1. RODGERS, G.M. Hemostatic properties of normal and perturbed vascular cells. FASEB J 2, 116-123, 1988. 2. ESMON, C.T. The regulation of natural anticoagulant pathways. Science 235, 1348-1352, 1987. 3. ERICKSON, L.A., SCHLEEF, R.R., NY, T., LOSKUTOFF, D.J. The fibrinolytic system of the vascular wall. Clin Hematol 14,

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513-530, 1985. SPRENGERS, E.D., KLUFT, C. Plasminogen 4. activator inhibitors. Blood 69, 381-387, 1987. M.H., KRUITHOF, E.K.O., CONARD, J., 5. NGUYEN, G., HORELLOU, SAMAMA, M.M. Residual plasminogen activator inhibitor activity after venous stasis as a criterion for hypofibrinolysis: A study in 83 patients with confirmed deep vein thrombosis. Blood 72, 601-605, 1988. PRINS, M.H., HIRSH, J. 6. A critical review of the evidence supporting a relationship between impaired fibrinolytic activity and venous thromboembolism. Arch Int Med 151, 1721-1731, 1991. 7. FAUCETTE, K.J., PARKER, C.J., MCCLUSKEY, T., BERNSHAW, N.J., RODGERS, G.M. Induction of tissue factor activity in endothelial cells and monocytes by a modified form of albumin present in normal human plasma. Blood 79, 2888-2895, 1992. 8. GARFIN, D.E. One-dimensional gel electrophoresis. Methods 425-441, 1990. Enzymol 182, R.L., BECKER, C.G., MINICK, C.R. 9. JAFFE, E.A., NACHMAN, Culture of human endothelial cells derived from umbilical Identification by morphologic and immunologic veins. J Clin Invest 52, 2745-2756, 1973. criteria. an atherogenic Homocysteine, 10. RODGERS, G.M., CONN, M.T. reduces protein C activation by arterial and venous stimulus, 895-901, 1990. endothelial cells. Blood 75, BRADFORD, M.M. A rapid and sensitive method for the 11. quantitation of microgram quantities of protein utilizing the Anal Biochem 72, 248principle of protein-dye binding. 254, 1976. Regulation of WILSON, B.D., PITAS, R.E., RODGERS, G.M. 12. endothelial cell protein C activation by native and oxidized 11-17, Sem Thromb Hemost IS, low density lipoprotein. 1992. Protein and cell membrane FRAKER, P.J., SPECK, J.C. 13. iodinations with a sparingly soluble chloroamide, 1,3,4,6Biochem Biophys Res 6a-diphenylglycoluril. tetrachloro-3a, 849-857, 1978. Comm 80, Inhibition of thrombomodulin LENTZ, S-R., SADLER, J.E. 14. surface expression and protein C activation by the J Clin Invest 88, 1906thrombogenic agent homocysteine. 1914, 1991. Bovine mercaptalbumin and nonNOEL, J.K.F., HUNTER, M.J. 15. 7391-7406, J Biol Chem 247, mercaptalbumin monomers. 1972. L.A., PARKER, C.J., RODGERS, G.M. 16. FAUCETTE, K.J., FITZGERALD, Characterization of the interactions between procoagulant 376a, Clin Res 40, albumin and human endothelial cells. 1992. Thrombin induction of R. 17. GELEHRTER, T-D., SZNYCER-LASZUK, plasminogen activator-inhibitor in cultured human endothelial 165-169, 1986. J Clin Invest 77, cells. Tumor necrosis factor MOORE, K-L., ESMON, C.T., ESMON, N.L. 18. leads to the internalization and degradation of thrombomodulin from the surface of bovine aortic endothelial cells in 159-165, 1989. culture. Blood 73,

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19. DITTMAN, W.A., KUMADA, T., SADLER, J.E., MAJERTJS, P.W. The structure and function of mouse thrombomodulin: Phorbol myristate acetate stimulates degradation and synthesis of thrombomodulin without affecting mRNA levels in hemangioma J Biol Chem 263, 15815-15822, 1988. cells. 20. HEATON, J-H., DAME, M.K., GELEHRTER, T.D. Thrombin induction of plasminogen activator inhibitor mRNA in human umbilical J Lab Clin Med 120, 222-228, vein endothelial cells in culture. 1992.