- Email: [email protected]
Antiheparin activity in human endothelial cells
THROMBOSIS RESEARCH 42; 355-362, 1986 0049-3848/86 $3.00 t .OO Printed in the USA. Copyright (c) 1986 Pergamon Press Ltd. All rights reserved.
ANTIHEPARIN ACTIVITY IN HUMAN ENDOTHELIAL CELLS
Hussain I. Saba*, Sabiha R. Saba**, Genevieve A. Morelli* Division of Hematology, Departments of Medicine* and Pathology**, University of South Florida College of Medicine and James A. Haley Veterans Hospital Medical Center, Tampa, Florida 33612 U.S.A. (Received 30.7.1985; Accepted in revised form 20.11.1985 by Editor K.M. Brinkhous (Received in final form by Executive Editorial Office 18.2.1986)
ABSTRACT
Human endothelial cells possess antiheparin activity that neutralizes the anticoagulant action of heparin as measured by different tests of the clotting system. The antiheparin activity appears to be associated with an acid-soluble basic protein present in the particulate fraction of the endothelial cell cytoplasm. This finding might have some relevance in the maintenance of hemostasis. Furthermore, it might also have a pharmacological role in terms of resistance to exogenously infused heparin in patients with thromboembolic disorders.
INTRODUCTION Endothelium's role in hemostasis and thrombosis is becoming increasingly significant (1,2). Heparin remains an important anticoagulant in widespread clinical use. When infused in man, heparin's anticoagulant action is modified by a number of cellular and humor-alfactors. Antiheparin factors have been reported in platelets (3,4) and in leukocytes (5,6). In the polymorphonuclear leukocytes, the antiheparin activity is localized in the acid-soluble cationic proteins of their cytoplasmic lysosomes (7). Although endothelium is reported to bind heparin (8-lo), it is not known whether human endothelial cells possess antiheparin factor(s). A heparin inhibitor has been reported in the mucosa of hog intestines (11). In this preliminary study we report the presence of a hitherto unknown antiheparin activity in human endothelial cells.
Key words:
hemostasis.
Heparin, antiheparin, endothelium, blood vessels, basic proteins, 355
356
ENDOTHELIUM AND HEPARIN
Vol. 42, No. 3
MATERIALS AND METHODS Heparin used in this study was obtained from Lypho-Med (Chicago, IL). Appropriate dilutions of heparin were made in Modified Tyrodes Buffer (MTS, pH 7.4) (12). Non-activated Partial Thromboplastin Time (PTT) (131 was done using Thrombofax reagent obtained commercially from Ortho Diagnostics (RariThrombin Clotting Time (TCT) (14) was done using bovine throebin tan, NJ). obtained from Parke-Davis (Morris Plains, NJ). Normal human plasma was used in both PTT and TCT systems, with some modification, to assay the anticoaguFor PTT studies, 0.05 ml aliquots of human lant activity of heparin. umbilical cord endothelial cell preparations (whole cells, freeze-thawed (FT) suspensions and acid-extracts of cytoplasmic and nuclear cell fractdons) were combined with 0.05 ml (0.05 units) of heparin and incubated at 37 C for 0.20 ml of normal human plasma and 0.10 ml of Thromboplastin 10 minutes. were added and the PTT measured by the addition of 0.10 ml of 0.02 M CaCl . For TCT studies, 0.05 ml of these endothelial cell prepagations were comb?ned with 0.05 ml (0.02 units) of heparin and incubated at 37 C for 10 minutes. 0.20 ml of normal human plasma was then added and the mixture clotted by Buffer replaced heparin and/or difadding 0.10 ml (10 units) of thrombin. ferent cell preparations for controls. Endothelial cells were harvested from human umbilical cords and grown in tissue culture as previously described (15,161. Demonstration of antiheparin activity in endothelial cells was carried out by using whole endothelial cell suspensions, F-T suspensions of these cells and also acid-extracts of the nuclear and cytoplasmic fractions from endothelial cells. These nuclear and cytoplasmic cell fractions were prepared by the method of Wiggins et al (171, Endothelial cells were disrupted by gentle homogeniwith some modification. zation for 3.5 minutes using a tight-fitting, Teflon pestle tissue homogenizer (Thomas Company, Philadelphia, PA). The endothelial cell homogenate was centrifuged at 600 g for 10 minutes in a Damon/IEC Division CRU-5000 The centrifuged pellet containing the nuclear fractions was excentrifuge. amined and then discarded, and the supernatant containing the cytoplasmic constituents was collected and ultracentrifuged at 150,000 g for one hour. Under electron microscopy, the cytoplasmic pellet was compared to the nuclear fraction and found to be free of nuclear contamination (Figure 1). The pooled cytoplasmic pellet fractions from different preparations were extracted with 0.20 N H SO dialysed against 0.01 N HCl and then against Tris Buffered Saline (?BS!'(pH 7.4) overnight (161. The protein was determined by the Lowry-Hartree method (181. Electrophoretic characterization of the lyophilized acid-extract of endothelial cells cytoplasmic fraction was performed on a gradient acrylamide gel slab containing 2.5 M urea on a Bio-Rad Dual Vertical Slab Gel Electrophoresis Cell, Model 221 (Bio-Rad Laboratories, Richmond, CA). This technique of Traub and Boeckmann (19) has been used for separation of basic proteins. Neutralization of heparin-mediated anticoagulant activity by endothelial cells and their fractions was demonstrated in PTT and TCT systems. Studies presented in this report represent a mean of a minimum of five experiments _+ standard deviation (S.D.). ERESUIX'S AND DISCUSSION Table 1 shows that heparin alone prolonged the clotting time in PTT system to 179.0 and in TCT system to > 180.0 seconds. When the same amougt of heparin was added to the whole endothelial cell suspensions (1.42 x 10
Vol. 42, No. 3
ENDOTHELIUM AND HEPARIN
FIG.
357
1
Samples of whole endothelial cells, nuclear debris and cytoplasmic constituents obtained from homogenized endothelial cells were pelleted and fixed with 1.0 ml of 2% gluteraldehyde in 0.1 M phosphate buffer. These samples were then examined under electron microswhole endothelial cells x copy. (A 2000; B = nuclear debris x 2000; C = cytoplasmic constituents x 3000). ??
cells/ml, 0.89 mg/ml protein), the anticoagulant activity was neutralized, as the clotting time was now shortened to 150.0 seconds in PTT and 35.0 seconds in TCT system. F-T suspensions of endothelial cells at the same concentrations, more effectively neutralized heparin anticoagulant activity (65.0 seconds and 18.0 seconds in PTT and TCT systems respectively, with control clotting times of 57.0 seconds and 17.0 seconds). Table 2 shows the effect of the acid-extract of the cytoplasmic fraction on heparin neutralization when compared to F-T suspensions of whole endothelial cells. When equivalent amounts (0.064 mg protein) of acid-extract and F-T suspensions of endothelial cells (wt/wt protein) were examined for heparin neutralization, the acid-extract of the cytoplasmic fraction demonstrated stronger ability to neutralize heparin (68.0 seconds vs. 87.0 seconds). Acid-extract of the cytoplasmic fraction per se exhibited mild but persistent anticoagulant activity. The PTT with acid-extract was 69.0 seconds as compared to 54.0 seconds with buffer control. Results of the electrophoretic studies are shown on Figure 2. A single band migrating towards the cathode is seen. For comparison, the nuclear fraction containing the histones is also shown. The cytoplasmic band is not shared by the nuclear fraction of the endothelial cells.
358
ENDOTHELIUM AND HEPARIN
Vol. 42, No. 3
TABLE 1 -Neutralization Suspensions
Buffer Buffer + Heparin Buffer + Whole Cells Heparin + Whole Cells Buffer + F-T Suspension Heparin + F-T Suspension
of Heparin by Intact and Freeze-Thawed ofEndotherr;i7 Cells -
(F-T)
PTT (in seconds) + S.D.
TCT (in seconds) + S.D.
59.00 179.00 58.00 150.00 57.00 65.00
18.00 180.00 17.00 35.00 17.00 18.00
2 2.5 & 4.4 2 2.3 222.5 f 8.4 flO.O
2 1.5 _+ 0 + 1.5 _+ 4.5 _+ 0.7 f 1.4
Endothelial cells were adjusted to 1.42 x lo6 cells/ml (0.89 mg/ml protein) and divided into two equal samples. One of these samples was used as whole cells, while the other sample was F-T three times. 0.05 ml aliquots (0.045 mg protein) of these two preparations were incubated with heparin (0.05 units heparin in PTT, 9.02 units in TCT) in these studies. Results are expressed as mean clotting time in seconds + standard deviation (S-D.).
TABLE '2 -Comparison of Freeze-Thawed (F-T) Endothelial Cells Suspension and Fractlo~ofndothellal -- the AcidrEx~ofasmlc Cells~n~parin Neutralization -Partial Thromboplastin Time (PTT) (in seconds _+ S.O.) Buffer Buffer + Heparin Buffer t F-T Suspension of Endothelial Cells Heparin + F-T Suspension of Endothelial Cells Buffer + Acid-Extract of Cytoplasmic Fraction Heparin + Acid-Extract of Cytoplasmic Fraction
54.00 107.00 56.00 37.00 69.00 68.00
2 0.6 _+ 1.2 2 8.4 210.0 _+ 0 _+ 1.7
Endothelial cell samples were F-T three times and the protein content adjusted to 1.28 mg/ml with buffer. Acid-extracts were also prepared and adjusted to the same protein concentration. 0.05 ml (0.064 mg of protein) aliquots of either the F-T or acid-extract were incubated with heparin (0.05 units) in this study. Results are expressed as mean clotting time in seconds f standard deviation (S.D.).
Studies presented in this report demonstrate that endothelial cells possess antiheparin activity. This activity is capable of neutralizing the anticoagulant action of heparin assayed in both the PTT and TCT systems. Although Lypho-Med heparin was used in these studies, other commercial sources
Vol. 42, No. 3
ENDOTHELIUM AND HEPARIN
359
FIG. 2
Electrophoresis of the acid-extracts of endothelial cell fractions (cytoplasmic and nuclear) were carried out on a gradient slab gel. Samples were applied at the anode of the gel containing 2.5 M urea. Electrophoresis was carried out at 20 mAmp/gel for 24 hours. Gels were stained with 0.25% Coomassie Brilliant Blue - 25% E-propanol - 10% acetic acid followed by destaining with 10% acetic acid. of heparin, when tested, exhibited similar neutralization. As presented, the antiheparin activity was exhibited by whole endothelial cells, but the activity became enhanced when the cells were disrupted and examined in the test systems. Further studies clearly show that antiheparin activity is pronounced in the acid-soluble fraction of the cytoplasmic constituents. Electrophoretic studies on the acid-extract of the cytoplasmic fraction showed a single band migrating towards the cathode, exhibiting the mobility pattern of a basic protein. This band was not shared by the electrophoretic bands of nuclear histones. This further suggested that the endothelial cell's antiheparin factor was specifically related to the cytoplasmic constituents of these cells and not due to contaminants from the nuclear histones. The histones are also basic proteins (20-22) and their antiheparin activity is known (23,24). Further studies are being done in order to conclusively prove that this antiheparin activity is definitely associated with the protein visualized on the electrophoretic gels. Our studies, therefore, showed that the antiheparin activity, although present in whole endothelial cells, was more pronounced when the cell cytoplasm was disrupted and when the extracts of cytoplasmic constituents were examined in the test systems. The cytosolar fraction (post 150,000 g supernate) demonstrated no antiheparin activity (not shown). It is not known at this time if the antiheparin activity present in the endothelial cells is synthesized and/or stored in their cytoplasmic organelles. Studies indicate that some antiheparin activity is also present on whole endothelial cells. Further studies are needed to clearly understand the relationship between the cytoplasmic and whole cell/surface distribution of antiheparin activity of endothelial cells. Further studies are in progress to characterize the biochemical nature of endothelial cells antiheparin factor. Electrophoretic analysis of the acid-extract of the cytoplasmic fraction, however, exhibited the mobility pattern of a basic protein. Although the precise biochemical nature of the acid-extract of the cytoplasmic fraction needs to be further determined, it is this fraction nevertheless which exhibited the highest antiheparin activity.
360
ENDOTHELIUM AND HEPARIN
Vol. 42, No. 3
The antiheparin activity in the PMN-leukocytes are known to be basic proteins (7). These basic proteins also possess anticoagulant activity (25,261. The antiheparin fraction of endothelial cells showed mild but per sistent anticoagulant action (Table 2). This further suggested its biochemical similarity with basic proteins. Some antigens affecting hemostasis are commonly shared by endothelial cells and platelets (27,281. Tested by imnunoassay (not shown), no antigenic similarity was found between endothelial cells antiheparin factor and platelet factor 4 (PF ) - a known antiheparin factor of a basic protein nature (29,301. Recentlj, a histidine-rich basic protein possessing.antiheparin activity has been reported in platelets (31) and also in plasma (32). Studies are in progress to determine if such basic proteins have any similarity with the antiheparin activity of endothel ial cells. The demonstration of antiheparin activity in endothelial cells is important and of clinical signficance, considering the large surface area a'f vascular tree in the human body. It is possible that, along with other antiheparin factors in the human body, the endothelial cells might also be involved in regulating the activity of heparin when infused in man. Heparinlike substances (heparinoids) have been identified in endothelial cells (33). Furthermore, heparin-like molecules with anticoagulant activity have recently been reported to be synthesized by endothelial cells (34). In view of this, it is possible that the antiheparin factor of endothelial cells as presented here might be involved in hemostatic balance with these endogenous heparinlike substances influencing the phenomena of hemostasis and thrombosis. ACKNOWLEDGMENTS We express our appreciation to Drs. Hasan I. Zeya, Paul Byvoet and Robert C. Hartmann for their valuable suggestions in this project and in the preparation of this manuscript. We also acknowledge Suzette Urban for her secretarial assistance. This work was supported by the VA Medical Research Service, Tampa , FL. REFERENCES
1.
SABA, H.I., Saba, S.R. Endothelium: Its role in hemostasis, thrombosis and atherosclerosis. In: Platelets and Prostaqlandins h Cardiovascu-_ lar Disease. J. Mehta, P. Mehta (Edsl New York: Futura Publishing Co, ml, pp. 125-148
2.
MASON R.G., Mohammad, In: Pathobiol;r zs, 1979, pp. l-
3.
CONLEY, C.L., Hartmann, R.C., Morse, W.I.. The clotting behavior of human "platelet-free" plasma: Evidence for the existance of a "plasma thromboplastin". J --Clin Invest 28, 340-352, 1949.
4.
VAN CREVELD, S., Paulssen, M.M.P. Isolation and properties of the third clotting factor in blood platelets. -Lancet 1, 23-25, 1952.
5.
CACCIOLA, E., Ferranto, A. Presence of antiheparin activity in leukemic cells. -----Bull Sot Ital Biol Sper 36, 1317-1319, 1960.
S.F., Saba, H.I., et al. Functions of endotheliAnnual. H.L. Ioachim (Edsl New York: Raven
Vol. 42, No. 3
ENDOTHELIUM AND HEPARIN
361
Tyg Lek 20 ---'
6.
Anti hepari n acti vi ty of normal leukocytes. LISIEWICZ, J. 1150-1151, 1965.
7.
SABA, H.I., Roberts, H.R., Herion, J.C. Antiheparin activity of lysosoma1 cationic proteins from polymorphonuclear leukocytes. -Blood 31, 369-380, 1968.
8.
HIEBERT, L.M., Jaques, L.B. 26-31, 1976.
9.
HIEBERT, L.M., Jaques , L.B. The observation of heparin on endothelium after injection. --Thromb Res 8, 195-204, 1976.
Heparin uptake on endothelium. Artery 2,
10.
GLIMELIUS , B., Busch, C., Hook, M. Binding of heparin on the surface of cultured endothelial cells. --Thromb Res 12, 773-782, 1978.
11.
GERTLER, M.M., Chen, M., Yue, R.H. Detection and partial purification of a natural heparin inhibitor from hog small intestine. Throm Haemost 41, 567-575, 1979. -
12.
SABA, S.R., Mason, R.G. Studies of an activity from endothelial cells that inhibit platelet aggregation, serotonin release and clot retraction. --Thromb Res 5, 747-757, 1974.
13.
LANGDELL, R.D., Wagner, R.H., Brinkhous, K.M. Effect of antihemophilic factor on the one-stage clotting tests. A presumptive test for hemophilia and a simple one-stage antihemophilic factor assay procedure. J Lab Clin Med 41, 637-647, 1953. _---
14.
SEEGERS, W.H. Preparation, purification and assay of thrombin. In: Preoaration, purification & ESSAY of thrombin. L.M. Tocantins, L.A. Kazal (Eds) New York: Grune and Stratton, 1964, pp. 181.
15.
SABA, S.R., Zucker, W.H., Mason, R.G. Some properties of endothelial cells isolated from human umbilical cord vein. Sem Hematol -6, 456-486, 1973.
16.
SABA, H.I., Hartmann, R.C., Saba, S.R. Effects of polymorphonuclear leukocytes on endothelial cell growth. --Thromb Res 12, 397-407, 1978.
17.
WIGGINS, R.C., Loskutoff, D.J., Cochrane, C.G., Griffin, J.H., Edgington, T.S. Activation of rabbit Hageman factor by homogenates of cultured rabbit endothelial cells. -J Clin Invest 65, 197-206, 1980.
18.
HARTREE, E.F. Determination of protein: A modification of the Lowry method that gives a linear photometric response. Ann Biochem 48, 422427, 1972.
19.
TRAUB, P., Boeckmann, G. High-resolution polyacrylamide gradient slab gel electrophoresis of histone Hl, H3 and H4. Hoppe-Seyler's -Z Physiol Chem 359, 571-579, 1978. --
20.
CRUFT, H.J., Hindley, J., Mauritzen, C.M., Stedman, E. Amino acid composition of the six histones of calf thymocytes. -Nature 180, 1107-11909, 1957.
362
ENDOTHELIUM AND HEPARIN
Vol. 42, No. 3
21.
In: Nucleohistones. J. Bonner, P. MURRAY, K. The Nucleohistones. 1964, Ts'ao (Edsl San Francisco, London, Amsterdam: Holden-Day, Inc., pp. 15.
22.
JOHNS, E.W.
Histones and Nucleohistones. In: The preparation a&i_ . af histone$. B.M.P. Phillips (Eds) London, New York: Plenum Press, 1971, pp. 4-5.
m
.
23.
HILDEBRANO, C.E., Tobey, R.A., Gurley, L.R., Walters, R.A. Action of II. Cell-cycle-specific changes in chromaheparin on mammalian nuclei. tin organization correlate temporally with histone Hl phosphorylation. Biochim Biophys -Acta 517, 486-499, 1978.
24.
PAL, P.K., Gertler, M.M. Neutralization of heparin by histone and its Thromb Res 31, 69-79, 1983. subfractions. ---
25.
Antibacterial and enzymatic basic proteins ZEYA, H.I., Spitznagel, J.K. Separation and identification. Science -' 142 from leukocyte lysosomes: 1085-1087, 1963.
26.
Roberts, H.R., Herion, J.C. The anticoagulant activity of SABA, H.I., lysosomal cationic proteins from polymorphonuclear leukocytes. J Clin -Invest 46, 580-589, 1967. --
27.
HOWARD, M.A., Montgomery, D.C., Yardisty, R.M. Factor VIII-related Thromb Res 4, 617-624, 1974. tigen in platelets. ---
28.
NACHMAN, R.L., Jaffe, E.A. Subcellular platelet factor VIII antigen and J Exp Med 141, 1101-1113, 1975. von Willebrand factor. ----
29.
NATH, H., Lowery, C.T., Niewiarowski, S. Antigenic and antiheparin properties of human platelet factor 4 (PF4). -Blood 45, 537-550, 1975.
30.
VARMA, K.G., Niewiarowski, S., Holt, S., Rucinski, B., Paul, D. Human platelet basic protein. Its relation to low affinity platelet factor 4 and B-thromboglobulin. Biochim Biophys -Acta 701, 7-11, 1982.
31.
LEUNG, L.L.K., Harpel, P.C., Nachman, P.L., Rabellino, E.M. Histidinerich glycoprotein is present in human platelets and is released following thrombin stimulation. Blood 62, 1016-1021, 1983. --
32.
HAUPT, N., Heimburger, N. Human serumproteine mit hoher affinitat zu carboxymethycellulose I. Yoppe-Seyler's Z Physiol -Chem 353, 1125-1132, 1972.
33.
BUONASSISI, V., Root, 1.1. Enzymatic degradation of heparin-related mucopolysaccharides from the surface of endothelial cell cultures. Biochim Biophys -Acta 385, l-10, 1975.
34.
MARCUM, J.A., Rosenberg, R.D. Heparin-like molecules with anticoagulant activity are synthesized by cultured endothelial cells. Biochim Biophys Res Comm 126, 365-372, 1985.
an-
ANTIHEPARIN ACTIVITY IN HUMAN ENDOTHELIAL CELLS
Hussain I. Saba*, Sabiha R. Saba**, Genevieve A. Morelli* Division of Hematology, Departments of Medicine* and Pathology**, University of South Florida College of Medicine and James A. Haley Veterans Hospital Medical Center, Tampa, Florida 33612 U.S.A. (Received 30.7.1985; Accepted in revised form 20.11.1985 by Editor K.M. Brinkhous (Received in final form by Executive Editorial Office 18.2.1986)
ABSTRACT
Human endothelial cells possess antiheparin activity that neutralizes the anticoagulant action of heparin as measured by different tests of the clotting system. The antiheparin activity appears to be associated with an acid-soluble basic protein present in the particulate fraction of the endothelial cell cytoplasm. This finding might have some relevance in the maintenance of hemostasis. Furthermore, it might also have a pharmacological role in terms of resistance to exogenously infused heparin in patients with thromboembolic disorders.
INTRODUCTION Endothelium's role in hemostasis and thrombosis is becoming increasingly significant (1,2). Heparin remains an important anticoagulant in widespread clinical use. When infused in man, heparin's anticoagulant action is modified by a number of cellular and humor-alfactors. Antiheparin factors have been reported in platelets (3,4) and in leukocytes (5,6). In the polymorphonuclear leukocytes, the antiheparin activity is localized in the acid-soluble cationic proteins of their cytoplasmic lysosomes (7). Although endothelium is reported to bind heparin (8-lo), it is not known whether human endothelial cells possess antiheparin factor(s). A heparin inhibitor has been reported in the mucosa of hog intestines (11). In this preliminary study we report the presence of a hitherto unknown antiheparin activity in human endothelial cells.
Key words:
hemostasis.
Heparin, antiheparin, endothelium, blood vessels, basic proteins, 355
356
ENDOTHELIUM AND HEPARIN
Vol. 42, No. 3
MATERIALS AND METHODS Heparin used in this study was obtained from Lypho-Med (Chicago, IL). Appropriate dilutions of heparin were made in Modified Tyrodes Buffer (MTS, pH 7.4) (12). Non-activated Partial Thromboplastin Time (PTT) (131 was done using Thrombofax reagent obtained commercially from Ortho Diagnostics (RariThrombin Clotting Time (TCT) (14) was done using bovine throebin tan, NJ). obtained from Parke-Davis (Morris Plains, NJ). Normal human plasma was used in both PTT and TCT systems, with some modification, to assay the anticoaguFor PTT studies, 0.05 ml aliquots of human lant activity of heparin. umbilical cord endothelial cell preparations (whole cells, freeze-thawed (FT) suspensions and acid-extracts of cytoplasmic and nuclear cell fractdons) were combined with 0.05 ml (0.05 units) of heparin and incubated at 37 C for 0.20 ml of normal human plasma and 0.10 ml of Thromboplastin 10 minutes. were added and the PTT measured by the addition of 0.10 ml of 0.02 M CaCl . For TCT studies, 0.05 ml of these endothelial cell prepagations were comb?ned with 0.05 ml (0.02 units) of heparin and incubated at 37 C for 10 minutes. 0.20 ml of normal human plasma was then added and the mixture clotted by Buffer replaced heparin and/or difadding 0.10 ml (10 units) of thrombin. ferent cell preparations for controls. Endothelial cells were harvested from human umbilical cords and grown in tissue culture as previously described (15,161. Demonstration of antiheparin activity in endothelial cells was carried out by using whole endothelial cell suspensions, F-T suspensions of these cells and also acid-extracts of the nuclear and cytoplasmic fractions from endothelial cells. These nuclear and cytoplasmic cell fractions were prepared by the method of Wiggins et al (171, Endothelial cells were disrupted by gentle homogeniwith some modification. zation for 3.5 minutes using a tight-fitting, Teflon pestle tissue homogenizer (Thomas Company, Philadelphia, PA). The endothelial cell homogenate was centrifuged at 600 g for 10 minutes in a Damon/IEC Division CRU-5000 The centrifuged pellet containing the nuclear fractions was excentrifuge. amined and then discarded, and the supernatant containing the cytoplasmic constituents was collected and ultracentrifuged at 150,000 g for one hour. Under electron microscopy, the cytoplasmic pellet was compared to the nuclear fraction and found to be free of nuclear contamination (Figure 1). The pooled cytoplasmic pellet fractions from different preparations were extracted with 0.20 N H SO dialysed against 0.01 N HCl and then against Tris Buffered Saline (?BS!'(pH 7.4) overnight (161. The protein was determined by the Lowry-Hartree method (181. Electrophoretic characterization of the lyophilized acid-extract of endothelial cells cytoplasmic fraction was performed on a gradient acrylamide gel slab containing 2.5 M urea on a Bio-Rad Dual Vertical Slab Gel Electrophoresis Cell, Model 221 (Bio-Rad Laboratories, Richmond, CA). This technique of Traub and Boeckmann (19) has been used for separation of basic proteins. Neutralization of heparin-mediated anticoagulant activity by endothelial cells and their fractions was demonstrated in PTT and TCT systems. Studies presented in this report represent a mean of a minimum of five experiments _+ standard deviation (S.D.). ERESUIX'S AND DISCUSSION Table 1 shows that heparin alone prolonged the clotting time in PTT system to 179.0 and in TCT system to > 180.0 seconds. When the same amougt of heparin was added to the whole endothelial cell suspensions (1.42 x 10
Vol. 42, No. 3
ENDOTHELIUM AND HEPARIN
FIG.
357
1
Samples of whole endothelial cells, nuclear debris and cytoplasmic constituents obtained from homogenized endothelial cells were pelleted and fixed with 1.0 ml of 2% gluteraldehyde in 0.1 M phosphate buffer. These samples were then examined under electron microswhole endothelial cells x copy. (A 2000; B = nuclear debris x 2000; C = cytoplasmic constituents x 3000). ??
cells/ml, 0.89 mg/ml protein), the anticoagulant activity was neutralized, as the clotting time was now shortened to 150.0 seconds in PTT and 35.0 seconds in TCT system. F-T suspensions of endothelial cells at the same concentrations, more effectively neutralized heparin anticoagulant activity (65.0 seconds and 18.0 seconds in PTT and TCT systems respectively, with control clotting times of 57.0 seconds and 17.0 seconds). Table 2 shows the effect of the acid-extract of the cytoplasmic fraction on heparin neutralization when compared to F-T suspensions of whole endothelial cells. When equivalent amounts (0.064 mg protein) of acid-extract and F-T suspensions of endothelial cells (wt/wt protein) were examined for heparin neutralization, the acid-extract of the cytoplasmic fraction demonstrated stronger ability to neutralize heparin (68.0 seconds vs. 87.0 seconds). Acid-extract of the cytoplasmic fraction per se exhibited mild but persistent anticoagulant activity. The PTT with acid-extract was 69.0 seconds as compared to 54.0 seconds with buffer control. Results of the electrophoretic studies are shown on Figure 2. A single band migrating towards the cathode is seen. For comparison, the nuclear fraction containing the histones is also shown. The cytoplasmic band is not shared by the nuclear fraction of the endothelial cells.
358
ENDOTHELIUM AND HEPARIN
Vol. 42, No. 3
TABLE 1 -Neutralization Suspensions
Buffer Buffer + Heparin Buffer + Whole Cells Heparin + Whole Cells Buffer + F-T Suspension Heparin + F-T Suspension
of Heparin by Intact and Freeze-Thawed ofEndotherr;i7 Cells -
(F-T)
PTT (in seconds) + S.D.
TCT (in seconds) + S.D.
59.00 179.00 58.00 150.00 57.00 65.00
18.00 180.00 17.00 35.00 17.00 18.00
2 2.5 & 4.4 2 2.3 222.5 f 8.4 flO.O
2 1.5 _+ 0 + 1.5 _+ 4.5 _+ 0.7 f 1.4
Endothelial cells were adjusted to 1.42 x lo6 cells/ml (0.89 mg/ml protein) and divided into two equal samples. One of these samples was used as whole cells, while the other sample was F-T three times. 0.05 ml aliquots (0.045 mg protein) of these two preparations were incubated with heparin (0.05 units heparin in PTT, 9.02 units in TCT) in these studies. Results are expressed as mean clotting time in seconds + standard deviation (S-D.).
TABLE '2 -Comparison of Freeze-Thawed (F-T) Endothelial Cells Suspension and Fractlo~ofndothellal -- the AcidrEx~ofasmlc Cells~n~parin Neutralization -Partial Thromboplastin Time (PTT) (in seconds _+ S.O.) Buffer Buffer + Heparin Buffer t F-T Suspension of Endothelial Cells Heparin + F-T Suspension of Endothelial Cells Buffer + Acid-Extract of Cytoplasmic Fraction Heparin + Acid-Extract of Cytoplasmic Fraction
54.00 107.00 56.00 37.00 69.00 68.00
2 0.6 _+ 1.2 2 8.4 210.0 _+ 0 _+ 1.7
Endothelial cell samples were F-T three times and the protein content adjusted to 1.28 mg/ml with buffer. Acid-extracts were also prepared and adjusted to the same protein concentration. 0.05 ml (0.064 mg of protein) aliquots of either the F-T or acid-extract were incubated with heparin (0.05 units) in this study. Results are expressed as mean clotting time in seconds f standard deviation (S.D.).
Studies presented in this report demonstrate that endothelial cells possess antiheparin activity. This activity is capable of neutralizing the anticoagulant action of heparin assayed in both the PTT and TCT systems. Although Lypho-Med heparin was used in these studies, other commercial sources
Vol. 42, No. 3
ENDOTHELIUM AND HEPARIN
359
FIG. 2
Electrophoresis of the acid-extracts of endothelial cell fractions (cytoplasmic and nuclear) were carried out on a gradient slab gel. Samples were applied at the anode of the gel containing 2.5 M urea. Electrophoresis was carried out at 20 mAmp/gel for 24 hours. Gels were stained with 0.25% Coomassie Brilliant Blue - 25% E-propanol - 10% acetic acid followed by destaining with 10% acetic acid. of heparin, when tested, exhibited similar neutralization. As presented, the antiheparin activity was exhibited by whole endothelial cells, but the activity became enhanced when the cells were disrupted and examined in the test systems. Further studies clearly show that antiheparin activity is pronounced in the acid-soluble fraction of the cytoplasmic constituents. Electrophoretic studies on the acid-extract of the cytoplasmic fraction showed a single band migrating towards the cathode, exhibiting the mobility pattern of a basic protein. This band was not shared by the electrophoretic bands of nuclear histones. This further suggested that the endothelial cell's antiheparin factor was specifically related to the cytoplasmic constituents of these cells and not due to contaminants from the nuclear histones. The histones are also basic proteins (20-22) and their antiheparin activity is known (23,24). Further studies are being done in order to conclusively prove that this antiheparin activity is definitely associated with the protein visualized on the electrophoretic gels. Our studies, therefore, showed that the antiheparin activity, although present in whole endothelial cells, was more pronounced when the cell cytoplasm was disrupted and when the extracts of cytoplasmic constituents were examined in the test systems. The cytosolar fraction (post 150,000 g supernate) demonstrated no antiheparin activity (not shown). It is not known at this time if the antiheparin activity present in the endothelial cells is synthesized and/or stored in their cytoplasmic organelles. Studies indicate that some antiheparin activity is also present on whole endothelial cells. Further studies are needed to clearly understand the relationship between the cytoplasmic and whole cell/surface distribution of antiheparin activity of endothelial cells. Further studies are in progress to characterize the biochemical nature of endothelial cells antiheparin factor. Electrophoretic analysis of the acid-extract of the cytoplasmic fraction, however, exhibited the mobility pattern of a basic protein. Although the precise biochemical nature of the acid-extract of the cytoplasmic fraction needs to be further determined, it is this fraction nevertheless which exhibited the highest antiheparin activity.
360
ENDOTHELIUM AND HEPARIN
Vol. 42, No. 3
The antiheparin activity in the PMN-leukocytes are known to be basic proteins (7). These basic proteins also possess anticoagulant activity (25,261. The antiheparin fraction of endothelial cells showed mild but per sistent anticoagulant action (Table 2). This further suggested its biochemical similarity with basic proteins. Some antigens affecting hemostasis are commonly shared by endothelial cells and platelets (27,281. Tested by imnunoassay (not shown), no antigenic similarity was found between endothelial cells antiheparin factor and platelet factor 4 (PF ) - a known antiheparin factor of a basic protein nature (29,301. Recentlj, a histidine-rich basic protein possessing.antiheparin activity has been reported in platelets (31) and also in plasma (32). Studies are in progress to determine if such basic proteins have any similarity with the antiheparin activity of endothel ial cells. The demonstration of antiheparin activity in endothelial cells is important and of clinical signficance, considering the large surface area a'f vascular tree in the human body. It is possible that, along with other antiheparin factors in the human body, the endothelial cells might also be involved in regulating the activity of heparin when infused in man. Heparinlike substances (heparinoids) have been identified in endothelial cells (33). Furthermore, heparin-like molecules with anticoagulant activity have recently been reported to be synthesized by endothelial cells (34). In view of this, it is possible that the antiheparin factor of endothelial cells as presented here might be involved in hemostatic balance with these endogenous heparinlike substances influencing the phenomena of hemostasis and thrombosis. ACKNOWLEDGMENTS We express our appreciation to Drs. Hasan I. Zeya, Paul Byvoet and Robert C. Hartmann for their valuable suggestions in this project and in the preparation of this manuscript. We also acknowledge Suzette Urban for her secretarial assistance. This work was supported by the VA Medical Research Service, Tampa , FL. REFERENCES
1.
SABA, H.I., Saba, S.R. Endothelium: Its role in hemostasis, thrombosis and atherosclerosis. In: Platelets and Prostaqlandins h Cardiovascu-_ lar Disease. J. Mehta, P. Mehta (Edsl New York: Futura Publishing Co, ml, pp. 125-148
2.
MASON R.G., Mohammad, In: Pathobiol;r zs, 1979, pp. l-
3.
CONLEY, C.L., Hartmann, R.C., Morse, W.I.. The clotting behavior of human "platelet-free" plasma: Evidence for the existance of a "plasma thromboplastin". J --Clin Invest 28, 340-352, 1949.
4.
VAN CREVELD, S., Paulssen, M.M.P. Isolation and properties of the third clotting factor in blood platelets. -Lancet 1, 23-25, 1952.
5.
CACCIOLA, E., Ferranto, A. Presence of antiheparin activity in leukemic cells. -----Bull Sot Ital Biol Sper 36, 1317-1319, 1960.
S.F., Saba, H.I., et al. Functions of endotheliAnnual. H.L. Ioachim (Edsl New York: Raven
Vol. 42, No. 3
ENDOTHELIUM AND HEPARIN
361
Tyg Lek 20 ---'
6.
Anti hepari n acti vi ty of normal leukocytes. LISIEWICZ, J. 1150-1151, 1965.
7.
SABA, H.I., Roberts, H.R., Herion, J.C. Antiheparin activity of lysosoma1 cationic proteins from polymorphonuclear leukocytes. -Blood 31, 369-380, 1968.
8.
HIEBERT, L.M., Jaques, L.B. 26-31, 1976.
9.
HIEBERT, L.M., Jaques , L.B. The observation of heparin on endothelium after injection. --Thromb Res 8, 195-204, 1976.
Heparin uptake on endothelium. Artery 2,
10.
GLIMELIUS , B., Busch, C., Hook, M. Binding of heparin on the surface of cultured endothelial cells. --Thromb Res 12, 773-782, 1978.
11.
GERTLER, M.M., Chen, M., Yue, R.H. Detection and partial purification of a natural heparin inhibitor from hog small intestine. Throm Haemost 41, 567-575, 1979. -
12.
SABA, S.R., Mason, R.G. Studies of an activity from endothelial cells that inhibit platelet aggregation, serotonin release and clot retraction. --Thromb Res 5, 747-757, 1974.
13.
LANGDELL, R.D., Wagner, R.H., Brinkhous, K.M. Effect of antihemophilic factor on the one-stage clotting tests. A presumptive test for hemophilia and a simple one-stage antihemophilic factor assay procedure. J Lab Clin Med 41, 637-647, 1953. _---
14.
SEEGERS, W.H. Preparation, purification and assay of thrombin. In: Preoaration, purification & ESSAY of thrombin. L.M. Tocantins, L.A. Kazal (Eds) New York: Grune and Stratton, 1964, pp. 181.
15.
SABA, S.R., Zucker, W.H., Mason, R.G. Some properties of endothelial cells isolated from human umbilical cord vein. Sem Hematol -6, 456-486, 1973.
16.
SABA, H.I., Hartmann, R.C., Saba, S.R. Effects of polymorphonuclear leukocytes on endothelial cell growth. --Thromb Res 12, 397-407, 1978.
17.
WIGGINS, R.C., Loskutoff, D.J., Cochrane, C.G., Griffin, J.H., Edgington, T.S. Activation of rabbit Hageman factor by homogenates of cultured rabbit endothelial cells. -J Clin Invest 65, 197-206, 1980.
18.
HARTREE, E.F. Determination of protein: A modification of the Lowry method that gives a linear photometric response. Ann Biochem 48, 422427, 1972.
19.
TRAUB, P., Boeckmann, G. High-resolution polyacrylamide gradient slab gel electrophoresis of histone Hl, H3 and H4. Hoppe-Seyler's -Z Physiol Chem 359, 571-579, 1978. --
20.
CRUFT, H.J., Hindley, J., Mauritzen, C.M., Stedman, E. Amino acid composition of the six histones of calf thymocytes. -Nature 180, 1107-11909, 1957.
362
ENDOTHELIUM AND HEPARIN
Vol. 42, No. 3
21.
In: Nucleohistones. J. Bonner, P. MURRAY, K. The Nucleohistones. 1964, Ts'ao (Edsl San Francisco, London, Amsterdam: Holden-Day, Inc., pp. 15.
22.
JOHNS, E.W.
Histones and Nucleohistones. In: The preparation a&i_ . af histone$. B.M.P. Phillips (Eds) London, New York: Plenum Press, 1971, pp. 4-5.
m
.
23.
HILDEBRANO, C.E., Tobey, R.A., Gurley, L.R., Walters, R.A. Action of II. Cell-cycle-specific changes in chromaheparin on mammalian nuclei. tin organization correlate temporally with histone Hl phosphorylation. Biochim Biophys -Acta 517, 486-499, 1978.
24.
PAL, P.K., Gertler, M.M. Neutralization of heparin by histone and its Thromb Res 31, 69-79, 1983. subfractions. ---
25.
Antibacterial and enzymatic basic proteins ZEYA, H.I., Spitznagel, J.K. Separation and identification. Science -' 142 from leukocyte lysosomes: 1085-1087, 1963.
26.
Roberts, H.R., Herion, J.C. The anticoagulant activity of SABA, H.I., lysosomal cationic proteins from polymorphonuclear leukocytes. J Clin -Invest 46, 580-589, 1967. --
27.
HOWARD, M.A., Montgomery, D.C., Yardisty, R.M. Factor VIII-related Thromb Res 4, 617-624, 1974. tigen in platelets. ---
28.
NACHMAN, R.L., Jaffe, E.A. Subcellular platelet factor VIII antigen and J Exp Med 141, 1101-1113, 1975. von Willebrand factor. ----
29.
NATH, H., Lowery, C.T., Niewiarowski, S. Antigenic and antiheparin properties of human platelet factor 4 (PF4). -Blood 45, 537-550, 1975.
30.
VARMA, K.G., Niewiarowski, S., Holt, S., Rucinski, B., Paul, D. Human platelet basic protein. Its relation to low affinity platelet factor 4 and B-thromboglobulin. Biochim Biophys -Acta 701, 7-11, 1982.
31.
LEUNG, L.L.K., Harpel, P.C., Nachman, P.L., Rabellino, E.M. Histidinerich glycoprotein is present in human platelets and is released following thrombin stimulation. Blood 62, 1016-1021, 1983. --
32.
HAUPT, N., Heimburger, N. Human serumproteine mit hoher affinitat zu carboxymethycellulose I. Yoppe-Seyler's Z Physiol -Chem 353, 1125-1132, 1972.
33.
BUONASSISI, V., Root, 1.1. Enzymatic degradation of heparin-related mucopolysaccharides from the surface of endothelial cell cultures. Biochim Biophys -Acta 385, l-10, 1975.
34.
MARCUM, J.A., Rosenberg, R.D. Heparin-like molecules with anticoagulant activity are synthesized by cultured endothelial cells. Biochim Biophys Res Comm 126, 365-372, 1985.
an-