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Effect of plasma histidine-rich glycoprotein on the inhibition by anionic polysaccharides of thrombin-triggered platelet aggregation
THROMBOSIS RESEARCH 33; 139-143, 1984 0049-3848/84 $3.00 + .OO Printed in the USA. Copyright (c) 1984 Pergamon Press Ltd. All rights reserved.
EFFECT OF PLASMA HISTIDINE-RICH GLYCOPROTEIN ON THE INHIBITION BY ANIONIC POLYSACCHARIDES OF THROMBIN-TRIGGERED PLATELET AGGREGATION G. Kindness*, W.F. ~rga;ltF;A~n~iBro~:;thFIQ. Long' Williamson', W.T. *
Department of Molecular and Life Sciences, Dupdee College of Technology, Dundee, Scotland, DDl lGH, U.K., Polysaccharide Research Laboratory, Department of Bio&emistry, University of Aberdeen, Scotland, AB9 lAS, U.K. and Department of Biochemistry, Louisiana State University Medical Center, New Orleans, Louisiana 70112, U.S.A. (Received 10.8.1983; Accepted in revised form 2.11.1983 by Editor P.J. Gaffney) INTRODUCTION
Histidine-rich glycoprotein, a plasma component the concentration of which changes in various physiological and clinical conditions (l-6) is of uncertain function (7,8),but exhibits a range of properties in vitro. These include its interactions with organic ions (9), with maliX-iiktal ions (10-12) and with the high-affinity lysine-binding site of plasminogen(l3), and its inhibition of autorosette formation between erythrocytes and lymphocytes (14,15). The glycoprotein also binds heparin strongly (8,16,17), possesses an N-terminal amino acid sequence homologous with that of antithrotiin III (18), and counteracts the anticoagulant activity of heparin i'nplasma (16,17). In this communication we report that histidine-rich glycoprotein alleviates the inhibition by heparin and other sulphated polysaccharides of thrombi'n(EC 3.4.21.5)-induced aggregation of platelets. METHODS Sources of bovine throtiin (19) and of sulphated polysaccharides (19-21) were as previously described. Human platelet-rich plasma (19) and purified rabbit histidine-rich glycoprotein (22) were prepared as previously described. Throdin, polysaccharides and histtdine-rich glycoprotein were dissolved and diluted in O.llM-NaCl/O.O5M-Tris/HCl, pH 7.7. Pglysaccharides and histidinerich glycoprotein (59 ug/ml) were incubated at 37 C for 120s; JO ~1 of the mixture were then added to 140 ~1 of platelet-rich plasma at 37 C. After 6Os, 10 l.11 of thrombin (0.3 units/ml) were added, and aggregation of platelets monitored in an aggregometer and microscopically as previously described (19). Quoted concentrations of thrombin, polysaccharides and histidine-rich glycoprotein are final concentrations in the plasma-containing reaction mix.
Keywords: histidine-rich glycoprotein,platelet aggregation,thrombin,heparin 139
HRG AND PLATELET AGGREGATION
140
ioa
a
1
Vo1.33, No.2
1
A-k Lk:_l! I -. . :ill 2
50
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63
34
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4
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1
1
1.2
2
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3
3
4
2
Reaction
time
(min.)
FIG. 1 Effect of polysaccharides and histidine-rich glycoprotein on thrombintriggered platelet aggregation Experimental details are given in the text. Addition of thrombin is indicated by the arrows. The left-hand box of each pair represents the effect of the polysaccharide alone, and the right-hand box the effect of polysaccharide which has been pretreated with histidine-rich glycoprotein. Polysaccharide
vo1.33, No.2
HffiAND PLATELET AGGREGATION
141
concentrations @g/ml) are: (a) He rin (1) 0, (2) 0.03, (3) 0.06, (4) 0.12; (b) -Carra eenan (1) 0, (2) 1.5, f3~'3O('l4)-~n (1) 0, m&73) 60; (d) -Carr~'(!~)04*~~)(~) (5)'6.0; if) Dexttan sulph sul hate (1) 0, (2)0.3, (3)h. ti 9 , (3) 0.6, (4) 1.5; (h) Aga,rose (1) 0, (2) 6.0. n%TsRecorder outputs from the aggregometerwere traced and superimposed graphically for convenient presentation. RESULTS The figure shows that preincubation of histidine-rich glycoprotein at a fixed concentration with various concentrations of heparin, -carrageenan, - carrageenan, -carrageenan, xylan sulphate, dextran sulphate or cellulose sulphate alleviated the inhibition of thrombin-induced platelet aggregation caused by these sulphated polysaccharides at the stated concentrations. The figure also indicates the lack of effect of non-sulphated agarose, with or without histidine-rich glycoprotein, on thrombin-induced aggregation. Under the conditions used, in the absence of thrombin, neither polysaccharides nor histidine-rich glycoprotein, together or separately directly affected platelet behaviour. Microscopic observations were in accord with results seen in the aggregometer. DISCUSSION The mechanism by which heparins (and structurally-related polysaccharides) inhibit thrombin-induced pbtelet aggregation is not clear. It may involve potentiation of antithrombin III action, and presumably does involve il.'eraction of thrombin with heparin, to which this proteinase is known to bind avidly (23). The neutralization by rabbit histidine-rich glycoprotein of heparin antithrombin activity reported here accords with the neutralization by human histidine-rich glycoprotein of heparin anti-thrombin, anti-factor Xa (EC 34.21. 6) and anticoagulant activity in other assay systems reported elsehwere (16,17) Since human histidine-rich glycoprotein and heparin interact with apparent 1:l stoichiometry to form a complex with a dissociation constant of 7nM (16), it is of interest that, in the system we describe, pre-incubation of heparin with a molar excess of rabbit histidine-rich glycoprotein does not necessarily completely neutralize the subsequent anti-thrombin effectofthe heparin in plasma. This may reflect a lower affinity of heparin for rabbit histidine rich glycoprotein than for human histidine-rich glycoprotein, or the dissoc' iation of, or partial anti-thrombin activity of the heparin-histidine-rich glycoprotein in our system. Histidine-rich glycoprotein is released from thrombin-treated platelets (24); functional counterparts of its in vitro heparin neutralizing activities mayinclude its modulation ofoperties of platelet and endothelial cell heparans. REFERENCES 1.
MORGAN, W.T., KOSKELO, P., KOENIG, H. and CONWAY, T.P. Human histidinerich glycoprotein. II. Serum levels in adults, pregnant women and neonates. ProcZSoc. Exp. Biol. Med. 158, 647-651, 1978.
2.
LIJNEN, H.R., DE COCK, F. and COLLEN, D. Turnover of human histidinerich glycoprotein in healthy subjects and during thrombolytic therapy. Thrombosis Res. 23, 121-131, 1981.
142
HRG AND PLATELET AGGREGATION
Vol.33, No.2
3.
LIJNEN, H.R., JACOBS, G. and COLLEN, D. Histidine-rich glycoprotein in a normal and a clinical population. _Thro-osis Res. 22, 519-523, 1981
4.
SAI‘TO,H., GOODNOLJGH,L.T., BOYLE, J.M. and HEIMBURGER, N. Reduced histidine-rich glycoprotein (HRG) levels in plasma of patients with advanced liver cirrhosis : possible implications for enhanced fibrinolysis. Clin.Res. 29,763A, 1981.
5.
SAITO, H., GOODNOUGH, L.T., BOYLE, J.M. and HEIMBURGER, N. Reduced histidine-rScR glycoprotein levels in plasma of patients with advanced liver cirrhosis. Possible implications for enhanced fibrinolysis. Am.J.Med. a, 179-182, 1982.
6.
JESPERSEN, J. and KLUFT, C. rndfvi'dual levels of plasma histidinerich glycoprotein (HRG) during the normal menstrual cycle and in women on oral contraceptives low in estrogen. Scand.J.ClPn. Lab. .--_Invest,42,563-566. 1982.
7.
HAUPT, H. and HEIMBURGER, N. Humanserumproteine mit hoher Affinitit zu Carboxymethylcellulose, I. Isolierung von Lysozym, Clq und zwei bisher unbekannten a-Globulinen.Hoppe-Seyler's Z.Physiol. Chem. 353, 1125-1132, 1972.
8.
HumanserumHEIMBURGER, N., HAUPT, H.,,,KRANZ,T. and BAUDNER, S. proteine mit hoher Affinitat zu Carbo~methylcellulose, 11. Physikalisch-chemische und immunoloatsche Charakterisieruno etnes Histidinrichen 3,8 S-as- Glykoprotefns (CM-Protein r). &~~&~y~_er’l~... Physiol. Chem. 353, 1133-1140, 1972.
9.
MORGAN, W.T. Human serum histidine-rich glycoprotetn. r. Interactions with heme metal ions and organic ligands. Biochim.Biophys.Acta.533, 319-333, 1978.
10.
GUTHANS, S.L. and MORGAN, W.T. Metal binding of rabbit histidinerich glycoprotein : equilibriurndialysis and tmmunoadsorbent chromatography. Fed.Proc.Fed.Am.Soc.Exo.Btol.40, 1799, 1981.
11.
GUTHANS, S.L. and MORGAN, W.T. The interaction of zinc, nickel and cadmium with serum albumin and histidine-rich glycoprotein assessed by equilibrium dialysis and immunoadsorbentchromatography. &c& Biochem. Biophys. 218, 320-328, 1982.
12.
MORGAN, W.T. Interactions of the histidine-rich glycoprotein of serum with metals. Biochemistry,g, 1054-1061, 1981.
13.
LIJNEN, H.R., HOYLAERTS, M. and COLLEN, D. Isolation and characterization of a human plasma protein with affinity for the lysine binding sites in plasminogen. Role in the regulation of fibrinolysis and identification as histidine-rich glycoprotein. J.Biol. m. 255, 10314-10222, 1980.
14.
RYLATT, D.B., SIA, D.Y., MUNDY, J.P. and PARISH, C.R. Autorosette inhibition factor : isolation and properties of the human plasma protein. Eur.J.Biochem. llg, 641-646, 1981.
Vo1.33, No.2
HRG AND PLATELET AGGREGATION
143
15.
LIJNEN, H.R., RYLATT, D.B. and COLLEN, D.
Physicochemical, imnunochemical and functional comparison of human histidine-rich glycoprotein 109-115, and autorosette inhibition factor. Biochim.Biophys. Acta,742, 1983.
16.
LIJNEN, H.R. HOYLAERTS, M. and COLLEN, D. Heparin binding properties of human histidine-rich glycoprotein. Mechanism and role in the neutralization of hepartn in plasma. J.Biol. ChenL.,23, 3803-3808, 1983.
17.
LIJNEN, H.R., HOYLAERTS, M. and COLLEN, D. Neutralization of heparin activity by binding to human histidine-rich glycoprotein. Thrombosis Res. 29, 443-446, 1983.
18.
KOIDE, T., ODANI, S. and ONO, T. The N-terminal sequence of human plasma histidine-rich glycoprotein homologous to antithrombin with high affinity for heparin. FEBS Lett. -141, 222-224, 1982.
19.
KINDNESS, G., LONG,W.F., WILLIAMSON, F.B. and BOYD, J. Effects of carrageenans on the aggregation of human blood platelets. Thrombosis Res. 15, 3-15, 1979.
20.
KINDNESS, G., LONG, W.F. and WILLIAMSON, F.B. Enhancement of antithrornbinIII activity by carrageenans. Thrombosis Res. 12,4960, 1979.
21.
KINDNESS, G., WILLIAMSON, F.B. and LONG, W.F. Effects of a sulphated xylan on aggregation of human Glood platelets. Thrombosis Res. l-6_, 97-105, 1979.
22.
MORGAN, W.T. and SMITH, A. Interaction of histidine-rich glycoprotein of serum with metalsPergamon and plasmin. Press Iyn prei+otides of Bioloqical Fluids. New York : . ,
23.
JORDAN, R.E., OOSTA, G.M., GARDNER, W.T. and ROSENBERG, R.D. The binding of low molecular weight heparin to hemostatic enzymes. J.Biol. Chem. 255, 10073-10080, 1980.
24.
LEUNG, L.L.K., HARPEL, P.C. and NACHMAN, R.L. Histidine-rich glycoprotein release from human platelets following thronbin stimulation. Clin.Res. 30, 505A, 1982.
EFFECT OF PLASMA HISTIDINE-RICH GLYCOPROTEIN ON THE INHIBITION BY ANIONIC POLYSACCHARIDES OF THROMBIN-TRIGGERED PLATELET AGGREGATION G. Kindness*, W.F. ~rga;ltF;A~n~iBro~:;thFIQ. Long' Williamson', W.T. *
Department of Molecular and Life Sciences, Dupdee College of Technology, Dundee, Scotland, DDl lGH, U.K., Polysaccharide Research Laboratory, Department of Bio&emistry, University of Aberdeen, Scotland, AB9 lAS, U.K. and Department of Biochemistry, Louisiana State University Medical Center, New Orleans, Louisiana 70112, U.S.A. (Received 10.8.1983; Accepted in revised form 2.11.1983 by Editor P.J. Gaffney) INTRODUCTION
Histidine-rich glycoprotein, a plasma component the concentration of which changes in various physiological and clinical conditions (l-6) is of uncertain function (7,8),but exhibits a range of properties in vitro. These include its interactions with organic ions (9), with maliX-iiktal ions (10-12) and with the high-affinity lysine-binding site of plasminogen(l3), and its inhibition of autorosette formation between erythrocytes and lymphocytes (14,15). The glycoprotein also binds heparin strongly (8,16,17), possesses an N-terminal amino acid sequence homologous with that of antithrotiin III (18), and counteracts the anticoagulant activity of heparin i'nplasma (16,17). In this communication we report that histidine-rich glycoprotein alleviates the inhibition by heparin and other sulphated polysaccharides of thrombi'n(EC 3.4.21.5)-induced aggregation of platelets. METHODS Sources of bovine throtiin (19) and of sulphated polysaccharides (19-21) were as previously described. Human platelet-rich plasma (19) and purified rabbit histidine-rich glycoprotein (22) were prepared as previously described. Throdin, polysaccharides and histtdine-rich glycoprotein were dissolved and diluted in O.llM-NaCl/O.O5M-Tris/HCl, pH 7.7. Pglysaccharides and histidinerich glycoprotein (59 ug/ml) were incubated at 37 C for 120s; JO ~1 of the mixture were then added to 140 ~1 of platelet-rich plasma at 37 C. After 6Os, 10 l.11 of thrombin (0.3 units/ml) were added, and aggregation of platelets monitored in an aggregometer and microscopically as previously described (19). Quoted concentrations of thrombin, polysaccharides and histidine-rich glycoprotein are final concentrations in the plasma-containing reaction mix.
Keywords: histidine-rich glycoprotein,platelet aggregation,thrombin,heparin 139
HRG AND PLATELET AGGREGATION
140
ioa
a
1
Vo1.33, No.2
1
A-k Lk:_l! I -. . :ill 2
50
A2
63
34
0
4
,.!-100
C
50
d
1
1
1.2
2
4
6
2
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0
l-3
1.2
4
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50
T
l-3
1,2
h3
c P
3
3
4
2
Reaction
time
(min.)
FIG. 1 Effect of polysaccharides and histidine-rich glycoprotein on thrombintriggered platelet aggregation Experimental details are given in the text. Addition of thrombin is indicated by the arrows. The left-hand box of each pair represents the effect of the polysaccharide alone, and the right-hand box the effect of polysaccharide which has been pretreated with histidine-rich glycoprotein. Polysaccharide
vo1.33, No.2
HffiAND PLATELET AGGREGATION
141
concentrations @g/ml) are: (a) He rin (1) 0, (2) 0.03, (3) 0.06, (4) 0.12; (b) -Carra eenan (1) 0, (2) 1.5, f3~'3O('l4)-~n (1) 0, m&73) 60; (d) -Carr~'(!~)04*~~)(~) (5)'6.0; if) Dexttan sulph sul hate (1) 0, (2)0.3, (3)h. ti 9 , (3) 0.6, (4) 1.5; (h) Aga,rose (1) 0, (2) 6.0. n%TsRecorder outputs from the aggregometerwere traced and superimposed graphically for convenient presentation. RESULTS The figure shows that preincubation of histidine-rich glycoprotein at a fixed concentration with various concentrations of heparin, -carrageenan, - carrageenan, -carrageenan, xylan sulphate, dextran sulphate or cellulose sulphate alleviated the inhibition of thrombin-induced platelet aggregation caused by these sulphated polysaccharides at the stated concentrations. The figure also indicates the lack of effect of non-sulphated agarose, with or without histidine-rich glycoprotein, on thrombin-induced aggregation. Under the conditions used, in the absence of thrombin, neither polysaccharides nor histidine-rich glycoprotein, together or separately directly affected platelet behaviour. Microscopic observations were in accord with results seen in the aggregometer. DISCUSSION The mechanism by which heparins (and structurally-related polysaccharides) inhibit thrombin-induced pbtelet aggregation is not clear. It may involve potentiation of antithrombin III action, and presumably does involve il.'eraction of thrombin with heparin, to which this proteinase is known to bind avidly (23). The neutralization by rabbit histidine-rich glycoprotein of heparin antithrombin activity reported here accords with the neutralization by human histidine-rich glycoprotein of heparin anti-thrombin, anti-factor Xa (EC 34.21. 6) and anticoagulant activity in other assay systems reported elsehwere (16,17) Since human histidine-rich glycoprotein and heparin interact with apparent 1:l stoichiometry to form a complex with a dissociation constant of 7nM (16), it is of interest that, in the system we describe, pre-incubation of heparin with a molar excess of rabbit histidine-rich glycoprotein does not necessarily completely neutralize the subsequent anti-thrombin effectofthe heparin in plasma. This may reflect a lower affinity of heparin for rabbit histidine rich glycoprotein than for human histidine-rich glycoprotein, or the dissoc' iation of, or partial anti-thrombin activity of the heparin-histidine-rich glycoprotein in our system. Histidine-rich glycoprotein is released from thrombin-treated platelets (24); functional counterparts of its in vitro heparin neutralizing activities mayinclude its modulation ofoperties of platelet and endothelial cell heparans. REFERENCES 1.
MORGAN, W.T., KOSKELO, P., KOENIG, H. and CONWAY, T.P. Human histidinerich glycoprotein. II. Serum levels in adults, pregnant women and neonates. ProcZSoc. Exp. Biol. Med. 158, 647-651, 1978.
2.
LIJNEN, H.R., DE COCK, F. and COLLEN, D. Turnover of human histidinerich glycoprotein in healthy subjects and during thrombolytic therapy. Thrombosis Res. 23, 121-131, 1981.
142
HRG AND PLATELET AGGREGATION
Vol.33, No.2
3.
LIJNEN, H.R., JACOBS, G. and COLLEN, D. Histidine-rich glycoprotein in a normal and a clinical population. _Thro-osis Res. 22, 519-523, 1981
4.
SAI‘TO,H., GOODNOLJGH,L.T., BOYLE, J.M. and HEIMBURGER, N. Reduced histidine-rich glycoprotein (HRG) levels in plasma of patients with advanced liver cirrhosis : possible implications for enhanced fibrinolysis. Clin.Res. 29,763A, 1981.
5.
SAITO, H., GOODNOUGH, L.T., BOYLE, J.M. and HEIMBURGER, N. Reduced histidine-rScR glycoprotein levels in plasma of patients with advanced liver cirrhosis. Possible implications for enhanced fibrinolysis. Am.J.Med. a, 179-182, 1982.
6.
JESPERSEN, J. and KLUFT, C. rndfvi'dual levels of plasma histidinerich glycoprotein (HRG) during the normal menstrual cycle and in women on oral contraceptives low in estrogen. Scand.J.ClPn. Lab. .--_Invest,42,563-566. 1982.
7.
HAUPT, H. and HEIMBURGER, N. Humanserumproteine mit hoher Affinitit zu Carboxymethylcellulose, I. Isolierung von Lysozym, Clq und zwei bisher unbekannten a-Globulinen.Hoppe-Seyler's Z.Physiol. Chem. 353, 1125-1132, 1972.
8.
HumanserumHEIMBURGER, N., HAUPT, H.,,,KRANZ,T. and BAUDNER, S. proteine mit hoher Affinitat zu Carbo~methylcellulose, 11. Physikalisch-chemische und immunoloatsche Charakterisieruno etnes Histidinrichen 3,8 S-as- Glykoprotefns (CM-Protein r). &~~&~y~_er’l~... Physiol. Chem. 353, 1133-1140, 1972.
9.
MORGAN, W.T. Human serum histidine-rich glycoprotetn. r. Interactions with heme metal ions and organic ligands. Biochim.Biophys.Acta.533, 319-333, 1978.
10.
GUTHANS, S.L. and MORGAN, W.T. Metal binding of rabbit histidinerich glycoprotein : equilibriurndialysis and tmmunoadsorbent chromatography. Fed.Proc.Fed.Am.Soc.Exo.Btol.40, 1799, 1981.
11.
GUTHANS, S.L. and MORGAN, W.T. The interaction of zinc, nickel and cadmium with serum albumin and histidine-rich glycoprotein assessed by equilibrium dialysis and immunoadsorbentchromatography. &c& Biochem. Biophys. 218, 320-328, 1982.
12.
MORGAN, W.T. Interactions of the histidine-rich glycoprotein of serum with metals. Biochemistry,g, 1054-1061, 1981.
13.
LIJNEN, H.R., HOYLAERTS, M. and COLLEN, D. Isolation and characterization of a human plasma protein with affinity for the lysine binding sites in plasminogen. Role in the regulation of fibrinolysis and identification as histidine-rich glycoprotein. J.Biol. m. 255, 10314-10222, 1980.
14.
RYLATT, D.B., SIA, D.Y., MUNDY, J.P. and PARISH, C.R. Autorosette inhibition factor : isolation and properties of the human plasma protein. Eur.J.Biochem. llg, 641-646, 1981.
Vo1.33, No.2
HRG AND PLATELET AGGREGATION
143
15.
LIJNEN, H.R., RYLATT, D.B. and COLLEN, D.
Physicochemical, imnunochemical and functional comparison of human histidine-rich glycoprotein 109-115, and autorosette inhibition factor. Biochim.Biophys. Acta,742, 1983.
16.
LIJNEN, H.R. HOYLAERTS, M. and COLLEN, D. Heparin binding properties of human histidine-rich glycoprotein. Mechanism and role in the neutralization of hepartn in plasma. J.Biol. ChenL.,23, 3803-3808, 1983.
17.
LIJNEN, H.R., HOYLAERTS, M. and COLLEN, D. Neutralization of heparin activity by binding to human histidine-rich glycoprotein. Thrombosis Res. 29, 443-446, 1983.
18.
KOIDE, T., ODANI, S. and ONO, T. The N-terminal sequence of human plasma histidine-rich glycoprotein homologous to antithrombin with high affinity for heparin. FEBS Lett. -141, 222-224, 1982.
19.
KINDNESS, G., LONG,W.F., WILLIAMSON, F.B. and BOYD, J. Effects of carrageenans on the aggregation of human blood platelets. Thrombosis Res. 15, 3-15, 1979.
20.
KINDNESS, G., LONG, W.F. and WILLIAMSON, F.B. Enhancement of antithrornbinIII activity by carrageenans. Thrombosis Res. 12,4960, 1979.
21.
KINDNESS, G., WILLIAMSON, F.B. and LONG, W.F. Effects of a sulphated xylan on aggregation of human Glood platelets. Thrombosis Res. l-6_, 97-105, 1979.
22.
MORGAN, W.T. and SMITH, A. Interaction of histidine-rich glycoprotein of serum with metalsPergamon and plasmin. Press Iyn prei+otides of Bioloqical Fluids. New York : . ,
23.
JORDAN, R.E., OOSTA, G.M., GARDNER, W.T. and ROSENBERG, R.D. The binding of low molecular weight heparin to hemostatic enzymes. J.Biol. Chem. 255, 10073-10080, 1980.
24.
LEUNG, L.L.K., HARPEL, P.C. and NACHMAN, R.L. Histidine-rich glycoprotein release from human platelets following thronbin stimulation. Clin.Res. 30, 505A, 1982.