Turnover of histidine-rich glycoprotein during heparin administration in man

THROMBOSIS RESEARCH 30; 671-676, 1983 0049-3848183 $3.00 + .OO Printed in the USA. Copyright (c) 1983 Pergamon Press Ltd. All rights reserved.

BRIEF COMMUNICATION

TURNOVER OF HISTIDINE-RICH GLYCOPROTEIN DURING HEPARIN ADMINISTRATION IN MAN

H.R. LIJNEN and D. COLLEN Center for Thrombosis and Vascular Research Department of Medical Research University of Leuven, Belgium (Received 3.1.1983; Accepted in original form 21.3.1983 by Editor G. de Gaetano) INTRODUCTION Histidine-rich glycoprotein was first isolated from human plasma in 1972 (1). The native molecule has a molecular weight of about 75,000 (2) and interacts with heparin (l), with divalent cations (3) and with the high affinity lysine-binding site of plasminogen (4). The concentration in plasma of healthy individuals is 100 + 45 mg/l (1,s). We have recently demonstrated that histidine-rich glycoprotein interacts strongly with heparin in plasma thereby neutralizing its anticoagulant activity (6,7). Because of the possible clinical significance of this interaction we studied the turnover of native histidine-rich glycoprotein during heparin administration in man.

MATERIALS AND METHODS Proteins and Reagents Histidine-richglycoprotein waspurified from human plasma as previously described (2,4). It was homogeneous on sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis performed on 7% gels, and the molecular weight was determined to be 75,000 by comparison with a protein calibration mixture (Pharmacia, Uppsala, Sweden). Clinical grade heparin was obtained from Roche (Brussels, Belgium) and Sephadex products from Pharmacia (Uppsala, Sweden). Laboratory Procedures Histidine-rich glycoprotein was determined by Laurel1 electroimmunoassay using a plasma pool of 80 healthy blood donors as a standard. Heparin was

Key words : histidine-rich glycoprotein, turnover, heparinization

671

672

,HJSTIDINE-RICH GLYCOPROTEIN

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measured using the activated partial thromboplastin time (APTT) (8). The histidine-rich glycoprotein was labeled with Na1251 (IRE, Fleurus, Belgium) according to the method of McFarlane (9). The substitution level of iodine in the protein was 0.35 atoms per molecule, and the free iodide (determined as non-trichloroacetic acid-precipitable radioactivity) was about 3.7%. On SDS-polyacrylamide gel electrophoresis 85% of the radioactivity was recovered in the homogenous protein band corresponding to the histidine-rich glycoprotein. Analytical gel filtration of 2 ml plasma samples was performed at room temperature on a Sephadex G-200 column (2.5 x 37 cm) equilibrated with 0.15 M NaCl-0.01 M phosphate-O.01 M citrate buffer pH 7.4 containing 0.02% sodium azide and run at a flow rate of 20 ml per hr. Precipitation with trichloroacetic acid (10% final concentration) was performed at 4OC for 2 hr on 0.5 ml plasma (diluted 5 times with phosphate buffered saline) or on 2 ml urine (undiluted). 1251 was measured with a Berthold scintillation counter BF 5300 (Benelux Analytical Instruments, Vilvoorde, Belgium). The histidine-rich glycoprotein preparation used for the turnover studies was checked for the absence of Australia antigen by a radioimmunoassay (Ausria, Abbott) prior to labeling. The labeled protein was sterilized by filtration through a 0.22 urnMillex-GS filter (Millipore, Molsheim, France) and pyrogen testing was performed in rabbits, according to the pharmacopeia regulations (USP XVII, p. 863). Turnover Studies The turnover of the histidine-rich glycoprotein was studied in 3 patients with deep vein thrombosis treated with heparin by continuous infusion of amounts resulting in a two- to three-fold prolongation of the APTT (20,000 55,000 IU per 24 hr). One to two days after the start of the heparinization about 2.5 uCi of the labeled material was injected intravenously. Blood was drawn into trisodium citrate (final concentration 10 mM) 10 min and 4 hr after the injection, and then every 12 hr during 7 days. Urine was collected in 24 hr pools throughout the experiment. The tracer data were analyzed using a two-compartment mammillary model (lo), which allows the calculation of physiologically meaningful turnover parameters. The plasma radioactivity data plotted versus time were approximated by a sum of two exponential terms -a t -a2t 1 x(t) = Cle + C2e , by graphic curve peeling (10).

RESULTS AND DISCUSSION Table I summarizes the clinical and laboratory data on the 3 heparinized patients with deep vein thrombosis in whom the turnover of 1251-labeled histidine-rich glycoprotein was studied. Fig. 1 shows a representative study in patient B.H. who received 40,000 IU of heparin per day. Daily fluctuations of the plasma histidine-rich glycoprotein levels were small during the turnover, indicating that the patients were in a steady state with respect to histidine-rich glycoprotein turnover. A steady exponential decay of radioactivity down to approximately 5% of the initial plasma radioactivity was observed after an equilibration period of about 2 days. Throughout the

HISTODTNE-RICHGLYCOPROTETN

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turnover experiment more than 95% of the plasma radioactivity and none of the urinary radioactivity was precipitable by trichloroacetic acid.

FIG. 1

110 100iX E -1

Histidine-Rich

e Gtycoprotrin

Turnover of 1251-labeled histidine-rich glycoprotein in a heparinized patient with deep vein thrombosis. A. The APTT value upon administration of 40,000 IU of heparin per day; B. The plasma level of histidine-rich glycoprotein during the experimental period; C. Tracer data: x(t), plasma radioactivity; u(t), fractional daily urinary excretion of label. Graphic curve peeling is performed as previously described (10).

The individual values obtained for the parameters of x(t) are given in Table II. The plasma radioactivity half-life was 2.00 + 0.14 days. This is significantly shorter (pCO.01) than the half-life of 2.93 + 0.36 days that we have previously determined for histidine-rich glycoprote& in healthy volunteers (11). The fractional catabolic rate constant (klo,p) is slightly higher than in the controls (0.66 + 0.14 of the plasma pool per day as compared to 0.51 + 0.06), the fractional transfer rate constant (k12) slightly lower (0.52 + 0.09 of the plasma pool per day as compared to 0.61 + O.ll), and the absolute catabolic (synthetic) rate is slightly higher (1.89 + 0.40 mg/kg/day as compared to 1.59 + 0.35 mg/kg/day). These differences are however not statistically significant. The fractional catabolic rate constant (klO,u) calculated from the daily urinary radioactivity (10,ll) corresponds well with the value obtained from the plasma radioactivity data.

55 51 46

B.H. A.G. J.J. 34.0 5.5

28.8 31.5 41.6

Plasma volume (ml/kg)

TABLE II

88 22

117 84 64

Before injection

92 19

119 84 74

After injection

Plasma Histidine-rich Glycoprotein mg/liter (mean + SD)

0.58

0.30 -----

0.44

0.11

A.G.

J.J.

Mean

SD

0.43

c1

B.H.

Subject

0.02

0.35

0.38

0.33

0.33

al

0.11

0.56

0.70

0.42

0.57

c2

0.12

1.81

1.73

1.73

1.98

a2

0.14

2.00

1.80

2.10

2.10

t1/2 for al

-alt -a2t x(t) = Cle +Ce 2 10,P

0.14

0.66

0.84 --

0.50

0.63

k 10,u

0.21

0.68

0.96

0.46

0.62

k

Fractional catabolic rate constant

0.40

1.89

2.23

1.33

2.12

Absolute catabolic (synthetic) rate constant (mg/kg/day)

0.09

0.52

0.49

0.42

0.64

Fractional transcapillary transfer rate constant (k12)

0.09

0.54

0.45

0.66

0.52

Intravascular fraction (IV)

Tracer Data and Calculated Turnover Parameters in the Heparinized Patients with Deep Vein Thrombosis

Mean SD

Age (yr)

Subject

Clinical and Laboratory Data on the Heparinized Patients with Deep Vein Thrombosis

TABLE I

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The distribution of radioactivity in serial plasma samples was studied by gel filtration on Sephadex G-200. In all samples the radioactivity eluted in one main peak (Fig. 2A) in the same position as that of 1251-labeled histidine-rich glycoprotein added to human plasma (Fig. 2B). This indicates that the disappearance of plasma radioactivity is representative of the turnover of histidine-rich glycoprotein (and its complex with heparin).

FIG. 2

1.0 m

yI.300 ret

Gel filtration on Sephadex G-200 of serial plasma samples in 0.15 M NaCl-0.01 M phosphate-0.01 M citrate buffer pH 7.4 containing 0.02% sodium aside. (o), absorbance at 280 nm. A. Plasma samples (2 ml) from a patient with deep vein thrombosis during heparin therapy. Radioactivity (cpm) in samples taken 10 min after injection of the 125I-labeled protein (* ), onday (m)andday3 (+). B.Normal human plasma (2 ml) with addition of purified 1251-histidine-rich glycoprotein (3,300 cpm) (+ ). The distribution of histidink-rich glycoprotein as measured immunologically is also shown (m ); the height of the peaks on Laurel1 electroimmunoassay is indicated in mm.

The shorter half-life of histidine-rich glycoprotein during heparin therapy may be due to a faster clearance of the heparin-histidine-rich glycoprotein complex as compared to the free protein. A similar change in the turnover of antithrombin III during heparin administration was previously observed (12). The finding that the plasma level of histidine-rich glycoprotein does not change significantly during the turnover experiment may be explained by a compensatory increase of the synthesis rate during heparin administration.

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ACKNOWLEDGEMENTS This study was supported by a grant from de Geconcerteerde Onderzoeksacties (Conventie 80/85-3).

REFERENCES 1. HEIMBURGER, N., HAUPT, H., KRANZ, T. and BAUDNER, S. Humanserumproteine mit hoher Affinitlt zu Carboxymethylcellulose,II. Physikalisch-chemische und immunologische Charakterisierung eines histidinreichen 3,8 S-a2353, 1133Glykoproteins (CM-Protein I). Hoppe-Seyler's, 1140, 1972. 2. LIJNEN, H.R., RYLATT, D.B. and COLLEN, D. Physicochemical, immunochemical and functional comparison of human histidine-rich glycoprotein and autorosette inhibition factor. Biochim.Biophys.Acta (in press). 3. MORGAN, W.T. with metals.

Interactions of the histidine-rich glycoprotein of serum Biochemistry, 20, 1054-1061, 1981.

4. 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.Chem., 255,.10214-10222, 1980. 5. LIJNEN, H.R., JACOBS, G. and COLLEN, D. Histidine-rich glycoprotein in a normal and a clinical population. Thrombos.Res., 22, 519-523, 1981. 6. LIJNEN, H.R., HOYLAERTS, M. and COLLEN, D. Neutralization of heparin activity by binding to human histidine-rich glycoprotein. Thrombos.Res. (in press). 7. LIJNEN, H.R., HOYLAERTS, M. and COLLEN, D. Heparin binding properties of human histidine-rich glycoprotein. Mechanism and role in the neutralization of heparin in plasma. J.Biol.Chem. (in press). 8. PROCTOR, R.R. and RAPAPORT, S.I. The partial thromboplastin time with kaolin. A simple screening test for first stage plasma clotting factor deficiencies. Amer.J.Clin.Pathol., 36, 212-219, 1961. 9. McFARLANE, A.S. Efficient trace-labeling of proteins with iodine. Nature, g, 53, 1958. 10. COLLEN, D., TYTGAT, G., CLAEYS, H., VERSTRAETE, M. and WALLEN, P. Metabolism of plasminogen in healthy subjects : effect of tranexamic acid. J.Clin.Invest., 51, 1310-1318, 1972. 11. LIJNEN, H.R., DE COCK, F. and COLLEN, D. Turnover of human histidinerich glycoprotein in healthy subjects and during thrombolytic therapy. Thrombos.Res., 23, 121-131, 1981. 12. COLLEN, D., SCHETZ, J., DE COCK, F., HOLMER, E. and VERSTRAETE, M. Metabolism of antithrombin III (heparin cofactor) in man : effects of venous thrombosis and of heparin administration. Eur.J.Clin.Invest., 7, 27-35. 1977.