Molecular Immunology, Vol. 20, No. 12, pp. 1401-1404, 1983 0161-5890/83 $3.00+0.00 Printed in Great Britain. © 1983 Pergamon Press Ltd

HEPARIN PREVENTS FORMATION OF THE H U M A N C3 AMPLIFICATION CONVERTASE BY

INHIBITING THE BINDING SITE FOR B ON C3b*

FRAN~OISE MA1LLET, MICHEL D. KAZATCHKINE,t DENIS GLOTZ, ELISABETH FISCHER and MATHEW ROWE

INSERM U28, H6pital Broussais, 75014 Paris, France

(First received 17 May 1983; accepted in revised form 25 July 1983)

Abstract--Fluid-phase heparin prevents generation of the C3 amplification convertase of human complement, C3b, Bb most likely by inhibiting the formation of the bimolecular complex between cell-bound C3b and B. The effect of heparin on the binding of B to C3b was examined using t25I-labelled B and C3b-bearing sheep erythrocytes (E~C3b). In the absence of heparin, B bound to E~C3b with an affinity of 0.5-1 x 106M -~ in the presence of 5mMMg 2+. Incremental amounts of heparin (100-700#g/107 E~C3b) inhibited the binding of 125I-B to C3b in a dose-dependent manner. Scatchard analysis of the binding data in the presence of four inhibitory concns of heparin revealed that heparin did not affect the binding affinity of B for C3b but decreased the number of C3b sites recognized by B on the cells. No inhibition of binding occurred in the presence of totally (N- and O-) desulfated heparin which has no anticomplementary activity. These results demonstrate that heparin prevents generation of the C3 amplification convertase by binding to cell-bound C3b and masking the binding site for B on C3b.

INTRODUCTION

Heparin, whether fluid-phase or particle-bound, in- hibits activation of the complement system by inter- fering with formation of the classical (Ecker and Gross, 1929; Raepple et al., 1976; Loos et al., 1976a, b; Rent et al., 1976) or alternative pathway C3 convertases (Weiler et al., 1978; Kazatchkine et al., 1979, 1981), as well as with the function of late components (Baker et al., 1975). Fluid-phase heparin glycosaminoglycan prevents generation of the human C3 amplification convertase C3b, Bb by inhibiting the interaction of cell-bound C3b with B in the presence and absence of D (Weiler et al., 1978); the action of heparin was therefore considered to be on the for- mation of the C3b, B bimolecular complex.

*This work was supported by grants 82.L.0670 from Ministrre de la Recherche et de l'Industrie, France, and CRL 815036 from INSERM.

tPlease address all correspondence: Dr M. Kazatchkine, Department of Nephrology, INSERM U28, H6pital Broussais, 96 rue Didot, 75014 Paris, France.

:~Abbreviations: E s, sheep erythrocytes; ESC3b, C3b-bearing ES; K, affinity constant at equilibrium; VBS, veronal- buffered saline; GVB, VBS containing 0.1~o gelatin; GVB 2+, GVB containing 0.5 mM Mg 2+ and 0.15 mM Ca ~+, DGVB 2+, GVB z+ mixed with an equal vol of 5~o dextrose in water; GVB-Mg, GVB containing 5 mM Mg2+; EDTA, ethylenediaminetetraacetate; GVB-EDTA, GVB containing 0.04M EDTA. The nomenclature used for the alternative activating path- way of complement is that recommended by IUIS/WHO [J. Immun. 127, 1261 (1981)].

Binding studies of radiolabeled purified factor B to C3b in the presence of heparin provided direct evi- dence for the fact that heparin inhibits formation of C3b, B by inhibiting the binding site for B on cell- bound C3b.

MATERIALS AND METHODS

B (Hunsicker et al., 1973), C3 (Tack and Prahl, 1976), D (Fearon and Austen, 1975) and P (Fearon and Austen, 1977) were purified to homogeneity as assessed by polyacrylamide gel electrophoresis of their reduced forms in the presence of sodium dode- cyl sulfate. B was trace labeled with t25I (Amersham, Les Ulis, France) by use of insoluble lactoperoxidase (Worthington Biochemicals Corp., Freehold, N J) to a sp . act. of 66,000-90,000cpm/gg without loss of functional activity; radiolabeled B was stored at -70°C in veronal-buffered saline (VBS).J~ Radio- activity was measured with an LKB gamma counter, model 1275 (LKB, Orsay, France). Hog intestine heparin (H-3125) with an anticoagulant activity of 170 U/mg was obtained from Sigma Chemical Co. (St. Louis, MO). Total desulfation of the glycos- aminoglycan (Nagasawa et al., 1977) was obtained by treating heparin with dimethylsulfoxide containing 10~o methanol for 18 hr at 100°C as described pre- viously (Kazatchkine et al., 1981). E s were collected in Alsever's solution, washed twice in 0.15 M NaCI and twice in GVB-EDTA and stored in this buffer at 4°C. GVB 2+, DGVB 2+ and GVB-Mg were prepared as previously described (Nelson et al., 1966; Kazatch- kine et al., 1979).

1401

1402 FRAN(;:OISE MAILLET e t a l .

E'C3b

1 × 109E ~ were incubated with 1000/~g of C3, 200pg of B in 1.0ml of GVB 2+ containing D and 5 mM additional Mg z+ for 45 min at 30°C. The cells were washed twice in GVB 2+, incubated with 80#g of B in 1.0 ml of DGVB 2+ containing D and 5 mM additional Mg 2+ for 30min at 30°C, washed in ice-cold DGVB 2+ and further incubated in 1.0 ml of DGVB 2+ containing 450 pg of C3 for 45 min at 30°C. The cells were washed twice in DGVB 2+ and the second step of C3b deposition was repeated once.

E~C3b were assessed for their capacity to form an amplification convertase with B, D and P and stored in GVB-EDTA at 4°C.

Assay for binding of 125I-B to ESC3b (Kazatchkine et al., 1979)

E'C3b (0.75-2.5 x 107) were incubated with vari- ous concns of ~25I-B in 0.2 ml of GVB-Mg for 30 rain at 30'~C; duplicate 70-pl samples from each reaction were layered over 300pl of a mixture of di- butylphtalate (Merck-Clevenot, France) and dino- nylphtalate (Coger, Paris, France) [7:3 (v/v)] in 0.5-ml polypropylene tubes and centrifuged for 1 min at 8000g in a Beckman microfuge (Beckman, Paris, France). The tubes were cut just above the pellets and the sections were assessed for the radioactivity of the bound ligand.

Binding of '25I-B to E ~ in the absence of C3b was also assessed. The specific C3b-dependent binding was calculated by substracting non-specific uptake by E ~ from binding to E'C3b. The K o f B for bound C3b (KB) and the number of C3b sites/EsC3b were calcu- lated from Scatchard plots (Scatchard, 1949) of the binding data. The stoichiometry of the binding of B to C3b is 1 to 1 (Kazatchkine et al., 1979). In the absence of heparin, the KB for C3b was found to be 0.5-2.5 × 106M i.

RESULTS

The binding to 2.5 × 107ESC3b of 60 x 10 -6 #moles of 12SI-B, an input of B sufficient to saturate approximately 50% of C3b sites on ESC3b, was assessed in the absence or in the presence of increas- ing amounts of heparin ranging from 100 to 700/~g. Non-specific uptake of ~251-B by E ~ was similar in the presence and absence of heparin. Heparin inhibited the specific binding of ~25I-B to C3b in a dose- dependent fashion (Fig. 1); 50% inhibition of binding was achieved with an input of heparin of 160#g/ 2.5 x 107 E'C3b.

When the binding to E'C3b of incremental amounts of '25I-B ranging from 20.3 to 162.6 × 10-6/~moles was examined in the absence and presence of 200 #g of heparin/assay, the Ks was found to be 2.5 x 106 and 2.08 x I06M -t, whereas the apparent number of C3b binding sites for B revealed by B on the same cells was 39,000 and 24,000/cell respectively (Fig. 2). Thus, heparin de-

,=

I00 90 80 70 60

50

40

20.

tO,

I I I I I I

ioo 200 3o0 40o 5oo 7oo

~g heporin/25 x 107ceLts

Fig. 1. Inhibition of specific binding of ~5I-B to C3b on E'C3b by increasing amounts of heparin. The fixed amount of '25I-B used in these experiments was calculated in pre- liminary experiments to saturate approximately 50% of the

C3b binding sites for B in the absence of heparin.

creased the number of binding sites for B on ESC3b without altering the binding affinity of B for acces- sible C3b sites.

The results of the binding of 20.3 to 162.6 x 10-6/zmoles of ~25I-B to another batch of E~C3b in the absence and presence of heparin were plotted according to Scatchard. The cells demon- strated 84,000 C3b sites for B in the absence of heparin. Increasing amounts of heparin from 50 to 300 # g decreased the number of C3b sites revealed by B without altering the affinity orB for cell-bound C3b (Table 1).

The logarithm of the number of C3b sites that were revealed by B in the presence of heparin was inversely related to the amount of heparin/assay (Fig. 3); a 50% decrease in the number of C3b sites was achieved with an input of 170/zg of heparin and the slope of the

Table 1. Effect of heparin on the binding of ~25I-B to ESC3b"

Heparin K s (#g/0.75 x 107 E'C3b) ( x 106 M - ' ) C3b sites/E~C3b h

0 0.90 84,000 (100~) 50 0.48 72,000 (85.7%)

100 0.66 56,000 (66.6%) 200 0.76 40,000 (47.6%) 300 0.90 24,000 (28.6%)

"Scatchard analysis of duplicate measurements of the binding of four different amounts of B to 0.75 x 107 ESC3b. Scatchard plots were linear (r = 0.96, 0.99, 0.94, 0.98 and 0.78).

/'The number of C3b sites revealed by B/E~C3b was calculated from the intercept of the Scatcbard plot with the abscissa.

Heparin inhbition of the C3 amplification convertase 1403

0.007 '

re 0 . 0 0 6 ' to

I.O o o O_ 0 .005 ' x

O~ 0004

0.003

c 0.002 • • o 8 rn

0001

i 0 5 0 0 0 ~0,000 20,000 50;300 40pO0

Mo(ecul.es b o u n d / E S C S b

Fig. 2. Scatchard plots of the binding of 125I-B to E'C3b in the absence (© C)) or presence (O 0 ) of 200#g of heparin/assay. The amounts of ~25I-B used in these experiments ranged from 20.3 to

162.6 x 10 -6 #moles/0.75 x 10 7 ESC3b. Each point is the mean of duplicate measurements.

curve was similar to that obtained from the results of the experiment depicted in Fig. 1.

In contrast to intact glycosaminoglycan, totally desulfated heparin (230 #g/assay) had no inhibitory effect on the binding of ~25I-B to cell-bound C3b.

DISCUSSION

Heparin in the fluid phase inhibits the effective interaction of cell-bound C3b with B and D and the

© ×

L)

+,

re to

"5

Z

I00 90

80

70

60

50

40

30

20

i I I I 0 50 I00 200 300

p.g heporin / 075 x lOFcells Fig. 3. Decrease in the apparent numbers of C3b sites that are recognized by 125I-B on E~C3b in the presence of increasing amounts of heparin. The number of C3b binding sites for B was calculated from Scatchard plots of the binding data of incremental amounts of J25I-B to E'C3b in the absence and presence of four different concns of heparin.

fluid-phase cleavage of B by D in the presence of C3b (Weiler et al., 1978). Heparin inhibited formation of the cell-bound amplification convertase C3b, Bb re- gardless of the presence of P and impaired generation of the convertase formed in the absence of D and containing uncleaved B (Daha et al., 1976; Weiler et al., 1978). Furthermore, heparin was most active in preventing convertase formation on cells bearing low numbers of C3b and developed with high doses of B (Weiler et al., 1978) suggesting that the gly- cosaminoglycan inhibits the association between C3b and B, most likely by acting on C3b.

Heparin inhibited in a dose-dependent manner the binding of 125I-B to C3b on E ~ (Fig. 1). The 50% inhibitory input of heparin was 160#g/2.5 x l07 E~C3b; it was approximately 80 times higher than the amount of heparin that is necessary to inhibit for- mation of an unstabilized convertase in the absence of D, possibly because binding experiments were performed in the presence of 5 m M Mg 2+ which artificially increased the KB for bound-C3b (Ka- zatchkine et al., 1979).

Since heparin did not alter the KB for cell-bound C3b but decreased the apparent number of C3b sites revealed by B upon Scatchard analysis of the binding data (Fig. 2 and Table 1), heparin inhibits formation of the C3b, B bimolecular complex by masking the binding site for B on C3b. The effect requires the presence of intact sulfate groups on the molecule. The amount of heparin that decreased by half the number available C3b binding sites for B was similar to that that inhibited 50% of B binding to E'C3b (Fig. 3); per cent inhibition of binding of B to C3b by heparin was inversely related to the number of residual C3b binding sites for B.

Since polycations also inhibit formation of the C3b, B complex (Maillet and Kazatchkine, 1983), the low-affinity interaction between C3b and B appears

1404 FRAN(~OISE MAILLET et al.

as a privileged site for na tura l or pharmacologic modula t ion of complement by polyelectrolytes.

Acknowledgements--We thank Lydie Jacot for fine secre- tarial assistance. This work was supported by grant "Bio- materiaux" from CNRS.

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