Polyions regulate the alternative amplification pathway of complement

Polyions Regulate the Alternative Amplification Pathway of Complement John M. Weiler Abstract: Polyanion haspreviously been shown to inhibit generation of amplification pathway convertase of complement and to inhibit H-mediated decay of this complex. Polycations, protamine sulfate, and poly-l-lysine (PLL) were examined in this study for direct effects on alternative amplification pathway mechanisms and for ability to regulatepolyanion-induced inhibition; they werefound to inhibit generation of EAC4b,3b,Bb, and EAC4b,3b,Bb, P in a dose-related manner. The generation of EAC4b,3b,B was also inhibited by higher doses but was augmented by lower doses of polycation. Polycation interfered with the effective consumption of B in the fluid phase, bound minimally to EAC4b,3b, and did not cause accelerated decay of a preformed convertase. Polycation diminished the ability of polyanion (heparin) to inhibit generation of both cell-bound and fluid-phose amplification pathway convertase. In contrast, polycation enhanced the ability of polyanion to cause decay of preforrned convertase even though polycation had no effect itself. These studies demonstrate that both polyionic substances have the capacity to regulate amplification pathway mechanisms directly. Key Words: Heparin; Poly-l-lysine; Polycations; Protamine sulfate; Polyions

INTRODUCTION

Polyanion has previously been shown to be capable of regulating the amplification convertase of complement by inhibiting C3b,Bb complex generation and by preventing H-mediated decay of C3b,Bb (Weiler et al., 1978; Kazatchkine et al., 1979; Kazatchkine et al., 1981). Polyanions have also been shown to be capable of regulating the classical pathway (Ecker et al., 1941), potentiating the action of C1 esterase inhibitor (Rent et al., 1976; Caughman et al., 1982), interfering with C l q binding to immune complexes (Raepple et al., 1976), inhibiting Cls interaction with C4 and C2 (Loos et al., 1976b) and C2 binding to C4 (Loos et al., 1976a), and suppressing reactive lysis (Baker et al., 1975). When polycations were studied they were found to be able to cause classical pathway activation in acute-phase sera (Siegel et al., 1974) and to cause potentiation of reactive lysis (Baker et al., 1975). Furthermore, polycations, in the presence of polyanions, cause total depletion of serum hemolytic complement (Rent et al., 1975; Fiedel et al., 1976), which is markedly enhanced by the presence of C-reactive protein (Claus et al., 1977).

Received December 13, 1982; revised and accepted June 8, 1983. From the Department of Internal Medicine, The Universityof Iowa and the Iowa City VA Medical Center, Iowa City, Iowa. Address requests for reprints to: J. M. Weiler, M.D., SW34E UniversityHospital, Universityof Iowa, Iowa City, IA 52242. © ElsevierSciencePublishingCo., Inc., 1983 52 Vanderbilt Ave., New York, N.Y. Immunopharmacology6,245-255 ( 1 9 8 3 )

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The present study was designed to determine whether anticomplementary effects of heparin on the alternative amplification pathway could be prevented by the presence of polycation, as is the case with the anticoagulant effects of heparin. Indeed, polycation was found to interfere with heparin's ability to inhibit generation of amplification convertase and to inhibit fluid-phase utilization of B. In contrast, polycation increased the ability of heparin to inhibit a preformed convertase. Polycation itself inhibited generation of the amplification convertase and interfered with the effective utilization of B in the fluid phase. These findings demonstrate that polycation serves an independent role in regulating complement activation and, in addition, is able to modify the ability of polyanion to regulate complement.

MATERIALS AND METHODS Preservative-free aqueous commercial heparin, 1000 units per ml, obtained from Fellows Medical Division, Chromalloy Pharmaceuticals, Oak Park, MI, was manufactured by Elkins-Sinn, Inc., Cherry Hill, NJ. Heparin concentration was quantitated as previously described (Weiler eta[., 1978; Yurt et al., 1977). Protamine sulfate, catalog number P4020, was obtained from Sigma Chemical Company, St. Louis, MO, and was dissolved in distilled water at a concentration of 2 mg/ml and diluted in buffer before use in the various assays. Poly-1-1ysine HBr (PLL), 3 0 , 0 0 0 - 7 0 , 0 0 0 MW, catalog number 7 1 - 1 2 0 Q , was obtained from Miles Laboratories Inc., Elkhart, IN, and was dissolved in distilled water to 10 mg/ml and diluted in buffer just prior to use.

Buffers, Complement Components, and Assays Half-isotonic veronal-buffered saline, pH 7.5, containing 0.1% gelatin and 2.5% dextrose (DGVB), DGVB with 0.5 mM magnesium and 0.15 mM calcium (DGVB+ +), DGVB containing 10 mM ethylenediamine tetraacetate (EDTA) (10 mM EDTA) or containing 20 mM EDTA (20 mM EDTA), and isotonic veronal-buffered saline containing 0.1% gelatin and 40 mM EDTA (40 mM EDTA) were used as buffers in various hemolytic assays. C3 (Tack et al., 1976), B (Hunsicker et al., 1973;), P (Fearon et al., 1977a), D (Fearon et al., 1975b), and H (Weiler et al., 1976) were purified to homogeneity and quantitated as described. C3b was generated from purified C3 as previously described (Gitlin et al., 1976; Weiler et al., 1976). Rat serum was obtained from Rockland Inc., Gilbertsville, PA. E, EA, EAC1,4b, and EAC4b,3b cellular intermediates were prepared as previously described (Fearon et al., 1973; Lachmann et al., 1978). B hemolytic activity in reaction mixtures of purified proteins was assayed as described (Fearon et al., 1975a; 1977b). Each experiment in the Results section was repeated at least three times and the data presented in this report represent typical results.

Agglutination of Cells It has long been known that polycations agglutinate red blood cells by cross linking surface sialic acid and that this is inhibited by the presence of heparin (Lalezarietal., 1961);therefore, initially the dose of polycation that would cause E or EAC to agglutinate was quantitated. Tubes containing 1 × 107 E, EA, EAC1,4b or EAC4b,3b and various concentrations of protamine,

Abbreviations. PLL: poly-l-lysine; DGVB: half-isotonic veronal-buffered saline, pH 7.5, containing 0.1% gelatin and 2.5% dextrose; DGVB++: DGVB with 0.5 mM magnesium and 0.15 mM calcium; EDTA: ethylenediamine tetraacetate; I0 mM EDTA: DGVB containing 10 mM EDTA; 20 mM EDTA: DGVB containing 20 mM EDTA; 40 mM EDTA: isotonic veronal buffered saline containing 0.1% gelatin and 40 mM EDTA; C-EDTA rat serum diluted I : 20 in 40 mM EDTA; EAC4b,3b (low): EAC4rb,3b prepared with a low input of C3; EAC4b,3b(high): EAC4b,3b prepared with a high input of C3.

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heparin, PLL or protamine and heparin were incubated in 0.2 ml DGVB + + at 30°C or at 37°C for 60 min and assessed for agglutination. Protamine alone at concentrations of 0.2/~g per 107 cells and above caused agglutination of E and of the cellular intermediates after incubation at both temperatures. Protamine, in concentrations that caused marked agglutination (10 ~g/107 EAC4b,3b) did not cause lysis of EAC4b,3b intermediates. No such agglutination was seen in tubes containing cells treated with heparin alone in doses as high as 20 ~g per 107 cells. Similarly, no agglutination was seen in tubes containing heparin and protamine at equivalent weights, at concentrations of protamine that alone caused significant agglutination. For example, protamine alone at 1.0 ~g per 107 cells caused E and the cellular intermediates to agglutinate, while the addition of heparin at I. I ~g per 107 cells or at greater concentrations inhibited this agglutination. PLL was even more active and caused agglutination of the cells at concentrations as low as 24 ng per 107 cells. PLL, at doses greater than 1 ~g/107 EAC4b,3b, appeared to be directly toxic to the cells.

RESULTS Inhibition of the Formation of the Amplification Pathway Convertases Protamine, or protamine and heparin, were examined for capacity to regulate cell-bound amplification pathway convertase generation. I ml reaction mixtures contained DGVB+ + a n d 1.8 ng B, 100 ng D, and 600 ng P per 10~ EAC4b,3b to form EAC4b,3b,Bb,P or 18 ng B and 100 ng D per 10SEAC4b,3b to form EAC 4b,3b,Bb, or 1.67 ~g B per 10 s EAC4b,3b to form EAC4b,3b,B. 0. I ml of each of the three mixtures above was added to 0.1 ml DGVB+ + alone or containing heparin (0.4 ~g), protamine at various concentrations, or protamine and heparin, and incubated for 30 min at 30 ° with shaking. Finally, 0.3 ml of a I : 20 dilution of rat serum in 40 mM DGVB was added to each tube and incubation continued for 60 min at 37 °. Saline ( 1.5 ml) was added to each tube, the tubes were well mixed and centrifuged, percent lysis was determined, and the average number of hemolytic sites per cell (Z) was calculated. As seen in Figure I(A), protamine inhibited generation of EAC4b,3b,Bb and EAC4b,3b,Bb,P in a dose-related manner. Protamine also inhibited generation of EAC4b,3b,B at higher concentrations, whereas at lower protamine concentrations there was augmentation of lysis. In this experiment, moderate agglutination of EAC4b,3b was seen at protamine concentrations of 1 - 2 ~g/107 EAC4b,3b. When heparin at 0.4 ~g per 107EAC4b,3b was present during convertase generation there was 37, 74, and 75% inhibition of generation of EAC4b,3b,B, EAC4b,3b,Bb, and EAC4b,3b,Bb,P respectively (Figure I(B)). The addition of protamine suppressed the ability of heparin to inhibit the generation of all three of the convertases (Figure I(B)). These experiments demonstrate that: 1) protamine itself had the capacity to inhibit generation of all three convertases; 2) protamine had the least effect on the generation of EAC4b,3b,B, and at low concentrations caused enhanced generation of EAC4b,3b,B; and 3) protamine and heparin were not synergistic in inhibiting generation of convertase, but rather they inhibited each other's action, which is consistent with the ability of heparin and protamine to neutralize each other stoichiometrically. PLL also had the capacity to inhibit generation of these convertases at doses comparable to protamine (0.12 ~g PLL per 107 EAC4b,3b inhibited by 50% the generation of EAC4b,3b,Bb,P) although PLL caused significantly more agglutination than protamine. The mechanism of inhibition was examined further in experiments that used EAC4b,3b that were prepared to have a low or high amount of C3 on the surface. EAC4b,3b were produced using an input of C3 of I ~g (low) or 100 ~g (high) per 109 EAC1,4b,2a. In developing the convertases, the B input was adjusted to produce about one hemolytic site per cell. Tubes containing I x 107 low or high EAC4b,3b were incubated with 60 ng P, I 0 ng D, and 7.8 and 0.3 ng B, respectively, in 0. I ml DGVB + +. Then 0. I ml DGVB + + alone or containing protamine was added to the tubes which were incubated 30 min at 30°C. C-EDTA was added and the convertase sites were

248

J . M . Weiler

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Figure I(A) Dose-response effects of protamine on the generation of EAC4b,3b,B (Q- 0), EAC4b,3b,Bb (m- B), and EAC4b,3b,Bb,P ( A - &). (B) Inhibition of the formation of EAC4b,3b,B ( 0 - 0), EAC4b,3b,Bb ( • - •), and EAC4b,3b,Bb,P ( • - •) by the presence of protamine and heparin.

developed. As seen in Figure 2, the cells prepared with the lower amount of C3 and requiring more B were more susceptible to inhibition by protamine, suggesting that polycation acted on C3b to inhibit c o n v e r t a s e generation. T h e r e was no agglutination of E A C 4 b , 3 b o b s e r v e d in this experiment. It was necessary to consider whether protamine had any effect on the terminal components

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249

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Figure 2. Dose-response effects of protamine on the generation of EAC4b,3b,Bb,P using EAC4b,3b prepared with low ( 0 - 0) or high ( 0 - O) C3 input. since such an effect would not be excluded by the studies presented so far. EAC4b,3b,Bb,P were therefore prepared by interacting I × 107 EAC4b,3b with 0.04 ng B, 10 ng D, and 60 ng P in 0.2 ml DGVB+ + for 30 min at 30°C. 0.1 ml of 10 mM EDTA alone or containing varying amounts of protamine or protamine and heparin was added and immediately thereafter C-EDTA was added. The incubation was continued for 60 min at 37°C and lysis was determined. In the absence of polyion there was an average of one hemolytic site formed (Z = 1) per cell. Protamine at concentrations as high as 20/.~g per 107 cells did not have any effect on lysis, although there was slight agglutination of EAC4b,3b at protamine concentrations greater than 0.6 p~g/107 EAC4b,3b. Similarly, the presence of heparin alone at 43 p~g per 107cellsor protamine and heparin did not modify this result.

Preincubation of Cells with Protamine before Generation of Amplification Pathway Convertase Protamine, heparin, or protamine and heparin were preincubated with EAC4b,3b to determine whether they would bind to the cell or to cell-bound components. Tubes containing I × 107 EAC4b,3b in 0.2 ml DGVB + + were incubated with various concentrations of protamine, heparin (0.4 p~g), or heparin and protamine for 30 min at 30°C. Then 2 ml DGVB + + were added and the tubes were centrifuged. The supernatant was removed and the cycle of washing repeated two additional times with DGVB + +. The cells were resuspended in 0.2 ml DGVB + + containing 0.12 ng B, 10 ng D, and 60 ng P and were incubated at 30°C for 30 min, after which C-EDTA was added to each tube. Incubation was continued an additional 60 min at 37°C, and percent lysis was determined. Protamine alone did not bind to the cells in a fashion that led to subsequent inhibition of convertase generation except at concentrations where there was marked agglutination (greater than I p~gprotamine/107EAC4b,3b), and then the binding was minimal (Figure 3). When there was no wash between the preincubation and the generation of the convertase, protamine caused significant inhibition. Interestingly, the presence of both protamine and heparin caused significantinhibition, whereas the heparin alone had no effect on the subsequent ability of the cells to be lysed, suggesting that heparin may have increased the amount of protamine that bound to the cells by simple crosslinking.

250

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Figure 3. Dose-response effects of protamine alone ( 0 - ~) and in the presence of heparin ( 0 - O) on ability of EAC4b,3b cellular intermediates to be lysed subsequently by complement. As a control, EAC4b,3b cellular intermediates were incubated with protamine ( • - •), but no wash was imposed between the incubation and the subsequent attempt to lyse the cellular intermediate. Effect of Polyion on Preformed Convertases Heparin has previously been shown to be able to cause inhibition of a formed convertase. Consequently, that same experiment was repeated with protamine and various combinations of protamine, heparin, and H. I ml reaction mixtures containing I × 108 EAC4b,3b, 0.5 ng B, 100 ng D, and 600 ng P were incubated 30 min at 30°C. 0.1 ml aliquots were then added to tubes containing 0. I ml of 20 mM EDTA alone or containing: a) protamine; b) heparin (2 p,g); c) H (17 ng); d) protamine and heparin; e) protamine, heparin and H; or f) protamine and H, and incubated for 15 rain at 30°C. Then C-EDTA was added to each tube and the convertase sites were developed. Protamine alone did not inhibit the previously formed convertase at any dose (Figure 4). Protamine did enhance the ability of heparin to cause inhibition of the previously formed convertase from 13% with heparin alone to 52% in the presence of I p,g protamine per 107 cells (Figure 4). Protamine did not modify the ability of H to inhibit (not shown) nor did it modify inhibitionseen with heparin and H (Figure 4). No agglutination of EAC4b,3b was seen at any dose of protamine in this experiment. Fluid Phase Experiments

The ability of polycations to cause agglutination made interpretation of the experiments using cellular intermediates somewhat difficult. Therefore, the following experiments were performed to determine if protamine inhibited convertase generation by impairing B utilization in the fluid phase, as has been observed with heparin (Weiler et al., 1976). Reaction mixtures containing 0.25 p,g C3b and 20 ng D alone or with 2.5 pLgheparin, or 4/.~g protamine, or 2.5 p,g heparin and 4/.Lg protamine in 150 p~l DGVB++ were prewarmed to 30°C. At time zero, 1.1 /.Lg B in 50 p,l DGVB+ + was added, and at timed intervals 10p,l aliquots were removed and added to 0.75 ml ice-cold DGVB+ + and assayed for B hemolytic activity. Protamine alone and heparin alone each caused inhibition of the consumption of B in the reaction mixture (Figure 5(A)). The presence of

Polyions and Complement

251

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both polyions together, however, led to an intermediate level of inhibition, suggesting that they inhibited each other's action possibly by charge neutralization. Similarly, using the experimental design described above, PLL (12.5 pg) and heparin (1.6 ~g) were studied in the fluid-phase interaction of the purified components. Again, the polycation (PLL) caused inhibition of the utilization of B, whereas the presence of both PLL and heparin led to no inhibition of the utilization of B (Figure 5(B)).

DISCUSSION

These studies demonstrate that polycation had the capacity to inhibit directly the alternative amplification pathway of complement (Table I). Polycation inhibited generation of P-stabilized (EAC4b,3b,Bb,P)and nonstabilized (EAC4b,3b,Bb) convertase in a dose-related manner (Figure I(A)). The generation of convertase formed without D input (EAC4b,3b,B) was also inhibited by higher concentration of polycation, whereas lower concentrations enhanced lysis of this cellular intermediate (Figure I(A)). When the mechanism of this inhibition was examined, using EAC4b,3b formed with a high (EAC4b,3b hgh) or a low (EAC4b,3bl°~) C3 input, polycation had more activity on EAC4b,3b1°Wthan on EAC4b,3b h.gh (Figure 2). This suggests that polycation impaired the ability of C3b to interact with B to form a convertase. When the fluid-phase interaction of C3b, B, and D was examined, polycation was inhibitory, again suggesting an effect on C3b (Figure 5). However, incubation of polycation with EAC4b,3B only minimally prevented subsequent formation of an effective convertase (Figure 4) on this cellular intermediate, demonstrafing that binding to C3b was probably not irreversible. Furthermore, the action of polycation was probably not due to an irreversible binding to B, since the B did not lose hemolytic activity merely by being incubated with polycation (Figure 5). Polycation also had no ability to inhibit preformed convertase (Figure 4), nor did it inhibit when present during the terminal phase of the complement system (when added with C-EDTA). Polycation had the capacity to inhibit convertase generation in a manner similar to, but not identical to, polyanion (Table I). Both polyions inhibited convertase formation, being least active

252

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Figure 5(,4). Kineticsof consumption of B hemolytic activity in fluid phase by C3b and D alone ( 0 - 0 ) and in the presence of hepafin (E]- El), protamine ( A - A), and protamine and hepa~in ( 0 - 0). (B) Kinetics of consumption of B hemolytic activity in fluid phase by C3b and D alone ( 0 - 0 ) and in the presence of heparin ([]- El), PLL ( 0 - Q ) , and PLL and heparin ( 0 - 0). on EAC4b,3b,B generation. Neither polyion bound irreversibly to EAC4b,3b to prevent the cellular intermediate from subsequently forming an effective convertase. Both polyions had more activity on the ability of EAC4b,3bI°~ than on EAC4b,3b high to generate a convertase. Both polyions were able to inhibit the fluid-phase consumption of B by C3b and D. In contrast, only polyanion inhibited the ability of preformed EAC4b,3b,Bb,P to be lysed. The presence of both polyions simultaneously produced a more complex response (Table I). For example, during convertase generation the polyions were inhibitory when present individually, whereas their simultaneous presence produced little or no inhibition. Similarly, when they were present simultaneously in the fluid phase there was much less inhibition of the consumption of B than when either was present alone. In contrast, whereas neither polyion had much capacity to inhibit subsequent lysis of an EAC4b,3b cell after preincubation with and

Polyions and Complement

Table I

253

Effects of polyions on alternative amplification pathway of complement

Assay

Polycation

Polyanion

Polycation and polyanion

Generation of convertase

Inhibition

Inhibition

Little or no effect

Preincubation with EAC4b,3b, washing, then generation of convertase

Little or no effect

No effect

Inhibition

Fluid phase consumption of B by C3b and D Inhibition

Inhibition

Little or no effect

Preformed EAC4b,3b,Bb,P

Inhibition

Synergistic effect (inhibition)

Generation of convertase using EAC4b,3bhighand EAC4b,3bl°w

No effect

Greater inhibitionof EAC4b,3bI°wthan of EAC4b,3bhigh

Not studied

washing of the cell, their presence together during this preincubation step lead to significant inhibition. Furthermore, they were synergistic in their ability to inhibit a preformed convertase. Protamine and heparin have previously been studied only for their effects together on the classical pathway and similar results were found. Protamine and heparin together cause serum to be totally depleted of early classical complement components (Rent et al., 1975; Fiedel et al., 1976), whereas neither substance at the same concentration is capable of such marked activation. This activity is greatly enhanced by C-reactive protein (Claus et al., 1977). In one study utilizing acute-phase serum, protamine had a biphasic dose-response curve, such that at low and at high concentrations there was significantly moreconsumption thanthere was at intermediate concentrations (Siegel et al., 1974). This might be explained by assuming that the protamine was interacting with a polyanion in the acute-phase serum (Siegel et al., 1974). The ability of these charged substances to inhibit complement may be important in vivo. Heparin is released as a proteoglycan from the mast cell as a result of complement activation and subsequent binding of anaphylatoxin to the cell (Yurt et al., 1977). Other mediators are also released, when the mast cell degranulates, and lead to the ingress of eosinophils to the area of inflammation. The eosinophils then release their substances, including major basic protein (Gleich et al., 1974). One might postulate that major basic protein and heparin could regulate each other's ability to inhibit the generation of convertase. Depending then upon the amount of each polyion present in the microenvironment, one would expect to see either inhibition of the ability to generate an alternative amplification pathway convertase or no effect at all. In any case, these studies demonstrate that the polyions serve a complex role in regulating alternative amplification pathway complement mechanisms. This investigation was supported in part by Biomedical Research Support Grant RR 05372 from the Biomedical Research Support Branch, Divisionof Research Facilitiesand Resources, National Institutesof Health.The author would liketo thank Ms.KatyLoeb Schmit for her technicalassistanceduring the conduct of these studies and Ms. Darla Bartals for her secretarial assistance in the preparation of this manuscript. Dr. Weiler is the recipient of a ClinicalInvestigatorCareer Award from the Veterans Administration.

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Claus DR, Siegel J, Petras K, Skor D, Osmand AP, Gewurz H (1977) Complement activation by interaction of polyanions and polycations. III.Complement activation by interaction of multiple polyanions and polycations in the presence of C-reactive protein. J Immunol 118:83. Ecker EE, Pillemer L (1941) Anti-coagulants and complementary activity. An experimental study. J Immunol 40:73. Fearon DT, Austen KF, Ruddy S (1973) Formation of a hemolytically active cellular intermediate by the interaction between properdin factors B and D and the activated third component of complement. J Exp Meal 138:1305. Fearon DT, Austen KF (1975a) Properdin: initiation of alternative-complement pathway. Proc Natl Acad Sci USA 72:3220. Fearon DT, Austen KF (1975b) Properdin: binding to C3b and stabilization of the C3b-dependent C3 convertase. J Exp Med 142:856. Fearon DT, Austen KF (1977a) Activation of the alternative complement pathway due to resistance of zymosan-bound amplification convertase to endogenous regulatory mechanisms. Proc Natl Acad Sci USA 74:1683. Fearon DT, Austen KF (1977b) Activation of the alternative complement pathway with rabbit erythrocytes by circumvention of the regulatory action of endogenous control proteins. J Exp Med 146:22. Fiedel BA, Rent R, Myhrman R, Gewurz H (1976) Complement activation by interaction of polyanions and polycations. II. Precipitation and role of IgG, Clq and CI-INH during heparinprotamine-induced consumption of complement. Immunology 30:161. Gitlin JD, Rosen FS, Lachmann PJ (1975) The mechanism of action the C3b inactivator (conglutinin-activating factor) on its naturally occurring substrate, the major fragment of the third component of complement (C3b). J Exp Meal 141:1221. Gleich GJ, Loering DA, Kueppers F, Bajaj SP, Mann KG (1974) Physicochemical and biological properties of the major basic protein from guinea pig eosinophil granules. J Exp Meal 140:313. Hunsicker LG, Ruddy S, Austen KF (1973) Alternate complement pathway: Factors involved in cobra venom factor (CoVF) activalion of the third component of complement (C3). JImmunol 110:128. Kazatchkine MD, Fearon DT, Silbert JE, Austen KF (1979) Surface-associated heparin inhibits zymosan-induced activation of the human alternative complement pathway by augmenting the regulatory action of the control proteins on particle bound C3b. J Exp Med 150:1202. Kazatchkine MD, Fearon DT, Metcalfe DD, Rosenberg RD, Austen KF (1981) Structural determinants of the capacity of heparin to inhibit the formation of the human amplification C3 convertase. J Clin Invest 67:223. Lachmann PJ, Hobart HJ (1978) Complement technology. In: Handbook of Experimental Immunology, 3rd Edition. Ed., Weir. Oxford: Blackwell Scientific Publications (Chapter 5A). Lalezari P, Spaet TH (1961) Antiheparin and hemagglutinating activities of polybrene. J Lab Clin Med 57:868. Loos M, Volanakis JE, Stroud RM (1976a) Mode of interaction of different polyanions with the first (C1, C1), the second (C2) and the fourth (C4) component of complement. II. Effect of polyanions on the binding of C2 to EAC4b. Immunochemistry 13:257. Loos M, Volanakis JE, Stroud RM (1976b) Mode of interaction of different polyanions with the first (C1, C1), the second (C2) and the fourth (C4) component of complement. III.Inhibition of C4 and C2 binding site(s) on Cls by polyanions. Immunochemistry 13:789. Raepple E, Hill H-U, Loos M (1976) Mode of interaction of different polyanions with the first (C1, CC~), the second (C2) and the fourth (C4) component of complement. I. Effect on fluid phase CI) and on C1) bound to EA or to EAC4. Immunochemistry 13:251. Rent R, Ertel N, Eisenstein R, Gewurz H (1975) Complement activation by interaction of polaynion and polycations. I. Heparin-protamine induced consumption of complement. J Ira m unol 114:120.

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Rent R, Myhrman R, Fiedel BA, Gewurz H (1976) Potentiation of Cl-esterase inhibitor activity by heparin. Clin Exp Immunol 23:264. Siegel J, Rent R, Gewurz H (1974) Interaction of C-reactive protein with the complement system. J Exp Med 140:631. Tack BF, Prahl JW (1976) Third component of human complement: Purification from plasma and physicochemical characterization. Biochemistry 15:4513. Weiler JM, Daha MR, Fearon DT, Austen KF (1976) Control of the amplification convertase of complement by the plasma protein betalH. Proc Natl Acad Sci USA 73:3268. Weiler JM, Yurt RW, Fearon DT, Austen KF (1978) Modulation of the formation of the amplification convertase of complement, C3b,Bb, by native and commercial heparin. J Exp Med 147:409. Yurt RW, Leid RW, Spragg J, Austen KF (1977) Immunologic release of heparin from purified rat peritoneal mast cells. J Immuno! 118:1201.