Interaction of Cls̄ and Cl inactivator in the presence of heparin, dextran sulfate and protamine sulfate

0049-3848/80/120847-13$02.00/O THROMBOSIS RESEARCH 18; 847-859, 1980 Printed in the USA. All rights reserved. Copyright (c) 1980 Pergamon Press Ltd

INTERACTION OF Cl: AND Cl INACTIVATOR IN THE PRESENCE OF HEPARIN, DEXTRAN SULFATE AND PROTAMINE SULFATE

Akikazu Takada, and Yumiko Takada Department of Physiology, Hamamatsu University, School of Medicine, Hamamatsu-shi, Shituoka 431-31, Japan (Received 21.1.1980. Accepted by Editor P. Harpel. Received in final form by Executive Editorial Office 29.4.1980)

ABSTRACT When Cls'was mixed with Cl inactivator (ClINA) and the mixture was added with ATEe (acetyl tyrosine ethyl ester), the hydrolysis of ATEe decreased. The addition of heparin or dextran sulfate resulted in more hydrolysis of ATEe by the mixture of Cl5 and Cl IIJA, thus indicating the inhibition of ClINA activity. When the mixture of Cls and ClINA was added to S-2238 (H-D-Phe-Pip&g-pNA). the hydrolysis of S-2238 by the mixture was the same as its hydrolysis by Cls alone, thus ClINA being unable to inhibit Cl<'s capacity to hydrolyze S-2238. The addition of heparin did not affect the extent When of hydrolysis of S-2238 by the mixture of Cls and ClINA. protamine sulfate was added to Cli, Cls activity was inhibited, but the addition of ClINA to the mixture of ClS and protamine sulfate resulted in the same extent of inhibition of Cl; as the inhibition of Cls by ClINA in the absence of protamine sulfate. It may be possible that Cli has two sites, one interacting with both Cl INA and protamine sulfate, the other being responsible for hydrolysis of S-2238 which is not inhibited by ClINA. As to influences of polycations and anions on hemolytic activities of the complement system, polyanions inhibited both the classical and alternative pathways to the same extent, but polycations primarily inhibited the classical pathway, and the extent of the inhibition of the alternative pathway by polycation was small.

INTRODUCTION Binding of immune complexes to Cla results in activation of Clr to Cl; and further-in proteolytic'conversion of Cls to ClS by Clr. Cl: is inhibited by ClINA (1) by forming irreversible stoichiometric complex between Cl5 and Key words: Cl:, ClINA, heparin dextran sulfate, protamine sulfate

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ClINA (2, 3). Furthermore, heparin or dextral sulfate was shown to potentiate ClINA activities (4). The interaction of Cls with ClINA is peculiar because inhibition of-Cl: by ClINA depended upon substrates. Kondo et al (5) have shown that Cls hydrolyzed AAMe in the presence of ClINA while hydrolysis of TAMe was inhibited by ClINA. Cl; behaved as if it had two active sites, one interacting with ClINA, and the -other being able to hydrolyze some substrate even if ClINA already interacted with the former. The action of heparin on ClINA is also strange because heparin is shown to potentiate ClINA activity with respect to inhibition of Cl: (4), but Nagaki and Inai (6) have shown that heparin did not accelerate ClINA activity with respect to inhibition of plasmin. Since heparin accerelates antithrombin activity regardless of the use of thrombin and plasmin (7, 8), the mechanisms of the effects of heparin and dextran sulfate on ClS seem to be different from those on thrombin. In the present research we used purified Cl; and ClINA, and commercially available heparin which has accelerating capacities for antithrombin III,and tried to know effect of heparin, dextran sulfate and some polycations on interactions of Cl$.and ClINA, and on classical and alternative pathways of complement system as a whole. MATERIALS AND METHODS Polyanions: (a) Dextran sulfate (Elr3,000) was kindly provided by Kowa Shinyaku Co. Ltd. (Tokyo, Japan), (b) heparin was obtained commercially from Shimizu Seiyaku Co. Ltd. (Shizuoka, Japan). Polycations: (a) Poly-L-lysine (M 475,000) was purchased from Protein Research Foundation (Osaka, Japan), (b! protamine sulfate (M 7,500) was purchased from Sigma Co. Ltd.(!?issouri,U.S.A.), and (c) histoKe was purchased from Sigma Co. Ltd. (Missouri, U.S.A.). Human plasma was obtained from out-dated human blood. Cl: was isolated according to the method of Sakai and Stroud (9). Isolation of ClINA: One hundred ml of plasma was dialyzed against 2 liter of water at 4°C for 18 hrs and the insoluble materials, which contained Cls, were removed by centrifugation at 3,000 rpm for 10 min. Soluble fraction was applied to lysine Sepharose (3 x 6 cm) prepared according to Deutsch and Mertz (10). After plasma passed through, the column was once washed with 0.2 M phosphate buffer, pH 7.4, then eluted with the same buffer. Fig. 1 shows the profile of protein and ClINA activity. Fractions 2 to 5 were collected and dialyzed against 0.01 M It was applied to lysine Sepharose phosphate buffer, pH 7.4 for overnight. column (2 x 30 cm) and eluted initially with 0.01 M phosphate buffer, and then with liniar gradient to 0.05 M of phosphate. Fig. 2 shows the elution profile. ClINA was eluted after the gradient started. This fraction was collected and passed through Sephadex G-200 (3 x 90 cm). Fractions having ClINA activity were collected and concentrated. The concentration of ClINA was measured by radial immuno-diffusion technic.

Cl:,

Cl

INACTIVATOR

INTERACTIONS

849

FIG. 1 Elution pattern of plasma through lysine-Sepharose column (3 x 6 cm). Solid line indicates OD , and dotted line indicates tBD activity of ClINA. Plasminogen appeared after the elution with 0.2 M EACA. Each fraction contains 10 ml.

Fraction

No.

FIG. 2 Elution pattern of Fr.2 to 5 in Fig. 1 through lysineSepharose column (2 x 30 cm). Solid line indicates OD280, and dotted line indicates the activity of ClIWA. Each fraction contains 10 ml.

10

20

30

40

50

Fraction No.

Cl INA activity: The unit of Cl; was tentatively determined as 5 units, which is equal to 1012 SFU, when Cl; hydrolyzed 5 p moles of ATEe in 30 min at 37°C. 0.2 ml of Cl: containing 5 units was mixed with either 0.1 ml of ClINA (1.5 M), or 0.1 ml of two times diluted human plasma, and 0.2 ml of buffer (Tris-H1 1, pH 7.4), dextran sulfate, or heparin etc. The mixture was immediately added with 0.5 ml of ATEe (10 ~1moles), and incubated for 30 or 60 min. After the incubation, 1.5 ml of the mixture of equal volume of 2 M NH Cl-HCl and 3.5 N NaOH, and incubated for 15 min at 37'C, before 2 ml of TCkHCl and 1 ml of 10 % FeC13 was added (11). 0D530 was measured.

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Hydrolysis of chromogenic substrates (AB KABI, Stockholm, Sweden): In order to find a substrate suitable for Cl;, we compared various chromogenic substrates with respect to hydrolysis by Cls. Table 1 shows the results of experiments in which 0.2 ml (5 u) of Cl,g,0.3 ml of tris buffer (pH 7.4), and 0.5 ml of substrate (100 yg) were incubated for 60 min, then 1 ml of 50 % acetic acid solution was added to stop the reaction. OD405 of the mixture was measured. TABLE 1 Hydrolysis of chromogenic substrates by ClJ Substrates

Chemical structure

0D405

s-2222

Bz-I le-Glu-Gly-Arg-pNA

0.099

S-2444

Pro-Glu-Gly-Arg-pNA

0.269

s-2238

H-D-Phe-Pip-Arg-pNA

3.302

S-2160

Bz-Phe-Val-Arg-pNA

0.230

S-2251

H-D-Val-Leu-Lys-pNA

0.102

Assay of inhibitory effects on CH50 and AP50: AP50 was performed CH50 was performed by the method of Mayer (12). according to the method of Platts-Mills and Ishizaka (13) modified by Takada et al (14). The inhibitory effects of inhibitors on CH50 and AP50 were measured according to the method of Takada et al (14). Assay of inhibitory effects on CV50: CV50 was determined by using a modified method (15) of Brai and Osler(16). 0.2 ml of cobra venom factor (CVF) from Naja naja (20 u), 0.1 ml of serum diluted serially with EGTA-VB and 0.1 ml of inhibitor were mixed and incubated for 30 min at 37"C, then 0.2 ml of out-dated human plasma diluted 5 times with 0.1 M EDTA-VB and 0.4 ml of guinea pig erythrocytes (1.5 x 107) were added. After incubation at 37°C for 60 min, 2 ml of saline was added. OD supernatant was measured after centrifugation. This assay was des!&$a0tefdt2F CV5OI. CVSOII was done by adding inhibitor after 30 min incubation of the mixture of CVF and diluted serum. The rest of the procedure was the same as CV501. RESULTS 1. Effects of heparin on Cls and ClINA activities. Fig. 3A shows the results of experiment in which 0.2 ml of Cl: (5 u) was mixed with either 0.05 ml of human plasma or buffer in the presence or absence of heparin (1 u). Heparin slightly inhibited Cl; activity, but the addition of plasma to Cls resulted in significant decrease in Cl; activity. Further addition of heparin to the mixture of Cl: and plasma resulted in less inhibition of Cl: activity, possibly inhibiting ClINA activity in the plasma. Fig. 38 shows that,similar results were obtained by using purified ClINA.

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0.15 ml of ClINA (1 PM) were added to 5 u of Cl: in the presence or absence of heparin. Heparin slightly inhibited Cl; activity, and ClINA activity to inhibit Cl; decreased in the presence of heparin.

8

P

36 +H

. ,

, .H /

+P

15

30

Incubation time

45

60

0

(min)

15

/

,,d’

30

Incubation

FIG. 3A

,orc

45

60

time (min)

FIG. 3B

Time course of the hydrolysis of ATEe by various solutions. C, P and H indicate Cl;, plasma and heparin, respectively. Fig. 4 shows the results of experiments in which S-2238 (best substrate for Cl:) was mixed with Cl; in the presence and absence of ClINA or heparin. ClINA hardly inhibited the hydrolysis of S-2238 by the mixture of Cls and ClINA. Thus ClINA was not an inhibitor of Cl; when S-2238 was used as a substrate. We wanted to know if substrate quickly interacted with Cl; before Cl: interacted with ClINA. So ClS was mixed with ClINA for various time intervals, and ATEe was added. Fig. 5A shows that Cl5 quickly interacted with ClINA, and that 10 min after the addition of ClINA to Clf, ATEe was hardly hydrolyzed by the mixture of Cl: and ClIHA. Ye then incubated ClS and ClINA for 10 min and S-2238 was added in the presence or absence of heparin.

0D405 1

0.4 I

9

15

30

Incubation

45 time

60 (min)

Time course of the hydrolysis of S-2238 by various solutions.

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Results shown in Fig. 56 indicate that the mixture of Cl: and ClINA was still able to hydrolyze S-2238. Heparin alone slightly inhibited Cl? but rather enhanced hydrolysis of S-2238 by the miture of Cl: and ClINA.

OD40

0

Preincubation

time (min)

FIG. 5A Effect of preincubation on ATEe hydrolysis. ATEe was hydrolyzed for 60 min after preincubation of (C +ClINA).

1-S

30

4-S

6b

Incubation time (min) after 10 min preincubation FIG. 58

Time course of the hydrolysis of S-2238 in the presence or absence of heparin after 10 min preincubation of the mixture of Cl: and ClINA.

Now we compared various concentrations of heparin with respect to effects on the capacity of ClINA to inhibit the hydrolysis of ATEe by Cl:. Fig. 6A shows the results of experiments in which ATEe was hydrolyzed by ClS for 30 or 60 min in the presence or absence of plasma and heparin. Hydrolysis of ATEe by Cl3 alone in the absence of heparin was shown on the ordinate. Heparin did no_taffect on the hydrolysis of ATEe by Clg, but the addition of plasma to Cls resulted in decrease in ATEe hydrolysis at 30 and 60 min incubations (1.8 p moles at 30 min , and 2.8 u moles at 60 min, respectively). Increase in the concentrations of heparin resulted in the larger extent of hydrolysis of ATEe by the mixture of Cli and ClINA. ATEe hydrolysis by the mixture of Cls and ClINA was largest at 10 u/ml heparin concentration. Fig. 6B shows the results of similar experiments in which purified ClINA was used instead of plasma.

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h

t

C + .H (60') 4&.&J'L# C +plasma +H (60')

G f-i B

36

‘I

C+ClINA+H

(30')

‘I . 0

1

1

0

100

10

FIG.6B

FIG.6A Effects of heparin on Cls in the presence or absence of plasma.

-em-*

100

Heparin (u/ml)

Heparin (u/ml)

2. Effects

10

Effects of heparin on Cl: in the presence or absence of ClINA.

of dextran sulfate on Clf and ClINA activities. 14

-&_!16 + MDS (60')

‘-,tCls' + MDS (60') ~&----_A6-----_~---A

LL

---*+Cl:

0

0.001

+ MDS (30')

0.01 HDS

0.1

1

(mf-1)

FIG. 7A Effects of MDS on Cl: in the presence or absence of plasma.

0

0.001

0.01

0.1

1

iIDS (mfi

FIG.78 Effects of MDS on Clf in the presence or absence of ClINA.

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Fig. 7A shows effects of various amounts of monodextran sulfate (MIX) on Cli and plasma. The addition of 0.05 ml of plasma to 5 u of ClS resulted in decrease in the extent of ATEe hydrolysis by Cl:, but the addition of various amounts of dextran sulfate resulted in more ATEe hydrolysis by the mixture of Cls and plasma,At 0.01 mM of dextran sulfate, ATEe hydrolysis by Cl: was the same as that by the mixture of Cls and plasma. Fig. 7B shows the results of similar experiments in which ClINA was used instead of plasma. The presence of 0.001 mM of dextran sulfate almost completely inhibited ClINA activities. 3. Effects of protamine sulfate on Cls and ClINA activities. When protamine sulfate was added to the mixture of Cl: and plasma or ClS and ClINA. ClINA activity was hardly influenced. On the other hand, ClT activity was inhibited (Fig. BA and 88). Since ClS activity did not change much in the presence of both ClINA and protamine sulfate in spite of inhibition of Cl: by protamine sulfate alone, ClINA and protamine sulfate may bind with the same site on Cl;, and ClINA bound Cl: can not interact with protamine sulfate. .----j \ \ \ \ \ \ ',tclZ

“‘_-.

I

+ P.S.

=--a.$&

(60’)

\ t

.\

+ p.s. (30') -0, \

xasm+

\

\

\ ‘0

p.s.(60’)

I &_-00 Cl5 + p.s. (so')

0

0.1

1

10

Protamine sulfate (mM)

FIG. BA

(30')

0

Oil

1

10

Protamine sulfate (mM) FIG. 88

Effects of protamine sulfate on Cls Effects of protamine sulfate on Cl; in the presence or absence of plasma. in the presence or absence of Cl INA.

4. Effects of heparin and dextran sulfate on hemolytic activities of classical and alternative pathways.

a) Heparin Fig. 9 shows inhibition of CHSO, AP50, CVSOI and CVSOIIby heparin. Heparin most effectively inhibited CV501. CV501 mainly indicates formation of CVF dependent C3/C5 convertase. CH50 and AP50 were inhibited to an identical extent. CV501 was inhibited 100 % at 1 u/ml but CH50 and AP50 were inhibited 100 % at 100 u/ml.

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b) Dextran sulfate Dextran sulfate inhibited CV501 and CV5OII to the same extent. Inhibition on CH50 and AP50 was less than CV50. CH50 seems to be inhibited more effectively than AP50.

1

0.01

100

0.0625

Cont. of heparin (u/ml)

FIG. 10

Inhibitory effects on hemolytic activities of complement system by heparin. o---o AP50,

1

Cont. of poly-L-lysine(xlO-3pM)

FIG. 9

O---O CH50,

0.25

b--e

Inhibitory effects on hemolytic activities of complement system by poly-L-lysine. CV501,

A-

cv5oI.I

5. Effects of poly-L-lysine, protamine sulfate and histone on hemolytic activities of classical and alternative pathways. a) Poly-L-lysine Poly-L-lysine mainly inhibited CH50 (Fig. 10). 1 x 10-j p?4of poly-Llysine_&ompletely intiibitedCH50, while no inhibition of AP50 was shown at )rMof poly-L-lysine inhibited 17 % of AP50, but did not @I. lx 10 5 x 10 inhibit CVSOI and CV5OII . b) Protamine sulfate Two uM of protamine sulfate completely inhibited CH50, slightly AP50, and did not inhibit CV501. Ten pM of protamine sulfate inhibited CV501 to the extent of 20 %.

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c) Calf thymus histone Histone completely inhibited CH50 at 50 pg/ml, but AP50 was inhibited less than 40 % at 1 mg/ml. DISCUSSION The activated form of-Cl (Cl:) was shown to cleave C4 to C4a and C4b, and C2 to C2a and C2b. Cls was also shown to hydrolyze tosyl arginine methyl ester (TAM) and acetyl tyrosine ethyl ester (ATEe) (17 - 20). Gigli and Austen (21, 22) showed that inhibition of activated Cl by heating or by ClINA had no influence on the inactivation of C4, but resulted in inactivation of c2. Kondo et al (5) further showed that heating of activated Cl or its interaction with ClINA did not affect the caPacity-of Cl to hydrolyze AAMe (acetyl arginine methyl ester) but resulted n decrease in the capacities of Cl to hydrolyze TAMe. These results seem to show that Cl has two active sites one for C2 which is heat-labile and reactive with ClINA, and the other for C4 which is heat stable and non-reactive with C1INA. As to ClINA, it is known that Cl; forms an irreversible mole to mole complex with ClINA (2, 3). Schreiber et al ( 23) have shown that ClINA may have two sites, one being enzymatic and inact ivating (possibly cleaving) Hageman factor fragments and the other being a site to interact with Cl INA. Rent et al_C4) have shown that heparin enhanced ClIHA activities to inhibit the capacities of Cl to hydrolyze ATEe and to inhibit Cl hemolysis. Nagaki and Inai (6), however, showed that heparin did not affect the extent of inhibition of plasmin by ClINA when caseinolysis was used. When antithrombin III(ATIII)was used, heparin accelerated the capacities of ATIII to interact with both thrombin and plasmin (7). Furthermore dextran sulfate do not have the acceleration of ATIIIwith respect to inactivation of thrombin. Thus the effects of heparin on Cl: or Cl IIIA may be due to its negative charges and not due to specific heparin activity which is shown for ATIII.

In the present paper, we indicated that the hydrolysis of ATEe by Cl? was inhibited by Cl INA, but the hydrolysis of S-2238 by Cl: was hardly inhibited by ClINA, even after Cl: interacted with ClINA sufficiently. Furthermore heparin and dextran sulfate inhibited the activity of Cl INA to inhibit Cli's capacity to hydrolyze ATEe, but heparin hardly affected ClINA when S-2238 was used as a substrate for Cls. It appears that Cl: has two active sites, one being reactive with Cl INA and able to hydrolyze ATEe, and the other being not reactive with ClINA and being able to hydrolyze S-2238 even if the first site was occupied with Cl INA. Heparin attached to ClINA inhibited the interaction of the first site of ClS and ClINA, but did not influence the activities of the second site. Now, how these results could be compatible with the results of Rent et al (4), who indicated that heparin and other polyanions enhanced ClINA activities by using ATEe hydrolysis and Cl hemolysis? There might be two possibilities; one being due to the differences of assay systems used. Rent et al (4) used pH stat monitoring ATEe hydrolysis. If H ions generated on hydrolysis of ATEe bind more with heparin bound-ClINA, pH stat measurement will give the results indicating apparent inhibition of Cl< activities. Since heparin inhibited complement induced hemolysis at multiple sites during reaction sequences (24 - 29), it might be possible that heparin bound to EA through ClIRA could not be easily washed out and later inhibited hemolysis. ClINA was shown to bind with other proteins such as fibrin (30). Another possibility is that lots of heprin used were different. Rosenberg (31) indicated that fractionation of heparin resulted in isolation of heparin fractions

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which did not interact with ATIII. There was also an indication that some lots of heparin added to the plasma completely negated ClINA activity (S. Nagasawa, personal corrmunication). Heparin preparation which was used in the present experiments had acceleration of ATIU activities (32), and also inhibited complement systems by using hemolysis. So it could be said that some heparin preparation might inhibit ClINA activity. As to cations, Fig. 8A and 8B show that Cl; was inhibited by protanine sulfate, but ClINA neutralized inhibitory activity of protamine sulfate. The mixture of Clf and ClINA hydrolyzed ATEe to the same extent regardless of the amounts of protamine sulfate added to the mixture. It appears that a site on Cl: which interacted with protamine sulfate did not interact with ClINA, which may mean that ClINA and protamine sulfate competed for the same site on Cl:. The activities of Cl: bound with ClINA were not influenced by the presence of protamine sulfate, thus the same extent of hydrolysis of ATEe at increasing concentrations of protamine sulfate. Then we compared various polyanions and cations on hemolytic activities. of the complement systems. Heparin, dextran sulfate, and other polyanions inhibited both CH50 and AP50 to the same extent. On 59" other hand polycations such as poly-L-lysine completely inhibited CH50 at 10 PM. The same results were obtained by using protamine sulfate and histone. Thus it could be said that the alternative pathway was hardly inhibited by polycations while the classical pathway was inhibited by both polycations and polyanions. REFERENCES 1. LEVY, L.R. and LEPOW, I.H. Assay and properties of serum inhibitor of C'l-esterase. Proc. Sot. Exp. Biol. Med. lOl, 608-611, 1959. 2. NAGAKI, K., IIDA, K. and INAI, S? The inactivator of the first component of human complement (ClINA). The complex formation with the activated first component of human complement (CT) or with its subcomponents. Int. Arch. Allerg. Appl. Immunol. 46. 935-948. 1974. 3. HARPEL, P.C. and COOPER N.R. Studies on human plasma CT inactivatorenzyme interactions. I. Mechanisms of interaction with Cl:, plasmin, trypsin. J. Clin. Invest. 55, 593-604, 1975.

and

4. RENT, R., MYHRMAN, R., FIEDEL, B.A. and GEWURZ, H. Potentiation of Clesterase inhibitor activity by heparin. Clin. Exp. Imnunol. 23, 264-271, 1976. 5. KO[lO, M., GIGLI, I. and AUSTEN, K.F. Fluid phase destruction of C2hu by III.Changes in activity for synthetic substrates upon cell binding, Cl heat-inactivation and interaction with CTINH. Immunology 22, 305-318, 1972. 6. NAGAKI, K. and INAI, S. The inactivator of the first component of human Enhancement of CHINA activity against Clg by acidic complement (CTINA). mucopolysaccharides. Int. Arch. Allerg. Appl. Immunol. 50, 172-180, 1976. 7. ROSENBERG, R.D. and DAMUS, P.S. The purification and mechanism of action of human antithrombin-heparin cofactor. J. Biol. Chem. d248 6490-6505. 1973 8. HIGHSMITH, R.F. and ROSENBERG, R.D. The inhibition of human plasmin by human antithrombin-heparin cofactor. J. Biol. Chem. 299, 4335-4338, 1974.

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9. SAKAI, K. and STRDUD, R.M. of Cl proesterase, Cls. J.

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Purification, molecular properties and activation Inanunol.110, 1010-1020, 1973.

10. DEUTSCH, D.G. and MERTZ, E.T. Plasminogen: Purification from human plasma by affinity chromatography. Science l70, 1095-1096, 1970.

11. ROBERTS, P.S.

Measurement of the rate of plasmin on synthetic substrates. J. Biol. Chem. 238, 285-291, 1958.

12. MAYER, M.M. %%?.

Complement and complement fixation. In Experimental Immuno2nd edn. E.A. Kabat and M.M. Mayer (Eds), Illinois, U.S.A.: Thomas, 1961, pp. 133-240.

13. PLATTS-MILLS, T.A.E. and ISHIZAKA, K. Activation of the alternative path348-358, 1974. way of human complement by rabbit cells. J. Immunol. ll3, 14. TAKADA, Y., ARIMDTO, Y., MINEDA, H. and TAKADA, A. Inhibition of the classical and alternative pathways by aminoacids and their derivatives. Immunology 34, 509- 515, 1978. 15. TAKADA, A., IMAMURA,Y. and TAKADA, Y. Relationships between hemolytic activities of human complement system and complement components. Clin. Exp. Immunol. 35, 324-328, 1979. 16. BRAI, M. and OSLER, A.G. Stlrdiesof the C3 shunt activation in cobra venom induced lysis of unsensitized erythrocytes. Proc. Sot. Exp. Biol. Med. 14Q, 1116- 1121, 1972. 17. LEPOW, I.H., RATNDFF, O.D., ROSEN, F.S., and PILLEMER, L. Observations on a pro-esterase associated with partially purified first component of human complement (C'l). Proc. Sot. Exp. Biol. Med. 92, 32-37, 1956. 18. RATNDFF, O.D. and LEPOW, I.H. Some properties of an esterase derived from preparations of the first componenr of complement. J. Exp. Med. 106, 327343, 1957. 19. HAINES, A.L. and LEPOW, I.H. and enzymatic properties. J.

Studies on human C'l-esterase. I. Immunol. 92, 456-467, 1964.

Purification

20. HAINES, A.L. and LEPOW, I.H. Studies on human C'l-esterase. II . Function of purified C'l-esterase in the human complement system. A J Imounol. 92, 468-478, 1964. 21. GIGLI, I. and AUSTEN, K.F. Fluid phase destruction of C2hU by Clhu. I. Its enhancement and inhibition by homologous and heterologous C4. J. Exp. Med. 129, 679-696, 1969. 22. GIGLI, I. and AUSTE#* phase destruct&n of CZhU by Clhu. IL Unmasking by C4i ifFClh"$&ificity for C2"". J. Exp. Med. l$, 833-846, 1969. 23. SCHREIBER, A.D., KAPLAN, A.P. and AUSTEN K.F. Inhibition by CTINH of Hageman factor fragment activation of coagulation, fibrinolysis, and kinin generation. J. Clin. Invest. 52, 1402-1409, 1973.

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24. RAEPPLE, E., HILL, H.-U. and LOOS, M. Mode of interaction of different polyanions with the first (Cl, CT), the second (C2) and the fourth (C4) component of complement. I. Effect on fluid phase CT and on CT bound to 13 251-255, 1976. EA or to EAC4. Inxnunochemistry -’ 25. LOOS, M., VOLANAKIS, J.E. and STROUD, R.M. Mode of interaction of different polyanions with the first (Cl, CT), the second (C2) and the fourth (C4) component of complement. II. Effect of polyanions on the binding of C2 to EAC4b. Immunochemistry 13, 257-261, 1976. 26. LOOS, M., VOLANAKIS, J.E. and STROUD, R.M. Mode of interaction of different polyanions with the first (Cl, CT), the second (C2) and the fourth (C4) component of complement. III.Inhibition of C4 and C2 binding site(s) on Cls by polyanions. Immunochemistry 12, 789-791, 1976. 27. BAKER, P.J., LINT, T.F, McLEOD, B.C., BEHRENDS, C.L. and GEWURZ, H. Studies on the inhibition of C56-induced lysis (reactive lysis). VI. Modulation of C56-induced lysis by polyanions and polycations. J. Irrmunol.114, 554558, 1975. LINT, T.F., SIEGEL, J., KIES, M.W. and GEWURZ, H. Potentiation 28. BAKER, P.J., of C56-induced lvsis bv leucocvte cationic proteins, myelin basis proteins and lysine-rich histones. Imnunoloqy 30, 467-473, 1976. 29. WEILER, J.M., YURT, R.M., FEARON, D.T. and AUSTEN, K.F. Modulation of the formation of the amplification convertase of complement, C3b,Bb, by native and commercial heparin. J. Exp. Med. 147, 409-421, 1978. 30. TRUMPI-KALSHOVEN, M.M. The relevance of CT inhibitor in the inhibition of the fibrinolytic activity of plasmin. In Progress in Chemical Fibrinolysis Davidson, R.M. Rowan, M.M. Samama and P.C. and Thrombol sis Vol. 3, J.F. , New York, U.S.A .: Raven Press, 1978, pp. 257-267. d 31. ROSENBERG, R.D. and JORDAN, R.E. The neutralizarion of thrombin and other serine proteases of the hemostatic mechanism. In Chemistry and Biology of Thrombin R.L. Lundblad, J.W. Fenton,II and K.G. Mann (Eds.), Michigan, U.S.A.: Ann Arbor Science Publishers, 1977, pp. 375-393. and TAKADA, Y. Interaction of thrombin with anti32. TAKADA, A., KOIDE, T. thrombin IIIand $zmacroglobulin in the plasma. Thrombosis Res. 16, 59-68, 1979.