Intrinsic plasma kinin formation in man, dog and rat

EUROPEAN JOURNAL OF PHARMACOLOGY 12 (1970) 359-364. NORTH-HOLLAND PUBLISHING COMPANY

INTRINSIC PLASMA KININ FORMATION

IN MAN, DOG AND RAT

A. J. COLLINS *, V. EISEN and Kitty L. A. GLANVILLE Rheumatology Research Department, The Middlesex Hospital Medical School, London, W1P 9PG, England

Received 26 May 1970

Accepted 29 June 1970

A.J. COLLINS, V. EISEN and K.L.A. GLANVILLE, Intrinsic plasma kinin formation in man, dog and rat, European J. Pharmacol. 12 (1970) 359-364. An attempt was made to confirm the postulated existence in human plasma (Vogt, 1966) of a kinin-forming system I (activated by acetone) and a kinin-forming System II (activated by glass). Human plasma kininogenases activated in solution either by glass or by acetone, formed in dog plasma (claimed to contain only system I) more kinin than in rat plasma (claimed to contain only system II). When adsorbed however, glass-activated human, as well as dog and rat kininogenases were more effective on rat than on dog substrate. Adsorption on to activating surfaces may change the enzymic properties of plasma kininogenases.

Kin~ formation Kininogenases

1. INTRODUCTION Several recent studies (Pierce, 1968) have provided biochemical evidence that human plasma contains two kinin-yielding substrates (kininogens). Jacobsen and Kriz (1967) described a large-molecular kininogen S~ which interacted with the specific kininforming enzymes (kininogenases or kallikreins) from plasma and from tissues, and a small molecular kininogen $2 responding to tissue kallikreins only. The findings agreed with earlier views put forward by Margolis and Bishop (1963). Chromatographic and electrophoretic fractionations also suggested the presence in human serum of two or more plasma kallikreins (Colman, Mattler and Sherry, 1969; Movat et al., 1969; Eisen and Glanville, 1969), and of several enzymic activators of these kallikreins (Webster, 1968; Eisen and Glanville, 1969); all these enzymes normally circulate as pre* Present address: School of Pharmacy, Bath University of Technology, Bath, BA2 7AY, En~and.

Kallikreins Kininogens

active precursors. Possible differences in the actions of the chemically separated kallikreins on kininogens S~ and $2 were not investigated in these studies. However, differences in the substrate specificities of two human plasma kininogenases have recently been postulated on the basis o f functional analyses of intrinsic plasma kinin formation (Vogt, 1966). According to these views (fig. 1) glass and other surfaces Factor XII Glass

etCFact!r XIIa

Trypain~. [ Ktninogenase II ,~',..=Z..=- KAnlnogenaseIIa Kintnogenase I (plasma prekaUikreLn)

J K i n i nogenase la ( ~ ) l a s m a kalltkrein)

Kintnogen I

YAninogenII o rdn

P KLnin

l

SaUvary and pancreatic kanikrein

Fig. 1. Kinin formation in human plasma according to Vogt (1966). The suffix a after the name of a plasma enzyme denotes its active form.

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A.J. Collins et al., Kinin formation in man, dog and rat

initiate plasma kinin formation in human plasma by activating clotting factor XII (Hageman factor) which in turn activates the precursor of a kininogenase II; the active kininogenase II releases kinin from a kininogen II, and also activates the precursor of a kininogenase 1 (identical with plasma kallikrein); kininogenase I is very labile and acts on kininogen I only briefly. Treatment of plasma with acetone or acid mainly activates the precursor of kininogenase I. Kininogenase II does not attack kininogen I directly, whilst kininogenase I acts only slightly on kininogen II. Pancreatic and salivary kallikreins interact only with kininogen I. It is clear that Vogt's (1966) concepts of kininogens I and II differ from those of Jacobsen and Kriz (1967). Vogt's hypothesis was supported by experiments with animal plasmas. On the basis of reports that it does not form kinin when exposed to glass (Armstrong et al., 1955), dog plasma was considered to contain only kininogenase I and kininogen I. Rat plasma responded to glass, but not to pancreatic kallikrein (Fasciolo and Halvorsen, 1964), which was interpreted as evidence that only kininogenase II and kininogen II were present. The present study examines the possibilities of differentiating human plasma kallikreins by their actions on rat and dog kininogens.

separated, thoroughly washed with cold saline, and then mixed with heated substrate plasma. Since this substrate does not respond to clean glass, observed kinin formation was due to the kininogenase adsorbed on to the glass. When required, the adsorbed enzyme was eluted with 1 M NaC1 buffered at pH 8.0. Salts were removed from eluates by passage through Sephadex G-25. Human kininogenase I1 was prepared as described by Vogt and Wawretschek (1968). The method is derived from the 'B-depletion' used by Margolis and Bishop (1963): plasma was mixed with glass powder (0.1 g/ml) for 10min, then separated and left at room temperature for 1 hr. The remaining prekininogenase was activated by renewed glass contact for 3 - 4 min. Kinin formation in activated plasma and in enzyme-substrate mixtures was measured on the isolated rat uterus suspended in a 5 ml organ bath. Contractions were recorded with an iso- or auxotonic writing lever (Eisen, 1963). Immediately before an aliquot containing trypsin was tested, it was mixed with soya bean trypsin inhibitor (SBT1; 1 mg/ml) so as to prevent sensitization of the uterus by trypsin (Edery, 1964).

3. RESULTS 2. MATERIALS AND METHODS The following enzyme preparations were used: human plasma kallikrein activated by acetone and purified according to Webster and Pierce (1961) or Moriya, Yamazaki and Fukushima (1965); hog pancreatic kallikrein (Bayer, Leverkusen); bovine trypsin (Boehringer Corp., London). Human, dog and rat blood was collected with plastic equipment avoiding contact with glass. To destroy plasma kinin forming enzymes and their activators, without destroying kininogen, heparinized plasma was heated at 60°C for 60 min (Eisen, 1963). Glass activation of fresh plasma was carried out by mixing plasma with clean glass powder (0.16 g/ml), and testing the supernatant. Active kinin-forming plasma kininogenase adsorbed on to glass was obtained by immersing glass powder in plasma for 15 min. The powder was then

Eisen and Glanville (1969) obtained two peaks of kininogenase activity by chromatography of human serum on DEAE-cellulose: the first was found in the 7-globulins and was probably identical with plasma kallikrein; the second was more anionic and migrated like a j3-globulin. If these two peaks corresponded to the kininogenases I and II postulated by Vogt (1966), the first should release kinin from dog, but not from rat plasma kininogen, and the second from rat but not from dog plasma kininogen. However, both peaks formed more kinin in heated dog plasma than in heated rat plasma. Thus the second peak did not correspond to kininogenase II as defined by Vogt (1966). The failure to differentiate in this way two chromatographically separated plasma kininogenases prompted us to study more fully the responses of human, dog and rat kininogens to kallikreins of their own and of other species. First, the actions of

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A.J.Collins et al., Kinin formation in man, dog and rat <

kallikrein not on rat kininogen. To test this, glass powder was immersed in fresh plasma of one species for 15 min; the powder - coated with adsorbed factor XII and plasma kallikrein (Margolis, 1960; Eisen, 1963) - was separated, washed with saline, and then mixed with heated substrate plasma of the desired species. Glass-activated plasma kallikrein of each species released kinin from kininogen of all three species (fig. 2). The human enzyme formed the largest amount in kininogens of all three species. In human and rat kininogen, rat kallikrein formed slightly more kinin than did dog kallikrein; both enzymes produced similar amounts of kinin in dog kininogen. Results of a typical experiment are shown in fig. 3.

glass-activated plasma kallikreins were examined. As expected, clean glass induced kinin formation in fresh human and rat plasma. Contrary to earlier reports (Armstrong et al., 1955) exposure to glass formed about 0.5 nmole in 1 ml o f dog plasma, which was less than 10 percent of the kinin formed in this plasma by hog pancreatic kallikrein or trypsin. In 1 ml of rat plasma, glass released approximately 0 . 5 - 0 . 7 nmole o f bradykinin, which was o f the same order as the amount produced by trypsin. As reported by Fasciolo and Halvorsen (1964), hog pancreatic kallikrein produced only traces of kinin in rat plasma. Vogt's (1966) hypothesis implied that rat plasma kallikrein should not act on dog kininogen, and dog

CLEAN

HUMAN

DOG

RAT

GLASS

KININOGENASE

KININOGENASE

KININOGENASE

BK

(10"9M) /

1.0 0.5

/

J

0.25

f J

I~

i

j t

BK

I

KININOGEN:

GA D

R

H

'

GA D

R

H

'

GA D

R

H

D

R

H

Fig. 2. Rat uterus. Bk = responses to 0.25, 0.5 and 1.0 X 10.9 M bradykinin (recorded at slower speed). GA = fresh heparinized human, dog or rat plasma (twice diluted) was mixed with glass powder (0.6 g/ml), and 0.1 ml of the supcrnatant tested after 5 min. After 15 min mixing the powder was separatexl, washed with 3 X 15 ml of saline, and then mixed with 1 part of heated dog (D), rat (R), or human (H) plasma plus 1 part of Locke's solution, so that the quantity of enzyme-coated powder was 0.6 g]ml; 0.15 ml of the supernatant was tested after 10 rain. Note that the kininogen in heated dog (D), rat (R) and human (H) plasma responded to glass-adsorbed kininogenases of all three species, but not to clean glass.

362

A.J.Collins et al., Kinin formation in man, dog and rat SUBSTRATE

HEATED PLASMA 160t)/80 i n i l }

KININ F O R M A T I O N DOG

7.0

MAN (M)

DOG (O)

HAT (a' 6.8

0.6

6.0 0.5 :Z 5.8 1.0

~o.~ ~,

IN HEATED PLASMA RAT

I

0.8

o.3 m m

!

0.6

f,, 0.4 Ol

0.2 M

R

D

M SOURCE

R OF

D

M

R

D

ENZYME

1

Fig. 3. Kinin-forming enzyme was adsorbed on to glass powder as in fig. 2, and then mixed with heated plasma in presence of disodium edetate 1 mg/ml. Final kinin levels were obtained by testing aliquots until no further increase in kinin concentrations occurred.

3.1. Experiments with 'kininogenase H ' Vogt (1966) and Vogt and Wawretschek (1968) have postulated that during extensive glass treatment of plasma all pre-kininogenase I (pre-kallikrein) is first activated, and then inactivated so rapidly that only little kininogen I is consumed. Only a small part of pre-kininogenase II is activated, but it survives much longer so that all kininogen II is digested. When the activated kininogenase II decays, plasma depleted by glass still contains the bulk of pre-kininogenase II and of kininogen I. Renewed brief contact with glass therefore generates active 'kininogenase II', but no kinin because no kininogen II is available (cf. fig. 1). According to the hypothesis, the active 'kininogenase II' should release kinin from rat, but not from dog kininogen. When 'kininogenase II' was prepared in this way, kinin-forming activity was detected in solution as well as adsorbed on to the activating glass powder. The adsorbed enzyme formed in most, but not all experiments more kinin in heated rat plasma than in heated dog plasma (fig. 4). The soluble enzyme formed in dog plasma as much or more kinin than in rat plasma

2

3

4

5

6

I

2

3

4

5

6

Fig. 4. Heated dog or rat plasma was activated in presence of disodium edetate 1 mg/ml until no further kinin was formed. The activating enzymes were: 1) human plasma kaUikrein activated and adsorbed by glass powder; 2) glass-activated human plasma kallikrein in solution (supernatant of 1); 3) human plasma kallikrein adsorbed on to glass (see No.l), eluted and desalted; 4) acetone-activated human plasma kallikrein (5 KU/ml); 5) hog pancreatic kallikrein (10 KU/ ml); 6) trypsin (0.5 mg/ml).

(fig. 4: 2). Thus, the action of the adsorbed human kallikrein on dog and rat kininogen corresponded to the pattern postulated for 'kininogenase II', whilst the enzyme in solution resembled in its actions acetone-activated kallikrein. This finding possibly suggested that the adsorbed enzyme was a distinct 'kininogenase II'. Another possible interpretation was that adsorption altered the character of kallikrein. To clarify this question, human kallikrein adsorbed on to glass powder or celite was eluted with M NaC1 buffered at pH 8, and then desalted. The eluate formed somewhat more kinin in dog plasma than in rat plasma (fig. 4: 3), and thus resembled human plasma kallikrein in solution, activated by glass (fig. 4: 2) or by acetone (fig. 4: 4). The adsorbed kallikreins of all three species formed in rat plasma similar amounts of kinin as did trypsin (fig. 3; fig. 4: 1 and 6), and more kinin than did any of the other tested kallikreins.

A.J.Collins et aL, Kinin formation in man, dog and rat

4. DISCUSSION The dog plasma which responded to surfaces and interacted with rat plasma, was obtained from greyhounds. It is not known whether the previously reported inability of dog plasma to produce kinins when exposed to glass (Armstrong et al., 1955) was due to the use of different strains or of smaller glass surfaces. The present results are in good agreement with the finding that dog plasma contains factor XII (Ratnoff, 1966), and a pre-kininogenase which is activated by highly purified bovine factor XII (Temme, Jahrreiss, Habermann and Zilliken, 1969). Rocha e Silva, Reis and Ferreira (1967) found that the kinin formation induced by glass in 1 ml of dog plasma was equivalent to about 0.3 nmole of bradykinin. The kinin levels formed in the present experiments in rat plasma by trypsin and by glass were about 50 percent lower than those found by Rothschild (1967). Both studies showed that glass-activated kallikrein and trypsin released similar amounts of kinin from fresh rat plasma. In contrast, Rocha e Silva et al. (1967) reported that trypsin formed in 1 ml of fresh rat plasma 4 gg, and glass 1 #g. In the present study, SBTI was added before any mixture containing trypsin was tested on the rat uterus, so as to avoid the potent sensitization of this organ, which trypsin produces (Edery, 1964); this may partly account for the lower levels found. In rat plasma denatured by boiling in acetic acid, trypsin produced much higher levels ( 3 - 4 X 10-6M bradykinin) in agreement with earlier reports by Diniz and Carvalho (1963) and by Rothschild (1967). The postulated greater effect on rat than on dog plasma was displayed by the human 'kininogenase II' preparation only when it was adsorbed on to glass or celite. When eluted, this enzyme resembled the plasma kallikrein activated in solution by glass or acetone. Adsorption may conceivably enhance the action of human kallikrein on heated rat plasma, possibly by protecting it from inhibitors, or by altering its tertiary structure. This possibility is supported by the Finding that adsorbed rat and dog kallikrein also formed large amounts of kinin in heated rat plasma. Adsorption on to surfaces has been claimed to affect other plasma proteases in similar manner, as for example plasmin (Sherry, Fletcher and

363

Alkjaersig, 1959), and clotting factor XII (Margolis, 1963; Donaldson and Ratnoff, 1965). However, the present results do not fully exclude the possibility that the kallikrein adsorbed on to glass differs functionally from the kallikrein which is activated when plasma is treated with acetone or acid. In conclusion, the present findings suggest that the kinin forming systems in dog and rat plasma are not so different as to be incapable of interacting with each other. It seems therefore that tests with dog and rat plasma can make only a limited contribution to the differentiation of human kininogenases. This was evident in the comparison of glass and acetone activated human plasma kininogenase. The experiments did not reveal a clear difference, but suggested that adsorption on to surfaces may change the enzyme characteristics of plasma kallikreins.

ACKNOWLEDGEMENTS A.J.C. received generous support from the Arthritis and Rheumatism Council, and V.E. and K.L.A.G. from the Wellcome Trust. We are grateful to Professor C.A. Keele for valuable suggestions and comments; to Dr. M.E. Webster for human serum kaUikrein; and to The Bayer Company (Leverkusen) for hog pancreatic kallikrein. REFERENCKS Armstrong, D., J.B. Jepson, C.A. Keele, J.W. Stewart, 1955, Activation by glass of pharmacologically active agents in blood of various species, J. Physiol. 129, 80P. Colman, R.W., L. Matfler and S. Sherry, 1969, Studies on the prekallikrein (kaUikreinogen) - kallikrein enzyme system of human plasma. 1. Isolation and purification of plasma kaUikreins, J. Clin. Invest. 48, 11. Diniz, C.R. and I.F. Carvalho, 1963, A micro-method for determination of bradykininogen under several conditions, Ann. N.Y. Acad. Sci. 104, 77. Donaldson, V.H.O.D.Ratnoff, 1965, Hageman factor: Alterations in physical properties during activation, Science 150, 754. Edery, H., 1964, Potentiation of the action of bradykinin on smooth muscle by chymotrypsin, chymotrypsinogen and trylisin, Brit. J. Pharmacol. 22, 371. Eisen, V., 1963, Observations on intrinsic kinin-forming factors in human plasma; the effect of acid, acetone, chloroform, heat and euglobulin separation on kinin formation, J. Physiol. 166,496. Eisen, J.C. and K.L.A. Glanville, 1969, Separation of kinin-

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A.J.Collins et al., Kinin f o r m a t i o n in man, dog and rat

forming factors in human plasma, Brit. J. Exptl. Pathol. 50, 427. Fasciolo, J.C. and K. Halvorsen, 1964, Specificity of mammalian kallidinogen, Am. J. Physiol. 207,901. Jacobsen, S. and M. Kriz, 1967, Some data on two purified kininogens from human plasma, Brit. J. Pharmacol. 29, 25. Margolis, J., 1960, The mode of action of Hageman factor in the release of plasma kinins, J. Physiol. 15 l, 238. Margolis, J. and E.A. Bishop, 1963, Studies on plasma kinins. 1. The composition of the kininogen complex, Australian J. Exptl. Biol. Med. Sci. 41,293. Movat, H.Z., M.P. Treloar, C.E. Burrowes and Y.Takeuchi, 1969, Isolation and partial purification of two kininogenases and of permeability factors from plasma. Pharmacol. Res. Commun. 1,176. Pierce, J.V., 1968, Structural features of plasma kinins and kininogens. Federation Proc. 27, 52. Ratnoff, O.D., 1966, The biology and pathology of the initial stages of blood coagulation, Progr. Hematol. 5,204. Rocha e Silva, M., M.L. Reis and S.H. Ferreira, 1967, Release

of kinins from fresh plasma under varying experimental conditions, Biochem. Pharmacol. 16,1665. Rothschild, M., 1968, Some pharmacodynamic properties of cellulose sulphate, a kininogen depleting agent in the rat, Brit. J. Pharmacol. 33,501. Sherry, S., A.P. Fletcher and N. Alkjaersig, 1959, Fibrinolysis and fibrinolytic activity in man, Physiol. Rev. 39, 343. Temme, H., R. Jahreiss, E. Habermann and F. Zilliken, 1969, Aktivierung yon Gerinnungs- und Kininsystem durch eine Plasmaesterase (Hageman Factor). Reinigung und Wirkungsbedingungen, Z. Physiol. Chem. 350, 519. Vogt, W., 1966, Demontration of the presence of two separate kinin-forming systems in human and other plasma, in: Hypotensive Peptides, eds. E.G. Erdos, N. Back, F. Sicuteri and A.F. Wilde (Springer, New York) p. 185. Vogt, W. and W. Wawretschek, 1968, Weitere Untersuchungen zur Existenz zweier Kininbildender Systeme in mensehlichem Plasma, Arch. Pharmakol. Exptl. Pathol. 256, 127. Webster, M.E., 1968, Human plasma kallikrein, its activation and pathological role. Federation Proc. 27, 84.