kinin-forming action of plasmin is thus an indirect one, mediated by thought it possible that the activity of the plasmin preparation could have

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1 J. Phy8iol. (1964), 170, pp With 6 text-ftgure Printed in Great Britain KININ FORMATION BY PLASMIN, AN INDIRECT PROCESS MEDIATED BY ACTIVATION OF KALLIKREIN BY W. VOGT From the Medizinische Forschung8anstalt, Pharmakologische Abteilung, Max Planck Gaesellschaft, GYttingen, Germany (Received 10 July 1963) It has been assumed that plasma kinins appearing during tissue damage are formed by the action of proteases such as plasmin, the activation of which accompanies such damage (Beraldo, 1950; Armstrong, Jepson, Keele & Stewart, 1957; Lewis, 1958). Also the production of kinins on contact of plasma with glass surfaces has been suggested to result from the activation of plasmin (Lewis, 1960). However, it is not generally accepted that plasmin is able to liberate kinins. Beraldo (1950) first observed that bovine plasmin released bradykinin-like material from dog serum globulin and similar results were obtained by Schachter (1956), who, however, thought it possible that the activity of the plasmin preparation could have been due to contamination with serum kallikrein. Lewis (1958) found that h an plasmin released kinin from dog pseudo-globulin. Since plasminogen was inactive as a kinin-releasing enzyme but was activated by streptokinase, an interaction of contaminating kallikrein was ruled out. In contrast, Bhoola, Calle & Schachter (1960) did not see any release of kinin on incubation of various globulins with human plasmin. Eisen & Keele (1960a) described a quick kinin-releasing effect of human plasmin which later, however, turned out to be due to a contaminating Hagemanfactor-like material (Eisen & Keele, 1960b). In the present experiments the action of human plasmin on dog plasma globulin treated in several ways and on purified kininogen has been investigated. It has been found that plasmin does, in fact, produce kinin. This action is, however, dependent on the precursor of a kinin-forming enzyme which is present in the substrate and is activated by the plasmin. The enzyme precursor is most likely identical with kallikreinogen. The kinin-forming action of plasmin is thus an indirect one, mediated by activation of kallikrein. Kinin formation by contact of plasma with glass surfaces is effected without interaction of plasmin.

2 154 W. VOGT METHODS Dog plasma globulin. The globuilins of two batches of fresh dog plasma (390 and 485 ml.) were precipitated by adding ammoniurm sulphate 0-22 g/ml. The precipitate was collected by centrifugation, dialysed for 36 hr against distilled water and concentrated in a freezedrying apparatus to a volume of 228 ml. To this suspension was added (g) NaCl 1-8; KCl 0-045; CaCl2 0045; NaH2PO40-011; NaHCO Afterthe addition of the salts fibrin precipitated; it was eliminated by centrifugation. A clear solution (200 ml.) was obtained containing 66 mg protein/ml. and 9-65 mg N/ml. The globulin solution contained kininogen equivalent to bradykinin 5.4 pg/ml. Kininogen was estimated by the method of Diniz, Carvalho, Ryan & Rocha e Silva (1961). The globulin solution was divided into portions of 10 ml. each which were kept at -20 C until used. Human peuminogen. A sample of human plasminogen was prepared from human serum essentially by the method of Ronwin (1962). This method includes heating for 20 min at 600 C, which procedure probably destroys any kallikreinogen present. The preparation contained N 20-8 %. It was kept as a stock solution of 23-6 mg/ml. in 0-01 N-HCa at -20 C. Human pla&min. One millilitre of plasminogen stock solution was neutralized with 0-3 N-NaOH and streptokinase 4000 u. (Bistreptase, Behring-Werke), dissolved in 0-1 ml. saline, was added. After standing for a few minutes at room temperature the mixture was used as 'undiluted plasmin'. On fibrin plates, 0-03 ml. of a 1: 30 dilution of this preparation produced fibrinolysis in a zone of 12 mm diameter, whereas plasminogen was inactive. Antipla8min. A preparation of antiplasmin was obtained by the method of Loomis, Ryder & George (1949). On fibrin plates the antiplasmin in a concentration of 10-' g/ml. completely inhibited the fibrinolysis by plasmin 1:30. In a tenfold lower concentration weak partial inhibition was seen. Purified kininogen. Two samples yielding respectively bradykinin 8 and 10 ug/mg kininogen were kindly supplied by Professor Habermann, Wurzburg. They had been purified from ox serum (Habermann & Rosenbusch, 1962). Kallikrein. A preparation purified from commercial Padutin (Habermann, 1961 a) was also a gift of Professor Habermann. In some early experiments a sample of pancreatic kallikrein, kindly supplied by Professor Werle, Munich, was used. Biological assay of kicnin formation. For assay isolated strips of guinea-pig ileum were used, in a bath containing 10 ml. Tyrode solution at 34-35' C. The kinin content of samples was standardized against synthetic bradykinin, obtained by courtesy of Dr Sturmer, Sandoz. The activity was expressed as Fg of bradykinin. RESULTS Action of plasmin on untreated dog globulin When 0-1 ml. of undiluted plasmin solution was incubated with 1 ml. of globulin solution, kinin activity was formed in the course of 2-10 mi, as seen by the contraction which ml. of the incubation mixture elicited in the guinea-pig ileum. After about 30 min this activity disappeared. Incubation of globulin solution with plasminogen or streptokinase or saline alone produced no such effect (Fig. 1). These results confirm those of Lewis (1958), and indicate that the kinin-forming principle in the plasmin preparation was plasmin itself. This is further borne out by the following finding. Plasma kinin formed much more slowly when plasminogen was added first to the globulin solution and the streptokinase

3 KININ FORMATION BY PLASMIN 155 was added afterwards, than when the plasminogen had been activated by addition of streptokinase before the incubation with the globulin. Apparently some time elapsed in the former case until plasmin became active. In its kinin-forming activity, 0 1 ml. of undiluted plasmin solution corresponded to about 1 unit of kallikrein (in 0 1 ml. saline), as is shown in Fig. 2. In dog globulin which had been treated with plasmin for 1 hr at 370 C no kininogen was detected by the method of idiniz et al. (1961). This indicates that the kinin formed by plasmin comes from the same source as bradykinin..c 06L ~06-0 bo E E 0*2/.10 / Time of incubation (min) Fig. 1. Kinin formation in dog globulin on incubation at 370 C with plasmin (Pl) and with saline (C). For quantitative data see text. Action of plasmin on heated globulin After heating at 560 C, dog globulin solution itself showed rather strong kinin activity. In globulin heated for 20 min plasmin was still able to form kinin in addition to that already present. Plasmin did not, however, form additional kinin in samples of globulin which had been heated to 560 C for 2 hr. These samples still formed kinin on incubation with kallikrein 1 unit/ml. In some experiments samples of globulin after heating at 560 C for 2 hr were ultrafiltered and washed with saline by ultrafiltration to eliminate the kinin which had developed during heating. These samples were then made up to the original volume with Tyrode solution and purified kininogen was added to compensate for the loss of substrate. Plasmin was not

4 156 W. VOGT 1-2. y 3 k.u. c1*0_ -o I 0 -EI.0 a, s 1 ~~~~~~1 k.u. E /0 02g k.u. Fig Time of incubation (min) Kinin formation in dog globulin by kallkrin 0 3, 1 and 3 u./ml. globulin; and by plasmin (O 1 ml./ml. globulin; Pl). able to liberate any kinin from these samples, whereas kallikrein was as active as on untreated globuilin solution. After heating globuhln at 100 C neither plasmin nor kallikrein released any kinin. Such treatment, however, provides a good substrate for kinin formation by trypsin (van Arman, 1955). Action of plasmin on puzrified kininogen A solution of 100,ug of purified kininogen in 1 ml. saline was incubated at 370 C with 0 1 ml. of undiluted plasmin. No knnformed during 30 min. The same solution liberated kinin in less than 2 -min when kallikrein was added. The inability of plasmin to liberate knnfrom purified lrininogen was not due to the bovine origin or to some chemical alteration of this substrate which might have occurred during the process of purifica-

5 KININ FORMATION BY PLASMIN 157 tion. For the same preparation of kininogen released kinin when acted upon by plasmin in the presence of dog globulin. This is shown by the following result. To 0 3 ml. of globulin solution was added 0.01 ml. of a 5 % solution of phenanthroline (to inhibit kininases, see Erd6s & Sloane, 1962), 0-5 mg of kininogen in 0-2 ml. saline (equivalent to 5,ug bradykinin) and 003 ml. of undiluted plasmin. After 20 min incubation at 370 C an activity equivalent to 7 utg bradykinin was found. In the control sample in which the kininogen solution had been replaced by saline an activity of 13,ug bradykinin developed, which originated from the kininogen contained in the globulin preparation. The surplus kinin formed in the first sample had thus been formed from the added bovine kininogen. Action of plasmin on acid-treated globulin The results described above indicated that plasmin needs some co-factor present in dog plasma globulin to liberate kinin. This co-factor is apparently destroyed by prolonged heating at 5600 and is absent from purified kininogen preparations. It seemed likely that the presumed co-factor was kallikreinogen, which would be activated to kallikrein by plasmin. Any kiniin formed by plasmin would then be the result of the action of endogenous kallikrein. That kallikrein was contained in the globulin preparation was shown by the fact that after treatment with acid the globulin 'spontaneously' formed kinin. It has been shown by Kraut, Frey & Werle (1933) that acidification of plasma activates kallikrein, and by Habermann & Okon (1961) that the 'spontaneous' formation of kinin in acid-treated globulin is due to this activation. If plasmin was able to liberate kinin only by activation of kallikrein it should have no additional kinin-releasing effect on globulin samples, the kallikrein of which had already been activated by acid. In the experiment shown in Fig. 3, pseudo-globulin solution 10 ml. was brought to ph 2-1 with 3 05 ml. of 0 3 N-HCI. After standing for half an hour the sample was titrated back to ph 7*3 with 0-3 N-NaOH. The volume was now 16 ml. Two millilitres of this acid-treated solution was incubated with 0-2 ml. of undiluted plasmin, at 370 C (sample P), another 2 ml. with 0*2 ml. saline (sample C) and a third sample of 2 ml. with 2 units of kallikrein dissolved in 0-2 ml. saline (sample K). Portions of the incubated samples were taken for kinin assay at various times. Whereas the concentrations of plasmin and kallikrein used were about equally active in untreated globulin solution (see Fig. 2), it can be seen from Fig. 3 that in the acid-treated preparation plasmin liberated kinin much more slowly than did kallikrein. In fact, the release of kinin in the sample containing plasmin was not much faster than the 'spontaneous' release in the control sample. In another experiment with acid-treated globulin there was no

6 158 W. VOGT difference at all between spontaneous formation and that occurring in the presence of plasmin. Again, kallikrein liberated kinin much more quickly. Apparently the action of kallikrein added to the globulin liberated kinin in addition to that formed by endogenous kallikrein, whereas plasmin had little or no additional effect. This result thus supports the assumption that plasmin liberates kinin indirectly by activation of endogenous kallikrein. ~0 b0/ 3 K 2 2 E 0- C P Time of incubation (min) Fig. 3. Kinin formation in acid-treated dog globulin by 1 unit kallikrein/ml. (K), plasmin (0 1 ml./ml., P) and without addition of enzymes (C). Demonstration that plasmin activates a kinin-forming enzyme in globulin A sample of 1 ml. globulin solution was activated with 0 1 ml. plasmin and the time course of kinin formation and destruction was followed by bioassay. As is seen from tracing I in Fig. 4 no kinin was formed during the first minute. Maximal activity was reached, in this experiment, after 4 min of incubation and after 60 min the activity had disappeared. To this sample was then added purified kininogen in an amount corresponding to that originally present in the globulin (equivalent to 5,ug bradykinin/ ml.). Again, kinin activity developed on incubation at 37 C. From tracing II of Fig. 4 it is seen that this second kinin formation proceeded much more quickly than the first, reaching a kinin concentration of more than half the maximum after only 30 sec. This result indicates that during

7 KININ FORMATION BY PLASMIN 159 the latent period which preceded the first kinin formation plasmin activated a kinin-forming enzyme which, being active at the beginning of the second incubation period, liberated kinin without latency. Whereas the kinin-forming action of plasmin can be prevented by antiplasmin (Lewis, 1958), the action of the kinin-forming enzyme activated 1-5o 0 e 1.0 Fv I/ C El ' Time of incubation (min) Fig. 4. Time course of kinin formation in dog globulin after incubation with plasmin (I). A latent period of about 1 min elapsed before kinin was formed. When this kinin had disappeared, after 60 min, the same sample was supplied with purified kininogen and incubated again. New kinin formed (II), this time without a latent period. by plasmin is only partly inhibited, if at all. This is shown by the experiments of Fig. 5. Plasmin, 0.1 ml., was mixed with 0*3 ml. of a 10 % antiplasmin solution and 3 ml. of globulin was added. No kinin developed on incubation at 3700 (B). Another sample of globuilin was incubated with plasmin alone. After 5 mi kinin had developed (A) which nearly disappeared after 60 min (0). After this time the antiplasmin was added

8 160 W. VOGT and, afterwards, kininogen. On incubation at 370 C new kinin formed in the course of 2 min (D), in spite of the antiplasmin present. The kinin formation seemed to have been a little less strong than in another sample which did not contain antiplasmin, but this difference was not very marked. Fig. 5. Guinea-pig ileum. A, contraction produced by 01 ml. of dog globulin incubated with plasmin (0-033 ml./ml.) for 5 min at 370 C. B, lack of kinin formation in globulin which besides plasmin contained antiplasmin (0.1 ml. 10 % solution/ml.). C, sample A, incubated for 60 min; the kinin has nearly disappeared. D, antiplasmin and kininogen were added to sample A after 60 mnm incubation, and the mixture was incubated for 2 min at 370 C. New kinin formed in spite of anti-plasmin present. Doses given at B, C, D were also 041 ml. Disappearance of kallikreinogen from globulin treated with plasmin In order to find evidence that the kinin-forming enzyme activated by plasmin was kallikrein, an experiment was set up to investigate whether kallikreinogen disappeared from the globulin after treatment with plasmin. In this experiment use was made of the property of di-isopropyl-fluorophosphate (DFP) to inactivate kallikrein irreversibly but to leave intact its inactive precursor, kallikreinogen (Habermann, 1961 b). One millilitre of globulin was incubated with 0nl ml. of plasmin for 60 min at 37 C. Then 0-01 ml. of a 1 % alcoholic solution of DFP was added and the mixture was left at room temperature for 2 hr. A control sample of normal globulin solution was treated with DFP in the same way. Both samples were then diluted 10 times with saline solution and ultrafiltered to eliminate excess DFP. The protein residue was washed twice with 9 ml.

9 KININ FORMATION BY PLASMIN 161 saline, again by ultraffitration. It was then made up to the original volume of 1 ml. by addition of saline, brought to ph 2 with 0-3 w-hol and left at this ph for 40 min. After neutralization, kininogen was added and rinin formation on incubation at 370C was measured. In the control sample (C in Fig. 6) kinin activity developed well, but in the sample treated with plasmin there was only a very weak liberation of kinin (P) E ~1.0 0 c/ ws10f/~~~~~~~~~ Time of Incubation (min) Fig. 6. Kialikreinogen content of globulin treated with plasmin (P) and of untreated globulin (C). For estimation the kailiinogen was converted to kallikrein by acid, and the kinin formation which subsequently occurred on incubation with kininogen was taken as a measure of the acid-activated kallikrein. Any kallikrein which was present before acid treatment was blocked by DFP. For further details see text. Obviously the kallikreinogen content of this sample had decreased considerably during the incubation with plasmin, so that only traces were left for subsequent activation by acid. Action of gla8s and of quartz powder on plasminogen and on dog globulin In an attempt to find out whether contact with glass or quartz would activate plasminogen to plasmin (Lewis, 1960) fibrin plates were spotted with 0-03 ml. each of human plasmin diluted 1: 30, human plasminogen 11 Physiol. 170

10 162 W. VOGT 1: 30, and plasminogen plus a little quartz powder (surface area: 9 m2/g). Whereas in the plasmin spot marked fibrinolysis was visible after 12 hr at 370 C, the plasminogen spot containing the quartz was as inactive as plasminogen alone. Similar results were obtained with a commercial preparation of bovine plasminogen (Mann). Two interesting observations were made during experiments intended to deplete dog globulin of its kallikreinogen content by contact with glass beads (0.1mm diameter) or quartz powder. Whereas in human or bovine plasma kallikreinogen is activated by such contact in the course of some minutes (Armstrong et al. 1957; Margolis, 1958; and author's own experiments) and the kallikrein thus formed is inactivated soon after activation (Frey, Kraut & Werle, 1950), neither event happens in dog globulin solution. Inability of glass to activate dog plasma has already been observed by Armstrong, Jepson, Keele & Stewart (1955). In the present experiments there was no marked loss of kininogen nor kallikreinogen, even after treatment of dog globulin solution for 5 hr at 370 C with glass beads coated with Hageman factor, according to the experiments of Margolis (1960). Further, when active kallikrein was added to dog globulin solution it remained active during more than 1 hr incubation at 370 C. Only after prolonged incubation for about 24 hr did dog globulin lose its total kininogen content and part of the kallikreinogen and kallikrein, indicating some kallikrein activation and subsequent inactivation. This slow process was, however, not dependent on extensive contact with glass. DISCUSSION The results show that human plasmin when acting upon dog plasma globulin, or on bovine kininogen dissolved in dog globulin solution, forms plasma kinin. This confirms the work of Lewis (1958). Not only was the appearance of kinin demonstrated but also the concomitant disappearance of kininogen. The failure of Bhoola et al. (1960) to find kinin formation by plasmin in unheated globuilin preparations can probably be explained by the fact that these authors added the plasmin and the globulin separately to the organ bath used for assay. While the nearly immediate formation of kinin by high doses of kallikrein can be measured in this way, plasmin acts only after a latent period. The time allowed in the organ bath was probably too short for plasmin to act. Eisen & Keele (1960b) were also dealing with a quick liberation of kinin induced by their plasmin preparation. While they found this effect to be due to some contaminating factor the plasmin itself might have had a slower kinin-forming action. On the other hand, the formation of kinin by plasmin need not be quite as slow as was described by Lewis (1958). In the present experiments

11 KININ FORMATION BY PLASMIN 163 plasmin, when activated by streptokinase well before addition to the substrate, formed kinin even after 2 min and maximal activity was reached after about 8-12 min, in some cases even after 5rnin at 370 C. From the figures shown by Lewis it is seen that some kinin was released after 5 min incubation with plasmin at 340 C. On the basis of slow or quick kinin formation a classification has been made between kallikrein-like and plasmin-like kinin-forming enzymes (Lewis, 1959). The time course of kinin formation in a globulin preparation is, however, not necessarily specific for a given enzyme. It is even not only dependent on the concentration of enzyme added to or activated in the substrate but, not less important, also on the concentration of active kininase present in the incubate. At higher concentrations of kininase maximal kinin activity will be lower but will be reached earlier than at lower concentrations, under otherwise identical conditions. In a system as complex as globulin solution or organ extracts, tissue fluids and crude enzyme preparations the time course of kinin formation and destruction will not give a safe indication as to the nature of the releasing enzyme. Plasmin is only able to form plasma kinin if a co-factor, probably identical with kallikreinogen, is present in the incubation mixture. Thus, pure kininogen cannot be used as a substrate for plasmin and globulin heated at 560 C is also unsuitable. By this treatment kallikreinogen is destroyed. The co-factor in dog globulin is converted by plasmin to the kinin-releasing enzyme proper, probably identical with kallikrein. When the endogenous kallikrein in dog globulin has already been activated by treatment of the globujlin with acid there is no co-factor left for activation by plasmin, and consequently plasmin has no kinin-releasing effect in addition to the release induced by the acid-activated kallikrein. In contrast to the action of plasmin, which can be prevented by antiplasmin, the release of kinin by the kallikrein-like activated enzyme is inhibited only partially, if at all, by antiplasmin. Whereas the results discussed above clearly show that plasmin releases kinin only indirectly, by activating a kinin-forming enzyme, it seems difficult to prove the identity of this enzyme with serum kallikrein in such small volumes of the complex incubation mixtures as have been used. To find evidence for this another approach was tried. If plasmin would activate kallikrein, the precursor kallikreinogen should disappear. This, in fact, has been demonstrated. Only traces of kallikreinogen were left after incubation of globulin with plasmin. Dog plasma globulin is a particularly good substrate for kinin formation by plasmin, not only because of the absence of antiplasmin (Lewis, 1958). It is suitable also because kallikrein, once activated, remains active for a

12 164 W. VOGT long time, unlike the behaviour in, for example, human plasma. Also pancreatic kallikrein is not inactivated by dog globulin in 1 hr at 370 C. A further peculiarity of dog globulin is that its kallikreinogen is not activated by contact with glass or quartz, nor even by glass coated with Hageman factor. Contact with glass does not activate purified human plasminogen. Though this does not exclude the possibility that in whole plasma plasminogen is activated by glass, by interaction of other factors, it is unlikely that plasmin activation is involved in kinin formation after contact with glass. Bovine plasma on contact with glass forms strong kinin activity in a few minutes but no kinin formation is seen in the same time after addition of plasmin. The assumption that plasmin, activated during inflammation or other types of tissue injury, may cause the release of active peptides (Beraldo, 1950; Armstrong et al. 1957; Lewis, 1958), which has been doubtful for some time, can now be maintained. However, plasmin should be regarded as an activator of kallikrein, not as a kinin-forming enzyme itself, kinin formation being a secondary step of plasmin activation only. This twostep process would make it possible that plasmin when activated at a locus free of antiplasmin (e.g. in a thrombus) might in turn activate kallikrein which after diffusion out of this locus would liberate kinin even in the circulating blood, in the presence of antiplasmin. There are a few hints in the literature that plasmin may activate serum kallikrein. Webster & Pierce (1960) found that a small quantity of kallikrein was activated by streptokinase in human plasma. Also Back, Guth & Munson (1963) recently stated that 'experiments in progress strongly suggest that kallikrein release is involved in plasmin hypotension'. Though these experiments do not deal with kinin formation by plasmin, they are in accord with our findings. It seems possible that activation of kallikrein is not only involved in plasmin hypotension, as one of several mechanisms, but is the only mechanism by which plasmin produces hypotension as it seems to be the only way by which plasmin forms kimin. According to recent investigations by Eisen (1963), kinin formation can be activated in human plasma with only little activation of fibrinolysis, e.g. by contact with glass. Vice versa, streptokinase activates fibrinolysis more than kinin formation. This led to the conclusion that the fibrinolytic system and the kallikrein system are independent kininforming systems, the latter being much the more potent. These findings could also be explained by applying the conclusions drawn from my experiments. Though kallikrein-induced kinin formation is independent of the fibrinolytic system, for the latter to form kinin activable kallikreinogen is indispensable. The finding of Eisen (1963) that, after activa-

13 KININ FORMATION BY PLASMIN 165 tion of plasmin in human plasma by streptokinase, contact with glass will still increase kinin formation may indicate only that in human plasma activation of kallikrein by plasmin is incomplete or slow. SUMMARY 1. Human plasmin liberates plasma kinin from normal dog plasma globulin but not from globulin heated at 560 C. Also plasmin does not release kinin from pure bovine kininogen, though this substrate is attacked by plasmin if dissolved in globulin solution. 2. Dog globulin contains a heat-labile co-factor which is essential for plasmin to release kinin. This co-factor is probably identical with serum kallikreinogen. The kallikreinogen is converted to kallikrein by plasmin. Plasmin liberates kinin only by this indirect mechanism. After incubation of globulin with plasmin there is a considerable decrease in the concentration of kallikreinogen. 3. Kinin formation by contact of plasma with glass is not dependent on activation of plasmin. 4. Kinin formation in dog globulin cannot be induced by contact of the globulin with glass nor with human Hageman factor, at least not in the course of several hours. I wish to thank Miss G. Schmidt and Miss 0. Niklas for skilful assistance and Miss G. John for the determinations of kininogen. My thanks are also due to Professor E. Habermann, Wurzburg, Professor E. Werle, Munich and Dr E. Sturmer, Basle, for kindly supplying preparations of kallikrein, kininogen and synthetic bradykinin. REFERENCES ARMSTRONG, D., JEPSON, J. B., KEELE, C. A. & STEWART, J. W. (1957). Pain-producing substance in human inflammatory exudates and plasma. J. Phyaiol. 135, ARMSTRONG, D., JEPSON, J. B., KEELE, C. A. & STEWART, J. W. (1955). Activation by glass of pharmacologically active agents in blood of various species. J. Physiol. 129, P. BACK, N., GUTH, P. S. & MuJNsoN, A. E. (1963). On the relationship between plasmin and kinin. Ann. N.Y. Acad. Sci. 104, BERATDO, W. T. (1950). Formation of bradykinin in anaphylactic and peptone shock. Amwr. J. Phy8iol. 163, BHOOLA, K. D., CAi, J. D. & ScETcEiR, M. (1960). The effect of bradykinin, serum kallikrein and other endogenous substances on capillary permeability in the guinea-pig. J. Phy8iol. 152, Dimz, C. R., CARVALHo, I. F., RYAN, J. & RocHA E SnvA, M. (1961). A micromethod for the determination of bradykininogen in blood plasma. Nature, Lond., 192, EisEN, V. (1963). Kinin formation and fibrinolysis in human plasma. J. Phy8iol. 166, EIsEN, V. & KEELE, C. A. (1960a). The formation of plasma kinin by the action of human plasmin on human plasma. J. Physiol. 150, P. EiSEN, V. & KEELE, C. A. (1960b). Intrinsic plasma kinin-forming factors in human plasma. J. Phy8iol. 154, P. ERD6s, E. G. & SLOANE, E. M. (1962). An enzyme in human blood plasma that inactivates bradykinin and kallidin. Biochem. Pharmaol. 11,

14 166 W. VOGT FREY, E. K., KRAUT, H. & WFaurx, E. (1950). KaUlikrein Padutin. Stuttgart. Ente Verlag. HA Ls[Ax, E. (1961 a). Trennung und Reinigung von Pankreas-kallikreinen. Hoppe- Seyl. Z. 328, HT&AERmANN, E. (1961 b). Unterscheidung von Kallikrein und Kallikreinvorstufe mittels Antiseren und Diisopropylfluorophosphats. Hoppe-Seyl. Z. 328, HABERXMNN, E. & OKON, M. E. (1961). Identifizierung eines 'spontan' aus Serumglobin entstehenden Peptids mit Bradykinin. Hoppe-Seyl. Z. 326, HABEEMANN, E. & ROSENBUSCH, G. (1962). Reinigung und Spaltung von Kininogen aus Rinderblut. Arch. exp. Path. Pharmak. 243, KRAUT, H., FREy, E. K. & WERIu, E. (1933). tyber den Nachweis und das Vorkommmen des Kallikeins im Blut. VIII. Mitteilung. Hoppe-Seyl. Z. 222, LEWIS, G. P. (1958). Formation of plasma kinins by plasmin. J. Physiol. 140, LEwis, G. P. (1959). Plasma-kinin-forming enzymes in body fluids and tissues. J. Physiol. 147, LEWIS, G. P. (1960). Active polypeptides derived from plasma protein. Physiol. Rev. 40, Looms, E. C., RYDER, A. & GEORGE, C. (1949). Fibrinolysin and antifibrinolysin: biochemical concentration of antifibrinolysin. Arch. Biochem. Biophys. 20, MARGoLis, J. (1958). Activation of plasma by contact with glass: evidence for a common reaction which releases plasma kinin and initiates coagulation. J. Physsol. 144, MA GoLIs, J. (1960). The mode of action of Hageman factor in the release of plasma kinin. J. Physiol. 151, RoNwn, E. (1962). Properties of human plasmin. Canad. J. Biochem. Physiol. 40, SCHAcETER, M. (1956). A delayed slow-contracting effect of serum and plasma due to the release of a substance resembling kallidin and bradykinin. Brit. J. Pharmacol. 11, va AAN, C. G. (1955). In: Polypeptides which Stimulate Plain Muscle. Edinburgh. Livingston. WEBSTER, M. E. & PIERcE, J. V. (1960). Studies on plasma kallikrein and its relationship to plasmin. J. Pharmacol. 130,