ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 10, No. 3 Copyright (£) 1980, Institute for Clinical Science, Inc.

Patho-Physiology of Kallikrein System

ROBERT W. COLMAN, M.D.

Thrombosis Center and Hematology ¡Oncology, Section of the Department of Medicine, Temple University School of Medicine, Philadelphia, PA 19140

ABSTRACT

The properties of the contact factors, factor XII, high molecular weight kininogen and prekallikrein are described as well as the abnormalities in the hereditary deficiencies of these proteins. The interactions of each of these proteins with the other as well as their regulation by plasma proteolytic inhibitors such as Cl inhibitor and III are delineated. Biochemical techniques for measuring this system are discussed. Condi­ tions associated with abnormal synthesis of these proteins are described. Diseases in which increased kinin formation has been documented as well as disorders where there is strong evidence for the activation of kallikrein are presented. Further knowledge of this system should increase our under­ standing of its pathophysiological alterations.

Introduction illustrates the various control mechanism possible in proteolysis, including activa­ The plasma proteolytic enzyme systems tion of inactive precursors, positive feed­ of coagulation, fibrinolysis and kinin for­ back, stochiometric inhibition, multistep mation have been categorized as a “tan­ amplification and enzymatic degradation gled web.” The three interlocking net­ of active products. works are linked together in two ways. Factor XII (a beta ), molecular First, the initiating pathways are common weight 90,000, is first converted to an ac­ and factor XII (Hageman) factor is a pro­ tive derivative of the same size, factor tein intimately involved in all. Second, at Xlla, upon exposure to negatively least four inhibitors with defined specific­ charged foreign surfaces which in vivo is ity each have the capacity to regulate more the subendothelial basement membrane. than one system. The current understand­ The importance of factor XII for initiation ing of the three interlocking networks that of the various plasma proteolytic systems mediate the vascular responses necessary is emphasized by the fact that Hageman for hemostasis, fibrinolysis and vasodila­ factor deficient plasm a not only has a very tion (kinin forming system) are displayed prolonged partial thromboplastin time in figure 1. The working of these enzymes (PTT), which measures the steps to the 220 0091-7370/80/0500-0220 $01.20 © Institute for Clinical Science, Inc. PATHO-PHYSIOLOGY OF KALLIKREIN SYSTEM 2 2 1 formation of thrombin, but it also fails to support surface initiated fibrinolysis as Bradykinn well as the generation of bradykinin.

These pathways have been shown to be mediated by the various Hageman factor substrates. I Plasminogli

Activation of Proteins

Activation of other proteins is required for the coagulation pathway to proceed. Hageman factor activation of prekalli- 1I------Prothrombinl—^ iSlPL.Ca ...... s- krein generates the enzyme kallikrein which digests kininogen to liberate the FIG U R E 1. Participation of components of the vasoactive peptide bradykinin. However, kallikrein-kinin system in coagulation and fibrinoly­ sis. Boxed components represent precursors. factor Xlla, though a potent coagulant, has Dashed arrows represent transformation of precur­ relatively less activity in initiating kalli­ sor to product. Solid arrows are actions of a proteoly­ krein or plasmin formation. To perform tic enzyme. Dotted arrows are nonproteolytic activa­ these latter functions effectively, factor tion. Kgn = kininogen. Xlla must be proteolysedto Hageman fac­ tor fragments (XII/). The formation of these activators are a site for control since tal abnormalities have disclosed new the eventual products of the reaction exert functions for old proteins. a positive feedback in their own formation. Thromboplastin Factor Kallikrein can in the fluid phase cleave factor XII to Hageman factor fragments In 1965, Fletcher trait deficiency11 was (molecular weight 30,000) which are described as an autosomal recessive dis­ much more potent in prekallikrein activa­ order characterized by a diminished rate tion than the intact molecule .1 H ig h of surface mediated coagulation. This molecular weight (HMW) kininogen has a plasma possessed an unusual abnormality central place in the scheme. It is the pre­ in that the partial thromboplastin time, a ferred substrate from which kallikrein re­ measure of overall intrinsic coagulation, leases bradykinin. It also potentiates the returned to normal as the time of incuba­ conversion of Hageman factor to activated tion with surface (kaolin) was increased. Hageman factor on a surface22 an d to The factor in normal plasma which cor­ Hageman factor fragments in the fluid rected the deficiency was partially puri­ phase.10 In addition, it potentiates the ac­ fied. In 1972, the corrective protein was tion of activated Hageman factor on clot­ identified as prekallikrein.24 The Fletcher ting factor XI and Hageman factor frag­ trait plasma also possessed an abnormality ments on the reciprocal activation of kal­ in the rate of surface mediated firinolysis likrein.14 Kallikrein also directly activates and generated no bradykinin upon incu­ plasminbgen to plasmin, the enzyme re­ bation with kaolin. This last abnormality sponsible for fibrinolysis.4 R ecently, it has is expected if the plasma contained no been demonstrated by us that Hageman prekallikrein, but one would not have factor fragments can activate factor VII.21 readily predicted an effect upon the Thus, a link is created between the intrin­ coagulation and fibrinolytic pathways. sic and extrinsic pathways. In the dissec­ The presence of both of these abnor­ tion of the system, patients with congeni- malities suggested an effect upon Hage- 222 COLMAN man factor, one protein common to each pathway. Correction of both the coagula­ tion and fibrinolytic defects of Fletcher trait plasma by activated Hageman factor in the absence of a surface demonstrated that the abnormality was a diminished rate of formation of activated Hageman factor. This positive feedback of kallikrein may account for the decrease of surface initiated fibrinolysis and coagulation in Fletcher factor trait. An additional protein was found to be required for surface activation of Hage­ man factor as a result of investigation of F ig u r e 2. Comparison of the kaolin-activated three patients in different laboratories. In euglobulin lysis time of Williams trait plasma, nor­ 1974, a 64-year-old woman Mrs. Williams, mal plasma, and Fletcher trait plasma. Precisely 0.5 was referred, owing to an abnormal intrin­ ml of each plasma or an equal mixture of Williams and Fletcher trait plasmas were added to 4.25 ml of sic coagulation system in which all known sodium acetate buffer, pH 4.8. Exactly 0.25 ml of factors were normal. The results of further kaolin (8 mg per m l) was then added, the mixture was incubated at 37° C, and aliquots of 0.2 ml were re­ investigation of her coagulation defect re­ moved at intervals. The plasmin generated was de­ vealed that the abnormal partial throm­ termined as the euglobulin lysis time. boplastin time test was corrected by one- quarter volume of Hageman trait or tion, the addition of Hageman factor frag­ Fletcher trait plasma, indicating that these ments or even purified plasma kallikrein plasmas contained adequate concen­ did not initiate kinin formation indicating trations of the missing factor in William’s complete lack of kininogen. This result trait plasma. Unlike Fletcher trait plasma, was confirmed by demonstrating that Wil­ prolonged incubation with kaolin failed to liam’s plasma did not react with an an­ correct the coagulation abnormality of tiserum against kininogen. However, low William’s trait.3 molecular weight kininogen did not cor- A profound defect was also found in the fibrinolytic system of William’s plasma. The addition of kaolin markedly in­ creased the fibrinolytic potential of nor­ mal plasma shown as an increase of the reciprocal of the euglobulin lysis time (figure 2). As previously reported, Fletcher plasma showed a decreased rate of activation by kaolin. In contrast, Wil­ liam’s plasma showed virtually no in­ crease of fibrinolytic activity after expo­ sure to kaolin. A mixture of equal volumes of William’s and Fletcher plasma com­ pletely corrected the defect. Similar de­ Slice No. fects were found in the conversion of pre- F ig u r e 3. Alkaline disc gel electrophoresis at kallikrein to kallikrein. pH 9.3 of 30 ixg of high molecular weight kininogen When kaolin was added to William’s (B4-) obtained from Bio-Gel 0.5 m. A replicate disc plasma, no bradykinin was detected by gel was sliced and eluted; this eluate was assayed for kininogen antigen by counterimmunoelectro- bioassay. Although this might have been phoresis and for its ability to correct the coagulation due to the failure of prekallikrein activa- defect of Williams trait plasma. PATHO-PHYSIOLOGY OF KALLIKREIN SYSTEM 2 2 3 rect the fibrinolytic, coagulation and pre- kallikrein activation defects in William’s plasma. About 15 percent of kininogen is in a high molecular weight form. To determine whether or not the HMW kininogen is identical to William’s factor, HMW kininogen was purified with the use of an immunoabsorbent column of monospecific kininogen antiserum. HMW kininogen ran as a single peak on disc electrophoresis. Elution of an un­ stained gel showed that the kininogen antigen and William’s factor, as measured by a coagulant assay, were in the same position (figure 3). Thus, the major coagulant factor missing in William’s plasma appears to be similar or identical to HMW kininogen which by itself cor­ rects all the defects.

Evaluation of Abnormalities (units/ml)

FIGURE 4. Prekallikrein (1.0 kallikrein esterase In evaluating the abnormalities in defi­ unit) was incubated with HF/ (0.6 ¡x g per ml) for 3 cient plasma, it became clear that prekal- min at 37°C in the presence of various concen­ likrein and HMW kininogen are coagula­ trations of high molecular weight kininogen. The results are a mean of triplicate experiments. tion factors, both of which are required for Hageman factor activation and function. HMW kininogen has no known enzymatic man factor fragments to convert prekallik­ activity, but it facilitates the generation of rein to kallikrein is a function of the con­ a proteolytic site upon surface bond centration of HMW kininogen .14 T h e Hageman factor. The Hageman factor ac­ maximum effect is seen at about 6 p ercent tivity generated is proportional to the of the high molecular weight kininogen in HMW kininogen input. Upon the surface, normal plasma as determined by a clotting HMW kininogen enhances the function of assay using William’s plasma as a sub­ Hageman factor upon its substrates17— strate. At this concentration more than a prekallikrein to form kallikrein (brady- three fold augmentation of the initial rate kinin release) and factor XI to accelerate is observed. the PTT. The effect of kallikrein upon The characteristics of a group of pro­ Hageman factor is presumed to be a direct teins which are mainly serine proteases one however since HMW kininogen aug­ and their cofactors have been discussed. It ments this interaction. should be no surprise that the most effec­ In this system HMW kininogen could tive inhibitors of these pathways are pro­ accelerate the formation of activated fac­ tease inhibitors. Seven naturally occur­ tor XI and/or enhance its activity on pre­ ring plasma inhibitors of proteolytic en­ kallikrein. One way to separate these two zymes have been described. Two of these effects is to use Hageman factor fragments proteins, a 2 and «! anti­ already active in the fluid phase. Figure 4 trypsin, make up about 10 percent of totai shows the dependence of the initial rate plasma proteins by weight. All of these prekallikrein activation on kininogen proteins are of widely varying concentration. The potentiation of Hage- molecular weight. In considering the role 2 2 4 COLMAN

ond is to study the interaction of purified inhibitors with the enzyme in question. To demonstrate the stoichiometry of the reaction of kallikrein with Cl inhibitor, 1600 units of purified human inhibitor were incubated with increasing concen­ trations of kallikrein for 30 minutes. The residual kallikrein and Cl inhibitor were then measured .9 The exact parallelism demonstrates stoichiometric interaction between kallikrein and C 1 inhibitor. Upon incubation with plasma kalli­ krein, the Cl inhibitor is depleted (as measured in a hemolytic assay) and the esterolytic activity of kallikrein is sup­ pressed. Similar results were shown for kallikrein release of bradykinin.9 Studies of the inhibition of purified kallikrein by plasma from normal patients and patients TIM E (min) with hereditary angioneurotic edema F ig u r e 5. Inhibition of kallikrein’s esterolytic showed that the ability of plasma to in­ activity by antithrombin-heparin cofactor. Kallikrein hibit kallikrein paralleled the C l inhibitor and antithrombin-heparin cofactor were incubated at 25°C in the presence (A) and absence (0) of hepa­ concentrations and was markedly de­ rin. The buffer used for these experiments was 0.15 creased in hereditary angioedema. M sodium chloride in 0.01 M sodium phosphate (pH The effect of another major inhibitor 7.6). All reactants were extensively diluted in or dialyzed against this buffer. The final concentrations anti-thrombin III with kallikrein has been of enzyme, inhibitor and acidic mucopolysaccharide examined by us.12 It is demonstrated in (if employed) were 250 f i g per ml (2.5 /limol of figure 5 that purified anti-thrombin III in- TAME hydrolyzed m in '1 ml *), 75 fj. g per ml (75 units per ml) and 1 unit per ml, respectively. At appropri­ activates the esterolytic activity of ate times, aliquots were withdrawn and residual kal­ purified kallikrein. The process with in­ likrein activity was determined by esterolytic assay hibitor in excess obeys pseudo first order as mentioned in Evaluation section. During the time course of these experiments, the esterolytic activity kinetics. Heparin (lower curve) appears to of kallikrein remained stable when incubated at accelerate the rate of inactivation. Similar 25°C with buffer. The results presented are repre­ results are obtained when the ability of sentative of five separate experiments and are ex­ pressed as percentages of zero time activity prior to kallikrein to release bradykinin measured the incubation of kallikrein with antithrombin- in a radioimmunoassay is measured indi­ heparin cofactor. cating that the anti-thrombin III affects the proteolytic activity of kallikrein as of these inhibitors in the factor XII in­ well as its esterolytic activity. itiated pathways, it becomes clear that al­ Once again, the accelerating effect of though the inhibitors have multiple heparin was seen. In normal plasma, anti­ specificities, they have preferential ef­ thrombin III apparently plays little role in fects. This can be illustrated by present­ inhibiting kallikrein. Thus, the degree of ing the effect of the two major plasma in­ inhibition by normal plasma is not altered hibitors of kallikrein, Cl inhibitor and by addition of as much as 50 units per ml anti-thrombin III. heparin. In contrast, plasma from patients One way to approach this problem is to with hereditary angioedema had little in­ compare the effect of plasma congenitally hibitory capacity measured by esterase or deficient in a single inhibitor with the in­ bradykinin release. This inhibitory capac­ hibitory capacity of normal plasma. A sec­ ity is markedly increased by adding PATHO-PHYSIOLOGY OF KALLIKREIN SYSTEM 2 2 5

TABLE I T A BLE III

Deficiency of Proteins Necessary Disorders Leading to Kallikrein Activation for Kinin Formation

I. Acquired (suspected) I. Hereditary A. Coronary artery disease A. Factor XII deficiency (Hageman trait) B. Intravascular coagulation B. Prekallikrein deficiency (Fletcher trait) C. Hyperlipoproteinemia C. High molecular weight kininogen deficiency D. Transfusion reactions (Williams, Fitzgerald and Flaujeac traits) E. Typhoid fever F. Nephrotic syndrome II. Acquired A. Dengue hemorrhagic fever B. Cirrhosis of the liver Kinin formation not documented. therapeutic concentrations of heparin. related to bradykinin release by either tis­ These observations suggest a potential sue or plasma kallikrein. Septic shock is role for heparin in treating the acute mani­ accompanied in man by decreased prekal­ festations of the disease. likrein and kallikrein inhibitory acitivty, formation of active kallikrein16 and e le ­ With this background, the role of the vated kinin levels.19 The latter may con­ kallikrein system can be surveyed in tribute to the early phases of reversible human disease. In table I are summarized hypotension. Elevated bradykinin levels conditions which are associated with ab­ in pancreatitis are probably due to release normal synthesis of one or more of the of trypsin and pancreatic kallikrein. proteins involved in this system. The In table III are listed disorders in which three hereditary syndromes have been there is a strong evidence for activation of previously discussed. In dengue hemor­ kallikrein, including decreased factor XII rhagic fever, a selective deficiency of and prekallikrein diminished kallikrein plasma prekallikrein occurs probably inhibitory activity and formation of kalli- owing to the abnormal liver function krein-Cl inhibitor complexes. In coronary studies.8 In cirrhosis of the liver, the mean artery disease,20 m yocardial iscehm ia is prekallikrein levels are depressed to accompanied by these changes in over about 25 percent of normal and in the sev­ half the cases. Dissemination intravascu- eral cases, concentrations ofOto 10 percent lar coagulation (DIC) is accompanied by are usual.2,23 In contrast, factor XII is de­ evidence for kallikrein activation when creased to about 40 percent of normal, and endothelial injury is involved, such as in HMW kininogen is reported reduced to a sepsis, but not in DIC owing to similar level. neo p lasin.15 At the time when positive Of greater interest are the conditions in blood cultures and pyrexia are found in which increased kinin formation has been typhoid fever, the kallikrein system is ac­ documented (table II). In carcinoid syn­ tivated with the changes disappearing drom e6,7 and postgastrectomy dumping during convalescence.5 In the nephrotic syndrom e,18 the vasomotor symptoms are syndrome, activation of Hageman factor

T A B L E II T A B L E IV Disorders Leading to Increased Kinin Formation Disorders in Which Kallikrein-Kallikrein Abnormalities Have Been Suggested I. Hereditary A. Hereditary angioedema I. Acquired II. Acquired (documented) A. Thermal injury A. Carcinoid syndrome B. Arthritis B. Postgastrectomy dumping syndrome C. Allergic disorders C. Septic stock D. Cystic fibrosis D. Hemorrhagic pancreatitis E. Migraine syndrome 226 COLMAN levels lead to kallikrein factor XI and Hageman factor. Proc. Nat. Acad. Sei 73:2554- plasminogen activation.13 2558, 1970. 11. H a t h a w a y , W . E., B e l h a n s o n , L. P., and Finally, kallikrein activation has been H a t h a w a y , M. S.: Evidence for a new throm­ suggested to occur in various conditions boplastin factor; Case report, coagulation studies, and physiochemical studies. Blood but none of these have been shown defi­ 26:521-532, 1965. nitely (table IV). Further investigation is 12. L a h ir i, B., Ba g d a s a r ia n , A., M it c h e l l , B., important to appreciate the role of this T a l a m o , R. C ., C o l m a n , R. W ., and R o s e n ­ b e r g , R. D.: Antithrombin-heparin cofactor: An defensive system in inflammatory inhibitor of plasma kallikrein. Arch. Biochem. disorders. Biophys. 175:737-747, 1976. 13. L a n g e , L. G ., C a r v a l h o , A. C ., Ba g d a s a r ia n References A., L a h ir i, B., and C o l m a n , R. W .: Activation of Hageman factor in the nephrotic syndrome. 1. Ba g d a s a r ia n , A., La h ir i, B., a n d C o l m a n , B. Amer. J. Med. 56:565-569, 1974. W.: Origin of high m olecular weight activator of 14. L iu , C . Y., Sc o t t , C. F., Ba g d a s a r ia n , A., prekallikrein. J. Biol. Chem. 248:7742-7747, P i e r c e , J. V., Ka p l a n , A. P., a n d C o l m a n , R. 1973. W.: Potentiation of function of Hageman factor 2. Ba g d a s a r ia n , A., L a h ir i, B., T a l a m o , B. C., fragments by high molecular weight kininogen. W o n g , P., and C o l m a n , B. W .: Immunochemi­ J. Clin. Invest. 60:7-17, 1977. cal studies of plasma kallikrein. J. Clin. Invest. 15. M a s o n , J. W. and C o l m a n , R. W.: The role of 54:1444-1454, 1974. Hageman factor in dissemination intravascular 3. C o l m a n , R. W., Ba g d a s a r ia n , A., T a l a m o , R. coagulation induced by septicemia, neoplasia C., Sc o t t , C. F„ Se a v e y , M„ G u im a r a e s , J. A., or liver disease. Thromb. Diath. Haemorrh. Pie r c e , J. V., and Ka p l a n , A. P.: Williams trait: 26:325-331, 1971. Human kininogen deficiency with diminished 16. M a s o n ,J . W ., Kl e e b e r g , U. R., D o l a n , P., and levels of plasminogen proactivator and prekal­ C o l m a n , R. W .: Plasma kallikrein and Hage­ likrein associated with abnormalities of the man factor in gram negative bacteremia. Ann. Hageman factor dependent pathways. J. Clin. Int. Med. 73:545-551, 1970. Invest. 56:1650-1662, 1975. 17. M e i e r , H. L ., S c o t t , C. F., M a n d l e , R., J r ., 4. C o l m a n , R. W., Br a u n , W. E., B u s c h , G. J., W e b s t e r , M ., P ie r c e , J. V., C o l m a n , R. W ., D a m m in , D ., and M e r r il , J. P.: Coagulation and Ka p l a n , A. P.: Requirements for the con­ studies in the hyperacute and other forms of tact activation of human Hageman factor. Ann. renal allograft rejection. New Engl. J. Med N. Y. Acad. Sei. 283:93-103, 1977. 281:685-691, 1969. 18. M in n a , J. D., R o b b o y , S. J., and C o l m a n , R. 5. C o l m a n , R. W ., E d e l m a n , R., Sc o t t , C . F ., W .: Disseminated Intravascular Coagulation in a n d GlLMAN, R. H.: Plasma kallikrein activation Man. Springfield, C. C Thomas, 1974. and inhibition during typhoid fever. J. C lin . 19. O ’D o n n e l l , T. F., J r ., C l o w e s , G . H . A., J r ., In v e st. 61:287-296, 1978. Ka p l a n , A. P ., a n d C o l m a n , R. W.: Kinin acti­ 6. C o l m a n , R. W., Ma s o n , J. W., and Sh e r r y , S.: vation in the blood of patients with septic man. Studies on the kallikreinogen kallikrein en­ Surg. Gyn. Obst. 143:539-544, 1976. zyme system of human plasma. Assays of com­ 20. P i t t , B., M a s o n , J., C o n t i, C . R., and C o l m a n , ponents and preliminary observations in dis­ R. W .: Observations on the plasma kallikrein ease states. Ann. Int. Med. 71:763-773, 1969. system during myocardial ischemia. Trans. 7. C o l m a n , R. W., W o n g , P. Y., and Ta l a m o , R. Amer. Assoc. Physicians 82:98-108, 1969. C . Kallikrein-kinin system in carcinoid and 21. Ra d c l if f e , R., B a g d a sa r ia n , A., C o l m a n , R. postgastrectomy dumping syndromes. Proceed­ W., and NEMEBSON, Y.: Activation of factor VII ings of the Conference on the Chemistry and by Hageman factor fragments. Blood 50:611- Biology of the Kallikrein-Kmin System in 618, 1977. Health and Disease. Pisano, J. J. and Austen, K. 22. W e b s t e r , M . E., G u im a r a e s , J. A., Ka p l a n , A. F., eds. Washington, U.S. Government Print­ P ., C o l m a n , R. W ., a n d P ie r c e , J. V.: Activation ing, 1977, pp. 4 8 7 -4 9 4 . of surface-bond Hageman factor: Preeminent 8. E d e l m a n , R., N im m a n n it y a , S., C o l m a n , R. role of high molecular weight kininogen and W ., T a l a m o , a n d T o p , F. H.: Evaluation of the evidence for a new factor. Kinins: Phar­ plasma kinin system in dengue hemorrhagic macodynamics and Biological Roles. Sicuteri, fever. J. Lab. Clin. Med. 86:410-421, 1975. F., Back, N., and Haberland, G. L., eds. New 9. G ig l i, I., M a s o n , J. W., C o l m a n , R. W., and York, Plenum Press, 1976, p p . 285-299. A u s t e n , K. F.: Interaction of plasma kallikrein 23. W o n g , P. Y., C o l m a n , R. W ., T a l a m o , R. C., with the C l inhibitor. J. Immunol. 104:5 7 4 - and Ba b io r , B. M.: The kallikrein bradykinin 581, 1970. system in chronic alcoholic liver disease. Ann. 10. G r if f in , J. M. and C o c h r a n e , C. G.: Mecha­ Int. Med. 77:205-209, 1972. nisms for involvement of high molecular weight 24. W u e p p e r , R. D.: Prekallikrein deficiency in kininogen in surface dependent reactions of man. J. Exp. Med. 138:1345-1355, 1973.