THE EFFECT IN PATIENTS OFSTREPTOCOCCAL FIBRINOLYSIN(STREPTOKINASE) AND STREPTOCOCCALDESOXYRIBONUCLEASE ON FIBRINOUS,PURULENT, AND SANGUINOUS PLEURALEXUDATIONS

William S. Tillett, Sol Sherry

J Clin Invest. 1949;28(1):173-190. https://doi.org/10.1172/JCI102046.

Research Article

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THE EFFECT IN PATIENTS OF STREPTOCOCCALFIBRINOLYSIN(STREPTOKINASE) AND STREPTOCOCCALDESOXYRIBO-

NUCLEASEON FIBRINOUS, PURULENT, AND SAN-GUINOUSPLEURAL EXUDATIONS'

By WILLIAM S. TILLETT AND SOL SHERRY

(From the Department of Medicine, New York University College of Medicine, and the ThirdMedical Division of Bellevue Hospital, New York City)

(Received for publication August 6, 1948)

The results described in this article were ob-tained by the injection of concentrated and par-tially purified preparations derived from brothcultures of hemolytic streptococci into the pleuralcavity of selected patients who were sufferingfrom different types of diseases that gave rise topleural exudations. The possibility has been ex-plored of utilizing two of the defined propertieselaborated by hemolytic streptococci that have theunique capacity of causing rapid lysis of the solidelements (fibrin and nucleoprotein) that are sig-nificant parts of exudates. In a companion ar-ticle, Christensen (1) has described additionalsteps in the purification of the materials employedand has given quantitative methods of measure-ment of various increments concerned in thefibrinolytic system, including trypsin inhibitor andspecific antibodies, which have been employed inthis study.

In the study of patients it was first necessaryto: (a) develop the investigation within non-toxic but effective ranges of doses of the materialemployed, and (b) determine whether or not theenzymatic activities of the streptococcal productswere effectively operative when introduced di-rectly into the site of the disease in the patients.This article is essentially limited to findings thatare pertinent to the two aspects of the study men-tioned above.

A brief description of the two streptococcalproducts that have been the subject of the in-vestigation is as follows:I. Streptococcal fibrinolysin (2) (Strepokinase)

This has been found to be elaborated character-istically by strains of hemolytic streptococci(Group A) causing acute infections in patients.It is also produced by some strains of strepto-

1 This study was supported by a grant from the Na-tional Institute of Health, United States Public HealthService.

coccal groups C and G (3). The product isabundantly excreted into the culture medium inwhich the organisms are grown and is readily ob-tainable free from the bacterial cells in sterilefiltrates.

The fibrinolytic action, in tests conducted un-der optimal laboratory conditions, is unusuallyrapid in action on the fibrin coagulum of normalhuman blood, requiring only a few minutes whenwhole plasma is employed as a source of fibrin,and an even shorter time when preparations offibrinogen and thrombin of human origin areused. The ultimate proteolytic nature of thefibrinolysis has been evident in the liberation ofbreakdown products of protein digestion (4).

Another interesting feature of the phenom-enon is the high degree of activity of the strepto-coccal lytic system in the presence of normal hu-man blood as contrasted with its inaction or de-layed effect when tests are conducted with theblood of various animal species with the exceptionof monkeys (3). Although studies of factors in-volved in streptococcal fibrinolysis have indicateda basis for the differences in the behavior of bloodfrom different animals, nevertheless the existenceof the difference has rendered impossible mostexperimental studies, in vivo, on animals and haslimited them to observations conducted in pa-tients, such as those initially described in thisarticle.

The mechanism of the reaction has been a sub-ject of study by various investigators.

In an earlier report (2) it was noted that fibrinformed from mixtures of either human fibrinogenand rabbit thrombin, or of rabbit fibrinogen andhuman thrombin were susceptible to streptococcallysis, whereas the fibrin derived from rabbitfibrinogen plus rabbit thrombin was unaffected.Milstone (5) demonstrated that an increment ofhuman serum present in the euglobulin fraction,

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was necessary for the reaction to occur and that ifpreparations of human fibrinogen and thrombinwere highly purified no lysis would occur whenthe streptococcal product was added. Further-more, if this human globulin fraction was addedto fibrin made up of rabbit components, lysis oc-

curred. He referred to the euglobulin constituentof human blood as "lysing factor" and suggestedthat streptococcal fibrinolysis was mediated by thesame system as that involved in other examples offibrinolysis.

Christensen (4), and Christensen and Mac-Leod (6) have presented results concerning themechanism, and have concluded that the strepto-coccal product to which they have given the name

streptokinase acts as a catalyst or kinase in caus-

ing the transformation of a zymogen of normalhuman blood, designated by them plasminogen,into a proteolytic enzyme, designated plasmin.

Kaplan (7) has made extensive studies on

several aspects of the mechanism of the strepto-coccal fibrinolysin-serum protease system. Thesestudies have in part dealt with the dissimilarityof serum protease and trypsin and also with theinhibition of streptococcal fibrinolysis by antipro-tease. The findings are predicated upon thekinase nature of the action of the streptococcalproduct and the identity of the protease of serum

as the lysing principle. Ratnoff (8), studying a

proteolytic enzyme in human plasma, gave specialattention to the fibrinolytic system activated bythe streptococcal product and obtained data indi-cating that the activation of plasma proteolyticenzyme by streptococcal fibrinolysin behaved as

if it involved a stoichometric reaction. Thisfinding implies that the streptococcal product may

take an active part in the lytic system rather thanexert only a catalytic effect.

Without developing the discussion of the mech-anism, it is sufficient to state that the objectivedemonstration of changes in constituents ofpleural exudates that have been effected by thestreptococcal fibrin-dissolving system, actingwithin the pleural cavity of patients, constitutesdefinitive results around which this report cen-

ters. The conditions, unavoidably inherent inthe several factors that may be simultaneously op-

erative in association with the processes of dis-ease within patients, makes it impossible at thepresent time to analyze and identify the mecha-

nism of fibrinolysis that may be responsible forthe end results that we have observed.

Nomenclature. The fibrinolytic principle de-rived from hemolytic streptococci is referred toin this article as streptokinase. The kinase typeof action of the streptococcal product has beenconsistently observed by several investigators.However, whether or not its complete activityis solely that of kinase remains to be determined.

The term fibrin-lysing system has been employedas an objectively descriptive expression whichavoids the different recommendations of variousinvestigators (6, 9-1 1). The activatable fibrin-ly-sing system may be closely identified with theplasminogen of Christensen and MacLeod (6),and the active fibrin-lysing system with theirterm plasmin.

II. Streptococcal desoxyribonucleaseIn recent articles the occurrence of desoxy-

ribonucleoprotein in significant amounts in puru-lent empyematous fluids was reported (12) andits depolymerization by a desoxyribosenucleasefound to be present in the concentrated strepto-coccal flltrates was also described (13).

The solid sediments of the samples of pus werefound to contain as much as 30% to 70% nucleo-protein. Since the physical characteristics ofdesoxyribose nucleoprotein are fibrous and gel-atinous, the similarity of its appearance to thatof fibrin became a matter of importance in identi-fying the dual enzymatic systems with which wewere dealing; namely, fibrin substrate in thefibrinolytic system, and nucleoprotein substrateacted upon by nuclease. In each instance rapidliquefaction of the respective solid substrateoccurred.

Each of the substrates mentioned has beenidentified in varying amounts in different types ofpleural exudation. For example, empyemal pusof bacterial origin is high in its content of nu-cleoprotein, which is presumably derived fromthe leucocytes, while the constituent most con-spicuously present in hemothorax is fibrin. Fi-brinous pleural exudates such as may be en-countered in association with pneumonia, pul-monary neoplasm, and other diseases, or the ef-fusions of tuberculous pleurisy have been studiedchiefly for the fibrinolytic effect, although from

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observations on preparations of the exudatesstained by the Feulgen method (12), it is evi-dent that varying amounts of nucleoprotein arepresent.

Streptococcal concentrates 2

The preparations employed in this study have been pre-pared by Christensen according to the method of partiallypurifying the concentrated filtrates of hemolytic strepto-cocci previously described and further elaborated in theaccompanying article (1, 14). A strain of hemolyticstreptococcus (H46A) belonging to Lancefield Group Chas been employed. This strain is potent in its produc-tion of both streptokinase and desoxyribose nuclease (13)and most of the lots of concentrated filtrate used in thisstudy have possessed both properties. In addition itseemed reasonable to assume that since the strain (agroup C) was not of patient origin its products mightpresumably be least likely to contain other noxious, butas yet unidentified, increments associated with acute in-fections in man and would, therefore, be more desirablefor preparing material to be used in patients than woulda Group A fibrinolytic strain. All the partially purifiedand concentrated preparations, with one exception, thatwere introduced into patients, contained both streptokinaseand nuclease. In an additional preparation used in asingle patient with hemothorax, the content of nucleasewas reduced substantially by Christensen (1) but thestreptokinase activity remained unimpaired.

The dosage of streptokinase has been based upon a unit,developed and described in the accompanying article byChristensen (1). The dosages per patient are given inthe individual protocols. In general they ranged from20,000 to 400,000 units. The concentration per cubiccentimeter ranged from 2,000 to 40,000 units. The totaldose was usually contained in 10 cc. of normal salinesolution.

The desoxyribonuclease content of the various prepara-tions of streptococcal concentrates has not yet been elab-orated so extensively as that of streptokinase. Christensen(1) has suggested a unit basis which is at present beingemployed. In general the concentrates contained a ratioof approximately 6,000 units of nuclease to 15,000 unitsof streptokinase. The nuclease was of special significancein the cases of empyema, and the dosage is given in thedescription of the results obtained in that group.

Toxicity of streptococcal concentrates. Among the in-itial studies (2) concerned with the characterization ofthe streptococcal fibrinolytic phenomenon, preliminaryobservations (unpublished) were made with respect tothe skin reactivity of active filtrates derived from strainsof fibrinolytic streptococci. Following intracutaneous in-jections, areas of local erythema developed that simulatedthe type of a positive reaction elicited by the erythrogeriicscarlatinal toxin. However, by comparative tests in

2Lederle and Co. have cooperated by growing largevolumes of culture and supplying the filtrate in its initialconcentrated form.

which both scarlatinal toxin and fibrinolytic filtrates wereinjected into Dick positive and Dick negative individuals,no correlation was detected. From the limited observa-tions, it seemed reasonable to assume that the primarytoxicity of the fibrinolytic filtrate was due either to thefibrinolytic principle itself or to some contaminating prod-uct other than the established erythrogenic toxin ofhemolytic streptococcal origin.

When the recent concentrates prepared by Christensen(1) in the process of purifying the streptococcal fibrinoly-sin were being considered for introduction into patients,intradermal tests were performed. Although the concen-tration of streptokinase was increased many hundred-foldover the preparations of earlier use, the degree and fre-quency of erythematous reactions were found not to beany greater than those previously encountered nor to berelated to the concentration.

Samples of the concentrates were then injected intra-muscularly. No reactions occurred that were detectableby the development of local redness, tenderness or swell-ing, or as a generalized pyrogenic effect. When increas-ingly large concentrations failed to produce untowardeffects intramuscularly, small amounts were introducedintrapleurally into patients with empyema, inflammatorypleural effusions, or hydrothorax.

Without describing the details of the progressive ob-servations following intrapleural injections it may bestated that as the procedures of purification and concen-tration conducted by Christensen developed, the evidencesof toxicity have diminished in relationship to amountinjected. The studies have, therefore, up to the presenttime indicated that the primary toxicity of the fibrinolyticprinciple or the accompanying desoxyribonuclease is notgreat and that the transient pyrogenic reaction may bedue particularly to contaminating substances still presentin even the purest preparations now available.

The manifestations of toxicity when they occurred haveconsisted of a pyrogenic reaction beginning approximatelysix to eight hours after the injection, reaching its peakof a rise of one to four degrees at 24 hours after injectionand decreasing gradually over the next 24 to 72 hours tothe pre-injection level which in some instances was anormal temperature and in others was elevated prior tothe injection due to the underlying disease. Generalmalaise accompanied the febrile period. Nausea andgastro-intestinal discomfort were noted in some patients.Local pain, although never severe, was present in someinstances, and not in others.

An outpouring of polymorphonuclear leucocytes oc-curred which was demonstrable 24 hours after the injec-tion and decreased to the pre-injection level within thenext two to ten days. The increase in the leucocytecounts ranged from 1,000 to 25,000 cells per cubic centi-meter.

The general pyrogenic reaction together with individ-ual symptoms as mentioned, and the local outpouring ofcellular constituents did not follow any uniform or cor-related pattern, since all of the manifestations were notpresent to the same degree and some of them wereentirely absent. In several instances in which two injec-

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tions were given, the first may have caused a reactionwhile the second elicited no toxic response, or the firstwas silent and the second evoked some reaction. Fur-thermore, the occurrence of reaction was not clearlyrelated to dosage of concentrate.

In this study a total of 34 injections was given to 23patients. Utilizing a rise of 1° F in fever as an indi-cation of a reaction-and irrespective of whether or notthe patient was febrile before the injection-on 17 (50%)occasions a pyrogenic response was noted.

The degree of reaction appeared to be most directlyrelated to the nature of the patient's disease. In instanceswhere considerable thickening of the pleura was presentor other circumstances that may have impaired the ab-sorbing surface, minimum or no toxic reactions occurred.In other cases where the contrary situation of a thinpleura or presumably a relatively freely acting absorbingsurface was present, the reactions as described were moreconsistently noted.

Other factors that may participate in the reaction ina manner as yet undetermined are the breakdown prod-ucts derived on the one hand from the fibrinogen andfibrin system and, on the other hand, from the nucleo-protein system through the action of nuclease. No spe-cific information on this phase of the subject is availableexcept the fact that the toxic reaction, whatever its exactcause may be, was a transient one of the classical briefpyrogenic type without any residual effects. No indica-tion of chronic alterations of an untoward nature havebeen evident. Because of the implications of hemolyticstreptococci in relation to rheumatic fever and acutehemorrhagic nephritis, detailed examinations for theirpossible presence have been frequently made with uni-formly negative findings.

It has been of special importance to make preliminarytests with each new lot of purified concentrates madeavailable for use in patients by injecting a small amountintrapleurally and noting the degree of reaction. Whenthe reaction appeared to be relatively greater than wouldbe expected from the small dose, the lot was not furtheremployed in the study of patients. This precautionaryprocedure of selecting individual preparations for intra-pleural use has been regularly adhered to, and, as a result,no contraindications for the development of the studyhave been encountered.

PROCEDURE

Observations on Patients. In the study of patientsnumerous quantitative determinations have been made onsamples of exudate obtained prior to the injection ofstreptococcal concentrates and at stated intervals there-after, usually one hour, 24, 48, 72 hours, etc., according tothe nature of the case and the findings.

The tests and procedures employed have included thefollowing: fibrinogen, N.P.N., total protein, formal titra-tion, amount of sediment, viscosity, pH, volume of pleuralexudate, free streptokinase, activatable fibrin-lysing sys-tem, active fibrin-lysing system, trypsin inhibitor, andspecific anti-fibrinolytic antibody (anti-streptokinase).

Cytological studies, including total and differentialcounts, determination of motility and viability of cellsand the appearance of Feulgen-stained preparations forthe purpose of identifying- intra- and extracellular de-soxyribose nucleoprotein have also been made. Theseresults will be referred to briefly in this article and bereported in detail in a subsequent communication.

The technical procedures in each instance have beenas follows:Streptokinase titre, activatable fibrin-lysing system

(equivalent to plasminogen), trypsin inhibitor, andanti-streptokinase titre were determined on chest fluidaccording to the methods described by Christensen (1).

Active fibrin-lysing system (equivalent to plasmin) wasdetermined as follows: To 0.1 cc. samples of chestfluid, brought to neutral pH, were added 0.5 cc. of0.25% Bovine Fibrinogen (Armour) in 0.01 M salinephosphate buffer (pH 7.4), and 0.5 cc. of 0.25%Human Plasma Fraction III (Cohn) in saline phos-phate buffer. One tenth ml. of 1/3 dilution of LederleHemostatic Globulin (or 1/500 dilution of Parke DavisBovine thrombin) in saline phosphate buffer was thenrapidly added. The tubes were incubated in a water bathat 37°. One tenth cc. of saline instead of chest fluid wasused for controls. The time of complete lysis of thefibrin clot was noted. Since the lysis of fibrin in theabsence of extra increments of streptokinase was rela-tively slow and, therefore, could not be expressed inthe same unitage scale, the results were expressed as0-++++ as follows:

lysis up to one hour ++++lysis one to six hours +++lysis six to 24 hours ++lysis 24 to 48 hours +lysis 48 to 72 hours but no lysis in controls ±No lysis 0

Estimations of the degree of activity of the fibrin-lysing system with respect to time have proved impor-tant since the period required for lysis, in vitro, re-flected to some degree the activity of the process, invivo, which was significant with respect to changes inthe exudates that continued for several days after theinitial injection.

Amount of sediment: Wintrobe hematocrit tubes werefilled to the mark with a heparinized sample of chestfluid and centrifuged at 2,500 rpm for one hour. Theresults are expressed as per cent.

pH was determined on separately collected heparinizedsamples by glass electrode after chest fluid was allowedto equilibrate with room temperature. A previouslydetermined temperature correction for the instrumentused was applied, so that the results are expressed aspH at 370 C.

Viscosity: The viscosity of thin fluids was determined onheparinized samples in an Ostwald viscosimeter at370 C. The viscosity of thick purulent fluids wasdetermined with an LV model Brookfield electricviscometer at 370 C.

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Total protein and N.P.N. were done on the supernatantof centrifuged oxalated specimens of chest fluid. Thetotal protein was determined by Kjeldahl digestion andnesslerization. The N.P.N. was determined by diges-tion and nesslerization of a trichloracetic acid filtrate.The results are expressed as milligrams per cent forN.P.N., and grams per cent for total protein.

Fibrinogen was determined on the supernatant of centri-fuged oxalated specimens. Five-cc. samples were di-luted to 30 cc. with normal saline, to which were added1 cc. of 2.5%o CaCI, solution and 1 cc. of a 1/100 dilu-tion in normal saline of Parke, Davis Bovine Thrombin.The tubes were then placed in the refrigerator over-night. The clots were removed with a stirring rod,washed with distilled water, and the nitrogen deter-mined by digestion and nesslerization. The results areexpressed as milligrams per cent.

Formol titration: Ten-cc. samples of heparinized fluidwere brought to pH 7.0 by addition of either 0.1 NNaOHor 0.1 N HCl. Five cc. of 40% formaldehydewere added, and the mixture titrated with standard0.1 N NaOHby glass electrode to pH 8.6. The vol-ume of alkali necessary to bring 5 cc. of 40% formalde-hyde to pH 8.6 was subtracted from the titration. Theresults are expressed as cubic centimeters of 0.1 NNaOHper 100 cc. of chest fluid.

Volume of pleural fluid: Since information concerningthe volume of pleural effusions was desirable, the fol-lowing method was developed. The fluid volume wasdetermined in pleural transudates and non-purulentexudates by injecting known quantities of T-1824 andsampling the pleural fluid from 15 to 60 minutes there-after. This method is not applicable to purulent em-pyemas where a significant portion of the dye is boundto the sediment of the fluids and probably to the shaggypleural surface, but quantitative determinations on non-purulent pleural effusions have yielded satisfactoryresults.

RESULTS

Twenty-three patients have been studied fol-lowing the injection of amounts of streptococ-cal concentrates sufficiently large to bringabout definitive and measurable biochemical andbiophysical changes in the area of exudation at thesite of the injection. Although the general planof study has been the same in all instances thepresentation of the results has been arranged ac-cording to a grouping of the cases based on thetypes of exudates which were present in thepleural cavities of the patients. The changes thathave been effected by the concentrates have beenfound to bear a relationship to the character ofthe exudates.

The three major groups were: acute fibrinous

pleurisy, bacterial empyema, and hemothorax.3Group I. Acute fibrinous pleurisy, 13 patients.

Five cases of tuberculous pleurisy with effusion.Six cases of primary or metastatic malignant

pulmonary neoplasm with pleural effusion.Two cases of congestive heart failure with

pleural effusion.

Figure 1 contains data derived from serial de-terminations of fibrinogen and nonprotein nitro-gen made on seven patients before and after theintroduction of streptococcal concentrate. Thedosages, given in the figure, ranged from 20,000to 200,000 units of streptokinase, and approxi-mately 20 to 200 units/cc. chest fluid. Three ofthe patients received a second injection.

The fibrinogen represents the chief parent pro-tein (plus an undetermined amount of solidfibrin) that was acted upon by the fibrin-lysingsystem, and the N.P.N. represents the end-prod-uct of the local proteolysis.

The findings in each of the seven patients dem-onstrate the rapidity of the beginning decreasein measurable fibrinogen and increase in N.P.N.,the samples taken as early as one hour after theinjection revealing changes in the values of each.In patient W. H., for example, the immediate dropin fibrinogen was from 41 to 20 mgm.%, and inpatient J. G., from 108 to 26 mgm.%o. The con-comitant rise in N.P.N. in patient W. H. wasfrom 45 to 52 mgm.%o, and in patient J. G., from31 to 41 mgm.%o.

In five of the seven patients of the chartedgroup, a further drop in fibrinogen was noted inthe specimen obtained 24 hours after the injection,and in each of the seven the N.P.N. either con-tinued to rise or maintained a higher level thanthat of the pre-injection figures.

Three of the patients received a second injec-tion of streptococcal concentrate; patient C. L. re-ceived 100,000 units of streptokinase four dayslater, patient E. H. received 40,000 units of strep-tokinase three days later, and patient J. G. re-ceived 200,000 units of streptokinase three dayslater. In each case there was a further abrupt

8 The authors wish to acknowledge the cooperation andmany helpful suggestions which they have received fromthe Attending and Resident Medical and Surgical Staffsof the Chest Service of Bellevue Hospital, Dr. J. BurnsAmberson, Director.

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WILLIAM S. TILLETT AND SOL SHERRY

HR. I HR.

DAYS

FIG. 1. THE EFFECT OF INTRAPLEURALLY INJECTED STREPTOKINASEON THE FIBRINOGEN ANDN.P.N. CONCENTRATIONSOF THE CHESTFLUID OF SEVEN PATIENTS WITH FIBRINOUS PLEURAL EFFUSIONS

The crosses represent N.P.N. (mgm.%), and the circles, fibrinogen (mgm.%).

decrease in fibrinogen and rise in N.P.N. follow-ing the second injection.

In five cases in which the fibrinogen content was

followed for long periods of time by examiningsamples of pleural fluid obtained at repeated in-tervals, the levels of fibrinogen were found to re-

turn to the control value only after ten days toone month. Why the fibrinogen remained lowfor this period of time is not readily apparent.

Continuing lytic action during this period was notevident by the measurements of the active fibrin-lysing system employed. Furthermore, the fibri-nogen levels remained low despite the appearanceof antistreptokinase, and was independent of thereappearance of additional increments of the ac-tivatable fibrin-lysing system through further exu-dation of serum into the pleural area. It mayrepresent the time necessary for the exudation of

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STREPTOCINASEAND STREPTONUCLEASEEFFECT IN PATIENTS

additional fibrinogen to occur into the area ofpleurisy, or slow proteolysis may have continuedbut at a rate not determinable by our presentmethods.

There were in the patients several uncontrolla-ble factors that altered the serial findings as theyextended over several days. For example, whenthe patient's disease was in an acute active state,

there was a continuing increase in the effusion to-gether with the inflammatory constituents. Whenthe disease was stationary or subsiding, the con-tents of the pleural cavity were not substantiallyaltered during the period of observation. In ad-dition, the variable degree of local irritationcaused either by the injection material itself orthe breakdown products of proteolysis contributed

IHR 1 2 3 4 H"R 5 6 7 a

DAYS

FIG. 2. THE EFFECT OF INTRAPLEURALLY INJECTED STREPTOKINASEON THE FREE STREPTOKINASE, ACTIVATABLE ANDAcTVE FIBRIN-LYSING SYSTEMSOF THE CHEST FLUID OF FvE PATIENTS WITH FIBRINOUS EXUDATES

In the graphs for each patient, in the top sections the crosses represent the activatable fibrin-lysing system (units/cc.) and the circles the active fibrin-lysing system; in the lower sections, the circles represent free streptokinase(units/cc.).

179

WILLIAM S. TILLETT AND SOL SHERRY

w(n

zByI0

a-LLJ

Ir-z

2 3 4 5 6 7 8 9 10 11 12 13 14 15 20 25 30 35 40I MR

DAYS

ANTISTREPTOKINASE. TITRE IN PLEURAL EFFUSIONS FOLLOWING INTRAPLEURAL ADMINISTRATION OFSTREPTOKINASEIN FIVE PATIENTS WITH FIBRINOUS PLEURAL EXUDATES

Streptokinase was injected at zero time.

an additional increment of exudate. Further-more, the rate of absorption of the newly formednon-protein nitrogen proceeded at variable rates.Consequently, the figures obtained by analysis ofthe exudate were only relative with respect tototal proteolysis.

In spite, however, of the variables outlinedabove, the intrapleural degradation of fibrinogenin significant amounts following each injection ofthe streptococcal concentrates has uniformly oc-

curred in each of the patients in this series, as

well as in those of the other groups. The extentto which solid preformed fibrin underwent lysiswas not measurable but its breakdown undoubtedlycontributed to the N.P.N. that was liberated.

In some of the patients, estimations of bloodN.P.N. were done but no significant rise abovenormal levels was noted, the rate of absorptionfrom the pleural area being insufficient to raise thecontent of the general circulation.

The data contained in Figure 2 concern threeelements involved in the streptococcal fibrin-lysingsystem, namely: free streptokinase, and both theactivatable and the active fibrin-lysing system.They are recorded for five patients of the acutefibrinous pleurisy group. Similar findings were

obtained from most of the patients studied ofall the groups.

Prior to injection the amount of activatablefibrin-lysing system present in the pleural exudatewas approximately the same as that found innormal serum but within one hour after the in-jection of streptokinase striking changes occurredin the elements of the fibrin-lysing system of thepleural exudate. When streptokinase was intro-duced, the activatable elements of the lytic sys-tem were replaced by active increments whichwere demonstrable immediately following injec-tion but progressively disappeared during thenext 24 to 48 hours. This implies that once thesystem assumes the active form it is subsequentlydestroyed, or disappears through combinationwith substrate. Its replenishment must await,through further exudation from the general cir-culation into the local area, the addition of new

components of the activatable system which were

rapidly depleted following the introduction ofstreptokinase. The rate at which the replenish-ment occurred has depended upon factors thatcontrol the reformation of exudate. In the seriesof cases used in this study, it has been demon-strable within a few days. These findings indi-

FIG. 3. THE

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STREPTOKINASEAND STREPTONUCLEASEEFFECT IN PATIENTS

J.G. - FIBRINOUS EXUDATE

IHR 2 31 R 4 5 6 7 8 9

INJECTION IIJECTION 2 DAYS

FIG. 4. THE EFFECT OF Two INTRAPLEURAL INJECTIONS OF STREPTOCOCCALCONCENTRATEIN A CASE OF Tu-BERCULOUSFIBRINoUs EXUDATEON THE FIBRINOGEN, N.P.N., FORMOLTITRATION, TOTAL PROTEIN, FREE STREPTOKI-NASE, ACTIVE AND ACTIVATABLE FIBRIN-LYSING SYSTEMS, AND THE ANTISTREPTOKINASE TITRE

100,000 units of streptokinase were given in injection l,and 200,000 units in injection 2.

cate the self-limiting nature of the activity fol-lowing a single injection of streptokinase.

In our observations *trypsin inhibitor has notchanged after streptokinase injections and theresults indicate that this inhibitor plays little

part under these circumstances in the inactivationof the system.

An excess of free streptokinase was found to bepresent one hour after the injection but was notdemonstrable 24 to 48 hours later. The extent

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WILLIAM S. TILLETT AND SOL SHERRY

to which it may be dissipated by combining di-rectly with substrate, or by inactivation by someinhibitor, or by neutralization with specific anti-body is not at present determined. Also suggestedby the data in Figure 2 is the finding of activefibrin-lysing elements only in the presence offree streptokinase. This observation may havea bearing on the mechanism of the phenomenonas it occurs in exudates and will be given furtherstudy. However, once the activatable system hasbeen depleted, new increments of free strepto-kinase alone produce very little new active fibrin-lysing elements.

The development of specific antifibrinolytic an-tibodies following acute streptococcal infectionshas been previously demonstrated (15, 16). Thatthe injection intrapleurally of streptococcal con-centrates containing streptokinase also evokes thespecific antistreptokinase response has been men-tioned by Christensen (1), and was observed inconnection with the earlier injections which weremade primarily to estimate degrees of toxicity.

In Figure 3 evidence is presented from fivecases of the development of antistreptokinase anti-bodies that were demonstrable in pleural exudatesand followed intrapleural injections of streptococ-cal concentrates.

The rise in titre in the exudates occurred be-tween the sixth and 11th days and was noted si-multaneously in the serum.

The exact significance of the presence of spe-cific antibody in nullifying the action of thefibrin-lysing system in local areas is not at pres-ent clearly established. Subsequent reference willbe made to this point in connection with patientA. M. of Figures 7 and 8.

Figure 4 is presented for the purpose of demon-strating the total findings with respect to theircorrelation obtained serially in a single patient.In addition to the data described in Figure 1(fibrinogen and N.P.N.) and Figure 2 (activa-table fibrin-lysing system, active fibrin-lysing sys-tem, and free streptokinase) and Figure 3 (spe-cific antistreptokinase), measurements of totalprotein and formol titratable substances are in-cluded.

The patient, J. G., had acute fibrinous pleurisyof tuberculous origin which had been active forten weeks when first studied. He received two in-jections of streptococcal concentrate, the first con-

training 100,000 units and the second, three dayslater, containing 200,000 units of streptokinase.

The individual findings are comparable tothose presented in Figures 1 to 3 and they dem-onstrate the rising and falling changes previouslydescribed. The fibrinogen drops and the N.P.N.rises; the activatable fibrin-lysing system dropsas it is rapidly transformed into the active fibrin-lysing system which attacks the fibrinogen and thelocal deposit of fibrin; antistreptokinase beingevident in increased titre in the exudate on the11th day.

The total proteins have exhibited no quantita-tive changes. They have been determined ingrams rather than milligrams as has been the casewith fiArinogen. This is in agreement withChristensen's findings (unpublished) of little orno in vitro digestion of serum albumins or globu-lins.

The formol titrations which have risen coin-cident with the rise in N.P.N. after each injec-tion were of the same magnitude and probablyrepresent further evidence of the intrapleuralbreakdown of fibrinogen and fibrin.

The findings in the case of patient J. G. ofFigure 4 are similar to those obtained in eachof the other cases of Group I. In addition to theresults noted in Figures 1 to 4, the following ob-servations have been noted in this group of pa-tients. The pH of the chest fluids fell significantly(0.2 to 0.3 units) in 24 hours after the injec-tion of the streptococcal concentrate, slowly re-turning to the control values, and has been con-sidered as part of the phenomenon of increasedproteolysis. Small but significant falls in viscositywere noted, which are consistent with the break-down of the long eccentric fibrinogen moleculesand depolymerization of any small amounts ofdesoxyribosenucleic acid that may be present.Twenty-four to 48 hours after injection, the sedi-ment increased from 0.5% to 2.0%, then slowlydecreased over the next two to ten days to thecontrol levels. This appeared to be directly re-lated to the increased cellular content occurringin the pleural fluids in association with the localirritation. As noted earlier, no significant changesin the amount of trypsin inhibitor were found inthe samples of pleural fluid. Finally, the valuesfor activatable fibrin-lysing system, trypsin in-hibitor and antistreptokinase were found to agree

182

STREPTOKINASEAND STREPTONUCLEASEEFFECT IN PATIENTS 183

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The striking change in the character of the fluid is seen in the accompanying photo (Figure 6).

WILLIAM S. TILLETT AND SOL SHERRY

closely for both chest fluid and serum prior to theinjection of streptococcal concentrates. The de-crease noted in the activatable fibrin-lysing systemafter streptokinase injections occurred only locallysince no changes in this value occurred in the serumat any time. Antibody changes, however, as pre-viously described, were evident simultaneouslyboth locally and in the general circulation.

Group II. Cases of bacterial empyema, Eightpatients.

Four cases of mixed infections associated withtuberculous empyema and bronchopleuralfistula.

One case of empyema due to Friedlander'sbacillus.

One case of pneumococcal (Type XVII)empyema.

One case of post-pneumonic empyema (sterile).One case of empyema due to anaerobic strep-

tococcus.

Interesting differences were observed betweenthe constituents of purulent empyemal fluid andthose of acute fibrinous pleurisy. For example,fibrinogen has not been found to be present inthe purulent material aspirated before the in-jection of the concentrates. The supernatant por-tion of centrifuged specimens failed to producecoagulum when thrombin was added. It seemsprobable that the formation of fibrin had previ-ously occurred to a considerable degree at thesite of infection and depleted the exudate of itsfibrinogen.

In the empyemal group, the presence of de-soxyribose nucleoprotein as a significant portionof the granular sediment was regularly noted,and, as previously reported (12), constituted from30% to 70% of the total solids. In addition, al-though not recorded in detail in this article, prep-arations stained by the Feulgen method have re-vealed the presence of nucleoprotein occurringextracellularly as fibrous reticulum, granules, andamorphous plaques.

In Figure 5 comprehensive data are presentedconcerning one of the patients with empyema.The patient, T. H., had chronic pulmonary tu-berculosis, with a left bronchopleural fistula,draining thoracotomy, and a mixed empyema.After aspirating a sample of pus, streptococcalconcentrate containing approximately 160,000

units of desoxyribonuclease and 400,000 units ofstreptokinase in 10 cc. of saline were injected.The rapid and extensive liquefaction of the nu-cleoprotein by the desoxyribonuclease was demon-strated by the marked fall in viscosity from 150 Xwater to 3 x water and decrease in measurablesediment from 70%c to 23% in one hour. Thesequantitative changes persisted over the 24 hoursof sampling. Subsequent drainage of the em-pyema was temporarily facilitated following theliquefying changes. However, the lysing pro-cedure was not continued and consequently theclinical value was not extensively tested.

FIG. 6. RAPID LYsIS BY STREPTOCOCCALCONCENTRATEOF PURULENTPLEURAL EXUDATEWITHIN PLEURAL CAV-ITY OF PATIENT

Patient T. H. Chr. Pulm. Tbc, It. bronchopleural fis-tula, pyopneumothorax and thoracotomy. Streptococcalconcentrate containing desoxyribonuclease and strepto-kinase injected intrapleurally 12/4/47.

Specimen at left: Taken immediately prior to injection.Ten cc. centrifuged for one hour. Sediment 70%o-thick,pink, viscid, and containing large cheesy shreds. Vis-cosity of whole fluid 150 X water.

Specimen at right: Removed from chest one hour afterinjection. Ten cc. centrifuged for one hour. Sediment23%o-separates readily into three layers. From bottomup: small viscid layer; large pink layer of mixed red andwhite blood cells; and white layer composed of whitecells and granules. Viscosity of whole fluid 4 X water.

184

STREPTOKINASEAND STREPTONUCLEASEEFFECT IN PATIENTS

Definite increases in N.P.N. and formol titra-table material were also observed. One hourfollowing the injection the free streptokinasetitre was 640 units/cc., and subsequently fell to10 units/cc. in 24 hours. As in the cases pre-viously described, the active fibrin-lysing systemappeared but to a less degree than in the fibrinouspleuritic group.

Even though the above data indicate some de-gree of activity of the fibrinolytic system, the find-ings in this patient demonstrate the significant ac-tion of the streptococcal desoxyribonuclease.The changes in the physical character of theempyemal pus are strikingly illustrated in thephotographs shown in Figure 6. The amount oflysis of sediment that occurred at the site of infec-tion within the patient's thorax is evident and itwas to the greatest degree due to the depolymeri-zation of the nucleoprotein of the pus by the nu-clease contained in the concentrate that was in-troduced.

Similar findings were obtained in six of theeight cases of empyema. One of them is worthyof further mention. The patient, T. P., enteredthe hospital with acute lobar pneumonia of fourdays' duration. He was treated with penicillinbut his course and X-ray were suggestive ofempyema. Aspiration of the chest performed onthe tenth day of hospitalization yielded only afew cubic centimeters of thick green fluid whichwas sterile on culture. Introduction of air intra-pleurally revealed the presence of several locu-lations within the empyemal cavity. Ten dayslater, 50 cc. of thick greenish pleural fluid waswithdrawn and streptococcal concentrate con-taining approximately 40,000 units of desoxyri-bose nuclease and 100,000 units of streptokinasein 10 cc. of saline was introduced into the site ofaspiration. Examination of specimens of fluid ob-tained by subsequent repeated aspirations revealedthat the viscosity of the pus fell from 38 x waterto 14 x water in one hour, and to 5 X water in 24hours. The sediment decreased from 35%So to 14%in one hour, and to 10% in 24 hours. The N.P.N.rose from 58 mgm.% to 73 mgm.% in one hour,and to 84 mgm.% in 24 hours. Twenty-fourhours after the injection 465 cc. of thin greenish-grey fluid was removed with ease. An X-ray ofthe chest at this time revealed that the latest as-piration had effected almost complete drainage of

the empyemal cavity and that the previous evi-dence of loculation had disappeared. Followingthis tap, the temperature fell rapidly to normal,the leucocytosis disappeared within a few days,and the patient made an excellent clinical re-covery.

In one case of mixed empyema and in the onedue to Friedlander's bacillus infection, the pHs ofthe fluids were found to be very acidic (5.50, and5.21) apparently due to extensive proteolysis anddigestion (N.P.N.'s 184, and 400) that had oc-curred spontaneously as a result of the diseasesthemselves. The injected streptococcal concen-trate had no effect due to the low pH since stud-ies, in vitro, have shown that both streptokinaseand desoxyribonuclease are rapidly inactivated atthis pH.-

In cytological studies of the sediment of exu-dates, both of the acute fibrinous and the purulentgroups, one feature of special interest has beenthe consistent breaking up of clumps of whiteblood cells. This change has been evident in thecontrast between the cells of the pre-injectionspecimen and those of the sample obtained onehour after the introduction of the streptococcalconcentrate. Whether or not the clumping wasdue to the fact that the cells were made ad-herent by a coating of fibrin or fibrous nucleo-protein is not yet established. However, sincethe concentrates contained both streptokinase andnuclease a final explanation of the cellular effectawaits further study. As mentioned previouslya detailed report of the cytological studies will bethe subject of an early communication.

From the results obtained in the observationson the empyemal cases there is ample evidencethat nucleoprotein depolymerization and probablyfibrinolysis occurred, the former by the extensivedecrease in the nucleoprotein sediment and fallin viscosity, the latter by changes in the fibrin-lysing system and changes in the N.P.N. andformol titration.Group III. Cases of hemothorax, two patients.In each, the intrathoracic blood coagulum de-veloped following pneumonectomy.

Figures 7 and 8 illustrate changes in a case ofloculated hemothorax that were brought aboutby the streptococcal fibrin-lysing system.

400,000 units of streptokinase were intro-duced into a single pocket containing 71 cc. of

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187STREPTOKINASEAND STREPTONUCLEASEEFFECT IN PATIENTS

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FIG. 8. THE EFFECT OF Two INTRAPLEURAL INJECTIONS OF STREPTOCOCCALCONCENTRATEIN A CASE OF POST-OPERATIVE HEMOTHORAXON THE CHEST FLUID VOLUME, FREE STREPTOKINASE, ACTIVE AND ACTIVATABLE FIBRIN-LYSING SYSTEMS, ANTISTREPTOKINASE, AND TRYPSIN INHIBITOR OF THE CHEST FLUID

400,000 units of streptokinase were given in injection 1, and 200,000 units in injection 2.The striking clinical change is described in the text, and seen in the accompanying X-rays (Figure 7).

fluid. The concentration in this isolated area was, effect. In this instance the postoperative coagu-therefore, 5,600 units/cc. before resolution of lum prevented drainage of the intrathoracic con-the loculating bands took place. tents. However, after dissolution of the fibrinous

The legend under Figure 7 gives the essential bands and a considerable amount of the associateddetails of the findings in this patient both before blood clot it was possible during the ensuing 48and after the introduction of streptococcal con- hours to drain by thoracentesis a large proportioncentrate and illustrates objectively the fibrinolytic of the hemothorax which had become liquefied.

WILLIAM S. TILLETT AND SOL SHERRY

The multilocular condition had been transformedinto a unilocular state. It seems likely that thefibrinolytic system was chiefly operative.

It is of special interest to note that this patienthad a high titre of antifibrinolysin (antistrepto-kinase) at the beginning of the study. Althoughnot proved by cultures it seems likely that he hadpreviously had one or more infections with hemo-lytic streptococci since the height of antibody titreis consonant with that previously found in patientsconvalescent from known infection with hemolyticstreptococci.

In spite of the presence of immune bodies, how-ever, impairment of the action of the streptococcalproduct was not apparent. It is not possible at thepresent time to offer an explanation of these find-ings. However, the possibilities suggest them-selves either that a sufficient amount of strepto-kinase was injected to bind with antibody andleave an excess, or that the active lysing systemwas put in operation before the antibody tookeffect. Further study of this phase of the prob-lem is required. Also evident from the data, asin the results with the previous cases, is the fallin the activatable fibrin-lysing system coincidentwith the appearance of an active fibrin-lysing sys-tem, and its slow return toward the control value.Trypsin inhibitor did not change despite the ac-tivation and rapid inactivation of the fibrin-lysingsystem.

The course in the patient just described ap-pears to represent an example of a beneficialtherapeutic result effected by the action of thestreptococcal fibrin-lysing system.

The second patient with postoperative hemo-thorax will be referred to only briefly since heis still under observation. Thirty-two days post-operatively the patient was transferred to ourService because it was impossible to obtain morethan a few cubic centimeters of fluid from thepostoperative side of the thorax. X-ray examina-tion revealed a large shadow with fluid levels oc-cupying approximately one-half of the thoraciccavity. From three aspirations at different sitesa total of 3 cc. was obtained. Into each of thesites streptokinase was injected up to a total of250,000 units. In the preparation of concentrateemployed in this case almost all of the nucleasehad been eliminated. Consequently only the fibrin-olytic system was operative to any significant

degree. Twenty-four hours later 600 cc. of thinbrownish fluid were removed by thoracentesisfrom a single site without difficulty. Immediatelyafter the aspiration, by fluoroscopy and X-ray,only a small amount of effusion remained. Al-though fluid reaccumulated over the next fewdays, successful aspirations were readily performedindicating a considerable reduction in the pres-ence of loculation, and the creation of a favorablesituation for further intercostal drainage.

DISCUSSION

A group of unique enzymatic activities havebeen utilized to produce changes in fibrinous,purulent and sanguinous exudations within thepleural cavities of patients. Thus through themediation of substances elaborated by hemolyticstreptococci, fibrin is caused to undergo lysis;fibrinogen is altered so that it no longer can as-sume the solid form of fibrin; and the coarse sedi-ment of purulent exudate (primarily desoxyribosenuceloprotein) is degraded to a thin solution..

Once it had been well established by detailedpreliminary observations that the methods ofpurification of the streptococcal products devel-oped extensively by Christensen (1) yieldedpreparations that were progressively less toxic,although maintaining constant or increased po-tency, a demonstration of the occurrence and de-gree of action in patients of the active principles-streptokinase and desoxyribose nuclease-be-came a center of interest. For this purpose quan-titative estimations were made of the results ofthe action of each enzymatic system on its re-spective substrate in the areas of disease. Therapidity of the beginning of action, and the dura-tion of continuing action after a single, and insome instances after a second injection, were de-termined.

Definitive enzymatic changes of a significantdegree were regularly obtained and were demon-strable within an hour after the injection and en-dured for several days before the effect sub-sided and disappeared. The phenomenon was aself-limiting one following each injection.

The clinical courses of the patients have beenfollowed by frequent physical, laboratory, andX-ray examinations over extended periods. Fol-lowing the injections a febrile response occurred

188

STREPTOKINASEAND STREPTONUCLEASEEFFECT IN PATIENTS

not infrequently and there was also evidence oflocal irritation. Both signs of reaction were,however, transient.

Since the periods, in which the action of theconcentrates was operative, were limited to a fewdays, striking evidence of favorable alterations ofa permanent nature was not expected nor defi-nitely demonstrated except for the results ob-tained in cases of loculated hemothorax, one ex-ample of which is described in detail, and in someof the instances of empyema. In patients withhemothorax, it is obvious that consideration mustbe given to the broad principles and problems ofthoracic surgery before the application of the pro-cedure may be most advantageously made.

The findings, however, constitute a backgroundagainst which the scope of the study is beingbroadened to include a consideration of the pos-sible usefulness of the procedure when utilizedunder appropriate circumstances. The desira-bility of causing the liquefaction of fibrin or ofpreventing its formation, or of resolving purulentnucleoprotein containing sediments, would ontheoretical grounds depend upon a variety of con-ditions associated with the pathogenesis and ex-pected evolution of the disease processes to whichthis method involving lytic activities might be ap-plied. Comparable principles would also be ap-plicable to exudative diseases involving locationsin the body other than the pleural area.

If the solid increments constitute part of anadvantageous walling off process then the lique-faction of the wall might promote a spread of theinfection in its acute phases before the patients'immunity was sufficient to restrain the dissemina-tion of the infection. On the other hand, the samewalling off may prevent the introduction of anti-bacterial reagents of an immunological nature, aswell as antibiotic substances, into the field ofdisease. Under the latter circumstances elimi-nation of the wall might be advantageous. Othersets of conditions concern factors that are involvedin the ultimate formation of scar tissue, or of ad-hesions, and the permanent thickenings that arerelated to the final organization of infected areas.

The results presented in this article serve as abasis for extending the study along the lines thathave been discussed.

SUMMARY

1. Twenty-three patients suffering from exu-dative pleurisies associated with different types ofdiseases have received intrapleural injections ofpartially purified concentrates containing strepto-coccal fibrinolysin (streptokinase) and desoxy-ribose nuclease.

2. Intrapleural fibrinolytic and proteolyticchanges due to the activity of the fibrin-lysingsystem were demonstrable in samples of the exu-dates taken at repeated intervals after the in-jection. The effects were self-limiting after eachinjection.

Intrapleural depolymerization of the nucleopro-tein of the solid sediment of the exudates wasalso demonstrable in a similar manner.

3. When toxic manifestations occurred follow-ing the injections, they were limited to transientfebrile reactions with general malaise, and a localoutpouring of leucocytes of a few days' duration.

4. The possibility is discussed of influencingfavorably by the enzymatic systems the course ofexudative types of diseases and it is being givenadditional study.

The authors wish to acknowledge the excellence of thetechnical assistance which they have received from MissHarriet Heffernan, Mr. John Goeller, and Miss DorisBitz.

BIBLIOGRAPHY

1. Christensen, L. R., Methods for measuring the ac-tivity of components of the streptococcal fibrino-lytic system, and streptococcal desoxyribonuclease.J. Clin. Invest., 1949, 28, 163.

2. Tillett, W. S., and Garner, R. L., The fibrinolyticactivity of hemolytic streptococci. J. Exper. Med.,1933, 58, 485.

Garner, R. L., and Tillett, W. S., Biochemical studieson the fibrinolytic activity of hemolytic strepto-cocci. I. Isolation and characterization of fibri-nolysin. II. Nature of reaction. J. Exper. Med.,1934, 60, 239 and 255.

3. Review article for references up to 1938: See Tillett,W. S., The fibrinolytic activity of hemolytic strep-tococci. Bact. Rev., 1938, 2, 161.

4. Christensen, L. R., Streptococcal fibrinolysis: a pro-teolytic reaction due to a serum enzyme activatedby streptococcal fibrinolysin. J. Gen. Physiol.,1945, 28, 363.

5. Milstone, H., A factor in normal human blood whichparticipates in streptococcal fibrinolysis. J. Im-munol., 1941, 42, 109.

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WILLIAM S. TILLETT AND SOL SHERRY

6. Christensen, L. R., and MacLeod, C. M., A proteo-lytic enzyme of serum: characterization, activation,and reaction with inhibitors. J. Gen. Physiol.,1945, 28, 559.

7. Kaplan, M. H., Studies of streptococcal fibrinolysis.I. The dissimilarity of serum protease and trypsinas indicated by the separate specificities of theirkinases, fibrinolysin and enterokinase. II. The in-hibition of streptococcal fibrinolysis by antifibri-nolysin and antiprotease. J. Clin. Invest., 1946,25, 331 and 337.

8. Ratnoff, 0. D., Studies on a proteolytic enzyme inhuman plasma. I. The probable identity of theenzymes activated by chloroform and by filtratesof cultures of beta hemolytic streptococci. II.Some factors influencing the enzymes activated bychloroform and by streptococcal fibrinolysin. J.Exper. Med., 1948, 87, 199 and 211.

9. Loomis, E. C., George, C., Jr., and Ryder, A.,Fibrinolysin: nomenclature, unit, assay, prepara-

tion and properties. Arch. Biochem., 1947, 12, 1.10. Ferguson, J. H., Travis, B. L., and Gerheim, E. B.,

Fibrinogenolytic demonstration of activation andinhibition of tryptase in plasma protein fraction-I("antihemophilic globulin"). Proc. Soc. Exper.Biol. & Med., 1947, 64, 285.

11. Astrup, T., and Permin, P. M., Fibrinolysis in animalorganism. Nature, 1947, 159, 681.

Permin, P. M., Properties of the fibrinokinase-fibrinolysin system. Nature, 1947, 160, 571.

12. Sherry, S., Tillett, W. S., and Christensen, L. R.,Presence and significance of desoxyribose nucleo-protein in the purulent pleural exudates of patients.Proc. Soc. Exper. Biol. & Med., 1948, 68, 179.

13. Tillett, W. S., Sherry, S., and Christensen, L. R.,Streptococcal desoxyribose nuclease: significance inlysis of purulent exudates and production by strainsof hemolytic streptococci. Proc. Soc. Exper. Biol.& Med., 1948, 68, 184.

14. Christensen, L. R., Protamine purification of strep-tokinase and effect of pH and temperature on

reversible inactivation. J. Gen. Physiol., 1947, 30,465.

15. Tillett, W. S., Edwards, L. B., and Garner, R. L.,Fibinolytic activity of hemolytic streptococci. Thedevelopment of resistance to fibrinolysis followingacute henmolytic streptococcal infections. J. Clin.Invest., 1934, 13, 47.

16. Tillett, W. S., The occurrence of antifibrinolyticproperties in the blood of patients with acute hemo-lytic streptococcus infections. J. Clin. Invest.,1935, 14, 276.

190