FIBRINOLYTIC, ANTICOAGULATING AND PLASMA-CLOTTING PROPERTIES OF STAPHYLOCOCCI

ERWIN NETERLaboratories of the Children's Hospital and the Department of Pathology and

Bacteriology, Medical School, University of Buffalo

Received for publication March 24, 1937

In 1908, Much showed that plasma which had been coagulatedby the plasma-clotting factor of staphylococci, may become re-dissolved. He attributed the lysis of the plasma clot to the actionof a staphylococcus fibrinolysin. His observations were con-firmed by Kleinschmidt (1909), Gonzenbach and Uemura (1916),Gratia (1921), Aoi (1932), Gengou (1933) ,Vanbreuseghem (1934),Madison (1935), and Fisher (1936). The production of powerfulfibrinolysin by hemolytic streptococci was demonstrated by Tillettand Garner (1933). Confirming Tillett and Garner's results,Dennis and Berberian (1934) found a second factor produced byhemolytic streptococci which inhibits coagulation of plasma.Both factors were considered by these authors to be antigenic.Furthermore, they reported that one strain of Streptococcusviridans produced the anticoagulant. According to Tunnicliff(1936), there is a relation between the production of anticoagulantby Streptococcus viridans and its smooth phase. Neter andWitebsky (1936) showed that the production of bacterial anti-coagulants is not limited to Streptococcus hemolyticus only. Theyfound that Streptococcus viridans, enterococci, pneumococci ofvarious types, some strains of Escherichia coli, Pseudomonas pyo-cyaneas and others may produce an anticoagulant. In a subse-quent paper (Witebsky and Neter, 1936) the differences in theproperties of the fibrinolysin and the bacterial anticoagulantswere described. The authors came to the conclusion that bothfactors are entirely independent of each other. Recently, Dart(1936) reported that the streptococcus anticoagulant may be

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separated from the streptococcus fibrinolysin by means of alcoholextraction. The streptococcus fibrinolysin and the bacterialanticoagulants are not artificial products obtained in vitro only,since both factors were found to be present in human exudates(Neter and Witebsky, 1936; Neter, 1936; Neter and Young, 1937).The following experiments are concerned with a comparative

study of the production of fibrinolysin and anticoagulant bystaphylococci in vitro and in vivo, and with the relation of thestaphylococcus anticoagulant to the plasma-clotting factor ofthese microorganisms.

TECHNIC

Various strains of staphylococci were isolated from humanlesions; they were tested for pigment production on plain agarplates and for hemolytic activity on 5 per cent human-bloodagar plates. For the determination of gelatin liquefaction, nutri-ent gelatin (Difco) was employed.For the production of fibrinolysin and anticoagulant, the re-

spective strains of staphylococci were cultured in plain and 1-per-cent-glucose infusion broth for 18 hours at 370C.1 The cultureswere centrifuged and the supernatant fluids tested according tothe technic of Tillett and Garner (1933). The plasma used forthe experiments was prepared by mixing 10 cc. of blood (human,rabbit or guinea pig blood) with 1 cc. of a 2 per cent potassiumoxalate solution. The blood was shaken thoroughly and centri-fuged. For the demonstration of either the fibrinolysin or theanticoagulant, the supernatant fluid of the respective culture inserial dilutions (volume 0.5 cc.) was mixed with 1 cc. of 1 :5 dilu-tion of plasma; then 0.25 cc. of a 0.25 per cent calcium chloridesolution in normal saline was added; the tubes were shaken thor-

1 The respective broths were always tested and found to be lacking in coagulat-ing and anticoagulating properties in the order of the experiment described. Forcomparative studies, culture media prepared by the Digestive Ferments Companywere also employed. Brain heart infusion, heart infusion broth and veal infusionmedium gave practically the same results as the meat infusion broth prepared inthis laboratory. Extract broth, however, sometimes inhibited the clotting ofhuman plasma and, therefore, could not be used for the study of the anticlottingfactor of bacteria.

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oughly and kept at 370C. The results were read at various inter-vals. The fibrinolysin is characterized by dissolution of theplasma clot, the anticoagulant by its continuous inhibition of thecoagulation of the plasma.For the demonstration of the plasma-clotting factor of staphylo-

cocci, serial dilutions of the supernatant fluid of the respectivecultures in the volume of 0.5 cc. were mixed with 0.25 cc. ofplasma, incubated at 370C. and read at various intervals.

RESULTS

Sixty strains of staphylococci of human origin were examined.Of 43 strains of Staphylococcus aureus-hemolyticus, 2 producedthe fibrinolysin in plain infusion broth, 35 the anticoagulant in1-per-cent-glucose infusion broth; 4 strains produced the fibrin-olysin as well as the anticoagulant and 2 lacked both properties.Seven strains of Staphylococcus albus-hemolyticus were tested;1 was found to produce the fibrinolysin, 4 the anticoagulant and2 strains failed to show fibrinolytic or anticlotting properties.Of 10 strains of non-hemolytic staphylococci (S. aureus and S.albus), none produced the fibrinolysin, 5 showed anticlottingproperties and 5 were negative. While the majority of staphylo-cocci isolated from human lesions showed anticlotting properties,only a relatively small percentage produced the fibrinolysin, incontradistinction to hemolytic streptococci. According to Aoi(1932) and Madison (1935), however, a higher percentage offibrinolytic staphylococci is obtained when the isolated fibrintechnic is employed; or, as in the experiments of Fisher (1936),when the observation period is extended over several days. Inthis connection it may be mentioned that on some occasions,staphylococcus fibrinolysin was found to be effective towardplasma coagulated by means of the plasma-clotting factor ofstaphylococci, but failed to dissolve the same plasma when clottedby the addition of calcium chloride solution.

Experiments were carried out to determine whether or not arelationship exists between the production of fibrinolysin andanticoagulant by staphylococci on the one hand, and their abilityto liquefy gelatin on the other. The majority of strains of

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staphylococci isolated from human lesions liquefied gelatin andproduced the staphylococcus anticoagulant. A few strains, how-ever, did not liquefy gelatin within 3 days, but produced the anti-coagulant. Two fibrinolytic strains of staphylococci tested, werefound to liquefy gelatin.The properties of Staphylococcus fibrinolysin of human strains

parallel those of the Streptococcus hemolyticus fibrinolysin withthe exception that the fibrinolysin obtained from staphylococciacts more slowly and may be effective toward human as well asanimal plasma. This latter observation corresponds to the find-ings of Madison and Dart (1936), who reported that someveterinary staphylo-fibrinolysins were effective also toward hu-man plasma. The antigenicity of the staphylococcus fibrinolysincould be demonstrated in the following ways: (1) Staphylococcusantiserum (Staphylococcus Antitoxin Lederle) neutralized spe-cifically the staphylococcus fibrinolysin as did the serum of a12-year-old boy with osteomyelitis of the right thigh of one yearduration; (2) the plasma of this patient was found to be resistanttoward the staphylococcus fibrinolysin in contradistinction toplasma obtained from normal individuals and from two childrenwith scarlet fever. The plasma of the above patient, sufferingfrom osteomyelitis, however, was susceptible toward the Strepto-coccus hemolyticus fibrinolysin.The staphylococcus anticoagulant has the same properties as

the anticoagulant produced by other microorganisms: it inhibitscoagulation continuously; it is effective toward human as well asanimal plasma; it is produced in broth containing carbohydrates,but not in plain infusion broth. When staphylococci were cul-tured in broths containing various carbohydrates, such as glucose,lactose, maltose, sucrose and mannitol, different strains producedthe anticoagulant in different carbohydrate media. The effec-tiveness of the staphylococcus anticoagulant may be equallyinhibited by either normal serum or spinal fluid, proving that afactor other than an antibody-function is responsible for thisinhibition.Experiments were carried out to elucidate whether or not the

staphylococcus anticoagulant is antigenic. For this purpose,

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Staphylococcus Antitoxin (Lederle) and the serum of the above-mentioned patient with chronic osteomyelitis were tested; bothsera failed to inhibit the effectiveness of the staphylococcus anti-coagulant to a greater extent than did normal serum or spinalfluid. Moreover, coagulation of the plasma of the above patientwith osteomyelitis was inhibited in the presence of the staphylo-coccus anticoagulant. It may be concluded, therefore, that thestaphylococcus anticoagulant is lacking in antigenicity.

According to their action on plasma, staphylococci may beclassified into one of the following four groups: (1) Staphylococcimay produce the specific fibrinolysin; (2) they may form an anti-clotting principle; (3) they may possess both properties or (4)they may lack both properties. The simultaneous presence ofstaphylococcus anticoagulant and fibrinolysin in glucose brothcould be demonstrated in two ways: (1) When the supernatantfluid of the glucose broth is titrated, the undiluted broth inhibitscoagulation, while the broth in higher dilutions causes lysis of theplasma clot. (2) When undiluted glucose broth is mixed withplasma which has been diluted with normal spinal fluid instead ofsaline solution, fibrinolysis occurs and not inhibition of plasmacoagulation, because normal spinal fluid inhibits the effectivenessof the anticoagulant only. The question arises whether a relationexists between the production of fibrinolysin or anticoagulantand the pathogenicity of staphylococci for man. The respectiveexperiments may be summarized as follows: Staphylococci pro-ducing the fibrinolysin as well as those producing the anticlottingfactor were found as causative agents in abscesses, exudates andin the blood stream in cases of staphylococcus sepsis. Both typesof staphylococci, therefore, may be virulent for man and mayinvade the blood stream.

In order to determine whether the staphylococcus anticoagulantand the staphylococcus fibrinolysin may be found in vivo, puru-lent exudates were examined. The staphylococcus fibrinolysincould be demonstrated in the supernatant fluid of a staphylo-coccus empyema in two cases as well as in the purulent exudate ofa case of osteomyelitis. The strains obtained from these lesionsalso produced the staphylococcus fibrinolysin in vitro. The

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staphylococcus anticoagulant too may be present in exudatescaused by staphylococci; it was found in the supernatant fluidof a staphylococcus empyema of a patient who suffered fromstaphylococcus sepsis; the strains isolated from the blood streamas well as from the empyema fluid in pure culture produced thestaphylococcus anticoagulant also in vitro.

Staphylococci are capable of clotting human as well as animalplasma. This plasma-clotting property has been known for manyyears and was extensively studied by Loeb, Much, Kleinschmidt,Gross, Gratia, Chapman, Fisher and others. A review of thissubject is given in the article by Fisher (1936). The question,whether the plasma-clotting factor of staphylococci might be ofsignificance in the development and course of staphylococcusinfections was recently discussed by Chapman, Berens, Petersand Curcio (1934), by Menkin and Walston (1935) and byPioan (1935).* It is of interest, therefore, to determine whetheror not the staphylococcus coagulant may be produced in naturalstaphylococcus infections. For this purpose, exudates obtainedfrom human beings suffering from staphylococcus infections wereexamined; they were -centrifuged immediately after the specimenswere obtained. The supernatant fluid in decreasing amounts(volume 0.5 cc.) was mixed with 0.25 cc. of human as well asguinea pig and rabbit plasma. Several specimens even in dilu-tions up to 1:100 caused coagulation of human and animalplasma. In one case of staphylococcus pericarditis, the super-natant fluid of the exudate (dilution 1: 2) caused clotting of humanand guinea pig plasma within 10 minutes. In this connection,it may be mentioned that the exudates in two cases also containedthe fibrinolysin and, in one case, the anticoagulant besides theplasma-clotting factor of staphylococci. It follows from theseexperiments that the staphylococcus coagulant may be found invivo and may be present simultaneously with the staphylococcusanticoagulant or fibrinolysin.

In view of the fact that staphylococci may cause as well asinhibit coagulation of plasma, the quantitative relation betweenstaphylococcus anticoagulant and the plasma-clotting factor ofthese microorganisms was examined. The following protocol

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represents a typical example: In part I of the experiment, thesupernatant fluids of (a) plain broth, and (b) glucose broth cul-tures of Staphylococcus aureus-hemolyticus in decreasing amounts(volume 0.5 cc.) were mixed with 0.25 cc. of human plasma andincubated at 370C. The resulting coagulation is recorded in

TABLE 1Relation of the plasma-clotting factor of Staphylococcus aureus-Hemolyticus to the

staphylococcus anticoagulantPart I

COAGULATION OF HUMAN PLASMA (0.25 cc.) BY SUPERNATANTDECREASING AMOUNTS OF SUPER- FLUID OF CULTURE OF STAPHYLOCOCCUS GROWN INNATANT FLUID OF BROTH CULTURE

(VOLUME 0.5 CC.) a b

Plain broth Glucose broth

(1) 0.5 cc. Coagulation No coagulation(2) 0.05 cc. Coagulation Coagulation(3) 0.005 cc. Coagulation Coagulation(4) 0 No coagulation No coagulation(5) Broth control No coagulation No coagulation

Read after one hour incubation at 370C.

Part II

INHIBITION OF COAGULATION OF HUMAN PLASMA (1.0 CC. OF A 1:5DILUTION) BY SUPERNATANT FLUID OF CULTURE OF STAPHYLO-

COCCUS GROWN INDECREASING AMOUNTS OFSUPERNATANT FLUID OF

BROTH CULTURE a b(VOLUME 0.5 CC.) Plain broth Glucose broth

After addition of 0.25 cc. CaCl2

(1) 0.5 cc. Coagulation No coagulation(2) 0.05 cc. Coagulation Coagulation(3) 0.005 cc. Coagulation Coagulation(4) 0 Coagulation Coagulation(5) Broth control Coagulation Coagulation

Read after one hour incubation at 37°C.

part I of table 1. In part II of the experiment, the same super-natant fluids were mixed with 1 cc. of 1:5 dilution of humanplasma and 0.25 cc. of 0.25 per cent solution of calcium chloride.The mixtures were incubated at 370C. and the resulting inhibitionof coagulation is recorded in part II of table 1.

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Table 1 shows that the plasma-clotting factor is present in thesupernatant fluid of plain and glucose infusion broth cultures upto a dilution of 1: 100. When undiluted glucose broth, however,is used, no coagulation is observed. Part II of the experimentoffers an explanation of these findings: The anticoagulant, presentin undiluted glucose broth, counteracts the effectiveness of theplasma-clotting factor. The staphylococcus coagulant, however,is not destroyed by the anticoagulant since it can easily be demon-strated in higher dilutions of glucose broth. The antagonisticeffect of the staphylococcus anticoagulant is not a specific onesince the enterococcus anticoagulant was also found to inhibitthe plasma-clotting factor of staphylococci.

DISCUSSION

Coagulation of exudates and dissolution of fibrin are importantfactors in bacterial infections, as for example the fibrinous exudatein pneumonia and the formation of fibrinous adhesions in certaincases of meningitis on the one hand, and the dissolution of fibrinin an infected thrombus on the other. A study of the influenceof bacteria and their products on coagulation of plasma and dis-solution of fibrin may, therefore, throw light on some phases inthe development and course of bacterial infections.

Staphylococci from human sources may produce a fibrinolysinas well as a factor which inhibits coagulation of plasma. Thestaphylococcus fibrinolysin acts upon human as well as animalplasma clots. It is an antigen and can be neutralized by specificstaphylococcus antisera. Patients with staphylococcus infec-tions may develop a staphylococcus antifibrinolysin. The staph-ylococcus fibrinolysin resembles the streptococcus fibrinolysinwhich was first described by Tillett and Garner. It is, however,antigenically different.The staphylococcus anticoagulant is produced by the majority

of strains of staphylococci isolated from lesions of man. It hasthe same properties as the anticoagulant of other microorganisms.The anticoagulant inhibits the coagulation of plasma continu-ously; it is produced in culture media containing carbohydrates.

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This anticoagulant differs distinctly from the staphylococcusfibrinolysin not only by its action upon plasma, but also by thefact that it is not antigenic.The nature of bacterial anticoagulants is not known as yet.

It is even questionable whether or not we are dealing with auniform substance. Inhibition of coagulation of plasma may beachieved in different ways: On the one hand, calcium may berendered ineffective; on the other hand, a substance may actupon fibrinogen or thrombin and its constituents. Furtherstudies in this direction are under way. The fact that the bac-terial anticoagulant is produced only in culture media whichcontain carbohydrates, suggests that the anticoagulating factormay be a derivative of carbohydrates. Various sugars are suit-able for the production of the staphylococcus anticoagulant.Because of the importance of the carbohydrates for the develop-ment of the anticoagulant, the question arises whether or not theresulting acidity of the culture medium may be responsible forthe inhibition of plasma coagulation. Preliminary experimentsby the use of the quinhydron-electrode, however, showed that achange of the pH cannot be the only factor. This is in agreementwith the recent report of Dennis and Adham (1937).Both bacterial fibrinolysin and anticoagulant are factors which

may be of significance in natural infections. It was possible todemonstrate the staphylococcus fibrinolysin as well as the staphy-lococcus anticoagulant in exudates from human beings, provingthat these substances may be produced in vivo. These resultsare in accord with the findings that fibrinolytic as well as anti-clotting staphylococci could be isolated as causative agents fromlesions in man. In this connection, it is of interest to state thatboth types of staphylococci could also be isolated from the bloodstream in cases of staphylococcus sepsis, proving that anti-coagulating as well as fibrinolytic staphylococci may exhibit inva-sive properties.

Staphylococci may produce not only fibrinolysin and anticoagu-lant; they may also form a factor which causes clotting of humanas well as animal plasma. This plasma-clotting factor of staphy-

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lococci may be of importance for the development and course ofstaphylococcus infections. It was possible to demonstrate thestaphylococcus coagulant in exudates obtained from humanbeings.The possible interaction of these three factors: fibrinolysin,

anticoagulant, and plasma-clotting factor, make an experimentalanalysis of staphylococcus infections difficult. The antagonisticeffect of the plasma-clotting factor of staphylococci toward thestaphylococcus anticoagulant could be demonstrated in vitro.The anticoagulant inhibits the plasma-clotting factor, but doesnot destroy it. The effectiveness of the plasma-clotting factorbecomes manifest in dilutions in which the bacterial anticoagulantis no longer effective.

SUMMARY

1. Staphylococci from human sources may produce eitherfibrinolysin or anticoagulant, or they may simultaneously exhibitor lack both properties.

2. The staphylococcus fibrinolysin dissolves human as well asanimal plasma clots. It can be specifically neutralized by staphy-lococcus antiserum. It is antigenically different from the Strep-tococcus hemolyticus fibrinolysin.

3. The staphylococcus anticoagulant has the same propertiesas the anticoagulant of other microorganisms: it inhibits coagula-tion of human and animal plasma continuously; it is produced inbroths containing carbohydrates; it can be rendered ineffectiveby normal serum or spinal fluid; and it is not antigenic.

4. Staphylococcus lesions in man may be due to strains pro-ducing either the fibrinolysin or the anticoagulant. Both typesof staphylococci may invade the blood stream.

5. Staphylococcus fibrinolysin as well as staphylococcus anti-coagulant may be produced in staphylococcus infections in man.

6. Patients suffering from staphylococcus infections may de-velop a specific antifibrinolysin.

7. The plasma-clotting factor of staphylococci could be demon-strated in exudates of man.

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PROPERTIES OF STAPHYLOCOCCI 253

8. The plasma-clotting factor of staphylococci may be in-hibited by anticoagulants of staphylococci or of other micro-organisms without being destroyed.

REFERENCESAoi, F. 1932 On fibrinolysis of staphylococcus. Kitasato Arch. Exper. Med.,

9, 171-201.CHAPMAN, G. H., BERENS, C., PETERS, A., AND CURCIO, L. 1934 Coagulase and

hemolysin tests as measures of the pathogenicity of staphylococci.Jour. Bact., 28, 343-363.

DART, E. E. 1936 Streptococcus anticoagulant. Proc. Soc. Exper. Biol. andMed., 35, 285-286.

DENNIS, E. W., AND BERBERIAN 1934 A study of the mechanism of invasivenessof streptococci. Jour. Exper. Med., 60, 581-598.

DENNIS, E. W., AND ADHAM, L. D. 1937 Nature of the anticlotting activity ofstreptococci in vitro. Proc. Soc. Exper. Biol. and Med., 36, 84-85.

FISHER, A. M. 1936 The plasma coagulating properties of staphylococci. Bull.Johns Hopkins Hosp., 59, 393-414.

FISHER, A. M. 1936 The fibrinolytic properties of staphylococci. Bull. JohnsHopkins Hosp., 59, 415-426.

GENGOU, P. 1933 Contribution A l'6tude de F'action du staphylocoque sur leplasma oxalat6 et sur le fibrinogene. Ann. Inst. Pasteur, 51, 14-31.

v. GONZENBACH, W., UND UEMURA, H. 1916 Beitrag zur Gerinnung von Plasmadurch Wirkung des Staphylococcus pyogenes aureus. Cntrlbl. f. Bakt.e. Parasitenkunde, 78, 97-103.

GRATIA, A. 1921 Action fibrinolytique de Staphylocoque. Arch. Internat. d.Physiol., 18, 355-357.

KLEINSCHMIDT, H. 1909 Fibrinbildende und aufloesende Wirkung von Staphy-lokokken. Ztschr. f. ImmunitAtsforsch, 3, 516524.

MADISON, R. R. 1935 Fibrinolytic staphylococci. Proc. Soc. Exper. Biol. andMed., 33, 209-211.

MADISON, R. R., AND- DART, E. E. 1936 Veterinary staphylo-fibrinolysin.Proc. Soc. Exper. Biol. and Med., 34, 299-300.

MENKIN, E., AND WALSTON, H. D. 1935 R6le of coagulating principle of Staph-ylococcus aureus in relation to invasiveness of this microorganism.Proc. Soc. Exper. Biol. and Med., 32, 1259-1263.

MUcH, H. 1908 Ueber eine Vorstufe des Fibrinfermentes in Kulturen vonStaphylokokkus aureus. Biochem. Ztschr., 14, 143-155.

NETER, E. 1936 Production of fibrinolysin in vivo. Proc. Soc. Exper. Biol.and Med., 34, 735-736.

NETER, E., AND WITEBSKY, E. 1936 On the presence of fibrinolytic substance inthe spinal fluid of patients with streptococcus meningitis. Jour. Bact.,31, 77-78.

NETER, E., AND WITEBSKY, E. 1936 Fibrinolytic activity of hemolytic strepto-cocci and other microorganisms. Proc. Soc. Exper. Biol. and Med., 34,'549-552.

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NETPOR, E., AND YOUNG, G. 1937 A case of Ludwig's angina due to Enterococcushemolyticue. Amer. Jour. Dis. Child. 53, 1531-1533.

PIJOAN, M. 1935 A study of the blood-coagulating substance produced bystaphylococci and its relation to disease. Can. Med. Assoc. Jour., 32,476-481.

TILLETT, W. S., AND GARNER, R. L. 1933 The fibrinolytic activity of hemolyticstreptococci. Jour. Exper. Med., 58, 485-502.

TUNNICLIFF, R. 1936 Effect of dissociation of streptococci on their fibrinolyticand anticlotting activity. Jour. Infect. Dis., 58, 92-97.

VANBREUS1EGHEM, R. 1934 Coagulation et Fibrinolyse du Plasma et du Fibrino-gene par la Staphylocoque et sa Staphylocoagulase. C. r. Soc. de Biol.,116, 344-346.

WITEBSKY, E., AND NETER, E. 1936 Properties of different fibrinolysins pro-duced by streptococci. Proc. Soc. Exper. Biol. and Med., 34, 858-863.

* Since this paper was submitted for publication, an article by R. Cruickshank(1937) on "Staphylocoagulase" was published in the Journal of Pathology andBacteriology, 45, 295-303.

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