FIBRINOGEN CLOTTING AND FIBRINO-PEPTIDE FORMATION BYSTAPHYLOCOAGULASE AND THE COAGULASE-REACTING FACTOR

MARGARET C. DRUMMOND AND MORRIS TAGERDepartment of Microbiology, Division of Basic Health Sciences, Emory University, Atlanta, Georgia

Received for publication 15 October 1962

ABSTRACT

DRUMMOND, MARGARET C. (Emory University,Atlanta, Ga.) AND MORRIS TAGER. Fibrinogenclotting and fibrino-peptide formation by staphyl-ocoagulase and the coagulase-reacting factor. J.Bacteriol. 85:628-635. 1963.-The mechanism offibrinogen clotting by staphylocoagulase and thecoagulase-reacting factor (CRF) was studied fromthe standpoint of the products of the reaction,and compared with thrombin clotting of fibrino-gen. Both clotting principles effect clotting withthe release of nonprotein nitrogenous material,which can be resolved into three peptides byDowex-50 chromatography. On the basis of thesedimentation constants, electrophoretic mobili-ties, and end-group analyses of these peptides, itappears that the products of fibrinogen clottingby coagulase-CRF and by thrombin are similar.

Since Loeb (1903) first demonstrated theclotting of goose plasma by staphylococci, con-siderable interest and study have centered aroundthis phenomenon. It is now accepted that co-agulase, derived from staphylococci, is capable ofeffecting the transformation of soluble fibrinogento insoluble fibrin only in the presence of anaccessory plasma factor, referred to as thecoagulase-reacting factor (CRF). Little, however,is known regarding the chemical nature or themechanism of action of the clotting principleproduced when coagulase and CRF interact.

In spite of certain as yet unexplained differ-ences, the analogy between the coagulase-CRFclotting principle and thrombin is striking, andhas been strengthened by recent findings withpurified materials. Like thrombin, and certainother proteins with coagulating capabilities, co-agulase incubated with the plasma factor pos-sesses esterase activity on a synthetic substrate,N-a-toluene-p-sulfonyl-L-arginine methyl ester(TAMe; Haughton and Duthie, 1959). Likethrombin, coagulase-CRF is inhibited in its

fibrinogen-clotting and TAMe-esterase activitiesby diisopropylfluorophosphate but is unaffectedby soybean trypsin inhibitor (Drummond andTager, 1962b).The chemical basis of thrombin action on

fibrinogen has been extensively studied in recentyears. Thrombin clotting is now generally ac-cepted as being initiated by limited proteolysis,which cleaves from fibrinogen certain acidicpeptides, and which renders the remainder of thefibrinogen molecule capable of secondary poly-merization and gel formation (Scheraga andLaskowski, 1957). Lorand (1952) demonstratedthe release of nonprotein nitrogen from fibrinogenunder the catalysis of thrombin, and others(Bailey and Bettleheim, 1955; Lorand andMiddlebrook, 1952) showed that bovine fibrino-gen possesses only glutamyl and tyrosyl N-termi-nal residues, whereas fibrin has tyrosyl and glycylresidues. Blomback and Vestermark (1958) suc-cessfully chromatographed the thrombin-inducedfibrino-peptides.The present studies deal with (i) the chemical

isolation of the reaction products resulting fromthe interaction between the coagulase-CRFclotting principle and purified bovine fibrinogen,and (ii) a comparison of these products withthose obtained from thrombin-catalyzed fibrino-gen clotting. On the basis of the reaction products,an analogy is made between coagulase-CRF-in-duced and thrombin-induced clotting of fibrino-gen.

MATERIALS AND METHODS

Staphylocoagulase. Partially purified staphylo-coagulase was prepared by the method of Tager(1948). The material, at a protein concentrationof approximately 2%, was homogeneous uponultracentrifugation at 259,700 X g in a Spincoultracentrifuge (model E), giving an uncorrectedsedimentation constant of 1.36 X 10-13. Despitethis apparent homogeneity, similar preparationshave been shown to possess several enzymatic

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activities (Drummond and Tager, 1959, 1962a),suggesting the partial nature of their purification.The final (NH4)2SO4 precipitate was subjected

to a limited dialysis only, because of the labilityof the purified coagulase, and was lyophilized.Clotting activity was assayed by adding 0.5 mlof noninhibitory plasma to 0.5 ml of serial two-fold dilutions of coagulase in 2% peptone-saline(Merthiolated, 1:5,000). (Plasma was determinednoninhibitory by the following procedure. Out-dated blood-bank human plasma containing cit-rate, dextrose, and anticoagulant was assayedagainst a 5-day culture filtrate of Staphylococcusaureus strain 104 by adding 0.5 ml of plasma to0.5 ml of doubling dilutions of the culture filtrate.Those plasmas which clotted the stock culturefiltrate at dilutions in excess of 1:640 after over-night incubation at 38 C were considered non-inhibitory.) The titer was taken as the reciprocalof the dilution of coagulase in the last tubeshowing a solid (invertible) clot after incubationat 38 C for 24 hr. The coagulase preparationemployed throughout these studies had a clottingtiter of 2,048 per mg of lyophilized powder, or40,960 per mg of protein.

Coagulase-reacting factor. CRF was purifiedfrom outdated blood-bank human plasma by thecolumn chromatographic method (Tager, 1956).After limited dialysis and lyophilization, thepreparations employed in these studies contained4.5 and 8.6% and 7.4 and 9.2% prothrombin asassayed by the conventional two-stage pro-thrombin method (Wagner et al., 1955). Throm-bin contamination was negligible.

Fibrinogen. Armour fraction I bovine fibrinogenwas further purified by (NH4)2SO4 precipitation(Laki, 1951). The final step was a prolonged (60hr) dialysis at 8 to 10 C against 0.3 M KC1 toensure the elimination of extraneous peptides.Measurement of peptides. Column fractions

were assayed for peptide content by a modifiedLowry method (Blombiick and Vestermark,1958). Samples (0.25 ml) were taken for analysis,the extinction being read at 700 m, in a ColemanJunior spectrophotometer. Peptide values areexpressed as equivalents of bovine albumin(Armour), determined by micro-Kjeldahl analysisto be 97% protein.Paper electrophoresis. Paper electrophoresis was

performed on a Spinco apparatus, employingWhatman no. 3 mM paper strips, 0.1 M phosphatebuffer (pH 7.0), and 220 v per eight strips of

paper. After air-drying, the strips were dipped orsprayed with a solution of 0.25% ninhydrinreagent in acetone containing 5% (v/v) aceticacid (Gladner et al., 1959).

Ultracentrifuge studies. For analysis by ultra-centrifugation, the peptide materials were driedat 30 C by use of a Rinco flash evaporator. Thetemperature of the water bath was then raisedto 40 C and maintained for several hours, to ridthe peptide material of excess ammonium acetate.The sample was then dissolved in 0.1 M acetatebuffer (pH 4.8). Measurements were made in aSpinco analytical ultracentrifuge (model E), usinga synthetic boundary cell.

Analysis of N-terminal amino acids. The fluoro-dinitrobenzene method of Sanger (1945) wasemployed for the determination of the N-terminalamino acids of the clot-induced peptides. Thepeptides (1 to 5 mg) were dissolved in 0.2 M(NH4)2CO3 and placed in reaction with anethanolic solution of fluorodinitrobenzene(FDNB; 2.5%, v/v) at 40 C for 2 to 4 hr. Afterether extraction to remove excess FDNB, andovernight lyophilization, the dried material washydrolyzed with constantly boiling HCl at 105 Cin an electric autoclave (American Sterilizer Co.,all-purpose junior autoclave) for periods of 4 and24 hr. The dinitrophenyl (DNP)-labeled aminoacids were extracted by ether and further freedfrom contaminating FDNB degradation productsby sublimation, using a cold finger apparatus,Dry Ice, and a water bath at 60 C. The DNP-amino acids were identified by two-dimensionalpaper chromatography, employing the toluene-pyridine-chloroethanol solvent and 1.5 M phos-phate recommended by Fraenkel-Conrat, Harris,and Levy (1958).

RESULTS

Preparation of peptide mixture derived fromfibrinogen clotting by coagulase-CRF. For clotting,the fibrinogen concentration was adjusted to 1 %,and the ionic strength of the KCl diluent to 0.15.The resulting pH was 6.7. The solution waswarmed to 38 C prior to the addition of theclotting agents.

Coagulase (712 mg of protein) and CRF (177mg of protein) were each dissolved separately in30 ml of 0.067 M phosphate buffer (pH 6.8) andwarmed to 38 C. The two solutions were subse-quently mixed and incubated for 20 min for

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maximal generation of the clotting principle(Haughton and Duthie, 1959).The entire contents of the coagulase-CRF

mixture were then added to 900 ml of the 1%fibrinogen, mixed, and incubated at 38 C. Thesolution became opaque in approximately 3 min,and semisolid in 6 min. Incubation was continuedfor 30 min post-clotting, to ensure the maximalliberation of fibrino-peptides (Lorand, 1952).

After coagulation was complete, the solid clotwas cut into smaller pieces and homogenizedbriefly in a Waring Blendor. Upon subsequentcentrifugation, the fibrin packed solidly at thetop of the centrifuge tubes and could be removedwith a spatula, leaving the clear clot liquorbeneath.The proteins of the clot liquor were precipitated

by the addition of one-fourth volume of 40%trichloroacetic acid. After gravity filtration(Whatman no. 2 paper), the clear filtrate wasextracted with diethyl ether to remove the tri-chloroacetic acid. Preliminary hand extractionwas followed by continuous cold liquid-liquidextraction for 8 to 16 hr. After extraction, theclear filtrate was taken to dryness on a Rincoflash evaporator, at a temperature never iDexcess of 27 to 28 C. The residue was reconstitutedwith 0.1 M ammonium formate buffer (pH 3.5)preparatory to chromatography.Removal of salts from the clot liquor. Column

chromatography was employed to remove thenonvolatile salts present in the clotting mixture.A column (5 by 15 cm) of Dowex-50 X 2 ion-exchange resin, in the ammonium form, wasequilibrated with 0.1 M ammonium formate(pH 3.2). After addition of the clot material, thecolumn was washed with 1 liter of 0.1 M am-monium formate (pH 3.5) to remove the re-maining nonvolatile salts. The protein content ofthis wash was essentially negative. The peptideswere eluted from the column with 0.2 M am-monium acetate (pH 5.1), 10-ml fractions beingcollected on an automatic fraction collector at arate of 5 ml per min. The nonprotein nitrogenousmaterial was collected without loss in a volumeof approximately 240 ml. Prior to separationchromatography, this material was evaporatedas before, and reconstituted in 0.1 M formate(pH 3.2).

Resolution of the fibrino-peptides by columnchromatography. A Dowex-50 X 2 ion-exchangeresin column (2 by 45 cm), equilibrated with

0.1 M formate (pH 3.2), was employed for thedifferential separation of the peptides comprisingthe desalted clot material. After addition of thepeptide material, the column was washed with0.1 M ammonium formate (pH 3.5) and sequen-tially eluted with 0.1 M ammonium acetate (pH4.4), 0.1 M ammonium acetate (pH 4.8), and 0.2 Mammonium acetate (pH 5.2; Blombiick andVestermark, 1958). Fractions (5 ml) were col-lected automatically by gravity flow at a rate of20 to 30 ml per hr.

After measurement of elution fractions forprotein, those fractions comprising a single peakwere pooled, evaporated to dryness, and warmedslightly (40 C) to remove the acetate salt. Theseveral fractions were reconstituted in 0.1 Macetate (pH 4.8) for subsequent analysis.

Figure 1 illustrates a typical chromatogramobtained by the methods described. Although theseparation is incomplete, it is apparent that fourpeptides have been separated from the clotliquor resulting from the clotting of purifiedfibrinogen by coagulase-CRF. Adopting a modifi-cation of Blomback's nomenclature, we havedesignated as E, Al, and A2 the three peptidessuccessively eluted by the acetate buffer at pH4.4, and as B the single peptide eluted by theacetate at pH 4.8. All tubes comprising a singlepeak were pooled and lyophilized several times.

In four separate experiments, employing differ-ent but similarly prepared samples of coagulase,

-NH4 FORMATE-4 - NH4 ACETATE -'I'Z QI M, pH alIM.pH4.4 IM,pH4E 0.2M,pH520

~ 2

"-.0.8 - - '.

0-a-~~~~~~~~~~~~AAf

FRACTION NUMBER

FIG. 1. Fibrino-peptides resulting from the clottingof purified bovine fibrinogen by coagulase-CRF, asseparated by Dowex-50 column chromatography.Concentration expressed as mg of protein (mg ofalbumin equivalents) per ml of each fraction. Thedisconnected graph line between subsequent buffersindicates that other fractions were collected whichwere essentially negative for protein. Experimentaldetails in text.

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reacting factor, and fibrinogen, the chromato-graphic profiles of the four fibrino-peptides were

completely reproducible, although the yields ofeach varied considerably. Because of the extremehygroscopic nature of the peptide materials andthe inherent inaccuracies of dry-weight determi-nations, yields were calculated on the basis of theprotein contents of all component tubes of eachindividual peptide peak. In the particular experi-ment cited in Fig. 1, the yields of peptides E,Al, A2, and B were 5.8, 22.0, 11.5, and 24.1 mgof protein, respectively.

Preparation of thrombin-inducedfibrino-peptides.For purposes of comparison, fibrino-peptidesderived from the clotting of fibrinogen by throm-bin were prepared under conditions essentiallysimilar to those described by Blombaick andVestermark (1958). Clotting was accomplishedby the addition of bovine thrombin (commercialproduct, The Upjohn Co., Kalamazoo, Mich.) ata final concentration of 1 unit per ml to 0.7%purified fibrinogen at pH 7.3 and 38 C. Afterclot retraction, trichloroacetic acid precipitationand ether extraction of the clot liquor were

carried out as described in detail above for thecoagulase-CRF system. Column chromatographicseparation of the resulting material, employingthe conditions described, confirmed the findingsof Blombiick and Vestermark. Figure 2 showsthe elution diagram of thrombin-induced fibrino-peptides from a Dowex-50 X 2 column. Threepeptides (E, A, and B) can be seen which re-

semble, in their chromatographic behavior, thosedescribed by Blombiick and Vestermark (1958).Comparison of the elution diagrams of the

1.2

08

0.4

20 40 70 90 150 160 210 230

FRACTION NUMBER

FIG. 2. Separation of thrombin-induced fibrino-peptides by Dowex-50 chromatography. Peptidedesignation according to Blomback and Vestermark(1958).

z

0

F-L.)< 1.2

-J

0.8

z

0:

0-

180 200 250 270 380

FRACTION NUMBER400 520 540

FIG. 3. Dowex-50 chromatographic separation ofnonprotein material resulting from the interactionof purified bovine fibrinogen and the coagulase-reacting factor (in the absence of coagulase).

fibrino-peptides released from fibrinogen bythrombin and by the coagulase-CRF clottingprinciple reveals the presence of an additionalpeptide (A2) component in the latter system. Inthree separate experiments, this additional com-

ponent was consistently present, but variedsomewhat in concentration and extent of sepa-ration from the adjacent peptide peak (Al).

It was essential to determine whether the ad-ditional (A2) peptide in the coagulase-CRF clotliquor arose from the clotting reaction itself or

originated in the participating proteins. Althoughall the proteins involved in the clotting reactionhad been dialyzed prior to use, the degree ofdialysis was limited by the lability of the ma-

terials. Also, since the reacting factor is purifiedfrom plasma and is known to possess traces ofprothrombin and thrombin, it was necessary todetermine whether the concentrations of thesecontaminating materials alone were sufficient toreact with fibrinogen in the absence of coagulasewith the release of peptides.A control experiment was carried out with

reactions of identical concentrations of the same

preparations of reacting factor and fibrinogen,under conditions of time and temperature identi-cal to those described above. There were no

visible evidences of clotting, even after incubationof the reacting factor-fibrinogen mixture at 38 C.Notwithstanding the absence of visible clotting,the mixture was subjected to the same conditionsof blending, protein precipitation, ether extrac-tion, and concentration as the solid-clot materialshad been in previous experiments.

Figure 3 shows the column-chromatographic

NH4 FORMATE < - NH4 ACETATE, -

0.IM, pH 3.5 0.IM, pH4.4 OJM,pH4.8 0.2M. pH 5.2

I - - - -@A)^.

z0

P0

-J

z

0.CD

NH4 FORMATE - NH4 ACETATE -

O.IM, pH 3.5 OjM,pH4.4 OJM, pH4.8 .2M. pH 5.2

A

E

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separation of the nonprotein material from anincubated mixture of reacting factor and fibrino-gen in the absence of coagulase. A rather broadand flat peak is apparent. This peptide waseluted under approximately the same chromato-graphic conditions of buffer and volume as theA2 peptide derived from the clotting of fibrinogenby coagulase-CRF. It is thus apparent that the"additional" peptide seen to result from co-agulase-CRF-catalyzed fibrin formation is in-herently not a specific product of the reactionbut represents either a contaminating materialinitially present in one of the reactants, or aproduct of the action of partially purified reactingfactor on fibrinogen.

Ultracentrifugal analysis. The coagulase-CRF-induced fibrino-peptides were reconstituted in0.1 M acetate buffer (pH 4.8) at a concentrationof 0.5 to 1.0% protein for analysis in the syn-thetic boundary cell of a Spinco ultracentrifuge(model E) at 259,700 X g. The sedimentationdiagrams (Fig. 4) reveal no evidence of hetero-..WW£;ww

PEPTIDE A2

PEPTIDE 42

FIG. 4. Schlieren diagrams of coagulase-CRF-indutced fibrino peptides Al, A2, and B at 16-minintervals at 259,700 X g in acetate buffer.

TABLE 1. Ultracentrifugal analyses of coagulase-CRF-induced and thrombin-induced

fibrino-peptides

Peptide S20 Mol wt

Coagulase-CRF E... 0.50 tThrombin E -t

Coagulase-CRF Al.. 0.45 -tCoagulase-CRF A2. 0.41-0.48 tThrombin A ......... 0.52 2,000

Coagulase-CRF B... 0.56-0.59 -tThrombinB......... 0.56 2,400

* Sedimentation constant values of coagulase-CRF peptides were calculated on 0.5 to 1.0% pro-tein concentration in 0.1 M ammonium acetate(pH 4.8). Values for thrombin peptides as (Sn')from Gladner et al. (1959).

t Insufficient material for determination.t Not reported.

geneity. A comparison of the sedimentation con-stants in these peptides with those of thrombin-induced peptides are shown in Table 1. Themolecular weights of the thrombin peptides,calculated from sedimentation and diffusionvalues, are included for purposes of comparison.Insufficient material in these studies preventedthe determination of molecular weights of thecoagulase-CRF-induced peptides.Paper electrophoresis. Electrophoresis of the

coagulase-CRF-induced fibrino-peptides wascarried out employing a Spinco paper electro-phoresis apparatus, Whatman no. 3 MM paper,and 0.1 M phosphate buffer (pH 7.0). Samples ofpeptides A and B, as well as of the original un-resolved clot liquor, were applied to 3-cm paperstrips and run at room temperature (22 C) for 16hr at a potential gradient of 9.2 v per cm-lwidth. After electrophoresis, the strips were driedand either sprayed with or dipped into a solutionof 0.25% ninhydrin in acetone-5% acetic acid(v/v; Gladner et al., 1959). From Fig. 5, it canbe seen that all peptides move toward the anode,the A peptides possessing greater mobilities thanthe B peptide. The two peptides A (1 and 2)failed to be resolved by this technique.

N-terminal amino acids of coagulase-CRF-in-duced fibrino-peptides. The FDNB method fordetermination of N-terminal amino acids was

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unfroctionated clot liquor

petiIa

peptide aI

peptide a2

pept

origin

ide b nrinin

5cm 0 5cm

FIG. 5. Paper electrophoresis of the unfractionatedclot liquor resulting from the clotting of purifiedfibrinogen by coagulase-CRF, and of the componentpeptides resolved by Dowex-50 chromatography.Electrophoresis was performed in 0.1 M phosphatebuffer (pH 7.0) for 16 hr.

applied to the fractionated peptides resultingfrom the clotting of fibrinogen by coagulase-CRF.After two-dimensional chromatography, bothpeptides Al and A2, as well as peptide E, revealedglutamic acid as their single N-terminal aminoacid. Peptide B failed to show any N-terminalamino acid, despite repeated attempts withseveral concentrations of the peptide. End-groupanalyses made on fibrino-peptides subsequentlyderived from two independent experiments con-

firmed these findings.Bettleheim (1956), using the FDNB method

of Sanger, and Blomback (1958), employing theEdman phenylthiohydantoin method, independ-ently determined the N-terminal amino acids ofthe thrombin-catalyzed fibrino-peptides, A andB. Peptide A contained glutamic acid as its soleN-terminal amino acid, whereas no N-terminalamino acid could be demonstrated for peptide B.These findings were later fully substantiated(Folk et al., 1959a; Sjoquist, Blombiick, andWallen, 1960) when degradation studies andsequential analyses were performed on the throm-

bin-induced peptides A and B. The structure ofpeptide A was completely elucidated, whereasthat of peptide B was only partially determined,due to the uncertainties regarding its N-terminalend. Sjoquist suggested that the failure of peptideB to reveal a free N-terminal amino acid may bedue to the presence of either an interfering ad-jacent radical or a ring structure involving theN-terminal end of the molecule.

DISCUSSIONThe question of whether thrombin clotting of

fibrinogen involves proteolysis, much argued formany years, has recently been resolved on afirm chemical basis. Mihalyi (1950) reporteddifferences in the electrophoretic mobilities offibrinogen and fibrin when both are dissolved inurea, and Lorand (1952) demonstrated thatlimited proteolysis accompanies the transfor-mation of fibrinogen to fibrin by showing a 3%increase in nonprotein nitrogen during the re-action. Independent work by others (Bailey andBettleheim, 1955; Lorand and Middlebrook,1952) established the fact that the glutamic acidN-terminal residues present in fibrinogen giverise to glycine in fibrin when the clotting ofpurified fibrinogen occurs. The glutamic acid wasrecoverable as the N-terminal amino acid of apeptide fraction split off during the transfor-mation. The cleaved peptide material presumablyrepresented the 3% nonprotein nitrogen fractionreported by Lorand.

Recent work (Blomback and Vestermark, 1958;Folk et al., 1959b) has demonstrated the presenceof two peptides (A and B) in this nonproteinnitrogen fraction intimately related to the clottingprocess, and has elucidated their amino acidsequences (Sj6quist et al., 1960). The exactnature and origin of the peptide E, demonstratedonly under the conditions of Dowex-50 chroma-tography described by Blomback and Vester-mark, is unknown. Blombiack and Vestermarksuggested that it may be "a contaminant of thefibrinogen preparation or ... a denaturation pro-duct of the peptides." It is of interest that peptideE was also seen in the present studies on co-agulase-CRF clotting. Its relation to the clottingreaction seems questionable, in view of the factthat it is not apparent by diethylaminoethyl-cellulose chromatography, which successfully sepa-rates peptides A and B (Folk et al., 1959a, b).With the establishment of the chemical basis

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of thrombin clotting, it seemed propitious toattempt to resolve the analogy between thrombinand coagulase-CRF clotting of fibrinogen. Re-semblances between the two clotting principles,aside from the obvious, have been established.Both possess TAMe-splitting activities. Botheffect the release of nonprotein nitrogen con-currently with clotting. The two clotting princi-ples differ, however, in the fact that coagulase-CRF clotting can occur in the presence of certainanticoagulants, indicating the lack of an absolutecalcium requirement, whereas the conversion ofprothrombin to thrombin, preliminary to throm-bin clotting, cannot.

There is precedent for the idea that solublefibrinogen might be converted to insoluble fibrinvia a clotting mechanism not predicated on therelease of peptides A and B. Reptilase, thecoagulating enzyme of the venom of Bothropsjararaca, effects fibrinogen clotting with theliberation of peptide A only.

In the present study, we examined the reactionbetween fibrinogen and the coagulase-CRFclotting principle with regard to the compositionof the nonprotein nitrogen material released whenclotting occurs, for the purpose of establishingthe relationship of this reaction to thrombinclotting. From this viewpoint, it appears that thetwo reactions are similar. Both effect clotting bya preliminary proteolysis which results in theliberation of two peptides, A and B. Sedimen-tation studies, electrophoresis, and end-groupanalysis fail to distinguish between the peptidesA, or between the peptides B, derived from theaction of the two clotting principles on fibrinogen.

ACKNOWLEDGMENT

This study was supported in part by researchgrant H-1895 from the National Heart Institute,U.S. Public Health Service.

LITERATURE CITED

BAILEY, K., AND F. R. BETTLEHEIM. 1955. Theclotting of fibrinogen. I. The liberation ofpeptide material. Biochim. Biophys. Acta18:495-503.

BETTLEHEIM, F. R. 1956. The clotting of fibrino-gen. II. Fractionation of peptide materialliberated. Biochim. Biophys. Acta 19:121-130.

BLOMBACK, B. 1958. Studies on the action ofthrombic enzymes on bovine fibrinogen as

measured by N-terminal analysis. Arkiv Kemi12 :321-334.

BLOMBACK, B., AND A. VESTERMARK. 1958. Iso-lation of fibrino-peptides by chromatography.Arkiv Kemi 12:173-182.

DRUMMOND, M. C., AND M. TAGER. 1959. Enzy-matic activity of staphylocoagulase. I. Char-acterization of an esterase associated withpurified preparations. J. Bacteriol. 78:407-412.

DRUMMOND, M. C., AND M. TAGER. 1962a. Sepa-ration of acid phosphatase from tributyrinaseand plasma clotting activities in partiallypurified staphylocoagulase. J. Bacteriol.83:432.

DRUMMOND, M. C., AND M. TAGER. 1962b. Enzy-matic activities associated with clotting offibrinogen by staphylocoagulase and coagu-lase-reacting factor and their inhibition bydiisopropylfluorophosphate. J. Bacteriol.83:975-980.

FOLK, J. E., J. A. GLADNER, AND K. LAKI. 1959a.The thrombin-induced formation of co-fibrin.II. Preliminary amino acid sequence studiesin peptides A and B. J. Biol. Chem. 234:67-70.

FOLK, J. E., J. A. GLADNER, AND Y. LEVIN. 1959b.Thrombin-induced formation of co-fibrin. III.Acid degradation studies and summary ofsequential evidence on peptide A. J. Biol.Chem. 234:2317-2320.

FRAENKEL-CONRAT, H., J. I. HARRIS, AND A. L.LEVY. 1958. Recent developments in tech-niques for terminal and sequence studies inpeptides and proteins, p. 359-425. In D. Glick[ed.], Methods of biochemical analysis, vol. 2.Interscience Publishers, Inc., New York.

GLADNER, J. A., J. E. FOLK, K. LAKI, AND W. R.CARROLL. 1959. Thrombin-induced formationof co-fibrin. I. Isolation, purification andcharacterization of co-fibrin. J. Biol. Chem.234:62-66.

HAUGHTON, G., AND E. S. DUTHIE. 1959. Theactivation of staphylococcal free coagulase byplasma constituents and the hydrolysis ofN-toluene-p-sulfonyl-L-arginine methyl ester(TAMe) by activated coagulase. Biochem. J.71:348-355.

LAKI, K. 1951. The polymerization of proteins:the action of thrombin on fibrinogen. Arch.Biochem. Biophys. 32:317-324.

LOEB, L. 1903. The influence of certain bacteriaon the coagulation of blood. J. Med. Res.10:407-419.

LORAND, L. 1952. Fibrino-peptide. Biochem. J.52:200-203.

LORAND, L., AND W. R. MIDDLEBROOK. 1952. Theaction of thrombin on fibrinogen. Biochem. J.52:196-199.

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MIHALYI, E. 1950. Electrophoretic investigationsof fibrin and fibrinogen dissolved in urea

solutions. Acta Chem. Scand. 4:351-358.SANGER, F. 1945. The free amino groups of insulin.

Biochem. J. 39:507-515.SCHERAGA, H. A., AND M. LASKOWSKI, JR. 1957.

The fibrinogen-fibrin conversion. Advan. Pro-tein Chem. 12:2-269.

SJOQUIST, J., B. BLOMBACK, AND P. WALLEN. 1960.Amino acid sequence of bovine fibrinopeptidesArkiv Kemi 16:425-436.

TAGER, M. 1948. Concentration, partial purifi-

cation, properties, and nature of staphylo-coagulase. Yale J. Biol. Med. 20:487-501.

TAGER, M. 1956. Studies on the nature and thepurification of the coagulase-reacting factorand its relation to prothrombin. J. Exptl.Med. 104:675-686.

WAGNER, R. A., J. B. GRAHAM, G. D. PENICK,AND K. M. BRINKHAUS. 1955. Estimation ofprothrombin by the two-stage method, p. 105-111. In L. M. Tocantins [ed.], The coagulationof blood; methods of study. Grune & Stratton,Inc., New York.

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