Activation of Coagulase Clotting by Trypsin Inhibitor

Transcription

1 APPLIED MICROBIOLOGY, Nov. 1969, p Copyright 1969 American Society for Microbiology Vol. 18, No. 5 Printed In U.S.A. Activation of Coagulase Clotting by Trypsin Inhibitor D. S. ORTH, A. W. ANDERSON, AND M. W. MONTGOMERY Department of Microbiology and Department of Food Science and Technology, Oregon State University, Corvallis, Oregon Received for publication 28 July 1969 Egg white trypsin inhibitor activated coagulase clotting when added to a final concentration between 2 and 6 mg/ml. The greatest increase in clotting rate was observed in reaction mixtures containing the lowest concentrations of serum and plasma. Maximal activation was reached with 4 mg of trypsin inhibitor per ml when either serum or plasma was used as the source of coagulase-reacting factor (CRF). The increased rate of clotting is partly due to inhibition of plasmin. Freeing and thawing reduced plasma clotting inhibition. Soybean trypsin inhibitor also activated the coagulase reaction. The increased rate of clotting was observed with a coagulase preparation from organisms which produced plasminogen activators and with the culture supernatant fraction from organisms which did not activate plasminogen to plasmin. The tube test for coagulase could be made more sensitive for some strains of staphylococci by increasing the concentration of CRF (added as plasma or serum) by adding trypsin inhibitor, or both. The ability of certain staphylococci to clot blood plasma was first reported by Loeb in 193 (1). Coagulase was shown by Smith and Hale (14) to coagulate fibrinogen only in the presence of a third factor found in the plasma of certain animals (3). This plasma factor was called coagulase-reacting factor (CRF) by Tager (15). The coagulase test is currently recognied as one of the best in vitro criteria for determining the potential pathogenicity of a staphylococcus. Although many variations of coagulase testing have been devised, i.e., the tube test, the plate test, and the serum soft agar test, all tests for free coagulase require a suitable plasma substrate. In 198, Much showed that diluted plasma was often more satisfactory than the same plasma used full strength, due to dilution of plasma inhibitors (4). The standard tube test for coagulase (5, 9) uses plasma diluted 1:3. The routine coagulase test has been found to be unable to detect small amounts of coagulase produced by certain strains of staphylococci. Sword et al. (C. P. Sword, T. Shikashio, M. G. Sword, and M. K. Martin, Bacteriol. Proc., p. 11, 1961) showed that several strains of staphylococci produced small amounts of coagulase which was demonstrable only after concentration procedures. These findings indicate the need for a more sensitive coagulase test. Plasmin is a blood serum protease which interferes with the coagulase reaction (16). Plasmin is inactivated by soybean trypsin inhibitor (13). It seemed likely that trypsin inhibitor could inactivate some of the plasma inhibitors referred to by Much. The purpose of this study was to determine if the coagulase assay system could be made more sensitive by adjusting the CRF level and adding trypsin inhibitor. MATERIALS AND METHODS Cultures. Staphylococcus aureus was received from E. P. Casman of the Food and Drug Administration. This strain produced coagulase, Muller factor, staphylokinase, and protease. S. aureus ATCC 1995 was received directly from the American Type Culture Collection. This strain was coagulase positive, but did not elaborate protease or plasminogen activators. Partial purification of coagulase. Difco Brain Heart Infusion (BHI) broth culture of S. aureus (9 ml) was grown at 35 C for 12 hr. This served as the inoculum for 3 liters of BH1 broth in a Fermacell Fermenter growth chamber. The culture was grown at 35 to 37 C for 48 hr with aeration at the rate of 3.5 liters/min. Sterile 1 N NaOH and HCO were added automatically to maintain the ph at 7.2, and Antifoam C (Dow Corning) was added to minimie foaming Ȧt the completion of the growth period, the culture liquid was largely cleared of cells by a Sharples supercentrifuge at approximately 62, X g. The supernatant fraction was treated by the method of Blobel et al. (1) to yield partially purified coagulase. After lyophiliation, the tan powder was stored at -15 C. The coagulase preparation was not handled aseptically, but no decrease in coagulase activity was observed after storage for one year. 96 Downloaded from on October 1, 218 by guest

2 VOL. 18, 1969 COAGULASE CLOTTING BY TRYPSIN INHIBITOR 97 Preparation of reactants. Partially purified coagulase was added to.1 M sodium citrate buffer, ph 7., to give a suspension containing 1 mg/ml. This buffer was used in all experiments. Desiccated rabbit coagulase plasma (Difco) was rehydrated according to the manufacturer's directions to a 1:3 dilution. Fresh rabbit serum was obtained from the blood of three adult rabbits. The sera were pooled and concentrated twofold by dialysis against 5% (w/v) polyethylene glycol 6 (Baker Chemical Co.) for 6 hr at 4 C. Citrated fibrinogen (Bovine fraction 1, 66% clottable; Sigma Chemical Co., St. Louis, Mo.) was added to the citrate buffer to give a suspension containing 5 mg/ml. Trypsin inhibitor (egg white, B grade; each milligram inhibiting approximately.7 mg of trypsin; Calbiochem) was added to the citrate buffer to give a suspension containing 2 mg/ml. All components of the reaction mixture were dialyed for 8 hr at 4 C by using a "continuous-flow" system in which the above citrate buffer was continuously replenished at the rate of 15 ml/hr. This buffer was used for dilutions to minimie ph and ionic strength differences of the reaction components. The reactants were maintained at 4 C or were froen (-15 C) and thawed immediately before use. During the preparations for the tests, the clotting mixture components were maintained in an ice-water bath ( C). Timing was started when each series of tubes was placed in the water bath (37 C). The tests were performed with a 1-ml reaction mixture in tubes (12 by 74 mm). Determination of coagulase reaction velocity. Clotting times were determined at 1-min intervals by gently tipping the individual tubes. The extent of clotting was estimated (from negative to 4+) by the procedure of Turner and Schwart (18). When fibrin gel formation was noticed, readings were made at.5- min intervals until the tube containing the reaction mixture could be inverted without the fibrin gel being dislodged-a 4+ reaction. The effect of egg white trypsin inhibitor (EWTI) on the rate of coagulase clotting was determined with EWTI concentrations from to 6 mg/ml. The substrates were 1, 2, 3, and 4% plasma, or 1, 2, 3, and 4% serum + 1% plasma. All tubes contained 1% fibrinogen preparation, 1% coagulase suspension, and sufficient citrate buffer to bring the volume of the reaction mixture to 1 ml. The reaction velocity data are presented by plotting the time taken to produce a 4+ fibrin gel as a function of serum or plasma concentration for each level of trypsin inhibitor used. S. aureus 1995 coagulase clotting. S. aureus 1995 was inoculated into 1 ml of BHI broth in a 25-ml DeLong culture flask. The culture was incubated at 35 C for 48 hr on a rotary shaker at 21 rev/min. After growth, the cells were removed by centrifuging at 4,8 X g for 1 min. The clarified supernatant (5 ml) was dialyed against continuously flowing citrate buffer, and coagulase tests were performed with a 1% serum reaction mixture as described above. The extent of clotting was determined at 15-mnn intervals. RESULTS The effect of EWTI concentration on the clotting reaction with froen and thawed plasma is shown in Fig. 1. All concentrations of trypsin inhibitor used increased the rate of clotting over Downloaded from CS~r\ 4-H w LLJ F- F- U 3Ot II * CONTROL 2 MG/ML EWTI A 1OMG/ML EWTI o 2MG/MLEWTI - * 4MG/MLEWTI a EOMG/MLEWTI : 2- bj -j i H * CONTROL o 2MG/MLEWTI * IOMG/MLEWrg 2MG/MLEWTI * 4MG/MLEWTI A 6MG/MLEWTI on October 1, 218 by guest % PLASMA % SERUM FIG. 1. EWTI activation of coagulase clotting using plasma (diluted 1:3) as the source of CRF. 4 5 FIG. 2. EWTI activation of coagulase clotting using serum (concn 2-fold) as the primary source of CRF.

3 98 ORTH, ANDERSON, AND MONTGOMERY APPL. MICROBIOL. 2 i 3. LUI 2 PLASMA FIG. 3. EWTI activation of coagulase clotting using fresh plasma (diluted 1:3) as the source of CRF. that of the control. When compared to the control, 4 and 6 mg of EWTI per ml decreased the time to produce a firm gel by 5%. Since the reaction velocities produced by 4 and 6 mg of EWTI per ml were nearly identical, the upper limit of activation was reached in these experiments. Figure 1 also shows that the increased plasma concentration resulted in more rapid clotting at each concentration of EWTI tested. The greatest increase was observed with the control, whereas the smallest increase was noted with 4 and 6 mg of EWTI per ml. The effect of EWTI concentration on coagulase clotting with 1 to 4% rabbit serum (concentrated twofold) and 1% plasma is shown in Fig. 2. All concentrations of EWTI tested activated coagulase clotting when serum was the principal source of CRF. The reaction velocities produced by 4 and 6 mg/ml were nearly identical; hence, maximal activation was reached with these concentrations using both serum and plasma. Figure 2 also shows that increased serum concentration allowed faster clotting at each concentration of EWTI tested. The fastest clotting observed with 1 mg of coagulase per ml in the reaction mixture was the production of a 4+ fibrin gel in 6.5 min with 4% serum and 4 mg of EWTI per ml. When 2 mg of coagulase per ml was used in this system, a firm gel was obtained in 5 min. 5 e t Z a I -, EWTI CONCENTRATION (MG/ ML) FIG. 4. EWTI activation of S. aureus 1995 coagulase clotting using 1% serum + 1% plasma as the sources of CRF. Figure 3 illustrates the activation of coagulase clotting by EWTI by using fresh plasma. Since the increase in reaction rates produced by 4 and 6 mg of EWTI per ml were similar, the upper limit of activation was approached. At all levels of trypsin inhibitor used, the clotting with 3% plasma was equal to or faster than that with 4% plasma. This inhibition at high substrate levels appeared to be most pronounced with the highest concentration of EWTI studied. The lowered activation with 4% plasma was not evident in the control or the 2 mg of EWTI per ml reaction times. The maximal increase in clotting rate was obtained with 3% plasma and 6 mg of EWTI per ml. Figure 4 shows the effect of trypsin inhibitor on the coagulase reaction by using the dialyed culture supernatant fraction of S. aureus 1995 as the source of coagulase. The fastest clotting, obtained with 4 and 6 mg of EWTI per ml, was the production of a firm gel in 15 min. This rate of fibrin formation was significantly faster than that of the control in which a firm gel was not observed before the 3-hr reading. DISCUSSION The initial step in coagulase clotting is believed to involve the reaction of CRF with coagulase to form activated coagulase. Haughton and Duthie (6) have shown that clotting time and esterase activity are dependent on the concentration of activated coagulase. Increasing the sensitivity of the coagulase test, by using undiluted (or concentrated) serum or plasma as an increased source of CRF, would be successful only if the serum inhibitors were not active. Downloaded from on October 1, 218 by guest

4 VOL. 18, 1969 COAGULASE CLOTrING BY TRYPSIN INHIBITOR Plasmin is known to inactivate coagulase (16). Staphylococcal Muller factor and staphylokinase are believed to activate plasminogen to plasmin (7, 17). This enyme has similar specificity to trypsin and is inactivated by trypsin inhibitor (13). Kunit and Northrop (8) suggested that one molecule of trypsin inhibitor reacts with one molecule of trypsin to form an addition compound. This linear relationship would be expected for plasmin inactivation. The inactivation of plasmin in coagulase studies is especially important in plasma and serum which have had plasminogen spontaneously activated or acted upon by the staphylococcal plasminogen activators. Soybean trypsin inhibitor (SBTI) has been used in studying the esterase activity of coagulase preparations (2, 6) since it inhibits fibrinolysis by plasmin. In studying the esterase activity of activated coagulase on N' -toluene-p-sulfonyl-larginine methyl ester, Haughton and Duthie (6) reported that activated coagulase was not detectably inhibited by 4,ug of SBTI per ml. In similar experiments, Drummond and Tager (2) used.4 mg of SBTI in a.3-ml reaction mixture and reported that their results confirmed the observations of Haughton and Duthie (6). These investigators used SBTI at lower levels than the trypsin inhibitor concentrations employed in the experiments reported here. Comparison of Fig. 1 and 3 with Fig. 2 reveals that concentrated serum increased the rate of reaction more than plasma. This difference was probably due to the approximately sixfold difference in CRF level between the concentrated serum and the plasma diluted 1:3. Another difference noted between Fig. 1 and 2 is that the activation by EWTI appeared to be less for the serum than for the plasma. For example, the activation at 2% serum + 1% plasma was 2 min for an increase in EWTI from 2 to 6 mg/ml, and the activation at 2% plasma was 13 min for the same increase in trypsin inhibitor. This reduced activation with the serum source of CRF may reflect the fact that the maximal rate of fibrin gel formation was approached at the concentrations of reactants used, or that the serum contained higher levels of clotting inhibitors than the plasma. When the reaction mixture contained 1 mg of coagulase per ml, the maximal rate of clotting was obtained with 4 mg of EWTI per ml with serum or plasma. The data plotted in Fig. 1 were obtained with plasma which had been froen (-15 C) and thawed before use. When identical experiments were performed by using the same substrate before freeing, the rates of clotting with 4% 99 plasma containing 1 to 6 mg of EWTI per ml were slower than those with 3% plasma. This decreased velocity at the highest level of CRF added was generally not observed when concentrated rabbit serum was used as the primary substrate. The freeing and thawing treatment may have reduced the inhibition observed with the 4% plasma. The partially purified coagulase used in these experiments was prepared from S. aureus This strain was a potent producer of both Muller factor and staphylokinase, and the coagulase preparation contained these plasminogen activators. To determine if the trypsin inhibitor activation of coagulase clotting was unique to Staphylococcus strains which produced these kinases, the dialyed culture supernatant fraction from S. aureus 1995 was assayed with a 1% serum reaction mixture containing to 6 mg of EWTI per ml. It was found that trypsin inhibitor nearly doubled the coagulase reaction rate for this strain. The mechanism of activation by trypsin inhibitor is not known. It seems likely that the effect is partly due to the inhibition of plasmin; however, it appears that more than plasmin inhibition is involved since similar activation was observed with S. aureus strains which did and did not produce plasminogen activators. The activation of coagulase clotting by trypsin inhibitor does not appear to involve a stoichiometric inactivation of plasmin by trypsin inhibitor. If only plasmin inactivation were involved, different concentrations of EWTI should have produced the maximal activation in the concentrated serum and the diluted plasma (due to the approximately sixfold difference in plasminogen concentration). This difference in levels of EWTI required to produce maximal activation in plasma and serum was not observed. Normal plasma has been found to contain between 2 and 6 mg of plasminogen per ml (12). By using these figures for calculation, it was determined that the 4% concentrated serum reaction mixture would have contained a maximum of 4.8 mg of plasminogen per ml. This mixture contained approximately 8% of the plasmin (or precursors) present in normal serum. Since the potency of the trypsin inhibitor was known, it was calculated that maximal plasmin inhibition would be expected with 7 mg of EWTI per ml. Experiments showed that activation increased up to a concentration of 4 mg/ml. Trypsin inactivation by plasma trypsin inhibitor does not always follow a linear relationship in the presence of blood plasma (11). Trypsin inhibitor activation of coagulation may be partly due to nonlinear inhibition of plasmin. Downloaded from on October 1, 218 by guest

5 91 ORTH, ANDERSON, AND MONTGOMERY APPL. MICROBIOL. Other possible mechanisms of action of trypsin inhibition were investigated. When added to plasma, EWTI did not cause clotting. Thus, the method of activation was not due to the ability of EWTI to cause clotting. When EWTI was added to fibrinogen + coagulase, no clotting occurred; hence, EWTI does not function as an auxiliary source of CRF. Preliminary studies indicate that SBTI and EWTI have a similar activating effect on the coagulase reaction. The activation by trypsin inhibitor appears to be greater with more dilute serum or plasma. This may be why previous reports (2, 6) failed to recognie the activating influence in reaction mixtures containing coagulase and purified CRF. The tube coagulase test can be made more sensitive by manipulation of plasma or serum (CRF) and trypsin inhibitor concentrations. The results obtained with S. aureus 1995 illustrate how the addition of trypsin inhibitor to the coagulase test system may be useful in detecting low levels of coagulase. Thus, the use of this activator would allow coagulase detection with a 4-hr test period, whereas the conventional procedure would be unable to detect clotting activity in this time. There would be fewer incorrect "coagulase negative" reports. Although the diagnostic significance of the low coagulase producer has not been studied, the more sensitive assay system described here may provide a tool for further investigation. ACKNOWLEDGMENTS This study was supported by the U.S. Atomic Energy Commission grant AT (45-1)-193. We thank E. P. Casman of the U.S. Department of Health, Education, and Welfare, Food and Drug Administration, for sending the culture of S. aureus Technical Paper no. 2731, Oregon Agriculture Experiment Station, Oregon State University, Corvallis, Oregon, LITERATURE CITED 1. Blobel, H., D. T. Berman, and J. Simon Purification of staphylococcal coagulase. J. Bacteriol. 79: Drummond, M. C., and M. Tager Enymatic activities associated with clotting of fibrinogen by staphylocoagulase and coagulase-reacting factor and their inhibition by diisopropylfluorophosphate. J. Bacteriol. 83: Duthie, E. S., and L. L. Loren Staphylococcal coagulase: mode of action and antigenicity. J. Gen. Microbiol. 6: Elek, S. D Staphylococcus pyogenes and its relation to disease, p. 21. E. & S. Livingstone Ltd., Edinburgh. 5. Gilden, M. M., E. F. Baer, and M. K. Franklin Comparative evaluation of a direct plating procedure and an enrichment isolation procedure for detecting coagulase positive staphylococci in foods. J. Ass. Offic. Anal. Chem. 49: Haughton, G., and E. S. Duthie The activation of staphyloccoccal free coagulase by plasma constituents and the hydrolysis of Na-toluene-p-sulphonyl-L-arginine methyl ester (TAME) by activated coagulase. Biochem. J. 71: Hutchison, J. G. P Muller's phenomenon and its relationship to plasminogen activation and to blood constituents producing opacity in an agar gel. J. Pathol. Bacteriol. 84: Kunit, M., and J. H. Northrop Isolation from beef pancrease of crystalline trypsinogen, trypsin, a trypsin inhibitor, and an inhibitor-trypsin compound. J. Gen. Physiol. 19: Litkenhous, C. J., E. F. Baer, M. M. Gilden, and M. B. Bromer False-positive coagulase reactions in the characteriation of microorganisms isolated from foods. J. Ass. Offic. Anal. Chem. 51: Loeb, L The influence of certain bacteria on the coagulation of blood. J. Med. Res. 1: McCann, S. F., and M. Laskowski Determination of trypsin inhibitor in blood plasma. J. Biol. Chem. 24: Schult, H. E., and J. F. Heremans Nature and metabolism of extracellular proteins, p In Molecular biology of human proteins with special reference to plasma proteins, vol. 1. Elsevier Publishing Co., New York. 13. Sherry, S., and W. Troll The action of thrombin on synthetic substrates. J. Biol. Chem. 28: Smith, W., and J. H. Hale Nature and mode of action of staphylococcus coagulase. Brit. J. Exp. Pathol. 25: Tager, M Studies on the coagulase-reacting factor. I. The reaction of staphylocoagulase with the components of human plasma. Yale J. Biol. Med. 2: Tager, M Problems in the coagulation of plasma by staphylocoagulase. Ann. N. Y. Acad. Sci. 65: Tauraso, N. M., and D. A. White Studies on staphylococcal infections. Amer. J. Dis. Child. 15: Turner, F. S., and B. S. Schwart The use of a lyophilied human plasma standardied for blood coagulation factors in the coagulase and fibrinolytic tests. J. Lab. Clin. Med. 52: Downloaded from on October 1, 218 by guest