Inhibitors of fibrinolysis in saliva after oral surgery measured by enzymic and immunological methods

Int. J. Oral Surg. 1979: 8: 212-221 iKey "ords: fibrinolysis:

.'WliVll;

surgery, oral,' plasminogen,' fibrin)

Inhibitors of fibrinolysis in saliva after oral surgery measured by enzymic and immunological methods N. GERSEL-PEDERSEN Department of Oral Surgery, Royal Dental College; Department of Oral Medicine and Oral Surgery; Department of Clinical Chemistry ML, University Hospital, Copenhagen, Denmark

Mixed saliva was collected from 25 healthy young people before oral surgery, immediately after the operation and at control 4, 5 or 6 days later. The concentration of albumin, aj-antitrypsin, antithrombin III, Cr-esterase inhibitor, a2-macroglobuIin, a2-plasmin inhibitor and plasminogen was measured in plasma (Cll) and saliva (Cs) samples from 12 patients by electroimmuno assay. The capacity of saliva samples to inhibit fibrinolytic activity in a euglobulin precipitate from a standard plasma was estimated on human fibrin plates (13 patients) and plasminogen-free fibrin plates (six patients). Normal saliva only showed a weak inhibitory effect on human fibrin and except albumin, none of the investigated proteins were found in measurable amounts. In postoperative saliva the concentration of recorded proteins varied among patients from 39 % to lower than 4 % of normal plasma concentrations. This contamination caused total neutralization of fibrinolytic activity during 6 hours, except in one patient. After 20 h the primary inhibitory effect decreased around 30 % on human fibrin. The ratio (Cs/C ll) of a2-plasmin inhibitor postoperative was significantly lower (P 0.01) than the ratio of other proteinase inhibitors. The control samples contained negligible amounts of aj-antitrypsin and a2-macroglobulin in a few patients and showed a weak inhibitory effect on human fibrin. No inhibitors of fibrinolysis in saliva with physiological importance were discovered. The generation of plasmin in saliva after the operation is completely controlled by the high inhibitor capacity from plasma. However, the mixing of blood and saliva activates the fibrinolytic system and, as the supplement of fibrinolytic inhibitors from the ulcer in the postoperative period is insignificant, increased local fibrinolysis can develop. ABSTRACT -

<

(Received for publication 19 November 1977, accepted 5 May 1978)

The mixing of blood and saliva on the sur· face of an oral ulcer may decrease the stability of a normal blood clot26 ,29. This is at-

tributed to the existence of an activator of plasminogen in saliva 1,11,26. However, SCHULTE & VORBAUER29 demonstrated that

0300-9785/79/030212-10$02.50/0 © 1979 Munksgaard, Copenhagen

INHIBITORS OF FIBRINOLYSIS IN SALIVA a mixture of equal volumes of saliva and blood from the same patients only seldom showed any fibrinolytic activity. A recent study suggests l1 that fibrinolytic inhibitors from the blood are responsible for the decrease of salivary fibrinolytic activity measured after oral surgery. A slight inhibitory effect against trypsin in parotid saliva is demonstrated in one study1, but no further attempts to explore the anti-fibrinolytic capacity of saliva have been done. The proteinase inhibitors ai-antitrypsin 10,13,28, antithrombin 1II7,8,14, C1-esterase inhibitor12 ,27,28 and az-macroglobulin 28 ,30 possess antHibrinolytic capacity in purified biochemical systems. However, a:l-macroglobulin 2,22,24 and the recently isolated a2-inhibitor of plasmin o,20,23 are the only plasmin inhibitors in plasma with physiological importance. The concentration of proteinase inhibitors measured immunochemically and the amount of biologic inhibition exerted are not always well correlated 23 ,25. Although several other proteins occurring in plasma are identified in saliva17, the author is not aware of any report dealing with measurements of proteolytic enzyme inhibitors in saliva. The purpose of the present investigation was to measure the biological importance of possible fibrinolytic inhibitors in saliva and the influence of plasma proteinase inhibitors contaminating saliva after oral surgery.

213

Material and methods Healthy young people whose mandibular lower molars were to be removed were selected tor the studies. None of the patients took any medication and no complications developed in the postoperative period. Mixed saliva was collected from 25 patients as described previouslyl1, and three samples were obtained from each person: 1) before oral surgery, 2) immediately after the operation, and 3) 4, 5 or 6 days postoperatively, the day of control. Immediately after surgery a venous blood sample was collected in 0.11 M sodium citrate at the same moment as saliva. The platelet-poor plasma was separated by centrifugation for 30 min at 3000 rev/min. Saliva and plasma samples were frozen at once and stored at -20 e until assay. Prior to freezing, all saliva samples used for immunochemical assays were mixed with bovine protease inhibitor (Trasylol®), 0.0125 pmol/m\. The patients were divided into two groups which had an equal distribution with respect to sex, age and the length of postoperative observation. The samples from one group (n = 12) were used for immunochemical measurements, whereas the samples of the other group (n ::;: 13) were analyzed by a fibrinolytic inhibitor assay (see below). D

lmmullochemical techniques The concentration of albumin, at-antitrypsin, antithrombin III, C1-esterase inhibitor, a2-macroglobulin, a2 -plasmin inhibitor and plasminogen was measured by electroimmuno assay in agarose gel containing antibodyl•. Specific rabbit immunoglobulins to the following human proteins were purchased: albumin, at-antitrypsin and a2-macroglobulin (Dakopatts, Copenhagen, Denmark), antithrombin III (Nyegaard

Table 1. Fibrinolytic activity of plasma euglobulin activator with 50 % barbital buffer measured after 6 and 20 h. The mean of 13 measurements during 2 months. Lysed area in mm2 (x ± s.d.) Incubation time (hours)

x ± s.d.

Human

6 20

109 396

±

Plasminogen-free

6 20

60 157

± 13.2 %

Fibrin plate

±

±

in percent 9.5 % 9.8 % 12.5 %

GERSEL-PEDERSEN

214

mm 2 450

PER CENT INHIBITION OF ACTIVATOR ACTIVITY

SALIVA / PLASMA ACTIVATOR

BUFFER / PLASMA ACTIVATOR

20 HOURS

DID

400

0

25

300

50

6 HOURS

"10 T 0

+25 I

75

I

I

t I

50

+75 I

I

1.100

0.75 1.0 LOG. DID

o

6.25

12.5

100

CONCENTRATION

PREOPERATIVE

I--

20 HOURS

POSTOPERATIVE .----t> CONTROL

6 HOURS

•

50

25

100

0

PLASMA POOL

Fig. 1. Inhibitor effect of serial dilutions of whole saliva when mixed with an equal volume of a

plasma activator standard. The mean of measurements after 6 and 20 h on human fibrin plates (11=13). Abscissa. Concentration of saliva dilution (logarithmic). Ordinate. (left). Fibrinolytic activity in mm2• Ordinate. (right). Inhibitor effect in percent of the mean activity of plasma activator mixed with

an equal volume of buffer.

and Co., Oslo, Norway), Cl-esterase inhibitor and plasminogen (Beringwerke, Germany). Human protein standard plasma was from Beringwerke. Rabbit immune serum against a2-

plasmin inhibitor 23 and a reference of pooled human plasma were supplied by the courtesy of S. MUllertz and I. Clemmensen, Department of Clinical Chemistry, Hvidovre Hospi-

INHIBITORS OF FIBRINOLYSIS IN SALlYA tal, Copenhagen. A ratio of protein contamination into saliva was calculated: concentration in saliva/concentration in plasma. The lowest amount of protein which was possible to measure with the electroimmuno assay corresponds to approximately 3-4 pg antigen/ml or a dilution of normal plasma between 0.1 and 2.9 % depending on the initial protein concentration in plasma. Some postoperative samples with a high albumin ratio were subjected to crossed immunoelectrophoresis in agarose gel as described by WBEICE35. Each sample was processed with immune serum against plasminogen, at-anti· trypsin, antithrombin III (first-dimension gel contained heparin, 20 units/ml), Ct-esterase inhibitor or a2-plasmin inhibitor present in the second-dimension gel. Fibrinolytic inhibitor assay The potential effect of fibrinolytic inhibitors in each saliva sample was estimated by the fibrin plate method according to BRAKMAN4 ,5. The solutions of bovine plasminogen-free fibrinogen, human fibrinogen and thrombin for the fibrin plates have been described previouslyll. Activator precipitated with the euglobulin fraction

mm 2 200

BUFFER / PLASMA ACTIVATOR

215

at pH 5.9 and low ionic strength from diluted human plasma was used as standard activator. Human plasma with high content of activator was obtained after moderate physical exercise, distributed into plastic tubes containing 1.5 ml and stored at -20°C. The mean fibrinolytic activity of the standard activator (x ± s.d. in percent) is shown in Table 1 and demonstrates the reproducibility of the used plasma activator. A solution of standard activator (Table 1) was mixed with equal volumes of 1) serial dilutions (100, 50, 25, 12.50 %) of saliva, 2) serial dilutions of a plasma pool obtained from 10 healthy donors and 3) saline barbital buffer, pH 7.75, I = 0.15 containing 0.25 % gelatine. All mixtures were applied in duplicate on the fibrin plates. The fibrinolytic activity of each mixture was measured after 6 and 20 h of incubation at 37"C. The inhibitory effect of the samples are expressed as percent reduction in fibrinolytic activity of standard activator with 50 % barbital buffer (Figs. 1 & 2). The inhibitory effect of the plasma pool used for comparison is shown in Fig. 1. The effect of fibrinolytic inhibitors in plasma vanished, independent of incubation time, when the

PER CENT INHIBITION OF ACTIVATOR ACTIVITY

SALIVA / PLASMA ACTIVATOR

20 HOURS

%

a

25

100

!

6 HOURS U!S

0.75 1.0

50

"10 TO I I

T 100

+50

75

.Ll00

100

I I

LOG. "10

o • 6

PREOPERATIVE I - - 20 HOURS 6 HOURS POSTOPERATIVE ~---­ CONTROL

Fig. 2. The mean of measurements after 6 and 20 h on bovine plasminogen-free fibrin plates (n

= 6). Detailed comments, see Fig. 1.

216

GERSEL-PEDERSEN

final plasma concentration decreased below 0.315 %, i.e. standard activator mixed with plasma diluted higher than 0.750 % showed the same fibrinolytic activity as standard activator with 50 % barbital buffer. This indicates the range of sensitivity of the inhibitor assay. Statistics

The differences were evaluated by the paired t-test; two-tailed probability (P < 0.01) was used as level of significance.

Results Immunochemical assays Small concentrations (x = 0.93 ~mol/l sCi') = 0.38 ,amoll) of albumin were always rf'corded in normal mixed saliva. The proteinase inhibitors and plasminogen were not observed in normal saliva with the present method, Le. aI-antitrypsin < 0.06 ~mol/l, antithrombin III < 0.08 ,umol/I, Ct-esterase inhibitor < 0.03 Jlmol/l, CLimacroglobulin 0.02 ,umol/l, CL2-plasmin inhibitor 0.04 ,llmol/l and plasminogen < 0.03 ,umol/l. In a few patients trace amounts of acantitrypsin and a2-macroglobulin were measured in samples collected 4, 5 or 6 days postoperatively. The saliva samples collected immediately after surgery were contaminated with all the measured proteins, but with great variations among patients. The ratio of protein contamination into saliva calculated is presented in Table 2. The ratio of albumin was higher than the ratio of other proteins (P 0.01) indicating a consumption of plasminogen and proteinase inhibitors. ailPlasmin inhibitor, having a significantly lower ratio than the other proteinase inhibitors, showed a selective decrease. Antithrombin III and plasminogen had a reduced ratio compared to at-antitrypsin and ail-macroglobulin (P < 0.01). Decreased ratios might have been produced by partial degradation of proteins in saliva, because this alteration changes the electrophoretic mobility of the measured proteinS 12 -14,2;1,

<

34. However, no complexes of significant magnitude between plasmin and proteinase inhibitors could be demonstrated by crossed immunoelectrophoresis. All the measured plasminogen had /I2-mobility showing that no proteolytic degradation had taken place. The immunoprecipitation arcs of CLt-anti-

A

<

<

Fig. 3. Crossed immunoelectrophoresis of 30 ,ul saliva collected after oral surgery (patient no. 7). The second-dimension gel contained immune serum against at-antitrypsin 10 ~d/ml gel (A) and against Cl-esterase inhibitor 20 ~d/ ml gel (B). The shoulders (arrows) demonstrate that a small fraction of the inhibitors have formed complexes with other proteins in the sample.

Table 2. The ratio (R) of plasma protein contamination in saliva after oral surgery, estimated from protein concentration (flmol/l) in whole saliva (Cs) and plasma (Cp ); R = CS/CI ) Patient No.

Albumin

at- anti trypsin

Antithrombin III

C1-esterase inhibitor

Cl2-macroglobulin

a2-plasmin inhibitor

plasmiDogen

1

R

0.19

0.17

0.14

0.15

0.18

0.08

<0.06

2

R

0.39

0.37

0.23

0.25

0.30

0.12

<0.07

3

R

0.23

0.18

0.16

0.09

0.18

0.09

<0.10

4

R

0.17

0.19

0.19

0.15

0.16

0.12

<0.10

5

R

0.22

0.19

<0.09

0.20

0.15

0.05

0.11

6

R

0.16

0.13

<0.08

0.17

0.11

0.U4

<0.11

7

R

0.23

0.19

0.19

0.31

0.16

0.15

0.13

8

R

0.18

0.15

<0.10

0.14

0.07

<0.04

<0.13

9

R

0.18

0.10

<0.09

<0.04

0.12

<0.05

<0.11

10

R

0.22

0.17

0.14

0.13

0.19

0.12

<0.10

11

R

0.18

0.14

<0.09

<0.06

0.14

0.04

<0.09

12

R

0.23

0.15

<0.07

0.06

0.16

0.04

<0.09

104.2 ±31.3

5.85 ±2.19

0.63 ±0.29

0.44 ±0.26

0.47 ±0.24

0.10 ±0.06

0.18 ±0.03

486.3 ±46.0

33.5 ±6.8

4.8 ±0.5

3.0 ±0.5

2.9 ±0.8

1.3 ±0.2

1.9 ±0.4

0.18 ±O.o7

0.13 ±0.05

0.15 ±0.08

0.16 ±0.06

0.08 ±0.04

0.10 ±0.02

Cs x ± s.d.

Cp

0.22 ±0.06

R

«)

Z

::r:

@ ...... >-:l 0

:;;l

en

0

'Tj

::::l

1:0 :;;l

Z

0

-r-' ...:: en en

Z

en

>-

r'

=2

>-

Ratio indicates C s below measurable range: Antithrombin III (C s < 0040), Cr-esterase inhibitor (C s < 0.13), a2-plasmin inhibitor (C s < 0.05), plasminogen (C s 0.17).

<

....to

--.l

GERSEL-PEDERSEN

218

Table 3. The fibrinolytic activity on human fibrin plates after 6 and 20 h, measured in reaction mixtures containing a standard plasma activator and an equal volume of serial dilutions (100, 50, 25, 12.50 %) of whole saliva collected before and after oral surgery (11= 13). Number of patients with saliva samples showing total neutralization of plasma activator. Lysed area in mm2, x ± s.d. in percent (no. of samples without activity) Sample dilution

6h 20 h

Preoperative

12.50%

101 362

25%

108 378

50%

108 383

100%

113 389

± 12 % ± 9% ± 17 % ± 14 % ± 11 %

± ±

9%

16 %

± 10 %

trypsin and Ct-esterase inhibitor both demonstrated a small shoulder (Fig. 3). Inhibitor assay The arithmetical means of the fibrinolytic activity in mixtures of saliva samples and standard activator, measured on human fibrin plates, are presented in Fig. 1 and Table 3. Normal mixed saliva does not seem to have any inhibitory effect though a weak and significant decrease of activity is measured at the 12.50 % dilution. The postoperative samples demonstrated a distinct inhibition of standard activator at all dilutions. The inhibitory effect in percent was higher after 6 h than after 20 h, but always lower than the inhibitory effect of the plasma pool. The number of patients distributed according to the dilutions where total neutralization of standard activator was recorded is shown in Table 3. Only one patient had a postoperative saliva sample which did not show 100 % inhibitory effect after 6 h. Complete neutralization after 20 h was caused only by one sample at 50 % concentration and in two samples at 100 % concentration (Table 3). The control sample, collected 4, 5 or 6 days postoperatively, showed slightly decreased ac-

Postoperative

Control

37 262

± 100 % (5) ± 24%

98 354

± 11 %

27 244

± 126 % (7)

102 366

± 12 % ± 11 % ± 13 % ± 12 % ± 13 % ± 13 %

± 30 % 17 ± 188 % (9) 188 ± 53 % (1) 7 ± 360 (% (12) 125 ± 80 % (2)

104 377 102 375

± 10 %

tivity at the 12.50 % and 25 % dilution (P after both 6 and 20 h, but the inhibitory effect was negligible. The assay of fibrinolytic inhibitors in saliva from six patients on plasminogen-free fibrin plates is presented in Fig. 2. The postoperative samples showed essentially the same inhibitory effect as was demonstratecl on the human fibrin, but the plasmin activity was nearly totally neutralized by the undiluted sample even after 20 h of incubation. In the control samples an increased fibrinolytic activity (P = 0.03) was observed in the 12.50 % dilution.

< 0.01)

Discussion The electroimmuno assay did not reveal any plasminogen (Le. Cs < 0.03 proal/I) in mixed saliva collected preoperatively and 4, 5 or 6 days postoperatively. However, trace amounts of plasminogen are most likely present, since an admixture of streptokinase increases the fibrinolytic activity of normal mixed saliva on bovine plasminogen-rich fibrin 1• Streptokinase alone does not generate plasmin from bovine plasminogen, but streptokinase and human plasminogen form a 1: 1 molar complex, which

INHIBITORS OF FIBRINOLYSIS IN SALIVA

is a potent activator of bovine plasminogen31 • Human native plasminogen and streptokinase were shown to form an activator of bovine plasminogen even at concentrations of human plasminogen, which were below the measurable range of the electroimmuno assayl1. A recent studylD, which used another method, demonstrated plasminogen equivalent to around 0.05 casein units/ml native mixed saliva. This amount corresponds approximately to a plasminogen concentration of 0.03 !tmol! 134 • The absence of anti-fibrinolytic capacity in normal mixed saliva is in agreement with the result from the electroimmuno assay, as no proteinase inhibitors were discovered. This is also in accordance with another studyl, where a slight inhibition of trypsin by parotid saliva was observed only in one of 10 patients investigated. The lack of fibrinolytic inhibitors in normal saliva obviously amplifies the significance of the salivary plasminogen activator studied previouslyl,11,26. The postoperative samples demonstrated that a consumption of proteinase inhibitors and plasminogen in the oral ulcer or during contact with saliva had taken place. Crossed immunoelectrophoresis revealed that the results of the electroimmuno assay were not influenced by any significant complex formation or partially degradation of the measured proteins in the samples. Defibrination of plasma causes no or only a small reduction in concentration of aI-antitrypsin, Ct-esterase inhibitor, a2-macroglobulin33 and native plasminogen 32 , whereas the level of antithrombin III decreases approximately 50 %8, Plasminogen pretreated with activators or plasmin is also strongly adsorbed (around 65 %) during fibrin formation32 • The low ratios of antithrombin III and plasminogen (Table 2) may therefore be explained by coagulation and fibrin formation in the oral ulcer with concomi-

219

tant activation of plasminogen. An activation of the fibrinolytic system with generation of plasmin is in accordance with the selective decrease of a2-plasmin inhibitor (Table 2), which is the primary plasmin inhibitor in plasma6,2o,23. at-antitrypsin, C t esterase inhibitor and a2-macroglobulin exhibit a broad range of specificity towards proteinases I2 ,13,27,3o, and the consumption of these inhibitors (Table 2) may be caused by salivary kaIIikrein 6 or other proteolytic enzymes present in mixed saliva 1B • Salivary enzymes probably also explain the shoulder of the immunoprecipitate of at-antitrypsin and C1-esterase inhibitor (Fig. 3). The alantitrypsin complex encountered is considered to be different from the complex between at-antitrypsin and IgA identified in normal plasmal6 . Despite the low inhibitor ratios and a selective consumption of ail-plasmin inhibitor, the capacity of the postoperative saliva samples to inhibit standard activator was nearly 100 % (Figs. 1, 2, Table 3). It is therefore likely that the proteinase inhibitor contamination in saliva postoperative is responsible for the decrease of salivary fibrinolytic activity after oral surgery reported earIierl1 • The inhibitory effect of the postoperative samples decrease obviously after 20 h, especially on the human fibrin plates (Fig. 1, 2). This was also observed by BRAKMAN 5 • The plasminogen activator is strongly adsorbed to fibrin 4 ,21,34, whereas inhibitors are not4 ,33. This may dissociate the enzyme from a possible activator - inhibitor complex4 ,33. During a prolonged incubation enough plasmin is formed by a diffusible plasminogen activator to overcome the initial inhibitory effect. In the oral blood clot, where plasminogen activators from saliva inevitably have the opportunity to form plasmin during the first postoperative days, it is evident that the time is in favor of fibrinolytic degradation of the coagulum.

220

GERSEL-PEDERSEN

This investigation shows that the content of fibrinolytic inhibitors in normal whole saliva is without physiological importance. The mixing of blood and saliva initiates an early activation of the fibrinolytic system. The resistance of a blood clot to local fibrinolysis in the oral cavity is influenced mainly by the genuine inhibitor capacity of plasma. The postoperative period is in favor of increased fibrinolysis as the local plasminogen activators may generate enough plasmin to overcome an initial inhibitory potential. Acknowledgments - The fulfilment of this study would have been impossible without the skilled assistance of Mrs. Edith Weber. The equipment for the electroimmuno assay was borrowed from Dr. V. Gerhard Nielsen, Department of Clinical Chemistry, Herlev Hospital; this is appreciated. The staff of the Department of Clinical Chemistry, Hvidovre Hospital, have contributed to the studies: Dr. S. Thorsen has made valuable suggestions during the study, Dr. S. MUllertz and Dr. I. Clemmensen kindly supplied antiserum to a2-plasmin inhibitor and several of the immunochemical measurements have been performed by this Department. Materials were kindly supplied by KABI, Stockholm, Sweden. The work was supported by a grant from the Danish Medical Research Council No. 512-5658.

References 1. ALBRECHTSEN, O. K. & THAYSEN, J. H.: Fibrinolytic activity in human saliva. Acta Physiol. Scal1d. 1955: 35: 138-145. 2. ARNESEN, H. & FAGERHOL, M. K.: U2Macroglobulin, uj-Antitrypsin, and Antithrombin III in plasma and serum during fibrinolytic therapy with urokinase. Scand. J. Clin. Lab. Invest. 1972: 29: 259-263. 3. BRAKMAN, P.: Fibrinolysis. A stanclnrdizec1 fibrin plate method and a fibrinolytic as.. say of plasminogen. Thesis. Sheltema & Holkema, Amsterdam 1967. 4. BRAKMAN, P. & ASTRUP, T.: Selective inhibition in human pregnancy blood of urokinnse induced fibrinolysis. Scand. J. CUn. Lab. Invest. 1963: 15: 603-609,

5. BRAKMAN, P., MOHLER, F. R. & ASTRUP, T.: A group of patients with impaired plasma fibrinolytic system and selective inhibition of tissue activator-induced fibrinolysis. Scand. J. Haemat. 1966: 3: 389-398. 6. COLLEN, D.: Identification and some properties of a new fast-reacting plasmin inhibitor in human plasma. Eur. J. Biochem. 1976: 69: 209-216. 7. CRAWFORD, G. P. M. & OGSTON, D.: The action of antithrombin III on plasmin and activators of plasminogen. Biochim. et Biophys. Acta 1975: pp. 189-192. 8. FAGERHOL, M. K. & ABILDGAARD, V.: Immunological studies on human antithrombin III. Influence of age, sex and use of oral contraceptives on serum concentration, Seand. .T. Haemat. 1970: 7: 1.0-17. 9. FUJIMOTO, Y., MORIYA, H. & MORIWAKI, C. Studies on human salivary kallikrein. .T. Biochem. 1973: 74: 239-246. 10. GANS, H. & TAN, B. H.: (WAntitrypsin, an inhibitor for thrombin and plasmin. CUll. Chim. Acta 1967: 17: 111-117. 11. GERSEL-PEDERSEN, N.: Salivary fibrinolytic activity befo,re and after oral surgery estimated on clifferent types of fibrin. Int. .T. Oral Surg. 1976: 5: 270-275. 12. HARPEL, P. C. & COOPER, N. R.: Studies on human plasma Cj inactivator-enzyme interactions. .1. CUll. Invest. 1975: 55: 593604. 13. HERCZ, A: The inhibition of proteinases by human at-antitrypsin. EIlI". J. Biochem. 1974: 49: 287-292. 14. HIGHSMITH, R. F, & ROSENBERG, R. D,: The inhibition of human plasmin by human antithrombin-heparin cofactor. J. Bioi. Chem. 1974: 249: 4335-4338. 15. LAURELL, C. B.: Electroimmuno assay. Scand. .T. CUn. Lab. Invest. 1972: 29: supp!. 124: 21-37, 16. LAURELL, C. B. & THULIN, E.: Complexes in human plasma between at-antitrypsin and IgA and aj-antitrypsin and fibrinogen. Scand . .T. Il1ullullol. 1975: 4: suppi. 2: 712. 17. LEVINE, M. 1. & ELLISON, S. A.: Immunoelectrophoretic and chemical analyses of human parotid saliva. Archs. Oral Biol, 1973: 18: 839-853. 18. MODEER, T.: Some properties of proteolytic enzymes in human saliva. Acta Dc/ant. Scand. 1974: 32: 321-328. 19. MOODY, G. M.: Investigations into the fibrinolytic system of human mixed native

INHIBITORS OF FIBRINOLYSIS IN SALlYA

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21.

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23. 24.

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26. 27.

28.

saliva. Thesis, June 1976. Faculty of Medicine, University of Edinburgh. MOROI, M. & AOKI, N.: Isolation and characterization of u2-plasmin inhibitor from human plasma. J. Bioi. Chem. 1976: 251: 5956-5965. MULLERTZ, S.: Mechanism of activation and effect of plasmin in blood. Acta Physiol. Scand. 1956: 38: Supp!. 130. MULLERTZ, S.: Different molecular forms of plasminogen and plasmin produced by urokinase in human plasma and their relation to protease inhibitors and lysis of fibrinogen and fibrin. Biochem. J. 1974: 143: 273-283. MUu.ERTZ, S. & CLEMMENSEN, I.: The primary inhibitor of plasmin in human plasma. Biochem. J. 1976: 159: 545-553. NILimN, I.-E. & GANROT, P.O.: Plasmin, plasmin inhibitors and degradation products of fibrinogen in human serum during and after intravenous infusion of streptokinase. Scand. J. CUn. Lab. Invest. 1967: 20: 113-121. PRENTICE, C. R. M., TtlOMSEN, C. & FORBES, C. D.: Endogenous inhibitors of the fibrinolytic enzyme system. In: DAVIDSON, J. F., SAMAMA, M. M. & DESNOYERS, P. D. (ed.): Progress in chemical fibrinolysis and thrombolysis. Raven Press, New York 1975, pp. 209-215. RAMSTROM, G.: Fibrinolytic activity in the saliVa of patients with coagulation disorders. Swed. Dent. J. 1975:68: 49-54. RATNOFF, O. D., PENSKY, J., OGSTON, D. & NAFF, G. B.: The inhibition of plasmin, plasma kallikrein, plasma permeability factor, and the subcomponent of the first component of complement by serum C1 esterase inhibitor. J. Exp. Med. 1969: 129: 315-331. SCHREIBER, A. D., KAPLAN, A. P. & AuSTEN, K. F.: Plasma inhibitors of the components of the fibrinolytic pathway in man. J. CUn. Invest. 1973: 52: 1394-1401.

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29. SCHULTE, VON W. & VORBAUER, J.: Fibn· nolytische Effekte beim Kontakt von Speichel und Blut. Deutsche Zahn, -Mundund Kieferheilkunde 1965: 44: 23-36. 30. STEINBUCR, M., BLATRIX, C., DROUET, J. & AMOVCR, F.: Study of the a2Macroglobulin/plasmin interaction mechanism. Pathol. Bioi. 1972: 20: Supp!.: p. 52-55. 31. SUMMARIA, L., LING, C., GROSKOPF, W. R. & ROBBINS, K. C.: The active side of bovine plasminogen activator. Interaction of streptokinase with human plasminogen and plasmin. J. Bioi. Chem. 1968: 243: 144-150. 32. THORSEN, S.: Differences in the binding to fibrin of native plasminogen and plasminogen modified by proteolytic degradation influence of OJ-amino-carboxylic acids. Blochim. ('t Biophys. Acta 1975: 393: 5565. 33. THORSEN, S.: Human urokinase and porcine tissue plasminogen activator. Thesis, Lregeforeningens Forlag, Sept. 1977. Faculty of Medicine. University of Copenhagen. 34. TtIoRSEN, S. & MULLERTZ, S.: Rate of activation and electrophoretic mobility of unmodified and partially degraded plasminogen. Effects of 6-Amino-hexanoic acid and related compounds. Scaml. J. CUn. Lab. Invest. 1974: 34: 167-176. 35. WEEKE, B.: Crossed immunoelectrophoresis. Scand. J. Immunol. 1973: 2: Suppl. 1: p. 47-56.

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Department of Oral Surgery Royal Dental College 4, Universitetsparken DK-2100 Copenhagen (2) Denmark