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A Study of Cytofibrinokinase and Fibrinolysin in Extracts of Tissue from Human Myometrium, Endometrium, Decidua, and Placenta*
A STUDY OF CYTOFIBRINOKINASE AND FIBRINOLYSIN IN EXTRACTS OF TISSUE FROM HUMAN MYOMETRIUM, ENDOMETRIUM, DECIDUA, AND PLACENTA* LouisF: LANG PHILLIPS, PH.D., BYRON C. BuTLER, M.D., 1\:IED.Sc.D.,** AND HowARD C. TAYLOR, J·R., M.D., NEw YoRK, N.Y. (From Columbia University, College of Physicians and Surgeons)
accompanied by massive hemorrhage in obstetrical AFIBRINOGENEMIA patients has been reported by a number of investigators.l-7 The condiin cases of abruptio placentae, long-standing
tion has been observed primarily fetal death, and amniotic fluid infusion. The theory has been advanced that this afibrinogenemia is caused by an extensive intravascular clotting due to the infusion of thromboplastin-like material into the blood stream of the mother from the placenta, amniotic :fluid, or decidua. 2 • 3 • 5 • 6 Schneider6 has suggested for this condition a mechanism shown in the following diagram : Prothrombin
Other factors
'fissuc
thromboplastin
l
Heparin and other inhibitors
'Thrombin
Fihrinogen
1
-------------clo Fihrin
Although small thrombi have been found in the tissues of patients who have died from afibrinogenemia6 ' 8 these are not sufficient to account for all of the fibrinogen in the body. An intravascular clotting of such an extent as to remove most of this fibrinogen, i.e., 8 to 12 Gm., must of necessity be aecom· panied by a subsequent lysis of the clots. In some cases of afibrinogenemia au accompanying fibrinolysin ha.s been detected in the blood of the patient"• '; in other eases the investigators have been unable to demonstrate the presence of an active fibrinolytic enzyme system in vitro. It is suggested, however, that a fibrinolytic enzyme may be either partially or wholly responsible for the disappearance of the fibrinogen in spite of the fact that its presence cannot always be demonstrated in vitro by the methods in general use. *This investigation was supported
(in Part) by a
research grant H-1512 from the
National Heart Institute of the National Institutes of Health, Public Health Service. ••Present address, Phoenix, Ariz.
342
Volume 71
Number 2
FIBRINOLYTIC ENZYME IN HUMAN TISSUE
343
A precursor of fibrinolysin (profibrinolysin) is present in all normal
human blood plasma. This proenzyme can be activated by a number of factors including cytofibrinokinase (a tissue activator) and streptokinase (a bacterial activator). The active enzyme (fibrinolysin) thus formed is capable of hydrolyzing fibrinogen, fibrin, and certain other proteins. This may be represented by the following scheme: Tissue activator or cytofibrinokinase
l
FiheT'yeiiJn----~
>'ibrinngen Fibrin --------
Amino acids and polypeptides
I
Inhibitors
For the fibrinolysin enzyme system to be important in these situations, the activating enzyme, cytofibrinokinase, must be found in the tissues involved, i.e., endometrium, myometrium, placenta, decidua. With the breakdown of the cells of these tissues as a result of catabolic and autolytic changes subsequent to menstruation, labor and the separation of the placenta, and long-standing fetal death, the intracellular cytofibrinokinase could initiate enzymatic conversion of profibrinolysin to fibrinolysin. When fibrinolysin is unopposed by inhibitors or antifibrinolysin, the hydrolysis of the body fibrinogen is rapid ana aangerous. This mechanism has been suggested in previous papers. 9 • 10 A comparable situation has been demonstrated by Tagnon11 in cases of metastatic cancer of the prostate. He has reported fibrinolytic activity in the primary and metastatic cancer tissue and suggested that release of enzyme from this tissue was responsible for the hemorrhagic syndrome in his five patients. In his cases there was a deficiency of fibrinogen in the blood and a prolongation of the prothrombin time. Margulis and associates12 have reported fibrinolysin in 14 out of 17 uncomplicated pregnancies within the first 24 hours after delivery, with no change in prothrombin time, bleeding time, or clotting time. The purpose of the work presented here was twofold: (1) the development of a more sensitive method for the detection of fibrinolysin in vitro, and (2) a study of the activation of profibrinolysin b:;r the cytofi'brinokinasc con= tained in such tissues as myometrium, placenta, decidua, and endometrium. The presence of such an activator would account for the fibrinolysin observed 'in obstetrical patients with both complicated and uncomplicated- deliveries. A cytofibrinokinase in endometrium would give a mechanism by which menstrual blood remains fluid. Finally, such an activator would suggest an alternate method for the removal of fibrinogen from the blood stream of
l>ltiLLil>S, BUTLER, AND TAYLOR
Am.
J. Obst. & Gynec.
February. 1956
patients suffering from obstetrical accidents in which tissue extracts may be forced back into the maternal circulation. The subsequent removal of tibrinogen by hydrolysis may then either be the principal cause of afibrinogenemia in these patients or may supplement the removal by fibrin deposition dne to the presence of tissue thromboplastin.
Materials Extracts from the myometrium, decidua, and placenta of 5 women who had had cesarean hysterectomies were prepared immediately after the operation and subsequently tested for fibrinogenolytic, fibrinolytic, and cytofibrinokinase activities. Results of similar extracts from one woman (M. H.) who had a therapeutic abortion and hysterectomy for carcinoma of the liver when approximately five months pregnant are also included as are those of a sample of placenta from a hysterotomy at sixteen weeks and sampleR or t•nclometrium obtained after hysterectomy. Tissue extracts were prepared as described by Tagnon 1:l and ~uspended an
Methods Fibrinogenolytic activity has been determined by a method adapted from that used by Kunitz 15 for the hydrolysis of casein by trypsin. Fibrinogen was ~ubstituted for casein, and streptokinase-activated fibrinolysin for trypsin in the preparation of standard curves relating optical density of the hydrolysis products measured at 280 mp. to units of fibrinogenolytic activity as shown in Fig. 1.§ The fibrinogenolytic activity of the tissue-activated fibrinolysin and of the tissue extracts alone can then be determined as follows: • Armour Laboratories. tParke, Davis an
Volume 71 Number 2
FIBRINOLYTIC ENZYME IN HUMAN TISSUE
345
Two tul,es containing 0.2 ml. profibrinolysin, 0.2 ml. tissue extract, 0.6 mi. buffer, and 1 mi. of fibrinogen solution were prepared. In one tube the proteins were precipitated immediately hy the addition of 3 ml. of 5 per cent trichloracetic a()id. 'I'he other tube was incubated for two hours at 37° C. Lefore the reaction was stopped with trichloracetic adJ. Aftrr ~tanding one l1onr tne optiral density at 2.80 mp of the supernatant fluid was determined in a Beckman spectrophotometer. 'I'he fibrinogenolytic activity was then obtained from the difference in optical density of the two tubes by referring to the standard curve. The activity of the tissue extract itself was determined in the same >vay, omitting the profibrinolysin and snlJstituting buffer to keep the volume constant.
The eytofibrinokinase activity of the tissue extract is the difference between the activity of the prolysin plus tissue extract and that of the tissue extract alone. The profihrinolyRin solution itself showed no hydrolysis of fibrinogen in two hom·s' inenbatio11.
0.01
O.Ot
O.OS
0.04
Fibrinopnolytic Activity
0.015
Fig. I. -·Colihration curve relating optical density of the trichloracetic acid-soluble hydrolysis products at 280 mJt to units of fibrinogeno1ytic activity.
Fibrinolytic acti1'ily of the tissue-activated enzyme and tissue extract is determined by a method similar to that used by Tagnon13 and is reported in terms of the reciprocal of the time of lysis of a standard fibdn clot.
Results Table I shows the fibrinogenolytic activity of the tissue extract, the profibrinolysin plus tissue extract, and the cytofibrinokinase activity as determined bv the differrnc(' between the other two values. Of the tissues examined, all groups showed both fibrinogenolytic activity and cytofibrinokinase activity. Considerable differences exist, however, among the individual samples in each group. The high figures obtained with endometrial extracts are in agreement with those reported by Smith and Smith. 16 It is of particular interest that endometrium in the secretory phase possesse3 greater activity than does that in the proliferative. Table II contains figures showing the fibrinolytic activity of these same extracts. Significant fibrinolytic and cytofihrinokinase activity is demonstrated only in the extracts of myometrium and endometrium.
PHILLIPS, BUTLER, AND TAYLOR
346
TABLE I.
FIBRINOGENOLYTIC ACTIVITIES
PROF'IBRINOLYSIN PI,US TISSUE EXTRACT OPTICAL DENSITY TISSUE
Myometrium. M. B.
M. C. M.D. R. G. J. v. M. II. J)ecid-u.a.M.B. M. C. M.D. R.. G. .T. v. M. H. Placenta.M.B. M. C.
M.D.
R. G. M. H. 16 weeks
(xl03) 60 23 29 26 32
30
57
53
61 25 22
44
49 75 47 32 47 82
Endometrium, PmliferaUve.50 c. s. E. A. R.B. G. G.
93 124 101
M. F. M. M. K. G.
134 100
Endometrium, Secretory.-
Fetal Tiss~te, Lung;:, LiV(:'T
Brain
221 lt!. H.60
gg 77
UNITS*
(x1Q3) 3.3
1.1
1.3 1.3 ].6 1.5 3.1 2.8 3.4 1.2 1.1 2.3
TISSUE EXTRACT ALONE OPTICAL DENSITY
( xl03)
29 26 19 25 25
17 23
32 44 21
23
24
2.6
40
2.5 1.6 2.5 5.6
45
4.8
2.7
Am. J. Ohst. & Gynec. February, 1956
47
23
49
48
38
CY'l'OFIBRINO· KINASE
T.:'NITS*
(x103) 1.5 1.3 0.9 1.2 1 •) 0.8
UNITSt
(x103) 1.8
-t
0.4 0.4 0.7
1.1
2.0 1.2 1J
1.2
1.1
2.0 2.5 2.3 1.1 2.6
0.6 2.3
2.4
3.2
].7 5.S
1.0 1.2
1.6 2.3 1.0 1.1
0.5
7.0 14.4 8.3
84 59
64
2.6
18.5 8.0 00.0
103
S.7
!1.8
4.1 21.5
68.5
Ul 3.0
'1.8
:u
7.8 4.9
69
141
36
55 46
2.\)
2.4
11.5 5.7
:l.H
1.3
2.5
*Determined from optical density by use of calibration curve (Fig. 1). tDetermined by subtracting column 4 from column 2. t'Where differences are within experimental error of the method the figures have been
omitted.
The case histories of the patients who had cesarean hysterectomies contribute little to the understanding of the problem and therefore have been omitted. A short summary of pertinent facts about the patients from whom the endometrium was obtained may be of value, however; this information is shown in Table III. Comment As is evident from a comparison of Tables I and II a considerable difference is :found between the fibrinogenolytic activity as measured by the optical density at 280 mp. of the hydrolysis products of fibrinogen, and the fibrinolytic activity as measured by the time of lysis of a standard fibrin clot. Most of the extracts of each group of tissue showed ability to stimulate a fibrinogenolytic activity in the profibrinolysin, as well as the presence o£ an active proteolytic enzyme. On the other hand, only extracts of myometrial
Volume71 Number 2
FIBRINOLYTIC ENZYME IN HUMAN TISSUE
347
TABLE II. FIBRINOLYTIC AcTIVlTIES
TISSUE
Myometrium.M. B. M. C.
M.D.
R. G. J. v. M. H.
Decidua.M.B. M. C. H. G. .J. v.
M. H.
Placenta.M.B. M. C. M.D. R. G.
M. H.
16 weeks
PROFIBRINOLYSIN PLUS TISSUE EXTRACT TIME OF LYSIS IN MINUTES UNITS*
TISSUE EXTRACT ALONE TIME OF LYSIS IN UNITS* MINUTES
105 600-1,320 240 68 1,320 65
0.95 0.10t 0.42 1.47 0.08 1.54
600-1,320 1,320 600-1,320 330 1,320 240
0.10 0.08 0.10 0.30 0.08 0.42
0.85 0.02 0.32 1.17
600-1,320 600-1.320 600-1:320 600-1;320 600-1,320
0.10 0.10 0.10 0.10 0.10
1,320 600-1,320 1,320 600-1,320 1,320
0.08 0.10 0.08 0.10 0.08
0.02
600 600 600 600-1,320 1,320 600
0.18 0.18 0.18 0.10 0.08 0.18
1,320 600 1,320 1,320 1,320 600-1,320
0.08 0.18 0.08 0.08 0.08 0.10
0.10 0.10 0.02
0.18 0.67 0.30
1,320 1,320 1,320
0.08 0.08 0.08
0.10 0.59 0 ,,,,
120 180 60
0.83 0.55 1.67
600 600 120
0.18 0.18 0.83
0.65 0.37 0.8!
600-1,320 600-1,320 600-1,320
0.10 0.10 0.10
600-1,320 600-1,320 600-1,320
0.10 0.10 0.10
Endometri-um, Proliferative.600 150 330
E. A.
R.B. G. G.
Endometrium, Secretory.M. F. M. M. K. G.
Fetal Tissue, M. H.Lungs Liver Brain
100.
CYTOFIBRINO· KINASE
UNITSt
1.12
0.02 0.02
0.08
1 *Fibrinolytic units are expressed as the reciprocal of the time of lysis times 100 or T X
+Determined by subtracting column 4 from column 2, tAn activity of 0.10 units is used as an approximation where lysis occurred between 600 and 1.320 minutes. TABLE III. DATA CONCERNING PATIENTS FROM WHOM ENDOMETRIUM WAS OBTAINED
E. A.
46
Proliferative
10
R. B.
38
Proliferative
G. G.
32
Proliferative
No period 2 months 11
K. G. M. M.
35
38
Late secretory Early secretory
34 21
M. F.
42
Secretory
26
Metrorrhagia Menorrhagia Fibroids Menometrorrhagia Fibroids Menorrhagia Fibroids Endometriosis Left ovarian cyst Irregular vaginal bleeding Menorrhagia Fibroids
348
PHILLIPS, BUTLER, AND TAYLOH
Am. ]. Obst. & Gynec. February. 1956
tissue and of endometrium were able to produce from the prolysin an Pnzyuu· capable of lysing a fibrin clot, and in only twu of the uterine extracts aud •me extrad of endometrium was sufficient activt~ enzylll(' Jn·esent to fll'udw•p l.nds in less than ten hours. Whether this represents two different enzymE's or a different mode of action on the two substrates it is impossible to d<"eide at the present tim('. lt is also !'('Cognized that while these methods prodde a ltlenns of comparinl.( t ht> proteolytic and cytofibrinokinase activities of tisslw t•xtraetH, tlwy at·•· 11ut necessarily a direct measure of these quanti! ies. 1\eganUess of the mtmbt>r of en:;;ynws involYed it haB lwt>n (!Pmonstmted that extracts of placenta, myometrium, nndH control. \Ylwn these inhibitors are low t n h(egin with oJ· al'P ust>d up to inactivate unusual quantities of active fibrinolysin, however, 1h1· ;.;y;-;1 t'lll may then get out of control and hemorrhage may n·Kult. ']'he presence of a proteol:vtic enzyme and all activator of th<• tibrinolytie system in endometrium extracts helps tn (•xplain the fluidity of lllen::;tnwl blood. Both of these activities apparently inct·ease as the (~ycle }H'ogr'eHses, !'<•aching a maximum just hefol'e menstr·nation. A furtheT' study of 1ht>sf• ~~hanges is in progress. Summary The presence of a fibrinogenolytic Pnzyme and a cytofibrinokina:>e has been demonstrated in extracts of human JHyometrium, placenta, deddna. and endometrium.
Volume 71 Number 2
FIBRINOLYTIC JmZYME IK HUJ\fAN TISSUE
349
It is suggested that the presence of these enzymes may be partially or wholly responsible for the lack of fibrinogen which occurs in the blood stream of obstetrical patients under certain conditions, and in menstrual blood. References 1. Dieckmann, W. J.: AM. J. OBST. & GY::
Schneider, C. L.: Obst. & Gynec. 4: 273, l!J5J. 7. Maloney, V. C., Ego11, \Y. ,T., and Gorman, H. J.: New Englv.nd .r. Med. 240: 596, 1949 . .~. McKay, D. G., Merrill, S. J., Weiner, A. E., Hertig, A. T., and Reid, D. E.: AM. J. OBS'l'. & GYNEC. 66: 507, 1953. li. Butler, B. C., Taylor, H. C., Jr., am! Graff, 8.: A:>f. J. OBST. & GYNDC. 60: 56+, 1950. 10. Butler, B. C., Graff, S., and Graff, A. B.: AM. J. OBST. & GYN~;c. C2: 50fi, 1951. 11. Tagnon, H. J.: Cancer 6: 63, 1953. 12. Margulis, R. R., Luzadre, J. H., and Hodgkinson, C. Paul: Obst. & G;nwc. 3: 487, 1954. 13. Tagnon, H. J., and Palade, G. E.: .T. Clin. Invest. 29: 317, 1!150. 14. Lewis, Jessica H., and Ferguson, John H.: .I. Clin. Invest. 2<): 1059, 1950. 15. Kunitz, M.: J. Gen. Physiol. 30: 291, 1947. Hi. Smith, 0. W., and Smith, G. VanS.: Seieuce 102: 253, 1!145. 17. Astrup, '1'.: Biochem. J. 50: 5, 1951. li.
accompanied by massive hemorrhage in obstetrical AFIBRINOGENEMIA patients has been reported by a number of investigators.l-7 The condiin cases of abruptio placentae, long-standing
tion has been observed primarily fetal death, and amniotic fluid infusion. The theory has been advanced that this afibrinogenemia is caused by an extensive intravascular clotting due to the infusion of thromboplastin-like material into the blood stream of the mother from the placenta, amniotic :fluid, or decidua. 2 • 3 • 5 • 6 Schneider6 has suggested for this condition a mechanism shown in the following diagram : Prothrombin
Other factors
'fissuc
thromboplastin
l
Heparin and other inhibitors
'Thrombin
Fihrinogen
1
-------------clo Fihrin
Although small thrombi have been found in the tissues of patients who have died from afibrinogenemia6 ' 8 these are not sufficient to account for all of the fibrinogen in the body. An intravascular clotting of such an extent as to remove most of this fibrinogen, i.e., 8 to 12 Gm., must of necessity be aecom· panied by a subsequent lysis of the clots. In some cases of afibrinogenemia au accompanying fibrinolysin ha.s been detected in the blood of the patient"• '; in other eases the investigators have been unable to demonstrate the presence of an active fibrinolytic enzyme system in vitro. It is suggested, however, that a fibrinolytic enzyme may be either partially or wholly responsible for the disappearance of the fibrinogen in spite of the fact that its presence cannot always be demonstrated in vitro by the methods in general use. *This investigation was supported
(in Part) by a
research grant H-1512 from the
National Heart Institute of the National Institutes of Health, Public Health Service. ••Present address, Phoenix, Ariz.
342
Volume 71
Number 2
FIBRINOLYTIC ENZYME IN HUMAN TISSUE
343
A precursor of fibrinolysin (profibrinolysin) is present in all normal
human blood plasma. This proenzyme can be activated by a number of factors including cytofibrinokinase (a tissue activator) and streptokinase (a bacterial activator). The active enzyme (fibrinolysin) thus formed is capable of hydrolyzing fibrinogen, fibrin, and certain other proteins. This may be represented by the following scheme: Tissue activator or cytofibrinokinase
l
FiheT'yeiiJn----~
>'ibrinngen Fibrin --------
Amino acids and polypeptides
I
Inhibitors
For the fibrinolysin enzyme system to be important in these situations, the activating enzyme, cytofibrinokinase, must be found in the tissues involved, i.e., endometrium, myometrium, placenta, decidua. With the breakdown of the cells of these tissues as a result of catabolic and autolytic changes subsequent to menstruation, labor and the separation of the placenta, and long-standing fetal death, the intracellular cytofibrinokinase could initiate enzymatic conversion of profibrinolysin to fibrinolysin. When fibrinolysin is unopposed by inhibitors or antifibrinolysin, the hydrolysis of the body fibrinogen is rapid ana aangerous. This mechanism has been suggested in previous papers. 9 • 10 A comparable situation has been demonstrated by Tagnon11 in cases of metastatic cancer of the prostate. He has reported fibrinolytic activity in the primary and metastatic cancer tissue and suggested that release of enzyme from this tissue was responsible for the hemorrhagic syndrome in his five patients. In his cases there was a deficiency of fibrinogen in the blood and a prolongation of the prothrombin time. Margulis and associates12 have reported fibrinolysin in 14 out of 17 uncomplicated pregnancies within the first 24 hours after delivery, with no change in prothrombin time, bleeding time, or clotting time. The purpose of the work presented here was twofold: (1) the development of a more sensitive method for the detection of fibrinolysin in vitro, and (2) a study of the activation of profibrinolysin b:;r the cytofi'brinokinasc con= tained in such tissues as myometrium, placenta, decidua, and endometrium. The presence of such an activator would account for the fibrinolysin observed 'in obstetrical patients with both complicated and uncomplicated- deliveries. A cytofibrinokinase in endometrium would give a mechanism by which menstrual blood remains fluid. Finally, such an activator would suggest an alternate method for the removal of fibrinogen from the blood stream of
l>ltiLLil>S, BUTLER, AND TAYLOR
Am.
J. Obst. & Gynec.
February. 1956
patients suffering from obstetrical accidents in which tissue extracts may be forced back into the maternal circulation. The subsequent removal of tibrinogen by hydrolysis may then either be the principal cause of afibrinogenemia in these patients or may supplement the removal by fibrin deposition dne to the presence of tissue thromboplastin.
Materials Extracts from the myometrium, decidua, and placenta of 5 women who had had cesarean hysterectomies were prepared immediately after the operation and subsequently tested for fibrinogenolytic, fibrinolytic, and cytofibrinokinase activities. Results of similar extracts from one woman (M. H.) who had a therapeutic abortion and hysterectomy for carcinoma of the liver when approximately five months pregnant are also included as are those of a sample of placenta from a hysterotomy at sixteen weeks and sampleR or t•nclometrium obtained after hysterectomy. Tissue extracts were prepared as described by Tagnon 1:l and ~uspended an
Methods Fibrinogenolytic activity has been determined by a method adapted from that used by Kunitz 15 for the hydrolysis of casein by trypsin. Fibrinogen was ~ubstituted for casein, and streptokinase-activated fibrinolysin for trypsin in the preparation of standard curves relating optical density of the hydrolysis products measured at 280 mp. to units of fibrinogenolytic activity as shown in Fig. 1.§ The fibrinogenolytic activity of the tissue-activated fibrinolysin and of the tissue extracts alone can then be determined as follows: • Armour Laboratories. tParke, Davis an
Volume 71 Number 2
FIBRINOLYTIC ENZYME IN HUMAN TISSUE
345
Two tul,es containing 0.2 ml. profibrinolysin, 0.2 ml. tissue extract, 0.6 mi. buffer, and 1 mi. of fibrinogen solution were prepared. In one tube the proteins were precipitated immediately hy the addition of 3 ml. of 5 per cent trichloracetic a()id. 'I'he other tube was incubated for two hours at 37° C. Lefore the reaction was stopped with trichloracetic adJ. Aftrr ~tanding one l1onr tne optiral density at 2.80 mp of the supernatant fluid was determined in a Beckman spectrophotometer. 'I'he fibrinogenolytic activity was then obtained from the difference in optical density of the two tubes by referring to the standard curve. The activity of the tissue extract itself was determined in the same >vay, omitting the profibrinolysin and snlJstituting buffer to keep the volume constant.
The eytofibrinokinase activity of the tissue extract is the difference between the activity of the prolysin plus tissue extract and that of the tissue extract alone. The profihrinolyRin solution itself showed no hydrolysis of fibrinogen in two hom·s' inenbatio11.
0.01
O.Ot
O.OS
0.04
Fibrinopnolytic Activity
0.015
Fig. I. -·Colihration curve relating optical density of the trichloracetic acid-soluble hydrolysis products at 280 mJt to units of fibrinogeno1ytic activity.
Fibrinolytic acti1'ily of the tissue-activated enzyme and tissue extract is determined by a method similar to that used by Tagnon13 and is reported in terms of the reciprocal of the time of lysis of a standard fibdn clot.
Results Table I shows the fibrinogenolytic activity of the tissue extract, the profibrinolysin plus tissue extract, and the cytofibrinokinase activity as determined bv the differrnc(' between the other two values. Of the tissues examined, all groups showed both fibrinogenolytic activity and cytofibrinokinase activity. Considerable differences exist, however, among the individual samples in each group. The high figures obtained with endometrial extracts are in agreement with those reported by Smith and Smith. 16 It is of particular interest that endometrium in the secretory phase possesse3 greater activity than does that in the proliferative. Table II contains figures showing the fibrinolytic activity of these same extracts. Significant fibrinolytic and cytofihrinokinase activity is demonstrated only in the extracts of myometrium and endometrium.
PHILLIPS, BUTLER, AND TAYLOR
346
TABLE I.
FIBRINOGENOLYTIC ACTIVITIES
PROF'IBRINOLYSIN PI,US TISSUE EXTRACT OPTICAL DENSITY TISSUE
Myometrium. M. B.
M. C. M.D. R. G. J. v. M. II. J)ecid-u.a.M.B. M. C. M.D. R.. G. .T. v. M. H. Placenta.M.B. M. C.
M.D.
R. G. M. H. 16 weeks
(xl03) 60 23 29 26 32
30
57
53
61 25 22
44
49 75 47 32 47 82
Endometrium, PmliferaUve.50 c. s. E. A. R.B. G. G.
93 124 101
M. F. M. M. K. G.
134 100
Endometrium, Secretory.-
Fetal Tiss~te, Lung;:, LiV(:'T
Brain
221 lt!. H.60
gg 77
UNITS*
(x1Q3) 3.3
1.1
1.3 1.3 ].6 1.5 3.1 2.8 3.4 1.2 1.1 2.3
TISSUE EXTRACT ALONE OPTICAL DENSITY
( xl03)
29 26 19 25 25
17 23
32 44 21
23
24
2.6
40
2.5 1.6 2.5 5.6
45
4.8
2.7
Am. J. Ohst. & Gynec. February, 1956
47
23
49
48
38
CY'l'OFIBRINO· KINASE
T.:'NITS*
(x103) 1.5 1.3 0.9 1.2 1 •) 0.8
UNITSt
(x103) 1.8
-t
0.4 0.4 0.7
1.1
2.0 1.2 1J
1.2
1.1
2.0 2.5 2.3 1.1 2.6
0.6 2.3
2.4
3.2
].7 5.S
1.0 1.2
1.6 2.3 1.0 1.1
0.5
7.0 14.4 8.3
84 59
64
2.6
18.5 8.0 00.0
103
S.7
!1.8
4.1 21.5
68.5
Ul 3.0
'1.8
:u
7.8 4.9
69
141
36
55 46
2.\)
2.4
11.5 5.7
:l.H
1.3
2.5
*Determined from optical density by use of calibration curve (Fig. 1). tDetermined by subtracting column 4 from column 2. t'Where differences are within experimental error of the method the figures have been
omitted.
The case histories of the patients who had cesarean hysterectomies contribute little to the understanding of the problem and therefore have been omitted. A short summary of pertinent facts about the patients from whom the endometrium was obtained may be of value, however; this information is shown in Table III. Comment As is evident from a comparison of Tables I and II a considerable difference is :found between the fibrinogenolytic activity as measured by the optical density at 280 mp. of the hydrolysis products of fibrinogen, and the fibrinolytic activity as measured by the time of lysis of a standard fibrin clot. Most of the extracts of each group of tissue showed ability to stimulate a fibrinogenolytic activity in the profibrinolysin, as well as the presence o£ an active proteolytic enzyme. On the other hand, only extracts of myometrial
Volume71 Number 2
FIBRINOLYTIC ENZYME IN HUMAN TISSUE
347
TABLE II. FIBRINOLYTIC AcTIVlTIES
TISSUE
Myometrium.M. B. M. C.
M.D.
R. G. J. v. M. H.
Decidua.M.B. M. C. H. G. .J. v.
M. H.
Placenta.M.B. M. C. M.D. R. G.
M. H.
16 weeks
PROFIBRINOLYSIN PLUS TISSUE EXTRACT TIME OF LYSIS IN MINUTES UNITS*
TISSUE EXTRACT ALONE TIME OF LYSIS IN UNITS* MINUTES
105 600-1,320 240 68 1,320 65
0.95 0.10t 0.42 1.47 0.08 1.54
600-1,320 1,320 600-1,320 330 1,320 240
0.10 0.08 0.10 0.30 0.08 0.42
0.85 0.02 0.32 1.17
600-1,320 600-1.320 600-1:320 600-1;320 600-1,320
0.10 0.10 0.10 0.10 0.10
1,320 600-1,320 1,320 600-1,320 1,320
0.08 0.10 0.08 0.10 0.08
0.02
600 600 600 600-1,320 1,320 600
0.18 0.18 0.18 0.10 0.08 0.18
1,320 600 1,320 1,320 1,320 600-1,320
0.08 0.18 0.08 0.08 0.08 0.10
0.10 0.10 0.02
0.18 0.67 0.30
1,320 1,320 1,320
0.08 0.08 0.08
0.10 0.59 0 ,,,,
120 180 60
0.83 0.55 1.67
600 600 120
0.18 0.18 0.83
0.65 0.37 0.8!
600-1,320 600-1,320 600-1,320
0.10 0.10 0.10
600-1,320 600-1,320 600-1,320
0.10 0.10 0.10
Endometri-um, Proliferative.600 150 330
E. A.
R.B. G. G.
Endometrium, Secretory.M. F. M. M. K. G.
Fetal Tissue, M. H.Lungs Liver Brain
100.
CYTOFIBRINO· KINASE
UNITSt
1.12
0.02 0.02
0.08
1 *Fibrinolytic units are expressed as the reciprocal of the time of lysis times 100 or T X
+Determined by subtracting column 4 from column 2, tAn activity of 0.10 units is used as an approximation where lysis occurred between 600 and 1.320 minutes. TABLE III. DATA CONCERNING PATIENTS FROM WHOM ENDOMETRIUM WAS OBTAINED
E. A.
46
Proliferative
10
R. B.
38
Proliferative
G. G.
32
Proliferative
No period 2 months 11
K. G. M. M.
35
38
Late secretory Early secretory
34 21
M. F.
42
Secretory
26
Metrorrhagia Menorrhagia Fibroids Menometrorrhagia Fibroids Menorrhagia Fibroids Endometriosis Left ovarian cyst Irregular vaginal bleeding Menorrhagia Fibroids
348
PHILLIPS, BUTLER, AND TAYLOH
Am. ]. Obst. & Gynec. February. 1956
tissue and of endometrium were able to produce from the prolysin an Pnzyuu· capable of lysing a fibrin clot, and in only twu of the uterine extracts aud •me extrad of endometrium was sufficient activt~ enzylll(' Jn·esent to fll'udw•p l.nds in less than ten hours. Whether this represents two different enzymE's or a different mode of action on the two substrates it is impossible to d<"eide at the present tim('. lt is also !'('Cognized that while these methods prodde a ltlenns of comparinl.( t ht> proteolytic and cytofibrinokinase activities of tisslw t•xtraetH, tlwy at·•· 11ut necessarily a direct measure of these quanti! ies. 1\eganUess of the mtmbt>r of en:;;ynws involYed it haB lwt>n (!Pmonstmted that extracts of placenta, myometrium,
Volume 71 Number 2
FIBRINOLYTIC JmZYME IK HUJ\fAN TISSUE
349
It is suggested that the presence of these enzymes may be partially or wholly responsible for the lack of fibrinogen which occurs in the blood stream of obstetrical patients under certain conditions, and in menstrual blood. References 1. Dieckmann, W. J.: AM. J. OBST. & GY::
Schneider, C. L.: Obst. & Gynec. 4: 273, l!J5J. 7. Maloney, V. C., Ego11, \Y. ,T., and Gorman, H. J.: New Englv.nd .r. Med. 240: 596, 1949 . .~. McKay, D. G., Merrill, S. J., Weiner, A. E., Hertig, A. T., and Reid, D. E.: AM. J. OBS'l'. & GYNEC. 66: 507, 1953. li. Butler, B. C., Taylor, H. C., Jr., am! Graff, 8.: A:>f. J. OBST. & GYNDC. 60: 56+, 1950. 10. Butler, B. C., Graff, S., and Graff, A. B.: AM. J. OBST. & GYN~;c. C2: 50fi, 1951. 11. Tagnon, H. J.: Cancer 6: 63, 1953. 12. Margulis, R. R., Luzadre, J. H., and Hodgkinson, C. Paul: Obst. & G;nwc. 3: 487, 1954. 13. Tagnon, H. J., and Palade, G. E.: .T. Clin. Invest. 29: 317, 1!150. 14. Lewis, Jessica H., and Ferguson, John H.: .I. Clin. Invest. 2<): 1059, 1950. 15. Kunitz, M.: J. Gen. Physiol. 30: 291, 1947. Hi. Smith, 0. W., and Smith, G. VanS.: Seieuce 102: 253, 1!145. 17. Astrup, '1'.: Biochem. J. 50: 5, 1951. li.