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On the conversion of high molecular weight urokinase to the low molecular weight form by plasmin
THROMBOSIS RESEARCH 23; 541-547, 1981 0049-3848/81/180541-07$02.00/O Copyright (c) 1981 Pergamon Press Ltd. All rights reserved.
ON
THE CONVERSION OF HIGH TO THE LOW MOLECULAR
Grant H. Barlow, Hematology University
of
Charles Unit,
MOLECULAR WEIGHT UROKINASE WEIGHT FORM BY PLASMIN
W.
Francis
Department
and Department of Rochester School Rochester, New
and of
Victor
J.
Marder
Medicine
Biochemistry of Medicine York 14642
and
Dentistry
(Received 29.6.1981; in revised form 31.8.1981. Accepted by Editor N. Alkjaersig) ABSTRACT The conversion of high molecular weight urokinase (HMW-UK) to low molecular weight urokinase (LMW-UK) by plasmin in vitro has been -studied. The two forms of urokinase were separated by SDS-polyacrylamide gradient gel electrophoresis and active enzyme was extracted from 5 mm gel segments into isotonic saline and analyzed by the fibrin plate method. Electrophoretlc separation of the two forms was complete, as indicated by comparison with the migration of purified standards and by the absence of lytic activity in extracts of HMW-UK was incubated with plasminogen and intervening gel segments. fibrinogen for various time intervals, after which enzymatic activity was inhibited with aprotinin and the samples subjected to electrophoresis. Conversion from HMW-UK to LMW-UK was apparent in the first sample at 2.5 minutes and continued for the 10 minute duration of the experiments. Similar experiments starting with LMW-UK showed no change in molecular weight. Incubation of HMW-UK without plasminogen resulted in no conversion to LMW-UK, indicating that plasmin was the active agent and that the reaction was not autocatalytic.
INTRODUCTION In 1960, Celander and Guest demonstrated that the plasminogen activator urokinase could exist in multiple molecular forms (1), an observation that was confirmed by White et al., who isolated a 54,000 dalton high molecular weight form (HMW-UK) and a 31,000 dalton low molecular weight form (LMW-UK) from a pool of human urine (2). Lesuk et al. showed that LMW-UK could be derived from HMW-UK by limited proteolysis with trypsin and postulated that a similar enzymatic step was responsible for the conversion In urine (3). Soberano et al
Key
words:
Plasminogen
activators,
Fibrinolysin,
541
Fibrinolysis
542
HMW-UK CONVERSION TO LMW-UK
Vo1.23, No.6
subsequently demonstrated that partially purified HMW-UK slowly converts to 17 hours (4). They confirmed that LMW-UK on standing at room temperature for proteolysis effects this conversion, since it was inhibited by the potent serine protease inhibitor benzamidine. The same conversion occurs in the urokinase-rich conditioned medium of human fetal kidney cell culture after prolonged incubation (5). The present study seeks to determine whether plasmin can mediate this conversion of HMW-UK --in vitro. MATERIALS
AND METHODS
Purified preparations of HMW-UK and LMW-UK were a generous gift of Dr. F. Toulemonde (Choay Laboratories, Paris, France). Purified lys-plasminogen was provided by Dr. William Holleman (Abbott Laboratories, N. Chicago, 111.). Bovine fibrinogen (75% clottable) was purchased from Miles Laboratories, Elkhardt, Ind. and aprotinin (TrasylolR) was purchased from the FBA Pharmaceutical Co. (New York, NY). Sample preparation. or urokinase plus were mixed prescribed moved
and
units)
.
at 4OC. intervals immediately
Samples containing either urokinase alone (2,000 lys-plasminogen (2 casein units/ml) and fibrinogen The of
mixture 0, 2.5,
mixed
with
was transferred 5 and 10 minutes, .025
ml
aprotinin
to
a 37OC aliquots (250
waterbath of .25
kallikrein
IU/ml) (3 mg/ml)
and at ml were reinhibitory
Fibrinolytic activity of HMW-UK and LMW-UK. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis was performed using gradient gels of 5-20% (6,7) as modified in our laboratory (8). Duplicate samples were electrophoresed, after which one gel strip was stained for protein and the other was sliced at 5 mm intervals. Each slice was pulverized in .25 ml of .15 M sodium chloride and extracted at 4OC overnight, and a 20 1 aliquot of each supernatant was placed on a fibrin plate prepared according to the technique of Astrup and Mu1 lertz (9). After incubation at room temperature overnight, the area of the lysis zones was determined; when activity was contained in contiguous slices, the areas were summed. The stained gel strip was examined for specific fibrinogen degradation products as further evidence that fibrinolytic activity had been produced in the reaction mixture. RESULTS Figure 1 shows the electrophoretic gel pattern of a synthetic mixture of HMW-UK and LMW-UK and the fibrinolytic activity of the sliced gel segments. The two molecular forms were well-separated in the stained gel strip and there was no overlap in fibrinolytic activity in each band. Commercial preparations of urokinase showed different distributions of HMW-UK and LMW-UK forms (Fig. 2)) some containing virtually all of one or the other and some containing varying proportions of both forms. Figure 3 shows the results of fibrinolytic activity of electrophoretic gel strip segments after increasing incubation intervals of HMW-UK in the The first sample at 2.5 min. showed presence of fibrinogen and plasminogen. an increased amount of the LMW-UK form, which progressively increased in quantity during the 10 minute duration of the experiment, concomitant with a
vo1.23,
No.6
HMW-UK CONVERSION TO LMW-UK
543
FIBRINOLYTIC ACTIVITY OF HIGH AND LOW MOLECULAR WEIGHT UROKINASE
HMW
LMW
Fig. 1 SDS-polyacrylamide gradient gel electrophoresis of a synthetic mixture of HMW-UK (6,000 IU/ml) and LMW-UK (6,000 IU/ml). The electrophoretic position of HMW and LMW forms of urokinase are shown in the stained gel strip at the top, and the fibrinolytic activity of 5 mm gel slices as measured on fibrin plates is shown at the bottom. Each bar represents the iysis area of a single slice; there were two slices devoid of activity between the regions corresponding to HMW-UK and LMW-UK.
..-_.
-_
E t
Fig.
2
SDS-gradient gel electrophoretlc patterns of four different commercial urokinase preparations. The first and third preparations are of human urinary source, while the second and fourth preparations are obtained from tissue culture of human fetal kidney cells.
1
2
Commercial
3
4
Product
HMW-UK CONVERSION TO LMW-UK
544
0
2.5
5
IO
0
HMW
Vo1.23, No.6
2.5
5
10
LMW
TIME
(min)
3 Fig. Fibrinolytic activity measured on fibrin plates of HMW-UK and LMW-UK regions from an SDS-polyacrylamide gradient gel electrophoresis after incubation fibrinogen
decrease diffusion
in
the
rate
of HMW-UK with lys-plasminogen for the incubation times as
HMW-UK content. of
the
activator,
Since the the areas
fibrin noted
and noted.
lysis area in Figures
depends upon the 1 and 3 were log-
and resulted in a much larger lysis arithmic functions of molecular weight, area for the LMW-UK form than for an equivalent amount of the HMW-UK form. Analysis of the stained gel strips showed that the fibrinogen present in the control reaction mixture had been converted to a mixture of fragments X, Y, D and E (Stage 2 digest) at 2.5 minutes and to fragments D and E (Stage 3 digest) at 10 minutes. Incubation of LMW-UK with fibrinogen and plasminogen showed no tendency toward change in electrophoretic position or fibrinolytic activity of the sliced gel segments, indicating that no degradation of this molecular form occurred. When HMW-UK was incubated in the absence or plasminogen, there was no evidence of conversion to LMW-UK, further indicating that plasmin was the active agent and Omitting ruling out an autocatalytic reaction which converted HMW-UK to LMW-UK. fibrinogen from the reaction mixture of HMW-UK with plasminogen did not alter the rate or amount of conversion to LMW-UK, indicating that the reaction did not depend upon a contaminant present in the fibrinogen preparation. DISCUSSION Our results LMW-UK concurrent
show that with the
HMW-UK is conversion
rapidly and progressively of plasminogen to plasmin
degraded by the
to activator.
545
HMW-UK CONVERSION TO LMW-UK
vo1.23, No.6
The expected conversion of plasminogen to plasmin was indicated by the demonstration of characteristic plasmic degradation of fibrinogen (IO) when this substrate was included in the incubation mixture. No conversion of urokinase was seen when HMW-UK was incubated alone, indicating that the reaction was not autocatalytic. Accurate quantitation of the kinetics of this conversion was difficult, because of variability In the slicing of the slab gels, the extraction of protein from the gel slices and especially in the mathematical conversion of fibrin lysis zones into concentrations of activator. Since the fibrin lysis area depends upon the diffusion rate of the activator, the areas noted in Figs. 1 and 3 were logarithmic functions of relative molecular size, and resulted in a much larger lysis area for the LMW-UK form than for an equivalent amount of the HMW-UK form. The role of plasmin in the conversion of HMW-UK has precedence in other components of the fibrinolytic system. For instance, plasmin converts gluplasminogen to lys-plasminogen by the release of a 6,000 dalton peptide (11). Furthermore, during streptokinase activation of plasminogen, the streptokinase moiety is degraded to a series of smaller fragments (12). Little is known concerning the structural changes involved in the urokinase conversion. While it is well-established that HMW-UK is a two-chain molecule held together by disulfide bonds (13,14), no comparable information is available in the LMW-UK It is possible that form, nor is the protease-sensitive area of HMW-UK known. there are a series of low molecular weight urokinases of similar size with intact disulfide bonds, It is possible that the presence of free plasmin in the blood during thrombolytic therapy (15) or even the conversion of plasminogen to plasmin on the surface of a fibrin mesh (16) creates a situation in which the same conversion of HMW-UK to LMW-UK can occur during thrombolytic therapy. This possibility would have important physiologic implications, since kinetic studies show that HMW-UK is more active than LMW-UK both in converting plasminogen to plasmin (17) and in accelerating clot lysis (IS), and the two forms also react at different rates with proteolytic inhibitors (19). While different commercial preparations of urokinase that are licensed for clinical use may contain markedly different proportions of HMW-UK and LMW-UK (Fig. 2), and variation in the blood concentration of various parameters of activator activity may reflect such differences in urokinase forms (20), there is still no firm evidence in the literature to indicate which type of urokinase is most important for the achievement of therapeutic thrombolysis. Certainly, large amounts of either form are capable of producing a lytic state and clinical thrombolysis (21,22). Since our findings demonstrate that HMW-UK is rapidly converted to LMW-UK by plasmin in vitro, it is possible that a similar conversion occurs and that throxom could --in vivo result mostly from the action of the lower molecular weight plasmin derivative.
ACKNOWLEDGEMENTS This National
work was supported in Heart, Lung and Blood
Bethesda, Maryland, Space Administration.
and
Contract
part by Institute,
Grants HL-00733 and HL-18208 from National Institutes of Health, NAS-g-16187 from the National Aeronautics
the and
546
HMW-UK CONVERSION TO LMW-UK
Vo1.23, No.6
REFERENCES 1.
CELANDER, D.R., Am. J. Cardiol.
and GUEST, M.M. The a, 409-419, 1960.
2.
WHITE, W.F., BARLOW, G.H., and MOZEN, of plasminogen activators (urokinase) 2169, 1966.
3.
LESUK, A., TERMINELLA, L., TRAVER, J.H., and GROSS, J.L. Biochemical and biophysical studies on human urokinase. Thromb. Diath. Haemorrh. 18, 293-294,
biochemistry
M.M. from
and
physiology
of
urokinase.
The isolation and characterization human urine. Biochemistry 5_, 2160-
1967. 4.
SOBERANO,
M.E.,
the catalytic 5.
ONG,
E.F.,
conversion
and
of
JOHNSON,
urokinase.
A.J. The effects of inhibitors Thromb. Res. Y_, 675-681, 1976.
ASTEDT, 8, BARLOW, G.H., an HDLMBERG, L. Time-related release of Thromb. Res. 11, molecular forms of urokinase in tissue culture.
on
various 149-153,
1977. 6.
MARGOLIS, molecular
J. and KENRICK, sieve gradient.
K.G. Polyacrylamide gel-electrophoresis Nature 214, 1334-1336, 1967.
7.
NEVILLE, D. M., JR. Molecular weight determination complexes by gel electrophoresis in a discontinuous 246, 6328-6334, 1971.
8.
Plasmic degradation of crossFRANCIS, C.W., MARDER, V.J. and MARTIN, S.E. analysis of the particulate clot and identilinked fibrin. I. Structural fication of new macromolecualr complexes. Blood 56, 456-464, 1980.
9.
ASTRUP, T. and lytic activity.
of protein-dodecyl buffer. J. 8101.
The fibrin plate method for estimating MULLERTZ, S. Arch. Biochem. Biophys. 40, 346-351, 1952. High and
across
molecular weight derivaimmunological character-
N.R. and CARROLL, W.R. MARDER, V-J., SHULMAN, tives of human fibrinogen. I. Physicochemical ization. J. Biol. Chem. 244, 2111-2119, 1969.
11.
WIMAN, B. plasminogen
12.
SIEFRING, G.E. and CASTELLINO, F.J. Interaction Isolation and characterization of plasminogen. product. J. Bidl. Chem. 251, 3913-3920, 1976.
13.
SOBERANO, M.E., ONG, E.G., JOHNSON, A.J., LEVY, M. and SCHOELLMANN, G. Biochim. Purification and characterization of two forms of urokinase. Biophys. Acta 445, 763-773, 1976.
14.
HOLMBERG, L., chromatography.
peptides released during activation J. Biochem. 2, l-9, 1973.
BLADH, B. and ASTEDT, B. Biochim. Biophys. Acta
sulfate Chem.
fibrino-
10.
Primary structure of Eur. by urokinase.
a
of
human
of streptokinase with a streptokinase degradation
Purification of urokinase 445, 215-222, 1976.
by affinity
~01.23,
No.6
HMW-UK
547
CONVERSION TO LMW-UK
15.
FLETCHER, A.P., ALKJAERSIG, N., SHERRY, S., GENTON, E., HIRSH, J. and BACHMANN, F. The development of urokinase as a thrombolytic agent. Maintenance of a sustained thrombolytic state in man by its intravenous infusion. J. Lab. Clin. Med. 65, 713-731, 1965.
16.
SHERRY, S. Fibrinolysis 343-382, 1959.
17.
LORMEAU, J.C., GOULAY, J., VAIREL, E.G., and CHOAY, J. A comment on the Fibrinolysis, activities of high and low molecular weight urokinase. In: Current Fundamental and Clinical Concepts. P.J. GAFFNEY and S. BALKUVULUTIN (Eds.) London: Acadmic Press, 1978, p. 77.
18.
SAMAMA, M., CAZENAVE, Thrombos. Haemost. &,
19.
L. and BROWN, MURANO, G., ARONSON, D.A., WILLIAMS, high and low molecular weight urokinase in plasma.
20.
Plasma urokinase levels measured by chromoBARLOW, G.H. and MARDER, V.J. genic assay after infusion of tissue culture or urinary source material. Thromb. Res. 3, 431-437, 1980.
21.
MARDER, V.J., DONAHOE, J.F., BELL, W.R., CRANLEY, J.J., KWAAN, H.C., SASAHARA, A.A. and BARLOW, G.H. Changes in the plasma fibrinolytic system during uroComparison of tissue culture urokinase with urinary source kinase therapy. J. Lab. Clin. Med. 92, 721urokinase in patients with pulmonary embolism. 729, 1978.
22.
The Urokinase Pulmonary Embolism Trial. Heart Association Monograph Number 39.
and
fibrinolytic
B. and OTERO, A.M. 578-580, 1978.
activity
Urokinase
A National Circulation
in man.
Physiol.
Rev. 2,
I and II activity. L. The inhibition of Blood 55, 430-436, 1980. --
Cooperative Study. 47_(Suppl 2): i-108,
American 1973.
ON
THE CONVERSION OF HIGH TO THE LOW MOLECULAR
Grant H. Barlow, Hematology University
of
Charles Unit,
MOLECULAR WEIGHT UROKINASE WEIGHT FORM BY PLASMIN
W.
Francis
Department
and Department of Rochester School Rochester, New
and of
Victor
J.
Marder
Medicine
Biochemistry of Medicine York 14642
and
Dentistry
(Received 29.6.1981; in revised form 31.8.1981. Accepted by Editor N. Alkjaersig) ABSTRACT The conversion of high molecular weight urokinase (HMW-UK) to low molecular weight urokinase (LMW-UK) by plasmin in vitro has been -studied. The two forms of urokinase were separated by SDS-polyacrylamide gradient gel electrophoresis and active enzyme was extracted from 5 mm gel segments into isotonic saline and analyzed by the fibrin plate method. Electrophoretlc separation of the two forms was complete, as indicated by comparison with the migration of purified standards and by the absence of lytic activity in extracts of HMW-UK was incubated with plasminogen and intervening gel segments. fibrinogen for various time intervals, after which enzymatic activity was inhibited with aprotinin and the samples subjected to electrophoresis. Conversion from HMW-UK to LMW-UK was apparent in the first sample at 2.5 minutes and continued for the 10 minute duration of the experiments. Similar experiments starting with LMW-UK showed no change in molecular weight. Incubation of HMW-UK without plasminogen resulted in no conversion to LMW-UK, indicating that plasmin was the active agent and that the reaction was not autocatalytic.
INTRODUCTION In 1960, Celander and Guest demonstrated that the plasminogen activator urokinase could exist in multiple molecular forms (1), an observation that was confirmed by White et al., who isolated a 54,000 dalton high molecular weight form (HMW-UK) and a 31,000 dalton low molecular weight form (LMW-UK) from a pool of human urine (2). Lesuk et al. showed that LMW-UK could be derived from HMW-UK by limited proteolysis with trypsin and postulated that a similar enzymatic step was responsible for the conversion In urine (3). Soberano et al
Key
words:
Plasminogen
activators,
Fibrinolysin,
541
Fibrinolysis
542
HMW-UK CONVERSION TO LMW-UK
Vo1.23, No.6
subsequently demonstrated that partially purified HMW-UK slowly converts to 17 hours (4). They confirmed that LMW-UK on standing at room temperature for proteolysis effects this conversion, since it was inhibited by the potent serine protease inhibitor benzamidine. The same conversion occurs in the urokinase-rich conditioned medium of human fetal kidney cell culture after prolonged incubation (5). The present study seeks to determine whether plasmin can mediate this conversion of HMW-UK --in vitro. MATERIALS
AND METHODS
Purified preparations of HMW-UK and LMW-UK were a generous gift of Dr. F. Toulemonde (Choay Laboratories, Paris, France). Purified lys-plasminogen was provided by Dr. William Holleman (Abbott Laboratories, N. Chicago, 111.). Bovine fibrinogen (75% clottable) was purchased from Miles Laboratories, Elkhardt, Ind. and aprotinin (TrasylolR) was purchased from the FBA Pharmaceutical Co. (New York, NY). Sample preparation. or urokinase plus were mixed prescribed moved
and
units)
.
at 4OC. intervals immediately
Samples containing either urokinase alone (2,000 lys-plasminogen (2 casein units/ml) and fibrinogen The of
mixture 0, 2.5,
mixed
with
was transferred 5 and 10 minutes, .025
ml
aprotinin
to
a 37OC aliquots (250
waterbath of .25
kallikrein
IU/ml) (3 mg/ml)
and at ml were reinhibitory
Fibrinolytic activity of HMW-UK and LMW-UK. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis was performed using gradient gels of 5-20% (6,7) as modified in our laboratory (8). Duplicate samples were electrophoresed, after which one gel strip was stained for protein and the other was sliced at 5 mm intervals. Each slice was pulverized in .25 ml of .15 M sodium chloride and extracted at 4OC overnight, and a 20 1 aliquot of each supernatant was placed on a fibrin plate prepared according to the technique of Astrup and Mu1 lertz (9). After incubation at room temperature overnight, the area of the lysis zones was determined; when activity was contained in contiguous slices, the areas were summed. The stained gel strip was examined for specific fibrinogen degradation products as further evidence that fibrinolytic activity had been produced in the reaction mixture. RESULTS Figure 1 shows the electrophoretic gel pattern of a synthetic mixture of HMW-UK and LMW-UK and the fibrinolytic activity of the sliced gel segments. The two molecular forms were well-separated in the stained gel strip and there was no overlap in fibrinolytic activity in each band. Commercial preparations of urokinase showed different distributions of HMW-UK and LMW-UK forms (Fig. 2)) some containing virtually all of one or the other and some containing varying proportions of both forms. Figure 3 shows the results of fibrinolytic activity of electrophoretic gel strip segments after increasing incubation intervals of HMW-UK in the The first sample at 2.5 min. showed presence of fibrinogen and plasminogen. an increased amount of the LMW-UK form, which progressively increased in quantity during the 10 minute duration of the experiment, concomitant with a
vo1.23,
No.6
HMW-UK CONVERSION TO LMW-UK
543
FIBRINOLYTIC ACTIVITY OF HIGH AND LOW MOLECULAR WEIGHT UROKINASE
HMW
LMW
Fig. 1 SDS-polyacrylamide gradient gel electrophoresis of a synthetic mixture of HMW-UK (6,000 IU/ml) and LMW-UK (6,000 IU/ml). The electrophoretic position of HMW and LMW forms of urokinase are shown in the stained gel strip at the top, and the fibrinolytic activity of 5 mm gel slices as measured on fibrin plates is shown at the bottom. Each bar represents the iysis area of a single slice; there were two slices devoid of activity between the regions corresponding to HMW-UK and LMW-UK.
..-_.
-_
E t
Fig.
2
SDS-gradient gel electrophoretlc patterns of four different commercial urokinase preparations. The first and third preparations are of human urinary source, while the second and fourth preparations are obtained from tissue culture of human fetal kidney cells.
1
2
Commercial
3
4
Product
HMW-UK CONVERSION TO LMW-UK
544
0
2.5
5
IO
0
HMW
Vo1.23, No.6
2.5
5
10
LMW
TIME
(min)
3 Fig. Fibrinolytic activity measured on fibrin plates of HMW-UK and LMW-UK regions from an SDS-polyacrylamide gradient gel electrophoresis after incubation fibrinogen
decrease diffusion
in
the
rate
of HMW-UK with lys-plasminogen for the incubation times as
HMW-UK content. of
the
activator,
Since the the areas
fibrin noted
and noted.
lysis area in Figures
depends upon the 1 and 3 were log-
and resulted in a much larger lysis arithmic functions of molecular weight, area for the LMW-UK form than for an equivalent amount of the HMW-UK form. Analysis of the stained gel strips showed that the fibrinogen present in the control reaction mixture had been converted to a mixture of fragments X, Y, D and E (Stage 2 digest) at 2.5 minutes and to fragments D and E (Stage 3 digest) at 10 minutes. Incubation of LMW-UK with fibrinogen and plasminogen showed no tendency toward change in electrophoretic position or fibrinolytic activity of the sliced gel segments, indicating that no degradation of this molecular form occurred. When HMW-UK was incubated in the absence or plasminogen, there was no evidence of conversion to LMW-UK, further indicating that plasmin was the active agent and Omitting ruling out an autocatalytic reaction which converted HMW-UK to LMW-UK. fibrinogen from the reaction mixture of HMW-UK with plasminogen did not alter the rate or amount of conversion to LMW-UK, indicating that the reaction did not depend upon a contaminant present in the fibrinogen preparation. DISCUSSION Our results LMW-UK concurrent
show that with the
HMW-UK is conversion
rapidly and progressively of plasminogen to plasmin
degraded by the
to activator.
545
HMW-UK CONVERSION TO LMW-UK
vo1.23, No.6
The expected conversion of plasminogen to plasmin was indicated by the demonstration of characteristic plasmic degradation of fibrinogen (IO) when this substrate was included in the incubation mixture. No conversion of urokinase was seen when HMW-UK was incubated alone, indicating that the reaction was not autocatalytic. Accurate quantitation of the kinetics of this conversion was difficult, because of variability In the slicing of the slab gels, the extraction of protein from the gel slices and especially in the mathematical conversion of fibrin lysis zones into concentrations of activator. Since the fibrin lysis area depends upon the diffusion rate of the activator, the areas noted in Figs. 1 and 3 were logarithmic functions of relative molecular size, and resulted in a much larger lysis area for the LMW-UK form than for an equivalent amount of the HMW-UK form. The role of plasmin in the conversion of HMW-UK has precedence in other components of the fibrinolytic system. For instance, plasmin converts gluplasminogen to lys-plasminogen by the release of a 6,000 dalton peptide (11). Furthermore, during streptokinase activation of plasminogen, the streptokinase moiety is degraded to a series of smaller fragments (12). Little is known concerning the structural changes involved in the urokinase conversion. While it is well-established that HMW-UK is a two-chain molecule held together by disulfide bonds (13,14), no comparable information is available in the LMW-UK It is possible that form, nor is the protease-sensitive area of HMW-UK known. there are a series of low molecular weight urokinases of similar size with intact disulfide bonds, It is possible that the presence of free plasmin in the blood during thrombolytic therapy (15) or even the conversion of plasminogen to plasmin on the surface of a fibrin mesh (16) creates a situation in which the same conversion of HMW-UK to LMW-UK can occur during thrombolytic therapy. This possibility would have important physiologic implications, since kinetic studies show that HMW-UK is more active than LMW-UK both in converting plasminogen to plasmin (17) and in accelerating clot lysis (IS), and the two forms also react at different rates with proteolytic inhibitors (19). While different commercial preparations of urokinase that are licensed for clinical use may contain markedly different proportions of HMW-UK and LMW-UK (Fig. 2), and variation in the blood concentration of various parameters of activator activity may reflect such differences in urokinase forms (20), there is still no firm evidence in the literature to indicate which type of urokinase is most important for the achievement of therapeutic thrombolysis. Certainly, large amounts of either form are capable of producing a lytic state and clinical thrombolysis (21,22). Since our findings demonstrate that HMW-UK is rapidly converted to LMW-UK by plasmin in vitro, it is possible that a similar conversion occurs and that throxom could --in vivo result mostly from the action of the lower molecular weight plasmin derivative.
ACKNOWLEDGEMENTS This National
work was supported in Heart, Lung and Blood
Bethesda, Maryland, Space Administration.
and
Contract
part by Institute,
Grants HL-00733 and HL-18208 from National Institutes of Health, NAS-g-16187 from the National Aeronautics
the and
546
HMW-UK CONVERSION TO LMW-UK
Vo1.23, No.6
REFERENCES 1.
CELANDER, D.R., Am. J. Cardiol.
and GUEST, M.M. The a, 409-419, 1960.
2.
WHITE, W.F., BARLOW, G.H., and MOZEN, of plasminogen activators (urokinase) 2169, 1966.
3.
LESUK, A., TERMINELLA, L., TRAVER, J.H., and GROSS, J.L. Biochemical and biophysical studies on human urokinase. Thromb. Diath. Haemorrh. 18, 293-294,
biochemistry
M.M. from
and
physiology
of
urokinase.
The isolation and characterization human urine. Biochemistry 5_, 2160-
1967. 4.
SOBERANO,
M.E.,
the catalytic 5.
ONG,
E.F.,
conversion
and
of
JOHNSON,
urokinase.
A.J. The effects of inhibitors Thromb. Res. Y_, 675-681, 1976.
ASTEDT, 8, BARLOW, G.H., an HDLMBERG, L. Time-related release of Thromb. Res. 11, molecular forms of urokinase in tissue culture.
on
various 149-153,
1977. 6.
MARGOLIS, molecular
J. and KENRICK, sieve gradient.
K.G. Polyacrylamide gel-electrophoresis Nature 214, 1334-1336, 1967.
7.
NEVILLE, D. M., JR. Molecular weight determination complexes by gel electrophoresis in a discontinuous 246, 6328-6334, 1971.
8.
Plasmic degradation of crossFRANCIS, C.W., MARDER, V.J. and MARTIN, S.E. analysis of the particulate clot and identilinked fibrin. I. Structural fication of new macromolecualr complexes. Blood 56, 456-464, 1980.
9.
ASTRUP, T. and lytic activity.
of protein-dodecyl buffer. J. 8101.
The fibrin plate method for estimating MULLERTZ, S. Arch. Biochem. Biophys. 40, 346-351, 1952. High and
across
molecular weight derivaimmunological character-
N.R. and CARROLL, W.R. MARDER, V-J., SHULMAN, tives of human fibrinogen. I. Physicochemical ization. J. Biol. Chem. 244, 2111-2119, 1969.
11.
WIMAN, B. plasminogen
12.
SIEFRING, G.E. and CASTELLINO, F.J. Interaction Isolation and characterization of plasminogen. product. J. Bidl. Chem. 251, 3913-3920, 1976.
13.
SOBERANO, M.E., ONG, E.G., JOHNSON, A.J., LEVY, M. and SCHOELLMANN, G. Biochim. Purification and characterization of two forms of urokinase. Biophys. Acta 445, 763-773, 1976.
14.
HOLMBERG, L., chromatography.
peptides released during activation J. Biochem. 2, l-9, 1973.
BLADH, B. and ASTEDT, B. Biochim. Biophys. Acta
sulfate Chem.
fibrino-
10.
Primary structure of Eur. by urokinase.
a
of
human
of streptokinase with a streptokinase degradation
Purification of urokinase 445, 215-222, 1976.
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