(Received for publication, May 22, 1995; and in revised form, August 18, 1995)
From the
The distinguishing characteristic of vampire bat (Desmodus
rotundus) salivary plasminogen activators (DSPAs) is their strict
requirement for fibrin as a cofactor. DSPAs consist of structural
modules known from urokinase (u-PA) and tissue-type plasminogen
activator (t-PA) such as finger (F), epidermal growth factor (E),
kringle (K), and protease (P), combining to four genetically and
biochemically distinct isoenzymes, exhibiting the formulas FEKP
(DSPA1 and
2) and EKP and KP (DSPA
and DSPA
). Only
DSPA
1 and
2 bind to fibrin. All DSPAs are single-chain
molecules, displaying substantial amidolytic activity. In a plasminogen
activation assay, all four DSPAs are almost inactive in the absence of
fibrin but strongly stimulated by fibrin addition. The catalytic
efficiency (k
/K
)
of DSPA
1 increases 10
-fold, whereas the corresponding
value of t-PA is only 550. The ratio of the bimolecular rate constants
of plasminogen activation in the presence of fibrin versus fibrinogen (fibrin selectivity) of DSPA
1,
2,
,
, and t-PA was found to be 13,000, 6500, 250, 90, and 72,
respectively. Whereas all DSPAs are therefore more fibrin dependent and
fibrin selective than t-PA, the extent depends on the respective
presence of the various domains. The introduction of a
plasmin-sensitive cleavage site in a position akin to the one in t-PA
partially obliterates fibrin cofactor requirement. Fibrin dependence
and fibrin selectivity of DSPAs are accordingly mediated by fibrin
binding, which involves the F domain, as yet undefined determinants
within the K and P domains, and by the absence of a plasmin-sensitive
activation site. These findings transcend the current understanding of
fibrin-mediated stimulation of plasminogen activation: in addition to
fibrin binding, specific protein-protein interactions come into play,
which stabilize the enzyme in its active conformation.
Plasminogen activators (PAs), ()such as t-PA and
u-PA, are highly specific serine proteases, which catalyze the
hydrolysis of the Arg
-Val
peptide bond of
Glu-plasminogen. The activation product, plasmin, is a potent protease,
which digests fibrin and several extracellular matrix proteins. Plasmin
also processes the single-chain precursors of t-PA and u-PA to the more
active two-chain forms. In contrast to u-PA, the rate of plasminogen
activation by t-PA increases by 2-3 orders of magnitude in the
presence of fibrin or fibrin(ogen) degradation products (Camiolo et
al., 1971; Hoylaerts et al., 1982; Ranby, 1982; Bergum
and Gardell, 1992). Both t-PA and its substrate, Glu-plasminogen, bind
to fibrin, forming a ternary complex that facilitates the conversion of
Glu-plasminogen (Hoylaerts et al., 1982; Ranby, 1982; Fears,
1989). t-PA consists of several structural motifs known by structural
homology from other proteins: an N-terminal fibronectin-like finger
(F), an epidermal growth factor (E), two kringles (K1 and K2), and a
serine protease domain (P) (Pennica et al., 1983; Patthy,
1990). Several authors suggested the F domain and the lysine binding
site (LBS) of the K2 domain to be the major contributors to t-PA fibrin
affinity and to the observed fibrin-mediated enhancement of plasminogen
activation (van Zonneveld et al., 1986; Verheijen et
al., 1986; de Munk et al., 1989). Recent results,
however, indicate that t-PA interacts with fibrin via a binding region
that comprises surface areas of other structural modules as well,
including the protease domain (Bennett et al., 1991).
In recent years, thrombolytic treatment with t-PA has emerged as state of the art therapy of acute myocardial infarction (Topol, 1991; Collen and Lijnen, 1991). However, when administered in therapeutic doses, t-PA, due to its limited fibrin selectivity, causes plasminemia that may contribute to bleeding complications (Rao et al., 1988; Arnold et al., 1989). Therefore, considerable efforts have been devoted to the design of new variants of t-PA exhibiting improved fibrin selectivity (Higgins and Bennett, 1990; Lijnen and Collen, 1991). Recently, a novel mutein of t-PA called TNK, which is more fibrin selective than t-PA, has been characterized (Keyt et al., 1994; Collen et al., 1994).
We and others
(Gardell et al., 1989; Krätzschmar et
al., 1991) have previously reported the cloning, expression, and
characterization of plasminogen activators derived from the saliva of
vampire bats. A total of four different Desmodus rotundus salivary plasminogen activators (DSPAs), which we named
DSPA1,
2,
, and
have been cloned, expressed, and
characterized. DSPA
1 and
2 encompass an F, E, K, and P
domain, while DSPA
lacks the finger module and DSPA
contains
only a K and a P domain. Apart from these differences, DSPAs are very
similar (88.7-99.5% amino acid sequence identity;
Krätzschmar et al.(1991)). The amino acid
sequence of human t-PA is similarly related (72.3% (DSPA
1) and
74.2% (DSPA
2) identity; Krätzschmar et
al.(1991)). Like u-PA, all DSPAs only contain a single K domain
rather than two, as is the case for t-PA. The K module of DSPAs is more
similar to the K1 domain of t-PA and does not exhibit an LBS.
Furthermore, a plasmin-sensitive activation site, present in the
N-terminal region of the t-PA protease domain is absent in DSPAs.
Therefore, DSPAs activate plasminogen as single chain molecules
(Gardell et al., 1989; Krätzschmar et
al., 1991).
Functionally, DSPAs differ from t-PA by their
strict requirement for a fibrin cofactor. This was studied in great
detail for Bat-PA (equivalent to DSPA2) by Bergum and
Gardell(1992) and has been reported for DSPA
1 as well (Schleuning et al., 1992). When compared to t-PA, Bat-PA and DSPA
1
demonstrated an equal or even higher thrombolytic potency in several
animal models of arterial thrombosis (Gardell et al., 1991;
Mellot et al., 1992; Witt et al., 1992, 1994;
Muschick et al., 1993). Importantly, while being equally
effective as t-PA, fibrinogen degradation or
2-antiplasmin
consumption were considerably lower with Bat-PA and DSPA
1 (Gardell et al., 1991; Mellot et al., 1992; Witt et
al., 1992; Muschick et al., 1993).
The present study evaluates the fibrin selectivities of the recombinant forms of all naturally occurring DSPAs and compares these data with those obtained for t-PA. Furthermore, we present data that suggest a molecular mechanism for the unique fibrin selectivity of DSPAs.
The dissociation constant for the binding of DSPAs to fibrin and the numbers of binding sites were calculated by nonlinear regression analysis of the data according to the Scatchard equation employing the programs EBDA (equilibrium binding data analysis) and LIGAND, originally written by Munson and Rodbard(1980) and modified by G. A. McPherson (V 2.0), which were obtained from Elsevier-Biosoft (Cambridge, United Kingdom).
DSPAs 1,
2,
, and
were expressed in BHK
cells as described (Krätzschmar et al.,
1992). Recombinant proteins were purified to homogeneity from cell
culture supernatants by affinity chromatography on immobilized Erythrina trypsin inhibitor (Heussen et al., 1984).
As judged from SDS-PAGE analysis, preparations of recombinant DSPAs
1,
2,
, and
were homogeneous, and the proteins
displayed an apparent molecular mass of 52, 52, 46, and 44 kDa,
respectively (Fig. 1, lanes 1-4).
Figure 1:
SDS-PAGE of purified recombinant DSPAs
and t-PA. Prior to electrophoresis on a SDS-gel containing 12.5%
polyacrylamide (Laemmli 1970), all samples were reduced by the addition
of dithiothreitol (12.5 mM). Approximately 3 µg of each
protein was loaded. The gel was stained with Coomassie Brilliant Blue
(G250). Proteins were produced as outlined under ``Materials and
Methods.'' Lane 1, rDSPA1; lane 2,
rDSPA
2; lane 3, rDSPA
; lane 4, rDSPA
; lane 5, marker proteins (M) whose molecular mass is
indicated on the right; lane 6, rt-PA
(Actilyse®); lane 7, [R275H,I276S,K277T] t-PA
lacking the plasmin-sensitive site; lane 8,
[H189R,S190I,T191K] DSPA
1 containing a plasmin-sensitive
site.
Figure 2:
Scatchard analysis of the interaction
between DSPA1 or DSPA
2 and forming fibrin clots. A,
Scatchard analysis of fibrin binding data obtained for
I-DSPA
1. The plot is based on a model involving one
type of binding site. Inset, equilibrium binding of
I-DSPA
1 to fibrin. Binding was determined at
variable concentrations of unlabeled DSPA
1 as described under
``Materials and Methods.'' Each point is represented by the
results of three independent determinations. The average standard error
for the equilibrium binding curve was approximately 7%. Specific
binding was calculated by subtracting nonspecific binding (e.g. in the presence of excess unlabeled DSPA
1) from total
binding. Nonspecific binding was 4% of total cpm. B, Scatchard
plot of fibrin binding data of
I-DSPA
2. The plot is
based on a model involving a sole type of binding site. Inset,
equilibrium binding of
I-DSPA
2 to fibrin. Binding
was determined as described under ``Materials and Methods.''
The results of three independent experiments are displayed. The average
standard error for the equilibrium binding curve was about 6%. Specific
binding was calculated by subtracting nonspecific binding (e.g. in the presence of excess unlabeled DSPA
2) from total
binding. Nonspecific binding was 6% of total
cpm.
In the presence of fibrin, however, the
catalytic efficiency of DSPAs was augmented by several orders of
magnitude. The steepest increase by a factor of 10,
resulting in a k
/K
value of
684,000 M
s
, was
observed for DSPA
1. The bimolecular rate constant of DSPA
2
was raised to 517,000 M
s
, corresponding to a 53,000-fold enhancement (Table 1). DSPAs
and
exhibited considerably smaller
catalytic efficiencies of 9900 M
s
and 3510 M
s
, reflecting a 1650- and 800-fold increase in
catalytic activity, respectively. This demonstrates that binding of the
DSPAs to fibrin significantly contributes to enhanced plasminogen
activation. For DSPA
1 and
2, the fibrin-mediated enhancement
of catalytic efficiency resulted from both a moderate decrease (25- and
34-fold) in K
and a concomitant profound increase
(4300- and 1550-fold) in k
, respectively.
Interestingly, despite their high degree of homology (89% identity in
amino acid sequence) (Krätzschmar et al.,
1991), the unstimulated activity of DSPA
2 was slightly higher than
that of DSPA
1, which, however, was more active in the presence of
fibrin (Table 1). The 2-fold higher fibrin-mediated enhancement
of the catalytic efficiency of DSPA
1 was due to a steeper
increment in the k
value of DSPA
1 rather
than a more pronounced decrease in its K
(Table 1). The reduction in K
and in
particular the increase in k
was significantly
smaller for DSPAs
and
(Table 1).
In comparison to
the absence of a cofactor, fibrinogen promoted the catalytic efficiency
of DSPAs by 7-9-fold, resulting in k/K
values ranging from 39
to 79 M
s
, which were
several orders of magnitude smaller than those observed in the presence
of fibrin (Table 1). The ratio of catalytic efficiencies in the
presence of fibrin to the corresponding values in the presence of
fibrinogen, which serves as a measure of ``fibrin
selectivity'', amounted to 12,900 for DSPA
1. The bimolecular
rate constant of DSPA
2 in the presence of fibrin was 6550-fold
higher than that in the presence of fibrinogen. The respective values
for DSPAs
and
were 235 and 90 (Table 1). DSPA
1
therefore exhibited the highest fibrin selectivity, and this was mainly
attributable to its superior stimulation by fibrin.
The data
summarized in Table 1also depict how the kinetic parameters of
DSPAs compare to those obtained for t-PA. In the absence of a
fibrin(ogen) cofactor, t-PA was 260-fold more efficient in activating
Glu-plasminogen than DSPA1. In the presence of fibrin, however,
both plasminogen activators were similarly effective (Table 1).
The enhancement of the bimolecular rate constant of t-PA in the
presence of fibrin was only 550-fold as compared to 10
-fold
for DSPA
1. In the presence of fibrinogen, the catalytic efficiency
of t-PA was increased to 13,600 M
s
, which was 260- and 170-fold higher than the
respective values measured for DSPA
1 and
2 (Table 1).
Fibrin increased the catalytic efficiency of t-PA by only 72-fold over
that in the presence of fibrinogen, meaning that DSPA
1 was about
180-fold more fibrin selective than t-PA. DSPA
2 exhibited a
90-fold higher fibrin selectivity than t-PA, and even the
finger-deficient variant DSPA
was still 3-fold more fibrin
selective. The latter strongly indicates that fibrin selectivity is not
merely a consequence of the plasminogen activator's affinity for
fibrin.
Figure 3:
Plasmin-mediated conversion of
[R275H,I276S,K277T] t-PA and [H189R,S190I,T191K]
DSPA1. Recombinant proteins were prepared as described under
``Materials and Methods.'' Approximately 5 µg each of
t-PA, [R275H,I276S,K277T] t-PA, and
[H189R,S190I,T191K] DSPA
1 were incubated for 30 min at
37 °C in the absence (lanes 2, 4, and 6)
or presence of Sepharose-immobilized plasmin (lanes 3, 5, and 7). The samples were analyzed by SDS-PAGE as
outlined in the legend to Fig. 2. Lane 1, marker
proteins (M) whose molecular mass is indicated on the left; Lanes 2, 4, and 6, t-PA,
[R275H,I276S,K277T] t-PA, and [H189R,S190I,T191K]
DSPA
1, respectively, incubated in the absence of plasmin, or as
shown in lanes 3, 5, and 7, treated with
plasmin.
In the absence of a
stimulator, the DSPA1 mutein exhibited a bimolecular rate constant
of 135 M
s
, which
reflected a 20-fold increase over that of wild-type DSPA
1. The
catalytic efficiency of sct-PA was reduced, in comparison to t-PA, by
50-fold to 34 M
s
(Table 1), which is in good agreement to the activity
decrease observed previously (Andreasen et al., 1991; Petersen et al., 1988; Tate et al., 1987). Fibrinogen raised
the k
/K
value of
plasmin-sensitive DSPA
1 mutein to 516 M
s
, which in comparison
to the wild type, corresponded to a 10-fold increase. The respective
value (638 M
s
) of
sct-PA was 20-fold decreased. In the presence of fibrin, however, both
muteins displayed catalytic efficiencies (DSPA
1 mutein, 565,000 M
s
, and t-PA mutein,
525,000 M
s
) that were
similar to the respective wild-type proteins (Table 1). In
comparison to fibrinogen, fibrin promoted the catalytic efficiency of
plasmin-sensitive DSPA
1 by 1100-fold, which was 12-fold less than
that of the uncleavable wild-type enzyme. In case of t-PA, the absence
of the cleavage site resulted in an 11-fold increase of its fibrin
selectivity. Importantly, DSPA
1 and DSPA
2 were still about
16- and 8-fold more fibrin selective than uncleavable sct-PA, and
plasmin-sensitive DSPA
1 was 15-fold more fibrin selective than
t-PA (Table 1), implying that other features apart from the lack
of the plasmin-sensitive cleavage site must contribute to the superior
fibrin selectivity of DSPAs.
In the absence of a fibrin(ogen) cofactor, DSPA1 and -
2
exhibited 17 and 37.5%, respectively, of t-PA catalytic efficiency.
While fibrinogen had only a very small effect on the k
/K
of t-PA, fibrin
promoted its bimolecular rate constant by 4-fold. In the presence of
the latter the activity of DSPA
2 was equivalent to that of t-PA,
whereas S-2765 hydrolysis by DSPA
1 was 2-fold less efficient (Table 2).
As observed for t-PA and DSPA1, the extent of
fibrin-mediated stimulation of S-2765 hydrolysis was dependent on
whether they occurred in their single or two-chain forms. The single
chain forms of DSPA
1 and t-PA were more highly stimulated than
their two-chain counterparts (Table 2). The intrinsic activity of
tct-PA was not significantly stimulated by fibrin, while tc
DSPA
1's catalytic efficiency was still enhanced (2.6-fold),
albeit 5-fold less than that of the single chain molecule. This effect
was attributed to an increased k
, which was not
observed for t-PA. By comparison to the wild-type molecule, the tc
DSPA
1 mutein exhibited a 4-fold higher catalytic efficiency in the
absence of a fibrin(ogen) cofactor (Table 2).
Since alterations in plasminogen conformation
only accounted for a small portion of fibrin's stimulatory
effect, it is evident that the major contribution to fibrin stimulation
is mediated by the ternary complex formation and/or the interaction of
DSPA1 and
2 with fibrin (Hoylaerts et al., 1982; Wu et al., 1990). To discriminate between these two mechanisms,
we have measured enzyme kinetics in the presence of both fibrin and
EACA. Under these conditions, only the plasminogen activator is able to
bind to fibrin, whereas binding of plasminogen to fibrin is inhibited
(Lucas et al., 1983; Nesheim et al., 1990). Binding
of t-PA is solely mediated by the finger domain because kringle
2-dependent binding of t-PA to fibrin is inhibited (van Zonneveld et al., 1986; Nesheim et al., 1990).
By comparison
to their catalytic efficiencies in the presence of fibrin alone, the
additional presence of EACA diminished the catalytic activities of
DSPAs 1,
2, tc DSPA
1, and t-PA by 90-24-fold (Table 4). As expected, this decrease was entirely due to an
increase in the apparent K
, since the k
values were similar to that obtained in the
presence of fibrin alone ( Table 1and Table 4). Hence, the
dissociation of plasminogen from the fibrin template caused similar
decrements, within the same order of magnitude, of plasminogen
activator catalytic efficiencies. However, a comparison of the
bimolecular rate constants in the presence of fibrin and EACA to those
in the presence of EACA alone revealed striking differences between
DSPAs and t-PA. The addition of fibrin to the reaction mixture that
already contained EACA raised DSPA
1's k
/K
from 26 to 7790 M
s
(about 300-fold),
whereas that of t-PA was promoted by only 6-fold from 7480 to 41,270 M
s
( Table 3and Table 4). In accordance with our earlier observation, the
activity of DSPA
1 was 2-fold more highly promoted by the addition
of fibrin than that of DSPA
2 (Table 4). In the presence of
fibrin and EACA, the catalytic efficiency of the plasmin-sensitive
DSPA
1 mutein was enhanced to 11,020 M
s
, which represented an only 12-fold increase over
that, 890 M
s
, in the
presence of EACA alone.
There are three plausible mechanisms pertinent to fibrin-mediated stimulation of plasminogen activation, all based on protein-protein interactions: 1) a template-mediated rendezvous mechanism furthering the physical encounter of both enzyme and substrate, 2) the exposure of the activation site of plasminogen, following a conformational change induced by fibrin binding, and (3) a stabilizing effect of fibrin on the active site of plasminogen activators, probably mediated by domain-domain interactions.
We have attempted to attribute the observed
stimulatory effects to one or the other of these mechanisms. In
contrast to t-PA, the major contribution of fibrin to its overall
stimulatory effect on plasminogen conversion by DSPAs 1 and
2
is mediated by its interaction with the plasminogen activator itself (Table 4). The template effect appears to be less important,
whereas it is paramount to fibrin-mediated enhancement of plasminogen
activation by t-PA (Hoylaerts et al., 1982). Corroborating the
results from direct measurements of fibrin binding, an interaction of
DSPA
1 or
2 and fibrin is also demonstrated by the enhancement
of S-2765 hydrolysis. Upon binding to fibrin, the catalytic activities
of DSPA
1 and
2 were raised by about 1 order of magnitude,
whereas those of DSPAs
and
were increased only marginally (Table 2). Therefore, in case of DSPA
and
, the
increase in the plasminogen activation rate is most likely due to a
conformational change in plasminogen induced by its interaction with
partially degraded fibrin (Suenson et al., 1984), although
domain-domain interactions occurring within the DSPA molecules might
also be involved.
The striking difference between DSPAs and t-PA, as
far as fibrin stimulation is concerned, is not a consequence of
disparate fibrin affinities. The K values of
DSPA
1 and DSPA
2 (Fig. 2) are within the range of
values published for t-PA (0.13-0.58 µM) (Higgins
and Vehar, 1987; Husain et al., 1989; Nesheim et al.,
1990; Bergum and Gardell, 1992; Horrevoets et al., 1994). The
data are particularly consistent, if only finger-dependent binding of
t-PA is analyzed. Under these conditions, Nesheim et al.(1990)
measured a K
of 0.13 µM and a molar
binding ration of 0.6, values that are almost identical to those of
DSPA
1 and
2 (Fig. 2). Furthermore, the dependence on
the fibrin concentration was very similar for DSPAs and t-PA.
Half-maximal velocities were achieved at 25 ± 3, 31 ± 5,
and 13 ± 2 µg/ml for DSPA
1, DSPA
2, and t-PA,
respectively (data not shown).
All DSPAs exhibited only marginal
activity in the absence of a fibrin(ogen) cofactor (Table 1).
Upon addition of fibrinogen, their second order rate constants
increased similarly by roughly 1 order of magnitude, which is in
contrast to the markedly diverging stimulatory effect mediated by
fibrin. For instance, in case of DSPA1 and DSPA
, the extent
of fibrin stimulation differs by a factor of 62 (Table 1). These
data therefore suggest that the stimulatory effect exerted by
fibrinogen is not conferred via the DSPAs but is rather mediated by an
interaction between plasminogen and fibrinogen (Lucas et al.,
1983).
The ratio of the bimolecular rate constants of plasminogen
activation in the presence of fibrin versus fibrinogen is
defined as fibrin selectivity. Since DSPA1,
2, and t-PA
exhibited very similar bimolecular rate constants in the presence of
fibrin, the significant difference in fibrin selectivity of
DSPA
1/
2 and t-PA is mainly caused by their unequal catalytic
efficiencies in the presence of fibrinogen (Table 1).
To
further understand the underlying structure-function relationship, we
have analyzed the properties of a mutein of DSPA1, whose protease
domain contained a plasmin-sensitive site (Table 1Table 2Table 3Table 4). In the presence
of fibrin, the catalytic efficiency of plasmin-sensitive DSPA
1 was
strikingly less (24-fold) increased than that of the wild-type protein (Table 1). This decrease in fibrin stimulation was entirely
attributable to a diminished stimulation via the plasminogen activator
protease domain because upon prevention of the template effect by
addition of
-amino caproic acid, fibrin stimulated the catalytic
efficiency only 12-fold as opposed to 300-fold as observed for
DSPA
1 (Table 4). Since the bimolecular rate constants of
DSPA
1 and its plasmin-sensitive mutein were almost identical in
the presence of fibrin (Table 1), the decreased stimulatory
effect was a consequence of the mutein's higher basal activity.
Further, the fibrin selectivity of cleavable DSPA
1 was decreased
about 12-fold (Table 1) and the fibrin-stimulated intrinsic
catalytic activity about 5-fold (Table 2). Hence, the lack of a
plasmin-sensitive site within the DSPA
1 protease domain
contributes significantly to both the impressive stimulation by fibrin
and its fibrin selectivity. Abolition of the plasmin-sensitive site of
t-PA brings a quantitatively similar effect into bearing, namely a
28-fold increase in fibrin stimulation (Table 1). Concomitantly,
the fibrin selectivity is improved 11-fold, again quantitatively equal
to the effect observed for the cleavable DSPA
1 mutein. The role of
the t-PA plasmin cleavage site for fibrin stimulation has been
investigated previously, and similar results have been obtained (Tate et al., 1987; Petersen et al., 1988; Higgins et
al., 1990). Nienaber et al.(1992) suggest that the
interaction of the single-chain form of t-PA with fibrin(ogen) induces
a conformational change at the active site stabilizing an ``active
locked'' conformation. Since the influence on fibrin stimulation
and on fibrin selectivity of a plasmin-sensitive site in the protease
domain of DSPA
1 or t-PA is almost identical, it is likely that the
stimulation of the DSPA
1 protease domain by fibrin is due to a
similar mechanism.
Notably, plasmin-sensitive DSPA1 was still
15-fold more fibrin selective than t-PA, and DSPA
1 discriminated
more than 7-fold better between fibrin and fibrinogen than sct-PA (Table 1). This difference is also corroborated by amidolytic
data. While fibrin raised the intrinsic catalytic activity of tc
DSPA
1 by 2-3-fold over that in the presence of fibrinogen,
the k
/K
of tct-PA remained
essentially unchanged (Table 2). Therefore, the heterotropic
effect conferred by fibrin does not depend solely on the lack of the
plasmin-sensitive cleavage site but involves other, yet unknown,
determinants within the molecule.
Only DSPA2 (Bat-PA) and t-PA
have been evaluated in a way comparable to this study by other authors.
Pertaining to t-PA, our data are similar to those previously reported
(Rånby, 1982; Urano et al., 1988; de Vries et
al., 1991; Bergum and Gardell, 1992). Our values were generally
higher than those published for Bat-PA (corresponding to DSPA
2) by
Bergum and Gardell(1992). This difference can be explained by use of
different k
values for plasmin-mediated
hydrolysis of the chromogenic substrate (FlavigenPli versus SpectrozymePl). The relative stimulation factors, however, are in
good agreement (the Bat-PA k
/K
43500-fold increase in the presence of fibrin, DSPA
2
52,700-fold). Fibrin (fibrin II) stimulated DSPA
2 catalytic
efficiency 10,900-fold more than fibrinogen as compared to the ratio of
6550 determined in our system. Our data, however, do not confirm that
in the absence of a stimulator the Bat-PA K
was
0.6 µM and therefore smaller than in the presence of
fibrinogen (Bergum and Gardell, 1992). By contrast, DSPA
2 affinity
for Glu-plasminogen was very low as indicated by a K
of 12.3 µM, a value very similar to those determined
for DSPA
1 (9.5 µM) and sct-PA (17.7 µM).
Also, our unstimulated K
value for t-PA, 5.2
µM, was similar to the 6.7 µM reported by
Bergum and Gardell(1992) and agreed very well with the values of 7.6
and 9 µM published by Rånby(1982) and Urano et
al.(1988), respectively.
In summary, we have provided a biochemical rationale for the striking fibrin selectivity of DSPAs: finger-dependent fibrin binding confers a heterotropic effect, which is conceivably mediated by domain-domain interactions on the protease domain to stabilize a preformed active site. Introduction of a plasmin-sensitive cleavage site partially obliterates the requirement for the fibrin cofactor. Further understanding of the molecular details of this interaction will depend on the results of structural analysis, which is currently underway.