(Received for publication, May 30, 1995; and in revised form, July 10, 1995)
From the
Annexin V is a human protein that binds with high affinity to the abundant phosphatidylserine molecules exposed on activated platelets and accumulates selectively in thrombi after intravenous administration in animal models of arterial thrombosis. We designed two chimeras that use annexin V as a means to target thrombolytic agents to platelet-containing thrombi: prourokinase (1-411)-annexin V (1-320); and prourokinase (144-411)-annexin V (1-320) (amino acid numbers of parent proteins given in parentheses). Chimeras were produced by cytoplasmic expression in Escherichia coli, refolded, and purified in single-chain form. Both chimeras had the same specific activity as annexin V in binding to cell membranes containing exposed phosphatidylserine. After activation with plasmin, both chimeras had specific amidolytic activity similar to that of urokinase. Both chimeras activated plasminogen in vitro with kinetic parameters similar to those for urokinase, and both showed full activity compared to urokinase in an assay of clot lysis in vitro. This study shows the feasibility of producing chimeric plasminogen activators in which annexin V provides the thrombus-targeting component; although not yet tested in vivo, such chimeras may have advantages over antibody-based targeting agents.
Thrombolytic therapy has been a major advance in the treatment of myocardial infarction over the last decade. However, as discussed in recent reviews(1, 2, 3, 4) , available thrombolytic agents have certain limitations. The thrombus may be resistant to lysis in some patients or may reform after initial lysis; and bleeding, particularly intracerebral, is a serious side effect. This has led to many efforts to improve the potency and safety of thrombolytic agents (reviewed in (4) ). One approach has been to target the thrombolytic agent by preparing chimeric proteins in which a thrombus-specific antibody is attached to a plasminogen activator (usually a form of urokinase). Antibodies against fibrin (5, 6, 7, 8) and platelet-specific antigens such as glycoprotein IIb/IIIa (9, 10) and thrombospondin (10) have been used.
Phosphatidylserine (PS) ()represents a potential activation-dependent binding site
on the platelet component of the thrombus. In resting platelets, PS is
nearly absent from the extracellular face of the membrane, but it
becomes exposed during normal platelet
activation(11, 12) . Annexin V, a member of the
annexin family of calcium-dependent phospholipid-binding
proteins(13) , binds with very high affinity to PS-containing
phospholipid
bilayers(14, 15, 16, 17) . We have
previously shown that annexin V also binds to human platelets with a K
of 7
nM(18, 19) . Binding to quiescent platelets in vitro is minimal, while maximally stimulated platelets
contain nearly 200,000 annexin V binding sites; this substantially
exceeds the number of binding sites for antibodies to glycoprotein
IIb/IIIa (
25,000 on activated platelets)(20) .
Radiolabeled annexin V, administered intravenously in animal models of
acute arterial thrombosis, accumulates in the thrombus to a level about
13-fold higher than a nonspecific control protein,
ovalbumin(21) . Annexin V labeled with
Tc can
also be used to image acute intracardiac thrombi in
vivo(22) . Annexin V is a relatively small protein (M
36,000) that is easily produced in quantity by
recombinant DNA methods and, as a human protein, would not be expected
to induce an immune response, unlike murine monoclonal antibodies. It
is also virtually absent from normal human plasma(23) . For
these reasons, annexin V is attractive as a potential
thrombus-targeting agent.
Based on these considerations, we felt that chimeric thrombolytic proteins based on annexin V might have certain advantages. Our goal in this study was to design and express a prourokinase-annexin V chimera and to test its activity in vitro. We also expressed a chimera lacking the first 143 amino acids of prourokinase, which encode the epidermal growth factor-like and kringle domains of prourokinase (24) ; this molecule, while retaining the proenzyme character of full-length prourokinase, might have certain advantages in production or clinical use due to its smaller size. We show that these proteins can be expressed and purified in active form; they fully retain the component enzymatic and membrane-binding activities of the parent molecules; and they are active in assays of in vitro clot lysis.
Figure 1:
Structures of expression units and
chimeric proteins. Chimeras contain the amino acid residues of the
parent wild-type proteins indicated in parentheses; selected portions
of the predicted amino acid sequence are shown in single-letter
code, with added amino acids indicated by underlining.
The arrow indicates the site cleaved by plasmin to convert
prourokinase to urokinase. The expression unit for the 82K chimera
encodes a protein with 733 amino acids and a calculated M of 82,546 (both including the initiator Met);
corresponding values for the 69K chimera are 610 amino acids and
68,598. The sequence at the N terminus of the 69K chimera is encoded by
the pET-14b expression vector, and consists of MGSSH HHHHH SSGLV PRGSH
(in single-letter amino-acid code). Selected restriction sites
separating the major functional units are shown. Both expression units
are cloned into one of the pET family of vectors to allow cytoplasmic
exression in E. coli (see ``Experimental
Procedures''). Reference sequences: annexin V(27) ,
GenBank
accession no. M18366; prourokinase(24) ,
GenBank
accession no. M15476.
The 82K
chimera was purified as follows. The 8 M urea extract was
applied to a Sephacryl S-400 gel filtration column (2.5 90 cm)
equilibrated and eluted with 5 M urea, 0.5 M NH
Cl, 50 mM Tris-HCl, pH 8.0, 0.5 mM
EDTA, 10 mM 2-mercaptoethanol. The fractions that contained
the chimera (based on SDS-PAGE) were pooled and stored frozen after
adding 2-mercaptoethanol to 0.1 M.
The sample was dialyzed
overnight against 10 volumes of 5 M urea, 0.5 M
NHCl, 50 mM Tris-HCl, pH 8.5, to reduce
2-mercaptoethanol to 10 mM. Oxygen was removed from this
buffer by degassing (10 min) followed by nitrogen flush (5 min), and
the flask was sealed with parafilm during dialysis. Then, the sample
was added dropwise to 100 volumes of 2 M urea, 0.5 M NaCl, 0.5 M NH
Cl, 10 mM
benzamidine-HCl, 0.5 mM EDTA, 1.25 mM reduced
glutathione, 0.5 mM oxidized glutathione (protein
concentration,
0.02 mg/ml) and stirred slowly for 24 h at 4
°C. Oxygen was removed from this buffer, and the flask was sealed
as above. After 24 h, oxidized glutathione was added at 0.5 mM
(total 1 mM), and the sample was stirred overnight open to the
atmosphere. After Tween-20 (Bio-Rad) was added to the sample at 0.01%,
it was extensively dialyzed against 12 volumes of 10 mM Tris-HCl, pH 8.0, with three buffer changes. After addition of
Tween-20 to 0.01%, the sample was concentrated 60-fold by
ultrafiltration with an Amicon XM-50 membrane.
Ten ml of the sample was applied to an AP-1 column (Waters) packed with Protein-Pak DEAE 15HR, which was connected to a Waters advanced protein purification system, model 650E. The column was equilibrated with 50 mM Tris-HCl, pH 8.0, and eluted with a salt gradient from 0 to 0.6 M in the same buffer at a flow rate of 1 ml/min. The fractions were assayed for prourokinase and anticoagulant activities as described below. Most of the unfolded proteins were found in nonadsorbed fractions, and the folded protein was eluted with a single peak at a salt concentration about 0.3 M. The yield of folded chimera was about 0.25 mg/liter of culture, or about 0.125 mg/g E. coli paste.
To detect prourokinase, samples from column fractions (50 µl) were placed onto a microtiter plate with 1 µl of plasmin (3 units/ml, Sigma) and incubated for 20 min at room temperature. Five µl of aprotinin (1 mg/ml, gift from Novo) were added to inactivate plasmin, followed by 20 µl of 20 mM S-2444; absorbance at 405 nm was measured after a 20-min incubation at room temperature. The anticoagulant activity was determined with a partial thromboplastin time assay as described earlier(25) .
The 69K chimera was purified as follows. The
unfolded protein was isolated from the 8 M urea extract of
inclusion bodies by affinity chromatography on chelating Sepharose
according to a manufacturer's protocol (His-bind buffer kit;
Novagen, Madison, WI) except that all buffers contained 5 M urea. The extract was dialyzed overnight against binding buffer
and applied to a chelating Sepharose column (2 ml). After the column
was washed with 10 ml of wash buffer, the 69K protein was eluted with
elution buffer. This sample (1 ml) was mixed with 10 ml of 50 mM Tris, pH 8.5, 6 M urea, 0.5 M NHCl,
1 mM EDTA, 0.1 M
-mercaptoethanol and left 4 h
at room temperature. The sample was then dialyzed at 4 °C overnight
successively against 250 ml of Tris-HCl, pH 8.5, 0.5 M NH
Cl, 10 mM benzamidine, 1 mM EDTA
containing different concentrations of urea and reducing or oxidizing
reagents: first, 6 M urea; second, 2 M urea and 0.5
mM cysteine; third, 2 M urea; fourth, 2 M urea and 1 mM oxidized glutathione; fifth, 1 M urea and 0.5 mM oxidized glutathione. Finally it was
dialyzed against 1 liter of 50 mM Tris-HCl, pH 8.0, 40
mM NaCl with one buffer change. The sample was then applied to
the DEAE column and eluted as described above, with the 69K protein
eluting at a slightly lower salt concentration.
Clot lysis was then assayed by adding the urokinase or chimera
(preactivated with plasmin as described above) at a concentration of
1.85 nM (determined by amidolytic assay) to the preformed clot
in 0.4 ml of a solution containing 0.2 mg/ml plasminogen, 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 2.5 mM
CaCl. The plasminogen concentration was comparable to its
normal level in human plasma; lysis was minimal in the absence of added
plasminogen. The sample was incubated at room temperature; it was
periodically mixed, and 20-µl aliquots were withdrawn to determine
the amount of radioactivity released from the clot. Clot lysis was
expressed as percentage of soluble radioactivity, corrected for the
change in volume over the course of the experiment.
Figure 2: SDS-PAGE analysis of chimeric proteins. Proteins were subjected to electrophoresis with or without prior reduction by 2-mercaptoethanol. Molecular masses of standards are indicated in kilodaltons.
Figure 3:
Membrane binding of chimeras. Proteins
were added at the indicated final concentration (determined by annexin
V immunoassay) along with I-annexin V and preserved
erythrocytes with exposed PS in the presence of 2.5 mM calcium. Error bars indicate the standard deviation of
three separate experiments. Symbols: annexin V (squares); 82K chimera (circles); 69K chimera (triangles).
The amidolytic activity of the chimeras before and after activation with plasmin was measured on substrate S-2444. The chimeras had virtually no amidolytic activity prior to activation with plasmin, consistent with the results of SDS-PAGE analysis indicating that both chimeras were present in proenzyme (single-chain) form. However, after activation with plasmin, the amidolytic activity of the chimeras increased approximately 100-fold. After activation with plasmin, the specific amidolytic activity of the 82K chimera was 9.5 ± 1.0 IU/pmol, while the 69K chimera had a specific activity of 4.9 ± 1.6 IU/pmol. These values are comparable to the specific activity of urokinase (9 IU/pmol).
The
chimeras were then tested on their intended physiological substrate,
plasminogen (Fig. 4). Activation of plasminogen followed
Michaelis-Menten kinetics, and both chimeras fully retained the ability
to activate plasminogen compared to natural urokinase. In fact, there
was a trend to lower K values, indicating that the
chimeras may have a slightly higher affinity for plasminogen than
urokinase alone. V
values were comparable (Table 1). Thus, the presence of the annexin V moiety does not
hinder the interaction of the urokinase moiety with its substrate,
plasminogen.
Figure 4: Plasminogen activation by chimeras. Chimeras were first activated by incubation with plasmin, and the amount of urokinase activity present was quantitated by S-2444 assay. Urokinase or activated chimera was then added to plasminogen at the indicated concentration, and the rate of plasmin generation was determined with substrate S-2251 as described under ``Experimental Procedures.'' The lines are the fitted functions used to determine the kinetic parameters. Symbols: urokinase (inverted triangles); 82K chimera (circles); 69K chimera (triangles).
The two chimeras were assayed for their ability to lyse a clot formed from human platelet-rich plasma in vitro (Fig. 5). Both the rate and extent of clot lysis were similar to urokinase, indicating that both chimeras were also able to activate plasminogen and promote clot lysis in a platelet-containing thrombus.
Figure 5:
Clot lysis in vitro by chimeras.
Chimeras were first activated with plasmin and quantitated by S-2444
assay. Chimera or urokinase was then added at a final concentration of
1.85 nM based on amidolytic activity to a clot prepared from
platelet-rich plasma and I-fibrinogen; plasminogen was
also added to 0.2 mg/ml. The samples were then incubated at room
temperature, and the percentage of soluble radioactivity was determined
by removing aliquots at the indicated times. Error bars indicate the range of duplicates in a representative experiment. Symbols: urokinase (inverted triangles); 82K chimera (circles); 69K chimera (triangles); control with
plasminogen but no activator (squares).
This study shows the feasibility of producing hybrid
thrombolytic agents in which annexin V provides the thrombus-targeting
component. We have constructed two chimeric plasminogen activators that
contain either full-length or truncated prourokinase attached to a
membrane-targeting component, annexin V. In vitro assays show
that these molecules fully retain the component activities of the
parent molecules, i.e. membrane binding, activation of
plasminogen, and in vitro fibrinolysis. This study and our
related work using chemical conjugation procedures ()provide
the first example of the use of annexin V as an agent to target
chimeric proteins to cell membranes that are rich in exposed PS, such
as those on activated platelets.
Our results indicate that the
presence of the annexin V moiety does not prevent activation of the
prourokinase moiety by plasmin, or decrease the activity of the
urokinase moiety toward its physiological substrate, plasminogen.
Likewise, the attachment of the large prourokinase moiety at the N
terminus of the annexin V does not alter its affinity for cell
membranes. This is consistent with the expectations used to design the
chimera: structural evidence shows that the N terminus of annexin V is
located at the surface of the protein and faces away from the
membrane-binding side of the
molecule(35, 36, 37) . Thus the two major
functional units of the chimera are still able to act independently
when attached to each other. We also saw no evidence that the annexin V
moiety was degraded by plasmin or urokinase; this was expected from
previous work, which has shown that native annexin V has a compact
structure that is resistant to proteolysis. ()
The functional properties of the 82K and the 69K chimeras appear to be equivalent in the assays tested so far. Thus, both molecules are candidates for further testing in vivo in animal models of clot lysis. The smaller size of the 69K chimera might be advantageous in facilitating penetration into the thrombus. However, this molecule was somewhat harder to produce and purify than the 82K chimera, perhaps because absence of the N-terminal 143 amino acids of prourokinase decreases protein stability.
Despite initial production of
relatively large amounts of insoluble protein after expression of the
82K chimera was induced, the final yield of folded, soluble, monomeric
protein was rather low. The low yield probably reflects the difficulty
of forming all the correct disulfide bonds in the serine-protease
domain of the prourokinase moiety, since annexin V has no disulfide
bonds. Consistent with this view, yields were lower for the 69K
chimera, which retains the serine-protease domain but lacks the first
144 amino acids of prourokinase. Also, presence of the N-terminal
20-residue polyhistidine sequence did not affect yields, since removing
it from the construct used to express the 82K chimera did not improve
yield. Previous studies have also reported moderately low yields of
active prourokinase (0.15-0.3 mg of prourokinase per g of E. coli paste) after refolding of insoluble E.
coli-derived material(32, 33) . We were not able
to achieve production levels of active chimera as high as the levels
reported for prourokinase alone. It is possible that presence of the
annexin V moiety may further reduce the efficiency of folding and/or
disulfide bond formation in the prourokinase moiety. Thus, while the
present expression system produces adequate amounts of protein for in vitro characterization, production of large amounts of
protein for animal studies will probably require an alternative
expression host such as yeast or mammalian cells with naturally
catalyzed disulfide bond formation, or production via chemical
conjugation of separately produced proteins.
The fact that the
chimeras were similar to urokinase when tested for clot lysis activity in vitro (Fig. 5) might raise doubts about the
practical value of such chimeras for potential clinical use. However,
it is difficult to predict the in vivo potency of modified
plasminogen activators from in vitro data, since factors such
as blood flow past the thrombus and plasma clearance rate can greatly
affect the results compared those obtained in static test-tube assays.
In several instances, conjugates of urokinase with antiplatelet or
antifibrin antibodies showed much higher in vivo potency than
would be predicted from their in vitro potency(7, 8, 10) . But more
importantly, preliminary animal studies with an annexin V-urokinase
conjugate produced by chemical conjugation indicate that it has
3-4-fold higher in vivo thrombolytic potency than
urokinase alone. Thus, although further animal studies will
be needed to evaluate the efficacy of annexin V-prourokinase chimeras,
these initial results, together with previous results showing
accumulation of annexin V in thrombi in
vivo(21, 22) , provide grounds for optimism that
annexin V may be useful for targeting thrombolytic agents in
vivo.
Extracellular exposure of PS has long been recognized as an integral part of the platelet procoagulant response (11, 12) and may also be involved in augmenting the procoagulant potential of certain tumors (38, 39) . Recently, increased exposure of PS has also been implicated in other physiological events, such as marking oxidatively damaged (40) or apoptotic (41) cells for phagocytic removal. Thus, annexin V may have broader uses as a targeting agent beyond the field of blood coagulation wherever diagnostic or therapeutic agents need to be targeted to cells undergoing events that increase extracellular exposure of PS.