©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Prourokinase-Annexin V Chimeras
CONSTRUCTION, EXPRESSION, AND CHARACTERIZATION OF RECOMBINANT PROTEINS (*)

(Received for publication, May 30, 1995; and in revised form, July 10, 1995)

Jonathan F. Tait (1) (2)(§) Shelley Engelhardt (1) Christina Smith (1) Kazuo Fujikawa (3)

From the  (1)Departments of Laboratory Medicine, (2)Pathology, and (3)Biochemistry, University of Washington, Seattle, Washington 98195

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) (^1)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(r) 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.


EXPERIMENTAL PROCEDURES

Materials

Chromogenic substrates S-2444 (L-pyroglutamyl-glycyl-L-arginine-p-nitroaniline hydrochloride) and S-2251 (H-D-valyl-L-leucyl-L-lysine-p-nitroaniline dihydrochloride) were from Pharmacia Hepar Inc. (Franklin, OH). Human plasminogen (E = 17.0 at 280 nm; M(r) taken as 92,000); human urokinase (specific activity, 167,000 IU/mg; E = 15.1 at 280 nm; M(r) taken as 54,000), and the plasmid pSV-G1-preUK (encoding human prourokinase) were from The Green Cross Corp. (Osaka, Japan). Human fibrinogen was from Calbiochem (La Jolla, CA) and was labeled with I by the IODO-GEN method. Vectors pET-12a and pET-14b and Escherichia coli strain BL21(DE3) were from Novagen (Madison, WI). Vector pCRII and the TA cloning kit were from Invitrogen (San Diego, CA). Preserved whole blood (Coulter 4CPlus Normal Control) was from Coulter Corp. (Hialeah, FL). Chelating Sepharose and SDS-PAGE standards were from Pharmacia Biotech Inc. Placental annexin V was prepared as described elsewhere(25) .

Construction and Verification of Expression Vectors

Standard molecular biology methods were followed(26) . PCR was performed on the plasmid pPAP-I-1.6, which encodes full-length annexin V(27, 28) , with Pfu DNA polymerase (Stratagene) and oligonucleotide primers JT-248 (5`-ctc gag atg gca cag gtt ctc a-3`) and JT-249 (5`-gga tcc tta gtc atc ttc tcc aca-3`) to introduce XhoI and BamHI sites at the 5` and 3` ends of the annexin V-coding sequence (amino acids 1-320 of the primary translation product); the product was then cloned into the pCRII vector and completely sequenced to rule out any artifactual mutations introduced by PCR. PCR was also performed on the plasmid pSV-G1-preUK, which encodes full-length prourokinase, to introduce NdeI and XhoI sites at the 5` and 3` ends of the prourokinase coding sequence using primers JT-244 (5`-cat atg agc aat gaa cct cat caa gtt cca-3`) and JT-245 (5`-ctc gag ggc cag gcc att ctc-3`) for full-length prourokinase (amino acids 1-411 of the mature protein), and primers JT-253 (5`-cat atg tta aaa ttt cag tgt ggc caa aag act-3`) and JT-245 for truncated prourokinase (amino acids 144-411). Each product was cloned into pCRII and also fully sequenced. The insert encoding annexin V was then removed from the pCRII vector and cloned into the pET-14b vector at the XhoI and BamHI sites to produce plasmid pET-14b-PAP. Finally, the inserts encoding the prourokinase moieties were removed from the pCRII vector and cloned into the NdeI and XhoI sites of the pET-14b-PAP vector to produce the two desired constructs: pET-14b-UK(1-411)-PAP and pET-14b-UK(144-411)-PAP (see Fig. 1). The primary translation products of these vectors include a 20-amino acid polyhistidine-containing sequence present at the N terminus (sequence in single-letter code: MGSSH HHHHH SSGLV PRGSH). A plasmid pET-12a-UK(1-411)-PAP was also produced by transferring the expression unit between the NdeI and HindIII sites from pET-14b-UK(1-411)-PAP to the corresponding sites of pET-12a; this plasmid encodes the same protein except that it lacks the 20-amino acid polyhistidine-containing sequence present at the N terminus of the pET-14b product. Regions around the cloning sites in the final vectors were resequenced to verify that no undesired mutations had been inadvertently introduced. GenBank reference sequences were M18366 for the annexin V cDNA and M15476 for the prourokinase cDNA.


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(r) 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.



Expression and Purification of Chimeric Proteins

Plasmids were transformed into E. coli strain BL21(DE3) for large-scale expression. Cells were grown at 37 °C with shaking in 2% LB broth base (Life Technologies, Inc.) in 50 mM potassium phosphate buffer, pH 7.4. When the turbidity (OD) reached 0.4-0.5, isopropyl-beta-D-thiogalactoside was added to 1 mM, and growth was continued for 2 h; the final OD was about 1. Cells were then harvested by centrifugation, washed once with 50 mM Tris-HCl, pH 8.0, to give a pellet with wet weight of about 2 g per liter of culture, and stored frozen at -20 °C. The inclusion body fraction was prepared according to Nagai and Thogersen (29) from the cells obtained from 3 liters of culture. The inclusion body fraction, which contained chimera with about 70% purity, was suspended in 5 ml of 8 M urea, 0.5 M NH(4)Cl, 50 mM Tris-HCl, pH 8.5, 0.1 M 2-mercaptoethanol, and shaken for 4 h at room temperature. Insoluble materials were then removed by centrifugation.

The 82K chimera was purified as follows. The 8 M urea extract was applied to a Sephacryl S-400 gel filtration column (2.5 times 90 cm) equilibrated and eluted with 5 M urea, 0.5 M NH(4)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 NH(4)Cl, 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(4)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 NH(4)Cl, 1 mM EDTA, 0.1 M beta-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(4)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.

Membrane Binding Assay

The affinity of chimeras for cell membranes containing exposed PS was determined by a modification of the binding assay of Tait and Gibson(30) , using a commercial preparation of preserved blood in which the erythrocytes have high levels of exposed PS. Proteins to be tested were incubated at various concentrations with 5 nMI-annexin V (30) and 5.3 times 10^6 erythocytes/ml in the presence of 2.5 mM calcium for 15 min at 37 °C; the assay buffer consisted of 10 mM HEPES-Na, pH 7.4, 136 mM NaCl, 2.7 mM KCl, 2 mM MgCl(2), 1 mM NaH(2)PO(4), 5 mM glucose, 5 mg/ml bovine serum albumin (fatty-acid free; Sigma). Bound and free ligand were then separated by centrifugation through a silicone-oil barrier as described previously. Nonspecific binding was measured in the presence of 5 mM EDTA and was less than 1% of total binding.

Measurement of Amidolytic Activity (S-2444)

Where necessary, chimeras were first activated by incubation with 1 µg/ml plasmin for 60 min at 37 °C. Samples were then added to a solution of chromogenic substrate S-2444 (1 mM) in 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.1% Tween-80 in a final volume of 100 µl and incubated for 10 min at 37 °C. The reaction was then stopped with 500 µl of 10% acetic acid, and the absorbance was read against a substrate blank. Activity (expressed as equivalent concentration of urokinase) was calculated from a standard curve prepared using human urokinase (Green Cross), which was linear from 0 to 2.5 µg/ml. Control experiments showed that under these conditions the traces of plasmin present from the activation reaction had a negligible effect on the observed absorbance changes.

Plasminogen Activation Assay

Chimeras were first activated by incubation with 1 µg/ml plasmin for 60 min at 37 °C in 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.1% Tween-80. The amount of urokinase activity generated was then quantitated by S-2444 assay as described above. Urokinase or activated chimera was then added at 1.85 nM final concentration to plasminogen (0-27 µM) at room temperature in the same buffer. Aliquots were then taken at 1, 2, and 3 min and diluted 25-fold at room temperature in the same buffer containing 0.5 mM of chromogenic substrate S-2251. The rate of absorbance change was measured over a 2-min period after dilution; under these conditions the rate of substrate hydrolysis was linear with time, indicating that no new plasmin was being generated during the S-2251 assay. The amount of plasmin present was determined by reference to a standard curve. The rate of plasmin generation at each concentration of plasminogen was then determined graphically from the values of plasmin present at 1, 2, and 3 min after addition of the activator to the plasminogen. Kinetic parameters were determined by fitting the data to a standard Michaelis-Menten function by nonlinear least-squares analysis (RS/1 software, Bolt-Beranek-Newman, Cambridge, MA). Uncertainties of the parameters are given as standard deviations estimated by the curve-fitting program.

In Vitro Clot Lysis Assay

Venous blood was drawn from healthy volunteer donors into 0.1 volume of 109 mM sodium citrate, pH 6.5, and platelet-rich plasma prepared by centrifugation at room temperature for 5 min at 450 times g; the platelet count ranged from 250,000 to 450,000/µl, depending on the donor. Individual clots were then formed from 100 µl of platelet-rich plasma by adding calcium chloride (10 mM), human thrombin (10 units/ml), and I-fibrinogen (0.8 µg/ml, 1 µCi/µg) and incubating for 10 min at room temperature in 12 times 75-mm glass test tubes. Clots were then washed for 5 min at room temperature in 200 µl of 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 2.5 mM CaCl(2), transferred to fresh glass tubes, and total radioactivity present was determined.

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(2). 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.

Miscellaneous Procedures

Concentrations of refolded immunoreactive chimeras were determined by competitive fluorescence polarization immunoassay on a TDx Analyzer (Abbott Laboratories). Assays were performed at 34 °C in phosphate-buffered saline, pH 7.4, containing 0.1% Triton X-100, 2 nM fluorescein-annexin V(14) , 200 nM of an IgG fraction of rabbit polyclonal antiserum raised against placental annexin V, and placental annexin V as the standard. Western blotting was performed with the same polyclonal antiserum against annexin V, with detection based on alkaline-phosphatase-conjugated antibody. SDS-PAGE was performed with a Laemmli system with 12.5% gels; where necessary, samples were concentrated prior to electrophoresis with Microcon-30 concentrators (Amicon).


RESULTS

Design and Construction of Expression Vectors

Vectors were designed to produce two chimeras (Fig. 1). One chimera (82K) contains full-length prourokinase at the N terminus. The second chimera (69K) contains a truncated form of prourokinase beginning at amino acid 144; this derivative, although smaller than the full-length prourokinase, retains the full serine-protease domain as well as the plasmin activation site at Lys-158-Ile-159(24) . At the C terminus, both chimeras contain the full annexin V sequence (amino acids 1-320, including initiator Met), separated from the prourokinase by a single Glu residue. The 69K chimera also has a 20-amino acid N-terminal extension with a polyhistidine sequence for use in purification by metal-ion-dependent affinity chromatography. The expression vectors included a provision for optional insertion of a spacer sequence at the unique XhoI site between the two protein units, although so far we have not used this feature because subsequent work showed no evidence for steric interference between the two functional protein units. Both expression constructs are placed in one of the pET series of vectors(31) , allowing for high level expression in E. coli under control of the T7 promoter. We chose to express the chimeras cytoplasmically in E. coli since previous work has shown that both prourokinase (32, 33) and annexin V (34) can be produced by this method.

Expression and Purification of Chimeric Proteins

When expression was carried out as described under ``Experimental Procedures,'' Western blot analysis indicated that greater than 95% of the 82K chimera was present in single-chain form in the insoluble fraction after cell lysis, indicating that it was being packaged in inclusion bodies. The initial yield of chimera was approximately 10-25 mg/liter of culture, based on Western blot analysis. The inclusion body fraction was therefore isolated and subjected to a variety of denaturation and refolding procedures. We varied the denaturant (urea or guanidine), its rate of removal, the pH, the salt concentration, the reducing agent (2-mercaptoethanol or glutathione), the presence or absence of protein-disulfide isomerase, and the protein concentration during refolding. The yield of renatured material was then determined by S-2444 assay and by solution-phase immunoassay for native annexin V; yields of active protein ranged from about 0.1 to 1 mg/liter of culture prior to purification, depending on the procedure followed. The yield of the 69K chimera after refolding was about 2-fold lower. No method gave a high absolute yield of active chimera (see ``Discussion''). Once refolding had been optimized, the chimeric proteins were then purified as described under ``Experimental Procedures.'' SDS-PAGE analysis of the purified chimeras showed that both proteins were present in single-chain form, and their observed molecular weights were consistent with calculated values (Fig. 2). The purity of the 69K chimera was less than that of the 82K chimera due to lower initial yields and greater losses during refolding and purification.


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.



Measurement of Component Activities

The affinity of the chimeras for cell membranes with exposed PS was determined by competition assay against I-annexin V (Fig. 3). Both chimeras were able to fully displace the I-annexin V with affinities identical to native annexin V, indicating that the membrane-binding activity of the annexin V moiety was fully retained in the chimeric proteins.


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(m) values, indicating that the chimeras may have a slightly higher affinity for plasminogen than urokinase alone. V(max) 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).




DISCUSSION

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 (^2)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. (^3)

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.^2 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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant HL-47151. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Laboratory Medicine, Health Sciences Rm. NW-120, Box 357110, University of Washington, Seattle, WA 98195-7110. Tel.: 206-548-6131; Fax: 206-548-6189.

(^1)
The abbreviations used are: PS, phosphatidylserine; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.

(^2)
K. Tanaka, K. Einaga, H. Tsuchiyama, J. F. Tait, and K. Fujikawa, manuscript submitted for publication.

(^3)
J. F. Tait and K. Fujikawa, unpublished observations.


ACKNOWLEDGEMENTS

We thank Carlene Urrutia for assistance with bacterial preparations, and Donald Gibson for assistance in preparing the figures.


REFERENCES

  1. Anderson, H. V., and Willerson, J. T. (1993) N. Engl. J. Med. 329,703-709 [Free Full Text]
  2. Collen, D. (1993) Lancet 342,34-36 [Medline] [Order article via Infotrieve]
  3. deBono, D. (1994) in Haemostasis and Thrombosis (Bloom, A. L., Forbes, C. D., Thomas, D. P., and Tuddenham, E. G. D., eds) 3rd Ed., pp. 1459-1472, Churchill-Livingstone, Edinburgh
  4. Lijnen, H. R., and Collen, D. (1994) in Haemostasis and Thrombosis (Bloom, A. L., Forbes, C. D., Thomas, D. P., and Tuddenham, E. G. D., eds) 3rd Ed., pp. 625-637, Churchill Livingstone, Edinburgh
  5. Bode, C., Matsueda, G. R., Hui, K. Y., and Haber, E. (1985) Science 229,765-767 [Medline] [Order article via Infotrieve]
  6. Runge, M. S., Quertermous, T., Zavodny, P. J., Love, T. W., Bode, C., Freitag, M., Shaw, S. Y., Huang, P. L., Chou, C. C., Mullins, D., Schnee, J. M., Savard, C. E., Rothenberg, M. E., Newell, J. B., Matsueda, G. R., and Haber, E. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,10337-10341 [Abstract]
  7. Bode, C., Runge, M. S., Schonermark, S., Eberle, T., Newell, J. B., Kubler, W., and Haber, E. (1990) Circulation 81,1974-1980 [Abstract]
  8. Holvoet, P., Laroche, Y., Stassen, J. M., Lijnen, H. R., Van Hoef, B., De Cock, F., Van Houtven, A., Gansemans, Y., Matthyssens, G., and Collen, D. (1993) Blood 81,696-703 [Abstract]
  9. Bode, C., Meinhardt, G., Runge, M. S., Freitag, M., Nordt, T., Arens, M., Newell, J. B., Kubler, W., and Haber, E. (1991) Circulation 84,805-813 [Abstract]
  10. Dewerchin, M., Lijnen, H. R., Stassen, J. M., De, C. F., Quertermous, T., Ginsberg, M. H., Plow, E. F., and Collen, D. (1991) Blood 78,1005-1018 [Abstract]
  11. Bevers, E. M., Comfurius, P., and Zwaal, R. F. A. (1983) Biochim. Biophys. Acta 736,57-66 [Medline] [Order article via Infotrieve]
  12. Bevers, E. M., Comfurius, P., and Zwaal, R. F. A. (1991) Blood Rev. 5,147-154
  13. Raynal, P., and Pollard, H. B. (1994) Biochim. Biophys. Acta 1197,63-93 [Medline] [Order article via Infotrieve]
  14. Tait, J. F., Gibson, D., and Fujikawa, K. (1989) J. Biol.Chem. 264,7944-7949 [Abstract/Free Full Text]
  15. Andree, H. A., Reutelingsperger, C. P., Hauptmann, R., Hemker, H. C., Hermens, W. T., and Willems, G. M. (1990) J. Biol. Chem. 265,4923-4928 [Abstract/Free Full Text]
  16. Meers, P., Daleke, D., Hong, K., and Papahadjopoulos, D. (1991) Biochemistry 30,2903-2908 [Medline] [Order article via Infotrieve]
  17. Tait, J. F., and Gibson, D. (1992) Arch. Biochem. Biophys. 298,187-191 [Medline] [Order article via Infotrieve]
  18. Thiagarajan, P., and Tait, J. F. (1990) J. Biol. Chem. 265,17420-17423 [Abstract/Free Full Text]
  19. Thiagarajan, P., and Tait, J. F. (1991) J. Biol. Chem. 266,24302-24307 [Abstract/Free Full Text]
  20. Shattil, S. J., Hoxie, J. A., Cunningham, M., and Brass, L. F. (1985) J. Biol. Chem. 260,11107-11114 [Abstract/Free Full Text]
  21. Tait, J. F., Cerqueira, M. D., Dewhurst, T. A., Fujikawa, K., Ritchie, J. L., and Stratton, J. R. (1994) Thrombosis Res. 75,491-501 [Medline] [Order article via Infotrieve]
  22. Stratton, J. R., Dewhurst, T. A., Kasina, S., Reno, J. M., Cerqueira, M. D., Baskin, D. G., and Tait, J. F. (1995) Circulation , in press
  23. Flaherty, M. J., West, S., Heimark, R. L., Fujikawa, K., and Tait, J. F. (1990) J. Lab. Clin. Med. 115,174-181 [Medline] [Order article via Infotrieve]
  24. Holmes, W. E., Pennica, D., Blaber, M., Rey, M. W., Guenzler, W. A., Steffens, G. J., and Heyneker, H. L. (1985) Bio/Technology 3,923-929
  25. Funakoshi, T., Heimark, R. L., Hendrickson, L. E., McMullen, B. A., and Fujikawa, K. (1987) Biochemistry 26,5572-5578 [Medline] [Order article via Infotrieve]
  26. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  27. Funakoshi, T., Hendrickson, L. E., McMullen, B. A., and Fujikawa, K. (1987) Biochemistry 26,8087-8092 [Medline] [Order article via Infotrieve]
  28. Cookson, B. T., Engelhardt, S., Smith, C., Bamford, H. A., Prochazka, M., and Tait, J. F. (1994) Genomics 20,463-467 [CrossRef][Medline] [Order article via Infotrieve]
  29. Nagai, K., and Thogersen, H. C. (1987) Methods Enzymol. 153,461-481 [Medline] [Order article via Infotrieve]
  30. Tait, J. F., and Gibson, D. (1994) J. Lab. Clin. Med. 123,741-748 [Medline] [Order article via Infotrieve]
  31. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) Methods Enzymol. 185,60-89 [Medline] [Order article via Infotrieve]
  32. Winkler, M. E., and Blaber, M. (1986) Biochemistry 25,4041-4045 [Medline] [Order article via Infotrieve]
  33. Orsini, G., Brandazza, A., Sarmientos, P., Molinari, A., Lansen, J., and Cauet, G. (1991) Eur. J. Biochem. 195,691-697 [Abstract]
  34. Iwasaki, A., Suda, M., Nakao, H., Nagoya, T., Saino, Y., Arai, K., Mizoguchi, T., Sato, F., Yoshizaki, H., Hirata, M., Miyata, T., Shidara, Y., Murata, M., and Maki, M. (1987) J. Biochem. (Tokyo) 102,1261-1273 [Abstract]
  35. Huber, R., Berendes, R., Burger, A., Schneider, M., Karshikov, A., Luecke, H., Romisch, J., and Paques, E. (1992) J. Mol. Biol. 223,683-704 [Medline] [Order article via Infotrieve]
  36. Concha, N. O., Head, J. F., Kaetzel, M. A., Dedman, J. R., and Seaton, B. A. (1993) Science 261,1321-1324 [Medline] [Order article via Infotrieve]
  37. Voges, D., Berendes, R., Burger, A., Demange, P., Baumeister, W., and Huber, R. (1994) J. Mol. Biol. 238,199-213 [CrossRef][Medline] [Order article via Infotrieve]
  38. Rao, L. V., Tait, J. F., and Hoang, A. D. (1992) Thromb. Res. 67,517-531 [Medline] [Order article via Infotrieve]
  39. Sugimura, M., Donato, R., Kakkar, V. V., and Scully, M. F. (1994) Blood Coagul. & Fibrinolysis 5,365-373
  40. Sambrano, G. R., and Steinberg, D. (1995) Proc. Natl. Acad. Sci. U. S. A. 92,1396-1400 [Abstract]
  41. Fadok, V. A., Voelker, D. R., Campbell, P. A., Cohen, J. J., Bratton, D. L., and Henson, P. M. (1992) J. Immunol. 148,2207-2216 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.