Development of Annexin V Mutants Suitable for Labeling with Tc (I

Bioconjugate Chem. 2002, 13, 1119−1123

1119

Development of Annexin V Mutants Suitable for Labeling with Tc(I)-Carbonyl Complex Jonathan F. Tait,*,†,‡ Christina Smith,† and Donald F. Gibson† Departments of Laboratory Medicine, Medicine (Medical Genetics), and Pathology, University of Washington, Seattle, Washington 98195-7110 . Received May 1, 2002; Revised Manuscript Received July 16, 2002

99mTc-annexin

V can be used to image organs undergoing cell death during cancer chemotherapy and organ transplant rejection. We investigated whether the novel Tc-carbonyl labeling method would be suitable for annexin V. Two mutant molecules of annexin V, called annexin V-122 and annexin V-123, were constructed with N-terminal extensions containing either three or six histidine residues. These molecules were expressed cytoplasmically in E. coli and purified with a final yield of 33 mg of protein/L of culture. Analysis by SDS-PAGE, isoelectric focusing, gel filtration chromatography, and mass spectrometry confirmed the purity and homogeneity of the protein preparations. Both mutant proteins retained full binding affinity for cell membranes with exposed phosphatidylserine. Using the Tc-carbonyl reagent, both proteins could be labeled with 99mTc to specific activities of at least 10-20 µCi/µg with full retention of bioactivity. The radiolabeled proteins were stable when incubated with phosphate-buffered saline or serum in vitro, and there was no transchelation of label to serum proteins during in vitro incubation. In conclusion, annexin V can be modified near its N-terminus to incorporate sequences that form specific chelation sites for 99mTc-carbonyl without altering its high affinity for cell membranes.

INTRODUCTION

The phospholipid binding protein annexin V can be used to detect the exposure of PS1 at the extracellular face of the plasma membrane (1). Alterations in PS exposure occur in both blood coagulation and apoptosis (2, 3). We have developed annexin V as a means to detect PS exposure in vivo. Annexin V labeled with radioiodine and 99mTc was used to detect thrombus formation in experimental animals (4, 5). 99mTc-annexin V was used to detect cell death in vivo in animal models (6-10). Because of the clinical applicability of this work in monitoring cancer chemotherapy, rejection of transplanted organs (11), and mycoardial infarction (12), there is interest in developing new annexin V derivatives with improved imaging properties or greater ease of use in the clinical setting. There have been several methods developed so far for labeling annexin V with technetium. The N2S2 preformed chelate method (13) was used in the earliest studies (5). More recently, annexin V was chemically derivatized with HYNIC (6, 7) or iminomercaptobutylate (12). Most recently, we developed novel forms of annexin V that contain genetically engineered sequences that can directly chelate technetium in the +V oxidation state * Address correspondence to this author at the Department of Laboratory Medicine, University of Washington, Room NW120, Box 357110, Seattle, WA 98195-7110. Telephone: (206)598-6131; Fax: (206)598-6189; Email: [email protected]. † Department of Laboratory Medicine. ‡ Departments of Medicine (Medical Genetics) and Pathology. 1 Abbreviations: HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HYNIC, 6-hydrazinopyridine-3-carboxylate; IC50, 50% inhibitory concentration; ITLC, instant thin-layer chromatography; PBS, phosphate-buffered saline; PS, phosphatidylserine; TcCO, [99mTc(OH2)3(CO)3]+; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; wt, wildtype.

without need for any chemical derivatization with exogenous chelation groups (14). Although all these technetium-labeled forms of annexin have certain benefits and uses, none is ideal, and there is still a need to explore additional ways of radiolabeling the protein to provide derivatives with high specific activity and radiostability. A novel reagent was recently developed for Tc labeling of biomolecules. Alberto et al. (15) developed a convenient aqueous synthesis of [99mTc(OH2)3(CO)3]+ (abbreviated TcCO). Egli et al. (16) used this reagent to label peptides to high specific activities, and they showed that among the amino acids histidine formed complexes of highest stability with TcCO. Waibel et al. (17) used TcCO to label a recombinant single-chain Fv antibody containing five histidine residues at the C-terminus; this derivative showed high radiochemical stability in vitro and in vivo. Other groups have recently applied this method to label the peptides somatostatin (18) and bitistatin (19). In view of the potential advantages of this radiolabeling method, we investigated whether it would be suitable for labeling annexin V. Since histidine is the amino acid likely to form complexes of highest stability with TcCO, and trivalent complexes are the most stable (20), we added peptide sequences containing either three or six histidines to annexin V. Although the C-terminal carboxyl group may help to stabilize histidine-TcCO complexes (17), we chose to add the histidine residues to the N-terminus of annexin V because alterations in this region are much less likely to affect either membrane binding affinity or protein folding in E. coli (14, 21, 22). We show that these novel derivatives can be produced in high yield and purity by expression in E. coli, and they can be readily labeled with TcCO. MATERIALS AND METHODS

Materials. Recombinant annexin V was produced in E. coli as described (23). Fluorescein-annexin V was

10.1021/bc025545s CCC: $22.00 © 2002 American Chemical Society Published on Web 08/16/2002

1120 Bioconjugate Chem., Vol. 13, No. 5, 2002

prepared as described (24). Lyophilized carbonyl reaction mixture was a gift from Mallinckrodt (St. Louis, MO). Erythrocytes with exposed PS (4CPlus Normal Control, part number 7546923) were from Beckman-Coulter (Miami, FL). Construction and Verification of Plasmids. Two plasmids were constructed to express mutant forms of human annexin V in E. coli under control of the phage T7 promoter. Plasmid pJ122 was constructed with oligonucleotides JT-319 (5′-ATGGCCCATCATCATGCACAGGTTCTCAGA-3′) and JT-321 (5′-TTAGTCATCTTCTCCACAGAGCAGCAG-3′); plasmid pJ123 with JT320 (5′-ATGGCCCATCATCATCATCATCACGCACAGGTTCTCAGA-3′) and JT-321. These oligonucleotides were designed to amplify the entire protein-coding region of wild-type annexin V and add the desired coding sequences to the 5′ end of the gene. PCR of the template pET9a-PAP1, which encodes wild-type annexin V (25), was performed with proofreading Pfu DNA polymerase (Stratagene) and with annealing steps first at 45 °C for 15 cycles and next at 65 °C for 15 cycles. PCR products were purified by phenol/chloroform extraction and ethanol precipitation; the purified PCR products were amplified again for three cycles with the same primer sets with annealing steps at 65 °C using Taq DNA polymerase to add the 3′-dA nucleotide overhangs necessary for ligation. The PCR product was ligated into pETBlue-1 Acceptor Vector (Novagen, Madison, WI), and the ligation mixture was transformed into E. coli strain Nova Blue (Novagen) and plated on LB agar containing 50 µg/mL carbenicillin, 70 µg/mL X-gal, and 80 µM IPTG. Plasmid DNA was prepared from white, carbenicillin-resistant colonies and screened by restriction mapping for the presence of the desired insert. Positive clones were subjected to DNA sequence analysis with the Big-Dye kit (Perkin-Elmer) to verify the presence of the desired insertion and the absence of other adventitious mutations. Expression and Purification of Proteins. Plasmids pJ122 and pJ123 were each transformed into E. coli strain Tuner(DE3)pLac I (Novagen) for cytoplasmic expression. Each clone was grown overnight to saturation at 37 °C with shaking in Terrific Broth containing carbenicillin (50 µg/mL). The cells were separated from culture medium by centrifugation for 10 min at 2560g and washed in ice-cold buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 8.0). Bacteria were disrupted by sonication in ice-cold 50 mmol/L Tris-HCl, pH 7.2, 10 mmol/L CaCl2 and then centrifuged for 20 min at 22530g in a Sorvall SS-34 rotor. The supernatant was discarded, and the annexin V bound to bacterial membranes was released by resuspending the pellet in 50 mmol/L TrisHCl, pH 7.2, 20 mmol/L EDTA. Bacterial membranes were removed by centrifugation for 20 min at 22530g; the supernatant containing the annexin V was treated with 10 units/mL Benzonase (Novagen) plus 25 mM MgCl2 for 1 h on ice, to digest DNA and RNA. After centrifugation for 20 min at 4800g, the supernatant was filtered with a 0.45 µm filter and then dialyzed against 20 mmol/L Tris-HCl, pH 8.0, at 4 °C. The dialysand of annexin V-122 was further dialyzed against 20 mmol/L sodium acetate, pH 5.2, filtered through a 0.2 µm filter, applied to a Mono S HR 10/10 column (Pharmacia, Piscataway, NJ), and eluted with a gradient of 0-1 mol/L NaCl in the same buffer. The annexin V-122 eluted at approximately 0.170 mol/L NaCl. The dialysand of annexin V-123 was dialyzed against 20 mmol/L HEPES-Na, pH 7.0, filtered with a 0.2 µm filter, loaded onto a Mono Q HR 10/10 column (Pharmacia), and eluted with 0-1 mol/L NaCl in the same buffer. Annexin

Tait et al.

V-123 eluted at 0.170 mol/L NaCl. Each purified protein was dialyzed against 20 mmol/L HEPES, pH 7.4, 100 mmol/L NaCl, and stored in aliquots at -70 °C. The final yield of each protein was approximately 33 mg/L of bacterial culture (or 3.3 mg/g wet weight of cell paste), with a purity of ∼98% as judged by SDS-PAGE. Electrophoresis using 10-15% gradient SDS-PAGE and IEF 4-6.5 gels was performed on a PHAST System (Amersham-Pharmacia). Electrospray mass spectrometry was performed on a Micromass Quattro II tandem quadrupole mass spectrometer (Micromass Ltd., Manchester, U.K.) in the Mass Spectrometry Center (Department of Medicinal Chemistry, University of Washington). Labeling of Proteins with 99mTc. The 99mTcCO was made by adding 1 mL of TcO4 (usually 1-10 mCi) in 0.9% deoxygenated NaCl to a 10 mL vial of lyophilized carbonyl reaction mixture (13 mg of L-tartaric acid; 7.6 mg of KBH4; 20 mg of lactose; borate buffer, pH 11.6; carbon monoxide in headspace). The vial was incubated for 15 min in a boiling water bath, cooled to room temperature, vented with a syringe needle, and neutralized with 0.25 mL of 1 N HCl. Except where noted otherwise, labeling reactions were set up with 100 µL (100 µg) of annexin V-122 or annexin V-123 in 20 mM HEPES-Na, pH 7.4, 100 mM NaCl; 25 µL of 0.5 M HEPES-Na, pH 7.4; 125 µL of 99mTcCO in 0.9% saline and incubated at 37 °C for 1 h. Percent incorporation of 99mTc was determined by ITLC on oven-dried silica gel plates with PBS as solvent. Radiolabeled proteins were purified by applying the reaction mixture (diluted to 400 µL with PBS) to a Sephadex G-25 column (0.9 × 2.8 cm) previously equilibrated with PBS and eluting the column with 1000 µL of PBS; radiolabeled protein eluted in the last 600 µL. Percent protein-bound radioactivity after gel filtration was determined by ITLC as above. For studies of radiolabel stability in serum, percent protein-bound radioactivity was determined by centrifugal ultrafiltration (3 min at 1860g) through a membrane with a 10 000 dalton cutoff (Centricon-10 filter, Amicon, Danvers, MA). Radiolabel stability was also assessed by gel filtration on a Superose-12 column (1 × 30 cm). The column was equilibrated with PBS and eluted at a flow rate of 0.5 mL/min at room temperature; 0.5 mL fractions were collected. Cell Binding Assays. The affinity of nonradioactive proteins for cell membranes was determined by their ability to compete with fluorescein-annexin V for binding to erythrocytes with exposed phosphatidylserine (21). Mutant proteins were added at various concentrations to 1 mL of a solution containing 5 nmol/L fluoresceinannexin V and 8.3 × 106 erythrocytes in a buffer consisting of 10 mmol/L HEPES-Na, pH 7.4, 136 mmol/L NaCl, 2.7 mmol/L KCl, 5 mmol/L glucose, 1 mg/mL BSA (buffer HNKGB) plus 2.5 mmol/L CaCl2. The sample was incubated for 15 min at room temperature. The cells were centrifuged for 3 min at 8300g, the supernatant was removed, and the fluorescein-annexin V bound to the pelleted cells was released by resuspension in 980 µL of HNKGB buffer containing 5 mmol/L EDTA. The sample was centrifuged again, the supernatant removed, and the concentration of fluorescein-annexin V in the supernatant determined by fluorometry. The results of this assay are summarized as the concentration of competitor protein that inhibits 50% of the binding of fluorescein-annexin V, abbreviated IC50. The ability of radiolabeled proteins to bind to cell membranes was determined by incubating them with a large excess of cells in a similar procedure. Radiolabeled protein at 10 nmol/L final concentration was added to

Annexin V Mutants Labeled with Tc-Carbonyl

Bioconjugate Chem., Vol. 13, No. 5, 2002 1121 Table 2. Radiochemical Purity and Bioactivity of Labeled Proteinsa

Figure 1. Primary structures of annexin V mutants. Each mutant protein contains the indicated N-terminal extension (underlined) followed by amino acids 2-320 of human annexin V (25). The initiator methionine is not shown because it is removed posttranslationally. The calculated molecular mass (Mr) and calculated isoelectric point (pI) are listed to the right. For convenience, the mutant molecules are given arbitrary numerical designations.

Figure 2. SDS-PAGE analysis of mutant proteins. Recombinant wild-type annexin V (wt) and the two mutant annexin V molecules (122 and 123) were analyzed on a nonreducing 1015% gradient gel and stained with Coomassie Brilliant Blue. Molecular mass standards (with Mr in parentheses) were phosphorylase b (94 000), albumin (67 000), ovalbumin (43 000), carbonic anhydrase (30 000), trypsin inhibitor (20 100), and R-lactalbumin (14 400). Table 1. Membrane Binding Activity of Mutant Proteinsa

a

protein

IC50 (nmol/L)

annexin V-wt annexin V-122 annexin V-123

9(4 7(1 6(2

Results are given as mean ( SD (n ) 3).

duplicate tubes containing a final volume of 1 mL of buffer HNKGB plus 2.5 mmol/L CaCl2. One tube then received 4.2 × 108 erythrocytes in 100 µL; the other tube received an equal volume of buffer. After a 15 min incubation at room temperature, both tubes were centrifuged for 3 min at 8320g. Radioactivity was measured in 800 µL of the supernatants. The percentage of radioactivity bound to the cells was calculated from 100 × [1 - (supernatant counts in the presence of cells)/(supernatant counts in absence of cells)]. RESULTS

Protein Production and Characterization. Two mutant proteins were designed to have N-terminal sequences potentially suitable for labeling with TcCO (Figure 1). Annexin V-122 has three adjacent His residues, and annexin V-123 has six. The structures of the expression constructs were verified by DNA sequencing. Both proteins could be expressed cytoplasmically in E. coli and purified in reasonable yield (33 mg of pure protein per liter of bacterial culture) under conditions similar to those used to produce wild-type and mutant annexin V (14, 23, 26). SDS-PAGE analysis (Figure 2) showed that both mutants could be produced in highly pure form. As expected, both mutant proteins (323 and 326 amino acids in length) also migrated with the same apparent molecular weight as wild-type annexin V (319 amino acids). Isoelectric focusing showed that the iso-

protein

labeling yield (%)

radiochemical purity (%)

radioactivity bound to erythrocytes (%)

annexin V-wt annexin V-122 annexin V-123

43.0 ( 3.9 60.6 ( 6.4 80.6 ( 0.6

91.2 ( 1.7 96.7 ( 0.9 98.4 ( 0.5

78.3 80.7 ( 0.9 85.0 ( 2.8

a Labeling was performed with 100 µg of protein and 1 mCi of TcCO for 60 min. Results are given as mean ( SD for two experiments.

Figure 3. Time course of radiolabeling. Protein (100 µg) was reacted with 99mTcCO (1 mCi) at 37 °C, and ITLC was performed on aliquots taken at the indicated times. Results are mean ( SD for two experiments.

electric points of the mutant proteins were consistent within 0.1 pH unit with the calculated values given in Figure 1. Gel filtration analysis (described further below) indicated that both proteins were pure and migrated as monomers. Mass spectrometry showed a single major peak at 36 287 (calculated MW 36 288) for annexin V-122, and a single major peak at 36 696 (calculated MW 36 700) for annexin V-123. These results indicate that the initiator methionine was removed from both proteins, and that there were no posttranslational modifications such as N-terminal acetylation. The bioactivity of the mutant proteins was verified by measuring their ability to compete with wild-type annexin V for binding to erythrocyte membranes with exposed PS (Table 1). Both proteins displaced fluorescein-annexin V with the same relative affinity as wild-type annexin V, indicating that attachment of the N-terminal peptide did not interfere with membrane binding. Radiolabeling with 99mTc. Wild-type and mutant proteins were radiolabeled with TcCO (Table 2). There was some labeling of the wild-type protein, presumably due to its three endogenous histidine residues, but the two mutant proteins incorporated substantially more radioactivity. The radiolabel remained attached to protein after separation from labeling reagents, as indicated by the high radiochemical purity of gel-filtered proteins. To verify that the radiolabeled proteins retained bioactivity, radiolabeled proteins were incubated with erythrocytes in the presence of calcium, and the percentage of radioactivity bound to cells was measured. All proteins showed comparable levels of binding, and this level was comparable to the binding of 99mTc-HYNIC-annexin V and 99m Tc-annexin V-117 previously tested under the same conditions (14). (Even with fully bioactive protein, binding never exceeds about 85% under these assay conditions because of the finite amounts of cells and protein that can be added to the assay.) The time course of labeling was fairly rapid, with most of the Tc being incorporated

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Tait et al.

Figure 4. Radiolabeling as a function of technetium/protein ratio. Protein (10-100 µg) was added to TcCO (1 mCi). After 60 min at 37 °C, ITLC was performed on the reaction mixture to determine percent incorporation of technetium into protein. Results are mean ( SD for two experiments. Table 3. Stability of Radiolabeled Proteins in Vitroa protein

% protein-bound 99mTc in PBSb

% protein-bound 99mTc in serumc

annexin V-122 at 1 h annexin V-122 at 4 h annexin V-123 at 1 h annexin V-123 at 4 h

93.9 ( 0.8 90.1 ( 1.5 96.8 ( 1.4 95.3 ( 2.5

98.4 ( 0.1 98.3 ( 0.6 99.1 ( 0.6 98.8 ( 0.4

Results are given as mean ( SD for two experiments. b Determined by ITLC. c Determined by centrifugal ultrafiltration. a

by 60 min (Figure 3). To determine if proteins could be labeled to higher specific activity, the technetium/protein ratio was varied. As shown in Figure 4, the specific activity could be increased by increasing the 99mTcCO/ protein ratio, although at a cost of lower percent incorporation; specific activities of ∼10-20 µCi/µg were readily obtainable for both mutant proteins. Stability of Radiolabeled Proteins in Vitro. The stability of the radiolabeled protein in vitro was determined after challenge with phosphate-buffered saline and serum. ITLC or ultrafiltration analysis showed that the mutant proteins retained the radiolabel over a period of several hours, indicating that the Tc-protein chelate was of high affinity (Table 3). These results were confirmed by gel filtration chromatography (Figure 5). After incubation of 99mTc-annexin V-122 or -123 with PBS for 2 h, almost all of the radioactivity eluted in the same position as monomeric annexin V (Mr 36 000); there was no evidence for either multimeric protein aggregates or large-scale release of free Tc. Similarly, gel filtration chromatography of 99mTc-annexin V-122 or -123 after a 2 h incubation with human serum showed that the radioactivity still eluted in the same position as monomeric annexin V (Figure 5). Thus, there was no evidence for either degradation or transchelation of 99mTc to other serum proteins over a time period consistent with the normal blood clearance time of annexin V. DISCUSSION

We have developed and evaluated two mutant forms of annexin V that contain clusters of histidine residues suitable for labeling with TcCO reagent. Both proteins can be produced in good yield and high purity by recombinant-DNA methods. These mutant molecules retain bioactivity both before and after labeling with 99mTc, as measured by their ability to bind with high affinity to cell membranes with exposed PS. Labeling of these molecules with 99mTcCO is simple, and high specific

Figure 5. Gel filtration analysis. Labeled protein (50 µg in 300 µL) was mixed with either 300 µL of human serum (circles) or 300 µL of PBS (squares with x), incubated for 2 h at room temperature, and then analyzed on a Superose-12 column as described under Materials and Methods. An aliquot of unlabeled protein (500 µg; open squares) was chromatographed separately to determine the purity and elution position of unlabeled protein. Panel A: 99mTc-annexin V-122. Panel B: 99mTc-annexin V-123.

activities can be achieved. The mutant annexins show good radiolabel stability in vitro in PBS and serum, and there is no evidence for transchelation of 99mTc to other serum proteins during in vitro incubation (Figure 5). Since annexin V does not bind to normal erythrocytes, leukocytes, or platelets in whole blood (27), these results imply that TcCO-labeled annexin V will be confined to the plasma fraction of blood in vivo, and it will be stable in this compartment until cleared into other body compartments. Although both proteins are suitable for further development as potential in vivo imaging agents, annexin V-123 may be better than annexin V-122 since it labels to higher specific activity and has slightly higher in vitro radiostability. Presumably, the annexin V-123 mutant labels better than the V-122 mutant because it has six rather than three adjacent histidine residues. Wild-type annexin V labeled less well than either mutant protein, and would thus be the least desirable molecule for further development. As mentioned in the introduction, there are now several methods available for technetium labeling of annexin V. Each of these methods has certain advantages and disadvantages. Recombinant proteins with endogenous labeling sites are much simpler to produce, since they can be used directly for 99mTc labeling without the need for attachment of organic Tc chelation groups either during production or at the point of use. The annexin V mutants are also chemically homogeneous, in contrast to the heterogeneous labeling that usually occurs with amine-directed bifunctional organic labeling reagents used on proteins with multiple lysine residues. On the other hand, methods involving derivatization with chemical groups such as N2S2 or HYNIC have longer track records to date, which may simplify clinical testing and regulatory approval. The possible immunogenicity of both

Annexin V Mutants Labeled with Tc-Carbonyl

chemically modified proteins and recombinant proteins is unknown. The HYNIC-annexin V and annexin V-117 derivatives are the simplest to use in the clinical setting, since both can be labeled with short one-step reactions performed at room temperature or 37 °C; in contrast, the TcCO labeling is a two-step procedure in which the TcCO compound must be first formed in a separate reaction at high temperature, while the N2S2 method is a rather complex multistep procedure. In conclusion, we have shown the feasibility of labeling annexin V with the TcCO reagent, and the novel mutant molecules reported here are promising for future imaging studies of apoptosis, cancer chemotherapy, and myocardial infarction. ACKNOWLEDGMENT

We thank Alex Pagon for excellent assistance in initial efforts to construct expression vectors; Ross Lawrence of the Department of Medicinal Chemistry for performing mass spectrometry measurements; and Dr. J.-L. Vanderheyden for helpful advice on preparation of the TcCO reagent. This work was supported by a grant from Theseus Medical Imaging, Inc., and by NIH Grant HL61717. LITERATURE CITED (1) Thiagarajan, P., and Tait, J. F. (1990) Binding of annexin V/placental anticoagulant protein I to platelets. Evidence for phosphatidylserine exposure in the procoagulant response of activated platelets. J. Biol. Chem. 265, 17420-17423. (2) Zwaal, R. F., and Schroit, A. J. (1997) Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. Blood 89, 1121-1132. (3) Bevers, E. M., Comfurius, P., Dekkers, D. W., and Zwaal, R. F. (1999) Lipid translocation across the plasma membrane of mammalian cells. Biochim. Biophys. Acta 1439, 317-330. (4) Tait, J. F., Cerqueira, M. D., Dewhurst, T. A., Fujikawa, K., Ritchie, J. L., and Stratton, J. R. (1994) Evaluation of annexin V as a platelet-directed thrombus targeting agent. Thromb. Res. 75, 491-501. (5) Stratton, J. R., Dewhurst, T. A., Kasina, S., Reno, J. M., Cerqueira, M. D., Baskin, D. G., and Tait, J. F. (1995) Selective uptake of radiolabeled annexin V on acute porcine left atrial thrombi. Circulation 92, 3113-3121. (6) Blankenberg, F. G., Katsikis, P. D., Tait, J. F., Davis, R. E., Naumovski, L., Ohtsuki, K., Kopiwoda, S., Abrams, M. J., Darkes, M., Robbins, R. C., Maecker, H. T., and Strauss, H. W. (1998) In vivo detection and imaging of phosphatidylserine expression during programmed cell death. Proc. Natl. Acad. Sci. U.S.A. 95, 6349-6354. (7) Blankenberg, F. G., Katsikis, P. D., Tait, J. F., Davis, R. E., Naumovski, L., Ohtsuki, K., Kopiwoda, S., Abrams, M. J., and Strauss, H. W. (1999) Imaging of apoptosis (programmed cell death) with 99mTc annexin V. J. Nucl. Med. 40, 184-191. (8) Vriens, P. W., Blankenberg, F. G., Stoot, J. H., Ohtsuki, K., Berry, G. J., Tait, J. F., Strauss, H. W., and Robbins, R. C. (1998) The use of technetium Tc 99m annexin V for in vivo imaging of apoptosis during cardiac allograft rejection. J. Thorac. Cardiovasc. Surg. 116, 844-853. (9) Blankenberg, F. G., Robbins, R. C., Stoot, J. H., Vriens, P. W., Berry, G. J., Tait, J. F., and Strauss, H. W. (2000) Radionuclide imaging of acute lung transplant rejection with annexin V. Chest 117, 834-840. (10) Ogura, Y., Krams, S. M., Martinez, O. M., Kopiwoda, S., Higgins, J. P., Esquivel, C. O., Strauss, H. W., Tait, J. F., and Blankenberg, F. G. (2000) Radiolabeled annexin V imaging: diagnosis of allograft rejection in an experimental rodent model of liver transplantation. Radiology 214, 795800. (11) Narula, J., Acio, E. R., Narula, N., Samuels, L. E., Fyfe, B., Wood, D., Fitzpatrick, J. M., Raghunath, P. N., Tomaszewski, J. E., Kelly, C., Steinmetz, N., Green, A., Tait, J. F.,

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