Inhibition of platelet function by recombinant soluble ecto-ADPase/CD39.

Excessive platelet accumulation and recruitment, leading to vessel occlusion at sites of vascular injury, present major therapeutic challenges in cardiovascular medicine. Endothelial cell CD39, an ecto-enzyme with ADPase and ATPase activities, rapidly metabolizes ATP and ADP released from activated platelets, thereby abolishing recruitment. Therefore, a soluble form of CD39, retaining nucleotidase activities, would constitute a novel antithrombotic agent. We designed a recombinant, soluble form of human CD39, and isolated it from conditioned media from transiently transfected COS-1 cells and from stably transfected Chinese hamster ovary (CHO) cells. Conditioned medium from CHO cells grown under serum-free conditions was subjected to anti-CD39 immunoaffinity column chromatography, yielding a single approximately 66-kD protein with ATPase and ADPase activities. Purified soluble CD39 blocked ADP-induced platelet aggregation in vitro, and inhibited collagen-induced platelet reactivity. Kinetic analyses indicated that, while soluble CD39 had a Km for ADP of 5.9 microM and for ATP of 2.1 microM, the specificity constant kcat/Km was the same for both substrates. Intravenously administered soluble CD39 remained active in mice for an extended period of time, with an elimination phase half-life of almost 2 d. The data indicate that soluble CD39 is a potential therapeutic agent for inhibition of platelet-mediated thrombotic diatheses.

The endothelial cell ecto-ADPase responsible for inhibition of platelet function is CD39.

We previously demonstrated that when platelets are in motion and in proximity to endothelial cells, they become unresponsive to agonists (Marcus, A.J., L.B. Safier, K.A. Hajjar, H.L. Ullman, N. Islam, M.J. Broekman, and A.M. Eiroa. 1991. J. Clin. Invest. 88:1690-1696). This inhibition is due to an ecto-ADPase on the surface of endothelial cells which metabolizes ADP released from activated platelets, resulting in blockade of the aggregation response. Human umbilical vein endothelial cells (HUVEC) ADPase was biochemically classified as an E-type ATP-diphosphohydrolase. The endothelial ecto-ADPase is herein identified as CD39, a molecule originally characterized as a lymphoid surface antigen. All HUVEC ecto-ADPase activity was immunoprecipitated by monoclonal antibodies to CD39. Surface localization of HUVEC CD39 was established by confocal microscopy and flow cytometric analyses. Transfection of COS cells with human CD39 resulted in both ecto-ADPase activity as well as surface expression of CD39. PCR analyses of cDNA obtained from HUVEC mRNA and recombinant human CD39 revealed products of the same size, and of identical sequence. Northern blot analyses demonstrated that HUVEC express the same sized transcripts for CD39 as MP-1 cells (from which CD39 was originally cloned). We established the role of CD39 as a prime endothelial thromboregulator by demonstrating that CD39-transfected COS cells acquired the ability to inhibit ADP-induced aggregation in platelet-rich plasma. The identification of HUVEC ADPase/CD39 as a constitutively expressed potent inhibitor of platelet reactivity offers new prospects for antithrombotic therapeusis.

Ligands for the receptor tyrosine kinases hek and elk: isolation of cDNAs encoding a family of proteins.

Hek is a member of the eph subfamily of receptor tyrosine kinases whose members include elk, hek2, sek, eph and eck among others. Using a soluble form of hek consisting of the extracellular region of the receptor fused to the Fc domain of human IgG1 and an expression cloning strategy, we have isolated two different but related cDNAs from the human T-lymphoma line HSB-2 that encode ligands for hek. The cDNAs encode proteins of 238 and 201 amino acids (44% amino acid identity) that are anchored to the membrane by glycosylphosphatidylinositol (GPI)-linkage. The proteins encoded by these cDNAs are bound by hek with affinity constants of 2 x 10(8) M-1. These proteins also bind the elk tyrosine kinase receptor. These cDNAs are related to other cDNAs that we have recently isolated from a human placental library that encode ligands for both hek and elk and define an emerging family of ligands for eph-related kinases (LERKs).

Induction of recombinant gene expression in Escherichia coli using an alkaline pH shift.

A commonly used approach to control recombinant protein production in Escherichia coli utilizes the lambda pL promoter-operator and the lambda repressor. Inactivation of the lambda repressor function allows transcription to proceed. However, induction of the RecA-mediated cleavage of lambda repressor by the addition of nalidixic acid or inactivation of a temperature-sensitive lambda repressor by growth at the nonpermissive temperature can have detrimental effects on protein production. This paper describes the use of an alkaline shift in the pH of the growth medium that allows expression of genes from the pL promoter in a RecA-independent manner. This procedure results in high-level production of recombinant protein. The pH shift is performed in the stationary phase of cell growth, using culture volumes ranging from 1-1000 ml. This method can result in the production of over 15-fold more active protein than when using a temperature shift.

Construction and characterization of pBR322-derived plasmids with deletions of the RNA I region.

A region upstream from the origin of replication in ColE1-type plasmids has been shown to be necessary for replication. Two RNA transcripts are produced from this area, RNA II, which yields the primer for DNA polymerase initiation at the origin and RNA I, which is complementary to the 5' end of RNA II and acts to inhibit primer formation. We have constructed plasmids which do not possess the nucleotide sequence for RNA I, or the normal 5' terminus and promoter of RNA II. The RNA II analog, in these plasmids, is believed to be synthesized by the readthrough transcription of the upstream trimethoprim-resistant dihydrofolate reductase (DHFR) gene at a level comparable to that produced by the tryptophan promoter. These plasmids have a copy number of about tenfold higher than that of pBR322 during logarithmic growth and are compatible with other ColE1-type plasmids. These plasmids are stably maintained in several strains when selective pressure is present and the plasmids are stably maintained during exponential growth in W3110 strains without selective pressure. In all strains examined, the dimeric form of the plasmid was lost from cells much more rapidly than those containing the monomeric form.

Formation of MboII vectors and cassettes using asymmetric MboII linkers.

Class-IIS restriction endonucleases such as MboII cleave DNA at a specified distance away from their recognition sequences. This feature was exploited to cleave DNA at previously inaccessible locations by preparing special asymmetric linker/adapters containing the MboII recognition sequence. These could be joined to DNA fragments and subsequently cleaved by MboII. Attachment of a 3' phosphate to one of the two different oligodeoxynucleotides comprising the asymmetric duplex prevented ligation at the improper end of the linker. Plasmids were constructed containing a unique BamHI or BclI site between the recognition and cleavage site of MboII. These sites were used to introduce a foreign fragment into the plasmid at a position permitting MboII to cleave within the newly inserted fragment. Once cleaved at the unique MboII site, another DNA fragment was inserted. DNA was thus inserted at a sequence not previously accessible to specific cleavage by a restriction enzyme. A cassette containing an identifiable marker, the lac operator, between two oppositely oriented MboII/BamHI linkers was made and tested in a random insertion linker mutagenesis experiment.

Construction of interleukin-1 alpha mutants using unequal contamination of synthetic oligonucleotides.

Proteins without readily available three-dimensional structural data present a difficult problem in the exploration of structure/function relationships. Saturation mutagenesis using contaminated oligonucleotides can identify potentially interesting regions of such a protein. This technique, in which synthesized oligonucleotides contain low-level base substitutions, allows random mutations to be placed throughout a gene sequence. Using double-stranded cassettes, a region of the human interleukin-1 alpha gene has been altered using such mutagenic oligonucleotides. However, instead of contaminating both strands of the gene sequence at the same level, each strand of the insert was contaminated at a different level. Several recombinants were sequenced and the effects of the mutations on the activity of the proteins were examined. Contaminating the two oligonucleotides at different levels produced a significantly different distribution of nucleotide changes from that seen if both strands were contaminated at the same level. The observed distribution followed the average of the distributions for each of the two contamination levels. This resulted in roughly equal frequencies of 1 to 5 nucleotide changes per clone with very few clones containing the wild-type nucleotide sequence. This helped overcome the redundancy in the genetic code, resulting in a high frequency of amino acid changes, and allowed changes at every amino acid to be sampled in a small number of mutants. This procedure can allow a gene sequence to be screened rapidly by removing most wild-type sequences from analysis while making sure that there are many amino acid changes in the resultant mutants.

Site-directed mutagenesis of human endothelial cell ecto-ADPase/soluble CD39: requirement of glutamate 174 and serine 218 for enzyme activity and inhibition of platelet recruitment.

Endothelial cell CD39/ecto-ADPase plays a major role in vascular homeostasis. It rapidly metabolizes ADP released from stimulated platelets, thereby preventing further platelet activation and recruitment. We recently developed a recombinant, soluble form of human CD39, solCD39, with enzymatic and biological properties identical to CD39. To identify amino acids essential for enzymatic/biological activity, we performed site-directed mutagenesis within the four highly conserved apyrase regions of solCD39. Mutation of glutamate 174 to alanine (E174A) and serine 218 to alanine (S218A) resulted in complete and approximately 90% loss of solCD39 enzymatic activity, respectively. Furthermore, compared to wild-type, S57A exhibited a 2-fold increase in ADPase activity without change in ATPase activity, while the tyrosine 127 to alanine (Y127A) mutant lost 50-60% of both ADPase and ATPase activity. The ADPase activity of wild-type solCD39 and each mutant, except for R135A, was greater with calcium as the required divalent cation than with magnesium, but for ATPase activity generally no such preference was observed. Y127A demonstrated the highest calcium/magnesium ADPase activity ratio, 2.8-fold higher than that of wild-type, even though its enzyme activity was greatly reduced. SolCD39 mutants were further characterized by correlating enzymatic with biological activity in an in vitro platelet aggregation system. Each solCD39 mutant was similar to wild-type in reversing platelet aggregation, except for E174A and S218A. E174A, completely devoid of enzymatic activity, failed to inhibit platelet responsiveness, as anticipated. S218A, with 91% loss of ADPase activity, could still reverse platelet aggregation, albeit much less effectively than wild-type solCD39. Thus, glutamate 174 and serine 218 are essential for both the enzymatic and biological activity of solCD39.

Role of the Cro repressor carboxy-terminal domain and flexible dimer linkage in operator and nonspecific DNA binding.

A series of mutations comprising single and multiple substitutions, deletions, and extensions within the carboxy-terminal domain of the bacteriophage lambda Cro repressor have been constructed. These mutations generally affect the affinity of repressor for specific and nonspecific DNA. Additionally, substitution of the carboxy-terminal alanine with several amino acids capable of hydrogen-bonding interactions leads to improved specific binding affinities. A mutation is also described whereby cysteine links the two Cro monomers by a disulfide bond. As a consequence, a significant improvement in nonspecific binding and a concomitant reduction in specific binding are observed with this mutant. These results provide evidence that the carboxy terminus of Cro repressor is an important DNA binding domain and that a flexible connection between the two repressor monomers is a critical factor in modulating the affinity of wild-type repressor for DNA.

Identification of regions in interleukin-1 alpha important for activity.

Saturation mutagenesis of the mature human interleukin-1 alpha (IL-1 alpha) gene has been performed. Following expression in Escherichia coli, the biological and receptor binding activities of the mutant proteins were examined. Most of the molecule could be altered with little effect on either function. More than 3,500 mutants were examined, and only 23 unique amino acid sequences were identified which resulted in an altered ratio of biological to binding activity when compared with wild-type IL-1 alpha. These proteins possessed mutations at 38 of the 159 amino acid residues in IL-1 alpha. Random mutagenesis at several of these positions identified further substitutions that affected activity. Examination of a model for IL-1 alpha localized most of the residues which altered activity along one face of the molecule. This region appears to be distinct from areas of IL-1 which have been postulated to make contact with IL-1 receptor.

Importance of the amino terminus of the interleukin-8 receptor in ligand interactions.

Interleukin-8 (IL-8) and growth regulatory gene/melanoma growth stimulatory activity (GRO/MGSA) are small polypeptide molecules involved in the chemotactic response of certain cell types. Two receptors have been described which interact with IL-8, designated type 1 and type 2. IL-8 binds with high affinity to both receptors, whereas GRO/MGSA and neutrophil-activating peptide-2 demonstrate a high degree of binding only to the type 2 receptor. The two forms of IL-8 receptor are members of the rhodopsin seven-helix membrane-spanning superfamily, and share a high degree of overall homology, although the amino termini are very divergent. By using conserved restriction enzyme sites, a series of chimeric IL-8 receptor molecules were constructed between the type 1 and type 2 receptors and transfected into human 293 kidney epithelial cells. These chimeric molecules altered regions of the receptor presented to the ligand. The ability of the chimeric receptors to bind IL-8 was determined, as well as the ability of IL-8 and GRO/MGSA to inhibit radiolabeled IL-8 binding. The amino terminus of the IL-8 receptors was found to be important for differential binding of GRO/MGSA and IL-8. In addition, a series of peptides was also constructed to further investigate which residues of IL-8 receptor interact with IL-8. These peptides also identified the amino-terminal sequence of the IL-8 receptors as being important in interacting with IL-8.

Molecular characterization of murine and human OX40/OX40 ligand systems: identification of a human OX40 ligand as the HTLV-1-regulated protein gp34.

A ligand was cloned for murine OX40, a member of the TNF receptor family, using a T cell lymphoma cDNA library. The ligand (muOX40L) is a type II membrane protein with significant identity to human gp34 (gp34), a protein whose expression on HTLV-1-infected human leukemic T cells is regulated by the tax gene. The predicted structures of muOX40L and gp34 are similar to, but more compact than, those of other ligands of the TNF family. Mapping of the muOX40L gene revealed tight linkage to gld, the FasL gene, on chromosome 1. gp34 maps to a homologous region in the human genome, 1q25. cDNAs for human OX40 receptor were cloned by cross-hybridization with muOX40, and gp34 was found to bind the expressed human receptor. Lymphoid expression of muOX40L was detected on activated T cells, with higher levels found on CD4+ rather than CD8+ cells. The cell-bound recombinant ligands are biologically active, co-stimulating T cell proliferation and cytokine production. Strong induction of IL-4 secretion by muOX40L suggests that this ligand may play a role in regulating immune responses. In addition, the HTLV-1 regulation of gp34 suggests a possible connection between virally induced pathogenesis and the OX40 system.