P-SELECTIN LIGANDS AND RELATED MOLECULES AND METHODS

Abstract: Disclosed herein are organic molecules to which are covalently bonded a sialyl-Le<x> determinant and a sulfated determinant, at least one of these determinants being positioned at a non-naturally occurring site on the molecule. Also disclosed are particular P-selectin ligands and P-selectin ligand-antibody fusions. These moecules, ligands, and fusion proteins find use in methods of reducing or protecting against inflammation and extravasation-dependent adverse reactions, such as organ damage and clotting (for example, associated with adult respiratory distress syndrome or ischemic myocardial injury).

Description: P-SELECTIN LIGANDS AND RELATED MOLECULES AND METHODS Statement as to Federally Sponsored Research This invention vas made with Government support under NIH grant DK43031, and the Government therefore has certain rights in this invention. Background of the Invention This invention relates to P-selectin ligand molecules, DNAs, and uses thereof. P-selectin is an integral membrane C-type lectin found within the eibel-Palade bodies of endothelial cells and the alpha granules of platelets (McEver et al., J. Clin. Invest., 84:92-99, 1989; Bonfanti et al., Blood, 73:1109-1112, 1989; Hsu-Lin et al., J. Biol. Chem., 259:9121-9126, 1984; Stenberg et al., J. Cell Biol., 101:880-886, 1985). Its translocation to the plasma membrane can be induced by thrombin, histamine and other mediators released by mast cell activation, complement C5b-9 complex or C5a fragment, peroxides, and oxidized low-density lipoprotein (Hsu-Lin et al., J. Biol. Chem., 259:9121-9126, 1984; Stenberg et al., J. Cell Biol., 101:880-886, 1985; Hattori et al., J. Biol. Chem., 264:9053-9060, 1989; Kubes and Kanwar, J. Immunol., 152:3570-2577, 1994; Thorlacius et al., Biochem. Biophys. Res. Communications, 203:1043-1049, 1994; Foreman et al., J. Clin. Invest., 94:1147-1155, 1994; Patel et al., J. Cell Biol., 112:749-759, 1991; Lehr et al.. Laboratory Invest., 71:380-386, 1994; Gebuhrer et al., Biochem. J., 306:293-298, 1995). Once displayed on the cell surface, P-selectin supports the attachment of myelomonocytes to platelets or endothelial cells (Larsen et al., Cell, 59:305-312, 1989; Hamburger and McEver, Blood, 75:550-554 1990; Geng et al., Nature, 343:757-760, 1990; Gamble et al., Science, 249:414-417, 1990) . In the latter setting, its appearance heralds an underlying tissue insult and supports the initial step in leukocyte extravasation, the rolling of neutrophils along the postcapillary venule wall (Lawrence and Springer, Cell, 65:859-873, 1991). Mice which are homozygously deficient for the P-selectin structural gene exhibit decreased leukocyte rolling and show delayed recruitment of granulocytes to sites of experimentally induced inflammation (Mayadas et al., Cell, 74:541-554, 1993). Generally, the mediators which induce P-selectin expression are involved in signaling trauma or wounding. One of the first recognized responses to tissue trauma is mast cell activation, which is accompanied by release of histamine, serotonin, and other diffusible mediators. Other common events include thrombus formation at sites of vascular rupture and complement alternative pathway engagement by foreign bodies. P-selectin expression is induced by signals generated in each of these contexts. Although induction of P-selectin mediated neutrophil rolling has been thought to be an inevitable consequence of surgical intervention, cromolyn, an agent which blocks mast cell degranulation, has been shown to prevent such rolling, thereby providing an elegant demonstration of the role of the mast cell as the link between trauma and extravasation (Kubes and Kanwar, J. Immunol., 152:3570- 3577, 1994). Summary of the Invention In a first aspect, the invention features an organic molecule to which there is covalently bonded a sialyl-Le x determinant and a sulfated determinant, at least one of these determinants being positioned at a non-naturally occurring site on the molecule. In a second aspect, the invention features a P- selectin ligand selected from the group consisting essentially of: (a) amino acids 21-57 of Fig. 8A and (b) amino acids 38-57 of Fig. 8A. In a third aspect, the invention features fusion proteins that include a P-selectin ligand joined to an antibody domain (for example, one or more of the hinge, CH2, and CH3 domains). In related aspects, the invention features purified nucleic acid encoding a protein containing sites for the attachment of a sialyl-Le x determinant and a sulfated determinant, at least one of these determinants being positioned at a non-naturally occurring site on the protein; purified nucleic acid encoding any one of the P- selectin ligands of the invention; purified nucleic acid encoding a P-selectin-antibody fusion protein; and vectors and recombinant cells including any of these nucleic acids. Also included in the invention is the use of P-selectin ligands or organic molecules bearing such ligands (if desired, in combination with other proteins such as antibody or α 1 -acid glycoprotein domains) in the manufacture of a medicament for the treatment of any of the conditions described below. In another related aspect, the invention features a method of inhibiting the binding of a cell bearing a P- selectin protein to a molecule or cell bearing a sialyl- Le x determinant and a sulfated determinant. The method involves contacting the P-selectin protein-bearing cell with either an organic molecule bearing sialyl-Le x and sulfated determinants, at least one of these determinants being positioned at a non-naturally occurring site on the molecule; a P-selectin-antibody fusion protein; or any of the P-selectin ligands of the invention. In another related aspect, the invention features a method of reducing inflammation in a mammal involving administering to the patient a therapeutically-effective amount of either an organic molecule bearing sialyl-Le x and sulfated determinants, at least one of these determinants being positioned at a non-naturally occurring site on the molecule; a P-selectin-antibody fusion protein; or any one of the P-selectin ligands of the invention. In yet another related aspect, the invention features a method of reducing or protecting a mammal against any extravasation-dependent adverse reaction (including, without limitation, extravasation-dependent organ damage and/or clotting associated with adult respiratory distress syndrome, glomerular nephritis, and ischemic myocardial injury) . The method involves administering to the mammal a therapeutically-effective amount of either an organic molecule to which there is covalently bonded a sialyl-Le x and a sulfated determinant, at least one of these determinants being positioned at a non-naturally occurring site on the molecule; a P-selectin-antibody fusion protein; or any of the P-selectin ligands of the invention. In a final aspect, the invention features a method of reducing or protecting a mammal against an adverse immune reaction, involving administering to the mammal a therapeutically-effective amount of either an organic molecule to which there is covalently bonded a sialyl-Le x and a sulfated determinant, at least one of these determinants being positioned at a non-naturally occurring site on the molecule; a P-selectin-antibody fusion protein; or any of the P-selectin ligands of the invention. Preferably, this method involves treating the mammal for an adverse immune reaction w ich is induced by a microbial factor. Such microbial factors include, without limitation, gram-negative bacteria lipopolysaccharides (LPS) , peptidoglycans from gram- positive organisms, mannan from fungal cell walls, polysaccharides, extracellular enzymes (e.g., streptokinase) and toxins (e.g., toxic shock enterotoxins of staphylococci) . In other preferred embodiments, the method involves treating a mammal for any adverse immune reaction which is induced by a host factor. Such host factors include, without limitation, metabolites of complement, kinin, and coagulation systems, factors released from stimulated cells (e.g., cytokines such as interleukin 1 (IL-1) and tumor necrosis factor-o (TNF)) , enzymes and oxidants from polymorphonuclear leukocytes (PMNs), vasopeptides (e.g., histamine), and products of the metabolism of arachidonic acid. In other preferred embodiments, the adverse immune reaction is induced by recombinant TNF-α or is induced by recombinant IL-1. In yet other preferred embodiments, the adverse immune reaction is septic shock or is septicemia. In preferred embodiments of each of the above aspects, the organic molecule or protein also inhibits the binding of a cell bearing an E-selectin (ELAM-1) protein to a molecule or cell bearing a sialyl-Le x determinant and thus inhibits E-selectin-mediated inflammation, extravasation-dependent adverse reactions, and adverse immune reactions; the sialyl-Le and sulfated determinants are present on a P-selectin ligand consisting essentially of: amino acids 21-57 of Fig. 8A (for example, amino acids 38-57 of Fig. 8A) ; the sialyl- Le x determinant is N-linked or o-linked; the molecule or protein contains multiple sialyl-Le x and/or multiple sulfated determinants; the organic molecule is a protein (for example, an antibody (for example, IgG or IgM) , a λ - acid glycoprotein (AGP) , or an antibody fusion protein (for example, an AGP-antibody fusion protein) ; the protein is an antibody, AGP, or an antibody fusion protein (for example, an AGP-antibody fusion protein) to which any of the P-selectin ligands described herein is appended (for example, at the protein's amino-terminus) ; the antibody or antibody fusion protein (for example, the AGP-antibody fusion protein) includes, as an antibody portion, an IgGl CH2, CH3, and/or hinge domain; the antibody, AGP, or antibody fusion protein includes one or more of the N-linked glycan addition sites of o^-acid glycoprotein; the antibody portion of the molecule bears one or more non-naturally occurring sialyl-Le x determinants; the sialyl-Le x determinant interferes with the antibody's ability to fix complement or bind an F c receptor (for example, due to a sialyl-Le x determinant attached to one or more of amino acids 274, 287, or 322 of the sequence shown in Fig. 10) ; and the organic molecule is soluble. By a P-selectin ligand", as used herein, is meant any amino acid sequence capable of mediating an interaction with the P-selectin receptor and includes those proteins referred to as P-selectin counter- receptors. Preferable P-selectin ligands include, without limitation, amino acids 21-57, and more preferably amino acids 38-57, of Fig. 8A. P-selectin ligands according to the invention may be used in conjunction with additional protein domains (for example, antibody domains) to produce fusion proteins useful in the invention. By "non-naturally occurring" is meant a sialyl-Le x or sulfated determinant that is not one which is naturally bound to the molecule at that amino acid location. By "inflammation" is meant a pathologic process consisting of cytologic and histologic reactions that occur in the affected blood vessels and adjacent tissues in response to an injury or abnormal stimulation caused by a physical, chemical, or biologic agent. Inflammation, as used herein, includes any acute inflammatory response (for example, during or following adult respiratory distress syndrome or ischemic myocardial injury) as well as any chronic inflammatory response (for example, rheumatoid arthritis, psoriasis, or pemphigus vulgaris) . By "purified nucleic acid" is meant DNA that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence. By "N-linked" is meant bonded to the amide nitrogen of an asparagine residue of a protein. By "O-linked" is meant bonded to the hydroxy1- group oxygen of a serine, threonine, or hydroxylysine residue of a protein. By an "extravasation-dependent adverse reaction" is meant any reaction which is detrimental to the host and which results directly or indirectly from the inappropriate attachment of neutrophils to endothelium at or proximate to a site of inflammation, tissue damage, or thrombus formation and results in migration of those neutrophils into the attached blood vessel or organ. Organs which may be affected by such damage include, without limitation, the heart, lungs, and kidneys. By an "adverse immune reaction" is meant any reaction mediated by an immune cell (i.e., any B cell, T cell, monocyte/macrophage, natural killer cell, mast cell, basophil, or granulocyte) and which is detrimental to the host. Detailed Description The drawings will first be briefly described. Fig. IA is a schematic representation of the structure of the PSGL-1 deletion mutants. Systematic deletion of the ectodomain of PSGL-1 was accomplished with conventional PCR methods. A representative 10-residue repeat (stippled; SEQ ID NO:l) and the transmembrane domain (hatched) are illustrated. Fig. IB is histogram which represents P-selectin binding activity of transfected COS cells expressing the deletions shown in Fig. IA. 51 Cr-labeled cells were allowed to adhere to soluble P-selectin adsorbed to microtiter wells. The cells were washed, the bound cells were then lysed, and 51 Cr levels were counted. Deletion constructs were introduced into cells either in the absence (bar 2) or presence (remaining 7 bars) of the human FTVII fucosyltransferase. Fig. 2A is a schematic representation of chimeras of PSGL-1 and CD43. The membrane proximal extracellular domain, transmembrane, and intracellular domains of PSGL-1 were replaced with the cognate sequences of CD43. The resulting molecule lacks cysteines and thus cannot form a disulfide linked dimer. Fig. 2B is a histogram representing P-selectin binding activity of transfected COS cells expressing the chimeras shown in Fig. 2A. FTVIIh, cotransfection with the human FTVII fucosyltransferase. Fig. 3A is a schematic representation of chimeric mucins bearing the PSGL-1 apical domain appended to intact or truncated mucin C-termini. The PSGL-1 N-terminus (stippled; SEQ ID NO:l) and the transmembrane (TM) domains (hatched) are illustrated. The sequence of PSGL-1-NH 2 /CD43 "repeats" are represented by SEQ ID NO:2. PSGL-1 was fused to the N-terminus of the predicted mature CD34 and GlyCAM-1 molecules, and to the N-terminus of the repeat region of CD43. Fig. 3B is a histogram representing P-selectin binding activity of transfected COS cells expressing the constructs shown in Fig. 3A. FTVIIh, human FTVII fucosyltransferase. Fig. 4A is a schematic representation of PSGL deletion mutants. The amino terminal domain was appended to PSGL molecules having varying numbers of the repeated element. Fig. 4B is a histogram representing P-selectin binding activity of transfected COS cells expressing the chimeras illustrated in Fig. 4A. Fig. 5 is a photograph of an autoradiogram of mucin:immunoglobulin fusion proteins labeled with 35 S-sul ate and electrophoresed on an 8% denaturing polyacrylamide gel under reducing conditions. Lane A, supernatant of CDM8 transfected cells; Lane B, supernatant of cells transfected with Ig expression vector (no mucin insert) ; Lane C, supernatant of cells expressing PSGL-l:Ig; Lane D, supernatant of cells expressing CD43:Ig; Lane E, supernatant of cells expressing CD34:Ig; and Lane F, supernatant of cells expressing GlyCAM-l:Ig. Fig. 6A and Fig. 6B are histograms representing binding to immobilized P- and E-selectin of COS cells expressing PSGL-1 with or without fucosyltransferase and in the presence or absence of 10 mM NaCl0 3 . Fig. 6A is a histogram representing binding of cells to P-selectin. Fig. 6B is a his ogram representing binding of cells to E-selectin. Fig. 7 is a photograph of an autoradiogram of PSGL-1:immunoglobulin fusion proteins labeled with 35 S-sulfate in the presence or absence of 10 mM NaC10 3 and electrophoresed on an 8% denaturing polyacrylamide gel under reducing conditions. The photograph indicates that chlorate inhibits incorporation of 35 S-sulfate into soluble mucin chimeras. Lane A, supernatant of CDM8 transfected cells in the absence of chlorate; Lane B, supernatant of cells expressing PSGL-l:Ig in the absence of chlorate; Lane C, supernatant of CDM8 in the presence of chlorate; and Lane D, supernatant of cells expressing PSGL-l:Ig in the presence of chlorate. Fig. 8A is a listing of the sequence endpoints of various PSGL-1 deletion mutants (indicated by the arrows) The uppermost sequence is SEQ ID NO:3; the middle sequence is SEQ ID NO:13; the lowermost sequence is SEQ ID NQ:14. Fig. 8B is a histogram representing P-selectin binding activity of transfected COS cells expressing the deletion mutants having the endpoints shown in Fig. 8A. Fig. 9A is a schematic diagram of the constructs employed to measure the effect of appending wildtype and mutant variants of PSGL-1 residues 38-57 to deleted PSGL-1 or CD43. The inserted sequences are shown at bottom left. Fig. 9B is a histogram representing P-selectin binding activity of transfected COS cells expressing the chimeras illustrated in Fig. 9A. Fig. 10 is a listing of the nucleotide sequence (SEQ ID NO:8) encoding IgGl (SEQ ID NO:9) and mutations designed to create N-linked glycan addition sites (SEQ ID NO:12). Fig. IIA is the nucleotide sequence (SEQ ID NO:10) and Fig. IIB is the amino acid sequence (SEQ ID NO:11) of an AGP-IgGl fusion protein. Fig. 12A is a schematic diagram of immunoglobulin fusion proteins consisting of either intact PSGL-1 (SEQ ID NO:4) or 20 residue peptides joined to the hinge, CH2, and CH3 domains of human IgGl. Construct Y/F-hlgG bears SEQ ID NO 5; construct T/AhlgG bears SEQ ID NO:6; construct Y/F-T/A-hlgG bears SEQ ID NO:7. Fig. 12B is a photograph of an 8% polyacrylamide gel used to assess incorporation of [ 35 S]cysteine and methionine by the fusion proteins shown in Fig. 12A following transfection into COS cells. Lane A, supernatant of cells transfected with CDM8 control. Lane B, supernatant of cells transfected with PSGL-1-immunoglobulin fusion protein. Lane C, supernatant of cells transfected with T-hlgG. Lane D, supernatant of cells transfected with Y/F-hlgG. Lane E, supernatant of cells transfected with T/A-hlgG. Lane F, supernatant of cells transfected with Y/F-T/A- hlgG. Fig. 12C is a photograph of an 8% polyacrylamide gel used to assess incorporation of [ 35 S]sulfate by the fusion proteins shown in Fig. 12A following transfection of COS cells. In addition, a control fusion protein bearing no amino-terminal addition was included (Lane B) . Lanes C through G correspond to Lanes B through F in Fig. 12B. Fig. 13 is a bar graph of interacting HL-60 cells per video-captured field. The cells were infused into a parallel plate flow chamber precoated with either P- selectin-immunoglobulin chimera or a CD4-immunglobulin chimera control. The cells were subjected to a shear stress of 0.75 dynes/cm 2 . Each bar represents the average number of cells (±SEM) per field from eight frames taken at 15 second intervals. Cells rolling or flowing appear as streaks on the video image. The bars represent, from left to right: HL-60 cells rolling or flowing over P-selectin-immunoglobin chimera, HL-60 cells pretreated in a sulfate-free medium with 10 mM sodium chlorate, and HL-60 cells flowing over CD4-immunoglobulin chimera. Sialyl-Lewis X (sialyl-Le x ) and sulfated determinants were shown to interact with P-selectin and facilitate binding by the following experiments. These examples are presented to illustr te, not limit, the invention. The methods used in the following experiments will first be described. Production of Soluble P-Selectin P-selectin and E-selectin Ig chimeras were prepared by transient expression in COS cells of an expression plasmid encoding the lectin, EGF-related, and first two short consensus repeat related domains of P-selectin joined to the hinge, CH2, and CH3 domains of human IgGl (Aruffo et al., EMBO J., 6:3313-3316, 1991; alz et al.. Science, 250:1132-1135, 1990). The PSGL-1 cDNA coding sequence was obtained by PCR amplification of an HL-60 cDNA library, and the sequence confirmed by DNA sequencing. The coding segment for the mature extracellular, transmembrane, and intracellular domain was inserted into an expression vector based on CDM8 which lacks the polyoma virus origin of replication and contains the leader sequence for the CD5 antigen positioned just upstream of the coding region for an influenza hemagglutinin (flu) peptide (Field et al., Mol. Cell. Biol. 8:2159-2165, 1988) epitope tag. Construction of PSGL-1 Deletions Amino terminal PSGL-1 deletion constructs were prepared by PCR amplification using primers encoding the desired endpoint of the deletion mutant located downstream of an Xbal site in frame two (encodes Leu Asp) . The resulting sequences encoded a polypeptide in which the residues listed below immediately followed the aspartic acid (D) of the Xba site: A118, A128, A138, A148, A158, A168, G178, A188, A198, A208, A218, A228, A238, A248, A258, and T268 of the PSGL-1 precursor. The PCR fragments were then inserted in the CD5 leader flu tag expression vector used for expression of the intact PSGL-1. The flu tag terminates in an Xbal site in the frame described above. Sequences at the flu tag junction were verified, and expression was confirmed in COS cells by indirect immunofluorescence microscopy and flow cytometry. A series of internal deletions with an EcoRI site at the site of the deletion in frame one (encodes glutamic acid phenylalanine) was also prepared by first creating deletion variants with amino termini (residues immediately following phenylalanine [F] of the EcoRI site corresponding to A118, A128, A138, A148, A158, A168, G178, A188, A198, A208, A218, A228, A238, A248, and A258 of the peptide sequence of the precursor. To each of these deleted variants was appended a flu-tagged amino¬ terminal PSGL-1 domain ending with an EcoRI site in the glutamic acid phenylalanine frame immediately downstream of PSGL-1 precursor A117. The resulting constructs contained deletions between A117 and the various endpoints above. Mucin Domain Interchanges CD34, CD43, and GlyCAM-1 mucins were prepared for addition of the PSGL-1 amino-terminal domain by appending an EcoRI site to either the mature amino terminus (CD34 or GlyCAM-1) , or to the beginning of a region of threonine/proline-rich repeats (CD43) . As above, the EcoRI site was in the frame glutamic acid phenylalanine (frame 1) . The CD34 sequence began at residue F30 of the precursor, the Gly-CAM-1 at precursor L19, and the CD43 at precursor 1135. To each of these was appended the flu-tagged PSGL-1 domain terminating in EcoRI as above. The amino terminus and repeat elements of PSGL-1 were appended to the membrane proximal, transmembrane, and intracellular domains of CD43 through an EcoRI site in the glutamic acid phenylalanine frame positioned immediately upstream of the sequences S225 of the CD43 precursor. The complementary fragment from PSGL-1 corresponded to the amino-terminal residues of the precursor up to T267. Fine Structure Mapping of the Amino-Terminal Domain A similar strategy was employed for the construction of deletions in the amino-terminal domain, in which PCR generated deletions were formed using primers bearing an Xbal site in the leucine aspartic acid frame (frame 2) . Immediately downstream of the residues encoding aspartic acid were the PSGL-1 sequences corresponding to precursor R38, E58, P78, and A98. For the definition of the amino-terminal domain, duplex oligonucleotides were synthesized corresponding to the residues between 38 and 57 with the indicated sequence changes to mutate threonine or tyrosine residues to alanine or phenylalanine. All constructs were confirmed by dideoxy sequencing. Cell Adhesion Assays Transfected cells were detached from culture dishes with 0.5 mM EDTA in phosphate buffered saline (PBS) 48 to 60 hours after transfection. The cells were then loaded with 100 μl 51 Cr0 4 (1 mCi/ l; DuPont, Boston, MA) in 0.9% NaCl plus 100 ml medium by incubating them at 37°C for 1 hour. Loaded cells were washed twice in PBS and resuspended in 0.2% BSA, 0.15 M NaCl, 3 mM CaCl 2 . Variation in labeling rate (counts incorporated per cell) between cells prepared in parallel with the same batch of labeled chromate was typically minimal. The labeled cells were incubated in wells of 96-well microculture plates which had been coated with affinity purified goat anti-human IgG antibody (100 μl of 20 μg/ml anti-human IgG Fc (heavy chain specific) in PBS) for 2 hours in a humid chamber at room temperature. After the plate was washed twice with PBS, additional protein-binding sites were blocked by an overnight incubation with 200 μl 3% BSA in PBS. The plate was washed with PBS four times and incubated with 200 μl of fusion protein supernatants for 2 hours. Following three PBS washes and one additional wash (in 0.2% BSA, 0.15 M NaCl, 3 mM CaCl 2 ) , 2 X 10 s cells/well (in 200 μl 0.2% BSA, 0.15 M NaCl, 3 mM CaCl 2 ) were added and allowed to bind for 15 minutes at room temperature while the plate rotated on a rotary platform (80 rpm) . The plate was washed three times by filling the wells with 200 μl 0.15 M NaCl/3 mM CaCl 2 and then inverting the plate. Adherent cells were lysed by the addition of 200 μl 2% SDS, and labeled chromate was counted with a gamma ray spectrometer. Immunofluorescence Analysis Cells were prepared for cytometry by incubation with the primary monoclonal antibody (a 1:200 dilution of ascites or 5 μg/ml of purified antibody is suitable) in PBS containing 3% BSA for 30 to 45 minutes. The cells were washed twice with PBS and incubated with 2 μg/ml FITC-conjugated affinity purified antibody to either mouse IgG (12CA5) or mouse IgM (CSLEX-1) for 30 to 45 minutes in PBS/3% BSA. The cells were then washed twice with PBS and resuspended in 1 ml of 1% freshly depolymerized paraformaldehyde in PBS prior to analysis. For immunofluorescence microscopy, transfected cells were fixed with 4% freshly depolymerized paraformaldehyde, washed, exposed to BSA at 3% in PBS for 30 minutes, and then incubated with primary antibody (ascites, 1:250) for 30-45 minutes. The cells were then washed twice with PBS and incubated for 30-45 minutes with FITC-conjugated affinity-purified antibody to mouse IgG (Cappel1; 2 /ng/ml in PBS containing 3% BSA) . Finaly, the cells were washed twice with PBS and analyzed. Metabolic Labeling with 35 S0 4 COS cells transfected with expression plasmids encoding mucin:immunoglobulin chimeras were trypsinized one day after transfection and transferred to new plates in complete medium (DMEM with 10% calf serum) . Prior to labeling, the medium was removed, the cells were washed once with PBS, and the medium was replaced with either cysteine and methionine-free medium for labeling with [ 35 S]cysteine and methionine (TransLabel, ICN) or with sulfate-free CRCM-30 medium (Sigma Chemical Co.) for labeling with 35 S0 4 . Serum was not added, and radionuclide was typically present at a concentration of 200 μCi/ml. After a labeling interval of 12 to 16 hours, the supernatants were harvested, and the fusion proteins were collected by adsorption to goat anti-human IgG agarose (Cappel) . Adsorbed proteins were subjected to denaturing electrophoresis on 8% polyacrylamide gels under reducing conditions. Chlorate Inhibition of Adhesion COS cells were transfected with DEAE dextran and incubated immediately in DMEM containing 10% calf serum and 10 mM sodium chlorate. One day after transfection the cells were trypsinized and incubated in fresh dishes in the same medium for 6 hours. The medium was then removed, the cells were washed with PBS, and then incubated for 18 additional hours in a custom prepared DMEM medium (Life Technologies) lacking sulfate and containing 2% of the conventional levels of cysteine and methionine with 10% dialyzed fetal bovine serum in the presence of 10 mM sodium chlorate (Baeuerle and Huttner, Biochem. Biophys. Res. Comm., 141:870-877, 1986). Cells were then harvested for use in the adhesion and immunofluorescence assays. Control cells were treated similarly but were incubated in DMEM containing undialyzed serum. HL-60 Cell Rolling Video images of HL-60 cells rolling through a parallel plate rectangular flow chamber (FCS2, Bioptechs, Incorporated, Butler, PA) with a temperature controlled stage set at 37°C were acquired with an AIMS Technology (Bronx, NY) CCD camera mounted on a Zeiss ICM 405 inverted microscope equipped with a 2.5x objective. The chamber height was 250 μm. Cells were withdrawn through the chamber at a defined flow rate with the aid of a Harvard Apparatus (South Natick, MA) model l/w 22 syringe pump. Images were analyzed using NIH Image. To inhibt sulfation, HL-60 cells were washed once with PBS and grown for 18 hours in sulfate-free medium containing 2% of the normal levels of cysteine and methionine, 10 mM sodium chlorate, and dialyzed serum as described above. For each experiment, IO 6 cells were suspended in 1 ml of 0.15 M NaCl, 3 mM CaCl 2 and drawn through the chamber. Glass coverslips were coated with affinity-purified goat anti-human IgG antibody at a concentration of 10 μg/ml in 50 mM Tris-HCl (pH 9.0) for 2 hours, washed twice with PBS, and blocked overnight with 0.2% BSA in PBS. The treated coverslips were then immersed in supernatants of COS cells transfected with the appropriate immunoglobulin chimera expression plasmids, washed twice with PBS, and assembled in the flow chamber. The Amino Terminus of PSGL-1 is Necessary for P-Selectin bjp inq Deletions of the amino terminus of the PSGL-1 mucin were created with PCR techniques, and the resulting truncated cDNAs were inserted downstream of a secretory peptide sequence which had been fused to a short oligopeptide tag derived from influenza hemagglutinin (HA) . Expression plasmids encoding the truncated molecules (Fig. IA) were transfected into COS cells in the presence of a specific myeloid fucosyltransferase, designated FTVII, which directs the expression of sLe x determinants exclusively (Sasaki et al., J. Biol. Chem., 269:14730-14737, 1994; Natsuka et al., [published erratum appears in J. Biol. Chem, 269:20806, 1994], J. Biol. Chem., 269:16789-16794, 1994). Expression of the deletion mutants at the cell surface was confirmed by indirect immunofluorescence using anti-HA monoclonal antibodies. The presence of sLe x on the cell surface was similarly confirmed using the monoclonal antibody CSLEX-1. The ability of radiolabeled transfected cells to bind to plastic wells precoated with P-selectin:immunoglobulin fusion protein was determined. These experiments revealed that deletion of the amino terminal 100 residues (referred to herein as the apical domain) of PSGL-1 was sufficient to abolish binding of the transfectants to immobilized P-selectin (Fig. IB) . These experiments also demonstrate that sLe x mediates P- selectin binding, as expression of FTVII was required for P-selectin binding (Fig. IB; compare bar 2 with bar 3) . Expression of the deletion variants at the cell surface was confirmed by indirect immunofluorescence using anti- HA monoclonal antibodies, and the presence of sLe x on the cell surface was confirmed using the monoclonal antibody CSLEX-1. Table 1 shows the mean fluorescence intensity (MFI) of COS cells that were cotransfected with human FTVIIh and the deletion constructs (shown in Fig. IA) , and subjected to indirect immunofluorescence with antibody against the amino terminal flu peptide or sLe x . Table 1 Construct Expression (MFI. In the Context of Large. Sulfated Mucins. the Amino Terminus of PSGL-1 is Sufficient for P-Selectin Binding To determine whether PSGL-1 sequences other than those found in the first 100 N-terminal amino acids (i.e., the apical domain) of PSGL-1 were required for binding to P-selectin, the transmembrane and cytoplasmic regions of PSGL-1 were replaced with those of the CD43 antigen (Pallant et al., Proc. Natl. Acad. Sci., 86:1328- 1332, 1989; Shelley et al., Proc. Natl. Acad. Sci., 86:2819-2823, 1989). The resulting molecule, which did not contain cysteine residues, bound P-selectin with the same .efficiency as PSGL-1 did (Fig. 2A and Fig. 2B) . Thus, neither disulfide bond formation nor a specific membrane anchoring segment is required for P-selectin binding activity. The predicted first 100 amino acids of PSGL-1 were then genetically grafted onto the amino termini of ucin-like repeat elements of several unrelated mucins to determine whether or not the PSGL-l apical domain is sufficient for P-selectin ligand (i.e., counterreceptor) activity (Fig. 3A) . Certain of these chimeric mucins were able to support P-selectin binding in this setting. CD34 and CD43, two relatively large mucins found predominantly on human hematopoietic cells, were both able to support binding. In contrast, an artificially anchored variant of GlyCAM-1, a mucin expressed on high endothelial venules that has L-selectin ligand activity (Lasky et al.. Science, 258:964-969, 1992), was inactive in this assay (Fig. 3B) . The GlyCAM-1 mucin domain in these experiments was tethered to the cell surface via the extracellular stalk, transmembrane domain, and cytoplasmic anchoring segments of CD7 (Aruffo et al., EMBO J., 6:3313-3316, 1987). Cell surface expression of the different mucins and mucin chimeras was confirmed by indirect immunofluorescence using antibodies agains flu tag, sLe x , or the respective mucins. Table 2 shows mean fluorescence intensity (MFI) measurements of expression of flu tag or sLe x by COS cells transfected with the constructs analyzed in Fig. 3B. CD34 and CD43 constructs were positive for expression by indirect immunofluorescence using cognate anti-CD antibodies. Table 2 Construct Expression (MFI. Ω_ SLeX FTVIIh 52 PSGL-1-flu 9.8 43 CD43 60 PSGL-1-NH 2 /CD43 rep. 12 67 CD34 50 PSGL-l-NH 2 /CD34-COOH 8.0 33 Glycam-flu 12 32 PSGL-1-NH 2 /Glycam-COOH 8.0 28 The apparent molecular masses of CD43 and CD34 expressed in COS cells are reported to be 100-130 kD (Shelley et al., Proc. Natl. Acad. Sci., 86:2819-2823, 1989) and 100 kD (Simmons et al., J. Immunol., 148:267- 271, 1992), respectively; the PSGL-1 monomer exhibits an effective molecular mass of 110 kD (Sako et al., Cell, 75:1179-1186, 1993). GlyCAM-1, in its native (untethered) state comigrates with 50 kD proteins, suggesting that it is substantially smaller (Lasky et al., Science, 258:964- 969, 1992). In our studies, the larger mucins were able to εupport P-selectin binding when the apical domain of PSGL-1 was appended to the amino erminus of the mucins. Sequential deletion of the internal repeat elements of PSGL-1 allowed us to shorten the molecule in a systematic manner without compromising potential global tertiary associations (Fig. 4A) . As these repeat elements were deleted, the binding activity of PSGL-1 declined, consistent with the conclusion that distance from the plasma membrane is an important determinant of P-selectin binding activity (Fig. 4B) . Our data also indicate that sulfation is one determinant of the ability of mucins to support apical domain-directed binding. We assessed the ability of various mucins to undergo sulfation in COS cells. PSGL-1, CD34, CD43, and GlyCAM-1 soluble mucin chimeras readily incorporated sodium 35 S-sulfate when expressed in COS cells (Fig. 5) . Inibition of Sulfation Blocks PSGL-1 Binding to P- Selectin We have found that inhibition of sulfation blocks PSGL-1 binding to P-selectin. COS cells were cotransfected with PSGL-1 and FTVII, or transfected with PSGL-1 and FTVII separately. During the time period in which maximum synthesis of PSGL-1 was expected, the cells were incubated in a modified DMEM medium lacking sulfate and containing 10 mM sodium chlorate, a relativelly selective inhibitor of sulfation (NaC10 3 ) . We observed a significant decrease in the ability of chlorate-treated cotransfected cells to bind to immobilized P-selectin (Fig. 6A) , whereas the same cells showed little or no decrement in binding to immobilized E-selectin (Fig. 6B) . Cell surface expression of either the sLe x antigen and the PSGL-1 amino terminal tag sequence was not inhibited by NaC10 3 treatment. In fact, as shown in Table 3, an increase in the mean fluorescence intensity of the transfected cells, representing both anti-sLe x and anti-flu tag, was o. served following chlorate treatment, suggesting that chlorate may affect internalization or cell surface export. Table 3 Expression (MFI) w/o NaC10 3 w/10 mM NaC10 3 sLe x Flu sLe x Flu FTVIIh 23 --._-_ 30 1 PSGL-1-flu -_-__ 9 -._--. 22 PSGL-lflu + FTVIIh 15 10 35 34 | A soluble PSGL-1 immunoglobulin chimera synthesized under comparable conditions showed essentially complete inhibition of 35 S-sulfate incorporation (Fig. 7) , under conditions in which protein synthesis as measured by [ 35 S]cysteine and methionine incorporation was not inhibited. These data demonstrate that sulfation of the P-selectin ligand is required for P-selectin binding activity. Fine Structure Deletion Analysis of the Apical Domain of PSGL-1 To localize the elements within the 100 amino acid apical domain which contribute to P-selectin ligand activity, we prepared a collection of deletion mutants in which various regions of the apical domain were deleted (Fig. 8A) . Each amino terminal deletion mutant was then placed downstream of the CD5 leader/flu tag element to monitor cell surface expression. The fine structure deletion mutants showed little variability in their ability to express the epitope tag, as assessed by indirect immunofluorescence. Removal of the first 20 amino acids of the N-terminus of the mature PSGL-1 did not affect P-selectin binding activity. In contrast, removal of the first 40 amino acids of the N-terminus abrogated binding (Fig. 8B) . Further deletions of PSGL did not affect P-selectin binding activity. Accordingly, amino acid residues 20 to 40 of PSGL (i.e., residues 38 to 57 of the predicted precursor having the signal sequence) are required for P-selectin binding. To demonstrate that residues 38 to 57 are sufficient for PSGL-1 apical domain-directed activity, we appended this segment to the amino termini of PSGL-1 and CD43 mucin cores from which the apical domains had been deleted (Fig. 9A) . In both cases, addition of amino acids 38-57 of PSGL-1 peptide element conferred P-selectin binding activity upon the mucin core. In both cases, the level of P-selectin binding activity was equivalent to that of native PSGL-1 (Fig. 9B) . Specific Residues Within the Amino Terminal Peptide are Recruired for P-selectin Binding Activity The 20 amino acid region which is necessary for P-selectin binding contains three potential tyrosine sulfation sites and two threonine residues for O-linked glycosylation. To assess the importance of these residues, the tyrosines were converted to phenylalanine (Fig. 9A) . In a second peptide, the threonines were converted to alanines. In addition, a third peptide, containing a quintuple mutation, was prepared such that both conversions were made in a single peptide. Each mutated peptide was then positioned, separately, downstream of the flu tag and upstream of either (1) the truncated PSGL-1 lacking the apical domain, or (2) the CD43 repeat elements and transmembrane domain. Cells expressing the resulting chimeras were tested for their ability to bind to immobilized P-selectin (Fig. 9A) . Conversion of the tyrosines to phenylalanines resulted in a loss of binding activity to P-selectin. Replacement of the threonine residues with alanine diminished binding, but did not abolish it entirely. Expression of the flu tag or sLe x epitope was not affected in these cells. Binding mediated by the apical 20 residues was, like that of native PSGL-1, dependent on the presence of calcium. These data indicate that sulfation of tyrosines at positions 46, 48, and 51 is required for P-selectin binding activity. E-selectin binding was unaffected under the same condition. In addition, these data indicate that the threonines at positions 44 and 57 are required. These threonine residues can serve as sites for O-linked glycan addition. These experiments, in conjunction with our experiments showing that FTVII expression is necessary for P-selectin binding, provide evidence that P-selectin binding requires sLe x at threonines 44 and 57. In sum, the above-described experiments demonstrate that amino acids 38-57, containing three residues for sulfation and two residues for sLe x addition, are sufficient to confer P-selectin binding activity. Residues Within the Amino-Terminal 20 Amino Acids are Sulfated on Tyrosine To determine whether the amino-terminal segment was capable of being sulfated in vivo, we created fusion proteins consisting of the native or mutant peptide sequences joined to human immunoglobulin Gl (IgGl) (Fig. 12A) . The resulting fusion proteins were expressed in COS cells, and their ability to assimilate inorganic sulfate was assessed (Fig. 12B) . Immunoglobulin chimeras bearing the native peptide sequences were capable of incorporating sulfate, whereas those bearing phenylalanine substituted for tyrosine were not (Fig. 12C) . Replacement of threonine with alanine had no effect on sulfate incorporation (Fig. 12C) . Inhibitors of Sulfation Block HL-60 Rolling on P- Selectin-Immunoglobulin Chimeras To explore whether inhibition of sulfation would compromise a physiologically relevant adhesion, we subjected HL-60 cells to growth in medium containing chlorate and examined the ability of the resulting cells to attach and roll on coverslips coated with P-selectin- immunoglobulin chimeras under conditions of defined fluid shear stress (Lawrence et al.. Blood, 75:227-237, 1990). HL-60 cells were capable of attaching to and rolling upon coverslips precoated with P-selectin-immunoglobulin chimeras, whereas no such interaction was observed with coverslips coated with a CD4-immunoglobulin chimera (Fig. 13) . Growth of HL-60 cells in chlorate dramatically reduced the frequency of cell interaction with the substrate (Fig. 13). Antibodies and Antibodv Fusion Proteins Bearing Sialyl- Le x and Sulfated Determinants In one embodiment, the invention features an antibody bearing sialyl-Le x and sulfated determinants. Such an antibody may be created by introducing sulfation sites (i.e., a tyrosine in an acidic context) into an existing antibody molecule in the vicinity of an introduced or existing sialyl-Le x addition site (for example, by standard site-directed mutagenesis) . Alternatively, appropriate sialyl-Le x and/or sulfation sites may be added by appending any P-selectin ligand sequence (for example, any P-selectin domain described herein) to a naturally-occurring antibody sequence (for example, IgG or IgM) by standard recombinant DNA techniques to produce a P-selectin-antibody fusion protein. Preferably, the P-selectin ligand sequence is appended to the amino-terminus of the antibody molecule. Such antibodies are useful for disrupting undesirable interactions between cells or proteins, or, generally, for disrupting any interaction between two moleculeε, one of which bears a determinant carried by the antibody. Because these determinants normally act to facilitate interactions involving E-selectin and P-selectin (e.g., interactions between neutrophils and endothelial cells lining the blood vessel walls) , the ability to disrupt such interactions provides many therapeutic applications, for example, in minimizing inflammation and decreasing extravasation-dependent organ damage and/or clotting. In addition, if desired, one or more sialyl-Le x moieties which mask the CH2 portion of the immunoglobulin molecule and thus inhibit complement fixation and F c receptor binding may also be incorporated into the antibody sequence. Because the carbohydrate moieties block the immunoglobulin domain which triggers complement fixation and F c receptor binding, such antibodies do not elicit the undesirable side effects (i.e., those resulting from complement fixation and F c receptor binding) frequently associated with antibody-based therapies. Preferably, the carbohydrate groups serve not only to inhibit undesirable complement fixation and F c receptor binding, but also perform the function of competitively inhibiting an E-selectin and/or P-selectin mediated intracellular interaction. To inhibit complement fixation and F c receptor binding, sialyl-Le x determinants may be added to the antibody molecule at any appropriate site. N-linked glycan addition sites are well known to be: N X S/T (where N is asparagine, S is serine, T is threonine, and X is any amino acid except proline) . Accordingly, an exemplary molecule may be designed that includes several such sites for attachment of sialyl-Le x side chains. Inspection of the IgGl sequence (Fig. 10) reveals at least five sites at which N-linked glycan addition sites may be introduced into the molecule in advantageous locations, where complement fixing and F c receptor binding ability will be impaired by the process. These sites include amino acid residues 274, 287, 295, 322, and 335. Although these are preferred sites of N-linked glycan addition, they are not the only candidates; other useful sites may be identified and incorporated into the IgGl sequence using, as guidance, the following criteria: (1) the siteε are, preferably, located in the CH2 region of the immunoglobulin molecule, i.e., in the portion of the molecule responsible for complement fixation and F c receptor binding; (2) the siteε are located in regions of the sequence, predicted by their hydrophilic nature, to be present on the outside of the immunoglobulin molecule and therefore accessible to the enzymes responsible for attachment of carbohydrate side chains; (3) the sites are located in a region which is minimally disruptive to the primary amino acid sequence and, thus, the predicted secondary amino acid structure. For example, a naturally-occurring site which differs from an N-linked glycan addition site by a single amino acid would be preferable to a site requiring two alterations in the amino acid sequence. Moreover, it is preferable to create an N-linked glycan addition site by substituting amino acids of similar charge or polarity (e.g., substitution of one uncharged amino acid for another) . One or more N-linked glycan addition site substitutions may be engineered into a particular IgGl-encoding sequence; such sequences (i.e., those which encode an antibody molecule to which sialyl-Le x moieties are attached) are termed IgGl-sialyl-Le x or IgGl-Le x . The introduction of additional glycosylation sites at amino acids #274, #287, and #322 within the CH2 domain created a molecule that was unrecognized by F c receptor or complement using assays that are standard in the art; exemplary complement fixation assays include Weir et al., Handbook of Experimental Immunology, Blackwell, Oxford; and Coligan et al. Current Protocols In Immunology, Wiley Interscience, 1995. A particular IgGl molecule bearing sialyl-Le x moieties iε produced aε followε. The IgGl gene iε publically available, and itε εequence iε shown in Fig. 10. The gene is mutagenized by standard methods of in vitro site-directed mutagenesis in order to introduce one or more N-linked glycan addition sites (e.g., those described above and shown above the naturally-occurring sequence in Fig. 10) . The gene is then inserted into a vector designed to express the protein in a eukaryotic cell (see, e.g., those vectors described in Gillies et al., U.S. Patent No. 4,663,281, hereby incorporated by reference) . The eukaryotic host cell is preferably a mammalian cell (e.g., a CHO or lecll cell), and the expression vector containing the mutated IgGl-Le x - encoding sequence is introduced into the host cell by transient or stable transfection using standard techniques. Such host cells are also transfected (transiently or stably) with a vector capable of expresεing an α(1,3)fucosyltransferase capable of attaching the sialyl-Le x groups to the antibody molecule at the glycosylation siteε. The α(1,3)fucosyltransferase gene may be expresεed from a vector diεtinct from that encoding IgGl-Le x , or both genes may be carried on, and expressed from, a common vector. Mammalian cells are particularly uεeful hosts for the synthesis of IgGl-Le x because they provide all required precursors for sialyl- Le x production. To produce the sialyl-Le x -modified and sulfated antibodies of the invention, the gene encoding the antibody sequence is preferably expressed in a cell which also expresses an α(1,3)fucosyltransferase that exclusively catalyzes α(1,3)fucose linkages; εuch an enzyme iε described in Walz et al., Science 250:1132-1135 (1990) and in Seed, U.S.S.N. 08/483,151, entitled "Fucosyltransferase Genes and Useε Thereof," filed June 7, 1995 (hereby incorporated by reference). Leεε preferably, the α(1,3)fucosyltransferaεe cDNA described in Lowe et al. (Cell 6.2:475, 1990) may be utilized. Thiε fucoεyltransferase recognizeε a sialylated precursor molecule and adds either an α(l,3)- or an α(1,4)-linked fucose moiety to N-acetylglucosamine εide chainε. The sialyl-Le x determinant iε characterized by an α(l,3)- linkage, and, aε such, the α(1,3)fucosyltransferase enzyme of Lowe (supra) produces both the desired εialyl- Le x -modified molecules and products bearing α(1,4)-linked fucose which, although not active in binding to P- selectin and E-selectin, do not interfere with the action of the sialyl-Le -modified molecules nor produce other undesirable side effects. Host cells expressing α(1,3)fucosyltransferase and the antibody to be modified are grown by standard methods, and the antibody iε purified from a cell lyεate baεed on its affinity for a Protein A column or any other standard technique of antibody isolation and purification. o^-Acid Glycoprotein-Antibody Fusion Proteins Bearing Sialyl-Le and Sulfated Determinants As discuεεed herein, antibody fusion proteins modified by sulfation and sialyl-Le x addition have important therapeutic and diagnostic uses. Previouε work haε demonεtrated that large amounts of antibody fusion proteins may be generated and secreted transiently from transfected mammalian cells (for example, COS cells) . In general, to produce an AGP antibody fusion protein according to the invention, DNA encoding an AGP and a P- selectin ligand domain are fused in-frame to human IgG domains (for example, constant domains) by standard techniques, and the fuεion protein iε expressed, also by standard techniques. The antibody portion of the molecule facilitates fusion protein purification and also prolongs the plasma half-life of otherwise short-lived polypeptides or polypeptide domains. Preferably, antibody fusion proteins are expressed according to the methods disclosed in Seed et al., U.S.S.N. 08/483,151 entitled "Fucosyltransferase Genes and Useε Thereof," filed June 7, 1995 (which iε hereby incorporated by reference), e.g., uεing IgG or IgM antibodieε or portionε thereof (see also Zettlemeisl et al., DNA Cell Biol. 9:347 (1990) for IgM fusion proteinε) . Recombinant plasmids expressing particular AGP- antibody fusion proteins (e.g., AGP-Hinge-CH2-CH3 and AGP-CH2-CH3 proteins) have been conεtructed aε follows. A cDNA encoding the acute phase ot j -AGP gene was cloned from,a human liver cDNA library by polymerase chain reaction (PCR) using oligonucleotide primers corresponding to the 5' and 3' coding regions of o^-AGP (Board et al.. Gene 4_: 121 , 1986) according to standard techniques. The 5' AGP primer was designed to contain a Hindlll restriction site and the 3' primer was designed to contain a BamHI restriction site rather than the AGP stop codon. The PCR-amplified product was digested with Hindlll/BamHI and cloned into a Hindlll/BamHI-cut plasmid expression casεette (see Aruffo et al.. Cell, 61:1303. 1990) containing constant domains of human IgGl (i.e., Hinge-CH2-CH3 or CH2-CH3) . A nucleotide sequence and amino acid sequence of this AGP-IgG fusion protein are shown in Fig. IIA and Fig. IIB, respectively. To create a molecule that blocks P-selectin- mediated interactions, sites for sulfation and, if necessary, sialyl-Le x addition are introduced into the antibody fusion protein sequence (for example, the antibody fusion proteins described above) . Such siteε may be incorporated into an exiεting fusion molecule, for example, by introducing one or more sulfation sites (i.e., a tyrosine in an acidic context) in the vicinity of an introduced or existing sialyl-Le x addition site (for example, by standard techni .ies of εite-directed mutagenesis) , or a P-selectin ligand sequence (for example, any of the P-selectin ligand sequences described herein) may be appended to the antibody fusion protein sequence using standard techniques of recombinant DNA technology. The P-selectin-AGP-antibody fusion genes are then introduced into expresεion plasmids, and the plasmids are transfected into any appropriate fucosyltransferase- expressing cell for the production of soluble antibody fusion proteins. To prepare an antibody fusion protein capable of inhibiting complement fixation and F c receptor binding, additional sialyl-Le x conεenεuε glycoεylation εiteε (N-X- T/S) may be introduced into the CH2 domain of human IgGl as described above. Based on this construction strategy, any number of recombinant P-selectin-AGP-antibody fusion proteins may be designed having long plaεma half-liveε and the ability to inhibit undeεirable cell-cell interactionε (for example, the interactionε between leukocytes and selectin-bearing cells) . To generate molecules with heightened inhibitory potency, candidate molecules are designed and screened using the assays described above. In one particular example, molecules may be screened for their ability to incorporate εialyl-Le x and εulfated determinants and block the binding of neutrophils to activated endothelial cellε; εuch moleculeε find uεe in the inhibition of εelectin-dependent inflammatory reactions and tisεue injury inflicted by invading leukocytes. Molecules Capable of Interfering with P-Selectin-Mediated and E-Selectin-Mediated Interactions Because both P-selectin- and E-selectin-mediated intracellular interactions are involved in inflammation and because the crucial determinants involved in thoεe interactionε have now been identified, it iε possible to design a single molecule capable of interfering with both types of deleterious interactions. In particular, molecules (for example, proteins) may be constructed that include both a P-selectin ligand domain (i.e., a domain bearing sialyl-Le x and εulfated moietieε) and an E- selectin ligand domain (i.e., a domain bearing a sialyl- Le x moiety) . Such a molecule may be constructed by combining domainε, for example, by appending a P-selectin ligand domain to a sialylated molecule (for example, a sialylated antibody or antibody fusion protein described herein) . Alternatively, appropriate sialyl-Le x and/or sulfation sites may be introduced into an existing sequence, for example, by site directed mutagenesis. Glycosylation or sulfation of an engineered molecule may be tested, for example, as described herein and in Walz et al.. Science 250:1132-1135 (1990). The ability of a sialyl-Le x -modified and/or εulfated molecule to interfere with intracellular interactionε may also be tested aε deεcribed in Walz et al., εupra. or by any standard technique, for example, by assaying the ability of increasing concentrations of the determinant-bearing molecule to inhibit adherence of T lymphocytes or myeloid cells to immobilized P-selectin and/or E-selectin. Use For administering a protein or organic molecule of the invention to a patient, the pharmaceutically-pure protein or molecule is suspended in an acceptable carrier, e.g., physiological saline, and is delivered to the patient by any appropriate route (for example, intravenously) in a single dose or in multiple doses. Optimally, a sufficient quantity of the therapeutic is provided to saturate all P-selectin and, for a dual function molecule, all E-selectin binding siteε on an endothelial cell. Typically, thiε may be achieved with doses of 0.1 mg/kg or greater. The preferred dosage is in the range of 0.1-2.0 mg/kg. The sialyl-Le x -modified and sulfated molecules and proteins of the invention (for example, the modified antibodies and antibody fuεion proteins described herein) may be used, in one example, for the treatment of extravasation-dependent organ damage and/or clotting. In particular, because P-selectin mediates the attachment of neutrophils overlying sites of inflammation or tissue damage or proximate to thrombus formation, the molecules and proteins of the invention provide useful therapeutics for blocking such interactions. For example, P-selectin likely mediates the migration of neutrophils into the lung following adult respiratory distress syndrome and into the heart following ischemic myocardial injury (i.e., infarction) , and may play a role in glomerular damage to the kidneys under certain conditions. Accordingly, a sialyl-Le x -modified and sulfated molecule or protein of the invention may be administered to a patient suffering from such a disease or condition. Such treatment attenuates extravasation-dependent damage by competitively inhibiting the interaction between the invading neutrophils and the endothelial cells of the blood vessel or organ. The compounds of the invention, particularly, P-selectin ligand-AGP fusion proteins and P-selectin ligand-AGP-antibody fusion proteins may also be used, as described above, for the treatment of septic shock or septicemia. In addition, antibodieε or antibody fuεion proteins according to the invention may be used in conventional techniques of antibody-based therapies or jn vivo diagnostics, taking advantage of the antibody's εpecificity to target therapeutic or diagnoεtic sites. In one particular example, the P-selectin ligand domain of an antibody fusion protein according to the invention targetε that protein to a εite of inflammation and provideε both a therapeutic (uεeful for blocking deleteriouε P-εelectin-mediated intracellular interactionε) and a diagnoεtic (uεeful for tagging the site of inflammation) . Again, attached sialyl-Le x determinants may be used to mask the CH2 domain of the antibody and block the undesirable effects of complement fixation and F c receptor binding. Other Embodiments Other embodiments are within the claimε. For example, for the purpose of blocking interactions between cells or proteins, any other appropriate carrier molecule to which a εialyl-Le x and a sulfated determinant may be attached may be utilized in the invention. Generally, proteins are preferred becauεe of their relatively long half-liveε in serum. One clasε of carrier proteins are serum proteins such as albumin (e.g., bovine serum albumin or human serum albumin) , transferrin, or α-2 macroglobulin. The carrier proteins may contain endogenous sulfation and glycan addition siteε in addition to which εites are introduced into the DNA sequence of the carrier protein (as described above) by, for example, site-directed mutagenesiε. The carrier molecule, leεε preferably, may be a lipid. In one example, the lipid, with one or more attached εialyl-Le x and sulfated determinants is delivered aε a liposome to a target cell wall (e.g., an endothelial cell wall). The liposome may block a cell or protein interaction or may be uεed to deliver a drug to itε appropriate εite of action. Production of carrier moleculeε bearing εialyl-Le x and sulfated determinants may be carried out in a cell, preferably, a eukaryotic cell other than yeaεt. Mammalian cells, e.g., mammalian cell lines, provide particularly εuitable hosts. These cellε generally synthesize the necesεary precursor molecules and produce or can be engineered to produce the enzymes responsible for sulfation and carbohydrate attachment. For the attachment of sialyl-Le x determinants, mammalian cell lines such as CHO and lecll are particularly suitable. Alternatively, either or both of the sialyl-Le x and sulfated determinants may be attached to a carrier molecule in vitro, i.e., extracellularly. In one example, α(1,3)fucosyltransferase would be bound to a solid support (e.g., a column) and a sulfated carrier molecule pasεed over the bound fucoεyltranεferaεe enzyme, under conditionε which facilitate attachment of sialyl- Le x groups to their appropriate site(ε) on the carrier molecule. The invention also encompasses the use of sulfated and sialyl-Le x -modified AGP-antibody fusion proteins for protecting against, inhibiting, or treating a shock- inducing event, the clinical manifestations of shock, or both which are caused by microbial factors (e.g., lipopolysaccharides (LPS)), microbial toxins (e.g., toxic shock enterotoxins) , host mediators (e.g., cytokines), or anti-tumor therapies (e.g., administration of tumor necrosis factor (TNF) or interleukin-l (IL-1)) , or any combination thereof. For example, such an antibody fusion protein can be administered to a human patient to alleviate the effects of septic shock induced by microbial LPS. The ability of an antibody fusion protein to protect against, treat, or inhibit the effects of shock (e.g., septicemia or toxic shock syndrome) is evaluated according to standard methods known in the art (e.g., those described in Libert et al. (1994) J. Exp. Med. 180: 1571-1575). All publications, patents, and patent applications mentioned in thiε εpecification are herein incorporated by reference to the same extent as if each individual publication, patent, and patent application was specifically and individually indicated to be incoporated by reference. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: The General Hospital Corporation (li) TITLE OF INVENTION: P-SELECTIN LIGANDS AND RELATED MOLECULES AND METHODS (ill) NUMBER OF SEQUENCES: 14 ( v) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Fish & Richardson P.C. (B) STREET: 225 Franklin Street (C) CITY: Boston (D) STATE: MA (E) COUNTRY: USA (F) ZIP: 02210-2804 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy d sk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.30 (vll) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 60/000,213 (B) FILING DATE: 14-JUN-1995 (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Lech, Karen F. (B) REGISTRATION NUMBER: (C) REFERENCE/DOCKET NUMBER: 00786/28WO1 ( x) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 617/542-5070 (B) TELEFAX: 617/542-8906 (C) TELEX: 200154 (2) INFORMATION FOR SEQ ID NO:l: (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (11) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: Ala Thr Glu Ala Gin Thr Thr Pro Pro Ala 1 5 10 (2) INFORMATION FOR SEQ ID NO:2: - Sβ - t ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: am no ac d (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Ala Thr Asn Ser Leu Glu Thr Ser Thr Gly Thr Ser Gly Pro Pro 1 5 10 15 Val Thr (2) INFORMATION FOR SEQ ID NO:3: (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 amino acids (B) TYPE: amino acid .(C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Gin Leu Trp Asp Thr Trp Ala Aβp Glu Ala Glu Lys Ala Lβu Gly Pro 1 5 10 15 Leu Lβu Ala Arg Asp Arg Arg Gin Ala Thr Glu Tyr Glu Tyr Lβu Asp 20 25 30 Tyr Aβp Phe Leu Pro Glu Thr Glu Pro Pro 35 40 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Arg Asp Arg Arg Gin Ala Thr Glu Tyr Glu Tyr Leu Asp Tyr Asp Phe 1 5 10 15 Leu Pro Glu Thr 20 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 -imino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: Arg Aβp Arg Arg Gin Ala Thr Glu Phe Glu Phe Leu Aβp Phe Aβp Phe 1 5 10 15 Leu Pro Glu Thr 20 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acidβ (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Arg Asp Arg Arg Gin Ala Ala Glu Tyr Glu Tyr Leu Aβp Tyr Aβp Phe 1 5 10 15 Leu Pro Glu Ala 20 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acidβ (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Arg Aβp Arg Arg Gin Ala Ala Glu Phe Glu Phe Leu Asp Phe Asp Phe 1 5 10 15 Leu Pro Glu Ala 20 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2287 baβe pairβ (B) TYPE: nucleic acid (C) STRANDEDNESS: βingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: AAGCTTACCA CCATGGACTG GACCTGGAGG TTCCTCTTCT TTGTGGTGGC AGCAGCTACA 60 GGTGTCCAGT CCCAGGTGCA GCTGGTGCAG TCTGGGGCTG AGGTGAAGAA GCCTGGGTCC 120 TCGGTGAAGG TCTCCTGCAA GGCTTCTGGA GGCACCTTCA GCAGCTATGC TATCAGCTGG 180 GTGCGACAGG CCCCTGGACA AGGGCTTGAG TGGATGGGAG GGATCATCCC TATCTTTGGT 240 ACAGCAAACT ACGCACAGAA GTTCCAGGGC AGAGTCACGA TTACCGCGGA CGAATCCACG 300 AGCACAGCCT ACATGGAGCT GAGCAGCCTG AGATCTGAGG ACACGGCCGT GTATTACTGT 360 GCGAGAGATA ATGGAGCGTA TTGTAGTGGT GGTAGCTGCT ACTCGGGCTG GTTCGACCCC 420 TGGGGCCAGG GAACCCTGGT CACCGTCTCT TCAGGTGAGT ACTGAATTCT AGCTTTCTGG 480 GGCAGGCCAG GCCTGACCTT GGCTTTGGGG CAGGGAGGGG GCTAAGGTGA GGCAGGTGGC 540 GCCAGCAGGT GCACACCCAA TGCCCATGAG CCCAGACACT GGACGCTGAA CCTCGCGGAC 600 AGTTAAGAAC CCAGGGGCCT CTGCGCCTGG GCCCAGCTCT GTCCCACACC GCGGTCACAT 660 GGCACCACCT CTCTTGCAGC CTCCACCAAG GGCCCATCGG TCTTCCCCCT GGCACCCTCC 720 TCCAAGAGCA CCTCTGGGGG CACAGCGGCC CTGGGCTGCC TGGTCAAGGA CTACTTCCCC 780 GAACCGGTGA CGGTGTCGTG GAACTCAGGC GCCCTGACCA GCGGCGTGCA CACCTTCCCG 840 GCTGTCCTAC AGTCCTCAGG ACTCTACTCC CTCAGCAGCG TGGTGACCGT GCCCTCCAGC 900 AGCTTGGGCA CCCAGACCTA CATCTGCAAC GTGAATCACA AGCCCAGCAA CACCAAGGTG 960 GACAAGAAAG TTGGTGAGAG GCCAGCACAG GGAGGGAGGG TGTCTGCTGG AAGCAGGCTC 1020 AGCGCTCCTG CCTGGACGCA TCCCGGCTAT GCAGCCCCAG TCCAGGGCAG CAAGGCAGGC 1080 CCCGTCTGCC TCTTCACCCG GAGCCTCTGC CCGCCCCACT CATGCTCAGG GAGAGGGTCT 1140 TCTGGCTTTT TCCCAGGCTC TGGGCAGGCA CAGGCTAGGT GCCCCTAACC CAGGCCCTGG 1200 ACACAAAGGG GCAGGTGCTG GGCTCAGACC TGCCAAGAGC CATATCCGGG AGGACCCTGC 1260 CCCTGACCTA AGCCCACCCC AAAGGCCAAA CTCTCCACTC CCTCAGCTCG GACACCTTCT 1320 CTCCTCCCAG ATTCCAGTAA CTCCCAATCT TCTCTCTGCA GAGCCCAAAT CTTGTGACAA 1380 AACTCACACA TGCCCACCGT GCCCAGGTAA GCCAGCCCAG GCCTCGCCCT CCAGCTCAAG 1440 GCGGGACAGG TGCCCTAGAG TAGCCTGCAT CCAGGGACAG GCCCCAGCCG GGTGCTGACA 1500 CGTCCACCTC CATCTCTTCC TCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT 1560 TCCCCCCAAA ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG 1620 TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA CTGGTACGTG GACGGCGTGG 1680 AGGTGCATAA TGCCAAGACA AAGCCGCGGG AGGAGCAGTA CAACAGCACG TACCGGGTGG 1740 TCAGCGTCCT CACCGTCCTG CACCAGGACT GGCTGAATGG CAAGGAGTAC AAGTGCAAGG 1800 TCTCCAACAA AGCCCTCCCA GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGTGGGA 1860 CCCGTGGGGT GCGAGGGCCA CATGGACAGA GGCCGGCTCG GCCCACCCTC TGCCCTGAGA 1920 GTGACCGCTG TACCAACCTC TGTCCTACAG GGCAGCCCCG AGAACCACAG GTGTACACCC 1980 TGCCCCCATC CCGGGATGAG CTGACCAAGA ACCAGGTCAG CCTGACCTGC CTGGTCAAAG 2040 GCTTCTATCC CAGCGACATC GCCGTGGAGT GGGAGAGCAA TGGGCAGCCG GAGAACAACT 2100 ACAAGACCAC GCCTCCCGTG CTGGACTCCG ACGGCTCCTT CTTCCTCTAC AGCAAGCTCA 2160 CCGTGGACAA GAGCAGGTGG CAGCAGGGGA ACGTCTTCTC ATGCTCCGTG ATGCATGAGG 2220 CTCTGCACAA CCACTACACG CAGAAGAGCC TCTCCCTGTC TCCGGGTAAA TGAGTGCGAC 2280 GGCCGGC 2287 (2) INFORMATION FOR SEQ ID NO: : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 442 amino acidβ (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Lye Leu Thr Thr Met Aβp Trp Thr Trp Arg Phe Leu Phe Phe Val Val 1 5 10 15 Ala Ala Ala Thr Gly Val Gin Ser Gin Val Gin Leu Val Gin Ser Gly 20 25 30 Ala Glu Val Lye Lye Pro Gly Ser Ser Val Lye Val Ser Cyβ Lye Ala 35 40 45 Ser Gly Gly Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val Arg Gin Ala 50 55 60 Pro Gly Gin Gly Leu Glu Trp Met Gly Gly Ile Ile Pro Ile Phe Gly 65 70 75 80 Thr Ala Aβn Tyr Ala Gin Lys Phe Gin Gly Arg Val Thr Ile Thr Ala 85 90 95 Asp Glu Ser Thr Ala Arg Aβp Aβn Gly Ala Tyr Cyβ Ser Gly Gly Ser 100 105 110 Cyβ Tyr Ser Gly Trp Phe Asp Pro Trp Gly Gin Gly Thr Leu Val Thr 115 120 125 Val Ser Ser Ala Ser Thr Lye Gly Pro Ser Val Phe Pro Leu Ala Pro 130 135 140 Ser Ser Lye Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cyβ Leu Val 145 150 155 160 Lye Aβp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Aβn Ser Gly Ala 165 170 175 Leu Thr Ser Gly Val Hie Thr Phe Pro Ala Val Leu Gin Ser Ser Gly 180 185 190 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser λβp Lye 195 200 205 Lye Val Glu Pro Lys Ser Cyβ Aβp Lye Thr Hie Thr Cyβ Pro Pro Cyβ 210 215 220 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 225 230 235 240 Lye Pro Lye Aβp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cyβ 245 250 255 Val Val Val Aβp Val Ser Hie Glu Asp Pro Glu Val Lye Phe λβn Trp 260 265 270 Tyr Val Aβp Gly Val Glu Val His Aβn Ala Lye Thr Lye Pro Arg Glu 275 280 285 Glu Gin Tyr Aβn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300 Hie Gin Aβp Trp Leu Asn Gly Lys Glu Tyr Lye Cyβ Lys Val Ser Asn 305 310 315 320 Lye Ala Leu Pro Ala Pro Ile Glu Lye Thr Ile Ser Lye Ala Lye Gly 325 330 335 Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Aβp Glu 340 345 350 Leu Thr Lye Aβn Gin Val Ser Leu Thr Cyβ Leu Val Lye Gly Phe Tyr 355 360 365 Pro Ser Aep Ile Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Aβn 370 375 380 Aβn Tyr Lye Thr Thr Pro Pro Val Lβu Aβp Ser Aβp Gly Ser Phe Phe 385 390 395 400 Leu Tyr Ser Lye Leu Thr Val Asp Lye Ser Arg Trp Gin Gin Gly Aβn 405 410 415 Val Phe Ser Cyβ Ser Val Met Hie Glu Ala Leu Hie Aβn His Tyr Thr 420 425 430 Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1894 base pairβ (B) TYPE: nucleic acid (C) STRANDEDNESS: βingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: ATGGCGCTGT CCTGGGTTCT TACAGTCCTG AGCCTCCTAC CTCTGCTGGA AGCCCAGATC 60 CCATTGTGTG CCAACCTAGT ACCGGTGCCC ATCACCAACG CCACCCTGGA CCAGATCACT 120 GGCAAGTGGT TTTATATCGC ATCGGCCTTT CGAAACGAGG AGTACAATAA GTOGGTTCAG 180 GAGATCCAAG CAACCTTCTT TTACTTCACC CCCAACAAGA CAGAGGACAC GATCTTTCTC 240 AGAGAGTACC AGACCCGACA GGACCAGTGC ATCTATAACA CCACCTACCT GAATGTCCAG 300 CGGGAAAATG GGACCATCTC CAGATACGTG GGAGGCCAAG AGCATTTCGC TCACTTGCTG 360 ATCCTCAGGG ACACCAAGAC CTACATGCTT GCTTTTGACG TGAACGATGA GAAGAACTGG 420 GGGCTGTCTG TCTATGCTGA CAAGCCAGAG ACGACCAAGG AGCAACTGGG AGAGTTCTAC 480 GAAGCTCTCG ACTGCTTGCG CATTCCCAAG TCAGATGTCG TGTACACCGA TTGGAAAAAG 540 GATAAGTGTG AGCCACTGGA GAAGCAGCAC GAGAAGGAGA GGAAACAGGA GGAGGGGGAA 600 TCGGATCCCG AGGGTGAGTA CTAAGCTTCA GCGCTCCTGC CTGGACGCAT CCCGGCTATG 660 CAGCCCCAGT CCAGGGCAGC AAGGCAGGCC CCGTCTGCCT CTTCACCCGG AGCCTCTGCC 720 CGCCCCACTC ATCCTCAGGG AGAGGGTCTT CTGGCTTTTT CCCAGGCTCT GGGCAGGCAC 780 AGGCTAGGTG CCCCTAACCC AGGCCCTGCA CACAAAGGGG CAGGTGCTGG GCTCAGACCT 840 GCCAAGAGCC ATATCCGGGA GGACCCTGCC CCTGACCTAA GCCCACCCCA AAGGCCAAAC 900 TCTCCACTCC CTCAGCTCGG ACACCTTCTC TCCTCCCAGA TTCCAGTAAC TCCCAATCTT 960 CTCTCTGCAG AGCCCAAATC TTGTGACAAA ACTCACACAT GCCCACCGTG CCCAGGTAAG 1020 CCAGCCCAGG CCTCGCCCTC CAGCTCAAGG CGGGACAGGT GCCCTAGAGT AGCCTGCATC 1080 CAGGGACAGG CCCCAGCCGG GTGCTGACAC GTCCACCTCC ATCTCTTCCT CAGCACCTGA 1140 ACTCCTGGGG GGACCGTCAG TCTTCCTCTT CCCCCCAAAA CCCAAGGACA CCCTCATGAT 1200 CTCCCGGACC CCTGAGGTCA CATGCGTGGT GGTGGACGTG AGCCACGAAG ACCCTGAGGT 1260 CAAGTTCAAC TGGTACGTGG ACGGCGTGGA GGTGCATAAT GCCAAGACAA AGCCGCGGGA 1320 GGAGCAGTAC AACAGCACGT ACCGGGTGGT CAGCGTCCTC ACCGTCCTGC ACCAGGACTG 1380 GCTGAATGGC AAGGAGTACA AGTGCAAGGT CTCCAACAAA GCCCTCCCAG CCCCCATCGA 1440 GAAAACCATC TCCAAAGCCA AAGGTGGGAC CCGTGGGGTG CGAGGGCCAC ATGGACAGAG 1500 GCCGGCTCGG CCCACCCTCT GCCCTGAGAG TGACCGCTGT ACCAACCTCT GTCCTACAGG 1560 GCAGCCCCGA GAACCACAGG TGTACACCCT GCCCCCATCC CGGGATGAGC TGACCAAGAA 1620 CCAGGTCAGC CTGACCTGCC TGGTCAAAGG CTTCTATCCC AGCGACATCG CCGTGGAGTG 1680 GGAGAGCAAT GGGCAGCCGG AGAACAACTA CAAGACCACG CCTCCCGTGC TGGACTCCGA 1740 CGGCTCCTTC TTCCTCTACA GCAAGCTCAC CGTGGACAAG AGCAGGTGGC AGCAGGGGAA 1800 CGTCTTCTCA TGCTCCGTGA TGCATGAGGC TCTGCACAAC CACTACACGC AGAAGAGCCT 1860 CTCCCTGTCT CCGGGTAAAT GAGTGCGACG GCCG 1894 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 437 amino acidβ (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Met Ala Leu Ser Trp Val Leu Thr Val Leu Ser Leu Leu Pro Lβu Lβu 1 5 10 15 Glu Ala Gin Ile Pro Leu Cyβ Ala Aβn Leu Val Pro Val Pro lie Thr 20 25 30 Aβn Ala Thr Leu Aβp Gin lie Thr Gly Lye Trp Phe Tyr Ile Ala Ser 35 40 45 Ala Phe Arg λβn Glu Glu Tyr Aβn Lye Ser Val Gin Glu Ile βln Ala 50 55 60 Thr Phe Phe Tyr Phe Thr Pro λβn Lye Thr Glu Aβp Thr Ile Phe Leu 65 70 75 80 Arg Glu Tyr Gin Thr Arg Gin Asp Gin Cys lie Tyr Asn Thr Thr Tyr 85 90 95 Leu Asn Val Gin Arg Glu Aβn Gly Thr Ile Ser Arg Tyr Val Gly Gly 100 105 110 Gin Glu Hie Phe Ala Hie Leu Leu Ile Leu Arg λβp Thr Lye Thr Tyr 115 120 125 Met Leu Ala Phe Aβp Val Aβn Asp Glu Lys Asn Trp Gly Leu Ser Val 130 135 140 Tyr Ala Aβp Lye Pro Glu Thr Thr Lye Glu Gin Leu Gly Glu Phe Tyr 145 150 155 160 Glu Ala Leu Aβp Cyβ Leu Arg Ile Pro Lye Ser Aβp Val Val Tyr Thr 165 170 175 Aβp Trp Lye Lye Aβp Lye Cyβ Glu Pro Leu Glu Lye Gin His Glu Lye 180 185 190 Glu Arg Lye Gin Glu Glu Gly Glu Ser Aβp Pro Glu Gly Glu Pro Lye 195 200 205 Ser Cyβ Aβp Lye Thr Hie Thr Cyβ Pro Pro Cyβ Pro Ala Pro Glu Leu 210 215 220 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lye Pro Lye Aβp Thr 225 230 235 240 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cyβ Val Val Val Aβp Val 245 250 255 Ser Hie Glu Aβp Pro Glu Val Lye Phe Aβn Trp Tyr Val Asp Gly Val 260 265 270 Glu Val Hie Aβn Ala Lye Thr Lye Pro Arg Glu Glu Gin Tyr λβn Ser 275 280 285 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu Hie Gin Aβp Trp Leu 290 295 300 λβn Gly Lye Glu Tyr Lye Cys Lys Val Ser λβn Lye λla Leu Pro λla 305 310 315 320 Pro Ile Glu Lys Thr Ile Ser Lye λla Lye Gly Gin Pro Arg Glu Pro 325 330 335 Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lye λβn Gin 340 345 350 Val Ser Leu Thr Cyβ Leu Val Lye Gly Phe Tyr Pro Ser λβp Ile λla 355 360 365 Val Glu Trp Glu Ser Asn Gly Gin Pro Glu λβn λβn Tyr Lye Thr Thr 370 375 380 Pro Pro Val Leu λβp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lye Leu 385 390 395 400 Thr Val λβp Lys Ser Arg Trp Gin Gin Gly Aβn Val Phe Ser Cyβ Ser 405 410 415 Val Met Hie Glu λla Leu Hie λsn His Tyr Thr Gin Lye Ser Leu Ser 420 425 430 Leu Ser Pro Gly Lye 435 (2) INFORMATION FOR SEQ ID NO:12: (1) SEQUENCE CHARACTERISTICS: (λ) LENGTH: 442 amino acidβ (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Lye Leu Thr Thr Met λβp Trp Thr Trp λrg Phe Leu Phe Phe Val Val 1 5 10 15 λla λla λla Thr Gly Val Gin Ser Gin Val Gin Leu Val Gin Ser Gly 20 25 30 λla Glu Val Lye Lye Pro Gly Ser Ser Val Lye Val Ser Cyβ Lye λla 35 40 45 Ser Gly Gly Thr Phe Ser Ser Tyr λla Ile Ser Trp Val λrg Gin λla 50 55 60 Pro Gly Gin Gly Leu Glu Trp Met Gly Gly Ile Ile Pro Ile Phe Gly 65 70 75 80 Thr λla λβn Tyr λla Gin Lye Phe Gin Gly λrg Val Thr Ile Thr λla 85 90 95 λβp Glu Ser Thr λla λrg λβp λβn Gly λla Tyr Cyβ Ser Gly Gly Ser 100 105 110 Cyβ Tyr Ser Gly Trp Phe Asp Pro Trp Gly Gin Gly Thr Leu Val Thr 115 120 125 Val Ser Ser λla Ser Thr Lye Gly Pro Ser Val Phe Pro Leu λla Pro 130 135 140 Ser Ser Lye Ser Thr Ser Gly Gly Thr λla λla Leu Gly Cyβ Leu Val 145 150 155 160 Lye λβp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp λβn Ser Gly λla 165 170 175 Leu Thr Ser Gly Val Hie Thr Phe Pro λla Val Leu Gin Ser Ser Gly 180 185 190 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser λβp Lye 195 200 205 Lye Val Glu Pro Lye Ser Cyβ λβp Lye Thr Hie Thr Cyβ Pro Pro Cyβ 210 215 220 Pro λla Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 225 230 235 240 Lye Pro Lye λβp Thr Lβu Met Ile Ser λrg Thr Pro Glu Val Thr Cyβ 245 250 255 Val Val Val Asp Val Ser Hie Glu Aβp Pro Glu Val λβn Phe Ser Trp 260 265 270 Tyr Val λβp Gly Val Glu Val His λsn λβn Lye Thr Lye Pro λrg Glu 275 280 285 Glu λβn Tyr Ser Ser Thr Tyr λrg Val Val Ser Val Leu Thr Val Leu 290 295 300 Hie Gin λβp Trp Leu λβn Gly Lye Glu Tyr Lye Cyβ λβn Val Ser λβn 305 310 315 320 Lye λla Leu Pro λla Pro Ile Glu Lye λβn Ile Ser Lye λla Lye Gly 325 330 335 Gin Pro λrg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser λrg λβp Glu 340 345 350 Lβu Thr Lye λβn Gin Val Ser Leu Thr Cyβ Leu Val Lye Gly Phe Tyr 355 360 365 Pro Ser Asp Ile λla Val Glu Trp Glu Ser Asn Gly Gin Pro Glu λsn 370 375 380 λβn Tyr Lye Thr Thr Pro Pro Val Leu λβp Ser λβp Gly Ser Phe Phe 385 390 395 400 Leu Tyr Ser Lye Leu Thr Val λβp Lye Ser λrg Trp Gin Gin Gly λβn 405 410 415 Val Phe Ser Cyβ Ser Val Met Hie Glu λla Leu Hie λβn Hie Tyr Thr 420 425 430 Gin Lye Ser Leu Ser Leu Ser Pro Gly Lye 435 440 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 amino acidβ (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Pro Glu Met Lβu λrg λβn Ser Thr λsp Thr Thr Pro Leu Thr Gly 1 5 10 15 Pro Gly Thr Pro Glu Ser Thr Thr Val Glu Pro λla λla λrg λrg Ser 20 25 30 Thr Gly Leu λβp λla Gly Gly λla Val Thr Glu 35 40 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acidβ (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Leu Thr Thr Glu Leu Ala λsn Met Gly λβn Leu Ser Thr λβp Ser λla 1 5 10 15

Claims: CLAIMS - 48 - 1. An organic molecule to which there is covalently bonded a sialyl-Le x determinant and a sulfated determinant, at least one of these determinants being positioned at a non-naturally occurring site on said molecule. 2. The organic molecule of claim 1, wherein said molecule contains multiple sialyl-Le x determinants or multiple sulfated determinants. 3. The organic molecule of claim 1, wherein said molecule is soluble. 4. The organic molecule of claim 1, wherein said molecule compriεeε a P-εelectin ligand conεiεting essentially of amino acids 21-57 of Fig. 8A, or a portion thereof which is capable of mediating an interaction with the P selectin receptor. 5. The organic molecule of claim 4, wherein said molecule compriseε a P-εelectin ligand conεiεting essentially of amino acids 38-57 of Fig. 8A. 6. The organic molecule of claims 1 or 4, wherein said molecule compriseε α 1 -acid glycoprotein (AGP) . 7. The organic molecule of claimε 1 or 4, wherein εaid molecule compriεeε an antibody molecule. 8. A purified nucleic acid encoding any of the organic molecule of claims 4-7. 9. The purified nucleic acid of claim 8, wherein said nucleic acid further encodes either (a) α 1 -acid glycoprotein (AGP) or (b) an antibody molecule. 10. A vector comprising the nucleic acid of claim 8. 11. A cell comprising the purified nucleic acid of claim 8. 12. A method of inhibiting the binding of a cell bearing a P-selectin protein to a molecule or cell bearing a sialyl-Le x determinant and a εulfated determinant, said method comprising contacting said P- selectin protein-bearing cell with an organic molecule of claim 1. 13. The method of claim 12, wherein said organic molecule also inhibits the binding of a cell bearing an E-selectin protein to a molecule or cell bearing a sialyl-Le determinant. 14. A method of reducing inflammation in a mammal comprising administering to said mammal a therapeutically-effective amount of an organic molecule of claim 1. 15. A method for reducing or protecting a mammal against an extravasation-dependent adverse reaction, said method comprising administering to said mammal a therapeutically-effective amount of an organic molecule of claim 1. 16. The method of claim 15, wherein said extravasation-dependent adverse reaction is extravasation-dependent organ damage or clotting aεεociated with adult respiratory distress syndrome, glomerular nephritis, or ischemic myocardial injury. 17. A method for reducing or protecting a mammal against an adverse immune reaction, said method comprising administering to said mammal a therapeutically-effective amount of an organic molecule of claim 1. 18. The method of claim 17, wherein said adverse immune reaction is induced by a microbial factor or a host factor. 19. The method of claim 17, wherein said adverse immune reaction is septic shock or septicemia.

Inventors: POUYANI TARA, SEED BRIAN, POUYANI, TARA, SEED, BRIAN
Applicants: GEN HOSPITAL CORP, THE GENERAL HOSPITAL CORPORATION
Application year: 1996
Main classification: A61K38
IPC codes: A61K38, A61P5, A61P9, A61P11, A61P13, A61P15, A61P29, A61P31, A61P37, C07K14, C07K16, C12N15