WO2019178526A1 - Recombinant smnpp5 and methods of use - Google Patents

Abstract

Described herein are methods and compositions for reducing coagulation, e.g., in a subject having a coagulation disease or disorder. Aspects of the invention relate to administering to a subject a recombinant SmNPP-5 protein or pharmaceutical composition described herein. Other aspects of the invention relate to methods for producing a recombinant SmNPP-5 protein.

Description

RECOMBINANT SMNPP5 AND METHODS OF USE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional

Application No. 62/644,039 filed March 16, 2018, the contents of which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

[0002] The field of the invention relates to methods and compositions for reducing blood coagulation.

GOVERNMENT SUPPORT

[0003] This invention was made with Government support under Grant No. AI056273 awarded by the National Institutes of Health. The Government has certain rights in the invention.

SEQUENCE LISTING

[0004] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on March 15, 2019, is named 700355-09l550WOPT_Seq_ST25.txt and is 12,285 bytes in size.

BACKGROUND

[0005] Current anticoagulation therapies for treatment and/or prevention of thromboembolic disorders (such as deep vein thrombosis, pulmonary embolism and stroke prevention in patients with atrial fibrillation) include heparins (e.g., unfractionated heparin, low-molecular-weight heparins) and oral vitamin K antagonists. Though these therapies have proven benefits, they also have important limitations that result in their underuse in routine clinical practice. Heparins require parenteral administration and pose the risk of heparin-induced thrombocytopenia.

Vitamin K antagonists have a narrow separation of antithrombotic and hemorrhagic effects and numerous food and drug-drug interactions, and require frequent coagulation monitoring and dose adjustment to ensure effective antithrombotic protection while minimizing the risk of bleeding complications. In response to these limitations, it is important to develop new anticoagulants to treat patients who receive no, or inadequate treatment.

SUMMARY

[0006] The invention described herein, is due in part to the discovery that functional expression of an ectonucleotide pyrophosphatase/phosphodiesterase homolog, e.g., SmNPP5, that is expressed at the tegumental surface of intravascular Schistosoma mansoni provides an anti-coagulation effect. SmNPP-5 cleaves ADP, an essential step in preventing platelet aggregation and clot formation around the intravascular worm. SmNPP5 is a virulence factor for the Schistosoma mansoni worms, found to be expressed in all stages of development of the worm. Described herein is work that indicates that SmNPP5 inhibits platelet aggregation in a dose dependent manner, as measured by multiple electrode aggregometry (MEA) using whole blood. Unlike its mammalian homolog, NPP5, the schistosome protein cleaves ADP (with a Km of 246+/-34 mM) to act as an anti-coagulant. Additionally, provided herein are methods for producing recombinant SmNPP-5, e.g., for the use of reducing blood coagulation.

[0007] Accordingly, one aspect of the invention described herein provides a method for reducing blood coagulation in a subject, comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of a recombinant SmNPP-5 protein.

[0008] In one embodiment of any aspect, the recombinant SmNPP-5 is derived from the helminth Schistosoma mansoni. In one embodiment of any aspect, the recombinant SmNPP-5 is derived from the helminth Schistosoma japonicum or Schistosoma haematobium.

[0009] In one embodiment of any aspect, the recombinant SmNPP-5 protein comprises a sequence of SEQ ID NO: 1 or a polypeptide which has at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 1 and retains ADPase activity.

[0010] In one embodiment of any aspect, the recombinant SmNPP-5 protein is a truncated recombinant SmNPP-5 protein. In one embodiment of any aspect, the truncated recombinant SmNPP-5 protein comprises a sequence of SEQ ID NO: 2 or a polypeptide which has at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 2. In one embodiment of any aspect, truncated recombinant SmNPP-5 protein comprises at least 50% of the endogenous ADPase activity of a full length SmNPP-5 protein. [0011] In one embodiment of any aspect, the subject has a coagulation disorder. In one embodiment of any aspect, the coagulation disorder results in increased clotting. In one embodiment of any aspect, the coagulation disorder is selected from the group consisting of Factor V Leiden, Anti -thrombin III (A Till) deficiency, Protein C or Protein S deficiency, Prothrombin (PT) gene mutation, or Antiphospholipid antibody syndrome.

[0012] In one embodiment of any aspect, administration does not cause an immune response.

[0013] A second aspect of the invention described herein provides a polypeptide composition comprising a SmNPP-5 protein.

[0014] In one embodiment of any aspect, the SmNPP-5 protein is a recombinant SmNPP-5 protein.

[0015] A third aspect of the invention described herein provides a pharmaceutical composition comprising any of the SmNPP-5 polypeptide compositions described herein and a pharmaceutically acceptable carrier.

[0016] A fourth aspect of the invention described herein provides a nucleic acid encoding any of the polypeptide compositions described herein.

[0017] A fifth aspect of the invention described herein provides a vector comprising any of the nucleic acids described herein.

[0018] A sixth aspect of the invention described herein provides a cell comprising any of the nucleic acids described herein.

[0019] A seventh aspect of the invention described herein provides a cell comprising any of the vectors described herein.

[0020] An eighth aspect of the invention described herein provides a method of producing a recombinant protein, comprising providing a cell or an in vitro cell free transcription reaction mixture with a nucleic acid encoding a SmNPP-5 polypeptide under conditions suitable for transcription and/or translation of the nucleic acid. In one embodiment of any aspect, the method further comprises the step of purifying the SmNPP-5 polypeptide produced in this manner. In one embodiment of any aspect, the cell is any of the cells described herein.

[0021] A ninth aspect of the invention described herein provides a method for treating generalized arterial calcification of infancy (GACI) in a subject, comprising administering to a subject having GACI a composition comprising a therapeutically effective amount of a recombinant SmNPP-5 protein. [0022] A tenth aspect of the invention described herein provides a method for reducing blood coagulation in a subject, comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of a recombinant human NPP-5 protein, wherein the human NPP-5 protein comprises a tyrosine 73 to phenylalanine mutation. In one

embodiment, the human NPP-5 has ADPase activity and anti-coagulant activity.

[0023]

Definitions

[0024] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed technology, because the scope of the technology is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail

[0025] As used herein, the term "administering," refers to the placement of a therapeutic (e.g., a recombinant SmNPP-5 protein) or pharmaceutical composition as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent to the subject. Pharmaceutical compositions comprising agents as disclosed herein can be administered by any appropriate route which results in an effective result (e.g., a reduction of blood coagulation) in the subject.

[0026] As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include, for example, chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include, for example, mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include, for example, cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms,“individual,”“patient” and“subject” are used

interchangeably herein. [0027] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of disease e.g., blood coagulation disease or disorder. A subject can be male or female.

[0028] A subject can be one who has been previously diagnosed with or identified as suffering from or having a disease or disorder in need of treatment (e.g., a blood coagulation disease or disorder) or one or more complications related to such a disease or disorder, and optionally, have already undergone treatment for the disease or disorder or the one or more complications related to the disease or disorder. Alternatively, a subject can also be one who has not been previously diagnosed as having such disease or disorder (e.g., blood coagulation disease or disorder) or related complications. For example, a subject can be one who exhibits one or more risk factors for the disease or disorder or one or more complications related to the disease or disorder or a subject who does not exhibit risk factors.

[0029] As used herein,“blood coagulation” refers to the formation of a blood clot, whether in response to an injury (e.g., damage to a blood vessel) or as a result of a clotting disorder. As used herein, a“coagulation disease or disorder” refers to a disease or disorder characterized by a defect in the coagulation process resulting in either a failure to coagulate (e.g., in response to an injury), or excess or inappropriate clotting occurring, e.g., not in response to an injury. In one embodiment of any aspect, the coagulation disease or disorder results in increased or excess clotting in a subject. Exemplary coagulation diseases or disorders that result in increased coagulation include, but are not limited to thromboembolic disorders (such as deep vein thrombosis, pulmonary embolism, and stroke in patients with atrial fibrillation), Factor V Leiden, Anti-thrombin III (A Till) deficiency, Protein C or Protein S deficiency, Prothrombin (PT) gene mutation, and Antiphospholipid antibody syndrome.

[0030] As used herein, the terms“protein" and“polypeptide" are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein", and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. "Protein" and“polypeptide” are often used in reference to relatively large

polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

[0031] In some embodiments, a polypeptide described herein (or a nucleic acid encoding such a polypeptide) can be truncated, e.g., a functional fragment of one of the amino acid sequences described herein. As used herein, a“functional fragment” is a fragment or segment of a peptide which retains at least 50% of the wild type reference polypeptide’s activity according to an assay known in the art or described below herein. A functional fragment can comprise conservative substitutions of the sequences disclosed herein.

[0032] In some embodiments of any of the aspects, a nucleic acid encoding an SmNPP-5 protein can be a DNA or mRNA. In some embodiments of any of the aspects, a nucleic acid encoding an SmNPP-5 protein can be a modified DNA or mRNA, e.g., chemically modified to enhance stability or other beneficial characteristics. The nucleic acids described herein may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference. Modifications include, for example, (a) end modifications, e.g., 5’ end modifications (phosphorylation, conjugation, inverted linkages, etc.) 3’ end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases, (c) sugar modifications (e.g., at the 2’ position or 4’ position) or replacement of the sugar, as well as (d) backbone modifications, including modification or replacement of the phosphodiester linkages.

[0033] As used herein, the term“pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and/or ointment. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient would not be found to occur in in nature.

[0034] Methods and compositions described herein relate to the administration of SmNPP-5 protein or SmNPP-5 polypeptide. As used herein,“ Schistosoma mansoni NPP-5 (SmNPP-5)” refers to an ecto-phosphodiesterase that has ADPase activity and anti -coagulant activity encoded and expressed by the S. mansoni parasite. Though the“Sm” of SmNPP-5 is indicative of Schistosoma mansoni , the term“SmNPP-5” encompasses homologous proteins that have ADPase activity and anti-coagulant activity, e.g., from other Schistosome species, such as Schistosoma japonicum and Schistosoma haematobium. Sequences encoding a SmNPP-5 protein are known for a number of species, e.g., Schistosoma mansoni (GenBank Accession

#ACT29972), Schistosoma japonicum (GenBank Accession # CAX72650) and Schistosoma haematobium (GenBank Accession # XP_012799978). As used herein, an“SmNPP-5 polypeptide” refers to a polypeptide that has ADPase activity and anti-coagulant activity and has at least 50% sequence identity relative to SEQ ID NO: 1. The SmNPP-5 polypeptide can be a variant, a fragment, or an analog of SmNPP-5 that has ADPase and anti-coagulant activity, determined, for example, as described herein. A SmNPP-5 homolog described herein does not include a human SmNPP-5 homolog, e.g., ENPP-5, which comprises 38.2% sequence identity to S. mansoni SmNPP-5 and does not have ADPase activity or anti-coagulant activity. In one embodiment, a SmNPP-5 polypeptide comprises or consists essentially of the S. mansoni NPP-5 polypeptide fragment of SEQ ID NO: 2, or a polypeptide from another Schistosome species that corresponds to SEQ ID NO: 2.

[0035] The term“reduced”,“reduction”, or“inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments,“reduced”,“reduction”, or“inhibit” typically means a decrease by at least 10% as compared to an appropriate control (e.g. the absence of a recombinant SmNPP-5 protein or a pharmaceutical composition described herein) and can include, for example, a reduction by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein,“reduction” or

“inhibition” does not encompass a complete inhibition or reduction as compared to a reference level.“Complete reduction” is a 100% reduction as compared to an appropriate control.

[0036] As used herein, an“appropriate control” refers to an untreated, otherwise identical cell, population, or sample (e.g., a patient who was not administered a recombinant SmNPP-5 protein or composition described herein).

[0037] The term“statistically significant" or“significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.

[0038] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not. The term "consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0039] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is

synonymous with the term "for example."

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] This application file contains at least one drawing executed in color. Copies of this patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0041] FIG. 1A and FIG. IB present data that show purification of recombinant SmNPP-5 (rSmNPPS) by immobilized metal affinity chromatography (IMAC). (FIG. 1A) Fractions from the rSmNPPS purification scheme were resolved by SDS-PAGE and stained with Coomassie Blue. Lane 1, starting material - 48 hr culture medium; lane 2, flow through; lane 3, material washed from the column; lane 4, column eluate; lane 5 column eluate, after dialysis and concentration. The arrow shows rSmNPPS. M represents molecular size markers (kDa). (FIG

IB) Western blot analysis of SmNPPS glycosylation status. Recombinant SmNPPS or adult worm lysate (8m) was resolved by SDS-PAGE following treatment with PNGase F (+) or after no treatment (-), as indicated. The native protein resolves as a prominent band at ~55kDa (lane Sm right arrow) with a second band at ~60 kDa (lane Sm right arrowhead). Following incubation of the worm extract with PNGase F, this larger band disappears and a new, smaller band (at ~50 kDa) appears (lane Sm +). Incubation of rSmNPPS with PNGase F also leads to a notable shift in its migration profile; the broad band at ~ 60 kDa in the rSmNPPS lane (left arrow) disappears and a band running at ~ 50 kDa is now seen (rSmNPPS +, left arrowhead).

[0042] FIG. 2A- FIG. 2C present data that show characterization of rSmNPP-5. (FIG. 2A) Activity of rSmNPPS (filled circles) as measured by hydrolysis of the artificial substrate p- Nph-5'-TMP (whose chemical structure is depicted) at OD405. Heat treated rSmNPPS (triangles) displays no activity. (FIG. 2B) Relative activity of rSmNPPS in the presence of 0.5 - 5 mM of added metal cations (Mg2+, Ca2+ or Zn2+) or 5 mM ethyl enediaminetetraacetic acid (EDTA), as indicated. Activity of rSmNPPS in buffer lacking added metal cations (0) is set at 100%. (FIG. 2C) pH preference of rSmNPPS in the hydrolysis of p-Nph-5'~TMP.

[0043] FIG. 3A and FIG. 3B present data that show impact of rSmNPPS on platelet aggregation as determined by multiple electrode aggregometry (MEA). (FIG. 3 A)

Representative MEA traces following pre-incubation of blood samples with different

concentrations of rSmNPP-5 (0-40 pg) or heat inactivated (HI) rSmNPP-5 (40 pg), as indicated. Aggregation is measured in arbitrary' units (Y axis) over time (X axis). In the left panel, ADP (6 mM) is used as agonist and in the right panel, collagen (2 pg). Numbers at right represent pg SmNPPS added to the assay. (FIG. 3B) Combined MEA responses of blood exposed to different concentrations of r8mNPP-5 (0-40 pg) or of heat inactivated (HI) rSmNPP-5 (40 m_§), as indicated. Samples were either preincubated with rSmNPP5 for 45 min (+) or not (-), prior to addition of agonist ADP (6 isM) or collagen (2 pg). Results are expressed as Area-Under-the- Curve (AUC +/- SD, n 5) normalized to control (no recombinant protein added (0), set at 100 %). ANOVA, **P <0.001, ***P < 0.0001, versus control. [0044] FIG. 4A- FIG. 4C present data that show SmNPP-5 cleaves ADP. Decrease in ADP levels (mM +/- SD) (FIG. 4 A) and concurrent increase in free phosphate (Pi ) levels (pM +/- SD) (FIG. 4B) with time in the presence of different concentrations of rSmNPPS (0-5 pg), as indicated, but not in the presence of 5 _ug heat inactivated rSmNPPS (5, HI). In A, the chemical staicture of ADP is depicted. (FIG. 4C) Michaelis-Menton plot of SmNPPS-mediated .ADP cleavage kinetics; the Km of rSmNPPS for ADP is 245.9 ± 34 mM, derived from three independent experiments.

[0045] FIG. 5 presents data that show immimoloealization of SmNPPS in 1-day (left), 7- day (center) and 14-day (right) cultured schistosomu!a. Clear tegumental staining is evident in each case. The 7-day sehistosomulum is also stained with DAPI to reveal nuclei (blue). The bar represents 30 pm.

[0046] FIG. 6A and FIG. 6B present data that show SmNPPS activity. (FIG. 6A) SmNPPS activity (p-Nph-5 -TMP cleavage, OD405 +/- SD) observed in live schistosomula (groups of 1,000, filled triangles) compared to that detected in total lysates of equivalent numbers of schistosomula (open triangles). (FIG. 6B) SmNPPS activity detected in individual living adult parasites (males, filled circles; females, filled squares) compared to that detected in total lysates of individuals (males, open circles; females, open squares). N >10 in each case.

[0047] FIG. 7 A and FIG. 7B present data that show adult male schistosomes cleave exogenous ADP. Decrease in ADP levels (mM +/- SD) (FIG. 7A) and concurrent increase in free phosphate (Pi) levels (mM +/- SD) (FIG. 7B) with time only in the presence (+) of individual adult male worms (N =5).

[0048] FIG. 8 presents the amino acid sequence alignment for .S’, mansoni, S.

haematobium , and S. japonicum.

[0049] FIG. 9A-FIG. 9C present data that SmNPPS cleaves Ap3A (diadenosine triphosphate). FIG. 9A shows thin layer chromatographic resolution of Ap3A cleavage by SmNPPS. FIG. 9B is a cropped version of Fig. 9A, showing thin layer chromatographic resolution of Ap3 A before (-) and after (+) 1 and 24 h incubation in the presence of SmNPP5, as indicated. After 1 h incubation with SmNPPS Ap3 A is no longer seen while reaction products (AMP and ADP) are visible. After 24 h incubation much of the ADP too has been cleaved by SmNPPS yielding increased levels of AMP. The left panel shoves the pattern of resolution of standards, and the right panel shows reaction products. FIG. 9€ is a schema depicting the reactions described in FIG. 9A and FIG. 9B. Without wishing to be bound by theory, it is proposed that SmNPPS cleaves Ap3 A (dotted line) into ADP and AMP, and then SmNPPS cleaves ADP (dotted line) into AMP and Pi.

[0050] FIG. 10A-FIG. IOC present data that SmNPPS cleaves Ap4A (diadenosine tetraphosphate). FIG. 10A shows thin layer chromatographic resolution of Ap4A cleavage by SmNPPS. FIG. 10B is a cropped version of Fig. 10A, showing thin layer chromatographic resolution of Ap4A before (-) and after (+) 1 and 24 h incubation in the presence of SmNPPS, as indicated. After 0.5 h incubation with SmNPP5 Ap4A is no longer seen while reaction products (AMP and ATP) are visible. With increasing incubation time (1 h and 24 h) much of the ATP too has been cleaved by SmNPPS yielding increased levels of AMP. The left portion of the panel shows the pattern of resolution of standards and on the right is the reaction products. FIG. 10C is a schema depicting the reactions described in FIG. 10A and FIG. 10B. Without wishing to be bound by theory, it is proposed that SmNPPS cleaves Ap4A (dotted line) into ATP and AMP, and then SmNPPS cleaves ATP (dotted line) into AND⁵ and PPL

DETAILED DESCRIPTION

[0051] Degradation of ADP has been shown to be essential to prevent platelet aggregation and clot formation around the intravascular Schistosoma mansoni worms. The invention described herein, is based in part on the discovery that functional expression of an ecto- nucleotide pyrophosphatase/phosphodiesterase homolog, e.g., SmNPP5, that is expressed at the tegumental surface of intravascular Schistosoma mansoni, provides an anti-coagulation effect.

[0052] SmNPP5, a known virulence factor for Schistosoma mansoni worms, which are the etiological agent of the parasitic disease, schistosomiasis, is found to be expressed in all stages of development of the worm. Described herein is work indicating SmNPP5 is a Type One glycoprotein that cleaves the artificial substrate / Nph-5’-TMP in a reaction that requires cations and an optimal pH of 9. SmNPP5 inhibits platelet aggregation in a dose dependent manner, as measured by multiple electrode aggregometry (MEA) using whole blood. Inhibition is apparent when either collagen or ADP is used as agonist of SmNpPP-5 but is lost following heat treatment of recombinant SmNPP5 protein. Unlike its mammalian homolog, NPP5, which is inactive against both p-nitrophenyl-TMP and ADP, the Schistosome protein cleaves ADP and with a Km of 246+/-34 mM, to act as an anti-coagulant. A second mammalian homolog, NPP4, acts to promote, rather than impede, blood clotting. [0053] Additionally, provided herein are methods for producing recombinant SmNPP-5. Work described herein shows that expression of recombinant full length SmNPP-5 and truncated SmNPP-5 that retains the ADPase function as anti-coagulants and can be used therapeutically to reduce blood coagulation.

[0054] Hypercoagulation

[0055] One aspect of the invention described herein is a method for reducing blood coagulation comprising administering a recombinant SmNPP-5 protein to a subject in need thereof.“Blood coagulation” involves the activation, adhesion, and aggregation of platelets, e.g., a thrombocyte. As used herein, a“coagulation disease or disorder” refers to defects in the coagulation process resulting in a failure to coagulate (e.g., in response to an injury), or excess or inappropriate clotting occurring, (e.g., not in response to an injury). In one embodiment of any aspect, the coagulation disease or disorder results increased or excess clotting in a subject.

Exemplary coagulation disease or disorders that result in increased coagulation include, but are not limited to Factor V Leiden, Anti -thrombin III (A Till) deficiency, Protein C or Protein S deficiency, Prothrombin (PT) gene mutation, and Antiphospholipid antibody syndrome.

[0056] Excessive blood clotting, e.g., hypercoagualtion, is a condition in which blood clots form too easily or don't dissolve properly. Normally, blood clots form to seal small cuts or breaks on blood vessel walls and stop bleeding. Slow blood flow in the blood vessels also can cause blood clots to form. For example, if a blood vessel narrows, blood may slow down as it moves through the vessel. Excessive blood clotting has many causes, e.g., problems with the blood, blood vessel defects or damage, or other factors described herein can cause the condition. Blood clots can limit or block blood flow, resulting in damage organs, e.g., heart, or brain, and can result in death.

[0057] Factors that promote excessive blood clotting can be acquired or genetic; acquired causes of excessive blood clotting are more common than genetic causes. As used herein, an "acquired clotting disorder" is one in which another disease, condition, or factor triggers the condition. For example, atherosclerosis, e.g., a disease which results in plaque build-up inside the arteries, can damage the blood vessels, resulting in the formation of blood clots. Other acquired causes of excessive blood clotting include smoking, overweight and obesity, and being unable to move around much (for example, if you're in the hospital). [0058] Genetic defects that cause excessive blood clotting typically result from a mutation in the genes and/or proteins required for blood clotting. Genetic mutations can also occur with the substances that delay or dissolve blood clots. Exemplary coagulation disease or disorders caused by genetic mutations that result in increased coagulation include, but are not limited to Factor V Leiden, Anti-thrombin III (A Till) deficiency, Protein C or Protein S deficiency, Prothrombin (PT) gene mutation, and Antiphospholipid antibody syndrome.

[0059] A subject at highest risk for excessive blood clotting have both acquired and genetic causes.

[0060] Subjects at risk of a blood clot

[0061] In one embodiment, any treatment described herein, e.g., to reduce blood coagulation, can be administered to a subject who is at risk of developing a coagulation disorder, e.g., that results in hypercoagulation.

[0062] Risk factors for hypercoagulation, e.g., blood clot formation, include, but are not limited to immobility or lack of exercise (e.g., due to hospitalization, bed rest, paralysis, or prolonged sitting, e.g., during long airplane rides), surgery or trauma (e.g., major surgery

(especially of the pelvis, abdomen, hip, or knee), bone fracture or cast, or catheter in a large vein, e.g., PICC line, central venous catheter, or port), increased estrogen (e.g., due to birth control, e.g., pills, patches, or rings, pregnancy or post-pregnancy, or estrogen and/or progestin hormone therapy), certain medical conditions (e.g., cancer and chemotherapy, heart failure, atrial fibrillation, inflammatory disorders (e.g., lupus, rheumatoid arthritis, inflammatory bowel disease) or nephrotic syndrome), previous blood clots, family history of blood clots, clotting disorder (inherited or acquired), obesity, older age, cigarette and/or tobacco use, and varicose veins. A subject having genetic mutations in proteins and/or genes known to promote blood coagulation (e.g., blood clot formation) or dissolve a formed blood clot is at risk of developing a coagulation disease or disorder.

[0063] A skilled practitioner can identify a subject at risk of developing a coagulation disorder using techniques known in the art.

[0064] Reducing Blood Coagulation

[0065] One aspect of the invention described herein provides a method for reducing blood coagulation in a subject in need thereof comprising administering a therapeutically effective amount of recombinant SmNPP-5 protein. [0066] In one embodiment, the subject has been diagnosed with a coagulation disease or disorder. In one embodiment, the coagulation disease or disorder results in excessive clotting.

[0067] In one embodiment, a therapeutically effective amount of recombinant SmNPP-5 protein is administered systemically to a subject prior to, during, or after surgery (e.g., to prevent excess or inappropriate clotting). In one embodiment, a therapeutically effective amount of recombinant SmNPP-5 protein is administered locally prior to, during or after surgery (e.g., to prevent excess or inappropriate clotting in the area in which the protein was administered).

[0068] In one embodiment, administration of the recombinant SmNPP-5 protein results in a reduction of blood coagulation by at least 10% as compared to an appropriate control. In another embodiment, administration of the recombinant SmNPP-5 protein results in a reduction of blood coagulation by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more as compared to an appropriate control. As used herein, an appropriate control can refer, for example, to the level of blood coagulation in the subject prior to the administration. A skilled practitioner can measure the level of coagulation in a subject, e.g., prior to, during, and/or after administration of the recombinant SmNPP-5 using standard techniques known in the art, e.g., blood work to measure platelet count, or clotting time.

[0069] In one embodiment, a polypeptide composition comprising a SmNPP-5 protein is administered to a subject to reduce blood coagulation. In another embodiment, a pharmaceutical composition comprising polypeptide composition comprising a SmNPP-5 protein is administered to subject to reduce blood coagulation. In another embodiment, a nucleic acid encoding a polypeptide composition comprising a SmNPP-5 protein is administered to a subject to reduce blood coagulation. In one embodiment, administration results in a reduction of blood coagulation by at least 10% as compared to an appropriate control.

[0070] Generalized arterial calcification of infancy (GACI)

[0071] In one embodiment, a therapeutically effective amount of recombinant SmNPP-5 protein is administered to a subject having GACI. Generalized arterial calcification of infancy (GACI) is a rare, genetic disease characterized by an abnormal buildup of calcium in the walls of the blood vessels, including those that carry blood from the heart to the rest of the body. GACI affects infant and young children, and can be diagnosed by a skilled person, e.g., by the presence of symptoms including, but not limited to high blood pressure, heart failure, or kidney failure. Tests for diagnosing a subject with GACI include, but are not limited to standard X-rays, computerized axial tomography (CT scan), or MRI, e.g., to determine if calcification or stenosis (narrowing) in the arteries is present.

[0072] Two forms of GACI, Type 1 and Type 2, exist. Type 1, the more common form, is caused by mutations in the ENPP1 gene. Type 2 is caused by mutations in the ABCC6 gene. In one embodiment, the recombinant SmNPP-5 protein is administered to treat Type 1 GACI. In another embodiment, the recombinant SmNPP-5 protein is administered to treat Type 2 GACI.

[0073] In one embodiment, the recombinant SmNPP-5 protein is administered in

combination with at least one additional treatment for GACI. Treatments for GACI include, but are not limited to a bisphosphonate, a class of drugs typically used to treat osteoporosis, to reduce the calcium buildup.

[0074] Recombinant SmNPP-5 protein

[0075] In one aspect of the invention described herein, a subject is administered a recombinant SmNPP-5 protein. In one embodiment, the recombinant SmNPP-5 protein is derived from the Schistosome genus. In one embodiment, the recombinant SmNPP-5 protein is derived from the helminth Schistosoma mansoni. The SmNPP-5 protein can alternatively be derived, for example, from other Schistosome species, e.g., S. japonicum or S. haematobium.

[0076] In one embodiment, the recombinant SmNPP-5 protein corresponds to the amino acid sequence encoding the SmNPP-5 protein (SEQ ID NO: 1; S. mansoni SmNPP-5; see GenBank Accession #ACI29972).

MYCIETMQKMI ILLLICFFPYIERIYASGVVGKEQFSKVILISLDGFRYDYFDMAKQRNINM SAFDKI INQGVYIRRIENEFPTLTFPSHFSIVTGLHPGSHGIVDNVFYDPI INATFSSRNQS TATDSRFYDVGAEPIWVTNQFHGHKSGVTFWIGSEAI IKGERPTHYLTPYNESITFTQRIDI LMDWFEHENINLGLMYYHQPDRAGHIHGAASDEVFKAIEEINHGLEYLLTSIEMRPSLSCCL NLI ITSDHGMTNISSDRVIYLHDYIHPNEYISAPKKSAEIWTLWPKQGYTVRSLYNKLKDRH FRLNVYLKEELPTRFFYGSSDRVGPVVVYADIGWTI IADRTSGITLKNKGAHGYDPDYKEMS PFLMAMGPQIAKSQPTELKESIKLIDIYSLICLMLDLEPAPNNGSVCRVLPLLSQGSFANIN RLS11FI IKFI ILSIFMVHNGLYY (SEQ ID NO: 1)

[0077] In one embodiment, the recombinant SmNPP-5 protein comprises a sequence of SEQ ID NO: 1, or a polypeptide which has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater sequence identity to the sequence of SEQ ID NO: 1 and retains anti-coagulation activity and/or ADP cleavage activity.

[0078] In one embodiment, the recombinant SmNPP-5 protein is a truncated recombinant SmNPP-5 protein. In one embodiment, the truncated recombinant SmNPP-5 protein comprises at least 85% of the endogenous ADPase activity of full length S. Mansoni SmNPP-5 protein. In another embodiment, the truncated recombinant SmNPP-5 protein comprises at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the endogenous ADPase activity of a full length SmNPP-5 protein. One skilled in the art will be able to measure the ADPase activity of a truncated recombinant SmNPP-5 protein and full length SmNPP-5 protein, and compare the two, using a biological assay, e.g., an assay for ADPase activity as described herein or multiple electrode aggregometry using, e.g., a whole blood sample.

[0079] In one embodiment, the truncated recombinant SmNPP-5 protein corresponds to the amino acid sequence of (SEQ ID NO: 2)

GKEQFSKVILISLDGFRYDYFDMAKQRNINMSAFDKI INQGVYIRRIENEFPTLTFPSHFSI VTGLHPGSHGIVDNVFYDPI INATFSSRNQSTATDSRFYDVGAEPIWVTNQFHGHKSGVTFW IGSEAI IKGERPTHYLTPYNESITFTQRIDILMDWFEHENINLGLMYYHQPDRAGHIHGAAS DEVFKAIEEINHGLEYLLTSIEMRPSLSCCLNLI ITSDHGMTNISSDRVIYLHDYIHPNEYI SAPKKSAEIWTLWPKQGYTVRSLYNKLKDRHFRLNVYLKEELPTRFFYGSSDRVGPWVYAD IGWTI IADRTSGITLKNKGAHGYDPDYKEMSPFLMAMGPQIAKSQPTELKESIKLIDIYSLI CLMLDLEPAPNNGSVCRVLPLLSQGS (SEQ ID NO: 2)

[0080] In one embodiment, the truncated recombinant SmNPP-5 protein comprises a sequence of SEQ ID NO: 2, or a polypeptide which has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater sequence identity to the sequence of SEQ ID NO: 2.

[0081] The conserved phenylalanine (F 87, e.g., the 87^th amino acid of SEQ ID NO: 1) is required for the ADPase activity and anti-coagulant activity of SmNPP-5. F-87 is conserved amongst the S. mansoni, S. haematobium, and S. japonicum. It is contemplated herein that truncations, fragments, variants, or analogs of SmNPP-5 or SmNPP-5 polypeptides must contain the conserved F 87. [0082] The human homolog of NPP-5 does not comprise ADPase activity or anti-coagulant activity, however a mutation that changes tyrosine 73 to phenylalanine (Y73F) results in the capacity for the human NPP-5 homolog to hydrolyze ADP. It is contemplated herein that the recombinant human NPP-5^Y73F protein can be used in the same manner described herein for SmNPP-5.

[0083] In one aspect of the invention described herein is a method for reducing blood coagulation in a subject, comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of a recombinant human NPP-5 protein, wherein the recombinant human NPP-5 protein comprises a tyrosine 73 to phenylalanine (Y73F) mutation. In one embodiment, the recombinant human NPP-5^Y73F protein has ADPase activity and anti-coagulant activity.

[0084] In one embodiment, the recombinant human NPP-5^Y73F protein corresponds to the amino acid sequence of (SEQ ID NO: 5).

MTSKFLLVSF ILAALSLSTT FSLQPDQQKV LLVSFDGFRW DYLYKVPTPH FHYIMKYGVH VKQVTNVFIT KTFPNHYTLV TGLFAENHGI VANDMFDPIR NKSFSLDHMN IYDSKFWEEA TPIWITNQRA GHTSGAAMWP GTDVKIHKRF PTHYMPYNES VSFEDRVAKI IEWFTSKEPI NLGLLYWEDP DDMGHHLGPD SPLMGPVISD IDKKLGYLIQ MLKKAKLWNT LNLI ITSDHG MTQCSEERLI ELDQYLDKDH YTLIDQSPVA AILPKEGKFD EVYEALTHAH PNLTVYKKED VPERWHYKYN SRIQPIIAVA DEGWHILQNK SDDFLLGNHG YDNALADMHP IFLAHGPAFR KNFSKEAMNS TDLYPLLCHL LNITAMPHNG SFWNVQDLLN SAMPRWPYT QSTILLPGSV KPAEYDQEGS YPYFIGVSLG SIIVIVFFVI FIKHLIHSQI PALQDMHAEI AQPLLQA (SEQ ID NO: 5)

[0085] The underlined text in SEQ ID NO: 5 highlights the Y73F mutation.

[0086] In one embodiment, the recombinant human NPP-5^Y73F protein comprises a sequence of SEQ ID NO: 5, or a polypeptide which has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater sequence identity to the sequence of SEQ ID NO: 5.

[0087] In one embodiment, the recombinant human NPP- 5^Y73F protein is a truncated recombinant human NPP-5^Y73F protein that retains its ADPase activity or anti-coagulant activity. In one embodiment, the recombinant human NPP-5^Y73F protein is a functional fragment thereof that retains its ADPase activity or anti-coagulant activity.

[0088] In one embodiment, administration of recombinant SmNPP-5 does not cause an immune response in the subject. An immune response can be for example raising antibodies to the recombinant protein or provoking an allergic or inflammatory response. One of skilled in the art would know how to determine if any given SmNPP-5 protein provokes such a response.

[0089] Diadenosine polyphosphates

[0090] Diadenosine polyphosphates (APnAs, n = 3-6) are a family of endogenous vasoactive purine dinucleotides which have been isolated from thrombocytes. APnAs have been

demonstrated to be involved in the control of vascular tone as well as the growth of vascular smooth muscle cells and hence, possibly, in atherogenesis. APnAs isolated substances are Ap3 A, Ap4A, Ap5A, and Ap6A. APnAs are naturally occurring substances that facilitate tear secretion; they are released from the corneal epithelium, they stimulate tear production and therefore they may be considered as physiological modulators of tear secretion.

[0091] APnAs have emerged as intracellular and extracellular signaling molecules implicated in the maintenance and regulation of vital cellular functions and become considered as second messengers. The role of ApnAs as a second messenger has recently been discovered in The LysRS-Ap4A-MITF signaling pathway. APnAs are polyphosphated nucleotidic substances which are found in the CNS and are known to be released in a calcium-dependent manner from storage vesicles in brain synaptosomes. AP3 A and AP4A are canonical ATPase inhibitors.

[0092] Ap3 A is a platelet-dense granule component released into the extracellular space during the second wave of platelet aggregation on activation. AP3 A is a primer for

oligoadenylate synthesis catalyzed by interferon-inducible 2-5A synthetase. AP3 A is synthesized in cells by tryptophanyl-tRNA synthetase (WRS); cellular level of AP3 A significantly increases after interferon treatment. AP3 A is an avid inhibitor of eosinophil-derived neurotoxin (EDN).

[0093] AP4A is the only APnA that can induce a considerable increase in [Ca²⁺] in endothelial cells, indicating that its vasoactive effects are comparable to the known effects of arginine vasopressin, Angiotensin II, and ATP. AP4A is a ubiquitous ApnA is a signal molecule for DNA replication in mammalian cells. AP4A is a primer for oligoadenylate synthesis catalyzed by interferon-inducible 2-5A synthetase. AP4A is an avid inhibitor of eosinophil- derived neurotoxin (EDN). Ap4A is a putative alarmone, ubiquitous in nature being common to everything from bacteria to humans. AP4A has been shown to be a competitive inhibitor of ADP-induced platelet aggregation.

[0094] Polypeptide compositions

[0095] One aspect of the invention described herein provides a polypeptide composition comprising a SmNPP-5 protein.

[0096] A SmNPP-5 polypeptide can comprise SEQ ID NO: 1 or a homolog, variant, and/or functional fragment thereof that retains ADPase activity and anti-coagulant activity. In some embodiments, a SmNPP-5 polypeptide is a truncated SmNPP-5 polypeptide and can comprise amino acids 32 to 429 of SEQ ID NO: 1 (i.e. contains essentially only the ADPase activity domain of the SmNPP-5 protein), or a homolog, variant, and/or functional fragment thereof. In some embodiments, a SmNPP-5 polypeptide as described herein can be a homolog, derivative, variant, conservative substitution variant, deletion mutant, insertion mutant, or functional fragment of the amino acid sequences described above herein, that retains ADPase activity and anti-coagulation activity.

[0097] As used herein, a“truncated polypeptide” (e.g., a“functional fragment") of, e.g. SEQ ID NO: 1, is a fragment or segment of that polypeptide which comprises at least 50% of the ADPase activity as the reference polypeptide (i.e. SEQ ID NO: 1), e.g. at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% of its ADPase activity. A functional fragment can comprise conservative substitutions of the sequences disclosed herein.

[0098] Variants of the isolated peptides described herein can be obtained by mutations of native nucleotide or amino acid sequences, for example SEQ ID NO: 1 or a nucleotide sequence encoding a peptide comprising SEQ ID NO: 1. A“variant," as referred to herein, is a polypeptide substantially homologous to a SmNPP-5 polypeptide described herein (e.g. SEQ ID NO: 1), but which has an amino acid sequence different from that of one of the sequences described herein because of one or a plurality of deletions, insertions or substitutions.

[0099] The amino acid sequence conservation between S. mansoni, S. haematobium, and S. japonicum is shown in FIG. 8. Those amino acids conserved between the three species would be less likely to tolerate a substitution with non-conservative amino acids. Those amino acids that are not conserved between the three species would be more likely to tolerate substitutions with non-conservative amino acids. [00100] A homolog of a SmNPP-5 polypeptide as described herein can also comprise amino acid sequences that are homologous to the regions of SmNPP-5 comprised by the SmNPP-5 polypeptide described herein.

[00101] A variant amino acid or DNA sequence preferably is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to the sequence from which it is derived (referred to herein as an“original" sequence). The degree of homology between an original and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web. The variant amino acid or DNA sequence preferably is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, similar to the sequence from which it is derived (referred to herein as an“original" sequence). The degree of similarity (percent similarity) between an original and a mutant sequence can be determined, for example, by using a similarity matrix. Similarity matrices are well known in the art and a number of tools for comparing two sequences using similarity matrices are freely available online, e.g. BLASTp (available on the world wide web at http://blast.ncbi.nlm.nih.gov).

[00102] Alterations of the original amino acid sequence can be accomplished by any of a number of known techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations include those disclosed by Walder et al. (Gene 42: 133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are herein incorporated by reference in their entireties. Other approaches are known to those of skill in the art. In some embodiments, an isolated peptide as described herein can be chemically synthesized and mutations can be incorporated as part of the chemical synthesis process.

[00103] Variants can comprise conservatively substituted sequences, meaning that one or more amino acid residues of an original peptide are replaced by different residues with similar properties to the residue(s) replaced, and that the conservatively substituted peptide retains a desired biological activity, i.e., the ability to cleave ADP or inhibits coagulation, that is at least 50% of the original peptide. Examples of conservative substitutions include substitutions that do not change the overall or local hydrophobic character, substitutions that do not change the overall or local charge, substitutions by residues of equivalent sidechain size, or substitutions by sidechains with similar reactive groups.

[00104] A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Val, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gln and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics or substitutions of residues with similar sidechain volume are well known. Isolated peptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity, e.g. the ability to cleave ADP, is retained, as determined by the assays described elsewhere herein.

[00105] Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile, Phe, Trp; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln, Ala, Tyr, His, Pro, Gly; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe, Pro, His, or hydroxyproline. Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[00106] Particularly preferred conservative substitutions for use in the variants described herein are as follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gln or into His; Asp into Glu or into Asn; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gln; Ile into Leu or into Val; Leu into Ile or into Val; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or into Ile; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr or into Phe; Tyr into Phe or into Trp; and/or Phe into Val, into Tyr, into Ile or into Leu. In general, conservative substitutions encompass residue exchanges with those of similar physicochemical properties (i.e. substitution of a hydrophobic residue for another hydrophobic amino acid).

[00107] Any cysteine residue not involved in maintaining the proper conformation of the isolated peptide as described herein can also be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to the isolated peptide as described herein to improve its stability or facilitate multimerization.

[00108] One aspect of the invention described herein provides a pharmaceutical composition comprising any of the polypeptide compositions described herein and a pharmaceutically acceptable carrier.

[00109] Nucleic acids and expression systems

[00110] One aspect of the invention described herein provides a nucleic acid encoding any of the polypeptide compositions described herein. Nucleic acid molecules encoding any of the polypeptide compositions described herein are prepared by a variety of methods known in the art. These methods include, but are not limited to, PCR, ligation, and direct synthesis. A nucleic acid sequence encoding a polypeptide as described herein can be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are well known and disclosed, e.g., by Maniatis et ah, Molecular Cloning, Lab. Manual (Cold Spring Harbor Lab. Press, NY, 1982 and 1989), and Ausubel, 1987, 1993, and can be used to construct nucleic acid sequences which encode a SmNPP-5 polypeptide composition as described herein.

[00111] The term“vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells. A vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al. , Proc. Natl. Acad. Sci. USA (1995) 92: 1292).

[00112] In one aspect, the technology described herein relates to an expression vector comprising a nucleic acid encoding any of polypeptide compositions described herein. Such vectors can be used, e.g. to transform a cell in order to produce the encoded polypeptide. As used herein, the term "expression vector” refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian cells for expression and in a prokaryotic host for cloning and amplification. The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The gene may or may not include regions preceding and following the coding region, e.g. 5’ untranslated (5’UTR) or "leader" sequences and 3’ UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[00113] By“recombinant vector" is meant a vector that includes a heterologous nucleic acid sequence, or“transgene" that is capable of expression in vivo. It should be understood that the vectors described herein can, in some embodiments, be combined with other suitable

compositions and therapies. Vectors useful for the delivery of a sequence encoding an isolated peptide as described herein can include one or more regulatory elements (e.g., promoter, enhancer, etc.) sufficient for expression of the isolated peptide in the desired target cell or tissue. The regulatory elements can be chosen to provide either constitutive or regulated/inducible expression. As used herein, the term“viral vector" refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain the nucleic acid encoding an antibody or antigen binding portion thereof as described herein in place of non-essential viral genes. The vector and/or particle may be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[00114] Examples of vectors useful in delivery of nucleic acids encoding isolated peptides as described herein include plasmid vectors, non-viral plasmid vectors (e.g. see 6,413,942, 6,214,804, 5,580,859, 5,589,466, 5,763,270 and 5,693,622, all of which are incorporated herein by reference in their entireties); retroviruses (e.g. see U.S. Pat. No. 5,219,740; (1991) Virology 180:849-52; Miller et ah, Meth. Enzymol. 217:581-599 (1993); Burns et al. (1993) Proc. Natl. Acad. Sci. ETSA 90:8033-37; Boris-Lawrie and Temin (1993) Curr. Opin. Genet. Develop. 3: 102-09. Boesen et al ., Biotherapy 6:291-302 (1994); Clowes et al ., J. Clin. Invest. 93:644-651 (1994); Kiem et al ., Blood 83: 1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4: 129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993), the contents of each of which are herein incorporated by reference in their entireties); lentiviruses (e.g., see U.S. Patent Nos. 6,143,520; 5,665,557; and 5,981,276, the contents of which are herein incorporated by reference in their entireties; adenovirus-based expression vectors (e.g., see Bett e/ al. (1993) J. Virol. 67:5911-21; Mi ttereder e/ al. (1994) Human Gene Therapy 5:717-29; Seth et al. (1994) J. Virol. 68:933-40; Barr et al. (1994) Gene Therapy 1 :51- 58; Berkner, K. L. (1988) BioTechniques 6:616-29; and Rich et al. (1993) Human Gene Therapy 4:461-76; Wu et al. (2001) Anesthes. 94: 1119-32; Parks (2000) Clin. Genet. 58: 1-11; Tsai et al. (2000) Curr. Opin. Mol. Ther. 2:515-23; and U.S. Pat. No. 6,048,551; 6,306,652and 6,306,652, incorporated herein by reference in their entireties); Adeno-associated viruses (AAV) (e.g. see U.S. Pat. Nos. 5,139,941; 5,622,856; 5,139,941; 6,001,650; and 6,004,797, the contents of each of which are incorporated by reference herein in their entireties); and avipox vectors (e.g. see WO 91/12882; WO 89/03429; and WO 92/03545; which are incorporated by reference herein in their entireties).

[00115] Useful methods of transfection can include, but are not limited to electroporation, sonoporation, protoplast fusion, peptoid delivery, or microinjection. See, e.g., Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories, New York, for a discussion of techniques for transforming cells of interest; and Felgner, P. L. (1990) Advanced Drug Delivery Reviews 5: 163-87, for a review of delivery systems useful for gene transfer. Exemplary methods of delivering DNA using electroporation are described in U.S. Pat. Nos. 6,132,419; 6,451,002, 6,418,341, 6,233,483, U.S. Patent Publication No. 2002/0146831, and International Publication No. WO/0045823, all of which are incorporated herein by reference in their entireties.

[00116] The recombinant polypeptides described herein can be produced in, e.g., eukaryotic or prokaryotic cells.

[00117] Non-limiting examples of vectors useful for expression in prokaryotic cells can include plasmids. Plasmid vectors can include, but are not limited to, pBR322, pBR325, pACYCl77, pACYCl84, pUC8, pUC9, pUCl8, pUCl9, pLG339, pR290, pKC37, pKClOl, SV 40, pBluescript II SK +/- or KS +/- (see“Stratagene Cloning Systems" Catalog (1993) from Stratagene, La Jolla, Calif, which is hereby incorporated by reference), pQE, pIH82l, pGEX, pET series (see Studier et. ah,“Use of T7 RNA Polymerase to Direct Expression of Cloned Genes," Gene Expression Technology , vol. 185 (1990), which is hereby incorporated by reference in its entirety).

[00118] In some embodiments, the polypeptide can be constitutively expressed. In some embodiments, nucleic acids encoding the polypeptide can be operatively linked to a constitutive promoter. In some embodiments, the polypeptide can be inducibly expressed. In some embodiments, nucleic acids encoding the polypeptide can be operatively linked to an inducible promoter. As described herein, an“inducible promoter" is one that is characterized by initiating or enhancing transcriptional activity when in the presence of, influenced by, or contacted by an inducer or inducing agent than when not in the presence of, under the influence of, or in contact with the inducer or inducing agent. An“inducer" or“inducing agent" may be endogenous, or a normally exogenous compound or protein that is administered in such a way as to be active in inducing transcriptional activity from the inducible promoter. In some embodiments, the inducer or inducing agent, e.g., a chemical, a compound or a protein, can itself be the result of transcription or expression of a nucleic acid sequence (e.g., an inducer can be a transcriptional repressor protein), which itself may be under the control or an inducible promoter. Non-limiting examples of inducible promoters include but are not limited to, the lac operon promoter, a nitrogen-sensitive promoter, an IPTG-inducible promoter, a salt-inducible promoter, and tetracycline, steroid-responsive promoters, rapamycin responsive promoters and the like. Inducible promoters for use in prokaryotic systems are well known in the art, see, e.g. the beta- lactamase and lactose promoter systems (Chang et ah, Nature, 275: 615 (1978, which is incorporated herein by reference); Goeddel et ah, Nature, 281 : 544 (1979), which is incorporated herein by reference), the arabinose promoter system, including the araBAD promoter (Guzman et ah, J. Bacteriol., 174: 7716-7728 (1 992), which is incorporated herein by reference; Guzman et ah, J. Bacteriol., 177: 4121-4130 (1995), which is incorporated herein by reference; Siegele and Hu, Proc. Natl. Acad. Sci. USA, 94: 8168-8172 (1997), which is incorporated herein by reference), the rhamnose promoter (Haldimann et ah, J. Bacteriol., 180: 1277-1286 (1998), which is incorporated herein by reference), the alkaline phosphatase promoter, a tryptophan (trp) promoter system (Goeddel, Nucleic Acids Res., 8: 4057 (1980), which is incorporated herein by reference), the PLtetO-l and Plac/are-l promoters (Lutz and Bujard, Nucleic Acids Res., 25: 1203-1210 (1997), which is incorporated herein by reference), and hybrid promoters such as the tac promoter. deBoer et ah, Proc. Natl. Acad. Sci. USA, 80: 21-25 (1983), which is incorporated herein by reference.

[00119] An inducible promoter useful in the methods and systems as disclosed herein can be induced by one or more physiological conditions, such as changes in pH, temperature, radiation, osmotic pressure, saline gradients, cell surface binding, and the concentration of one or more extrinsic or intrinsic inducing agents. The extrinsic inducer or inducing agent may comprise amino acids and amino acid analogs, saccharides and polysaccharides, nucleic acids, protein transcriptional activators and repressors, cytokines, toxins, petroleum-based compounds, metal containing compounds, salts, ions, enzyme substrate analogs, hormones, and combinations thereof. In specific embodiments, the inducible promoter is activated or repressed in response to a change of an environmental condition, such as the change in concentration of a chemical, metal, temperature, radiation, nutrient or change in pH. Thus, an inducible promoter useful in the methods and systems as disclosed herein can be a phage inducible promoter, nutrient inducible promoter, temperature inducible promoter, radiation inducible promoter, metal inducible promoter, hormone inducible promoter, steroid inducible promoter, and/or hybrids and combinations thereof. Appropriate environmental inducers can include, but are not limited to, exposure to heat (i.e., thermal pulses or constant heat exposure), various steroidal compounds, divalent cations (including Cu2+ and Zn2+), galactose, tetracycline, IPTG (isopropyl -b-D thiogalactoside), as well as other naturally occurring and synthetic inducing agents and gratuitous inducers.

[00120] Inducible promoters useful in the methods and systems as disclosed herein also include those that are repressed by“transcriptional repressors" that are subject to inactivation by the action of environmental, external agents, or the product of another gene. Such inducible promoters may also be termed“repressible promoters" where it is required to distinguish between other types of promoters in a given module or component of the biological switch converters described herein. Preferred repressors for use in the present invention are sensitive to inactivation by physiologically benign agent. Thus, where a lac repressor protein is used to control the expression of a promoter sequence that has been engineered to contain a lacO operator sequence, treatment of the host cell with IPTG will cause the dissociation of the lac repressor from the engineered promoter containing a lacO operator sequence and allow transcription to occur. Similarly, where a tet repressor is used to control the expression of a promoter sequence that has been engineered to contain a tetO operator sequence, treatment of the host cell with tetracycline will cause the dissociation of the tet repressor from the engineered promoter and allow transcription of the sequence downstream of the engineered promoter to occur.

[00121] Cells

[00122] One aspect of the invention described herein provides a cell comprising any of the nucleic acids described herein.

[00123] Another aspect of the invention described herein provides a cell comprising any of the vectors described herein.

[00124] In one embodiment, the cell is a cell useful in amplification of a nucleic acid or a vector it comprises. In one embodiment, the cell is a prokaryotic cell. In one embodiment, the cell is a eukaryotic cell. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is a microbial cell. In one embodiment, the cell is a fungal cell, e.g., a yeast cell. In one embodiment, the cell is a primary cell. In one embodiment, the cell is an isolated primary cell. In one embodiment, the cell is a transformed cell. In one embodiment, the cell is an established human cell line. In one embodiment the cell is a Chinese Hamster (CHO) cell. [00125] Methods for expressing a nucleic acid or vector in a cell are known in the art and include, but are not limited to transduction, nucleofection, electroporation, direct injection, and/or transfection.

[00126] In one embodiment, the cell transiently expresses the nucleic acid or vector. In another embodiment, the cell stably expresses the nucleic acid or vector.

[00127] Producing recombinant protein

[00128] One aspect of the invention described herein is a method for producing a recombinant protein comprising providing a cell or an in vitro cell free transcription reaction, e.g., a kit, with a nucleic acid encoding SmNPP-5 under conditions suitable for transcription and/or translation of the nucleic acid.

[00129] In one embodiment, the cell is any of the cells described herein.

[00130] In some embodiments, the cell as described herein is cultured under conditions suitable for the expression of SmNPP-5 polypeptide. Such conditions can include, but are not limited to, conditions under which the cell is capable of growth and/or polypeptide synthesis. Conditions may vary depending upon the species and strain of cell selected. Conditions for the culture of cells, e.g. prokaryotic and eukaryotic, e.g., mammalian or insect cells, are well known in the art. If the recombinant polypeptide is operatively linked to an inducible promoter, such conditions can include the presence of the suitable inducing molecule(s).

[00131] Exemplary in vitro cell free transcription kits include but are not limited to

PURExpress. In vitro protein synthesis kit (e.g., commercially available by New England Biolabs; Ipswich, MA), AccuRapid cell-free protein expression kit (e.g., commercially available by Bioneer; Daejeon, Korea), In vitro LEXSY translation system (e.g., commercially available by Jena Bioscience; Jena, Germany), and the like.

[00132] In one embodiment, the method further comprises purifying the polypeptide. As used herein,“purifying” refers to the process of isolating a particular molecule or composition and/or treating a sample comprising a particular molecule or composition such that the molecule or composition is more isolated than before the treatment (e.g. is present at a higher level of purity). The term " isolated" or "partially purified" as used herein refers to a molecule or composition separated from at least one other component (e.g., nucleic acid or polypeptide) that is present with the molecule as found in its natural source and/or that would be present with the molecule when expressed by a cell, or secreted in the case of secreted polypeptides. A chemically synthesized nucleic acid or polypeptide or one synthesized using in vitro transcription/translation can be considered "isolated." Further processing to separate a derived polypeptide product from other components of its synthesis reaction can provide a purified preparation.

[00133] Administration

[00134] In some embodiments, the methods described herein relate to reducing coagulation in a subject having or diagnosed as having a coagulation disease or disorder, or GACI, comprising administering a treatment for reducing coagulation as described herein (e.g., a recombinant SmNPP-5 protein, a polypeptide composition comprising such SmNPP-5, a nucleic acid encoding such SmNPP-5, or a pharmaceutical composition described herein). Subjects having a coagulation disease or disorder, or GACI can be identified by a physician using current methods of diagnosing a condition.

[00135] Symptoms and/or complications of a coagulation disease or disorder, which characterize this disease and aid in diagnosis are well known in the art and include but are not limited to the presence of an inappropriate blood clot, or symptoms related to blood clots, e.g., chest pain, shortness of breath, heart attack, stroke, and/or swelling of legs. Tests that may aid in a diagnosis of, e.g., a coagulation disease or disorder, include but are not limited to blood tests, e.g., to measure platelet counts and/or clotting time, and are known in the art for a given coagulation disease or disorder. A family history of coagulation diseases or disorders will also aid in determining if a subject is likely to have the condition or in making a diagnosis of a coagulation disease or disorder.

[00136] The recombinant SmNPP-5 protein and/or pharmaceutical compositions described herein can be administered to a subject having or diagnosed as having a coagulation disease or disorder, or GACI to reduce coagulation in the subject. In some embodiments, the methods described herein comprise administering an effective amount of a treatment for reducing coagulation as described herein (e.g., a recombinant SmNPP-5 protein, a polypeptide composition comprising SmNPP-5, a nucleic acid encoding SmNPP-5, or pharmaceutical composition described herein) to a subject in order to alleviate at least one symptom of the coagulation disease or disorder (e.g., reduce coagulation). As used herein, "alleviating at least one symptom of the condition,” e.g., coagulation disease or disorder is ameliorating any condition or symptom associated with, e.g., the coagulation disease or disorder (e.g., increased coagulation or clotting of blood). As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the treatment described herein to subjects are known to those of skill in the art. In one embodiment, the treatment is administered systemically. In one embodiment, the treatment is administered intravenously. In one embodiment, the treatment is administered continuously, in intervals, or sporadically. The route of administration of the treatment will be optimized for the type of treatment being delivered (e.g., a recombinant SmNPP-5 protein, a polypeptide composition comprising SmNPP-5, a nucleic acid encoding SmNPP-5, or pharmaceutical composition described herein), and can be determined by a skilled practitioner.

[00137] The term“effective amount" as used herein refers to the amount of a treatment for reducing coagulation as described herein (e.g., a recombinant SmNPP-5 protein, a polypeptide composition comprising SmNPP-5, a nucleic acid encoding SmNPP-5, or pharmaceutical composition described herein) that can be administered to a subject having or diagnosed as having a coagulation disease or disorder needed to alleviate at least one or more symptom of a coagulation disease or disorder, or GACI. The term "therapeutically effective amount" therefore refers to an amount of the treatment that is sufficient to provide a particular anti-coagulation effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount of the treatment sufficient to delay the development of a symptom of the coagulation disease or disorder, alter the course of a symptom of, for example, a coagulation disease or disorder (e.g., reducing increased coagulation, or blood clotting), or reverse a symptom of the coagulation disease or disorder, or GACI (e.g., reducing increased coagulation, or blood clotting). Thus, it is not generally practicable to specify an exact“effective amount". However, for any given case, an appropriate“effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

[00138] In one embodiment, a treatment for reducing coagulation as described herein (e.g., a recombinant SmNPP-5 protein, a polypeptide composition comprising SmNPP-5, a nucleic acid encoding SmNPP-5, or pharmaceutical composition described herein) is administered continuously (e.g., at constant levels over a period of time). Continuous administration of the treatment can be achieved, e.g., by epidermal patches, continuous release formulations, or on- body injectors. [00139] Effective amounts, toxicity, and therapeutic efficacy can be evaluated by standard pharmaceutical procedures in cell cultures or experimental animals. The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (z.e., the concentration of the agent, which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay, e.g., measuring the anti coagulation effect. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

[00140] Dosage

[00141] "Unit dosage form" as the term is used herein refers to a dosage for suitable one administration. By way of example a unit dosage form can be an amount of therapeutic disposed in a delivery device, e.g., a syringe or intravenous drip bag. In one embodiment, a unit dosage form is administered in a single administration. In another, embodiment more than one unit dosage form can be administered simultaneously.

[00142] The dosage of a treatment for reducing coagulation as described herein (e.g., a recombinant SmNPP-5 protein, a polypeptide composition comprising SmNPP-5, a nucleic acid encoding SmNPP-5, or pharmaceutical composition described herein) can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to administer further cells, discontinue treatment, resume treatment, or make other alterations to the treatment regimen. The dosage should not be so large as to cause adverse side effects, such as cytokine release syndrome. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication.

[00143] Combinational therapy [00144] In one embodiment, a treatment for reducing coagulation as described herein (e.g., a recombinant SmNPP-5 protein, a polypeptide composition comprising SmNPP-5, a nucleic acid encoding SmNPP-5, or pharmaceutical composition described herein) is used as a monotherapy. In one embodiment, the treatment can be used in combination with other known agents and therapies for a coagulation disease or disorder, or GACI. Administered "in combination," as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject's affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder (e.g., a coagulation disease or disorder) and before the disorder has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as "simultaneous" or "concurrent delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered. The treatment described herein (e.g., a recombinant SmNPP-5 protein, a polypeptide composition comprising SmNPP-5, a nucleic acid encoding SmNPP-5, or pharmaceutical composition described herein) and the at least one additional therapy can be administered simultaneously, in the same or in separate compositions, or sequentially. For sequential administration, the treatment described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed. The treatment described herein and/or other therapeutic agents, procedures or modalities can be administered during periods of active disorder, or during a period of remission or less active disease. The treatment described herein can be administered before another treatment, concurrently with the treatment, post-treatment, or during remission of the disorder.

[00145] Therapeutics currently used to treat a coagulation disease or disorder include, but are not limited to, antiplatelet drugs, e.g., aspirin, clopidogrel, and dipyridamole, oral anticoagulants, e.g., warfarin, and injected anticoagulants, e.g., dalteparin, enoxaparin, heparin, and tinzaparin.

In one embodiment, a recombinant SmNPP-5 protein is administered with tissue plasminogen activator (tPA). Therapeutics currently used to treat GACI are described herein above.

[00146] When administered in combination, a treatment for reducing coagulation as described herein (e.g., a recombinant SmNPP-5 protein, a polypeptide composition comprising SmNPP-5, a nucleic acid encoding SmNPP-5, or pharmaceutical composition described herein) and the additional agent (e.g., second or third agent), or all, can be administered in an amount or dose that is higher, lower or the same as the amount or dosage of each agent used individually, e.g., as a monotherapy. In certain embodiments, the administered amount or dosage of the treatment described herein, the additional agent (e.g., second or third agent), or all, is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each agent used individually. In other embodiments, the amount or dosage of the treatment described herein, the additional agent (e.g., second or third agent), or all, that results in a desired effect (e.g., treatment of a coagulation disease or disorder) is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower) than the amount or dosage of each agent individually required to achieve the same therapeutic effect.

[00147] Parenteral Dosage Forms

[00148] Parenteral dosage forms of a treatment for reducing coagulation as described herein (e.g., a recombinant SmNPP-5 protein, a polypeptide composition comprising SmNPP-5, a nucleic acid encoding SmNPP-5, or pharmaceutical composition described herein) can be administered to a subject by various routes, including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to

administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, controlled- release parenteral dosage forms, and emulsions.

[00149] Suitable vehicles that can be used to provide parenteral dosage forms of the disclosure are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl my ri state, and benzyl benzoate.

[00150] Controlled and Delayed Release Dosage Forms

[00151] In some embodiments of the aspects described herein, a treatment for reducing coagulation as described herein (e.g., a recombinant SmNPP-5 protein, a polypeptide

composition comprising SmNPP-5, a nucleic acid encoding SmNPP-5, or pharmaceutical composition described herein) is administered to a subject by controlled- or delay ed-release means. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance;

4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions. (Kim, Chemg-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000)). Controlled-release formulations can be used to control a compound of formula (I)'s onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels. In particular, controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of an agent is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug. [00152] A variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with any agent described herein. Examples include, but are not limited to, those described in ET.S. Pat. Nos. : 3,845,770; 3,916,899; 3,536,809; 3,598, 123;

4,008,719; 5674,533; 5,059,595; 5,591 ,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556;

5,733,566; and 6,365,185, each of which is incorporated herein by reference in their entireties. These dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS® (Alza Corporation, Mountain View, Calif. ETSA)), multilayer coatings, microparticles, liposomes, or microspheres or a combination thereof to provide the desired release profile in varying proportions. Additionally, ion exchange materials can be used to prepare immobilized, adsorbed salt forms of the disclosed compounds and thus effect controlled delivery of the drug. Examples of specific anion exchangers include, but are not limited to, DEiOLITE® A568 and DETOLITE® AP143 (Rohm&Haas, Spring House, Pa. USA).

[00153] Efficacy

[00154] The efficacy of a treatment for reducing coagulation as described herein (e.g., a recombinant SmNPP-5 protein, a polypeptide composition comprising SmNPP-5, a nucleic acid encoding SmNPP-5, or pharmaceutical composition described herein), e.g., for reducing coagulation in a subject, e.g., having a coagulation disease or disorder, or GACI, can be determined by the skilled practitioner. However, a treatment is considered“effective treatment," as the term is used herein, if one or more of the signs or symptoms of the coagulation disease or disorder are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and/or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate, e.g., symptoms related to blood clots, e.g., chest pain, shortness of breath, heart attack, stroke, and/or swelling of legs. Efficacy can also be measured by a failure of an individual to worsen as assessed by

hospitalization, or need for medical interventions (i.e., increased coagulation, or decreased blood clot formation). Methods of measuring these indicators are known to those of skill in the art and/or are described herein. [00155] Efficacy can be assessed in animal models of a condition described herein, for example, a mouse model or an appropriate animal model of coagulation disease or disorder, as the case may be. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed, e.g., symptoms related to blood clots, e.g., chest pain, shortness of breath, heart attack, stroke, and/or swelling of legs.

[00156] All patents, patent applications, and publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the

correctness of the dates or contents of these documents.

EXAMPLES

Example 1

Introduction

[00157] Schistosomes are intravascular parasitic platyhelminths, commonly known as blood flukes that cause the debilitating disease schistosomiasis. Pathological changes associated with this disease can impact multiple organ systems, including the kidneys and urinary tract, the liver and spleen, the gastrointestinal tract, and the uterus, especially in individuals with longstanding or heavy infections. Three major species infect humans; these are Schistosoma mansoni, S. japonicum , and S. haematobium. Over 200 million people are infected with these worms around the world and > 800 million live at risk of infection (1, 2). The parasites have a complex life cycle; adult female worms release eggs that hatch in freshwater. Larval forms emerge that penetrate intermediate snail hosts and replicate asexually to generate new infectious forms called cercariae. These invade the skin of the final human host and transform into juveniles called schistosomula. Schistosomula find and penetrate a blood vessel, then circulate to the liver where they mature into adult males and females. The adults mate and migrate to the blood vessels around the intestines or bladder (depending on the species) where egg laying commences. Work described herein focuses on S. mansoni, a prominent schistosome of humans that can also be found in rodents and dogs (3,

4)·

[00158] Schistosomes can live for many years, up to a decade or more, within the human blood stream (5, 6). The adult worms are relatively large; mature male worms are -10 mm long and possess a groove in their ventral surface called the gynaecophoric canal in which the longer, more cylindrical, adult female resides. In cross section, the male/female couple spans about 1 mm. These adults may enter blood vessels whose diameter is equivalent to their own and can elongate considerably to enter even smaller vessels to lay eggs (7). Despite their large size and long lives, and the disruptions they cause in local blood flow, the parasites do not provoke overt thrombus formation around them in vivo (8). It has been hypothesized that host interactive proteins found in the parasite’s tegument (skin) are central to the worm’s ability to impede blood clot formation (9, 10). For instance, the worms possess an ATP diphosphohydrolase, e.g., SmATPDasel (11, 12), that was detected in the adult tegument by immunolocalization (13, 14) and in adult tegument extracts by proteomic analysis (15-17). SmATPDasel was cloned and expressed, and found to comprise the capacity to degrade the platelet agonist ADP (18). Further, schistosomes whose SmATPDasel gene is suppressed using RNAi are significantly impaired in their ability to degrade ADP (18). Thus, SmATPDasel prevents platelet aggregation and clot formation around the intravascular worms.

[00159] Proteomic analysis of tegument preparations revealed a second potential ADP hydrolyzing enzyme there, e.g., the ecto-nucleotide pyrophosphatase / phosphodiesterase homolog designated SmNPP5 (16, 19). SmNPP5 is a ~53 kDa type I transmembrane protein that is available for biotinylation at the adult parasite surface (17). The SmNPP5 gene is rapidly turned on in the intravascular parasitic life stages, following invasion of the definitive host (20) and the protein is expressed exclusively in the intra-mammalian life stages (21). Highest expression is found in mated adult males where the protein is found predominantly in the dorsal tegument (20). The relative amount of SmNPP5 in the adult worm tegument is estimated to be about five-fold lower than that of SmATPDasel (22). Parasites whose SmNPP5 gene is suppressed by RNAi exhibit an impaired ability to hydrolyze the artificial phosphodiesterase substrate /i-nitrophenyl 5’-TMP (p- Nph-5’-TMP) (20). The literature on the broader NPP5 family provides few clues as to the physiological role of the Schistosome homolog. For instance, SmNPP5’s closest mammalian counterpart, NPP5, is inactive against both p-nitrophenyl-TMP (23) and ADP (24) and a second mammalian homolog, NPP4, acts to promote, not impede, blood clotting (25). SmNPP5 does provide an essential function for the parasites since schistosomula whose SmNPP5 gene is suppressed are significantly impaired in their ability to establish infection in experimental animals; thus, the protein is considered a virulence factor for the worms (20). Work described herein uncovers the physiological function of SmNPP5 by expressing and characterizing the active enzyme.

Material and Methods

[00160] Parasites and mice. Schistosoma mansoni- infected Biomphalaria glabrata snails

(strain NMRI) were obtained from the Schistosomiasis Resource Center, at the Biomedical Research Institute (BRI), Rockville MD. Larval schistosomes (< cercariae , strain NMRI) were obtained from the infected snails and schistosomula were prepared (26). Adult male and female parasites were recovered by perfusion from Swiss Webster mice that were infected with -100 S. mansoni cercariae about 6 weeks previously. All parasites were cultured in complete

DMEM/F12 medium supplemented with 10% heat-inactivated fetal bovine serum, 200 U/ml penicillin and 200 pg/ml streptomycin, 0.2 mM Triiodo-L-thyronine, 1 pM serotonin and 8 pg/ml human insulin and were maintained at 37oC, in an atmosphere of 5% C02 (27). All protocols involving animals were approved by the Institutional Animal Care and Use Committees

(IACUC) of Tufts University under protocol G2015-113. All experimental procedures were carried out in accordance with approved guidelines of the IACUC.

[00161] I mmunolocalization of SmNPP5 in S. mansoni schistosomula. Schistosomula cultured for 1, 7 or 14 days were fixed in acetone for 30 min, dried and incubated for 1 hr in blocking buffer (1% bovine serum albumin (BSA) in Hank's Balanced Salt Solution (HBSS)). Parasites were then incubated for 1 hr with affinity purified, rabbit anti-SmNPP5 antibody (1 :50) (20). After washing 4 times with blocking buffer, schistosomula were incubated with Alexa Fluor 488 goat anti -rabbit IgG (Molecular Probes) diluted 1 : 100 in blocking buffer. Samples were washed 4 times and viewed using an inverted fluorescent microscope (Eclipse Ti-E; Nikon) using NIS- Elements advanced software (version 4.50) to capture images.

[00162] Expression and purification of recombinant SmNPP5. The full-length coding sequence of SmNPP5 (accession number, EU769293), including the predicted signal peptide and GPI anchor domain, was codon optimized using hamster codon preferences and synthesized commercially (Genscript). Next, the region encoding amino acids G32-S429 (e.g., lacking the amino terminal signal peptide and the carboxyl terminal GPI anchoring signal) was generated by PCR using the primers SmNPP-5 Fw (5 -TAC TGA ATT CGG TGT TGT TGG GAA GGA ACA GTT TTC-3' (SEQ ID NO: 3)), SmNPP-5 Rv (5 -TAC TCT CGA GTC AGG GAA GAA CTC GAC AAA CAC TAC CAT-3' (SEQ ID NO: 4)) and adult S. mansoni cDNA as template. The amplified product was cloned into the pSecTag2A plasmid (Invitrogen) at the Ascl and Xhol sites in frame with the IgK leader sequence at the 5’ -end and the 6-histidine tag at the 3’ -end. Successful in-frame cloning was confirmed by sequencing at the Tufts ETniversity Core Facility.

[00163] To express recombinant SmNPP5 (rSmNPP5), suspension-adapted FreeStyle Chinese Hamster Ovary Cells were transfected with plasmid using Free-Style Max Reagent according to the manufacturer’s instructions (Invitrogen). Cells were harvested at various time points post transfection to monitor viability (by Trypan Blue exclusion) and rSmNPP5 expression (by western blotting). To facilitate protein production, stable cell line clones secreting rSmNPP5 were selected by treating transfected cells with 250 pg/ml Zeocin for two weeks; individual clones that produced high yields (5-10 mg) of purified active rSmNPP5/L were maintained.

[00164] Recombinant SmNPP5 was purified from 48 hr cell culture medium by standard Immobilized Metal Affinity Chromatography (IMAC) using HisTrap™ Excel columns, following the manufacturer’s instructions (GE Healthcare Life Sciences). Fractions from each step in the purification procedure were analyzed by SDS-PAGE. Purified recombinant protein, eluted from the column, was dialyzed overnight at 4°C against 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, then concentrated by ultrafiltration centrifugation (Pierce Protein Concentrators, 10K MWCO, Thermo Scientific). Final protein concentration was determined using a BCA Protein Assay Kit (Pierce).

[00165] Deglycosylation of SmNPP5. N-deglycosylation of recombinant and native SmNPP5 proteins was undertaken using peptide-N-Glycosidase F (PNGase F) according to the

manufacturer’s instructions (New England Biolabs). Briefly, 10 pg of rSmNPP5, or 50 pg of adult male worm lysate (prepared by homogenizing adult worms in ice cold PBS containing protease inhibitors), was denatured at l00°C for 10 min in the presence of 0.5% SDS and 40 mM DTT. Then, NP-40 was added to 1%. Deglycosylation buffer and PNGase F were added and incubated at 37oC for 3 hr. Next, the PNGase F-treated, as well as control, untreated protein samples were resolved by 4-20% SDS-PAGE (BioRad), transferred to PVDF membrane and probed by standard western blotting with anti-SmNPP5 antibody (20). Briefly, the membrane was blocked with TBST (Tris-buffered saline, pH 7.5, 0.05% Tween 20) containing 5% dry, non fat milk powder for 1 hr at room temperature. The membrane was then incubated with anti- SmNPP5 antibody (1 :500) for 1 hr at room temperature, followed by washing with TBST buffer for 30 minutes and incubation with horseradish peroxidase-labeled donkey anti-rabbit IgG (1 :5000) (GE Healthcare, NJ) for 1 hr at room temperature. Signals were detected using ECL Western Blotting Detection Reagents (GE Healthcare). The membrane was exposed to X-ray film. Films were then scanned using a Kodak Image Station 2000RT.

[00166] SmNPP5 activity assays. To measure SmNPP5 activity in live parasites, -1,000 schistosomula or individual adult male or female worms (in replicate) were incubated in assay buffer (50 mM Tris-HCl buffer (pH 9), 120 mM NaCl, 5 mM KC1, 30 mM glucose, 2 mM CaCl2, 10 mM MgCl2) containing substrate (0.5 mM p-nitrophenyl 5-dTMP (p-Nph-5’-TMP) or 0-20 mM ADP), as described previously (20). Recombinant SmNPP5 was used at 0.5 - 5 pg/assay, as indicated. To monitor p-Nph-5’-TMP cleavage, changes in optical density at 405 nm over time were monitored using a Synergy HT spectrophotometer (Bio-Tek Instruments). ADP cleavage was monitored by two methods: using an ADP Colorimetric Assay Kit II (BioVision) to detect changes in ADP levels and using a Phosphate Colorimetric Assay Kit (BioVision) to monitor Pi levels, following the manufacturer’s instructions. Aliquots were recovered from each incubation at selected time points and substrate cleavage was measured. As a negative control, rSmNPP5 was heat treated at 95°C for 10 min before being included in activity assays.

[00167] Biochemical characterization of rSmNPP5. To determine the pH preference of rSmNPP5, the hydrolysis of p-Nph-5'-TMP was measured over a pH range from 5.5 to 12 in either MES, (pH 5-6.5), MOPS, (pH 6.5-7.5), HEPES, (pH 7.0-8.0), Tris-HCl, (pH 7.5-9.0), Trizma, (pH 9.0) or glycine-NaOH, (pH 9.0-12) buffers. The reaction mixture (200 mΐ) contained 50 mM of the appropriate buffer, 10 mM MgCl2, 2 mM CaCl2, 0.5 pg rSmNPP5 and 2 mM p- Nph-5'-TMP.

[00168] To monitor the need of rSmNPP5 for divalent ions, the standard p-Nph-5'-TMP hydrolysis assay was conducted, as described above, but with the 50 mM Tris-HCl pH 9 buffer modified to contain different concentrations of MgCl2, CaCl2, ZnCl2, as indicated, or 5 mM ethylenediaminetetraacetic acid (EDTA). The“0” cation control condition indicates the standard assay buffer without cations (i.e. 50 mM Tris-HCl buffer (pH 9), 120 mM NaCl, 5 mM KC1, 30 mM glucose). Enzyme specific activity was expressed as pmol p-nitrophenol released/min/mg of protein, using a molar extinction coefficient of l8.5mM-l/cm-l. The Michaelis-Menten equation was applied to determine the enzyme’s Michaelis constant (Km) for ADP hydrolysis. Data were analyzed and plotted using GraphPad Prism 5.0.

[00169] Impact of rSmNPP5 on platelet aggregation in whole blood. The impact of rSmNPP5 on platelet aggregation was analyzed by whole blood impedance, multiple electrode

aggregometry (MEA) using a Multiplate Analyzer (Roche Diagnostics). Whole blood was collected by venipuncture into lithium heparin tubes from healthy dogs at the Foster Hospital, Tufts University. Tests were performed in disposable cells with two independent sensor units, each a silver-coated highly conductive copper electrode (Roche Diagnostics). Recombinant SmNPP5 (0, 5, 20 or 40 pg), diluted in 0.9% NaCl, was added to 500 mΐ of blood. In some experiments, the mixture was incubated for 45 min at 37°C prior to conducting the assay. In other experiments, no such preincubation was carried out. Additionally, in some experiments, heat-inactivated rSmNPP5 was used. In each test cell, 300 mΐ blood was diluted 1 : 1 (vol/vol) with 0.9% saline and incubated at 37°C for 3 min. Then the reaction was started by adding 30 mΐ of agonist (either ADP, 6 mM final concentration, ADPtest Roche Diagnostics or collagen, 2 pg, COLtest; Roche Diagnostics). During this test, platelets adhere and aggregate on the sensor electrode surfaces, resulting in an increase in electrical resistance (impedance). The signal is recorded, plotted as an aggregometer trace and quantified as Area-Under-the-Curve (AUC). The mean values of the two independent determinations per sample are expressed as the AUC of the tracing.

[00170] Statistical analysis. Data are compared using a two-tailed, unpaired t-test or by ANOVA with post hoc Bonferroni multiple comparison testing, as appropriate, using GraphPad Prism 5.0. A probability (P) value of less than 0.05 was considered significant.

Results

[00171] Expression of recombinant SmNPP5.

[00172] As described herein, plasmid encoding a his-tagged, secreted form of SmNPP5 was transfected into CHO-S cells. Approximately 48 hours (hr) later, the culture supernatant was collected and rSmNPP5 was purified using standard immobilized metal affinity chromatography (IMAC). Different fractions from the rSmNPP5 purification scheme, analyzed by SDS-PAGE and Coomassie Blue staining, are shown in FIG. 1 A. Lane 4 shows purified rSmNPP5, eluted from the column and lane 5 is the same material after dialysis and concentration. The arrow (right) highlights a single prominent rSmNPP5 band in lanes 4 and 5 running at ~60 kDa. This band can also be seen in lane 1 - the 48 hr cell culture medium - showing that the recombinant protein is a major secreted protein. Since rSmNPP5 binds to the column, it cannot be clearly seen in the flow through fraction (lane 2) or in the column wash (lane 3). The estimated molecular weight of the recombinant protein (~60 kDa) is greater than the predicted molecular weight of SmNPP5 based on its amino acid sequence (49 kDa). Since this is suggestive of post- translational modification of the protein, the glycosylation status of SmNPP5 was examined.

FIG. 1B compares rSmNPP5 with the native schistosome protein by western blotting both before (-) and after (+) treatment with PNGase F. In extracts of adult male worms, the native protein resolves as a prominent band at ~55 kDa (FIG. 1B, lane Sm -, arrow) with a second band at ~60 kDa (FIG. 1B, lane Sm -, arrowhead). Following incubation of the worm extract with PNGase F, this larger band disappears and a new band (at ~50 kDa) appears (FIG. 1B, lane Sm +).

Incubation of rSmNPP5 with PNGase F also leads to a notable shift in its migration profile; the broad band at ~ 60 kDa in the rSmNPP5 (-) lane (arrow) disappears and a band running at ~ 50 kDa is now seen (rSmNPP5 +, arrowhead).

[00173] Biochemical characterization of rSmNPP5.

[00174] Purified rSmNPP5 displays robust enzymatic activity; in FIG 2A the ability of 0.2 pg rSmNPP5 to hydrolyze the artificial substrate p-Nph-5'-TMP over time is shown. (The chemical structure of p-Nph-5'-TMP is illustrated.) The enzyme displays a specific activity of 7.56 pmol p-nitrophenol generated/min/mg rSmNPP5. Heat treatment destroys enzyme activity (FIG. 2 A, lower line,“triangles”).

[00175] As shown in FIG. 2B, adding divalent cations in the form of Ca2+ or Mg2+ to the assay buffer changes rSmNPP5 activity little (by 0-10%). However, adding Zn2+ significantly increases activity by 35-40% (ANOVA, p < 0.001). Removing cations from the assay buffer by the addition of the divalent ion chelator EDTA (5 mM) abolishes rSmNPP5 activity (FIG. 2B, EDTA). The pH preference of rSmNPP5 for p-Nph-5'-TMP hydrolysis was tested in the range of 5.5 to 12 and FIG. 2C shows that the enzyme displays optimal activity at pH 9.

[00176] Impact of rSmNPP5 on platelet aggregation.

[00177] Platelet function in the presence of rSmNPP5 was assessed in whole blood using multiple electrode aggregometry (MEA) and findings are shown in FIGs 3A and 3B. Samples were preincubated with rSmNPP5 for 45 min prior to the addition of agonist. Representative tracings of samples treated with ADP as agonist and containing 0, 5, 20 or 40 pg of active rSmNPP5 are depicted in FIG. 3 A, left panel. The area under the curve decreases with increasing rSmNPP5. Heat inactivating rSmNPP5 completely abolishes the enzyme’s impact in this assay (FIG. 3 A,“40, HI”). Representative tracings of samples treated with collagen as agonist and containing 0 or 40 pg of active rSmNPP5 versus 40 pg of heat inactivated enzyme are depicted in FIG. 3A, right panel. MEA data from replicate experiments are compiled in FIG. 3B. The Area-Under-the-Curve (AUC) of the control sample (no rSmNPP5) is set to represent“100% aggregation” in FIG. 3B. From these data, it is clear that the addition of rSmNPP5 has a profound impact on platelet function and the more rSmNPP5 used the greater the effect. Adding 5, 20 or 40 pg of rSmNPP5 results in ~20, 60 and 90% reduction in platelet aggregation versus control, respectively (t test, P = 0.0064, 0.0002 and 0.0001). In contrast, treating samples with 40 pg of heat inactivated rSmNPP5 (HI) yields no significant change in platelet aggregation versus control (t test, P = 0.11).

[00178] In order to determine how important the preincubation phase was to the aggregation inhibition observed, the MEA experiment was repeated without preincubation. Instead, rSmNPP5 was added to blood at the same time as agonist, not before. Results of this work are also compiled in FIG. 3B (“Preincubation series). The data with or without preincubation are essentially the same - similar decreased platelet aggregation is observed as the amount of added rSmNPP5 increases. Again, treating samples with 40 pg of heat inactivated rSmNPP5 shows no significant change versus control.

[00179] To determine if rSmNPP5-driven aggregation inhibition only occurs when ADP is used as agonist, the assay was repeated using collagen (and following pretreatment of blood with 40 pg rSmNPP5). Representative tracings are shown in FIG. 3 A (right panel) and data compiled from replicate experiments are shown in FIG. 3B (+, Collagen). Significant inhibition of aggregation is again seen versus control (t test, P = 0.0096) but the degree of aggregation inhibition (23%) is substantially less using collagen than for equivalent experiments using ADP (-90% inhibition).

[00180] SmNPPS is an ADPase.

[00181] The capacity of SmNPP5 to degrade ADP (whose chemical structure is shown in figure 4A) was monitored using two assays. The ADP Colorimetric Assay Kit II was used to measure ADP levels and the Phosphate Colorimetric Assay Kit was used to measure inorganic phosphate (Pi) levels. ADP cleavage is predicted to lead to both a diminution in intact ADP levels and an increase in free Pi levels. As shown in FIG. 4A-4C, incubating rSmNPP5 with ADP leads to both outcomes; diminished ADP with time and with increasing enzyme concentration (FIG. 4A, ANOVA P<0.000l) along with concurrent increase in Pi release (FIG. 4B, ANOVA P<0.000l). Heat inactivating SmNPP5 abrogates ADP cleavage activity (“HI”, FIG. 4A and 4B). The kinetics of rSmNPP5 mediated cleavage of ADP is presented in FIG. 4C. The Km of rSmNPP5 for ADP is 246 ± 34 mM.

[00182] SmNPP5 immunolocalization in schistosomula.

[00183] As seen in FIG. 5, staining of schistosomula with anti-SmNPP5 antibodies reveals that the protein is localized predominantly in the parasite tegument. Staining is seen in the periphery of the larval worms at both 1 day post cercarial transformation (left), at 7 days (middle) and at 14 days (right). The 7-day sample is counterstained with DAPI to reveal stained parasite nuclei (blue). The scale bar represents 30 pM.

[00184] SmNPP5 in schistosomes: enzyme activity.

[00185] FIGs 6A and 6B illustrate the ability of living schistosomula to cleave p-Nph-5'- TMP. Further, the activity of live parasites (FIG. 6A, filled triangles), representing surface enzyme activity, is indistinguishable from that of total lysates of an equivalent numbers of parasites (open triangles). Lysate activity represents surface enzyme action as well as activity exhibited by the internal tissues. Similarly, the activity of living individual adult worms (FIG.

6B, males - filled circles, females - filled squares) resembles that of lysates of individuals (males - open circles, females - open squares). Individual adult female worms display significantly less activity (FIG. 6B, lower lines) as compared to their male counterparts (FIG. 6B, upper lines). FIGs. 7A and 7B confirms the ability of schistosomes (male parasites) to cleave exogenous ADP; in the presence of live worms ADP levels diminish (FIG. 7A) while levels of Pi generated increase (FIG. 7B).

Discussion

[00186] Work presented herein show purified, functionally active recombinant SmNPP5 in a CHO cell expression system. SDS-PAGE and Coomassie staining reveal that the recombinant protein is of high purity and reacts with commercially generated anti-SmNPP5 antibodies.

Analysis of the recombinant protein by SDS-PAGE shows it to be larger than the predicted molecular size, based on the amino acid sequence (~60 kDa vs 49 kDa). This indicates that the protein is post translationally modified; it was found herein that the recombinant protein, like its native counterpart, is glycosylated. Treatment with the N-glycan cleaving enzyme, PNGase F, results in a clear protein mobility shift on SDS-gels, with the (originally ~60 kDa) rSmNPP5 protein now running at ~50 kDa. The native protein in worm extracts resolves as two bands running at 60 and 55 kDa. Both bands are SmNPP5 since earlier gene knockdown experiments led to a clear diminution in both (20). Without wishing to be bound by a particular theory, it is hypothesized that the two bands may represent two isoforms of SmNPP5, perhaps with different degrees and patterns of glycosylation. In agreeance with this hypothesis, analysis of the amino acid sequence of SmNPP5 shows that the protein contains several potential N-glycosylation and O-glycosylation sites.

[00187] It is shown herein that rSmNPP5 can cleave the artificial substrate p-nitrophenyl-5’- TMP (p-Nph-5’-TMP) in a reaction that requires divalent ions. Removing cations by treating the protein extract with the chelator EDTA eliminates enzyme activity. These data are consistent with the known requirement of NPP protein family members for Zn2+ in the bi-metallo-catalytic core (28). Increasing zinc cation concentration here enhances enzyme activity; heating the protein to 95oC for 5 minutes destroys its activity. The enzyme’s pH optimum is 9. How this fact impacts enzyme function in vivo is unclear, given that the pH of blood, the schistosome’s natural habitat, is neutral (pH 7.35 - 7.45). Other schistosome ectoenzymes expressed at the host-parasite interface similarly display highest activity under alkaline conditions (e.g. SmAP and

SmATPDasel) (18, 29, 30). It is possible that worms in the vasculature maintain an alkaline environment immediately around them in which SmNPP5, and other ectoenzymes, optimally act and which is selectively advantageous for them. How the worms might generate such an alkaline environment is unclear given the strong buffering capacity of blood plasma coupled with the fact that the adult worms excrete large amounts of lactate (31).

[00188] To assess whether SmNPP5 plays a role in the known ability of schistosomes to impede blood coagulation (32), recombinant SmNPP5 was added to whole blood and platelet function was monitored by multiple electrode aggregometry (MEA) using ADP as agonist. These experiments reveal that the protein has a profoundly inhibitory effect on platelet aggregation in a dose-dependent manner. Adding rSmNPP5 to the blood sample 45 minutes before, or at the same time as, the addition of ADP had no appreciable impact on the outcome; substantial inhibition of platelet aggregation was observed in both cases. Significant inhibition of aggregation is also seen if collagen is used as agonist instead of ADP. However, the degree of rSmNPP5-driven aggregation inhibition in the collagen experiments (23%) is substantially less than for equivalent experiments using ADP (-90% inhibition). Since heat inactivation of rSmNPP5 essentially abolishes the protein’s inhibitory properties, it is clear that active enzyme is key. These data led to the hypothesis that SmNPP5 mediates its inhibitory effects by cleaving ADP, an agonist used here to trigger platelet activation. In vitro analysis reveals that rSmNPP5 can indeed hydrolyze ADP to liberate Pi with a Km for ADP of 246±34 mM. Heat treated rSmNPP5 can no longer cleave ADP.

[00189] Unlike schistosome SmNPP5, mammalian NPP5 is inactive against both p- nitrophenyl-5'-TMP (23) and ADP (24). It has been suggested the SmNPP5 is more likely an ortholog of mammalian NPP4 (24). Like SmNPP5, mammalian NPP4 is exposed to blood, being present on the surface of vascular endothelium. However, unlike SmNPP5, NPP4 acts as a procoagulant; hydrolysis of adenosine(5')triphospho(5')adenosine (Ap3 A) by NPP4 is thought to sustain ADP generation at the site of vascular injury and so augment platelet aggregation (25). In contrast, SmNPP5 is a type one glycoprotein, expressed in the intravascular environment where it can degrade ADP and act as an anti-coagulant.

[00190] As revealed here by immunolocalization on schistosomula, SmNPP5 is expressed largely in the parasite tegument. This agrees with earlier tegumental localization data from the adult life stages (20, 21) and with proteomic analyses which revealed the presence of SmNPP5 in tegumental extracts (16, 19). Such a localization would be key for an enzyme that, as

hypothesized, modulates the biochemistry of the immediate external environment of the worms.

[00191] The rate of cleavage of p-Nph-5'-TMP reported here is about the same whether live schistosomula or schistosomula lysates are tested. Similarly, in adults, the rate of p-Nph-5'-TMP cleavage is largely indistinguishable whether individual live worms or worm lysates (equivalent to a single worm) are tested. These data indicate that essentially all the active SmNPP5 in schistosomes is at the parasite/host interface and that the enzyme’s role involves interacting with host intravascular components.

[00192] The amount of activity measured in adult females is low and this agrees with gene expression data which show the level of SmNPP5 mRNA in females at -20% that of males (20, 21). It was previously shown that single (unmated) females express SmNPP5 to about the same degree as single (unmated) males but this changes following mating; mated males upregulate the SmNPP5 gene while mated females downregulate it (20). This has led to the hypothesis that males assume the function performed by SmNPP5 once they have found a partner. This permits the mated female to divert the resources required to express SmNPP5 to undertake other tasks, notably to express egg-laying genes. The sexual dimorphism of schistosome adults suggests that adult males and females have separate and distinctive functions in vivo. Work reported herein indicates that a major role for males, by upregulating surface SmNpp5, is to assume greater involvement in the control of blood flow around the paired couple.

[00193] SmNPP5 is not the only tegumental ectoenzyme possessed by schistosomes that can degrade ADP; an ecto-ATP diphosphohydrolase (SmATPDasel) has also been shown capable of performing this function (18). Like SmNPP5, the SmATPDasel gene is well expressed in the intravascular life stages, with highest relative expression in adult males (33). The SmATPDasel protein has been immunolocalized on the external surface,“conspicuously” in adults (13).

Recombinant SmATPDasel (like rSmNPP5) works optimally at alkaline pH and cleaves ADP with a similar Km (252 ±20 mM) (18). SmATPDasel is found in ~5 fold greater relative abundance compared to SmNPP5 in adult males (22) suggesting that SmATPDasel plays the greater role in regulating ADP levels around the parasites in vivo. Thus, parasites whose

SmNPP5 gene is suppressed (that exhibit a reduced ability to hydrolyze p-Nph-5'-TMP) still exhibit considerable ADP-cleaving capability (18). Since such SmNPP5-suppressed larval worms are greatly impaired in their ability to establish infection in experimental animals, functions of SmNPP5 in addition to (or instead of) its ability to cleave exogenous ADP are likely responsible. While ADP can activate platelets, which can then participate in thrombus formation, platelets have additionally been shown to be, by themselves, directly cytotoxic to schistosomes (34). Therefore, schistosome mediated ADP cleavage may not only curtail blood clot formation but may also impede direct, platelet-mediated attack focused on the worms themselves.

Certainly, examination of worms fixed and stained in situ reveals that they can fit snugly into some vessels yet provoke no obvious thrombus formation or accumulation of platelets around them (8, 35).

[00194] It is concluded herein that schistosomes and their hosts utilize similar ecto-enzymes - ectonucleoside triphosphate diphosphohydrolases (like SmATPDasel) and ectonucleotide pyrophosphatase/phosphodiesterases (like SmNPP5) - to modulate levels of extracellular nucleotides and thereby control local purinergic signaling pathways. For schistosomes, this ability serves to impede platelet activation and thrombus formation and contributes to the longevity of these globally important and debilitating parasitic worms.

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Example 2

Additional SmNPP5 Substrates

[00196] SmNPP5 has additional promiscuity and has been shown to cleave substrates in addition to ADP. As a non-limiting example, SmNPP5 has been shown to cleave Ap3 A

(diadenosine triphosphate) into AMP and Pi (see e.g., FIG. 9A-FIG. 9C). As another non limiting example, SmNPP5 has been shown to cleave Ap4A (diadenosine tetraphosphate) into AMP and PPi (see e.g., FIG. 10A-FIG. 10C). Ap3A and Ap4A are both canonical ATPase inhibitors. Without wishing to be bound by theory, it is proposed that cleavage of Ap3 A or Ap4A by SmNPP5 can have an effect on coagulation, such as an anti-coagulation effect.

Claims

1) A method for reducing blood coagulation in a subject, comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of a recombinant SmNPP-5 protein.

2) The method of claim 1, wherein the recombinant SmNPP-5 is derived from the helminth Schistosome mansoni.

3) The method of claim 1, wherein the recombinant SmNPP-5 is derived from the helminth Schistosoma japonicum or Schistosoma haematobium.

4) The method of claim 1, wherein the recombinant SmNPP-5 protein comprises a sequence of SEQ ID NO: 1 or a polypeptide which has at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 1.

5) The method of claim 1, wherein the recombinant SmNPP-5 protein is a truncated

recombinant SmNPP-5 protein.

6) The method of claim 4, wherein the truncated recombinant SmNPP-5 protein comprises a sequence of SEQ ID NO: 2 or a polypeptide which has at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 2.

7) The method of claims 4 and 5, wherein the truncated recombinant SmNPP-5 protein

comprises at least 85% of the endogenous ADPase activity of a full length SmNPP-5 protein.

8) The method of claims 1-7, wherein the subject has a coagulation disorder.

9) The method of claim 8, wherein the coagulation disorder results in increased clotting.

10) The method of claim 8, wherein the coagulation disorder is selected from the group

consisting of Factor V Leiden, Anti-thrombin III (A Till) deficiency, Protein C or Protein S deficiency, Prothrombin (PT) gene mutation, or Antiphospholipid antibody syndrome.

11) The method of claims 1-7, wherein administration does not cause immune response.

12) A polypeptide composition comprising a SmNPP-5 protein.

13) The polypeptide composition of claim 12, wherein the SmNPP-5 protein is a recombinant SmNPP-5 protein.

14) The polypeptide composition of claim 13, wherein the recombinant SmNPP-5 protein comprises a sequence of SEQ ID NO: 1 or a polypeptide which has at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 1. 15) The polypeptide composition of claim 13, wherein the SmNPP-5 protein is a truncated recombinant SmNPP-5 protein.

16) The polypeptide composition of claim 15, wherein the truncated recombinant SmNPP-5 protein comprises a sequence of SEQ ID NO: 2 or a polypeptide which has at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 2.

17) The polypeptide composition of claims 15 and 16, wherein the truncated recombinant SmNPP-5 protein comprises at least 50% of the endogenous ADPase activity of the full length SmNPP-5 protein.

18) A pharmaceutical composition comprising any of the polypeptide compositions of claim 12-17 and a pharmaceutically acceptable carrier.

19) A nucleic acid encoding any of the polypeptide compositions of claim 12-17.

20) A vector comprising the nucleic acid of claim 19.

21) A cell comprising the nucleic acid of claim 19.

22) A cell comprising the vector of claim 20.

23) A method of producing a recombinant protein, comprising providing a cell or an in vitro cell free transcription reaction mixture with a nucleic acid encoding SmNPP-5 under conditions suitable for transcription and/or translation of the nucleic acid.

24) The method of claim 23, further comprising the step of purifying the nucleic acid.

25) The method of claim 23, wherein the cell is the cell of claim 21 or 22.

26) A method for treating generalized arterial calcification of infancy (GACI) in a subject, comprising administering to a subject having GACI a composition comprising a therapeutically effective amount of a recombinant SmNPP-5 protein.

27) A method for reducing blood coagulation in a subject, comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of a recombinant human NPP-5 protein, wherein the human NPP-5 protein comprises a tyrosine 73 to phenylalanine mutation.

28) The method of claim 27, wherein the human NPP-5 has ADPase activity and anti

coagulant activity.