US20070015703A1 - ADAMTS13-containing compositions having thrombolytic activity - Google Patents

ADAMTS13-containing compositions having thrombolytic activity Download PDF

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US20070015703A1
US20070015703A1 US11/454,615 US45461506A US2007015703A1 US 20070015703 A1 US20070015703 A1 US 20070015703A1 US 45461506 A US45461506 A US 45461506A US 2007015703 A1 US2007015703 A1 US 2007015703A1
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adamts13
mice
thrombus
pharmaceutical composition
biologically active
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Denisa Wagner
Anil Chauhan
Friedrich Scheiflinger
Barbara Plaimauer
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Baxter Healthcare SA
Baxter International Inc
Childrens Medical Center Corp
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Baxter Healthcare SA
Baxter International Inc
Immune Disease Institute Inc
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Priority to US13/747,307 priority patent/US10233436B2/en
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Priority to US16/262,180 priority patent/US11124787B2/en
Priority to US17/478,520 priority patent/US20220002703A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6489—Metalloendopeptidases (3.4.24)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4886—Metalloendopeptidases (3.4.24), e.g. collagenase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • This invention relates to pharmaceutical compositions having thrombolytic activity comprising a pharmaceutically effective amount of ADAMTS13, to methods of treating or preventing disorders associated with the formation and/or the presence of one or more thrombus as well as to methods for disintegrating one or more thrombus in a patient in need thereof. Furthermore, the invention relates to the use of a pharmaceutically effective amount of ADAMTS13 for the preparation of a pharmaceutical composition for treating or preventing a disorder associated with the formation and/or the presence of one or more thrombus and for disintegrating one or more thrombus in a patient in need thereof.
  • Thrombotic thrombocytopenic purpura is a disorder characterized by thrombotic microangiopathy, thrombocytopenia and microvascular thrombosis that can cause various degrees of tissue ischemia and infarction.
  • TTP patients are diagnosed by symptoms such as thrombocytopenia, schistocytes (fragments of erythrocytes) and elevated levels of lactate dehydrogenase (Moake J L. Thrombotic microangiopathies. N Engl J Med. 2002; 347:589-600; Moake J L. von Willebrand factor, ADAMTS-13, and thrombotic thrombocytopenic purpura. Semin Hematol.
  • the protease belongs to the ADAMTS family and is designated as ADAMTS13 LA Disintegrin-like And Metalloprotease with Thrombospondin type I repeats), a 190 kDa glycosylated protein produced predominantly by the liver (Levy G G, Nichols W C, Lian E C, Foroud T, McClintick J N, McGee B M, Yang A Y, Siemieniak D R, Stark K R, Gruppo R, Sarode R, Shurin S B, Chandrasekaran V, Stabler S P, Sabio H, Bouhassira E E, Upshaw J D, Jr., Ginsburg D, Tsai H M.
  • Idiopathic TTP is a more common disorder that occurs in adults and older children and can recur at regular intervals in 11-36% of patients (Tsai H M, Lian E C. Antibodies to von Willebrand factor-cleaving protease in acute thrombotic thrombocytopenic purpura. N Engl J Med. 1998; 339:1585-1594; Furlan M, Lammle B. Deficiency of von Willebrand factor-cleaving protease in familial and acquired thrombotic thrombocytopenic purpura. Baillieres Clin Haematol. 1998; 11:509-514).
  • Non neutralizing autoantibodies could also inhibit ADAMTS activity by inducing clearance from circulation (Scheiflinger F, Knobl P, Trattner B, Plaimauer B, Mohr G, Dockal M, Dorner F, Rieger M. Nonneutralizing IgM and IgG antibodies to von Willebrand factor-cleaving protease (ADAMTS-13) in a patient with thrombotic thrombocytopenic purpura. Blood. 2003; 102:3241-3243). Plasma ADAMTS13 activity in healthy adults ranges from 50% to 178% (Moake J L. Thrombotic thrombocytopenic purpura and the hemolytic uremic syndrome. Arch Pathol Lab Med. 2002; 126:1430-1433).
  • ADAMTS13 activity is absent or less than 5% of the normal. Without treatment the mortality rate exceeds 90%, but plasma therapy has reduced mortality to about 20% (Moake J L. Thrombotic thrombocytopenic purpura and the hemolytic uremic syndrome. Arch Pathol Lab Med. 2002; 126:1430-1433).
  • vWF synthesized in megakaryocytes and endothelial cells is stored in platelet ⁇ -granules and Weibel-Palade bodies, respectively, as ultra large vWF (UL-vWF) 5.
  • UL-vWF ultra large vWF
  • Endothelial cell-derived high molecular weight von Willebrand factor is converted into the plasma multimer pattern by granulocyte proteases. Biochem Biophys Res Commun. 1989; 158:980-985; Tsai H M, Nagel R L, Hatcher V B, Sussman, I I. Multimeric composition of endothelial cell-derived von Willebrand factor. Blood. 1989; 73:2074-2076).
  • these UL-vWF multimers are cleaved by ADAMTS13 in circulation into a series of smaller multimers at specific cleavage sites within the vWF molecule (Tsai H M, Nagel R L, Hatcher V B, Sussman, I I. Endothelial cell-derived high molecular weight von Willebrand factor is converted into the plasma multimer pattern by granulocyte proteases. Biochem Biophys Res Commun. 1989; 158:980-985; Dent J A, Galbusera M, Ruggeri Z M. Heterogeneity of plasma von Willebrand factor multimers resulting from proteolysis of the constituent subunit. J Clin Invest.
  • vWF exists in “ball-of-yarn” and filamentous form as seen by electron microscopy (Slayter H, Loscalzo J, Bockenstedt P, Handin R I. Native conformation of human von Willebrand protein. Analysis by electron microscopy and quasi-elastic light scattering. J Biol. Chem. 1985; 260:8559-8563). Furthermore, atomic force microscopy confirms that vWF exits in a globular conformation under static conditions and an unfolded filamentous state after exposure to shear stress (Siedlecki C A, Lestini B J, Kottke-Marchant K K, Eppell S J, Wilson D L, Marchant R E. Shear-dependent changes in the three-dimensional structure of human von Willebrand factor. Blood. 1996; 88:2939-2950). This could occur also in vivo when one end of the vWF filament is anchored to a surface.
  • UL-vWF multimers present in Weibel-Palade bodies, when released by activated endothelial cells bind platelets more tightly (through GPIb ⁇ ) than plasma vWF (27. Arya M, Anvari B, Romo G M, Cruz M A, Dong J F, McIntire L V, Moake J L, Lopez J A. Ultralarge multimers of von Willebrand factor form spontaneous high-strength bonds with the platelet glycoprotein Ib-IX complex: studies using optical tweezers. Blood. 2002; 99:3971-397).
  • ADAMTS13 ADAMTS-13 metalloprotease interacts with the endothelial cell-derived ultra-large von Willebrand factor. J Biol. Chem. 2003; 278:29633-29639).
  • Thrombi of TTP patients consist of little fibrin and mainly of vWF and platelets, suggesting vWF-mediated platelet aggregation as a cause of thrombosis (30. Asada Y, Sumiyoshi A, Hayashi T, Suzumiya J, Kaketani K. Immunohistochemistry of vascular lesion in thrombotic thrombocytopenic purpura, with special reference to factor VIII related antigen. Thromb Res. 1985; 38:469-479). Patients with relapsing TTP have ultra-large multimers in the plasma. The UL-vWF multimers accumulate over time because the persistence of the inhibitor (Anti-ADAMTS13 Ab) decreases ADAMTS13 activity.
  • the UL-vWF multimers are hyperactive and unfold as a result of shear stress causing platelet aggregation, resulting in intravascular thrombosis (Tsai H M. Von Willebrand factor, ADAMTS13, and thrombotic thrombocytopenic purpura. J Mol Med. 2002; 80:639-647; Tsai H M. Deficiency of ADAMTS-13 in thrombotic and thrombocytopenic purpura. J Thromb Haemost. 2003; 1:2038-2040; discussion 2040-2035).
  • An object of the present invention is to provide a pharmaceutical composition having thrombolytic activity.
  • the pharmaceutical composition comprises a pharmaceutically effective amount of ADAMTS13 or a biologically active derivative thereof, and optionally one or more pharmaceutically acceptable carrier and/or diluent. Said composition may also comprise one or more additional active ingredient. Further, the present invention relates to a method of treating or preventing a disorder associated with the formation and/or presence of one or more thrombus and to a method of disintegrating one or more thrombus in a patient in need thereof.
  • a pharmaceutically effective amount of ADAMTS13 or a biologically active derivative thereof can be used for the preparation of a pharmaceutical composition for treating or preventing a disorder associated with the formation and/or the presence of one or more thrombus and for disintegrating one or more thrombus in a patient in need thereof.
  • Said pharmaceutically effective amount of ADAMTS13 or a biologically active derivative thereof may range, for example, from 0.1 to 20 mg/kg body weight.
  • FIG. 1 Thrombus formation in microvenules.
  • Mesenteric venules of approx. 25-30 ⁇ m in diameter were visualized after an incision was made through the abdominal wall to expose the mesentery of live mice.
  • the arrows indicate the microthrombi.
  • stimulation of Weibel-Palade body secretion can lead to spontaneous thrombus formation in Adamts13 ⁇ / ⁇ mice in the absence of vascular injury.
  • Inset time points in lower right corner refer to the time after superfusion of A23187. Bar shown in the middle panel is (approx.) 50 ⁇ m.
  • Inset time points in lower right corner refer to the time after superfusion of A23187. Bar shown in the middle panel is (approx.) 25 ⁇ m.
  • FIG. 4 Recombinant ADAMTS13 inhibits platelet strings in Adamts13 ⁇ / ⁇ mice. Rhodamin 6G was used to label endogenous platelets and leukocytes. 1 mM histamine (200 ⁇ l) was administered i.p. 15 mins before surgery and 3 mesenteric venules of approx. 200-300 ⁇ m in diameter were visualized per mouse.
  • A: No platelet strings are seen in Adamts13 WT mice (n 5).
  • C The platelet strings could form platelet aggregates in Adamts13 ⁇ / ⁇ mice as indicated by arrowhead.
  • FIG. 5 Quantitative analysis of platelet adhesion and thrombi formation in arterioles of WT and Adamts 13 ⁇ / ⁇ mice.
  • A The number of fluorescent platelets deposited per minute was determined in the interval 2-3 min after injury (total number of adherent platelets counted in 1 min interval). For statistics and mean, platelets greater than 100 was considered as 100. Absence of ADAMTS13 in the plasma clearly influences the early platelet interaction with the subendothelium. Compared with WT, in Adamts 13 ⁇ / ⁇ more platelets were deposited on the vessel wall (P ⁇ 0.05).
  • FIG. 6 Inhibition of integrin ⁇ IIb ⁇ 3 blocks thrombus formation of ADAMTS13 ⁇ / ⁇ platelets on collagen under arterial shear rate conditions.
  • Adamts13 +/+ or Adamts13 ⁇ / ⁇ whole blood was perfused for 2 min over a collagen surface at a shear rate of 1500 s ⁇ 1.
  • FIG. 7 Infusion of recombinant human ADAMTS13 inhibits thrombus growth.
  • Recombinant human ADAMTS13 was infused (i.v.) in the Adamts13 ⁇ / ⁇ mice 15 min before the ferric chloride injury. The occlusion time (blood flow completely stopped for 10 sec) was determined.
  • C Representative fluorescent images of injured arteriole of an Adamts13 ⁇ / ⁇ mouse treated with r-hu ADAMTS13 are shown. Arrowheads indicate a disintegrating thrombus.
  • FIG. 8 Infusion of recombinant mouse ADAMTS13 inhibits thrombus growth in WT mice.
  • Recombinant mouse ADAMTS13 (2.6 mg/kg mouse) was infused (i.v.) into the WT mice 5 min before the ferric chloride injury.
  • FIG. 9 Table 1: Hemodynamic parameters prior to and after application of A23187 on venules ( FIG. 1 ); Table 2: Hemodynamic parameters prior to and after application of ferric chloride on arterioles ( FIG. 5 ).
  • One aspect of the present invention relates to a pharmaceutical composition having thrombolytic activity, comprising a pharmaceutically effective amount of ADAMTS13 or a biologically active derivative thereof.
  • thrombolytic activity means the disintegration of one or more thrombus. Suitable methods for the determination of thrombolytic activity are well known in the art. For example, suitable methods are the determination of the lysis of a whole blood clot using tPA or streptokinase, or the determination of the thrombus lysis in vivo.
  • disintegration includes the partial or complete disintegration, dissolving, dissolution, destruction and/or lysis of a thrombus.
  • thrombus as used herein comprises a blood clot, especially a platelet-comprising blood clot, a microthrombus, and/or an embolus. Said thrombus may be attached to an arterial or venous blood vessel or not, and may partially or completely block the blood flow in an arterial or venous blood vessel.
  • biologically active derivative means any polypeptides with substantially the same biological function as ADAMTS13.
  • the polypeptide sequences of the biologically active derivatives may comprise deletions, additions and/or substitution of one or more amino acids whose absence, presence and/or substitution, respectively, do not have any substantial negative impact on the biological activity of polypeptide.
  • the biological activity of said polypeptides may be measured, for example, by the reduction or delay of platelet adhesion to the endothelium, the reduction or delay of platelet aggregation, the reduction or delay of the formation of platelet strings, the reduction or delay of thrombus formation, the reduction or delay of thrombus growth, the reduction or delay of vessel occlusion, the proteolytical cleavage of vWF, and/or the disintegration of thrombi.
  • ADAMTS13 and “biologically active derivative”, respectively, also include polypeptides obtained via recombinant DNA technology.
  • the recombinant ADAMTS13 (“rADAMTS13”), e.g. recombinant human ADAMTS13 (“r-hu-ADAMTS13”), may be produced by any method known in the art.
  • rADAMTS13 e.g. recombinant human ADAMTS13
  • r-hu-ADAMTS13 recombinant human ADAMTS13
  • r-hu-ADAMTS13 recombinant human ADAMTS13
  • r-hu-ADAMTS13 recombinant human ADAMTS13
  • This may include any method known in the art for (i) the production of recombinant DNA by genetic engineering, e.g.
  • RNA and/or amplification of DNA via reverse transcription of RNA and/or amplification of DNA, (ii) introducing recombinant DNA into prokaryotic or eukaryotic cells by transfection, i.e. via electroporation or microinjection, (iii) cultivating said transformed cells, e.g. in a continous or batchwise manner, (iv) expressing ADAMTS13, e.g. constitutively or upon induction, and (v) isolating said ADAMTS13, e.g. from the culture medium or by harvesting the transformed cells, in order to (vi) obtain substantially purified recombinant ADAMTS13, e.g. via anion exchange chromatography or affinity chromatography.
  • biologically active derivative includes also chimeric molecules such as e.g. ADAMTS13 (or a biologically active derivative thereof) in combination with Ig, in order to improve the biological/pharmacological properties such as e.g. half life of ADAMTS13 in the circulation system of a mammal, particularly human.
  • the Ig could have also the site of binding to an optionally mutated Fc receptor.
  • the rADAMTS13 can be produced by expression in a suitable prokaryotic or eukaryotic host system characterized by producing a pharmacologically effective ADAMTS13 molecule.
  • eukaryotic cells are mammalian cells, such as CHO, COS, HEK 293, BHK, SK-Hep, and HepG2.
  • reagents or conditions used for producing or isolating ADAMTS13 according to the present invention and any system known in the art or commercially available can be employed.
  • rADAMTS13 is obtained by methods as described in the state of the art.
  • vectors can be used for the preparation of the rADAMTS13 and can be selected from eukaryotic and prokaryotic expression vectors.
  • vectors for prokaryotic expression include plasmids such as pRSET, pET, pBAD, etc., wherein the promoters used in prokaryotic expression vectors include lac, trc, trp, recA, araBAD, etc.
  • vectors for eukaryotic expression include: (i) for expression in yeast, vectors such as pAO, pPIC, pYES, pMET, using promoters such as AOX1, GAP, GAL1, AUG1, etc; (ii) for expression in insect cells, vectors such as pMT, pAc5, pIB, pMIB, pBAC, etc., using promoters such as PH, p10, MT, Ac5, OpIE2, gp64, polh, etc., and (iii) for expression in mammalian cells, vectors such as pSVL, pCMV, pRc/RSV, pcDNA3, pBPV, etc., and vectors derived form viral systems such as vaccinia virus, adeno-associated viruses, herpes viruses, retroviruses, etc., using promoters such as CMV, SV40, EF-1, UbC, RSV, ADV, BPV,
  • the pharmaceutical composition of the present invention also comprises one or more pharmaceutically acceptable carrier and/or diluent.
  • the pharmaceutical composition of the present invention may also comprise one or more additional active ingredients such as e.g. anti-thrombotic agents, agents that stimulate ADAMTS13 production/secretion by the treated patient/individual, agents that inhibit the degradation of ADAMTS13 and thus prolonging its half life, agents that enhance ADAMTS13 activity (for example by binding to ADAMTS13, thereby inducing an activating conformational change), or agents that inhibit ADAMTS13 clearance from circulation, thereby increasing its plasma concentration.
  • additional active ingredients such as e.g. anti-thrombotic agents, agents that stimulate ADAMTS13 production/secretion by the treated patient/individual, agents that inhibit the degradation of ADAMTS13 and thus prolonging its half life, agents that enhance ADAMTS13 activity (for example by binding to ADAMTS13, thereby inducing an activating conformational change), or agents that inhibit ADAMTS13 clearance from circulation, thereby increasing its plasma concentration.
  • anti-thrombotic agents include anti-platelets, t-
  • the present invention further relates to a method of treating or preventing a disorder associated with the formation and/or the presence of one or more thrombus, comprising the step of administering a composition according to the invention to a patient.
  • Said disorder may be due to hereditary defects, inflammatory diseases, stroke or septic conditions.
  • disorders associated with the formation and/or the presence of one or more thrombus are hereditary thrombotic thrombocytopenic purpura (TTP), acquired TTP, arterial thrombosis, acute myocardial infarction (AMI), stroke, sepsis, disseminated intravascular coagulation (DIC), and venous thrombosis, such as e.g. deep vein thrombosis or pulmonary embolism.
  • Another aspect of the present invention relates to a method of disintegrating one or more thrombus in a patient, comprising the step of administering a composition according to the present invention to said patient.
  • the route of administration of the composition of the present invention does not exhibit a specific limitation and can be, for example, subcutaneous or intravenous.
  • the term “patient” as used in the present invention includes mammals, particularly human.
  • the present invention relates to the use of a pharmaceutically effective amount of ADAMTS13 or a biologically active derivative thereof for the preparation of a pharmaceutical composition for treating or preventing a disorder associated with the formation and/or the presence of one or more thrombus.
  • Another aspect of the present invention is the use of a pharmaceutically effective amount of ADAMTS13 or a biologically active derivative thereof for the preparation of a pharmaceutical composition for disintegrating one or more thrombus in a patient in need thereof.
  • the pharmaceutically effective amount of ADAMTS13 or a biologically active derivative thereof may range, for example, from 0.1 to 20 mg/kg body weight.
  • A23187 a secretagouge of Weibel-Palade bodies activated with calcium ionophore A23187 (a secretagouge of Weibel-Palade bodies) at low shear rate (approx. 100 s ⁇ 1 ) in Adamts13 ⁇ / ⁇ mice has been observed compared with WT. It was investigated whether activation of microvenule endothelium by A23187 could result in platelet aggregation resulting in thrombus formation. A23187 does not denude the endothelium (Andre P, Denis C V, Ware J, Saffaripour S, Hynes R O, Ruggeri Z M, Wagner D D.
  • r-hu ADAMTS13 r-hu ADAMTS13
  • Ferric chloride (FeCl 3 ) injury leads to deendothelization and exposes sub endothelium (Ni H, Denis C V, Subbarao S, Degen J L, Sato T N, Hynes R O, Wagner D D. Persistence of platelet thrombus formation in arterioles of mice lacking both von Willebrand factor and fibrinogen. J Clin Invest. 2000; 106:385-392). Platelet subendothelial interactions after injury at arterial shear are initiated by GPIb-vWF interaction and then propagated by other receptors (Ni H, Denis C V, Subbarao S, Degen J L, Sato T N, Hynes R O, Wagner D D.
  • vWF present in the plasma is either constitutively synthesized by endothelial cells or is secreted in the form of unusually large multimers from the platelet ⁇ -granules and endothelial Weibel-Palade bodies upon activation.
  • UL-vWF is the most adhesive and reactive form of vWF and may lead to platelet aggregation resulting in a thrombus if not processed by ADAMTS13.
  • ferric chloride thrombosis model in the Adamts13 ⁇ / ⁇ mice thrombi grew faster as thrombi greater than 30 ⁇ m were seen at 6.64 ⁇ 0.93 min compared to 10.78 ⁇ 0.80 min in the WT mice and the results were statistically significant (P ⁇ 0.005, FIG.
  • ADAMTS13-Deficiency Enhances Thrombus Growth in a ⁇ lIb ⁇ 3 Integrin-Dependent Manner
  • Beta3-integrin-deficient mice are a model for Glanzmann thrombasthenia showing placental defects and reduced survival J Clin Invest 1999; 103:229-238) would induce thrombus formation after ferric chloride injury.
  • r-hu ADAMTS13 corrects the vWF cleavage defect in hereditary TTP plasma (Antoine G, Zimmermann K, Plaimauer B, Grillowitzer M, Studt J D, Laemmle B, Scheiflinger F. ADAMTS13 gene defects in two brothers with constitutional thrombotic thrombocytopenic purpura. Br J Haematol 2003; 120:821-824). Since accelerated growth of thrombi in Adamts13 ⁇ / ⁇ mice has been observed, it is hypothesized that ADAMTS13 negatively modulates thrombus growth and therefore infusion of r-hu ADAMTS-13 in Adamts 13 ⁇ / ⁇ mice could delay thrombus formation.
  • the concentration of the circulating human protein was approximately 8.8 U/ml 17 min after infusion and 1.1 U/ml 53 min after infusion of r-hu ADAMTS13 into Adamts 13 ⁇ / ⁇ mice. These times correspond approximately to the onset of ferric chloride injury and the termination of the experiment.
  • injured arterioles did not occlude for up to 40 min when the experiment was terminated ( FIG. 7A ).
  • the effect of the infused r-hu ADAMTS13 was more than that of endogenous ADAMTS13 in WT mice as in this injury model all WT vessels occluded at less than 24 min ( FIG. 5C ).
  • mice ADAMTS13 appears to have the same anti-thrombotic potential as the human ADAMTS13 in WT mice with normal levels of endogenous ADAMTS protein.
  • mice used in the examples were siblings obtained from crosses of Adamts13 +/ ⁇ mice on C57BL/6J/129Sv background (Motto D G, Chauhan A K, Zhu G, Homeister J, Lamb C B, Desch K C, Zhang W, Tsai H M, Wagner D D, Ginsburg D. Shigatoxin triggers thrombotic thrombocytopenic purpura in genetically susceptible ADAMTS13-deficient mice. J Clin Invest 2005; 115:2752-2761).
  • the mice of pure C57BI/6J background were purchased from the Jackson Laboratory, Bar Harbor, Me. and beta3 integrin ⁇ / ⁇ mice on Balb/C bacjground were a gift from Richard Hynes (MIT).
  • mice used for intravital microscopy were young mice (approx. 4 weeks old), both male and female, weighing 14 to 18 grams. Infused platelets were isolated from 4 to 6 months old mice of the same genotype. Animals were bred and housed at CBR Institute for Biomedical Research and all experimental procedures were approved by its Animal Care and Use Committee.
  • Calcium ionophore A23187 and ferric chloride were from Sigma Chemicals, St. Louis, Mo.
  • Blood was harvested from the retro-orbital venous plexus by puncture and collected in 1.5 ml polypropylene tubes comprising 300 ⁇ l of heparin (30 U/ml).
  • Platelet rich plasma (PRP) was obtained by centrifugation at 1200 rpm for 5 min.
  • the plasma and buffy coat comprising some RBCs were gently transferred to fresh polypropylene tubes and recentrifuged at 1200 rpm for 5 min.
  • the PRP was transferred to fresh tubes comprising 2 ⁇ L of PGI 2 (2 ⁇ g/ml) and incubated at 37° C. for 5 min.
  • pellets were resuspended in 1 ml modified Tyrode's-HEPES buffer (137 mM NaCl, 0.3 mM Na 2 HPO 4 , 2 mM KCl, 12 mM NaHCO 3 , 5 mM HEPES, 5 mM glucose, 0.35% BSA) comprising 2 ⁇ l of PGI 2 and incubated at 37° C. for 5 min.
  • the suspended pellet was centrifuged at 2800 rpm for 5 min.
  • the washing step was repeated twice and platelets were fluorescently labeled with calcein AM 0.25 mg/mL (Molecular Probes, Eugene, Oreg.) for 10 min at room temperature.
  • Polyclonal rabbit anti-human ADAMTS13 IgG was produced by Baxter Bioscience, Vienna Austria. The antibody was obtained by immunization of New Zealand white rabbits with purified r-hu ADAMTS13, C-terminally tagged with six His residues. Two rabbits were immunized by injection of 20 ⁇ g of r-hu ADAMTS13 (6-His) in 200 ⁇ l of complete Freund's adjuvant. The animals were boostered after two, four and six weeks by injecting 20 ⁇ g of r-hu ADAMTS13 (6-His) in 200 ⁇ l of incomplete Freund's adjuvant. After eight weeks the rabbits were sacrificed and bled. IgG antibodies were purified by Protein G affinity chromatography (HiTrap Protein G HP column; Amersham Bioscience, Piscataway, N.J., USA) and formulated in PBS.
  • mice were anesthetized with 2.5% tribromoethanol (0.15 ml/10 g) and an incision was made through the abdominal wall to expose the mesentery and mesenteric venule of 25 to 30- ⁇ m diameter was studied. Exposed mesentery was kept moist by periodic superfusion using PBS (without Ca 2+ or Mg 2+ ) warmed to 37° C.
  • the mesentery was transluminated with a 12 V, 100 W, DC-stabilized source.
  • the shear rate was calculated using an optical Doppler velocity meter as described in Frenette P S, Moyna C, Hartwell D W, Lowe J B, Hynes R O, Wagner D D. Platelet-endothelial interactions in inflamed mesenteric venules. Blood. 1998; 91:1318-1324.
  • Venule was visualized using a Zeiss (Germany) Axiovert 135 inverted microscope (Objective 10 ⁇ and 32 ⁇ ) connected to an SVHS video recorder (AG-6730; Panasonic, Tokyo, Japan).
  • One venule was chosen per mouse and filmed for 3 min for the baseline before the A23187 superfusion (30 ⁇ l of a 10 Mmol/L solution) and monitored for 10 min.
  • Intravital microscopy was done as described above except mesenteric venules of 200 to 300- ⁇ m diameters were studied. Fluorescent platelets (1.25 ⁇ 10 9 platelets/kg) were infused through the tail vein. One venule per animal was filmed for 3 minutes for the baseline before the A23187 superfusion (30 ⁇ l of a 10 ⁇ mol/L solution) and filming continued until after the platelet sticking and rolling returned to baseline. Purified rabbit polyclonal anti-human ADAMTS13 antibody (5 mg/kg mouse) was dissolved in PBS. Control rabbit IgG (Sigma, St. Louis, Mo.) was in PBS. 200 ⁇ l of 1 mM histamine (Sigma) was injected i.p. to stimulate the endothelium. 100 ⁇ l (0.2 mg/ml) of Rhodamine 6G (Sigma) was injected i.v. to label the endogenous platelets and leukocytes prior to surgery and imaging.
  • mice were anesthetized with 2.5% tribromoethanol (0.15 ml/10 g) and fluorescent platelets (1.25 ⁇ 10 9 platelets/kg) were infused through the retro-orbital plexus of the eye.
  • An incision was made through the abdominal wall to expose the mesentery and arterioles of approx. 100 ⁇ m diameters were studied.
  • Exposed mesentery was kept moist by periodic superfusion using PBS (without Ca 2+ or Mg 2+ ) warmed to 37° C. The mesentery was transluminated and the shear rate was calculated as described above.
  • Arterioles were visualized using the same microscope described above, equipped with a 100-W HBO fluorescent lamp source (Optic Quip, Highland Mills, N.Y.) with a narrow band fluorescein isothiocyanate filter set (Chroma Technology, Brattleboro, Vt.) and a silicon-intensified tube camera C2400 (Hamamatsu, Tokyo, Japan). Whatman paper saturated with ferric chloride (10%) solution was applied topically which induced vessel injury and denudation of the endothelium. The paper was removed after 5 min and vessel was monitored for 40 min after injury or until occlusion. One arteriole was chosen per mouse.
  • thrombus formation was performed blinded to the genotype.
  • the parameters that were applied to describe the characteristics of thrombus formation were: (1) Single platelet-vessel wall interaction within 2-3 min, determined as the number of fluorescent platelets that deposited on the 250 ⁇ m vessel wall (seen on the video monitor) during 1 min. (2) The time required for formation of a thrombus larger than 30 ⁇ m. (3) Thrombus stability by determining the number of thrombi of diameter larger than 30 ⁇ m embolizing away from the viewing field before vessel occlusion. (4) Occlusion time of the vessel, that is, time required for blood to stop flowing for 10 sec and (5) site of vessel occlusion, that is, at the site of injury or downstream.
  • Recombinant human ADAMTS13 has been obtained by the methods as described in Plaimauer B, Zimmermann K, Volkel D, Antoine G, Kerschbaumer R, Jenab P, Furlan M, Gerritsen H, Lämmle B, Schwarz H P, and Scheiflinger F. Cloning, expression, and functional characterization of the von Willebrand factor-cleaving protease (ADAMTS13). Blood 2002; 100(10):3626-3632. Recombinant human ADAMTS13 protein was dissolved in 150 mmol NaCl/20 mmol Histidin/2% Sucrose/0.05% Crillet 4HP (Tween 80), pH 7.4 (Baxter Bioscience, Vienna, Austria).
  • ADAMTS13 Recombinant human ADAMTS13 was injected i.v. (3460 U/kg mouse). Levels of human ADAMTS13 antigen were determined by a slight modification of the ELISA method described by Rieger (Rieger M, Kremer Hovinga J A, Konetschny C, Herzog A, Koller L, Weber A, Remuzzi G, Dockal M, Plaimauer B and Scheiflinger F. Relation between ADAMTS13 Activity and ADAMTS13 Antigen Levels in Healthy Donors and Patients with Thrombotic Microangiopathies (TMA).
  • TMA Thrombotic Microangiopathies
  • Thrombosis and Hemostasis 2006; 95(2):212-20) and r-hu ADAMTS13 activity was determined according to Gerritsen (Gerritsen H E, Turecek P L, Schwarz H P, Laemmle B, and Furlan M. Assay of von Willebrand factor (vWF)-cleaving protease based on decreased collagen binding affinity of degraded vWF: a tool for the diagnosis of thrombotic thrombocytopenic purpura (TTP). Thromb Haemost 1999; 82:1386-1389). 1 U corresponds to the level of ADAMTS13 activity in pooled normal human plasma.
  • vWF von Willebrand factor
  • Recombinant mouse ADAMTS13 has been obtained by the methods as described in Bruno K, Völkel D, Plaimauer B, Antoine G, Pable S, Housing D G, Lemmerhirt H L, Dorner F, Zimmermann K, and Scheiflinger F. Cloning, expression and functional characterization of the full-length murine ADAMTS-13 . J Thromb Haemost 2005; 3(5):1064-1073. Recombinant mouse ADAMTS13 protein was dissolved in 150 mmol NaCl/20 mmol Histidin/2% Sucrose/0.05% Crillet 4HP (Tween 80), pH 7.4 (Baxter Bioscience, Vienna, Austria).
  • Platelet poor whole blood was reconstituted with labeled platelets before perfusion in a parallel-plate flow chamber system coated with 100 ⁇ g/mL collagen Horm (NYCOMED, Munich, Germany) for 1 h at RT. Where indicated, samples were pretreated with 30 ⁇ g/ml JON/A (emfret Analytics, Wuerzburg, Germany) for 10 min prior to perfusion. Platelet adhesion was visualized with an Axiovert 135 inverted microscope (Zeiss). The percentage of surface area covered by fluorescent platelets was analyzed using NIH Image 1.61 software by an individual blinded to genotypes.
  • Results are reported as the mean ⁇ SEM. The statistical significance of the difference between means was assessed by the Student's t test.
  • the experimental results have defined a key role for ADAMTS13 in preventing thrombi formation in activated microvenules and excessive thrombus formation in the injured arterioles of mice.
  • activating venules 25-30 ⁇ m
  • platelet aggregation leading to microthrombi formation
  • FIG. 1 Microthrombi do not form in the venules of WT mice treated identically. When released from the activated endothelium, these microthrombi can travel downstream and cause occlusion elsewhere in the small capillaries where they cannot pass, thus leading to organ ischemia.
  • TTP patients suffering from TTP often have microthrombi that are formed in the microvasculature of organs: like brain, heart, pancreas, spleen, kidney and mesentery.
  • Various agents including viruses, bacterial shiga toxins, drugs such as ticlopidine and clopidogrel, antibodies and immune complexes can trigger vascular activation perhaps inducing Weibel-Palade body release.
  • arterioles which have higher shear stress
  • no thrombi could be seen. This is because either Weibel-Palade bodies were not released in these vessels or, more likely, vWF is washed too quickly from the endothelial surface to promote platelet adhesion.
  • Platelet strings and aggregates were frequently seen in the Adamts13 ⁇ / ⁇ mice when challenged with Weibel-Palade body secretagogues such as histamine, inflammatory cytokine TNF- ⁇ and activated platelets.
  • Weibel-Palade body secretagogues such as histamine, inflammatory cytokine TNF- ⁇ and activated platelets.
  • Infusion of the r-hu ADAMTS13 protein in the Adamts13 ⁇ / ⁇ mice challenged with histamine inhibits the platelet strings formation. These strings are cleaved at the upstream end.
  • microvenules 25 to 30 ⁇ m
  • A23187 Activation of microvenules (25 to 30 ⁇ m) with A23187 results in platelet aggregates leading to thrombi formation in the Adamts13 WT mice infused with anti-ADAMTS13-Ab ( FIG. 3 ).
  • these thrombi embolized reapidly, similar to those in the Adamts13 ⁇ / ⁇ mice.
  • Microthrombi formation can be also induced in the WT mice on a C57BL/6 background infused with inhibitory antibody.
  • the mouse infused with anti-ADAMTS 13 Ab is in many aspects a good model for acquired TTP.
  • it demonstrates the role of the ADAMTS13 in preventing platelet aggregation in the circulation.
  • vWF through its receptors, GPIb ⁇ and ⁇ IIb ⁇ 3, contributes to platelet function in initiating platelet aggregation and progression of thrombus formation.
  • ADAMTS13 Since early platelet deposition in arterioles is vWF dependent, it means that either plasma ADAMTS13 reduces vWF incorporation into the basement membrane when it is exposed to blood, or that it digests vWF already present in the extra-cellular matrix.
  • ADAMTS13 cleavage of VWF domain A2 by ADAMTS13 is facilitated by the binding of VWF to GPIb ⁇ .
  • the VWF-GPIb interaction within the thrombus may negatively regulate thrombus growth.
  • Thrombus formation under venous and arterial flow conditions also depends on major integrin ⁇ IIb ⁇ 3.
  • the present examples at arteriolar shear rates show that ADAMTS13 modulates the growing thrombus only when platelets in the thrombus express an active beta3 integrin. Under these in vitro and in vivo experimental conditions, ADAMTS13-deficiency did not promote thrombus growth if the major platelet integrin was absent or inhibited ( FIG. 6 ).
  • r-hu ADAMTS13 was infused into Adamts13 ⁇ / ⁇ and WT mice prior to injury.
  • the anti-thrombotic effect of the r-hu ADAMTS13 although highly statistically significant, varied among the animals ( FIGS. 7A and 7B ).
  • Some mice did not respond to r-hu ADAMTS13 treatment. It is possible that in these mice r-hu ADAMTS13 was proteolytically inactivated by thrombin and plasmin produced at the sites of vascular injury. IL-6 and high amounts of vWF released after inflammation or injury could also reduce ADAMTS13 activity.
  • ADAMTS13 have both anti-thrombotic and thrombolytic activity.
  • a possible mechanism is ADAMTS13 cleaving the UL-vWF multimers into smaller fragments that are less adhesive or directly cleaving the vWF molecules bridging platelets in a thrombus as is the case in cleavage of platelets attached to strings.
  • ADAMTS13 is active at both low venous and high arterial shear stress conditions. It cleaves platelet strings and regulates platelet interaction with the “activated” vessel wall in the venules, prevents thrombi in activated microvenules and modulates the thrombotic response in injured arterioles.
  • ADAMTS13 Congenital TTP patients are currently treated by plasma exchange, whereas acquired TTP patients are subjected to plasmapheresis where autoantibodies are removed from the plasma.
  • the antithrombotic and thrombolytic effect of ADAMTS13 protein indicates that besides TTP, e.g. recombinant ADAMTS13 can be used to treat patients suffering with thrombotic disorders due to hereditary defects, inflammatory disease, septic conditions, or venous thrombosis, such as e.g. deep vein thrombosis or pulmonary embolism.
  • the thrombolytic activity of the ADAMTS13 shows that this protein can be used in combination with other therapies to disintegrate thrombi in clogged arteries or after minor stroke.
  • the experimental data show that the metalloprotease ADAMTS13 negatively regulates thrombosis, which indicates that the molecule has anti-thrombic activity.
  • recombinant human ADAMTS13 protein having thrombolytic activity can be used as a therapeutic agent to treat thrombotic disorders.

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US11124787B2 (en) 2021-09-21
WO2006133955A8 (en) 2007-11-29
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US20220002703A1 (en) 2022-01-06
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HK1118579A1 (en) 2009-02-13
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CA2606351C (en) 2016-12-13
AU2006257073B2 (en) 2011-08-04
CA2606351A1 (en) 2006-12-21
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US20130136732A1 (en) 2013-05-30
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