Methods for treating an ischemic disorder and improving stroke outcome

The present invention provides for a method for treating an ischemic disorder in a subject which comprises administering to the subject a pharmaceutically acceptable Factor IXa compound in a sufficient amount over a sufficient time period so as to treat the ischemic disorder in the subject. The invention further provides a method for treating an ischemic disorder in a subject which comprises administering to the subject a pharmaceutically acceptable form of inactivated Factor IXa in a sufficient amount over a sufficient period of time to inhibit coagulation so as to treat the ischemic disorder in the subject.

Throughout this application, various publications are referenced following
 certain Examples and within the Detailed Description of the Invention
 section. The disclosures of these publications in their entireties are
 hereby incorporated by reference into this application in order to more
 fully describe the state of the art as known to those skilled therein as
 of the date of the invention described and claimed herein.
 BACKGROUND OF THE INVENTION
 As described in Colman et al., Editors, Hemostasis and Thrombosis, Third
 Edition, J.B. Lippincott Company, Philadelphia, 1994, pages 33-36, 62-63
 and 94-105, human Factor IX is a 415 amino acid glycoprotein
 (Mr.apprxeq.57,000, 17% carbohydrate). Factor IX is a proenzyme that has
 no catalytic activity. During the coagulation cascade, it is cleaved by
 Factor XIa to produce catalytically active Factor IXa. A wide variety of
 Factor IX gene mutations are found in patients with hemophilia B. Among
 these are mutations in the enzyme active site, including a Ser365 to Arg
 mutation and mutations near His221. (Colman et al., page 63) These
 mutations affect the ability of the active site to proteolytically cleave
 its Factor X substrate. Mutations of Gly363 to Val were found to be
 functionally normal but unable to activate Factor X (Colman et al., page
 104).
 The gene for Factor IX has been identified, cDNA for Factor IX has been
 isolated, sequenced, and cloned into expression vectors, and recombinant
 Factor IX has been expressed. See, for example, Durachi et al., "Isolation
 and characterization of a cDNA coding for human Factor IX," Proc. Natl.
 Acad. Sci. USA 79: 6461, 1982 (GenBank Accession Nos. J00136 and 182690);
 Choo et al., "Molecular cloning of the gene for human anti-haemophilic
 factor Factor IX," Nature 299: 178, 1982; Anson et al., "the gene
 structure of human anti-haemophilic factor Factor IX," EMBO J. 3:1053,
 1984; Yshitake et al., "Nucleotide Sequence of the gene for human Factor
 IX," Biochemistry 24:3736, 1985 (GenBank Accession No. 182,613); Anson et
 al., "Expression of active human clotting Factor IX from recombinant DNA
 clones in mammalian cells," Nature 315:683,1985; Busby et al., "Expression
 of active human Factor IX in transfected cells," Nature 316:271, 1985; de
 la Salle et al., "Active gamma carboxylated human Factor IX expressed
 using recombinant DNA techniques," Nature 316: 268, 1985; and Kaufman et
 al., "Expression, purification, and characterization of recombinant
 gamma-carboxylated factor IX synthesized in Chinese hamster ovary cells,"
 J. Biol. Chem. 261:9622, 1986. See also Brownlee et al., UK Patent
 Application GB 2 125 409 A, published Mar. 7, 1984; Anson et al., U.S.
 Pat. No. 5,171,569, issued Dec. 15, 1992; Muelien U.S. Pat. No. 5,521,070,
 issued May 284 1996; Kaufman et al., U.S. Pat. No. 4,770,999, issued Sep.
 13, 1988; and Barr et al., U.S. Pat. No. 5,460,950, issued Oct. 24, 1995.
 In addition, Benedict et al. (1994) Texas Heart Institute Journal Vol 21,
 No. 1, pp 85-90 disclose that infusion of Factor IXai at concentrations
 sufficient to inhibit intravenous coagulation did not produce bleeding
 significantly different from that in control animals. Therefore, the
 invention disclosed herein was unexpected in view of this report.
 SUMMARY OF THE INVENTION
 The present invention provides a method for treating an ischemic disorder
 in a subject which comprises administering to the subject a
 pharmaceutically acceptable form of a Factor IXa compound in a sufficient
 amount over a sufficient period of time to inhibit coagulation so as to
 treat the ischemic disorder in the subject. The present invention provides
 a method for treating an ischemic disorder in a subject which comprises
 administering to the subject a pharmaceutically acceptable form of
 inactivated Factor IXa in a sufficient amount over a sufficient period of
 time to inhibit coagulation so as to treat the ischemic disorder in the
 subject.

DETAILED DESCRIPTION OF THE INVENTION
 The present invention provides a method for treating an ischemic disorder
 in a subject which comprises administering to the subject a
 pharmaceutically acceptable form of a Factor IXa compound in a sufficient
 amount over a sufficient period of time to inhibit coagulation so as to
 treat the ischemic disorder in the subject.
 The present invention also provides a method for treating an ischemic
 disorder in a subject which comprises administering to the subject a
 pharmaceutically acceptable form of a Factor IXa compound in a sufficient
 amount over a sufficient period of time to inhibit coagulation so as to
 treat the ischemic disorder in the subject.
 The present invention provides a method for treating an ischemic disorder
 in a subject which comprises administering to the subject a
 pharmaceutically acceptable form of a Factor IXa compound and a
 pharmaceutically acceptable form of an indirect or direct fibrinolytic
 agent, each in a sufficient amount over a sufficient period of time to
 inhibit coagulation so as to treat the ischemic disorder in the subject.
 In another embodiment, the ischemic disorder comprises a peripheral
 vascular disorder, a pulmonary embolus, a venous thrombosis, a myocardial
 infarction, a transient ischemic attack, unstable angina, a reversible
 ischemic neurological deficit, sickle cell anemia or a stroke disorder.
 In another embodiment, the ischemic disorder is iatogenically induced. In
 another embodiment, the subject is undergoing angioplasty, heart surgery,
 lung surgery, spinal surgery, brain surgery, vascular surgery, abdominal
 surgery, or organ transplantation surgery. In another embodiment, the
 organ transplantation surgery comprises heart, lung, pancreas or liver
 transplantation surgery.
 In another embodiment, the period of time comprises from about 5 days
 before surgery or onset of the disorder to about 5 days after surgery or
 the onset of the disorder. In another embodiment, the period of time
 comprises from about 1 hour before surgery or the onset of the disorder to
 about 12 hours after surgery or the onset of the disorder. In another
 embodiment, the period of time comprises from about 12 hours before
 surgery or the onset of the disorder to about 1 hour after surgery or the
 onset of the disorder. In another embodiment, the period of time comprises
 from about 1 hour before surgery or the onset of the disorder to about 1
 hour after surgery or the onset of the disorder.
 In one embodiment, the subject is a mammal. In another embodiment, the
 mammal is a human. In another embodiment, the amount comprises from about
 75 .mu.g/kg to about 550 .mu.g/kg. In another embodiment, the amount
 comprises 300 .mu.g/kg.
 In one embodiment, the direct fibrinolytic agent comprises plasmin or viper
 venom. In another embodiment, the indirect fibrinolytic agent comprises
 tissue plasminogen activator, urokinase, streptokinase, RETROVASE.RTM., or
 recombinant tissue plasminogen activator.
 The present invention also provides for a method for identifying a compound
 that is capable of improving an ischemic disorder in a subject which
 comprises: a) administering the compound to an animal, which animal is a
 stroke animal model; b) measuring stroke outcome in the animal, and c)
 comparing the stroke outcome in step (b) with that of the stroke animal
 model in the absence of the compound so as to identify a compound capable
 of improving an ischemic disorder in a subject. In another embodiment, the
 compound is a Factor IXa compound.
 In one embodiment, the stroke animal model comprises a murine model of
 focal cerebral ischemia and reperfusion. In another embodiment, the stroke
 outcome is measured by physical examination, magnetic resonance imaging,
 laser doppler flowmetry, triphenyl tetrazolium chloride staining, chemical
 assessment of neurological deficit, computed tomography scan, or cerebral
 cortical blood flow.
 The present invention provides a method for treating a reperfusion injury
 in a subject which comprises administering to the subject a Factor IXa
 compound in a sufficient amount over a sufficient period of time to
 inhibit coagulation so as to treat the reperfusion injury in the subject.
 In one embodiment, the Factor IXa compound comprises recombinant
 inactivated Factor IXa. In another embodiment, the Factor IXa compound is
 a peptide, a peptidomimetic, a nucleic acid, a small molecule, a mutated
 peptide or nucleic acid, a mutein, an antibody or fragment thereof. In
 another embodiment, the Factor IXa compound is a synthetic molecule.
 The present invention provides for a proteolytically inactive recombinant
 mutein of Factor IX, which has substantially the same amino acid sequence
 as normal Factor IX but which has an amino acid substitution for one or
 more of His221, Asp269 or Ser365. In one embodiment, the mutein has a
 Ser365 to Ala substitution.
 The present invention also provides a proteolytically inactive recombinant
 mutein of Factor IXa which has substantially the same amino acid sequence
 as normal human Factor IXa but which has an amino acid substitution for
 one or more of His41, Asp89 or Ser185 in the heavy chain of Factor IXa. In
 one embodiment, the mutein. has a Ser185 to Ala substitution.
 In another embodiment, an isolated cDNA encodes the mutein. In another
 embodiment, a replicable vector comprises the cDNA. In another embodiment,
 a microorganism is transfected with the vector. In another embodiment, an
 expression vector comprises DNA which encodes the mutein. In another
 embodiment, a microorganism is transfected with the vector. In one
 embodiment, the Factor IXa compound comprises the mutein.
 The present invention provides a method of inhibiting clot formation in a
 subject which comprises adding to blood an amount of an inactive
 recombinant mutein in an amount effective to inhibit clot formation in the
 subject but which does not significantly interfere with hemostasis when
 the blood is administered to a patient. In another embodiment, the patient
 has experienced an ischemic event.
 The present invention provides for an assay to monitor the effect of a
 Factor IXa compound administered to a subject to treat an ischemic
 disorder in the subject which comprises: a) measuring the ischemic
 disorder in the subject; b) administering the Factor IXa compound to the
 subject and measuring the ischemic disorder, and c) comparing the
 measurement of the ischemic disorder in step (b) with that measured in
 step (a) so as to monitor the effect of the Factor IXa compound. In one
 embodiment, the ischemic disorder is measured by physical examination,
 magnetic resonance imaging, laser doppler flowmetry, triphenyl tetrazolium
 chloride staining, chemical assessment of neurological deficit, computed
 tomography scan, or cerebral cortical blood flow.
 As used herein, the "ischemic disorder" encompasses and is not limited to a
 peripheral vascular disorder, a venous thrombosis, a pulmonary embolus, a
 myocardial infarction, a transient. ischemic attack, lung ischemia,
 unstable angina, a reversible ischemic neurological deficit, adjunct
 thromolytic activity, excessive clotting conditions, reperfusion injury,
 sickle cell anemia, a stroke disorder or an iatrogenically induced
 ischemic period such as angioplasty.
 In one embodiment of the present invention, the subject is undergoing heart
 surgery, angioplasty, lung surgery, spinal surgery, brain surgery,
 vascular surgery, abdominal surgery, or organ transplantation surgery. The
 organ transplantation surgery may include heart, lung, pancreas or liver
 transplantation surgery.
 In the intrinsic pathway, Factor XIa cleaves Factor IX between Arg145 and
 Ala146 and between Arg 180-Val18l, releasing a 35 amino acid peptide and
 producing Factor IXa having a 145 amino acid light chain (amino acids
 1-145) and a 235 amino acid heavy chain (amino acids 181-415) joined by a
 disulfide bond between cysteine residues at positions 132 and 289. Factor
 IXa is a serine protease which, when complexed with Factor VIIIa on
 membrane surfaces, converts Factor X to its active form Factor Xa. The
 enzyme active site of Factor IXa is located on the heavy chain. Three
 amino acids in the heavy chain are principally responsible for the
 catalytic activity, His221, Asp269 and Ser365 (H221, D269 and S365, the
 catalytic triad). If the amino acids of the heavy chain are numbered from
 1 to 235, the catalytic triad is His41, Asp89 and Ser185, and the
 disulfide bond joining the heavy chain to the light chain is at Cys109 on
 the heavy chain.
 As used herein "a Factor IXa compound" means a compound which inhibits or
 reduces the conversion of Factor X to Factor Xa by naturally occurring
 Factor IX. As used herein, a Factor IXa compound may be chosen from one of
 several subsets. One subset is a chemically modified form of naturally
 occurring Factor IXa which chemical modification results in the
 inactivation of Factor IXa (e.g., inactivated Factor IXa, active-site
 blocked Factor IXa or Factor IXai). Another subset of a Factor IXa
 compound is any recombinant mutated form of Factor IXa (e.g., a mutein
 form of Factor IXa, a recombinant Factor IXa with a deletion or Factor
 IXami). In addition, there are other subsets of a Factor IXa compound
 which include but are not limited to, for example: (1) nucleic acids, (2)
 anti-Factor IXa antibodies or fragments thereof, (3) saccharides, (4)
 ribozymes, (5) small organic molecules, or (6) peptidomimetics.
 Thus, a Factor IXa compound may encompass the following: a Glu-Gly-Arg
 chloromethyl ketone-inactivated human factor IXa, an inactive Christmas
 factor, a Glu-Aly-Arg chloromethyl ketone-inactivated factor IXa, a
 glutamyl-glycyl-arginyl-Factor IXa, a dansyl Glu-Gly-Arg chloromethyl
 ketone-inactivated bovine factor IXa (IXai), a Factor IXai, a competitive
 inhibitor of Factor IXa, a peptide mimetic of Factor IXa, a carboxylated
 Christmas factor, a competitive inhibitor of the formation of a Factor
 IXa/VIIIa/X complex, a des-.gamma.-carboxyl Factor IX, Factor IX lacking a
 calcium-dependent membrane binding function, inactive Factor IX including
 only amino acids 1-47, apoFactor IX including amino acids 1-47, Factor IX
 Bm Kiryu, a Val-313-to-Asp substitution in the catalytic domain of Factor
 IX, a Gly-311-to-Glu substitution in the catalytic domain of Factor IX, a
 Gly-311 to Arg-318 deletion mutant of Factor IX, an anti-Factor IXa
 antibody, an anti-Factor IXa monoclonal or polyclonal antibody. The Factor
 IXa compound may also include inactive species of Factor IX described in
 the references provided herein, especially Freedman et al., 1995; Furie
 and Furie, 1995; Miyata et al., 1994 and Wacey et al., 1994. Factor IX or
 Factor IXa may be obtained from blood.
 Thus, a Factor IXa compound may be Factor IXa in which the active site is
 blocked and may be prepared as described in Experimental Details below.
 The Factor IXa compound may be a Factor IXa which includes
 post-translational modifications including glycosylation,
 .beta.-hydroxylation of aspartic acid, .gamma.-carboxylation of glutamic
 acid and propeptide cleavage. The Factor IXa compound may be concentrated
 via heparin affinity chromatography or hydrophobic interaction
 chromatography. The Factor IXa compound may be a genetically engineered, a
 recombinant Factor IXa in which amino acids at the active site, especially
 the serine amino acid at the active site, have been altered to render the
 recombinant Factor IXa functionally inactive, but still capable of
 competing with intact, native Factor IXa for cell surface binding. In
 another embodiment, the Factor IXa compound is a synthetic molecule. In
 another embodiment, the carrier comprises an aerosol, intravenous, oral or
 topical carrier.
 In one embodiment of the present invention the Factor IXa compound is a
 form of Factor IXa inactivated by the standard methods known to one of
 skill in the art, such as mutation of the gene which encodes Factor IXa.
 As used herein an "indirect fibrinolytic agent" is an agent whose activity
 indirectly results in fibrin lysis. In one embodiment, an indirect
 fibrinolytic agent comprises tissue plasminogen activator (tPA),
 urokinase, streptokinase, RETROVASE.RTM., or recombinant tissue
 plasminogen activator. As used herein "direct fibrinolytic agent" is an
 agent that is capable of fibrinolysis. In one embodiment, a direct
 fibrinolytic agent is plasmin or viper venom. In one embodiment, the
 amount of fibrinolytic agent administered to a subject is up to the amount
 necessary to lyse an intravascular fibrin clot or an amount to cause lysis
 of a formed intravascular fibrin clot.
 One embodiment of the present invention is wherein the Factor IXa compound
 is inactivated by the standard methods known to one of skill in the art,
 such as mutation. Factor IXa compound may be an antagonist of Factor IXa.
 Such antagonist may be a peptide mimetic, a nucleic acid molecule, a
 ribozyme, a polypeptide, a small molecule, a carbohydrate molecule, a
 monosaccharide, an oligosaccharide or an antibody.
 A preferred embodiment of the present invention is wherein the Factor IXa
 compound is an active site-blocked Factor IXa or a Glu-Gly-Arg
 chloromethyl ketone-inactivated human factor IXa. In a preferred
 embodiment, the effective amount is from about 0.1 .mu.g/ml plasma to
 about 250 .mu.g/ml plasma or from about 0.5 .mu.g/ml plasma to about 25
 .mu.g/ml plasma or preferably from 0.7 .mu.g/ml plasma to about 5 g/ml
 plasma.
 Another embodiment of present invention is where the sufficient amount
 includes but is not limited to from about 75 .mu.g/kg to about 550
 .mu.g/kg. The amount may be 300 .mu.g/kg.
 In an embodiment of the present invention the Factor IXa compound is an
 inactive mutein form of Factor IXa which is useful as selective
 antithrombotic agent. As used herein, "mutein form" of Factor IXa means a
 protein which differs from natural factor IXa by the presence of one or
 more amino acid additions, deletions, or substitutions which reduce or
 eliminate the ability of the protein to participate in the conversion of
 Factor X to Factor Xa.
 In another embodiment of the present invention the Factor IXa compound is a
 proteolytically inactive, recombinant mutein form of Factor IX, which has
 substantially the same amino acid sequence as normal or native human
 Factor IX but in which a different amino acid has been substituted for one
 or more of His221, Asp269 and Ser365. The present invention also provides
 a proteolytically inactive, recombinant mutein form of Factor IXa, which
 has substantially the same amino acid sequence as normal or native human
 factor IXa but in which a different amino acid has been substituted for
 one or more of His41, Asp89 or Ser185 in the heavy chain of Factor IXa.
 The term "proteolytically inactive" means that the muteins are incapable
 of converting Factor X to Factor Xa.
 The invention also provides a method of inhibiting thrombosis in a human
 patient which comprises administering to the patient, or adding to the
 blood which is to be administered to the patient, an amount of an inactive
 recombinant mutein of this invention which is effective to inhibit
 thrombosis but which does not significantly interfere with hemostasis in
 the patient.
 Recombinant muteins of Factor IX useful in this invention are referred to
 collectively as Factor Ixmi (i.e., Factor IX mutationally inactivated).
 Recombinant muteins of Factor IXa useful in this invention are referred to
 collectively as Factor IXami. Examples of Factor IXa compounds which are
 recombinant muteins are as follows:
 Factor IXmi (Ser365.fwdarw.Xxx)
 Factor IXmi (Asp269.fwdarw.Yyy)
 Factor IXmi (His221.fwdarw.Zzz)
 Factor IXmi (Ser365.fwdarw.Xxx, Asp269.fwdarw.Yyy)
 Factor IXmi (Ser 365.fwdarw.Xxx, His221Zzz)
 Factor IXmi (Asp269.fwdarw.Yyy, His.fwdarw.Zzz)
 Factor IXmi (Ser365.fwdarw.Xxx, Asp269.fwdarw.Yyy, His.fwdarw.Zzz)
 Factor IXami (Ser365.fwdarw.Xxx)
 Factor IXami (Asp269.fwdarw.Yyy)
 Factor IXami (His221.fwdarw.Zzz)
 Factor IXami (Ser365.fwdarw.Xxx, Asp269.fwdarw.Yyy)
 Factor IXami (Ser365.fwdarw.Xxx, His221.fwdarw.Zzz)
 Factor IXami (Asp269.fwdarw.Yyy, His.fwdarw.Zzz)
 Factor IXami (Ser365.fwdarw.Xxx, Asp269.fwdarw.Yyy, His.fwdarw.Zzz)
 wherein Xxx is any one of the standard amino acids other than serine, Yyy
 is any one of the standard amino acids other than aspartic acid, and Zzz
 is any of the standard amino acids other than histidine. Preferred
 recombinant muteins are Factor IXmi(Ser365.fwdarw.Ala) and Factor IXami
 (Ser365.fwdarw.Ala).
 Factor IXmi and Factor IXami are functionally similar to Factor IXai in
 terms of their ability to establish effective anti-coagulation
 intravascularly and in ex vivo equipment connected to the blood stream
 while permitting retention of effective hemostasis. The advantages of
 Factor IXmi and Factor IXami over Factor IXai are the following:
 Factor IXmi and Factor IXami can be produced directly in a genetically
 engineered organism, thus avoiding several processing and purification
 steps with their attendant losses, thereby improving yield of product.
 The cost of production of Factor IXmi and Factor IXami in an appropriate
 genetically engineered organism is lower than the cost of production of
 Factor IXai from human plasma.
 Factor IXmi and Factor IXami, produced in a genetically engineered
 organism, will not be subject to the risk cf contamination with various
 infectious agents such as viruses or prions (for example the agents for
 HIV disease and for bovine and/or human spongiform encephalopathies).
 Factor IXmi and Factor IXami, being less different from wild-type human
 Factor IX and Factor IXa than is the chemically modified Factor IXai, will
 have a lower probability of eliciting an immune response in patients who
 are dosed with the modified protein for extended periods of time, thereby
 reducing the risk of delayed type hypersensitivity reactions and improving
 the safety for indications such as anticoagulation in hemodialysis that
 will require repeated, long-term use.
 The recombinant muteins of this invention can be produced by known genetic
 engineering techniques, using as starting material recombinant cDNA for
 Factor IX in an appropriate cloning vector. For example, starting
 materials which may be used in the production of a Factor IXa compound may
 be the product of Example 5 of U.S. Pat. No. 4,770,9990 which are
 recombinant plaques of E. coli infected with bacteriophage M12mp11 Pst
 vector containing the entire sequence of recombinant Factor IX cDNA
 ligated to Pst: adapters. The recombinant plaques are used to prepare
 single-stranded DNA by either the small-scale or large-scale method
 described in Sambrook et al., Molecular Cloning, A Laboratory Manual,
 Second Edition, Cold Spring Harbor Press, 1989, pages 4.29-4.30 and 4.32.
 The single-stranded M13mp11 containing Factor IX cDNA is then used to carry
 out oligonucleotide-mediated mutagenesis using the double primer method of
 Zoller and Smith as described in Sambrook et al., 1989, pages 15.51-15.73.
 Mutagenic primers which can be used include the following:
 1) Oligonucleotides for producing Factor IXmi(Ser365.fwdarw.Xxx)
 3'-W ACA GTT CCT CTA XXX CCC CCT GGG GTA V-5' (SEQ ID NO: 1-9)
 where
 W is T, 3'-GT or 3'-AGT
 V is C, 3'-CA, or 3'-CAA
 XXX is the complement to a DNA codon for any one of the standard amino
 acids other than serine.
 2) Oligonucleotides for producing FACTOR IXmi (Asp269.fwdarw.Yyy)
 3'-W TTC ATG TTA GTA YYY TAA CGC GAA GAC V-5' (SEQ ID NO: 10-18)
 where
 W IS A, 3'=TA, OR 3'-TTA
 V is C, 3'-CT, or 3'-CTT
 YYY is the complement to a DNA codon for any one of the standard amino
 acids other than aspartic acid and cysteine.
 3) Oligonucleotides for producing Factor IXmi (His221Zzz)
 3'-TTA CAT TGA CGA CGG ZZZ ACA CAA CTT TGA CCA-5' (SEQ ID NO: 19)
 where
 W is A, 3'-AA, or 3'-TAA
 V is C, 3'-CC, or 3'-CCA
 ZZZ is the complement to a DNA codon for any one of the standard amino
 acids other than histidine and cysteine.
 Oligonucleotide primers for producing the preferred Factor IXmi of this
 invention, Factor IXmi(Ser365.fwdarw.Ala), are those of No. 1 above,
 wherein XXX is the complement of a codon for alanine, i.e., 3'-CGA,
 3'-CGC, 3'-CGT or 3'-CGC. A specific primer for producing Factor IXmi
 (Ser365.fwdarw.Ala) is:
 3'-GT ACA GTT CCT CTA CGA CCC CCT GGG GTA C-5' (SEQ ID NO: 20)
 A skilled artisan would recognize and know how to carry out the remaining
 steps of oligonucleotide-mediated mutagenesis as follows:
 Hybridization of mutagenic oligonucleotides to the target DNA.
 Extension of the hybridized oligonucleotides to the target DNA.
 Transfection of susceptible bacteria.
 Screening of plaques for the desired mutation.
 Preparation of single-stranded DNA from a mutant plaque.
 Sequencing the single-stranded DNA.
 Recovery of double-stranded Factor IXmi cDNA.
 Inserting the double-stranded Factor IXmi cDNA into the expression vector
 used by Kaufman (for example).
 Expression of Factor IXmi.
 Treating the Factor IXmi with Factor XIa to produce Factor IXami.
 Another embodiment of the present invention wherein the Factor IXa compound
 is capable of inhibiting the active site of Factor IXa. Such a compound is
 obtainable from the methods described herein. The Factor IXa compound may
 be a peptide, a peptidomimetic, a nucleic acid or a small molecule. The
 agent may be an antibody or portion thereof. The antibody may be a
 monoclonal antibody or a polyclonal antibody. The portion of the antibody
 may include a Fab.
 One embodiment of the present invention is wherein the Factor IXa compound
 is a peptidomimetic having the biological activity of a Factor IXa or a
 Glu-Gly-Arg chloromethyl ketone-inactivated human Factor IXa wherein the
 compound has a bond, a peptide backbone or an amino acid component
 replaced with a suitable mimic. Examples of unnatural amino acids which
 may be suitable amino acid mimics include .beta.-alanine, L-.alpha.-amino
 butyric acid, L-.gamma.-amino butyric acid, L-.alpha.-amino isobutyric
 acid, L-.epsilon.-amino caproic acid, 7-amino heptanoic acid, L-aspartic
 acid, L-glutamic acid, cysteine (acetamindomethyl),
 N-.epsilon.-Boc-N-.alpha.-CBZ-L-lysine,
 N-.epsilon.-Boc-N-.alpha.-Fmoc-L-lysine, L-methionine sulfone,
 L-norleucine, L-norvaline, N-.alpha.-Boc-N-.delta.CBZ-L-ornithine,
 N-.delta.-Boc-N-.alpha.-CBZ-L-ornithine, Boc-p-nitro-L-phenylalanine,
 Boc-hydroxyproline, Boc-L-thioproline. (Blondelle, et al. 1994; Pinilla,
 et al. 1995).
 The present invention incorporates U.S. Pat. Nos. 5,446,128, 5,422,426 and
 5,440,013 in their entireties as references which disclose the synthesis
 of peptidomimetic compounds and methods related thereto. The compounds of
 the present invention may be synthesized using these methods. The present
 invention provides for peptidomimetic compounds which have substantially
 the same three-dimensional structure as those compounds described herein.
 In addition to the compounds disclosed herein having naturally-occurring
 amino acids with peptide or unnatural linkages, the present invention also
 provides for other structurally similar compounds such as polypeptide
 analogs with unnatural amino acids in the compound. Such compounds may be
 readily synthesized on a peptide synthesizer available from vendors such
 as Applied Biosystems, Dupont and Millipore.
 Another embodiment of the present invention is a pharmaceutical composition
 which may include an effective amount of a Factor IXa compound and a
 pharmaceutically acceptable carrier. The carrier may include a diluent.
 Further, the carrier may include an appropriate adjuvant, a herpes virus,
 an attenuated virus, a liposome, a microencapsule, a polymer encapsulated
 cell or a retroviral vector. The carrier may include an aerosol,
 intravenous, oral or topical carrier.
 The present invention provides for a method for identifying a compound that
 is capable of improving an ischemic disorder in a subject which includes:
 a) administering the compound to an animal, which animal is a stroke
 animal model; b) measuring stroke outcome in the animal, and c) comparing
 the stroke outcome in step (b) with that of the stroke animal model in the
 absence of the compound so as to identify a compound capable of improving
 an ischemic disorder in a subject. The stroke animal model includes a
 murine model of focal cerebral ischemia and reperfusion. The stroke
 outcome may be measured by physical examination, magnetic resonance
 imaging, laser doppler flowmetry, triphenyl tetrazolium chloride staining,
 clinical assessment of neurological deficit, computed tomography scan, or
 cerebral cortical blood flow. The stroke outcome in a human may be
 measured also by clinical measurements, quality of life scores and
 neuropsychometric testing.
 The present invention provides for treatment of ischemic disorders by
 inhibiting the ability of the neutrophil, monocyte or other white blood
 cell to adhere properly. This may be accomplished removing the counter
 ligand, such as CD18. It has been demonstrated as discussed hereinbelow,
 that "knock-out" CD18 mice (mice that do not have expression of the normal
 CD18 gene) are protected from adverse ischemic conditions. The endothelial
 cells on the surface of the vessels in the subject may also be a target
 for treatment. In a mouse model of stroke, administration of TPA as a
 thrombolytic agent caused some visible hemorrhaging along with improvement
 of the stroke disorder. The present invention may be used in conjunction
 with a thrombolytic therapy to increase efficacy of such therapy or to
 enable lower doses of such therapy to be administered to the subject so as
 to reduce side effects of the thrombolytic therapy.
 As used herein, the term "suitable pharmaceutically acceptable carrier"
 encompasses any of the standard pharmaceutically accepted carriers, such
 as phosphate buffered saline solution, water, emulsions such as an
 oil/water emulsion or a triglyceride emulsion, various types of wetting
 agents, tablets, coated tablets and capsules. An example of an acceptable
 triglyceride emulsion useful in intravenous and intraperitoneal
 administration of the compounds is the triglyceride emulsion commercially
 known as Intralipid.RTM..
 Typically such carriers contain excipients such as starch, milk, sugar,
 certain types of clay, gelatin, stearic acid, talc, vegetable fats or
 oils, gums, glycols, or other known excipients. Such carriers may also
 include flavor and color additives or other ingredients.
 This invention also provides for pharmaceutical compositions including
 therapeutically effective amounts of protein compositions and compounds
 capable of treating ischemic disorder or improving stroke outcome in the
 subject of the invention together with suitable diluents, preservatives,
 solubilizers, emulsifiers, adjuvants and/or carriers useful in treatment
 of neuronal degradation due to aging, a learning disability, or a
 neurological disorder. Such compositions are liquids or lyophilized or
 otherwise dried formulations and include diluents of various buffer
 content (e.g., Tris-HCl., acetate, phosphate), pH and ionic strength,
 additives such as albumin or gelatin to prevent absorption to surfaces,
 detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts),
 solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants
 (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g.,
 Thimerosal, benzyl alcohol, parabens), bulking substances or tonicity
 modifiers (e.g., lactose, mannitol), covalent attachment of polymers such
 as polyethylene glycol to the compound, complexation with metal ions, or
 incorporation of the compound into or onto particulate preparations of
 polymeric compounds such as polylactic acid, polglycolic acid, hydrogels,
 etc, or onto liposomes, micro emulsions, micelles, unilamellar or multi
 lamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions
 will influence the physical state, solubility, stability, rate of in vivo
 release, and rate of in vivo clearance of the compound or composition. The
 choice of compositions will depend on the physical and chemical properties
 of the compound capable of alleviating the symptoms of the stroke disorder
 or improving the stroke outcome in the subject. Controlled or sustained
 release compositions include formulation in lipophilic depots (e.g., fatty
 acids, waxes, oils). Also comprehended by the invention are particulate
 compositions coated with polymers (e.g., poloxamers or poloxamines) and
 the compound coupled to antibodies directed against tissue-specific
 receptors, ligands or antigens or coupled to ligands of tissue-specific
 receptors. Other embodiments of the compositions of the invention
 incorporate particulate forms protective coatings, protease inhibitors or
 permeation enhancers for various routes of administration, including
 parenteral, pulmonary, nasal and oral.
 Portions of the compound of the invention may be "labeled" by association
 with a detectable marker substance (e.g., radiolabeled with 125I or
 biotinylated) to provide reagents useful in detection and quantification
 of compound or its receptor bearing cells or its derivatives in solid
 tissue and fluid samples such as blood, cerebral spinal fluid or urine.
 When administered, compounds are often cleared rapidly from the circulation
 and may therefore elicit relatively short-lived pharmacological activity.
 Consequently, frequent injections of relatively large doses of bioactive
 compounds may by required to sustain therapeutic efficacy. Compounds
 modified by the covalent attachment of water-soluble polymers such as
 polyethylene glycol, copolymers of polyethylene glycol and polypropylene
 glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol,
 polyvinylpyrrolidone or polyproline are known to exhibit substantially
 longer half-lives in blood following intravenous injection than do the
 corresponding unmodified compounds (Abuchowski et al., 1981; Newmark et
 al., 1982; and Katre et al., 1987). Such modifications may also increase
 the compound's solubility in aqueous solution, eliminate aggregation,
 enhance the physical and chemical stability of the compound, and greatly
 reduce the immunogenicity and reactivity of the compound. As a result, the
 desired in vivo biological activity may be achieved by the administration
 of such polymer-compound adducts less frequently or in lower doses than
 with the unmodified compound.
 Attachment of polyethylene glycol (PEG) to compounds is particularly useful
 because PEG has very low toxicity in mammals (Carpenter et al., 1971). For
 example, a PEG adduct of adenosine deaminase was approved in the United
 States for use in humans for the treatment of severe combined
 immunodeficiency syndrome. A second advantage afforded by the conjugation
 of PEG is that of effectively reducing the immunogenicity and antigenicity
 of heterologous compounds. For example, a PEG adduct of a human protein
 might be useful for the treatment of disease in other mammalian species
 without the risk of triggering a severe immune response. The compound of
 the present invention capable of alleviating symptoms of a cognitive
 disorder of memory or learning may be delivered in a microencapsulation
 device so as to reduce or prevent an host immune response against the
 compound or against cells which may produce the compound. The compound of
 the present invention may also be delivered microencapsulated in a
 membrane, such as a liposome.
 Polymers such as PEG may be conveniently attached to one or more reactive
 amino acid residues in a protein such as the alpha-amino group of the
 amino terminal amino acid, the epsilon amino groups of lysine side chains,
 the sulfhydryl groups of cysteine side chains, the carboxyl groups of
 aspartyl and glutamyl side chains, the alpha-carboxyl group of the
 carboxy-terminal amino acid, tyrosine side chains, or to activated
 derivatives of glycosyl chains attached to certain asparagine, serine or
 threonine residues.
 Numerous activated forms of PEG suitable for direct reaction with proteins
 have been described. Useful PEG reagents for reaction with protein amino
 groups include active esters of carboxylic acid or carbonate derivatives,
 particularly those in which the leaving groups are N-hydroxysuccinimide,
 p-nitrophenol, imidazole or 1-hydroxy-2-nitrobenzene-4-sulfonate. PEG
 derivatives containing maleimido or haloacetyl groups are useful reagents
 for the modification of protein free sulfhydryl groups. Likewise, PEG
 reagents containing amino hydrazine or hydrazide groups are useful for
 reaction with aldehydes generated by periodate oxidation of carbohydrate
 groups in proteins.
 By means of well-known techniques such as titration and by taking into
 account the observed pharmacokinetic characteristics of the agent in the
 individual subject, one of skill in the art can determine an appropriate
 dosing regimen. See, for example, Benet, et al., "Clinical
 Pharmacokinetics" in ch. 1 (pp. 20-32) of Goodman and Gilman's The
 Pharmacological Basis of Therapeutics, 8th edition, A. G. Gilman, et al.
 eds. (Pergamon, New York 1990).
 The present invention provides for a pharmaceutical composition which
 comprises an agent capable of treating an ischemic disorder or improving
 stroke outcome and a pharmaceutically acceptable carrier. The carrier may
 include but is not limited to a diluent, an aerosol, a topical carrier, an
 aqueous solution, a nonaqueous solution or a solid carrier.
 This invention is illustrated in the Experimental Detail section which
 follows. These sections are set forth to aid in an understanding of the
 invention but are not intended to, and should not be construed to, limit
 in any way the invention as set forth in the claims which follow
 thereafter.
 Experimental Details
 Abbreviations:
 EC, endothelial cell; PMN, polymorphonuclear leukocyte; WP, Weibel-Palade
 body; vWF, von Willebrand factor; EGTA, ethyleneglycol bis
 (aminoethylether) tetraacetic acid; HBSS, Hank's balanced salt solution;
 CS, coronary sinus; IL, interleukin; PAF, platelet activating factor;
 HUVEC, human umbilical vein EC; LR, lactated Ringer's solution; MCAO,
 middle cerebral artery occlusion; rt-PA, recombinant tissue plasminogen
 activator; ICH, intracerebral hemorrhage; OD, optical density; MCA, middle
 cerebral artery; rt-PA, recombinant tissue-type plasminogen activator;
 TIA, transient ischemic attack; TTC, triphenyltetrazolium chloride.
 EXAMPLE 1
 Procedural and Strain-Related Variables Significantly Effect Outcome in a
 Murine Model of Focal Cerebral Ischemia
 The recent availability of transgenic mice has led to a burgeoning number
 of reports describing the effects of specific gene products on the
 pathophysiology of stroke. Although focal cerebral ischemia models in rats
 have been well-described, descriptions of a murine model of middle
 cerebral artery occlusion are scant, and sources of potential experimental
 variability remain undefined. It was hypothesized that slight technical
 modifications would result in widely discrepant results in a murine model
 of stroke, and that controlling surgical and procedural conditions could
 lead to reproducible physiologic and anatomic stroke outcomes. To test
 this hypothesis, a murine model was established which would permit either
 permanent or transient focal cerebral ischemia by intraluminal occlusion
 of the middle cerebral artery (MCA). This study provides a detailed
 description of the surgical technique, and reveals important differences
 between strains commonly used in the production of transgenic mice. In
 addition to strain-related differences, infarct volume, neurologic
 outcome, and cerebral blood flow appear to be importantly affected by
 temperature during the ischemic and post-ischemic periods, mouse size, and
 size of the suture which obstructs the vascular lumen. When these
 variables were kept constant, there was remarkable uniformity of stroke
 outcome. These data emphasize the protective effects of hypothermia in
 stroke, and should help to standardize techniques among different
 laboratories to provide a cohesive framework for evaluating the results of
 future studies in transgenic animals.
 Introduction:
 The recent advent of genetically altered mice provides a unique opportunity
 to evaluate the role of single gene products in the pathophysiology of
 stroke. Although there is an increasing number of reports about the effect
 of cerebral ischemia in transgenic mice, to date, there exists no detailed
 description of the murine models involved, nor is there a detailed
 analysis of potentially important procedural variables which may effect
 stroke outcome. Most descriptions of a murine model
 (1,4,8,9,14,17-19,23,24; see references listed at end of Example 1) are
 devolved descriptions of the widely used rat models of focal cerebral
 ischemia (22,26). Although there has been some attention paid to strain
 related differences in the susceptibility of mice to cerebral ischemia
 (4), few technical considerations have been addressed in published
 studies. Because pilot data demonstrated that minor differences in
 operative procedure or postoperative care translated into major
 differences in stroke outcome, the current study was undertaken to
 systematically identify important surgical, technical, and anatomic
 considerations required to obtain consistent results in a murine model of
 focal cerebral ischemia. When strokes are created in a rigidly controlled
 manner, differences, due to the absence (or overexpression) of a single
 gene product, should be readily discernable.
 This study presents a detailed rendering of a reproducible murine model of
 focal cerebral infarction based on modifications of the original rat model
 (26). This study identifies procedural variables that have a large impact
 on stroke outcome which have not been previously reported in technical
 descriptions of murine stroke models. These variables include suture
 length and gauge, methods of vascular control, temperature regulation in
 mice, and differences between strains commonly used in the breeding of
 transgenic animals. As the model described lends itself to the study of
 either permanent or transient focal cerebral ischemia, evidence is
 presented that with carefully chosen ischemia times, infarct volume and
 mortality in reperfused animals can be made to approximate those seen with
 permanent occlusion. Understanding potential model-dependent sources of
 variability in stroke outcome can help to clarify divergent results
 between different laboratories. Adoption of a standardized model which
 yields consistent results is an important first step towards the use of
 transgenic mice in the study of the pathophysiology of stroke.
 Materials and Methods:
 Animal Purchase and Anesthesia: Male mice of three different strains (C57
 BlackJ6, CD-1 and 129J) were purchased from Jackson Laboratories (Bar
 Harbor, Me.). Animals were eight to ten weeks of age and weighed between
 18-37 grams (as indicated) at the time of experiments. Mice were
 anesthetized with an intraperitoneal injection of 0.3 ml of ketamine (10
 mg/cc) and xylazine (0.5 mg/cc). An additional dose of 0.1 cc was given
 prior to withdrawal of the catheter in animals undergoing transient
 ischemia. On the day following surgery, anesthesia was repeated
 immediately prior to laser doppler flow measurement and humane euthansia.
 These procedures have been approved by the Institutional Animal Care and
 Use Committee at Columbia University, and are in accordance with AALAC
 guidelines for the humane care and use of laboratory animals.
 Surgical Set-up:
 The animal was positioned supine on a gauze pad which rests on a
 temperature controlled operating surface (Yellow Springs Instruments,
 Inc.[YSI], Yellow Springs, Ohio). A rectal temperature probe (YSI) was
 inserted, in order to regulate the temperature of the operating surface to
 maintain a constant animal core temperature of 36-38.degree. C. To
 facilitate exposure, the right hindpaw and left forepaw were taped to the
 operating surface, the right forepaw was taped to the animal's chest, and
 the tail was taped to the rectal probe (FIG. 1A). A midline neck incision
 was made by gently lifting the loose skin between the manubrium and the
 jaw and excising a 1 cm.sup.2 circle of skin. The paired midline
 submandibular glands directly underlying this area were bluntly divided,
 with the left gland left in situ. The right gland was retracted cranially
 with an small straight Sugita aneurysm clip (Mizutto America, Inc.,
 Beverly, Mass) secured to the table by a 4.0 silk and tape. The
 sternocleidomastoid muscle was then identified, and a 4.0 silk ligature
 placed around its belly. This ligature was drawn inferolaterally, and
 taped to the table, to expose the omohyoid muscle covering the carotid
 sheath. The exposure is shown in FIG. 1B.
 Operative Approach:
 Once the carotid sheath was exposed, the mouse and the temperature control
 surface were placed under an operating microscope (16-25.times.zoom,
 Zeiss, Thornwood, N.Y.), with a coaxial light source used to illuminate
 the field. Under magnification, the omohyoid muscle was carefully divided
 with pickups. The common carotid artery (CCA) was carefully freed from its
 sheath, taking care not to apply tension to the vagus nerve (which runs
 lateral to the CCA). Once freed, the CCA was isolated with a 4.0 silk,
 taped loosely to the operating table. Once proximal control of the CCA was
 obtained, the carotid bifurcation was placed in view. The occipital
 artery, which arises from the proximal external carotid artery and courses
 postero-laterally across the proximal internal carotid artery (ICA) to
 enter the digastric muscle, was isolated at its origin, and divided using
 a Malis bipolar microcoagulator (Codman-Schurtleff, Randolph, Mass.). This
 enabled better visualization of the ICA as it courses posteriorly and
 cephalad underneath the stylohyoid muscle towards the skull base. Just
 before the ICA enters the skull it gives off a pterygopalatine branch,
 which courses laterally and cranially. This branch was identified,
 isolated, and divided at its origin, during which time the CCA-ICA axis
 straightens. A 4.0 silk suture was then placed around the internal carotid
 artery for distal control, the end of which was loosely taped to the
 operating surface.
 Next, the external carotid artery was placed in view. Its cranio-medial
 course was skeletonized and its first branch, the superior thyroid artery,
 was cauterized and divided. Skeletonization was subsequently carried out
 distally by elevation of the hyoid bone to expose the artery's bifurcation
 into the lingual and maxillary arteries. Just proximal to this bifurcation
 the external carotid was cauterized and divided. Sufficient tension was
 then applied to the silk sutures surrounding the proximal common, and
 distal internal, carotid arteries to occlude blood flow, with care taken
 not to traumatize the arterial wall. Tape on the occluding sutures was
 readjusted to maintain occlusion.
 Introduction and Threading of the Occluding Intraluminal Suture:
 Immediately following carotid occlusion, an arteriotomy was fashioned in
 the distal external carotid wall just proximal to the cauterized area.
 Through this arteriotomy, a heat-blunted 5.0 or 6.0 nylon suture (as
 indicated in the Results section) was introduced (FIGS. 1C and 1D). As the
 suture was advanced to the level of the carotid bifurcation, the external
 stump was gently retracted caudally directing the tip of the suture into
 the proximal ICA. Once the occluding suture entered the ICA, tension on
 the proximal and distal control sutures was relaxed, and the occluding
 suture was slowly advanced up the ICA towards the skull base under direct
 visualization (beyond the level of the skull base, sight of the occluding
 suture is lost). Localization of the distal tip of the occluding suture
 across the origin of the middle cerebral artery (MCA) (proximal to the
 origin of the anterior cerebral artery) was determined by the length of
 suture chosen (12 mm or 13 mm as indicated in the Results section, shown
 in FIG. 1C), by laser doppler flowmetry (see Ancillary physiological
 procedures section), and by post-sacrifice staining of the cerbral
 vasculature (see below). After placement of the occluding suture was
 complete, the external carotid artery stump was cauterized to prevent
 bleeding through the arteriotomy once arterial flow was reestablished.
 Completion of Surgical Procedure:
 For all of the experiments shown, the duration of carotid occlusion was
 less than two minutes. To close the incision, the sutures surrounding the
 proximal and distal CCA, as well as the sternocleidomastoid muscle, were
 cut and withdrawn. The aneurysm clip was removed from the submandibular
 gland and the gland was laid over the operative field. The skin edges were
 then approximated with one surgical staple and the animal removed from the
 table.
 Removal of the Occluding Suture to Establish Transient Cerebral Ischemia:
 Transient cerebral ischemia experiments required reexploration of the wound
 to remove the occluding suture. For these experiments, initial wound
 closure was performed with a temporary aneurysm clip rather than a
 surgical staple to provide quick access to the carotid. Proximal control
 with a 4-0 silk suture was reestablished prior to removal of the occluding
 suture to minimize bleeding from the external carotid stump. During
 removal of the occluding suture, cautery of the external carotid artery
 stump was begun early, before the distal suture has completely cleared the
 stump. Once the suture was completely removed, the stump is more
 extensively cauterized. Reestablishment of flow in the extracranial
 internal carotid artery was confirmed visually and the wound was closed as
 for permanent focal ischemia described above. Confirmation of intracranial
 reperfusion was accomplished with laser doppler flowmetry (see Ancillary
 physiological procedures section).
 Calculation of Stroke Volume:
 Twenty-four hours after middle cerebral artery occlusion, surviving mice
 were reanesthetized with 0.3 cc of ketamine (10 mg/ml) and xylazine (0.5
 mg/ml). After final weights, temperatures and cerebral blood flow readings
 were taken (as described below), animals were perfused with 5 ml of a
 0.15% solution of methylene blue and saline to enhance visualization of
 the cerebral arteries. Animals were then decapitated, and the brains were
 removed. Brains were then inspected for evidence of correct catheter
 placement, as evidenced by negative staining of the vascular territory
 subtended by the MCA, and placed in a mouse brain matrix (Activational
 Systems Inc., Warren, Minn.) for 1 mm sectioning. Sections were immersed
 in 2% 2,3,5-triphenyltetrazolium chloride (TTC) in 0.9% phosphate-buffered
 saline, incubated for 30 minutes at 37.degree. C., and placed in 10
 formalin (5). After TTC staining, infarcted brain was visualized as an
 area of unstained (white) tissue in a surrounding background of viable
 (brick red) tissue. Serial sections were photographed and projected on
 tracing paper at a uniform magnification; all serial sections were traced,
 cut out, and the paper weighed by a technician blinded to the experimental
 conditions. Under these conditions, infarct volumes are proportional to
 the summed weights of the papers circumscribing the infarcted region, and
 were expressed as a percentage of the right hemispheric volume. These
 methods have been validated in previous studies (3,12,15,16).
 Ancillary Physiological Studies:
 Ancillary physiogical studies were performed on each of the three different
 strains used in the current experiments, immediately prior to and after
 the operative procedure. Systemic blood pressures were obtained by
 catheterization of the infrarenal abdominal aorta, and measured using a
 Grass Model 7 polygraph (Grass Instrument Co., Quincy, Mass.). An arterial
 blood sample was obtained from this infrarenal aortic catheter; arterial
 pH, PCO.sub.2 (mm Hg), pO.sub.2 (mm Hg) and hemoglobin oxygen saturation
 (%) were measured using a Blood Gas Analyser and Hemoglobinometer (Grass
 Instrument Co., Quincy, Mass.). Because of the need for arterial puncture
 and abdominal manipulation to measure these physiologic parameters,
 animals were designated solely for these measurements (stroke volumes,
 neurologic outcome, and cerebral blood flows were not measured in these
 same animals).
 Transcranial measurements of cerebral blood flow were made using laser
 doppler flowmetry (Perimed, Inc., Piscataway, N.J.) after reflection of
 the skin overlying the calvarium, as previously described (10)
 (transcranial readings were consistently the same as those made after
 craniectomy in pilot studies). To accomplish these measurements, animals
 were placed in a stereotactic head frame, after which they underwent
 midline skin incision from the nasion to the superior nuchal line. The
 skin was swept laterally, and a 0.7 mm straight laser doppler probe (model
 #PF2B) was lowered onto the cortical surface, wetted with a small amount
 of physiologic saline. Readings were obtained 2 mm posterior to the
 bregma, both 3 mm and 6 mm to each side of midline using a sterotactic
 micromanipulator, keeping the angle of the probe perpendicular to the
 cortical surface. Relative cerebral blood flow measurements were made
 immediately after anesthesia, after occlusion of the MCA, and immediately
 prior to euthanasia, and are expressed as the ratio of the doppler signal
 intensity of the ischemic compared with the nonischemic hemisphere. For
 animals subjected to transient cerebral ischemia, additional measurements
 were made just before and just after withdrawal of the suture, initiating
 reperfusion.
 The surgical procedure/intraluminal MCA occlusion was considered to be
 technically adequate if .gtoreq.50% reduction in relative cerebral blood
 flow was observed immediately following placement of the intraluminal
 occluding catheter (15 of the 142 animals used in this study [10.6%] were
 exluded due to inadequate drop in blood flow at the time of occlusion).
 These exclusion criteria were shown in preliminary studies to yield levels
 of ischemia sufficient to render consistent infarct volumes by TTC
 staining. Reperfusion was considered to be technically adequate if
 cerebral blood flow at catheter withdrawal was at least twice occlusion
 cerebral blood flow (13/17 animals in this study [76%]).
 Temperature:
 Core temperature during the peri-infarct period was carefully controlled
 throughout the experimental period. Prior to surgery, a baseline rectal
 temperature was recorded (YSI Model 74 Thermistemp rectal probe, Yellow
 Springs Instruments, Inc., Yellow Springs, OH). Intraoperatively,
 temperature was controlled using a thermocouple-controlled operating
 surface. Following MCA occlusion, animals were placed for 90 minutes in an
 incubator, with animal temperature maintained at 37.degree. C. using the
 rectal probe connected via thermocouple to a heating source in the
 incubator. Temperature was similarly controlled in those animals subjected
 to transient ischemia, including a 45 minute (ischemic) period as well as
 a 90 minute post-ischemic period in the incubator. Following placement in
 the core-temperature incubator, animals were returned to their cages for
 the remaining duration of pre-sacrifice observation.
 Neurological Exam: Prior to giving anesthesia at the time of euthanasia,
 mice were examined for obvious neurological deficit using a four-tiered
 grading system: (1) normal spontaneous movements, (2) animal circling
 towards the right, (3) animal spinning to the right, (4) animal crouched
 on all fours, unresponsive to noxious stimuli. This system was shown in
 preliminary studies to accurately predict infarct size, and is based on
 systems developed for use in rats (6).
 Data Analysis:
 Stroke volumes, neurologic outcome scores, cerebral blood flows and
 arterial blood gas data were compared using an unpaired Student's t-test.
 Values are expressed as means.+-.SEM, with a p&lt;0.05 considered
 statistically significant. Mortality data, where presented was evaluated
 using chi-squared analysis.
 Results:
 Effects of Strain:
 Three different commonly used mouse strains (CD1, C57/Bl6, and 129J) were
 used to compare the variability in stroke outcome following permanent
 focal cerebral ischemia. To establish that there were no gross anatomic
 differences in collateralization of the cerebral circulation, the Circle
 of Willis was visualized using India ink in all three strains (FIG. 2).
 These studies failed to reveal any gross anatomic differences. Mice of
 similar sizes (20.+-.0.8 g, 23.+-.0.4 g, and 23.+-.0.5 g for 129J, CD1,
 and C57Bl mice, respectively) were then subjected to permanent focal
 ischemia under normothermic conditions using a 12 mm length of 6-0 nylon
 occluding suture. Significant strain-related differences in infarct volume
 were noted, with infarcts in 129J mice being significantly smaller than
 those observed in CD1 and C57/Bl6 mice despite identical experimental
 conditions (FIG. 3A). Differences in infarct size were paralleled by
 neurological exam, with the highest scores (i.e., most severe neurologic
 damage) being seen in the C57/Bl6 and CD1 mice (FIG. 3B).
 To determine the relationship between infarct volume and cerebral blood
 flow to the core region, laser doppler flowmetry was performed through the
 thin murine calvarium. No preoperative strain-related differences in
 cerebral blood flow were observed, corresponding to the lack of gross
 anatomic differences in vascular anatomy (FIG. 2). Measurement of cerebral
 blood flow immediately following insertion of the occluding catheter
 revealed that similar degees of flow reduction were created by the
 procedure (the percentage of ipsilateral/contralateral flow immediately
 following insertion of the obstructing catheter was 23.+-.2%, 19.+-.2%,
 17.+-.3% for 129J, CD1, and C57/Bl6 mice, respectively). Not surprisingly,
 blood flow to the core region measured at 24 hours just prior to
 euthanasia demonstrated the lowest blood flows in those animals with the
 most severe neurologic injury (FIG. 3C).
 Anatomic and Physiologic Characteristics of Mice:
 Baseline arterial blood pressures, as well as arterial blood pressures
 following middle cerebral artery occlusion, were nearly identical for all
 animals studied, and were not effected by mouse strain or size (Table I).
 Analysis of arterial blood for pH, pCO.sub.2, and hemoglobin oxygen
 saturation (%) similarly revealed no significant differences (Table I).
 Effect of Animal Size and Bore of the Occluding Suture:
 To investigate the effects of mouse size on stroke outcome, mice of two
 different sizes (23.+-.0.4 g and 31.+-.0.7 g) were subjected to permanent
 focal cerebral ischemia. To eliminate other potential sources of
 variability in these experiments, experiments were performed under
 normothermic conditions in mice of the same strain (CD1), using occluding
 sutures of identical length and bore (12 mm 6-0 nylon). Under these
 conditions, small mice (23.+-.0.4 g) sustained consistently large infarct
 volumes (28.+-.9% of ipsilateral hemisphere). Under identical experimental
 conditions, large mice (31.+-.0.7 g) demonstrated much smaller infarcts
 (3.2.+-.3%, p=0.02, FIG. 4A), less morbidity on neurological exam (FIG.
 4B), and a tendency to maintain higher ipsilateral cerebral blood flow
 following infarction than smaller animals (FIG. 4C).
 Because it was hypothesized that the reduction in infarct size infarcts in
 these large animals was related to a mismatch in diameter/length between
 occluding suture and the cerebral blood vessels, longer/thicker occluding
 sutures were fashioned (13 mm, 5-0 nylon) for use in these larger mice.
 Large CD1 mice (34.+-.0. 8 g) which underwent permanent occlusion with
 these larger occluding sutures sustained a marked increase in infarct
 volumes (50.+-.10% of ipsilateral hemisphere, p&lt;0.0001 compared with
 large mice infarcted with the smaller occluding suture, FIG. 4A). These
 larger mice infarcted with larger occluding sutures demonstrated higher
 neurologic deficit scores (FIG. 4B) and lower ipsilateral cerebral blood
 flows (FIG. 4C) compared with similarly large mice infarcted with smaller
 occluding sutures.
 Effects of Temperature:
 To establish the role of perioperative hypothermia on the stroke volumes
 and neurologic outcomes following MCA occlusion, small C57/Bl6 mice
 (22.+-.0.4 g) were subjected to permanent MCA occlusion with 12 mm 6-0
 gauge suture, with normothermia maintained for two different durations;
 Group 1 ("Normothermia") was operated as described above, maintaining
 temperature at 37.degree. C. from the preoperative period until 90 minutes
 post-occlusion. Group 2 animals ("Hypothermia") were maintained at
 37.degree. C. from preop to only 10 minutes post-occlusion, as has been
 described previously (14). Within 45 minutes following removal from the
 thermocouple-controlled warming incubator, core temperature in this second
 group of animals dropped to 33.1.+-.0.4.degree. C. (and dropped further to
 31.3.+-.0.2.degree. C. at 90 minutes). Animals operated under conditions
 of prolonged normothermia (Group 1) exhibited larger infarct volumes
 (32.+-.9%) than hypothermic (Group 2) animals (9.2.+-.5%, p=0.03, FIG.
 5A). Differences in infarct volume were mirrored by differences in
 neurological deficit (3.2.+-.0.4 vs. 2.0.+-.0.8, p=0.02, FIG. 5B), but
 were largely independent of cerebral blood flow (52.+-.5 vs. 52.+-.7,
 p=NS, FIG. 5C).
 Effects of Transient MCA Occlusion:
 Because reperfusion injury has been implicated as an important cause of
 neuronal damage following cerebrovascular occlusion (25), a subset of
 animals was subjected to a transient (45 minute) period of ischemia
 followed by reperfusion as described above, and comparisons made with
 those animals which underwent permanent MCA occlusion. The time of
 occlusion was chosen on the basis of preliminary studies (not shown) which
 demonstrated unacceptibly high mortality rates (&gt;85%) with 180 minutes
 of ischemia and rare infarction (&lt;15%) with 15 minutes of ischemia. To
 minimize the confounding influence of other variables, other experimental
 conditions were kept constant (small (22.5.+-.0.3 g) C57/Bl6 mice were
 used, the occluding suture consisted of 12 mm 6-0 nyon, and experiments
 were performed under normothermic conditions). The initial decline in CBF
 immediately post-occlusion were similar in both groups (16.+-.2% vs
 17.+-.3%, for transient vs permanent occlusion groups, respectively,
 p=NS). Reperfusion was confirmed both by laser doppler (2.3-fold increase
 in blood flow following removal of the occluding suture to 66.+-.13%), and
 visually by intracardiac methylene blue dye injection in representative
 animals. Infarct sizes (29.+-.10% vs. 32.+-.9%), neurologic deficit scores
 (2.5.+-.0.5 vs. 3.2.+-.0.4), and sacrifice cerebral blood flow (46.+-.18%
 vs. 53.+-.5%) were quite similar between animals subjected to transient
 cerebral ischemia and reperfusion and those subjected to permanent focal
 cerebral ischemia (p =NS, for all groups) (FIGS. 6A-6C).
 Discussion:
 The growing availability of genetically altered mice has led to an
 increasing use of murine models of focal cerebral ischemia to impute
 specific gene products in the pathogenesis of stroke. Although recent
 publications describe the use of an intraluminal suture to occlude the
 middle cerebral artery to create permanent and/or transient cerebral
 ischemia in mice, there has been only scant description of the necessary
 modifications of the original technical report in rats (8,14,17-19,24,26).
 The experiments described herein not only provide a detailed technical
 explanation of a murine model suitable for either permanent or transient
 focal middle cerebral artery ischemia, but also address potential sources
 of variability in the model.
 Importance of Strain:
 One of the most important potential sources of variability in the murine
 cerebral ischemia model described herein is related to the strain of
 animal used. The data suggest that, of the three strains tested, 129J mice
 are particularly resistant to neurologic injury following MCA occlusion.
 Although Barone similarly found differences in stroke volumes between 3
 strains of mice (BDF, CFW and BALB/C), these differences were ascribed to
 variations in the posterior communicating arteries in these strains (4).
 As anatomical differences in cerebrovascular anatomy were not grossly
 apparent in the study (FIG. 2), the data suggests that non-anatomic
 strain-related differences are also important in outcome following MCA
 occlusion.
 As stroke outcome differs significantly between 2 strains of mice (129J and
 C57/Bl6) commonly used to produce transgenic mice via homologous
 recombination in embryonic stem cells (11), the data suggest an important
 caveat to experiments performed with transgenic mice. Because early
 founder progeny from the creation of transgenic animals with these strains
 have a mixed 129J/C57/Bl6 background, ideally experiments should be
 performed either with sibling controls or after a sufficient number of
 backcrossings to ensure strain purity.
 Importance of Size:
 Larger animals require a longer and thicker intralumenal suture to sustain
 infarction volumes which are consistent with those obtained in smaller
 animals with smaller occluding sutures. Size matching of animal and suture
 appear to be important not only to produce consistent cerebral infarction,
 but whereas too small a suture leads to insufficient ischemia, too large a
 suture leads to frequent intracerebral hemorrhage and vascular trauma.
 The use of animals of similar size is important not only to minimize
 potential age-related variability in neuronal susceptability to ischemic
 insult, but also to ensure that small differences in animal size do not
 obfuscate meaningful data comparison. In this example, it is demonstrated
 that size differences of as little as 9 grams can have a major impact on
 infarct volume and neurologic outcome following cerebral ischemia. Further
 experiments using larger bore occluding suture in larger animals suggest
 that the increased propensity of smaller animals to have larger strokes
 was not due to a relative resistance of larger animals to ischemic
 neuronal damage, but was rather due to small size of the suture used to
 occlude the MCA in large animals. Although these data were obtained using
 CD1 mice, similar studies have been performed and found these results to
 be true with other mouse strains as well, such as C57/Bl6. Previously
 published reports use mice of many different sizes (from 21 g to 35 g), as
 well as different suture diameters and lengths which are often unreported
 (14,17). The studies indicate that animal and suture size are important
 methodological issues which must be addressed in scientific reports.
 Importance of Temperature:
 It has long been recognized that hypothermia protects a number of organs
 from ischemic injury, including the brain. Studies performed in rats have
 demonstrated that intraischemic hypothermia up to 1 hour post-MCA
 occlusion is protective (2,15), reducing both mortality and infarct
 volumes with temperatures of 34.5 degrees. Although these results have
 been extrapolated to murine models of cerebral ischemia in that studies
 often describe maintenance of normothermia in animals, the post-MCA
 occlusion temperature monitoring periods have been extremely brief
 ("immediately after surgery" or "10 minutes after surgery") (4,14). The
 results indicate that animals fail to autoregulate their temperature
 beyond these brief durations, becoming severely hypothermic during the
 postoperative period, and that temperature differences up to 90 minutes
 following MCA occlusion can have a profound effect on indices of stroke
 outcome following MCA occlusion (longer durations of normothermia were not
 studied). While others have ensured normothermia using a feedback system
 based on rectal temperature similar to the one described herein, the
 duration of normothermia is often not specified (17). The results argue
 for clear identification of methods for monitoring and maintaining
 temperature, as well as the durations involved, so that experimental
 results can be compared both within and between Centers studying the
 pathophysiology of stroke.
 Transient vs Permanent Occlusion:
 The pathophysiology of certain aspects of permanent cerebral ischemia may
 well be different from that of cerebral ischemia followed by reperfusion,
 so it was important that a model be described which permitted analysis of
 either condition. Although differences between these two models were not
 extensively tested in the current series of experiments, under the
 conditions tested (45 minutes of ischemia followed by 23 hours of
 reperfusion), no significant differences were found in any index of stroke
 outcome. Variable durations of ischemia and reperfusion have been reported
 in other murine models of transient cerebral ischemia, with ischemic times
 ranging from 10 minutes to 3 hours and reperfusion times ranging from 3 to
 24 hours (17,24). Studies in rats have shown that short periods of
 ischemia followed by reperfusion are associated with smaller infarcts than
 permanent occlusion (21,25). However as the duration of ischemia increases
 beyond a critical threshold (between 120 and 180 minutes), reperfusion is
 associated with larger infarcts (7,21,26). For the current series of
 experiments, the durations of ischemia and reperfusion were chosen so as
 to obtain infarcts comparable to those observed following permanent MCA
 occlusion, which is likely to explain why the data failed to show
 differences between permanent and transient ischemia. These durations in
 the transient model were chosen after pilot experiments revealed that
 shorter ischemic durations (15 minutes) rarely led to infarction, whereas
 180 minutes of occlusion followed by reperfusion led to massive infarction
 and nearly 100% mortality within 4-6 hours in normothermic animals
 (unpublished observation). Although indices of stroke outcome may be
 measured earlier than 24 hours, the 24 hour observation time was elected
 because observation at this time permits the study of delayed penumbral
 death, which is likely to be clinically relevant to the pathophysiology of
 stroke in humans. Furthermore, 24 hours has been shown in a rat model to
 be sufficient for full infarct maturation (3,12,15,16).
 Technical Aspects of the Murine Model:
 Technical aspects of the surgery needed to create focal cerebral ischemia
 in mice differ in certain important respects from that in rats.
 Self-retaining retractors, which have been advocated in previous reports
 in rats (26), are unweildy in mice. Suture-based retraction secured with
 tape provides a superior alternative. In rats, clip occlusion of the
 proximal and distal carotid artery after mobilization of the external
 carotid artery has been reported (26), but creates more carotid trauma and
 hemmorhage in mice. Without distal internal carotid control, which has not
 been previously described in mice, backbleeding from the external carotid
 artery is consistently uncontrollable. Using the techniques described in
 this paper, surgery can be completed with virtually no blood loss, which
 is especially important given the small blood volume in mice.
 Unlike the rat model, the occlusion and transection of the external carotid
 artery branches and the pterygopalatine artery in the murine model is
 achieved with electrocautery alone. Previous reports of murine surgery
 have been unclear as to whether or not the pterygopalatine artery was
 taken (17,24). Others have described a method with permanent occlusion of
 the common carotid artery and trans-carotid insertion of the suture
 without attention to either the external carotid system or the
 pterygopalatine artery. While effective for permanent occlusion, this
 latter method makes reperfusion studies impossible.
 The method of reperfusion originally described in the rat requires blind
 catheter withdrawal without anesthesia (26). When attempted in pilot
 studies in mice, several animals hemorrhaged. Therefore, a method of
 suture removal under direct visualization in the anesthetized animal was
 developed, which not only allows visual confirmation of extracranial
 carotid artery reperfusion, but also affords meticulous hemostasis.
 Further, the method permits immediate pre- and post-reperfusion laser
 doppler flowmetry readings in the anesthetized animal.
 These laser doppler flowmetry readings are similar to those described by
 Kamii et al. and Yang et al. in that the readings are made intermittantly
 and with the use of a stereotactic micromanipulator (17,24). The readings
 differ, however, in that the coordinates used (2 mm posterior and 3 and 6
 mm lateral to the bregma) are slightly more lateral and posterior than the
 previously published core and penumbral coordinates (1 mm posterior and 2
 mm and 4.5 mm lateral to the bregma). These coordinates, which were
 adopted based on pilot studies, are the same as those used by Huang et al
 (14).
 Conclusion:
 These studies demonstrate specific technical aspects of a murine model of
 focal cerebral ischemia and reperfusion which permits reproducibility of
 measurements between different laboratories. In addition, these studies
 provide a framework for understanding important procedural variables which
 can greatly impact on stroke outcome, which should lead to a clear
 understanding of non-procedure related differences under investigation.
 Most importantly, this study points to the need for careful control of
 mouse strain, animal and suture size, and temperature in experimental as
 well as control animals. Conditions can be established so that stroke
 outcome is similar between models of permanent focal cerebral ischemia and
 transient focal cerebral ischemia, which should facilitate direct
 comparison and permit the study of reperfusion injury. The model described
 in this study should provide a cohesive framework for evaluating the
 results of future studies in transgenic animals, to facilitate an
 understanding of the contribution of specific gene products in the
 pathophysiology of stroke.
 Table I.
 Pre- and post-operative physiologic parameters. MAP, mean arterial
 pressure; pCO.sub.2, partial pressure of arterial CO.sub.2 (mm Hg);
 O.sub.2 Sat, O.sub.2 saturation (%); Hb, hemoglobin concentration (g/dl);
 Preoperative, anesthetized animals prior to carotid dissection; Sham,
 anesthetized animals undergoing the surgical described in the text,
 immediately prior to introduction of the occluding suture; Stroke,
 anesthetized animals undergoing the surgical described in the text,
 immediately after introduction of the occluding suture. p=NS for all
 between-group comparisons. (data shown is for small 22 gram C57/Bl6 mice).

AMETER PREOPERATIVE SHAM STROKE
 MAP 102 .+-. 5.5 94 .+-. 1.9 88 .+-. 4.9
 pH 7.27 .+-. 0.02 7.23 .+-. 0.04 7.28 .+-. 0.01
 pCO.sub.2 46 .+-. 1.3 44 .+-. 1.3 47 .+-. 3.5
 O.sub.2 Sat 89 .+-. 1.6 91 .+-. 1.8 85 .+-. 2.2
 Hb 14.6 .+-. 0.42 14.3 .+-. .12 14.2 .+-. 0.12
 REFERENCES
 1. Backhaub C, et al. (1992) J Pharmacol Methods 27:27-32.
 2. Baker C J, et al. (1992) J Neurosurg 77:438-444.
 3. Baker C J, et al. (1995) Neurosurgery 36:1-9.
 4. Barone F C, et al. (1993) J Cereb Blood Flow Metab 13:683-692.
 5. Bederson J B, et al. (1986) Stroke 17:1304-1308.
 6. Bederson J B, et al. (1986) Stroke 17:472-476.
 7. Buchan A M, et al. (1992) Stroke 23:273-279.
 8. Chan P H, et al. (1993) NeuroReport 5:293-296.
 9. Chiamulera C, et al. (1993) Brain Res 606:251-258.
 10. Dirnagl U, et al. (1989) J Cereb Blood Flow Metab 9:589-596.
 11. Donehower L A, et al. (1992) Nature 356:215-221.
 12. Frazzini V I, et al. (1994) Neurosurgery 34:1040-1046.
 13. Ginsberg M D and Busto R (1989) Stroke 20:1627-1642.
 14. Huang Z, et al. (1994) Science 265:1883-1885.
 15. Kader A, et al. (1992) Neurosurgery 31:1056-1061.
 16. Kader A, et al. (1993) Stroke 24:1709-1716.
 17. Kamii H, et al. (1994) J Cereb Blood Flow Metab 14:478-486.
 18. Kinouchi H, et al. (1991) Proc Natl Acad Sci 88: 11158-11162.
 19. Martinou J-C, et al. (1994) Neuron 13:1017-1030.
 20. Memezawa H, et al. (1992) Stroke 23:552-559.
 21. Menzies S A, et al. (1992) Neurosurgery 31:100-107.
 22. Tamura A, et al. (1981) J Cereb Blood Flow Metabol 1: 53-60.
 23. Welsh F A, et al. (1987) J Neurochem 49:846-851.
 24. Yang G, et al. (1994) Stroke 25:165-170.
 25. Yang G-Y and Betz A L (1994) Stroke 25: 1658-65.
 26. Zea-Longa E, et al. (1989) Stroke 20:84-91.
 EXAMPLE 2
 Factor IXai
 Factor IX is a clotting factor which exists in humans and other mammals,
 and is an important part of the coagulation pathway. In the normal scheme
 of coagulation, Factor IX is activated by either Factor XIa or a tissue
 factor/VIIa complex to its active form, Factor IXa. Factor IXa then can
 activate Factor X, which triggers the final part of the coagulation
 cascade, leading to thrombosis. Because Factor X can be activated by one
 of two pathways, either the extrinsic (via VIIa/tissue factor) or the
 intrinsic pathways (via Factor IXa), we hypothesized that inhibiting
 Factor IXa might lead to impairment of some forms of hemostasis, but leave
 hemostasis in response to tissue injury intact. In other words, it might
 lead to blockade of some types of clotting, but might not lead to
 excessive or unwanted hemorrhage. Factor IXai is Factor IXa which has been
 chemically modified so as to still resemble Factor IXa (and therefore, can
 compete with native Factor IXa), but which lacks its activity. This can
 "overwhelm" or cause a competitive inhibition of the normal Factor
 IXa-dependent pathway of coagulation. Because Factor IXa binds to
 endothelium and platelets and perhaps other sites, blocking the activity
 of Factor IXa may also be possible by administering agents which interfere
 with the binding of Factor IXa (or by interfering with the activation of
 Factor IX).
 In stroke and other ischemic disorders, there may be clinical benefit
 derived by lysing an existing thrombus, but there is also the potentially
 devastating complication of hemorrhage. In the current experiments, the
 mouse model of cerebral ischemia and reperfusion (stroke) was used. Mice
 received an intravenous bolus of 300 .mu.g/kg of Factor IXai just prior to
 surgery. Strokes were created by intraluminal occlusion of the right
 middle cerebral artery. When stroke outcomes were measured 24 hours later,
 animals that had received Factor IXai had smaller infarct volumes,
 improved cerebral perfusion, less neurological deficits, and reduced
 mortality compared with controls which underwent the same surgery but
 which did not receive Factor IXai. (See Table II.) It was also noted that
 the Factor IXai animals were free of apparent intracerebral hemorrhage. By
 contrast, intracerebral hemorrhage was occasionally noted in the control
 animals not receiving Factor IXai.
 TABLE II
 Control Experimental (Factor IXai)
 mean sd mean sd stats
 weight 26.91 3.21 25.25 2.49 0.14
 dopp 0.96 0.24 1.04 0.35 0.52
 occ dop 1 0.18 0.07 0.16 0.08 0.60
 occ dop 2 0.40 0.22 0.43 0.20 0.68
 reper dop 0.55 0.42 0.53 0.30 0.89
 sac dop 0.38 0.25 0.75 0.31 0.02
 grade 2.22 0.67 1.67 0.49
 I/C Ratio 1.18 0.20 1.08
 inf vol 21.16 25.14 3.47 12.03 0.0452
 count 11 16
 Abbreviations:
 dopp = doppler;
 occ dop = occlusion doppler;
 reper dop = reperfusion doppler;
 sac dop = sacrifice doppler.
 EXAMPLE 3
 Active-site Blocked Factor IXa Limits Microvascular Thrombosis and Cerebral
 Injury in Murine Stroke Without Increasing Intracerebral Hemorrhage
 The clinical dilemma in stroke treatment is that agents which restore
 vascular patency increase the risk of intracerebral hemorrhage.
 Active-site blocked Factor IXa (IXai) competes with native Factor IXa to
 inhibit assembly of Factor IXa into the intrinsic Factor X activation
 complex. When pretreated with Factor IXai, mice subjected to focal
 cerebral ischemia and reperfusion demonstrated reduced microvascular
 fibrin and platelet deposition, increased cerebral perfusion, and
 significantly smaller cerebral infarcts than vehicle-treated controls.
 Factor IXai-mediated cerebroprotection was dose-dependent, not associated
 with intracerebral hemorrhage at therapeutically effective doses, and was
 seen even when Factor IXai was administered after the onset of cerebral
 ischemia. Administration of Factor IXai represents a new strategy to treat
 stroke in evolution without increasing the risk of intracerebral
 hemorrhage.
 Introduction
 Timely reestablishment of blood flow to ischemic brain represents the
 current treatment paradigm for acute stroke.sup.1-3. Administration of a
 thrombolytic agent, even when given under optimal conditions, may not
 achieve this desired clinical result. Perfusion often fails to return to
 preischemic levels (postischemic hypoperfusion), suggesting that ischemic
 injury is not produced solely by the original occlusion, but that there is
 also an element of microcirculatory failure. In addition, thrombolysis of
 acute stroke is associated with an increased risk of intracerebral
 hemorrhage (ICH).sup.1-4, indicating that there remains a clear need to
 identify new agents which can promote reperfusion without increasing the
 risk of ICH.
 Following an ischemic event, the vascular wall is modified from its
 quiescent, anti-adhesive, antithrombotic state, to one which promotes
 leukocyte adhesion and thrombosis. In acute stroke, active recruitment of
 leukocytes by adhesion receptors expressed in the ipsilateral
 microvasculature, such as ICAM-1.sup.5 and P-selectin.sup.6, potentiates
 postischemic hypoperfusion. However, experiments with mice deletionally
 mutant for each of these genes demonstrate that even in their absence,
 postischemic cerebral blood flow (CBF) returns only partially to baseline,
 suggesting the existence of additional mechanisms responsible for
 postischemic cerebrovascular no-reflow. To explore this possibility, the
 first set of experiments was designed to test the hypothesis that local
 thrombosis occurs at the level of the microvasculature (distal to the site
 of primary occlusion) in stroke.
 To assess the deleterious consequences of microvascular thrombosis in
 stroke, the second set of experiments tested the hypothesis that selective
 blockade of the intrinsic pathway of coagulation could limit microvascular
 thrombosis, thereby protecting the brain in stroke. The strategy of
 selective inhibition of the intrinsic pathway of coagulation was chosen
 because it is primarily responsible for intravascular thrombosis. Heparin,
 hirudin, and fibrinolytic agents interfere with the final common pathway
 of coagulation to inhibit the formation or accelerate the lysis of fibrin,
 and therefore increase the propensity for ICH. We hypothesized that
 selective blockade of IXa/VIIIa/X activation complex assembly might
 provide a novel mechanism to limit intravascular thrombosis while
 preserving mechanisms of extravascular hemostasis by the extrinsic/tissue
 factor pathway of coagulation which may be critical in infarcted brain
 tissue or adjacent regions where small vessels are friable and subject to
 rupture. We used a novel strategy in which a competitive inhibitor of
 Factor IXa (active-site blocked IXa, or IXai) was given to mice subjected
 to stroke to test the hypothesis that it would improve stroke outcome
 without increasing ICH.
 Methods
 Murine stroke model: Transient focal cerebral ischemia was induced in mice
 by intralumenal occlusion of the middle cerebral artery (45 minutes) and
 reperfusion (22 hrs) as previously reported.sup.7. Serial measurements of
 relative cerebral blood flow (CBF) were recorded via laser doppler
 flowmetry.sup.7, and infarct volumes (k ipsilateral hemisphere) determined
 by planimetric/volumetric analysis of triphenyl tetrazolium chloride
 (TTC)-stained serial cerebral sections.sup.7.
 .sup.111 Indium-platelet studies: Platelet accumulation was determined
 using .sup.111 Indium labeled platelets, collected and prepared as
 previously described.sup.8. Immediately prior to surgery, mice were given
 5.times.10.sup.6 111 In-labeled-platelets intravenously; deposition was
 quantified after 24 hours by as ipsilateral cpm/contralateral cpm.
 Fibrin Immunoblotting/Immunostaining:
 The accumulation of fibrin was measured following sacrifice (of fully
 heparinized animals) using immunoblotting/immunostaining procedures which
 have been recently described and validated.sup.9. Because fibrin is
 extremely insoluble, brain tissue extracts were prepared by plasmin
 digestion, then applied to a standard SDS-polyacrylamide gel for
 electrophoresis, followed by immunoblotting using a polyclonal rabbit
 anti-human antibody prepared to gamma--gamma chain dimers present in
 cross-linked fibrin which can detect murine fibrin, with relatively little
 cross-reactivity with fibrinogen.sup.10. Fibrin accumulation was reported
 as an ipsilateral to contralateral ratio. In additional experiments,
 brains were embedded in paraffin, sectioned, and immunostained using the
 same anti-fibrin antibody.
 Spectrophotometric Hemoglobin Assay and Visual ICH Score:
 ICH was quantified by a spectrophotometric-based assay which we have
 developed and validated.sup.11,12 In brief, mouse brains were homogenized,
 sonicated, centrifuged, and methemoglobin in the supernatants converted
 (using Drabkin's reagent) to cyanomethemoglobin, the concentration of
 which was assessed by measuring O.D. at 550 nm against a standard curve
 generated with known amounts of hemoglobin. Visual scoring of ICH was
 performed on 1 mm serial coronal sections by a blinded observer based on
 maximal hemorrhage diameter seen on any of the sections [ICH score 0, no
 hemorrhage; 1, &lt;1 mm; 2, 1-2 mm; 3, &gt;2-3 mm; 4, &gt;3 mm].
 Preparation of Factor IXai.sup.13 :
 Factor IXai was prepared by selectively modifying the active site histidine
 residue on Factor IXa, using dansyl-glu-gly-arg-chloromethylketone.
 Proplex was applied to a preparative column containing immobilized
 calcium-dependent monoclonal antibody to Factor IX. The column was washed,
 eluted with EDTA-containing buffer, and Factor IX in the eluate (confirmed
 as a single band on SDS-PAGE) was then activated by applying Factor XIa
 (incubating in the presence of CaCl.sub.2). Purified Factor IXa was
 reacted with a 100-fold molar excess of dansyl-glu-gly-arg
 chloromethylketone, and the mixture dialyzed. The final product (IXai),
 devoid of procoagulant activity, migrates identically to IXa on SDS-PAGE.
 This material (Factor IXai) was then used for experiments following
 filtration (0.2 .mu.m) and chromatography on DeToxi-gel columns, to remove
 any trace endotoxin contamination (in sample aliquots, there was no
 detectable lipopolysaccharide). IXai was subsequently frozen into aliquots
 at -80.degree. C. until the time of use. For those experiments in which
 IXai was used, it was given as a single intravenous bolus at the indicated
 times and at the indicated doses.
 Results
 To create a stroke in a murine model, a suture is introduced into the
 cerebral vasculature so that it occludes the orifice of the right middle
 cerebral artery, rendering the subtended territory ischemic. By
 withdrawing the suture after a 45 minute period of occlusion, a reperfused
 model of stroke is created; mice so treated demonstrate focal neurological
 deficits as well as clear-cut areas of cerebral infarction. Because the
 occluding suture does not advance beyond the major vascular tributary (the
 middle cerebral artery), this model provides an excellent opportunity to
 investigate "downstream" events that occur within the cerebral
 microvasculature in response to the period of interrupted blood flow.
 Using this model, the role of microvascular thrombosis was investigated as
 follows. To demonstrate that platelet-rich thrombotic foci occur within
 the ischemic cerebral hemisphere, .sup.111 In-labeled platelets were
 administered to mice immediately prior to the introduction of the
 intraluminal occluding suture, to track their deposition during the
 ensuing period of cerebral ischemia and reperfusion. In animals not
 subjected to the surgical procedure to create stroke, the presence of
 platelets was approximately equal between the right and left hemispheres,
 as would be expected [FIG. 7A, left bar]. However, when animals were
 subjected to stroke (and received only vehicle to control for subsequent
 experiments), radiolabeled platelets preferentially accumulated in the
 ischemic (ipsilateral) hemisphere, compared with significantly less
 deposition in the contralateral (nonischemic) hemisphere [FIG. 7A, middle
 bar]. These data support the occurrence of platelet-rich thrombi in the
 ischemic territory. When Factor IXai is administered to animals prior to
 introduction of the intraluminal occluding suture, there is a significant
 reduction in the accumulation of radiolabelled platelets in the
 ipsilateral hemisphere [FIG. 7A, right bar].
 Another line of evidence also supports the occurrence of microvascular
 thrombosis in stroke. This data comes from the immunodetection of fibrin,
 using an antibody directed against a neoepitope on the gamma--gamma chain
 dimer of cross-linked fibrin. Immunoblots demonstrate a band of increased
 intensity in the ipsilateral (right) hemisphere of vehicle-treated animals
 subjected to focal cerebral ischemia and reperfusion [FIG. 7B, "Vehicle"].
 In animals treated with Factor IXai (300 .mu.g/kg) prior to stroke, there
 is no apparent increase in the ipsilateral accumulation of fibrin [FIG.
 7B, "Factor IXai"]. To demonstrate that fibrin accumulation was due to the
 deposition of intravascular fibrin (rather than due to nonspecific
 permeability changes and exposure to subendothelial matrix), fibrin
 immunostaining clearly localized the increased fibrin to the lumina of
 ipsilateral intracerebral microvessels [FIG. 7C].
 To investigate whether Factor IXai can limit intracerebral thrombosis and
 restore perfusion, IXai was given to mice immediately prior to stroke (300
 .mu.g/kg). These experiments demonstrate both a reduction in .sup.111
 In-platelet accumulation in the ipsilateral hemisphere [FIG. 8A] as well
 as decreased evidence of intravascular fibrin by immunostaining.
 Furthermore, there is a significant increase in CBF by 24 hours,
 suggesting the restoration of microvascular patency by Factor IXai [FIG.
 8A]. The clinical relevance of this observation is underscored by the
 ability of Factor IXai to reduce cerebral infarct volumes [FIG. 8B]. These
 beneficial effects of Factor IXai were dose dependent, with 600 .mu.g/kg
 being the optimal dose [FIG. 8C]. Because the development of ICH is a
 major concern with any anticoagulant strategy in the setting of stroke,
 the effect of IXai on ICH was measured using our recently validated
 spectrophotometric method for quantifying ICH.sup.11,12. These data
 indicate that at the lowest doses (and the most effective ones), there is
 no significant increase in ICH [FIG. 9A]. At the highest dose tested (1200
 .mu.g/kg), there is an increase in ICH, which was corroborated by a
 semiquantitative visual scoring method which we have also recently
 reported [FIG. 9B].sup.11,12.
 Because therapies directed at improving outcome from acute stroke must be
 given after clinical presentation, and because fibrin continues to form
 following the initial ischemic event in stroke, we tested whether IXai
 might be effective when given following initiation of cerebral ischemia.
 IXai given after middle cerebral artery occlusion (following removal of
 the occluding suture) provided significant cerebral protection judged by
 its ability to significantly reduce cerebral infarction volumes compared
 with vehicle-treated controls [FIG. 10].
 Discussion
 The data in these studies demonstrate clear evidence of intravascular
 thrombus formation (both platelets and fibrin) within the post-ischemic
 cerebral microvasculature. The pathophysiological relevance of
 microvascular thrombosis in stroke is underscored by the ability of Factor
 IXai to reduce microvascular thrombosis (both platelet and fibrin
 accumulation are reduced, with an attendant increase in postischemic CBF)
 and to improve stroke outcome. These potent antithrombotic actions of
 Factor IXai are likely to be clinically significant in the setting of
 stroke, because Factor IXai not only reduces infarct volumes in a
 dose-dependent manner, but it does so even when given after the onset of
 stroke. In addition, at clinically relevant doses, treatment with Factor
 IXai does not cause an increase in ICH, making selective inhibition of
 Factor IXa/VIIIa/X activation complex assembly with Factor IXai an
 attractive target for stroke therapy in humans.
 There are a number of reasons why targetted anticoagulant strategies might
 be an attractive alternative to the current use of thrombolytic agents in
 the management of acute stroke, because of their checkered success in
 clinical trials. Theoretically, an ideal treatment for acute stroke would
 prevent the formation or induce dissolution of the fibrin-platelet mesh
 that causes microvascular thrombosis in the ischemic zone without
 increasing the risk of intracerebral hemorrhage. However, thrombolytic
 agents which have been studied in clinical trials of acute stroke have
 consistently increased the risk of intracerebral hemorrhage.sup.1-4.
 Streptokinase, given in the first several (&lt;6) hours following stroke
 onset, was associated with an increased rate of hemorrhagic transformation
 (up to 67%); although there was increased early mortality, surviving
 patients suffered less residual disability. Administration of tissue-type
 plasminogen activator (tPA) within 7 hours (particularly within 3 hours)
 of stroke onset resulted in increased early mortality and increased rates
 of hemorrhagic conversion (between 7-20%), although survivors demonstrated
 less residual disability. In order to develop improved anticoagulant or
 thrombolytic therapies, several animal models of stroke have been
 examined. These models generally consist of the administration of clotted
 blood into the internal carotid artery followed by administration of a
 thrombolytic agent. In rats, tPA administration within 2 hours of stroke
 improved cerebral blood flow and reduced infarct size by up to
 77%.sup.14,15. In a similar rabbit embolic stroke model, tPA was effective
 at restoring blood flow and reducing infarct size, with occasional
 appearance of intracerebral hemorrhage.sup.16,17. However, although there
 are advantages to immediate clot dissolution, these studies (as well as
 the clinical trials of thrombolytic agents) indicate that there is an
 attendant increased risk of intracerebral hemorrhage with this therapeutic
 approach.
 Because of the usually precipitous onset of ischemic stroke, therapy has
 been targetted primarily towards lysing the major fibrinous/atheroembolic
 debris which occludes a major vascular tributary to the brain. However, as
 the current work demonstrates, there is an important component of
 microvascular thrombosis which occurs downstream from the site of original
 occlusion, which is likely to be of considerable pathophysiological
 significance for post-ischemic hypoperfusion (no-reflow) and cerebral
 injury in evolving stroke. This data is in excellent agreement with that
 which has been previously reported, in which microthrombi have been
 topographically localized to the ischemic region in fresh brain
 infarcts.sup.18. The use of an agent which inhibits assembly of the Factor
 IXa/VIIIa/X activation complex represents a novel approach to limiting
 thrombosis which occurs within microvascular lumena, without impairing
 extravascular hemostasis, the maintenance of which may be critical for
 preventing ICH. In the current studies, treatment with Factor IXai reduces
 microvascular platelet and fibrin accumulation, improves postischemic
 cerebral blood flow, and reduces cerebral infarct volumes in the setting
 of stroke without increasing ICH.
 The potency of Factor IXai as an anticoagulant agent stems from the
 integral role of activated Factor IX in the coagulation cascade. Not only
 does a strategy of Factor IXa blockade appear to be effective in the
 setting of stroke, but it also appears to be effective at preventing
 progressive coronary artery occlusion induced following the initial
 application of electric current to the left circumflex coronary artery in
 dogs.sup.13. As in those studies, in which Factor IXai did not prolong the
 pro time.
 The data which demonstrate that IXai given after the onset of stroke is
 effective leads to another interesting hypothesis, that the formation of
 thrombus represents a dynamic equilibrium between the processes of ongoing
 thrombosis and ongoing fibrinolysis. Even under normal (nonischemic)
 settings, this dynamic equilibrium has been shown to occur in man.sup.19.
 The data in the current studies, which show that Factor IXai is effective
 even when administered after the onset of stroke, suggests that this
 strategy restores the dynamic equilibrium, which is shifted after cerebral
 ischemia to favor thrombosis, back towards a more quiescent
 (antithrombotic) vascular wall phenotype.
 As a final consideration, even if thrombolysis successfully removes the
 major occluding thrombus, and/or anticoagulant strategies are effective to
 limit progressive microcirculatory thrombosis, blood flow usually fails to
 return to pre-ischemic levels. This is exemplified by data in the current
 study, in which although CBF is considerably improved by Factor IXai
 (which limits fibrin/platelet accumulation), CBF still does not return to
 preischemic levels. This data supports the existence of multiple effector
 mechanisms for postischemic cerebral hypoperfusion, including postischemic
 neutrophil accumulation and consequent microvascular plugging, with
 P-selectin and ICAM-1 expression by cerebral microvascular endothelial
 cells being particularly germane in this regard.sup.5,6. When looked at
 from the perspective of leukocyte adhesion receptor expression, even when
 these adhesion receptors are absent, CBF levels are improved following
 stroke compared with controls but do not return to preischemic levels.
 Taken together, these data suggests that microvascular thrombosis and
 leukocyte adhesion together contribute to postischemic cerebral
 hypoperfusion.
 In summary, administration of a competitive inhibitor of Factor IXa,
 active-site blocked Factor IXa, represents a novel therapy for the
 treatment of stroke. This therapy not only reduces microcirculatory
 thrombosis, improves postischemic cerebral blood flow, and reduces
 cerebral tissue injury following stroke, but it can do so even if given
 after the onset of cerebral ischemia and without increasing the risk of
 ICH. This combination of beneficial properties and relatively low downside
 risk of hemorrhagic transformation makes this an extremely attractive
 approach for further testing and potential clinical trials in human
 stroke.
 REFERENCES
 1. New Engl. J. Med. (1995)333:1581-1587.
 2. Hacke W, et al. (1995) JAMA 274(13):1017-1025.
 3. del Zoppo G J (1995) N. Engl. J. Med. 333(13):1632-1633.
 4. Hommel M, et al. (1996) N. Engl. J. Med. 335:145-150.
 5. Connolly E S Jr, et al. (1996) J. Clin. Invest. 97:209-216.
 7. Connolly E S Jr, et al. (1996) Neurosurg 38(3):523-532.
 8. Naka Y, et al. (1995) Circ. Res. 76:900-906.
 9. Lawson C A, et al. (1997) J. Clin. Invest. 99:1729-1738.
 10. Lahiri B, et al. (1981) Thromb. Res. 23:103-112.
 12. Choudhri T F, et al. (1997) Annual Meeting Joint Section on
 Cerebrovascular Surgery.
 13. Benedict C R, et al. (1991) J Clin Invest 88:1760-1765.
 14. Papadopoulos S M, et al. (1987) J Neurosurg 67:394-398.
 15. Overgaard K, et al. (1993) Neurol Res 15:344-349.
 16. Carter L P, et al.(1992) Stroke 23:883-888.
 17. Phillips D A, et al. (1990) Stroke 21:602-605.
 18. Heye N, et al. (1992) Acta Neurologica Scandinavica 86:450-454.
 19. Nossel H L (1981) Nature 1981;291:165-167
 EXAMPLE 4
 Active-site Blocked Factor IXa Limits Microvascular Thrombosis and Cerebral
 Injury in Murine Stroke Without Increasing Intracerebral Hemorrhage
 [Please note the following abbreviations: CBF, cerebral blood flow; Factor
 IXai, active-site blocked factor IXa; ICAM-1, intercellular adhesion
 molecule-1; ICH, intracerebral hemorrhage; tPA, tissue plasminogen
 activator; TTC, triphenyl tetrazolium chloride.]
 The clinical dilemma in stroke treatment is that agents which restore
 vascular patency increase the risk of intracerebral hemorrhage (ICH). It
 was hypothesized that inhibiting cerebral microvascular thrombosis by
 inhibiting intrinsic Factor IX-dependent coagulation may restore vascular
 patency in stroke without impairing extrinsic hemostatic mechanisms that
 may limit ICH. Active-site blocked Factor IXa (IXai) was formed from
 purified factor IXa by dansylation of its active site, to compete with
 native Factor IXa to inhibit assembly of Factor IXa into the intrinsic
 Factor X activation complex. Although in vitro, Factor IXai had little
 effect on the PT or PTT, it prolonged clotting time in an assay in which
 Factor IX-deficient plasma was reconstituted with Factor IX. When
 pretreated with Factor IXai, mice subjected to middle cerebral artery
 occlusion and reperfusion demonstrated an 1.8-fold reduced microvascular
 fibrin and platelet deposition, 2.4-fold increased cerebral perfusion, and
 significantly smaller cerebral infarcts 3.5-fold than vehicle-treated
 controls (p&lt;0.05, 0.05, and 0.05, respectively). Factor IXai-mediated
 cerebroprotection was not associated with ICH at therapeutically effective
 doses, and was seen even when Factor IXai was administered after the onset
 of cerebral ischemia. In contrast, a less targeted anticoagulant strategy
 with heparin reduced cerebral infarction volumes only at doses which
 increased ICH. Administration of Factor IXai represents a new strategy to
 treat stroke in evolution without increasing the risk of ICH. The apparent
 efficacy of Factor IXai when given after stroke suggests that
 microvascular thrombosis continues to evolve (and may be inhibited) even
 after occlusion of a major vascular tributary, thereby broadening the
 potential therapeutic window for its administration.
 Timely reestablishment of blood flow to ischemic brain represents the
 current treatment paradigm for acute stroke (1-3). Administration of a
 thrombolytic agent, even when given under optimal conditions, may not
 achieve this desired clinical result. Perfusion often fails to return to
 preischemic levels (postischemic hypoperfusion), suggesting that ischemic
 injury is not produced solely by the original occlusion, but that there is
 also an element of microcirculatory failure. Small early trials of a
 general anticoagulant strategy involving heparin in stroke were
 disappointing in that the use of heparin was either ineffective and/or
 associated with an unacceptably high incidence of hemorrhagic conversion
 (in up to 14% of treated patients) (4-7,7-9). Although the current vogue
 is to use recombinant tissue plasminogen activator (tPA) to achieve
 thrombolysis in ischemic stroke, this approach is also associated with an
 increased risk of intracerebral hemorrhage (ICH) (1-3,10). Consequently,
 there remains a clear need to identify new agents which can promote
 reperfusion without increasing the risk of ICH.
 Following an ischemic event, the vascular wall is modified from its
 quiescent, anti-adhesive, antithrombotic state, to one which promotes
 leukocyte adhesion and thrombosis. In acute stroke, active recruitment of
 leukocytes by adhesion receptors expressed in the ipsilateral
 microvasculature, such as intercellular adhesion molecule-1 (ICAM-1) (11)
 and P-selectin (12), potentiates postischemic hypoperfusion. However,
 experiments with mice deletionally mutant for each of these genes
 demonstrate that even in their absence, postischemic cerebral blood flow
 (CBF) returns only partially to baseline after removal of an intraluminal
 middle cerebral artery occluding suture. This indicates that there exist
 additional mechanisms responsible for postischemic cerebrovascular
 no-reflow, especially the possibility that local thrombosis occurs at the
 level of the microvasculature (distal to the site of primary occlusion) in
 stroke. Furthermore, if the ischemic insult is particularly severe, reflow
 continues to worsen over the time subsequent to withdrawal of the
 occluding suture, suggesting ongoing vascular obstructive processes (such
 as de novo thrombosis).
 These observations provide the basis for exploring the role of general
 thrombolytic and/or anticoagulant strategies in the murine model of
 stroke. However, compelling clinical data indicate that agents which
 selectively limit thrombosis in stroke without increasing ICH will offer
 unique advantages which are not seen with any agent tested so far. Because
 the subendothelial vascular matrix in brain tissue is a rich source of
 tissue factor, we hypothesized that anticoagulant strategis which do not
 impair tissue-factor mediated hemostatic events might provide a novel
 means to reduce thrombosis in the microvascular lumen, yet not impair the
 ability of friable postischemic cerebral microvessels to form effective
 hemostatic plugs to limit ICH. Heparin or hirudin, which interfere with
 the final common pathway of coagulation, or thrombolytic agents, which
 nonselectively lyse fibrin, do not offer the theroretical advantage
 offered by targeting the intrinsic limb of the coagulation cascade. The
 current experiments test the hypothesis that selective blockade of
 IXa/VIIIa/X activation complex assembly using a novel strategy in which a
 competitive inhibitor of Factor IXa (active-site blocked IXa, Factor
 IXai), might provide a novel mechanism to limit intravascular thrombosis
 while preserving mechanisms of extravascular hemostasis, thereby improving
 stroke outcome without increasing ICH.
 Methods
 Murine Stroke Model:
 Transient focal cerebral ischemia was induced in mice by intralumenal
 occlusion of the middle cerebral artery (45 minutes) and reperfusion (24
 hrs) as previously reported (13). Serial measurements of relative cerebral
 blood flow (CBF) were recorded via laser doppler flowmetry (13), and
 infarct volumes (% ipsilateral hemisphere) determined by
 planimetric/volumetric analysis of triphenyl tetrazolium chloride
 (TTC)-stained serial cerebral sections (13).
 .sup.111 Indium-platelet Studies:
 Platelet accumulation was determined using .sup.111 Indium labeled
 platelets, collected and prepared as previously described (14).
 Immediately prior to surgery, mice were given 5.times.10.sup.6 111
 In-labeled-platelets intravenously; deposition was quantified after 24
 hours by as ipsilateral cpm/contralateral cpm.
 Fibrin Immunoblotting/Immunostaining:
 The accumulation of fibrin was measured following sacrifice (of fully
 heparinized animals) using immunoblotting/immunostaining procedures which
 have been recently described and validated (15). Because fibrin is
 extremely insoluble, brain tissue extracts were prepared by plasmin
 digestion, then applied to a standard SDS-polyacrylamide gel for
 electrophoresis, followed by immunoblotting using a polyclonal rabbit
 anti-human antibody prepared to gamma--gamma chain dimers present in
 cross-linked fibrin which can detect murine fibrin, with relatively little
 cross-reactivity with fibrinogen (16). Fibrin accumulation was reported as
 an ipsilateral to contralateral ratio. In additional experiments, brains
 were embedded in paraffin, sectioned, and immunostained using the same
 anti-fibrin antibody.
 Spectrophotometric Hemoglobin Assay and Visual ICH Score:
 ICH was quantified by a spectrophotometric-based assay which we have
 developed and validated (17). In brief, mouse brains were homogenized,
 sonicated, centrifuged, and methemoglobin in the supernatants converted
 (using Drabkin's reagent) to cyanomethemoglobin, the concentration of
 which was assessed by measuring O.D. at 550 nm against a standard curve
 generated with known amounts of hemoglobin.
 Preparation of Factor IXai (18):
 Factor IXai was prepared by selectively modifying the active site histidine
 residue on Factor IXa, using dansyl-glu-gly-arg-chloromethylketone.
 Proplex was applied to a preparative column containing immobilized
 calcium-dependent monoclonal antibody to Factor IX. The column was washed,
 eluted with EDTA-containing buffer, and Factor IX in the eluate (confirmed
 as a single band on SDS-PAGE) was then activated by applying Factor XIa
 (incubating in the presence of CaCl.sub.2). Purified Factor Ixa was
 reacted with a 100-fold molar excess of dansyl-glu-gly-arg
 chloromethylketone, and the mixture dialyzed. The final product (IXai),
 devoid of procoagulant activity, migrates identically to IXa on SDS-PAGE.
 This material (Factor IXai) was then used for experiments following
 filtration (0.2 .mu.m) and chromatography on DETOXI-GEL.TM. columns, to
 remove any trace endotoxin contamination (in sample aliquots, there was no
 detectable lipopolysaccharide). Factor IXai was subsequently frozen into
 aliquots at -80.degree. C. until the time of use. For those experiments in
 which Factor IXai was used, it was given as a single intravenous bolus at
 the indicated times and at the indicated doses.
 Modified Cephalin Clotting Time
 Equal volumes of factor IX-deficient plasma (American Diagnostica Inc.) and
 0.024M celite in 0.05M barbital buffer (Sigma) were combined in
 silicone-coated glass tubes (Sigma) for 2 minutes at 37.degree. C. To this
 mixture, an equal volume of 1:16 (v/v) cephalin (10 mg/ml, Sigma) in 0.05M
 barbital buffer was added, followed by a one-half volume of sample plasma.
 After the addition of calcium chloride to a final concentration of 0.001M,
 the time required for clot formation was determined.
 Results
 To create a stroke in a murine model, a suture is introduced into the
 cerebral vasculature so that it occludes the orifice of the right middle
 cerebral artery, rendering the subtended territory ischemic. By
 withdrawing the suture after a 45 minute period of occlusion, a reperfused
 model of stroke is created; mice so treated demonstrate focal neurological
 deficits as well as clear-cut areas of cerebral infarction. Because the
 occluding suture does not advance beyond the major vascular tributary (the
 middle cerebral artery), this model provides an excellent opportunity to
 investigate "downstream" events that occur within the cerebral
 microvasculature in response to the period of interrupted blood flow.
 Using this model, the role of microvascular thrombosis was investigated as
 follows. To demonstrate that platelet-rich thrombotic foci occur within
 the ischemic cerebral hemisphere, .sup.111 In-labeled platelets were
 administered to mice immediately prior to the introduction of the
 intraluminal occluding suture, to track their deposition during the
 ensuing period of cerebral ischemia and reperfusion. In animals not
 subjected to the surgical procedure to create stroke, the presence of
 platelets was approximately equal between the right and left hemispheres,
 as would be expected [FIG. 11A, left bar]. However, when animals were
 subjected to stroke (and received only vehicle to control for subsequent
 experiments), radiolabeled platelets preferentially accumulated in the
 ischemic (ipsilateral) hemisphere, compared with significantly less
 deposition in the contralateral (nonischemic) hemisphere [FIG. 11A, middle
 bar]. These data support the occurrence of platelet-rich thrombi in the
 ischemic territory. Another line of evidence also supports the occurrence
 of microvascular thrombosis in stroke. This data comes from the
 immunodetection of fibrin, using an antibody directed against a neoepitope
 on the gamma--gamma chain dimer of cross-linked fibrin. Immunoblots
 demonstrate a band of increased intensity in the ipsilateral (right)
 hemisphere of vehicle-treated animals subjected to focal cerebral ischemia
 and reperfusion [FIG. 11B, "Vehicle"]. To demonstrate that fibrin
 accumulation was due to the deposition of intravascular fibrin (rather
 than due to nonspecific permeability changes and exposure to
 subendothelial matrix), fibrin immunostaining clearly localized the
 increased fibrin to the lumina of ipsilateral intracerebral microvessels
 [FIG. 11C]. As an in vivo physiological correlate of microvascular
 thrombosis, relative cerebral blood flow was measured by laser doppler
 during the occlusive period as well as after stroke. These data [FIG. 11D,
 bars labelled "Vehicle"] show that the intraluminal suture technique
 significantly reduces ipsilateral cerebral blood flow during the occlusive
 period [FIG. 11D, middle panel]. Blood flow remains depressed even 24
 hours after removing the intraluminal occluding suture [FIG. 11D, right
 panel], corresponsding to the platelet, fibrin immunoblot, and fibrin
 immunostaining data indicating the presence of postischemic microvascular
 thrombosis.
 To help establish a functionally deleterious role of microvascular
 thrombosis in stroke, experiments were performed to test the effect of
 inhibiting assembly of the Factor IXa/VIIIa/X activation complex in vivo.
 This particular strategy was selected based upon the hypothesis that
 relatively selective inhibition of the intrinsic pathway of coagulation
 might inhibit intravascular thrombosis yet not impair tissue
 factor/VIIa-mediated extravascular hemostasis (and hence, may not increase
 intracerebral hemorrhage at clinically effective doses). Active-site
 blocked factor IXa (Factor IXai), formed by dansylation of the active site
 of Factor IXa, demonstrated antithrombotic potency similar to that of
 heparin when measured in a modified cephalin clotting time assay [FIG.
 12], in which the activity of Factor IXa is a rate-limiting step in
 thrombus formation. To achieve this goal, Factor IXai was administered to
 mice immediately prior to stroke in various doses. When Factor IXai is
 administered to animals prior to introduction of the intraluminal
 occluding suture, there is a significant reduction in the accumulation of
 radiolabelled platelets in the ipsilateral hemisphere [FIG. 11A, rightmost
 bar], no apparent increase in the ipsilateral accumulation of fibrin [FIG.
 11B, "Factor IXai"], as well as decreased evidence of intravascular fibrin
 by immunostaining. In addition, there is a significant increase in
 postischemic blood flow by this treatment, albeit not completely to
 preischemic levels [FIG. 11D].
 The clinical relevance of these observations is underscored by the striking
 ability of Factor IXai to reduce cerebral infarct volumes [FIG. 13A]. To
 test whether this infarct size-reducing property of Factor IXai was unique
 to this compound, or whether a nonspecific anticoagulant would also
 demonstrate efficacy in this regard, intravenous heparin was also examined
 at two doses. Only at the highest dose tested (100 U/kg) did heparin
 reduce cerebral infarct volumes, however, this was at the cost of a
 significant increase in intracerebral hemorrhage, measured with a recently
 validated spectrophotometric assay (17)[FIG. 13B]. In sharp contrast,
 Factor IXai caused an increase in ICH only at the highest dose tested, but
 did not do so at doses which demonstrated striking efficacy to reduce
 cerebral infarct volumes [FIG. 13B]. Because a desirable therapeutic agent
 in stroke will not only reduce cerebral infarction volumes, but will also
 minimize ICH, the data shown in FIGS. 13A and 13B are displayed with
 infarct volumes plotted along the ordinate and intracerebral hemorrhage
 plotted along the abscissa [FIG. 13C]. As can be seen in the figure,
 Factor IXai appears to be therapeutically superior to heparin, because
 with heparin, it was a trade-off between infarct volume-reducing efficacy
 and increasing ICH, which was not the case with Factor IXai (minimized
 both infarction volumes and ICH). Pilot experiments in which tPA was
 administered to mice subjected to stroke resulted in reduced cerebral
 infarction volumes at the cost of increased ICH.
 Because therapies directed at improving outcome from acute stroke must be
 given after clinical presentation, and because fibrin continues to form
 following the initial ischemic event in stroke, we tested whether Factor
 IXai might be effective when given following initiation of cerebral
 ischemia. Factor IXai given after middle cerebral artery occlusion
 (following removal of the occluding suture) provided significant cerebral
 protection judged by its ability to significantly reduce cerebral
 infarction volumes compared with vehicle-treated controls [FIG. 14].
 Discussion
 The data in these studies demonstrate clear evidence of intravascular
 thrombus formation (both platelets and fibrin) within the post-ischemic
 cerebral microvasculature. In fact, the ability of an anticoagulant such
 as Factor IXai to improve outcome even when given after the onset of the
 reperfusion phase suggests that the process of microvascular thrombosis is
 not limited to that which occurs during the major occlusive event. Rather,
 microvascular thrombosis appears to be a dynamic process which continues
 to evolve even after recanalazition of the major vascular tributary. The
 pathophysiological relevance of microvascular thrombosis in stroke is
 underscored by the ability of Factor IXai to reduce microvascular
 thrombosis (both platelet and fibrin accumulation are reduced, with an
 attendant increase in postischemic CBF) and to improve stroke outcome.
 These potent antithrombotic actions of Factor IXai are likely to be
 clinically significant in the setting of stroke, because Factor IXai not
 only reduces infarct volumes in a dose-dependent manner, but it does so
 even when given after the onset of stroke. In addition, at clinically
 relevant doses, treatment with Factor IXai does not cause an increase in
 ICH, making selective inhibition of Factor IXa/VIIIa/X activation complex
 assembly with Factor IXai an attractive target for stroke therapy in
 humans.
 There are a number of reasons why targetted anticoagulant strategies might
 be an attractive alternative to the current use of thrombolytic agents in
 the management of acute stroke, because of their checkered success in
 clinical trials. Theoretically, an ideal treatment for acute stroke would
 prevent the formation or induce dissolution of the fibrin-platelet mesh
 that causes microvascular thrombosis in the ischemic zone without
 increasing the risk of intracerebral hemorrhage. However, thrombolytic
 agents which have been studied in clinical trials of acute stroke have
 consistently increased the risk of intracerebral hemorrhage (1-3,10).
 Streptokinase, given in the first several (&lt;6) hours following stroke
 onset, was associated with an increased rate of hemorrhagic transformation
 (up to 67%); although there was increased early mortality, surviving
 patients suffered less residual disability. Administration of tissue-type
 plasminogen activator (tPA) within 7 hours (particularly within 3 hours)
 of stroke onset resulted in increased early mortality and increased rates
 of hemorrhagic conversion (between 7-20%), although survivors demonstrated
 less residual disability. In order to develop improved anticoagulant or
 thrombolytic therapies, several animal models of stroke have been
 examined. These models generally consist of the administration of clotted
 blood into the internal carotid artery followed by administration of a
 thrombolytic agent. In rats, tPA administration within 2 hours of stroke
 improved cerebral blood flow and reduced infarct size by up to 77%
 (19,20). In a similar rabbit embolic stroke model, tPA was effective at
 restoring blood flow and reducing infarct size, with occasional appearance
 of intracerebral hemorrhage (21,22). However, although there are
 advantages to immediate clot dissolution, there are several potential
 disadvantages of tPA; in murine models, tPA has been shown to directly
 mediate excitotoxic neuronal cell injury via extracellular tPA-catalyzed
 proteolysis of nonfibrin substrates (23-28). Moreover, animal studies (as
 well as the clinical trials of thrombolytic agents) indicate that there is
 an attendant increased risk of intracerebral hemorrhage with this
 therapeutic approach. In preliminary studies in which tPA was given after
 removal of the MCA occluding suture, doses of tPA which tended to reduce
 infarct volumes also increased the degree of ICH (Huang, Kim, Pinsky,
 unpublished observation).
 Because of the usually precipitous onset of ischemic stroke, therapy has
 been targeted primarily towards lysing the major fibrinous/atheroembolic
 debris which occludes a major vascular tributary to the brain. However, as
 the current work demonstrates, there is an important component of
 microvascular thrombosis which occurs downstream from the site of original
 occlusion, which is likely to be of considerable pathophysiological
 significance for post-ischemic hypoperfusion (no-reflow) and cerebral
 injury in evolving stroke. This data is in excellent agreement with that
 which has been previously reported, in which microthrombi have been
 topographically localized to the ischemic region in fresh brain infarcts
 (29). The use of an agent which inhibits assembly of the Factor
 IXa/VIIIa/X activation complex represents a novel approach to limiting
 thrombosis which occurs within microvascular lumena, without impairing
 extravascular hemostasis, the maintenance of which may be critical for
 preventing ICH. In the current studies, treatment with Factor IXai reduces
 microvascular platelet and fibrin accumulation, improves postischemic
 cerebral blood flow, and reduces cerebral infarct volumes in the setting
 of stroke without increasing ICH.
 The potency of Factor IXai as an anticoagulant agent stems from the
 integral role of activated Factor IX in the coagulation cascade. Not only
 does a strategy of Factor IXa blockade appear to be effective in the
 setting of stroke, but it also appears to be effective at preventing
 progressive coronary artery occlusion induced following the initial
 application of electric current to the left circumflex coronary artery in
 dogs (18).
 The data which demonstrate that IXai given after the onset of stroke is
 effective leads to another interesting hypothesis, that the formation of
 thrombus represents a dynamic equilibrium between the processes of ongoing
 thrombosis and ongoing fibrinolysis. Even under normal (nonischemic)
 settings, this dynamic equilibrium has been shown to occur in man (30).
 The data in the current studies, which show that Factor IXai is effective
 even when administered after the onset of stroke, suggests that this
 strategy restores the dynamic equilibrium, which is shifted after cerebral
 ischemia to favor thrombosis, back towards a more quiescent
 (antithrombotic) vascular wall phenotype.
 As a final consideration, even if thrombolysis successfully removes the
 major occluding thrombus, and/or anticoagulant strategies are effective to
 limit progressive microcirculatory thrombosis, blood flow usually fails to
 return to pre-ischemic levels. This is exemplified by data in the current
 study, in which although CBF is considerably improved by Factor IXai
 (which limits fibrin/platelet accumulation), CBF still does not return to
 preischemic levels. This data supports the existence of multiple effector
 mechanisms for postischemic cerebral hypoperfusion, including postischemic
 neutrophil accumulation and consequent microvascular plugging, with
 P-selectin and ICAM-1 expression by cerebral microvascular endothelial
 cells being particularly germane in this regard (11,12). When looked at
 from the perspective of leukocyte adhesion receptor expression, even when
 these adhesion receptors are absent, CBF levels are improved following
 stroke compared with controls but do not return to preischemic levels.
 Taken together, these data suggests that microvascular thrombosis and
 leukocyte adhesion together contribute to postischemic cerebral
 hypoperfusion.
 In summary, administration of a competitive inhibitor of Factor IXa,
 active-site blocked Factor IXa, represents a novel therapy for the
 treatment of stroke. This therapy not only reduces microcirculatory
 thrombosis, improves postischemic cerebral blood flow, and reduces
 cerebral tissue injury following stroke, but it can do so even if given
 after the onset of cerebral ischemia and without increasing the risk of
 ICH. This combination of beneficial properties and relatively low downside
 risk of hemorrhagic transformation makes this an extremely attractive
 approach for further testing and potential clinical trials in human
 stroke.
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 29. Heye, N., et al. (1992) Acta Neurologica Scandinavica 86:450-454.
 30. Nossel, H. L. (1981) Nature 291:165-167.
 EXAMPLE 5
 Microvascular Thrombosis as a Pathophysiological Mechanism in Ischemic
 Stroke and Use of Active-site Blocked Factor IX as a Novel Treatment
 Ischemic stroke is the third leading cause of death in the United States.
 Current treatments aim to reestablish perfusion to ischemic brain by
 thrombolysis, however, they can increase the risk of hemorrhage,
 particularly in the setting of ischemia. Studies of acute stroke thus far
 have focused on ischemia associated with thromboembolic occlusion of
 cerebral vascular tributary. We hypothesize, however, that ischemic injury
 is not produced solely by the original occlusion, but that the initial
 ischemic event modifies the microvasculature to trigger further
 local/microvascular thrombosis which contributes to post-ischemic
 hypoperfusion (no-reflow). An ideal treatment would overcome post-ischemic
 microvascular thrombosis and allow reperfusion without increasing the risk
 of hemorrhage.
 Materials and Methods:
 We studied post-ischemic microvascular thrombosis in a murine model of
 ischemic stroke in which the right middle cerebral artery (MCA) is
 transiently occluded for 45 minutes. The role of platelets and fibrin were
 investigated using 111-Indium-labeled platelets and fibrin immunostaining.
 We studied the efficacy of a novel anticoagulant, active-site blocked
 factor IX (IXAI, 150-300 .mu.g/kg IV), which inhibits the Factor
 IXA/VIIIa/X activation complex. Outcome indices were platelet accumulation
 (measured as an ipsilateral to contralateral ratio), relative cerebral
 blood flow measured by laser doppler (CBF, ratio of ipsilateral to
 contralateral hemispheric flow), and infarct volume (Inf Volume, %
 ipsilateral hemisphere by triphenyltetrazolium chloride staining). In
 addition, intracerebral hemorrhage (ICH) was quantified in homogenized
 brain tissue using a method which we developed and validated, based on the
 conversion of hemoglobin to cyanomethemoglobin (OD measured at 550 nm; the
 amount of intracerebral blood is linearly related to OD).
 Results:
 TABLE III
 Platelets Fibrin CBF Inf Volume ICH
 No Stroke (n = 11) 1.1 .+-. 0.1 0 110 .+-. 8 0.0 .+-. 0 0.07
 .+-. 0.0
 Stroke + Placebo 2.9 .+-. 0.3* ++ 37 .+-. 5* 26 .+-. 3.7* 0.15
 .+-. 0.04*
 (n = 62)
 Stroke + IXai (n = 48) 1.6 .+-. 0.2* + 61 .+-. 6** 7.4 .+-. 3.0**
 0.12 .+-. 0.02
 (Results are expressed as means .+-.SEM.
 *p &lt; 0.05 vs. no stroke,
 **o &lt; 0.01 vs. stroke + placebo)
 These data, along with immunohistochemical evidence of intravascular fibrin
 only in the ischemic hemisphere, show that thrombus accumulates within the
 post-ischemic cerebral microvasculature. Furthermore, IXai reduces both
 this platelet and fibrin accumulation, improves CBF, and reduces infarct
 volumes in a dose-dependent manner. The advantage of IXai in treating
 stroke without increasing ICH was shown in experiments where it did not
 increase ICH when compared with controls (0.12.+-.0.02 vs. 0.15.+-.0.04,
 p=NS). The benefit of IXai was also observed when given after the onset of
 stroke (placebo infarct volume 39.+-.5.5% vs. IXai 14.+-.2.4%, p&lt;0.05).
 Conclusions:
 In ischemic regions of brain, platelets and fibrin accumulate to form
 microvascular thrombosis, contributing to post-ischemic hypoperfusion
 (no-reflow). Treatment with IXai reduces platelet and fibrin accumulation,
 improves CBF, and reduces infarct volume without increasing ICH.
 EXAMPLE 6
 Active-Site Blocked Factor Ixai: An Alternative Anticoagulant for Use in
 Hemodialysis
 Significant bleeding complications during hemodialysis (HD) in high-risk
 patients (GI/intracerebral hemorrhage) have been reported with an
 incidence as high as 26%. Patients with increased risk of bleeding as well
 as those with specific contraindications to heparin would greatly benefit
 from an alternative anticoagulant for use in HD. Active-site blocked
 factor IXA (Ixai) has previously been shown to selectively block the
 intrinsic/contact mediated pathway of coagulation in the setting of
 contact of blood with an extracorporeal circuit, while maintaining
 extravascular/tissue factor-mediated hemostasis. In order to investigate
 the use of this novel anticoagulant strategy in the setting of HD and
 chronic uremia, obstructive renal failure was induced in 11 female mongrel
 dogs by bilateral ureteral ligation through a midline laparotomy. Renal
 failure, as indicated by a rise in BUN&gt;65 mg/dl, was reliably induced
 within 48 hours at which time the animals underwent standard HD using COBE
 Centrysystem 3 equipped with 300 HG hemodialyzers and standard bicarbonate
 dialysate (BiCart). Venovenous HD lasted for three hours and was performed
 on three consecutive days at flows of 300-350 ml/min. HD was successfully
 completed using Ixai (400-460 .mu.g/kg given at 0 min & 90 min) or
 standard heparin with equivalent efficacy as reflected by the urea
 reduction ratio (74.86%.+-.3.43% vs. 78.16%.+-.2.49%, p=43). There was no
 evidence of gross clot formation in the tubing or resultant increase in
 circuit pressure. Analysis of data from incisional wound models at 15 min
 suggested a decreased bleeding tendency in IXai treated animals as
 compared to those treated with heparin (0.05.+-.0.11 gm vs. 0.38.+-.0.17
 gm closed wound, p=0.004; 4.59.+-.1.74 gm vs. 8.75.+-.2.09 gm open wound,
 p=17). IXai, a selective anticoagulant which confers extracorporeal
 circuit anticoagulation without compromising extravascular hemostasis, may
 therefore represent a novel alternative anticoagulant strategy for use in
 chronic HD.
 EXAMPLE 7
 Role of Factor IXai in Pulmonary Ischemia and Reperfusion and Role of
 Factor IXai as an Adjunct to Tissue-type Plasminogen Activator (tPA) in
 Stroke.
 (1) Factor IXai can be effective at lower doses with the lower doses being
 less likely to cause intracerebral hemorrhage. This Example includes data
 regarding the dose response range of Factor IXai with respect to its
 effect on clotting time in the modified cephalin clotting time assay. The
 dose/response data with respect to intracerebral hemorrhage can be found
 in the data provided in Example 4.
 (2) Factor IXai is effective in other types of ischemia (and reperfusion.
 New data shown in this example show that when the lungs are subjected to
 ischemia and reperfusion (by cross-clamping their blood supply, waiting a
 bit, and then releasing the clamp), Factor IXai is protective. Both the
 lung function (oxygenation of blood) and survival of the animal which had
 received Factor IXai was better than that seen in vehicle-treated animals.
 (3) Factor IXai may be effective after the thrombotic event; i.e., it is
 effective when given after stroke, not just beforehand. This data can be
 found in the information hereinabove in Example 4.
 (4) Factor IXai may be useful to lower the dose of thrombolytic therapy
 necessary to achieve reperfusion (for instance, in heart attacks, stroke,
 pulmonary emboli, etc.). The data which shows this point is in Table IV
 hereinbelow. In a stroke model, a dose of tissue-type plasminogen
 activator (an example of a commonly used thrombolytic agent) which itself
 did not protect the brain In stroke was given in combination with a dose
 of Factor IXai which was too low by itself to confer protection; however,
 the combination was significantly protective (reduced cerebral infarction
 volume) without causing any excess in intracerebral hemorrhage.
 Role of Factor IXai in Pulmonary Ischemia and Reperfusion:
 Seven C57BL mice (male 25 gm) were anesthetized with ketamine and xylazine,
 and a bilateral thoracotomy was performed using a clam-shell incision. A
 loose suture was placed around the right pulmonary artery, and the left
 pulmonary hilum was exposed. An intravenous injection was given (0.3 mL of
 either saline [control, n=4] or Factor IXai [300 .mu.g, n=3]. After 3
 minutes, the left pulmonary hilum (pulmonary artery, vein, and bronchus)
 was cross-clamped for 1 hour to create ischemia, after which the
 cross-clamp was released and the left lung reperfused and ventilated for 1
 hour. After this reperfusion period, the loose suture around the right
 pulmonary artery was tightened, so that the animal's arterial oxygenation
 and survival depended solely on the function of the postischemic left
 lung. The data revealed that in the control group, the mean arterial
 oxygenation was 66 mm Hg, whereas in the Factor IXai-treated group, it was
 120 mm Hg. Factor IXai also improved survival, in that 100% of control
 animals failed to survive the right pulmonary artery ligation procedure
 (mean time to death, 10 minutes), whereas 2/3 of the Factor IXai-treated
 animals survived for 30 minutes (at which time they were sacrificed for
 arterial blood gas analysis). Taken together, these data show that Factor
 IXai can protect against ischemia reperfusion injury in this model, and
 extend the previous data which showed that Factor IXai was protective
 after middle cerebral artery ischemia and reperfusion.
 Role of Factor IXai as an Adjunct to Tissue-type Plasminogen Activator
 (tPA) in Stroke:
 For these data, 17 mice were used, and subjected to middle cerebral artery
 occlusion (45 minutes) and reperfusion as described hereinabove. Because
 Factor IXai by itself has been shown to have a dose-related
 cerebroprotective effect in stroke, a dose was chosen which we had
 previously shown to be below the protective threshold (50 .mu.g/kg). In
 the experimental group, mice were given 50 .mu.g/kg of Factor IXai
 preoperatively, and tPA was given immediately after withdrawal of the
 occluding suture at a dose of 0.5 mg/kg. Either of these agents when given
 by themselves at these low doses did not confer cerebral protection.
 However, compared to control animals which received vehicle alone (n=7),
 when tPA 0.5 mg/kg and Factor IXai (50 .mu.g/kg) (n=10) were combined,
 there was significant protection; relative cerebral blood flows are
 expressed as an ipsilateral/contralateral blood flow ratio (.times.100),
 Infarct volumes are expressed as the percent of the ipsilateral hemisphere
 which was infarcted, and intracerebral hemorrhage was recorded as the
 optical density at 550 nm (higher numbers mean more hemorrhage, using our
 recently validated spectrophotometric method for quantifying intracerebral
 hemorrhage). ***=p&lt;0.001 vs. control.
 TABLE IV
 Interacerebral
 Relative cerebral blood flow Infarct Volume Hemorrhage
 Control 39 .+-. 6.4% 29.6 .+-. 8.4% 0.112 .+-. 0.013
 IXai + tPA 72 .+-. 4.1%*** 10.0 .+-. 2.6%*** 0.110 .+-. 0.014
 We conclude that administration of Factor IXai even at low doses can make
 tPA effective and cerebroprotective, at doses of tPA which otherwise
 showed no beneficial effects in previous experiments. Note that the
 combination treatment did not increase the degree of intracerebral
 hemorrhage.
 SEQUENCE LISTING
 &lt;100&gt; GENERAL INFORMATION:
 &lt;160&gt; NUMBER OF SEQ ID NOS: 22
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 1
 &lt;211&gt; LENGTH: 29
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 Oligonucleotides for producing Factor IXmi.
 &lt;400&gt; SEQUENCE: 1
 tacagttcct ctannncccc ctggggtac 29
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 2
 &lt;211&gt; LENGTH: 30
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 Oligonucleotides for producing Factor IXmi.
 &lt;400&gt; SEQUENCE: 2
 tacagttcct ctannncccc ctggggtaca 30
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 3
 &lt;211&gt; LENGTH: 31
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 Oligonucleotides for producing Factor IXmi.
 &lt;400&gt; SEQUENCE: 3
 tacagttcct ctannncccc ctggggtaca a 31
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 4
 &lt;211&gt; LENGTH: 30
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 Oligonucleotides for producing Factor IXmi.
 &lt;400&gt; SEQUENCE: 4
 gtacagttcc tctannnccc cctggggtac 30
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 5
 &lt;211&gt; LENGTH: 31
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 Oligonucleotides for producing Factor IXmi.
 &lt;400&gt; SEQUENCE: 5
 gtacagttcc tctannnccc cctggggtac a 31
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 6
 &lt;211&gt; LENGTH: 32
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 Oligonucleotides for producing Factor IXmi.
 &lt;400&gt; SEQUENCE: 6
 gtacagttcc tctannnccc cctggggtac aa 32
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 7
 &lt;211&gt; LENGTH: 32
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 Oligonucleotides for producing Factor IXmi.
 &lt;400&gt; SEQUENCE: 7
 agttacagtt cctctannnc cccctggggt ac 32
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 8
 &lt;211&gt; LENGTH: 33
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 Oligonucleotides for producing Factor IXmi.
 &lt;400&gt; SEQUENCE: 8
 agttacagtt cctctannnc cccctggggt aca 33
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 9
 &lt;211&gt; LENGTH: 34
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 Oligonucleotides for producing Factor IXmi.
 &lt;400&gt; SEQUENCE: 9
 agttacagtt cctctannnc cccctggggt acaa 34
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 10
 &lt;211&gt; LENGTH: 29
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 Oligonucleotides for producing Factor IXmi.
 &lt;400&gt; SEQUENCE: 10
 attcatgtta gtannntaac gcgaagacc 29
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 11
 &lt;211&gt; LENGTH: 30
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 Oligonucleotides for producing Factor IXmi.
 &lt;400&gt; SEQUENCE: 11
 attcatgtta gtannntaac gcgaagacct 30
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 12
 &lt;211&gt; LENGTH: 31
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 Oligonucleotides for producing Factor IXmi.
 &lt;400&gt; SEQUENCE: 12
 attcatgtta gtannntaac gcgaagacct t 31
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 13
 &lt;211&gt; LENGTH: 30
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 Oligonucleotides for producing Factor IXmi.
 &lt;400&gt; SEQUENCE: 13
 tattcatgtt agtannntaa cgcgaagacc 30
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 14
 &lt;211&gt; LENGTH: 31
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 Oligonucleotides for producing Factor IXmi.
 &lt;400&gt; SEQUENCE: 14
 tattcatgtt agtannntaa cgcgaagacc t 31
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 15
 &lt;211&gt; LENGTH: 32
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 Oligonucleotides for producing Factor IXmi.
 &lt;400&gt; SEQUENCE: 15
 tattcatgtt agtannntaa cgcgaagacc tt 32
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 16
 &lt;211&gt; LENGTH: 31
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 Oligonucleotides for producing Factor IXmi.
 &lt;400&gt; SEQUENCE: 16
 ttattcatgt tagtannnta acgcgaagac c 31
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 17
 &lt;211&gt; LENGTH: 32
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 Oligonucleotides for producing Factor IXmi.
 &lt;400&gt; SEQUENCE: 17
 ttattcatgt tagtannnta acgcgaagac ct 32
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 18
 &lt;211&gt; LENGTH: 33
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 Oligonucleotides for producing Factor IXmi.
 &lt;400&gt; SEQUENCE: 18
 ttattcatgt tagtannnta acgcgaagac ctt 33
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 19
 &lt;211&gt; LENGTH: 33
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 Oligonucleotides for producing Factor IXmi.
 &lt;400&gt; SEQUENCE: 19
 ttacattgac gacggnnnac acaactttga cca 33
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 20
 &lt;211&gt; LENGTH: 30
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 Oligonucleotide Primer for producing Factor IXmi.
 &lt;400&gt; SEQUENCE: 20
 gtacagttcc tctacgaccc cctggggtac 30
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 21
 &lt;211&gt; LENGTH: 461
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Homo Sapien
 &lt;400&gt; SEQUENCE: 21
 Met Gln Arg Val Asn Met Ile Met Ala Glu Ser Pro Gly Leu Ile Thr
 1 5 10 15
 Ile Cys Leu Leu Gly Tyr Leu Leu Ser Ala Glu Cys Thr Val Phe Leu
 20 25 30
 Asp His Glu Asn Ala Asn Lys Ile Leu Asn Arg Pro Lys Arg Tyr Asn
 35 40 45
 Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg Glu Cys
 50 55 60
 Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe Glu Asn
 65 70 75 80
 Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val Asp Gly Asp Gln
 85 90 95
 Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys Lys Asp Asp Ile
 100 105 110
 Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe Glu Gly Lys Asn Cys
 115 120 125
 Glu Leu Asp Val Thr Cys Asn Ile Lys Asn Gly Arg Cys Glu Gln Phe
 130 135 140
 Cys Lys Asn Ser Ala Asp Asn Lys Val Val Cys Ser Cys Thr Glu Gly
 145 150 155 160
 Tyr Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val Pro Phe
 165 170 175
 Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr Arg Ala
 180 185 190
 Glu Thr Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu Ala Glu
 195 200 205
 Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser Phe Asn Asp Phe
 210 215 220
 Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe Pro Trp
 225 230 235 240
 Gln Val Val Leu Asn Gly Lys Val Asp Ala Phe Cys Gly Gly Ser Ile
 245 250 255
 Val Asn Glu Lys Trp Ile Val Thr Ala Ala His Cys Val Glu Thr Gly
 260 265 270
 Val Lys Ile Thr Val Val Ala Gly Glu His Asn Ile Glu Glu Thr Glu
 275 280 285
 His Thr Glu Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His His Asn
 290 295 300
 Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu Leu Glu
 305 310 315 320
 Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile Cys Ile
 325 330 335
 Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe Gly Ser Gly Tyr
 340 345 350
 Val Ser Gly Trp Gly Arg Val Phe His Lys Gly Arg Ser Ala Leu Val
 355 360 365
 Leu Gln Tyr Leu Arg Val Pro Leu Val Asp Arg Ala Thr Cys Leu Arg
 370 375 380
 Ser Thr Lys Phe Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly Phe His
 385 390 395 400
 Glu Gly Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro His Val
 405 410 415
 Thr Glu Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser Trp Gly
 420 425 430
 Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys Val Ser
 435 440 445
 Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr
 450 455 460
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 22
 &lt;211&gt; LENGTH: 2775
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Homo Sapien
 &lt;400&gt; SEQUENCE: 22
 atgcagcgcg tgaacatgat catggcagaa tcaccaggcc tcatcaccat ctgcctttta 60
 ggatatctac tcagtgctga atgtacagtt tttcttgatc atgaaaacgc caacaaaatt 120
 ctgaatcggc caaagaggta taattcaggt aaattggaag agtttgttca agggaacctt 180
 gagagagaat gtatggaaga aaagtgtagt tttgaagaag cacgagaagt ttttgaaaac 240
 actgaaagaa caactgaatt ttggaagcag tatgttgatg gagatcagtg tgagtccaat 300
 ccatgtttaa atggcggcag ttgcaaggat gacattaatt cctatgaatg ttggtgtccc 360
 tttggatttg aaggaaagaa ctgtgaatta gatgtaacat gtaacattaa gaatggcaga 420
 tgcgagcagt tttgtaaaaa tagtgctgat aacaaggtgg tttgctcctg tactgaggga 480
 tatcgacttg cagaaaacca gaagtcctgt gaaccagcag tgccatttcc atgtggaaga 540
 gtttctgttt cacaaacttc taagctcacc cgtgctgaga ctgtttttcc tgatgtggac 600
 tatgtaaatt ctactgaagc tgaaaccatt ttggataaca tcactcaaag cacccaatca 660
 tttaatgact tcactcgggt tgttggtgga gaagatgcca aaccaggtca attcccttgg 720
 caggttgttt tgaatggtaa agttgatgca ttctgtggag gctctatcgt taatgaaaaa 780
 tggattgtaa ctgctgccca ctgtgttgaa actggtgtta aaattacagt tgtcgcaggt 840
 gaacataata ttgaggagac agaacataca gagcaaaagc gaaatgtgat tcgaattatt 900
 cctcaccaca actacaatgc agctattaat aagtacaacc atgacattgc ccttctggaa 960
 ctggacgaac ccttagtgct aaacagctac gttacaccta tttgcattgc tgacaaggaa 1020
 tacacgaaca tcttcctcaa atttggatct ggctatgtaa gtggctgggg aagagtcttc 1080
 cacaaaggga gatcagcttt agttcttcag taccttagag ttccacttgt tgaccgagcc 1140
 acatgtcttc gatctacaaa gttcaccatc tataacaaca tgttctgtgc tggcttccat 1200
 gaaggaggta gagattcatg tcaaggagat agtgggggac cccatgttac tgaagtggaa 1260
 gggaccagtt tcttaactgg aattattagc tggggtgaag agtgtgcaat gaaaggcaaa 1320
 tatggaatat ataccaaggt atcccggtat gtcaactgga ttaaggaaaa aacaaagctc 1380
 acttaatgaa agatggattt ccaaggttaa ttcattggaa ttgaaaatta acagggcctc 1440
 tcactaacta atcactttcc catcttttgt tagatttgaa tatatacatt ctatgatcat 1500
 tgctttttct ctttacaggg gagaatttca tattttacct gagcaaattg attagaaaat 1560
 ggaaccacta gaggaatata atgtgttagg aaattacagt catttctaag ggcccagccc 1620
 ttgacaaaat tgtgaagtta aattctccac tctgtccatc agatactatg gttctccact 1680
 atggcaacta actcactcaa ttttccctcc ttagcagcat tccatcttcc cgatcttctt 1740
 tgcttctcca accaaaacat caatgtttat tagttctgta tacagtacag gatctttggt 1800
 ctactctatc acaaggccag taccacactc atgaagaaag aacacaggag tagctgagag 1860
 gctaaaactc atcaaaaaca ctactccttt tcctctaccc tattcctcaa tcttttacct 1920
 tttccaaatc ccaatcccca aatcagtttt tctctttctt actccctctc tcccttttac 1980
 cctccatggt cgttaaagga gagatgggga gcatcattct gttatacttc tgtacacagt 2040
 tatacatgtc tatcaaaccc agacttgctt ccatagtgga gacttgcttt tcagaacata 2100
 gggatgaagt aaggtgcctg aaaagtttgg gggaaaagtt tctttcagag agttaagtta 2160
 ttttatatat ataatatata tataaaatat ataatataca atataaatat atagtgtgtg 2220
 tgtgtatgcg tgtgtgtaga cacacacgca tacacacata taatggaagc aataagccat 2280
 tctaagagct tgtatggtta tggaggtctg actaggcatg atttcacgaa ggcaagattg 2340
 gcatatcatt gtaactaaaa aagctgacat tgacccagac atattgtact ctttctaaaa 2400
 ataataataa taatgctaac agaaagaaga gaaccgttcg tttgcaatct acagctagta 2460
 gagactttga ggaagaattc aacagtgtgt cttcagcagt gttcagagcc aagcaagaag 2520
 ttgaagttgc ctagaccaga ggacataagt atcatgtctc ctttaactag cataccccga 2580
 agtggagaag ggtgcagcag gctcaaaggc ataagtcatt ccaatcagcc aactaagttg 2640
 tccttttctg gtttcgtgtt caccatggaa cattttgatt atagttaatc cttctatctt 2700
 gaatcttcta gagagttgct gaccaactga cgtatgtttc cctttgtgaa ttaataaact 2760
 ggtgttctgg ttcat 2775