AU766910B2 - Prevention of brain damage in stroke - Google Patents

Description

WO 00/60943 PCT/US00/10002 PREVENTION OF BRAIN DAMAGE IN STROKE Technical Field of the Invention The present invention provides a method and a pharmaceutical composition useful to mitigate tissue damage associated with ischemia, particularly of the brain (stroke, brain attack) and heart (myocardial infarction, heart attack).

Background of the Invention Cerebral ischemia, also termed "stroke" or "brain attack," is a leading cause of mortality and neurologic disability worldwide, but proven treatment options are severely limited. Recent clinical results indicate that the timely administration ofthrombolytic agents can improve the outcome from stroke by restoring blood flow to the ischemic brain, for instance, but this approach has its limitations and additional therapeutic modalities are urgently needed (Anonymous, NEngl JMed 333:1581-1587, 1995). The timing of therapeutic intervention against stroke damage is critical because outside the most profoundly ischemic zone where all cells are destined to die (the "ischemic core"), lies a "penumbral zone" where brain cell death slowly continues to occur for minutes, hours and days after the onset of ischemia. Delayed cell death in the under-perfused penumbral region is caused by a poorly understood cascade of cytotoxic mediators that kill otherwise potentially viable cells. The time course of progressive brain damage within this penumbra limits the duration of the therapeutic window, and new therapeutic approaches will depend first on the identification of responsible cytotoxic mediators and secondly on the identification of antagonists that can be administered within the therapeutic window. Thus the goal of therapy for cerebral infarction is to prevent the loss of potentially viable brain tissue in the early hours after the onset of ischemia, and there is a need to identify both target mechanisms of cytotoxicity and suitable antagonist agents to minimize brain damage in stroke.

alpha-2HS-Glycoprotein (a2-HS), sometimes called human fetuin, is the human homolog of the bovine protein originally isolated as fetuin. Alpha-2HS-Glycoprotein is a major protein occurring in human blood and calciferous tissues (where has been known as "bone resorptive protein-2," or BRP-2). Due to extensive sequence identity, a2-HS has been grouped with the fetuins, a family of proteins that occur in fetal plasma in high concentrations.

Native a2-HS undergoes a series ofposttranslational modifications including proteolytic processing, multiple N-glycosylations and O-glycosylations, sulfation of the carbohydrate side chains, and phosphorylation, such that slightly differing mature forms may be present. a2-HS is generally considered to comprise two polypeptide chains, the A chain (282 amino acids) with five internal disulfide bridges forming it into a series of loops, and the B chain (27 amino acids) linked by a single disulfide bridge tothe A chain. Human fetuin, or a2-HS, is generally considered to arise from a single mRNA transcript encoding a 367 amino acid peptide known 1WO 00/60943 PCT/US00/10002 as the "alpha-2-HS-glycoprotein precursor" (SEQ ID NO. Amino acids 1-18 (SEQ ID NO.

2) comprise a signal sequence domain. Amino acids 19-300 comprise the a2-HS-glycoprotein A chain domain (SEQ ID NO. Amino acids 341-367 comprise the a2-HS-glycoprotein B chain domain (SEQ ID NO. By inference, amino acids 301-340 comprise a 40 amino acid connecting sequence (SEQ ID NO. 5) that is not present in the mature form, although single chain forms of c2-HS have been isolated (Jahnen-Dechent et al., Eur. J. Biochem. 226:59-69, 1994).

Fetuin was first identified more than 50 years ago as a major protein component of bovine fetal serum but its biological function remains unclear, particularly as a circulating protein. Bovine fetuin occurs as a single chain, globular 341 amino acid polypeptide (amino acids 19-359 of the 359 amino acid bovine fetuin precursor) with six internal disulfide bonds and three N-linked and two O-linked oligosaccharides (SEQ ID NO. Primary amino acid sequence and the position of cysteine residues are well conserved across species, human, bovine, sheep, rat and mouse (Dziegielewska et al., J. Biol. Chem. 265:4354, 1990; Rauth et al., Eur. J. Biochem. 205:321, 1992; Lee et al., Proc. Natl. Acad. Sci. USA 84:4403, 1987; and Brown et al., Eur. J. Biochem. 205:321, 1992). Fetuin (a2-HS) levels in human plasma are regulated in the manner of a negative acute phase reactant (Lebreton et al., J. Clin. Invest.

64:1118, 1979). IL-1 was shown to suppress a2-HS transcript levels in cultured hepatocytes (Akhoundi et al., J. Biol. Chem. 268:15925, 1994). a2-HS appears to be expressed in bone because transcripts have been detected in both chondrocytes and osteoblasts (Yang et al., Blood 12:7, 1991), and a2-HS influences the mineral phase of bone. The a2-HS glycoprotein is the human homolog of fetuin and is secreted in high levels by adult liver into the peripheral circulation (Triffitt et al., Nature 262:226, 1976).

Human fetuin (a2-HS) has 2 N-linked oligosaccharide chains (attached to the amine nitrogen atom of asparagine), and 3 O-linked oligosaccharide chains (attached to the oxygen atom of serine or threonine). The sugar moiety directly attached to the a2-HS polypeptide is usually a N-acetylglucosamine residue. The terminal sugar residue is usually a sialic acid, in particular a N-acetylneuraminic acid (NANA) residue, which bears a net negative charge. If one removes the terminal sialic acid residue from a2-HS by neuraminidase treatment, the resulting glycoprotein is an asialofetuin. Fetuin (a2-HS) is also a carrier protein for growth factors and cytokines. The synthesis of human a2-HS -glycoprotein is down-regulated by cytokines (hIL-1 P, hIL-6) (Lebreton et al., J. Clin. Invest. 64:1118-1129, 1979). Human fetuin (a2-HS) levels are decreased (25-50%) in trauma patients (van Oss et al., J. Trauma 15:451, 1975). a2-HS is structurally related to the cystatins and kininogens.

Summary of the Invention.

"Fetuin" as used herein refers, in the context of the human protein, to the glycoprotein referred to variously as "a2-HS-glycoprotein" or "a2-Z-globulin" or "human fetuin" or "human fetuin glycoprotein," and in broader context to any of the fetuin family of proteins, WO 00/60943 PCT/US00/10002 with members occurring in various species and closely related in sequence to bovine fetuin and human a2-HS. Two common alleles are known for a2-HS: one has threonine at position 248 and 256, the other has methionine at 248 and serine at 256. Use of"fetuin" or "a2-HS" according to the teachings herein shall correspondingly include use of allelic variants, glycosylation, sulfation and phosphorylation variants, reduced and native forms, precursor and proteolytically processed forms, and sequence variants substantially homologous to the polypeptides described by SEQ ID NO.'s 1-6 or to the fetuins of other (non-human) species, and fragments of any of the above. Such variants, whether naturally occurring, intentionally introduced or spontaneously arising, are conveniently tested for activity (and thereby evaluated for clinical utility) in accordance with the Detailed Description and Examples described herein.

The present invention arose out of a series of experiments wherein brain damage (cell death) subsequent to induced focal ischemia was found to be ameliorated by treatment with the glycoprotein a2-HS. The present invention identifies for the first time the cellular and tissue protective effects of administering a2-HS in the setting of ischemia. The present invention further provides methods and pharmaceutical compositions for preventing tissue damage in ischemia, particularly brain damage attendant to stroke or cerebral ischemia, comprising administering an effective amount of an a2-HS glycoprotein. Preferably, the a2-HS glycoprotein is a human a2-HS glycoprotein comprising a primary sequence according to SEQ ID NO. 1 through SEQ ID NO. 5 or a shorter fragment thereof. Highly homologous sequence variants are also useful in this regard, particularly such homologous glycoproteins as have effects quantitatively indistinguishable from a2-HS in the assays described herein. Such variant glycoproteins are conveniently produced according to techniques of molecular biology well-known in the art and are readily compared to human a2-HS glycoprotein in the assays described herein, or in comparable assays for cellular or tissue protection in the setting of ischemia.

The utility of the methods and compositions involving a2-HS glycoproteins as taught herein are directly extended to other instances of tissue ischemia, particularly heart attack or myocardial infarction. Stroke (ischemia and associated tissue damage) is well-known to arise from a variety of distal causes, giving rise to such clinical characterizations as: stroke, cerebral infarction, cerebrovascular accident, thrombotic stroke, embolic stroke, occlusive cerebrovascular lesion, apoplexy (of various types), apoplectic stroke, paralytic stroke, intracranial hemorrhage, hemorrhagic stroke, ruptured aneurysm, post-traumatic stroke, transient ischemic attack, and stroke syndrome. Likewise, heart attack may arise variously, giving rise to such representative clinical characterizations as: cardiac infarction, myocardial infarction (various types), coronary artery occlusion, coronary thrombosis, coronary embolism, periarteritis nodosa, and obliterating endarteritis. Similarly, other organs and tissues may become afflicted by ischemia involving, among other conditions, anemic, pale, white or bland infarction, various embolic disorders including embolic infarction, various thrombotic disorders including thrombotic infarction, hemorrhagic or red infarction, and coagulation WO 00/60943 PCT/US00/10002 necrosis. Any of these conditions of ischemia, and their clinical relations, is amenable to treatment according to the methods and with the pharmaceutical compositions taught herein.

Brief Description of the Drawings Figure 1 demonstrates the expression of a2-HS in infarct regions of brain cortex from rats subjected to experimental focal cerebral ischemia. Figure 1A is a Western blot showing a2-HS protein expression in regions of infarct induced in the brain cortex of rats subjected to experimental focal ischemia. Lane 1, sampled from normal brain; lane 2, two hours post onset of ischemia; lane 3, six hours post onset of ischemia; lane 4, 24 hours post onset of ischemia; lane 5, 48 hours post onset of ischemia; lane 6, 96 hours post onset of ischemia. Figure 1B is a plot of scanning densitometric values derived from the Western blots of Figure 1A plotted against hours post onset of ischemia.

Figure 2 shows the effects of c2-HS treatment on infarct size in experimental focal cerebral ischemia. a2-HS treatment significantly reduced infarct volume when administered intravenously 15 min after the induction of ischemia. Histograms represent infarct volume as a percentage of half cortical volume 24 hours after onset of ischemia. Asialofetuin worsened brain damage. Values are mean SD, n 6; **P<0.01 compared to control and to asialofetuin; *P<0.05 compared to control.

Figure 3 shows that the therapeutic benefits ofa2-HS treatment are dose-dependent.

Histograms represent infarct volume as percent of half-cortical volume of brains collected 24 hours after ischemic challenge. Treatments were intravenous 15 minutes after onset of ischemia with from left to right, 50 mg/kg asialofetuin, no treatment control, 5 mg/kg a2-HS, mg/kg a2-HS, 50 mg/kg a2-HS, 500 mg/kg a2-HS. Values represent mean SD, n 6; **P<0.01 compared to control; *P<0.05 compared to control.

Figure 4 shows a time course of the therapeutic benefit of a2-HS treatment in stroke.

The histograms represent cortical infarct volume as a percentage of half-cortex volume at 24 hours after induction of focal cerebral ischemia in rats. a2-HS was administered at a dose of mg /kg intravenously 60 min (a2-HS 30 min (a2-HS 2) or 15 min (a2-HS 3) after induction of experimental focal cerebral ischemia in rats. Leftmost bar is control (no a2-HS).

Values represent mean SD, n 6; *P<0.01 compared to control.

Detailed Description of the Invention The present invention is based on the discovery that a human plasma glycoprotein, a2- HS, is beneficial in preventing tissue damage associated with ischemia, and specifically in treating stroke. Although fetuin was discovered more than 50 years ago as a component of fetal bovine serum, and subsequently found to share high homology with a human a2-HS counterpart (a2-HS-glycoprotein), no role for a2-HS in the natural history, etiology or treatment of stroke (cerebral infarction, cerebral ischemia, brain attack) or other tissue ischemia had been suspected. After recognizing the activity of a2-HS to potentiate the anti- WO 00/60943 PCT/US00/10002 inflammatory activity of certain low molecular weight compounds and metabolites, we tested for the occurrence and activity of a2-HS in a predictive animal model of human stroke, in experimentally induced focal cerebral ischemia in rats. We discovered that a2-HS occurs in increasing amounts in the areas of brain damage following permanent focal cerebral ischemia, and that administration of a2-HS alone, even after the onset of ischemia, dose-dependently decreased the volume of total brain damage in stroke. Asialofetuin (a2-HS glycoprotein treated to remove sialic acid residues) was ineffective or exacerbative in this regard.

The present invention provides novel methods (a2-HS administration as adjunctive or monotherapy) and pharmaceutical compositions (comprising a2-HS or glycoprotein sequence variants) to treat and mitigate against tissue damage in ischemia, particularly in stroke and heart attack. Effective doses of the therapy are determined by routine procedures in the art with reference to the findings described herein, and may be formulated in suitable pharmacological carriers for administration by any appropriate means including, but not limited to, injection (such as, intravenous, intramuscular, intrathecal, and intracranial) and other means available within the pharmaceutical arts. Treatment may be accomplished by administration of the a2-HS glycoprotein alone or in a pharmaceutical composition where it is mixed with suitable carriers or excipients to treat tissue ischemia or mitigate against ischemic tissue damage. Preferably, administration of a2-HS is systemic to provide organ sites of treatment including the brain, CNS, myocardium, or other organ site experiencing ischemia. A therapeutically effective dose refers to that amount of the active agent sufficient to treat tissue ischemia or to mitigate against ischemic tissue damage. Therapeutically effective doses may be administered alone or as adjunctive therapy in combination with other treatments or supportive measures for tissue ischemia, particularly for stroke or for heart attack. In particular, a2-HS may be co-administered with spermine or otherwise in accordance with the teachings of USSN: 08/780,311 (filed), the disclosure of which is incorporated herein by reference in its entirety. Techniques for the formulation and administration of the compounds of the instant application may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Company, Easton, PA, latest edition.

Example 1: Appearance of a2-HS in stroke We first sought to assess the appearance of a2-HS in ischemic areas of brain, relative to its appearance in the normally perfused brain. A widely used model for human stroke is achieved by permanently occluding the middle cerebral artery in rats, and subsequently measuring the volume of the resultant cortical brain damage after 24 hours. The resulting infarction in this model is highly reproducible and provides a reasonable model of the typical brain damage that occurs in the setting of human focal cerebral ischemia or stroke. Moreover, the clinical efficacy of experimental therapeutics can be conveniently assessed in this model because quantitative volumetric estimates of brain damage can be made, allowing direct comparison between various cerebroprotective strategies and agents. Moreover, therapeutic WO 00/60943 PCT/US00/10002 modalities identified to mitigate against ischemic tissue damage in this model can be directly extended to other tissue ischemias, such as heart attack.

Permanent focal cerebral ischemia: Rats were subjected to a microsurgical right frontal craniotomy and permanent occlusion of the middle cerebral artery as described previously in detail (Zimmerman, et al., Proc Natl Acad Sci USA, 92:3744-3748, 1995; Cocroft, et al., Stroke, 27:1393-1398, 1996). Briefly, the ipsilateral common carotid artery was ligated and divided, the middle cerebral artery was coagulated and divided distally to the lenticulostriate branch, and the contralateral common carotid artery temporarily occluded (one hour). The onset of ischemia was defined as the time the middle cerebral artery was cut. Twenty-four hours later, the animals were euthanized, fresh brain sections were prepared (1 mm), immersed in a solution of 2,3,5-triphenyl -2H-tetrazolium chloride in 154 mM NaCI) for 30 min at 37 and total cerebral infarct volume was estimated by computerized quantitative planimetry. The volume of stroke damage in this model is relatively modest and limited to the cortex such that, behavioral deficits are not readily observable and the animals exhibit normal ambulation, feeding and grooming without seizure or paralysis.

To determine the appearance of a2-HS in normal and in stroke-damaged brain areas, rats were subject to focal cerebral ischemia as above, and brains were collected at various time points after the onset of ischemia. Brain sections corresponding to damaged and normal tissue were isolated and solubilized proteins were analyzed by Western blot using anti-a2-HS antibodies according to well-known protocols. As shown in Figure 1, immunoreactive (2-HS protein was present in normal brain tissue, and the amount of immunoreactive c2-HS protein increased for up to 48 hours after ischemic insult, with above normal levels persisting for at least 96 hours.

Example 2: Effects of a2-HS treatment on stroke damage Since ischemic brain is known to contain elevated levels ofpolyamines, and since cc2- HS is known to enhance the anti-inflammatory properties of spermine (USSN 08/932,871, incorporated herein in its entirety), we sought to assess the therapeutic benefit of a2-HS treatment in stroke. As shown in Figure 2, rats subjected to experimental focal cerebral ischemic challenge suffered smaller infarcts if they were treated intravenously with a2-HS at mg/kg than if they were untreated. Under these conditions, treatment with asialofetuin exacerbated stroke damage. This brain damage-ameliorating effect of a2-HS treatment is predictive of a therapeutic benefit in response to a2-HS treatment in the context of human stroke or cerebrovascular accident, and in other conditions of ischemic tissue damage heart attack).

Example 3: Inhibition of brain damage by a2-HS treatment is dose-dependent To determine the dose-dependency of a2-HS treatment to prevent tissue damage in the setting of ischemia, we examined various doses of a2-HS for efficacy in a stroke model WO 00/60943 PCT/US00/10002 involving focal cerebral ischemia. Stroke was induced and animals treated as described above, and a2-HS was administered 15 minutes after the onset of ischemia at a dose of 5, 25, 50 or 500 mg/kg. As shown in Figure 3, a2-HS improved the outcome of stroke at all doses tested, with doses of 50 or 500 mg/kg providing the most significant benefits. This example shows how therapeutically effective dose ranges are initially estimated. Final determination of effective dosages for specific clinical conditions such as stroke or heart attack is readily accomplished by those skilled in the medical and pharmaceutical arts by reference to these preclinical results in experimentally induced tissue ischemia.

Example 4: Timing of a2-HS treatment for stroke We next sought to establish the therapeutic window during which a2-HS treatment would be most beneficial as a treatment for ischemic damage. Rats were subjected to unilateral permanent focal cerebral ischemia and treated intravenously with a2-HS at 50 mg/kg at different latencies after onset of ischemia: 15 minutes, 30 minutes or 60 minutes after permanent occlusion of the middle cerebral artery. As shown in Figure 4, treatment with a2- HS within 30 minutes following the onset of ischemia was of the greatest benefit in minimizing stroke damage.

In that clinical treatment for stroke (or heart attack or other ischemic conditions) cannot be initiated until after the underlying insult occurs, it is important that a2-HS treatment is of benefit when initiated after the onset of ischemia; this requisite therapeutic window has not always been apparent for other candidate therapeutics against ischemic damage, as some must be initiated before or simultaneously with the onset of ischemia in comparable experimental models. This example shows how the temporal range of therapeutically effective treatment latency is initially estimated. Final determination of the "therapeutic window" during which a2-HS monotherapy or adjunctive therapy is effective for specific clinical conditions such as stroke or heart attack is readily accomplished by those skilled in the medical and pharmaceutical arts, with reference to these pre-clinical results in experimentally induced tissue ischemia.

Example 5 Measurement of TNF levels in the stroke model Brain tissues were fixed by sequential intracardiac perfusion with 0.05 M phosphate buffer saline (PBS, pH 7.4) containing 0.1% sodium nitrate and heparin, followed by infusion with 2% paraformaldehyde in 0.1M PBS (pH 7.4) containing 5% sucrose (for TNF staining), or 4% paraformaldehyde in 0.1M PB (pH 7.4, for a2-HS staining). Following perfusion, the brains were removed and stored in the same fixative solution for 15 min at 4 °C (staining for TNF) and overnight (staining for a2-HS) and then transferred to a solution of 20% sucrose in PBS overnight, at 4 The frozen sections of the samples were cut para-sagittally and coronally in alternate series of 20 |m thick with the cryostat. The sections were attached to gelatin-coated slides, air dried, and stored at -20 °C until use. After quenching endogenous WO 00/60943 PCT/US00/10002 peroxidase activity with 0.3% H 2 0 2 solution, sections were incubated in a 1:20 dilution of either normal horse or goat serum (ACCURATE) for 1 hour. Using an avidin-biotinylated horseradish peroxidase system (DBS), the following were used as primary antibodies for overnight incubation at 4 °C in a humidified chamber. ED1, which is a mouse monoclonal IgG antibody (Accurate Chemical Scientific Corp.), was used at a dilution of 1:2000; a polyclonal rabbit antimouse TNF-a, (RDI), was used at dilution of 1:100. With intervening washes in PBST, the following steps were performed: biotinylated horse anti-mouse adsorbed (for ED 1) and biotinylated goat anti-rabbit (for TNF-a antisera) antibodies (1:150 dilution in PBST) for 1 hour at 25 avidin-biotinylated horseradish peroxidase complex (DBS) in PBST, pH 7.2, for 1 hour at 25 and a 0.1 M solution of 3,3'-diaminobenzidine (DAB) in 0.05 M Tris-HCl buffer, pH 7.4, for 10 min, to which bad been added 0.75 ml of 3% HO 2 (for ED1), or 3% 3-amino-9-ethylcarbazole (AEC) in N,N-dimethylformamide (for TNF-a and fetuin), for 15 min.

Administration of a2-HS suppressed TNF production. TNF was not detected in brain sections of normal brain by immunostaining with anti-TNF antibodies. After the onset of cerebral ischemia in the present model, however, TNF immunoreactivity was significantly increased in the ischemic core and penumbra area, but remained undetectable in the contralateral hemisphere. Most TNF-a positive cells in the ipsilateral cortical neuronal layer showed typical morphology of neuronal cells; whereas some TNF-positive cells in the surrounding ventricles, the out most layer of the core, and the corpus collosum in the ischemic hemisphere revealed morphology of microglia cells. Most TNF-positive neronal cells were located in the focal ischemic region (as opposed to the perifocal ischemic area). Treatment of rat with a2-HS at 50 mg/kg 15 minutes after onset of cerebral ischemia significantly decreased TNF immunoreactivity both in the ischemic core and penumbra regions. However, treatment of animals with asialofetuin did not affect the TNF immunoreactivity as compared to controls that treated with control (vehicle) alone, indicating that a2-HS protected against cerebral ischemic injury. Without being bound by theory the mechanism of the therapeutic activity is thought to be at least partially through down-regulation of TNF expression during ischemia.

WO 00/60943 PCT/US00/10002 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Tracey, Kevin et al.

(ii) TITLE OF INVENTION: Prevention of Brain Damage in Stroke (iii) NUMBER OF SEQUENCES: 6 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: DAVIS WRIGHT TREMAINE LLP STREET: 2600 Century Square, 1101 Fourth Avenue CITY: Seattle STATE: Washington COUNTRY: USA ZIP: 98101-1688 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: Pentium PC OPERATING SYSTEM: Windows SOFTWARE: Word (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: to be assigned FILING DATE: 13 April 2000

CLASSIFICATION:

(vii) ATTORNEY/AGENT INFORMATION: NAME: Oster, Jeffrey B.

REGISTRATION NUMBER: 32,585 REFERENCE/DOCKET NUMBER: 0604WO (viii) TELECOMMUNICATION INFORMATION TELEPHONE: (206) 628-7711 TELEFAX: (206) 628-7699 INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 367 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no FRAGMENT TYPE: (vi) ORIGINAL SOURCE:a2-HS-glycoprotein precursor ORGANISM: human (ix) SEQUENCE DESCRIPTION: SEQ ID Met Lys Ser Leu Val Leu Leu Leu Cys His Ser Ala Pro His Gly Pro Gly Leu Leu 10 Ile NO: 1: Ala Gin Tyr Arg Leu Trp Gly Cys Gin Pro Asn Cys Asp Tyr Ile Asp Asp Pro Asn Gin Asn Glu Thr Glu Glu Ala Leu Val Ala Ile Leu Pro Trp Val Gly 55 Gin Tyr Lys His Thr Leu Asn Gin Ile Asp Glu Glu Lys Val Trp Gin Pro Ser Glu Leu Phe Glu Ile Asp Thr Leu Ser Glu Thr Thr Cys His 90 Lys Val Leu Asp Glu His Ala Val Ala Arg Cys Val Arg Gin 100 Phe Leu 105 Leu Pro Thr Pro Val Glu Gly 110 Ser Val Val Val Arg Lys Asp Cys Asp 115 Tyr Ala Lys Gin Leu Leu Lys 120 Pro Asp Gly Lys Phe 125 Asp Cys Asp Ser 130 Val Cys Ser 135 Leu Asp Ser Ala Glu 140 Asn Gln Asp Cys Pro Leu Ala Pro Leu Asp Thr Arg Val WO 00/60943 145 Val His Ala Gly Ser Asn Leu Pro Pro 195 Val Ala Lys 210 Lys Gin Tyr 225 Ala Glu Val Ser Gin Pro Val Asp Pro 275 Pro Pro Ala 290 Pro Gly His 305 Met Gly Val Arg Lys Thr Pro Val Val 355 Ala Phe 180 Ser Glu Gly Ala Gin 260 Asp Gly Gin Val Arg 340 Pro Lys 165 Gin Thr Ala Phe Val 245 Pro Ala Ser Leu Ser 325 Thr Pro 150 Ala Leu Tyr Thr Cys 230 Thr Glu Pro Pro His 310 Leu Val Cys Leu Glu Glu 200 Ala Ala Thr Ala Ser 280 Asp Ala Ser Gin Gly 360 Ala 170 Ser Thr Lys Leu Phe 250 Glu Pro His Tyr Ser 330 Ser Ile Asn Ala Ser Asn 220 Glu Thr Val Gly Leu 300 Leu Glu Gly His Ala Gin Gly 205 Leu Lys Gin Pro Ala 285 Leu Arg Val Ala Phe 365 Gin Leu 190 Thr Leu Leu Pro Thr 270 Pro Ala His Ser Ala 350 Lys PCT/US00/10002 160 Asn Asn 175 Val Pro Asp Cys Ala Glu Gly Gly 240 Val Thr 255 Pro Val Gly Leu Ala Pro Thr Phe 320 His Pro 335 Ala Gly Val INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 18 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no FRAGMENT TYPE: N-terminal fragment (vi) ORIGINAL SOURCE: ORGANISM: uncertain (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Lys Ser Leu Val Leu Leu Leu Cys Leu Ala Gin Leu Trp Gly Cys 10 His Ser INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 282 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no FRAGMENT TYPE: fragment residues 19-300 of :a2-HS-glycoprotein precursor (vi) ORIGINAL SOURCE: ORGANISM: uncertain (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 3: WO 00/60943 Ala Pro His Pro Glu Thr Asn Leu Pro Lys Val Trp Asp Thr Leu 65 Arg Cys Ser Asp Phe Gin Lys Cys Asp 115 Gin Asp Cys 130 Ala Ala Lys 145 Asn Phe Gin Pro Ser Thr Lys Glu Ala 195 Tyr Gly Phe 210 Val Ala Val 225 Pro Gin Pro Pro Asp Ala Ala Gly Ser 275 Gly Glu Trp Pro Glu Val Leu 100 Ser Pro Ala Leu Tyr 180 Thr Cys Thr Glu Pro 260 Pro Pro Glu Gly Gin Thr Arg Leu Ser Leu Ala Glu 165 Val Glu Lys Cys Gly 245 Pro Pro Gly Ala Tyr Gin Thr 70 Gin Lys Pro Leu Leu 150 Glu Glu Ala Ala Thr 230 Ala Ser Asp Leu Ala Lys Pro Cys Leu Leu Asp Ala 135 Ala Ile Phe Ala Thr 215 Val Asn Pro Ser Ile Leu His 40 Ser His Lys Asp Ser 120 Pro Ala Ser Thr Lys 200 Leu Phe Glu Pro His 280 Tyr Va 1 25 Thr Gly Val Glu Gly 105 Ala Leu Phe Arg Val 185 Cys Ser Gin Ala Leu 265 Val Arg 10 Ala Leu Glu Leu His 90 Lys Glu Asn Asn Ala 170 Ser Asn Glu Thr Val 250 Gly Leu Gin Ile Asn Leu Asp 75 Ala Phe Asp Asp Ala 155 Gin Gly Leu Lys Gin 235 Pro Ala Asn Tyr Ile Glu Thr Glu Val Arg 125 Arg Asn Val Asp Ala 205 Gly Val Pro Gly Cys Ile Asp Ile Pro Gly Val 110 Lys Val Asn Pro Cys 190 Glu Gly Thr Val Leu 270 PCT/US00/10002 Asp Asp Asn Gin Glu Val Glu Ile Val Ala Asp Cys Tyr Ala Val Cys Val His Gly Ser 160 Leu Pro 175 Val Ala Lys Gin Ala Glu Ser Gln 240 Val Asp 255 Pro Pro INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 27 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no FRAGMENT TYPE: fragment residues 341-367 of :a2-HS-glycoprotein precursor (vi) ORIGINAL SOURCE: ORGANISM: uncertain (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Thr Val Val Gin Pro Ser Val Gly Ala Ala Ala Gly Pro Val Val Pro 10 Pro Cys Pro Gly Arg Ile Arg His Phe Lys Val 20 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 40 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear WO 00/60943 PCT/US00/10002 (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: no (iv) ANTI-SENSE:. no FRAGMENT TYPE: fragment residues 301-340 of :a2-HS-glycoprotein precursor (vi) ORIGINAL SOURCE: ORGANISM: uncertain (ix) SEQUENCE DESCRIPTION: SEQ ID NO: Leu Ala Ala Pro Pro Gly His Gin Leu His Arg Ala His Tyr Asp Leu 5 10 Arg His Thr Phe Met Gly Val Val Ser Leu Gly Ser Pro Ser Gly Glu 25 Val Ser His Pro Arg Lys Thr Arg INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 341 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no FRAGMENT TYPE: fragment residues 19-359 of bovine fetuin (vi) ORIGINAL SOURCE: ORGANISM: uncertain (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Ile Pro Leu Asp Pro Val Ala Gly Tyr Lys Glu Pro Ala Cys Asp Asp 10 Pro Asp Thr Glu Gin Ala Ala Leu Ala Ala Val Asp Tyr Ile Asn Lys 25 His Leu Pro Arg Gly Tyr Lys His Tyr Leu Asn Gin Ile Asp Ser Val 35 40 Lys Val Trp Pro Arg Arg Pro Thr Gly Glu Val Tyr Asp Ile Glu Ile 55 Asp Thr Leu Glu Thr Thr Cys His Val Leu Asp Pro Thr Pro Leu Ala 70 75 Asn Cys Ser Val Arg Gin Gin Thr Gin His Ala Val Glu Gly Asp Cys 90 Asp Ile His Val Leu Lys Gin Asp Gly Gin Phe Ser Val Leu Phe Thr 100 105 110 Lys Cys Asp Ser Ser Pro Asp Ser Ala Glu Asp Val Arg Lys Leu Cys 115 120 125 Pro Asp Cys Pro Leu Leu Ala Pro Leu Asn Asp Ser Arg Val Val His 130 135 140 Ala Val Glu Val Ala Leu Ala Thr Phe Asn Ala Glu Ser Asn Gly Ser 145 150 155 160 Tyr Leu Gin Leu Val Glu Ile Ser Arg Ala Gin Phe Val Pro Leu Pro 165 170 175 Val Ser Val Ser Val Glu Phe Ala Val Ala Ala Thr Asp Cys Ile Ala 180 185 190 Lys Glu Val Val Asp Pro Thr Lys Cys Asn Leu Leu Ala Glu Lys Gin 195 200 205 Tyr Gly Phe Cys Lys Gly Ser Val Ile Gin Lys Ala Leu Gly Gly Glu 210 215 220 Asp Val Arg Val Thr Cys Thr Leu Phe Gin Thr Gin Pro Val Ile Pro 225 230 235 240 Gin Pro Gin Pro Asp Gly Ala Glu Ala Glu Ala Pro Ser Ala Val Pro 245 250 255 Asp Ala Ala Gly Pro Thr Pro Ser Ala Ala Gly Pro Pro Val Ala Ser 12 WO 00/60943 PCTIUSOO/10002 Val Val Val 275 Ala His Tyr 290 Ser Ser Ser 305 Gin Pro Ser Arg Tyr Phe 260 Gly Asp Gly Ile Lys 340 265 270 Pro Ser Val Vai Ala Val Pro Leu Pro Leu His Arg 280 285 Leu Arg His Thr Phe Ser Giy Val Ala Ser Val Glu 295 300 Giu Ala Phe His Val Gly Lys Thr Pro Ile Val Gly 310 315 320 Pro Giy Gly Pro Val Arg Leu Cys Pro Gly Arg Ile 325 330 335 Ile

Claims (11)

1. A method of treating tissue ischemia in a subject, comprising administering an effective amount of a human a2-HS glycoprotein.

2. The method of claim 1 wherein the human a2-HS glycoprotein in selected from the group consisting of human a2-HS glycoproteins comprising a primary polypeptide sequence according to SEQ ID NO. 1 through, SEQ ID NO. 5, shorter fragments thereof, and mixtures thereof.

3. The method of claim 1 wherein the tissue to be treated for ischemia is brain or heart.

4. A method of inhibiting tissue damage caused by ischemia, comprising administering to a subject an effective amount of a human a2-HS glycoprotein. The method of claim 4 wherein the human a2-HS glycoprotein is selected from the group consisting of human a2-HS glycoproteins comprising a primary polypeptide sequence according to SEQ ID NO. 1 through SEQ ID NO.

5, shorter fragments thereof, and mixtures thereof.

6. The method of claim 4 wherein the tissue damage is manifest as stroke or as myocardial infarction.

7. A pharmaceutical composition for treating tissue ischemia or preventing ischemic tissue damage comprising a human a2-HS glycoprotein and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition of claim 7 wherein the human a2-HS glycoprotein is selected from the group consisting of human a2-HS glycoproteins comprising a primary polypeptide sequence according to SEQ ID NO. 1 through SEQ ID NO. 5, shorter fragments thereof, and mixtures thereof.

9. A method for treating tissue ischemia or preventing ischemic tissue damage comprising administering to a subject a combination of human a2-HS glycoprotein and at least one additional treatment to mitigate ischemic tissue damage.

The method of claim 9 wherein the human a2-HS glycoprotein is selected from the group consisting of human a2-HS glycoproteins comprising a primary polypeptide sequence according to SEQ ID NO. 1 through SEQ ID NO. 5, shorter fragments thereof, and mixtures thereof.

11. The method of claim 9 wherein the additional treatment to mitigate ischemic tissue damage is an enzymatic clot-dissolving agent.