EP1191845A1

EP1191845A1 - Prevention of brain damage in stroke

Info

Publication number: EP1191845A1
Application number: EP00923342A
Authority: EP
Inventors: Kevin J. Tracey; Haichao Wang
Original assignee: North Shore Long Island Jewish Research Institute
Current assignee: North Shore Long Island Jewish Research Institute
Priority date: 1999-04-13
Filing date: 2000-04-13
Publication date: 2002-04-03
Also published as: WO2000060943A1; AU4348500A; JP2002541162A; EP1191845A4; CA2368937A1; AU766910B2

Abstract

The present invention provides methods and pharmaceutical compositions of the human glycoprotein fetuin, or α2-HS glycoprotein, or fragments thereof to mitigate tissue damage associated with ischemia, particularly in stroke or in myocardial infarction.

Description

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 of thrombolytic 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, N Engl J Med 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 (α2-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, α2-HS has been grouped with the fetuins, a family of proteins that occur in fetal plasma in high concentrations. Native α2-HS undergoes a series of posttranslational 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. 2-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 to the A chain. Human fetuin, or α2-HS, is generally considered to arise from a single mRNA transcript encoding a 367 amino acid peptide known as the "alpha-2-HS-glycoρrotein precursor" (SEQ ID NO. 1). Amino acids 1-18 (SEQ ID NO. 2) comprise a signal sequence domain. Amino acids 19-300 comprise the α2-HS-glycoprotein A chain domain (SEQ ID NO. 3). Amino acids 341-367 comprise the α2-HS-glycoprotein B chain domain (SEQ ID NO. 4). 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 α2-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. 6). Primary amino acid sequence and the position of cysteine residues are well conserved across species, e.g., 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 (α2-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 α2-HS transcript levels in cultured hepatocytes (Akhoundi et al., J. Biol Chem. 268: 15925, 1994). α2-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 α2-HS influences the mineral phase of bone. The α2-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 (ct2-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 α2-HS polypeptide is usually a Ν-acetylglucosamine residue. The terminal sugar residue is usually a sialic acid, in particular a Ν-acetylneuraminic acid (ΝAΝA) residue, which bears a net negative charge. If one removes the terminal sialic acid residue from α2-HS by neuraminidase treatment, the resulting glycoprotein is an asialofetuin. Fetuin (α2-HS) is also a carrier protein for growth factors and cytokines. The synthesis of human α2-HS -glycoprotein is down-regulated by cytokines (hIL-lβ, hIL-6) (Lebreton et al., J. Clin. Invest. 64:1118-1129, 1979). Human fetuin (α2-HS) levels are decreased (25-50%) in trauma patients (van Oss et al, J. Trauma 15:451, 1975). α2-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 "α2-HS-glycoprotein" or "α2-Z-globulin" or "human fetuin" or "human fetuin glycoprotein," and in broader context to any of the fetuin family of proteins, with members occurring in various species and closely related in sequence to bovine fetuin and human ct2-HS. Two common alleles are known for α2-HS: one has threonine at position 248 and 256, the other has methionine at 248 and serine at 256. Use of "fetuin" or "α2-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 α2-HS. The present invention identifies for the first time the cellular and tissue protective effects of administering α2-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 α2-HS glycoprotein. Preferably, the α2-HS glycoprotein is a human α2-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 α2-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 α2-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 α2-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 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 α2-HS in infarct regions of brain cortex from rats subjected to experimental focal cerebral ischemia. Figure 1 A is a Western blot showing α2-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 IB 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 α2-HS treatment on infarct size in experimental focal cerebral ischemia. α2-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 of α2-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 α2-HS, 25 mg/kg α2-HS, 50 mg/kg α2-HS, 500 mg/kg α2-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 α2-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. α2-HS was administered at a dose of 50 mg /kg intravenously 60 min (cc2-HS 1), 30 min (α2-HS 2) or 15 min (α2-HS 3) after induction of experimental focal cerebral ischemia in rats. Leftmost bar is control (no α2-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, α2- 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 α2-HS counterpart (α2-HS-glycoprotein), no role for α2-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 α2-HS to potentiate the anti- inflammatory activity of certain low molecular weight compounds and metabolites, we tested for the occurrence and activity of α2-HS in a predictive animal model of human stroke, i.e., in experimentally induced focal cerebral ischemia in rats. We discovered that α2-HS occurs in increasing amounts in the areas of brain damage following permanent focal cerebral ischemia, and that administration of α2-HS alone, even after the onset of ischemia, dose-dependently decreased the volume of total brain damage in stroke. Asialofetuin (α2-HS glycoprotein treated to remove sialic acid residues) was ineffective or exacerbative in this regard.

The present invention provides novel methods (α2-HS administration as adjunctive or monotherapy) and pharmaceutical compositions (comprising α2-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 α2-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 α2-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, α2-HS may be co-administered with spermine or otherwise in accordance with the teachings of USSN: 08/780,31 1 (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 α2-HS in stroke

We first sought to assess the appearance of α2-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 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., Pro c 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 (2% in 154 mM NaCl) for 30 min at 37 °C, 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 α2-ΗS 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 con-esponding to damaged and normal tissue were isolated and solubilized proteins were analyzed by Western blot using anti-α2-HS antibodies according to well-known protocols. As shown in Figure 1 , imnumoreactive α2-HS protein was present in normal brain tissue, and the amount of immunoreactive α2-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 α2-HS treatment on stroke damage

Since ischemic brain is known to contain elevated levels of polyamines, and since α2- 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 α2-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 α2-HS at 50 mg/kg than if they were untreated. Under these conditions, treatment with asialofetuin exacerbated stroke damage. This brain damage-ameliorating effect of α2-HS treatment is predictive of a therapeutic benefit in response to α2-HS treatment in the context of human stroke or cerebrovascular accident, and in other conditions of ischemic tissue damage (e.g., heart attack).

Example 3: Inhibition of brain damage by α2-HS treatment is dose-dependent

To determine the dose-dependency of α2-HS treatment to prevent tissue damage in the setting of ischemia, we examined various doses of α2-HS for efficacy in a stroke model involving focal cerebral ischemia. Stroke was induced and animals treated as described above, and α2-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, α2-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 pre- clinical results in experimentally induced tissue ischemia.

Example 4: Timing of α2-HS treatment for stroke

We next sought to establish the therapeutic window during which α2-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 α2-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 α2- 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 α2-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 α2-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 α2-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 α2-HS) and then transferred to a solution of 20% sucrose in PBS overnight, at 4 °C. The frozen sections of the samples were cut para-sagittally and coronally in alternate series of 20 μm thick with the cryostat. The sections were attached to gelatin-coated slides, air dried, and stored at -20 °C until use. After quenching endogenous peroxidase activity with 0.3% H₂O 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. EDI , which is a mouse monoclonal IgG antibody (Accurate Chemical & Scientific Corp.), was used at a dilution of 1 :2000; a polyclonal rabbit antimouse TNF- , (RDI), was used at dilution of 1 TOO. With intervening washes in PBST, the following steps were performed: biotinylated horse anti-mouse adsorbed (for EDI) and biotinylated goat anti-rabbit (for TNF-α antisera) antibodies (1 :150 dilution in PBST) for 1 hour at 25 °C; avidin-biotinylated horseradish peroxidase complex (DBS) in PBST, pH 7.2, for 1 hour at 25 °C; 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% HO₂ (for EDI), or 3% 3-amino-9-ethylcarbazole (AEC) in N,N-dimethylformamide (for TNF-α and fetuin), for 15 min.

Administration of α2-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-α 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 α2-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 α2-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.

SEQUENCE LISTING (1) GENERAL INFORMATION:

(i) APPLICANT: Tracey, Kevin et al .

(ii) TITLE OF INVENTION: Prevention of Brain Damage in Stroke (iϋ) NUMBER OF SEQUENCES: 6

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: DAVIS WRIGHT TREMAINE LLP

(B) STREET: 2600 Century Square, 1101 Fourth Avenue

(C) CITY: Seattle (D) STATE: Washington

(E) COUNTRY: USA

(F) ZIP: 98101-1688 (v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: Pentium PC

(C) OPERATING SYSTEM: Windows 95

(D) SOFTWARE: Word

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: to be assigned (B) FILING DATE: 13 April 2000

(C) CLASSIFICATION: (vii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Oster, Jeffrey B.

(B) REGISTRATION NUMBER: 32,585 (C) REFERENCE/DOCKET NUMBER: 0604WO

(viii) TELECOMMUNICATION INFORMATION

(A) TELEPHONE: (206) 628-7711

(B) TELEFAX: (206) 628-7699 (2) INFORMATION FOR SEQ ID NO : 1:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 367 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS : single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE : α2 -HS -glycoprotein precursor

(A) ORGANISM: human (ix) SEQUENCE DESCRIPTION: SEQ ID NO : 1: Met Lys Ser Leu Val Leu Leu Leu Cys Leu Ala Gin Leu Trp Gly Cys

5 10 15 His Ser Ala Pro His Gly Pro Gly Leu He Tyr Arg Gin Pro Asn Cys

20 25 30

Asp Asp Pro Glu Thr Glu Glu Ala Ala Leu Val Ala He Asp Tyr He

35 40 45

Asn Gin Asn Leu Pro Trp Gly Tyr Lys His Thr Leu Asn Gin He Asp 50 55 60

Glu Val Lys Val Trp Pro Gin Gin Pro Ser Gly Glu Leu Phe Glu He

65 70 75 80

Glu He Asp Thr Leu Glu Thr Thr Cys His Val Leu Asp Pro Thr Pro

85 90 95 Val Ala Arg Cys Ser Val Arg Gin Leu Lys Glu His Ala Val Glu Gly

100 105 110

Asp Cys Asp Phe Gin Leu Leu Lys Leu Asp Gly Lys Phe Ser Val Val

115 120 125

Tyr Ala Lys Cys Asp Ser Ser Pro Asp Ser Ala Glu Asp Val Arg Lys 130 135 140

Val Cys Gin Asp Cys Pro Leu Leu Ala Pro Leu Asn Asp Thr Arg Val 145 150 155 160

Val His Ala Ala Lys Ala Ala Leu Ala Ala Phe Asn Ala Gin Asn Asn

165 170 175

Gly Ser Asn Phe Gin Leu Glu Glu He Ser Arg Ala Gin Leu Val Pro 180 185 190

Leu Pro Pro Ser Thr Tyr Val Glu Phe Thr Val Ser Gly Thr Asp Cys

195 200 205

Val Ala Lys Glu Ala Thr Glu Ala Ala Lys Cys Asn Leu Leu Ala Glu

210 215 220 Lys Gin Tyr Gly Phe Cys Lys Ala Thr Leu Ser Glu Lys Leu Gly Gly

225 230 235 240

Ala Glu Val Ala Val Thr Cys Thr Val Phe Gin Thr Gin Pro Val Thr

245 250 255

Ser Gin Pro Gin Pro Glu Gly Ala Asn Glu Ala Val Pro Thr Pro Val 260 265 270

Val Asp Pro Asp Ala Pro Pro Ser Pro Pro Leu Gly Ala Pro Gly Leu

275 280 285

Pro Pro Ala Gly Ser Pro Pro Asp Ser His Val Leu Leu Ala Ala Pro

290 295 300 Pro Gly His Gin Leu His Arg Ala His Tyr Asp Leu Arg His Thr Phe

305 310 315 320

Met Gly Val Val Ser Leu Gly Ser Pro Ser Gly Glu Val Ser His Pro

325 330 335

Arg Lys Thr Arg Thr Val Val Gin Pro Ser Val Gly Ala Ala Ala Gly 340 345 350

Pro Val Val Pro Pro Cys Pro Gly Arg He Arg His Phe Lys Val 355 360 365

(2) INFORMATION FOR SEQ ID NO : 2: (1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 amino acids

(B) TYPE: amino acid

(C) STRAIN.DEDNESS : single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: no (iv) ANTI- SENSE: no

(v) FRAGMENT TYPE: N-terminal fragment (vi) ORIGINAL SOURCE: (A) 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

5 10 15

His Ser

(2) INFORMATION FOR SEQ ID NO : 3:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 282 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: no (iv) ANTI -SENSE: no

(v) FRAGMENT TYPE: fragment residues 19-300 of :α2-HS -glycoprotein precursor

(vi) ORIGINAL SOURCE:

(A) ORGANISM: uncertain (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Ala Pro His Gly Pro Gly Leu He Tyr Arg Gin Pro Asn Cys Asp Asp

5 10 15

Pro Glu Thr Glu Glu Ala Ala Leu Val Ala He Asp Tyr He Asn Gin 20 25 30 Asn Leu Pro Trp Gly Tyr Lys His Thr Leu Asn Gin He Asp Glu Val 35 40 45

Lys Val Trp Pro Gin Gin Pro Ser Gly Glu Leu Phe Glu He Glu He

50 55 60

Asp Thr Leu Glu Thr Thr Cys His Val Leu Asp Pro Thr Pro Val Ala 65 70 75 80

Arg Cys Ser Val Arg Gin Leu Lys Glu His Ala Val Glu Gly Asp Cys

85 90 95

Asp Phe Gin Leu Leu Lys Leu Asp Gly Lys Phe Ser Val Val Tyr Ala 100 105 110 Lys Cys Asp Ser Ser Pro Asp Ser Ala Glu Asp Val Arg Lys Val Cys 115 120 125

Gin Asp Cys Pro Leu Leu Ala Pro Leu Asn Asp Thr Arg Val Val His

130 135 140

Ala Ala Lys Ala Ala Leu Ala Ala Phe Asn Ala Gin Asn Asn Gly Ser 145 150 155 160

Asn Phe Gin Leu Glu Glu He Ser Arg Ala Gin Leu Val Pro Leu Pro

165 170 175

Pro Ser Thr Tyr Val Glu Phe Thr Val Ser Gly Thr Asp Cys Val Ala 180 185 190 Lys Glu Ala Thr Glu Ala Ala Lys Cys Asn Leu Leu Ala Glu Lys Gin 195 200 205

Tyr Gly Phe Cys Lys Ala Thr Leu Ser Glu Lys Leu Gly Gly Ala Glu

210 215 220

Val Ala Val Thr Cys Thr Val Phe Gin Thr Gin Pro Val Thr Ser Gin 225 230 235 240

Pro Gin Pro Glu Gly Ala Asn Glu Ala Val Pro Thr Pro Val Val Asp

245 250 255

Pro Asp Ala Pro Pro Ser Pro Pro Leu Gly Ala Pro Gly Leu Pro Pro 260 265 270 Ala Gly Ser Pro Pro Asp Ser His Val Leu 275 280

(2) INFORMATION FOR SEQ ID NO : 4:

(1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: no

(iv) ANTI-SENSE: no

(v) FRAGMENT TYPE: fragment residues 341-367 of :0C2 -HS-glycoprotein precursor

(vi) ORIGINAL SOURCE: (A) 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

5 10 15

Pro Cys Pro Gly Arg He Arg His Phe Lys Val 20 25

(2) INFORMATION FOR SEQ ID NO : 5:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: no (iv) ANTI-SENSE:- no

(v) FRAGMENT TYPE: fragment residues 301-340 of :α2 -HS-glycoprotein precursor

(vi) ORIGINAL SOURCE:

(A) ORGANISM: uncertain (ix) SEQUENCE DESCRIPTION: SEQ ID NO : 5: Leu Ala Ala Pro Pro Gly His Gin Leu His Arg Ala His Tyr Asp Leu 5 10 15

Arg His Thr Phe Met Gly Val Val Ser Leu Gly Ser Pro Ser Gly Glu

20 25 30

Val Ser His Pro Arg Lys Thr Arg 35 40

(2) INFORMATION FOR SEQ ID NO : 6:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 341 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(v) FRAGMENT TYPE: fragment residues 19-359 of bovine fetuin (vi) ORIGINAL SOURCE:

(A) ORGANISM: uncertain (ix) SEQUENCE DESCRIPTION: SEQ ID NO : 6: He Pro Leu Asp Pro Val Ala Gly Tyr Lys Glu Pro Ala Cys Asp Asp

5 10 15

Pro Asp Thr Glu Gin Ala Ala Leu Ala Ala Val Asp Tyr He Asn Lys

20 25 30

His Leu Pro Arg Gly Tyr Lys His Tyr Leu Asn Gin He Asp Ser Val 35 40 45

Lys Val Trp Pro Arg Arg Pro Thr Gly Glu Val Tyr Asp He Glu He

50 55 60

Asp Thr Leu Glu Thr Thr Cys His Val Leu Asp Pro Thr Pro Leu Ala 65 70 75 80 Asn Cys Ser Val Arg Gin Gin Thr Gin His Ala Val Glu Gly Asp Cys

85 90 95

Asp He 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 He 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 He 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 He Gin Lys Ala Leu Gly Gly Glu

210 215 220

Asp Val Arg Val Thr Cys Thr Leu Phe Gin Thr Gin Pro Val He 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 260 265 270

Val Val Val Gly Pro Ser Val Val Ala Val Pro Leu Pro Leu His Arg

275 280 285

Ala His Tyr Asp Leu Arg His Thr Phe Ser Gly Val Ala Ser Val Glu 290 295 300

Ser Ser Ser Gly Glu Ala Phe His Val Gly Lys Thr Pro He Val Gly 305 310 315 320

Gin Pro Ser He Pro Gly Gly Pro Val Arg Leu Cys Pro Gly Arg He 325 330 335 Arg Tyr Phe Lys He 340

Claims

We claim:

1. A method of treating tissue ischemia in a subject, comprising administering an effective amount of a human α2-HS glycoprotein.

2. The method of claim 1 wherein the human α2-HS glycoprotein in selected from the group consisting of human α2-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 α2-HS glycoprotein.

5. The method of claim 4 wherein the human α2-HS glycoprotein is selected from the group consisting of human α2-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 α2-HS glycoprotein and a phannaceutically acceptable carrier.

8. The pharmaceutical composition of claim 7 wherein the human α2-HS glycoprotein is selected from the group consisting of human α2-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 α2-HS glycoprotein and at least one additional treatment to mitigate ischemic tissue damage.

10. The method of claim 9 wherein the human α2-HS glycoprotein is selected from the group consisting of human α2-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.