US20060045853A1 - Cross-beta structure comprising amyloid-binding proteins and methods for detection of the cross-beta structure, for modulating cross-beta structures fibril formation and for modulating cross-beta structure-mediated toxicity - Google Patents

Cross-beta structure comprising amyloid-binding proteins and methods for detection of the cross-beta structure, for modulating cross-beta structures fibril formation and for modulating cross-beta structure-mediated toxicity Download PDF

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US20060045853A1
US20060045853A1 US11/033,105 US3310505A US2006045853A1 US 20060045853 A1 US20060045853 A1 US 20060045853A1 US 3310505 A US3310505 A US 3310505A US 2006045853 A1 US2006045853 A1 US 2006045853A1
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cross
tpa
binding
albumin
amyloid
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Louise Kroon-Batenburg
Barend Bouma
Onno Kranenburg
Martijn Frans Ben Gerard Gebbink
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Crossbeta Biosciences BV
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Publication of US20060045853A1 publication Critical patent/US20060045853A1/en
Assigned to UNIVERSITAIR MEDISCH CENTRUM UTRECHT, UNIVERSITEIT UTRECHT HOLDING B.V. reassignment UNIVERSITAIR MEDISCH CENTRUM UTRECHT CORRECTIVE ASSIGNMENT TO CORRECT THE ASSIGNEE NAME, PREVIOUSLY RECORDED ON REEL 016754 FRAME 0418. Assignors: BOUMA, BAREND, KRANENBURG, ONNO W., GEBBINK, MARTIN F.B.G., KROON, LOUISE M.
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Priority to US11/982,161 priority patent/US8158585B2/en
Priority to US13/437,807 priority patent/US20120189615A1/en
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Definitions

  • the invention relates to the fields of biotechnology, biochemistry, molecular biology, structural biology and medicine. More in particular, the invention relates to cross- ⁇ structure, their binding proteins and their biological roles.
  • Unfolded proteins can initiate protein aggregation and fibrillization by adopting a partially structured conformation. Such fibrillar aggregates can (slowly) accumulate in various tissue types and are associated with a variety of degenerative diseases.
  • the term “amyloid” is used to describe these fibrillar deposits (or plaques).
  • Diseases characterized by amyloids are referred to as amyloidosis and include Alzheimer disease (AD), light-chain amyloidosis, type II diabetes and spongiform encephalopathies. It has been found recently that toxicity is an inherent property of misfolded proteins. According to the present invention, this is the common mechanism for these conformational diseases. 1
  • a cross- ⁇ structure is a secondary structural element in peptides or proteins.
  • a cross- ⁇ structure can be formed upon denaturation, proteolysis or unfolding of proteins. 2 These secondary structure elements are typically absent in globular regions of proteins.
  • the cross- ⁇ structure is found in amyloid fibrils. Amyloid peptides or proteins are cytotoxic to cells.
  • a cross- ⁇ structure includes stacked ⁇ -sheets. In a cross- ⁇ structure, the individual ⁇ -strands either run perpendicular to the long axis of a fibril or run in parallel to the long axis of a fiber. The direction of the stacking of the ⁇ -sheets in cross- ⁇ structures is perpendicular to the long fiber axis.
  • cross- ⁇ structure pathway a novel pathway involving a cross- ⁇ structure, which pathway will be called a “cross- ⁇ structure pathway.”
  • This pathway includes several cross- ⁇ structure-binding proteins, including so-called multiligand receptors, and is involved in protein degradation and/or protein clearance.
  • novel cross- ⁇ -binding proteins that contain a cross- ⁇ structure-binding module.
  • the present invention discloses that proteolyzed, denatured, unfolded, glycated, oxidized, acetylated or otherwise structurally altered proteins adopt cross- ⁇ structures.
  • cross- ⁇ structure-forming proteins are all proteins that cause amyloidosis or proteins that are found in disease-related amyloid depositions, for example, but not restricted to, Alzheimer ⁇ -amyloid (A ⁇ ) and Islet Amyloid PolyPeptide (IAPP).
  • a ⁇ Alzheimer ⁇ -amyloid
  • IAPP Islet Amyloid PolyPeptide
  • fibrin, glycated proteins for example, glycated albumin and glycated hemoglobin
  • endostatin are also capable of adopting a cross- ⁇ structure.
  • the invention furthermore discloses the identification of the formation of a cross- ⁇ structure as a signal for protein degradation and/or protein clearance.
  • the serine protease tissue plasminogen activator induces the formation of plasmin through cleavage of plasminogen. Plasmin cleaves fibrin and this occurs during lysis of a blood clot. Although not essential for fibrinolysis in mice, 3,4 tPA has been recognized for its role in fibrinolysis for a long time. 5,6 Activation of plasminogen by tPA is stimulated by fibrin or fibrin fragments, but not by its precursor, fibrinogen. 7-10 This can be in part explained by the strong binding of tPA to fibrin and weak binding to fibrinogen. The binding sites in fibrin and in tPA responsible for binding and activation of tPA have been mapped and studied in detail.
  • tissue-type plasminogen activator as a protein capable of binding cross- ⁇ structures.
  • the invention discloses the finger domain (also named fibronectin type I domain) and other comparable finger domains as a cross- ⁇ structure-binding module.
  • the present invention further discloses that proteins which bind to these fingers will be typically capable of forming cross- ⁇ structures.
  • the present invention also discloses that the generation of cross- ⁇ structures plays a role in physiological processes.
  • the invention discloses that the generation of cross- ⁇ structures is part of a signaling pathway, the “cross- ⁇ structure pathway,” that regulates protein degradation and/or protein clearance. Inadequate function of this pathway may result in the development of diseases, such as conformational diseases 37 and/or amyloidosis.
  • the present invention furthermore discloses that the cross- ⁇ structure is a common denominator in ligands for multiligand receptors. 38 The invention discloses, therefore, that multiligand receptors belong to the “cross- ⁇ structure pathway.”
  • RAGE receptor for a cross- ⁇ structure
  • ligands for RAGE are A ⁇ , protein-advanced glycation end-products (AGE) adducts (including glycated-BSA), amphoterin and S100.
  • AGE protein-advanced glycation end-products
  • S100 amphoterin
  • RAGE is a member of a larger family of multiligand receptors 38 that includes several other receptors, some of which, including CD36, are known to bind cross- ⁇ structure-containing proteins (see also FIG. 1 ). At present, it is not clear what the exact nature of the structure or structures is in the ligands of these receptors that mediates the binding to these receptors.
  • glycation of proteins also induces the formation of a cross- ⁇ structure. Therefore, it is disclosed that all of these receptors form part of a mechanism to deal with the destruction and removal of unwanted or even damaging proteins or agents. These receptors play a role in recognition of infectious agents or cells, recognition of apoptotic cells and in internalization of protein complexes and/or pathogens. It is furthermore disclosed that all of these receptors recognize the same or similar structure, the cross- ⁇ structure, to respond to undesired molecules. It is shown herein that tPA binds cross- ⁇ structures, providing evidence that tPA belongs to the multiligand receptor family.
  • tPA and the other multiligand receptors bind the cross- ⁇ structure and participate in the destruction of unwanted biomolecules.
  • a prominent role of the protease tPA in the pathway lies in its ability to initiate a proteolytic cascade that includes the formation of plasmin. Proteolysis is likely to be essential for the degradation and subsequent removal of extracellular matrix components. The effect of tPA on the extracellular matrix will affect cell adhesion, cell migration, cell survival and cell death, through, for example, integrin-mediated processes.
  • FXII factor XII
  • HGFa hepatocyte growth factor activator
  • fibronectin fibronectin
  • FXII The role of FXII is especially important, since it activates the intrinsic coagulation pathway. Activation of the intrinsic pathway, the resulting formation of vasoactive peptides, and the activation of other important proteins contribute to the process of protection and/or clearance of undesired proteins or agents.
  • the “cross- ⁇ structure pathway” is modulated in many ways. Factors that regulate the pathway include modulators of synthesis and secretion, as well as modulators of activity. The pathway is involved in many physiological and pathological processes. Therefore, the invention furthermore provides a method for modulating extracellular protein degradation and/or protein clearance comprising modulating the activity of a receptor for cross- ⁇ structure-forming proteins.
  • receptors for cross- ⁇ structure-forming proteins include RAGE, CD36, Low density lipoprotein-Related Protein (LRP), Scavenger Receptor B-1 (SR-B1), and SR-A.
  • LRP Low density lipoprotein-Related Protein
  • SR-B1 Scavenger Receptor B-1
  • SR-A SR-A
  • FXII, HGFa and fibronectin are also receptors for cross- ⁇ structures.
  • tissue-type plasminogen activator is a cross- ⁇ structure-binding protein, a multiligand receptor and a member of the “cross- ⁇ structure pathway.”
  • tPA tissue-type plasminogen activator
  • the invention discloses that tPA mediates cross- ⁇ structure-induced cell dysfunction and/or cell toxicity.
  • the invention discloses that tPA mediates, at least in part, cell dysfunction and/or toxicity through activation of plasminogen.
  • the plasminogen-dependent effects are inhibited by B-type carboxypeptidase activity B and, thus, a role for carboxyterminal lysine residues in the cross- ⁇ structure pathway is disclosed.
  • the present invention relates, amongst others, to the structure(s) in fibrin and other proteins that bind tPA, to the binding domain in tPA, and to the pathway(s) regulated by this structure.
  • the present invention discloses a presence of cross- ⁇ structures in proteins and peptides that are capable of binding tPA.
  • the herein-disclosed results indicate a strong correlation between the presence of a cross- ⁇ structure and the ability of a molecule to bind tPA.
  • the results indicate the presence of an amyloid structure in fibrin. This indicates that under physiological conditions, a cross- ⁇ structure can form, a phenomenon that has been previously unrecognized. The formation of cross- ⁇ structures has thus far only been associated with severe pathological disorders.
  • tPA binds denatured proteins, which indicates that a large number of proteins, if not all proteins, can adopt a conformation containing cross- ⁇ structures or cross- ⁇ -like structure(s). Taken together, the formation of cross- ⁇ structures is likely to initiate and/or participate in a physiological cascade of events necessary to adequately deal with removal of unwanted molecules, i.e., misfolded proteins, apoptotic cells or even pathogens.
  • FIG. 1 shows a schematic representation of the “cross- ⁇ structure pathway.”
  • This pathway regulates the removal of unwanted biomolecules during several processes, including fibrinolysis, formation of neuronal synaptic networks, clearance of used, unwanted and/or destroyed (denatured) proteins, induction of apoptosis and clearance of apoptotic cells and pathogens. If insufficiently or incorrectly regulated or disbalanced, the pathway may lead to severe disease.
  • the invention discloses a method for modulating extracellular protein degradation and/or protein clearance comprising modulating cross- ⁇ (beta) structure formation (and/or cross- ⁇ structure-mediated activity) of the protein present in the circulation.
  • a cross- ⁇ structure is composed of stacked ⁇ -sheets. In a cross- ⁇ structure, the individual ⁇ -strands either run perpendicular to the long axis of a fibril or run in parallel to the long axis of a fiber.
  • the direction of the stacking of the ⁇ -sheets in cross- ⁇ structures is perpendicular to the long fiber axis.
  • a broad range of proteins is capable of adopting a cross- ⁇ structure and, moreover, these cross- ⁇ structure-comprising proteins are all capable of binding and stimulating tPA, thus promoting destruction of unwanted or damaging proteins or agents.
  • An extracellular protein is typically defined as a protein present outside a cell or cells.
  • Protein degradation and/or protein clearance includes the breakdown and removal of unwanted proteins, for example, unwanted and/or destroyed (for example, denatured) proteins. Also included is the removal of unwanted biomolecules during several processes, including fibrinolysis, formation of neuronal synaptic networks, clearance of used, unwanted and/or destroyed (denatured) proteins, induction of apoptosis and clearance of apoptotic cells and pathogens.
  • the term “in the circulation” is herein defined as a circulation outside a cell or cells, for example, but not restricted to, the continuous movement of blood.
  • the invention discloses a method for increasing extracellular protein degradation and/or protein clearance comprising increasing cross- ⁇ structure formation and/or cross- ⁇ structure-mediated activity of the protein present in the circulation.
  • Increase of cross- ⁇ structure formation of a particular protein leads, for example, to activation of tPA, which, in turn, induces the formation of plasmin through cleavage of plasminogen and thus results in an increase in the degradation and/or protein clearance.
  • the invention discloses a method for increasing extracellular protein degradation and/or protein clearance comprising providing a compound capable of increasing cross- ⁇ structure formation (and/or cross- ⁇ structure-mediated activity) of the protein present in the circulation.
  • the compound capable of increasing cross- ⁇ structure formation is glucose.
  • the addition of glucose to a protein leads to an irreversible, non-enzymatic glycation reaction in which predominantly a glucose molecule is attached to the free amino groups of lysine residues in a protein.
  • N-termini and free amino groups of arginine residues are prone to glycation. It is disclosed herein within the experimental part that glycation leads to cross- ⁇ structure formation.
  • the invention discloses a method for increasing extracellular protein degradation and/or protein clearance comprising providing a compound capable of increasing cross- ⁇ structure formation of the protein present in the circulation.
  • a method for increasing extracellular protein degradation and/or protein clearance comprising increasing cross- ⁇ structure formation of the protein present in the circulation via any of the above-described methods to degrade and/or remove, preferably, the protein which comprises the cross- ⁇ structure
  • This is, for example, accomplished by providing a compound comprising a cross- ⁇ structure and a compound comprising tPA-like activity at or near the protein which needs to be degraded and/or removed.
  • An example of a compound comprising a cross- ⁇ structure is fibrin or a fragment thereof comprising the cross- ⁇ structure.
  • An example of a compound comprising tPA-like activity is tPA.
  • the invention discloses a method for decreasing extracellular protein degradation and/or protein clearance comprising decreasing cross- ⁇ structure formation of the protein present in the circulation.
  • the invention discloses a method for decreasing extracellular protein degradation and/or protein clearance comprising providing a compound capable of decreasing cross- ⁇ structure formation of the protein present in the circulation.
  • Decreasing of cross- ⁇ structure formation is, for example, accomplished by shielding or blocking of the groups involved in the formation of a cross- ⁇ structure.
  • Examples of compounds capable of decreasing cross- ⁇ structure formation are Congo red, antibodies, ⁇ -breakers, phosphonates, heparin, amino-guanidine or laminin. 45
  • Yet another way to decrease cross- ⁇ structure formation in a protein is by removal of a glucose group involved in the glycation of the protein.
  • the invention discloses a method for modulating extracellular protein degradation and/or protein clearance comprising modulating tPA or tPA-like activity.
  • tPA induces the formation of plasmin through cleavage of plasminogen. Plasmin cleaves fibrin and this occurs during lysis of a blood clot. Activation of plasminogen by tPA is stimulated by fibrin or fibrin fragments, but not by its precursor fibrinogen.
  • tPA-like activity is herein defined as a compound capable of inducing the formation of plasmin, possibly in different amounts, and/or other tPA-mediated activities.
  • tPA-like activity is modified such that it has a higher activity or affinity towards its substrate and/or a cofactor. This is, for example, accomplished by providing the tPA-like activity with multiple binding domains for cross- ⁇ structure-comprising proteins.
  • the tPA-like activity is provided with multiple finger domains. It is herein disclosed that the three-dimensional structures of the tPA finger domain and the fibronectin finger domains 4-5 reveal striking structural homology with respect to local charge-density distribution.
  • Both structures contain a similar solvent-exposed stretch of five amino acid residues with alternating charge; for tPA, Arg7, Glu9, Arg23, Glu32, Arg30, and for fibronectin, Arg83, Glu85, Lys87, Glu89, Arg90, located at the fifth finger domain, respectively.
  • the charged-residue alignments are located at the same side of the finger module.
  • the tPA-like activity is provided with one or more extra finger domain(s) which comprise(s) ArgXGlu(X)13Arg(X)8GluXArg (SEQ ID NO: 1) or ArgXGluXLysXGluArg (SEQ ID NO: 2).
  • B-type carboxypeptidases including, but not limited to, carboxypeptidase B (CpB) or Thrombin Activatable Fibrinolysis Inhibitor (TAFI, also named carboxypeptidase U or carboxypeptidase R), are enzymes that cleave off carboxy-terminal lysine and arginine residues of fibrin fragments that would otherwise bind to tPA and/or plasminogen and stimulate plasmin formation.
  • the invention discloses a method for increasing extracellular protein degradation and/or protein clearance comprising providing a compound capable of increasing tPA-like and/or tPA-mediated activity or activities.
  • the invention discloses a method for increasing extracellular protein degradation and/or protein clearance comprising providing a compound capable of increasing tPA-like activity, wherein the compound comprises a cross- ⁇ structure.
  • the invention discloses a method for increasing extracellular protein degradation and/or protein clearance comprising providing a compound capable of inhibiting B-type carboxypeptidase activity.
  • the compound comprises carboxypeptidase inhibitor (CPI) activity.
  • CPI carboxypeptidase inhibitor
  • the invention discloses a method for decreasing extracellular protein degradation and/or protein clearance comprising providing a compound capable of decreasing tPA-like activity.
  • the invention discloses a method for decreasing extracellular protein degradation and/or protein clearance comprising providing a compound capable of decreasing tPA-like activity or tPA-mediated activity or activities, wherein the compound is a protein and/or a functional equivalent and/or a functional fragment thereof.
  • a compound capable of decreasing tPA-like activity is an inhibitor of tPA or a substrate of tPA which binds and does not let go.
  • Examples of a compound capable of decreasing tPA-like activity or tPA-mediated activity include, but are not limited to, lysine, arginine, e-amino-caproic acid or tranexamic acid, serpins (for example, neuroserpin, PAI-1), tPA-Pevabloc, antibodies that inhibit tPA-like activity or tPA-mediated activity or B-type carboxypeptidase(s).
  • providing lysine results in the prevention or inhibition of binding of a protein comprising a C-terminal lysine-residue to the Kringle domain of plasminogen. Hence, tPA activation is prevented or inhibited.
  • the compound capable of decreasing tPA-like activity or tPA-mediated activity or activities reduce the tPA-like activity or tPA-mediated activity or activities and, even more preferably, the tPA-like activity or tPA-mediated activity or activities is completely inhibited.
  • a functional fragment and/or a functional equivalent are typically defined as a fragment and/or an equivalent capable of performing the same function, possibly in different amounts.
  • a functional fragment of an antibody capable of binding to a cross- ⁇ structure would be the Fab′ fragment of the antibody.
  • the invention discloses a method for modulating extracellular protein degradation and/or protein clearance comprising modulating an interaction between a compound comprising a cross- ⁇ structure and a compound comprising tPA-like activity.
  • the invention discloses a method for decreasing extracellular protein degradation and/or protein clearance comprising decreasing an interaction between a compound comprising a cross- ⁇ structure and a compound comprising tPA-like activity.
  • a compound is, for example, a chemical, a proteinaceous substance or a combination thereof.
  • the invention discloses a method for decreasing extracellular protein degradation and/or protein clearance comprising providing a compound capable of decreasing an interaction between a compound comprising a cross- ⁇ structure and a compound comprising tPA-like activity.
  • the invention discloses a method for decreasing extracellular protein degradation and/or protein clearance according to the invention, wherein the compound is a protein and/or a functional equivalent and/or a functional fragment thereof.
  • the protein is an antibody and/or a functional equivalent and/or a functional fragment thereof.
  • the invention also discloses a method for decreasing extracellular protein degradation and/or protein clearance comprising decreasing an interaction between a compound comprising a cross- ⁇ structure and a compound comprising tPA-like activity, wherein the interaction is decreased by providing a compound capable of competing with the interaction.
  • the compound capable of competing with the interaction comprises a finger domain and, even more particularly, the finger domain comprises a stretch of at least 5 amino acid residues with alternating charge, for example, ArgXGlu(X) 13 Arg(X) 8 GluXArg (SEQ ID NO: 1) or ArgXGluXLysXGluArg (SEQ ID NO: 2).
  • the compound is fibronectin, FXII, HGFa or tPA.
  • the invention also comprises a method for increasing extracellular protein degradation and/or protein clearance comprising increasing an interaction between a compound comprising a cross- ⁇ structure and a compound comprising tPA-like activity. This is, for example, accomplished by providing a compound capable of increasing an interaction between a compound comprising a cross- ⁇ structure and a compound comprising tPA-like activity.
  • the compound capable of increasing an interaction between a compound comprising a cross- ⁇ structure and a compound comprising tPA-like activity is a protein and/or a functional equivalent and/or a functional fragment thereof.
  • an antibody which stabilizes the interaction between a compound comprising a cross- ⁇ structure and a compound comprising tPA-like activity, rendering the tPA-like activity in a continuous activated state results in increased protein degradation and/or protein clearance.
  • increasing an interaction between a compound comprising a cross- ⁇ structure and a compound comprising tPA-like activity is also accomplished by mutations in either the compound comprising a cross- ⁇ structure or in the compound comprising tPA-like activity, like swapping of domains (for example, by providing the compound comprising tPA-like activity with other or more finger domains obtainable from tPA, fibronectin, FXII or HGFa), or by including binding domains of, for example, RAGE or CD36.
  • the invention discloses a method for modulating extracellular protein degradation and/or protein clearance comprising modulating an interaction of a compound comprising tPA-like activity and the substrate of the activity. It is clear that there are multiple ways by which the interaction can either be increased or decreased. An increase in the interaction between a compound comprising tPA-like activity and the substrate of the activity is, for example, accomplished by providing the compound comprising tPA-like activity with a mutation or mutations which improve the affinity of the compound with tPA-like activity for its substrate.
  • the invention discloses a method for removing cross- ⁇ structures from the circulation, using a compound comprising a cross- ⁇ structure-binding domain.
  • the compound is tPA or the finger domain of tPA.
  • the invention also comprises other cross- ⁇ structure-binding domains, including, but not limited to, the finger domains of HGFa, FXII and fibronectin (SEQ ID NOs: 3-17). It is clear that the invention also comprises antibodies that bind cross- ⁇ structures.
  • the present invention further discloses the use of a novel strategy to prevent the formation of, or to decrease/diminish, (amyloid) plaques involved in a conformational disease, type II diabetes and/or aging (e.g., Alzheimer's disease). Plaques are typically defined as extracellular fibrillar protein deposits (fibrillar aggregates) and are characteristic of degenerative diseases.
  • the “native” properties of the constituent amyloid proteins may vary: some are soluble oligomers in vivo (e.g., transthyretin in familial amyloid polyneuropathy), whereas others are flexible peptides (e.g., amyloid-b in Alzheimer's disease (AD)).
  • conformational diseases for example, neurodegenerative disorders (AD, prion disorders)
  • AD neurodegenerative disorders
  • prion disorders abnormal pathologic protein conformation, i.e., the conversion of a normal cellular and/or circulating protein into an insoluble, aggregated, ⁇ -structure-rich form which is deposited in the brain. These deposits are toxic and produce neuronal dysfunction and death.
  • the formation of cross- ⁇ structures has thus far only been associated with severe pathological disorders.
  • the results herein show that tPA and other receptors for cross- ⁇ structure-forming proteins can bind denatured proteins, indicating that a large number of proteins are capable of adopting a conformation containing cross- ⁇ or cross- ⁇ -like structures.
  • cross- ⁇ structure initiates or participates in a physiological cascade of events necessary to adequately deal with removal of unwanted molecules, i.e., misfolded proteins, apoptotic cells or even pathogens.
  • unwanted molecules i.e., misfolded proteins, apoptotic cells or even pathogens.
  • the pathway for protein degradation and/or protein clearance is activated and the protein is degraded, resulting in a decreasing plaque or, in another aspect, the plaque is completely removed.
  • the effects of the conformational disease are diminished or, alternatively, completely abolished.
  • the invention discloses the use of a compound capable of increasing cross- ⁇ structure formation for diminishing plaques involved in a conformational disease.
  • the invention discloses the use of a compound capable of binding to a cross- ⁇ structure for diminishing plaques and/or inhibiting cross- ⁇ structure-mediated toxicity involved in a conformational disease.
  • the compound is a protein and/or a functional equivalent and/or a functional fragment thereof and, in another aspect, the protein is tPA, a finger domain, an antibody and/or a functional equivalent and/or a functional fragment thereof. Examples of such antibodies are 4B5 or 3H7.
  • the invention discloses the use of a compound capable of increasing tPA-like activity for diminishing plaques involved in a conformational disease.
  • the tPA-like activity is modified such that it has a higher activity or affinity towards its substrate and/or cofactor. This is, for example, accomplished by providing the tPA-like activity with multiple binding domains for cross- ⁇ structure-comprising proteins.
  • the binding domain comprises a finger domain and, in an additional aspect, the finger domain comprises a stretch of at least five amino acid residues with alternating charge, for example ArgXGlu(x) 13 Arg(X) 8 GluXArg (SEQ ID NO: 1) or ArgXGluXLysXGluArg (SEQ ID NO: 2).
  • the finger domain is derived from fibronectin, FXII, HGFa or tPA.
  • the invention discloses the use of a compound capable of binding to a cross- ⁇ structure for the removal of cross- ⁇ structures.
  • the compound is a protein and/or a functional equivalent and/or a functional fragment thereof.
  • the compound comprises tPA or tPA-like activity and/or a functional equivalent and/or a functional fragment thereof.
  • the functional fragment comprises a finger domain.
  • the finger domain comprises a stretch of at least five amino acid residues with alternating charge, for example, ArgXGlu(X) 13 Arg(X) 8 GluXArg (SEQ ID NO: 1) or ArgXGluXLysXGluArg (SEQ ID NO: 2).
  • the finger domain is derived from fibronectin, FXII, HGFa or tPA.
  • the protein is an antibody and/or a functional equivalent and/or a functional fragment thereof.
  • the invention discloses, for example, a therapeutic method to remove cross- ⁇ structure-comprising proteins from, for example, the circulation, such as via extracorporeal dialysis.
  • a patient with sepsis is subjected to such use by dialysis of the blood of that patient through means which are provided with, for example, immobilized finger domains.
  • cross- ⁇ structure-comprising proteins will be removed from the blood stream of the patient, thus, relieving patients of the negative effects caused by the cross- ⁇ structure-comprising proteins.
  • finger domain-comprising compounds it is also possible to use other cross- ⁇ structure-binding compounds, like antibodies or Congo Red. It is also clear that the use could be applied in hemodialysis of kidney patients.
  • the invention discloses the use of a compound capable of increasing or stabilizing an interaction of a compound comprising a cross- ⁇ structure and a compound comprising tPA-like activity for diminishing plaques involved in a conformational disease.
  • a compound capable of increasing or stabilizing an interaction of a compound comprising a cross- ⁇ structure and a compound comprising tPA-like activity are given herein.
  • the invention is used to treat the conformational disease Alzheimer or diabetes. It is clear that the invention not only discloses a use to decrease/diminish plaques involved in a conformational disease, but also that the onset of the disease can also be inhibited or even completely prevented.
  • diseases which can be prevented and/or treated according to the invention are conformational disease, amyloidosis-type diseases, atherosclerosis, diabetes, bleeding, thrombosis, cancer, sepsis and other inflammatory diseases, Multiple Sclerosis, auto-immune diseases, disease associated with loss of memory or Parkinson and other neuronal diseases (epilepsy).
  • the invention discloses the use of an antibody capable of recognizing a cross- ⁇ structure epitope for determining the presence of plaque involved in a conformational disease.
  • the invention discloses the use of a cross- ⁇ structure-binding domain (such as a finger domain from, for example, tPA) for determining the presence of a plaque involved in a conformational disease.
  • a finger domain of, for example, tPA
  • a label radioactive, fluorescent, etc.
  • This labeled finger domain may be used either in vitro or in vivo for the detection of cross- ⁇ structure-comprising proteins and, thus, for determining the presence of a plaque involved in a conformational disease.
  • the invention discloses a recombinant tPA comprising an improved cross- ⁇ structure-binding domain or multiple cross- ⁇ structure-binding domains.
  • tPA is provided with multiple, possibly different, finger domains.
  • a recombinant tPA comprising an improved cross- ⁇ structure-binding domain or multiple cross- ⁇ structure-binding domains is used for different purposes, for example, in a method for the improved treatment of thrombolysis or for the removal of cross- ⁇ structure-comprising proteins from the circulation of a patient in need thereof.
  • a recombinant tPA comprising an improved cross- ⁇ structure-binding domain or multiple cross- ⁇ structure-binding domains is in diagnostic assays such as, for example, in a BSE detection kit or in imaging experiments.
  • This imaging with a recombinant tPA comprising an improved cross- ⁇ structure-binding domain or multiple cross- ⁇ structure-binding domains is, for example, useful for detection of apoptosis.
  • labeled tPA such as, but not limited to, radio-labeled tPA, is inoculated in an individual, followed by detection and localization of labeled tPA in the body. It is clear that recombinant tPA comprising a cross- ⁇ structure-binding domain or multiple cross- ⁇ structure-binding domains are also useful in therapeutic applications.
  • cross- ⁇ structure-mediated effects comprising providing an effective amount of a protein comprising a finger domain to block the binding sites of the cross- ⁇ structure for tPA.
  • Cross- ⁇ structure-mediated effects may even be further diminished by providing an effective amount of B-type carboxypeptidase activity to inhibit the tPA activity.
  • the local cross- ⁇ structure-mediated effect can be used against tumors.
  • cross- ⁇ structure-mediated effects are locally induced to increase local cytotoxicity and/or fibrinolysis comprising locally administering an effective amount of cross- ⁇ structures and/or cross- ⁇ structure-inducing compounds in conjunction with tPA or a compound with tPA-like activity and/or CPI or a compound with CPI-like activity.
  • the present invention discloses in a further embodiment a method which is carried out ex vivo, e.g., by dialysis.
  • the circulating fluid (blood) of a subject is brought in a system outside the body for clearing cross- ⁇ structures from the circulation.
  • a system is a flow-through system connected to the body circulation with an inlet and an outlet.
  • the cross- ⁇ structures are cleared by binding to a cross- ⁇ binding compound as defined hereinbefore. It is very important that no elements, such as the cross- ⁇ binding compounds from the system, are brought into the subject's circulation. For that reason, among others, preferred systems are dialysis systems.
  • the invention further discloses devices for carrying out methods as disclosed herein.
  • the invention discloses a separation device for carrying out a method according to the invention wherein the apparatus comprises a system for transporting circulation fluids ex vivo, the system provided with means for connecting to a subject's circulation for entry into the system and return from the system to the subject's circulation, the system comprising a solid phase, the solid phase comprising at least one compound capable of binding cross- ⁇ structures.
  • the device is a dialysis apparatus.
  • the invention also provides for detection of cross- ⁇ structures in samples.
  • samples may be tissue samples, biopsies and the like, body fluid samples, such as blood, serum, liquor, CSF, urine, and the like.
  • the invention thus discloses a method for detecting cross- ⁇ structures in a sample, comprising contacting the sample with a compound capable of binding cross- ⁇ structures, allowing for binding of cross- ⁇ structures to the compound and detecting the complex formed through binding.
  • Cross- ⁇ binding compounds have been defined hereinbefore. Detection of the complex or one of its constituents can be done through any conventional means involving antibodies or other specific binding compounds, further cross- ⁇ binding compounds, etc. Detection can be direct such as by labeling the complex or a binding partner for the complex or its constituents, or even by measuring a change in a physical or chemical parameter of the complex versus unbound material. It may also be indirect by further binding compounds provided with a label.
  • a label may be a radioactive label, an enzyme, a fluorescent molecule, etc.
  • the invention further discloses devices for carrying out the diagnostic methods.
  • a diagnostic device for carrying out a method according to the invention comprising a sample container, a means for contacting the sample with a cross- ⁇ binding compound, a cross- ⁇ binding compound and a means for detecting bound cross- ⁇ structures.
  • the device comprises a means for separating unbound cross- ⁇ structures from bound cross- ⁇ structures which can be typically done by providing the cross- ⁇ binding compounds on a solid phase.
  • FIG. 1 is a schematic representation of the “cross- ⁇ structure pathway.”
  • the cross- ⁇ structure is found in a number of proteins (1).
  • the formation of a cross- ⁇ structure can be triggered by several physiological or pathological conditions and subsequently initiates a cascade of events, the “cross- ⁇ structure pathway.”
  • factors that trigger or regulate the formation of a cross- ⁇ structure within a given protein are: 1) the physicochemical properties of the protein, 2) proteolysis, 3) regulated post-translational modification, including cross-linking, oxidation, phosphorylation, glycosylation and glycation, 4) glucose, and 5) zinc.
  • Certain mutations within the sequence of a protein are known to increase the ability of the protein to adopt a cross- ⁇ structure and form amyloid fibrils. These mutations are often found in hereditary forms of amyloidosis, for example in AD.
  • the present invention discloses multiple novel examples of proteins capable of adopting a cross- ⁇ structure.
  • proteins are known to bind cross- ⁇ -containing proteins (2). These proteins are part of the herein disclosed signaling cascade (“cross- ⁇ structure pathway”) that is triggered upon formation of a cross- ⁇ structure.
  • the “cross- ⁇ structure pathway” is modulated in many ways (3, 4, 5). Factors that regulate the pathway include modulators of synthesis and secretion including NO regulators, as well as modulators of activity, including protease inhibitors.
  • the pathway is involved in many physiological and pathological processes including, but not limited to, atherosclerosis, diabetes, amyloidosis, bleeding, inflammation, multiple sclerosis, Parkinson's disease, sepsis, hemolytic uremic syndrome (7).
  • the “cross- ⁇ structure pathway” may also be involved in learning.
  • FIG. 2 illustrates a cross- ⁇ structure in fibrin.
  • Panel A depicts Thioflavin T fluorescence of a fibrin clot. A fibrin clot was formed in the presence of Thioflavin T and fluorescence was recorded at indicated time points. Background fluorescence of buffer, Thioflavin T and a clot formed in the absence of Thioflavin T, was substracted.
  • Panel B is a graph depicting circular dichroism analysis of fibrin-derived peptides 85, 86 and 87. Ellipticity (Dg.cm 2 /dmol) is plotted against wavelength (nm). The CD spectra demonstrates that peptides 85 and 86, but not peptide 87, contain ⁇ -sheets.
  • Panel C shows that X-ray fiber diffraction analysis of peptide 85 reveals that the peptide forms cross- ⁇ sheets.
  • Panel D is a graph showing plasminogen activation assay with fibrin peptides 85, 86 and 87. It is seen that peptides 85 and 86, both containing a cross- ⁇ structure, stimulate the formation of plasmin by tPA, whereas peptide 87, which lacks a cross- ⁇ structure, does not.
  • FIG. 3 is a set of graphs depicting binding of tPA, plasminogen and plasmin to A ⁇ .
  • a ⁇ was coated onto plastic 96-well plates. Increasing concentrations of either (A) tPA or (B) plasmin(ogen) were allowed to bind to the immobilized peptide. After extensive washing, tPA and plasmin(ogen) binding was assessed by enzyme-linked immunosorbent assays using anti-tPA and anti-plasminogen antibodies. Binding of (C) tPA and (D) plasmin to A ⁇ in the presence of 50 mM ⁇ -aminocaproic acid ( ⁇ -ACA) was assessed as in A and B.
  • ⁇ -ACA ⁇ -aminocaproic acid
  • FIG. 4 is a set of graphs illustrating stimulation of tPA-mediated plasmin formation by A ⁇ and synergistic stimulation of cell detachment by plasminogen and A ⁇ .
  • Panel A depicts that plasminogen (200 ⁇ g/ml) and tPA (200 pM) were incubated with A ⁇ (5 ⁇ M) or control buffer. Samples were taken from the reaction mixture at the indicated periods of time and plasmin activity was measured by conversion of the chromogenic plasmin substrate S-2251 at 405 nm.
  • Panel B shows that N1E-115 cells were differentiated and received the indicated concentrations of plasmin in the presence or absence of 25 ⁇ M A ⁇ .
  • Panel C illustrates that N1E-115 cells were differentiated and received the indicated concentrations of plasminogen in the presence or absence of 10 ⁇ M A ⁇ . After 24 hours, cell detachment was assessed. A ⁇ or plasminogen alone does not affect cell adhesion, but cause massive cell detachment when added together.
  • Panel D is an immunoblot analysis of plasmin formation and laminin degradation. Differentiated N1E-115 cells were treated with or without A ⁇ (10 ⁇ M) in the absence or presence of added plasminogen. Addition of A ⁇ results in the formation of plasmin (bottom panel) and in degradation of laminin (top panel).
  • FIG. 5 depicts graphs illustrating that carboxypeptidase B inhibits A ⁇ -stimulated tPA-mediated plasmin formation and cell detachment.
  • Panel A shows that plasminogen (200 ⁇ g/ml) and tPA (200 pM) were incubated with A ⁇ (5 ⁇ M) or control buffer. Samples were taken from the reaction mixture at the indicated periods of time and plasmin activity was measured by conversion of the chromogenic plasmin substrate S-2251 at 405 nm. The reaction was performed in the absence or the presence of 50 ⁇ g ml ⁇ 1 carboxypeptidase B (CpB) and in the absence or presence of 3.5 ⁇ M carboxypeptidase inhibitor (CPI).
  • CpB carboxypeptidase B
  • CPI carboxypeptidase inhibitor
  • CpB greatly attenuates A-stimulated plasmin formation.
  • Panel B shows that N1E-115 cells were differentiated and treated with A ⁇ (10 ⁇ M), plasminogen (Plg, 20 ⁇ g ml ⁇ 1 ) and/or CpB (1 ⁇ M) as indicated. After 24 hours, the cells were photographed.
  • Panel C illustrates that, subsequently, the cells were washed once with PBS and the remaining cells were quantified as percentage-adhered cells by methylene blue staining.
  • Panel D the cells were treated as in Panels B and C and medium and cell fractions were collected and analyzed by Western blot using an anti-plasmin(ogen) antibody. A ⁇ stimulates plasmin formation that is inhibited by CpB.
  • FIG. 6 is a set of graphs illustrating that endostatin can form fibrils comprising cross- ⁇ structure and stimulates plasminogen activation.
  • Panel A TEM shows the formation of endostatin fibrils.
  • Panel B contains an X-ray analysis that reveals the presence of cross- ⁇ structure in precipitated (prec.) endostatin.
  • Panel C is a plasminogen activation assay demonstrating the stimulating activity of cross- ⁇ structure-containing endostatin on tPA-mediated plasmin formation. A ⁇ is shown for comparison.
  • Panel D is an analysis of endostatin-induced cell death by methylene blue staining. It is seen that only the precipitated form is capable of efficiently inducing cell death. Direct cell death, but not cell detachment, is protected in the presence of sufficient glucose. Buffer prec. indicates control buffer.
  • FIG. 7 is a graph showing that IAPP stimulates tPA-mediated plasminogen activation. Both full length (fl-hIAPP) and truncated amyloid core ( ⁇ -hIAPP), but not mouse IAPP ( ⁇ -mIAPP), stimulate tPA-mediated plasminogen activation.
  • FIG. 8 is a set of graphs illustrating glycated albumin: Thioflavin T and tPA binding, TEM images, X-ray fiber diffraction.
  • Panel A is an ELISA showing binding of tPA to albumin-g6p.
  • Panel B shows competition of tPA binding to albumin-g6p by Congo red as determined using ELISA.
  • Panel C shows fluorescence measurements of Thioflavin T binding to albumin-g6p, which is incubated for two, four, or 23 weeks.
  • Panel D shows that inhibition of the fluorescent signal is obtained upon incubation of 430 nM of albumin-g6p with 19 ⁇ M of Thioflavin T by tPA.
  • Panels E and F illustrate that spectrophotometric analysis at 420 nm shows that increasing amounts of tPA result in a decrease of the specific absorbance obtained upon incubation of 500 nM of albumin-g6p with 10 ⁇ M of Thioflavin T.
  • Panels G, H and I are electron micrographs showing (G) amorphous precipitates of four weeks glycated albumin-g6p, (H) bundles of fibrillar aggregates of 23 weeks incubated albumin-g6p, and (I) two weeks glycated albumin-g6p.
  • Panel J is an X-ray scattering of albumin-g6p (23 weeks).
  • Scattering intensities are color coded on a linear scale and decreases in the order white-grey-black. Scattering from amorphous control albumin is substracted, as well as scattering from the capillary glass wall and from air. d-spacings and the direction of the fiber axis are given and preferred orientations are indicated with arrows.
  • Panel K is radial scan of albumin control and albumin-g6p (23 weeks).
  • Panel L is a radial scan of albumin-g6p (23 weeks), showing repeats originating from fibrous structure, after subtracting background scattering of amorphous precipitated albumin. d-spacings (in ⁇ ) are depicted above the peaks.
  • Panel M contains tangential scans along the 20 scattering-angles corresponding to indicate d-spacings. The scans show that the 4.7 ⁇ repeat, which corresponds to the hydrogen-bond distance within individual ⁇ -sheets, and the 6 ⁇ repeat, are oriented perpendicular to the 2.3 ⁇ repeat that runs parallel to the fiber axis.
  • Panel N is a schematic drawing of the orientation of the cross- ⁇ structures in albumin-g6p (23 weeks) amyloid fibrils.
  • FIG. 9 illustrates fibril formation of human hemoglobin.
  • Panel A depicts binding of tPA to in vitro glycated Hb-g6p.
  • Panel B is an electron micrograph showing in vitro glycated Hb, which aggregates in an amorphous and fibrous manner.
  • FIG. 10 shows that amyloid properties of albumin-AGE are introduced irrespective of the carbohydrate or carbohydrate derivative used for glycation.
  • Panels A-I illustrate Congo red fluorescence of air-dried albumin preparations. Fluorescence was measured with albumin incubated with buffer (Panel A) or with buffer and NaCNBH 3 (Panel B), with amyloid core peptide of human IAPP (Panel C), A ⁇ (Panel D), with albumin incubated with g6p (Panel E), glucose (Panel F), fructose (Panel G), glyceraldehyde (Panel H), and glyoxylic acid (Panel I).
  • Panel J shows that Thioflavin T—amyloid fluorescence was measured in solution with the indicated albumin preparations.
  • Panels K and L show that binding of amyloid-binding serine protease tPA to albumin preparations was assayed using an ELISA set-up.
  • Panel K binding of tPA to albumin-glucose, -fructose, -glyceraldehyde, -glyoxylic acid, and albumin-buffer controls is shown.
  • Panel L binding of tPA to positive controls albumin-g6p, A ⁇ and IAPP is shown, as well as to albumin incubated with control buffer.
  • FIG. 11 illustrates analysis of Congo red and tPA binding to A ⁇ .
  • Panel A shows binding of tPA to immobilized A ⁇ as measured using an ELISA.
  • Panel B illustrates the influence of increasing concentrations of Congo red on binding of tPA to A ⁇ .
  • 10 ⁇ g ml ⁇ 1 of A ⁇ (1-40) was coated and incubated with 40 nM of tPA and 0-100 ⁇ M of Congo red.
  • FIG. 12 illustrates binding of human FXII to amyloid peptides and proteins that contain the cross- ⁇ structure fold.
  • Panels A and B show binding of FXII to prototype amyloid peptides hA ⁇ (1-40) and human fibrin fragment ⁇ 147-159 FP13, and albumin-AGE and Hb-AGE, that all contain cross- ⁇ structure, were tested in an ELISA.
  • FXII does not bind to negative controls mouse ⁇ islet amyloid polypeptide ( ⁇ mIAPP), albumin-control and Hb-control, all three lacking the amyloid-specific structure.
  • k D 's for hA ⁇ (1-40), FP13, albumin-AGE and Hb-AGE are approximately 2, 11, 8 and 0.5 nM, respectively.
  • ACTILYSE® full-length tPA
  • K2P-tPA RETEPLASE®
  • Panels E and F show that coated amyloid albumin-AGE was incubated with 15 nM FXII in binding buffer, in the presence of a concentration series of f.l. tPA or K2P-tPA.
  • the tPA concentration was, at maximum, 150 times the k D for tPA binding to albumin-AGE (1 nM).
  • Panel G illustrates that binding of FXII to hA ⁇ (1-40) and the prototype amyloid human amylin fragment h ⁇ IAPP was tested using dot blot analysis. 10 ⁇ g of the peptides that contain cross- ⁇ structure as well as the negative control peptide m ⁇ IAPP and phosphate-buffered saline (PBS) were spotted in duplicate. FXII specifically bound to hA ⁇ (1-40) as well as to h ⁇ IAPP.
  • FIG. 13 illustrates that finger domains bind to amyloid (poly)peptides.
  • Panel A depicts binding of tPA and K2-P tPA to albumin-g6p.
  • Panel B shows binding of tPA and K2-P tPA to A ⁇ (1-40). The tPA antibody used for detection recognizes both tPA and K2-P-tPA with equal affinity (not shown).
  • Panel C shows binding of tPA-F-GST and tPA to immobilized A ⁇ (1-40) and albumin-g6p. Control RPTP ⁇ -GST does not bind A ⁇ or albumin-g6p.
  • Panel D is a pull-down assay with insoluble A ⁇ fibrils and tPA domains.
  • m ⁇ IAPP was coated as non-amyloid negative control (Panel E).
  • Peptides were immobilized on ELISA plates and overlayed with concentration series of tPA and F-EGF-GST. GST was used as a negative control. Binding was detected using rabbit anti-GST antibody Z-5.
  • Panels H-M depict immunohistochemical analysis of binding of tPA F-EGF-GST to amyloid deposits in human brain inflicted by AD. Brain sections were overlayed with tPA F-EGF-GST (Panels H and J) or negative control GST (Panel L). The same sections were incubated with Congo red (Panels I, K and M) to locate amyloid deposits.
  • Panels N and O are pull-down assays with insoluble A ⁇ fibrils and finger domains.
  • Recombinant F domains with a C-terminal GST tag were expressed by stably transfected BHK cells.
  • Samples were analyzed on Western blot using rabbit anti-GST antibody Z-5.
  • FIG. 14 illustrates the finger module.
  • Panel A is a schematic representation of the location of the finger domain in tPA, factor XII, HGFa and fibronectin.
  • Panel B is an alignment of the amino acid sequence of the finger domain of the respective proteins. Specifically: tPA, FXII, HGFa, FN1-1, FN1-2, FN1-3, FN1-4, FN1-5, FN1-6, FN1-7, FN1-8, FN1-9, FN1-10, FN1-11, and FN1-12 (SEQ ID NOs: 3-17 respectively).
  • Panel C is a representation of the peptide backbone of the tPA finger domain and the fourth and fifth finger domain of FN. conserveed disulfide bonds are shown in ball and stick.
  • FIG. 15 shows that antibodies elicited against amyloid peptides cross-react with glycated proteins, and vice versa.
  • Panels A-C are ELISA with immobilized g6p-glycated albumin-AGE:23 and Hb-AGE, their non-glycated controls (Panel A), A ⁇ (1-40) (Panel B), and IAPP and m ⁇ IAPP (Panel C).
  • a ⁇ ELISA polyclonal anti-human vitronectin antibody ⁇ -hVn K9234 was used as a negative control.
  • Panel D shows binding of ⁇ -AGE1 to immobilized A ⁇ (1-40) on an ELISA plate after pre-incubation of ⁇ -AGE 1 with IAPP fibrils.
  • Panels F and G depict that in an ELISA set-up, immobilized A ⁇ (1-40) (Panel F) and IAPP (Panel G) are co-incubated with tPA and 250 or 18 nM ⁇ -AGE1, respectively.
  • Panel H shows that in an ELISA set-up binding of ⁇ -A ⁇ (1-42) H-43 to immobilized positive control A ⁇ (1-40), and to IAPP and albumin-AGE:23 is tested.
  • Albumin-control:23 and m ⁇ IAPP are used as negative controls.
  • Panel I depicts binding of 100 nM ⁇ -A ⁇ (1-42) H-43 to IAPP, immobilized on an ELISA plate, in the presence of a concentration series of tPA.
  • Panels J and K are ELISA showing binding of a polyclonal antibody in mouse serum elicited against albumin-AGE:23 and A ⁇ (1-40) (ratio 9:1) (“poab anti-amyloid”) and of a polyclonal antibody elicited against a control protein (“control serum”) to immobilized IAPP (Panel J) and albumin-AGE:23 (Panel K). Serum was diluted in PBS with 0.1% v/v Tween 20.
  • Panel L is an ELISA showing binding of mouse poab anti-amyloid serum to amyloid A ⁇ (1-40), h ⁇ IAPP and fibrin fragment ⁇ 148-160 FP13.
  • Panel M is an immunohistochemical analysis of the binding of rabbit anti-AGE2 to a spherical amyloid plaque (arrow) in a section of a human brain afflicted by AD. Magnification 400 ⁇ .
  • Panel N is a Congo red fluorescence of the same section. Magnification 630 ⁇ .
  • FIG. 16 illustrates that monoclonal anti-cross- ⁇ structure antibody 3H7 detects glycated hemoglobin, A ⁇ , IAPP and FP13.
  • ELISA showing binding of mouse monoclonal anti-cross- ⁇ structure antibody 3H7 to (Panel A) glycated hemoglobin vs. control unglycated hemoglobin or (Panel B) A ⁇ , hIAPP, ⁇ mIAPP and fibrin fragment ⁇ 148-160 FP13.
  • FIG. 17 is a sandwich ELISA for detection of amyloid albumin-AGE or amyloid hemoglobin in solution. Immobilized recombinant tPA on Exiqon protein Immobilizers was overlayed with albumin-AGE:23 solution or albumin-control:23 solution at the indicated concentrations. Bound amyloid structures were detected with anti-A ⁇ (1-42) H-43 (A).
  • the invention discloses (i) the identification of a “cross- ⁇ structure pathway,” (ii) the identification of multiligand receptors as being cross- ⁇ structure receptors, (iii) the identification of the finger domain as a cross- ⁇ -binding module and (iv) the identification of finger-containing proteins, including tPA, FXII, HGFa and fibronectin as part of the “cross- ⁇ structure pathway.”
  • This invention further discloses compounds not previously known to bind cross- ⁇ structure.
  • the invention describes compounds and methods for the detection and treatment of diseases associated with the excessive formation of a cross- ⁇ structure.
  • diseases include known conformational diseases including Alzheimer disease and other types of amyloidosis.
  • the present invention also discloses that other diseases not yet known to be associated with excessive formation of cross- ⁇ structures are also caused by excessive formation of cross- ⁇ structures.
  • diseases include atherosclerosis, sepsis, diffuse intravascular coagulation, hemolytic uremic syndrome, preeclampsia, rheumatoid arthritis, autoimmune diseases, thrombosis and cancer.
  • the compound or means for binding the cross- ⁇ structure is a cross- ⁇ structure-binding molecule, such as a finger domain or a molecule containing one or more finger domains, or is a peptidomimetic analog of one or more finger domains.
  • the compound can also be an antibody or a functional fragment thereof directed to the cross- ⁇ structure.
  • the compound or means for binding the cross- ⁇ structure may also be a multiligand receptor or fragment thereof.
  • the compound may be, e.g., RAGE, CD36, Low density lipoprotein Related Protein (LRP), Scavenger Receptor B-1 (SR-B1), SR-A, or a fragment of one of these proteins.
  • the finger domains, finger-containing molecules or antibodies may be human, mouse, rat or from any other species.
  • amino acids of the respective proteins may be replaced by other amino acids which may increase/decrease the affinity, the potency, bioavailability and/or half-life of the peptide.
  • Alterations include conventional replacements (acid-acid, bulky-bulky and the like), introducing D-amino acids, making peptides cyclic, etc.
  • This invention also discloses methods for preparing an assay to measure cross- ⁇ structure in sample solutions.
  • This invention also discloses methods for detecting cross- ⁇ structure in tissue samples or other samples obtained from living cells or animals.
  • This invention further discloses compounds and methods for preparing a composition for inhibiting cross- ⁇ structure fibril formation.
  • This invention still further discloses compounds and methods for preparing a composition for modulating cross- ⁇ structure-induced toxicity.
  • a ⁇ beta-amyloid peptide
  • AD Alzheimer disease
  • AGE advanced glycation end-products
  • CpB carboxypeptidase B
  • COI carboxypeptidase inhibitor
  • ELISA enzyme-linked immunosorbent assay
  • FN fibronectin
  • FXII factor XII
  • HGFa hepatocyte growth factor activator
  • IAPP islet amyloid polypeptide
  • PCR polymerase chain reactions
  • RAGE receptor for AGE
  • tPA tissue-type plasminogen activator.
  • the invention discloses compounds and methods for the detection and treatment of diseases associated with the excessive formation of cross- ⁇ structure.
  • the cross- ⁇ structure can be part of an A ⁇ fibril or part of another amyloid fibril.
  • the cross- ⁇ structure can also be present in denatured proteins.
  • a cross- ⁇ structure-binding compound or means for binding the cross- ⁇ structure such as a finger domain or a molecule comprising one or more finger modules, is bound or affixed to a solid surface, such as a microtiter plate.
  • a solid surface such as a microtiter plate.
  • the solid surfaces useful in this embodiment would be known to one of skill in the art.
  • a solid surface is a bead, a column, a plastic dish, a plastic plate, a microscope slide, a nylon membrane, etc. After blocking, the surface is incubated with a sample.
  • bound molecules comprising the cross- ⁇ structure are subsequently detected using a second cross- ⁇ structure-binding compound, such as an anti-cross- ⁇ structure antibody or a molecule containing a finger module.
  • the second cross- ⁇ structure compound is bound to a label such as an enzyme, i.e., peroxidase.
  • the detectable label may also be a fluorescent label, a biotin, a digoxigenin, a radioactive atom, a paramagnetic ion, and a chemiluminescent label. It may also be labeled by covalent means such as chemical, enzymatic or other appropriate means with a moiety such as an enzyme or radioisotope.
  • Portions of the above-mentioned compounds of the invention may be labeled by association with a detectable marker substance (e.g., radiolabeled with 125 I or biotinylated) to provide reagents useful in detection and quantification of a compound or its receptor-bearing cells or its derivatives in solid tissue, and fluid samples such as blood, cerebral spinal fluid, urine or others.
  • a detectable marker substance e.g., radiolabeled with 125 I or biotinylated
  • Such samples may also include serum used for tissue culture or medium used for tissue culture.
  • the solid surface can be microspheres, for example, for agglutination tests.
  • the compound containing a finger module is used to stain tissue samples.
  • the compound or means for binding the cross- ⁇ structure is fused to a protein or peptide, such as glutathion-S-transferase.
  • the compound is coupled to a label.
  • the detectable label may be a fluorescent label, a biotin, a digoxigenin, a radioactive atom, a paramagnetic ion, or a chemiluminescent label. It may also be labeled by covalent means such as chemical, enzymatic or other appropriate means with a moiety such as an enzyme or radioisotope.
  • Portions of the above-mentioned compounds of the invention may be labeled by association with a detectable marker substance (e.g., radiolabeled with 125 I, 99m Tc, 131 I, chelated radiolabels, or biotinylated) to provide reagents useful in detection and quantification of a compound or its receptor-bearing cells or its derivatives in solid tissue, and fluid samples such as blood, cerebral spinal fluid or urine.
  • a detectable marker substance e.g., radiolabeled with 125 I, 99m Tc, 131 I, chelated radiolabels, or biotinylated
  • the compound or means for binding the cross- ⁇ structure is incubated with the sample and after washing, is visualized with antibodies directed against the fused protein or polypeptide, such as glutathion-S-transferase.
  • the sample is tissue from patients with or expected to suffer from a conformational disease.
  • the tissue is derived from animals or from cells cultured in vitro.
  • the methods of the invention disclose a new diagnostic tool. It was not until the present invention that a universal ⁇ -structure epitope was disclosed and that a diagnostic assay could be based on the presence of the cross- ⁇ structure. Such use is particularly useful for diagnostic identification of conformational diseases or diseases associated with amyloid formation, such as Alzheimer or diabetes. It is clear that this diagnostic use is also useful for other diseases which involve cross- ⁇ structure formation, like all amyloidosis-type diseases, atherosclerosis, diabetes, bleeding, cancer, sepsis and other inflammatory diseases, Multiple Sclerosis, auto-immune diseases, disease associated with loss of memory or Parkinson and other neuronal diseases (epilepsy).
  • a finger domain of, for example, tPA
  • a label radioactive, fluorescent, etc.
  • This labeled finger domain may be used either in vitro or in vivo for the detection of cross- ⁇ structure-comprising proteins, hence, for determining the presence of a plaque involved in a conformational disease.
  • this invention discloses a method for inhibiting the formation of amyloid fibrils or to modulate cross- ⁇ structure-induced toxicity.
  • the compound is a cross- ⁇ -binding module, such as a finger domain, a finger domain-containing molecule, a peptidomimetic analog, and/or an anti-cross- ⁇ structure antibody, and/or a multiligand receptor or a fragment thereof.
  • the inhibition of fibril formation has the consequence of decreasing the load of fibrils.
  • the inhibition of fibril formation or modulating cross- ⁇ structure toxicity may also have the consequence of modulating cell death.
  • the cell can be any cell, but may be a neuronal cell, an endothelial cell, or a tumor cell.
  • the cell can be a human cell or a cell from any other species.
  • the cell may typically be present in a subject.
  • the subject to which the compound is administered may be a mammal or a human.
  • the subject may be suffering from amyloidosis, from another conformational disease, from prion disease, from chronic renal failure and/or dialysis-related amyloidosis, from atherosclerosis, from cardiovascular disease, from autoimmune disease, or the subject may be obese.
  • the subject may also be suffering from inflammation, rheumatoid arthritis, diabetes, retinopathy, sepsis, diffuse intravascular coagulation, hemolytic uremic syndrome, and/or preeclampsia.
  • the diseases which may be treated or prevented with the methods of the present invention include, but are not limited to, diabetes, Alzheimer disease, senility, renal failure, hyperlipidemic atherosclerosis, neuronal cytotoxicity, Down's syndrome, dementia associated with head trauma, amyotrophic lateral sclerosis, multiple sclerosis, amyloidosis, an autoimmune disease, inflammation, a tumor, cancer, male impotence, wound healing, periodontal disease, neuropathy, retinopathy, nephropathy or neuronal degeneration.
  • the administration of compounds according to the invention may be constant or for a certain period of time.
  • the compound may be delivered hourly, daily, weekly, monthly (e.g., in a time release form) or as a one time delivery.
  • the delivery may also be continuous, e.g., intravenous delivery.
  • a carrier may also be used to deliver the compound to a subject.
  • the carrier may be a diluent, an aerosol, an aqueous solution, a non-aqueous solution, or a solid carrier.
  • This invention also discloses pharmaceutical compositions including therapeutically effective amounts of polypeptide compositions and compounds, together with suitable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers.
  • compositions may be 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), antioxidants (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, hydro
  • the administration of compounds according to the invention may comprise intralesional, intraperitoneal, intramuscular or intravenous injection; infusion; liposome-mediated delivery; topical, intrathecal, gingival pocket, per rectum, intrabronchial, nasal, oral, ocular or otic delivery.
  • the administration includes intrabronchial administration, anal, intrathecal administration or transdermal delivery.
  • the compounds may be administered hourly, daily, weekly, monthly or annually.
  • the effective amount of the compound comprises from about 0.000001 mg/kg body weight to about 100 mg/kg body weight.
  • Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils).
  • particulate compositions coated with polymers e.g., poloxamers or poloxalenes
  • the agent coupled to antibodies directed against tissue-specific receptors, ligands or antigens or coupled to ligands of tissue-specific receptors e.g., IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, etc.
  • 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/or oral.
  • the effective amount of the compounds according to the invention may comprise 1 ng/kg body weight to about 1 gr/kg body weight.
  • the actual effective amount will be based upon the size of the compound and its properties.
  • B-type carboxypeptidases including, but not limited to, carboxypeptidase B (CpB) or Thrombin Activatable Fibrinolysis Inhibitor (TAFI, also named carboxypeptidase U or carboxypeptidase R), are enzymes that cleave off carboxy-terminal lysine and arginine residues of fibrin fragments that would otherwise bind to tPA and/or plasminogen and stimulate plasmin formation.
  • cross- ⁇ structures are harmful when present in certain parts of the body like, for example, the brain and the damage is effected by the combination of cross- ⁇ structures with tPA
  • a method is disclosed to inhibit cross- ⁇ structure-mediated effects comprising providing an effective amount of a protein comprising a finger domain to block the binding sites of the cross- ⁇ structure for tPA.
  • the cross- ⁇ structure-mediated effects may even be further diminished comprising providing an effective amount of B-type carboxypeptidase activity to inhibit the tPA activity.
  • the invention discloses the use of a compound capable of binding to a cross- ⁇ structure for the removal of cross- ⁇ structures.
  • the compound or means for binding the cross- ⁇ structure is a cross- ⁇ -binding molecule, such a protein and/or a functional equivalent and/or a functional fragment thereof.
  • the compound comprises a finger domain or a finger domain-containing molecule or a functional equivalent or a functional fragment thereof.
  • the finger domain is derived from fibronectin, FXII, HGFa or tPA. It is clear that the invention also comprises antibodies that bind cross- ⁇ structures.
  • the protein is an antibody and/or a functional equivalent and/or a functional fragment thereof.
  • the invention discloses, for example, a therapeutic method to remove cross- ⁇ structure-comprising proteins from, for example, the circulation, such as via extracorporeal dialysis.
  • a patient with sepsis is subjected to such use by dialysis of blood of the patient through means which are provided with, for example, immobilized finger domains.
  • all cross- ⁇ structure-comprising proteins will be removed from the bloodstream of the patient, thus, relieving the patient of the negative effects caused by the cross- ⁇ structure-comprising proteins.
  • finger domain-comprising compounds it is also possible to use other cross- ⁇ structure-binding compounds, like antibodies or soluble multiligand receptors. It is also clear that the use could be applied in hemodialysis of kidney patients.
  • finger encompasses a sequence that fulfills the criteria outlined in FIG. 14 .
  • the sequence encompasses approximately 50 amino acids, containing four cysteine residues at distinct spacing.
  • the finger domains of tPA, FXII, HGFa or fibronectin are used (SEQ ID NOs: 3-17).
  • the “finger” may be a polypeptide analog or peptidomimetic with similar function, e.g., by having three-dimensional conformation. It is feasible that such analogs have improved properties.
  • Bovine serum albumin (BSA) fraction V pH 7.0 and D-glucose-6-phosphate di-sodium (g6p), D, L-glyceraldehyde, and chicken egg-white lysozyme were from ICN (Aurora, Ohio, USA).
  • Rabbit anti-recombinant tissue-type plasminogen activator (tPA) 385R and mouse anti-recombinant tPA 374B were purchased from American Diagnostica (Veenendaal, The Netherlands).
  • Anti-laminin (L9393) was from Sigma.
  • Swine anti-rabbit immunoglobulins/HRP (SWARPO) and rabbit anti-mouse immunoglobulins/HRP (RAMPO) were from DAKO Diagnostics B.V.
  • Congo red was obtained from Aldrich (Milwaukee, Wisc., USA).
  • Thioflavin T and lyophilized human hemoglobin (Hb) were from Sigma (St. Louis, Mo., USA). Lyophilized human fibrinogen was from Kordia (Leiden, The Netherlands).
  • Chromogenic plasmin substrate S-2251 was purchased from Chromogenix (Milan, Italy).
  • Oligonucleotides were purchased from Sigma-Genosys (U.K.). Boro glass capillaries (0.5 mm ⁇ ) were from Mueller (Berlin, Germany).
  • Peptide A ⁇ (1-40), containing amino acids as present in the described human Alzheimer peptide (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV) (SEQ ID NO: 18), fibrin peptides 85 (or FP13) (KRLEVDIDIKIRS) (SEQ ID NO: 19), 86 (or FP12) (KRLEVDIDIKIR) (SEQ ID NO: 20) and 87 (or FP10) (KRLEVDIDIK) (SEQ ID NO: 21), derived from the sequence of human fibrin(ogen) and the islet amyloid polypeptide (IAPP) peptide or derivatives (fl-hIAPP: KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY (SEQ ID NO: 22), ⁇ hIAPP (SNNFGAILSS) (SEQ ID NO:23 ), ⁇ mIAPP (SNNLGPVLPP) (SEQ ID NO: 24) were obtained from Pepscan
  • Apparatus settings excitation at 435 nm (slit 10 nm), emission at 485 nm (slit 10 nm), PMT voltage 950 V, measuring time 10 seconds, delay 0 seconds.
  • a fibrin clot was formed at room temperature as described above (Thioflavin T was omitted in the buffer). The clot was incubated with Congo red solution and washed according to the manufacturer's recommendations (Sigma Diagnostics, MO, USA). The clot was analyzed under polarized light.
  • albumin-g6p For preparation of advanced glycation end-product modified bovine serum albumin (albumin-g6p), 100 mg ml ⁇ 1 of albumin was incubated with PBS containing 1 M of g6p and 0.05% m/v NaN 3 , at 37° C. in the dark. One albumin solution was glycated for two weeks, a different batch of albumin-was glycated for four weeks. Glycation was prolonged up to 23 weeks with part of the latter batch. Human Hb at 5 mg ml ⁇ 1 was incubated for ten weeks at 37° C. with PBS containing 1 M of g6p and 0.05% m/v of NaN 3 .
  • a Hb solution of 50 mg ml ⁇ 1 was incubated for eight weeks with the same buffer.
  • glyceraldehyde-modified albumin albumin-glyceraldehyde
  • chicken egg-white lysozyme lysozyme-glyceraldehyde
  • filter-sterilized protein solutions 15 mg ml ⁇ 1 were incubated for two weeks with PBS containing 10 mM of glyceraldehyde.
  • g6p or glyceraldehyde was omitted in the solutions.
  • albumin and lysozyme solutions were extensively dialyzed against distilled water and subsequently stored at ⁇ 20° C.
  • Protein concentrations were determined with Advanced protein-assay reagent ADV01 (Cytoskeleton, Denver, Colo., USA). Glycation was confirmed by measuring intrinsic fluorescent signals from advanced glycation end-products; excitation wavelength 380 nm, emission wavelength 435 nm.
  • Bovine albumin has 83 potential glycation sites (59 lysine and 23 arginine residues, N-terminus). Albumin was glycated for two weeks (albumin-AGE:2), four weeks (albumin-AGE:4) or 23 weeks (albumin-AGE:23).
  • albumin was incubated for 86 weeks with 1 M g6p, 250 mM DL-glyceraldehyde (ICN, Aurora, Ohio, USA)/100 mM NaCNBH 3 , 1 M ⁇ -D-( ⁇ )-fructose (ICN, Aurora, Ohio, USA), 1 M D(+)-glucose (BDH, Poole, England), 500 mM glyoxylic acid monohydrate (ICN, Aurora, Ohio, USA)/100 mM NaCNBH 3 , and corresponding PBS and PBS/NaCNBH 3 buffer controls.
  • Human Hb was isolated from erythrocytes in EDTA-anticoagulated blood of three healthy individuals and of 16 diabetic patients. 100 ⁇ l of whole blood was diluted in 5 ml of physiological salt (154 mM NaCl), cells were gently spun down, and resuspended in 5 ml of physiological salt. After 16 hours incubation at room temperature, cells were again spun down. Pelleted cells were lysed by adding 2 ml of 0.1 M of boric acid, pH 6.5 and subsequently, cell debris was spun down. Supernatant was collected and stored at ⁇ 20° C.
  • physiological salt 154 mM NaCl
  • Endostatin was purified from Escherichia coli essentially as described. 47
  • B121.DE3 bacteria expressing endostatin were lysed in a buffer containing 8 M urea, 10 mM Tris (pH 8.0), 10 mM imidazole and 10 mM ⁇ -mercapto-ethanol.
  • the protein sample was extensively dialyzed against H 2 O. During dialysis, endostatin precipitates as a fine white solid. Aliquots of this material were either stored at ⁇ 80° C. for later use, or were freeze-dried prior to storage. Soluble endostatin produced in the yeast strain Pichia pastoris was kindly provided by Dr.
  • Aggregated endostatin was prepared from soluble endostatin as follows. Soluble yeast endostatin was dialyzed overnight in 8 M urea and subsequently three times against H 2 O. Like bacterial endostatin, yeast endostatin precipitates as a fine white solid.
  • Freeze-dried bacterial endostatin was resuspended in either 0.1% formic acid (FA) or in dimethyl-sulfoxide and taken up in a glass capillary. The solvent was allowed to evaporate and the resulting endostatin material was stained with Congo red according to the manufacturer's protocol (Sigma Diagnostics, St. Louis, Mo., USA).
  • UV circular dichroism (CD) spectra of peptide and protein solutions were measured on a JASCO J-810 CD spectropolarimeter (Tokyo, Japan). Averaged absorption spectra of five or ten single measurements from 190-240 nm or from 190-250 nm, for fibrin peptides 85, 86, 87 or for albumin, glycated albumin and human A ⁇ (16-22), respectively, are recorded.
  • the CD spectrum of A ⁇ (16-22) was measured as a positive control.
  • a ⁇ (16-22) readily adopts amyloid fibril conformation with cross- ⁇ structure when incubated in H 2 O 45 .
  • relative percentage of the secondary structure elements present was estimated using k2d software. 48
  • Endostatin, hemoglobin and albumin samples were applied to 400 mesh specimen grids covered with carbon-coated collodion films. After five minutes, the drops were removed with filter paper and the preparations were stained with 1% methylcellulose and 1% uranyl acetate. After washing in H 2 O, the samples were dehydrated in a graded series of EtOH and hexanethyldisilazane. Transmission electron microscopy (TEM) images were recorded at 60 kV on a JEM-1200EX electron microscope (JEOL, Japan).
  • TEM Transmission electron microscopy
  • Enzyme-Linked Immunosorbent Assay Binding of tPA to Glycated Albumin, Hb and A ⁇ (1-40)
  • Binding of tPA to albumin-g6p (four-week and 23-week incubations), albumin-glyceraldehyde, control albumin, human Hb-g6p (ten-week incubation), Hb control, or to A ⁇ (1-40) was tested using an enzyme-linked immunosorbent assay (ELISA) set-up.
  • Albumin-g6p and control albumin (2.5 ⁇ g ml ⁇ 1 in coat buffer, 50 mM Na 2 CO 3 /NaHCO 3 pH 9.6, 0.02% m/v NaN 3 , 50 ⁇ l/well) were immobilized for one hour at room temperature in 96-well protein Immobilizer plates (Exiqon, Vedbaek, Denmark).
  • a ⁇ (1-40) (10 ⁇ g ml ⁇ 1 in coat buffer) was immobilized for 75 minutes at room temperature in a 96-well peptide Immobilizer plate (Exiqon, Vedbaek, Denmark). Control wells were incubated with coat buffer only. After a wash step with 200 ⁇ l of PBS/0.1% v/v Tween 20, plates were blocked with 300 ⁇ l of PBS/1% v/v Tween 20, for two hours at room temperature while shaking. All subsequent incubations were performed in PBS/0.1% v/v Tween 20 for one hour at room temperature while shaking, with volumes of 50 ⁇ l per well.
  • ⁇ ACA ⁇ -amino caproic acid
  • Albumin-g6p 500 nM
  • Thioflavin T 10 ⁇ M
  • tPA 50 mM glycine-NaOH pH 9
  • Absorbance measurements were performed at the albumin-g6p Thioflavin T absorbance maximum at 420 nm. Samples were prepared in four-fold. For blank readings, albumin-g6p was omitted in the solutions. Absorbance was read in a quartz cuvette on a Pharmacia Biotech Ultrospec 3000 UV/visible spectrophotometer (Cambridge, England).
  • Plasminogen 200 ⁇ g ml ⁇ 1 was incubated with tPA (200 pM) in the presence or the absence of a co-factor (5 ⁇ M of either endostatin, A ⁇ (1-40), or one the fibrin-derived peptides 85, 86 and 87).
  • a co-factor 5 ⁇ M of either endostatin, A ⁇ (1-40), or one the fibrin-derived peptides 85, 86 and 87.
  • samples were taken and the reaction was stopped in a buffer containing 5 mM EDTA and 150 mM ⁇ ACA.
  • a chromogenic plasmin substrate S-2251 was added and plasmin activity was determined kinetically in a spectrophotometer at 37° C.
  • N1E-115 mouse neuroblastoma cells were routinely cultured in DMEM containing 5% FCS, supplemented with antibiotics. Cells were differentiated into post-mitotic neurons. 52 The cells were exposed to A ⁇ (50 ⁇ g ml ⁇ 1 ) for 24 hours in the presence or absence of 20 ⁇ g ml ⁇ 1 plasminogen in the presence or absence of 50 ⁇ g ml ⁇ 1 CpB. Cells were photographed, counted and lysed by the addition of 4 ⁇ sample buffer (250 mM Tris pH 6.8, 8% SDS, 10% glycerol, 100 mM DTT, 0.01% w/v bromophenol blue) to the medium.
  • 4 ⁇ sample buffer 250 mM Tris pH 6.8, 8% SDS, 10% glycerol, 100 mM DTT, 0.01% w/v bromophenol blue
  • FXII binding buffer included 10 mM HEPES pH 7.3, 137 mM NaCl, 11 mM D-glucose, 4 mM KCl, 1 mg ml ⁇ 1 albumin, 50 ⁇ M ZnCl 2 , 0.02% (m/v) NaN 3 and 10 mM ⁇ -amino caproic acid ( ⁇ ACA).
  • Lysine analogue ⁇ ACA was added to avoid putative binding of FXII to cross- ⁇ structure via the FXII kringle domain.
  • binding of FXII to hA ⁇ (1-40) and the prototype amyloid human amylin fragment h ⁇ IAPP was tested using dot blot analysis. 10 ⁇ g of the peptides that contain cross- ⁇ structure, as well as the negative control peptide m ⁇ IAPP and phosphate-buffered saline (PBS) were spotted in duplicate onto methanol-activated nitrocellulose. Spots were subsequently incubated with 2 nM FXII in FXII buffer or with FXII buffer alone, anti-FXII antibody, and SWARPO.
  • Coated hA ⁇ (1-40) or amyloid albumin-AGE were incubated with 2.5 nM or 15 nM FXII in binding buffer in the presence of a concentration series of human recombinant tissue-type plasminogen activator (ACTILYSE®, full-length tPA) or RETEPLASE® (K2P-tPA).
  • RETEPLASE® is a truncated form of tPA that includes the second kringle domain and the protease domain.
  • the f.l. tPA and K2P-tPA concentration was at maximum 135 times the k D for tPA binding to hA ⁇ (1-40) (50 nM) or 150 times the k D for tPA binding to albumin-AGE (1 nM).
  • Oligonucleotides used were 5′AAAAGTCGACAGCCGCCACCATGGATGCAATGAAGAGA (SEQ ID NO: 25) (1) and 3′AAAAGCGGCCGCCCACTTTTGACAGGCACTGAG (SEQ ID NO: 26) (2) comprising a SalI or a NotI restriction site, respectively (underlined).
  • the PCR product was cloned in a SalI/NotI-digested expression vector, pMT2-GST.
  • a construct is generated that contains a SalI restriction site, the coding sequence for the finger domain of tPA, a NotI and a KpnI restriction site, a thrombin cleavage-site (TCS), a glutathion-S-transferase (GST) tag and an EcoRI restriction site.
  • TCS thrombin cleavage-site
  • GST glutathion-S-transferase
  • HindIII-SalI-tPA pro-peptide-BglII-F-NotI-KpnI-TCS-GST-EcoRI construct was used as a cloning cassette for preparation of constructs containing tPA K1, F-EGF-K1, EGF, as well as human hepatocyte growth factor activator F and F-EGF, human factor XII F and F-EGF, and human fibronectin F4, F5, F4-5 and F10-12. Subsequently, constructs were ligated HindIII-EcoRI in the pcDNA3 expression vector (Invitrogen, Breda, The Netherlands).
  • 293T cells were grown in RPMI1640 medium (Invitrogen, Scotland, U.K.) supplemented with 5% v/v fetal calf-serum, penicillin, streptomycin and guanidine, to 15% confluency. Cells were transiently transfected using Fugene-6, according to the manufacturer's recommendations (Roche, Ind., USA).
  • pMT2-tPA-F-GST containing the tPA fragment, or a control plasmid, pMT2-RPTP ⁇ -GST, containing the extracellular domain of receptor-like protein tyrosine phosphatase [ (RPTP ⁇ ) 54 were transfected, and medium was harvested after 48 hours transfection.
  • tPA-F-GST and RPTP ⁇ -GST in 293T medium were verified by immunoblotting. Collected samples were run out on SDS-PAA gels after the addition of 2 ⁇ sample buffer. Gels were blotted on nitrocellulose membranes. Membranes were blocked in 1% milk (Nutricia) and incubated with primary monoclonal anti-GST antibody 2F3 54 and secondary HRP-conjugated rabbit anti-mouse IgG (RAMPO). The blots were developed using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, MA, USA).
  • Baby hamster kidney cells were seeded in DMEM/NUT mix F-12 (HAM) medium (Invitrogen, U.K.) supplemented with 5% v/v fetal calf-serum (FCS), penicillin, streptomycin and guanidine, to 1% confluency.
  • Cells were stably transfected by using the Ca 3 (PO 4 ) 2 —DNA precipitation method. After 24 hours, medium was supplemented with 1 mg ml ⁇ 1 geneticin G-418 sulphate (Gibco, U.K.). Medium with G-418 was refreshed several times during ten days to remove dead cells. After this period of time, stable single colonies were transferred to new culture flasks and cells were grown to confluency.
  • constructs were verified by immunoblotting. Collected samples were run out on SDS-PAA gels after the addition of 2 ⁇ sample buffer. Gels were blotted on nitrocellulose membranes. Membranes were blocked in 5% milk (Nutricia) with 1.5% mn/v BSA and incubated with primary monoclonal anti-GST antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., USA, catalogue # Z-5), and secondary HRP-conjugated rabbit anti-mouse IgG (RAMPO). The blots were developed using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, MA, USA). Stable clones were grown in the presence of 250 ⁇ g ml ⁇ 1 G-418.
  • conditioned medium with 5% FCS of stable clones that produce constructs of interest was used.
  • cells of a stable clone of tPA F-EGF-GST were transferred to triple-layered culture flasks and grown in medium with 0.5% v/v Ultroser G (ITK Diagnostics, Uithoorn, The Netherlands). Medium was refreshed every three to four days.
  • TPA F-EGF-GST was isolated from the medium on a Glutathione Sepharose 4B (Amersham Biosciences, Uppsala, Sweden) column and eluted with 100 mM reduced glutathione (Roche Diagnostics, Mannheim, Germany).
  • Conditioned medium of BHK cells expressing GST-tagged tPA F, F-EGF, EGF, K1, F-EGF-K1, FXII F, HGFa F, Fn F4, Fn F5, Fn F4-5 and GST was used for amyloid binding assays.
  • constructs were adjusted to approximately equal concentration using Western blots.
  • Qualitative binding of the recombinant fragments are evaluated using a “pull-down” assay.
  • the recombinantly made fragments are incubated with either A ⁇ or IAPP fibrils. Since these peptides form insoluble fibers, unbound proteins can be easily removed from the fibers following centrifugation. The pellets containing the bound fragments are subsequently washed several times. Bound fragments are solubilized in SDS-sample buffer and analyzed by PAGE, as well as unbound proteins in the supernatant fraction and starting material. The gels are analyzed using immunoblotting analysis with the anti-GST antibody Z-5.
  • Amyloid ELISA with tPA F-EGF-GST Amyloid ELISA with tPA F-EGF-GST
  • ELISAs were performed with immobilized amyloid (poly)peptides and non-amyloid control ⁇ mIAPP. Twenty-five ⁇ g ml ⁇ 1 of A ⁇ , FP13, IAPP or ⁇ mIAPP was immobilized on Exiqon peptide immobilizer plates. A concentration series of tPA F-EGF-GST in the presence of excess ⁇ ACA was added to the wells and binding was assayed using anti-GST antibody Z-5. As a control, GST (Sigma-Aldrich, St. Louis, Mo., USA, catalogue # G-5663) was used at the same concentrations.
  • Paraffin brain sections of a human inflicted with AD was a kind gift of Prof. Slootweg (Dept. of Pathology, UMC Utrecht). Sections were deparaffinized in a series of xylene-ethanol. Endogenous peroxidases were blocked with methanol/1.5% H 2 O 2 for 15 minutes. After rinsing in H 2 O, sections were incubated with undiluted formic acid for ten minutes, followed by incubation in PBS for five minutes. Sections were blocked in 10% HPS in PBS for 15 minutes. Sections were exposed for two hours with 7 nM of tPA F-EGF-GST or GST in PBS/0.3% BSA.
  • Sections were cleared in xylene and mounted with D.P.X. Mounting Medium (Nustain, Nottingham, U.K.). Analysis of sections was performed on a Leica DMIRBE fluorescence microscope (Rijswijk, The Netherlands). Fluorescence of Congo red was analyzed using an excitation wavelength of 596 nm and an emission wavelength of 620 nm.
  • ELISA Binding of tPA-F-GST and RPTP ⁇ -GST to Human Ab (1-40) and Glycated Albumin
  • Binding of tPA-F-GST and RPTP ⁇ -GST to fibrous amyloids human A ⁇ (1-40) and albumin-g6p was assayed with an ELISA.
  • human A ⁇ (1-40), albumin-g6p, or buffer only were coated on a peptide I Immobilizer or a protein I Immobilizer, respectively.
  • Wells were incubated with the purified GST-tagged constructs or control medium and binding was detected using primary anti-GST monoclonal antibody 2F3 and RAMPO. The wells were also incubated with 500 mM of tPA in the presence of 10 mM of ⁇ ACA. Binding of tPA is independent of the lysyl binding site located at the kringle 2 domain. Binding of tPA was measured using primary antibody 374B and RAMPO. Experiments were performed in triplicate and blank readings of non-coated wells were substracted.
  • Antibodies against glucose-6-phosphate glycated bovine serum albumin were elicited in rabbits using standard immunization schemes.
  • Anti-AGE1 was obtained after immunization with two-week glycated albumin-AGE (Prof. Dr. Ph.G. de Groot/Dr. I. Bobbink; unpublished data). The antibody was purified from serum using a Protein G column.
  • Anti-AGE2 was developed by Davids Biotechnologie (Regensburg, Germany). After immunization with albumin-AGE:23, antibodies were affinity purified on human A ⁇ (1-40) conjugated to EMD Epoxy-activated beads (Merck, Darmstadt, Germany).
  • Polyclonal mouse anti-AGE antibody was obtained after immunization with albumin-AGE:23 and human A ⁇ (1-40) in a molar ratio of 9:1.
  • Polyclonal serum was obtained using standard immunization procedures, which were performed by the Academic Biomedical Cluster Hybridoma Facility (Utrecht University, The Netherlands). Subsequently, monoclonal antibodies were generated using standard procedures.
  • ELISA Binding of Antibodies Against Amyloid Peptides or Glycated Protein to Protein-AGE and Amyloid Fibrils
  • amyloid compounds were immobilized on Exiqon peptide or protein Immobilizers (Vedbaek, Denmark), as described before.
  • Anti-AGE antibodies and commercially available anti-A ⁇ (1-42) H-43 were diluted in PBS with 0.1% v/v Tween 20.
  • Rabbit anti-human vitronectin K9234 was a kind gift of Dr. H. de Boer (UMC Utrecht) and was used as a negative control.
  • UMC Utrecht UMC Utrecht
  • Binding of mouse polyclonal anti-albumin-AGE/A ⁇ was performed using a dilution series of serum in PBS/0.1% Tween 20.
  • IAPP immunosorbent protein
  • anti-AGE1 was pre-incubated with varying IAPP concentrations.
  • the IAPP fibrils were spun down and the supernatant was applied in triplicate to wells of an ELISA plate coated with A ⁇ .
  • Competitive binding assays with multiligand cross- ⁇ structure binding serine protease tPA were performed in a slightly different way. Coated A ⁇ and IAPP are overlayed with an anti-AGE1 or anti-A ⁇ (1-42) H-43 concentration related to the k D , together with a concentration series of tPA.
  • Anti-AGE1 was incubated with amyloid aggregates of A ⁇ (16-22), A ⁇ (1-40) and IAPP. After centrifugation, pellets were washed three times with PBS/0.1% Tween 20, dissolved in non-reducing sample buffer (1.5% (mn/v) sodium dodecyl sulphate, 5% (v/v) glycerol, 0.01% (m/v) bromophenol blue, 30 mM Tris-HCl pH 6.8). Supernatant after pelleting of the amyloid fibrils was diluted 1:1 with 2 ⁇ sample buffer. Samples were applied to a polyacrylamide gel and after Western blotting, anti-AGE1 was detected with SWARPO.
  • Rabbit anti-AGE2 affinity purified on an A ⁇ column, was used for assaying binding properties towards amyloid plaques in brain sections of a human with AD. The procedure was performed essentially as described above. To avoid eventual binding of 11 ⁇ g ml ⁇ 1 anti-AGE2 to protein-AGE adducts or to human albumin in the brain section, 300 nM of g6p-glycated dipeptide Gly-Lys was added to the binding buffer, together with 0.3% m/v BSA. After the immunohistochemical stain, the section was stained with Congo red.
  • binding of amyloid structures was detected upon incubation with 1 ⁇ g ml ⁇ 1 anti-A ⁇ (1-42) H-43 (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) and subsequently 0.3 ⁇ g ml ⁇ 1 SWARPO, followed by ortho-phenylene-diamine/H 2 O 2 /H 2 SO 4 stain.
  • a ⁇ Contains Cross- ⁇ Structure, Binds Plasmin(ogen) and tPA, Stimulates Plasminogen Activation, Induces Matrix Degradation and Induces Cell Detachment that is Aggravated by Plasminogen and Inhibited by CpB
  • FIG. 3 Panel A, shows that tPA binds to A ⁇ with a k D of about 7 nM, similar to the k D of tPA binding to fibrin. 55 In contrast, No detectable binding of plasminogen to A ⁇ was found ( FIG. 3 , Panel B).
  • activated plasminogen does bind to A ⁇ and does so with a k D of 47 nM.
  • the fact that (active) plasmin, but not (inactive) plasminogen, binds to A ⁇ suggests that plasmin activity and, hence, the generation of free lysines, is important for binding of plasmin to A ⁇ .
  • ⁇ ACA lysine analogue ⁇ -aminocaproic acid
  • ⁇ ACA has no effect on the tPA-A ⁇ interaction ( FIG. 3 , Panel C).
  • plasmin binds to free lysines that were generated during the incubation period, presumably via its lysine-binding Kringle domain(s).
  • the k D of plasminogen Kringle domain binding to free lysines in fibrin is similar to the k D for plasmin binding to A ⁇ .
  • plasminogen In the absence of A ⁇ , plasminogen has no effect on cell adhesion ( FIG. 4 , Panel C). However, plasminogen has a dramatic potentiating effect on A ⁇ -induced cell detachment. The minimal levels of plasminogen that are required to potentiate A ⁇ -induced cell detachment (10-20 ⁇ g/ml) are well below those found in human plasma (250 ⁇ g/ml). Plasmin-mediated degradation of the extracellular matrix molecule laminin precedes neuronal detachment and cell death in ischemic brain. Whether A ⁇ -stimulated plasmin generation leads to laminin degradation was tested. Cell detachment was accompanied by degradation of the extracellular matrix protein laminin ( FIG. 4 , Panel D).
  • CpB carboxypeptidase B
  • FIG. 5 Panel A, shows that in the presence of CpB, the generation of plasmin is greatly diminished. Furthermore, this effect depends on CpB activity as it is abolished by co-incubation with CPI.
  • FIG. 5 Panel A, also shows that CpB does not completely abolish A ⁇ -stimulated plasmin generation, but that the reaction proceeds with slow first-order kinetics.
  • Endostatin can Form Amyloid Fibrils Comprising Cross- ⁇ Structure
  • endostatin is an example of a denatured protein that is able to stimulate the suggested cross- ⁇ pathway.
  • Amyloid deposits of IAPP are formed in the pancreas of type II diabetic patients. 59 IAPP can cause cell death in vitro and is, therefore, thought to contribute to destruction of ⁇ -cells that is seen in vivo, which leads to insufficient insulin production. IAPP forms fibrils comprising cross- ⁇ structure. 60
  • IAPP could stimulate tPA-mediated plasminogen activation was tested ( FIG. 7 ). Indeed, similar to A ⁇ , IAPP can enhance the formation of plasmin by tPA.
  • glycation of several proteins can induce or increase the ability of these proteins to bind tPA and stimulate tPA-mediated plasmin formation. 22,61 It is shown herein that glycation of albumin with g6p not only confers high-affinity tPA binding to albumin ( FIG. 8 , Panel A), but also leads to its ability to bind Thioflavin T ( FIG. 8 , Panel C). Binding of tPA can be competed with Congo red ( FIG. 8 , Panel B). In addition, binding of Thioflavin T to glycated albumin can be competed by tPA ( FIG. 8 , Panels D and E). The fact that Congo red and/or Thioflavin T and tPA compete illustrates that they have either the same or overlapping binding sites.
  • albumin-g6p (23 weeks) comprises a significant amount of crystalline fibers ( FIG. 8 , Panels J and L), whereas diffraction patterns of albumin-g6p (2 weeks) and albumin-g6p (4 weeks) show features originating from amorphous precipitated globular protein very similar to the patterns obtained for albumin controls ( FIG. 8 , Panel K).
  • the 4.7 ⁇ repeat corresponds to the characteristic hydrogen-bond distance between ⁇ -strands in ⁇ -sheets.
  • the 2.3 and 3.3 ⁇ repeats have a preferred orientation perpendicular to the 4.7 ⁇ repeat ( FIG. 8 , Panel M).
  • Amyloid Albumin is Formed Irrespective of the Original Carbohydrate (Derivative)
  • Glyceraldehyde and glyoxylic acid are carbohydrate derivatives that are precursors of AGE in Maillard reactions. 63,64 After 86 weeks, albumin-glyceraldehyde and albumin-fructose were light-yellow/brown suspensions. Controls were colorless and clear solutions.
  • Albumin-glucose and albumin-glyoxylic acid were clear light-yellow to light-brown solutions.
  • Albumin-g6p:86 was a clear and dark brown solution.
  • AGE formation was confirmed by autofluorescence measurements using AGE-specific excitation/emission wavelengths (not shown), binding of moab anti-AGE 4B5 (not shown) and binding of poab anti-AGE (not shown).
  • albumin-glyoxylic acid did not show an autofluorescent signal due to the fact that (mainly) non-fluorescent carboxymethyl-lysine (CML) adducts are formed.
  • CML carboxymethyl-lysine
  • FIG. 10 Panels A, B and I.
  • These ThT and Congo red fluorescence data show that, in addition to albumin-g6p, albumin-glyceraldehyde, albumin-glucose and albumin-fructose have amyloid-like properties.
  • binding of amyloid-specific serine protease tPA in an ELISA was tested for.
  • the enzyme bound specifically to albumin-g6p, albumin-glyceraldehyde, albumin-glucose and albumin-fructose ( FIG. 10 , Panels K and L) and to positive controls A ⁇ and IAPP, as was shown before. 49 No tPA binding is observed for albumin-glyoxylic acid and buffer controls.
  • the graphs in FIG. 12 show that FXII binds specifically to all amyloid compounds tested.
  • k D s for hA ⁇ (1-40), FP13, albumin-AGE and Hb-AGE are approximately 2, 11, 8 and 0.5 nM, respectively.
  • the data obtained with the competitive FXII—tPA ELISA show that tPA efficiently inhibits binding of FXII to amyloid (poly)peptides ( FIG. 12 ). From these data, it is concluded that FXII and f.l. tPA compete for overlapping binding sites on hA ⁇ (1-40). K2P-tPA does not inhibit FXII binding.
  • ELISA Binding of tPA-F-GST and RPTP-GST to human A ⁇ (1-40) and Glycated Albumin
  • cDNA constructs in pcDNA3 of the F, F-EGF, EGF, F-EGF-K1 and K1 fragments of human tPA was prepared.
  • Recombinant proteins with a C-terminal GST tag were expressed in BHK cells and secreted to the medium.
  • Medium from BHK cells expressing the GST tag alone was used as a control.
  • Conditioned medium was used for pull-down assays using A ⁇ and IAPP fibrils, followed by Western blot analyses. Efficient binding to A ⁇ is evident for all three tPA mutants that contain the finger domain, i.e., F-GST, F-EGF-GST and F-EGF-K1-GST ( FIG. 13 , Panel D).
  • the K1-GST and EGF-GST constructs, as well as the GST tag alone, remain in the supernatant after A ⁇ incubation.
  • a similar pattern was obtained after IAPP pull-downs (not shown).
  • tPA F-EGF-GST Binding of purified tPA F-EGF-GST, recombinant f.l. Actilyse tPA and a GST control to immobilized amyloid A ⁇ , amyloid fibrin fragment ⁇ 148-160 FP13, amyloid IAPP and to non-amyloid m ⁇ IAPP control was compared ( FIG. 13 , Panels E-G).
  • Full-length tPA and tPA F-EGF-GST bind to all three amyloid peptides; for A ⁇ k D s for tPA and F-EGF are 2 and 2 nM, respectively; for FP13, 5 and 2 nM; for IAPP, 2 and 13 nM.
  • tPA based on sequential and structural homology, next to tPA, three proteins are known that contain one or more finger domains, i.e., HGFa (one F domain), FXII (one F domain), Fn (one stretch of six F domains, two stretches of three F domains). From the above-listed results, it was concluded that the F domain of tPA (SEQ ID NO: 3) plays a crucial role in binding of tPA to amyloid (poly)peptides. It was hypothesized that the finger domain could be a general cross- ⁇ structure-binding module. Presently, four proteins, tPA, FXII, HGFa and fibronectin, are known that contain a finger motif. FIG.
  • FIG. 14 Panel A, schematically depicts the localization of the finger module in the respective proteins.
  • FIG. 14 , Panel B shows an alignment of the human amino acid sequences of the finger domains in these four proteins. (SEQ ID NOs: 3-17)
  • FIG. 14 , Panel C shows a schematic representation of the three-dimensional structure of the finger domain of tPA (SEQ ID NO: 3), and of the fourth and fifth finger domain of fibronectin (SEQ ID NOs: 9 and 10).
  • Fn F5-GST binds to A ⁇ to some extent, however, it is extracted less efficiently from the medium and seems to be party released during the washing procedure of the amyloid pellet ( FIG. 13 , Panel M).
  • No construct was left in the medium after extraction of positive control tPA F-EGF-GST, whereas no negative control GST was detected in the pellet fraction (not shown).
  • SEQ ID NO: 3 binding to amyloid (poly)peptides is not a unique capacity of the tPA F domain (SEQ ID NO: 3), yet a more general property of the F domains tested.
  • these data indicate that observed binding of FXII to amyloid (poly)peptides, as shown in FIG. 13 , Panels A and H, and by Shibayama et al., 65 is regulated via the F domain.
  • the finger domain of tPA has been shown to be of importance for high-affinity binding to fibrin. 12,66
  • RETEPLASE® K2-P tPA
  • F-tPA, F-EGF-tPA and F-EGF-K1-tPA indicate an important role for the N-terminal finger domain of tPA in binding to stimulatory factors other than fibrin.
  • all of these factors bind Congo red and contain cross- ⁇ structure.
  • the binding site of fibronectin for fibrin has been mapped to the finger domain tandem F4-F5.
  • plasminogen activation by full-length tPA, in the presence of fibrin fragment FCB2 can be inhibited by fibronectin.
  • Negative controls were non-glycated albumin and Hb, non-amyloid peptide mouse ⁇ IAPP for IAPP and polyclonal anti-human vitronectin antibody ⁇ -hVn K9234 for A ⁇ .
  • ELISAs with polyclonal mouse anti-albumin-AGE/A ⁇ show that the antibody not only binds to these antigens, but that it specifically binds to other amyloid peptides than those used for immunization ( FIG. 15 , Panels J-L). Similar to the rabbit anti-AGE1 antibody and anti-A ⁇ (1-42) H-43, anti-albumin-AGE/A ⁇ displays affinity for the amyloid peptides tested, irrespective of amino acid sequence. This suggests that also mouse anti-albumin-AGE/A ⁇ is a multiligand amyloid-binding antibody.
  • anti-amyloid and anti-AGE antibodies display affinity for a broad range of sequentially unrelated (poly)peptides as long as the cross- ⁇ structure fold is present, is in agreement with the recently published data by O'Nuallain and Wetzel 70 and Kayed et al. 71 From several older reports in literature, it can be distilled that anti-cross- ⁇ antibodies can be obtained. For example, cross-reactive antibodies against fibrin and A ⁇ and against A ⁇ and hemoglobin are described.
  • fibrinogen and hemoglobin-AGE adopt the cross- ⁇ structure fold, which suggests that the cross-reactivity observed for anti-A ⁇ antibodies was, in fact, binding of anti-cross- ⁇ structure antibodies to similar structural epitopes on A ⁇ , fibrinogen and hemoglobin.
  • the three-dimensional structures of the tPA finger domain 74,75 and the fibronectin finger domains 4-5 75,76 reveal striking structural homology with respect to local charge-density distribution. Both structures contain a similar solvent-exposed stretch of five amino acid residues with alternating charge; for tPA, Arg7, Glu9, Arg23, Glu32, Arg30; and for fibronectin, Arg83, Glu85, Lys87, Glu89, Arg90, located at the fifth finger domain, respectively.
  • the charged-residue alignments are located at the same side of the finger module. These alignments may be essential for fibrin binding.
  • cross- ⁇ structure pathway a general system, which is termed “cross- ⁇ structure pathway,” to remove unwanted biomolecules.
  • Hb A1c concentration is given as a percentage of the total amount of Hb present in erythrocytes of diabetes mellitus patients and of healthy controls. The s.d. is 2.3% of the values given. ⁇ Presence of fibers as determined with TEM.

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