METHODS OF REDUCING FACTOR VIII CLEARANCE AND COMPOSITIONS THEREFOR

Abstract: The present invention provides methods of increasing the half-life of factor VIII. More specifically, the invention provides methods of increasing the half-life of factor VIII by substituting amino acids in the A2 domain or in the C2 domain of factor VIII or in both domains. It further provides factor VIII mutants produced by these methods. The invention also provides a method of using receptor-associated protein (RAP) to increase the half-life of factor VIII. The invention also provides polynucleotides encoding the mutant factor VIII, polynucleotides encoding RAP, and methods of treating hemophilia using the polypeptides and polynucleotides of the invention.

Description: Methods of Reducing Factor VIII Clearance and Compositions Therefor Statement as to Rights to Inventions Made Under Federally-Sponsored Research and Development Part of the work performed during development of this invention utilized U.S. Government funds. The U.S. Government has certain rights in this invention. Background of the Invention Field of the Invention This invention relates generally to a mutant factor VIII having increased half-life, methods of production, pharmaceutically acceptable compositions and uses thereof. This invention also relates to a method of using receptor associated protein to increase the half-life of factor VIII, methods of production, pharmaceutically acceptable compositions and uses thereof. Related Art Coagulation of blood occurs by either the "intrinsic pathway" or the "extrinsic pathway," whereby certain blood proteins interact in a cascade of proteolytic activations to ultimately convert soluble fibrinogen to insoluble fibrin. These threads of fibrin are cross-linked to form the scaffolding of a clot; without fibrin formation, coagulation cannot occur. The intrinsic pathway consists of seven steps: ( 1 ) the proteolytic activation of factor XII; (2) activated factor XII cleaves factor XI to activate it; (3) activated factor XI cleaves factor IX, thereby activating it; (4) activated factor IX interacts with activated factor VIII to cleave and activate factor X; (5) activated factor X binds to activated factor V on a membrane surface, which complex proteoly tically cleaves prothrombin to form thrombin; (6) thrombin proteolytically cleaves fibrinogen to form fibrin; (7) fibrin monomers assemble into fibrils, which are then cross-linked by factor XIII. The extrinsic pathway consists of the following steps: (1) upon rupture of a blood vessel, factor VII binds to tissue factor, a lipoprotein present in tissues outside the vascular system; (2) factor VII is activated to factor Vila by proteolytic cleavage; and (3) the factor Vila-tissue factor complex cleaves and activates factor X. Thereafter, the extrinsic pathway is identical to the intrinsic pathway, i.e. the two pathways share the last three steps described above. The plasma glycoprotein factor VIII circulates as an inactive precursor in blood, bound tightly and non-covalently to von Willebrand factor. Factor VIII (fVIII) is proteolytically activated by thrombin or factor Xa, which dissociates it from von Willebrand factor (vWf) and activates its procoagulant function in the cascade. In its active form, factor Villa (fVIIIa) functions as a cofactor for the factor X activation enzyme complex in the intrinsic pathway of blood coagulation, and it is decreased or nonfunctional in patients with hemophilia A. In hemophilia, blood coagulation is impaired by a deficiency in certain plasma blood coagulation factors. People with deficiencies in factor VIII or with antibodies against factor VIII suffer uncontrolled internal bleeding that may cause a range of serious symptoms unless they are treated with factor VIII. Symptoms range from inflammatory reactions in joints to early death. The classic definition of factor VIII, in fact, is that substance present in normal blood plasma that corrects the clotting defect in plasma derived from individuals with hemophilia A. A deficiency in vWf can also cause phenotypic hemophilia A because vWf is an essential component of functional factor VIII. In these cases, the half-life of factor VIII is decreased to such an extent that it can no longer perform its particular functions in blood-clotting. The fVIII protein consists of a homologous A and C domains and a unique B domain which are arranged in the order A1-A2-B-A3-C1-C2 (Vehar, G.A., et al.. Nature 372:337-340 (1984)). It is processed to a series of Me 2+ linked heterodimers produced by cleavage at the B-A3 junction (Fay. PJ., et al, Biochem. Biophys. Ada. 871:268-218 (1986)), generating a light chain (LCh) consisting of an acidic region (AR) and A3, CI, and C2 domains and a heavy chain (HCh) which consists of the Al, A2, and B domains (Fig. 1). Activation of fVIII by thrombin leads to dissociation of activated fVIII (fVHIa) from vWf and at least a 100-fold increase of the cofactor activity. The fNIIIa is a A1/A2/A3-C1-C2 heterotrimer (Fay, PJ., et al, J. Biol. Chem 266:8957-8962 (1991)) in which domains Al and A3 retain the metal ion linkage (Fig. 1) and the stable dimer A1/A3-C1-C2 is weakly associated with the A2 subunit through electrostatic forces (Fay, PJ., et al. , J. Biol. Chem 266:8957-8962 (1991)). Spontaneous dissociation of the A2 subunit from the heterotrimer results in non-pro teoly tic inactivation of fVIIIa. Infusion of fVIII/vWf complex or purified plasma or recombinant fVIII into patients with severe hemophilia A who do not have fVIII (Fijnvandraat, K., et al, Thromb. Haemostas. 77:298-302 (1997); Morfini, M., et al, Thromb. Haemostas 65:433-435 (1992)) or in normal individuals (Over, J., et al.,J. Clin. Invest. 62:223-234 (1978)) results in a similar fVIII disappearance with a half-life of 12-14 hours. Although the complex between fVIII and vWf is crucial for normal half-life and level of factor VIII in the circulation, the mechanisms associated with turnover of fVIII/vWf complex are not wel) defined. The human factor VIII gene was isolated and expressed in mammalian cells (Toole, J. J., et al. , Nature 372:342-347 ( 1984); Gitschier, j.. et al. , Nature 312:326-330 (1984); Wood. W. I., et al. Nature 312:330-331 (1984); Vehar, G. A., et al., Nature 372:337-342 (1984); WO 87/04187; WO 88/08035; WO 88/03558; U.S. Pat. No. 4,757,006), and the amino acid sequence was deduced from cDNA. Capon etal., U.S. Pat. No. 4,965,199, disclose a recombinant DNA method for producing factor VIII in mammalian host cells and purification of human factor VIII. Human factor VIII expression in CHO (Chinese hamster ovary) cells and BHKC (baby hamster kidney cells) has been reported. Human factor VIII has been modified to delete part or all of the B domain (U.S. Pat. No. 4,868,1 12). and replacement of the human factor VIII B domain with the human factor V B domain has been attempted (U.S. Pat. No. 5.004,803). The cDNA sequence encoding human factor VIII and predicted amino acid sequence are shown in SEQ ID NOs: 1 and 2, respectively. U.S. Patent No.5,859,204, Lollar, J.S., describes mutants of human factor VIII having reduced antigenicity and reduced immunoreactivity. Porcine factor VIII has been isolated and purified from plasma (Fass, D. N., et al., Blood 59:594 (1982)). Partial amino acid sequence of porcine factor VIII corresponding to portions of the N-terminal light chain sequence having homology to ceruloplasmin and coagulation factor V and largely incorrectly located were described by Church, et al., Proc. Natl. Acad. Sci. USA 57:6934 (1984). Toole, J. J., et al., Nature 372:342-347 (1984) described the partial sequencing of the N-terminal end of four amino acid fragments of porcine factor VIII but did not characterize the fragments as to their positions in the factor VIII molecule. The amino acid sequence of the B and part of the A2 domains of porcine factor VIII were reported by Toole, J. J., et al., Proc. Natl. Acad. Sci. USA 53:5939-5942 (1986). The cDNA sequence encoding the complete A2 domain of porcine factor VIII and predicted amino acid sequence and hybrid human/porcine factor VIII having substitutions of all domains, all subunits, and specific amino acid sequences were disclosed in U.S. Pat. No. 5.364,771 by Lollar and Runge, and in WO 93/20093. More recently, the nucleotide and corresponding amino acid sequences of the A 1 and A2 domains of porcine factor VIII and a chimeric factor VIII with porcine Al and/or A2 domains substituted for the corresponding human domains were reported in WO 94/1 1503. U.S. Patent No. 5,859,204, Lollar, J.S., discloses the porcine cDNA and deduced amino acid sequences. Cellular endocytosis mediated by LRP was shown to be a mechanism of removal of a number of structurally unrelated ligands including several proteins related to coagulation or fibrilolysis. These ligands are: complexes of thrombin with antithrombin III (ATIII), heparin cofactor II (HC1 1) (Kounnas, M.Z., et al., J. Biol. Chem. 271:6523-6529 (1996)), protease nexin I (Knauer, M.F., et al, J. Biol. Chem. 272:12261-12264 (1997)), complexes of urokinase-type and tissue- type plasminogen activators (u-PA and t-PA, respectively) with plasminogen activator inhibitor (PAI-1) (Nykjaer, A., et al. , J. Biol. Chem. 267: 14543-14546 (1992); Orth, K., et al, Proc. Natl. Acad. Sci. 59:7422-7426 (1992)), thrombospondin (Mikhailenko, I., et al, J. Biol. Chem. 272:6784-6791 (1997)), tissue factor pathway inhibitor (TFPI) (Warshawsky, I., et al. , Proc. Natl. Acad. Sci. 97:6664-6668 (1994)), and factor Xa (Narita, M., et al, Blood 97:555-560 (1998); Ho, G., et al, J. Biol. Chem 277:9497-9502 (1996)). LRP, a large cell-surface glycoprotein identical to α 2 -macroglobulin receptor (Strickland, D.K., et al., J. Biol. Chem. 265:17401-17404 (1990)), is a member of the low density lipoprotein (LDL) receptor family which also includes the LDL receptor, very low density lipoprotein (VLDL) receptor, vitellogenin receptor and glycoprotein 330 receptor. LRP receptor consists of the non- covalently linked 515 kDa -chain(Herz, J.. et α/., E RO . 7:41 19-4127 (1988)) containing binding sites for LRP ligands, and the 85 kDa transmembrane β-chain. Within the -chain, cluster of cysteine-rich class A repeats is responsible for ligand binding (Moestrup, S. K., et al. J. Biol. Chem 265:13691-13696 (1993)). In contrast to the acidic ligand binding region in LRP, ligands of LRP expose regions rich in positively charged amino acid residues (Moestrup. S.K., Biochim. Biophys. Acta 1197: 197-213 (1994)). This type of binding and 31 class A repeats present in LRP may be responsible for its wide ligand diversity and ability to serve as a multi-ligand clearance receptor. LRP is expressed in many cell types and tissues including placenta, lung and brain (Moestrup, S.K., et al. , Cell Tissue Res. 269:315-382 (1992)) and is a major endocytic receptor in the liver (Strickland, D.K.. et al, FASEB J 9:890-898 (1995)). A 39 kDa receptor-associated protein (RAP) binds to LRP with high affinity (K d =4 nM (27)) and inhibits binding and LRP-mediated internalization and degradation of all ligands (Moestrup. S.K., Biochim. Biophys. Acta 1197:197- 213 (1994): Williams, S.Ε., et al, J. Biol. Chem. 267:9035-9040 (1992)). therefore serving as a useful tool for testing whether LRP is involved in endocytosis of a given ligand. Severe hemophiliacs, who number about 10,000 in the United States, can be treated with infusion of human factor VIII, vWf/factor VIII complex or vWf which will restore the blood's normal clotting ability if administered with sufficient frequency and concentration. However, supplies have been inadequate and problems in therapeutic use occur due to difficulty in isolation and purification, immunogenicity, and the necessity of removing the AIDS and hepatitis infectivity risk. Several preparations of human plasma-derived factor VIII of varying degrees of purity are available commercially for the treatment of hemophilia A. These include a partially-purified factor VIII derived from the pooled blood of many donors that is heat- and detergent-treated for viruses but contains a significant level of antigenic proteins; a monoclonal antibody-purified factor VIII that has lower levels of antigenic impurities and viral contamination; and recombinant human factor VIII, clinical trials for which are underway. Unfortunately, human factor VIII is unstable at physiologic concentrations and pH, is present in blood at an extremely low concentration (0.2 μg/ml plasma), and has low specific clotting activity. The problems associated with the commonly used, commercially available, plasma-derived factor VIII have stimulated significant interest in the development of a better factor VIII product. There is a need for a more potent factor VIII; a factor VIII that is stable at a selected pH and physiologic concentration; a factor VIII that is has a longer half-life in circulating blood. Summary of the Invention The present invention relates to a method of increasing the half-life of factor VIII. More specifically, the present invention relates to a mutant of factor VIII having reduced clearance from plasma. In one embodiment, the mutant factor VIII has one or more amino acid substitutions in the A2 domain. In a preferred embodiment, the substituted amino acid(s) are important for receptor-dependent clearance of factor VIII, such that the resulting mutant factor VIII has a longer (increased) circulating half-life. In another embodiment, the mutant factor VIII has one or more amino acid substitutions in the C2 domain. In a preferred embodiment, the substituted amino acid(s) are important for receptor-independent clearance of factor VIII, such that the resulting mutant factor VIII has a longer (increased) circulating half-life. In yet another preferred embodiment, amino acid(s) important for receptor-dependent clearance in the A2 domain and amino acid(s) important for receptor-independent clearance in the C2 domain are substituted, such that the resulting mutant factor VIII has an increased circulating half-life. The invention also relates to a method of using receptor associated protein (RAP) to increase the half-life of factor VIII. Further aspects of the invention include a method of producing factor VIII mutants having an increased half-life, pharmaceutically acceptable compositions thereof, and a method of treating factor VIII deficiency using mutant factor VIII of the invention and/or RAP. Brief Description of the Figures FIG. 1. Domain structure of fVIII and its fragments. The domain structure of mature fVIII protein is shown in line 1. The LCh acidic region is labeled as AR. Thrombin-cleaved LCh (A3-C1-C2), heterotrimeric fVIIIa (A1/A2/3-C1-C2) and heterodimer A1/A3-C1-C2 are shown in lines 2, 3 and 4. FIGs. 2A and 2B. The amino acid sequence of mature, B-domainless fVIII (SEQ IDNO:5; composed from GenBank Accession No. X01 179). The A2 sequence within fVIII is underlined and the sequence of the LRP binding site (residues 484-509) within A2 is indicated with asterisks. The amino acid residues shown as one-letter amino acid abbreviations. FIGS. 3 A and 3B. The deduced amino acid sequence of full-length factor VIII (SEQ ID NO:2; from GenPep Accession No. CAA25619J and GenBank Accession No. X01179). FIG. 4. The deduced amino acid sequence of RAP (SEQ ID NOJ; GenBank Accession No. M63959). The signal sequence (amino acids 1-34) is underlined and the LDL receptor binding region (amino acids 237-353) is indicated with asterisks. FIGS. 5A and 5B. Binding of ,25 I-fVIII to purified LRP by ligand competition assay. 12 J-fVIII (1 nM) was incubated for 1 h at 37 °C in wells coated with LRP (•) or BSA (o) in the presence of increasing concentrations of unlabeled competitors, fVIII (•, o) or vWf (A), panel A, and RAP (•, o), panel B. In the experiment (Δ), ,25 I-fVIII was preincubated with vWf for 30 min at 37 °C, prior to its addition to the wells. Following incubation, the wells were washed and 125 I-fVIII binding was determined. Binding of 125 I-fVIII in the presence of unlabeled fVIII, vWf, or RAP is expressed as the percentage of l25 fVIII binding, when no competitor was added. Each point represents the mean value of triplicates and the error bars display the standard deviation. The curves show a best fit of the data to a model describing heterologous ligand displacement from a single class of binding sites using the program LIGAND. FIG. 6. Effect of fragments of fVIII on its binding to LRP. 125 fVIII (1 nM) and increasing concentrations of unlabeled HCh (•), A2 (A), LCh (o) or A1/A3- C1-C2 (Δ) were incubated with LRP as described in Fig. 5. Each point represents the mean value and the standard deviation of the triplicates. The data were fitted as in Fig. 5 to a model describing heterologous ligand displacement from a single class of binding sites with K, values of 120 and 132 nM for HCh and A2, respectively. FIGS. 7A and 7B. Effect of monoclonal antibodies and synthetic peptides on 125 fVIII binding to purified LRP. Panel A, l 25 fVIII (1 nM) and increasing concentrations of mAbs 413 (•) or T5 (o) were added to LRP coated wells as described in Fig. 5. In the control experiment (Δ), l25 If-VIII and increasing concentrations of mAb 413 were added to BSA coated wells. Panel B, 125 I-fVIII and increasing concentrations of synthetic peptides consisting of the A2 domain residues 484-509 (•) or 432-456 (o) were added to LRP coated wells. In the control experiment (Δ), l25 I-fVIII and increasing concentrations of the peptide 484-509 were added to BSA coated wells. In the panels A and B, binding of 125 I- fNIII in the presence antibodies or peptides is expressed as the percentage of its binding, when no competitor was added. The mean and standard deviation of the triplicate measurements are presented. FIGS. 8 A and 8B. Internalization and degradation of ,25 I-fVIII/vWf complex by LRP-expressing (MEF) and LRP-deficient (PEA 13) fibroblasts. Wells containing 2x10 of each MEF (o, •) or PEA 13 cells (Δ, A) were incubated with 1 nM ,25 I-fVIII/vWf in the absence (closed symbols) or presence (opened symbols) of RAP (1 μM). 125 I-fVIII/vWf complex was prepared by incubation of 125 I-fVIII with unlabeled vWf at a molar ratio 1 :50 for 30 min at 37 °C. At the indicated times, the amounts of internalized ,25 1 -fVIII (panel A) and degraded ,25 I-fVIII (panel B) by the MEF and PEA 13 fibroblasts were determined as described under Experimental Procedures. In the experiment (V), degradation of , 25 I-fVIII (1 nM) by MEF cells in the presence of (0J mM) chloroquine is shown. Each data point represents the mean and standard deviation of duplicate determinations. FIGS. 9A and 9B. Comparison of internalization of isolated 125 I-fVHI and components of fVIII/vWf complex. Wells containing 2xl0 5 of each MEF and PEA 13 cells were incubated with 1 nM of isolated l25 I-fVIII or 1 nM of fNIII/vWf complex formed by mixing either 125 I-fVIII (1 nM) with unlabeled vWf (50 nM) or 125 I-vWf (50 nM) with unlabeled fVIII (1 nM). Following incubation for 6 hours with MEF cells in the absence of RAP (open bars) or in the presence of 1 μM RAP (solid bars) or after incubation with PEA 13 cells (hatched bars) the amounts of internalized (panel A) and degraded (panel B) isolated I25 I-JNIII, and l 5 I-fVIII or 12 J-vWf from the fVIII/vWf complex were determined as described in Fig. 8. The data shown are an average of duplicate determinations ± standard deviation. FIGS. 10A and 10B. The A2 domain of fVIII inhibits the internalization and degradation of 12 J-fVIII/vWf complex by MEF fibroblasts. One nM of 12 J- fVIII/vWf complex was prepared as in Fig. 8 and incubated with 2xl0 5 of MEF cells in presence of 1 μM of A2 (o), 1 μM of A1/A3-C1-C2 (Δ), or in the absence of any competitor (•). At the indicated times, the amounts of internalized (panel A) and degraded 125 I-fNIII (panel B) were determined as in Fig. 8. Each data point represents the mean and standard deviation of duplicate determinations. FIGS. 1 1 A-D. Internalization and degradation of 12 J-A2 by MEF fibroblasts and by LRP-expressing smooth muscle cells (SMC) and alveolar epithelial cells (T2). In the panels A andB, 2xl 0 5 of MEF (o, •) or PEA 13 cells (Δ. A) were incubated with 10 nM 125 I-A2 in the absence (closed symbols) or presence (opened symbols) of RAP ( 1 μM). At the indicated times, the amounts of internalized l25 I-A2 (panel A) and degraded 125 I-A2 (panel B) by the MEF and PEA 13 fibroblasts were determined as described in Fig. 8. In the experiment (V), degradation of 125 I-A2 by MEF cells in the presence (OJ mM) chloroquine is shown. Each data point represents the mean and standard deviation of duplicate determinations. In the, panels C and D, 125 I-A2 ( 10 nM) was incubated for 4 h at 37 °C inthe wells containing 3xl0 5 SMC (solid bars) or T2 (open bars) cells inthe presence or absence of RAP (1 mM). The amount of 125 I-A2 internalized (panel C) and degraded (panel D) by the cells was determined as in Fig. 8. The data shown are an average of duplicate determinations ± standard deviation. FIGS. 12A and 12B. The effect of RAP on clearance of ,25 I-A2 (A) or l2 J-fVIII/vWf (B) from plasma of mice. BALB/c mice were injected into the tail vein by sample containing ,2 J-A2 (36 nM panel A, or 12 J-fNIII/vWf (20 nM). panel B, in the absence (• ) or presence (O) of RAP (267 μM). At indicated time points, blood (50 μl) was collected into 10 μl of 100 mM EDTA and an aliquot (50 μl) was counted for radioactivity. The percentage of ligand remaining in circulation was calculated considering radioactivity of the aliquot taken at 1 min after injection as 100%. The clearance of each preparation was examined in two mice, and the data plotted represent the average value ± standard deviation. Detailed Description of the Preferred Embodiments "Factor VIII" (or "coagulation factor VIII"), as used herein, refers to a plasma glycoprotein that is a member of the intrinsic coagulation pathway and is essential to blood coagulation. A congenital X-linked deficiency of biologically active factor VIII results in Hemophilia A, a potentially life-threatening disorder. Unless otherwise specified or indicated, as used herein, "factor VIII" denotes any functional human factor VIII protein molecule in its normal role in coagulation, including any fragment, analog derivative or modified factor VIII. The human factor VIII cDNA nucleotide and full-length predicted amino acid sequences are shown in SEQ ID NOs: 1 and 2, respectively. Human factor VIII peptides of the invention include full-length factor VIII. full-length factor VIII minus Met at the N-terminus, mature factor VIII (minus the signal sequence), mature factor VIII with an additional Met at the N-terminus, and/or factor VIII with or without a B domain. Factor VIII of the invention may also include porcine factor VIII. The cDNA and predicted amino acid sequences of the porcine factor VIII are disclosed in U.S. Patent No..859,204. "Subunits" of factor VIII, as used herein, are the heavy and light chains of the protein. The heavy chain of factor VIII contains three domains, Al , A2, and B. The light chain of factor VIII also contains three domains, A3, CI, and C2. Factor VIII is synthesized as an approximately 300 kDa single chain protein with internal sequence homology that defines the "domain" sequence NH 2 -A1-A2-B-A3-C1-C2-COOH. In a factor VIII molecule, a "domain", as used herein, is a continuous sequence of amino acids that is defined by internal amino acid sequence identity and sites of proteolytic cleavage by thrombin. Unless otherwise specified, factor VIII domains include the following amino acid residues: Al, residues Alal-Arg372; A2, residues Ser373-Arg740; B, residues Ser741-Argl648; A3, residues Serl690-Ile2032; CI, residues Arg2033-Asn2172; C2, residues Ser2173-Tyr2332. The A3-C1-C2 sequence includes residues Serl690-Tyr2332. The remaining sequence, residues Glul 649-Arg 1689, is usually referred to as the factor VIII light chain activation peptide. A "B-domainless" factor VIII or "B (-)" factor VIII, or fragment of thereof, as used herein, refers to any one of the factor VIII mutants described herein that lacks the B domain. The amino acid sequence of mature, B (-) factor VIII as constructed from GenBank Accession No. X01 179 is shown in Figure 2 (SEQ ID NO:5). B (-) factor VIII of the invention includes B (-) factor VIII with or without a signal sequence and with or without a Met at the N-terminus. As used herein, a "mutant factor VIII or fragment thereof or "factor VIII mutant or fragment thereof is an active factor VIII molecule or fragment thereof comprising at least one amino acid substitution. "RAP," as used herein, refers to the receptor-associated protein, also called the 2 macroglobulin receptor-associated protein. RAP reduces receptor- dependent clearance of factor VIII. The human RAP deduced amino acid sequence is shown in Figure 4 (SEQ ID NOJ; GenBank Accession No. P30533). The RAP cDNA sequence is shown in SEQ ID NOJ and GenBank Accession No. M63959. Mutant RAP proteins of the invention may have an amino acid substitution at one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more positions of RAP. An amino acid substitution at "position" 327, for example, of RAP, refers to an amino acid substitution at amino acid 327 of the RAP amino acid sequence in GenBank Accession No. P30533. By "amino acid substitution" is meant a substitution of one amino acid for one of the remaining 19 naturally occurring amino acids. By an amino acid substitution at any one of positions "484 to 509," for example, is meant an amino acid substitution any position in the range, including at positions 484 and 509. The mutant factor VIII or RAP proteins of the invention may have an amino acid substitution at one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more positions. An amino acid substitution at "position" 499, for example, of factor VIII, refers to an amino acid substitution at position 499 according to the numbering system of Wood et al, Nature 312:330-331 (1984). "Half-life," as used herein, refers to the half-life of factor VIII in circulation, as determined in animals such as mice, for example, using the method of Examples 1 and 2. Factor VIII has a half-life of 12-14 hours. As provided herein, methods to increase the half-life of factor VIII would lead to a factor VIII half-life of longer than 12-14 hours. "Receptor-dependant clearance ' as used herein, refers to the receptor- mediated removal of factor VIII from circulation. As described in the examples, receptor-dependant clearance is exhibited by MEF cells, and is inhibited by RAP. Receptor-dependent clearance includes, but is not limited, to LRP-mediated clearance of factor VIII clearance. Additional receptors may be involved in receptor-dependent clearance. "Receptor-independent clearance/' as used herein, refers to the removal of factor VIII from circulation by means different from receptor-dependant clearance. RAP does not inhibit receptor-independent clearance. "Factor VIII deficiency ' as used herein, includes deficiency in clotting activity caused by production of defective factor VIII, by inadequate or no production of factor VIII, or by partial or total inhibition of factor VIII by inhibitors. Hemophilia A is a type of factor VIII deficiency resulting from a defect in an X-linked gene and the absence or deficiency of the factor VIII protein it encodes. A deficiency in vWf can also cause phenotypic hemophilia A because vWf is an essential component of functional factor VIII. In these cases, the half-life of factor VIII is decreased to such an extent that it can no longer perform its particular functions in blood-clotting. "Plasma," as used herein, refers to the fluid, non-cellular portion of the blood of humans or animals as found prior to coagulation. It is distinguished from serum, which is obtained after coagulation. "Pharmaceutically acceptable carrier," as used herein, refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. "Patient," as used herein, refers to human or animal individuals receiving medical care and/or treatment. "Congenital deficiency," as used herein, refers to the condition of an individual that lacks, as a result of heredity, a compound found in normal individuals. Congenital deficiencies are permanent absent transplantation or genetic intervention, which at this time are not guaranteed cures. "Acquired deficiency," as used herein, refers to the condition of an individual that lacks, as a result of a non-congenital influence, a compound found in normal individuals. Acquired deficiencies are frequently the transient result of other conditions or their treatment, but are nonetheless debilitating and life threatening. A "fusion protein," as used herein, is the product of a gene in which the coding sequence for one protein is extensively altered, for example, by fusing part of it to the coding sequence for a second protein from a different gene to produce a gene that encodes the fusion protein. As used herein, a fusion protein is a subset of the factor VIII protein or RAP protein described in this application. A "corresponding" nucleic acid or amino acid or corresponding sequence of either, as used herein, is one present at a site in a factor VIII or mutant factor VIII molecule or fragment thereof that has the same structure and/or function as a site in the factor VIII molecule of another species, although the nucleic acid or amino acid number may not be identical. "Procoagulant activity," as used herein, refers to factor VIII coagulation activity exhibited in a human factor VIII assay. "Specifϊc activity," as used herein, refers to the activity that will correct the coagulation defect of human factor VIII deficient plasma. Specific activity is measured in units of clotting activity per milligram total factor VIII protein in a standard assay in which the clotting time of human factor VIII deficient plasma is compared to that of normal human plasma. One unit of factor VIII activity is the activity present in one milliliter of normal human plasma. In the assay, the shorter the time for clot formation, the greater the activity of the factor VIII being assayed. Mutant factor VIII has coagulation activity in a human factor VIII assay. This activity may be less than, equal to, or greater than that of either plasma-derived or recombinant human factor VIII. "Polypeptides," "molecules" and "proteins," as used herein, includes all polypeptides as described below. The basic structure of polypeptides is well known and has been described in innumerable textbooks and other publications in the art. In this context, the term is used herein to refer to any peptide or protein comprising two or more amino acids joined to each other in a linear chain by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and ohgomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. It will be appreciated that polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids, and that many amino acids, including the terminal amino acids, may be modified in a given polypeptide, either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques which are well known to the art. Even the common modifications that occur naturally in polypeptides are too numerous to list exhaustively here, but they are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art. Among the known modifications which may be present in polypeptides of the present invention are, to name an illustrative few. acetylation. acylation, ADP-ribosylation, amidation, PEGylation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidyhnositol, cross-linking, cychzation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formulation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. Such modifications are well known to those of skill and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in most basic texts, such as, for instance Proteins - Structure and Molecular Properties, 2nd Ed.. T. E. Creighton, W.H. Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as, for example, those provided by Wold, F.. Posttranslational Protein Modifications: Perspectives and Prospects, pp. 1 - 12 in Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press. New York (1983); Seifter etal., Analysis for protein modifications and nonprotein cofactors, Meth. Enzymol. 182 : 626-646 (1990) and Rattan et al.. Protein Synthesis: Post translational Modifications and Aging, Ann. NN. Acad. Sci. 663: 48-62 (1992). In general, as used herein, the term polypeptide encompasses all such modifications, particularly those that are present in polypeptides synthesized by expressing a polynucleotide in a host cell. The invention also relates to fragments, "derivatives" and analogs of these polypeptides. The terms "fragment," "derivative" and "analog" when referring to the polypeptides of FIGS. 2, 3 or 4, means a polypeptide which retains essentially the same biological function or activity as such polypeptide. A mutant, fragment derivative or analog of factor VIII refers to a polypeptide that retains factor VIII procoagulant activity. A mutant, fragment derivative or analog of RAP refers to a polypeptide that retains the ability to reduce receptor-dependent clearance of factor VIII. Thus, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide. Fragments, derivatives and analogs are described in detail herein. A fragment, derivative or analog of the polypeptide of the invention may be (i) one in which one or more of the amino acid residues includes a substituent group, or (ii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iii) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein. The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide. In certain preferred embodiments it is a recombinant polypeptide. Further particularly preferred in this regard are mutants, analogs and fragments; and mutants and analogs of the fragments, having the defined activity and/or having the amino acid sequence of the polypeptides of FIGS. 2, 3 or 4. The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity. "Polynucleotide(s)" generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotides as used herein refers to, among others, single-and double-stranded DNA, DNA that is a mixture of single-and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, polynucleotide as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. As used herein, the term polynucleotide includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabohcally modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia. Polynucleotides of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced by chemical synthetic teclmiques or by a combination thereof. The DNA may be double-stranded or single-stranded. Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand. Polynucleotides of the present invention may include, but are not limited to the coding sequence for the mature polypeptide, by itself; the coding sequence for the mature polypeptide and additional coding sequences, such as those encoding a leader or secretory sequence, such as a pre-, or pro- or prepro- protein sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing— including splicing and polyadenylation signals, for example— ribosome binding and stability of mRNA; additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities. Thus, for instance, the polypeptide may be fused to a marker sequence, such as a peptide, which facilitates purification of the fused polypeptide. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexahistidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al, Proc. Natl. Acad. Sci. , USA 86: 821 -824 ( 1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. The HA tag corresponds to an epitope derived of influenza hemagglutinin protein, which has been described by Wilson et al.. Cell 37: 767 (1984), for instance. An "effective amount" of an agent, as used herein, is an amount of such agent that is sufficient to bring about a desired result, especially upon administration of such agent to an animal or human. The term "administration" is meant to include introduction of polypeptides or polynucleotides of the invention into an animal or human by any appropriate means known to the medical art, including, but not limited to, injection, oral, enteral, transdermal and parenteral (e.g., intravenous) administration. The term "pharmaceutically acceptable salt" is intended to include salts of the mutant factor VIII or RAP of the invention. Such salts can be formed from pharmaceutically acceptable acids or bases, such as, for example, acids such as sulfuric, hydrochloric, nitric, phosphoric, etc., or bases such as alkali or alkaline earth metal hydroxides, ammonium hydroxides, alkyl ammonium hydroxides, etc. The term "pharmaceutically acceptable composition" is intended to include solvents, carriers, diluents, and the like, which are utilized as additives or vehicles to preparations of the mutant factor VIII or RAP of the invention so as to provide a carrier or adjuvant for the administration of such compounds to patients (human or animal) in need of the same. Such additives can perform certain functions, such as, for example, provide the proper ionic conditions for administration, stabilize the mutant factor VIII or RAP against inactivation or degradation, and/or increase the half-life of the mutant factor VIII or RAP. A pharmaceutically acceptable composition is medically compatible with the host to which it is being administered. The term "treatment" or "treating" is intended to include the administration of the pharmaceutically acceptable compositions of the invention comprising effective amounts of mutant factor VIII or RAP (polypeptides or polynucleotides) of the invention to a patient for purposes which may include prophylaxis, amelioration, prevention or cure of a medical disorder. A material is said to be "substantially free of natural contaminants" if it has been substantially purified from materials with which it is normally and naturally found before such purification and those contaminants normally and naturally found with the substance in vivo or in vitro are substantially absent from the final preparation of the material. When administered to a subject in need of treatment, the mutant factor VIII or RAP of the invention is substantially free of natural contaminants which associate with the mutant factor VIII or RAP either in vivo (in the host from which the mutant factor VIII or RAP was isolated), or in vitro (as a result of a chemical synthesis). By "substantially absent" is meant that such contaminants are either completely absent or are present at such low concentrations that their presence (1) does not interfere with the desired therapeutic effect of the active agent in the therapeutically acceptable composition when such composition is administered to a patient in need of same and (2) does not harm the patient as the result of the administration of such composition. Since current information indicates that the B domain has no known effect on factor VIII function, in some embodiments the B domain is deleted ("B domain (-)" or "B domainless") in the mutant factor VIII molecule or fragments thereof ("B(-) factor VIII" or "B domainless factor VIII") prepared by any of the methods described herein. Generation of mutant(s) with a prolonged lifetime may be a promising approach to increase the efficacy and reduce the cost of fNIII infusion therapy. The invention provides methods of increasing the half-life of factor VIII by mutating factor VIII, and further provides methods of increasing the half-life of factor VIII using receptor-associated protein (RAP). Factor VIII Mutants: A2 Domain A recombinant mutant factor VIII having reduced receptor-dependent clearance and/or reduced receptor-independent clearance, and/or having superior coagulant activity, compared to human factor VIII, may be less expensive to make than plasma-derived factor VIII and may decrease the amount of factor VIII required for effective treatment of factor VIII deficiency. The present invention provides active recombinant mutant factor VIII molecules or fragments thereof comprising at least one amino acid substitution in the A2 domain, polynucleotides encoding these, methods of producing and isolating them, and methods for characterizing their coagulant and plasma clearance properties. The A2 domain is necessary for the procoagulant activity of the factor VIII molecule. Studies show that porcine factor VIII has six-fold greater procoagulant activity than human factor VIII (Lollar, P., and E. T. Parker 266 J. Biol. Chem. 12481-12486 (1991)), and that the difference in coagulant activity between human and porcine factor VIII appears to be based on a difference in amino acid sequence between one or more residues in the human and porcine A2 domains (Lollar, P., et al, 267 J. Biol. Chem. 23652-23657 (1992)). In one embodiment, the invention provides a method of increasing the half-life of factor VIII by substituting amino acids in the factor VIII A2 domain. In another embodiment, the invention provides mutant factor VIII and fragments thereof, and the polynucleotides encoding same, which have an increased circulating half-life than human factor VIII. The increased circulating half-life is due to a reduction in receptor-dependent clearance of factor VIII. As shown in the examples, amino acids in the factor VIII A2 domain interact with at least one receptor that mediates A2 clearance and factor VIII clearance from plasma. Thus, factor VIII mutants of the invention include mutants with one or more substitutions within the A2 domain. In a preferred embodiment, the factor VIII mutants have an amino acid substitution at one or more positions from 484 to 509. This region includes the following sequence: NH 2 - Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys His Leu Lys Asp Phe Pro He Leu Pro Gly Glu He Phe -COOH. In another preferred embodiment, the factor VIII mutants have an amino acid substitution at one or more of positions 484, 489, 490, 493, 496 or 499. The amino acid at a particular position is substituted with any of the 19 other naturally occurring amino acids. A2 amino acid substitutions of the invention are those that inhibit the interaction of factor VIII with its clearance receptor(s). Thus, nonconservative A2 amino acid substitutions are preferred over conservative substitutions. Conservative amino acid substitutions include, for example, the substitution of an acidic amino acid with another acidic amino acid, a basic amino acid with another basic amino acid, a hydrophobic amino acid with a another hydrophobic amino acid, a polar amino acid with another polar amino acid, or an aromatic amino acid with another aromatic amino acid. Conservative amino acid substitutions are well known in the art. Thus, an example of a conservative substitution is the substitution of Lys with Arg, while an example of a preferred nonconservative substitution is the substitution of Lys with Asp, Glu, Tyr, Asn, Gin, Thr, Ser, Cys, Tip, Phe, Pro, Met, Val, Leu, He, Tip, Gly or Ala. Preferred A2 amino acid substitutions of the invention are the substitution of Lys or Arg with Leu, He or Val. Additional preferred A2 amino acid substitutions of the invention are the substitutions of Lys or Arg with Asp or Glu. Further preferred amino acid substitutions of the invention are the substitution of Lys or Arg with Ala, Ser, Thr, Met or Gly. In another embodiment, amino acids at positions outside 484-509 are substituted, such as at positions 480, 481, 482, 483, 510, 51 1, 512 or 513. Preferred substitutions at these positions are those that reduce receptor-dependent clearance of factor VIII, such as introducing bulky or negatively charged amino acids. Specifically provided as an exemplary and a preferred embodiment is active recombinant human factor VIII having substituted amino acids in the A2 domain, the polynucleotide encoding it, and the methods of producing, isolating, and characterizing its activity. The methods by which this mutant is prepared can also be used to prepare active recombinant factor VIII or fragments thereof having substituted amino acids in domains other than A2. One skilled in the art will recognize that these methods also demonstrate how other recombinant mutant factor VIII molecules or fragments thereof can be prepared in which amino acids are substituted. Additionally, recombinant methods are described in Current Protocols in Molecular Biology, F. M. Ausubel et al, eds. ( 1991 ): and Sambrook, J., et al, Molecular Cloning. A Laboratory Manual. Mutant factor VIII is prepared starting with humaj; cDNA (Biogen, Inc.) encoding the factor VIII sequence. In a preferred embodiment, the factor VIII encoded by this cDNA includes domains A 1-A2-A3-C1-C2, lacking the entire B domain, and corresponds to amino acid residues 1-740 and 1649-2332 of single chain human factor VIII (see SEQ ID NO:2), according to the numbering system of Wood et al, 312 Nature 330-337 (1984). The mutant factor VIII cDNA are cloned into expression vectors for ultimate expression of active factor VIII protein molecules in cultured cells by established techniques, as described by Selden, R.F., "Introduction of DNA into mammalian cells," in Current Protocols in Molecular Biology, F.M. Ausubel et al, eds (1991). In a preferred embodiment, a cDNA encoding mutant factor VIII is inserted in a mammalian expression vector, such as ReNeo, to form a mutant factor VIII construct. Preliminary characterization of the mutant factor VIII is accomplished by insertion of the mutant cDNA into the mammalian expression vector and transient expression of the mutant protein in COS-7 cells. A determination of whether active protein is expressed can then be made. The expression vector construct is used further to stably transfect cells in culture, such as baby hamster kidney cells, using methods that are routine in the art, such as liposome-mediated transfection (Lipofectin™, Life Technologies, Inc.). Expression of recombinant mutant factor VIII protein can be confirmed, for example, by sequencing, Northern and Western blotting, or polymerase chain reaction (PCR). Mutant factor VIII protein in the culture media in which the transfected cells stably expressing the protein are maintained can be precipitated, pelleted, washed, and resuspended in an appropriate buffer, and the recombinant mutant factor VIII protein purified by standard techniques, including immunaffinity chromatography using, for example, monoclonal anti-A2-Sepharose™. In a further embodiment, the mutant factor VIII comprising amino acid substitutions is expressed as a fusion protein from a recombinant molecule in which sequence encoding a protein or peptide that enhances, for example, stability, secretion, detection, isolation, or the like is inserted in place adjacent to the factor VIII encoding sequence. Established protocols for use of homologous or heterologous species expression control sequences including, for example, promoters, operators, and regulators, in the preparation of fusion proteins are known and routinely used in the art. (See Current Protocols in Molecular Biology, Ausubel, F.M.. et al, eds, Wiley Inlerscience, NN.) Other vectors, including both plasmid and eukaryotic viral vectors, may be used to express a recombinant gene construct in eukaryotic cells depending on the preference and judgment of the skilled practitioner (see, for example, Sambrook et al, Chapter 16). Other vectors and expression systems, including bacterial, yeast, and insect cell systems, can be used but are not preferred due to differences in, or lack of, glycosylation. The purified mutant factor VIII or fragment thereof can be assayed for amount and for coagulation activity by standard assays including, for example, the plasma-free factor VIII assay, the one-stage clotting assay, and the enzyme-linked immunosorbent assay using purified recombinant human factor VIII as a standard. Recombinant mutant factor VIII protein can be expressed in a variety of cells commonly used for culture and recombinant mammalian protein expression. A preferred cell line, available from the American Type Culture Collection, Rockville, Md., is baby hamster kidney cells, which are cultured using routine procedure and media. Any mutant factor VIII construct having an amino acid substitution at one or more positions in the A 2 domain as described can be assayed by standard procedures for coagulant activity and may be assayed for receptor-dependent clearance as described herein to identify mutant factor VIII molecules with enhanced coagulant activity and/or reduced receptor-mediated clearance. Mutant molecules may also be identified that have reduced coagulant activity compared to human or porcine factor VIII but also have reduced receptor-mediated clearance. One skilled in the art will recognize that mutant factor VIII molecules or fragments thereof having less, equal, or greater coagulant activity, compared to human or porcine factor VIII, is useful for treating patients who have a factor VIII deficiency. The methods described herein to prepare active recombinant mutant factor VIII with amino acid substitution(s) in the A2 domain can be used to prepare active recombinant mutant factor VIII protein with amino acid substitution(s) in the C2 domain or fragments thereof. These molecules can be expressed in COS-7 cells and baby hamster kidney cells as described above. They can be purified to homogeneity using methods known in the art, such as heparin-Sepharose™ and immunoaffinity chromatography. Protein concentration can be estimated by absorption of ultraviolet light at A 280 , and the specific activity of the constructs can be determined by dividing coagulant activity (measured in units per ml by single stage clotting assay) by A 280 . Human factor VIII has a specific activity of approximately 3000-4000 U/A 280 , whereas porcine factor VIII has a specific activity of approximately 20,000 U/A 280 . In a preferred embodiment, the coagulant mutant factor VIII has a specific activity of 3000 U/A 280 . In a preferred embodiment, the coagulant mutant factor VIII has a specific activity of 3000 U/A 280 . The a specific activity of mutant factor VIII may be anywhere in the range of 1000-20,000 U/A 280 . As described herein, site-directed mutagenesis techniques are used to identify mutant protein with coagulant activity that can be enhanced, equal to, or reduced, compared to human factor VIII, but preferably is enhanced. Oligonucleotide-directed mutagenesis can be used as described in Kunkel, T.A., et al, Meth. Enzymol. 204:125-139 (1991). The mutant factor VIII proteins of the invention may have an amino acid substitution at one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, twenty or more positions of factor VIII. The mutant factor VIII molecules of the invention may have amino acid substitutions in more than one domain, such as having an amino acid substitution both in the A2 domain and in the C2 domain. The present invention contemplates that mutant factor VIII cDNA and protein can be characterized by methods that are established and routine, such as DNA sequencing, coagulant activity assays, mass by ELISA and by UV absorbency at 280 nm of purified mutant factor VIII, specific coagulant activity (U/mg), SDS-PAGE of purified mutant factor VIII, and the like. Other known methods of testing for clinical effectiveness may be required, such as amino acid, carbohydrate, sulfate, or metal ion analysis. Factor VIII Mutants: C2 Domain The same methods employed for preparing mutant human factor VIII having A2 domain amino acid substitution(s) can be used to prepare other recombinant mutant factor VIII protein and fragments thereof and the polynucleotides encoding these, such as mutant factor VIII having amino acid substitutions in the C2 domain. Mutant human factor VIII molecules with amino acid substitution(s) in the C2 domain, which have reduced or no receptor-independent clearance can be identified. More specifically, the procedures can be the same or similar to those described herein for amino acid substitution in the A2 domain (by alanine scanning mutagenesis, site-directed mutagenesis, etc.,) substituting amino acids in the C2 domain of B (-) factor VIII: insertion into an expression vector, such as pBluescript; expression in cultured cells; and routine assay for coagulant activity and receptor-independent clearance. In one embodiment, the invention provides mutant factor VIII and fragments thereof, and the polynucleotides encoding same, which have an increased circulating half-life than human factor VIII. The increased circulating half-life of mutant factor VIII is due to a reduction in receptor-independent clearance of factor VIII. The C2 domain consists of amino acid residues 2173-2332. Within this 154 amino acid region, positions 2303-2332 are involved in both phospholipid binding and vWf binding. A synthetic peptide of factor VIII amino acids 2310- 2320 (in which residues 2310 and 2320 are covalently linked) competes with factor VIII for phospholipid binding. A comparison of factor V. which does not bind vWf, and factor VIII reveals 5 amino acids within positions 2311-2319 that are unique to factor VIII. Although not being bound by any theory, these unique positions (Gln231 1 , Ser 2312, Val 2314, His2315 and Gln2316) are important for receptor-independent clearance, but are not critical for vWf binding. Thus, one embodiment of the present invention is a mutant factor VIII having an amino acid substitution at one or more of positions 2173-2332 in the C2 domain. In another preferred embodiment, the mutant factor VIII has an amino acid substitution at one or more positions 231 1-2319 in the C2 domain. The amino acid at a particular position is substituted with any of the 19 other naturally occurring amino acids. C2 amino acid substitutions of the invention are those that inhibit the interaction of factor VIII with phospholipid. Thus, nonconservative C2 amino acid substitutions are preferred over conservative substitutions. Conservative amino acid substitutions include, for example, the substitution of an acidic amino acid with another acidic amino acid, a basic amino acid with another basic amino acid, a hydrophobic amino acid with a another hydrophobic amino acid, a polar amino acid with another polar amino acid, or an aromatic amino acid with another aromatic amino acid. Conservative amino acid substitutions are well known in the art. Thus, an example of a conservative substitution is the substitution of Leu with He or Val, while an example of a preferred nonconservative substitution is the substitution of Leu with Asp, Glu, Arg, Lys, His, Tyr, Asn, Gin, Thr, Ser, Cys, Trp, Phe, Pro, Met, Tip, Gly or Ala. One preferred substitution is Ala. Additional embodiments of the present invention include a method of treating hemophilia by administering a C2 domain mutant of factor VIII, pharmaceutically acceptable compositions comprising a C2 domain mutant of factor VIII either alone or in combination with RAP, and polynucleotides encoding a C2 domain mutant of factor VIII. Furthermore, the amino acid substitution(s) in the C2 domain can be combined with amino acid substitution(s) in the A2 domain, to produce a mutant factor VIII with increased half-life. Receptor Associated Protein A preferred embodiment of the present invention is directed to a method of increasing the half-life of factor VIII by administering RAP. Preferably, the RAP binds LRP, more preferably, the RAP has an increased affinity for LRP as compared to the naturally occurring RAP. In another preferred embodiment of the present invention, RAP is a fragment, mutant or analog. Preferably, the RAP fragment, mutant or analog retains LRP binding activity. More preferably, the RAP fragment, mutant or analog has increased affinity for LRP as compared to the naturally occurring RAP. In one embodiment, the RAP is a fragment having LRP binding activity. Such RAP fragments may comprise 10, 20, 30, 40, 50, 60, 75, 100, 125, 150, 175, 200, 250, 300 or 350 or more contiguous amino acids. In one embodiment, RAP comprises amino acids 1 to 357 of Figure 4 (full-length RAP; amino acids -19 to 323 of SEQ ID NOJ). RAP contains a signal sequence 34 amino acids in length. Thus, in another embodiment. RAP comprises amino acids 35 to 357 of Figure 4 (mature RAP: amino acids 1 to 323 of SEQ ID NOJ.). In another embodiment of the present invention, RAP contains an N-terminal or a C-terminal deletion, or a combination of N- and C-terminal deletions. N-terminal deletions often result in a protein with increased stability. Thus, for example, deleting between 1 and 50 amino acids from the N-terminus of mature RAP is useful to produce a more stable RAP. Therefore, additional embodiments of the present invention include, for example, RAP comprising amino acids 36-357. 37-357, 38-357, 39-357, 40-357, 41-357, 42-357, 43-357, 44-357, 45-357, 46-357, 47-357, 48-357, 49-357, 50-357, 51-357, 52-357, 53- 357, 54-357, 55-357, 56-357, 57-357, 58-357, 59-357. 60-357, 61-357, 62-357, 63-357, 64-357, 65-357, 66-357, 67-357. 68-357, 69-357, 70-357, 71-357, 72- 357, 73-357, 74-357, 75-357, 76-357. 77-357, 78-357, 79-357, 80-357. 81-357, 82-357, 83-357. 84-357 and 85-357 of Figure 4 (positions 1-323, 2-323, 3-323, 4-323, 5-323, 6-323. 7-323, 8-323. 9-323. 10-323, 1 1-323. 12-323, 13-323, 14- 323, 15-323, 16-323, 17-323, 18-323, 19-323, 20-323, 21-323, 22-323, 23-323, 24-323, 25-323, 26-323, 27-323, 28-323, 29-323, 30-323, 31-323, 32-323, 33- 323, 34-323, 35-323, 36-323, 37-323, 38-323, 39-323, 40-323, 41 -323, 42-323. 43-323, 44-323, 45-323, 46-323, 47-323, 48-323, 49-323 and 50-323 of SEQ ID NOJ). The LDL receptor binding domain encompasses amino acids 237 to 353 of Figure 4 (amino acids 203 to 319 of SEQ ID NOJ). Thus, a preferred embodiment of the present invention is RAP comprising amino acids 237 to 353 (amino acids 203 to 319 of SEQ ID NOJ). Another embodiment of the present invention is a polynucleotide encoding RAP. In another embodiment of the present invention, RAP or a polynucleotide encoding RAP is used to treat hemophilia either alone or in combination with a factor VIII mutant. Additional embodiments of the present invention include pharmaceutically acceptable compositions comprising RAP alone or in combination with one or more factor VIII mutants. Pharmaceutically Acceptable Compositions Pharmaceutically acceptable compositions comprising mutant factor VIII or RAP, alone or in combination with appropriate pharmaceutical stabilization compounds, delivery vehicles, and/or carrier vehicles, are prepared according to known methods, as described in Remington's Pharmaceutical Sciences by E.W. Martin. In one preferred embodiment, the preferred carriers or delivery vehicles for intravenous infusion are physiological saline or phosphate buffered saline. In another preferred embodiment, suitable stabilization compounds, delivery vehicles, and carrier vehicles include but are not limited to other human or animal proteins such as albumin. Phospholipid vesicles or liposomal suspensions are also preferred as pharmaceutically acceptable carriers or delivery vehicles. These can be prepared according to methods known to those skilled in the art and can contain, for example, phosphatidylserine/phosphatidylcholine or other compositions of phospholipids or detergents that together impart a negative charge to the surface, since factor VIII binds to negatively charged phospholipid membranes. Liposomes may be prepared by dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the container. An aqueous solution of the mutant factor VIII or RAP is then introduced into the container. The container in then swirled by hand to free lipid material from the sides of the container and to disperse lipid aggregates, thereby forming the liposomal suspension. Mutant factor VIII or RAP can be combined with other suitable stabilization compounds, delivery vehicles, and/or carrier vehicles, including vitamin K dependent clotting factors, tissue factor, and von Willebrand factor (vWf) or a fragment of vWf that contains the factor VIII binding site, and polysaccharides such as sucrose. Mutant factor VIII can be stored bound to vWf to increase the shelf-life of the mutant molecule. Additionally, lyophilization of factor VIII can improve the yield of active molecules in the presence of vWf. Lyophilization can also improve the yield of RAP. Current methods for storags. of human and animal factor VIII used by commercial suppliers can be employed for storage of mutant factor VIII or RAP. These methods include: (1) lyophilization of factor VIII in a partially-purified state (as a factor VIII "concentrate" that is infused without further purification); (2) immunoaffinity-purification of factor VIII by the Zimmerman method and lyophilization in the presence of albumin, which stabilizes the factor VIII; (3) lyophilization of recombinant factor VIII in the presence of albumin. Additionally, factor VIII has been indefinitely stable at 4°C in 0.6 M NaCl, 20 mM MES. and 5 mM CaCL at pH 6.0 and also can be stored frozen in these buffers and thawed with minimal loss of activity. Methods of Treatment Mutant factor VIII or RAP is used to treat uncontrolled bleeding due to factor VIII deficiency (e.g., intraarticular, intracranial, or gastrointestinal hemorrhage) in hemophiliacs with and without inhibitory antibodies and in patients with acquired factor VIII deficiency due to the development of inhibitory antibodies. The active materials are preferably administered intravenously. Factor VIII is classically defined as that substance present in normal blood plasma that corrects the clotting defect in plasma derived from individuals with hemophilia A. The coagulant activity in vitro of purified and partially-purified forms of factor VIII is used to calculate the dose of factor VIII for infusions in human patients and is a reliable indicator of activity recovered from patient plasma and of correction of the in vivo bleeding defect. There are no reported discrepancies between standard assay of novel factor VIII molecules in vitro and their behavior in the dog infusion model or in human patients, according to Lusher, J. M., et al, New. Engl. J. Med. 325:453-459 (1993); Pittman, D. D., et al, Blood 79:389-397 (1992), and Brinkhous et al, Proc. Natl. Acad. Sci. 52:8752-8755 (1985). Usually, the desired plasma factor VIII level to be achieved in the patient through administration of the mutant factor VIII is in the range of 30-100%) of normal. In a preferred mode of administration of the mutant factor VIII, the composition is given intravenously at a preferred dosage in the range from about 5 to 50 units/kg body weight, more preferably in a range of 10-50 units/kg body weight, and most preferably at a dosage of 20-40 units/kg body weight; the interval frequency is in the range from about 8 to 24 hours (in severely affected hemophiliacs); and the duration of treatment in days is in the range from 1 to 10 days or until the bleeding episode is resolved. See, e.g., Roberts, H. R., and M. R. Jones, "Hemophilia and Related Conditions - Congenital Deficiencies of Prothrombin (Factor II, Factor V, and Factors VII to XII)," Ch. 153, 1453-1474, 1460, in Hematology, Williams, W. J., et al, ed. (1990). Administration of an effective amount of RAP will result in similar levels of factor VIII in patient blood as indicated above. Patients with inhibitors may require more mutant factor VIII, or patients may require less mutant factor VIII because of its higher specific activity than human factor VIII or increased plasma half-life. Likewise, patients may require more or less RAP, depending on RAP's binding affinity to LRP or other factor VIII clearance receptor, or depending on its stability in circulating blood. As in treatment with human or porcine factor VIII, the amount of mutant factor VIII or RAP infused is defined by the one-stage factor VIII coagulation assay and, in selected instances, in vivo recovery is determined by measuring the factor VIII in the patient's plasma after infusion. It is to be understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. Administration In a preferred embodiment, pharmaceutically acceptable compositions of mutant factor VIII or RAP alone or in combination with stabilizers, delivery vehicles, and/or carriers are infused into patients intravenously according to the same procedure that is used for infusion of human or animal factor VIII. The treatment dosages of mutant factor VIII or RAP composition that must be administered to a patient in need of such treatment will vary depending on the severity of the factor VIII deficiency. Generally, dosage level is adjusted in frequency, duration, and units in keeping with the severity and duration of each patient's bleeding episode. Accordingly, the mutant factor VIII or RAP is included in the pharmaceutically acceptable carrier, delivery vehicle, or stabilizer in an amount sufficient to deliver to a patient a therapeutically effective amount of the mutant protein to stop bleeding, as measured by standard clotting assays. Treatment can take the form of a single intravenous administration of the composition or periodic or continuous administration over an extended period of time, as required. Alternatively, mutant factor VIII or RAP can be administered subcutaneously or orally with liposomes in one or several doses at varying intervals of time. Mutant factor VIII or RAP can also be used to treat uncontrolled bleeding due to factor VIII deficiency in hemophiliacs who have developed antibodies to human factor VIII. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the protein, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogens, e.g., poly (2-hydroxyethyl-methacry late) as described by Langer et al, J. Biomed. Mater. Res. 15:161-211 (1981) and Langer, Chem. Tech. 12: 98-105 (1982) or poly(vinylalcohol), polylactides (U.S. Pat. No. 3,773,919. EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 ( 19831 )), non-degradable ethylene-vinyl acetate (Langer et al, supra), degradable lactic acid-glycolic acid copolymers such as the Lupron Depot™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid (EP 133,988). While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated proteins remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37 °C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for protein stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions. Sustained-release blood factor compositions also include liposomally entrapped blood factor or antibody. Liposomes containing the claimed blood factor or antibody are prepared by methods known per se: DE 3,218,121 ; Epstein et al, Proc. Natl. Acad. Sci. USA, 82: 3688-3692 (1985); Hwang et al, Proc. Natl. Acad. Sci. USA, 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patent application 83-118008; U.S. Pat. No. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily the liposomes are of the small (about 200-800 Angstroms) unilamelar type, the selected proportion being adjusted for the optimal blood factor therapy. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. Additionally, Giles, A. R., et al. Brit. J. Hematol. 69:491-497 (1988) describe the formulation of factor Xa in phosphatidylcholine-phosphatidylserine vesicles. Additionally, mutant factor VIII or RAP can be administered by transplant of cells genetically engineered to produce the protein or by implantation of a device containing such cells, as described below. Gene Therapy Polynucleotides encoding the mutant factor VIII or R AP may be employed in accordance with the present invention by expression of such mutant factor VIII or RAP in vivo, in treatment modalities often referred to as "gene therapy." Mutant factor VIII or RAP can also be delivered by gene therapy in the same way that human factor VIII can be delivered, using delivery means such as retroviral vectors. This method consists of incorporation of factor VIII cDNA into human cells that are transplanted directly into a factor VIII deficient patient or that are placed in an implantable device, permeable to the factor VIII molecules but impermeable to cells, that is then transplanted. The preferred method will be retroviral-mediated gene transfer. In this method, an exogenous gene (e.g., a factor VIII cDNA) is cloned into the genome of a modified retrovirus. The gene/cDNA is inserted into the genome of the host cell by viral machinery where it will be expressed by the cell. The retroviral vector is modified so that it will not produce virus, preventing viral infection of the host. The general principles for this type of therapy are known to those skilled in the art and have been reviewed in the literature (e.g., Kohn, D.B., and P.W. Kantoff, Transfusion 29:812-820 (1989)). Thus, for example, cells from a patient may be engineered with a polynucleotide, such as a DNA or RNA, encoding a polypeptide ex vivo, and the engineered cells then can be provided to a patient to be treated with the polypeptide. For example, cells may be engineered ex vivo by the use of a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention. Such methods are well-known in the art and their use in the present invention will be apparent from the teachings herein. Similarly, cells may be engineered in vivo for expression of a polypeptide in vivo by procedures known in the art. For example, a polynucleotide of the invention may be engineered for expression in a replication defective retroviral vector, as discussed above. The retroviral expression construct then may be isolated and introduced into a packaging cell is transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest. These producer cells may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo. These and other methods for administering a polypeptide of the present invention by such method should be apparent to those skilled in the art from the teachings of the present invention. Retroviruses from which the retroviral plasmid vectors herein above mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus. In one embodiment, the retroviral plasmid vector is derived from Moloney Murine Leukemia Virus. Such vectors well include one or more promoters for expressing the polypeptide. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller et al., Biotechniques 7: 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, RNA polymerase III, and β-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein. The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501 , PA317, Y-2, Y-AM, PA 12, T19-14X, VT-19-17-H2, YCRE, YCRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, A., Human Gene Therapy 7:5-14 (1990). The vector may be transduced into the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO 4 precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host. The producer cell line will generate infectious retroviral vector particles, which include the polynucleotide(s) encoding the polypeptides. Such retroviral vector particles then may be employed to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express the polynucleotide(s) encoding the polypeptide. Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes. fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells. The following examples are illustrative only and are not intended to limit the scope of the invention as defined by the appended claims. It will be apparent to those skilled in the art that various modifications and variations can be made in the methods of the present invention without departing from the spirit and scope of the invention. Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. All patents, publications and publicly available sequences referred to herein are expressly incoφorated by reference. Examples Example I Activated factor VIII (fVIIIa) functions in the intrinsic pathway of blood coagulation as a cofactor for factor IXa in the conversion of factor X to activated factor X (Xa). When IXa is bound to membrane and fVIII the rate of factor X to IXa conversion increases 100,000-1,000.000 fold. The procoagulant activity of fVIIIa is regulated by rapid and potentially reversible dissociation of the A2 subunit from the A1/A3C 1 C2 dimer and by activated protein C (APC) proteolysis of the residual fVIIIa. Removal of the A2 and A1/A3C1C2 fragments is an additional in vivo mechanism to control factor Villa activity at the site of blood coagulation. We tested this in a model system using mouse embryonic fibroblasts (MEF) that express low density lipoprotein receptor related protein (LRP) a multi ligand endocytic receptor and PEA 13 fibroblasts that are genetically deficient in LRP. Using the above model system we studied the mechanisms of cellular uptake and degradation of thrombin activated fVIII subunits to evaluate the role of these mechanisms in regulation of fVIIIa level. Methods Cell mediated ligand internalization and degradation assays. Cells were seeded into 24 well dishes and allowed to grow for 24 hours at 37°C. 5% CO 2 MEF and PEA 13 cells were incubated for selected time intervals at 37°C with ' 25 I-labeled fVIIIa fragments in the presence and absence of unlabeled competitors as described in the figure legends. Radioactivity appearing in the cell culture medium that was soluble after precipitation with 10% trichloroacetic acid (TCA) was taken to represent degraded ligand. Total ligand degradation was corrected by subtracting the amount of 10% TCA soluble radioactivity occurred in control wells lacking cells. The amount of labeled ligand bound to the cell surface or that was internalized by cells was determined as follows. Cells were washed with cold phosphate buffered saline and treated with a trypsin EDTA proteinase K solution. Surface bound material was defined as the amount of radioactive ligand released by this treatment and the amount of internalized ligand was defined as the amount of radioactivity which remained associated with the cell pellet following the treatment. Determining of the A2 affinity for LRP. LRP (3.5 μg/ml ) in 0.1 M NaHCO 3 . pH 9.6 was incubated in Immulon I microtiter well strips for 16 hours at 4°C. After washing with TBS, 5 mM CaCl 2 , 0.05% Tween 20 buffer (TBS-T) and blocking with 3% BSA, ,25 I-A2 (5 nM) and increasing concentrations unlabeled A2 (0-1750 nM) were added. Following the incubation for 1 hour at 37 °C and washing with TBS-T, the radioactivity bound to the wells was counted. 125 I-A2 binding in the presence of unlabeled A2 was plotted using the computer program "Ligand." The K d value for A2/LRP binding was calculated from the displacement curve, showing a best fit of the data to a single class of sites. Effect of RAP on the clearance of 12 I-A2 domain from the plasma of mice. To elucidate the role of LRP receptor in the clearance of the A2 domain from ^10- plasma in vivo we tested the plasma level of l25 I-labeled A2 in the presence and absence of RAP after tail vein injection in mice. 250 μl samples of A2 (36 nM), in the presence and absence of RAP (267 μM) were injected into the tail vein of BALB/c mice. At the indicated times, blood (50 μl) was collected into 10 μl of 0.5 M EDTA and counted for its 125 I content. RAP significantly delays the plasma elimination of A2 domain. This experiment indicates that a RAP dependent hepatic receptor, LRP, plays a major role in the removal of A2 from circulation. LRP receptor mediated internalization and degradation of the Ϊ25 I-A2 domain by fibroblast cells. The cellular uptake and degradation of activated factor VIII fragments was studied using mouse embryonic fibroblast (MEF) cells expressing low density lipoprotein receptor - related protein (LRP), a multi ligand endocytic receptor, and PEA 13 cells represents fibroblasts lacking LRP. The fVIIIa subunits interaction with MEF and PEA 13 cells represent an adequate model for in vivo processes because fibroblast cells became exposed to coagulation site upon vascular injury. LRP mediated internalization and degradation of some proteins (Thrombin: ATIII complex and other complexes of thrombin with inhibitors, tissue factor pathway inhibitor involved in coagulation cascade is known. l25 I-A2 (10 nM) was incubated with cells for several times and amount of surface bound, internalized and degraded 125 I-labeled protein were determined as described under "Methods." The A2 domain was internalized and degraded by MEF cells but not by PEA 13 cells suggesting that expression of LRP receptor is required for these processes. The internalization and degradation of A2 was blocked by RAP, an inhibitor of LRP binding to its ligands. Internalization of the 125 I-A2 and APC cleaved A2 domain, by LRP presenting MEF cells and control PEA 13 cells, lacking LRP. Inactivation of fVIIIa by APC leads to a cleavage of the A2 at Arg 562 . Since cofactor activity cannot be reconstituted from A2 N /A2 C and A1/A3C1C2 dimer, we proposed that A2 N /A2 C removal from circulation may occur by a mechanism different than that for intact A2. To examine the effect of proteolysis by APC on cellular internalization of the A2 domain, we compared the 125 I-A2 and l25 I-A2 N /A2 c uptake by MEF and PEA 13 cells. We found that in contrast to A2 domain, the internalization of 125 I-A2 N /A2 C is not mediated by LRP receptor. Binding the A2 domain to the immobilized LRP. To the microtiter wells with immobilized LRP 125 I-A2 (5 nM) and increasing concentrations of unlabeled A2 (0-1750 nM) were added. After incubation for 1 hour at 37°C the wells were washed with TBS-T and radioactivity bound to the wells was counted. I25 I-A2 binding in the presence of unlabeled A2 is expressed as the percentage of 125 I-A2 binding, when no competitor was added. The data was analyzed using the computer program "Ligand". The K d value for A2/ LRP binding calculated from the displacement data was 130 nM. Internalization of ,25 I-labeled A1/A3C1C2 and A1 33 7A3C1C2 by fibroblast cells. We proposed that phospholipid binding site previously localized to the C2 domain of fVIII light chain mediates the cellular surface binding and internalization of A1/A3C1C2 and A1 336 /A3C1C2 dimers. To test this hypothesis we determined internalization ,25 I-A1/A3C1C2 and I25 I-A1 336 /A3C1C2 by MEF cells in the presence and absence of anti-C2 domain monoclonal antibody NMC- VIII/5, which blocks the membrane binding sites of the C2 domain. Wells containing 2x10 5 MEF cells were incubated with 3 nM of 125 I- A1/A3C1C2 or 3 nM of 12 J-A1 33 7A3C1C2 at 37°C in the presence or absence of 30 nM monoclonal antibody NMC-VIII/5. In the control experiments, PEA 13 cells lacking LRP were incubated as above with 125 I-A1/A3C1C2 and 125 I- A1 336 /A3C1C2. At several times internalization of the dimers was described under "Methods." Since internalization of both ,2 J-A1/A3C1C2 and ,2 J-A1 336 /A3C1C2 dimers was completely inhibited hy monoclonal antibody NMC-VIII/5, that recognizes the membrane binding site of fVIII C2 domain, we concluded that membrane binding of C2 is a critical step required for internalization of the above dimers. The rate of internalization was similar for MEF and PEA 13 cells, which indicates that LRP receptor is not involved in this process. Degradation of 125 I-A1/A3C1C2 and 12 I-A1 336 /A3C1C2 by MEF cells. MEF cells were incubated with ,25 I-A1/A3C1C2 (3 nM) or 12 JA1 336 /A3C1C2 (3 nM) for 22 hours at 37 °C in the presence and absence PAP (1 μM). The degradation of dimers was measured as described under "Methods". The degradation of A1/A3C1C2 dimer is RAP dependent. In contrast, degradation of APC cleaved Af 36 /A3ClC2 dimer is RAP independent and does not correlate with LRP expression. Conclusions The A2 domain was internalized and degraded by mouse embryonic fibroblasts (MEF) which are expressing low density lipoprotein receptor - related protein (LRP), a multi ligand endocytic receptor. The internalization and degradation of A2 was blocked by RAP, an inhibitor of LRP binding to its ligands. In vivo clearance studies in mice demonstrated that RAP inhibited the clearance of 125 I-A2 from circulation. The radioactivity was preferentially accumulated in liver in the absence but not in the presence of RAP. This indicate that a RAP sensitive hepatic receptor most likely LRP, plays a major role in the removal of 125 I-A2 from the circulation. The phospholipid binding site previously localized to the C2 domain of fVIII light chain mediates the cellular membrane binding and internalization of A1/A3C1C2 and A1 336 /A3C1C2 dimers. LRP receptor does not participate in cellular uptake and degradation of fragments A2 N /A2 C and A1 336 /A3C1C2, produced by irreversible inactivation of fVIIIa by APC. A2 and A1/A3C1C2 fragments produced by reversible inactivation of fVIIIa are removed by LRP-mediated and LRP-independent mechanisms, respectively. LRP is involved in the regulation of coagulation processes in vivo, by removal of A2 domain and A1/A3C1C2 dimer, the fragments from which active factor Villa can be reconstituted. Example 2 The plasma glycoprotein factor VIII (fVIII) serves as a cofactor for the factor X activation complex in the intrinsic pathway of blood coagulation. FVIII circulates in plasma in a tight noncovalent complex with its carrier protein von Willebrand factor (vWf). Although the complex formation of fVIII with vWf is critical for maintenance of a normal half-life and level of fVIII in circulation, the mechanisms associated with fVIII turnover are not well defined. In the present study, we found that catabolism of fVIII is mediated by the low density lipoprotein receptor-related protein/α 2 -macroglobulin receptor (LRP), a liver endocytic, receptor responsible for in vivo clearance of a number of structurally unrelated ligands. A specific binding between fVIII and LRP was demonstrated by homologous ligand competition experiments, where a K d of 116 nM was determined for fNIII binding to LRP. A 39 kDa receptor-associated protein (RAP), an antagonist of ligand binding by LRP, completely inhibited fVIII binding to purified LRP. The region of fNIII involved in its binding to LRP was localized to the A2 domain residues 484-509, based on the ability of the isolated A2 domain and the synthetic A2 domain peptide 484-509 to prevent fVIII interaction with LRP. Since vWf did not inhibit fVIII binding to LRP, we proposed that LRP receptor may internalize fVIII from its complex with vWf. In agreement with this, mouse embryonic fibroblasts (MEF) that express LRP, but not fibroblasts genetically deficient in LRP (PEA 13), were able to internalize and degrade 125 I-fVIII/vWf complex. The latter processes were competed by RAP and A2 subunit of fVIII, indicating that cellular internalization and subsequent degradation were mediated by interaction of the A2 domain of fVIII with LRP. MEF cells were not able to internalize 125 I-vWf from 125 I-vWf /fVIII complex. This indicates that vWf does not follow fVIII in the LRP-mediated pathway and dissociates from fVIII at the early stage of endocytosis. In vivo clearance studies of 125 I-fNIII/vWf complex in mice demonstrated that RAP prolonged the half-life of 125 I-fVIII in circulation by 2.5-fold, indicating that RAP-sensitive receptor, most likely LRP, is responsible for the plasma clearance of fVIII. Introduction The plasma glycoprotein factor VIII (fVIII) functions as a cofactor for the factor X activation enzyme complex in the intrinsic pathway of blood coagulation, and it is decreased or nonfunctional in patients with hemophilia A. The fVIII protein consists of a homologous A and C domains and a unique B domain which are arranged in the order A1-A2-B-A3-C1-C2 (Vehar, G.A., et al. , Nature 372:337-340 (1984)). It is processed to a series of Me 2 " linked heterodimers produced by cleavage at the B-A3 junction (Fay, P. J., et al, Biochem. Biophys. Acta. 577:268-278 (1986)), generating a light chain (LCh) consisting of an acidic region (AR) and A3, CI, and C2 domains and a heavy chain (HCh) which consists of the Al, A2, and B domains (Fig. 1). Transplantational studies both in animals and in humans demonstrated that the liver hepatocytes are the major fVIII-producing cells (Lewis, J. H.. et al, N. Engl. J. e 372:1189-1 191 (1985); Bontempo, F. A., et al. Blood 69:1721- 1724 (1987)). Immediately after release into circulation, fVIII binds with high affinity (K d < 0.5 nM (MacGregor, I.R., et al, Vox. Sang. 69:319-327 (1995); Saenko, E.L. and Scandella, D., J. Biol Chem 272: 18007-18014 (1995)) to its carrier protein vWf to form a tight, noncovalent complex, which is required for maintenance of a normal fVIII level in the circulation. Complex formation with vWf stabilizes association of the LCh and HCh within fVIII molecule (Wise, RJ., et al, J. Biol. Chem. 266:21948-21955 (1991)) and prevents fVIII from C2- domain mediated binding to phospholipid membranes (Gilbert, G.E., et al, J. Biol. Chem. 267:1586115868 (1992)), activation by activated factor X (Koppelman, S.J., et al, J. Lab. Clin. Med. 123:585-593 (1994)) and from protein C-catalyzed inactivation (Fay, P.J., et al., J. Biol. Chem 266:2172-2177 (1991)). vWf comprises a series of high molecular weight, disulfide-bonded multimers with molecular weight values as high as 2 x 10 7 Da (Hoyer, L.W. and Shainoff, J.R., Blood 55: 1056- 1059 (1980)) and circulates in plasma at 10 μg/ml or 50 nM, assuming a molecular mass of 270 kDa for vWf monomers (Girma, J.- P., et al, Biochemistry 25:3156-3163 (1986)). Since the concentration of fVIII in plasma is approximately 1 nM (Wion, K., et al, Nature 377:726-730 (1985)), one fVIII molecule is bound per 50 vWf monomers (Vlot, A.J., et al, Blood 55:3150-3157 (1995)). Activation of fVIII by thrombin leads to dissociation of activated fNIII (fVIIIa) from vWf and to at least 100-fold increase of the cofactor activity. The fVIIIa is a A1/A2/A3-C1-C2 heterotrimer (Fay, P.J., et al, J. Biol. Chem 266:8957-8962 (1991)) in which domains Al and A3 retain the metal ion linkage (Fig. 1) and the stable dimer A1/A3-C1-C2 is weakly associated with the A2 subunit through electrostatic forces (Fay, P.J., et al. , J. Biol. Chem 266: 8957-8962 (1991)). Spontaneous dissociation of the A2 subunit from the heterotrimer results in non-proteolytic inactivation of fVIIIa. Infusion of fVIII/vWf complex or purified plasma or recombinant fVIII into patients with severe hemophilia A who do not have fNIII (Fijnvandraat, K., et al, Thromb. Haemostas. 77:298-302 (1997); Morfini, M.. et al, Thromb. Haemostas. 65:433-435 (1992)) or in normal individuals (Over, J., et al. , J. Clin. Invest. 62:223-234 (1978)) results in a similar f III disappearance with a half-life of 12-14 hours. Although the complex between fVIII and vWf is crucial for normal half-life and level of fVIII in the circulation, the mechanisms associated with turnover of fVIII/vWf complex are not well defined. We proposed that fVIII/vWf complex is eliminated from plasma via clearance receptor and tested the possibility that this receptor is low density lipoprotein related protein receptor (LRP). Cellular endocytosis mediated by LRP was shown to be a mechanism of removal of a number of structurally unrelated ligands including several proteins related to coagulation or fibrilolysis. These ligands are: complexes of thrombin with antithrombin III (ATIII), heparin cofactor II (HC11) (Kounnas, M.Z., et al, J. Biol. Chem. 271:6523-6529 (1996)), protease nexin I (Knauer, M.F., et al, J. Biol. Chem. 272:12261-12264 (1997)), complexes of urokinase-type and tissue- type plasminogen activators (u-PA and t-PA, respectively) with plasminogen activator inhibitor (PAI-1) (Nykjaer, A., et al, J. Biol. Chem. 267:14543-14546 (1992);Orth, K., et al, Proc. Natl. Acad. Sci. 59:7422-7426 (1992)), thrombospondin (Mikhailenko, I., et al, J. Biol. Chem. 272:6784-6791 (1997)), tissue factor pathway inhibitor (TFPI) (Warshawsky, I., et al, Proc. Natl. Acad. Sci. 97:6664-6668 (1994)), and factor Xa (Narita, M., et al. Blood 97:555-560 (1998); Ho, G., et al, J. Biol. Chem 277:9497-9502 (1996)). LRP, a large cell-surface glycoprotein identical to oc 2 -macroglobulin receptor (Strickland, D.K., et al, J. Biol. Chem. 265:17401-17404 (1990)), is a member of the low density lipoprotein (LDL) receptor family which also includes the LDL receptor, very low density lipoprotein (VLDL) receptor, vitellogenin receptor and glycoprotein 330 receptor. LRP receptor consists of the non- covalently linked 515 kDaα-chain(Herz, J., etα/.,E 73O . 7:41 19-4127 (1988)) containing binding sites for LRP ligands, and the 85 kDa transmembrane β-chain. Within the -chain, cluster of cysteine-rich class A repeats is responsible for ligand binding (Moestrup, S. K., et al, J. Biol. Chem 265:13691-13696 (1993)). In contrast to the acidic ligand binding region in LRP. its ligands expose regions rich in positively charged amino acid residues (Moestrup, S.K., Biochim. Biophys. Acta 7797:197-213 (1994)). This type of binding and 31 class A repeats present in LRP may be responsible for its wide ligand diversity and ability to serve as a multi-ligand clearance receptor. LRP is expressed in many cell types and tissues including placenta, lung and brain (Moestrup, S.K., et al , Cell Tissue Res. 269:375-382 (1992)) and is a major endocytic receptor in the liver (Strickland, D.K., et al, FASEB J. 9:890-898 (1995)). A 39 kDa receptor-associated protein (RAP) binds to LRP with high affinity (K d =4 nM (27)) and inhibits binding and LRP-mediated internalization and degradation of all ligands (Moestrup, S.K. Biochim. Biophys. Acta 7797:197-213 (1994); Williams, S.E., et al, J. Biol. Chem. 267:9035-9040 (1992)), therefore serving as a useful tool for testing whether LRP is involved in endocytosis of a given ligand. In the present study we demonstrated that fVIII specifically binds to LRP, and that LRP mediates the internalization and subsequent degradation of fNIII in cultured fibroblasts and appears to be responsible for in vivo clearance of fNIII from circulation. We also demonstrated that interaction of the A2 domain of fVIII with LRP is responsible for mediating catabolism of fVIII. Experimental Procedures Monoclonal Antibodies. The monoclonal antibodies (mAbs) C4 (epitope within the fVIII light chain residues 1670-1684 (Foster, P.A., et al, J. Biol Chem 263:5230-5234 (1988))). C5 (epitope within Al residues 351 -361) and T5 (epitope within the residues 701 -740 (Fulcher, C. A., et al. , J. Clin. Invest. 76: 117- 124 (1985))) were kindly provided by Dr. Carol Fulcher (Scripps Clinic and Research Foundation, La Jolla. CA). The anti-A2 mAb 8860 was generously provided by Baxter/Hyland. Mab 413 (epitope within A2 domain residues 484- 509 (Healey, et al, .. F., J. Biol. Chem 270: 14505-14509 (1995))) was prepared as described previously (Saenko, E.L., et al, J. Biol. Chem 269:1 1601-1 1605 (1994)). Proteins. LRP was isolated from human placenta as described (Ashcom, J.D., et al , J. Cell Biol. 770: 1041-1048 (1990)). Human RAP was expressed in bacteria and purified as described (Williams, S.E., et al, J. Biol. Chem. 267:9035-9040 (1992)). FVIII was purified from therapeutic concentrates of Method M, American Red Cross (Saenko, E.L., et al, J. Biol. Chem 77:27424-27431 (1996)). HCh and LCh were prepared from fVIII as described previously (Saenko, E.L. and Scandella, D., J. Biol Chem 272, 18007-18014 (1995)). Purification of the A1/A3-C1-C2 dimer and A2 subunit was performed using ion exchange chromatography of thrombin activated fVIII on a Resource S column (Pharmacia) (Fay, ?.T., etal,J. Biol Chem 268, 17861-17866 (1993)). Residual A2 present in the A1/A3-C1-C2 preparation was removed by its passage over an immobilized mAb 8860 column equilibrated in 20 mM Tris, pH 7.4, 0J 5 M NaCl, 5 mM CaCl 2 . Radiolabeling of fVIII and synthetic peptides. Prior to iodination fVIII and A2 were dialyzed into 0.2 M sodium acetate, 5 mM calcium nitrate, pH 6.8 (iodination buffer). Five μg of fVIII in 30 μl of iodination buffer were added to lactoperoxidase beads (Worthington Biochemical Corp.), 5 μl of Na ,25 I (100 mCi/ml, Amersham), and 5 μl of 0.03% H 2 O 2 (Mallincrodt) and incubated for 4 min. Free Na' 25 I was removed by chromatography on a PD10 column (Pharaiacia). The specific radioactivity of fVIII and A2 was 3.5-5 μCi/μg of protein. The activity of I25 I-fVIII determined in the one-stage clotting assay (3740 units/μg) was similar to that of unlabeled fVIII. Solid-phase binding assays. Homologous and heterologous ligand displacement assays were performed as previously described (Williams, S.E., et al, J. Biol. Chem. 267:9035-9040 (1992)). Microtiter wells were coated with purified LRP or BSA (3 μg/ml) in 50 mM TrJs, 0. 15 M NaCl, pH 8.0, for 16 h and then blocked with 3 % BSA in TBS. Coated wells were incubated with 125 I-A2 or ,25 I- fVIII in 20 mM Tris-buffered saline pH 7.4, containing 5 mM CaCl 2 , 0.05 % Tween-20 in the presence or absence of unlabeled competitors for 1 h at 37 °C. The radioactivity bound to the wells was counted using a γ -counter (Pharmacia). Affinity constants were derived from homologous and heterologous displacement data using the computer program LIGAND (Munson, PT and Rodbard, D. Anal. Biochem. 707:220-239 (1980)). Cell-mediated ligand internalization and degradation assays. A normal mouse embryonic fibroblast line (MEF) and a mouse embryonic fibroblast cell line that is genetically deficient in LRP biosynthesis (PEA 13) were obtained from Dr. Joachim Herz (University of Texas Southwestern Medical Center, Dallas, TX) and maintained as described (Willnow, T.E. and Herz, J., J. Cell Sci. 107:1X9-126 (1994)). Cells were seeded at lxlO 5 cells/well and allowed to grow for 24 h at 37 °C, 5% CO 2 . Cellular internalization and degradation assays were conducted as described previously (Kounnas, M.Z., et al, J. Biol. Chem. 270:9307-9312 (1995)). Internalization and degradation of the 125 Mabeled fVIII and A2 was measured after incubation for indicated time intervals at 37 °C in 0.5 ml of Dulbecco's modified medium (Gibco BRL) containing 2% BSA. Internalization was defined as radioactivity that is resistant to release from cells by trypsin (50 μg/ml) and proteinase K (50 μg/ml) (Sigma) in a buffer containing 5 mM EDTA. This treatment was previously shown to release radioligand bound to cell surface (Kounnas, M.Z., et al., J. Biol. Chem. 270:9301-93 X2 (1995)) and therefore the ligand remained associated with cells after this treatment was considered as internalized. Degradation was defined as radioactivity in the medium that is soluble in 10% trichloroacetic acid. The value of degradation was corrected for non-cellular mediated degradation by subtracting the amount of degradation products generated in parallel wells lacking cells. Clearance of 125 I-A2 domain and I25 I-fVIII/vWf complex from mouse plasma. The complex of ,25 I-labeled fVIII with vWf in the presence or absence of RAP (in a total volume 250 μl) was injected in a tail vein of BALB/C mice over a period of approximately 20 seconds. At selected time intervals following injection (1, 3, 6, and 18 min), blood (50 μl) was withdrawn from the orbital plexus into 10 μl of 100 mM EDTA, and the radioactivity of the aliquot was determined. The percentage of ligand remaining in circulation was calculated considering radioactivity of the aliquot taken at 1 min after injection as 100%. The clearance of each preparation was examined in two mice and the results were averaged. At the end of experiment, animals were sacrificed, liver lobules and kidneys were excised and weighed, followed by measuring the radioactivity in these tissues. Results Factor VIII binds to LRP and its binding is prevented by RAP. The ability of fNIII to bind to LRP in vitro was examined in homologous displacement binding assay. In the assay, binding of l25 I-fVIII (1 nM) to purified LRP, but not to BSA-coated wells, was competed (> 90%) by excess of unlabeled fVIII (Fig. 5A). The quantitative data regarding fVIII interaction with LRP were derived from the homologous displacement of 125 I-fVIII by unlabeled fVIII, which was adequately described by a model containing a single class of fNIII binding sites with K d of 1 16 nM. To elucidate whether fVIII in a complex with vWf is also able to bind to LRP, we tested the effect of vWf on l25 I-fVIII binding to immobilized LRP. In this experiment, 125 I-fVIII was preincubated with vWf for 30 min at 37 °C to allow complex formation prior to its addition to LRP coated wells. As shown in Fig. 5A, ,25 I-fVIII binding to LRP was not inhibited by vWf up to the concentration of 1000 nM, which is 20-fold higher than its concentration in plasma (50 mM (Vlot, A.J., et al, Blood 55:3150-3157 (1995))). This indicates that the complex formation with vWf does not affect fVIII ability to bind to LRP. RAP, the antagonist of LRP-ligand binding, completely inhibited the binding of ,25 I-fVIII to LRP-coated wells with K, of 2.5 nM (Fig. 5B), a value similar to the previously determined affinity (4 nM) of RAP for LRP (Strickland, D.K., et al, J. Biol. Chem. 265:17401-17404 (1990)). Together, these results demonstrate specific fVIII binding to LRP. The amino acid residues 484-509 within the fVIII A2 domain are responsible for fVIII binding to purified LRP. In order to localize fNIII region(s) involved in interaction with LRP, binding between 125 I-fVIII and immobilized LRP was competed by unlabeled fVIII fragments. As shown in Fig. 6, HCh and A2 domain of fVIII, but not LCh (AR-A3-C1-C2) or A1/A3-C 1 -C2 dimer, displaced ,25 I-fNJII from LRP in the heterologous ligand displacement assay. The K, values determined for the HCh and A2 were similar, 120 nM and 132 nM, respectively. The similarity of the above K d value for fVIII binding to LRP and the K, value for inhibition of this binding by isolated A2 subunit indicates that A2 domain of HCh is responsible for fNIII binding to LRP. To localize the region of the A2 domain responsible for the interaction with LRP, we tested the effect of anti-A2 monoclonal antibodies with known epitopes on fVIII/LRP binding. Fig. 7A shows that mAb 413 (epitope within the A2 domain residues 484-509 (Healey, J.F., etal.,J. Biol. Chem 270: 14505-14509 (1995))) but not mAb T5 (epitope within the A2 domain residues 701-740 (35)) is able to block fVIII/LRP interaction. The concentration of mAb 413 required for 50%) inhibition of l25 I-fVIII/LRP binding was 2.5 nM. The low molar excess (2.5-fold) of mAb 413 over fVIII required for 50% inhibition of fVIII/LRP binding is consistent with a previously reported high affinity of m Ab 413 for fVIII (Lollar. P., et al. , J. Clin. Invest. 93:2497-2504 (1994)). In a control experiment, mAbs C5 (epitope within Al residues 351-361) and C4 (epitope within LCh residues 1670-1684 (Foster, P.A., et al, J. Biol. Chem 263:5230-5234 (1988))) did not have any effect on fVIII binding to LRP (data not shown), which is consistent with the lack of participation of Al and LCh in fVIII binding to LRP. Since it was previously demonstrated that mAb 413 recognizes synthetic peptide with a human fNIII sequence 484-509 (Healey, J.F., et al. , J. Biol. Chem 270:14505- 14509 (1995)), we tested if the region of the A2 domain encompassed by peptide 484-509 is involved in binding to LRP. As seen from Fig. 7B, the synthetic peptide 484-509, but not the control A2 peptide 432-456, inhibited fVIII binding to LRP in a dose-dependent fashion, indicating that the region 484-509 of the A2 domain contains critical residues for fVIII binding to LRP. In a control experiment, no binding of ,25 I-fVIII to BSA-coated wells was observed in the presence of peptide 484-509 (Fig. 7B). Internalization and degradation of I25 l-fVIII complex with vWf by cultured fibroblasts is mediated by LRP. Since the data presented above demonstrated specific interaction between fVIII and LRP, and vWf does not interfere with this interaction, we hypothesized that LRP may be also capable of mediating the cellular internalization of ,25 I-fVIII from its complex with vWf. To examine this hypothesis, cellular uptake and degradation experiments were conducted in mouse embryonal fibroblasts (MEF) which express LRP and in PEA 13 fibroblasts that are genetically deficient in LRP (Willnow, T.E. and Herz, J. J. Cell Sci. 107:1X9- 726 (1994)). The 12 T-fNIII/vWf complex was prepared by 30 min (37 °C) incubation of ,25 I-fVIII with vWf at their plasma concentrations of 1 nM and 50 nM, respectively. As shown in Figs. 8A and B, MEF cells, but not PEA 13 cells lacking LRP, were capable of internalizing and degrading of l25 I-fVIII in the presence of vWf. Further, internalization and degradation of 125 I-fNIII by MEF but not by PEA 13 fibroblasts was inhibited by RAP, an antagonist of ligand binding to LRP. The ability of RAP to block the uptake and degradation of 125 I- fVIII/vWf in MEF cells and inability of PEA 13 cells to efficiently mediate uptake and degradation indicates that LRP is the mediator of 125 I-fNIII/vWf catabolism. To further characterize the degradation pathway of fVIII in the MEF cells, we tested the effect of chloroquine (an agent that blocks lysosomal degradation) on 125 I-fVIII degradation. As seen from Fig. 8B, the degradation of l25 I-fVIII is completely inhibited by chloroquine. To elucidate if fVIII internalization in the absence of vWf is also mediated by LRP, we measured the internalization and degradation of isolated 125 I-fVIII (Fig. 9). As seen from Figs. 9A and B. both internalization and degradation of isolated ,25 I-fVIII by MEF fibroblasts is approximately 2-fold higher than that in the presence of vWf. RAP inhibited internalization and degradation of 125 I-fVIII to a lesser degree than those of 125 I-fVIII/vWf complex and, in addition, LRP- deficient PEA 13 fibroblasts were able to internalize and degrade isolated ,25 I- fVIII. This indicates that LRP-mediated pathway is not the sole mechanism of fVIII internalization and degradation in the absence of vWf. To determine whether vWf bound to fNIII is also internalized and degraded by MEF cells, internalization and degradation of ,25 I-labeled vWf complexed with fVIII was measured. As shown in Figs. 9A and B, the amounts of internalized and degraded ,25 I-vWf by both MEF and PEA 13 cells were less than 5 %> of the corresponding amounts of 125 I-fNIII catabolized from its complex with vWf under the same experimental conditions. This indicates that vWf does not follow fNIII in the LRP-mediated pathway and possibly dissociates from fVIII at early stage of endocytosis, prior to entry of the complex into endosomal compartments. The A2 subunit of fVIII inhibits endocytosis and degradation of ,2 I- fVIII/vWf by MEF cells. Since we have demonstrated above that the A2 subunit of fVIII prevents an in vitro interaction between LRP and fNIII, we examined if A2 can also inhibit LRP-mediated internalization and degradation of fVIII/vWf complex by MEF cells. Figs. 10A and B demonstrate that 1000-fold excess of the A2 subunit over 125 I-fVIII/vWf complex effectively inhibit internalization (by >70%> after 4 hours) and degradation (by >60%> after 4 hours) of this complex. In contrast, A1/A3-C1-C2 heterodimer, which did not inhibit fVIII interaction with purified LRP in the above experiments, did not have any effect on 125 I-fNIII endocytosis and degradation by MEF cells (Fig. 10). To confirm that the inhibitory effect of the A2 subunit results from its direct competition with 125 I-fVIII/vWf complex for LRP-mediated internalization and degradation, we tested whether MEF cells are able to internalize and degrade isolated A2 subunit. As shown in Figs. 11 A and B, 125 I-A2 is readily internalized and degraded by LRP-expressing MEF cells. Both the internalization and degradation of the 125 I-labeled A2 were blocked in the presence of RAP. In contrast, LRP-deficient PEA 13 cells were unable to internalize or degrade 125 I-A2 (Fig. 11), confirming that catabolism of the A2 subunit is LRP-mediated. To verify that LRP-mediated internalization and degradation of the A2 domain was not the unique feature of the MEF cells, we tested 125 I-labeled A2 internalization and degradation by smooth muscle cells (SMC) and alveolar epithelial cells (T2), which also express LRP on their surfaces (Moestrup, S.K., Ce7/ Tissue Res. 269:315-382 (1992)). As shown in Figs. 1 1C and D, RAP effectively inhibited both internalization of ,25 I-A2 by SMC and T2 (by 81 %> and 64 %>, respectively), and its degradation (by 78 % and 68 %), indicating that these processes were mediated by LRP. Thus, the data shown in Figs. 10 and 1 1 demonstrate that LRP is capable of binding fVIII via its A2 domain and of mediating fVIII endocytosis leading to lysosomal degradation. Effect of RAP on the plasma clearance of , 5 I-fVIII and 125 I-A2. To determine whether LRP is capable of catabolizing the isolated fNIII A2 subunit and whole fNIII from its complex with vWfm vivo, the effect of RAP on the clearance rates of 125 I-fNIII/vWf complex and 125 I-A2 in mice was tested. As shown in Fig. 12A, RAP increased the half-life of both 125 I-A2 and 125 I-fVIII in mouse plasma by approximately 4 and 2.5-fold, respectively. In addition, in the absence of RAP, most of radioactivity was found in the liver but not in kidney, consistent with LRP presence in high abundance in hepatic tissues (Strickland, D.K., et al. , FASEB J. 9:890-898 ( 1995)). Thus, our data indicate that a RAP-sensitive hepatic receptor, LRP, plays a major role in the removal of fVIII and its A2 subunit from circulation. Discussion In the present study we demonstrated that LRP mediates the internalization and degradation of human fVIII in a model system using LRP- expressing cells and is responsible for fVIII clearance in vivo. This conclusion is based on several independent observations. First of all, we found that fNIII directly binds to purified LRP immobilized on microtiter wells, and that this binding is competed by RAP, an antagonist of ligands binding to LRP. Second, ,25 I-fVIII is internalized from its complex with vWf by mouse fibroblasts expressing LRP (MEF cells) , but not by mouse fibroblasts genetically deficient in LRP (PEA 13 cells). Third, we demonstrated that RAP effectively inhibited the cellular uptake and degradation of I25 I-fVIII from its complex with vWf by MEF cells and in vivo clearance of ,25 I-fVIII from circulation in mice. Our studies revealed that the A2 domain of fVIII is responsible for its interaction with LRP, since only A2 domain and HCh, which contains the A2 domain, were able to inhibit the interaction of l25 I-fVIII with LRP in a purified system. Thus, it was concluded that A2 is responsible for fVIII binding to LRP. Based on the observation that vWf did not inhibit fVIII binding to LRP, we proposed that LRP may internalize fVIII from its complex will. vWf. Indeed, mouse embryonic fibroblasts (MEF) that express LRP, but not fibroblasts genetically deficient in LRP, were able to internalize and degrade 125 I-fVIII in the presence of vWf. These processes were competed by RAP and A2 subunit of fNIII, indicating that cellular internalization and degradation were mediated by interaction of the A2 domain of fNIII with LRP. The physiological relevance of the observations utilizing the LRP-expressing cell model system was supported by in vivo clearance studies of 125 I-fVIII/vWf complex in mice which demonstrated that RAP prolonged the half-life of 125 I-fNIII in circulation by 2.5- fold, indicating that a RAP-sensitive receptor, most likely LRP, is responsible for the clearance of fVIII from plasma. Further localization of the region within the A2 domain responsible for its binding to purified LRP was initiated by the finding that monoclonal antibody with an epitope within A2 domain residues 484-509 completely inhibited fVIII interaction with LRP. Inhibition of fVIII/LRP binding by synthetic peptide with a human fNIII sequence 484-509 indicated that the region of the A2 domain is likely to be directly involved in fNIII binding to purified LRP. The region 484-509 contains 6 positively charged residues, Lys at positions 493, 496 and 499 and Arg at positions 484, 489 and 490. Basic residues in lipoprotein lipase (Chappell, D.A., et al, J. Biol. Chem. 265:14168-14175 (1993)), u-PA-PAI-1 complex (Rodenburg, K.W., et al, Biochem. J. 329:55-63 (1998)), and 2 -macroglobulin (Howard, G. C, et al, J. Biol. Chem 277:14105- 1411 1 (1996)) were previously shown to be critical for electrostatic interaction with LRP. Alanine substitution of the basic amino acid residues in lipoprotein lipase (Williams, S.E., etal, J. Biol. Chem. 269:8653-8658 (1994)), u-PA/PAI-I complex (Rodenburg, K.W., et al, Biochem. J. 329:55-63 (1998)) and in the receptor binding fragment from α 2 -macroglobulin (Howard, G.C., et al. J. Biol. Chem 277:14105-1411 1 (1996)) lead to a considerable reduction of affinity for ligand binding to LRP and partial (Rodenburg, K.W., et al, Biochem. J. 329:55- 63 (1998)) or complete (Howard, G.C., et al., J. Biol. Chem 277:14105-14111 (1996)) inhibition of internalization and degradation of the mutants. Therefore, Ala or other amino acid substitutions within the 484-509 region of the recombinant fVIII are useful for reduction of the rate of its LRP-mediated endocytosis and generation of the fVIII mutants with a longer life in the circulation. FVIII binds to purified LRP with affinity 1 16 nM, which is much lower than the concentration of fVIII/vWf complex in plasma (1 nM; Wion, K., et al, Nature 377:726-730 (1985)). FVIII affinity for LRP is similar to that of the complexes of serine proteases with inhibitors such as ATIII/thrombin (Kounnas, M.Z., et al. J. Biol. Chem. 277:6523-6529 (1996)), HCII/thrombin and α,- antitrypsin/trypsin (Koumιas, M.Z., etα/., J. Biol. Chem. 277:6523-6529 (1996)), which also bind to LRP with affinities 80-120 nM, and weaker than measured for other LRP ligands. It was shown (Kounnas, M.Z., et al, J. Biol. Chem. 271:6523-6529 (1996)) that internalization and degradation of the above low affinity LRP ligands at their 1 nM concentration by MEF cells occur at a lower rate than that of the u-PA/PAI- 1 complex which binds to LRP with high affinity (K d < 1 nM). Therefore, relatively low affinity of fVIII for LRP is responsible for a slow rate of fVIII internalization and degradation by MEF cells, which is comparable to the rate of ATIII/thrombin, HCII/thrombin and l- antitrypsin/trypsin degradation at 1 nM concentration of each ligand. The low affinity of fVIII for LRP may also be a necessary requirement for the relatively long fVIII half-life (12-14 h) in plasma of normal individuals (Over, J., et al, J. Clin. Invest. 62:223-234 (1978)). Alternatively, the low fVIII affinity for LRP may be compensated by concentration of fVIII molecules on the membrane of LRP-expressing cells, for example, via interaction with cell-surface proteoglycans which have been shown to facilitate the uptake of a number of LRP ligands including lipoprotein lipase (Chappell, D.A., et al, J. Biol. Chem. 265:14168- 14175 (1993)), hepatic lipase (Kounnas, M.Z.. et al, J. Biol. Chem. 270:9301- 9312 (1995)), and thrombospondin (Mikhailenko, I., et al , J. Biol. Chem. 270:9543-9549 (1995); Mikhailenko, I., et al, J. Biol. Chem. 272:6784-6791 (1997)). We found that internalization and degradation of isolated fVIII by MEF cells was greater than the corresponding processes for fVIII bound to vWf. In addition, catabolism of the isolated fVIII by MEF cells was only partially inhibited by RAP, indicating that LRP-mediated endocytosis of fVIII is not the sole mechanism of fNIII clearance in the absence of vWf. Our data suggest that in the presence of vWf, which blocks C2 domain-mediated fVIII binding to phospholipid membranes (Saenko, E.L. and Scandella, D., J. Biol. Chem 270:13826-13833 (1995)), fVIII binds only to LRP, whereas in the absence of vWf, fVIII binds both to LRP and to an unidentified cell membrane component. The latter binding may lead to fVIII internalization via RAP-independent pathway, which may be mediated by unidentified receptor as it was previously proposed for hepatic lipase (Kounnas, M.Z., etal, J. Biol. Chem. 270:9307-9312 (1995)). Since we found that l25 I-vWf is not internalized by MEF cells, we propose the model for fVIII endocytosis where fVIII/vWf complex binds to LRP and then vWf dissociates from fVIII during the early stage of fVIII endocytosis, i. e. during formation of the coated pits. Since the half-life for the dissociation of fVIII/vWf complex is about 1 hour (Saenko, E.L. and Scandella, D., J. Biol Chem 272, 18007-18014 (1995)), vWf may delay LRP-mediated endocytosis of fVIII according to the proposed model. Faster catabolism of fVIII in the absence of vWf is consistent with a demonstrated shorter half-life of fVIII in patients with severe von Willebrand disease (vWD) lacking plasma vWf than that in hemophilia A patients, who have normal levels of vWf (Morfini, M., et al. Thromb. Haemostas. 70:210-212 (1993); Lethagen, S., et al, Ann. Hematol 65:253-259 (1992)). Moreover, the half-life of fVIII in vWD patients was prolonged by the presence of vWf in the infused fVIII preparation (Lethagen. S . , etal, Ann. Hematol. 65 :253 -259 (1992)). The above observations were previously explained by vWf-mediated stabilization of fVIII by binding to vWf (Wise. RJ., et al. J. Biol. Chem. 266:21948-21955 (1991)) and via secondary vWf-mediated release of endogenous fNIII (Wise, RJ. , et al, J. Biol. Chem. 266:21948-21955 (1991); Kaufman, RJ., Mol. Cell. Biol. 9: 1233-1242 (1989)). Our data suggest that in addition to the above effects, vWf may reduce the rate of fVIII clearance by preventing LRP-independent pathway and limiting fVIII clearance to LRP-mediated pathway. The activity of the factor X activation complex (factor Xase), consisting of membrane-bound activated fVIIIa and factor IXa, can be down regulated by inactivation of fVIIIa. The latter occurs via proteolytic degradation of fVIII by activated protein C, factor Xa and factor IXa. and via spontaneous but reversible dissociation of the A2 subunit from fVIIIa heterotrimer (Fay, P. J. and Smudzin, T. M., J. Biol. Chem 267:13246-13250 (1992)). Dissociation of the fVIIIa heterotrimer may be accelerated by LRP mediated internalization of the A2 domain, and therefore complement regulation of fVIIIa activity at the sites of coagulation. This hypothesis is supported by availability of LRP at these sites, since LRP is exposed on the surface of monocytes and macrophage (Moestrup, S.K., et al, Exp. Cell. Res. 790:195-203 (1990); Moestrup, S.K., et al, Cell Tissue Res. 269:375-382 (1992)) and upon vascular injury on fibroblasts and smooth muscle cells (Moestrup, S.K., et al, Cell Tissue Res. 269:375-382 (1992)). In addition, it was recently shown that isolated A2 but not isolated Al and A3-C1-C2 subunits of activated fVIII is able to accelerate factor IXa- catalyzed conversion of factor X by approximately 100-fold (Fay, PJ. and Koshibu, K., Blood 92:353a (abstract) (1998)). Even though acceleration of the factor X activation by A2 is only 1 % of that in the presence of heterotrimeric activated fNIII (A1/A2/A3-C1-C2) (Fay, PJ. and Koshibu, K., Blood 92:353a (abstract) (1998)), it is possible that LRP-mediated removal of the A2, dissociated from fVIIIa bound to a phospholipid membrane at the site of coagulation, is important to prevent activation of factor X not in the place of the coagulation event. In summary, the current study demonstrates that LRP can bind fVIII/vWf complex and mediate uptake of fVIII from it. In vivo clearance studies underscored the likelihood that LRP indeed functions to remove LRP from plasma. Example 3 Experiments on the development of recombinant fVIII molecule with extended lifetime in circulation. Since recombinant fNIII products are widely used for fVIII replacement therapy in hemophiliacs who have decreased or nonfunctional fVIII, generation of mutant(s) with a prolonged lifetime is a promising approach to increase the efficacy and reduce the cost of fNIII infusion therapy. A 39 kDa receptor associated protein (RAP) binds reversibly to LRP and inhibits the binding of other ligands and therefore serves as a useful tool for testing whether LRP is involved in endocytosis of a given ligand. We found that fNIII binding to LRP is inhibited by RAP, confirming the specificity of this interaction. Since von Willebrand factor (vWf), bound to fNIII in the circulation, does not inhibit fVIII binding to purified LRP, we proposed that removal of the fVIII/vWf complex from the circulation may also be LRP-mediated. This role of LRP was supported by our finding that the lifetime of human ,25 I-fVIII/vWf complex in mice was 2.5 -times prolonged in the presence of RAP. Based on our finding that fVIII amino acids 484-509 were important for JNIII binding to LRP, these amino acids are also important for LRP-mediated endocytosis. To identify the key fVIII amino acids required for endocytosis, single residues 484-509 are mutated to Ala in the B- domain deleted fVIII (B(-) fVIII). Since the basic residues are commonly involved in ligand binding to LRP. six basic residues within 484-509 (3 Lys and 3 Arg) are mutated. U.S. Patent No. 55,859,204 discloses the substitution to Ala of three of these residues (Arg 484 - Lys 493 and Arg 490 ); however the other 3 residues - Arg 490 , Lys 496 and Lys 499 - were not substituted. Thus, these residues, individually and in combination, are mutated to Ala. In particular, each of three Arg and each of three Lys are mutated by pairs (this implies preparation of 9 additional fVIII Ala double-mutants). It is then determined whether endocytosis of the vWf complexes with B(-) fVIII mutant(s) by LRP-expressing cells is reduced compared to that of wild-type B (-) fVIII/vWf. Some mutations result in a decreased rate of internalization and a longer in vivo half-life of the complex of the B- fVIII mutant with vWf in plasma of mice compared to that of wild type B- fVIII/vWf complex. The data of the in vivo experiments performed in normal and fVIII-deficient mice is mathematically analyzed using biphasic time-course clearance model and equations approximating interspecies scaling which allow to predict fVIII half-life in humans (Toxicology and Applied Pharmacology 136:75-78 (1996)). Clearance of mutant fVIII in vWf-deficient mice which lack fVIII in circulation (a mouse model for severe von Willebrand disease is described in Proc. Natl. Acad. Sci. USA 95:9524-9529 (1998)) is also analyzed. These experiments are aimed at determining mutant fNIII' s prolonged half-life in the absence of vWf. Factor VIII interaction with endothelial cells is also analyzed, since this interaction leads to fVIII internalization. In experiments using fluorescent microscopy techniques we observed uptake of fVIII by endothelial cells. Since a fine equilibrium exists in circulation between fVIII bound to vWf and fNIII bound and internalized by endothelial cells, fVIII interaction with phospholipid endothelial cell membrane is an important factor influencing concentration of fVIII (and hence its half-life) in circulation following fVIII injection. Therefore, individual amino acids within the previously localized fNIII phospholipid binding site (C2 domain region 2303-2332) which play a role in fVIII binding to vWf and to phospholipid are identified. We identify the amino acids playing a key role in fVIII binding to phospholipid, but not to vWf. The amino acids which participate in fVIII binding to vWf and to phospholipids are selected based on the following observations. The homology search between the C2 domain of fNIII and the corresponding region of the discoidin and a family of homologous proteins, containing the so called DS domain, has revealed the fVIII C2 domain sequences involved in the formation of β-structures. In addition, it has been shown that the synthetic fVIII peptide 2310-2320 in which residues 2310 and 2320 are covalently linked to reproduce the corresponding loop structure within the C2 domain, competes for fVIII binding with vWf or phospholipid. Therefore, residues within the 2311-2319 region are mutated to Ala, and other amino acids. Since fN, a fVIII homolog, does not bind to vWf, we mutate only five residues which are unique within the 2311-2319 region of fNIII. The mutants are tested for binding to vWf and phospholipid, which identifies the fVIII residues playing a key role in binding to these ligands. Clearance of the fVIII mutants with reduced phospholipid binding was compared with that of wt- fNIII in normal and hemophilic mice to determine the contribution of the phospholipid-dependent fVIII clearance component to total fNIII clearance. The mutations within the C2 domain region 2310-2320 prove to be effective for extension of fNIII lifetime in circulation, so we generate mutant fVIII in which both the C2 domain mutation(s) (positions 2310-2320) and mutation(s) within the A2 (positions 484-509) are combined. We test the designed extended lifetime fVIII for gene therapy purposes. The extended lifetime fNIII gene is inserted in a virus-based vector, and delivered into hemophilia A mice. The time course of the fVIII in vivo expression level is assessed as follows: the number of the gene copies per cell (hepatic), the gene transcription level, fVIII activity and the antigen level are determined. Since it was shown that high titer antibodies increase clearance of fVIII (Br. J. Hematol. 93:688-693 (1996)), we examine the immune response against the extended lifetime fVIII. We also compare its half-life in circulation in hemophilia A mice which formed antibodies against wild type fNIII.

Claims: What Is Claimed Is: 1. A mutant factor VIII comprising an amino acid substitution at two or more positions in the A2 domain; wherein at least one of said amino acid substitutions is not at any of positions 484, 485, 487, 488, 489, 492, 493, 495, 501 or 508; wherein the mutant factor VIII has reduced receptor-dependent clearance; and wherein the mutant factor VIII has procoagulant activity. 2. The mutant factor VIII of claim 1 , which lacks the B domain. 3. The mutant factor VIII of claim 2, comprising an amino acid substitution at two or more of positions 484 to 509. 4. The mutant factor VIII of claim 3, comprising an amino acid substitution at two or more of positions 490, 496 or 499. 5. The mutant factor VIII of claim 3, comprising an amino acid substitution at one or more of positions 490, 496 or 499; and at one or more of positions 484, 489 or 493. 6. The mutant factor VIII of claim 5, comprising an amino acid substitution at position 490; and at one or more of positions 484, 489 or 493. 7. The mutant factor VIII of claim 5, comprising an amino acid substitution at position 496; and at one or more of positions 484, 489 or 493. 8. The mutant factor VIII of claim 5, comprising an amino acid substitution at position 499; and at one or more of positions 484, 489 or 493. 9. The mutant factor VIII of claim 2, comprising SEQ ID NO:5. 10. A pharmaceutically acceptable composition comprising the mutant factor VIII of claim 2. 11. A method of treating hemophilia which comprises administering to a patient in need thereof an effective amount of the mutant factor VIII of claim 2. 12. The method of claim 11 , which further comprises administering an effective amount of receptor associated protein (RAP). 13. A polynucleotide encoding the mutant factor VIII of claim 2. 14. A method of treating hemophilia which comprises administering to a patient in need thereof an effective amount of the polynucleotide of claim 13. 15. The method of claim 14, which further comprises administering an effective amount of a polynucleotide encoding RAP. 16. A mutant factor VIII comprising an amino acid substitution at one or more positions in the A2 domain, which is not at any of positions 484, 485, 487, 488, 489, 492. 493, 495, 501 or 508; wherein the mutant factor VIII has reduced receptor-dependent clearance; and wherein the mutant factor VIII has procoagulant activity. 17. The mutant factor VIII of claim 16, which lacks the B domain. 18. The mutant factor VIII of claim 17, comprising an amino acid substitution at one or more of positions 484 to 509. 19. The mutant factor VIII of claim 18, comprising an amino acid substitution at one or more of positions 490, 496 or 499. 20. The mutant factor VIII of claim 19, comprising an amino acid substitution at position 490. 21. The mutant factor VIII of claim 19, comprising an amino acid substitution at position 496. 22. The mutant factor VIII of claim 19, comprising an amino acid substitution at position 499. 23. The mutant factor VIII of claim 17, comprising SEQ ID NO:5. 24. A pharmaceutically acceptable composition comprising the mutant factor VIII of claim 17. 25. A method of treating hemophilia which comprises administering to a patient in need thereof an effective amount of the mutant factor VIII of claim 17. 26. The method of claim 25, which further comprises administering an effective amount of RAP. 27. A polynucleotide encoding the mutant factor VIII of claim 17. 28. A method of treating hemophilia which comprises administering to a patient in need thereof an effective amount of the polynucleotide of claim 27. 29. The method of claim 28, which further comprises administering an effective amount of a polynucleotide encoding RAP. 30. A mutant factor VIII comprising an amino acid substitution at one or more positions in the C2 domain; wherein the mutant factor VIII has reduced receptor-independent clearance; and wherein the mutant factor VIII has procoagulant activity. 31. The mutant factor VIII of claim 30, which lacks the B domain. 32. The mutant factor VIII of claim 31, comprising an amino acid substitution at one or more of positions 2303 to 2332. 33. The mutant factor VIII of claim 32, comprising an amino acid substitution at one or more of positions 231 1 to 2319. 34. The mutant factor VIII of claim 31 , comprising SEQ ID NO: 1. 35. A pharmaceutically acceptable composition comprising the mutant factor VIII of claim 31. 36. A method of treating hemophilia which comprises administering to a patient in need thereof an effective amount of the mutant factor VIII of claim 31. 37. The method of claim 36, which further comprises administering an effective amount of RAP. 38. A polynucleotide encoding the mutant factor VIII of claim 31. 39. A method of treating hemophilia which comprises administering to a patient in need thereof an effective amount of the polynucleotide of claim 38. 40. The method of claim 39, which further comprises administering an effective amount of a polynucleotide encoding RAP. 41. A mutant factor VIII comprising: (i) an amino acid substitution at two or more positions in the A2 domain; wherein at least one of said amino acid substitutions is not at any of positions 484, 485, 487, 488, 489, 492, 493, 495, 501 or 508; and (ii) an amino acid substitution at one or more positions in the C2 domain as numbered in SEQ ID NO: 1 ; wherein the mutant factor VIII has reduced clearance; and wherein the mutant factor VIII has procoagulant activity. 42. The mutant factor VIII of claim 41, which lacks the B domain. 43. The mutant factor VIII of claim 42, comprising SEQ ID NO:5. 44. A pharmaceutically acceptable composition comprising the mutant factor VIII of claim 42. 45. A method of treating hemophilia which comprises administering to a patient in need thereof an effective amount of the mutant factor VIII of claim 42. 46. The method of claim 45, which further comprises administering an effective amount of RAP. 47. A polynucleotide encoding the mutant factor VIII of claim 42. 48. A method of treating hemophilia which comprises administering to a patient in need thereof an effective amount of the polynucleotide of claim 47. 49. The method of claim 48, which further comprises administering an effective amount of a polynucleotide encoding RAP. 50. A mutant factor VIII comprising: (i) an amino acid substitution at one or more positions in the A2 domain, which is not at any of positions 484, 485, 487, 488, 489, 492, 493, 495, 501 or 508; and (ii) an amino acid substitution at one or more positions in the C2 domain as numbered in SEQ ID NO : 1 ; wherein the mutant factor VIII has reduced clearance; and wherein the mutant factor VIII has procoagulant activity. 51. The mutant factor VIII of claim 50, which lacks the B domain. 52. The mutant factor VIII of claim 51 , comprising SEQ ID NO:5. 53. A pharmaceutically acceptable composition comprising the mutant factor VIII of claim 51. 54. A method of treating hemophilia which comprises administering to a patient in need thereof an effective amount of the mutant factor VIII of claim 51. 55. The method of claim 54, which further comprises administering an effective amount of RAP. 56. A polynucleotide encoding the mutant factor VIII of claim 51. 57. A method of treating hemophilia which comprises administering to a patient in need thereof an effective amount of the polynucleotide of claim 56. 58. The method of claim 57, which further comprises administering an effective amount of a polynucleotide encoding RAP. 59. A polypeptide selected from the group consisting of: (a) a polypeptide comprising a fragment of receptor-associated protein (RAP) which binds LRP; (b) a polypeptide comprising a mutant of RAP which binds LRP; (c) a polypeptide comprising an analog of RAP which binds LRP; (d) a polypeptide comprising 20 contiguous amino acids of the sequence of SEQ ID NOJ, which binds LRP; and (e) a polypeptide comprising amino acids 203 to 319 of SEQ ID NOJ. 60. A pharmaceutically acceptable composition comprising the polypeptide of claim 59. 61. A method of treating hemophilia which comprises administering to a patient in need thereof an effective amount of the polypeptide of claim 59. 62. The method of claim 61, which further comprises administering a mutant factor VIII having an amino acid substitution at one or more positions in the A2 domain. 63. The method of claim 61, which further comprises administering a mutant factor VIII having an amino acid substitution at one or more positions in the C2 domain. 64. The method of claim 61, which further comprises administering a mutant factor VIII having an amino acid substitution at one or more positions in the A2 domain and an amino acid substitution at one or more positions in the C2 domain. 65. A method of increasing the half-life of factor VIII, selected from the group consisting of: (a) a method which comprises substituting an amino acid at two or more positions in the A2 domain; wherein at least one of said amino acid substitutions is not at any of positions 484, 485, 487, 488, 489, 492, 493, 495, 501 or 508; wherein the resulting factor VIII has reduced receptor-dependent clearance; and wherein the resulting factor VIII has procoagulant activity; (b) method which comprises substituting an amino acid at one or more positions in the A2 domain, which is not at any of positions 484, 485, 487, 488, 489, 492, 493, 495, 501 or 508; wherein the resulting factor VIII has reduced receptor-dependent clearance; and wherein the resulting factor VIII has procoagulant activity; (c) a method which comprises substituting an amino acid at one or more positions in the C2 domain; wherein the resulting factor VIII has reduced receptor-independent clearance; and wherein the resulting factor VIII has procoagulant activity; (d) a method which comprises administering to a patient in need thereof an effective amount of a fragment of RAP, wherein said fragment binds LRP; and (e) a method comprising two or more of methods (a), (b), (c) or (d).

Inventors: SAENKO EVGUENI L, STRICKLAND DUDLEY K, SAENKO, EVGUENI, L., STRICKLAND, DUDLEY, K.
Applicants: AMERICAN NAT RED CROSS, SAENKO EVGUENI L, STRICKLAND DUDLEY K, SAENKO, EVGUENI, L., STRICKLAND, DUDLEY, K., THE AMERICAN NATIONAL RED CROSS
Application year: 2000
Main classification: C12N15
IPC codes: A61K38, A61K48, C07K14, C12N15