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• W2022686807 abstract "Low-density lipoprotein receptor-related protein (LRP) is an endocytic receptor that binds multiple distinct ligands, including blood coagulation factor VIII (FVIII). FVIII is a heterodimeric multidomain protein that consists of a heavy chain (domains A1, a1, A2, a2, and B) and a light chain (domains a3, A3, C1, and C2). Both chains contribute to high-affinity interaction with LRP. One LRP-interactive region has previously been located in the C2 domain, but its affinity is low in comparison with that of the entire FVIII light chain. We now have compared a variety of FVIII light chain derivatives with the light chain of its homolog FVa for LRP binding. In surface plasmon resonance studies employing LRP cluster II, the FVa and FVIII light chains proved different in that only FVIII displayed high-affinity binding. Because the FVIII a3-A3-C1 fragment was effective in associating with LRP, this region was explored for structural elements that are exposed but not conserved in FV. Competition studies using synthetic peptides suggested that LRP binding involves the FVIII-specific region Lys1804–Ala1834 in the A3 domain. In line with this observation, LRP binding was inhibited by a recombinant antibody fragment that specifically binds to the FVIII sequence Glu1811–Lys1818. The role of this sequence in LRP binding was further tested using a FVIII/FV chimera in which sequence Glu1811–Lys1818 was replaced with the corresponding sequence of FV. Although this chimera still displayed residual binding to LRP cluster II, its affinity was reduced. This suggests that multiple sites in FVIII contribute to high-affinity LRP binding, one of which is the FVIII A3 domain region Glu1811–Lys1818. This suggests that LRP binding to the FVIII A3 domain involves the same structural elements that also contribute to the assembly of FVIII with FIXa. Low-density lipoprotein receptor-related protein (LRP) is an endocytic receptor that binds multiple distinct ligands, including blood coagulation factor VIII (FVIII). FVIII is a heterodimeric multidomain protein that consists of a heavy chain (domains A1, a1, A2, a2, and B) and a light chain (domains a3, A3, C1, and C2). Both chains contribute to high-affinity interaction with LRP. One LRP-interactive region has previously been located in the C2 domain, but its affinity is low in comparison with that of the entire FVIII light chain. We now have compared a variety of FVIII light chain derivatives with the light chain of its homolog FVa for LRP binding. In surface plasmon resonance studies employing LRP cluster II, the FVa and FVIII light chains proved different in that only FVIII displayed high-affinity binding. Because the FVIII a3-A3-C1 fragment was effective in associating with LRP, this region was explored for structural elements that are exposed but not conserved in FV. Competition studies using synthetic peptides suggested that LRP binding involves the FVIII-specific region Lys1804–Ala1834 in the A3 domain. In line with this observation, LRP binding was inhibited by a recombinant antibody fragment that specifically binds to the FVIII sequence Glu1811–Lys1818. The role of this sequence in LRP binding was further tested using a FVIII/FV chimera in which sequence Glu1811–Lys1818 was replaced with the corresponding sequence of FV. Although this chimera still displayed residual binding to LRP cluster II, its affinity was reduced. This suggests that multiple sites in FVIII contribute to high-affinity LRP binding, one of which is the FVIII A3 domain region Glu1811–Lys1818. This suggests that LRP binding to the FVIII A3 domain involves the same structural elements that also contribute to the assembly of FVIII with FIXa. Coagulation factor VIII (FVIII) 1The abbreviations used are: FVIII, factor VIII; FIXa, factor IXa; FV, factor V; FXa, factor Xa; LRP, low-density lipoprotein receptor-related protein; scFv, single-chain variable domain antibody fragment; HSA, human serum albumin; SPR, surface plasmon resonance serves its role in the intrinsic coagulation pathway as a cofactor for factor IXa (FIXa) in the proteolytic activation of factor X (for reviews, see Refs. 1Mann K.G. Nesheim M.E. Church W.R. Haley P. Krishnaswamy S. Blood. 1990; 76: 1-16Google Scholar and2Lenting P.J. van Mourik J.A. Mertens K. Blood. 1998; 92: 3983-3996Google Scholar). Functional absence of FVIII is associated with the bleeding disorder hemophilia A. The cofactor is a 300-kDa glycoprotein that comprises a discrete domain structure (A1-a1-A2-a2-B-a3-A3-C1-C2) (2Lenting P.J. van Mourik J.A. Mertens K. Blood. 1998; 92: 3983-3996Google Scholar,3Vehar G.A. Keyt B. Eaton D. Rodriguez H. O'Brien D.P. Rotblat F. Opperman H. Keck R. Wood W.I. Harkins R.N. Tuddenham E.G.D. Lawn R.M. Capon D.J. Nature. 1984; 312: 337-342Google Scholar). The A and C domains share 30–40% homology with the A and C domains of the structurally related protein factor V (FV), whereas the B domain and the short acidic regions a1, a2, and a3 are unique to FVIII (4Church W.R. Jernigan R.L. Toole J. Hewick R.M. Knopf J. Knutson G.J. Nesheim M.E. Mann K.G. Fass D.N. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 6934-6937Google Scholar). In plasma, FVIII circulates as a metal ion-linked heterodimer consisting of a 90–220-kDa heavy chain (A1-a1-A2-a2-B) and an 80-kDa light chain (a3-A3-C1-C2) (5Rotblat F. O'Brien D.P. O'Brien F.J. Goodall A.H. Tuddenham E.G.D. Biochemistry. 1985; 24: 4294-4300Google Scholar, 6Kaufman R.J. Wasly L.C. Dorner A.J. J. Biol. Chem. 1988; 263: 6352-6362Google Scholar). The inactive protein is tightly associated with its carrier protein, von Willebrand factor (7Lollar P. Hill-Eubanks D.C. Parker C.G. J. Biol. Chem. 1988; 263: 10451-10455Google Scholar). Limited proteolysis by either thrombin or factor Xa (FXa) converts the FVIII precursor into its activated derivative (8Lollar P. Knutson G.J. Fass D.N. Biochemistry. 1985; 24: 8056-8064Google Scholar, 9Eaton D. Rodriguez H. Vehar G.A. Biochemistry. 1986; 25: 505-512Google Scholar). The B domain and the acidic region that borders the A3 domain are then removed from the molecule (10Fay P.J. Haidaris P.J. Smudzin T.M. J. Biol. Chem. 1991; 266: 8957-8962Google Scholar), which leads to the loss of high-affinity binding to von Willebrand factor (7Lollar P. Hill-Eubanks D.C. Parker C.G. J. Biol. Chem. 1988; 263: 10451-10455Google Scholar). The resulting FVIIIa molecule consists of a heterotrimer comprising the A2-a2 domain that is noncovalently associated with the metal ion-linked A1-a1/A3-C1-C2 moiety (10Fay P.J. Haidaris P.J. Smudzin T.M. J. Biol. Chem. 1991; 266: 8957-8962Google Scholar). Within the heavy and light chains of FVIII, several regions have been identified as FIXa-interactive sites (11Fay P.J. Beattie T. Huggins C.F. Regan L.M. J. Biol. Chem. 1994; 269: 20522-20527Google Scholar, 12Bajaj S.P. Schmidt A.E. Mathur A. Padmanabhan K. Zhong D. Mastri M. Fay P.J. J. Biol. Chem. 2001; 276: 16302-16309Google Scholar, 13Lenting P.J. van de Loo J.W.H.P. Donath M.J.S.H. van Mourik J.A. Mertens K. J. Biol. Chem. 1996; 271: 1935-1940Google Scholar). A2 domain residues Arg484–Phe509, Ser558–Gln565, and Arg698–Asp712 contribute to binding of the heavy chain to FIXa (11Fay P.J. Beattie T. Huggins C.F. Regan L.M. J. Biol. Chem. 1994; 269: 20522-20527Google Scholar, 12Bajaj S.P. Schmidt A.E. Mathur A. Padmanabhan K. Zhong D. Mastri M. Fay P.J. J. Biol. Chem. 2001; 276: 16302-16309Google Scholar, 14Fay P.J. Scandella D. J. Biol. Chem. 1999; 274: 29826-29830Google Scholar). Within the FVIII light chain, the A3 domain region Glu1811–Lys1818 has been identified as a FIXa-interactive site (13Lenting P.J. van de Loo J.W.H.P. Donath M.J.S.H. van Mourik J.A. Mertens K. J. Biol. Chem. 1996; 271: 1935-1940Google Scholar). In addition, FVIII regions Arg484–Phe509 and Lys1804–Lys1818 have also been identified as target epitopes for antibodies that may occur in hemophilia A patients. Such antibodies inhibit FVIII activity by interfering with the complex assembly of FVIIIa and FIXa (15Haeley J.F. Lubin I.M. Nakai H. Saenko E.L. Hoyer L.W. Scandella D. Lollar P. J. Biol. Chem. 1995; 270: 14505-14509Google Scholar, 16Fijnvandraat K. Celie P.H.N. Turenhout E.A.M. van Mourik J.A. ten Cate J.W. Mertens K. Peters M. Voorberg J. Blood. 1998; 91: 2347-2352Google Scholar, 17Zhong D. Saenko E.L. Shima M. Felch M. Scandella D. Blood. 1998; 92: 136-142Google Scholar). Recently, it has been demonstrated that FVIII interacts with the multifunctional endocytic receptor low-density lipoprotein receptor-related protein (LRP) (18Lenting P.J. Neels J.G. van den Berg B.M.M. Clijsters P.P.F.M. Meijerman D.W.E. Pannekoek H. van Mourik J.A. Mertens K. van Zonneveld A.-J. J. Biol. Chem. 1999; 274: 23734-23739Google Scholar, 19Saenko E.L. Yakhyaev A.V. Mikhailenko I. Strickland D.K. Sarafanov A.G. J. Biol. Chem. 1999; 274: 37685-37692Google Scholar). It is suggested that this receptor plays a role in the clearance of FVIII from the circulation (19Saenko E.L. Yakhyaev A.V. Mikhailenko I. Strickland D.K. Sarafanov A.G. J. Biol. Chem. 1999; 274: 37685-37692Google Scholar, 20Schwarz H.P. Lenting P.J. Binder B. Mihaly J. Denis C. Dorner F. Turecek P.L. Blood. 2000; 95: 1703-1708Google Scholar). LRP is a member of the low-density lipoprotein receptor family, which also includes the low-density lipoprotein receptor, the very low-density lipoprotein receptor, apoE receptor-2, and megalin (for reviews, see Refs. 21Neels J.G. Horn I.R. van den Berg B.M.M. Pannekoek H. van Zonneveld A.-J. Fibrinolysis Proteolysis. 1998; 12: 219-240Google Scholar and 22Herz J. Strickland D.K. J. Clin. Invest. 2001; 108: 779-784Google Scholar). It is expressed in a variety of tissues, including liver, lung, placenta, and brain (23Moestrup S.K. Gliemann J. Pallesen G. Cell Tissue Res. 1992; 269: 375-382Google Scholar). The receptor consists of an extracellular 515-kDa α-chain that is noncovalently linked to a transmembrane 85-kDa β-chain (24Herz J. Kowal R.C. Goldstein J.L. Brown M.S. EMBO J. 1990; 9: 1769-1776Google Scholar). The α-chain contains four clusters of a varying number of complement-type repeats that mediate the binding of many structurally and functionally unrelated ligands (25Moestrup S.K. Hotlet T.L. Etzerodt M. Thogersen H.C. Nykjaer A. Andreasen P.A. Rasmussen H.H. Sottrup-Jensen L. Gliemann J. J. Biol. Chem. 1993; 268: 13691-13696Google Scholar, 26Willnow T.E. Orth K. Herz J. J. Biol. Chem. 1994; 269: 15827-15832Google Scholar, 27Neels J.G. van den Berg B.M.M. Lookene A. Olivecrona G. Pannekoek H. van Zonneveld A.-J. J. Biol. Chem. 1999; 274: 31305-31311Google Scholar). The FVIII light chain has been demonstrated to interact with recombinant LRP clusters II and IV, whereas no binding was observed to LRP clusters I and III (27Neels J.G. van den Berg B.M.M. Lookene A. Olivecrona G. Pannekoek H. van Zonneveld A.-J. J. Biol. Chem. 1999; 274: 31305-31311Google Scholar). Within FVIII, both the heavy and light chains contain LRP-interactive sites. Both the A2 domain region Arg484–Phe509and a so far unidentified region within the light chain are involved in the high-affinity interaction with LRP (18Lenting P.J. Neels J.G. van den Berg B.M.M. Clijsters P.P.F.M. Meijerman D.W.E. Pannekoek H. van Mourik J.A. Mertens K. van Zonneveld A.-J. J. Biol. Chem. 1999; 274: 23734-23739Google Scholar, 19Saenko E.L. Yakhyaev A.V. Mikhailenko I. Strickland D.K. Sarafanov A.G. J. Biol. Chem. 1999; 274: 37685-37692Google Scholar). Previously, we showed that an anti-C2 domain antibody inhibits high-affinity binding of the FVIII light chain to LRP, suggesting a major role for the C2 domain in the interaction (18Lenting P.J. Neels J.G. van den Berg B.M.M. Clijsters P.P.F.M. Meijerman D.W.E. Pannekoek H. van Mourik J.A. Mertens K. van Zonneveld A.-J. J. Biol. Chem. 1999; 274: 23734-23739Google Scholar). However, the isolated recombinant C2 domain demonstrates low-affinity binding to LRP compared with the intact FVIII light chain (18Lenting P.J. Neels J.G. van den Berg B.M.M. Clijsters P.P.F.M. Meijerman D.W.E. Pannekoek H. van Mourik J.A. Mertens K. van Zonneveld A.-J. J. Biol. Chem. 1999; 274: 23734-23739Google Scholar). In the present study, we investigated the apparently paradoxical role of the C2 domain in the interaction of the FVIII light chain with LRP. To this end, the interaction between the FVIII light chain and LRP is addressed using purified recombinant FVIII light chain fragments, synthetic peptides, recombinant antibody fragments, and a chimeric FVIII light chain variant. This approach allowed us to identify the FVIII light chain region Glu1811–Lys1818 as a sequence that contributes to the interaction with LRP. CNBr-Sepharose 4B was from Amersham Biosciences (Uppsala, Sweden). Microtiter plates (Maxisorp), cell culture flasks, Opti-MEM I medium, penicillin, and streptomycin were from Invitrogen (Breda, The Netherlands). Grace's insect medium, Insect-XPRESS medium, and fetal calf serum were purchased from BioWhittaker (Alkmaar, The Netherlands). Plasma-derived FVIII light chain and its FXa-cleaved derivative were prepared as described previously (28Lenting P.J. Donath M.J.S.H. van Mourik J.A. Mertens K. J. Biol. Chem. 1994; 269: 7150-7155Google Scholar, 29Donath M.S.J.H. Lenting P.J. van Mourik J.A. Mertens K. J. Biol. Chem. 1995; 270: 3648-3655Google Scholar). Anti-FVIII monoclonal antibodies CLB-CAgA, CLB-CAg117, and CLB-CAg12 have been described previously (28Lenting P.J. Donath M.J.S.H. van Mourik J.A. Mertens K. J. Biol. Chem. 1994; 269: 7150-7155Google Scholar, 30Leyte A. Mertens K. Distel B. Evers R.F. de Keyzer-Nellen M.J. Groenen-van Dooren M.M. de Bruin J. Pannekoek H. van Mourik J.A. Verbeet M.P. Biochem. J. 1989; 263: 187-194Google Scholar). Single-chain variable domain antibody fragments (scFv fragments) directed against the light chain of FVIII were expressed in Escherichia coli strain TG1 and purified by metal chelate chromatography (QIAGEN, Hilden, Germany) as described previously (31van den Brink E.N. Turenhout E.A.M. Bovenschen N. Heijnen B.G. Mertens K. Peters M. Voorberg J. Blood. 2001; 97: 966-972Google Scholar, 32van den Brink E.N. Turenhout E.A.M. Davies J. Bovenschen N. Fijnvandraat K. Ouwehand W.H. Peters M. Voorberg J. Blood. 2000; 95: 558-563Google Scholar), with the exception that scFv fragments KM36 and KM41 were eluted in 150 mm NaCl, 100 mm imidazole, and 20 mm Hepes (pH 7.4). The anti-FVa light chain monoclonal antibody CLB-FV5 was obtained by standard hybridoma techniques and will be described in detail elsewhere. 2M. H. A. Bos, D. W. E. Meijerman, C. van der Zwaan, and K. Mertens, manuscript in preparation. Synthetic peptides encompassing human FVIII regions Trp1707–Arg1721 (WDYGMSSSPHVLRNR), Lys1804–Lys1818 (KNFVKPNETKTYFWK), Tyr1815–Ala1834 (YFWKVQHHMAPTKDEFDCKA), His1822–Ala1834 (HMAPTKDEFDCKA), Thr1892–Ala1901 (TENMERNCRA), Glu1908–His1919 (EDPTFKENYRFH), Thr1964–Lys1972 (TVRKKEEYK), Lys2049–Gly2057 (KLARLHYSG), and Asp2108–Gly2117 (DGKKWQTYRG) were synthesized by Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry following the manual “T-bag” method (33Houghton R.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5131-5135Google Scholar) or employing an Applied Biosystems Model 430A instrument (Amersham Biosciences, Roosendaal, The Netherlands; Medprobe AS, Oslo, Norway). Peptides were >95% pure as determined by high-pressure liquid chromatography analysis, and their identity was confirmed by mass spectrometry. Purified placenta-derived LRP (34Moestrup S.K. Gliemann J. J. Biol. Chem. 1991; 266: 14011-14017Google Scholar) was a generous gift from Dr. S. K. Moestrup (University of Aarhus, Aarhus, Denmark). The bacterial vector encoding glutathioneS-transferase-fused receptor-associated protein was kindly provided by Dr. J. Kuiper (Leiden University, Leiden, The Netherlands). Glutathione S-transferase-fused receptor-associated protein was expressed in E. coli strain DH5α and purified on glutathione-Sepharose as described (35Herz J. Goldstein J.L. Strickland D.K. Ho Y.K. Brown M.S. J. Biol. Chem. 1991; 266: 21232-21238Google Scholar). Baby hamster kidney cells expressing recombinant LRP ligand-binding clusters II and IV have been described previously (27Neels J.G. van den Berg B.M.M. Lookene A. Olivecrona G. Pannekoek H. van Zonneveld A.-J. J. Biol. Chem. 1999; 274: 31305-31311Google Scholar) and were kindly provided by Dr. H. Pannekoek (Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands). Human serum albumin (HSA) was from the Division of Products of CLB. Protein was quantified by the method of Bradford (36Bradford M.M. Anal. Biochem. 1976; 72: 248-254Google Scholar) using HSA as a standard. The plasmid pCLB-BPVdB695, encoding the FVIII B domain deletion variant FVIII-Δ(868–1562), has been described previously (37Mertens K. Donath M.J.S.H. van Leen R.W. de Keyzer-Nellen M.J.M. Verbeet M.P. Klaasse Bos J.M. Leyte A. van Mourik J.A. Br. J. Haematol. 1993; 85: 133-142Google Scholar) and was used as a template to construct the plasmid coding for the FVIII-(1811–1818)/FV chimera. Oligonucleotide primers derived from the FVIII light chain sequence containing the FVIII/FV codon replacements (see Table II) were employed to construct the plasmids using the overlap extension PCR mutagenesis method (38Tao B.Y. Lee K.C.P. Griffin H.G. Griffin A.M. PCR Technology Current Innovations. CRC Press LLC, Boca Raton, FL1994: 71-72Google Scholar). Sequence analysis was performed to verify the presence of the mutations in the plasmid. Transfection of FVIII-encoding plasmids into murine fibroblasts (C127) cells was performed as described previously (37Mertens K. Donath M.J.S.H. van Leen R.W. de Keyzer-Nellen M.J.M. Verbeet M.P. Klaasse Bos J.M. Leyte A. van Mourik J.A. Br. J. Haematol. 1993; 85: 133-142Google Scholar). Stable cell lines expressing wild-type FVIII or the FVIII-(1811–1818)/FV chimera were maintained in cell factories in RPMI 1640 medium supplemented with 5% fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 1 μg/ml amphotericin B, and 0.8 μg/ml deoxycholate. FVIII-containing medium was harvested three times/week. The medium was subsequently filtered to remove cell debris and concentrated ∼10-fold employing a hollow fiber cartridge (Hemoflow F5, Fresenius, Bad Homburg, Germany). Benzamidine was added to a final concentration of 10 mm, and concentrates were stored at −20 °C. FVIII was purified from the concentrated medium by immunoaffinity chromatography employing antibody CLB-CAg117 and Q-Sepharose chromatography according to an established procedure (37Mertens K. Donath M.J.S.H. van Leen R.W. de Keyzer-Nellen M.J.M. Verbeet M.P. Klaasse Bos J.M. Leyte A. van Mourik J.A. Br. J. Haematol. 1993; 85: 133-142Google Scholar). FVIII light chains were prepared by incubating the purified FVIII-(1811–1818)/FV chimera and wild-type FVIII in buffer containing 40 mm EDTA, 100 mm NaCl, and 50 mmTris (pH 7.4) for 4 h at 25 °C. Subsequently, the FVIII-(1811–1818)/FV and wild-type FVIII light chains were purified by Q-Sepharose chromatography. Recombinant proteins were eluted in buffer containing 1 m NaCl and 50 mm Tris (pH 7.4), dialyzed against 150 mm NaCl and 50 mm Tris (pH 7.4), and stored at 4 °C. The construction of the plasmid encoding the recombinant C2 domain (i.e.Ser2173–Tyr2332) has been described previously (16Fijnvandraat K. Celie P.H.N. Turenhout E.A.M. van Mourik J.A. ten Cate J.W. Mertens K. Peters M. Voorberg J. Blood. 1998; 91: 2347-2352Google Scholar). The plasmid pACgp67b-His-a3-A3-C1, encoding the FVIII a3-A3-C1 fragment (i.e. Glu1649–Asn2172), was constructed by PCR employing the oligonucleotide primers 5′-TTACTCGAGGAAATAACTCGTACTACTC-3′ (sense) and 5′-AATGCGGCCGCTTCAATTTAAATCACAGCCCAT-3′ (antisense) using pCLB-BPVdB695 as a template (37Mertens K. Donath M.J.S.H. van Leen R.W. de Keyzer-Nellen M.J.M. Verbeet M.P. Klaasse Bos J.M. Leyte A. van Mourik J.A. Br. J. Haematol. 1993; 85: 133-142Google Scholar). The amplified DNA fragment was purified, digested with XhoI and NotI, and ligated into pBluescript. The resulting construct was verified by sequencing. Subsequently, pBluescript-a3-A3-C1 was digested with EspI andNotI, and the obtained fragment was purified and ligated into the EspI/NotI-digested pACgp67b-80K plasmid (39Fijnvandraat K. Turenhout E.A.M. van den Brink E.N. ten Cate J.W. van Mourik J.A. Peters M. Voorberg J. Blood. 1997; 89: 4371-4377Google Scholar). A DNA fragment encoding a polyhistidine tag (5′-ATTGGATCCGGCCATCATCATCATCATCATGGCGGCAGCCCCCGCAGCTTTCAAAAGCCCGGGGCCATGGGA-3′) was digested with BamHI and NcoI and cloned into the BamHI/NcoI-digested pACgp67b-a3-A3-C1 plasmid. Using the baculovirus expression system, recombinant a3-A3-C1 and C2 fragments were obtained by infection of insect cells as described (16Fijnvandraat K. Celie P.H.N. Turenhout E.A.M. van Mourik J.A. ten Cate J.W. Mertens K. Peters M. Voorberg J. Blood. 1998; 91: 2347-2352Google Scholar). The a3-A3-C1 fragment was purified from Insect-XPRESS medium by immunoaffinity chromatography using the anti-A3 domain antibody CLB-CAgA coupled to CNBr-Sepharose 4B as an affinity matrix. CLB-CAgA-Sepharose was incubated with medium containing the a3-A3-C1 fragment for 16 h at 4 °C. After binding, the immunoaffinity matrix was collected; washed with buffer containing 1 mNaCl and 50 mm Tris (pH 7.4); and eluted with 150 mm NaCl, 55% (v/v) ethylene glycol, and 50 mmlysine (pH 11). Elution fractions were immediately neutralized with 1m imidazole (pH 6); dialyzed against 150 mmNaCl, 50% (v/v) glycerol, and 50 mm Tris (pH 7.4); and stored at −20 °C. The recombinant C2 domain was purified employing the same immunoaffinity chromatography technique, except that the anti-C2 domain antibody CLB-CAg117 was used instead of CLB-CAgA.Table IKinetic parameters for binding of the FVIII light chain and its derivatives to immobilized LRPk offk onK ds −1m −1 s −1nmFVIII light chainClass 1(2.8 ± 0.7) × 10−3(1.6 ± 0.3) × 10518 ± 6Class 2(6.9 ± 1.4) × 10−2(1.2 ± 0.2) × 10659 ± 15FXa-cleaved light chainClass 1(3.0 ± 0.3) × 10−3(1.3 ± 0.2) × 10522 ± 4Class 2(5.6 ± 0.7) × 10−2(0.9 ± 0.3) × 10660 ± 23a3-A3-C1 fragmentClass 1(4.1 ± 0.7) × 10−3(1.6 ± 0.4) × 10526 ± 7Class 2(8.0 ± 0.7) × 10−2(1.1 ± 0.3) × 10674 ± 20C2 domain Class 1(1.3 ± 0.1) × 10−1(3.6 ± 1.7) × 1043602 ± 1743Association and dissociation of various concentrations of the FVIII light chain (10–250 nm), the 67-kDa fragment (10–250 nm), the a3-A3-C1 fragment (10–250 nm), or the C2 domain (500–2000 nm) to immobilized LRP (16 fmol/mm2) were assessed as described under “Experimental Procedures.” The data obtained were analyzed to calculate association (k on) and dissociation (k off) rate constants using a one- or two-site binding model. Each class of binding sites is referred to as 1 and 2, respectively. Affinity constants (K d) were calculated from the ratiok off/k on. Data are based on three to six measurements using at least five different concentrations for each measurement. Data represent the means ± S.D. Open table in a new tab Table IIEffect of FVIII a3-A3-C1 fragment-derived synthetic peptides on the interaction between the FVIII light chain and LRP cluster IIDomainResiduesIC50SequenceA31707–1721>1 mmWDYGMSSSPHVLRNRA31804–18181.9 ± 0.2 μmKNFVKPNETKTYFWKA31815–183416.8 ± 0.4 μmYFWKVQHHMAPTKDEFDCKAA31822–1834>1 mmHMAPTKDEFDCKAA31892–1901>1 mmTENMERNCRAA31908–1919>1 mmEDPTFKENYRFHA31964–1972>1 mmTVRKKEEYKC12049–2057>1 mmKLARLHYSGC12108–21170.9 ± 0.3 mmDGKKWQTYRGThe FVIII light chain (25 nm) was incubated with immobilized LRP cluster II (1 pmol/well) in a volume of 50 μl of 150 mm NaCl, 5 mm CaCl2, 1% (w/v) HSA, 0.1% Tween 20, and 50 mm Tris (pH 7.4) in the presence or absence of various concentrations of synthetic peptide (0–1 mm) for 2 h at 37 °C. After washing with the same buffer, bound FVIII light chain was quantified by incubation with peroxidase-conjugated anti-FVIII antibody CLB-CAg12 for 15 min at 37 °C. Half-maximum inhibition constants (IC50) represent the mean ± S.D. of three experiments. Open table in a new tab Association and dissociation of various concentrations of the FVIII light chain (10–250 nm), the 67-kDa fragment (10–250 nm), the a3-A3-C1 fragment (10–250 nm), or the C2 domain (500–2000 nm) to immobilized LRP (16 fmol/mm2) were assessed as described under “Experimental Procedures.” The data obtained were analyzed to calculate association (k on) and dissociation (k off) rate constants using a one- or two-site binding model. Each class of binding sites is referred to as 1 and 2, respectively. Affinity constants (K d) were calculated from the ratiok off/k on. Data are based on three to six measurements using at least five different concentrations for each measurement. Data represent the means ± S.D. The FVIII light chain (25 nm) was incubated with immobilized LRP cluster II (1 pmol/well) in a volume of 50 μl of 150 mm NaCl, 5 mm CaCl2, 1% (w/v) HSA, 0.1% Tween 20, and 50 mm Tris (pH 7.4) in the presence or absence of various concentrations of synthetic peptide (0–1 mm) for 2 h at 37 °C. After washing with the same buffer, bound FVIII light chain was quantified by incubation with peroxidase-conjugated anti-FVIII antibody CLB-CAg12 for 15 min at 37 °C. Half-maximum inhibition constants (IC50) represent the mean ± S.D. of three experiments. Human FV was obtained from human plasma provided by our institute (Sanquin Plasma Products). Full-length FV was purified by immunoaffinity chromatography.2 The FVa light chain was prepared by incubating FV (10 μm) with thrombin (2 μm) in buffer containing 100 mm NaCl, 5 mmCaCl2, 5% (v/v) glycerol, and 50 mm Tris (pH 7.4) for 2 h at 37 °C. Thrombin was inactivated by hirudin (Sigma), and the FVa light chain was purified by immunoaffinity chromatography on CNBr-Sepharose 4B coupled to the anti-FV light chain monoclonal antibody CLB-FV5 (3 mg/ml). The immunoaffinity matrix was washed with 100 mm NaCl, 50 mm EDTA, and 50 mm Tris (pH 7.4) and eluted with 100 mm NaCl, 5 mm CaCl2, 55% (v/v) ethylene glycol, and 50 mm Tris (pH 7.4). Purified FVa light chain was dialyzed against 150 mm NaCl, 5 mm CaCl2, 50% (v/v) glycerol, and 50 mm Tris (pH 7.4) and stored at −20 °C. Recombinant LRP clusters II and IV were expressed in baby hamster kidney cells using Opti-MEM I medium supplemented with 100 units/ml penicillin and 100 μg/ml streptomycin (27Neels J.G. van den Berg B.M.M. Lookene A. Olivecrona G. Pannekoek H. van Zonneveld A.-J. J. Biol. Chem. 1999; 274: 31305-31311Google Scholar). After harvesting of the medium, CaCl2 was added to a final concentration of 10 mm. Purification of LRP clusters II and IV from the conditioned medium was performed by a single purification step using glutathione S-transferase-fused receptor-associated protein coupled to CNBr-Sepharose 4B as an affinity matrix. The matrix was collected in a column; washed with buffer containing 150 mmNaCl, 5 mm CaCl2, and 50 mm Hepes (pH 7.4); and eluted with 150 mm NaCl, 20 mmEDTA, and 50 mm Hepes (pH 7.4). Subsequently, purified LRP cluster preparations were concentrated in Centricon 10 concentrators (Millipore Corp., Bedford, MA) by successive rounds of centrifugation at 4000 × g for 1 h at 4 °C. Finally, the preparations were dialyzed against 150 mm NaCl, 2 mm CaCl2, and 20 mm Hepes (pH 7.4) and stored at 4 °C. Recombinant LRP cluster II or IV (1 pmol/well) was adsorbed onto microtiter wells in 50 mmNaHCO3 (pH 9.8) in a volume of 50 μl for 16 h at 4 °C. Wells were blocked with 2% (w/v) HSA, 150 mmNaCl, 5 mm CaCl2, and 50 mm Tris (pH 7.4) in a volume of 200 μl for 1 h at 37 °C. Subsequently, the FVIII light chain was incubated at various concentrations in a volume of 50 μl of buffer containing 150 mm NaCl, 5 mm CaCl2, 1% (w/v) HSA, 0.1% (v/v) Tween 20, and 50 mm Tris (pH 7.4) for 2 h at 37 °C. After three rapid washes (<5 s each) with 150 mm NaCl, 5 mm CaCl2, 0.1% (v/v) Tween 20, and 50 mm Tris (pH 7.4), bound ligand was detected by incubation with peroxidase-conjugated monoclonal antibody CLB-CAg12 in the same buffer for 15 min at 37 °C. During this latter incubation period, one would expect that the FVIII light chain would completely dissociate from the immobilized LRP clusters. However, subsequent rebinding of the FVIII light chain to the LRP clusters allows the formation of new FVIII·LRP cluster complexes, which can be detected by this sensitive method. In surface plasmon resonance (SPR), this is prevented by a continuous buffer flow. Because of these differences in experimental approach, the solid-phase binding assay is compared only with SPR analysis in a qualitative manner. Antibody CLB-CAg12 did not interfere with binding of FVIII fragments to LRP or its clusters (data not shown). In competition experiments, the FVIII light chain (25 nm) was incubated with wells containing immobilized LRP clusters either in the presence or absence of serial dilutions of competitor in a volume of 50 μl for 2 h at 37 °C. Residual FVIII binding was detected as described above. Data were corrected for binding to empty microtiter wells, which was <5% relative to binding to wells containing immobilized LRP clusters. The kinetics of protein interactions was determined by SPR analysis using a BIAcoreTM 2000 biosensor system (BIAcore AB, Uppsala). LRP (16 fmol/mm2), the FVIII light chain (71 fmol/mm2), the a3-A3-C1 fragment (67 fmol/mm2), the FVa light chain (76 fmol/mm2), or scFv EL14 (67 fmol/mm2) was covalently coupled to the dextran surface of an activated CM5 sensor chip via primary amino groups using the amine coupling kit (BIAcore AB, Uppsala, Sweden) as recommended by the supplier. One control flow channel was routinely activated and blocked in the absence of protein. Association of analyte was assessed in 150 mm NaCl, 2 mm CaCl2, 0.005% (v/v) Tween 20, and 20 mm Hepes (pH 7.4) for 2 min at a f" @default.
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• W2022686807 date "2003-03-01" @default.
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• W2022686807 title "Low Density Lipoprotein Receptor-related Protein and Factor IXa Share Structural Requirements for Binding to the A3 Domain of Coagulation Factor VIII" @default.
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• W2022686807 doi "https://doi.org/10.1074/jbc.m212053200" @default.
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