1.  Abbreviations

aPTT      :               Activated Partial Thrombo-plastin assay

BMP       :               Bone morphogenetic protein

ER          :               Endoplasmic reticulum

FVIII      :               Factor VIII

HASA    :               Human serum albumin

MTX      :               Methotrexate

PC           :               Proprotein convertase

SDS-PAGE:         Sodium dodecyl sulfate polyacrylamide gel electrophoresis

TGN       :               trans Golgi network

2.  Introduction

Many extracellularly-destined proteins and peptides are synthesized as protein precursors and undergo proteolytic cleavage before or upon reaching their final locations. A family of proteases, termed Proprotein Convertases (PCs) [1-5], which share sequence homology with bacterial subtilases and yeast kexin, has been found to process many of these precursors. Most of these specific cleavages occur at relatively conserved sites often with single or pairs of basic amino acids (aa) at the general motif (R/K)-2nX-R↓ (n=0-3 aa) [3,4]. The cleavages are likely carried out by one or more of seven basic-residue-specific mammalian PC family members known as PC1/3, PC2, Furin, PC4, PC5/6, PACE4, and PC7 [1-5]. The PCs family also comprises two non-basic-residue-specific family members, SKI-1 [6,7] and PCSK9 [8,9]. SKI-1 cleaves substrates like sterol regulatory element binding protein SREBP 1/2 [6] at the motif (R/K)-X (hydrophobic)-X↓ [9], whereas PCSK9 was found to cleave auto-catalytically its own propeptide at VFAQ152↓ [8].

The PCs family members themselves are produced as inactive zymogens [1,2,4,5]. Auto-cleavage in Endoplasmic Reticulum (ER) or in immature secretory granules (PC2 only) results in an inactive heterodimer of the mature protein associated with inhibitory propeptide. A second cleavage event takes place on the prosegment at specific subcellular compartments to free the active enzyme from the inhibitory propeptide. This might ensure that these PCs are only active at specific intracellular sites. Furin, PACE4, PC5/6, PC7, and SKI-1, have been shown to be active at the Golgi complex, whereas PC1/3 and PC2 are maximally active in secretory granules [1,2,5]. PCSK9 is secreted as catalytically inactive heterodimer [8].

When overexpressed in cell lines, basic-residue-specific PC members have been found to display some functional redundancy, yet inactivation studies in mice or human indicate that each of them seems to have unique processing functions and participate different biological events in vivo [4]. PC1/3 [10] and PC2 [11] disruption in mice reveals their dominant neuroendocrine functions, whereas PC4 deficiency in rodents leads to severe impairment of fertility in homozygous mutant males [12]. Furin, PC5/6, PACE4, and SKI-1 are essential genes. Furin Knock-out (KO) in mice causes numerous embryonic malfunctions such as absence of axial rotation and heart loop, implying its critical role in cardiac development [13]. PC5/6 KO results in death at birth with an altered antero-posterior pattern, kidney agenesis, hemorrhages, and retarded ossification [14,15]. PACE4 KO develops incompletely penetrant left-right patterning defects and cardiac malformations in some embryos [16]. Tissue-specific KO of SKI-1 confirms its regulatory roles in cholesterol and fatty acid synthesis [17]. Though not being essential genes, PC7 and PCSK9’s KO mice exhibit interesting phenotypes, with anxiolytic and novelty seeking phenotype for PC7 KO mice [18] and significantly less circulating total cholesterol/ LDLc for PCSK9 KO mice [19,20]. Different phenotypic consequences of PC family member inactivation are likely due to the inefficient processing of their targeted substrates. Identifying these individual protein substrates should provide a further understanding of the physiological roles of these proteases.

Factor VIII (FVIII) is a nonenzymatic cofactor in the intrinsic pathway of blood coagulation for the activated factor IX-mediated activation of Factor X [21]. FVIII gene defects may result in the bleeding disorder hemophilia A. In plasma, FVIII contains a heavy chain sized heterogeneously up to 200kDa in a metal ion complex with an 80kDa light chain. FVIII has a domain organization of A1-A2-B-A3-C1-C2 [22,23], with A domains similar to the A domains of the copper-binding protein ceruloplasmin [24], C domains homogenous to the phospholipid binding domains present in discoidins and milk fat globule protein [25], and B domain diverged among species [26-28]_.

FVIII is a complex plasma protein, undergoing extensive post-translational modifications [29], such as disulfide bond formation, N-linked and O-linked glycosylation, tyrosine sulfation, and phosphorylation. It is also subjected to proteolytic processing. A 19-amino-acid signal sequence is removed from its single chain precursor of 2351 amino acids by signal peptidase upon protein translocation across endoplasmic reticulum membranes. The mature polypeptide is further processed at R1313 and R1648 of the B domain within the secretory pathway in cells to yield a heterodimer. Additional minor cleavage sites in B-domain (R740, S817, K1115, S1657) have also been detected in purified FVIII preparations [30]. The predominant heavy chain (residue 1-1313) is composed of domains A1-A2-B* while the major light chain (residue 1649-2332) has domains A3-C1-C2. Both R1313 and R1648 major processing sites contain an Arg-X-X-Arg motif which can be cleaved by some PC family members [31-34], but it remains unknown whether or not these cleavages are enzyme-specific and whether or not other family members can also process FVIII. In this study, nine known members of the PC family were examined for their activities on the processing of a B-domain deleted FVIII-albumin fusion protein stably expressed in Chinese Hamster Ovary (CHO) cells. Furin and Proprotein convertase-5/6 (PC5/6) were found to process FVIII. The data also shows that this cleavage event has no substantial effect on FVIII functional activity in vitro [31].

3.  Materials and Methods

3.1.  DNA Constructs

A PCR fragment, encoding a human B-domain-deleted form of FVIII (LAFVIII) [28,35] fused to N-terminus of Human Serum Albumin (HSA) with a linker of GGSGGSGG-EDENQSPR (thrombin cleavage site)-GSGSGG in between (Figure 1B), was cloned into a murine cytomegalovirus promoter containing vector pSMED2 (pSMED2-LAFVIII-HSA). A similar PCR fragment encoding LAFVIII-HSA with Arg-to-Ile variant of R1648I was cloned into pSMED2 (pSMED2-LAFVIII-HSA-R1648I based on full length FVIII). To generate a catalytically inactive variant of human PC5/6 [36], a PCR fragment encoding a full-length PC5/6 with Ser 386 changed to Ala was cloned into pSMED2 (PC5/6-S386A). To generate a catalytically inactive mutant of human Furin [37], a PCR fragment encoding a full-length Furin with Ser 368 changed to Ala was cloned into pSMED2 (Furin-S368A). All constructs were confirmed by DNA sequencing.

3.2.  Cell lines and Cell Culture

Mammalian cell lines were grown and maintained in a humidified incubator with 5% CO₂ at 37^ºC. CHO cells lacking dihydrofolate reductase (CHO-DUKX) were grown in alpha medium supplemented with adenosine (10mg/L), deoxyadenosine (10mg/L), thymidine (10mg/L), and 10% fetal bovine serum (FBS). The CHO-DUKX cells were stably transfected with pSMED2-LAFVIII-HSA or pSMED2-LAFVIII-HSA-R1648I, linearized with PvuI and subjected to selection with 10nM, or 20nM, or 50nM methotrexate (MTX). The stable colony formation was allowed to undergo selection for 3 weeks. The colonies were picked and expanded in media with MTX. Following selection, condition medium was harvested and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Immunoblot. One clone designated for each construct was chosen for further analysis. The stable CHO-DUKX cells were maintained in alpha medium supplemented with 10% FBS and 20nM MTX. Conditioned media was harvested at the end of 3-day culturing, cleared by centrifugation prior to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and analyzed by Immunoblot.

3.3.  Immunoblot Analysis

Conditioned media were separated by SDS-PAGE, and analyzed by anti-human serum albumin antibody HRP conjugated (Abcam, Cambridge, MA, 1.4µg/ml).

3.4.  Assay for FVIII Activity

FVIII activity was measured as described [31]. Conditioned medium was diluted, added to the Activated Partial Thrombo-plastin Assay (aPTT) reagent containing Actin^® FSL (Siemens) and FVIII deficient plasma (George King Bio-Medical, Inc., Overland Park, KS). The time to clot was measured using a StarT4 coagulation instrument (Diagnostica Stago, Parsippany, NJ). Standard curves were prepared by dilution of pooled normal plasma (FACT, George King Bio-Medical, Inc.). One International Unit (IU) of FVIII activity was defined as that amount measured in 1 mL of normal human pooled plasma.

4.  Results

·     Nine members of proprotein convertase family in mammalian expression vector and stable CHO expression of a FVIII B-domain deletion mutant with C-terminal human serum albumin fusion.

To study FVIII processing, human PC1/3, PC2, PC4, PC5/6, PC7, PACE4, Furin, SKI-1, and rat PCSK9 (rPCSK9), were cloned into a mammalian expression vector (Figure1A). They all contain a signal peptide, a pro-domain, and a catalytic domain.

To simplify processing study, we have made use of a B-domain-deleted form of FVIII (LA-FVIII) [28,35]. This FVIII molecule has a deletion from residue 760 through 1639 and exhibits activity similar to that of wild type recombinant FVIII, as B-domain is dispensable for procoagulant activity. LA-FVIII is expressed at 10- to 20- fold greater level compared with wild type FVIII, likely due to increased levels of mRNA and secretion efficiency [28,35]. To extend half-life of FVIII for potential in vivo studies, human serum albumin (HSA, aa25-609) [38,39] was fused to the C-terminus of the protein with a linker in between (LAFVIII-HSA, 2060aa, Figure1B). One major processing site at R1313 is consequentially eliminated along with the 880aa B-domain deletion, whereas another major processing site at R1648 remains in this construct [31]. The resulting heterodimer after cleavage contains LAFVIII-HC (759aa, A1-A2-B*) and FVIII-LC-HSA (1291aa, A3-C1-C2-HSA).

To generate a CHO-DUKX stable clone constitutively expressing LAFVIII-HSA, the DNA construct was transfected into CHO-DUKX cells for methotrexate selection. A positive clone was isolated by screening the conditioned medium from a number of colonies with anti-HSA SDS-PAGE and Immunoblot. This stable clone of FVIII-HSA (Figure 2. lane 1) produced one 220kDa-band and one 120kDa-band which were absent in the negative CHO clone (lane 2). The 220kDa-band corresponds to the full length unprocessed based on detection with an HSA antibody that recognizes the C-terminal albumin fusion. The 120kDa-band corresponds to FVIII-LC-HSA (A3-C1-C2-HSA). The processing of LAFVIII-HSA was not completed, as the full-length LAFVIII-HSA was still detected. This observation is consistent with reported findings [27,30,31,35]. Wild type FVIII contains both R1313 and R1648 sites, which might ensure efficient processing, as no full-length unprocessed polypeptide is measurable [30].

The detection of unprocessed LAFVIII-HSA is consistent with the notion that processing of FVIII is not required for its extracellular secretion. As shown in Figure 2, cleavage site variant of LAFVIII-HSA-R1648I was also stably transfected into CHO-DUKX cells. Positive clones were isolated with anti-HSA immunoblotting. As shown in Figure 2 Lanes 3 &4, the 220kDa-unprocessed LAFVIII-HSA band was prominent while the 120kDa-band was also present. The data suggested that R1648 was the major processing site for LAFVIII-HSA.

·    Furin and PC5/6 can specifically process LAFVIII-HSA.

To address which PC family members may cleave FVIII, DNAs of the PC constructs were transiently transfected into the CHO-DUKX cell line stably expressing LAFVIII-HSA. Conditioned media was harvested after 3 days and analyzed by SDS-PAGE and detected by anti-HSA Immunoblot. As shown in Figure 3A, when human PC1/3, PC2, PC4, PC7, PACE4, SKI-1, or rat PCSK9, were transfected into stable CHO cells, the 220kDa band of LAFVIII-HSA was still detectable (Lanes 3-6, & 8-10). In contrast, the 220kDa-band disappeared completely when the cells were transfected with Furin (Lane 2) or PC5/6 (Lane 7). To confirm that the disappearance of the 220kDa band is associated with the enzymatic activity of Furin or PC5/6, catalytically-inactive mutation S368A in Furin [37] or S386A in PC5/6 [36] were generated. These mutations completely inactivated the processing activity of Furin or PC5/6 by destroying the key Ser residue within the catalytic pockets [36,37]. When the DNA constructs were transfected into CHO cells, processing of LAFVIII-HSA was no longer detectable (Figure3B, Lane 1 vs 2, lanes 3, 4 vs 5, 6). This data indicates that LAFVIII-HSA processing was associated with the activities of Furin and PC5/6.

·   There is no activity difference between Furin-treated or PC5/6-treated LAFVIII-HSA and the R1648I LAFVIII-HSA variant.

CHO-DUKX cells stably expressing R1648I variant produced predominantly full-length LAFVIII-HSA (Figure 2. lanes 3 &4), whereas Furin-transfected (Figure 3B, lane 3) or PC5/6-transfected (Figure 3B, lane 1) CHO-DUKX cells stably expressing wild type LAFVIII-HSA produced no detectable full-length protein. These samples could allow us to do a direct comparison to ascertain if there is a functional activity difference among them. As shown in Table I, R1648I and PC5/6-treated or Furin-treated LAFVIII-HSA exhibited a similar activity in a one stage clotting assay.

5.  Discussion

In this study, we have shown that Furin and PC5/6 of the PC family members could process a B-domain-deleted form of FVIII with a C-terminal HSA fusion, while seven other PC family members appear not to possess such activity. The cleavage is associated with the enzymatic activity of Furin or PC5/6 because the corresponding catalytically-inactive mutants lost this capability. Native mature FVIII is a processed heterodimeric cofactor, but under recombinant conditions both processed and unprocessed molecules have been found secreted out of producing cells. The finding that PC5/6 or Furin can process full-length LAFVIII-HSA fusion efficiently has allowed us to compare the activity between fully processed molecules    and predominantly unprocessed R1648I variant in an in vitro assay. Both forms of FVIII molecules seem similarly active, consistent with the previous finding [27,31].

The involvements of Furin and PC5/6 in FVIII processing are aligned with the notion that each PC family member has unique substrates in vivo, even though they exhibit a certain degree of functional redundancy during overexpression. Among these nine PC members, PCSK9 and SKI-1 are least likely involved in FVIII processing, as PCSK9 is secreted as catalytically inactive enzyme and SKI has a unique cleavage site sequence. The not-involvement of PC1/3 and PC2 seems possible, because they are dominant in the regulated secretory pathway of endocrine and neural cells [1,4]. For the processing in the constitutive secretory pathway, Furin, PC5/6, PACE4, and PC7 are the most common [2,4]. PC7 is unique in its ability to cleave transferrin receptor 1 [40], whereas PACE4 shares some substrates with Furin such as TGFβ-related Nodal precursor [41]. Some protein substrates like angiopoietin-like 3 were found to be processed by Furin, PC5/6, and PACE4 in vitro [42]. Our data might provide another substrate that is shared by PC5/6 and Furin.

The data suggesting that FVIII can be processed by PC5/6 and Furin might imply possible FVIII’s processing location in cells, because these two PC enzymes are only active at specific intracellular sites. Furin activation occurs in trans Golgi Network (TGN), cell surface, as well as recycling endosome [43]. PC5/6 is activated predominantly at the cell surface, and is localized by binding to heparin sulfate proteoglycans [2]. Since both PC5/6 and Furin could process FVIII, the cleavage event might take place at TGN or cell surface where these two enzymes are both active.

Identifying the involvement of PC5/6 and Furin in FVIII cleavage adds to the list of a number of therapeutically important proteins or peptides processed by the PCs. Proinsulin is processed by PC1/3 and PC2 [1]. Bone morphogenetic protein (BMP)-2 precursor [44] is processed by PC5/6 [45], whereas BMP10 is mainly cleaved by Furin [46]. For hemostasis pathway, presegments of Pro-factor VII [1] and Pro-factor IX (FIX) [47] are processed by Furin. Different from that of FVIII, processing of Pro-FIX to the zymogen FIX is critical for activated FIX to form a phospholipid interaction [48]. These findings together emphasize the critical roles of the PC family members in therapeutic protein biogenesis. Further investigation on the potential roles of PC5/6 and Furin in FVIII processing in vivo should shed new light on their molecular mechanisms.

Figures 1(A,B): Nine members of proprotein convertase family and a FVIII B-domain deletion mutant with C-terminal human serum albumin fusion. A. Diagram of nine members of proprotein convertase family. SP, signal sequence; Pro, prodomain; AH, amphipathic helix; CRD, cysteine rich domain; TM, transmembrane domain; CTD, C-terminal domain. B. Diagram for wild type FVIII and a B-domain deletion mutant FVIII (LAFVIII) with a C-terminal human serum albumin fusion. Major processing sites as well as some reported minor clipping sites in FVIII are annotated.

Figure 2: Stable CHO expression of LAFVIII-HSA and R1648I LAFVIII-HSA. with a C-terminal human serum albumin fusion. Conditioned media from stable CHO-DUKX cell clones established for LAFVIII-HSA were analyzed by SDS-PAGE and immunoblotted with anti-HSA-HRP, as described in Materials & Methods. Lanes 1 is for LAFVIII-HSA CHO clone and lane 2 is negative CHO clone. Lane 3 & 4 are individual clones for R1648I LAFVIII-HSA.

Figures 3(A,B): Furin and PC5/6 process FVIII-HSA. A. Furin and PC5/6, but not the other seven PC family members, can process LAFVIII-HSA. DNAs of nine PC constructs (Figure 1) were transiently transfected into the CHO-DUKX cell line stably expressed LAFVIII-HSA. Conditioned medium (20µL) after three days was analyzed by SDS-PAGE and anti-HSA Immunoblot. B. Processing of FVIII-HSA by Furin and PC5/6 is associated with their enzymatic activities. Wild Type (WT) and catalytically-inactive mutant (Mut) constructs of Furin and PC5/6 were transiently transfected into the CHO-DUKX cell line stably expressing LAFVIII-HSA, and analyzed as described in Materials & Methods. Catalytically-inactive mutants of PC5/6 (S386A) and Furin (S368A) did not effectively process LAFVIII-HSA precursor.

Table I: FVIII activity of conditioned medium of LAFVIII-HSA. CHO-DUKX cells stably expressing wild type LAFVIII-HSA, LAFVIII-HSA R1648I variant or those transfected with Furin or PC5/6, were collected after three days’ culturing. The conditioned media were analyzed for its FVIII activity as described in Materials & Methods.

1. Seidah NG, Day R, Marcinkiewicz M, Chretien M (1998) Precursor convertases: an evolutionary ancient, cell-specific, combinatorial mechanism yielding diverse bioactive peptides and proteins. Ann N Y Acad Sci 839:  9-24.
2. Seidah NG, Mayer G, Zaid A, Rousselet E, Nassoury N, et al. (2008) The activation and physiological functions of the proprotein convertases. Int J Biochem Cell Biol 40: 1111-1125.
3. Seidah NG, Prat A (2012) The biology and therapeutic targeting of the proprotein convertases. Nat Rev Drug Discov 11: 367-383.
4. Seidah NG,    Sadr MS, Chretien M, Mbikay M (2013) The Multifaceted Proprotein Convertases: Their Unique, Redundant, Complementary, and Opposite Functions. J Biol Chem 288: 21473-21481.
5. Steiner DF (1998) The proprotein convertases. Curr Opin Chem Biol 2: 31-39.
6. Cheng D, Espenshade PJ, Slaughter CA, Jaen JC, Brown MS, et al. (1999) Secreted site-1 proteas cleaves peptides corresponding to luminal loop of sterol regulatory element-binding proteins.  J Biol Chem 274: 22805-22812.
7. Seidah NG, Mowla SJ, Hamelin J, Mamarbachi AM, Benjannet S, et al. (1999) Mammalian subtilisin/kexin isozyme SKI-1: A widely expressed proprotein convertase with a unique cleavage specificity and cellular localization. Proc Natl Acad Sci USA 96: 1321-1326.
8. Seidah NG, Benjannet S, Wickham L, Marcinkiewicz J, Jasmin SB, et al. (2003) The secretory proprotein convert as neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation. Proc Natl Acad Sci USA 100: 928-933.
9. Pasquato A,  Pullikotil P, Asselin MC, Vacatello M, Paolillo L, et  al. (2006 ) The proprotein convertase SKI-1/S1P. In vitro analysis of Lassa virus glycoprotein-derived substrates and ex vivo validation of irreversible peptide inhibitors. J Biol Chem 281: 23471-23481.
10. Zhu X, Zhou A, Dey A, Norrbom C, Carroll R, et al. (2002) Disruption of PC1/3 expression in mice causes dwarfism and multiple neuroendocrine peptide processing defects. Proc Natl Acad Sci USA 99: 10293-10298.
11. Furuta M, Yano H, Zhou A, Rouillé Y, Holst JJ, et al. (1997) Defective prohormone processing and altered pancreatic islet morphology in mice lacking active SPC2. Proc Natl Acad Sci USA 94: 6646-6651.
12. Mbikay M, Tadros H, Ishida N, Lerner CP, De Lamirande E, et al. (1997) Impaired fertility in mice deficient for the testicular germ-cell protease PC4. Proc Natl Acad Sci USA 94: 6842-6846.
13. Roebroek AJ, Umans L, Pauli IG, Robertson EJ, van Leuven F, et al. (1998) Failure of ventral closure and axial rotation in embryos lacking the proprotein convertase Furin. Development 125:  4863-4876.
14. Essalmani R, Zaid A, Marcinkiewicz J, Chamberland A, Pasquato A, et al. (2008) In vivo functions of the proprotein convertase PC5/6 during mouse development: Gdf11 is a likely substrate. Proc Natl Acad Sci USA 105: 5750-5755.
15. Szumska D, Pieles G, Essalmani R, Bilski M, Mesnard D, et al. (2008) VACTERL/caudal regression/Currarino syndrome-like malformations in mice with mutation in the proprotein convertase Pcsk5. Genes Dev 22: 1465-1477.
16. Constam DB, Robertson EJ (2000) SPC4/PACE4 regulates a TGFbeta signaling network during axis formation. Genes Dev 14: 1146-1155.
17. Yang J, Goldstein JL, Hammer RE, Moon YA, Brown MS, et al. (2001) Decreased lipid synthesis in livers of mice with disrupted Site-1 protease gene. Proc Natl Acad Sci USA 98: 13607-13612.
18. Besnard J, Ruda GF, Setola V, Abecassis K, Rodriguiz RM, et al. (2012) Automated design of ligands to polypharmacological profiles. Nature 492: 215-220.
19. Rashid S, Curtis DE, Garuti R, Anderson NN, Bashmakov Y, et al. (2005) Decreased plasma cholesterol and hypersensitivity to statins in mice lacking Pcsk9. Proc Natl Acad Sci USA 102: 5374-5379.
20. Zhao Z, Tuakli-Wosornu Y, Lagace TA, Kinch L, Grishin NV, et al. (2006) Molecular characterization of loss-of-function mutations in PCSK9 and identification of a compound heterozygote. American journal of human genetics 79: 514-523.
21. Kaufman RJ (1992) Biological regulation of factor VIII activity. Annual review of medicine 43: 325-339.
22. Toole JJ, Knopf JL, Wozney JM, Sultzman LA, Buecker JL, et al. (1984) Molecular cloning of a cDNA encoding human antihaemophilic factor. Nature 312: 342-347.
23. Vehar GA, Keyt B, Eaton D, Rodriguez H, O'Brien DP, et al. (1984) Structure of human factor VIII. Nature 312: 337-342.
24. Ortel TL, Takahashi N, Putnam FW (1984) Structural model of human ceruloplasmin based on internal triplication, hydrophilic/hydrophobic character, and secondary structure of domains. Proc Natl Acad Sci USA 81: 4761-4765.
25. Stubbs JD, Lekutis C, Singer KL, Bui A, Yuzuki D, et al. (1990) cDNA cloning of a mouse mammary epithelial cell surface protein reveals the existence of epidermal growth factor-like domains linked to factor VIII-like sequences. Proc Natl Acad Sci USA 87: 8417-8421.
26. Andersson LO, Forsman N, Huang K, Larsen K, Lundin A, et al. (1986) Isolation and characterization of human factor VIII: molecular forms in commercial factor VIII concentrate, cryoprecipitate, and plasma. Proc Natl Acad Sci USA 83: 2979-2983.
27. Pittman DD, Marquette KA, Kaufman RJ (1994) Role of the B domain for factor VIII and factor V expression and function. Blood 84: 4214-4225.
28. Toole JJ, Pittman DD, Orr EC, Murtha P, Wasley LC, et al. (1986) A large region (approximately equal to 95 kDa) of human factor VIII is dispensable for in vitro procoagulant activity. Proc Natl Acad Sci USA 83: 5939-5942.
29. Kaufman RJ (1998) Post-translational modifications required for coagulation factor secretion and function. Thromb Haemost 79:  1068-1079.
30. Jankowski MA, Patel H, Rouse JC, Marzilli LA, Weston SB, et al. (2007) Defining 'full-length' recombinant factor VIII: a comparative structural analysis. Haemophilia: the official journal of the World Federation of Hemophilia 13: 30-37.
31. Pittman DD, Tomkinson KN, Kaufman RJ (1994) Post-translational requirements for functional factor V and factor VIII secretion in mammalian cells. J Biol Chem 269: 17329-17337.
32. Peters RT, Toby G, Lu Q, Liu T, Kulman JD, et al. (2013) Biochemical and functional characterization of a recombinant monomeric factor VIII-Fc fusion protein. Journal of thrombosis and haemostasis 11: 132-141.
33. Nguyen GN, George LA, Siner JI, Davidson RJ, Zander CB, et al. (2017) Novel factor VIII variants with a modified furin cleavage site improve the efficacy of gene therapy for hemophilia A. Journal of thrombosis and haemostasis 15: 110-121.
34. Demasi MA, de S Molina E, Bowman-Colin C, Lojudice FH, Muras A, et al. (2016) Enhanced Proteolytic Processing of Recombinant Human Coagulation Factor VIII B-Domain Variants by Recombinant Furins. Mol Biotechnol 58: 404-414.
35. Pittman DD,  Alderman EM, Tomkinson KN, Wang JH, Giles AR, et al. (1993) Biochemical, immunological, and in vivo functional characterization of B-domain-deleted factor VIII. Blood 81: 2925-2935.
36. Lusson J, Vieau D, Hamelin J, Day R, Chrétien M, et al. (1993) cDNA structure of the mouse and rat subtilisin/kexin-like PC5: a candidate proprotein convertase expressed in endocrine and nonendocrine cells. Proc Natl Acad Sci USA 90: 6691-6695.
37. Creemers JW, Vey M, Schäfer W, Ayoubi TA, Roebroek AJ, et al. (1995) Endoproteolytic cleavage of its propeptide is a prerequisite for efficient transport of furin out of the endoplasmic reticulum. J Biol Chem 270: 2695-2702.
38. Lawn RM, Adelman J, Bock SC, Franke AE, Houck CM, et al. (1981) The sequence of human serum albumin cDNA and its expression in E. coli. Nucleic Acids Res 9: 6103-6114.
39. Anderson CL, Chaudhury C, Kim J, Bronson CL, Wani MA, et al. (2006) Perspective-- FcRn transports albumin: relevance to immunology and medicine. Trends in immunology 27: 343-348.
40. Guillemot J, Canuel M, Essalmani R, Prat A, Seidah NG (2013) Implication of the proprotein convertases in iron homeostasis: proprotein convertase 7 sheds human transferrin receptor 1 and furin activates hepcidin. Hepatology 57: 2514-2524.
41. Mesnard D, Donnison M, Fuerer C, Pfeffer PL, Constam DB (2011) The microenvironment patterns the pluripotent mouse epiblast through paracrine Furin and Pace4 proteolytic activities. Genes Dev 25: 1871-1880.
42. Essalmani R, Susan-Resiga D, Chamberland A, Asselin MC, Canuel M, et al. (2013) Furin is the primary in vivo convertase of angiopoietin-like 3 and endothelial lipase in hepatocytes. J Biol Chem 288: 26410-26418.
43. Thomas G (2002) Furin at the cutting edge: from protein traffic to embryogenesis and disease. Nature reviews. Molecular cell biology 3: 753-766.
44. Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, et al. (1988) Novel regulators of bone formation: molecular clones and activities. Science 242: 1528-1534.
45. Heng S, Paule S, Hardman B, Li Y, Singh H, et al. (2010) Posttranslational activation of bone morphogenetic protein 2 is mediated by proprotein convertase 6 during decidualization for pregnancy establishment. Endocrinology 151: 3909-3917.
46. Susan-Resiga D, Essalmani R, Hamelin J, Asselin MC, Benjannet S, et al. (2011) Furin is the major processing enzyme of the cardiac-specific growth factor bone morphogenetic protein 10. J Biol Chem 286: 22785-22794.
47. Wasley LC, Rehemtulla A, Bristol JA, Kaufman RJ (1993) PACE/furin can process the vitamin K-dependent pro-factor IX precursor within the secretory pathway. J Biol Chem 268: 8458-8465.
48. Diuguid DL, Rabiet MJ, Furie BC, Liebman HA, Furie B (1986) Molecular basis of hemophilia B: a defective enzyme due to an unprocessed propeptide is caused by a point mutation in the factor IX precursor. Proc Natl Acad Sci USA 83: 5803-5807.