Factor VIII Arg2304 → His Binds to Phosphatidylserine and Is a Functional Cofactor in the Factor X-ase Complex

2001 ◽  
Vol 85 (02) ◽  
pp. 260-264 ◽  
Author(s):  
Deborah Lewis ◽  
Karen Moore ◽  
Thomas Ortel
Keyword(s):  
Factor Viii ◽  
Light Chain ◽  
Mammalian Cells ◽  
Exchange Chromatography ◽  
Light Chains ◽  
Factor X ◽  
Wild Type ◽  
Functional Protein ◽  
Type Factor ◽  
Circulating Levels

SummaryFour factor VIII light chain constructs containing hemophilia A mutations at R2304 and R2307 were prepared and expressed in mammalian cells. These mutations are located in a putative phosphatidylserine binding site identified by peptide studies (spanning amino acids 2303-2332). The levels of all four mutants in conditioned medium were significantly less than wild type by immunoprecipitation and ELISA. R2304H and wild type factor VIII light chains were concentrated by cation exchange chromatography from medium. R2304H and wild type factor VIII light chains bound immobilized phosphatidylserine similarly. The reconstituted cofactor activity of R2304H factor VIII light chain was slightly greater than wild type factor VIII light chain. These results are consistent with the recently reported crystal structure of factor VIII C2 domain that suggests R2304H is not directly involved in phospholipid binding. The observed clinical phenotype is probably due to decreased circulating levels of a functional protein.

Thrombin-Catalyzed Cleavages of Factor VIII at Arg372 and Arg1689 Are Facilitated by the Acidic Residues Glu720-Asp725.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2686-2686
Author(s):  
Jennifer Newell ◽  
Qian Zhou ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Specific Activity ◽  
Factor Xa ◽  
Factor X ◽  
Wild Type ◽  
Factor Viiia ◽  
N Terminus ◽  
Acidic Residues ◽  
Type Factor ◽  
A2 Domain

Abstract Factor VIIIa acts as an essential cofactor for the serine protease factor IXa, together forming the Xase complex which catalyzes the conversion of factor X to factor Xa. The procofactor, factor VIII circulates as a heterodimeric protein comprised of a heavy chain (A1–A2-B domains) and a light chain (A3-C1-C2 domains) and is activated by proteolytic cleavage by thrombin at Arg372 (A1–A2 junction), Arg740 (A2-B junction), and Arg1689 (near the N-terminus of A3). The regions adjacent to the A1, A2, and A3 domains contain high concentrations of acidic residues and are designated a1 (residues 337–372), a2 (residues 711–740), and a3 (residues 1649–1689). In addition, the N-terminus of the A2 domain (residues 373–395) is rich in acidic residues, and results from a previous study revealed that this region contributes to the rate of thrombin-catalyzed cleavage at Arg740 (Nogami et. al., J. Biol. Chem. 280:18476, 2005). In this study we reveal a role for the acidic region following the A2 domain (a2, residues 717–725) in thrombin-catalyzed cleavage at both Arg372 and Arg1689. The factor VIII mutations Asp717Ala, Glu720Ala, Asp721Ala, Glu724Ala, Asp725Ala, and the double mutations of Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala were constructed, expressed, and purified from stably-transfected BHK cells as B-domainless protein. Specific activity values for the variants, relative to the wild type value were reduced to 70% for Asp717Ala; ∼50% for Glu720Ala, Asp721Ala, Glu724Ala, and Asp725Ala; and ∼30% for Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala. SDS-PAGE and western blotting of reactions containing the factor VIII variants and thrombin showed reductions in the rates of thrombin cleavage at both Arg372 and Arg1689 as compared to wild-type factor VIII. The cleavage rates for the single mutations comprising acidic residues 720–724 of factor VIII were reduced from ∼3-5-fold at Arg372, whereas this rate for the Asp717Ala mutant was similar to the wild-type value. The double mutations of Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala showed rate reductions of ∼7- and ∼27-fold, respectively at Arg372. While the rate for thrombin-catalyzed cleavage at Arg1689 in the Glu720Ala variant was similar to wild-type, rates for cleavage at this site were reduced ∼30-fold compared to wild-type factor VIII for the Asp721Ala, Glu724Ala, Asp725Ala, and Glu720Ala/Asp721Ala mutants, and ∼50-fold for the Glu724Ala/Asp725Ala variant. Furthermore, the generation of factor VIIIa activity following reaction with thrombin as assayed by factor Xa generation showed that all the mutants possessed peak activity values that were ∼2-3-fold reduced compared to wild type factor VIIIa. Moreover, in all the mutants the characteristic peak of activation was replaced with a slower forming, broad plateau of activity, with the double mutants showing the broadest activation profiles. These results suggest that residues Glu720, Asp721, Glu724, and Asp725 following the A2 domain modulate thrombin interactions with factor VIII facilitating cleavage at Arg372 and Arg1689 during procofactor activation.

A membrane-interactive surface on the factor VIII C1 domain cooperates with the C2 domain for cofactor function

Blood ◽  
2011 ◽  
Vol 117 (11) ◽  
pp. 3181-3189 ◽  
Author(s):  
Junhong Lü ◽  
Steven W. Pipe ◽  
Hongzhi Miao ◽  
Marc Jacquemin ◽  
Gary E. Gilbert
Keyword(s):  
Monoclonal Antibody ◽  
Factor Viii ◽  
Membrane Binding ◽  
Factor X ◽  
Wild Type ◽  
C2 Domains ◽  
C1 Domain ◽  
Interactive Surface ◽  
Type Factor ◽  
Membrane Interactive

Abstract Factor VIII binds to phosphatidylserine (PS)-containing membranes through its tandem, lectin-homology, C1 and C2 domains. However, the details of C1 domain membrane binding have not been delineated. We prepared 4 factor VIII C1 mutations localized to a hypothesized membrane-interactive surface (Arg2090Ala/Gln2091Ala, Lys2092Ala/Phe2093Ala, Gln2042Ala/Tyr2043Ala, and Arg2159Ala). Membrane binding and cofactor activity were measured using membranes with 15% PS, mimicking platelets stimulated by thrombin plus collagen, and 4% PS, mimicking platelets stimulated by thrombin. All mutants had at least 10-fold reduced affinities for membranes of 4% PS, and 3 mutants also had decreased apparent affinity for factor X. Monoclonal antibodies against the C2 domain produced different relative impairment of mutants compared with wild-type factor VIII. Monoclonal antibody ESH4 decreased the Vmax for all mutants but only the apparent membrane affinity for wild-type factor VIII. Monoclonal antibody BO2C11 decreased the Vmax of wild-type factor VIII by 90% but decreased the activity of 3 mutants more than 98%. These results identify a membrane-binding face of the factor VIII C1 domain, indicate an influence of the C1 domain on factor VIII binding to factor X, and indicate that cooperation between the C1 and C2 domains is necessary for full activity of the factor Xase complex.

scholarly journals A Spacer Sequence Inserted between the A1 and A2 Domains of Factor VIII Overcomes the Requirement for Proteolytic Cleavage at Arg 372 in the Generation of Cofactor Activity

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 21-21
Author(s):  
Manjunath Goolyam Basavaraj ◽  
Sriram Krishnaswamy
Keyword(s):  
Factor Viii ◽  
Western Blotting ◽  
Light Chain ◽  
Specific Activity ◽  
Factor X ◽  
Physiological Concentration ◽  
Proteolytic Activation ◽  
Linker Sequence ◽  
One Stage ◽  
Cofactor Activity

Factor VIII (FVIII) with a multi-domain structure (A1-a1-A2-a2-B-a3-A3-C1-C2) is a procofactor and precursor for the anti-hemophilic cofactor protein, FVIIIa. Following the intracellular processing within the B domain, secreted FVIII circulates as a heterodimer with variably sized (90K-200K) heavy chain (A1-a1-A2-a2-B) and an 80K light chain (a3-A3-C1-C2). Proteolytic activation of FVIII by thrombin that yields heterotrimeric FVIIIa (A1-a1/A2-a2/A3-C1-C2), the cofactor for intrinsic tenase, involves cleavage of three peptide bonds between Arg372-Ser373, Arg740-Ser741, and Arg1689-Ser1690. Cleavage at Arg740 removes the B-domain, and cleavage at Arg1689 removes the a3-acidic region and releases FVIII from vWF, its carrier protein, and exposes membrane binding sites within the FVIII light chain. Cleavage at Arg372 separates A1-a1 and A2-a2 domains and is implicated in the cofactor-dependent recognition and enhancement in the rate of factor X (FX) activation by intrinsic tenase. Subsequently, the separated A2-a2 domain dissociates spontaneously from the heterotrimeric FVIIIa resulting in the rapid loss of cofactor activity. We speculated that the requirement for cleavage at Arg372 might be obviated by the insertion of an optimized linker sequence between A1-a1 and A2-a2 domains on an uncleavable Gln372 backbone. To investigate this possibility, we prepared cDNA constructs of B-domain deleted FVIII variants; FVIII wild-type (FVIIIWT), FVIII372Q, and FVIII372Q followed by a rigid (Ala-Pro)5 linker sequence (FVIII372Q-AP5). All three FVIII constructs were stably transfected into BHK cells and high expressing clones were selected by one stage aPTT and western blotting of expression media. Selected stable clones were further expanded to collect 15L of expression media over 5-day period, and recombinant FVIII variants were purified using a three-step chromatographic approach. These FVIII variants were studied using SDS-PAGE, western blotting, aPTT assays, thrombin generation assay (TGA) and purified assays to assess kinetics of FX activation and spontaneous loss of cofactor activity. In contrast to FVIIIWT, FVIII372Q and FVIII372Q-AP5 were completely resistant to cleavage at Gln372 by thrombin, yielding bands corresponding to A1-a1-A2-a2 (90K) and A3-C1-C2 (73K). In one stage aPTT assays, FVIII372Q showed prolonged clotting times with specific activity in the range of 200-400 U/mg, while FVIIIWT and FVIII372Q-AP5 displayed comparable clotting times with specific activities ranging between 8000-10000 U/mg and 4500-5500 U/mg, respectively. In TGA initiated with either 0.1 pM tissue factor or 1 pM factor XIa, both FVIIIWT and FVIII372Q-AP5 displayed similar TGA profiles. In steady state kinetic studies of FX activation using limiting concentrations of factor IXa, saturating concentrations of FVIII variants pretreated with thrombin, membranes and increasing concentrations of FX, the cofactor function of thrombin-cleaved FVIII372Q was severely impaired. However, despite lack of cleavage at Gln372 in FVIII372Q-AP5, catalytic efficiency for FX activation by intrinsic tenase assembled by this variant was comparable to that seen with FVIIIaWT. At the physiological concentration of FX, the initial velocity for Xa formation (v/E) for intrinsic tenase assembled with FVIIIa372Q-AP5 was within a factor of 2 of that observed with FVIIIaWT while the rate observed with FVIIIa372Q was >10-fold lower. Following rapid activation with thrombin, loss of cofactor function was significantly slower for FVIIIa372Q-AP5(t1/2 ~ 10 min) compared to FVIIIaWT (t1/2 ~ 2 min). Our findings indicate that the requirement for cleavage at Arg372 for the development of full FVIIIa cofactor function can be overcome by modulating the A1-A2 connector with an optimized linker sequence. Failure to yield an infinitely stable cofactor in the case of FVIIIa372Q-AP5 suggests that cleavage at Arg372 does not solely explain the spontaneous loss of FVIIIa cofactor function. Disclosures Krishnaswamy: Bayer: Research Funding.

An Essential Role of the Factor VIII Light Chain in Facilitating Heavy Chain Secretion.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4034-4034
Author(s):  
Lingxia Chen ◽  
Juan Li ◽  
Hui Lu ◽  
Haiyan Jiang ◽  
Rita Sarkar ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Hek293 Cells ◽  
Light Chains ◽  
Trans Splicing ◽  
In Cis

Abstract Blood coagulation Factor VIII (FVIII) is secreted as a heterodimer consisting of a heavy and light chain. Both in vitro and in vivo studies have demonstrated that these chains can be expressed independently. The expressed heavy and light chains can reassociate with recovery of biological activities. These observations have been particularly useful in a gene therapy setting since vector packaging capacity for adeno-associated virus (AAV) is a limiting factor. However, it has been demonstrated that the FVIII heavy chain is expressed ~10–100-fold less efficiently compared to the light chain when expressed independently. Previously the FVIII F309S mutation in the context of B-domainless FVIII (FVIII-BDD) and enhanced glycosylations within the B-domain have been shown to improve factor VIII expression and secretion. However, our in vitro studies indicate that these improvements in secretion were not retained when expressing the heavy chain alone with the same modifications. Other sequences, possibly in the light chain, may facilitate secretion. To investigate this further, we designed an intein trans-splicing strategy to control the addition of light chain to the heavy chain before secretion. Using HEK293 cells, we cotransfected seperate intein light chain and intein heavy chain plasmids and compared results to single plasmid transfected cells. 48 hours post-transfection, FVIII-specific ELISA results demonstrated that cotransfection of intein heavy chain and intein light chain had a significant influence on total heavy chain secretion compared to intein heavy chain expression alone. The co-transfected intein heavy chain and intein light chain were efficiently ligated together yielding a biologically active single chain FVIII derivative as demonstrated by clotting assays and Western blot analysis. Therefore, heavy chain secretion was directly enhanced by the attachment of the light chain to the C-terminus of the heavy chain. A similar phenomenon was not found when heavy and light chains were simply co-expressed in the same cell. It suggested that light chain functioned in cis. Hydrodynamic injection of plasmids with intein heavy chain and intein light chain into hemophilia A mice led to a much higher level of FVIII secretion. The amount of functional FVIII expression reached 3–6 units/ml at peak level. In the absence of intein light chain, FVIII heavy chain secretion was approximately 100 fold less efficient in vivo. To map the key elements of FVIII light in helping FVIII secretion, we made deletion variants in the light chain. These mutants had a dominant negative effect in reducing FVIII and FVIII heavy chain secretion while increasing the level of intracellular FVIII accumulation. Collectively our results are consistent with the conclusion that the FVIII light chain plays a critical role in facilitating heavy chain secretion in cis; probably through helping FVIII heavy chain maintain correct configuration and folding. The strategy to manipulate FVIII light chain addition through intein mediated trans-splicing reaction may also be explored for human gene therapy.

B-domain deleted factor VIII is aggregated and degraded through proteasomal and lysosomal pathways

2005 ◽  
Vol 93 (05) ◽  
pp. 824-832 ◽  
Author(s):  
Benoit Guillet ◽  
Cécile Ducasse ◽  
Nathalie Enjolras ◽  
Marie-Hélène Rodriguez ◽  
Véronique Rolli ◽  
...  
Keyword(s):  
Factor Viii ◽  
Light Chain ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Mrna Synthesis ◽  
Extracellular Production ◽  
Single Chain ◽  
Cho Cell ◽  
Over Expression ◽  
Triton X

SummaryFactor VIII (FVIII) processing within mammalian cells is demonstrated to be much less efficient than proteins of similar size. The deletion of the B-domain from FVIII improves the level of production, due partly to the increase in mRNA synthesis. We aimed to characterise the cellular fate and the intracellular processing of the FVIII molecule devoid of B-domain. A B-domain deleted factor VIII (BDD-FVIII) possessing a furin consensus cleavage site in the connecting segment between the heavy and the light chain, was produced in CHO cell line. In such cells, FVIII was retained as two single chain products from which a majority was aggregated. The two species were located in Triton X-100 soluble (for 60–80%) and insoluble fractions (for 20–40%). The incubation of the expressing cells with tunicamycin (5 μg/ml) and the treatment of the intracellular species with a mixture of Neuraminidase and N-glycosidase-F revealed that both intracellular species were N-glycosylated. Furin over-expression neither diminished the intracellular FVIII contents nor improved its extracellular production. Intracellular FVIII was degraded through both lysosomal and proteasomal pathways as evidenced by inhibitor treatments (e.g. NH4Cl, leupeptin, clasto-Lactacystin β-lactone and MG-132), pulse-chase analysis and confocal observations. This study demonstrates that a BDD-FVIII expressed in CHO cells is inefficiently processed consecutively to intracellular aggregation, proteasomal degradation, and routage to lysosomes.

scholarly journals Structural and functional characterization of Factor VIII-Δ II, a new recombinant Factor VIII lacking most of the B-domain

1991 ◽  
Vol 277 (1) ◽  
pp. 23-31 ◽  
Author(s):  
N Bihoreau ◽  
P Paolantonacci ◽  
C Bardelle ◽  
M P Fontaine-Aupart ◽  
S Krishnan ◽  
...  
Keyword(s):  
Factor Viii ◽  
Mammalian Cells ◽  
Functional Characterization ◽  
Activated Protein C ◽  
Recombinant Factor Viii ◽  
Coagulation Cascade ◽  
Molecular Forms ◽  
Regulation System ◽  
Factor X ◽  
Active Peak

A recombinant Factor VIII (Factor VIII-delta II) consists of a unique polypeptide chain of 165 kDa deleted from the major part of the B-domain and from the cleavage site at Arg-1648-Glu-1649 found in plasma-derived Factor VIII. It was expressed in mammalian cells in serum-free medium containing von Willebrand factor and purified by a one-step immunopurification. The recombinant Factor VIII was characterized as a single active peak when subjected to f.p.l.c., in contrast with the plasma-derived molecule. Its coagulant activity was decreased in the presence of EDTA, suggesting that a bivalent ion is required, as for plasma-derived Factor VIII. The activation by thrombin and the inactivation by activated protein C were studied and the resulting molecular forms were analysed by f.p.l.c. and SDS/PAGE. The results clearly demonstrate that, despite the structural differences between plasma-derived and recombinant Factor VIII, activation and inactivation of Factor VIII-delta II generate proteolysed complexes similar to that described for plasma-derived Factor VIII. Thus this deleted recombinant Factor VIII, which is processed similarly to plasma-derived Factor VIII, should be normally integrated in the regulation system of Factor X activation in the blood-coagulation cascade.

Role of the Direct Interaction between Factor VIII C2 and Factor IXa Gla Domain in the Factor Xase Complex.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2687-2687
Author(s):  
Tetsuhiro Soeda ◽  
Keiji Nogami ◽  
Masahiro Takeyama ◽  
Kenichi Ogiwara ◽  
Kazuhiko Tomokiyo ◽  
...  
Keyword(s):  
Factor Viii ◽  
Light Chain ◽  
Dependent Manner ◽  
C2 Domain ◽  
Factor X ◽  
Calcium Dependent ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Gla Domain

Abstract Factor VIII functions as a cofactor for factor IXa in the anionic phospholipid surface-dependent conversion of factor X to Xa. It is well-known that the A2 and A3 domains of factor VIII interact with the catalytic domain and EGF2 domain of factor IXa, respectively. Recently, Furie et al. have reported that the Gla domain of factor IXa (factor IXa-GD) interacts with the light chain of factor VIII. However, the factor IXa-GD-interactive site on the light chain remained to be investigated. In the current study, the recombinant C2 (rC2) domain of factor VIII was prepared using a yeast secretion system. ELISA-based assay in the absence of phospholipid showed the Glu-Gly-Arg-active site modified factor IXa (EGR-factor IXa) bound to the immobilized rC2 domain dose-dependently, and the binding ability was maximum under the condition of 150 mM NaCl/1 mM CaCl2. This binding was competitively inhibited by the addition of excess of factor VIII or rC2 domain, supporting the specificity of this interaction. Furthermore, the presence of high ionic strength and the metal-ion chelator EDTA blocked this binding by ∼95 and ∼75%, respectively. Surface plasmon resonance-based assay showed that the binding affinity (Kd) of rC2 domain for EGR-factor IXa was 108 ± 15.5 nM. GD less-factor IXa, deleting the GD completely, failed to bind to rC2 domain. A monoclonal antibody against factor IXa-GD specific for calcium-dependent conformation (mAbIXa-GD) also inhibited (∼ 95%) the rC2 domain binding to EGR-factor IXa in a dose-dependent manner (IC50; 758 nM), suggesting the authentic of the C2 domain and factor IXa-GD interaction. The addition of rC2 domain or mAbIXa-GD inhibited the factor IXa-catalyzed factor X activation with factor VIIIa in the absence of phospholipid (IC50; 15.7 μM or 43.2 nM, respectively), whilst both any little affected in the absence of factor VIIIa. In addition, the ∼8-kDa C2 fragment obtained by V8 protease digestion (residues 2182–2259) bound directly to EGR-factor IXa. Taken together, these results indicate that factor VIII C2 domain directly interacts with factor IXa-GD via both the electrostatic- and calcium-dependent interactions. Furthermore, our results provide the first evidence for an essential role of the C2 domain in the association between factor VIII and factor IXa in the factor Xase complex.

Residues Surrounding Arg372, Arg740, and Arg1689 Contribute to the Rates of Thrombin-Catalyzed Cleavage of Factor VIII.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 847-847
Author(s):  
Jennifer L. Newell ◽  
Amy E. Griffiths ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Cleavage Site ◽  
Conflicts Of Interest ◽  
Specific Activity ◽  
Factor Xa ◽  
Wild Type ◽  
Cleavage Rate ◽  
Thrombin Cleavage ◽  
Type Factor ◽  
Bold Type

Abstract Abstract 847 Hemophilia A results from defects or deficiencies in the blood coagulation protein, factor VIII. Factor VIII circulates as an inactive procofactor that must be cleaved by thrombin or factor Xa at Arg740 (A2-B junction), Arg372 (A1-A2 junction), and Arg1689 (a3-A3 junction) to yield the active cofactor, factor VIIIa. Activation of factor VIII by thrombin is exosite-dependent yielding rates of cleavage at Arg740 ∼20-fold faster than Arg372, while cleavage at Arg1689 appears intermediary to Arg740 and Arg372. The contribution of P3-P3' residues flanking each cleavage site to the mechanism of thrombin-catalyzed cleavage of factor VIII has not been extensively studied. The P3-P3' residues for the 372, 1689, and 740 factor VIII sites are 370QIR*↓SVA375, 1687SPR*↓SFQ1692, and 738EPR*↓SFS743, respectively. Residues flanking Arg372 are considered non-optimal for thrombin cleavage with only two residues optimal (in bold type) for cleavage in the P3-P3' sequence, while residues flanking at the two other P1 sites are considered near-optimal with four out of six residues optimal (in bold type). Therefore, we investigated whether the P3-P3'residues surrounding Arg740, Arg372, and Arg1689 affect activation of factor VIII by thrombin. We constructed, stably transfected, and expressed four recombinant P3-P3' factor VIII mutants designated 372(P3-P3')740, 372(P3-P3')1689, 372(P3-P3')740/740(P3-P3')372, and 372(P3-P3')740/1689(P3-P3')372. For example, the 372(P3-P3')740 variant has replaced the non-optimal P3-P3' residues flanking Arg372 with the near-optimal P3-P3' residues flanking Arg740. The specific activities of the 372(P3-P3')740 and 372(P3-P3')740/740(P3-P3')372 mutants were 98% and 122% the wild-type factor VIII value, respectively. In comparison, the 372(P3-P3')1689 and 372(P3-P3')740/1689(P3-P3')372 showed reductions in specific activity with values that were 14% and 17% of wild-type factor VIII, consistent with possible impaired rates of activation by thrombin. SDS-PAGE and Western blotting of the three variants possessing the 372(P3-P3')740 mutation showed cleavage rates at Arg372 increased 11- to 14-fold compared with wild-type factor VIII as judged by rates of generation of the A1 subunit. Furthermore, these variants revealed 11-21-fold rate increases in the generation of the A2 subunit as compared to wild-type factor VIII. The rates of A1 and A2 subunit generation were moderately increased from 2-3-fold for the 372(P3-P3')1689 mutant. These results indicate that replacing the non-optimal residues flanking Arg372 with near-optimal residues enhances rates of cleavage at this site. Furthermore, since the P2-P2' residues flanking Arg740 and Arg1689 are identical, these results also suggest that the P3 and/or P3' residues from the Arg740 cleavage site make a greater contribution to the enhanced cleavage rate when inserted at Arg372 than the equivalent residues from the Arg1689 site. Thrombin cleavage of light chain showing the largest effect was obtained for the 372(P3-P3')740/1689(P3-P3')372 mutant which yielded a reduced rate of A3-C1-C2 subunit generation by 33-fold. This result suggests that replacing near-optimal P3-P3' residues at Arg1689 with non-optimal residues at Arg372 significantly reduces the rate of thrombin cleavage at Arg1689, an effect that may contribute to its low specific activity. There was no observed defect in Arg1689 cleavage in the 372(P3-P3')740 mutant and moderate 2-3-fold reductions in thrombin-catalyzed cleavage rates at Arg1689 in the 372(P3-P3')1689, 372(P3-P3')740/740(P3-P3')372, and 372(P3-P3')740 variants. Overall, these results suggest that faster cleavage rates at Arg740 and Arg1689 can be attributed to more optimal residues in the P3-P3' region, while the relatively slower cleavage rate at Arg372 can be accelerated by replacement with more optimal residues for thrombin cleavage. Thus, the P3-P3' residues surrounding Arg740, Arg1689, and Arg372 in factor VIII impact rates of thrombin proteolysis at each site and contribute to the mechanism for thrombin activation of the procofactor. Disclosures: No relevant conflicts of interest to declare.

Purification and characterization of factor VIII 1,689-Cys: a nonfunctional cofactor occurring in a patient with severe hemophilia A

Blood ◽  
1989 ◽  
Vol 73 (8) ◽  
pp. 2117-2122
Author(s):  
DP O'Brien ◽  
EG Tuddenham
Keyword(s):  
Factor Viii ◽  
Light Chain ◽  
Hemophilia A ◽  
Specific Activity ◽  
Exchange Chromatography ◽  
Heavy Chains ◽  
Thrombin Cleavage ◽  
Before And After ◽  
Functional Defect ◽  
Thrombin Activation

We have purified the factor VIII from a CRM+ Hemophilia A plasma (90 U/dL VIII:Ag but 0 U/dL VIII:C) and analyzed the protein before and after thrombin activation by Western blotting with monoclonal antibodies (MoAbs). Normal or patient citrated plasma was ultracentrifuged, cryo-ethanol-precipitated and chromatographed on Sepharose 6B. The void volume fractions were reduced and subjected to ion exchange chromatography yielding material of specific activity approximately 1,000 U/mg protein (VIII:C or VIII:Ag). Factor VIII purified in this way from normal plasma is fully activatable by thrombin with proteolytic fragmentation as previously described by F. Rotblat et al (Biochemistry 24: 4294, 1985). Factor VIII 1,689-Cys has the normal distribution of factor VIII light and heavy chains prior to thrombin activation. After exposure to thrombin the heavy chain polypeptides were fully proteolysed but the light chain was totally resistant to cleavage. This is consistent with the demonstration in the patient's leucocyte DNA of a C to T transition in codon 1,689 converting Arg to Cys at the light chain thrombin cleavage site as previously described by J. Gitschier et al (Blood 72:1022, 1988). Uncleaved light chain of Factor VIII 1,689-Cys is not released from von Willebrand factor (vWF) by thrombin, but this is not the sole cause of the functional defect since the protein purified free of vWF has no coagulant activity. We conclude that the functional defect in factor VIII 1,689-Cys is a consequence of failure to release the acidic peptide from the light chain upon thrombin activation.

scholarly journals Mlc1p Is a Light Chain for the Unconventional Myosin Myo2p in Saccharomyces cerevisiae

1998 ◽  
Vol 142 (3) ◽  
pp. 711-722 ◽  
Author(s):  
Richard C. Stevens ◽  
Trisha N. Davis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Light Chain ◽  
Essential Gene ◽  
Direct Interaction ◽  
Neck Region ◽  
Light Chains ◽  
Polarized Growth ◽  
Wild Type ◽  
Uncharacterized Protein ◽  
Unconventional Myosin

In Saccharomyces cerevisiae, the unconventional myosin Myo2p is of fundamental importance in polarized growth. We explore the role of the neck region and its associated light chains in regulating Myo2p function. Surprisingly, we find that precise deletion of the six IQ sites in the neck region results in a myosin, Myo2-Δ6IQp, that can support the growth of a yeast strain at 90% the rate of a wild-type isogenic strain. We exploit this mutant in a characterization of the light chains of Myo2p. First, we demonstrate that the localization of calmodulin to sites of polarized growth largely depends on the IQ sites in the neck of Myo2p. Second, we demonstrate that a previously uncharacterized protein, Mlc1p, is a myosin light chain of Myo2p. MLC1 (YGL106w) is an essential gene that exhibits haploinsufficiency. Reduced levels of MYO2 overcome the haploinsufficiency of MLC1. The mutant MYO2-Δ6IQ is able to suppress haploinsufficiency but not deletion of MLC1. We used a modified gel overlay assay to demonstrate a direct interaction between Mlc1p and the neck of Myo2p. Overexpression of MYO2 is toxic, causing a severe decrease in growth rate. When MYO2 is overexpressed, Myo2p is fourfold less stable than in a wild-type strain. High copies of MLC1 completely overcome the growth defects and increase the stability of Myo2p. Our results suggest that Mlc1p is responsible for stabilizing this myosin by binding to the neck region.

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