scholarly journals Compound heterozygosity of the gamma-glutamyl carboxylase mutants V255M and S300F causes pseudoxanthoma elasticum-like disease through impaired processivity

2019 ◽  
Author(s):  
Mark A. Rishavy ◽  
Kevin W. Hallgren ◽  
Kurt W. Runge ◽  
Kathleen L. Berkner
Keyword(s):  
Protein Complexes ◽  
Pseudoxanthoma Elasticum ◽  
Factor Ix ◽  
Clotting Factor ◽  
Severe Bleeding ◽  
Compound Heterozygosity ◽  
Wild Type ◽  
Important Advance ◽  
Gamma Glutamyl ◽  
Gla Domain

ABSTRACTVitamin K-dependent (VKD) protein activities require carboxylated Glus (Glas) generated by the gamma-glutamyl carboxylase. Some carboxylase mutations cause severe bleeding, while others cause pseudoxanthoma elasticum (PXE)-like associated with excessive calcification. How carboxylase mutations cause PXE-like was unknown. We analyzed two mutants (V255M and S300F) whose compound heterozygosity causes PXE-like. Substrates derived from VKD proteins important to calcification (MGP) or clotting (factor IX) were studied, which contained the Gla domain and exosite-binding domain that mediates carboxylase binding. Surprisingly, the V255M mutant was more active (4-5 fold) than wild type carboxylase, while S300F activity was low. The V255M results suggested faster substrate release, which could impact carboxylase processivity, where the carboxylase remains bound to VKD proteins throughout multiple Glu to Gla conversions. To assess mutant processivity, we performed a novel challenge assay in which MGP-carboxylase and factor IX-carboxylase complexes were reacted in the presence of excess challenge VKD peptide. Tight complexes between VKD proteins and wild type carboxylase excluded access of the challenge peptide during the carboxylation of VKD protein in the complex. In contrast, VKD protein complexes with V255M or S300F allowed promiscuous access of challenge peptide. Both mutants therefore impair processivity. Most of the V255M product was carboxylated challenge peptide, which could explain mild PXE-like observed in the proband’s mother and aunt. Both have wild type and V255M carboxylase alleles; however, higher V255M production of a potentially defective MGP product could account for their phenotype. The results are an important advance in understanding why carboxylase mutations cause the PXE-like disease.

EXPRESSION IN MAMMALIAN CELLS OF FUSION PROTEINS BETWEEN HUMAN FACTORS IX AND VII

1987 ◽  
Author(s):  
K L Berkner ◽  
S J Busby ◽  
J Gambee ◽  
A Kumar
Keyword(s):  
Fusion Protein ◽  
Fusion Proteins ◽  
Factor Ix ◽  
Biologically Active ◽  
Factor Vii ◽  
Clotting Factor ◽  
Clotting Factors ◽  
Mammalian Expression ◽  
Plasma Factor ◽  
Gla Domain

The vitamin K-dependent plasma proteins demonstrate remarkable similarities in their structures: all have multiple domains in common and extensive homology is observed within many of these domains. In order to investigate the structure-function relationship of these proteins, we have interchanged domains of one protein (factor IX) with that of another (factor VII) and have compared the expression of these fusion proteins with recombinant and native factors IX and VII. Oligonucleotide-directed mutagenesis was used to generate four fusion proteins: factor IX/VII-1, which contains the factor IX leader and gla domain fused to the growth factor and serine protease of factor VII; factor VII/IX-1, a reciprocal fusion protein of factor IX/VII-1; factor IX/VII-2, which contains the factor IX leader adjoined to the mature factor VII protein sequence; and factor VII/IX-2, the reciprocal fusion protein of factor IX/VII-2. The cDNAs encoding all four proteins were cloned into mammalian expression vectors, and to date three of these (factors IX/VII-1, 2 and VII/IX-1) have been transfected into baby hamster kidney (BHK) cells or 293 cells and characterized. Factors IX/VII-1 and VII/IX-1 were both secreted at levels comparable to recombinant factors IX and VII. The factor IX/VII-1 was identical in molecular weight to native or recombinant factor VII (i.e., 53 K). Factor VII/IX-1 was expressed as two proteins with molecular weights around 68 kd, as observed with recombinant factor IX. The factor IX/VII-1 protein has been purified to homogeneity and has been found to possess factor VII biological activity, but at a specific activity approximately 20% that of plasma factor VII. Thus, the gla domain of one clotting factor is capable of directing the activation of another and of generating biologically active protein. In contrast, no activity was observed with the factor IX/VII-2 fusion protein, indicating that there are limits to the interchanges which can generate functional blood clotting factors.

An Analysis of Cleavage of the Factor IX Activation Sites by Factor XIa

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3088-3088 ◽  
Author(s):  
David Gailani ◽  
Stephen B. Smith ◽  
Sayeh Agah ◽  
S. Paul Bajaj
Keyword(s):  
Active Site ◽  
Factor Viia ◽  
Exchange Chromatography ◽  
Factor Ix ◽  
Severe Bleeding ◽  
Western Blots ◽  
Factor Xia ◽  
Gla Domain ◽  
Activation Peptide ◽  
Time Courses

Abstract During blood coagulation, the plasma zymogen factor IX (fIX) is converted to the active protease factor Ixaβ (fIXaβ). The severe bleeding disorder associated with deficiency of fIX (hemophilia B) attests to the importance of this protein in hemostasis. Conversion of fIX to fIXaβ requires two proteolytic cleavages after Arg145 and Arg180, releasing an activation peptide. This process is mediated by the proteases factor VIIa (fVIIa) and factor XIa (fXIa). FVIIa in complex with tissue factor initially cleaves fIX after Arg145 forming an intermediate, factor IXα (fIXα), which is then cleaved after Arg180 to form fIXaβ. Western blots of activation time courses demonstrate fIXα accumulation during this process, indicating cleavage at Arg180 is rate limiting. In contrast, little intermediate accumulation occurs during fIX activation by fXIa. Previously, we showed that fXIa also cleaves fIX initially after Arg145, generating fIXα (Smith et al., J. Biol. Chem.283;6696:2008). To account for the lack of intermediate accumulation, then, the subsequent cleavage after Arg180 must occur at least as rapidly as the initial cleavage. We examined the relative rates of conversion of fIX, fIXα, and the alternative intermediate factor IXaα (fIXaα - cleaved after Arg180) to fIXaβ by fXIa. FIXα or fIXaα were prepared from tritium-labeled fIX by incubation with fXIa-Pro192 (discussed below) or Russell’s Viper Venom protease, respectively, and purified by anion exchange chromatography. Conversion to fIXaβ was determined by measuring release of the tritiated activation peptide. FXIa converted fIX to fIXaβ with a kcat of 29.4 ± 0.4/min, a value reflecting cleavage at both activation sites. Kcat for conversion of fIXα and fIXaα to fIXaβ were 29.9 ± 0.5 and 30.0 ± 1.0/min, respectively. The rate of conversion of fIX to fIXα, estimated by measuring tritiated activation products separated by SDS-PAGE, was 30.0 ± 0.4/min. Recently, we showed that amino acid substitutions in fXIa for the conserved active site residue Gly193 (chymotrypsin numbering) decreased kcat for fIX activation 7–1000 fold, with residues with long branched side-chains having the greatest effect (Schmidt et al. Biochemistry47;1326:2008). Gly193 substitutions had a modestly larger detrimental effect (1.2–1.5 fold) on cleavage of fIX after Arg180 compared to Arg145 that was associated with varying degrees of fIXα accumulation. Similar effects were noted with substitutions for the adjacent residue Lys192. FXIa with Pro192 cleaved fIX after Arg180 >10-fold more slowly than after Arg145, generating fIXα with little subsequent conversion to fIXaβ. Cumulatively, these data support the premise that the rates for the two sequential reactions required for normal fIX activation by fXIa are comparable. Therefore, perturbations causing a greater effect on cleavage after Arg180 compared to Arg145, even if relatively small, result in fIXα accumulation. Initial recognition of fIX by fXIa involves substrate binding exosites distinct from the enzyme active site. At least one exosite appears to be located in the fXIa third apple (A3) domain, and may interact with an epitope on the fIX Ca2+-binding Gla-domain. The rate of fIX activation to fIXaβ by fXIa was significantly reduced in the presence of an antibody to the fXIa A3 domain or by mutations in the A3 domain. Similarly, rates of activation were decreased in the absence of Ca2+, in the presence of an antibody to the fIX Gla-domain, or when fIX with a decarboxylated Gla-domain was the substrate. In all cases, significant fIXα accumulation was noted in time courses, indicating that interfering with this particular substrate-exosite interaction has a significantly greater effect on cleavage after Arg180 than after Arg145. These findings raise the possibility that the exosite on fXIa A3 plays a larger role in conversion of fIXα to fIXaβ than in initial fIX conversion to fIXα, and are consistent with the possibility, recently proposed by Sinha et al. (Biochemistry46;9830:2007), that a second fIX binding exosite is present on fXIa.

Circulating and binding characteristics of wild-type factor IX and certain Gla domain mutants in vivo

Blood ◽  
2002 ◽  
Vol 100 (1) ◽  
pp. 153-158 ◽  
Author(s):  
Tong Gui ◽  
Hui-Feng Lin ◽  
Da-Yun Jin ◽  
Maureane Hoffman ◽  
David L. Straight ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Collagen Iv ◽  
Factor Ix ◽  
Blood Levels ◽  
Wild Type ◽  
Binding Characteristics ◽  
Gla Domain ◽  
Type Factor

Abstract Residue K5 in factor IX γ-carboxyglutamic acid (Gla) domain participates in binding endothelial cells/collagen IV. We injected recombinant factor IX containing mutations at residue 5 (K5A, K5R) into factor IX–deficient mice and compared their behavior with that of wild-type factor IX. The plasma concentration of factor IX that binds to endothelial cells/collagen IV (recombinant wild type and K5R) was consistently lower than that of the one that does not bind (K5A). Mice treated with wild type or K5R had 79% of the injected factor IX in the liver after 2 minutes, whereas 17% remained in circulation. In mice injected with K5A, 59% of the injected factor IX was found in liver and 31% was found in plasma. When we blocked the liver circulation before factor IX injection, 74% of K5A and 64% of K5R remained in the blood. When we treated the mouse with EDTA after injecting exogenous factor IX, the blood levels of factor IX that bind to endothelial cells/collagen IV increased, presumably because of release from endothelial cell/collagen IV binding sites. In contrast, the levels of the mutants that do not bind were unaffected by EDTA. In immunohistochemical studies, factor IX appears on the endothelial surfaces of mouse arteries after factor IX injection and of human arteries from surgical specimens. Thus, we have demonstrated that factor IX binds in vivo to endothelial cell–collagen IV surfaces. Our results suggest that factor IX Gla-domain mediated binding to endothelial cells/collagen IV plays a role in controlling factor IX concentration in the blood.

Inhibitor treatment by rituximabin congenital haemophilia A

2009 ◽  
Vol 29 (02) ◽  
pp. 151-154 ◽  
Author(s):  
Escuriola Ettingshausen ◽  
R. Linde ◽  
G. Kropshofer ◽  
L.-B. Zimmerhackl ◽  
W. Kreuz ◽  
...  
Keyword(s):  
B Cell ◽  
Immune Tolerance ◽  
High Titre ◽  
Clotting Factor ◽  
Severe Bleeding ◽  
High Morbidity ◽  
Haemophilia A ◽  
Concomitant Treatment ◽  
Bleeding Tendency ◽  
Severe Haemophilia

SummaryThe development of neutralizing alloanti-bodies (inhibitors) to factor VIII (FVIII) is one of the most serious complications in the treatment of haemophiliacs. Inhibitors occur in approximately 20 to 30% of previously untreated patients (PUPs), predominantly children, with severe haemophilia A within the first 50 exposure days (ED). Immune tolerance induction (ITI) leads to complete elimination of the inhibitor in up to 80% of the patients and offers the possibility to restore regular FVIII prophylaxis. However, patients with high titre inhibitors, in whom standard ITI fails, usually impose with high morbidity and mortality and therefore prompting physicians to alternate therapy regimens. Rituximab, an anti-CD 20 monoclonal antibody has been successfully used in children and adults for the management of B-cell mediated disorders. We report on the use of a new protocol including rituximab in two adolescents with severe haemophilia A and high titre inhibitors, severe bleeding tendency and high clotting factor consumption after failing standard ITI. Both patients received a concomitant treatment with FVIII according to the Bonn protocol, cyclosporine A and immunoglobulin. Treatment with rituximab resulted in a temporary B-cell depletion leading to the disappearance of the inhibitor. FVIII recovery and half-life turned towards normal ranges. In patient 1 the inhibitor reappeared 14 months after the last rituximab administration. In patient 2 complete immune tolerance could be achieved for 60 months. Bleeding frequency diminished significantly and clinical joint status improved in both patients. In patient 1 the treatment course was complicated by aspergillosis and hepatitis B infection. Conclusion: Rituximab may be favourable for patients with congenital haemophilia, high-titre inhibitors and a severe clinical course in whom standard ITI has failed. Prospective studies are required to determine safety, efficacy and predictors of success.

Clinical Factors Associated with Progression to AIDS in the Italian Cohort of HIV-Positive Hemophiliacs

1994 ◽  
Vol 72 (01) ◽  
pp. 033-038 ◽  
Author(s):  
N Schinaia ◽  
A M G Ghirardini ◽  
M G Mazzucconi ◽  
G Tagariello ◽  
M Morfini ◽  
...  
Keyword(s):  
Cumulative Incidence ◽  
National Registry ◽  
Factor Ix ◽  
Clotting Factor ◽  
Coagulation Disorders ◽  
Factors Associated ◽  
Kaplan Meier ◽  
Significant Difference ◽  
Congenital Coagulation Disorders ◽  
Clotting Factor Concentrates

SummaryThis study updates estimates of the cumulative incidence of AIDS among Italian patients with congenital coagulation disorders (mostly hemophiliacs), and elucidates the role of age at seroconversion, type and amount of replacement therapy, and HBV co-infection in progression. Information was collected both retrospectively and prospectively on 767 HIV-1 positive patients enrolled in the on-going national registry of patients with congenital coagulation disorders. The seroconversion date was estimated as the median point of each patient’s seroconversion interval, under a Weibull distribution applied to the overall interval. The independence of factors associated to faster progression was assessed by multivariate analysis. The cumulative incidence of AIDS was estimated using the Kaplan-Meier survival analysis at 17.0% (95% Cl = 14.1-19.9%) over an 8-year period for Italian hemophiliacs. Patients with age greater than or equal to 35 years exhibited the highest cumulative incidence of AIDS over the same time period, 32.5% (95% Cl = 22.2-42.8%). Factor IX recipients (i.e. severe B hemophiliacs) had higher cumulative incidence of AIDS (23.3% vs 14.2%, p = 0.01) than factor VIII recipients (i.e. severe A hemophiliacs), as did severe A hemophiliacs on less-than-20,000 IU/yearly of plasma-derived clotting factor concentrates, as opposed to A hemophiliacs using an average of more than 20,000 IU (18.8% vs 10.9%, p = 0.02). No statistically significant difference in progression was observed between HBsAg-positive vs HBsAg-negative hemophiliacs (10.5% vs 16.4%, p = 0.10). Virological, immunological or both reasons can account for such findings, and should be investigated from the laboratory standpoint.

Mutations which Introduce Free Cysteine Residues in the Gla-Domain of Vitamin K Dependent Proteins Result in the Formation of Complexes with α1-Microglobulin

1996 ◽  
Vol 75 (01) ◽  
pp. 070-075 ◽  
Author(s):  
E G C Wojcik ◽  
P Simioni ◽  
M v d Berg ◽  
A Girolami ◽  
R M Bertina
Keyword(s):  
Vitamin K ◽  
Protein C ◽  
Cysteine Residue ◽  
Factor Ix ◽  
Disulphide Bond ◽  
Clotting Factors ◽  
High Molecular Weight Complex ◽  
Low Concentrations ◽  
Gla Domain ◽  
Formation Of Complexes

SummaryWe have previously described a genetic factor IX variant (Cys18→Arg) for which we demonstrated that it had formed a heterodimer with armicroglobulin through formation of a disulphide bond with the remaining free cysteine residue of the disrupted disulphide bond in the Gla-domain of factor IX. Recently, we observed a similar high molecular weight complex for a genetic protein C variant (Arg-1→Cys). Both the factor IX and the protein C variants have a defect in the calcium induced conformation. In this study we show that the aminoterminus of this protein C variant is prolonged with one amino acid, cysteine. This protein C variant, as well as protein C variants with Arg9→Cys and Ser12→Cys mutations which also carry a free cysteine residue, are shown to be present in plasma as a complex with α1-microglobulin. A prothrombin variant with a Tyr44→Cys mutation, had not formed such a complex. Furthermore, complexes between normal vitamin K-dependent clotting factors and α1-microglobulin were shown to be present in plasma at low concentrations. The data suggest that the presence of an unpaired cysteine residue in the propeptide or the N-terminal half of the Gla-domain has strongly promoted the formation of a complex with α1-microglobulin in the variants.

The Effect of Adrenaline Infusions on Blood Coagulation in Normal and Haemophilia B Dogs

1966 ◽  
Vol 15 (03/04) ◽  
pp. 349-364 ◽  
Author(s):  
A.H Özge ◽  
H.C Rowsell ◽  
H.G Downie ◽  
J.F Mustard
Keyword(s):  
Body Weight ◽  
Factor Viii ◽  
Factor Ix ◽  
Clotting Factor ◽  
Blood Ph ◽  
Order Of Magnitude ◽  
Dose Dependent ◽  
Generation Test ◽  
Plasma Activity

SummaryThe addition of trace amounts of adrenaline to whole blood in plasma in vitro increased factor VIII, factor IX and whole plasma activity in the thromboplastin generation test. This was dose dependent.Adrenaline infusions less than 22 (μg/kg body weight in normal dogs accelerated clotting, increased factor IX, factor VIII and whole plasma activity in the thromboplastin generation test and caused a fall in blood pH. In a factor IX deficient dog, there was no increase in factor IX activity. After adrenaline infusions, however, the other changes occurred and were of the same order of magnitude as in the normal. Adrenaline in doses greater than 22 μg/kg body weight did not produce as great an effect on clotting in normal or factor IX deficient dogs. The platelet count in the peripheral blood was increased following the infusion of all doses of adrenaline. These observations suggest that the accelerating effect of adrenaline on clotting is not mediated through increase in activity of a specific clotting factor.

Inherent hepatocytic heterogeneity determines expression and retention of edited F9 alleles post-AAV/CRISPR infusion

2021 ◽  
Vol 118 (42) ◽  
pp. e2110887118
Author(s):  
Qiang Wang ◽  
Lin Zhang ◽  
Guo-Wei Zhang ◽  
Jian-Hua Mao ◽  
Xiao-Dong Xi ◽  
...  
Keyword(s):  
Viral Vectors ◽  
Gene Editing ◽  
Regulatory Region ◽  
Factor Ix ◽  
Host Cells ◽  
Clotting Factor ◽  
Dependent Manner ◽  
Coding Region ◽  
Therapeutic Benefits

Infusing CRISPR/donor-loaded adeno-associated viral vectors (AAV/CRISPR) could enable in vivo hepatic gene editing to remedy hemophilia B (HB) with inherited deficiency of clotting factor IX (FIX). Yet, current regimens focus on correcting HB with simple mutations in the coding region of the F9, overlooking those carrying complicated mutations involving the regulatory region. Moreover, a possible adverse effect of treatment-related inflammation remains unaddressed. Here we report that a single DNA cutting-mediated long-range replacement restored the FIX-encoding function of a mutant F9 (mF9) carrying both regulatory and coding defects in a severe mouse HB model, wherein incorporation of a synthetic Alb enhancer/promoter-mimic (P2) ensured FIX elevation to clinically meaningful levels. Through single-cell RNA sequencing (scRNA-seq) of liver tissues, we revealed that a subclinical hepatic inflammation post-AAV/CRISPR administration regulated the vulnerability of the edited mF9-harboring host cells to cytotoxic T lymphocytes (CTLs) and the P2 activity in a hepatocytic subset–dependent manner via modulating specific sets of liver-enriched transcription factors (LETFs). Collectively, our study establishes an AAV/CRISPR-mediated gene-editing protocol applicable to complicated monogenetic disorders, underscoring the potentiality of improving therapeutic benefits through managing inflammation.

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