In vitro production of coenzyme A using thermophilic enzymes

Coenzyme A (CoA) is an essential cofactor present in all domains of life and is involved in numerous metabolic pathways, including fatty acid metabolism, pyruvate oxidation through the TCA cycle, and production of secondary metabolites. This characteristic makes CoA a commercially valuable compound in the pharmaceutical, cosmetic, and clinical industries. However, CoA is difficult to accumulate in living cells at a high level as it is consumed in multiple metabolic pathways, hampering its manufacturing by typical cell cultivation and extraction approaches. The feedback inhibition by CoA to a biosynthetic enzyme, pantothenate kinase (PanK), is also a serious obstacle for high-titer production of CoA. To overcome this challenge, in vitro production of CoA, in which the CoA biosynthetic pathway was reconstructed outside of cells using recombinant thermophilic enzymes, was performed. The in vitro pathway was designed to be insensitive to the feedback inhibition of CoA using a CoA-insensitive type-III PanK from the thermophilic bacterium Thermus thermophilus. Furthermore, a statistical approach using Design of Experiments was employed to rationally determine the enzyme loading ratio to maximize CoA production rate. Consequently, 0.94 mM CoA could be produced from 2 mM d-pantetheine through the designed pathway. We hypothesized that the insufficient conversion yield is attributed to the high Km value of T. thermophilus PanK towards ATP. Based on these observations, possible CoA regulation mechanisms in T. thermophilus and approaches to improve the feasibility of CoA production through the in vitro pathway have been investigated. IMPORTANCE The biosynthesis of coenzyme A (CoA) in bacteria and eukaryotes is regulated by feedback inhibition targeting type-I and type-II pantothenate kinase (PanK). Type-III PanK is only found in bacteria and is generally insensitive to CoA. Previously, type-III PanK from the hyperthermophilic bacterium Thermotoga maritima was shown to defy this typical characteristic, and instead shows inhibition towards CoA. In the present study, phylogenetic analysis combined with functional analysis of type-III PanK from thermophiles revealed that the CoA-sensitive behavior of type-III PanK from T. maritima is uncommon. We cloned type-III PanKs from Thermus thermophilus and Geobacillus sp. 30 and showed that neither enzyme’s activities were inhibited by CoA. Furthermore, we utilized type-III PanK for a one-pot cascade reaction to produce CoA.

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Regulation of Coenzyme A Biosynthesis in the Hyperthermophilic Bacterium Thermotoga maritima

Journal of Bacteriology ◽

10.1128/jb.00077-16 ◽

2016 ◽

Vol 198 (14) ◽

pp. 1993-2000 ◽

Cited By ~ 2

Author(s):

Takahiro Shimosaka ◽

Hiroya Tomita ◽

Haruyuki Atomi

Keyword(s):

Coenzyme A ◽

Feedback Inhibition ◽

Thermotoga Maritima ◽

Single Type ◽

Type I ◽

Type Ii ◽

Pantothenate Kinase ◽

Content Type ◽

Type Iii ◽

Hyperthermophilic Bacterium

ABSTRACTRegulation of coenzyme A (CoA) biosynthesis in bacteria and eukaryotes occurs through feedback inhibition targeting type I and type II pantothenate kinase (PanK), respectively. In contrast, the activity of type III PanK is not affected by CoA. As the hyperthermophilic bacteriumThermotoga maritimaharbors only a single type III PanK (Tm-PanK), here we examined the mechanisms that regulate CoA biosynthesis in this organism. We first examined the enzyme responsible for the ketopantoate reductase (KPR) reaction, which is the target of feedback inhibition in archaea. A classical KPR homolog was not present on theT. maritimagenome, but we found a homolog (TM0550) of the ketol-acid reductoisomerase (KARI) fromCorynebacterium glutamicum, which exhibits KPR activity. The purified TM0550 protein displayed both KPR and KARI activities and was designatedTm-KPR/KARI. WhenT. maritimacell extract was subjected to anion-exchange chromatography, the fractions containing high levels of KPR activity also displayed positive signals in a Western blot analysis using polyclonal anti-TM0550 protein antisera, strongly suggesting thatTm-KPR/KARI was the major source of KPR activity in the organism. The KPR activity ofTm-KPR/KARI was not inhibited in the presence of CoA. We thus examined the properties ofTm-PanK and the pantothenate synthetase (Tm-PS) of this organism.Tm-PS was not affected by CoA. Surprisingly however,Tm-PanK was inhibited by CoA, with almost complete inhibition in the presence of 400 μM CoA. Our results suggest that CoA biosynthesis inT. maritimais regulated by feedback inhibition targeting PanK, althoughTm-PanK is a type III enzyme.IMPORTANCEBacteria and eukaryotes regulate the biosynthesis of coenzyme A (CoA) by feedback inhibition targeting type I or type II pantothenate kinase (PanK). The hyperthermophilic bacteriumThermotoga maritimaharbors a single type III PanK (Tm-PanK), previously considered to be unaffected by CoA. By examining the properties of three enzymes involved in CoA biosynthesis in this organism, we found thatTm-PanK, although a type III enzyme, is inhibited by CoA. The results provide a feasible explanation of how CoA biosynthesis is regulated inT. maritima, which may also apply for other bacteria that harbor only type III PanK enzymes.

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Crystal Structure of a Type III Pantothenate Kinase: Insight into the Mechanism of an Essential Coenzyme A Biosynthetic Enzyme Universally Distributed in Bacteria

Journal of Bacteriology ◽

10.1128/jb.00469-06 ◽

2006 ◽

Vol 188 (15) ◽

pp. 5532-5540 ◽

Cited By ~ 27

Author(s):

Kun Yang ◽

Yvonne Eyobo ◽

Leisl A. Brand ◽

Dariusz Martynowski ◽

Diana Tomchick ◽

...

Keyword(s):

Crystal Structure ◽

Active Site ◽

Coenzyme A ◽

Feedback Inhibition ◽

Thermotoga Maritima ◽

Type I ◽

Pantothenate Kinase ◽

E Coli ◽

Type Iii ◽

Sugar Kinase

ABSTRACT Pantothenate kinase (PanK) catalyzes the first step in the five-step universal pathway of coenzyme A (CoA) biosynthesis, a key transformation that generally also regulates the intracellular concentration of CoA through feedback inhibition. A novel PanK protein encoded by the gene coaX was recently identified that is distinct from the previously characterized type I PanK (exemplified by the Escherichia coli coaA-encoded PanK protein) and type II eukaryotic PanKs and is not inhibited by CoA or its thioesters. This type III PanK, or PanK-III, is widely distributed in the bacterial kingdom and accounts for the only known PanK in many pathogenic species, such as Helicobacter pylori, Bordetella pertussis, and Pseudomonas aeruginosa. Here we report the first crystal structure of a type III PanK, the enzyme from Thermotoga maritima (PanKTm), solved at 2.0-Å resolution. The structure of PanKTm reveals that type III PanKs belong to the acetate and sugar kinase/heat shock protein 70/actin (ASKHA) protein superfamily and that they retain the highly conserved active site motifs common to all members of this superfamily. Comparative structural analysis of the PanKTm active site configuration and mutagenesis of three highly conserved active site aspartates identify these residues as critical for PanK-III catalysis. Furthermore, the analysis also provides an explanation for the lack of CoA feedback inhibition by the enzyme. Since PanK-III adopts a different structural fold from that of the E. coli PanK—which is a member of the “P-loop kinase”superfamily—this finding represents yet another example of convergent evolution of the same biological function from a different protein ancestor.

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The 2′-O-methylation status of a single guanosine controls transfer RNA–mediated Toll-like receptor 7 activation or inhibition

Journal of Experimental Medicine ◽

10.1084/jem.20111075 ◽

2012 ◽

Vol 209 (2) ◽

pp. 235-241 ◽

Cited By ~ 61

Author(s):

Stefanie Jöckel ◽

Gernot Nees ◽

Romy Sommer ◽

Yang Zhao ◽

Dmitry Cherkasov ◽

...

Keyword(s):

Influenza A ◽

Mononuclear Cells ◽

Human Peripheral Blood ◽

Thermus Thermophilus ◽

Methylation Status ◽

Transfer Rna ◽

Type I ◽

Peripheral Blood Mononuclear ◽

Molecular Pattern

Foreign RNA serves as pathogen-associated molecular pattern (PAMP) and is a potent immune stimulator for innate immune receptors. However, the role of single bacterial RNA species in immune activation has not been characterized in detail. We analyzed the immunostimulatory potential of transfer RNA (tRNA) from different bacteria. Interestingly, bacterial tRNA induced type I interferon (IFN) and inflammatory cytokines in mouse dendritic cells (DCs) and human peripheral blood mononuclear cells (PBMCs). Cytokine production was TLR7 dependent because TLR7-deficient mouse DCs did not respond and TLR7 inhibitory oligonucleotides inhibited tRNA-mediated activation. However, not all bacterial tRNA induced IFN-α because tRNA from Escherichia coli Nissle 1917 and Thermus thermophilus were non-immunostimulatory. Of note, tRNA from an E. coli knockout strain for tRNA (Gm18)-2′-O-methyltransferase (trmH) regained immunostimulatory potential. Additionally, in vitro methylation of this immunostimulatory Gm18-negative tRNA with recombinant trmH from T. thermophilus abolished its IFN-α inducing potential. More importantly, Gm18-modified tRNA acted as TLR7 antagonist and blocked IFN-α induction of influenza A virus–infected PBMCs.

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Bacterial Enzymes Catalyzing the Synthesis of 1,8-Dihydroxynaphthalene, a Key Precursor of Dihydroxynaphthalene Melanin, fromSorangium cellulosum

Applied and Environmental Microbiology ◽

10.1128/aem.00258-18 ◽

2018 ◽

Vol 84 (9) ◽

Cited By ~ 8

Author(s):

Yusuke Sone ◽

Shuto Nakamura ◽

Makoto Sasaki ◽

Fumihito Hasebe ◽

Seung-Young Kim ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Optical Purity ◽

Brown Pigment ◽

Type I ◽

Enzymatic System ◽

Type Iii Polyketide Synthase ◽

Content Type ◽

Type Iii ◽

Bacterial Origin

ABSTRACT1,8-Dihydroxynaphthalene (1,8-DHN) is a key intermediate in the biosynthesis of DHN melanin, which is specific to fungi. In this study, we characterized the enzymatic properties of the gene products of an operon consisting ofsoceCHS1,bdsA, andbdsBfrom the Gram-negative bacteriumSorangium cellulosum. Heterologous expression ofsoceCHS1,bdsA, andbdsBinStreptomyces coelicolorcaused secretion of a dark-brown pigment into the broth. High-performance liquid chromatography (HPLC) analysis of the broth revealed that the recombinant strain produced 1,8-DHN, indicating that the operon encoded a novel enzymatic system for the synthesis of 1,8-DHN. Simultaneous incubation of the recombinant SoceCHS1, BdsA, and BdsB with malonyl-coenzyme A (malonyl-CoA) and NADPH resulted in the synthesis of 1,8-DHN. SoceCHS1, a type III polyketide synthase (PKS), catalyzed the synthesis of 1,3,6,8-tetrahydroxynaphthalene (T4HN)in vitro. T4HN was in turn converted to 1,8-DHN by successive steps of reduction and dehydration, which were catalyzed by BdsA and BdsB. BdsA, which is a member of the aldo-keto reductase (AKR) superfamily, catalyzed the reduction of T4HN and 1,3,8-tetrahydroxynaphthalene (T3HN) to scytalone and vermelone, respectively. The stereoselectivity of T4HN reduction by BdsA occurred on thesi-face to give (R)-scytalone with more than 99% optical purity. BdsB, a SnoaL2-like protein, catalyzed the dehydration of scytalone and vermelone to T3HN and 1,8-DHN, respectively. The fungal pathway for the synthesis of 1,8-DHN is composed of a type I PKS, naphthol reductases of the short-chain dehydrogenase/reductase (SDR) superfamily, and scytalone dehydratase (SD). These findings demonstrated 1,8-DHN synthesis by novel enzymes of bacterial origin.IMPORTANCEAlthough the DHN biosynthetic pathway was thought to be specific to fungi, we discovered novel DHN synthesis enzymes of bacterial origin. The biosynthesis of bacterial DHN utilized a type III PKS for polyketide synthesis, an AKR superfamily for reduction, and a SnoaL2-like NTF2 superfamily for dehydration, whereas the biosynthesis of fungal DHN utilized a type I PKS, SDR superfamily enzyme, and SD-like NTF2 superfamily. Surprisingly, the enzyme systems comprising the pathway were significantly different from each other, suggesting independent, parallel evolution leading to the same biosynthesis. DHN melanin plays roles in host invasion and adaptation to stress in pathogenic fungi and is therefore important to study. However, it is unclear whether DHN biosynthesis occurs in bacteria. Importantly, we did find that bacterial DHN biosynthetic enzymes were conserved among pathogenic bacteria.

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Heavily Armed Ancestors: CRISPR Immunity and Applications in Archaea with a Comparative Analysis of CRISPR Types in Sulfolobales

Biomolecules ◽

10.3390/biom10111523 ◽

2020 ◽

Vol 10 (11) ◽

pp. 1523

Author(s):

Isabelle Anna Zink ◽

Erika Wimmer ◽

Christa Schleper

Keyword(s):

Comparative Analysis ◽

Molecular Mechanisms ◽

Model Organisms ◽

Special Focus ◽

Natural Environments ◽

Type I ◽

Accessory Proteins ◽

Type Iii

Prokaryotes are constantly coping with attacks by viruses in their natural environments and therefore have evolved an impressive array of defense systems. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is an adaptive immune system found in the majority of archaea and about half of bacteria which stores pieces of infecting viral DNA as spacers in genomic CRISPR arrays to reuse them for specific virus destruction upon a second wave of infection. In detail, small CRISPR RNAs (crRNAs) are transcribed from CRISPR arrays and incorporated into type-specific CRISPR effector complexes which further degrade foreign nucleic acids complementary to the crRNA. This review gives an overview of CRISPR immunity to newcomers in the field and an update on CRISPR literature in archaea by comparing the functional mechanisms and abundances of the diverse CRISPR types. A bigger fraction is dedicated to the versatile and prevalent CRISPR type III systems, as tremendous progress has been made recently using archaeal models in discerning the controlled molecular mechanisms of their unique tripartite mode of action including RNA interference, DNA interference and the unique cyclic-oligoadenylate signaling that induces promiscuous RNA shredding by CARF-domain ribonucleases. The second half of the review spotlights CRISPR in archaea outlining seminal in vivo and in vitro studies in model organisms of the euryarchaeal and crenarchaeal phyla, including the application of CRISPR-Cas for genome editing and gene silencing. In the last section, a special focus is laid on members of the crenarchaeal hyperthermophilic order Sulfolobales by presenting a thorough comparative analysis about the distribution and abundance of CRISPR-Cas systems, including arrays and spacers as well as CRISPR-accessory proteins in all 53 genomes available to date. Interestingly, we find that CRISPR type III and the DNA-degrading CRISPR type I complexes co-exist in more than two thirds of these genomes. Furthermore, we identified ring nuclease candidates in all but two genomes and found that they generally co-exist with the above-mentioned CARF domain ribonucleases Csx1/Csm6. These observations, together with published literature allowed us to draft a working model of how CRISPR-Cas systems and accessory proteins cross talk to establish native CRISPR anti-virus immunity in a Sulfolobales cell.

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Separation of the Binding Step from the Collagen Platelet Reaction

10.1055/s-0039-1682724 ◽

1977 ◽

Author(s):

P.L. Kronick ◽

S.A. Jimenez

Keyword(s):

Type I Collagen ◽

Specific Activity ◽

Adenine Nucleotides ◽

High Specific Activity ◽

Type Iii Collagen ◽

Type I ◽

Type Iii ◽

Valid Comparison ◽

Platelet Reaction

Determination of activity of most agents in stimulating platelets to aggregate or release adenine nucleotides is conveniently done by titrating the platelet reaction with the agent. Platelets have previously been titrated with different types of collagen (types I, II, and III) in this way to compare the activities of the collagens. It has been concluded that the order of activity is type III>I>II. Whether this order is due to differences in binding was not obvious from these experiments because the binding was not determined directly. We have developed a method of comparing activities by measuring the targeted dose for each point in the titration - the amount of collagen which actually binds to platelets. The collagens used in these experiments were prepared in vitro from embryonic chick tissue to give labelled products of extremely high specific activity without structural alteration. We find that type I collagen is at least 20 times as active as previously reported, and that the activity of Type III collagen is not significantly higher when the amounts bound are taken into account. The fraction of the labelled tendon collagen which was bound to platelets was identified as type I by its hydroxyproline/proline ratio. Direct measurement of the bound fraction in dose-response studies is required for valid comparison of collagen activities.

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Assessment of Mycobacterium tuberculosis Pantothenate Kinase Vulnerability through Target Knockdown and Mechanistically Diverse Inhibitors

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00140-14 ◽

2014 ◽

Vol 58 (6) ◽

pp. 3312-3326 ◽

Cited By ~ 12

Author(s):

B. K. Kishore Reddy ◽

Sudhir Landge ◽

Sudha Ravishankar ◽

Vikas Patil ◽

Vikas Shinde ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Present Report ◽

Kinetic Studies ◽

Antimycobacterial Activity ◽

Type I ◽

Pantothenate Kinase ◽

Content Type ◽

Rate Limiting Step

ABSTRACTPantothenate kinase (PanK) catalyzes the phosphorylation of pantothenate, the first committed and rate-limiting step toward coenzyme A (CoA) biosynthesis. In our earlier reports, we had established that the type I isoform encoded by thecoaAgene is an essential pantothenate kinase inMycobacterium tuberculosis, and this vital information was then exploited to screen large libraries for identification of mechanistically different classes of PanK inhibitors. The present report summarizes the synthesis and expansion efforts to understand the structure-activity relationships leading to the optimization of enzyme inhibition along with antimycobacterial activity. Additionally, we report the progression of two distinct classes of inhibitors, the triazoles, which are ATP competitors, and the biaryl acetic acids, with a mixed mode of inhibition. Cocrystallization studies provided evidence of these inhibitors binding to the enzyme. This was further substantiated with the biaryl acids having MIC against the wild-typeM. tuberculosisstrain and the subsequent establishment of a target link with an upshift in MIC in a strain overexpressing PanK. On the other hand, the ATP competitors had cellular activity only in aM. tuberculosisknockdown strain with reduced PanK expression levels. Additionally,in vitroandin vivosurvival kinetic studies performed with aM. tuberculosisPanK (MtPanK) knockdown strain indicated that the target levels have to be significantly reduced to bring in growth inhibition. The dual approaches employed here thus established the poor vulnerability of PanK inM. tuberculosis.

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The use of naturally occurring hybrid variants of chloramphenicol acetyltransferase to investigate subunit contacts

Biochemical Journal ◽

10.1042/bj1930541 ◽

1981 ◽

Vol 193 (2) ◽

pp. 541-552 ◽

Cited By ~ 7

Author(s):

L C Packman ◽

W V Shaw

Keyword(s):

Chloramphenicol Acetyltransferase ◽

Alpha Subunit ◽

Type I ◽

Chloramphenicol Resistance ◽

Type Iii ◽

Naturally Occurring ◽

Alpha Beta ◽

Beta 2

1. Hybrids of the tetrameric enzyme chloramphenicol acetyltransferase (EC 2.3.1.28) were formed in vivo in a strain of Escherichia coli which harbours two different plasmids, each of which normally confers chloramphenicol resistance and specifies an easily distinguished enzyme variant (type I or type III) which is composed of identical subunits. Cell-free extracts of the dual-plasmid strain were found to contain five species of active enzyme, two of which were the homomeric enzymes corresponding to the naturally occurring tetramers of the type-I (beta 4) and type-III (alpha 4) enzymes. The other three variants were judged to be the heteromeric hybrid variants (alpha 3 beta, alpha 2 beta 2, alpha beta 3). 2. The alpha 3 beta and alpha 2 beta 2 hybrids of chloramphenicol acetyltransferase were purified to homogeneity by combining the techniques of affinity and ion-exchange chromatography. The alpha beta 3 variant was not recovered and may be unstable in vitro. 3. The unique lysine residues that could not be modified with methyl acetimidate in each of the native homomeric enzymes were also investigated in the heteromeric tetramers. 4. Lysine-136 remains buried in each beta subunit of the parental (type I) enzyme and in each of the hybrid tetramers. Lysine-38 of each alpha subunit is similarly unreactive in the native type-III chloramphenicol acetyltransferase (alpha 4), but in the alpha 2 beta 2 hybird lysine-38 of each alpha subunit is fully exposed to solvent. Another lysine residue, fully reactive in the alpha 4 enzyme, was observed to be inaccessible to modification in the symmetrical hybrid. The results obtained for the alpha 3 beta enzyme suggest that lysine-38 in two subunits and a different lysine group (that identified in the alpha 2 beta 2 enzyme) in the third alpha subunit are buried. 5. A tentative model for the subunit interactions of chloramphenicol acetyltransferase is proposed on the basis of the results described.

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Lack of BIC and microRNA miR-155 Expression in Primary Cases of Burkitt Lymphoma.

Blood ◽

10.1182/blood.v106.11.1922.1922 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1922-1922

Author(s):

Joost Kluiver ◽

Eugenia Haralambieva ◽

Debora de Jong ◽

Tjasso Blokzijl ◽

Susan Jacobs ◽

...

Keyword(s):

Hodgkin Lymphoma ◽

Cell Lines ◽

Burkitt Lymphoma ◽

High Expression ◽

Common Finding ◽

Type I ◽

Pediatric Leukemia ◽

Normal Peripheral Blood ◽

Type Iii

Abstract We previously demonstrated a high expression of primary-microRNA BIC (pri-miRNA-155) in Hodgkin lymphoma (HL) and lack of expression in most non-Hodgkin lymphoma subtypes including some Burkitt lymphoma (BL) cases. Recently, a high expression of BIC was reported in BL in comparison to pediatric leukemia and normal peripheral blood samples. In this study we extended our series of BL cases and cell lines for BIC expression by RNA in-situ hybridization (ISH) and quantitative (q)RT-PCR. Both BIC RNA-ISH and qRT-PCR revealed no or only low levels of BIC in 25 BL tissues, including 7 Epstein-Barr virus (EBV) positive cases, compared to HL and normal controls. In agreement with these findings, Northern blotting revealed absence of miR-155 in BL tissues. EBV negative and EBV latency type I BL cell lines also showed very low BIC and miR-155 expression levels as compared to HL cell lines. Higher levels of BIC and miR-155 were detected in in vitro transformed lymphoblastoid EBV latency type III BL cell lines. An association of latency type III infection and induction of BIC was supported by consistent expression of BIC in 11 and miR-155 in 2 posttransplantation lymphoproliferative disorder (PTLD) cases. In summary, we demonstrated that expression of BIC and miR-155 is not a common finding in BL. Expression of BIC and miR-155 in 3 latency type III EBV positive BL cell lines and in all primary PTLD cases suggests a possible role for EBV latency type III specific proteins in the induction of BIC expression.

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A Subset of High Titer Acquired Hemophilia Patients May Benefit from Treatment with High Dose Factor VIII.

Blood ◽

10.1182/blood.v114.22.3476.3476 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3476-3476

Author(s):

Shannon Meeks ◽

John F Healey ◽

Ernest T Parker ◽

Pete Lollar

Keyword(s):

Factor Viii ◽

Hemophilia A ◽

High Titer ◽

High Dose ◽

Type I ◽

C2 Domain ◽

Type Ii ◽

Acquired Hemophilia ◽

Proteolytic Activation

Abstract Abstract 3476 Poster Board III-413 Approximately 30% of patients with severe hemophilia A will develop inhibitory antibodies (Abs) to factor VIII (fVIII inhibitors). The immune response to fVIII currently is the most significant complication in the management of patients with hemophilia A. In addition, autoimmune Abs to fVIII can develop in non-hemophiliacs, producing acquired hemophilia A, which frequently produces life- or limb-threatening bleeding. Patients with autoimmune hemophilia often have Abs with type II kinetics in which there is incomplete inactivation of fVIII at saturating concentrations of inhibitor. We have characterized the antibody response to the C2 domain of human fVIII in a murine hemophilia model and described 5 structural groups of Abs. Groups A, AB, and B are classical anti-C2 Abs that block fVIII and fVIIIa binding to phospholipid. Groups BC and C consist of non-classical anti-C2 Abs that inhibit the proteolytic activation of fVIII but do not block the binding of fVIII to phospholipid. Subsequently, we identified classical and non-classical anti-C2 Abs in human fVIII inhibitor plasmas. Most murine non-classical Abs have inhibitor titers greater than 10,000 Bethesda units/mg IgG. In a murine in vivo bleeding model, both type I classical C2 Abs, type II non-classical C2 Abs, and a type I anti-A2 Ab produced similar amounts of blood loss that were significantly greater than control mice injected with 180 U/kg of fVIII alone. Increasing the dose of fVIII to 360 U/kg overcame the bleeding diathesis produced by the type II MAbs, but not the type I Abs. These results were consistent with the in vitro Bethesda assay in which a type I anti-A2 Ab, 4A4, completely inhibited both 1 U/mL and 3 U/mL fVIII, while there was 40% residual activity at saturating concentrations of a type II anti-C2 Ab, 2-77, at either concentration of fVIII. To determine if similar in vitro characteristics exist in patients with acquired hemophilia, plasmas from 3 patients with high titer type II inhibitors were studied. All 3 plasmas primarily had C2 domain epitope specificity that included non-classical Abs. Plasma A7 additionally had detectable anti-A2 activity. Recovery of fVIII activity after a 2 h incubation at 37 °C at nominal added concentrations of 1 mL and 3 U/mL fVIII was compared (Table 1). At 3 U/mL added fVIII, recovery of activity in plasmas A4 and A5 was 1.1 U/mL and 0.51 U/mL, respectively, despite the presence of inhibitor titers of 18 and 11 Bethesda units (BU) per mL. The presence of anti-A2 Abs, which typically have type I kinetics, may have contributed to the overall lower recovery of activity in plasma A7. These results suggest that treatment with high-dose fVIII rather than bypassing agents may be warranted in patients with an inhibitor response dominated by non-classical anti-C2 Abs. Table 1 Patient Plasma Inhibitor Titer (BU/mL) Recovered Activity at 1U/mL FVIII (U/mL) Recovered Activity at 3 U/mL FVIII (U/mL) A4 18 0.31 1.1 A5 11 0.18 0.51 A7 62 0.07 0.12 Disclosures: No relevant conflicts of interest to declare.

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