A Monoclonal Antibody Recognizing an Iscosapeptide Sequence in the Heavy Chain of Human Factor XII Inhibits Surface-Catalyzed Activation

Pixley, Robin A.; Stumpo, L G; Birkmeyer, K.; Silver, Lee D.; Colman, Robert W.

doi:10.1007/978-1-4615-9543-4_72

Cited by 29 publications

(34 citation statements)

References 10 publications

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“…Human studies show enzymatic inhibition properties using both agents. 16,28 When the purified and concentrated fractions of FXII protein from normal cats were added to a homozygous cat plasma pool, the APTT was corrected to near-normal values (from >200 seconds corrected to 33.6-43.1 seconds), indicating that the purified FXII was intact and active (data not shown). Testing of the mutant factor XII protein isolated from deficient cat plasma revealed no correction of the APTT (>200 after adding purified mutant FXII), suggesting this abnormal protein lacks FXII catalytic activity reflective of a primary defect in FXII protein (Suppl .…”

Section: Resultsmentioning

confidence: 95%

“…To define the functional enzymatic activity of normal and mutant feline FXII, we performed plasma mixing studies using human prekallikrein-deficient (PKd) plasma (Sigma Diagnostics, St Louis, MO) and FXII-deficient plasma (George King Biomedicals, Overland Park, KS), corn trypsin inhibitor (CTI; Enzyme Research Laboratories, South Bend, IN), and a monoclonal mouse anti-human FXII heavy chain antibody, B7C9, directed to amino acids p.I19-V38 and p.T153-R172 of the human FXII protein 5,28 (a gift from Dr Robin Pixley) with cat plasmas prior to assaying coagulant activity using the Stago ST4 and the modified APTT procedure. Human PKd plasma was used as a diluent instead of a HZY cat plasma pool to assess for a prekallikrein deficiency in our colony.…”

Section: Protein Isolation and Identification Techniquesmentioning

confidence: 99%

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Molecular Characterization of Cat Factor XII Gene and Identification of a Mutation Causing Factor XII Deficiency in a Domestic Shorthair Cat Colony

et al. 2014

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Coagulation factor XII (FXII) may be important in cardiovascular and inflammatory diseases. We have identified and characterized a naturally occurring mutation in the feline FXII gene that results in a mutant protein and enzymatic loss of activity. Feline intron/ exon gene structure and sequence were acquired by comparing DNA sequences obtained from a fragmented Felis catus genomic sequence and the National Center for Biotechnology Information's Cross Species Megablast of multiple species' FXII gene sequences. Fourteen exons ranging in size from 57 to 222 base pairs were confirmed spanning 8 Kb on chromosome A1. The 1828-base pair feline FXII messenger RNA (mRNA) sequence contains an open reading frame that encodes a protein of 609 amino acids with high homology to human FXII protein. Total RNA and mRNA purified from liver tissue of 4 wild-type/normal and 8 FXII-deficient cats confirmed the predicted mRNA sequence and identified one important single-nucleotide polymorphism (SNP). A single base deletion in exon 11 of the FXII coding gene in our colony of cats results in deficient FXII activity. Translation of the mRNA transcript shows a frame shift at L441 (C441fsX119) resulting in a nonsense mutation and a premature stop codon with a predicted 560-amino acid protein. The mutant FXII protein is truncated in the 3 0 proteolytic light chain region of the Cterminus, explaining its loss of enzymatic activity. This study is the first molecular characterization of the feline FXII gene and the first identification of an FXII mutation in the domestic cat, providing insights into the origin and nature of feline FXII deficiency.Keywords coagulation, technology, cat, domestic mammals, species, factor XII, FXII, Hageman factor, gene, mutation, mRNA Deficiency of factor XII (FXII or Hageman factor) was first described in 1955 in a human patient who lacked plasma coagulant activity. 31 In the intervening time, FXIIs from multiple species have been genetically characterized and various mutations have been reported in humans. Although FXII deficiency does not present as a bleeding tendency, recent evidence indicates FXII may be important in maintaining blood clot stability and pregnancy. 22,25,32 A feline FXII-deficient case was first described in 1977, 10 and later Kier et al 18 established a colony in the early 1980s to study FXII deficiency.Today, these offspring continue to provide information about the in vivo role of FXII and are the source of this study's molecular characterization of a mutation in the FXII gene with a similar autosomal recessive inheritance pattern and coagulation profile as seen in humans. Studies in human populations indicate that the prevalence of moderate to severe FXII deficiency is between 2% and 5% of a given white population.11 Similar to humans, the prevalence of FXII deficiency in domestic cats of the United States is 2%. 3Factor XII is primarily produced in the liver and circulates in the plasma as an inactive precursor enzyme or zymogen. 40The FXII protein is a 2-chain molecule consis...

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Section: Resultsmentioning

confidence: 95%

Section: Protein Isolation and Identification Techniquesmentioning

confidence: 99%

Molecular Characterization of Cat Factor XII Gene and Identification of a Mutation Causing Factor XII Deficiency in a Domestic Shorthair Cat Colony

et al. 2014

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“…Several other domains, located at the N-terminal end, may have regulatory functions and comprise a leader peptide, a fibronectin-11 domain, a growth-factor-like domain, a fibronectin-I domain, a second growth-factor-like domain, a kringle domain and a proline-rich region which is unique to this protein. The functions of these domains have not been explored in relation to the multiple physiological roles of FXII and can only be assigned by analogy with similar domains of other serine proteases (Cool et al, 1985;Chien et al, 1988;Pixley et a]., 1987;Clarke et al, 1989). Their importance for the activation of the contact phase of blood coagulation is inferred from the inability of a fragment of FXII, comprising only the catalytic region and nine amino acids of the FXII heavy chain (the FXII fragment, also known as P-FXII), to bind to negative charges and to efficiently promote blood clotting (Kaplan and Silver ber g , 1 98 7).…”

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confidence: 99%

Control of human coagulation by recombinant serine proteases

Citarella

Aiuti

Porta

et al. 1992

European Journal of Biochemistry

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The availability of engineered serine proteases allows one to study the activation, substrate specificity and regulation of human coagulation and fibrinolytic activities. Human coagulation factor XI1 is composed of the protease catalytic region at the C-terminus, a hinge proline-rich region and regulatory domains at the N-terminus. From cDNA clones coding for factor XII, two DNA molecules were constructed, one being full length and the other being deleted of exons coding for the regulatory domains. Engineered factor-XI1 cDNA species were inserted by a homologous recombination technique into vaccinia viruses. which were used to infect the human hepatoma cell line HepG2. Two recombinant proteins were prepared from the culture media and identified by their antigenic properties and electrophoretic mobilities. The recombinant protein of larger size was identified as the full-length factor XI1 of 80 kDa and its specific activities and activation patterns, determined both by the coagulation and the amidolytic assays, are very similar to these of native human factor XII. The recombinant protein of smaller size was identified as a 319-amino-acid-deleted factor-XI1 protein of 32 kDa, containing only the entire protease region and part of the proline-rich hmge. This protein was expected to be the 'minimal' portion of Factor XI1 able to sustain protease activity, but unable to recognize substrates and surfaces necessary to activate the contact phase of coagulation. However, this 'minimal' Factor-XI1 protein displays a marked protease activity and, although lacking five regulatory domains of factor XLI, is bound and activated by negative charges and promotes coagulation with high efficiency.Events relevant to cellular and extracellular processes are regulated by the sequential enzymatic activation of proteins. One example of this is protein kinase activity leading to the regulation of important functions in the cell cycle and differentiation. Cascades of serine protease activities bring into effect and regulate extracellular processes of great importance to vertebrate organisms, such as blood coagulation, fibrinolysis and inflammation. The multiple and composite functions of serine proteases may be ascribed to structural domains able to recognize activating surfaces, inhibitors, cofactors and other serine proteases. The present study is aimed at producing and characterizing recombinant (r) human factor XI1 (FXII) proteins in order to clarify the functional role of the different domains composing human FXII.

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“…Several monoclonal antibodies that recognize different regions of FXII were used to purify, quantify, and characterize the recombinant FXII proteins: mAbs F1, KOKS, and B7C9, which are directed against epitopes localized on the heavy-chain region of FXII, and mAbs OT2 and C6B7 recognizing epitopes on the light-chain region. mAbs B7C9 and C6B7 have been described before [27,301 and were kindly provided by Dr Robin A. Pixley (Temple University, Philadelphia, PA). mAb F1 also has been described previously 1311. mAb OT2 binds to an epitope near the catalytic centre on the light-chain region, since it inhibits the amidolytic activity of FXIIa [32], and, when coupled to Sepharose beads, is able to bind '*'I-PFXIIa (see Results).…”

Section: Methodsmentioning

confidence: 99%

“…Increasing evidence suggests that, in general, activation of serine proteases is regulated by their structural domains [22-241. However, the precise role of the domains of FXII is unknown. The only functional site on the heavy chain of FXII identified so far, is the putative binding site for negatively charged surfaces, which has been mapped at the Nterminus of FXII and presumably consists of the sequences covering residues 1-28 [25, 261 and 134-153 [27] which belongs to the fibronectin type I domain. Our work is aimed at testing this hypothesis by the use of recombinant FXII proteins lacking these sequences.…”

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confidence: 99%

Structure/Function Analysis of Human Factor XII Using Recombinant Deletion Mutants

Citarella

Ravon

Pascucci

et al. 1996

European Journal of Biochemistry

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The binding site of human factor XI1 (FXII) for negatively charged surfaces has been proposed to be localized in the N-terminal region of factor XII. We have generated two recombinant factor XI1 proteins that lack this region : one protein consisting of the second growth-factor-like domain, the kringle domain, the proline-rich region and the catalytic domain of FXII (rFXII-U-like), and another consisting of only 16 amino acids of the proline-rich region of the heavy-chain region and the catalytic domain (rFXII-lpc). Each recombinant truncated protein, as well as recombinant full-length FXII (rFXII), were produced in HepG2 cells and purified by immunoaffinity chromatography. The capability of these recombinant proteins to bind to negatively charged surfaces and to initiate contact activation was studied. Radiolabeled rFXII-U-like and, to a lesser extent, rFXTT-lpc bound to glass in a concentration-dependent manner, yet with lower efficiency than rFXII. The binding of the recombinant proteins was inhibited by a 100-fold molar excess of non-labeled native factor XII. On native polyacrylamide gel electrophoresis, both truncated proteins appeared to bind also to dextran sulfate, a soluble negatively charged compound. Glassbound rFXII-U-like was able to activate prekallikrein in FXII-deficient plasma (assessed by measuring the generation of kallikrein-C1-inhibitor complexes), but less efficiently than rFXII. rFXIT-U-like and rFXII-lpc exhibited coagulant activity, but this activity was significantly lower than that of rFXII. These data confirm that the N-terminal part of the heavy-chain region of factor XI1 contains a binding site for negatively charged activating surfaces, and indicate that other sequences, possibly located on the second epidermal-growth-factor-like domain and/or the kringle domain, contribute to the binding of factor XI1 to these surfaces.

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A Monoclonal Antibody Recognizing an Iscosapeptide Sequence in the Heavy Chain of Human Factor XII Inhibits Surface-Catalyzed Activation

Cited by 29 publications

References 10 publications

Molecular Characterization of Cat Factor XII Gene and Identification of a Mutation Causing Factor XII Deficiency in a Domestic Shorthair Cat Colony

Molecular Characterization of Cat Factor XII Gene and Identification of a Mutation Causing Factor XII Deficiency in a Domestic Shorthair Cat Colony

Control of human coagulation by recombinant serine proteases

Structure/Function Analysis of Human Factor XII Using Recombinant Deletion Mutants

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