scholarly journals Characterization of the catalytic subunit of factor XIII by radioimmunoassay

Blood ◽  
1982 ◽  
Vol 60 (5) ◽  
pp. 1089-1095
Author(s):  
C Skrzynia ◽  
HM Reisner ◽  
J McDonagh
Keyword(s):  
Factor Xiii ◽  
Protein A ◽  
Catalytic Subunit ◽  
Molecular Forms ◽  
Plasma Factor ◽  
Correlate Factor ◽  
Polyclonal Antisera ◽  
High Concentrations ◽  
A Chain

Plasma factor XIII is composed of two subunits, a and b, whereas platelet and other intracellular zymogens have only a-subunits. The catalytic subunit, a, is the same in all forms. In order to characterize the interactions of 1- with b-chains in the plasma zymogen and a-chains with other molecules and to correlate factor XIII activity with a-protein, a specific, sensitive radioimmunoassay was developed. With the polyclonal antisera used, the assay recognizes all molecular forms of a (zymogens, activation intermediates, enzyme) equally well. The assay can be used to determine a-chain concentration in plasma and serum and in purified test systems. Fibrinogen in high concentrations affects the assay, probably by interfering with the interactions of 125I-a with antibody. However, at the plasma dilutions used in the assay, there is no significant fibrinogen effect. With this assay, the a-chain concentration in normal plasma is approximately 15 micrograms/ml. This compares with 14 micrograms/ml b-chain in plasma and indicates that all of the a- and b-chains in plasma probably circulate in the form of an equimolar zymogen complex. The serum concentration of a-protein is about 6% of the plasma concentration. There is a high correlation between a-protein and factor XIII activity.

scholarly journals Characterization of the catalytic subunit of factor XIII by radioimmunoassay

Blood ◽  
1982 ◽  
Vol 60 (5) ◽  
pp. 1089-1095 ◽  
Author(s):  
C Skrzynia ◽  
HM Reisner ◽  
J McDonagh
Keyword(s):  
Factor Xiii ◽  
Protein A ◽  
Catalytic Subunit ◽  
Molecular Forms ◽  
Plasma Factor ◽  
Correlate Factor ◽  
Polyclonal Antisera ◽  
High Concentrations ◽  
A Chain

Abstract Plasma factor XIII is composed of two subunits, a and b, whereas platelet and other intracellular zymogens have only a-subunits. The catalytic subunit, a, is the same in all forms. In order to characterize the interactions of 1- with b-chains in the plasma zymogen and a-chains with other molecules and to correlate factor XIII activity with a-protein, a specific, sensitive radioimmunoassay was developed. With the polyclonal antisera used, the assay recognizes all molecular forms of a (zymogens, activation intermediates, enzyme) equally well. The assay can be used to determine a-chain concentration in plasma and serum and in purified test systems. Fibrinogen in high concentrations affects the assay, probably by interfering with the interactions of 125I-a with antibody. However, at the plasma dilutions used in the assay, there is no significant fibrinogen effect. With this assay, the a-chain concentration in normal plasma is approximately 15 micrograms/ml. This compares with 14 micrograms/ml b-chain in plasma and indicates that all of the a- and b-chains in plasma probably circulate in the form of an equimolar zymogen complex. The serum concentration of a-protein is about 6% of the plasma concentration. There is a high correlation between a-protein and factor XIII activity.

scholarly journals Monoclonal Antibody to Noncatalytic Subunit of Bovine Plasma Factor XIII: Selective Isolation of Catalytic Subunit

1987 ◽  
Vol 51 (2) ◽  
pp. 523-530
Author(s):  
Koji Ikura ◽  
Hiroshi Sakurai ◽  
Hiroyuki Yokota ◽  
Ryuzo Sasaki ◽  
Hideo Chiba
Keyword(s):  
Monoclonal Antibody ◽  
Factor Xiii ◽  
Catalytic Subunit ◽  
Selective Isolation ◽  
Bovine Plasma ◽  
Plasma Factor

RAPID FORMATION OF LARGE MOLECULAR WEIGHT O-P0LYMERS IN CROSSLINKED FIBRIN INDUCED BY HIGH FACTOR XIII CONCENTRATIONS: ROLE OF PLATELET FACTOR XIII.

1987 ◽  
Author(s):  
C W Francis ◽  
V J Marder
Keyword(s):  
Factor Xiii ◽  
Factor Xiiia ◽  
Freezing And Thawing ◽  
Platelet Factor ◽  
Polymer Formation ◽  
Wide Range ◽  
Sequential Addition ◽  
High Concentrations ◽  
A Chain

Following fibrin polymerization, activated factor XIII stabilizes the clot by catalyzing the formation of specific intermolecular covalent crosslinks between pairs of y chains to form dimers and also among two or more a chains to form polymers. We have identified a series of previously uncharacterized a chain polymers with a wide range of sizes, including some with apparent Mr in excess of several million. Additionally, we establish the role of high concentrations of factor XIII in the extent and rate of α-polymer formation and provide evidence that the factor XIII required can be provided by platelets. Using SDS gel electrophoresis, we find that fibrin prepared from purified fibrinogen or from platelet-deficient plasma contains a series of 21 factor XIIIa crosslinked a chain polymers with Mr from 140,000 to 770,000. The mean Mr difference between individual polymers of 32,000 is consistent with a staggered, overlapping sequential addition of monomers to the growing α-polymer chain. In plasma containing no platelets, α-polymer formation was incomplete with residual α-monomer remaining. Progressively higher platelet counts facilitated more rapid crosslinking of a chains into larger polymers. Intact platelets were not required to promote crosslinking, since platelets lysed by freezing and thawing were also effective. Enrichment of plasma with placental factor XIII in an amount equal to that contained in platelets was as effective as platelets in accelerating the rate of formation and increasing the size of α-chain polymers. We conclude that platelets are a principal source of factor XIII for maximal fibrin stabilization, providing a larger quantity than is available from plasma alone and regulating both the rate and extent of α-polymer formation in thrombi or hemostatic plugs at sites of vascular injury.

Characteristics of Platelet Protransglutaminase (Factor XIII)-Binding Activity in Human Plasma

1975 ◽  
Author(s):  
D. Bannerjee ◽  
M. W. Mosesson
Keyword(s):  
Human Plasma ◽  
Ammonium Sulfate ◽  
Factor Xiii ◽  
Binding Activity ◽  
Platelet Factor ◽  
Catalytic Potential ◽  
Plasma Factor ◽  
Exclusion Chromatography ◽  
A Chain ◽  
Polypeptide Chains

Human plasma Factor XIII (F. XIII) complex is composed of two types of noncovalently linked polypeptide chains termed a and b; only the a chain possesses catalytic potential. Platelet Factor XIII is comprised solely of a chains which are identical to those found in plasma. In this study platelets were utilized as a source of unbound a chains to characterize F. XIII (a chain)-binding activity in plasma and its subfractions. Upon exclusion chromatography of unheated plasma or of an unheated ammonium sulfate (20% sat.) subfraction, F. XIII activity emerged in a peak corresponding to a mol. wt. of < 500,000 (region 1). If these samples had first been heated at 60° to precipitate fibrinogen, F. XIII was eluted in a peak corresponding to a mol wt. of about 300,000 (region 2). Chromatography of the platelet zymogen alone resulted in a F. XIII peak corresponding in position to that of monomeric a chain (mol. wt. 80,000, region 3).Exclusion chromatography of the unheated ammonium sulfate fraction yielded, in addition to the F. XIII peak in region 1, a protein peak (peak II) in region 2 which contained no F. XIII. When peak II was mixed with platelet F. XIII, and again subjected to exclusion chromatography, the platelet F. XIII peak shifted from its expected position in region 3 and emerged instead in region 2 ; this behavior demonstrated F. XIII (a chain)-binding activity within peak II. The same chromatographic shift was observed in mixtures of platelet F. XIII and normal plasma or that from a patient with congenital F. XIII (a chain) deficiency. Immunochemical analyses of chromatographic fractions indicated that a chain-binding was due to complexing of a chains with freely circulating b chains. Since a chains and b chains have different biosynthetic sites we conclude that b chains serve as an extracellular F. XIII (a chain)-binding protein.Supported by NHLI grant HL-11409.

Fatal Bleeding Caused by Congenital Factor XIII Deficiency and Development of An Inhibitor in a Hispanic Male

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4515-4515
Author(s):  
Filipe R. Lorenzo ◽  
Derrick Haslem ◽  
Josef T. Prchal ◽  
Charles Greenberg
Keyword(s):  
Factor Xiii ◽  
Factor Xiiia ◽  
Bleeding Complications ◽  
Factor Xiii Deficiency ◽  
Catalytic Core ◽  
Fatal Bleeding ◽  
Plasma Factor ◽  
A Chain ◽  
Hispanic Male ◽  
Congenital Factor

Abstract We report a case of fatal bleeding in a patient with mild congenital Factor XIII deficiency who also developed an inhibitor that interfered with fibrin stabilization. Plasma Factor XIII circulates as a tetramer composed of two A-chain and two B-chains and is bound to fibrinogen. After thrombin cleaves the A-chain at Arg 37, the B-chain dissociate producing Factor XIIIa. Factor XIIIa catalyzes covalent linkages between the fibrin molecules aligned in the fibrin clot. Factor XIIIa makes the fibrin resistant to disruption by urea and plasmin. Factor XIIIa levels greater than 1 % are needed to covalently stabilize a clot. We studied a 39 year old Hispanic male that presented with altered mental status, headache and a large left frontal parietal hemorrhage. Ultimately, he became non-responsive and was intubated. He had no major bleeding history but did have frequent nosebleeds. Routine blood counts and coagulation laboratory tests were normal. However, a qualitative Factor XIII assay was abnormal and the clot dissolved in 5M Urea. Furthermore, a 1:1 mixing study did not correct the defect suggesting the presence of an inhibitor. A small dose of cryoprecipitate was administered to the patient and this corrected the clot stability defect. Four days later, the defect recurred and 150 ml of cryoprecipitate was administered and despite correction of the fibrin stabilizing abnormality the patient died. Two of his sisters had a history of repeated hemorrhagic miscarriages in Mexico a finding consistent with congenital Factor XIII deficiency. DNA sequencing of the factor XIII a-chain gene was performed from the propositus’ mononuclear cells. A reverse transcription was done using an oligo(dT) 12–18 primer followed by nested PCR amplification and a heterozygous missense mutation was observed at codon 35 (G226T; Val35Leu). This mutation was than confirmed in propositus’ platelet cDNA. The Val35Leu substitution is close to the thrombin cleavage site, the G226T substitution is in the catalytic core domain. This case demonstrates that in some cases of Factor XIII deficiency there is residual Factor XIIIa activity in the mutant molecule that can prevent serious bleeding. However, patients may develop autoantibodies that further interfere with Factor XIII function and may suffer serious bleeding complications. Additional cases of Factor XIII deficiency may exist in patients that have mild bleeding problems.

Purification and Immunochemical Characterization of Human Plasma Factor XIII

1976 ◽  
Vol 5 (6) ◽  
pp. 329-340 ◽  
Author(s):  
M. Kazama ◽  
Jan McDonagh ◽  
R.H. Wagner ◽  
R.D. Langdell ◽  
R.P. McDonagh
Keyword(s):  
Human Plasma ◽  
Factor Xiii ◽  
Immunochemical Characterization ◽  
Plasma Factor

Serological characterization of surface proteins of Spiroplasma citri

1991 ◽  
Vol 37 (1) ◽  
pp. 28-33 ◽  
Author(s):  
Jacqueline Fletcher ◽  
Chandi Wijetunga
Keyword(s):  
Protein A ◽  
Surface Proteins ◽  
Spiroplasma Citri ◽  
Phase Partitioning ◽  
Serological Characterization ◽  
Polypeptide Patterns ◽  
Polyclonal Antisera ◽  
Hydrophilic Treatment ◽  
Triton X

Four surface proteins of the phytopathogenic Mollicute Spiroplasma citri were characterized using monospecific polyclonal antisera. Antiserum specific for P29 (spiralin) caused growth inhibition, spiral deformation, and metabolism inhibition, while anti-P89, anti-P77, anti-P58, and preimmune sera did not. In Triton X-114 phase partitioning, P29, P89, and one of two P77 bands were hydrophobic, indicating the probability that they are integral membrane proteins. P58 partitioned into the hydrophilic phase and may be an extrinsic membrane protein. A second band which reacts with anti-P77 serum was also hydrophilic. Treatment of the two P77 components with trypsin and chymotrypsin resulted in very different polypeptide patterns, suggesting that the two bands represent unrelated proteins. These results demonstrate the variability in hydrophobicity among surface-exposed proteins in S. citri and describe procedures that should prove useful for characterizing and separating other proteins of interest in spiroplasmas. Key words: Spiroplasma, mollicute, surface proteins, serology, Triton X-114.

Characterization of the kinetic pathway for fibrin promotion of .alpha.-thrombin-catalyzed activation of plasma factor XIII

Biochemistry ◽  
1991 ◽  
Vol 30 (4) ◽  
pp. 934-941 ◽  
Author(s):  
Michael C. Naski ◽  
Laszlo Lorand ◽  
Jules A. Shafer
Keyword(s):  
Factor Xiii ◽  
Plasma Factor

Characterization of a Monoclonal Antibody Directed Against the Carboxyl-Terminus of Human Factor XIII

1994 ◽  
Vol 71 (01) ◽  
pp. 062-067 ◽  
Author(s):  
Yeqing Song ◽  
Stephen M Taubenfeld ◽  
DeQuan Sheng ◽  
Gary R Matsueda
Keyword(s):  
Monoclonal Antibody ◽  
Factor Xiii ◽  
Human Factor ◽  
Carboxyl Terminus ◽  
Heat Denaturation ◽  
Structural Element ◽  
Assay Format ◽  
Plasma Factor ◽  
Carboxyl Terminal

SummaryBy deriving an anti-peptide monoclonal antibody, mAb 7A4, we characterized the relatively unstudied carboxyl-terminal end of the α-chain of human factor XIII, the plasma transglutaminase. MAb 7A4 was directed against the last eight amino acids (Gln-Ile-Gln-Arg-Arg-Pro-Ser-Met) and bound with a dissociation constant of 3.4 × 10−8 M. In a solid assay format, mAb 7A4 bound equally well to factor XIII obtained from human plasma, platelets or placenta. However, in a solution-phase assay format, the epitope was largely unavailable but could be readily exposed by heat denaturation. Tmmunoblotting showed that this epitope is conserved among all species of plasma factor XIII tested except rabbit suggesting that the carboxyl-terminus might be an important structural element. Other competitive binding experiments with synthetic peptides as inhibitors pointed toward the final carboxyl-terminal amino acid, Met-731, as an immunochemically important determinant. This was used advantageously to confirm the finding that the caiboxyl-leiminal Mel-731 is largely absent from placental factor XIII (1) as compared to platelet or plasma factor XIII.

scholarly journals Non-proteolytic activation of cellular protransglutaminase (placenta macrophage factor XIII)

1990 ◽  
Vol 267 (2) ◽  
pp. 557-560 ◽  
Author(s):  
J Polgár ◽  
V Hidasi ◽  
L Muszbek
Keyword(s):  
Factor Xiii ◽  
Specific Activity ◽  
Activation Process ◽  
Proteolytic Activation ◽  
Plasma Factor ◽  
Nacl Concentration ◽  
Catalytically Active ◽  
Thrombin Activation ◽  
A Subunit ◽  
High Concentrations

Plasma Factor XIII is a zymogen (plasma protransglutaminase) with the tetrametric structure A2B2, whereas the cellular protransglutaminase, i.e. Factor XIII in the platelet and monocyte/macrophage, consists exclusively of A subunits (A2). It is generally accepted that at Ca2+ concentrations comparable with that in plasma the proteolytic removal of an N-terminal activation peptide is the prerequisite for the Ca2(+)-induced formation of a catalytically active configuration of subunit A. In this study it was demonstrated that at high concentrations NaCl or KCl induced a non-proteolytic activation of cellular (placental macrophage) but not plasma protransglutaminase. The activation depended on time and salt concentration, and Ca2+, in the range 0-20 mM, greatly enhanced the activation process. At 1.25 M-NaCl maximal activation occurred within 60 min in the presence of 2 mM-CaCl2, and even at physiological NaCl concentration a slow progressive activation could be observed in the presence of Ca2+. The specific activity of salt-activated Factor XIII was 1.5-2.0-fold higher than that obtained after thrombin activation. The non-proteolytic activation of cellular protransglutaminase was abolished by the addition of subunit B of plasma Factor XIII in stoichiometric amount, which suggests that (one of) the physiological function(s) of the B subunit in plasma Factor XIII is to prevent the slow spontaneous activation of A subunit that would occur in a plasmatic environment.

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