scholarly journals Protein-protein interactions in blood clotting. The use of polarization of fluorescence to measure the dissociation of plasma factor XIIIa

1978 ◽  
Vol 169 (2) ◽  
pp. 403-410 ◽  
Author(s):  
J M Freyssinet ◽  
B A Lewis ◽  
J J Holbrook ◽  
J D Shore
Keyword(s):  
Protein Interactions ◽  
Factor Xiii ◽  
Protein Concentration ◽  
Fluorescein Isothiocyanate ◽  
Biochemical Properties ◽  
Factor Xiiia ◽  
Protein Protein Interactions ◽  
Plasma Factor ◽  
Polarization Of Fluorescence ◽  
A Subunit

1. Human plasma Factor XIII (the precursor of fibrin-glutamine-fibrin-lysine endo-gamma-glutamyltransferase) was randomly labelled by incubation with fluorescein isothiocyanate. The biochemical properties of the system were unaltered by the label. The polarization of the fluorescein fluorescence attached to the plasma protein was measured and the following conclusions were reached. 2. Factor XIII (a'2b2) does not dissociate in the protein-concentration range 10-500 microgram/ml either with or without added Ca2+. 3. Factor XIIIa (a'2b2) does not dissociate in the absence of Ca2+ in the protein-concentration range 10-500 microgram/ml. 4. Additions of Ca2+ to Factor XIIIa result in a decreased polarization of fluorescence as the tetramer dissociates. The decrease in polarization was the same amplitude at protein concentrations 10-500 microgram/ml and Ca2+ concentrations 2-66 mM and indicates that the overall process is essentially irreversible. The decrease in polarization consisted of fast and slow exponential phases. Both the rate of the fast phase and the proportion of the reaction it represented increased with Ca2+ concentration. 5. A comparison of the rate of dissociation measured by fluorescence polarization and the rate of appearance of enzyme activity in the presence of a protein substrate suggests that the Factor XIII is autoactivated by a soluble a-subunit-containing molecular forming a tight complex with the substrate.

scholarly journals Val34Leu polymorphism of plasma factor XIII: biochemistry and epidemiology in familial thrombophilia

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2479-2486 ◽  
Author(s):  
István Balogh ◽  
Gabriella Szôke ◽  
Levente Kárpáti ◽  
Ulla Wartiovaara ◽  
Éva Katona ◽  
...  
Keyword(s):  
Protective Effect ◽  
Venous Thrombosis ◽  
Factor Xiii ◽  
Coagulation Factor ◽  
Wild Type ◽  
Thrombin Cleavage ◽  
Plasma Factor ◽  
Thrombin Activation ◽  
A Subunit ◽  
The Mean

Abstract Val34Leu polymorphism of the A subunit of coagulation factor XIII (FXIII-A) is located in the activation peptide (AP) just 3 amino acids away from the thrombin cleavage site. This mutation has been associated with a protective effect against occlusive arterial diseases and venous thrombosis; however, its biochemical consequences have not been explored. In the current study it was demonstrated that the intracellular stability and the plasma concentration of FXIII of different Val34Leu genotypes are identical, which suggests that there is no difference in the rate of synthesis and externalization of wild-type and mutant FXIII-A. In contrast, the release of AP by thrombin from the Leu34 allele proceeded significantly faster than from its wild-type Val34 counterpart. By molecular modeling larger interaction energy was calculated between the Leu34 variant and the respective domains of thrombin than between the Val34 variant and thrombin. In agreement with these findings, the activation of mutant plasma FXIII by thrombin was faster and required less thrombin than that of the wild-type variant. Full thrombin activation of purified plasma FXIII of different genotypes, however, resulted in identical specific transglutaminase activities. Similarly, the mean specific FXIII activity in the plasma was the same in the groups with wild-type, heterozygous, and homozygous variants. Faster activation of the Leu34 allele hardly could be associated with its presumed protective effect against venous thrombosis. No such protective effect was observed in a large group of patients with familial thrombophilia.

scholarly journals Four novel mutations in deficiency of coagulation factor XIII: consequences to expression and structure of the A-subunit

Blood ◽  
1996 ◽  
Vol 87 (1) ◽  
pp. 141-151 ◽  
Author(s):  
H Mikkola ◽  
VC Yee ◽  
M Syrjala ◽  
R Seitz ◽  
R Egbring ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Nonsense Mutation ◽  
Factor Xiii ◽  
Three Dimensional ◽  
Factor Xiiia ◽  
Missense Mutations ◽  
Dimensional Model ◽  
Three Dimensional Model ◽  
Core Domain ◽  
A Subunit

Abstract The characterization of naturally occurring mutations is one way to approach functionally significant domains of polypeptides. About 10 mutations have been reported in factor XIII (FXIII) A-subunit deficiency, but very little is known about the effects of the mutations on the expression or the structure of this enzyme. In this study, the recent crystallization of FXIII A-subunit and determination of the three-dimensional model were used for the first time to pursue the structural consequences of mutations in the A-subunit. The molecular analysis of four families from Sweden, Germany, and Denmark revealed four previously unreported point mutations. Three of the mutations were missense mutations, Arg326-->Gln, Arg252-->Ile, and Leu498-->Pro, and one was a nonsense mutation, a deletion of thymidine in codon for Phe8 resulting in early frameshift and premature termination of the polypeptide chain. In the case of the nonsense mutation, delT Phe8, the steady-state mRNA level of FXIII A-subunit was reduced, as quantitated by reverse transcriptase-polymerase chain reaction and solid-phase minisequencing. In contrast, none of the missense mutations affected mRNA levels, indicating the possible translation of the mutant polypeptides. However, by enzyme-linked immunosorbent analysis and immunofluorescence, all the patients demonstrated a complete lack of detectable factor XIIIA antigen in their platelets. In the structural analysis, we included the mutations described in this work and the Met242-->Thr mutation reported earlier by us. Interestingly, in the three-dimensional model, all four missense mutations are localized in the evolutionarily conserved catalytic core domain. The substitutions are at least 15 A away from the catalytic cleft and do not affect any of the residues known to be directly involved in the enzymatic reaction. The structural analyses suggest that the mutations are most likely interfering with proper folding and stability of the protein, which is in agreement with the observed absence of detectable FXIIIA antigen. Arg326, Arg252, and Met242 are all buried within the molecule. The Arg326-->Gln and Arg252-->Ile mutations are substitutions of smaller, neutral amino acids for large, charged residues. They disrupt the electrostatic balance and hydrogen-bonding interactions in structurally significant areas. The Met242-->Thr mutation is located in the same region of the core domain as the Arg252-->Ile site and is expected to have a destabilizing effect due to an introduction of a smaller, polar residue in a tightly packed hydrophobic pocket. The substitution of proline for Leu498 is predicted to cause unfavorable interatomic contacts and a disruption of the alpha-helix mainchain hydrogen-bonding pattern; it is likely to form a kink in the helix next to the dimer interface and is expected to impair proper dimerization of the A-subunits. In the case of all four missense mutations studied, the knowledge achieved from the three-dimensional model of crystallized FXIII A-subunit provides essential information about the structural significance of the specific residues and aids in understanding the biologic consequences of the mutations observed at the cellular level.

scholarly journals Calcium-induced dissociation of human plasma factor XIII and the appearance of catalytic activity

1974 ◽  
Vol 141 (3) ◽  
pp. 683-691 ◽  
Author(s):  
Rodney D. Cooke
Keyword(s):  
Steady State ◽  
Protein Concentration ◽  
Factor Xiiia ◽  
Lag Phase ◽  
Conformation Change ◽  
Platelet Factor ◽  
Plasma Enzyme ◽  
Plasma Factor ◽  
Steady State Rate ◽  
State Rate

1. The Ca2+dependence of the activity of plasma Factor XIIIa was studied by using the continuous assay based on the incorporation of dansylcadaverine into dephosphorylated acetylated β-casein (β-substrate). The Km for Ca2+is about 0.170mm. 2. At low concentrations of Ca2+there was a lag in attaining the steady-state rate. The size of the lag was decreased and eventually abolished if the enzyme was preincubated with a high concentration of Ca2+before assay. The concentration of Ca2+required to decrease the lag phase by 50% in 10min depended on the protein concentration: at 0.87mg of protein/ml it required 17mm-Ca2+and at 0.44mg/ml it needed 10mm-Ca2+. 3. The concentrations of Ca2+required either to abolish the lag phase in the appearance of enzyme activity or to activate the essential thiol for reaction with 5,5′-dithiobis-(2-nitrobenzoate) in 10min incubation were similar at the same protein concentration. This indicated that Ca2+induces a conformation change that is responsible for both phenomena. A model is proposed that links this conformation change to the dissociation of the tetrameric enzyme. 4. This was supported by the observation that the addition of excess of b chains to the Factor XIIIa (a′2b2) increased the concentration of Ca2+required to expose the reactive thiol, and inhibited the Ca2+-dependent aggregation of a′ chains. 5. Platelet Factor XIIIa (a′2) was inhibited by 5,5′-dithiobis-(2-nitrobenzoate) in the absence of Ca2+, and no lag phases were observed in attaining the steady-state rate at low Ca2+concentrations, thus confirming the model for the activation of the plasma enzyme. 6. The Ca2+dependence of platelet Factor XIIIa indicated that Ca2+has an additional role in the enzyme mechanism of the plasma enzyme, perhaps being involved in substrate binding. 7. The dependence of the stability of plasma Factor XIIIa on Ca2+and protein concentration indicates that the decay in activity is related to the tetramer dissociation. 8. β-Substrate decreased the Ca2+concentration required for (1) abolition of the lag phase and (2) enzyme inhibition by thiol reagents. The effect on the former is greater than on the latter. 9. The role of the b chains of the plasma Factor and the evolutionary significance of the plasma and platelet Factors are considered.

scholarly journals Regulation of plasma factor XIII binding to fibrin in vitro

Blood ◽  
1985 ◽  
Vol 66 (5) ◽  
pp. 1028-1034 ◽  
Author(s):  
CS Greenberg ◽  
JV Dobson ◽  
CC Miraglia
Keyword(s):  
Factor Xiii ◽  
Specific Binding ◽  
Divalent Cations ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protamine Sulfate ◽  
Factor Xiiia ◽  
Plasma Sodium ◽  
Platelet Factor ◽  
Plasma Factor

Abstract The binding of plasma factor XIII to fibrinogen or fibrin that has been chemically or enzymatically induced to polymerize was studied. Factor XIII binding was assayed using a 3H-putrescine incorporation assay and an 125I-plasma factor XIII binding assay. More than 80% of the native and radiolabeled plasma factor XIII was bound to fibrin I formed by reptilase in EDTA, citrate, or heparin anticoagulated plasma. Plasma factor XIII and 125I-factor XIII was bound (89.6% to 92.5%) to fibrin II formed by thrombin in either citrate or EDTA anticoagulated plasma. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of 125I-plasma factor XIII bound to fibrin I or fibrin II formed by reptilase or thrombin in the presence of EDTA demonstrated the b2- subunit remained bound to the a-chains or thrombin-cleaved a-chains. In the presence of calcium chloride and thrombin, the b2-subunit dissociated and factor XIIIa was bound. Protamine sulfate caused fibrinogen polymerization in the absence of divalent cations and reduced both plasma factor XIII and immunologic fibrinogen levels. Fibrinogen polymerized by protamine sulfate bound plasma factor XIII and the a2-subunit of 125I-platelet factor XIII. Plasma factor XIII was also bound to sonicated non-cross-linked fibrin II in either normal plasma or afibrinogenemic plasma. Plasma levels of several coagulation proteins were unchanged after the addition of reptilase, protamine sulfate, or sonicated fibrin to plasma. These results demonstrate that a specific binding site for the a2-subunit of plasma factor XIII is present on polymerized fibrinogen, fibrin I, and fibrin II. Furthermore, the presence of divalent cations, thrombin-cleavage of plasma factor XIII, and release of fibrinopeptides A or B are not required for plasma factor XIII binding to polymerized fibrinogen and fibrin.

The Paradoxical Hemorrhagic and Thrombotic Nature of the Fibrinogen Aα R16C Mutation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1047-1047
Author(s):  
Veronica H. Flood ◽  
Hamid A. Al-Mondhiry ◽  
David H. Farrell
Keyword(s):  
Factor Xiii ◽  
Biochemical Properties ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Dimer Formation ◽  
Critical Position ◽  
Fibrin Polymerization ◽  
Dual Nature ◽  
Sds Page ◽  
Significant Difference

Abstract The Aα R16C mutation is a common cause of dysfibrinogenemia, but the complete implications of this mutation on the process of hemostasis have not been fully characterized. Because of its critical position at the fibrinopeptide A cleavage site, this mutation leads to delayed fibrinopeptide release and subsequent delayed fibrin polymerization. The point mutation responsible for this dysfibrinogen leads to a clinical paradox, however, with both hemorrhage and thrombosis as reported complications. Of previously identified patients with this dysfibrinogen, approximately 30% have experienced bleeding and 15% thrombosis, with the remainder asymptomatic. In this report, the biochemical properties of Aα R16C dysfibrinogens that contribute to either hemorrhage or thrombosis are characterized. Blood samples were obtained from two young siblings who presented with excessive trauma-induced bleeding. Functional fibrinogen levels were 46–55 mg/dL and fibrinogen antigen levels were 427–429 mg/dL, consistent with the diagnosis of dysfibrinogenemia (Fibrinogen Hershey III). DNA sequencing demonstrated both siblings to be heterozygous for the Aα R16C mutation. Fibrinogen was then purified from plasma by classical glycine precipitation. In order to determine if this dysfibrinogen has altered rates of factor XIIIa cross-linking, cross-linking kinetics were assessed by incubating normal or mutant fibrinogen with factor XIII and thrombin and quantifying band intensity at successive timepoints for the resultant γ-γ dimers and α multimers by SDS-PAGE. Analysis of factor XIIIa cross-linking showed a significant decrease in the amount of γ-γ dimer formation when compared to normal fibrinogen (p<0.05 for both siblings) but no significant difference in the rate or quantity of α multimer formation. After an initial lag, the rate of γ-γ dimer formation was not appreciably different from that of the control. This decreased amount of cross-linking, which may also reflect the delay in fibrin polymerization, likely contributes to the hemorrhagic phenotype sometimes seen with this dysfibrinogen. Fibrinolysis kinetics were next measured by monitoring the optical density of purified Fibrinogen Hershey III clotted with thrombin in the presence of factor XIII, tissue plasminogen activator, and Glu-plasminogen. For the propositus, fibrinolysis was significantly delayed, with t1/2 of 51 ± 3 minutes (mean ± SEM) compared to 38 ± 0.2 minutes for normal fibrinogen. Similar results were obtained for the second sibling. The decreased rate of fibrinolysis could explain the paradoxical thrombotic phenotype sometimes seen with this dysfibrinogen. Thus the dual nature of the Aα R16C mutation is demonstrated by the simultaneous presence of deficient fibrinolysis and deficient fibrin cross-linking. Slower clot formation results from the delays in fibrinopeptide cleavage and fibrin polymerization. The delay in fibrinolysis, however, represents a hypercoagulable state leading to potential thrombosis. For this particular dysfibrinogen, the balance of procoagulant versus fibrinolytic factors may be most important in determining its clinical phenotype.

scholarly journals Detection of factor XIIIa (active fibrin-stabilizing factor) in normal plasma

Blood ◽  
1978 ◽  
Vol 52 (3) ◽  
pp. 581-591 ◽  
Author(s):  
JC Nelson ◽  
RG Lerner
Keyword(s):  
Factor Xiii ◽  
Specific Activity ◽  
Proteolytic Enzymes ◽  
High Specific Activity ◽  
Factor Xa ◽  
Factor Xiiia ◽  
Calcium Dependent ◽  
Normal Plasma ◽  
Plasma Factor

Abstract Factor XIIIa (active fibrin-stabilizing factor) generated in heat- defibrinated plasma by the addition of thrombin can be measured by 14C- putrescine incorporation into casein. Modification of this assay be substituting 3H-putrescine of high specific activity as the donor amine permits measurement of amine incorporation by plasma even in the absence of added thrombin. Incorporation is calcium dependent, inhibited by iodoacetamide, and absent from congenital factor XIII- deficient plasma and from normal platelets. The transamidating activity detected by radioenzymatic assay catalyzed the formation of gamma-gamma dimers and alpha polymers of fibrin and was thus biologically functional. This fibrin cross-linking activity was absent from factor XIII-deficient plasma. These experiments show (1) some factor XIII is present in plasma as factor XIIIa; (2) this factor XIIIa can cross-link fibrin and thus has biologic activity as well; and (3) this activity is not present in factor XIII-deficient plasma. Factor XIIIa in normal plasma is possibly activated in vivo, perhaps by circulating thrombin, factor Xa, or other proteolytic enzymes.

The Effect Of Plasmin On Factor XIII (Fibrin Stabilizing Factor)

1981 ◽  
Author(s):  
D M Rider ◽  
J M McDonagh
Keyword(s):  
Factor Xiii ◽  
Activate Factor ◽  
Polyacrylamide Gels ◽  
Clotting Factors ◽  
Human Fibrinogen ◽  
Platelet Factor ◽  
Plasma Factor ◽  
Before And After ◽  
A Subunit ◽  
Almost All

The action of plasmin on several blood clotting factors has been studied; however, controversy exists concerning the effect of plasmin on factor XIII. Factor XIII was purified from plasma and platelets and then exposed to plasmin for up to 6 hours. Plasmin to factor XIII ratios ranged from 0.03-0.1 casein units plasmin per mg factor XIII. These plasmin levels exhibited strong proteolytic activities against B-casein and purified human fibrinogen Following incubation of factor XIII (activated and unactivated) with plasmin the mixtures were electrophoresed on 7% SDS-polyacrylamide gels. The factor XIII preparations were assayed for 14C-putrescine incorporating activity before and after exposure to plasmin. Platelet factor XIII was,labeled With 125Iodine and lableled a subunit (activated and unactivated) was exposed to plalmin for up to 2 hours. These mixtures were electrophoresed on 12.5% Urea-SDS Polyacrylamide gels and a radioactivity profile was determined for each gel.Following extensive exposure to Plasmin the relative molecular weights of the factor XIII subunits (a, a* and b)remained constant and almost all (90-100%) of the 14C-put-rescine incorporating activity was recovered. The radio-activity profiles of the gels of 125I-labeled platelet factor XIII were identical before and after incubation with plasmin. Plasmin did not activate factor XIII in the assay system nor did factor XIII inactivate plasmin by crosslinking it. These experiments indicate that plasmin does not activate or degrade factor XIII and that the b subunit of plasma factor XIII plays no role in protecting the a subunit from the action of plasmin.

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.

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