scholarly journals Factor XIII A-Subunit V34L Variant Affects Thrombus Cross-Linking in a Murine Model of Thrombosis

2016 ◽  
Vol 36 (2) ◽  
pp. 308-316 ◽  
Author(s):  
Cédric Duval ◽  
Majid Ali ◽  
Waleed W. Chaudhry ◽  
Victoria C. Ridger ◽  
Robert A.S. Ariëns ◽  
...  
Keyword(s):  
Murine Model ◽  
Factor Xiii ◽  
Cross Linking ◽  
A Subunit

An Investigation of Factor XIII Binding to Recombinant γ′/γ′ and γ/γ′ Fibrinogen.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1705-1705 ◽  
Author(s):  
Kathryn C. Gersh ◽  
Susan T. Lord
Keyword(s):  
Factor Xiii ◽  
Polymerization Rate ◽  
Cross Linking ◽  
Critical Element ◽  
C Terminus ◽  
Rate Of Polymerization ◽  
Alternatively Spliced ◽  
A Subunit ◽  
Artery Disease ◽  
The Stability

Abstract Increased levels of the alternatively spliced γ′ chain of fibrinogen have been measured in patients suffering from coronary artery disease (Lovely et al. Thromb. Haemost.2002;88:26–31). Others have suggested that the γ′ chain serves as a binding site for factor XIII (Siebenlist et al. Biochemistry1996;35:10448–10453), thus increasing the amount of cross-linking and therefore the stability of the γ′-containing clots (Falls and Farrell JBC1997;272:14251–14256). We have expressed recombinant γ′/γ′ homodimer and γ/γ′ heterodimer fibrinogen variants in CHO cells and characterized their polymerization by turbidity. Compared to normal recombinant γ/γ fibrinogen, γ′/γ′ fibrinogen displayed a slower rate of polymerization and a lower final absorbance. γ/γ′ Heterodimer fibrinogen had both a polymerization rate and final absorbance that was intermediate between γ/γ and γ′/γ′. These observations suggest that the presence of the γ′ chain slows lateral aggregation and leads to the formation of thinner fibers. These variants were then used to investigate the binding of factor XIII to fibrinogen. We designed an ELISA assay to measure this binding by immobilizing fibrinogen on a 96-well plate, incubating with factor XIII, and measuring bound factor XIII with an anti-factor XIII A-subunit antibody. Normal recombinant γ/γ fibrinogen bound factor XIII zymogen (A2B2) with a KD of 4.1 x 10−8 M +/− 1.3 x 10−8 M, similar to previous data for plasma fibrinogen. Surprisingly, both γ′/γ′ and γ/γ′ fibrinogen variants bound factor XIII zymogen with the same KD as did γ/γ fibrinogen. As shown by ELISAs with antibodies specific for either γ or γ′, the C-termini of both the γ and the γ′ chains are accessible on this immobilized fibrinogen. These findings suggest that the C-terminus of the γ′ chain is not a critical element in the binding of factor XIII zymogen. Our findings differ from previously reported findings. Further studies are in progress to reconcile these differences.

Platelet Calmodulin Mediates The Calcium Dependent Activation Of Factor XIII

1981 ◽  
Author(s):  
D Kahn ◽  
N Crawford ◽  
I Cohen
Keyword(s):  
Factor Xiii ◽  
Sulfhydryl Group ◽  
Cross Linking ◽  
Platelet Factor ◽  
Subunit A ◽  
Calcium Dependent ◽  
B Subunit ◽  
A Subunit ◽  
Subunit B

Transglutaminases are ubiquitous in cells and require calcium for their activation. The factor XIII zymogen, present in plasma and in the platelet cytosol, requires for its activation both a limited proteolytic activity on the catalytic subunit,“a”, and, in the use of the plasma enzyme, calcium for dissociating subunit“a” from the carrier subunit“b”. Calcium is also necessary for exposing the reactive sulfhydryl group. We have recently suggested a role for the platelet factor XIII in the generation of calcium-dependent cross-linking processes in platelets. Since calmodulin is present in considerable amounts in the platelet cytosol and is known to regulate the activity of various calcium-dependent enzymes and cellular reactions, we have investigated its possible role in factor XIII activation. Since the“a” subunit of platelet factor XIII is identical to its plasma counterpart, the more easily purified plasma zymogen was used. The effect of calmodulin on the two calcium-dependent steps of factor XIII activation was investigated following thrombin-stimulated hydrolysis of the“a” subunit. Platelet calmodulin was found to enhance by at least 3 fold the calcium-dependent unmasking of the reactive sulfhydryl groups which were titrated with 14C-iodoacetamide. Calmodulin also enhanced by at least 4 fold the calcium-dependent dissociation of the b subunit from its complex with the thrombin-hydrolyzed“a” subunit. The calmodulin mediation of the calcium-dependent steps of factor XIII activation may be important for regulating the factor XIII-dependent cross-linking reactions in platelets and is reminiscent of the calcium-related regulatory role of fibrinogen on factor XIII activation which could prevail in plasma. An investigation of the possible role of calmodulin on other tissue transglutaminases is warranted.

Abstract 1653: The Role of the Fibrinogen Alpha Chain in Controlling the Rate of Factor XIII Activation: Implications for Coronary Artery Thrombus Formation

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Kerrie A Smith ◽  
Penelope J Adamson ◽  
Robert A Ariens ◽  
Helen Philippou ◽  
Peter J Grant
Keyword(s):  
Factor Xiii ◽  
Plaque Rupture ◽  
Thrombus Formation ◽  
Coagulation Factor ◽  
Cross Linking ◽  
Active Site Cysteine ◽  
B Subunit ◽  
Thrombin Cleavage ◽  
A Subunit ◽  
Α Chain

Background: The development of an obstructive arterial thrombus is dependent on the generation of a platelet rich fibrin mesh secondary to plaque rupture. Fibrin itself is formed by the cleavage of fibrinogen by thrombin with subsequent fibrin cross-linking by activated Factor XIII. Critical to these processes, fibrinogen α-chain residues 242– 424 have a major regulatory role in the dissociation of coagulation Factor XIII A subunit (FXIIIA) from the Factor XIII B subunit (FXIIIB), however the mechanisms for this enhancing effect have not been determined. Methods: α-Chain residues 242– 424 were expressed as a GST-fusion protein in E .coli, along with six further truncations; αC fragments α242– 402, α242–387, α242–374, α242–341, α242–289 and α242–265. FXIII-A and an inactive double thrombin cleavage mutant (R37A/K513A) were also expressed as GST-fusion proteins in E . coli . Results: Using surface plasmon resonance (SPR) and ELISA we can confirm the presence of at least two binding sites on the αC for activated FXIII-A within residues α388 – 424 and α242–265 with an overall KD of 4.9 ± 2.29 μM. This interaction was specific for activated FXIII-A, as the inactive FXIII-A double thrombin cleavage mutant did not bind to the αC as indicated by SPR and ELISA. We demonstrated that the binding of the αC is dependent on a conformational change which occurs during activation of FXIII-A with calcium and thrombin. FXIII-A activated with thrombin alone did not bind αC as determined by SPR and ELISA. Competition studies showed that the αC competitively inhibits binding with a K I of 1.2 μM confirming the specificity of this interaction. Iodoacetamide blocking of FXIII-A active site cysteine did not prevent binding of the αC suggesting this interaction is independent of FXIII-A cross-linking. Furthermore we have identified a novel interaction between FXIIIB and α-chain by ELISA. Utilising the αC fragments we have localised two binding regions between α290–341 and α242–265 with a K D of 123nM ± 11 and 286nM ± 24.7 respectively. Conclusions: The binding of both FXIII A- and B-subunits to this area of the fibrinogen α-chain suggests a crucial role in controlling the amount of activated FXIII generated for clot stabilisation and presents a potential target for therapeutic intervention.

Cross-Linked αsγt-Chain Hybrids in Plasma Clots Studied by 1D- and 2D Electrophoresis and Western Blotting

1993 ◽  
Vol 70 (03) ◽  
pp. 438-442 ◽  
Author(s):  
B Grøn ◽  
C Filion-Myklebust ◽  
S Bjørnsen ◽  
P Haidaris ◽  
F Brosstad
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
Human Plasma ◽  
Western Blotting ◽  
Isoelectric Point ◽  
Factor Xiii ◽  
Cross Linking ◽  
2D Electrophoresis ◽  
Complex Phenomenon ◽  
Fibrinopeptide A

SummaryFibrinogen and fibrin related chains in reduced human plasma as well as the bonds interlinking partially cross-linked fibrin from plasma clots have been studied by means of 1D- and 2D electrophoresis and Western blotting. Immunovisualization of reduced plasma or partially cross-linked fibrin with monoclonal antibodies specific for the α-chains or the γ-chains have shown that several bands represent material belonging to both chains. In order to decide whether these bands constitute αγ-chain hybrids or superimposed α- and γ-chain dimers, the cross-linked material was separated according to both isoelectric point (pI) and molecular weight (MW) using Pharmacia’s Multiphor II system. Western blotting of the second dimension gels revealed that partially cross-linked fibrin contains αsγt-chain hybrids and γ- polymers, in addition to the well-known γ-dimers and α-polymers. The main αsγt-chain hybrid has a pI between that of the α- and the γ-chains, a MW of about 200 kDa and contains Aα-chains with intact fibrinopeptide A (FPA). It was also observed that soluble fibrinogen/fibrin complexes as well as partially cross-linked fibrin contain degraded α-dimers with MWs close to the γ-dimers. These findings demonstrate that factor XIII-catalyzed cross-linking of fibrin is a more complex phenomenon than earlier recognized.

Platelet Accumulation on Fibrin-coated Polyethylene: Role of Platelet Activation and Factor XIII

1995 ◽  
Vol 73 (05) ◽  
pp. 850-856 ◽  
Author(s):  
F D Rubens ◽  
D W Perry ◽  
M W C Hatton ◽  
P D Bishop ◽  
M A Packham ◽  
...  
Keyword(s):  
Factor Xiii ◽  
Cross Linking ◽  
Internal Diameter ◽  
Static System ◽  
Graft Occlusion ◽  
Platelet Binding ◽  
Coated Surface ◽  
Platelet Accumulation ◽  
Polyethylene Tubing

SummaryPlatelet accumulation on small- and medium-calibre vascular grafts plays a significant role in graft occlusion. We examined platelet accumulation on the surface of fibrin-coated polyethylene tubing (internal diameter 0.17 cm) during 10 min of flow (l0ml/min) at high wall shear rate (764 s-1). Washed platelets labelled with 51Cr were resuspended in Tyrode solution containing albumin, apyrase and red blood cells (hematocrit 40%). When the thrombin that was used to form the fibrin-coated surface was inactivated with FPRCH2C1 before perfusion of the tubes with the platelet:red blood cell suspension, the accumulation of platelets was 59,840 ± 27,960 platelets per mm2, whereas accumulation on fibrin with residual active thrombin was 316,750 ± 32,560 platelets per mm2 (n = 4). When the fibrin on the surface was cross-linked by including recombinant factor XIII (rFXIII) in the fibrinogen solution used to prepare the fibrin-coated surface, platelet accumulation, after thrombin neutralization, was reduced by the cross-linking from 46,974 ± 9702 to 36,818 ± 7964 platelets per mm2 (n = 12, p <0.01). Platelet accumulation on tubes coated with D-dimer was ten times less than on tubes coated with D-domain; this finding also supports the observation that cross-linking of fibrin with the formation of γ-γ dimers reduces platelet accumulation on the fibrin-coated surface. Thrombin-activated platelets themselves were shown to cross-link fibrin when they had adhered to it during perfusion, or in a static system in which thrombin was used to form clots from FXIII-free fibrinogen in the presence of platelets. Thus, cross-linking of fibrin by FXIII in plasma or from platelets probably decreases the reactivity of the fibrin-containing thrombi to platelets by altering the lysine residue at or near the platelet-binding site of each of the γ-chains of the fibrinogen which was converted into the fibrin of these thrombi.

Molecular and Genetic Mechanisms of Factor XIII A Subunit Deficiency

2000 ◽  
Vol Volume 26 (Number 01) ◽  
pp. 005-010 ◽  
Author(s):  
Akitada Ichinose ◽  
Masayoshi Souri ◽  
Tomonori Izumi ◽  
Nobumasa Takahashi
Keyword(s):  
Factor Xiii ◽  
Genetic Mechanisms ◽  
A Subunit

Cross-linking of fibrinogen by factor XIII zymogen is not apparent in vivo

2003 ◽  
Vol 89 (05) ◽  
pp. 943-944 ◽  
Author(s):  
Patricia DiBello ◽  
John Shainoff
Keyword(s):  
Factor Xiii ◽  
Cross Linking

scholarly journals Recombinant factor XIII: a safe and novel treatment for congenital factor XIII deficiency

Blood ◽  
2012 ◽  
Vol 119 (22) ◽  
pp. 5111-5117 ◽  
Author(s):  
Aida Inbal ◽  
Johannes Oldenburg ◽  
Manuel Carcao ◽  
Anders Rosholm ◽  
Ramin Tehranchi ◽  
...  
Keyword(s):  
Factor Xiii ◽  
Autosomal Recessive Disorder ◽  
Allergic Reactions ◽  
Open Label ◽  
Impaired Wound Healing ◽  
Bleeding Rate ◽  
Life Threatening ◽  
A Subunit ◽  
Bleeding Episodes ◽  
Congenital Factor

Congenital factor XIII (FXIII) deficiency is a rare, autosomal-recessive disorder, with most patients having an A-subunit (FXIII-A) deficiency. Patients experience life-threatening bleeds, impaired wound healing, and spontaneous abortions. In many countries, only plasma or cryoprecipitate treatments are available, but these carry a risk for allergic reactions and infection with blood-borne pathogens. The present study was a multinational, open-label, single-arm, phase 3 prophylaxis trial evaluating the efficacy and safety of a novel recombinant FXIII (rFXIII) in congenital FXIII-A subunit deficiency. Forty-one patients ≥ 6 years of age (mean, 26.4; range, 7-60) with congenital FXIII-A subunit deficiency were enrolled. Throughout the rFXIII prophylaxis, only 5 bleeding episodes (all trauma induced) in 4 patients were treated with FXIII-containing products. The crude mean bleeding rate was significantly lower than the historic bleeding rate (0.138 vs 2.91 bleeds/patient/year, respectively) for on-demand treatment. Transient, non-neutralizing, low-titer anti-rFXIII Abs developed in 4 patients, none of whom experienced allergic reactions, any bleeds requiring treatment, or changes in FXIII pharmacokinetics during the trial or follow-up. These non-neutralizing Abs declined below detection limits in all 4 patients despite further exposure to rFXIII or other FXIII-containing products. We conclude that rFXIII is safe and effective in preventing bleeding episodes in patients with congenital FXIII-A subunit deficiency. This study is registered at http://www..clinicaltrials.gov as number NCT00713648.

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