Enzyme-linked immunosorbent assay for the determination of blood coagulation factor XIII A-subunit in plasma and in cell lysates

Katona, Éva; Ajzner, Éva; Tóth, Katalin; Kárpáti, Levente; Muszbek, László

doi:10.1016/s0022-1759(01)00479-3

Cited by 66 publications

(64 citation statements)

References 22 publications

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“…These samples contained over 90% promyelocytes and no measurable monocytes at diagnosis by both flow cytometry and hematology analyzer, therefore, the possibility that the measured FXIII-A originated from monocytes was excluded. In these selected APL samples the following amounts of FXIII-A were measured: 29 and 80 fg/cell; thus in these cases leukemic promyelocytes contained FXIII-A antigen in amounts similar to that described previously in platelets (mean: 60 AE 10 fg/platelet) [9]. (Fig.…”

Section: Detection Of Fxiii-a By Elisa and Immunoblottingsupporting

confidence: 68%

“…Detection of FXIII-A in cell lysates from APL samples was carried out as described earlier, by Katona et al [9] with slight modification. To avoid platelet contamination, promyelocytic cells were washed three times in PBS containing 20 mM of EDTA at 1,100g for 4 min.…”

Section: Elisa Assaymentioning

confidence: 99%

“…Platelets contain huge amounts of FXIII-A, 150-fold more per volume than plasma, while FXIII-A concentration in monocytes is at least one magnitude less than that in platelets [9]. It has been demonstrated that normal bone marrow precursor cells of monocytes and megakaryocytes also express FXIII-A [10].…”

Section: Introductionmentioning

confidence: 99%

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Expression of coagulation factor XIII subunit A in acute promyelocytic leukemia

Simon¹,

Bagoly²,

Hevessy³

et al. 2012

Cytometry Part B Clinical

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Leukemic cells often express markers, which are not characteristic of their particular cell lineage. In this study, we identified the ''A'' subunit of coagulation factor XIII (FXIII-A) in leukemic promyelocytes in de novo AML M3 cases. The cytoplasmic presence of factor XIII-A has previously been shown only in platelets/megakaryocytes and monocytes/macrophages. Furthermore, more recently we described the presence of FXIII-A in leukemic lymphoblasts. We studied 14 patients with this rare type of acute leukemia in a period of 4 years and investigated their bone marrow samples by 3-color flow cytometry upon diagnosis, mainly focusing on FXIII-A expression of leukemic cells. We detected FXIII-A also by ELISA, Western-blot, and confocal laser scanning microscopy. This was a homogenous group of AML M3 patients with translocation t(15;17)(q22;q21) detected by fluorescence in situ hybridization (FISH). In 10 out of 14 samples, FXIII-A was detectable by flow cytometry and was coexpressed with markers characteristic for leukemic promyleocytes (CD45dim/CD131/CD331/ CD1171/cyMPO1 and HLA-DR-/CD342/CD142/CD152). Staining for the markers GPIIb and GPIX were negative, and FXIII-A was identified in the cytoplasm of the cells by confocal microscopy in a relatively high quantity, as measured by ELISA. By Western blot analysis we could identify FXIII-A in the native 82 kDa form and in cleaved forms corresponding to cleavage products observed when purified FXIII-A was treated by human neutrophil elastase. This novel expression site of FXIII-A in AML M3 can be considered as a leukemia associated immunophenotype and may have pathophysiological significance. V C 2012 International Clinical Cytometry Society

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Section: Detection Of Fxiii-a By Elisa and Immunoblottingsupporting

confidence: 68%

Section: Elisa Assaymentioning

confidence: 99%

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Expression of coagulation factor XIII subunit A in acute promyelocytic leukemia

Simon¹,

Bagoly²,

Hevessy³

et al. 2012

Cytometry Part B Clinical

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“…For activity measurement, washed platelet suspension was solubilized in 1% Triton X-100. FXIII-A 2 B 2 complex antigen in plasma 17 and FXIII-A antigen in plasma and platelets 18 were measured by sandwich enzyme-linked immunosorbent assay (ELISA) (R-ELISA FXIII and R-ELISA FXIII-A; Reanal-ker). In platelets FXIII-A was also detected by Western blotting.…”

Section: Methodsmentioning

confidence: 99%

Severe bleeding complications caused by an autoantibody against the B subunit of plasma factor XIII: a novel form of acquired factor XIII deficiency

Ajzner

Schlammadinger

Kerényi

et al. 2009

Blood

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Acquired factor XIII (FXIII) deficiency due to autoantibody against FXIII is a very rare severe hemorrhagic diathesis. Antibodies directed against the A subunit of FXIII, which interfere with different functions of FXIII, have been described. Here, for the first time, we report an autoantibody against the B subunit of FXIII (FXIII-B) that caused lifethreatening bleeding in a patient with systemic lupus erythematosus. FXIII activity, FXIII-A 2 B 2 complex, and individual FXIII subunits were undetectable in the plasma, whereas platelet FXIII activity and antigen were normal. Neither FXIII activation nor its activity was inhibited by the antibody, which bound to structural epitope(s) on both free and complexed FXIII-B. The autoantibody highly accelerated the elimination of FXIII from the circulation. FXIII supplementation combined with immunosuppressive therapy, plasmapheresis, immunoglobulin, and anti-CD20 treatment resulted in the patient's recovery. FXIII levels returned to around 20% at discharge and after gradual increase the levels stabilized above 50%. IntroductionBlood coagulation factor XIII (FXIII) is a protransglutaminase of tetrameric structure (FXIII-A 2 B 2 ). 1,2 Its potentially active A subunit (FXIII-A) is synthesized in cells of bone marrow origin; it is also present in platelets and monocytes/macrophages in dimeric form (FXIII-A 2 ). The noncatalytic B subunit (FXIII-B) is in excess and it is essential for the stabilization of FXIII-A 2 in plasmatic conditions. FXIII is converted into an active transglutaminase (FXIIIa) by limited proteolysis of FXIII-A and by Ca 2ϩ -induced dissociation of FXIII-B. Cross-linking of fibrin ␣-and ␥-chains and ␣ 2 -plasmin inhibitor to fibrin by FXIIIa stabilizes fibrin and protects it from prompt elimination by plasmin. 3 Inherited FXIII-A deficiency is a severe bleeding diathesis with the high risk of intracranial bleeding in nonsupplemented patients. 4 Only 5 cases of inherited FXIII-B deficiency with mild-to-moderate bleeding tendency have been reported. [5][6][7][8] In the absence of FXIII-B, plasma FXIII activity and FXIII-A concentration were considerably decreased, whereas in platelets a normal amount of FXIII-A was measured. 9 Thirty-six cases of severe FXIII deficiency due to an autoantibody against FXIII-A have been reported. In a few cases, the autoantibody was characterized and classified into subgroups according to its inhibition of FXIII activation, FXIIIa activity, or binding to fibrin. [10][11][12][13][14][15] In about one third of the cases the autoantibody was associated with systemic lupus erythematosus (SLE). No report on an autoantibody directed against FXIII-B has been published so far. MethodsA 28-year-old woman suffering from SLE with end-stage kidney disease was on hemodialysis. For preparation of cubital fistula she was admitted to a county hospital. In the proximity of the surgical wound, hematomas developed that were explored. The wounds showed no tendency of healing and remained open. A few days later extensive intramuscular hematoma ...

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“…22 The crucial role of crosslinked a 2 AP in stabilizing fibrin is demonstrated by the rapid lysis of clots formed in the absence of a 2 AP, 1,3,17 or when a 2 AP activity is neutralized. 1 FXIII-A is present in platelets [22][23][24][25][26][27] in large quantities; a single platelet contains 60 6 10 fg, 28 making local platelet FXIII-A concentration around 150-fold greater than that in plasma. 29 Platelet FXIII-A, also known as cellular FXIII, 30 is largely stored in the cytoplasm 23,24 and, in contrast to FXIII-A 2 B 2 , can be nonproteolytically activated by Ca 21 alone.…”

Section: Introductionmentioning

confidence: 99%

Functional factor XIII-A is exposed on the stimulated platelet surface

Mitchell

Lionikiene

Fraser

et al. 2014

Blood

105

141

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Key Points• Factor XIII-A is exposed in protruding caps on the activated platelet surface.• Platelet FXIII-A exerts antifibrinolytic function by cross-linking a 2 AP to fibrin.Factor XIII (FXIII) stabilizes thrombi against fibrinolysis by cross-linking a 2-antiplasmin (a 2 AP) to fibrin. Cellular FXIII (FXIII-A) is abundant in platelets, but the extracellular functions of this pool are unclear because it is not released by classical secretion mechanisms. We examined the function of platelet FXIII-A using Chandler model thrombi formed from FXIIIdepleted plasma. Platelets stabilized FXIII-depleted thrombi in a transglutaminase-dependent manner. FXIII-A activity on activated platelets was unstable and was rapidly lost over 1 hour. Inhibiting platelet activation abrogated the ability of platelets to stabilize thrombi. Incorporating a neutralizing antibody to a 2 AP into FXIII-depleted thrombi revealed that the stabilizing effect of platelet FXIII-A on lysis was a 2 AP dependent. Platelet FXIII-A activity and antigen were associated with the cytoplasm and membrane fraction of unstimulated platelets, and these fractions were functional in stabilizing FXIII-depleted thrombi against lysis. Fluorescence confocal microscopy and flow cytometry revealed exposure of FXIII-A on activated membranes, with maximal signal detected with thrombin and collagen stimulation. FXIII-A was evident in protruding caps on the surface of phosphatidylserine-positive platelets. Our data show a functional role for platelet FXIII-A through exposure on the activated platelet membrane where it exerts antifibrinolytic function by cross-linking a 2 AP to fibrin. (Blood. 2014;124(26):3982-3990) IntroductionFactor XIII (FXIII) plays an essential role in normal hemostasis where it contributes to the regulation of fibrinolysis, 1-3 the maintenance of pregnancy, 4 wound healing, and angiogenesis. 5 The function of FXIII in hemostasis is further emphasized in its deficiency, which results in hemorrhage 6,7 and slow wound healing. 4,7 FXIII circulates in plasma as a heterotetramer (FXIII-A 2 B 2 ) where the catalytic A (FXIII-A 2 ) subunits are almost exclusively in complex with the inhibitory carrier B (FXIII-B 2 ) subunits. 8 FXIII is activated by the combined action of thrombin and Ca 21 to form the active transglutaminase (TG) FXIIIa. 9,10 FXIIIa confers stability to the fibrin matrix by cross-linking fibrin, substantially altering its rheologic properties.11,12 TG catalyzes formation of covalent e-(g-glutamyl) lysyl bonds 13 in which the lysine e-amino group is cross-linked to the glutamine g-carboxymide group. 14The cross-linking of g-chains confers a degree of rigidity to the fibrin network, which is further stabilized by the cross-linking of a-chains 15 to generate high-molecular-weight polymers. 10,16 FXIIIa also cross-links inhibitors of fibrinolysis to fibrin, further increasing its insolubility and resistance to plasmin. These inhibitors include a 2 -antiplasmin (a 2 AP), 17 thrombin activatable fibrinolysis inhibitor, 18 and plasminogen ac...

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Enzyme-linked immunosorbent assay for the determination of blood coagulation factor XIII A-subunit in plasma and in cell lysates

Cited by 66 publications

References 22 publications

Expression of coagulation factor XIII subunit A in acute promyelocytic leukemia

Expression of coagulation factor XIII subunit A in acute promyelocytic leukemia

Severe bleeding complications caused by an autoantibody against the B subunit of plasma factor XIII: a novel form of acquired factor XIII deficiency

Functional factor XIII-A is exposed on the stimulated platelet surface

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