Introduction : Factor X (FX) is a vitamin K dependent serine protease that plays a key role in the coagulation cascade as the first pro-enzyme of the common pathway of fibrin formation. FX deficiency is one of the most severe among recessively inherited coagulation disorders. The G222D mutation, located in the loop-40 of catalytic domain, was previously identified in homozygous state in 4 unrelated patients affected by severe FX deficiency, FX:C <1% and FX:Ag 10-15%. In vitro expression studies showed a secretion defect, confirmed by metabolic labelling and immunofluorescence studies. The present study analyzes why the trace amount of secreted mutant protein did not conserve its procoagulant activity Methods : recombinant FX222D was produced and activated by 1nM RVV-X and 11pM rFVIIa (with 20 mul lipidated human rTF, by IL, Milan) and results were compared to recombinant FXWT. FX activation was monitored by quenching samples over time into assay buffer with 25mM EDTA. The rate of hydrolysis of 100mM S2765 chromogenic substrate by the activated WT and mutant FX was measured in a kinetic microplate reader (absorbance 405nm) Results : a preliminary SSP prevision showed that the G222D might lead to a conformational rearrangement of the FX222D hiding the peptidic bond usually hydrolyzed during the activation process. A further conformational transition of the loop-40 could allosterically inhibit the catalytic activity of the enzyme. Kinetic studies showed that the rate of FX222D activation by RVV-X and FVIIa/TF was modestly reduced (2-3 folds). Moreover, the S2765 hydrolysis by FX222D showed a 11-fold reduction of the apparent kcat/Km, compared to WT, being kcat severely and Km modestly reduced (kcat 1.1 vs 14.6 sec-1, Km 26 vs 40 muM) Conclusions : the G222D mutation is thus responsible for the severe deficiency of FX, impairing both the secretion of the zymogen FX and the activation of the trace amount of the secreted protein, as well as the catalytic competence of the mutant enzyme toward chromogenic substrate

Kinetics studies of a naturally occurring mutation on Factor X (FX) gene (G222D) / M. Menegatti, R. Palla, A. Afrasiabi, M. Karimi, R. De Cristofaro, F. Peyvandi. - In: JOURNAL OF THROMBOSIS AND HAEMOSTASIS. - ISSN 1538-7933. - 5:suppl. 2(2007 Aug), pp. OM.019-OM.019. ((Intervento presentato al 21. convegno International Society of Thrombosis and Haemostasis tenutosi a Genève nel 2007.

Kinetics studies of a naturally occurring mutation on Factor X (FX) gene (G222D)

M. Menegatti
Primo
;
R. Palla
Secondo
;
F. Peyvandi
Ultimo
2007

Abstract

Introduction : Factor X (FX) is a vitamin K dependent serine protease that plays a key role in the coagulation cascade as the first pro-enzyme of the common pathway of fibrin formation. FX deficiency is one of the most severe among recessively inherited coagulation disorders. The G222D mutation, located in the loop-40 of catalytic domain, was previously identified in homozygous state in 4 unrelated patients affected by severe FX deficiency, FX:C <1% and FX:Ag 10-15%. In vitro expression studies showed a secretion defect, confirmed by metabolic labelling and immunofluorescence studies. The present study analyzes why the trace amount of secreted mutant protein did not conserve its procoagulant activity Methods : recombinant FX222D was produced and activated by 1nM RVV-X and 11pM rFVIIa (with 20 mul lipidated human rTF, by IL, Milan) and results were compared to recombinant FXWT. FX activation was monitored by quenching samples over time into assay buffer with 25mM EDTA. The rate of hydrolysis of 100mM S2765 chromogenic substrate by the activated WT and mutant FX was measured in a kinetic microplate reader (absorbance 405nm) Results : a preliminary SSP prevision showed that the G222D might lead to a conformational rearrangement of the FX222D hiding the peptidic bond usually hydrolyzed during the activation process. A further conformational transition of the loop-40 could allosterically inhibit the catalytic activity of the enzyme. Kinetic studies showed that the rate of FX222D activation by RVV-X and FVIIa/TF was modestly reduced (2-3 folds). Moreover, the S2765 hydrolysis by FX222D showed a 11-fold reduction of the apparent kcat/Km, compared to WT, being kcat severely and Km modestly reduced (kcat 1.1 vs 14.6 sec-1, Km 26 vs 40 muM) Conclusions : the G222D mutation is thus responsible for the severe deficiency of FX, impairing both the secretion of the zymogen FX and the activation of the trace amount of the secreted protein, as well as the catalytic competence of the mutant enzyme toward chromogenic substrate
Settore MED/09 - Medicina Interna
ago-2007
International Society of Thrombosis and Haemostasis
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/69039
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