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Modeling of human factor Va inactivation by activated protein C.

Bravo MC, Orfeo T, Mann KG, Everse SJ - BMC Syst Biol (2012)

Bottom Line: Reaction mechanisms, rate constants and equilibrium constants informing these model constructs were initially derived from various research groups reporting on APC inactivation of FVa in isolation, or in the presence of FXa or prothrombin.Our work integrates previously published findings and through the cooperative analysis of in vitro experiments and mathematical constructs we are able to produce a final validated model that includes 24 chemical reactions and interactions with 14 unique rate constants which describe the flux in concentrations of 24 species.This study highlights the complexity of the inactivation process and provides a module of equations describing the Protein C pathway that can be integrated into existing comprehensive mathematical models describing tissue factor initiated coagulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cell and Molecular Biology Program, University of Vermont, 89 Beaumont Ave, Burlington, VT 05405, USA.

ABSTRACT

Background: Because understanding of the inventory, connectivity and dynamics of the components characterizing the process of coagulation is relatively mature, it has become an attractive target for physiochemical modeling. Such models can potentially improve the design of therapeutics. The prothrombinase complex (composed of the protease factor (F)Xa and its cofactor FVa) plays a central role in this network as the main producer of thrombin, which catalyses both the activation of platelets and the conversion of fibrinogen to fibrin, the main substances of a clot. A key negative feedback loop that prevents clot propagation beyond the site of injury is the thrombin-dependent generation of activated protein C (APC), an enzyme that inactivates FVa, thus neutralizing the prothrombinase complex. APC inactivation of FVa is complex, involving the production of partially active intermediates and "protection" of FVa from APC by both FXa and prothrombin. An empirically validated mathematical model of this process would be useful in advancing the predictive capacity of comprehensive models of coagulation.

Results: A model of human APC inactivation of prothrombinase was constructed in a stepwise fashion by analyzing time courses of FVa inactivation in empirical reaction systems with increasing number of interacting components and generating corresponding model constructs of each reaction system. Reaction mechanisms, rate constants and equilibrium constants informing these model constructs were initially derived from various research groups reporting on APC inactivation of FVa in isolation, or in the presence of FXa or prothrombin. Model predictions were assessed against empirical data measuring the appearance and disappearance of multiple FVa degradation intermediates as well as prothrombinase activity changes, with plasma proteins derived from multiple preparations. Our work integrates previously published findings and through the cooperative analysis of in vitro experiments and mathematical constructs we are able to produce a final validated model that includes 24 chemical reactions and interactions with 14 unique rate constants which describe the flux in concentrations of 24 species.

Conclusion: This study highlights the complexity of the inactivation process and provides a module of equations describing the Protein C pathway that can be integrated into existing comprehensive mathematical models describing tissue factor initiated coagulation.

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Related in: MedlinePlus

Effect of Factor Xa on Activated Protein C Inactivation of Factor Va. Empircal time courses of FVa inactivation measured by activity analysis (Panels A and B) or by monitoring proteolytic cleavages (Panel C) are presented along with corresponding simulated time courses. All data points are presented as averages ± S.D. of 4–6 experiments. (A,B) Empirical time courses of APC (0.5 nM – black circles; 2.0 nM – red circles) inactivation of preformed prothrombinase (200 pM FVa and 100 pM FXa). Corresponding simulated time courses for 0.5 nM (black) or 2.0 nM (red) APC are presented with three potential KD values for the formation of the prothrombinase complex (100 pM – dotted lines; 500 pM – dashed lines; 750 pM – solid line): in Panel A each equilibrium process was described using a kon (4.0 x 108 M-1 s-1) and adjusting the koff constant; in Panel B each equilibrium process was described using a kon (1.5 x 108 M-1 s-1) and adjusting the koff constant. (C) Empirical time course of 2.0 nM APC inactivation of preformed prothrombinase (20 nM FVa and 30 nM FXa*) anaylzed by Western blotting. FVaHC densitometric data are expressed as relative to its value at time 0 (red circles). Corresponding simulated time courses for 2.0 nM (red) APC are presented using three potential KD values for the formation of the prothrombinase complex with each equilibrium process described by using a kon (1.5 x 108 M-1 s-1) and adjusting the koff constant (100 pM – dotted lines; 500 pM – dashed lines; 750 pM – solid line).
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Figure 4: Effect of Factor Xa on Activated Protein C Inactivation of Factor Va. Empircal time courses of FVa inactivation measured by activity analysis (Panels A and B) or by monitoring proteolytic cleavages (Panel C) are presented along with corresponding simulated time courses. All data points are presented as averages ± S.D. of 4–6 experiments. (A,B) Empirical time courses of APC (0.5 nM – black circles; 2.0 nM – red circles) inactivation of preformed prothrombinase (200 pM FVa and 100 pM FXa). Corresponding simulated time courses for 0.5 nM (black) or 2.0 nM (red) APC are presented with three potential KD values for the formation of the prothrombinase complex (100 pM – dotted lines; 500 pM – dashed lines; 750 pM – solid line): in Panel A each equilibrium process was described using a kon (4.0 x 108 M-1 s-1) and adjusting the koff constant; in Panel B each equilibrium process was described using a kon (1.5 x 108 M-1 s-1) and adjusting the koff constant. (C) Empirical time course of 2.0 nM APC inactivation of preformed prothrombinase (20 nM FVa and 30 nM FXa*) anaylzed by Western blotting. FVaHC densitometric data are expressed as relative to its value at time 0 (red circles). Corresponding simulated time courses for 2.0 nM (red) APC are presented using three potential KD values for the formation of the prothrombinase complex with each equilibrium process described by using a kon (1.5 x 108 M-1 s-1) and adjusting the koff constant (100 pM – dotted lines; 500 pM – dashed lines; 750 pM – solid line).

Mentions: In the first set of experiments, prothrombinase was preformed under non-saturating concentrations with FVa in excess (0.2 nM FVa and 0.1 nM FXa) on 20 μM PC:PS vesicles. Under these conditions, if the KD = 0.5 nM for the (FXaFVa) complex (estimate based on previous studies [45]), only 13 % of the FVa would be expected to be bound to FXa. Inactivation reactions were initiated with 0, 0.5, or 2.0 nM APC. For these experiments, the amount of thrombin generated in the 0 nM APC control was set at 100 % prothrombinase activity, with thrombin generation levels represented relative to the prothrombinase activity in the 0 nM APC control. The loss of prothrombinase activity following the addition of APC at either concentration (Figure 4, Panel A, filled circles) appeared to follow a monophasic decay (fits not shown).


Modeling of human factor Va inactivation by activated protein C.

Bravo MC, Orfeo T, Mann KG, Everse SJ - BMC Syst Biol (2012)

Effect of Factor Xa on Activated Protein C Inactivation of Factor Va. Empircal time courses of FVa inactivation measured by activity analysis (Panels A and B) or by monitoring proteolytic cleavages (Panel C) are presented along with corresponding simulated time courses. All data points are presented as averages ± S.D. of 4–6 experiments. (A,B) Empirical time courses of APC (0.5 nM – black circles; 2.0 nM – red circles) inactivation of preformed prothrombinase (200 pM FVa and 100 pM FXa). Corresponding simulated time courses for 0.5 nM (black) or 2.0 nM (red) APC are presented with three potential KD values for the formation of the prothrombinase complex (100 pM – dotted lines; 500 pM – dashed lines; 750 pM – solid line): in Panel A each equilibrium process was described using a kon (4.0 x 108 M-1 s-1) and adjusting the koff constant; in Panel B each equilibrium process was described using a kon (1.5 x 108 M-1 s-1) and adjusting the koff constant. (C) Empirical time course of 2.0 nM APC inactivation of preformed prothrombinase (20 nM FVa and 30 nM FXa*) anaylzed by Western blotting. FVaHC densitometric data are expressed as relative to its value at time 0 (red circles). Corresponding simulated time courses for 2.0 nM (red) APC are presented using three potential KD values for the formation of the prothrombinase complex with each equilibrium process described by using a kon (1.5 x 108 M-1 s-1) and adjusting the koff constant (100 pM – dotted lines; 500 pM – dashed lines; 750 pM – solid line).
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Figure 4: Effect of Factor Xa on Activated Protein C Inactivation of Factor Va. Empircal time courses of FVa inactivation measured by activity analysis (Panels A and B) or by monitoring proteolytic cleavages (Panel C) are presented along with corresponding simulated time courses. All data points are presented as averages ± S.D. of 4–6 experiments. (A,B) Empirical time courses of APC (0.5 nM – black circles; 2.0 nM – red circles) inactivation of preformed prothrombinase (200 pM FVa and 100 pM FXa). Corresponding simulated time courses for 0.5 nM (black) or 2.0 nM (red) APC are presented with three potential KD values for the formation of the prothrombinase complex (100 pM – dotted lines; 500 pM – dashed lines; 750 pM – solid line): in Panel A each equilibrium process was described using a kon (4.0 x 108 M-1 s-1) and adjusting the koff constant; in Panel B each equilibrium process was described using a kon (1.5 x 108 M-1 s-1) and adjusting the koff constant. (C) Empirical time course of 2.0 nM APC inactivation of preformed prothrombinase (20 nM FVa and 30 nM FXa*) anaylzed by Western blotting. FVaHC densitometric data are expressed as relative to its value at time 0 (red circles). Corresponding simulated time courses for 2.0 nM (red) APC are presented using three potential KD values for the formation of the prothrombinase complex with each equilibrium process described by using a kon (1.5 x 108 M-1 s-1) and adjusting the koff constant (100 pM – dotted lines; 500 pM – dashed lines; 750 pM – solid line).
Mentions: In the first set of experiments, prothrombinase was preformed under non-saturating concentrations with FVa in excess (0.2 nM FVa and 0.1 nM FXa) on 20 μM PC:PS vesicles. Under these conditions, if the KD = 0.5 nM for the (FXaFVa) complex (estimate based on previous studies [45]), only 13 % of the FVa would be expected to be bound to FXa. Inactivation reactions were initiated with 0, 0.5, or 2.0 nM APC. For these experiments, the amount of thrombin generated in the 0 nM APC control was set at 100 % prothrombinase activity, with thrombin generation levels represented relative to the prothrombinase activity in the 0 nM APC control. The loss of prothrombinase activity following the addition of APC at either concentration (Figure 4, Panel A, filled circles) appeared to follow a monophasic decay (fits not shown).

Bottom Line: Reaction mechanisms, rate constants and equilibrium constants informing these model constructs were initially derived from various research groups reporting on APC inactivation of FVa in isolation, or in the presence of FXa or prothrombin.Our work integrates previously published findings and through the cooperative analysis of in vitro experiments and mathematical constructs we are able to produce a final validated model that includes 24 chemical reactions and interactions with 14 unique rate constants which describe the flux in concentrations of 24 species.This study highlights the complexity of the inactivation process and provides a module of equations describing the Protein C pathway that can be integrated into existing comprehensive mathematical models describing tissue factor initiated coagulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cell and Molecular Biology Program, University of Vermont, 89 Beaumont Ave, Burlington, VT 05405, USA.

ABSTRACT

Background: Because understanding of the inventory, connectivity and dynamics of the components characterizing the process of coagulation is relatively mature, it has become an attractive target for physiochemical modeling. Such models can potentially improve the design of therapeutics. The prothrombinase complex (composed of the protease factor (F)Xa and its cofactor FVa) plays a central role in this network as the main producer of thrombin, which catalyses both the activation of platelets and the conversion of fibrinogen to fibrin, the main substances of a clot. A key negative feedback loop that prevents clot propagation beyond the site of injury is the thrombin-dependent generation of activated protein C (APC), an enzyme that inactivates FVa, thus neutralizing the prothrombinase complex. APC inactivation of FVa is complex, involving the production of partially active intermediates and "protection" of FVa from APC by both FXa and prothrombin. An empirically validated mathematical model of this process would be useful in advancing the predictive capacity of comprehensive models of coagulation.

Results: A model of human APC inactivation of prothrombinase was constructed in a stepwise fashion by analyzing time courses of FVa inactivation in empirical reaction systems with increasing number of interacting components and generating corresponding model constructs of each reaction system. Reaction mechanisms, rate constants and equilibrium constants informing these model constructs were initially derived from various research groups reporting on APC inactivation of FVa in isolation, or in the presence of FXa or prothrombin. Model predictions were assessed against empirical data measuring the appearance and disappearance of multiple FVa degradation intermediates as well as prothrombinase activity changes, with plasma proteins derived from multiple preparations. Our work integrates previously published findings and through the cooperative analysis of in vitro experiments and mathematical constructs we are able to produce a final validated model that includes 24 chemical reactions and interactions with 14 unique rate constants which describe the flux in concentrations of 24 species.

Conclusion: This study highlights the complexity of the inactivation process and provides a module of equations describing the Protein C pathway that can be integrated into existing comprehensive mathematical models describing tissue factor initiated coagulation.

Show MeSH
Related in: MedlinePlus