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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 ofProthrombinaseComponents on APC Proteolysis of FVa. Normalized densitometry data measuring the disappearance of the FVaHC are from reactions containing 20 μM PC:PS, 2.0 nM APC and 20 nM FVa (filled circles, N = 6), with either 30 nM active site blocked FXa (open circles, N = 5), 1.4 μM prothrombin (filled triangles, N = 4), or 30 nM active site blocked FXa + 1.4 μM prothrombin (open triangles, N = 4). Solid lines are fits of the empirical data to monophasic exponential decay curves (FVa, FVa + FXa*, and FVa + PT) or a linear decay (FVa + FXa* + PT).
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Figure 8: Effect ofProthrombinaseComponents on APC Proteolysis of FVa. Normalized densitometry data measuring the disappearance of the FVaHC are from reactions containing 20 μM PC:PS, 2.0 nM APC and 20 nM FVa (filled circles, N = 6), with either 30 nM active site blocked FXa (open circles, N = 5), 1.4 μM prothrombin (filled triangles, N = 4), or 30 nM active site blocked FXa + 1.4 μM prothrombin (open triangles, N = 4). Solid lines are fits of the empirical data to monophasic exponential decay curves (FVa, FVa + FXa*, and FVa + PT) or a linear decay (FVa + FXa* + PT).

Mentions: In order to measure the cumulative protection prothrombin and saturating levels of active site blocked FXa have on the APC inactivation of FVa, reactions were carried out at physiological concentrations of FVa (20 nM) and prothrombin (1.4 μM), with saturating levels of FXa* (30 nM) on phospholipid vesicles (20 μM). Figure 8 summarizes the time course data for FVaHC proteolysis by 2.0 nM APC for reactions constructed with FVa, FVa + FXa*, FVa + PT, and FVa + FXa* + PT. The combined presence of prothrombin and FXa* provided maximum protection against APC inactivation.


Modeling of human factor Va inactivation by activated protein C.

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

Effect ofProthrombinaseComponents on APC Proteolysis of FVa. Normalized densitometry data measuring the disappearance of the FVaHC are from reactions containing 20 μM PC:PS, 2.0 nM APC and 20 nM FVa (filled circles, N = 6), with either 30 nM active site blocked FXa (open circles, N = 5), 1.4 μM prothrombin (filled triangles, N = 4), or 30 nM active site blocked FXa + 1.4 μM prothrombin (open triangles, N = 4). Solid lines are fits of the empirical data to monophasic exponential decay curves (FVa, FVa + FXa*, and FVa + PT) or a linear decay (FVa + FXa* + PT).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3403913&req=5

Figure 8: Effect ofProthrombinaseComponents on APC Proteolysis of FVa. Normalized densitometry data measuring the disappearance of the FVaHC are from reactions containing 20 μM PC:PS, 2.0 nM APC and 20 nM FVa (filled circles, N = 6), with either 30 nM active site blocked FXa (open circles, N = 5), 1.4 μM prothrombin (filled triangles, N = 4), or 30 nM active site blocked FXa + 1.4 μM prothrombin (open triangles, N = 4). Solid lines are fits of the empirical data to monophasic exponential decay curves (FVa, FVa + FXa*, and FVa + PT) or a linear decay (FVa + FXa* + PT).
Mentions: In order to measure the cumulative protection prothrombin and saturating levels of active site blocked FXa have on the APC inactivation of FVa, reactions were carried out at physiological concentrations of FVa (20 nM) and prothrombin (1.4 μM), with saturating levels of FXa* (30 nM) on phospholipid vesicles (20 μM). Figure 8 summarizes the time course data for FVaHC proteolysis by 2.0 nM APC for reactions constructed with FVa, FVa + FXa*, FVa + PT, and FVa + FXa* + PT. The combined presence of prothrombin and FXa* provided maximum protection against APC inactivation.

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