Dataset Information

The roles of factor Va and protein S in formation of the activated protein C/protein S/factor Va inactivation complex.

ABSTRACT:

Background

Activated protein C (APC)-mediated inactivation of factor (F)Va is greatly enhanced by protein S. For inactivation to occur, a trimolecular complex among FVa, APC, and protein S must form on the phospholipid membrane. However, direct demonstration of complex formation has proven elusive.

Objectives

To elucidate the nature of the phospholipid-dependent interactions among APC, protein S, and FVa.

Methods

We evaluated binding of active site blocked APC to phospholipid-coated magnetic beads in the presence and absence of protein S and/or FVa. The importance of protein S and FV residues were evaluated functionally.

Results

Activated protein C alone bound weakly to phospholipids. Protein S mildly enhanced APC binding to phospholipid surfaces, whereas FVa did not. However, FVa together with protein S enhanced APC binding (>14-fold), demonstrating formation of an APC/protein S/FVa complex. C4b binding protein-bound protein S failed to enhance APC binding, agreeing with its reduced APC cofactor function. Protein S variants (E36A and D95A) with reduced APC cofactor function exhibited essentially normal augmentation of APC binding to phospholipids, but diminished APC/protein S/FVa complex formation, suggesting involvement in interactions dependent upon FVa. Similarly, FVa^Nara (W1920R), an APC-resistant FV variant, also did not efficiently incorporate into the trimolecular complex as efficiently as wild-type FVa. FVa inactivation assays suggested that the mutation impairs its affinity for phospholipid membranes and with protein S within the complex.

Conclusions

FVa plays a central role in the formation of its inactivation complex. Furthermore, membrane proximal interactions among FVa, APC, and protein S are essential for its cofactor function.

SUBMITTER: Gierula M

PROVIDER: S-EPMC6916587 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

The roles of factor Va and protein S in formation of the activated protein C/protein S/factor Va inactivation complex.

Gierula Magdalena M Salles-Crawley Isabelle I II Santamaria Salvatore S Teraz-Orosz Adrienn A Crawley James T B JTB Crawley James T B JTB Lane David A DA Ahnström Josefin J

Journal of thrombosis and haemostasis : JTH 20190809 12

<h4>Background</h4>Activated protein C (APC)-mediated inactivation of factor (F)Va is greatly enhanced by protein S. For inactivation to occur, a trimolecular complex among FVa, APC, and protein S must form on the phospholipid membrane. However, direct demonstration of complex formation has proven elusive.<h4>Objectives</h4>To elucidate the nature of the phospholipid-dependent interactions among APC, protein S, and FVa.<h4>Methods</h4>We evaluated binding of active site blocked APC to phospholip ...[more]

PMID: 31364267

Similar Datasets

Project description: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.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.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.

Dataset Information

The roles of factor Va and protein S in formation of the activated protein C/protein S/factor Va inactivation complex.

Background

Objectives

Methods

Results

Conclusions

Publications

The roles of factor Va and protein S in formation of the activated protein C/protein S/factor Va inactivation complex.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets