Prothrombin amino terminal region helps protect coagulation factor Va from proteolytic inactivation by activated protein C

Yegneswaran, Subramanian; Nguyen, Phuong Mai; Gale, Andrew J.; Griffin, John H.

doi:10.1160/th08-07-0491

Cited by 11 publications

(18 citation statements)

References 46 publications

(41 reference statements)

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“…Inhibition of prothrombinase by APC(S360A) is enhanced at low FXa concentrations. Prothrombin effectively abolished the anticoagulant effect APC(S360A) in the model system, which is in agreement with recent results (34). These observations are in line with an anticoagulant mechanism in which APC(S360A) inhibits the cofactor activity of FVa by competing with FXa and prothrombin for binding to FVa.…”

Section: Discussionsupporting

confidence: 92%

“…The binding regions on FVa for prothrombin have been less well described. However, the fact that prothrombin inhibits APC-catalyzed FVa inactivation in the absence of FXa (33)(34) suggests that APC and prothrombin also share binding sites on FVa. The protective effect of prothrombin in the presence of OG-FXa can be explained by formation of a nonproductive ternary OG-FXa⅐FVa⅐prothrombin complex.…”

Section: Discussionmentioning

confidence: 99%

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Inhibition of Thrombin Formation by Active Site Mutated (S360A) Activated Protein C

Nicolaes

Bock

Segers

et al. 2010

Journal of Biological Chemistry

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Activated protein C (APC) down-regulates thrombin formation through proteolytic inactivation of factor Human activated protein C (APC)2 is formed after activation of zymogen (protein C) on endothelial cells by the thrombin⅐ thrombomodulin (TM) complex. APC is a serine protease that, together with its cofactor, protein S, down-regulates thrombin formation by inactivating the activated forms of the coagulation factors V and VIII (FVa and FVIIIa) via limited proteolysis. The protein C pathway is vital to normal hemostasis, as is indicated by the severe thrombotic events observed in individuals with homozygous or compound homozygous protein C deficiency (1, 2). Heterozygous individuals have an increased risk for thromboembolic events (3, 4). Another commonly observed (ϳ5% of Caucasians) impairment of the anticoagulant properties of protein C is associated with the factor V Leiden missense mutation (FV(R506Q)), causing the ablation of one of two APC cleavage sites, the other being at Arg 306 . Carriership of the FV Leiden mutation is the most common hereditary risk factor of thromboembolic disease.Interactions between APC and its substrates (FVa and FVIIIa) are largely dependent on three exposed surface loops (the 37-loop: Lys 191(37) ; the 60-loop: Lys 217(62) and Lys 218(63) ; and the 70 -80-loop: Arg 229(74) , Arg 230(75) , and Lys 233(78) ) that together form an electropositive exosite on APC (5-9). Protein C numbering is used throughout the text, followed by chymotrypsinogen numbering in parentheses. Several epitopes on the surface of FVa contribute to the interaction with APC at the Arg 506 and the Arg 306 cleavage sites (10, 11). Mutagenesis studies suggest the presence of an extended exosite on FVa near Arg 506 , which is much less prominent around the Arg 306 cleavage site (10). These data are in line with three-dimensional molecular models for the complexes between FVa and APC (at Arg 506 and Arg 306 ), which suggest that exosite-mediated contacts are more important for docking of APC at Arg 506 than for docking at Arg 306 , by virtue of the extended character of the surface loop in which Arg 306 is located (12). Much less information is available about the interaction between APC and FVIIIa. Manithody et al. (13), however, have reported that the same basic residues in the 37-loop and the calcium-binding 70 -80-loop of APC are also critical for recognition and inactivation of factor VIIIa.The affinity of APC for FVa has not been measured directly but can be inferred from the apparent K m for the proteolysis at Arg 506 in native FVa of between 2 and 20 nM and for cleavage at

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Inhibition of Thrombin Formation by Active Site Mutated (S360A) Activated Protein C

Nicolaes

Bock

Segers

et al. 2010

Journal of Biological Chemistry

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“…Apart from protein S, several other factors modulate FVa inactivation in vivo . In particular, when FVa is incorporated in the prothrombinase complex, it is protected from APC‐mediated inactivation by interactions with FXa, which specifically protects the Arg 506 cleavage site [18,19], and with prothrombin, which inhibits cleavage at both Arg 306 and Arg 506 [20–22]. These protective effects are abrogated by protein S [19,21].…”

Section: The Protein C Anticoagulant Pathwaymentioning

confidence: 99%

APC resistance: biological basis and acquired influences

Castoldi

Rosing

2010

Journal of Thrombosis and Haemostasis

109

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To cite this article: Castoldi E, Rosing J. APC resistance: biological basis and acquired influences. J Thromb Haemost 2010; 8: 445-53.Summary. Proteolytic inactivation of factors Va (FVa) and VIIIa (FVIIIa) by activated protein C (APC) and its cofactors protein S and factor V (FV) is a key process in the physiological down-regulation of blood coagulation. Functional abnormalities of this pathway, which manifest themselves in vitro as a poor anticoagulant response of plasma to added APC (APC resistance), are prevalent in the general population and are associated with an increased risk of venous thrombosis. APC resistance was originally discovered in thrombophilic families and later shown to be associated with the common FV Arg506Gln (FV Leiden ) mutation, which abolishes one of the APC-cleavage sites in FV. Although FV Leiden is the major cause of hereditary APC resistance, it is becoming increasingly clear that several other genetic and acquired conditions contribute to APC resistance and thereby increase the risk of venous thrombosis. This paper reviews the multifactorial etiology of APC resistance and discusses its clinical implications.

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“…Guinto and Esmon reported that prothrombin protects the intact FVa molecule by competing with APC for binding to FVa [58]; more recent studies have reported a K D of between 500–700 nM for FVa and prothrombin [39,40]. …”

Section: Resultsmentioning

confidence: 99%

Modeling of human factor Va inactivation by activated protein C

et al. 2012

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BackgroundBecause 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.ResultsA 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.ConclusionThis 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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Prothrombin amino terminal region helps protect coagulation factor Va from proteolytic inactivation by activated protein C

Cited by 11 publications

References 46 publications

Inhibition of Thrombin Formation by Active Site Mutated (S360A) Activated Protein C

Inhibition of Thrombin Formation by Active Site Mutated (S360A) Activated Protein C

APC resistance: biological basis and acquired influences

Modeling of human factor Va inactivation by activated protein C

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