Role of Proexosite I in Factor Va-dependent Substrate Interactions of Prothrombin Activation

Anderson, Patricia J.; Nesset, Anna; Dharmawardana, Kumudini R.; Bock, Paul E.

doi:10.1074/jbc.m001255200

Cited by 75 publications

(133 citation statements)

References 35 publications

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“…The Gla domain of PT is known to contribute to the FVa binding (12). In addition, both kringle domains and several sites in the serine protease domain of prothrombin have been shown to be involved in the PT-FVa interaction (11,13,15,17,18). To date, there are no reports suggesting that APC interacts directly with PT, and it appears valid to conclude that the PT-mediated inhibition of FVa degradation depends on interactions between FVa and PT rather than interactions between APC and PT.…”

Section: Discussionmentioning

confidence: 99%

“…The two proteins bind to each other with a K d of about 8 M in the absence of negatively charged phospholipids (10,11), but the affinity is probably enhanced by the presence of a negatively charged phospholipid surface. In PT, the Gla domain, both kringle domains, and the serine protease domain participate in the binding of FVa (12)(13)(14)(15)(16)(17)(18). In FVa, the heavy chain is involved in the binding to PT, but a more detailed mapping of the binding site in FVa has not been accomplished (14).…”

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confidence: 99%

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Effects of Prothrombin on the Individual Activated Protein C-mediated Cleavages of Coagulation Factor Va

Tran¹,

Norström²,

Dahlbäck

2008

Journal of Biological Chemistry

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The factor Va (FVa) inactivation by activated protein C (APC), mediated by cleavages at Arg 306 and Arg 506 in FVa, is inhibited by both factor Xa (FXa) and prothrombin. Although FXa is known to specifically inhibit the Arg 506 cleavage, the effect of prothrombin has not been confined to one cleavage site. We used recombinant FV variants, FV:R506Q/R679Q and FV:R306Q/R679Q, to investigate the effect of prothrombin on the individual cleavage sites. The APC-mediated FVa inhibition was monitored by a prothrombinase-based FVa assay, and apparent first order rate constants were calculated for each of the cleavage sites both in the presence and absence of prothrombin. Prothrombin impaired cleavages at both Arg 306 and Arg 506 and the inhibition correlated with a delayed appearance of proteolytic products on Western blots. Almost complete inhibition was obtained at around 3 M prothrombin, whereas half-maximal inhibition was obtained at 0.7 M prothrombin. After cleavage of prothrombin by thrombin, the inhibitory activity was lost. The inhibitory effect of prothrombin on APC-mediated inhibition of FVa was seen both in the presence and absence of protein S, but in particular for the Arg 306 sites, it was more pronounced in the presence of protein S. Thus, prothrombin inhibition of APC inactivation of FVa appears to be due to both impaired APC function and decreased APC cofactor function of protein S. In conclusion, FVa, being part of the prothrombinase complex, is protected from APC by both FXa and prothrombin. Release of products of prothrombin activation from the prothrombinase complex would alleviate the protection, allowing APC-mediated inactivation of FVa.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Effects of Prothrombin on the Individual Activated Protein C-mediated Cleavages of Coagulation Factor Va

Tran¹,

Norström²,

Dahlbäck

2008

Journal of Biological Chemistry

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“…Therefore, proexosite 1 consists in a binding site for FVa (Chen et al 2003), and the interaction with C-terminal hirudin decreases the productive recognition of prothrombin as substrate by FXa in the presence but not in the absence of its protein cofactor (Anderson et al 2000b).…”

Section: Prothrombin and Thrombinmentioning

confidence: 99%

Targeting exosites on blood coagulation proteases

Monteiro

2005

An. Acad. Bras. Ciênc.

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The high specificity of blood coagulation proteases has been attributed not only to residues surrounding the active site but also to other surface domains that are involved in recognizing and interacting with macromolecular substrates and inhibitors. Specific blood coagulation inhibitors obtained from exogenous sources such as blood sucking salivary glands and snake venoms have been identified. Some of these inhibitors interact with exosites on coagulation enzymes. Two examples are discussed in this short revision. Bothrojaracin is a snake venom-derived protein that binds to thrombin exosites 1 and 2. Complex formation impairs several exosite-dependent activities of thrombin including fibrinogen cleavage and platelet activation. Bothrojaracin also interacts with proexosite 1 on prothrombin thus decreasing the zymogen activation by the prothrombinase complex (FXa/FVa). Ixolaris is a two Kunitz tick salivary gland inhibitor, that is homologous to tissue factor pathway inhibitor. Recently it was demonstrated that ixolaris binds to heparin-binding exosite of FXa, thus impairing the recognition of prothrombin by the enzyme. In addition, ixolaris interacts with FX possibly through the heparin-binding proexosite. Differently from FX, the ixolaris-FX complex is not recognized as substrate by the intrinsic tenase complex (FIXa/FVIIIa). We conclude that these inhibitors may serve as tools for the study of coagulation exosites as well as prototypes for new anticoagulant drugs.

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“…In contrast, positive charges within proexosite-1 of prothrombin are essential for its activation by prothrombinase (36,37), whereas E39K prothrombin is normally activated (17). In addition, proexosite-1-specific ligands such as the sulfated C-terminal tail of hirudin block prothrombin activation by prothrombinase (13,38).…”

Section: Plasma (Ex Vivo) Studies Of Fxa and Derivatives-mentioning

confidence: 99%

The Elusive Role of the Potential Factor X Cation-binding Exosite-1 in Substrate and Inhibitor Interactions

Bianchini

Pike

Bonniec

2004

Journal of Biological Chemistry

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Coagulation factor X (FX) 1 is a vitamin K-dependent zymogen activated into FXa by cleavage at a single bond (Arg 15 -Ile 16 in the chymotrypsin numbering system 2 ) hydrolyzed by either of two complexes: FVIIa bound to tissue factor (TF), which triggers coagulation after injury, or FIXa associated with FVIIIa, which amplifies thrombin production after initiation of coagulation. FXa is the only endogenous prothrombin activator, but substantial thrombin production occurs only within the prothrombinase complex, where FXa binds to FVa in the presence of calcium on an appropriate phospholipid surface. Two inhibitors control FXa activity, viz. the tissue factor pathway inhibitor (TFPI) and antithrombin.Extended binding sites (exosites) play a crucial role in virtually any substrate, cofactor, or inhibitor recognition within the coagulation cascade. Perhaps best characterized is the case of thrombin taking advantage of two exosites for extended interactions in numerous functions (1). Exosite-1 is formed by a set of basic residues comprising loops 34 -40 and 70 -80, which neighbor each other in the folded structure (2); it interacts with fibrinogen, thrombomodulin, platelet activator receptor-1, FV, FVa, heparin cofactor II, and hirudin. Exosite-2, comprising loop 91-102 and helices 165-173 and 233-245 (3), is also formed by a set of basic residues that interact with heparin, with the chondroitin sulfate of thrombomodulin, and within prothrombin with kringle-2 (4). In FVII, the region corresponding to exosite-1 of thrombin is critical for FX activation (5, 6); in FIXa, this region is also important for FX activation in the absence of FVIII and for its inhibition by antithrombin in the absence of heparin (7). In contrast to thrombin, FVIIa, FIXa, and FXa share a negatively charged patch within segment 70 -80, whereas FXa is unique in that both loops 34 -40 and 70 -80 are negatively charged. Thus, the region of FXa corresponding to exosite-1 of thrombin forms a negative cluster, raising the question as to whether it could constitute a functional exosite. FXa has a unique macromolecule substrate, but exosites may also participate in its inhibition and/or in the activation of its zymogen. In fact, a number of studies have established that exosite(s) must be critical in several FXa functions. Electrostatic interactions involving loop 34 -40 of FXa appear to play a significant role in binding to the second Kunitz domain of TFPI (8,9). FXa also appears to interact with a complementary surface on pentasaccharide-activated antithrombin (10), and the region of FXa topologically equivalent to exosite-2 of thrombin seems to be involved in the binding of heparin and FVa (11). Above all, prothrombin recognition by prothrombinase undoubtedly involves one or more exosites on .In a previous study (15), we reported that the catalytic groove of FXa is minimally selective with unconstrained peptides and that FVa has little effect, if any, on this selectivity. These two observations therefore also favor the hypothesis that FXa uses seconda...

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Role of Proexosite I in Factor Va-dependent Substrate Interactions of Prothrombin Activation

Cited by 75 publications

References 35 publications

Effects of Prothrombin on the Individual Activated Protein C-mediated Cleavages of Coagulation Factor Va

Effects of Prothrombin on the Individual Activated Protein C-mediated Cleavages of Coagulation Factor Va

Targeting exosites on blood coagulation proteases

The Elusive Role of the Potential Factor X Cation-binding Exosite-1 in Substrate and Inhibitor Interactions

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