Factor Va Is Inactivated by Activated Protein C in the Absence of Cleavage Sites at Arg-306, Arg-506, and Arg-679

Kolfschoten, Marijn van der Neut; Dirven, Richard; Vos, Hans L.; Tans, Guido; Rosing, Jan; Bertina, Rogier M.

doi:10.1074/jbc.m308574200

Cited by 21 publications

(14 citation statements)

References 43 publications

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“…During this work, it was observed that mutagenesis of Arg306, Arg506 and Arg679, did not give complete resistance to APC [22]. In fact, at high APC concentrations, the cleavage pattern appeared to be identical to that of wild-type FVa.…”

Section: Discussionmentioning

confidence: 92%

“…In the study by van der Neut Kolfschoten et al [22], the novel cleavage sites were not identified and conclusions were based on APC degradation of a FVa variant being mutated at positions 306, 506 and 679 and an equation for two cleavage sites. They identified a rapid phase, during which a FVa i intermediate with 80% activity was formed, followed by a slow phase resulting in complete loss of activity.…”

Section: Discussionmentioning

confidence: 99%

“…During studies of the FVa variants, the unexpected observation was made that even when all three APC-cleavage sites in FVa were mutated, the FVa was still sensitive to cleavage by APC [22]. This suggested the presence of at least two previously unknown APC-cleavage sites in FVa, the cleavage pattern on Western blots suggesting that the sites are close to the Arg506 and Arg306 sites.…”

Section: Introductionmentioning

confidence: 99%

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Novel APC‐cleavage sites in FVa provide insights into mechanisms of action of APC and its cofactor protein S

Tran

Dahlbäck

2010

Journal of Thrombosis and Haemostasis

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To cite this article: Tran S, Dahlbä ck B. Novel APC-cleavage sites in FVa provide insights into mechanisms of action of APC and its cofactor protein S. J Thromb Haemost 2010; 8: 129-36.Summary. Background: Activated protein C (APC) inhibits factor Va (FVa) by cleaving at Arg306, Arg506 and Arg679. Protein S serves as cofactor, in particular for the Arg306 site, and a protein S-mediated relocation of the active site of APC closer to the membrane has been proposed as a mechanism. Recently, it was demonstrated that FVa, which was mutated at all three APC-cleavage sites (FVa-306Q/506Q/679Q), could still be cleaved by APC. These sites were close to Arg306 and Arg506 but not further defined. Objective: To identify and characterize the additional APC-cleavage sites in FVa. Methods: The cDNA for FV-306Q/506Q/679Q was used as a template to create FV variants with one or more possible cleavage sites being mutated. The FV variants were expressed and their sensitivity for APC characterized functionally and with Western blotting. Results: The additional APC-cleavage sites were located at Lys309, Arg313, Arg316, Arg317 and Arg505. FVa-306Q/309Q/313Q/316Q/317Q/505Q/506Q/679Q (denoted 8M-FVa) was APC resistant. To investigate individual sites, they were mutated back using 8M-FV as a template. The kinetics of APC-degradation of these variants demonstrated that protein S was equally efficient in enhancing the APC effect for all the novel sites. Conclusions: Multiple APC-cleavage sites close to Arg306 and a single site close to Arg506 were identified. Protein S was equally efficient as APC cofactor for all novel sites. The stimulation by protein S of the Arg505 cleavage argues against a specific protein S-mediated stimulation of cleavage at Arg306 due to relocation of the APC active site closer to the membrane.

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Section: Discussionmentioning

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Novel APC‐cleavage sites in FVa provide insights into mechanisms of action of APC and its cofactor protein S

Tran

Dahlbäck

2010

Journal of Thrombosis and Haemostasis

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“…The 14E11 antibody was described earlier. 24 Anti-human fV monoclonal antibody AHV-5146 25 was obtained from Haematologic Technologies (Essex Junction, VT).…”

Section: Chemicals and Reagentsmentioning

confidence: 99%

Coagulation factor V mediates inhibition of tissue factor signaling by activated protein C in mice

Liang

Kerschen

Basu

et al. 2015

Blood

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Key Points• Factor V and protein S are required for sepsis mortality reduction and suppression of inflammatory gene expression by activated protein C.• The R506Q mutation (Leiden mutation) abrogates the antiinflammatory cofactor function of factor V for activated protein C.The key effector molecule of the natural protein C pathway, activated protein C (aPC), exerts pleiotropic effects on coagulation, fibrinolysis, and inflammation. Coagulation-independent cell signaling by aPC appears to be the predominant mechanism underlying its highly reproducible therapeutic efficacy in most animal models of injury and infection. In this study, using a mouse model of Staphylococcus aureus sepsis, we demonstrate marked disease stage-specific effects of the anticoagulant and cell signaling functions of aPC. aPC resistance of factor (f)V due to the R506Q Leiden mutation protected against detrimental anticoagulant effects of aPC therapy but also abrogated the anti-inflammatory and mortalityreducing effects of the signaling-selective 5A-aPC variant that has minimal anticoagulant function. We found that procofactor V (cleaved by aPC at R506) and protein S were necessary cofactors for the aPC-mediated inhibition of inflammatory tissue-factor signaling. The antiinflammatory cofactor function of fV involved the same structural features that govern its cofactor function for the anticoagulant effects of aPC, yet its anti-inflammatory activities did not involve proteolysis of activated coagulation factors Va and VIIIa. These findings reveal a novel biological function and mechanism of the protein C pathway in which protein S and the aPC-cleaved form of fV are cofactors for anti-inflammatory cell signaling by aPC in the context of endotoxemia and infection. (Blood. 2015;126(21):2415-2423 Introduction Natural aPC exerts pleiotropic effects on coagulation, fibrinolysis, and inflammation (for review, see Esmon, 1 Mosnier and Griffin,2 and Dahlbäck and Villoutreix 3 ) that are predominantly mediated by 2 mechanistically discernable pathways: (1) aPC degrades activated coagulation factor (f)Va and fVIIIa to curtail the formation of thrombin by the plasma coagulation system and (2) when bound to the endothelial protein C receptor (EPCR, ProcR) or the integrin aMb2 (Mac1)(CD11b/CD18), aPC becomes competent for activating the G protein-coupled protease activated receptor (PAR) 1, 2, or 3. PAR activation and crosstalk with other receptors mediate anti-inflammatory and cytoprotective effects of aPC in innate immune cells, vascular endothelium, and other cells (for review, see Weiler,4 Mosnier et al, 5 and Dahlbäck and Villoutreix 6). In animal models of endotoxemia and sepsis, the cell signaling activity of aPC on vascular endothelium and innate immune cells is the predominant therapeutic mechanism of action, and recombinant variants of aPC that are largely devoid of anticoagulant activity but retain signaling function confer vasoprotection, inhibit inflammation, and reduce mortality. 7-9 Although the incidence of microvascular coagulopathies in p...

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“…The quantification of the 1-506 fragment in the experiment containing QGNSEDY-APC and protein S demonstrated a peak at 3 min and then a decline, which may be due to slow cleavage at an unknown site. The existence of one or more such sites in FVa was recently proposed (31).…”

Section: Fig 2 Inactivation Of 306q679qmentioning

confidence: 99%

Enhanced Rate of Cleavage at Arg-306 and Arg-506 in Coagulation Factor Va by Gla Domain-mutated Human-activated Protein C

Sun¹,

Tran²,

Norström

et al. 2004

Journal of Biological Chemistry

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A Gla domain-mutated protein C variant, QGNSEDY, modified at positions 10 -12, 23, 32-33, and 44, having enhanced affinity for negatively charged phospholipid and increased anticoagulant potential, was used to elucidate the importance of the interaction between the Gla domain and the phospholipid for the ability of activated protein C (APC) to inactivate factor Va (FVa). FVa degradation by wild type (WT)-APC and QGNSEDY-APC yielded similar fragments on Western blotting; QGNSEDY-APC was, however, considerably more efficient. The kinetic parameters for individual APC-mediated cleavages in FVa, i.e. at Arg-306 and Arg-506, were investigated at high and low phospholipid concentrations in the presence and absence of protein S. FVa variants 306Q679Q and 506Q679Q, which can only be cleaved at Arg-506 and Arg-306, respectively, were used. In the absence of protein S, QGNSEDY-APC was 17.8-and 4-fold more efficient than WT-APC in cleaving at Arg-306 and Arg-506, respectively, at high phospholipid. Similar values were obtained at low phospholipid. In the presence of protein S, QGNSEDY-APC was 6.8-and 3.2-fold more active than WT-APC in cleaving at Arg-306 and Arg-506, respectively, at high phospholipid. At low phospholipid, the corresponding values were 14-and 6.5-fold. In conclusion, the modification of the Gla domain in QGNSEDY-APC yielded increased rates of cleavage at both sites in FVa, the increase being particularly pronounced for the Arg-306 site in the absence of protein S. The results obtained with QGNSEDY-APC provide insights into the importance of the APC-phospholipid interaction for the APC-mediated cleavages at Arg-306 and Arg-506 in FVa.Blood coagulation comprises a series of enzymatic reactions, many of which take place on negatively charged phospholipid membranes upon which enzymes and cofactors form highly efficient complexes (1-3). Several of the coagulation enzymes, e.g. factor IX (FIX), factor X (FX) and prothrombin, are vitamin K-dependent and thus contain a ␥-carboxyl glutamic acid (Gla)-rich domain, which binds to negatively charged phospholipid (4). Activated factor IX and activated factor X (FXa) 1 interact with their respective cofactors, activated factor VIII (FVIIIa) and activated factor V (FVa), on the phospholipid surface, thus forming the tenase and prothrombinase complexes that activate FX and prothrombin, respectively (1, 3, 5). The cofactors FVIIIa and FVa provide several orders of magnitude enhancement to the efficiency of their respective enzyme.The protein C anticoagulant pathway provides efficient and specific regulation of the coagulation system by inhibiting the activity of the two cofactors, FVIIIa and FVa (3,[6][7][8]. Protein C, the key component of the pathway, is converted to activated protein C (APC) on the surface of endothelial cells by thrombin bound to the membrane protein thrombomodulin. The specific proteolysis of FVIIIa and FVa by APC is stimulated by protein S, which serves as an APC cofactor (9 -11). Protein C and protein S are both vitamin K-dependent proteins contain...

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Factor Va Is Inactivated by Activated Protein C in the Absence of Cleavage Sites at Arg-306, Arg-506, and Arg-679

Cited by 21 publications

References 43 publications

Novel APC‐cleavage sites in FVa provide insights into mechanisms of action of APC and its cofactor protein S

Novel APC‐cleavage sites in FVa provide insights into mechanisms of action of APC and its cofactor protein S

Coagulation factor V mediates inhibition of tissue factor signaling by activated protein C in mice

Enhanced Rate of Cleavage at Arg-306 and Arg-506 in Coagulation Factor Va by Gla Domain-mutated Human-activated Protein C

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