Importance of Protein S and Phospholipid for Activated Protein C-mediated Cleavages in Factor Va

Norström, Eva; Steen, Mårten; Tran, Sinh; Dahlbäck, Björn

doi:10.1074/jbc.m303829200

Cited by 66 publications

(82 citation statements)

References 37 publications

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“…To study the influence of PT on the Arg 306 and Arg 506 cleavages, we used FV variants FV:R506Q/R679Q and FV:R306Q/ R679Q, respectively (32,33,40). These mutants enabled us to investigate each cleavage site separately and to calculate cleavage rates in the presence and absence of PT.…”

Section: Discussionmentioning

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%

“…Protein S is an important cofactor to APC, mainly enhancing the cleavage at Arg 306 (23,32,33). In contrast, FXa inhibits the APC-mediated cleavage at Arg 506 (23,33).…”

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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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“…Protein S enhancement of APC-mediated inactivation of a previously prepared and reported double mutant of FV, FV R506Q/R679Q, 31 was assessed by determining the loss of FVa activity as described previously. 26,27 Briefly, FV R506Q/R679Q was activated by human thrombin followed by the addition of hirudin.…”

Section: Protein S-dependent Apc-mediated Fva Inactivation Assaymentioning

confidence: 99%

TFPI cofactor function of protein S: essential role of the protein S SHBG-like domain

Reglińska-Matveyev

Andersson

Rezende

et al. 2014

Blood

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Key Points• The protein S SHBG-like domain and, more specifically, its LG1 subunit are important for binding and enhancement of TFPI.• TFPI binding to the protein S SHBG-like domain likely positions TFPI Kunitz domain 2 for optimal interaction with the active site of FXa.Protein S is a cofactor for tissue factor pathway inhibitor (TFPI), accelerating the inhibition of activated factor X (FXa). TFPI Kunitz domain 3 residue Glu226 is essential for enhancement of TFPI by protein S. To investigate the complementary functional interaction site on protein S, we screened 44 protein S point, composite or domain swap variants spanning the whole protein S molecule for their TFPI cofactor function using a thrombin generation assay. Of these variants, two protein S/growth arrestspecific 6 chimeras, with either the whole sex hormone-binding globulin (SHBG)-like domain (Val243-Ser635; chimera III) or the SHBG laminin G-type 1 subunit (Ser283-Val459; chimera I), respectively, substituted by the corresponding domain in growth arrest-specific 6, were unable to enhance TFPI. The importance of the protein S SHBG-like domain (and its laminin G-type 1 subunit) for binding and enhancement of TFPI was confirmed in FXa inhibition assays and using surface plasmon resonance. In addition, protein S bound to C4b binding protein showed greatly reduced enhancement of TFPI-mediated inhibition of FXa compared with free protein S. We show that binding of TFPI to the protein S SHBG-like domain enables TFPI to interact optimally with FXa on a phospholipid membrane. (Blood. 2014;123(25):3979-3987) IntroductionTissue factor pathway inhibitor (TFPI) is a ;41 kDa Kunitz-type protease inhibitor that consists of an acidic amino-terminal polypeptide, followed by 3 tandem Kunitz-type domains (Kunitz domains 1, 2, and 3) and a basic carboxy (C)-terminal tail. 1 It circulates in plasma at a concentration of ;1.6 nM. 2,3 The majority (;80%) of plasma TFPI is truncated and bound to lipoproteins, ;5% is localized in storage granules within platelets, ;5% circulates as free truncated variants, and only around 10% is considered to be free full-length TFPI, 4 with this having the greatest anticoagulant activity. [5][6][7] TFPI and its cofactor protein S downregulate tissue factor (TF)-induced thrombin generation, and a deficiency of either protein has been linked to an increased risk of venous thrombosis. [8][9][10] TFPI specifically inhibits the initiation of coagulation through direct binding and inhibition of factor Xa (FXa) and, in a FXa-dependent manner, inhibition of the TF/factor VIIa (FVIIa) complex by forming a quarternary TF/FVIIa/FXa/TFPI complex. 11,12 The P1 residue in the Kunitz domain 2 of TFPI is required for binding to FXa, whereas the P1 residue in Kunitz domain 1 is required for binding and inhibition of TF/FVIIa. 13 The kinetic mechanism of FXa inhibition by TFPI is described as a 2-step process where TFPI first forms an immediate encounter complex with FXa (FXa/TFPI), followed by a slow isomerization into a final tight complex (FXa/TFPI*...

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“…The importance of specific cleavages has been clarified using point mutants of fVa where Arg 506 and Arg 306 have been replaced with Gln, and the A2 domain has been covalently cross-linked to the A3 domain (7)(8)(9)(10). A partial loss of activity occurs when APC cleaves fVa at Arg 506 (ϳ30% loss in fXa cofactor activity), whereas complete inactivation of fVa requires the cleavage at Arg…”

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Factor Va Residues 311–325 Represent an Activated Protein C Binding Region

Yegneswaran

Kojima

Nguyen

et al. 2007

Journal of Biological Chemistry

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Importance of Protein S and Phospholipid for Activated Protein C-mediated Cleavages in Factor Va

Cited by 66 publications

References 37 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

TFPI cofactor function of protein S: essential role of the protein S SHBG-like domain

Factor Va Residues 311–325 Represent an Activated Protein C Binding Region

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