Enhancing the Activity of Protein C by Mutagenesis To Improve the Membrane-Binding Site:  Studies Related to Proline-10

Shen, Lei; Shah, Anup; Dahlbäck, Björn; Nelsestuen, Gary L.

doi:10.1021/bi971730v

Cited by 32 publications

(54 citation statements)

References 41 publications

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“…Human ␣-thrombin was from Haematologic Technologies, Inc. (Essex Junction, VT). Recombinant human wild-type protein C was expressed, isolated, and activated as described (37), and the APC concentration was determined by chromogenic substrate S2366. Human protein S was purified as reported (38) with minor modifications, as described (39).…”

Section: Methodsmentioning

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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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: Methodsmentioning

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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“…6 We have previously reported that site-directed mutagenesis of positions 10 to 12, 32, and 33 in protein C result in protein C variants with enhanced membranebinding ability and anticoagulant activity. 7,8 We now report on novel Gla domain mutations resulting in the creation of APC variants with very high anticoagulant activity. The results provide insights into the link between sequence variations of the Gla domain and the membrane-binding properties and the importance of membrane binding for expression of APC anticoagulant activity.…”

Section: Introductionmentioning

confidence: 99%

Gla domain–mutated human protein C exhibiting enhanced anticoagulant activity and increased phospholipid binding

Sun

Shen

Dahlbäck

2003

Blood

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Protein C is a member of the vitamin Kdependent protein family. Proteins in this family have similar ␥-carboxyglutamic acid (Gla)-rich domains, but their affinities for negatively charged phospholipid membranes vary more than 1000-fold. We have shown that it is possible to enhance anticoagulant activity and membrane affinity of protein C by selective mutagenesis of the Gla domain. In this study, 3 new mutants, Q10G11N12 ( IntroductionNegatively charged phospholipid membranes play an important role in blood coagulation, providing a suitable surface for the assembly of enzyme-cofactor complexes, which efficiently convert circulating zymogens to active enzymes. Phosphatidylserine (PS) is a key component in the negatively charged membrane that supports the clotting reactions. [1][2][3] In most cells, PS is normally mainly present in the inner leaflet of the cell membrane, but certain cellular processes such as platelet activation result in the exposure of PS on the cell surface. The vitamin K-dependent coagulation proteins in plasma bind to the surface of negatively charged phospholipid with a common mechanism involving the ␥-carboxyglutamic acid (Gla)-rich domains of these proteins. 4 Vitamin K is required in the posttranslational carboxylation of glutamic acid residues to Gla. 5 The Gla domains comprise approximately 45 amino acid residues, and they are structurally very similar in the different vitamin K-dependent proteins. 6 Despite the extensive structural similarity between different Gla domains, they bind negatively charged phospholipid with very different affinity, with dissociation constants (K d 's) ranging more than 1000-fold. 6 This variation in phospholipid binding ability is related to amino acid sequence differences at relatively few positions. This has made it possible to modulate the phospholipid binding abilities of this group of proteins by site-directed mutagenesis. [7][8][9] The nonvitamin K-dependent cofactors of blood coagulation also interact with the phospholipid membranes, certain proteins being membrane spanning, whereas others, such as factor V (FV) and factor VIII (FVIII), contain binding sites for negatively charged phospholipid membranes. 2,10 FV circulates as a procofactor to activated FV (FVa), which functions as a cofactor to activated FX (FXa) in the activation of prothrombin to thrombin. Activated FVIII (FVIIIa) serves a similar function in the FIXa-mediated activation of FX. 2,3 The protein C anticoagulant system, which includes the vitamin K-dependent proteins protein C and protein S, regulates the activities of FVa and FVIIIa by limited proteolysis (reviewed by Dahlbäck et al 11 and Esmon et al 12 ). Protein C is activated on the endothelial cell surface by the thrombin-thrombomodulin complex, and the serine protease activated protein C (APC) down-regulates coagulation by cleaving a limited number of peptide bonds in each of FVa and FVIIIa. Protein S serves as a cofactor to APC by increasing the affinity of APC for the membrane 13 and also by changing the distance of the activ...

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“…This suggestion has been supported by mutations of the Gla domain that affect membrane affinity. For example, the P10H mutant of bovine protein C shows an ϳ10-fold enhancement in membrane affinity, whereas the H10P mutant of human protein C shows an ϳ3-fold decline in membrane affinity (9). Although a major difference among the vitamin K-dependent proteins is a Gla residue versus another amino acid at position 32, the Q32E mutation of protein C has no impact on membrane affinity (10,11).…”

mentioning

confidence: 99%

Mutagenesis of the γ-Carboxyglutamic Acid Domain of Human Factor VII to Generate Maximum Enhancement of the Membrane Contact Site

Harvey

Stone

Martinez

et al. 2003

Journal of Biological Chemistry

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Site-directed mutagenesis of the 40 N-terminal residues (␥-carboxyglutamic acid domain) of blood clotting factor VII was carried out to identify sites that improve membrane affinity. Improvements and degree of change included P10Q (2-fold), K32E (13-fold), and insertion of Tyr at position 4 (2-fold). Two other beneficial changes, D33F (2-fold) and A34E (1.5-fold), may exert their impact via influence of K32E. The modification D33E (5.2-fold) also resulted in substantial improvement. The combined mutant with highest affinity, (Y4)P10Q/K32E/D33F/ A34E, showed 150 -296-fold enhancement over wild-type factor VIIa, depending on the assay used. Undercarboxylation of Glu residues at positions 33 and 34 may result in an underestimate of the true contributions of ␥-carboxyglutamic acid at these positions. Except for the Tyr 4 mutant, all other beneficial mutations were located on the same surface of the protein, suggesting a possible membrane contact region. An initial screening assay was developed that provided faithful evaluation of mutants in crude mixtures. Overall, the results suggest features of membrane binding by vitamin K-dependent proteins and provide reagents that may prove useful for research and therapy.

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Enhancing the Activity of Protein C by Mutagenesis To Improve the Membrane-Binding Site: Studies Related to Proline-10

Cited by 32 publications

References 41 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

Gla domain–mutated human protein C exhibiting enhanced anticoagulant activity and increased phospholipid binding

Mutagenesis of the γ-Carboxyglutamic Acid Domain of Human Factor VII to Generate Maximum Enhancement of the Membrane Contact Site

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