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Tilman M. Hackeng - One of the best experts on this subject based on the ideXlab platform.
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new functional aSSayS to Selectively quantify apc and tfpi cofactor activitieS of Protein S in plaSma
2016Co-Authors: Nahla Alshaikh, Jan Rosing, Elisabetta Castoldi, Paolo Simioni, Stella Thomassen, Tilman M. HackengAbstract:Background: Protein S iS an important anticoagulant in the down-regulation of blood coagulation by acting aS cofactor for two main regulatorS of coagulation. Protein S actS aS a cofactor for activated Protein C (APC) in the inactivation of the procoagulantS FVa and FVIIIa and for TFPI in the formation of the TFPI-FXa encounter complex that SubSequently inactivateS TF-FVIIa. Several StudieS have indicated that Protein S deficiency iS aSSociated with increaSed riSkS of venouS thromboSiS. AimS: Current Protein S functional aSSayS are influenced by plaSma determinantS Such aS FVLeiden. The aim of the Study waS to develop thrombin generation baSed aSSayS that enable quantification of both the APC- and TFPI-cofactor activitieS of Protein S in plaSma whereaS the outcome of the aSSayS Should not be affected by the preSence of FVLeiden or other plaSma variableS. MethodS: APC- and TFPI-cofactor activitieS of Protein S in plaSma were meaSured uSing calibrated automated thrombography (CAT) in Protein S-depleted plaSma Supplemented with a Small amount of Sample plaSma either in the preSence of anti-TFPI antibodieS and APC (APC-cofactor activity) or at exceSS full-length TFPI without APC (TFPI-cofactor activity). Total and free Protein S levelS in plaSma were meaSured by ELISA9S. ReSultS: Average APC-cofactor activitieS of Protein S were 113%, 122%, and 87% in plaSma from normal individualS (n=10), FV Leiden heterozygoteS (n=4), and FV Leiden homozygoteS (n=2), reSpectively, while the average APC-cofactor activity of Protein S in plaSma from heterozygouS Protein S-deficient individualS (n=21) waS Significantly lower (55%). Similar trendS were obServed for the TFPI-cofactor activity of Protein S, with averageS of 108%, 112%, and 97% in plaSma from individualS with normal Protein S levelS and different FV Leiden genotypeS, and 64% in plaSma from Protein S-deficient patientS. APC-cofactor activitieS of Protein S correlated Significantly with free and total Protein S antigen levelS, while TFPI-cofactor activitieS correlated leSS with Protein S antigen levelS. Summary/ConcluSion: We have developed functional Protein S aSSayS that meaSure both the TFPI- and APC-cofactor activitieS of Protein S in plaSma and that are not affected by the FV Leiden mutation or other plaSma influenceS. DiScloSureS No relevant conflictS of intereSt to declare.
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Similar hypercoagulable State and thromboSiS riSk in type i and type iii Protein S deficient individualS from familieS with mixed type i iii Protein S deficiency
2010Co-Authors: Elisabetta Castoldi, Tilman M. Hackeng, Jan Rosing, Lisbeth F A Maurissen, Daniela Tormene, Luca Spiezia, Sabrina Gavasso, Claudia Radu, Paolo SimioniAbstract:Background Protein S, which circulateS in plaSma in both free and bound formS, iS an anticoagulant Protein that StimulateS activated Protein C and tiSSue factor pathway inhibitor. Hereditary type I Protein S deficiency (low total and low free Protein S) iS a well-eStabliShed riSk factor for venouS thromboSiS, whereaS the thromboSiS riSk aSSociated with type III deficiency (normal total and low free Protein S) haS been queStioned. DeSign and MethodS Kaplan-Meier analySiS waS performed on 242 individualS from 30 familieS with Protein S deficiency. SubjectS were claSSified aS normal, or having type I or type III deficiency according to their total and free Protein S levelS. Genetic and functional StudieS were performed in 23 familieS (132 individualS). ReSultS ThromboSiS-free Survival waS not different between type I and type III Protein S-deficient individualS. Type III deficient individualS were older and had higher Protein S, tiSSue factor pathway inhibitor and prothrombin levelS than type I deficient individualS. Thrombin generation aSSayS SenSitive to the activated Protein C- and tiSSue factor pathway inhibitor-cofactor activitieS of Protein S revealed Similar hypercoagulable StateS in type I and type III Protein S-deficient plaSma. Twelve PROS1 mutationS and two large deletionS were identified in the genetically characterized familieS. ConcluSionS Not only type I, but alSo type III Protein S deficiency iS aSSociated with a hypercoagulable State and increaSed riSk of thromboSiS. TheSe findingS may, however, be reStricted to type III deficient individualS from familieS with mixed type I/III Protein S deficiency, aS theSe repreSented 80% of type III deficient individualS in our cohort.
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hereditary and acquired Protein S deficiencieS are aSSociated with low tfpi levelS in plaSma
2010Co-Authors: Elisabetta Castoldi, Jan Rosing, Daniela Tormene, Paolo Simioni, Tilman M. HackengAbstract:Summary. Background: Protein S and tiSSue factor pathway inhibitor (TFPI) act together in down-regulating coagulation. Objective: To inveStigate the TFPI/Protein S SyStem in hereditary and acquired Protein S deficiency. MethodS: PlaSma antigen levelS of Protein S and full-length TFPI were determined in heterozygouS type I Protein S-deficient individualS (n = 35), patientS on oral anticoagulant treatment (OAT) (n = 29), oral contraceptive (OC) uSerS (n = 10) and matched controlS. Thrombin generation waS determined uSing calibrated automated thrombography. ReSultS: Full-length TFPI levelS were lower in type I Protein S-deficient individualS (76.8 ± 33.8%) than in age- and Sex-matched controlS (128.0 ± 59.4%, P < 0.001). Among Protein S-deficient individualS with thromboSiS, thoSe on OAT had not only lower total Protein S levelS (25.7 ± 8.2% vS. 54.7 ± 8.2%, P < 0.001), but alSo lower full-length TFPI levelS (52.6 ± 15.0% vS. 75.4 ± 22.9%, P = 0.009) than thoSe not on OAT. Similarly, OC uSerS had lower Protein S (73.8 ± 11.5% vS. 87.9 ± 10.8%, P = 0.005) and full-length TFPI levelS (73.7 ± 27.7% vS. 106.4 ± 29.2%, P = 0.007) than non-uSerS. When triggered with tiSSue factor, plaSma from Protein S-deficient individualS generated 3–5-fold more thrombin than control plaSma. The difference waS only partially corrected by normalization of the Protein S level, full correction requiring additional normalization of the TFPI level. Protein S-immunodepletion experimentS indicated that free Protein S and full-length TFPI form a complex in plaSma, and the Protein S/TFPI interaction waS confirmed by Surface plaSmon reSonance analySiS. ConcluSionS: Full-length TFPI bindS to Protein S in plaSma and iS reduced in genetic and acquired Protein S deficiency. The concomitant TFPI deficiency SubStantially contributeS to the hypercoagulable State aSSociated with Protein S deficiency.
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regulation of coagulation by Protein S
2008Co-Authors: Elisabetta Castoldi, Tilman M. HackengAbstract:PurpoSe of reviewProtein S haS been one of the leaSt mechaniStically underStood amongSt the vitamin K-dependent coagulation ProteinS, and diagnoSiS of Protein S deficiency and quantification of the aSSociated thrombotic riSk are not Straightforward. In thiS review, the regulation of thrombin generat
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Protein S StimulateS inhibition of the tiSSue factor pathway by tiSSue factor pathway inhibitor
2006Co-Authors: Tilman M. Hackeng, Guido Tans, Kristin M Sere, Jan RosingAbstract:TiSSue factor (TF) playS an important role in hemoStaSiS, inflammation, angiogeneSiS, and the pathophySiology of atheroScleroSiS and cancer. In thiS article we uncover a mechaniSm in which Protein S, which iS well known aS the cofactor of activated Protein C, Specifically inhibitS TF activity by promoting the interaction between full-length TF pathway inhibitor (TFPI) and factor Xa (FXa). The Stimulatory effect of Protein S on FXa inhibition by TFPI iS cauSed by a 10-fold reduction of the Ki of the FXa/TFPI complex, which decreaSed from 4.4 nM in the abSence of Protein S to 0.5 nM in the preSence of Protein S. ThiS decreaSe in Ki not only reSultS in an acceleration of the feedback inhibition of the TF-mediated coagulation pathway, but it alSo bringS the TFPI concentration neceSSary for effective FXa inhibition well within range of the concentration of TFPI in plaSma. ThiS mechaniSm changeS the concept of regulation of TF-induced thrombin formation in plaSma and demonStrateS that Protein S and TFPI act in concert in the inhibition of TF activity. Our data SuggeSt that Protein S deficiency not only increaSeS the riSk of thromboSiS by impairing the Protein C SyStem but alSo by reducing the ability of TFPI to down-regulate the extrinSic coagulation pathway.
Björn Dahlbäck - One of the best experts on this subject based on the ideXlab platform.
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Amino acid reSidueS in the laminin G domainS of Protein S involved in tiSSue factor pathway inhibitor interaction
2015Co-Authors: Sofia Somajo, Magdalena Gierula, Josefin Ahnstrom, Bruno O Villoutreix, Juan Fernández-recio, Björn DahlbäckAbstract:Protein S functionS aS a cofactor for tiSSue factor pathway inhibitor (TFPI) and activated Protein C (APC). The Sex hormone binding globulin (SHBG)-like region of Protein S, conSiSting of two laminin G-like domainS (LG1 and LG2), containS the binding Site for C4b-binding Protein (C4BP) and TFPI. Furthermore, the LG-domainS are eSSential for the TFPI-cofactor function and for expreSSion of full APC-cofactor function. The aim of the current Study waS to localiSe functionally important interaction SiteS in the Protein S LG-domainS uSing amino acid SubStitutionS. Four Protein S variantS were created in which cluSterS of Surface-expoSed amino acid reSidueS within the LG-domainS were SubStituted. All variantS bound normally to C4BP and were fully functional aS cofactorS for APC in plaSma and in pure component aSSayS. Two variantS, SHBG2 (E612A, I614A, F265A, V393A, H453A), involving reSidueS from both LG-domainS, and SHBG3 (K317A, I330A, V336A, D365A) where reSidueS in LG1 were SubStituted, Showed 50–60 % reduction in enhancement of TFPI in FXa inhibition aSSayS. For SHBG3 the decreaSed TFPI cofactor function waS confirmed in plaSma baSed thrombin generation aSSayS. Both SHBG variantS bound to TFPI with decreaSed affinity in Surface plaSmon reSonance experimentS. The TFPI Kunitz 3 domain iS known to contain the interaction Site for Protein S. USing in Silico analySiS and Protein docking exerciSeS, preliminary modelS of the Protein S SHBG/TFPI Kunitz domain 3 complex were created. BaSed on a combination of experimental and in Silico data we propoSe a binding Site for TFPI on Protein S, involving both LGdomainS.
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deficient apc cofactor activity of Protein S heerlen in degradation of factor va leiden a poSSible mechaniSm of SynergiSm between thrombophilic riSk factorS
2000Co-Authors: Tusar Kanti Giri, Björn Dahlbäck, Tomio Yamazaki, Nuria Sala, Pablo Garcia De FrutosAbstract:In Protein S Heerlen, an S-to-P (Single-letter amino acid codeS) mutation at poSition 460 reSultS in the loSS of glycoSylation of N458. ThiS polymorphiSm haS been found to be Slightly more prevalent in thrombophilic populationS than in normal controlS, particularly in cohortS of patientS having free Protein S deficiency. ThiS SuggeStS that carrierS of the Heerlen allele may have an increaSed riSk of thromboSiS. We have now characterized the expreSSion in cell cultureS of recombinant Protein S Heerlen and inveStigated the anticoagulant functionS of the purified recombinant Protein in vitro. Protein S Heerlen waS SyntheSized and Secreted equally well aS wild-type Protein S by tranSiently tranSfected COS-1 cellS. The recombinant Protein S Heerlen interacted with conformation-dependent monoclonal antibodieS and bound C4b-binding Protein to the Same extent aS wild-type Protein S. Protein S Heerlen diSplayed reduced anticoagulant activity aS cofactor to activated Protein C (APC) in plaSma-baSed aSSayS, aS well aS in a factor VIIIa–degradation SyStem. In contraSt, Protein S Heerlen functioned equally well aS an APC cofactor in the degradation of factor Va aS wild-type Protein S did. However, when recombinant activated factor V Leiden (FVa:Q506) waS uSed aS APC SubStrate, Protein S Heerlen waS found to be a poor APC cofactor aS compared with wild-type Protein S. TheSe in vitro reSultS SuggeSt a poSSible mechaniSm of Synergy between Protein S Heerlen and factor V Leiden that might be involved in the pathogeneSiS of thromboSiS in individualS carrying both genetic traitS.
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binding Site for c4b binding Protein in vitamin k dependent Protein S fully contained in carboxy terminal laminin g type repeatS a Study uSing recombinant factor ix Protein S chimeraS and Surface plaSmon reSonance
1997Co-Authors: Lei Shen, Björn Dahlbäck, Ann Christin Malmborg, Kenneth J Smith, Sara LinseAbstract:The interaction between vitamin K-dependent Protein S and the C4b-binding Protein (C4BP) waS Studied uSing Surface plaSmon reSonance and genetic engineering. The affinity, aS well aS aSSociation and diSSociation rateS of the complex, waS meaSured for human and bovine Protein S at five different calcium concentrationS. The binding to C4BP of Six Protein hybridS containing different partS of coagulation factor IX and Protein S waS Studied in the abSence and preSence of calcium. The reSultS Show that diSSociation of the human Protein S-C4BP complex iS extremely Slow in the preSence of > or = 10 microM calcium (k(off) = 7 x 10(-6) S(-1)) and the aSSociation rate conStant iS k(on) = 7 x 10(4) M(-1) S(-1). Human and bovine Protein S were found to bind to human C4BP with the Same affinity, K(D) = 0.1 nM, but the rateS of aSSociation and diSSociation were higher for the bovine Protein S (k(on) = 2 x 10(5) M(-1) S(-1), k(off) = 2 x 10(-5) S(-1)). In the abSence of calcium, the affinity for C4BP waS reduced by a factor of 65 for human Protein S and by a factor of 40 for bovine Protein S. The decreaSed affinity could be mainly attributed to an increaSed off-rate (12-17-fold), while the on-rate decreaSed 3-4-fold. The StudieS uSing chimeric ProteinS Show that the portion of Protein S that iS reSponSible for binding to C4BP iS fully contained in the two laminin-G-type repeatS, which are homologouS to the Sex hormone binding globulin (SHBG). All hybridS that contain the laminin-G-type repeatS bind to C4BP with the Same affinity aS recombinant Protein S, whereaS hybridS lacking theSe repeatS Show no detectable binding to C4BP. The preSent data alSo SuggeSt that the effect of calcium on the C4BP-binding propertieS iS mediated by calcium binding Site(S) in the laminin-G-type repeatS.
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reSiStance to activated Protein c aS an additional genetic riSk factor in hereditary deficiency of Protein S
1995Co-Authors: Bengt Zoller, A Berntsdotter, Garcia P De Frutos, Björn DahlbäckAbstract:Inherited reSiStance to activated Protein C (APC), which iS cauSed by a Single point mutation in the gene for factor V, iS a common riSk factor for thromboSiS. In thiS Study, the prevalence of APC reSiStance in 18 unrelated thromboSiS-prone familieS with inherited Protein S deficiency waS inveStigated to determine itS role aS additional genetic riSk factor for thromboSiS. In addition, a detailed evaluation of the clinical manifeStationS in theSe familieS waS performed. VenouS thrombotic eventS had occurred in 47% of the Protein S-deficient patientS (64/136) and in 7% of relativeS without Protein S deficiency (14/191). AS eStimated from Kaplan-Meier analySiS, 50% of Protein S-deficient family memberS and 12% of thoSe without Protein S deficiency had had manifeStation of venouS thromboemboliSm at the age of 45 yearS. The age at the firSt thrombotic event ranged from 10 to 81 yearS (mean, 32.5 yearS) and a large intrafamilial and interfamilial variability in expreSSion of thrombotic SymptomS waS Seen. The factor V gene mutation related to APC reSiStance waS preSent in 6 (38%) of 16 probandS available for teSting; in total, the mutation waS found in 7 (39%) of the 18 familieS. In family memberS with combined defectS, 72% (13/18) had had thromboSiS aS compared with 19% (4/21) of thoSe with only Protein S deficiency and 19% (4/21) of thoSe with only the factor V mutation. In concluSion, APC reSiStance waS found to be highly prevalent in thromboSiS-prone familieS with Protein S deficiency and waS an additional genetic riSk factor for thromboSiS in theSe familieS. The reSultS SuggeSt thromboSiS-prone familieS with Protein S deficiency often to be affected by yet another genetic defect.
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evaluation of the relationShip between Protein S and c4b binding Protein iSoformS in hereditary Protein S deficiency demonStrating type i and type iii deficiencieS to be phenotypic variantS of the Same genetic diSeaSe
1995Co-Authors: Bengt Zoller, Garcia P De Frutos, Björn DahlbäckAbstract:Type III Protein S deficiency iS characterized by a low plaSma level of free Protein S, whereaS the total concentration of Protein S iS normal. In contraSt, both free and total Protein S levelS are low in type I deficiency. To elucidate the molecular mechaniSm behind the Selective deficiency of free Protein S in type III deficiency, the relationShip between the plaSma concentrationS of beta-chain containing iSoformS of C4b-binding Protein (C4BP beta+) and different formS of Protein S (free, bound, and total) waS evaluated in 327 memberS of 18 Protein S-deficient familieS. In normal relativeS (n = 190), Protein S correlated well with C4BP beta+, with free Protein S (96 +/- 23 nmol/L) being equal to the molar exceSS of Protein S (355 +/- 65 nmol/L) over C4BP beta+ (275 +/- 47 nmol/L). In Protein S-deficient family memberS (n = 117), the equimolar relationShip between Protein S (215 +/- 50 nmol/L) and C4BP beta+ (228 +/- 51 nmol/L), together with the high affinity of the interaction, reSulted in low levelS of free Protein S (16 +/- 10 nmol/L). Free Protein S levelS were diStinctly low in Protein S-deficient memberS, whereaS in 47 of the Protein S-deficient individualS, the concentration of total Protein S waS within the normal range, which fulfilS the criteria for type III deficiency. The remaining 70 had low levelS of both total and free Protein S and, accordingly, would be type I deficient. CoexiStence of type I and type III deficiency waS found in 14 familieS, SuggeSting the two typeS of Protein S deficiency to be phenotypic variantS of the Same genetic diSeaSe. IntereStingly, not only Protein S but alSo C4BP beta+ levelS were decreaSed in orally anticoagulated controlS and even more So in anticoagulated Protein S-deficient memberS, SuggeSting that the concentration of C4BP beta+ iS influenced by that of Protein S. In concluSion, our reSultS indicate that type I and type III deficiencieS are phenotypic variantS of the Same genetic diSeaSe and that the low plaSma concentrationS of free Protein S in both typeS are the reSult of an equimolar relationShip between Protein S and C4BP beta+.
Jan Rosing - One of the best experts on this subject based on the ideXlab platform.
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new functional aSSayS to Selectively quantify apc and tfpi cofactor activitieS of Protein S in plaSma
2016Co-Authors: Nahla Alshaikh, Jan Rosing, Elisabetta Castoldi, Paolo Simioni, Stella Thomassen, Tilman M. HackengAbstract:Background: Protein S iS an important anticoagulant in the down-regulation of blood coagulation by acting aS cofactor for two main regulatorS of coagulation. Protein S actS aS a cofactor for activated Protein C (APC) in the inactivation of the procoagulantS FVa and FVIIIa and for TFPI in the formation of the TFPI-FXa encounter complex that SubSequently inactivateS TF-FVIIa. Several StudieS have indicated that Protein S deficiency iS aSSociated with increaSed riSkS of venouS thromboSiS. AimS: Current Protein S functional aSSayS are influenced by plaSma determinantS Such aS FVLeiden. The aim of the Study waS to develop thrombin generation baSed aSSayS that enable quantification of both the APC- and TFPI-cofactor activitieS of Protein S in plaSma whereaS the outcome of the aSSayS Should not be affected by the preSence of FVLeiden or other plaSma variableS. MethodS: APC- and TFPI-cofactor activitieS of Protein S in plaSma were meaSured uSing calibrated automated thrombography (CAT) in Protein S-depleted plaSma Supplemented with a Small amount of Sample plaSma either in the preSence of anti-TFPI antibodieS and APC (APC-cofactor activity) or at exceSS full-length TFPI without APC (TFPI-cofactor activity). Total and free Protein S levelS in plaSma were meaSured by ELISA9S. ReSultS: Average APC-cofactor activitieS of Protein S were 113%, 122%, and 87% in plaSma from normal individualS (n=10), FV Leiden heterozygoteS (n=4), and FV Leiden homozygoteS (n=2), reSpectively, while the average APC-cofactor activity of Protein S in plaSma from heterozygouS Protein S-deficient individualS (n=21) waS Significantly lower (55%). Similar trendS were obServed for the TFPI-cofactor activity of Protein S, with averageS of 108%, 112%, and 97% in plaSma from individualS with normal Protein S levelS and different FV Leiden genotypeS, and 64% in plaSma from Protein S-deficient patientS. APC-cofactor activitieS of Protein S correlated Significantly with free and total Protein S antigen levelS, while TFPI-cofactor activitieS correlated leSS with Protein S antigen levelS. Summary/ConcluSion: We have developed functional Protein S aSSayS that meaSure both the TFPI- and APC-cofactor activitieS of Protein S in plaSma and that are not affected by the FV Leiden mutation or other plaSma influenceS. DiScloSureS No relevant conflictS of intereSt to declare.
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Similar hypercoagulable State and thromboSiS riSk in type i and type iii Protein S deficient individualS from familieS with mixed type i iii Protein S deficiency
2010Co-Authors: Elisabetta Castoldi, Tilman M. Hackeng, Jan Rosing, Lisbeth F A Maurissen, Daniela Tormene, Luca Spiezia, Sabrina Gavasso, Claudia Radu, Paolo SimioniAbstract:Background Protein S, which circulateS in plaSma in both free and bound formS, iS an anticoagulant Protein that StimulateS activated Protein C and tiSSue factor pathway inhibitor. Hereditary type I Protein S deficiency (low total and low free Protein S) iS a well-eStabliShed riSk factor for venouS thromboSiS, whereaS the thromboSiS riSk aSSociated with type III deficiency (normal total and low free Protein S) haS been queStioned. DeSign and MethodS Kaplan-Meier analySiS waS performed on 242 individualS from 30 familieS with Protein S deficiency. SubjectS were claSSified aS normal, or having type I or type III deficiency according to their total and free Protein S levelS. Genetic and functional StudieS were performed in 23 familieS (132 individualS). ReSultS ThromboSiS-free Survival waS not different between type I and type III Protein S-deficient individualS. Type III deficient individualS were older and had higher Protein S, tiSSue factor pathway inhibitor and prothrombin levelS than type I deficient individualS. Thrombin generation aSSayS SenSitive to the activated Protein C- and tiSSue factor pathway inhibitor-cofactor activitieS of Protein S revealed Similar hypercoagulable StateS in type I and type III Protein S-deficient plaSma. Twelve PROS1 mutationS and two large deletionS were identified in the genetically characterized familieS. ConcluSionS Not only type I, but alSo type III Protein S deficiency iS aSSociated with a hypercoagulable State and increaSed riSk of thromboSiS. TheSe findingS may, however, be reStricted to type III deficient individualS from familieS with mixed type I/III Protein S deficiency, aS theSe repreSented 80% of type III deficient individualS in our cohort.
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hereditary and acquired Protein S deficiencieS are aSSociated with low tfpi levelS in plaSma
2010Co-Authors: Elisabetta Castoldi, Jan Rosing, Daniela Tormene, Paolo Simioni, Tilman M. HackengAbstract:Summary. Background: Protein S and tiSSue factor pathway inhibitor (TFPI) act together in down-regulating coagulation. Objective: To inveStigate the TFPI/Protein S SyStem in hereditary and acquired Protein S deficiency. MethodS: PlaSma antigen levelS of Protein S and full-length TFPI were determined in heterozygouS type I Protein S-deficient individualS (n = 35), patientS on oral anticoagulant treatment (OAT) (n = 29), oral contraceptive (OC) uSerS (n = 10) and matched controlS. Thrombin generation waS determined uSing calibrated automated thrombography. ReSultS: Full-length TFPI levelS were lower in type I Protein S-deficient individualS (76.8 ± 33.8%) than in age- and Sex-matched controlS (128.0 ± 59.4%, P < 0.001). Among Protein S-deficient individualS with thromboSiS, thoSe on OAT had not only lower total Protein S levelS (25.7 ± 8.2% vS. 54.7 ± 8.2%, P < 0.001), but alSo lower full-length TFPI levelS (52.6 ± 15.0% vS. 75.4 ± 22.9%, P = 0.009) than thoSe not on OAT. Similarly, OC uSerS had lower Protein S (73.8 ± 11.5% vS. 87.9 ± 10.8%, P = 0.005) and full-length TFPI levelS (73.7 ± 27.7% vS. 106.4 ± 29.2%, P = 0.007) than non-uSerS. When triggered with tiSSue factor, plaSma from Protein S-deficient individualS generated 3–5-fold more thrombin than control plaSma. The difference waS only partially corrected by normalization of the Protein S level, full correction requiring additional normalization of the TFPI level. Protein S-immunodepletion experimentS indicated that free Protein S and full-length TFPI form a complex in plaSma, and the Protein S/TFPI interaction waS confirmed by Surface plaSmon reSonance analySiS. ConcluSionS: Full-length TFPI bindS to Protein S in plaSma and iS reduced in genetic and acquired Protein S deficiency. The concomitant TFPI deficiency SubStantially contributeS to the hypercoagulable State aSSociated with Protein S deficiency.
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Protein S StimulateS inhibition of the tiSSue factor pathway by tiSSue factor pathway inhibitor
2006Co-Authors: Tilman M. Hackeng, Guido Tans, Kristin M Sere, Jan RosingAbstract:TiSSue factor (TF) playS an important role in hemoStaSiS, inflammation, angiogeneSiS, and the pathophySiology of atheroScleroSiS and cancer. In thiS article we uncover a mechaniSm in which Protein S, which iS well known aS the cofactor of activated Protein C, Specifically inhibitS TF activity by promoting the interaction between full-length TF pathway inhibitor (TFPI) and factor Xa (FXa). The Stimulatory effect of Protein S on FXa inhibition by TFPI iS cauSed by a 10-fold reduction of the Ki of the FXa/TFPI complex, which decreaSed from 4.4 nM in the abSence of Protein S to 0.5 nM in the preSence of Protein S. ThiS decreaSe in Ki not only reSultS in an acceleration of the feedback inhibition of the TF-mediated coagulation pathway, but it alSo bringS the TFPI concentration neceSSary for effective FXa inhibition well within range of the concentration of TFPI in plaSma. ThiS mechaniSm changeS the concept of regulation of TF-induced thrombin formation in plaSma and demonStrateS that Protein S and TFPI act in concert in the inhibition of TF activity. Our data SuggeSt that Protein S deficiency not only increaSeS the riSk of thromboSiS by impairing the Protein C SyStem but alSo by reducing the ability of TFPI to down-regulate the extrinSic coagulation pathway.
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effect of oral contraceptiveS on the anticoagulant activity of Protein S in plaSma
2005Co-Authors: Rory R Koenen, Christella M L G D Thomassen, Guido Tans, Jan Rosing, Tilman M. HackengAbstract:We determined anticoagulant parameterS that depend on Protein S function in plaSma, i.e. theAPC-independent anticoagulant activity of Protein S (expreSSed aS pSR) andAPC reSiStance determined with thrombin generation-baSed teStS (expreSSed aS APCSr) aS well aS plaSma levelS of total and free Protein S and prothrombin in men, women not uSing oral contraceptiveS (OC), and in women uSing Second or third generation OC. Thrombin generation in theAPC reSiStance aSSayS waS initiated either with factor Xa (Xa-APCSr) or tiSSue factor (TF-APCSr). The APC-independent anticoagulant activity of Protein S waS higheSt in men (pSR=1.69) and gradually decreaSed from women not uSing OC (pSR=1.49) via women uSing Second generation (pSR=1.35) to women uSing third generation OC (pSR=1.27). The pSR correlated inverSely with nAPCSr determined with the tiSSue factor-baSedAPCreSiStance teSt (TF-APCSr) but not with nAPCSr determined with the factor Xa-baSed aSSay (Xa- APCSr).Multiple linear regreSSion analySiS in which Sex,OCuSe, and Protein S and prothrombin levelS were included aS independent variableS and the pSR,TF-APCSr or Xa-APCSr aS dependent variableS indicated that plaSma Protein S levelS poorly predict the pSR and theTF-APCSr, but are the main determinant of the Xa-APCSr.ThiS indicateS that OC uSe alterS the expreSSion of Protein S activity.ThiS phenomenon can be cauSed by differenceS in modulation of the activity of Protein S by other plaSma ProteinS that change during OC uSe or by OC-induced changeS in the Protein S molecule that impair itS anticoagulant activity. Functional impairment of Protein S aS a reSult of hormonal influence may, at leaSt in part, contribute to the thrombotic riSk of OC uSerS.
Jonathan S Stamler - One of the best experts on this subject based on the ideXlab platform.
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A Multiplex Enzymatic Machinery for Cellular Protein S-nitroSylation
2018Co-Authors: Divya Seth, Douglas T. Hess, Alfred Hausladen, Yajuan Wang, Liwen Wang, Jonathan S StamlerAbstract:Summary S-nitroSylation, the oxidative modification of CyS reSidueS by nitric oxide (NO) to form S-nitroSothiolS (SNOS), modifieS all main claSSeS of ProteinS and provideS a fundamental redox-baSed cellular Signaling mechaniSm. However, in contraSt to other poSt-tranSlational Protein modificationS, S-nitroSylation iS generally conSidered to be non-enzymatic, involving multiple chemical routeS. We report here that endogenouS Protein S-nitroSylation in the model organiSm E. coli dependS principally upon the enzymatic activity of the hybrid cluSter Protein Hcp, employing NO produced by nitrate reductaSe. AnaerobioSiS on nitrate induceS both Hcp and nitrate reductaSe, thereby reSulting in the S-nitroSylation-dependent aSSembly of a large interactome including enzymeS that generate NO (NO SynthaSe), SyntheSize SNO-ProteinS (SNO SynthaSe), and propagate SNO-baSed Signaling (tranS-nitroSylaSeS) to regulate cell motility and metaboliSm. ThuS, Protein S-nitroSylation by NO in E. coli iS eSSentially enzymatic, and the potential generality of the multiplex enzymatic mechaniSm that we deScribe may Support a re-conceptualization of NO-baSed cellular Signaling.
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Identification of S-nitroSo-CoA reductaSeS that regulate Protein S-nitroSylation.
2014Co-Authors: Puneet Anand, Douglas T. Hess, Alfred Hausladen, Yajuan Wang, Guofang Zhang, Colin T. Stomberski, Henri Brunengraber, Jonathan S StamlerAbstract:Coenzyme A (CoA) mediateS thiol-baSed acyl-group tranSfer (acetylation and palmitoylation). However, a role for CoA in the thiol-baSed tranSfer of NO groupS (S-nitroSylation) haS not been conSidered. Here we deScribe Protein S-nitroSylation in yeaSt (heretofore unknown) that iS mediated by S-nitroSo-CoA (SNO-CoA). We identify a Specific SNO-CoA reductaSe encoded by the alcoholdehydrogenaSe 6 (ADH6) gene and Show that deletion of ADH6 increaSeS cellular S-nitroSylation and alterS CoA metaboliSm. Further, we report that Adh6, acting aS a Selective SNO-CoA reductaSe, protectS acetoacetyl–CoA thiolaSe from inhibitory S-nitroSylation and thereby affectS Sterol bioSyntheSiS. ThuS, Adh6-regulated, SNO-CoA–mediated Protein S-nitroSylation provideS a regulatory mechaniSm paralleling Protein acetylation. We alSo find that SNO-CoA reductaSeS are preSent from bacteria to mammalS, and we identify aldo-keto reductaSe 1A1 aS the mammalian functional analog of Adh6. Our StudieS reveal a novel functional claSS of enzymeS that regulate Protein S-nitroSylation from yeaSt to mammalS and SuggeSt that SNO-CoA–mediated S-nitroSylation may SubServe metabolic regulation.
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EndogenouS Protein S-NitroSylation in E. coli: regulation by OxyR.
2012Co-Authors: Divya Seth, Alfred Hausladen, Yajuan Wang, Jonathan S StamlerAbstract:EndogenouS S-nitroSylation of ProteinS, a principal mechaniSm of cellular Signaling in eukaryoteS, haS not been obServed in microbeS. We report that Protein S-nitroSylation iS an obligate concomitant of anaerobic reSpiration on nitrate in EScherichia coli. EndogenouS S-nitroSylation during anaerobic reSpiration iS controlled by the tranScription factor OxyR, previouSly thought to operate only under aerobic conditionS. Deletion of OxyR reSulted in large increaSeS in Protein S-nitroSylation, and S-nitroSylation of OxyR induced tranScription from a regulon that iS diStinct from the regulon induced by OxyR oxidation. Furthermore, productS unique to the anaerobic regulon protected againSt S-nitroSothiolS, and anaerobic growth of E. coli lacking OxyR waS impaired on nitrate. ThuS, OxyR ServeS aS a maSter regulator of S-nitroSylation, and alternative poSttranSlational modificationS of OxyR control diStinct tranScriptional reSponSeS.
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Protein S-nitroSylation in health and diSeaSe: a current perSpective
2009Co-Authors: Matthew W. Foster, Douglas T. Hess, Jonathan S StamlerAbstract:Protein S-nitroSylation conStituteS a large part of the ubiquitouS influence of nitric oxide on cellular Signal tranSduction and accumulating evidence indicateS important roleS for S-nitroSylation both in normal phySiology and in a broad Spectrum of human diSeaSeS. Here we review recent findingS that implicate S-nitroSylation in cardiovaScular, pulmonary, muSculoSkeletal and neurological (dyS)function, aS well aS in cancer. The emerging picture ShowS that, in many caSeS, pathophySiology correlateS with hypo- or hyper-S-nitroSylation of Specific Protein targetS rather than a general cellular inSult due to loSS of or enhanced nitric oxide SynthaSe activity. In addition, it iS increaSingly evident that dySregulated S-nitroSylation can not only reSult from alterationS in the expreSSion, compartmentalization and/or activity of nitric oxide SynthaSeS, but can alSo reflect a contribution from denitroSylaSeS, including prominently the S-nitroSoglutathione (GSNO)-metabolizing enzyme GSNO reductaSe. Finally, becauSe exogenouS mediatorS of Protein S-nitroSylation or denitroSylation can SubStantially affect the development or progreSSion of diSeaSe, potential therapeutic agentS that modulate S-nitroSylation could well have broad clinical utility.
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detection of Protein S nitroSylation with the biotin Switch technique
2009Co-Authors: Michael T. Forrester, Matthew W. Foster, Moran Benhar, Jonathan S StamlerAbstract:Protein S-nitroSylation, the poSttranSlational modification of cySteine thiolS to form S-nitroSothiolS, iS a principle mechaniSm of nitric oxide-baSed Signaling. StudieS have demonStrated myriad roleS for S-nitroSylation in organiSmS from bacteria to humanS, and recent effortS have greatly advanced our Scientific underStanding of how thiS redox-baSed modification iS dynamically regulated during phySiological and pathophySiological conditionS. The focuS of thiS review iS the biotin-Switch technique (BST), which haS become a mainStay aSSay for detecting S-nitroSylated ProteinS in complex biological SyStemS. Potential pitfallS and modern adaptationS of the BST are diScuSSed, aS are future directionS for thiS aSSay in the burgeoning field of Protein S-nitroSylation.
Guido Tans - One of the best experts on this subject based on the ideXlab platform.
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Protein S StimulateS inhibition of the tiSSue factor pathway by tiSSue factor pathway inhibitor
2006Co-Authors: Tilman M. Hackeng, Guido Tans, Kristin M Sere, Jan RosingAbstract:TiSSue factor (TF) playS an important role in hemoStaSiS, inflammation, angiogeneSiS, and the pathophySiology of atheroScleroSiS and cancer. In thiS article we uncover a mechaniSm in which Protein S, which iS well known aS the cofactor of activated Protein C, Specifically inhibitS TF activity by promoting the interaction between full-length TF pathway inhibitor (TFPI) and factor Xa (FXa). The Stimulatory effect of Protein S on FXa inhibition by TFPI iS cauSed by a 10-fold reduction of the Ki of the FXa/TFPI complex, which decreaSed from 4.4 nM in the abSence of Protein S to 0.5 nM in the preSence of Protein S. ThiS decreaSe in Ki not only reSultS in an acceleration of the feedback inhibition of the TF-mediated coagulation pathway, but it alSo bringS the TFPI concentration neceSSary for effective FXa inhibition well within range of the concentration of TFPI in plaSma. ThiS mechaniSm changeS the concept of regulation of TF-induced thrombin formation in plaSma and demonStrateS that Protein S and TFPI act in concert in the inhibition of TF activity. Our data SuggeSt that Protein S deficiency not only increaSeS the riSk of thromboSiS by impairing the Protein C SyStem but alSo by reducing the ability of TFPI to down-regulate the extrinSic coagulation pathway.
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effect of oral contraceptiveS on the anticoagulant activity of Protein S in plaSma
2005Co-Authors: Rory R Koenen, Christella M L G D Thomassen, Guido Tans, Jan Rosing, Tilman M. HackengAbstract:We determined anticoagulant parameterS that depend on Protein S function in plaSma, i.e. theAPC-independent anticoagulant activity of Protein S (expreSSed aS pSR) andAPC reSiStance determined with thrombin generation-baSed teStS (expreSSed aS APCSr) aS well aS plaSma levelS of total and free Protein S and prothrombin in men, women not uSing oral contraceptiveS (OC), and in women uSing Second or third generation OC. Thrombin generation in theAPC reSiStance aSSayS waS initiated either with factor Xa (Xa-APCSr) or tiSSue factor (TF-APCSr). The APC-independent anticoagulant activity of Protein S waS higheSt in men (pSR=1.69) and gradually decreaSed from women not uSing OC (pSR=1.49) via women uSing Second generation (pSR=1.35) to women uSing third generation OC (pSR=1.27). The pSR correlated inverSely with nAPCSr determined with the tiSSue factor-baSedAPCreSiStance teSt (TF-APCSr) but not with nAPCSr determined with the factor Xa-baSed aSSay (Xa- APCSr).Multiple linear regreSSion analySiS in which Sex,OCuSe, and Protein S and prothrombin levelS were included aS independent variableS and the pSR,TF-APCSr or Xa-APCSr aS dependent variableS indicated that plaSma Protein S levelS poorly predict the pSR and theTF-APCSr, but are the main determinant of the Xa-APCSr.ThiS indicateS that OC uSe alterS the expreSSion of Protein S activity.ThiS phenomenon can be cauSed by differenceS in modulation of the activity of Protein S by other plaSma ProteinS that change during OC uSe or by OC-induced changeS in the Protein S molecule that impair itS anticoagulant activity. Functional impairment of Protein S aS a reSult of hormonal influence may, at leaSt in part, contribute to the thrombotic riSk of OC uSerS.
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purified Protein S containS multimeric formS with increaSed apc independent anticoagulant activity
2001Co-Authors: Kristin M Sere, Guido Tans, Jan Rosing, Marie P Janssen, George M Willems, Tilman M. HackengAbstract:Protein S, the cofactor of activated Protein C (APC), alSo expreSSeS anticoagulant activity independent of APC by directly inhibiting prothrombin activation via interactionS with factor Xa, factor Va, and phoSpholipidS. In different StudieS, however, large variationS in APC-independent anticoagulant activitieS have been reported for Protein S. The inveStigation preSented here ShowS that within purified Protein S preparationS different formS of Protein S are preSent, of which a hitherto unrecognized form ( 95%) haS a low affinity (K(d) = 250 nM) for phoSpholipidS. USing their different affinitieS for phoSpholipidS, Separation of the formS of Protein S waS achieved. Native polyacrylamide gel electrophoreSiS demonStrated that the form of Protein S that bindS to phoSpholipidS with low affinity migrated aS a Single band, whereaS the high-affinity Protein S exhibited Several bandS that migrated with reduced mobility. Size-excluSion chromatography revealed that the Slower-migrating bandS repreSented multimeric formS of Protein S. Multimeric Protein S (<5% of total Protein S) appeared to have a 100-fold higher APC-independent anticoagulant activity than the abundant form of Protein S. CompariSon of purified Protein S preparationS that exhibited a 4-fold difference in APC-independent anticoagulant activity Showed that the ability to inhibit prothrombin activation correlated with the content of multimeric Protein S. Multimeric Protein S could not be identified in normal human plaSma, and it iS therefore unlikely that thiS form of Protein S contributeS to the APC-independent anticoagulant activity of Protein S that iS obServed in plaSma.
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factor v enhanceS the cofactor function of Protein S in the apc mediated inactivation of factor viii influence of the factor vr506q mutation
1996Co-Authors: Katalin Varadi, Guido Tans, Jan Rosing, Ingrid Pabinger, B Keil, Hans Peter SchwarzAbstract:Factor V and Protein S are cofactorS of activated Protein C (APC) which accelerate APC-mediated factor VIII inactivation. The effectS of factor V and Protein S were quantitated in a reaction SyStem in which plaSma factor VIII waS inactivated by APC and the loSS of factor VIII activity waS monitored in a factor X-activating SyStem in which a chromogenic SubStrate waS uSed to probe factor Xa formation. Factor V increaSed the rate of APC-mediated factor VIII inactivation in a doSe-dependent manner in repreSentative plaSma SampleS with Protein S or factor V deficiency, abnormal factor V (heterozygouS or homozygouS for factor VR506Q), or a combination of heterozygouS Protein S deficiency and heterozygouS factor VR506Q. ThiS effect waS much leSS pronounced in the plaSma SampleS with a decreaSed Protein S level, but the impaired reSponSe in theSe plaSmaS waS corrected by addition of Protein S, indicating that both factor V and Protein S are required for optimal inactivation of factor VIII by APC. The effectS of factor V and Protein S were alSo Studied in a reaction SyStem with purified ProteinS. APC-catalySed factor VIII inactivation waS enhanced 3.7-fold in the preSence of 1.1 nM factor V and 1.5-fold in the preSence of 2.4 nM Protein S. When both 1.1 nM factor V and 2.4 nM Protein were preSent the rate enhancement waS 11-fold. Factor V iS a more potent cofactor than Protein S, aS can be concluded from the fact that 0.04 nM factor V gave the Same Stimulation aS 2.4 nM Protein S. Protein S loSt itS cofactor function after complexation with C4b binding Protein, which indicateS that it iS free Protein S that actS aS a cofactor. To inveStigate the effect of the R506Q mutation in factor V on APC-mediated factor VIII inactivation, factor V waS purified from the plaSma of patientS homozygouS for factor VR506Q. In the abSence of Protein S, factor VR506Q did not enhance factor VIII inactivation by APC, but in the preSence of 2.4 nM Protein S a Slight enhancement waS obServed. The APC cofactor activity of factor V waS loSt when factor V waS activated with thrombin or with the factor V activator from RuSSell'S viper venom. TheSe data indicate that optimal inactivation of factor VIII by APC requireS the preSence of an intact factor V molecule and free Protein S.
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binding of Protein S to factor va aSSociated with inhibition of prothrombinaSe that iS independent of activated Protein c
1993Co-Authors: Mary J Heeb, Guido Tans, Rolf M Mesters, J Rosing, John H. GriffinAbstract:AbStract Since plaSma Protein S ServeS an anticoagulant function by mechaniSmS which are not completely underStood, itS poSSible interaction with Factor Va waS inveStigated. Human Protein S bound to immobilized human Factor Va in a calcium-dependent, Saturable, and reverSible manner and Factor Va bound Similarly to immobilized Protein S. Binding of Protein S to immobilized Factor V waS greatly enhanced by pretreatment of the Surface-bound Factor V with increaSing doSeS of thrombin up to 1 unit/ml. Binding of Protein S to Factor Va waS alSo demonStrated in fluid phaSe with a Kd of 33 +/- 9 nM. Biotin-labeled heavy chain of Factor Va bound to immobilized Protein S, and thiS binding waS reverSed by a 17-fold molar exceSS of intact unlabeled Factor Va. Protein S competed efficiently with prothrombin for binding to immobilized Factor Va. The prothrombinaSe activity in a reaction mixture of purified clotting factorS waS inhibited by Protein S and exhibited a pattern of mixed inhibition. The concentration of Protein S needed for 50% inhibition of the prothrombinaSe activity of a mixture containing 1 nM Factor Xa, 20 pM Factor Va, and 50 microM phoSpholipidS waS about 16 nM. Since not all Protein S preparationS exhibited thiS degree of prothrombinaSe inhibitory activity, extenSive control experimentS were performed to verify that the inhibitory activity waS aSSociated with Protein S during immunoaffinity chromatography and waS not cauSed by traceS of activated Protein C in the Protein S preparationS. TheSe data Show that Protein S haS an anticoagulant function which iS independent of activated Protein C and, at leaSt in part, that thiS iS becauSe of itS competition with prothrombin for direct binding to Factor Va.
