Padua FIXa resistance to Protein S and a potential therapy for hyperactive FIXa.

INTRODUCTION: Abnormalities in the levels and functions of proteins that maintain hemostasis can cause thrombosis. Factor IX (FIX) R338L, i.e., Factor IX Padua, is a hyperactive clotting factor that promotes thrombosis. The R338L mutation increases the clotting rate by 8-fold despite increasing the Factor IXa enzymatic activity by only 2-fold. Protein S (PS) is a natural anticoagulant that directly inhibits FIXa. Because individuals affected by the R338L mutation have normal concentrations of PS, we speculated that the Padua hypercoagulation phenotype is due to decreased inhibition of FIXa R338L by PS. METHODS: We measured the ability of PS to inhibit FIX R338L, and we assessed the ability of PS to mitigate the prothrombotic effect FIX R338L. RESULTS: Plasma clotting assays demonstrated that 3-fold more PS was required to inhibit FIXa R338L compared with inhibition of wild type FIXa. Thrombin generation assays with Padua patient plasma recapitulated this biochemical consequence of the R338L mutation. Importantly, the less efficient inhibition of FIXa R338L was reversed by increasing PS concentration. Binding and co-immunoprecipitation studies revealed that the decrease in the inhibition of FIXa R338L by PS was caused by a 3- to 4-fold reduction in FIXa R338L affinity for PS. CONCLUSION: In summary, the resistance of FIXa R338L to inhibition by PS likely contributes to the unexpectedly high clotting rate in Padua individuals. Moreover, PS-mediated reversal of the pathological properties of FIXa R338L suggests that PS administration may be a novel and effective means to mitigate thrombophilia caused by any source of elevated FIXa activity.

Cholesterol crystals increase vascular permeability by inactivating SHP2 and disrupting adherens junctions.

To understand the adverse effects of cholesterol crystals on vascular homeostasis, we have studied their effects on endothelial barrier function. Cholesterol crystals increased endothelial barrier permeability in a dose and time dependent manner. In addition, cholesterol crystals induced tyrosine phosphorylation of VE-cadherin and α-catenin, disrupting endothelial AJ and its barrier function and these effects required xanthine oxidase-mediated H2O2 production, SHP2 inactivation and Frk activation. Similarly, feeding C57BL/6 mice with cholesterol-rich diet increased xanthine oxidase expression, H2O2 production, SHP2 inactivation and Frk activation leading to enhanced tyrosine phosphorylation of VE-cadherin and α-catenin, thereby disrupting endothelial AJ and increasing vascular permeability. Resolvin D1, a specialized proresolving mediator, prevented all these adverse effects of cholesterol crystals and cholesterol-rich diet in endothelial cells and mice, respectively. Based on these observations, it is likely that cholesterol crystals via disrupting AJ increase vascular permeability, a critical event of endothelial dysfunction and specialized proresolving mediators such as Resolvin D1 exert protection against these effects.

Anticoagulant Protein S Targets the Factor IXa Heparin-Binding Exosite to Prevent Thrombosis.

OBJECTIVE: PS (protein S) is a plasma protein that directly inhibits the coagulation FIXa (factor IXa) in vitro. Because elevated FIXa is associated with increased risk of venous thromboembolism, it is important to establish how PS inhibits FIXa function in vivo. The goal of this study is to confirm direct binding of PS with FIXa in vivo, identify FIXa amino acid residues required for binding PS in vivo, and use an enzymatically active FIXa mutant that is unable to bind PS to measure the significance of PS-FIXa interaction in hemostasis. APPROACH AND RESULTS: We demonstrate that PS inhibits FIXa in vivo by associating with the FIXa heparin-binding exosite. We used fluorescence tagging, immunohistochemistry, and protein-protein crosslinking to show in vivo interaction between FIXa and PS. Importantly, platelet colocalization required a direct interaction between the 2 proteins. FIXa and PS also coimmunoprecipitated from plasma, substantiating their interaction in a physiological milieu. PS binding to FIXa and PS inhibition of the intrinsic Xase complex required residues K132, K126, and R170 in the FIXa heparin-binding exosite. A double mutant, K132A/R170A, retained full activity but could not bind to PS. Crucially, Hemophilia B mice infused with FIXa K132A/R170A displayed an accelerated rate of fibrin clot formation compared with wild-type FIXa. CONCLUSIONS: Our findings establish PS as an important in vivo inhibitor of FIXa. Disruption of the interaction between PS and FIXa causes an increased rate of thrombus formation in mice. This newly discovered function of PS implies an unexploited target for antithrombotic therapeutics.

Resolvin D1 blocks H2O2-mediated inhibitory crosstalk between SHP2 and PP2A and suppresses endothelial-monocyte interactions.

In recent years, various studies have demonstrated a role for endogenously derived specialized proresolving mediators such as resolvins in the resolution of inflammation. In exploring the signaling mechanisms, in the present study we show that Resolvin D1 (RvD1) reduces LPS-induced endothelial cell (EC)-monocyte interactions via blocking H2O2-mediated PP2A inactivation, NFκB activation and ICAM1 and VCAM1 expression. In addition, we found that H2O2-mediated SHP2 inhibition leads to tyrosine phosphorylation and inactivation of PP2A by LPS, which in turn, accounts for increased NFκB activation and ICAM1 and VCAM1 expression facilitating EC-monocyte interactions and all these LPS-mediated responses were reduced by RvD1. Furthermore, the suppression of NFκB activation, ICAM1 and VCAM1 expression and EC and monocyte interactions by RvD1 involved its receptors ALX/FPR2 and GPR32 as inhibition or neutralization of these receptors negated its effects. Besides, pertussis toxin completely prevented the effects of RvD1 on inhibition of LPS-induced H2O2 production, SHP2 and PP2A inactivation, NFκB activation, ICAM1 and VCAM1 expression and EC and monocyte interactions. Together, these observations suggest that RvD1 via activation of Gi-coupled ALX/FPR2 and GPR32 receptors blocks LPS-induced H2O2-mediated SHP2 and PP2A inactivation, NFκB activation, ICAM1 and VCAM1 expression and EC-monocyte interactions, which could be one of the several possible mechanisms underlying the anti-inflammatory actions of this specialized proresolving mediator.

Resolvin D1 via prevention of ROS-mediated SHP2 inactivation protects endothelial adherens junction integrity and barrier function.

Resolvins are a novel class of lipid mediators that play an important role in the resolution of inflammation, although the underlying mechanisms are not very clear. To explore the anti-inflammatory mechanisms of resolvins, we have studied the effects of resolvin D1 (RvD1) on lipopolysaccharide (LPS)-induced endothelial barrier disruption as it is linked to propagation of inflammation. We found that LPS induces endothelial cell (EC) barrier disruption via xanthine oxidase (XO)-mediated reactive oxygen species (ROS) production, protein tyrosine phosphatase SHP2 inactivation and Fyn-related kinase (Frk) activation leading to tyrosine phosphorylation of α-catenin and VE-cadherin and their dissociation from each other affecting adherens junction (AJ) integrity and thereby increasing endothelial barrier permeability. RvD1 attenuated LPS-induced AJ disassembly and endothelial barrier permeability by arresting tyrosine phosphorylation of α-catenin and VE-cadherin and their dislocation from AJ via blockade of XO-mediated ROS production and thereby suppression of SHP2 inhibition and Frk activation. We have also found that the protective effects of RvD1 on EC barrier function involve ALX/FPR2 and GPR32 as inhibition or neutralization of these receptors negates its protective effects. LPS also increased XO activity, SHP2 cysteine oxidation and its inactivation, Frk activation, α-catenin and VE-cadherin tyrosine phosphorylation and their dissociation from each other leading to AJ disruption with increased vascular permeability in mice arteries and RvD1 blocked all these effects. Thus, RvD1 protects endothelial AJ and its barrier function from disruption by inflammatory mediators such as LPS via a mechanism involving the suppression of XO-mediated ROS production and blocking SHP2 inactivation.

12/15-Lipoxygenase-dependent ROS production is required for diet-induced endothelial barrier dysfunction.

To understand the mechanisms of 15(S)-HETE-induced endothelial cell (EC) barrier dysfunction, we examined the role of xanthine oxidase (XO). 15(S)-HETE induced junction adhesion molecule A (JamA) phosphorylation on Y164, Y218, and Y280 involving XO-mediated reactive oxygen species production and Src and Pyk2 activation, resulting in its dissociation from occludin, thereby causing tight junction (TJ) disruption, increased vascular permeability, and enhanced leukocyte and monocyte transmigration in vitro using EC monolayer and ex vivo using arteries as models. The phosphorylation of JamA on Y164, Y218, and Y280 appears to be critical for its role in 15(S)-HETE-induced EC barrier dysfunction, as mutation of any one of these amino acid residues prevented its dissociation from occludin and restored TJ integrity and barrier function. In response to high-fat diet (HFD) feeding, WT, but not 12/15-lipoxygenase (LO)(-/-), mice showed enhanced XO expression and its activity in the artery, which was correlated with increased aortic TJ disruption and barrier permeability with enhanced leukocyte adhesion and these responses were inhibited by allopurinol. These observations provide novel insights on the role of XO in 12/15-LO-induced JamA tyrosine phosphorylation and TJ disruption leading to increased vascular permeability in response to HFD.

Factor Xa dimerization competes with prothrombinase complex formation on platelet-like membrane surfaces.

Exposure of phosphatidylserine (PS) molecules on activated platelet membrane surface is a crucial event in blood coagulation. Binding of PS to specific sites on factor Xa (fXa) and factor Va (fVa) promotes their assembly into a complex that enhances proteolysis of prothrombin by approximately 105. Recent studies demonstrate that both soluble PS and PS-containing model membranes promote formation of inactive fXa dimers at 5 mM Ca²âº. In the present study, we show how competition between fXa dimerization and prothrombinase formation depends on Ca²âº and lipid membrane concentrations. We used homo-FRET measurements between fluorescein-E-G-R-chloromethylketone (CK)-Xa [fXa irreversibly inactivated by alkylation of the active site histidine residue with FEGR (FEGR-fXa)] and prothrombinase activity measurements to reveal the balance between fXa dimer formation and fXa-fVa complex formation. Changes in FEGR-fXa dimer homo-FRET with addition of fVa to model-membrane-bound FEGR-fXa unambiguously demonstrated that formation of the FEGR-fXa-fVa complex dissociated the dimer. Quantitative global analysis according to a model for protein interaction equilibria on a surface provided an estimate of a surface constant for fXa dimer dissociation (K(fXa×fXa)(d, σ)) approximately 10-fold lower than K(fXa×fVa)(d,σ) for fXa-fVa complex. Experiments performed using activated platelet-derived microparticles (MPs) showed that competition between fXa dimerization and fXa-fVa complex formation was even more prominent on MPs. In summary, at Ca²âº concentrations found in the maturing platelet plug (2-5 mM), fVa can compete fXa off of inactive fXa dimers to significantly amplify thrombin production, both because it releases dimer inhibition and because of its well-known cofactor activity. This suggests a hitherto unanticipated mechanism by which PS-exposing platelet membranes can regulate amplification and propagation of blood coagulation.

Soluble phosphatidylserine binds to two sites on human factor IXa in a Ca2+ dependent fashion to specifically regulate structure and activity.

Clinical studies have demonstrated a correlation between elevated levels of FIX and the risk of coronary heart disease, while reduced plasma FIX causes hemophilia B. FIXa interacts with FVIIIa in the presence of Ca2+ and phosphatidylserine (PS)-containing membranes to form a factor X-activating complex (Xase) that is key to propagation of the initiated blood coagulation process in human. We test the hypothesis that PS in these membranes up-regulates the catalytic activity of this essential enzyme. We used a soluble form of phosphatidylserine, 1, 2-dicaproyl-sn-glycero-3-phospho-L-serine (C6PS), as a tool to do so. C6PS and PS in membranes are reported to regulate the homologous FXa nearly identically. FIXa binds a molecule of C6PS at each of with two sites with such different affinities (∼100-fold) that these appear to be independent. A high affinity C6PS binding site (Kd∼1.4 µM) regulates structure, whereas a low-affinity binding site (Kd∼140 µM) regulates activity. Equilibrium dialysis experiments were analyzed globally with four other data sets (proteolytic and amidolytic activities, intrinsic fluorescence, ellipticity) to unequivocally demonstrate stoichiometries of one for both sites. Michaelis-Menten parameters for FIXa proteolytic activity were the same in the presence of C6PS or PS/PC membranes. We conclude that the PS molecule and not a membrane surface is the key regulator of both factors Xa and IXa. Despite some minor differences in the details of regulation of factors Xa and IXa, the similarities we found suggest that lipid regulation of these two proteases may be similar, a hypothesis that we continue to test.

Vascular endothelial tight junctions and barrier function are disrupted by 15(S)-hydroxyeicosatetraenoic acid partly via protein kinase C ε-mediated zona occludens-1 phosphorylation at threonine 770/772.

Disruption of tight junctions (TJs) perturbs endothelial barrier function and promotes inflammation. Previously, we have shown that 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), the major 15-lipoxygenase 1 (15-LO1) metabolite of arachidonic acid, by stimulating zona occludens (ZO)-2 tyrosine phosphorylation and its dissociation from claudins 1/5, induces endothelial TJ disruption and its barrier dysfunction. Here, we have studied the role of serine/threonine phosphorylation of TJ proteins in 15(S)-HETE-induced endothelial TJ disruption and its barrier dysfunction. We found that 15(S)-HETE enhances ZO-1 phosphorylation at Thr-770/772 residues via PKCε-mediated MEK1-ERK1/2 activation, causing ZO-1 dissociation from occludin, disrupting endothelial TJs and its barrier function, and promoting monocyte transmigration; these effects were reversed by T770A/T772A mutations. In the arteries of WT mice ex vivo, 15(S)-HETE also induced ZO-1 phosphorylation and endothelial TJ disruption in a PKCε and MEK1-ERK1/2-dependent manner. In line with these observations, in WT mice high fat diet feeding induced 12/15-lipoxygenase (12/15-LO) expression in the endothelium and caused disruption of its TJs and barrier function. However, in 12/15-LO(-/-) mice, high fat diet feeding did not cause disruption of endothelial TJs and barrier function. These observations suggest that the 12/15-LO-12/15(S)-HETE axis, in addition to tyrosine phosphorylation of ZO-2, also stimulates threonine phosphorylation of ZO-1 in the mediation of endothelial TJ disruption and its barrier dysfunction.

Inhibition of intrinsic Xase by protein S: a novel regulatory role of protein S independent of activated protein C.

OBJECTIVE: Protein S is a vitamin K-dependent plasma protein that functions in the feedback regulation of thrombin generation. Our goal was to determine how protein S regulates the intrinsic pathway of blood coagulation. METHODS AND RESULTS: We used plasma, including platelet-rich plasma, and in vitro methods to determine how the intrinsic pathway of blood coagulation is regulated by protein S. We obtained the following results: (1) activated partial thromboplastin time assays with protein S-supplemented plasma confirmed that protein S prolongs clotting time; (2) a modified activated partial thromboplastin time assay with factor IX (fIX)-deficient plasma confirmed that protein S affects fIX-initiated clotting; (3) a fIXa/factor VIIIa (fVIIIa)-mediated thrombin generation assay with either platelet-rich plasma or factor-deficient plasma, initiated with a limiting amount of tissue factor, was regulated by protein S; (4) in the presence of phosphatidylserine vesicles, protein S inhibited fIXa in the absence and presence of fVIIIa; and (5) protein S altered only the K(M) for factor X activation by fIXa in the absence of fVIIIa and both k(cat) and K(M) in the presence of fVIIIa. CONCLUSIONS: From our findings, it can be concluded that protein S inhibits fIXa in the presence or absence of fVIIIa in an activated protein C-independent way.

Functional and structural characterization of factor Xa dimer in solution.

Previous studies showed that binding of water-soluble phosphatidylserine (C6PS) to bovine factor Xa (FXa) leads to Ca2+-dependent dimerization in solution. We report the effects of Ca2+, C6PS, and dimerization on the activity and structure of human and bovine FXa. Both human and bovine dimers are 10(6)- to 10(7)-fold less active toward prothrombin than the monomer, with the decrease being attributed mainly to a substantial decrease in k(cat). Dimerization appears not to block the active site, since amidolytic activity toward a synthetic substrate is largely unaffected. Circular dichroism reveals a substantial change in tertiary or quaternary structure with a concomitant decrease in alpha-helix upon dimerization. Mass spectrometry identifies a lysine (K(270)) in the catalytic domain that appears to be buried at the dimer interface and is part of a synthetic peptide sequence reported to interfere with factor Va (FVa) binding. C6PS binding exposes K(351) (part of a reported FVa binding region), K(242) (adjacent to the catalytic triad), and K(420) (part of a substrate exosite). We interpret our results to mean that C6PS-induced dimerization produces substantial conformational changes or domain rearrangements such that structural data on PS-activated FXa is required to understand the structure of the FXa dimer or the FXa-FVa complex.

Cyclic AMP-dependent functional forms of cyclic AMP receptor protein from Vibrio cholerae.

The cyclic AMP receptor protein (CRP) from Escherichia coli, involved in the transcriptional regulation of a number of genes and operons, works by binding to specific sites upstream of promoters. CRP also binds cyclic AMP (cAMP), and this binding, which causes conformational changes in CRP, is mandatory for its activity. A cAMP-dependent variation in the conformation as well as biological activity of E. coli CRP has been reported, with the cAMP-CRP complex formed at high cAMP concentrations resembling the uncomplexed apoprotein CRP. CRP from Vibrio cholerae, which plays an important role in the regulation of virulence gene expression, has a 95% sequence identity with the E. coli protein. We have purified and characterized V. cholerae CRP and studied its transcription activation properties as a function of increasing cAMP concentrations. A biphasic dependence on cAMP levels was observed, similar to that found for E. coli CRP. The implications of these results on regulation of cAMP-CRP dependent promoters in V. cholerae has been discussed.