P2Y12 Inhibition in Patients Requiring Oral Anticoagulation After Percutaneous Coronary Intervention: The SWAP-AC-2 Study.

BACKGROUND: Among patients treated with a novel oral anticoagulant (NOAC) undergoing percutaneous coronary intervention (PCI), combination therapy with clopidogrel (ie, known as dual antithrombotic therapy [DAT]) is the treatment of choice. However, there are concerns for individuals with impaired response to clopidogrel. OBJECTIVES: The authors sought to assess the pharmacodynamic (PD) effects of clopidogrel vs low-dose ticagrelor in patients with impaired clopidogrel response assessed by the ABCD-GENE score. METHODS: This was a prospective, randomized PD study of NOAC-treated patients undergoing PCI. Patients with an ABCD-GENE score ≥10 (n = 39), defined as having impaired clopidogrel response, were randomized to low-dose ticagrelor (n = 20; 60 mg twice a day) or clopidogrel (n = 19; 75 mg once a day). Patients with an ABCD-GENE score <10 (n = 42) were treated with clopidogrel (75 mg once a day; control cohort). PD assessments at baseline and 30 days post-randomization (trough and peak) were performed to assess P2Y12 signaling (VerifyNow P2Y12 reaction units [PRU], light transmittance aggregometry, and vasodilator-stimulated phosphoprotein); makers of thrombosis not specific to P2Y12 signaling were also assessed. The primary endpoint was PRU (trough levels) at 30 days. RESULTS: At 30 days, PRU levels were reduced with ticagrelor-based DAT compared with clopidogrel-based DAT at trough (23.0 [Q1-Q3: 3.0-46.0] vs 154.5 [Q1-Q3: 77.5-183.0]; P < 0.001) and peak (6.0 [Q1-Q3: 4.0-14.0] vs 129.0 [Q1-Q3: 66.0-171.0]; P < 0.001). Trough PRU levels in the control arm (104.0 [Q1-Q3: 35.0-167.0]) were higher than ticagrelor-based DAT (P = 0.005) and numerically lower than clopidogrel-based DAT (P = 0.234). Results were consistent by light transmittance aggregometry and vasodilator-stimulated phosphoprotein. Markers measuring other pathways leading to thrombus formation were largely unaffected. CONCLUSIONS: In NOAC-treated patients undergoing PCI with an ABCD-GENE score ≥10, ticagrelor-based DAT using a 60-mg, twice-a-day regimen reduced platelet P2Y12 reactivity compared with clopidogrel-based DAT. (Tailoring P2Y12 Inhibiting Therapy in Patients Requiring Oral Anticoagulation After PCI [SWAP-AC-2]; NCT04483583).

Changes in fibrin clot properties in patients after Roux-en-Y gastric bypass surgery.

Background: Obesity is a complex condition associated with prothrombotic fibrin networks that are resistant to fibrinolysis. Altered fibrin clot properties enhance cardiovascular risk and associate with a poorer prognosis following acute ischemic events. Bariatric surgery is commonly employed to improve cardiometabolic outcomes in individuals with obesity. However, the effects of this surgical intervention on fibrin clot properties have not been comprehensively studied. Objectives: To examine fibrin clot and lysis parameters in Roux-en-Y gastric bypass (RYGB) patients before and after surgery. Methods: The fibrin clot properties of 32 individuals living with obesity before and 9 months after RYGB surgery were determined using turbidimetric analysis. Correlation and regression analyses were used to identify relationships between clot properties and anthropomorphic and clinical measures. Results: RYGB surgery resulted in a significant reduction in adiposity-associated anthropometric measures as well as improvements in glycemia and lipid profile. Clot maximum absorbance was reduced from 0.43 ± 0.11 at baseline to 0.29 ± 0.10 at 9 months postsurgery (P < .0001), while fibrin clot lysis time failed to show a difference. The change in maximum absorbance was not caused by alterations in fibrinogen levels, while plasminogen activator inhibitor-1 concentration was significantly increased after surgery from 10,560 ± 6681 pg/mL to 15,290 ± 6559 pg/mL (P = .009). Correlation and regression analyses indicated that maximum absorbance was influenced by markers of adiposity as well as glycated hemoglobin and high-sensitivity C-reactive protein concentrations. Conclusion: RYGB surgery led to a decrease in the maximum absorbance of the fibrin clot. Values of maximum absorbance were associated with measures of glycemic control and inflammation. In contrast to previous reports, fibrin clot lysis time was not affected after surgery.

Affinity purification of fibrinogen using an Affimer column.

BACKGROUND: Fibrinogen is an abundant plasma protein with an essential role in blood coagulation and haemostasis thus receiving significant research interest. However, protein purification is time consuming and commercial preparations often have protein contaminants. The aim of this study was to develop a new method to purify high quality and functional fibrinogen. METHODS: Fibrinogen-specific Affimer protein, isolated using phage display systems, was immobilised to SulfoLink resin column and employed for fibrinogen purification from plasma samples. Fibrinogen was eluted using a high pH solution. Commercial human fibrinogen was also further purified using the Affimer column. Fibrinogen purity was determined by SDS-PAGE and mass spectrometry, while functionality was assessed using turbidimetric analysis. RESULTS: Affimer-purified fibrinogen from human plasma showed purity at least comparable to commercially available preparations and was able to form physiological fibrin networks. Further purification of commercially available fibrinogen using the Affimercolumn eliminated multiple contaminant proteins, a significant number of which are key elements of the coagulation cascade, including plasminogen and factor XIII. CONCLUSIONS: The Affimercolumn represents a proof of concept novel, rapid method for isolating functional fibrinogen from plasma and for further purification of commercially available fibrinogen preparations. GENERAL SIGNIFICANCE: Our methodology provides an efficient way of purifying functional fibrinogen with superior purity without the need of expensive pieces of equipment or the use of harsh conditions.

Albumin-mediated alteration of plasma zinc speciation by fatty acids modulates blood clotting in type-2 diabetes.

Zn2+ is an essential regulator of coagulation and is released from activated platelets. In plasma, the free Zn2+ concentration is fine-tuned through buffering by human serum albumin (HSA). Importantly, the ability of HSA to bind/buffer Zn2+ is compromised by co-transported non-esterified fatty acids (NEFAs). Given the role of Zn2+ in blood clot formation, we hypothesise that Zn2+ displacement from HSA by NEFAs in certain conditions (such as type 2 diabetes mellitus, T2DM) impacts on the cellular and protein arms of coagulation. To test this hypothesis, we assessed the extent to which increasing concentrations of a range of medium- and long-chain NEFAs reduced Zn2+-binding ability of HSA. Amongst the NEFAs tested, palmitate (16 : 0) and stearate (18 : 0) were the most effective at suppressing zinc-binding, whilst the mono-unsaturated palmitoleate (16 : 1c9) was markedly less effective. Assessment of platelet aggregation and fibrin clotting parameters in purified systems and in pooled plasma suggested that the HSA-mediated impact of the model NEFA myristate on zinc speciation intensified the effects of Zn2+ alone. The effects of elevated Zn2+ alone on fibrin clot density and fibre thickness in a purified protein system were mirrored in samples from T2DM patients, who have derranged NEFA metabolism. Crucially, T2DM individuals had increased total plasma NEFAs compared to controls, with the concentrations of key saturated (myristate, palmitate, stearate) and mono-unsaturated (oleate, cis-vaccenate) NEFAs positively correlating with clot density. Collectively, these data strongly support the concept that elevated NEFA levels contribute to altered coagulation in T2DM through dysregulation of plasma zinc speciation.

Prothrombotic fibrin network characteristics in patients with acromegaly: a novel mechanism for vascular complications.

OBJECTIVE: There remains increased cardiovascular mortality in patients with acromegaly. This study aims to evaluate whether GH/IGF-1 excess increases vascular disease by adversely affecting fibrin network characteristics. DESIGN: Cross-sectional study in 40 patients with acromegaly (21 males, age 53 ± 13 years) and 40 age/gender-matched controls. METHODS: Clot structure was analysed using a validated turbidimetric assay and fibrin networks were visualised by laser scanning confocal microscopy (LSCM). Metabolic profile parameters, body composition, plasma fibrinogen and PAI-1 were also assessed. RESULTS: Twenty-two patients had active acromegaly and 18 were in remission. There was no difference in qualitative patient characteristics between the two groups. Both groups had less favourable body composition and cardiovascular risk profile compared with controls. Despite no difference in clot formation and lysis parameters between the two patient groups, active disease patients had higher fibrinogen and clot maximum absorbance compared with controls, after adjusting for BMI (3.8 ± 0.2 vs 2.6 ± 0.2 mg/mL, P < 0.001; and 0.39 ± 0.02 vs 0.33 ± 0.01 arbitrary units, P = 0.03, respectively). Patients in remission had higher fibrinogen compared with controls following adjustment for BMI (3.3 ± 0.2 vs 2.6 ± 0.2 mg/mL, P = 0.02) but not clot maximum absorbance (0.35 ± 0.03 vs 0.33 ± 0.02 arbitrary units, P = 0.6). LSCM showed increased fibrin network density only in active disease patients, consistent with turbidimetric analysis. In addition to active disease, BMI, fat mass and skinfold thickness were associated with higher clot density and longer lysis time. CONCLUSIONS: Patients with active acromegaly have more compact clots, thus conferring increased thrombosis risk. Prothrombotic fibrin networks may represent one mechanism for enhanced vascular risk in active acromegaly.

Reduced Plasma Magnesium Levels in Type-1 Diabetes Associate with Prothrombotic Changes in Fibrin Clotting and Fibrinolysis.

Individuals with type-1 diabetes mellitus (T1DM) have a higher risk of thrombosis and low plasma magnesium concentrations. As magnesium is a known regulator of fibrin network formation, we investigated potential associations between fibrin clot properties and plasma magnesium concentrations in 45 individuals with T1DM and 47 age- and sex-matched controls without diabetes. Fibrin clot characteristics were assessed using a validated turbidimetric assay and associations with plasma magnesium concentration were examined. Plasma concentrations of fibrinogen, plasminogen activator inhibitor-1 (PAI-1), and lipids were measured and fibrin fiber diameters assessed using scanning electron microscopy. Fibrin clot maximum absorbance was unchanged in subjects with T1DM compared with controls, while lysis time was prolonged (p = 0.0273). No differences in fibrin fiber diameters or in lipid profile were observed between T1DM and controls. PAI-1 concentration was lower in the T1DM group compared with the controls (p = 0.0232) and positively correlated with lysis time (p = 0.0023). Plasma magnesium concentration was lower in the T1DM group compared with controls (p < 0.0001). Magnesium concentration negatively correlated with clot maximum absorbance (p = 0.0215) and lysis time (p = 0.0464). A turbidimetric fibrin clot lysis assay performed in a purified system that included PAI-1 and 0 to 3.2 mM Mg2+ showed a shortening of lysis time with increasing Mg2+ concentrations (p = 0.0004). Our findings reveal that plasma magnesium concentration is associated with changes in fibrin clot and lysis parameters.

Aspirin, clopidogrel and prasugrel monotherapy in patients with type 2 diabetes mellitus: a double-blind randomised controlled trial of the effects on thrombotic markers and microRNA levels.

BACKGROUND: Despite increased atherothrombotic risk in type 2 diabetes mellitus, (T2DM) the best preventative antithrombotic strategy remains undetermined. We defined the effects of three antiplatelet agents on functional readout and biomarker kinetics in platelet activation and coagulation in patients with T2DM. MATERIALS AND METHODS: 56 patients with T2DM were randomised to antiplatelet monotherapy with aspirin 75 mg once daily (OD), clopidogrel 75 mg OD or prasugrel 10 mg OD during three periods of a crossover study. Platelet aggregation (PA) was determined by light-transmittance aggregometry and P-selectin expression by flow cytometry. Markers of fibrin clot dynamics, inflammation and coagulation were measured. Plasma levels of 14 miRNA were assessed by quantitative polymerase chain reactions. RESULTS: Of the 56 patients, 24 (43%) were receiving aspirin for primary prevention of ischaemic events and 32 (57%) for secondary prevention. Prasugrel was the strongest inhibitor of ADP-induced PA (mean ± SD maximum response to 20µmol/L ADP 77.6 ± 8.4% [aspirin] vs. 57.7 ± 17.6% [clopidogrel] vs. 34.1 ± 14.1% [prasugrel], p < 0.001), P-selectin expression (30 µmol/L ADP; 45.1 ± 21.4% vs. 27.1 ± 19.0% vs. 14.1 ± 14.9%, p < 0.001) and collagen-induced PA (2 µg/mL; 62.1 ± 19.4% vs. 72.3 ± 18.2% vs. 60.2 ± 18.5%, p < 0.001). Fibrin clot dynamics and levels of coagulation and inflammatory proteins were similar. Lower levels of miR-24 (p = 0.004), miR-191 (p = 0.019), miR-197 (p = 0.009) and miR-223 (p = 0.014) were demonstrated during prasugrel-therapy vs. aspirin. Circulating miR-197 was lower in those cardiovascular disease during therapy with aspirin (p = 0.039) or prasugrel (p = 0.0083). CONCLUSIONS: Prasugrel monotherapy in T2DM provided potent platelet inhibition and reduced levels of a number of platelet-associated miRNAs. miR-197 is a potential marker of cardiovascular disease in this population. Clinical outcome studies investigating prasugrel monotherapy are warranted in individuals with T2DM. Trial registration EudraCT, 2009-011907-22. Registered 15 March 2010, https://www.clinicaltrialsregister.eu/ctr-search/trial/2009-011907-22/GB.

Affimer proteins as a tool to modulate fibrinolysis, stabilize the blood clot, and reduce bleeding complications.

Bleeding complications secondary to surgery, trauma, or coagulation disorders are important causes of morbidity and mortality. Although fibrin sealants are considered to minimize blood loss, this is not widely adopted because of its high cost and/or risk for infection. We present a novel methodology employing nonantibody fibrinogen-binding proteins, termed Affimers, to stabilize fibrin networks with the potential to control excessive bleeding. Two fibrinogen-specific Affimer proteins, F5 and G2, were identified and characterized for their effects on clot structure/fibrinolysis, using turbidimetric and permeation analyses and confocal and electron microscopy. Binding studies and molecular modeling identified interaction sites, whereas plasmin generation assays determined effects on plasminogen activation. In human plasma, F5 and G2 prolonged clot lysis time from 9.8 ± 1.1 minutes in the absence of Affimers to 172.6 ± 7.4 and more than 180 minutes (P < .0001), respectively, and from 7.6 ± 0.2 to 28.7 ± 5.8 (P < .05) and 149.3 ± 9.7 (P < .0001) minutes in clots made from purified fibrinogen. Prolongation in fibrinolysis was consistent across plasma samples from healthy control patients and individuals at high bleeding risk. F5 and G2 had a differential effect on clot structure and G2 profoundly altered fibrin fiber arrangement, whereas F5 maintained physiological clot structure. Affimer F5 reduced fibrin-dependent plasmin generation and was predicted to bind fibrinogen D fragment close to tissue plasminogen activator (tPA; residues γ312-324) and plasminogen (α148-160) binding sites, thus interfering with tPA-plasminogen interaction and representing 1 potential mechanism for modulation of fibrinolysis. Our Affimer proteins provide a novel methodology for stabilizing fibrin networks with potential future clinical implications to reduce bleeding risk.

Prolonged Prothrombotic Effects of Antecedent Hypoglycemia in Individuals With Type 2 Diabetes.

OBJECTIVE: Hypoglycemia has been linked to persistent increases in cardiovascular (CV) mortality in type 2 diabetes after the event. Our aim was to examine acute and downstream effects of hypoglycemia on markers of thrombosis risk and inflammation in type 2 diabetes. RESEARCH DESIGN AND METHODS: Twelve individuals with type 2 diabetes with no history of CV disease and 11 age- and BMI-matched volunteers without diabetes underwent paired hyperinsulinemic-euglycemic (glucose 6 mmol/L for two 60-min periods) and hypoglycemic (glucose 2.5 mmol/L for two 60-min periods) clamps on separate occasions on day 0. Fibrin clot properties, platelet reactivity, and inflammatory markers were measured at baseline, end of and after recovery from the initial clamp, day 1, and day 7 using validated assays and electron microscopy. RESULTS: Euglycemic hyperinsulinemia reduced platelet reactivity, decreased fibrin clot density, and improved fibrinolytic efficiency in both groups. Platelet reactivity and aggregation increased during acute hypoglycemia in both groups, resolving at recovery. In type 2 diabetes, clot lysis times and clot maximum absorbance increased up to day 7 (P = 0.002 and 0.001 vs. euglycemia, respectively), but clots from control subjects without diabetes showed limited changes. Fibrin network density increased Δ 1.15 ± 0.28 fibers/µm2 at day 7 after the hypoglycemic clamp (P < 0.01 for glycemic arm), whereas fibrinogen and complement C3 increased after hypoglycemia up to day 7 in type 2 diabetes only. CONCLUSIONS: Antecedent hypoglycemia has acute and persistent prothrombotic effects, lasting at least 7 days, that were enhanced in individuals with type 2 diabetes. These findings identify mechanisms by which hypoglycemia might increase short- and medium-term risk of CV mortality.

Effects of dabigatran on the cellular and protein phase of coagulation in patients with coronary artery disease on dual antiplatelet therapy with aspirin and clopidogrel. Results from a prospective, randomised, double-blind, placebo-controlled study.

There is growing interest in understanding the effects of adding an oral anticoagulant in patients on dual antiplatelet therapy (DAPT). Vitamin K antagonists (VKAs) and clopidogrel represent the most broadly utilised oral anticoagulant and P2Y12 receptor inhibitor, respectively. However, VKAs can interfere with clopidogrel metabolism via the cytochrome P450 (CYP) system which in turn may result in an increase in platelet reactivity. Dabigatran is a direct acting (anti-II) oral anticoagulant which does not interfere with CYP and has favourable safety and efficacy profiles compared with VKAs. The pharmacodynamic (PD) effects on platelet reactivity and clot kinetic of adjunctive dabigatran therapy in patients on DAPT are poorly explored. In this prospective, randomised, double-blind, placebo-controlled PD study, patients (n=30) on maintenance DAPT with aspirin and clopidogrel were randomised to either dabigatran 150 mg bid or placebo for seven days. PD testing was performed before and after treatment using four different assays exploring multiple pathways of platelet aggregation and fibrin clot kinetics: light transmittance aggregometry (LTA), multiple electrode aggregometry (MEA), kaolin-activated thromboelastography (TEG) and turbidimetric assays. There were no differences in multiple measures of platelet reactivity investigating purinergic and non-purinergic signaling pathways assessed by LTA, MEA and TEG platelet mapping. Dabigatran significantly increased parameters related to thrombin activity and thrombus generation, and delayed fibrin clot formation, without affecting clot structure or fibrinolysis. In conclusion, in patients on DAPT with aspirin and clopidogrel, adjunctive dabigatran therapy is not associated with modulation of profiles of platelet reactivity as determined by several assays assessing multiple platelet signalling pathways. However, dabigatran significantly interferes with parameters related to thrombin activity and delays fibrin clot formation.

Platelet P2Y12 Inhibitors Reduce Systemic Inflammation and Its Prothrombotic Effects in an Experimental Human Model.

OBJECTIVE: Clinical studies suggest that platelet P2Y12 inhibitors reduce mortality from sepsis, although the underlying mechanisms have not been clearly defined in vivo. We hypothesized that P2Y12 inhibitors may improve survival from sepsis by suppressing systemic inflammation and its prothrombotic effects. We therefore determined whether clopidogrel and the novel, more potent P2Y12 inhibitor, ticagrelor, modify these responses in an experimental human model. APPROACH AND RESULTS: We randomized 30 healthy volunteers to ticagrelor (n=10), clopidogrel (n=10), or no antiplatelet medication (controls; n=10). We examined the effect of P2Y12 inhibition on systemic inflammation, which was induced by intravenous injection of Escherichia coli endotoxin. Both P2Y12 inhibitors significantly reduced platelet-monocyte aggregate formation and peak levels of major proinflammatory cytokines, including tumor necrosis factor α, interleukin-6, and chemokine (C-C motif) ligand 2. In contrast to clopidogrel, ticagrelor also significantly reduced peak levels of IL-8 and growth colony-stimulating factor and increased peak levels of the anti-inflammatory cytokine IL-10. In addition, ticagrelor altered leukocyte trafficking. Both P2Y12 inhibitors suppressed D-dimer generation and scanning electron microscopy revealed that ticagrelor also suppressed prothrombotic changes in fibrin clot ultrastructure. CONCLUSIONS: Potent inhibition of multiple inflammatory and prothrombotic mechanisms by P2Y12 inhibitors demonstrates critical importance of platelets as central orchestrators of systemic inflammation induced by bacterial endotoxin. This provides novel mechanistic insight into the lower mortality associated with P2Y12 inhibitors in patients with sepsis in clinical studies.

Gender-specific alterations in fibrin structure function in type 2 diabetes: associations with cardiometabolic and vascular markers.

CONTEXT: Diabetes is associated with increased incidence of atherothrombotic disease. The fibrin network forms the backbone of the arterial thrombus, and fibrin clot structure determines predisposition to cardiovascular events. OBJECTIVES: The aim of the study was to investigate fibrin clot structure/fibrinolysis in the largest type 2 diabetes cohort and analyze associations with cardiometabolic risk factors and vascular pathology. DESIGN: Clot structure/fibrinolysis was assessed in 875 participants of the Edinburgh Type 2 Diabetes Study [age, 68 (range, 60-75) yr; 450 males] by turbidimetric assays, and clots were visualized by confocal microscopy. Four parameters of clot structure/fibrinolysis were analyzed, and plasma levels of fibrinogen and plasminogen activator inhibitor-1 were studied by Clauss assay and ELISA, respectively. RESULTS: Clot maximum absorbance was increased in females compared with males (0.37 ± 0.005 and 0.34 ± 0.005 arbitrary unit, respectively; P < 0.001), and took longer to lyse (803 ± 20 and 665 ± 12 sec, respectively; P < 0.001). These gender differences in clot structure and fibrinolysis were still evident after correcting for fibrinogen and plasminogen activator inhibitor-1 plasma levels. A prothrombotic fibrin structure profile was associated with increased body mass index and low levels of high-density lipoprotein in women and with inadequate diabetes control in men. Clot formation time was related to previous cardiac ischemic events in both men and women after adjusting for traditional risk factors [odds ratio, 1.22 (95% confidence interval, 1.07, 1.38); and 1.33 (1.15, 1.50), respectively], and prothrombotic clots were associated with low ankle brachial index, renal impairment, and smoking, regardless of gender. CONCLUSIONS: Women with type 2 diabetes have compact clots with compromised fibrinolysis compared with men. There are gender-specific associations between clotting parameters and cardiometabolic risk factors in this population, whereas vascular abnormalities, impaired renal function, and smoking are associated with prothrombotic clot structure profile regardless of gender.

Effects of MASP-1 of the complement system on activation of coagulation factors and plasma clot formation.

BACKGROUND: Numerous interactions between the coagulation and complement systems have been shown. Recently, links between coagulation and mannan-binding lectin-associated serine protease-1 (MASP-1) of the complement lectin pathway have been proposed. Our aim was to investigate MASP-1 activation of factor XIII (FXIII), fibrinogen, prothrombin, and thrombin-activatable fibrinolysis inhibitor (TAFI) in plasma-based systems, and to analyse effects of MASP-1 on plasma clot formation, structure and lysis. METHODOLOGY/PRINCIPAL FINDINGS: We used a FXIII incorporation assay and specific assays to measure the activation products prothrombin fragment F1+2, fibrinopeptide A (FPA), and activated TAFI (TAFIa). Clot formation and lysis were assessed by turbidimetric assay. Clot structure was studied by scanning electron microscopy. MASP-1 activated FXIII and, contrary to thrombin, induced FXIII activity faster in the Val34 than the Leu34 variant. MASP-1-dependent generation of F1+2, FPA and TAFIa showed a dose-dependent response in normal citrated plasma (NCP), albeit MASP-1 was much less efficient than FXa or thrombin. MASP-1 activation of prothrombin and TAFI cleavage were confirmed in purified systems. No FPA generation was observed in prothrombin-depleted plasma. MASP-1 induced clot formation in NCP, affected clot structure, and prolonged clot lysis. CONCLUSIONS/SIGNIFICANCE: We show that MASP-1 interacts with plasma clot formation on different levels and influences fibrin structure. Although MASP-1-induced fibrin formation is thrombin-dependent, MASP-1 directly activates prothrombin, FXIII and TAFI. We suggest that MASP-1, in concerted action with other complement and coagulation proteins, may play a role in fibrin clot formation.

Phosphorylation at serines 104 and 106 by Erk1/2 MAPK is important for estrogen receptor-alpha activity.

Phosphorylation of estrogen receptor-alpha (ERalpha) at specific residues in transcription activation function 1 (AF-1) can stimulate ERalpha activity in a ligand-independent manner. This has led to the proposal that AF-1 phosphorylation and the consequent increase in ERalpha activity could contribute to resistance to endocrine therapies in breast cancer patients. Previous studies have shown that serine 118 (S118) in AF-1 is phosphorylated by extracellular signal-regulated kinases 1 and 2 (Erk1/2) mitogen-activated protein kinase (MAPK) in a ligand-independent manner. Here, we show that serines 104 (S104) and 106 (S106) are also phosphorylated by MAPK in vitro and upon stimulation of MAPK activity in vivo. Phosphorylation of S104 and S106 can be inhibited by the MAP-erk kinase (MEK)1/2 inhibitor U0126 and by expression of kinase-dead Raf1. Further, we show that, although S118 is important for the stimulation of ERalpha activity by the selective ER modulator 4-hydroxytamoxifen (OHT), S104 and S106 are also required for the agonist activity of OHT. Acidic amino acid substitution of S104 or S106 stimulates ERalpha activity to a greater extent than the equivalent substitution at S118, suggesting that phosphorylation at S104 and S106 is important for ERalpha activity. Collectively, these data indicate that the MAPK stimulation of ERalpha activity involves the phosphorylation not only of S118 but also of S104 and S106, and that MAPK-mediated hyperphosphorylation of ERalpha at these sites may contribute to resistance to tamoxifen in breast cancer.

Common variation in the C-terminal region of the fibrinogen beta-chain: effects on fibrin structure, fibrinolysis and clot rigidity.

Fibrinogen BbetaArg448Lys is a common polymorphism, positioned within the carboxyl terminus of the Bbeta-chain of the molecule. Studies suggest that it is associated with severity of coronary artery disease and development of stroke. The effects of the amino acid substitution on clot structure remains controversial, and the aim of this study was to investigate the effect(s) of this polymorphism on fibrin clot structure using recombinant techniques. Permeation, turbidity, and scanning electron microscopy showed that recombinant Lys448 fibrin had a significantly more compact structure, with thin fibers and small pores, compared with Arg448. Clot stiffness, measured by means of a novel method using magnetic tweezers, was significantly higher for the Lys448 compared with the Arg448 variant. Clots made from recombinant protein variants had similar lysis rates outside the plasma environment, but when added to fibrinogen-depleted plasma, the fibrinolysis rates for Lys448 were significantly slower compared with Arg448. This study demonstrates for the first time that clots made from recombinant BbetaLys448 fibrinogen are characterized by thin fibers and small pores, show increased stiffness, and appear more resistant to fibrinolysis. Fibrinogen BbetaArg448Lys is a primary example of common genetic variation with a significant phenotypic effect at the molecular level.

T:G mismatch-specific thymine-DNA glycosylase (TDG) as a coregulator of transcription interacts with SRC1 family members through a novel tyrosine repeat motif.

Gene activation involves protein complexes with diverse enzymatic activities, some of which are involved in chromatin modification. We have shown previously that the base excision repair enzyme thymine DNA glycosylase (TDG) acts as a potent coactivator for estrogen receptor-alpha. To further understand how TDG acts in this context, we studied its interaction with known coactivators of nuclear receptors. We find that TDG interacts in vitro and in vivo with the p160 coactivator SRC1, with the interaction being mediated by a previously undescribed motif encoding four equally spaced tyrosine residues in TDG, each tyrosine being separated by three amino acids. This is found to interact with two motifs in SRC1 also containing tyrosine residues separated by three amino acids. Site-directed mutagenesis shows that the tyrosines encoded in these motifs are critical for the interaction. The related p160 protein TIF2 does not interact with TDG and has the altered sequence, F-X-X-X-Y, at the equivalent positions relative to SRC1. Substitution of the phenylalanines to tyrosines is sufficient to bring about interaction of TIF2 with TDG. These findings highlight a new protein-protein interaction motif based on Y-X-X-X-Y and provide new insight into the interaction of diverse proteins in coactivator complexes.

T:G mismatch-specific thymine-DNA glycosylase potentiates transcription of estrogen-regulated genes through direct interaction with estrogen receptor alpha.

Nuclear receptors (NR) classically regulate gene expression by stimulating transcription upon binding to their cognate ligands. It is now well established that NR-mediated transcriptional activation requires the recruitment of coregulator complexes, which facilitate recruitment of the basal transcription machinery through direct interactions with the basal transcription machinery and/or through chromatin remodeling. However, a number of recently described NR coactivators have been implicated in cross-talk with other nuclear processes including RNA splicing and DNA repair. T:G mismatch-specific thymine DNA glycosylase (TDG) is required for base excision repair of deaminated methylcytosine. Here we show that TDG is a coactivator for estrogen receptor alpha (ERalpha). We demonstrate that TDG interacts with ERalpha in vitro and in vivo and suggest a separate role for TDG to its established role in DNA repair. We show that this involves helix 12 of ERalpha. The region of interaction in TDG is mapped to a putative alpha-helical motif containing a motif distinct from but similar to the LXXLL motif that mediates interaction with NR. Together with recent reports linking TFIIH in regulating NR function, our findings provide new data to further support an important link between DNA repair proteins and nuclear receptor function.