Ual to 5.4 and 8.7 , respectively. In a subgroup of subjects (28 cases) from

Ual to 5.4 and 8.7 , respectively. In a CAL-120 subgroup of subjects (28 cases) from type 2 diabetes patients with the highest Fruquintinib plasma protein carbonyls level (.200 pmol/mg), it was reliably possible to measure the specific carbonyl content of VWF, purified fromOxidized von Willebrand Factor and DiabetesTable 3. Haemostatic and oxidative biomarker levels in T1- and T2-DM patients and respective controls.Patameter n Fibrinogen (mg/dl) D-dimer (ng/ml) VWF:ag ( ) VWF:act ( ) VWF-R (Act/Ag) ADAMTS13 Antigen (ng/ml) ADAMTS13 Activity ( ) ADAMTS13/VWF:act ratio Log MMW Protein carbonyls (pmol/mg)T1DM 41 2506311 196?87 110642 108?1 0.92?.2 595?57 83?1 0.8?.5 6.1?.7 167?Controls T1DM 41 245634 160?7 108624 97?8 0.91?.1 629?43 111?5 1.14?.5 6.160.6P*T2 DMControls T2DM 42 264?0 171?9 115?6 102?2 0.9?.13 589?85 100?2 0.98?.5 6.2?.7 128?P*P**0.546 0.289 0.789 0.156 0.051 0.292 ,0.001 0.038 0.63 0.352?18 306?43 165?5 148?2 0.9460.5 592?98 95?7 0.6?.5 7.3?.8 340?,0.001 ,0.001 ,0.001 ,0.001 0.618 0.936 0.8243 0.021 0.0062 ,0.0.0169 0.0008 ,0.001 0.0056 0.589 0.976 0.332 0.214 0.0065 ,0.*Comparison with respective controls; **Comparison between type 1 and type 2 patients. 1 The values of mean6SD are listed in the table. doi:10.1371/journal.pone.0055396.tplasma according to a previously described method, with some modifications [5]. Frozen plasma from each subject (20 ml) was thawed at room temperature, added with 0.2 g of PEG [poly(ethylene glycol)]-6000 (Sigma) to a final 1 (w/v) concentration, and gently stirred for 15 min. This solution was added with 1 mM PMSF (0.2 ml, 0.1 M) and 10 mM EDTA (0.4 ml, 0.5 M), as protease inhibitors and left overnight at 4uC under gentle magnetic stirring. The suspension was then centrifuged at 3000 rev./min for 1 h at 4uC. In each test tube, the supernatant was discarded and the pellet resuspended under gentle stirring with 0.2 ml of 110 mM sodium citrate buffer, pH 7.4, and 0.75 ml of 25 mM Tris/HCl, pH 6.8, containing 0.35 M NaCl and 2.6 M glycine. The suspension was centrifuged at 3000 rpm for 45 min at room temperature. The pellet was discarded, while the supernatant was added with solid NaCl to a final concentration of 1.55 M. The suspension was stirred for 30 min and then centrifuged at 6000 rpm for 30 min at 25uC. The supernatant was discarded, and the pellet (derived from the 20 ml plasma pool) wasdissolved in citrate buffer (1 ml), divided into aliquots and stored at 280uC. Finally, the solution was fractionated by size exclusion HPLC (SE-HPLC), using a TSK gel Super SW3000 column equilibrated with 20 mM phosphate buffer, 0.15 M NaCl, pH = 7.40, at a flow rate of 0.2 ml/min. The fractions contained in the void volume were collected and analyzed by SDS/PAGE and western blot, using a polyclonal anti-VWF Ab (Dako). No contaminating protein was observed in the SDS-PAGE gel. The concentration of purified VWF was determined spectrophotometrically by measuring the absorbance value at 280 nm, using a molar absorption coefficient of 0.846 mg21 ? cm2, calculated on the 1317923 amino acid sequence of VWF monomer. The quality of VWF preparations was also assessed by measuring VWF concentration as antigen (VWF:Ag) and RiCof (ristocetin cofactor) (VWF:RiCof), according to the immunoturbidometric assays `VWF antigen’ and `VWF activity’ (Instrumentation Laboratory), as detailed previously [5]. The purified VWF was concentrated with a Sartorius Vivaspin 500 centrifugal disposable ultrafiltration device at 14000 rpm in a Thermo microfuge an.Ual to 5.4 and 8.7 , respectively. In a subgroup of subjects (28 cases) from type 2 diabetes patients with the highest plasma protein carbonyls level (.200 pmol/mg), it was reliably possible to measure the specific carbonyl content of VWF, purified fromOxidized von Willebrand Factor and DiabetesTable 3. Haemostatic and oxidative biomarker levels in T1- and T2-DM patients and respective controls.Patameter n Fibrinogen (mg/dl) D-dimer (ng/ml) VWF:ag ( ) VWF:act ( ) VWF-R (Act/Ag) ADAMTS13 Antigen (ng/ml) ADAMTS13 Activity ( ) ADAMTS13/VWF:act ratio Log MMW Protein carbonyls (pmol/mg)T1DM 41 2506311 196?87 110642 108?1 0.92?.2 595?57 83?1 0.8?.5 6.1?.7 167?Controls T1DM 41 245634 160?7 108624 97?8 0.91?.1 629?43 111?5 1.14?.5 6.160.6P*T2 DMControls T2DM 42 264?0 171?9 115?6 102?2 0.9?.13 589?85 100?2 0.98?.5 6.2?.7 128?P*P**0.546 0.289 0.789 0.156 0.051 0.292 ,0.001 0.038 0.63 0.352?18 306?43 165?5 148?2 0.9460.5 592?98 95?7 0.6?.5 7.3?.8 340?,0.001 ,0.001 ,0.001 ,0.001 0.618 0.936 0.8243 0.021 0.0062 ,0.0.0169 0.0008 ,0.001 0.0056 0.589 0.976 0.332 0.214 0.0065 ,0.*Comparison with respective controls; **Comparison between type 1 and type 2 patients. 1 The values of mean6SD are listed in the table. doi:10.1371/journal.pone.0055396.tplasma according to a previously described method, with some modifications [5]. Frozen plasma from each subject (20 ml) was thawed at room temperature, added with 0.2 g of PEG [poly(ethylene glycol)]-6000 (Sigma) to a final 1 (w/v) concentration, and gently stirred for 15 min. This solution was added with 1 mM PMSF (0.2 ml, 0.1 M) and 10 mM EDTA (0.4 ml, 0.5 M), as protease inhibitors and left overnight at 4uC under gentle magnetic stirring. The suspension was then centrifuged at 3000 rev./min for 1 h at 4uC. In each test tube, the supernatant was discarded and the pellet resuspended under gentle stirring with 0.2 ml of 110 mM sodium citrate buffer, pH 7.4, and 0.75 ml of 25 mM Tris/HCl, pH 6.8, containing 0.35 M NaCl and 2.6 M glycine. The suspension was centrifuged at 3000 rpm for 45 min at room temperature. The pellet was discarded, while the supernatant was added with solid NaCl to a final concentration of 1.55 M. The suspension was stirred for 30 min and then centrifuged at 6000 rpm for 30 min at 25uC. The supernatant was discarded, and the pellet (derived from the 20 ml plasma pool) wasdissolved in citrate buffer (1 ml), divided into aliquots and stored at 280uC. Finally, the solution was fractionated by size exclusion HPLC (SE-HPLC), using a TSK gel Super SW3000 column equilibrated with 20 mM phosphate buffer, 0.15 M NaCl, pH = 7.40, at a flow rate of 0.2 ml/min. The fractions contained in the void volume were collected and analyzed by SDS/PAGE and western blot, using a polyclonal anti-VWF Ab (Dako). No contaminating protein was observed in the SDS-PAGE gel. The concentration of purified VWF was determined spectrophotometrically by measuring the absorbance value at 280 nm, using a molar absorption coefficient of 0.846 mg21 ? cm2, calculated on the 1317923 amino acid sequence of VWF monomer. The quality of VWF preparations was also assessed by measuring VWF concentration as antigen (VWF:Ag) and RiCof (ristocetin cofactor) (VWF:RiCof), according to the immunoturbidometric assays `VWF antigen’ and `VWF activity’ (Instrumentation Laboratory), as detailed previously [5]. The purified VWF was concentrated with a Sartorius Vivaspin 500 centrifugal disposable ultrafiltration device at 14000 rpm in a Thermo microfuge an.