ATO reduces collagen, ADP, and thrombin induced platelet aggregation, and intraperitoneal injection of ATO reduces circulating platelets.

<p>(A) PRP was incubated with ATO (2 μM) or vehicle for 1 hr, washed platelets was incubated with ATO (16 μM) or vehicle for 2 hrs. Platelet aggregation assay was performed by addition of collagen (5 μg/mL) or ADP (10 μmol/L) into PRP, or thrombin (0.5 U/mL) into washed platelets under stirring condition, and recorded by a turbidometric platelet aggregometer. Results are representative of 3 separate experiments with different donors. (B) Two groups of mice were intraperitoneally injected with ATO (dissolved in 0.9% NS, 5 mg/kg) or vehicle once a day for 5 continuous days. Blood samples were taken from the mice before injection and 24 hrs after the final injection, and then subjected to platelet count assay using Sysmex KX-21N Blood Cell Analyser. Platelet count (% of baseline) equals platelet count after injection/platelet count before injection ×100%. Quantitation data from 12 mice per group are illustrated as mean ± SD. **<i>P</i><0.01, compared with vehicle controls.</p

ATO dose-dependently induces ΔΨm depolarization in platelets.

<p>Washed platelets were pre-treated with 2, 4, 8, or 16 μM of ATO or vehicle at 37°C for 5 hrs. JC-1 was added into the pre-treated platelets to a final concentration of 5 μg/mL and then incubated at 37°C in the dark for 20 min. Then the treated samples were detected using flow cytometry. (A) Typical flow cytometric histograms represented of 3 separate experiments are shown. FL1 and FL3 stand for green and red fluorescence, respectively. Dots inside R1 indicate that platelets are out of the main population, bearing ΔΨm depolarization. (B) Quantitation data from 3 separate experiments with different donors are illustrated (mean ± SD). *<i>P</i><0.05, **<i>P</i><0.01, compared with vehicle controls.</p

Additional file 1: of A novel monoclonal antibody against the von Willebrand Factor A2 domain reduces its cleavage by ADAMTS13

Supplemental data. Detailed methods and materials are shown. (DOC 115 kb

Additional file 3: Figure S2. of A novel monoclonal antibody against the von Willebrand Factor A2 domain reduces its cleavage by ADAMTS13

SZ-179 inhibits cleavage of VWF by ADAMTS13 in plasma under denaturing conditions. (A, B) Pooled normal human plasma was pre-incubated with SZ-179 or isotype control IgG1 for 2 h at 37°C, and then incubated with 1.5M urea for 16 h. The proteolytic products were separated by electrophoresis in a 1.3% agarose gel and detected by anti-VWF. (C) Dose–response curve for inhibition of plasma ADAMTS13-mediated cleavage of plasma-VWF. (D) Dose–response curve for inhibition of rADAMTS13-mediated GST-VWF73-H cleavage. Results represented as mean ± SD of four independent experiments. (DOCX 1519 kb

ATO dose-dependently induces up-regulation of Bax, down-regulation of Bcl-2 and Bcl-X_L, and caspase-3 activation in platelets.

<p>Platelets were incubated with 2, 4, or 8 μM of ATO or vehicle at 37°C for 5 hrs. (A) The treated platelets were subjected to Western blot analysis with anti-Bax, anti-Bcl-2, and anti-Bcl-X_L antibodies. (B) Pre-treated platelets were subjected to Western blot analysis using anti-caspase-3 antibody. The 32 kDa caspase-3 fragment indicates nonactivated caspase-3, the 17 kDa caspase-3 fragment indicates activated caspase-3. GAPDH levels demonstrate similar loading. Results are representative of 3 separate experiments with different donors.</p

Additional file 2: Figure S1. of A novel monoclonal antibody against the von Willebrand Factor A2 domain reduces its cleavage by ADAMTS13

Characterization of mAb SZ-179. (A) Quantification of ELISA analyses detecting SZ-179 binding to IgG1, IgG2a, IgG2a, IgG3, or IgM. (B) Quantification of ELISA analyses for SZ-179 or murine IgG1 binding to VWFα5. Dose–response curves are shown. (C) Quantification of ELISA analyses for SZ-179 or murine IgG1 binding to plasma-derived VWF. Dose–response curves are shown. Data are mean ± SD of four independent experiments. (DOCX 974 kb

ATO induces JNK activation and dicumarol reduces ATO induced ΔΨm depolarization in platelets.

<p>(A) Washed platelets were pre-treated with or without dicumaol (2 μM) or vehicle at 37°C for 15 min, then further incubated with various concentration of ATO at 37°C for 5 hrs. The treated platelets were subjected to Western blot analysis using anti-nonphosphorylated JNK1 and JNK2 antibody (anti-JNK 1/2) and anti-phosphorylated JNK1 and JNK2 antibody (anti-p-JNK 1/2). GAPDH levels demonstrate similar loading. Results are representative of 3 separate experiments with different donors. (B) Washed platelets were pre-treated with or without dicumarol (2 μM) or vehicle at 37°C for 15 min, then further incubated with or without ATO at 37°C for 5 hrs. JC-1 was added into the treated platelets to a final concentration of 5 μg/mL and then incubated at 37°C in the dark for 20 min. The treated samples were detected using flow cytometry. Dots inside R1 are platelets out of the main population bearing ΔΨm depolarization. (C) Quantitation data from 3 separate experiments with different donors are illustrated (mean ± SD). **<i>P</i><0.01 compared with vehicle controls.</p

Analysis of plasma and platelet VWF multimers.

<p>Plasma (A) and platelet (B) VWF multimers were assessed by 1.6% SDS-agarose gel electrophoresis and Western blotting. Normal plasma samples were diluted 1∶20 and patient plasma samples were diluted 1∶5. Samples from family members and a normal control (N) were indicated. Platelet samples were not available from IV-4.</p

A novel nonsense NBEAL2 gene mutation causing severe bleeding in a patient with gray platelet syndrome

<p>Gray platelet syndrome (GPS) is a rare, inherited bleeding disorder characterized by the defect of platelet α-granule. Up to date, these are only four studies identifying NBEAL2 gene correlated with GPS. In the current report, we present a Chinese GPS patient who had severe bleeding tendency, abnormalities of platelet functions, and absence of platelet α-granules. Genomic DNA sequencing for the patient identified a nonsense mutation (g.27713C>A) of NBEAL2 gene (g.NG__031914.1) resulting in a premature protein (p.Glu1726*). In comparison with the reported patients, we conclude that homozygotes with nonsense or deletion mutation leading to a premature stop codon exhibit more serious bleeding problem than those with missense mutations.</p

Identification of a VWF gene mutation.

<p>Sequence analysis of the VWF gene in the proband detected a 6-bp nucleotides deletion in exon 28. The mutation caused D1529V1530 deletion (ΔD1529V1530) in VWF A2 domain. The ADAMTS13 cleavage site is indicated by an arrow.</p