May gr nwald solution

To perform cone and plate(let) analysis (CPA) we employed the Impact-R device [18] (link), [19] (link) (DiaMed, Switzerland). Citrated whole blood was collected from at least three volunteers and allowed to rest for 45 min at RT. Then, 16 µM InsPs (InsP₃, InsP₅ or InsP₆) or the corresponding volume of PBS in absence or presence of 2.8 µg/ml abciximab (ReoPro®, Lilly Deutschland GmbH, Bad Homburg, Germany) were added and mixed for 1 min at 10 rpm on the tube rotator of the Impact-R device.
When using washed blood, fibrinogen (1.5 mg/ml) was added to facilitate aggregate adhesion. CPA samples contained 16 µM InsPs (InsP₃, InsP₅ or InsP₆) with or without either additional 1.5 mg/ml fibrinogen, 10 µg/ml rVWF (produced as previously described [20] (link)), or 3 µg/ml human collagen type III (Southern Biotech, Biozol, Eching, Germany). After an incubation of 5 min, the samples were mixed at 10 rpm for 1 min.
For CPA, 130 µl of each sample were subjected to flow for 2 min at a defined shear rate of 720 rpm (corresponding to 1800 s⁻¹). Subsequently, the wells were washed with PBS, stained with May-Grünwald solution (Roth, Karlsruhe, Germany) for 1 min and further analyzed with the internal image analyzer of the Impact-R device. Platelet aggregation was evaluated by examining the average size (AS) of surface-bound objects [21] .

InsP₅ (triethylammonium salt) was synthesised as previously reported [12] (link). InsP₃ (potassium salt) was purchased from Buchem B.V. (Apeldoorn, The Netherlands)_, InsP₆ (potassium salt) from Millipore (Darmstadt, Germany), apyrase from potato and human fibrinogen (purified from plasma) were purchased from Sigma Aldrich (Munich, Germany), fibrinogen from human plasma Alexa Fluor 488 conjugate from Thermo Fisher Scientific (Darmstadt, Germany), human α-thrombin from Haematologic Technologies Inc (Essex Junction, VT, USA), t-PA from Haemachrom (Essen, Germany), Glu-Plasminogen from Enzyme Research Laboratories (South Bend, IN, USA), human collagen type III from Southern Biotech (Birmingham, AL, USA) and May-Grünwald solution from Carl Roth (Karlsruhe, Germany). Chemicals for all buffers described below were purchased from Sigma Aldrich (Munich, Germany).

A total of 4 × 10⁴ cells were resuspended in 150 μL of phosphate-buffered saline (PBS) and centrifuged on glass slides using a Cytofuge^® (Medite, Burgdorf, Germany) at 700× g for 10 min. Afterwards, May–Grünwald Giemsa staining was performed. In short, cells were stained in 0.25% (w/v) May–Grünwald solution (Roth, Karlsruhe, Germany) for 5 min, washed in distilled water and stained with 1:20 diluted GIEMSA solution (Roth) for 20 min. After a second wash step, the slides were dried overnight and embedded in Roti-Histokitt mounting solution (Roth, Karlsruhe, Germany). Pictures were taken using an Olympus IX71 with the CellSens Dimension imaging software (Olympus, Shinjuku, Japan).