Cd42b fitc

Platelet surface GPIX was measured by whole blood flow cytometry as previously described^{43 (link),45 (link),71 (link)–73} . Briefly, citrate-anticoagulated whole blood (10 µL) was incubated 15 min RT with an antibody cocktail consisting of CD42a-PE (phycoerythrin, to detect GPIX, catalog # 558819, BD Pharmingen, San Diego, CA), CD42b-FITC (fluorescein isothiocyanate, catalog # 555472, BD Pharmingen) and CD41-PerCP-Cy5.5 (peridinin chlorophyll protein-Cyanine5.5, catalog # 340930, BD Biosciences, San Diego, CA) in the presence of vehicle (10 mM HEPES, 0.15 M NaCl, pH 7.4, “HEPES-saline”), adenosine diphosphate (ADP, catalog # 384, ChronoLog, Havertown, PA) 20 µM, or thrombin receptor activating peptide (TRAP, catalog # 4031274, BaChem, Torrance, CA) 20 µM (total volume 25 µL). Samples were then fixed by addition of 1% formaldehyde in HEPES-saline. Control samples with FITC and PE conjugated normal IgG were used to establish levels of non-specific staining. Samples were analyzed in a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA) (threshold on FL3 CD41-PerCP-Cy5.5) and gated for platelet forward and side light scatter. Compensation for fluorescence overlap of the channels was determined using single stained samples.

Arterial blood samples were collected in citrate (3.2%) anticoagulated tubes (BD Biosciences) prior to intervention and processed directly after collection to prevent any potential blood storage effects.
Platelet reactivity was quantified by stimulation with an increasing concentration of ADP, ranging from 8 nM to 125 µM. The serial dilutions were prepared in 50 µL HEPES buffered saline (HBS; 10 mM HEPES, 150 mM NaCl, 1 mM MgSO₄, 5 mM KCl, pH 7.4) to which 2 µL CD62P-PE (550561, BD Biosciences) and 2 µL CD42b-FITC (555472, BD Biosciences) mouse anti-human antibodies were added. The platelet reactivity assay was initiated by the addition of 5 µL whole blood to each sample of the serial dilution for 20 minutes at room temperature. Samples were fixated with 500 µL of 0.2% formaldehyde in 0.9% NaCl solution. Using flow cytometry, 10.000 platelets were identified by scatter gating and CD42b-FITC binding, after which CD62P-PE mean fluorescence intensity (MFI) was measured. We used the area under the curve (AUC) to integrate the sensitivity and the maximum response of platelets towards agonist concentrations into one quantitative value (Figure 1). All data acquisition was performed with the CXP SYSTEM software.

Histology and Immunohistochemistry: Lungs were flushed with PBS then inflation‐fixed at 25 cm H2O for 30 min with 4% paraformaldehyde for paraffin embedding. Immunohistochemistry was performed for CD41 using the rabbit polyclonal antibody (1:200, GTX113758, GeneTex) diluted in Dako Antibody Diluent (S0809 Agilent Dako). Sections were developed with Dako EnVision+ Dual Link System‐HRP (DAB+) (K4065 Agilent Dako) and counterstained with Light Green (STLGC100 American MasterTech Scientific).
FACS: 100 µl of a 1:10 dilution of CD41‐BV421 (Biolegend, Clone # MWReg30) in PBS was retro‐orbitally injected into the mice 5 min before collecting lungs to label intravascular platelets. Studies were performed on a single day with an n of 5–7. Lungs were homogenized as previously described (PMID 30024304). Whole lung digests were then stained with CD41‐APC (Clone # MWReg30, BD Biosciences) and CD42b‐FITC (Clone # Xia.G5, Emfret) to label whole lung platelets. Interstitial platelets were defined for positive staining for CD41‐APC and CD42b‐FITC but negative staining for CD41‐BV421 (CD41‐APC^Hi, CD42b‐FITC^Hi, CD41‐BV421^Lo). Quantification was performed using 123eCount beads (Thermofisher) as previously described (Good et al., 2018).