Jon a pe

Mice were injected in the retro-orbital plexus with 100 μg Jaq-1 GPVI depletion antibody (Emfret Analytics. Eibelstadt, Germany) for 4-5 days. Depletion of GPVI was confirmed by measurement of JON/A (PE, Emfret Analytics, Eibelstadt, Germany) binding to platelets stimulated with either the GP VI agonist, convulxin (0.6 μg/mL), or the PAR4 agonist peptide, AYPGKF (200 μM), from either Jaq-1- or vehicle-treated mice. JON/A binding was measured using a BD FACSCanto cytofluorimeter. Data were acquired with BD FACSDiva software and analyzed with FlowJo v10.

Freshly washed platelets were incubated in HEPES Tyrode’s (HT) buffer with 5mM glucose ± 1mM pyruvate and 2mM glutamate for 30 minutes. Platelets were then treated with agonists at the indicated concentrations in the presence of JonA-PE, CD62p-FITC, (Emfret Analytics, Germany) and CD41-APC (Ebioscience, San Diego, CA) for 10 minutes at 37° C. Annexin V binding was determined in a similar manner, except staining was conducted for 15 minutes. Samples were analyzed using flow cytometry LSR II (Beckman Dickson, San Jose, CA) gating for CD41 positive events. Detailed protocols can be found in supplemental methods.

Hybridoma cells were obtained from American Type Culture Collection (Manassas, VA). M90 (monoclonal anti-human CD40L IgG1 mAb, ATCC HB-12055) was purified into azide-free PBS from conditioned media by protein-G chromatography. Polyclonal goat anti-human β2GPI antibody (which does not bind mouse β2GPI) was from Affinity Biologicals (Ancaster, ON, Canada). Anti-human-FcγRIIa mAb, IV.3, was from StemCell Technologies (Vancouver, BC, Canada). The phycoerythrin (PE)-labeled anti-mouse α_IIbβ₃ mAb, JON/A-PE, was from Emfret Analytics (Eibelstadt, Germany). Antibodies against mouse P-selectin were obtained from BD Biosciences and labeled with Alexa-800 (Invitrogen, Grand Island, NY). Purified human β2GPI was from Haemotologic Technologies (Essex Junction, VT). Recombinant soluble human CD40L was from PeproTech (Rocky Hill, NJ). Clopidogrel (Plavix) was from Sanofi-Aventis (Bridgewater NJ). 2-methylthio-adenosine 5′-monophosphate triethylammonium salt hydrate (2-MeSAMP, P2Y12 inhibitor) was from Biolog (Farmingdale, NY). Preformed ICs were prepared in a microfuge tube by mixing the antibody (anti-CD40L or anti- β2GPI) with antigen (CD40L or β2GPI) in PBS at balanced stoichiometry and incubated 5 minutes at room temperature prior to use.

Whole blood was obtained from adult mice by retroorbital bleeding and from newborn mice (P2 and P5) by decapitation and bleeding into a tube containing 20 U/mL heparin. For flow cytometric analysis of surface protein expression, platelets were stained for 15 min at RT with CD41-FITC (clone MWReg30; BD Pharming, San Jose, CA) or GPIbα-FITC (clone: Xia.G5; Emfret analytics, Eibelstadt, Germany). Subsequently, samples were quantified in a flow cytometer. To measure platelet activation the blood was washed twice in Ca²⁺-free modified Tyrode’s buffer containing apyrase (0.02 U/ml) and PGI₂ (0.1 μg/ml). Finally, the blood was resuspended in Ca²⁺-containing modified Tyrode’s buffer. The washed blood was left untreated or incubated with agonists at the indicated concentrations for 15 min in the presence of JON/A-PE (clone: JON/A, emfret analytics, Eibelstadt., Germany) and APC-conjugated anti-mouse P-selectin (clone: RB40.34; BD Pharming, San Jose, CA) antibody, followed by analysis on a FACS-Fortessa.