Prostaglandin i2 pgi2

Bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), fibrinogen,
thrombin, and Tyrode’s salt solution were purchased from Sigma-Aldrich
(St. Louis, MO, USA). Adenosine diphosphate (ADP) was purchased from Chrono-Log
(Havertown, PA, USA). Paraformaldehyde (PFA) was purchased from Electron
Microscopy Sciences (Hatfield, PA, USA). Prostaglandin I₂(PGI₂) was purchased from Cayman Chemical (Ann Arbor, MI, USA).
The following Hsp inhibitors were kindly provided to us by several
collaborators: C86 (Hsp40 inhibitor; Jane Trepel, Developmental Therapeutics
Branch, NCI, Bethesda, MD, USA)[21 (link)], JG98
(Hsp70 inhibitor; Jason Gestwicki, University of California San Francisco, San
Francisco, CA, USA)[23 ], Kung65 (Grp94
inhibitor; Brian Blagg, University of Notre Dame, Notre Dame, IN, USA), and
TAS116 (Hsp90 inhibitor; Taiho Pharmaceutical Co., Tsukuba, Ibaraki,
Japan)[24 (link)]. Phalloidin AF488, and
AlexaFluor AF594 were purchased from Invitrogen Life Technologies (Carlsbad, CA,
USA). The flow cytometry antibodies CD61 AF647, CD62P BV421 (also known as
P-selectin), PAC-1 AF647 (the activated form of glycoprotein IIb/IIIa;
GPIIb/IIIa), and Annexin V Pacific Blue were purchased from Bio-Rad (Hercules,
CA, USA), BD Biosciences (San Jose, CA, USA), BioLegend (San Diego, CA, USA),
and Invitrogen Life Technologies (Carlsbad, CA, USA), respectively.

Apheresis platelets were incubated with Prostaglandin I₂ (PgI₂) [1 ng/ml] (Cayman Chemical Company, Ann Arbor, MI) to inhibit aggregation, centrifuged at 1000g for 5 minutes, resuspended in Tyrode’s Buffer, and diluted to a final concentration of 300×10⁶ platelets/ml. 22×10⁶ platelets plus Seahorse media for a final volume of 100 μl were loaded onto the sensor cartridge for the Seahorse XFe24 Analyzer. To measure mitochondrial metabolism the Seahorse XF Cell Mito Stress Kit (Agilent) was used to assess oxygen consumption and pH changes. After the initial baseline measurement, either hormone (testosterone or estradiol diluted into seahorse media) or vehicle control (seahorse media/0.0001% DMSO) was injected. This was followed by sequential addition of 1.5 μM Oligomycin, 1.0 μM Carbonyl cyanide-4 phenylhydrazone (FCCP), and 0.5 μM Rotenone/Antimycin for oxygen and pH measurements, per assay protocol. The oxygen consumption rate (OCR) was calculated in pmol/min for Basal Respiration, ATP-linked Respiration, Maximal Respiration, Spare Respiratory Capacity, and Nonmitochondrial Respiration. Extracellular Acidification Rate (ECAR) was calculated in mpH/min for non-sensitive/basal and oligomycin sensitive.

Soluble collagen was from Corning (Corning, NY, USA). Prostaglandin I2 (PGI₂) was from Cayman Chemical Company (Ann Arbor, MN, USA). Crosslinked collagen related peptide (CRP-XL) was from CambCol Laboratories (Cambridge, UK) and human fibrinogen (FIB3) was from Enzyme Research Labs (South Bend, IN, USA). Chrono-lume detection agent was from Chrono-Log Corporation (Havertown, PA, USA). TRITC-phalloidin (P1951) was from Sigma-Aldrich (St. Louis, MO, USA). Rhodocytin was kindly provided by Dr. Johannes Eble (University of Münster, Germany).