GPRP 10 mM

Dilute GPRP (Gly-Pro-Arg-Pro) in modified HT buffer (see recipe) to a concentration of 10 mM. Store up to 1 week at 4°C or 1 year at -20°C. Bring to room temperature prior to use.

Use for diluting thrombin and in assays containing CaCl2 to prevent fibrin polymerization and clot formation (Michelson, 1994).

ModifiedHEPES/Tyrode's (HT) buffer 10 mM HEPES 137 mM NaCl 2.8 mM KCl 1 mM MgCl2 12 mM NaHCO3 0.4 mM Na2HPO4 0.35% bovine serum albumin 5.5 mM glucose

Adjust pH to 7.4 with 0.1 M NaOH or 0.1 M HCl Store up to 1 week at 4°C or 1 year at -20°C Bring to room temperature prior to use

The pH may need readjustment after storage at 4°C. Platelet agonists

Prepare all platelet agonist working solutions (concentrations determined empirically) by diluting stocks in modified HT buffer (see recipe), with or without 6 mM CaCl2 as appropriate, just prior to use. Discard left-over working solutions daily. ADP: Adenosine diphosphate (ADP) is typically used at concentrations of 20 ^M (maximal platelet activation in diluted whole blood) to sub-maximal doses of 0.5 ^M. Store stock ADP according to manufacturer's instructions (often frozen at -20°C).

Collagen: Collagen is typically used at concentrations of 10 to 20 ^g/ml alone or in combination with sub-maximal doses of thrombin (see below). Dilute in modified HT buffer (see recipe) supplemented with 6 mM (2x) CaCl2. Use in the evaluation of procoagulant platelets and platelet-derived microparticles in the presence of 2.5 mM (final concentration) GPRP (see recipe for 10 mM). Store stock collagen according to manufacturer's instructions.

Epinephrine: Epinephrine is often used in combination with ADP at concentrations of 10 to 20 ^M (maximal platelet activation in diluted whole blood). Store stock epinephrine according to manufacturer's instructions.

Human a-thrombin: Thrombin is typically used at concentrations of 1 to 2 U/ml (maximal platelet activation in diluted whole blood) to sub-maximal doses of 0.1 U/ml. The concentration of thrombin required to achieve maximal platelet activation in plasma-free systems is ~0.1 U/ml. Dilute working concentration of thrombin in modified HT buffer (see recipe) containing sufficient 10 mM GPRP (see recipe for 10 mM) to give a final assay concentration of 2.5 mM. Store 200 U/ml stock concentration up to 6 months at -80°C or up to 1 week at 4°C.

Phenotypic Analysis

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