Human α thrombin

Human α-thrombin and adenosine 5’-diphosphate sodium salt (ADP) were purchased from Sigma-Aldrich (St Louis, MO, USA). PAR1-activating peptide (PAR1-AP; TFLLR-NH₂) and PAR4-activating peptide (PAR4-AP; AYPGKF-NH₂) were synthesised at Monash Institute of Pharmaceutical Sciences (Melbourne, Australia) by Assoc Professor Philip Thompson. The mouse anti-P-selectin (FITC anti-mouse CD62P) and human anti-P-selectin (PE anti-human CD62P) antibodies were purchased from BD Biosciences (San Jose, CA, USA). PE anti-CD45.2, PE anti-CD41a (human) antibody and PE anti-CD41 antibody (mouse) were purchased from Abcam (Melbourne, Australia). The PE-anti-GPIb-tail and non-immune rabbit IgG isotype were a generous gift from Assoc Professor Robert Andrews (Monash University, Melbourne, Australia). The anti-PAR1 antibody (ATAP2), anti-PAR3 antibody (8E8), HRP-anti-actin (I-19) and HRP- anti-mouse IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Clot permeability (K_s), an indication of the intrinsic pore size of a fibrin network, was measured as described previously in [34 (link),36 (link)]. Plasma clots (1 U/mL human α-thrombin, Merck, Darmstadt, Germany, and 20 mM CaCl₂) were prepared in triplicate in 3 cm sections of 1 mL plastic serological pipettes. Buffer was permeated through at a pressure height of Δh = 4 cm and K_s was calculated from $$K_s=\frac{\Delta Q\times L\times \eta }{\Delta T \times A \times \Delta P}$$ .
ΔQ/ΔT is the flow rate (flow-through volume/time), $$\eta$$ is the dynamic viscosity (η_water = 1.0 cP at 20 °C), L is the clot length, A is the cross-sectional area of the clot, and $$\Delta P$$ is the pressure drop. ΔP = ρ_water × g × Δh, where the density of water, ρ_water = 1 g/cm³, the acceleration due to gravity, g = 980 cm/s², and Δh = 4 cm is the height from the meniscus of the buffer to the bottom of the clot.