Subcellular fractionation of HUVECs on Percoll gradients.

Resting HUVECs (A,B), or HUVECs stimulated with A23187 (C,D) were treated with DPDPB, homogenized, and fractionated on a 35% Percoll gradient. (A) Fractions were collected and analyzed by ELISA for the presence of vWf, which is present in 2 peaks: the first represents the ER/Golgi fraction (I) and the second represents Weibel-Palade bodies (II). Fractions 1 to 4 from peak I and 9 to 12 from peak II were analyzed by immunoblotting with a vWf polyclonal antibody. The Weibel-Palade fraction is recognized by the presence of only the mature subunit of vWf in this fraction, whereas both the precursor (260 kd) and mature subunit (220 kd) are present in the ER/Golgi fractions. (B) P-selectin was immunoprecipitated with a polyclonal antibody from pooled fractions 3 to 5 (peak I) and fractions 9 and 10 (peak II) and then immunoblotted with monoclonal P-selectin antibody AC1.2. P-selectin dimer (d) and monomer (m) are detected in both samples. (C) vWf ELISA profile obtained from A23187-treated HUVEC homogenates fractionated on a Percoll gradient. (D) P-selectin immunoblot of early fractions from a Percoll gradient of cross-linked, A23187-treated HUVECs. Fractions 1 and 2 contain P-selectin precursor (p) and mature subunit (m). Fractions 3 and 4 contain mature subunit and P-selectin dimer (d).