Figure 2. PP2B inhibitors induced release of ULVWF/VWF multimers from HUVECs.

(A) ULVWF/VWF multimeric patterns. HUVECs were treated with PP2B inhibitors and ULVWF/VWF multimers from the cell supernatants analyzed by unreduced 1% agarose/SDS electrophoresis/anti-VWF antibody immunoblotting. VWF multimeric patterns from normal platelet-poor plasma or endothelial cell supernatant (EC sup) from histamine-treated cells are shown for comparison. ULVWF multimers are indicated by a vertical line. Compared to the normal platelet-poor plasma lane, ULVWF multimers were prominently enhanced in lane containing the PP2B inhibitor-treated samples. Occasionally, DMSO treated lane also showed some VWF and ULVWF forms. (N=3). (B) Perfused platelets adhered instantaneously to ULVWF strings secreted by HUVECs in the presence of PP2B inhibitors under flowing conditions. HUVECs were preincubated with the control reagents or PP2B inhibitors before the perfusion of normal fresh washed human platelets using a flow chamber (wall shear stress, 2.5 dyne/cm²). The number of ULVWF-platelet strings that formed was counted over the course of 2 min in 20 different microscope fields. Data are presented as +/- SEM of 2-6 experiments. Compared to DMSO, ^‡P=0.02 (for 1 nM CsA), *P=0.0024 (for 10 nM CsA), ^∞P=0.05 (for 1 nM FK506), ⁺P=0.002 (for 10 nM FK506) and ^#P=0.0069 (for histamine). Compared to Tyrode's buffer treated cells, †P=0.05 (for 1 μM AI peptide), ^♣P<0.01 for 2 μM AI peptide).