The results indicate that the promoter activity of the 760/+19 region of the vWF gene depends on transcription factors of the Ets family of which several members like Ets-1, Ets-2 and Erg are expressed in endothelium. Cotransfection of Ets-1 and Erg expression plasmids is su cient to induce the 760/+19 vWF promoter activity in HeLa cells.

Objective-Relationships between intracellular Ca 2ϩ concentration ([Ca 2ϩ ] cyt ) and apoptotic events, such as mitochondrial depolarization (⌬⌿m loss) and Bcl-2 and Bad phosphorylation, were analyzed in platelets and Jurkat cells in relation to rapid procoagulant phosphatidylserine (PS) exposure. Methods and Results-Platelets were stimulated with A23187, thapsigargin (TG) and thrombin plus convulxin (Thr/Cvx), andJurkat cells with ionomycin, in the presence or absence of cyclosporin A (CsA), a mitochondrial permeability transition pore inhibitor. ⌬⌿m loss occurred when platelets were stimulated in Ca 2ϩ medium in conditions exposing PS, but also in EGTA medium. CsA inhibited PS exposure, [Ca 2ϩ ] cyt increase, and ⌬⌿m loss in platelets stimulated with TG and Thr/Cvx, but had no inhibitory effect on A23187 stimulation. CsA reduced TG-induced Ca 2ϩ release from the endoplasmic reticulum and, consequently, external Ca 2ϩ influx. In ionomycin-stimulated Jurkat cells, rapid PS exposure was evidenced but not ⌬⌿m loss, and CsA did not inhibit the process. The status of phosphorylated Bad and Bcl-2 in both cell types remained unchanged on stimulation. Conclusions-Whether ⌬⌿m loss occurs or not, PS exposure is triggered by a high [Ca 2ϩ ] cyt increase. Data further demonstrate that CsA prevents membrane scrambling by inhibiting the high [Ca 2ϩ ] cyt increase, independently of its effect on mitochondrial permeability transition pore. Key Words: platelets Ⅲ Jurkat Ⅲ apoptosis Ⅲ procoagulant phosphatidylserine Ⅲ thrombin plus convulxin P hospholipid scrambling, leading to phosphatidylserine (PS) exposure to the external leaflet of cell membranes, characterizes blood platelet procoagulant activation. The essential role of phospholipid reorganization on the surface of stimulated blood cells is illustrated by the existence of Scott syndrome, a rare bleeding disorder caused by defective membrane scrambling in platelets and hematopoietic cells such as lymphocytes (reviewed in 1 ).PS exposure also characterizes apoptosis and occurs during platelet storage with other typical morphological and biochemical features of apoptosis (reviewed in 2 ), including loss of the mitochondrial transmembrane potential (⌬⌿m). [3][4][5] ⌬⌿m loss is caused by opening of a channel, the mitochondrial permeability transition pore (mPTP), and requires an increase in mitochondrial Ca 2ϩ . 6 The mPTP is composed of several proteins, including the adenine nucleotide translocator (ANT), the voltage-dependent anion channel, and cyclophilin D (CypD). 7 mPTP opening is blocked by cyclosporin A (CsA), which binds to CypD and dissociates it from the translocator. 8 In platelets, the physiological procoagulant activity is triggered by dual agonist stimulation with thrombin (Thr) plus convulxin (Cvx), the latter stimulating GPVI, a platelet collagen receptor. 9 This leads a subpopulation of platelets, named coated-platelets, to express PS and high levels of several procoagulant proteins on their surface. 10 Studies have shown that both ⌬⌿m loss and ...

The expression of the von Willebrand factor (vWF) gene by cultured endothelial cells from the porcine pulmonary artery, aorta, and lung was compared at the levels of messenger (m)RNA and antigen. Steady-state levels of vWF mRNA were determined by dot-blot analysis using a partial human vWF cDNA as the hybridization probe; vWF mRNA from cultured aortic endothelial cells, and vWF antigen secreted into the culture supernatants were barely detectable. In contrast, vWF mRNA and antigen from the pulmonary artery endothelial cells were approximately eight to nine times that demonstrated by aortic cells. Levels of vWF mRNA and antigen in cultured lung cells were intermediate of those found in the pulmonary artery and aorta and correlated with the estimated number of cells demonstrated to be of endothelial origin in the mixed cell populations grown from the lung. Differences between the levels of vWF mRNA found in cultured cells from the pulmonary artery and those found in the aorta were maintained in cells processed directly from these vessels. Correlation between the levels of vWF mRNA and antigen in endothelial cells from different vessels of the pig suggests that the differential control of vWF synthesis is at the level of transcription. Furthermore, maintenance in cultured cells of the difference in transcription rates that were observed in vivo suggests that vWF gene expression is not exclusively regulated through environmental factors.