Enoxaparin

Fondaparinux sodium was kindly provided by Alchemia Limited, Australia. Chow supplemented with dabigatran etexilate (DE) and matched vehicle was kindly supplied by Boehringer Ingelheim Pharma GmbH & Co KG, Germany. Enoxaparin was purchased from Sanofi-aventis.

Platelets were obtained from one day expired human platelet-rich plasma concentrates (anticoagulated with citrate) by centrifugation at 670× g for 10 min. Isolated platelet pellets were resuspended in platelet buffer (10 mM N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid, 140 mM NaCl, 3 mM KCl, 0.5 mM MgCl₂, 5 mM NaHCO₃, 10 mM glucose) and the concentration was adjusted to 4 × 10⁸ platelets/mL. CaCl₂ was added to a concentration of 1 mM. Prior to activation, in some cases preincubation of platelets was carried out with UFH (UFH, Ratiopharm GmbH, Ulm, Germany) and Enoxaparin (Sanofi, Paris, France) for 30 min. The anticoagulant exposure was performed in platelet buffer at a final concentration of 1 IU/mL which corresponds to therapeutic concentrations in anticoagulated patients. Platelets were stimulated with 42.5 µM Thrombin Receptor Activating Peptide-6 (TRAP-6) (Tocris Bioscience, Bristol, UK) or 1 × 10⁴ tumor cells/mL for 12 min at 37 °C. Activation by tumor cells was carried out with PC-3 and AsPC-1 cells in presence or absence of anticoagulants. Platelet supernatants/releasates were obtained by sample centrifugation for 20 min at 1000× g.

Treatment included subcutaneous injection of low molecular weight heparin (LMWH) either Fraxiparin (GlaxoSmithKline) or Enoxaparin (Sanofi-Aventis), intravenous dexamethasone, and intravenous fluids with or without furosemide when PVOS with acute respiratory distress occurred. Further use of chemotherapy or targeted therapy or hormone therapy depended on the patient's condition. CT scans were obtained from the hospital picture archiving and communication system (PACS).

Enoxaparin (Clexane, Sanofi, France) and EGb 761 (Ginaton, Germany) were purchased from the Affiliated Zhongshan Hospital of Dalian University (Dalian, China).
The EGb 761 used in our study was standardized to 9.6 mg Ginkgo flavone glycosides and 2.4 mg terpene lactones (Ginkgolides, Bilobalide).

To investigate cytoskeleton organization, trophoblast cells from PE (n = 9) or control placentae (n = 9) were seeded on glass bottom dishes (Ibidi, Gräfelfing, Germany) at a concentration of 5 × 10⁴ cell/dish in DMEM with 10% FBS and incubated with medium only or with enoxaparin (10 IU/mL; Clexane^® Sanofi, Paris, France) for 24 h at 37 °C. Cells were then rinsed twice in PBS, fixed with 4% PFA for 10 min at R/T, and permeabilized with 0.1% Triton-X for 5 min. Next, cells were incubated with Phalloidin Fluorescein Isothiocyanate labeled according to manufacturer’s instructions for 30 min at R/T (Sigma-Aldrich, St. Louis, MO, USA). F-actin fibers were visualized by inverted confocal microscope as previously described. Phalloidin was excited with a single-photon laser (excitation wavelength: 488 nm) and fluorescence was collected with an emission filter 525/50 nm.