title: Internalization of Tissue Factor-Rich Microvesicles by Platelets Occurs Independently of GPIIb-IIIa, and Involves CD36 Receptor, Serotonin Transporter and Cytoskeletal Assembly.
abstract: Platelets are important in hemostasis, but also detect particles and pathogens in the circulation. Phagocytic and endocytic activities of platelets are widely recognized; however, receptors and mechanisms involved remain poorly understood. We previously demonstrated that platelets internalize and store phospholipid microvesicles enriched in human tissue factor (TF+MVs) and that platelet-associated TF enhances thrombus formation at sites of vascular damage. Here, we investigate the mechanisms implied in the interactions of TF+MVs with platelets and the effects of specific inhibitory strategies. Aggregometry and electron microscopy were used to assess platelet activation and TF+MVs uptake. Cytoskeletal assembly and activation of phosphoinositide 3-kinase (PI3K) and RhoA were analyzed by western blot and ELISA. Exposure of platelets to TF+MVs caused reversible platelet aggregation, actin polymerization and association of contractile proteins to the cytoskeleton being maximal at 1 min. The same kinetics were observed for activation of PI3K and translocation of RhoA to the cytoskeleton. Inhibitory strategies to block glycoprotein IIb-IIIa (GPIIb-IIIa), scavenger receptor CD36, serotonin transporter (SERT) and PI3K, fully prevented platelet aggregation by TF+MVs. Ultrastructural techniques revealed that uptake of TF+MVs was efficiently prevented by anti-CD36 and SERT inhibitor, but only moderately interfered by GPIIb-IIIa blockade. We conclude that internalization of TF+MVs by platelets occurs independently of receptors related to their main hemostatic function (GPIIb-IIIa), involves the scavenger receptor CD36, SERT and engages PI3-Kinase activation and cytoskeletal assembly. CD36 and SERT appear as potential therapeutic targets to interfere with the association of TF+MVs with platelets and possibly downregulate their prothrombotic phenotype.
authors.affiliation: Department of Hemotherapy and Hemostasis, Hospital Clinic, Centre of Biomedical Diagnosis (CDB), Institute of Biomedical Research August Pi i Sunyer (IDIBAPS), University of Barcelona, 170 Villarroel Street, Barcelona 08036, Spain.
authors.forename: Irene
authors.initials: I
authors.lastname: Lopez-Vilchez
authors.affiliation: Department of Hemotherapy and Hemostasis, Hospital Clinic, Centre of Biomedical Diagnosis (CDB), Institute of Biomedical Research August Pi i Sunyer (IDIBAPS), University of Barcelona, 170 Villarroel Street, Barcelona 08036, Spain.
authors.forename: Maribel
authors.initials: M
authors.lastname: Diaz-Ricart
authors.affiliation: Department of Hemotherapy and Hemostasis, Hospital Clinic, Centre of Biomedical Diagnosis (CDB), Institute of Biomedical Research August Pi i Sunyer (IDIBAPS), University of Barcelona, 170 Villarroel Street, Barcelona 08036, Spain.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Galan
authors.affiliation: Institute Clinic of Thorax, Hospital Clinic, Institute of Biomedical Research August Pi i Sunyer (IDIBAPS), University of Barcelona, 170 Villarroel Street, Barcelona 08036, Spain.
authors.forename: Merce
authors.initials: M
authors.lastname: Roque
authors.affiliation: Department of Hemotherapy and Hemostasis, Hospital Clinic, Centre of Biomedical Diagnosis (CDB), Institute of Biomedical Research August Pi i Sunyer (IDIBAPS), University of Barcelona, 170 Villarroel Street, Barcelona 08036, Spain.
authors.forename: Carolina
authors.initials: C
authors.lastname: Caballo
authors.affiliation: Department of Hemotherapy and Hemostasis, Hospital Clinic, Centre of Biomedical Diagnosis (CDB), Institute of Biomedical Research August Pi i Sunyer (IDIBAPS), University of Barcelona, 170 Villarroel Street, Barcelona 08036, Spain.
authors.forename: Patricia
authors.initials: P
authors.lastname: Molina
authors.affiliation: Departments of Laboratory and Clinical Medicine and Pediatrics, University of Minnesota, 420 Delaware Street S.E., Minneapolis, Minnesota, 55455.
authors.forename: James G
authors.initials: JG
authors.lastname: White
authors.affiliation: Department of Hemotherapy and Hemostasis, Hospital Clinic, Centre of Biomedical Diagnosis (CDB), Institute of Biomedical Research August Pi i Sunyer (IDIBAPS), University of Barcelona, 170 Villarroel Street, Barcelona 08036, Spain.
authors.forename: Gines
authors.initials: G
authors.lastname: Escolar
mesh_terms: D001792:Blood Platelets / Q000502:physiology*; D018955:CD36 Antigens / Q000378:metabolism*; D055252:Cell-Derived Microparticles / Q000378:metabolism*; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D039661:Integrin beta3 / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010974:Platelet Aggregation; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism; D021381:Protein Transport; D050486:Serotonin Plasma Membrane Transport Proteins / Q000378:metabolism*; D015398:Signal Transduction; D013925:Thromboplastin / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D018955:CD36 Antigens; C548810:CD36 protein (93-110); D039661:Integrin beta3; D010446:Peptide Fragments; D019038:Platelet Glycoprotein GPIb-IX Complex; D050486:Serotonin Plasma Membrane Transport Proteins; C032193:glycoprotein receptor GPIb-IX; C066737:RHOA protein, human; D013925:Thromboplastin; D019869:Phosphatidylinositol 3-Kinases; D020742:rhoA GTP-Binding Protein
keywords: CELL-DERIVED MICROPARTICLES; CYTOSKELETON; INTERNALIZATION; PLATELETS; TISSUE FACTOR