iloprost and Hemorrhagic-Disorders

Thrombospondin-1 (TSP-1) is released by platelets upon activation and can increase platelet activation, but its role in hemostasis in vivo is unclear. We show that TSP-1 is a critical mediator of hemostasis that promotes platelet activation by modulating inhibitory cyclic adenosine monophosphate (cAMP) signaling. Genetic deletion of TSP-1 did not affect platelet activation in vitro, but in vivo models of hemostasis and thrombosis showed that TSP-1-deficient mice had prolonged bleeding, defective thrombosis, and increased sensitivity to the prostacyclin mimetic iloprost. Adoptive transfer of wild-type (WT) but not TSP-1-/- platelets ameliorated the thrombotic phenotype, suggesting a key role for platelet-derived TSP-1. In functional assays, TSP-1-deficient platelets showed an increased sensitivity to cAMP signaling, inhibition of platelet aggregation, and arrest under flow by prostacyclin (PGI2). Plasma swap experiments showed that plasma TSP-1 did not correct PGI2 hypersensitivity in TSP-1-/- platelets. By contrast, incubation of TSP-1-/- platelets with releasates from WT platelets or purified TSP-1, but not releasates from TSP-1-/- platelets, reduced the inhibitory effects of PGI2. Activation of WT platelets resulted in diminished cAMP accumulation and downstream signaling, which was associated with increased activity of the cAMP hydrolyzing enzyme phosphodiesterase 3A (PDE3A). PDE3A activity and cAMP accumulation were unaffected in platelets from TSP-1-/- mice. Platelets deficient in CD36, a TSP-1 receptor, showed increased sensitivity to PGI2/cAMP signaling and diminished PDE3A activity, which was unaffected by platelet-derived or purified TSP-1. This scenario suggests that the release of TSP-1 regulates hemostasis in vivo through modulation of platelet cAMP signaling at sites of vascular injury.

Topics: Animals; Bleeding Time; Blood Platelets; CD36 Antigens; Cells, Cultured; Chlorides; Cyclic AMP; Cyclic Nucleotide Phosphodiesterases, Type 3; Cytoplasmic Granules; Epoprostenol; Ferric Compounds; Hemorrhagic Disorders; Hemostasis; Humans; Iloprost; Mice; Mice, Inbred C57BL; Platelet Transfusion; Second Messenger Systems; Thrombosis; Thrombospondin 1

Alternatively spliced GNAS1 and XL-GNAS1, encoding respectively the stimulatory G-protein alpha-subunit (Gsalpha) and the extra-large stimulatory G-protein alpha-subunit (XLsalpha), are located on the imprinted chromosomal region 20q13.12-13. We presently report a functional polymorphism in the imprinted XL-GNAS1 gene. In three patients, a 36 bp insertion and two basepair substitutions flanking this insertion were found in the paternally inherited XL-GNAS1 exon 1. They clinically manifest an enhanced trauma-related bleeding tendency and a variable degree of mental retardation. A platelet aggregation inhibition test to evaluate Gs function was developed. Their platelets display Gs hyperfunction and an enhanced cAMP generation upon stimulation of Gs-coupled receptors. The prevalence of the XLsalpha insertion in a normal control group was 2.2%. Normal controls, inheriting the insertion maternally, had a normal platelet Gs activity, whereas controls inheriting the insertion paternally had increased inducible platelet Gs activity, defining the insertion as a functional polymorphism. This paternally inherited XLsalpha insertion represents a new genetic cause of an inherited bleeding tendency, although to a variable degree.

Topics: Abnormalities, Multiple; Adenosine; Adenylyl Cyclases; Adolescent; Alprostadil; Alternative Splicing; Amino Acid Sequence; Amino Acid Substitution; Bleeding Time; Blood Platelets; Child; Chromogranins; Cyclic AMP; Female; Fingers; Gene Frequency; Genomic Imprinting; GTP-Binding Protein alpha Subunits, Gs; Hemorrhagic Disorders; Heterotrimeric GTP-Binding Proteins; Humans; Hyperkinesis; Hypoparathyroidism; Iloprost; Intellectual Disability; Male; Molecular Sequence Data; Muscle Hypotonia; Mutagenesis, Insertional; Nerve Tissue Proteins; Phenotype; Platelet Aggregation; Platelet Function Tests; Polymorphism, Genetic; Protein Subunits; Repetitive Sequences, Amino Acid; Risk Factors; Second Messenger Systems; Structure-Activity Relationship; Wounds and Injuries