acid-phosphatase and Thrombosis

This study evaluates the effect of the H2S donor GYY4137 (GYY) on adhesion molecule expression, protein S-sulfhydration and morphology of platelets in vitro and on kinetics of microvascular thrombus formation in vivo. Using flowcytometry, untreated resting, TRAP-activated, or TRAP-activated and GYY-exposed human platelets were studied for expression of P-selectin, GPIb and GPIIb/IIIa as well as for fibrinogen binding. By means of electron microscopy, platelet morphology and intracellular granule numbers were assessed. Platelet shape change was studied using immunohistochemistry for P-selectin, NSF and F-actin by SR-SIM. Biotin switch assay served for the analysis of platelet protein S-sulfhydration by GYY. Using the FeCl3 and the light/dye model in dorsal skinfold chamber-equipped mice, the effect of GYY and its vehicle DMSO was studied on venular thrombus formation and tail-vein bleeding time. Soluble (s)P-selectin plasma concentrations were measured in GYY- or DMSO-treated animals. Exposure to GYY increased the S-sulfhydration of platelet proteins. GYY reduced dose-dependently the TRAP-induced adhesion molecule expression and attenuated the morphological signs of TRAP-associated platelet activation. In mice, GYY caused a significant prolongation of venular thrombus formation and tail-vein bleeding time. Application of an anti-P-selectin antibody in DMSO-exposed animals prolonged thrombosis formation comparably as GYY did. GYY reversed the TRAP-induced distribution of P-selectin at the plasma membrane of platelets. This indicates reduced exocytosis and shedding of P-selectin, which is supported by significantly lower sP-selectin concentrations in GYY- vs. DMSO-treated mice. H2S acts anti-thrombotic and seems to regulate thrombogenesis by interference with platelet activation and adhesion molecule-mediated aggregation.

Topics: Acid Phosphatase; Animals; Blood Platelets; Humans; Hydrogen Sulfide; Isoenzymes; Male; Mice; Mice, Inbred C57BL; Microscopy, Electron; Morpholines; Organothiophosphorus Compounds; P-Selectin; Platelet Activation; Platelet Glycoprotein GPIIb-IIIa Complex; Tartrate-Resistant Acid Phosphatase; Thrombosis

Platelet activation requires rapid remodeling of the actin cytoskeleton which is regulated by small GTP-binding proteins. By using the Rac1-specific inhibitor NSC23766, we have recently found that Rac1 is a central component of a signaling pathway that regulates dephosphorylation and activation of the actin-dynamising protein cofilin, dense and α-granule secretion, and subsequent aggregation of thrombin-stimulated washed platelets.. To study whether NSC23766 inhibits stimulus-induced platelet secretion and aggregation in blood.. Human platelet aggregation and ATP-secretion were measured in hirudin-anticoagulated blood and platelet-rich plasma (PRP) by using multiple electrode aggregometry and the Lumi-aggregometer. Platelet P-selectin expression was quantified by flow cytometry.. NSC23766 (300 μM) inhibited TRAP-, collagen-, atherosclerotic plaque-, and ADP-induced platelet aggregation in blood by 95.1%, 93.4%, 92.6%, and 70%, respectively. The IC50 values for inhibition of TRAP-, collagen-, and atherosclerotic plaque-, were 50 ± 18 μM, 64 ± 35 μM, and 50 ± 30 μM NSC23766 (mean ± SD, n = 3-7), respectively. In blood containing RGDS to block integrin αIIbβ3-mediated platelet aggregation, NSC23766 (300 μM) completely inhibited P-selectin expression and reduced ATP-secretion after TRAP and collagen stimulation by 73% and 85%, respectively. In ADP-stimulated PRP, NSC23766 almost completely inhibited P-selectin expression, in contrast to aspirin, which was ineffective. Moreover, NSC23766 (300 μM) decreased plaque-stimulated platelet adhesion/aggregate formation under arterial flow conditions (1500s-1) by 72%.. Rac1-mediated signaling plays a central role in secretion-dependent platelet aggregation in blood stimulated by a wide array of platelet agonists including atherosclerotic plaque. By specifically inhibiting platelet secretion, the pharmacological targeting of Rac1 could be an interesting approach in the development of future antiplatelet drugs.

Topics: Acid Phosphatase; Adenosine Diphosphate; Adenosine Triphosphate; Aminoquinolines; Blood Platelets; Collagen; Humans; Isoenzymes; P-Selectin; Plaque, Atherosclerotic; Platelet Aggregation; Platelet-Rich Plasma; Prostaglandin-Endoperoxide Synthases; Pyrimidines; rac1 GTP-Binding Protein; Signal Transduction; Tartrate-Resistant Acid Phosphatase; Thrombosis

Topics: Acid Phosphatase; Arterial Occlusive Diseases; Capillaries; Dihydrolipoamide Dehydrogenase; Glucosephosphate Dehydrogenase; Hemodynamics; Humans; Leg; Lipid Metabolism; Succinate Dehydrogenase; Thrombosis

Topics: Acid Phosphatase; Adenosine Triphosphatases; Adult; Alkaline Phosphatase; Electron Transport Complex IV; Fibrinolysis; Gastrointestinal Diseases; Histocytochemistry; Humans; Intestinal Diseases; Male; Microscopy, Electron; Middle Aged; Skin; Skin Diseases; Skin Tests; Skin Ulcer; Succinate Dehydrogenase; Syndrome; Thrombosis

1. Autografts and homografts of full thickness skin were made on a hind limb of rabbits. During the following days the appearance and histological changes of the grafts were studied; the lymph flow from the limb, and the enzyme activities in the supernatant and cell pellet of the lymph after centrifugation were determined, as well as the enzyme activities in the graft roof and the underlying host tissue. It was further examined whether a lymphatic and vascular connexion occurred between graft and host tissue.2. During the first 5 days the grafts changed from pale blue to bright pink, became swollen, soft and had a mild cellular inflammatory exudate. Autografts then became pale, took on the appearance of normal skin with the inflammatory changes subsiding, whereas homografts became firm, showed heavy mononuclear cell infiltration, had a blotchy purple appearance due to thrombosis and haemorrhage, developed widespread necrosis and changed into a black hard scab which was eventually shed. With high dose homografts (6-8 grafts) these changes occurred 1-2 days earlier than with low dose (2-4) grafts.3. The flow of lymph increased during the first 5 days after grafting, then returned to normal with autografts but remained increased with homografts.4. In the supernatant of the lymph the activities of LDH and beta-glucuronidase did not change during the first 5 days but activities of cathepsin, acid phosphatase, GOT and GPT increased. With the autografts the increase in the activities of these four enzymes then subsided, but with the homografts they increased further and there was an increase in the activities of LDH and beta-glucuronidase, even greater than in those of the other four enzymes.5. In the cell pellets of the lymph the activities of the six enzymes did not increase during the first 5 days; with homografts, but not with autografts, they then increased. These increases occurred even though the cell count in the pellet remained unchanged. Thus some of the lymphocytes must have become ;activated' to contain higher enzyme activities.6. The enzyme activities in the roof tissue did not parallel those in lymph. They did not change during the first three days. During the following three days the activities of acid phosphatase, LDH, beta-glucuronidase and cathepsin increased, but not those of GOT and GPT which remained low. From then onwards the behaviour was different with auto- and homografts. With autografts only the activity of acid phosphatase continued t

Topics: Acid Phosphatase; Alanine Transaminase; Animals; Aspartate Aminotransferases; Cathepsins; Glucuronidase; Graft Rejection; Hemorrhage; Hindlimb; Inflammation; L-Lactate Dehydrogenase; Lymph; Lymphocytes; Necrosis; Rabbits; Skin; Skin Transplantation; Thrombosis; Time Factors; Transplantation, Autologous; Transplantation, Homologous; Wound Healing

Topics: Acid Phosphatase; Adenoma; Aged; Alkaline Phosphatase; Bilirubin; Blood Coagulation Disorders; Blood Coagulation Tests; Blood Protein Electrophoresis; Blood Proteins; Blood Sedimentation; Bone and Bones; Heparin; Humans; Lung; Lymphatic Metastasis; Male; Neoplasm Metastasis; Prostatectomy; Prostatic Neoplasms; Thrombosis; Urea

Topics: Acid Phosphatase; Animals; Aorta; Bone Marrow; Endotoxins; Female; Infusions, Parenteral; Kidney Diseases; Kidney Glomerulus; Leukocytes; Male; Nitrogen Mustard Compounds; Pulmonary Artery; Rabbits; Salmonella; Shwartzman Phenomenon; Thrombosis; Time Factors