Introduction: In vitro platelets activation induced by Collagen is a good model that mimics the events occuring "in vivo" by consequence of the exposure of the subendothelial matrix to the blood stream due to vessel damage. The interaction of Collagen with the platelet membrane receptor VLA-2 (the alfa2-beta1 integrin) is followed by intraplatelet processes leading to the secretion of granules content and formation of aggregates. The correct sequence of these events is yet unknown. "Active metabolites" of PLC (IP3, DG) and PL-A2 (Arachidonic Acid) are produced after the agonist-receptor complex formation but it is uncertain whether their rise is due to direct transduction of the external signal or is induced by the activation of phospholipases by second intracellular messengers. Aggregation induced by Collagen is preceded by a lag phase (20-40 sec) during which the shape of platelets changes from discoid to spiny sphere. Methods: Aggregation was followedby the light transmission increase measured by Aggrecoder II (Menarini); TBARS were determined by the method reported in (1); Phospholipids were determined after thin-layer chromatography as in (2). Results and Discussion: We investigated the time courses of the production of TBARS (an indication of TX-A2 formation by Arachidonic Acid oxidative metabolism) and of the changes in the membrane phospholipids composition (namely PC, PE and PI). TBARS appearance was slightly faster than aggregates formation but was markedly preceded by membrane phospholipids degradation. The phospholipid undergoing the fastest metabolism was PI (which reached the maximal changes in a few seconds) followed by PE and PC. PI consumption is considered a good indication for the metabolism of all the phosphatidyl inositides (including PIP and PIP2). Being these phospholipids the preferred substrates for PL-C, we conclude that the first, fast, effect of the agonist-receptor complex formation is activation of PL-C. The consequence of PL-C activation is the pro-duction of IP3 and DG, the former being necessary to liberate Ca2+ and the latter to activate PK-C (and consequently the contractile system). The delay of the consumption of PE and PC could be due to the fact that these phospholipids are the preferred substrates for PL-A2: Ca2+ could be the second intracellular messenger necessary to trigger the Arachidonic Acid relase from which TX-A2 is produced. TX-A2 acts by two different ways: 1) it amplifies the intracellular signal by its strong activity in dissequestering Ca2+ from stores; 2) it propagates the stimulus to other platelets by its strong agonist activity. Bibliography 1-Slater T.F. (1984, Methods in Enzymology 105, 283-293, 2-Jork H., Wimmer H. Quantitative Auswertung von Duennschicht Chromatogrammen. Darmstadt: GIT-Verlag, p.III./ 3-82.
MEMBRANE PHOSPHOLIPIDS METABOLISM IN ACTIVATED PLATELETS
CALZA, Roberta;
1994
Abstract
Introduction: In vitro platelets activation induced by Collagen is a good model that mimics the events occuring "in vivo" by consequence of the exposure of the subendothelial matrix to the blood stream due to vessel damage. The interaction of Collagen with the platelet membrane receptor VLA-2 (the alfa2-beta1 integrin) is followed by intraplatelet processes leading to the secretion of granules content and formation of aggregates. The correct sequence of these events is yet unknown. "Active metabolites" of PLC (IP3, DG) and PL-A2 (Arachidonic Acid) are produced after the agonist-receptor complex formation but it is uncertain whether their rise is due to direct transduction of the external signal or is induced by the activation of phospholipases by second intracellular messengers. Aggregation induced by Collagen is preceded by a lag phase (20-40 sec) during which the shape of platelets changes from discoid to spiny sphere. Methods: Aggregation was followedby the light transmission increase measured by Aggrecoder II (Menarini); TBARS were determined by the method reported in (1); Phospholipids were determined after thin-layer chromatography as in (2). Results and Discussion: We investigated the time courses of the production of TBARS (an indication of TX-A2 formation by Arachidonic Acid oxidative metabolism) and of the changes in the membrane phospholipids composition (namely PC, PE and PI). TBARS appearance was slightly faster than aggregates formation but was markedly preceded by membrane phospholipids degradation. The phospholipid undergoing the fastest metabolism was PI (which reached the maximal changes in a few seconds) followed by PE and PC. PI consumption is considered a good indication for the metabolism of all the phosphatidyl inositides (including PIP and PIP2). Being these phospholipids the preferred substrates for PL-C, we conclude that the first, fast, effect of the agonist-receptor complex formation is activation of PL-C. The consequence of PL-C activation is the pro-duction of IP3 and DG, the former being necessary to liberate Ca2+ and the latter to activate PK-C (and consequently the contractile system). The delay of the consumption of PE and PC could be due to the fact that these phospholipids are the preferred substrates for PL-A2: Ca2+ could be the second intracellular messenger necessary to trigger the Arachidonic Acid relase from which TX-A2 is produced. TX-A2 acts by two different ways: 1) it amplifies the intracellular signal by its strong activity in dissequestering Ca2+ from stores; 2) it propagates the stimulus to other platelets by its strong agonist activity. Bibliography 1-Slater T.F. (1984, Methods in Enzymology 105, 283-293, 2-Jork H., Wimmer H. Quantitative Auswertung von Duennschicht Chromatogrammen. Darmstadt: GIT-Verlag, p.III./ 3-82.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
