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RAB27 REGULATES THE CA2+-INDUCED DENSE-GRANULE SECRETION IN PLATELETS
S122
Poster Abstracts / Cardiovascular Pathology 13 (2004) S80–S138
investigated plasma Lp(a) concentrations in 1200 subjects, including 340 healthy elderly controls, 260 coronary artery disease (CAD), 200 peripheral arterial disease (PAD) and 400 abdominal aortic aneurysm (AAA) patients. High Lp(a) ( > 75nmol/L) carried a statistically significant independent odds ratio (OR) for all three forms of arterial disease (CAD & PAD OR 1.63, 95%CI 1.1-2.4, AAA OR 1.45, 95%CI 1.01-2.10). Several groups have shown that Lp(a) is present within the intima of established atherosclerotic plaques. We therefore investigated the presence of Lp(a) and its two component proteins (apolipoprotein (apo) B-100 and apo(a)) within human arteries, and in particular, at sites of early intimal alteration using immunohistochemistry. Specifically we aimed to determine any associations between Lp(a) and arterial cellular or connective tissue components. In early intimal thickenings, from adolescents and young adults, the plasminogen homologue apo(a) showed a strong affinity for the newly formed intimal elastic tissues and accumulated at such sites independent of the presence of macrophage/foam cells. This localisation was most prominent within intimal thickenings at the lateral angle of flow dividers and at the distal shoulders of developing lesions. As the intima thickened staining also included diffuse deposits throughout the intima and occasional foam cells. Staining within advanced atherosclerotic plaques was dominated by abundant Lp(a) deposits within the foam cell laden intimal lipid core. Staining for other plasma proteins such as albumin did not show any associations with intimal elastic tissue deposits or other connective tissues in early lesions. Though the exact physiological role of Lp(a) remains elusive its localisation within very early intimal lesions along with an association between its circulating concentration and the development of cardiovascular disease support the hypothesis that Lp(a) plays a role in the early pathogenesis of atherosclerosis. The study was supported by a project grant from the Health Research Council of New Zealand.
P334 ENHANCED TYROSINE-PHOSPHORYLATION OF PLATELET SUBSTRATES INCLUDING PP60C-SRC AND PP125FAK IS INDICATIVE OF ENDOGENOUS PLATELET ACTIVATION IN PULMONARY VASO-OCCLUSIVE DISEASE. Nair Maeda, Se´rgio Bydlowski, Augusto Lopes. Fundac¸a˜o Pro´-Sangue Hemocentro de Sa˜o Paulo, Dept. of Hematology,University of Sa˜o Paulo School of Medicine, Heart Institute,University of Sa˜o Paulo School of Medicine. Controversy exists about the occurrence of chronic endogenous platelet activation in pulmonary vaso-occlusive disease. This is due to the fact that platelets may release their content of proaggregating substances in vivo, making it unrealistic to test their ability to undergo activation and aggregation ex-vivo. We therefore decided to look at the pattern of tyrosine phosphorylation of several platelet substrates as an evidence for endogenous activation. We examined this in 10 adult patients with moderate to severe precapillary pulmonary hypertension by densitometric analysis of Western blots performed with anti-phosphotyrosine antibodies. We also used anti-beta-3 integrin, anti-pp60c-src and anti-pp125FAK antibodies to identify these proteins. In comparison with controls, platelets from patients had equivalent amounts of beta-3 integrin, pp60c-src and pp125FAK. Also there was a similar increase in the cytoskeletal incorporation of these proteins following thrombin stimulation. We observed a two-fold increase in beta-3 integrin, a three to four-fold increase in pp60csrc and 13 to 20-fold increase in pp125FAK in cytoskeletal preparation of stimulated platelet in patients and controls. However, the level of tyrosine phosphorylation of all 12 platelet substrates analyzed was higher in patients (1.2 to 3.5-fold increase versus controls, p = .0022). This included a strong signal in the region correspoding to pp120/Ras-GAP, known to be substrates of pp60c-src. Also, there was a two-fold increase in phosphorylated pp125FAK compared with controls ( p < 0.05) after normalization for the amount of pp125FAK protein present in platelets. These findings support the assumption that platelets undergo chronic endogenous activation in pulmonary hypertension, an observation with obvious therapeutic implications. FAPESP and CNPq, Brazil
P333 RAB27 REGULATES THE CA2+-INDUCED DENSE-GRANULE SECRETION IN PLATELETS. Hirokazu Kondo, Hisanori Horiuchi, Ryutaro Shirakawa, Tomohito Higashi, Arata Tabuchi, Mitsunori Fukuda, Toru Kita. The Department of Geriatric Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan, Initiative Research Unit, RIKEN, Wako, Japan, The Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan. Dense-core granule secretion in activated platelets plays a critical role in positive-feedback activation of platelets. Here, we demonstrate that densecore granule exocytosis is regulated by small GTPase Rab27. All the Rab27 effectors so far identified use the synaptotagmin-like protein homology domain (SHD) for the interaction with GTP-Rab27. Incubation of permeabilized platelets with the SHD of Slac2-b, a Rab27-specific effector, but not its GTP-Rab27-binding-defective mutant SHD-E10A, inhibited the Ca2+-induced secretion. Incubation with unprenylated active mutant Rab27A-Q78L and wild-type Rab27A and Rab27B also inhibited the secretion, whereas inactive mutant Rab27A-T23N or other GTPases had no effects. Thus, Rab27 is involved in the regulation of the secretion. Furthermore, we identified a novel type of Rab27 effector in platelets by affinity chromatography which specifically bound to GTP gamma SRab27A, but not GDP-Rab27A , GTP gamma S-Rab4 or GTP gamma S-Rab5. We are characterizing the Rab27A effector in the regulation of the secretion.
P335 INSULIN INDUCES THE RELEASE OF VASODILATOR COMPOUNDS FROM PLATELETS BY A NITRIC OXIDE/G KINASE/VAMP-3-DEPENDENT PATHWAY: A NOVEL MECHANISM OF INSULIN-INDUCED VASODILATATION. Voahanginirina Randriamboavonjy, Rudi Busse, Ingrid Fleming. Cardiovascular Physiology, Frankfurt am Main, Germany. Insulin-induced vasodilatation is sensitive to nitric oxide (NO) synthase (NOS) inhibitors. However, insulin is unable to relax isolated arteries or to activate eNOS in endothelial cells. Since insulin can enhance platelet eNOS activity, we determined whether insulin-induced vasodilatation can be attributed to a NO-dependent, platelet-mediated process. Insulin failed to relax endothelium-intact rings of porcine coronary artery. The supernatant from insulin-stimulated human platelets induced complete relaxation which was prevented by preincubation of platelets with a NOS inhibitor, the soluble guanylyl cyclase inhibitor, NS 2028, or the G kinase inhibitor, KT 5823 and was abolished by an adenosine A2A receptor antagonist. Insulin induced the release of ATP, adenosine and serotonin from platelet dense granules in a NO-dependent manner. This response was not detected using insulin-stimulated platelets from eNOS-/mice, although an NO donor elicited ATP release. Insulin-induced ATP release from human platelets correlated with the association of syntaxin 2 with the vesicle-associated membrane protein-3 (VAMP-3) but was not associated with the activation of aIIb-b3 integrin, the release of the
Poster Abstracts / Cardiovascular Pathology 13 (2004) S80–S138
investigated plasma Lp(a) concentrations in 1200 subjects, including 340 healthy elderly controls, 260 coronary artery disease (CAD), 200 peripheral arterial disease (PAD) and 400 abdominal aortic aneurysm (AAA) patients. High Lp(a) ( > 75nmol/L) carried a statistically significant independent odds ratio (OR) for all three forms of arterial disease (CAD & PAD OR 1.63, 95%CI 1.1-2.4, AAA OR 1.45, 95%CI 1.01-2.10). Several groups have shown that Lp(a) is present within the intima of established atherosclerotic plaques. We therefore investigated the presence of Lp(a) and its two component proteins (apolipoprotein (apo) B-100 and apo(a)) within human arteries, and in particular, at sites of early intimal alteration using immunohistochemistry. Specifically we aimed to determine any associations between Lp(a) and arterial cellular or connective tissue components. In early intimal thickenings, from adolescents and young adults, the plasminogen homologue apo(a) showed a strong affinity for the newly formed intimal elastic tissues and accumulated at such sites independent of the presence of macrophage/foam cells. This localisation was most prominent within intimal thickenings at the lateral angle of flow dividers and at the distal shoulders of developing lesions. As the intima thickened staining also included diffuse deposits throughout the intima and occasional foam cells. Staining within advanced atherosclerotic plaques was dominated by abundant Lp(a) deposits within the foam cell laden intimal lipid core. Staining for other plasma proteins such as albumin did not show any associations with intimal elastic tissue deposits or other connective tissues in early lesions. Though the exact physiological role of Lp(a) remains elusive its localisation within very early intimal lesions along with an association between its circulating concentration and the development of cardiovascular disease support the hypothesis that Lp(a) plays a role in the early pathogenesis of atherosclerosis. The study was supported by a project grant from the Health Research Council of New Zealand.
P334 ENHANCED TYROSINE-PHOSPHORYLATION OF PLATELET SUBSTRATES INCLUDING PP60C-SRC AND PP125FAK IS INDICATIVE OF ENDOGENOUS PLATELET ACTIVATION IN PULMONARY VASO-OCCLUSIVE DISEASE. Nair Maeda, Se´rgio Bydlowski, Augusto Lopes. Fundac¸a˜o Pro´-Sangue Hemocentro de Sa˜o Paulo, Dept. of Hematology,University of Sa˜o Paulo School of Medicine, Heart Institute,University of Sa˜o Paulo School of Medicine. Controversy exists about the occurrence of chronic endogenous platelet activation in pulmonary vaso-occlusive disease. This is due to the fact that platelets may release their content of proaggregating substances in vivo, making it unrealistic to test their ability to undergo activation and aggregation ex-vivo. We therefore decided to look at the pattern of tyrosine phosphorylation of several platelet substrates as an evidence for endogenous activation. We examined this in 10 adult patients with moderate to severe precapillary pulmonary hypertension by densitometric analysis of Western blots performed with anti-phosphotyrosine antibodies. We also used anti-beta-3 integrin, anti-pp60c-src and anti-pp125FAK antibodies to identify these proteins. In comparison with controls, platelets from patients had equivalent amounts of beta-3 integrin, pp60c-src and pp125FAK. Also there was a similar increase in the cytoskeletal incorporation of these proteins following thrombin stimulation. We observed a two-fold increase in beta-3 integrin, a three to four-fold increase in pp60csrc and 13 to 20-fold increase in pp125FAK in cytoskeletal preparation of stimulated platelet in patients and controls. However, the level of tyrosine phosphorylation of all 12 platelet substrates analyzed was higher in patients (1.2 to 3.5-fold increase versus controls, p = .0022). This included a strong signal in the region correspoding to pp120/Ras-GAP, known to be substrates of pp60c-src. Also, there was a two-fold increase in phosphorylated pp125FAK compared with controls ( p < 0.05) after normalization for the amount of pp125FAK protein present in platelets. These findings support the assumption that platelets undergo chronic endogenous activation in pulmonary hypertension, an observation with obvious therapeutic implications. FAPESP and CNPq, Brazil
P333 RAB27 REGULATES THE CA2+-INDUCED DENSE-GRANULE SECRETION IN PLATELETS. Hirokazu Kondo, Hisanori Horiuchi, Ryutaro Shirakawa, Tomohito Higashi, Arata Tabuchi, Mitsunori Fukuda, Toru Kita. The Department of Geriatric Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan, Initiative Research Unit, RIKEN, Wako, Japan, The Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan. Dense-core granule secretion in activated platelets plays a critical role in positive-feedback activation of platelets. Here, we demonstrate that densecore granule exocytosis is regulated by small GTPase Rab27. All the Rab27 effectors so far identified use the synaptotagmin-like protein homology domain (SHD) for the interaction with GTP-Rab27. Incubation of permeabilized platelets with the SHD of Slac2-b, a Rab27-specific effector, but not its GTP-Rab27-binding-defective mutant SHD-E10A, inhibited the Ca2+-induced secretion. Incubation with unprenylated active mutant Rab27A-Q78L and wild-type Rab27A and Rab27B also inhibited the secretion, whereas inactive mutant Rab27A-T23N or other GTPases had no effects. Thus, Rab27 is involved in the regulation of the secretion. Furthermore, we identified a novel type of Rab27 effector in platelets by affinity chromatography which specifically bound to GTP gamma SRab27A, but not GDP-Rab27A , GTP gamma S-Rab4 or GTP gamma S-Rab5. We are characterizing the Rab27A effector in the regulation of the secretion.
P335 INSULIN INDUCES THE RELEASE OF VASODILATOR COMPOUNDS FROM PLATELETS BY A NITRIC OXIDE/G KINASE/VAMP-3-DEPENDENT PATHWAY: A NOVEL MECHANISM OF INSULIN-INDUCED VASODILATATION. Voahanginirina Randriamboavonjy, Rudi Busse, Ingrid Fleming. Cardiovascular Physiology, Frankfurt am Main, Germany. Insulin-induced vasodilatation is sensitive to nitric oxide (NO) synthase (NOS) inhibitors. However, insulin is unable to relax isolated arteries or to activate eNOS in endothelial cells. Since insulin can enhance platelet eNOS activity, we determined whether insulin-induced vasodilatation can be attributed to a NO-dependent, platelet-mediated process. Insulin failed to relax endothelium-intact rings of porcine coronary artery. The supernatant from insulin-stimulated human platelets induced complete relaxation which was prevented by preincubation of platelets with a NOS inhibitor, the soluble guanylyl cyclase inhibitor, NS 2028, or the G kinase inhibitor, KT 5823 and was abolished by an adenosine A2A receptor antagonist. Insulin induced the release of ATP, adenosine and serotonin from platelet dense granules in a NO-dependent manner. This response was not detected using insulin-stimulated platelets from eNOS-/mice, although an NO donor elicited ATP release. Insulin-induced ATP release from human platelets correlated with the association of syntaxin 2 with the vesicle-associated membrane protein-3 (VAMP-3) but was not associated with the activation of aIIb-b3 integrin, the release of the