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3P-0835 Effects of cholesterol extraction from platelet membrane on the thrombus formation under blood flow conditions
Wednesday October 1, 2003: Poster Session Thrombosis and thrombolysis 3P-0832
The prevalence and treatment of hypercholesterolaemia in an Australian population sample
B. Blades 1 , P. Roach 1 , P. Lewis 2 . 1 University of Newcastle, School of Applied Sciences, Ourimbah; 2 Central Coast Health Public Health Unit, Australia Objectives: To determine the prevalence of hypercholesterolaemia (HC) in a sample of the New South Wales Central Coast population and the extent of pharmaceutical treatment for this cardiovascular risk factor. Methods: Plasma samples were collected from 391 subjects (215 females; 176 males) aged 18 years and over. Plasma cholesterol was measured enzymatically and pharmaceutical treatment was ascertained by interview. HC was defined as either being on cholesterol-lowering medication or having a plasma cholesterol higher than 5.5 mmol/L. Results: The overall prevalence of HC was 54% (55% in females and 51% in males). Of the 391 subjects, 68 (17%) were on cholesterol-lowering medication: 35 (16%) of the 215 females and 33 (19%) of the 176 males. Of the 323 subjects not on medication, 142 (44%) and 32 (10%) had plasma cholesterols higher than 5.5 and 6.5 mmol/L respectively. Of the 180 females not on medication, 83 (46%) and 16 (9%) had plasma cholesterols higher than 5.5 and 6.5 mmol/L respectively. Of the 143 males not on medication, 57 (40%) and 16 (11%) had plasma cholesterols higher than 5.5 and 6.5 mmol/L respectively. For subjects not on medication, the mean age of the females with cholesterols higher than 5.5 and 6.5 mmol/L was 55.3 ± 13.8 and 64.5 ± 13.8 years respectively compared to 54.5 ± 13.4 and 51.7 ± 12.8 years for males, respectively. Also, for subjects 65 years and over and not on medication, 67% and 20% of females had cholesterols greater than 5.5 and 6.5 mmol/L respectively compared to 44% and 8% of males respectively. Conclusions: The prevalence of HC in this population sample was similar to that of other western population groups. Many were receiving pharmaceutical treatment for this cardiovascular risk factor, however compared to males, there was a higher proportion of females 65 years and over with HC who were not receiving treatment. 3P-0833
Established and emerging coronary risk factors in patients with heterozygous familial hypercholesterolaemia
A. Neil 1 , V. Seagroatt 1 , J. Betteridge 2 , M. Cooper 2 , P. Durrington 3 , P. Miller 3 , M. Seed 4 , R. Naoumova 2 , G. Thompson 2 , R. Huxley 1 , S. Humphries 2 . 1 University of Oxford; 2 University of London; 3 University of Manchester; 4 Imperial College, United Kingdom
3P-0834
Transcriptional regulation of the apolipoprotein A5 gene by insulin
M. Nowak 1 , A. Helleboid-Chapman 1 , H. Jakel 1 , C. Rommens 1 , E. Baugé 1 , P. Gervois 1 , N. Vu-Dac 1 , G. Martin 2 , D. Duran-Sandoval 1 , B. Staels 1 , M.-R. Taskinen 3 , L. Pennacchio 4 , E. Rubin 4 , J. Fruchart-Najib 1 , J.-C. Fruchart 1 . 1 Institut Pasteur de Lille; 2 Genfit, France; 3 Department of Internal Medicine, Finland; 4 Genome Sciences Department, USA The recently discovered APOA5 gene has been shown to be very important in determining plasma triglycerides levels. This apolipoprotein represents the first apolipoprotein where over expression lowers triglycerides levels in mice. Since several studies have shown that hyperinsulinemia is associated with high triglycerides levels, we addressed the question whether APOA5 gene is
regulated by insulin. To investigate the role of insulin in the regulation of APOA5 gene expression, HepG2 cells, human and rat primary hepatocytes were treated with different insulin concentrations. The results demonstrated that in all cell models the insulin treatment induced a down-regulation of APOA5 expression in dose-dependent manner. These data were confirmed in vivo in humans after repeated injections of insulin, and in insulin-treated mice. The transcriptional activity of the human APOA5 promoter transfected into HepG2 cells is decreased by insulin. Deletion analysis of the promoter showed that a fragment of the promoter mediating the effects of insulin. These data show strong evidence for the repression of APOA5 gene expression by insulin, thus providing a potential mechanism for the elevation of triglycerides in hyperinsulinemic patients.
THROMBOSIS AND THROMBOLYSIS 3P-0835
Effects of cholesterol extraction from platelet membrane on the thrombus formation under blood flow conditions
S. Goto 1 , N. Tamura 1 , H. Ishida 2 , S. Handa 1 . 1 Division of Cardiology, of Physiology, Tokai Univesity School of Medicine, Japan
2 Department
Introduction: We attempted to clarify the effects of cholesterol extraction from platelet membrane on the thrombus formation occurring on the immobilized type I collagen and von Willebrand factor (VWF) surface under blood flow conditions. Methods: Blood specimen drawn from 6 normal volunteers are anticoagulated by addition of the specific antithrombin agent Argatroban (100µM). Platelets were rendered fluorescent by mepacrine. To visualize platelet shape changes occurring in response to its interaction with immobilized matrix, outer surface of platelets were fluorescinated by addition of FITC-conjugated anti-GP IIb/IIIa (not influencing platelet aggregation). Whole blood sample containing fluorescinated platelets were perfused on either immobilized type I collagen or immobilized VWF surface for 9 minutes at a wall shear rate of 1,500 s-1 controlled by rectangular flow chamber. Surface area covered by platelets were calculated using NIH Image. Effects of cholesterol extraction from platelet membrane was achieved by addition of various concentration of methyl-β-cyclodextrin (MBCD), which is known to preferentially extracts plasma membrane cholesterol. Results: Platelets firmly adhered on the collagen surface with multiple small psudopod formation under control situation, while only transient tethering without firm adhesion could be seen when cholesterol was extracted from platelet membrane by addition of MBCD. Surface area coverage by platelets after 9 minutes perfusion of 37±6% was decreased to 6±3% by MBCD (p=0.0012). Firm platelet adhesion on the immobilized VWF surface was also inhibited by MBCD, while transient tethering was not influenced at all. Conclusions: Our results suggest that the presence of cholesterol in platelet membrane is crucial to form thrombus formation. 3P-0836
Biological characterization of ER129614-06, a novel, non-peptide protease-activated receptor-1 (PAR-1) antagonist
M. Kogushi, T. Matsuoka, H. Kobayashi, N. Sato, S. Suzuki, T. Kawahara, A. Kajiwara, I. Hishinuma. Eisai Co., Ltd., Tsukuba, Ibaraki, Japan Objectives: Thrombin plays a central role in haemostasis, as well as thrombotic disorders and atherosclerosis. Thrombin stimulates various cell types including platelet and smooth muscle cells. Many of the cellular actions of thrombin are mediated by PAR-1. Through optimization of a lead compound obtained from high throughput screening, the iminoisoindoline derivative, ER129614-06 was identified as a potent PAR-1 antagonist. The aim of this study was to clarify the biological profiles of ER129614-06. Methods: PAR-1 binding assay was performed using human platelet membranes and high-affinity thrombin receptor activating peptide (haTRAP) as a radio-ligand. Effects on platelet aggregations were evaluated using plateletrich plasma (PRP) obtained from humans. The anti-thrombotic effects were evaluated in a photochemically induced thrombosis model (PIT) using guinea pigs. Results: ER129614-06 inhibits haTRAP binding to human platelets with an IC50 value of 64 nM. ER129614-06 shows potent inhibitory effects against thrombin-induced human PRP aggregation. This inhibitory effect was increased when the preincubation time was prolonged (IC50 = 230 nM [3 min] and 14 nM [60 min]). Furthermore, the inhibitory effects on thrombin-induced PRP aggregation were sustained in ER129614-06-treated platelets after com-
XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan
WEDNESDAY
Aim: To assess clinical and biochemical factors associated with interindividual variation in susceptibility to coronary artery disease (CAD) in patients with treated heterozygous familial hypercholesterolaemia (FH). Methods and Results: A cross-sectional study was conducted of 211 men and 199 women recruited from six lipid clinics in the United Kingdom. CAD was documented in 159 (39%) of patients (104 men) with a mean age of onset in men and women of 43.1 and 46.5 years respectively. CAD was more common in men than women (49% vs 28%, p<0.001) and in patients who had ever smoked cigarettes vs never (51% vs 28%, p<0.001). After adjusting for age, sex and current smoking status, there were no significant differences between patients with or without CAD in lipoprotein (a), homocysteine, fibrinogen, PAI-1, glucose, white blood cell count, body mass index, triglyceride or total cholesterol. However, HDL cholesterol concentrations were significantly lower (7%, 95% CI 12 to 1%, p=0.047) and this difference was greater in women than men (12.1% vs 2.3%, p=0.041). Conclusion: Emerging coronary risk factors do not appear to be associated with CAD in adults with treated FH. The strong association with smoking suggests that FH should be diagnosed early in childhood and patients discouraged from ever starting to smoke.
245
The prevalence and treatment of hypercholesterolaemia in an Australian population sample
B. Blades 1 , P. Roach 1 , P. Lewis 2 . 1 University of Newcastle, School of Applied Sciences, Ourimbah; 2 Central Coast Health Public Health Unit, Australia Objectives: To determine the prevalence of hypercholesterolaemia (HC) in a sample of the New South Wales Central Coast population and the extent of pharmaceutical treatment for this cardiovascular risk factor. Methods: Plasma samples were collected from 391 subjects (215 females; 176 males) aged 18 years and over. Plasma cholesterol was measured enzymatically and pharmaceutical treatment was ascertained by interview. HC was defined as either being on cholesterol-lowering medication or having a plasma cholesterol higher than 5.5 mmol/L. Results: The overall prevalence of HC was 54% (55% in females and 51% in males). Of the 391 subjects, 68 (17%) were on cholesterol-lowering medication: 35 (16%) of the 215 females and 33 (19%) of the 176 males. Of the 323 subjects not on medication, 142 (44%) and 32 (10%) had plasma cholesterols higher than 5.5 and 6.5 mmol/L respectively. Of the 180 females not on medication, 83 (46%) and 16 (9%) had plasma cholesterols higher than 5.5 and 6.5 mmol/L respectively. Of the 143 males not on medication, 57 (40%) and 16 (11%) had plasma cholesterols higher than 5.5 and 6.5 mmol/L respectively. For subjects not on medication, the mean age of the females with cholesterols higher than 5.5 and 6.5 mmol/L was 55.3 ± 13.8 and 64.5 ± 13.8 years respectively compared to 54.5 ± 13.4 and 51.7 ± 12.8 years for males, respectively. Also, for subjects 65 years and over and not on medication, 67% and 20% of females had cholesterols greater than 5.5 and 6.5 mmol/L respectively compared to 44% and 8% of males respectively. Conclusions: The prevalence of HC in this population sample was similar to that of other western population groups. Many were receiving pharmaceutical treatment for this cardiovascular risk factor, however compared to males, there was a higher proportion of females 65 years and over with HC who were not receiving treatment. 3P-0833
Established and emerging coronary risk factors in patients with heterozygous familial hypercholesterolaemia
A. Neil 1 , V. Seagroatt 1 , J. Betteridge 2 , M. Cooper 2 , P. Durrington 3 , P. Miller 3 , M. Seed 4 , R. Naoumova 2 , G. Thompson 2 , R. Huxley 1 , S. Humphries 2 . 1 University of Oxford; 2 University of London; 3 University of Manchester; 4 Imperial College, United Kingdom
3P-0834
Transcriptional regulation of the apolipoprotein A5 gene by insulin
M. Nowak 1 , A. Helleboid-Chapman 1 , H. Jakel 1 , C. Rommens 1 , E. Baugé 1 , P. Gervois 1 , N. Vu-Dac 1 , G. Martin 2 , D. Duran-Sandoval 1 , B. Staels 1 , M.-R. Taskinen 3 , L. Pennacchio 4 , E. Rubin 4 , J. Fruchart-Najib 1 , J.-C. Fruchart 1 . 1 Institut Pasteur de Lille; 2 Genfit, France; 3 Department of Internal Medicine, Finland; 4 Genome Sciences Department, USA The recently discovered APOA5 gene has been shown to be very important in determining plasma triglycerides levels. This apolipoprotein represents the first apolipoprotein where over expression lowers triglycerides levels in mice. Since several studies have shown that hyperinsulinemia is associated with high triglycerides levels, we addressed the question whether APOA5 gene is
regulated by insulin. To investigate the role of insulin in the regulation of APOA5 gene expression, HepG2 cells, human and rat primary hepatocytes were treated with different insulin concentrations. The results demonstrated that in all cell models the insulin treatment induced a down-regulation of APOA5 expression in dose-dependent manner. These data were confirmed in vivo in humans after repeated injections of insulin, and in insulin-treated mice. The transcriptional activity of the human APOA5 promoter transfected into HepG2 cells is decreased by insulin. Deletion analysis of the promoter showed that a fragment of the promoter mediating the effects of insulin. These data show strong evidence for the repression of APOA5 gene expression by insulin, thus providing a potential mechanism for the elevation of triglycerides in hyperinsulinemic patients.
THROMBOSIS AND THROMBOLYSIS 3P-0835
Effects of cholesterol extraction from platelet membrane on the thrombus formation under blood flow conditions
S. Goto 1 , N. Tamura 1 , H. Ishida 2 , S. Handa 1 . 1 Division of Cardiology, of Physiology, Tokai Univesity School of Medicine, Japan
2 Department
Introduction: We attempted to clarify the effects of cholesterol extraction from platelet membrane on the thrombus formation occurring on the immobilized type I collagen and von Willebrand factor (VWF) surface under blood flow conditions. Methods: Blood specimen drawn from 6 normal volunteers are anticoagulated by addition of the specific antithrombin agent Argatroban (100µM). Platelets were rendered fluorescent by mepacrine. To visualize platelet shape changes occurring in response to its interaction with immobilized matrix, outer surface of platelets were fluorescinated by addition of FITC-conjugated anti-GP IIb/IIIa (not influencing platelet aggregation). Whole blood sample containing fluorescinated platelets were perfused on either immobilized type I collagen or immobilized VWF surface for 9 minutes at a wall shear rate of 1,500 s-1 controlled by rectangular flow chamber. Surface area covered by platelets were calculated using NIH Image. Effects of cholesterol extraction from platelet membrane was achieved by addition of various concentration of methyl-β-cyclodextrin (MBCD), which is known to preferentially extracts plasma membrane cholesterol. Results: Platelets firmly adhered on the collagen surface with multiple small psudopod formation under control situation, while only transient tethering without firm adhesion could be seen when cholesterol was extracted from platelet membrane by addition of MBCD. Surface area coverage by platelets after 9 minutes perfusion of 37±6% was decreased to 6±3% by MBCD (p=0.0012). Firm platelet adhesion on the immobilized VWF surface was also inhibited by MBCD, while transient tethering was not influenced at all. Conclusions: Our results suggest that the presence of cholesterol in platelet membrane is crucial to form thrombus formation. 3P-0836
Biological characterization of ER129614-06, a novel, non-peptide protease-activated receptor-1 (PAR-1) antagonist
M. Kogushi, T. Matsuoka, H. Kobayashi, N. Sato, S. Suzuki, T. Kawahara, A. Kajiwara, I. Hishinuma. Eisai Co., Ltd., Tsukuba, Ibaraki, Japan Objectives: Thrombin plays a central role in haemostasis, as well as thrombotic disorders and atherosclerosis. Thrombin stimulates various cell types including platelet and smooth muscle cells. Many of the cellular actions of thrombin are mediated by PAR-1. Through optimization of a lead compound obtained from high throughput screening, the iminoisoindoline derivative, ER129614-06 was identified as a potent PAR-1 antagonist. The aim of this study was to clarify the biological profiles of ER129614-06. Methods: PAR-1 binding assay was performed using human platelet membranes and high-affinity thrombin receptor activating peptide (haTRAP) as a radio-ligand. Effects on platelet aggregations were evaluated using plateletrich plasma (PRP) obtained from humans. The anti-thrombotic effects were evaluated in a photochemically induced thrombosis model (PIT) using guinea pigs. Results: ER129614-06 inhibits haTRAP binding to human platelets with an IC50 value of 64 nM. ER129614-06 shows potent inhibitory effects against thrombin-induced human PRP aggregation. This inhibitory effect was increased when the preincubation time was prolonged (IC50 = 230 nM [3 min] and 14 nM [60 min]). Furthermore, the inhibitory effects on thrombin-induced PRP aggregation were sustained in ER129614-06-treated platelets after com-
XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan
WEDNESDAY
Aim: To assess clinical and biochemical factors associated with interindividual variation in susceptibility to coronary artery disease (CAD) in patients with treated heterozygous familial hypercholesterolaemia (FH). Methods and Results: A cross-sectional study was conducted of 211 men and 199 women recruited from six lipid clinics in the United Kingdom. CAD was documented in 159 (39%) of patients (104 men) with a mean age of onset in men and women of 43.1 and 46.5 years respectively. CAD was more common in men than women (49% vs 28%, p<0.001) and in patients who had ever smoked cigarettes vs never (51% vs 28%, p<0.001). After adjusting for age, sex and current smoking status, there were no significant differences between patients with or without CAD in lipoprotein (a), homocysteine, fibrinogen, PAI-1, glucose, white blood cell count, body mass index, triglyceride or total cholesterol. However, HDL cholesterol concentrations were significantly lower (7%, 95% CI 12 to 1%, p=0.047) and this difference was greater in women than men (12.1% vs 2.3%, p=0.041). Conclusion: Emerging coronary risk factors do not appear to be associated with CAD in adults with treated FH. The strong association with smoking suggests that FH should be diagnosed early in childhood and patients discouraged from ever starting to smoke.
245