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Risk of ischaemic heart disease or ischaemic cerebral stroke and levels of fibrinogen and von Willebrand factor
Monday June 26, 2000: Poster Abstracts P : W2 Thrombosis and Fibrinolysis
22
Results: a-Thrombin time-dependently and concentration-dependently increased VEGF mRNA levels, mainly that coding for the soluble splice variant VEGF165, and stimulated the release of VEGF protein. These effects required the proteolytic activity of thrombin and were mimicked by a thrombin receptor activating peptide and reduced by inhibitors of either protein kinase C, protein tyrosine kinases or phosphatidylinositol 3~-kinase, and by antioxidant treatments. Upregulation of VEGF expression was also induced by conditioned medium from a-thrombin-stimulated SMC. Both a PDGF-neutralizing antibody and a TGF-b-neutralizing antibody significantly attenuated the a-thrombin-induced release of VEGE Conclusions: Thrombin upregulates the expression of VEGF in vascular SMC through a direct effect, which is dependent on the activation of protein kinase C, protein tyrosine kinases and phosphatidylinositol 31-kinase, and the formation of reactive oxygen species, and an indirect effect, which is dependent on the endogenous formation of PDGF and TGF-b.
MoP18:W2 ] Effects of homocysteine on platelet aggregation in vitro B. Fan, B. Tomlinson, N. Thomas, J. Critchley. Division of Clinical Pharmacology, Department of Medicine & Therapeutics, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong, China Objective: High levels of plasma homocysteine are considered to be an independent risk factor for athero-thrombotic disease. One possible mechanism may be through activation of platelet aggregation. This study was performed to explore the effects of homocysteine on platelet aggregation using various concentrations of homocysteine. Methods: Platelets were obtained from 16 healthy subjects not taking drugs known to modify platelet activity. Platelet rich plasma (PRP) and whole blood, were incubated with homocysteine at concentrations of 30 # M , 0.5 mM and 1 mM at 37°C for 15 min before aggregation was assessed with and without the addition of other agonists. Changes in impedance were measured over 8 min and were expressed as amplitude (ohms) and slope (ohms/rain). Results: Homocysteine alone failed to induce platelet aggregation, both in the whole blood and PRP. Homocysteine at 30 # M slightly increased (p < 0.05) platelet aggregation induced by ADP 20/zM in whole blood (slope 3.0 4- 0.8 vs 5.0 4- 2.0, ohms/min, n = 9) and by collagen 2/zg/ml in PRP (slope 8.5 4- 1.5 vs 11.2 4- 2.5, ohms/rain, n = 6). However, homocysteine at 1 mM decreased (p < 0.05) platelet aggregation induced by ADP 10/zM and 20/zM in whole blood (amplitude 7.0 4- 4.0 vs 3.6 4- 2.6 and 6.8 4- 2.2 vs 4.1 4- 3.3 ohms respectively, n = 9). Conclusion: Homocysteine alone did not induce platelet aggregation in vitro. However, at 30 #M, a concentration similar to moderately elevated in vivo levels, homocysteine potentiated agonist-induced platelet aggregation in whole blood and PRP, but very high concentrations of homocysteine appeared to decrease platelet aggregation in vitro.
MoP19:W2 [ Adrenomedullin modulates the expression of tissue factor [
and tissue factor pathway inhibitor by human aortic endothelial cells
K. Marutsuka, Y. Asada, A. Yamashita, K. Hatakeyama, Y. Sato, A. Sumiyoshi. First Department of Pathology, Miyazaki Medical College, Miyazaki, Japan
Objective: Adrenomedullin (AM) is a potent hypotensive peptide, recently isolated from pheochromocytoma tissue. Recent reports have shown some physiological roles of AM in the cardiovascular system. However, there is no report concerning the role of AM on blood coagulation system. We examined the effect of AM on expression of tissue factor (TF) and tisue factor pathway inhibitor (TFPI) in cultured human aortic endothelial cells (HAoECs). Methods: HAoECs were originally isolated from a 52-year-old caucasian female. The subconfluent cells, between passages 4 to 6, were exposed to various concentrations of AM with serum-free medium. Cell growth was examined by colorimetric assay and apoptosis by TUN-EL assay. TF and TFPI antigen levels in conditioned medium (CM) and cell lysates (CL) were measured with ELIZA. The effects of AM receptor antagonists (AM22-52, AM1-25, CGRP8-37), specific anti-AM monoclonal antibodies, and inhibitors of second messengers for cAMP; Rp-8-Br-cAMP and for MAP kinase; PD98059 on AM action were also examined. Results: Apoptosis was rapidly induced by serum-free condition, which was prevented by AM with dose dependent manner. Increase of TF antigen level in CL, probably induced by apoptosis, was suppressed by AM. TFPI antigen level in CM increased rapidly after AM exposure and also gradually until 24 hr in time- and dose-dependent manner. These effects of AM were
inhibited by AM receptor antagonists, anti-AM antibodies, and inhibitors of cAMP and MAP kinase. Conclusions: These findings suggest that AM could maintain the hypocoagulable status of human aortic endothelial cell surface.
MoP20:W2 I
Risk oflschaemic heart disease or ischaemic cerebral stroke and levels of fibrinogen and von Willebrand factor
M. Jastrzfbska, B. Torbus-Lisiecka, J. Pieczul-Mrtz, K. Honczarenko, K. Chelstowski, M. Naruszewicz. Pomeranian Medical University, Szczecin, Poland
Objective: To study levels of fibrinogen (Fb), yon Willebrand factor (vWF) and arterial blood pressure in patients with ischaemic heart disease (IHD) or ischaemic cerebral stroke (ICS) and their children. Material and Methods: The study was performed in 50 families (100 parents and 90 children) with a history of IHD (36 parents, 27 children; fathers with myocardial infarction, mothers healthy), ICS (30 parents, 34 children; one parent father or mother with cerebral stroke) or as healthy controls (34 parents and 29 children). The mean age of parents and children was 43.5 46.1 and 12.2 + 3.1 years, respectively. Results: Fb and vWF levels in children of the IHD and ICS groups did not differ significantly from control values. Systolic (SP) and diastolic (DP) blood pressures in children of the IHD and ICS groups were significantly higher (IHD: 119 4- 19/76 4- 8); ICS: 108 -4- 12/73 4- 8; control 98 4- 9/63 48). Parents of the ICS group had elevated levels of Fb, vWF, SP and DP, as compared with controls (Fb: mothers 3.64 4- 0.84 vs. 2.98 4- 0.35 g/l, fathers 4.02 4- 0.78 vs. 3.08 4- 0.54 g/l; vWF: mothers 110.7 4- 40.1 vs. 88.2 421.5%, fathers 118.4 4- 41.5 vs. 82.9 4- 23.7%; SP: mothers 140 4- 25 vs. 116 4- 15, fathers 151 4- 28 vs. 127 4- 18 rnmHg; DP: mothers 90 4- 13 vs. 74 49, fathers 98 4- 14 vs. 79 4- 13 mmHg). A positive correlation was noted only for vWF levels in children and their mothers, being strongest in the ICS group (r = 0.43, 0.48 and 0.55 for control, IHD and ICS group, respectively). Conclusion: It appears from our results that Fb and vWF are risk factors of particular importance for the progression of ICS, chiefly in females with accompanying arterial hypertension.
I MOP21:W2 ] Procoagulant activity of T lymphocytes due to exposure of negatively charged phospholipids - Role of lipid oxidation T.W. Barrowchffe 1, M. Jardi, N. Rodriguez-Lambies, P. Fabregas, J. Felez. 1NIBSC, Potters Bar, UK; 1170, Barcelona, Spain Our previous studies have described procoagulant activity (PCA) of a variety of cell lines due to exposure of negatively charged phospholipids, as confirmed by binding studies with Annexin V and coagulation FVIII. In the present study the ability of two T-lymphoblastoid cell lines, Molt 4 & Jurkat, to promote generation of Factor Xa (FXa) was compared to that of other cell lines, to normal lymphocytes and neutrophils, and to a standard procoagulant phospholipid (PL). Cell lines Molt 4, Jurkat, Nalm 6, THP- 1. U-937 & NB4 were cultured under standard conditions, washed in RPMI, and suspended in 0.05 M Tris/0.15 M NaCl, pH 7.4 at a concentration of 4 x 106/ml. Factor Xa generation was measured by incubating purified human FX, FIXa, FVIII with CaC12 & cells or PL, and subsampling at intervals into $2765. The activity of the cells, measured as initial rate of FX activation, was converted to phospholipid units (PLU) by comparison with a PL dilution curve. The T lymphoblastoid cells Molt 4 (28 4- 6 PLU/ml) and Jurkat (17 4- 2 PLU/ml) had most activity; the other cell lines gave activity ranging from 5.6-16 PLU/ml. Normal fresh lymphocytes and neutrophils had no detectable activity but both cell types developed activity (6-12 PLU/ml) on incubation in serum-free medium. Incorporation of vitamin E into cell cultures inhibited by 68% the increase in PCA on incubation of neutrophils but had no effect on the activity of normal lymphocytes or Molt 4. These results demonstrate that T lymphocytes can develop potent PCA due to exposure of negatively charged phospholipids and this is unlikely to be due to lipid oxidation. T lymphocytes occur in atherosclerotic plaques and it is possible that their PCA could play a role in thrombotic episodes associated with plaque rupture.
Xlhh International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000
22
Results: a-Thrombin time-dependently and concentration-dependently increased VEGF mRNA levels, mainly that coding for the soluble splice variant VEGF165, and stimulated the release of VEGF protein. These effects required the proteolytic activity of thrombin and were mimicked by a thrombin receptor activating peptide and reduced by inhibitors of either protein kinase C, protein tyrosine kinases or phosphatidylinositol 3~-kinase, and by antioxidant treatments. Upregulation of VEGF expression was also induced by conditioned medium from a-thrombin-stimulated SMC. Both a PDGF-neutralizing antibody and a TGF-b-neutralizing antibody significantly attenuated the a-thrombin-induced release of VEGE Conclusions: Thrombin upregulates the expression of VEGF in vascular SMC through a direct effect, which is dependent on the activation of protein kinase C, protein tyrosine kinases and phosphatidylinositol 31-kinase, and the formation of reactive oxygen species, and an indirect effect, which is dependent on the endogenous formation of PDGF and TGF-b.
MoP18:W2 ] Effects of homocysteine on platelet aggregation in vitro B. Fan, B. Tomlinson, N. Thomas, J. Critchley. Division of Clinical Pharmacology, Department of Medicine & Therapeutics, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong, China Objective: High levels of plasma homocysteine are considered to be an independent risk factor for athero-thrombotic disease. One possible mechanism may be through activation of platelet aggregation. This study was performed to explore the effects of homocysteine on platelet aggregation using various concentrations of homocysteine. Methods: Platelets were obtained from 16 healthy subjects not taking drugs known to modify platelet activity. Platelet rich plasma (PRP) and whole blood, were incubated with homocysteine at concentrations of 30 # M , 0.5 mM and 1 mM at 37°C for 15 min before aggregation was assessed with and without the addition of other agonists. Changes in impedance were measured over 8 min and were expressed as amplitude (ohms) and slope (ohms/rain). Results: Homocysteine alone failed to induce platelet aggregation, both in the whole blood and PRP. Homocysteine at 30 # M slightly increased (p < 0.05) platelet aggregation induced by ADP 20/zM in whole blood (slope 3.0 4- 0.8 vs 5.0 4- 2.0, ohms/min, n = 9) and by collagen 2/zg/ml in PRP (slope 8.5 4- 1.5 vs 11.2 4- 2.5, ohms/rain, n = 6). However, homocysteine at 1 mM decreased (p < 0.05) platelet aggregation induced by ADP 10/zM and 20/zM in whole blood (amplitude 7.0 4- 4.0 vs 3.6 4- 2.6 and 6.8 4- 2.2 vs 4.1 4- 3.3 ohms respectively, n = 9). Conclusion: Homocysteine alone did not induce platelet aggregation in vitro. However, at 30 #M, a concentration similar to moderately elevated in vivo levels, homocysteine potentiated agonist-induced platelet aggregation in whole blood and PRP, but very high concentrations of homocysteine appeared to decrease platelet aggregation in vitro.
MoP19:W2 [ Adrenomedullin modulates the expression of tissue factor [
and tissue factor pathway inhibitor by human aortic endothelial cells
K. Marutsuka, Y. Asada, A. Yamashita, K. Hatakeyama, Y. Sato, A. Sumiyoshi. First Department of Pathology, Miyazaki Medical College, Miyazaki, Japan
Objective: Adrenomedullin (AM) is a potent hypotensive peptide, recently isolated from pheochromocytoma tissue. Recent reports have shown some physiological roles of AM in the cardiovascular system. However, there is no report concerning the role of AM on blood coagulation system. We examined the effect of AM on expression of tissue factor (TF) and tisue factor pathway inhibitor (TFPI) in cultured human aortic endothelial cells (HAoECs). Methods: HAoECs were originally isolated from a 52-year-old caucasian female. The subconfluent cells, between passages 4 to 6, were exposed to various concentrations of AM with serum-free medium. Cell growth was examined by colorimetric assay and apoptosis by TUN-EL assay. TF and TFPI antigen levels in conditioned medium (CM) and cell lysates (CL) were measured with ELIZA. The effects of AM receptor antagonists (AM22-52, AM1-25, CGRP8-37), specific anti-AM monoclonal antibodies, and inhibitors of second messengers for cAMP; Rp-8-Br-cAMP and for MAP kinase; PD98059 on AM action were also examined. Results: Apoptosis was rapidly induced by serum-free condition, which was prevented by AM with dose dependent manner. Increase of TF antigen level in CL, probably induced by apoptosis, was suppressed by AM. TFPI antigen level in CM increased rapidly after AM exposure and also gradually until 24 hr in time- and dose-dependent manner. These effects of AM were
inhibited by AM receptor antagonists, anti-AM antibodies, and inhibitors of cAMP and MAP kinase. Conclusions: These findings suggest that AM could maintain the hypocoagulable status of human aortic endothelial cell surface.
MoP20:W2 I
Risk oflschaemic heart disease or ischaemic cerebral stroke and levels of fibrinogen and von Willebrand factor
M. Jastrzfbska, B. Torbus-Lisiecka, J. Pieczul-Mrtz, K. Honczarenko, K. Chelstowski, M. Naruszewicz. Pomeranian Medical University, Szczecin, Poland
Objective: To study levels of fibrinogen (Fb), yon Willebrand factor (vWF) and arterial blood pressure in patients with ischaemic heart disease (IHD) or ischaemic cerebral stroke (ICS) and their children. Material and Methods: The study was performed in 50 families (100 parents and 90 children) with a history of IHD (36 parents, 27 children; fathers with myocardial infarction, mothers healthy), ICS (30 parents, 34 children; one parent father or mother with cerebral stroke) or as healthy controls (34 parents and 29 children). The mean age of parents and children was 43.5 46.1 and 12.2 + 3.1 years, respectively. Results: Fb and vWF levels in children of the IHD and ICS groups did not differ significantly from control values. Systolic (SP) and diastolic (DP) blood pressures in children of the IHD and ICS groups were significantly higher (IHD: 119 4- 19/76 4- 8); ICS: 108 -4- 12/73 4- 8; control 98 4- 9/63 48). Parents of the ICS group had elevated levels of Fb, vWF, SP and DP, as compared with controls (Fb: mothers 3.64 4- 0.84 vs. 2.98 4- 0.35 g/l, fathers 4.02 4- 0.78 vs. 3.08 4- 0.54 g/l; vWF: mothers 110.7 4- 40.1 vs. 88.2 421.5%, fathers 118.4 4- 41.5 vs. 82.9 4- 23.7%; SP: mothers 140 4- 25 vs. 116 4- 15, fathers 151 4- 28 vs. 127 4- 18 rnmHg; DP: mothers 90 4- 13 vs. 74 49, fathers 98 4- 14 vs. 79 4- 13 mmHg). A positive correlation was noted only for vWF levels in children and their mothers, being strongest in the ICS group (r = 0.43, 0.48 and 0.55 for control, IHD and ICS group, respectively). Conclusion: It appears from our results that Fb and vWF are risk factors of particular importance for the progression of ICS, chiefly in females with accompanying arterial hypertension.
I MOP21:W2 ] Procoagulant activity of T lymphocytes due to exposure of negatively charged phospholipids - Role of lipid oxidation T.W. Barrowchffe 1, M. Jardi, N. Rodriguez-Lambies, P. Fabregas, J. Felez. 1NIBSC, Potters Bar, UK; 1170, Barcelona, Spain Our previous studies have described procoagulant activity (PCA) of a variety of cell lines due to exposure of negatively charged phospholipids, as confirmed by binding studies with Annexin V and coagulation FVIII. In the present study the ability of two T-lymphoblastoid cell lines, Molt 4 & Jurkat, to promote generation of Factor Xa (FXa) was compared to that of other cell lines, to normal lymphocytes and neutrophils, and to a standard procoagulant phospholipid (PL). Cell lines Molt 4, Jurkat, Nalm 6, THP- 1. U-937 & NB4 were cultured under standard conditions, washed in RPMI, and suspended in 0.05 M Tris/0.15 M NaCl, pH 7.4 at a concentration of 4 x 106/ml. Factor Xa generation was measured by incubating purified human FX, FIXa, FVIII with CaC12 & cells or PL, and subsampling at intervals into $2765. The activity of the cells, measured as initial rate of FX activation, was converted to phospholipid units (PLU) by comparison with a PL dilution curve. The T lymphoblastoid cells Molt 4 (28 4- 6 PLU/ml) and Jurkat (17 4- 2 PLU/ml) had most activity; the other cell lines gave activity ranging from 5.6-16 PLU/ml. Normal fresh lymphocytes and neutrophils had no detectable activity but both cell types developed activity (6-12 PLU/ml) on incubation in serum-free medium. Incorporation of vitamin E into cell cultures inhibited by 68% the increase in PCA on incubation of neutrophils but had no effect on the activity of normal lymphocytes or Molt 4. These results demonstrate that T lymphocytes can develop potent PCA due to exposure of negatively charged phospholipids and this is unlikely to be due to lipid oxidation. T lymphocytes occur in atherosclerotic plaques and it is possible that their PCA could play a role in thrombotic episodes associated with plaque rupture.
Xlhh International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000