001 THROMBIN INHIBITION WITH MELEGOTRAN PREVENTS PLAQUE RUPTURE IN APOLIPOPROTEIN E KNOCKOUT MICE

*S. Karanam¹, R. F. Danielson², C. Jackson¹.¹Bristol Heart Institute, University of Bristol, Bristol, UK; ²AstraZeneca R&D, Mölndal, Sweden

Atherosclerotic plaque rupture and subsequent thrombogenicity cause the majority of acute coronary events. Therefore, the effect of the oral direct thrombin inhibitor Melagatran on atherosclerotic plaques was investigated in the fat-fed apolipoprotein E (apoE) knockout mouse, in which plaque ruptures occur in the brachiocephalic artery after 8 weeks on a high fat diet. Eighty male apoE knockout mice, 6–8 weeks old, were fed a diet containing 21% fat and 0.15% cholesterol for 8 weeks. Forty mice were treated with 500 μmol/Kg body weight/day of Melagatran throughout the 8 weeks. The brachiocephalic artery was removed following perfusion fixation with formalin at a constant pressure of 100 mmHg and embedded in paraffin. Computerised morphometry of serial sections was used to measure the areas of the plaque, the media, and the lumen. The number of buried fibrous layers within the plaque and presence or absence of acute plaque rupture was also recorded. Immunostaining was used to detect the expression of markers of inflammation and thrombogenicity. Melagatran treatment at 500 μmol/Kg bodyweight/day significantly reduced plaque size from 38.5 ± 5.6 to 7.3 ± 1.3 × 10³ μm² (−81%; p < 0.001) and the incidence of plaque rupture from 0.43 ± 0.10 to 0.10 ± 0.05 (−77%; p < 0.05). Immunohistochemical analysis of serial sections showed reduced staining for various inflammatory and thrombogenic markers. This study supports the hypothesis that serial accumulation of thrombus through repeated episodes of non-fatal plaque rupture contributes to atherosclerotic plaque growth in the apoE knockout mouse and also that inhibition of thrombin activity may be a useful strategy for inhibiting plaque rupture.

002 EXPRESSION OF PEROXISOME PROLIFERATORS-ACTIVATOR RECEPTOR-δ AND RETINOID X RECEPTORS IN ENDOTHELIAL CELLS

*L. Piqueras, T. D. Warner, D. Bishop-Bailey.William Harvey Research Institute, Bart’s and the London Queen Mary’s School of Medicine, London, UK

The endothelium plays a critical role in vascular homeostasis in part by secreting potent mediators such as nitric oxide and prostacyclin that both control vascular tone and inhibit the activity of circulating blood cells. Peroxisome proliferators-activated receptors (PPARs) are a family of three nuclear receptors: α, β/δ, and γ, which heterodimerize with the retinoid X receptors (RXR)s.¹ Prostacyclin is a putative endogenous ligand for PPARδ.² In this study we evaluated the expression and role of PPAR delta and RXR in endothelial cells. PPARδ and RXR protein expression were measured by Western blot analysis and indirect immunofluorescence. PPARδ and RXR were expressed in human aortic endothelial cells (HAEC) and the “endothelial”-A549 fusion cell line EaHy 926, and localised mainly to the nucleus and peri-nuclear region of the cell. Furthermore, we used the MTT assay to test the effects of selective PPARδ and RXR/RAR ligands on endothelial cell growth. The selective PPARδ agonist GW501516 (0.01–1 μM) induced a concentration–dependent increased in cell number (215 ± 18% at 72 h with 1 μM; n  =  4). In contrast, the RXR/RAR ligand 9-cis retinoid acid (1–20 μM) inhibited endothelial cell growth (51 ± 3% at 72 h; n  =  3). PPARδ and RXR are expressed in endothelial cells. Activation of PPAR delta leads to increased proliferation of endothelial cells while RXR/RAR activation reduces cell viability. Activation of PPARδ by endogenous or synthetic ligands may represent a new target for endothelial cell re-growth in damaged vessels.

LP is funded by a postdoctoral fellowship from the Spanish Ministry of Education.

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003 EFFECTS OF APOPTOSIS AND LIPID PEROXIDATION ON LEUCOCYTE PROCOAGULANT ACTIVITY (PCA)

W. M. Pickering¹, E. Gray¹, A. H. Goodall², *T. W. Barrowcliffel¹.¹National Institute for Biological Standards and Control, Potters Bar, Herts, UK; ²Department of Cardiovascular Sciences, University of Leicester, Leicester, UK

Leucocyte PCA is recognised as a major cause of thrombosis in cancer and an important contributor to the atherothrombotic process. Monocytes’ expression of tissue factor (TF) is well known, but our recent studies have shown that T lymphocyte cell lines can potentiate PCA via expression of negatively charged phospholipids (NCPL). Apoptosis results in exposure of NCPL and is also associated with lipid peroxidation; in the present study we have investigated the relationship between apoptosis, lipid peroxidation, and leucocyte PCA. T cell lines Molt4, Jurkat, CEM-CCRF, A3.01, and monocyte line THP-1 were cultured under standard conditions, and tested for PCA at a concentration of 4 × 10⁶/ml in assays of TF, Factor Xa, and thrombin generation. Apoptosis was monitored 0–24 hours after treatment with staurosporine, by measuring DNA fragmentation (Sub G_o), and by flow cytometry of cells binding to Annexin V^FITC and anti-phosphatidyl serine (PS) antibody. Lipid peroxidation was monitored by measurement of thio-barbituric acid reactive substances (TBARS), either during apoptosis or after treatment with 4 mM H₂O₂ and 40 μM CuSO₄. Basal levels of apoptosis of the cells were low (2.7–4.6%), but increased to 37.9–61.2% during 24 hours with staurosporine. Binding of annexin V and anti-PS increased to a maximum of 45% and 29% respectively. TF bearing cells THP-1 and Jurkat showed increases in TF activity of 60 and 67%. Factor Xa generation increased by 38–250% in the various cell lines, and increases in thrombin generation were 50–420%. Detectable TBARS were found in the basal cells, and these increased during apoptosis by up to 128%. In separate experiments in which lipid peroxidation was induced by treatment with H₂O₂ and CuSO_4, PCA increased in all tests: 112–290% for TF; 90–285% for Factor Xa generation; 190–412% for thrombin generation. These results show that apoptosis and lipid peroxidation, which both occur in plaques, give marked increases in leucocyte PCA; this could be a contributory mechanism to thrombosis following plaque instability and rupture.

004 CD14 DEPENDENT ADHESION OF MONOCYTES TO OXIDISED LDL AND HSP60 VIA LIPID RAFTS

*R. N. Poston^1,2, M. Marshall², H. Louis².¹Department of Histopathology, Guy’s Campus, King’s College, London, UK; ²Centre for Cardiovascular Biology & Medicine, Guy’s Campus, King’s College, London, UK

To determine whether oxidised LDL and HSP60 (heat shock protein 60) could act as monocyte adhesion molecules, and be involved in atheroslerosis. Monocytes were isolated from human blood by dextran sedimentation and a NycoPrep gradient,¹ and labelled with carboxyfluorescein ester. Plastic wells were coated with oxidised LDL, LDL, recombinant human HSP60 and fibronectin. Monocytes were incubated for 1 hour, and adhesion with and without inhibitors was measured in a fluorescent spectrophotometer. Surface expression of HSP60 on HUVEC was assayed by ELISA and the expression of ox-LDL, LDL, and HSP60 on atherosclerotic human endothelium by immunohistochemistry. Effects of antibodies to HSP60 on the adhesion of U937 monocytes to histological sections of human arteries were assessed as described by Poston et al, 1996. Monocytes bound selectively ox-LDL on plastic wells, and this was inhibited by antibodies to LDL, ox-LDL, and CD14. Likewise the monocytes bound to HSP60, and was inhibited by antibodies to HSP60 and CD14. LDL was expressed universally at a high level in carotid endothelium, and ox-LDL focally in plaques. HSP60 was expressed on the surface of TNF activated HUVEC, and antibodies to HSP60 inhibited the adhesion of U937 cells to the plaques. Nystatin and methylcyclodextrin, inhibitors of lipid raft function, completely inhibited monocyte adhesion to both ligands. In conclusion, ox-LDL and HSP60 are CD14/lipid raft dependent monocyte adhesion ligands that are expressed in atherosclerotic endothelium.

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005 PPAR-ALPHA ACTIVATION DOWNREGULATES COX-2 EXPRESSION IN HUMAN MONOCYTES

*J. A. Wray, N. P. Williams, T. D. Warner, D. Bishop-Bailey.William Harvey Research Institute, Barts and The London Queen Mary’s School of Medicine and Dentistry, London, UK

Monocyte/macrophages are the predominant inflammatory leucocyte in atherosclerotic lesions, contributing to disease progression and plaque instability. Peroxisome proliferator-activated receptor-α (PPARα) is expressed in macrophages and human atherosclerotic lesions where its ligands, such as the clinically used fibrates, reduce tissue factor and matrix malloprotease-9 expression. PPARα ligands also inhibit inflammatory responses in vascular smooth muscle and endothelial cells. The aim of this investigation was to study directly the effects of PPARα on inflammatory responses in human monocytes. Human monocytic cells (THP-1) were maintained in DMEM supplemented with antibiotics/antimycotics and 10% FCS (37°C; 5% CO₂; 95% air). Cells were incubated (24 h) with IL-1β (10 ng/ml), to induce an inflammatory type response, together with vehicle, the selective synthetic PPARα agonist GW7647 (10 nM) or bezafibrate (300 μM). Conditioned medium was then removed and stored for PGE₂ measurements by RIA, while the cells were lysed, protein content measured, and samples (10 μg) subjected to Western blot analysis.¹ GW7657 or bezafibrate alone had no effect. In the presence of vehicle IL-1β induced the expression of COX-2 protein and the release of PGE₂, both of which were reversed by incubation with GW7647 or bezafibrate (eg PGE₂: control, 1.8 ± 0.1 ng/ml; IL-1β 6.0 ± 1.6 ng/ml; bezafibrate 1.9 ± 0.2 ng/ml; IL-1β + bezafibrate 1.4 ± 0.3 ng/ml, n  =  3). The levels of PPARα and β-actin were unaffected by any treatment. In conclusion, activation of PPARα downregulates expression of COX-2 in activated human monocytes so reducing the production of PGE₂. These direct anti-inflammatory effects of PPARα in monocytes further highlight the potential for PPARα ligands as therapeutics for cardiovascular disease.

JAW and DB-B are funded by the British Heart Foundation.

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006 DIFFERENTIAL UPREGULATION OF TISSUE FACTOR AND TISSUE FACTOR PATHWAY INHIBITOR IN MONOCYTES BY ACTIVATED PLATELETS AND LIPOPOLYSACCHARIDE

*J. A. Appleby, J. R. Wright, A. H. Goodall.Department of Cardiovascular Sciences, University of Leicester, Leicester, UK

Monocytes can express both tissue factor (TF), the principal initiator of coagulation and its inhibitor, tissue factor pathway inhibitor (TFPI). This study explored the role of activated platelets in regulating monocyte TF and TFPI expression. Whole blood was incubated at 37°C with the GPVI-specific agonist, cross-linked collagen-related peptide (XL-CRP), with and without 9E1—a P-selectin antibody that blocks platelet-monocyte aggregation. Monocytes activated with lipopolysaccharide (LPS) served as a control. Monocyte mRNA was extracted at 0, 1, 2, 4, and 6 h. TF and TFPI expression was determined by RT-PCR and the PCR products compared using densitometry with reference to GAPDH. mRNA was also extracted from washed platelets and cDNA for TFPI and TF amplified. P-selectin and CD11b served as positive and negative controls respectively. Monocyte TF mRNA was significantly higher after 1 h in blood in which the platelets were activated with XL-CRP (p  =  0.036; n  =  3) and remained elevated above the control for the next 5 h. Upregulation of TFPI mRNA was, however, not seen in XL-CRP stimulated blood until 4 h (p  =  0.027; n  =  3), remaining above control at 6 h (p  =  0.007). Blocking direct platelet-monocyte interaction in XL-CRP stimulated blood did not abolish either TF or TFPI expression at 4 hours. Whilst LPS induced persistent monocyte TF expression between 1 and 6 h, TFPI mRNA remained undetectable in any LPS-stimulated samples. mRNA for TFPI and TF could not be detected in platelet lysates except in the presence of contaminating leucocytes, demonstrated by amplification of CD11b mRNA. In conclusion, activated platelets can upregulate both TF and TFPI in monocytes but TFPI appears much later. This effect is at least in part due to soluble mediators. This may have implications for sepsis in which adverse outcomes may result from rapid upregulation of TF without a concomitant increase in its inhibitor.

007 RETINOID X RECEPTOR LIGANDS INHIBIT HUMAN PLATELET AGGREGATION INDUCED BY ADP AND U46619

*L. A. Moraes, T. D. Warner, D. Bishop-Bailey.William Harvey Research Institute, Barts and The London Queen Mary’s School of Medicine and Dentistry, London

The 9 cis-retinoic X receptors (RXRs) are a family of three nuclear receptors (α, β, and γ) that can activate transcription as homodimers or as obligate heterodimeric partners of a number of other non-steroidal nuclear receptors. RXRs play important regulatory roles in cell proliferation, differentiation and development, metabolic signalling pathways, hyperlipidemia, and atherosclerosis. We have examined the expression of RXR in platelets. Platelet rich plasma (PRP) and washed platelets were isolated from the citrated blood of healthy volunteers by centrifugation. Responses were measured using light-transmission aggregometry. Western blotting and immunohistochemistry demonstrated clear expression of RXR in human PRP and washed platelets. ADP (4 μM) induced PRP aggregation was inhibited by the RXR ligands 9-cis retinoic acid (10.0 μM; 70 ± 11%; n  =  6) and methoprene acid (10.0 μM; 45 ± 8%; n  =  6) but not by the retinoic acid receptor ligand all trans RA (10.0 μM; 0 ± 3%; n  =  6). 9-cis RA also inhibited aggregation induced by the thromboxane A2 mimetic U46619 (1 μM; 90 ± 13%; n  =  6), but not collagen (1 μM). Human platelets express functional RXR. Inhibition of platelet aggregation by RXR ligands represents both a novel mechanism of action for this nuclear receptor and a potential new cellular target for its actions.

LAM is funded by a grant from the Bart’s and the London Research Advisory Board. DB-B is funded by the British Heart Foundation.

008 FARNESOID X RECEPTOR LIGANDS INHIBITED HUMAN PLATELET AGGREGATION INDUCED BY ADP

*M. E. Haque, L. A. Moraes, T. D. Warner, D. Bishop-Bailey.Cardiac, Vascular and Inflammation Research, William Harvey Research Institute, Bart’s and The London Queen Mary’s School of Medicine and Dentistry, London, UK

The farnesoid X receptor/bile acid receptor (FXR) is a recently discovered member of the nuclear hormone superfamily. FXR ligands have been proposed as targets in cardiovascular disease, regulating cholesterol metabolism as well as bile acid transport and metabolism in the liver and gastrointestinal tract. Here we have investigated the expression and activation of FXR in human platelets. Human platelet rich plasma (PRP) and washed platelets were isolated from the citrated blood of healthy volunteers by centrifugation, and responses were measured using light-transmission aggregometry. Aggregation of PRP induced by ADP (2 μM) was inhibited in a concentration-dependent manner by the synthetic FXR ligand, 6-α-ethyl-chenodeoxycholic acid (0.1–20 μM), with an inhibition of 67 ± 13% (n  =  3) at 20 μM. In contrast, 300 μM of the “natural” FXR ligand chenodeoxycholic acid was required to produce an equivalent of ADP-induced aggregation (73 ± 19%; n  =  3). These relative potencies of 6-ECDCA and CDCA as inhibitors of ADP-induced platelet aggregation are similar to their relative potencies on FXR.¹ Inhibition of platelet aggregation by FXR ligands represents both a novel mechanism of action for this nuclear receptor and a potential new “cellular” target for its actions.

Funded by Bart’s and the London Research Advisory Board, and the British Heart Foundation

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009 POTENT AND SPECIFIC EFFECTS OF GRAPE SEED EXTRACT ON PLATELET AGGREGATION

S. S. Devasurendra, L. A. Moraes, M. J. Carrier, R. Corder, *L. S. Harrington.William Harvey Research Institute, Barts and The London Queen Mary’s School of Medicine and Dentistry, London, UK

The beneficial anti-platelet effects of red wine drinking have been widely reported, and have largely been credited to the grape seeds and skins being an excellent source of flavonoids. However, there is little understanding of the mechanism by which flavonoids act on platelets. This study investigated changes in aggregation in response to ADP and phorbol myristate acetate (PMA) induced by grape seed extract (GSE). Venous blood (50 ml) was obtained from healthy subjects who were not taking any medication and had refrained from flavonoid consumption that day. Whole blood was mixed with 10% volume of 3.2% sodium citrate to prevent clotting, and the plasma rich platelet (PRP) fraction was isolated by centrifugation at 1,100 rpm for 15 mins. Platelet poor plasma (PPP) was prepared by centrifugation of the PRP at 2000 rpm for 5 mins. Platelet aggregation was performed using a dual chamber Payton aggregation model and the instrument calibrated for each individual using the PRP compared to PPP fractions. PRP samples were incubated with 10 μg/ml GSE or vehicle for 4 minutes prior to aggregation with an EC₅₀ of either ADP or PMA. The extracts were dissolved in vehicle consisting of: 2% ethanol, 1 mM citric acid, 5 mM HCl in 5 mM ascorbic acid. ADP induces a biphasic aggregation response; the initial phase was not affected by the presence of either extract. However, the second phase was significantly inhibited by pre-incubation with GSE (74.3 ± 13.4%; n  =  3) compared with vehicle control. PMA induced aggregation was not changed by incubation with either extract. This data not only demonstrates that grape extracts are potent inhibitors of aggregation ex vivo, but also suggests that flavonoids may act on the PLC second messenger pathway.

010 THE ROLE OF PERIPHERAL TACHYKININS IN PLATELET FUNCTION

*S. Jones, G. Graham, J. M. Gibbins.School of Animal and Microbial Sciences, University of Reading, Reading, UK

We have reported that the tachykinin substance P (SP) stimulates functional effects in platelets, however, this peptide is predominantly expressed in the central nervous system raising questions of its significance in vivo. The recent discovery of genes encoding new, peripherally expressed tachykinins revealed two peptides, endokinin A and endokinin B, with an identical C-terminal sequence, homologous to that of SP. Platelets were stimulated with increasing concentrations of EKA/B in the presence and absence of other platelet agonists, platelet inhibitors, and the calcium chelator, BAPTA-AM. Aggregation of the platelets was then measured using optical aggregometry. Secretion assays were carried out using platelets loaded with [³H]-5HT and arachidonic acid. Intracellular calcium mobilisation was measured using platelets preloaded with FURA2-AM in a spectrofluorimeter EKA/B was found to produce significant secretion and aggregation of platelets in a concentration-dependent manner (between 2 and 10 µM). This was accompanied by raised intracellular calcium levels, which proved necessary for aggregation. At sub-aggregatory concentrations (1.25 µM), EKA/B produced synergistic effects with other platelet agonists to mediate full aggregation. The release of other platelet agonists such as ADP and thromboxane A₂ from activated platelet is also necessary for EKA/B mediated aggregation to occur. In conclusion, EKA/B activates human platelets in vitro at micromolar concentrations and synergises with other agonists at lower concentration to stimulate aggregation. We propose that EKA/B may be a peripheral target for newly identified peripheral tachykinins.

011 STUDIES OF POSSIBLE ATHEROGENIC EFFECTS OF PHOSPHOLIPID CHLOROHYDRINS

*G. Dever^1,2, S. Kennedy², C. M. Spickett¹, C. L. Wainwright³.¹Department of Bioscience, University of Strathclyde, Glasgow, UK; ²Department of Physiology and Pharmacology, University of Strathclyde, Glasgow, UK; ³School of Pharmacy, The Robert Gordon University, Aberdeen, UK

The oxidation of low-density lipoprotein (LDL) is thought to contribute to atherogenesis. Phagocytes produce a variety of oxidants as part of the innate immune defense, which react both with proteins and lipids, and could contribute to the oxidation of LDL and plaque formation in vivo. Myeloperoxidase, an enzyme which is able to produce hypochlorous acid (HOCl), is released from these phagocytes. HOCl can oxidise lipids to give chlorohydrins. The aim of this study was to investigate the biological effects of chlorohydrins of stearoly-oleoyl phosphatidylcholine (SOPC)—a phospholipid found in LDL—on: (1) ATP levels and viability of human myeloid cells; and (2) leucocyte adhesion to the arterial endothelium in a murine model. 16-week old ApoE -/- mice were euthanised by carbon dioxide asphyxiation and the aorta removed and cut into two segments (aortic arch and thoracic), and each segment was pinned out luminal-side up. Leucocytes were isolated from the homologous spleen and radiolabelled with ⁵¹Cr. The artery segments were pre-treated with SOPC chlorohydrin and incubated with a 5 μl aliquot of the labelled leucocytes for 30 minutes at 37°C, washed, and assayed for ⁵¹Cr on a gamma counter. In addition, U937 cells (human myeloid cell line) were treated with SOPC chlorohydrin in a 96-well plate, and a bioluminescence assay and MTT assay were employed to determine cellular ATP levels and viability, respectively. SOPC chlorohydrin was found to deplete ATP levels in human myeloid cells, which was both time and concentration dependent, and correlated with a loss of viability using an MTT assay. Furthermore, sub-toxic concentrations of the chlorohydrin decreased leucocyte adhesion to homologous artery segments isolated from atherosclerotic (ApoE -/-) mice, and this was also in a time and concentration dependent manner. These are the first data showing effects of a phospholipid chlorohydrin on leucocyte adhesion and, together with the effects on cell viability, suggest a possible atherogenic/inflammatory role for SOPC chlorohydrin.

012 ANTIOXIDANT ENZYME ACTIVITY IN CULTURED HUMAN ARTERIAL AND VENOUS ENDOTHELIAL CELLS

*A. R. Conant¹, W. C. Dihimis¹, A. W. M. Simpson².¹The Cardiothoracic Centre, Liverpool NHS Trust, Thomas Drive, Liverpool, UK; ²Dept Human Anatomy and Cell Biology, University of Liverpool, Liverpool, UK

In coronary artery bypass grafting (CABG) arterial grafts have shown a significantly higher long term patency rate than their venous counterparts. There is evidence that oxidant stress plays an important role in endothelial dysfunction and it is this dysfunction that precedes the development of atherosclerosis and intimal hyperplasia. Our aim was to determine whether endothelial cells cultured from human arterial vessels have a greater ability to protect themselves from oxidant stress than human venous graft endothelial cells. Endothelial cells (ECs) were isolated by collagenase digestion from sections of radial artery (RA) and saphenous vein (SV) obtained surplus to CABG, with informed patient consent and ethical committee approval. Confluent cells were treated with 20 nM TNFα 24 hours prior to cell extraction and compared with untreated controls. Superoxide dismutase (SOD) and catalase activity and total glutathione (GSH) were determined in cell lysates. Cu/ZnSOD or MnSOD activity, measured as the component of activity sensitive or insensitive to 2 mM NaCN, respectively, showed no significant difference between the two groups. Extracellular SOD, determined as the amount of activity removed by washing cells with excess heparin prior to isolation, was only measurable in RAECs. In SVECs and RAECs TNFα increased total SOD activity 1.6 ± 0.2 times and 1.5 ± 0.1 times respectively, solely by increases in MnSOD activity. Catalase activity and GSH levels were not significantly different between the two cell types and were not increased by TNFα. Extracellular SOD, found only in RAECs may confer a first line of antioxidant defence not available to SVEC.

Funded by the Garfield-Weston Trust.

013 NOVEL GENETIC PREDISPOSITION TO CORONARY HEART DISEASE ASSOCIATED WITH THE PROTEIN C PATHWAY

*H. Ireland¹, C. J. Konstantoulas¹, S. E. Humphries¹, A. K. Ohlin², H. Mather³, A. H. Goodall⁴, J. Hogwood⁴, D. J. Stearns-Kurosawa⁵, S. Kurosawa⁵, C. T. Esmon⁶.¹University College London, London, UK; ²Lund University, Lund, Sweden; ³Ealing Hospital, London, UK; ⁴University of Leicester, Leicester, UK; ⁵Oklahoma Medical Research Foundation, OK, USA; ⁶Howard Hughes Medical Centre, MD, USA

The protein C (PC) pathway is both anti-thrombotic and anti-inflammatory. We have been studying genetic variants and proteins that may influence this pathway, to determine the consequences for coronary heart disease (CHD). We have previously identified risk for CHD associated with genetic variants in two proteins that contribute to the pathway; thrombomodulin (Tm)¹² and endothelial protein C receptor (EPCR)³ particularly in patients with metabolic syndrome or Type 2 diabetes. We have now measured plasma levels of three participants in this pathway, soluble Tm (n  =  550), soluble EPCR (n  =  495) and activated protein C (APC, n  =  240) in a cross-sectional study of Type 2 diabetes. A high percentage (43.8%) of individuals in the lowest quintile of the APC distribution had a family history of CHD (⩾1 family member), declining to the 4^th quintile (16.7%), chi-square p  =  0.022. Similar results were obtained for the lowest quintile of sTm, (36.0% decreasing to 22.0%, chi-squarer p  =  0.006). Low levels of APC were found in individuals homozygous for the rare allele of a variant site in EPCR that we have shown to be associated with high soluble EPCR levels (Ser219Gly) and increased risk for CHD. These results point to the defective PC pathway as a novel pathway for CHD risk.

Supported by The Coronary Thrombosis Trust.

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014 INCREASED LEVELS OF PROCOAGULANT FACTORS ASSOCIATED WITH INCREASED POTENTIAL FOR THROMBIN GENERATION IN PATIENTS WITH PREMATURE MYOCARDIAL INFARCTION

*J. R. Wright, J. Thompson, R. K. Singh, S. Brouilette, J. Pasi, N. J. Samani, A. H. Goodall.Department of Cardiovascular Sciences, University of Leicester, Leicester, UK

The extent of the haemostatic response may determine the risk of forming an occlusive thrombus, particularly in patients who suffer myocardial infarction (MI) at an early age, when the atherosclerotic burden is relatively low. While levels of individual haemostatic factors have been linked to increased risk of MI, measurement of the endogenous thrombotic potential (ETP) may better indicate the overall haemostatic response of an individual. By relating variations in individual coagulation factors to ETP insight may be provided into regulation of the overall thrombotic potential. ETP was measured by chromogenic assay in plasma from 162 individuals with an MI <50 years, and 186 age/sex-matched controls with no history of CHD. Individual coagulation factors and inhibitors were measured in clotting assays. Tissue Factor (TF) and Tissue Factor Pathway Inhibitor (TFPI) activity were measured by chromogenic assay for FXa generation. The mean ETP was significantly higher in the cases (118 ± 36 vs 101 ± 23%; p < 0.0001) as were TF:act and the components of the tenase complex (TF:act 11.3 ± 7.7 vs 9.3 ± 6.4 pmol/L; FIX 1.33 ± 0.26 vs 1.19 ± 0.21 IU; FVIII 1.61 ± 0.56 vs 1.15 ± 0.41 IU; FX 1.36 ± 0.22 vs 1.28 ± 0.18 IU respectively; p ⩽ 0.0003 for all). Levels of prothrombin were also higher in the cases, although not reaching significance (p  =  0.053), whereas FVII, FV, and FXI were not significantly different (p ⩾ 0.3 for all). Of the inhibitors, TFPI and protein C were lower in the cases (p ⩽ 0.04 respectively) but antithrombin did not differ between cases and controls (p  =  0.12). Statistical modelling demonstrated that in the case cohort TF, prothrombin and FIX contributed most to ETP (r > 0.38; p < 0.0001), whereas in the control group the level of prothrombin has the major effect (r  =  0.43; p < 0.0001). In conclusion subjects who suffer MI at an early age have a greater overall haemostatic response. This is linked to elevated levels of procoagulant factors that drive the propagation phase of coagulation.

Funded by the British Heart Foundation. RKS was a British Cardiac Society Research Fellow.

015 TIME DEPENDANT VARIATIONS IN RESISTANCE TO THROMBOLYSIS IN ARTERIAL THROMBI

*C. I. Jones, V. Chudasama, N. J. Samani, A. H. Gershlick, A. H. Goodall.Department of Cardiovascular Science, University of Leicester, Leicester, UK

Effective thrombolysis is known to decrease with the age of the thrombus. Understanding the changes that occur within the thrombus over time is important to improving the efficacy of thrombolytic therapy. Here we investigate changes in thrombus composition and resistance to thrombolysis over time in thrombi formed in vitro from whole blood under flow in a Chandler loop. Thrombi were formed from recalcified citrated blood for 0.5, 1.5, 3, 6, 12, and 24 hours under flow, then rinsed in Hank’s buffered saline (HBS), blotted, and weighed. Thrombus structure was visualised in formalin-fixed, paraffin-embedded sections using FITC-labelled antibodies against GPIIbIIIa, fibrinogen, and PAI-1. Thrombolysis was quantified by adding FITC-fibrinogen (50 μg) to the blood prior to re-calcification and measuring the release of FITC from the thrombus over the subsequent 24 hours, at 37°C, in the presence of tPA (15 μg/ml; Alterplase). There were no differences in the weight of thrombi formed for different times. However, thrombolysis decreased significantly as the age of the thrombus increased. Immunohistological examination revealed that the thrombi exhibited the features of arterial thrombi with a platelet rich head (PRH) containing foci of tightly aggregated platelets, discrete leucocytes aggregates, and tight fibrin bundles. Within the PHR the binding of anti-GPIIbIIIa antibody, a marker of platelet number, increased between 0.5 and 1.5 hrs (p  =  0.04) but then remained unchanged over time. Anti-fibrinogen antibody bound to the PRH in thrombi formed over 0.5 hours but failed to react with the PRH in thrombi formed for between 1.5 and 6 hours. However, fibrin was present in these regions as antibodies added to the blood prior to thrombus formation were strongly bond in the PRH region. Levels of PAI-1 detected in the PRH rose significantly (p  =  0.04) over the first 3 hours, then fell by 6 hours (p  =  0.02), rising again at 12 and 24 hours (p  =  0.01). This method enables examination of variations within thrombi over time, demonstrate both quantitative and qualitative differences in the presence of key determinants in resistance to thrombolysis.

Supported by the British Heart Foundation.

016 RAC1 IS ESSENTIAL FOR PLATELET LAMELLIPODIA AND AGGREGATE STABILITY UNDER FLOW

J. Owen, T. McCarty¹, M. K. Larson¹, *J. M. Auger¹, B. T. Atkinson¹, A. C. Pearce¹, S. Ruf³, R. B. Henderson³, V. L. J. Tybulewicz³, L. M. Machesky², S. P. Watson¹.¹Centre for Cardiovascular Sciences, University of Birmingham, Birmingham, UK; ²School of Biosciences, University of Birmingham, Birmingham, UK; ³Division of Immune Cell Biology, National Institute for Medical Research, London UK

The Rho family of small GTPases, which includes Rac, Rho, and Cdc42 proteins, play distinct roles in regulating actin assembly and motility. The aim of the present study was to investigate the role of Rac in mediating platelet morphologic changes upon a fibrinogen surface in the absence and presence of G protein-coupled receptor agonists. Furthermore, we sought to investigate the functional role of Rac on platelet adhesion to and platelet aggregation on collagen under shear conditions using a flow-based assay. We demonstrate that murine platelets form filopodia and undergo limited spreading on fibrinogen independently of Rac1 and Rac2. In the presence of thrombin or ADP, marked lamellipodia formation is observed on fibrinogen, which is abrogated in the absence of Rac1. Neither Rac1 nor Rac2 are required for aggregation or elevation of F-actin levels in thrombin-stimulated platelets, although an ADP-induced increase in F-actin content is Rac1 dependent. Rac1 is required for platelet aggregate stability at arterial rates of shear, as evidenced by a dramatic increase in platelet embolisation. The results demonstrate a central role for Rac1 in supporting formation of lamellipodia but not filopodia in thrombin-stimulated platelets on a fibrinogen surface. However, Rac is not required for thrombin-induced increase in F-actin. Furthermore, Rac is required for sustaining shear-resistant platelet aggregates under dynamic flow conditions. Taken together, this study demonstrates a cooperative role of integrin and G protein coupled receptors in regulating Rac activation and lamellipodia formation in platelets that enables them to withstand the high shear forces present in the vasculature.