Anti-endothelial antibodies interfere in apoptotic cell clearance and promote thrombosis in patients with antiphospholipid syndrome.

Antiphospholipid syndrome is an important cause of recurrent thrombotic events. The pathogenesis of the thrombosis remains unclear, but it has been suggested that anti-phospholipid Abs, which are laboratory markers for the disease and include species capable of binding to vascular endothelial cells, play an important role. We hypothesized that these anti-endothelial Abs promote thrombosis through interference with clearance of dying cells. We show that healthy endothelial cell monolayers effectively remove apoptotic endothelial cells, but this clearance is markedly inhibited by serum or IgG from patients with antiphospholipid syndrome and anti-endothelial Abs. In addition, patient sera or IgG opsonize apoptotic endothelial cells and cause enhanced Fc-mediated uptake by professional phagocytes. Importantly, the delayed clearance of apoptotic cells by healthy endothelial cells and the enhanced Fc-mediated macrophage uptake each result in procoagulant consequences, as judged by increased thrombin generation. The effects on apoptotic cell clearance were reproduced by a mAb derived from a patient with antiphospholipid syndrome, which binds to endothelial cells and is thrombogenic in experimental models. Taken together, our data support a novel, dual mechanism by which anti-endothelial Abs are prothrombotic in antiphospholipid syndrome by inhibiting removal of procoagulant apoptotic cells and by diverting their clearance to provoke inflammatory and prothrombotic changes in professional phagocytes.

Randomized controlled trial of aspirin and clopidogrel versus aspirin and placebo on markers of smooth muscle proliferation before and after peripheral angioplasty.

OBJECTIVE: In peripheral arterial disease (PAD) patients, a limiting factor in the success of percutaneous transluminal angioplasty (PTA) is the development of restenosis secondary to vascular smooth muscle cell (SMC) proliferation. Following endothelial damage and platelet activation, there is release of factors and adhesion molecules which affect SMC proliferation. The aim of this study was to determine the effect of combination antiplatelet therapy (clopidogrel and aspirin compared with aspirin and placebo) on the ability of plasma from PAD patients undergoing PTA to stimulate SMCs in vitro. We further aimed to investigate the effect of combination treatment on the levels of circulating adhesion molecules and factors, which are known to mediate SMC proliferation in experimental models. METHODS: Fifty patients were randomized to receive blinded clopidogrel or placebo, for thirty days, in addition to their daily 75 mg aspirin. To measure proliferative capacity, diluted plasma was incubated for 15 minutes with 24 hour-growth-arrested rat vascular smooth muscle cells, and extracellular regulated kinase (ERK)1/2 activation was analyzed by Western blotting at baseline, one hour pre-PTA, one hour, 24 hours and 30 days post-PTA. Plasma platelet-derived growth factor (PDGF), sE-selectin, intracellular adhesion molecule-1 (sICAM-1), and von Willebrand factor (vWF) were measured by ELISA, at the same five timepoints. Platelet activation was measured by flow cytometry of ADP-stimulated platelet fibrinogen binding at baseline and one hour post-PTA. RESULTS: ADP-stimulated platelet fibrinogen binding was significantly inhibited by clopidogrel before and after PTA. ERK 1/2 activation was significantly increased post-PTA in both the aspirin/clopidogrel and aspirin/placebo groups (P < .001). There was a statistically significant decrease in PDGF (P = .004), and increase in vWF (P = .026), following loading with clopidogrel. sICAM-1 levels significantly decreased (P = .016) in the aspirin/placebo group following PTA. There were no other significant changes and also there was no statistically significant difference between the two treatment groups for each of ERK 1/2, sICAM-1, sE-selectin, or vWF. CONCLUSIONS: This is the first study to show in-vitro ERK 1/2 activation (a surrogate marker of SMC proliferation) increases post-PTA. Combination antiplatelet therapy had no significant effect on this, although it did reduce PDGF. Further work is required to evaluate potential therapeutic treatments, which may reduce peripheral PTA-induced smooth muscle cell activation. CLINICAL RELEVANCE: High rates of restenosis remain the major limitation of peripheral arterial angioplasty and stenting.The restenotic lesion occurs secondary to platelet activation, released circulating factors, and subsequent smooth musclecell proliferation and migration into the intima. Methods to limit the restenotic lesion are poorly understood. This paperinvestigates the effect of PTA on smooth muscle cell activation and the release of factors in plasma which mediate SMCproliferation. It also examines the effect of combination antiplatelet therapy as a potential therapeutic strategy.

Effect of Antiplatelet Therapy (Aspirin + Dipyridamole Versus Clopidogrel) on Mortality Outcome in Ischemic Stroke.

The optimal regimen of antiplatelet therapy for secondary prevention in noncardioembolic ischemic stroke remains controversial. We aimed to determine which regimen was associated with the greatest reduction in adverse outcomes. We analysed prospectively collected data from the Norfolk and Norwich University Hospital Stroke Register. The sample population consisted of 3,572 participants (mean age 74.96 ± 12.67) with ischemic stroke, who were consecutively admitted between 2003 and 2015. Patients were placed on one of three antiplatelet regimens at hospital discharge; aspirin monotherapy, aspirin plus dipyridamole and clopidogrel. Clopidogrel and aspirin plus dipyridamole were compared to aspirin. A direct comparison between clopidogrel and aspirin plus dipyridamole was also performed. Outcomes included all-cause mortality and a combined end point of all-cause mortality and incidence of major adverse cardiac events (stroke or myocardial infarction). Cox-regression models adjusted for potential confounders at the following time periods after discharge; 0 to 90 days, 91 to 365 days, and 1 to 3 years. Aspirin plus dipyridamole was associated with a lower risk of mortality at 0 to 90 days; hazard ratio (HR) 0.62 (0.43 to 0.91). Clopidogrel was associated with a lower risk of mortality at 1 to 3 years; HR of 0.39 (0.26 to 0.60). Similar HRs were observed for the corresponding time points in the composite outcome. In conclusion, patients with noncardioembolic stroke may gain maximum benefits from aspirin plus dipyridamole initially (≤1 year) with a subsequent switch to clopidogrel, with regard to mortality and major adverse cardiac eventsoutcomes.

Haemostasis, inflammation and renal function following exercise in patients with intermittent claudication on statin and aspirin therapy.

BACKGROUND: Previous studies have suggested that exercise in patients with intermittent claudication (IC) may induce a systemic thrombo-inflammatory response. The effect of secondary prevention therapy on this response is unknown. This study aimed to investigate the effects of treadmill exercise on markers of coagulation activation, inflammation and renal function in patients with IC, receiving aspirin and statin therapy compared to healthy controls. METHODS: Samples were taken before, immediately and 1 hour after exercising on a treadmill in 20 patients with IC and 20 healthy volunteers. Interleukin-6 (IL-6), thrombin-anti-thrombin complex (TAT) and fibrin D-dimer were measured by ELISA. High sensitivity CRP (HsCRP) and urinary albumin were measured via a nephelometric technique, urinary protein via a turbidometric assay and N-acetyl-beta-D-glucosaminidase (NAG) via a colorimetric assay. RESULTS: Elevated baseline levels of Hs-CRP, IL-6, white cell counts, D-dimer and urinary NAG occurred in patients with IC compared to volunteers (p > 0.05). Following exercise there was no increase in Hs CRP or IL-6. D-dimer levels significantly increased following exercise in the patients and volunteers. TAT levels increased immediately after exercise in the patient group only and were significantly increased at 1 hour in both patients and volunteers. A transient rise in the protein creatinine ratio occurred in both groups (p < 0.007), and in albumin creatinine ratio in the patient group. There was no change in urinary NAG. CONCLUSION: Elevated markers of inflammation occurred in patients with IC on statin and aspirin therapy but these did not increase following exercise. However, acute exercise resulted in a prothrombotic state evident in both groups, although this was more prolonged in patient with IC. The clinical significance of these findings in patients who are known to be at an increased risk of cardiac and other thrombotic event are unclear.

Effect of hypobaric hypoxia, simulating conditions during long-haul air travel, on coagulation, fibrinolysis, platelet function, and endothelial activation.

CONTEXT: The link between long-haul air travel and venous thromboembolism is the subject of continuing debate. It remains unclear whether the reduced cabin pressure and oxygen tension in the airplane cabin create an increased risk compared with seated immobility at ground level. OBJECTIVE: To determine whether hypobaric hypoxia, which may be encountered during air travel, activates hemostasis. DESIGN, SETTING, AND PARTICIPANTS: A single-blind, crossover study, performed in a hypobaric chamber, to assess the effect of an 8-hour seated exposure to hypobaric hypoxia on hemostasis in 73 healthy volunteers, which was conducted in the United Kingdom from September 2003 to November 2005. Participants were screened for factor V Leiden G1691A and prothrombin G20210A mutation and were excluded if they tested positive. Blood was drawn before and after exposure to assess activation of hemostasis. INTERVENTIONS: Individuals were exposed alternately (> or =1 week apart) to hypobaric hypoxia, similar to the conditions of reduced cabin pressure during commercial air travel (equivalent to atmospheric pressure at an altitude of 2438 m), and normobaric normoxia (control condition; equivalent to atmospheric conditions at ground level, circa 70 m above sea level). MAIN OUTCOME MEASURES: Comparative changes in markers of coagulation activation, fibrinolysis, platelet activation, and endothelial cell activation. RESULTS: Changes were observed in some hemostatic markers during the normobaric exposure, attributed to prolonged sitting and circadian variation. However, there were no significant differences between the changes in the hypobaric and the normobaric exposures. For example, the median difference in change between the hypobaric and normobaric exposure was 0 ng/mL for thrombin-antithrombin complex (95% CI, -0.30 to 0.30 ng/mL); -0.02 [corrected] nmol/L for prothrombin fragment 1 + 2 (95% CI, -0.03 to 0.01 nmol/L); 1.38 ng/mL for D-dimer (95% CI, -3.63 to 9.72 ng/mL); and -2.00% for endogenous thrombin potential (95% CI, -4.00% to 1.00%). CONCLUSION: Our findings do not support the hypothesis that hypobaric hypoxia, of the degree that might be encountered during long-haul air travel, is associated with prothrombotic alterations in the hemostatic system in healthy individuals at low risk of venous thromboembolism.

Coming safely to a stop: a review of platelet activity after cessation of antiplatelet drugs.

The platelet P2Y12 antagonists are widely used, usually in combination with aspirin, to prevent atherothrombotic events in patients with acute coronary syndromes during percutaneous coronary intervention and after placement of arterial stents. Inhibition by clopidogrel or prasugrel lasts for the lifetime of the affected platelets and platelet haemostatic function gradually recovers after stopping the drug, as new unaffected platelets are formed. The optimal durations for dual antiplatelet therapy are prescribed by clinical guidelines. Continuation beyond the recommended duration is associated with an increased mortality, mainly associated with major bleeding. Fear of a 'rebound' of prothrombotic platelet activity on stopping the drug has provoked much discussion and many studies. However, review of the available literature reveals no evidence for production of hyper-reactive platelets after cessation of clopidogrel in patients who are stable. Any increase in acute coronary and other vascular events after stopping seems most likely therefore to be due to premature discontinuation or disruption of treatment while thrombotic risk is still high. No difference in rebound was found with the newer P2Y12 inhibitors, although ticagrelor and prasugrel are more potent platelet inhibitors than clopidogrel. Recent randomized controlled trials confirm it is safe to stop the thienopyridine and continue with aspirin alone in most patients after the duration of treatment recommended by the guidelines. Decisions on when to stop therapy in individuals, however, remain challenging and there is a growing rationale for platelet testing to assist clinical judgement in certain situations such as patients stopping dual antiplatelet therapy before surgery or in individuals at highest bleeding or thrombotic risk.

Intraoperative changes in platelet function in relation to moderate haemorrhage.

BACKGROUND: Haemorrhage is associated with haemostatic dysfunction. Previous studies have focused on coagulation factors, but platelet function plays an equally important role. The time course of alterations in platelet function in relation to injurious stimuli is not known. AIM: To evaluate short-term, intra-operative changes in platelet function, by whole blood impedance aggregometry in patients undergoing hip arthroplasty. The primary outcome was platelet aggregation in response to adenosine diphosphate (ADP) stimulation. We also evaluated other agonists, and the feasibility of conducting platelet aggregometry measurement in the operating theatre. METHODS: Patients undergoing primary cemented hip arthroplasty had six peri-operative arterial blood samples analysed at pre-set stages of the operation, using the Multiplate Impedance Aggregometer. Four agonists were used: ADP, TRAP, Collagen and Arachidonic Acid. RESULTS: There was a statistically significant change (p<0.05, ANOVA) in platelet response to ADP over the course of the operations. The trend demonstrated an initial decrease in responsiveness, followed by increased platelet responsiveness in the later stages. Other agonists (TRAP, COL, ASPItest) demonstrated a similar pattern of changes. Of 360 tests conducted, 12 (3.3%) had to be re-run due to poor intra-assay variability. Satisfactory values were obtained on the second attempt in all 12 samples. CONCLUSION: Platelet function, as measured by impedance aggregometry, changes in response to a surgical stimulus involving blood loss. The clinical significance of these changes, and the potential of manipulating them for therapeutic purposes, remains to be elucidated.

Supplementation with a 9c,11t-rich conjugated linoleic acid blend shows no clear inhibitory effects on platelet function in healthy subjects at low and moderate cardiovascular risk: a randomized controlled trial.

SCOPE: The 9cis,11trans-conjugated linoleic acid (9c,11t-CLA) is reported to have anti-atherogenic properties in animal models and to modulate protein expression in unstimulated human platelets in vivo. Platelet function was therefore investigated after dietary supplementation with 9c,11t-CLA enriched oil (CLA80:20) in a randomized, baseline-controlled cross-over trial. METHODS AND RESULTS: Forty-three healthy adults at low to moderate risk of cardiovascular disease received 4 g/day of CLA80:20 or placebo for two weeks each. Platelet function, inflammation, and endothelial activation were assessed before and after each phase. Compared with placebo, supplementation had no significant effects on platelet function measured by Platelet Function Analyzer-100. Inhibitory effects on collagen-induced aggregation were sex-dependent (p = 0.005) that reached significance only in women (p = 0.045). Thrombin receptor-activating peptide (TRAP)-induced P-selectin expression was higher after supplementation in all subjects (p = 0.017). TRAP-induced platelet fibrinogen binding was also dependent on sex (p = 0.015), with fibrinogen binding after CLA80:20 being higher in males (p = 0.035). Plasma monocyte chemoattractant protein-1 was higher (p = 0.041) after CLA80:20. CONCLUSION: No clear evidence was found for inhibition or activation of platelet function as well as inflammation by CLA80:20 in a low to moderate cardiovascular risk group.

Markers of endothelial activation and atherothrombosis in women with history of preeclampsia or gestational hypertension.

Women who have had preeclampsia (PE) or gestational hypertension (GH) exhibit relatively high rates of circulatory diseases. PE is a disease associated with inflammation and vascular endothelial dysfunction. We therefore hypothesised that women with a history of PE or GH might have abnormal levels of markers of endothelial activation or inflammation, reflecting either an innate predisposition to preeclampsia or changes induced by the eclamptic process. Levels of von Willebrand factor, fibrinogen and C-reactive protein were compared in 392 women with a history of PE between 1951 and 1970, 297 women with a history of GH and 163 matched controls. Although no significant differences between those with either PE or GH and controls were noted, subjects with a history of PE had significantly higher CRP values than those with GH. No significant differences were found when the three groups were compared for von Willebrand factor or fibrinogen. Overall, the data do not support our hypothesis. In addition, our data document increasing von Willebrand factor levels increase with age, which may help explain the age dependent increase in venous or arterial thrombosis. Moderate alcohol consumption was also associated with lower levels of inflammatory markers.

A randomized controlled trial of platelet activity before and after cessation of clopidogrel therapy in patients with stable cardiovascular disease.

OBJECTIVES: The aim of this randomized placebo-controlled trial was to determine if withdrawing clopidogrel therapy leads to increased platelet activity compared with pre-treatment values in patients with stable coronary artery or peripheral arterial disease. BACKGROUND: Reports of increased cardiovascular events after planned cessation of clopidogrel therapy have raised concerns over the possible existence of a rebound in platelet activity. METHODS: In all, 171 patients receiving established aspirin therapy were randomly assigned to placebo or clopidogrel (75 mg daily) for 28 days. Blood samples were taken at pre-treatment baseline, on treatment just before discontinuation of study drug, and on days 7, 14, and 28 after discontinuation. The primary outcome measure was adenosine diphosphate (ADP)-stimulated platelet fibrinogen binding. Six secondary outcomes were assessed: ADP-stimulated platelet P-selectin, unstimulated platelet fibrinogen binding, and light transmission aggregometry with ADP 5 and 10 µmol/l recorded at maximum and at 6 min. RESULTS: The ADP-stimulated platelet fibrinogen binding, P-selectin expression, and platelet aggregation were lower on treatment with clopidogrel compared with baseline (p < 0.0001), but returned to baseline levels by 7 days after discontinuation. Mixed model analyses excluding the on-treatment timepoint showed no overall differences between the clopidogrel and placebo groups (p > 0.05). Furthermore, there was no evidence of an interaction between platelet inhibition over time and treatment allocation. CONCLUSIONS: This trial found no evidence for rebound of platelet activity to above baseline after stopping clopidogrel in patients with stable coronary artery disease or peripheral arterial disease. (Is Cessation of Clopidogrel Therapy Associated With Rebound of Platelet Activity in Stable Vascular Disease Patients?; ISRCTN77887299/77887299).

Thrombin-anti-thrombin levels and patency of arterio-venous fistula in patients undergoing haemodialysis compared to healthy volunteers: a prospective analysis.

BACKGROUND: Patients on haemodialysis (HD) are at an increased risk of sustaining thrombotic events especially to their vascular access which is essential for maintenance of HD. OBJECTIVES: To assess whether 1) markers of coagulation, fibrinolysis or endothelial activation are increased in patients on HD compared to controls and 2) if measurement of any of these factors could help to identify patients at increased risk of arteriovenous (AVF) access occlusion. PATIENTS/METHODS: Venous blood samples were taken from 70 patients immediately before a session of HD and from 78 resting healthy volunteers. Thrombin-antithrombin (TAT), D-dimer, von Willebrand factor (vWF), plasminogen activator inhibitor-1 antigen (PAI-1) and soluble p-selectin were measured by ELISA. C-reactive protein (hsCRP) was measured by an immunonephelometric kinetic assay. Determination of the patency of the AVF was based upon international standards and was prospectively followed up for a minimum of four years or until the AVF was non-functioning. RESULTS: A total of 70 patients were studied with a median follow-up of 740 days (range 72-1788 days). TAT, D-dimer, vWF, p-selectin and hsCRP were elevated in patients on HD compared with controls. At one year follow-up, primary patency was 66% (46 patients). In multivariate analysis TAT was inversely associated with primary assisted patency (r = -0.250, p = 0.044) and secondary patency (r = -0.267, p= 0.031). CONCLUSIONS: The novel finding of this study is that in patients on haemodialysis, TAT levels were increased and inversely correlated with primary assisted patency and secondary patency. Further evaluation is required into the possible role of TAT as a biomarker of AVF occlusion.

Effect of Omega-3 fatty acid supplementation on markers of platelet and endothelial function in patients with peripheral arterial disease.

OBJECTIVE: Omega-3 fatty acids have been shown to reduce platelet and endothelial activation in patients with or at risk of cardiac disease. We aimed to determine if Omega-3 fatty acid supplementation in addition to best medical therapy can reduce the increased platelet and endothelial activity that is present in patients with intermittent claudication. METHODS: One hundred and fifty patients who were receiving aspirin and statin therapy were recruited into a randomised cross-over double blind study involving 6 week supplementation with OMACOR fish oil (850-882 mg eicosapentaenoic and docosahexaenoic acid) versus placebo. A 12 week washout period occurred between treatments. Patients with diabetes were excluded. For each outcome a random effects model was fitted in which treatment and period were fixed effects and patients were random effects. RESULTS: Omega-3 supplementation had no effect on the primary outcome measure von Willebrand factor. Similarly Omega-3 supplementation resulted in no change in unstimulated or stimulated P-selectin expression and fibrinogen binding, or platelet aggregation (Ultegra point of care). Pulse wave velocity was also unchanged. High-sensitivity C-reactive protein, s-ICAM and IL-6 were also unchanged. CONCLUSION: Supplementation with Omega-3 fatty acids had no affect on platelet and endothelial activation or markers of inflammation in patients with peripheral arterial disease.

Effect of supplementation with an 80:20 cis9,trans11 conjugated linoleic acid blend on the human platelet proteome.

SCOPE: The dietary fatty acid cis9,trans11 conjugated linoleic acid (cis9,trans11 CLA) has been shown to modify the function of endothelial cells, monocytes, and platelets, all of which are involved in the development of atherosclerosis. Potential mechanisms for the platelet effects have not been assessed previously. In this study, we assessed how supplementation of the diet with an 80:20 cis9,trans11 CLA blend affects the platelet proteome. METHODS AND RESULTS: In a double-blind, randomized, placebo-controlled, parallel-group trial, 40 overweight but apparently healthy adults received either 4 g per day of cis9,trans11 CLA-enriched oil or placebo oil, consisting of palm oil and soybean oil, for 3 months. Total platelet proteins were extracted from washed platelets, separated using two-dimensional gel electrophoresis and differentially regulated protein spots were identified by LC-ESI-MS/MS. Supplementation with the CLA blend, compared with placebo, resulted in significant alterations in levels of 46 spots (p < 0.05), of which 40 were identified. Network analysis revealed that the majority of these proteins participate in regulation of the cytoskeleton and platelet structure, as well as receptor action, signaling, and focal adhesion. CONCLUSION: The platelet proteomics approach revealed novel insights into regulation of cellular biomarkers of atherogenic and thrombotic pathways by an 80:20 cis9,trans11 CLA blend.

Prothrombotic changes in platelet, endothelial and coagulation function following hemodialysis.

PURPOSE: Patients on hemodialysis (HD) have an increased risk of thrombotic events, including myocardial infarction and vascular access thrombosis. The study hypothesis is that a single session of dialysis leads to platelet, endothelial and coagulation activation. Our aim is to determine the effect of a single HD session on prothrombotic vascular biomarkers before and after a single session of hemodialysis. METHODS: Blood samples were taken from the vascular access of 55 patients immediately before and after a hemodialysis session. Platelet function was assessed by (1) flow cytometric measurement of P-selectin expression and fibrinogen binding +/- ADP stimulation, (2) Ultegra rapid platelet function assay (RPFA) using the agonists thrombin receptor activating peptide (TRAP) and arachidonic acid (AA), (3) soluble P-selectin, and (4) soluble CD40L. Coagulation (thrombin-antithrombin III [TAT] and D-dimer), endothelial von Willebrand factor (vWF) and high sensitivity C-Reactive protein (hsCRP) were assessed by ELISA. RESULTS: Unfractionated heparin was given to all patients during dialysis and 30 patients (55%) were on antiplatelet agents. Post-hemodialysis there were significant increases in unstimulated platelet P-selectin (p=.037), stimulated P-selectin (p<.001), soluble P-selectin (p<.001) and soluble CD40L (p=.036). Stimulated platelet fibrinogen binding was increased post-hemodialysis (p<.001) but unstimulated fibrinogen binding was unchanged. TRAP- (p<.001] and AA-(p=.009) stimulated aggregation were reduced post-hemodialysis. There were increases post-hemodialysis in TAT (p<.001), D-dimer (p<.001), vWF (p<.001) and hsCRP (p=.011). CONCLUSION: This study has shown that despite heparin therapy, a single session of HD induced increases in platelet, endothelial, and coagulation activation. More effective medical strategies to reduce the prothrombotic state of patients on hemodialysis should be investigated.

Aspirin failure in patients presenting with acute cerebrovascular ischaemia.

Aspirin is the most commonly used antiplatelet drug for prevention of ischaemic stroke. In order to determine the prevalence and nature of aspirin failure, we studied 51 adults admitted with suspected ischaemic stroke and already prescribed daily aspirin. Within 48 hours (h) of onset, blood and urine samples were collected to assess platelet aggregation, activation and aspirin response by a range of methods. All tests were then repeated on a second sample taken 24 h after witnessed administration of 75 mg or 150 mg aspirin. At entry to the study, incomplete response to aspirin, measured by arachidonic acid (AA)-stimulated platelet aggregation, was found in 43% of patients. Following in-hospital aspirin administration, there was a significant decrease in AA-aggregation (p=0.001) suggesting poor adherence to therapy prior to admission. However, residual aggregation (10-15%) persisted in 11 subjects - suggesting alternative causes. In incomplete responders on admission, platelet aggregation with adenosine diphosphate (ADP) was significantly higher compared with responders (p<0.05) but there were no significant differences in collagen aggregation, platelet fibrinogen binding or P-selectin expression, plasma von Willebrand factor, fibrinogen, high-sensitivity C-reactive protein, or the urinary metabolite, 11-dehydro-TxB2. Incomplete platelet inhibition is common around the time of acute cerebrovascular ischaemic events in patients prescribed aspirin. Up to 50% of these observations appear due to incomplete adherence to aspirin therapy. Intervention studies are required to determine the clinical relevance of measured platelet response to aspirin in terms of outcome, and the effectiveness of improved pharmacotherapy for stroke prevention.

Platelet activation increases with the severity of peripheral arterial disease: implications for clinical management.

INTRODUCTION: Patients with peripheral arterial disease (PAD) have increased mortality from cardiovascular events compared with age and sex matched controls Platelets play a major role in atherosclerosis and thrombotic vascular events. Platelet reactivity is increased in patients with PAD compared with healthy controls. We aimed to determine the relationship, if any, between platelet activation and severity of disease. METHODS AND RESULTS: One hundred eighty-two patients with intermittent claudication (IC) or subcritical limb ischemia (SLI), defined as the presence of rest pain or ulceration, had the following investigations performed: platelet P-selectin expression and bound fibrinogen by flow cytometric analysis and platelet aggregation using the rapid platelet function assay with arachidonic acid (AA) and thrombin receptor activation peptide (TRAP) as agonists. Patients with SLI compared with IC had significantly enhanced ADP stimulated P-selectin expression (median 42.45% [inter-quartile range 33.32% to 58.5%] vs 35.2% [26.07% to 46.32%], P = .002) and bound fibrinogen (73.7% [54.3% to 83.2%] vs 63.7% [43.8% to 76.5%], P = .001). TRAP stimulated aggregation was higher (207 [153 to 238] PAU vs 183[155 to 199] PAU, P = .04) but AA mediated aggregation was not significantly different. An ankle-brachial pressure index (ABPI) of less than 0.6 was associated with increased ADP stimulated P-selectin and bound fibrinogen (P < .05). ABPI correlated inversely with ADP stimulated P-selectin expression (r = -0.228, P = .003), ADP stimulated fibrinogen binding (r = -0.156, P = .043) and TRAP stimulated aggregation (r = -0.179, P = .04). CONCLUSION: We have demonstrated for the first time that progression of severity of PAD is not only reflected by symptoms, signs, and ABPI but also by increased platelet activity as assessed by both flow cytometry and aggregation. As patients with more severe PAD have increased cardiovascular mortality, our findings suggest that new strategies for platelet inhibitory therapy are indicated in these patients.

The influence of anti-endothelial/antiphospholipid antibodies on fibrin formation and lysis on endothelial cells.

The prothrombotic mechanisms associated with antiphospholipid antibodies remain incompletely defined. Antibody binding to endothelial cells in vitro is a feature of antiphospholipid antibody-positive sera. We hypothesised that impairment of endothelium-dependent fibrinolysis by antiphospholipid/anti-endothelial antibodies is a contributory factor in the pathogenesis of thrombosis. We also aimed to confirm the displacement of annexin-V from endothelial cells and enhanced fibrin formation. Binding of immunoglobulin (Ig) from antiphospholipid antibody-positive sera to endothelial cells was examined using a cell-based enzyme-linked immunosorbent assay. Effects on fibrin formation and lysis were examined on cultured endothelial cell monolayers. Plasminogen activator inhibitor-1 (PAI-1) was assayed in supernatants. We confirmed antibody binding to endothelial cells. With four of 14 antiphospholipid antibody-positive sera there was some prolongation of fibrin clot lysis time, consistent with impairment of endothelial fibrinolytic activity. Secretion of PAI-1 was significantly correlated with clot lysis time on endothelial cell monolayers incubated with antiphospholipid/anti-endothelial antibody-positive sera, but not with control sera. IgG from antiphospholipid antibody-positive sera had little effect on endothelial cell surface annexin-V expression. We conclude that impaired endothelial fibrinolysis is a potential prothrombotic mechanism in subjects with antiphospholipid antibodies. We were unable to confirm enhanced displacement of annexin-V from endothelium by antiphospholipid antibodies.

Clopidogrel has no effect on D-dimer and thrombin-antithrombin III levels in patients with peripheral arterial disease undergoing peripheral percutaneous transluminal angioplasty.

OBJECTIVE: Coagulation activation markers are significantly elevated in patients with peripheral arterial disease compared with healthy controls. The more severe the disease, the higher the markers. Increased coagulation activation may contribute to the disease process and the risk of complications in patients with peripheral arterial disease, particularly after endovascular intervention. Animal studies have shown that clopidogrel significantly inhibits coagulation activation. The aim of this study was to determine whether combination of aspirin and clopidogrel affects thrombin-antithrombin III and D-dimer in patients with intermittent claudication undergoing angioplasty, compared with aspirin alone. METHODS: This was a double blind, randomized placebo-controlled trial conducted in a vascular unit in a tertiary referral center. One hundred thirty-two patients with intermittent claudication were randomized to clopidogrel and aspirin or placebo and aspirin, with a loading dose 12 hours before endovascular intervention. D-dimer and thrombin-antithrombin III (TAT) levels were measured using enzyme-linked immunosorbent assay at baseline, 1 hour before, and 1 hour, 24 hours, and 30 days after intervention in 103 patients who underwent endovascular intervention. RESULTS: There was a significant rise in D-dimer levels at 1 hour and 24 hours after angioplasty in both groups (placebo group: 63.69, 141.45, 122.18 ng/mL; clopidogrel group: 103.79, 159.95, 134.69 ng/mL), but no difference between the two groups (P = .514). Similarly there was a significant rise in TAT levels at 1 hour after angioplasty in both groups (placebo group: 2.93, 6.16 microg/L; clopidogrel group: 3.39, 5.27 microg/L), with no significant difference between the two groups (P = .746). CONCLUSION: Endovascular intervention results in a significant increase in TAT and D-dimer. The addition of clopidogrel to aspirin has no effect on TAT and D-dimer before or after endovascular intervention.

Helicobacter pylori seropositivity is associated with enhanced platelet activation in patients with intermittent claudication.

OBJECTIVE: Patients with intermittent claudication have a significantly increased risk of mortality from cardiovascular and cerebrovascular causes. Helicobacter pylori infection and abnormal platelet function have been shown to be associated with atherosclerosis as well as with acute ischemic events. The aim of this study was to assess for the first time the relation between H pylori serology status, platelet activation, and endothelial injury in patients with intermittent claudication. Design of study : A prospective observational study of 125 patients with intermittent claudication suitable for angioplasty was conducted at the Vascular Unit of the Aberdeen Royal Infirmary. Main outcome measures Main outcome measures were (1) H pylori serology using ELISA kit for immunoglobulin G antibody to H pylori and (2) whole blood flow cytometric analysis of resting platelet P-selectin expression and fibrinogen binding as measures of platelet activation. Results are presented as platelet percentage. von Wildebrand factor levels were measured using ELISA as a marker of endothelial injury. Carstair deprivation scores were calculated for all patients. RESULTS: H pylori serology was positive in 62 patients (49.6%), negative in 56 (44.8%) and equivocal in 7 (5.6%). Median P-selectin expression was significantly increased in H pylori-positive patients compared with seronegative patients (0.815 vs 0.65; P =.039). Median platelet fibrinogen binding was higher in seropositive patients, but this failed to reach statistical significance (2.135 vs 1.85%; P =.11). There was no difference in von Wildebrand factor levels between the two groups (P =.51). There was no difference in socioeconomic status between the two groups. CONCLUSION: Patients with intermittent claudication who are H pylori positive show enhanced platelet activation that does not appear to be mediated by means of endothelial cell injury.