Isobel Ford

The procoagulant potential of environmental particles (PM10)

Background and Aims: Epidemiology studies have shown that cardiovascular (CV) disease is primarily responsible for the mortality associated with increased pulmonary environmental particle (PM10) exposure. The mechanisms involved in PM10 mediated CV effects are unknown although changes in plasma viscosity and in the homoeostasis of blood coagulation have been implicated. It was hypothesised that PM10 exposure would result in an inflammatory response and enhance the activation of the extrinsic coagulation mechanisms in pulmonary and vascular cells in culture. Methods: Primary human monocyte derived macrophages and human umbilical cord vein endothelial, human alveolar type II epithelial (A549), and human bronchial epithelial (16HBE) cells were tested for their inflammatory and procoagulant response to PM10 exposure. IL-8, tissue factor (TF), and tissue plasminogen activator (tPA) gene expression and protein release, and coagulation enhancing ability of culture media were determined 6 and 24 hours following exposure. Results: The culture media from macrophages and 16HBE bronchial epithelial cells, but not A549 cells, exposed to PM10 had an enhanced ability to cause clotting. Furthermore, H2O2 also increased the clotting activity. Apoptosis was significantly increased in macrophages exposed to PM10 and LPS as shown by annexin V binding. TF gene expression was enhanced in macrophages exposed to PM10, and HUVEC tissue factor and tPA gene and protein expression were inhibited. Conclusions: These data indicate that PM10 has the ability to alter macrophage, epithelial, and endothelial cell function to favour blood coagulation via activation of the extrinsic pathway and inhibition of fibrinolysis pathways.

The role of immunoglobulin G and fibrinogen in platelet aggregation by Streptococcus sanguis

Previous work has shown that the type strain of Streptococcus sanguis, NCTC 7863, induces aggregation of normal platelets by a complement‐dependent mechanism. We investigated the roles of IgG and fibrinogen in the aggregation process. Plasma depleted of IgG by passage through protein A–sepharose failed to support platelet aggregation, as did plasma absorbed at 0°C with whole bacteria. However, absorption of plasma with a non‐aggregating strain of S. sanguis, SK96, did not remove aggregating activity for NCTC 7863. Supplementing 0°C‐absorbed plasma with purified IgG restored the aggregation supporting activity. A monoclonal antibody to the FcγRII receptor inhibited platelet aggregation by the bacteria, indicating a requirement for bacteria–IgG complexes interacting with the Fc receptor in platelet aggregation. There was a lag time to the onset of platelet aggregation of 7–19 min depending upon the platelet donor, but the length of this lag did not correlate with either total IgG concentration recognizing NCTC 7863 in subjects’ plasma, or the concentration any of the four IgG subclasses or with IgG avidity levels.

Platelet and coagulation activation markers in myeloproliferative diseases: relationships with JAK2 V6I7 F status, clonality, and antiphospholipid antibodies

Summary.  Background and objectives: Patients with myeloproliferative disease (MPD) have an increased risk of thrombosis. We studied markers of platelet and coagulation activation in a large cohort of patients with MPD (n = 118) and related this to Janus Kinase 2 (JAK2) V617 F mutation status, a marker of clonality, and the presence of antiphospholipid antibodies (APA), all of which have been associated with thrombosis in MPD. Methods: D‐dimer, thrombin–antithrombin complexes (TAT), prothrombin fragments 1 + 2 (F1+2), soluble E‐selectin (sE‐selectin), and soluble P‐selectin (sP‐selectin) levels were compared between patients and hypertensive controls (n = 127). Assays for lupus anticoagulant (LA), anticardiolipin antibodies (ACA), antibeta2 glycoprotein 1 antibodies (anti‐β2GP1), and antiprothrombin antibodies (α‐Pro) were also performed. The JAK2 V617F mutation status was determined in the cohort using amplification refractory mutation system (ARMS) polymerase chain reaction. Disease clonality was determined in 54 patients using the HUMARA assay. Results: sP‐selectin was significantly increased in patients with MPD (P ≤ 0.001). sP‐selectin levels were significantly elevated in JAK2 V617F‐positive patients compared to wild‐type (P = 0.006), or controls (P < 0.001). There was no correlation between proportion clonality and any activation marker. We found no significant difference in the incidence of APA between patients and controls (11% vs. 14%, P = 0.46), and no significant association between APA status and any activation marker. Conclusions: The JAK2 V617F mutation is associated with platelet activation, as measured by elevated sP‐selectin levels, in MPD. In contrast to previous reports, we found no excess of APA in patients with MPD.

Randomized clinical trial of the antiplatelet effects of aspirin–clopidogrel combination versus aspirin alone after lower limb angioplasty

There is a high risk of reocclusion after successful lower limb angioplasty. Platelets play a central role in this process. The aim of this study was to investigate the antiplatelet effect of a combination of aspirin and clopidogrel compared with aspirin alone in patients with claudication undergoing endovascular revascularization.

Mechanisms of platelet aggregation by Streptococcus sanguis, a causative organism in infective endocarditis

Summary. The ability of certain strains of Streptococcus sanguis to aggregate human platelets in vitro may be related to their virulence in the pathogenesis of infective endocarditis. We have studied the mechanisms of aggregation of human platelets by S. sanguis strain NCTC 7863. Platelet aggregation follows incubation of S. sanguis cells with platelet‐rich plasma from normal, healthy adults, after a lag of 7–19 min. Platelet aggregation was accompanied by 5‐hydroxytryptamine release and thromboxane B2 production. Aggregation was prevented by aspirin and by EDTA. Platelets from two patients with Glanzmanns thrombasthenia did not respond to bacteria. Fixed, washed platelets resuspended in normal plasma were not agglutinated by S. sanguis.

Platelet activation, myocardial ischemic events and postoperative non-response to aspirin in patients undergoing major vascular surgery.

Summary.  Objectives: Myocardial ischemia is the leading cause of postoperative mortality and morbidity in patients undergoing major vascular surgery. Platelets have been implicated in the pathogenesis of acute thrombotic events. We hypothesized that platelet activity is increased following major vascular surgery and that this may predispose patients to myocardial ischemia.Methods: Platelet function in 136 patients undergoing elective surgery for subcritical limb ischemia or infrarenal abdominal aortic aneurysm repair was assessed by P‐selectin expression and fibrinogen binding with and without adenosine diphosphate (ADP) stimulation, and aggregation mediated by thrombin receptor‐activating peptide and arachidonic acid (AA). Cardiac troponin‐I (cTnI) was performed.Results: P‐selectin expression increased from days 1 to 3 after surgery [median increase from baseline on day 3: 53% (range: −28% to 212%, P < 0.01) for unstimulated and 12% (range: −9% to 45%, P < 0.01) for stimulated]. Fibrinogen binding increased in the immediate postoperative period [median increase from baseline: 34% (range: −46% to 155%, P < 0.05)] and decreased on postoperative day 3 (P < 0.05). ADP‐stimulated fibrinogen binding increased on day1 (P < 0.05) and thereafter decreased. Platelet aggregation increased on days 1–5 (P < 0.05). Twenty‐eight (21%) patients had a postoperative elevation (> 0.1 ng mL−1) of cTnI. They had significantly increased AA‐stimulated platelet aggregation in the immediate postoperative period and on day 2 (P < 0.05), and non‐response to aspirin (48% vs. 26%, P = 0.036).Conclusions: This study has shown increased platelet activity and the existence of non‐response to aspirin following major vascular surgery. Patients with elevated postoperative cTnI had significantly increased AA‐mediated platelet aggregation and a higher incidence of non‐response to aspirin compared with patients who did not.

Evidence for the involvement of complement proteins in platelet aggregation by Streptococcus sanguis NCTC 7863.

We investigated the mechanisms of platelet aggregation by the type strain of Streptococcus sanguis (NCTC 7863). This species is one of the major aetiological agents of infective endocarditis. S. sanguis NCTC 7863 caused aggregation of normal human platelets in vitro following a lag period that varied between donors (7–19 min). Platelet aggregation was dependent on one or more plasma consistuents and all the necessary factors gradually became bound to the bacterial surface during the lag period. The length of the lag period was determined by the plasma of the donor and not by a feature of their platelets. Platelet aggregation by S. sanguis NCTC 7863 could be inhibited by heating plasma at 56°C, by treating plasma with cobra venom factor, or by incubating with soluble Complement Receptor 1, all of which inhibit or deplete complement. Complement activation required Mg2+, but not Ca2+ ions and the the cleavage fragment, Ba, of factor B was produced, indicating that the alternative pathway was operative. Zymosan‐ and S. sanguis‐induced aggregation showed similarities, including the same variability in lag times among donors, and absorption of plasma with zymosan prevented the plasma from supporting platelet aggregation by S. sanguis. C3, C9 and vitronectin were found to bind to S. sanguis NCTC 7863, but the latter two were present at very low levels on a non‐aggregating strain of S. sanguis, SK96. The rate of assembly of the C5b–9 complex on the NCTC 7863 bacterial surface correlated with the lag time. These data suggest a role for the complement pathway in platelet aggregation by the type strain of S. sanguis.

The role of platelets in infective endocarditis

There is growing evidence that platelets play an important role in the development of infective endocarditis (IE). This review focuses on interactions between bacteria and platelets. Many types of microorganism are capable of causing endocarditis. The oral streptococci, particularly Streptococcus sanguis and S. oralis, remain the most common causative bacteria in IE but Staphylococcus aureus is becoming an increasingly important agent. Several species of bacteria and fungi are able to cause platelet aggregation in vitro, and there are indications that this ability is associated with the production of severe disease. Different bacteria appear to utilise different mechanisms of aggregation, and mechanisms for adhesion may be distinct from those responsible for aggregation. At least one commonly studied strain of S. sanguis apparently utilises the hosts IgG and complement responses to induce platelet activation. IE may therefore be among the number of diseases in which immunological activation of platelets is thought to be of importance. Other bacterial strains have their own adhesins and agonists. The more virulent bacteria, notably the staphylococci, also possess proteolytic enzymes and may directly activate coagulation pathways, whereas less virulent, opportunistic pathogens, such as some streptococci and Candida albicans, may rely on interactions with host factors for colonisation and proliferation. It is proposed that platelets are involved in formation of the non-bacterial thrombotic vegetation (NBTV) and are necessary for the effective adhesion of bacteria to the NBTV, and that platelets also influence the persistence and clinical outcome of the disease through proliferation of the thrombotic vegetation. It is hypothesised that successful colonisation of the heart valves by a micro-organism depends on effective adhesion of the bacteria or fungi to platelets, in combination with the ability to induce platelet activation and aggregation.

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.

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.