Patrizia Ferroni

Platelet activation in type 2 diabetes mellitus

Summary.  The abnormal metabolic state that accompanies diabetes renders arteries susceptible to atherosclerosis, being capable of altering the functional properties of multiple cell types, including endothelium and platelets. In particular, an altered platelet metabolism and changes in intraplatelet signaling pathways may contribute to the pathogenesis of atherothrombotic complications of diabetes. A variety of mechanisms may be responsible for enhanced platelet aggregation. Among them, hyperglycemia may represent a causal factor for in vivo platelet activation, and may be responsible for nonenzymatic glycation of platelet glycoproteins, causing changes in their structure and conformation, as well as alterations of membrane lipid dynamics. Furthermore, hyperglycemia‐induced oxidative stress is responsible for enhanced peroxidation of arachidonic acid to form biologically active isoprostanes, which represents an important biochemical link between impaired glycemic control and persistent platelet activation. Finally, increased oxidative stress is responsible for activation of transcription factors and expression of redox‐sensitive genes leading to a phenotypic switch of endothelium toward an adhesive, pro‐thrombotic condition, initial platelet activation, adhesion and subsequent platelet aggregate formation. All this evidence is strengthened by the results of clinical trials documenting the beneficial effects of metabolic control on platelet function, and by the finding that aspirin treatment may even be more beneficial in diabetic than in high‐risk non‐diabetic patients. Attention to appropriate medical management of diabetic patients will have great impact on long‐term outcome in this high‐risk population.

Serum metalloproteinase 9 levels in patients with coronary artery disease: a novel marker of inflammation.

Background The finding that expression of metalloproteinases (MMPs) is induced in atherosclerotic plaques prone to rupture suggests the possibility that patients with atherosclerotic diseases would show enhanced blood levels of MMPs and that MMPs might represent a potential inflammatory risk factor for atherosclerosis. Therefore, the present study was aimed at verifying whether MMPs may represent sensitive markers of inflammation in patients with coronary artery disease. Methods MMP-2, MMP-9, interleukin (IL)-6, C-reactive protein (CRP), and fibrinogen levels were measured in blood samples obtained from 66 cases with previous acute myocardial infarction and 66 control subjects similar for age, sex, and major atherosclerotic risk factors but without history or evidence of atherothrombotic diseases. Results Biohumoral markers of inflammation and MMP-9 levels were significantly elevated in cases compared with controls (median values 40.6 versus 9.8 ng/mL; p < .0001), whereas MMP-2 levels did not differ between the two groups (median values 839 versus 873 ng/mL; p = .53). A direct correlation was found among MMP-9, CRP, IL-6, and fibrinogen levels. Conditional logistic regression analysis showed that MMP-9 is related to myocardial infarction (p = .006) even after adjusting for cardiovascular medications and CRP. Conclusion These findings suggest that measurement of serum MMP-9 levels may represent a novel marker of inflammation in patients with known coronary artery disease and might provide an index of plaque activity in this clinical setting.

Genetics of ischemic stroke, stroke-related risk factors, stroke precursors and treatments

Stroke remains a leading cause of death worldwide and the first cause of disability in the western world. Ischemic stroke (IS) accounts for almost 80% of the total cases of strokes and is a complex and multifactorial disease caused by the combination of vascular risk factors, environment and genetic factors. Investigations of the genetics of atherosclerosis and IS has greatly enhanced our knowledge of this complex multifactorial disease. In this article we sought to review common single-gene disorders relevant to IS, summarize candidate gene and genome-wide studies aimed at discovering genetic stroke risk factors and subclinical phenotypes, and to briefly discuss pharmacogenetics related to stroke treatments. Genetics of IS is, in fact, one of the most promising research frontiers and genetic testing may be helpful for novel drug discoveries as well as for appropriate drug and dose selection for treatment of patients with cerebrovascular disease.

Biomarkers of platelet activation in acute coronary syndromes

The most convincing evidence for the participation of platelets in arterial thrombosis in humans comes from studies of platelet activation in patients with acute coronary syndromes (ACS) and from trials of antiplatelet drugs. Both strongly support the concept that repeated episodes of platelet activation over the thrombogenic surface of a vulnerable plaque may contribute to the risk of death from coronary causes. However, the relation of in vivo platelet activation and adverse clinical events to results of platelet function tests remains largely unknown. A valuable marker of in vivo platelet activation should be specific, unaltered by pre-analytical artefacts and reproducibly measured by easily performed methods. This article describes current biomarkers of platelet activation in ACS, reviews their advantages and disadvantages, discusses their potential pitfalls, and demonstrates emerging data supporting the positive clinical implications of monitoring in vivo platelet activation in the setting of ACS.

Soluble P-selectin as a marker of in vivo platelet activation.

BACKGROUND Platelets are the major source of circulating sP-selectin. Elevated levels of this protein have been found in many atherothrombotic disorders. Thus, we investigated whether sP-selectin dosage might reflect platelet function in patients with risk factors for or with established cardiovascular diseases and whether its levels can be modulated by aspirin therapy. METHODS Plasma sP-selectin levels and light transmission platelet aggregometry (LTA) were analyzed in 152 outpatients. The effects of a 6-month aspirin therapeutic course on sP-selectin levels and LTA in 51 consecutive patients have been also investigated. RESULTS Significant correlations were observed between sP-selectin and Mx% LTA in response to epinephrine (p=0.022) and arachidonic acid (p=0.006), or between sP-selectin and collagen lag-phase (p=0.016). Multiple regression analysis showed that the only predictors of sP-selectin levels were platelet number (p<0.001) and collagen-induced lag-phase (p<0.01). Aspirin-treated patients showed a significant reduction of sP-selectin levels by 13% (p=0.021) which significantly correlated with collagen-induced lag-phase (p=0.005). CONCLUSIONS sP-selectin dosage could be proposed as a reliable marker of platelet activation in patients with major atherosclerotic risk factors either in the absence of clinically overt disease, and might represent a valid tool to asses in vivo platelet behavior.

Platelet function in health and disease: From molecular mechanisms, redox considerations to novel therapeutic opportunities

Increased oxidative stress appears to be of fundamental importance in the pathogenesis and development of several disease processes. Indeed, it is well known that reactive oxygen species (ROS) exert critical regulatory functions within the vascular wall, and it is, therefore, plausible that platelets represent a relevant target for their action. Platelet activation cascade (including receptor-mediated tethering to the endothelium, rolling, firm adhesion, aggregation, and thrombus formation) is tightly regulated. In addition to already well-defined platelet regulatory factors, ROS may participate in the regulation of platelet activation. It is already established that enhanced ROS release from the vascular wall can indirectly affect platelet activity by scavenging nitric oxide (NO), thereby decreasing the antiplatelet properties of endothelium. On the other hand, recent data suggest that platelets themselves generate ROS, which may evoke pro-thrombotic responses, triggering many biological processes participating in atherosclerosis initiation, progression, and complication. That oxidative stress may alter platelet function is conceivable when considering that antioxidants play a role in the prevention of cardiovascular disease, although the precise mechanism accounting for changes attributable to antioxidants in atherosclerosis remains unknown. It is possible that the effects of antioxidants may be a consequence of their enhancing or promoting the antiplatelet effects of NO derived from both endothelial cells and platelets. This review focuses on current knowledge regarding ROS-dependent regulation of platelet function in health and disease, and summarizes in vitro and in vivo evidence for their physiological and potential therapeutic relevance.

Vascular endothelial growth factor (VEGF-A) plasma levels in non-small cell lung cancer: Relationship with coagulation and platelet activation markers

Platelet activation, commonly found in lung cancer patients, may cause the release of angiogenic factors, such as vascular endothelial growth factor (VEGF-A). The present study was designed to investigate whether plasma VEGF-A levels were associated to different stages of non-small cell lung cancer (NSCLC). Moreover, sP-selectin, prothrombin fragment 1+2 (F1+2), thrombin-antithrombin III complex (TATc) and D-dimer levels were measured to test the hypothesis of an involvement of platelet and coagulation activation in tumor angiogenesis. VEGF-A, sP-selectin, F1+2, TATc and D-dimer levels were elevated in 65 patients with NSCLC, particularly in metastatic patients. sP-selectin (p <0.003) and F1+2 (p <0.005) levels were independently associated to VEGF-A. In addition, patients with positive levels of both sP-selectin and F1+2 had the highest levels of VEGF-A. In conclusion, our findings support the hypothesis that thrombin generation might induce platelet activation and VEGF-A release in NSCLC.

Contribution of Platelet-Derived CD40 Ligand to Inflammation, Thrombosis and Neoangiogenesis

CD40-CD40L interactions have been involved in inflammation and thrombosis. Several diseases are characterized by inflammation, hypercoagulability and increased prevalence of thromboembolic events. In the past decade, a series of preclinical and clinical studies has provided more insight into the pathogenetic mechanisms linking inflammatory mediators to the activation and regulation of the haemostatic system. In particular, the study of CD40-CD40L interactions has greatly contributed to understanding the role of platelets in a variety of pathophysiological conditions, including atherothrombosis, immuno-inflammatory diseases and, possibly, cancer. A wide variety of preclinical and clinical studies have generated clinical interest in the use of CD40L as a prognostic marker of thrombotic risk. However, the use of sCD40L in clinical studies requires reliable methods. For the correct interpretation of results, clinical and research laboratories and physicians must be aware of the limitations of immunoassays for this cytokine, which underlines the need for standardization of preanalytic conditions. This review will focus on biochemical evidence of CD40L involvement in platelet activation, contribution of platelet-derived CD40L to inflammation, thrombosis and neoangiogenesis, and possible methodological pitfalls regarding the appropriate specimen and preparation for laboratory evaluation of blood soluble CD40L as a biomarker in various human diseases characterized by underlying inflammation, such as atherothrombosis, cancer and immuno-inflammatory diseases.

Overexpression of the EMT driver brachyury in breast carcinomas: association with poor prognosis.

BACKGROUND The epithelial-mesenchymal transition (EMT) has been implicated as an important process in tumor cell invasion, metastasis, and drug resistance. The transcription factor brachyury has recently been described as a driver of EMT of human carcinoma cells. METHODS Brachyury mRNA and protein expression was analyzed in human breast carcinomas and benign tissues. The role of brachyury in breast tumor prognosis and drug resistance and the ability of brachyury-specific T cells to lyse human breast carcinoma cells were also evaluated. Kaplan-Meier analyses were used to evaluate the association between brachyury expression and survival. All statistical tests were two-sided. RESULTS The level of brachyury expression in breast cancer cells was positively associated with their ability to invade the extracellular matrix, efficiently form mammospheres in vitro, and resist the cytotoxic effect of docetaxel. A comparison of survival among breast cancer patients treated with tamoxifen in the adjuvant setting who had tumors with high vs low brachyury mRNA expression demonstrated that high expression of brachyury is associated as an independent variable with higher risk of recurrence (hazard ratio [HR] = 7.5; 95% confidence interval [CI] = 2.4 to 23.5; P = 5.14×10(-4)) and distant metastasis (HR = 15.2; 95% CI = 3.5 to 66.3; P = 3.01×10(-4)). We also demonstrated that brachyury-specific T cells can lyse human breast carcinoma cells. CONCLUSIONS The studies reported here provide the rationale for the use of a vaccine targeting brachyury for the therapy of human breast cancer, either as a monotherapy or in combination therapies.

Enhanced Interleukin-1β in Hypercholesterolemia Effects of Simvastatin and Low-Dose Aspirin

Background—This study was aimed at verifying whether activation of platelets might represent a source of interleukin (IL)-1&bgr; levels in hypercholesterolemia. To this purpose, we compared the effects of a short-term treatment with simvastatin or low-dose aspirin on circulating levels of this cytokine. Methods and Results—Fifty patients with hypercholesterolemia were randomly allocated to receive an 8-week therapeutic course of simvastatin 20 mg daily (n=25) or aspirin 100 mg daily (n=25). Baseline soluble (s) P-selectin directly correlated with IL-1&bgr; (P <0.0001) and C-reactive protein (CRP) (P <0.05) but not with von Willebrand factor, total cholesterol, or LDL cholesterol levels. Furthermore, sP-selectin (P <0.02) and IL-1&bgr; (P <0.0001) levels were independently related to CRP by multiple regression analysis. Both drugs were associated with comparable, significant reductions in IL-1&bgr; and sP-selectin. Simvastatin, but not aspirin treatment, significantly lowered CRP levels (P <0.05). The change in IL-1&bgr; levels correlated with the change in sP-selectin in patients randomized to either simvastatin (Rho, 0.42; P <0.05) or aspirin (Rho, 0.42; P <0.05). In contrast, the simvastatin-induced change in IL-1&bgr; did not correlate with the change in CRP levels. Conclusions—This study suggests that platelets might contribute to IL-1&bgr; production in hypercholesterolemia, thus providing an additional link between inflammation and the prothrombotic state in this setting.