Gemma Arderiu

Evaluation of effects of rofecoxib on platelet function in an in vitro model of thrombosis with circulating human blood.

Background  Cyclooxygenase (COX)‐2‐selective non‐steroidal anti‐inflammatory drugs have been used for anti‐inflammatory therapy. However, it has also been described that they may increase risk of cardiovascular events.

Differential Expression of Proteins From Cultured Endothelial Cells Exposed to Uremic Versus Normal Serum

BACKGROUND Deficient hemostasis and accelerated atherosclerosis coexist in patients with chronic kidney disease. Endothelial dysfunction may be involved in the high incidence of atherothrombotic events in these patients. We established an in vitro model of endothelial dysfunction by exposing endothelial cells to uremic media and applied a proteomic approach to characterize endothelial cell dysfunction in uremia. STUDY DESIGN Cross-sectional study. SETTING AND PARTICIPANTS Serum samples from 8 patients with chronic kidney disease on hemodialysis treatment were collected. PREDICTOR Exposure of cultured endothelial cells to normal and uremic serum. OUTCOME AND MEASUREMENTS: Proteins from lysed cells were characterized by isoelectric point and molecular weight by using 2-dimensional gel electrophoresis. Spots were visualized by means of silver staining and identified by using mass spectrometry. RESULTS Identification of the most prominent proteins showed molecules related to inflammation (high mobility group box 1, aldose reductase, and proteasome components) and oxidative stress (superoxide dismutase and glutathione peroxidase), both associated with chronic kidney disease. These changes may be caused by activation of the nuclear factor-kappaB transcription factor. Changes in expression of cytoskeletal proteins (destrin and vimentin) also were detected. LIMITATIONS In vitro study. CONCLUSION Proteomic techniques proved to be a powerful tool to investigate endothelial dysfunction in uremia. A more exhaustive analysis will provide answers and potential therapeutic targets in the near future.

Inhibition of Cytoskeletal Assembly by Cytochalasin B Prevents Signaling Through Tyrosine Phosphorylation and Secretion Triggered by Collagen but Not by Thrombin

Activation of platelets leads to cytoskeletal assembly that is responsible for platelet motility and internal contraction. We have evaluated the involvement of the cytoskeleton in platelet activation by two strong agonists, collagen and thrombin. Activation was assessed by measuring changes in cytoskeletal assembly, externalization of activation-dependent markers and expression of procoagulant activity, and tyrosine phosphorylation of proteins, in both the absence and the presence of cytochalasin B. Activation of platelets with collagen and thrombin induced morphological changes and increased the expression of CD62P, CD63, glycoprotein IV, and binding of annexin V to platelets. Moreover, both activating agents induced actin polymerization, increased the association of other contractile proteins, and promoted tyrosine phosphorylation of multiple proteins, some of which were associated with the cytoskeleton. The presence of cytochalasin B blocked the previous events when collagen was used as the activating agent, although binding of annexin V still occurred. In contrast, platelet response to thrombin was not completely prevented by the presence of cytochalasin B. Thus, activation by collagen requires a functional cytoskeleton to trigger signaling through tyrosine phosphorylation and secretion. This is not the case for thrombin, which is capable of activating signaling mechanisms in the presence of strong inhibitors of cytoskeletal assembly. Moreover, the expression of a procoagulant surface in platelets still occurs even when platelet motility has been inhibited.

Antithrombotic effect of a new nitric oxide donor (LA419) on experimental thrombogenesis

Background  The ability of nitrous compounds to donate nitric oxide (NO), an agent with vasodilating and inhibitory effects on platelet function, has been considered a useful pharmacologic strategy for cardiovascular complications. The purpose of this study was to investigate the effects of a new NO donor, LA419, on platelet interaction in an ex vivo model with human blood circulating through collagen‐rich surfaces.

Effects on primary haemostasis of an anti-inflammatory agent with 5-lipoxygenase and cyclooxygenase inhibitory activity.

ObjectiveLicofelone ([2,2-dimethyl-6-(4-chlorophenyl)-7-phenyl-2,3-dihydro-1H-pyrrolizine-5-yl]-acetic acid) has been demonstrated to inhibit cyclooxygenase (COX)-1, COX-2, and 5-lipoxygenase. The aim of this study was to investigate the in-vitro effects of licofelone on platelet function. Effects observed were compared with those produced by the classic COX-1 inhibitor aspirin (ASA). MethodsPlatelet aggregation was assessed by a turbidimetric method. Platelet haemostatic performance was studied with the platelet function analyser (PFA-100), using collagen epinephrine and collagen ADP cartridges. Interaction of platelets with thrombogenic surfaces was analysed by perfusion experiments performed under flow conditions using both parallel and annular chambers. ResultsLicofelone prolonged the lag time of platelet aggregation induced by arachidonic acid and reduced maximal platelet aggregation induced by ADP or collagen. Studies using PFA-100 demonstrated that licofelone (0.1, 1 and 10 μM) significantly prolonged closure times (P < 0.05) with both types of cartridges. In studies with the parallel chamber exposing purified collagen, both licofelone and ASA significantly reduced (P < 0.05) overall platelet interaction with the thrombogenic surface. In studies performed in annular chamber exposing a highly thrombogenic vessel surface, licofelone reduced height and area of the platelet masses deposited (7.0 ± 0.5 μm; P < 0.005 and 80.2 ± 17.3 μm2; P < 0.05 vs. control 10.6 ± 0.9 μm and 194.8 ± 44.7 μm2, respectively). ASA also impaired thrombus formation but differences did not reach the levels of statistical significance. ConclusionsUnder our experimental in-vitro conditions, licofelone interfered with platelet function as demonstrated by a diminished platelet aggregation, being more powerful than ASA and reducing the interaction of platelets with thrombogenic surfaces.

Differences and similarities in tyrosine phosphorylation of proteins in platelets from human and pig species

Summary.  Background: Pigs have been widely used as animal models to study hemostasis. However, there are significant differences when comparing the hemostatic behavior of pig and human platelets. Objective: To investigate signaling through tyrosine‐phosphorylation of proteins in pig platelets after activation in suspension or by adhesion under flow conditions, in comparison with human platelets. Methods: Activation of platelet suspensions was performed with thrombin (T; 0.1 and 1 U mL−1) and type I collagen (Col‐I; 20 µg mL−1), at two different time points (30 and 90 s). Activation by adhesion was carried out on Col‐I‐coated coverslips, using citrated whole blood samples perfused through a parallel‐plate chamber. Results and conclusions: Significant differences between pig and human platelets were detected before and after activation. Activation of pig platelets required higher concentrations of thrombin, as well as increased activation times, to achieve similar levels of tyrosine phosphorylation. Proteins p160, p140, p85 and pp62, present in human platelets, were not detected in profiles corresponding to activated pig platelets. A protein of 70 kDa appeared only in pig platelet profiles, p55 was highly phosphorylated, and the phosphorylation levels of some proteins were significantly different from those found in human platelet profiles. In profiles corresponding to adhered pig platelets, p85 and p62 were absent, and p115 appeared highly phosphorylated. As observed in suspension studies, p70 and p55 appeared specifically in adhered pig platelets. Our study shows that the phosphotyrosine proteins involved in the activation of pig platelets are significantly different from those observed in activated human platelets. These findings may help to explain the differing adhesive and cohesive properties of platelets from both species, which should be considered when extrapolating results.

Adherence of Platelets Under Flow Conditions Results in Specific Phosphorylation of Proteins at Tyrosine Residues

Collagen is a powerful platelet activating agent that promotes adhesion and aggregation of platelets. To differentiate the signals generated in these processes we have analyzed the tyrosine phosphorylation occurring in platelets after activation with collagen in suspension or under flow conditions. For the suspension studies, washed platelets were activated with different concentrations of purified type I collagen (Coll). Studies under flow conditions were performed using two different adhesive substrata: Coll and endothelial cells extracellular matrix (ECM). Coverslips coated with Coll or ECM were perfused through a parallel-plate perfusion chamber at 800s−1 for 5 min. After activation of platelets either in suspension or by adhesion, samples were solubilized and proteins were resolved by electrophoresis. Tyrosine-phosphorylated proteins were detected in immunoblots by specific antibodies. Activation of platelet suspensions with collagen induced tyrosine phosphorylation before aggregation could be detected. Profiles showing tyrosine-phosphorylated proteins from platelets adhered on Coll or on ECM were almost identical and lacked proteins p95, p80, p66, and p64. which were present in profiles from platelets activated in suspension. The intensity of phosphorylation was quantitatively weaker in those profiles from platelets adhered on ECM. Results from the present work indicate that activation of platelets in suspension or by adhesion induces differential tyrosine phosphorylation patterns. Phosphorylation of proteins p90 and p76 may be related to early activation events occurring during initial contact and spreading of platelets. Considering that adhesion is the first step of platelet activation, studies on signal transduction mechanisms under flow conditions may provide new insights to understand the signaling processes taking place at earliest stages of platelet activation.

External calcium facilitates signalling, contractile and secretory mechanisms induced after activation of platelets by collagen

Platelet activation leads to the initiation of intracellular signalling processes, many of which are triggered by Ca2+. We have studied the involvement of exogenous Ca2+ in platelet response to collagen activation. Platelet suspensions were prepared with and without adding external calcium in the suspension buffers. Activation with collagen (Col-I) was carried out, before and after incubation with cytochalasin B (Cyt-B) to block the actin assembly and the cytoskeletal reorganization. We evaluated changes in (i) tyrosine phosphorylation of proteins, in platelet lysates and associated with the cytoskeletal fraction, (ii) the association of contractile proteins to the cytoskeleton, (iii) expression of intraplatelet substances at the surface, and (iv) cytosolic Ca2+ levels ([Ca2+]i). Ultrastructural evaluation of platelets by electron microscopy was also performed. Platelet activation by Col-I in the absence of added Ca2+ was followed by mild association of actin and other contractile proteins, low phosphorylation of proteins at tyrosine residues, lack of expression of intraplatelet substances at the membrane, and absence of aggregation. In the presence of millimolar Ca2+, Col-I induced intense actin filament formation with association of contractile proteins with the cytoskeleton, resulting in profound morphological changes. Under these conditions, Col-I induced signalling through tyrosine phosphorylation, with increases in the [Ca2+]i, release of intragranule content and aggregation. Inhibiting actin polymerization with Cyt-B prevented all these events. Our data indicates that platelet activation by collagen requires external Ca2+. Studies with Cyt-B indicate that assembly of new actin and cytoskeleton-mediated contraction, both dependent on exogenous Ca2+, are key events for platelet activation by collagen. In addition, our results confirm that entrance of exogenous Ca2+ depends on a functional cytoskeleton.