Berta Fuste

Effects of a new pathogen-reduction technology (Mirasol PRT) on functional aspects of platelet concentrates.

BACKGROUND: Several strategies are being developed to reduce the risk of pathogen transmission associated with platelet (PLT) transfusion.

High-throughput sequence analysis of turbot (Scophthalmus maximus) transcriptome using 454- pyrosequencing for the discovery of antiviral immune genes

Background Turbot (Scophthalmus maximus L.) is an important aquacultural resource both in Europe and Asia. However, there is little information on gene sequences available in public databases. Currently, one of the main problems affecting the culture of this flatfish is mortality due to several pathogens, especially viral diseases which are not treatable. In order to identify new genes involved in immune defense, we conducted 454-pyrosequencing of the turbot transcriptome after different immune stimulations. Methodology/Principal Findings Turbot were injected with viral stimuli to increase the expression level of immune-related genes. High-throughput deep sequencing using 454-pyrosequencing technology yielded 915,256 high-quality reads. These sequences were assembled into 55,404 contigs that were subjected to annotation steps. Intriguingly, 55.16% of the deduced protein was not significantly similar to any sequences in the databases used for the annotation and only 0.85% of the BLASTx top-hits matched S. maximus protein sequences. This relatively low level of annotation is possibly due to the limited information for this specie and other flatfish in the database. These results suggest the identification of a large number of new genes in turbot and in fish in general. A more detailed analysis showed the presence of putative members of several innate and specific immune pathways. Conclusions/Significance To our knowledge, this study is the first transcriptome analysis using 454-pyrosequencing for turbot. Previously, there were only 12,471 EST and less of 1,500 nucleotide sequences for S. maximus in NCBI database. Our results provide a rich source of data (55,404 contigs and 181,845 singletons) for discovering and identifying new genes, which will serve as a basis for microarray construction, gene expression characterization and for identification of genetic markers to be used in several applications. Immune stimulation in turbot was very effective, obtaining an enormous variety of sequences belonging to genes involved in the defense mechanisms.

Transcriptomics of In Vitro Immune-Stimulated Hemocytes from the Manila Clam Ruditapes philippinarum Using High-Throughput Sequencing

Background The Manila clam (Ruditapes philippinarum) is a worldwide cultured bivalve species with important commercial value. Diseases affecting this species can result in large economic losses. Because knowledge of the molecular mechanisms of the immune response in bivalves, especially clams, is scarce and fragmentary, we sequenced RNA from immune-stimulated R. philippinarum hemocytes by 454-pyrosequencing to identify genes involved in their immune defense against infectious diseases. Methodology and Principal Findings High-throughput deep sequencing of R. philippinarum using 454 pyrosequencing technology yielded 974,976 high-quality reads with an average read length of 250 bp. The reads were assembled into 51,265 contigs and the 44.7% of the translated nucleotide sequences into protein were annotated successfully. The 35 most frequently found contigs included a large number of immune-related genes, and a more detailed analysis showed the presence of putative members of several immune pathways and processes like the apoptosis, the toll like signaling pathway and the complement cascade. We have found sequences from molecules never described in bivalves before, especially in the complement pathway where almost all the components are present. Conclusions This study represents the first transcriptome analysis using 454-pyrosequencing conducted on R. philippinarum focused on its immune system. Our results will provide a rich source of data to discover and identify new genes, which will serve as a basis for microarray construction and the study of gene expression as well as for the identification of genetic markers. The discovery of new immune sequences was very productive and resulted in a large variety of contigs that may play a role in the defense mechanisms of Ruditapes philippinarum.

Tissue factor-enriched vesicles are taken up by platelets and induce platelet aggregation in the presence of factor VIIa

We investigated the interactions of vesicles containing human tissue factor (TF) with platelets and evaluated responses induced by rFVIIa using standard aggregometry, ultrastructural and flow-cytometry techniques. Washed platelets were exposed to a preparation of placental human TF (pTF) or to a relipidated formulation of recombinant human TF (rTF). Under stirring conditions, pTF induced reversible aggregation with platelets returning to their resting state after 5 minutes. This reversible response to pTF was partially inhibited by antibodies against CD62-P, but not by antithrombin agents, and was not observed with rTF. Sequential ultrastructural studies revealed uptake of both TF preparations by platelets involving traffic of vesicles through channels of the open canalicular system (OCS). Immunocytochemical studies on cryosections identified TF in the OCS, and occasionally in the alpha-granules of the platelets. These processes were faster with pTF than with rTF, but both TF preparations accumulated in platelets at the end of incubation periods. Flow cytometry studies revealed the presence of other cellular antigens (CD62-P, CD14 and CD45) associated to the pTF. Addition of rFVIIa to washed platelets exposed to pTF or rTF, caused a thrombin dependent irreversible platelet aggregation. Our studies demonstrate that platelets possess mechanisms to capture and incorporate TF-rich vesicles. These processes are accelerated by the presence of other cellular antigens in the vesicles. Our findings may explain the hemostatic action of rFVIIa in severely hemodiluted patients, but are also relevant for the understanding of potential implications of TF-associated to platelets in the propagation of thrombus.

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.

TRAP Induces More Intense Tyrosine Phosphorylation than Thrombin with Differential Ultrastructural Features

We have analyzed modifications on platelet ultrastructural morphology, cytoskeletal assembly, and tyrosine phosphorylation developing in platelets activated by both thrombin and the thrombin receptor-activating peptide (TRAP). Washed platelets exposed to various concentrations of thrombin or TRAP, for different periods, were: fixed and examined by electron microscopy, or lysed and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under similar activating conditions, thrombin and TRAP induced different sequences of activation causing distinctive morphological and biochemical changes. Platelets exposed to thrombin showed centralized organelles encircled by constricted microtubule coils and granules secreting their contents through narrow channels of the open canalicular system. In contrast, activation by TRAP induced swelling of the open canalicular system with organelles remaining randomly dispersed and microtubules peripherally distributed. Compared to thrombin activation, TRAP induced higher rates of actin polymerization; increased association of actin-binding protein, myosin, and alpha-actinin; and higher association of tyrosine-phosphorylated proteins with the insoluble cytoskeletal fraction. Secretion of intragranule substances, measured as expression of P-selectin and lysosomal integral membrane protein at the surface level, were similar for both agonists at equivalent concentrations. Our biochemical observations indicate that TRAP causes more intense changes in signaling through tyrosine phosphorylation of proteins associated with the cytoskeletal fraction than thrombin. However, as derived from ultrastructural observations, TRAP seems to be less efficient in triggering cytoskeletal assembly and internal contraction in an organized manner in contrast with the natural protease.

Platelet adhesion onto a polystyrene surface under static conditions: role of specific platelet receptors and effect of divalent cations

An experimental model was used to elucidate the basic mechanisms involved in the interaction of platelets with an artificial surface. The role of divalent cations and the involvement of specific platelet membrane receptors were evaluated. Isolated platelets were allowed to interact with a polystyrene surface for 20 min in the presence of divalent cations (Ca 2+ , Mg 2+ or Zn 2+ ), a chelating agent (ethylenediaminetetraacetic, EDTA), and specific antibodies to the main platelet receptors, glycoproteins (GP) Ib and IIb-IIIa. The degree of platelet interaction was evaluated using light and electron microscopy. Morphometric analysis was performed to follow up the progression of platelet shape changes after surface activation. Neither Ca 2+ nor Mg 2+ influenced the number of adherent platelets or the degree of spreading on the polymer. Only Zn 2+ induced a statistically significant increase in the rate of platelet adhesion ( P <0.01) with higher proportion of fully spread platelets ( P <0.01). Chelation of internal pools of divalent cations did not modify the rates of platelet adhesion but prevented platelet spreading. Presence of monoclonal antibodies to GPIb and GP IIb-IIIa did not result in significant differences in the studied parameters. These results suggest that platelet adhesion onto artificial surfaces, in the absence of flow and plasma proteins, is more dependent on cellular motility, where Zn 2+ could play an important role, and less dependent on major receptorial mechanisms.

Effect of Two Different Dialysis Membranes on Leukocyte Adhesion and Aggregation

Aims: We evaluated modifications in formation of heterotypic platelet-leukocyte aggregation induced by dialysis through cellulosic or synthetic membranes and evaluated the effects of such procedures promoting adhesive interactions between leukocytes and normal endothelial cells (ECs). Methods: Samples were obtained from arterial and venous lines at baseline, after 15 and 120 min of hemodialysis. Heterotypic aggregation was assessed using flow-cytometric techniques. Experiments to determine leukocyte adhesion to ECs were performed in parallel plate perfusion chambers at 450 s–1. Results: Patients dialyzed with a cellulosic membrane showed a significantly higher baseline granulocyte heterotypic aggregation (median 22.5%, range 8.6–32%) versus healthy subjects (median 10%, range 3.2–14.6%; p < 0.05). Granulocyte heterotypic aggregation values remained increased throughout the hemodialysis session not only in the arterial line (median 18 and 24.5%, range 7–30 and 8.7–36% at 15 and 2 h, respectively) but also in the venous line (median 20 and 25%, range 8.6–32 and 11.5–35% at 15 min and 2 h, respectively). Basal lymphocytes heterotypic aggregation values observed in uremic patients were 6% (0.1–7.1%) versus 1.0% (0.5–2.8%) in the control group (p < 0.05). The increase remained during the hemodialysis session both in the arterial line (median 5 and 4%, range 0.2–14 and 0.5–7.1 % at 15 min and 2 h, respectively) and in the venous line (median 7 and 7%, range 1.4–14 and 0.5–10.6% at 15 min and 2 h). In contrast, patients dialyzed with a synthetic membrane showed a decreased basal granulocyte heterotypic aggregation compared to healthy subjects (median 3.5 vs. 10%, range 2.8–7 vs. 3.2–14.6%, respectively; p < 0.05). For lymphocytes, basal heterotypic aggregation values were 0.2% (range 0.1–0.5%) in dialyzed patients vs. 0.98% (range 0.5–2.8%) in healthy subjects (p < 0.05), without changes throughout the dialysis session. Changes in leukocyte adhesion during hemodialysis did not reach statistical significance with either hemodialysis membrane. Our studies confirm a differential activation of platelets and leukocytes depending on the nature of the dialysis membranes. However, activation of circulating cellular elements by hemodialysis procedures did not enhance cross-talk interactions between leukocytes and unaltered ECs.

Efficient tyrosine phosphorylation of proteins after activation of platelets with thrombin depends on intact glycoprotein Ib.

The role of platelet glycoprotein Ib as a thrombin receptor has been often a subject of controversy. We have investigated the role of the thrombin receptors, GPIb and protease-activated receptor (PAR)-1. Tyrosine phosphorylation in whole platelet lysates and in cytoskeletal extracts was evaluated after activation with thrombin and with the thrombin receptor-activating peptide (TRAP). Different experimental approaches were applied including: (i) congenital deficiency of platelet GPIb (Bernard Soulier syndrome, BSS), (ii) antibody to GPIb (AP1), (iii) selective protease cleavage (metalloprotease), and (iv) antibody to (PAR)-1. After activation of control platelets with thrombin or TRAP, multiple proteins became tyrosine phosphorylated in platelet lysates and some of them associated with the cytoskeletal fraction. These effects were absent in BSS platelets. Presence of AP1 or metalloprotease treatment showed an inhibitory effect when platelets were activated with a low concentration of thrombin or TRAP. Blockade of PAR-1 with a specific antibody, SPAN 12, inhibited platelet response to both agonists. This study reinforces the hypothesis that GPIb is the high-affinity receptor for thrombin. The signaling mechanisms occurring through tyrosine phosphorylation of proteins triggered by thrombin seem to be dependent on intact GPIb. Moreover, our results indicate that both receptors, GPIb and PAR-1, are necessary to achieve a full platelet response to thrombin.

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.