Maria Rosa Hernandez

Biocompatibility of Cellulosic and Synthetic Membranes Assessed by Leukocyte Activation

Background/Aims: The contact of blood with artificial surfaces may activate blood leukocytes and platelets and initiate the leukocyte inflammatory response. We have investigated the effect of a hemodialysis (HD) with a cellulosic- and a synthetic-based membrane on circulating leukocyte activation. Methods: Samples were obtained from patients with ESRD at baseline, and at 15 and 120 min of a hemodialysis session from both the arterial and venous lines. Leukocyte respiratory burst was analyzed by luminol chemiluminescence. Actin polymerization, expression of CD11b, and heterotypic aggregation were studied by flow cytometry, leukocyte labeling with NBD phallacidin and monoclonal antibodies, respectively. Results: HD with a cellulosic membrane induced a transient fall in neutrophil (1.2 ± 0.5 × 109 vs. 3.6 ± 0.6 × 109 cells/l; p < 0.05) and monocyte counts (0.2 ± 0.1 × 109 vs. 0.7 ± 0.1 × 109 cells/l; p < 0.05). There was also an increase in respiratory burst in the venous line during a HD with a cellulosic membrane, at 15 and 120 min (100 ± 41 and 143.2 ± 45.3 vs. 23.8 ± 15.7; p < 0.05). Polymerized actin, expressed as fluorescence arbitrary units, was increased in baseline samples from uremic patients versus control subjects (327.8 ± 60.8 for a cellulosic membrane, p < 0.005, and 205 ± 26.5 for a synthetic one, p < 0.05 vs. 97.8 ± 27.6 in controls). The percentage of CD11b+ cells increased in samples during a HD with a cellulosic membrane at the venous line at 15 and 120 min (9.6 ± 4.5 and 18.4 ± 7.1% vs. 3.3 ± 1.9%; p < 0.05%). Changes in heterotypic aggregation during HD did not reach statistical significance, but levels were higher in patients treated with a cellulosic membrane at all points than in patients dialyzed with a synthetic one. Conclusion: There is evidence of a priming state of leukocytes from uremic patients, which is more evident in patients dialyzed with a cellulosic membrane. Cellulosic membranes also induce greater leukocyte activation than synthetic membranes during hemodialysis.

Hemostatic effect of activated recombinant factor VII (rFVIIa) in liver disease: studies in an in vitro model

BACKGROUND/AIMS There is clinical evidence for the efficacy of activated recombinant factor VII (rFVIIa) in patients with cirrhosis. The exact mechanism of action of rFVIIa in this clinical condition is unknown. We have explored effects of rFVIIa on hemostasis in cirrhotic patients using an in vitro perfusion technique. METHODS Blood samples were drawn from control donors or from 11 patients previously diagnosed with cirrhosis (seven Child-Pugh B and four Child-Pugh C) and anticoagulated with low molecular weight heparin. rFVIIa was added to blood samples at therapeutic concentrations (0.5 or 1 microg/ml of plasma) and blood was recirculated through annular chambers containing damaged vascular segments. Presence of platelets and fibrin on the subendothelium were morphometrically quantified. RESULTS Cirrhotic patients showed a diminished platelet interaction with the subendothelium compared to healthy donors (17.3% (9.28-28.88%) vs. 26.16% (19.96-54.5%), P<0.05). After addition of rFVIIa to cirrhotic samples, no differences in platelet covered surface were observed. However, fibrin formation was significantly improved after the addition of rFVIIa (from 51.81% (3.02-86.68%) to 86.94% (30.03-93.18%) and 89.05% (45.65-93.84%), respectively, P<0.05). CONCLUSIONS Our data confirm a defective interaction of platelets with the subendothelium in cirrhotic patients. rFVIIa improved local fibrin formation at damaged sites and this mechanism could explain the beneficial action of rFVIIa in cirrhotic patients.

In vitro Evaluation of the Hemostatic Effectiveness of Non Viable Platelet Preparations: Studies with Frozen‐Thawed, Sonicated or Lyophilized Platelets

Objectives: Because of the limited life span of platelets in regular storage systems, we were interested in investigating the effects on hemostasis of nonliving platelet derivatives. Methods: We evaluated the effects of different platelet preparations on primary hemostasis in a well‐established perfusion model. Studies were carried out with blood anticoagulated with low molecular weight heparin. Similar amounts of frozen‐thawed, sonicated or lyophilized platelets were added to normal blood or to blood which had been experimentally depleted of platelets. Platelet interaction with the subendothelium and fibrin deposition were morphometrically evaluated. Results: Addition of nonviable platelet preparations to thrombocytopenic blood always promoted a statistically significant increase in the deposition of fibrin on the subendothelium, but only lyophilized platelets retained some ability to interact with the subendothelium. Flow cytometry studies demonstrated the presence of GPIb, GPIIIa and P‐selectin on lyophilized platelets. Conclusions: Preparations containing nonviable platelets may still retain some hemostatic properties.

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.

The hemostatic agent ethamsylate enhances P-selectin membrane expression in human platelets and cultured endothelial cells

Ethamsylate possesses antihemorrhagic properties, but whether or not it directly activates blood platelets is unclear. Here we investigated the platelet activation potential of ethamsylate, by measuring membrane P-selectin expression with flow cytometry in human whole blood and also by immunofluorescence imaging of isolated human platelets. Moreover, we measured membrane P-selectin expression in the SV40-transformed aortic rat endothelial cell line (SVAREC) and 14C-ethamsylate membrane binding and/or uptake in platelets and endothelial cells. Whole blood flow cytometry showed a modest, but statistically significant increase by ethamsylate in the percentage of platelets expressing P-selectin (from 2% to 4-5%, p < 0.05). Immunofluorescence showed a sizable (39%) and significant (p < 0.01) enhancement of P-selectin expression at the lowest concentration of ethamsylate tested (1 microM), with maximal enhancement of P-selectin expression (75-90%) at 10 microM ethamsylate. Similar results were obtained in SVAREC endothelial cells. 14C-ethamsylate specifically bound to platelets and endothelial cell membranes, without significant uptake into the cell interior. In conclusion, ethamsylate enhances membrane P-selectin expression in human platelets and in cultured endothelial cells. Ethamsylate specifically binds to some protein receptor in platelet and endothelial cell membranes, receptor which can signal for membrane P-selectin expression. These results support the view that ethamsylate acts on the first step of hemostasis, by improving platelet adhesiveness and restoring capillary resistance.

The hemostatic agent ethamsylate promotes platelet/leukocyte aggregate formation in a model of vascular injury.

The hemostatic agent ethamsylate enhances membrane expression of P‐selectin in human platelets, but whether this promotes platelet–leukocyte aggregate formation is unknown. Here we investigated this point by flow cytometry determination of human platelet–leukocyte aggregates under basal conditions and after whole‐blood perfusion through a damaged rabbit aorta segment. Actions of ethamsylate on adhesive molecules of platelets and leukocytes were investigated in parallel. Under basal conditions, ethamsylate was unable to modify whole‐blood platelet–leukocyte aggregation, but following whole‐blood perfusion through a damaged vessel, ethamsylate produced a modest, but significant increase in platelet–leukocyte aggregates (48 ± 21 and 45 ± 26% above control levels at ethamsylate 20 and 40 μm respectively). In isolated leukocyte plasma membranes, 14C‐ethamsylate specifically bound up to an amount of 660 pmol/mg protein. Moreover, at concentrations ≥1 μm, ethamsylate induced an important (100–200%) and significant increase in the P‐selectin glycoprotein ligand 1 (PSGL‐1) fluorescence signal in isolated leukocytes and was unable to significantly modify the percentage of CD11b‐positive cells. However, no significant changes in aggregate formation were found when ethamsylate was incubated with isolated leukocytes and blood was reconstituted and perfused. In isolated platelet cell membranes, anti‐P‐selectin antibody and the anti‐integrin RGD‐containing pentapeptide (GRDGS) were unable to displace 14C‐ethamsylate binding. In conclusion, ethamsylate specifically binds to plasma membranes of leukocytes, enhances membrane PSGL‐1 expression and promotes leukocyte–platelet aggregation in whole‐blood perfused through a damaged vascular segment. These results together with the previously observed enhancement of platelet P‐selectin membrane expression [Thromb. Res. (2002)107:329–335] confirms and extends the view that ethamsylate acts on the first step of hemostasis, by improving platelet homo‐ and heterotypic adhesiveness.

Platelet-associated tissue factor enhances platelet reactivity and thrombin generation in experimental studies in vitro

INTRODUCTION The thrombogenic potential of tissue factor (TF) associated to platelets is controversial. We have investigated the in vitro contribution of platelet-associated TF to thrombus formation. MATERIALS AND METHODS Platelets suspensions were exposed to human TF-rich microvesicles (TF-MV) from placental or recombinant origin. Platelet-associated TF was quantified through coagulometric assays. Adhesive and cohesive properties of platelets containing TF were assessed in perfusion models using two thrombogenic surfaces: 1) type-I collagen, or 2) damaged vascular segments. Perfusion studies were performed with heparinized blood enriched with a 30% of washed platelets exposed to TF-MV vs. washed control platelets. Thrombin generation and thromboelastometric properties of clots were also assessed using a fluorometric assay and ROTEM analysis, respectively. Inhibitory strategies with an antibody to TF were performed in some cases. RESULTS The addition of 30% of platelets containing TF to blood perfusates resulted in a statistically significant increase in the platelet coverage (%CS) vs. non-exposed platelets on collagen surfaces (%CS: 19.7 ± 0.6 and 23.9 ± 0.7 respectively, vs.14.5 ± 1.4; p<0.01) and on the vascular subendothelium (%CS: 54.0 ± 1.5 and 47.2 ± 6.8 respectively vs. 38.0 ± 3.5, p<0.05), with a statistically significant increase in the size of large platelet aggregates (p<0.05) vs. control platelets. These effects on collagen surfaces were almost totally prevented by an antibody to TF. Platelet-associated TF significantly accelerated thrombin generation and clot formation (p<0.05), effects that were partially prevented by a neutralizing anti-TF. CONCLUSIONS Platelet-associated TF potentiated adhesive and aggregating properties in in vitro studies with flowing blood and accelerated thrombin generation and clot formation time under steady conditions.

Platelets interact with tissue factor immobilized on surfaces: effects of shear rate

Background  While procoagulant activities of Tissue Factor (TF) have been widely investigated, its possible pro‐adhesive properties towards platelets have not been studied in detail.

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 of aspirin and indomethacin separately in red blood cells and platelets. Modulation of the adhesive and cohesive functions of platelets under flow conditions

We treated red cells and platelet sepatately in vitro with aspirin or indomethacin to inhibit platelet function. The excess of drug was removed by profuse washing prior to blood reconstitution. We examined the influence of such treatments on platelet interaction with vascular subendothelium employing a perfusion system. Treatment of red cells or platelets with aspirin showed a similar pattern of platelet deposition onto subendothelium. However, platelet adhesion was significantly increased in treated red cells (21.7 ± 2.7% vs 13.7 ± 1.9% in controls; P < 0.05) whereas thrombus was significantly decreased in treated platelets (6.2 ± 1.62% vs 13.8 ± 1.7% in controls; P < 0.05). Treatment of red cells or platelets with indomethacin strongly inhibited platelet interaction. Thrombus and covered surface were decreased in experiments with treated red cells (2.3 ± 0.73% and 14.9 ± 2.3%, respectively; P < 0.05). Adhesion, thrombus and covered surface were decreased in experiments with treated platelets (4.3 ± 0.7%, 2.3 ± 0.9%, 9.2 ± 1.5%, respectively; P < 0.05). Platelet aggregation experiments performed with aspirin-treated red cells showed a progressive inhibition of platelet function. Testing levels of drug in plasma samples from the perfusates showed that levels of drug were very similar to those obtained if treated red blood cells were not washed after treatment. All these results suggest that red cells retained some quantities of drug. Our data highlight the potential effect of red cells interfering with platelet function inhibitors.