Raul Tonda

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.

Increased local procoagulant action: a mechanism contributing to the favorable hemostatic effect of recombinant FVIIa in PLT disorders

BACKGROUND: Recombinant FVIIa (rFVIIa) has been shown to improve hemostasis in patients with thrombocytopenia and to prevent or control bleeding episodes in patients with inherited deficiencies of major PLT glycoproteins, but the mechanism of action is not well understood.

Recombinant Factor VIIa (NovoSeven@) Restores Deficient Coagulation: Experience From an Ex Vivo Model

The action of recombinant factor VIIa (rFVIIa) in coagulation deficiencies with increased risk of bleeding was investigated using in vitro perfusion. Blood samples were drawn from healthy donors, a patient with hemophilia A and inhibitors, and six patients undergoing oral anticoagulant treatment. Fragmin 10 U/mL was used as anticoagulant. rFVIIa (10 microg/mL in plasma) was added to blood samples, incubated for 1 minute at 37 degrees C, and perfusion studies performed for 10 minutes at 600 x s(-1) through annular chambers containing damaged vascular segments. Subendothelial fibrin and platelets were expressed as a percentage of subendothelial surface screened. Under different conditions, rFVIIa consistently restored or improved fibrin formation on the damaged vascular subendothelium exposed to circulating blood. It restored fibrin deposition in blood from the hemophilia A patient; in patients undergoing acenocoumarol treatment, it reduced the international normalized ratio (INR) from 2.47 to 1.25 with a significant increase in fibrin deposition. Platelet deposition varied slightly between clinical conditions but was less evident in the hemophilia A patient. These data support the concept that rFVIIa facilitates fibrin formation in these clinical situations, promoting procoagulant activity at sites of vascular damage where tissue factor is exposed. This could improve hemostasis in patients with hemophilia A and inhibitors, and in patients treated with oral anticoagulants.

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.

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.

Hemostatic effect of activated recombinant factor VIIa in Bernard‐Soulier syndrome: studies in an in vitro model

To the Editor: Bernard-Soulier syndrome (BSS) is a rare disorder associated with the lack or dysfunction of the platelet (PLT) glycoprotein complex Ib-V-IX. These PLTs have impaired interaction with the subendothelium and affected persons experience repeated mucosal bleeding. Transfusion of PLT concentrates may be required to control severe bleeding. Unfortunately, some patients develop antibodies and refractoriness to further PLT transfusions. Recombinant factor VIIa (rFVIIa) is indicated for treating bleeding episodes in patients with hemophilia A or B with inhibitors. A recent study from our group found that rFVIIa facilitates fibrin formation on damaged vasculature using blood from patients with hemophilia and inhibitors. 1 Clinical experience suggests that rFVIIa may have a potential role in the treatment of bleeding in patients with quantitative and qualitative PLT disorders. 2

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.

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.