Jordi Bozzo

Infusible platelet membranes improve hemostasis in thrombocytopenic blood: experimental studies under flow conditions.

BACKGROUND : The potential hemostatic effect of infusible platelet membranes (IPM; Cyplex, Cypress Bioscience) prepared from outdated human platelets is investigated.

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.

Prohemorrhagic potential of dipyrone, ibuprofen, ketorolac, and aspirin: mechanisms associated with blood flow and erythrocyte deformability.

Summary: Dipyrone, ibuprofen, ketorolac, and aspirin were tested in a well‐defined perfusion system (shear rates: 300/s, 800/s, and 1,800/s). Whole blood samples were treated with the drugs at analgesic doses and platelet interaction with damaged subendothelium was measured. All the drugs fully inhibited platelet cyclooxygenase, as assessed by classic aggregometry. Perfusion studies showed that there was a general tendency to reduce the percentage of large aggregates (thrombus; %T), to increase the percentage of adhered platelets (adhesion; %A), and to reduce the height of thrombi with respect to control. Aspirin significantly increased %A and reduced %T at all shear rates tested, whereas dipyrone had the same effect at 800/s, and ketorolac and ibuprofen at 1,800/s. In addition, aspirin significantly reduced erythrocyte deformability with respect to the other drugs. In conclusion, under our experimental conditions, aspirin showed the most remarkable effects on platelet function, closely followed by dipyrone. The effects of ketorolac were moderate, whereas ibuprofen had a minor impact on platelet function.

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.

Platelet concentrates promote procoagulant activity: evidence from experimental studies using a perfusion technique

BACKGROUND: Evaluation of the hemostatic effectiveness of platelet transfusions is difficult. Perfusion methods have been employed to test the quality and function of platelet concentrates, allowing differentiation between platelet‐platelet and platelet‐surface interactions.

Reduced red cell deformability associated with blood flow and platelet activation: improved by dipyridamole alone or combined with aspirin

OBJECTIVES To explore the role of blood flow in red blood cell deformability and determine whether it is associated with the release of ATP by red cells. METHODS A perfusion system was employed to mimic the blood flow conditions. Red cell treatment in vitro with dipyridamole or aspirin, either alone or combined, was performed either on red cells alone prior to blood reconstitution or on already reconstituted blood. Blood viscosity at shear rates of 90 and 450 s-1 was measured, before and after perfusion, by means of a cone/plate viscometer. Red cell deformability was estimated as the magnitude of the slope between these two values of viscosity. ATP level in platelet-free plasma was measured by the firefly luciferase assay, and was used to monitor dipyridamole-induced blockade of ATP efflux by red cells. RESULTS In control experiments, red cells became more rigid after perfusion (slope values: 0.62 +/- 0.09 vs 0.40 +/- 0.04; P < 0.05). Absence of platelets or treatment of red cells with aspirin and/or dipyridamole prevented dipyridamole prevented the increased stiffness of red cells observed after exposure of blood to flow conditions. Moreover, a significant increase in red cell deformability was observed when blood was treated with dipyridamole alone or combined with aspirin (0.69 +/- 0.07 and 0.65 +/- 0.05 respectively vs. 0.40 +/- 0.04; P < 0.05). Treatment with dipyridamole alone or combined with aspirin caused a decrease in ATP levels. Statistical significance was reached when red cells alone were treated with dipyridamole alone (45.8 +/- 2.8 vs. 173.3 +/- 47.6 ng ATP/ml; P < 0.05). CONCLUSIONS Flow stress induced a decrease in red cell deformability probably linked to platelet activation. Treatment with dipyridamole alone or associated with aspirin eliminated the effect of flow, increasing red cell deformability. Under our experimental conditions, modifications could not be ascribed to red cell-released ATP. Our results emphasize the importance of blood flow conditions to evaluate RBC deformability.

Preparations of synthetic phospholipids promote procoagulant activity on damaged vessels: studies under flow conditions

BACKGROUND: The possibility of developing synthetic platelet substitutes that could promote hemostasis with prolonged shelf‐life and increased safety is an appealing one. STUDY DESIGN AND METHODS: Preparations containing synthetic phospholipids were incorporated into blood samples (1.15 mg/mL) in which platelets and white cell counts had been experimentally reduced by a filtration procedure. Vesicles containing phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), or combinations of PC and PE and of PC and PS were tested in this system. Blood was recirculated (10 min; shear rate, 250/sec) through a perfusion chamber containing vascular segments. The ability of the various phospholipid preparations to promote fibrin formation on the damaged subendothelium was evaluated morphometrically and expressed as the percentage of fibrin coverage. Generation of thrombin in the system was monitored through the measurement of prothrombin fragments 1 and 2. RESULTS: Vesicles containing PC, PI, PE:PC (1:1), or PS:PC (1:3) increased fibrin deposition on the subendothelium (64.5 +/− 9.8%, 32.7 +/− 6.3%, 58.3 +/− 6.5%, and 46.6 +/− 15.2%, respectively; p < 0.01 vs. 11.5 +/− 1.2% in thrombocytopenic blood). Vesicles containing PE, PS, or PS:PC (3:1) did not show procoagulant effect. CONCLUSION: Synthetic phospholipid preparations promote a local procoagulant activity at sites of vascular damage when they are incorporated into thrombocytopenic blood maintained under flow conditions.

Assessment of Potential Thrombogenicity of Coagulation Factor IX Concentrates in an in Vitro Model of Human Thrombogenesis

We have investigated the potential use of perfusion techniques in the evaluation of the thrombogenic profile of factor IX concentrates. Blood from healthy donors was anticoagulated with low molecular weight heparin and incubated with one of the following: (a) diluent (DIL); (b) a prothrombin complex concentrate (PCC); (c) an intermediate-purity concentrate (FIX/X); or (d) a high-purity concentrate (HPFIX). The thrombogenic potential was assessed as: (1) fibrin formation on subendothelium (Baumgartners perfusion) and (2) prothrombin activation fragment 1+2 (F1+2, nM) determination. The percentage of fibrin deposition on the subendothelium was only significantly increased after incubation with PCC (62.0+/-3.6% vs. DIL 35.0+/-6.1%;p<0.05). None of the FIX concentrates modified platelet interaction versus control blood (DIL: 26.7+/-2.1%). F1+2 baseline values in anticoagulated blood were 0.6+/-0.1 nM. Preperfusion levels of F1+2 reached values of 4.4+/-0.1 nM for PCC and 5.4+/-0.1 nM for FIX/X. After perfusion, F1+2 values were 2.7+/-0.2 nM for DIL, 5.6+/-0.1 nM for PCC and FIX/X, and 3.3+/-0.2 nM for HPFIX. While measurement of F1+2 was influenced by residual contaminants present in the concentrates, the morphometric evaluation of fibrin deposition on perfused vascular surfaces could be more closely related to the net thrombogenic profile of each FIX preparation.

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.

Influence of co-incubation and cell number of platelets and polymorphonuclear leukocytes in cellular inhibition and activation phenomena

Evidence indicates that complex interactions occur between blood platelets and polymorphonuclear leukocytes (PMN) referring to both activation and inhibition phenomena. We studied the influence of co-incubating activated PMN and platelets at varying cell counts in the production of two active metabolites, thromboxane A2 (TxA2) measured as thromboxane B2 (TxB2) and platelet activating factor (PAF). The decrease observed in the quantity of TxA2 synthesized by 2 x 10(8) platelets/ml in the presence of physiological PMN counts clearly indicates an inhibitory effect of PMN in AA-derived metabolites. This suggests there is an influence of PMN-released products on activated platelets, decreasing their capacity to synthesize TxA2. Moreover, we found that the addition of platelets, which do not make measurable quantities of PAF, to PMN suspensions significantly decreased the capacity of PMN to synthesize this mediator. Together with this inhibiting action we also observed an activating effect, in such a way that the more PMN were added to platelet suspensions the more TxA2 was produced. Conversely, the maximal amounts of PAF were synthesized when the highest platelet count suspensions were added to PMN. In summary, the results we present here clearly show cellular interactions between platelets and PMN that directly influence the amounts of metabolites synthesized by both cells.