Ana M. Galan

Reversal of apixaban induced alterations in hemostasis by different coagulation factor concentrates: significance of studies in vitro with circulating human blood.

Apixaban is a new oral anticoagulant with a specific inhibitory action on FXa. No information is available on the reversal of the antihemostatic action of apixaban in experimental or clinical settings. We have evaluated the effectiveness of different factor concentrates at reversing modifications of hemostatic mechanisms induced by moderately elevated concentrations of apixaban (200 ng/ml) added in vitro to blood from healthy donors (n = 10). Effects on thrombin generation (TG) and thromboelastometry (TEM) parameters were assessed. Modifications in platelet adhesive, aggregating and procoagulant activities were evaluated in studies with blood circulating through damaged vascular surfaces, at a shear rate of 600 s−1. The potential of prothrombin complex concentrates (PCCs; 50 IU/kg), activated prothrombin complex concentrates (aPCCs; 75 IU/kg), or activated recombinant factor VII (rFVIIa; 270 μg/kg), at reversing the antihemostatic actions of apixaban, were investigated. Apixaban interfered with TG kinetics. Delayed lag phase, prolonged time to peak and reduced peak values, were improved by the different concentrates, though modifications in TG patterns were diversely affected depending on the activating reagents. Apixaban significantly prolonged clotting times (CTs) in TEM studies. Prolongations in CTs were corrected by the different concentrates with variable efficacies (rFVIIa≥aPCC>PCC). Apixaban significantly reduced fibrin and platelet interactions with damaged vascular surfaces in perfusion studies (p<0.05 and p<0.01, respectively). Impairments in fibrin formation were normalized by the different concentrates. Only rFVIIa significantly restored levels of platelet deposition. Alterations in hemostasis induced by apixaban were variably compensated by the different factor concentrates investigated. However, effects of these concentrates were not homogeneous in all the tests, with PCCs showing more efficacy in TG, and rFVIIa being more effective on TEM and perfusion studies. Our results indicate that rFVIIa, PCCs and aPCCs have the potential to restore platelet and fibrin components of the hemostasis previously altered by apixaban.

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.

Serotonergic mechanisms enhance platelet-mediated thrombogenicity.

Although it is generally acknowledged that serotonin (5-HT) is a weak agonist for human platelets, recent information suggests an association between serotonergic mechanisms and cardiovascular risk. We investigated the action of 5-HT on adhesive, cohesive and procoagulant properties of human platelets. Impact of 5-HT on whole blood coagulation and thrombin generation was measured by modified thromboelastometry (TEM) and specific fluorogenic assays. We evaluated the effects of 5-HT on thrombus formation in an in-vitro model of thrombosis using human flowing blood. In platelet-rich plasma (PRP), 5-HT favoured the expression of CD62-P, and procoagulant molecules on platelet membranes. These effects were potentiated in the presence of Ca(++) and/or ADP. Incubation with 5-HT accelerated clotting times and augmented clot strength in whole blood TEM, and enhanced thrombin generation in PRP. In perfusion studies, 5-HT significantly increased fibrin deposition at low shear (300s(-1)) and enhanced platelet thrombus formation on the damaged vascular surface at high shear (1,200s(-1)). Selective inhibition of serotonin reuptake (SSRI) attenuated effects of 5-HT on platelet activation and downregulated the prothrombotic tendencies observed in the previous experimental conditions. In general, reductions of thrombogenic patterns observed with SSRI were more evident under shear conditions (aggregation and perfusion systems) and less evident under steady conditions (TEM and thrombin generation assays). In conclusion, 5-HT is not a weak agonist for human platelets; instead it accentuates platelet activation, potentiates procoagulant responses on human blood and increases thrombogenesis on damaged vascular surfaces. The remarkable antithrombotic actions achieved through SSRI deserve further mechanistic and clinical investigations.

Impact of pathogen reduction technology and storage in platelet additive solutions on platelet function.

BACKGROUND: There are limited studies on the effect of pathogen reduction technology (PRT) on platelet (PLT) products stored in PLT additive solutions (PASs). Our study has evaluated the effect of Mirasol PRT (CaridianBCT) followed by storage in PASs (PAS‐III or PAS‐IIIM) on analytical and functional characteristics of PLT concentrates (PCs).

Physical stability of liposomes bearing hemostatic activity.

The physical stability of six liposome systems designed as platelet substitutes was determined on storage at 4 degrees C over a 3-month period under quiescent conditions. Liposomes used were large unilamellar vesicles. Correlation of the n-average mean diameter, polydispersity, zeta-potential and the presence of aminophospholipid on liposome surface (in those preparations which contain phosphatidylethanolamine (PE) and phosphatidylserine (PS)) led to the conclusion that liposomes that mimicked the composition of platelets were the most stable. When a net charge was present in the vesicles (liposomes with PS), the likelihood of aggregation was extremely low. In the period studied, a proportion of 25% of charged lipid (PS) conferred sufficient electrostatic stabilization to prevent vesicle fusion. An increase in this charge did not modify the stability characteristics. PE-containing liposomes behaved in a particular way: when PE content was 50%, the stability of the preparation was limited to 1 month; whereas if the content was 25%, the zeta-potential rose with time, as did the presence of PE in the liposome surface.

Serotonin enhances platelet procoagulant properties and their activation induced during platelet tissue factor uptake

AIMS Circulating tissue factor (TF) has been linked to thrombus propagation. Our group demonstrated that platelets possess mechanisms to capture TF-rich microvesicles (TF-MVs). Serotonin facilitates the development of platelets with increased procoagulant activity. An enhanced platelet serotonin uptake has been identified with increased cardiovascular risk. We have investigated the involvement of serotonergic mechanisms facilitating the interaction of human platelets with TF-MVs. Inhibitory strategies aimed at blocking serotonin and coagulation mechanisms were also studied. METHODS AND RESULTS Standard aggregometry, flow cytometry, electron microscopy, and thrombin generation assays were performed. TF-MVs induced platelet aggregation in heparinized platelet-rich plasma (PRP) samples; this aggregation was further accelerated by serotonin. In washed platelets, serotonin enhanced platelet aggregation to TF-MVs with a maximum peak of 55.9 +/- 1.8 vs. 48.7 +/- 2.1% (P < 0.05). Inhibitory strategies with a selective serotonin re-uptake inhibitor and with lepirudin decreased these aggregations. Ultrastructural analysis revealed that serotonin induced platelet pseudopodia formation, thus facilitating the engulfment of TF-MVs. In general, serotonin significantly enhanced (P < 0.05) thrombin generation and the expression of activation markers and procoagulant activity in platelets measured for TF-MVs alone. CONCLUSION Serotonin enhances the interaction of platelets with TF-MVs, increases platelet activation, and potentiates their overall procoagulant activity. The present results could have significant implications in thrombus formation and in the thrombogenic profile of pathological situations with increased cardiovascular risk.

Leukoreduced buffy coat–derived platelet concentrates photochemically treated with amotosalen HCl and ultraviolet A light stored up to 7 days: assessment of hemostatic function under flow conditions

BACKGROUND: Amotosalen plus ultraviolet A light photochemical treatment (PCT) inactivates high titers of bacteria, and other pathogens, in platelet concentrates (PCs) potentially allowing the storage of platelets (PLTs) for up to 7 days. Adhesion and aggregation of PLTs to injured vascular surfaces are critical aspects of PLT hemostatic function.

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.

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.