Irene Lopez-Vilchez

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.

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.

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.

Reversal of rivaroxaban-induced alterations on hemostasis by different coagulation factor concentrates - In vitro studies with steady and circulating human blood -

BACKGROUND Despite the good safety of rivaroxaban, there is limited information on strategies for urgent reversal of its antihemostatic effects. METHODS AND RESULTS Alterations of hemostasis induced by rivaroxaban (230 ng/ml) were assessed by using several tests applied to steady and circulating human blood. Effects on thrombin generation (TG) and thromboelastometry (TEM) parameters were measured. Modifications in platelet adhesive, aggregating and procoagulant activities were evaluated in studies with circulating blood. The potential reversal of prothrombin complex concentrates (PCCs; 50 IU/kg), activated PCCs (aPCCs; 75 IU/kg), or recombinant factor VIIa (rFVIIa; 270 μg/kg) was evaluated. Impairment of TG parameters induced by rivaroxaban were corrected by the different concentrates (aPCC≥PCC>rFVIIa). Prolonged clotting times and reduced clot firmness caused by rivaroxaban on TEM tests were improved by different concentrates (rFVIIa≥aPCC>PCC). Rivaroxaban significantly reduced platelets and fibrin interactions with damaged vascular surfaces in perfusion studies. While alterations of platelet interactions were favourably counteracted by rFVIIa or aPCCs, reductions in fibrin formation were only partially restored by the different factor concentrates (rFVIIa>aPCC≥PCC). CONCLUSIONS Rivaroxaban-induced alterations on coagulation parameters measured through assays performed under static conditions were easily reversed by the different concentrates. Studies under flow conditions revealed that these concentrates normalized the action of rivaroxaban on platelets, and significantly improved fibrin formation; although in the later case, levels were not restored to the pre-treatment value.

Impact of experimental haemodilution on platelet function, thrombin generation and clot firmness: effects of different coagulation factor concentrates

BACKGROUND Haemodilution during resuscitation after massive haemorrhage may worsen the coagulopathy and perpetuate bleeding. MATERIALS AND METHODS Blood samples from healthy donors were diluted (30 and-60%) using crystalloids (saline, Ringers lactate, Plasmalyte(TM)) or colloids (6% hydroxyethylstarch [HES130/0.4], 5% human albumin, and gelatin). The effects of haemodilution on platelet adhesion (Impact R), thrombin generation (TG), and thromboelastometry (TEM) parameters were analysed as were the effects of fibrinogen, prothrombin complex concentrates (PCC), activated recombinant factor VII (FVIIa), and cryoprecipates on haemodilution. RESULTS Platelet interactions was already significantly reduced at 30% haemodilution. Platelet reactivity was not improved by addition of any of the concentrates tested. A decrease in TG and marked alterations of TEM parameters were noted at 60% haemodilution. HES130/0.4 was the expander with the most deleterious action. TG was significantly enhanced by PCC whereas rFVIIa only caused a mild acceleration of TG initiation. Fibrinogen restored the alterations of TEM parameters caused by haemodilution including those caused by HES 130/0.4. Cryoprecipitates significantly improved the alterations caused by haemodilution on TG and TEM parameters; the effects on TG disappeared after ultracentrifugation of the cryoprecipitates. DISCUSSION The haemostatic alterations caused by haemodilution are multifactorial and affect both blood cells and coagulation. In our in vitro approach, HES 130/0.4 had the most deleterious effect on haemostasis parameters. Coagulation factor concentrates did not improve platelet interactions in the Impact R, but did have favourable effects on coagulation parameters measured by TG and TEM. Fibrinogen notably improved TEM parameters without increasing thrombin generation, suggesting that this concentrate may help to preserve blood clotting abilities during haemodilution without enhancing the prothrombotic risk.

Redistribution and Hemostatic Action of Recombinant Activated Factor VII Associated with Platelets

Clinical evidence accumulated from hemophilic patients during prophylaxis with recombinant activated factor VII (rFVIIa) suggests that the duration of the hemostatic action of rFVIIa exceeds its predicted plasma half-life. Mechanisms involved in this outcome have not been elucidated. We have investigated in vitro the redistribution of rFVIIa in platelets from healthy donors, patients with FVII deficiency, and one patient with Bernard-Soulier syndrome. Platelet-rich plasma was exposed to rFVIIa (3 to 60 μg/mL). Flow cytometry, immunocytochemistry, and coagulation tests were applied to detect and quantify rFVIIa. The hemostatic effect of rFVIIa associated to platelets was evaluated using perfusion models. Our studies revealed a dose-dependent association of rFVIIa to the platelet cytoplasm with redistribution into the open canalicular system, and α granules. Mechanisms implicated in the internalization are multiple, involve GPIb and GPIV, and require phospholipids and cytoskeletal assembly. After platelet activation with thrombin, platelets exposed rFVIIa on their membrane. Perfusion studies revealed that the presence of 30% of platelets containing FVIIa improved platelet aggregate formation and enhanced fibrin generation (P < 0.01 versus control). Our results indicate that, at therapeutic concentrations, rFVIIa can be internalized into platelets, where it is protected from physiological clearance mechanisms and can still promote hemostatic activity. Redistribution of rFVIIa into platelets may explain the prolonged prophylactic effectiveness of rFVIIa in hemophilia.

Prothrombotic platelet phenotype in major depression: Downregulation by antidepressant treatment

BACKGROUND Serotonergic mechanisms have been suggested as a link between major depression and cardiovascular risk. We investigated the existence of a prothrombotic condition in depressed patients and its possible modulation during treatment with a selective serotonin-reuptake inhibitor (SSRI). METHODS Modifications in a series of biomarkers of platelet and coagulation activation were evaluated in blood from 19 patients with a major depression disorder (MDD) at the time of diagnosis, and at 8 and 24 weeks of treatment with escitalopram. Response of blood aliquots recirculated through a thrombogenic surface was assessed in a thrombosis model. Results were compared with those of 20 healthy-matched controls. RESULTS In comparison with controls, platelets from MDD patients showed elevated volumes (p<0.01), significantly enhanced aggregating response to arachidonic acid and augmented expression of GPIb, fibrinogen, factor V, and anionic phospholipids by flow cytometry (p<0.05). Clot firmness and procoagulant activity of platelet-associated tissue factor were also significantly elevated (p<0.05). Studies with circulating blood revealed increased fibrin formation in early diagnosed patients (71.1±9.5% vs. 45.8±5.3%; p<0.05 vs. controls). After 24 weeks of treatment with escitalopram, the majority of the alterations observed were normalized, except for a residual increased expression of GPIIbIIIa (p<0.05) and persistent alterations in thromboelatometic parameters. LIMITATIONS Despite the reduced number of followed-up patients our findings were consistent reaching statistical significance. CONCLUSIONS Our results reveal a prothrombotic phenotype in MDD patients. While continuous treatment with an SSRI downregulated the majority of the biomarkers analyzed, alterations in viscoelastic parameters of clot formation remained unaffected by the antidepressant treatment.

Internalization of tissue factor by platelets

Tissue factor (TF) has been found associated with platelets. Mechanisms responsible for TF-platelet interactions and transport are not fully understood. We explored the response of isolated washed platelets to preparations of recombinant (rTF) or placental (pTF) human TF, exposed on lipid microvesicles (MVs). Sequential ultrastructural and immunocytochemical studies revealed trafficking of these TF preparations, being endocytosed by platelets into channels of the open canalicular system (OCS) and accumulating in the cytoplasm and occasionally in alpha-granules. The process of internalization of TF-MVs was accomplished in less than 30 min, being faster for the placental (pTF) than for the recombinant (rTF) preparation. Signs of mild platelet activation with pseudopodia formation were observed at early stages of internalization. Platelets returned to an apparent resting state after 5-10 min. All of these observations paralleled with modifications on patterns of tyrosine phosphorylation for several signaling proteins. Our studies demonstrate that platelets possess mechanisms to capture and incorporate TF-rich vesicles. These processes were accelerated by the presence of other contaminating cellular antigens in the vesicles (pTF). TF carried by platelets could play a potential role in platelet thrombus formation and by extension in the development of ischemic complications.