Prediction of Oscillations in Glycolysis in Ethanol-Consuming Erythrocyte-Bioreactors.

A mathematical model of energy metabolism in erythrocyte-bioreactors loaded with alcohol dehydrogenase and acetaldehyde dehydrogenase was constructed and analyzed. Such erythrocytes can convert ethanol to acetate using intracellular NAD and can therefore be used to treat alcohol intoxication. Analysis of the model revealed that the rate of ethanol consumption by the erythrocyte-bioreactors increases proportionally to the activity of incorporated ethanol-consuming enzymes until their activity reaches a specific threshold level. When the ethanol-consuming enzyme activity exceeds this threshold, the steady state in the model becomes unstable and the model switches to an oscillation mode caused by the competition between glyceraldehyde phosphate dehydrogenase and ethanol-consuming enzymes for NAD. The amplitude and period of metabolite oscillations first increase with the increase in the activity of the encapsulated enzymes. A further increase in these activities leads to a loss of the glycolysis steady state, and a permanent accumulation of glycolytic intermediates. The oscillation mode and the loss of the steady state can lead to the osmotic destruction of erythrocyte-bioreactors due to an accumulation of intracellular metabolites. Our results demonstrate that the interaction of enzymes encapsulated in erythrocyte-bioreactors with erythrocyte metabolism should be taken into account in order to achieve the optimal efficacy of these bioreactors.

Filterability of Erythrocytes in Patients with COVID-19.

For the first time, the influence of COVID-19 on blood microrheology was studied. For this, the method of filtering erythrocytes through filters with pores of 3.5 µm was used. Filterability was shown to significantly decrease with the increasing severity of the patient's condition, as well as with a decrease in the ratio of hemoglobin oxygen saturation to the oxygen fraction in the inhaled air (SpO2/FiO2). The filterability of ≤ 0.65, or its fast decrease during treatment, were indicators of a poor prognosis. Filterability increased significantly with an increase in erythrocyte count, hematocrit and blood concentrations of hemoglobin, albumin, and total protein. The effect of these parameters on the erythrocyte filterability is directly opposite to their effect on blood macrorheology, where they all increase blood viscosity, worsening the erythrocyte deformability. The erythrocyte filterability decreased with increasing oxygen supply rate, especially in patients on mechanical ventilation, apparently not due to the oxygen supplied, but to the deterioration of the patients' condition. Filterability significantly correlates with the C-reactive protein, which indicates that inflammation affects the blood microrheology in the capillaries. Thus, the filterability of erythrocytes is a good tool for studying the severity of the patient's condition and his prognosis in COVID-19.

Ammonium removal by erythrocyte-bioreactors based on glutamate dehydrogenase from Proteus sp. jointly with porcine heart alanine aminotransferase.

Excessive ammonium blood concentration causes many serious neurological complications. The medications currently used are not very effective. To remove ammonium from the blood, erythrocyte-bioreactors containing enzymes that processing ammonium have been proposed. The most promising bioreactor contained co-encapsulated glutamate dehydrogenase (GDH) and alanine aminotransferase (ALT). However, a low encapsulation of a commonly used bovine liver GDH (due to high aggregation), makes clinical use of such bioreactors impossible. In this study, new bioreactors containing ALT and non-aggregating GDH at higher loading were first produced using the flow dialysis method and the new bacterial GDH enzyme from Proteus sp. The efficacy of these erythrocyte-bioreactors and their properties (hemolysis, osmotic fragility, intracellular and extracellular activity of included enzymes, erythrocyte indices, and filterability) were studied and compared with native cells during 1-week storage. The ammonium removal rate in vitro by such erythrocyte-bioreactors increased linearly with an increase in encapsulated GDH activity. Alanine in vitro increased in accordance with ammonium consumption, which indicated the joint functioning of both included enzymes. Thus, novel bioreactors for ammonium removal containing GDH from Proteus sp. are promising for clinical use, since they have a more efficient GDH encapsulation and their properties are not inferior to previously obtained erythrocyte-bioreactors.

Theoretical Analysis of the Built-in Metabolic Pathway Effect on the Metabolism of Erythrocyte-Bioreactors That Neutralize Ammonium.

The limitations of the efficiency of ammonium-neutralizing erythrocyte-bioreactors based on glutamate dehydrogenase and alanine aminotransferase reactions were analyzed using a mathematical model. At low pyruvate concentrations in the external medium (below about 0.3 mM), the main limiting factor is the rate of pyruvate influx into the erythrocyte from the outside, and at higher concentrations, it is the disappearance of a steady state in glycolysis if the rate of ammonium processing is higher than the critical value (about 12 mM/h). This rate corresponds to different values of glutamate dehydrogenase activity at different concentrations of pyruvate in plasma. Oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) by glutamate dehydrogenase decreases the fraction of NADPH in the constant pool of nicotinamide adenine dinucleotide phosphates (NADP + NADPH). This, in turn, activates the pentose phosphate pathway, where NADP reduces to NADPH. Due to the increase in flux through the pentose phosphate pathway, stabilization of the ATP concentration becomes impossible; its value increases until almost the entire pool of adenylates transforms into the ATP form. As the pool of adenylates is constant, the ADP concentration decreases dramatically. This slows the pyruvate kinase reaction, leading to the disappearance of the steady state in glycolysis.

Erythrocytes as Carriers: From Drug Delivery to Biosensors.

Drug delivery using natural biological carriers, especially erythrocytes, is a rapidly developing field. Such erythrocytes can act as carriers that prolong the drug's action due to its gradual release from the carrier; as bioreactors with encapsulated enzymes performing the necessary reactions, while remaining inaccessible to the immune system and plasma proteases; or as a tool for targeted drug delivery to target organs, primarily to cells of the reticuloendothelial system, liver and spleen. To date, erythrocytes have been studied as carriers for a wide range of drugs, such as enzymes, antibiotics, anti-inflammatory, antiviral drugs, etc., and for diagnostic purposes (e.g. magnetic resonance imaging). The review focuses only on drugs loaded inside erythrocytes, defines the main lines of research for erythrocytes with bioactive substances, as well as the advantages and limitations of their application. Particular attention is paid to in vivo studies, opening-up the potential for the clinical use of drugs encapsulated into erythrocytes.

Kaolin, used to trigger coagulation in thrombin generation test, increases sensitivity of the method in hemophilia patients.

: Thrombin generation test (TGT) is well established tool to research blood coagulation in plasma of hemophilia patients. Traditionally coagulation in this test is triggered by a tissue factor (TF), an extrinsic coagulation pathway activator. However, it is known that disorders of the intrinsic pathway are most important for coagulation in hemophilia. In this study, we hypothesized that triggering coagulation via the intrinsic pathway could increase a sensitivity of the TGT to monitor hemophilia treatment. The aim of this study was to compare thrombin generation in hemophilia A patients with inhibitors to factor VIII before and after infusion of bypassing agent [recombinant-activated factor VIIa (rVIIa)] using standard activation of coagulation by TF or by kaolin, an activator of coagulation by intrinsic pathway. Endogenous thrombin potential (ETP) in nine patients was measured. ETP before (ETP0) and 60 min after rVIIa infusion (ETP60) were compared. It was shown that ETP0 and ETP60 were significantly different when using any coagulation activator (paired Student's t test, P = 0.017 and 3.7 × 10 for clotting activation by TF and kaolin, respectively). The ratios of ETP60/ETP0 were 1.2 ±â€Š0.2 or 30.0 ±â€Š22.4 (mean ±â€ŠSD, n = 9) for coagulation activated by TF or kaolin, respectively, and were significantly different (paired Student's t test, P < 0.005). The TGT clearly distinguished between ETP0 and ETP60 in the case of any coagulation activator, but ETP increasing after rVIIa infusion was significantly higher when activated with kaolin. This provided increased sensitivity of this method for monitoring hemophilia therapy.

Erythrocytes as bioreactors to decrease excess ammonium concentration in blood.

Increased blood ammonium concentrations cause neurological complications. Existing drugs are not always sufficiently effective. Alternatively, erythrocytes-bioreactors (EBRs) loaded with enzymes utilizing ammonium, were suggested for ammonium removal from blood. However all they worked only for a short period of time. The reasons for this were not investigated. In this study, EBR mathematical models were developed and analysed based on the reactions of glycolysis and different enzymes utilizing ammonium, which showed that the efficiency and duration of EBRs' functioning could be limited due to low permeability of the cell membrane for some key substrates and products. A new enzyme system including glutamate dehydrogenase and alanine aminotransferase was proposed and realised experimentally, which was not limited by cell membrane permeability for glutamate and α-ketoglutarate due to creating metabolic pathway where these metabolites were produced and consumed cyclically. New bioreactors removed ammonium in vitro at the rate of 1.5 mmol/h × lRBCs (for human bioreactors) and in vivo in a model of hyperammoniemia in mice at the rate of 2.0 mmol/h × lRBCs (for mouse bioreactors), which correlated with model calculations. Experimental studies proved the proposed mathematical models are correct. Mathematical simulation of erythrocyte-bioreactors opens new opportunities for analysing the efficiency of any enzyme included in erythrocytes.

Thrombodynamics, a new global coagulation test: Measurement of heparin efficiency.

The actual coagulation status may be reliably measured using only highly sensitive global functional tests; however, they are not numerous and all of them have disadvantages. Thrombodynamics (TD), a novel global coagulation test, is sensitive to hypo- and hypercoagulable states. The main properties of this test were investigated, and its capabilities for hemostasis analysis were verified through pharmacodynamic monitoring of the most widely used anticoagulants, heparins. The anticoagulant effects in the plasma of donors (n = 20) and patients after hip replacement (n = 20) spiked with unfractionated heparin or enoxaparin were measured in vitro to eliminate the influence of pharmacokinetic factors. Sensitivity for heparins was compared for activated partial thromboplastin time, thrombin generation tests and TD. TD was shown to reliably characterize the pharmacodynamics of any heparin in the entire range of its prophylactic and therapeutic concentrations. Inter-individual variability for the anticoagulant action of heparins was also calculated using the TD data. This variability did not differ between the investigated groups and did not exceed 12% and 20% for the stationary clot growth rate in the presence of unfractionated heparin and enoxaparin, respectively. That finding was in accordance with the values determined earlier using the thrombin generation test. The study results showed that TD has advantages over the other global methods of coagulation analysis. These advantages are good standardization, high reproducibility, independence of the parameter values from patient age and gender, and a narrower parameter distribution in a normal population. These results indicate that TD is a promising universal assessment method that improves the quality of hemostasis analysis because it more reliably detects deviations from the parameters' reference values.

Rapid Elimination of Blood Alcohol Using Erythrocytes: Mathematical Modeling and In Vitro Study.

Erythrocytes (RBCs) loaded with alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALD) can metabolize plasma ethanol and acetaldehyde but with low efficiency. We investigated the rate-limiting factors in ethanol oxidation by these enzymes loaded into RBCs. Mathematical modeling and in vitro experiments on human RBCs loaded simultaneously with ADH and ALD (by hypoosmotic dialysis) were performed. The simulation showed that the rate of nicotinamide-adenine dinucleotide (NAD+) generation in RBC glycolysis, but not the activities of the loaded enzymes, is the rate-limiting step in external ethanol oxidation. The rate of oxidation could be increased if RBCs are supplemented by NAD+ and pyruvate. Our experimental data verified this theoretical conclusion. RBCs loaded with the complete system of ADH, ALD, NAD+, and pyruvate metabolized ethanol 20-40 times faster than reported in previous studies. The one-step procedure of hypoosmotic dialysis is the optimal method to encapsulate ADH and ALD in RBCs after cell recovery, encapsulation yield, osmotic resistance, and RBC-indexes. Consequently, transfusion of the RBCs loaded with the complete metabolic system, including ADH, ALD, pyruvate, and NAD+ in the patients with alcohol intoxication, may be a promising method for rapid detoxification of blood alcohol based on metabolism.

Moderate plasma dilution using artificial plasma expanders shifts the haemostatic balance to hypercoagulation.

Artificial plasma expanders (PEs) are widely used in modern transfusion medicine. PEs do not contain components of the coagulation system, so their infusion in large volumes causes haemodilution and affects haemostasis. However, the existing information on this effect is contradictory. We studied the effect of the very process of plasma dilution on coagulation and tested the hypothesis that moderate dilution with a PE should accelerate clotting owing to a decrease in concentration of coagulation inhibitors. The standard clotting times, a thrombin generation test, and the spatial rate of clot growth (test of thrombodynamics) were used to assess donor plasma diluted in vitro with various PEs. The pH value and Ca+2 concentration were maintained strictly constant in all samples. The effect of thrombin inhibitors on dilution-induced hypercoagulation was also examined. It was shown that coagulation was enhanced in plasma diluted up to 2.0-2.5-fold with any PE. This enhancement was due to the dilution of coagulation inhibitors in plasma. Their addition to plasma or PE could partially prevent the hypercoagulation shift.

The modification of the thrombin generation test for the clinical assessment of dabigatran etexilate efficiency.

A new oral anticoagulant, dabigatran etexilate (DE, a prodrug of direct thrombin inhibitor (DTI) dabigatran), has been used clinically to prevent thrombosis. The assessment of dabigatran efficiency is necessary in some clinical cases, such as renal insufficiency, risk of bleeding, and drug interactions. However, a specific thrombin generation test (TGT) that is one of the most informative and sensitive to anticoagulant therapy (calibrated automated thrombinography (САТ)) shows a paradoxical increase of test parameters, such as endogenous thrombin potential (ETP) and peak thrombin, in patients receiving DE. The paradoxical behaviour of ETP and peak thrombin in these patients in the presence of DTIs is mostly caused by a decrease in the activity of thrombin in the α2-macroglobulin-thrombin complex that is used as a calibrator in CAT. For a correct estimation of the TGT parameters in patient's plasma containing DTIs we proposed to use our previously described alternative calibration method that is based on the measurement of the fluorescence signal of a well-known concentration of the reaction product (7-amino-4-methylcoumarin). In this study, the validity of such approach was demonstrated in an ex vivo study in patients with knee replacement and two special patients with multiple myeloma, who received DE for thrombosis prophylaxis.

Application of Molecular Modeling to Development of New Factor Xa Inhibitors.

In consequence of the key role of factor Xa in the clotting cascade and absence of its activity in the processes that do not affect coagulation, this protein is an attractive target for development of new blood coagulation inhibitors. Factor Xa is more effective and convenient target for creation of anticoagulants than thrombin, inhibition of which may cause some side effects. This study is aimed at finding new inhibitors of factor Xa by molecular computer modeling including docking SOL and postdocking optimization DISCORE programs. After validation of molecular modeling methods on well-known factor Xa inhibitors the virtual screening of NCI Diversity and Voronezh State University databases of ready-made low molecular weight species has been carried out. Seventeen compounds selected on the basis of modeling results have been tested experimentally in vitro. It has been found that 12 of them showed activity against factor Xa (IC50 = 1.8-40 µM). Based on analysis of the results, the new original compound was synthesized and experimentally verified. It shows activity against factor Xa with IC50 value of 0.7 µM.

Thromboelastography, thrombin generation test and thrombodynamics reveal hypercoagulability in patients with multiple myeloma.

Patients with multiple myeloma (MM) are at increased risk of venous thromboembolism. Therefore, adequate laboratory control of hemostasis and subsequent adjustments of anticoagulant therapy are necessary. We studied hemostasis changes using thromboelastography (TEG), thrombin generation test (TGT) and thrombodynamics (TD) in primary MM patients (PMMpt, n=25) and patients in remission (RMMpt, n=34) during blood stem cell (BSC) mobilization. TD and TEG reveal hypercoagulability in PMMpt (*p<0.05) in relation to healthy volunteers. There was no difference in any of the tests between PMMpt and RMMpt. We detected no heparin effect in 22% of patients one day after the onset of the prophylactic heparin treatment (500 IU/h) during BSC mobilization; tests shifted toward the hypercoagulability in 75% of patients one day after cyclophosphamide (4 g/m2) chemotherapy. Global hemostasis tests were in good agreement with each other, revealed hypercoagulability and heparin "resistance" in patients with MM and may be useful for therapy individualization.

Thrombin inhibitors based on single-stranded DNA aptamers.

Investigation of inhibitory effect of two single-stranded DNA thrombin-inhibiting aptamers (15TBA and 31TBA) on fibrin polymerization in fibrinogen solutions and comparison of anticoagulant properties of these aptamers by a new global coagulation test of thrombodynamics. Measurement of aptamers' functional stability in human plasma and blood in vitro in order to investigate the involvement of 3'-exonuclease in fast decrease of aptamers' functional activity in vivo. Thrombin inhibition activity was measured in a buffer system in vitro as effects of aptamers on fibrin polymerization. Anticoagulant activity was investigated by measuring the spatial clot growth rate in the presence of aptamers. The stability of aptamers during incubation in human plasma was investigated in vitro by measuring activated partial thromboplastin time. Both aptamers dose-dependently inhibit fibrin polymerization in a buffer solution (IC50=10 nm for 15TBA and 3 nm for 31TBA) and are effective anticoagulants in human plasma (IC50 for spatial clot growth rate decreasing are 9.5 µmol/l and 4.0 µmol/l for 15TBA and 31TBA, correspondingly). Both aptamers remain stable in plasma or whole blood in vitro for at least 4 h. It was shown that 31TBA was 2-3 times more effective than 15TBA. Both aptamers were stable in human plasma and whole blood in vitro. So, the 3'-exonuclease could not be the reason for fast decrease of aptamers' functional activity in vivo. The main role in the removal of oligonucleotides from the circulation is played obviously by the liver.

Antiplatelet agents can promote two-peaked thrombin generation in platelet rich plasma: mechanism and possible applications.

BACKGROUND: Thrombin generation assay is a convenient and widely used method for analysis of the blood coagulation system status. Thrombin generation curve (TGC) is usually bell-shaped with a single peak, but there are exceptions. In particular, TGC in platelet-rich plasma (PRP) can sometimes have two peaks. OBJECTIVE: We sought to understand the mechanism underlying the occurrence of two peaks in the PRP thrombin generation curve. METHODS: Tissue factor-induced thrombin generation in PRP and platelet-poor plasma (PPP) was monitored using continuous measurement of the hydrolysis rate of the thrombin-specific fluorogenic substrate Z-Gly-Gly-Arg-AMC. Expression of phosphatidylserine (PS) and CD62P on the surface of activated platelets was measured by flow cytometry using corresponding fluorescently labeled markers. RESULTS: The addition of the P(2)Y(12) receptor antagonist MeS-AMP (160 µM), 83 nM prostaglandin E(1) (PGE(1)), or 1.6% DMSO to PRP caused the appearance of two peaks in the TGC. The PS exposure after thrombin activation on washed platelets in a suspension supplemented with DMSO, PGE(1) or MeS-AMP was delayed, which could indicate mechanism of the second peak formation. Supplementation of PRP with 1.6% DMSO plus 830 nM PGE(1) mediated the disappearance of the second peak and decreased the amplitude of the first peak. Increasing the platelet concentration in the PRP promoted the consolidation of the two peaks into one. CONCLUSIONS: Procoagulant tenase and prothrombinase complexes in PRP assemble on phospholipid surfaces containing PS of two types--plasma lipoproteins and the surface of activated platelets. Thrombin generation in the PRP can be two-peaked. The second peak appears in the presence of platelet antagonists as a result of delayed PS expression on platelets, which leads to delayed assembly of the membrane-dependent procoagulant complexes and a second wave of thrombin generation.

Anticoagulant therapy: basic principles, classic approaches and recent developments.

The standard multipotent anticoagulants (unfractionated and low molecular weight heparins, antagonists of vitamin K) are commonly used for treatment and/or prophylaxis of different thrombotic complications, such as deep vein thrombosis, thrombophilia, pulmonary embolism, myocardial infarction, stroke and so on. Advantages and shortcomings of these anticoagulants are considered. The modern tendencies to use small selective direct inhibitors of thrombin or factor Xa are surveyed. The search of the new targets in the coagulation cascade for development of new promising anticoagulants and improvement in antithrombotic therapy is discussed.

New synthetic thrombin inhibitors: molecular design and experimental verification.

BACKGROUND: The development of new anticoagulants is an important goal for the improvement of thromboses treatments. OBJECTIVES: The design, synthesis and experimental testing of new safe and effective small molecule direct thrombin inhibitors for intravenous administration. METHODS: Computer-aided molecular design of new thrombin inhibitors was performed using our original docking program SOL, which is based on the genetic algorithm of global energy minimization in the framework of a Merck Molecular Force Field. This program takes into account the effects of solvent. The designed molecules with the best scoring functions (calculated binding energies) were synthesized and their thrombin inhibitory activity evaluated experimentally in vitro using a chromogenic substrate in a buffer system and using a thrombin generation test in isolated plasma and in vivo using the newly developed model of hemodilution-induced hypercoagulation in rats. The acute toxicities of the most promising new thrombin inhibitors were evaluated in mice, and their stabilities in aqueous solutions were measured. RESULTS: New compounds that are both effective direct thrombin inhibitors (the best K(I) was <1 nM) and strong anticoagulants in plasma (an IC(50) in the thrombin generation assay of approximately 100 nM) were discovered. These compounds contain one of the following new residues as the basic fragment: isothiuronium, 4-aminopyridinium, or 2-aminothiazolinium. LD(50) values for the best new inhibitors ranged from 166.7 to >1111.1 mg/kg. A plasma-substituting solution supplemented with one of the new inhibitors prevented hypercoagulation in the rat model of hemodilution-induced hypercoagulation. Activities of the best new inhibitors in physiological saline (1 µM solutions) were stable after sterilization by autoclaving, and the inhibitors remained stable at long-term storage over more than 1.5 years at room temperature and at 4°C. CONCLUSIONS: The high efficacy, stability and low acute toxicity reveal that the inhibitors that were developed may be promising for potential medical applications.

A new drug form of blood coagulation factor IX: red blood cell-entrapped factor IX.

BACKGROUND: Deficiency of factor IX causes hemophilia B, and primary treatment for hemophilia B is based on recurrent infusions of deficient factor IX. Frequent infusions of foreign protein diminish patients' quality of life, and increase the risk of development of immune reaction. We entrapped factor IX into erythrocytes-carriers (pharmacocytes) to prolong the drug's circulation life time, and to prevent immune response to the drug. MATERIAL/METHODS: Factor IX was biotynilated by standard method and then loaded aseptically into volunteers' erythrocytes with our gentle procedure of stepwise dialysis. The comparison of pharmacokinetics for free and autologous erythrocytes-entrapped biotinylated factor IX (FIXbiot) was done. Concentrations of factor IXbiot in plasma and lysates of erythrocytes were quantitatively assessed with a sandwich ELISA. RESULTS: Stepwise dialysis method allowed stable loading of factor IXbiot into erythrocytes. Elimination of the loaded erythrocytes followed the first-order kinetics. The mean half-time of elimination for free FIXbiot was 8.8±5.6 hours, and for RBC-entrapped factor IXbiot 73.9±16.0 hours. Elimination of FIXbiot from plasma did not follow the first order kinetics because this factor concentration depended not only on the rate of its elimination, but also on the rate of factor appearance in plasma as a result of pharmackocytes' degradation. A rough estimate of the feasibility of the approach was done. CONCLUSIONS: The life time of the erythrocyte-based form of FIXbiot in the circulation is significantly (5-10 times) prolonged compared with its free form, suggesting that this form has potential clinical applications.

Mechanisms of action of recombinant activated factor VII in the context of tissue factor concentration and distribution.

Supraphysiological concentrations of recombinant activated factor VII (rVIIa, NovoSeven) are used to control bleeding in hemophilia. Current experimental evidence suggests that rVIIa may increase thrombin generation via two pathways: one being tissue factor (TF)-dependent and another being activated platelet-dependent. Contribution of TF to the rVIIa action may justify different administration profiles of rVIIa. In the present study, thrombin and fibrin generation and spatial clot formation assays in platelet-free hemophilia A and normal plasma were used to investigate this contribution. By varying the concentration of TF and the way it becomes available to plasma, we obtained the following results. Activation of clotting with less than 5 pmol/l of TF facilitates thrombin and fibrin generation at low, but not at supraphysiological rVIIa concentrations. Activation with more than 13 pmol/l of TF saturates thrombin and fibrin generation kinetics, making it insensitive to rVIIa. rVIIa minimally modulates clot growth on the surface of TF-expressing fibroblasts. On the contrary, rVIIa produces spontaneous clot formation in nonflowing platelet-free plasma far away from fibroblasts via plasma lipid particles. Therefore, both the concentration and the distribution of TF determine relevance of a particular experimental system for the studies of rVIIa action. The results indicate that 300-1600 nmol/l (megadoses) of rVIIa may deliver coagulation outside of the TF-rich areas of blood vessel damage via the platelet-derived microparticles. Therefore, rate and extent of platelet-derived microparticles generation might be important with regard to rVIIa treatment safety.