Marian Tomasiak

Combination of LC–MS- and GC–MS-based Metabolomics to Study the Effect of Ozonated Autohemotherapy on Human Blood

Ozonated autohemotherapy (O3-AHT) is a medical approach during which blood obtained from the patient is ozonated and injected back into the body. Despite an increasing number of evidence that O3-AHT is safe, this type of therapy remains controversial. To extend knowledge about the changes in blood evoked by O3-AHT, LC-MS- and GC-MS-based metabolic fingerprinting was used to compare plasma samples obtained from blood before and after the treatment with potentially therapeutic concentrations of ozone. The procedure was performed in PVC bags utilized for blood storage to study also possible interactions between ozone and plastic. By use of GC-MS, an increase in lactic acid and pyruvic acid was observed, which indicated an increased rate of glycolysis. With LC-MS, changes in plasma antioxidants were observed. Moreover, concentrations of lipid oxidation products (LOP) and lysophospholipids were increased after ozone treatment. This is the first report of increased LOPs metabolites after ozonation of blood. Seven metabolites detected by LC-QTOF-MS only in ozonated samples could be considered as novel biomarkers of oxidative stress. Several plasticizers have been detected by both techniques in blood stored in PVC bags. PVC is known to be an ozone resistant material, but ozonation of blood in PVC bags stimulates leaching of plasticizers into the blood.

Inflammatory markers and acid-base equilibrium in exhaled breath condensate of stable and unstable asthma patients.

Background: Nitrosative and acid stress play an important role in the pathogenesis of asthma. The aim of this study was to evaluate whether, in asthmatics, a link exists between the concentrations of nitrite/nitrate, ammonia and pH values in exhaled breath condensate (EBC) and asthma severity, lung function, exhaled nitric oxide (FENO), total IgE, eosinophil cationic protein (ECP) and blood eosinophilia. Methods: The above-mentioned parameters were measured in 19 healthy volunteers and 91 allergic asthmatics divided into three groups, i.e. 22 subjects with steroid-naïve stable asthma, 35 with inhaled corticosteroid (ICS)-treated stable asthma and 34 with ICS-treated unstable asthma. Results: Compared with healthy subjects, EBC from asthmatics had significantly lower pH values and ammonia concentrations and significantly higher levels of nitrite/nitrate. The extent of these changes was higher in patients with unstable than in patients with steroid-naïve and stable ICS-treated asthma. The EBC pH was positively correlated with ammonia and negatively correlated with nitrite/nitrate, FENO or blood eosinophilia in all three groups of asthmatics. Significant positive correlations between EBC nitrite/nitrate and blood eosinophilia, ECP levels or FENO were observed in all groups of asthmatics. Significant negative correlations between EBC ammonia and nitrite/nitrate, FENO, ECP concentrations or blood eosinophilia were demonstrated in the groups of ICS-naïve and ICS-treated stable asthmatics. Conclusions: In asthmatic patients there is a relationship between EBC pH, ammonia and nitrite/nitrate concentrations and other recognized markers of airway inflammation. EBC pH values, ammonia and nitrite/nitrate levels measured together may help to assess airway inflammatory status and asthma severity.

The importance of Na + /H + exchanger for the generation of procoagulant activity by porcine blood platelets

This study addresses the role of the Na + /H + exchanger (NHE) in the generation of procoagulant activity in blood platelets. It was found that monensin (simulating the action of NHE) and gramicidin (causing sodium influx without concomitant H + efflux) produced a dose- and time-dependent increase in platelet procoagulant activity. Alkalinization of platelet cytosol by NH 4 Cl failed to evoke a procoagulant response. Collagen-induced procoagulant response was diminished in the absence of external Na + and in the presence of EIPA (NHE inhibitor) or GF 109203X (protein kinase C inhibitor). Phorbol ester (PMA) produced a dose- and time-dependent generation of procoagulant response which was inhibited in the absence of the external Na+ and in the presence of EIPA. Platelets stimulated by collagen and PMA accumulated 22 Na + , a phenomenon inhibited in the presence of EIPA. The data indicate that development of procoagulant activity in platelets may occur as a result of Na + influx via Na + /H + exchanger.

Peroxynitrite – altered platelet mitochondria—A new link between inflammation and hemostasis

Using porcine blood, we tested the hypothesis that peroxynitrite (ONOO(-)) may affect platelet-fibrin clot formation, clot retraction rate (CRR) and fibrinolysis through the inhibition of platelet energy production. It was found that ONOO(-) reduces CRR and enlarges final clot size in platelet rich plasma (PRP) (IC(50)=100μM) and in whole blood (IC(50)=200μM) dose-dependently. In a reconstituted system (washed platelets+fibrinogen), CRR was inhibited by 5-100nM ONOO(-) (IC(50)=25nM). Concentrations of ONOO(-) reducing CRR in PRP, inhibited platelet oxygen consumption, augmented lactate production and decreased total ATP contents in clots derived from PRP. In washed platelets ONOO(-) (5-20nM) produced a drop of the mitochondrial transmembrane potential (ΔΨ(m)). Blocking of mitochondrial energy production resulted in a reduction of CRR, whereas inhibition of glycolysis failed to affect CRR. ONOO(-), up to 300μM, failed to affect coagulation in platelet free plasma. Fibrinolysis of platelet-fibrin clots was enhanced by ONOO(-) (25-300μM), cytochalasin B and following the reduction of platelet energy production. Fibrinolysis of plasma clots was resistant to ONOO(-) treatment up to a concentration of 500μM. Tromboelastometry (ROTEM) measurements performed in PRP show that inhibition of platelet energy production or treatment with ONOO(-) (100-300μM) diminishes MCF, alpha angle and MCE parameters. Blockage the platelet contractile apparatus by cytochalasin B resulted in reduction of CRR and ROTEM variables (MCF, alpha angle, MCE). We conclude that physiologically relevant ONOO(-) concentrations may inhibit clot retraction, reduce clot stability and accelerate its lysis through the inhibition of platelet mitochondrial energy production.

Vasopressin acts on platelets to generate procoagulant activity.

The aim of our study was to examine whether arginine vasopressin (AVP) is able to evoke in human platelets a procoagulant response due to activation of an Na+/H+ exchanger. It was found that treatment of platelets with AVP (20–100 nmol/l) results in generation of a weak calcium signal, activation of Na+/H+ exchanger, aggregation, and development of a procoagulant response. The AVP-evoked procoagulant response was dose and time dependent, weaker than that produced by collagen or monensin (mimics Na+/H+ exchanger), and less pronounced following the inhibition of Na+/H+ exchanger by 5-(N-ethyl-N-isopropyl) amiloride or genistein. Flow cytometry studies reveal that in-vitro platelet treatment with AVP results in an unimodal left shift in the forward and side scatter of the entire platelet population, indicating morphological changes on the plasma membrane. The shift was dose related, weaker than that evoked by collagen, similar to that produced by monensin and strongly reduced in the presence of 5-(N-ethyl-N-isopropyl) amiloride or genistein. Using flow cytometry, we demonstrated enhanced expression of phosphatidylserine on the AVP-treated platelets. AVP-evoked phosphatidylserine exposure was dose dependent, inhibited by 5-(N-ethyl-N-isopropyl) amiloride or genistein and weaker than that produced by collagen. AVP in a dose-dependent manner produced a rise in platelet volume. The swelling was inhibited by 5-(N-ethyl-N-isopropyl) amiloride, and its kinetics was similar to that observed in the presence of monensin. We conclude that prolonged treatment of platelets with AVP results in a procoagulant response, which may occur as a consequence of Na+ influx mediated by Na+/H+ exchanger.

[Pathophysiological consequences of hemolysis. Role of cell-free hemoglobin].

Abundant hemolysis is associated with a number of inherent and acquired diseases including sickle-cell disease (SCD), polycythemia, paroxysmal nocturnal hemoglobinuria (PNH) and drug-induced hemolytic anemia. Despite different etiopathology of hemolytic diseases, many concomitant symptoms are comparable and include e.g. hypertension, hemoglobinuria and hypercoagulation state. Studies in the last years have shown a growing list of mechanisms lying at the basis of those symptoms, in particular irreversible reaction between cell-free hemoglobin (Hb) and nitric oxide (NO) - endogenous vasorelaxant and anti-thrombotic agent. Saturation of protective physiological cell-free Hb-scavenging mechanisms results in accumulation of Hb in plasma and hemoglobinemia. Extensive hemoglobinemia subsequently leads to hemoglobinuria, which may cause kidney damage and development of Fanconi syndrome. A severe problem in patients with SCD and PNH is pulmonary and systemic hypertension. It may lead to circulation failure, including stroke, and it is related to abolition of NO bioavailability for vascular smooth muscle cells. Thrombotic events are the major cause of death in SCD and PNH. It ensues from lack of platelet inhibition evoked by Hb-mediated NO scavenging. A serious complication that affects patients with excessive hemolysis is erectile dysfunction. Also direct cytotoxic, prooxidant and proinflammatory effects of cell-free hemoglobin and heme compose the clinical picture of hemolytic diseases. The pathophysiological role of plasma Hb, mechanisms of its elimination, and direct and indirect (via NO scavenging) deleterious effects of cell-free Hb are presented in detail in this review. Understanding the critical role of hemolysis and cell-free Hb is important in the perspective of treating patients with hemolytic diseases and to design new effective therapies in future.

Platelet-related fibrinolysis resistance in patients suffering from PV. Impact of clot retraction and isovolemic erythrocytapheresis

Using patients with polycythemia vera (PV) as an experimental model, we evaluated the impact of clot retraction (CR) and architecture of the clot on fibrinolysis. We studied the kinetics of clot retraction and the fibrinolysis rate in whole blood from 48 PV patients and 48 healthy controls. Measurements were performed before and after isovolemic eryhrocytapheresis (ECP). CR was measured by optical method. Clot lysis time (CLT) and maximum clot firmness (MCF) were measured by thromboelastometry in recalcified blood supplemented with t-PA and tissue factor. Compared with healthy controls, CR rate in PV patients was higher (0.0219 vs. 0.0138; p<0.001), the clot volume smaller and MCF elevated (64 vs. 58 mm). CR rate correlated with platelet count (r=0.546; p=0.001) but not with erythrocyte concentration (r=0.192; p>0.3). Compared with healthy controls, CLT in PV patients was significantly prolonged (158 min vs. 71 min). Fibrinolysis rate inversely correlated with CR rate (r=-0.566; p<0.001); MCF (r=-0.704; p<0.001) and platelet count (r=-0.461; p<0.001). As judged by confocal microscope, in comparison to healthy controls, clots formed in blood from PV patients demonstrated booth a distinctly higher degree of crosslinking and possessed thinner fibers. Altered CR, MCF and fibrinolysis speeds were not normalized following the ECP procedure. Tirofiban (a blocker of platelet GPIIb/IIIa receptors), unlike aspirin, normalized abnormal CR and fibrinolysis in blood from PV patients. Collectively, our data indicate that in PV patients, abnormal CR may result in formation of thrombi that are more resistant to fibrinolysis. ECP and aspirin failed to normalize platelet-related fibrinolysis resistance.

Peroxynitrite may affect clot retraction in human blood through the inhibition of platelet mitochondrial energy production

Peroxynitrite (ONOO(-)) contributes to hemostasis abnormalities associated with inflammatory states by a poorly understood mechanism. Here we show that ONOO(-) may affect clot retraction (CR), an important step in hemostasis, by reducing contractility of human platelets resulting from the inhibition of mitochondrial energy production. Reduced CR may result in thromboembolic and hemorrhage events. The results show that in human blood, in vitro, physiologically relevant ONOO(-) concentrations reduce clot retraction rate and enlarge final clot size. The stressor was more effective in reconstituted system consisting of washed platelets and fibrinogen, (IC₅₀=25 nM) than in platelet rich plasma (IC₅₀=75 μM) or in whole blood (IC₅₀=120 μM), indicating that its effect depends on the number of targets. Retardation of CR by lower concentrations of ONOO(-) resulted in reduction of platelet energy production due to impairment of mitochondria but not from tyrosine nitration or inhibition of actin polymerization. In washed platelets nanomolar ONOO(-) concentrations produced a drop of the mitochondrial transmembrane potential (ΔΨm) explaining high sensitivity of CR (a large consumer of platelet energy) to stressor. Thromboelastometry measurements showed that ONOO(-) may diminish clot stability and elasticity through the reduction of platelet contractility. Our findings suggest that in humans ONOO(-)- altered platelet mitochondria represent a new link between inflammation and hemostasis.

The involvement of the Na(+)/H(+) exchanger in the formation of microvesicles by porcine platelets.

Activated platelets release microvesicles, which express procoagulant activity. The mechanism by which vesicles are formed is not entirely clear. This study was undertaken to determine whether a link exists between the operation of the plasma membrane Na(+)/H(+) exchanger (NHE) and vesiculation. It was found, that platelets treated with NHE-simulating monensin and the sodium influx-inducing gramicidin (without concomitant H+ efflux) produced vesicles demonstrating procoagulant activity. Alkalinization of platelet cytosol by NH4Cl failed to evoke vesicle release. Collagen and phorbol ester (PMA)-evoked vesiculation was diminished in the presence of 5-(N-ethyl-N-isopropyl amiloride) (EIPA, inhibitor of NHE) or GF 109203X (inhibitor of protein kinase C). Vesicle formation induced by collagen, PMA, and the calcium ionophore A23187 was less pronounced in the absence of external Na+. In comparison with collagen, thrombin was a stronger inducer of vesiculation. Platelets stimulated by thrombin, collagen, and PMA accumulated 22Na+, a phenomenon inhibited in the presence of EIPA. Collagen-evoked vesicle formation started with aggregation but culminated after its completion. The data indicate a significant contribution of the Na(+)/H(+) exchanger in the formation of microvesicles by porcine platelets.

Nitric oxide scavenging by cell-free hemoglobin may be a primary factor determining hypertension in polycythemic patients

Abstract We tested the hypothesis that hypertension associated with polycythemia vera (PV) may be related to hemoglobin released from erythrocytes (cell-free hemoglobin, fHb). We assessed hematocrit, mean arterial pressure (MAP), blood viscosity, and the level of fHb and nitrite/nitrate (NOx) in the plasma of 73 PV patients and 38 healthy controls. The effect of isovolemic erythrocytapheresis (ECP) on the considered parameters was also studied. From the whole group of PV patients a subset of subjects with normal (normotensive patients, n = 16) and elevated MAP (hypertensive patients, n = 57) can be subtracted. It was found that in comparison with healthy controls, PV patients have significantly (p ≤ 0.01) elevated Hct (0.567 vs. 0.422), blood viscosity (5.45 vs. 3.56 cP), MAP (106.8 vs. 93.8 mmHg), plasma fHb (9.7 vs. 2.8 mg/dL), and NOx levels (34.1 vs. 27.5 μM). Compared with normotensive patients, hypertensive PV patients demonstrated a higher rise in fHb (10.2 vs. 8.0) and plasma NOx levels (35.8 vs. 31.0). In PV patients, fHb positively correlates with MAP (r = 0.489), NOx levels (r = 0.461), hematocrit (r = 0.428), and viscosity (r = 0.393). Blood viscosity positively correlated with hematocrit (r = 0.894), but not with other considered parameters. In PV patients MAP poorly correlated with hematocrit, whereas the correlation between MAP and NOx altered from − 0.325 (healthy control) to + 0.268 (PV patients). ECP procedure was associated with a significant (p < 0.01) reduction of hematocrit, fHb, blood viscosity, and MAP. In the normotensive subgroup of PV patients the ECP procedure did not affect MAP. It can be concluded that accelerated scavenging of nitric oxide by fHb rather than high Hct may be a key factor determining the development of hypertension in PV patients.