HAuCl4, Putative General Aquaporins Blocker, Reduces Platelet Spreading, Filopodia Formation, Procoagulant Response, and Thrombus Formation Under Flow.

Background: Recent studies indicate that aquaporin (AQP) water channels have a regulatory function in human platelet secretion and in procoagulant response of murine platelets. However, the engagement of AQPs in morphological changes, procoagulant response, and thrombus formation in human blood has never been investigated. Methods: Confocal microscopy was used to study platelet spreading, filopodia formation, ballooning, and thrombus formation under flow. Flow cytometry was utilized to assess platelet phosphatidylserine (PS) exposure and microparticles shedding. Kinetics of clot formation in vitro was evaluated by thromboelastometry. Mouse model of ferric chloride (III) (FeCl3)-induced thrombosis was used to investigate thrombus formation in vivo. Results: We found that chloroauric(III) acid (HAuCl4), a classical AQP inhibitor (10-100 µM), reduced spreading of human platelets on collagen-coated surfaces and inhibited filopodia formation in a fluid phase. Under flow conditions, HAuCl4 (100 µM) attenuated thrombi growth on collagen, platelet secretion, and PS exposure. Thrombus formation was restored by the addition of exogenous adenosine diphosphate (ADP). Collagen-evoked platelet procoagulant response (evaluated as PS exposure, shedding of microparticles, platelet-dependent thrombin generation, and membrane ballooning) was distinctly reduced by HAuCl4 (25-200 µM), as well as the dynamics of clot formation. In mouse model of thrombosis, reduction of surface of PS-positive cells within thrombus was observed in the presence of HAuCl4 (1-10 mg/kg). Conclusion: These results suggest that in human platelets AQPs are crucial for agonist-evoked morphological changes, thrombus formation under flow, and in development of procoagulant response. Antithrombotic effect in vivo suggests that nontoxic inhibitors of AQPs may be considered as potential candidates for a novel class of antiplatelet drugs.

Aquaporins in human platelets: intracellular localization and possible role in granule and lysosome secretion.

This study was undertaken to establish the presence and the role of aquaporins (AQPs) in human platelets. Immunodetection with polyclonal antibodies and fluorescent microscopy suggest the presence of AQP isoforms - 0-7 and 9-12 - localized (in resting platelets) in the plasma membrane and in the dense and alpha granules. In thrombin- or monensin-treated platelets, the granules' AQPs become visible in the whole cell body, indicating the granules' swelling. In our studies on the role of AQPs in platelet responses we used tetrachloroauric acid (HAuCl4), a classical water channel blocker. We found that 10-100 µM of Au(III) inhibited the hypotonicity-, monensin (simulating the action of Na+/H+ exchanger)-, and collagen-evoked platelet swelling and reduced tritiated water uptake by platelets treated by collagen or monensin, indicating its ability to block water channels in these cells. HAuCl4, at the concentrations reducing water influx, did not induce cell lysis, alter the plasma membrane shape or the -SH group content. The inhibitor also failed to affect Na+ and Cl--related osmotic gradient formation and protein kinase D2 phosphorylation. In platelets activated by threshold concentrations of collagen, the thrombin receptor activating peptide, ADP, calcium ionophore A23187, phorbol ester and arachidonic acid, HAuCl4 (100 µM) completely inhibited secretion of ATP from dense granules but failed to reduce platelet aggregation. In collagen-stimulated platelets, HAuCl4 (10-100 µM) reduced secretion from dense and alpha granules, as well as lysosomes, in a dose-dependent manner. We conclude that human platelets possess numerous AQPs subtypes localized in the plasma and granule membranes. AQP-mediated water fluxes may be crucial for platelet volume regulation as well as secretion from dense and alpha granules and lysosomes.

Involvement of hyperglycemia in the development of platelet procoagulant response: the role of aldose reductase and platelet swelling.

: Rise in mean platelet volume (MPV) has been demonstrated to be associated with increased platelet reactivity. In diabetes patients, augmented MPV was proposed to contribute to increased risk of thrombotic complications. Therefore, the aim of this study was to investigate whether under hyperglycemic conditions, aldose reductase (AR)-mediated sorbitol formation and associated rise in cell volume, which subsequently results in platelet hyperactivation. Platelets were obtained from 30 healthy volunteers and 13 patients with diabetes. We evaluated changes in platelet size, their reactivity (measured as aggregation and secretion), and sorbitol content evoked by glucose. Measurement of procoagulant activity and thromboelastography were performed to assess how hyperglycemia affects coagulation. We have found that incubation of platelets with glucose (>10 mmol/l) leads to increased MPV, potentiation of collagen-evoked platelet aggregation, secretion, and procoagulant response (measured as platelet-dependent thrombin generation and phosphatidylserine expression). Glucose-treated platelets had higher sorbitol content and demonstrated enhanced tubulin polymerization. All the above-mentioned phenomena were reduced following the blocking of AR or by vincristine (microtubule destabilizing agent). Thromboelastography measurements demonstrated that hyperglycemia is associated with reduction of clotting time (R) and increase in the alpha angle (reflects platelet activation). Addition of sorbinil (AR inhibitor) or vincristine normalized R variable and alpha angle. The hyperglycemic conditions may accelerate platelet-related thrombin generation through the activation of polyol pathway, enhanced tubulin polymerization and associated with it rise in platelet volume.

Reduced clot retraction rate and altered platelet energy production in patients with asthma.

OBJECTIVE: Asthma enhances the risk of pulmonary embolism. The mechanism of this phenomenon is unclear. METHODS: We evaluated the kinetics of clot formation, clot retraction rate (CRR), clot volume at 40 min, the rate of lactate production (a marker of aerobic glycolysis in platelets in contracting clots), blood eosinophil count (EOS), nitric oxide in exhaled breath (FENO), and spirometry (FEV1) in 50 healthy controls and in 81 allergic asthmatics (41 subjects with steroid-naïve asthma and 40 with steroid-treated asthma). RESULTS: Thromboelastometry revealed that only steroid-treated asthmatics had slightly activated coagulation. Compared with healthy controls, whole asthmatics demonstrated (p < 0.05) reduced CRR, higher clot volume at 40 minutes, higher FENO, decreased FEV1, elevated EOS, and augmented lactate production in retracting clots. Reduced CRR was observed also in the absence of native plasma. In whole study population (asthmatics and healthy controls), CRR positively correlated with spirometry (rS = 0.668, p = <0.001) and negatively with FENO (rS = -0.543; p < 0.001), EOS (rS = -0.367, p < 0.002), and lactate production (rS = -0.791; p < 0.001). However, in steroid-treated asthmatics, the CRR did not correlate with FENO and EOS. In all study patients lactate production negatively correlated with FEV1 and positively with FENO. CONCLUSION: Collectively, this data is consistent with the hypothesis that, in asthmatics, reactive nitrogen species produced in the lungs may reduce platelet contractility (and CRR) through the diminution of platelet energy production. CRR inhibition would predispose asthmatics to pulmonary embolism.

Peroxynitrite may affect fibrinolysis via the reduction of platelet-related fibrinolysis resistance and alteration of clot structure.

We tested the hypothesis that in vitro peroxynitrite (ONOO(-), a product of activated inflammatory cells) may affect fibrinolysis in human blood through the reduction of platelet-related fibrinolysis resistance. It was found that ONOO(-) (25-300 µM) accelerated lysis of platelet-fibrin clots (in PRP) dose-dependently, whereas fibrinolysis of platelet-free clots was slightly inhibited by ≥ 1000 µM stressor. Concentrations of ONOO(-) affecting the lysis of platelet-rich clots, inhibited clot retraction (CR) in a dose-dependent manner. Thromboelastometry (ROTEM) measurements performed in PRP showed that treatment with ONOO(-) (threshold conc. 100 µM) prolongs clotting time, and reduces alpha angle, and clot formation velocity parameters indicating for reduced thrombin formation rate. In PRP, ONOO(-) (threshold conc. 100 µM) reduced the collagen-evoked exposure of phosphatidylserine (PS) on platelets' plasma membrane, the shedding of platelet-derived microparticles (PMP), and inhibited platelet-dependent thrombin generation (measured in artificial system), dose-dependently. As judged by confocal microscopy, similar ONOO(-) concentrations altered the architecture of clots formed in collagen-treated PRP. Clots formed in the presence of ONOO(-) were less dense and were composed of thicker fibers, which make them more susceptible to lysis. In platelet-depleted plasma, ONOO(-) (up to milimolar concentration) did not alter clot structure. Blockage of PS exposed on platelets resulted in an alteration of clot architecture toward more prone to lysis. ONOO(-), at lysis-affecting concentrations, inhibited the collagen-evoked secretion of fibrinolytic inhibitors from platelets. We conclude that physiologically relevant ONOO(-) concentrations may accelerate the lysis of platelet-fibrin clots predominantly via downregulation of platelet-related mechanisms including: platelet secretion, clot retraction, platelet procoagulant response, and the alteration in clot architecture associated with it.

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.

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.

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 (F(ENO)), 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, F(ENO) or blood eosinophilia in all three groups of asthmatics. Significant positive correlations between EBC nitrite/nitrate and blood eosinophilia, ECP levels or F(ENO) were observed in all groups of asthmatics. Significant negative correlations between EBC ammonia and nitrite/nitrate, F(ENO), 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.

Evaluation of hemostatic balance in blood from patients with polycythemia vera by means of thromboelastography: the effect of isovolemic erythrocytapheresis.

Polycythemia vera (PV) is associated with an increased frequency of thrombotic complications. This study was undertaken to evaluate the hemostatic balance in the blood of PV patients by means of thromboelastography (TEG). The effect of isovolemic erythrocytapheresis (ECP) on the hemostasis of PV patients was also studied. We assessed the coagulation status of 76 PV patients undergoing ECP and 50 of healthy controls. TEG measurements were performed immediately before and after the ECP procedure. Coagulation was triggered by recalcification in freshly collected citrated blood. We recorded clotting time (R), alpha angle, and maximum amplitude (MA) of the clot. The results presented here show that, compared with healthy controls, PV patients demonstrated an increase in alpha angle (p<0.005) and in MA (p=0.14). In the subgroup of PV patients with high (>440 × 10(9)l(-1)) platelet (PLT) count, differences in MA (p<0.01) and alpha angle (p<0.001) were more significant. Following ECP procedure, a significant (p ≤ 0.01) reduction of R time, a rise of alpha angle, and MA were observed, indicating augmentation of a hypercoagulable state. In PV patients, the rise in alpha angle positively correlated (r=0.549) with platelet count but not with the number of erythrocytes and leukocytes. Following ECP, this correlation was reduced (r=0.382). Dilution (with saline) of blood from PV patients and of healthy controls, to a degree similar to that used during the ECP procedure, resulted in reduction of R and rise of the alpha angle. In conclusion, TEG measurements show that the majority of PV patients demonstrate abnormal hemostasis in which a major role is played by platelets rather than plasma factors. The hypercoagulable state in PV patients is significantly augmented following the ECP and may be related to the hemodilution intrinsically included in this procedure. TEG may help to assess the thrombotic risk in individual PV patients.

[Pathophysiological consequences of hemolysis. Role of cell-free hemoglobin]. / Patofizjologiczne konsekwencje hemolizy. Rola wolnej hemoglobiny.

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.

[The role of interleukin 13 and interleukin 5 in asthma]. / Rola interleukin 13 i 5 w astmie.

Asthma is a chronic inflammatory disorder of the airways in which many cells and cellular elements play roles. Interleukins 5 (IL-5) and 13 (IL-13) are cytokines which play important roles in the pathophysiology of asthma. Selective accumulation and activation of eosinophils in the bronchial mucosa is considered a central event in the pathogenesis of asthma. IL-5 acts as a mediator of activation of eosinophils, influencing adhesion, membrane receptor expression, chemotaxis, and mediator synthesis. Airway eosinophilia has been related to bronchial hyperreactivity, asthma symptoms, and airway narrowing in subjects with asthma. IL-13 has a great influence on bronchial hyperreactivity, inflammation, and airway remodeling. Moreover, this cytokine drives many cellular responses relevant in asthma, including epithelial cell maturation and mucus production, synthesis of extracellular matrix proteins, and enhanced contractility of airway smooth muscle cells. In recent years, efforts have been underway to use substances acting as antagonists of these cytokines in the treatment of asthma. Many studies are being performed to assess the efficacy of anti-IL-5 and anti-IL-13 antibodies as well as substances inactivating receptors of these cytokines in asthma therapy. The results of these studies seem very interesting and induced the authors to discuss this issue.

The in vitro effect of eptifibatide, a glycoprotein IIb/IIIa antagonist, on various responses of porcine blood platelets.

The current study systematically evaluates the in vitro effect of eptifibatide, a GPIIb/IIIa blocker, on various responses of porcine platelets evoked by principal physiological stimulators. Eptifibatide at concentrations up to 40 mg/mL did not affect the calcium signal produced by thrombin, partly reduced the procoagulant response evoked by collagen, and strongly inhibited (IC50 approximately 11 mg/mL) adhesion of these cells to fibrinogen coated surfaces. Eptifibatide in a concentration-dependent manner reduced ADP, collagen, and thrombin-induced platelet aggregation (IC50 = 16-27 mg/mL), dense granule secretion (IC50 = 22-31 mg/mL) and lysosome secretion (IC50 = 25-50 mg/mL). Substantial (up to 30-40%) collagen or thrombin-evoked platelet aggregation still occurred at high (52 mg/mL) eptifibatide concentrations. Direct comparison of the susceptibility of platelet aggregation and dense granule secretion to the inhibitory action of eptifibatide indicates that aggregation is appreciably more sensitive than secretion. Eptifibatide (8 mg/mL) added together with a low (70 ng/mL) concentration of bivalirudin (a direct thrombin inhibitor) effectively (approximately 90%) reduced platelet aggregation induced by thrombin (0.2 U/mL). Based on these results, eptifibatide is not expected to reduce efficiently thrombus formation initiated by rapid local production of large amounts of thrombin. One practical consequence of our in vitro studies is the suggestion that the anti-thrombotic efficacy of eptifibatide, especially in preventing acute thrombotic events, may be largely improved by its combination with direct thrombin inhibitors.

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.

The in-vitro effect of tirofiban, glycoprotein IIb/IIIa antagonist, on various responses of porcine blood platelets.

The present study systematically evaluates the in-vitro effect of tirofiban, glycoprotein IIb/IIIa (integrins alphaIIbbetaIII) antagonist, on porcine blood platelets. It was found that tirofiban at concentrations up to 5,000 ng/ml did not affect the calcium signal produced by thrombin. Tirofiban, in a concentration-dependent manner reduced platelet aggregation evoked by ADP (IC50 approximately 70 ng/ml), collagen (IC50 approximately 200 ng/ml), and thrombin (IC50 approximately 5,000 ng/ml). Substantial thrombin-evoked platelet aggregation still occurred at high (5,000 ng/ml) tirofiban concentrations. The concentrations of tirofiban completely blocking the optical aggregation evoked by ADP or collagen failed to eliminate microaggregate formation totally. Tirofiban strongly inhibited the dense-granule and lysosome secretion induced by ADP (IC50 approximately 70-170 ng/ml), moderately inhibited that induced by collagen (IC50 approximately 420-500 ng/ml) and very poorly inhibited that elicited by thrombin (IC50 approximately 1,500-5,000 ng/ml). The extent of the inhibition of aggregation and secretion rose as concentrations of the stimulus lowered. Tirofiban was a moderate inhibitor (IC50 approximately 200 ng/ml) of adhesion and a poor inhibitor of platelet procoagulant response induced by collagen. Thromboelastography measurements indicate that, in whole blood, tirofiban, up to concentrations of 2,000 ng/ml, did not affect the kinetics of tissue factor induced clot formation. The obtained results reveal that in porcine platelets, the maximal concentrations of tirofiban used in human medicine (250 ng/ml), effectively block platelet responses triggered by ADP, partly block those induced by collagen and very poorly block those evoked by thrombin. The reason for this phenomenon seems to be the inability of tirofiban to reduce platelet secretion completely.

N-acetyl-beta-hexosaminidase activity in asthma.

BACKGROUND: N-acetyl-beta-hexosaminidase (beta-hex) is a lysosomal hydrolase, which is selectively secreted into the extracellular space by inflammatory cells. The aim of our study was to assess the activity of beta-hex in the plasma of asthmatic patients, and to establish whether it correlates with asthma severity and airway inflammation. METHODS: The study was conducted in a group of 46 asthmatic patients and 13 healthy volunteers. All study participants underwent analysis of exhaled nitric oxide and flow-volume spirometry. beta-hex activity, peripheral blood eosinophils, total serum IgE and eosinophil cationic protein were analyzed in blood samples from all asthmatic patients and healthy volunteers. RESULTS: beta-hex activity was significantly higher in patients with severe or moderate asthma compared with healthy volunteers and was positively correlated with exhaled nitric oxide levels and serum eosinophil cationic protein in these groups of patients. There was no correlation between beta-hex activity and forced expiratory volume in 1 s, blood eosinophil count or total serum IgE in these groups of asthmatics. CONCLUSIONS: Our results suggest that beta-hex could take part in airway inflammation and remodeling in asthma. Our study is the first report in which the elevated activity of beta-hex in subjects with asthma has been observed. However, more studies are needed to establish the precise role of this enzyme in asthma in humans.

The involvement of Na+/K(+)-ATPase in the development of platelet procoagulant response.

In circulation, platelets may come into contact with both exogenous (cardiac glycoside treatment) and endogenously produced inhibitors of Na+/K(+)-ATPase. We examined whether blocking of platelet Na+/K(+)-ATPase by ouabain results in generation of procoagulant activity. It was shown that an in vitro treatment of platelets with ouabain (20-200 microM for 20 to 60 min) is associated with an intracellular accumulation of sodium ([Na+](i)), generation of a weak calcium signal, and expression of procoagulant activity. The ouabain-induced procoagulant response was dose- and time-related, less pronounced than that evoked by collagen and similar to that produced by gramicidin, not affected by EDTA or aspirin, and strongly reduced in the absence of extracellular Na+ or by hyperosmolality. Flow cytometry studies revealed that ouabain treatment results in a unimodal left shift in the forward and side scatter of the entire platelet population indicating morphological changes of the plasma membrane. The shift was dose related, weaker than that evoked by collagen and similar to that produced by gramicidin. Ouabain-treated platelets express phosphatidylserine (PS). The ouabain-evoked PS expression was dose- and time-dependent, weaker than that produced by collagen and similar to that evoked by gramicidin. Electronic cell sizing measurements showed a dose-dependent increase in mean platelet volume upon treatment with ouabain. Hypoosmotically-evoked platelet swelling resulted in the appearance of procoagulant activity. Thromboelastography measurements indicate that, in whole blood, nanomolar (50-1000 nM, 15 min) concentrations of ouabain significantly accelerate the rate of clot formation initiated by contact and high extracellular concentration of calcium. We conclude that inefficiently operating platelet Na+/K(+)-ATPase results in a rise in [Na+](i). An increase in [Na+](i) and the swelling associated with it may produce PS exposure and a rise in membrane curvature leading to the generation of a procoagulant activity.

Cyclosporine enhances platelet procoagulant activity.

BACKGROUND: Clinical use of cyclosporine (CsA) was suggested to be associated with an increased risk of thromboembolic complications. The molecular mechanisms underlying these effects remain unresolved. METHODS: We tested the hypothesis that CsA may produce platelet procoagulant activity due to its interaction with the platelet plasma membrane. To verify this hypothesis the possible relationship between platelet morphology, exposure to platelet phosphatidylserine (PS) and platelet procoagulant activity (measured as phospholipid-dependent thrombin generation) was studied. RESULTS: It was found that CsA (1-100 microg/ml) potentiates collagen-evoked platelet procoagulant response. Platelets treated in vitro with CsA (20-200 microg/ml 20-60 min) expressed procoagulant activity. The CsA-induced platelet procoagulant response was both dose- and time-related and weaker than that produced by collagen. Flow cytometry studies revealed that CsA treatment results in a left shift (decrease) in the forward and side scatter of the entire platelet population. The shift was unimodal, dose-dependent and less pronounced than that elicited by collagen. Using flow cytometry and fluorescein isothiocyanate-labelled annexin V as a probe for PS, we demonstrated an increased binding of this marker to a CsA-treated platelet population. CsA-evoked PS-expression was dose- and time-dependent and smaller than that produced by collagen. CsA, at concentrations similar to those affecting platelet procoagulant response, released lactate dehydrogenase from platelets. CONCLUSIONS: These observations indicate that the thrombogenic properties of CsA may result from the alteration of lipid organization in platelet plasma membrane, leading to externalization of PS and accelerated thrombin generation.

Peroxynitrite can affect platelet responses by inhibiting energy production.

Peroxynitrite (ONOO-) strongly inhibits agonist-induced platelet responses. However, the mechanisms involved are not completely defined. Using porcine platelets, we tested the hypothesis that ONOO- reduces platelet aggregation and dense granule secretion by inhibiting energy production. It was found that ONOO- (25-300 microM) inhibited collagen-induced dense granule secretion (IC50 = 55 +/- 7 microM) more strongly than aggregation (IC(50) = 124 +/- 16 microM). The antiaggregatory and antisecretory effects of ONOO- were only slightly (5-10%) reduced by 1H-[1,2,4]-oxadiazolo-[4,3-alpha]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase. In resting platelets ONOO- (50-300 microM) enhanced glycolysis rate and reduced oxygen consumption, in a dose dependent manner. The ONOO- effects on glycolysis rate and oxygen consumption were not abolished by ODQ. The extent of glycolysis stimulation exerted by ONOO- was similar to that produced by respiratory chain inhibitors (cyanide and antimycin A) or an uncoupler (2,4-dinitrophenol). Stimulation of platelets by collagen was associated with a rise in mitochondrial oxygen consumption, accelerated lactate production, and unchanged intracellular ATP content. In contrast to resting cells, in collagen-stimulated platelets, ONOO- (200 microM) distinctly decreased the cellular ATP content. The glycolytic activity and oxygen consumption of resting platelets were not affected by 8-bromoguanosine 3',5'-cyclic monophosphate. Blocking of the mitochondrial ATP production by antimycin A slightly reduced collagen-induced aggregation and strongly inhibited dense granule secretion. Treatment of platelets with ONOO- (50-300 microM) resulted in decreased activities of NADH : ubiquinone oxidoreductase, succinate dehydrogenase and cytochrome oxidase. It is concluded that the inhibitory effect of ONOO- on platelet secretion and to a lesser extent on aggregation may be mediated, at least in part, by the reduction of mitochondrial energy production.