Asthma is associated with reduced fibrinolytic activity, abnormal clot architecture, and decreased clot retraction rate.

The aim of this study was to assess whether steroid-naïve asthma modulates hemostasis. We evaluated the clot retraction rate (CRR), fibrinolysis rate (FR), clot density (CD) (by confocal microscopy), plasma levels of plasminogen activator inhibitor (PAI-1), and factor XIII (FXIII), NO in exhaled breath (FENO ), spirometry (FEV1 ) and eosinophil count (EOS) in 36 patients with allergic, steroid-naïve asthma and in 34 healthy controls. We observed significantly (P < 0.001) reduced CRR, FR, and FEV1 and increased FENO , EOS, PAI-1, FXIII, and CD in patients with asthma compared with controls. In patients with asthma, FR negatively correlated with CD, FXIII, PAI-1, FENO , and EOS and positively with FEV1 . FXIII positively correlated with CD. Clot retraction rate negatively correlated with FENO and positively with FEV1 (all P < 0.001). These novel findings suggest that asthma itself is associated with decreased CRR and reduced fibrinolytic potential resulting from alterations in clot architecture and elevated levels of plasma FXIII and PAI-1.

Bronchial hyperresponsiveness and airway inflammation in patients with seasonal allergic rhinitis.

BACKGROUND: Allergic rhinitis is a chronic inflammatory disease induced by an immunoglobulin (Ig) E-mediated reaction after exposure to an allergen. Many patients with allergic rhinitis and no clinical evidence of asthma show a heightened response to histamine. OBJECTIVES: The aims of the study were to measure changes in markers of airway inflammation in patients with seasonal allergic rhinitis and estimate changes in bronchial reactivity before and during the pollen season. METHODS: The study sample comprised 22 patients sensitized to grass pollen and 10 healthy volunteers. Based on the results of the bronchial provocation test (BPT) during the pollen season, we divided patients into those with and without bronchial hyperresponsiveness (BHR). We determined changes in nitrite and pH in exhaled breath concentrate (EBC), fraction of exhaled nitric oxide (FE(NO)), blood eosinophil count, and BPT results before and during the pollen season. RESULTS: In allergic rhinitis patients with BHR, we observed an increase in EBC nitrite (5.44 [2.33] vs 8.57 [3.35] nmol/mL, P = .02) and FE(NO) (20.90 [13.68] vs. 43.40 [31.60] ppb, P = .02) and a decrease in EBC pH (7.07 [0.33] vs. 6.74 [0.28], P = .01) during the pollen season. In allergic rhinitis patients with BHR, the increase in BHR was negatively correlated with increased FE(NO) and EBC nitrite and positively correlated with a decrease in EBC pH during the pollen season. CONCLUSIONS: Our results revealed a relationship between increased BHR in patients with seasonal allergic rhinitis and changes in airway inflammation markers. EBC pH, EBC nitrite concentration, and FE(NO) could act as prognostic markers for identifying patients at risk of developing asthma.

Changes in RANTES and beta-thromboglobulin after intensive exercise in patients with allergic asthma.

BACKGROUND: There is increasing evidence that exercise-induced bronchoconstriction (EIB) is associated with eosinophilic airway inflammation. In the pathogenesis of EIB the role of chemokines - responsible for promoting the migration and activation of inflammatory cells - as well as blood platelets, a potential source of those chemokines, remains unclear. METHODS: The study was conducted in a group of 19 asthmatics (11 with EIB, 8 without EIB) and 8 healthy volunteers. Changes in the plasma concentrations of RANTES and beta-thromboglobulin (beta-TG) induced by intensive exercise were determined. Moreover, the possible correlation of these measurements with the results of other tests used in the diagnosis of asthma as well as laboratory tests commonly associated with asthma were investigated. RESULTS: A comparison of the concentrations of beta-TG in all groups studied at rest did not reveal any significant differences. In all groups studied, 30 min after exercise elevated beta-TG concentrations were observed; the most significant increase was revealed in asthmatics with EIB. The baseline concentrations of RANTES before exercise in both groups of asthmatics were significantly higher in comparison to the group of healthy volunteers. After exercise, in the group of patients with EIB, a significant increase in RANTES concentrations was observed. These changes correlated with an increase in other markers of airway inflammation 24 h after exercise. CONCLUSIONS: We suggest that platelet activation, resulting in elevated RANTES release, could be one of the factors responsible for the increase of airway inflammation observed in consequence of EIB in asthmatics.

Platelet thromboxane synthetase inhibitors with low doses of aspirin: possible resolution of the "aspirin dilemma".

Selective pharmacological inhibition of thromboxane A2 synthesis did not prevent arachidonate-induced aggregation of human platelets in vitro. Prevention was instead achieved by a combination of thromboxane A2 inhibitors with low concentrations of aspirin. The latter partially reduced the proaggregatory cyclooxygenase products that accumulated when thromboxane A2 synthesis was blocked. The aspirin concentrations did not affect per se either platelet aggregation or prostacyclin synthesis in cultured human endothelial cells. The combination of thromboxane synthetase inhibitors with low doses of aspirin may offer greater antithrombotic potential than either drug alone.

RANTES in exhaled breath condensate of stable and unstable asthma patients.

RANTES has been implicated in the allergic inflammation of asthma by promoting the migration and activation of the inflammatory cells, including eosinophils. The study was undertaken to evaluate RANTES levels in the exhaled breath condensate (EBC) of asthmatics with different degrees of asthma severity. EBC was collected from 33 patients with allergic asthma (11 with steroid-naïve mild asthma, 10 with ICS-treated, stable mild-to-moderate asthma, 12 with ICS-treated unstable, severe asthma) and seven healthy volunteers. In the three groups of asthmatics, RANTES concentrations in EBC were significantly higher compared with healthy volunteers. RANTES levels were significantly higher in patients with unstable asthma than in the two groups with stable disease. We observed statistically significant correlations between the concentrations of RANTES in EBC and F(ENO) in the three studied groups of asthmatics; notably, the correlation between the parameters described above was strong positive in the group of unstable and steroid-naïve stable asthmatics. We also discovered a significantly positive correlation between RANTES in EBC and the serum ECP or blood eosinophil count in the groups of asthmatics with severe, unstable asthma and between RANTES and serum ECP in the group of steroid-naïve stable asthmatics. Measurements of RANTES in EBC may provide another useful diagnostic tool for detecting and monitoring inflammation in patients with asthma.

Endothelin-1 in exhaled breath condensate of stable and unstable asthma patients.

Endothelins are proinflammatory, profibrotic, broncho- and vasoconstrictive peptides, which play an important role in the development of airway inflammation and remodeling in asthma. The study was undertaken to evaluate the endothelin-1 (ET-1) levels in exhaled breath condensate (EBC) of asthmatics with different degree in asthma severity. EBC was collected from 31 patients with allergic asthma (11 with steroid-naïve mild asthma, 10 with ICS-treated, stable mild-to-moderate asthma, 10 with ICS-treated unstable, severe asthma) and 7 healthy volunteers. In the three groups of asthmatics, ET-1 concentrations in EBC were significantly higher than in healthy volunteers. ET-1 levels were significantly higher in patients with unstable asthma than in the two groups with stable disease. There was a significant correlation between ET-1 levels and FENO in the three groups of asthmatics and between ET-1 and blood eosinophil counts in the group of patients with unstable asthma. Measurements of ET-1 in EBC may provide another useful diagnostic tool for detecting and monitoring inflammation in patients with asthma.

Soluble CD40 ligand and soluble P-selectin in allergic asthma patients during exercise-induced bronchoconstriction.

BACKGROUND: The interactions between CD40 and its ligand, CD40L, control humoral and cell-mediated immune responses. CD40 ligation may promote asthma-associated inflammatory responses in the airways. Many reports confirm the inflammatory basis of exercise-induced bronchoconstriction (EIB) in asthmatics. METHODS: The study was conducted in a group of 19 asthmatic patients (11 with EIB, 8 without EIB) and 8 healthy volunteers. We analyzed the changes in plasma concentrations of soluble CD40 ligand (sCD40L) and soluble P-selectin (sP-selectin) induced by intensive exercise. We also studied possible correlations with the results of measurements commonly associated with asthmatic inflammation. RESULTS: The study revealed statistically significant higher baseline concentrations of sCD40L--but not sP-selectin--in the group of asthmatics with EIB than in those without. In the asthmatic patients with EIB, sCD40L and sP-selectin concentrations increased significantly 30 minutes after exercise and returned to baseline 24 hours after exercise. Baseline concentrations of sCD40L correlated with baseline sP-selectin or fractional exhaled nitric oxide concentration (FE(NO)), an increase in sP-selectin 30 minutes after exercise, and changes in FE(NO) or bronchial hyperresponsiveness 24 hours after exercise. A statistically significant correlation between an increase in sCD40L concentrations 30 minutes after exercise and an increase in FE(NO) 24 hours after exercise or baseline eosinophil cationic protein was observed. CONCLUSION: After exercise in the group of allergic asthmatics with EIB, upregulation of CD40L by increased expression of inflammatory molecules and improved sensitivity of CD40-responsive cell types to the effects of proinflammatory cytokines may play an important role in the increased airway inflammation observed after postexercise bronchoconstriction.

Comparison of exhaled nitric oxide measurement with conventional tests in steroid-naive asthma patients.

BACKGROUND: Nitric oxide (NO) is a molecule with potent biological activity that plays an important role in the physiology of the respiratory system. Increased expression of inducible nitric oxide synthase (iNOS) and elevated fractional concentration of exhaled nitric oxide (F(ENO)) are seen in asthmatic patients. Measurement of F(ENO) has become increasingly recognized for use in the evaluation of bronchial inflammation during monitoring of antiinflammatory treatment. OBJECTIVES: The aim of this study was to evaluate F(ENO) in a group of steroid-naive asthmatics and assess the relationship of this parameter with the results of other tests used in the diagnosis of asthma and monitoring of antiinflammatory treatment in asthmatic patients. METHODS: The study was conducted in a group of 101 steroid-naive asthmatics (56 allergic and 45 nonallergic) and 39 healthy volunteers. All patients underwent measurement of F(ENO), skin prick tests with common inhaled allergens, analysis of serum eosinophil cationic protein (ECP) and blood eosinophilia, and flow-volume spirometry. When the forced expiratory volume in the first second (FEV1) was less than 80% of predicted, reversibility of airway obstruction with a beta2-agonist was assessed. A nonspecific bronchial provocation test with histamine was carried out in asthmatic patients with a baseline FEV1 of more than 70% of predicted. RESULTS: Compared to the healthy volunteers, F(ENO) was elevated in both groups of asthmatics. F(ENO) in the allergic asthma group was higher than in the group of nonallergic asthmatics. In allergic and nonallergic asthmatics, F(ENO) was significantly correlated with bronchial hyperresponsiveness to histamine, reversibility of airway obstruction, serum ECP levels, and blood eosinophilia. F(ENO) did not correlate with baseline FEV, in either group of asthmatics. In 31% of nonallergic and 9% of allergic patients, F(ENO) was less than 20 parts per billion. CONCLUSIONS: We suggest that measurement of F(ENO) could be clinically useful in steroid-naive asthmatics and should be more widely used in clinical practice. Measurement of F(ENO) is a noninvasive, simple, and reproducible procedure, the results of which correlate with other routinely used methods in the diagnosis of asthma. However, it is worth noting that some patients, especially those with nonallergic asthma, do not display elevated F(ENO).

SQ 22536, an adenylate-cyclase inhibitor, prevents the antiplatelet effect of dazoxiben, a thromboxane-synthetase inhibitor.

This study shows that dazoxiben, a selective inhibitor of thromboxane A2-synthetase in human platelets, inhibited arachidonic acid-induced platelet aggregation in platelet-rich plasma samples from four out of 16 healthy volunteers. In these four "responder" samples, the anti-aggregating effect of dazoxiben was prevented by the compound SQ 22536, a 9-substituted adenine analogue, endowed with an inhibitory activity on adenylate-cyclase. The compound SQ 22536 also counteracted the antiaggregating effect of prostaglandin D2, a known activator of platelet adenylate-cyclase. When platelet thromboxane A2-synthetase was blocked by dazoxiben, a marked increase of prostaglandin D2 was concomitantly observed both in "responder" and "non responder" samples. The compound SQ 22536 blunted the increase in platelet cAMP caused by either dazoxiben and sodium arachidonate or prostaglandin D2. It is suggested that the antiaggregating effect of dazoxiben is mediated by newly synthesized prostaglandin D2. The latter acts by stimulating adenylate-cyclase and increasing cAMP levels. The compound SQ 22536 prevents both phenomena. In "non responder" samples some factors--still to be defined--might counteract similarly to the compound SQ 22536 the antiaggregating activity of PGD2.

Importance of the malate-aspartate shuttle for the reoxidation of glycolytically produced NADH and for cell aggregation in porcine blood platelets.

Malate dehydrogenase (EC 1.1.1.37) and aspartate aminotransferase (EC 2.6.1.1) are present in porcine blood platelets in both mitochondria and the cytosol. The latter enzyme is inhibited in a typical way by aminooxycompounds and cycloserine. Blocking of aminotransferase or inhibition of the mitochondrial dicarboxylate carrier by butylmalonate stimulates lactate production by intact platelets and inhibits their aggregation induced by ADP or collagen. These results indicate that the reoxidation of cytosolic NADH via the malate-aspartate shuttle is important for covering the energy demand of platelets necessary for their stimulation.

On the mechanism of glycolysis stimulation by mitochondria from Guérin epithelioma.

The experiments were performed to determine the factor(s) responsible for the stimulatory effect on glycolysis in the cytosol (post-microsomal supernatant) of mitochondria isolated from Guérin epithelioma. It was found that epithelioma mitochondria contain bound hexokinase which constitutes about 50% of the total cellular hexokinase activity. The solubilized and partially purified enzyme, when added to the cytosol, stimulated glycolysis. The stimulatory effect of mitochondria on glycolysis was associated with the decrease of adenylate energy charge which was caused by an apparently very fast production of ADP in the hexokinase reaction. A large part of ATP hydrolyzed in this process was converted to IMP and NH3, which can additionally stimulate glycolysis through its stimulatory effect on phosphofructokinase. It is therefore suggested that the stimulatory effect of epithelioma mitochondria on glycolysis can be explained by production of ADP by the hexokinase associated with these mitochondria.

Exocrine cell mitochondria of the rat pancreas after lead intoxication.

The aim of the study was to evaluate alterations in exocrine cell mitochondria of the rat pancreas after lead acetate intoxication. The experiment used 45 rats divided into 2 experimental groups receiving lead acetate to drink, of lead concentration 50 and 500 mg/dm3 (ppm), and a control group given tap water. The animals from the experimental group were decapitated after 2, 4, 6 and 8 weeks, 5 rats from the control group after 8 weeks of the experiment. Rats from experimental groups decapitated after 8 weeks had lead administration stopped after six weeks and then, for two weeks tap water was given. Pancreatic sections were examined with biochemical methods for the activity of cytochrome oxidase and succinic dehydrogenase. Ultrastructural and morphometric examinations were also performed. It was demonstrated that: a) exocrine cell mitochondria are particularly predisposed to lead effect, b) intoxication of rats with lower lead doses (50 ppm) causes reversible adaptative or compensatory changes in these organelles, c) intoxication of rats with higher lead doses (500 ppm) induces irreversible ultrastructural alterations in numerous mitochondria, including damage to inner and to outer mitochondrial membranes, d) structural changes in the mitochondria in the course of lead intoxication are the morphological expression of the impairment of metabolic processes, associated with the inhibited activity of the respiratory enzymes: succinic dehydrogenase and cytochrome oxidase.

[Adaptation of the spectrocolorimeter "Spekol 10" for measuring platelet aggregation]. / Adaptacja spektrokolorymetru "Spekol 10" do pomiaru agregacji plytek krwi.

Adaptation of "Spekol 10" with Ti Mi attachment for measurement of platelet aggregation (by the turbidimetric method) in small samples of platelet suspensions (0.2-0.4 ml) is described. This set can be further easily modified for simultaneous recording of aggregation and ion concentration in the same platelet sample.

The effect of quercetin and lithium ions on platelet aggregation.

Quercetin inhibited aggregation of porcine blood platelets induced by collagen (IC50 = 0.2 mM), ADP (IC50 = 0.5 mM), thrombin (IC50 = 0.02 mM) and ionophore A23187 (IC50 = 1.5 mM). Preincubation of platelets with 10 mM LiCl abolished the inhibitory effect of quercetin on the aggregation of these cells induced by thrombin. Lithium ions per se caused potentiation of the aggregation of platelets induced by thrombin. Neither potentiation of aggregation by Li+, nor the abolishing of the inhibitory effect of quercetin by Li+ was observed when platelets were activated by ionophore A23187. Since lithium ions inhibit activity of enzymes degradating inositol phosphate, the obtained results can be interpreted to mean that quercetin affect platelet aggregation by the inhibition of inositol phosphates production.

AMP deaminase from porcine blood platelets, regulation by adenine nucleotides, phosphate and by Na+ and K+ ions.

Porcine blood platelets contain a relatively high amount of AMP deaminase (14.4 U per 10 11 cells). The enzyme showed sigmoidal behaviour as a function of AMP concentration with a S0.5 value (substrate concentration required for half-maximum velocity) of 3.6 and 4.0 mM in the presence of Na+ and K+ respectively. MgATP and MgADP at micromolar concentration activated the enzyme. Activation by saturating MgATP and MgADP in the presence of Na+ or K+ converted the rate versus substrate plots to hyperbolic with a dramatic decrease of S0.5. Phosphate at milimolar concentrations inhibited the enzyme and this inhibitory effect was totally reversed as the concentrations of MgATP and MgADP rised to physiologically high levels. Na+ and K+ activated the enzyme in the absence of MgATP and MgADP. Both cations largely enhanced the Vmax with Na+ being more potent. A comparison of the kinetic behaviour of the enzyme in vitro with the metabolite concentrations in vivo suggest that a substantial regulation can occur through changes in AMP and Na+ concentrations.

[The role of endothelium in normal pregnancy and pregnancy complicated by preeclampsia]. / Rola sródblonka naczyniowego w ciazy prawidlowej i powiklanej EPH gestoza.

Endothelial cell dysfunction is thought to play a role in preeclampsia and the reduced production by vascular endothelial cells of the antiaggregatory and vasodilatory factors is well documented. The present study was designed to evaluate endothelial cells function in preeclamptic and healthy pregnant subjects. The nitric oxide plasma concentration in women with preeclampsia was significantly lower as compared with normotensive pregnant women. A significant increase in ET concentration was found in preeclamptic women as compared with normal pregnant patients and normal non-pregnant. The plasma concentrations of von Willebrand factor were significantly increased in healthy pregnancy as compared with preeclamptic patients. The results of our study demonstrate a significant endothelial cells damage in preeclamptic patients. Whether these observations contribute to the vascular pathophysiologic features of preeclampsia remains to be proved.

[The role of nitric oxide and blood platelets in pathogenesis of preeclampsia]. / Rola tlenku azotu (NO) i plytek krwi w patogenezie EPH gestozy.

The aim of this study was to evaluate the concentration of nitric oxide and the platelet function in preeclamptic and normal pregnant women. The patients with preeclampsia had new hypertension (diastolic blood pressure consistently > or = 90 mmHg with previously lower readings), new proteinuria and generalized oedema that subsequently regressed after delivery. Blood was collected by routine forearm venipuncture before delivery. The following parameters were evaluated: nitric oxide, beta-TG and PF4. The nitric oxide plasma concentration in women with preeclampsia was significantly lower compared with normotensive pregnant women. beta-TG and PF4 concentrations were significantly increased in patients with preeclampsia. Whether these observations contribute to the vascular pathophysiologic features of preeclampsia remains to be proved.

Clinically relevant HOCl concentrations reduce clot retraction rate via the inhibition of energy production in platelet mitochondria.

Using porcine blood, we examined the impact of hypochlorite, product of activated inflammatory cells, on clot retraction (CR), an important step of hemostasis. We found that, in vitro, HOCl is able to reduce CR rate and enlarge final clot size in whole blood (t.c. 100 µM), platelet-rich plasma (PRP) threshold concentration (t.c. 50 µM), and an artificial system (washed platelets and fibrinogen) (t.c. 25 nM). Combination of low HOCl and peroxynitrite concentrations resulted in synergistic inhibition of CR by these stressors. Concentrations of HOCl completely inhibiting CR failed to affect the kinetics of coagulation measured in PRP and in platelet-free plasma. Concentrations of HOCl reducing CR rate in PRP augmented production of lactate, inhibited consumption of oxygen by platelets, and decreased total adenosine triphosphate (ATP) content in PRP-derived clots. In an artificial system, concentrations of HOCl resulting in inhibition of CR (25-100 nM) reduced mitochondrial transmembrane potential and did not affect actin polymerization in thrombin-stimulated platelets. These concentrations of HOCl failed to affect the adhesion of washed platelets to fibrinogen and to evoke sustained calcium signal, thus excluding stressor action on glycoprotein IIb/IIIa receptors. Exogenously added Mg-ATP almost completely recovered HOCl-mediated retardation of CR. Concentrations of HOCl higher than those affecting CR reduced thromboelastometric variables (maximum clot firmness and α angle). We conclude that low clinically relevant HOCl concentrations may evoke the inhibition of CR via the reduction of platelet contractility resulted from malfunction of platelet mitochondria. At the inflammatory conditions, CR may be the predominant HOCl target.

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

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.

The importance of aspartate aminotransferase for platelet aggregation.

In bovine platelets aspartate aminotransferase has a high activity. The enzyme in vitro is inhibited in a dose dependent manner by aminooxyacetate (IC50 = 10(-4) M), hydroxylamine (IC50 = 10(-4) M), and cycloserine (IC50 = 5 X 10(-3). The inhibitory effect of all the three compounds is strongest at low substrate (aspartate) concentration. Blocking of aspartate aminotransferase activity by these compounds in intact platelets is accompanied by the inhibition of ADP and collagen-induced aggregation. Among the three compounds the strongest inhibitor of platelet aggregation was hydroxylamine, which was also the most effective inhibitor of aspartate aminotransferase. Other metabolic blockers, i.e. dinitrophenol (DNP), rotenone and antimycin also inhibited the aggregation of platelets, and a synergism has been demonstrated between DNP, rotenone and antimycin A action on platelet aggregation and blockade of aspartate aminotransferase activity. The results are interpreted to mean that transamination is of importance in the energy production in the activated platelet, probably through its participation in reducing equivalents transport from the cytosol to the mitosol via the malate: oxaloacetate: aspartate shuttle.