Role of platelet-activating factor in the ovine heparin-protamine reaction.

Platelet-activating factor (PAF) infusion into sheep, as well as protamine reversal of heparin anticoagulation, causes thromboxane release into plasma, pulmonary hypertension, hypoxemia, and leukopenia. We investigated the possible role of PAF in the heparin-protamine reaction. Intravenous protamine was administered to neutralize heparin anticoagulation in five awake sheep and caused an increase of mean pulmonary arterial pressure from 16.6 +/- 1 (SE) mmHg at base-line to 47 +/- 9 mmHg at 1 min after protamine injection (P < 0.01) because of a 4.5-fold increase of pulmonary vascular resistance. This neutralization reaction induced a 25% reduction of circulating leukocyte count and arterial PO2. Undetectable blood levels of PAF were measured by bioassay and high-performance liquid chromatography during these heparin-protamine reactions. Infusion of BN 52021 (20 mg/kg), a PAF receptor antagonist, before rechallenging the same sheep with heparin and then protamine did not reduce the level of peak pulmonary hypertension or the degree of hypoxemia and leukopenia. We conclude that the leukopenia and thromboxane-mediated pulmonary vasoconstriction occurring after rapid intravascular formation of heparin-protamine complexes in sheep are not due to the release of PAF.     

Effect of leukopenia on pulmonary hypertension after heparin-protamine in pigs.

Heparin neutralization by protamine after cardiac surgery and cardiopulmonary bypass may be associated with complement activation, transient leukopenia, thromboxane A2 release, and severe pulmonary hypertension. The role of leukocytes in the heparin-protamine reaction was studied in leukopenic pigs (n = 9) and a control group (n = 8). Leukopenia was induced by pretreatment with cyclophosphamide (30 mg.kg-1.day-1) for 6-7 days. During general anesthesia and after catheterization, baseline recordings of hemodynamics were performed and blood samples were withdrawn. Heparin (250 IU/kg) was injected and measurements were repeated after 10 min. Protamine sulfate (100 mg) was then infused over 2 min and measurements were performed after 2, 5, and 15 min. Prostanoid concentrations were measured by radioimmunoassays. In additional in vitro experiments, the release of thromboxane B2 from washed platelets and leukocytes after heparin-protamine stimulation was measured. Pretreatment with cyclophosphamide reduced leukocyte counts by 95.5% and the number of neutrophils by greater than 99.9%. Protamine infusion increased mean pulmonary arterial pressure by 74 and 46% and pulmonary vascular resistance by 185 and 384% in control and leukopenic animals, respectively. Thromboxane B2 concentrations increased in both groups. Stimulation by heparin, protamine, or heparin and protamine in sequence did not induce any thromboxane A2 release from washed blood cells. It is concluded that leukocytes do not contribute to pulmonary hypertension after heparin-protamine.     

Effects of thromboxane synthase inhibition on air emboli lung injury in sheep

We tested the effects of OKY-046, a thromboxane synthase inhibitor, on lung injury induced by 2 h of pulmonary air infusion (1.23 ml/min) in the pulmonary artery of unanesthetized sheep with chronic lung lymph fistula so as to assess the role of thromboxane A2 (TxA2) in the lung injury. We measured pulmonary hemodynamic parameters and the lung fluid balance. The concentrations of thromboxane B2 (TxB2) and 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) in plasma and lung lymph were determined by radioimmunoassay. Air infusion caused sustained pulmonary hypertension and an increase in pulmonary vascular permeability. The levels of TxB2 and 6-keto-PGF1 alpha in both plasma and lung lymph were significantly elevated during the air infusion. TxB2 concentration in plasma obtained from the left atrium was higher than that from the pulmonary artery at 15 min of air infusion. When sheep were pretreated with OKY-046 (10 mg/kg iv) prior to the air infusion, increases in TxB2 were prevented. The pulmonary arterial pressure, however, increased similarly to that of untreated sheep (1.8 X base line). The increase in lung lymph flow was significantly suppressed during the air infusion. Our data suggest that the pulmonary hypertension observed during air embolism is not caused by TxA2.     

Plasma thromboxane B2 levels and thromboxane B2 production by platelets are increased in patients during spinal and epidural anesthesia

Concentrations of thromboxane (Tx) B2 in plasma and its production by platelets were measured in 20 spinal and 10 epidural anesthesia patients scheduled for small operations in the lower extremities. The main metabolite of prostacyclin, 6-keto-PGF1 alpha and prostaglandin (PG) E2 in plasma were also determined. Plasma TxB2 and TxB2 production by platelets increased during both spinal and epidural anesthesia. Plasma TxB2 levels also remained elevated 1 h after anesthesia. The plasma concentrations of 6-keto-PGF1 alpha and PGE2 did not change during spinal or epidural anesthesia. In in vitro studies, only low concentrations of lidocaine (0.5-1.0 micrograms/ml) and bupivacaine (0.5-3.0 micrograms/ml) increased platelet TxB2 production. In platelet rich plasma, neither lidocaine nor bupivacaine in concentrations of 0.5-3.0 micrograms/ml caused constant changes in ADP-induced platelet aggregation, but they inhibited it in toxic concentrations (12 micrograms/ml). The results suggest that the increased TxB2 plasma levels and platelet TxB2 production during regional anesthesia are not caused by local anesthetics itself but by other factors, e.g. tissue trauma. In clinically found concentrations, local anesthetics do not cause any constant changes in platelet aggregation.     

Nafamstat mesilate attenuates pulmonary hypertension in heparin-protamine reactions

Rapid protamine reversal of heparin anticoagulation in awake sheep caused, after 1 min, a approximately 15-fold increase of arterial plasma thromboxane B2 (TxB2) levels, a 4-fold rise of pulmonary vascular resistance (PVR), a 2-fold rise of pulmonary arterial pressure, and after 3 min, a 2-fold rise of ovine arterial plasma complement C3a levels (P less than 0.05). Infusion of nafamstat mesilate (FUT-175), a protease and complement pathway inhibitor, before protamine reduced these increases by approximately 60-90% (P less than 0.05). FUT-175 did not modify heparin + protamine-induced leukopenia, suggesting that FUT-175 incompletely blocked C5a production. We also learned that infusing protamine first and heparin 5 min later did not increase either plasma C3a or TxB2 levels or PVR while the activated clotting time increased only minimally. Thus, in awake sheep, the sequence of heparin and protamine infusion influences complement activation and pulmonary vasoconstriction. FUT-175 pretreatment reduces thromboxane release and pulmonary vasoconstriction probably by limiting complement activation.     

Plasma lipoproteins affect platelet malondialdehyde and thromboxane B2 production

Platelet interaction with plasma lipoproteins was studied using gel-filtered platelets free of plasma constituents and purified lipoproteins. On incubation of gel-filtered platelets with plasma lipoproteins at 30 degrees C for 30 min, 100 micrograms of protein/ml of very-low as well as low-density lipoprotein caused 10% increment in platelet aggregation and [14C]serotonin release in parallel to elevation of around 15% of malondialdehyde and thromboxane B2 production. High-density lipoprotein showed the opposite effect and reduced platelet aggregation as well as thromboxane B2 synthesis by 17 and 32%, respectively. Lipoprotein-deficient plasma enhanced platelet function. Preincubation of the platelet suspension with prostacyclin did not prevent the effect of the lipoproteins on the in vitro platelet response as well as on the platelet prostaglandin pathway. Our results suggest that the formation of thromboxane B2 and malondialdehyde is influenced by plasma lipoproteins and that these, in turn, affect platelet aggregation and the release reaction. The possible significance of these results to platelet function in hyperlipidemic patients is discussed.     

Effects of thromboxane synthase inhibition on tumor necrosis factor-induced lung injury in sheep.

To examine the role of thromboxane (Tx) A2 in the pathogenesis of acute lung injury caused by tumor necrosis factor alpha (TNF), we tested the effects of OKY-046, a selective thromboxane synthase inhibitor, on pulmonary hemodynamics, lung lymph balance, circulating leukocytes, arterial blood gas analysis, and TxA2 (as TxB2) and prostacyclin (as 6-keto-prostaglandin F1 alpha) levels in plasma and lung lymph after TNF infusion in awake sheep. Infusion of human recombinant TNF (3.5 micrograms/kg) into a chronically instrumented awake sheep caused a transient increase in pulmonary arterial pressure (Ppa). The Ppa peaked within 15 min of the start of TNF infusion from 23.3 +/- 1.1 (SE) cmH2O of baseline to 42.3 +/- 2.3 cmH2O and then decreased toward baseline. The pulmonary hypertension was accompanied by transient hypoxemia, peripheral leukopenia, and the increases in TxB2 in plasma and lung lymph. These changes were followed by an increase in flow of protein-rich lung lymph, consistent with an increase in pulmonary microvascular permeability. OKY-046 significantly prevented the rises of Ppa and TxB2 concentrations in plasma and lung lymph during early phase after TNF infusion. OKY-046, however, did not attenuate the increase of lung lymph flow, transient hypoxemia, and leukopenia. From these data, and by comparison with our previous studies of OKY-046-pretreated sheep during endotoxemia, we conclude that TxA2 has an important role of the increase in the early pulmonary hypertension, but it is not related to the early hypoxemia, leukopenia, and lung lymph balances in TNF-induced lung injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extract of a spice--omum (Trachyspermum ammi)-shows antiaggregatory effects and alters arachidonic acid metabolism in human platelets

An ethereal extract of omum (Trachyspermum ammi; Hindustani: ajwan)--a frequently consumed spice--was found to inhibit platelet aggregation induced by arachidonic acid (AA), epinephrine and collagen; in this respect it was most effective against AA-induced aggregation. Inhibition of aggregation by omum could be explained by its effect on platelet thromboxane production as suggested by the following experimental observation. (i) Omum reduced TxB2 formation in intact platelet preparations from added arachidonate, and (ii) it reduced the formation of TxB2 from AA-labelled platelets after stimulation with Ca2+-ionophore A23187 by a direct action on cyclooxygenase as it did not affect the release of AA from labelled platelets. An increased formation of lipoxygenase-derived products from exogenous AA in omum-treated platelets was apparently due to redirection of AA from cyclooxygenase to the lipoxygenase pathway.     

Role of circulating platelets and granulocytes in PAF-induced pulmonary dysfunction in awake sheep

The effects of a single intravascular bolus injection of platelet-activating factor (PAF) on pulmonary hemodynamics, lung mechanics, and lung fluid and solute exchange were studied in 13 chronically instrumented unanesthetized sheep. Since PAF has profound effects on both platelets and granulocytes, we investigated the effects of platelet and granulocyte depletion on the sheep's response to exogenous PAF. Sheep received PAF when granulocyte and platelets counts were normal and after platelet depletion with rabbit antisheep platelet antibodies (n = 5) or granulocyte depletion with hydroxyurea (n = 5). Sheep served as their own controls, and the order of experimentation was varied. Bolus injections of PAF had reproducible effects on pulmonary hemodynamics (pulmonary arterial pressure increased acutely to 85 +/- 3.7 cmH2O) and lung mechanics (dynamic compliance of the lungs decreased to 24.5 +/- 3.8% of base line and resistance to airflow across the lungs increased greater than 10-fold) and caused marked increases in lung lymph concentrations of thromboxane B2 and 6-ketoprostaglandin F1 alpha. The single bolus injection of PAF also caused marked prolonged elevations in lung lymph flow and increases in the lymph-to-plasma protein concentration ratio for 3 h after PAF. PAF had profound effects despite platelet and granulocyte depletion. Platelet depletion slightly attenuated the pulmonary hypertension observed after PAF injection. Platelet depletion also attenuated the increases in thromboxane B2 concentrations in lung lymph, and lung mechanics normalized more rapidly in platelet-depleted sheep. There were no statistically significant effects of granulocyte depletion to less than 200 granulocytes/mm3 on any of the measured variables.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics of various synthetic peptide calpain inhibitors and their application for the analysis of platelet reaction

Calpeptin (a cell permeable synthetic peptide calpain inhibitor) inhibited the generation of thromboxane B2 (TxB2) by the direct inhibition on Tx synthetase in platelets at the concentrations more than 30 microM. Calpeptin, its analogues and E-64d (EST) were further examined with regard to cell permiability and inhibitory spectra. Among all compounds, only calpeptin inhibited the degradation of substrate proteins of calpain with negligible effect on TxB2 generation in intact platelets at the concentrations less than 30 microM. These concentrations of calpeptin did not inhibit the platelet aggregation, the elevation of [Ca2+], nor the formation of inositol 1,4,5-trisphosphate (IP3) in thrombin or collagen activated platelets. These results indicate that calpain dose not participate in the process of platelet activation induced by thrombin or collagen.     

Influence of propranolol on platelet aggregation and thromboxane B2 production from platelet-rich plasma and whole blood

The beta-adrenoceptor antagonist propranolol is used in the therapy of hypertension and ischemic heart disease. The aim of our study was to evaluate the effects of this drug on platelet aggregation and on synthesis of thromboxane B2 (the stable metabolite of Thromboxane A2) from platelet rich plasma (PRP), whole blood samples and during spontaneous clotting. The results indicate that propranolol at concentrations near the therapeutic range, significantly inhibit collagen and thrombin-induced platelet aggregation and TxB2 synthesis from PRP. Furthermore the drug demonstrates inhibitory activity on B-TG release and TxB2 production from whole blood samples and on spontaneous clotting. The results suggest that some benefits of propranolol in the treatment of patients with coronary artery disease or cardiovascular conditions associated with platelet hyperaggregability may also be related to interference with platelet activation "in vivo" and with TxA2 generation.     

Mode of action of 2-(diethylamino)-7-ethoxychromone on human platelets.

The in vitro effect of 2-(diethylamino)-7-ethoxychromone (RC39XVIII) on human platelet aggregation induced by several agonists and on thromboxane B2 formation, granule release and intracellular cAMP elevation has been studied. The chromosome-derivative exerts a dose-dependent inhibitory effect on aggregation produced by U46619, arachidonic acid, thrombin, collagen and ADP. RC39XVIII inhibits aggregation, TxB2 formation and granule release in parallel. Moreover the drug potentiates cAMP accumulation induced by iloprost and forskolin. The drug also inhibits soluble cAMP phosphodiesterase in a dose-dependent manner. No effect on adenylate cyclase activity measured in platelet membranes was evident.     

Intravenous injection of propylene glycol causes pulmonary hypertension in sheep

Propylene glycol (30%) is the carrier base for pentobarbital sodium in preparations often used in research laboratories. It has caused pulmonary hypertension in calves, and we found it caused pulmonary hypertension in sheep as well. To investigate the mechanism of pulmonary hypertension with propylene glycol, we injected an average loading dose of 30% propylene glycol (0.5 ml/kg) into adult sheep, which was followed by a rise in thromboxane levels (P less than 0.05) in systemic arterial plasma and lung lymph and by a dramatic increase in pulmonary arterial pressure (17 +/- 1 to 35 +/- 4 mmHg, P less than 0.05) and a fall in cardiac output (2.7 +/- 0.5 to 1 +/- 0.2 l/min). Indomethacin pretreatment blocked the rise in thromboxane in lung lymph and arterial plasma and substantially, although not entirely, blocked the rise in pulmonary arterial pressure. Pulmonary intravascular macrophages (PIMS), which are present in sheep and calves, can release thromboxane in response to a stimulus. To test whether PIMS might be the source of the thromboxane and pulmonary hypertension, we injected propylene glycol into guinea pigs and dogs, which are reported to have no PIMS, as well as into newborn lambs, which are not believed to develop many PIMS until the 2nd wk of life. In dogs and guinea pigs there was no response to propylene glycol. In lambs there was a rise in pulmonary vascular resistance but significantly less than in adult sheep; indomethacin blocked this response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thromboxane-induced pulmonary vasoconstriction: involvement of calcium

Infusion of tert-butyl hydroperoxide (t-bu-OOH) or arachidonic acid into rabbit pulmonary arteries stimulated thromboxane B2 (TxB2) production and caused pulmonary vasoconstriction. Both phenomena were blocked by cyclooxygenase inhibitors or a thromboxane synthase inhibitor. The increase in pulmonary arterial pressure caused by either t-bu-OOH or arachidonic acid infusion correlated with the concentration of TxB2 in the effluent perfusate. The concentration of TxB2 in the effluent perfusate, however, was always 10-fold greater after arachidonic acid infusion. In the rabbit pulmonary vascular bed lipoxygenase products did not appear involved in the vasoactive response to t-bu-OOH or exogenous arachidonic acid infusion. Calcium entry blockers or a calcium-free perfusate prevented the thromboxane-induced pulmonary vasoconstriction. Calmodulin inhibitors also blocked the pulmonary vasoconstriction induced by t-bu-OOH without affecting the production of TxB2 or prostacyclin. These results suggest that thromboxane causes pulmonary vasoconstriction by increasing cytosol calcium concentration.     

Cyclooxygenase pathway mediates lung injury induced by phorbol and platelets

The role of platelets in lung injury has not been well defined. In the present study of isolated perfused rat lungs, phorbol myristate acetate (PMA; 0.15 microgram/ml) or platelets (6.7 X 10(4)/ml) alone did not discernibly change the pulmonary arterial pressure (PAP) or lung weight (LW). However, the combination of platelets and PMA drastically increased the PAP and LW (delta PAP 26.2 +/- 1.0 mmHg, delta LW 2.7 +/- 0.4 g). delta PAP was positively correlated with the increase in thromboxane B2 produced by infusion of platelets and PMA (thromboxane B2 = 35.6 + 0.97 delta PAP, r = 0.67, P less than 0.01). The hypertension and edema formation induced by PMA and platelets were strongly attenuated by indomethacin, an inhibitor of platelet cyclooxygenase (delta PAP 5.6 +/- 2.0 mmHg, P less than 0.001; delta LW 0.0 +/- 0.1 g, P less than 0.001), and by imidazole, an inhibitor of thromboxane A2 synthase (PAP 8.0 +/- 2.5 mmHg, P less than 0.001; LW 0.0 +/- 0.3 g, P less than 0.01). Inactivation of platelet lipoxygenase with nordihydroguaiaretic acid mildly depressed pulmonary pressure but did not affect delta LW (delta PAP 18.9 +/- 1.6 mmHg, P less than 0.05; delta LW 3.1 +/- 0.3 g, P greater than 0.05). In vitro experiments showed that the capacity of platelets to release oxygen radicals was only 2.6% of that found for granulocytes. These results suggest that platelets may be activated by PMA to increase PAP and vascular permeability.(ABSTRACT TRUNCATED AT 250 WORDS)     

The roles of platelet function, thromboxane, blood lipids and nitric oxide in hypertension of children and adolescents

The roles of platelet function, plasma lipids and nitric oxide (NO) were studied in adolescent patients with essential hypertension (JEHT group), with chronic renal failure (CRF) associated with hypertension (CRFH group), and CRF patients with normal blood pressure (CRF group), as compared with normal controls (cont. group). Platelet aggregation and the thromboxane B(2)(TxB(2)) level were significantly higher in the JEHT and CRFH groups as compared with the cont. group, whereas they were significantly lower in the CRF group. On the other hand, the platelet cAMP level was significantly lower in the JEHT and CRFH groups than in the cont. group. The plasma NO level was significantly higher only in the JEHT as compared with the cont. group (120 +/- 39 and 89 +/- 21 microM, respectively). The plasma total cholesterol, triglyceride and LDL cholesterol concentrations were normal in the JEHT group, but high in the CRF and CRFH group, the HDL cholesterol level was lower in the CRF and CRFH groups as compared with the cont. and JEHT groups. There was a positive correlation between the platelet aggregation and the TxB(2)level and between the BP and the platelet aggregation. In conclusion, hyperlipidaemia is commonly present in uraemia with haemodialysis, but is not specific for hypertension in children, while an increased platelet function is frequently associated with hypertension. The increased NO level might play a compensatory role in JEHT.     

Protective actions of a new thromboxane synthetase inhibitor in arachidonate induced sudden death

A new thromboxane synthetase inhibitor, OKY-046, at doses of 0.5 and 1.0 mg/kg prevented mortality induced by sodium arachidonate in 100% of the rabbits studied. Sodium arachidonate at a dose of 1.25 mg/kg uniformly decreased mean arterial blood pressure to values approximately 0 mm Hg, stopped respiration and produced sudden death within 3-5 minutes in all rabbits studied. OKY-046 prevented all these sequelae of the sodium arachidonate. Untreated rabbits challenged with sodium arachidonate develop large increases in circulating thromboxane B2 (TxB2) and 6-keto PGF1 alpha of about 12- to 18-fold. In contrast, OKY-046 prevented the increase in TxB2 concentrations and the pulmonary thrombosis, but did not block the rise in 6-keto PGF1 alpha following arachidonate injection. These results suggest that the protective mechanism of OKY-046 in arachidonate induced sudden death is via selective inhibition of thromboxane synthesis.     

Activation of protein kinase C inhibits sodium fluoride-induced elevation of human platelet cytosolic free calcium and thromboxane B2 generation

Addition of NaF to washed platelets produces a dose-dependent and transient elevation of the intracellular free calcium concentration ([Ca++]i), thromboxane B2 (TxB2) generation and dense granule release, all of which are significantly inhibited when the extracellular calcium concentration ([Ca++]e) is reduced with EGTA. Inhibition of platelet cyclo-oxygenase by acetylsalicylic acid (ASA) does not affect NaF-induced elevation of [Ca++]i and dense granule release in the presence of 1 mM [Ca++]e. Pre-incubation of the platelets with the phorbol ester TPA produces a marked inhibition of NaF-induced elevation of [Ca++]i and TxB2 generation without affecting dense granule release. Thus, NaF may have more than one site of action. Pretreatment of the platelets with the selective protein kinase C inhibitor H7 prevents TPA induced inhibition of NaF mediated rise in [Ca++]i and TxB2 generation. Thus we propose that NaF induced calcium mobilisation is analogous to receptor-operated calcium mobilisation in platelets, as it is readily inhibited by protein kinase C activation or by the reduction of [Ca++]e and is independent of platelet cyclo-oxygenase activity.     

A species difference in platelet aggregation induced by tumour cells

Ehrlich ascites tumour cells (EATC) induced the aggregation of human platelets but not of sheep or rabbit platelets in native platelet-rich plasma. Aggregation was initiated by the interaction of EATC with a component(s) of human plasma, possibly related to the complement system, which led to the release of cellular ADP, a potent platelet aggregating agent. EATC previously incubated with human platelet-poor plasma induced immediate aggregation in platelet-rich plasma from all three species. The species difference in platelet aggregation by EATC is therefore related to the activity or availability of plasma component(s) responsible for release of cellular ADP rather than to intrinsic differences in platelet responsiveness to the tumour cells.     

Effects of Welsh onion extracts on human platelet function in vitro

Welsh onion has been consumed for prevention of cardiovascular disorders. However, its underlying mechanisms are still unclear. This study investigated whether Welsh onion extracts can alter human platelet function (ie, platelet adhesion, aggregation, and thromboxane release). To clarify the underlying mechanisms, we also measured the intracellular calcium ([Ca2+]i) and cyclic nucleotide levels in platelets. Our results showed that 1) boiled extracts directly induced platelet aggregation in a dose-dependent manner; 2) raw extracts inhibited platelet adhesion and ADP-evoked platelet aggregation, while boiled extracts enhanced them; 3) raw green extract suppressed ADP-stimulated platelet [Ca2+]i elevation and thromboxane production, whereas boiled green extract enhanced them; 4) raw green extract elevated platelet cAMP level, whereas boiled green extract had no effect on cAMP level. Furthermore, the boiled green extract, but not the raw extract, induced pronounced platelet morphological changes. In conclusion, raw extracts of Welsh onion inhibit platelet function in vitro while boiled extracts activate platelets.