Human rhabdosarcoma cell-induced aggregation of blood platelets.

The ability of tumor cells shed into the circulation to cause adhesion and aggregation of blood platelets may be involved in successful metastasis of primary tumors. Rhabdosarcoma is a rare, early metastasizing tumor previously uncharacterized for ability to alter platelet function. It was found that human rhabdosarcoma cells (American Type Culture Collection) dose dependently induce biphasic aggregation of human blood platelets in heparinized platelet-rich plasma; aggregation responses could also be elicited in citrated plasma. Aggregation caused by rhabdosarcoma can be inhibited by apyrase treatment of either rhabdosarcoma or platelets, and by pretreatment of platelets with prostacyclin, cilostamide, inhibitors of thromboxane A2 production, or TMB-8; only apyrase and prostacyclin inhibited both phases of aggregation. Tumor cell supernatant contained only enough ADP to cause a negligible, reversible aggregation response. Hirudin, verapamil, and triazolam do not inhibit rhabdosarcoma-induced aggregation. Aggregation of platelets by rhabdosarcoma cells thus appears to involve ADP, from tumor cells and/or platelets, and platelet calcium mobilization and thromboxane A2 synthesis and release.

Kinetics of ibuprofen effect on platelet and endothelial prostanoid release.

Reciprocal control of platelet function in the circulation has been proposed for the platelet-produced platelet proaggregatory prostanoid thromboxane A2 (TxA2) and the vascular endothelium-produced antiaggregatory prostanoid prostacyclin (PGI2). Forty drug-free healthy subjects were given a single dose of ibuprofen (0, 8, 10, 12, or 14 mg/kg) in a randomized, double-blind study. Blood samples were drawn 0, 2, 4, and 6 hours and 7 days after dosing for determination in serum (from untreated or in vitro indomethacin-treated portions of the blood) of TxA2 and PGI2 by radioimmunoassay of their stable metabolites (TxB2 and 6-keto-PGF1 alpha). Maximal platelet release of TxA2 (untreated serum) was lower in all drug groups 2, 4, and 6 hours after dosing. There was no significant decrease in PGI2 release. All doses of ibuprofen (except 0 mg/kg) induced essentially identical plasma levels at the times of measurement (postpeak decline), and effects could not be distinguished by dose for 8, 10, 12, or 14 mg/kg at these times. It is concluded that ibuprofen induces antiplatelet effects for at least 6 hours while preserving normal antiplatelet mechanisms.

Characterization of polymorphonuclear leukocyte aggregation in vitro induced by heat-inactivated group B streptococcus.

We studied the aggregatory characteristics of human polymorphonuclear leukocytes (PMNs) in response to heat-inactivated group B streptococcus. PMNs suspended in physiologic salt solution do not aggregate to heat-inactivated group B streptococcus (GBS) unless the GBS is previously opsonized in autologous plasma. The aggregating activity of both opsonized GBS and activated plasma are reduced if the plasma is decomplemented before incubation with GBS. Pretreatment of PMNs with pronase inhibited opsonized GBS-induced aggregation, suggesting aggregation via cell membrane receptors for opsonic fragments of C3. Pronase pretreatment had no significant effect on aggregation induced by activated plasma or arachidonic acid. Unlike PMNs in physiologic salt solution, PMNs suspended in plasma aggregate when stimulated by unopsonized GBS. GBS aggregates PMNs via complement cascade activation, opsonization, and interaction with cell membrane receptors to stimulate cellular mechanisms resulting in PMN aggregation.

In vitro inhibition of group B streptococcus-induced polymorphonuclear leukocyte aggregation.

Evidence suggests that part of the pathophysiologic response seen in group B streptococcal (GBS) sepsis may be due to polymorphonuclear leukocyte (PMN) activation. Indomethacin (INDO), which inhibits eicosanoid metabolism, attenuates the pathophysiologic response stimulated by GBS, possibly due to inhibition of PMN aggregation. We examined the capability of two eicosanoid metabolism inhibitors, INDO and nordihydroguaiaretic acid (NDGA), to inhibit PMN aggregation induced by heat-inactivated opsonized GBS and GBS-activated plasma. Opsonized GBS-induced PMN aggregation was inhibited by INDO (50-500 microM) and NDGA (1-100 microM). Over similar concentration ranges, INDO and NDGA had no significant effect on PMN aggregation induced by GBS-activated plasma. PMNs in plasma aggregate in response to unopsonized GBS. The stimuli for aggregation are opsonized GBS and GBS-activated plasma. INDO (50-500 microM) was unable to inhibit aggregation under this condition. Over the same concentration range in which INDO inhibited opsonized GBS-induced PMN aggregation, INDO was unable to inhibit opsonized GBS-induced superoxide production in PMNs. NDGA was examined but was found to interfere with the assay. The above evidence suggests PMN aggregation via eicosanoid metabolism may play a role in GBS-induced sepsis, which may be attenuated by agents such as INDO and NDGA.

Effects of acetaldehyde condensation products on human platelet aggregation.

Condensation products (CP) of the ethanol metabolite, acetaldehyde, and endogenous amines, such as dopamine and serotonin, have been proposed to be effectors of some symptoms of chronic ethanol use. Since hemostatic defects are known to occur in chronic ethanol use, the effects of CP on in vitro human platelet aggregation responses induced by several agents were determined. Both isoquinoline and beta carboline type CP significantly inhibited aggregation responses induced by epinephrine, with the concentrations to produce 50% inhibition ranging from 8-347 uM. The beta-carbolines significantly inhibited ADP-induced aggregation and also inhibited aggregation induced by collagen or arachidonic acid, but at high concentrations. Effects on epinephrine aggregation and ADP aggregation were reversible. Potential mechanisms of the inhibitory effects were briefly examined. Concomitant use of the phosphodiesterase inhibitor theophylline potentiated the effect of some but not other CP, possibly indicating an involvement of cyclic AMP. Concomitant use of the non-specific beta-adrenergic inhibitor propranolol had no effect on CP inhibition, indicating that CP probably do not stimulate platelet adenylyl cyclase-coupled beta 2-adrenoceptors. Thus, general inhibition by CP of platelet responses in the circulation is unlikely, except, possibly, for epinephrine-induced aggregation, because of the high concentrations of CP required. However, local regulation of platelet responses by release of stored CP during aggregation is possible since CP are stored in platelet dense granules.

Tetrahydroisoquinolines and beta-carbolines: specific binding to human platelet alpha 2-receptors in vitro.

Previous human platelet aggregation studies indicated that tetrahydroisoquinoline (TIQ) and beta-carboline (BC) compounds act as competitive antagonists for epinephrine for the platelet alpha 2-adrenergic receptor. In this study, the relative binding affinities of 1,2,3,4-TIQ (THIQ), salsolinol (SAL), norharman (NH), harmaline (HAR) and tetrahydronorharman (THN) for the platelet alpha 2-receptor were determined using the selective antagonist [3H]-yohimbine. The order of potency in yohimbine-displacing ability was THIQ = THN greater than HAR greater than NH greater than SAL, with IC50's ranging from 10 microM to 170 microM. A structure-activity relationship could also be identified for the condensation products examined.

Alteration of platelet reactivity following a shock-inducing procedure in pigs.

Changes (usually increases) in platelet reactivity occur concomitantly with circulatory shock. This suggests both effects of the shock state on platelets and participation by platelets in sequelae of shock such as shock-lung syndrome. Changes in platelet reactivity have not been documented previously for splanchnic artery occlusion (SAO)-induced shock. SAO shock in pigs produced a decrease in platelet reactivity in the postrelease period. This decreased reactivity was not accompanied by changes in platelet counts or size distribution. Since exposure of the platelets to a submaximal aggregating stimulus prior to the sampling time at 15 minutes postrelease may cause this type of relative refractoriness, it is possible that the platelets may have been in a hyperreactive state very early in the postrelease period or prior to release. Exposure to increased but submaximal ADP levels, or to other submaximal stimuli, could cause the observed decrease in reactivity. Further examination of the time sequence and causes of changes of platelet reactivity in shock are necessary and desirable.

Sodium arachidonate induced in vitro polymorphonuclear leukocyte aggregation.

Human polymorphonuclear leukocytes (PMNs) aggregated to sodium arachidonate (AA) in a dose dependent manner. Other agents such as epinephrine, or ADP were unable to initiate aggregation. The cells released lactate dehydrogenase (LDH) during aggregation, and electron microscopy demonstrated distinct morphologic changes in PMNs aggregated with AA. Indomethacin enhanced AA induced aggregation, whereas 1-methylimidazole inhibited aggregation and LDH release. Preincubation with cytochalasin B (CTB) did not enhance AA induced aggregation, nor did it reduce LDH release. Also, the effects of indomethacin or 1-methylimidazole on AA induced aggregation were unaltered by the presence of CTB. The results indicate that 1) LDH release occurs as a function of the aggregation process rather than a non-specific effect, 2) competition between cyclooxygenase and lipoxygenase occurs and 3) shifting this competition in favor of lipoxygenase enhances the PMN aggregation process.

Measurement of malonyldialdehyde production during sodium arachidonate-induced polymorphonuclear leukocyte aggregation.

Sodium arachidonate stimulated canine polymorphonuclear leukocytes (PMNs) to aggregate and produce malonyldialdehyde (MDA). The MDA production was due to cellular processes during the aggregation, as boiled PMN suspension neither aggregated nor produced MDA. Aggregation and MDA production were not due to platelet contamination because epinephrine and ADP were unable to stimulate either of these responses in the PMN suspensions. Finally, use of the aggregatory modifiers indomethacin, 1-methylimidazole, and vitamin E dissociated aggregation from MDA production. These results suggest that the cellular process(es) resulting in MDA production is/are not responsible for canine PMN aggregation.

Platelet regulatory prostanoids and platelet release products in sickle cell disease.

Changes in platelet function have been observed for sickle cell disease (SCD). Levels of the arachidonic acid metabolites, thromboxane A2 (released by stimulated platelets) and prostacyclin (released from vascular endothelium), which stimulate and inhibit platelets, respectively, have been implicated in overall regulation of platelet function. Circulating basal levels of thromboxane and prostacyclin were determined in 1) a group of SCD volunteers (n = 21; at half-yearly steady state intervals and also at 24 hr, 72 hr, and 7 days after start of pain crisis) and 2) an age-, sex-, and race-matched control group (n = 18; single determinations). Circulating levels of beta-thromboglobulin (beta-TG), as well as thrombin (clotting)-stimulated platelet release of thromboxane, were also determined. Statistically significant decreases were found for prostacyclin, basal thromboxane, and thrombin-induced (maximal) thromboxane (alone or per platelet), for steady state SCD vs. normal controls. In addition, significant increases in maximal thromboxane were identified in crises (24, 72 hr) compared with steady state. Crisis beta-TG (24 hr) was significantly elevated compared with controls or steady state SCD. The ratio of basal thromboxane to prostacyclin was increased in crisis, but not significantly. Crisis frequency may correlate in part with changes in platelet function: steady state maximal thromboxane and released thromboxane per platelet were significantly lower in SCD volunteers who had crises during the study vs. those who did not (equivalent study time). The data support altered platelet function in SCD, possibly refractoriness (desensitization), manifest as decreased thromboxane release, to thrombin and/or other stimuli: alternate explanations are discussed.

Lung edema caused by high peak inspiratory pressures in dogs. Role of increased microvascular filtration pressure and permeability.

Mechanical ventilation with high peak airway pressures (Paw) has been shown to induce pulmonary edema in animal experiments, but the relative contributions of transvascular filtration pressure and microvascular permeability are unclear. Therefore, we examined the effects of positive-pressure ventilation on two groups of open-chest dogs ventilated for 30 min with a peak Paw of 21.8 +/- 2.3 cm H2O (Low Paw) or 64.3 +/- 3.5 cm H2O (High Paw). No hemodynamic changes were observed in the Low Paw group during ventilation, but mean pulmonary artery pressure (Ppa) increased by 9.9 cm H2O, peak inspiratory Ppa by 24.6 cm H2O, and estimated mean microvascular pressure by 12.5 cm H2O during High Paw ventilation. During the same period, lung lymph flow increased by 435% in the High Paw and 35% in the Low Paw groups, and the terminal extravascular lung water/blood-free dry weight ratios were 5.65 +/- 0.27 and 4.43 +/- 0.13 g/g, respectively, for the two groups. Lung lymph protein clearances and minimal lymph/plasma ratios of total protein were significantly higher (p less than 0.05) after 2 h of increased left atrial pressure (PLA) in the High Paw group versus the Low Paw group, which indicates a significant increase in microvascular permeability. Lymph prostacyclin concentration in pulmonary lymph, measured as the stable metabolite 6-0-PGF1 alpha, was increased significantly by 70 to 150% from baseline (p less than 0.05) in both groups during the periods of increased Paw and increased PLA, but it was not significantly different between the groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Aortic prostacyclin release is lowered by superior mesenteric artery occlusion (SMAO) shock in pigs.

We have previously demonstrated altered platelet reactivity subsequent to splanchnic artery occlusion (SAO) shock in pigs: decreased reactivity, such as might be seen subsequent to submaximal stimulation, was observed in the postrelease period. Prostacyclin (PGI2) formed and released by vessel endothelium may function as a circulating hormone and regulate platelet reactivity by opposing the stimulatory influence of other factors. We have therefore investigated PGI2 release by aortas taken at "death" (systolic blood pressure = 30 mmHg) from pigs subjected to shock induced by a 2-hr occlusion of the superior mesenteric artery (SMAO). This shock is analogous to, but with a longer time course than, SAO shock. Aortas from sham-operated animals (matched for "death" time to the SMAO animals) served as controls. Thoracic aortas were removed, placed in cold 50 mM pH 7.5 tris buffer, cleaned of adhering tissue or clots, cut into rings and suspended in room temperature 50 mM pH 7.5 tris buffer into which PGI2 was released. Aliquots of the latter were quantitated for PGI2 by bioassay using human platelets for which a dose-response relationship for pure, synthetic PGI2 had been established: aliquot PGI2 quantities were determined graphically from the log dose-response (percentage inhibition of aggregation) curves, and converted arithmetically to amount per weight tissue. After 10 min incubation of tissue in buffer, the mean +/- SEM PGI2 for the shock animals (N = 8) was 0.053 +/- 0.02 ng/mg, and for the sham animals (N = 7), 0.174 +/- .06; the P value for t-test of means was 0.083. For 30-min incubation, the mean PGI2 for the shock group was 0.052 +/- 0.02 and for the sham, 0.283 +/- 0.11; the P value was 0.029. Inhibitory activity of the buffer aliquots declined in parallel with authentic PGI2. Thus, the occurrence of SMAO shock resulted in a significant decrease in aortic release of PGI2. Altered platelet reactivity as a consequence of shock, such as demonstrated by us and by others, could thus be in part a result of lowered endothelial release of PGI2, either alone or in combination with the occurrence of activation by other factors.

Pulmonary embolism: analysis of endothelial pore sizes in canine lung.

The effect of glass-bead microemboli (diameter 100 micron, range 77-125 micron) in the absence of fibrinolysis inhibition on pulmonary hemodynamics and microvascular permeability was determined in anesthetized, microfilaria-free dogs acutely prepared for the collection of lung lymph. Pulmonary vascular resistance, pulmonary capillary pressure (Pc), lymph flow (QL), and the ratio of lymph (CL) to plasma (Cp) protein concentrations were measured after 0.2 (n = 4), 0.4 (n = 6), or 0.6 (n = 3) g/kg beads. In all cases, emboli increased resistance and QL severalfold (P less than 0.05), while CL/Cp remained unchanged. In part, the increase in QL could be attributed to an increase in Pc compared with control (12.4 +/- 2.2 vs. 6.7 +/- 0.6 mmHg, P less than 0.05). Furthermore, since the solvent-drag reflection coefficient (sigma f) for total proteins approaches the osmotic reflection coefficient (sigma d) at high QL, sigma d was estimated under these conditions with sigma f approximately equal to sigma d approximately equal to 1 - (CL/Cp)min. The sigma d was decreased (P less than 0.05) after 0.4 and 0.6 g/kg beads to 0.55 +/- 0.03 and 0.50 +/- 0.07, respectively, when compared with that in control lungs (sigma d = 0.62 +/- 0.02; Parker et al., Circ. Res. 48: 549-561, 1981). A pore-stripping analysis demonstrated that after emboli the pulmonary endothelial barrier could be described by a population of small (80 A) and large (350 A) pores. However, the number of large to small pores was 1:1,195, compared with 1:195 in control lungs, suggesting an increased contribution of extra-alveolar vessels upstream of the emboli.(ABSTRACT TRUNCATED AT 250 WORDS)

Ibuprofen in experimental group B streptococcal shock.

A rabbit model was used to characterize the effects of high (Group II, 100 mg/kg) and low (Group III, 10 mg/kg) dose ibuprofen in modulating the hemodynamic and hematologic manifestations of group B streptococcal shock. Short-term survival was significantly increased with ibuprofen pretreatment. Ibuprofen failed to prevent GBS-induced shock, although shock was favorably modified in a dose dependent manner. Likewise, GBS-induced increases in 6KPGF1a and TxB2 were not prevented but were modified in Group II at 120 min. However, neutropenia, thrombocytopenia, and acidosis were not prevented by pretreatment with ibuprofen and may have been exacerbated. Thus, ibuprofen modifies but does not prevent GBS-induced hemodynamic and hematologic manifestation.