Human platelets modulate edema formation in isolated rabbit lungs.

The role of platelet glucose-6-phosphate dehydrogenase (G-6-PD) in mediating the effects of human platelets on oxidant-induced edema in the isolated perfused rabbit lung was investigated using dehydroepiandrosterone, a specific steroidal inhibitor of G-6-PD. Xanthine oxidase (0.003 and 0.012 U/ml) caused lung edema that was attenuated by coinfusion of washed human platelets. Platelets that were incubated with DEA to inhibit G-6-PD activity augmented xanthine oxidase-induced lung edema and pulmonary hypertension at both doses of xanthine oxidase. Infusion of papaverine to maintain stable pulmonary artery (PA) pressures, incubation of G-6-PD-inhibited platelets with acetylsalicylate, or infusion of a thromboxane-prostaglandin endoperoxide receptor site antagonist, SQ 29548, into the lung perfusate prevented augmentation of lung edema and the PA pressor response by G-6-PD-inhibited platelets. It was concluded that antioxidant-intact platelets attenuate oxidant-induced lung edema by preventing increased membrane permeability, and that G-6-PD-inhibited platelets augment lung edema through hydrostatic mechanisms mediated by release of platelet cyclooxygenase products.     

Acetyl glyceryl ether phosphorylcholine-stimulated human platelets cause pulmonary hypertension and edema in isolated rabbit lungs. Role of thromboxane A2.

Macrophages, neutrophils, and platelets may play a role in acute edematous lung injury, such as that seen in the adult respiratory distress syndrome (ARDS), but their potential actions and interactions are unclear. Because stimulated human macrophages and neutrophils can release acetyl glyceryl ether phosphorylcholine (AGEPC), a potent platelet activator, we hypothesized that in ARDS, leukocyte release of AGEPC might stimulate platelets to release thromboxane A2 (TXA2), which then produces pulmonary hypertension and lung edema. In support of this premise, we found that pulmonary hypertension and edema occurred in isolated rabbit lungs perfused with human platelets and AGEPC, but not with platelets or AGEPC alone. Infusion of a vasodilator (nitroglycerin) to maintain base-line pulmonary artery pressures in lungs perfused with platelets and AGEPC prevented the development of lung edema suggesting that platelet and AGEPC-induced edema was hydrostatic in nature. Additional experiments suggested that the increased pressure was a result of TXA2 release from platelets stimulated by AGEPC. Specifically, preincubation of platelets with imidazole, a thromboxane synthetase blocker, prior to infusion with AGEPC significantly diminished pulmonary hypertension and prevented lung edema. Furthermore, pretreating lung preparations with 13-azaprostanoic acid, a TXA2 antagonist, before infusion of AGEPC and untreated platelets also reduced the pulmonary hypertension and blocked the lung edema. The role of TXA2 was further suggested when perfusates from lungs infused with platelets and AGEPC developed high levels of TXA2, whereas perfusates from controls did not. These results suggest that platelet aggregation induced by AGEPC may contribute to ARDS by releasing TXA2, which raises microvascular pressure and increases edema formation, especially when an underlying permeability defect is present.     

Enhancement of platelet function by superoxide anion.

During the aerobic conversion of xanthine to uric acid by xanthine oxidase, superoxide anion and hydrogen peroxide are produced along with the hydroxyl radical. Our studies demonstrate that washed human platelets incubated with xanthine and xanthine oxidase aggregated and released [14C]serotonin. Aggregation and release were dependent on the duration of exposure to xanthine oxidase as well as the concentration of enzyme. Both reactions were inhibited by the superoxide scavenger enzyme superoxide dismutase but not by catalase, or the free radical scavenger mannitol, suggesting that they were induced by superoxide anion. Superoxide-dependent release was inhibited by prior incubation of platelets with 1 mM EDTA, 1 micronM prostaglandin E1, or 1 mM dibutyryl cyclic AMP, but was unaffected by 1 mM acetylsalicylic acid or 1 micronM indomethacin. After prolonged incubation with xanthine and xanthine oxidase there was also efflux of up to 15% of intraplatelet 51Cr, a cytosol marker. This leakage was prevented by the addition of catalase to the media but not by superoxide dismutase. Incubation with xanthine and xanthine oxidase did not produce malonyldialdehyde, the three-carbon fatty acid fragment produced during prostaglandin endoperoxide synthesis and lipid peroxidation. Prior exposure of platelets to low fluxes of superoxide anion lowered the threshold for release by subsequent addition of thrombin, suggesting a synergistic effect. We conclude that superoxide-dependent aggregation and release may be a physiologically important method to modulate hemostatic reactions particularly in areas of inflammation or vessel injury which could have high local concentrations of superoxide anion.     

Glutathione disulfide formation occurring during hypoxia and reoxygenation of rat lung

To study changes in glutathione redox status as an indicator of oxidant stress during hypoxia and reoxygenation, we perfused isolated rat lungs with a high or low oxygen perfusate and measured the release of total glutathione and glutathione disulfide (GSSG) into the perfusate. Lungs were perfused for a 20-minute baseline period with a perfusate equilibrated with 95% O2 and 5% CO2 and ventilated with a 95% O2 and 5% CO2 gas mixture. Only very low amounts of oxygen were measurable in this hypoxic perfusate. The lungs were then perfused from a second reservoir containing perfusate equilibrated with 95% N2 and 5% CO2 and ventilated with a 95% N2 and 5% CO2 gas mixture. After the period of hypoxia, the lungs were reperfused with the 95% O2 and 5% CO2 equilibrated perfusate and ventilated with a 95% O2 and 5% CO2 gas mixture for the remainder of the experiment. Glutathione was measured in the perfusate serially throughout the experiment, and lactic dehydrogenase (LDH) was also measured to assess cell membrane rupture during the infusion. GSSG release remained stable in the baseline and hypoxic period but rose significantly in the reoxygenation period, to concentrations approximately two times basal release. Lung tissue concentrations of GSSC also rose in the reoxygenation period. Decreasing lung glutathione reductase activity by pretreating animals with 1,3-bis-2-chloroethyl)-1-nitrosourea (BCNU) increased GSSG release into the perfusate during reoxygenation. We conclude that GSSG formation and release is increased in the lung during the reoxygenation period after lung hypoxia, suggesting the presence of hydroperoxide and free radical metabolism. These data support the hypothesis that alterations in lung metabolism occur during hypoxia that allow free GSSG formation and release during the reintroduction of oxygen.     

Binding of collagen alpha1 chains to human platelets.

We previously reported that purified alpha1 chains of type 1 chick skin collagen induce platelet aggregation. We now describe immunological and biochemical evidence that the peptide binds to intact platelets as an early event in the induction of platelet aggregation and the release reaction. Antibody against alpha1 (I) was obtained by immunizing rabbits with complete Freund's adjuvant mixed with purified alpha1. Immunofluorescence studies showed that alpha1(I)-treated platelets exhibited strong immunofluorescence. The intensity of fluorescence was markedly decreased by the pretreatment of platelets with alpha1-CB5 and glucosylgalactosylhydroxylysine. Dose-response curves of platelet aggregation induced by alpha1 and the binding of alpha1 by washed intact platelets are correlated. The biochemical studies showed that the binding of the alpha1 chain to washed intact platelets was platelet concentration and temperature dependent, and that it reached a maximum in 10 min. The process was reversible and specific, with an association constant of 1.7 muM. The inhibitor of alpha1-induced platelet aggregation, glucosylgalactosyl hydroxylysine, inhibited the alpha1 binding. These results suggest that alpha1(I) chains bind to specific receptor site(s) on platelet membranes to trigger aggregation and the release reaction.     

Interaction of prostaglandins E1 and E2 in regulation of cyclic-AMP and aggregation in human platelets: evidence for a common prostaglandin receptor

The effects of (PGE) prostaglandins E1 and E2 on the aggregation and release reaction induced in human platelets by ADP have been investigated. Measurements of cyclic-AMP content in (PRP) platelet-rich plasma were made concurrently. Although both PGE1 and PGE2 independently increased platelet cyclic-AMP and inhibited 1st phase ADP-induced aggregation (order of potency, PGE1 PGE2), the effect of a fixed concentration of PGE2 in the presence of PGE1 varied. At low PGE1 concentrations, the effects were additive, but at higher PGE1 concentrations PGE2 lowered the efficacy of PGE1. These results suggest that PGE2 may be a "partial agonist" of PGE1. PGE2 enhanced and PGE1 inhibited the 2nd phase of ADP-induced aggregation and the release of serotonin by a mechanism which appeared to be independent of cyclic-AMP content. A mixture of the 2 PGs produced responses intermediate between those observed with each PG independently. Binding of PGE1-3H to platelets was demonstrated in PRP and in concentrated platelet suspensions. PGE1 and PGE2 inhibited binding in a simular manner. It is proposed that PGE1 and PGE2 compete for a common receptor on the platelet membrane.     

Protection of rabbit lungs from endotoxin injury by in vivo hyperexpression of the prostaglandin G/H synthase gene.

A recombinant prostaglandin G/H (PGH) synthase gene has been expressed in vitro in bovine pulmonary artery endothelial cells and in vivo in rabbits by transfection with a plasmid using cationic liposomes. Transfection of bovine pulmonary artery endothelial cells with the PGH synthase cDNA resulted in increased intracellular PGH synthase protein (determined by Western blot analysis) and increased release of prostacyclin. Rabbits intravenously transfected with the PGH synthase gene had increased plasma levels of prostacyclin and PGE2, and their lungs produced increased amounts of the same eicosanoids. In an in situ, perfused preparation of PGH synthase transfected rabbit lungs, the pressor response to endotoxin was markedly attenuated. In addition, pulmonary edema and release of thromboxane B2 into the perfusate after endotoxin infusion were markedly decreased in transfected lungs compared to controls (animals transfected with a pCMV4 construct that did not contain a cDNA insert). The data suggest that augmented endogenous production of prostacyclin and PGE2, achieved by liposome-mediated gene transfer, protects the lungs from endotoxin. This may be caused in part by suppression of endotoxin-stimulated thromboxane B2 production. Modification of lipid mediator responses by in vivo transfection is a potential approach to the therapy of acute lung injury.     

Staphylococcal superantigen-like 5 activates platelets and supports platelet adhesion under flow conditions, which involves glycoprotein Ibα and αIIbβ3

Summary.  Objectives:  Staphylococcal superantigen-like 5 (SSL5) is an exoprotein secreted by Staphylococcus aureus that has been shown to inhibit neutrophil rolling over activated endothelial cells via a direct interaction with P-selectin glycoprotein ligand 1 (PSGL-1). Methods and Results:  When purified recombinant SSL5 was added to washed platelets in an aggregometry set-up, complete and irreversible aggregation was observed. Proteolysis of the extracellular part of GPIbα or the addition of dRGDW abrogated platelet aggregation. When a mixture of isolated platelets and red cells was perfused over immobilized SSL5 at a shear rate of 300 s⁻¹, stable platelet aggregates were observed, and platelet deposition was substantially reduced after proteolysis of GPIb or after addition of dRGDW. SSL5 was shown to interact with glycocalicin, a soluble GPIbα fragment, and binding of SSL5 to platelets resulted in GPIb-mediated signal transduction as evidenced by translocation of 14-3-3ζ. In addition, SSL5 was shown to interact with endothelial cell matrix (ECM) and this interaction enhanced aggregation of platelets from whole blood to this ECM. Conclusions:  SSL5 activates and aggregates platelets in a GPIbα-dependent manner, which could be important in colonization of the vascular bed and evasion of the immune system by S. aureus .     

Cyclic adenosine 3'',5''-monophosphate inhibits the availability of arachidonate to prostaglandin synthetase in human platelet suspensions.

When thrombin is added to washed human platelets, one of its actions results in activation of a phospholipase that hydrolyzes arachidonic acid from phospholipids. The arachidonate is converted to the cyclic endoperoxides (prostaglandin G2 and prostaglandin H2) by fatty acid cyclo-oxygenase. These compounds are then converted to thromboxane A2, also called rabbit aorta-contracting substance, by thromboxane synthetase. These labile, pharmacologically active compounds then break down to inactive products including thromboxane B2 and malonaldehyde. Incubation of platelets with either dibutyryl cyclic adenosine 3',5'-monophosphate (dBcAMP) or prostaglandin E1 (PGE1) before thrombin addition blocks the subsequent formation of oxygenated products of arachidonic acid including thromboxane A2, thromboxane B2, and malonaldehyde. In contrast, when arachidonic acid is added directly to platelets, prior incubation with dBcAMP or PGE1 does not inhibit production of the prostaglandins or their metabolites. Thrombin treatment of platelets also blocks the acetylation of cyclo-oxygenase by aspirin since the hydrolyzed arachidonic acid competes with aspirin for the active site on cyclo-oxygenase. Prior treatment of platelets with dBcAMP or PGE1 reverses the thrombin inhibition of the acetylation of cyclo-oxygenase. We conclude that agents which elevate platelet cAMP levels inhibit the hydrolysis of arachidonic acid from platelet phospholipids. We also find that prostaglandin synthesis can be dissociated, in part, from platelet aggregation and release, and that cAMP has separate actions on these processes. Higher thrombin concentrations are required to stimulate prostaglandin synthesis (0.05-2 U/ml) than are required to induce [14C]serotonin release (0.02-0.1 U/ml). Furthermore, dBcAMP and PGE1 both inhibit platelet aggregation induced by either arachidonic acid or prostaglandin H2 without affecting the production of prostaglandin metabolites from these compounds.     

Degradation of sulfated proteoglycans in the subendothelial extracellular matrix by human platelet heparitinase

Cultured vascular and corneal endothelial cells produce an underlying extracellular matrix (ECM) which induces platelet adherence, aggregation, and release reaction. Incubation of a metabolically (35S)O = 4-labeled ECM with platelet-rich plasma or washed platelets, but not with platelet-poor plasma, resulted in degradation of its heparan sulfate-containing proteoglycans into labeled fragments four to five times smaller than intact glycosaminoglycan side chains. These fragments were sensitive to deamination with nitrous acid and were not produced in the presence of heparin, indicating that heparan sulfate in the ECM is susceptible to cleavage by the platelet heparitinase. This degradation required adhesion of platelets to the ECM rather than aggregation since it was not inhibited by aspirin, which prevented platelet aggregation but not adherence. The enzyme was not released during aggregation of platelets on the ECM but was readily liberated upon their exposure to thrombin. This liberation was inhibited in the presence of prostacyclin (PGI2). Isolated high molecular weight proteoglycans first released from the ECM by incubation with platelet poor plasma served as a substrate for further degradation by the platelet heparitinase, suggesting a cascade mechanism for degradation of heparan sulfate in the ECM. Heparitinase, although to a lower level, was also active when washed platelets were added on top of a confluent endothelial cell monolayer covering the (35S)O = 4-labeled ECM. It is suggested that the platelet heparitinase may be involved in the impairment of the integrity of the vessel wall and thus facilitate the extravasation of blood-borne cells.     

Primed stimulation of isolated perfused rabbit lung by endotoxin and platelet activating factor induces enhanced production of thromboxane and lung injury.

Bacterial sepsis often precedes the development of the adult respiratory distress syndrome (ARDS) and bacterial endotoxin (LPS) produces a syndrome similar to ARDS when infused into experimental animals. We determined in isolated, buffer-perfused rabbit lungs, free of plasma and circulating blood cells that LPS synergized with platelet activating factor (PAF) to injure the lung. In lungs perfused for 2 h with LPS-free buffer (less than 100 pg/ml), stimulation with 1, 10, or 100 nM PAF produced transient pulmonary hypertension and minimal edema. Lungs perfused for 2 h with buffer containing 100 ng/ml of Escherichia coli 0111:B4 LPS had slight elevation of pulmonary artery pressure (PAP) and did not develop edema. In contrast, lungs exposed to 100 ng/ml of LPS for 2 h had marked increases in PAP and developed significant edema when stimulated with PAF. LPS treatment increased capillary filtration coefficient, suggesting that capillary leak contributed to pulmonary edema. LPS-primed, PAF-stimulated lungs had enhanced production of thromboxane B2 (TXB) and 6-keto-prostaglandin F1 alpha (6KPF). Indomethacin completely inhibited PAF-stimulated production of TXB and 6KPF in control and LPS-primed preparations, did not inhibit the rise in PAP produced by PAF in control lungs, but blocked the exaggerated rise in PAP and edema seen in LPS-primed, PAF-stimulated lungs. The thromboxane synthetase inhibitor dazoxiben, and the thromboxane receptor antagonist, SQ 29,548, similarly inhibited LPS-primed pulmonary hypertension and edema after PAF-stimulation. These studies indicate that LPS primes the lung for enhanced injury in response to the physiologic mediator PAF by amplifying the synthesis and release of thromboxane in lung tissue.     

Salutary effects of prostaglandin E1 in perfused rat lungs injured with hydrogen peroxide

Studies were conducted in isolated, buffer-perfused rat lungs to determine if prostaglandin (PG) E1 attenuated pulmonary edema provoked by hydrogen peroxide (H2O2). When lungs were challenged by 60 min of perfusion with H2O2 (generated by the reaction between glucose and glucose oxidase) the wet weight-to-dry weight ratio increased from control by 54%, indicating development of pulmonary edema. In contrast, lungs treated simultaneously with H2O2 plus PGE1 (1 microgram/min) failed to exhibit an elevated wet-to-dry weight ratio. H2O2-injured lungs demonstrated a modest 2 torr increase in pulmonary arterial perfusion pressure that was not influenced by simultaneous treatment with PGE1. Both radioimmunoassay (RIA) and high-performance liquid chromatographic (HPLC) analysis detected increased amounts of (5S)-5-hydroxy-6,8,11,14 eicosatetraenoic acid in the perfusion medium of H2O2-injured lungs (RIA, 48.0 +/- 14.7; HPLC, 54.8 +/- 13.5) relative to controls (RIA, 6.6 +/- 1.6; HPLC, 6.8 +/- 1.9), and simultaneous treatment with PGE1 tended to blunt this increase (RIA, 29.2 +/- 8.3; HPLC, 29.8 +/- 7.6). PGE1 abolished the increase in wet weight-to-dry weight ratio induced by exogenous leukotriene C4. Production of H2O2 by the glucose-glucose oxidase reaction was not influenced by PGE1. Taken together, these observations indicate that PGE1 attenuates H2O2-induced pulmonary edema formation in buffer-perfused rat lungs by mechanisms that may relate to inhibition of lung 5'-lipoxygenase activation and/or to inhibition of the injurious effects of endogenously produced lipoxygenase products.     

Stimulus-induced release of endogenous catecholamines from human washed platelets

Using high-performance liquid chromatography with electrochemical detection, we have studied the release of endogenous catecholamines from washed platelets induced to aggregate by ADP and thrombin. Washed platelets exhibit irreversible aggregation with 10 mumol/l ADP and 0.3 unit of thrombin/ml, extents of aggregation being 27% and 76% respectively. ADP (10 mumol/l) increased noradrenaline release to the medium by 20% and adrenaline by 28%. As observed with aggregation, 0.3 unit of thrombin/ml produced a more marked effect on release than ADP, noradrenaline and adrenaline being increased by 570% and 169% respectively. Values for platelet noradrenaline content were found to mirror those for the release from platelets induced by thrombin. A correlation was observed between catecholamine release and the concentration of thrombin present in incubations. These data reflect the changes observed with platelet aggregation. This is the first study to determine catecholamine release from platelets by direct measurement. Local catecholamine release after platelet aggregation in vivo may have important consequences for tissue perfusion.     

Immunoinhibition of ristocetin-induced platelet aggregation.

Human platelets washed and fixed in paraformaldehyde aggregate in the presence of the antibiotic ristocetin and normal plasma. This aggregation response is abolished after digestion of the fixed platelets with chymotrypsin. Antisera to fixed washed platelets were produced in rabbits and absorbed with chymotrypsin-treated, fixed washed platelets. Monovalent Fab fragments obtained from the isolated gamma-globulin fractions of the antisera blocked ristocetin-induced aggregation of fixed washed platelets in buffer and normal platelets in platelet-rich plasma. By double-antibody immunoprecipitation, it was shown that the antibody which blocked the ristocetin reaction interacted with a platelet membrane surface protein of mol wt 155,000. The results suggest that the glycoprotein I complex on the surface of the human platelet mediates ristocetin-induced von Willebrand factor-dependent platelet aggregation.     

Comparison of equimolar concentrations of iloprost, prostacyclin, and prostaglandin E1 on human platelet function

Prostaglandins E1 and I2 (PGE1 and PGI2) have been shown to be potent inhibitors of platelet aggregation. We compared the antiaggregatory effect of equimolar concentrations of these two agents with that of a newly synthesized prostacyclin analogue, iloprost, and measured the effects of these agents on intracellular levels of cyclic adenosine monophosphate (cAMP) in human platelets. In addition, because the platelet inhibitory properties of prostanoids are associated with increased vasoactivity, we assessed the effects of each prostanoid on coronary flow in isolated perfused rat hearts. Concentrations ranging from 0.0001 mumol/L to 1 mumol/L of iloprost, PGI2, and PGE1 were incubated with either platelet-rich plasma or gel-filtered platelets. Greater than 90% inhibition of platelet aggregation in response to threshold concentrations of adenosine diphosphate (n = 6) and epinephrine (n = 6) was observed in all donors when 0.01 mumol/L iloprost, 0.1 mumol/L PGI2, and 1 mumol/L PGE1 were added to platelet-rich plasma. In gel-filtered platelets, at threshold concentrations of thrombin (n = 6), 90% inhibition was observed with 0.01 mumol/L iloprost. In contrast, similar inhibition to thrombin required 0.1 mumol/L PGI2, and with PGE1 it was never achieved in two donors. At 0.01 mumol/L of prostanoid, cAMP levels (n = 6) rose from a baseline value of 439 +/- 99 pmol/10(9) platelets to 1857 +/- 454 pmol/10(9) platelets for iloprost, 758 +/- 99 pmol/10(9) platelets for PGI2, and 692 +/- 199 pmol/10(9) platelets for PGE1. In addition, at 6 mumol/L, alterations in coronary flow (P greater than 0.05) were noted to be 127% of baseline values for iloprost (n = 5) and 153% for PGI2 (n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)     

alphaIIbbeta3 antagonism vs. antiadhesive treatment to prevent platelet interactions with vascular subendothelium.

Platelets adhering to blood vessels promote coagulation and inflammation, and release growth factors that trigger smooth muscle cell activation. We have therefore studied the pharmacological modification of platelet deposition quantitatively by comparing adhesion of flowing platelets to various subendothelial ligands in the absence or presence of an antialpha(IIb)beta(3) antagonist with the effects of antiadhesive treatment consisting of von Willebrand factor (VWF) and fibronectin neutralization or of the combined inhibition of platelet adhesion and aggregation. In vitro, perfusion of anticoagulated human blood over calf skin collagen reiterated that alpha(IIb)beta(3) antagonism prevents platelet aggregation, but not adhesion per se: single platelets strongly bound to collagen at wall shear rates of both 1300 and 2700 s(-1), largely VWF-independent. When perfused over a human umbilical vein endothelial cell-derived extracellular matrix, single alpha(IIb)beta(3)-antagonized platelets primarily adhered to matrix-bound VWF when perfused at 2700 s(-1), but at 1300 s(-1) they also adhered significantly to fibronectin. During perfusion of anticoagulated rabbit blood over de-endothelialized rabbit aorta at a wall shear rate of 1100 s(-1), alpha(IIb)beta(3) antagonism even increased the absolute numbers of adhering platelets and VWF neutralization redirected alpha(IIb)beta(3)-antagonized platelets towards other vascular ligands. Finally, in vivo, following photochemically induced blood vessel injury in mice, alpha(IIb)beta(3) antagonism inhibited platelet-rich thrombus formation, but platelet adhesion was only significantly inhibited when associated with fibronectin neutralization. In conclusion, antiadhesive platelet treatment more potently interferes with platelet deposition on injured blood vessels than alpha(IIb)beta(3) antagonism, but abrogating platelet adhesion can only be achieved by carefully selected antiplatelet drug combinations.     

Internalization of microparticles by endothelial cells promotes platelet/endothelial cell interaction under flow

Summary. Background: Microparticles (MPs) released by activated or apoptotic cells increase in number in the blood of subjects with vascular or metabolic diseases and may contribute to thrombotic complications. Objectives: In this study, we investigated whether MPs promoted platelet recruitment to endothelial cells in flow conditions, and by which mechanism. Methods: Human umbilical vein endothelial cells (HUVECs) grown in microslide perfusion chambers were exposed to MPs prepared in vitro from HUVECs, monocytes or platelets. Results: Videomicroscopy of DIOC‐labelled blood perfused at arterial rate on human umbilical vein ECs demonstrated that, irrespective of their cell origin, MPs promoted the formation of platelet strings at the surface of HUVECs. This platelet/endothelial cell interaction was dependent on von Willebrand factor (VWF) expression at the HUVEC surface and involved Glycoprotein Ib and P‐selectin. Interestingly, HUVECs internalized MPs within a few hours through a process involving anionic phospholipids, lactadherin and αvβ3 integrin. This uptake generated the production of reactive oxygen species via the xanthine/xanthine oxidase system (inhibited by allopurinol and the ROCK inhibitor Y‐27632) and the NADPH oxidase (inhibited by SOD). Reactive oxygen species appeared essential for VWF expression at the endothelial cell surface and subsequent platelet/endothelial cell interaction under flow. The pathophysiological relevance of this process is underlined by the fact that circulating MPs from Type I diabetic patients induced platelet/endothelial cell interaction under flow, with an intensity correlated with the severity of the vasculopathy.     

αIIbβ3-mediated outside-in signaling induced by the agonist peptide LSARLAF utilizes ADP and thromboxane A2 receptors to cause α-granule secretion by platelets

Summary.  The peptide LSARLAF (LSA) causes αIIbβ3-dependent platelet activation that results in α-granule secretion and aggregation. LSARLAF-induced, αIIbβ3-mediated outside-in signaling causing α-granule secretion and platelet aggregation was studied using washed mouse platelets. ADP receptor antagonists, enzyme inhibitors, normal platelets and platelets from mice that lack either Gαq or thromboxane (Tx) A2 receptors were used for this investigation. The results demonstrate that LSA-induced αIIbβ3-mediated signaling producing aggregation of washed platelets is mediated through the release of ADP and thromboxane, which cause α-granule release by mediating their effects though Gαq and/or Gi depending on the level of LSA used to activate the platelets. Specifically, αIIbβ3 elicited aggregation of washed platelets in response to a low level of LSA requires signaling through the ADP receptor P2Y1 and Gαq, and the ADP receptor P2Y12 and Gi as well as TxA2 receptors. However, this aggregation is independent of Gαq and TxA2 signaling in response to high LSA concentrations, but is dependent on ADP signaling through its receptor P2Y12, and therefore presumably Gi, regardless of the level of LSA used to activate the platelets. PKC function is required for ADP secretion and the subsequent signaling through P2Y12 regardless of the level of LSA used to activate the platelets. The end point of the LSA-induced αIIbβ3-mediated signaling characterized in this study is α-granule secretion, which provides the fibrinogen required for aggregation of washed platelets.     

Intravascular filarial parasites inhibit platelet aggregation. Role of parasite-derived prostanoids.

The nematode parasites that cause human lymphatic filariasis survive for long periods in their vascular habitats despite continual exposure to host cells. Platelets do not adhere to blood-borne microfilariae, and thrombo-occlusive phenomena are not observed in patients with circulating microfilariae. We studied the ability of microfilariae to inhibit human platelet aggregation in vitro. Brugia malayi microfilariae incubated with human platelets caused dose-dependent inhibition of agonist-induced platelet aggregation, thromboxane generation, and serotonin release. As few as one microfilaria per 10(4) platelets completely inhibited aggregation of platelets induced by thrombin, collagen, arachidonic acid, or ionophore A23187. Microfilariae also inhibited aggregation of platelets in platelet-rich plasma stimulated by ADP, compound U46619, or platelet-activating factor. The inhibition required intimate proximity but not direct contact between parasites and platelets, and was mediated by parasite-derived soluble factors of low (less than 1,000 Mr) molecular weight that were labile in aqueous media and caused an elevation of platelet cAMP. Prior treatment of microfilariae with pharmacologic inhibitors of cyclooxygenase decreased both parasite release of prostacyclin and PGE2 and microfilarial inhibition of platelet aggregation. These results indicate that microfilariae inhibit platelet aggregation, via mechanisms that may include the elaboration of anti-aggregatory eicosanoids.     

In vitro evidence of an endothelial cell-dependent antiplatelet activity for isosorbide dinitrate, but not for its 2- and 5-mononitrate metabolites

In an experimental model in which cultured endothelial cells (EC) and platelets were incubated with autologous plasma, we investigated the pharmacological modulations by isosorbide nitrates (ISN) [isosorbide dinitrate (ISDN) + 2-isosorbide mononitrate (2-ISMN) + 5-ISMN] of the EC-induced inhibition of platelet aggregation; and the associated changes in prostanoid profile of these mixed EC-platelet suspensions. ISDN antiplatelet activities were found to be magnified profoundly by EC, being dependent upon both ISDN concentration and EC number, e.g., 5.10(-5) M ISDN in the presence of 2.10(4) cells, fully arrested ADP-induced aggregation, whereas the same ISDN concentration induced 30% inhibition in control platelet activities. In contrast, there were no significant changes in 2- and 5-ISMN antiaggregating properties, whether incubated in the presence or absence of EC. Thromboxane B2 accumulated noticeably after aggregation, whereas 6-keto-prostaglandin (PG) F1 alpha and PGE2 accumulated poorly in the medium. In the presence of EC, thromboxane B2 accumulation fell in parallel to the extent of aggregation, whereas 6-keto-PGF2 alpha and PGE2 accumulated in the medium. Aspirin-treated, washed ECs still inhibited platelet aggregation. ISDN was the only ISN capable of inducing PG-accumulation profile changes. These results demonstrate the existence of an endothelium-dependent ISDN antiplatelet activity. Furthermore, this effect is specific to ISDN not being shown by its mononitrate metabolites. These results suggest that PG accumulation changes may be a consequence rather than a cause of the inhibition of platelet activity by (ISDN-stimulated) EC.