Role of chondroitin 4-sulphate as a receptor for polycation induced human platelet aggregation

1. Proteoglycans provide negatively charged sites on the surface of platelets, leukocytes and endothelial cells. Since chondroitin 4-sulphate is the main proteoglycan present on the platelet surface, the role of this molecule in mediating the activation of human platelets by polylysine was studied. 2. Platelets were desensitized with phorbol 12-myristate 13-acetate (PMA, 10 nM) 5 min before the addition of polylysine to platelet-rich plasma (PRP). Changes in the intracellular Ca2+ concentration were measured in fura2-am (2 microM) loaded platelets and protein phosphorylation was assessed by autoradiography of the electrophoretic profile obtained from [32P]-phosphate labelled platelets. The release of dense granule contents was measured in [14C]-5-hydroxytryptamine loaded platelets and the synthesis of thromboxane (TXA2) was assessed by radioimmunoassay. Surface chondroitin 4-sulphate proteoglycan was degraded by incubating platelets with different concentrations of chondroitinase AC (3 min, 37 degrees C). The amount of chondroitin 4-sulphate remaining in the platelets was then quantified after proteolysis and agarose gel electrophoresis. 3. The addition of PMA to PRP before polylysine inhibited the aggregation by 88 +/- 18% (n = 3). Staurosporine (1 microM, 5 min) prevented the PMA-induced inhibition. Chondroitinase AC (4 pu ml-1 to 400 muu ml-1, 3 min) abolished the polylysine-induced aggregation in PRP but caused only a discrete inhibition of ADP-induced aggregation. The concentration of chrondroitin 4-sulphate in PRP (0.96 +/- 0.2 microgram/10(8) platelets, n = 3) and in washed platelets (WP; 0.35 +/- 0.1 microgram/10(8) platelets, n = 3) was significantly reduced following incubation with chondroitinase AC (PRP = 0.63 +/- 0.1 microgram/10(8) platelets and WP = 0.08 +/- 0.06 microgram/10(8) platelets). 4. Washed platelets had a significantly lower concentration of chondroitin 4-sulphate than platelets in PRP. The addition of polylysine to WP induced a rapid increase in light transmission which was not accompanied by TXA2 synthesis or the release of dense granule contents. This effect was not inhibited by sodium nitroprusside (SNP), iloprost, EDTA or the peptide RGDS. This event was accompanied by the discrete phosphorylation of plekstrin and myosin light chain, which were inhibited by staurosporine (10 microM, 10 min). The hydrolysis of platelet surface chondroitin 4-sulphate strongly reduced the polylysine-induced phosphorylation. 5. Our results indicate that polylysine activates platelets through a specific receptor which could be the proteoglycan chondroitin 4-sulphate present on the platelet membrane.     

Inhibition of rat platelet aggregation by mycalolide-B, a novel inhibitor of actin polymerization with a different mechanism of action from cytochalasin-D

In vitro effects of mycalolide-B (MB), isolated from marine sponge, were investigated with regard to the activation of rat platelets. Collagen-induced platelet aggregation in platelet-rich plasma (PRP) was slightly but significantly potentiated by lower concentrations of MB (0.3 and 1 microM) but was inhibited by higher concentrations (3 and 10 microM). ADP-induced platelet aggregation in PRP was also significantly prevented by MB (1-10 microM). Potentiation of ADP-induced aggregation by MB (0.3 microM) was hardly observed. G-actin contents, determined by DNase I inhibition assay, were increased in resting washed platelets incubated with MB (3 microM). In contrast, cytochalasin-D (CD) at 3 microM slightly reduced G-actin contents in resting platelets. After platelet aggregation with collagen (3 microg/ml) or ADP (10 microM), G-actin contents in platelets were reduced, indicating de novo actin polymerization. MB (3 microM) and CD (3 microM) abolished both ADP (10 microM)- and collagen (3 microg/ml)-induced platelet aggregation and actin polymerization in washed platelets. MB (1-10 microM) had no effects on intracellular Ca2+ concentrations in ADP (10 microM)-stimulated platelets. [125I]-fibrinogen binding to activated platelets with ADP (10 microM)(was inhibited by MB (0.3-3 microM) in a concentration-dependent manner. Thrombin-induced platelet-fibrin clot retraction was inhibited by MB (1 and 10 microM). These results suggest that MB inhibits platelet activation by interfering with actin polymerization through a different mechanism of action from CD. MB may be a useful tool for studying the role of actin polymerization in various cells.     

Dual effects of nimesulide, a COX-2 inhibitor, in human platelets

Nimesulide (CAS 51803-78-2) has been shown to exert marked anti-inflammatory effect in several in vivo models of inflammation. Since nimesulide is considered to be a selective inhibitor of COX-2, it has not been studied in detail in relation to its mechanistic effects on platelets, which express COX-1. This study was conducted to investigate the effects of nimesulide in platelet aggregation. We show that nimesulide (1-100 microM) inhibited platelet aggregation induced by adrenaline (20-200 microM). It also inhibited thromboxane A2 (TXA2) formation by platelets at low concentration (IC50; 1 microM). However, much lower concentrations of nimesulide (0.01-0.1 microM) potentiated the aggregatory response of subthreshold concentrations of adrenaline (0.2-2 microM). Such an effect was blocked by Ca2+-channel blockers, verapamil and diltiazem (IC50: 7 and 46 microM, respectively), nitric oxide donor, SNAP (IC50; 2 microM) and cinchonine (10 nM) but not by genistein (up to 10 microM). These results are indicative of the concentration-dependent dual effects of nimesulide on human platelet aggregation. The synergistic effect of low doses of nimesulide and adrenaline seems to be mediated through inhibition of multiple signalling pathways.     

Effects of some antineoplastic drugs (vincristine, doxorubicin and epirubicin) on human platelet aggregation

The effects of some antineoplastic drugs (vincristine, doxorubicin and epirubicin) on collagen- and ADP-induced human platelet aggregation are investigated. Platelet rich plasma (PRP) and platelet poor plasma (PPP) from healthy male and female donors were used. The PRP was adjusted with analogous PPP to 300,000 platelets/microliters. Platelet aggregation was studied according to Born's turbidimetric technique using an Aggrecorder II PA 3220 with collagen at a concentration of 10 micrograms/ml and ADP at a concentration of 30 microM. Vincristine, doxorubicin and epirubicin significantly (p < 0.01) inhibited collagen- and ADP-induced platelet aggregation. The vincristine induced inhibition was higher than that induced by doxorubicin or epirubicin. The effects of doxorubicin and epirubicin were more intense on ADP-induced platelet aggregation than on the collagen induced one. Moreover, the doxorubicin inhibition of ADP-induced platelet aggregation was greater than the epirubicin one. In conclusion, our study shows that vincristine, doxorubicin and epirubicin inhibit human platelet aggregation. The present results may improve the therapeutic use of these drugs since it has been clearly shown that drugs with antiplatelet activity could block metastases.     

Competitive inhibition of phospholipase A2 activity by the platelet-activating factor antagonist Ro 19-3704 and evidence for a novel suppressive effect on platelet activation

The compound Ro 19-3704 [3-4(R)-2-(methoxycarbonyl) oxy-3-(octadecylcarbamoyl)oxy-propoxy butylthiazolium iodide], initially described as an antagonist of platelet-activating factor, is reported here to directly inhibit rabbit platelet phospholipase (PL) A2 activity, with an IC50 value of 4 to 7 microM. Classical Michaelis-Menten analysis showed that inhibition was reversible and competitive, inasmuch as apparent Km values increased in the presence of Ro 19-3704 (from 0.2-0.4 to 2 microM), whereas Vmax values remained constant (200 +/- 20 nmol/min/10(9) cells). Ro 19-3704 inhibited platelet aggregation, PLA2 release and thromboxane B2 formation induced by thrombin (0.25 U/ml), with IC50 values of 8, 15 and below 5 microM, respectively. Aggregation and PLA2 release by arachidonic acid (100 microM) were also inhibited, but thromboxane B2 formation was unaffected, indicating that Ro 19-3704 does not inhibit cyclooxygenase. Platelet activation by collagen (5 micrograms/ml), the thromboxane mimetic U46619 ([15(S)-hydroxy-11,9(epoxymethano)-prosta-5Z,13E-dienoic acid] 1 microM) and low concentrations of thrombin (0.05-0.1 U/ml) was also inhibited by Ro 19-3704. Inhibition of platelet activation was reversible, suggesting that its suppressive effect was not due to cytotoxicity. Finally, Ro 19-3704 did not stimulate cyclic AMP formation or inhibit phosphodiesterase activity. Ro 19-3704 is a competitive inhibitor of PLA2 activity, and is also endowed with a potent suppressive effect on platelet activation induced by different agonists.     

XV454, a novel nonpeptide small-molecule platelet GIIb/IIIa antagonist with comparable platelet alpha(IIb)beta3-binding kinetics to c7E3

XV454 demonstrated high potency (IC50 = 14-25 nM) in inhibiting human platelet aggregation induced by adenosine diphosphate (ADP, 10 microM), thrombin receptor agonist peptide (TRAP) (10 microM), or collagen (20 microg/ml). XV454 exhibited a high degree of selectivity for platelet alpha(IIb)beta3 in comparison with c7E3, which is a nonspecific antagonist for both alpha(IIb)beta3 and alpha(v)beta3. Both XV454 and c7E3 bind with high affinity to either activated (A) or unactivated (U) human, baboon, or canine platelets. XV454 binds with a relatively higher affinity [Kd = 0.5 nM (A), 0.6 nM (U)] as compared with c7E3 [Kd = 9.1 nM (A), 9.2 (U) nM]. XV454 demonstrated a tight association with human, baboon, and, to a lesser extent, with canine platelets (t(1/2) of dissociation = 110 +/- 6, 80 +/- 10, and 23 +/- 2 min, respectively). Both c7E3 and XV454 associate tightly with a slower dissociation rate with unactivated human platelets: t(1/2) of 42 and 116 min, respectively. In non-human primates, oral (0.1 mg/kg, p.o.) and intravenous (0.05 mg/kg, i.v. bolus administration of XV454 methyl ester pro-drug resulted a long-lasting maximal antiplatelet efficacy for < or = 72 h with significant but reversible prolongation of bleeding time and without effects on platelet count, clinical chemistry, or hemodynamic profile. In conclusion, XV454 represents a potent antiplatelet agent in inhibiting platelet aggregation along with a high affinity and relatively slow dissociation rate from human platelet GPIIb/IIIa receptors that allow a long-lasting antiplatelet efficacy after single i.v. or oral administration.     

Disease severity, physical limitations and depression in HIV-infected men

The demand for stored platelet concentrates (PC) for therapeutic transfusions has been increasing for the past three decades. Loss of platelet functionality increases with the length of storage time due to a multitude of factors collectively referred to as a platelet storage lesion. As more of the causes of the storage lesion have been defined, storage conditions have improved along with the therapeutic value of the transfused platelet samples. This report addressed new aspects of the storage lesion correlated with the pH of the storage medium. Platelet function was evaluated as aggregation induced by the synergistic agonist pair, U46619 (TXA2 mimetic) plus epinephrine or the strong agonists SFLLRNP (a peptide thrombin receptor agonist) or thrombin each added alone. Stored PC were compared to freshly prepared platelets as platelet-rich plasma (PRP) or washed platelets re-suspended in hepes Tyrode's buffer. The pH of the storage plasma, was inversely proportional to the cell count with platelets stored for 6 days. Agonist-induced platelet aggregation was significantly impaired by storage for 6-7 days as PRP; however, upon washing, a significant level of activity was restored. Washed platelets more accurately reflect conditions of transfused platelets that may regain activity in vivo. There appeared to be two subpopulations of stored PRP samples--one that retained activity and one that lost virtually all activity with the agonists tested. However, the lack of response to agonist observed with one subpopulation was reversed to the same level obtained with the active subpopulation upon washing. The subpopulation of stored PRP samples that were inactive with U46619-plus-epinephrine did not correspond to the subpopulation of samples that were nonresponsive to SFLLRNP, indicating that loss of activity with selected samples was possibly receptor specific. Loss of agonist-induced aggregation with PRP samples did not correlate with storage pH; however, the level of aggregation with washed platelets correlated significantly with pH. Results implied that pH caused a permanent storage lesion that could only be detected with washed platelets. A partially reversible lesion was superimposed on the pH lesion and was only detectable with PRP samples. Results indicate that continued attention must be paid to regulate the pH of stored PC even in the second generation plastic bags.     

The P2Y1 receptor, necessary but not sufficient to support full ADP-induced platelet aggregation, is not the target of the drug clopidogrel

Recently we showed that the P2Y1 receptor coupled to calcium mobilization is necessary to initiate ADP-induced human platelet aggregation. Since the thienopyridine compound clopidogrel specifically inhibits ADP-induced platelet aggregation, it was of interest to determine whether the P2Y1 receptor was the target of this drug. Therefore we studied the effects of clopidogrel and of the two specific P2Y1 antagonists A2P5P and A3P5P on ADP-induced platelet events in rats. Although clopidogrel treatment (50 mg/kg) greatly reduced platelet aggregation in response to ADP as compared to untreated platelets, some residual aggregation was still detectable. In contrast, A2P5P and A3P5P totally abolished ADP-induced shape change and aggregation in platelets from both control and clopidogrel-treated rats. A2P5P and A3P5P (100 microM) totally inhibited the [Ca2+]i rise induced by ADP (0.1 microM) in control and clopidogrel-treated platelets, whereas clopidogrel treatment had no effect. Conversely, the inhibition of adenylyl cyclase induced by ADP (5 microM) was completely blocked by clopidogrel but not modified by A2P5P or A3P5P (100 microM). A3P5P (1 mM) reduced the number of [33P]2MeSADP binding sites on control rat platelets from 907 +/- 50 to 611 +/- 25 per platelet. After clopidogrel treatment, binding of [33P]2MeSADP decreased to 505 +/- 68 sites per platelet and further decreased to 55 +/- 12 sites in the presence of A3P5P (1 mM). In summary, these results demonstrate that the platelet P2Y1 receptor responsible for the initiation of aggregation in response to ADP is not the target of clopidogrel. Platelets may express another, as yet unidentified, P2Y receptor, specifically coupled to the inhibition of adenylyl cyclase and necessary to induce full platelet aggregation, which could be the target of this drug.     

Rat platelet aggregation by ATP. Aggregometrical and ultrastructural comparison with aggregations induced by ADP and collagen

This paper describes the aggregation of rat platelets by adenosine triphosphate (ATP). The aggregometry of ATP-induced aggregation and the ultrastructure of ATP-aggregated platelets were compared and contrasted with those of adenosine diphosphate (ADP)-treated and collagen-treated samples. Human platelets were also studied alongside with rat specimens. Several lines of evidence indicate that the ATP-induced aggregation of rat platelet-rich plasma (PRP) is not a result of contaminating ADP in the ATP preparation. ATP did not cause aggregation of human platelets; it inhibited ADP- and collagen-induced human platelet aggregation. ATP pretreated with a creatine phosphate/creatine phosphokinase system caused similar rat platelet aggregation as did ATP not treated with this system. The aggregometry of ATP-induced aggregation of rat PRP was similar to that of collagen-induced aggregation but markedly different from that of ADP-induced aggregation. However, the nature of ATP-induced aggregation was similar to that induced by ADP. Both ATP- and ADP-induced rat platelet aggregations were not affected by adenosine, adenosine monophosphate, or acetylsalicylic acid. The ultrastructure of ATP-aggregated platelets was similar to that of ADP-aggregated ones. It appears that either platelets of rats possess specific ATP receptors or the rat plasma contains a material, lacking or insufficiently present in human plasma, that converts ATP to ADP in a fashion similar to the release of ADP from platelet storage granules.     

Influence of a selective 5HT1-receptor agonist GR43175 on platelet responsiveness

The possible interaction of sumatriptan, a selective 5HT1-receptor agonist, with platelet responsiveness has been investigated. Stimulation of platelet rich plasma with sumatriptan (1-100 microM) did not induce shape change, aggregation or modification of intraplatelet cytosolic calcium levels. Total inhibition of aggregation induced by 20 microM 5HT was observed in platelets preincubated for 20 min with 100 microM sumatriptan. In the same model, platelet stimulation with 4 microM adenosine 5'-diphosphate (ADP), concentration known to induce an irreversible single-phase curve, determined a decrease of aggregatory response. Concentrations from 1 microM to 50 microM of sumatriptan did not influence the aggregatory response induced by 5HT and ADP. These effects appear not to be determined by modifications of platelet calcium homeostasis. The possibility to modulate platelet responsiveness by sumatriptan offers a further approach for evaluating the probable link between platelet behaviour and pathophysiology of migraine.     

Thromboxane A2 synthase inhibition and thromboxane A2 receptor blockade by 2-[(4-cyanophenyl)amino]-3-chloro-1,4-naphthalenedione (NQ-Y15) in rat platelets

The effects of 2-[(4-acetylphenyl)amino]-3-chloro-1,4-naphthalenedione (NQ-Y15), a synthetic 1,4-naphthoquinone derivative, on platelet activity and its mechanism of action were investigated. NQ-Y15 caused a concentration-dependent inhibition of the aggregation induced by thrombin, collagen, arachidonic acid (AA), and A23187. The IC50 values of NQ-Y15 on thrombin (0.1 U/mL)-, collagen (10 microg/mL)-, AA (50 microM)-, and A23187 (2 microM)-induced aggregation were 36.2 +/- 1.5, 6.7 +/- 0.7, 35.4 +/- 1.7, and 93.1 +/- 1.4 microM, respectively. NQ-Y15 also inhibited thrombin-, collagen-, AA-, and A23187-stimulated serotonin secretion in a concentration-dependent manner. However, a high concentration (100 microM) of NQ-Y15 showed no significant inhibitory effect on ADP-induced primary aggregation, which is independent of thromboxane A2 (TXA2) production in rat platelets. In fura-2-loaded platelets, the elevation of intracellular free calcium concentration stimulated by AA, thrombin, and 4-bromo-A23187 was inhibited by NQ-Y15 in a concentration-dependent manner. The formation of TXA2 caused by AA, thrombin, and collagen was inhibited significantly by NQ-Y15. NQ-Y15 inhibited TXA2 synthase in intact rat platelets, since this agent reduced the conversion of prostaglandin (PG) H2 to TXA2. Similarly, NQ-Y15 selectively inhibited the TXA2 synthase activity in human platelet microsomes, whereas it had no effect on activity of phospholipase A2, cyclooxygenase, and PGI2 synthase in vitro. NQ-Y15 inhibited platelet aggregation induced by the endoperoxide analogue U46619 in human platelets, indicating TXA2 receptor antagonism, possibly of a competitive nature. These results suggest that the antiplatelet effect of NQ-Y15 is due to a combination of TXA2 synthase inhibition with TXA2 receptor blockade, and that it may be useful as an antithrombotic agent.     

Stability of metallothionein isoforms by capillary zone electrophoresis

Advanced glyco-oxidation end products (AGEs) generate oxygen free radicals that potentiate the development of atherosclerosis. Thus, AGEs may potentiate the aggregation of human platelets through oxidative stress. AGE-bovine serum albumin (BSA) and AGE-poly-L-lysine were evaluated for aggregation of human platelets. Superoxide in platelet-rich plasma (PRP) was measured using lucigenin-derived chemiluminescence. The platelet aggregation induced by ADP or U46619 was potentiated by preincubation with AGE-BSA, by 40% and by 59%, P < .05, respectively, vs BSA. Aggregation was increased by AGEs in a dose-dependent manner. The production of superoxide was significantly greater in PRP incubated with AGE-BSA vs BSA. The other Maillard reaction products, such as Amadori-, pentosidine-, and carboxymethyl lysine (CML)-BSA had no effect. Superoxide dismutase or indomethacin abolished the enhancing effect of AGEs on the platelet aggregation. AGEs potentiate platelet aggregation possibly with superoxide anions and prostanoids. AGE-induced potentiation of platelet aggregation may be involved in the development of atherosclerosis.     

Triflavin potentiates the antiplatelet activity of platelet activating factor receptor antagonist on activated neutrophil-induced platelet aggregation

In this study, specific platelet activating factor (PAF) receptor antagonist ginkgolide B (BN52021) was tested for its antiplatelet activity in zymosan activated polymorphonuclear neutrophil-induced platelet aggregation. Triflavin was also tested for its antiplatelet activity compared with PAF receptor antagonist. Triflavin, an Arg-Gly-Asp-containing disintegrin purified from venom peptide inhibited platelet aggregation by interfering with the interaction of fibrinogen with the glycoprotein IIb/IIIa complex. Furthermore, we also report an efficient high resolution method for quantitative analysis of PAF using high-performance capillary electrophoresis (HPCE). The supernatant of polymorphonuclear neutrophils after their activation by opsonized zymosan induces the aggregation of washed rabbit platelets. In rabbit platelets, BN52021 (100-1000 microM) only partially inhibited activated polymorphonuclear neutrophil-induced platelet aggregation, and its maximal inhibition was estimated to be about 79%. Triflavin also partially inhibited platelet aggregation about 82% induced by activated polymorphonuclear neutrophils. Furthermore, after treatment with a combination of triflavin (0.26 microM) with various concentrations of BN52021 (4-1000 microM), the inhibitory effect of platelet aggregation was almost completely. This inhibition was greater than that produced by the individual drugs alone. These results indicate that a combination of glycoprotein IIb/IIIa complex and PAF receptor antagonist could completely inhibit activated polymorphonuclear neutrophil-induced platelet aggregation. In addition, the amount of PAF released from zymosan (6 mg/ml)-activated polymorphonuclear neutrophils was accurately calculated about 11.8+/-1.5 ng/10(6) cells, and did not further increase even at a high concentration of zymosan (10 mg/ml). These results suggest that PAF play a major role in the interaction between platelets and polymorphonuclear neutrophils. This interaction may be important in the pathogenesis of thrombosis and inflammatory diseases. Our present findings support the hypothesis that combination therapy with glycoprotein IIb/IIIa complex antagonists and PAF receptor antagonists may represent a new approach to the treatment of ischemic disorders.     

Inhibitory mechanisms of naloxone on human platelets

1. In the present study, naloxone was tested for its antiplatelet activities in human platelet-rich plasma (PRP). In human PRP, naloxone (0.1-0.5 mmol/L) inhibited aggregation stimulated by a variety of agonists (i.e. collagen, adenosine diphosphate (ADP), U46619 and adrenaline). 2. Naloxone (0.1-0.5 mmol/L) did not significantly affect cyclic adenosine monophosphate and cGMP levels in human washed platelets, whereas naloxone (0.5 mmol/L) significantly inhibited thromboxane B2 formation stimulated by collagen (5 micrograms/mL) in human washed platelets. 3. Naloxone (0.5 mmol/L) significantly inhibited [3H]-inositol monophosphate formation of [3H]-myoinositol-loaded platelets stimulated by collagen and U46619. Moreover, naloxone did not influence the binding of 125I-triflavin to platelet membranes. Triflavin is an Arg-Gly-Asp-containing specific fibrinogen receptor antagonist. 4. Addition of naloxone (0.5 mmol/L) to platelet preparations tagged with diphenylhexatriene (DPH) resulted in a considerable decrease in relative fluorescence intensity. 5. It is suggested that the anti-platelet effects of naloxone may be caused, at least partly, by the induction of conformational changes in the platelet membrane initially, followed by the inhibition of thromboxane A2 formation and phosphoinositide breakdown of platelets stimulated by agonists.     

Proliferative responses to PHA, anti-CD3 and antigens in patients with lymphoproliferative disease of granular lymphocytes. Mannoproteins of Candida albicans induce proliferation and cytotoxicity

The effect of the specific PAF-antagonist WEB 2086, a thieno-triazolo-diazepine, and ketoprofen, a NSAID, was investigated on PAF-induced bovine platelet aggregation measured ex vivo in platelet-rich plasma (PRP). WEB 2086 was infused intravenously over 1 min followed immediately by ketoprofen administration over 1 s (both drugs = 3 mg/kg), in a group of six healthy male Friesian calves. Depending on the PAF concentration, a reversible (10(-8)-10(-9) mol/l) and irreversible (10(-5)-10(-7) mol/l) platelet aggregation was observed. The reversible aggregation was completely blocked by pretreatment of the animal with WEB 2086 and ketoprofen. The inhibitory effects observed during the irreversible aggregation were 47.22%, 54.00% and 88.00% at 10(-5), 10(-6) and 10(-7) mol/l PAF, respectively. Moreover, the aggregation obtained in these condition became reversible. Maximal inhibitory effect of WEB 2086 and ketoprofen on PAF-induced platelet aggregation in calves was observed within 30 min after administration of both drugs. This inhibition persisted even after 24 h and was significantly different from control with P < 0.05. The combined effect of both drugs exceeded the sum of the individual effects (synergism). It was concluded that WEB 2086 and ketoprofen very effectively blocked PAF-induced bovine platelet aggregation in platelet-rich plasma. The study also suggested a synergism between both substances.     

Transcellular activation of platelets and endothelial cells by bioactive lipids in platelet microparticles

Microparticles are released during platelet activation in vitro and have been detected in vivo in syndromes of platelet activation. They have been reported to express both pro- and anticoagulant activities. Nevertheless, their functional significance has remained unresolved. To address the mechanism(s) of cellular activation by platelet microparticles, we examined their effects on platelets and endothelial cells. Activation of human platelets by diverse stimuli (thrombin, 0.1 U/ml; collagen, 4 microg/ml; and the calcium ionophore A23187, 1 microM) results in shedding of microparticles. Pretreatment of these particles, but not membrane fractions from resting platelets, with (s)PLA2 evokes a dose-dependent increase in platelet aggregation, intracellular [Ca2+] movement, and inositol phosphate formation. These effects localize to the arachidonic acid fraction of the microparticles and are mimicked by arachidonic acid isolated from them. However, platelet activation requires prior metabolism of microparticle arachidonic acid to thromboxane A2. Thus, pretreatment of platelets with the cyclooxygenase (COX) inhibitor, indomethacin (20 microM), the thromboxane antagonist SQ29,548 (1 microM), or the protein kinase C inhibitor GF109203X (5 microM) prevents platelet activation by microparticles. However, platelet microparticles fail to evoke an inositol phosphate response directly, via either of the cloned thromboxane receptor isoforms stably expressed in human embryonic kidney (HEK) 293 cells. Prelabeling platelets with [2H(8)] arachidonate was used to demonstrate platelet metabolism of the microparticle-derived substrate to thromboxane. Platelet microparticles can also induce expression of COX-2 and prostacyclin (PGI2) production, but not expression of COX-1, in human endothelial cells. These effects are prevented by pretreatment with actinomycin D (12 microM) or cycloheximide (5 microg/ml). Expression of COX-2 is again induced by the microparticle arachidonate fraction, which it may then use to synthesize PGI2. Both PGE2 and iloprost, a stable PGI2 analog, evoke human umbilical vein endothelial cell COX-2 expression, albeit with kinetics that differ from the response to platelet microparticles. These studies indicate a novel mechanism of transcellular lipid metabolism whereby platelet activation may be amplified or modulated by concentrated delivery of arachidonic acid to adjacent platelets and endothelial cells.     

Synthesis and antiplatelet evaluation of alpha-methylene-gamma-butyrolactone bearing 2-methylquinoline and 8-hydroxyquinoline moieties

In a search for inhibitors of platelet aggregation, some alpha-methylene-gamma-butyrolactones bearing 2-methylquinoline and 8-hydroxyquinoline moieties were synthesized and evaluated for antiplatelet activities against thrombin (Thr)-, arachidonic acid (AA)-, collagen (Col)-, and platelet-activating factor (PAF)-induced aggregation in washed rabbit platelets. With the exception of 2-[[2,3,4,5-tetrahydro-4-methylene-5-oxo-2-(4-phenylphenyl)-2 -furanyl]methoxy]-8-hydroxyquinoline (8f), these alpha-methylene-gamma-butyrolactones completely inhibited the platelet aggregation induced by AA and Col. The 2-methylquinoline derivatives were also active against Thr- and PAF-induced aggregation, while their 8-hydroxyquinoline counterparts were relatively inactive.     

Tunable alignment of macromolecules by filamentous phage yields dipolar coupling interactions

The action of lipoprotein lipase on chylomicrons (CM) and very low density lipoproteins (VLDL) produces remnant lipoproteins (RLP) which are rich in triglycerides, cholesterol and apolipoprotein E (apo E). Apo E serves as a ligand for uptake of RLP by macrophages, platelets, endothelial cells and other cells expressing the LDL-receptor or the remnant receptor, thus having a major role in the clearance of plasma cholesterol and triglycerides, but at the same time, uptake of apo E-bearing RLP can profoundly alter the physiology of these cells and promote atherosclerosis. Like RLP, blood platelets also have roles in atherosclerosis and thrombosis, hence it is likely that RLP influence platelet activity as well. RLP derived from normal human plasma VLDL and CM were prepared using two monoclonal antibodies, anti-apo B-100 (JI-H) and anti-apo A-I (H-12) coupled to Sepharose 4B gel to form an immunoaffinity column. Lipoproteins containing apo B-100 including VLDL and LDL adsorb to (JI-H)-gel, while CM and HDL with apo A-I adsorb to (H-12)-gel. The particles in the unbound fraction (RLP) are rich in apo B-48, apo E and apo B-100 containing particles with multiple molecules of apo E. The RLP fraction with a total triglyceride of 14+/-3.2 mg/ml; cholesterol, 0.39+/-0.1 mg/ml and protein, 0.78+/-0.24 mg/ml (n=19) was added to aliquots of blood of man, rabbits, guinea pigs, mice, and rats at protein equivalents of 0.98 to 78 microg/ml blood and agitated gently at 37 degrees C for 40 sec. Platelet aggregation was measured as a fall in single platelet count. RLP induced aggregation of platelets in man (p<0.005) rabbit (p<0.0005), guinea pig (p<0.002) and mouse (p<0.0001), but no RLP induced platelet aggregation was observed in the rat blood. Scanning electron microscopy revealed that in the presence of RLP, platelets had adhered to and formed aggregates on red cells. The platelet response to RLP was inhibited by apyrase known to scavenge ADP, by 5 microM 2-chloroadenosine, a platelet ADP receptor antagonist and by 3.4 microM cilostazol, a phosphodiesterase type III inhibitor known to raise cyclic AMP level in platelets. It is thought that RLP cause leakage of ADP from red cells which then mediates platelet aggregation.     

Cyclopiazonic acid and thapsigargin induce platelet aggregation resulting from Ca2+ influx through Ca2+ store-activated Ca2+-channels

The effects of cyclopiazonic acid and thapsigargin, selective inhibitors of the endoplasmic reticulum Ca2+-ATPase pump, on the platelet aggregation were investigated using washed rat platelets prepared by chromatography on Sepharose 2B columns. In Ca2+-free medium, cyclopiazonic acid and thapsigargin did not induce aggregation, but in the presence of 1 mM Ca2+, platelet aggregation was induced in a concentration-dependent manner. Cyclopiazonic acid- and thapsigargin-induced platelet aggregation was blocked by 1 mM Ni2+ but not by 100 microM indomethacin or 1 microM nifedipine. In aequorin-loaded platelets, cyclopiazonic acid and thapsigargin caused sustained elevation of the cytosolic Ca2+ concentration, an effect which was blocked by Ni2+, a non-selective Ca2+ channel blocker and SK&F 96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenyl]-1H-imidazole hydrochloride), a putative receptor-operated Ca2+ channel antagonist. The above results indicated that both cyclopiazonic acid and thapsigargin induced platelet aggregation and elevation of cytosolic Ca2+ concentration, that extracellular Ca2+ was essential for cyclopiazonic acid- and thapsigargin-induced platelet aggregation, and that platelet aggregation may be associated with Ca2+ influx through Ca2+ store-activated Ca2+ channels.     

The antiplatelet activity of rutaecarpine, an alkaloid isolated from Evodia rutaecarpa, is mediated through inhibition of phospholipase C

In this study, the mechanism involved in the antiplatelet activity of rutaecarpine in human platelet suspensions was investigated. In platelet suspensions (4.5 x 10(8)/ml), rutaecarpine (100 and 200 microM) did not influence the binding of FITC-triflavin to platelet glycoprotein IIb/IIIa complex. Additionally, rutaecarpine (200 microM) did not significantly change the fluorescence of platelet membrane labeled with diphenylhexatriene (DPH). On the other hand, rutaecarpine (50 and 100 microM) dose-dependently inhibited the increase in intracellular free Ca2+ of Fura 2-AM loaded platelets stimulated by collagen. Moreover, rutaecarpine (100 and 200 microM) did not significantly affect the thromboxane synthetase activity of aspirin-treated platelet microsomes. Furthermore, retaecarpine (100 and 200 microM) significantly inhibited [3H]arachidonic acid released in collagen-activated platelets but not in unactivated-platelets. Nitric oxide (NO) production in human platelets was measured by a chemiluminesence detection method in this study. Rutaecarpine (100 and 200 microM) did not significantly affect nitrate production in collagen (10 microg/ml)-induced human platelet aggregation. On the other hand, various concentrations of rutaecarpine (50, 100, and 200 microM) dose-dependently inhibited [3H]inositol monophosphate formation stimulated by collagen (10 microg/ml) in [3H]myoinositol-loaded platelets at different incubation times (1, 2, 3, and 5 minutes). It is concluded that the antiplatelet activity of rutaecarpine may possibly be due to the inhibition of phospholipase C activity, leading to reduce phosphoinositide breakdown, followed by the inhibition of thromboxane A2 formation, and then inhibition of [Ca2+]i mobilization of platelet aggregation stimulated by agonists.