A Scanning Electron Microscopic Investigation of Effects of Prostacyclin on Platelet Retention in Glass Bead Columns

As whole blood passes through a column of glass beads, platelets are activated and all 3 basic platelet properties (adhesion, release reaction and aggregation) displayed, an aspect similar to platelets being involved in a hemostatic process. For this reason, we chose the platelet retention test as the tool to study the effect of synthetic prostacyclin (PGI₂) on platelet function and behavior. PGI₂ inhibited platelet retention with its major impact on platelet aggregation. The extent of inhibition was not proportional to the concentration of PGI₂. It appears that 2 levels of platelet aggregation take place inside the column. One, triggered by a weak stimulus, is readily inhibitable by low concentrations of PGI². The other, brought about by a strong stimulus, requires higher concentrations of PGI₂ to achieve complete inhibition. Even with as much as 100 ng PGI₂/ml blood, a 15% retention remained which probably represented platelet adhesion to glass beads. Scanning electron microscopic examination of glass beads showed massive platelet aggregation and adhesion in control blood samples. No platelet aggregate was observed on beads filtered with blood containing 20 ng/ml or more PGI₂. Individual platelets were found on glass beads filtered with samples containing any concentration of PGI₂; these platelets exhibited extensive morphologic changes.     

Thrombin-, Epinephrine- and Collagen-Induced Platelet Aggregation Inhibited by α1-Acid Glycoprotein

α₁-acid glycoprotein (α₁-acid GP) isolated from human plasma was found to inhibit thrombin-induced aggregation of washed human platelets (final thrombin concentration 0.05 NIH U/ml), and inhibition was complete with physiological concentrations of the glycoprotein (1.0–1.5 g/l final conc.). The inhibitory effect seemed to occur immediately on thrombin addition, thus being similar to the effect of heparin previously observed. As opposed to heparin, however, α₁-acid GP did not affect spontaneous platelet aggregation. Furthermore, α₁-acid GP (in optimal concentrations) reduced the combined inhibitory effect of heparin and antithrombin III on thrombin-induced platelet aggregation, thus being consistent with the previous findings using heparin thrombin clotting time. Snyder & Coodley (1976) found α₁-acid GP to inhibit platelet aggregation induced by epinephrine and adenosine diphosphate in platelet-rich plasma. As we also found α₁-acid GP to inhibit collagen-induced platelet aggregation, this acid glycoprotein may possibly act as an inhibitor of the release reaction although fairly high concentrations (10 mg/ml final conc.) were needed for complete inhibition.     

Tumor Cells Induce Platelet Aggregation and Intraplatelet Calcium Ion Movements

We studied platelet aggregation and changes in cytosolic Ca⁺⁺ concentrations induced by cells isolated from 5 human tumor tissues (2 hepatocellular carcinomas, 1 colon carcinoma, 1 gastric carcinoma and 1 pancreatic carcinoma). A Platelet Ionized Calcium Aggregometer was used and washed, aequorin loaded platelets were employed. Tumor cells were able to induce aggregation and an increase in cytoplasmic Ca⁺⁺ concentrations in the presence of trace amounts (10 µl) of PPP, while no aggregating response was found after addition of fibrinogen alone to washed platelets. The platelet aggregating activity of tumor cells was maintained in the presence of factor VII deficient plasma or of factor VIII deficient plasma, and disappeared completely when factor X deficient plasma was added to washed platelets. Furthermore, tumor cell induced platelet aggregation and Ca ⁺⁺ movements were inhibited by hirudin (100 U/ml), a specific thrombin inhibitor, while concanavalin A (100 µg/ml), a tissue factor inhibitor, had no effect. Finally, preincubation of neoplastic cells with HgCl₂ (0.5 mM), a cysteine protease inhibitor, markedly decreased their ability to induce aggregation and Ca⁺⁺ movements; on the contrary, incubation of cells with soybean trypsin inhibitor (10 µg/ml), a serine protease inhibitor, or with concanavalin A (100 µg/ml) had no effect. These data suggest that cells isolated from human tumor tissues activate platelet function through the generation of thrombin, due to a cysteine protease which directly activates factor X.     

Platelet-activating factor is a weak platelet agonist: Evidence from normal human platelets and platelets with congenital secretion defects

We have examined the effects of a novel platelet agonist, platelet activating factor (PAF), on human platelets. Irreversible aggregation and ¹⁴C-serotonin secretion in response to PAF (10^?5 M) was found to be dependent on both thromboxane production and secreted adenosine diphosphate (ADP). Liberation of arachidonic acid (AA) from membrane-bound phospholipids is a prerequisite step in platelet thromboxane production. Studies with ³H-AA-labeled platelets revealed that PAF (10^?5 M) was a weak stimulus for the mobilization of AA. In addition, PAF (10^?5M) was found to be a weak inducer of thromboxane synthesis (mean = 6 pmol/10⁸ platelets) as compared to thrombin 5 U/ml (mean = 177 pmol/10⁸ platelets), measured using a radioimmunoassay for thromboxane B₂. Formation of phosphatidic acid is an early step in stimulus-response coupling in platelets. Our studies indicate that PAF is a weak stimulus for phosphatidic acid formation as well. To obtain further insights into its action, we examined the effect of PAF on platelets from three groups of patients with congenital secretion defects: patients with the storage pool deficiency, those with impaired thromboxane synthesis due to impaired liberation of AA from phospholipids, and those with impaired secretion despite normal granule stores and thromboxane production. The response to PAF was impaired in all patients, providing further evidence that PAF-induced platelet activation is dependent on secreted ADP and thromboxane A₂ synthesis, and occurs by mechanisms common to a number of agonists. Overall, these studies indicate that PAF is a weak platelet agonist.     

Epinephrine induces a late thromboxane-dependent platelet shape change and enhances synergistically the shape change induced by other platelet agonists

Epinephrine is the only physiological platelet activator which induces platelet aggregation without a preceding change in platelet shape. The reason why epinephrine cannot induce this shape change is not known. Electron microscopically, we could show that during the first phase of epinephrine-induced platelet aggregation, the platelet aggregate is composed of discoid platelets, lying in rather loose contact with neighbouring platelets. During the second wave of epinephrine-induced aggregation (this is when thromboxane (TX)A₂ production has taken place), platelets have completely lost their discoid shape and are very tightly bound. In EDTA-platelet rich plasma (PRP), we could demonstrate a clear synergistic action of epinephrine 10–20 μM on the first phase of shape change (disc-to-sphere transformation), induced by low concentrations of arachidonic acid (AA), collagen, adenosine diphosphate (ADP) and platelet activating factor (PAF). In combination with moderate concentrations of AA or collagen, epinephrine induced a clear aggregation-independent secretion of platelet granules, which in the absence of epinephrine, only takes place with higher inducer concentrations. All these synergistic actions could be demonstrated in the aggregometer and electron microscopically. To explain these findings, we hypothesize that the inability of epinephrine to induce a shape change that precedes aggregation is due to slow generation of TXA₂ which is only formed as a positive feedback mechanism of aggregation. This TXA₂ will bind to its own receptor and produce a shape change coinciding with the second wave of epinephrine-induced aggregation. Collagen, in contrast, induces very rapid TXA₂ generation, causing Ca²⁺ mobilization and myosin light chain-phosphorylation, leading to shape change, clearly before aggregation starts.     

SIRT1 prevents pulmonary thrombus formation induced by arachidonic acid via downregulation of PAF receptor expression in platelets

SIRT1, a class III histone deacetylase, is critically involved in cellular response to stress and modulates cardiovascular risk factors. However, its role in thrombus formation is largely unknown. Thus, this study investigated the effect of SIRT1 on pulmonary thrombus formation, and then identified its role in the modulation of platelet aggregation. In isolated human platelets, cell aggregation was increased by various platelet activators, such as platelet activating factor (PAF), arachidonic acid (AA), ADP, and thrombin. AA- and PAF-mediated platelet aggregations were suppressed by WEB2086, a PAF receptor (PAFR) antagonist. Pulmonary thrombus formation induced by PAF or AA was also attenuated by WEB2086, suggesting that PAFR plays a key role in AA-induced platelet aggregation. In platelets isolated from SIRT1-TG mice as well as in platelets treated with resveratrol or reSIRT1, PAFR expression was decreased, whereas this expressional downregulation by SIRT1 activators was inhibited in platelets treated with MG132 (a proteasome inhibitor) or NH₄Cl (a lysosome inhibitor). Furthermore, platelet aggregation induced by AA was markedly attenuated by resveratrol and reSIRT1. Likewise, the increased pulmonary thrombus formation in mice treated with AA was also attenuated by SIRT1 activators. In line with these results, pulmonary thrombus formation was markedly attenuated in SIRT1-TG mice. Taken together, this study showed that SIRT1 downregulates PAFR expression on platelets via proteasomal and lysosomal pathways, and that this downregulation inhibits platelet aggregation in vitro and pulmonary thrombus formation in vivo.     

Polyphosphoinositide Changes in Rabbit Platelets Stimulated with Platelet Activating Factor During the Formation of Platelet-fibrin Clots

Phosphoinositide metabolism in rabbit platelets prelabelled with [³²P]phosphate and [³H]inositol was stimulated by platelet activating factor (PAF, 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) with stirring at 200 rpm for 120 s in the presence of polymerising fibrin produced by the action of batroxobin (B. atrox) (also referred to by the proprietary name Reptilase) on fibrinogen. Under these conditions platelet-fibrin clots formed and retracted around the stirring bar. Phosphoinositides were extracted with chloroform: methanol: HC1. The role of the secretion of platelet granule contents in the phosphoinositide changes was examined by comparison of the effects of 1 nM PAF which did not cause secretion, with 50 nM PAF which caused extensive secretion. Stimulation of platelets with PAF in the presence of polymerising fibrin caused a greater decrease in the amount and labelling of extractable phosphatidylinositol 4,5-bisphosphate (PIP₂) than was observed with platelets stimulated in the presence of fibrinogen. With 1 nM PAF, the decrease (1.26 ± 0.11 nmol/10⁹ platelets) in amount of extractable PIP₂ when platelets were stimulated in the presence of polymerising fibrin compared with in the presence of fibrinogen was accounted for by an increase in the amount of phosphatidylinositol 4-phosphate (PIP). With 50 nM PAF, the decrease in amount of extractable PIP₂ (1.09±0.11 nmol/10⁹ platelets) was not accounted for by an increase in the amount of PIP; the decrease in the amount of [³H]inositol label in PIP₂ in platelets stimulated in the presence of polymerising fibrin was accounted for by the sum of the increases in PIP labelling and the label associated with interfacial protein from the lipid extractions. When fibrin polymerisation was blocked with glycyl-L-prolyl-L-arginyl-L-proline (GPRP), the large decrease in extractable PIP₂ and the increase in the association of label with the interfacial protein did not occur. Thus, both the formation of a fibrin network, and the changes that accompany the secretion of granule contents, are necessary for the association of the ³H-labelled material with interfacial protein. Blocking thromboxane A₂ formation had no effect on the changes in response to 50 nM PAF. Although PAF stimulated phospholipase C, resulting in increases in amount and ³²P-labelling of phosphatidic acid and ³H-labelling of inositol bisphosphate and inositol phosphate, the increases were similar in the presence of polymerising fibrin or fibrinogen. Thus, further stimulation of phospholipase C does not occur in association with clot formation. The specific radioactivities of labelling with [³H]inositol of the phosphoinositides in unstimulated platelets differed (PIP₂> phosphatidylinositol (PI) > PIP). Upon stimulation of the platelets with 1 nM PAF, the specific radioactivity of PIP rose above that of PI and toward that of PIP₂, indicating that the increase in PIP was due to degradation of PIP₂. Thus, the large decrease in extractable PIP₂ and increase in formation of PIP caused by the presence of polymerising fibrin appear to be due to increased degradation of PIP₂ to PIP.     

Heterogeneity of Platelet Fc-receptor-dependent Response to Activating Monoclonal Antibodies

Platelet activation induced by monoclonal antibodies (mAB) was studied using three stimulatory mAB (all IgG₁) against different platelet surface glycoproteins: VM58 against GPIV, LeoAl against PTA1, and FMC 56 against CD9. F(ab')₂ fragments of these antibodies failed to activate platelets themselves but blocked platelet aggregation induced by the relevant intact antibody. Platelet aggregation was also completely blocked by the anti-FcγRII (Fc-receptor) monoclonal antibody, IV.3. A heterogeneity of platelet response to stimulatory mAB was observed amongst normal donors. All three antibodies added to platelet-rich plasma (PRP) from responders induced full platelet aggregation and dense body release. However, in PRP from nonresponders, VM58 and LeoAl did not induce platelet activation whilst FMC 56 activated platelets but to a lesser extent than in responders (longer lag phase and reduced release). The ratio of responders to nonresponders was ～ 1:1 (n = 110). The heterogeneity was not due to differences in the copy number of either the antigen (VM58) or FcγRII. The ability of donor platelets to be aggregated by stimulatory mAB in PRP correlated with the ability of these platelets to respond to aggregated murine IgG₁ (mAB irrelevant to platelets). The combined results suggest that both the Fab and Fc region of stimulatory mAB are necessary in order to induce a platelet response and that this response is mediated through FcγRII. The difference between responders and nonresponders can be explained by the known polymorphism of FcγRII (Looney et al, 1988) and the capacity of the polymorphic forms of FcγRII to bind and to respond to murine IgG₁.     

Effect of ADP-induced Shape Change on Incorporation of 32P into Platelet Phosphatidic Acid and Mono-, Di- and Triphosphatidyl Inositol

Summary . Experiments were carried out to determine whether the ADP-induced increase in ³²P-content of phosphatidic acid (PA), monophosphatidylinositol (MPI), diphosphatidylinositol (DPI) or triphosphatidylinositol (TPI) of washed ³²PO₄ labelled platelets is related to platelet aggregation or to the change in shape induced by ADP. Washed platelets were labelled with ³²PO₄, washed, and resuspended in medium containing unlabelled PO₄. With rabbit platelets, either the omission of Ca⁺⁺ from the suspending medium or the addition of ethyleneglycoltetraacetic acid (EGTA) in amounts sufficient to chelate Ca ⁺⁺ in the suspending medium, prevented ADP-induced platelet aggregation. Lack of free Ca⁺⁺ in the suspending medium did not prevent the ADP-induced shape change or the increased labelling of PA and DPI. In suspensions containing EGTA, the increase in ³²P-content of PA induced by varying amounts of ADP was compared to the extent of the shape change. ADP in a final concentration of 10^?6m or greater, produced both the maximum increase in ³²P-content of PA and the maximum degree of shape change. Washed platelets from pigs or humans without fibrinogen in the suspending medium showed a shape change following the addition of ADP but no aggregation, whereas in suspensions containing fibrinogen, the platelets showed both a shape change and aggregation. In suspension with and without fibrinogen, the ADP induced increase in ³²P-content of PA in human platelets and of PA+ MPI and DPI in pig platelets was the same. Prostaglandin E₁ (PGE₁), 10^?4m final concentration, added to suspensions of rabbit platelets prevented both the ADP-induced shape change and the increase in ³²P incorporation into PA and DPI. PGE₁, 10^?4m , also caused a decrease in labelling of TPI. PGE₁, 10^?7m final concentration, prevented platelet aggregation but did not prevent the ADP-induced shape change or the increase in ³²P-content of PA. Dibutyrylcyclic AMP (DBcAMP) inhibited both the ADP-induced shape change and the increase in ³²P-content of PA. With rabbit platelets, the time course of the ADP-induced shape change was compared to the time course of the increase in ³²P-content of PA, MPI and DPI. The increase in labelling of PA was detected 2 s after the addition of ADP and was near maximum at 8 s, the time of maximum shape change. The increase in labelling of DPI was not significant until 30 s. The regaining of the disc shape was associated with an increase in labelling of MPI and a decrease in labelling of PA and DPI. The results show that the changes in platelet PA are related to the ADP-induced shape change rather than to aggregation.     

Platelet Lesion of Collection

In the early stages of blood collection for transfusion, whole blood is subjected to trauma from the anticoagulant. The blood – anticoagulant mixture does not attain a pH at which platelets can remain functional (approximately pH 6.0) until about 25% of the unit has been collected. We have examined platelet ultrastructure and in vitro aggregation responses to adenosine diphosphate (ADP) and epinephrine (EPN), to assess the platelet trauma of collection into citrate-phosphate-dextrose. For comparison blood was collected by 2 methods: one was conventional (CC), the other metered anticoagulant into the blood as it was collected (MC). Platelets from CC blood showed disruption of organelles and depletion of the dense bodies, whereas platelets from MC blood showed no ultrastructural damage. In addition, biphasic aggregation in response to ADP or EPN was seen in MC platelets but not in CC platelets.     

Angiotensin Receptor Blocker Losartan Suppresses Platelet Activity by Interfering with Thromboxane Signaling

Enhanced platelet activity and platelet endothelial interaction are hallmarks of different vascular and metabolic diseases with subsequent thrombus formation. In atherosclerosis, coronary artery disease, congestive heart failure, nitrate tolerance, chronic inflammation, or diabetic states, platelet activation may in part be due to a stimulation of the renin-angiotensin-aldosteron system, which also contributes to enhanced oxidant stress in these conditions. Aims. We examined the putative role of the angiotensin receptor (AT₁) and of phospholipase A₂ (PLA₂) in mediating platelet activation under defined in vitro conditions using the AT₁ receptor antagonists losartan, EXP 3174, candesartan, and the PLA₂ inhibitor arachidonyltrifluoromethyl ketone (AACOCF3), respectively. Results. In washed human or canine platelet suspensions, losartan (10^–4–10^–6 mol/L) dose-dependently suppressed thrombin-induced calcium transients as well as thromboxane (TxA₂) release. In both species, aggregation of washed platelets in response to thrombin or ADP was substantially diminished by different doses of losartan. This inhibition of platelet aggregation was even maintained in ADP-stimulated platelet-rich plasma. While the PLA₂ inhibitor AACOCF3 effectively inhibited thrombin-induced TxA₂ release from washed human or canine platelets (similar to the effects observed with losartan), the AT₁ agonist angiotensin II elicited platelet TxA₂ release only at high supra-physiological doses (e.g., at 10^–4 mol/L). The AT₁ specific antagonist candesartan did not diminish stimulated platelet aggregation, TxA₂ formation, or calcium transients. By contrast, the active losartan metabolite EXP 3174 dose-dependently inhibited stimulated platelet calcium transients as well as TxA₂ release at 1–100 mol/L. Conclusions. Losartan significantly counteracts ex vivo platelet activation, probably via the blockade of TxA₂ receptor-dependent signaling (e.g. implying activation of phospholipase A₂) rather than acting at the AT₁ receptor itself. This implies that the TxA₂ signaling pathway plays a significant role during platelet activation, which may be successfully antagonized in vivo under different pathological states with enhanced thrombus formation or platelet-endothelium interactions.     

Blockade of receptor-operated calcium channels by mibefradil (Ro 40-5967): Effects on intracellular calcium and platelet aggregation

Summary The goal of the present study was to evaluate if mibefradil, a novel nondihydropyridine Ca²⁺ antagonist, could block receptor-operated calcium channels (ROCC) present in human platelets and to determine the functional consequences of this blockade. Therefore, the effect of mibefradil on increases in intracellular Ca²⁺ concentrations and aggregation of human platelets induced by platelet activating factor (PAF) was examined. In order to differentiate effects on Ca²⁺ mobilization from intracellular stores from those on Ca²⁺ influx through ROCC, intracellular Ca²⁺ concentrations were measured either in fura-2-loaded platelets or in cells loaded with both BAPTA and fura-2. Mibefradil totally and dose dependently inhibited PAF-induced Ca²⁺ influx with a maximal effective concentration of 10 µM, but at this concentration only reduced Ca²⁺ mobilization from intracellular stores. A similar effect was observed when platelets were stimulated with ADP, suggesting that mibefradil was indeed interfering with ROCC and not specifically with PAF receptors. In the same range of concentrations, mibefradil inhibited Ca²⁺-dependent platelet aggregation induced by PAF. This effect was most likely due to the inhibition of ROCC, as Ca²⁺-independent aggregation induced by phorbolmyristyl-acetate (PMA) was insensitive to mibefradil. We conclude that mibefradil, which has previously been described to be an antagonist for L- and T-Type Ca²⁺ channels, also blocks receptor-operated Ca²⁺ channels. This blockade seems to be functionally relevant for platelet aggregation.     

Dextran sulfate triggers platelet aggregation via direct activation of PEAR1

Dextran sulfate (DxS; Mr 500 kD) induces fibrinogen receptor (α_IIbβ₃) activation via CLEC-2/Syk signaling and via a Syk-independent SFK/PI3K/Akt-dependent tyrosine kinase pathway in human and murine platelets. The platelet surface receptor, responsible for the DxS-induced Syk-independent Akt-activation, has hitherto not been identified. We found that DxS elicited a concentration-dependent aggregation of human platelets resulting from direct PEAR1 activation by DxS. Blocking the PEAR1 receptor, in combination with a selective Syk-inhibitor, completely abrogated the DxS-driven platelet aggregation. The DxS-induced Syk-phosphorylation was not affected in Pear1^?/? platelets, but Akt-phosphorylation was largely abolished. As a result, the aggregation of Pear1^?/? platelets was reduced and reversible, i.e. aggregates were less stable compared to wild-type platelet aggregates. Moreover, DxS-induced Pear1^?/? platelet aggregation was fully abrogated by Syk inhibition, indicating that the remaining platelet aggregation of Pear1^?/? platelets was Syk dependent. Hence, the Pear1/c-Src/PI3K/Akt- and CLEC-2/Syk-signaling pathways are independently and additively activated during platelet aggregation by DxS. Conclusion. The DxS-induced aggregation of human and murine platelets is the result of activation of PI3K/Akt through direct PEAR1 phosphorylation and parallel Syk-signaling through CLEC-2.     

Evaluation of the platelet storage pool deficiency in the feline counterpart of the chediak-higashi syndrome

Cats with the Chediak-Higashi (CH) syndrome have abnormal hemostasis with prolonged bleeding times and normal coagulation times. Platelet aggregation induced by serotonin, ADP, and collagen was impaired. Platelets from normal and CH cats were incubated with ¹⁴C-adenine and then gel-filtered. Gel-filtered platelets (GFP) from CH cats contained 63% of the ATP, 38% of the ADP, 100% of the Ca²⁺, and 75% of the Mg²⁵ of normal platelets. Serotonin could not be detected in CH platelets. Acid hydrolase and total platelet protein of CH platelets was similar to normal platelets. Gel-filtered platelets were treated with thrombin to induce maximal secretion. Secretion of ATP, Ca²⁺, and Mg²⁺ was 1.9%, 12.4%, and 16% respectively of normal platelets. ADP secretion by CH platelets was not detectable. The ATP/ADP ratio in the ¹⁴C-labeled metabolic pool of normal platelets was similar to that of total measured nucleotide pool of CH platelets. These findings suggest that in feline CH platelets, as in platelets from CH mink and cattle, there is storage pool deficiency that is virtually complete, and the virtual absence of ADP and 5HT may in part account for the abnormal hemostasis. Aggregation of platelets from CH cats was impaired, but these platelets did aggregate to arachidonate, serotonin-induced biphasic aggregation, and the aggregation response to ADP and collagen varied according to the amount of serotonin-induced TxB₂ formed. These findings support a major role for arachidonate in platelet activation.     

Tumor Cell-Induced Platelet Aggregation in Vitro by Human Pancreatic Cancer Cell Lines

Background: Tumor cell-induced platelet aggregation (TCIPA) is considered to be a critical step in hematogenous metastasis. Methods: TCIPA was studied in vitro in six human pancreatic carcinoma cell lines (PC 3, PC 44, AsPC1, BxPC3, Capan2, Pane1). Results: Whereas all cell lines induced aggregation of washed platelets in the presence of minimal amounts of platelet-poor plasma, five cell lines also induced aggregation of platelets in platelet-rich plasma. The thrombin-antagonist hirudin inhibited TCIPA in all cell lines, indicating that TCIPA is thrombin-dependent. Since pretreatment of tumor cells with phospholipase A₂ or C inhibited TCIPA, the thrombin-generating activity might be confined to the tumor cell surface. Further support for a prothrombinase activity was provided by the observation that all cell lines were able to induce the aggregation of washed platelets after addition of purified coagulation factors II and V. Conclusions: Pancreatic carcinoma cells are able to induce platelet aggregation via activation of thrombin. This might support metastasis in pancreatic cancer and possibly explain the incidence of thrombosis in this tumor.     

Differential Expression of Platelet Activation Markers CD62P and CD63 Following Stimulation with PAF,Arachidonic Acid and Collagen

The effects of varying concentrations of platelet-activating factor (PAF), arachidonic acid (AA) and collagen on the expression of the platelet activation markers CD63 and CD62P were assessed in 10 normal subjects using flow cytometry. CD63 expression was significantly greater than CD62P expression, with PAF (80 nM) inducing mean maximum CD63 expression of 32.9 ± 6.4% and mean maximum CD62P expression of 5.5 ± 1.8%. AA (1 mM) induced maximum CD63 expression of 37.7 ± 7% and maximum CD62P expression of 9.3 ± 1%. Collagen (2-80 pg/ml) induced minimal expression but 800 pg/ml induced mean CD63 expression of 33.1 ± 4.1% and mean CD62P expression of 6.1 ± 0.8%. Greater CD63 and CD62P expression were induced by phorbol myristate acetate (1.6 pM, 70.9 ± 11% and 69.4 ± 9.9%, respectively) and thrombin (0.1 U/ml, 70.7 ± 9.3% and 73.5 ± 5.4%, respectively). With PAF and collagen only one platelet population was detected whereas with 1 mM AA two populations were observed. These results indicate that expression of platelet adhesion receptors depends on the nature and concentration of agonist and that subpopulations of platelets may exist. Importantly, PAF concentrations inducing moderate CD63 and CD62P expression did not induce platelet aggregation, suggesting that platelets can be activated independently of aggregation.     

Thrombin Binding to Platelets Defines Functional Receptors: Inhibition of Thrombin-Induced Platelet Activation by Catalytically-Inactivated Thrombin

It has been widely questioned as to whether the observed binding of a-thrombin to intact platelets defines receptors coupled to signal transduction or merely thrombin binding sites. We have now shown that at α-thrombin concentrations sufficient to induce a full shape change response without aggregation (0.1 nM), PPACK-thrombin (that is, α-thrombin treated with the irreversible active site inhibitor D-phenylalanyl-L-prolyl-L-arginine chloromethylketone) dose-dependently inhibits platelet shape change (IC₅₀～70 nM), the concomitant increases in [Ca²⁺Ii (IC₅₀～75 nM) and ATP secretion (IC₅₀～50 nM). Since PPACK-thrombin competes fully in the binding of a-thrombin to high, moderate and low affinity sites on intact platelets, these results show that this binding defines functional receptors coupled to platelet activation.     

Platelet receptors and signaling in the dynamics of thrombus formation

Hemostasis and pathological thrombus formation are dynamic processes that require a co-ordinated series of events involving platelet membrane receptors, bidirectional intracellular signals, and release of platelet proteins and inflammatory substances. This review aims to summarize current knowledge in the key steps in the dynamics of thrombus formation, with special emphasis on the crucial participation of platelet receptors and signaling in this process. Initial tethering and firm adhesion of platelets to the exposed subendothelium is mediated by glycoprotein (GP) Ib/IX/V complex and collagen receptors, GP VI and α₂β₁ integrin, in the platelet surface, and by VWF and fibrillar collagen in the vascular site. Interactions between these elements are largely influenced by flow and trigger signaling events that reinforce adhesion and promote platelet activation. Thereafter, soluble agonists, ADP, thrombin, TxA₂, produced/released at the site of vascular injury act in autocrine and paracrine mode to amplify platelet activation and to recruit circulating platelets to the developing thrombus. Specific interactions of these agonists with their G-protein coupled receptors generate inside-out signaling leading to conformational activation of integrins, in particular α_IIbβ₃, increasing their ligand affinity. Binding of α_IIbβ₃ to its ligands, mainly fibrinogen, supports processes such as clot retraction and platelet aggregation. Stabilization of thrombi is supported by the late wave of signaling events promoted by close contact between aggregated platelets. The best known contact-dependent signaling is outside-in signaling through αIb β₃, but new ones are being clarified such as those mediated by interaction of Eph receptors with ephrins, or by Sema 4D and Gas-6 binding to their receptors. Finally, newly identified mechanisms appear to control thrombus growth, including back-shifting of activated integrins and actuation of compensatory molecules such as ESAM or PECAM-1. The expanding knowledge of thrombotic disease is expected to translate into the development of new drugs to help management and prevention of thrombosis.     

Effects of Ethanol on Human Platelets Stimulated with Platelet-Activating Factor, a Biologically Active Ether Phospholipid

The interaction between ethanol and 1-0-alkyl-2-acetyl-sn-glycerol-3-phosphocholine (platelet activating factor, PAF) was addressed using platelets obtained from normal nonalcoholic volunteers. Ethanol at concentrations of 20 to 100 mM inhibited PAF activation of human platelets. Ethanol inhibited prominently the second or arachidonic acid metabolite dependent wave of platelet aggregation, which occurs with human platelets in citrated plasma. It also inhibited serotonin release and thromboxane A2 formation associated with this secondary phase of aggregation. Ethanol did not readily inhibit the primary wave of PAF-induced aggregation. The incorporation of PAF into platelets or metabolism of PAF was not influenced by up to 100 mM ethanol. Since ethanol inhibited only the secondary response, a direct interaction between PAF, ethanol, and a platelet PAF receptor is unlikely. The effect of ethanol on PAF-induced platelet aggregation shows a selectivity similar to that demonstrated by other investigators for epinephrine and adenosine diphosphate.     

Inhibition of cyclooxygenase-independent platelet aggregation by low vitamin E concentration

Platelet aggregation induced by threshold concentrations of agonists such as collagen, PAF or epinephrine was inhibited in vitro by 100 microM aspirin but was restored by stimulating platelets with high concentrations of collagen, PAF or by a combination of epinephrine and PAF. Incubating aspirin-treated platelets with 50-100 microM vitamin E or vitamin E acetate inhibited platelet aggregation by high concentrations of collagen and PAF and by the combination of epinephrine and PAF; platelet thromboxane A2 formation was less than 10% in samples incubated with 100 microM aspirin. Apyrase, added to aspirin-treated platelet, did not influence platelet aggregation induced by epinephrine and PAF. The present study suggests that concentrations of vitamin E as low as 50-100 microM inhibit cyclooxygenase-independent platelet aggregation when combined with an inhibitor of the arachidonate pathway.