Effect of calcium ion concentration on the ability of fibrinogen and von Willebrand factor to support the ADP-induced aggregation of human platelets

To investigate the suggestion that von Willebrand factor (vWf) can substitute for fibrinogen in supporting ADP-induced aggregation of human platelets, we studied platelet reactions in two media: (1) a high calcium medium, Tyrode-albumin solution containing calcium ions in the physiological range of 2 mmol/L, and (2) a low calcium medium, modified Tyrode-albumin solution from which calcium salt was omitted (calcium ion concentration approximately 20 mumol/L). In the high calcium medium vWf even at concentrations up to six times as high as physiological, showed little or no potentiation of ADP-induced platelet aggregation, whereas fibrinogen strongly potentiated reversible aggregation without thromboxane formation or release of granule contents. In the low calcium medium, either vWf or fibrinogen supported biphasic aggregation in response to ADP, with thromboxane formation and release of granule contents. Aspirin and the thromboxane receptor blocker BM 13.177 inhibited these secondary responses to von Willebrand factor, indicating that they require thromboxane A2 formation and feedback amplification by thromboxane A2. A monoclonal antibody, 10E5, to the platelet glycoprotein IIb/IIIa complex inhibited both primary and secondary aggregation. Although vWf supports ADP-induced aggregation when the concentration of ionized calcium is in the micromolar range, it does not support ADP-induced aggregation in the presence of a concentration of ionized calcium in the physiological range, indicating that vWf probably cannot substitute for fibrinogen in supporting ADP- induced aggregation in vivo.     

Immunocytochemical localization of fibrinogen on washed human platelets. Lack of requirement for fibrinogen during adenosine diphosphate-induced responses and enhanced fibrinogen binding in a medium with low calcium levels

The association of fibrinogen with washed human platelets was examined by immunocytochemistry during aggregation induced by adenosine diphosphate (ADP) and during deaggregation. The platelets were suspended either in a medium containing 2 mmol/L Ca2+ or in a medium containing no added Ca2+ (20 mumol/L Ca2+). Platelets were fixed at several times during aggregation and deaggregation, embedded in Lowicryl K4M, sectioned, incubated with goat antihuman fibrinogen, washed, reacted with gold-labeled antigoat IgG, and prepared for electron microscopy. To determine whether the method detected fibrinogen associated with the platelets, the platelets were pretreated with chymotrypsin (10 U/mL) and aggregated by fibrinogen; gold particles were apparent not only in the alpha granules but on the platelet surface and between adherent platelets as well. In the medium with 2 mmol/L Ca2+, ADP caused extensive aggregation of normal platelets in the presence of fibrinogen (0.4 mg/mL), and gold particles were evident between the adherent platelets and on the platelet surface; when the platelets deaggregated, gold was no longer present on the surface. In a medium without added Ca2+, ADP caused extensive aggregation in the presence of fibrinogen, and large numbers of gold particles were on the platelet surface and even more between adherent platelets. In this medium, the platelets did not deaggregate, and by five minutes, the granules appeared to be swollen or fused. In the absence of external fibrinogen, ADP caused the formation of small aggregates, and fibrinogen was not detected between adherent platelets. Thus, the association of fibrinogen with the platelet surface enhances platelet aggregation but is not essential for the ADP-induced formation of small aggregates. The association of fibrinogen with platelets is greater under conditions in which platelets release their granule contents and do not deaggregate because both endogenous and exogenous fibrinogen take part in aggregation.     

Platelet hypersensitivity in cholesterol-fed rabbits: enhancement of thromboxane A2-dependent and thrombin-induced, thromboxane A2-independent platelet responses

Hypercholesterolemia (mean plasma cholesterol: 15 mM) was induced in rabbits by the feeding of a chow diet enriched with a low amount (0.25%, w/w) of cholesterol only. Platelet size and protein content decreased significantly, but the whole blood platelet count did not change. The platelets became enriched in cholesterol, as indicated by a significant increase in the cholesterol:phospholipid molar (C/P) ratio. Specific responses of washed platelets stimulated with various agonists were studied to determine the effects of hypercholesterolemia on the various pathways of platelet aggregation in the absence of plasma components. In platelets from hypercholesterolemic rabbits compared with controls: aggregation induced by ADP was not altered; collagen-induced responses (aggregation, secretion of [14C]serotonin from prelabelled platelets, thromboxane A2 (TXA2) formation, mobilization of [3H]arachidonate from prelabelled platelets) were enhanced; with aspirin-treated platelets, aggregation induced by the TXA2 mimetic U46619 was enhanced: and thrombin-induced responses of both untreated platelets (aggregation, secretion of granule contents, TXA2 formation) and aspirin-treated platelets (aggregation) were enhanced. Thus, platelets from cholesterol-fed rabbits not only form more TXA2, but they aggregate more extensively when stimulated by its mimetic. In addition, it has not been previously recognized that these platelets are also hypersensitive to thrombin-induced aggregation that is independent of TXA2.     

Pathogenetic analysis of three cases with a bleeding disorder characterized by defective platelet aggregation induced by Ca2+ ionophores

We report three cases of platelet dysfunction characterized by defective Ca2+ ionophore-induced platelet aggregation without impaired production of thromboxane A2 (TXA2). The patients had mild to moderate bleeding tendencies, and their platelet aggregation and secretion induced by ADP, collagen, arachidonic acid, stable TXA2 (STA2) and Ca2+ ionophore A23187 was defective or much reduced. However, ristocetin- or thrombin-induced platelet aggregation was normal. The analysis of second messenger formation showed that inositol 1,4,5-triphosphate formation or Ca2+ mobilization induced by thrombin, STA2 or A23187 was normal. Furthermore, the phosphorylation of 47 kDa protein (pleckstrin) and 20 kDa protein (myosin light chain, MLC) in response to those agonists was normal. These findings suggest that the defective site in the patients' platelets lies in the process distal to or independent of protein kinase C activation, Ca2+ mobilization and MLC phosphorylation.     

Effect of extracellular magnesium on platelet activation and intracellular calcium mobilization.

A dose-dependent effect of magnesium on the inhibition of platelet aggregation and release of ATP from dense granules was observed in human platelets (in whole blood, platelet-rich plasma, or washed platelets) against various aggregation agents (ADP, U46619, collagen, or thrombin). The synthesis and release of the proaggregatory cyclooxygenase (CO) and lipoxygenase (LO) products, thromboxane A2 (TXA2) and 12-hydroxyeicosatetraenoic acid (12-HETE), respectively, in platelets were also inhibited by Mg in a dose-dependent manner (IC50 4 to 6 mmol/L). These Mg-mediated activities were further enhanced when platelets were preincubated with insulin (100 microU/mL). The effect of extracellular Mg on the change of intracellular calcium concentration ([Ca2+]i) was assessed using Fura-2/AM loaded cells in the presence or absence of extracellular Ca. Thrombin-stimulated influx of Ca ions decreased from 194 +/- 30 nmol/L to 156 +/- 21 nmol/L in the presence of 5 mmol/L Mg and to 111 +/- 16 nmol/L in 10 mmol/L Mg. However, the intracellular Ca release (as determined in the presence of 5 mmol/L EGTA) was not affected by Mg. The intracellular Ca-dependent protein kinase C and myosin light chain kinase activities on the phosphorylation of endogenous p47 and p20 proteins studied after 2 min of thrombin addition decreased only 10 to 25% in the presence of 5 to 10 mmol/L Mg. Similar results were obtained when EGTA was added prior to the initiation of protein phosphorylation. We conclude that Mg can dose dependently inhibit a wide variety of agonists on platelet aggregation. Furthermore, insulin can potentiate the inhibitory effects of Mg on platelet activation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Defective signal transduction induced by thromboxane A2 in a patient with a mild bleeding disorder: impaired phospholipase C activation despite normal phospholipase A2 activation

A patient with a mild bleeding disorder whose platelets responded defectively to thromboxane A2 (TXA2) was identified, and the mechanism of this dysfunction was analyzed. The platelets were defective in shape change, aggregation, and release reaction in response to synthetic TXA2 mimetic (STA2). When the platelet TXA2 receptor was examined with both a 125I-labeled derivative of a TXA2 receptor antagonist ([125I]-PTAOH) and [3H]-labeled TXA2 agonist ([3H]U-46619), the equilibrium dissociation rate constants (kd) and the maximal concentrations of binding sites (Bmax) of the platelets to both ligands were within normal ranges, suggesting that the binding capacity of their TXA2 receptor was normal. STA2 could not induce IP3 formation and intracellular Ca2+ mobilization, whereas these responses to thrombin were within normal ranges. GTPase activity was also decreased when the patient's platelet membrane was challenged with STA2. On the other hand, lysophosphatidylinositol formation, which is a direct indicator of phospholipase A2 (PLA2) activation, was found to be normal when the [3H]-inositol-labeled platelets were challenged with STA2. Thromboxane B2 (TXB2) was also produced in response to STA2. These results suggested that the abnormality in these platelets was impaired coupling between TXA2 receptor and phospholipase C (PLC) activation. Furthermore, it is also suggested that the activation of PLA2 and PLC are separable events in thromboxane-induced platelet activation.     

Pathogenesis of a bleeding disorder characterized by platelet unresponsiveness to thromboxane A2

A platelet disorder characterized by the absence of thromboxane A2 (TXA2)-induced platelet aggregation is a new clinical entity of platelet dysfunction. The platelets of three patients had the ability to bind exogenous TXA2, but synthetic TXA2 mimetic-induced postreceptor biochemical events, such as IP3 formation, Ca2+ mobilization, phosphatidic acid formation, and GTPase activities, were selectively defective, suggesting impaired coupling between the TXA2 receptor and phospholipase C activation. Gene analysis of the TXA2 receptor showed a substitution of Leu for Arg60 in the first cytoplasmic loop in all patients, and this mutation seemed to be responsible for this platelet disorder.     

Activation of Phospholipase C as a Primary Target of the Thromboxane A(2)-mediated Amplification Mechanism in Thrombin-induced Rabbit Platelet Activation

The inhibitory effects of a cyclooxygenase inhibitor, indomethacin, and a thromboxane (TX) A(2) receptor antagonist, S-145, on thrombin-stimulated rabbit platelet responses were examined for their contribution to the TXA-mediated amplification mechanism. Although thrombin (0.01-0.3 U/ml) induced the dosedependent aggregation and release of TXB(2) from washed rabbit platelets, indomethacin (30 μM) inhibited only the aggregation induced by a threshold dose of thrombin, even though it completely inhibited the formation of TXB(2). Indomethacin inhibited both the secretion of ATP and the elevation of the intracellular concentration of Ca(2+) ([Ca(2+)](i)) induced by all doses of thrombin used and induced a considerable rightward shift of the dose-response curves. S-145 also significantly inhibited the elevation of [Ca(2+)](i). Thrombin caused rapid accumulation of [(3)H]-InsP(3) or of inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)]. This accumulation was also inhibited by indomethacin to about 70% of the control level. STA(2), a stable analogue of TXA(2), and arachidonic acid caused accumulation of InsP, and that induced by the latter was completely inhibited by indomethacin (1 μM). Thrombin-induced aggregation peaked at a lower level of [Ca(2+)](i) than that required for the secretion of ATP. The apparent contribution of TXA(2) to aggregation therefore appears to be restricted to that induced by lower doses of thrombin. These results suggest that in thrombin-stimulated rabbit platelets, activation of phospholipase C, which is regulated by TXA(2) receptors, is a primary target of the TXA(2)-mediated amplification mechanism. Through its effect on the accumulation of Ins(1,4,5)P(3), this amplification mechanism may contribute to about 25-30% of the elevation of (Ca(2+)](i), in addition to the thrombin receptor-mediated mechanism.     

Evidence for a role of glycoprotein IIb-IIIa, distinct from its ability to support aggregation, in platelet activation by ionophores in the presence of extracellular divalent cations

Ionophore A23187-induced 14C-serotonin secretion and thromboxane B2(TxB2) formation were found to be absent in citrated platelet-rich plasma (PRP) from thrombasthenic subjects and in normal PRP treated with glycoprotein (GP) IIb-IIIa complex-specific monoclonal antibodies. Both responses were restored to normal levels when 5 mmol/L EDTA was present, indicating that their absence was not caused by the absence of aggregation per se. In gel-filtered platelets (GFP) incubated with various additions, the blockade or absence of GPIIb-IIIa resulted in reduced A23187-induced secretion and TxB2 formation in media containing 1 mmol/L Ca2+ with or without fibrinogen and 1 mmol/L Mg2+ plus fibrinogen, but not when Ca, Mg-free buffer alone, 1 mmol/L EDTA, or fibrinogen alone were present. In contrast, no such dependence or GPIIb- IIIa was seen in GFP stimulated with thrombin or phorbol myristate acetate in the presence of 1 mmol/L Ca2+, 1 mmol/L EDTA, or buffer alone. The inhibition of ionophore-induced responses seen in both normal GFP treated with antibodies and thrombasthenic GFP was not associated with any significant alteration of the ionophore-mediated [Ca2+]i increase, as measured in both aequorin-loaded GFP stimulated with A23187 and fura-2-loaded GFP stimulated with ionomycin. Incubation of normal GFP with either the monoclonal antibodies or the ligand binding site peptide RGDS in the presence of 1 mmol/L Ca2+ caused virtually complete inhibition of A23187-induced aggregation, measured as the loss of single platelets, but RGDS, in contrast to the antibodies, did not inhibit secretion or TxB2 formation. We conclude that platelet activation induced by ionophores in the presence, but not in the absence, of extracellular divalent cations involves a GPIIb-IIIa- dependent process that most likely involves a property of the ligand- occupied form of the complex distinct from its ability to support aggregation. This could represent another example of an aggregation- independent activity of the receptor-occupied state of the GPIIb-IIIa complex in signal transduction.     

Mechanisms involved in the antiplatelet activity of magnesium in human platelets

In this study, magnesium sulphate dose-dependently (0.6-3.0 mmol/l) inhibited platelet aggregation in human platelets stimulated by agonists. Furthermore, magnesium sulphate (3.0 mmol/l) markedly interfered with the binding of fluorescein isothiocanate-triflavin to the glycoprotein (GP)IIb/IIIa complex in platelets stimulated by collagen. Magnesium sulphate (1.5 and 3.0 mmol/l) also inhibited phosphoinositide breakdown and intracellular Ca+2 mobilization in human platelets stimulated by collagen. Magnesium sulphate (3.0 mmol/l) significantly inhibited thromboxane A2 formation stimulated by collagen in platelets. Moreover, magnesium sulphate (1.5 and 3.0 mmol/l) obviously increased the fluorescence of platelet membranes tagged with diphenylhexatriene. In addition, magnesium sulphate (1.5 and 3.0 mmol/l) increased the formation of cyclic adenosine monophosphate (AMP) in platelets. Phosphorylation of a protein of Mr 47 000 (P47) was markedly inhibited by magnesium sulphate (1.5 mmol/l). In conclusion, the antiplatelet activity of magnesium sulphate may involve the following two pathways. (1) Magnesium sulphate may initially induce membrane fluidity changes with resulting interference of fibrinogen binding to the GPIIb/IIIa complex, followed by inhibition of phosphoinositide breakdown and thromboxane A2 formation, thereby leading to inhibition of both intracellular Ca2+ mobilization and phosphorylation of P47. (2) Magnesium sulphate might also trigger the formation of cyclic AM, ultimately resulting in inhibition of the phosphorylation of P47 and intracellular Ca+2 mobilization.     

The central role of the P(2T) receptor in amplification of human platelet activation, aggregation, secretion and procoagulant activity

Adenosine diphosphate (ADP) is an important platelet agonist and ADP released from platelet dense granules amplifies responses to other agonists. There are three known subtypes of ADP receptor on platelets: P2X(1), P2Y(1) and P(2T) receptors. Sustained ADP-induced aggregation requires co-activation of P2Y(1) and P(2T) receptors. AR-C69931MX, a selective P(2T) receptor antagonist and novel antithrombotic agent, was studied to characterize further the function of the P(2T) receptor. The roles of the P2Y(1) receptor and thromboxane A(2) were assessed using the selective P2Y(1) antagonist A2P5P and aspirin respectively. Aggregation was measured by whole blood single-platelet counting and platelet-rich plasma turbidimetry, using hirudin anticoagulation. Dense granule release was estimated using ([14)C]-5-hydroxytryptamine (HT)-labelled platelets. Ca(2+) mobilization, P-selectin expression, Annexin V binding and microparticle formation were determined by flow cytometry. P(2T) receptor activation amplified ADP-induced aggregation initiated by the P2Y(1) receptor, as well as amplifying aggregation, secretion and procoagulant responses induced by other agonists, including U46619, thrombin receptor-activating peptide (TRAP) and collagen, independent of thromboxane A(2) synthesis, which played a more peripheral role. P(2T) receptor activation sustained elevated cytosolic Ca(2+) induced by other pathways. These studies indicate that the P(2T) receptor plays a central role in amplifying platelet responses and demonstrate the clinical potential of P(2T) receptor antagonists.     

Regulation of cytosolic PlA2 activity by PP1/PP2A serine/threonine phosphatases in human platelets

Platelet thromboxane A2 (TXA2) synthesis is an important pathway of platelet reactivity. We report that in thrombin-stimulated platelets, PP1/PP2A serine/threonine phosphatases regulate phospholipase A2 (cPLA2) activity, which is required for TXA2 synthesis. Two mechanisms are involved: (a) constitutively active PP1/PP2A regulate cPLA2 phosphorylation, and (b) PP1/PP2A activity mediates agonist-induced increase in cytosolic Ca2+ ([Ca2+]i). Inhibition of PP1/PP2A with okadaic acid (OA) induces cPLA2 phosphorylation but reduces Ca2+ responses: release from intracellular stores and influx through the plasma membrane, particularly that mediated by store-mediated Ca2+ entry (SMCE). A significant correlation (r = 0.64) exists between OA-regulated [Ca2+]i and TXA2 synthesis. Okadaic acid-induced decrease in SMCE and the associated TXA2 synthesis are mediated by a reduction in protein-tyrosine phosphorylation. This reduction is not due to inhibition of tyrosine kinases but rather to an OA-mediated increase in tyrosine phosphatases. This is the first study to report that PP1/PP2A phosphatases are involved in the regulation of the two key elements in eicosanoid synthesis, [Ca2+]i and cPLA2 phosphorylation. Moreover, PP1/PP2A regulation of [Ca2+]i and tyrosine phosphorylation may be important for other calcium-dependent processes and/or signal transduction mechanisms in platelets.     

Inhibitory mechanism of human platelet aggregation by nafamostat mesilate

We found that nafamostat mesilate (NM) inhibits platelet aggregation induced by all agonists tested, including ADP, collagen, arachidonic acid, thromboxane A analog, A23187, phorbol 12-myristate 13-acetate (PMA), NaF and thrombin. The IC50 values were in the range of 9.3-17.8 microM. NM inhibited agonists-induced aspirin-treated platelet aggregation at >10 microM, suggesting that the action site lies beyond thromboxane (TXA)2 formation. However, NM inhibited thrombin (0.5 IU/ml)-induced TXB2 formation (IC50 = 1.9 +/- 0.6 microM, mean +/- SD). Intracellular Ca2+ mobilization was also inhibited only when platelets were challenged by thrombin, but the effect was found at NM concentrations >50 microM. This finding suggests that NM reduces the responses to thrombin by inhibiting its proteolytic activity on the platelet thrombin receptor (PAR1). NM did not affect the intracellular cAMP concentration or A-kinase activity. Agonists-induced surface expression of activated glycoprotein (GP)IIb-IIIa was inhibited by 10 microM NM and was completely inhibited by 50 microM NM. Since this inhibitory effect was parallel to the inhibition of platelet aggregation, the main inhibitory mechanism of NM against platelet aggregation seemed to be the suppression of activated GPIIb-IIIa expression, which makes it able to bind fibrinogen.     

Inhibitory effects of endothelial cells and calcium channel blockers on platelet aggregation

This study examined the effects of human umbilical vein endothelial cells (ECs) and calcium channel blockers (Ca2+ blockers), diltiazem, verapamil, and nicardipine, on platelet aggregation in vitro. ECs markedly inhibited the platelet aggregation induced by ADP, collagen, thromboxane A2 (TXA2), or thrombin. When platelets were incubated with ECs, the antiaggregatory activity reached a plateau within 5 to 10 min. As the number of ECs added to the platelet-rich plasma was increased, platelet aggregation declined progressively. The antiaggregatory activity of ECs was attenuated considerably by the addition of aspirin. All three Ca2+ blockers inhibited platelet aggregation in a dose-dependent manner. The combination of ECs and a Ca2+ blocker resulted in more potent inhibition of platelet aggregation than either alone, but the effects were not synergistic. Both ECs and Ca2+ blockers inhibited the synthesis of TXA2 during platelet aggregation. However, Ca2+ blockers did not significantly influence the production of prostaglandin I2 (PGI2) by ECs during incubation and aggregation. These results suggest that PGI2 is an important factor in endothelial antiaggregatory activity. Ca2+ blockers directly inhibit platelet aggregation by suppressing TXA2 formation, but do not appear to enhance the antiaggregatory activity of ECs.     

Thromboxane A2/prostaglandin endoperoxide (TXA2/PG-END) receptor binding properties in human platelets of ridogrel, a combined TXA2 synthase inhibitor--TXA2/PG-END receptor antagonist.

Ridogrel, a potent thromboxane A2 (TXA2) synthase inhibitor, also has thromboxane A2 prostaglandin endoperoxide (TXA2/PG-END) receptor antagonistic properties as documented in functional studies of human platelets. In the present study, the binding affinities of the TXA2 synthase inhibitors, ridogrel, dazoxiben, dazmegrel and pirmagrel, and the TXA2/PG-END receptor antagonists, GR32191, L670596, SQ29548, ICI159995, AH69212 and sulotroban, for the TXA2/PG-END receptor labelled with [3H]SQ29548 on intact human platelets were assessed. The potencies of the TXA2/PG-END receptor antagonists to inhibit specific [3H]SQ29548 binding to intact human platelets ranged between 1.2 nM and 6,200 nM and corresponded to the ability of the drugs to suppress human platelet aggregation induced by TXA2/PG-END receptor stimulation with U46619 and collagen. The TXA2 synthase inhibitors dazoxiben, dazmegrel and pirmagrel could not inhibit specific [3H]SQ29548 binding to intact human platelets, tested up to 10(-5) M, nor suppress human platelet aggregation, indicating lack of any receptor antagonistic properties. Ridogrel, however, directly bound to the TXA2/PG-END receptor with micromolar affinity (IC50 = 5.2 microM) and inhibited U46619-27, or collagen-induced platelet aggregation, with ED50-values of 27 microM and 4.7 microM respectively. The present study thus demonstrates that antagonism by ridogrel of TXA2/PG-END receptor activation on platelets as defined in functional tests, coincides with inhibition of specific ligand binding to the receptors.     

Sodium fluoride mimics effects of both agonists and antagonists on intact human platelets by simultaneous modulation of phospholipase C and adenylate cyclase activity

Using intact human platelets, we studied the effect of sodium fluoride (NaF) on platelet aggregation and release reaction and correlated the functional changes to intracellular events specific for either agonist- induced or antagonist-induced platelet responses. At lower concentrations, with a peak activity between 30 and 40 mmol/L, NaF induced aggregation and release of adenosine 5'-triphosphate (ATP) that was associated with increased formation of inositol phosphates, a rise in cytosolic free Ca2+, and phosphorylation of 20-kd and 40-kd proteins. At NaF concentrations greater than 40 mmol/L, aggregation and ATP release decreased dose-dependently in parallel with a decrease in Ca2+ mobilization, whereas neither inositol phosphate formation nor 40- kd protein phosphorylation was reduced. At these concentrations, NaF caused a dose-dependent transient rise in platelet cyclic adenosine 3',5'-monophosphate (cAMP) levels that was sufficient to account for the observed reduction in Ca2+ mobilization, aggregation, and ATP release. Stimulated cAMP levels started declining rapidly within 30 seconds of addition of NaF, however. Similarly, prostacyclin (PGI2)- induced cAMP accumulation was temporarily enhanced but subsequently suppressed by NaF, suggesting either stimulation of a cAMP phosphodiesterase or delayed inhibition of adenylate cyclase. Evidence for the latter was provided by the finding that NaF pretreatment of platelets resulted in partial inhibition of PGI2-stimulated cAMP formation in the presence of the cAMP phosphodiesterase inhibitor 3- isobutyl-1-methyl-xanthine (MIX). We conclude that NaF exerts a dual (stimulatory and inhibitory) effect on adenylate cyclase in intact platelets that is accompanied by simultaneous activation of a phosphoinositide-specific phospholipase C; in addition, a cAMP phosphodiesterase may be activated.     

Rabbit and rat platelets do not respond to thrombin receptor peptides that activate human platelets

Human platelets are aggregated and induced to release their granule contents and form thromboxane by peptides as short as 6-amino acid residues (SFLLRN) corresponding to the newly released N-terminus of the thrombin receptor that is cleaved by thrombin. Using washed platelets, we found that these responses to SFLLRN (2 to 6 mumol/L) were enhanced by fibrinogen. However, neither SFLLRN nor SFLLRNPNDKYEPF had any effect on washed rabbit or rat platelets, although they were fully responsive to human thrombin. Concentrations of the peptides as high as 100 mumol/L did not cause the platelets of rabbits or rats to change shape, aggregate, release granule contents, or form thromboxane. SFLLRN did not affect the extent of aggregation induced by adenosine diphosphate (ADP) or a low concentration of thrombin. Pig platelets responded to 50 mumol/L SFLLRN with reversible aggregation, which was enhanced by fibrinogen, but not accompanied by the release of dense granule contents. Guinea pig platelets aggregated and released granule contents in response to 25 or 50 mumol/L of SFLLRN, but responded with only shape change to lower concentrations. Thus, these experiments indicate that rabbit and rat platelets lack a functional response to human thrombin receptor peptides that fully activate the previously described human thrombin receptor, despite a full response of both rabbit and rat platelets to human thrombin, and that pig and guinea pig platelets have incomplete responses to these human thrombin receptor peptides. The results suggest that platelets of rabbits and rats, and perhaps guinea pigs and pigs, respond to thrombin through an alternative receptor that has also been suggested to be present on human platelets.     

Effect of losartan on human platelet activation by thromboxane A2

INTRODUCTION AND OBJECTIVES: Previous studies have demonstrated that losartan, an AT-1 receptor antagonist of angiotensin II (Ang II) could block the receptor of thromboxane A2 (TXA2) in the vascular wall. The aim of the present study was to assess the effect of losartan on human platelet activation. MATERIALS AND METHODS: Platelets were obtained from 15 healthy men between the age 26 and 40. Platelet activation was measured by changes in the light transmission of platelet-rich plasma stimulated by a synthetic TXA2 analogue, U46619 (5 x 10(-6) mol/l). RESULTS: The U46619-stimulated platelet aggregation was significantly inhibited by losartan in a dose-response manner. Only a high dose of EXP 3174 (5 10-5 mol/l), the in vivo active metabolite of losartan, was able to attenuate U46619-induced platelet activation. Captopril, an angiotensin I-converting inhibitor failed to modify U46619-induced platelet aggregation. Despite the platelets expressing AT-1 type receptors, of Ang II exogenous Ang II did not modify platelet aggregation induced by U46619. The binding of U46619 to platelets was competitively inhibited by losartan in dose-dependent manner. However, only a high dose of EXP 3174 reduced the binding of U46619. Captopril failed to modify the binding of U46619 to platelets. CONCLUSIONS: Losartan decreased platelet aggregation by a TXA2-dependent mechanism. EXP 3174 showed a lesser potency than losartan to reduce TXA2-platelet activation. Captopril and exogenous angiotensin II had no effect on human platelet activation. These results suggest that losartan reduced TXA2-dependent platelet activation independently of the blockade of AT-1 receptors.     

Platelet receptors for adenine nucleotides and thromboxane A2

Adenosine diphosphate (ADP) and thromboxane A (2) (TXA (2)) are important physiological activators of platelets and exert their effects by acting on cell surface receptors. Platelet nucleotide receptors can be distinguished as three separate subtypes of the P2 receptor family. The P2X (1) receptor is a ligand-gated adenosine triphosphate (ATP) receptor that was originally mistaken for an ADP receptor. This calcium-influx-causing receptor mediates platelet shape change and plays an important role in thrombus formation in small arterioles. The P2Y (1) receptor, through activation of G (q) and phospholipase C, is required for ADP-induced platelet shape change, fibrinogen receptor activation, and TXA (2) generation. The G (i)-coupled P2Y (12) receptor plays an important role in platelet aggregation, potentiation of dense granule release, and TXA (2) generation. Both the P2Y receptors are crucial for in vivo thrombus formation. TXA (2) stimulates two subtypes of G protein-coupled TP receptor, TPalpha and TPbeta, but its effects in platelets are mediated predominantly through the alpha isoform. Although interference with the activation of G protein-coupled ADP or TP receptors results in increased bleeding times and protection from thromboembolism, TP receptor antagonists did not translate into effective antiplatelet drugs. Blockade of ADP receptor is a mode of newer classes of antithrombotic drugs in the coming era. This review focuses on the contribution of different nucleotide receptors and TP receptors to platelet function and their potential as antithrombotic agents.     

Inhibitory mechanism of human platelet aggregation by nafamostat mesilate

We found that nafamostat mesilate (NM) inhibits platelet aggregation induced by all agonists tested, including ADP, collagen, arachidonic acid, thromboxane A analog, A23187, phorbol 12-myrisate 13-acetate (PMA), NaF and thrombin. The IC50 values were in the range of 9.3-17.8 mu M. NM inhibited agonists-induced aspirin-treated platelet aggregation at >10 mu M, suggesting that the action site lies beyond thromboxane (TXA)2 formation. However, NM inhibited thrombin (0.5 IU/ml)-induced TXB2 formation (IC50 = 1.9 +/- 0.6 mu M, mean +/- SD). Intracellular Ca2+ mobilization was also inhibited only when platelets were challenged by thrombin, but the effect was found at NM concentrations >50 mu M. This finding suggests that NM reduces the responses to thrombin by inhibiting its proteolytic activity on the platelet thrombin receptor (PAR1). NM did not affect the intracellular cAMP concentration or A-kinase activity. Agonists-induced surface expression of activated glycoprotein (GP)IIb-IIIa was inhibited by 10 mu M NM and was completely inhibited by 50 mu M NM. Since this inhibitory effect was parallel to the inhibition of platelet aggregation, the main inhibitory mechanism of NM against platelet aggregation seemed to be the suppression of activated GPIIb-IIIa expression, which makes it able to bind fibrinogen.