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.     

Subsecond calcium dynamics in ADP- and thrombin-stimulated platelets: a continuous-flow approach using indo-1

The regulation and kinetics (less than 5 seconds) of cytosolic calcium changes ([Ca2+]i) in stimulated blood platelets have been investigated under physiological blood flow conditions. Using a newly-developed continuous-flow approach with indo-1-loaded human platelets, adenosine diphosphate (ADP, 10 mumol/L) and thrombin (5 U/mL) were equally effective in significantly increasing [Ca2+]i by 0.5 seconds. ADP induced a transient [Ca2+]i peak of 1 to 2 mumol/L near 2 seconds, whereas thrombin caused a sustained and larger response. The first phase (less than 2 seconds) was not influenced by a lack of extracellular Ca2+, in contrast to the subsequent [Ca2+]i increase that only reached about 0.7 mumol/L for either ADP or thrombin. The shear rates used in our continuous-flow apparatus were physiological (less than 1,258 sec-1) and only slightly increased the basal [Ca2+]i of 0.1 mumol/L. Platelet aggregation (less than 5 seconds), assessed by single- particle counting, was not altered in platelets loaded with indo-1/AM (2.5 mumol/L).     

Platelet secretion defect associated with impaired liberation of arachidonic acid and normal myosin light chain phosphorylation

We describe four patients with impaired platelet aggregation and 14C- serotonin secretion during stimulation with adenosine diphosphate (ADP), epinephrine, collagen, and platelet-activating factor. The response to arachidonic acid was normal in all patients with regard to aggregation and in three of the four with regard to 14C-serotonin secretion. The total platelet adenosine triphosphate (ATP) and ADP content and the ATP to ADP ratio was normal in all patients, thereby excluding storage pool deficiency as the cause of the secretion defect. Studies with 3H-arachidonic acid-labeled platelets revealed that the thrombin-induced liberation of arachidonic acid from membrane-bound phospholipids was impaired in these patients. Further, platelet thromboxane B2 production, measured using a radioimmunoassay, was diminished during stimulation with ADP and thrombin, but was normal with arachidonic acid, indicating that the oxygenation of arachidonic acid was normal and that the diminished thromboxane production was due to a defect in the liberation of arachidonic acid. Release of arachidonic acid is mediated by phospholipases that are Ca++ dependent. To examine whether these patients may have a defect in making intracellular Ca++ available, another Ca++-dependent process, myosin light chain phosphorylation, was studied during thrombin stimulation. Platelets from three of the patients were found to behave the same as normal ones, suggesting that the deficiency in phospholipase activity may not be due to impaired Ca++ mobilization. Our studies demonstrate a novel group of patients with platelet secretion defects associated with impaired liberation of arachidonic acid from phospholipids. These patients exemplify a congenital defect, other than deficiencies of cyclooxygenase and thromboxane synthetase, by which thromboxane production may be impaired in platelets.     

Prostaglandin E2 potentiates platelet aggregation by priming protein kinase C

Prostaglandin E2 (PGE2) is produced by activated platelets and by several other cells, including capillary endothelial cells. PGE2 exerts a dual effect on platelet aggregation: inhibitory, at high, supraphysiologic concentrations, and potentiating, at low concentrations. No information exists on the biochemical mechanisms through which PGE2 exerts its proaggregatory effect on human platelets. We have evaluated the activity of PGE2 on human platelets and have analyzed the second messenger pathways involved. PGE2 (5 to 500 nmol/L) significantly enhanced aggregation induced by subthreshold concentrations of U46619, thrombin, adenosine diphosphate (ADP), and phorbol 12-myristate 13-acetate (PMA) without simultaneously increasing calcium transients. At a high concentration (50 mumol/L), PGE2 inhibited both aggregation and calcium movements. PGE2 (5 to 500 nmol/L) significantly enhanced secretion of beta-thromboglobulin (beta TG) and adenosine triphosphate from U46619- and ADP-stimulated platelets, but it did not affect platelet shape change. PGE2 also increased the binding of radiolabeled fibrinogen to the platelet surface and increased the phosphorylation of the 47-kD protein in 32P- labeled platelets stimulated with subthreshold doses of U46619. Finally, the amplification of U46619-induced aggregation by PGE2 (500 nmol/L) was abolished by four different protein kinase C (PKC) inhibitors (calphostin C, staurosporine, H7, and TMB8). Our results suggest that PGE2 exerts its facilitating activity on agonist-induced platelet activation by priming PKC to activation by other agonists. PGE2 potentiates platelet activation at concentrations produced by activated platelets and may thus be of pathophysiologic relevance.     

Comparison of the relative activities of inducing platelet apoptosis stimulated by various platelet-activating agents

Apoptosis-like events are known to occur in anuclear platelets. Although the mechanisms responsible for these events are still not completely understood, studies suggested that some platelet agonists can activate platelet apoptosis. However, the relative activities of various platelet agonists in inducing apoptosis have not yet been investigated. In the present study we explored this issue, and attempted to identify the correlation between platelet activation and apoptosis. In a platelet aggregation study, there were no significant differences respectively stimulated by arachidonic acid (AA; 100 µM), ADP (20 µM), collagen (10 µg/mL), thrombin (0.1 U/mL), U46619 (10 µM), and A23187 (5 µM). In a subsequent study, we fixed these concentrations of agonists to further compare their relative activities in inducing platelet apoptosis. Our results found that thrombin, U46619, and A23187 possess stronger activities than the other agonists in inducing platelet apoptosis (i.e., phosphatidylserine exposure, mitochondrial membrane potential depolarization, eukaryotic initiation factor (eIF)2α, and caspase activation). On the other hand, AA induced no apoptotic events in platelets. Based on this approach, we demonstrated for the first time that thrombin, U46619, and A23187, but not AA, possess stronger activity in inducing platelet apoptosis. In addition, we also found that platelet activation might not necessarily be associated with the occurrence of platelet apoptosis. The in vivo physiological function of the apoptotic machinery in platelets is not yet clearly understood, and needs to be further investigated in the future.     

High molecular weight kininogen inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets.

In this study we show that high molecular weight kininogen (HK) inhibited alpha-thrombin-induced aggregation of human platelets in a dose-dependent manner with complete inhibition occurring at plasma concentration (0.67 mumol/L) of HK. HK (0.67 mumol/L) also completely inhibited thrombin-induced cleavage of aggregin (Mr = 100 Kd), a surface membrane protein that mediates adenosine diphosphate (ADP)-induced shape change, aggregation, and fibrinogen binding. The inhibition of HK was specific for alpha- and gamma-thrombin-induced platelet aggregation, because HK did not inhibit platelet aggregation induced by ADP, collagen, calcium ionophore (A23187), phorbol myristate acetate (PMA), PMA + A23187, or 9,11-methano derivative of prostaglandin H2 (U46619). These effects were explained by the ability of HK, at physiologic concentration, to completely inhibit binding of 125I-alpha-thrombin to washed platelets. As a result of this action of HK, this plasma protein also completely inhibited thrombin-induced secretion of adenosine triphosphate, blocked intracellular rise in Ca2+ in platelets exposed to alpha- and gamma-thrombin, inhibited thrombin-induced platelet shape change, and blocked the ability of thrombin to antagonize the increase in intracellular cyclic adenosine monophosphate (cAMP) levels induced by iloprost. Because elevation of cAMP is known to inhibit binding of thrombin to platelets, we established that HK did not increase the intracellular concentration of platelet cAMP. Finally, HK did not inhibit enzymatic activity of thrombin. To study the role of HK in the plasma environment, we used gamma-thrombin to avoid fibrin formation by alpha-thrombin. Platelet aggregation induced by gamma-thrombin was also inhibited by HK in a dose-dependent manner. The EC50 (concentration to produce 50% of the maximum rate of aggregation) of gamma-thrombin for washed platelets was 7 nmol/L and increased to 102 nmol/L when platelets were suspended in normal human plasma. The EC50 for platelet aggregation induced by alpha-thrombin in plasma deficient in total kininogen was 40 nmol/L. When supplemented with HK at plasma concentration (0.67 mumol/L), the EC50 increased to 90 nmol/L, a value similar to that for normal human plasma. These results indicate that (1) HK inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets; (2) HK is a specific inhibitor of platelet aggregation induced by alpha- and gamma-thrombin; and (3) HK plays a role in modulating platelet aggregation stimulated by alpha-thrombin in plasma.     

The role of ADP secretion and thromboxane synthesis in factor VIII binding to platelets

Following stimulation with arachidonic acid, collagen, U-46619 (a stable analogue of prostaglandin endoperoxide/thromboxane-A2), thrombin, or adenosine diphosphate (ADP), unstirred human platelet suspensions bound labeled factor VIII in a reaction that reached equilibrium within 10 min. Apyrase inhibited binding induced by arachidonic acid, collagen, U-46619, and thrombin by less than 40%, but inhibited ADP-induced binding by 95%. Binding to aspirin-treated platelets was normal in response to U-46619, reduced by 60%-70% in response to ADP, collagen, and thrombin, and absent in response to arachidonic acid. Binding in response to U-46619 was not altered by the combination of apyrase and aspirin. Binding of factor VIII was decreased by 90% when 10 mM EDTA was added before each agonist, but it was inhibited less than 30% when EDTA was added following platelet stimulation. We conclude that arachidonic acid, collagen, and thrombin can expose binding sites for factor VIII independently of released ADP; that Ca++ is required for activation but probably not for binding of factor VIII to platelets; and that platelet thromboxane synthesis plays a major role in the binding of factor VIII to platelets induced by thrombin, ADP, or collagen.     

Differential requirements for platelet aggregation and inhibition of adenylate cyclase by epinephrine. Studies of a familial platelet alpha 2-adrenergic receptor defect

We describe a family whose members have impaired platelet aggregation and secretion responses to epinephrine with normal responses to adenosine diphosphate and collagen. Platelet alpha 2-adrenergic receptors (measured using 3H methyl-yohimbine) were diminished in the propositus (78 sites per platelet), his two sisters (70 and 27 sites per platelet), and parents (37 and 63 sites per platelet), but not in two maternal aunts (12 normal subjects, 214 +/- 18 sites per platelet; mean +/- SE). However, the inhibition of cyclic adenosine monophosphate (cAMP) levels by epinephrine in platelets exposed to 400 nmol/L PGI2 was similar in the patients and five normal subjects (epinephrine concentration for 50% inhibition, 0.04 +/- 0.01 mumol/L v 0.03 +/- 0.01 mumol/L; P greater than .05). In normal platelets, the concentration of yohimbine (0.18 mumol/L) required for half maximal inhibition of aggregation induced by 2 mumol/L epinephrine was lower than that for inhibition of its effect on adenylate cyclase (1.6 mumol/L). In quin2 loaded platelets, thrombin (0.1 U/mL) stimulated rise in cytoplasmic Ca2+ concentration, [Ca2+]i, was normal in the two patients studied. The PGI2 analog ZK 36,374 completely inhibited thrombin-induced rise in [Ca2+]i; the reversal of this inhibition by epinephrine was normal in the two patients. Thus, despite the impaired aggregation response to epinephrine, platelets from these patients have normal ability to inhibit PGI2-stimulated cAMP levels. These patients with an inherited receptor defect provide evidence that fewer platelet alpha 2-adrenergic receptors are required for epinephrine-induced inhibition of adenylate cyclase than for aggregation.     

Modulation of platelet and leucocyte function by a Chinese herbal formulation as compared with conventional antiplatelet agents

Platelet and leucocyte activity are important in the acute development of thrombosis and in the pathogenesis of ischaemic vascular disease. Dan Shen Di Wan (DS, Cardiotonic Pill or Dantonic(R) Pill) is one of the most commonly used Chinese herbal formulations for treating patients with atherosclerotic disease in China and several Asian countries. We studied the effect of DS on platelet and leucocyte function and compared the effects with conventional antiplatelet agents, cangrelor (ADP P2Y(12) receptor antagonist) and aspirin (acetyl salicylic acid, ASA). Measurements were made by platelet aggregation (%) and activation (CD62P %), platelet-monocyte conjugate formation (P/M, CD42a median fluorescence, mf), platelet-neutrophil conjugate formation (P/N, mf), and leucocyte activation (CD11b median fluorescence on monocytes and neutrophils, mf) in response to 3.3 micromol/L adenosine diphosphate (ADP), 1.0 micromol/L platelet activating factor (PAF), 5.0 micromol/L adrenaline and 0.5 microg/mL collagen. We also evaluated the effect of its main component, water soluble extract of salvia miltiorrhiza (SME) on intracellular calcium mobilization in platelets triggered by 10 micromol/L ADP, 10 micromol/L PAF, 2 microg/mL collagen and 15 micromol/L thrombin receptor activating peptide (TRAP). Overall DS showed inhibition of platelet aggregation, platelet activation, platelet-leucocyte conjugate formation and leucocyte activation in response to all the agonists apart from adrenaline (all p < 0.01). DS showed inhibition of platelet aggregation and leucocyte activation equivalent to cangrelor 100 nmol/L and ASA 100 micromol/L. SME dose-dependently inhibited intracellular calcium mobilization in platelets following stimulation with all the platelet agonists with maximum effective at 0.36 mg/mL (all p < 0.01). When used at 0.18 mg/mL its inhibitory effect was equivalent to cangrelor and ASA. We conclude that DS is a potential inhibitor of both platelet and leucocyte activation.     

Abnormal inside-out signal transduction-dependent activation of glycoprotein IIb-IIIa in a patient with impaired pleckstrin phosphorylation

Platelet-agonist interaction results in activation of glycoprotein (GP) IIb-IIIa complex and fibrinogen binding, a prerequisite for platelet aggregation. Fibrinogen binding exposes new antibody binding sites on GPIIb-IIIa (ligand-induced binding sites: LIBS). Signal transduction events, including pleckstrin phosphorylation by protein kinase C (PKC), are considered to regulate GPIIb-IIIa activation. We studied a 16-year- old white male with lifelong mucocutaneous bleeding manifestations and abnormal platelet aggregation and secretion in response to multiple agonists. Pleckstrin phosphorylation was diminished in response to platelet-activating factor (PAF; 4 and 400 nmol/L) and thrombin (0.05 U/mL). Binding of monoclonal antibodies (MoAbs) 10E5 and A2A9, which bind to both resting and activated GPIIb-IIIa, was normal. Binding of MoAb PAC1, which binds to only activated GPIIb-IIIa, was diminished upon activation with PAF, adenosine diphosphate (ADP), thrombin receptor agonist peptide (SFLLRN), A23187, and 1,2-dioctonylglycerol (DiC8). Signal transduction-dependent LIBS expression (studied using MoAb 62) induced by ADP, SFLLRN, and DiC8 and signal transduction- independent LIBS expression induced by RGDS peptide or disintegrin albolabrin were normal or minimally decreased, indicating the presence of intact ligand binding sites. We conclude that the patient's platelets have a defect in inside-out signal transduction-dependent GPIIb-IIIa activation due to an upstream defect in the signal transduction mechanisms rather than in the GPIIb-IIIa complex itself. Our findings extend the spectrum of congenital mechanisms leading to impaired aggregation from defects in GPIIb-IIIa per se to aberrations in signaling mechanisms.     

Removal of extraplatelet Na+ eliminates indomethacin-sensitive secretion from human platelets stimulated by epinephrine, ADP, and thrombin.

We have previously observed that removal of extraplatelet Na+ markedly diminishes human platelet aggregation and secretion in response to epinephrine. The present studies demonstrate that this effect of the removal of extraplatelet Na+ on platelet function is not unique to activation of platelets by alpha 2-adrenergic agents but represents a phenomenon also evident for other platelet stimuli. Thus, platelet aggregation and secretion in response to maximal concentrations of ADP and lower concentrations of thrombin (less than 0.04 unit/ml) were also markedly reduced in platelets in "Na+-free" medium, suggesting that these agents share an effector mechanism that is similarly inhibited by the removal of extraplatelet Na+. In contrast, platelet aggregation and secretion in response to higher concentrations of thrombin (greater than or equal to 0.04 unit/ml) and to 0.04-1.0 microM (15S)-hydroxy-11 alpha, 9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619), an endoperoxide analog, were identical in control platelets and in those suspended in "Na+-free" medium, indicating that platelets suspended in "Na+-free" medium are functionally intact, at least in response to some stimuli. Furthermore, the observation that U46619 can elicit platelet aggregation and secretion independently of extraplatelet Na+ indicates that the loss of platelet responsiveness to epinephrine, ADP, and low concentrations of thrombin cannot be attributed to a loss of sensitivity to the stimulus-provoked secondary mediator(s) of platelet function, endoperoxides or thromboxane A2. Treatment with indomethacin to block the secondary aggregation and secretion pathways of platelets reduced the aggregatory and secretory responses of control platelets induced by epinephrine, ADP, and low concentrations of thrombin to those characteristic of platelets suspended in "Na+-free" medium. In contrast, indomethacin did not alter the functional responses induced by these agents in platelets suspended in "Na+-free" medium, suggesting that "primary" aggregation is intact but that the "secondary" aggregation and secretion mediated by arachidonic acid metabolites are eliminated by removal of extraplatelet Na+. Consistent with this interpretation is the observation that the indomethacin-insensitive aggregation and secretion induced by U46619 and higher concentrations of thrombin were retained in platelets suspended in "Na+-free" medium. Thus, the responses eliminated by removal of extraplatelet Na+ are those eliminated by treating control platelets with indomethacin, suggesting a strong link between the presence of extraplatelet Na+ and the operation of platelet function mediated by the cyclooxygenase pathway.     

Original Article: Intracellular Ca(2+) Mobilization and Aggregatory Response to ADP and Thrombin in Aged Rat Platelets

We previously reported that thrombin-induced Ca(2+) mobilization was enhanced in aged rat platelets. Since Ca(2+) mobilization in platelets is believed to be closely associated with platelet activation, we examined Ca(2+) mobilization and the aggregatory response to ADP and thrombin in young (3 months) and aged (24 months) rat platelets. Blood levels of fibrinogen and Ca(2+) in aged rats were higher than those in young rats. ADP-induced platelet aggregation in aged rats was significantly enhanced in platelet rich plasma and in washed platelet suspension, suggesting that age-associated hyperaggregability to ADP is attributable to changes in platelets themselves. On the other hand, thrombin (0.03-0.3 unit/ml)-induced aggregation in washed platelet suspension from aged rats was comparable to that from young rats. But, thrombin (0.3 unit/ml)-induced intracellular Ca(2+)mobilization was enhanced in aged rat platelets in the presence of extracellular Ca(2+). Likewise, ADP-induced Ca(2+) mobilization was enhanced in aged rat platelets. These results suggest that enhanced Ca(2+) mobilization in aged rat platelets is associated with hyperaggregability to ADP but not to thrombin.     

Modulation of thrombin-mediated activation of factor VIII:C by calcium ions, phospholipid, and platelets

The activation of factor VIII:C by thrombin appears to be an important prerequisite for the function of factor VIII:C as a cofactor in factor X activation in coagulation. The possible modulation of factor VIII:C activation by potential cofactors such as calcium ions, phospholipid, and platelets was studied systematically. Factor VIII:C activation could not be studied in the complete absence of Ca2+, since factor VIII:C activity decayed rapidly in calcium-free buffers, EDTA, or ethylene glycol tetra-acetic acid (EGTA), with only partial or no recovery of activity after readdition of Ca2+, Mn2+, or Mg2+. Added calcium chloride at 1.25, 2.5, 4, 10, 50, and 200 mmol/L produced progressive inhibition of factor VIII:C activation, with complete inhibition achieved by 50 mmol/L. Crude phospholipid preparations gave varying results, while purified phospholipids either had no effect or inhibited activation. This paper reports the new finding that fresh washed human platelets markedly potentiated factor VIII:C activation by a low concentration of thrombin (0.02 U/mL), even with prostaglandin E1 (PGE1) or dibutyryl cyclic AMP (cAMP) added to the washed platelets. However, the activity of platelets in factor VIII:C activation was inhibited by inclusion of PGE1 or dibutyryl cAMP during platelet washing, and ionophore A23187 increased this platelet activity; these data suggest that platelet stimulation is involved in the development of this activity. When platelets were maximally stimulated by thrombin (0.5 U/mL), the external calcium concentration increased 55 to 160 mumol/L, as measured with murexide, supporting the possible modulation of factor VIII:C activation by a transient increase in Ca2+ at the platelet surface.     

Glycoprotein IIb-IIIa complex and Ca2+ influx into stimulated platelets

Changes in intracellular Ca2+ concentrations [( Ca2+]i) in platelets stimulated with aggregating agents were measured with the fluorescent indicator dye quin 2. Ca2+ influx, but not intracellular mobilization, in response to adenosine diphosphate (ADP), platelet aggregating factor (PAF-acether), and sodium arachidonate was significantly inhibited by monoclonal antibodies against the glycoprotein (GP) IIb-IIIa complex; inhibition of thrombin-stimulated influx was inhibited to a lesser extent and reached statistical significance only at thrombin concentrations of 0.1 U/mL and below. Anti-GP Ib and HLA-ABC monoclonal antibodies had no effect on Ca2+ influx in response to any agonist. Thrombasthenic platelets gave normal [Ca2+]i responses to ADP and thrombin, which were not inhibited by an anti-GP IIb-IIIa antibody. It is suggested that Ca2+ influx in response to weak agonists occurs predominantly via a channel closely adjacent to the GP IIb-IIIa complex, but that higher concentrations of thrombin and A23187 also stimulate influx via another pathway.     

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)     

ADP-induced platelet shape change and mobilization of cytoplasmic ionized calcium are mediated by distinct binding sites on platelets: 5'- p-fluorosulfonylbenzoyladenosine is a weak platelet agonist

Platelet stimulation with ADP results in several responses, including shape change, increase in cytoplasmic ionized calcium concentration [Ca2+]i, an inhibition of adenylate cyclase. 5'-p-Fluorosulphonyl benzoyladenosine (FSBA), which covalently labels an ADP binding site on platelets, blocks platelet shape change but not the inhibition of cyclic AMP levels by ADP, whereas p-chloromercuribenzenesulfonate (pCMBS), a nonpenetrating thiol reagent, has the opposite effects. We examined the effect of FSBA and pCMBS on ADP-induced increase in [Ca2+]i using platelets loaded with fluorescent Ca2+ indicators quin2 and fura-2. FSBA (50 to 200 mumol/L) induced a dose-dependent rise in [Ca2+]i, indicating that it is a weak platelet agonist. Under conditions of covalent labeling of the ADP binding sites, FSBA (50 to 100 mumol/L) did not inhibit the ADP-induced increase in [Ca2+]i or its inhibition of adenylate cyclase, whereas pCMBS (up to 1 mmol/L) abolished both these responses but not shape change. These findings suggest that ADP-induced Ca2+ mobilization and inhibition of adenylate cyclase are mediated by platelet binding sites distinct from those mediating shape change.     

Identification of high-affinity (Kd 0.35 mumol/L) and low-affinity (Kd 7.9 mumol/L) platelet binding sites for ADP and competition by ADP analogues

Steady-state binding of ADP to blood platelets and isolated membranes has not previously been obtained because of complications arising from metabolism of the ligand and dilution due to its secretion from storage granules. In the present studies, competition binding isotherms (n = 9) using paraformaldehyde-fixed platelets showed that [2-3 H]ADP bound to two sites with a small amount (approximately 5% of total) of nonspecific binding: 410,000 +/- 40,000 sites of low affinity (Kd 7.9 +/- 2.0 mumol/L) and 160,000 +/- 20,000 sites of high affinity (Kd 0.35 +/- 0.04 mumol/L) corresponding to the ADP concentration required for activation in fresh platelets (0.1-0.5 mumol/L). All agonists and antagonists examined were able to compete with ADP at the high-affinity site. The strong platelet agonists 2-methylthio ADP and 2-(3- aminopropylthio)ADP competed with ADP at the high-affinity site with dissociation constant values of 7 mumol/L and 200 mumol/L, respectively. The partial agonist 2',3'-dialdehyde ADP and the weak agonist GDP also competed at the high-affinity site with Kd values of 5 mumol/L and 49 mumol/L, respectively. The sequence of binding affinities of other adenine nucleotides at the high-affinity site corresponded to their relative activities as known antagonists of platelet activation by ADP; namely, ADP(Kd 0.35 mumol/L) approximately equal to ATP (Kd 0.45 mumol/L) much greater than AMP (Kd 360 mumol/L). Adenosine and 2-chloroadenosine did not compete with ADP. ADP binding to the high-affinity site was inhibited by p-mercuribenzene sulfonate (Ki 250 mumol/L) but only very weakly by 5'-p- fluorosulfonylbenzoyladenosine (Ki 1 mmol/L). All the above nucleotides also competed with ADP at the low-affinity sites but, because of the high concentrations of competing nucleotide required, dissociation constants at this site were obtained only for ATP (21 mumol/L), 2-MeS ADP (200 mumol/L) and 2',3'-dialdehyde ADP (270 mumol/L). 8-Bromo ADP competed strongly with ADP at the high-affinity site (Kd 0.40 mumol/L) but weakly if at all at the low-affinity site. 8-Bromo ADP inhibited platelet activation induced by ADP (EC50 approximately 100 mumol/L) but not by collagen, thrombin, or ionophore A23187.(ABSTRACT TRUNCATED AT 400 WORDS)     

Defective platelet aggregation in polycythaemia vera is not caused by impaired calcium signaling, phospholipase D activation or decreased amounts of focal adhesion proteins

We have previously demonstrated that platelets in polycythaemia vera (PV) exhibit decreased aggregation after stimulation with platelet activating factor (PAF) and reduced expression of GPIIIa on both resting and stimulated platelets. In the present study, we investigated if these results were related to changes in the mobilization of intracellular calcium, activation of phospholipase D (PLD) or amounts of GPIIIa and the intracellular tyrosine kinases Fak, Syk, Grb2, Shc and rhoA. Intracellular calcium levels were not different in resting platelets from 14 PV patients and 15 healthy controls (median 43 nmol/L, range 10-114, vs. 36 nmol/L, range 10-119). After stimulation with PAF (1 micromol/L) an equal increase was seen (125 nmol/L for PV platelets, range 67-257, vs. 113 nmol/L for controls, range 60-250). Also formation of phosphatidyl ethanol (PEt) was similar after exposure to 0.5 U/ml thrombin (0.28% PEt of total phospholipid, range 0.16-1.10, vs. 0.24 for controls, range 0.11-2.3) and 1 micromol/L PMA (0.25, range 0.16-0.32, vs. 0.14, range 0.09-0.6). In contrast to the reduced amount of GPIIIa on the surface of PV platelets, immunoblotting on whole cell lysates showed no reduction in PV patients compared to controls, indicating the possibility of an impaired incorporation of GPIIIa to the cell membrane. Levels of Fak, Syk, Shc, Grb2 and rhoA appeared equal in patients and controls. Similar intracellular proteins were tyrosine phosphorylated after stimulation with thrombin, PAF and PMA. In summary, defective platelet aggregation after stimulation with PAF is caused by neither defective mobilization of intracellular calcium nor, in contrast to the situation in PV granulocytes, an impaired activation of PLD. Moreover, no apparent differences in the intracellular amounts of Fak, Syk Shc, Grb2 and rhoA could be detected between PV and control platelets.     

野黄芩苷抗血小板活化效应的评价

目的评价野黄芩苷对血小板的聚集与活化的影响。方法在人洗涤血小板中进行血小板聚集实验,分别用胶原(1.00μg/ml)、U46619(0.30μM)、ADP(10.00μM)、凝血酶(0.04U/ml)作为诱导剂,对比在0.04mg/ml、0.10mg/ml、0.20mg/ml三种不同预孵浓度状态下野黄芩苷对血小板聚集的影响。并进一步研究三种不同浓度的野黄芩苷对血栓素类似物(U46619)引起的血小板纤维蛋白原结合的影响。结果胶原、U46619、ADP、凝血酶均可明显促进血小板聚集,而血小板预孵育野黄芩苷可以抑制诱导剂引起的聚集,并且抑制作用呈现浓度依赖关系。血小板预孵育野黄芩苷可以减少U46619引起的血小板纤维蛋白结合,并且抑制作用呈现浓度依赖关系。结论野黄芩苷可以抑制血小板的聚集和活化,很可能是通过阻断血栓素A2诱导血小板聚集的某一个中间环节产生作用。     

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.