The serotonergic mechanisms are not involved in the inhibitory effect of captopril on rat platelet aggregation

The influence of captopril on blood platelet aggregation and on serotonergic mechanisms in blood platelets were studied. In rats pretreated with captopril (10.0 mg/kg p.o.) platelet aggregation induced by ADP and collagen was significantly reduced. In vitro this drug had no inhibitory effect. Besides, captopril did not change the amplifying effect of serotonin on platelet aggregation. Captopril also had no influence on the uptake and storage of 5-hydroxytryptamine by rat blood platelets. These results show that serotonergic mechanisms are not involved in the suppressive effect of captopril on platelet aggregation.     

Storage Pool Disease: Comparative Fluorescence Microscopical, Cytochemical and Biochemical Studies on Amine-Storing Organelles of Human Blood Platelets

The platelet content of ATP, ADP, serotonin (5-HT), dopamine, noradrenaline and adrenaline as well as the net uptake of radiolabelled 5-HT and mepacrine were subnormal in six patients with Storage Pool Disease (SPD). Fewer amine-storing organelles were found by fluorescence microscopy with the fluorescent probe mepacrine and by electron microscopy with a cytochemical (uranaffin) reaction specific for 5'-phosphonucleotides. Both methods showed that SPD platelets have atypical organelles with a reduced capacity to store amines, 5'-phosphonucleotides and mepacrine. The changes were most marked in platelets of the two patients who also had oculocutaneous albinism.     

Grey platelet syndrome: studies on platelet alpha-granules, lysosomes and defective response to thrombin

The platelets of a young man with the grey platelet syndrome were severely depleted of all seven alpha-granule proteins assayed as well as partially deficient in alpha-mannosidase and alpha-fucosidase; four other lysosomal enzymes were present in normal concentrations. Total platelet 5-hydroxytryptamine (5HT) and adenine nucleotides were normal, and 14C-5HT uptake reached normal levels only slightly more slowly than a control. Aggregation and dense body secretion occurred normally in response to ADP, adrenaline, collagen, PAF-acether, sodium arachidonate, A23187, Ionomycin, TPA and U44069, but were very delayed in response to thrombin. The increase in cytosolic free calcium in response to thrombin was very slow and much reduced in amplitude, whether in the presence or absence of extracellular Ca2+. These defects in response to thrombin were not corrected by the separate addition of purified alpha-granule proteins or by a whole releasate from normal platelets. It is suggested that these platelets, in addition to their alpha-granule deficiency, may have a specific defect of thrombin receptor-mediated activation of phospholipase C.     

Inhibition of platelet function by cis-unsaturated fatty acids

The uptake of free fatty acids has previously been shown to affect the capping of lymphocytes, and there is evidence that different types of fatty acids may partition into separate lipid domains in cell surface membranes. In studies of gel-filtered human platelets, we found that cis-unsaturated fatty acids (1-35 microM) inhibited platelet shape change, aggregation, and secretion of 5-hydroxytryptamine induced by thrombin, adenosine diphosphate (ADP), collagen, U46619 (a thromboxane A2 analog), or plant lectins, but not that induced by A23187, a calcium ionophore. Trans-unsaturated and saturated fatty acids had little or no inhibitory effect. The inhibitory effects of cis-unsaturated fatty acids were not affected by inhibition of adenylate cyclase or cyclooxygenase. 14C-labeled fatty acids were taken up into platelet lipids. The maximum platelet-inhibitory effect of cis-unsaturated fatty acids was seen when over 90% of the platelet label was still in the form of free fatty acids. Platelet inhibition could be reversed by washing the platelets by gel filtration. Binding of platelet agonists to the platelet was not inhibited by the fatty acids. Cis-unsaturated fatty acids, but not trans-unsaturated or saturated fatty acids, decreased fluorescence polarization of platelets or isolated platelet membranes monitored with 1,6-diphenyl- 1,3,5-hexatriene. The potency of the fatty acids as inhibitors of platelet aggregation was inversely correlated with their melting points. These data suggest that inhibition of receptor-mediated platelet responses by cis-unsaturated fatty acids results from perturbation of the platelet membrane in specific lipid domains.     

A new congenital defect of platelet secretion: impaired responsiveness of the platelets to cytoplasmic free calcium

Summary . A 16-year-old boy with a bleeding disorder since infancy has a long bleeding time, normal platelet count and morphology and normal plasma factor-VIII activities. His platelets undergo normal shape change and primary aggregation in response to ADP but show defective 5-hydroxytryptamine (5-HT) secretion and aggregation in response to adrenaline, sodium arachidonate, U44069, PAF-acether, A23187 and low concentrations of collagen. Thrombin and higher concentrations of collagen produce a normal response. Secretion of β-thromboglobulin and platelet factor 4 parallels that of 5-HT. Thromboxane B₂ is produced normally in response to exogenous arachidonate and to stimulation by thrombin, collagen and A23187 in all concentrations tested. The patient's endoperoxides and thromboxane A₂ aggregate aspirin-treated platelets, though his platelets are themselves unresponsive. Cyclic AMP is present at normal concentration in the patient's unstimulated platelet-rich plasma, and PGI₂ inhibits platelet aggregation by ADP and thrombin in a normal dose-related manner. Platelet ultrastructure, 5-HT uptake and content of adenine nucleotides, platelet factor 4 and β-thromboglobulin are all within normal limits. When the patient's platelets were loaded with the fluorescent dye quin 2, which serves as an indicator of cytoplasmic free calcium ions, their responses to thrombin, whether in the presence or virtual absence of extracellular Ca2+, were entirely normal in respect of free calcium ions, secretion, shape-change and aggregation. In response to ionomycin, however, a normal increase in free calcium ions was accompanied by normal shape-change but virtually no aggregation or 5-HT secretion. The platelet calmodulin content was normal. These findings show that the defect in this patient's platelets is of utilization of cytoplasmic Caz+ for secretion and aggregation, rather than of Ca2+ uptake or mobilization of Ca2+ from intracellular storage sites. It is suggested that the most likely site of the defect is the phosphorylation of one of the proteins concerned in the secretory mechanism.     

The effect of aggregation and release on platelet prothrombin-converting activity.

Platelets provide a procoagulant activity for the conversion of prothrombin to thrombin during normal hemostatis. This activity designated as platelet prothrombin-converting activity (PPCA) was monitored as rate of thrombin production in a two-stage assay using gel-filtered bovine platelets, factor Xa, and prothrombin. Expression of PPCA was not associated with ADP-induced release or platelet shape change but was associated with aggregation. Release of the contents of dense bodies, measured by release of 14C-5-hydroxytryptamine, was not required for expression of PPCA during platelet aggregation. During the PPCA assay, 5-hydroxytrypamine was released, but only after onset of thrombin production. Furthermore, the release of 5-hydroxytryptamine was retarded during the assay by the addition of 2 mM theophylline and 100 nM prostaglandin E1 without a comparable reduction in PPCA. In addition, 125I-factor-Xa was bound in greater amounts to platelets (aspirin-treated) after ADP-induced aggregation (without detectable release) than to unactivated control platelets. Finally, the PPCA of the ADP-activated platelets was saturated with respect to factors Xa and Va at less than 1 nM concentrations, indicating that the aggregation induced by ADP leads to the exposure of specific procoagulant sites by some process other than dense body secretion.     

Metabolism and Function of Human Platelets Washed by Albumin Density Gradient Separation

A method for washing platelets by albumin density gradient separation, originally designed for the study of platelet coagulant activities, has been modified for platelet aggregation and metabolic studies. Platelets are sedimented into a continuous density gradient of isosmolar albumin containing apyrase to protect them from clumping and physical injury and are resuspended in calcium-free Tyrode's solution. The mean recovery of platelets after two separations relative to plateletrich plasma (PRP) was 90.3%. When small amounts of plasma were added to washed platelet suspensions, aggregation and release of [¹⁴C]5-hydroxytryptamine (5HT) in response to adenosine diphosphate (ADP) or 5HT were similar to results obtained with PRP. When fibrinogen was substituted for plasma, ADP-induced aggregation occurred but was feeble. Without added plasma or fibrinogen, platelets were refractory to ADP and insensitive to the cyclic endoperoxide analogue U44619. When both ADP and U44619 were added simultaneously, in low concentrations, to washed platelets without added plasma or fibrinogen, aggregation occurred immediately. Washed platelets were not aggregated by adrenaline, which potentiated ADP-induced aggregation. Several biochemical measurements which are sensitive indicators of cellular damage were normal in washed platelets, including [¹⁴C]adenine uptake, adenylate energy charge, hypoxanthine formation and the response of adenylate cyclase to stimulation by PGE₁ or PGD₂. Platelet coagulant activities were not made available and heparin-neutralizing activity (HNA) was not spontaneously released by the washing procedure, but the washed platelets responded normally to appropriate agents by developing coagulant activities and releasing HNA. The ultrastructure of washed platelets was similar to those in control PRP. Inclusion of apyrase in the first albumin gradient had a beneficial effect on platelet morphology, aggregation and metabolism, but washing at 37deg;C compared with 25deg;C did not. Albumin density gradient separation is a useful method for isolating platelets for aggregation and metabolic studies.     

Function of the serotoninergic system of blood platelets in patients with hypertension]

To investigate if there can be alterations in the 5-hydroxytryptamine (5-HT) uptake system in the sensitivity of platelets in patients with essential hypertension, 38 hypertensive patients and 37 normotensive healthy subjects were compared. In patients, the maximal 5-HT uptake velocity was reduced. The density of binding sites for 3H-imipramine was elevated in hypertensive females, but unchanged in males. The sensitivity of 5-HT uptake to trazodone was unchanged in patients. Half-maximal concentrations of 5-HT for inducing a shape change reaction of platelets were positively correlated with diastolic blood pressure in male patients and were reduced in female mild hypertensives. It is suggested that these changes are likely to be involved in the pathogenesis of hypertension.     

Platelet von Willebrand's antigen II: active release by aggregating agents and a marker of platelet release reaction in vivo

von Willebrand's antigen II (vW AgII), a plasma and platelet antigen immunologically and biochemically distinct from factor-VIII-related antigen (VIIIR:Ag), is decreased in von Willebrand's disease. vW AgII is released from platelets during aggregation and clotting. The release of platelet vW AgII was studied in washed platelets following aggregation by thrombin, collagen, and adenosine diphosphate (ADP). Total platelet vW AgII was 3.39 U/10(9) platelets. Thrombin and collagen yielded release of 85% and 86% of platelet vW AgII, respectively, at the highest concentrations. Release of platelet vW AgII was correlated with the release of 5-hydroxytryptamine (5HT). Release of vW AgII by collagen and thrombin was inhibited by metabolic inhibitors. In addition, collagen release of vW AgII was inhibited by aspirin. In clinical syndromes associated with intravascular platelet destruction, marked elevations of plasma vW AgII were noted. Thus, vW AgII is released by a metabolically active process from platelets during aggregation. In addition, vW AgII appears to be a marker of intravascular platelet destruction.     

Differential alterations of spontaneous and stimulated 45Ca(2+) uptake by platelets from patients with type I and type II diabetes mellitus

Diabetes mellitus (DM) is associated with hyperaggregability of platelets. Although the mechanisms underlying this abnormality remain unknown, Ca²⁺ imbalance has been implicated. Both activators (-adrenoceptor agonists, collagen, and ADP) and inhibitors (β-adrenoceptor agonists, iloprost and dibutyryl cAMP) of platelet function, respectively, elicit the uptake of [⁴⁵Ca²⁺] in human platelets. It was determined that the [⁴⁵Ca²⁺] uptake methods employed reflected signal transduction events at the plasma membrane rather than absolute changes of Ca²⁺ fluxes or levels of cytosolic Ca²⁺. In the present study, basal (unstimulated) [⁴⁵Ca²⁺] uptake by platelets from both type I and type II diabetic patients was significantly enhanced when compared to age-matched controls. When basal values were subtracted from stimulated values, there were highly significant decreases in [⁴⁵Ca²⁺] uptake in platelets from type I diabetic patients compared to controls when stimulated with adrenaline, isoprenaline, noradrenaline, collagen, A23187, or iloprost. In contrast, when basal values were subtracted from stimulated values there were significant increases in [⁴⁵Ca²⁺] uptake by platelets from type II diabetic patients when stimulated with adrenaline, isoprenaline, noradrenaline, A23187, iloprost, and collagen. It is concluded that in type I and type II DM there are differential alterations in [⁴⁵Ca²⁺] sequestration linked to inhibitors and stimulators of platelet activation. These data indicate that the hyperaggregability of platelets that is associated with both type I and type II DM may be due to an aetiology other than Ca²⁺ mobilization linked to signal transduction.     

Differential changes in alpha- and beta-adrenoceptor linked [45Ca2+] uptake in platelets from patients with anorexia nervosa.

[45Ca2+] Uptake was studied in response to adrenaline, isoprenaline, noradrenaline, and (Bu)2cAMP in platelets from patients with anorexia nervosa. In both controls and anorectics, adrenaline, isoprenaline, noradrenaline, and (Bu)2cAMP stimulated [45Ca2+] uptake. In receptor subtype characterisation studies on control platelets, adrenaline-stimulated [45Ca2+] uptake was blocked by yohimbine (an alpha 2-adrenoceptor antagonist) and the specific beta 2-adrenoceptor antagonist ICI 118,551, but not by atenolol (a beta 1-antagonist). Isoprenaline action was blocked by ICI 118,551, but not by yohimbine. Noradrenaline-stimulated [45Ca2+] uptake was blocked by yohimbine but not by ICI 118,551. In platelets from anorectic patients, there was a significant increase in noradrenaline-stimulated [45Ca2+] uptake, a significant diminution in adrenaline and isoprenaline-stimulated [45Ca2+] uptake, but no significant difference in (Bu)2cAMP-stimulated [45Ca2+] uptake, when compared with controls. Basal uptake was also significantly enhanced in anorectics and was found to be inhibited with verapamil but not adrenoceptor antagonist. These data firstly indicate that both alpha 2- and beta 2-adrenoceptor activation elicits [45Ca2+] uptake by platelets. It is proposed that this stimulated [45Ca2+] uptake does not reflect changes in cytosolic Ca2+ but to localized changes of Ca2+ at the plasma membrane, possibly associated with receptor activation, per se. The respective increase and decrease of alpha- and beta-adrenoceptor activity in platelets from anorectic patients is in accord with other reports of changes of adrenoceptor number and type in platelets and other cells from anorectic patients. There may also be an increase in calcium channel activity in platelets from anorectics.     

Characterization of platelets from normal mink and mink with the Chediak-Higashi syndrome

Bleeding times of mink with the Chediak-Higashi (CH) syndrome was markedly prolonged. Platelet counts were normal but there was an impaired platelet aggregation response to collagen. The metabolic adenine nucleotide pool of platelets from normal and CH mink was labeled with 14C-adenine and the platelets were gel-filtered. Gel-filtered platelets (GFP) from CH mink contained only 37.9% of the adenosine triphosphate (ATP) and 9.6% of the adenosine diphosphate (ADP) found in normal platelets and the ATP/ADP ratio was similar to the 14C-ATP/14C-ADP ratio. Platelet content of Ca2+, Mg2+, and in particular 5-hydroxytryptamine was decreased. When GFP were incubated with thrombin to induce maximal secretion, only negligible amounts of ATP and ADP were released. The specific activity of the extracellular nucleotides approximated that within the platelet. These findings suggest that the stored nucleotide pool in CH platelets is virtually absent and that the abnormalities in platelet function may be due, in part, to the essential absence of secretable ADP and serotonin. The release of Ca2+ and Mg2+ by CH platelets was 56% and 27.8% of normal, respectively.     

Inhibition of intravascular platelet aggregation by endothelium-derived relaxing factor: reversal by red blood cells

Studies were performed to determine whether endothelium-derived relaxing factor (EDRF) can inhibit platelet aggregation within the vascular lumen, and if so, whether the inhibition persists in the presence of red blood cells (RBCs). Canine femoral arteries mounted in an organ bath were perfused with physiologic saline solution to which acetylsalicylic acid was added to block prostacyclin formation. During contraction with phenylephrine, addition of acetylcholine to the perfusing solution to evoke EDRF release relaxed the vessel wall. Washed human platelets labeled with 14C-5-hydroxytryptamine were added to the perfusing solution, and activated by thrombin infused via a branch vessel. The perfusate was collected downstream and centrifuged; the fraction of 14C-5-hydroxytryptamine appearing in the supernatant reflected the degree of platelet activation. Stimulation of EDRF release with acetylcholine inhibited 14C-5-hydroxytryptamine release. Hemoglobin (Hb) (10(-5) mol/L) blocked vascular relaxation and platelet-inhibition. RBCs at a hematocrit of 10% (treated with echothiophate to block erythrocyte cholinesterase) did not prevent relaxation but reversed the platelet inhibition. Lower hematocrits did not completely block the inhibition. Thus, erythrocyte Hb may modulate the inhibition of intraluminal platelet aggregation by EDRF.     

Platelet activation by a synthetic hydrophobic polymer, polymethylmethacrylate

Platelets adhere to artificial surfaces in the initial stage of thrombus formation, but the subsequent steps in signal transduction that lead to platelet activation by artificial surfaces are not understood. When 0.325-micron diameter beads composed of a hydrophobic polymer, polymethylmethacrylate (PMMA), were added to gel-filtered aequorin-loaded platelets suspended in media containing Ca2+, the platelets aggregated; addition of fibrinogen was not required. Platelet aggregation was preceded by an increase in cytoplasmic Ca2+ and was accompanied by phosphorylation of the 47-Kd substrate of protein kinase C (PKC), 5-hydroxytryptamine (5-HT) release, and accumulation of phosphatidic acid. All these effects were partially inhibited by apyrase and aspirin. Monoclonal antibodies (MoAbs) 7E3 and M148 and the synthetic peptides RGDS and fibrinogen gamma chain fragment 400-411, all of which bind to the platelet fibrinogen receptor glycoprotein IIb-IIIa (GPIIb-IIIa) and inhibit fibrinogen binding, prevented PMMA-induced aggregation but did not inhibit the Ca2+ increase. Chymotrypsin-treated platelets aggregated after addition of fibrinogen, but not PMMA. We conclude that platelets interact initially with PMMA at membrane sites other than those required for fibrinogen binding, leading to activation of membrane phospholipases and PKC, an increase in cytoplasmic Ca2+, release of 5-HT, ADP, and fibrinogen from storage granules, and to platelet aggregation.     

Canine lung uptake of plasma and platelet serotonin

Platelet-endothelial interaction and serotonin release were studied in 20 in vitro pump perfused lung lobes. Indicator dilution studies were performed in sequence using an injectate composed of indocyanine green and platelets labelled with either ¹⁴C-serotonin (¹⁴C-5-HT) or ⁵¹Cr. In control experiments all tracer transit times were equivalent (¹⁴C-5-HT, ⁵¹Cr and indocyanine green) without entrapment of the tracer or platelets by the system. In six lungs with normal pulmonary function platelet transit times were not prolonged and no platelet entrapment was observed. These lungs took up to 72% of the total ¹⁴C-5-HT contained in the injected platelets and plasma. The remaining 14 lobes showed variable degrees of pulmonary insufficiency such as elevated intrapulmonary shunt, low compliance and high vascular resistance; platelet entrapment was related to these phenomena (p 0.01). Six lobes were studied with ¹⁴C-5-HT, and despite entrapment of 22% of the injected platelets and transit time prolongations no 5-HT uptake was observed. The platelet-plasma ¹⁴C-5-HT ratio was higher in the effluent blood than in the injectate. The data suggest that in the pump perfused lung lobe with normal pulmonary function: (1) Serotonin released from platelets is rapidly taken up by the pulmonary circulation during a single transit through the lung lobe; (2) the serotonin uptake occurs without any observable prolongation of platelet time. In the pump perfused lung lobe with abnormal pulmonary function: (1) Serotonin released from the platelets is not taken up by the pulmonary circulation despite significant platelet entrapment; (2) the released serotonin is removed by the circulating platelets.     

Effects of nicotine on platelet function

In non-smokers, only high concentrations of nicotine (10 mM) caused platelet aggregation in platelet-rich plasma and release of 5-hydroxytryptamine (5-HT). Both responses to ADP and 5-HT were enhanced at 1 and 10 mM nicotine while they were inhibited to collagen, ristocetin, adrenaline and arachidonic acid. Lower concentrations of nicotine had no effect with any agent other than 5-HT, with which a variable enhancement of 5-HT-induced aggregation was observed. Uptake of 14C-5-HT was inhibited by nicotine at 100 microM or higher while platelet factor 3 availability was unaffected. Thus it is unlikely that direct effects of nicotine on platelets are responsible for smoking-related changes in platelet reactivity.     

Platelet serotonin absorption in Turner's syndrome

Functional activity of platelets was studied in 20 patients with Shereshevsky-Turner's syndrome. There was revealed a significant rise of platelet serotonin (5-hydroxytryptamine) content and also a reduction of exogenous serotonin absorption by platelets. The noted disturbances pointed to dysfunction of these cells, this serving as an auxiliary factor pointing out that the ageing process involved the blood elements. Reduction of platelet functional activity in patients with monosomia X was apparently due to chromosomic unbalance and with the associated hormonal control disturbances.     

Aggregation, membrane potential and Na+ and Ca2+ in the thrombocytes of rats with spontaneous hypertension

Platelet aggregation, plasmatic membrane potential and sodium and calcium uptake were investigated in spontaneously hypertensive rats (SHR). The sensitivity of SHR's platelets to effects of all aggregational agents examined (ADP, collagen, ADP + noradrenaline) was enhanced. Ca-ionophore A 23187 caused complete SHR platelet aggregation when Ca2+ concentration in the incubation medium was 20-30% below the level of normotensive control specimens. The membrane potential of SHR platelets was reduced by 8-12 mV, which may be attributed to increased content of metabolizing sodium as well as a higher rate of its transmembrane exchange. A higher rate of Ca2+ uptake by SHR platelets may be related to partial depolarization of the membrane and the cation's diffusion along potential-dependent Ca-channels.     

Platelet–adenovirus vs. inert particles interaction: Effect on aggregation and the role of platelet membrane receptors

Platelets are involved in host defense via clearance of bacteria from the circulation, interaction with virus particles, and uptake of various size particulates. There is a growing interest in micro- and nanoparticles for drug delivery and there is evidence that the properties of these particles critically influence their interaction and uptake by various tissues and cells including platelets. Virus mediated gene therapy applications are still challenged by the resultant thrombocytopenia and the mechanism(s) of platelet–foreign particles interaction remains unclear. We studied the specifics of platelet interaction with an active biological agent (adenovirus) and inert latex microspheres (MS) and investigated the role of platelet proteins in this interaction. We show that activated and not resting platelets internalize MS, without influencing platelet aggregation. In contrast, adenovirus induces and potentiates ADP-induced platelet aggregation and results in rapid expression of P-selectin. Platelets then internalize adenovirus and viral particles appear inside the open canalicular system. Inhibition of platelet αIIbβ3, GPIbα, and P-selectin decreases both platelet aggregation and internalization of MS. Inhibition of αIIbβ3 and αVβ3 does not abolish adenovirus platelet internalization and adenovirus-induced platelet activation is maintained. Our study demonstrates that platelets react differentially with foreign particles and that αIIbβ3 is a key player in platelet engulfing of foreign particles but not in mediating adenovirus internalization. Other platelet candidate molecules remain to be investigated as potential targets for management of adenovirus-induced thrombocytopenia.     

The Hermansky-Pudlak Syndrome

A Dutch kindred with the Hermansky-Pudlak syndrome (HPS) is described. We show for the first time evidence of a lowered platelet 5-hydroxytryptamine content in obligate heterozygotes. Platelet ATP and ADP levels and ATP/ADP ratio were normal in these patients. Platelet aggregation with ADP, collagen and adrenaline was within the normal range. In contrast to the homozygous HPS patients the heterozygotes are normally pigmented and none has diaphanous irides, nystagmus or a bleeding tendency. All homozygous HPS patients have the typical triad of oculocutaneous albinism, pigmented macrophages in the bone marrow and a bleeding disorder, based on a platelet dysfunction. The platelets showed the typical characteristics of a storage pool deficiency. Their platelet factor 3 availability was decreased and the aggregation patterns showed an absent second wave with ADP, adrenaline and absent collagen aggregation. Platelet ADP levels were strongly decreased in all homozygous HPS patients, whereas ATP was lowered only in 3 out of 6 HPS patients. The 5-hydroxytryptamine content of their platelets was very low (15–20% of normal).