Human platelet electrorotation changed induced by activation: inducer specificity and correlation to serotonin release

Electrorotation of single platelets was compared with [¹⁴C]serotonin release, aggregation and electron microscopy. Activation of washed and degranulated platelets was induced by thrombin, arachidonic acid, collagen, adrenaline, platelet activation factor (PAF (PA), ADP and A23187. A strong correlation between electrorotation decrease and serotonin release was found. Electrorotation did not correlate with aggregation. It was concluded that an increase of the specific conductivity of the platelet membrane by three orders of magnitude (approx. 1.0–10⁻⁷ S · m⁻¹ to 1.0·10⁻⁴ S · m⁻¹) upon activation was responsible for the observed decrease of anti-field rotation and the shift of the first characteristic frequency towards higher values. Electrorotation allowed for time-dependent measurements of activation. Characteristic activation times in the order of minutes were found. There was the following sequence of activators classified by increasing activation time constants: A23187 was the fastest followed by thrombin, collagen, PAF, arachidonic acid, adrenaline, and ADP.     

Human platelet aggregation induced by prostaglandin endodisulfide

The effect of human platelet functions of 9,11-dithio analogues of prostaglandin endoperoxide was investigated. Methyl (5z,9α,11α,13e,15S)-9,11-epidithio-15-hydroxyprosta- 5,13-dienoate induced platelet aggregation, while the 9β,11β-epimer was inactive. The platelet aggregation caused by the 9α,11α-dithio analogue was associated with serotonin release from platelets, and was inhibited by methyl ester of prostaglandin I₂ (prostacyclin) but not by indomethacin.     

The platelet activation induced by wheat germ agglutinin

In human platelets, wheat germ agglutinin (WGA) induced serotonin release without cell agglutination. WGA induced the phosphorylation of both 40-kDa and 20-kDa proteins in a parallel manner, and at least, the phosphorylation of 40-kDa protein was preceded by transient formation of endogenous diacylglycerol (DG) accompanied by a decrease in phosphatidylinositol (PI). Both phosphorylation of these two proteins and serotonin release were inhibited by prior treatment of platelets with dibutyryl cyclic AMP, W-7, or TMB-8. These results suggest that both phosphatidylinositol turnover and Ca2+ mobilization play an essential role in WGA-induced platelet activation.     

Human platelet aggregation induced by prostaglandin endodisulfide.

The effect on human platelet functions of 9,11-dithio analogues of prostaglandin endoperoxide was investigated. Methyl (5Z, 9alpha, 11alpha, 13E, 15S)-9,11-epidithio-15-hydroxyprosta-5,13-dienoate induced platelet aggregation, while the 9beta,11beta-epimer was inactive. The platelet aggregation caused by the 9alpha,11alpha-dithio analogue was associated with serotonin release from platelets, and was inhibited by methyl ester of prostaglandin I2 (prostacyclin) but not by indomethacin.     

Human platelet activation by an alkylacetyl analogue of phosphatidylcholine

The present study has evaluated the influence of semi-synthetic platelet-aggregating factor, (PAF) i.e., alkylacetylglycerophosphocholine, on human platelet morphology, biochemistry and function in order to determine if PAF serves as the corrective factor restoring sensitivity to refractory platelets after treatment with epinephrine. Threshold concentrations of PAF caused irreversible platelet aggregation which could be blocked by agents elevating endogenous levels or cyclic AMP or inhibited by antagonists of platelet prostaglandin synthesis and secretion. PAF did not stimulate platelets through α-adrenergic receptors or receptors for arachidonate, endoperoxides or thromboxanes. 24 h after aspirin ingestion, platelets could be aggregated irreversibly by high concentrations, but not by threshold amounts of PAF, even though they were still insensitive to arachidonate. Another less potent PAF derivative, alkenylacetylglycerophosphocholine, blocked aggregation of 24-h aspirin platelets by PAF, but did not inhibit restoration of arachidonate sensitivity and irreversible aggregation when the samples were treated first with epinephrine. Our findings indicate that threshold amounts of PAF activate human platelets in a physiologic manner and cause irreversible aggregation which is dependent on prostaglandin synthesis and the release reaction. The results do not support the concept that PAF is the mediator of the mechanism of membrane modulation through which epinephrine induces correction of the refractory state in prostaglandin I₂-treated or dissociated platelets, or cells obtained from individuals following aspirin ingestion. Thus, the mechanism of platelet membrane modulation is capable of securing irreversible aggregation of secretion, prostaglandin synthesis or PAF formation.     

Effects of propranolol and pindolol on platelet aggregation and serotonin release

The aggregation of human platelets by adrenaline and adenosine di-phosphate (ADP) and its inhibition by β-blockers was studied by measuring the light transmission of plateletrich plasma (PRP) and suspensions of washed platelets exposed to these agents. Inhibition of aggregation of PRP and washed platelets was dose related in the two β-blockers tested: propranolol and pindolol. The potent β-blockers pindolol was less inhibitory than propranolol when adrenaline and ADP were used to induce platelet aggregation. The aggregation of platelets by adrenaline has two phases. With low doses of the blockers only the second phase was inhibited whereas higher doses blocked both phases. Preincubation of human platelets (PRP and washed platelets) with both blockers per se resulted in release of ¹⁴C-labelled serotonin. Propranolol released more serotonin than pindolol. There was no concomitant release of lactic dehydrogenase. It is concluded that the effects of propranolol and pindolol on platelets do not correlate with the β-blocking activity of these agents. Rather, the more lypophilic agent, propranolol, is more active both in inhibition of aggregation and in releasing platelet serotonin. It is suggested that these actions of the drugs are related to their non-specific membrane effects.     

Whole blood serotonin and platelet activation in depressed post-myocardial infarction patients

Depression is an independent risk factor for post myocardial infarction (MI) mortality. Abnormalities in platelet function have been proposed as one of the mechanisms involved in increased cardiovascular risk among patients with depression post-MI. Depression in somatically healthy patients has been associated with increased platelet activation. Some but not all studies showed changes in blood serotonin level. Increased platelet activation and blood serotonin level have been associated with increased risk of cardiac events in patients with MI. The goal of this study was to investigate whether 1) depressed post-MI patients have higher markers of platelet activation as measured by plasma levels of beta-thromboglobulin (betaTG), platelet factor 4 (PF4) and soluble CD40 ligand (sCD40L) and higher serotonin (5-HT) levels than non-depressed post-MI patients and 2) treatment with the antidepressant mirtazapine decreases platelet activation. In this study, 25 depressed post-MI patients were asked for blood collection before start as well as after 8 weeks treatment with mirtazapine or placebo. The control group (n=22) consisted of non-depressed post-MI patients, matched for age, gender and time elapsed since MI. Plasma levels of betaTG, PF4 and sCD40L were not statistically different between the groups, but 5-HT levels were significantly higher in depressed patients. Treatment with mirtazapine resulted in a non-significant decrease in betaTG and PF4 and platelet 5-HT levels. Platelet and whole blood 5-HT, but not platelet activation was significantly increased in depressed post-MI patients. Treatment with mirtazapine showed a non-significant decrease in platelet activation and platelet 5-HT.     

Endogenous platelet serotonin release monitored during aggregation by means of a new electrochemical technique

Differential pulse voltammetry combined with electrochemically treated carbon fibre microelectrodes was used to monitor endogenous serotonin release occurring during platelet aggreagtion. After platelet stimulation by thrombin, an oxidation peak was recorded at +280 mV. HPLC analyses performed with fluorimetric detection have shwon that this released electroactive compound was essentially serotonin. Moreover, serotonin measurements in the same samples by the technique reported here and by fluorimetry were found to be very similar (1.15 ± 0.30 μM and 1.17 ± 0.15 μM (mean ± dS.D., n = 6), respectively). Extracellular serotonin concentrations could be estimated either directly during aggregation or in supernatants obtained from stimulated or lysed platelets. Maximal serotonin concentrations have been found to be 6.93 ± 0.37 and 3.28 ± 0.39 nmol/10⁹ platelets from rat and human, respectively. Using the reported procedure, we have observed that no serotonin was released from thrombin-stimulated platelets prepared from rats treated with reserpine. Our new technique represents a selective and performant tool for rapid determination of endogenous serotonin platelet secretion.     

Endogenous platelet serotonin release monitored during aggregation by means of a new electrochemical technique

Differential pulse voltammetry combined with electrochemically treated carbon fibre microelectrodes was used to monitor endogenous serotonin release occurring during platelet aggregation. After platelet stimulation by thrombin, an oxidation peak was recorded at +280 mV. HPLC analyses performed with fluorimetric detection have shown that this released electroactive compound was essentially serotonin. Moreover, serotonin measurements in the same samples by the technique reported here and by fluorimetry were found to be very similar (1.15 +/- 0.30 microM and 1.17 +/- 0.15 (mean +/- S.D., n = 6), respectively). Extracellular serotonin concentrations could be estimated either directly during aggregation or in supernatants obtained from stimulated or lysed platelets. Maximal serotonin concentrations have been found to be 6.93 +/- 0.37 and 3.28 +/- 0.39 nmol/10(9) platelets from rat and human, respectively. Using the reported procedure, we have observed that no serotonin was released from thrombin-stimulated platelets prepared from rats treated with reserpine. Our new technique represents a selective and performant tool for rapid determination of endogenous serotonin platelet secretion.     

Human platelet calmodulin-binding proteins: identification and Ca2+-dependent proteolysis upon platelet activation

Calmodulin-binding proteins have been identified in human platelets by using Western blotting techniques and 125I-calmodulin. Ten distinct proteins of 245, 225, 175, 150, 90, 82 (2), 60, and 41 (2) kilodaltons (kDa) bound 125I-calmodulin in a Ca2+-dependent manner; the binding was blocked by ethylene glycol bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA), trifluoperazine, and nonradiolabeled calmodulin. Proteins of 225 and 90 kDa were labeled by antisera against myosin light chain kinase; 60- and 82-kDa proteins were labeled by antisera against the calmodulin-dependent phosphatase and caldesmon, respectively. The remaining calmodulin-binding proteins have not been identified. Calmodulin-binding proteins were degraded upon addition of Ca2+ to a platelet homogenate; the degradation could be blocked by either EGTA, leupeptin, or N-ethylmaleimide which suggests that the degradation was due to a Ca2+-dependent protease. Activation of intact platelets by thrombin, adenosine 5'-diphosphate, and collagen under conditions which promote platelet aggregation (i.e., stirring with extracellular Ca2+) also resulted in limited proteolysis of calmodulin-binding proteins including those labeled with antisera against myosin light chain kinase and the calmodulin-dependent phosphatase. Activation by the Ca2+ ionophores A23187 and ionomycin also promoted degradation of the calmodulin-binding proteins in the presence of extracellular Ca2+; however, degradation in response to the ionophores did not require stirring of the platelet suspension to promote aggregation. Many Ca2+/calmodulin-regulated enzymes are irreversibly activated in vitro by limited proteolysis. Our data indicate that limited proteolysis of Ca2+/calmodulin-regulated enzymes also occurs in the intact platelet and suggest that the proteolysis is triggered by an influx of extracellular Ca2+ associated with platelet aggregation.     

Quantitation of paf-acether by release of endogenous platelet serotonin assessed by liquid chromatography with electrochemical detection

A new method to quantitate paf-acether (paf) was developed. It is based on the measurement of serotonin released from washed rabbit platelets challenged with paf. Platelets (1 X 10(8)/ml) were exposed with or without stirring to various concentrations of paf (26-130 pM) at 37 degrees C or at room temperature. Supernatants were submitted to a 4-min liquid chromatography run and serotonin was measured by electrochemical detection. We quantitated paf from three different biological sources, human neutrophils, mouse peritoneal macrophages, and cultured mast cells, comparing a classical method, i.e., platelet aggregation with the electrochemical detection of endogenous serotonin. We found similar results since, when compared with the aggregation method, the results differed by 12 to 47%. The sensitivity of both methods was 26 pM. The between-day variation coefficient was 23 and 14% (n = 12) for the aggregation method and the serotonin release, respectively, whereas the within-day variation coefficient for serotonin quantitation was less than 5% (n = 12). The superiority of the new method lies in its simplicity, the economy of platelets, and its possibility of automation. It can be applied to any agonist or any mechanism capable of releasing serotonin from platelets and more generally when a simple and fast method for measuring serotonin is desirable.     

Molecular mechanisms of platelet activation and aggregation induced by breast cancer cells

Tumor cell-induced platelet aggregation represents a critical process both for successful metastatic spread of the tumor and for the development of thrombotic complications in cancer patients. To get further insights into this process, we investigated and compared the molecular mechanisms of platelet aggregation induced by two different breast cancer cell lines (MDA-MB-231 and MCF7) and a colorectal cancer cell line (Caco-2). All the three types of cancer cells were able to induce comparable platelet aggregation, which, however, was observed exclusively in the presence of CaCl₂ and autologous plasma. Aggregation was supported both by fibrinogen binding to integrin αIIbβ3 as well as by fibrin formation, and was completely prevented by the serine protease inhibitor PPACK. Platelet aggregation was preceded by generation of low amounts of thrombin, possibly through tumor cells-expressed tissue factor, and was supported by platelet activation, as revealed by stimulation of phospholipase C, intracellular Ca²⁺ increase and activation of Rap1b GTPase. Pharmacological inhibition of phospholipase C, but not of phosphatidylinositol 3-kinase or Src family kinases prevented tumor cell-induced platelet aggregation. Tumor cells also induced dense granule secretion, and the stimulation of the P2Y12 receptor by released ADP was found to be necessary for complete platelet aggregation. By contrast, prevention of thromboxane A₂ synthesis by aspirin did not alter the ability of all the cancer cell lines analyzed to induce platelet aggregation. These results indicate that tumor cell-induced platelet aggregation is not related to the type of the cancer cells or to their metastatic potential, and is triggered by platelet activation and secretion driven by the generation of small amount of thrombin from plasma and supported by the positive feedback signaling through secreted ADP.     

Kinetics of thrombin-induced release and activation of platelet factor V

The kinetics of thrombin-induced platelet factor V activation were studied in suspension of washed human platelets. The effect of thrombin in stimulating the release reaction could be separated from its effect on factor V activation by use of a potent inhibitor of the release reaction, the prostacyclin analogue ZK 36374. When platelets were incubated with ZK 36374 prior to stimulation with thrombin, the amount of ZK 36374 required to inhibit 50% of factor Va formation was 15 pM. ZK 36374 at a final concentration of 1 nM was found to block instantaneously and completely the release of factor Va, whereas it has no effect neither on platelet factor V activation nor on the factor Va assay. By varying the time interval between the addition of thrombin (0.5 nM) and ZK 36374 to suspensions of 4.6 X 10(6) platelets/ml the rate of factor V release was found to be 12 pM factor V/min. In the absence of ZK 36374 the total amount of factor V released was 8 pM, whereas Triton X-100-treated platelets gave 13 pM factor V. It appeared that the amount of factor V that could be released was dependent on the thrombin concentration. Maximum release was obtained at 1 nM thrombin. The rate of factor V release increased in proportion to the thrombin concentration. The rate of factor V activation was found to be proportional to the thrombin concentration as well as to the amount of released factor V. When 4.6 X 10(6) platelets/ml were activated by 0.5 nM thrombin, the rates of factor V activation were found to be 0.3 pM and 1.2 pM factor Va/min at 20% and 90% completion of the release reaction. Therefore, the rate of factor V release was at least one order of magnitude faster than the rate of factor V activation. The kinetics of thrombin-induced platelet factor V activation were compared to those of plasma factor V activation in platelet-rich and platelet-free plasma. The results clearly demonstrate that platelets have no effect on the rate of factor V activation and that the kinetics of plasma factor V activation are identical to those of platelet factor V activation.     

Proteomic analysis of the porcine platelet proteome and alterations induced by thrombin activation

Platelets are enucleated cells derived from bone marrow megakaryocytes and defects in platelet functions could be involved in many cardiovascular diseases. Proteomics can be used to provide a new insight in the study of these platelet functions and can help to identify the biochemical events underlying platelet activation. In this study, we have obtained a reference 2-DE map of porcine platelet proteins. A large number of cytoskeletal and metabolic proteins were found as well as some proteins related to cell mobility and immunological functions. Other proteins implicated in the cell signalling process, transport or apoptosis were also identified. Moreover, we have analysed, by 2D-DIGE methodology, quantitative modifications of platelet proteins following their activation by thrombin. 26 spots exhibited statistically significant differences, and a total of 16 spots corresponding to 13 different proteins were successfully identified. Using Ingenuity Pathway Analysis, the association of the deregulated proteins with canonical pathways highlighted two major pathways; coagulation system and integrin signalling. These results confirm that this proteomic approach (based on 2D-DIGE, mass spectrometry and bioinformatic and pathway databases) has proved to be a powerful tool when applied to studying signalling pathways that could play a relevant role in the activation of platelets.     

Role of the recombinant non-integrin platelet collagen receptor P65 on platelet activation induced by convulxin

Convulxin (Cvx) isolated from Crotalus durissus terrificus venom selectively binds with a high affinity to platelets and induces platelet aggregation by a mechanism that resembles that induced by collagen. Taking advantage that P65 has been recently cloned and expressed as a recombinant soluble protein (rec-P65), we examined the role of this non-integrin collagen receptor in platelet activation induced by Cvx. Rec-P65 blocked platelet adhesion to collagen-coated surfaces and inhibited platelet aggregation and ATP secretion induced by type I collagen. On the other hand, rec-P65 did not inhibit platelet aggregation and ATP secretion induced by Cvx, and it did not affect platelet adhesion to Cvx. In addition, ligand-blotting indicated that the Cvx binding to the collagen receptor GPVI was preserved in the presence of rec-P65. These observations indicate that P65 does not play a significant role in platelet activation by Cvx; in contrast, platelet response to collagen involves multiple receptors.     

Activation of phospholipase C is dissociated from arachidonate metabolism during platelet shape change induced by thrombin or platelet-activating factor. Epinephrine does not induce phospholipase C activation or platelet shape change

The present study compares the molecular mechanism by which thrombin, platelet-activating factor, and epinephrine induce platelet activation. Thrombin and platelet-activating factor induce an initial activation of phospholipase C, as measured by formation of 1,2-diacylglycerol and phosphatidic acid, during platelet shape change which is independent of and dissociated from metabolism of arachidonic acid. Phospholipase C activation and shape change are independent of extracellular Ca2+ and Mg2+. Formation of cyclooxygenase products occurs subsequent to the initial activation of phospholipase C and those metabolites are associated with platelet aggregation and further activation of phospholipase C. On the other hand, epinephrine is an unique platelet stimulus since it requires extracellular divalent cations and does not induce platelet shape change or activation of phospholipase C. Our results indicate that activation of phospholipase C may be a mechanism by which physiological agonists can activate platelets independently of extracellular divalent cations.     

Function of glycoprotein Ib alpha in platelet activation induced by alpha-thrombin.

We have obtained evidence that selective inhibition of high affinity thrombin-binding sites located in the amino-terminal domain of the membrane glycoprotein (GP) Ib alpha results in impaired platelet activation, as shown by abrogation or reduction of the following responses induced in normal platelets by exposure to less than 1 nM alpha-thrombin: (i) increase in intracellular ionized calcium concentration ([Ca2+]i), (ii) release of dense granule content, (iii) binding of fibrinogen, (iv) aggregation. An anti-GP Ib monoclonal antibody, LJ-Ib 10, which does not inhibit von Willebrand factor binding to platelets, obliterated the high affinity alpha-thrombin-binding sites on normal platelets. Isotherms of alpha-thrombin binding to normal platelets treated with saturating amounts of the antibody were virtually identical to those obtained with platelets from a patient with classical Bernard-Soulier syndrome. In parallel with decreased binding of the agonist, this antibody caused 50% inhibition of the maximal extent of platelet aggregation and 90% inhibition of ATP release induced by 0.3 nM alpha-thrombin. By inhibiting alpha-thrombin binding to GP Ib, the antibody prevented the activation of platelets exposed to low concentrations of the agonist, as demonstrated by abrogation of the increase in intraplatelet ionized calcium concentration induced in control platelets by 0.18 nM alpha-thrombin; under these conditions, fibrinogen binding was inhibited by 84%. Therefore, there is a correlation between occupancy of the high affinity sites for alpha-thrombin on GP Ib alpha and platelet activation, secretion, and aggregation, suggesting that GP Ib alpha is part of an alpha-thrombin receptor relevant for platelet function.