Human Blood Platelets Research Articles

Light scattering and absorption model for the quantitative interpretation of human blood platelet spectral data.

Multiwavelength ultraviolet-visible (UV-Vis) transmission spectroscopy is a relatively simple technique that can provide considerable quantitative information on the properties of micron and submicron particle suspensions. Two important particle properties are particle size distribution (PSD) and chemical composition. These properties provide characteristics for the identification and classification of biological systems ranging in size and composition from proteins and nucleic acids to cells. By measuring the complete UV-Vis spectrum, the combined scattering and absorption properties are obtained as a function of wavelength. The quantitative evaluation of the size distribution and chemical composition is accomplished through the application of light-scattering theory. This paper reports on the estimation of the optical properties of human blood platelets and their use in the interpretation of platelet UV-Vis spectra within the context of Mie theory. The model developed herein provides reliable and accurate estimates for the PSD and particle number of platelet suspensions. One potential application of this characterization method is in the analysis of platelet activation by thrombin. Quantification of spectral data with respect to average particle size and particle number provides a real-time description of the dramatic changes that accompany the platelet activation process.

Platelet-dependent modulation of polymorphonuclear leukocyte chemiluminescence

Human blood platelets decreased luminol-enhanced chemiluminescence of human polymorphonuclear leukocytes (PMNL) stimulated with FMLP or Ca2+-ionophore A23187 by 56 or 47%, respectively. Horseradish peroxidase potentiated the decreasing effect of platelets on A23187-stimulated PMNL (92% inhibition) or reversed inhibition of FMLP-induced chemiluminescence to 94% potentiation, indicating dependence of platelet activity on availability of extracellular peroxidase. Moreover, platelet activity may depend also on the extent of platelet activation, as non-activated platelets (in the presence of FMLP) were found to potentiate PMNL-generated chemiluminescence, while platelets activated with A23187 displayed the opposite effect. Interference of platelets with formation and liberation of superoxide anion was indicated by platelet-modified isoluminol chemiluminescence. Superoxide dismutase with catalase and sodium azide were used, respectively, to differentiate the intracellular and the extracellular part of the chemiluminescence signal. Platelets were found to be capable of modifying both components of chemiluminescence, i.e., oxygen metabolites produced on the plasma membrane as well as on membranes of intracellular granules.

A Key Role of Adenosine Diphosphate in the Irreversible Platelet Aggregation Induced by the PAR1-Activating Peptide Through the Late Activation of Phosphoinositide 3-Kinase

Although adenosine diphosphate (ADP), per se, is a weak platelet agonist, its role as a crucial cofactor in human blood platelet functions has now been clearly demonstrated in vitro and in vivo. The molecular basis of the ADP-induced platelet activation is starting to be understood since the discovery that 2 separate P2 purinergic receptors may be involved simultaneously in the activation process. However, little is known about how ADP plays its role as a cofactor in platelet activation and which signaling pathway initiated by a specific agonist can be modulated by the released ADP. To investigate these points, we took advantage of a model of platelet activation through the thrombin receptor PAR1 in which both ADP scavengers and phosphoinositide 3-kinase (PI 3-kinase) inhibitors have been shown to transform the classical irreversible aggregation into a reversible one. We have observed that, among the different PI 3-kinase products, the accumulation of phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2] was dramatically and specifically attenuated when ADP was removed by apyrase treatment. A comparison between the effects of PI 3-kinase inhibitors and apyrase strongly suggest that the late, ADP-dependent, PtdIns(3,4)P2accumulation is necessary for PAR1-induced irreversible aggregation. Using selective antagonists, we found that the effect of ADP was due to the ADP receptor coupled to inhibition of adenylyl cyclase. Finally, we found that both ADP and PI 3-kinase play an important role in PAR1-dependent reorganization of the cytoskeleton through a control of myosin heavy chain translocation and the stable association of signaling complexes with the actin cytoskeleton.

A Key Role of Adenosine Diphosphate in the Irreversible Platelet Aggregation Induced by the PAR1-Activating Peptide Through the Late Activation of Phosphoinositide 3-Kinase

AbstractAlthough adenosine diphosphate (ADP), per se, is a weak platelet agonist, its role as a crucial cofactor in human blood platelet functions has now been clearly demonstrated in vitro and in vivo. The molecular basis of the ADP-induced platelet activation is starting to be understood since the discovery that 2 separate P2 purinergic receptors may be involved simultaneously in the activation process. However, little is known about how ADP plays its role as a cofactor in platelet activation and which signaling pathway initiated by a specific agonist can be modulated by the released ADP. To investigate these points, we took advantage of a model of platelet activation through the thrombin receptor PAR1 in which both ADP scavengers and phosphoinositide 3-kinase (PI 3-kinase) inhibitors have been shown to transform the classical irreversible aggregation into a reversible one. We have observed that, among the different PI 3-kinase products, the accumulation of phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2] was dramatically and specifically attenuated when ADP was removed by apyrase treatment. A comparison between the effects of PI 3-kinase inhibitors and apyrase strongly suggest that the late, ADP-dependent, PtdIns(3,4)P2accumulation is necessary for PAR1-induced irreversible aggregation. Using selective antagonists, we found that the effect of ADP was due to the ADP receptor coupled to inhibition of adenylyl cyclase. Finally, we found that both ADP and PI 3-kinase play an important role in PAR1-dependent reorganization of the cytoskeleton through a control of myosin heavy chain translocation and the stable association of signaling complexes with the actin cytoskeleton.

Effects of catechins on human blood platelet aggregation and lipid peroxidation.

The activity of catechins was studied for inhibitory activity in human blood platelets. Platelet aggregation and peroxidation were evaluated in platelet rich plasma (PRP) obtained from samples of healthy volunteers. Human blood platelets were submitted to stimulation with 300 microM arachidonic acid, 3 microM adenosine diphospate (ADP) and 6 microM epinephrine. Treatment with (200 microg/mL) catechin or epicatechin was sufficient to exhibit a potent inhibitory effect of the three agents. The inhibitory effect was dose dependent at concentrations of 20-200 microg/mL. Using malondialdehyde (MDA) as an index of total lipid peroxidation capacity, decreased production of MDA of the platelets treated with catechin or epicatechin after stimulation by arachidonic acid was observed. These findings suggest that catechins protect platelets from peroxidative stress and their aggregation.

Effects of catechins on human blood platelet aggregation and lipid peroxidation

Effects of catechins on human blood platelet aggregation and lipid peroxidation

Effects of ligand-mimetic peptides Arg-Gly-Asp-X (X = Phe, Trp, Ser) on alphaIIbbeta3 integrin conformation and oligomerization.

The purpose of this investigation was to determine what structural changes convert "inert" alphaIIbbeta3 integrins into "activated" high-affinity receptors for adhesive proteins. Light scattering, analytical ultracentrifugation, electron microscopy, and molecular modeling were used to probe the conformational states of the alphaIIbbeta3 integrin. Isolated from human blood platelets in octyl glucoside, the alphaIIbbeta3 complex behaved as an asymmetric 230 kDa macromolecule with a z-average translational diffusion coefficient of 2.9 F and a weight-average sedimentation coefficient of 7.7 S. Dynamic light scattering showed that ligand-mimetic peptides (RGDX, X = F, W, S) caused prompt, concentration-dependent increases in the Stokes radius (R(s)) of the alphaIIbbeta3 complex, whereas control peptides of reversed sequence (XDGR, X = F, W, S) had no significant effect. Sedimentation velocity data coupled with time-derivative analyses showed that RGDX peptides shifted the distribution of alphaIIbbeta3 sedimenting species toward smaller s values. Sedimentation equilibrium measurements indicated that a slower increase in the alphaIIbbeta3 molecular weight distribution took place in the presence of RGDX ligand-mimetics. Electron microscopy showed a split of alphaIIbbeta3's globular domain into two distinct nodules in the presence of RGDX peptides; oligomers joined through their stalk regions were seen frequently. These observations suggest that receptor occupancy by ligand-mimetic RGDX peptides is tightly coupled to relatively large changes in the structure of the alphaIIbbeta3 complex. alphaIIbbeta3 bead models were developed to describe quantitatively the ligand-induced transition from a "closed" to an "open" integrin conformation and the limited oligomerization that follows. This provides a new mechanistic framework for understanding integrin activation and the formation of signaling clusters on the surface of stimulated platelets.

Identification and characterization of LRP8 (apoER2) in human blood platelets

Recently, we reported that apoE inhibits platelet reactivity by stimulating NO release and postulated apoE-receptor activation of intracellular NO synthase (eNOS). Here, we implicate a low density lipoprotein receptor (LDL-R) family member by studying ligand requirements using purified apoE isoforms, synthetic peptides, and the receptor antagonist, receptor-associated protein (RAP). Then, using a homology cloning approach and degenerate PCR primers to amplify the conserved Cys-rich binding domain of the LDL-R family, this receptor was identified as LRP8 (formerly termed, apoER2), a newly described brain protein with several splice variants. Immunoprecipitation of platelet membranes with anti-peptide antisera confirmed protein expression, while analysis of RNA from platelets and two megakaryocytic cell lines (Meg-01 and HEL) disclosed that the major LRP8 transcript lacked binding repeats 4-6 (LRP8delta4-6) but contained the full-length cytoplasmic tail. Sequence analysis of cytoplasmic LRP8 revealed several peptide motifs with potential for cellular signaling and we propose this as a rational mechanism through which apoE inhibits platelet aggregation.

Blood compartmental metabolism of docosahexaenoic acid (DHA) in humans after ingestion of a single dose of [13C]DHA in phosphatidylcholine

Blood compartmental metabolism of docosahexaenoic acid (DHA) in humans after ingestion of a single dose of [13C]DHA in phosphatidylcholine

Aggregation of human blood platelets in the presence of the pyridoindole stobadine

The antiarrhythmic and cardioprotective drug stobadine, possessing antioxidant and neuroprotective properties, was studied as to its in vitro effect on aggregation of human blood platelets. Pretreatment of platelets with stobadine for 30 s inhibited stimulated platelet aggregation in a dose-dependent way. Depending on the aggregation stimulus used, the minimal effective concentrations of the drug were 1 μmol/l (adrenaline), 200 μmol/l (ADP), and 1,000 μmol/l (PMA). Aggregation induced with thrombin or Ca 2+-ionophore A23187 was not changed in the presence of stobadine even in the concentration of 1,000 μmol/l. Addition of stobadine 30 s after adrenaline was also effective and terminated aggregation (100 and 1,000 μmol/l) or prolonged onset of its second phase (10 μmol/l). The presented experiments showed stobadine as a potent inhibitor of adrenaline-induced aggregation, indicating its involvement in the observed antithrombotic and cytoprotective activity.

Effect of the crude extract of Cestrum parqui on carrageenin-induced rat paw oedema and aggregation of human blood platelets.

An extract of Cestrum parqui aerial parts in methanol:water (1:1) showed inhibition of carrageenin-induced oedema. The aggregation of human blood platelets induced by adenosine diphosphate and platelet activating factor was also inhibited (IC(50)s were 3 and 2 mg/mL, respectively). On the contrary, the extract did not inhibit arachidonic acid-mediated platelet aggregation.

Differential effects of adenine nucleotide analogues on shape change and aggregation induced by adnosine 5-diphosphate (ADP) in human platelets.

Adenosine 5'-diphosphate (ADP) induces human blood platelets to aggregate and change shape, and it has been suggested that these two responses are mediated by more than one subtype of ADP receptor. The structure-activity relationships for several analogues of adenine nucleotides in causing aggregation and shape change were measured and compared in washed platelets using an aggregometer. ADP and its analogues 2-methylthioadenosine 5'-diphosphate (2-methylthio-ADP), adenosine 5'(alpha,beta-methylene)diphosphonate (AMPCP), S(P)-adenosine 5'-O-(1-thiodiphosphate) (AD-P alphaS) and adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS) were used as agonists. Adenosine 5'-triphosphate (ATP) and its analogues, P1, P5-diadenosine pentaphosphate (ApsA), adenosine (5'-(alpha,beta-methylene)triphosphonate (AMPCPP), 2-methylthioadenosine 5'-triphosphate (2-methylthio-ATP) and uridine 5'-triphosphate (UTP), as well as the trypanocidal drug suramin, were used as antagonists. In general, the structure-activity relationships for both responses were similar, but for some analogues differences were observed. ADPalphaS and ADPbetaS were much more potent agonists relative to ADP for shape change than for aggregation and indeed ADPalphaS antagonized ADP-induced aggregation with an apparent pK(B) value of 5.5+/-0.1. 2-Methylthio-ATP also had different effects in aggregation and shape change, being a much higher affinity antagonist of aggregation than of shape change with an apparent pK(B) value of 7.0+/-0.2 for aggregation and 5.2+/-0.2 for shape change. These results support the suggestion that these two responses are mediated by multiple ADP receptors on human platelets, and are consistent with shape change being mediated via one receptor (the P2Y1 receptor) with aggregation requiring the activation of two receptors (the P2Y1 and another P2Y receptor).

Pharmacological effects of the leaf-nosed viper snake ( Eristocophis macmahoni) venom and its HPLC fractions

Crude venom from Eristocophis macmahoni was demonstrated to exert a potent inhibition of human blood platelet aggregation mediated by adenosine diphosphate (ADP), platelet activating factor (PAF) and arachidonic acid (AA). The venom caused lysis of the platelets, however, the red blood cells were not lysed by the venom. Substantial oedema was produced upon injection of the venom into the rat hind paw. Contrarily, the intraperitoneal injection of the venom to the rats caused an inhibition of the carrageenin-induced rat paw oedema. However, an 100% lethality within 24 h was observed with a dose of 40 mg/kg body weight. The venom was fractionated by reverse phase high pressure liquid chromatography (HPLC) and the fractions were analyzed for their effect on ADP-induced platelet aggregation. The fraction eluted at 15.5 min (20% acetonitrile concentration) exhibited an inhibitory effect of several-fold greater potency than that of the crude venom. Fractions eluted at 18.5 min (25.4% acetonitrile concentration) and onward showed a pro-aggregatory but insignificant effect. It is suggested that although the venom contains pro-aggregatory components, inhibition of platelet aggregation seems to be its predominant activity.

Are Lipid Phase Transitions Responsible for Chilling Damage in Human Platelets?

In previous studies we have proposed that the well-known chilling-induced activation of human blood platelets can be ascribed at least in part to a thermotropic phase transition in membrane lipids. The evidence that this is the case is reviewed and amplified in this review, followed by an examination of the available physical data concerning phase transitions in lipid mixtures that mimic the mixture found in platelet membranes. Assuming complete mixing at all temperatures and equal contributions of the members of the mixture to the phase transition, the lipid mixture found in platelets should give values forTmranging from about 1°C to about 16°C, depending on the isomers present in the mixture. (The former value is not in agreement with the observedTm, but the latter is in excellent agreement.) However, examination of the phase diagram for a binary pair of lipids found in platelet membranes shows that ideal mixing almost certainly does not occur; instead of a linear phase diagram, a convex one was obtained. This shape for the phase diagram, which would displaceTmto an unexpectedly elevated temperature, is in agreement with previously published phase diagrams for mixtures of this type. The prediction, based on thermodynamic properties of lipids found in the platelets, is thatTmwill be displaced upward in more complex mixtures of the composition found in platelets, a prediction that requires experimental testing.

Determination of Human Platelet Membrane Permeability Coefficients Using the Kedem–Katchalsky Formalism: Estimates from Two- vs Three-Parameter Fits

Attempts to cryopreserve human blood platelets have resulted in poor postthaw survival rates and have been inadequate for routine clinical application. As a result, most blood banks maintain platelets in nonfrozen solutions. Using this approach, platelets can be stored for only about 5 days and are then discarded. This situation greatly limits the use of platelet transfusion in clinical practice. Information regarding fundamental cryobiological characteristics can be applied to predict platelet response to cryoprotective agent (CPA) addition/removal and to cooling/warming. Methods can then be engineered to optimize cryopreservation procedures, thereby minimizing platelet damage and maximizing postthaw recovery. It was therefore the purpose of this study to determine some of the necessary biophysical parameters required for this process: (i) plasma membrane hydraulic conductivity (Lp), (ii) cryoprotectant solute permeability coefficient (Ps), (iii) the associated reflection coefficient (ς), and (iv) their activation energies. The CPAs studied included dimethyl sulfoxide (Me2SO) and propylene glycol at 1.5 M concentration. Permeability was measured at 22, 10, and 4°C using a modified Coulter counter in conjunction with a water-jacketed beaker system for temperature regulation. The Kedem–Katchalsky formalism was used to estimate the parameters using: (1) a three-parameter fit and (2) a two-parameter fit in which a noninteracting value of ς was calculated. Two-parameter estimates were in closer agreement with previously published values, and these were used in a model to simulate addition and removal of 0.64 M (5%) and 1.0 M (7.8%) Me2SO, the most common CPA currently used in empirically determined platelet cryopreservation protocols.

Inhibitory effect of andrographolide from Andrographis paniculata on PAF-induced platelet aggregation

Andrographolide, an active principle of the Chinese drug Andrographis paniculata, used for prevention and treatment of common cold in Scandinavia and known as an antiinflammatory, antiviral, antithrombotic, hypotensive and antiatherosclerotic drug, was investigated for its suggested influence on the biosynthesis of eicosanoids and the platelet-activating factor (PAF). Whereas in isolated human polymorph-nuclear leukocytes (PMNL) no influence on the biosynthesis was found, it could be shown that andrographolide inhibits PAF-induced human blood platelet aggregation in a dose dependent manner (IC50-5 microM). These results indicate that andrographolide has a mechanism of action different from that of non-steroidal antiinflammatory drugs (NSAID) and most likely associated with the cardiovascular and antithrombotic activity described of Andrographis paniculata.

Genetic variation in the serotonin transporter promoter region affects serotonin uptake in human blood platelets

Genetic variation in the serotonin transporter promoter region affects serotonin uptake in human blood platelets

2E4 (Kaptin): A novel actin-associated protein from human blood platelets found in lamellipodia and the tips of the stereocilia of the inner ear

Platelet activation, crucial for hemostasis, requires actin polymerization, yet the molecular mechanisms by which localized actin polymerization is mediated are not clear. Here we report the characterization of a novel actin-binding protein, 2E4, originally isolated from human blood platelets and likely to be involved in the actin rearrangements occurring during activation. 2E4 binds to filamentous (F)-actin by F-actin affinity chromatography and is eluted from F-actin affinity columns and extracted from cells with ATP. Its presence at the leading edge of platelets spread on glass and in the lamellipodia of motile fibroblasts suggests a role in actin dynamics. Using localization to obtain clues about function, we stained the sensory epithelium of the embryonic inner ear to determine whether 2E4 is at the barbed end of actin filaments during their elongation. Indeed, 2E4 was present at the tips of the elongating stereocilium. 2E4 is novel by DNA sequence and has no identifiable structural motifs. Its unusual amino acid sequence, its ATP-sensitive actin association and its location at sites of actin polymerization in cells suggest 2E4 plays a unique role in the actin rearrangements that accompany platelet activation and stereocilia formation.

Genetic variation in the serotonin transporter promoter region affects serotonin uptake in human blood platelets

The human serotonin transporter (5-HTT), encoded by a single gene on chromosome 17q11.2, is expressed in brain and blood cells. 5-HTT is implicated in mood and anxiety regulation, and is where antidepressant and antianxiety drugs initially act in the brain. A 5-HTT-linked promoter region (5-HTTLPR) insertion/deletion polymorphism with long (l) and short (s) forms affects transporter expression and function. The s variant reduced 5-HTT gene transcription in a reporter gene construct and human lymphoblasts, resulting in reduced transporter levels and 5-HT uptake, acting as a dominant allele. In this study, we investigated the expression and function of 5-HTT in platelets from healthy male volunteers. The l variant was associated with more rapid initial platelet 5-HT uptake (Vmax), the index of platelet 5-HTT function most clearly heritable, while the s allele was dominant. The 5-HTTLPR genotype had no effect on platelet [3H]paroxetine binding (Bmax), affinity for [3H]5-HT or [3H]paroxetine, or 5-HT content. The 5-HT uptake findings support a functional difference in the two 5-HTTLPR variants, reinforcing their attractiveness as candidate genes in neuropsychiatric research.

Inhibition of platelet aggregation by S-nitroso-cysteine via cGMP-independent mechanisms: evidence of inhibition of thromboxane A 2 synthesis in human blood platelets

S-Nitroso-cysteine (SNC), a putative endothelium-derived relaxing factor, potently inhibited collagen- and arachidonic acid-induced platelet aggregation (IC 50=100 nM) and thromboxane A 2 (TxA 2) synthesis of human blood platelets. ODQ, a selective inhibitor of the soluble guanylyl cyclase, inhibited SNC-induced formation of cGMP but did not reverse inhibition by SNC of collagen- and arachidonic acid-induced platelet aggregation. Combination of ODQ with SQ-29548, a specific platelet TxA 2 receptor antagonist, did not modify the antiaggregatory action of SNC. Our study shows that SNC inhibits platelet aggregation by cGMP-independent mechanisms that may involve inhibition of TxA 2 synthesis in human platelets.