Thrombin-stimulated Platelets Research Articles

AMPK inhibition protects against arterial thrombosis while sparing hemostasis through differential modulation of platelet responses

AMP-activated protein kinase (AMPK) is a metabolic master switch that has critical role in wide range of pathologies including cardiovascular disorders. As AMPK-α2 knockout mice exhibit impaired thrombus stability, we asked whether pharmacological inhibition of AMPK with a specific small-molecule inhibitor, compound C, could protect against arterial thrombosis without affecting hemostasis. Mice pre-administered with compound C exhibited decreased mesenteric arteriolar thrombosis but normal tail bleeding time compared to vehicle-treated animals. Compound C potently restricted platelet aggregation, clot retraction and integrin activation induced by thrombin and collagen. It impaired platelet spreading on both immobilized fibrinogen and collagen matrices; it, however, had no significant effect on thrombin-induced phosphatidylserine exposure that is characteristic of procoagulant platelets. In parallel, compound C brought about significant drop in thrombin-induced phosphorylation of myosin light chain (MLC) and MLC phosphatase (MYPT1) as well as abrogated rise in level of RhoA-GTP in thrombin-stimulated platelets. Thus, effects of compound C on agonist-induced platelet responses could be at least in part attributed to modulation of cytoskeletal changes mediated by RhoA-MYPT1-MLC signaling. An ideal antithrombotic drug would spare hemostatic responses that maintain vascular integrity while preferentially protecting against thrombosis. The present study suggests that AMPK could be one such potential therapeutic target.

The antithrombotic effect of caffeic acid is associated with a cAMP-dependent pathway and clot retraction in human platelets

Caffeic acid (CA) is a polyphenol widely distributed in the plant kingdom. Studies have shown CA possesses antithrombotic activity in mouse cerebral arterioles in vivo and inhibits platelet aggregation in vitro. However, little is known regarding the effects of CA on platelet-mediated clot retraction. We investigated the effects of CA on platelet activation and clot retraction in response to thrombin. CA inhibited thrombin-induced platelet aggregation, calcium mobilization, adenosine 1,4,5-tri-phosphate (ATP) release, P-selectin expression and fibrinogen binding to integrin αIIbβ3 activation without inducing any cytotoxic effect, and inhibited the phosphorylations of protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) in thrombin-stimulated platelets. In addition, CA enhanced cyclic adenosine monophosphate (cAMP) generation, which led to the phosphorylations of vasodilator-stimulated phosphoprotein (VASP) and inositol trisphosphate (IP3) receptor, and reduced clot retraction without any anticoagulation effect. Dipyridamole, a phosphodiesterase 3 (PDE3) inhibitor, reduced clot retraction, which suggested CA-mediated cAMP generation is the main signaling pathway responsible for its inhibition of clot retraction. Taken together, the findings of the present study suggest that CA may have potential as a therapeutic for the prevention of thrombotic disorders.

Salidroside inhibits platelet function and thrombus formation through AKT/GSK3β signaling pathway.

Salidroside is the main bioactive component in Rhodiola rosea and possesses multiple biological and pharmacological properties. However, whether salidroside affects platelet function remains unclear. Our study aims to investigate salidroside’s effect on platelet function. Human or mouse platelets were treated with salidroside (0-20 μM) for 1 hour at 37°C. Platelet aggregation, granule secretion, and receptors expression were measured together with detection of platelet spreading and clot retraction. In addition, salidroside (20 mg/kg) was intraperitoneally injected into mice followed by measuring tail bleeding time, arterial and venous thrombosis. Salidroside inhibited thrombin- or CRP-induced platelet aggregation and ATP release and did not affect the expression of P-selectin, glycoprotein (GP) Ibα, GPVI and αIIbβ3. Salidroside-treated platelets presented decreased spreading on fibrinogen or collagen and reduced clot retraction with decreased phosphorylation of c-Src, Syk and PLCγ2. Additionally, salidroside significantly impaired hemostasis, arterial and venous thrombus formation in mice. Moreover, in thrombin-stimulated platelets, salidroside inhibited phosphorylation of AKT (T308/S473) and GSK3β (Ser9). Further, addition of GSK3β inhibitor reversed the inhibitory effect of salidroside on platelet aggregation and clot retraction. In conclusion, salidroside inhibits platelet function and thrombosis via AKT/GSK3β signaling, suggesting that salidroside may be a novel therapeutic drug for treating thrombotic or cardiovascular diseases.

Stepping Up to the Plate(let) against Candida albicans.

Candida albicans is a pervasive commensal fungus that is the most common pathogen responsible for invasive fungal infection (IFI). With incidence of IFI on the rise due to increasing susceptible populations, it is imperative that we investigate how Candida albicans interacts with blood components. When stimulating either human or mouse whole blood with thrombin, we saw a significant decrease in C. albicans survival. We then repeated Candida killing assays with thrombin-stimulated or unstimulated washed platelets and saw a similar decrease in CFU. To investigate whether killing was mediated through surface components or releasable products, platelets were pretreated with an inhibitor of actin polymerization (cytochalasin D [CytoD]). CytoD was able to abrogate C. albicans killing. Moreover, dilution of releasates from thrombin-stimulated platelets showed that the toxicity of the releasates on C. albicans is concentration dependent. We then investigated C. albicans actions on platelet activation, granule release, and aggregation. While C. albicans does not appear to affect alpha or dense granule release, C. albicans exerts a significant attenuation of platelet aggregation to multiple agonists. These results illustrate for the first time that platelets can directly kill C. albicans through release of their granular contents. Additionally, C. albicans can also exert inhibitory effects on platelet aggregation.

Time-dependent interactions of blood platelets and cancer cells, accompanied by extramedullary hematopoiesis, lead to increased platelet activation and reactivity in a mouse orthotopic model of breast cancer - implications for pulmonary and liver metastasis.

Aging has become a significant risk factor for several diseases, including breast cancer.Platelet activation and platelet-cancer cell aggregate fractions were found to increase with tumor progression in a mouse model of breast cancer. At advanced stages of tumor development, platelets from mice with breast cancer were hyperreactive to low agonist concentrations and hyporeactive to high ones. Platelet activation and reactivity were strongly associated with breast cancer metastasis in the lungs and extramedullary hematopoiesis in the liver. A greater fraction of platelet aggregates was observed in 4T1-injected mice at the advanced stages of breast cancer. In vitro, platelet activation was elevated after incubation with 4T1 cells, and thrombin-stimulated platelets formed aggregates with 4T1 cells. Neither GPIbα, nor GPIIb/IIIa blocking antibodies, were able to affect platelet-cancer cell aggregation in vitro.The primed circulating platelets became more sensitive to subthreshold stimuli at advanced stages of tumor development, and the formation of platelet-cancer cell aggregates increased with cancer progression. Our findings demonstrate that the age-associated progression of breast cancer cells is connected with increased platelet functioning, and that it can be manifested by the increased number of metastases and extramedullary hematopoiesis in a time-dependent-manner.

The Cell Cycle Checkpoint System MAST(L)-ENSA/ARPP19-PP2A is Targeted by cAMP/PKA and cGMP/PKG in Anucleate Human Platelets.

The cell cycle is controlled by microtubule-associated serine/threonine kinase-like (MASTL), which phosphorylates the cAMP-regulated phosphoproteins 19 (ARPP19) at S62 and 19e/α-endosulfine (ENSA) at S67and converts them into protein phosphatase 2A (PP2A) inhibitors. Based on initial proteomic data, we hypothesized that the MASTL-ENSA/ARPP19-PP2A pathway, unknown until now in platelets, is regulated and functional in these anucleate cells. We detected ENSA, ARPP19 and various PP2A subunits (including seven different PP2A B-subunits) in proteomic studies of human platelets. ENSA-S109/ARPP19–S104 were efficiently phosphorylated in platelets treated with cAMP- (iloprost) and cGMP-elevating (NO donors/riociguat) agents. ENSA-S67/ARPP19-S62 phosphorylations increased following PP2A inhibition by okadaic acid (OA) in intact and lysed platelets indicating the presence of MASTL or a related protein kinase in human platelets. These data were validated with recombinant ENSA/ARPP19 and phospho-mutants using recombinant MASTL, protein kinase A and G. Both ARPP19 phosphorylation sites S62/S104 were dephosphorylated by platelet PP2A, but only S62-phosphorylated ARPP19 acted as PP2A inhibitor. Low-dose OA treatment of platelets caused PP2A inhibition, diminished thrombin-stimulated platelet aggregation and increased phosphorylation of distinct sites of VASP, Akt, p38 and ERK1/2 MAP kinases. In summary, our data establish the entire MASTL(like)–ENSA/ARPP19–PP2A pathway in human platelets and important interactions with the PKA, MAPK and PI3K/Akt systems.

Modulation of Thrombin-Induced Platelet Activation By Defibrotide

Defibrotide is a mixture of single-stranded phosphodiester oligonucleotides 9-80 bases in length derived from mammalian tissue and it is the only medication that is FDA approved for the prevention and treatment of post-transplant veno-occlusive disease, also known as sinusoidal obstructive syndrome (VOD/SOS). The mechanism of action of defibrotide is not well understood. A better understanding of the molecular mechanism by which defibrotide prevents and reverses the microvascular occlusion seen in VOD/SOS will be critical in the development of novel therapeutics for VOD/SOS and other related disorders. Our central hypothesis is that single stranded phosphodiester oligonucleotide mixtures such as defibrotide alter thrombin-induced platelet and endothelial cell activation primarily via altered signaling through protease activated receptors (PARs). To determine the effect of defibrotide on thrombin-induced platelet activation we performed flow cytometry on washed platelets from humans and mice. Washed platelets were incubated with agonist (1 nM thrombin, 100 μM synthetic PAR1 agonist TRAP6, or 100 μM synthetic PAR4 agonist PAR4AP) in the presence or absence of 1 mg/mL of defibrotide. Fluorescent antibodies against P-selectin (anti-CD62P antibody) and against the high affinity conformation of the αIIβ3 receptor (PAC1 antibody for human samples and JON/A antibody for murine samples) were added to assess for inhibition of platelet activation by defibrotide. Fluorescent anti-PAR1 antibodies SPAN12 (which spans the canonical thrombin cleavage site) and WEDE15 (which binds at the hirudin-like domain) were also added to separate samples to assess whether defibrotide inhibits cleavage of the PAR1 N-terminal domain by thrombin. After twenty minutes of incubation at room temperature the samples were analyzed by flow cytometry. We calculated the geometric mean of the fluorescence intensity (MFI) for three replicate samples and data are shown as the fold change compared to the washed platelet control without addition of agonist or defibrotide. For thrombin-stimulated human platelets, both anti-CD62P binding (MFI fold over control 5.7 vs. 1.2) and PAC1 antibody binding (MFI fold over control 7.1 vs. 1.7) are significantly reduced in the presence of defibrotide (See Figure 1a). For thrombin-stimulated murine platelets there is a small decrease in anti-CD62P antibody binding (MFI fold over control 8.4 vs. 6.6), but no significant change in JON/A antibody binding (See Figure 1b). Binding of both anti-CD62P and PAC1 antibodies to human platelets stimulated with synthetic PAR agonists was unaffected by the presence of defibrotide (See Figure 1c). There is no significant difference in SPAN12 or WEDE15 binding in thrombin-stimulated platelets in the absence or presence of defibrotide. However, defibrotide alone does appear to interfere with WEDE15 binding in unstimulated platelets (See Figure 1d). In conclusion, we showed that defibrotide inhibits thrombin-induced human platelet activation at low concentrations of thrombin, as shown by a decrease in surface platelet activation markers measured by flow cytometry. This effect is much less pronounced under the same conditions if mouse platelets are used, supporting the hypothesis that defibrotide inhibits platelet activation by alteration of PAR1 receptor signaling, since PAR1 receptors are not present on mouse platelets. Defibrotide does not interfere with TRAP6-induced platelet activation, evidence against interference with the intracellular portion of the signaling pathway, since TRAP6 is a synthetic PAR1 agonist that binds directly into the ligand-binding pocket of the receptor, eliminating the need for cleavage of the PAR1 extracellular domain. The decrease in WEDE15 antibody binding to washed platelets seen with defibrotide alone suggests that the mechanism may involve alterations in the hirudin-like domain of the PAR1 receptor which is crucial for thrombin binding and cleavage site specificity. Ongoing studies including investigating the effect of defibrotide on peptide mimics of the extracellular N-terminal domain of the PAR1 receptor and the effect of defibrotide on the activation of endothelial cells will likely shed light on the exact mechanism of platelet response modulation by defibrotide and may provide new targets for treatment of the microvascular occlusion that characterizes VOD/SOS. Disclosures No relevant conflicts of interest to declare.

Fatal dysfunction and disintegration of thrombin-stimulated platelets.

Platelets play a key role in the formation of hemostatic clots and obstructive thrombi as well as in other biological processes. In response to physiological stimulants, including thrombin, platelets change shape, express adhesive molecules, aggregate, and secrete bioactive substances, but their subsequent fate is largely unknown. Here we examined late-stage structural, metabolic, and functional consequences of thrombin-induced platelet activation. Using a combination of confocal microscopy, scanning and transmission electron microscopy, flow cytometry, biochemical and biomechanical measurements, we showed that thrombin-induced activation is followed by time-dependent platelet dysfunction and disintegration. After ~30 minutes of incubation with thrombin, unlike with collagen or ADP, human platelets disintegrated into cellular fragments containing organelles, such as mitochondria, glycogen granules, and vacuoles. This platelet fragmentation was preceded by Ca2+ influx, integrin αIIbβ3 activation and phosphatidylserine exposure (activation phase), followed by mitochondrial depolarization, generation of reactive oxygen species, metabolic ATP depletion and impairment of platelet contractility along with dramatic cytoskeletal rearrangements, concomitant with platelet disintegration (death phase). Coincidentally with the platelet fragmentation, thrombin caused calpain activation but not activation of caspases 3 and 7. Our findings indicate that the late functional and structural damage of thrombin-activated platelets comprise a calpain-dependent platelet death pathway that shares some similarities with the programmed death of nucleated cells, but is unique to platelets, therefore representing a special form of cellular destruction. Fragmentation of activated platelets suggests that there is an underappreciated pathway of enhanced elimination of platelets from the circulation in (pro)thrombotic conditions once these cells have performed their functions.

Fatal Dysfunction and Fragmentation of Thrombin-Stimulated Platelets

Introduction: Platelets play a key role in formation of protective hemostatic blood clots and pathological obstructive thrombi. Under (patho)physiological conditions, chemically activated platelets change their morphology, express adhesive molecules, undergo aggregation, secrete procoagulant substances, and induce mechanical contraction (retraction) of the blood clots. Despite the vital importance of these platelet functions, the subsequent fate of activated platelets is largely unknown. We hypothesize that activated platelets undergo late alterations that determine their fate and may have a pathogenic importance in thrombotic and hemostatic disorders.Methods: We used a combination of confocal microscopy, immunofluorescence, scanning and transmission electron microscopy, flow cytometry, biochemical and biomechanical measurements to study deferred structural, metabolic, and functional consequences of thrombin-induced activation of viable human platelets, either suspended in platelet-rich plasma or isolated by gel-filtration.Results: Visualized by confocal microscopy, fluorescently labeled platelets in thrombin-induced plasma clots initially underwent shape changes characteristic of platelet activation, but in about 30 min many platelets and platelet aggregates broke up into organelle-containing vesicular fragments. There were two types of platelet-derived vesicles differing in their size and cellular origin: one smaller type was shedding from the tips of filopodia, while the other type resulted from fragmentation of platelet bodies. Concurrently with the fragmentation, thrombin-activated platelets displayed dramatically altered intracellular distribution of F-actin and septins detected as intense fluorescent clusters with a ~2-fold increase in the intensity of septins 2 and 9 and a ~300-fold increase in the F-actin staining. Synchronously with the structural alterations, thrombin induced a time-dependent reduction of the mitochondrial membrane potential (Δψm) in platelets. The overall fluorescence intensity of the Δψm-sensitive MitoTracker dye in freshly formed thrombin-initiated plasma clots dropped 2- and 4-fold after 60 min and 90 min, respectively. A drop of Δψm inversely correlated with an increase of the fraction of disintegrated platelets (r=-0.93, p<0.01). Flow cytometry showed enhanced phosphatidylserine exposure in thrombin-activated platelets, either with or without mitochondrial depolarization. Thrombin caused a significant 59% decrease of the average ATP content in activated platelets relative to untreated platelets after 60 min of incubation. Remarkably, the initial drop of Δψm and ATP content was almost concurred with the termination of contraction of the platelet-rich plasma clot measured as a 90%-decrease of platelet-generated contractile stress. Unexpectedly, no activation of caspase 3/7 was detected in platelets after 90 min of treatment with thrombin. Meanwhile, calpain activity detected in platelets 90 min after thrombin treatment was 6.5-fold higher compared to untreated platelets. Moreover, calpain inhibition caused a ~30-min delay in the commencement of thrombin-induced platelet fragmentation.Conclusions: Our findings indicate that following thrombin-induced platelet activation, a substantial fraction of platelets undergo time-dependent dysfunction and structural disintegration into subcellular particles. The fragmentation of platelets is accompanied by dramatic rearrangements of platelet cytoskeletal components, including polymerization, clustering, and redistribution of actin and septins. Thrombin-induced platelet fragmentation is concurrent with severe impairment of platelet functionality, including mitochondrial depolarization, ATP depletion, and loss of platelet contractility. The lack of caspase activity and increased calpain activity in energetically exhausted thrombin-treated platelets undergoing fragmentation suggests a calpain-dependent platelet death pathway. These studies indicate that such a form of platelet death may be an underappreciated mechanism for enhanced elimination of platelets from the circulation in (pro)thrombotic conditions or under other conditions once they have performed their functions. Work supported by the Program for Competitive Growth at KFU and AHA grant 17SDG33680177. DisclosuresNo relevant conflicts of interest to declare.

Platycodin D inhibits platelet function and thrombus formation through inducing internalization of platelet glycoprotein receptors

BackgroundPlatycodin D (PD) is one of the major bioactive components of the roots of Platycodon grandiflorum and possesses multiple biological and pharmacological properties, such as antiviral, anti-inflammatory, and anti-cancer activities. However, whether it affects platelet function remains unclear. This study aims to evaluate the role of PD in platelet function and thrombus formation.MethodsPlatelets were treated with PD followed by measuring platelet aggregation, activation, spreading, clot retraction, expression of glycoprotein receptors. Moreover, mice platelets were treated with PD and infused into wild-type mice for analysis of in vivo hemostasis and arterial thrombosis.ResultsPlatycodin D treatment significantly inhibited platelet aggregation in response to collagen, ADP, arachidonic acid and epinephrine, reduced platelet P-selectin expression, integrin αIIbβ3 activation, spreading on fibrinogen as well as clot retraction, accompanied with decreased phosphorylation of Syk and PLCγ2 in collagen-related peptide or thrombin-stimulated platelets. Moreover, PD-treated mice platelets presented significantly impaired in vivo hemostasis and arterial thrombus formation. Interestingly, PD induced internalization of glycoprotein receptors αIIbβ3, GPIbα and GPVI. However, GM6001, cytochalasin D, BAPTA-AM and wortmannin did not prevent PD-induced internalization of receptors.ConclusionsOur study demonstrates that PD inhibits platelet aggregation, activation and impairs hemostasis and arterial thrombosis, suggesting it might be a potent anti-thrombotic drug.

SNARE-dependent membrane fusion initiates α-granule matrix decondensation in mouse platelets

AbstractPlatelet α-granule cargo release is fundamental to both hemostasis and thrombosis. Granule matrix hydration is a key regulated step in this process, yet its mechanism is poorly understood. In endothelial cells, there is evidence for 2 modes of cargo release: a jack-in-the-box mechanism of hydration-dependent protein phase transitions and an actin-driven granule constriction/extrusion mechanism. The third alternative considered is a prefusion, channel-mediated granule swelling, analogous to the membrane “ballooning” seen in procoagulant platelets. Using thrombin-stimulated platelets from a set of secretion-deficient, soluble N-ethylmaleimide factor attachment protein receptor (SNARE) mutant mice and various ultrastructural approaches, we tested predictions of these mechanisms to distinguish which best explains the α-granule release process. We found that the granule decondensation/hydration required for cargo expulsion was (1) blocked in fusion-protein-deficient platelets; (2) characterized by a fusion-dependent transition in granule size in contrast to a preswollen intermediate; (3) determined spatially with α-granules located close to the plasma membrane (PM) decondensing more readily; (4) propagated from the site of granule fusion; and (5) traced, in 3-dimensional space, to individual granule fusion events at the PM or less commonly at the canalicular system. In sum, the properties of α-granule decondensation/matrix hydration strongly indicate that α-granule cargo expulsion is likely by a jack-in-the-box mechanism rather than by gradual channel-regulated water influx or by a granule-constriction mechanism. These experiments, in providing a structural and mechanistic basis for cargo expulsion, should be informative in understanding the α-granule release reaction in the context of hemostasis and thrombosis.

Rapid kinetics of changes in oxygen consumption rate in thrombin-stimulated platelets measured by high-resolution respirometry

Platelet activation plays a key role in normal haemostasis and pathological thrombosis. Platelet activation is rapid; within minutes of stimulation, platelets generate bioactive phospholipids, secrete their granule contents, activate integrins and aggregate together to form a haemostatic plug. These events are dependent on ATP synthesis. Mitochondrial function in platelets from healthy volunteers and patients with a range of diseases indicate an important role for oxygen consumption in oxidative phosphorylation in normal and pathological function. Platelets also consume oxygen during oxidation reactions, such as cyclooxygenase-dependent thromboxane A2 synthesis. In this study, we used high-resolution respirometry to investigate rapid changes in oxygen consumption during platelet activation. We demonstrated a rapid, transient increase in oxygen consumption rate within minutes of platelet stimulation by the physiological activator, thrombin. This was partly inhibited by aspirin and by oligomycin. This shows that high resolution respirometry can provide information regarding rapid and dynamic changes in oxygen consumption during platelet activation.

P3786The role of rho kinases in thrombin-stimulated platelets and monocyte platelet aggregates in atherothrombosis

P3786The role of rho kinases in thrombin-stimulated platelets and monocyte platelet aggregates in atherothrombosis

Abstract 015: PAR4 Ala120Thr Variant Alters PAR4 Desensitization, Sensitivity to Platelet Antagonists and Risk of Large Vessel Stroke

F2RL3 encodes protease-activated receptor 4 (PAR4) and harbors a A/G SNP (rs773902) with racially dimorphic allelic frequencies. This SNP mediates an alanine to threonine substitution at residue 120 that alters platelet PAR4 activation by the artificial PAR4-activation peptide (PAR4-AP), AYPGKF, which does not occur in vivo. Platelet function studies from healthy human donors demonstrated that compared to rs773902 GG donors, platelets from rs773902 AA donors had increased aggregation to low concentrations (&lt;1 nM) of thrombin, increased Ca 2+ signaling, increased granule secretion and decreased PAR4 desensitization. The increased thrombin sensitivity of rs773902 AA platelets required both PAR4 and PAR1. Whereas receptor cleavage, dimerization, or lipid raft localization were not different between platelets from the two F2RL3 genotypes, rs773902 AA platelets required more than 3-fold amounts of PAR4-AP for receptor desensitization. PAR4-AP stimulation of PAR4-transfected COS-7 cells increased PAR4 levels on the cell surface. Thrombin-stimulated platelets from rs773902 AA donors displayed reduced inhibition by P2Y12 and PAR1 blockers, but not aspirin. Finally, the association of rs773902 alleles with stroke was assessed using the Stroke Genetics Network (SiGN) study. Patients carrying at least one copy of the rs773902 A allele had an increased risk of both large vessel stroke and diabetes in the SiGN dataset. In summary, both PAR4 and PAR1 are required for platelet activation to sub-nanomolar thrombin concentrations. Compared to rs773902 GG platelets, rs773902 AA platelets have a lower EC50 for thrombin and are relatively resistant to receptor desensitization, perhaps due to anterograde trafficking of internal PAR4 to the plasma membrane post-stimulation. The association of the rs773902 A variant with large vessel stroke, diabetes and partial resistance to PAR1 and P2Y12 inhibition must be considered for development of personalized anti-platelet agents.

Mirabegron, a β3-adrenoceptor agonist reduced platelet aggregation through cyclic adenosine monophosphate accumulation

Mirabegron is a β3-adrenoceptor agonist and released on the marked for the treatment of overactive bladder. Because mirabegron is the only β3-adrenoceptor agonist available and substances that increase the levels of cyclic adenosine monophosphate (cAMP) inhibit platelet activity, we tested the hypothesis that mirabegron could have antiplatelet activity. Collagen- and thrombin induced platelet aggregation, thromboxane B2 (TXB2) and cyclic nucleotides quantification and calcium (Ca2+) mobilization were determined in the absence and presence of mirabegron in human washed platelets. Our results revealed that mirabegron (10−300 µM) produced significant inhibitions on platelet aggregation induced by collagen- or thrombin, accompanied by greater intracellular levels of cAMP. The β3-adrenoceptor antagonist L 748,337 (1 µM) and the adenylate cyclase inhibitor, SQ 22,536 (100 µM) reversed the inhibition induced by mirabegron in thrombin-stimulated platelets. The selective antagonists for β1-and β2-adrenoceptors, atenolol and ICI 117,551 (3 µM), respectively did not interfere on the inhibition induced by mirabegron. In Fluo-4 loaded platelets, mirabegron reduced the total and intracellular Ca2+ levels. Pre-incubation with mirabegron almost abolished the levels of TXB2. Mirabegron did not augment the intracellular levels of cyclic guanosine monophosphate. In conclusion, mirabegron inhibited human platelet aggregation through cAMP accumulation, thus suggesting that substances that activate β3-adrenoceptor could be beneficial as adjuvant antiplatelet therapy.

Functional links between Disabled‐2 Ser723 phosphorylation and thrombin signaling in human platelets

Background Disabled-2 (Dab2) is a platelet protein that is functionally involved in thrombin signaling in mice. It is unknown whether or not Dab2 undergoes phosphorylation during human platelet activation. Objectives To investigate the phosphorylation status of Dab2 and its functional consequences in thrombin-stimulated human platelets. Methods Dab2 was immunoprecipitated from resting and thrombin-stimulated platelet lysates for differential isotopic labeling. After enrichment of the phosphopeptides, the phosphorylation sites were analyzed by mass spectrometry. The corresponding phospho-specific antibody was generated. The protein kinases responsible for and the functional significance of Dab2 phosphorylation were defined by the use of signaling pathway inhibitors/activators, protein kinase assays, and various molecular approaches. Results Dab2 was phosphorylated at Ser227, Ser394, Ser401 and Ser723 in thrombin-stimulated platelets, with Ser723 phosphorylation being the most significantly increased by thrombin. Dab2 was phosphorylated by protein kinase C at Ser723 in a Gαq -dependent manner. ADP released from the stimulated platelets further activated the Gβγ -dependent pathway to sustain Ser723 phosphorylation. The Cbl-interacting protein of 85 kDa (CIN85) bound to Dab2 at a motif adjacent to Ser723 in resting platelets. The consequence of Ser723 phosphorylation was the dissociation of CIN85 from the Dab2-CIN85 complex. These molecular events led to increases in fibrinogen binding and platelet aggregation in thrombin-stimulated platelets by regulating αIIb β3 activation and ADP release. Conclusions Dab2 Ser723 phosphorylation is a key molecular event in thrombin-stimulated inside-out signaling and platelet activation, contributing to a new function of Dab2 in thrombin signaling.

Structure-activity relationship of three synthesized benzimidazole-based oligosaccharides in human platelet activation.

Antiplatelet agents have considerable benefits in the treatment of thromboembolic diseases; however, these agents still have substantial limitations due to their severe side-effects. In this study, the antiplatelet activity of three newly synthesized saccharide based benzimidazole derivatives, M3BIM, Malto-BIM and Melibio-BIM, in collagen and thrombin-stimulated human platelets invitro was examined. Among the compounds tested, only compound M3BIM exerted concentration(20-60µM)-dependent inhibitory effects against collagen(1µg/ml) and thrombin(0.01U/ml)-induced washed human platelet aggregation. Moreover, at a concentration of 60µM, M3BIM distinctly abolished collagen-induced adenosine triphosphate(ATP) release and intracellular Ca2+mobilization. Additionally, this compound attenuated the collagen-induced phosphorylation of p47, a marker of the activation of protein kinaseC(PKC) and p38mitogen-activated protein kinase(MAPK). However, Malto-BIM and Melibio-BIM were not effective in this regard. Moreover, the toxic effects of these compounds were evaluated using zebrafish embryo toxicity(ZET) assay, and the results revealed that all three compounds had no comparative cytotoxicity within the range of 25-200µM. Overall, the results of this study provide evidence for the inhibitory effects of M3BIM on collagen-induced platelet aggregation invitro compared to other imidazole derivatives. The presence of 1-imidazolyl moiety at one end with a longer chain length(three sugar moieties) may be mainly responsible for the observed effects of M3BIM. These results suggest that compound M3BIM may be used as a potential candidate for the treatment of aberrant platelet activation-related diseases as it inhibits the activation of p47 and p38MAPK, and reduces ATP release and Ca2+ mobilization.

Relative distribution and biological characterization of CXCL4L1 isoforms in platelets from healthy donors

CXCL4L1, a platelet-derived ELR-negative CXC chemokine, is a powerful angiostatic and anti-tumoral chemokine. We developed a mass spectrometric assay for the detection of different natural CXCL4L1 isoforms. Using this assay, we identified 4 different CXCL4L1 isoforms in the supernatant of thrombin-stimulated platelets from healthy volunteers: the classical isoform CXCL4L1(1-70), CXCL4L1(-4-70), which probably arises through alternative signal peptide removal and two COOH-terminally truncated isoforms CXCL4L1(1-69) and CXCL4L1(-4-69). CXCL4L1(1-70) was the most abundant isoform, whereas CXCL4L1(-4-70) was detected in 50% of the platelet preparations. Since alterations to the NH2-terminus of chemokines can have severe biological consequences, we investigated the impact of the extension with 4 NH2-terminal amino acids on the biological activity of CXCL4L1. In vitro, CXCL4L1(-4-70) was as potent as CXCL4L1(1-70) in inhibiting signal transduction and migration of human microvascular endothelial cells towards vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2). In a FITC-conjugated dextran cell permeability assay, both splice variants showed a strong but comparable anti-permeable effect upon VEGF stimulation of the endothelial cell monolayer. In vivo angiogenesis induced by FGF-2 was equally reduced by CXCL4L1(1-70) and CXCL4L1(-4-70). In chemotaxis assays with CXCR3A-transfected cells the CXCL4L1 isoforms both induced migration from 125ng/ml onward. Finally, CXCL4L1(1-70) and CXCL4L1(-4-70) showed the same affinity for heparin. In conclusion, the investigated biological activities of CXCL4L1 are not influenced by the four extra NH2-terminal residues present in the alternatively spliced isoform CXCL4L1(-4-70). Therefore, our results suggest that both isoforms equally interact with the CXCR3A and CXCR3B receptor.

Thrombin-induced cytoskeleton dynamics in spread human platelets observed with fast scanning ion conductance microscopy

Platelets are small anucleate blood cells involved in haemostasis. Platelet activation, caused by agonists such as thrombin or by contact with the extracellular matrix, leads to platelet adhesion, aggregation, and coagulation. Activated platelets undergo shape changes, adhere, and spread at the site of injury to form a blood clot. We investigated the morphology and morphological dynamics of human platelets after complete spreading using fast scanning ion conductance microscopy (SICM). In contrast to unstimulated platelets, thrombin-stimulated platelets showed increased morphological activity after spreading and exhibited dynamic morphological changes in the form of wave-like movements of the lamellipodium and dynamic protrusions on the platelet body. The increase in morphological activity was dependent on thrombin concentration. No increase in activity was observed following exposure to other activation agonists or during contact-induced activation. Inhibition of actin polymerization and inhibition of dynein significantly decreased the activity of thrombin-stimulated platelets. Our data suggest that these morphological dynamics after spreading are thrombin-specific and might play a role in coagulation and blood clot formation.

Antiplatelet Aggregation Activity of Walnut Hull Extract via Suppression of Reactive Oxygen Species Generation and Caspase Activation

Walnut hull (wal hull) is an agricultural by-product that is widely used in traditional medicine for alleviating pain and treating skin diseases, however, recently it has gained much attention in modern pharmacology due to its antioxidant properties. The current study was aimed to determine the total phenolic, flavonoid, and tannin content of Persian wal hull extract and evaluate its biological effects on platelet function. Experimental data showed that acetone extract of wal hulls has a high content of polyphenolic compounds and antioxidant properties. The analytical study of crude extract by gas chromatography–mass spectrometry demonstrated different types of high- and low-molecular-weight compounds that are basically and biologically important. Moreover, an in vitro study revealed that wal hull extract at a concentration of 50 μg/mL inhibited thrombin-induced platelet aggregation and protein secretion by 50%, without any cytotoxic effects on platelets. The examined extract suppressed reactive oxygen species generation and also caspase activation in thrombin-stimulated platelets. Identically, N-acetylcysteine inhibited the increase of reactive oxygen species level induced by thrombin in platelets, and supported a link between cellular redox status and caspase activation in activated platelets. Presumably, the antiplatelet activity of wal hull extract is related to its polyphenolic compounds and their antioxidant properties. Therefore, wal hulls can be considered as a candidate for thrombotic disorders.