Platelet Stimuli Research Articles

Pro-inflammatory sensitization of platelets after a mild course of COVID-19 infection

Abstract Background Coronavirus disease-2019 (COVID-19) is a viral respiratory illness caused by the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) which can lead to an increased risk of thromboembolic events but also complications even after recovery from the disease. Multiple factors may enhance the risk of thrombotic events in COVID-19 patients including excessive inflammatory responses and increased platelet activity. To date there are hardly any data on how platelet function is affected after a mild course of infection. Purpose In this study, we investigated whether platelet function is affected after recovery from mild COVID-19 disease. Besides the response of platelets to platelet stimuli, expression of the transporter protein multidrug resistance protein 4 (MRP4) and one of its substrates, the inflammatory lipid mediator sphingosine-1-phosphate (S1P), were measured. Methods Venous blood samples were collected from patients within 2-15 weeks after recovery from a mild SARS-CoV-2 infection (recovered) and matched individuals (controls) after written informed consent. Participants were invited again at 6-9 months after infection to reexamine platelet function. Parameters of platelet aggregation were determined by light transmission aggregometry (LTA) in platelet-rich plasma (PRP). Expression of platelet activation markers were measured by Flow Cytometry (FACS). In addition, concentrations of S1P were determined by LC-MS/MS and MRP4-expression in platelets via Western blotting. Results Within 2-15 weeks after recovery, no alterations in platelet aggregation were observed after stimulation of PRP with various ADP (1, 2, and 10 µM) or collagen (1 and 2.5 µg/mL) concentrations. However, regarding FACS analysis, platelet surface expression of CD62P and PAC1 from convalescents prior activation by ADP (2 µM) and by CRP-XL (0.125 ng/mL) was significantly upregulated in comparison to samples from controls (p<0.05 for both). Intriguingly, S1P levels in platelet-poor plasma (PPP) from COVID-19 convalescents were significantly elevated compared to controls, i.e. 0.75±0.01 vs 0.65±0.02 nmol/mL, respectively (p<0.001). S1P in PRP and serum samples was not altered. Furthermore, platelet MRP4 expression was significantly elevated in COVID-19 convalescents (p<0.05). These differences in S1P and MRP4 levels were only present after 2-15 weeks of recovery, but not 6-9 months after infection. Conclusions While after a mild course of COVID-19 infection robust platelet functions such as aggregation determined by LTA were not affected, subtle differences in platelet activation markers occurred. Enhanced platelet MRP4 expression, which may correlate with the observed elevated S1P in PPP, are in agreement with a pro-inflammatory sensitization of platelets even after a mild COVID-19 infection. The implications of this observation concerning long-term effects of COVID-19 await further studies.

Platelet-driven routes to chaos in a model of hepatitis

Hepatitis is a general term used to describe inflammation in the liver, the regulation of which involves a complex array of interactions between liver cells, inflammatory mediators and cells of the immune system (macrophages and neutrophils, in particular). There is increasing evidence that platelets (small blood cells that are primarily responsible for clotting) affect numerous mechanisms that underlie hepatitis, including the rates of cell migration, liver damage and mediator production, giving rise to a complex and diverse range of inflammatory outcomes. In particular, platelet activation has the scope to promote both healthy and chronic outcomes in a highly unpredictable and potentially state-dependent manner that is not currently well-understood. In this paper, we take an existing model of the inflammatory dynamics associated with hepatitis, and introduce platelets and their effects. We interrogate this new model via numerical simulations in Matlab and bifurcation analysis in XPPAUT, and find that amplification of certain key interactions by platelets can stimulate chaotic dynamics that correspond to complex chronic outcomes. We initially introduce a single parameter to represent the scale of the platelet stimulus and, through bifurcation analysis, illustrate that as this parameter is gradually increased we see the emergence of a period-doubling cascade that results in chaotic dynamics. We then explore individual mechanisms in more detail and illustrate that, while neutrophils can promote chaos in some settings, routes to chaos are primarily driven by macrophage-related platelet stimuli. Finally, we briefly comment upon the implications of our observations in terms of the ongoing hunt for new therapeutic interventions in hepatitis, as well as other inflammation-related medical conditions.

Profiling oxylipins released from human platelets activated through the GPVI collagen receptor

In addition to haemostasis, platelets are involved in pathological processes, often driven by material released upon activation. Interaction between collagen and glycoprotein VI (GPVI) is a primary platelet stimulus that liberates arachidonic acid and linoleic acid from membrane phospholipids. These are oxidised by cyclooxygenase-1 (COX-1) and 12-lipoxygenase (12-LOX) to eicosanoids and other oxylipins with various biological properties.Using liquid chromatography-tandem mass spectrometry we found that GPVI-stimulated platelets released significant levels of ten oxylipins; the well documented TxA2 and 12-HETE, PGD2 and PGE2, as well as 8-, 9-, 11-, and 15-HETE, 9- and 13-HODE.11TxA2 – thromboxane A2; HETE – Hydroxyeicosatetraenoic acid; PGD2 – prostaglandin D2; PGE2 – prostaglandin E2; HODE – hydroxyoctadecanoic acid. Levels of oxylipins released from washed platelets mirrored those from platelets stimulated in the presence of plasma, indicating generation from intracellular, rather than exogenous AA/LA. Inhibition of COX-1 with aspirin, as expected, completely abolished production of TxA2 and PGD/E2, but also significantly inhibited the release of 11-HETE (89 ± 3%) and 9-HODE (74 ± 6%), and reduced 15-HETE and 13-HODE by ∼33 %. Inhibition of 12-LOX by either esculetin or ML355 inhibited the release of all oxylipins apart from 15-HETE.These findings suggest routes to modify the production of bioactive molecules released by activated platelets.

Functional Assessment of Platelet Dense Granule ATP Release.

This study was undertaken to explore the feasibility of assessing platelet dense granule release in response to platelet stimuli, using less than 1 mL of whole blood (WB). Optimization of the luciferin-luciferase (LL) assay for ATP release, together with additional modifications, was applied to 1:10 diluted WB. LL assay optimization using nonstirred 1:10 diluted WB resulted in dense granule ATP release in response to thrombin receptor-activating peptide (TRAP) of similar magnitude to that observed using stirred platelet-rich plasma. Stirring of the 1:10 diluted WB restored collagen-induced dense granule secretion. Addition of lyophilized, formalin-fixed platelets, together with stirring, restored dense granule secretion responsiveness to ADP. TRAP, ADP, and collagen all stimulated ATP release in 1:10 diluted WB under the optimized conditions of this study at levels close to those observed using platelet-rich plasma. Blood sample reconstitution experiments offer hope that this assay may prove robust down to WB platelet counts as low as 50 × 103/μL. Platelet dense granule release in response to a number of classic stimuli, including ADP, was accomplished from less than 1 mL WB with minimal specimen processing, using widely available reagents and instrumentation.

Analysis of Platelet Alpha and Dense Granule Release Using Small Volumes of Blood

A significant limitation of the gold-standard platelet function test, light transmission aggregometry (LTA), is the relatively large volume of blood required, as well as need for a platelet count in the resulting platelet-rich plasma of not much less than 100,000/μL, thus potentially precluding this test in pediatric and thrombocytopenic patients. Flow cytometric assessment of platelet activation in diluted whole blood avoids these limitations, typically employing anti-CD62P (P-Selectin) binding reflecting alpha granule release and PAC-1 binding reflecting conversion of GPIIb/IIIa to its active conformation. We have recently reported the use of tandem mass spectrometry to assess platelet dense granule release of deuterated serotonin in comparably diluted whole blood samples (https://doi.org/10.1093/ajcp/aqz094). In the present study we examine the possibility of adapting and optimizing the luciferin-luciferase (L-L) approach often used together with LTA for the assay of platelet dense granule ATP release from 10-fold diluted whole blood in parallel with flow cytometric analysis. We optimized the L-L reaction with commercially available reagents and used a conventional lumi-aggregometer to measure real-time secretion of platelet dense granules following agonist stimulation in 10-fold diluted whole blood obtained from healthy adult volunteers, with or without specimen stirring at 1000 RPM. Direct comparison of optimized reagents to a commercial L-L reagent kit showed an improved lower limit of detection for exogenously added ATP by at least one order of magnitude. Initial studies were performed under non-stirring conditions. Dose-dependency of agonist was observed, with representative results for released ATP of 243.1 ± 29.7 pmol/107 platelets in response to 40 μM TRAP (thrombin receptor-activating peptide) and 18.9 ± 2.4 pmol/107 platelets in response to 1 nM convulxin (GPVI receptor activator) (mean ± SEM, N = 15). In contrast, virtually no ATP secretion was observed in response to 10 μM ADP or to 10 μg/mL collagen. Despite the quite low platelet count (as low as 11,000/μL to date) following the 10-fold dilution of whole blood, introducing 1000 RPM stirring to these samples now permitted robust ATP release in response to collagen as low as 2 μg/mL. There continued to be little or no ATP release, however, in response to 10 μM ADP even with the addition of stirring. Flow cytometry was used to interrogate agonist-stimulated alpha granule release and GPIIb/IIIa conformation change. Following incubations with agonist and fluorescently-labeled antibodies, the 10-fold diluted whole blood was further diluted an additional 10-fold, and gating based on light scatter and binding of anti-CD61 antibody used to identify individual platelets. Under non-stirring conditions, TRAP, ADP, and convulxin all produced strong P-selectin exposure, as well as conversion of GPIIb/IIIa to its active conformation. Analysis of samples that had undergone 1000 RPM stirring during incubation with agonist showed quite similar results in response to TRAP. While still strongly positive, the responses to ADP in the stirred specimens showed decreases typically of at least 50%. Without stirring, platelet responses to 2-10 μg/mL collagen were virtually absent, whereas in the stirred specimens significantly positive anti-CD62P and PAC-1 binding were now observed. The combined flow cytometric and L-L studies required <1 mL blood. The results demonstrate the dramatic differences observed in the release of platelet alpha and dense granules, depending upon conditions and assay systems employed. We have now shown that with agonist addition to the same 10-fold diluted whole blood samples employed in flow cytometric platelet analysis, dense granule release may also be assessed by modifications of the L-L methodology already employed for lumi-aggregation studies in many laboratories. We have additionally shown that by employing stirring of the specimen during incubation with agonist, collagen itself may be employed as platelet stimulus, rather than having to rely upon convulxin. The difference in requirements for alpha versus dense granule release, however, remains exemplified by ADP: While a potent stimulus of alpha granule release, ADP employed in the 10-fold diluted whole blood setting appeared incapable of producing significant dense granule release either in the absence or presence of sample stirring. Disclosures Wool: Diagnostica Stago: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Development of a Nonradioactive Platelet Serotonin Uptake and Release Assay by Micro-Liquid Chromatography Tandem Mass Spectrometry Using Minimal Blood Volume.

Analysis of platelet functional responses to stimuli is presently quite limited with respect to measurement of dense granule secretion. We sought to develop a nonradioactive assay of stimulated serotonin release using liquid chromatography tandem mass spectrometry (LC-MS/MS). Citrated whole blood (200 μL) was incubated with deuterated serotonin (d45-HT). Following uptake by platelets, blood was diluted 10-fold and aliquots were incubated with platelet stimuli. Following stimulation, blood was further diluted, centrifuged, and supernatant was assayed for released d45-HT by micro-LC-MS/MS. This study demonstrated a broad linear range of 50 to 2,000 pg/mL d45-HT, with a total precision of less than 15.0% coefficient of variation at all quality control levels and a limit of quantitation of 50 pg/mL. Quantification of d45-HT by micro-LC-MS/MS assay offers a highly sensitive, nonradioactive methodology for quantitating platelet serotonin uptake and dense granule secretion, requiring only small volumes of patient blood.

P2X1 Receptors Amplify FcγRIIa-Induced Ca2+ Increases and Functional Responses in Human Platelets.

Platelets express key receptors of the innate immune system such as FcγRIIa and Toll-like receptors (TLR). P2X1 cation channels amplify the platelet responses to several major platelet stimuli, particularly glycoprotein (GP)VI and TLR2/1, whereas their contribution to Src tyrosine kinase-dependent FcγRIIa receptors remains unknown. We investigated the role of P2X1 receptors during activation of FcγRIIa in human platelets, following stimulation by cross-linking of an anti-FcγRIIa monoclonal antibody (mAb) IV.3, or bacterial stimulation with Streptococcus sanguinis . Activation was assessed in washed platelet suspensions via measurement of intracellular Ca 2+ ([Ca 2+ ] i ) increases, ATP release and aggregation. P2X1 activity was abolished by pre-addition of α,β-meATP, exclusion of apyrase or the antagonist NF449. FcγRIIa activation evoked a robust increase in [Ca 2+ ] i (441 ± 33 nM at 30 μg/mL mAb), which was reduced to a similar extent (to 66–70% of control) by NF449, pre-exposure to α,β-meATP or apyrase omission, demonstrating a significant P2X1 receptor contribution. FcγRIIa activation-dependent P2X1 responses were partially resistant to nitric oxide (NO), but abrogated by 500 nM prostacyclin (PGI 2 ). Aggregation responses to bacteria and FcγRIIa activation were also inhibited by P2X1 receptor desensitization (to 66 and 42% of control, respectively). However, FcγRIIa-mediated tyrosine phosphorylation and ATP release were not significantly altered by the loss of P2X1 activity. In conclusion, we show that P2X1 receptors enhance platelet FcγRIIa receptor-evoked aggregation through an increase in [Ca 2+ ] i downstream of the initial tyrosine phosphorylation events and early dense granule release. This represents a further route whereby ATP-gated cation channels can contribute to platelet-dependent immune responses in vivo.

A novel flow cytometry assay using dihydroethidium as redox-sensitive probe reveals NADPH oxidase-dependent generation of superoxide anion in human platelets exposed to amyloid peptide β

Reactive oxygen species (ROS) generation is critical in the regulation of platelets, which has important implications in the modulation of hemostasis and thrombosis. Nonetheless, despite several assays have been described and successfully utilized in the past, the analysis of ROS generation in human platelets remains challenging.Here we show that dihydroethidium (DHE) allows the characterization of redox responses upon platelet activation by physiological and pathological stimuli. In particular, the flow cytometry assay that we describe here allowed us to confirm that thrombin, collagen-related peptide (CRP) and arachidonic acid but not adenosine diphosphate (ADP) stimulate superoxide anion formation in a concentration-dependent manner. 0.1unit/ml thrombin, 3 μg/ml CRP and 30 μM arachidonic acid are commonly used to stimulate platelets in vitro and here were shown to stimulate a significant increase in superoxide anion formation. The ROS scavenger N-acetylcysteine (NAC) abolished superoxide anion generation in response to all tested stimuli, but the pan-NADPH oxidase (NOX) inhibitor VAS2870 only inhibited superoxide anion formation in response to thrombin and CRP. The involvement of NOXs in thrombin and CRP-dependent responses was confirmed by the inhibition of platelet aggregation induced by these stimuli by VAS2870, while platelet aggregation in response to arachidonic acid was insensitive to this inhibitor.In addition, the pathological platelet stimulus amyloid β (Aβ) 1–42 peptide induced superoxide anion formation in a concentration-dependent manner. Aβ peptide stimulated superoxide anion formation in a NOX-dependent manner, as proved by the use of VAS2870. Aβ 1–42 peptide displayed only moderate activity as an aggregation stimulus, but was able to significantly potentiate platelet aggregation in response to submaximal agonists concentrations, such as 0.03 unit/ml thrombin and 10 μM arachidonic acid. The inhibition of NOXs by 10 μM VAS2870 abolished Aβ-dependent potentiation of platelet aggregation in response to 10 μM arachidonic acid, suggesting that the pro-thrombotic activity of Aβ peptides depends on NOX activity. Similar experiments could not be performed with thrombin or collagen, as NOXs are required for the signaling induced by these stimuli. These findings shed some new light on the pro-thrombotic activity of Aβ peptides.In summary, here we describe a novel and reliable assay for the detection of superoxide anion in human platelets. This is particularly important for the investigation of the pathophysiological role of redox stress in platelets, a field of research of increasing importance, but hindered by the absence of a reliable and easily accessible ROS detection methodology applicable to platelets.

Farnesoid X Receptor and Liver X Receptor Ligands Initiate Formation of Coated Platelets.

The liver X receptors (LXRs) and farnesoid X receptor (FXR) have been identified in human platelets. Ligands of these receptors have been shown to have nongenomic inhibitory effects on platelet activation by platelet agonists. This, however, seems contradictory with the platelet hyper-reactivity that is associated with several pathological conditions that are associated with increased circulating levels of molecules that are LXR and FXR ligands, such as hyperlipidemia, type 2 diabetes mellitus, and obesity. We, therefore, investigated whether ligands for the LXR and FXR receptors were capable of priming platelets to the activated state without stimulation by platelet agonists. Treatment of platelets with ligands for LXR and FXR converted platelets to the procoagulant state, with increases in phosphatidylserine exposure, platelet swelling, reduced membrane integrity, depolarization of the mitochondrial membrane, and microparticle release observed. Additionally, platelets also displayed features associated with coated platelets such as P-selectin exposure, fibrinogen binding, fibrin generation that is supported by increased serine protease activity, and inhibition of integrin αIIbβ3. LXR and FXR ligand-induced formation of coated platelets was found to be dependent on both reactive oxygen species and intracellular calcium mobilization, and for FXR ligands, this process was found to be dependent on cyclophilin D. We conclude that treatment with LXR and FXR ligands initiates coated platelet formation, which is thought to support coagulation but results in desensitization to platelet stimuli through inhibition of αIIbβ3 consistent with their ability to inhibit platelet function and stable thrombus formation in vivo.

Simultaneous Assay of Agonist-Induced Endogenous and Non-Radioactive Isotopic Serotonin Secretion from Small Numbers of Human Platelets By Mass Spectrometry

Background: Light transmission aggregometry (LTA) with simultaneous assay of ATP released from platelet dense granules (lumi-aggregation) provides the ability to assess platelet function in a variety of clinical settings. This methodology, however, requires the ability to obtain adequate sample volume from the patient, if a number of platelet agonists are to be assessed. The platelet count within the patient's blood is also a limiting factor, and LTA interpretations can become difficult when platelet counts in the sample fall significantly below 150,000/µL. Analysis of platelet function by flow cytometry circumvents the limitations of both blood volume and low platelet counts, typically providing information as to glycoprotein IIb/IIIa activation state and release of platelet alpha granules in response to platelet agonists. Flow cytometry, however, is not presently capable of providing quantitative analysis of the release of platelet dense granule constituents. Given the clinical importance of platelet dense granule secretion, we sought to develop a non-radioactive method for quantitation of agonist-induced release of serotonin (5-HT) from blood sample volumes obtainable from small infants and from patients with at least moderate thrombocytopenia.Methods: In the initial studies, total platelet content and released 5-HT following stimulation by a panel of agonists were assessed using 200 µL of freshly drawn, normal citrated blood. In subsequent studies, uptake and agonist-induced secretion of non-radioactive, deuterated serotonin (d-5HT) were additionally measured in parallel with the endogenous 5-HT in these 200 µL samples. In brief, the citrated blood was initially incubated for 15' at 37° with d-5HT, then diluted 10-fold with buffer, and platelet stimulus (collagen, TRAP, ADP, epinephrine, ionophore A23187, thromboxane analogue U46619, ristocetin, or buffer blank) added. Following stimulation, samples were further diluted with buffer, prostaglandin E1 added, and then centrifuged to yield supernatants for analysis. 5-HT and d-5HT total content in unstimulated platelets was additionally assayed. N-methyl-5HT was employed as an internal standard. 5-HT, d-5HT and N-methyl-5HT were subsequently detected and quantified simultaneously by liquid chromatography/tandem mass spectrometry (MS) (Eksigent MicroLC 200/QTrap 6500 Mass Spectrometer, Sciex, Framingham, MA).Results and Conclusions: MS analysis of 5-HT was linear over the range of 50-2000 pg/mL. For whole blood platelet counts of at least 120,000/µL, this sensitivity was sufficient to permit analysis of 5-HT released in response not only to strong stimuli, but even to weaker stimuli such as low concentration (2 µg/mL) collagen. Since the goal of this project, however, was to be able to extend functional analysis to patients with more significant degrees of thrombocytopenia, further efforts were made to improve sensitivity. Assay of agonist-induced release, following platelet uptake, of d-5HT was assessed in parallel with the assay of released endogenous 5-HT. Release of 5-HT and d-5HT in response to increasing concentrations of collagen showed generally similar kinetics. However, not only was the absolute signal increased for the d-5HT, but the analytic assay itself proved significantly more sensitive as compared to that for 5-HT. Combined, these effects increased the overall sensitivity of the assay by at least an order of magnitude. For example, d-5HT released in response to 2 µg/mL collagen in a donor blood having a platelet count of 188,000/µL produced an MS signal approximately 30-fold higher than the lower limit of detection for the assay. Although it will require actual study of thrombocytopenic patients to verify, these results suggest that by use of this MS approach with d-5HT it may be possible to measure platelet dense granule secretion to a panel of agonists from 200 µL of blood in patients with thrombocytopenia potentially as severe as 10,000/µL. DisclosuresNo relevant conflicts of interest to declare.

P2X1-initiated p38 signalling enhances thromboxane A2-induced platelet secretion and aggregation

ATP released by activated platelets can serve as a positive feedback machinery to amplify platelet responses by activating P2X1 receptors. It has, however, not been defined how P2X1 activities influence thromboxane A2 (TXA2)-stimulated platelet functional responses. Our aim was to elaborate the molecular mechanisms of P2X1 engagements in TXA2-induced platelet secretion and aggregation. P2X1 inhibition by 1 µM NF449 inhibited platelet P-selectin expression induced by a low concentration of the TXA2 analogue U46619 (0.3 µM) (32.0 ± 2.0% vs 43.4 ± 3.0%; n=5; p<0.05). p38 inhibition by SB203580, but not ERK inhibition by U0126, elicited a similar inhibition by NF499. The combination of NF449 and SB203580 provided, however, no additive effects. U46619-induced platelet aggregation was similarly decreased by NF449 and SB203580 alone or in combination, and by P2X1 pre-desensitisation with α,β-Me-ATP. U46619 caused rapid and reversible P2X1-dependent p38 phosphorylation. However, the P2X1-p38 pathway mainly enhanced mild platelet activation by U46619, because α,β-Me-ATP supplementation or p38 blockade had no effect on intense platelet activation induced by a higher concentration of U46619 (3 µM). In conclusion, P2X1 activation, via p38 signalling, potentiates platelet activation initiated by low doses of U46619. Hence, the P2X1-induced p38 signalling promotes more robust platelet activation in response to mild platelet stimuli.

Mechanism of platelet activation induced by endocannabinoids in blood and plasma

Platelets play a central role in atherosclerosis and atherothrombosis, and circulating endocannabinoids might modulate platelet function. Previous studies concerning effects of anandamide (N-arachidonylethanolamide) and 2-arachidonoylglycerol (2-AG) on platelets, mainly performed on isolated cells, provided conflicting results. We therefore investigated the action of three main endocannabinoids [anandamide, 2-AG and virodhamine (arachidonoylethanolamine)] on human platelets in blood and platelet-rich plasma (PRP). 2-AG and virodhamine induced platelet aggregation in blood, and shape change, aggregation and adenosine triphosphate (ATP) secretion in PRP. The EC50 of 2-AG and virodhamine for platelet aggregation in blood was 97 and 160 µM, respectively. Lower concentrations of 2-AG (20 µM) and virodhamine (50 µM) synergistically induced aggregation with other platelet stimuli. Platelet activation induced by 2-AG and virodhamine resembled arachidonic acid (AA)-induced aggregation: shape change, the first platelet response, ATP secretion and aggregation induced by 2-AG and virodhamine were all blocked by acetylsalicylic acid (ASA) or the specific thromboxane A2 (TXA2) antagonist daltroban. In addition, platelet activation induced by 2-AG and virodhamine in blood and PRP were inhibited by JZL184, a selective inhibitor of monoacylglycerol lipase (MAGL). In contrast to 2-AG and virodhamine, anandamide, a substrate of fatty acid amidohydrolase, was inactive. Synthetic cannabinoid receptor subtype 1 (CB1) and 2 (CB2) agonists lacked stimulatory as well as inhibitory platelet activity. We conclude that 2-AG and virodhamine stimulate platelets in blood and PRP by a MAGL-triggered mechanism leading to free AA and its metabolism by platelet cyclooxygenase-1/thromboxane synthase to TXA2. CB1, CB2 or non-CB1/CB2 receptors are not involved. Our results imply that ASA and MAGL inhibitors will protect platelets from activation by high endocannabinoid levels, and that pharmacological CB1- and CB2-receptor ligands will not affect platelets and platelet-dependent progression and complications of cardiovascular diseases.

Induction of platelet aggregation after a direct physical interaction with diesel exhaust particles

There is a proven link between exposure to traffic-derived particulate air pollution and the incidence of platelet-driven cardiovascular diseases. It is suggested that inhalation of small, nanosized particles increases cardiovascular risk via toxicological and inflammatory processes and translocation of nanoparticles into the bloodstream has been shown in experimental models. We therefore investigated the ability of diesel exhaust particles (DEP) to interact physically and functionally with platelets. The interaction of DEP and carbon black (CB) with platelets was examined by transmission electron microscopy (TEM), whereas the functional consequences of exposure were assessed by measuring in vitro and in vivo platelet aggregation via established methods. Both DEP and CB were internalized and seen in proximity with the open canalicular system in platelets. DEP induced platelet aggregation in vitro whereas CB had no effect. DEP induced Ca(2+) release, dense granule secretion and surface P-selectin expression, but not toxicologic membrane disruption. Low concentrations of DEP potentiated agonist-induced platelet aggregation in vitro and in vivo. DEP associate physically with platelets in parallel with a Ca(2+) -mediated aggregation response displaying the conventional features of agonist-induced aggregation. The ability of DEP to enhance the aggregation response to platelet stimuli would be expected to increase the incidence of platelet-driven cardiovascular events should they be inhaled and translocate into the blood. This study provides a potential mechanism for the increased thrombotic risk associated with exposure to ambient particulate air pollution.

The Collagen-binding Integrin α2β1 Is a Novel Interaction Partner of the Trimeresurus flavoviridis Venom Protein Flavocetin-A

Many snake venoms are known for their antithrombotic activity. They contain components that specifically target different platelet-activating receptors such as the collagen-binding integrin α2β1 and the von Willebrand factor receptor GPIb. In a search for an α2β1 integrin-blocking component from the venom of the habu snake (Trimeresurus flavoviridis), we employed two independent purification protocols. First, we used the integrin α2A domain, a major collagen-binding domain, as bait for affinity purification of an α2β1 integrin-binding toxin from the crude venom. Second, in parallel, we used classical protein separation protocols and tested for α2β1 integrin-inhibiting capabilities by ELISA. Using both approaches, we identified flavocetin-A as an inhibitor of α2β1 integrin. Hitherto, flavocetin-A has been reported as a GPIb inhibitor. However, flavocetin-A inhibited collagen-induced platelet aggregation even after GPIb was blocked with other inhibitors. Moreover, flavocetin-A antagonized α2β1 integrin-mediated adhesion and migration of HT1080 human fibrosarcoma cells, which lack any GPIb, on collagen. Protein chemical analyses proved that flavocetin-A binds to α2β1 integrin and its α2A domain with high affinity and in a cooperative manner, which most likely is due to its quaternary structure. Kinetic measurements confirmed the formation of a strong complex between integrin and flavocetin-A, which dissociates very slowly. This study proves that flavocetin-A, which has long been known as a GPIb inhibitor, efficiently targets α2β1 integrin and thus blocks collagen-induced platelet activation. Moreover, our findings suggest that the separation of GPIb- and α2β1 integrin-blocking members within the C-type lectin-related protein family is less strict than previously assumed.

Comparative analysis of platelet-derived microparticles reveals differences in their amount and proteome depending on the platelet stimulus

Platelet-derived microparticles (PMP) are elevated in a number of disorders (e.g. cardiovascular disease and cancer). In the present study, we have carried out a high-resolution 2-DE-based proteomic analysis of PMP originated following platelet activation with different stimulus. Flow cytometric analysis revealed a higher average number of microparticles when platelets are activated with shear (1800 s(-1)) compared to 0.5 U/mL thrombin. Regarding the proteomic analysis, 30 protein features were found to be differentially regulated between shear and thrombin groups in the pI 4-7 range, from which 28 were successfully identified by MS, corresponding to 26 open-reading frames. Signaling proteins constituted the major functional group, including membrane receptors, and adapters. Ingenuity Pathways Analysis (IPA) software revealed that 21 of the 26 differentially regulated unique proteins identified are part of a common network related to cell assembly and organization and cell morphology. Western blotting analyses confirmed that Dok-2 and the integrin α6 are differentially regulated in PMP depending on the platelet stimulus. Interestingly, both proteins participate in mechanisms regulating angiogenesis so could be part of PMP regulation of endothelial cells function. In conclusion, this study shows that platelets shed microparticles in different amounts and with a different proteome depending on the stimulus.This article is part of a Special Issue entitled: Integrated omics.

Distinct platelet packaging, release, and surface expression of proangiogenic and antiangiogenic factors on different platelet stimuli

AbstractThe present study characterized platelet secretion and surface expression of proangiogenic stromal cell-derived factor-1α (SDF-1α) and vascular endothelial growth factor (VEGF) and antiangiogenic PF4 and endostatin on activation. The angiogenic factors presented in randomly distributed granules in resting platelets, which were peripherized on activation. Confocal and immunogold electron microscopy demonstrated that SDF-1α/CXCL12 and PF4/CXCL4 mostly present in different granules. Platelet activation induced marked SDF-1α and endostatin but mild PF4 or no VEGF surface expression. PAR1-activating peptide (PAR1-AP), adenosine diphosphate (via P2Y1/P2Y12), and glycoprotein VI-targeting collagen-related peptide induced massive SDF-1α and VEGF but modest PF4 or no endostatin release. In contrast, PAR4-AP triggered marked PF4 and sole endostatin release but limited SDF-1α or VEGF secretion. Distinct platelet release of SDF-1α and endostatin involved different engagements of intracellular signaling pathways. In conclusion, different platelet stimuli evoke distinct secretion and surface expression of proangiogenic and antiangiogenic factors. PAR1, adenosine diphosphate, and glycoprotein VI stimulation favors proangiogenic, whereas PAR4 promotes antiangiogenic, factor release.

Cover Picture: Proteomics 17'09

AbstractBlue on blueFew protein analysis techniques approach the resolution of, or the tedium of preparing for, large‐format 2‐D gel electrophoresis. Then you find the first dimension gives poor resolution of samples rich in membrane‐bound proteins. A relatively new modification of the classic standard IPG method for analysis of membrane‐bound proteins is “Blue Native” gel electrophoresis. BN‐PAGE incorporates a dye such as Coomassie Blue G‐250 into membrane complexes to give them an electrophoretic mobility roughly proportional to their size. Conventionally SDS‐PAGE is used for a second dimension. Wessels et al. instead used in‐gel digestion followed by LCMS/MS for a higher resolution second dimension. The modified method gave faster identification of overlapping proteins and was able to detect assembly intermediates and transient interactions..Wessels, H. J. C. T. et al., Proteomics 2009, 9, 4221–4228.Making sense of senescenceThe ripening (senescence) of fruit is one of the final steps in the life cycle of a plant. It is also the step that makes many edible fruits palatable or sweet and so is of economic significance as well. Since ripening is an oxidative step, Qin et al. examine the effect of reactive oxygen species (ROS) on the proteome of apple mitochondria. Accelerating senescence by exposure to 100% oxygen led to under‐expression of Mn superoxide dismutase (MnSOD), an enzyme characteristic of mitochondrial membranes and primary scavenger of free radicals. In addition to previously recognized enzymes, two new spots of unknown function were identified that will require further study. High oxygen also increased the frequency of carbonylation of selected proteins. Reducing the oxygen to 2% markedly reduced the rate of senescence and decreased the number of proteins up‐ or down‐regulated.Qin, G. et al., Proteomics 2009, 9, 4241–4253.Pleckstrin, pleckstrin, wherefore art thou?It may not be quite as romantic as Juliet awaiting Romeo, but pleckstrin is rather shy about holding hands in public. Platelets lacking pleckstrin do not aggregate, secrete granules or polymerize actin, yet no direct signals between pleckstrin and actin have been seen. Pleckstrin is the major target of protein kinase C (PKC) in platelets and many platelet stimuli correlate with phosphorylation. Baig et al. set off to find out how the secret messages were being passed. They used recombinant and pseudo‐phosphorylated pleckstrin, immunoprecipitation, GST‐pulldown assays, PKC‐activated and PKC‐inhibited platelets, and quad‐TOF MS. Several unusual protein associations were found but no direct association with actin. The authors propose an indirect link between actin and pleckstrin (mediated by Juliet's maid), possibly through a‐actinin, factor XIIIA, moesin, radixin, or 17‐b‐hydroxysteroid dehydrogenase 4Baig, A. et al., Proteomics 2009, 9, 4254–4258.

In this issue

AbstractBlue on bluepp. 4221–4228Few protein analysis techniques approach the resolution of, or the tedium of preparing for, large‐format 2‐D gel electrophoresis. Then you find the first dimension gives poor resolution of samples rich in membrane‐bound proteins. A relatively new modification of the classic standard IPG method for analysis of membrane‐bound proteins is “Blue Native” gel electrophoresis. BN‐PAGE incorporates a dye such as Coomassie Blue G‐250 into membrane complexes to give them an electrophoretic mobility roughly proportional to their size. Conventionally SDS‐PAGE is used for a second dimension. Wessels et al. instead used in‐gel digestion followed by LCMS/MS for a higher resolution second dimension. The modified method gave faster identification of overlapping proteins and was able to detect assembly intermediates and transient interactions.. magnified imageMaking sense of senescencepp. 4241–4253The ripening (senescence) of fruit is one of the final steps in the life cycle of a plant. It is also the step that makes many edible fruits palatable or sweet and so is of economic significance as well. Since ripening is an oxidative step, Qin et al. examine the effect of reactive oxygen species (ROS) on the proteome of apple mitochondria. Accelerating senescence by exposure to 100% oxygen led to under‐expression of Mn superoxide dismutase (MnSOD), an enzyme characteristic of mitochondrial membranes and primary scavenger of free radicals. In addition to previously recognized enzymes, two new spots of unknown function were identified that will require further study. High oxygen also increased the frequency of carbonylation of selected proteins. Reducing the oxygen to 2% markedly reduced the rate of senescence and decreased the number of proteins up‐ or down‐regulated. magnified imagePleckstrin, pleckstrin, wherefore art thou?pp. 4254–4258It may not be quite as romantic as Juliet awaiting Romeo, but pleckstrin is rather shy about holding hands in public. Platelets lacking pleckstrin do not aggregate, secrete granules or polymerize actin, yet no direct signals between pleckstrin and actin have been seen. Pleckstrin is the major target of protein kinase C (PKC) in platelets and many platelet stimuli correlate with phosphorylation. Baig et al. set off to find out how the secret messages were being passed. They used recombinant and pseudo‐phosphorylated pleckstrin, immunoprecipitation, GST‐pulldown assays, PKC‐activated and PKC‐inhibited platelets, and quad‐TOF MS. Several unusual protein associations were found but no direct association with actin. The authors propose an indirect link between actin and pleckstrin (mediated by Juliet's maid), possibly through a‐actinin, factor XIIIA, moesin, radixin, or 17‐b‐hydroxysteroid dehydrogenase 4 magnified image

Heritability, Platelet Function, and Aspirin

Platelets play a critical role in the pathophysiology of atherothrombotic disease. A pivotal event contributing to the understanding of platelet-dependent clot formation was the development of the platelet aggregometer in 1962.1 An aggregometer specifically measures the ability of platelets to adhere via glycoprotein IIb/IIIa (integrin αIIbβ3), and thousands of articles using this technique have been published, characterizing platelet function; however, the usefulness of these measurements remains unclear. Whereas the aggregometer and related techniques that measure platelet aggregation or glycoprotein expression have led to large amounts of data characterizing platelet function in various settings, the clinical importance of measurable differences in platelet function is still debated.2 The use of platelet function testing is established in rarer platelet abnormalities, such as the autosomal recessive bleeding disorder Glanzmann thrombasthenia,3 but no clear consensus has been reached on its usefulness for highly prevalent diseases caused by platelet-dependent thrombosis, such as myocardial infarction. A major factor for this discrepancy is that many of the platelet function defects that lead to bleeding are known to be caused by a single defect, whereas thrombosis in the setting of cardiovascular disease is presumed to be multifactorial. Article p 2490 The evolution of platelet function studies in various clinical settings has led to the realization that wide interindividual variability exists in the platelet activation response.4,5 What accounts for this variability? Only a few studies have systematically examined this question. Platelet function has been established as markedly dependent on the type of agonist used, the agonist concentration, and the concomitant use of antiplatelet therapy.6 In addition, in the large population-based Framingham Heart Study, O’Donnell and colleagues7 have demonstrated that heritable factors play a major role in determining platelet aggregation, as opposed to measured covariates. Less clear from the current literature is the direct …

Unresponsiveness of Platelets Lacking Both Gαq and Gα13: IMPLICATIONS FOR COLLAGEN-INDUCED PLATELET ACTIVATION

The diffusible platelet stimuli ADP and thromboxane A(2) activate multiple G protein-mediated signaling pathways and function as important secondary mediators of platelet activation as they are released from activated platelets. Because they can also increase their own formation and release, their effects are amplified; eventually, all major G protein-mediated signaling pathways are activated. The multiple positive feedback mechanisms operating during platelet activation have obscured the exact analysis of the roles individual G protein-mediated signaling pathways play during the platelet activation process. In this report, we show that platelets lacking G(q) and G(13) are completely unresponsive to diffusible stimuli such as ADP, thromboxane A(2), or thrombin, even when applied at very high concentrations in combination, whereas all stimuli are able to induce platelet aggregation, shape change, and RhoA activation in platelets lacking only one Galpha subunit. This shows that G(q) or G(13) is required to induce some platelet activation, whereas the activation of G(i)-mediated signaling alone is not sufficient to induceactivation of mouse platelets. In addition, platelets lacking Galpha(q) and Galpha(13) adhered normally to collagen under high shearbut did not aggregate any more in response to collagen, indicating that collagen-induced platelet activation but not platelet adhesion requires intact G protein-mediated signaling pathways.