Strong Platelet Agonist Research Articles

Impaired glucose metabolism is associated with increased thrombin generation potential in patients undergoing angioplasty and stenting

BackgroundAs a strong platelet agonist on the one hand and key molecule in plasmatic coagulation on the other hand, thrombin connects primary and secondary hemostasis. Thrombin generation potential reflects the individual capacity to generate thrombin, and has been associated with the occurrence of thromboembolic events. In the current study, we sought to identify predictors of thrombin generation potential in patients undergoing angioplasty and stenting for atherosclerotic cardiovascular disease.MethodsPeak thrombin generation potential and area under the curve (AUC) of thrombin generation potential were determined with a commercially available assay in 315 patients on dual antiplatelet therapy 1 day after percutaneous intervention, and in 100 healthy individuals without cardiovascular disease.ResultsMedian (interquartile range) peak thrombin generation potential and AUC of thrombin generation potential in the study cohort (n = 315) were significantly higher than in healthy individuals (n = 100) without cardiovascular disease (peak thrombin generation potential: 445.4 nM [354.5–551.8 nM] vs. 174.5 nM [141.2–261.2 nM]; AUC of thrombin generation potential: 5262.7 nM thrombin [4806.6–5756.9 nM thrombin] vs. 3405.2 nM thrombin [3043.6–3747.3 nM thrombin]; both p < 0.001). In patients undergoing angioplasty and stenting, hemoglobin A1c (HbA1c) was the only variable that was independently associated with both, peak thrombin generation potential and AUC of thrombin generation potential (both p ≤ 0.007). In contrast, platelet count and high-sensitivity C-reactive protein were only associated with peak thrombin generation potential, and body mass index and serum creatinine were only associated with AUC of thrombin generation potential after adjustment for covariates by multivariate linear regression analyses (all p < 0.05). Patients with HbA1c ≥ 6% had significantly higher peak thrombin generation potential and AUC of thrombin generation potential than patients with HbA1c < 6% (peak thrombin generation potential: 476.9 nM [385.8–577.9 nM] vs. 423.9 nM [335.8–529.5 nM], p = 0.002; AUC of thrombin generation potential: 5371.8 nM thrombin [4903 – 5899 nM thrombin] vs. 5172.5 nM thrombin [4731.8–5664.7 nM thrombin], p = 0.01). HbA1c ≥ 6% remained independently associated with both parameters of thrombin generation potential after multivariate linear regression analyses (both p ≤ 0.02).ConclusionsImpaired glucose metabolism is associated with increased thrombin generation potential in patients undergoing angioplasty and stenting for cardiovascular disease.

Escitalopram Impairs Thrombin-Induced Platelet Response, Cytoskeletal Assembly and Activation of Associated Signalling Pathways.

Background Serotonin reuptake inhibitors (SSRIs) may impair platelet function. Thrombin is a strong platelet agonist causing irreversible aggregation, release of granules' contents, cytoskeletal rearrangement and activation of signalling pathways. We investigated the effects of the SSRI escitalopram (SCIT) on thrombin-induced platelet response. Methods Isolated platelets were exposed to SCIT and activated with thrombin. We evaluated (1) platelet response by aggregometry and flow cytometry; (2) modifications in cytoskeleton proteins and signalling pathways by electrophoresis and Western blot; and (3) ultrastructural changes in platelets by electron microscopy. Results SCIT inhibited platelet response to thrombin, measured as platelet aggregation and expression of activation markers CD62-P and CD63 from platelet granules. Platelet aggregation decreased in a dose-dependent manner, reaching statistical significance with SCIT ≥32 µg/mL (65.4 ± 6.8% vs. 77.7 ± 2.5% for controls; p < 0.05). Expression of activation markers was statistically reduced with SCIT ≥20 µg/mL (p < 0.05). SCIT impaired the polymerization of the actin cytoskeleton and association of contractile proteins during activation with thrombin (p < 0.05 with SCIT ≥50 µg/mL). Resting platelets incubated with SCIT became most spherical, with increased platelet roundness (p < 0.01, SCIT 50 µg/mL vs. control). SCIT interfered with signalling pathways modulated by thrombin (RhoA, PKC, Erk1/2 and PI3K/AKT). Conclusions Our data indicate that SCIT inhibits thrombin-induced platelet response and interferes with cytoskeletal assembly and related signalling pathways, thus resulting in compromised release of granules' contents, reduced platelet activation and aggregation. These mechanisms may explain the antithrombotic benefits observed in patients treated with this SSRI, and could become new therapeutic targets for future antithrombotic strategies.

Dual role of the p38 MAPK/cPLA 2 pathway in the regulation of platelet apoptosis induced by ABT-737 and strong platelet agonists

p38 Mitogen-activated protein (MAP) kinase is involved in the apoptosis of nucleated cells. Although platelets are anucleated cells, apoptotic proteins have been shown to regulate platelet lifespan. However, the involvement of p38 MAP kinase in platelet apoptosis is not yet clearly defined. Therefore, we investigated the role of p38 MAP kinase in apoptosis induced by a mimetic of BH3-only proteins, ABT-737, and in apoptosis-like events induced by such strong platelet agonists as thrombin in combination with convulxin (Thr/Cvx), both of which result in p38 MAP kinase phosphorylation and activation. A p38 inhibitor (SB202190) inhibited the apoptotic events induced by ABT-737 but did not influence those induced by Thr/Cvx. The inhibitor also reduced the phosphorylation of cytosolic phospholipase A2 (cPLA2), an established p38 substrate, induced by ABT-737 or Thr/Cvx. ABT-737, but not Thr/Cvx, induced the caspase 3-dependent cleavage and inactivation of cPLA2. Thus, p38 MAPK promotes ABT-737-induced apoptosis by inhibiting the cPLA2/arachidonate pathway. We also show that arachidonic acid (AA) itself and in combination with Thr/Cvx or ABT-737 at low concentrations prevented apoptotic events, whereas at high concentrations it enhanced such events. Our data support the hypothesis that the p38 MAPK-triggered arachidonate pathway serves as a defense mechanism against apoptosis under physiological conditions.

Abstract 489: Differential Phosphoproteomic Analysis of Platelet Activation by Specific Oxidized Phospholipids and Thrombin Reveals Mechanisms of Platelet Activation in Hyperlipidemia

We previously showed that specific oxidized phospholipids (oxPC CD36 ) activate platelets via the scavenger receptor CD36 and promote platelet hyper-reactivity in hyperlipidemia, however the signaling pathway(s) induced in platelets by oxPC CD36 are not defined. We employed mass spectrometry-based phosphoproteomics for the unbiased analysis of changes in protein phosphorylation induced by oxPC CD36 and thrombin, a strong platelet agonist, in human platelets. oxPC CD36 induced changes in phosphorylation of 148 unique phosphorylation sites (116 proteins) while thrombin induced changes of 297 unique sites (181 proteins). Most of the changes in phosphorylation induced by oxPC CD36 and thrombin identified in our study have never been reported before in platelets and include high- and low-abundant proteins with diverse molecular functions located in the plasma membrane, cytosol, or cytoskeleton. Analysis using multiple bioinformatic tools identified protein interaction networks, signaling pathways, activated kinases, and enriched phosphorylation motifs. Comparison between platelet agonists revealed multiple differences including the specific activation of a signaling pathway involving Src-family kinases (SFK), SYK kinase, and PLCγ2 by oxPC CD36 . Subsequent biochemical studies in human platelets demonstrated that this pathway is critical for platelet activation by oxPC CD36 and is downstream of CD36. In conclusion, systematic analysis of platelet activation pathways provided novel insights into the mechanism of platelet activation and specific signaling pathways induced by oxidized phospholipids that modulate platelet function in vivo in hyperlipidemia.

Transfusion of Cryopreserved Autologous Platelets to Overcome HLA Alloimmunization in Patients with Hematological Malignancies,

Abstract 3375Alloimmunization to platelet antigens occurs in up to 25% of mainly female patients with hematologic disorders receiving intensive chemotherapies. It leads to platelet transfusion refractoriness, increased bleeding risk and often compromises further treatment. Once alloimmunization is established, only HLA-matched platelets improve platelet count increments. However, in patients with rare HLA phenotypes, the identification of HLA matched platelets may be difficult. In this situation, transfusion of autologous platelets is a clinical option, but cryopreservation of platelets has been notoriously difficult. Here, we present our clinical experience with transfusion of cryopreserved autologous platelets in patients with hematological malignancies. The platelets were collected after recovery from chemotherapy. Per patient, 2.5 (1–5) platelet aphereses were performed collecting 298 (190–598) ml per apheresis resulting in 6 (4–8) concentrates. All platelet concentrates were irradiated with 50 Gy, cryopreserved with 10% DMSO and RPMI-1640 without Phenol Red as freezing medium. Platelets were frozen by computer-controlled rate freezing and afterwards kept in liquid nitrogen. For transfusion, platelet concentrates were thawed at bedside in a warming bath (37°C) and transfused immediately without removal of DMSO. Nine patients received 36 cryopreserved autologous platelet transfusions. All patients were female and received intensive chemotherapies for acute leukemias (8) or NHL (1). The median age at diagnosis was 50 (range 30–69) years and the patients had 2 (1–4) prior pregnancies. Alloimmunization occurred 25 (15–458) days after diagnosis with a panel reactivity of 73 (14–93)%. The platelets were transfused 26 (9–133) days after cryopreservation. The platelet count before transfusion was 7 (2–16) G/L and the platelets were transfused prophylactically (32) or therapeutically (4). The median number of platelets per concentrate was 1.7 (0.8–2.9) ×1011. The corrected count increment after 1 hour (1h-CCI) was 5.2 (−1.8–20.2) and after 18–24 hours 4.7 (−5.8–25.2). There was no DMSO toxicity after infusion. The phenotype and function of the cryopreserved platelets were further analyzed in preliminary in vitro experiments by flow-cytometry. As compared to fresh platelets, cryopreservation resulted in a reduction of the size and cytoplasmic complexity. The surface glycoproteins (gp) IIa and IIIb remained unchanged while the expression of the gp Ib was reduced to 54 (47–61)%. The cryopreserved platelets were partially activated, as demonstrated by the expression of negatively charged surface phospholipids (median 73%, normal <5%) and P-selectin (33%, normal <5%). However, the fibrinogen receptor was not activated, as demonstrated by PAC-1 binding (4%, normal <5%). Stimulation with strong platelet agonists (thrombin 0.5 U/ml, convulxin 50 ng/ml) induced full activation of about 1/3 of the cryopreserved platelets, as demonstrated by PAC-1 positivity (increased from 4% to 35%), and further degranulation of the alpha granula (increased from 33% to 55%) and expression of negatively charged phospholipids (increased from 73 to 99%). In conclusion, transfusion of cryopreserved autologous platelets is a feasible alternative to HLA-matched platelets offering an important clinical option for patients with HLA alloimmunization and rare HLA phenotypes. The experimental data demonstrate that the platelets are partially stimulated by the cryopreservation, but can be further activated in vitro by strong agonists, suggesting their functionality. Disclosures:No relevant conflicts of interest to declare.

Recently recognized platelet agonists

The story of platelet activation is complex, involving many intertwined and overlapping pathways and processes that continue to expand in complexity. This review will cover some of the recent novel mediators and receptors identified to be important in platelet activation. Many newly discovered platelet activators and receptors are not strong platelet agonists, but rather are important modifiers of platelet activation, and thus represent potentially novel targets to blunt, but not totally prevent, platelet activation and thrombus formation. The diversity of platelet activation mediators and receptors also expands as we learn more about the disease processes that are either initiated or accelerated by platelets. Although platelets are a unique cell type with a unique role in thrombosis, they share a surprising amount in common with the molecules and machinery present in synaptic termini and immune cells. Many of these secreted molecules have important roles in platelet activation. Many newly identified platelet activators are weak agonists. These represent intriguing targets for platelet inhibitor development or shed light on the overlap between inflammation and thrombosis.

Lipid-mediated membrane binding properties of Disabled-2

Disabled-2 (Dab2) is an adaptor protein involved in several biological processes ranging from endocytosis to platelet aggregation. During endocytosis, the Dab2 phosphotyrosine-binding (PTB) domain mediates protein binding to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) at the inner leaflet of the plasma membrane. As a result of platelet activation, Dab2 is released from α-granules and associates with both the αIIbβ3 integrin receptor and sulfatide lipids on the platelet surface through its N-terminal region including the PTB domain (N-PTB), thus, modulating platelet aggregation. Thrombin, a strong platelet agonist, prevents Dab2 function by cleaving N-PTB within the two basic motifs required for sulfatide association, a reaction that is prevented when Dab2 is bound to these sphingolipids. We have characterized the membrane binding properties of Dab2 N-PTB using micelles enriched with Dab2 lipid ligands, sulfatides and PtdIns(4,5)P2. Remarkably, NMR spectroscopy studies suggested differences in lipid-binding mechanisms. In addition, we experimentally demonstrated that sulfatide- and PtdIns(4,5)P2-binding sites overlap in Dab2 N-PTB and that both lipids stabilize the protein against temperature-induced unfolding. We found that whereas sulfatides induced conformational changes and facilitated Dab2 N-PTB penetration into micelles, Dab2 N-PTB bound to PtdIns(4,5)P2 lacked these properties. These results further support our model that platelet membrane sulfatides, but not PtdIns(4,5)P2, protect Dab2 N-PTB from thrombin cleavage.

Sulfatides Partition Disabled-2 in Response to Platelet Activation

BackgroundPlatelets contact each other at the site of vascular injury to stop bleeding. One negative regulator of platelet aggregation is Disabled-2 (Dab2), which is released to the extracellular surface upon platelet activation. Dab2 inhibits platelet aggregation through its phosphotyrosine-binding (PTB) domain by competing with fibrinogen for αIIbβ3 integrin receptor binding by an unknown mechanism.Methodology/Principal FindingsUsing protein-lipid overlay and liposome-binding assays, we identified that the N-terminal region of Dab2, including its PTB domain (N-PTB), specifically interacts with sulfatides. Moreover, we determined that such interaction is mediated by two conserved basic motifs with a dissociation constant (Kd) of 0.6 µM as estimated by surface plasmon resonance (SPR) analysis. In addition, liposome-binding assays combined with mass spectroscopy studies revealed that thrombin, a strong platelet agonist, cleaved N-PTB at a site located between the basic motifs, a region that becomes protected from thrombin cleavage when bound to sulfatides. Sulfatides on the platelet surface interact with coagulation proteins, playing a major role in haemostasis. Our results show that sulfatides recruit N-PTB to the platelet surface, sequestering it from integrin receptor binding during platelet activation. This is a transient recruitment that follows N-PTB internalization by an actin-dependent process.Conclusions/SignificanceOur experimental data support a model where two pools of Dab2 co-exist at the platelet surface, in both sulfatide- and integrin receptor-bound states, and their balance controls the extent of the clotting response.

No influence of the VAMP8 rs1010 single nucleotide polymorphism on platelet functions in vitro

No influence of the VAMP8 rs1010 single nucleotide polymorphism on platelet functions <i>in vitro</i>

Thromboxane A2Receptor Antagonism by Flavonoids: Structure−Activity Relationships

Thromboxane A2 (TxA2) is a strong platelet agonist involved in the pathogenesis of thrombotic diseases that elicits platelet aggregation and vasoconstriction through the activation of its specific membrane receptor (TP). Previous studies have demonstrated that certain flavonoids, naturally occurring phytochemicals, inhibit platelet function through several mechanisms, including antagonism of TP in these cells. However, the steric and inductive or mesomeric requirements underlying this effect are not fully understood. In this study, the ability of 20 naturally occurring flavonoids belonging to different structural subtypes to inhibit [3H]-SQ29548 binding to platelet-rich plasma was compared to establish the structural basis explaining their TP antagonistic activity. The results show a key contribution of C7 and C8 carbons in the A ring, gamma-pyrone structure conjugated with a double bond between C2 and C3 carbons in the C ring, and C2', C3', and C4' carbons in the B ring as the structural determinants that create the active flavonoid skeleton in TP blockade. These data might help in the design of new TP antagonists with potential antithrombotic effects and provide additional evidence for the correlation between biological properties of flavonoids and their structure.

Comparative anti-platelet and antioxidant properties of polyphenol-rich extracts from: berries of Aronia melanocarpa, seeds of grape and bark of Yucca schidigera in vitro

The aim of the present study was to investigate and compare the anti-platelet action of extracts from three different plants: bark of Yucca schidigera, seeds of grape and berries of Aronia melanocarpa (chokeberry). Anti-platelet action of tested extracts was compared with action of well characterized antioxidative and anti-platelet commercial monomeric polyphenol–resveratrol. The effects of extracts on platelet adhesion to collagen, collagen–induced platelet aggregation and on the production of in resting platelets and platelets stimulated by a strong platelet agonist–thrombin were studied. The in vitro experiments have shown that all three tested extracts (5–50 µg/ml) rich in polyphenols reduce platelet adhesion, aggregation and generation of in blood platelets. Comparative studies indicate that all three plant extracts were found to be more reactive in reduction of platelet processes than the solution of pure resveratrol. The tested extracts due to their anti-platelet effects may play an important role as components of human diet in prevention of cardiovascular or inflammatory diseases, where blood platelets are involved.

Pennogenin glycosides with a spirostanol structure are strong platelet agonists: structural requirement for activity and mode of platelet agonist synergism.

Steroidal saponins have long attracted scientific attention, due to their structural diversity and significant biological activities. For example, total steroidal saponins extracted from the rhizome of Paris polyphylla Sm. var. yunnanensis (TSSPs) constitute an effective treatment for abnormal uterine bleeding. To determine the active constituents in TSSPs and elucidate the mechanisms that underlie their in vivo pharmacologic actions on hemostasis. Steroidal saponins were purified by chromatography, and their effects upon hemostasis and platelet function were evaluated by tail bleeding time in mice and rats, aggregometry, flow cytometry and Western blotting. TSSPs promoted hemostasis in vivo and dose-dependently induced rat or human platelet aggregation in vitro. Using bioassay-guided separation, four known pennogenin glycosides with a spirostanol structure were identified as the active ingredients of TSSPs. A structure-activity assay showed that the aglycone and sugar moieties of pennogenin glycosides are both essential for their aggregatory activity. Their synergistic actions on platelet aggregation were observed with pennogenin glycosides and with other known platelet agonists, suggesting that these glycosides are platelet agonists. Aggregation in response to the pennogenin glycosides involved alpha(IIb)beta(3) activation, was inhibited by cAMP, was dependent upon extracellular calcium, secreted ADP and thromboxane synthesis, and was mediated by phosphatidylinositol-3-kinase. We identified pennogenin glycosides with a spirostanol structure as the active ingredients of Paris polyphylla Sm. var. yunnanensis in promoting hemostasis in vivo. Their mode of their action on platelets suggests that they represent a new type of platelet agonist.

The Design and Synthesis of Small Molecule Inhibitors of Collagen Binding to Integrin α2β1 as Antithrombotic Agents.

Vascular damage due to trauma or disease exposes circulating platelets to collagen in the subendothelial matrix. This is a critical event in the formation of a hemostatic plug or an occluding thrombus because collagen is not only a substrate for platelet adhesion but is also a strong platelet agonist. Platelets possess two physiologic collagen receptors: glycoprotein VI, a member of the immunoglobin superfamily, and the integrin α2β1. To design small molecule inhibitors of the interaction of platelets with collagen, we focused on α2β1 as a target because murine models of α2β1 deficiency display normal bleeding times and only a slight decrease in platelet activation by collagen and because the small number of reported patients with congenital α2β1 deficiency demonstrated only a mild bleeding diathesis. Thus, α2β1 antagonists could be effective anti-thrombotic agents with minimal toxicity, especially when combined with other anti-platelet drugs. We have developed a class of compounds that target the I-like domain of the β1 subunit, an allosteric site that regulates collagen binding to α2β1 by preventing the conversion of α2β1 from an inactive (low affinity) to an active (high affinity) conformation. This class of compounds is based on a proline-substituted 2,3-diaminopropionic acid scaffold. Structure-activity relationship studies of the scaffold have focused on optimization of the proline moiety, the urea functionality, and the sulfonyl group and have resulted in the development of potent inhibitors of α2β1-mediated platelet adhesion to collagen with IC50's in the high picomolar to low nanomolar range. In particular, optimization of the proline moiety lead to compounds with high potency: transitioning from proline (DB496, IC50 of 29–62 nM) to a thiazolidine (SB68A) improved the IC50 to 2–8 nM; adding a methyl group at the 2 position of the thiazolidine (SB68B) slightly improved the IC50 to 1–12 nM; adding two methyl groups at the 5 position of the thiazolidine (SW4-161) resulted in a lead compound with an IC50 of 0.33–8 nM. As expected, the compounds had no effect on the binding of isolated α2 I-domains to collagen, consistent with their I-like domain mode of activity. Further, they were specific for α2β1-mediated platelet adhesion to collagen because they had no impact on ADP-stimulated platelet aggregation when added at 2 μM, a concentration more than 100-fold greater than the IC50 for inhibition of platelet adhesion to collagen. The compounds were also strong inhibitors of murine platelet adhesion to collagen and when tested in the ferric chloride-initiated murine carotid artery injury model, displayed activity similar to aspirin. Thus, 71% of untreated mice in this thrombosis model developed occlusive thrombi that remained stable for the 30 min duration of the assay, whereas stable thrombi developed in only 32% of mice treated with 1g/kg aspirin orally and in 41% of mice receiving 60 mg/kg CSW4-161intravenously. In summary, we have developed a class of potent inhibitors of the integrin α2β1 that demonstrate both in vitro and in vivo anti-platelet activity. Further development of this class of compounds may result in novel and relatively non-toxic anti-thrombotic agents.

Inhibition of the Platelet P2Y12 Receptor Potentiates the Antiplatelet Effect of Prostacyclin.

Adenosine diphosphate (ADP) plays a key role in platelet function because, although itself a weak platelet agonist, when it is secreted from the platelet dense granules, it amplifies the platelet responses induced by strong platelet agonists. Concomitant activation of two platelet receptors for ADP is necessary for ADP-induced platelet aggregation (PA): the Gq-coupled P2Y1 receptor and the Gi-coupled P2Y12 receptor. P2Y12 is coupled to inhibition of adenylyl cyclase (AC) through activation of Gαi2. However, inhibition of AC bears no causal relationship to PA: other, partially identified signalling events downstream of P2Y12 are required for ADP-induced PA. P2Y12 plays an important role in thrombogenesis, because its irreversible inhibition by the thienopyridine drug clopidogrel effectively reduces the incidence of cardiovascular events in patients at risk. It is generally held that the antithrombotic effect of anti-P2Y12 drugs is uniquely mediated by inhibition of the P2Y12-dependent PA. However, prevention of inhibition of AC by ADP could play an additional, important antithrombotic role in vivo. In fact, P2Y12 inhibition could amplify the antiplatelet effect of prostacyclin (PGI2), which increases the platelet levels of cyclic adenosine monophosphate (cAMP), a potent inhibitor of PA, by stimulating AC. To test our hypothesis, we measured the effects of increasing concentrations of PGI2 (0.01-10uM) on PA of washed, normal human platelets induced by thrombin in the presence or absence of saturating concentrations of ARC69931MX (anti-P2Y12, 1μM) or MRS2500 (anti-P2Y1, 1μM). We used high concentration of thrombin (0.5 U/ml), which induced PA that was not inhibited by anti-P2Y1 or anti-P2Y12, both alone and in combination. PGI2 inhibited PA in the presence of anti-P2Y12, but did not inhibit PA in the presence of anti-P2Y1 or in the absence of inhibitors. In contrast to PGI2, dibutiryl-cAMP inhibited PA both in the presence and absence of anti-P2Y1 or anti-P2Y12. PGI2 increased platelet cAMP levels in the absence of thrombin or in the presence of thrombin plus anti-P2Y12, but it had no effect on cAMP levels in the presence of thrombin with or without anti-P2Y1. These results suggest that:PGI2, at the tested concentrations, does not inhibit PA induced by high concentration of thrombin, because its effect on AC is prevented by the interaction of released ADP with P2Y12;anti-P2Y12, by rescuing AC activity, facilitates the inhibitory effect of PGI2 on PA: this effect may contribute to the known antithrombotic effect of drugs inhibiting P2Y12.In the last years, attempts have been made to improve the antithrombotic efficacy of antiplatelet therapy by combined administration of clopidogrel and aspirin (which inhibits the thromboxane A2 pathway of PA). However, the results of recent clinical trials indicated that this therapeutic strategy is not superior to monotherapy with either clopidogrel or aspirin in some clinical conditions. The unexpected negative results of these clinical trials can be interpreted in light of the proposed, additional antithrombotic mechanism of anti-P2Y12: since aspirin, at the doses that are commonly used to prevent cardiovascular events, inhibits the production of PGI2 by vascular wall, it could abolish the antithrombotic effect of clopidogrel that is mediated by the potentiation of the inhibitory effect of PGI2 on PA.

TMVA, a snake C-type lectin-like protein from Trimeresurus mucrosquamatus venom, activates platelet via GPIb

TMVA is a C-type lectin-like protein with potent platelet activating activity from Trimeresurus mucrosquamatus venom. In the absence of von Willebrand factor (vWF), TMVA dose-dependently induced aggregation of washed platelets. Anti-GP Ib monoclonal antibodies (mAbs), HIP1, specifically inhibited TMVA-induced aggregation in a dose-dependent manner. The aggregation was also inhibited by mAb P2 (an anti-GP IIb mAb). Flow cytometric analysis revealed that FITC-TMVA bound to human formalin-fixed platelets in a saturable manner, and its binding was specifically blocked by HIP1 in a dose-dependent manner. Flow cytometric analysis showed that TMVA did not bind to platelet GPIX, GPIIb, GPIIIa, GPIa, GPIIa and GPIV. Moreover, the platelet aggregation induced by TMVA was partially inhibited when platelet was pretreated with mocarhagin, a snake venom protease that specifically cleaves human GPIb. These results suggest that TMVA is a strong platelet agonist via GPIb and it might have multiple functional binding-sites on GPIb molecule or on other unknown receptor.

Real-Time Detection of Activation Patterns in Individual Platelets during Thromboembolism in vivo: Differences between Thrombus Growth and Embolus Formation

Knowledge on single platelet behavior and intracellular mechanisms during thromboembolism in vivo is scarce. In the present study, we used a new method that enables real-time detection and quantification of activation of individual platelets participating in a thromboembolic process in vivo, using their intracellular free Ca²⁺ concentration ([Ca²⁺]_i) as a marker of activation. Isolated platelets were labeled with the Ca²⁺-sensitive fluorescent probe fluo-3 and injected into anesthetized rabbits so that 0.5–1% of their circulating platelets were labeled. Wall puncture of mesenteric arterioles resulted in thrombus formation followed by embolization. Fluorescence intensity changes of labeled platelets participating in this process were quantified. Within 30 min after injection, labeled platelets behaved similarly to native platelets, and fluorescence intensity was not influenced by dye leakage. Upon adherence to the stationary thrombus, platelets exhibited a prolonged [Ca²⁺]_i increase, accompanied by shape change and degranulation, which is consistent with a role for strong platelet agonists like collagen. In contrast, when platelets adhered to a growing embolus their [Ca²⁺]_i rise was transient, and they hardly showed shape change and degranulation, suggesting the involvement of weaker agonists like ADP. These results show, for the first time, the relation between single platelet activation patterns, which are different during thrombus growth and embolus formation, and their behavior in a thromboembolic process in vivo.

The inflammatory action of CD40 ligand (CD154) expressed on activated human platelets is temporally limited by coexpressed CD40

Recently, we have demonstrated that human platelets carry preformed CD40 ligand (CD154) molecules, which rapidly appear on the platelet surface following stimulation by thrombin. Once on the surface, platelet CD154 induces an inflammatory reaction of CD40-bearing endothelial cells. This study shows that strong platelet agonists other than thrombin also lead to the expression of CD154 on the platelet surface. At the same time, several lines of evidence are presented that together indicate that thrombotic events in the vasculature are generally accompanied by activation of the inflammatory potential of platelet CD154. This study also reports the constitutive expression of CD40, the receptor for CD154, on platelets. The binding of CD154 to coexpressed CD40 in the platelet aggregate leads within minutes to hours to the cleavage of membrane-bound surface CD154 and the release of an 18-kd soluble form of the molecule. Soluble CD154 (sCD154), in contrast to transmembrane CD154, can no longer induce an inflammatory reaction of endothelial cells. These findings indicate that the interaction of platelet CD154 with CD40 on neighboring cells is temporally limited to prevent an uncontrolled inflammation at the site of thrombus formation. Thus, similar to the very tight regulation of the CD154-CD40 interaction in the immune system, an effective mechanism controls the inflammatory potential of platelet CD154 in the vascular system. (Blood. 2001;98:1047-1054)

Flow cytometric kinetic assay of calcium mobilization in whole blood platelets using Fluo‐3 and CD41

Background: Platelet activation plays a major role in the physiology and pathology of hemostasis. Flow cytometry is a promising approach for the structural and functional analysis of platelets. However, the choice of adequate biological parameters and most technical issues are still under discussion. A rise in cytosolic free Ca2+ is a key early event that follows platelet stimulation and precedes several activation responses, including shape change, aggregation, secretion, and expression of procoagulant activity. Our objective was to set up a fast and sensitive flow cytometric method to determine the kinetics of intracellular Ca2+ mobilization in platelets, which could be performed with the least artifactual perturbation of platelet function. Methods: Anticoagulated blood was diluted in Tyrode's buffer and incubated with Fluo-3-acetoxymethyl ester prior to staining with phycoerytrin-conjugated antiplatelet GPIIb/IIIa complex monoclonal antibody. Platelets were identified by a gate including only CD41+ events. After the determination of baseline Fluo-3 green fluorescence on a flow cytometer (EPICS XL-MCL, Coulter Electronics, Hialeah, FL), adequate agonists were added and time-dependent changes in Fluo-3 fluorescence were recorded on-line for up to 3 min. Results: In these conditions, a very fast and transient increase of cytosolic-free Ca2+ was observed following the addition of thrombin, a strong platelet agonist. Stimulation with adenosine diphosphate (ADP), a weak agonist, also resulted in evident increase of Ca2+ levels. Conclusions: Our results show that this flow cytometric kinetic method provides a simple and sensitive tool to assess in vitro the time course and intensity of signal transduction responses to different platelet agonists under near physiological conditions. In this way, it may be useful to evaluate the degree of platelet reactivity and thus to monitor antiplatelet therapy. Cytometry 35:302–310, 1999. © 1999 Wiley-Liss, Inc.

Changes in blood platelets exposed to UV-B radiation.

The effects of UV-B radiation (312 nm) on the pig-blood platelet secretory process (platelet activation) and platelet lipid peroxidation have been studied. The responses of platelets to UV-B radiation are compared with the response of these cells to thrombin, which is a strong platelet agonist. The obtained results show that exposure of blood platelets to UV-B radiation (1.2 mW/cm2, 0.072-8.64 J/cm2) causes dose-dependent platelet lipid peroxidation (measured as thiobarbituric acid reactive substances, TBARS) and release of adenine nucleotides and proteins from irradiated platelets. The dose-dependent release of platelet compounds from irradiated platelet does not correlate with the activity of platelet lactic dehydrogenase (marker of cell lysis) in the extracellular medium. It seems that UV-B radiation can partly activate platelets by stimulating the platelet secretory process and metabolism of arachidonate.

Flow cytometric kinetic assay of calcium mobilization in whole blood platelets using Fluo-3 and CD41

Platelet activation plays a major role in the physiology and pathology of hemostasis. Flow cytometry is a promising approach for the structural and functional analysis of platelets. However, the choice of adequate biological parameters and most technical issues are still under discussion. A rise in cytosolic free Ca2+ is a key early event that follows platelet stimulation and precedes several activation responses, including shape change, aggregation, secretion, and expression of procoagulant activity. Our objective was to set up a fast and sensitive flow cytometric method to determine the kinetics of intracellular Ca2+ mobilization in platelets, which could be performed with the least artifactual perturbation of platelet function. Anticoagulated blood was diluted in Tyrode's buffer and incubated with Fluo-3-acetoxymethyl ester prior to staining with phycoerytrin-conjugated antiplatelet GPIIb/IIIa complex monoclonal antibody. Platelets were identified by a gate including only CD41+ events. After the determination of baseline Fluo-3 green fluorescence on a flow cytometer (EPICS XL-MCL, Coulter Electronics, Hialeah, FL), adequate agonists were added and time-dependent changes in Fluo-3 fluorescence were recorded on-line for up to 3 min. In these conditions, a very fast and transient increase of cytosolic-free Ca2+ was observed following the addition of thrombin, a strong platelet agonist. Stimulation with adenosine diphosphate (ADP), a weak agonist, also resulted in evident increase of Ca2+ levels. Our results show that this flow cytometric kinetic method provides a simple and sensitive tool to assess in vitro the time course and intensity of signal transduction responses to different platelet agonists under near physiological conditions. In this way, it may be useful to evaluate the degree of platelet reactivity and thus to monitor antiplatelet therapy.