Thrombin-induced Platelet Aggregation Research Articles

Effect of Biopolymers and Functionalized by Them Vaterite Microparticles on Platelet Aggregation

Vaterite microparticles, metastable form of calcium carbonate, are promising forms of delivery of medicinal compounds. For more efficient delivery of target molecules (increased incorporation and retention), vaterite microparticles must be functionalized with biopolymers. In this article the effect of polysaccharides, mucin and vaterite microparticles, as well as hybrid vaterite microparticles with the above-mentioned biopolymers was studied on platelet aggregation. It was found that fucoidan, heparin and dextran sulfate (when added to platelet-rich plasma) and mucin (when added to isolated platelets) initiated cell aggregation. Pectin and chondroitin sulfate inhibited ADP- and thrombin-induced aggregation in a dose-dependent manner, mucin suppressed ADP-induced, and dextran sulfate suppressed thrombin-induced platelet aggregation. Vaterite microparticles at a concentration of 100–1000 μg/ml did not affect the aggregation of isolated platelets, but caused 10–15% cell aggregation in plasma; at the same time, at a concentration of 1000 μg/ml vaterite microparticles prevented agonist-induced cell aggregation by ~30%. It has been established that hybrid vaterite microparticles with fucoidan or heparin, when added both to platelet-rich plasma and to isolated cells, are capable to initiate platelet aggregation. Vaterite microparticles functionalized with pectin or chondroitin sulfate had no effect on spontaneous cell aggregation, and did not affect (with chondroitin sulfate) or inhibit (with pectin) agonist-induced platelet aggregation. Thus, the use of hybrid vaterite microparticles with pectin or fucoidan/heparin may be promising for the delivery of drugs aimed at modulating (inhibition with pectin or activation with fucoidan/heparin) the platelet component of hemostasis.

G protein–coupled receptor kinase 5 regulates thrombin signaling in platelets

BackgroundOur prior genome-wide association study of thrombin-induced platelet aggregation identified a G protein–coupled receptor kinase 5 (GRK5) noncoding variant (rs10886430-G) that is strongly associated with increased platelet reactivity to thrombin. This variant predisposes to increased risk of stroke, pulmonary embolism, and venous thromboembolism. ObjectivesTo determine role of platelet specific GRK5 in platelet responses to agonists and injury. MethodsPlatelets from GRK5 mutant mice have been shown to have increased thrombin sensitivity, indicating that GRK5 may be a negative regulator of platelet activation. However, this has not been studied in a platelet-specific manner. We therefore used platelet-specific GRK5 mutant mice and models of thrombosis and pulmonary embolism. ResultsWe now demonstrate that mice lacking GRK5 specifically in platelets had a mild increase in thrombin responses in vitro and a shortened time to arterial thrombosis in vivo. In addition, platelet GRK5 mutant mice had increased thrombin but not collagen-induced thrombus burden in a mouse model of pulmonary embolism. ConclusionThese data indicate that platelet GRK5 has a significant role in limiting platelet responses to thrombin.

Small-Molecule Disruptors of the Interaction between Calcium- and Integrin-Binding Protein 1 and Integrin αIIbβ3 as Novel Antiplatelet Agents.

Thrombosis, a key factor in most cardiovascular diseases, is a major contributor to human mortality. Existing antithrombotic agents carry a risk of bleeding. Consequently, there is a keen interest in discovering innovative antithrombotic agents that can prevent thrombosis without negatively impacting hemostasis. Platelets play crucial roles in both hemostasis and thrombosis. We have previously characterized calcium- and integrin-binding protein 1 (CIB1) as a key regulatory molecule that regulates platelet function. CIB1 interacts with several platelet proteins including integrin αIIbβ3, the major glycoprotein receptor for fibrinogen on platelets. Given that CIB1 regulates platelet function through its interaction with αIIbβ3, we developed a fluorescence polarization (FP) assay to screen for potential inhibitors. The assay was miniaturized to 1536-well and screened in quantitative high-throughput screening (qHTS) format against a diverse compound library of 14,782 compounds. After validation and selectivity testing using the FP assay, we identified 19 candidate inhibitors and validated them using an in-gel binding assay that monitors the interaction of CIB1 with αIIb cytoplasmic tail peptide, followed by testing of top hits by intrinsic tryptophan fluorescence (ITF) and microscale thermophoresis (MST) to ascertain their interaction with CIB1. Two of the validated hits shared similar chemical structures, suggesting a common mechanism of action. Docking studies further revealed promising interactions within the hydrophobic binding pocket of the target protein, particularly forming key hydrogen bonds with Ser180. The compounds exhibited a potent antiplatelet activity based on their inhibition of thrombin-induced human platelet aggregation, thus indicating that disruptors of the CIB1- αIIbβ3 interaction could carry a translational potential as antithrombotic agents.

Investigating the role of rotenone on human blood platelets: Molecular insights into abnormal platelet functions in Parkinson's disease.

Parkinson's disease (PD) is a predominant neuromotor disorder characterized by the selective death of dopaminergic neurons in the midbrain. The majority of PD cases are sporadic or idiopathic, with environmental toxins and pollutants potentially contributing to its development or exacerbation. However, clinical PD patients are often associated with a reduced stroke frequency, where circulating blood platelets are indispensable. Although platelet structural impairment is evident in PD, the platelet functional alterations and their underlying molecular mechanisms are still obscure. Therefore, we investigated rotenone (ROT), an environmental neurotoxin that selectively destroys dopaminergic neurons mimicking PD, on human blood platelets to explore its impact on platelet functions, thus replicating PD conditions in vitro. Our study deciphered that ROT decreased thrombin-induced platelet functions, including adhesion, activation, secretion, and aggregation in human blood platelets. As ROT is primarily responsible for generating intracellular reactive oxygen species (ROS), and ROS is a key player regulating the platelet functional parameters, we went on to check the effect of ROT on platelet ROS production. In our investigation, it became evident that ROT treatment resulted in the stimulation of ROS production in human blood platelets. Additionally, we discovered that ROT induced ROS production by augmenting Ca2+ mobilization from inositol 1,4,5-trisphosphate receptor. Apart from this, the treatment of ROT triggers protein kinase C associated NADPH oxidase-mediated ROS production in platelets. In summary, this research, for the first time, highlights ROT-induced abnormal platelet functions and may provide a mechanistic insight into the altered platelet activities observed in PD patients.

Salvia miltiorrhiza ameliorates endometritis in dairy cows by relieving inflammation, energy deficiency and blood stasis.

Introduction: According to traditional Chinese veterinary medicine, endometritis is caused by a combination of Qi deficiency, blood stasis, and external evil invasion. Salvia miltiorrhiza is a traditional Chinese medicine that counteracts blood stasis and has additional demonstrated effects in boosting energy and restraining inflammation. Salvia miltiorrhiza has been employed in many traditional Chinese prescriptions that have proven effective in healing clinical dairy cow endometritis. Methods: the in vivo effect of Salvia miltiorrhiza in treating endometritis was evaluated in dairy cows. In addition, bovine endometrial epithelium cell inflammation and rat blood stasis models were employed to demonstrate the crosstalk between energy, blood circulation and inflammation. Network analysis, western blotting, qRT-PCR and ELISA were performed to investigate the molecular mechanism of Salvia miltiorrhiza in endometritis treatment. Results: The results demonstrate that treatment with Salvia miltiorrhiza relieves uterine inflammation, increases blood ATP concentrations, and prolongs blood clotting times. Four of the six Salvia miltiorrhiza main components (SMMCs) (tanshinone IIA, cryptotanshinone, salvianolic acid A and salvianolic acid B) were effective in reversing decreased ATP and increased IL-1β, IL-6, and IL-8 levels in an in vitro endometritis model, indicating their abilities to ameliorate the negative energy balance and external evil invasion effects of endometritis. Furthermore, in a blood stasis rat model, inflammatory responses were induced in the absence of external infection; and all six SMMCs inhibited thrombin-induced platelet aggregation. Network analysis of SMMC targets predicted that Salvia miltiorrhiza may mediate anti-inflammation via the Toll-like receptor signaling pathway; anti-aggregation via the Platelet activation pathway; and energy balance via the Thermogenesis and AMPK signaling pathways. Multiple molecular targets within these pathways were verified to be inhibited by SMMCs, including P38/ERK-AP1, a key molecular signal that may mediate the crosstalk between inflammation, energy deficiency and blood stasis. Conclusion: These results provide mechanistic understanding of the therapeutic effect of Salvia miltiorrhiza for endometritis achieved through Qi deficiency, blood stasis, and external evil invasion.

418 The antiplatelet effects of EPA, an omega-3 fatty acid, are mediated by its 12-lipoxygenase metabolite, 12-HEPE

OBJECTIVES/GOALS: To determine whether cardioprotective effects observed in individuals taking dietary supplementation with eicosapentaenoic acid (EPA), an ω-3 polyunsaturated fatty acid, are realized by altering platelet function, and if these effects are mediated through the 12-lipoxygenase derived metabolite, 12-hydroxyeicosapentaenoic acid (12-HEPE). METHODS/STUDY POPULATION: Washed platelets or platelet rich plasma from healthy human donors were treated with EPA and 12-HEPE to assess their ability to inhibit platelet activation. Platelets were stimulated with agonists targeting different steps of the hemostatic response to vascular injury. Platelet aggregation, dense granule secretion, surface expression of integrin αIIbβ3 and P-selectin, and clot retraction were analyzed. To assess signaling through Gαs-GPCRs and protein kinase A activity, phosphorylation of vasodilator-stimulated phosphoprotein (VASP) was examined via western blot following treatment with EPA or 12-HEPE. RESULTS/ANTICIPATED RESULTS: EPA and 12-HEPE dose-dependently inhibit both collagen and thrombin-induced platelet aggregation. Furthermore, 12-HEPE more potently attenuates dense granule secretion and surface expression of platelet activation markers, integrin αIIbβ3 and P-selectin, in comparison to EPA. Plasma treated with EPA delayed thrombin-induced clot retraction, while 12-HEPE had no effect. Additionally, treatment with 12-HEPE increases phosphorylation of VASP, suggesting it could signal through the activation of the eicosanoid Gαs-GPCRs. DISCUSSION/SIGNIFICANCE: Here, we show for the first time that EPA directly inhibits platelet activation through its 12-LOX metabolite, 12-HEPE. These findings provide further insight into the mechanisms underlying the cardioprotective effects of EPA. A better understanding of current PUFA supplementations can inform treatment and prevention of cardiovascular diseases.

4'-O-MethylbavachalconeB Targeted 14-3-3ζ Blocking the Integrin β3 Early Outside-In Signal to Inhibit Platelet Aggregation and Thrombosis.

14-3-3ζ protein, the key target in the regulation and control of integrin β3 outside-in signaling, is an attractive new strategy to inhibit thrombosis without affecting hemostasis. In this study, 4'-O-methylbavachalconeB (4-O-MB) in Psoraleae Fructus was identified as a 14-3-3ζ ligand with antithrombosis activity by target fishing combined with ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) analysis. The competitive inhibition analysis showed that 4-O-MB targeted 14-3-3ζ and blocked the 14-3-3ζ/integrin β3 interaction with inhibition constant (Ki) values of 9.98 ± 0.22 μM. Molecular docking and amino acid mutation experiments confirmed that 4-O-MB specifically bound to 14-3-3ζ through LSY9 and SER28 to regulate the 14-3-3ζ/integrin β3 interaction. Besides, 4-O-MB affected the integrin β3 early outside-in signal by inhibiting AKT and c-Src phosphorylation. Meanwhile, 4-O-MB could inhibit ADP-, collagen-, or thrombin-induced platelet aggregation function but had no effect on platelet adhesion to collagen-coated surfaces in vivo. Administration of 4-O-MB could significantly inhibit thrombosis formation without disturbing hemostasis in mice. These findings provide new prospects for the antithrombotic effects of Psoraleae Fructus and the potential application of 4-O-MB as lead compounds in the therapy of thrombosis by targeting 14-3-3ζ.

Bifunctional Sildenafil Diazeniumdiolates Acting as Phosphodiesterase 5 Inhibitors and Nitric Oxide Donors- Towards Wound Healing.

Inefficient wound healing poses a global health challenge with a lack of efficient treatments. Wound healing issues often correlate with low endogenous nitric oxide (NO) levels. While exogenous delivery with NO-releasing compounds represents a promising therapeutic strategy, controlling the release of the highly reactive NO remains challenging. Phosphodiesterase 5 (PDE5) inhibitors, like sildenafil, have also been shown to promote wound healing. This study explores hybrid compounds, combining NO-releasing diazeniumdiolates with a sildenafil-derived PDE5 inhibitor. One compound demonstrated a favorable NO-release profile, triggered by an esterase (prodrug), and displayed in vitro nanomolar inhibition potency against PDE5 and thrombin-induced platelet aggregation. Both factors are known to promote blood flow and oxygenation. Thus, our findings unveil promising prospects for effective wound healing treatments.

Design and synthesis of dabigatran etexilate derivatives with inhibiting thrombin activity for hepatocellular carcinoma treatment

Hepatocellular carcinoma (HCC) is one of the most fatal solid malignancies worldwide. Evidence suggests that thrombin stimulates tumor progression via fibrin formation and platelet activation. Meanwhile, we also found a correlation between thrombin and HCC through bioinformatics analysis. Dabigatran is a selective, direct thrombin inhibitor that reversibly binds to thrombin. Dabigatran was used as the lead agent in this study, and 19 dabigatran derivatives were designed and synthesized based on docking mode. The thrombin-inhibitory activity of the derivative AX-2 was slightly better than that of dabigatran. BX-2, a prodrug of AX-2, showed a fairly strong inhibitory effect on thrombin-induced platelet aggregation, and effectively antagonized proliferation of HCC tumor cells induced by thrombin at the cellular level. Furthermore, BX-2 reduced tumor volume, weight, lung metastasis, and secondary tumor occurrence in nude mouse models. BX-2 combined with sorafenib increased sorafenib efficacy. This study lays the foundation for discovering new anti-HCC mechanism based on thrombin. BX-2 can be used as an anti-HCC drug lead for further research.

Endogenous SIRT6 in platelets negatively regulates platelet activation and thrombosis.

Thromboembolism resulting from platelet dysfunction constitutes a significant contributor to the development of cardiovascular disease. Sirtuin 6 (SIRT6), an essential NAD+-dependent enzyme, has been linked to arterial thrombosis when absent in endothelial cells. In the present study, we have confirmed the presence of SIRT6 protein in anucleated platelets. However, the precise regulatory role of platelet endogenous SIRT6 in platelet activation and thrombotic processes has remained uncertain. Herein, we present compelling evidence demonstrating that platelets isolated from SIRT6-knockout mice (SIRT6-/-) exhibit a notable augmentation in thrombin-induced platelet activation, aggregation, and clot retraction. In contrast, activation of SIRT6 through specific agonist treatment (UBCS039) confers a pronounced protective effect on platelet activation and arterial thrombosis. Moreover, in platelet adoptive transfer experiments between wild-type (WT) and SIRT6-/- mice, the loss of SIRT6 in platelets significantly prolongs the mean thrombus occlusion time in a FeCl3-induced arterial thrombosis mouse model. Mechanistically, we have identified that SIRT6 deficiency in platelets leads to the enhanced expression and release of proprotein convertase subtilisin/kexin type 9 (PCSK9), subsequently activating the platelet activation-associated mitogen-activated protein kinase (MAPK) signaling pathway. These findings collectively unveil a novel protective role of platelet endogenous SIRT6 in platelet activation and thrombosis. This protective effect is, at least in part, attributed to the inhibition of platelet PCSK9 secretion and mitogen-activated protein kinase signaling transduction. Our study provides valuable insights into the intricate interplay between SIRT6 and platelet function, shedding light on potential therapeutic avenues for managing thrombotic disorders.

Preclinical Study on a Novel Fluoroderivative of Dabigatran Etexilate in Animal Models.

Here, the fluorinated derivative, R1, was synthesized from the fluorinated dabigatran derivative (R0). The in vivo pharmacokinetic characteristics of orally administered R1, R0 injection, and dabigatran etexilate in rats were compared. Safety evaluation results showed no significant changes in the QRS wave or PR and QT intervals in rat lead II electrocardiograms. The possible toxicity of R1 was studied using the limit test method, and no obvious toxicity occurred in mice after the acute oral administration of R1. R1 inhibited thrombin-induced platelet aggregation in a dose-dependent manner, had an inhibitory effect on platelet aggregation induced by arachidonic acid and adenosine diphosphate, could significantly prolong prothrombin time and activated partial thromboplastin time, and increased fibrinogen levels. R1 is the optimal candidate compound from among more than 100 candidate compounds designed and synthesized by our research group. It was first selected through preliminary in vitro anticoagulant activity screening and further through in vivo mouse activity testing. A systematic pharmacodynamic study showed that R1 was superior to the raw material drug dabigatran ester; particularly, the absolute bioavailability of R1 increased by 206%, and this can overcome the low bioavailability defect associated with the marketed drug dabigatran ester. Another safety assessment of R1 indicated that there were no risks of acute poisoning in rats and cardiac toxicity in mice or rats. Therefore, R1 can be considered a new candidate anticoagulant compound with great potential and significance for further clinical research.

Argania spinosa Leaves and Branches: Antiaggregant, Anticoagulant, Antioxidant Activities and Bioactive Compounds Quantification

Thrombocytes, also known as platelets, are crucial in maintaining the balance between blood clotting. Platelet hyperactivity and oxidative stress are the primary factors contributing to cardiovascular complications. Antithrombotic therapy remains one of the most effective treatments, but various potential side effects hinder its effectiveness, including the risk of haemorrhage. Intense research has been conducted on medicinal plants to discover the natural antithrombotic compounds. Argania spinosa, commonly known as the argan tree or argan oil tree, is a native species of southwestern Morocco. This study evaluated the primary and secondary hemostasis and antioxidant activity of leaf and branch aqueous extracts of A. spinosa and also assessed the phytochemical composition of these extracts. Platelet aggregation assay was performed using washed platelets stimulated with thrombin. For plasmatic coagulation, activated partial thromboplastin time and prothrombin time were measured using the poor plasma method. Bleeding time was evaluated by inducing bleeding at the tip of a mouse tail. The antioxidant activity of the extracts was determined through the DPPH, β-carotene, and FRAP methods. The presence or absence of the secondary metabolites was carried out with the help of specific reagents, and the quantitative analysis was carried out using spectrophotometric and colorimetric methods. The study results revealed the presence of phenols, total flavonoids, cardiac glycosides, tannins, and coumarins type of secondary metabolites in both types of aqueous extracts and a higher concentration of these was recorded in the leaves extracts. Both aqueous extracts significantly reduced in vitro thrombin-induced platelet aggregation, extended tail bleeding time, prolonged activated partial thromboplastin and prothrombin time and exhibited remarkable antioxidant activity. The leaf extract of A. spinosa exerts significant effects against thrombotic manifestations and could be a promising source of new antithrombotic compounds.

Daboxin P, a phospholipase A2 of Indian Daboia russelii venom, modulates thrombin-mediated platelet aggregation.

Daboxin P, reported earlier from the venom of Daboia russellii, disturbs the blood coagulation cascade by targeting factor X and factor Xa. The present study exhibits that Daboxin P also inhibits platelet aggregation induced by various agonists. The thrombin-induced platelet aggregation was inhibited maximum whereas inhibition of collagen-induced platelet aggregation was found to be 50% and no inhibition of adenosine diphosphate (ADP)and arachidonic acid-induced aggregation was observed. Daboxin P dose-dependently inhibited the thrombin-induced platelet aggregation with Anti-Aggregation 50 (AD50 ) dose of 55.166 nM and also reduced the thrombin-mediated calcium influx. In-silico interaction studies suggested that Daboxin P binds to thrombin and blocks its interaction with its receptor on the platelet surface. Quenching of thrombin's emission spectrum by Daboxin P and electrophoretic profiles of pull-down assay further reveals the binding between Daboxin P and thrombin. Thus, the present study demonstrates that Daboxin P inhibits thrombin-induced platelet aggregation by binding to thrombin.

The anti-platelet activity of panaxadiol fraction and panaxatriol fraction of Korean Red Ginseng in vitro and ex vivo

BackgroundThe anti-platelet activity of the saponin fraction of Korean Red Ginseng has been widely studied. The saponin fraction consists of the panaxadiol fraction (PDF) and panaxatriol fraction (PTF); however, their anti-platelet activity is yet to be compared. Our study aimed to investigate the potency of anti-platelet activity of PDF and PTF and to elucidate how well they retain their anti-platelet activity via different administration routes. MethodsFor ex vivo studies, Sprague-Dawley rats were orally administered 250 mg/kg PDF and PTF for 7 consecutive days before blood collection via cardiac puncture. Platelet aggregation was conducted after isolation of the washed platelets. For in vitro studies, washed platelets were obtained from Sprague-Dawley rats. Collagen and adenosine diphosphate (ADP) were used to induce platelet aggregation. Collagen was used as an agonist for assaying adenosine triphosphate release, thromboxane B2, serotonin, cyclic adenosine monophosphate, and cyclic guanosine monophosphate (cGMP) release. ResultsWhen treated ex vivo, PDF not only inhibited ADP and collagen-induced platelet aggregation, but also upregulated cGMP levels and reduced platelet adhesion to fibronectin. Furthermore, it also inhibited Akt phosphorylation induced by collagen treatment. Panaxadiol fraction did not exert any anti-platelet activity in vitro, whereas PTF exhibited potent anti-platelet activity, inhibiting ADP, collagen, and thrombin-induced platelet aggregation, but significantly elevated levels of cGMP. ConclusionOur study showed that in vitro and ex vivo PDF and PTF treatments exhibited different potency levels, indicating possible metabolic conversions of ginsenosides, which altered the content of ginsenosides capable of preventing platelet aggregation.

Role of Prednisolone in Platelet Activation by Inhibiting TxA2 Generation through the Regulation of cPLA2 Phosphorylation.

Glucocorticoids have been commonly used in the treatment of inflammation and immune-mediated diseases in human beings and small animals such as cats and dogs. However, excessive use can lead to Cushing's syndrome along with several thrombotic and cardiovascular diseases. Although it is well-known that glucocorticoids exert a significant effect on coagulation, the effect of cortisol on platelet function is much less clear. Thus, we aimed to study the effects of prednisolone, one of the commonly used glucocorticoids, on the regulation of platelet function using murine platelets. We first evaluated the concentration-dependent effect of prednisolone on 2-MeSADP-induced platelet function and found that the 2-MeSADP-induced secondary wave of aggregation and dense granule secretion were completely inhibited from 500 nM prednisolone. Since 2-MeSADP-induced secretion and the resultant secondary wave of aggregation are mediated by TxA2 generation, this result suggested a role of prednisolone in platelet TxA2 generation. Consistently, prednisolone did not affect the 2-MeSADP-induced aggregation in aspirinated platelets, where the secondary wave of aggregation and secretion were blocked by eliminating the contribution of TxA2 generation by aspirin. In addition, thrombin-induced platelet aggregation and secretion were inhibited in the presence of prednisolone by inhibiting the positive-feedback effect of TxA2 generation on platelet function. Furthermore, prednisolone completely inhibited 2-MeSADP-induced TxA2 generation, confirming the role of prednisolone in TxA2 generation. Finally, Western blot analysis revealed that prednisolone significantly inhibited 2-MeSADP-induced cytosolic phospholipase A2 (cPLA2) and ERK phosphorylation in non-aspirinated platelets, while only cPLA2 phosphorylation, but not ERK phosphorylation, was significantly inhibited by prednisolone in aspirinated platelets. In conclusion, prednisolone affects platelet function by the inhibition of TxA2 generation through the regulation of cPLA2 phosphorylation, thereby shedding light on its clinical characterization and treatment efficacy in dogs with hypercortisolism in the future.

Stoichiometry and architecture of the platelet membrane complex glycoprotein Ib-IX-V.

Glycoprotein (GP) Ib-IX-V is the second most abundant platelet receptor for thrombin and other ligands crucial for hemostasis and thrombosis. Its activity is involved in platelet adhesion to vascular injury sites and thrombin-induced platelet aggregation. GPIb-IX-V is a heteromeric complex composed of four subunits, GPIbα, GPIbβ, GPV and GPIX, in a stoichiometric ratio that has been wildly debated. Despite its important physiological roles, the overall structure and molecular arrangement of GPIb-IX-V are not yet fully understood. Here, we purify stable and functional human GPIb-IX-V complex from reconstituted EXPi293F cells in high homogeneity, and perform biochemical and structural characterization of this complex. Single-particle cryo-electron microscopy structure of GPIb-IX-V is determined at ∼11Å resolution, which unveils the architecture of GPIb-IX-V and its subunit organization. Size-exclusion chromatography-multi-angle static light scattering analysis reveals that GPIb-IX-V contains GPIb-IX and GPV at a 1:1 stoichiometric ratio and surface plasmon resonance assays show that association ofGPV leads to slow kinetics of thrombin binding to GPIb-IX-V. Taken together, our results provide the first three-dimensional architecture of the intact GPIb-IX-V complex, which extends our understanding of the structure and functional mechanism of this complex in hemostasis and thrombosis.

Activation of Hippo Pathway Proteins in Thrombin-Stimulated Platelets and Regutates Platelets Activation in Vitro

To investigate the phosphorylation levels of Hippo pathway proteins in thrombin stimulated platelets and to explore its effects on platelet activation. The phosphorylation levels of Hippo pathway proteins - Mammalian STE20-like kinase 1/2 (MST1/2), Nuclear Dbf2 related kinase 1/2 (NDR1/2) and Mps one binder 1(MOB1) in human thrombin stimulated platelets was detected by Western blot. The effect of MST1/2 inhibitor XMU-MP-1 on platelet aggregation was detected by Platelet Aggregometer. The effect of XMU-MP-1 on platelet integrin αIIbβ3 activation and CD62p expression was detected by flow cytometry. The effect of XMU-MP-1 on the "outside-in" signal of platelet integrin was detected by blood clot retraction test. The effects of XMU-MP-1 on platelet Hippo pathway proteins and p38 phosphorylation levels was detected by Western blot. The phosphorylation levels of MST1/2, NDR1/2 and MOB1 were significantly increased in thrombin activated platelets; XMU-MP-1 inhibited thrombin-induced platelet aggregation and αIIbβ3 activation, but did not affect α-granules release and clot retraction. In addition, thrombin induced phosphorylation of the Hippo proteins were decreased in XMU-MP-1 treated platelets, while the phosphorylation of p38 was not affected. In thrombin stimulated platelets, Hippo pathway proteins were activated and contributed to platelets activation.

Augmentation by resveratrol of the inhibitory effect of ethanol on platelet aggregation

Ethanol and resveratrol have been shown to inhibit platelet aggregation. The aim of this study was to determine whether resveratrol has an additional effect on ethanol-induced inhibition of platelet aggregation. Ca2+ entry and subsequent aggregation of human platelets were measured by the fluorescence method and light transmittance method, respectively. Thromboxane B2 concentrations in media containing platelets were measured by using the enzyme-linked immunosorbent assay. Platelet aggregation induced by thrombin (0.025 U/ml) was significantly inhibited by preincubation of platelets with ethanol (0.5%). Preincubation with resveratrol (3.125 µM), which did not affect thrombin-induced platelet aggregation, significantly augmented the inhibitory effect of ethanol on platelet aggregation. Similar synergic effects of ethanol and resveratrol were found on aggregatory responses to collagen (2 µg/ml) and arachidonic acid (0.25 mM). On the other hand, the thrombin-induced increase in intracellular Ca2+ concentration ([Ca2+]i) was not affected by ethanol alone, resveratrol alone or both ethanol and resveratrol together. In nominally Ca2+-free medium, arachidonic acid (0.75 mM) caused a potent platelet aggregation, which was not affected by the presence of ethanol alone, resveratrol alone, or both of them together. Thromboxane B2 formation induced by thrombin was significantly inhibited by ethanol (0.5%) alone and resveratrol (3.125 µM) alone, and these inhibitory effects were significantly augmented in the presence of both ethanol and resveratrol together. Resveratrol shows an additive effect on ethanol-induced inhibition of platelet aggregation. This effect by resveratrol is partly explained by its inhibitory action on thromboxane A2 production in platelets. In addition, both ethanol and resveratrol attenuate platelet aggregation through acting on the Ca2+-dependent intra-platelet pathway after an increase in [Ca2+]i induced by thrombin.

In Vivo Anticoagulant and Antithrombic Activity of Depolymerized Glycosaminoglycan from Apostichopus japonicus and Dynamic Effect-Exposure Relationship in Rat Plasma.

Glycosaminoglycan from Apostichopus japonicus (AHG) and its depolymerized fragments (DAHGs) are anticoagulant fucosylated chondroitin sulfate. The aim of this study was to further evaluate the anticoagulant and antithrombic activity of AHG and DAHGs, as well as reveal the dynamic relationship between exposure and effect in vivo. The results demonstrated that AHG100 (Mw~100 kDa), DAHG50 (Mw~50 kDa), and DAHG10 (Mw~10 kDa) exhibited potent anticoagulant activity by inhibiting intrinsic factor Xase complex (FXase) as well as antithrombin-dependent factor IIa (FIIa) and factor Xa (FXa). These glycosaminoglycans markedly prevented thrombosis formation and thrombin-induced platelet aggregation in a dose- and molecular weight-dependent manner in vitro and in vivo. The further bleeding time measurement indicated that DAHG10 exhibited obviously lower hemorrhage risks than native AHG100. Following oral administration, DAHG10 could be absorbed into blood, further dose-dependently prolonging activated partial thromboplastin time (APTT) and thrombin time (TT) as well as inhibiting FXa and FIIa partially through FXase. Anticoagulant activity was positively associated with plasma concentration following oral administration of DAHG10. Our study proposed a new point of view to understand the correlation between effects and exposure of fucosylated chondroitin sulfate as an effective and safe oral antithrombotic agent.

S298: CS585 IS A FIRST-IN-CLASS COMPOUND TARGETING THE IP RECEPTOR FOR PREVENTION OF THROMBOSIS WITHOUT INCREASED RISK OF BLEEDING

Background: Uncontrolled platelet activation leads to the formation of occlusive thrombi resulting in myocardial infarction, stroke, VTE and critical limb ischemia. There is an unmet need for more efficacious anti-thrombotics with less bleeding in this area. Our group recently identify a novel oxidized lipid in the blood that potently and selectively inhibits platelet activation through activation of the prostacyclin receptor. Aims: Develop a first-in-class antiplatelet drug, CS585, that potently and selectively inhibits platelet reactivity and thrombosis without altering hemostasis and bleeding risk. Methods: We developed a mimetic of the oxidized lipid shown to selectively bind to and activate the prostacyclin receptor. Using human platelets, we assessed the ability of CS585 to inhibit platelet activation by assessing 1)aggregometry, 2)adhesion under arterial flow, and 3) granule secretion and integrin activation using flow cytometry. We additionally assessed thrombosis in vivo in 2 different mouse models as well as bleeding. Finally, we assessed potential off-target effects using thromboelastography (TEG). Results: CS585 potently inhibited both collagen and thrombin-induced platelet aggregation. This inhibition was confirmed by measuring inhibition of αIIbβ3 activation, α-granule secretion, and dense-granule secretion by flow cytometry. Selectivity was confirmed in human platelets by demonstrating a full reversal of inhibition when the IP receptor was pharmacologically blocked or genetically eliminated in IP receptor knockout mice. In vivo inhibition of injury-induced thrombosis in the small (laser-induced cremaster thrombosis model) and large (FeCl3-induced carotid artery thrombosis model) vessels by CS585 was demonstrated and no increased risk for bleeding was observed using the tail vein bleeding model. Finally, TEG experiments in human blood spiked with CS585 demonstrated no delay or decrease in clot strength confirming the tail vein bleeding assay experiments in the mouse. Summary/Conclusion: We have shown for the first time that CS585, a first-in-class analog of an oxidized lipid in the blood potently and selectively activates the prostacyclin receptor resulting in inhibition of human and mouse platelet activation and thrombosis without an increased risk of bleeding. This discovery represents a new class of inhibitors for prevention of platelet activation and thrombosis and protection from myocardial infarction, stroke, VTE, and critical limb ischemia.