Collagen-activated Platelets Research Articles

NADPH oxidase 1/4 dual inhibitor setanaxib suppresses platelet activation and thrombus formation

AimsThe production of reactive oxygen species (ROS) by NADPH oxidase (NOX) is able to induce platelet activation, making NOX a promising target for antiplatelet therapy. In this study, we examined the effects of setanaxib, a dual NOX1/4 inhibitor, on human platelet function and ROS-related signaling pathways. Materials and methodsIn collagen-stimulated human platelets, aggregometry, assessment of ROS and Ca2+, immunoblotting, ELISA, flow cytometry, platelet adhesion assay, and assessment of mouse arterial thrombosis were performed in this study. Key findingsSetanaxib inhibited both intracellular and extracellular ROS production in collagen-activated platelets. Additionally, setanaxib significantly inhibited collagen-induced platelet aggregation, P-selectin exposure from α-granule release, and ATP release from dense granules. Setanaxib blocked the specific tyrosine phosphorylation-mediated activation of Syk, LAT, Vav1, and Btk within collagen receptor signaling pathways, leading to reduced activation of PLCγ2, PKC, and Ca2+ mobilization. Setanaxib also inhibited collagen-induced activation of integrin αIIbβ3, which is linked to increased cGMP levels and VASP phosphorylation. Furthermore, setanaxib suppressed collagen-induced p38 MAPK activation, resulting in decreased phosphorylation of cytosolic PLA2 and reduced TXA2 generation. Setanaxib also inhibited ERK5 activation, affecting the exposure of procoagulant phosphatidylserine. Setanaxib reduced thrombus formation under shear conditions by preventing platelet adhesion to collagen. Finally, in vivo administration of setanaxib in animal models led to the inhibition of arterial thrombosis. SignificanceThis study is the first to show that setanaxib suppresses ROS generation, platelet activation, and collagen-induced thrombus formation, suggesting its potential use in treating thrombotic or cardiovascular diseases.

Volume Regulation and Nonosmotic Volume of Individual Human Platelets Quantified by High-Speed Scanning Ion Conductance Microscopy.

Platelets are anucleate cells that play an important role in wound closure following vessel injury. Maintaining a constant platelet volume is critical for platelet function. For example, water-induced swelling can promote procoagulant activity and initiate thrombosis. However, techniques for measuring changes in platelet volume such as light transmittance or impedance techniques have inherent limitations as they only allow qualitative measurements or do not work on the single-cell level. Here, we introduce high-speed scanning ion conductance microscopy (HS-SICM) as a new platform for studying volume regulation mechanisms of individual platelets. We optimized HS-SICM to quantitatively image the morphology of adherent platelets as a function of time at scanning speeds up to 7 seconds per frame and with 0.1 fL precision. We demonstrate that HS-SICM can quantitatively measure the rapid swelling of individual platelets after a hypotonic shock and the following regulatory volume decrease (RVD). We found that the RVD of thrombin-, ADP-, and collagen-activated platelets was significantly reduced compared with nonactivated platelets. Applying the Boyle-van't Hoff relationship allowed us to extract the nonosmotic volume and volume fraction on a single-platelet level. Activation by thrombin or ADP, but not by collagen, resulted in a decrease of the nonosmotic volume, likely due to a release reaction, leaving the total volume unaffected. This work shows that HS-SICM is a versatile tool for resolving rapid morphological changes and volume dynamics of adherent living platelets.

Mammalian STE20-like kinase-1/2 are activated in human platelets stimulated by collagen or thrombin and play a vital role in collagen-activated platelets

IntroductionMammalian ste20-like kinases-1/2 (MST1/2), the core kinases of the Hippo pathway, play critical roles in the biology of hematopoietic cells via noncanonical mechanisms and contributes to megakaryocyte differentiation, polyploidization, and maturation to produce platelets. However, the role of MST1/2 in platelet functions remains unclear. Materials and methodsIn this study, we investigated this topic by determining platelet aggregation and through flow cytometry, ATP release assay, clot retraction assay, and immunoblotting analysis. ResultsWe found that MST1/2 were rapidly phosphorylated and activated upon platelet stimulation by thrombin and collagen. XMU-MP-1, a specific inhibitor of MST1/2, blocks the activation of MST1/2 in platelets. Inhibitor-pretreated platelets showed impaired platelet aggregation and dense-granule secretion mediated by collagen, thrombin, and U46619, whereas ristocetin or ADP mediated platelet aggregation was unaffected by XMU-MP-1. Although platelet-mediated clot retraction was not affected by MST1/2 inhibitors, integrin αIIbβ3 activation was significantly attenuated in XMU-MP-1-treated platelets. Moreover, MST1/2 inhibition significantly attenuated the mobilization of platelet calcium ions and the secretion of α-granules induced by convulxin. ConclusionsThis study is the first to demonstrate that MST1/2 play vital roles in human platelets and contributes to collagen-induced platelet activation and aggregation.

Fruitflow inhibits platelet function by suppressing Akt/GSK3\u03b2, Syk/PLC\u03b32 and p38 MAPK phosphorylation in collagen-stimulated platelets

BackgroundPlatelets play an important role in the progression of atherosclerosis and cardiovascular events. The inhibition of platelet function is a main strategy to reduce risk of cardiovascular events. Some studies have shown that tomato extracts inhibit platelet function, but the molecular mechanisms remain unclear. Fruitflow is a water-solute tomato extract and the main ingredients including flavonoids, adenosine, chlorogenic acid, phytosterols, naringenin, and carotenoids. The present study investigated the effects of fruitflow on adenosine diphosphate (ADP)- and collagen- stimulated platelet aggregation, platelet adhesion, and levels of thromboxane B2 (TXB2), 6-keto-prostaglandin F1α (PGF1α), and platelet factor 4 (PF4) and explored the underlying molecular mechanisms.MethodsPlatelet-rich plasma (PRP) was used for measurement of platelet aggregation, TXB2, 6-keto- PGF1α, and PF4 levels. Platelet aggregation was analyzed using a Chrono-Log aggregometer. TXB2, 6-keto- PGF1α, and PF4 levels were determined using enzyme-linked immunosorbent assay kits. Immunoblotting was used to detect protein expression and phosphorylation on washed platelets. Platelet adhesion and spreading were determined by immunofluorescence.ResultsFruitflow (1, 3, 10 and 100 μg/ml) dose-dependently inhibited platelet aggregation that was induced by ADP and collagen. Fruitflow (100 μg/ml) treatment completely suppressed ADP- and collagen-stimulated platelet aggregation. Fruitflow (100 μg/ml) significantly decreased TXB2 and 6-keto-PGF1α generation and PF4 release in ADP- and collagen-stimulated platelets. Treatment with fruitflow effectively blocked collagen-induced platelet spreading. To determine the potential molecule mechanism of action of fruitflow, we investigated the protein expression and phosphorylation of several signaling molecules in collagen-activated platelets. Fruitflow dose-dependently suppressed Akt, Glycogen synthase kinase-3β (GSK-3β), spleen tyrosine kinase (Syk) and phospholipase Cγ2 (PLCγ2) and p38 MAPK phosphorylation that was induced by collagen.ConclusionFruitflow inhibited platelet aggregation and reduced TXB2, 6-keto-PGF1α, and PF4 levels in ADP- and collagen-stimulated platelets. The mechanism of action of fruitflow may be associated with the suppression of Akt/GSK3β, Syk/PLCγ2, and p38 MAPK phosphorylation in collagen-activated platelets. Fruitflow is a natural product derived from tomato and can be used as a health food for decreasing platelet activity.

Water-Soluble Tomato Extract Fruitflow Alters the Phosphoproteomic Profile of Collagen-Stimulated Platelets.

Platelet hyperactivity is a risk factor for cardiovascular disease and thrombosis. Recent studies reported that the tomato extract Fruitflow inhibited platelet function, but the molecular mechanism is still unclear. The present study used proteomics to quantitatively analyze the effect of fruitflow on the inhibition of collagen-stimulated platelets and validated the involvement of several signaling molecules. Fruitflow significantly inhibited human platelet aggregation and P-selectin expression that were induced by collagen. Proteomics analysis revealed that compared fruitflow-treated collagen-stimulated platelets with only collagen-stimulated platelets, 60 proteins were upregulated and 10 proteins were downregulated. Additionally, 66 phosphorylated peptides were upregulated, whereas 37 phosphorylated peptides were downregulated. Gene Ontology analysis indicated that fruitflow treatment downregulated phosphoinositide 3-kinase (PI3K)/protein kinase B and guanosine triphosphatase-mediated signal transduction in collagen-activated platelets. Biological validation indicated that fruitflow decreased Akt, glycogen synthase kinase 3β, p38 mitogen-activated protein kinase (MAPK), and heat shock protein (Hsp27) phosphorylation in collagen-stimulated platelets. Fruitflow recovered cyclic adenosine monophosphate levels in collagen-activated platelets and reduced protein kinase A substrate phosphorylation that was induced by collagen. These findings suggest that fruitflow is a functional food that can inhibit platelet function, conferring beneficial effects for people who are at risk for platelet hyperactivity-associated thrombosis.

Platelet-Derived Microvesicles Promote VSMC Dedifferentiation After Intimal Injury via Src/Lamtor1/mTORC1 Signaling.

Phenotypic switch of vascular smooth muscle cells (VSMCs) is important in vascular remodeling which causes hyperplasia and restenosis after intimal injury. Platelets are activated at injured intima and secrete platelet-derived microvesicles (PMVs). Herein, we demonstrated the role of PMVs in VSMC phenotypic switch and the potential underlying mechanisms. In vivo, platelets were locally adhered and activated at intimal injury site, while Lamtor1 was promoted and VSMCs were dedifferentiated. PMVs, collected from collagen-activated platelets in vitro which mimicked collagen exposure during intimal injury, promoted VSMC dedifferentiation, induced Lamtor1 expression, and activated mTORC1 signaling, reflected by the phosphorylation of two downstream targets, i.e., S6K and 4E-BP1. Knockdown of Lamtor1 with small interfering RNA attenuated these processes induced by PMVs. Based on the previously published proteomic data, Ingenuity Pathway Analysis revealed that Src may participate in regulating effects of PMVs. Src inhibitor significantly reversed the effects of PMVs on VSMC dedifferentiation, Lamtor1 expression and mTORC1 activation. Furthermore, in SMC-specific Lamtor1 knockout mice, intimal hyperplasia was markedly attenuated after intimal injury compared with the wild type. Our data suggested that PMVs secreted by activated platelets promoted VSMC dedifferentiation via Src/Lamtor1/mTORC1 signaling pathway. Lamtor1 may be a potential therapeutic target for intimal hyperplasia after injury.

Pterostilbene, a Dimethylether Analogue of Resveratrol, Possesses High Potency in the Prevention of Platelet Activation in Humans and the Reduction of Vascular Thrombosis in Mice.

Platelets play a crucial role in cardiovascular disorders (CVDs); thus, development of a therapeutic target that prevents platelet activation reduces CVDs. Pterostilbene (PTE) has several remarkable pharmacological activities, including anticancer and neuroprotection. Herein, we examined the inhibitory mechanisms of PTE in human platelets and its role in the prevention of vascular thrombosis in mice. At very low concentrations (1-5 μmol/L), PTE strongly inhibited collagen-induced platelet aggregation, but it did not have significant effects against thrombin and 9,11-dideoxy-11α,9α-epoxymethanoprostaglandin (U46619). PTE markedly reduced P-selectin expression on isolated α-granules by a novel microchip. Moreover, PTE inhibited adenosine triphosphate (ATP) release, intracellular ([Ca2+]i) mobilization (resting, 216.6 ± 14.0 nmol/L; collagen-activated platelets, 396.5 ± 25.7 nmol/L; 2.5 μmol/L PTE, 259.4 ± 8.8 nmol/L; 5 μmol/L PTE, 231.8 ± 9.7 nmol/L), phospholipase C (PLC)γ2/protein kinase C (PKC), Akt, and mitogen-activated protein kinase (MAPK) phosphorylation. Neither 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536) nor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reversed platelet aggregation inhibited by PTE. PTE did not affect vasodilator-stimulated phosphoprotein phosphorylation. In mice, PTE obviously reduced the mortality (from 100 to 37.5%) associated with acute pulmonary thromboembolism without increasing the bleeding time. Thus, PTE could be used to prevent CVDs.

Platelets Promote Thromboinflammation in SARS-CoV-2 Pneumonia.

Pulmonary thrombosis is observed in severe acute respiratory syndrome coronavirus 2 pneumonia. Aim was to investigate whether subpopulations of platelets were programmed to procoagulant and inflammatory activities in coronavirus disease 2019 (COVID-19) patients with pneumonia, without comorbidities predisposing to thromboembolism. Approach and Results: Overall, 37 patients and 28 healthy subjects were studied. Platelet-leukocyte aggregates, platelet-derived microvesicles, the expression of P-selectin, and active fibrinogen receptor on platelets were quantified by flow cytometry. The profile of 45 cytokines, chemokines, and growth factors released by platelets was defined by immunoassay. The contribution of platelets to coagulation factor activity was selectively measured. Numerous platelet-monocyte (mean±SE, 67.9±4.9%, n=17 versus 19.4±3.0%, n=22; P<0.0001) and platelet-granulocyte conjugates (34.2±4.04% versus 8.6±0.7%; P<0.0001) were detected in patients. Resting patient platelets had similar levels of P-selectin (10.9±2.6%, n=12) to collagen-activated control platelets (8.7±1.5%), which was not further increased by collagen activation on patient platelets (12.4±2.5%, P=nonsignificant). The agonist-stimulated expression of the active fibrinogen receptor was reduced by 60% in patients (P<0.0001 versus controls). Cytokines (IL [interleukin]-1α, IL-1β, IL-1RA, IL-4, IL-10, IL-13, IL, 17, IL-27, IFN [interferon]-α, and IFN-γ), chemokines (MCP-1/CCL2 [monocyte chemoattractant protein 1]), and growth factors (VEGF [vascular endothelial growth factor]-A/D) were released in significantly larger amounts upon stimulation of COVID-19 platelets. Platelets contributed to increased fibrinogen, VWF (von Willebrand factor), and factor XII in COVID-19 patients. Patients (28.5±0.7 s, n=32), unlike controls (31.6±0.5 s, n=28; P<0.001), showed accelerated factor XII-dependent coagulation. Platelets in COVID-19 pneumonia are primed to spread proinflammatory and procoagulant activities in systemic circulation.

Regulation of Human Platelet Activation and Prevention of Arterial Thrombosis in Mice by Auraptene through Inhibition of NF-κB Pathway.

Platelets are major players in the occurrence of cardiovascular diseases. Auraptene is the most abundant coumarin derivative from plants, and it has been demonstrated to possess a potent capacity to inhibit platelet activation. Although platelets are anucleated cells, they also express the transcription factor, nuclear factor-κB (NF-κB), that may exert non-genomic functions in platelet activation. In the current study, we further investigated the inhibitory roles of auraptene in NF-κB-mediated signal events in platelets. MG-132 (an inhibitor of proteasome) and BAY11-7082 (an inhibitor of IκB kinase; IKK), obviously inhibited platelet aggregation; however, BAY11-7082 exhibited more potent activity than MG-132 in this reaction. The existence of NF-κB (p65) in platelets was observed by confocal microscopy, and auraptene attenuated NF-κB activation such as IκBα and p65 phosphorylation and reversed IκBα degradation in collagen-activated platelets. To investigate cellular signaling events between PLCγ2-PKC and NF-κB, we found that BAY11-7082 abolished PLCγ2-PKC activation; nevertheless, neither U73122 nor Ro31-8220 had effect on NF-κB activation. Furthermore, both auraptene and BAY11-7082 significantly diminished HO• formation in activated platelets. For in vivo study, auraptene prolonged the occlusion time of platelet plug in mice. In conclusion, we propose a novel inhibitory pathway of NF-κB-mediated PLCγ2-PKC activation by auraptene in human platelets, and further supported that auraptene possesses potent activity for thromboembolic diseases.

Modulation of human platelet activation and in vivo vascular thrombosis by columbianadin: regulation by integrin \u03b1IIb\u03b23 inside-out but not outside-in signals

BackgroundColumbianadin (CBN) is one of the main coumarin constituents isolated from Angelica pubescens. The pharmacological value of CBN is well demonstrated, especially in the prevention of several cancers and analgesic activity. A striking therapeutic target for arterial thrombosis is inhibition of platelet activation because platelet activation significantly contributes to these diseases. The current study examined the influence of CBN on human platelet activation in vitro and vascular thrombotic formation in vivo.MethodsAggregometry, immunoblotting, immunoprecipitation, confocal microscopic analysis, fibrin clot retraction, and thrombogenic animals were used in this study.ResultsCBN markedly inhibited platelet aggregation in washed human platelets stimulated only by collagen, but was not effective in platelets stimulated by other agonists such as thrombin, arachidonic acid, and U46619. CBN evidently inhibited ATP release, intracellular ([Ca2+]i) mobilization, and P-selectin expression. It also inhibited the phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), Akt (protein kinase B), and mitogen-activated protein kinases (MAPKs; extracellular signal-regulated kinase [ERK] 1/2 and c-Jun N-terminal kinase [JNK] 1/2, but not p38 MAPK) in collagen-activated platelets. Neither SQ22536, an adenylate cyclase inhibitor, nor ODQ, a guanylate cyclase inhibitor, reversed the CBN-mediated inhibition of platelet aggregation. CBN had no significant effect in triggering vasodilator-stimulated phosphoprotein phosphorylation. Moreover, it markedly hindered integrin αIIbβ3 activation by interfering with the binding of PAC-1; nevertheless, it had no influences on integrin αIIbβ3-mediated outside-in signaling such as adhesion number and spreading area of platelets on immobilized fibrinogen as well as thrombin-stimulated fibrin clot retraction. Additionally, CBN did not attenuate FITC-triflavin binding or phosphorylation of proteins, such as integrin β3, Src, and focal adhesion kinase, in platelets spreading on immobilized fibrinogen. In experimental mice, CBN increased the occlusion time of thrombotic platelet plug formation.ConclusionThis study demonstrated that CBN exhibits an exceptional activity against platelet activation through inhibition of the PLCγ2-PKC cascade, subsequently suppressing the activation of Akt and ERKs/JNKs and influencing platelet aggregation. Consequently, this work provides solid evidence and considers that CBN has the potential to serve as a therapeutic agent for the treatment of thromboembolic disorders.

Cigarette Smoking Cessation Temporarily Enhances the Release of Phosphorylated-HSP27 from Human Platelets

Objective Cigarette smoking is a risk factor for arteriopathy, including acute coronary syndrome, stroke and peripheral vascular disease. Thus, cessation is strongly recommended in order to reduce these risks. We recently demonstrated that smoking cessation causes temporary hyper-aggregability of human platelets. We previously showed that heat shock protein 27 (HSP27) is released from human platelets stimulated by collagen, accompanied by its phosphorylation. Accumulating evidence indicates potent roles of extracellular HSP27 as a modulator of inflammation. In the present study, using the stored samples obtained in the previous study, we investigated the effect of cigarette smoking cessation on the release of phosphorylated-HSP27 from collagen-activated human platelets (n=15 patients). Methods We enrolled patients who visited smoking cessation outpatient services between January 2012 and November 2014. Platelet-rich plasma, chronologically obtained before and after the cessation, was stimulated by collagen using a PA-200 aggregometer in the previous study. The levels of phosphorylated-HSP27 released from platelets were determined by an enzyme-linked immunosorbent assay. The phosphorylation of HSP27 in platelets was evaluated by a Western blot analysis. Results Cessation of cigarette smoking significantly upregulated the levels of collagen-stimulated release of phosphorylated-HSP27 at four and eight weeks after quitting smoking compared to before cessation. However, there was no significant difference between the levels before cessation and those at 12 weeks after cessation. The levels of phosphorylated-HSP27 stimulated by collagen in the platelets at four weeks after smoking cessation were remarkably enhanced compared to before cessation. Conclusion Cigarette smoking cessation temporarily enhances the collagen-stimulated release of phosphorylated-HSP27 from human platelets in the short term.

Platelet-derived microvesicles promote endothelial progenitor cell proliferation in intimal injury by delivering TGF-β1.

Intimal injury is an early stage of several cardiovascular diseases. Endothelial progenitor cells (EPCs) play a significant role in endothelial repair following vascular injury. Once the intima is damaged, EPCs are mobilized from the bone marrow to the injury site. Meanwhile, the injury to the intimal surface triggers platelet degranulation, aggregation, and adhesion to the damaged endothelium, and exposed collagen stimulates platelet to secrete platelet-derived microvesicles (PMVs). However, the role of PMVs in EPC function during this process remains unknown. In an invivo study, EPCs and platelets were found to adhere to the injury site in Sprague-Dawley (SD) rat vascular injury model. Invitro, collagen stimulation induced the release of PMVs, and collagen-activated PMVs (ac.PMVs) significantly promoted EPC proliferation. Transforming growth factor-β1 (TGF-β1) content was increased in ac.PMVs. Activated PMVs significantly upregulated Smad3 phosphorylation in EPCs and increased Smad3 nuclear translocation from the cytoplasm. TGF-β1 knockdown ac.PMVs downregulated EPC proliferation. Recombinant TGF-β1 enhanced EPC proliferation. The TGF-β1 inhibitor SB431542 significantly repressed the intracellular signal triggered by ac.PMVs. Furthermore, the Smad3-specific phosphorylation inhibitor SIS3 effectively reversed the cell proliferation induced by ac.PMVs. Smad3 translocated to the nucleus and enhanced EPC proliferation via its downstream genes tenascin C (TNC), CDKN1A, and CDKN2A. r-TGF-β1 promoted reendothelialization and EPC proliferation invivo. Our data demonstrate that activated PMVs deliver TGF-β1 from collagen-activated platelets to EPCs, which in turn activates Smad3 phosphorylation and regulates TNC, CDKN1A, and CDKN2A expression to promote EPC proliferation, suggesting that PMVs act as a key transporter and a potential therapeutic target for vascular injury.

Synthetic Platelets for Treatment of Traumatic Hemorrhage and Thrombocytopenia

Platelets are primarily responsible for staunching bleeding by forming a 'platelet plug' and further amplifying thrombin generation on its surface to facilitate fibrin formation, leading to hemostatic clot formation at the site of vascular breach. Therefore, platelet transfusions are clinically used to mitigate bleeding risks in thrombocytopenia (prophylactic transfusion) and to mitigate hemorrhage in traumatic injuries (emergency transfusion). Currently these transfusions utilize donor-derived platelets, stored at 20-24oC with gentle agitation. In this condition, platelets have high risk of bacterial contamination and very short shelf-life (~ 5 days), which severely limit their logistical availability and use. Several parallel strategies are currently undergoing research to address these issues, including platelet storage at reduced temperatures (chilled or freeze-dried), pathogen reduction technologies and bioreactor-based in vitro platelet production from precursor cells. An alternative (and complimentary) approach that is the focus of our research is the engineering of I.V.-administrable synthetic hemostat nanoparticles that functionally mimic platelet's clotting mechanisms. These 'synthetic platelet' nanoparticle systems can be manufactured at large scale, sterilized without compromising functions and stored for long periods of time (6-9 months), thereby allowing significant logistical advantages in transfusion applications. Here we present in vitro and in vivo evaluation of such technology. For these studies, the 'synthetic platelet' nanoparticles were manufactured by decorating liposomes with a combination of VWF-binding, collagen-binding and fibrinogen-mimetic peptides, for integrative mimicry of platelet's hemostasis-relevant adhesive and aggregatory mechanisms. The nanoparticles were stored at room temperature in aqueous suspension as well as lyophilized powder, and particle stability was assessed over 6-9 months by dynamic light scattering (DLS). The nanoparticles were also exposed to E-beam sterilization, and particle stability as well platelet-mimetic bioactivity was assessed by DLS, aggregometry, microfluidics and rotational thromboelastometry (ROTEM). The systemic safety and targeted hemostatic efficacy of I.V.-administered nanoparticles were evaluated in mouse model of thrombocytopenia, and in mouse, rat and pig models of traumatic hemorrhage. DLS and electron microscopy confirmed that the synthetic platelet nanoparticles have a size of 150-200 nm diameter, and they remain stable over 6-9 months in storage. Microfluidic studies showed that these nanoparticles could rapidly adhere to 'vWF + collagen'-coated surfaces and enhance the recruitment and aggregation of active platelets on these surfaces. Aggregometry studies showed that the nanoparticles did not affect resting platelets but enhanced aggregation of ADP- or collagen-activated platelets (i.e. no thrombotic risk towards resting platelets). Flow cytometry studies confirmed this specificity of nanoparticle binding to active platelets. ROTEM studies showed that the 'synthetic platelet' nanoparticles significantly improved clot kinetics and firmness. In vivo, in all animal models, the nanoparticles showed no systemic pro-thrombotic effects, as assessed by hemodynamics as well as organ histology. In thrombocytopenic mice, prophylactically administered 'synthetic platelet' nanoparticles dose-dependently reduced tail bleeding time. In mouse, rat and pig trauma models, post-injury administration of 'synthetic platelet' nanoparticles reduced blood loss, stabilized blood pressure, delayed hypotension and thereby significantly improved survival. The nanoparticles could be further utilized as a platform for targeted presentation of phosphatidylserine (PS) to augment thrombin generation, or targeted delivery of tranexamic acid (TXA) for anti-fibrinolytic effect or delivery of inorganic polyphosphate (PolyP) to augment clot stability. These studies not only establish the potential of these nanoparticles as a platelet surrogate for transfusion applications, but also demonstrate their utilization as a platform for modular augmentation of various hemostatic outputs in prophylactic and emergency applications. Figure Disclosures Sen Gupta: Haima Therapeutics LLC: Equity Ownership.

Esculetin, a Coumarin Derivative, Prevents Thrombosis: Inhibitory Signaling on PLCγ2-PKC-AKT Activation in Human Platelets.

Esculetin, a bioactive 6,7-dihydroxy derivative of coumarin, possesses pharmacological activities against obesity, diabetes, renal failure, and cardiovascular disorders (CVDs). Platelet activation plays a major role in CVDs. Thus, disrupting platelet activation represents an attractive therapeutic target. We examined the effect of esculetin in human platelet activation and experimental mouse models. At 10–80 μM, esculetin inhibited collagen- and arachidonic acid-induced platelet aggregation in washed human platelets. However, it had no effects on other agonists such as thrombin and U46619. Esculetin inhibited adenosine triphosphate release, P-selectin expression, hydroxyl radical (OH·) formation, Akt activation, and phospholipase C (PLC)γ2/protein kinase C (PKC) phosphorylation, but did not diminish mitogen-activated protein kinase phosphorylation in collagen-activated human platelets. Platelet function analysis indicated that esculetin substantially prolonged the closure time of whole blood. In experimental mice, esculetin significantly increased the occlusion time in thrombotic platelet plug formation and reduced mortality associated with acute pulmonary thromboembolism. However, it did not prolong the bleeding time. This study demonstrates that esculetin inhibits human platelet activation via hindering the PLCγ2–PKC cascade, hydroxyl radical formation, Akt activation, and ultimately suppressing platelet activation. Therefore, esculetin may act as an essential therapeutic agent for preventing thromboembolic diseases.

BFPI: A Minimal Prothrombinase Inhibitor from the Saliva of Simulium Vittatum

Background: The black fly, Simulium Vittatum, has an anticoagulant protein in its saliva that allows it to feed on mammalian blood (black fly protease inhibitor; BFPI). Remarkably, BFPI is similar to the human anticoagulant tissue factor pathway inhibitor alpha (TFPIα). TFPIα contains three Kunitz-type protease inhibitor domains (K1, K2, K3), which inhibit factor VIIa (FVIIa) and factor Xa (FXa) and bind the co-factor protein S (PS), respectively; BFPI contains a single Kunitz domain that inhibits FXa. In addition, TFPIα and BFPI contain homologous basic regions (BRs) near their C-termini (252LIKTKRKRKK261 in human TFPIα, LIKTRKRKPKK in BFPI). The TFPIα BR binds a regulatory acidic region (AR) in factor Va (FVa). The AR is present in forms of FVa released by collagen-activated platelets and generated through limited proteolysis by FXa (FVaXa), and is removed by thrombin (FVaIIa). We hypothesized that BFPI, through its Kunitz domain and basic C-terminus, inhibits early forms of the prothrombinase complex, but does not possess the other inhibitory functions of TFPIα: (1) K1-dependent inhibition of the tissue factor (TF)-FVIIa complex; and (2) PS/K3-dependent FXa inhibition.Results: Recombinant BFPI inhibited FXa in an amidolytic activity assay, and PS did not promote this inhibition. BFPI did not inhibit TF-FVIIa-mediated FX activation. As described with TFPIα, FV promoted FXa inhibition by BFPI but FVaIIa did not, suggesting that the BFPI BR is capable of binding the FVa AR. In a purified protein assay, BFPI inhibited prothrombinase assembled with FVaXa (IC50=4.9nM), but not FVaIIa. Similarly, 5nM BFPI increased the lag time for FXa-initiated plasma thrombin generation by 10.4±1.5%.We next used BFPI as a backbone to evaluate a reported human mutation in the TFPIα BR, K254E. Every mammalian, avian, or reptilian TFPIα sequence available contains either a Lys or Arg residue at this position, suggesting that this residue is functionally important. In purified protein assays, BFPI-K254E inhibited FXa amidolytic activity identically to BFPI, but FV did not promote this inhibition, suggesting that BFPI-K254E has a specific defect in its interaction with FV. Consistent with this, BFPI-K254E was a weaker inhibitor of prothrombinase assembled with FVaXa (IC50 = 15.8nM) and FXa-initiated plasma thrombin generation.The results obtained with BFPI-K254E were confirmed using peptides and full-length TFPIα proteins. First, a peptide mimicking the wild type TFPIα BR (LIKTKRKRKK) inhibited prothrombinase assembled with FVaXa (IC50 = 1.0 µM), while the substituted peptide (LIETKRKRKK) was substantially weaker (20% inhibition observed with 340 µM peptide). Second, full-length TFPIα-K254E was a weaker inhibitor of prothrombinase containing FXa-activated FVa (IC50 = 14.8 nM, vs. 1.8 nM for TFPIα) and had greatly reduced anticoagulant activity in plasma-based thrombin generation assays.Conclusions: In summary, the anticoagulant effect of BFPI is mediated through inhibition of early forms of prothrombinase, independent of TF-FVIIa inhibition or PS-dependent FXa inhibition. The natural mutation TFPIα K254E disrupts prothrombinase inhibition, despite the presence of six other conserved basic residues, and is thus procoagulant in human plasma. The absolute conservation of the TFPIα BR, and its usurpation to allow feeding by black flies, point to formation of the initial prothrombinase complex as a key regulatory step in blood coagulation. DisclosuresMast:Novo Nordisk: Research Funding.

Activated platelets promote an osteogenic programme and the progression of calcific aortic valve stenosis.

Calcific aortic valve stenosis (CAVS) is characterized by a fibrocalcific process. Studies have shown an association between CAVS and the activation of platelets. It is believed that shear stress associated with CAVS promotes the activation of platelets. However, whether platelets actively participate to the mineralization of the aortic valve (AV) and the progression of CAVS is presently unknown. To identify the role of platelets into the pathobiology of CAVS. Explanted control non-mineralized and mineralized AVs were examined by scanning electron microscope (SEM) for the presence of activated platelets. In-depth functional assays were carried out with isolated human valve interstitial cells (VICs) and platelets as well as in LDLR-/- apoB100/100 IGFII (IGFII) mice. Scanning electron microscope and immunogold markings for glycoprotein IIb/IIIa (GPIIb/IIIa) revealed the presence of platelet aggregates with fibrin in endothelium-denuded areas of CAVS. In isolated VICs, collagen-activated platelets induced an osteogenic programme. Platelet-derived adenosine diphosphate induced the release of autotaxin (ATX) by VICs. The binding of ATX to GPIIb/IIIa of platelets generated lysophosphatidic acid (LysoPA) with pro-osteogenic properties. In IGFII mice with CAVS, platelet aggregates were found at the surface of AVs. Administration of activated platelets to IGFII mice accelerated the development of CAVS by 2.1-fold, whereas a treatment with Ki16425, an antagonist of LysoPA receptors, prevented platelet-induced mineralization of the AV and the progression of CAVS. These findings suggest a novel role for platelets in the progression of CAVS.

Caffeic Acid Diminishes the Production and Release of Thrombogenic Molecules in Human Platelets

Caffeic acid (CA) is a well-known to inhibit thrombogenic thromboxane A2 (TXA2) production, but there are no reports for the supply and usage of its precursor arachidonic acid (AA) and the release of serotonin, another thrombogenic molecule. In this study, we investigated the effect of CA on the phosphorylation of AA supply enzymes [cytosolic phospholipase A2α (cPLA2α), and phospholipase Cγ2 (PLCγ2)] and AA utilization enzymes [cyclooxygenase (COX)-1, TXA2 synthase (TXAS)] in collagen-activated human platelets. p38 MAPK phosphorylation associated with TXA2 production and c-Jun NH2-terminal kinase 1 (JNK1) phosphorylation associated with serotonin release were also determined. CA inhibited collagen-induced TXA2 production in a dose-dependent manner. It acted by counteracting the phosphorylation of AA release enzyme (PLCγ2) and AA utilization enzymes (COX-1 and TXAS) without affecting p38 MAPK and cPLA2α in collagenactivated human platelets. CA decreased collagen-released serotonin by inhibiting JNK1 phosphorylation. Therefore, our data demonstrate that CA might protect TXA2- and serotonin-mediated thrombogenesis.

Novel Therapeutic Agent against Platelet Activation In Vitro and Arterial Thrombosis In Vivo by Morin Hydrate.

Morin hydrate, a bioactive flavonoid, has been proven to prevent inflammation and apoptosis of cells. Flavonoids can reduce the risk of cardiovascular diseases, in which platelet activation plays a major role. This study investigated the effect of morin hydrate on platelet activation in vitro and in vivo. Morin hydrate markedly inhibited platelet aggregation stimulated by collagen in human platelets but not that stimulated by other agonists. In collagen-activated platelets, morin hydrate inhibited adenosine triphosphate (ATP) release; intracellular Ca2+ mobilization; P-selectin expression; and phosphorylation of phospholipase Cγ2 (PLCγ2), protein kinase C (PKC), and Akt. In mitogen-activated protein kinase (MAPK) activation, morin hydrate evidently diminished ERK2 or JNK1 activation, except for p38 MAPK. Additionally, morin hydrate markedly reduced the OH· signals in platelet suspensions but not in the cell-free system (Fenton reaction solution). Moreover, morin hydrate substantially increased the occlusion time of thrombotic platelet plug formation but had no effect on bleeding time in mice. In conclusion, morin hydrate crucially inhibits platelet activation through inhibition of the PLCγ2–PKC cascade and subsequent suppression of Akt and MAPK activation, thereby ultimately inhibiting platelet aggregation. Therefore, this paper suggests that morin hydrate constitutes a novel and potential natural therapeutic product for preventing or treating thromboembolic disorders.

Hemotoxic and wound healing potential of coelomic fluid of sea-star Astropecten indicus

BackgroundThe coelomic fluid of echinoderms bathes all the internal organs and is a natural reservoir of various bio-active compounds. However, coelomic fluid from Indian sea-star Astropecten indicus is not well characterized for its biochemical and pharmacological profiles. Sea-star (or starfish) Astropecten indicus was collected from the coast of Goa, India during low tides. Sea-star coelomic fluid (SCF) extracted from the sea-stars was concentrated, filtered, and assayed for various hemotoxic activities such as fibrinogenolysis, fibrinolysis, hemolysis, and effects on platelet aggregation.ResultsStudies on effects of SCF on A-549 human lung cancer cell line showed its non-cytotoxic and wound healing abilities in vitro. SCF was found to possess fibrinogen and fibrin digesting ability at a dose of 4 μg/ml within 30 min of incubation with the substrate. Additionally, SCF could hemolyze goat RBC by 50% and human RBC by only 5%. SDS-PAGE analysis showed that SCF contains various proteins and peptides. Two protein components of SCF yielded strong fibrinolytic, fibrinogenolytic, caseinolytic, and gelatinase activities as revealed by zymography. SCF enhanced the aggregation of ADP and collagen-activated platelets in a dose-dependent manner and had a specific proteolytic activity of 2.5 μg/ml in 30 min at 37 °C as assayed using azocasein as substrate.ConclusionThis study focuses on the anticoagulant, pro-platelet aggregation and cell proliferative potential of SCF in vitro and is the first report on hemotoxic and wound healing potential of coelomic fluid of Astropecten indicus.

Novel iridium (III)‑derived organometallic compound for the inhibition of human platelet activation.

Since cisplatin achieved clinical success, transition metal platinum (Pt) drugs have been effectively used for the treatment of cancer. Iridium (Ir) compounds are considered to be potential alternatives to Pt compounds, as they possess promising anticancer effects with minor side effects. Platelet activation is associated with the metastasis and progression of cancer, and also with arterial thrombosis. Therefore, it is necessary to develop novel, effective antithrombotic agents. An Ir (III)-derived complex, [Ir (Cp*) 1-(2-pyridyl)-3-(3-me-thoxyphenyl)imidazo[1,5-a]pyridine Cl]BF4 (Ir-3), was developed as a novel antiplatelet drug. Ir-3 exerted more potent inhibitory activity on platelet aggregation stimulated by collagen compared with other agonists, including thrombin. In collagen-activated platelets, Ir-3 also inhibited adenosine trisphosphate release, intracellular Ca+2 mobilization and surface P-selectin expression, as well as the phosphorylation of phospholipase Cγ2 (PLCγ2), protein kinase C (PKC), protein kinase B (Akt) and c-Jun N-terminal kinase (JNK) 1, but not p38 mitogen-activated protein kinase or extracellular signal-regulated kinases. Ir-3 did not markedly affect phorbol 12, 13-dibutyrate-stimulated platelet aggregation. Neither the adenylate cyclase inhibitor SQ22536 nor the guanylate cyclase inhibitor 1H-[1, 2, 4] oxadiazolo [4,3-a]quinoxalin-1-one significantly reversed the Ir-3-mediated inhibition of platelet aggregation. Furthermore, Ir-3 had no considerable diminishing effects on OH radical signals in collagen-stimulated platelets or Fenton reaction solution. In conclusion, Ir-3 serves a novel function in the inhibition of platelet aggregation through inhibiting the PLCγ2-PKC cascade, and the subsequent suppression of Akt and JNK1 activation. Therefore, Ir-3 may be a potential novel therapeutic agent for the treatment of thromboembolic disorders, or the interplay between platelets and tumor cells which contributes to tumor cell proliferation and progression.