Platelet Thrombus Formation Research Articles

Ischemia-Reperfusion Injury with Pringle's Maneuver Induces Increasing of Unusually Large Von Willebrand Factor Multimers after Hepatectomy

Background/Aims: Von Willebrand factor (VWF) is exclusively synthesized in vascular endothelial cells and released into the plasma as unusually large VWF multimers (UL-VWFM). ADAMTS13 is a metalloproteinase produced primarily in hepatic stellate cells and specifically cleaves the VWF. Since UL-VWFM have a strong activity of platelet aggregation, increase of UL-VWFM leads to platelet clumping and/or thrombus formation, resulting in microcirculatory disturbance. On the other hand, ischemia-reperfusion injury of the liver is an imperative event in liver surgery due to compression of the liver and/or Pringle's maneuver. Many previous studies have indicated that vascular endothelial cell is a major target of ischemia-reperfusion injury of the liver. Thus, we hypothesized UL-VWFM increases markedly by stimulating vascular endothelial cells with ischemia-reperfusion injury. However, little information is available regarding the alterations in VWF, ADAMTS13 and especially in UL-VWFM after hepatectomy. The aim of this study is to clarify the effect of hepatectomy on UL-VWFM.Methods: A total 35 patients who underwent a hepatectomy were eligible for the study. Blood samples were obtained for analysis at the following time points: preoperation, intraoperative period, and on the first, second, third, fifth, seventh postoperative days. Plasma levels of ADAMTS13 activity and VWF antigen were measured by the ELISA method. Multimer analysis of plasma VWF was performed according to the method of Ruggeri and Zimmerman. High molecular weight bands that were not detected in normal plasma were defined as UL-VWFM. UL-VWFM was quantified from images of VWF multimer analysis by NIH image. We calculated the UL-VWFM index, proposed in this study, which was defined as the ratio of the UL-VWFM area of a patient to that of normal plasma. All UL-VWFM indexes of 40 healthy volunteers were less than 1.00%. Therefore, UL-VWFM positive was defined as a UL-VWFM index of more than 1.00%. The patients were categorized and analyzed with respect to the positive or negative of UL-VWFM after hepatectomy. The values were expressed as median (range).Results: Plasma levels of ADAMTS13 activity were significantly decreased from 61.0% (27.7-126.2%) in preoperation to 37.4% (20.2-71.4%) in postoperative day 7 after hepatectomy (p <0.001). Plasma levels of VWF antigen were significantly increased from 172.1% (80.5-412.8%) in preoperation to 361.0% (154.7-745.8%) in postoperative day 2 after hepatectomy and remained continuously high until postoperative day 7 (p <0.001). The intraoperative plasma levels of ADAMTS13 activity, VWF antigen were not significantly altered. The UL-VWFM index were significantly increased from 0.2% (0.0-7.8%) in preoperation to 4.2% (0.1-16.3%) in postoperative day 7 after hepatectomy (p <0.001). The patients were divided into 2 groups with respect to the positive or negative of UL-VWFM after operation. Seven patients remained UL-VWFM negative after operation. UL-VWFM index were changed from negative in preoperation to positive after operation in 22 patients. Preoperative characteristics including ADAMTS13 activity, VWF antigen levels and liver functional reserve were similar in the two groups of UL-VWFM positive and negative. In surgery-related factors, the time of duration of Pringle's maneuver in UL-VWFM positive group were longer than those in UL-VWFM negative group (p=0.001). In addition, the volume of blood loss in UL-VWFM positive group was greater than those in UL-VWFM negative group (p=0.003). Multivariable analysis revealed that the time of duration of Pringle's maneuver [OR, 1.049 95%CI, 1.001-1.098; p=0.043] was significantly associated with increasing UL-VWFM after hepatectomy. Moreover, UL-VWFM index was significantly correlated with time of duration of Pringle's maneuver (r=0.444, p=0.017).Conclusion: In this study, we demonstrated that plasma UL-VWFM increases after hepatectomy due to ischemia-reperfusion injury with Pringle's maneuver. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Protein Disulfide Isomerase Negatively Regulates Platelet Function through Modulation of the Thromboxane A2 Receptor

Protein disulfide isomerase (PDI) is an intracellular oxidoreductase that cleaves and forms disulfide bonds in substrate proteins to facilitate their proper folding. However, platelets and endothelium release PDI upon vascular injury. This extracellular PDI serves a critical function in platelet activation and thrombus formation. A PDI antagonist is currently in phase II/III clinical trials for thrombosis prevention. Yet the mechanisms of how PDI enables platelets to respond to the local redox environment remain poorly understood. In particular, the substrates on which PDI acts to modify platelet function have yet to be elucidated.To identify platelet receptors that are controlled by PDI, we evaluated the ability of PDI to modify the effect of different platelet agonists on a platelet aggregation. Recombinant PDI (rPDI) had no effect on platelet aggregation induced by a dose curve of a PAR1 activating peptide, a PAR4 activating peptide, thrombin, collagen, or epinephrine. Unexpectedly, rPDI markedly inhibited aggregation induced by the stable thromboxane A2 analog U46619 (13.8±5.6% of control). In contrast, ERp57, a related thiol isomerase, had little effect and a mutant PDI that lacks the active site cysteines responsible for disulfide bond rearrangement failed to inhibit U46619-induced platelet aggregation, implicating disulfide bond rearrangement. rPDI also blocked U46619-induced dense granule secretion (15.0±5.5% of control), α-granule secretion (24.4±0.1% of control) and α2β3 activation (65.0±0.1% of control). Conversely, inhibition of endogenous PDI augmented U46619-mediated platelet activation. To confirm that PDI was acting upstream in the U46619 activation pathway, we evaluated proximal signaling events. rPDI significantly decreased U46619-induced p38 MAPK phosphorylation in both platelets and HEK cells overexpressing the thromboxane prostanoid (TP) receptor, indicating inhibition of Gα12/13 pathways. In contrast, there was little inhibition of Ca2+ flux, indicating lack of inhibition of Gαq-mediated signaling. These results suggest the presence of a functional disulfide bond within the TP receptor that is sensitive to PDI and required for Gα12/13 signaling.Evaluation of the amino sequence of the TP receptor demonstrates two cysteines, Cys-105 in the extracellular loop I and Cys-183 in the extracellular loop II, that form a disulfide bond and may be modified by redox environment. To determine if the platelet TP receptor is sensitive to modification of redox environment, we evaluated U46619-induced signaling in the presence of GSH and GSSG. Incubation with >0.25 mM GSH reduced U46619-induced platelet aggregation to 11.0±0.6% of control, whereas GSSG did not alter platelet aggregation in response to U46619. These results indicate a functional disulfide bond in the TP receptor that responds to changes in redox environment.Evaluation of additional agonists demonstrated that ADP-induced aggregation was sensitive to rPDI. rPDI did not affect the initial response to 3.5 μM ADP, but completely abrogated the secondary wave of aggregation. This defect in the secondary wave could be overcome by using 5 μM ADP, thus suggesting impaired endogenous ADP release. Indeed, rPDI blocked dense granule release even in response to 5 μM ADP. This result is consistent with the known reliance of dense granule release on TP stimulation and suggests that the negative regulation of PDI on ADP-induced platelet activation is secondary to the inhibition of PDI on the TP receptor.These are the first studies to our knowledge that demonstrate the ability of a thiol isomerase to modulate the function of a G-protein coupled receptor via disulfide bond rearrangement. This mechanism could provide a means to modify signaling through the TP receptor in respond to changes in redox environment. DisclosuresNo relevant conflicts of interest to declare.

CD36 Promotes Platelet Phosphatidylserine Exposure through MAP Kinase ERK5

Dyslipidemic conditions can promote atherosclerotic plaque instability and rupture, leading to thrombosis that can cause life threatening complications. Thrombosis in this setting is augmented by circulating oxidized lipids on oxidized low density lipoprotein particles (oxLDL) that are recognized by key scavenger receptors. CD36 is a major platelet scavenger receptor that recognizes oxLDL and consequently promotes a prothrombotic platelet phenotype through multiple signaling pathways. We previously reported that recognition of oxLDL by CD36 generates specific reactive oxygen species through Src family kinases and NADPH oxidase. These species modulate redox sensitive signaling, such as the activation of the oxidant sensor MAP kinase ERK5, leading to platelet integrin activation, aggregation, and thrombus formation. Although ERK5 was a key effector for CD36 to promote thrombosis, the downstream signaling mechanisms are unclear. We hypothesize that CD36 promotes a procoagulant phenotype that requires ERK5. Platelets isolated and washed from healthy human donors were stimulated with buffer, native low-density lipoprotein (LDL), or oxLDL, and were stained with fluorophore-conjugated Annexin V or Lactadherin to detect exposure of procoagulant phosphatidylserine (PS). OxLDL-stimulated platelets showed dose-dependent 13.4 ± 2.8% and 18.5 ± 3.5% increases in Lactadherin binding at 50 and 100μg/mL oxLDL compared to control LDL (p=0.026; p<0.01), and a rapid binding within 5 min (11.0±0.35%, p<0.001). Furthermore, pre-treatment with oxLDL followed by addition of convulxin, a snake venom that activates platelets through the collagen signaling receptor GPVI, showed a 58.1 ± 4.62% increase in Lactadherin binding compared to oxLDL and convulxin alone (p<0.0001). Addition of ADP or thrombin, agonists that activates specific G-protein coupled receptors, following pre-treatment with oxLDL did not enhance Lactadherin binding. Platelets treated with FA6, a monoclonal CD36 antibody which prevents oxLDL binding, or BIX02188, an ERK5 inhibitor, showed an 80-90% decrease in Annexin V binding (p=0.039; p=0.02) after oxLDL exposure compared to a control non-immunizing IgG or DMSO. To link known pathways for PS exposure with oxLDL-induced PS exposure, platelets were stimulated with oxLDL followed by treatment with Z-VAD-FMK, a “pan” caspase inhibitor, or cyclosporin A, a cyclophilin D/mitochondrial permeability transition pore inhibitor. Z-VAD-FMK treated platelets showed 68% decrease in Lactadherin binding compared to control DMSO (p=0.0196), whereas cyclosporin A did not inhibit Lactadherin binding. Relevance of CD36/ERK5-mediated PS exposure was demonstrated using an in vivo arterial thrombosis model that relies on autologous transplantation of the epigastric artery, which has an adventitial surface that serves as a collagen-rich thrombogenic substrate for thrombosis, into the carotid artery. This model of thrombosis was chosen due to the potential impact of free radical generation in the standard ferric chloride or Rose Bengal-mediated arterial thrombosis models. Bone marrows from platelet ERK5-deficient mice (ERK5 flox/Pf4-cre+) or control mice (ERK5 flox/flox) were transplanted into irradiated atherogenic apoE null mice fed a high fat or control diet. Fibrin deposition, detected by a fluorophore-conjugated antibody to fibrin, was enhanced in hyperlipidemic conditions for mice with platelet ERK5. High fat diet-fed chimeric mice without ERK5 or animals on control diet do not show enhanced fibrin deposition. In conclusion, oxLDL promoted PS exposure through a CD36-Src family kinases-ERK5-caspase-dependent mechanism. PS exposure by this pathway potentially enhances fibrin deposition in an atherogenic arterial in vivo thrombosis model, which suggests a role for CD36 and ERK5 in thrombosis and coagulation in dyslipidemic conditions. DisclosuresNo relevant conflicts of interest to declare.

Pharmacologic targeting of Cdc42 GTPase by a small molecule Cdc42 activity-specific inhibitor prevents platelet activation and thrombosis

Gene targeting of Cdc42 GTPase has been shown to inhibit platelet activation. In this study, we investigated a hypothesis that inhibition of Cdc42 activity by CASIN, a small molecule Cdc42 Activity-Specific INhibitor, may down regulate platelet activation and thrombus formation. We investigated the effects of CASIN on platelet activation in vitro and thrombosis in vivo. In human platelets, CASIN, but not its inactive analog Pirl7, blocked collagen induced activation of Cdc42 and inhibited phosphorylation of its downstream effector, PAK1/2. Moreover, addition of CASIN to washed human platelets inhibited platelet spreading on immobilized fibrinogen. Treatment of human platelets with CASIN inhibited collagen or thrombin induced: (a) ATP secretion and platelet aggregation; and (b) phosphorylation of Akt, ERK and p38-MAPK. Pre-incubation of platelets with Pirl7, an inactive analog of CASIN, failed to inhibit collagen induced aggregation. Washing of human platelets after incubation with CASIN eliminated its inhibitory effect on collagen induced aggregation. Intraperitoneal administration of CASIN to wild type mice inhibited ex vivo aggregation induced by collagen but did not affect the murine tail bleeding times. CASIN administration, prior to laser-induced injury in murine cremaster muscle arterioles, resulted in formation of smaller and unstable thrombi compared to control mice without CASIN treatment. These data suggest that pharmacologic targeting of Cdc42 by specific and reversible inhibitors may lead to the discovery of novel antithrombotic agents.

Hemocompatibility of polymers for use in vascular endoluminal implants

AbstractImplanted polymers for cardiovascular applications may function as structural supports, barriers, or provide a means for local drug delivery. Several thermoplastic biodegradable drug delivery polymers are potential candidates for blood‐contacting implant applications. For intravascular applications specifically, a criterion for material selection is the intrinsic hemocompatibility of the baseline polymer. As an initial screening approach for selection of polymers for in vivo use, thin films of polyesters: poly(ɛ‐caprolactone) (PCL), poly(lactic acid) (PLA), poly(lactic‐co‐glycolic acid) (PLGA); polyanhydrides: poly(fatty acid dimer‐co‐sebacic acid) (PFAD:SA) and poly(biscarboxyphenoxypropane‐co‐sebacic acid) (PCPP:SA); and poly(ethylene glycol) (PEG)‐ylated polyesters: PLA:PEG, PCL:PEG and PCL:PLA:PEG polymers were spin‐cast on glass cover slips and placed in an in vitro flow system exposing them at a controlled shear to overflowing human whole blood. Platelet adherence, aggregate formation, and thrombus formation, as well as leukocyte adherence were assessed following 5 min of flow. At 5 min of flow the rank order of materials, in terms of least to most thrombogenic was: PCL &lt; PFAD:SA &lt; PCPP:SA &lt; PLGA &lt; PLA. All PEGylated materials, in general, had less thrombus formation than baseline unmodified materials.

Tyrosine Kinase Inhibitor Sunitinib Delays Platelet-Induced Coagulation: Additive Effects of Aspirin.

Sunitinib is a multitarget tyrosine kinase inhibitor (TKI) used for cancer treatment. In platelets, sunitinib affects collagen-induced activation under noncoagulating conditions. We investigated (1) the effects of sunitinib on thrombus formation induced by other TK-dependent receptors, and (2) the effects under coagulating conditions. Cardiovascular disease is a comorbidity in cancer patients, resulting in possible aspirin treatment. Sunitinib and aspirin are associated with increased bleeding risk, and therefore we also investigated (3) the synergistic effects of these compounds on thrombus and fibrin formation. Blood or isolated platelets from healthy volunteers or cancer patients were incubated with sunitinib and/or aspirin or vehicle. Platelet activation was determined by TK phosphorylation, flow cytometry, changes in [Ca2+]i, aggregometry, and whole blood perfusion over multiple surfaces, including collagen with(out) tissue factor (TF) was performed. Sunitinib reduced thrombus formation and phosphatidylserine (PS) exposure under flow on collagen type I and III. Also, sunitinib inhibited glycoprotein VI-induced TK phosphorylation and Ca2+ elevation. Upon TF-triggered coagulation, sunitinib decreased PS exposure and fibrin formation. In blood from cancer patients more pronounced effects of sunitinib were observed in lung and pancreatic as compared to neuroglioblastoma and other cancer types. Compared to sunitinib alone, sunitinib plus aspirin further reduced platelet aggregation, thrombus formation, and PS exposure on collagen under flow with(out) coagulation. Sunitinib suppresses collagen-induced procoagulant activity and delays fibrin formation, which was aggravated by aspirin. Therefore, we urge for awareness of the combined antiplatelet effects of TKIs with aspirin, as this may result in increased risk of bleeding.

Actin polymerization regulates glycoprotein Ibα shedding

Glycoprotein (GP) Ibα shedding mediated by ADAM17 (a disintegrin and metalloproteinase 17) plays an important role in negatively regulating platelet function and thrombus formation. However, the mechanism of GPIbα shedding remains elusive. Here, we show that jasplakinolide (an actin-polymerizing peptide)-induced actin polymerization results in GPIbα shedding and impairs platelet function. Thrombin and A23187-induced GPIbα shedding is increased by jasplakinolide; in contrast, GPIbα shedding is reduced by a depolymerization regent (cytochalasin B). We find that actin polymerization activates calpain leading to filamin A hydrolyzation. We further demonstrate that the interaction of filamin A with the cytoplasmic domain of GPIbα plays a critical role in regulating actin polymerization-induced GPIbα shedding. Taken together, these data demonstrate that actin polymerization regulates ADAM17-mediated GPIbα shedding, suggesting a novel strategy to negatively regulate platelet function.

MTOR regulates GPVI-mediated platelet activation

BackgroundDue to mTOR (mammalian/mechanistic target of rapamycin) gene-loss mice die during embryonic development, the role of mTOR in platelets has not been evaluated using gene knockout technology.MethodsA mouse model with megakaryocyte/platelet-specific deletion of mTOR was established, and be used to evaluate the role of mTOR in platelet activation and thrombus formation.ResultsmTOR−/− platelets were deficient in thrombus formation when grown on low-concentration collagen-coated surfaces; however, no deficiency in thrombus formation was observed when mTOR−/− platelets were perfused on higher concentration collagen-coated surfaces. In FeCl3-induced mouse mesenteric arteriole thrombosis models, wild-type (WT) and mTOR−/− mice displayed significantly different responses to low-extent injury with respect to the ratio of occluded mice, especially within the first 40 min. Additionally, mTOR−/− platelets displayed reduced aggregation and dense granule secretion (ATP release) in response to low doses of the glycoprotein VI (GPVI) agonist collagen related peptide (CRP) and the protease-activated receptor-4 (PAR4) agonist GYPGKF-NH2; these deficiencies were overcame by stimulation with higher concentration agonists, suggesting dose dependence of the response. At low doses of GPVI or PAR agonist, the activation of αIIbβ3 in mTOR−/− platelets was reduced. Moreover, stimulation of mTOR−/− platelets with low-dose CRP attenuated the phosphorylation of S6K1, S6 and Akt Ser473, and increased the phosphorylation of PKCδ Thr505 and PKCε Ser729. Using isoform-specific inhibitors of PKCs (δ, ɛ, and α/β), we established that PKCδ/ɛ, and especially PKCδ but not PKCα/β or PKCθ, may be involved in low-dose GPVI-mediated/mTOR-dependent signaling.ConclusionThese observations indicate that mTOR plays an important role in GPVI-dependent platelet activation and thrombus formation.

Cellulose acetate-polyvinyl alcohol blend hemodialysis membranes integrated with dialysis performance and high biocompatibility

Hemodialysis considered as therapy of end-stage renal disease (ESRD) for the separation of protein and uremic toxins based on their molecular weights using semi-permeable membranes. Cellulose Acetate (CA) hemodialysis membrane has been widely used in the biomedical field particularly for hemodialysis applications. The main issue of CA membrane is less selectivity and hemocompatibility. In this study, to enhance the filtration capability and biocompatibility of CA hemodialysis membrane modified by using Polyvinyl Alcohol (PVA) and Polyethylene Glycol (PEG) as additives. CA-PVA flat sheet membranes were cast by phase inversion method, and separation was done by dead-end filtration cell. The synthesized membranes were described in terms of chemical structure using Fourier Transform Infrared Spectroscopy (FTIR) and morphology by Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM), pure water flux, solute permeation, and protein retention. Biocompatibility of the membranes was tested by the platelet adherence, hemolysis ratio, thrombus formation, and plasma recalcification time. SEM images exposed that the CA-PVA membrane has a uniform porous structure. 42.484 L/m2 h is the maximum pure water flux obtained. The CA-PVA rejected up to 95% of bovine serum albumin (BSA). A similar membrane separated 93% of urea and 89% of creatinine. Platelet adhesion and hemolysis ratio of casted membranes were less than the pure CA membrane. Increased clotting time and less thrombus formation on the membrane's surface showed that the fabricated membrane is biocompatible. CA-PVA hemodialysis membranes are more efficient than conventional reported hemodialysis membranes. It revealed that CA-PVA is high performing biocompatible hemodialysis membrane.

Sanguinarine Attenuates Collagen-Induced Platelet Activation and Thrombus Formation.

Sanguinarine, a benzophenanthridine alkaloid, has been described to have an antiplatelet activity. However, its antithrombotic effect and the mechanism of platelet inhibition have not thoroughly been explored. The current study found that sanguinarine had an inhibitory effect on thrombus formation. This inhibitory effect was quite evident both in the flow-chamber assays as well as in a murine model of FeCl3-induced carotid artery thrombosis. Further investigations also revealed that sanguinarine inhibited the collagen-induced human platelet aggregation and granule release. At the same time, it also prevented platelet spreading and adhesion to immobilized fibrinogen. The molecular mechanisms of its antiplatelet activity were found to be as follows: 1. Reduced phosphorylation of the downstream signaling pathways in collagen specific receptor GPVI (Syk-PLCγ2 and PI3K-Akt-GSK3β); 2. Inhibition of collagen-induced increase in the intracellular Ca2+ concentration ([Ca2+]i); 3. Inhibition of integrin αIIbβ3 outside-in signaling via reducing β3 and Src (Tyr-416) phosphorylation. It can be concluded that sanguinarine inhibits collagen-induced platelet activation and reduces thrombus formation. This effect is mediated via inhibiting the phosphorylation of multiple components in the GPVI signaling pathway. Current data also indicate that sanguinarine can be of some clinical value to treat cardiovascular diseases involving an excess of platelet activation.

Antiplatelet and Antithrombotic Effects of Epimedium koreanum Nakai.

Background and Objective. Epimedium koreanum Nakai is a medicinal plant known for its health beneficial effects on impotence, arrhythmia, oxidation, aging, osteoporosis, and cardiovascular diseases. However, there is no report available that shows its effects on platelet functions. Here, we elucidated antiplatelet and antithrombotic effects of ethyl acetate fraction of E. koreanum. Methodology. We analyzed the antiplatelet properties using standard in vitro and in vivo techniques, such as light transmission aggregometry, scanning electron microscopy, intracellular calcium mobilization measurement, dense granule secretion, and flow cytometry to assess integrin αIIbβ3 activation, clot retraction, and Western blot, on washed platelets. The antithrombotic effects of E. koreanum were assessed by arteriovenous- (AV-) shunt model in rats, and its effects on hemostasis were analyzed by tail bleeding assay in mice. Key Results. E. koreanum inhibited platelet aggregation in agonist-stimulated human and rat washed platelets, and it also reduced calcium mobilization, ATP secretion, and TXB2 formation. Fibrinogen binding, fibronectin adhesion, and clot retraction by attenuated integrin αIIbβ3-mediated inside-out and outside-in signaling were also decreased. Reduced phosphorylation of extracellular signal-regulated kinases (ERK), Akt, PLCγ2, and Src was observed. Moreover, the fraction inhibited thrombosis. HPLC results revealed that the fraction predominantly contained icariin. Conclusion and Implications. E. koreanum inhibited platelet aggregation and thrombus formation by attenuating calcium mobilization, ATP secretion, TXB2 formation, and integrin αIIbβ3 activation. Therefore, it may be considered as a potential candidate to treat and prevent platelet-related cardiovascular disorders.

Platelet autophagic machinery involved in thrombosis through a novel linkage of AMPK-MTOR to sphingolipid metabolism

ABSTRACT Basal macroautophagy/autophagy has recently been found in anucleate platelets. Platelet autophagy is involved in platelet activation and thrombus formation. However, the mechanism underlying autophagy in anucleate platelets require further clarification. Our data revealed that LC3-II formation and SQSTM1/p62 degradation were noted in H2O2-activated human platelets, which could be blocked by 3-methyladenine and bafilomycin A1, indicating that platelet activation may cause platelet autophagy. AMPK phosphorylation and MTOR dephosphorylation were also detected, and block of AMPK activity by the AMPK inhibitor dorsomorphin reversed SQSTM1 degradation and LC3-II formation. Moreover, autophagosome formation was observed through transmission electron microscopy and deconvolution microscopy. These findings suggest that platelet autophagy was induced partly through the AMPK-MTOR pathway. In addition, increased LC3-II expression occurred only in H2O2-treated Atg5 f/f platelets, but not in H2O2-treated atg5 −/− platelets, suggesting that platelet autophagy occurs during platelet activation. atg5 −/− platelets also exhibited a lower aggregation in response to agonists, and platelet-specific atg5 −/− mice exhibited delayed thrombus formation in mesenteric microvessles and decreased mortality rate due to pulmonary thrombosis. Notably, metabolic analysis revealed that sphingolipid metabolism is involved in platelet activation, as evidenced by observed several altered metabolites, which could be reversed by dorsomorphin. Therefore, platelet autophagy and platelet activation are positively correlated, partly through the interconnected network of sphingolipid metabolism. In conclusion, this study for the first time demonstrated that AMPK-MTOR signaling could regulate platelet autophagy. A novel linkage between AMPK-MTOR and sphingolipid metabolism in anucleate platelet autophagy was also identified: platelet autophagy and platelet activation are positively correlated. Abbreviations: 3-MA: 3-methyladenine; A.C.D.: citric acid/sod. citrate/glucose; ADP: adenosine diphosphate; AKT: AKT serine/threonine kinase; AMPK: AMP-activated protein kinase; ANOVA: analysis of variance; ATG: autophagy-related; B4GALT/LacCS: beta-1,4-galactosyltransferase; Baf-A1: bafilomycin A1; BECN1: beclin 1; BHT: butylate hydrooxytoluene; BSA: bovine serum albumin; DAG: diacylglycerol; ECL: enhanced chemiluminescence; EDTA: ethylenediamine tetraacetic acid; ELISA: enzyme-linked immunosorbent assay; GALC/GCDase: galactosylceramidase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GBA/GluSDase: glucosylceramidase beta; GPI: glycosylphosphatidylinositol; H2O2: hydrogen peroxide; HMDB: human metabolome database; HRP: horseradish peroxidase; IF: immunofluorescence; IgG: immunoglobulin G; KEGG: Kyoto Encyclopedia of Genes and Genomes; LAMP1: lysosomal associated membrane protein 1; LC-MS/MS: liquid chromatography-tandem mass spectrometry; mAb: monoclonal antibody; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MPV: mean platelet volume; MTOR: mechanistic target of rapamycin kinase; ox-LDL: oxidized low-density lipoprotein; pAb: polyclonal antibody; PC: phosphatidylcholine; PCR: polymerase chain reaction; PI3K: phosphoinositide 3-kinase; PLS-DA: partial least-squares discriminant analysis; PRP: platelet-rich plasma; Q-TOF: quadrupole-time of flight; RBC: red blood cell; ROS: reactive oxygen species; RPS6KB/p70S6K: ribosomal protein S6 kinase B; SDS: sodium dodecyl sulfate; S.E.M.: standard error of the mean; SEM: scanning electron microscopy; SGMS: sphingomyelin synthase; SM: sphingomyelin; SMPD/SMase: sphingomyelin phosphodiesterase; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; UGT8/CGT: UDP glycosyltransferase 8; UGCG/GCS: UDP-glucose ceramide glucosyltransferase; ULK1: unc-51 like autophagy activating kinase 1; UPLC: ultra-performance liquid chromatography; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: phosphatidylinositol-3-phosphate; WBC: white blood cell; WT: wild type

Изменения функционального состава тромбоцитов у пациентов с политравмой

Акутальность. Политравма остается главной причиной глобальной заболеваемости и смертности и является причиной более 10 % смертей. Цель: изучить данные литературы об изменениях показателей сосудисто-тромбоцитарного гемостаза у пациентов с политравмой, исследовать динамику морфофункционального состояния тромбоцитов, проанализировать изменения показателей внутрисосудистой активации тромбоцитов у пациента с политравмой. Результаты. Для нормального свертывания крови нужны как минимум 4 компонента — кровеносные сосуды, тромбоциты, способность крови к свертыванию и фибринолиз. Определение таких компонентов, как показатели внутрисосудистой активации тромбоцитов, может стать важным шагом оценки нарушений тромбоцитарного звена гемостаза у пациентов с политравмой. Сосудисто-тромбоцитарный гемостаз начинается рефлекторным первичным спазмом артериол, после чего возникает вторичный спазм артериол, дальше образуется первичная тромбоцитарная пробка (адгезия и агрегация) и, соответственно, консолидация тромба, в результате чего формируется окончательный тромбоцитарный тромб. Еще до контакта тромбоцитов с обнаженным коллагеном происходит первичная активация тромбоцитов. Сначала меняется форма интактных тромбоцитов с дискоидной формы к активированым клеткам — дискоэхиноцитам, сфероцитам и/или сфероэхиноцитам. Нами установлено, что на 3-и сутки после травмы при нормальном количестве тромбоцитов в венозной крови уменьшается количество интактных тромбоцитов — дискоцитов, растет количество активных форм тромбоцитов — дискоэхиноцитов и сфероэхиноцитов, соответственно, растет общая сумма активных форм тромбоцитов. Нормальный уровень тромбоцитов у пациентов с политравмой может замаскировать выраженность коагулопатии, а проведение исследований показателей внутрисосудистой активации тромбоцитов может стать диагностическим компонентом сосудисто-тромбоцитарного звена гемостаза у пациентов с политравмой. Выводы. У пациентов с коагулопатией вследствие политравмы на 3 сутки наблюдаются изменения показателей внутрисосудистой активации тромбоцитов и агрегации тромбоцитов, индуцированной адреналином и АДФ.

Anti-platelet activity of phytocompounds in various dandelion organs in human whole blood model in vitro

• The anti-platelet potential of four fractions from various parts of dandelion in whole blood was studied. • Fraction C reduced: the thrombus formation and platelets’ activation upon stimulation with collagen. • None of the tested fractions caused changes in the platelet proteome. Numerous studies indicate that the usage of natural compounds, such as those found in dandelion, might modulate blood platelets’ function. Our previous investigations indicated that fractions obtained from different organs of dandelion possessed anti-platelet properties and inhibited activation of washed platelets. However, the impact of those fractions on platelets in whole blood is still unknown. Therefore, the aim of the presented study was to evaluate the anti-platelet potential of four fractions obtained from various parts of dandelion (fractions A and B - roots; fraction C - leaves; fraction D - petals) on platelets activation and thrombus formation in whole blood and an impact of tested fractions on platelets’ proteome. The level of activation of resting or agonist-stimulated (ADP or collagen) whole blood platelets after incubation with different dandelion fractions was determined based on their cell-surface expression of P-selectin (CD62P) and presence of an active form of GPIIb/IIIa (PAC-1 binding) by flow cytometry technique. The influence of the tested fractions on whole blood thrombus formation was estimated by the thrombus-formation analysis system (T-TAS). The changes in platelets’ proteome after incubation with dandelion fractions were analyzed using gel electrophoresis (both, native and SDS-PAGE). We found that fraction C from dandelion leaves reduced: 1) the thrombus formation and 2) platelets’ activation upon stimulation with collagen (statistically significant decrease in P-selectin expression and presence of an active form of GPIIb/IIIa) in whole blood. None of the tested fractions caused changes in the platelet proteome. This may represent a novel avenue of research on antiplatelet supplementation in the prevention and treatment of cardiovascular diseases caused by hyperactivation of platelets.

The metabolic enzyme pyruvate kinase M2 regulates platelet function and arterial thrombosis

Very little is known about the role of metabolic regulatory mechanisms in platelet activation and thrombosis. Dimeric pyruvate kinase M2 (PKM2) is a crucial regulator of aerobic glycolysis that facilitates the production of lactate and metabolic reprogramming. Herein, we report that limiting PKM2 dimer formation, using the small molecule inhibitor ML265, negatively regulates lactate production and glucose uptake in human and murine stimulated platelets. Furthermore, limiting PKM2 dimer formation reduced agonist-induced platelet activation, aggregation, clot retraction, and thrombus formation under arterial shear stress in vitro in both human and murine platelets. Mechanistically, limiting PKM2 dimerization downregulated phosphatidylinositol 3-kinase (PI3K)-mediated protein kinase B or serine/threonine-specific protein kinase (Akt)/glycogen synthase kinase 3 (GSK3) signaling in human and murine platelets. To provide further evidence for the role of PKM2 in platelet function, we generated a megakaryocyte or platelet-specific PKM2−/− mutant strain (PKM2fl/flPF4Cre+). Platelet-specific PKM2-deficient mice exhibited impaired agonist-induced platelet activation, aggregation, clot retraction, and PI3K-mediated Akt/GSK3 signaling and were less susceptible to arterial thrombosis in FeCl3 injury–induced carotid- and laser injury–induced mesenteric artery thrombosis models, without altering hemostasis. Wild-type mice treated with ML265 were less susceptible to arterial thrombosis with unaltered tail bleeding times. These findings reveal a major role for PKM2 in coordinating multiple aspects of platelet function, from metabolism to cellular signaling to thrombosis, and implicate PKM2 as a potential target for antithrombotic therapeutic intervention.

Derrone Inhibits Platelet Aggregation, Granule Secretion, Thromboxane A2 Generation, and Clot Retraction: An In Vitro Study.

Cudrania tricuspidata (C. tricuspidata) is widespread throughout East Asia and in China and Korea, and it is widely used as a traditional remedy against eczema, mumps, and tuberculosis. With regard to the aforementioned medical efficacy, various studies are continuously being conducted, and it has been reported that C. tricuspidata extract has various actions against inflammation, diabetes, obesity, and tumors. Therefore, we evaluated antiplatelet effects using derrone in C. tricuspidata. We examined the effect of derrone on the phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and inositol 1, 4, 5-triphosphate receptor I (IP3RI), and on the dephosphorylation of cytosolic phospholipase A2 (cPLA2), mitogen-activated protein kinases p38 (p38MAPK), and Akt, which affects platelet function and thrombus formation. Various agonists-induced human platelets were inhibited by derrone without cytotoxicity, and it also decreased the intracellular calcium level through the signaling molecule phosphorylations. In addition, derrone inhibited glycoprotein IIb/IIIa (αIIb/β3) affinity. Thus, in the present study, derrone suppressed human platelet aggregation and thrombin-induced clot formation.

Prostacyclin Analogues Inhibit Platelet Reactivity, Extracellular Vesicle Release and Thrombus Formation in Patients with Pulmonary Arterial Hypertension.

(1) Background: Prostacyclin analogues (epoprostenol, treprostinil, and iloprost) induce vasodilation in pulmonary arterial hypertension (PAH) but also inhibit platelet function. (2) Objectives: We assessed platelet function in PAH patients treated with prostacyclin analogues and not receiving prostacyclin analogues. (3) Methods: Venous blood was collected from 42 patients treated with prostacyclin analogues (49.5 ± 15.9 years, 81% female) and 38 patients not receiving prostacyclin analogues (55.5 ± 15.6 years, 74% female). Platelet reactivity was analyzed by impedance aggregometry using arachidonic acid (AA; 0.5 mM), adenosine diphosphate (ADP; 6.5 µM), and thrombin receptor-activating peptide (TRAP; 32 µM) as agonists. In a subset of patients, concentrations of extracellular vesicles (EVs) from all platelets (CD61+), activated platelets (CD61+/CD62P+), leukocytes (CD45+), and endothelial cells (CD146+) were analyzed by flow cytometry. Platelet-rich thrombus formation was measured using a whole blood perfusion system. (4) Results: Compared to controls, PAH patients treated with prostacyclin analogues had lower platelet reactivity in response to AA and ADP (p = 0.01 for both), lower concentrations of platelet and leukocyte EVs (p ≤ 0.04), delayed thrombus formation (p ≤ 0.003), and decreased thrombus size (p = 0.008). Epoprostenol did not affect platelet reactivity but decreased the concentrations of platelet and leukocyte EVs (p ≤ 0.04). Treprostinil decreased platelet reactivity in response to AA and ADP (p ≤ 0.02) but had no effect on the concentrations of EVs. All prostacyclin analogues delayed thrombus formation and decreased thrombus size (p ≤ 0.04). (5) Conclusions: PAH patients treated with prostacyclin analogues had impaired platelet reactivity, EV release, and thrombus formation, compared to patients not receiving prostacyclin analogues.

DHA 12‐LOX‐derived oxylipins regulate platelet activation and thrombus formation through a PKA‐dependent signaling pathway

BackgroundThe effects of docosahexaenoic acid (DHA) on cardiovascular disease are controversial and a mechanistic understanding of how this omega‐3 polyunsaturated fatty acid (ω‐3 PUFA) regulates platelet reactivity and the subsequent risk of a thrombotic event is warranted. In platelets, DHA is oxidized by 12‐lipoxygenase (12‐LOX) producing the oxidized lipids (oxylipins) 11‐HDHA and 14‐HDHA. We hypothesized that 12‐LOX DHA‐oxylipins may be involved in the beneficial effects observed in dietary supplemental treatment with ω‐3 PUFAs or DHA itself. ObjectivesTo determine the effects of DHA, 11‐HDHA, and 14‐HDHA on platelet function and thrombus formation, and to elucidate the mechanism by which these ω‐3 PUFAs regulate platelet activation. Methods and resultsDHA, 11‐HDHA, and 14‐HDHA attenuated collagen‐induced human platelet aggregation, but only the oxylipins inhibited ⍺IIbβ3 activation and decreased ⍺‐granule secretion. Furthermore, treatment of whole blood with DHA and its oxylipins impaired platelet adhesion and accumulation to a collagen‐coated surface. Interestingly, thrombus formation was only diminished in mice treated with 11‐HDHA or 14‐HDHA, and mouse platelet activation was inhibited following acute treatment with these oxylipins or chronic treatment with DHA, suggesting that under physiologic conditions, the effects of DHA are mediated through its oxylipins. Finally, the protective mechanism of DHA oxylipins was shown to be mediated via activation of protein kinase A. ConclusionsThis study provides the first mechanistic evidence of how DHA and its 12‐LOX oxylipins inhibit platelet activity and thrombus formation. These findings support the beneficial effects of DHA as therapeutic intervention in atherothrombotic diseases.

The Total Thrombus Formation (T-TAS) platelet (PL) assay, a novel test that evaluates whole blood platelet thrombus formation under physiological conditions

Dual antiplatelet therapy (DAPT, aspirin, and a P2Y12 inhibitor) reduces thrombotic events in patients with coronary artery disease (CAD). The T-TAS PL assay uses arterial shear flow over collagen surface, better mimicking in vivo conditions compared to conventional agonist-based platelet function assays, to evaluate platelet function. Here, the platelet function in patients taking DAPT is evaluated with the T-TAS PL assay. In 57 patients with CAD, taking DAPT ≥7 days (n = 22 for clopidogrel, n = 15 for prasugrel, n = 20 for ticagrelor), T-TAS PL assessments were performed in duplicate, and expressed as area under the flow pressure curve within a 10-minute period (AUC10). The duplicate measurements were strongly correlated (r = 0.90, p < .001), with an intra-assay coefficient of variation (CV) of 11,5%. For clopidogrel, the median AUC10 was 11.5 (IQR5.9–41.8), for prasugrel 28.8 (IQR10.3–37.6), and for ticagrelor 8.9 (IQR 6.4–10.9). All measurements were below the AUC10 cutoff of 260 measured in healthy volunteers, suggesting excellent discrimination of DAPT-treated and untreated persons. The new T-TAS PL assay demonstrated complete discrimination of platelet function in patients on DAPT based on an established cutoff. Ticagrelor showed lower levels of platelet function and a more uniform response compared to prasugrel and clopidogrel.

Selatogrel, a reversible P2Y12 receptor antagonist, has reduced off-target interference with haemostatic factors in a mouse thrombosis model

IntroductionSelatogrel is a reversible antagonist of the P2Y12 receptor. In rat thrombosis/haemostasis models, selatogrel was associated with lower blood loss than clopidogrel or ticagrelor at equivalent anti-thrombotic effect. Material and methodsWe sought to elucidate the mechanism underlying the observed differences in blood loss, using real-time intravital microscopy in mouse. ResultsSelatogrel, ticagrelor and clopidogrel dose-dependently inhibited laser-induced platelet thrombus formation. At maximal antithrombotic effect, only small mural platelets aggregates, corresponding to hemostatic seals, were present. The phenotype of these hemostatic seals was dependent on the type of P2Y12 receptor antagonist. In the presence of clopidogrel and ticagrelor, detachment of platelets from the hemostatic seals was increased, indicative of reduced stability. In contrast, in the presence of selatogrel, platelet detachment was not increased. Moreover, equivalent antithrombotic dosing regimens of ticagrelor and clopidogrel reduced laser-induced calcium mobilization in the endothelium, restricted neutrophil adhesion and subsequent fibrin formation and thus reduced fibrin-mediated stabilization of the hemostatic seals. The effects of ticagrelor were also observed in P2Y12 receptor deficient mice, indicating that the effects are off-target and independent of the P2Y12 receptor. In contrast, selatogrel did not interfere with these elements of haemostasis in wild-type or in P2Y12 receptor deficient mice. ConclusionIn the presence of selatogrel the stability of hemostatic seals was unperturbed, translating to an improved blood loss profile. Our data suggest that the mechanism underlying the differences in blood loss profiles of P2Y12 receptor antagonists is by off-target interference with endothelial activation, neutrophil function and thus, fibrin-mediated stabilization of haemostatic seals.