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In in vitro model of short-term therapeutic inhalation of Xe/O2 mixture, xenon in millimolar concentrations led to a pronounced decrease in induced platelet aggregation in the platelet-enriched blood plasma. The maximum and statistically significant decrease occurred in response to induction by collagen (by ≈30%, p≤0.01) and ADP (by ≈25%, p≤0.01). A slightly weaker but statistically significant reduction in aggregation appeared in response to ristocetin (by ≈12%, p≤0.01) and epinephrine (by ≈9%, p≤0.01). It should be noted that the spontaneous aggregation exceeded the reference values in the control group. Nevertheless, even at minimal absolute values, spontaneous platelet aggregation decreased by 2 times in response to xenon (p≤0.01). The reasons for the decrease of spontaneous and induced aggregation are xenon accumulation in the lipid bilayer of the membrane with subsequent nonspecific (mechanical) disassociation of membrane platelet structures and specific block of its distinct from neuronal NMDA receptor.

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The processes of blood coagulation and fibrinolysis that in part maintain the physical integrity of the circulatory system and fluidity of its contents are complex as they are critical for life. While the roles played by cellular components and circulating proteins in coagulation and fibrinolysis are widely acknowledged, the impact of metals on these processes is at best underappreciated. In this narrative review we identify twenty-five metals that can modulate the activity of platelets, plasmatic coagulation, and fibrinolysis as determined by in vitro and in vivo investigations involving several species besides human beings. When possible, the molecular interactions of the various metals with key cells and proteins of the hemostatic system were identified and displayed in detail. It is our intention that this work serve not as an ending point, but rather as a fair evaluation of what mechanisms concerning metal interactions with the hemostatic system have been elucidated, and as a beacon to guide future investigation.

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BACKGROUND: Vaccine-induced thrombotic thrombocytopenia (VITT) is a rare coagulation disorder reported after administration of COVID-19 adenovirus-vectored vaccines. VITT is mediated by anti-platelet factor 4 (PF4) antibodies activating platelets through the Fcγ-receptor II (FcγRII), and it is associated with strong fibrin turnover. The complement system is involved in several other immunothrombotic entities, but its impact on VITT is not established. OBJECTIVE: To assess antibodies in interaction with the activation of platelets and complement triggered by VITT. METHODS: Antibodies against adenovirus type 2 hexon protein, ChAdOx1 adenoviral vector-specific IgG and PF4 were analyzed by enzyme immunoassays from VITT patients (n = 5). The EDTA plasma samples of the patients and controls were used to measure both terminal complement complexes (TCC) by ELISA and aggregation of healthy donor platelets. We studied the effects of human immunoglobulin (IVIG) and glycoprotein IIb/IIIa inhibitor (GPIIb/IIIa) on spontaneous and collagen-induced platelet aggregation supplemented with VITT plasma. RESULTS: None of the patients had experienced a COVID-19 infection. Antibody analyses confirmed the immunogenicity of the adenovirus-vectored ChAdOx1 vaccine. Moreover, VITT plasma had anti-PF4 antibodies and elevated TCC levels as a sign of complement activation. In isolated healthy donor platelets, VITT patient plasma caused marked, spontaneous aggregation of platelets, which was abolished by eptifibatide and high-dose therapeutic IVIG. CONCLUSIONS: Our findings suggest that VITT is triggered by antibodies against adenovirus vector and PF4-polyanion complexes which strongly co-activate complement and platelets. The spontaneous platelet aggregation was suppressed by IVIG or eptifibatide, indicating that besides FcγRII, also GPIIb/IIIa receptor exerts platelet procoagulant role in VITT.

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Perfluoro-alkyl substances (PFAS), particularly perfluoro-octanoic acid (PFOA), are persisting environmental chemicals showing bioaccumulation in human tissues. Recently, exposure to PFAS has been associated with increased prevalence of cardiovascular diseases (CVDs). However, a causal role of PFAS in atherosclerosis pathogenesis is under-investigated. Here, we investigated the effect of PFOA exposure on platelets' function, a key player in atherosclerosis process. PFOA accumulation in platelets was evaluated by liquid chromatography-mass spectrometry. Changes in platelets' membrane fluidity and activation after dose-dependent exposure to PFOA were evaluated by merocyanine 540 (MC540) and anti P-Selectin immune staining at flow cytometry, respectively. Intracellular calcium trafficking was analyzed with Fluo4M probe, time-lapse live imaging. Platelets' aggregation state was also evaluated with Multiplate® aggregometry analyzer in 48 male subjects living in a specific area of the Veneto region with high PFAS environmental pollution, and compared with 30 low-exposure control subjects. Platelets' membrane was the major target of PFOA, whose dose-dependent accumulation was associated in turn with increased membrane fluidity, as expected by a computational model; increased activation at resting condition; and both calcium uptake and aggregation upon activation. Finally, exposed subjects had higher serum and platelets levels of PFOA, together with increased aggregation parameters at Multiplate®, compared with controls. These data help to explain the emerging association between PFAS exposure and CVD.

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Heterofunctionalized gold nanoparticles (AuNPs) were obtained in a one pot reaction of gold precursor with cationic carbosilane dendrons (first to third generations, 1-3G) and (polyethylene)glycol (PEG) ligands in the presence of a reducing agent. The final dendron/PEG proportion on AuNPs depends on the initial dendron/PEG ratio (3/1, 1/1, 1/3) and dendron generation. AuNPs were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), ultraviolet spectroscopy (UV-VIS), thermogravimetric analysis (TGA), nuclear magnetic resonance (1H NMR) and zeta potential (ZP). Several assays have been carried out to determine the relevance of PEG/dendron ratio and dendron generation in the biomedical properties of PEGylated AuNPs and the results have been compared with those obtained for non-PEGylated AuNPs. Finally, analyses of PEG recognition by anti-PEG antibodies were carried out. In general, haemolysis, platelet aggregation and toxicity were reduced after PEGylation of AuNPs, the effect being dependent on dendron generation and dendron/PEG ratio. Dendron generation determines the exposure of PEG ligand and the interaction of this ligand with AuNPs environment. On the other hand, increasing PEG proportion diminishes toxicity but also favors interaction with antibodies.

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Microvesicles (MVs) released from leukocytes, platelets and endothelial cells are elevated in patients with acute coronary syndrome (ACS). In the present study, we assessed the potential pro-aggregatory properties of MVs obtained from ACS patients. Thus, we divided the patients into two groups based on clopidogrel-responsiveness, i.e. high on-treatment platelet reactivity (HPR; n = 16), and low or normal on-treatment platelet reactivity (non-HPR; n = 14), respectively. MVs from patients were obtained by high-speed centrifugation, and the pro-aggregatory effect of MVs added to fresh isolated platelets from healthy subjects were analyzed by 96-well microplate aggregometry. MVs from HPR patients significantly enhanced spontaneous platelet aggregation around two times more than MVs from non-HPR patients. The pro-aggregatory effect of three out of four MV phenotypes correlated to MV-concentrations as determined by flow cytometry. Furthermore, MVs from patients with diabetes mellitus (n = 9) had a stronger pro-aggregatory effect compared to MVs from those without diabetes (n = 21; p = .025 between groups). In conclusion, MVs from ACS patients with clopidogrel non-responsiveness enhance platelet aggregation, as do MVs from ACS patients with diabetes. Thus, MVs from patients with hyperreactive platelets boost platelet aggregation. Blocking MV-formation may reduce platelet hyperreactivity.

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The disorders of hemostasis and coagulation were believed to be the main contributors to the pathogenesis of pulmonary thromboembolism (PTE), and platelets are the basic factors regulating hemostasis and coagulation and play important roles in the process of thrombosis. This study investigated the proteome of human umbilical vein endothelial cells (HUVECs) with platelet endothelial aggregation receptor-1 (PEAR1) knockdown using the isobaric tags for relative and absolute quantitation (iTRAQ) method and analyzed the role of differential abundance proteins (DAPs) in the regulation of platelets aggregation. Our results showed that the conditioned media-culturing HUVECs with PEAR1 knockdown partially suppressed the adenosine diphosphate (ADP)-induced platelet aggregation. The proteomics analysis was performed by using the iTRAQ technique, and a total of 215 DAPs (124 protein was upregulated and 91 protein were downregulated) were identified. The Gene Ontology (GO) enrichment analysis showed that proteins related to platelet α granule, adenosine triphosphate metabolic process, and endocytosis were significantly enriched. Further, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis also identified the significant enrichment of endocytosis-related pathways. The real-time polymerase chain reaction assay confirmed that the expression of P2Y12 , mitochondrial carrier 2, NADH dehydrogenase (ubiquinone) iron-sulfur protein 3, and ubiquinol-cytochrome c reductase hinge protein are significantly downregulated in the HUVECs with PEAR1 knockdown. In conclusion, our in vitro results implicated that DAPs induced by PEAR1 knockdown might contribute to the platelet aggregation. Proteomic studies by employing GO enrichment and KEGG pathway analysis suggested that the potential effects of DAPs on platelet aggregation may be linked to the balance of ADP synthesis or degradation in mitochondria.

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The Phaseolus vulgaris (common bean), a worldwide vegetable of high consumption, can act as a nutritional supplement in the diet of oversized individuals to reduce weight. Studies have demonstrated the existence of molecules capable of inhibiting the breakdown of carbohydrates via inhibition of both α-amylases and glycosidases. Here, we describe a novel property of the Phaseolus vulgaris: inhibition of thrombotic cardiovascular events. Using assays to test platelet aggregation and secretion, and flow cytometry against the surface expression of P-Selectin. We show that bean extracts significantly reduced adenosine 5'-diphosphate and arachidonic acid induced-platelet aggregation. The mechanism underlying such effect appears to be mediated by AKT, since AKT hypo-phosphorylation decreases the "inside out" activation of platelets. In sum, our results support the hypothesis that common beans are nutritional ingredients that help reduce the risk of cardiovascular diseases associated with platelet hyper-reactivity.

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In this article we present the synthesis of enantiomerically pure sulfoxide and study the influence of this compound on hemostasis. Detailed NMR studies and molecular dynamics simulations using sodium dodecyl sulfate (SDS) membrane models indicated that the bicyclic fragment of sulfoxide was embedded into the SDS micelle whereas the -SO(CH2)2OH fragment remained on the surface of the micelle and was in contact with the solvent. We also found that the pro-coagulative activity of sulfoxide was due to its ability to inhibit platelet activation and inhibited the catalytic activity of phospholipid surface which was involved in formation of coagulation clotting factor complexes.

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ETHNOPHARMACOLOGICAL RELEVANCE: Moslae Herba (MH) is broadly used as an antiviral, antipyretic and anticoagulant drug which effectively treats respiratory diseases including cough, asthma, throat, cold and flu. AIM OF THIS STUDY: The excessive inflammation of the lungs is the hallmark of severe influenza A virus (IAV) infection, while platelet aggregation and its subsequent microvascular thrombosis can exacerbate IAV-induced lung injury. Thus, inhibition of platelet aggregation can be a potential target for IAV treatment. Previous studies focus on the flavonoids from MH and their anti-inflammatory activities, but the anticoagulant compounds and potential molecular mechanism of MH remains unclear. This study was to isolate and characterize diketopiperazines (DKPs) from MH and to explore the underlying anticoagulant mechanism on IAV infection models. MATERIALS AND METHODS: EtOAc sub-extract separated from MH ethanolic extract was subjected to fractionation through column chromatography. The chemical structures of pure compounds were characterized by the spectral analysis. Antiviral activities of DKPs were assayed in IAV-infected Madin-Darby canine kidney (MDCK) cells and mice. Anticoagulant effects of DKPs were investigated on adenosine 5'-diphosphate (ADP)-induced acute pulmonary embolism and IAV-induced lung injury in vivo, as well as the inhibition on platelet activating factor (PAF), arachidonic acid (AA) and ADP-induced platelet aggregation in vitro. The serum levels of thromboxane B2 (TXB2) and 6-keto-PGF1α were detected by ELISA. The expressions of key proteins in CD41-mediated PI3K/AKT pathways were determined by western blotting analysis. RESULTS: Six DKPs were, for the first time, isolated from MH and identified as cyclo(Tyr-Leu) (1), cyclo(Phe-Phe) (2), cyclo(Phe-Tyr) (3), cyclo(Ala-Ile) (4), cyclo(Ala-Leu) (5) and Bz-Phe-Phe-OMe (6). Among these DKPs, cyclo(Ala-Ile) and Bz-Phe-Phe-OMe possessed low cytotoxicities and significant inhibition against cytopathic effects induced by IAV (H1N1 and H3N2) replication in MDCK cells. Furthermore, cyclo(Ala-Ile) and Bz-Phe-Phe-OMe significantly alleviated IAV-induced platelet activation and lung inflammation in mice. They could reduce the expression of CD41 and the phosphorylation of PI3K and AKT in PLTs of IAV-infected mice. CONCLUSION: These results suggested that cyclo(Ala-Ile) and Bz-Phe-Phe-OMe isolated from MH have antiviral and anticoagulant effects against IAV-induced PLT aggregation and lung inflammation via regulating CD41/PI3K/AKT pathway, and could be used as the potential agents for IAV treatment.

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Background: Systemic lupus erythematosus (SLE) is a relatively uncommon disease of young females in Pakistan. Usually, it has a relapsing-remitting course with variable severity and disease activity. Amongst the different clinical and laboratory parameters used to monitor disease activity in lupus, mean platelet volume (MPV) is a novel biomarker. Although MPV has been studied in other rheumatological conditions like rheumatoid arthritis, its role in adult SLE needs to be defined, especially in Pakistan.  Methods: The aim of this study was to evaluate the role of MPV as a biomarker of disease activity in SLE. This study included 25 patients with active SLE, and another 25 participants with stable, inactive lupus. MPV was measured in each group and compared using SPSS version 16. MPV was also correlated with SLE disease activity index (SLEDAI) and erythrocyte sedimentation rate (ESR). Independent sample t-test and Pearson's correlation tests were applied. Sensitivity and specificity of MPV were checked through ROC analysis.  Results: The MPV of patients with active SLE (n=25, mean [M]=7.12, SD=1.01) was numerically lower than those in the inactive-SLE group (n=25, M= 10.12, SD=0.97), and this was statistically significant ( P<0.001). MPV had an inverse relationship with both ESR (r=-0.93, P<0.001) and SLEDAI (r= -0.94, P<0.001). However, there was a strong positive correlation between ESR and SLEDAI (r=0.95, P<0.001). For MPV, a cutoff value of less than 8.5fl had a sensitivity of 92% and a specificity of 100% ( P< 0.001).  Conclusions: Higher disease activity in SLE is associated with a correspondingly low MPV.

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In previous work, a snake venom arginine esterase (SVAE), agkihpin from the venom of Gloydius halys Pallas, was isolated and its biochemical data including Mr, PI, amino acid components and sugar content was collected. Here, the agkihpin was cloned and further characterized and we found that agkihpin could promote ADP-induced platelets aggregation, hydrolyze fibrin, cleave Aα and Bß chains of fibrinogen and reduce the thrombosis induced by thrombin. Moreover, agkihpin hydrolyzed TAME with optimum temperatures at 30 °C-45 °C, and the hydrolysis was inhibited by EDTA, PMSF, DTT and promoted by Ca2+, Fe3+, Mg2+, Zn2+. The sequence features of agkihpin were detected as follows: the N-terminal residues was determined as I(V)L(Y)GDDECNINE by protein sequencing; the ORF was determined as 705 bp, and the deduced amino acid sequence was identified by peptide mass fingerprinting; the cysteines, cleavage sites, active sites and substrate binding sites of snake venom thrombin-like enzyme (SVTLE), were all conserved in amino acid sequence of agkihpin; 2 Leu(Tyr), 4 Asn and 121 Ile in amino acid sequence of agkihpin were first found in the amino acid sequences of SVTLEs. These findings indicated that agkihpin is a novel SVTLE. What's more, due to its several advantages of fibrino(gen)olytic and thrombosis-reduced activities, and devoid of bleeding risk, agkihpin may be developed into a thrombolytic drug in the future.

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Objective To explore the interaction of streptococcus suis serotype 2 recombinant suilysin ( SLY ) with platelets, and provide the theoretical basis for clinic treatment of patients infected with S.suis.Methods The nickel column affinity chromatography was used to purify the recombinant SLY.The hemolytic acivity was identified by optical density before the platelets aggregation induced by a SLY was detected by a platelet aggregometer or electron microscope and the effect of aspirin on platelets aggregation was analyzed.The impact of wild type 05ZYH33 and sly-deficient mutant strainΔSLY on platelets of mice was compared to predict the interaction of the SLY with platelets in vivo.Results and Conclusion Hemolytic activity of recombinant SLY was 2000 hemolytic units( HU) and platelets aggregation was induced at 1 μg/ml.The aggregation can be inhibited by aspirin in 5 mmol/L.SLY can also increase the volume and reduce the amount of platelets in mice.

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OBJECTIVE: The aim of this study was to determine the effect of long-term physical load on the changes in the fibrinogen concentration and platelet aggregation. MATERIAL AND METHODS: Platelet aggregation was investigated in 144 patients while fibrinogen concentration in 138 patients with CHF. The patients were divided into the groups of the trained patients and the controls and were investigated as follows: on admission to the hospital (stage 1); after treatment in the hospital (stage 2); after 3 months (stage 3); after 6 months (stage 4); and after 1 year (stage 5). The indices were investigated before and after physical load. RESULTS: It was determined that fibrinogen concentration significantly increased after physical load in all the treatment stages in both groups of the patients (P=0.045). In the course of the treatment, fibrinogen concentration gradually decreased in the group of the trained patients (P=0.02). Platelet aggregation investigated with ADP significantly increased after physical load in all the stages in both groups of the patients and decreased during the different investigation stages in the groups of the untrained (P=0.02) and trained patients. Platelet aggregation investigated with ADR consistently decreased before physical load during the different investigation stages in the groups of the trained (difference is not significant) and untrained patients (P=0.02). CONCLUSIONS: Physical training reduces fibrinogen concentration in patients with CHF. It remains unclear whether physical training can have an effect on the decrease in platelet aggregation in patients who have long-term physical training applied.

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Several changes in platelets have been reported in patients with iron-deficiency anemia (IDA), so a relationship between iron metabolism and thrombopoiesis should be considered. We aimed to study the alterations of platelet functions in patients with IDA by assessment of platelet aggregation with epinephrine, adenosine diphosphate (ADP) and ristocetin and by measuring platelet function analyzer-100 (PFA-100) closure time together with the effect of iron therapy on the same tests. A follow-up study was conducted in Ain Shams University Children's hospital in the period from June 2011 to June 2012 including 20 patients with confirmed IDA and 20 healthy age- and sex-matched control. Bleeding manifestations were reported. Laboratory analysis included complete blood count, assessment of iron status by measuring serum iron, TIBC and ferritin, assessment of platelet functions by PFA-100 closure time and platelet aggregation with collagen, ADP and ristocetin. Patients with IDA were treated by oral iron therapy 6 mg/kg/day of ferrous sulfate and post-therapeutic re-assessment was done. Mean age of IDA patients was 5.7 ± 4.2 years. Bleeding manifestations were more common in patients group. Mean PFA-100 closure times (with epinephrine) were significantly longer in patients (179.1 ± 86.4 seconds) compared to control group (115 ± 28.5 seconds) (p < 0.05). Platelet aggregation by ADP (38.1 ± 22.2%), epinephrine (19.7 ± 14.2%) and ristocetin (58.8 ± 21.4%) were significantly reduced in patients compared to control (62.7 ± 6.2, 63.3 ± 6.9, 73.8 ± 8.3, respectively; p < 0.001). After treatment platelet aggregation tests induced by ADP (64.78 ± 18.25%), and epinephrine (55.47 ± 24%) were significantly increased in patients with IDA compared to before treatment (39.44 ± 21.85%, 20.33 ± 14.58%; p < 0.001). PFA-100 closure time as well showed significant decreased after treatment (118.4 ± 27.242) compared to before treatment (186.2 ± 90.35; p < 0.05). A negative correlation between platelet aggregation induced by ADP and mean values of serum ferritin before treatment (r = 0.042, p < 0.05) was found. A mutual effect is considered between iron deficiency and platelet functions. Subtle bleeding manifestations can occur in patients with IDA with delay in platelet aggregation and prolongation in PFA-100 closure times which can be reversed by iron therapy.

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Snake venom toxins have been reportedly used as a rich source of a number of proteins of biotechnological interest due to their wide range of effects on haemostasis. These effects vary greatly: coagulant, anticoagulant, platelet-activating, anti-platelet, fibrinolytic and hemorrhagic, in either enzymatic or non-enzymatic pathways. Agkistrodon venom contains a variety of proteins that possess antiplatelet activities. This study presents recent development in our laboratory to produce and purify antiplatelet proteins derived from Agkistrodon blomhoffi ussuriensiss nake venom. Different matrices of HPLC (size exclusion, ion exchange and affinity chromatography) were employed for purifying the proteins and their biological and biochemical properties were characterized by SDSPAGE, 2-D electrophoresis, platelet aggregation assay and enzyme activity assay.A purified disintegrin was a single chain glycoprotein with Mr of 13 kDа and рІ 4.7, whereas PLA2 had Mr of 14 kDa and pI of 4.17. A dose-dependent activity curve analysis shows that the platelet aggregation inhibitory activity of disintegrin and PLA2 in the rabbit plateletrich plasma were ID50 of 0.25 μМ and 0.65 μМ, respectively. Bioprocesses to produce and purify active antiplatelet agents from A. blomhoffi ussuriensis venom have been developed, using modern liquid chromatography matrices. Ongoing work to optimize large-scale production process is being undertaken.

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The aim of this study was to investigate the effectiveness of tetrapeptide pankragen-forte in older people with prediabetic state. A total of 150 healthy people of different ages, including 39 young people (20-39 years), 42 people. middle-aged (40-59 years) and 69 older adults (60 years and older). As a result of screening, 69 seniors were selected 12 patients with impaired glucose tolerance, which, for the correction of glucose metabolism used Pankragen-forte as follows: 500 mkg 2 times a day (morning and evening) for 4 weeks. All included surveyed was defined indicator of glycosylated hemoglobin, conducted a standard oral glucose tolerance test with determination of glucose, insulin in plasma glucose and venous blood at 30, 60 and 120 min after ingestion of 75 g glucose. Pro-insulin levels were determined in plasma, venous blood glucose and 30-min OGTT. Also calculated an index of insulin resistance (HOMA-IR), insulin sensitivity index (Matsuda index) and functional activity of ß cells of the pancreas (HOMA- %). In 50 percent older people with impaired glucose tolerance use of pankragen forte led to a significant decrease in the concentration of plasma glucose during a standard oral glucose tolerance test, which indicates the normalizing effect of peptide drug in the violation of carbohydrate metabolism, which is consistent with the improvement of the lipid composition of serum blood, endothelial function, decreased platelet aggregation. The use of peptide drug pankragen forte is promising correction prediabetic status in the elderly.

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Objective To investigate the changes and clinical significance of platelets aggregation rate of patients with coronary heart disease before and after percutaneous transluminal coronary stenting Methods There are 13 patients underwent coronary angiography and 20 patients underwent percutaneous transluminal coronary stenting Their blood samples were taken from the coronary sinus and coronary artery before and after interventional therapy Results The level of platelets aggregation rate in percutaneous transluminal coronary stenting group was higher after interventional therapy ( P

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In the control rabbits 6 h after smoke inhalation injury, the pulmonary vascular permeability and the circulating platelet aggregation percentage increased distinctly, pathological changes of trachea and lung were marked. In the other 2 groups, ginsenosides (25 mg/kg, iv) or ketoprofen (12 mg/kg, iv) were given 15 min after injury, 6 h after treatment, although both drugs decreased, the aggregation percentage of circulating platelets, only ginsenosides did alleviate the augmented pulmonary vascular permeabil ity and the pathological changes of trachea and lung. In the similar model of smoke inhalation injury in rats, ginsensi-des increased the PGI2 level in arterial blood and reduced the ratio of TXA2/ PGI2 1 h after treatment.

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S AIM To study the protective effects of trigonella foenum graecum(TFGs) on acute cerebral ischemia. METHODS Acute incomplete ischemia was induced by ligaturing bilateral carotid arterise and cutting heads in mice, the survival time and asthmatic time were observed; the coagulation time was measured by sheet glass method,and the blood viscosity was also assayed; the platelet aggregation induced by collagen were studied by turbdimetry in vitro . RESULTS TFGs prolonged survival time, coagulation time and asthmatic time significantly and inhibited platelet aggregation ratio in rabbit,and decreased the blood viscosity. CONCLUSION TFGs has the protective effects on acute cerebral ischemia.