Thrombus-formation Analysis System Research Articles

Response of human blood platelets to preparations from leaves of Paulownia Clon in Vitro 112

Paulownia Clon in Vitro 112, also called the Oxytree, is a fast-growing hybrid of two trees belonging to the Paulowniaceae family – P. elongata and P. fortunei. It demonstrates a wide range of biological effects (including antioxidant, anti-inflammatory, antibacterial, and neuroprotective) due to the high concentration of secondary metabolites. Our previous results showed an in vitro antioxidant and antiplatelet activity of the extract and four fractions (A–D) from the leaves of Paulownia Clon in Vitro 112 in human plasma and washed blood platelets. Here, we used a microchip flow chamber-based thrombus formation analysis system (T-TAS) and flow cytometry to assess the anticoagulant and antiplatelet activity of the extract and four fractions with different chemical content (A-D) from Paulownia Clon in Vitro 112 leaves in human whole blood. Two tested fractions: fraction C and D (at the concentrations of 5 and 50 μg/mL) inhibited the exposition of the active form of GPIIb/IIIa (integrin αIIbβ3) on the surface of blood platelets stimulated by ADP and collagen. The antiplatelet activity of fraction C is likely due to its high verbascoside content and the presence of apigenin’s derivatives. Fraction D contains triterpenoids, including ursolic, pomoleic, and maslinic acid, which could be responsible for decreased activation of ADP- and collagen-stimulated blood platelets. These results suggest that fractions C and D might be promising sources of phytochemicals with antiplatelet activity, which are important for prophylaxis and treatment of cardiovascular diseases associated with hyperactivation of blood platelets. However, further research is needed to ascertain which exact compounds and mechanisms are responsible for this phenomenon.

Antiplatelet and Anticoagulant Activity of Isorhamnetin and Its Derivatives Isolated from Sea Buckthorn Berries, Measured in Whole Blood.

Blood platelets play a crucial role in hemostasis, the process responsible for keeping blood flowing in the circulatory system. However, unnecessary platelet activation can lead to aggregation at the site of atherosclerotic plaque rapture and the formation of a thrombus, which promotes atherothrombotic diseases. Various dietary components, such as phenolic compounds, are known to demonstrate antiplatelet and anticoagulant properties, and it is possible that these could form an important element in the prophylaxis and therapy of cardiovascular diseases. Our present study examined the biological activity of isorhamnetin (1) and two isorhamnetin derivatives, (2): 3-O-beta-glucoside-7-O-alpha-rhamnoside and (3): 3-O-beta-glucoside-7-O-alpha-(3″′-isovaleryl)-rhamnoside, isolated from the phenolic fraction of sea buckthorn fruit, against human washed blood platelets and human whole blood in vitro. The anti-platelet and anticoagulant potential was determined using (A) flow cytometry, (B) the thrombus-formation analysis system (T-TAS) and (C) colorimetry. The results of the T-TAS test indicate that the AUC10 (Area Under the Curve) of the tested phenolic compounds (compounds 1, 2 and 3; 50 µg/mL) was markedly reduced compared to the control values. Moreover, flavonol demonstrated anti-platelet potential, including anti-adhesive activity, with these effects being more intense in compound 2 than isorhamnetin. Different actions of flavonol on platelet activation may depend on their binding ability to various receptors on blood platelets. However, the mechanism of their anti-platelet potential requires further additional studies, including in vitro and in vivo experiments.

Qualitative and Quantitative Analysis of Secondary Metabolites in Morphological Parts of Paulownia Clon In Vitro 112® and Their Anticoagulant Properties in Whole Human Blood.

It is not easy to find data in the scientific literature on the quantitative content of individual phytochemicals. It is possible to find groups of compounds and even individual compounds rather easily, but it is not known what their concentration is in cultivated or wild plants. Therefore, the subject of this study was to determine the content of individual compounds in the new Paulownia species, Oxytree, developed in a biotechnology laboratory in 2008 at La Mancha University in Spain. Six secondary metabolites were isolated, and their chemical structure was confirmed by spectral methods. An analytical method was developed, which was then used to determine the content of individual compounds in leaves, twigs, flowers and fruits of Paulownia Clon in Vitro 112®. No flavonoids were found in twigs and fruits of Oxytree, while the highest phenylethanoid glycosides were found in twigs. In this study, we also focused on biological properties (anticoagulant or procoagulant) of extract and four fractions (A–D) of different chemical composition from Paulownia Clon in Vitro 112 leaves using whole human blood. These properties were determined based on the thrombus-formation analysis system (T-TAS), which imitates in vivo conditions to assess whole blood thrombogenecity. We observed that three fractions (A, C and D) from leaves decrease AUC10 measured by T-TAS. In addition, fraction D rich in triterpenoids showed the strongest anticoagulant activity. However, in order to clarify the exact mechanism of action of the active substances present in this plant, studies closer to physiological conditions, i.e., in vivo studies, should be performed, which will also allow to determine the effects of their long-term effects.

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.

Influence of high-dose antithrombin on platelet function and blood coagulation.

AimIn healthy adults, there are sufficient amounts of antithrombin in the blood to regulate thrombin. However, the effects of high concentrations of antithrombin on dose‐dependent anticoagulation and platelet function have not been reported. In this study, we assessed platelet function and blood coagulation following high‐dose antithrombin supplementation in vitro.MethodsBlood samples were collected from 10 healthy volunteers, and samples with different antithrombin concentrations were prepared by adding an antithrombin agent (Neuart). Blood coagulation was assessed by the Thrombus‐Formation Analysis System (T‐TAS) and Rotational Thromboelastometry (ROTEM) using whole blood samples.ResultsThe data obtained by the platelet chip, exclusively representing platelet function, revealed that the onset of thrombus formation was significantly delayed in a dose‐dependent manner (100%–200%, P = 0.021; 100%–500%, P = 0.011; 200%–500%, P = 0.047). In measurements using the atheroma chip, which enables assessment of blood coagulation, the thrombus formation ability was found to be reduced (100%–200%, P = 0.022; 100%–500%, P = 0.05). In the ROTEM measurements, clotting time was prolonged in a dose‐dependent manner (100%–200%: P = 0.203, 200%–500%: P = 0.005, 500%–1000%: P = 0.022), except when comparing with 100% and 200%. Although antithrombin is reportedly saturated in healthy blood, its anticoagulant ability appears to be enhanced depending on its concentration. Furthermore, data obtained from the platelet chip showed that antithrombin might reduce platelet function.ConclusionsAntithrombin suppressed platelet function and blood coagulation in a dose‐dependent manner.

Abstract 12614: Total Thrombus Formation Analysis System Can Predict High Bleeding Risk in Coronary Artery Disease Patients Undergoing Percutaneous Coronary Intervention

Introduction: Antithrombotic therapy is established for the treatment in various cardiovascular events, however, it has shown to increase the bleeding risk. Total Thrombus-formation Analysis System (T-TAS) is reported to be useful for evaluating thrombogenicity. Hypothesis: We examined whether T-TAS might predict 1-year bleeding risk in patients undergoing percutaneous coronary intervention (PCI). Methods: This was a retrospective, observational study at Kumamoto University Hospital between April 2017 and March 2019. Blood samples obtained on the day of PCI were used in T-TAS to compute the thrombus formation area under the curve (AUC) (AR10-AUC30, AUC for AR chip). We divided the study population into 2 groups according to the Academic Research Consortium for High Bleeding Risk (ARC-HBR) (182 patients in ARC-HBR positive, 118 in ARC-HBR negative). The primary endpoint was 1-year bleeding events that were defined by Bleeding Academic Research Consortium type2, 3, or 5. Results: The AR10-AUC30 levels were significantly lower in the ARC-HBR positive group than in the ARC-HBR negative group (median [interquartile range] 1568.1 [1258.5-1744.1] vs. 1723.1 [1567.0-1799.5], p<0.001). The combination of ARC-HBR and AR10-AUC30 could discriminate the bleeding risk, and improved predictive capacity compared with ARC-HBR by c-statistics and integrated discrimination improvement. In multivariate Cox hazards analyses, combining ARC-HBR and lower AR10-AUC30 levels were significantly associated with 1-year bleeding events. Decision curve analysis revealed that combining AR10-AUC30 with ARC-HBR ameliorated risk-prediction of bleeding events. Conclusions: The results highlighted that AR10-AUC30 could be a potentially useful marker for predicting high bleeding risk in patients undergoing PCI.

高用量アンチトロンビン投与が血小板機能と凝固機能に及ぼす影響(The influence of a high dose antithrombin on the platelet function and blood coagulability)

要旨【目的】KyberSept trialのサブグループ解析では，アンチトロンビン高用量投与群はプラセボ群に比べて有意に死亡率が低かった。しかし，高用量アンチトロンビン投与による，凝固系への影響に関しては詳しく知られていない。今回アンチトロンビン高濃度検体の凝固特性について調査を行った。【対象】健常者10人より血液検体を採取し，アンチトロンビン製剤を添加することでアンチトロンビン濃度が異なる検体を作成した。凝固特性の調査には生体内での反応に近づけるため，全血を利用するT–TAS®およびROTEM®を使用した。【結果】血小板機能を反映するPL測定では，血栓閉塞開始時間はアンチトロンビン濃度に依存し有意差をもって延長した。AR測定でも同様に，血栓閉塞開始時間はアンチトロンビン濃度に依存し延長した。また血栓閉塞時間もアンチトロンビン濃度に依存し延長した。健常検体内では十分量存在し飽和状態と考えられていたアンチトロンビンであるが，高用量投与された場合には抗凝固作用が増強することが証明された。さらに，T–TAS®を用いたPL測定を行うことで，血小板血栓形成能にも抑制効果が認められることが証明された。【結語】アンチトロンビンは濃度依存性に抗凝固作用を発揮する。また血小板粘着能に対しても影響を及ぼしており，濃度依存性に血小板血栓形成能を抑制する。

Influence of hyperbaric oxygen therapy on thrombus formation ability in humans

Background: Hyperbaric oxygen (HBO2) therapy was introduced nearly 300 years ago. However, its effect on thrombus formation is unclear. This may be because platelet and coagulation functions are unstable, yielding variable results; hence, accurate measurement is difficult. Our study aimed to analyze changes in thrombus formation before and after HBO2 therapy by using a total thrombus formation analysis system (T-TAS). Methods: Six patients were prescribed HBO2 therapy for skin and soft tissue ulcers, and necrotic fasciitis. Blood samples were collected immediately before and after treatment. Then samples were put into a reservoir that connected to AR-chip to assess changes in the thrombus formation ability of both platelets and coagulation factors. We examined the differences in the thrombus formation ability using T-TAS. Time until the onset of white thrombus formation (T10) and complete occlusion of the capillary (T80) were analyzed by a two-way repeated measure analysis of variance (ANOVA). Results: The duration to pressure increase of samples after HBO2 therapy was longer than the duration before HBO2 therapy (p<0.05). This suggests decreased clot adhesiveness to the inner surface of the simulated blood vessel and reduced clot formation ability. Conclusions: The results for T10 and T80 suggest that HBO2 therapy reduced thrombus formation ability in the enrolled patients. We believe that T-TAS is a promising method to predict the efficacy of HBO2 therapy.