• 한국식품위생안전성학회지
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• 제9권1호
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• pp.23-30
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• 1994

When three systems commonly used for screening(washed platelets suspended in uffer containing Ca2+, washed platelets in buffer without Ca2+and platelet rich plasma) are compared by studying the anti-aggregating capacity of garlic extract, platelet rich plasma was the least sensitive system. The most sensitive assay system, based on the IC50s of garlic extract and 2 other platelet aggregation-inhibiting agents(vitamin K3 and propranolol), was the platelet preparation without Ca2+in the suspension buffer. This system was confirmed as the most sensitive during subsequent investigation of garlic extract's capacity to inhibit platelet ATP release. These results suggest that applying the system with washed platelet without Ca2+is most effective to screen for the anti-affregating capacity of natural food.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1994년도 춘계학술대회 and 제3회 신약개발 연구발표회
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• pp.280-280
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• 1994

혈행 장애 예방 및 치료 약물의 개발을 목적으로 혈소판 기능에 대한 약물의 영향을 연구하는데 있어, 효율적인 실험 조건의 선택은 중요하다. 본 연구에서는 마늘 메탄을 추출물의 혈소판 응집 억제능율 platelet rich plasma(PRP), washed platelet(Ca$^2$비첨가), washed platelet(Ca$^{2+}$ 첨가) 등 세가지 분리 조건을 사용하여 실험하였다. 그 결과 마늘 추출물의 $IC_{50}$/이 각각 2348 $\mu\textrm{g}$/ml, 221 $\mu\textrm{g}$/ml, 851 $\mu\textrm{g}$/ml로서, washed platelet(Ca$^{2+}$ 비첨가)가 마늘 추출물의 혈소판 응집 억제 작용에 가장 민감한 것으로 나타났다. 또한 마늘 추출물과는 다른 기전으로 혈소판 응집 억제 효과를 나타내는 menadione과 propranolol도 이와 동일한 결과를 보였다. 실험 조건에 따른 이러한 차이가 PRP 중에 존재하는 알부민 등과의 단백 결합에의한 약물의 유효농도 감소에 기인한다고 추정되어, washed platelet에 여러 농도의 알부민을 첨가하여 혈소판 응집 억제 실험을 실시한 결과 약물의 $IC_{50}$/은 알부민 농도 의존적으로 증가하였다. 혈소판 응집과 함께 혈소판 기능 변화를 측정하는 지표인 혈소판 분비능을 분비된 ATP를 Luciferin-Luciferase로 측정하여 알아보았다. 그 결과 thrombin에 의한 혈소판의 ATP 유리에 미치는 마늘 추출물의 영향을 위의 세 실험조건에서 실험하여 $IC_{50}$/을 비교한 결과 그 값이 washed platelet에서 가장 낮고 PRP에서 가장 높아, 혈소판 응집 억제 실험과 동일한 결과를 나타내었다. 이상의 결과는 세가지 혈소판 분리 조건 중 washed platelet(Ca$^{2+}$비첨가)가 약물에 의한 혈소판 기능 억제 실험에 가장 효율적임을 제시하고 있으며, 그 주요한 원인은 알부민 등 단백 결합에 의한 유효 약물 농도 감소때문 이라고 추정된다.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1993년도 제2회 신약개발 연구발표회 초록집
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• pp.152-152
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• 1993

혈전 등에 의한 혈행 장애 예방 및 치료 약물의 개발을 목적으로 혈소판 기능에 대한 약물의 영향을 연구하는데 있어, 효율적인 실험 조건의 선택은 중요하다. 본 연구에서는 마늘 메탄올 추출물의 혈소판 응집 억제능을 platelet rich plasma(PRP), washed platelet (Ca$^2$비첨가), washed platelet(Ca$_{2＋}$ 첨가) 등 세가지 분리 조건을 사용하여 실험하였다. 그 결과 마늘 추출물의 $IC_{50}$/이 각각 2348 $\mu\textrm{g}$/ml, 221 $\mu\textrm{g}$/ml, 851 $\mu\textrm{g}$/ml로서, washed platelet(Ca$_{2＋}$ 비첨가)가 마늘 추출물의 혈소판 응집 억제 작용에 가장 민감한 것으로 나타났다. 또한 마늘 추출물과는 다른기전으로 혈소판 응집 억제 효과를 나타내는 menadione과 propranolol도 이와 동일한 결과를 보였다. 실험 조건에 따른 이러한 차이가 PRP 중에 존재하는 알부민 등과의 단백 결합에 의한 약물의 유효농도 감소에 기인한다고 추정되어, washed platelet에 여러 농도의 알부민을 첨가하여 혈소판 응집 억제 실험을 실시한 결과 약물의 $IC_{50}$/은 알부민 농도 의존적으로 증가하였다. 혈소판 응집과 함께 혈소판 기능 변화를 측정하는 지표인 혈소판 분비능을 분비된 ATP를 Luciferin-Luciferase로 측정하여 알아보았다. 그 결과 thrombin에 의한 혈소판의 ATP 유리에 미치는 마늘 추출물의 영향을 위의 세 실험 조건에서 실험하여 $IC_{50}$/을 비교한 결과 그 값이 washed platelet에서 가장 낮고 PRP에서 가장 높아, 혈소판 응집 억제 실험과 동일한 결과를 나타내었다. 이상의 결과는 세가지 혈소판 분리 조건 중 washed platelet(Ca$_{2＋}$ 비첨가)가 약물에 의한 혈소판 기능 억제실험에 가장 효율적임을 제시하고 있으며, 그 주요한 원인은 알부민 등 단백 결합에 의한 유효 약물 농도 감소 때문이라고 추정된다.

• Journal of Ginseng Research
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• 제47권5호
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• pp.638-644
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• 2023

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

• 생약학회지
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• 제52권3호
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• pp.127-133
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• 2021

Platelet activation plays a major role in cardiovascular disorders (CVDs). Thus, disrupting platelet activation represents an attractive therapeutic target on CVDs. Esculetin, a bioactive 6,7-dihydroxy derivative of coumarin, possesses pharmacological activities against obesity, diabetes, renal failure, and CVDs. In other report, the effect of esculetin has been examined in human platelet activation and experimental mouse models, and 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, and its mechanism is not also clearly known. This study investigated the effect of esculetin on collagen-induced human platelet aggregation, and we clarified the mechanism. Esuletin has effects on the down regulation of PI3K/Akt and MAPK, phosphoproteins that act in the signaling process in platelet aggregation. The effects of esculetin reduced of TXA₂ production and phospholipase A₂ activation, and intracellular granule secretion including ATP and serotonin, leading to inhibit platelet aggregation. These results clearly clarified the effect of esculetin in inhibiting platelet activity and thrombus formation in humans.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1998년도 Proceedings of UNESCO-internetwork Cooperative Regional Seminar and Workshop on Bioassay Guided Isolation of Bioactive Substances from Natural Products and Microbial Products
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• pp.177-177
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• 1998

In the previous report, tetrandrine (TET) and fangchinoline (FAN) showed antithrombotic and antiplatelet aggregation activities. The present study was undertaken to investigate the effects of tetrandrine and fangchinoline on human platelet aggregation, formation of thromboxane B$_2$ and coagulation of platelet poor plasma. TET and FAN inhibited platelet activating factor (PAF) induced human platelet aggregation, but didn't inhibit the specific binding of PAF to its receptor. Meanwhile, TET and FAN also inhibited PAF, thrombin and arachidonic acid induced thromboxane B$_2$ formation in human washed platelets. In addition, neither TET nor FAN showed any anticoagulation activities in the measurement of the activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) using human platelet poor plasma. These results suggest that antithrombotic effects of TET and FAN in mice may be mainly related to the antiplatelet aggregation activities, and the antiplatelet aggregation effects may be related to the intracellular messenger system such as TXA$_2$ formation etc., but not to the binding of PAF to PAF-receptor on the platelet membrane directly.

• Toxicological Research
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• 제16권4호
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• pp.311-315
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• 2000

Previous studies showed that benzoquinone derivatives inhibited platelet aggregation. but there is no information available on their cytotoxicity to platelets. 1n the present study. washed platelets isolated from rats were treated with 1.4-benzoquinone. a representative benzoquinone derivative. to examine its antiaggregating effect and cytotoxicity. 1.4-Benzoquinone significantly inhibited thrombin-induced platelet aggregation. Consistent with this finding. 1.4-benzoquinone suppressed cytosolic calcium increase induced by thrombin. To examine the cytotoxicity by 1 A-benzoquinone in platelets. turbidometry and lactate dehydrogenase release were measured. Treatment with 1.4-benzoquinone resulted in slight cytotoxicity (30% release at 60 min) to platelets. However. the cytotoxicity was not correlated with increase of cytosolic calcium levels in platelets. All these data suggested that 1.4-benzoquinone inhibited thrombin-induced platelet aggregation mediated by inhibition elf calcium level increase and that 1.4-benzoquinone reveals cytotoxicity to some extent without alteration of calcium level in platelets.

• 약학회지
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• 제56권6호
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• pp.382-389
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• 2012

Effects of citrate-capped silver nanoparticles (AgNPs) on the blood coagulation and platelet aggregation were investigated using whole blood, platelet rich plasma (PRP) and washed platelet obtained from SD male rats. To confirm the stability of AgNPs in the test, size distribution of the nanoparticles was measured in the vehicles including distilled water, serum, and platelet buffers. The average size of AgNPs was 20 nm in the vehicles, which means that the stability was maintained during the whole experimental period. When blood coagulation was monitored by using whole blood impedance aggregometer, coagulation was not observed at the concentration of 1, 10 and 50 ppm. Platelets in plasma or in buffer were not aggregated by AgNPs at the concentration of 1, 2 and 4 ppm, respectively. The test concentration of AgNPs could not be increased because the dark color of the nanoparticles impeded the transmission of light, which is an indicator of aggregation. Although the blood or platelets were pre-activated by collagen, thrombin, or ADP with sub-threshold level, aggregation was not observed at the test concentration. Microscopic observation also supported the result obtained by the aggregometer.

• Journal of Applied Biological Chemistry
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• 제64권3호
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• pp.317-322
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• 2021

Among the different cardiovascular disorders (CVDs), the activation of platelets is a necessary step. Based on this knowledge, therapeutic treatments for CVDs that target the disruption of platelet activation are proving to be worthwhile. One such substance, a bioactive 6,7-dihydroxy derived from coumarin, is 6,7-Dihydroxy-2H-1-benzopyran-2-one (esculetin). This compound has demonstrated several pharmacological effects on CVDS as well as various other disorders including diabetes, obesity, and renal failure. In various reports, esculetin and its effect has been explored in experimental mouse models, human platelet activation, esculetin-inhibited collagen, and washed human platelets exhibiting aggregation via arachidonic acid. Yet, esculetin affected aggregation with agonists like U46619 or thrombin in no way. This study investigated esculetin and how it affected human platelet aggregation activated through U46619. Ultimately, we confirmed that esculetin had an effect on the aggregation of human platelets when induced from U46619 and clarified the mechanism. Esculetin interacts with the downregulation of both phosphoinositide 3-kinase/Akt and mitogen-activated protein kinases, important phosphoproteins that are involved in activating platelets and their signaling process. The effects of esculetin reduced TXA₂ production, phospholipase A₂ activation, and platelet secretion of intracellular granules (ATP/serotonin), ultimately causing inhibition of overall platelet aggregation. These results clearly define the effect of esculetin in inhibiting platelet activity and thrombus formation in humans.

• Archives of Pharmacal Research
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• 제29권5호
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• pp.375-383
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• 2006

The anti platelet effects of a novel guanidine derivative, KR-32570 ([5-(2-methoxy-5-chlorophenyl) furan-2-ylcarbonyl]guanidine), were investigated with an emphasis on the mechanisms underlying its inhibition of collagen-induced platelet aggregation. KR-32570 significantly inhibited the aggregation of washed rabbit platelets induced by collagen $(10{\mu}g/mL)$, thrombin (0.05 U/mL), arachidonic acid $(100{\mu}M)$, a thromboxane (TX) $A_2$ mimetic agent U46619 (9,11-dideoxy-9,11-methanoepoxy-prostaglandin $F_2,\;1{\mu}M$) and a $Ca^{2+}$ ATPase inhibitor thapsigargin $(0.5{\mu}M)$ ($IC_{50}$ values: $13.8{\pm}1.8,\;26.3{\pm}1.2,\;8.5{\pm}0.9,\;4.3{\pm}1.7\;and\;49.8{\pm}1.4{\mu}M$, respectively). KR-32570 inhibited the collagen-induced liberation of $[^3H]$arachidonic acid from the platelets in a concentration dependent manner with complete inhibition being observed at $50{\mu}M$. The $TXA_2$ synthase assay showed that KR-32570 also inhibited the conversion of the substrate $PGH_2$ to $TXB_2$ at all concentrations. Furthermore, KR-32570 significantly inhibited the $[Ca^{2+}]_i$ mobilization induced by collagen at $50{\mu}M$, which is the concentration that completely inhibits platelet aggregation. KR-32570 also decreased the level of collagen $(10{\mu}g/mL)$induced secretion of serotonin from the dense-granule contents of platelets, and inhibited the NHE-1-mediated rabbit platelet swelling induced by intracellular acidification. These results suggest that the antiplatelet activity of KR-32570 against collagen-induced platelet aggregation is mediated mainly by inhibiting the release of arachidonic acid, $TXA_2$ synthase, the mobilization of cytosolic $Ca^{2+}$ and NHE-1.