• 대한의생명과학회지
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• 제27권3호
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• pp.170-176
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• 2021

Thapsigargin (TG), a sarco/endoplasmic reticulum (ER) Ca²⁺-ATPase (SERCA) inhibitor, has been widely used as an agonist for platelet aggregation for decades. In this study, we investigated the effect of TG on the release of lactate dehydrogenase (LDH) for platelets and elucidated its mechanism. Platelet LDH release and platelet aggregation were increased by TG treatment; 1,000 nM of TG induced the complete lysis of platelets. Other agonists such as collagen (2.5 ㎍/mL), thrombin (0.1 U/mL), and ADP (10 mM) did not induce significant platelet LDH release despite platelet aggregation. Finally, we investigated the effects of pharmacological inhibitors on TG-induced platelet aggregation and LDH release. SP600125, a JNK inhibitor, and LY294002, a PI-3K inhibitor, inhibited TG-induced platelet LDH release but not platelet aggregation. Forskolin, an adenylyl cyclase activator, also inhibited LDH release without affecting platelet aggregation by TG. These results suggest that the TG-induced platelet aggregation was accompanied by LDH release but regulated by a different signaling pathway.

• Journal of Microbiology and Biotechnology
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• 제21권10호
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• pp.1053-1056
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• 2011

Optimal conditions for a high cell density fermentation were investigated in a recombinant Escherichia coli producing salmosin, a platelet aggregation inhibitor. The optimized carbon and nitrogen sources were glycerol 10 g/l, yeast extract 30 g/l, and bacto-tryptone 10 g/l, yielding the dry cell weight (DCW) of 10.61 g/l in a 500 ml flask culture. The late-stage induction with 1% L-arabinose in a 5 l jar fermentor showed the highest DCW of 65.70 g/l after 27 h of the fed-batch fermentation. Around 2,200 mg/l of the protein was expressed as an inclusion body that was then refolded to obtain the active salmosin of 96 mg/l. We also confirmed the inhibitory activity against platelet aggregation of the active salmosin from the high cell density fermentation.

• Preventive Nutrition and Food Science
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• 제12권3호
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• pp.141-147
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• 2007

Cordycepin (3'-deoxyadenosine), which comes from Cordyceps militaris, the Chinese medicinal fungal genus Cordyceps, is used in the treatment of various diseases such as cancer and chronic inflammation. We recently reported that cordycepin has a novel antiplatelet effect through the down regulation of $[Ca^{2+}]_{i}$ and the elevation of cGMP/cAMP production. In this study, we further investigated the effect of cordycepin on collagen-induced platelet aggregation in the presence of cGMP-dependent protein kinase (PKG)- or cAMP-dependent protein kinase (PKA)-inhibitor. PKG inhibitor Rp-8-pCPT-cGMPS potentiated the collagen-induced platelet aggregation, but PKA inhibitor Rp-8-Br-cAMPS did not. However, both Rp-8-pCPT-cGMPS and Rp-8-Br-cAMPS reduced inhibition by cordycepin of collagen-induced platelet aggregation. Moreover, cordycepin inhibited $Ca^{2+}-dependent$ phosphorylation of both 47 kDa- and 20 kDa-protein in the presence of both PKG inhibitor and PKA inhibitor. Taken altogether, these results suggest that the inhibitory effect of cordycepin on collagen-induced platelet aggregation is associated with cGMP/PKG- and cAMP/PKA-pathways, and thus cordycepin may be an efficacious intervention against platelet aggregation-mediated thrombotic disease.

• 대한의생명과학회지
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• 제20권3호
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• pp.129-138
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• 2014

In this study, we investigated the effect of water extract from rice bran (RB) on ADP ($20{\mu}M$)-stimulated platelet aggregation. RB dose-dependently inhibited ADP-induced platelet aggregation, and its $IC_{50}$ value was $224.0{\mu}g/mL$, which was increased by adenylate cyclase inhibitor SQ22536 and cAMP-dependent protein kinase (A-kinase) inhibitor Rp-8-Br-cAMPS. RB elevated the phosphorylation of VASP ($Ser^{157}$) which was also inhibited by SQ22536 and Rp-8-Br-cAMPS. It is thought that RB-elevated cAMP contributed to the phosphorylation of VASP ($Ser^{157}$) to inhibit ADP-induced platelet aggregation. Therefore, we demonstrate that RB has an antiplatelet effect via cAMP-dependent phosphorylation of VASP ($Ser^{157}$), and RB may have preventive or therapeutic potential for platelet aggregation-mediated diseases, such as thrombosis, myocardial infarction, atherosclerosis, and ischemic cerebrovascular disease.

• 대한의생명과학회지
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• 제21권2호
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• pp.103-114
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• 2015

In this study, we investigated the effect of rice bran water extract fermented with Lactobacillus plantarum KCCM-12116 (RBLp) on ADP ($20{\mu}M$)-, collagen ($10{\mu}g/mL$)-, and thrombin (0.2 U/mL)-stimulated platelet aggregation. RBLp dose-dependently inhibited ADP-, collagen-, and thrombin-induced platelet aggregation, with $IC_{50}$ values of 501.1, 637.2, and > $2,000{\mu}g/mL$, respectively. The platelet aggregation induced by ADP plus RBLp ($750{\mu}g/mL$) was increased by the adenylate cyclase inhibitor, SQ22536, and the cAMP-dependent protein kinase (A-kinase) inhibitor, Rp-8-Br-cAMPS. Treatment with RBLp increased the phosphorylation of VASP ($Ser^{157}$), an A-kinase substrate, which was also inhibited by SQ22536 and Rp-8-Br-cAMPS. It is thought that the RBLp-induced increases in cAMP contributed to the phosphorylation of VASP ($Ser^{157}$), which in turn resulted in an inhibition of ADP-induced platelet aggregation, thereby indicating that RBLp has an antiplatelet effect via cAMP-dependent phosphorylation of VASP ($Ser^{157}$). Thus, RBLp may have therapeutic potential for the treatment (or prevention) of platelet aggregation-mediated diseases, such as thrombosis, myocardial infarction, atherosclerosis, and ischemic cerebrovascular disease.

• 한국식품위생안전성학회:학술대회논문집
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• 한국식품위생안전성학회 2004년도 제3회 식품안전의 날 기념 학술 세미나
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• pp.231-231
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• 2004

• Food Science and Biotechnology
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• 제16권2호
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• pp.218-222
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• 2007

The anticoagulant properties of Cinnamomum cassia bark-derived materials were evaluated against platelet aggregation induced by arachidonic acid (AA), collagen, platelet activating factor (PAF), or thrombin, and these effects were then compared to those of three commercially available compounds (cinnamic acid, cinnamyl alcohol, and aspirin). The active constituent obtained from C. cassia barks was isolated by silica gel column chromatography and high pressure liquid chromatography (HPLC), and was characterized as trans-cinnamaldehyde by MS, $^1H-NMR$, $^{13}C-NMR$, and IR spectroscopy. With regard to 50% inhibitory concentration ($IC_{50}$) values, cinnamaldehyde was found to effectively inhibit platelet aggregation induced by AA ($IC_{50},\;43.2\;{\mu}M$) and collagen ($IC_{50},\;3.1\;{\mu}M$). By way of comparison, cinnamaldehyde proved to be a significantly more potent platelet inhibitor against platelet aggregation induced by collagen than aspirin. The effect exerted by cinnamaldehyde against platelet aggregation induced by AA was 1.2 times less than that of aspirin. These results indicate that cinnamaldehyde may prove useful as a lead compound for the inhibition of platelet aggregation induced by AA and collagen.

• 한국식품위생안전성학회지
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• 제19권3호
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• pp.161-166
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• 2004

We investigated the antiplatelet effect of a newly synthesized guanidine derivative KR-32558, a sodium-hydrogen exchanger-1 (NHE-1) inhibitor, together with the elucidation of the possible mechanisms of action. KR-32558 concentration -dependently inhibited the aggregation of washed rabbit platelets induced by collagen (10 ${\mu}g/ml$) with an $IC_{50}$ value of 85.9 ${\mu}M$, but with much weaker potency against aggregation induced by thapsigargin (0.5 ${\mu}M$) or A23187 (5 ${\mu}M$). And had no effect on platelet aggregation induced by arachidonic acid (100 ${\mu}M$), thrombin (0.05 U/ml) and U46619 (1 ${\mu}M$) up to 100 ${\mu}M$. KR-32558 completely inhibited the $[Ca^{2+}]_i$ mobilization induced by collagen at concentration of 100${\mu}iM$. Taken together, these observation suggest that KR-32558 selectively inhibited collagen-mediated platelet aggregation by blocking the cytoplasmic calcium mobilization in addition to NHE-1 inhibition.

• BMB Reports
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• 제40권5호
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• pp.678-683
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• 2007

Extracts from the leaves of the Ginkgo biloba are becoming increasingly popular as a treatment that is claimed to reduce atherosclerosis, coronary artery disease, and thrombosis. In this study, the effect of ginkgolide B (GB) from Ginkgo biloba leaves in collagen (10 ${\mu}g/ml$)-stimulated platelet aggregation was investigated. It has been known that human platelets release matrix metallo-proteinase-9 (MMP-9), and that it significantly inhibited platelet aggregation stimulated by collagen. Zymographic analysis confirmed that pro-MMP-9 (92-kDa) was activated by GB to form an MMP-9 (86-kDa) on gelatinolytic activities. And then, activated MMP-9 by GB dose-dependently inhibited platelet aggregation, intracellular $Ca^{2+}$ mobilization, and thromboxane $A_2$ ($TXA_2$) formation in collagen-stimulated platelets. Activated MMP-9 by GB directly affects down-regulations of cyclooxygenase-1 (COX-1) or $TXA_2$ synthase in a cell free system. In addition, activated MMP-9 significantly increased the formation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which have the anti-platelet function in resting and collagen-stimulated platelets. Therefore, we suggest that activated MMP-9 by GB may increase the intracellular cAMP and cGMP production, inhibit the intracellular $Ca^{2+}$ mobilization and $TXA_2$ production, thereby leading to inhibition of platelet aggregation. These results strongly indicate that activated MMP-9 is a potent inhibitor of collagen-stimulated platelet aggregation. It may act a crucial role as a negative regulator during platelet activation.

• 대한의생명과학회지
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• 제10권1호
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• pp.1-8
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• 2004

Cordycepin separated from Cordyceps militaris is a major physiologic active component in Cordyceps militaris. The platelet aggregation is stimulated by $Ca^{2+}$, which is either mobilized from intracellular endoplasmic reticulum or transported from extracellular space. cGMP antagonizes the actions of $Ca^{2+}$. Based on these facts, we have investigated the effects of cordycepin on the mobilization of $Ca^{2+}$ and the production of cGMP on collagen ($10\mu$g/ml)-induced human platelet aggregation. Cordycepin potently stimulated the human platelet aggregation induced by collagen ($10\mu$g/ml) in a dose-dependent manner. Cordycepin (500 $\mu$M) inhibited also the collagen-induced human platelet aggregation in the presence both 1 mM and 2 mM of $CaCl_2$. These are in accord with the results that cordycepin inhibited the $Ca^{2+}$- influx on collagen-induced human platelet aggregation. These results suggest that cordycepin decrease the intracellular $Ca^{2+}$ concentration to inhibit collagen-induced human platelet aggregation. Besides, cordycepin increased the level of cGMP on collagen-induced human platelet aggregation. This result is related with the decrease of intracellular $Ca^{2+}$ concentration, because cGMP inhibits the mobilization of $Ca^{2+}$. In addition, cordycepin inhibited the human platelet aggregation induced by LY -83583, inhibitor of guanylate cyclase. This result suggested that cordycepin inhibit the platelet aggregation by stimulating the activity of guanylate cyclase. In conclusion, we demonstrated that cordycepin might have the antiplatelet function by inhibiting $Ca^{2+}$-mobilization via the stimulation of the production of cGMP.