Matrine Impairs Platelet Function and Thrombosis and Inhibits ROS Production

Matrine is a naturally occurring alkaloid and possesses a wide range of pharmacological properties, such as anti-cancer, anti-oxidant, anti-inflammatory effects. However, whether it affects platelet function and thrombosis remains unclear. This study aims to evaluate the effect of matrine on platelet function and thrombus formation. Human platelets were treated with matrine (0–1 mg/ml) for 1 h at 37°C followed by measuring platelet aggregation, granule secretion, receptor expression by flow cytometry, spreading and clot retraction. In addition, matrine (10 mg/kg) was injected intraperitoneally into mice to measure tail bleeding time, arterial and venous thrombus formation. Matrine dose-dependently inhibited platelet aggregation and ATP release in response to either collagen-related peptide (Collagen-related peptide, 0.1 μg/ml) or thrombin (0.04 U/mL) stimulation without altering the expression of P-selectin, glycoprotein Ibα, GPVI, or αIIbβ3. In addition, matrine-treated platelets presented significantly decreased spreading on fibrinogen or collagen and clot retraction along with reduced phosphorylation of c-Src. Moreover, matrine administration significantly impaired the in vivo hemostatic function of platelets, arterial and venous thrombus formation. Furthermore, in platelets stimulated with CRP or thrombin, matrine significantly reduced Reactive oxygen species generation, inhibited the phosphorylation level of ERK1/2 (Thr202/Tyr204), p38 (Thr180/Tyr182) and AKT (Thr308/Ser473) as well as increased VASP phosphorylation (Ser239) and intracellular cGMP level. In conclusion, matrine inhibits platelet function, arterial and venous thrombosis, possibly involving inhibition of ROS generation, suggesting that matrine might be used as an antiplatelet agent for treating thrombotic or cardiovascular diseases.

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Effect of alteplase on platelet function and receptor expression

Journal of International Medical Research ◽

10.1177/0300060519829991 ◽

2019 ◽

Vol 47 (4) ◽

pp. 1731-1739 ◽

Cited By ~ 1

Author(s):

Jun Lu ◽

Peng Hu ◽

Guangyu Wei ◽

Qi Luo ◽

Jianlin Qiao ◽

...

Keyword(s):

Arachidonic Acid ◽

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Function ◽

Adenosine Diphosphate ◽

Receptor Expression ◽

Light Transmittance ◽

Dependent Manner ◽

Clot Retraction ◽

Platelet Receptors

Objective To investigate the role of alteplase, a widely-used thrombolytic drug, in platelet function. Methods Human platelets were incubated with different concentrations of alteplase followed by analysis of platelet aggregation in response to adenosine diphosphate (ADP), collagen, ristocetin, arachidonic acid or epinephrine using light transmittance aggregometry. Platelet activation and surface levels of platelet receptors GPIbα, GPVI and αIIbβ3 were analysed using flow cytometry. The effect of alteplase on clot retraction was also examined. Results This study demonstrated that alteplase significantly inhibited platelet aggregation in response to ADP, collagen and epinephrine in a dose-dependent manner, but it did not affect ristocetin- or arachidonic acid-induced platelet aggregation. Alteplase did not affect platelet activation as demonstrated by no differences in P-selectin levels and PAC-1 binding being observed in collagen-stimulated platelets after alteplase treatment compared with vehicle. There were no changes in the surface levels of the platelet receptors GPIbα, GPVI and αIIbβ3 in alteplase-treated platelets. Alteplase treatment reduced thrombin-mediated clot retraction. Conclusions Alteplase inhibits platelet aggregation and clot retraction without affecting platelet activation and surface receptor levels.

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Naringenin Inhibits Platelet Activation and Arterial Thrombosis Through Inhibition of Phosphoinositide 3-Kinase and Cyclic Nucleotide Signaling

Frontiers in Pharmacology ◽

10.3389/fphar.2021.722257 ◽

2021 ◽

Vol 12 ◽

Author(s):

Manting Huang ◽

Minzhen Deng ◽

Wenqiang Nie ◽

Dezhi Zou ◽

Huanlin Wu ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Cyclic Nucleotide ◽

Ex Vivo ◽

Thrombus Formation ◽

Antiplatelet Agent ◽

Protective Effects ◽

Clot Retraction ◽

Vasp Phosphorylation ◽

Nucleotide Signaling

Citrus flavanoids intake can reduce the risk of cardiovascular diseases. Naringenin, a natural predominant flavonoid abundant in citrus fruits, possesses protective effects against atherothrombotic diseases. As platelet activation plays central roles in atherothrombogenesis, we studied the effects of naringenin on platelet activation, signaling, thrombosis and hemostasis. Naringenin dose-dependently inhibited agonist-induced platelet aggregation in vitro, and exhibited more-potent efficacy on ADP-induced platelet aggregation. It also suppressed platelet aggregation stimulated by ADP ex vivo. Naringenin inhibited ADP-induced platelet α-granule secretion, fibrinogen binding, intracellular calcium mobilization and platelet adhesion on collagen-coated surface. Naringenin also inhibited platelet spreading on fibrinogen and clot retraction, processes mediated by outside-in integrin signaling. Mechanism studies indicated that naringenin suppressed PI3K-mediated signaling and phosphodiesterase activity in platelets, in addition to increasing cGMP levels and VASP phosphorylation at Ser239. Furthermore, naringenin-induced VASP phosphorylation and inhibition of platelet aggregation were reversed by a PKA inhibitor treatment. Interestingly, naringenin inhibited thrombus formation in the (FeCl3)-induced rat carotid arterial thrombus model, but not cause a prolonged bleeding time in mice. This study suggests that naringenin may represent a potential antiplatelet agent targeting PI3K and cyclic nucleotide signaling, with a low bleeding risk.

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All-Trans Retinoic Acid Impairs Platelet Function and Thrombus Formation and Inhibits Protein Kinase CßI/δ Phosphorylation

Thrombosis and Haemostasis ◽

10.1055/s-0039-1693737 ◽

2019 ◽

Vol 119 (10) ◽

pp. 1655-1664 ◽

Cited By ~ 1

Author(s):

Qi Luo ◽

Guangyu Wei ◽

Xiamin Wang ◽

Xiaoqi Xu ◽

Wen Ju ◽

...

Keyword(s):

Retinoic Acid ◽

Protein Kinase ◽

Platelet Function ◽

Thrombus Formation ◽

Receptor Expression ◽

Dependent Manner ◽

Clot Retraction ◽

Trans Retinoic Acid ◽

All Trans Retinoic Acid ◽

Arterial Thrombus

AbstractAll-trans retinoic acid (ATRA) is widely used for induction of complete remission in patients with acute promyelocytic leukemia (APL). ATRA also regulates protein kinase C (PKC) activity. Therapeutic use of ATRA reportedly interferes with hemostatic function in APL patients, including effects on coagulation or other vascular cells, although effects of ATRA on platelets remain unclear. This study aims to investigate the effect of therapeutic-relevant doses of ATRA on platelet function. Human platelets were preincubated with ATRA (0–20 μM) for 1 hour at 37°C, followed by analysis of aggregation, granule secretion, receptor expression by flow cytometry, platelet spreading, or clot retraction. Additionally, ATRA (10 mg/kg) was injected intraperitoneally into mice and tail bleeding time and arterial thrombus formation were evaluated. ATRA inhibited platelet aggregation and adenosine triphosphate release induced by collagen (5 μg/mL) or thrombin (0.05 U/mL) in a dose-dependent manner without affecting P-selectin expression or surface levels of glycoprotein (GP) Ibα, GPVI, or αIIbβ3. ATRA-treated platelets demonstrated reduced spreading on immobilized fibrinogen or collagen and reduced thrombin-induced clot retraction together with reduced phosphorylation of Syk and PLCγ2. In addition, ATRA-treated mice displayed significantly impaired hemostasis and arterial thrombus formation in vivo. Further, in platelets stimulated with either collagen-related peptide or thrombin, ATRA selectively inhibited phosphorylation of PKCßI (Ser661) and PKCδ (Thr505), but not PKCα or PKCßII phosphorylation (Thr638/641). In conclusion, ATRA inhibits platelet function and thrombus formation, possibly involving direct or indirect inhibition of PKCßI/δ, indicating that ATRA might be beneficial for the treatment of thrombotic or cardiovascular diseases.

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Targeting The PI3K/AKT Pathway To Inhibit Platelet Activation and Thrombus Formation

Blood ◽

10.1182/blood.v122.21.3513.3513 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3513-3513

Author(s):

Wenxiu Yi ◽

Wei Li ◽

Lijie Ren ◽

Xinliang Mao ◽

Li Zhu

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Function ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Venous Blood ◽

Pi3k Pathway ◽

Dependent Manner ◽

Clot Retraction ◽

Occlusion Time

Abstract The phosphatidylinositol 3' –kinase (PI3K)-Akt signaling pathway has been shown to be critical in modulating platelet function and increasing number of studies have been focusing on the development of PI3K inhibitors to modulate platelet function. We recently identified a novel small molecule compound S14161, namely 8-ethoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene, displaying potent antileukemia and antimyeloma activity via inhibition of the PI3K pathway (Mao et al, Blood, 2011, 117:1986). In the present study, we evaluated the effect of S14161 on platelet activation and the underlying mechanisms. Gel-filtered human platelets were isolated from venous blood of healthy adults and the effect of S14161 on platelet aggregation in response to agonists was determined. Results showed that S14161 inhibited platelet aggregation induced by collagen, convulxin, thrombin, PAR1 agonist peptide SFLLRN, and U46619 in a dose dependent manner (2.5-10μM) with the most striking inhibition for collagen by 89.8% (P<0.001, n=3) and for U46619 by 94.3% (P<0.001, n=3), respectively compared to vehicle-treated samples when 10μM S14161 was used. Flow cytometry studies showed that S14161 inhibits convulxin- or thrombin-induced P-selectin expression and fibrinogen binding of single platelet. S14161 also inhibited platelet spreading on fibrinogen and clot retraction, processes mediated by outside-in signaling. Using a microfluidic chamber we demonstrated that incubation of S14161 decreases platelet adhesion on collagen-coated surface by about 80% at various time points of blood flow in the chambers. Western blot showed that similar to LY294002, the classic PI3K inhibitor, S14161 inhibited phosphorylation of Akt Ser473 and Akt Thr308 in response to collagen, thrombin, or U46619, implying the involvement of PI3K pathway. Additionally, S14161 inhibited MAPK/ERK1/2 phosphorylation. Finally, the effects of S14161 on thrombus formation in vivo were measured using a ferric chloride-induced carotid artery injury model in mice. The intraperitoneal injection of S14161 (2mg/kg) to male C57BL6/J mice significantly extended the first occlusion time (5.05±0.99 min, N=9) compared to the vehicle controls (3.72±0.95 min, N=8) (P<0.05), but did not increase the bleeding time (P>0.05). Taken together, our data showed that S14161 inhibits platelet activation and thrombus formation, and may be developed as a novel therapeutic agent for the prevention of thrombotic disorders. (This study was supported by National Natural Science Foundation of China 81170132 to Li Zhu) Disclosures: No relevant conflicts of interest to declare.

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Abstract 13598: The G-protein BetaGamma Subunits Regulate Platelet Function

Circulation ◽

10.1161/circ.142.suppl_3.13598 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Ahmed Alarabi ◽

Zubair Karim ◽

Victoria Hinojos ◽

Patricia A Lozano ◽

Keziah Hernandez ◽

...

Keyword(s):

G Protein ◽

Platelet Function ◽

Thrombus Formation ◽

Human Platelets ◽

Inhibitory Effects ◽

Clot Retraction ◽

Betagamma Subunits

Platelet activation involves tightly regulated processes to ensure a proper hemostasis response, but when unbalanced, can lead to pathological consequences such as thrombus formation. G-protein coupled receptors (GPCRs) regulate platelet function by interacting with and mediating the response to various physiological agonists. To this end, an essential mediator of GPCR signaling is the G protein Gαβγ heterotrimers, in which the βγ subunits are central players in downstream signaling pathways. While much is known regarding the role of the Gα subunit in platelet function, that of the βγ remains poorly understood. Therefore, we investigated the role of Gβγ subunits in platelet function using a Gβγ (small molecule) inhibitor, namely gallein. We observed that gallein inhibits platelet aggregation and secretion in response to agonist stimulation, in both mouse and human platelets. Furthermore, gallein also exerted inhibitory effects on integrin αIIbβ3 activation and clot retraction. Finally, gallein’s inhibitory effects manifested in vivo , as documented by its ability to modulate physiological hemostasis and delay thrombus formation. Taken together, our findings demonstrate, for the first time, that Gβγ directly regulates GPCR-dependent platelet function, in vitro and in vivo . Moreover, these data highlight Gβγ as a novel therapeutic target for managing thrombotic disorders.

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FUNDC2 regulates platelet activation through AKT/GSK-3β/cGMP axis

Cardiovascular Research ◽

10.1093/cvr/cvy311 ◽

2018 ◽

Vol 115 (11) ◽

pp. 1672-1679 ◽

Cited By ~ 1

Author(s):

Qi Ma ◽

Weilin Zhang ◽

Chongzhuo Zhu ◽

Junling Liu ◽

Quan Chen

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Rich Plasma ◽

Thrombus Formation ◽

Mitochondrial Protein ◽

Dependent Manner ◽

Clot Retraction ◽

Akt Phosphorylation ◽

Gsk 3Β

Abstract Aims AKT kinase is vital for regulating signal transduction in platelet aggregation. We previously found that mitochondrial protein FUNDC2 mediates phosphoinositide 3-kinase (PI3K)/phosphatidylinositol-3,4,5-trisphosphate (PIP3)-dependent AKT phosphorylation and regulates platelet apoptosis. The aim of this study was to evaluate the role of FUNDC2 in platelet activation and aggregation. Methods and results We demonstrated that FUNDC2 deficiency diminished platelet aggregation in response to a variety of agonists, including adenosine 5′-diphosphate (ADP), collagen, ristocetin/VWF, and thrombin. Consistently, in vivo assays of tail bleeding and thrombus formation showed that FUNDC2-knockout mice displayed deficiency in haemostasis and thrombosis. Mechanistically, FUNDC2 deficiency impairs the phosphorylation of AKT and downstream GSK-3β in a PI3K-dependent manner. Moreover, cGMP also plays an important role in FUNDC2/AKT-mediated platelet activation. This FUNDC2/AKT/GSK-3β/cGMP axis also regulates clot retraction of platelet-rich plasma. Conclusion FUNDC2 positively regulates platelet functions via AKT/GSK-3β/cGMP signalling pathways, which provides new insight for platelet-related diseases.

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Antiplatelet and Antithrombotic Effects of Epimedium koreanum Nakai

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2021/7071987 ◽

2021 ◽

Vol 2021 ◽

pp. 1-11

Author(s):

Muhammad Irfan ◽

Tae-Hyung Kwon ◽

Dong-Ha Lee ◽

Seung-Bok Hong ◽

Jae-Wook Oh ◽

...

Keyword(s):

Platelet Aggregation ◽

Light Transmission ◽

Thrombus Formation ◽

Calcium Mobilization ◽

Potential Candidate ◽

Clot Retraction ◽

Integrin Αiibβ3 ◽

Atp Secretion ◽

Antithrombotic Effects ◽

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.

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Doxorubicin contributes to thrombus formation and vascular injury by interfering with platelet function

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00456.2019 ◽

2020 ◽

Vol 319 (1) ◽

pp. H133-H143 ◽

Cited By ~ 1

Author(s):

Haichen Lv ◽

Ruopeng Tan ◽

Jiawei Liao ◽

Zhujing Hao ◽

Xiaolei Yang ◽

...

Keyword(s):

Endothelial Cells ◽

Platelet Aggregation ◽

Vascular Injury ◽

Platelet Function ◽

Thrombus Formation ◽

Platelet Activity ◽

Vascular Toxicity ◽

Platelet Functions

Doxorubicin therapy in mice (antitumor dosage) markedly enhanced platelet functions measured as agonist-induced platelet aggregation, degranulation, and adhesion to endothelial cells, actions leading to thrombus formation and thrombosis-independent vascular injury. Clopidogrel treatment ameliorated thrombus formation and vascular toxicity induced by doxorubicin via inhibiting platelet activity.

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Pharmacological Evaluation of Ticlopidine, A Novel Inhibitor of Platelet Function

10.1055/s-0039-1680487 ◽

1977 ◽

Author(s):

M. Johnson ◽

P. L. Walton ◽

R. C. Cotton ◽

C. J. L. Strachau

Keyword(s):

Survival Time ◽

Platelet Function ◽

Thrombus Formation ◽

Antiplatelet Agent ◽

Dose Effect ◽

Onset Of Action ◽

Duration Of Action ◽

Platelet Survival ◽

Induced Aggregation

Ticlopidine (T), 5-(2-chlorobenzyl)-4,5,6,7-tetrahydrothieno(3,2-C)pyridine hydrochloride (a product of Parcor Research) has been evaluated as an antiplatelet agent in various animal species and in human volunteers. T was inactive in vitro, but inhibited platelet aggregation induced by ADP, collagen, thrombin, arachidonic acid and prostaglandin (PG) endoperoxide, when administered orally to mice, rats, rabbits, guinea pigs, pigs, dogs and baboons. Platelet adhesiveness was reduced but platelet survival time was normal in treated animals. Basal PG synthesis and platelet ultra-structure were unaffected by T.T protected against acute thrombocytopenia and death from pulmonary embolism induced by i. v. injection of ADP or collagen. Thrombus formation in experimental models of extra-corporeal circulation and deep venous thrombosis was also impaired.In man, a single oral dose of 500mg was shown to be a potent inhibitor of ADP, collagen, adrenaline, ristocetin, bovine fibrinogen and 5HT-induced aggregation. A dose-effect relationship was apparent, 250 and 500mg resulting in ~47 and 75% inhibition of ADP-induced aggregation respectively. Inhibition was sustained by chronic daily dosing.There was a delay in the onset of action of T in vivo, but which then persisted after withdrawal for at least 48 hours, with no evidence of rebound hyperactivity. The duration of action of T correlated with platelet survival time, suggesting an irreversible modification of platelet function. T is a potent platelet inhibitor, exhibiting a novel mode of action and lack of agonist specificity, which may be of value in the treatment of thrombotic conditions.

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“M-AAA-Thrombin”, a New Thrombin Mutant with Potent Anticoagulant and Antiplatelet Properties.

Blood ◽

10.1182/blood.v106.11.4154.4154 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4154-4154

Author(s):

Kazuya Hosokawa ◽

Tomoko Ohnishi ◽

Hiroyuki Matsuda ◽

Kousuke Kashima ◽

Takehiko Koide

Keyword(s):

Platelet Aggregation ◽

Thrombus Formation ◽

Antiplatelet Agent ◽

Dependent Manner ◽

In Vivo Experiments ◽

Thrombosis Model ◽

Venous Shunt ◽

Prolonged Aptt ◽

Antithrombotic Efficacy

Abstract Thrombosis is a major cause of morbidity and mortality, and thrombin is a major inducer of thrombus formation. Thus several antithrombotic agents targeting thrombin have been developed. We previously reported an anticoagulant and antiplatelet thrombin derivative, ‘M-anhydrothrombin’ prepared by chemical modifications. In this study, we prepared a new thrombin mutant, specificity of which was highly modulated with substantially improved antithrombotic efficacy. The thrombin mutant designated “AAA-Thrombin” in which Lys65, His43 and Ser205 in B-chain have been replaced by Ala revealed higher affinity and specificity for factor VIII with no enzymatic activity. AAA-Thrombin prolonged APTT much more than anhydrothrombin in a dose dependent manner without affecting PT and TT. Platelet aggregation induced by activation of PAR-1 was also effectively suppressed by AAA-Thrombin. “M-AAA-Thrombin” prepared by further chemical modification of carboxyl groups in AAA-Thrombin enhanced its antithrombotic efficacy. M-AAA-Thrombin (250nM) prolonged APTT approx. two times, and suppressed platelet aggregation by PAR-1 activation, while AAA-Thrombin did not at the same concentration. M-AAA-Thrombin also suppressed ristocetin-induced platelet aggregation. In vivo experiments, M-AAA-Thrombin demonstrated significant antithrombotic property in the arterio-venous shunt thrombosis model and the FeCl3-induced carotid artery thrombosis model in guinea pigs. These results indicate that M-AAA-Thrombin would be a candidate for quite an innovative anticoagulant and antiplatelet agent for both arterial and venous thromboses. Further optimization of mutagenesis and modification, in terms of efficacy and safety is in progress in our laboratory.

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