scholarly journals Antithrombotic Effects of Paeoniflorin from Paeonia suffruticosa by Selective Inhibition on Shear Stress-Induced Platelet Aggregation

2019 ◽  
Vol 20 (20) ◽  
pp. 5040 ◽  
Author(s):  
Thien Ngo ◽  
Keunyoung Kim ◽  
Yiying Bian ◽  
Hakjun Noh ◽  
Kyung-Min Lim ◽  
...  
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Ex Vivo ◽  
Antiplatelet Agents ◽  
Human Platelets ◽  
Platelet Glycoprotein ◽  
High Shear ◽  
High Shear Stress ◽  
Paeonia Suffruticosa ◽  
Induced Aggregation

Antiplatelet agents are important in the pharmacotherapeutic regime for many cardiovascular diseases, including thrombotic disorders. However, bleeding, the most serious adverse effect associated with current antiplatelet therapy, has led to many efforts to discover novel anti-platelet drugs without bleeding issues. Of note, shear stress-induced platelet aggregation (SIPA) is a promising target to overcome bleeding since SIPA happens only in pathological conditions. Accordingly, this study was carried out to discover antiplatelet agents selectively targeting SIPA. By screening various herbal extracts, Paeonia suffruticosa and its major bioactive constituent, paeoniflorin, were identified to have significant inhibitory effects against shear-induced aggregation in human platelets. The effects of paeoniflorin on intraplatelet calcium levels, platelet degranulation, and integrin activation in high shear stress conditions were evaluated by a range of in vitro experiments using human platelets. The inhibitory effect of paeoniflorin was determined to be highly selective against SIPA, through modulating von Willebrand Factor (vWF)-platelet glycoprotein Ib (GP Ib) interaction. The effects of paeoniflorin on platelet functions under high shear stress were confirmed in the ex vivo SIPA models in rats, showing the good accordance with the anti-SIPA effects on human platelets. Treatment with paeoniflorin significantly prevented arterial thrombosis in vivo from the dose of 10 mg/kg without prolonging bleeding time or blood clotting time in rats. Collectively, our results demonstrated that paeoniflorin can be a novel anti-platelet agent selectively targeting SIPA with an improved safety profile.

Effect of a New Monoclonal Anti-Glycoprotein IX Antibody, KMP-9, on High Shear-Induced Platelet Aggregation

1997 ◽  
Vol 78 (02) ◽  
pp. 902-909 ◽  
Author(s):  
Tetsuya Miyake ◽  
Shosaku Nomura ◽  
Yutaka Komiyama ◽  
Yasuhiko Miyazaki ◽  
Hideo Kagawa ◽  
...  
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Physiological Role ◽  
Inhibitory Effect ◽  
Platelet Glycoprotein ◽  
High Shear ◽  
High Shear Stress ◽  
Receptor Component ◽  
Induced Aggregation

SummaryHuman platelet glycoprotein Ib/IX complex acts as a receptor for von Willebrand factor. It is widely accepted that glycoprotein lb is the essential receptor component, but the role of glycoprotein IX is still unclear. We produced a new monoclonal anti-glycoprotein IX antibody (KMP-9) by the hybridoma technique using platelets from a patient with Glanzmann’s thrombasthenia. The epitope of KMP-9 was localized to the C-terminal 8 kD fragment of glycoprotein IX using ELISA analysis of polyethylene-pin-synthesized peptides, as well as Western blot analysis of platelets after digestion with N-glycosidase and Staphylococcus aureus V8 protease. KMP-9 partially inhibited high shear stress-induced platelet aggregation, but had no effect on aggregation induced by ristocetin or low shear stress. Its inhibitory effect on high shear stress-induced aggregation was weaker than that of antiglycoprotein lb or anti-glycoprotein Ilb/IIIa monoclonal antibodies. A 21-mer synthetic peptide (glycoprotein IX L110-G130) inhibited the binding of KMP-9 to platelets. It also competively inhibited the suppression of high shear stress-induced platelet aggregation by KMP-9, but had no direct effect on this aggregation. KMP-9 may be useful to clarify the physiological role of GPIX.

Novel Platelet Antagonists

2006 ◽  
Author(s):  
Richard C. Becker ◽  
Frederick A. Spencer
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Adenosine Monophosphate ◽  
Stress Conditions ◽  
Platelet Glycoprotein ◽  
High Shear ◽  
High Shear Stress ◽  
Surface Receptor ◽  
Pharmacologic Agents

The development of pharmacologic agents that inhibit platelet performance could not have proceeded without a fundamental knowledge of normal biology and a clear understanding of the laws that govern cellular events in the circulatory system. The adhesion of platelets to a site of vessel wall injury is mediated by von Willebrand factor (vWF), which binds to the platelet glycoprotein (GP) Ib/IX-V complex receptor (and the GPIIb/IIIa receptor under high shear stress conditions). Monoclonal antibodies to vWF have been developed and tested in animal models, as has aurintricarboxylic acid (Strony et al., 1990), a triphenylmethyl compound that inhibits vWF binding. To date, investigation in humans has not taken place, perhaps because of concerns regarding the potential risk for hemorrhagic complications. Nevertheless, the scientific community remains interested in vWF and its platelet surface receptor as potential pharmacology-directed targets. Although the GPIIb/IIIa receptor antagonists are best known for their ability to inhibit platelet aggregation, under high shear stress conditions vWF can also bind the GPIIb/IIIa receptor, facilitating adhesion. As a result, GPIIb/IIIa antagonists may have an impact on both platelet adhesion and aggregation. As previously discussed, platelet activation is followed by a series of intracellular events that culminate in the release of calcium and substances that augment platelet aggregation and support coagulation protease binding. Thus, pharmacologic agents that inhibit initial surface receptor–mediated activation may also impair platelet aggregation. Several natural prostanoids (prostaglandin [PG] E1 and PGI2) can inhibit platelet activation and aggregation by elevating cyclic adenosine monophosphate (cAMP) levels. Although the mechanism is complex, the primary mode of inhibition is through the activation of adenylate cyclase (with a subsequent rise in cAMP concentrations), which in turn prevents calcium mobilization. The clinical application of PGE1 and PGI2 has been limited by their effect on vascular tone, producing substantial systemic hypotension (Emmons et al., 1967; Terres et al., 1989), and by extensive first-pass metabolism in the lungs (70% of the active compound is rapidly cleared) (Kleiman et al., 1994).

scholarly journals High shear stress can initiate both platelet aggregation and shedding of procoagulant containing microparticles

Blood ◽  
1996 ◽  
Vol 88 (9) ◽  
pp. 3456-3464 ◽  
Author(s):  
Y Miyazaki ◽  
S Nomura ◽  
T Miyake ◽  
H Kagawa ◽  
C Kitada ◽  
...  
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Coagulation Cascade ◽  
Platelet Glycoprotein ◽  
High Shear ◽  
High Shear Stress ◽  
Microparticle Formation ◽  
High Level ◽  
Von Willebrand ◽  
Willebrand Factor

Previous studies have demonstrated that a high level of shear stress can produce platelet aggregation without the addition of any agonist. We investigated whether high shear stress could cause both platelet aggregation and shedding of microparticles from the platelet plasma membrane. A coneplate viscometer was used to apply shear stress and microparticle formation was measured by flow cytometry. It was found that microparticle formation increased as the duration of shear stress increased. Both microparticles and the remnant platelets showed the exposure of procoagulant activity on their surfaces. Investigation of the mechanisms involved in shear-dependent microparticle generation showed that binding of von Willebrand factor (vWF) to platelet glycoprotein lb, influx of extracellular calcium, and activation of platelet calpain were required to generate microparticles under high shear stress conditions. Activation of protein kinase C (PKC) promoted shear-dependent microparticle formation. Epinephrine did not influence microparticle formation, although it enhanced platelet aggregation by high shear stress. These findings suggest the possibility that local generation of microparticles in atherosclerotic arteries, the site that pathologically high shear stress could occur, may contribute to arterial thrombosis by providing and expanding a catalytic surface for the coagulation cascade.

scholarly journals Shear stress activation of platelet glycoprotein IIb/IIIa plus von Willebrand factor causes aggregation: filter blockage and the long bleeding time in von Willebrand's disease

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1354-1361 ◽  
Author(s):  
JR O'Brien ◽  
GP Salmon
Keyword(s):  
Shear Stress ◽  
Bleeding Time ◽  
Platelet Rich Plasma ◽  
Divalent Cations ◽  
Mean Transit Time ◽  
Platelet Glycoprotein ◽  
High Shear ◽  
The Difference ◽  
Von Willebrand’S Factor ◽  
Induced Aggregation

The article explores the finding that high shear alone applied to normal, native blood results in platelet aggregation. A filter with tortuous capillary-sized channels permits a study of the effect of shearing forces at different pressures. Native, heparinized, citrated and EDTA blood and platelet-rich plasma (PRP) were forced through the filter. Normal and von Willebrand's blood were studied, as were the effects of antibodies to platelet glycoproteins (GPr) and to von Willebrand's factor (vWf) and of “membrane-active” drugs. Normally, the filter blocked at 40 mmHg but not at 5 mmHg. Transmission electronmicroscopy of the filter at 40 mmHg showed blockage by platelet aggregates. Initially, the mean transit time through the filter was 8 milliseconds. Platelet retention in the filter occurred in two phases. From 0 to 3 seconds, only high-shear, vWf, and GPrIIb/IIIa were required. From 10 to 20 seconds, retention presumably involved these three attributes, but divalent cations were also essential. Only this phase was inhibited by some membrane-active drugs. ADP- and thrombin- induced aggregation requires GPrIIb/IIIaand fibrinogen. Shear-induced blocking of the filter by blood with a normal concentration of fibrinogen requires GPrIIb/IIIa and vWf. This indicates a different type of exposure of GPrIIb/IIIa. The long bleeding time in vW disease highlights the absolute requirement for vWf and emphasizes the difference in exposure of GPrIIb/IIIa induced by shear stress. Evidently, a process similar to that occurring in the filter is required in normal capillary hemostasis.

A critical role for thrombin in platelet aggregation under high shear stress

2004 ◽  
Vol 113 (5) ◽  
pp. 311-318 ◽  
Author(s):  
Yoshihiko Sakurai ◽  
Midori Shima ◽  
John Giddings ◽  
Masahiro Takeyama ◽  
Shogo Kasuda ◽  
...  
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Critical Role ◽  
High Shear ◽  
High Shear Stress

Monosubstituted Coumarins Inhibit Epinephrine-Induced Platelet Aggregation Antiplatelet Effect of Monosubstituted Coumarins

2021 ◽  
Vol 19 ◽  
Author(s):  
Fausto Alejandro Jiménez-Orozco ◽  
Sergio Galicia-Zapatero ◽  
Edgar López-López ◽  
José L. Medina-Franco ◽  
Fernando León Cedeño ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
In Silico ◽  
Drug Targets ◽  
Platelet Reactivity ◽  
Antiplatelet Agents ◽  
Human Platelets ◽  
In Silico Studies ◽  
Vitro Effect ◽  
Induced Aggregation

Aim: Evaluate the in vitro effect of coumarin and 15 monosubstituted derivatives on the inhibition of human platelet aggregation induced by various pro-aggregatory agonists, particularly by epinephrine. Background: The emergence of residual platelet reactivity during the use of conventional antiplatelet agents (acetylsalicylic acid and clopidogrel) is one of the main causes of double therapy´s therapeutic failure. Platelet adrenoceptors participate in residual platelet reactivity. Therefore, it is necessary to develop new antiplatelet agents that inhibit epinephrine-induced platelet aggregation as a new therapeutic strategy. Information on the antiplatelet activity of coumarins in inhibiting epinephrine-induced aggregation is limited. Objective: Establish the structure-activity relationship (SAR) of coumarin derivatives with hydroxy, methoxy, and acetoxy groups in different positions of the coumarin nucleus to identify the most active molecules. Using in silico studies, suggest potential drug targets to which the molecules bind to produce antiplatelet effects. Methods: The platelet aggregation was performed using a Lumi-aggregometer; the inhibitory activity of 16 compounds were evaluated by inducing the aggregation of human platelets (250 × 103/μl) with epinephrine (10 µM), collagen (2 µg / ml) or ADP (10 µM). The aggregation of controls platelets was considered 100% of the response for each pro-aggregatory agonists. Results: Eleven molecules inhibited epinephrine-induced aggregation, with 3-acetoxycoumarin and 7-methoxycoumarin being the most active. Only coumarin inhibited collagen-induced platelet aggregation, but no molecule showed activity when using ADP as an inducer. Conclusions : In silico studies suggest that most active molecules might have antagonistic interactions in the adrenoceptors α2 and β2. The antiplatelet actions of these coumarins have the potential to reduce residual platelet reactivity and thus contribute to the development of future treatments for patients who do not respond adequately to conventional agents.

Abstract 33: Platelet 12-lipoxygenase is a Key Regulator of Platelet Reactivity and Thrombus Formation in vivo

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Reheman Adili ◽  
Katherine Mast ◽  
Theodore R Holman ◽  
Michael Holinstat
Keyword(s):  
Platelet Aggregation ◽  
Ex Vivo ◽  
Platelet Reactivity ◽  
Thrombus Formation ◽  
High Shear ◽  
Vessel Occlusion ◽  
12 Lipoxygenase ◽  
Induced Aggregation

Background: Platelet reactivity is required to maintain hemostasis, however high platelet reactivity leads to thrombus formation, myocardial infarction, and stroke. Platelet 12-lipoxygenase (12-LOX) has been demonstrated by our lab and others to regulate agonist-mediated platelet reactivity suggesting a role for 12-LOX in regulation of in vivo thrombosis. The ability to target 12-LOX in vivo has not been established to date. Therefore, we sought to determine if 12-LOX regulates platelet reactivity and thrombus formation in vivo using the selective 12-LOX inhibitor ML355 to determine whether platelet 12-LOX is an effective target for anti-platelet therapeutics. Methods: ML355 effects on human platelet function was assessed in vitro by platelet aggregometry, ex vivo by perfusion chamber, and in vivo by thrombus formation and vessel occlusion in small and large vessels in 12-LOX -/- , WT mice, and mice treated with ML355 via intravital microscopy using the FeCl 3 and laser injury models. Results: In in vitro platelet aggregation, ML355 dose-dependently inhibited agonist-induced aggregation. In ex vivo flow chamber assays, platelet adhesion and thrombus formation on collagen-coated surfaces at high shear was attenuated in both mouse and human whole blood after incubation with ML355. Further, platelet aggregation and thrombus growth in 12-LOX -/- mice were impaired in both laser and FeCl 3 -induced mesenteric, carotid artery and cremaster arteriole thrombosis models. Thrombi in 12-LOX -/- mice were unstable and frequently formed emboli, which resulted in impaired vessel occlusion or reopening. Additionally, thrombus formation and vessel occlusion was impaired in ML355 treated WT mice. Conclusions: The 12-LOX inhibitor ML355 inhibits platelet aggregation induced by a number of platelet agonists. Ex vivo high shear conditions in both mice and human was attenuated in the presence of ML355. Thrombus formation and vessel occlusion were impaired in mice deficient in 12-LOX. Finally, ML355 attenuates thrombus formation and prevents vessel occlusion in vivo . Our data strongly indicates 12-LOX is an important determinant of platelet reactivity and inhibition of platelet 12-LOX may represent a new target for anti-platelet therapeutics.

Cytoplasmic domains of GpIbα and GpIbβ regulate 14-3-3ζ binding to GpIb/IX/V

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 551-557 ◽  
Author(s):  
Shuju Feng ◽  
Nicolaos Christodoulides ◽  
Julio C. Reséndiz ◽  
Michael C. Berndt ◽  
Michael H. Kroll
Keyword(s):  
Shear Stress ◽  
Amino Acid ◽  
Fusion Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Human Platelets ◽  
Platelet Glycoprotein ◽  
Structural Determinants ◽  
Ovary Cells ◽  
Induced Aggregation

Shear stress causes the platelet glycoprotein (Gp) Ib/IX/V to bind to von Willebrand factor, resulting in platelet adhesion. GpIb/IX/V also functions to stimulate transmembranous signaling, leading to platelet activation and the expression of a ligand-receptive GpIIb-IIIa complex. The highly conserved cytoplasmic domain of GpIb binds directly to a dimeric 14-3-3 adapter protein ζ isoform. To explore structural determinants of GpIb/IX/V binding to 14-3-3ζ, the authors examined 14-3-3ζ interactions with GpIb and GpIbβ in heterologous cells and platelets. Truncations of GpIb at amino acid 542 or 594, or deletions of residues 542 through 590, inhibited binding of 14-3-3ζ. Deletion of GpIb from Trp570 to Ser590 eliminated 14-3-3ζ binding, and deletion of the sequence from Arg542-Trp570 enhanced binding of 14-3-3ζ to GpIb. All GpIb mutations that eliminated GpIb binding to the GST-14-3-3ζ fusion protein also eliminated GpIbβ binding to the fusion protein. Forskolin treatment of Chinese hamster ovary cells expressing wild-type GpIb/β/IX resulted in the phosphorylation of GpIbβ associated with enhanced binding of GpIbβ to GST-14-3-3ζ fusion protein and increased 14-3-3ζ coimmunoprecipitated with GpIb. When intact human platelets aggregated in response to 90 dynes/cm2 shear stress, 14-3-3ζ disassociated from GpIb. Prostacyclin treatment of platelets inhibited shear stress-induced aggregation and the release of 14-3-3ζ from GpIb. These data demonstrate that amino acid residues in the cytoskeletal interaction domains of GpIb regulate 14-3-3ζ binding to GpIb/β/IX, and suggest that protein kinase A-dependent phosphorylation of GpIbβ enhances 14-3-3ζ binding to the GpIb/IX/V complex in human platelets.

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