Multiparameter microfluidics assay of thrombus formation reveals increased sensitivity to contraction and antiplatelet agents at physiological temperature

Vertebral artery dissection (VAD) is caused by an intimal tear that leads to bleeding into the vascular wall, which may cause vascular occlusion by thrombus formation and subsequent distal emboli (leading to ischemic stroke), aneurysm formation and subarachnoid hemorrhage. Cervical artery dissections (either carotid or vertebral artery dissection) are an important cause of stroke in patients under 50-years of age. Headache with or without neck pain is a common symptom. Usually, it occurs with focal neurological signs but sometimes it may occur without any neurological deficits and may mimic migraine. Often it occurs spontaneously without trauma but sometimes there is history of minor traumas, sudden neck movements or chiropractic manipulation. Imaging modalities include magnetic resonance imaging (MRI) brain, magnetic resonance angiography (MRA), and computed tomography angiography (CTA). Treatment involves anticoagulation or antiplatelet agents.

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Black Sorghum Phenolic Extract Modulates Platelet Activation and Platelet Microparticle Release

Nutrients ◽

10.3390/nu12061760 ◽

2020 ◽

Vol 12 (6) ◽

pp. 1760 ◽

Cited By ~ 3

Author(s):

Borkwei Ed Nignpense ◽

Kenneth A Chinkwo ◽

Christopher L Blanchard ◽

Abishek B Santhakumar

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Conformational Changes ◽

Thrombus Formation ◽

Antiplatelet Agents ◽

Limited Information ◽

Platelet Activity ◽

Dietary Polyphenols ◽

High Antioxidant Activity ◽

Phenolic Extract

Platelet hyper-activation and platelet microparticles (PMPs) play a key role in the pathogenesis of cardiovascular diseases. Dietary polyphenols are believed to mimic antiplatelet agents by blunting platelet activation receptors via its antioxidant phenomenon. However, there is limited information on the anti-platelet activity of grain-derived polyphenols. The aim of the study is to evaluate the effects of sorghum extract (Shawaya short black 1 variety), an extract previously characterised for its high antioxidant activity and reduction of oxidative stress-related endothelial dysfunction, on platelet aggregation, platelet activation and PMP release. Whole blood samples collected from 18 healthy volunteers were treated with varying non-cytotoxic concentrations of polyphenol-rich black sorghum extract (BSE). Platelet aggregation study utilised 5 µg/mL collagen to target the GPVI pathway of thrombus formation whereas adenine phosphate (ADP) was used to stimulate the P2Y1/P2Y12 pathway of platelet activation assessed by flow cytometry. Procaspase-activating compound 1 (PAC-1) and P-selectin/CD62P were used to evaluate platelet activation- related conformational changes and degranulation respectively. PMPs were isolated from unstimulated platelets and quantified by size distribution and binding to CD42b. BSE treatment significantly reduced both collagen-induced platelet aggregation and circulatory PMP release at 40 µg/mL (p < 0.001) when compared to control. However, there was no significant impact of BSE on ADP-induced activation-dependent conformational change and degranulation of platelets. Results of this study suggest that phenolic rich BSE may confer cardio-protection by modulating specific signalling pathways involved in platelet activation and PMP release.

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YM150, An Oral Direct Factor Xa Inhibitor: Combination with Aspirin or Clopidogrel In Arteriovenous Shunt Thrombosis In Rats

Blood ◽

10.1182/blood.v116.21.3318.3318 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3318-3318

Author(s):

Yoshiyuki Iwatsuki ◽

Chinatsu Sakata ◽

Yumiko Moritani

Keyword(s):

Platelet Aggregation ◽

Arterial Thrombosis ◽

Bleeding Time ◽

Thrombus Formation ◽

Control Level ◽

Factor Xa ◽

Antiplatelet Agents ◽

Factor Xa Inhibitor ◽

Direct Factor ◽

Silk Thread

Abstract Abstract 3318 Background: YM150, an oral direct factor Xa inhibitor, is currently in clinical development for the prevention of venous thromboembolism in patients undergoing orthopedic surgery, prevention of stroke in patients with atrial fibrillation, and prevention of ischemic events after recent acute coronary syndrome (ACS). The antiplatelet agents aspirin or clopidogrel will likely be co-prescribed with YM150 in ACS. Here, we report the effects of YM150 in combination with aspirin or clopidogrel on thrombus formation, bleeding, platelet aggregation, and coagulation in rats. Methods: The antithrombotic effect was estimated in a rat arteriovenous shunt model. The shunt was formed by attaching a polyethylene tube containing a silk thread to the carotid artery and the contralateral carotid vein. Blood was allowed to circulate in this shunt for 15 min, and then the silk thread was withdrawn from the tube to assess the thrombus weight. YM150, aspirin, or clopidogrel was orally administered 0.5, 1, or 2 h before shunt formation, respectively. At the same time as shunt formation, an incision was made at the sole of the left foot using a template bleeding device (Surgicutt®) to measure bleeding time. To avoid interference with the thrombosis model, blood samples to assess platelet aggregation and prothrombin time were obtained from separate animals at the same time point as shunt formation in the thrombus study. Platelet aggregation was induced using 10 μg/mL of collagen and 5 μM of adenosine 5`-diphosphate (ADP) to assess the effects of aspirin and clopidogrel, respectively. Results: YM150 alone inhibited thrombus formation, with significance at 10 mg/kg and more (P < 0.05). Respective thrombus weights in the control, 3, 10, and 30 mg/kg groups of YM150 were 4.8, 3.6, 2.4, and 2.0 mg. Aspirin alone inhibited thrombus formation, with significance at 100 mg/kg and more (P < 0.01). Respective thrombus weights in the control, 30, 100, and 300 mg/kg group of aspirin were 6.2, 4.2, 2.8, and 1.5 mg. Clopidogrel alone inhibited thrombus formation, with significance at 1 mg/kg and more (P < 0.01). Respective thrombus weights in the control, 0.3, 1, and 3 mg/kg group of clopidogrel were 4.8, 3.6, 2.9, and 1.3 mg. When administered concomitantly with 100 mg/kg of aspirin, YM150 (3, 10, 30 mg/kg) further inhibited thrombogenesis, with significance at 30 mg/kg of YM150 (P < 0.05) and thrombus weights of 2.4, 1.5, and 1.3 mg, respectively. When administered concomitantly with 1 mg/kg of clopidogrel, YM150 (3, 10, 30 mg/kg) further inhibited thrombogenesis, with significance at 30 mg/kg of YM150 (P < 0.05) and thrombus weights of 3.0, 2.0, and 1.5 mg, respectively. Collagen-induced platelet aggregation was reduced to 16.7% of the control level by 100 mg/kg of aspirin, and ADP-induced platelet aggregation was reduced to 74.4% of the control level by 1 mg/kg of clopidogrel. These effects were not changed in the presence of YM150. Prothrombin time and bleeding time were not prolonged by any of the agents alone, and further, these parameters were not affected by combined use of YM150 with either aspirin or clopidogrel. Conclusions: The thrombosis study suggests that both the platelet aggregation and coagulation cascade participate in thrombus formation in this model since both antiplatelet agents and the anticoagulant YM150 were effective. Thus, the thrombosis induced in this model can be considered similar to arterial thrombosis in humans where both platelets and fibrin are involved. Taken together, YM150 is a promising antithrombotic agent that augments the effects of antiplatelet agents against arterial thrombosis without increasing bleeding risk. Disclosures: Iwatsuki: Astellas Phama Inc.: Employment. Sakata:Astellas Phama Inc.: Employment. Moritani:Astellas Phama Inc.: Employment.

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Mechanisms of platelet activation: Need for new strategies to protect against platelet-mediated atherothrombosis

Thrombosis and Haemostasis ◽

10.1160/th09-03-0192 ◽

2009 ◽

Vol 102 (08) ◽

pp. 248-257 ◽

Cited By ~ 195

Author(s):

Lisa Jennings

Keyword(s):

Antiplatelet Therapy ◽

Platelet Activation ◽

Platelet Reactivity ◽

Thrombus Formation ◽

Antiplatelet Agents ◽

Bleeding Risk ◽

Adenosine Diphosphate ◽

Adp Receptor ◽

Activation Pathways ◽

New Strategies

SummaryPlatelets are central mediators of haemostasis at sites of vascular injury, but they also mediate pathologic thrombosis. Activated platelets stimulate thrombus formation in response to rupture of an atherosclerotic plaque or endothelial cell erosion, promoting atherothrombotic disease. They also interact with endothelial cells and leukocytes to promote inflammation, which contributes to atherosclerosis. Multiple pathways contribute to platelet activation, and current oral antiplatelet therapy with aspirin and a P2Y12 adenosine diphosphate (ADP) receptor antagonist target the thromboxane A2 and ADP pathways, respectively. Both can diminish activation by other factors, but the extent of their effects depends upon the agonist, agonist strength, and platelet reactivity status. Although these agents have demonstrated significant clinical benefit, residual morbidity and mortality remain high. Neither agent is effective in inhibiting thrombin, the most potent platelet activator. This lack of comprehensive inhibition of platelet function allows continued thrombus formation and exposes patients to risk for recurrent thrombotic events. Moreover, bleeding risk is a substantial limitation of antiplatelet therapy, because these agents target platelet activation pathways critical for both protective haemostasis and pathologic thrombosis. Novel antiplatelet therapies that provide more complete inhibition of platelet activation without increasing bleeding risk could considerably decrease residual risk for ischemic events. Inhibition of the protease-activated receptor (PAR)-1 platelet activation pathway stimulated by thrombin is a novel, emerging approach to achieve more comprehensive inhibition of platelet activation when used in combination with current oral antiplatelet agents. PAR-1 inhibition is not expected to increase bleeding risk, as this pathway does not interfere with haemostasis.

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Effect of antiplatelet therapy on inflammatory markers in atherothrombotic patients

Thrombosis and Haemostasis ◽

10.1160/th09-03-0177 ◽

2010 ◽

Vol 103 (01) ◽

pp. 71-82 ◽

Cited By ~ 81

Author(s):

Joseph Muhlestein

Keyword(s):

Antiplatelet Therapy ◽

Inflammatory Markers ◽

Thrombus Formation ◽

Inflammatory Marker ◽

Vascular Inflammation ◽

Antiplatelet Agents ◽

Cd40 Ligand ◽

Reactive Protein ◽

Atherosclerotic Lesions

SummaryInflammation is central to the pathogenesis and progression of atherosclerosis and thrombosis, the underlying cause of major cardiovascular disease. Platelets, in addition to their role in haemostasis, play a key role in both thrombus formation and inflammation following vascular injury, especially atherosclerotic lesions. An increasing body of evidence suggests that inhibition of platelet function can modulate inflammatory markers, particularly those associated with activated platelets, such as CD40 ligand, P-selectin, and C-reactive protein. The currently available antiplatelet agents aspirin, clopidogrel, prasugrel, abciximab, and eptifibatide have shown varying effects on inflammatory markers. These effects seem to be mostly indirect, i.e. mediated primarily through reduced platelet activation that results in reduced inflammatory marker expression. However, there is some evidence that suggests direct effects (i.e. those independent of platelets) may also play a role in modulating inflammatory markers. Evidence linking inflammation and thrombosis supports the hypothesis that agents with both anti-inflammatory and antiplatelet effects may reduce vascular inflammation and limit acute and long-term thrombotic events. An assessment of the involvement of inflammatory mediators in atherosclerosis may provide further insight into important predictive markers of cardiovascular outcomes that may also serve as potential therapeutic targets. This review examines the evidence for and potential clinical relevance of the effects of antiplatelet therapy on inflammatory markers.

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A Naphthalenic Derivative ND-1 Inhibits Thrombus Formation by Interfering the Binding of Fibrinogen to Integrin αIIbβ3

BioMed Research International ◽

10.1155/2016/8587164 ◽

2016 ◽

Vol 2016 ◽

pp. 1-8 ◽

Cited By ~ 2

Author(s):

Xue Ding ◽

Tong-dan Liu ◽

Zhou-ling Xie ◽

Qi Zhao ◽

Yuan Cao ◽

...

Keyword(s):

Platelet Aggregation ◽

Thrombus Formation ◽

Antiplatelet Agents ◽

Bleeding Risk ◽

Dependent Manner ◽

Antithrombotic Effect ◽

Thrombosis Model ◽

Prolonged Bleeding Time ◽

Cutting Model

Integrin αIIbβ3 plays a crucial role in the process of platelet aggregation. Three integrin αIIbβ3 antagonists (abciximab, eptifibatide, and tirofiban) have been approved by FDA for clinical use. Unfortunately, they all showed severe side effects such as thrombocytopenia and bleeding risk. Thus, researches on the development of more effective and safer antiplatelet agents are needed. In this manuscript we reported a novel naphthalenic derivative compound ND-1 with potent antithrombotic effect and lower bleeding risk. ND-1 inhibited ADP-, collagen-, thrombin-, and U46619-induced platelet aggregation with IC50 values of 1.29, 14.46, 12.84, and 40.24 μM, respectively. Mechanism studies indicated that ND-1 inhibited the binding of fibrinogen to integrin αIIbβ3 in a dose-dependent manner with an IC50 value of 3.12 μM. ND-1 inhibited P-selectin expression induced by ADP, collagen, thrombin, and U46619 on the surface of platelets. Additionally, this compound reduced platelets spreading to the immobilized fibrinogen. In vivo, ND-1 potently decreased thrombus formation in an arteriovenous shunt thrombosis model in rats and slightly prolonged bleeding time in a tail cutting model in mice. Taken together, our results reveal that ND-1 is a novel antagonist of αIIbβ3 with strong antithrombotic effect and lower bleeding risk.

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Novel Antiplatelet Agents in Cardiovascular Disease

Journal of Cardiovascular Pharmacology and Therapeutics ◽

10.1177/1074248419899314 ◽

2020 ◽

Vol 25 (3) ◽

pp. 191-200 ◽

Cited By ~ 2

Author(s):

Maximilian Tscharre ◽

Alan D. Michelson ◽

Thomas Gremmel

Keyword(s):

Cardiovascular Disease ◽

Antiplatelet Therapy ◽

Animal Studies ◽

Randomized Clinical Trials ◽

Thrombus Formation ◽

Antiplatelet Agents ◽

Adenosine Diphosphate ◽

Clinical Routine ◽

New Agents ◽

Antithrombotic Efficacy

Antiplatelet therapy reduces atherothrombotic risk and has therefore become a cornerstone in the treatment of cardiovascular disease. Aspirin, adenosine diphosphate P2Y12 receptor antagonists, glycoprotein IIb/IIIa inhibitors, and the thrombin receptor blocker vorapaxar are effective antiplatelet agents but significantly increase the risk of bleeding. Moreover, atherothrombotic events still impair the prognosis of many patients with cardiovascular disease despite established antiplatelet therapy. Over the last years, advances in the understanding of thrombus formation and hemostasis led to the discovery of various new receptors and signaling pathways of platelet activation. As a consequence, many new antiplatelet agents with high antithrombotic efficacy and supposedly only moderate effects on regular hemostasis have been developed and yielded promising results in preclinical and early clinical studies. Although their long journey from animal studies to randomized clinical trials and finally administration in daily clinical routine has just begun, some of the new agents may in the future become meaningful additions to the pharmacological armamentarium in cardiovascular disease.

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Combined Dabigatran and Antiplatelet Agents Assessed by the Novel Microchip-Based Flow Chamber System

Blood ◽

10.1182/blood.v120.21.1167.1167 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1167-1167

Author(s):

Kenichi Tanaka ◽

Kazuya Hosokawa ◽

Tomoko Ohnishi ◽

Hisayo Sameshima ◽

Takehiko Koide ◽

...

Keyword(s):

Whole Blood ◽

Platelet Rich Plasma ◽

Thrombus Formation ◽

Antiplatelet Agents ◽

Lag Time ◽

Flow Chamber ◽

Chamber System ◽

Shear Rates ◽

Antithrombotic Effects ◽

Flow Pressure

Abstract Abstract 1167 Evaluation of the overall antithrombotic activity of dabigatran in combination with antiplatelet agents is difficult because plasma-based clotting for dabigatran, and platelet aggregometry in anticoagulated blood are two separate tests which do not reflect physiological interactions between soluble factors and platelets. The use of a flow chamber could be more suitable in evaluating a flow-dependent platelet activation and coagulation responses. The aim of the current study was to comparatively evaluate antithrombotic effects of dabigatran in combination with dual antiplatelet therapy (aspirin plus P2Y12 blockade) using the microchip-based flow chamber (T-TAS, Fujimori Kogyo, Japan)(1), and thrombin generation (TG) assay (Thrombinoscope, Maastricht, the Netherlands)(2). After the local ethics committee approval, blood samples were obtained from consented 5 healthy volunteers in the tubes containing 3.2% sodium citrate. Whole blood samples were mixed with dabigatran (250, 500, 1000 nM), aspirin (100 nM) plus ARC-66096 (P2Y12 inhibitor, 1000 nM) at 25¡C for 10 min. Corn trypsin inhibitor (50 μg/ml) was used to prevent contact activation. The whole blood sample was perfused in the capillary pre-coated with collagen and thromboplastin at the shear rate of 240 or 600 s−1. The process of thrombus formation was monitored by flow pressure increases inside the capillary; (i) lag time before it reaches 10 kPa (T10), (ii) occlusion time (OT) is the lag time before it reaches 80 kPa as thrombus completely occludes the capillary, and (iii) AUC30 is an area under the flow pressure curve (under 80 kPa) after 30 min of perfusion. For TG assay, platelet-rich plasma (platelet count 150 × 103/μl) was prepared from citrated whole blood. TG was triggered by adding 20 μl of CaCl2-fluorogenic substrate buffer to 80 μl of the sample mixed with tissue factor (1 pM) in each well. The lag time (min), and peak thrombin concentration (nM) were evaluated. In the flow chamber, dabigatran inhibited white thrombus formation in a concentration dependent manner at shear rates of 240 and 600 s−1(Fig. 1). At 500 nM of dabigatran, OT was prolonged by ∼2-fold from the (non-treated) control at both shear rates. The combination of aspirin and AR-C66096 only weakly suppressed thrombus formation, but it enhanced the antithrombotic efficacy of dabigatran at both shear rates (Fig. 1). In TG measurements using platelet-rich plasma, dabigatran at 500 nM prolonged the by 3.17-fold, and reduced the peak by 57.6% compared to the untreated control (Table 1). Aspirin and AR-C66096 weakly prolonged the lag time without affecting the peak height. There were relatively small changes in these parameters when antiplatelet agents were combined with dabigatran (Table 1). Our results suggest that combined antithrombotic effects of dabigatran, aspirin, and P2Y12inhibition can be demonstrated in the whole blood using the flow chamber system compared without additional plasma preparation required for TG assay. The re-calcified whole blood was perfused at the shear rate of 240 s−1 or 600 s−1. Asp/AR-C=aspirin and AR-C66096 Table 1. Lag time (min) Peak (nM) Native Asp/AR-C Native Asp/AR-C Control 6.8 ± 0.8 9.4 ± 3.2 92.1 ± 23.7 91.2 ± 29.5 Dabi 250 nM 18.6 ± 5.4 21.1 ± 4.5 69.3 ± 20.6 52.2 ± 13.6 Dabi 500 nM 21.6 ± 5.3 26.2 ± 10.2 53.0 ± 5.8 47.8 ± 9.1 Dabi 1000 nM 30.2 ± 5.6 35.1 ± 6.3 23.0 ± 6.9 22.0 ± 8.4 Dabi=dabigatran, Native=no antiplatelet agents, Asp/AR-C=aspirin and AR-C66096 Disclosures: Hosokawa: Fujimori Kogyo: Employment. Ohnishi:Fujimori Kogyo: Employment. Sameshima:Fujimori Kogyo: Employment.

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Heparin-Coated versus Uncoated Palmaz-Schatz Stent in Native Coronary Circulation. A Randomized Study with Blind Angioscopic Assessment

The International Journal of Artificial Organs ◽

10.1177/039139880202500517 ◽

2002 ◽

Vol 25 (5) ◽

pp. 461-469 ◽

Cited By ~ 4

Author(s):

S. Kedev ◽

G. Guagliumi ◽

O. Valsechi ◽

M. Tespili

Keyword(s):

Stent Thrombosis ◽

Thrombus Formation ◽

Randomized Study ◽

Quantitative Coronary Angiography ◽

Antiplatelet Agents ◽

Stent Deployment ◽

Group 2 ◽

To Receive ◽

Group 1

The increasing use of stenting to treat more complex lesions and highly thrombogenic situations still carries higher risk for subacute stent thrombosis. To assess new heparin-coated stents in a more stringent reality, 40 consecutive patients were randomized in 1:1 ratio to receive either heparin-coated (group 1, 25 stents) or uncoated Palmaz-Schatz stents (group 2, 32 stents). The two groups were similar in baseline clinical, pre-and post-procedural angiographic and angioscopic characteristics. High pressure stent deployment without intravascular ultrasound guidance was used. All pts received antiplatelet agents alone. We applied serial angioscopy (baseline and on 7th day) to evaluate thrombus formation and quantitative coronary angiography (QCA) to define late (6 months, n=39, 100% the eligible pts) neointimal regrowth. There was one subacute stent thrombosis with subsequent acute myocardial infarction and death in the uncoated group. Conclusion The implantation of heparin-coated stents in nonselected population is well tolerated and associated with no clinical or angioscopic evidence of new thrombus formation, resulting in favorable long-term clinical and angiographic outcome.

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