Towards the Therapeutic Use of TSP-1 (Thrombospondin-1)/CD47 Targeting TAX2 Peptide as an Antithrombotic Agent

Objective: TSP-1 (thrombospondin 1) is one of the most expressed proteins in platelet α-granules and plays an important role in the regulation of hemostasis and thrombosis. Interaction of released TSP-1 with CD47 membrane receptor has been shown to regulate major events leading to thrombus formation, for example, platelet adhesion to vascular endothelium, nitric oxide/cGMP signaling, platelet activation as well as aggregation. Therefore, targeting TSP-1:CD47 axis may represent a promising antithrombotic strategy. Approach and Results: A CD47-derived cyclic peptide was engineered, namely TAX2, that targets TSP-1 and selectively prevents TSP-1:CD47 interaction. Here, we demonstrate for the first time that TAX2 peptide strongly decreases platelet aggregation and interaction with collagen under arterial shear conditions. TAX2 also delays time for complete thrombotic occlusion in 2 mouse models of arterial thrombosis following chemical injury, while Thbs1 −/− mice recapitulate TAX2 effects. Importantly, TAX2 administration is not associated with increased bleeding risk or modification of hematologic parameters. Conclusions: Overall, this study sheds light on the major contribution of TSP-1:CD47 interaction in platelet activation and thrombus formation while putting forward TAX2 as an innovative antithrombotic agent with high added-value.

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A Short Half-Life αIIbβ3 Antagonist ANTP266 Reduces Thrombus Formation

International Journal of Molecular Sciences ◽

10.3390/ijms19082306 ◽

2018 ◽

Vol 19 (8) ◽

pp. 2306 ◽

Cited By ~ 1

Author(s):

Tong-Dan Liu ◽

Shen-Hong Ren ◽

Xue Ding ◽

Zhou-Ling Xie ◽

Yi Kong

Keyword(s):

Half Life ◽

Bleeding Time ◽

Thrombus Formation ◽

Bleeding Risk ◽

Effective Dosage ◽

Enzyme Linked Immunosorbent Assay ◽

Severe Bleeding ◽

Antithrombotic Agent ◽

Integrin Αiibβ3 ◽

Plasma Half Life

Integrin αIIbβ3 plays a pivotal role in platelet aggregation. Three αIIbβ3 antagonists have been approved by the Food and Drug Administration (FDA) for the treatment of cardiovascular diseases. Unfortunately, all of these three drugs can cause the side effect of severe bleeding. Therefore, developing a new αIIbβ3 antagonist with low bleeding was needed. In the present study, we screened compounds by using a fibrinogen/integrin αIIbβ3 enzyme-linked immunosorbent assay (ELISA), and a novel αIIbβ3 antagonist ANTP266 was attained. The antithrombotic effects of ANTP266 were estimated by using two animal models, the bleeding risk was estimated by using a mice tail cutting assay, and the plasma half-life time was tested by LC-MS/MS. The results showed that ANTP266 potently decreased thrombosis formation, while not prolonging bleeding time at its effective dosage. The bleeding of ANTP266 reduced rapidly as time went on from 5 to 60 min, but tirofiban produced high bleeding continuously. The plasma half-life of ANTP266 in rats was 10.8 min. Taken together, ANTP266 is an effective antithrombotic agent with a low bleeding risk. The shorter bleeding time benefits from its short plasma half-life. ANTP266 could be a candidate for developing the αIIbβ3 antagonist of rapid elimination for a patient undergoing percutaneous coronary intervention.

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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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Oxidized LDL activates blood platelets through CD36/NOX2–mediated inhibition of the cGMP/protein kinase G signaling cascade

Blood ◽

10.1182/blood-2014-05-574491 ◽

2015 ◽

Vol 125 (17) ◽

pp. 2693-2703 ◽

Cited By ~ 73

Author(s):

Simbarashe Magwenzi ◽

Casey Woodward ◽

Katie S. Wraith ◽

Ahmed Aburima ◽

Zaher Raslan ◽

...

Keyword(s):

Protein Kinase ◽

Tyrosine Kinase ◽

Platelet Activation ◽

Thrombus Formation ◽

Blood Platelets ◽

Oxidized Ldl ◽

Protein Kinase G ◽

Signaling Cascade ◽

Key Points ◽

Cgmp Signaling

Key Points oxLDL binds platelet CD36 to stimulate tyrosine kinase– and PKC-dependent activation of NOX2 and generation of ROS. oxLDL- and hyperlipidemia-induced ROS mediate platelet desensitization to inhibitory cGMP signaling to facilitate platelet activation and thrombus formation.

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GRK6 Regulates the Hemostatic Response to Injury through Its Rate-Limiting Effects on GPCR Signaling in Platelets

Blood ◽

10.1182/blood-2019-129175 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3611-3611

Author(s):

Xi Chen ◽

Shuchi Gupta ◽

Matthew Cooper ◽

Daniel Dehelian ◽

Xuefei Zhao ◽

...

Keyword(s):

Platelet Activation ◽

Conflicts Of Interest ◽

Early Stage ◽

Thrombus Formation ◽

Surface Expression ◽

Cerebrovascular Diseases ◽

Human Platelets ◽

Akt Activation ◽

Granule Secretion ◽

First Time

Inappropriate platelet activation remains a major cause of cardiovascular and cerebrovascular diseases. Most agonists activate platelets through G protein-coupled receptors (GPCRs). However, questions remain about mechanisms that provide negative feedback towards activated GPCRs to limit platelet activation and thrombus formation. Here we provide the first evidence that GPCR kinase 6 (GRK6) serves this role in platelets, using GRK6-/- mice generated by CRISPR-Cas9 genome editing to examine the consequences of GRK6 knockout on GPCR-dependent signaling. Hemostatic thrombi formed in GRK6-/- mice are larger than in WT controls during the early stages of thrombus formation, with a rapid increase of platelet accumulation at site of injury. Platelet activation in the absence of GRK6 is enhanced, but in an agonist-selective manner. Responses to PAR4 agonist peptide or ADP stimulation in GRK6-/- platelets are increased compared to WT control littermates, while the response to TxA2 is normal. Underlying these changes in GRK6-/- platelets is an increase in Ca2+ mobilization, Akt activation, and granule secretion. Furthermore, deletion of GRK6 in human MEG-01 cells causes an increase in Ca2+ response and PAR1 surface expression in response to thrombin. Finally, we show that in human platelets, platelet activation in response to thrombin causes an increase in binding of GRK6 to PAR1, as well as an increase of the phosphorylation of PAR1. Deletion of GRK6 in MEG-01 cells causes a decrease in PAR1 phosphorylation. Collectively, these observations, for the first time, show that 1) GRK6 regulates the hemostatic response to injury by thrombin and ADP, 2) it mediates platelet activation by reducing PAR1/4- and P2Y12-dependent signaling, and 3) GRK6 limits the rate of platelet activation during early stage of thrombus growth and helps prevent inappropriate platelet activation. Disclosures No relevant conflicts of interest to declare.

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An Antithrombotic Strategy by Targeting Phospholipase D in Human Platelets

Journal of Clinical Medicine ◽

10.3390/jcm7110440 ◽

2018 ◽

Vol 7 (11) ◽

pp. 440 ◽

Cited By ~ 3

Author(s):

Wan Lu ◽

Chi Chung ◽

Ray Chen ◽

Li Huang ◽

Li Lien ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Phospholipase D ◽

Thrombus Formation ◽

Human Platelets ◽

Platelet Activity ◽

Clot Retraction ◽

First Time

Phospholipase D (PLD) is involved in many biological processes. PLD1 plays a crucial role in regulating the platelet activity of mice; however, the role of PLD in the platelet activation of humans remains unclear. Therefore, we investigated whether PLD is involved in the platelet activation of humans. Our data revealed that inhibition of PLD1 or PLD2 using pharmacological inhibitors effectively inhibits platelet aggregation in humans. However, previous studies have showed that PLD1 or PLD2 deletion did not affect mouse platelet aggregation in vitro, whereas only PLD1 deletion inhibited thrombus formation in vivo. Intriguingly, our data also showed that the pharmacological inhibition of PLD1 or PLD2 does not affect mouse platelet aggregation in vitro, whereas the inhibition of only PLD1 delayed thrombus formation in vivo. These findings indicate that PLD may play differential roles in humans and mice. In humans, PLD inhibition attenuates platelet activation, adhesion, spreading, and clot retraction. For the first time, we demonstrated that PLD1 and PLD2 are essential for platelet activation in humans, and PLD plays different roles in platelet function in humans and mice. Our findings also indicate that targeting PLD may provide a safe and alternative therapeutic approach for preventing thromboembolic disorders.

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Intracellular cyclophilin A is an important Ca2+ regulator in platelets and critically involved in arterial thrombus formation

Blood ◽

10.1182/blood-2011-12-398438 ◽

2012 ◽

Vol 120 (6) ◽

pp. 1317-1326 ◽

Cited By ~ 40

Author(s):

Margitta Elvers ◽

Annika Herrmann ◽

Peter Seizer ◽

Patrick Münzer ◽

Sandra Beck ◽

...

Keyword(s):

Platelet Activation ◽

Arterial Thrombosis ◽

Thrombus Formation ◽

Critical Role ◽

Cyclophilin A ◽

Primary Hemostasis ◽

Chaperone Protein ◽

Extracellular Compartment ◽

Arterial Thrombus ◽

First Time

Abstract Platelet adhesion and aggregation play a critical role in primary hemostasis. Uncontrolled platelet activation leads to pathologic thrombus formation and organ failure. The decisive central step for different processes of platelet activation is the increase in cytosolic Ca2+ activity ([Ca2+]i). Activation-dependent depletion of intracellular Ca2+ stores triggers Ca2+ entry from the extracellular space. Stromal interaction molecule 1 (STIM1) has been identified as a Ca2+ sensor that regulates store-operated Ca2+ entry through activation of the pore-forming subunit Orai1, the major store-operated Ca2+ entry channel in platelets. In the present study, we show for the first time that the chaperone protein cyclophilin A (CyPA) acts as a Ca2+ modulator in platelets. CyPA deficiency strongly blunted activation-induced Ca2+ mobilization from intracellular stores and Ca2+ influx from the extracellular compartment and thus impaired platelet activation substantially. Furthermore, the phosphorylation of the Ca2+ sensor STIM1 was abrogated upon CyPA deficiency, as shown by immunoprecipitation studies. In a mouse model of arterial thrombosis, CyPA-deficient mice were protected against arterial thrombosis, whereas bleeding time was not affected. The results of the present study identified CyPA as an important Ca2+ regulator in platelets, a critical mechanism for arterial thrombosis.

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BF061, a novel antiplatelet and antithrombotic agent targeting P2Y12 receptor and phosphodiesterase

Thrombosis and Haemostasis ◽

10.1160/th11-06-0400 ◽

2011 ◽

Vol 106 (12) ◽

pp. 1203-1214 ◽

Cited By ~ 21

Author(s):

Liang Hu ◽

Zhichao Fan ◽

Hongguang Du ◽

Ran Ni ◽

Si Zhang ◽

...

Keyword(s):

Arterial Thrombosis ◽

Thrombus Formation ◽

Antiplatelet Agent ◽

Atp Release ◽

Bleeding Risk ◽

P2y12 Receptor ◽

Antithrombotic Agent ◽

Thrombosis Model ◽

P2y12 Antagonists ◽

Pde Inhibition

SummaryThe addition of phosphodiesterase (PDE) inhibitors has been reported to potentiate the antithrombotic effects of P2Y12 antagonists without increasing bleeding risk. In this study, we report that a potent antiplatelet agent, 2-ethylthio-6-phenethylaminoadenosine (BF061), inhibits platelet activation and thrombosis via P2Y12 antagonism and PDE inhibition. We explored the antiplatelet mechanism of BF061 by measuring cAMP, cGMP levels, PDE activity, and the interaction between ADP and P2Y12 using atomic force microscopy. The antithrombotic effect of BF061 was evaluated in mice using intravital microscopy in FeCl3-induced mesenteric and laser-induced cremasteric arterial thrombosis models. BF061 robustly inhibited platelet aggregation and ATP release induced by multiple platelet agonists via P2Y12 antagonism and PDE inhibition. Interestingly, despite being structurally similar to BF061, P2Y12 receptor antagonist AR-C69931MX had no effect on human platelet PDE. In FeCl3-induced mesenteric arterial thrombosis model, BF061 effectively prevented thrombus formation similarly to clopidogrel; it also reduced thrombus volume in laser-injured cremaster arteriole model. In contrast, BF061 induced dramatically less bleeding at an antithrombotic dose compared to clopidogrel. In summary, we developed a novel antiplatelet and antithrombotic agent targeting both P2Y12 and PDE. Given the prevalence of combined antiplatelet therapy in clinical practice, an antiplatelet agent bearing dual activities may have therapeutic advantage as a potential antithrombotic drug.

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Doxepin inhibits GPVI-dependent platelet Ca2+ signaling and collagen-dependent thrombus formation

AJP Cell Physiology ◽

10.1152/ajpcell.00262.2016 ◽

2017 ◽

Vol 312 (6) ◽

pp. C765-C774 ◽

Cited By ~ 2

Author(s):

Sascha Geue ◽

Britta Walker-Allgaier ◽

Daniela Eißler ◽

Roland Tegtmeyer ◽

Malte Schaub ◽

...

Keyword(s):

Platelet Activation ◽

Platelet Adhesion ◽

Thrombus Formation ◽

Tricyclic Antidepressant ◽

Thrombotic Occlusion ◽

Shear Rates ◽

Glycoprotein Vi ◽

Primary Hemostasis ◽

Intracellular Ca

Platelet adhesion, activation, and aggregation are essential for primary hemostasis, but are also critically involved in the development of acute arterial thrombotic occlusion. Stimulation of the collagen receptor glycoprotein VI (GPVI) leads to phospholipase Cγ2-dependent inositol triphosphate (IP3) production with subsequent platelet activation, due to increased intracellular Ca2+ concentration ([Ca2+]i). Although tricyclic antidepressants have been shown to potentially impair platelet activation, nothing is hitherto known about potential effects of the tricyclic antidepressant doxepin on platelet Ca2+ signaling and thrombus formation. As shown in the present study, doxepin significantly diminished the stimulatory effect of GPVI agonist collagen-related peptide (CRP) on intracellular Ca2+ release as well as subsequent extracellular Ca2+ influx. Doxepin was partially effective by impairment of CRP-dependent IP3 production. Moreover, doxepin abrogated CRP-induced platelet degranulation and integrin αIIbβ3 activation and aggregation. Finally, doxepin markedly blunted in vitro platelet adhesion to collagen and thrombus formation under high arterial shear rates (1,700−s). In conclusion, doxepin is a powerful inhibitor of GPVI-dependent platelet Ca2+ signaling, platelet activation, and thrombus formation.

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Antithrombotic Therapy in Acute Coronary Syndromes: Current Evidence and Ongoing Issues Regarding Early and Late Management

Thrombosis and Haemostasis ◽

10.1055/s-0040-1722188 ◽

2021 ◽

Author(s):

Paul Guedeney ◽

Jean-Philippe Collet

Keyword(s):

Thrombus Formation ◽

Antiplatelet Agents ◽

Bleeding Risk ◽

Coronary Intervention ◽

Therapeutic Strategies ◽

Added Value ◽

Current Evidence ◽

Onset Of Action ◽

P2y12 Inhibitors ◽

Coronary Syndrome

AbstractA few decades ago, the understanding of the pathophysiological processes involved in the coronary artery thrombus formation has placed anticoagulant and antiplatelet agents at the core of the management of acute coronary syndrome (ACS). Increasingly potent antithrombotic agents have since been evaluated, in various association, timing, or dosage, in numerous randomized controlled trials to interrupt the initial thrombus formation, prevent ischemic complications, and ultimately improve survival. Primary percutaneous coronary intervention, initial parenteral anticoagulation, and dual antiplatelet therapy with potent P2Y12 inhibitors have become the hallmark of ACS management revolutionizing its prognosis. Despite these many improvements, much more remains to be done to optimize the onset of action of the various antithrombotic therapies, for further treating and preventing thrombotic events without exposing the patients to an unbearable hemorrhagic risk. The availability of various potent P2Y12 inhibitors has opened the door for individualized therapeutic strategies based on the clinical setting as well as the ischemic and bleeding risk of the patients, while the added value of aspirin has been recently challenged. The strategy of dual-pathway inhibition with P2Y12 inhibitors and low-dose non-vitamin K antagonist oral anticoagulant has brought promising results for the early and late management of patients presenting with ACS with and without indication for oral anticoagulation. In this updated review, we aimed at describing the evidence supporting the current gold standard of antithrombotic management of ACS. More importantly, we provide an overview of some of the ongoing issues and promising therapeutic strategies of this ever-evolving topic.

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Regulation of Key Antiplatelet Pathways by Bioactive Compounds with Minimal Bleeding Risk

International Journal of Molecular Sciences ◽

10.3390/ijms222212380 ◽

2021 ◽

Vol 22 (22) ◽

pp. 12380

Author(s):

Eduardo Fuentes ◽

Sergio Wehinger ◽

Andrés Trostchansky

Keyword(s):

Platelet Activation ◽

Bioactive Compounds ◽

Fruits And Vegetables ◽

Plaque Rupture ◽

Thrombus Formation ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Bleeding Risk ◽

Activation Process ◽

Mitogen Activated Protein

Cardiovascular disease is strongly influenced by platelet activation. Platelet activation and thrombus formation at atherosclerotic plaque rupture sites is a dynamic process regulated by different signaling networks. Therefore, there are now focused efforts to search for novel bioactive compounds which target receptors and pathways in the platelet activation process while preserving normal hemostatic function. The antiplatelet activity of numerous fruits and vegetables and their multiple mechanisms of action have recently been highlighted. In this review, we review the antiplatelet actions of bioactive compounds via key pathways (protein disulfide isomerase, mitogen-activated protein kinases, mitochondrial function, cyclic adenosine monophosphate, Akt, and shear stress-induced platelet aggregation) with no effects on bleeding time. Therefore, targeting these pathways might lead to the development of effective antiplatelet strategies that do not increase the risk of bleeding.

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