Studying the efficacy of antiplatelet drugs on atherosclerosis by optofluidic imaging on a chip.

Vascular stenosis caused by atherosclerosis instigates activation and aggregation of platelets, eventually resulting in thrombus formation. Although antiplatelet drugs are commonly used to inhibit platelet activation and aggregation, they unfortunately cannot prevent recurrent thrombotic events in patients with atherosclerosis. This is partially due to the limited understanding of the efficacy of antiplatelet drugs in the complex hemodynamic environment of vascular stenosis. Conventional methods for evaluating the efficacy of antiplatelet drugs under stenosis either fail to simulate the hemodynamic environment of vascular stenosis characterized by high shear stress and recirculatory flow or lack spatial resolution in their analytical techniques to statistically identify and characterize platelet aggregates. Here we propose and experimentally demonstrate a method comprising an in vitro 3D stenosis microfluidic chip and an optical time-stretch quantitative phase imaging system for studying the efficacy of antiplatelet drugs under stenosis. Our method simulates the atherogenic flow environment of vascular stenosis while enabling high-resolution and statistical analysis of platelet aggregates. Using our method, we distinguished the efficacy of three antiplatelet drugs, acetylsalicylic acid (ASA), cangrelor, and eptifibatide, for inhibiting platelet aggregation induced by stenosis. Specifically, ASA failed to inhibit stenosis-induced platelet aggregation, while eptifibatide and cangrelor showed high and moderate efficacy, respectively. Furthermore, we demonstrated that the drugs tested also differed in their efficacy for inhibiting platelet aggregation synergistically induced by stenosis and agonists (e.g., adenosine diphosphate, and collagen). Taken together, our method is an effective tool for investigating the efficacy of antiplatelet drugs under vascular stenosis, which could assist the development of optimal pharmacologic strategies for patients with atherosclerosis.

The Pharmacological Effects of Silver Nanoparticles Functionalized with Eptifibatide on Platelets and Endothelial Cells.

Purpose: In the search for new drug delivery platforms for cardiovascular diseases and coating of medical devices, we synthesized eptifibatide-functionalized silver nanoparticles (AgNPs-EPI) and examined the pharmacological activity of AgNPs-EPI on platelets and endothelial cells in vitro and ex vivo. Methods: Spherical AgNPs linked to eptifibatide were synthesized and characterized. Cytotoxicity was measured in microvascular endothelial cells (HMEC-1), platelets and red blood cells. Platelet mitochondrial respiration was measured using the Oxygraph-2k, a high-resolution modular respirometry system. The effect of AgNPs-EPI on the aggregation of washed platelets was measured by light aggregometry and the ex vivo occlusion time was determined using a reference laboratory method. The surface amount of platelet receptors such as P-selectin and GPIIb/IIIa was measured. The influence of AgNPS-EPI on blood coagulation science was assessed. Finally, the effect of AgNPs-EPI on endothelial cells was measured by the levels of 6-keto-PGF1alpha, tPa, cGMP and vWF. Results: We describe the synthesis of AgNPs using eptifibatide as the stabilizing ligand. The molecules of this drug are directly bonded to the surface of the nanoparticles. The synthesized AgNPs-EPI did not affect the viability of platelets, endothelial cells and erythrocytes. Preincubation of platelets with AgNPs-EPI protected by mitochondrial oxidative phosphorylation capacity. AgNPs-EPI inhibited aggregation-induced P-selectin expression and GPIIb/IIIa conformational changes in platelets. AgNPs-EPI caused prolongation of the occlusion time in the presence of collagen/ADP and collagen/adrenaline. AgNPs-EPI regulated levels of 6-keto-PGF1alpha, tPa, vWf and cGMP produced in thrombin stimulated HMEC-1 cells. Conclusion: AgNPs-EPI show anti-aggregatory activity at concentrations lower than those required by the free drug acting via regulation of platelet aggregation, blood coagulation, and endothelial cell activity. Our results provide proof-of-principle evidence that AgNPs may be used as an effective delivery platform for antiplatelet drugs.

Unraveling the role of the homoarginine residue in antiplatelet drug eptifibatide in binding to the αIIbß3 integrin receptor.

Eptifibatide is an αIIbß3 inhibitor that is currently used in the clinic. More than 10 scientific communications indicate that eptifibatide has a Lys-Gly-Asp or Arg-Gly-Asp sequence, while it actually has a hArg-Gly-Asp sequence. We aimed to unravel the importance of the homoarginine residue in eptifibatide in platelet activation and aggregation. Arg- and Lys-eptifibatide were synthesized by solid-phase peptide synthesis and measured in light transmission aggregometry, flow cytometry and whole blood thrombus formation under flow. Interactions of eptifibatide and its variants with αIIbß3 integrin were studied using molecular dynamics simulations. Eptifibatide showed inhibition of collagen- and ADP-induced platelet aggregation, while Arg- and Lys-eptifibatide did not. Multiparameter assessment of thrombus formation showed suppressed platelet aggregate and fibrin formation upon eptifibatide treatment, in contrast to the other variants. Molecular dynamics simulations revealed that the hArg residue in eptifibatide is crucial to its activity, since the substitution of the hArg to Arg or Lys resulted in the inability to form double H-bonds with Asp224 in the αIIb chain of the αIIbß3 receptor. The hArg is pivotal for the interaction of eptifibatide for the αIIbß3 receptor and efficient inhibition of platelet aggregation.

Inhibition of Src but not Syk causes weak reversal of GPVI-mediated platelet aggregation measured by light transmission aggregometry.

Src tyrosine kinases and spleen tyrosine kinase (Syk) have recently been shown to contribute to sustained platelet aggregation on collagen under arterial shear. In the present study, we have investigated whether Src and Syk are required for aggregation under minimal shear following activation of glycoprotein VI (GPVI) and have extended this to C-type lectin-like receptor-2 (CLEC-2) which signals through the same pathway. Aggregation was induced by the GPVI ligand collagen-related peptide (CRP) and the CLEC-2 ligand rhodocytin and monitored by light transmission aggregometry (LTA). Aggregation and tyrosine phosphorylation by both receptors were sustained for up to 50 min. The addition of inhibitors of Src, Syk or Bruton's tyrosine kinase (Btk) at 150 sec, by which time aggregation was maximal, induced rapid loss of tyrosine phosphorylation of their downstream proteins, but only Src kinase inhibition caused a weak (~10%) reversal in light transmission. A similar effect was observed when the inhibitors were combined with apyrase and indomethacin or glycoprotein IIb-IIIa (GPIIb-IIIa) antagonist, eptifibatide. On the other hand, activation of GPIIb-IIIa by GPVI in a diluted platelet suspension, as measured by binding of fluorescein isothiocyanate-labeled antibody specific for the activated GPIIb-IIIa (FITC-PAC1), was reversed on the addition of Src and Syk inhibitors showing that integrin activation is rapidly reversible in the absence of outside-in signals. The results demonstrate that Src but not Syk and Btk contribute to sustained aggregation as monitored by LTA, possibly as a result of inhibition of outside-in signaling from GPIIb-IIIa to the cytoskeleton through a Syk-independent pathway. This is in contrast to the role of Syk in supporting sustained aggregation on collagen under arterial shear.

Platelet integrin αIIbß3: signal transduction, regulation, and its therapeutic targeting.

Integrins are a family of transmembrane glycoprotein signaling receptors that can transmit bioinformation bidirectionally across the plasma membrane. Integrin αIIbß3 is expressed at a high level in platelets and their progenitors, where it plays a central role in platelet functions, hemostasis, and arterial thrombosis. Integrin αIIbß3 also participates in cancer progression, such as tumor cell proliferation and metastasis. In resting platelets, integrin αIIbß3 adopts an inactive conformation. Upon agonist stimulation, the transduction of inside-out signals leads integrin αIIbß3 to switch from a low- to high-affinity state for fibrinogen and other ligands. Ligand binding causes integrin clustering and subsequently promotes outside-in signaling, which initiates and amplifies a range of cellular events to drive essential platelet functions such as spreading, aggregation, clot retraction, and thrombus consolidation. Regulation of the bidirectional signaling of integrin αIIbß3 requires the involvement of numerous interacting proteins, which associate with the cytoplasmic tails of αIIbß3 in particular. Integrin αIIbß3 and its signaling pathways are considered promising targets for antithrombotic therapy. This review describes the bidirectional signal transduction of integrin αIIbß3 in platelets, as well as the proteins responsible for its regulation and therapeutic agents that target integrin αIIbß3 and its signaling pathways.