Whole Blood Based Multiparameter Assessment of Thrombus Formation in Standard Microfluidic Devices to Proxy In Vivo Haemostasis and Thrombosis

Provenzale, Isabella; Brouns, Sanne L. N.; Meijden, Paola E. J. van der; Swieringa, Frauke; Heemskerk, Johan W. M.

doi:10.3390/mi10110787

Cited by 18 publications

(16 citation statements)

References 90 publications

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“…After initial compound screening, the further validation of hit antagonists involves a long process by applying several methodologies. In research to platelet GPCRs or ILR signaling inhibitors with antithrombotic potential, conventional further assays in hit-to-lead identification are light transmission aggregometry and whole-blood microfluidics assays ( Provenzale et al., 2019 ). Bioavailability and toxicity assays as well as medicinal chemical improvements also need to be done before continuing the drug development process ( Choi and Choi, 2017 ).…”

Section: Discussionmentioning

confidence: 99%

Ultra-high-throughput Ca2+ assay in platelets to distinguish ITAM-linked and G-protein-coupled receptor activation

et al. 2022

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Summary Antiplatelet drugs targeting G-protein-coupled receptors (GPCRs), used for the secondary prevention of arterial thrombosis, coincide with an increased bleeding risk. Targeting ITAM-linked receptors, such as the collagen receptor glycoprotein VI (GPVI), is expected to provide a better antithrombotic-hemostatic profile. Here, we developed and characterized an ultra-high-throughput (UHT) method based on intracellular [Ca 2+ ] i increases to differentiate GPVI and GPCR effects on platelets. In 96-, 384-, or 1,536-well formats, Calcium-6-loaded human platelets displayed a slow-prolonged or fast-transient [Ca 2+ ] i increase when stimulated with the GPVI agonist collagen-related peptide or with thrombin and other GPCR agonists, respectively. Semi-automated curve fitting revealed five parameters describing the Ca 2+ responses. Verification of the UHT assay was done with a robustness compound library and clinically relevant platelet inhibitors. Taken together, these results present proof of principle of distinct receptor-type-dependent Ca 2+ signaling curves in platelets, which allow identification of new inhibitors in a UHT way.

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Section: Discussionmentioning

confidence: 99%

Ultra-high-throughput Ca2+ assay in platelets to distinguish ITAM-linked and G-protein-coupled receptor activation

et al. 2022

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“…To investigate how the novel collagen receptor GPR56 contributes to platelet adhesion and thrombus formation, we treated blood from healthy donors with the synthetized peptide pGRP (TYFAVLM, 50 μg/mL), corresponding to the hidden tethered ligand of GPR56, and hence mimicking the shear-dependent activation of this receptor [ 32 ]. Perfusion of the blood was performed at an arterial wall-shear rate of 1600 s −1 [ 11 , 35 ]. For platelet interaction, microspots of collagen I, collagen III, and collagen IV were used with decreasing GPVI dependency.…”

Section: Resultsmentioning

confidence: 99%

Roles of Focal Adhesion Kinase PTK2 and Integrin αIIbβ3 Signaling in Collagen- and GPVI-Dependent Thrombus Formation under Shear

Huang

Jooss

Fernández

et al. 2022

IJMS

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Glycoprotein (GP)VI and integrin αIIbβ3 are key signaling receptors in collagen-dependent platelet aggregation and in arterial thrombus formation under shear. The multiple downstream signaling pathways are still poorly understood. Here, we focused on disclosing the integrin-dependent roles of focal adhesion kinase (protein tyrosine kinase 2, PTK2), the shear-dependent collagen receptor GPR56 (ADGRG1 gene), and calcium and integrin-binding protein 1 (CIB1). We designed and synthetized peptides that interfered with integrin αIIb binding (pCIB and pCIBm) or mimicked the activation of GPR56 (pGRP). The results show that the combination of pGRP with PTK2 inhibition or of pGRP with pCIB > pCIBm in additive ways suppressed collagen- and GPVI-dependent platelet activation, thrombus buildup, and contraction. Microscopic thrombus formation was assessed by eight parameters (with script descriptions enclosed). The suppressive rather than activating effects of pGRP were confined to blood flow at a high shear rate. Blockage of PTK2 or interference of CIB1 no more than slightly affected thrombus formation at a low shear rate. Peptides did not influence GPVI-induced aggregation and Ca2+ signaling in the absence of shear. Together, these data reveal a shear-dependent signaling axis of PTK2, integrin αIIbβ3, and CIB1 in collagen- and GPVI-dependent thrombus formation, which is modulated by GPR56 and exclusively at high shear. This work thereby supports the role of PTK2 in integrin αIIbβ3 activation and signaling.

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“…[33,41] According to previous studies, when anticoagulated blood is used, the thrombi are formed because of platelet activation only, as the generation of thrombin and fibrin is prevented. [18,42] Collagen type-I is the major ECM protein of human body with a characteristic right-handed triple helical structure and the most thrombogenic component of the sub-endothelium. Resting platelets react with insoluble collagen fibers or an immobilized collagen surface.…”

Section: Discussionmentioning

confidence: 99%

Atherothrombosis‐on‐Chip: A Site‐Specific Microfluidic Model for Thrombus Formation and Drug Discovery

et al. 2022

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Atherothrombosis, an atherosclerotic plaque disruption condition with superimposed thrombosis, is the underlying cause of cardiovascular episodes. Herein, a unique design is presented to develop a microfluidic site‐specific atherothrombosis‐on‐chip model, providing a universal platform for studying the crosstalk between blood cells and plaque components. The device consists of two interconnected microchannels, namely main and supporting channels: the former mimics the vessel geometry with different stenosis, and the latter introduces plaque components to the circulation simultaneously. The unique design allows the site‐specific introduction of plaque components in stenosed channels ranging from 0% to above 50%, resulting in thrombosis, which has not been achieved previously. The device successfully explains the correlation between vessel geometry and thrombus formation phenomenon as well as the influence of shear rate on platelet aggregation, confirming the reliability and the effectiveness of the design. The device exhibits significant sensitivity to aspirin. In therapeutic doses (50 × 10−6 and 100 × 10−6 m), aspirin delays and prevents platelet adhesion, thereby reducing the thrombus area in a dose‐dependent manner. Finally, the device is effectively employed in testing the targeted binding of the RGD (arginyl‐glycyl‐aspartic acid) labeled polymeric nanoparticles on the thrombus, extending the use of the device to examine targeted drug carriers.

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Whole Blood Based Multiparameter Assessment of Thrombus Formation in Standard Microfluidic Devices to Proxy In Vivo Haemostasis and Thrombosis

Cited by 18 publications

References 90 publications

Ultra-high-throughput Ca2+ assay in platelets to distinguish ITAM-linked and G-protein-coupled receptor activation

Ultra-high-throughput Ca2+ assay in platelets to distinguish ITAM-linked and G-protein-coupled receptor activation

Roles of Focal Adhesion Kinase PTK2 and Integrin αIIbβ3 Signaling in Collagen- and GPVI-Dependent Thrombus Formation under Shear

Atherothrombosis‐on‐Chip: A Site‐Specific Microfluidic Model for Thrombus Formation and Drug Discovery

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