Abstract
Background: Recent studies indicate that aquaporin (AQP) water channels have a regulatory function in human platelet secretion and in procoagulant response of murine platelets. However, the engagement of AQPs in morphological changes, procoagulant response, and thrombus formation in human blood has never been investigated.Methods: Confocal microscopy was used to study platelet spreading, filopodia formation, ballooning, and thrombus formation under flow. Flow cytometry was utilized to assess platelet phosphatidylserine (PS) exposure and microparticles shedding. Kinetics of clot formation in vitro was evaluated by thromboelastometry. Mouse model of ferric chloride (III) (FeCl3)-induced thrombosis was used to investigate thrombus formation in vivo.Results: We found that chloroauric(III) acid (HAuCl4), a classical AQP inhibitor (10–100 μM), reduced spreading of human platelets on collagen-coated surfaces and inhibited filopodia formation in a fluid phase. Under flow conditions, HAuCl4 (100 μM) attenuated thrombi growth on collagen, platelet secretion, and PS exposure. Thrombus formation was restored by the addition of exogenous adenosine diphosphate (ADP). Collagen-evoked platelet procoagulant response (evaluated as PS exposure, shedding of microparticles, platelet-dependent thrombin generation, and membrane ballooning) was distinctly reduced by HAuCl4 (25–200 μM), as well as the dynamics of clot formation. In mouse model of thrombosis, reduction of surface of PS-positive cells within thrombus was observed in the presence of HAuCl4 (1–10 mg/kg).Conclusion: These results suggest that in human platelets AQPs are crucial for agonist-evoked morphological changes, thrombus formation under flow, and in development of procoagulant response. Antithrombotic effect in vivo suggests that nontoxic inhibitors of AQPs may be considered as potential candidates for a novel class of antiplatelet drugs.
Highlights
To test whether Au(III) may affect activation-dependent morphological changes of platelets, we examined its effect on lamellipodia and filopodia formation
To check potential effect of Au(III) on thrombus formation and platelet activation status under flow conditions, we performed flow chamber assay under arterial shear rate (1,000 s−1) with concomitant visualization of platelet bound P-selectin and exposed PS
Au(III) (100 μM) did not modulate PAC-1 antibody binding to GPIIb/IIIa receptors (PAC-1 binds to these receptors only in their active conformation) on adenosine diphosphate (ADP)- or collagen-stimulated platelets (Figure 3), ruling out the possibility that Au(III)related arrest of thrombi growth is a result of direct inhibition of GPIIb/IIIa activation
Summary
Aquaporin (AQP) water channels are members of a large, evolutionarily conserved family of integral membrane proteins that mediate rapid and selective transport of water and small solutes (e.g., glycerol) across cell and organelle membranes in response to osmotic gradient generated by ion transporters and exchangers (Agre et al, 2002; Kato et al, 2006; Hub and de Groot, 2008; Carbrey and Agre, 2009; Verkman, 2012).Topologically, AQPs are homotetramers forming four channels within the membrane, where each of them acts as independent water transductor (Sui et al, 2001; Khalili-Araghi et al, 2009; Walz et al, 2009). Deletion of AQP1 has been shown to be related with a reduction of platelet procoagulant response and thrombus formation in vivo but had minimal effect on platelet secretion and aggregation triggered by collagen in vitro (Agbani et al, 2018). Homozygosity in the AQP7 gene (G264V mutation) has been connected with the reduction of adrenalineevoked platelet aggregation and secretion from dense granules triggered by collagen or adenosine diphosphate (ADP; Goubau et al, 2013). Recent studies indicate that aquaporin (AQP) water channels have a regulatory function in human platelet secretion and in procoagulant response of murine platelets. The engagement of AQPs in morphological changes, procoagulant response, and thrombus formation in human blood has never been investigated
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