Abstract
Platelets play a major role in hemostasis and thrombosis, by binding to the underlying extracellular matrix around injured blood vessels, via integrin receptors. In this study, we investigated the effects of adhesive ligand spacing on the stability of platelets’ adhesion and the mode of their spreading on extracellular surfaces. Toward this end, we have examined the differential adhesion and spreading of human platelets onto nanogold-patterned surfaces, functionalized with the αIIbβ3 integrin ligand, SN528. Combining light- and scanning electron-microscopy, we found that interaction of platelets with surfaces coated with SN528 at spacing of 30–60 nm induces the extension of filopodia through which the platelets stably attach to the nanopatterned surface and spread on it. Increasing the nanopattern-gold spacing to 80–100 nm resulted in a dramatic reduction (>95%) in the number of adhering platelets. Surprisingly, a further increase in ligand spacing to 120 nm resulted in platelet binding to the surface at substantially larger numbers, yet these platelets remained discoid and were essentially devoid of filopodia and lamellipodia. These results indicate that the stimulation of filopodia extension by adhering platelets, and the consequent spreading on these surfaces depend on different ligand densities. Thus, the extension of filopodia occurs on surfaces with a ligand spacing of 100 nm or less, while the sustainability and growth of these initial adhesions and induction of extensive platelet adhesion and spreading requires lower ligand-to-ligand spacing (≤60 nm). The mechanisms underlying this differential ligand-density sensing by platelets, as well as the unexpected retention of discoid platelets on surfaces with even larger spacing (120 nm) are discussed.
Highlights
Platelets are cell fragments, derived from bone marrow megakaryocytes, which play an essential role in arresting bleeding from injured vessels.[1,2] They are involved in multiple physiological processes, including the regulation of vascular integrity, angiogenesis, inflammation, and wound healing.[3]
It had been extensively demonstrated that the functionality of platelets is tightly regulated by their selective adhesion processes, whereby they adhere to sites of injury, where endothelial cells are altered, the subendothelial extracellular matrix (ECM) is exposed, and a blood clot is formed.[4−7] Initial adhesive interaction with extracellular surfaces, such as a fibrin clot or basement membrane-associated collagen, trigger platelet activation, which further regulates the adhesion to and spreading on the external surfaces.[8,9]
We address the effect of ligand spacing on the formation and stability of platelet adhesions to surfaces, functionalized with a biomimetic ligand (SN528), highly specific for αIIbβ[3] integrin, with IC50 = 3.58 nM.[50]
Summary
Silane, which binds the exposed glass surface, and serves as cell repellant layer; (C) Representative HR-SEM micrograph of nanostructured glass surfaces with interparticle spacing of 30, 60, 80, 100, and 120 nm ±10; (D) Immunofluorescence images of actin-labeled platelets seeded on (left to right) 30, 60, 80, 100, and 120 nm nanopatterned surfaces, bioactivated with SN528; (E) Quantification of the number of platelets per 1 mm[2] of substrate. The vast majority of platelets adhering to the 120 nm surfaces remained discoid (Figure 4C) This observation is consistent with the interpretation that platelets interacting with nanopatterned surfaces with spacing of up to 100 nm can induce filopodia extension, yet a higher ligand density (spacing of up to 60 nm) is needed to further sustain stable adhesion (Figure 4A). We further show that substrates with ligand spacing of 30 and 60 nm, support platelet adhesion and spreading, comparable to those obtained with platelets seeded on uniform SN528 or fibrinogen, though, the 30 nm surfaces are more efficient in stimulating full lamellipodial spreading compared to the 60 nm surfaces, which induced mainly filopodial adhesion and were more effective in recruiting zyxin to the adhesion site, suggesting a higher contractile activity.
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