Abstract
Introduction
Soluble plasma fibronectin (Fn) with its inactive compact structure needs to be unfolded to assemble into active fibrils. Fibril formation of Fn is cell-mediated and depends on interactions between Fn and integrin receptors, through binding to αIIbβ3, α5β1, or αvβ3. Less is known about the contribution of biomechanical forces on the fibrillogenesis of Fn. Mechanical forces assessed by atomic force microscopy were shown to regulate the transformation of Fn from its compact structure to its extended fibrillar state when cryptic binding sites of Fn type III repeats were substituted (Gao et al., 2003). The aim of this study was to investigate conformational changes of Fn, as induced (1) by platelet integrin receptors and/or (2) by shear rates simulating venous or arterial flow conditions.
Methods
Fn isolated from fresh frozen human plasma was added, at different concentrations (50 or 100 μg/ml), to plates pre-coated with 100 μg/ml of soluble Fn or BSA. Subsequently, the solutions were exposed to shear generated by different stainless steel cones with bare surfaces, which were used as controls, or which were pre-grafted with O,O’-Bis(amino-propyl) polyethylene glycol (PEG) once or twice to reduce non-specific adsorption of plasma proteins. To study the effects of platelet on conformational changes of Fn, in the absence or presence of washed platelets (2.5 x 107/ml or 2.5 x 106/ml), soluble Fn (100 μg/ml) was exposed to shear generated by cones grafted with PEG twice. To examine the role of distinct platelet integrins on fibril formation of Fn, washed platelets (2.5 x 107/ml) were incubated with the monoclonal antibodies LM609, P1D6, 10E5, or abciximab (10 μg/ml, each) for 30 min at room temperature to block αvβ3, α5β1, αIIbβ3, or both αIIbβ3 and αvβ3, respectively, prior to the addition of Fn (100 μg/ml) and shear exposure.
In all experiments, flow conditions were simulated by dynamic shear rates stepwise increasing from 50 s-1 to 5000 s-1 within 5 min and subsequently decreasing from 5000 s-1 to 50 s-1 within 5 min using a cone-plate rheometer (Haake Rheostress 1). Viscosities (mPa s) of shear-exposed solutions were recorded over 10 min. To quantify the amount of fibril formation, DOC solubility assays and Western blotting were performed. Control experiments were conducted under static conditions.
Results
Upon exposure to shear stress, the viscosity in the sample increased, suggesting conformational changes in Fn. Western blotting and densitometric analyses revealed that, under shear generated by untreated cones, the ratios of insoluble to soluble Fn (indicative of fibril formation) increased significantly from 0.018 ± 0.012 (mean ratio ± SD) to 0.121 ± 0.08 (p < 0.05, n = 4) (Fn-immobilized plates) and from 0.021 ± 0.009 to 0.059 ± 0.022 (p < 0.05, n = 4) (BSA-immobilized plates) when the concentration of added soluble Fn was elevated from 50 μg/ml to 100 μg/ml. The observed fibril formation of Fn was significantly lower when using cones grafted with PEG once or twice, in comparison to bare steel cones (p < 0.05, n = 4). Addition of washed platelets to Fn solution (100 μg/ml) resulted in significant increases of 8- and 20-fold in fibril formation of Fn, generated by shear on BSA- and Fn-immobilized plates, respectively (p < 0.05, n = 3). In contrast, using 10E5 or abxicimab to block αIIbβ3, or both αIIbβ3 and αvβ3 caused a reduction by 82% or 74% in fibril formation of Fn, respectively (n = 3), in comparison to samples without antibodies. Blocking α5β1 or αvβ3 by P1D6 or LM609 only caused a reduction by 17% or 56%, respectively (n = 3). Under static conditions, no fibril formation was detected.
Conclusions
Our results indicate that the fibril formation of Fn solution under shear can be monitored by changes in its viscosity. In addition, fibrillogenesis of Fn is modulated by shear conditions and physical properties of stainless steel. Furthermore, the formation of fibrils depends on the Fn concentration and is modulated by platelet integrins. Hereby, αIIbβ3 plays a predominant role, while α5β1 has a minor part among the three examined platelet integrins, in terms of fibril formation. Our system provides useful information regarding (1) surface- and shear-induced alterations of unfolding of Fn and (2) the contribution of its binding partners, including β3-integrins and α5β1.
Disclosures:
No relevant conflicts of interest to declare.
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