Platelet integrin-dependent fibrillogenesis of fibronectin: Impact of shear stress

Introduction: Soluble plasma fibronectin (Fn) with its inactive compact structure requires unfolding to assemble into active fibrils. Fibril formation of Fn is a cell-mediated process. Less is known about the contribution of biomechanical forces on the fibrillogenesis of Fn. The aim of this study was to investigate conformational changes of Fn, as induced by platelet integrins and/or shear rates simulating venous or arterial flow conditions.

Methods: Human plasma Fn, in the presence or absence of washed platelets, was added to plates pre-coated with Fn. Subsequently, the solutions were exposed to shear using a cone-plate rheometer (Haake Rheostress 1). For microscopic analysis (LSM 510, Carl Zeiss), Alexa flour 488-conjugated Fn was used. In parallel experiments, a N-terminal 70kDa fragment of Fn was incubated with soluble Fn before exposure to shear. To examine the role of distinct platelet integrins on fibril formation of Fn, washed platelets were incubated with monoclonal antibodies LM609, P1D6, 10E5, or abciximab (10 µg/ml, each) to block αvβ3, α5β1, αIIbβ3, or both αIIbβ3 and αvβ3, prior to the addition of Fn and subsequent exposure to shear. In all experiments, flow conditions were simulated by shear rates, stepwise increasing from 50 1/s to 5000 1/s within 5 min and subsequently decreasing from 5000 1/s to 50 1/s within 5 min. To study the structure of Fn fibrils, solutions were examined by laser scanning microscopy after exposure to shear. To quantify the amount of fibril formation, deoxycholate solubility assays and densitometric analysis of Western blots were performed. Control experiments were conducted under static conditions.

Results: Microscopic analyses showed that exposing Fn solutions to shear resulted in fibril formation. Fn fibril diameter varied from 0.5 to 5 µm. Observed fibrils were linked with each other and varied in length (from 50 to 300 µm). Treatment of Fn with the N-terminal 70 kDa fragment of Fn, which is known to inhibit Fn matrix assembly, blocked fibril formation of Fn. Western blotting and densitometric analyses revealed that addition of washed platelets to Fn solution resulted in increases of 20-fold in fibril formation of Fn (calculated as the ratio of insoluble to soluble Fn), generated by shear (p < 0.05, n = 3). In contrast, 10E5 or abxicimab blocking αIIbβ3, or both αIIbβ3 and αvβ3 caused a reduction by 82% or 74% in fibril formation of Fn (p < 0.05, n = 3 each), in comparison to samples without antibodies. Blocking α5β1 or αvβ3 by P1D6 or LM609 only caused a reduction by 17% or 56% (p > 0.05, n = 3 each). Under static conditions, no fibril formation was detected.

Conclusions: Our results indicate that fibrillogenesis of Fn is modulated by shear conditions. Furthermore, formation of fibrils is induced by platelet integrins. Hereby, αIIbβ3 plays a predominant role, while α5β1 has a minor part among the three examined platelet integrins with regards to Fn fibril formation.

Corresponding author: Nguyen, Huong T. T.

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