Characterization of Fibronectin Assembly by Adherent Platelets Under Flow Conditions: Effect of Shear Stress and Role of β3 Integrins

To fulfill their role in hemostasis, circulating platelets need to irreversibly adhere to the site of vascular injury and to resist to shear stress generated by the flowing blood. We previously reported that there is a relationship between the conformation of fibronectin (Fn) and its role in platelet adhesion and aggregation (Huynh, K. C. et al., ASH Annual Meeting Abstract, 2011. 118(21): p. 2209). In the present study, we examined the effect of shear stress on the assembly of Fn by adherent platelets. Moreover, we studied the role of β3 integrins (αIIbβ3 and αvβ3) in Fn assembly under flow conditions.

Alexa fluor 488-conjugated fibronetin (Fn488) was added to suspensions of washed platelets (10⁸/ml) in HEPES Tyrode buffer. CaCl₂ (2 mM) and ADP (10 μM) were added immediately prior to the experiments. The samples (150 μl) were subsequently applied onto plates precoated with 50 μg/ml Fn. A DiaMed Impact-R device was used to generate shear rates of 500 s⁻¹ or 5000 s⁻¹ for 2 min or 10 min. Nonadherent platelets were removed by washing with PBS buffer followed by addition of 150 μl of 2 % DOC lysis buffer. Lysates were collected and total protein concentrations were determined by Bradford assay. The DOC-insoluble pellets containing Fn fibrils were isolated by centrifugation at 13, 500 rpm for 20 min. Pellets were then solubilized with 100 μl of 1 % SDS buffer. Equal amounts of samples based on total protein concentrations were loaded onto wells of 96-well microplates. Fluorescence signals from Fn488 of samples were recorded by a Fluoroskan microplate reader. In some experiments, abciximab (anti-β3, 10 μg/ml) or LM609 (anti-αvβ3, 5 μg/ml) antibody, were added to platelet mixtures before loading onto Fn precoated plates. All data were collected from at least three different experiments and analyzed using GraphPad Quickcals. To test for statistical differences, student's t-test was used.

Fn assembly by adherent platelets was strongly affected by the applied shear rate but not by the exposure time to shear. At a shear rate of 500 s⁻¹, there were no insoluble Fn fibrils detectable in samples with adherent platelets after 2 or 10 min. When shear rates increased from 500 s⁻¹ to 5000 s⁻¹, the amount of insoluble Fn detectable on platelets after 2 and 10 min increased significantly (p < 0. 05) suggesting that adherent platelets exposed to high shear rates assemble more Fn fibrils on their surface. However, prolongation of exposure time to shear from 2 to 10 min did not result in significantly more Fn assembled by adherent platelets. By contrast, there were no insoluble fibrils that could be detected with adherent platelets under static conditions for 2 and 10 min. After 2 min at a shear rate of 5000 s⁻¹, platelets blocked with abciximab showed a significant decrease in the amounts of insoluble Fn fibrils in comparison with control experiments (no antibody) (p = 0. 02). Similar inhibitory effects could be seen with platelets treated with LM609. In parallel experiments in which 10 min at 5000 s⁻¹ were applied, both abciximab and LM609 had an inhibitory effect on Fn fibrillogenesis with a stronger effect by abciximab. Taken together, these data show that αvβ3 even at the low expression on platelets plays a major role in initiating the fibrillogenesis of Fn under high shear rate conditions, whereas αIIbβ3 contributes to the progression of Fn fibrils formation subsequently.

Our observations document that the assembly of Fn on the surface of adherent platelets is strongly affected by shear rate conditions. In addition, our data imply that, despite its lower expression on platelet surface, αvβ3 provides a significant contribution in initiating the Fn assembly under high flow conditions, as compared with αIIbβ3. By contrast, αIIbβ3 with its abundant amount on the platelet surface probably exerts its effect in the later phase of Fn fibrillogenesis. The present findings support the contention that not a single integrin or Fn binding domain, but multiple interaction steps including different molecules and Fn domains may be involved in assembling Fn.

No relevant conflicts of interest to declare.