Fibronectin Unfolding by Platelets and Its Effect on Platelet Adhesion and Aggregation
Authors: Huynh Khon C. , Stoldt Volker R. , Gyenes Marianna , El-Khattouti Abdelouahid , Scharf Rudiger E. 
Published: October 2019

Abstract
Abstract 2209

Introduction:
Fibronectin (Fn), a dimeric adhesive glycoprotein of 230 to 250 kDa monomers, is present both in plasma and the extracellular matrix. Fn has been suggested to interact with platelets, subsequently being unfolded and forming fibrillar-like networks that contribute to platelet adhesion and aggregation. In our study, we examined the effect of Fn isolated from plasma on platelet adhesion and aggregation in vitro. Specifically, we explored the effect of Fn unfolding while interacting with platelets.

Methods:
For adhesion experiments, mepacrine-labeled washed platelets in the absence or presence of exogenous Fn (100 μg/ml) were incubated in wells pre-coated with collagen type I, fibrinogen (Fg) or Fn (10 μg/ml each) for 30 min at 37°C. For aggregation experiments, washed platelets were stimulated with 40 nM PMA or 10 μg/ml collagen in the absence or presence of Fn (300 μg/ml). For fluorescence resonance energy transfer (FRET) experiments, Fn isolated from human plasma was doubly conjugated with alexa fluor 488 and 546. Labeled Fn was mixed with 10-fold excess of unlabeled Fn to prevent energy transfer between adjacent protein molecules. Fn mixtures (20 or 100 μg/ml) were incubated for 3 h at 22°C with washed platelets in suspension (108/ml) or with platelets adherent onto immobilized Fn (50 μg/ml). In both settings, platelets were stimulated by 40 nM PMA. In some experiments, platelets were pre-incubated with the monoclonal antibodies LM609 or 10E5 (10 μg/ml) to block αvβ3 or αIIbβ3, respectively, prior to the addition of labeled Fn. For control, FRET signals of Fn mixtures without platelets were recorded.

Results:
Upon addition of soluble Fn (100 μg/ml) to washed platelets and subsequent co-incubation in wells pre-coated with collagen, Fg, or Fn (10 μg/ml) for 30 min, the percentage (mean % ± SD) of platelets adherent onto one of the immobilized ligands increased significantly by 228±33 (p=0.0112, n=3), 249±42 (p=0.005, n=3), or 198±21 (p=0.0017, n=3), respectively, as compared to adhesion experiments without addition of soluble Fn. By contrast, Fn had an opposing effect on platelet aggregation. Thus, addition of Fn (300 μg/ml) to washed platelets resulted in a reduction of 25 % or 50 % in platelet aggregation induced by PMA (40nM) or collagen (10 μg/ml), respectively. To determine Fn unfolding, the protein was doubly labeled with alexa fluor 488 (donor) randomly at 7–9 amine residues and alexa fluor 546 (acceptor) specifically at 4 free cysteine residues for FRET analyses. To access the sensitivity of FRET for conformational changes in Fn, we exposed labeled Fn to increasing concentrations of GdnHCl (1–4 M) and measured FRET. FRET signals, defined by the ratio of acceptor to donor fluoresecence intensity, varied over the range of GdnHCl concentrations indicating the conformational changes in Fn from its compact to its unfolded state. Fn in its compact conformation (0 M GdnHCl) had a FRET signal of 0.55 (100%) which decreased to 0.34 (63%), as Fn extended in 1 M GdnHCl solution. Further unfolding of Fn in 2 M, 3M and 4 M GdnHCl reduced the FRET signal to 0.27 (50%), 0.23 (44%) and 0.21 (39%), respectively. Addition of labeled Fn to PMA-activated platelets adherent onto immobilized unlabled Fn caused a slow but progressive decrease in FRET signal by 4% at 1 h, 5 % at 2 h and 6% at 3 h incubation. The decrease in FRET signal was reduced to 2% when platelet αvβ3 was blocked by LM609. By contrast, FRET remained unchanged in control experiments without platelets. The same was true when labeled Fn was incubated with PMA-activated platelets in suspension or in the presence of 10E5 (blocking αIIbβ3).

Conclusion:
Our in vitro studies strongly suggest that fibronectin can play a dual role in hemostasis by promoting platelet adhesion onto immobilized ligands but reducing platelet aggregation. We also demonstrate that activated adherent but not suspended platelets can indeed progressively unfold fibronectin, thereby inducing profound conformational changes that may explain its oppositional effects in platelet adhesion and aggregation. Moreover, our data suggest that unfolding of fibronectin caused by adherent platelets is governed by β3 integrins. Hereby, αIIbβ3 plays a predominant role in comparison to αvβ3.

Disclosures:
No relevant conflicts of interest to declare.