Characterization of fibronectin assembly by platelets adherent to adsorbed laminin-111

Background: Various types of laminin (LN) are ubiquitous components of basementmembrane and exposed to blood upon localized damage of vascular endothelial cells. Fibronectin is a plasma protein that is insolubilized into fibrils in a regulated fashion by, for example, lysophosphatidic acid (LPA)-stimulated fibroblasts or platelets spread on supportive adhesive ligands. Objective: To study assembly of plasma fibronectin by LPA-activated platelets adherent to LN-111 via α6β1 integrin. Results: Platelets adherent to LN-111-bound plasma fibronectin or itsN-terminal 70 kD fragment in fibrillar arrays at the periphery of spread platelets under static but not shear conditions. Formation of fibronectin arrays under static conditions was inhibited by co-incubation with the N-terminal 70 kD fragment or with a 49-amino acid peptide that binds to the N-terminal region of fibronectin. Approximately 7000 fibronectin dimers bound per adherent platelet with a K_d of 50 nM. Bound 70 kD fragment was readily olubilized with deoxycholate (DOC), whereas bound fibronectin became progressively insoluble. Bound 70 kD fragment became resistant to DOC extraction after treatment with a cell-impermeable, reducible crosslinker. Crosslinked 70 kD fragment was found in a high molecular weight complex. As with fibroblasts, signaling molecules modulating actin cytoskeletal organization controlled expression of binding sites for the N-terminal 70 kD region of fibronectin on adherent platelets. Conclusions: These results indicate that platelets adherent to LN-111 via α₆β₁ support subsequent assembly of fibronectin, but possibly only under conditions of intermittent or stagnant blood flow.