Fibrillar collagen

For platelet adhesion assays, video microscopy and immunofluorescence microscopy experiments, #1.5 glass coverslips (Warner Instruments) or #1.5 Glass Bottom Dishes (MatTek) were coated with 50 μg/ml human fibrinogen (FIB3, Enzyme Research Labs) or 100 μg/ml fibrillar collagen (Chronolog) and blocked with fatty-acid free BSA (Sigma) prior to platelet seeding, as described [18 (link)]. Platelet static adhesion, immunofluorescence and live video microscopy were performed using Kohler-illuminated Nomarski differential interference contrast (DIC) optics with a Zeiss 63× oil immersion 1.40 NA plan-apochromat lens on a Zeiss Axiovert 200M microscope as previously described [19 (link)-21 (link)]. The surface areas of individual platelets were measured using Image J software and plotted as previously described [18 (link)]. For acK immunofluorescence experiments, fixed platelets were blocked, permeablized and stained in blocking buffer (PBS + 0.1% SDS + 1% BM). Super resolution-structured illumination microscopy (SR-SIM) experiments were carried out on a Zeiss Elyra SR-SIM microscope as previously described [17 (link), 18 (link)].

Reagents were purchased from Sigma-Aldrich (St Louis, MO, USA) unless specified otherwise. Recombinant human nidogen-1 was obtained from R&D Systems (Minneapolis, MN, USA), soluble collagen from Corning (Corning, NY, USA), fibrillar collagen from Chrono-Log (Havertown, PA, USA), Collagen-related peptide (CRP-XL) was from R. Farndale (Cambridge University, UK) and human fibrinogen from Enzyme Research (South Bend, IN, USA). U73122 and U73343 were obtained from Tocris (Bristol, UK). Anti-GPVI ACT017 blocking antibody was donated by Acticor Biotech (Paris, France). Anti–β1 (clone: AIIB2) blocking antibody was purchased from Millipore (Burlington, MA, USA).