Neutrophil granulocyte-dependent proteolysis enhances platelet adhesion to the arterial wall under high-shear flow.

SUMMARY BACKGROUND: Under high shear stress platelets adhere preferentially to the adventitia layer of the arterial vessel wall in a von Willebrand factor (VWF)-dependent manner. OBJECTIVE: The present study was undertaken in an attempt to characterize the structural background of the relative thromboresistance of the media and the impact of neutrophil leukocyte-derived proteases (matrix metalloproteinases, neutrophil elastase) on platelet adhesion in this layer of the arteries. METHODS AND RESULTS: Platelet adhesion to cross-sections of the human iliac artery was monitored by indirect immunofluorescent detection of GpIIb/IIIa antigen. Exposure of the vessel wall to activated neutrophils or neutrophil-derived proteases increased platelet adhesion to the media about tenfold over the control level at 3350 s(-1) surface shear rate. In parallel with this enhanced thrombogenicity morphological changes in the media were evidenced by atomic force microscopy (AFM) and scanning electron microscopy (SEM). The fine proteoglycan meshwork seen with Cupromeronic Blue enhancement of the SEM images was removed by the proteolytic treatment and the typical collagen fiber structure was exposed on the AFM images of the media. CONCLUSION: Through their proteases activated neutrophils degrade proteoglycans, unmask VWF binding sites and thus abolish the thromboresistance of the media in human arteries.

Supramolecular assemblies of adsorbed collagen affect the adhesion of endothelial cells.

The behavior of endothelial cells (HUVECs) in contact with thin collagen films presenting different supramolecular organizations was investigated. Collagen was adsorbed on polystyrene (PS) and plasma-oxidized PS (PSox) in conditions ensuring the formation of continuous layers presenting an increasing density of fibrillar structures. Discontinuous collagen layers were also prepared on PS by adsorption followed by dewetting. The morphology of the obtained collagen films was checked by using atomic force microscopy. HUVECs adhesion was evaluated in terms of cell number, cell area, cell shape, and actin structure after 4 h of contact with the prepared collagen layers. In the presence of serum, no adhesion was observed on PS, whereas a substantial adhesion was found on PSox. This is explained by the competition for adsorption, which turns in favor of adhesive proteins secreted by the cells on the hydrophilic PSox, but turns in favor of serum albumin on the hydrophobic PS. The progressive coating of PS by smooth collagen films increased cell adhesion and spreading. However, cell spreading and cytoskeleton organization were adversely affected by the appearance of a high density of collagen fibrillar structures. This latter trend was similarly observed on PSox. On the other hand, HUVECs spreading and cytoskeleton organization were clearly enhanced on discontinuous collagen layers compared with continuous ones. A possible explanation for these observations lies in the modification of exposure and/or spatial distribution of recognition sequences due to spontaneous collagen self-assembly on fibril formation or to collagen aggregation on dewetting.