Digital Image Analysis of Platelet-Extracellular Matrix Interactions: Studies in von Willebrand Disease Patients and Aspirin-Treated Donors

Abstract

We have developed an automated image analysis method which provides en face morphometric evaluation of platelet interactions with coverslips coated with extracellular matrices. This procedure gives also additional information on the intensity of platelet-surface interactions in the same specimens. To standardize the method, anticoagulated human blood was flowed at 800 s-1 during 10 min at 37°C through a parallel perfusion chamber in which extracellular matrix (ECM)-coated coverslips were placed. The sensitivity of the new method was tested in similar studies performed in patients with von Willebrand disease or treated with aspirin (500 mg/ day). The extent of platelet deposition on the ECMs was evaluated by both standard and new morphometric procedures. Coverslips were successively embedded in resin and processed for cross-sectional analysis and results were compared with those provided by the new automated procedure. A good correlation was found between results of covered surface obtained by both methods (r = 0.86; p < 0.005). Results from perfusions performed with blood from patients with platelet defects confirmed the correspondence of grey levels in the digitized images with the heights of groups of interacting platelets calculated from cross sections. The new automated method, in only one stage, allows a reliable evaluation of the global interaction of platelets with the ECM and effectively detected qualitative modifications in platelet-platelet interactions in patients with known platelet defects. The method described could be applied to the evaluation of the interactions of platelets with purified components of the subendothelium.