Ex vivo observation of granulocyte activity during thrombus formation

Infiltration of growing thrombi by leukocytes, being the key part of the thromboinflammation, is well established in vivo. The study was aimed at the development of an ex vivo simulation of this phenomenon. Thrombus formation in anticoagulated whole blood from healthy volunteers and patients was visualized by fluorescent microscopy in parallel-plate flow chambers with fibrillar collagen type I coverslips. Moving CD66b-positive cells (granulocytes) were observed in hirudinated or recalcified blood under low wall shear rate conditions (<200 s-1). These cells crawled around thrombi in a step-wise manner with an average rate of 70 nm/s. Pre-incubation of blood with leukocyte priming agents lead to a significant increase in average cell velocity. On the contrary, leukocytes from Wiskott-Aldrich syndrome patients demonstrated a 1.5-fold lower average velocity, in line with their impaired actin polymerization. Thereby, the observed features of granulocytes crawling are consistent with the neutrophil chemotaxis phenomenon. We conclude that the proposed ex vivo experimental setting allows us to observe granulocytes activity in near-physiological conditions.