Ex vivo observation of granulocyte activity during thrombus formation

Abstract

Background The process of thrombus formation is thought to involve interactions between platelets and leukocytes. Leukocyte incorporation into growing thrombi has been well established in vivo, and a number of properties of platelet-leukocyte interactions critical for thrombus formation have been characterized in vitro in thromboinflammatory settings and have clinical relevance. Leukocyte activity can be impaired in distinct hereditary and acquired disorders of immunological nature, among which is Wiskott-Aldrich Syndrome (WAS). However, a more quantitative characterization of leukocyte behavior in thromboinflammatory conditions has been hampered by lack of approaches for its study ex vivo. Here, we aimed to develop an ex vivo model of thromboinflammation, and compared granulocyte behavior of WAS patients and healthy donors. Results 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 granulocytes were observed in hirudinated or sodium citrate-recalcified blood under low wall shear rate conditions (100 s−1). These cells crawled around thrombi in a step-wise manner with an average velocity of 90–120 nm/s. Pre-incubation of blood with granulocyte priming agents lead to a significant decrease in mean-velocity of the cells and increase in the number of adherent cells. The leukocytes from patients with WAS demonstrated a 1.5-fold lower mean velocity, in line with their impaired actin polymerization. It is noteworthy that in an experimental setting where patients’ platelets were replaced with healthy donor’s platelets the granulocytes’ crawling velocity did not change, thus proving that WASP (WAS protein) deficiency causes disruption of granulocytes’ behavior. Thereby, the observed features of granulocytes crawling are consistent with the neutrophil chemotaxis phenomenon. As most of the crawling granulocytes carried procoagulant platelets teared from thrombi, we propose that the role of granulocytes in thrombus formation is that of platelet scavengers. Conclusions We have developed an ex vivo experimental model applicable for observation of granulocyte activity in thrombus formation. Using the proposed setting, we observed a reduction of motility of granulocytes of patients with WAS. We suggest that our ex vivo approach should be useful both for basic and for clinical research.

Novel hemizygous CORO1A variant leads to combined immunodeficiency with defective platelet calcium signaling and cell mobility

“…To assess their motility, we visualized granulocytes’ activity during thrombus formation in a flow chamber. 19 The patient’s granulocyte crawling velocity was decreased in comparison with that of healthy donors, and the numbers of motionless or slowly crawling cells were increased ( Fig 3 , F ). These data correspond with the described defects in mouse neutrophils deficient in CORO1A.…”

Flow Chamber Analyses in Cardiovascular Research: Impact of Platelets and the Intercellular Crosstalk with Endothelial Cells, Leukocytes, and Red Blood Cells

“…66 In addition, neutrophils are able to form heterotypic aggregates with annexin V-positive platelets and thereby contribute to the clearance of PS-positive platelets within the growing thrombus. 67 68…”

“…66 In addition, neutrophils are able to form heterotypic aggregates with annexin V-positive platelets and thereby contribute to the clearance of PS-positive platelets within the growing thrombus. 67,68 Beyond the aspect that all three types of blood cells (platelets, RBCs, WBCs) are in a resting, low-adhesive state while circulating in blood under physiological conditions, they could interact with each other, with vascular ECs and the subendothelium after cell-specific stimulation in Hämostaseologie Vol. 43 No.…”

Platelet lifespan and mechanisms for clearance

“…At the same time, these structures are also thrombogenic, thereby activating platelets and contributing to immune thrombosis [100]. Neutrophils interact with the thrombus to dissolve it by binding to procoagulant platelets and releasing them from the thrombus [101,102]. Consequently, thrombus size is increased in neutropenia, and thrombus resolution takes more time under this condition [103].…”