Dataset Information

Cationic zinc is required for factor XII recruitment and activation by stimulated platelets and for thrombus formation in vivo.

ABSTRACT:

Background

Although divalent zinc (Zn²⁺ ) is known to bind factor (F)XII and affect its sensitivity to autoactivation, little is known about the role of Zn²⁺ in the binding of FXII to platelets, where FXII activation is thought to occur in vivo, and the function of Zn²⁺ during thrombus formation following vascular injury remains poorly understood.

Objectives

To evaluate the role of Zn²⁺ in platelet-dependent FXIIa generation.

Methods

FXII binding to platelets and FXII activation by stimulated platelets were assessed using flow cytometry and a platelet-dependent thrombin generation assay. The mouse cremaster laser injury model was used to evaluate the impact of Zn²⁺ chelation on thrombus formation in vivo.

Results

Our data demonstrate that stimulated platelets support FXII-dependent thrombin generation and that FXII activation by platelets requires the presence of Zn²⁺ . By contrast, thrombin generation by stimulated endothelial cells occurred independently of FXII and Zn²⁺ . Using flow cytometry, we found that FXII-fluorescein-5-isothiocyanate binds to the surfaces of stimulated platelets in a specific and Zn²⁺ -dependent manner, whereas resting platelets demonstrated minimal binding. Other physiologically-relevant divalent cations are unable to support this interaction. Consistent with these findings, the Zn²⁺ -specific chelator ethylenediaminetetraacetic acid calcium disodium salt confers thromboprotection in the mouse cremaster laser injury model without causing increased bleeding. We observed an identical phenotype in FXII null mice tested in the same system.

Conclusions

Our results suggest a novel role for Zn²⁺ in the binding and activation of FXII at the platelet surface, an interaction that appears crucial to FXII-dependent thrombin generation but dispensable for hemostasis.

SUBMITTER: Chaudhry SA

PROVIDER: S-EPMC8147875 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe 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.ResultsThrombus 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.ConclusionsWe 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.