Ex vivo observation of granulocyte activity during thrombus formation
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Summary: Specifically, the proposed model system is valuable to study ex vivo the infiltration of the growing thrombus by granulocytes using fluorescent microscopy. In addition, this system has the potential to facilitate investigations on the role of granulocytes in thrombus growth and immune-thrombosis. Granulocytes have been classified into two different types based on their DiOC6 staining pattern, namely, type A with uniform DiOC6 and type B with cluster-like DiOC6. However, it remains unclear if the staining pattern is homogeneously so clear-cut and if the type A and B granulocytes are in addition defined by their velocity. Granulocyte activation process by "priming agents" has to be validated and the rationale for using the chosen agents needs to be provided. Finally, better-defined controls for the part of the paper dedicated to the synergistic effect between granulocytes and platelets during thrombus growth are necessary. Because in the Wiskott-Aldrich syndrome (WAS) granulocyte motility as well as platelet number and function are impaired, blood from patients with WAS is not an appropriate control for this study. For example, for the control experiments, the following controls might be used in replacement of WAS blood: (1) blood from thrombocytopenic patients or platelet-depleted blood and (2) blood in which granulocyte mobility/activation is inhibited. Finally, it would be interesting to see if neutrophil extracellular traps (NETs) develop in this model system.
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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.
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Reviewer #3:
The manuscript named "Ex vivo observation of granulocyte activity during thrombus formation "submitted by Morozova and colleagues try to demonstrate the implication of deux different types of granulocytes in thrombus formation. Author study thrombus formation in anticoagulated whole blood from healthy and Wiskott-Aldrich patients in parallel-plate flow under collagen type I and low shear rate (100 s-1). They identified a CD66/CD11 cell population defined as granulocytes able to interact with growing thrombus. Two types of granulocytes were observed and differentiated with their fluorescent patterns: type A (uniform DiOC6 staining) and type B (cluster-like DiOc6 staining). Authors studied granulocytes behavior under several kinds of inflammation mediator. The manuscript should be improved, please see my following comments.
Au…
Reviewer #3:
The manuscript named "Ex vivo observation of granulocyte activity during thrombus formation "submitted by Morozova and colleagues try to demonstrate the implication of deux different types of granulocytes in thrombus formation. Author study thrombus formation in anticoagulated whole blood from healthy and Wiskott-Aldrich patients in parallel-plate flow under collagen type I and low shear rate (100 s-1). They identified a CD66/CD11 cell population defined as granulocytes able to interact with growing thrombus. Two types of granulocytes were observed and differentiated with their fluorescent patterns: type A (uniform DiOC6 staining) and type B (cluster-like DiOc6 staining). Authors studied granulocytes behavior under several kinds of inflammation mediator. The manuscript should be improved, please see my following comments.
Authors should clarify the technical part of the manuscript and the figure 1, essentially the use of anticoagulant to perform follow chamber. It is not obvious which anticoagulant was used to performed flow chamber: citrate, heparin, hirudin. Does recalcification was performed in all experiments?
The authors should explain why the figure 1 demonstrates that granulocytes need free calcium ions to adhere to the growing thrombus. This is not the conclusion of figure 1. Moreover, all the growing thrombi seem different (more compact in citrate than with hirudin, w/o granulocyte in citrate and with granulocytes in hirudin) the authors should discuss this point.
This following sentence is confusing (last sentence of 3.1): “Hirudin- and heparin-anticoagulated blood was used in all further experiments because citrated blood recalcification causes local fibrin formation and platelet activation.” Platelets activation is essential to growing thrombus.
Author hypothesized that type B are more activated than type A essentially based on crawling and velocity cells. Could they do supplemental experiments to prove this point (increased of CD11 active form) and to differentiate neutrophils from eosinophils and basophils?
It will be great to perform a competition experiment to prove that platelets are interacting with granulocytes through CD11.
Did authors find NETs in this setting?
In all pictures platelets seem not well represented, only two and three platelets in figure 2. How the authors could be sure that granulocytes interact with platelets and not collagen?
Some platelets seem inactivated (round form) and annexin V positive. Could the authors discuss this point?
Concerning the last figure, it will be great to use healthy platelets and WAS granulocytes to conclude that crawling is altered.
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Reviewer #2:
The authors report on a model system to study the infiltration of growing thrombi by leukocytes using fluorescent microscopy. Such a system will facilitate studies on the role of leukocytes in the context of immuno-thrombosis and thrombus growth. I like the proposed model and I'm convinced about the utility of such a system. However, although the model is sound, I do have a couple of major concerns:
The authors describe crawling neutrophils; which methodology has been applied to guarantee that the crawling of the neutrophil is a general phenomenon in this system and not a feature of selected neutrophils? Has there been used a method of quantification (e.g. 86% of the neutrophils are crawling)?
The authors identify two types of granulocytes (uniform DiOC6-type A vs cluster-like DiOC6-type B granulocytes. Although the pictures …
Reviewer #2:
The authors report on a model system to study the infiltration of growing thrombi by leukocytes using fluorescent microscopy. Such a system will facilitate studies on the role of leukocytes in the context of immuno-thrombosis and thrombus growth. I like the proposed model and I'm convinced about the utility of such a system. However, although the model is sound, I do have a couple of major concerns:
The authors describe crawling neutrophils; which methodology has been applied to guarantee that the crawling of the neutrophil is a general phenomenon in this system and not a feature of selected neutrophils? Has there been used a method of quantification (e.g. 86% of the neutrophils are crawling)?
The authors identify two types of granulocytes (uniform DiOC6-type A vs cluster-like DiOC6-type B granulocytes. Although the pictures provided (fig 2) do represent nice and clear examples of type A vs type B granulocytes, the experiment will reveal for sure staining patterns which have been less clear. What kind of systematic methodology has been applied to delineate type A from Type B neutrophils?
Figure 3 and 4 are nice figures showing differences in granulocytes/FOV and velocity- but it is not clear to the reader what percentage of the neutrophils visible in the FOV do move or not. Was there a minimal amount of neutrophils being in motion?
The authors want to point out the synergistic effects of neutrophils and platelets in the thrombus growth. To finally make the point they use whole blood of a WAS patient. However, since WAS affects neutrophil motility as well as platelet morphology/number the individual role of platelets and neutrophils in this process remains open. Therefore control experiments targeting either platelets (whole blood of a thrombocytopenic patient and/or platelet depletion) may contribute to identify the role of platelets in the model. On the other hand, inhibition of neutrophil activation/motility may illustrate the individual contribution of neutrophils in this setting.
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Reviewer #1:
The authors utilize a novel ex vivo system to visualize granulocytes migrating within experimentally induced blood clots. Granulocytes are labeled using CD11b and CD66b and visualized over time using fluorescence microscopy as they move within the ex vivo formed clots, which have been prepared under different anticoagulant conditions to generate varied clot structure. Leukocytes are activated using various agonists and behavior classified into 2 different phenotypes based on the staining pattern of DiOC6 as either diffuse or punctate. The experimental system allows for measure of individual cell velocity and clear images of cells showing changes in cell body structure. The use of cells from WAS patients provides a nice validation of the model system being presented in the study.
The authors state that citrated blood results …
Reviewer #1:
The authors utilize a novel ex vivo system to visualize granulocytes migrating within experimentally induced blood clots. Granulocytes are labeled using CD11b and CD66b and visualized over time using fluorescence microscopy as they move within the ex vivo formed clots, which have been prepared under different anticoagulant conditions to generate varied clot structure. Leukocytes are activated using various agonists and behavior classified into 2 different phenotypes based on the staining pattern of DiOC6 as either diffuse or punctate. The experimental system allows for measure of individual cell velocity and clear images of cells showing changes in cell body structure. The use of cells from WAS patients provides a nice validation of the model system being presented in the study.
The authors state that citrated blood results in local fibrin formation and platelet activation; would it not be relevant to compare granulocyte behavior in such a setting to the hiridin or heparin-anticoagulated samples? This could also provide a valuable setting to study platelet-granulocyte interactions.
The further elimination of heparin-anticoagulated blood in favor of hirudin is also not clear. How does heparin pre-activate granulocytes, and what experimental evidence is seen by the authors other than an increase in number?
Are type A and type B leukocytes defined only by the DiOC6 staining pattern, or also by their velocity? Please clarify in the text.
The choice of "leukocyte priming agents" is not clear, in particular myeloperoxidase and lactoferrin. Leukocyte activation caused by these agents should be validated and the rationale more clearly defined (i.e. by referencing previous work that provides the mechanism of binding for these leading to neutrophil activation).
Regarding the Annexin V stained smaller structures presented in Figure 4; have the authors ruled out that these could be procoagulant extracellular vesicles from other leukocytes, i.e. by performing a co-staining with a platelet marker for positive identification?
Have the fibrinogen (Sigma) and von Willebrand factor (an in-house preparation from an academic lab collaborator) been tested for endotoxin levels prior to use in this system?
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Summary: Specifically, the proposed model system is valuable to study ex vivo the infiltration of the growing thrombus by granulocytes using fluorescent microscopy. In addition, this system has the potential to facilitate investigations on the role of granulocytes in thrombus growth and immune-thrombosis. Granulocytes have been classified into two different types based on their DiOC6 staining pattern, namely, type A with uniform DiOC6 and type B with cluster-like DiOC6. However, it remains unclear if the staining pattern is homogeneously so clear-cut and if the type A and B granulocytes are in addition defined by their velocity. Granulocyte activation process by "priming agents" has to be validated and the rationale for using the chosen agents needs to be provided. Finally, better-defined controls for the part of the paper dedicated to …
Summary: Specifically, the proposed model system is valuable to study ex vivo the infiltration of the growing thrombus by granulocytes using fluorescent microscopy. In addition, this system has the potential to facilitate investigations on the role of granulocytes in thrombus growth and immune-thrombosis. Granulocytes have been classified into two different types based on their DiOC6 staining pattern, namely, type A with uniform DiOC6 and type B with cluster-like DiOC6. However, it remains unclear if the staining pattern is homogeneously so clear-cut and if the type A and B granulocytes are in addition defined by their velocity. Granulocyte activation process by "priming agents" has to be validated and the rationale for using the chosen agents needs to be provided. Finally, better-defined controls for the part of the paper dedicated to the synergistic effect between granulocytes and platelets during thrombus growth are necessary. Because in the Wiskott-Aldrich syndrome (WAS) granulocyte motility as well as platelet number and function are impaired, blood from patients with WAS is not an appropriate control for this study. For example, for the control experiments, the following controls might be used in replacement of WAS blood: (1) blood from thrombocytopenic patients or platelet-depleted blood and (2) blood in which granulocyte mobility/activation is inhibited. Finally, it would be interesting to see if neutrophil extracellular traps (NETs) develop in this model system.
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