Investigating von Willebrand Factor Pathophysiology Using a Flow Chamber Model of von Willebrand Factor-platelet String Formation

Michels, Alison; Swystun, Laura L.; Mewburn, Jeffrey; Albánez, Silvia; Lillicrap, David

doi:10.3791/55917

Cited by 6 publications

(3 citation statements)

References 48 publications

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“…The markedly prolonged closure times and reduced PL-AUC for the two dogs with VWF deficiency are consistent with this critical need for VWF to support platelet adhesion under flow conditions. Flow chamber assays have been instrumental in investigations of the pathogenesis of VWD and of the interactions between VWF and platelet surface receptors (47)(48)(49)(50). We also found a positive correlation between the thrombin generation parameters and the AUC for the PL-chip.…”

Section: Discussionsupporting

confidence: 59%

A Novel Microchip Flow Chamber (Total Thrombus Analysis System) to Assess Canine Hemostasis

et al. 2020

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Hemorrhagic diseases are common in dogs. Current coagulation assays do not model all aspects of in vivo hemostasis and may not predict bleeding risk. The Total-Thrombus Analysis System (T-TAS) is a novel hemostasis assay system in which whole blood flows through microfluidic channels at defined shear rates to provide qualitative and quantitative evaluation of platelet function (PL-chip) and coagulation function (AR-chip). The present study evaluated the T-TAS in dogs with hereditary bleeding disorders and with acquired hemorrhagic syndromes (Group 1), and healthy controls (Group 2). Hereditary defects included von Willebrand's disease (VWD; n = 4), hemophilia A (n = 2), and canine Scott syndrome (n = 2). Acquired hemorrhagic disorders included neoplastic hemoperitoneum (n = 2) and acute hemorrhagic diarrhea syndrome (n = 1). Citrate anticoagulated samples were collected from diseased dogs (Group 1, n = 11) and controls (Group 2, n = 11) for coagulation screening tests, fibrinogen analyses, D-dimer concentration, antithrombin activity, von Willebrand Factor antigen, PFA-100 closure time (PFA-CT), and thromboelastography (TEG). Citrate and hirudin anticoagulated samples were used for T-TAS analyses at two shear rates. Qualitative thrombus formation in each chip was recorded using the T-TAS video camera. Numeric parameters, derived from the instrument software, included occlusion start time (OST; time to 10 kPa), occlusion time (OT; time to 60 kPa (PL-chip) or 80 kPa (AR-chip)), and area under the pressure curve (AUC). Correlations between continuous variables were evaluated by Spearman's rank. Continuous variables were compared between groups by Student's t-test or the Mann-Whitney U-test. Alpha was set at 0.05. In combined analyses of all dogs, significant correlations were identified between T-TAS variables, between the PFA-CT and PL-chip parameters and between TEG variables and AR-chip parameters. The prothrombin time correlated with the AR-chip AUC at both shear rates. In Group 1 dogs, the AR-chip AUC at low shear was significantly reduced compared with Group 2 dogs. Aberrant thrombus formation was seen in video images recorded from dogs with VWD and hemophilia A. The T-TAS AR-chip analysis distinguished dogs with bleeding risk compared to healthy controls. Initial evaluations of the T-TAS suggest it may aid characterization of hemostasis in patients at-risk of bleeding and assist with delineating bleeding phenotypes.

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Section: Discussionsupporting

confidence: 59%

A Novel Microchip Flow Chamber (Total Thrombus Analysis System) to Assess Canine Hemostasis

et al. 2020

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“…A limitation of our fixed endothelial chip is that it is not suitable for the study of the effect of vWF on thromboinflammation, as this requires live endothelial cells for active secretion of vWF under shear. Published methods, using purified platelets and neutrophils to study their interaction with vWF, have been included in Table 1 [ 33 , 34 , 50 ]. A modification of our chip including live endothelial cells, for the study of endothelial-derived vWF in thromboinflammation, is provided in Supplementary Methods and Supplementary Figure S1 .…”

Section: Discussionmentioning

confidence: 99%

Thromboinflammation Model-on-A-Chip by Whole Blood Microfluidics on Fixed Human Endothelium

et al. 2021

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Microfluidic devices have an established role in the study of platelets and coagulation factors in thrombosis, with potential diagnostic applications. However, few microfluidic devices have assessed the contribution of neutrophils to thrombus formation, despite increasing knowledge of neutrophils’ importance in cardiovascular thrombosis. We describe a thromboinflammation model which uses straight channels, lined with fixed human umbilical vein endothelial cells, after treatment with tumour necrosis factor-alpha. Re-calcified whole blood is perfused over the endothelium at venous and arterial shear rate. Neutrophil adhesion, platelet and fibrin thrombus formation, is measured over time by the addition of fluorescent antibodies to a whole blood sample. Fixed endothelium retains surface expression of adhesion molecules ICAM-1 and E-Selectin. Neutrophils adhere preferentially to platelet thrombi on the endothelium. Inhibitors of neutrophil adhesion and anti-inflammatory agents, such as isoquercetin, decrease neutrophil adhesion. Our model offers the advantage of the use of (1) fixed endothelium, (2) whole blood, instead of isolated neutrophils, and (3) a small amount of blood (1 mL). The characteristics of this thromboinflammation model provide the potential for further development for drug screening and point-of-care applications.

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“…In contrast to other protocols, this system uses whole blood while others study EC interaction with a single blood component such as platelets and leukocytes 19,20,21 . There have been more advanced microfluidic models developed that allow the study of endothelial function in a vascular model with a round vessel geometry and soft extracellular matrix.…”

Section: Discussionmentioning

confidence: 99%

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

Manz

Albers

Symersky

et al. 2020

JoVE

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The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network. By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease. 15. In addition to lung emboli, it is postulated that the pulmonary endothelium provides a prothrombotic environment in CTEPH that

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Investigating von Willebrand Factor Pathophysiology Using a Flow Chamber Model of von Willebrand Factor-platelet String Formation

Cited by 6 publications

References 48 publications

A Novel Microchip Flow Chamber (Total Thrombus Analysis System) to Assess Canine Hemostasis

A Novel Microchip Flow Chamber (Total Thrombus Analysis System) to Assess Canine Hemostasis

Thromboinflammation Model-on-A-Chip by Whole Blood Microfluidics on Fixed Human Endothelium

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

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