The tyrosine kinase JAK2 protein is critical for the JAK/STAT cytokine signaling of hematopoietic cells.
The somatic JAK2 V617F mutation is common in myeloproliferative neoplasms (MPNs), on which venous and arterial thrombosis is the leading cause of death.
However, the role of JAK2 in hemostasis remains unclear. Thus, researchers at the Versiti Blood Research Institute investigated the role of this protein in platelet hemostasis in JAK2 knockout mice.
The study used mice lacking JAK2 in platelets and megakaryocytes (MKs). The researchers collected the animal´s blood and tissues to analyse complete blood count, blood smear, plasma Von Willebrand factor (WWF) levels, and immunohistochemistry of the femur. They also determined the tail-bleeding time.
For platelet preparation, the researchers collected blood from the retroorbital plexus and anticoagulated it in acid citrate dextrose. They isolated the platelets by centrifugation and resuspended them in a buffer solution.
They used these samples to analyse platelet surface glycoproteins, platelet proteins, and platelet aggregation.
Ex vivo perfusion assay
First, the researchers pre-coated the microchannels of Cellix’s Vena8 Fluoro+ biochips with Type 1 collagen. After washing and blocking, the researchers used Cellix’s VenaFlux platform to perfuse the blood collected from the mice through the collagen-coated biochip. The researchers analysed JAK2Plt-/-’s ability in whole blood to adhere to type 1 collagen and form thrombi under arterial shear rate (1500 s -1), see Fig. 3 A-B.
Reduced thrombus formation of JAK2 knockout platelets at arterial shear rates
After 3 minutes, the mean fluorescence intensity for JAK2Plt-/- platelets was 3.25% ±2.69% vs. 12.38 ± 4.07% in controls.
The platelets exhibited similar dwelling times.
Thrombus formation, rather than initial platelet adhesion, was impaired in JAK2Plt-/- mice.
Other experiments in this study
Ferric chloride-induced thrombus formation
The researchers positioned a microvascular flow probe attached to a perivascular flowmeter into the mice’s carotid artery to monitor blood flow. Then, they induced vascular injury using 10% ferric chloride (FeCl3). After that, they recorded blood flow and time to the first occlusion.
Laser-induced thrombus formation
The experiment involved using a laser to induce vascular injury. A high-speed camera captured fluorescent images, which the scientists used to analyse the data. Platelet and fibrin accumulation and/or extravasation confirmed consistent injury.
Main findings of these experiments:
Knockout mice developed severe thrombocytosis and increased immature platelet fraction. Platelet glycoprotein expression, fibrinogen, and VWF were normal.
Mice lacking the JAK2 protein showed severe splenomegaly and abnormal hematopoiesis.
JAK2Plt-/- mice showed severe bleeding diathesis with a median bleeding time of 8.20 minutes, compared with 2.29 minutes in the controls.
JAK2Plt-/- mice failed to show occlusion following FeCl3- induced injury in up to 30 minutes. Similar results were observed following laser-induced injury.
Mice lacking JAK2 showed defective thrombi formation.
JAK2Plt-/- platelets showed decreased spreading, aggregation, and impaired signaling under stimuli with different agonists.
These results show that JAK2 deletion impairs platelet activation and hemostatic function in mice.
If you´d like to learn more about these experiments, please access the complete study.
What do I need to get started?
Would you like to run similar experiments in your lab and don’t know how to start? This is what you´ll need :
Vena8 Fluoro+ biochip – to mimic human blood vessels and model blood clots, see further details below.
Mirus Evo pump – to control shear rates (flow rates) in the biochip; this enables you to set the shear rate at a setting which models flow rates for thrombosis in micropillaries or other vessels.
Microenvironmental chamber – this is a temperature-controlled frame, the biochip sits in this and it keeps everything at 370C. The microenvironmental chamber sits on the microscope stage.
Inverted microscope – we supply the Zeiss AxioVert A1 with the VenaFlux Pro option or the Zeiss AxioObserver7 with the VenaFlux Elite option.
Digital camera – to capture images and video recordings. We supply the Prime BSI Express with both the VenaFlux Pro and Elite options. This is an excellent camera with a high frame rate suitable for thrombosis studies.
Image Pro Cell Analysis software – to analyse the images and videos from your experiments.
If you already have some of these items (such as the inverted microscope, camera, or cell analysis software), we recommend the VenaFlux Starter kit. Our options suit all budgets. Take a look at them on our eShop.
Nathan Eaton, Saravanan Subramaniam, Marie L. Schulte, Caleb Drew, David Jakab, Sandra L. Haberichter, Hartmut Weiler, Hervé Falet. “Bleeding diathesis in mice lacking JAK2 in platelets.” Blood Adv 2021; 5 (15): 2969–2981. doi: https://doi.org/10.1182/bloodadvances.2020003032