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rJararacin, a recombinant disintegrin from Bothrops jararaca venom: Exploring its effects on hemostasis and thrombosis - David et al., 2023.


Background

Integrins are proteins involved in the control of cell proliferation, apoptosis, and differentiation. They contain two subunits, α and β, consisting of different domain structures. Integrins can bind to various molecules, including extracellular matrices and proteins on the surface of neighbouring cells. They recognize their ligands thanks to classical sequences such as the RGD motif in both vitronectin and fibronectin.


The integrin αIIbβ3 is mainly found in platelets and binds to fibronectin, fibrinogen, and von Willebrand factor. This protein plays a significant role in controlling bleeding and arterial thrombosis. Arterial thrombosis can occur when a blood clot forms at the site of a ruptured atherosclerosis plaque. This causes changes in blood flow that trigger platelet activation and aggregation, leading to thrombus formation. This process is mainly facilitated by integrin αIIbβ3. Snake venoms are a complex mixture of peptides, proteins, and other molecules that can act as toxins. Disintegrins are found in the venom of snakes belonging to the Vipirinae and Crotalinae subfamilies. Disintegrins with an RGD motif can bind to integrins αIIbβ3 in platelets and modulate their activity. Scientists have isolated and characterized jararacin and jarastatin, two RGD disintegrins from Bothrops jararaca venom with the ability to inhibit ADP-induced platelet aggregation. Moreover, jararacin has demonstrated potential as an αIIbβ3 antagonist in studies related to homeostasis and thrombosis. In this study, David and colleagues aimed to obtain the recombinant form of jararacin and evaluate the secondary structure and its effects on hemostasis and thrombosis.


Methods


Expression of rJararacin

First, the researchers expressed rJararacin in the P. pastoris expression system and purified the recombinant protein.


Platelet adhesion under flow

The researchers conducted a study on platelet adhesion under flow conditions. To do this, they prepared a Vena8 Fluoro+ biochip by coating it with fibrinogen (2.4 mg/mL) overnight at 4◦C. The next day, the researchers blocked the biochip with a 5% BSA solution for 1 hour and washed it once with Tyrode buffer (129 nM NaCl; 0.34 mM Na2HPO4; 2.9 mM KCl; 12 mM NaHCO3; 20 mM HEPES; 1 mM MgCl2; 5 mM Glucose; pH 7.3). Platelet-rich plasma (PRP) underwent pre-treatment with disintegrin at concentrations of 150, 300, 450, and 600 nM for 1 min. Following this, the scientists added 10 μM ADP, 2.5 mM CaCl2, and 1 mM MgCl2 to the platelets and perfused them over the fibrinogen-coated channels at a constant shear stress of 2.5 dyn/cm^2 for 6 min. Acetylsalicylic acid at 10 mM served as a control of platelet aggregation inhibition. The group recorded platelet adhesion using a microscope coupled to a digital camera and analyzed the results with Image Pro Premiere software.


In vitro thrombogenesis under flow

For the in vitro thrombogenesis assay, the researchers coated a Vena8 Fluoro+ biochip with collagen (0.1 mg/mL), blocked, and washed. The researchers collected whole blood in 3.8% sodium citrate solution from healthy volunteers and incubated it with 3.3′-dihexyloxacarbocyanine iodide – dioc6 (1 μM) in the dark for 10 min. They pre-incubated the whole blood with disintegrin at concentrations of 250, 500, 750, and 1000 nM for 1 min. Following that, they added 2.5 mM CaCl2 and 1 mM MgCl2 and perfused the blood over coated collagen channels at a constant shear rate of 67.5 dyn/cm^2. Acetylsalicylic acid at 10 mM served as a control of platelet aggregation inhibition. They performed the analysis as described previously for platelet adhesion.


Results

The main findings of these experiments were:

  • Platelets in the control group could adhere and form large aggregates over fibrinogen coats (Fig 1A). On the other hand, rJararacin inhibited the adhesion and formation of platelet aggregates in the concentration of 0.15 μM, resulting in a 67% decrease in the covered area (Fig. 1G)

Fig. 1. Effect of rjararacin on platelet adhesion and aggregation under flow. (A–F) Red marks represent the presence of platelets (blue dots) identified by the program (G) Calculated percentage area covered by platelets based on three independent experiments

  • Platelets adhered to the collagen matrix and formed stable aggregates (2A). In contrast, in the presence of rJararacin, the size and number of thrombi were decreased (Fig. 2 B-F), rJararacin decreased from 33 to 94% of the covered area (Fig. 2G).

Fig. 2. Effect of rjararacin on thrombosis in vitro. (A–F) Representative images of three different blood donors with comparable results. Red marks represent the presence of platelets (Green fluorescent dots) identified by the program (G). Calculated percentage area covered by platelets based on three independent experiments.

Other experiments in this study

Once purified, the researchers performed a static adhesion assay to evaluate the integrin specificity of rJararacin. Next, they evaluated rJararacin on human platelet aggregation assay.

To verify the effect of rJararacin in an animal model of thrombosis, the researchers evaluated its antiplatelet activity in rat PRP. Next, they assessed the side effects of rJararacin by bleeding time assay.


Main findings of these experiments:

  • rJararacin inhibited platelet aggregation induced by ADP or collagen in a dose-dependent manner.

  • In an FeCl3-induced arterial thrombosis model, rJararacin delayed occlusion for over 45 min, comparable to heparin's effect.

  • rJararacin induced less bleeding loss than heparin.


Conclusion

These data suggest that rJararacin is a potential αIIbβ3 antagonist, capable of preventing arterial thrombosis.


See more details here.


How to get started


Thinking about trying out similar experiments in your lab? Here's what you'll need:

  • VenaFlux Basic platform – a semi-automated microfluidic platform designed for conducting studies on cell adhesion, binding, rolling, and migration under shear flow conditions that replicate in vivo flow rates.

  • Vena8Fluoro+ biochips - to mimic the shear stress of arterial circulation.

  • ExiGo Microfluidic Pump – a pulse-free syringe pump for low-flow microfluidic applications.

  • Microenvironmental chamber – a temperature-controlled frame that keeps the biochip at 370˚ C.

  • 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 the VenaFlux Pro and Elite options.

  • Image Pro Cell Analysis software – for image and video analysis.

If you already have some of these items, we recommend the Vena Flux Starter Basic Kit. We have options that suit all budgets. Request a quote or check out more options on our eShop!


References

  1. David, Victor, et al. "rJararacin, a recombinant disintegrin from Bothrops jararaca venom: Exploring its effects on hemostasis and thrombosis." Archives of Biochemistry and Biophysics 738 (2023): 109557.








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