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Summary

Fibrin is responsible for clot formation during hemostasis and thrombosis. Turbidity assays and thromboelastograhy (TEG) can be utilized as synergistic tools that provide complementary assessment of a clot. These two techniques together can give more insight into how clotting conditions affect fibrin clot formation.

Abstract

Thrombosis is a leading cause of death worldwide. Fibrin(ogen) is the protein primarily responsible for clot formation or thrombosis. Therefore, characterizing fibrin clot formation is beneficial to the study of thrombosis. Turbidity and thromboelastography (TEG) are both widely utilized in vitro assays for monitoring clot formation. Turbidity dynamically measures the light transmittance through a fibrin clot structure via a spectrometer and is often used in research laboratories. TEG is a specialized viscoelastic technique that directly measures blood clot strength and is primarily utilized in clinical settings to assess patients' hemostasis. With the help of these two tools, this study describes a method for characterizing an in vitro fibrin clot using a simplified fibrinogen/thrombin clot model. Data trends across both techniques were compared under various clotting conditions. Human and bovine fibrin clots were formed side-by-side in this study as bovine clotting factors are often used as substitutes to human clotting factors in clinical and research settings. Results demonstrate that TEG and turbidity track clot formation via two distinct methods and when utilized together provide complementary clot strength and fiber structural information across diverse clotting conditions.

Introduction

Thrombosis is the pathological formation of a blood clot in the body that blocks blood circulation leading to high morbidity and mortality worldwide. There are 1 to 2 cases of venous thromboembolism and 2 to 3 cases of thrombosis-induced vascular diseases per 1000 people annually1,2. Presented here is a method leveraging thromboelastography (TEG) and turbidity to monitor clot formation under various clotting conditions. Fibrin(ogen) is the primary protein that is responsible for clot formation in the body. In the final steps of the coagulation cascade, fibrinopeptides are cleaved from fibrinogen by thromb....

Protocol

1. Preparation of phosphate buffer saline (PBS)

NOTE: PBS was used throughout this study as the described assays did not require the addition of calcium. It is important to note that when adding calcium, often utilized to re-calcify citrated blood products, PBS should be avoided as calcium is known to precipitate in phosphate buffers.

  1. Make a 0.01 M, pH 7.4 PBS buffer by mixing 137 mM sodium chloride, 1.8 mM potassium phosphate monobasic, 10 mM sodium phosphate dibasic and 2.7 mM po.......

Representative Results

The experiments shown in Figure 1 are representative turbidity tracing curves of human and bovine fibrin clots at different fibrinogen levels. Representative TEG tracing curves for fibrin clot formation at different fibrinogen levels are shown in Figure 2. Both tracing curves demonstrate that after a lag period following clot initiation, clot turbidity or clot amplitude increases over time and levels off at the end of clot formation. An endpoint value of maximum.......

Discussion

This protocol demonstrates the utilization of two distinct clot characterization tools testing a simplified fibrinogen/thrombin clot model using commercially available components. Both TEG and turbidity assays are easy to conduct. They not only provide end point clot examinations such as max clot formation (TurbMax and TEGMax) and clot formation times (TurbTime and TEGTime) but also assess the dynamic clot forming process. This makes TEG and turbidity valuable tools for clot ch.......

Acknowledgements

None.

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Materials

NameCompanyCatalog NumberComments
96-Well Clear Flat Bottom UV-Transparent MicroplateCorning3635Non-treated acrylic copolymer, non-sterile
Albumin from human serumMillipore SigmaA1653≥96%, lyophilized powder
Arium Mini Plus Ultrapure Water SystemSartoriusNADI water source
Bovine serum albuminMillipore SigmaA2153≥96%, lyophilized powder
Disposable Cups and Pins for TEG 5000 (Clear)HaemoneticsREF 6211
Fibrinogen, Bovine PlasmaMillipore Sigma341573contains more than 95% clottable protein
Fibrinogen, Plasmingogen-Depleted, Human PlasmaMillipore Sigma341578Contains ≥ 95% clottable proteins.
Phosphate buffered salineMillipore SigmaP3813Powder, pH 7.4, for preparing 1 L solutions
Potassium chlorideMillipore Sigma60130≥99.5% purity
Potassium phosphate monobasicMillipore SigmaP9791≥98% purity
SevenEasy pH MeterMettler ToledoS20
Sodium chlorideMillipore Sigma71378≥99.5% purity
Sodium phosphate dibasicMillipore Sigma71636≥99.5% purity
SpectraMax M5 multi-detection microplate reader systemMolecular DevicesM5
TEG 5000 Thrombelastograph Hemostasis analyzer systemHaemonetics07-022
Thrombin, BovineMillipore Sigma605157
Thrombin, Human Plasma, High ActivityMillipore Sigma605195

References

  1. Beckman, M. G., Hooper, W. C., Critchley, S. E., Ortel, T. L. Venous Thromboembolism. A Public Health Concern. American Journal of Preventive Medicine. 38, 495-501 (2010).
  2. Goldhaber, S. Z., Bounameaux, H. Pulmonary embolism and deep vein thrombosis.

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