Background: Venous thromboembolism (VTE) is one of major causes of sudden death. Prevention of VTE is hard because of lacking understanding of mechanical and physical property of venous thrombus. Thrombin generation is most important phase for fibrin clot formation. Thrombin is explosively generated by prothrombinase complex on activated platelets.

Aims: Our aim of this study is visualization of time course of thrombin generation on activated platelets and fibrin formation in high magnification to elucidate extract physical property of venous thrombus generation at the point of view of fibrin formation in low shear stress.

Methods: Human venous blood was obtained from healthy volunteers. We separated platelets by centrifugation to stain with Alexa Fluor 488 and mixed them into the whole blood composition again. Alexa fluor 546-conjugated fibrinogen was added to visualize fibrin formation. BOC-Val-Pro-Arg-AMC (Boc-VPA), a sensitive fluorogenic substrate for alpha-thrombin, was added to visualize thrombin generation.
To meet our aim, we made a microfluidic device system to observe thrombus structure formed under venous shear rate conditions. The blood sample was introduced to a home build microfluidic channels that ware fully siliconized and partly coated with collagen. The thrombus image and thrombin were observed by a fluorescence microscope with 60X magnification.

Results:
1. Thrombin was generated at a same time explosively on every cluster of activated platelets.
2. Thrombin generation was accelerated shortly before formation of fibrin network.

Conclusions: Thrombin generation on activated platelets was visualized by a fluorescence microscope.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/visualization-of-thrombin-formation-on-activated-platelets-and-fibrin-formation-in-venous-flow-condition/