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Domjan György

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Hársfalvi Jolán
Biophysical Characterization of Clot Retraction in Platelet Rich Plasma of Patients with Primary Anti-phospholipid Syndrome

Aug 29 - kedd

15:30 – 17:00

I. Poszterszekció

P11

Biophysical Characterization of Clot Retraction in Platelet Rich Plasma of Patients with Primary Anti-phospholipid Syndrome

Jolán Hársfalvi¹_, Tímea Feller¹, György Domjan³, Klára Gadó³, Katalin Várnai², Eszter Barabás², Miklós Kellermayer¹

¹Department of Biophysical and Radiation Biology, ²Department of Laboratory Medicine and ³Department of Internal Medicine and Oncology, Semmelweis University, Budapest, Hungary

Anti-phospholipid syndrome (APS) is an autoimmune process leading to thrombotic disorders [1], but with poorly understood mechanisms.

By using atomic force microscopy (AFM)-based nano-thrombelastography (nTEG) [2], we investigated the biophysical characteristics of the fibrin network during clot formation and degradation in platelet-rich plasma (PRP) of 38 APS patients compared with 18 controls. Patients with APS and venous thromboembolism (VTE) were selected as case and control, respectively.

Citrated blood was centrifuged at room temperature (150 g, 10 min) to obtain PRP, so that platelet count was set to 50G/L. An AFM cantilever was submerged in a 0.3-mL sample and cyclically moved up and down with an amplitude of 1 μm and a speed of 1 μm/s. In addition to PRP the sample was completed with 10mM Ca²⁺, and clotting was initiated with thrombin at a final activity of 1 IU/ml. As the sample clotted, the cantilever deflected progressively during its vertical travel, reflecting the onset and increase in the elastic and viscous properties of the clot. The onset of clot formation was determined by measuring the time delay until the first non-zero force signal appeared. Clot contractility was assessed by measuring the rate of force increase. Finally, the viscoelastic response of the clot was obtained by measuring the force hysteresis area and the peak force difference in the datasets collected in the opposite cantilever directions (up versus down). The median parameter values of the APS and control samples were compared.

We found that in the APS group, the delay until the first force signal increased 2-3-fold [sec]; the slope of the force generation decreased to about 1/2 [nN/sec]; and the maximal force difference in the mechanical cycles decreased to about 1/3 [nN]. These results compare well with recent observations in which macroscale methods were used [3].

In sum, we were able to characterize quantitatively the nanoscale changes in the viscoelastic properties during platelet contractility and fibrin network formation in human PRP in a distinct pathology. We expect that the rich dataset provided by the AFM-based measurement employed here will provide insights into the molecular mechanisms associated with the pathology of APS.

References

1. Vreede AP, Bockenstedt PL, McCune WJ, Knight JS (2019) Curr Opin Rheumatol. 31(3):231-240.

2. Feller T, Kellermayer MS, Kiss B. (2014) Journal of Structural Biology 462-71

3. Le Minh, G., A.D. Peshkova, I.A. Andrianova, T.B. Sibgatullin, A.N. Maksudova, J.W. Weisel, and R. Litvinov, (2018) Clinical Science 132: 243-254