Összes szerző

Hársfalvi Jolán

az alábbi absztraktok szerzői között szerepel:

Csányi Mária Csilla
Nanosurgical manipulation of extended von Willebrand factor multimers

Aug 29 - kedd

15:30 – 17:00

I. Poszterszekció

P07

Nanosurgical manipulation of extended von Willebrand factor multimers

Mária Csilla Csányi¹, Dominik Sziklai¹, Tímea Feller², Jolán Hársfalvi¹ and Miklós S.Z. Kellermayer¹

¹ Semmelweis University, Faculty of Medicine, Department of Biophysics and Radiation Biology

² University of Leeds, Leeds Institute of Cardiovascular and Metabolic Medicine, Discovery and Translational Science Department

The von Willebrand factor (VWF) is a varying-length multimeric chain of glycoprotein dimers/protomers with distinct domain structures. Conformational response of domains upon exposure to shear- and elongational forces ensures that VWF is able to mediate platelet adhesion to and aggregation on an injured vessel wall where it is explored or immobilized from the circulation. Human plasma-derived VWF multimers stretched by molecular combing extend through a specific hierarchy of structural intermediates of protomers as we have recently shown by using atomic force microscopy (AFM) [1]. However, the full scope of local domain stabilities and extensibilities remained hidden.

To uncover these cryptic structural details, in the present work, we used an in situ nanosurgical approach to probe whether targeted domains of VWF could be further extended and unfolded. To achieve this, surface-stabilized and pre-extended VWF multimers were manipulated at distinct spatial locations with the tip of the AFM cantilever. By moving the AFM tip in a direction perpendicular to the longitudinal axis of the VWF multimer, protein loops could be pulled out of the chain, the local extension of which was assessed following the acquisition of a subsequent topographic AFM image.

The extension resulted in ruptured and non-ruptured protein loops, with and without the appearance of hairpin-like thin sections and nodules, while the adjacent domains were non-displaced. Ruptures occurred in 27% of the VWF. Extension, which is the ratio of the length of the section following and prior to manipulation was 2.7 vs. 1.6, p= 0.0001 in non-ruptured and ruptured multimers. Nanomanipulation of protomers in which all the 5 nodules are separated, resulted in a mean final length of 345±69 nm, compared to their pre-manipulation length of 168±54 nm. None of the above results correlated with the protomer’s structural hierarchy showing the main role of the adhesion of certain domains to the mica surface. The nanosurgical manipulation used here demonstrates the VWF mechanical properties at the single domain level via extending further the C1-C6 and A1-A2-A3 domains or eventually rupturing them. The observed conformational changes indicate that VWF may have a large conformational force response potential in order to fine-tune the opening up of hidden epitopes for the different functions of the VWF.

Acknowledgment

TKP2021-EGA-23

References

[1] Csányi MC, Salamon P, Feller T, Bozó T, Hársfalvi J, Kellermayer MSZ. (2023) Protein Sci 32(1):e4535.

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