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Biomechanics

Biomechanics - Poster Session B

Poster D2 - Comparative Analysis of Force Generation by High-Force Platelets on von Willebrand Factor and Fibrinogen

Thursday, October 24, 2024
2:45 PM – 3:45 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Platelets are blood cells that join together during hemostasis to prevent the loss of blood in mammals. In the formation of a platelet plug, platelets send cell-to-cell signals, extend and bind to proteins and other platelets, and contract. Platelet contraction (i.e., force generation) is essential in the stopping of bleeding and in the reinforcement of hemostatic plugs. Two proteins capable of activating platelets and initiating contraction are fibrinogen and von Willebrand factor (VWF). Fibrinogen, known as coagulation factor I, is a glycoprotein complex produced within the liver that binds with and cross-links platelets to form a hemostatic plug and cease bleeding. VWF, like fibrinogen, is a glycoprotein produced by endothelial (cells found in blood vessels and the heart) and megakaryocytes (cells in bone marrow that produce platelets). VWF binds to collagen around a hemorrhage and then recruits platelets to that spot and promotes these platelets to aggregate together to form a clot. Much of platelet force research has examined the average forces generated by platelets as either a full clot or as an average of a large pool of single platelets. The aims of this research is to: (1) examine how high-force platelets, stored at various temperatures, interact with VWF and fibrinogen and (2) investigate whether it is important to look at both the average of all platelet forces and the individual platelet forces, as it may show us a new view on the data.

Materials and

Methods:  Black dots analysis (BDs) was used to measure single platelet cellular forces. BDs involves laying a fluorescent, flexible surface (created by polydimethylsiloxane, PDMS and Alexa Fluor 594) with a nonfluorescent pattern of microscopic circles. A master structure, casted via Sylgard 184, was pipetted with fluorescent bovine serum. The dots were transferred onto the PDMS surface using a vinyl alcohol film. This microcontact printing process resulted in a flexible substrate with a uniform pattern of circles that are 1 µm in diameter and 2 µm apart (center-to-center). This flexible substrate allowed for platelets to adhere and contract, creating deformation of the circles (Figure A). Platelets were then fixed, imaged and analyzed using a MATLAB code, which tracked the deformations of the dot pattern to calculate platelet contractile forces (Figure B). BDs is a high-yield, easily producible method that enables us to examine contraction forces and structure of platelets.

Blood samples were collected via apheresis, resulting in platelet rich plasma (PRP). Three conditions were examined: fresh (not stored), cold (stored at 4 °C for 5 days) and RT (stored at room temperature for 5 days). After meeting these conditions, platelets were isolated and washed from PRP via differential centrifugation. With the washed platelets the final steps of the BDs occurred as the platelets were then seeded and prepped for imaging. In summary, this research employed BDs, human blood samples, and different platelet storage techniques to examine how platelets with higher contractile forces interact with VWF and fibrinogen under various storage conditions.

Results, Conclusions, and Discussions: The original analysis of comparing platelet forces between the fresh, cold-stored, and room temperature-stored platelets, observed no differences among average platelet forces (Figure C). This resulted in the conclusion that storage temperatures do not influence platelet forces.

It was only when we looked at high-force platelets bound to VWF and fibrinogen did we find significant differences between the platelets in these conditions. On VWF, when looking at the top 15%, significantly higher forces were produced by fresh platelets compared to cold-stored or room temperature-stored (Figure D(i)). On fibrinogen, no significant differences were observed (Figure D(ii)). This trend helped us interject that high-force platelets may interact with VWF differently and/or that storage may change platelets’ ability to interact with VWF. These results suggested that analyzing this subpopulation of platelets might bring different insights opposed to solely focusing on the average forces of a whole platelet population.

In summary we concluded: that 1) high-force platelets may interact with fibrinogen and VWF differently at different storage temperatures and that 2) examining subpopulations of platelets may lead to distinct interpretation of data as compared to solely looking at average platelet forces.

This work is important because it may help the understanding of high-force platelets, and indicate that it may be important to account for subpopulations of platelets in addition to total average forces. There is still an ignorance about the function and role of high-force platelets in hemostasis. Within this analysis, we determined that populations of fresh and stored platelets had no significant differences between each other, but when inspecting a subpopulation, we found there is a significant difference between the groups. These differences could be related to Glycoprotein VI (GPVI), a membrane surface receptor that binds to collagen and fibrinogen and assists the aggregation of platelets under flow. GPVI expression varies in cold-stored platelets compared to fresh and room temperature-stored platelets, which could affect binding and contraction. With a higher understanding of high-force platelets and how they interact with other cells and proteins, we may be able to understand platelet contraction, interpret platelet function, and detect platelet dysfunction more sensitively.

Acknowledgements (Optional):  We would like to acknowledge Jeffrey Miles, Moritz Stolla, and Nathan Sniadecki. This work was partially funded by the American Heart Association (24CDA1268959).