Erythrocyte Rigidity Affects Blood Clot Contraction and Formation of Polyhedrocytes
Authors: Tutwiler Valerie , Litvinov Rustem I. , Nagaswami Chandrasekaran , Russell J. Eric , Siegel Don L. , Villa Carlos Hipolito , Pan Daniel , Muzykantov Vladimir R , Weisel John W. 
Published: October 2019

Abstract
Blood clot contraction or retraction has been implicated to play a significant role in hemostasis, reduction of thrombus volume, and wound healing. Clot contraction is driven by forces that are generated by platelets and transmitted by fibrin and results in volume shrinkage followed by the compaction of erythrocytes into the core of the blood clot, resulting in their mechanical deformation towards a polyhedral shape, giving rise to the term polyhedrocytes. Despite the fact that erythrocytes are a major component of blood clots, relatively little is known about the influence of the mechanical properties or deformability of erythrocytes on the process of clot contraction. Increased hematocrit reduces extent of clot contraction due to mechanical resilience of erythrocytes and it is likely that in addition to a volume fraction the stiffness of erythrocytes can also affect the extent and rate of clot contraction. Here we tested this assumption by using artificially or naturally stiffened erythrocytes that have pathophysiological implications. The reduced deformability of erythrocytes is associated with a number of pathological conditions, such as hypertension, diabetes mellitus, atherosclerosis and smoking, but perhaps one of the most well-known diseases associated with increased erythrocyte rigidity is sickle cell disease (SCD). Another example of naturally stiff erythrocyte membrane is that of llama or camel that have red blood cells with increased osmotic resistance.
To assess the extent of clot contraction, we used an optical tracking methodology that allows for the quantitative tracking for clot size. To assess the influence of erythrocyte rigidity on clot contraction we also used scanning electron microscopy to evaluate deformations of the erythrocytes, including the presence of polyhedrocytes. Centrifugation of citrated blood can be used to mimic the contractile forces generated by platelets and has been shown to cause polyhedrocyte formation. Increasing the erythrocyte rigidity through their treatment with a low concentration of glutaraldehyde resulted in a decrease in polyhedrocyte formation and the requirement of larger centrifugal forces to observe erythrocyte deformation, suggesting that the mechanical properties of erythrocytes could influence the process of clot contraction. As residual glutaraldehyde may have unwanted effects on platelets, clot contraction experiments were completed using naturally stiffer erythrocytes from SCD patients and llama ovalocytes, which are stiffer than human erythrocytes due to the increased amount of the membrane cytoskeletal protein spectrin. SCD patients were only included in this study if they had Sickle Trait, SCD Hb SS, SCD Hb SC and have not received recent transfusions. The blood samples of SCD patients were examined and on average had a 53% decrease (p<0.0001) in the extent of clot contraction compared to healthy subjects. Likewise, addition of llama ovalocytes to human platelet rich plasma resulted in a 28% decrease in extent of clot contraction compared to human erythrocytes and larger centrifugal forces were needed to see red cell deformation. SCD patients contracted 2.4X slower (p<0.001) during linear contraction (Phase 2) and 2.7X slower (p<0.05) during mechanical stabilization (Phase 3) when compared to healthy subjects. Clot contraction was impaired also by erythrocytes treated with antibodies that bind to the Wright b epitope on the erythrocytes and exert a rigidifying effect on the cells. The binding of the antibody to erythrocytes was determined by flow cytometry and the KD was ~50 nM. Increased red blood cell rigidity following exposure to antibodies was confirmed through mechanical and osmotic resistance and compared to unaltered erythrocytes.
Collectively, these results demonstrate that erythrocyte mechanical properties can influence the process of clot contraction so that stiffer cells reduce the rate and extent of clot contraction. A better understanding of the role of erythrocyte deformability in the process of clot contraction has the potential to inform the development of more targeted treatments for limiting bleeding and thrombosis in patients who are prone to having altered erythrocyte content and mechanical properties of these highly abundant cells embedded into blood clots and thrombi.

Disclosures
Weisel: Bayer: Research Funding.