Blood Clots are Mechanically Weak in Patients with Sickle Cell Disease

Introduction:: Sickle cell disease (SCD) is a genetic blood disorder associated with high morbidity and mortality.¹ In SCD, a mutation causes hemoglobin to polymerize when deoxygenated, giving red blood cells their characteristic sickle shape and making them stiff and rigid.² Ultimately, this leads to many downstream complications. For example, sickle cell blood clots more easily than healthy blood.³ Observational data has linked this increased tendency to form venous blood clots to an overall state of hypercoagulability within sickle cell blood, which naturally results in severe pulmonary complications. In fact, pulmonary complications, such as acute chest syndrome, pulmonary hypertension, and pulmonary embolism, are some of the most severe sequalae of SCD and are collectively the leading cause of mortality in patients. Patients with sickle cell disease are 3.5 times more likely to experience pulmonary embolism than hospitalized, race-matched controls, i.e., clinically, it appears that clots from sickle cell patients do not resist hemodynamic forces as well as those in other patients.⁴ Unfortunately, the mechanisms and disease specific factors that cause hypercoagulability and resulting pulmonary complications remain unknown. Since blood clot embolization is inherently a mechanical process, where clots embolize or “fracture” when hemodynamic forces exceed the internal “strength” of the clot, we submit that investigating the mechanical properties of sickle cell blood clots will reveal insights into the mechanisms and disease specific factors at play. We hypothesized that SCD clots are not only structurally abnormal, but that this abnormality informs an increased propensity for clot embolization and resulting severe pulmonary complications.


Materials and Methods:: Under the guidance and approval of the IRB, we acquired venipuncture derived blood samples from 7 healthy control subjects (males and females, ages 22-29) and 9 patients with sickle cell disease (males and females, ages 5-19). We coagulated blood into 3D-printed molds compatible with our mechanical testing machine, see figure 1. To do so, we added calcium chloride to a concentration of 20 mM to reverse the anticoagulant, cast samples into pure shear geometry following prior protocols, and incubated the samples for 60-minutes.⁵ Samples were then mounted to our tensile testing machine and two tests were conducted: extension-to-failure in a simple pure shear geometry and extension-to-failure in a mode I fracture geometry, as shown in figure 2A. We conducted all tests until failure at a strain rate of 1% s^-1.



Figure 2B illustrates three of our mechanical metrics, while we computed fracture toughness as Γ_C=W(λ_C)H, where H is the reference sample height and W(λ_C) is the strain energy density of the pure shear sample at the fracture stretch, λ_C. For each patient, we tested multiple samples (technical replicates). We utilized the afex library in R to use linear mixed models to account for random and fixed effects in our study. We defined differences to be statistically significant for p-values less than 0.05.


Results, Conclusions, and Discussions:: In our study, we collected various data in support of our central hypothesis by quantifying the structural abnormalities and propensity for embolization of SCD blood clots. Figure 3 shows that the stiffness of blood clots formed from healthy subjects and SCD patients varies greatly. In fact, the stiffness of healthy clots is 4.8± 0.9 kPa, which is significantly lower than the stiffness of SCD clots, 7.2 ± 1.7 kPa. This data suggests that sickle cell clots respond to external forces differently than clots made from healthy blood, indicating underlying structural abnormalities. What specific factors and mechanisms account for the stiffness difference and how this difference may contribute to SCD patients’ increased risk of pulmonary complications remain unknown.



Figure 4 compares the fracture toughness of sickle cell and healthy clots, which colloquially is a measure of whether a blood clot is strong or weak. A high fracture toughness means that a clot will resist embolization well, while a lower fracture toughness means that a clot will not resist embolization well. The blood from SCD patients makes clots with a fracture toughness of 2.8±1.0 J/m², while blood from healthy controls makes clots with a fracture toughness of 3.9±0.6 J/m². Thus, our data supports our hypothesis that structural abnormalities in SCD blood clots renders patients more susceptible to embolization, which likely explains the pulmonary complications so prevalent among SCD patients. Nonetheless, there is significant work to be done to fully elucidate the mechanisms behind sickle cell blood clots’ mechanical differences when compared to healthy clots. While data shows that clots from SCD patients have a lower fracture toughness, we do not know why this is the case, what disease specific factors may be responsible for this difference, nor whether this weakness is modifiable or treatable. Moving forward, we hope to continue studying SCD blood in hopes of discovering more about their clot structure, mechanics, and how these factors influence their high rates of pulmonary embolisms and other pulmonary complications.


Acknowledgements (Optional): :

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3. Connes, P. et al. The role of blood rheology in sickle cell disease. Blood Rev. 30, 111–118 (2016).
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5. Sugerman, G. et al. Preparation and Mounting of Whole Blood Clot Samples for Mechanical Testing. Curr. Protoc. 1, e197 (2021).