RBCs promote VWF tension-dependent activation and hemostatic clot formation under hemodilution: A combined in silico and ex vivo study

Abstract Introduction: Dilutional coagulopathy remains a major challenge in trauma and transfusion medicine, yet the mechanistic basis of impaired hemostasis under hemodilution is not fully understood. While red blood cells (RBCs) are traditionally regarded as passive carriers of hemoglobin and contributors to bulk blood viscosity, emerging evidence suggests that they may actively influence hemostasis by mechanically modulating thrombus formation. We hypothesize that RBCs mechanically promote von Willebrand factor (VWF) tension-dependent activation, and adding RBCs restores hemostatic clotting efficiency under dilutional hemorrhagic flow conditions. Methods: We developed and applied an in silico molecular and cellular blood flow and clotting model, run on supercomputers, to quantify the stretching dynamics and tensile mechanics of VWF multimers under hemorrhagic flow conditions with different hemodilution levels. The in silico investigation on VWF tension-dependent activation was complemented by ex vivo microfluidic experiments using healthy human whole blood and serially diluted blood (hemodilution) perfused through a hemostasis-on-a-chip system (250 µm height narrowing to 70 µm over 800 µm). Occlusion time (s) and blood loss volume (ml) were assessed as end points. RBCs were prepared from the same blood sample used for the dilution control. For the hemostatic restoration test, the packed RBCs were resuspended in 0.9% saline to restore the hematocrit to its original value. Results: In silico results show that RBCs at physiological hematocrit (~40%) generate squeezing-induced microscale stresses and elevate VWF multimer tension (>20 pN), promoting VWF-A1 activation under hemorrhagic shear conditions (>5000 1/s). Hemodilution to sub-physiological hematocrit (10%-30%) significantly reduces the probability of VWF tension-dependent activation by >50%. Hemodilution to ultra-low hematocrit (<10%) completely removes VWF tension-dependent activation needed for hemostasis. Correlated with in silico prediction, ex vivo hemodilution experiments show that reducing hematocrit to 24% prolongs the occlusion time by 2.1±0.6x (control group: 292±71s, p = 6.89 × 10⁻⁷, n = 17) and the blood loss (control group: 0.37±0.13 ml, p = 1.14 × 10⁻⁶, n = 17) by 3.8±1.9x. Reintroducing RBCs into the diluted samples significantly restores occlusion time and bleeding volume by ~95% without altering plasma protein levels. Additional replenishing platelet and plasma protein to physiological level only led to insignificant recovery of clotting efficiency compared to the RBC restoration case. Conclusions: Through an integrated in silico and ex vivo mechanistic framework, we demonstrate a novel biomechanical role of RBCs in promoting VWF tension-dependent activation and sustaining hemostatic clot formation under dilutional hemorrhagic flow conditions. These findings have direct implications for optimizing transfusion strategies and guiding therapeutic interventions for trauma-associated dilutional coagulopathy.