Stimulation of human red blood cells leads to Ca2+-mediated intercellular adhesion

Red blood cells (RBCs) are a major component of blood clots, which form physiologically as a response to injury or pathologically in thrombosis. The active participation of RBCs in thrombus solidification has been previously proposed but not yet experimentally proven. Holographic optical tweezers and single-cell force spectroscopy were used to study potential cell-cell adhesion between RBCs. Irreversible intercellular adhesion of RBCs could be induced by stimulation with lysophosphatidic acid (LPA), a compound known to be released by activated platelets. We identified Ca2+ as an essential player in the signaling cascade by directly inducing Ca2+ influx using A23187. Elevation of the internal Ca2+ concentration leads to an intercellular adhesion of RBCs similar to that induced by LPA stimulation. Using single-cell force spectroscopy, the adhesion of the RBCs was identified to be approximately 100 pN, a value large enough to be of significance inside a blood clot or in pathological situations like the vasco-occlusive crisis in sickle cell disease patients.

💡 Research Summary

This study investigates whether human red blood cells (RBCs) can actively participate in thrombus formation by adhering to one another, a phenomenon that has been hypothesized but not experimentally demonstrated. Using a combination of holographic optical tweezers (HOT) and single‑cell force spectroscopy (SCFS) based on atomic force microscopy, the authors examined cell‑cell adhesion under controlled conditions.

Stimulation with lysophosphatidic acid (LPA), a lipid mediator released by activated platelets, induced a rapid influx of extracellular Ca²⁺ through a non‑selective voltage‑dependent cation (NSVDC) channel. Intracellular Ca²⁺ elevation was confirmed by Fluo‑4 fluorescence imaging. Parallel experiments employed the Ca²⁺ ionophore A23187 to raise intracellular Ca²⁺ directly, bypassing LPA signaling. Both treatments caused a marked increase in intracellular Ca²⁺, accompanied by phosphatidylserine (PS) exposure on the outer membrane leaflet, as shown by annexin‑V‑FITC staining, indicating activation of a Ca²⁺‑dependent scramblase.

HOT experiments demonstrated that, after LPA (2.5 µM or 10 µM) exposure in the presence of extracellular Ca²⁺, RBCs formed irreversible contacts that could not be broken by the optical traps (≈5 mW per trap). Approximately 72 % of cell pairs adhered under these conditions, whereas control cells remained non‑adhesive. The adhesion was independent of cell orientation, as both discocyte and spherocyte morphologies displayed the effect.

To quantify the adhesion strength, SCFS was performed. In control measurements, the maximum unbinding force (F_max) averaged 28.8 ± 8.9 pN (n = 71). In LPA‑treated cells, F_max increased dramatically to 100 ± 84 pN (n = 193, three donors), a force magnitude that exceeds typical shear stresses in microcirculation and is therefore physiologically relevant for clot consolidation or pathological vaso‑occlusion.

The authors discuss a mechanistic cascade: LPA → NSVDC channel opening → Ca²⁺ influx → activation of Gardos (Ca²⁺‑activated K⁺) channel and scramblase → PS externalization → Ca²⁺‑dependent adhesion molecules (potentially integrin‑like proteins) mediate strong inter‑RBC binding. While the exact molecular mediators were not identified, the data convincingly show that Ca²⁺ elevation alone (via A23187) is sufficient to trigger adhesion, confirming Ca²⁺ as the pivotal second messenger.

Methodologically, the study benefits from rapid medium exchange using a PDMS microfluidic chip, allowing precise timing of stimulus delivery and immediate observation of Ca²⁺ dynamics. Statistical analysis employed Student’s t‑test, revealing highly significant differences (p < 0.001) between treated and control groups.

Limitations include the static nature of the experiments; shear flow, platelet‑RBC interactions, and the presence of plasma proteins were not examined. Moreover, only healthy donor blood was used, leaving open the question of how disease‑related alterations (e.g., in sickle cell disease or thalassemia) might modulate the observed adhesion.

In conclusion, the paper provides the first direct evidence that human RBCs can undergo Ca²⁺‑mediated, irreversible intercellular adhesion when stimulated by LPA or a Ca²⁺ ionophore. The measured adhesion forces (~100 pN) are sufficient to influence thrombus stability and may contribute to pathological vaso‑occlusive events. Future work should aim to identify the specific adhesion receptors involved and to validate these findings under physiologically relevant flow conditions.