Tyro3 is a key regulator of clot retraction mediated by platelet-derived protein S Free

Background:

Clot retraction is a crucial step in hemostasis that stabilizes the thrombus by consolidating the fibrin network and expelling excess fluid. This platelet-driven process is essential for wound healing and thrombus integrity. While integrin αIIbβ3 has been established as a key mediator of clot retraction, the upstream signaling pathways—particularly those involving endogenous platelet-derived modulators—remain poorly understood. Tyro3, AXL, and MER (TAM) receptors are known to transduce αIIbβ3 signals, and Protein S (PS), a vitamin K-dependent glycoprotein, serves as one of their ligands. Although PS is primarily synthesized in hepatocytes, emerging evidence shows that activated platelets secrete PS at sites of vascular injury, positioning it as a locally acting modulator of thrombus biology. However, its functional role within the clot microenvironment has not been fully explored.

We hypothesize that platelet-derived PS enhances clot retraction via TAM receptor signaling—particularly through Tyro3—beyond its known anticoagulant functions. Supporting this, PS shares significant structural similarity with Gas6, a TAM ligand known to promote platelet activation and clot dynamics. Notably, the TAM receptors exhibit differential affinities for PS and Gas6, raising the question of receptor-specific involvement in clot retraction.

Methods:

Human platelet-rich plasma (PRP) was prepared from citrated whole blood of healthy donors. Isolated platelets were washed and resuspended in PS-deficient plasma. Three experimental groups were established: control, Tyro3-inhibited (500 nM), and Axl/Mer-inhibited (100 nM). Coagulation was initiated by adding 0.5% red blood cells (RBCs) and thrombin (1 U/mL). Clot images were captured at defined time points, and clot mass was recorded to assess retraction kinetics. Fibrin architecture was analyzed using confocal microscopy. Clots were lysed and subjected to immunoblotting using Tyro3, AXL, and MER antibodies. In parallel, clot dynamics under physiological hematocrit (33%) were assessed using a microfluidic broadband dielectric spectroscopy sensor, which tracked changes in relative permittivity (εr) as a surrogate marker for clot compaction.

Results:Our findings reveal that Tyro3 is a critical mediator of PS-induced clot retraction. Pharmacological inhibition of TAM receptors delayed clot retraction and increased clot mass. Specifically, Tyro3 inhibition had the most pronounced effect, resulting in a clot weight of 57 mg at 60 minutes, compared to 19 mg in controls and Axl/Mer-inhibited samples. Dielectric spectroscopy corroborated these findings: the maximal change in relative permittivity (Δεr,max) was highest in controls (0.04295 ± 0.016), reduced in Axl/Mer-inhibited samples (0.01812 ± 0.014), and nearly abolished in Tyro3-inhibited samples (0.002846 ± 0.0004). Immunoblotting confirmed a significant reduction in Tyro3 protein levels in Tyro3-inhibited samples, validating the specificity of the inhibitor. These results strongly implicate Tyro3 as the principal TAM receptor involved in PS-mediated clot retraction.

Conclusion:

This study establishes platelet-derived Protein S as a novel regulator of clot retraction, functioning primarily through Tyro3-dependent TAM receptor signaling. The observed enhancement of fibrin deposition following exogenous PS treatment suggests a role for PS in amplifying integrin-mediated signaling cascades during thrombus maturation. However, the intracellular mechanisms linking PS-Tyro3 interaction to platelet inside-out signaling remain to be defined. Further studies are warranted to elucidate the downstream molecular pathways involved in this process.