A Reinforced Fibrin-Platelet Layer at the Hemostatic Blood Clot Surface In Vivo is Formed by Platelet Contraction

Spatial organization of the fibrin network in the hemostatic clots affects their mechanical performance and susceptibility to lysis. We developed a murine penetrating parenchymal organ injury model that produces large-scale, erythrocyte-rich hemostatic clots. Confocal scanning and two-photon fluorescent microscopy revealed a dense, reinforced fibrin–platelet surface layer covering the clot at the boundary with air. The thickness and density of this layer gradually increased over 1 hour post-injury, accompanied by a significant progressive narrowing of the wound edges. Scanning and transmission electron microscopy confirmed a tightly woven network of fibrin fibers within this outer zone. In contrast, the internal regions of the clot were packed with compressed polyhedral erythrocyte, indicating substantial global clot contraction. Preventing contact between blood and air by applying an oil overlay moderately reduced the thickness and density of the surface layer. Conversely, pharmacological inhibition of non-muscle myosin using blebbistatin profoundly disrupted the formation of this outer shell, causing its relative density to fall to the average level of the inner clot core. Blebbistatin also prevented the wound gap closure. Atomic force microscopy demonstrated that the outer layer possessed significantly lower deformability and drastically higher stiffness compared to the clot core. These findings reveal an essential physiological role of actomyosin-driven clot contraction in structurally reorganizing hemostatic clots to form a protective, mechanically resilient outer barrier that seals large injuries.