Protein Kinase C-related Kinase and ROCK Are Required for Thrombin-induced Endothelial Cell Permeability Downstream from G{alpha}12/13 and G{alpha}11/q

Increase in vascular permeability occurs under many physiological conditions and is central in diverse human pathologies. Thrombin is a pro-coagulant serine protease, which causes the local loss of endothelial barrier integrity thereby enabling the rapid extravasation of plasma proteins and the local formation of fibrin-containing clots. Available information suggests that thrombin induces endothelial permeability by promoting actomyosin contractility through the Rho/ROCK signaling pathway. Here we took advantage of pharmacological inhibitors, knockdown approaches, and the emerging knowledge on how permeability factors affect endothelial junctions to investigate in detail the mechanism underlying thrombin-induced endothelial permeability. We show that thrombin signals through PAR-1 and its coupled G proteins G(12/13) and G(11/q) to induce RhoA activation and intracellular calcium elevation, and that these events are interrelated. In turn, this leads to the stimulation of ROCK, which causes actin stress-fiber formation. However, this alone is not sufficient to account for thrombin-induced permeability. Instead, we found that protein kinase C-related kinase, a Rho-dependent S/T kinase, is activated in endothelial cells upon thrombin stimulation and that its expression is required for endothelial permeability and remodeling of cell-extracellular matrix and cell-cell adhesions. Our results demonstrate that the signal initiated by thrombin bifurcates at the level of RhoA to promote changes in the cytoskeletal architecture through ROCK and the remodeling of focal adhesion components through PRK. Ultimately, both pathways converge to cause cell-cell junction disruption and provoke vascular leakage.

💡 Research Summary

This study dissects the signaling cascade by which thrombin compromises endothelial barrier integrity, a process central to inflammation, hemostasis, and tumor metastasis. Using human endothelial cells, the authors demonstrate that thrombin activates the protease‑activated receptor‑1 (PAR‑1), which couples to two heterotrimeric G‑protein families: Gα12/13 and Gα11/q. Both G‑protein pathways are required for full activation of the small GTPase RhoA and for a rapid rise in intracellular calcium; inhibition of either Gα subunit blunts both responses, indicating that the two branches are interdependent rather than strictly parallel.

RhoA activation traditionally leads to the activation of Rho‑associated coiled‑coil containing protein kinase (ROCK). ROCK phosphorylates myosin light chain (MLC) and LIM‑kinase, promoting actin‑myosin contractility and the formation of stress fibers. Pharmacological blockade of ROCK with Y‑27632 abolishes stress‑fiber formation but does not fully prevent thrombin‑induced permeability, suggesting that additional downstream effectors are required.

The authors identify Protein Kinase C‑related Kinase (PRK), a Rho‑dependent serine/threonine kinase, as this missing component. Within minutes of thrombin stimulation, PRK becomes phosphorylated and catalytically active. siRNA‑mediated knockdown of PRK or selective PRK inhibition (e.g., BIM‑1) leaves stress‑fiber formation intact but markedly reduces phosphorylation of focal‑adhesion proteins such as paxillin, focal adhesion kinase (FAK), and vinculin. Moreover, PRK inhibition stabilizes VE‑cadherin/β‑catenin complexes at cell‑cell borders, preventing the junctional disassembly that normally follows thrombin exposure. Consequently, endothelial monolayer permeability is dramatically reduced despite ongoing ROCK activity.

These findings support a bifurcated model downstream of RhoA: one arm proceeds through ROCK to increase contractile tension, while the other arm proceeds through PRK to remodel focal adhesions and adherens junctions. Both arms must be engaged simultaneously to generate the intercellular gaps that underlie vascular leakage. The study further shows that simultaneous inhibition of Gα12/13 (with YM‑254890) and Gα11/q (with pertussis toxin) suppresses both RhoA activation and calcium signaling, thereby preventing activation of both ROCK and PRK and fully protecting the barrier.

Clinically, the work expands the therapeutic landscape beyond ROCK inhibitors, which have shown efficacy in pulmonary hypertension and certain inflammatory conditions. Targeting PRK, either alone or in combination with ROCK blockade, could provide a more comprehensive strategy to curb pathological permeability without the side‑effects associated with high‑dose ROCK inhibition. Likewise, drugs that disrupt Gα12/13 or Gα11/q coupling to PAR‑1 may serve as upstream “dual‑node” inhibitors, simultaneously dampening contractile and adhesive remodeling pathways.

In summary, the paper elucidates a dual‑pathway mechanism by which thrombin, via PAR‑1‑coupled Gα12/13 and Gα11/q, activates RhoA, which then diverges to ROCK‑mediated actomyosin contractility and PRK‑mediated focal‑adhesion remodeling. The convergence of these pathways leads to adherens‑junction disassembly and increased endothelial permeability. This mechanistic insight not only clarifies long‑standing ambiguities about thrombin‑induced barrier dysfunction but also identifies PRK as a novel, druggable target for diseases characterized by excessive vascular leakage.