Date Published: December 16, 2013
Publisher: Public Library of Science
Author(s): Swapan K. Dasgupta, Anhquyen Le, Sandra B. Haudek, Mark L. Entman, Rolando E. Rumbaut, Perumal Thiagarajan, Kathleen Freson.
The transbilayer movement of phosphatidylserine mediates the platelet procoagulant activity during collagen stimulation. The Rho-associated coiled-coil kinase (ROCK) inhibitor Y-27632 inhibits senescence induced but not activation induced phosphatidylserine exposure. To investigate further the specific mechanisms, we now utilized mice with genetic deletion of the ROCK1 isoform.
ROCK1-deficient mouse platelets expose significantly more phosphatidylserine and generate more thrombin upon activation with collagen compared to wild-type platelets. There were no significant defects in platelet shape change, aggregation, or calcium response compared to wild-type platelets. Collagen-stimulated ROCK1-deficient platelets also displayed decreased phosphorylation levels of Lim Kinase-1 and cofilin-1. However, there was no reduction in phosphorylation levels of myosin phosphatase subunit-1 (MYPT1) or myosin light chain (MLC). In an invivo light/dye-induced endothelial injury/thrombosis model, ROCK1-deficient mice presented a shorter occlusion time in cremasteric venules when compared to wild-type littermates (3.16 ± 1.33 min versus 6.6 ± 2.6 min; p = 0.01).
These studies define ROCK1 as a new regulator for collagen-induced phosphatidylserine exposure in platelets with functional consequences on thrombosis. This effect was downstream of calcium signaling and was mediated by Lim Kinase-1 / cofilin-1-induced cytoskeletal changes.
The Rho-like small GTPases such as RhoA, Rac, and Cdc42 regulate cytoskeletal remodeling by binding to downstream effectors in a variety of cells [1–3]. Two closely related kinases, Rho-associated coiled-coil serine/threonine kinase-1 (ROCK1) and -2 (ROCK2) have been identified as key downstream effectors of RhoA . Though ROCK1 and ROCK2 share 92% amino acid sequence identity across their kinase domains, they have distinct biological effects . In addition, genetic deletion of ROCK2 is embryonically lethal, as ROCK1 cannot compensate for the loss of the other .
Results of this paper show that ROCK1-deficiency accentuated collagen-induced phosphatidylserine exposure in murine platelets. ROCK1-deficient mouse platelets had increased phosphatidylserine exposure and generated more thrombin following activation with collagen. These changes accounted for a shorter occlusion time in a light/dye-induced endothelial injury/thrombosis model and supported the concept that ROCK1 negatively regulated platelet procoagulant responses. It is also possible that changes in the endothelium may play an additional role in the procoagulant response. The most striking effect of ROCK1 deficiency in platelets resulted in the collagen-induced transbilayer movement of phosphatidylserine. There was no significant effect on platelet shape change or aggregation. Several investigators have noticed that the signal transduction pathways for phosphatidylserine exposure were different from those from platelet aggregation and secretion . Patients with impaired secretion have normal phosphatidylserine exposure and platelets from patients with Scott Syndrome, an isolated deficiency of platelet procoagulant activity due to defective phosphatidylserine exposure, have normal aggregation response .