Rho associated coiled-coil kinase-1 regulates collagen-induced phosphatidylserine exposure in platelets

Abstract

Background: 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.

Methods and results: 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 in vivo 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).

Conclusions: 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.

Figure 1. Collagen-induced platelet aggregation and ATP secretion are normal in ROCK1-deficient mice.

Platelets were isolated from ROCK1-deficient mice and littermate wild-type controls and stimulated with the indicated concentrations of collagen. Aggregation and ATP secretion were measured in a lumi-aggregometer. The traces shown are representative of at least 3 independent experiments.

Figure 2. ROCK1 deficiency does not affect platelet shape change.

Isolated murine (A, B) or human (C) platelets were stimulated with collagen (10 µg/ml,) and aggregation and ATP release (where indicated) were measured in a lumi aggregometer. (A) Wild-type and ROCK1-deficient platelets responded similarly to collagen. (B) Addition of non-specific ROCK inhibitor Y-27632 to ROCK1-deficient platelets abolished platelet shape change, similar to its effect on human platelets, though it did not alter ATP secretion. (C) The traces shown are representative of at least 3 independent experiments.

Figure 3. ROCK inhibition and ROCK1-deficiency increases collagen-induced phosphatidylserine expression and thrombin generation.

Isolated human (A and B) or murine (C) platelets were stimulated with collagen (10 µg/mL), and FITC-lactadherin and PE-labeled anti-CD42b were added. The exposure of phosphatidylserine was analyzed by flow cytometry. Platelets were incubated with Y-27632 or latrunculin A for 20 minutes before collagen. (D) ROCK1-deficient and wild-type murine platelets were activated with collagen in the presence of prothrombin, factor Xa, calcium, and factor V; thrombin generation was measured by the amiodolyis of thrombin substarte S-2238. (n= 3/group). * denotes a p value of < 0.05.

Figure 4. Collagen-induced calcium signaling I s independent of ROCK-1.

Fura-2 AM loaded platelets were incubated with buffer, Y-27632 and BAPTA-AM as described in materials and method and activated with collagen. The increase in calcium levels was quantified by measuring Fura-2 fluorescence. (A) Representative traces of Ca²⁺ signaling induced by collagen in isolated murine platelets. (B) Group data of intracellular Ca²⁺ levels in resting and activated platelets (n= 3/group) in presence of buffer, Y-27632 and BAPTA-AM. .

Figure 5. ROCK1-deficiency results in lower F-actin content.

Isolated platelets from ROCK1^-/- and wild-type mice were stimulated with collagen (10 µg/ml) for 2 minutes; then were fixed, permeabilized, and stained with Alexa Fluor 488-phalloidin, and the F-actin content was quantified by flow cytometry (n= 3/group).

Figure 6. ROCK1-deficieny results in reduced phosphorylation levels.

Isolated platelets from ROCK1^−/− and wild-type mice were stimulated with collagen (10 µg/ml). Platelets were solubilized at the indicated time intervals, subjected to SDS-PAGE and immunoblotted with antibodies against phospho-Lim Kinase-1 (LMK), phospho-cofilin-1, phospho-myosin phosphatase target subunit-1 (MYPT1); phospho-myosin light chain (threonine 18, P-18 MLC), and phospho-myosin light chain (serine 19; P-19 MLC). Immunoblot of β-actin was used as a loading control. One representative blot and group data (n= 3/group) are shown. * denotes a P value of less than 0.05 compared to wild-type.

Figure 7. Effect of ROCK1-deficiency on hemostasis in vivo.

(A) Tail-bleeding time. (B and C) Endothelial injury was induced by light/dye in the cremasteric venules and thrombus onset (B) and occlusion time (flow cessation; C) were monitored by intravital microscopy in ROCK1^-/- (n= 7) and wild-type mice (n= 8). The P value in (C) was determined by one-way ANOVA.