Signaling pathways regulating LIM-kinase-1 activation and cofilin phosphorylation in activated platelets

The activation of platelets is a central step during the
physiological process of hemostasis and its understanding may lead
us to control the pathophysiological process of intra-arterial
thrombus formation and vascular occlusion, which can cause acute
coronary syndrome and myocardial infarction. One of the important
aspects of platelet activation is to understand the dynamic
regulation and rearrangement of the cytoskeleton after stimulation.
The morphological and functional changes of platelets require a
drastic remodeling of the actin cytoskeleton regulated by numerous
actin-binding proteins and signaling molecules such as the family
of Rho-GTPases. The small GTPase Rho can regulate several aspects
of cellular function, predominantly through its downstream effector
Rho-kinase. One of the well established Rho-kinase-mediated
signaling pathways is the phosphorylation of myosin light chain
(MLC) and its counteracting MLC phosphatase. Rho-kinase regulates a
second pathway that involves activation of LIM-kinases (LIMKs) and
subsequent phosphorylation and inactivation of cofilin, an actin
dynamizing protein. Dephosphorylation and activation of cofilin
lead to severing and depolymerization of existing actin filaments.
The signaling pathway Rho-kinase/LIMKs/cofilin phosphorylation
during platelet activation and the question, how the
phosphorylation of cofilin affects the actin dynamics underlying
platelet activation, has not previously been studied. The
physiological agonist thrombin and the pathophysiological relevant
agonist lysophosphatidic acid (LPA), which is the main
platelet-activating lipid in atherosclerotic plaque, were used as
platelet stimuli to address these questions. It was found that the
activation of Rho-kinase is important for an increase in F-actin
content underlying Ca2+-independent platelet shape change. The
activation of Rho-kinase was found to be upstream to secretion and
integrin IIbβ3 activation. The rapid activation of Rho-kinase
during secretion leads to a further increase in F-actin content as
compared to shape change. It was observed that LPA-stimulated dense
granule secretion is mainly regulated by Rho-kinase, whereas
secretion induced by thrombin was only in part
Rho-kinase-dependent. Together, these results show that Rho-kinase
regulates the F-actin increase underlying shape change and
secretion, but it is not directly involved in aggregation. This
study for the first time demonstrates that platelet expresses only
LIMK-1 and not LIMK-2. LIMK-1 can be activated by Rho-kinase as
well as by p21-activated kinases (PAKs). Our study shows that
LIMK-1 activation was mainly Rho-kinase dependent in LPA- and
thrombin-stimulated platelets. Although, PAK-1/2 activation was
observed during LPA-stimulated platelet shape change, PAKs are
unlikely to be involved in LIMK-1 activation in these cells. Like
Rho-kinase activation, it was also found that LIMK-1 activation was
independent and upstream of integrin IIbβ3 activation.
Surprisingly, the activation of LIMK-1 failed to increase cofilin
phosphorylation during shape change induced by LPA as well as by
thrombin. Inhibition of the Rho-kinase/LIMK-1 pathway unmasked
cofilin dephosphorylation suggesting that during shape change the
simultaneous activation of a cofilin phosphatase counteracts the
effect of LIMK-1 for phosphorylating cofilin. During secretion and
aggregation induced by LPA and thrombin, cofilin was rapidly
dephosphorylated and subsequently rephosphorylated; the latter
phase was due to Rho-kinase/LIMK-1 activation. After stimulation
with LPA and thrombin under conditions, where platelet aggregation
could not occur, the kinetics of cofilin de- and rephosphorylation
were unperturbed indicating their independence of integrin IIbβ3
engagement. Furthermore, the results clearly showed that cofilin
dephosphorylation is also independent and upstream of secretion,
since the onset of cofilin dephosphorylation was as rapid as
secretion in thrombin-stimulated platelets and also occurred in the
absence of dense granule secretion in LPA (10 µM)-stimulated
platelets. Since the kinetics of cofilin phospho-cycle was similar
during secretion and platelet aggregation in LPA- and
thrombin-stimulated cells, I propose a general two-step regulatory
process for cofilin phospho-cycle underlying primarily secretion,
and subsequently platelet aggregation: dephosphorylation by a
cofilin phosphatase and then rephosphorylation by the
Rho-kinase/LIMK-1 pathway. Our results showing that only
dephosphorylated (activated) cofilin binds with F-actin support
previous observations that the state of cofilin phosphorylation
determines its association with F-actin. The effect of Y-27632 in
resting platelets showing a reduction in cofilin phosphorylation,
and an increase of F-actin content and cofilin association with
F-actin suggested that LIMK-1-mediated cofilin phosphorylation
reduces the F-actin content and cofilin association with F-actin in
resting platelets. In contrast, during shape change, cofilin that
showed no change in its phosphorylation was rapidly associated with
the actin cytoskeleton. The maximal cofilin association with actin
cytoskeleton occurred before the maximal F-actin increase,
suggesting that cofilin association with F-actin might regulate the
turnover and actin polymerization during platelet shape change. It
is an open question, whether cofilin is locally dephosphorylated
before binding to F-actin during shape change. Previous studies in
other cells could correlate cofilin dephosphorylation (activation)
with the depolymerization of F-actin. However, in our studies
cofilin dephosphorylation during the initial phase of
thrombin-induced secretion (up to 30 seconds) was associated with a
large increase of F-actin and a high amount of cofilin association
with F-actin. Cofilin rephosphorylation after 30 seconds did not
decrease F-actin content and cofilin association with F-actin.
Together, in activated platelets the association of cofilin with
F-actin and the F-actin increase do not simply correlate to the
cofilin phosphorylation state: it seems to be more complex. It is
assumed that the cofilin phosphorylation and actin dynamics are
regulated in specific compartments during platelet activation. The
rapid cofilin dephosphorylation in platelets was mediated by an
okadaic-acid insensitive phosphatase. The activation of the cofilin
phosphatase seemed to be regulated at least in part by an increase
in intracellular Ca2+ and by PI3-kinase. Cofilin de- and
rephosphorylation occurring upstream of secretion and platelet
aggregation suggests that the enzymes regulating the cofilin
phospho-cycle could be potential targets for the development of
anti-thrombotic drugs.