Primary platelet signaling cascades and integrin-mediated signaling control ADP-ribosylation factor (Arf) 6-GTP levels during platelet activation and aggregation |
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Authors: | Karim Zubair A Choi Wangsun Whiteheart Sidney W |
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Institution: | Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky 40536-0509, USA. |
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Abstract: | Previous studies showed that ADP-ribosylation factor 6 (Arf6) is important
for platelet function; however, little is known about which signaling events
regulate this small GTP-binding protein. Arf6-GTP was monitored in platelets
stimulated with a number of agonists (TRAP, thrombin, convulxin, collagen,
PMA, thapsigargin, or A23187) and all led to a time-dependent decrease in
Arf6-GTP. ADP and U46619 were without effect. Using inhibitors, it was shown
that the decrease of Arf6-GTP is a direct consequence of known signaling
cascades. Upon stimulation via PAR receptors, Arf6-GTP loss could be blocked
by treatment with U-73122, BAPTA/AM, Ro-31-8220, or Gö6976, indicating
requirements for phospholipase C, calcium, and protein kinase C (PKC)
α/β, respectively. The Arf6-GTP decrease in convulxin-stimulated
platelets showed similar requirements and was also sensitive to piceatannol,
wortmannin, and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, indicating additional requirements for Syk and
phosphatidylinositol 3-kinase. The convulxin-induced decrease was sensitive to
both PKCα/β and δ inhibitors. Outside-in signaling,
potentially via integrin engagement, caused a second wave of signaling that
affected Arf6. Inclusion of RGDS peptides or EGTA, during activation, led to a
biphasic response; Arf6-GTP levels partially recovered upon continued
incubation. A similar response was seen in β3 integrin-null platelets.
These data show that Arf6-GTP decreases in response to known signaling
pathways associated with PAR and GPVI. They further reveal a second,
aggregation-dependent, process that dampens Arf6-GTP recovery. This study
demonstrates that the nucleotide state of Arf6 in platelets is regulated
during the initial phases of activation and during the later stages of
aggregation.Platelet activation is initiated through several classes of membrane
receptors, which are stimulated by agonists produced at the vascular lesion
(1–3).
A second wave of signaling, caused by engagement of integrins, occurs as
platelets bind to the lesion surface and aggregate
(4). Together, these plasma
membrane proteins initiate the platelet processes important for thrombosis
(e.g. adhesion, spreading, secretion, and clot retraction). Small
GTP-binding proteins, specifically members of the Ras superfamily, link
signaling events from various platelet receptors to defined outcomes, such as
shape change
(5–7),
aggregation (8,
9), and secretion
(10–12).
Rab proteins play roles in granule secretion, with Rab4 and Rab6 being
involved in alpha granule release
(10,
11) and Rab27a/b in dense core
granule release (12,
13). RalA is activated in
response to various stimuli
(14–16)
and may play a role in secretion by anchoring the exocyst complex to specific
membrane sites (17). Rap1
plays a role in integrin αIIbβ3 activation
(8,
9). Rho family GTPases (Rho,
Rac, and Cdc42) play roles in platelet phosphoinositide signaling and in the
regulation of the actin cytoskeleton
(5–7).
While these small GTP-binding proteins are clearly important to platelet
function, it is equally clear that other small G proteins are present and
functional in platelets
(18).The ADP-ribosylation factor
(Arf)2 family are
Ras-related, small GTPases that affect both vesicular transport and
cytoskeletal dynamics (19,
20). Based on their primary
sequences, this family is divided into three classes, with Arf6 as the only
member of class III (19).
Arf6-GTP is considered the “active state” and can interact with
downstream effectors, such as phospholipase D (PLD)
(21), phosphatidylinositol
4-phosphate 5-kinase type α
(22), and arfaptin 2
(23,
24), resulting in the
recruitment of these effectors to the plasma membrane. The Arf6 GTP/GDP cycle
is mediated by interactions with guanine nucleotide exchange factors (GEFs)
and GTPase-activating proteins (GAPs). The large number of Arf-GEF and -GAP
proteins have been discussed in recent reviews where it was noted that, unlike
other small GTPases, Arf functions are generally not mediated solely by the
GTP-bound state but through its cycling between states
(19,
20,
25,
26).The effects that Arf6 has on the secretion and actin dynamics in nucleated
cells make it an ideal candidate for function in platelets. Arf6 influences
cortical actin and is important for spreading, ruffling, migration, and
phagocytosis (reviewed in Ref.
19). Our previous work
(27) showed that Arf6 is
present on platelet membranes and is important for platelet function. Unlike
other small G proteins, the Arf6 GTP-bound form is readily detectible in
resting platelets and upon activation with collagen or convulxin there is a
rapid conversion to the GDP-bound form. Acylated peptides, which mimic the
myristoylated N terminus of Arfs have been used as isoform-specific inhibitors
(28). In platelets, a
myristoylated-Arf6 (myr-Arf6) peptide specifically blocks the
activation-dependent loss of Arf6-GTP. This peptide also blocks aggregation,
spreading on collagen, and activation of the Rho family of GTPases. Other
GTPases, such as Ral and Rap, were unaffected. The simplest explanation for
these data is that platelet activation stimulates the GTPase activity of Arf6,
perhaps through activation of an Arf6-GAP. Alternatively, platelet activation
could affect an Arf6-GEF thus reducing the production of Arf6-GTP. Regardless
of mechanism, disruption of the activation-dependent loss of Arf6-GTP, with
the myr-Arf6 peptide, profoundly affects the actin-based cytoskeletal
rearrangements associated with platelet activation. While our initial report
(27) established a role for
Arf6 in platelet function, it was not clear what platelet signaling events
were required to induce the loss of Arf6-GTP.In this article, we delineate the signaling cascades required for the
activation-dependent loss of Arf6-GTP. We show that the Arf6-GTP to -GDP
conversion was stimulated by primary agonists (thrombin, TRAP, collagen, or
convulxin) but not by ADP or U46619. The decrease in Arf6-GTP, downstream of
thrombin and convulxin, required PLC, and PKC activity. Loss of Arf6-GTP, via
stimulation of GPVI with convulxin, additionally required Syk and PI3K
activities. Pretreatment with passivators, nitric oxide (NO), and
prostaglandin I2 (PGI2) blocked thrombin- and
convulxin-induced loss of Arf6-GTP. Further experiments suggested a role for
“outside-in” signaling, especially once platelet aggregates begin
to form. Inclusion of RGDS peptide, EGTA, or the deletion of the β3
integrin had only minimal effects on the initial loss of Arf6-GTP but led to
the partial recovery of Arf6-GTP levels. This biphasic change in Arf6-GTP
levels was not seen when aggregation was allowed to occur normally. Taken
together, these data show that the Arf6 nucleotide state is responsive to both
initial agonist-mediated signaling and to a second wave of integrin-mediated
signaling that occurs upon aggregation. |
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Keywords: | |
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