?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 2 February 2009
PKCα regulates platelet granule secretion
and thrombus formation in mice
Olga Konopatskaya,1 Karen Gilio,2 Matthew T. Harper,1 Yan Zhao,3 Judith M.E.M. Cosemans,2
Zubair A. Karim,4 Sidney W. Whiteheart,4 Jeffery D. Molkentin,5 Paul Verkade,6 Steve P. Watson,3
Johan W.M. Heemskerk,2 and Alastair W. Poole1
1Department of Physiology & Pharmacology, School of Medical Sciences, University of Bristol, Bristol, United Kingdom. 2Department of Biochemistry, CARIM,
University of Maastricht, Maastricht, The Netherlands. 3Centre for Cardiovascular Sciences, Institute for Biomedical Research, Division of Medical Sciences,
The Medical School, University of Birmingham, Birmingham, United Kingdom. 4Department of Molecular and Cellular Biochemistry,
University of Kentucky College of Medicine, Lexington, Kentucky, USA. 5Division of Molecular Cardiovascular Biology,
Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA. 6Department of Biochemistry and Department of Physiology & Pharmacology,
Wolfson Bioimaging Facility, School of Medical Sciences, University of Bristol, Bristol, United Kingdom.
The PKC family comprises 10 isozymes grouped into 3 classes: con-
ventional (α, βI, βII, γ), novel (δ, ε, η/L, θ), and atypical (ζ, ι/λ) (1).
PKCs have long been known to play major roles in a number of
platelet processes (2), most importantly aggregation and secretion,
in which stimulation of platelets with diacylglycerol (DAG) or phor-
bol ester can induce aggregation and agonist-induced secretion
can be prevented by broad spectrum pharmacological inhibition
of PKC isoforms (3–6). Recently, it has been shown pharmacologi-
cally that PKC isoforms may exert a dual-control role in thrombus
formation by mediating secretion and integrin activation under
flow while also suppressing phosphatidylserine exposure and sub-
sequent thrombin generation and coagulation (7).
At least 4 PKC isoforms (α, β, δ, θ) are expressed in human plate-
lets (8–14), and it is becoming clear that each isoform may play a
different role in platelet function. Using genetic and pharmacolog-
ical approaches, we have shown PKCδ to play a negative role in reg-
ulating filopodia formation and platelet aggregation in response
to collagen through a functional interaction with the actin regula-
tory protein VASP (15, 16). Using biochemical approaches, PKCα
has been identified as an essential factor in positively regulating
α-granule and dense-granule secretion in platelets (17) as well as
platelet aggregation (18). We have recently shown, using biochemi-
cal and pharmacological approaches, that 2 tyrosine kinases, Syk
and Src, physically and functionally interact with PKCα to regu-
late each other and cellular activities in platelets (19). It is now
important that the function of PKCα in platelets be addressed
definitively by a genetic approach.
Genetic and molecular approaches have revealed a wide range of
roles for PKCα in other cell types. Cell proliferation, differentia-
tion, apoptosis, motility, and adhesion are all regulated by PKC
(1, 20), with consequent roles for PKCα in regulation of tumor pro-
gression. Although there is evidence that in some tumors, PKCα
may have a suppressor role (21, 22), in the majority of cases, PKCα
expression and activity are higher in tumors than in normal tis-
sues (23, 24) and PKCα activity promotes a more aggressive pheno-
type in breast cancer cells, with an increased metastatic potential
(25–27). PKCα has therefore become a major target for therapeutic
intervention in various cancers (28). In immune regulation, PKCα
mediates T cell–dependent interferon generation (29) and is also
critical for T cell receptor downregulation (30). Insulin signaling
to PI3K and subsequently to glucose transport is enhanced in mice
lacking PKCα (31), suggesting a negative feedback role for PKCα
in metabolic processes. Finally, in the heart, PKCα regulates car-
diac contractility and Ca2+ handling in myocytes, and deficiency of
PKCα protects against heart failure induced by pressure overload
and against dilated cardiomyopathy (32).
In a recent elegant study reconstructing the signaling pathway
regulating platelet integrin αIIbβ3 in a nucleated cell system, it was
shown that expression levels of PKCα equivalent to those found
in platelets are required for activation of the integrin through the
Rap1 pathway (33). This is in agreement with previous biochemi-
cal evidence suggesting an essential role for PKCα in regulating
integrin (18). Importantly also, genome-wide association analysis
of coronary artery disease revealed a cluster of SNPs in the PRKCA
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Nonstandard?abbreviations?used: CRP, collagen-related peptide; DIC, differential
interference contrast; GPVI, glycoprotein VI; PB, phosphate buffer.
Citation?for?this?article: J. Clin. Invest. 119:399–407 (2009). doi:10.1172/JCI34665.
400?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 2 February 2009
gene, with a maximal P value association of just over 10–3 (SNP
rs12600582) (34, 35). This analysis marks the gene as one requir-
ing further phenotypic analysis. It was essential to identify defini-
tively by a genetic approach the role of PKCα in regulating platelet
function and thrombus formation. Here, we report that genetic
ablation of PKCα in platelets reveals nonredundant roles in regu-
lating platelet dense-granule biogenesis and exocytosis and in the
control of thrombus formation in vitro and in vivo.
Major role for PKCα in inside-out regulation of αIIbβ3 but no significant role
in outside-in signaling through this integrin. The PKC family of kinases
has been implicated in agonist-induced αIIbβ3 integrin activation
(36), and although an elegant reconstruction study showed a criti-
cal role for PKCα in regulating the integrin (33), an involvement of
PKCα in this event in platelets has not yet been addressed directly
and definitively. Here, we report for what we believe is the first
time in platelet functional and thrombosis analysis, data from a
mouse gene knockout lacking PKCα. Supplemental Figure 1 (sup-
plemental material available online with this article; doi:10.1172/
JCI34665DS1) shows baseline data demonstrating lack of expres-
sion of PKCα but normal expression of other platelet PKCs (β, δ,
and θ) in Prkca–/– platelets. Figure 1A shows that both collagen-
related peptide (CRP) (5 μg/ml) and thrombin (1 U/ml) produced
significantly greater αIIbβ3 activation in WT platelets compared
with Prkca–/–. This suggests that PKCα is a major regulator of
inside-out signaling to αIIbβ3, although the residual response in
the absence of PKCα suggests a component independent of this
kinase. By way of control, Supplemental Figure 2 shows no signifi-
cant difference in surface expression of αIIb (CD41) in Prkca–/– ver-
sus WT irrespective of activation state of the platelets and irrespec-
tive of agonist (CRP or thrombin).
It has been shown recently that PKCβ and PKCθ play critical
roles in regulating outside-in signaling from αIIbβ3 (37, 38). Hence,
in mouse platelets lacking either PKCβ or PKCθ, cell spreading
on fibrinogen-coated surfaces was ablated. Additionally, we had
recently shown that absence of PKCδ caused platelets to spread on
CRP- or collagen-coated surfaces with markedly more sustained
generation of filopodia than WT control platelets. It was impor-
tant therefore to assess the role of PKCα in adhesion and spread-
ing on either fibrinogen or CRP, and so we measured the number
and surface area of adherent platelets by differential interference
contrast (DIC) microscopy over a 40-minute period. Figure 1, B–D,
shows that platelets lacking PKCα had no significant defect in
their ability to spread on either fibrinogen-coated or CRP-coated
surfaces, and importantly in the case of CRP, there was no evident
sustained development of filopodia. In addition, although Figure
1, C and D, shows a trend toward decreased adhesion of platelets to
both surfaces in Prkca–/–, significance was achieved only at a single
time point (30 minutes; 90.31 ± 6.9 platelets/field of view in WT
PKCα plays a major role in regulating
inside-out signaling to integrin αIIbβ3
but no significant role in outside-in sig-
naling or in adhesion to fibrinogen or
collagen. (A) Washed platelets from
WT or Prkca–/– mice were labeled with
PE–anti-αIIbβ3 antibody and stimulated
with CRP (5 μg/ml) or thrombin (1 U/ml)
for 15 minutes; immunofluorescence
intensity was measured by flow cytom-
etry. The data presented are geometric
means as percentages of basal non-
stimulated levels. Error bars represent
SEM. n = 3. *P < 0.05. (B–D) Washed
platelets from WT or Prkca–/– mice were
added to CRP- or fibrinogen-coated
coverslips, and the levels of spread-
ing and adherence were analyzed.
(B) Representative images of murine
platelets 40 minutes after addition to
CRP- or fibrinogen-coated surfaces as
indicated. Images were taken under
oil immersion. Original magnification,
×63. Scale bars: 5 μm. (C and D) Time
course of WT and Prkca–/– platelet
adherence (number of platelets esti-
mated per field of view; upper panel)
and spreading (as assessed by mea-
surement of cell surface area; lower
panel) on CRP-coated (C) and fibrino-
gen-coated (D) surfaces. Data shown
are mean ± SEM. n = 4.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 2 February 2009
compared with 62.18 ± 5.6 platelets/field of view in Prkca–/– mice;
mean ± SEM, P < 0.05) in platelets adherent to fibrinogen.
PKCα is a major regulator of dense- and α-granule secretion. Figure
2A shows that Prkca–/– platelets also exhibit a marked reduction
in CRP- and thrombin-induced ATP secretion relative to WT con-
trols (40.9 ± 7.0 nM ATP in WT vs. 8.9 ± 3.4 nM ATP in Prkca–/–
for CRP, P < 0.05; and 61.1 ± 14.7 nM in WT vs. 10.8 ± 5.7 in
Prkca–/– for thrombin, P < 0.05). The defect in granule secretion
was not restricted to dense granules, however, but also involved
regulation of α-granules. Stimulation with CRP induced P selec-
tin surface expression in WT platelets, which was significantly
greater than that in Prkca–/– platelets (411.9% ± 145.3% over basal
in WT vs. 130.7% ± 14.6% over basal in Prkca–/–). A similar marked
knockdown of P selectin exposure was observed after thrombin
activation (Figure 2B) (705% ± 235.7% in WT vs. 113.9% ± 5.5% in
KO; P < 0.05), suggesting that PKCα plays a major role in platelet
α-granule and dense-granule release.
It has been demonstrated that SNAP23, a member of SNARE
traffic and fusion protein complex, undergoes PKC-dependent
phosphorylation during agonist-stimulated platelet degranula-
tion, and in vitro PKCα was one of the isoforms able to phosphor-
ylate recombinant SNAP23 (39). We therefore assessed the contri-
bution of PKCα to phosphorylation of SNAP23 on Ser95, the site
reported to be phosphorylated following thrombin stimulation
(40). Supplemental Figure 3A shows a time course of phosphory-
lation of SNAP23 from WT mouse platelets. This time course cor-
related well with release of ATP from those platelets stimulated
with thrombin. We then showed that both CRP and thrombin
activation led to a potent increase in SNAP23 phosphorylation at
Ser95, which was considerably although not completely inhibited
by absence of PKCα (Supplemental Figure 3B). PKCα-mediated
phosphorylation of SNAP23 may therefore play a role in regulat-
ing secretion or SNARE recycling.
It was possible that the secretory defect seen in Prkca–/– platelets
may be a result of a granule developmental problem. Transmission
electron microscopy analysis of platelet ultrastructure showed an
abnormality in the number of dense granules in Prkca–/– platelets,
characterized by an approximately 3.7-fold reduction compared
with WT platelets (Figure 3). Dense-granule biogenesis in mega-
karyocytes or peripheral distribution during proplatelet develop-
ment could be the key points of regulation by PKCα, and this may
additionally explain the major defect in dense-granule secretion
seen in Prkca–/– platelets. Transmission electron microscopy quan-
tification of α-granule numbers, however, did not detect any sub-
stantial difference between WT and Prkca–/– platelets, indicating
that the defect in α-granule secretion is due to an essential role
played by PKCα in regulation of secretion of these granules rather
than their biogenesis.
Platelet aggregation deficit in Prkca–/– platelets is rescued by exogenous
ADP. It was now important to determine whether the deficits in
secretion and activation of integrin αIIbβ3 would translate into
functional deficits in platelet aggregation and thrombus forma-
tion. Figure 4 shows concentration-response relationships for
Key role for PKCα in secretion of dense granules and α-granules. (A)
Washed platelets from WT or Prkca–/– mice were stimulated with CRP
(5 μg/ml) or thrombin (0.25 U/ml) and secretion of ATP assessed by
luminometry. Data shown represent maximal increase in ATP concen-
tration and represent mean ± SEM. n = 3. *P < 0.05. (B) Platelets from
WT or Prkca–/–mice were labeled with FITC-C62P antibody and stimu-
lated with CRP (5 μg/ml) or thrombin (1 U/ml) for 15 minutes. Fluo-
rescence intensity was measured by flow cytometry. Data presented
represent geometric means as percentages of basal nonstimulated
levels. Error bars represent SEM. n = 3.
PKCα regulates dense-granule but not α-granule biogenesis. Washed
platelets from WT or Prkca–/– mice were examined by transmission
electron microscopy, and the number of dense granules (black arrows)
and α-granules (white arrows) was quantified as described in Methods.
(A) Images of WT (left panel) or Prkca–/– (right panel) platelets are rep-
resentative of 3 independent experiments. Scale bar: 1 μm. Original
magnification, ×19,000. (B) Dense granules (left panel) and α-granules
(right panel) were counted per field of view (25–30 fields of view per
preparation) and shown as mean ± SEM for number of granules per
μm2. n = 3. *P < 0.05.
402? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 2 February 2009
platelet aggregation activated by CRP (Figure 4A) or thrombin (Fig-
ure 4B). At submaximal concentrations of either agonist, absence
of PKCα had a marked effect upon the aggregation response, such
that the aggregation responses to 1.25 μg/ml CRP or 0.065 U/ml
thrombin were almost ablated in Prkca–/– platelets. Importantly,
however, responses may be effectively rescued by addition of exog-
enous ADP (10 μM) simultaneously with CRP or thrombin. This
indicates that the important functional deficit in Prkca–/– platelets
is the lack of dense-granule secretion. The effect upon aggrega-
tion was also relative, since higher concentrations of either CRP or
thrombin were able to restore aggregations to wild-type values.
Pharmacological inhibition of PKCβ reveals redundancy with PKCα for
regulation of platelet aggregation. Given that higher concentrations of
agonists are capable of overcoming the aggregation deficits seen
in the absence of PKCα, it was important to address whether there
may be functional redundancy between PKCα and its closely relat-
ed family member PKCβ. To assess this possibility, we preincubated
platelets with the inhibitor of classical PKC isoforms Gö6976 at its
maximally effective concentration (1 μM), which completely abro-
gated CRP-induced aggregation both in WT and Prkca–/–mouse
platelets (Figure 5A). This confirmed an indispensable role for clas-
sical PKC isoforms in glycoprotein VI–mediated
(GPVI-mediated) aggregation and suggested a
potential redundancy between PKCα and PKCβ
in regulating aggregation. This redundancy
was demonstrated by using the PKCβ-selective
inhibitor LY333531 (ruboxistaurin), which at
a maximally effective concentration (10 μM)
had no significant effect upon aggregation of
WT platelets to CRP but was markedly inhibi-
tory to this response in platelets lacking PKCα
(Figure 5B). This was in parallel with thrombin
(0.5 U/ml), for which addition of 10 μM
LY333531 had no effect upon WT platelet
aggregation response but markedly inhibited
the response in Prkca–/– platelets (data not
shown). Interestingly, for weaker agonists such
as the thromboxane analogue U46619, aggre-
gation was ablated in platelets lacking PKCα,
although shape change in these platelets was
unaffected (Figure 5C). This is consistent
with pharmacological data that show a critical
role for PKC in platelet aggregation induced
through the thromboxane receptor system (41). These data there-
fore suggest that for weak agonists, PKCα plays an essential role,
whereas redundancy between PKCα and PKCβ exists for strong
agonists in regulating the platelet aggregation response.
Ablation of PKCα markedly attenuates thrombus formation in vitro
and in vivo. Finally, it was important to determine the role played
by PKCα in thrombus formation both in vitro and in vivo. Figure
6A shows a major role for PKCα in vitro in mediating this func-
tion, where the images reveal no reduction in primary adhesion
of platelets to the collagen-coated surface, consistent with the
absence of effect upon static adhesion shown in Figure 1, but a
marked reduction in aggregated platelet thrombus formation on
top of the initially adherent cells. In order to confirm in a quan-
titative manner the lack of effect of PKCα ablation on primary
adhesion, fluo-4–labeled platelets were added to the blood and
used for measurements of platelet adhesion during flow over a
collagen surface, as we previously described (42). Fluo-4–labeled
cells flowing across the field of view were counted, and the per-
centage of these cells that adhered stably (adherent for >20 sec-
onds) was estimated. At a shear rate of 1000 s–1, 81.7% ± 3.6% of
WT and 78.1% ± 3.0% of Prkca–/– platelets became stably adherent
ADP rescues deficient aggregation
responses to CRP or thrombin in Prkca–/–
platelets. Washed platelets from WT or
Prkca–/– mice were stimulated with vari-
ous concentrations of CRP (A) or throm-
bin (B) with or without simultaneous
addition of ADP (10 μM) and aggrega-
tion assessed turbidimetrically. Aggre-
gation traces shown are representative
of 4 independent experiments.
Combined pharmacological and genetic approaches reveal redundancy between PKCα and
PKCβ for regulation of platelet aggregation. (A and B) Washed platelets from WT or Prkca–/–
mice were pretreated (10 minutes) with the classical PKC isoform inhibitor Gö6976 (1 μM)
(A), the PKCβ-selective inhibitor LY333531 (10 μM) (B), or DMSO as vehicle control and
stimulated with CRP (5 μg/ml). Aggregation was monitored by turbidimetric aggregometry.
Traces shown are representative of 5 independent experiments. (C) Washed platelets from
WT or Prkca–/– mice were stimulated with U46619 (U4) (10 μM) and aggregation assessed
turbidimetrically. Traces shown are representative of 4 independent experiments.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 2 February 2009
(mean ± SEM, n = 5; P > 0.05), indicating no significant difference
in initial stable adhesion to collagen. Also, in order to confirm
that in the absence of PKCα, the 3D buildup of thrombus forma-
tion was markedly impaired, reconstructed images were generated
from confocal Z-stacks (as described in Methods) and platelet
aggregate (thrombus) volumes estimated. The mean volume of
representative platelet aggregates on the coverslips after flow at
1000 s–1 was 570 ± 168 μm3 and 72.3 ± 7.4 μm3 for WT and Prkca–/–
blood, respectively (mean ± SEM, n = 5; P < 0.05). The major secre-
tory defect seen in Prkca–/– platelets was likely to be responsible for
the impairment of thrombus formation, since addition of ADP
(20 μM) to the blood in the flow chamber was capable of rescuing
thrombus formation (Figure 6, A and B). This indicated that the
thrombotic process under flow conditions markedly relies upon
the contribution to secretion made by PKCα.
It was important finally to assess the role in vivo of PKCα in regu-
lating thrombus formation. Figure 7 shows data reflecting applica-
tion of a laser-induced endothelial injury model in cremaster mus-
cle arterioles, described previously (43, 44). A marked difference is
evident between WT controls and mice lacking PKCα (Figure 7),
PKCα regulates thrombus formation
in vitro. Heparin/D-phenylalanyl-
prolyl-arginyl chloromethyl ketone–
anticoagulated) blood from WT or
Prkca–/– mice was passed over col-
lagen (shear rate 1000 s–1). (A and
B) ADP solution was coinfused at a
10% flow rate (20 μM ADP, final con-
centration) (lower panels). (A) Rep-
resentative phase-contrast images
after 4 minutes. Images were taken
under oil immersion. Scale bars: 20
μm. Original magnification, ×63. (B)
Surface area coverage with thrombi.
Mean ± SEM. n ≥ 3.*P < 0.0001.
In vivo thrombus formation is impaired in the absence of PKCα, but tail bleeding time is normal. (A–D) Mice were either Prkca–/– or littermate-
matched wild-type controls. Platelets were labeled in vivo with Alexa Fluor 488, as described in Methods. (A) Platelets (green) composited with
bright field images (black/white) of the cremaster arteriole were viewed, and images were acquired using a digital CCD camera (SensiCam II;
Cooke Corp.) with a 640 × 480 pixel array. Original magnification, ×40. (B) Traces shown are median integrated platelet fluorescence of 15
thrombi induced in 3 or more mice for each group. Fluorescent intensity of platelets in arbitrary units is presented as a function of time. (C) Data
are presented as a scatter diagram. Horizontal bar represents mean of 15 thrombi induced in at least 3 mice for each group. There is a signifi-
cant reduction in thrombus intensity in Prkca–/– in comparison with WT controls (mean Prkca+/+, 652200; mean Prkca–/–, 279600; P < 0.05). (D)
Time to reach peak thrombus size also significantly differed from WT controls, with mean data shown by the horizontal bars (mean Prkca+/+,
68.1 seconds; mean Prkca–/–, 115.2 seconds; P < 0.01). (E) Mice were anesthetized and a transverse incision made with a scalpel at a position
where the diameter of the tail was 2.25 to 2.5 mm. The tail was immersed in normal saline (37°C) in a hand-held test tube. The time from incision
to cessation of bleeding was recorded, and mean times are shown as horizontal bars. No significant difference was seen comparing WT mice
(mean 140 seconds) with Prkca–/– (mean 117 seconds; P > 0.05).
404? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 2 February 2009
since in the absence of PKCα, thrombus formation is delayed and
results in fewer accumulated platelets (see Supplemental Videos 1
and 2). Importantly, however, the ability of Prkca–/– to undergo nor-
mal hemostasis was demonstrated by the lack of alteration in tail-
bleed time (Figure 7E) and by the lack of any fecal occult blood (data
not shown). This may indicate that compensatory mechanisms are
sufficient to allow normal hemostasis but are not sufficient to sup-
port normal thrombus formation and may reflect the observation
that Prkca–/– platelets adhere normally to collagen under static and
flow conditions (Figures 1 and 6) but have a marked reduction in
subsequent platelet-platelet interaction (as evidenced by the lack of
thrombus accumulation shown in Figure 6A).
Platelets play a central role in mediating atherothrombosis and
are therefore the target of numerous therapies aimed at reduc-
ing their activity, particularly in the prevention of coronary artery
thrombosis in heart attacks (45). PKC is established, largely by
pharmacological studies, as a major regulator of multiple platelet
activities (2), and it is increasingly clear that the different isozymes
of PKC expressed in platelets perform distinct functions. There is
a difficulty of interpretation of data from some pharmacological
studies, however, because of the lack of selectivity of the reagents
available to target specific PKC isozymes. Here, we used a genetic
approach to demonstrate definitively, for what we believe is the
first time, the role played by PKCα in regulating platelet func-
tion and thrombus formation. Importantly, the study revealed a
key role for PKCα in regulating granule biogenesis and exocyto-
sis, which was essential for thrombus formation, since ablation
of thrombus formation in Prkca–/– platelets could be rescued by
addition of exogenous ADP. The findings reveal PKCα to be a
potential drug target for antithrombotic therapy, since selective
inhibitors would exert a major effect upon thrombus formation
while sparing primary platelet adhesive functions.
It has recently been shown pharmacologically that PKC isoforms
exert dual control of thrombus formation by mediating secretion
and integrin activation under flow while suppressing phosphati-
dylserine exposure and subsequent thrombin generation and coag-
ulation (7). There is incomplete and largely indirect evidence that,
of the various isoforms of PKC expressed in platelets, PKCα plays
a role in regulation of integrin αIIbβ3 activation (36). The study
by Han et al. (33) elegantly showed that PKCα was required for
integrin αIIbβ3 activation in a reconstituted cell system rather than
in the platelet. The study by Tabuchi et al. (18) used an in vitro
permeabilized platelet system to investigate receptor-independent
calcium-mediated activation of platelet aggregation. Although
this latter study demonstrated that added purified PKCα was able
to support a calcium-induced platelet aggregation response, the
approach has limitations in that receptor-mediated activation is
lost. Nonetheless, these studies provided indications that PKCα
may regulate platelet integrin activation and aggregation, and it
was therefore essential to perform a genetic-based definitive study
of the role played by PKCα in mediating these events.
The marked knockdown in activation of integrin αIIbβ3 in
response either to thrombin or CRP in platelets lacking PKCα par-
alleled a deficit in the ability of these platelets to undergo aggrega-
tion at submaximal concentrations of agonist. This was consistent
with the impaired ability of Prkca–/– platelets to form a thrombus
in blood flowing over a collagen-coated surface. The effect on
aggregation could, however, be overcome by increasing the concen-
tration of agonist, and the reasons for this may derive from several
factors. First, it is known that integrin αIIbβ3 activation in response
to strong agonists is in part regulated independently of PKC (3,
46). Further, studies with integrin blockers have shown that not
all αIIbβ3 receptors are required to be activated (e.g., by the PKC-
independent pathway) for a full platelet aggregation response in
the platelet aggregometer (47). Thus, it may be possible to achieve
a maximal aggregation response with a markedly reduced expres-
sion of activated integrin. In contrast, in thrombus formation in
flowing whole blood, stable platelet aggregation critically depends
on limited levels of autocrine-produced ADP and hence limited
and reversible integrin activation (48). Under these more physio-
logical conditions, the number of activated integrin receptors may
be low, and therefore the marked reduction in integrin activation
seen in Prkca–/– platelets may cause a significant reduction in the
ability of platelets to form thrombi. Additionally, we provide evi-
dence for redundancy between PKCα and PKCβ in Figure 5, which
shows that selective inhibition of PKCβ only significantly attenu-
ated aggregation responses in Prkca–/– platelets, not WT platelets,
indicating redundant functions of these 2 isoforms. It is also sig-
nificant to note that in assays for occult blood in feces, no gastro-
intestinal bleeding was detected in either WT (n = 5) or Prkca–/–
(n = 6) mice. Together with the data shown in Figure 7, where no
difference in tail bleeding time is seen in Prkca–/– compared with
WT controls, this also implies redundancy of signaling molecules
for regulation of hemostasis in vivo.
There is significant contribution to the effects of PKCα ablation
on aggregation and thrombus formation by regulation of granule
secretion, since these processes greatly rely upon autocrine ADP
release (49). Clearly, both dense- and α-granule secretion are mark-
edly disrupted in platelets lacking PKCα, and evidence that this is
functionally critical is provided by the rescue experiments depicted
in Figure 4 for aggregation and Figure 6 for in vitro thrombus for-
mation, where addition of exogenous ADP recovers the deficits in
these responses seen in Prkca–/– platelets.
The role of unspecified PKC isoforms as a family in the regu-
lation of secretion has been shown pharmacologically by several
groups (3–6). The role of the PKCα isoform in agonist-indepen-
dent secretion has been demonstrated previously in an artificial
permeabilized platelet system (17). Although valuable to indicate
the role of PKCα, it was important to address the issue by a defini-
tive genetic approach. Indeed, the only study to date to address
specific PKC isoform function in secretion in platelets in a com-
bined genetic and pharmacological approach had shown a major
discrepancy in results obtained by the 2 approaches for PKCδ. Pula
et al. (15) showed that genetic ablation of PKCδ had no significant
effect upon dense-granule secretion, whereas in that study and
previous studies (9, 16), the PKCδ-selective inhibitor rottlerin had
been shown to enhance dense-granule secretion in response to
collagen and alboaggregin, operating through the GPVI receptor.
The present results provide direct evidence that PKCα is a major
regulator of secretion of α-granules, since P selectin expression
is reduced almost to basal levels in Prkca–/– platelets in response
either to CRP or thrombin. Importantly, we show that α-granule
numbers are equivalent between Prkca–/– and WT platelets and
therefore the secretion defect for this granule type reflects a genu-
ine deficiency in the secretory pathway. For dense granules, this
study shows an additional knockdown in numbers of granules in
Prkca–/– platelets. This suggests a role for PKCα in biogenesis of
dense granules and is therefore an area that requires further analy-
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 2 February 2009
sis. A number of different elements of the secretory machinery,
including SNAP23, SNAP25, Munc18a and Munc18b, syntaxin 2,
and Rab6, have been reported to be PKC substrates (40, 50–52),
and therefore their phosphorylation may influence secretion. In
this study then, phosphorylation of SNAP23 serves as a potential
marker for these events and may contribute functionally to the
process of secretion. However, although SNAP23 phosphorylation
on Ser95 is markedly reduced in Prkca–/– platelets and this may
play a role in regulating α-granule secretion, its role in dense-gran-
ule secretion cannot be clarified from this study.
In summary, we have shown PKCα to play major roles in regu-
lating platelet secretion of α-granules, biogenesis and secretion
of dense granules, regulation of integrin αIIbβ3, and thrombus
formation in vitro and in vivo. PKCα also regulates platelet
aggregation, although the defect in Prkca–/– platelets is relative,
since high concentrations of agonists overcome the deficit and
there is evident redundancy of the action of PKCα with the other
expressed classical PKC isoform in platelets, PKCβ. PKCα plays
no significant role in regulating adhesion of platelets to collagen-
coated surfaces under static or flow conditions and does not reg-
ulate platelet spreading response or outside-in signaling through
integrin αIIbβ3. It could be argued that, because PKCα is the most
highly expressed of the PKC isoforms in platelets, its role in
platelets may be wide ranging and effectively mask that of other
PKC isoforms that are expressed at lower levels. The evidence
presented here, however, suggests highly specific roles for PKCα,
since, for instance, its absence has no significant effect upon
platelet adhesion and spreading on collagen but markedly sup-
presses thrombosis. Additionally, if PKCα were so predominant
functionally in platelets, knockout of the other PKC isoforms
would be predicted not to have functional effects. This is clearly
not the case, since we have shown absence of PKCδ to enhance
platelet responses to collagen through enhanced filopodia for-
mation (15) and we have more recently shown absence of PKCθ
to enhance α-granule secretion and integrin αIIbβ3 activation in
response to GPVI agonists (53). Shattil’s group has also shown
absence of PKCs β and θ to ablate outside-in signaling through
integrin αIIbβ3 (37, 38). These data therefore demonstrate specif-
ic functional roles for the different PKC isoforms, including for
PKCα in regulating secretion in particular, such that redundancy
of activity is not apparent for specific functions.
Genome-wide association analyses of major human diseases
have been conducted recently, based upon technical advances
in high throughput microarray analyses of SNPs. These studies
are introducing major new leads in genes that may be related to
disease, and although PKCα is not in the top ranking of genes
associated with coronary artery disease, a cluster of SNPs in this
gene with a maximal P value of just over 10–3 (SNP rs12600582,
intronic, minor allele frequency of 0.233 in Europeans) (34,
35) may indicate some significance in this disease of polygenic
cause. For these reasons, PKCα may represent a drug target for
antithrombotic therapy, with inhibitors exerting an effect upon
thrombus formation but sparing primary platelet adhesive func-
tions. PKCα is already a target for the drug aprinocarsen, an anti-
sense oligonucleotide therapy used in the treatment of specific
neoplastic conditions (54, 55), and it will now be important to
assess whether this or other small molecule–based approaches
may represent opportunities in the development of platelet-based
antithrombotic drugs in the management of coronary artery dis-
ease and other arterial thrombotic diseases.
Materials. CRP was from Richard Farndale (University of Cambridge, Cam-
bridge, United Kingdom). Thrombin, BSA, ADP, protein G-Sepharose, and
fibrinogen were purchased from Sigma-Aldrich. Complete Mini Protease
Inhibitor Tablets were from Roche Applied Science. Secondary HRP-con-
jugated anti-rabbit antibody was obtained from Santa Cruz Biotechnol-
ogy Inc. SNAP23 (P-T95) antibody was a generous gift from Paul Roche
(NIH). JON/A (anti-αIIbβ3) and WugE9 (anti–P selectin) antibodies were
from Emfret Analytics. PE-conjugated anti-CD41 antibody and its iso-
type-matched control, PE-conjugated rat IgG1, were from AbD Serotec.
Isoform-specific anti-PKC mouse monoclonal antibodies were from BD
Biosciences. LY333531 was supplied by A.G. Scientific Inc. (Fluorochem
Ltd.), U46619 was from ALEXIS Biochemicals, Gö6976 was from Calbio-
chem, and CHRONO-LUME reagent came from Chrono-log (Labmedics).
The P2Y12 antagonist AR-C69931MX was a gift from AstraZeneca. The
P2Y1 antagonist MRS2500 was from Tocris Bioscience.
Platelet preparation. Mice were bred and maintained in the University of
Bristol animal facility in accordance with United Kingdom Home Office
regulations. All procedures were undertaken with United Kingdom Home
Office approval in accordance with the Animals (Scientific Procedures) Act
of 1986 (project license numbers: 40/2212, 40/2749 and 30/2386). Prkca–/–
mice were generated as previously described (32). Blood was drawn by cardi-
ac puncture under terminal anesthesia into acid citrate dextrose (20 mM cit-
ric acid, 110 mM sodium citrate, 5 mM glucose), 1:7 ratio, v/v. Platelets were
prepared as previously described (15). In brief, blood was diluted with 250
μl of modified Tyrode’s-HEPES buffer (134 mM NaCl, 0.34 mM Na2HPO4,
2.9 mM KCl, 12 mM NaHCO3, 20 mM HEPES, 5 mM glucose, and 1 mM
MgCl2, pH 7.3) and centrifuged at 180 g for 6 minutes at room temperature.
Platelet-rich plasma was removed, and platelets were isolated by centrifuga-
tion at 550 g for 6 minutes in the presence of PGE1 (140 nM) and indometh-
acin (10 μM). Pelleted platelets were resuspended to the required density in
modified Tyrode’s-HEPES buffer and rested for 30 minutes at 37°C in the
presence of 10 μM indomethacin prior to stimulation.
Platelet aggregation. Platelets were resuspended in Tyrode’s–HEPES buffer
at a final concentration of 2 × 108 per ml. Platelets were preincubated with
different inhibitors or vehicle solution (0.1% Me2SO final concentration)
for 10 minutes at 37°C, and aggregation of agonist-stimulated platelets
was monitored in an optical aggregometer (Chrono-log; Labmedics) at
37°C, with continuous stirring at 800 rpm.
Flow cytometry. Two-color analysis of mouse platelet activation was con-
ducted using PE-conjugated JON/A, an antibody that preferentially binds
to the active form of αIIbβ3 integrin, and with a FITC-conjugated antibody
specific for CD62P (P selectin). 25 μl of washed platelets (4 × 107/ml in
Tyrode’s-HEPES buffer supplemented with 1 mM CaCl2 and 0.35% BSA)
was mixed with 10 μl of antibody and subsequently stimulated either with
5 μg/ml CRP or 1 U/ml thrombin for 15 minutes at room temperature.
The reaction was stopped by addition of 400 μl ice-cold PBS, and samples
were analyzed within 30 minutes. For estimation of surface expression
levels of total αIIbβ3 integrin, CD41 was stained with PE-conjugated anti-
CD41. Flow cytometry was performed on a FACSCalibur flow cytometer
(BD Biosciences), using CellQuest version 3.1f software (BD Biosciences),
and a total of 20,000 events per sample were collected.
Measurement of ATP secretion. ATP secretion was measured using
CHRONO-LUME reagent according to the manufacturer’s protocol. 5 μl
luciferase-luciferin was added directly to the platelets, which were being
continually stirred (1000 rpm), and 5 μg/ml CRP or 1 U/ml thrombin
was added to activate platelets for 1 minute. The luminescence intensity
was measured at a setting of ×0.05.
Platelet spreading and adhesion assay. Coverslips were coated with either
0.1 mg/ml fibrinogen or 50 μg/ml CRP and left overnight, followed by
406? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 2 February 2009
nonspecific blocking step with 2% BSA for 1 hour. Platelets were resus-
pended to 2 × 107/ml in modified Tyrode’s-HEPES buffer, and 500 μl was
dispensed onto the coverslip mounted in a live-cell microscopy chamber.
Adhesion and spreading of platelets was observed by differential interfer-
ence contrast (DIC) microscopy, with a wide-field DM IRB microscope
attached to an ORCA ER camera (63×/1.40 NA oil objective) (Leica). Five
images in different random parts of the coverslip area were taken at 15,
30, and 45 minutes and processed with OpenLab 4.03 (Improvision) and
Adobe Photoshop software. The surface area and number of adherent
platelets was estimated using ImageJ software (http://rsbweb.nih.gov/ij/).
Immunoblotting. For stimulated samples, 2 × 108/ml of washed platelets
were activated with 5 μg/ml CRP or 1 U/ml thrombin in the presence of
2 mM CaCl2 for 3 minutes and solubilized in Laemmli sample buffer. Pro-
teins were resolved by electrophoresis in 9.5% (for PKC isoform estimation)
or 16% (for SNAP23 Ser95 phosphorylation estimation) SDS-PAGE. Sam-
ples were then transferred to PVDF membranes, blocked with 10% BSA,
and subjected to immunoblotting with anti-PKCα, -β, -θ or -δ antibodies,
SNAP antibody, or phospho–SNAP23-Ser95 antibody.
Thrombus formation under flow in vitro. Flow-induced thrombus formation
was assessed basically as described before (56). Heparin/D-phenylalanyl-
prolyl-arginyl chloromethyl ketone–anticoagulated (Heparin/PPACK-anti-
coagulated) mouse blood was passed over immobilized collagen through
a parallel plate perfusion chamber at a shear rate of 1000 s–1 for 4 minutes.
Where indicated, ADP (200 μM in saline) was coinfused at a 10% rate imme-
diately before the blood reached the flow chamber. For each perfusion
surface, phase-contrast images from 10 random microscopic fields were
collected. Surface coverage was analyzed using Image-Pro software, version
4.1 (Media Cybernetics). The average percentage area covered by adherent
platelets was measured by automated setting of masks for the ranges of
gray levels corresponding to the presence of platelets and thrombi. For
each image, the (blinded) observer needed to approve the mask settings.
Averaged pixel fractions of the masks from 10 images were considered as a
best estimate of the surface area coverage with thrombi.
Aggregate volume was also estimated by confocal laser scanning micros-
copy, using a Bio-Rad 2100 multiphoton system (Bio-Rad Laboratories),
as described before (57). Platelet aggregates that formed on collagen
(4 minutes, shear rate 1000 s–1) were postlabeled with FITC–anti-mouse
CD62 mAb (1:100). Confocal stacks were recorded for measurement of the
volume of individual platelet aggregates. Analysis of confocal images (gray
level bit maps) and 3D reconstruction of images were with LaserPix soft-
ware (Media Cybernetics).
Detection of occult fecal blood. The presence of occult fecal blood was
detected by Hemdetect (DIPRO, supplied by Autogen Bioclear) on freshly
obtained stool samples.
Electron microscopy. Platelet-rich plasma was collected and spun at 590 g
for 5 minutes. Supernatant was removed from the platelet pellet, and the
pellet was fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB) (pH
7.4). The pellet was washed in PB and then incubated in 1% osmium tetrox-
ide in PB for 30 minutes. After washing in PB and deionized water, the pel-
let was incubated in 3% uranyl acetate in deionized water for 30 minutes.
After washing with deionized water, the pellet was dehydrated in a graded
series of increasing amounts of ethanol (70%, 80%, 90%, 96%, 100%, and
100%, with each step lasting for 10 minutes). After removal of the 100%
ethanol, the pellet was incubated with pure Epon for 2 hours at room
temperature. Thereafter, the Epon was replaced with fresh Epon, and this
was hardened overnight in a 60°C oven. Ultrathin counterstained sections
were imaged on a Philips CM100 equipped with a side-mount MegaView
III camera (Olympus Soft Imaging Solutions).
To determine the dense-granule and α-granule content, total numbers of
granules in equivalent-sized fields of view were counted. For each genotype,
25–30 randomly chosen fields of view were examined. All microscopic images
were taken at the same magnification (×19,000), and the number of cells per
field of view between WT and Prkca–/– preparations were equivalent.
Analysis of bleeding time. Experiments were conducted on 25–35 g male
and female mice. Mice were anesthetized (75 mg/kg ketamine and 1 mg/kg
medetomidine intraperitoneally), and a transverse incision was made with
a scalpel at a position where the diameter of the tail was 2.25 to 2.5 mm.
The tail was immersed in normal saline (37°C) in a hand-held test tube,
and the time from incision to cessation of bleeding was recorded.
Intravital microscopy of thrombus formation in vivo. Intravital microscopy was
performed essentially as described previously (44). Experiments were con-
ducted on 25–30 g male Prkca–/– mice and their littermate-matched con-
trol WTs. Anesthesia was induced by intraperitoneal ketamine (100 mg/kg
Vetalar; Pharmacia & Upjohn Ltd.) and 2% xylazine (20 mg/kg; Millpledge
Pharmaceuticals). The left cremaster muscle was exteriorized and spread
flat over an optically clear coverslip and continuously superfused. High-
speed intravital microscopy experiments were performed as previously
described by Falati et al. (43).
Platelets were labeled fluorescently with Alexa Fluor 488–conjugated
goat anti-rat antibody (Molecular Probes) and rat anti-murine CD41
antibody (BD Biosciences — Pharmingen) infused via a carotid cannula.
Thrombi were induced in arterioles with a diameter of 25–35 μm by a
nitrogen ablation laser (MicroPoint; Photonic Instruments), which was
introduced through the microscope objective. Bright field and fluores-
cent images were captured simultaneously for 4–5 minutes, and multiple
thrombi were generated with a distance of at least 200 μm between them
and upstream of previous injuries. The integrated intensity value for the
growing thrombus was plotted against time and peak size of thrombus,
and time taken to reach peak was determined.
Statistics. Statistical analyses were carried out on raw data using unpaired,
2-tailed Student’s t test, and P < 0.05 was considered statistically signifi-
cant. Values are expressed as mean ± SEM. For all data, n indicates number
of mice tested.
We thank Elizabeth Aitken for expert technical assistance sup-
porting this work. The authors would like to thank AstraZeneca
for the kind gift of AR-C69931MX. SNAP23 (P-T95) antibody was
a generous gift from Paul Roche (NIH). We thank Mark Jepson
and Alan Leard for their assistance within the School of Medical
Sciences Cell Imaging Facility. We are also grateful to Gini Tilly
and Deborah Carter for their assistance with electron microscopy.
We thank Ian Day and Tom Gaunt (University of Bristol) for valu-
able discussions and advice regarding genome-wide–association
analyses. We thank Majd Protty, University of Birmingham, for
help with analysis of in vivo thrombosis data. The work was sup-
ported by grants from the British Heart Foundation (RG/05/015,
FS/04/023, and FS/05/017 to A.W. Poole; CH/03/003 to S.P. Wat-
son); the Medical Research Council (MRC-65282 to S.P. Watson);
and the NIH (NIHLB HL56652 to S.W. Whiteheart). A.W. Poole is a
Biotechnology and Biological Sciences Research Council Research
Received for publication September 30, 2008, and accepted in
revised form December 3, 2008.
Address correspondence to: Alastair W. Poole, Department of
Physiology & Pharmacology, School of Medical Sciences, Univer-
sity Walk, Bristol BS8 1TD, United Kingdom. Phone: 44-117-331-
1435; Fax: 44-117-331-2288; E-mail: firstname.lastname@example.org.
research article Download full-text
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 2 February 2009
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