?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 6 June 2007
Mice lacking the signaling molecule
CalDAG-GEFI represent a model for
leukocyte adhesion deficiency type III
Wolfgang Bergmeier,1,2 Tobias Goerge,1,2 Hong-Wei Wang,1,2
Jill R. Crittenden,3,4 Andrew C.W. Baldwin,1 Stephen M. Cifuni,1
David E. Housman,4 Ann M. Graybiel,3 and Denisa D. Wagner1,2
1CBR Institute for Biomedical Research and 2Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA.
3Department of Brain and Cognitive Sciences and McGovern Institute for Brain Research and
4Center for Cancer Research, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA.
Integrins are heterodimeric cell-surface receptors that mediate
adhesion to the extracellular matrix and cell-cell interactions. On
circulating blood cells, most integrins are expressed in a resting,
low-affinity state. Cellular stimulation induces a high-affinity
state in the integrins, which enables them to bind to their ligands.
The molecular basis of this inside-out activation of integrins is
only partially understood (1). In blood cells, integrins are critical
for the formation of the immunological synapse (2), in the extrava-
sation of circulating immune cells from the bloodstream (3), and
in the formation of platelet plugs at sites of vascular damage (4).
The functional importance of integrins expressed on blood cells is
best documented in patients with germline mutations in the gene
encoding β2 integrins (leukocyte adhesion deficiency type I; LAD-I;
ref. 5), who experience recurrent infections, and in patients with
mutations in the genes encoding αIIb or β3 integrins (Glanzmann
thrombasthenia; ref. 6), who have bleeding diathesis. Similar phe-
notypes have been observed in mice with specific deletions in the
genes coding the β2 (7) or β3 (8) integrins.
Recently, patients with normal expression but defective activa-
tion of β1, β2, and β3 integrins have been identified (9–13). Affected
patients exhibit clinical symptoms such as severe recurrent infec-
tions, a heightened tendency to bleed, and marked leukocytosis. It
has been proposed that this group of integrin activation disorders
be designated LAD-III (9) based on the nomenclature for LAD-I
and LAD-II, which describes patients with impaired expression
of β2 integrins (5) and defective fucosylation of selectin ligands
(14), respectively. Because the expression of β1, β2, and β3 integrins
appears to be normal in LAD-III patients, it seems likely that a
genetic defect in 1 or more intracellular signaling molecules
involved specifically in the activation of leukocyte and platelet
integrins is the basis of the LAD-III syndrome.
We have recently identified Ca2+ and diacylglycerol-regulated gua-
nine nucleotide exchange factor I (CalDAG-GEFI; also referred to
as RasGRP2) as crucial for β3 integrin activation in platelets (15).
CalDAG-GEFI is a member of the CalDAG-GEF/RasGRP family of
intracellular signaling molecules, containing binding sites for cal-
cium and diacylglycerol as well as a guanine nucleotide exchange
factor (GEF) domain that catalyzes the exchange of GTP for GDP
bound to Rap1 or Rap2 (16, 17). Rap1 is the major isozyme in
both platelets and neutrophils (18, 19). Interestingly, in contrast
to Rap1, which is ubiquitously expressed in hematopoietic and
nonhematopoietic cells, CalDAG-GEFI appears to be specifically
expressed in platelets and megakaryocytes as well as neutrophils
within the hematopoietic system as well as in neurons, especially in
the striatum of the basal ganglia (15, 17). CalDAG-GEFI–/– mice are
characterized by severely impaired hemostasis, caused by defective
activation of Rap1 and integrin αIIbβ3 in platelets, and by mild neu-
trophilia (15). This neutrophilia indicated to us that neutrophils
from CalDAG-GEFI–/– mice might have a defect in extravasation
similar to that observed in selectin-deficient mice (20), causing neu-
trophil accumulation in the bloodstream. Therefore, we evaluated
the role of CalDAG-GEFI in the activation of leukocyte integrins,
specifically members of the β2 integrin family. Our results point-
ed to significant defects in β1 integrin– and β2 integrin–mediated
Nonstandard?abbreviations?used: CalDAG-GEFI, Ca2+ and diacylglycerol-regulated
guanine nucleotide exchange factor I; fMLP, formyl-methionylleucylphenylalanine;
GEF, guanine nucleotide exchange factor; LAD, leukocyte adhesion deficiency; LTB4,
leukotriene B4; PAF, platelet activating factor; PAR4p, PAR4-activating peptide;
PSGL-1, P-selectin glycoprotein ligand–1; TG, thioglycollate.
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Citation?for?this?article: J. Clin. Invest. 117:1699–1707 (2007). doi:10.1172/JCI30575.
1700?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 6 June 2007
adhesion of CalDAG-GEFI–deficient neutrophils in vitro and in
vivo, which caused a markedly impaired response to acute inflam-
mation. Furthermore, we examined the function of CalDAG-GEFI
in platelets and found that CalDAG-GEFI was essential for the
activation of β1 integrins on platelets and that arterial thrombus
formation was completely abolished in CalDAG-GEFI–/– mice. Thus
CalDAG-GEFI regulates β1, β2, and β3 integrins, which suggests this
gene may be defective in patients with LAD-III.
Normal expression of agonist receptors, calcium flux, and ROS formation
in CalDAG-GEFI–deficient neutrophils. Integrin activation plays a key
role in the firm adhesion and extravasation of PBLs (3). We have
previously shown that CalDAG-GEFI is expressed in platelets and
neutrophils and that it is critical for β3 integrin activation in plate-
lets (15). To determine whether CalDAG-GEFI
plays a role in integrin activation in neutrophils,
we studied neutrophil responses to activation
both in vitro and in vivo in CalDAG-GEFI–/– mice
and their littermate WT controls.
We first compared the surface expression of
key adhesion receptors in neutrophils from
CalDAG-GEFI–/– and WT mice. As shown in Table 1,
CalDAG-GEFI–deficient and WT neutrophils
expressed comparable levels of P-selectin glyco-
protein ligand–1 (PSGL-1) and β2 integrins on
their surface. No difference in agonist-induced
upregulation of αMβ2 (Mac-1) on the cell sur-
face was observed, demonstrating that knock-
out neutrophils were fully capable of recruiting
intracellular pools of integrins by granule release (Figure 1A). Com-
pared with WT neutrophils, CalDAG-GEFI–deficient neutrophils
expressed less L-selectin and β1 integrin on their cell surface.
To study signaling responses triggered by surface-expressed ago-
nist receptors, we tested several agonists for their ability to induce
calcium flux in neutrophils from WT and CalDAG-GEFI–/– mice.
No differences were observed in calcium flux in response to vari-
ous doses of C5a or leukotriene B4 (LTB4; Figure 1B) or formyl-
methionylleucylphenylalanine (fMLP; data not shown) between
WT and mutant cells, nor did we detect significant differences in
the formation of ROS between WT and CalDAG-GEFI–deficient
neutrophils stimulated with fMLP or PMA (Figure 1C). This find-
ing is in line with previous studies showing that the small GTPase
Rac (not a target of CalDAG-GEFI) is required for the activation of
the respiratory burst in neutrophils (21, 22). These data establish
Expression of adhesion receptors on peripheral blood neutrophils
CalDAG-GEFI–/– 10.9 ± 1.7 392 ± 61 62 ± 9.9
9.5 ± 1.7 456 ± 81 62 ± 10.7 54.2 ± 8.3 1839 ± 135 804 ± 46.4
35 ± 2.7 1662 ± 246 647 ± 18.9
0.03 0.6 0.5 0.9 0.01
Peripheral blood neutrophils were stained with fluorophore-labeled antibodies against the
indicated adhesion receptors and analyzed by flow cytometry. Results are mean fluorescence
intensity ± SEM, n = 7. ATwo populations of neutrophils were detected in CalDAG-GEFI–/– mice:
one with mean fluorescence similar to that observed in WT neutrophils, the other with mean
fluorescence less than 50% that of WT. A similar decrease in L-selectin was previously
observed in our lab in circulating neutrophils from P/E-selectin–deficient mice, which also show
a defect in neutrophil extravasation (20). LFA-1, lymphocyte function-associated antigen 1.
Degranulation, calcium flux, and ROS production in stimulated neutrophils. (A) Mac-1 expression. PBLs from WT and CalDAG-GEFI–/– mice were
kept resting or were activated for 10 minutes with LTB4 (300 nM), C5a (50 ng/ml), or PMA (200 nM), stained with antibodies against Mac-1, and
immediately analyzed by flow cytometry. n = 6. (B) Calcium flux. PBLs from WT and CalDAG-GEFI–/– mice were loaded with the calcium-sensi-
tive dye Fluo-3, activated with the indicated doses of LTB4 or C5a, and immediately analyzed by flow cytometry. Results are representative of 6
individual experiments. (C) ROS formation. PBLs from WT and CalDAG-GEFI–/– mice were loaded with the ROS-sensitive agent DCFDA, acti-
vated with PMA (2 μM) or fMLP (5 μM) for 30 minutes at 37°C, and immediately analyzed by flow cytometry. n = 5. ROS production is expressed
as fold increase of mean fluorescence intensity over unstimulated cells. No significant differences in Mac-1 expression, calcium flux, or ROS
production were observed between WT and CalDAG-GEFI–deficient neutrophils.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 6 June 2007
that the expression and function of agonist receptors and
intracellular signaling molecules upstream of calcium flux are nor-
mal in CalDAG-GEFI–deficient neutrophils and that cellular func-
tions such as degranulation and ROS formation are also intact.
CalDAG-GEFI mediates Rap1 activation in stimulated neutrophils. After
establishing that agonist-triggered events upstream of calcium flux
were normal in CalDAG-GEFI–deficient neutrophils, we next stud-
ied Rap1 activation in these cells. Within seconds, stimulation of leu-
kocytes triggers the activation of Rap1, i.e., the exchange of GDP for
GTP bound to the small GTPase (23, 24). We detected trace amounts
of activated Rap1 in lysates of resting WT and CalDAG-GEFI–defi-
cient neutrophils. Upon stimulation with LTB4, C5a, or a low dose
of platelet activating factor (PAF; 3 nM), a strong increase in Rap1-
GTP was observed in lysates from WT but not CalDAG-GEFI–defi-
cient neutrophils (Figure 2). The defect in PAF-induced activation of
Rap1 observed in CalDAG-GEFI–deficient neutrophils was partially
overcome by increasing the concentration of the agonist to 30 nM.
Furthermore, we precipitated Rap1-GTP from lysates preincubated
with GTP-γS, a form of GTP that cannot be hydrolyzed to GDP by
the intrinsic GTPase activity of Rap1. We detected equivalent levels
of GTP-γS–loaded Rap1 in mutant and WT samples, demonstrat-
ing that Rap1 from the neutrophils of CalDAG-GEFI–/– mice did
not have an intrinsic activation defect. The levels of total Rap1 were
comparable in whole cell lysates from WT and CalDAG-GEFI–defi-
cient neutrophils (Figure 2). Together, these data demonstrate that
CalDAG-GEFI plays an important role in Rap1 activation in neu-
trophils stimulated by various agonists and that the defect in Rap1
activation observed in CalDAG-GEFI–deficient neutrophils may be
overcome by increasing the concentration of the agonist.
Impaired activation of β1 and β2 integrins in CalDAG-GEFI–deficient
neutrophils in vitro. To determine whether CalDAG-GEFI deficiency
also affected integrin activation in neutrophils, we first studied β1
integrin–mediated adhesion of isolated CalDAG-GEFI–deficient
neutrophils to fibronectin. Neutrophils isolated from the bone mar-
row of WT and CalDAG-GEFI–/– mice were activated with 300 nM
LTB4, and adhesion to fibronectin-coated plates was monitored.
As shown in Figure 3A, adhesion of CalDAG-GEFI–deficient neu-
trophils was significantly impaired compared with that of WT
neutrophils. We next examined β2 integrin–mediated adhesion to
fibrinogen, as it was shown previously that leukocyte binding to
this ligand is mediated by the β2 integrin receptors αMβ2 (Mac-1)
and αXβ2 (25). Compared with WT neutrophils, CalDAG-GEFI–
deficient neutrophils activated with LTB4 or PAF showed impaired
adhesion to fibrinogen (Figure 3B).
CalDAG-GEFI is critical for β2 integrin–mediated firm adhesion of neu-
trophils in vivo. We next examined the adhesion of leukocytes to
activated mesenteric venules in the CalDAG-GEFI–/– and WT mice.
Venules were superfused with 300 nM LTB4 in PBS, and rolling
as well as firmly adherent rhodamine 6G–labeled leukocytes were
counted over a period of 20 minutes. Significantly more roll-
ing leukocytes were observed in CalDAG-GEFI–/– than in WT mice
(P < 0.04; Figure 4A). However, this may not be a result of increased
adhesiveness of the mutant cells, but rather the approximately
2-fold greater number of peripheral neutrophils circulating in these
animals (15). It is not surprising that CalDAG-GEFI–deficient leu-
kocytes rolled normally along stimulated venules, as this process
depends largely on the binding of leukocyte PSGL-1 to endothelial
selectins (26), and PSGL-1 is a constitutively active receptor that we
found to be expressed normally in CalDAG-GEFI–/– mice (Table 1).
In contrast, firm leukocyte adhesion induced by LTB4 requires the
rapid activation of integrins (27, 28) via intracellular signaling path-
ways. We found that WT leukocytes adhered firmly to mesenteric
venules within minutes of LTB4 superfusion, while firm adhesion of
Impaired Rap1 activation in CalDAG-GEFI–deficient neutrophils. West-
ern blots of affinity-precipitated Rap1-GTP showing strongly decreased
Rap1 activation in CalDAG-GEFI–deficient neutrophils (–/–) stimulated
with LTB4 (300 nM for 30 seconds), C5a (75 ng/ml for 30 seconds),
or PAF (3 or 30 nM for 30 seconds) relative to that of WT neutrophils
(+/+). Equivalent loading of GTP onto Rap1 in neutrophils from WT
and CalDAG-GEFI–/– mice was shown by preincubation of lysates with
GTP-γS. Total Rap1 levels were determined in whole cell lysates of
WT and CalDAG-GEFI–deficient neutrophils. Results are representa-
tive of 3 individual experiments.
CalDAG-GEFI deficiency causes impaired β1 integrin– and β2 integrin–mediated adhesion of neutrophils in vitro. (A and B) Neutrophil adhesion
to fibronectin (A) or fibrinogen (B) in vitro. WT or CalDAG-GEFI–deficient neutrophils isolated from bone marrow were added to fibronectin- or
fibrinogen-coated plates and incubated for 30 minutes in the presence or absence (i.e., resting) of 300 nM LTB4 or 3 or 30 nM PAF. Plates were
washed and adherent neutrophils were counted. n = 4. *P < 0.05, **P < 0.01, ***P < 0.001.
1702?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 6 June 2007
CalDAG-GEFI–deficient leukocytes was reduced by more than 90%
(Figure 4B). Infusion of WT mice with a blocking antibody against
β2 integrins reduced the firm adhesion of leukocytes by more than
80% (Figure 4C), confirming the important role of β2 integrins in
leukocyte adhesion previously shown in venules of CD18–/– (29) and
Mac1–/– mice (28). Our results indicate that CalDAG-GEFI is needed
for the activation of β2 integrins both in vitro and in vivo.
Impaired inflammatory response in CalDAG-GEFI–/– mice. To test
the ability of CalDAG-GEFI–/– mice to respond to acute inflam-
mation, we challenged WT and mutant mice by intraperitoneal
injection of thioglycollate (TG). Using this model, several groups
have shown a key role for β2 integrins in the rapid recruitment of
neutrophils to the peritoneal cavity (30–32). Others did not find
a marked reduction in the total number of neutrophils recruited
to the peritoneum of CD18–/– mice (7). However, CD18–/– mice
are characterized by neutrophil counts elevated by greater than
10-fold compared with controls, suggesting that the efficiency of
TG-induced neutrophil recruitment was also impaired in these
studies. We found few neutrophils in the unchallenged peritoneal
cavities of WT and CalDAG-GEFI–/– mice (Figure 5A). In WT mice,
we found robust intraperitoneal infiltration of neutrophils 4
hours after challenge. In contrast, CalDAG-GEFI–/– mice exhibited
approximately 80% reduction in neutrophil infiltration compared
with WT mice after challenge (P < 0.001; Figure 5A), demonstrat-
ing a pivotal function for CalDAG-GEFI in neutrophil recruit-
ment to inflamed peritoneum.
To test CalDAG-GEFI–/– mice in a specifically β2 integrin–depen-
dent inflammation model (7), we studied neutrophil infiltration into
croton oil–irritated ears. As shown in Figure 5B, the number of neu-
trophils counted in the croton oil–painted ears of CalDAG-GEFI–/–
mice was about 60% that of WT controls (P < 0.001). These findings
strengthen the case for CalDAG-GEFI as a critical intracellular sig-
naling molecule upstream of β2 integrin activation.
Impaired β1 integrin activation in CalDAG-GEFI–deficient platelets. To
determine whether CalDAG-GEFI also regulates β1 integrin func-
tion in platelets, we examined platelet adhesion to established sub-
strates of β1 integrins. Platelets express various members of the β1
integrin subfamily, including α2β1 (collagen as the main ligand),
α5β1 (fibronectin as the main ligand), and α6β1 (laminin as the
main ligand) (33, 34). The expression levels of β1 integrins were
comparable in platelets from CalDAG-GEFI–/– and WT mice (mean
fluorescence intensity, 240.5 ± 8.0 and 242.7 ± 14.8, respectively).
We studied the adhesion of activated WT and CalDAG-GEFI–
deficient platelets to a laminin-coated surface by stimulating plate-
lets with PAR4-activating peptide (PAR4p; GYPGKF) or a combi-
nation of ADP and the thromboxane A2 mimetic U46619. In order
to avoid adhesion/aggregation of platelets mediated by αIIbβ3 inte-
grin, the experiments were performed in the presence of a block-
ing antibody against this receptor (35). In contrast to activated
WT platelets, which showed robust adhesion to laminin, the adhe-
sion of CalDAG-GEFI–deficient platelets stimulated with either
agonist was significantly inhibited (Figure 6A). The dependence
of the adhesion process on α6β1 integrin was demonstrated by
blocking this receptor on WT platelets (Figure 6A). With the same
experimental setup, we also tested the adhesion of CalDAG-GEFI–
deficient platelets to fibronectin, a process mediated by integrins
α5β1 and αIIbβ3 (34, 36). Because we are not aware of any specific
reagents that inhibit αIIbβ3 integrin–mediated adhesion of mouse
platelets to fibronectin, we used EDTA in control experiments to
inhibit both β1 integrin– and β3 integrin–mediated adhesion. The
specificity of the adhesion process for β3 and β1 integrins was veri-
fied in studies with activated platelets lacking β3 integrins, which
adhered to fibronectin in an α5β1 integrin–dependent manner
(data not shown). Again, U46619/ADP-stimulated CalDAG-GEFI–
deficient platelets showed significantly impaired adhesion to
fibronectin (Figure 6B). No significant difference in the adhe-
sion of WT and CalDAG-GEFI–deficient platelets stimulated
with PAR4p was observed, indicating that 1 or more alternative
signaling pathways mediated β1 integrin activation in platelets
stimulated by PAR4. These results provide strong evidence that
Impaired firm adhesion of leukocytes to mesenteric venules in CalDAG-GEFI–/– mice. (A) Leukocyte rolling in vivo. The number of rolling leu-
kocytes in WT or CalDAG-GEFI–/– mice was determined by intravital microscopy. Mice were infused with rhodamine 6G to label circulating leu-
kocytes. Leukocyte rolling was quantified 1–5 minutes after superfusion with 300 nM LTB4. (B) Firm adhesion. WT or CalDAG-GEFI–deficient
leukocytes were considered firmly adherent when they remained stationary for more than 30 seconds. n = 6. (C) Blocking antibodies to β2 integrin
markedly reduce leukocyte adhesion to mesenteric venules. Leukocyte adhesion within the first 5 minutes after LTB4 superfusion was studied
in WT mice infused with PBS or 40 μg anti-β2 antibodies (β2 Ab). n = 3. *P < 0.05, ***P < 0.001. No significant difference in leukocyte adhesion
was observed between untreated and control IgG–treated WT mice (not shown).
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 6 June 2007
CalDAG-GEFI is an important signaling molecule mediating acti-
vation of α6β1 and α5β1 integrins in platelets activated by some,
but not all, platelet agonists.
CalDAG-GEFI–/– mice do not form thrombi in an experimental arte-
rial thrombosis model. To determine the effect of defective β1 and
β3 integrin activation on thrombus formation in vivo, we studied
WT and CalDAG-GEFI–/– mice in a model of FeCl3-induced arte-
rial thrombosis (37, 38). In this model, FeCl3 causes endothelial
denudation leading to the exposure of extracellular matrix, which
in turn promotes the adhesion and activation of circulating
platelets. We did not observe a significant difference between WT
and CalDAG-GEFI–/– mice in the frequency of platelet tethering
(Figure 7, A and B). This was not surprising, as the initial tethering
of platelets depends on the constitutively active GPIb-V-IX receptor
complex (33), which was expressed in high copy numbers on both WT
and mutant platelets (mean fluorescence intensity, CalDAG-GEFI–/–,
907 ± 37.8; WT, 886.7 ± 35.9). Firm adhesion and/or aggrega-
tion of platelets at sites of vascular injury, however, requires ago-
nist-induced inside-out activation of platelet integrins. The first
thrombi formed in WT mice approximately 10 minutes after
application of FeCl3 (data not shown), and vessel occlusion was
observed in all WT mice within 20 minutes (mean occlusion time,
14 ± 1.3 minutes), whereas thrombus formation was completely
absent in CalDAG-GEFI–/– mice for the duration of the 40-minute
observation period (Figure 7, A and C). Video of real-time platelet
adhesion/thrombus formation in injured arterioles is provided as
supplemental data (Supplemental Video 1; available online with
this article; doi:10.1172/JCI30575DS1). Thus, CalDAG-GEFI is a
major regulator of β1 and β3 integrin activation, and its absence
has a profound impact on platelet function in vivo.
We found that CalDAG-GEFI was a major regulator of the activa-
tion of β1, β2, and β3 integrins on platelets and neutrophils both
in vitro and in vivo. As a consequence, mice lacking CalDAG-GEFI
had defective inflammatory responses and markedly impaired
ability to form thrombi in response to vascular injury. This phe-
notype is strikingly similar to that described for LAD-III patients,
which combines a mild LAD with a Glanzmann-like bleeding dis-
order (9–13). In all cases reported to date, the clinical symptoms of
LAD-III seem to be caused by a defect in the activation, but not the
expression or structure, of β1, β2, and β3 integrins on leukocytes and
platelets (9). The successful treatment of LAD-III patients by bone
marrow transplantation strongly suggests that the genetic defect
CalDAG-GEFI regulates neutrophil extravasation. (A)
TG-induced peritonitis. Infiltrating leukocytes were iso-
lated from the peritoneum of WT and CalDAG-GEFI–/–
mice 4 hours after injection of PBS or 3% TG. Neutro-
phil counts were determined by morphological analy-
ses of Diff-Quik–stained cytocentrifuge preparations
by an observer blinded to the genotype. (B) Croton oil–
induced dermatitis. Ears of WT and CalDAG-GEFI–/–
mice were painted with croton oil, and infiltrating neu-
trophils were counted 6 hours later in H&E-stained
ear sections. Few neutrophils were found in vehicle-
treated ears. n = 3–5. ***P < 0.001.
Impaired activation of β1 integrins in CalDAG-GEFI–deficient platelets. (A and B) Biotinylated WT and CalDAG-GEFI–deficient platelets were
stimulated with PAR4p (2 mM) or U46619/ADP (5 or 10 μM) in the presence of a blocking antibody to αIIbβ3 and allowed to adhere for 30 minutes
under static conditions to laminin (A) or fibronectin (B) in microtiter plates. A separate group of WT platelets was pretreated with a blocking
antibody to α6 (adhesion to laminin) or with EDTA (adhesion to fibronectin) to demonstrate the integrin dependency of the adhesion process.
Adherent platelets were quantified colorimetrically for peroxidase activity. Data are mean ± SEM of 3 individual experiments in triplicate wells.
**P < 0.01, ***P < 0.001. Similar results were observed with nonbiotinylated platelets when the number of adhesive platelets was determined by
light microscopy (not shown).
1704?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 6 June 2007
underlying the disease resides in hematopoietic cells (9). Thus, it
has been speculated that the absence or malfunction of a key inte-
grin regulator expressed in hematopoietic cells is responsible for
the phenotype observed in LAD-III patients (9). We propose here
that CalDAG-GEFI is such a regulator and that genetic deficiency
of CalDAG-GEFI may be present in patients with LAD-III.
Like leukocytes from LAD-III patients (10–12), neutrophils from
CalDAG-GEFI–/– mice behaved normally in many functional assays
including ROS formation, intracellular calcium flux, and granule
release (Figure 1), demonstrating that the cells were fully capable
of activating intracellular signaling pathways. CalDAG-GEFI–
deficient neutrophils expressed normal levels of β2 integrins and
PSGL-1 on the cell surface, while the surface expression of β1
integrins and L-selectin was significantly reduced (Table 1). The
decrease in L-selectin expression was similar to that previously
observed in P/E-selectin–deficient mice, which also show a defect
in neutrophil extravasation (20). Thus, it may reflect shedding of
the receptor from activated blood leukocytes that are unable to
extravasate into tissue. Interestingly, reduced L-selectin expression
was also reported for leukocytes from 1 LAD-III patient (13). As a
consequence of their defective integrin activation, CalDAG-GEFI–
deficient neutrophils showed an impaired response to acute
inflammation. Compared with controls, significantly fewer
CalDAG-GEFI–deficient neutrophils migrated into the inflamed
tissues of mice subjected to experimental peritonitis or dermati-
tis (Figure 5). It is interesting to note, however, that neutrophil
recruitment in the dermatitis model was only partially reduced
in CalDAG-GEFI–/– mice (approximately 40% of control), while
previous studies showed a complete inhibition in neutrophil
recruitment in this model in CD18–/– mice (7). These data sug-
gest that CalDAG-GEFI plays an important and specific role in
the activation of β2 integrins in neutrophils and that, at least in
some inflammatory situations, other signaling molecules such as
PKC family members (39, 40) or other Rap-GEFs (41) can serve
as alternative pathways leading to β2 integrin activation in the
absence of CalDAG-GEFI. Similarly, we have previously shown
robust aggregation of CalDAG-GEFI–deficient platelets stimu-
lated with thrombin or collagen (15). In further studies, we iden-
tified signaling by PKC as an independent pathway allowing for
αIIbβ3 activation in the absence of CalDAG-GEFI (Bergmeier et
al., unpublished observations).
We now provide evidence that CalDAG-GEFI was also critical for the
activation of platelet β1 integrins (Figure 6). Activation of β1 integrins
was almost completely inhibited in CalDAG-GEFI–deficient platelets
stimulated with ADP and the thromboxane A2 analog U46619, while
it was only partially inhibited in CalDAG-GEFI–deficient platelets
activated by PAR4 receptors (Figure 6). Our results confirm observa-
tions by Gruner et al., who showed that β1 integrins are expressed on
resting mouse platelets in a low-affinity state and that cellular activa-
tion is required for these integrins to shift to a high-affinity state (34).
Previous work with human platelets suggested that integrins α5β1
and α6β1 may be expressed constitutively in a high-affinity state, as
these cells spontaneously adhere to fibronectin and laminin, respec-
tively (36, 42). However, these studies only analyzed the adhesion of
unstimulated platelets and did not investigate whether platelet acti-
vation would further increase the number of adherent cells. Based on
our findings that strong agonists induced significant activation of
β1 and β3 integrins in knockout platelets in vitro, we were surprised
that CalDAG-GEFI deficiency led to complete inhibition of arterial
thrombus formation (Figure 7). Probably the simplest explanation
for this finding is that the collagen and thrombin concentrations
that a platelet encounters at sites of vascular damage are much lower
than those used in our in vitro studies. Alternatively, the strong defect
in platelet adhesion observed in CalDAG-GEFI–/– mice could reflect
the critical role that CalDAG-GEFI plays in outside-in signaling by
ligand-occupied integrins in platelets (43) or that CalDAG-GEFI–
deficient platelets are not capable of activating their integrins on the
subsecond scale required for successful adhesion under conditions of
flow. Such a defect has been suggested for leukocytes from LAD-III
patients, which showed a prominent adhesion deficiency under flow
(13). We similarly found the most striking defects in the adhesion
of CalDAG-GEFI–deficient neutrophils in vivo rather than in vitro
under static conditions (Figures 3 and 4).
Although we observed strong defects in platelet and neutrophil
adhesion in CalDAG-GEFI–/– mice, we did not observe spontaneous
bleeding or apparent infections in these mice. These findings may
Platelet adhesion and thrombus formation in FeCl3-injured arterioles. (A) WT and CalDAG-GEFI–/– mice were injected with calcein-green-labeled
platelets of the respective genotype, and platelet adhesion was monitored in mesenteric arterioles (diameter, WT, 90.7 ± 3.5 μm; CalDAG-GEFI–/–,
96.4 ± 4.0 μm) upon application of FeCl3. Images show platelet adhesion and thrombus formation in arterioles at the indicated times after appli-
cation of FeCl3. The direction of blood flow is from top to bottom. (B and C) Comparison of the number of tethering platelets (B) and the time of
occlusion (C) in FeCl3-treated mesenteric arterioles of WT and CalDAG-GEFI–/– mice. No thrombi formed in the mutant mice during the 40-minute
observation period. ***P < 0.001.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 6 June 2007
appear in contrast to the phenotype described for patients with
LAD-III, who have mild to severe bleeding complications as well as
recurrent infections (9). However, in patients, bleeding or infection
is most likely caused by accidental tissue damage or exposure to
pathogens, challenges that mice held in a restricted, pathogen-free
living space hardly encounter. Spontaneous bleeding complica-
tions are also rare in mouse models of Bernard-Soulier syndrome
or severe thrombocytopenia, while strongly impaired hemostasis
is observed in tail bleed assays in these mice (44, 45).
The small GTPase Rap1 has recently been identified as a critical
molecule regulating integrin activation in many cell types, including
platelets, megakaryocytes, and neutrophils (23, 24). However, the
widespread expression of Rap1 and its reported importance in non-
hematopoietic cells (23, 24) make it unlikely that Rap1 itself is mutat-
ed in patients with LAD-III (9). In addition, a recent study by Kinashi
et al. demonstrated normal expression but impaired activation of
Rap1 in transformed PBLs derived from a patient with LAD-III (46).
The authors concluded that a Rap1-GEF activity essential for
Rap1 and integrin activation was defective in these cells. We
have shown that CalDAG-GEFI is a major regulator of Rap1 and
β1 and β3 integrin activation in platelets (Figure 6 and ref. 15)
and that it is also critical for activation of Rap1 (Figure 2) and β1 and
β2 integrin (Figures 3 and 4) in neutrophils. In addition, the expres-
sion of CalDAG-GEFI is much more tissue specific than that of
Rap1 (15, 17), which may explain why mice lacking CalDAG-GEFI
do not show obvious defects during embryonic development,
whereas mice lacking Rap1b display approximately 85% embryonic
lethality (47). Thus, CalDAG-GEFI could be the defective Rap-GEF
in platelets and leukocytes of LAD-III patients.
Other proteins involved in the inside-out activation of more
than 1 integrin family include cytoskeletal proteins such as talin
or filamin (48, 49), integrin-linked kinase (ILK) (50), or mem-
bers of the PKC family (39, 40). These proteins, however, are not
restricted to cells of the hematopoietic lineage; thus, mutations
in the genes encoding them are not likely responsible for LAD-III.
Furthermore, normal expression of various cytoskeletal adapt-
ers, PKC isoforms, and ILK were reported in leukocytes from 1
patient with LAD-III (12).
Different phenotypes have been described in individual patients
with LAD-III (10–13), which suggests that there are variations in
disease penetrance or that more than 1 gene may be implicated.
For example, Harris et al. (11) found that a patient’s integrins had
an intrinsic defect in their avidity to bind ligands that could not be
overcome by activating antibodies or exogenous cations and that
neutrophils from this patient showed an impaired chemotactic
response. Such defects have not to our knowledge been report-
ed by other groups. Alon et al. (13) found that leukocytes from
another patient with LAD-III exhibited impaired adhesion under
physiological flow conditions, but that their adhesion under static
conditions was similar to that of controls. Adhesion under static
conditions has been found to be severely impaired in all other
reported cases of LAD-III (10–12). In CalDAG-GEFI–/– mice, the
defect in neutrophil adhesion was most prominent under condi-
tions of flow, but it was also observed under static conditions.
Our finding that CalDAG-GEFI was critical to the activation of
β1, β2, and β3 integrins in mouse platelets and neutrophils sug-
gests this gene may be defective in patients with LAD-III. With
their defects in thrombus formation and leukocyte recruitment to
sites of inflammation, mice deficient in CalDAG-GEFI represent
an animal model for this disease.
CalDAG-GEFI–/– (15) and littermate control WT mice were obtained from
the mouse facility at MIT and were bred in the mouse facility of the CBR
Institute for Biomedical Research. Itgb3–/– mice on a BALB/c background
were a generous gift from R. Hynes (MIT). Experimental procedures were
approved by the Animal Care and Use Committee of the CBR Institute
for Biomedical Research.
Blood was drawn from the retroorbital plexus into heparinized tubes. One
milliliter of whole blood was incubated in 10 ml of red blood cell lysis buffer
(155 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA, pH 7.4) for 10 minutes
and then centrifuged for 8 minutes at 185 g. The supernatant was discarded,
and the pellet resuspended in a modified Tyrode buffer (15).
Expression of adhesion receptors on resting PBLs. Resting cells were stained
with fluorescently labeled antibodies for 30 minutes at 4°C and immedi-
ately analyzed by flow cytometry. Neutrophils were identified by determin-
ing forward/side scatter characteristics and staining with the neutrophil-
specific monoclonal antibody Gr-1 (BD Biosciences).
Mac-1 expression on resting and activated neutrophils. PBLs were kept rest-
ing or were stimulated with 300 nM LTB4, 200 nM PMA, or 50 ng/ml C5a
for 10 minutes at room temperature. Cells were stained with 2 μg/ml
FITC-conjugated anti–Mac-1 (BD Biosciences) and were immediately
analyzed by flow cytometry.
Calcium flux measurements in peripheral blood neutrophils. PBLs were incubated
with 5 μM of the calcium-sensing dye Fluo-3 (Invitrogen) for 15 minutes,
washed in modified Tyrode buffer containing 1 mM CaCl2 and 1 mM MgCl2,
activated with the indicated agonists, and analyzed immediately by FACS.
Data were analyzed with FlowJo software (version 6.4.3; Tree Star Inc.).
Production of ROS in neutrophils. PBLs were resuspended in RPMI and incu-
bated with 7.5 μM ROS-sensing dye DCFDA-H2 (Invitrogen) for 15 min-
utes. After spinning labelled cells at 185 g for 5 minutes, excessive dye was
discarded and cells were resuspended in RPMI buffer. PBS, 5 μM fMLP, or
2 μM PMA was added to DCFDA-loaded PBLs at time point 0. Mean fluo-
rescence values in channel 1 (FL1) of approximately 2,000 Gr-1–positive
cells were assessed in 5-minute intervals for up to 60 minutes. To normal-
ize individual experiments, the ratio between mean FL1 values of stimu-
lated and unstimulated cells was determined.
Amounts of activated Rap1 in neutrophils were measured using a protocol
similar to the one previously described for platelets (15). Neutrophils were
isolated from bone marrow of WT and CalDAG-GEFI–/– mice using nega-
tive sorting with MACS separation columns (Miltenyi Biotec). After red
cell lysis, bone marrow cells were incubated for 10 minutes at 4°C with
10 μg/ml anti-CD45R, 10 μg/ml anti-CD5, 5 μg/ml anti-CD8, and 5 μg/ml
anti-CD4 (all from BD Biosciences). After 2 washing steps, cells were incu-
bated with anti–rat Ig beads for 10 minutes at 4°C, washed again, and run
through 25 LD magnetic columns (Miltenyi Biotec). The eluted cells were
stained with Gr-1 antibody, and the percentage of Gr-1–positive cells was
determined by flow cytometry. Only samples containing more than 90%
Gr-1–positive cells were used for adhesion studies. Neutrophils (5 × 106) were
activated for 30 seconds with 300 nM LTB4, 75 ng/ml C5a, or 3 or 30 nM
PAF and immediately lysed with ice-cold lysis buffer (25 mM Tris-HCl,
pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 5 mM MgCl2, 5% glycerol, and
complete protease inhibitor cocktail lacking EDTA; Roche Diagnos-
tics). Detection of activated Rap1 (Rap1-GTP) in neutrophil lysates was
performed according to the instructions of the manufacturer (Pierce).
1706?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 6 June 2007
Briefly, Rap1-GTP (or Rap1–GTP-γS) was precipitated from lysates using a
GST-RalGDS-RBD fusion protein. Precipitated proteins were separated
on a 15% SDS-PAGE gel and transferred to PVDF membranes (Millipore).
Rap1 was detected with rabbit polyclonal antibodies followed by anti-rab-
bit antibodies conjugated to horseradish peroxidase (Vector Laboratories).
Immunoreactivity was detected by Western Lightning enhanced chemilu-
minescence (PerkinElmer Life Sciences).
To determine the level of total Rap1, 1 × 106 WT or CalDAG-GEFI–defi-
cient neutrophils were lysed with reducing SDS sample buffer, and pro-
teins were separated on a 15% SDS-PAGE gel. Rap1 was detected with rab-
bit polyclonal antibodies as described above.
Neutrophil adhesion assay
Ninety-six-well plates (Costar) were coated with 2 mg/ml fibrinogen or
10 μg/ml fibronectin (both from Sigma-Aldrich) overnight and blocked
with 1% BSA for 2 hours. Isolated bone marrow neutrophils (1 × 106/ml)
were added in the presence or absence of the indicated agonists and incu-
bated for 30 minutes at 37°C. The supernatant was discarded, the plate
was washed with PBS, and the neutrophils adhered to the plate were count-
ed using a Nikon TMS microscope. Adherent neutrophils in the region of
interest were counted by 2 individuals blinded to their source. Alternatively,
neutrophil adhesion was analyzed by quantifying myeloperoxidase (MPO)
activity in the lysate of adherent cells (51). Briefly, adherent neutrophils
were lysed in a potassium phosphate buffer containing 0.5% hexadecyl tri-
methyl ammonium bromide (HTAB), and MPO activity was determined
by adding tetramethylbenzidine (TMB) as a substrate. OD readings were
performed in a 96-well microplate reader at a wavelength of 630 nm.
Platelet adhesion assay
For platelet adhesion studies, 96-well plates (Nunc) were coated overnight
at 4°C with either laminin or fibronectin (both from Sigma-Aldrich) at a
concentration of 4 μg/ml and then incubated with PBS plus 5% BSA for
2 hours at 37°C. Platelet-rich plasma (PRP) was obtained from heparin-
ized whole blood by centrifugation at 100 g for 10 minutes and then cen-
trifuged at 700 g in the presence of PGI2 (2 μg/ml) for 7 minutes at room
temperature. After 2 washing steps, pelleted platelets were resuspended in
modified Tyrode-HEPES buffer. Platelets (2 × 109/ml) were incubated with
NHS-biotin (1 mM; Pierce) for 10 minutes, washed twice, and resuspend-
ed in a modified Tyrode buffer containing Ca2+ and Mg2+ (1 mM each).
The cells were either left unstimulated or activated with 2 mM PAR4p or
5 or 10 μM U46619/ADP and immediately plated. After incubation for
30 minutes at 37°C, the plate was rinsed 3 times with 100 μl PBS, and 50 μl
of HRP-labeled streptavidin solution (1 μg/ml; Jackson ImmunoResearch
Laboratories) was added to each well for 15 minutes. After extensive wash-
ing, 100 μl HRP substrate (ABTS; Roche Diagnostics) was added to each
well, and the OD at a wavelength of 405 nm was determined after 5 min-
utes. To inhibit β3 integrin–mediated platelet aggregation, all experiments
were performed in the presence of a blocking antibody to αIIbβ3 (50 μg/ml;
emfret Analytics). Blocking antibodies to α6 integrin (50 μg/ml; BD Biosciences
— Pharmingen) or EDTA (15 mM) were used as inhibitors in adhesion
studies of laminin and fibronectin, respectively. Calcium flux and integrin
activation studies did not show significant differences between biotinyl-
ated and nonbiotinylated platelets (data not shown).
Thrombosis model. Platelets were labeled for 10 minutes with calcein-green
(5 μg/ml; Invitrogen) and infused into 3- to 5-week-old anesthetized male
CalDAG-GEFI–/– mice or their littermate WT controls. The mesentery was
exposed through a midline abdominal incision. Vessels with a shear rate
of 1,000–1,500 s–1 were selected by use of an Optical Doppler Velocimeter
(Cardiovascular Research Institute, Texas A&M University System
Health Science Center). Arterioles were examined with a Zeiss Axiovert
135 inverted microscope (Zeiss), and adhesion of fluorescently labeled
platelets was monitored with a silicon-intensified tube black and white
camera (C2400-08; Hamamatsu) connected to an S-VHS video recorder
(AG-6730; Panasonic). Vessel injury was generated by placing a filter
paper (1 × 4 mm) soaked with 10% FeCl3 over the vessel for 5 minutes.
The filter paper was then removed, and the vessel was superfused with
saline at 37°C. Vessels were monitored for 40 minutes after FeCl3 treat-
ment or until blood flow had stopped for longer than 10 seconds (identi-
fied as occlusion time).
Leukocyte adhesion. Male mice were infused with rhodamine 6G
(100 μg/ml in PBS) to label circulating leukocytes. Exposed mesenteric
venules (shear rates ranging 50–150 s–1) were superfused with PBS con-
taining 300 nM LTB4 to initiate leukocyte adhesion. We made 30-second
recordings of fluorescent cells in different parts of 2–4 venules per animal
during 4 consecutive 5-minute intervals. The number of rolling leukocytes
was determined by counting the cells passing through a perpendicular
plane in 10 seconds. To determine firm adhesion of cells, we counted the
number of leukocytes that remained stationary for more than 30 seconds.
To study the role of β2 integrins in leukocyte adhesion under these
experimental conditions, mice were infused with 40 μg inhibitory anti-β2
antibodies (BD Biosciences) 15 minutes prior to the start of the experiment
and analyzed as described above.
Irritant dermatitis was induced by topical application of croton oil (7).
Mice were anesthetized by isoflurane inhalation, and each side of 1 ear was
treated with 10 μl of 2% croton oil (Sigma-Aldrich) in 4:1 acetone/olive oil.
After 6 hours, mice were sacrificed by an overdose of halothane inhalation.
Ears were removed, fixed in 10% formalin, and embedded in paraffin, and
sections were stained with H&E for examination by light microscopy.
Mice were injected intraperitoneally with 1 ml of 3% thioglycollate (Sigma-
Aldrich), and peritoneal lavage was performed after 4 hours as described
previously (52). Neutrophil counts were determined by morphological
analyses of Diff-Quik–stained (VWR) cytocentrifuge preparations by an
observer blind to the genotype.
Results are reported as mean ± SEM. Statistical significance was
assessed by unpaired 2-tailed Student t test. A P value less than 0.05
was considered significant.
Note added in proof. It was brought to our attention that the loss
of CalDAG-GEFI expression in 2 LAD-III patients resulted in
aberrant G protein–coupled receptor–triggered αIIbβ3 activation
in platelets and absent β2 integrin activation in neutrophils (R.
Alon and A. Etzioni, unpublished observations).
We are grateful to Tanya Mayadas for advice and helpful discussions
and Richard Hynes for critical reading of the manuscript and advice.
We thank Lesley Cowan for help with preparing the manuscript.
This work was funded by a Scientist Development grant from the
American Heart Association (to W. Bergmeier); by stipend GO1360
from Deutsche Forschungsgemeinschaft (to T. Goerge); by National
Heart, Lung, and Blood Institute, NIH, grant R37-HL41002 (to
D.D. Wagner); by National Institute of Child Health and Develop-
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 6 June 2007
ment, NIH, grant R01-HD28341 (to A.M. Graybiel); by the Simons
Foundation (to A.M. Graybiel); and by National Institutes of Mental
Health, NIH, grant F32-MH065815 (to J.R. Crittenden).
Received for publication October 9, 2006, and accepted in revised
form February 27, 2007.
Address correspondence to: Denisa D. Wagner or Wolfgang Berg-
meier, CBR Institute for Biomedical Research, 800 Huntington Ave-
nue, Boston, Massachusetts 02115, USA. Phone: (617) 278-3344;
Fax: (617) 278-3368; E-mail: email@example.com
(D. Wagner). Phone: (617) 278-6617; Fax: (617) 278-3368; E-mail:
firstname.lastname@example.org (W. Bergmeier).
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