IIIa apparently does not act as a platelet LDL binding site [7,12,13,17] and that other receptors, as CD36
[18,19] are implicated in the LDL-platelet interaction. Overall, these new data provide conflicting results, and
further studies are needed to explore the implication of GPIIb – IIIa in platelet HDL
3
binding. In fact, HDL
3
binding characteristics clearly differ from those of fibrinogen binding [4 – 6]. Moreover, rather than induc-
ing inhibition, HDL
3
particles did not alter the binding of fibrinogen to activated platelets [7]. The aim of this
study was to investigate the hypothetical involvement of platelet integrin a
IIb
b
3
on the binding of HDL
3
to intact human resting platelets.
2. Materials and methods
2
.
1
. Materials
125
I-Na from New England Nuclear Boston, MA; Iodo-Gen was purchased from Sigma Chemical Co. St
Louis, MO, and highly purified B 95 by SDS – PAGE vitronectin and fibronectin, from Calbiochem
Corp and Chemicon Intl. Polyclonal antibodies against the GPIIb – IIIa complex edu-3 and 5B12 [the latter
from Dako] and human antiserums against platelet alloantigens anti-Bak
aB
[ = Lek
ab
, HPA-3, or anti- GPIIb] and anti-PL
A12
[ZW
ab
, HPA-1, or anti-GPI- IIa] were a gift from Dr E. Mun˜iz Hospital de la
Santa Creu i Sant Pau, Barcelona, Spain. A panel of pure murine mAbs against GPIIb M3, M4, M5, M6,
M8 and M95-2b and against GPIIIa P23-7, P33, P37, P40, and P97, which recognize different epitopes of
GPIIb and GPIIIa, respectively, were a gift from Dr J. Gonzalez-Rodriguez Instituto de Quı´mica Fı´sica ‘Ro-
casolano’ CSIC, Madrid, Spain; polyclonal antibodies against a
V
a-vitronectin receptor and b
3
anti-IIIa integrin subunits and their corresponding nonimmune
rabbit sera were kindly donated by Dr E. Dejana Vascular Biology Laboratory, Mario Negri Institut,
Milan, Italy. A mouse immunoglobulin G used as a negative control in binding studies was from Menarini
Diagnostics.
2
.
2
. Isolation of lipoproteins HDL
3
density range, 1.125 – 1.210 gml was isolated from pooled sera of normolipidemic volunteers and
separated by sequential ultracentrifugation [20] follow- ing the procedure previously reported [6]. Serum was
treated with NaN
3
7.5 mmoll, gentamicin sulfate 0.1 mmoll, PMSF 1 mmoll, EDTA 3 mmoll, chlo-
ramphenicol 0.25 mmoll and aprotinin 0.25 mmoll. Lipoproteins were washed during their flotation in a
potassium bromide solution of the respective lipo- protein density containing EDTA 1 mmoll, NaN
3
3 mmoll, gentamicin sulfate 0.1 mmoll, chloram-
phenicol 0.25 mmoll, glutathione 0.65 mmoll and NaCl 150 mmoll. HDL
3
was dialyzed against buffer A 150 mmoll NaCl, 20 mmoll Tris, and 0.3 mmoll
EDTA, pH 7.4 and filtered through a Millipore filter 0.22 mm before use. The concentration of HDL
3
was expressed in terms of their protein content gl mea-
sured by Bradford’s method [21] using BSA as stan- dard. The purity, composition and specific mobility of
HDL
3
were assessed by previously reported methods [6].
2
.
3
. Iodination of HDL
3
Freshly isolated HDL
3
were labeled with Na
125
I by the Iodo-Gen method [22] and the solution was subse-
quently treated to remove unbound iodine as previously described [6]. In all cases, more than 75 of trichlo-
racetic acid-precipitable radioactivity was associated with the protein component of lipoproteins. The
125
I- HDL
3
was used immediately at a specific radioactivity of 1.7 9 0.1 Bqng protein. The chromatographic profi-
les of HDL
3
before and after iodination, obtained in a fast protein liquid chromatography device [6], indicated
that this iodination method did not cause aggregation or breakdown products of HDL
3
.
2
.
4
. Modification of HDL
3
particles Tyrosine Tyr residues of HDL
3
were modified by tetranitromethane TNM nitrosylation according to
the method described by Chacko et al. [23]. HDL
3
at a protein concentration of 2 mgml was incubated with 5
mlml of a freshly prepared solution of TNM 0.6 M in ethanol for 1 h at room temperature. TNM – HDL
3
was then dialyzed and filtered before use 0.45 mm,
Millipore
2
.
5
. Platelets Human platelets were isolated from freshly drawn 60
ml citrated sodium citrate 3.8 blood from healthy volunteers who had taken no medication for at least 10
days before sampling. Blood from well-characterized patients [24] with type I I AIo, patient 23b and type II
A Dom, patient 40 Glanzmann’s thrombasthenia GT with less than 5 and 12 of the total platelet
content of GPIIb – IIIa, respectively, was kindly pro- vided by Dr M. Pico´ of the Hospital Vall de Hebro´n,
Barcelona, Spain. PRP was obtained by centrifugation at 300 × g for 10 min. EDTA 5.6 mmoll was then
added and platelets were sedimented at 900 × g for 6 min. The platelet pellet was resuspended with binding
incubation buffer 0.02 moll Tris – HCl, pH 7.45 con- taining 0.15 moll NaCl and 5 gl of BSA to give a
final platelet count of 10
12
cellsl. Platelets isolated and
washed under our experimental conditions showed ‘swirling’ as a strong indication that discoid shape was
maintained and fully responded to agonists in the pres- ence of fibrinogen.
2
.
6
. Ligand binding studies and competition experiments
Washed platelets 10
8
from healthy volunteers were incubated at room temperature for 25 min with
125
I- HDL
3
up to 1.5 gl in a total volume of 0.25 ml of incubation buffer.
125
I-HDL
3
binding to platelets was determined as described previously [6] and nonspecific
binding was defined as binding that was not displaced by a 100-fold excess of unlabeled HDL
3
. Displacement of
125
I-HDL
3
0.05 gl in the presence of varying protein concentrations of unlabeled HDL
3
particles native and TNM – HDL
3
or the main GPIIb – IIIa ligands fibrinogen, fibronectin, von Willebrand factor
and vitronectin was measured as described above. Cal- culation of the bound lipoprotein was based on the
specific activity of the labeled ligand, and the results were expressed as nanograms of protein bound per 10
8
platelets dissociation constants K
d
for the competing ligands nmoll were determined according to the
method of Cheng and Prussof [25]. Displacement curves were analyzed and the N
H
obtained [26]. Finally, B
max
and K
d
were calculated by Scatchard analysis [27] of the specific binding data using the
KINETICEBDALIGAND
program [28]. To investigate whether platelet GPIIb and GPIIIa are implicated in the binding of HDL
3
to intact resting platelets, we used the following ap-
proaches: 1 the specificity of HDL
3
binding sites for platelet integrin a
IIb
b
3
was evaluated by displacement experiments of
125
I-HDL
3
by several purified GPIIb – IIIa ligands, such as fibrinogen, fibronectin, von Wille-
brand factor and vitronectin, as mentioned above; 2 with a human model of inherited lack of GPIIb – IIIa,
the relationship between HDL
3
binding sites and platelet integrin a
IIb
b
3
was investigated on intact platelets from GT patients type I and II by binding
and functional studies HDL-mediated activation of PKC and the HDL-mediated inhibition of thrombin-in-
duced inositoltriphosphate formation and calcium [Ca
2 +
] mobilization; and 3 immunological ligand- binding assays were performed after washed human
platelets had been preincubated with the indicated con- centrations of a wide panel of polyclonal antibodies
and mAbs or their respective negative antisera for 10 min at room temperature before the addition of
125
I- HDL
3
0.25 gl. The polyclonal antibodies tested were the following: anti-GPII – IIIa complex edu-3 and
5B12, anti-integrin subunits anti-a
V
or anti-a-vit- ronectin receptor, and anti-b
3
, and human antiserums anti-Bak
aB
and anti-PL
A12
. We also tested pure murine mAbs that recognize different epitopes of
GPIIb M3, M4, M5, M6, M8 and M95-2b and GPI- IIa P23-7, P33, P37, P40 and P97 and strongly inhibit
both platelet aggregation and fibrinogen binding; some of them P40, P37, P23-7 and M5 cross react with b-
and a-subunits of the vitronectin receptor in endothelial cells [29].
2
.
7
. Measurement of cytosolic free Ca
2 +
concentration Basal platelet cytosolic free Ca
2 +
concentration [Ca
2 +
]
i
using the Ca
2 +
-sensitive fluorophore Fura 2 Fura 2AM was determined in control and thrombas-
thenic platelets. Briefly, fura 2AM 2 mmoll in DMSO and PGE
1
5 mmoll were added to 10 ml of PRP. Next, PRP was incubated for 1 h at 37°C and
centrifuged at 1100 × g for 15 min. The supernatant plasma was discarded and the pellet was resuspended in
1 ml HEPES – Tyrode’s buffer containing 5 mmoll PGE
1
and 5 mmoll EGTA. The platelet count was adjusted to 1 × 10
8
ml using HEPES – Tyrode’s buffer without PGE
1
and EGTA. In some experiments, washed platelet suspension was loaded with 2 mmoll
fura 2AM and data were similar to those of the PRP loaded method results not shown. Aliquots of 1.6 ml
were incubated at 37°C for 5 – 10 min, in the presence and absence of thrombin 0.1 Uml, HDL
3
1.0 gl and both agents, in a thermostatized quarz cuvette with
constant stirring. Fluorescence intensity was measured with excitation set at 340 and 380 nm and emission at
510 nm.
2
.
8
. Determination of inositol
1
,
4
,
5
-triphosphate IP
3
release After
stimulating control
and thrombasthenic
platelets with thrombin 0.1 Uml, HDL
3
1.0 gl, and both agents, the reaction was stopped by adding 1 gl
TCA 1:5 vv. The precipitate was removed by cen- trifugation and the water soluble-phase was neutralized.
Finally, IP
3
release was determined by a commercial radioreceptor assay NEN, Barcelona, Spain.
2
.
9
. Platelet membrane isolation and protein kinase PKC
assay Platelets from controls and thrombasthenic patients
were washed twice with ice-cold sonication buffer 20 mmoll Tris – HCl, pH 7.4, 0.5 mmoll EDTA, 0.5
mmoll EGTA, 0.25 moll sucrose, 50 mgml leupeptin, 5 mgml antipain, 5 mgml aprotinin, 50 mgml PMSF
and 10 mmoll 2-b-mercaptoethanol. Washed platelets were then homogenized, centrifuged for 3 min at
1000 × g at 4°C, and the pellet was resuspended in 1 ml of the same buffer. Membranes were then separated
following the method described previously [6]. The involvement of PKC in the signaling induced by HDL
3
was investigated following the method described previ- ously [6]. The PKC enzyme activity assay was assayed
using a commercially available kit Amersham.
2
.
10
. Statistical tests The results are expressed as mean 9 S.E.M. Statisti-
cal analysis was performed with Student’s t-test for paired data with P B 0.05 considered significant.
3. Results
