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
3
.
1
. Binding of
125
I-HDL
3
to washed human platelets To determine whether platelet GPIIb – IIIa or fibrino-
gen receptors are implicated in the binding of HDL
3
to platelets, we carried out binding studies of
125
I-HDL
3
to platelets from type I I AIo and type II A Dom
thrombasthenic patients
with abnormal
levels of
GPIIb – IIIa and fibrinogen. We found that both type I and type II GT platelets bound
125
I-HDL
3
and pre- sented similar values 2978 binding sites per platelet
with a K
d
of 236 nmoll and 3356 binding sites per platelet with a K
d
of 189 nmoll, respectively to those of control platelets 2773 9 432 binding sites with a K
d
of 355 9 120 nmoll.
3
.
2
.
[
Ca
2 +
]
i
transients in human platelets The basal value of cytosolic free [Ca
2 +
]
i
in resting control and thrombasthenic platelets incubated in a
low-Ca
2 +
medium was 115 9 16 and 126 9 21 nmoll mean 9 S.E.M., n = 3, respectively. This response was
not changed when platelet cytosolic free [Ca
2 +
]
i
was determined in a medium containing 1 mmoll external
Ca
2 +
111 9 14 and 119 9 17 nmoll; mean 9 S.E.M., n = 3. In a low-Ca
2 +
medium, HDL
3
1.0 gl did not change the basal cytosolic free [Ca
2 +
]
i
, in control and thrombasthenic platelets. Thrombin 0.1 Uml induced
in control and thrombasthenic platelets a significant increase in cytosolic free [Ca
2 +
] in a low-Ca
2 +
medium 489 9 78 and 501 9 76 nmoll; mean 9 S.E.M., n = 3,
respectively and in 1 mmoll [Ca
2 +
] medium 805 9 101 and 824 9 89 nmoll; mean 9 S.E.M., n = 3, respec-
tively. When control and thrombasthenic platelets were pretreated with HDL
3
1,0 gl for 2 min at 37°C, the thrombin-induced rise in [Ca
2 +
]
i
decreased significantly to 65 and 72, respectively P B 0.001. This inhibitory
effect in the presence and absence of external Ca
2 +
was dose-dependent results not shown. Finally, TNM –
HDL
3
1.0 gl had no effect and cytosolic free [Ca
2 +
]
i
mobilization induced by thrombin was not significantly different from that observed in the absence of native
HDL
3
.
3
.
3
. Inositol
1
,
4
,
5
-triphosphate IP
3
formation On stimulation with HDL
3
1.0 gl the amount of IP
3
did not change the resting level 16.3 9 4.1 pmol10
9
platelets in both control and thrombasthenic platelets 15.6 9 5.1 and 18.3 9 3.3 pmol10
9
platelets, respec- tively. Thrombin 0.1 Uml rapidly enhanced IP
3
for- mation
in control
and thrombasthenic
platelets, reaching the maximum effect within 15 s 58 9 6.6 and
65 9 12 pmol10
9
platelets, respectively. IP
3
returned to basal levels within 45 s and no increase was observed
thereafter. When platelets were preincubated in the presence of HDL
3
1.0 gl, the thrombin-induced IP
3
formation in control and thrombasthenic platelets was inhibited to 80 and 75, respectively. In contrast, in the
presence of TNM – HDL
3
1.0 gl, the thrombin-in- duced IP
3
formation was unaltered in control and thrombasthenic platelets 63.4 9 7.1 59.6 9 6.6 pmol
10
9
platelets, mean 9 S.E.M., n = 3, respectively.
3
.
4
. Protein kinase C acti6ation In resting control and thrombasthenic platelets, 15 9
8 and 17 9 5 of total PKC activity was membrane associated. When platelets were preincubated for 2 min
in the presence of HDL
3
1.0 gl the total PKC activity associated with membranes rapidly increased 44 9
3.1 in control platelets and 47 9 3 in thrombas- thenic platelets, with no return to basal level within the
10-min stimulation period. Exposure to TNM – HDL
3
1.0 gl produced no significant effect in control and thrombasthenic platelets.
3
.
5
. Competition experiments To determine whether increasing concentrations of
unlabeled HDL
3
and specific GPIIb – IIIa ligands fibrinogen, vitronectin, von Willebrand factor and
fibronectin interfere with
125
I-HDL
3
, we carried out competition experiments of HDL
3
binding to intact resting platelets at 22°C for 25 min. Fig. 1 shows that
the binding of
125
I-HDL
3
0.05 g of proteinl was fully inhibited by unlabeled HDL
3
. Half-maximal IC
50
and inhibition constant K
i
values were 0.04 gl and 34 mgml, respectively. The Hill coefficient, calculated from
the slope of displacement curves analyzed by Hill plots, was − 1.05 which suggests that a single set of binding
sites is involved. In contrast, purified ligands of GPIIb – IIIa, such as fibrinogen up to 10 g of proteinml,
fibronectin up to 10 mmoll, von Willebrand factor up to 10 mmoll and vitronectin up to 10 mmoll were not
effective competitors of
125
I-HDL
3
and there was only a slight unspecific decrease in the total binding. Finally,
TNM – HDL
3
up to 1.5 gl had no effect, indicating that modification of Tyr residues by nitrosylation fully
abolished the ability of HDL
3
particles to interact with its specific binding sites.
Fig. 1. Inhibition of
125
I-HDL
3
binding to intact resting platelets by specific ligands of GPIIb – IIIa. Platelets were preincubated for 25 min
at room temperature with
125
I-HDL
3
0.05 gl both in the presence and in the absence of unlabeled HDL
3
, up to 0.5 gl, fibrinogen , up to 10 mgl, fibronectin
, up to 10 mmoll, von Willebrand
factor , up to 10 mmoll and vitronectin + , up to 10 mmoll. Specific binding was determined as indicated in Section 2. Results are
expressed as the mean of four independent experiments per duplicate S.E.M. never exceeds more than 10 of the mean.
Fig. 3. Plot showing the effect of human antiserums against platelet alloantigens. + , anti-Bak
aB
[ = Lek
ab
, HPA-3, or anti-GPIIb]; , anti-PL
A12
[ZW
ab
, HPA-1, or anti-GPIIIa] and polyclonal antibod- ies against integrin subunits
, anti-b
3
; , anti-a
V
or against the GPIIb – IIIa complex; , 5B12. Unlabeled HDL
3
was used as a positive inhibitor in all experiments. Results are the mean of four
independent experiments S.E.M. never exceeds more than 10 of the mean.
showed any specific inhibitory effect at any protein concentration. However, with some mAbs against
GPIIb, a maximum inhibition 35 was found with low concentrations of P37. A similar unspecific effect
was found with antiserums against platelet alloantigens and polyclonal antibodies against anti-a
V
anti-a-vit- ronectin receptor, and anti-b
3
anti-GPIIIa integrin subunits. All these experiments clearly indicate that
neither the GPIIb – IIIa complex nor GPIIb or GPIIIa individually are the GPs implicated in the binding of
HDL
3
to intact resting platelets.
4. Discussion
