Blood, Vol. 92 No. 12 (December 15), 1998:
pp. 4874-4877
CORRESPONDENCE
Distribution of GPIb on and in Resting Platelets
 |
LETTER |
To the Editor:
Several laboratories have proposed that glycoprotein (GP) Ib, the
receptor for von Willebrand factor (vWF) on platelet external surfaces,
is rapidly redistributed to internal membranes during cardiopulmonary
bypass surgery, induction of bleeding time wounds, or exposure to
thrombin in vitro (for review, see Nurden1). The recent
article by van Zanten et al2 confirms the observation that
GPIb decreases on the surfaces of activated platelets, but shows that a
50% reduction in the vWF receptor does not affect platelet adhesion
under flow conditions. The findings contrast with those of earlier
investigators who presumed that clearance of GPIb resulted in decreased
adhesive capacity,3,4 but agree with reports showing that
carriers of the Bernard-Soulier syndrome (BSS) with half the normal
number of GPIb copies on their platelets do not bleed.5,6
Yet, following the logic of previous investigations, BSS carrier
platelets should also lose 60% to 80% of the half normal number of
GPIb receptors present on their resting cells after activation in vitro
or in vivo.1 Thus, the assumption that the data obtained in
this study explain why carriers of BSS syndrome do not bleed seems open
to question. A more inviting rationale regarding the lack of bleeding
symptoms in carriers of BSS might be that the half normal number of
GPIb receptors remain on exposed membranes of their activated platelets
for interaction with damaged vascular surfaces.
Results of the ultrastructural studies performed by van Zanten et
al2 are pertinent to that concern. Normal platelets treated with thrombin receptor activation peptide (TRAP) in suspension showed a
marked reduction in immunogold particles on external membranes and
increased frequency of particles associated with membranes of the open
canalicular system (OCS) when compared with resting control cells.
However, when TRAP-treated platelets were allowed to interact with
collagen under conditions of high shear, the frequency of immunogold
particles bound to GPIb on external and internal membrane surfaces was
the same as that on resting control platelets. The investigators
suggested that GPIb receptors cleared to the OCS after exposure to TRAP
in suspension were rapidly returned to the exterior membrane during
spreading on collagen.
An alternative explanation for the results obtained by van Zanten et
al2 deserves consideration. There appears to be
considerably more internal membrane in the TRAP-treated platelet shown
in Fig 4B of their report than in the control cell shown in Fig 4A. The number of OCS channels does not increase after platelet activation in
suspension, but due to shape change and internal transformation, OCS
channels become dilated and indistinguishable from the surface membranes with which they are continuous.7
Our experience indicates that it is always easier to label the
receptors of dilated channels than those on membranes of narrow canaliculi in well-preserved, discoid platelets.8 Others
are beginning to make similar observations.1 As an example,
we have noted the same distribution of GPIb on external and internal OCS membranes of platelets from patients with giant platelet disorders, other than the BSS, whose OCS channels are usually dilated (Figs 1 and
2). Another
confirmation is based on studies with cytochalasin B (CB), an agent
that inhibits new actin filament assembly and is reputed to prevent
translocation of GPIb. CB causes dilation of the OCS of resting cells.
Immunogold staining for GPIb is as prominent on membranes of the OCS in
CB-treated discoid platelets as on the exposed surface (Figs 3 and
4).
View larger version (182K):
[in this window]
[in a new window]
| Fig 1.
Giant platelet from peripheral blood of a patient with
May-Hegglin anomaly. Channels of the surface-connected OCS are
usually dilated in these large cells. (Original
magnification ×20,000.)
|
|
View larger version (169K):
[in this window]
[in a new window]
| Fig 2.
Giant platelet from a patient with Epstein's syndrome. A
frozen thin section from one of the large platelets has been stained by
the immunogold technique for GPIb/IX. Gold particles are 5 nm in
diameter. Membranes lining OCS channels are stained by as many
gold particles identifying GPIb/IX as are present on the exterior
surface (S). (Original magnification ×104,000.)
|
|
View larger version (142K):
[in this window]
[in a new window]
| Fig 3.
Frozen thin section of a CB-treated normal discoid
platelet stained by the immunogold technique for GPIb/IX. The
gold particles coupled to anti-IgG are 10 nm in diameter. Dilated OCS
channels are as heavily stained by gold particles indicating
sites of GPIb/IX as are present on the exposed surface. (Original
magnification ×44,000.)
|
|
View larger version (153K):
[in this window]
[in a new window]
| Fig 4.
Frozen thin section of another CB-treated resting
platelet stained for GPIb/IX. Again, gold particles identifying sites
of GPIb/IX are as frequent on membranes lining OCS channels as are on
the exposed surface. (Original magnification ×44,000.)
|
|
Accepting the possibility that immunocytochemical techniques have
limitations in their ability to quantitate antigens on narrow internal
channels would lead to a second view of the morphometric data presented
in Table 2 of the report by van Zanten et al.2 According to
the data, gold labeling for GPIb in resting platelets is distributed
70% versus 25% (external v internal membranes). After TRAP
activation, the distribution is modified to 35% versus 58% (external
v internal membranes).
Studies of giant platelets and CB-treated normal platelets described
above would lead to the assumption that the frequency of GPIb/IX
receptors on internal and external membranes of resting discoid
platelets should be the same. Thus, a starting point of 47.5% versus
47.5% (external v internal membranes) would be expected. Current literature suggests that activation with TRAP should cause disappearance of 60% to 80% of GPIb/IX receptors from the platelet surface as a result of translocation to internal
membranes.1 If that was the case, immunolabeling on
TRAP-activated platelets should bring immunolabeling distributions
close to 20% versus 75% (external v internal membranes). This
would represent a real internalization of GPIb receptors.
Interestingly, the results in Table 2 of van Zanten et al2
show that the immunolabeling on the internal membranes of
TRAP-activated platelets is still below that on external membranes of
resting cells (57.9% v 69.0%; activated v resting).
We believe that the proposed increase in the percentage of gold
particles identifying GPIb on internal membranes of TRAP-treated
platelets may be due to their increased availability for staining in
dilated channels rather than to translocation.
Restoration of the percentages of gold particles marking GPIb on
internal and external membranes of TRAP-treated platelets after
spreading on collagen to values found on resting platelets in
suspension is also of interest. During the course of surface interaction, channels of the OCS are evaginated and become part of the
exposed surface, accounting for a greater than 400% increase in
surface area on spread platelets.9 This should result in a
decreased percentage of gold particles on internal membranes, because
few OCS channels remain inside. It should also cause a marked increase
in the relative percentage of particles on the spread cell surface for
the same reason. It is unlikely that fully spread cells would have the
same percentages of GPIb on internal and external membranes as found in
suspended control cells. Evidence has been presented to support that
point of view.10 Perhaps van Zanten et al2
could respond to these concerns.
Ginés Escolar
University of Barcelona
Servicio de Hemoterapia y
Hemostasia
Hospital Clinic Barcelona
Barcelona, Spain
James
G. White
Departments of Laboratory Medicine and Pathology,
Pediatrics
University of Minnesota
Minneapolis,
MN
| |
REFERENCES |
1.
Nurden P:
Bidirectional trafficking of membrane glycoproteins following platelet activation in suspension.
Thromb Haemost
78:1305, 1997[Medline]
[Order article via Infotrieve]
2.
van Zanten GH, Heijnen HFG, Win Y, Schut-Hese KM, Slootweg PJ, de Groot PG, Sixma JJ, Niewwland R:
A fifty percent reduction of platelet surface glycoprotein Ib does not affect platelet adhesion under flow conditions.
Blood
91:2353, 1998[Abstract/Free Full Text]
3.
van Oeveren W, Harder MP, Roozendaal KJ, Eijsman L, Wildevuur CRH:
Aprotinin protects platelets against the initial effect of cardiopulmonary bypass.
J Thorac Cardiovasc Surg
99:788, 1990[Abstract]
4.
Kondo C, Tanaka K, Takagi K, Shimono T, Shinop H, Yada I, Yasa H, Kusagawa M, Akamatsu N, Tanoue K:
Platelet dysfunction during cardiopulmonary bypass surgery: With special reference to platelet membrane glycoproteins.
ASAIO J
39:M550, 1993[Medline]
[Order article via Infotrieve]
5.
George JN, Reimann TA, Moake JL, Morgan RK, Cimo PL, Sears DA:
Bernard-Soulier disease: A study of four patients and their parents.
Br J Haematol
48:459, 1981[Medline]
[Order article via Infotrieve]
6.
Ingerslev J, Stenbjerg S, Taaning E:
A case of Bernard-Soulier syndrome: Study of platelet glycoprotein Ib in a kindred.
Eur J Haematol
39:182, 1987[Medline]
[Order article via Infotrieve]
7.
White JG, Leistikow El, Escolar G:
Platelet membrane responses to surface and suspension activation.
Blood Cells
16:43, 1990[Medline]
[Order article via Infotrieve]
8.
White JG, Escolar G:
Restoring platelet adhesion function.
Br J Haematol
87:439, 1994[Medline]
[Order article via Infotrieve]
9.
Escolar G, Leistikow E, White JG:
The fate of the open canalicular system in surface and suspension activated platelets.
Blood
74:1983, 1989[Abstract/Free Full Text]
10.
White JG, Krumwiede MD, Cocking-Johnson DJ, Burris S, Rao GHR:
Influence of cytochalasin B (CB) on GPIb distribution after thrombin or TRAP and before surface activation.
Platelets
8:53, 1997
Response
We thank Drs Escolar and White for their interest in our study
reporting that a 50% reduction of platelet surface GPIb expression does not affect platelet adhesion. When we activate platelets in vitro,
a reduced expression of GPIb on the platelet membrane was found, both
using semiquantitative immuno electron microscopy (IEM) as well as FACS
analysis. We have much experience in immunogold cytochemistry and we
are aware of the pitfalls in quantifying gold particles. Indeed, a
dilated open canalicular system (OCS) is better accessible to luminal
antibodies than a narrow OCS. However, limited access is generally not
a problem for antigens present on the cell surface. Because we found
comparable results with both EM and FACS techniques, we are convinced
that in in vitro experiments GPIb expression on the surface is reduced.
The impact of our article is that reduction of GPIb on the platelet surface does not influence platelet adhesion. It was not our aim to
enter the endless discussion of whether GPIb expression on the cell
surface is reduced or not.
At present, it is still unclear whether platelet activation in vivo
results in diminished surface expression of GPIb. Unpublished studies
performed in our laboratory during cardio-pulmonary bypass surgery did
not show a reduced GPIb surface expression, which is in line with other
studies that showed only a minor reduction. An interesting question is
what happens with platelets of carriers of Bernard-Soulier syndrome
when their platelets are activated and have lost 50% of the remaining
GPIb on the cell surface. Obviously, the carriers do not bleed. There
are two explanations for the lack of bleeding: the reduction of
GPIb after activation only occurs in vitro and not in vivo or, as we
have shown in our report, there is an immediate redistribution of GPIb
after adhesion of the platelets, resulting in a restored GPIb
expression on the platelet surface.
We agree that, in the case of fully spread platelets, most of the OCS
membrane become part of the cell surface. The complete spreading of the
platelets to which Drs Escolar and White refer is only achieved after
20 minutes of static adhesion to formvar and is never observed during a
5-minute perfusion over type III collagen, especially not when dRGDW is
used to avoid platelet aggregation.
Ginés Escolar
University of Barcelona
Servicio de Hemoterapia y
Hemostasia
Hospital Clinic Barcelona
Barcelona, Spain
James
G. White
Departments of Laboratory Medicine and Pathology,
Pediatrics
University of Minnesota
Minneapolis,
MN
