Blood, Vol. 93 No. 7 (April 1), 1999:
pp. 2422-2425
CORRESPONDENCE
Platelet Secretory Process
 |
LETTER |
To the Editor:
A recent exchange of letters appearing in Blood1,2
was of particular interest to our group. Lemons et al3 had
published a report in Blood that stimulated a letter from Dr
Morgenstern, to which one of the authors responded. Dr Morgenstern had
no objections to the biochemical results of the article. Rather, he was
concerned that the authors chose to discuss their molecular findings in the context of a morphological design with which Dr Morgenstern disagreed. In the introduction to their article the authors stated: "In one model of platelet activation, these stimuli (collagen, thrombin, and ADP) trigger morphological changes in the platelet resulting in the apparent movement of the secretory granules to the
center of the cell and their subsequent fusion with the surface connected canalicular system (SCCS)."
Dr Morgenstern objected to this model. His letter stated, "The
disadvantage with this model is that the fusion of membranes of
secretory organelles with membranes of the SCCS (ie, open canalicular system, OCS) has never been demonstrated. In contrast, it was clearly
shown in studies using rapid freezing with a time resolution in the
range of milliseconds to capture fusion events, the secretory organelles (granules and dense cored bodies) fuse with the plasma membranes when the platelets were stimulated before." He bolsters his argument with several citations of his own work4-7 and
that of others,8 including one of our
publications9 showing that 
granules in bovine platelets
that lack the OCS of human cells do fuse with the plasma membrane to
secrete products to the exterior following activation in suspension.
The first paragraph of the responding letter by Whiteheart2
answers the concerns of Dr Morgenstern very well. He points out: "It
is well established that, subsequent to stimulation, human platelets
undergo a series of morphological rearrangements that include the
formation of pseudopods and centralization of secretory granules. The
process culminates in the release of granule contents into the extra
platelet space. . . . ."
What Whiteheart is saying, in essence, is that it doesn't matter
whether secretory organelles fuse directly with the plasma membrane or
channels of the OCS because they are the same membranes. The network of
surface membrane invaginations making up the OCS is unique. It is not
found in any other type of blood cell. Behnke's early
studies10,11 and the work of other
investigators12,13 have shown that OCS channels are not
only continuous with the cell surface but are identical
morphologically. That the platelet surface and linings of the OCS are
identical has also been shown by ultrastructural immunocytochemistry.
Monoclonal and polyclonal antibodies, together with immunogold
techniques, have shown that the exposed surface and OCS channels in
frozen thin sections are uniformly covered by GPIIb/IIIa14
and GPIb/IX/V15 receptor complexes.
Morgenstern1 did not appear concerned about whether the
SCCS and exposed surface are identical membranes. He points out: "It
was concluded (ie, from his studies) that the fate of the SCCS during
stimulation is to become evaginated within seconds to allow the surface
enlargement necessary for formation of pseudopodia or spreading. Fully
stimulated platelets do not show SCCS, but do show the membranes of
degranulating organelles." His point seems to be that OCS channels
return to the exposed surface in the earliest stages of shape change
after activation and are unavailable for fusion with granules and
dense bodies to facilitate secretion. These observations are
incorrect.16,17
The SCCS is evaginated during platelet spreading on surfaces, but the
process takes minutes, not microseconds, and elements of the SCCS
remain in over 20% of fully spread platelets.17 Activation
in suspension does not cause evagination of a significant number of OCS
channels. Channels of the OCS do become dilated in platelets activated
in suspension, and may appear to be part of the exposed surface, but
are not evaginated. Had Morgenstern carefully read the articles from
our laboratory cited in his letter, and others referred to in those
citations, he would not have made the statements he has on this subject.
Nor would Morgenstern have made the comment that fusion of membranes of
secretory organelles with membranes of the OCS has never been shown.
The reports from our laboratory that he cited, and
others,18 answer this concern quite well. In one of the experiments, platelets in suspension were combined with 20-nm colloidal
gold particles coated with fibrinogen (Fgn/Au), then exposed to
thrombin for 1, 3, and 5 minutes without stirring.19 At
these intervals the platelets were fixed, frozen, and frozen thin
sections prepared. The sections were stained with a polyclonal antifibrinogen antibody and then staph protein A bound to 10-nm gold
particles. Thrombin stimulated uptake of 20 nm Fgn/Au gold particles
into OCS channels and their transfer to granules in the process of
discharge into the OCS (Fig 1). Protein A 10-nm gold
particles demonstrated fibrinogen in nonlabilized granules and
those in the process of discharge into the OCS. The work showed that
the OCS was truly a final common pathway; hence, the name of the
article.19
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| Fig 1.
Cryosection of blood platelet from a washed cell
suspension incubated with 18- to 20-nm colloidal gold particles coated
with Fgn/Au for 5 minutes, then exposed to 1 U/mL of thrombin for 60 seconds. The Fgn/Au particles bind to the cell surface and penetrate
into peripheral channels of the OCS. After fixation, freezing, and
cryotomy, the frozen thin section was stained with a polyclonal
antifibrinogen antibody and protein A bound to 5-nm gold particles.
Immunogold beads detecting endogenous fibrinogen are concentrated in
intact  granules (G3) and in granules
(G1, G2) in the process of discharging their
contents into channels of the OCS. Some of the Fgn/Au particles
entering from the outside are mixed with the immunogold beads in the
same OCS channels (OCS) communicating with the exterior surface.
Original magnification ×60,000. (Reprinted with
permission.18)
|
|
The second experiment also involved human platelets.18,20
Cells in suspension were combined with thrombin for 15-second to
5-minute intervals and fixed in solutions containing tannic acid.
Tannic acid combines with the surface membrane glycoproteins to form a
mordant dye that binds and converts osmic acid to osmium black, an
electron dense stain. Tannic acid under these conditions binds only to
those membranes exposed to the exterior, or membranes continuous with
the outside. Thus, it stains the platelet surfaces and membranes lining
channels of the OCS. It has one other desirable feature. Tannic acid
also selectively stains fibrinogen and fibrin strands in the same
manner as the glycocalyx.20
The surface and OCS membranes are the only structures stained by osmium
black in resting platelets. After exposure to thrombin, the dense stain
also enters granules that have become labelized and communicate
with the OCS.20 Fibrinogen and fibrin in the process of
extrusion from granules fill channels of the OCS. During this
process the OCS becomes dilated, as do the labelized granules,
yielding the appearance of swollen vacuoles (Fig 2). In
experiments where the platelets were combined with fibrinogen-coated gold particles before exposure to thrombin and fixation through the
tannic acid staining procedure, Fgn/Au was present in OCS channels and
in swollen granules. The findings showed that the OCS is a two-way
street, and granules communicate with channels of the OCS to
secrete their contents.
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| Fig 2.
Thin section of a platelet from a sample of washed cells
incubated with Fgn/Au particles for 15 minutes, then exposed to 5 U/mL
of thrombin for 60 seconds before fixation in glutaraldehyde-tannic
acid-osmium to selectively stain the platelet glycocalyx, fibrinogen,
and fibrin. This example is one of several serial sections through the
same platelet. It and the other serial sections show the typical
features of shape change and internal transformation caused by
thrombin. Fgn/Au particles are bound to the irregular surface and are
in the process of entering channels of the OCS. The OCS channel
indicated by an arrow ( ) is filled with fibrinogen and fibrin
stained by tannic acid-osmium black. The tortuous channel is connected
directly to several granules (G1, G2,
G3) in various stages of labelization. Residual fibrinogen
is stained by the reaction product. Fgn/Au particles carried by the OCS
have entered the granules (G1, G3,
G4). The direct connections between OCS channels and
granules in this example and its serial sections is indisputable.
Original magnification ×45,000. (Reprinted with
permission.19)
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As far as Lemons et al3 are concerned, it may matter little
whether granules fuse with the exposed surface or channels of the
OCS, because their membranes are identical. However, the morphological
background serving as a palette for their work on the molecular
machinery should be as accurate as possible. I hope this letter will
help to clarify the background.
James G. White
University of Minnesota
School of Medicine
Minneapolis,
MN
| |
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