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Platelet aggregation in flowing blood at a site of injury to an endothelial
cell monolayer: quantitation and real-time imaging with the TAB monoclonal
antibody
EF Grabowski
Department of Pediatrics, Cornell University Medical Center, New York, New
York 10021.
Epifluorescence videomicroscopy permits real-time imaging of platelet
adhesion/aggregation to a defined microinjury of a monolayer of endothelial
cells exposed to flowing blood. The fluorescent label is the TAB murine
monoclonal antibody directed against human platelet GP IIB, together with a
fluorescein-conjugated goat F(ab')2 against murine immunoglobulin. The
combination assures specificity for platelet membranes, yet leaves platelet
function intact. TAB is first added to gently mixed, citrated human blood;
the second antibody is added 1 hour after the first, mixing continuing for
a second hour. Bovine aortic endothelial cell monolayers (ECMs), grown on
rectangular cover glasses precoated with microfibrillar collagen, comprise
one wall of a flow chamber mounted on a vertical microscope stage. A loop
of 6-0 sterile suture is drawn across the ECM in order to create
microinjuries of width 70 +/- 15 microns (mean +/- SD) oriented in a
direction transverse to flow. Platelet adhesion/aggregation is virtually
absent on intact and confluent regions of the monolayer. On micro-injury
sites and at shear rates of 60 to 1,080 sec-1, however, computer-enhanced
images obtained by means of videomicroscopy show arrival and adherence of
single platelets resulting in the formation of platelet aggregates
elongated in the flow direction. When the monolayers are pretreated with
1.0 mmol/L lysine acetylsalicylate, the mean aggregate thickness increases
(2P less than .05) to 260 +/- 58% (mean +/- SE, N = 6) of control,
aggregates are regularly shed downstream, and the surface area of the
injury site covered by platelets is augmented (2P less than .05) from 14.8
+/- 3.9% to 49.2 +/- 4.7% (mean +/- SE, N = 6). Donor ingestion of aspirin,
on the other hand, leads to an increase (2P less than .01) in percent
surface coverage to 42.7 +/- 8.5 without a concomitant increase in mean
aggregate thickness. In parallel with the above, outflow levels of serum
thromboxane and prostacyclin are measured by radioimmunoassays (RIAs) for
thromboxane B2 and 6-Keto-PGF1 alpha, respectively. Thromboxane B2 is
increased (2P less than .01) by monolayer pretreatment with lysine
acetylsalicylate from 5.08 +/- 1.47 to 9.35 +/- 2.42, but decreased (2P
less than .05) after oral aspirin to 1.21 +/- 0.38 ng/mL (mean +/- SE, N =
6). Levels of 6-Keto-PGF1 alpha were reduced (2P less than .05) by
monolayer pretreatment from 0.48 +/- 0.046 to 0.36 +/- 0.016 ng/mL.
Platelet adhesion/aggregation at a site of injury to an endothelial cell
monolayer, therefore, can be imaged in flowing blood in real time using a
monoclonal antibody approach.(ABSTRACT TRUNCATED AT 400 WORDS)
Volume 75,
Issue 2,
pp. 390-398,
01/15/1990
Copyright © 1990 by The American Society of Hematology
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