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Prepublished online as a Blood First Edition Paper on June 21, 2002; DOI 10.1182/blood-2002-03-0902.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Division of Thrombosis Research, Mount Sinai
School of Medicine, and the New York Blood Center and Department of
Pathology, Columbia University College of Physicians and Surgeons, New
York, NY; and the Cardiovascular Thrombosis Laboratory, Massachusetts
General HospitalHarvard Medical School, Boston.
Although it is generally accepted that the initial event in
coagulation and intravascular thrombus formation is the exposure of
tissue factor (TF) to blood, there is still little agreement about the
mechanisms of thrombus propagation and the identities of the molecular
species participating in this process. In this study, we characterized
the thrombotic process in real-time and under defined flow conditions
to determine the relative contribution and spatial distribution of 3 components of the thrombi: circulating or blood-borne TF (cTF), fibrin,
and platelets. For this purpose, we used high-sensitivity, multicolor
immunofluorescence microscopy coupled with a laminar flow chamber.
Freshly drawn blood, labeled with mepacrine (marker for platelets and
white cells), anti-hTF1Alexa.568 (marker for tissue
factor), and anti-T2G Tissue factor (TF) is an integral membrane protein
that is found primarily on the surface of certain cell types that are
located outside the vasculature.1,2 Because of its ideal
physical location in the vasculature, vessel wall TF remains separated from the various coagulation proteins that circulate in the blood, thus
preventing thrombus formation in intact vessels. The most widely
accepted view of coagulation and thrombosis is that following vascular
injury, vessel wall TF is exposed to flowing blood, whereupon it forms
a complex with factor VII/VIIa
(FVII/VIIa), thus initiating the coagulation
cascade and eventually resulting in clot or thrombus formation.
Therefore, in this current view of arterial thrombosis, propagation of
thrombi requires that various coagulation reactions as well as platelet
deposition occur on the luminal thrombus surface. However, thrombi are
relatively large structures extending 1 to 3 mm above the vessel wall.
Vessel wall-derived activated coagulation factors would have to
diffuse from the injured vessel wall site through the deposited
thrombus to the luminal surface to be present at the site of the
growing thrombus. Even in an ideal situation of free diffusion, a
protein of molecular weight 50 000 would require hours to reach the
apex of a 1-mm thrombus. Circulating or blood-borne TF (cTF), carried
by unidentified cells or vesicles in flowing blood, would permit the
activation of FIX and FX right on the luminal surface of a growing
thrombus, and thereby would not be subject to the same diffusional limitation.
Thrombi formed in vitro by flowing whole blood over
collagen-coated glass slides, in the absence of vascular TF, were found to be intensely immunostained with anti-TF antibodies; whereas addition
of anti-TF antibodies and inhibited FVIIa
(FVIIai) to the flowing blood markedly inhibited
the formation of thrombi.3 These authors suggested that
blood-borne TF, possibly leukocyte derived, may be involved in the
thrombus propagation at the site of vascular injury. The studies
conducted thus far, however, have been limited to an end product of the
thrombotic process and have yielded little information about the
dynamics of thrombus initiation and propagation. The primary objective
of this study was therefore to characterize the thrombotic process in
real-time and under defined flow conditions, to determine the relative
contribution and spatial distribution of 3 critical components of the
thrombi: cTF, fibrin, and platelets.
Reagents
Preparation of collagen-coated glass slides
Blood collection and processing Ca++ is required for the production of TF in whole blood.7 To prevent TF production after collection, human blood was collected into 1:10 (vol/vol) 3.4% sodium citrate. The blood was then incubated with the following markers for 90 minutes at 37°C with gentle mixing: mepacrine (10 µM), hTF1Alexa568 (20 µg/mL), T2G Perfusion experiments The parallel plate chamber (Grabowski chamber) used in this perfusion system has been described.8 Collagen-coated cover slips were mounted in this chamber. Labeled, recalcified blood, placed in a reservoir was connected to the inlet of the chamber via Tygon tubing (DOW Corning, Midland, MI). A syringe pump connected to the outlet was used to aspirate the blood through the chamber at desired flow rates. Wall shear rates of 100 and 650 s 1 were used in this study. Chamber occlusion was taken
as the end-point of the experiment.
Image capture and analysis The laminar flow chamber was coupled with a high-sensitivity, multicolor immunofluorescence microscope similar in design to that previously described by Grabowski,8 which incorporates mounting of the chamber with the direction of blood flow antiparallel to the direction of gravity so as to minimize red cell sedimentation transverse to flow streamlines. We therefore constructed the apparatus shown in Figure 1.
In this configuration, the set-up yielded one 3-wavelength composite image per 5 seconds. Images were acquired at a magnification of ×20. Image acquisition was performed using Image-1 (Universal Imaging, Downingtown, PA). ASYST was used for microscope control. Captured images were corrected for background, uneven illumination and sensitivity, as well as image shift between wavelengths. Imaging software Paint Shop Pro was used to add pseudo-color to the obtained images. Analysis included determination of the time-dependence of the cross-sectional area of thrombi in the plane of view and of the spatial distribution of the 3-labeled components. Controls The probes were selected for their relatively high photostability and the wide spectral separation, which maximizes the sensitivity while minimizing the cross-talk among individual fluorescence images obtained with the filter set appropriate for each chromophore. To determine the stability as well as specificity of each of these probes, the following control experiments were performed: (1) ChromPure mouse IgG was individually labeled with the Alexa 568 and Cy-5 probes. In one set of experiments whole blood was labeled with mepacrine, IgGAlexa568 and anti-fibrinT2G
Low wall shear rates Freshly drawn blood was labeled with mepacrine (platelet marker) and hTF1Alexa568 (cTF marker) and perfused over collagen-coated slides at wall shear rate ( w) of 100 s1. Real-time videomicroscopy demonstrated the presence
of cTF deposited in the areas of large platelet aggregates/thrombi
during 25 minutes of blood flow. "Particles" of cTF moving past the
glass slide were visible within 5 minutes of initiation of blood flow.
Figure 2 shows a series of images of
platelets (green) and cTF (red) deposited onto the cover slides at
various time points of flow. The chamber occluded after about 30 minutes of blood flow. Thereafter, the camera was moved along the
length of the chamber to capture the presence of platelets and cTF in
the various deposited thrombi. One such thrombus demonstrating
colocalization of platelet aggregates and deposited cTF is shown in
Figure 3. Although colocalization is
intuitively obvious in Figure 2, superpositioning of platelet aggregates (panel 1) and cTF (panel 2) demonstrates significant colocalization of these 2 blood components (panel 3). Figure
4 shows a large number of platelet
aggregates imaged downstream, near the exit outlet of the chamber. cTF
was found anchored to such large aggregates.
Physiologic arterial wall shear rate Preliminary experiments having demonstrated that the proposed real-time imaging studies were indeed experimentally feasible, the next experiments were designed to study the incorporation of platelets, TF, and fibrin in a growing platelet thrombus, as described above. To mimic the rheologic conditions of intact arteries, the flow experiments were performed at a wall shear rate of 650 s1. At this wall
shear rate, too, cTF was found primarily colocalized with large
platelet aggregates. Platelets were seen anchored to the
collagen-coated cover glass in about 10 minutes of blood flow. Moving
platelet and fibrin particles were observed within a few minutes of
perfusion (Figure 5); platelet adhesion
and aggregation commenced within 10 minutes. After 30 minutes the
chamber completely occluded. Shown in Figure
6 are the pseudo-colored digitized images from the same experiment of platelets, cTF, and fibrin incorporated into growing thrombi. The deposition pattern of each of these components, although colocalized, is unique within the
thrombi.
Control experiments The antibodies (anti-TF hTF1 and anti-fibrin T2G1) and mepacrine were used at a final concentration of 20 µg/mL and 10 µM, respectively, to label their target antigens in circulating blood. These values were chosen by performing a titration to determine the concentration at which antibodies (or mepacrine) did not inhibit platelet aggregation or thrombus formation. Immunohistochemical staining as well as real-time visualization techniques were used for this purpose.ChromPure mouse IgG labeled with each of the fluorophores was used to
test the specificity of the antibodies used in these studies. Only
platelet aggregates and cTF incorporated into such platelet clusters,
were recorded by perfusing whole blood labeled with mepacrine, anti-TF
hTF1Alexa568, and IgGCy-5 at 650 s Does cTF circulate with platelets as a complex? To ascertain whether cTF circulates as a complex with resting as well as single activated platelets, labeled whole blood (mepacrine for platelets and anti-TF hTF1Alexa 568 for cTF), not recalcified was perfused at 100 and 650 s1 over glass
slides that were not coated with collagen. In both cases, a number of
platelets moving past the surface were recorded. cTF moving past the
slide was also visible (not recordable due to the small amounts present
in blood). The spatial distribution of these components was dissimilar;
that is, colocalization was not seen in flowing blood in the absence of
platelet adhesion/aggregation. Due to the absence of a "sticky"
substrate, platelets scantily adhered to the flow area of the plain
glass slides in this experiment. Adherent cTF molecules were not found
in this case (Figure 7). After about 20 minutes, calcium was introduced into the reservoir containing the
blood. Chamber occlusion occurred when large masses of platelets
aggregated downstream, near the exit outlet of the flow chamber. The
flow field tends to be 3-dimensional with important effects of fluid
inertia in the exit (and inlet) region of the flow chambers. In
contrast to no colocalization with the sparse population of adherent
platelets on the glass substrate, the cTF marker was found incorporated
into these large platelet masses downstream after recalcification.
Although it is generally accepted that the initial event in coagulation and intravascular thrombus formation is the exposure of TF to blood, there is still little agreement about the mechanism of thrombus propagation and the identities of the molecular species participating in this process. The order in which platelets, fibrin, and circulating TF are deposited onto natural as well as artificial substrates to enable thrombus formation is largely unknown. Therefore, we characterized the thrombotic process in real-time and under defined flow conditions, to determine the relative contribution and spatial distribution of 3 components of the thrombi: circulating TF, fibrin, and platelets. Flowing cTF was observed within a few minutes of onset of perfusion of blood over collagen-coated cover glass placed in a parallel plate flow chamber. Real-time video recordings obtained during each of 10 experiments show rapid deposition of platelets and fibrin onto collagen-coated glass (Figure 5). Overlay images of platelets, fibrin, and cTF clearly demonstrate colocalization of these 3 components in growing thrombi (Figure 6). cTF was recruited only by areas where large platelet aggregates and thrombi had been deposited, thus clearly indicating the thrombogenic potential of cTF. Our images show unambiguous colocalization of platelet aggregates, cTF, as well as fibrin; the pattern of deposition of each of these components within the thrombi being unique (Figure 6). Blood was perfused at 100 and 650 s The obtained circulating TF signals were categorized based on the
obtained colocalized signals. One pool of cTF molecules seemed to align
well with the platelets as well as fibrin and was found to be present
even when the fluorescent tags were incubated with whole blood for
short time periods. In some images, particularly the perfusion
experiments where the incubation was carried out for 90 minutes, in
addition to colocalized signals, we imaged cTF that seemed not to
perfectly colocalize with platelets. A shift in registers between the
platelet and tissue factor was observed. Although the tissue factor
signal lies in the same region as the platelet clusters, overlay images
of these 2 components did not result in 100% alignment. It is possible
that in addition to a plasma-derived fraction of TF, circulating
microparticles containing TF were also imaged. TF is a transmembrane
protein and this transcellular transfer (eg, to the membrane of
activated platelets) would involve TF "residing" in vesicles. An
important question then arises as to what may be the source of such
circulating TF containing microparticles in short-term ex vivo
perfusion experiments performed in the absence of TF-containing
tissues. A variety of events (apoptosis, acute coronary
syndromes),12-14 agents (cytokines, lipopolysaccharides),15,16 and pathologic conditions
(lupus, meningococcal sepsis)17,18 have been reported to
trigger release of microparticles containing TF activity. Because TF is
expressed on a variety of extravascular cells even under normal
conditions,18,19 it appears then that these particles may
have been synthesized and released into circulation prior to the
withdrawal of blood in our experiments. Cells such as granulocytes
might have captured these microparticles and become their active
carriers, providing the adhesion molecules necessary for the
recruitment of such particles into growing thrombi. Recent data suggest
that monocytes and possibly polymorphonuclear leukocytes are one
possible source of circulating TF, which is transferred to platelets,
thereby making TF+ platelets capable of perhaps triggering
and subsequently propagating thrombosis.20 These authors
demonstrated that CD15 (a leukocyte membrane-bound carbohydrate known
as sialyl Lewis) and P-selectin (an A large part of the obtained TF signals appears to be derived from plasma. Strong TF signals were obtained from platelet-rich plasma as well as platelet-poor plasma fractions of blood, whereas washed suspension of resting as well as activated platelets emitted baseline levels of fluorescence signal (data not shown). Recent evidence from our laboratory suggests that there is indeed a plasma protein form of tissue factor circulating in the human vascular system (V. Bogdanov, V.B., O. Vele, et al, manuscript submitted, May 2002). Studies are currently underway to determine the nature of this plasma protein and its functional significance with respect to thrombosis as well as hemostasis. A series of human studies suggest that blood-borne tissue factor (cTF) might be an important factor in the etiology of several diseases. Plasma TF levels quantified by enzyme-linked immunosorbent assay have been reported to be increased in patients with unstable angina, myocardial infarction, trauma, sepsis, disseminated intravascular coagulation, antiphospholipid antibody syndrome, and sickle cell disease.21-24 Levels of blood-borne TF were reported to correlate with the severity of the coronary artery disease state. Irrespective of the origin of cTF, an interesting question that warrants investigation is how blood-borne TF circulates in a dormant state until thrombotic events commence. Our studies show that cTF is incorporated into large platelet clusters only as the thrombotic events commence. Prior to that cTF does not appear to anchor onto the platelet membrane surface and circulate as complex (Figure 7). Therefore, bringing the TF/FVIIa activity into close proximity to the activated platelet surfaces appears to be a key step in the propagation of thrombosis. Whether circulating (blood-borne) TF provides an effective signal to initiate thrombosis, in addition to propagation, remains to be determined. To address this question, perfusion experiments using a faster imaging system that provides temporal resolution in the order of milliseconds are currently being conducted in our laboratory.
The support of Heiki Vaananen (Department of Physiology and Biophysics) for his extensive technical support with the imaging work (imaging system construction as well as image acquisitioning) is gratefully acknowledged. The authors would like to thank Dan Wu for excellent technical help in the preparation of monoclonal antibody T2G1.
Submitted March 22, 2002; accepted May 15, 2002.
Prepublished online as Blood First Edition Paper, June 21, 2002; DOI 10.1182/blood-2002-03-0902.
Supported by National Institutes of Health grants 5 P50 HL54469, 5 P01 HL29019, and HL 33095.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Yale Nemerson, Division of Thrombosis Research, Mount Sinai School of Medicine, Box 1269, Annenberg 24-92, 1 Gustave L. Levy Pl, New York, NY 10029; e-mail: yale.nemerson{at}mssm.edu.
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© 2002 by The American Society of Hematology.
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Top
Abstract
Introduction
chain (
granule-derived
activation-dependent adhesion molecule found on platelets) interactions
mediate the formation of highly procoagulant platelet aggregates
containing TF particles from leukocytic cells. Whether these cells are
able to express TF themselves or acquire TF from other cells and
deposit them into thrombi warrants investigation.