Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0405193 on October 4, 2005

Published online before print October 4, 2005
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(Journal of Leukocyte Biology. 2005;78:1318-1326.)
© 2005 by Society for Leukocyte Biology

Leukocyte-versus microparticle-mediated tissue factor transfer during arteriolar thrombus development

Peter L. Gross1, Barbara C. Furie, Glenn Merrill-Skoloff, Janet Chou2 and Bruce Furie3

Center for Hemostasis and Thrombosis Research, Center for Vascular Biology Research, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts

3 Correspondence: BI-Deaconess, Harvard Medical School, Medicine, 330 Brookline Avenue, Boston, MA 02215. E-mail: bfurie{at}bidmc.harvard.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Circulating tissue factor accumulates in the developing thrombus and contributes to fibrin clot formation. To determine whether tissue factor derived from hematopoietic cells is delivered to the thrombus via tissue factor-bearing microparticles or circulating leukocytes expressing tissue factor on the plasma membrane, we compared the kinetics of tissue factor accumulation in the developing arteriolar thrombus with the time course of leukocyte-thrombus interaction and microparticle-thrombus interaction in the microcirculation of a living mouse using intravital high-speed widefield and confocal microscopy. Tissue factor rapidly accumulated in the developing thrombus, appearing immediately following vessel wall injury, reaching a first peak in ~100 s. In contrast, leukocyte-thrombus interaction was not observed until after 2–3 min following vessel wall injury. Maximal leukocyte rolling and firm leukocyte adherence on thrombi in wild-type mice were observed after ~8 min and were dependent on P-selectin and P-selectin glycoprotein ligand-1. This delay in P-selectin-dependent leukocyte rolling is a result of time-dependent platelet activation and P-selectin expression on the luminal surface of the thrombus. In contrast, microparticle accumulation in the developing arteriolar thrombus was rapid, and peak accumulation was within 60 s. The accumulation of hematopoietic cell-derived tissue factor in the developing thrombus correlates to the kinetics of microparticle accumulation and does not correlate temporally with leukocyte-thrombus interaction. These results indicate that tissue factor derived from hematopoietic cells is delivered by microparticles during the initial phase of thrombus development in vivo.

Key Words: intravital microscopy • P-selectin • blood coagulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tissue factor is a membrane protein which is critical for the initiation of blood coagulation. In complex with factor VIIa, tissue factor catalyzes the activation of factors IX and X, leading to the generation of thrombin and the conversion of fibrinogen to fibrin. Tissue factor is present within the vessel wall, separated from the flowing blood, and comes in contact with blood following vascular injury. In addition, tissue factor in concentrations of 100–150 pg per ml circulates in blood [1 2 3 4 5 ], although others have recently challenged the validity of these observations [6 ]. Some leukocytes in the circulation are reported to constitutively express tissue factor [7 ], whereas de novo tissue factor biosynthesis is induced in other leukocytes. For example, lipopolysaccharide and P-selectin activate monocytes, leading to the de novo synthesis of tissue factor [8 , 9 ].

We have described previously a P-selectin-dependent pathway of blood coagulation [10 ]. Leukocytes accumulated in the developing thrombus over time, and this accumulation was P-selectin-dependent. One possible mechanism for fibrin deposition in this animal model involved leukocytes that deliver tissue factor to the developing thrombus. More recently, we have observed P-selectin-dependent microparticle incorporation into a thrombus during initial thrombus development [4 ]. These tissue factor-bearing microparticles, hematopoetic in origin [11 ], offer a second mechanism for tissue factor delivery to the thrombus. Del Conde et al. [5 ] have recently shown that tissue factor-bearing microparticles are generated from lipid rafts and are transferred into the platelet membrane in an interaction mediated by P-selectin and P-selectin glycoprotein ligand 1 (PSGL-1) [5 ].

We have developed an imaging system for high-speed confocal and widefield intravital microscopy of the microcirculation of a living mouse [12 ]. Using this system, we have observed arterial thrombus formation in which platelets, tissue factor, and fibrin are rapidly incorporated into the developing thrombus. We use this system in the current study to determine whether leukocyte-associated tissue factor or microparticle-associated tissue factor is the source of hematopoietic cell-derived tissue factor, which accumulates within the thrombus in the initial phase of thrombus development. We demonstrate that the microparticle tissue factor rapidly accumulates in the developing thrombus, whereas leukocyte-thrombus interaction is delayed by 2–3 min following vessel wall injury. These results indicate that initial tissue factor accumulation in the developing arteriolar thrombus is mediated by microparticles.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Genetically deficient mice
PSGL-1 null mice [13 ], from a fifth generation backcross into a C57BL/6J background, were bred from mice previously prepared by gene targeting. These mice have been deposited at Jackson Laboratories (Bar Harbor, ME; B6.Cg-Selpltm1Fur). P-selectin null mice and L-selectin null mice in a C57BL/6J background as well as wild-type C57BL/6J mice were obtained from Jackson Laboratories, whereas E-selectin null mice in a C57BL/6J background [14 ] were the generous gift of Dr. David Bullard (University of Alabama, Birmingham, AL). Chimeric mice, prepared by transplantation of bone marrow from wild-type donor mice into low tissue factor mice, have been described previously [11 ].

Intravital videomicroscopy
The intravital technique, adapted from Ley [15 ], is based on the analysis of leukocyte rolling in the mouse cremaster muscle described previously [13 ]. Mice were pre-anesthetized with intraperitoneal ketamine (125 mg/kg, Abbott Laboratories, North Chicago, IL), xylazine (12.5 mg/kg, Phoenix Pharmaceuticals, St. Joseph, MO), and atropine (0.25 mg/kg, American Pharmaceutical Partners, Los Angeles, CA). A tracheal tube was inserted, and the mouse maintained at 37°C on a thermo-controlled rodent blanket. To maintain anesthesia, nembutal (Abbott Laboratories) was administered through a cannulus placed in the jugular vein. After the scrotum was incised, the testicle and surrounding cremaster muscle were exteriorized onto an intravital microscopy tray. The cremaster muscle was stretched and pinned across the intravital microscopy stage. The cremaster preparation was superfused with thermo-controlled (36°C) and aerated (95% N2, 5% CO2) bicarbonate-buffered saline throughout the experiment. Microvessel data were obtained using an Olympus AX-70 microscope with a 40x 0.8 NA or 60x 0.9 NA water immersion objective. Centerline erythrocyte velocity was measured in real-time with a photo-diode velocimeter running a digital cross-correlation program on the computer (Microvessel Velocity OD-RT, CircuSoft Instrumentation, Hockessin, DE). The images were acquired using a Sony Model SSC-S20 charged-coupled device (CCD) camera connected to a monitor and a SVHS recorder (Sony, SVA-9500MD). The digital confocal and widefield fluorescence microscopy system has been described previously [12 , 16 ]. In experiments using high-speed confocal microscopy, a Yokagawa CSU-10 confocal scanner was used. Confocal sections were obtained using a piezo-electric focusing device to rapidly adjust the focal plane. Digital images were captured with a Cooke Sensicam CCD camera in 640 x 480 format. Four channel experiments were performed as described previously [11 ]. The system was controlled, and the images were analyzed using Slidebook (Intelligent Imaging Innovations, Denver, CO).

Labeling of exogenous leukocytes
Blood obtained by carotid cannulation was anticoagulated in sodium citrate buffer. Leukocytes were isolated by sedimentation through Dextran and differential centrifugation. Residual red blood cells were lysed with a hypotonic solution as described previously [17 ]. Typically, more than 2 x 106 leukocytes per mouse were obtained at a yield of ~50%. Isolated leukocytes were fluorescently labeled by incubation with carboxyfluorescein diacetate-succinimidyl ester (Molecular Probes, Eugene, OR) for 15 min at room temperature, and these exogenous leukocyte suspensions (0.5x106 in 200 µL) were injected into the jugular vein of an anesthetized mouse prior to laser injury. Analog recordings of microscope images were made with a silicon-intensified camera (Dage-MTI100).

Laser-induced injury
Vessel wall injury was induced with a nitrogen dye laser (Micropoint System, Chicago, IL), focused through the microscope objective parfocal with the focal plane and aimed at the vessel wall [12 , 16 ].

Microparticle preparation and visualization in thrombi
Microparticles were generated from calcein-labeled WEHI 274.1 cells with A23187 as described previously [4 ]. Immediately after the infusion of microparticles (5x105) into mice, arteriolar thrombi were generated and observed by intravital microscopy. In some experiments, mice were pretreated with hirudin (2 U/g mouse) to eliminate complications from fibrin formation.

Rolling and adherence parameters
Arterioles with a centerline velocity in excess of 4000 µm/s and a diameter of 40–60 µm were selected for induction of experimental thrombosis. These arterioles have shear rates of at least 800 s–1. The endogenous leukocytes rolling on the thrombus in each 1-min interval were recorded for 15 min after the time of injury, and the number of leukocytes rolling in each 1-min time interval was determined. In separate experiments, the number of exogenous, fluorescently labeled leukocytes rolling on the thrombus was recorded for 10 min after the time of injury, and the number of fluorescently labeled leukocytes rolling was determined in each 1-min interval. In addition, the number of leukocytes that were adherent to the thrombus for greater than 2 min over a 60-min period of observation was also determined.

Antibodies
Mice were infused with 1.9 µg/g body weight rat anti-mouse P-selectin antibody (RB40.34), 0.1 µg/g body weight rat anti-mouse CD41 antibody (MWREG30, BD Biosciences, San Jose, CA), 2 µg/g body weight human immunoglobulin g (IgG; Serotec, Oxford, UK), 1 µg/g body weight chicken anti-rat IgG antibody, 4 µg/g body weight chicken anti-rabbit IgG antibody, 4 µg/g body weight chicken anti-human Fc antibody (Molecular Probes), 8 µg/g body weight rabbit anti-mouse tissue factor peptide antibody [4 ], 2 µg/g body weight mouse antifibrin ß-II chain antibody (Accurate, Hicksville, NY), or 1 µg/g body weight soluble intercellular adhesion molecule-1 (sICAM-1)/human IgG chimera (R&D Systems, Minneapolis, MN), where indicated. Alexa 350, Alexa 488, and Alexa 660 were conjugated to purified antibodies or Fab fragments according to the Alexa Fluor protein-labeling kit (Molecular Probes). Fab fragments of anti-CD41 antibody were prepared as described by the manufacturer’s product manual (Pierce, Rockford, IL).

Image analysis
Every image frame was analyzed using Slidebook. An arbitrary area upstream of the injury site was used for measurement of a dynamic background. The average intensity of each fluorochrome in this background image at each time-point was subtracted from each pixel intensity in each image. Data were then expressed as an integrated intensity.

Statistical analysis
Data are expressed as mean ± SEM or as the median value where indicated. Data sets were compared using Student’s t-test.

Fluorescence analysis
Fluorescence image analysis and quantitation were performed as described previously [11 , 16 ]. In experiments performed in which multiple thrombi were made to quantitate the accumulation of tissue factor, fibrin, or microparticles, we determined the median response from among a large number of thrombi to compensate for the variation in the magnitude of vessel injury associated with laser-induced vessel wall injury.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Kinetics of tissue factor accumulation in the developing arteriolar thrombus
The rate and magnitude of tissue factor antigen accumulation into the developing arteriolar thrombus were determined following laser-induced vessel wall injury in the microcirculation of a living wild-type mouse. The fluorescence associated with the developing thrombus, detected using Alexa 488-conjugated chicken anti-rabbit antibody and a rabbit anti-mouse tissue factor antibody, imaged tissue factor expressed on the vessel wall and tissue factor accumulating within the thrombus in a single representative experiment (Fig. 1A ) [4 ]. As tissue factor resides in the vessel wall as well as in microparticles and leukocytes in the blood, we determined the contribution of blood-borne tissue factor to tissue factor accumulation in and around the developing thrombus using mice deficient in vessel wall tissue factor. We have previously generated chimeric mice by transplanting bone marrow from wild-type mice into low tissue factor mice that express ~1% of the normal level of tissue factor [11 ]. These mice have minimal amounts of tissue factor in the vessel wall but normal levels of tissue factor in blood components of hematopoietic origin. Using these mice, we examined the kinetics of tissue factor accumulation into the developing thrombus in the absence of a contribution from vessel wall tissue factor (Fig. 1B) . During the initial several minutes, tissue factor accumulates in the thrombus on a linear time scale. No tissue factor is observed on the vessel wall or in the thrombus–vessel wall interface 1 min after injury, as shown (Fig. 1C) . At a later time-point, tissue factor continued to accumulate within the thrombus but was also visualized on the vessel wall in the thrombus–vessel wall interface 4.5 min after injury, as shown (Fig. 1D) . We speculate that this tissue factor is associated with microparticles accumulating on the vessel wall in an interaction mediated by endothelial cell P-selectin. These experiments indicate the rapid time-frame in which tissue factor antigen of hematopoietic origin initially becomes associated with the developing thrombus.



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  		Figure 1. Tissue factor antigen accumulation in the developing arteriolar thrombus. (A) Tissue factor antigen fluorescence (blue) and the brightfield image of the cremaster muscle arteriole are composited from videos acquired over 15 min. Images at 0 s (preinjury), 5 s, 30 s, 2 min, 5 min, and 15 min are depicted. Blood flow is from left to right. (B) Accumulation of tissue factor antigen of hematopoietic origin in the developing arteriolar thrombus. In this representative experiment using chimeric mice prepared by transplanting wild-type bone marrow into low tissue factor mice, we quantitated tissue factor antigen fluorescence in the developing thrombus in an animal where vessel wall tissue factor could only minimally contribute to the observed tissue factor antigen. The tissue factor observed during the initial 120 s was located solely within the thrombus. (C) Representative image of thrombus development 1 min after vessel wall injury in the arteriole of a chimeric mouse. (D) Representative image of thrombus development 4.5 min after vessel wall injury in the arteriole of a chimeric mouse. Intravital, four-channel video images depict the accumulation of platelets (red), tissue factor (green), and fibrin (blue) as well as the colocalization of tissue factor and platelets (yellow), platelets and fibrin (magenta), tissue factor and fibrin (turquoise), and all three components (white). Tissue factor antigen is indicated by green, yellow, and white. To simplify analysis of the composite image, the dynamic range of the intensity of each pseudocolor was minimized and thus, does not directly reflect the concentration of the targeted antigen. The methods used in these experiments have been described previously [11 ].

 
Leukocyte interaction with the developing arteriolar thrombus
Blood-borne tissue factor, which accumulates in the developing arteriolar thrombus, may be derived from circulating microparticles or from tissue factor associated with leukocytes, specifically monocytes and possibly granulocytes, although the latter case remains controversial [7 , 18 ]. To determine whether leukocytes are the direct source of tissue factor in the developing thrombus, we analyzed the kinetics of leukocyte rolling and firm adherence with the arteriolar thrombus in vivo.

To analyze leukocyte–thrombus interaction, we visualized endogenous leukocytes rolling on the thrombus using brightfield analog videomicroscopy during a 15-min period of observation following vessel wall injury. No rolling leukocytes were visible on the arterial wall or on the platelet thrombus during the initial phase of thrombus formation. However, leukocyte rolling on the arteriolar thrombus was observed after an interval of 3–8 min following the initiation of thrombus formation (mean 4.5±0.7 min; 17 thrombi in five mice; Fig. 2 ). Within the 15 min of observation after vessel wall injury, the number of rolling leukocytes per minute stabilized at 24.1 ± 4.4 per minute (average±SEM). The velocity of leukocyte rolling on the thrombus was 10 µm per s (range: 8–12 µm per s).



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  		Figure 2. Time course of leukocyte rolling on arteriolar thrombi of wild-type mice. The number of endogenous rolling leukocytes per 1-min intervals following thrombus formation was determined by brightfield analog videomicroscopy. Data represent the mean ± SEM of 17 thrombi in five mice.

 
To allow direct comparison of the kinetics of fluorescently labeled leukocytes and fluorescently labeled microparticles into the developing thrombus, we isolated mouse leukocytes, labeled them with a fluorescent dye, and then infused them into the circulation of an anesthetized mouse. Following vessel wall injury, the number of exogenous leukocytes rolling on a developing thrombus per 1-min time interval over 10 min was determined by fluorescence analog videomicroscopy. The kinetic profile of rolling of exogenous leukocytes was similar to that of endogenous leukocytes, with delayed onset of rolling following vessel wall injury (data not shown).

We asked whether endogenous leukocytes rolling on the thrombus would firmly adhere to the thrombus—an interaction defined as lasting for more than 2 min. We visualized the formation of a thrombus over 1 h following vessel wall injury and observed leukocytes that firmly adhered to the thrombus as early as 8 min after thrombus formation (Fig. 3A ). However, the actual number of adherent leukocytes is small compared with the number of rolling leukocytes. The presence of certain chemokines and intracellular signals during the inflammatory response results in a conformational change in the CD18 integrins on leukocyte membranes [19 ]. TNF-{alpha} stimulation of vessels leads to firm adhesion of leukocytes (Fig. 3B) . These leukocytes express the activated form of the CD18 integrins as they bind the ICAM-1/human Fc chimera. To assess whether adherent leukocytes bound to the laser-induced thrombus express the form of the CD18 integrins capable of high-affinity interaction with ICAM-1, we infused a mouse sICAM-1/human Fc chimera into the mouse circulation and detected the bound chimera using an Alexa 488-labeled chicken anti-human Fc antibody (Fig. 3C) . Leukocytes firmly adherent to the thrombus were labeled with the ICAM-1/human Fc chimera. When arterioles were imaged with identical parameters after the infusion of human IgG and the same concentration of Alexa 488-labeled chicken anti-human Fc antibody, firmly adherent leukocytes were not visualized. This result indicates that the firmly adherent leukocytes express receptors that bind the mouse sICAM-1/Fc chimera.



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  		Figure 3. Leukocyte firm adhesion on arteriolar thrombi. (A) Using analog brightfield intravital microscopy, the number of leukocytes firmly adhering to the thrombus was determined. Firm adhesion was defined as interaction of a single leukocyte with a thrombus for longer than 2 min. The cumulative number of firmly adherent leukocytes per thrombus over 5-min intervals following thrombus formation is shown. Wild-type mice (•; 12 thrombi in five mice); E-selectin-deficient mice ({circ}; seven thrombi in four mice); P-selectin-deficient mice ({blacktriangledown}; five thrombi in two mice). (B) Firmly adherent leukocytes in an inflamed venule bind ICAM-1. One hour after intrascrotal injection of tumor necrosis factor {alpha} (TNF-{alpha}; 0.5 µg in 200 µL saline), ICAM-1 binding was imaged (green) against a brightfield background after the infusion of mouse ICAM-1/human Ig chimera (1 µg/g mouse) and Alexa 488-conjugated chicken anti-human Fc antibody (4 µg/g mouse). Rolling leukocytes showed no fluorescence. Blood flow is right to left. (C) Firmly adherent leukocyte in an arteriolar thrombus binds ICAM-1. A 45-µm arteriole of the cremaster muscle circulation after the infusion of 1 µg/g mouse sICAM-1/human Ig chimera and 4 µg/g Alexa-488-conjugated chicken anti-human Fc is shown 35 min after vessel wall injury. The thrombus is visualized in the brightfield image (arrows). A firmly adherent leukocyte (green) is observed fluorescently stained with ICAM-1/human Ig chimera and anti-human Fc antibody. Under identical conditions, no fluorescence was observed following infusion of 1 µg/g human IgG and 4 µg/g Alexa 488-conjugated chicken anti-human Fc.

 
Selectin and selectin-ligand dependence of leukocyte–thrombus interaction
We and others have described P-selectin-mediated leukocyte accumulation into thrombi [10 , 20 ]. To determine whether P-selectin is required for leukocyte rolling on arterial thrombi, we examined thrombi in the arterioles of P-selectin null mice. In thrombi generated in five P-selectin null mice, few rolling leukocytes were observed in thrombi during the 15-min of observation. The average number of rolling leukocytes per minute between 8 and 15 min after injury was 0.20 ± 0.05 per minute (17 thrombi in five mice; Fig. 4 ). These results suggest that leukocyte rolling on an arterial thrombus is almost entirely P-selectin-mediated. No firmly adherent leukocytes were observed in seven thrombi generated in P-selectin null mice. Infusion of wild-type platelets into a P-selectin null mouse rescued leukocyte rolling on the arterial thrombus (data not shown). This observation indicates that platelet P-selectin and not endothelial cell P-selectin is important for leukocyte rolling on the arteriolar thrombus.



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  		Figure 4. Role of selectins and selectin ligands in leukocyte rolling. Selectin and selectin ligand requirement for leukocyte rolling on arteriolar thrombi. The average number of leukocytes rolling per minute on arteriolar thrombi between 8 and 15 min after vessel wall injury in mice of various genotypes is shown. Mean ± SEM of 17 thrombi in wild-type (WT) mice, 17 thrombi in P-selectin null mice (P-selectin–/–), 16 thrombi in PSGL-1 null mice (PSGL-1–/–), 14 thrombi in E-selectin null mice (E-selectin–/–), and 14 thrombi in L-selectin null mice (L-selectin–/–). In comparison with the number of rolling leukocytes in wild-type mice, the number of rolling leukocytes was significantly less in PSGL-1 and P-selectin null mice (P<0.0001).

 
To evaluate the contributions of E-selectin and L-selectin to leukocyte rolling on arteriolar thrombi, we quantitated leukocyte rolling on arteriolar thrombi following vessel wall injury in E-selectin null mice and in L-selectin null mice. The time of onset of leukocyte rolling on thrombi in E-selectin null mice (4.4±0.3 min; 14 thrombi in four mice) and L-selectin null mice (4.5±0.8 min; 14 thrombi in four mice) was not different compared with that in wild-type mice. The average number of leukocytes that rolled on thrombi between 8 and 15 min after arterial injury of E-selectin null mice and L-selectin null mice was 21.5 ± 4.3 per min and 14.1 ± 3.7 per min, respectively (Fig. 4) . Similarly, the number of firmly adherent leukocytes in E-selectin null mice was comparable with the number found in wild-type mice.

To determine the leukocyte counter-receptor required for P-selectin-mediated leukocyte rolling on arteriolar thrombi, we examined thrombus formation in PSGL-1 null mice. In 16 thrombi generated in PSGL-1 null mice, few leukocytes were seen rolling during the 15 min of observation. Between 8 and 15 min following injury, the average number of leukocytes rolling on the thrombi was 0.07 ± 0.03 per min (average of 16 thrombi in four mice; Fig. 4 ). These results indicate that PSGL-1 is required for leukocyte rolling on an arteriolar thrombus.

Kinetics of P-selectin expression in thrombi in vivo
Having determined that platelet P-selectin interacts with leukocyte PSGL-1 during leukocyte rolling and firm adherence to the arteriolar thrombus, we analyzed the kinetics of P-selectin expression on the thrombus to understand the time course of leukocyte–thrombus interaction. We hypothesized a requirement for two time-dependent events prior to leukocyte rolling: platelet activation with concomitant P-selectin expression and increases in P-selectin density on the luminal surface of the thrombus to a threshold luminal P-selectin density which can support leukocyte rolling. To determine the kinetics of expression of P-selectin in the developing thrombus, we used high-speed confocal, two-color fluorescence microscopy to localize platelets and P-selectin over time following vessel wall injury. Initial P-selectin expression was limited to the interior portion of the thrombus adjacent to the vessel wall (Fig. 5A ). A wave of P-selectin, correlating to platelet activation, was observed over time from the vessel wall–thrombus interface to the thrombus–lumen interface. Although some unactivated platelets may not stably bind to the growing thrombus and may embolize downstream within approximately 5 min, the expression of P-selectin was visible throughout the entire remaining thrombus, including the luminal surface of the thrombus (representative of 13 thrombi generated in five wild-type mice). A fluorescent-tagged, isotype-control monoclonal antibody did not incorporate into these thrombi.



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  		Figure 5. P-selectin expression in the developing arteriolar thrombus. (A) High-speed confocal microscopy was used to generate an image through the center of the developing thrombus. A sequence of images of the confocal plane as a function of time starting with initial injury demonstrates a wave of P-selectin initiating at the vessel wall and ultimately extending to the thrombus–lumen interface. Platelets are labeled with Alexa 660-conjugated CD41 Fab fragments (red); P-selectin is labeled with Alexa 488-conjugated anti-P-selectin antibody (blue). Colocalization of P-selectin and platelets is displayed as magenta. To simplify viewing and analysis, the dynamic range of the intensities of the two colors was compressed. Blood flow is from top to bottom. Thrombus–vessel wall interface (solid arrowhead); thrombus–lumen interface (open arrowhead). (B) Kinetics of development of P-selectin density in the arteriolar thrombus along the vessel wall–thrombus interface and on the thrombus at the luminal surface. Confocal images over time were generated through a single plane. The kinetics of P-selectin expression on the luminal surface of the thrombus was measured by determining the ratio of the integrated fluorescence intensity associated with anti-P-selectin antibodies to the integrated fluorescence intensity associated with anti-CD41 antibodies, P-selectin density, using a mask located over the portion of the thrombus adjacent to the luminal surface ({circ}). The kinetics of P-selectin expression at the vessel wall–thrombus interface was measured by determining the ratio of the integrated fluorescence intensity associated with anti-P-selectin antibodies to the integrated fluorescence intensity associated with anti-CD41 antibodies using a mask located over a portion of the thrombus adjacent to the vessel wall (•).

 
To determine the kinetics of exposure of platelet P-selectin to the vessel lumen, we quantitated P-selectin at the thrombus–lumen interface in a wild-type mouse, as it is this exposed P-selectin that is available to interact with PSGL-1 on the leukocyte surface and support leukocyte rolling on the thrombus. As the size of the platelet thrombus changes with time, we corrected for these changes by measuring the relative density of P-selectin to platelets, as the integrated fluorescence intensity associated with P-selectin divided by the integrated fluorescence intensity associated with CD41. This ratio was calculated as a function of time for a series of confocal images generated through the middle of the thrombus during the initial 5–6 min after vessel wall injury. As shown in Figure 5B (•), there was an immediate linear increase in P-selectin density at the vessel wall–thrombus interface, which leveled off at ~3 min. The P-selectin density on the luminal surface of the thrombus was undetectable above background fluorescence for the initial 2 min and then increased linearly over time for the next 4 min (Fig. 5B , {circ}). These in vivo experiments demonstrate that there is no P-selectin at 0–120 s on the luminal surface of the thrombus. However, between 2 and 6 min, there is the development of significant P-selectin density.

Kinetics of microparticle accumulation in the developing thrombus
We had previously shown that leukocyte-derived microparticles accumulate in the thrombus via an interaction that is dependent on platelet P-selectin [4 ]. To compare the rate of accumulation of microparticles into the developing thrombus with the kinetics of tissue factor accumulation in the developing thrombus, we examined microparticle accumulation into the thrombus over a 3-min period (Fig. 6 ). Microparticles were prepared from calcein-acetoxymethyl ester-labeled WEHI 274.1 cells. Immediately after the infusion of calcein-labeled microparticles into the mouse circulation, a thrombus was generated in an arteriole of a wild-type mouse. Fluorescently labeled microparticles were observed in the thrombus immediately after vessel wall injury. The integrated fluorescence intensity associated with these microparticles peaked within the first minute and then decreased over the next 3 min. This decrease relates to the rapid clearance of the proportionately small numbers of labeled microparticles infused into the circulation. This result indicates that microparticles accumulate rapidly on the developing thrombus and precede leukocyte–thrombus interaction by several minutes. The rapid accumulation of tissue factor in the developing thrombus closely correlates to microparticle accumulation and does not correlate temporally with leukocyte–thrombus interaction. These results indicate that tissue factor is delivered by microparticles during the initial phase of thrombus development.



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  		Figure 6. Microparticle accumulation in the developing arteriolar thrombus. Time course from time of injury to 3 min, demonstrating the accumulation of microparticles in arteriolar thrombi of wild-type mice. Calcein-labeled microparticles derived from WEHI cells were infused into wild-type mice just prior to thrombus formation. To prevent complications from the infusion of tissue factor associated with the infusion and monitoring of microparticles over a significant period of time, hirudin (2 U/g mouse) was infused prior to the microparticles. Images were captured as a function of time. The integrated fluorescence intensity colocalized with the thrombus was determined. A representative experiment from among four experiments is shown.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Tissue factor is an integral transmembrane protein with a molecular weight of ~43,000, which is located on the plasma membrane. A single polypeptide chain, the protein includes a 219-residue extracellular domain, a 23-residue transmembrane domain, and a 21-residue cytoplasmic tail [21 , 22 ]. This protein, a receptor for factor VII, is required for the initiation of blood coagulation. Although tissue factor is expressed constitutively in most nonvascular cells, monocytes and endothelial cells express tissue factor following cell stimulation [23 24 25 ]. Monocytes can be activated by endotoxin, immune complexes, certain cytokines, platelets, and P-selectin, leading to tissue factor expression, whereas endothelial cells express tissue factor when stimulated by certain cytokines [8 , 9 , 26 27 28 ]. De novo synthesis of tissue factor in monocytes can be measured after 2–4 h following induction of synthesis. However, neutrophils and macrophages in normal peripheral blood have also been reported to stain positively for tissue factor antigen [7 ]. In addition to this cellular pool within blood leukocytes, tissue factor is also found at low concentrations within the plasma fraction [1 2 3 ], although others have recently challenged the amount of tissue factor detected [6 ]. This pool of tissue factor is at least in part associated with leukocyte-derived microparticles [4 ].

We have previously demonstrated the presence of a P-selectin-dependent pathway of blood coagulation [10 ]. In an arteriovenous shunt model of thrombosis, fibrin deposition and leukocyte accumulation in the developing thrombus were inhibited using a blocking antibody to P-selectin. Circulating leukocytes expressing tissue factor antigen in normal blood suggests a potential role for these tissue factor-bearing leukocytes in thrombus formation [7 ]. Antibodies to P-selectin would inhibit leukocyte binding via PSGL-1 to the platelet thrombus expressing P-selectin, precluding tissue factor delivery to the thrombus. Tissue factor delivery from leukocytes might take two different forms. Leukocytes could interact directly with the platelet thrombus, rolling on, and then becoming firmly adherent to the thrombus. The presence of leukocytes within the architecture of the mature thrombus is consistent with this view. Alternatively, leukocyte rolling over the platelet thrombus might be associated with membrane transfer from the leukocyte to the platelet, with concomitant transfer of tissue factor. Such membrane tethers between leukocytes and platelets have been observed [29 ]. In this construct, leukocyte rolling might be adequate for tissue factor delivery in the absence of firm leukocyte adhesion.

Although the existence of blood-borne tissue factor has recently been challenged [6 ], we believe that most of the reports about this subject are consistent with our current model. In this construct, tissue factor in whole blood cannot be detected because of the limits of sensitivity of the assay, but it is present; the accumulation of blood-borne tissue factor into the thrombus involves a significant concentration of tissue factor of multiple orders of magnitude; and the concentrated tissue factor in the thrombus is within the levels of detection by fluorescence microscopy and also reaches the threshold to initiate blood coagulation.

Giesen et al. [7 ] showed the incorporation of tissue factor into the developing thrombus in an in vitro model of thrombosis. Tissue factor, distinct from cells, and tissue factor associated with circulating neutrophils and macrophages from normal blood are incorporated into the thrombus. We have shown in in vivo experiments that microparticles expressing tissue factor deliver tissue factor that accumulates into the developing thrombus in a mechanism that is P-selectin and PSGL-1-dependent [4 ]. To identify whether microparticles or leukocytes are the primary delivery vehicles for tissue factor, which accumulates into the initial phase of the developing thrombus and is distinct from the tissue factor contribution from the injured vessel wall, we have compared the kinetics of microparticle–thrombus interaction and leukocyte–thrombus interaction to the kinetics of tissue factor incorporation.

In these studies, we, by necessity, generated fluorescent, dye-labeled microparticles from an exogenous monocyte-like cell line and tracked these particles by fluorescence imaging following infusion into the mouse. The question remains open as to how similar these exogenous-prepared microparticles compare with natural microparticles in the circulating blood. Although we cannot be certain that ionophore-generated microparticles behave identically to natural microparticles, we have previously demonstrated that natural microparticles deliver tissue factor to the developing thrombus and lead to fibrin formation.

Tissue factor begins to appear in the developing thrombus of a wild-type mouse immediately following platelet accumulation and significantly before leukocyte rolling is observed. Similarly, microparticles rapidly accumulate in the thrombus. As microparticle–thrombus interaction and leukocyte–thrombus interaction are dependent on P-selectin–PSGL-1 binding, why do microparticles accumulate in the thrombus faster than leukocytes? These microparticles, whose size varies between 200 and 800 nm (unpublished data), may flow through the interstices of the developing thrombus and have access to P-selectin on activated platelets within internal aspects of the thrombus before P-selectin is exposed on the luminal thrombus surface. Given their small size, microparticles expressing PSGL-1 can interact with P-selectin expressed on activated platelets within the thrombus, thus leading to microparticle capture during the initial phase of thrombus formation—significantly before the P-selectin density on the luminal surface of the thrombus is sufficient to support leukocyte–thrombus interaction and leukocyte rolling at arteriolar shear rates. Although no data are available about the density of P-selectin necessary to support microparticle adherence, we have previously estimated the P-selectin density necessary to support leukocyte binding. In a static system, we estimated that ~100 molecules of P-selectin per µm2 are required to support HL60 cell binding [30 ]. Furthermore, in vitro, activated platelets contain 200–250 P-selectin molecules per µm2.

In sum, initial hematopoietic cell-derived tissue factor accumulation in the developing thrombus is generated from microparticles. Although leukocytes and microparticles circulate in the blood and bear tissue factor and PSGL-1, the time course of leukocyte–thrombus interaction is delayed because of the relative size of the leukocyte, precluding its flow through the thrombus and the time-dependent appearance of P-selectin on the luminal surface of the thrombus. For these reasons, microparticles play a functional role in the initial delivery of tissue factor to the developing thrombus. The contributions of microparticles and leukocytes to the delivery of tissue factor during later thrombus development are an independent question and remain unknown.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the National Institutes of Health. We are grateful to Dr. Nigel Mackman for providing low tissue factor mice.


   FOOTNOTES
 
1 Current address: St. Michael’s Hospital, University of Toronto, 30 Bond Street, Toronto Canada. Back

2 Current address: Children’s Hospital Medical Center, Boston, MA 02115. Back

Received April 13, 2005; revised July 26, 2005; accepted August 3, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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