Immunoneutralization of Glycoprotein Ibα Attenuates Endotoxin-Induced Interactions of Platelets and Leukocytes With Rat Venular Endothelium In Vivo
Abstract—This study aimed to examine molecular mechanisms for endotoxin-induced adhesive changes in platelets in vivo. Platelets labeled with carboxyfluorescein diacetate succinimidyl ester were visualized in rat mesenteric venules through intravital microscopy assisted by a high-speed fluorescence video imager at 1000 frames per second or by a normal-speed intensifier under monitoring of erythrocyte velocity. Leukocyte rolling was examined by normal-speed transmission video images. The velocity of platelets traveling along the centerline of venules followed that of erythrocytes, whereas that measured at the periendothelial space was significantly smaller than the erythrocyte velocity; a majority of these cells exhibited transient but notable rolling with endothelium. Administration of endotoxin increased the density of periendothelial platelets and reduced the rolling velocities of platelets and leukocytes in venules: All events were attenuated by anti–rat P-selectin monoclonal antibody s789G or by anti–human glycoprotein (GP) Ibα monoclonal antibody GUR83/35, which blocks ristocetin-induced aggregation of rat platelets. Isolated rat platelets injected into endotoxin-pretreated rats were able to roll on the venules. This event was attenuated by pretreatment of platelets in vitro with GUR83/35 but not with s789G, suggesting involvement of endothelial P-selectin and platelet GP Ibα in the endotoxin-induced responses. Furthermore, isolated human platelets showed similar rolling interactions with endotoxin-preexposed rat venules, and pretreatment of the platelets with GUR83/35, but not with s789G, significantly reduced such interactions. Our results provide the first evidence for involvement of GP Ibα in endotoxin-induced microvascular rolling of platelets and leukocytes, and this system serves as a potentially useful tool to examine GP Ibα–associated function of human platelets in vivo.
Under physiological conditions, platelets are thought to circulate in close contact with microvascular endothelial cells without adhering to their surface. The cells have been thought to neither adhere to the site of injury nor undergo activation until these cells are exposed to platelet-activating subendothelial substances such as collagen.1 On the initial adhesive process, platelets can release varied mediators and activate other platelets.2 Once the first layer of adhered platelets forms the stable thrombogenic surface, growth of the hemostatic plug occurs dependently on the mediator-elicited platelet-to-platelet interactions.2 These 2 events represent adhesion and aggregation of platelets, respectively.
Detailed knowledge of the density or velocity distribution of platelets flowing along the periendothelial space in vivo could help us understand mechanisms for the initial process of thrombogenesis and subsequent inflammatory responses involving leukocyte adhesion for several aspects: First, determination of the density of platelets in the local area allows us to estimate the rate of local platelet delivery. Second, analyses of velocity distribution of the periendothelial platelets make it possible to estimate alterations in adhesive force between platelets and endothelial cells under disease conditions and thus provide important information on changes in their adhesivity under flow conditions. Third, considering the presence of varied adhesion molecules on platelets that might bind to ligand molecules on the surface of leukocytes, the density of platelets in the periendothelial space could determine venular leukocyte recruitment in acute inflammatory processes.3 4 However, little knowledge of the behavior of individual platelets circulating close to the periendothelial space has been available because of technical difficulties in visualizing these cells in a reliable manner. We applied intravital ultrahigh-speed intensified microscopy assisted by carboxyfluorescein diacetate succinimidyl ester, a fluorochrome that can stain platelets intravitally. The present results provide evidence that under disease conditions such as endotoxemia, circulating platelets display the periendothelial rolling in postcapillary venules through mechanisms involving endothelial P-selectin and glycoprotein (GP) Ibα on platelets and help leukocyte rolling and adhesion in vivo.
Materials and Methods
Our study protocol was approved by the Animal Care and Utilization Committee of Keio University School of Medicine. Male Wistar rats were anesthetized for intravital microscopy of mesenteric microvessels in vivo5 6 7 8 and treated with an intravenous injection of carboxyfluorescein diacetate succinimidyl ester, a nonfluorescent precursor taken up into platelets and partly into leukocytes, forming a stable fluorochrome, carboxyfluorescein succinimidyl ester (CFSE), in the cells.5 6 CFSE-labeled platelets were visualized through multifunctional intravital fluorescence microscopy as described previously.5 7 Advantages of the present approach compared with previous methods for platelet visualization are described in the online-only Materials and Methods (see http://www.circresaha.org). Six main protocols were used: Rats in group 1 were intravenously perfused with physiological saline as a vehicle for 30 minutes. In group 2, rats were injected with lipopolysaccharide (LPS, O111B4; 1.0 mg · kg−1 · h−1 IV for 30 minutes) according to the previous experimental model of endotoxemia.9 Animals in groups 3 and 4 were pretreated with new monoclonal antibodies (mAbs) against rat P-selectin s789G and s84F. Rats in groups 5 and 6 were pretreated with anti–human GP Ibα mAbs, GUR83/35 and WGA3. GUR83/35 is reported to recognize a conformation-specific epitope between residues 1 and 302 of GP Ibα and to block the binding of von Willebrand factor to human platelets in the presence of ristocetin, whereas WGA3 binds to the same region but does not block the ristocetin-induced aggregation of platelets.10 11 These mAbs were injected at 1.5 mg/kg IV 5 minutes before the start of the LPS infusion. Individual erythrocytes and platelets were visualized through an ultrahigh-speed intensified video microscope. This system allowed us to visualize platelets and erythrocytes at 1000 frames per second for 1 second under epi-illumination and transillumination, respectively. Velocities of individual platelets were normalized by dividing the values by the regional erythrocyte velocity in the centerline or the periendothelial region, with the result designated as the relative platelet velocity (VP/VR) in each region. This value serves as a semiquantitative index of the adhesion energy between platelets and endothelial cells. We examined differences in circulating platelet counts and the density of platelets between the periendothelial and centerline regions of microvessels as shown in previous studies using acridine red–assisted intravital microscopy.12 The normalized densities of CFSE-positive platelets in the centerline (DPC) and periendothelial (DPE) regions were defined as described in the online-only Materials and Methods (see http://www.circresaha.org). The relative rolling velocity of leukocytes (VW/VR) and the density of adherent cells were determined separately with normal-speed transmission video images as described elsewhere.5 7 In other experiments, isolated rat and human platelets were injected intravenously into rats undergoing 30-minute exposure to the LPS infusion, and their behavior in venules was examined with a silicon-intensified target (SIT) camera.8 Function of isolated platelets was examined with a laser-scattering platelet aggregometer. Anti–P-selectin mAbs were generated, and their ability to block P-selectin–mediated cell adhesion in vitro was characterized. Differences among groups were examined by 1-way ANOVA with Fisher’s multiple comparison test.
An expanded Materials and Methods section is available online at http://www.circresaha.org.
Individual Platelets Labeled With CFSE
Figure 1⇓ illustrates the effects of in vivo fluorescence labeling with CFSE on platelet aggregation. As seen in Figures 1A⇓ and 1B⇓, the distribution of rat platelets on a culture dish corresponded to that of the cells tagged with fluorescence, whereas not all platelets were able to be visualized under the same optical conditions as used for intravital microscopy. As depicted in Figure 1C⇓, platelets labeled or unlabeled with CFSE were equally activated and displayed similar extents of microaggregate formation in response to ristocetin and ADP. Peak signal intensities indicating small and large aggregates and peak percentage values of light transmission (%T) elicited by ADP were not statistically different between the groups (Table 1⇓). Furthermore, time to observe these peak values did not differ significantly. Circulating counts of platelets were not different between the animals treated or untreated with CFSE (data not shown). Collectively, the present method for labeling of platelets allowed us to observe individual platelets through fluorescence microscopy without notable alterations in platelet functions in vitro.
Millisecond Interactions of Platelets With Microvascular Endothelium
As shown in Figure 2A⇓, videorecording at 1000 frames per second under transillumination allowed us to visualize individual erythrocytes packed in microvessels and recognized as granular patterns of images. Regional velocities of single erythrocytes in venules were analyzed by tracing movement of each cell per unit frame with a frame-by-frame analysis, as shown in Figure 3⇓. At this recording rate, CFSE-labeled platelets can be visualized, but their exact localization inside the vessels could hardly be identified because of their rapidity of movement, as seen in Figure 2B⇓. Conversely, their frame-by-frame reproduction by replaying at 30 frames per second allowed us to visualize individual platelets as pinpoint dots and to trace movement of these cells along vessel walls. As seen in serial fluorographs with 20- or 40-ms intervals in Figures 2C⇓ through 2H, a single platelet moving in the periendothelial space of the venule exhibited quite heterogeneous changes in its velocity. The distance of movement per unit time in 1 cell (arrows) differed greatly from that in another cell (asterisks), indicating a heterogeneity in velocities among different platelets.
Spatial and Temporal Alterations in Erythrocyte Velocity in Microvessels
On the basis of the frame-by-frame analysis of transillumination images, we examined temporal and spatial profiles of the erythrocyte velocity in postcapillary venules. As seen in Figure 3A⇑, showing the velocity of single erythrocytes (z axis) as a function of time (x axis) and position (y axis) of measurements, the velocity measured in the venular centerline region did not fluctuate greatly. The velocity of erythrocytes flowing in venules was greater in the centerline region than in the periendothelial region. This observation led us to hypothesize that the heterogeneity of movement of platelets in the periendothelial region illustrated in Figure 2⇑ could result from transient adhesive interactions of the cells with the endothelial surface rather than from local changes in erythrocyte velocities. To test this hypothesis, we examined alterations in the relative velocity of individual platelets versus erythrocyte velocity (VP/VR) in venules of the control rats. As shown in 3B, the VP/VR values of a single platelet flowing at the centerline of venules was almost constant, at ≈100%, in venules. Conversely, the velocity of platelets flowing in the periendothelial regions exhibited a fluctuating pattern: The VP/VR values of these platelets occasionally became as low as 50% of the local erythrocyte velocity, suggesting the presence of adhesive interactions between the periendothelial platelets and microvascular endothelium.
Such a baseline level of the platelet-endothelium interactions was enhanced by treatment of rats with LPS. Figure 3C⇑ illustrated histograms showing distribution of the VP/VR values in the periendothelial space of venules. In the controls, the periendothelial platelet velocity varied greatly among different cells, whereas the mean VP value was ≈92% of the mean VR value, and ≈50% of the cells exhibited a velocity <90% of the regional erythrocyte velocity. At 30 minutes after the start of the LPS administration, venular wall shear rates did not change significantly, but the VP/VR histogram exhibited a marked shift to the left side. Under the present experimental conditions, venular wall shear rates in the LPS-untreated and -treated rats were 486±39 s−1 and 476±52 s−1 (mean±SD of 7 experiments), respectively, showing no statistical difference. These results suggest that LPS increases the adhesion energy between periendothelial platelets and venular endothelium in vivo.
P-Selectin–Mediated Microvascular Rolling of Platelets in LPS-Treated Rats
We determined the effects of mAbs against P-selectin and GP Ibα and then applied them to the in vivo systems. Figure 4A⇓ illustrates the effects of the anti–rat P-selectin mAbs on the intercellular adhesion between the P-selectin–expressing transfectant and HL-60 cells. The mAb s789G, but not s84F, dose-dependently attenuated the cell adhesion, indicating that the former mAb serves as a reagent to block leukocyte adhesion. Figure 4B⇓ depicted effects of anti–human GP Ibα mAbs on ristocetin-induced aggregation of rat platelets. As seen, GUR83/35, but not WGA3, significantly suppressed ristocetin-induced responses of rat cells, indicating that the former mAb can be used to block the rat GP Ibα–mediated events.
Figure 4C⇑ shows LPS-induced alterations in circulating platelet counts and density (DPE/DPC) and velocity (VP/VR) of periendothelial platelets in venules and depicts the effects of the aforementioned mAbs against P-selectin or GP Ibα on these parameters. Administration of LPS significantly reduced the number of platelets in peripheral blood samples. Conversely, the DPE/DPC values exhibited an ≈20% elevation on the administration of LPS. The LPS-induced elevation of the periendothelial platelet density coincided with a marked reduction of the mean VP/VR values, indicating an increasing adhesivity between the platelets and endothelial cells. Collectively, the LPS-induced thrombocytopenic changes appeared to occur concurrently with margination of circulating platelets to the periendothelial space. Pretreatment with s789G but not with s84F significantly attenuated the LPS-induced changes in these platelet parameters, suggesting that P-selectin mediates the LPS-induced microvascular margination of platelets. Most importantly, pretreatment with the mAb GUR83/35 but not with WGA3 elicited similar inhibitory effects on the LPS-induced changes, suggesting that functional blockade of GP Ibα on circulating platelets cancels platelet-endothelium interactions in LPS-exposed venules. Conversely, the administration of 1 of the 2 mAbs to the LPS-untreated rats did not evoke alterations in these parameters (data not shown).
Another important event observed during the administration of LPS was a reduction of the rolling velocity of leukocytes in venules. As seen in Table 2⇓, the administration of LPS induced an ≈60% reduction of the VW/VR. Pretreatment with s789G abolished the LPS-elicited alterations, suggesting involvement of P-selectin in leukocyte rolling in the stimulated venules, which was in good agreement with previous studies.3 8 The LPS-elicited reduction of the rolling velocity was also attenuated significantly by pretreatment with GUR83/35, suggesting that functional blockade of GP Ibα attenuates the LPS-induced elevation of adhesion energy. The LPS infusion also elicited a marked increase in the density of adherent leukocytes that was attenuated by pretreatment with s789G. The blockade of GP Ibα by GUR83/35 also attenuated the LPS-induced venular leukocyte adhesion significantly, although its effect was smaller than that of s789G. Collectively, these results suggest that the platelet adhesion molecule GP Ibα is a determinant involved in mechanisms for the LPS-induced microvascular leukocyte recruitment.
Role of GP Ibα in Rolling of Rat and Human Platelets in LPS-Pretreated Venules
Observations shown in Figure 4⇑ led us to further examine the roles of P-selectin and GP Ibα in the LPS-induced interactions between platelets and venular endothelium. To that effect, differences in adhesivity of isolated rat platelets pretreated or not treated with mAbs against these adhesion molecules were examined in LPS-pretreated rats under observation with an SIT camera. Once injected, the platelets displayed a transient adhesion and rolling, and some cells exhibited stationary adhesion to the venular endothelium (Figure 5⇓). A subpopulation of the platelets flowing at the periendothelial space often attached to rolling leukocytes and made them slow down further. Formation of leukocyte-platelet complex was seen occasionally. As summarized in Figure 5⇓, right, CFSE-labeled platelet rolling on venules was enhanced by pretreatment with LPS. When the isolated platelets were pretreated with the mAb s789G, their adhesive responses were not greatly changed. By contrast, pretreatment of the isolated platelets with GUR83/35 almost abolished their adhesion to the LPS-preexposed venules. These results showed that blockade of constitutively expressed GP Ibα on platelets is involved in mechanisms through which this mAb administered in vivo attenuated the LPS-induced platelet interactions with the endothelium.
This observation tempted us to examine whether human platelets could utilize GP Ibα–mediated mechanisms to adhere to rat venular endothelium in vivo. As seen in Figure 6A⇓, the platelets administered into the LPS-treated rats exhibited rolling with greater frequency than those administered into the untreated controls over the whole ranges of the velocities. The population of the slow rollers with velocities <0.2 mm/s became markedly elevated in the LPS-treated group. As shown in Figure 6B⇓, in vitro pretreatment of human platelets with the mAb GUR83/35, but not with WGA3, significantly attenuated such adhesive changes observed in vivo. Thus, the present findings demonstrated that human platelets exogenously administered into the rat vascular system are able to roll on microvascular endothelium through GP Ibα–mediated mechanisms.
High-speed microfluorography allowed us to show that platelets not only can traverse through the centerline region of microvessels in a free-flowing manner but also can move along the periendothelial region and display notable interactions with the endothelial surface at millisecond intervals even under unstimulated, ordinary conditions. Such interactions of platelets are distinct from the slow rolling behavior observed only in a subpopulation of platelets that has been detected by normal-speed videomicroscopy.13 14 Although this varied among individual cells, a majority of periendothelial platelets displayed rapid but detectable attachment to the endothelium, and some of them exhibited a velocity <50% of the erythrocyte velocity measured in situ. Such interactions of platelets were markedly enhanced by LPS treatment. The present data indicate that unstimulated platelets are able to roll on the LPS-preexposed venules and suggest a crucial role of P-selectin expressed at the side of the stimulated endothelium, as also shown in previous reports using different experimental models.14 15 Moreover, this adhesion molecule appears to be involved in the LPS-induced shift of platelet distribution toward the periendothelial space. However, we were unable to address molecular determinants for the baseline adhesive interactions of platelets with the unstimulated venules: Inasmuch as the s789G pretreatment did not alter the baseline platelet interactions in vivo, the role of P-selectin in vivo appeared to be small or none.
Most importantly, the present study provided a novel insight into mechanisms for platelet adhesion mediated by GP Ibα in vivo. Two parameters were significantly altered by LPS through mechanisms involving GP Ibα: increases in the adhesion energy of platelets to venular endothelium and the density of the cells traveling along the periendothelial space. Because 1 of the mAbs s789G and GUR83/35 at the present dose sufficiently attenuated the LPS-induced changes in these platelet parameters, the interaction between P-selectin expressed on the LPS-stimulated venular endothelium and GP Ibα on platelets appears to play a role in the adhesive responses of platelets. This notion is fully supported by recent studies in vitro showing that GP Ibα serves as a platelet counterreceptor for P-selectin.16
Another important event observed under GP Ibα immunoneutralization is downregulation of LPS-induced venular leukocyte rolling and adhesion: Several different patterns of platelet-leukocyte interactions were observed in the LPS-treated microvessels. First, as also shown elsewhere,3 17 circulating platelets adhere to the surface of leukocytes and thereby help them slow down to roll and adhere. Second, platelets moving along the periendothelial space may by chance collide with leukocytes rolling directly along venular endothelium and help them slow down further for stationary adhesion. The GP Ibα–mediated elevation of the periendothelial platelet density and increase in the adhesion energy to the LPS-exposed venules could enhance both of these mechanisms for intercellular interactions and secondarily activate leukocyte adhesivity. Detailed molecular mechanisms for the GP Ibα–mediated direct interactions between platelets and leukocytes in vivo (eg, L-selectin) and involvement of plasma factors reacting with GP Ib (eg, von Willebrand factor) should be examined further.18 19
Finally, our study revealed that the rat system serves as a potentially useful tool to examine GP Ibα–associated function of human platelets in vivo, in that they can utilize GP Ibα to roll in a manner similar to that of rat platelets on the stimulated microvascular endothelial cells. GP Ib has been known to play important roles in a variety of functional alterations of platelets in vitro, such as shear-dependent aggregation19 and adhesion to immobilized von Willebrand factor under flow conditions in vitro.20 In humans, conversely, there are genetic disorders of this adhesion molecule, such as Bernard-Soulier syndrome21 or cases of heightened platelet aggregation by ristocetin.10 22 However, a pathophysiological link of such in vitro events to actual behavior of functionally impaired platelets in vivo is largely unknown because of lack of the intravital system to examine human platelet functions. Determination of impaired adhesive properties of human platelets collected from disease patients deserves further studies to understand the whole mechanisms for platelet-dependent hemostatic and thrombotic disorders from microvascular viewpoints, and such studies are under way in our laboratories.
This work was supported by Advanced Medical Technology in Health Sciences Research Grants from Ministry of Health and Welfare and in part by Keio Medical Science Fund.
- Received January 31, 2000.
- Accepted March 29, 2000.
- © 2000 American Heart Association, Inc.
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