J.-F. Dong, Thrombosis Research Section, Department of Medicine, BCM286, N1319, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. Tel.: 713 798 1078; fax: 713 798 1384; e-mail: email@example.com
Summary. Leukocyte rolling on vascular endothelium is mediated by an interaction between P-selectin expressed on endothelial cells and P-selectin glycoprotein ligand-1 on leukocytes. This interaction reduces the velocity of leukocyte movements to allow subsequent firm adhesion and transmigration. However, the interaction has so far been observed only under low venous shear stress and cannot explain the accumulation of monocytes in atherosclerotic plaques found in arteries, where shear stress is much higher. We have previously shown that newly released ultra-large von Willebrand factor (ULVWF) forms extremely long string-like structures to which platelets tether. Here, we investigated whether platelets adhered to ULVWF strings are activated and form aggregates. We also determined whether activated platelets on ULVWF strings can support leukocyte tethering and rolling under high shear stresses. We found that platelets adhered to ULVWF expressed P-selectin and bound PAC-1, suggesting their rapid activation. We also found that leukocytes tethered to and rolled on these platelet-decorated ULVWF strings, but not directly on endothelial cells, under high shear stresses of 20 and 40 dyn/cm2 in a P-selectin dependent manner. These results suggest that the endothelial cell-bound ULVWF provide an ideal matrix to aggregate platelets and recruit leukocytes to endothelial cells under high shear stress. The observed phenomenon delineates a mechanism for leukocytes to be tethered to arterial endothelial cells under high shear, providing a potential link between inflammation and thrombosis.
Inflammation has been increasingly recognized as a precondition for atherosclerosis and thrombosis, but mechanisms linking it to the thrombotic events remain largely unknown [1,2]. One hallmark event of vascular inflammation is leukocyte adhesion and transmigration [3–5], where leukocytes circulating in blood are tethered to, rolled on, and transmigrate through endothelial cells. This series of events is initiated by a ligand–receptor interaction between P-selectin expressed on the surface of endothelial cells and P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes [6,7]. Leukocyte rolling on vascular endothelial cells reduces the velocity of cell movements to allow other ligand–receptor interactions between leukocytes and endothelial cells, such as those of integrins, to firmly arrest leukocytes . Interestingly, leukocyte tethering and rolling have so far largely been observed in the venous environment, where blood shear stress is significantly lower than that in arterial vessels [8–10], the primary locations for atherosclerosis. This preferred location is due probably to weaker bonds formed between selectins and their ligands, compared to a GP Ib-VWF bond that mediates platelet tethering and rolling on von Willebrand factor (VWF) under high shear stress . However, this relatively weak bond can be enhanced by an increase in the density of either selectins or their ligands [12–15]. Upon activation, platelets express P-selectin in a density that is significantly higher than that on endothelial cells [6,16–18], to provide an ideal matrix for leukocytes recruitments under high arterial shear stresses . Previous studies have shown that neutrophils tether to and roll on adherent activated platelets in a P-selectin dependent manner [16,20]. The obvious question is what mediates platelet adhesion to endothelial cells under high shear stress?
We have recently demonstrated that, in the absence of the VWF-cleaving metalloprotease ADAMTS-13 (A Disintegrin and Metalloprotease with ThromboSpondin motif) [21–23], the ultra-large form (UL) of VWF newly released from endothelial cells forms extremely long string-like structures. These ULVWF strings can be induced not only by histamine, but also by inflammatory cytokines such as interleukin (IL)-8 and tumor necrosis factor (TNF)-α. We have further shown that platelets tethered and adhered to these ULVWF strings through the interaction of GP Ib , consistent with previous findings that ULVWF is functionally hyper-reactive, capable of spontaneously binding its platelet receptor, the GP Ib-IX-V complex, to aggregate platelets [25–27]. These ULVWF strings are normally cleaved by ADAMTS-13 [24,28,29]. It is therefore not surprising that deficiency of this metalloprotease results in the systemic thrombotic microangiopathy called thrombotic thrombocytopenic purpura [30,31]. We hypothesize that platelets adhered to ULVWF strings on endothelial cells can also provide an ideal matrix for leukocyte tethering under high arterial shear stress because they express a higher density of P-selectin [16–18].
To test this hypothesis, we conducted experiments to specifically answer two questions. First, are platelets that adhere to ULVWF strings activated and express P-selectin? Secondly, if they do, will these ULVWF-platelet strings support leukocyte tethering and rolling under high shear stress? The results of these studies could provide a potential mechanism to allow that leukocyte–endothelial cell interaction to occur under high shear stress and serve as a link between inflammation and atherosclerosis.
Platelet and plasma preparations
Washed platelets were obtained from freshly drawn blood obtained strictly under a protocol approved by the Institutional Review Board of the Baylor College of Medicine for using human subjects in biomedical research. All donors signed consent forms before blood was drawn. The donor pool consisted of 28 healthy donors; among them are 18 females and 10 males with age 22–45 years.
To obtain washed platelets, whole blood drawn into 10% acid–citrate dextrose buffer (85 mmol L−1 sodium citrate, 111 mmol L−1 glucose and 71 mmol L−1 citric acid) was centrifuged at 150 g for 15 min at 24 °C to obtain platelet-rich plasma (PRP), which was then centrifuged at 900 g for 10 min to separate platelets from plasma. Platelet pellets were washed once with a CGS buffer (13 mmol L−1 sodium citrate, 30 mmol L−1 glucose and 120 mmol L−1 sodium chloride, pH 7.0) and then resuspended in Ca2+ and Mg2+-free Tyrode's buffer (138 mmol L−1 sodium chloride, 5.5 mmol L−1 glucose, 12 mmol L−1 sodium bicarbonate, 2.9 mmol L−1 potassium chloride and 0.36 mmol L−1 dibasic sodium phosphate, pH 7.4).
For experiments using reconstituted blood (to remove plasma, the source of ADAMTS-13), citrated whole blood was centrifuged at 150 g for 15 min at 24 °C to remove PRP. Erythrocytes and leukocytes were washed twice with phosphate buffered saline (PBS) to remove residual plasma to deplete ADAMTS-13. Blood was then reconstituted by mixing erythrocytes, leukocytes, washed platelets and Tyrode's buffer to restore original hematocrit and platelet counts.
Preparation of peripheral leukocytes
To prepare washed leukocytes, 30 mL of citrate blood were laid on the top of the Ficoll plaque (15 mL) and centrifuged for 30 min at 1500 g at 25 °C. Leukocytes were then collected between PRP and erythrocytes. They were washed twice with and then resuspended in phosphate buffered saline (PBS) for flow cytometry cell sorting.
To further determine the types of white blood cells that tethered and rolled on ULVWF strings, we perfused purified monocytes and neutrophils over preformed ULVWF strings.
These cells were isolated from total white blood cells by fluorescent-activated cell sorting using FITC conjugated monoclonal CD14 antibody (for monocytes ) and PE-conjugated monoclonal CD16 antibody (for neutrophils and natural killer cells ).
Endothelial cells were obtained from human umbilical veins (HUVECs) under a protocol approved by the Institutional Review Board of the Baylor College of Medicine for using human subjects . The umbilical cords were first washed with phosphate buffer (140 mmol L−1 NaCl, 0.4 mmol L−1 KCl, 1.3 mmol L−1 NaH2PO4, 1.0 mmol L−1 Na2HPO4, 0.2% glucose, pH 7.4), and then infused with a collagenase solution (0.02%, Invitrogen Life Technologies, Carlsbad, CA, USA). After 30 min incubation at room temperature, the cords were rinsed with 100 mL of the phosphate buffer. Elutes containing endothelial cells were centrifuged at 250 g for 10 min. The cell pellets were resuspended in medium 199 (Invitrogen Life Technologies) containing 20% heat-inactivated fetal calf serum and 0.2 mmol L−1 of l-glutamine, and plated on a culture dish coated with 1% gelatin until confluent.
To induce the release of ULVWF, endothelial cells were stimulated with 25 µmol L−1 histamine (Sigma-Aldrich, St Louis, MO, USA) for 10 min at room temperature immediately before the perfusion experiments.
Preparation of polystyrene beads coated with antibodies
Polystyrene beads with green fluorescence (0.5 µmol L−1 in diameter, fluoresbrite YG microspheres, Polysciences, Inc., Warrington, PA, USA) were coated with antibody against either P-selectin (BD Biosciences, San Diego, CA, USA) or integrin αIIbβ3 (PAC-1, BD Biosciences) according to the manufacturer's instructions. Briefly, beads were incubated with 200 µL of purified antibody (100 µg mL−1) overnight at room temperature with gentle shaking. Coated beads were washed twice with 0.5 mL borate buffer (pH 8.5) and then incubated with 1% bovine serum albumin (BSA) for 30 min at room temperature with constant shaking. After blocking, the beads were washed with borate buffer twice and resuspended in PBS buffer containing 1% BSA. Beads with isotype controls were coated with mouse IgG (5 µg mL−1 coating density, Zymed, South San Francisco, CA, USA).
Parallel-plate flow chamber
We induced the formation of ULVWF strings on endothelial cells through a previously described method in a parallel-plate flow chamber . Briefly, HUVECs growing in a 35-mm cell culture dish were stimulated with histamine and then assembled to form the bottom of the parallel-plate flow chamber (Glycotech, Rockville, MD, USA). The assembled chamber was connected to a syringe pump so that Tyrode's buffer containing washed platelets with or without leukocytes were drawn through the chamber at defined flow rates to generate specific wall shear stresses. Leukocytes were also perfused in the absence of platelets to determine whether they could be directly tethered to ULVWF. The flow chamber was mounted onto an inverted-stage microscope (Nikon, Eclipse TE300, Garden City, NY, USA) equipped with a high-speed digital camera (Photometrics, Model Quantix, Tucson, AR, USA) and kept at 37 °C with a thermostatic air bath during the experiments. The formation of VWF strings was recorded after 2 min perfusion of platelet suspension and acquired images were analyzed offline using MetaMorph software (Universal Images, West Chester, PA, USA).
To determine direct leukocyte rolling on endothelial cells, citrate whole blood was perfused over stimulated HUVECs for 40 s at 2.5 dyn/cm2 and then switched to a high shear stress of 40 dyn/cm2 for an additional 40 s. As whole blood contained ADAMTS-13, there were no ULVWF strings formed and leukocytes rolled directly on endothelial cells. Leukocytes in whole blood were labeled with the fluorescent dye mepacrine (final concentration 250 µg mL−1) in order to be visualized.
To calculate rolling velocity , images of cell rolling were first acquired at one frame s−1. When these images were overlaid, the paths traveled by individual cells were illustrated and the distance of two positions of any given cell in two adjacent frames measured. This distance indicated the cell rolling velocity because these images were acquired at a time interval of 1 frame s−1. Cell rolling was defined for these experiments as cells moving continuously in the direction of flow while maintaining contact with ULVWF strings for at least 4 s.
All experimental data are presented as mean ± SEM. The unpaired two-tailed Student's t-test was used for data analysis and a P-value less than 0.05 was considered to be statistically significant.
Platelets adhered to VWF strings are activated
Previous studies have demonstrated that platelets and CHO cells expressing the GP Ib-IX-V complex roll on immobilized VWF [11,34–36]. To determine platelet movements on these ULVWF strings, we monitored platelet adhesion to and their movements on ULVWF strings over a period of 10 min. We found that platelets tethered and adhered to the ULVWF strings instantaneously without any rolling movement under both low and high shear stresses (Fig. 1).
To determine whether platelets that adhered to VWF strings were activated, we first perfused washed platelets over histamine-stimulated HUVECs to allow platelets to adhere to ULVWF strings and then polystyrene beads coated with either PAC-1, which binds specifically to the activated platelet integrin αIIbβ3, or an anti-P-selectin antibody under both low (2.5 dyn/cm2) and high (20 and 40 dyn/cm2) shear stresses. We found that both anti-P-selectin and PAC-1 beads adhered to platelets on ULVWF strings, whereas beads coated with mouse IgG did not (Fig. 2).
Platelets aggregated on VWF strings
In the previous experiments, ULVWF-platelet strings were induced by perfusing platelet suspended in calcium and magnesium-free Tyrode's buffer in the absence of fibrinogen. To further determine whether platelets were able to aggregate on the VWF strings, we perfused washed platelets in the presence of human purified fibrinogen and calcium, and monitored the course of platelet aggregation on ULVWF strings. We found that platelets aggregated in the presence, but not the absence, of fibrinogen within 3–5 min after perfusion started (Fig. 3a,b). In comparison, perfusion of washed platelets in the presence of VWF purified from plasma showed minimal aggregation over the same period (Fig. 3c). The platelet aggregates grew in size and changed positions on endothelial cells constantly under flow (Fig. 3d,e). When grown sufficiently large, these platelet aggregates were eventually released from the surface of endothelial cells by the shear force directly into the buffer flow (data not shown).
Furthermore, we also perfused platelets that were preactivated by 10 µmol L−1 of ADP. In this case, preformed platelet aggregates were able to tether to and continued to grow on ULVWF strings [Fig. 4]. These aggregates were again released from the surface of endothelial cells by fluid shear stress (Fig. S1).
Leukocyte rolling on VWF-platelet strings
One of the pathological consequences of the VWF string formation may be to provide a matrix to capture leukocytes. Previous studies suggest that P-selectin mediated neutrophil tethering and rolling on stimulated endothelial cells occur only in a low shear environment . We hypothesize that activated platelets adhered to ULVWF strings may provide a suitable matrix for leukocyte tethering and rolling under much higher arterial shear stress because platelets express significantly more P-selectin (and at higher density) compared to endothelial cells. To test this hypothesis, we perfused reconstituted blood that lacked ADAMTS-13 (using buffer to replace plasma, the source of metalloprotease) over the stimulated endothelial cells under high shear stresses of 20 and 40 dyn/cm2. We found that leukocytes tethered to and rolled on platelets adhered to ULVWF strings under both shear stresses (Fig. 5 and Fig. S2). To further demonstrate the role of platelets on leukocyte rolling on ULVWF-platelet strings, we also perfused purified leukocyte over stimulated endothelial cells in the absence of platelets under 40 dyn cm2 shear stress and found no leukocyte tethering and rolling (Fig. 5d). When whole blood was perfused over activated endothelial cells, no ULVWF strings were formed because of ADAMTS-13 activity in plasma. Under this condition, leukocytes were tethered to and rolled on endothelial cells only under a shear stress of 2.5 dyn cm2(Fig. 6a), but very few leukocyte tethering and no rolling at a high shear stress of 40 dyn cm2 (Fig. 6b, Fig. S3). The rolling cells included both monocytes and neutrophils (data not shown).
We have also measured velocities of leukocytes rolling under different shear stresses. As shown in Fig. 7, rolling velocity of leukocytes on ULVWF-platelet strings under a shear stress of 40 dyn cm2 was 61.23 ± 26.9 µm s−1, similar to that on a monolayer of activated platelets under same shear stress (62.47 ± 22.3 µm s−1), which was created by perfusing whole blood preincubated with 20 µg mL−1 of ReoPro over immobilized collagen. It was significantly slower than that under a low shear of 2.5 dyn/cm2 on the surface of endothelial cells (84.26 ± 19.7 µm s−1, Student's t-test, n = 47, P < 0.01).
In the current study, we showed that platelets are captured and firmly adhered to ULVWF strings under flow conditions. This finding is clearly different from previous observations that plasma VWF, once immobilized onto a solid surface, supports rolling, but not firm adhesion, of platelets and CHO cells expressing the GP Ib-IX-V complex [11,34–36]. The firm adhesion is due probably to the high-strength bond formed between ULVWF and GP Ibα, as we have demonstrated previously . The extraordinarily high bond strength between ULVWF and platelets, as well as the strength of ULVWF strings themselves, can be further demonstrated by the finding that preformed platelet aggregates (by treating platelets with ADP) can be captured onto these ULVWF strings under high arterial shear stress. Our results are different from those of Andre et al. , showing that platelets transiently tether to endothelial cells of mouse mesenteric venules stimulated with calcium ionophor. The difference may be due to ADAMTS-13 because we perfused washed platelets over endothelial cells in the absence of plasma, the source of ADAMTS-13. In comparison, ULVWF released and anchored to endothelial cells is likely to be rapidly cleaved and released by plasma ADAMTS-13 in the ex vivo experiments described in the mouse study using intravital microscopy. Having demonstrated the formation of the platelet-decorated strings on endothelial cells, we have further investigated the potential thrombotic consequences of these ULVWF-platelet strings.
We first determined whether the adhered platelets were activated by measuring the expression of P-selectin on platelet surface and by binding PAC-1, which interacts only with the activated form of platelet integrin αIIbβ3, to these platelets. Both PAC-1 and P-selectin antibodies specifically bind to the ULVWF-bound platelets, but not to resting platelets suspended in buffer, suggesting that these platelets are indeed activated upon adhesion through a GP Ib–VWF interaction. Consistent with this notion, we found that platelets also aggregate on ULVWF strings in the presence of fibrinogen. The aggregates grow during the course of perfusion until they become sufficiently large and can no longer be held onto the ULVWF strings against the pulling force generated by a buffer flow. They are then released from endothelial cells into the buffer flow. This observation has a clear clinical implication of microvascular thrombosis often seen in patients with TTP. Upon release from endothelial cells, these thrombi can travel downstream and stop in microvasculatures where they can no longer pass through because of their sizes. It is unclear how these platelet aggregates are released from the surface of endothelial cells: by detaching from the ULVWF strings or by breaking them. We did not attempt to determine whether these platelet aggregates contain endothelial cell-derived ULVWF because of its extremely small amount. Furthermore, because platelets also contain ULVWF, a traditional immunoblotting assay may not be able to distinguish the source of ULVWF detected in these platelet clumps.
Because of P-selectin expression, the platelets on ULVWF strings may provide a matrix to capture leukocytes from bloodstream under high shear stress found in arterial environment. Leukocytes have previously been demonstrated to tether and roll on activated endothelial cells through the P-selectin–PSGL-1 interaction. However, cell rolling occurs primarily in the low shear venous environment, due to the weak strength of P-selectin–PSGL-1 bonds . This venous location is inconsistent with the predominant arterial location of atherosclerosis, the hallmark of which is the accumulation of macrophages filled with lipid droplets (foam cells) [39,40], suggesting that leukocytes can be captured to the arterial endothelial cells under significantly higher fluid shear stress. The question is how leukocytes are captured under such high shear stresses. Platelets may play a key role in this process because they express significantly higher numbers of P-selectin on their surface [6,17–19]. Consistent with this hypothesis, platelet adhesion to endothelial cells are found in disease processes such as inflammation and atherosclerosis [16,41] and endothelium-bound platelets have been found to capture lymphocytes to high endothelial venules through P-selectin-peripheral node addressin . Here, we show that platelets adhered to ULVWF strings on the surface of endothelial cells are rapidly activated and the activated platelets support leukocyte rolling on ULVWF strings under high shear stresses of 20 and 40 dyn/cm2, which are commonly found in arteries and arterioles. The observation is consistent with a previous study showing that monocyte infiltration into the fatty streaks formed in aortic sinus was significantly less in mice deficient in VWF synthesis . The phenomenon we describe here may therefore explain why activated platelets mediated lymphocytes homing in high endothelial venules  and monocyte tethering to endothelial cells . Leukocyte rolling on ULVWF strings is significantly slower than their rolling on endothelial cells (Fig. 7), consistent with previous studies [7,46], suggesting that the strength of bonds formed between leukocytes and platelets is much greater than that of leukocytes and endothelial cells. Our results delineate a new mechanism through which leukocytes may be captured to vascular endothelial cells under a fluid shear stress that is much higher than it has been demonstrated previously. For this, platelets are first tethered to and activated on endothelial cells. Upon activation, the endothelial cell-bound platelets tether leukocytes to endothelial cells under high shear stress.
One obvious question is whether this mechanism also operates in the vascular system of conditions other than TTP. One possibility is that rapid release of a large amount of ULVWF may temporarily deplete ADAMTS-13 activity, resulting in transient and consumptive deficiency of the metalloprotease. This hypothesis is supported by several lines of evidence. First, several studies have shown that inflammation could result in mild to moderate ADAMTS-13 deficiency [47–49]. Secondly, acquired TTP are found in patients with severe infections, such as Staphylococcus aureus infection  and HIV-AIDS . Thirdly, a recent study by Reiter et al.  showed that intravenous administration of DDAVP, which stimulates the release of ULVWF from endothelial cells, resulted in transient increase in ULVWF and decreased in ADAMTS-13 activity. Such a transient deficiency of ADAMTS-13 may be sufficient to allow accumulation of ULVWF on endothelial cells and capture platelets and leukocytes. Finally, we have demonstrated recently that inflammatory cytokine IL-6 directly inhibits ADAMTS-13 activity, further suggesting that ADAMTS-13 deficiency could occur during systemic inflammation.
In summary, we have demonstrated that platelets firmly adhere to, instead of roll on, ULVWF strings formed on the surface of endothelial cells under both low and high shear stresses. Platelets, once adhered to ULVWF strings, are rapidly activated. The activated platelets form aggregates that grow locally, but are eventually released from endothelial cells. The ULVW-platelet strings provide a matrix to recruit leukocytes to the surface of endothelial cells under high shear stresses. The results of these studies provide further insights into thrombotic consequences of deficiency in ULVWF proteolysis and demonstrate a potential role for ADAMTS-13 in linking inflammation to thrombosis.
This work was supported by NIH grants P50-HL65967 and HL71895, a Grant-in-Aid from the American Heart Association–Texas Affiliate, and the Mary R. Gibson Foundation. J. F. D is an Established Investigator of the American Heart Association