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(Circulation. 1998;98:873-882.)
© 1998 American Heart Association, Inc.

Clinical Investigation and Reports

Venous Levels of Shear Support Neutrophil-Platelet Adhesion and Neutrophil Aggregation in Blood via P-Selectin and ß₂-Integrin

Konstantinos Konstantopoulos, PhD; Sriram Neelamegham, PhD¹; Alan R. Burns, PhD; Eric Hentzen, BS; Geoffrey S. Kansas, PhD; Karen R. Snapp, PhD; Ellen L. Berg, PhD; J. David Hellums, PhD; C. Wayne Smith, MD; Larry V. McIntire, PhD; ; Scott I. Simon, PhD

From the Cox Laboratory for Biomedical Engineering, Institute of Biosciences and Bioengineering, Rice University, Houston, Tex (K.K., J.D.H., L.V.M.); Speros P. Martel Section of Leukocyte Biology, Department of Pediatrics, Baylor College of Medicine, Houston, Tex (S.N., A.R.B., E.H., C.W.S., S.I.S.); Northwestern Medical School, Chicago, Ill (G.S.K., K.R.S.); and Protein Design Labs Inc, Mountain View, Calif (E.L.B.)

Correspondence to Scott I. Simon, PhD, Children's Nutrition Research Center, 1100 Bates St, Room 6014, Houston, TX 77030-2600. E-mail ssimon{at}bcm.tmc.edu

	Abstract

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Background—After activation, platelets adhere to neutrophils via P-selectin and ß₂-integrin. The molecular mechanisms and adhesion events in whole blood exposed to venous levels of hydrodynamic shear in the absence of exogenous activation remain unknown.

Methods and Results—Whole blood was sheared at 100 s^-1. The kinetics of neutrophil-platelet adhesion and neutrophil aggregation were measured in real time by flow cytometry. P-selectin was upregulated to the platelet surface in response to shear and was the primary factor mediating neutrophil-platelet adhesion. The extent of neutrophil aggregation increased linearly with platelet adhesion to neutrophils. Blocking either P-selectin, its glycoprotein ligand PSGL-1, or both simultaneously by preincubation with a monoclonal antibody resulted in equivalent inhibition of neutrophil-platelet adhesion (30%) and neutrophil aggregation (70%). The residual amount of neutrophil adhesion was blocked with anti-CD11b/CD18. Treatment of blood with prostacyclin analogue ZK36374, which raises cAMP levels in platelets, blocked P-selectin upregulation and neutrophil aggregation to baseline. Complete abrogation of platelet-neutrophil adhesion required both ZK36374 and anti-CD18. Electron microscopic observations of fixed blood specimens revealed that platelets augmented neutrophil aggregation both by forming bridges between neutrophils and through contact-mediated activation.

Conclusions—The results are consistent with a model in which venous levels of shear support platelet adherence to neutrophils via P-selectin binding PSGL-1. This interaction alone is sufficient to mediate neutrophil aggregation. Abrogation of platelet adhesion and aggregation requires blocking Mac-1 in addition to PSGL-1 or P-selectin. The described mechanisms are likely of key importance in the pathogenesis and progression of thrombotic disorders that are exacerbated by leukocyte-platelet aggregation.

Key Words: blood cells • neutrophils • platelets • glycoproteins • integrins

	Introduction

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Several studies have demonstrated that P-selectin plays a predominant role in mediating binding of activated platelets to neutrophils, monocytes, basophils, eosinophils, and a subpopulation of T lymphocytes.^{1 2 3 4 5 6 7 8 9} Adhesion between platelets and leukocytes represents an important process in hemostasis and thrombosis. Thrombotic stimuli induce platelets to aggregate via glycoprotein (GP) IIb/IIIa and to express P-selectin on their surfaces at sites of vascular injury. Under normal conditions, upregulated P-selectin mediates leukocyte accumulation and fibrin formation¹⁰ and accelerates clotting. However, under pathological conditions, these adhesive interactions may promote thrombosis and vascular occlusion, thereby impairing blood flow and exacerbating ischemia.¹¹

Neutrophil-platelet aggregation may occur in the circulation and be pathophysiologically significant. Enhanced neutrophil-platelet adhesion has been observed in the circulation of patients with acute myocardial infarction (AMI)¹² or stroke¹³ and after coronary angioplasty.¹⁴ Furthermore, increased monocyte and neutrophil adhesion to platelets has been reported after cardiopulmonary bypass.¹⁵ Leukocyte-platelet adhesion increases in parallel with the extent of platelet activation.^{5 13 15 16} The mechanisms by which platelets become activated in vivo under pathological conditions such as stroke or heart disease remain largely unknown. Studies in vitro have shown that platelet activation can be induced by chemical agonists such as thrombin, ADP, and phorbol myristate.¹⁷ In the absence of exogenously added chemical stimuli, high levels of shear stress can also activate platelets^{13 18 19} and induce platelet aggregation via GP IIb/IIIa, GP Ib, and von Willebrand factor.^{19 20 21}

Published data indicate that the adhesive interactions between activated platelets and neutrophils under shear occur via a multistep process analogous to that described for neutrophil adhesion to stimulated endothelial cells. P-selectin expressed on the surface of immobilized, activated platelets supports tethering and rolling of neutrophils from the free stream.^{2 7 22 23} Subsequent firm adhesion and transmigration across adherent platelets in a chemotactic gradient is mediated through the ß₂-integrin Mac-1 (CD11b/CD18) but not LFA-1 (CD11a/CD18).⁷ The transition from rolling to stable adhesion on immobilized platelets^{7 23} suggests that on contact with platelets, neutrophils undergo activation that is dependent on platelet-activating factor (PAF).²⁴ Further evidence of a contact-dependent mechanism of neutrophil activation is corroborated by the increased binding of monoclonal antibody (mAb) 24, a reporter of ß₂-integrin activation epitope on neutrophil.⁶

Most studies of neutrophil-platelet adhesion have been performed under conditions in which isolated neutrophils interact with surface adherent, activated platelets in shear flow.^{2 7 8 23 24} Such models may simulate events that take place after severe injury of a blood vessel, as may occur after coronary angioplasty, in which initial platelet deposition to denuded endothelial cell surfaces is followed by leukocyte accumulation. We wished to extend these studies and to develop an assay to monitor the transition of platelets from the unactivated to activated state, as may occur in the circulating blood of patients experiencing AMI or stroke. Our primary objective was to measure the time course of receptor-mediated neutrophil-platelet adhesion and neutrophil aggregation in real time. There have been no reports on the molecular mechanisms underlying the process of neutrophil-neutrophil adhesion under conditions in which platelets were the primary participants in the process. We endeavored to investigate the dynamics and molecular constituents that support neutrophil-platelet adhesion and neutrophil aggregation in freshly isolated whole blood in the absence of exogenous stimulus.

To simulate the intercellular collisions and shear rates and stresses (100 s^-1 and 0.1 Pa, respectively) that are prevalent in the venous microcirculation, we exposed blood suspensions to hydrodynamic shear. Freshly isolated whole blood was diluted in endotoxin-free buffer at 37°C, and activation was derived from exposure to rapid rotational mixing in a test tube. During the course of the experiment, samples were directly fed into a flow cytometer,^{25 26} thus allowing real-time measurement of the kinetics of neutrophil-platelet adhesion. The approach that we used has several important advantages over previous studies: (1) use of pristine whole blood specimens instead of isolated and reconstituted cell suspensions; (2) analysis of live cells without the need for fixation; (3) study of endogenous activation of blood specimens, thereby avoiding thrombin-activated platelets and/or chemotactic-stimulated leukocytes; and (4) evaluation of receptor-mediated adhesion events induced by shear in real time using flow cytometry. Our results suggest that hydrodynamic shear induces platelet-neutrophil adhesion, which subsequently drives neutrophil aggregation. These adhesive interactions are initiated by platelet P-selectin and subsequent activation of CD11b/CD18, which is involved in but not required for neutrophil aggregation in blood.

	Methods

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Reagents/Antibodies
Anti-L-selectin mAb LAM1–3 (IgG₁) was obtained from Cell Genesys. A humanized form of mAb 60.1, which blocks CD11b function, was kindly provided by Lora Whitehorse (Repligen Corp, Cambridge, Mass). Anti-CD11a mAb R3.1 (IgG₁) was a gift from Dr Robert Rothlein (Boehringer-Ingelheim Pharmaceuticals), and anti-CD18 mAb IB4 (IgG_2a) was provided by Drs J.D. Chambers and K. Arfors (La Jolla Cancer Institute, La Jolla, Calif). Humanized anti–P/E-selectin–blocking mAb EP5C7 (IgG₂) was produced by Dr Ellen Berg and Protein Design Labs (Mountain View, Calif).²⁷ S12 is a functionally nonblocking mAb directed against P-selectin (Centocor Inc). Blocking mAb against P-selectin GP ligand-1 (PSGL-1, CD162), PSL 275 (IgG₁), was kindly supplied by Dr Joe Sypek (Genetics Institute, Cambridge, Mass), and rabbit polyclonal mAb 4RB was a generous gift from Dr Michelle Mariscalco (Baylor College of Medicine, Houston, Tex). Anti–PSGL-1 mAb KPL-1 was produced by Drs Geoffrey Kansas and Karen Snapp.²⁸ Chimeric 7E3 Fab (ReoPro; Centocor Inc) is an anti–GP IIb/IIIa mAb. LAM1–3 Fab, EP5C7, PSL 275, S12, and c7E3 Fab were used at 20 µg/mL. IB4, R3.1 Fab, KPL-1, and humanized 60.1 were used at 30 µg/mL. 4RB was used at 100 µg/mL.

CD61-FITC mAb (Dako) is specific for platelet GP IIIa. LDS-751 (Molecular Probes) is a vital nucleic acid that homogeneously stains leukocytes but not platelets or erythrocytes. This dye is excited at 488 nm and has a peak emission at 670 nm.²⁹

Measurement of Neutrophil-Platelet Conjugate Formation by Flow Cytometry
Venous blood from 16 healthy volunteers (aged 25 to 47 years, equal distribution of male and female subjects) and patients with leukocyte-adhesion deficiency I (LAD-I) was drawn into porcine heparin (Elkins-Sinn Inc) at a final concentration of 10 U/mL. Specimens were stored at room temperature (RT) in capped polypropylene tubes and used within 1 to 1.5 hours of collection. Anticoagulated blood (100 µL) was diluted 1:5 with a HEPES buffer (containing 110 mmol/L NaCl, 10 mmol/L KCl, 10 mmol/L glucose, 1 mmol/L MgCl₂, and 30 mmol/L HEPES, pH 7.4; all buffers tested negative for endotoxin). This dilution was determined to be the minimum level that enabled real-time flow cytometric measurements of neutrophil events, eliminating the coincident measurement caused by the presence of >1000-fold excess of red blood cells and platelets in diluted blood. Platelets were labeled by incubating diluted blood with 10 µL of CD61-FITC for 10 minutes at RT in polypropylene cytometry test tubes (12x75 mm; Falcon Tubes; Becton Dickinson). After an additional 2-minute incubation with 1.0 µg/mL LDS-751 at 37°C, shear was initiated with a small magnetic bar (2x7 mm rotating at 700 rpm). The shear field is proportional to the rotation rate of the magnetic bar. The average shear rate was estimated at 100 s^-1. However, there are apparently regions of the flow field, such as at the surface of the rotating bar, that reach much higher shear rates (up to 3000 s^-1).³⁰

During the course of the experiment, samples were injected directly into a FACScan flow cytometer (Becton Dickinson),^{25 26} thus allowing real-time measurement of neutrophil-platelet adhesion kinetics. The forward and side scatter and FITC fluorescence profiles were acquired on a logarithmic scale, whereas LDS-751 fluorescence intensity (FL3) was obtained with linear detection settings. A fluorescence threshold was set to detect only those cells that were labeled with the leukocyte-specific marker LDS-751, thus excluding unbound erythrocytes and single platelets from the display. Acquisition of 2000 events at each time point required 20 s. Neutrophils were distinguished from the other leukocyte subpopulations on the basis of their characteristic forward and side scatter profiles. Neutrophil-platelet aggregates were considered those particles that expressed CD61-FITC fluorescence above a background level (Figure 1a and 1b), as previously described.^{5 13} The extent of neutrophil-platelet adhesion was expressed as the percentage of total neutrophils that were bound to platelets. The mean platelet fluorescence intensity of single neutrophils with adherent platelets (located in the bottom right quadrant of Figure 1a and 1b) was quantitated by gating this population on the basis of the LDS-751 fluorescence (Figure 1c). This fluorescence is directly correlated to the relative number of platelets attached to the neutrophil surface.

View larger version (34K): [in this window] [in a new window]   Figure 1. Flow cytometric analysis of neutrophil-platelet and neutrophil-neutrophil aggregates. Whole blood was diluted 1:5 and incubated with CD61-FITC (to label platelets) for 10 minutes at RT. Leukocytes were labeled with LDS-751 for 2 minutes, and the sample was sheared at 100 s-1 for 14 minutes at 37°C. Live 2-color flow cytometric analysis enabled quantification of neutrophil-platelet and neutrophil-neutrophil aggregates. a, Unsheared blood specimen at 0 minutes (baseline) showing single neutrophils with and without adherent platelets. b, Blood sample subjected to continuous shear mixing for 14 minutes. The vertical line in a and b corresponds to the FITC-fluorescence threshold that separates nonadherent neutrophils (left) from those bound to platelets (right). The horizontal line separates single neutrophils from neutrophil aggregates. c, Mean platelet fluorescence histograms of single neutrophils associated with platelets at 0 and 14 minutes. d, Histograms of neutrophil aggregation after shear for the data in b. LDS-751 mean fluorescence values of neutrophil aggregates including doublets (D), triplets (T), and quartets and larger aggregates (Q+) are integral multiples of the singlet (S) value.

Neutrophil-neutrophil aggregates were defined as those particles that expressed LDS-751 fluorescence levels greater than those of single neutrophils in the presence or absence of platelets (Figure 1b). Neutrophil aggregation was quantitated based on LDS-751 histograms (Figure 1d), whereas mean fluorescence intensity of aggregates was an integral multiple of the singlet neutrophil value.^{25 29} The extent of aggregation was determined by dividing the number of neutrophils in aggregates by the total number of neutrophils detected:

where the neutrophil aggregate sizes are given by S (singlets), D (doublets), T (triplets), and Q⁺ (quartets and larger unresolved aggregates).^{25 26}

Treatment of Blood Specimens with ZK36374
The stable prostacyclin (PGI₂) derivative ZK36374 (ZK) was a generous gift from Schering Co (Berlin, Germany). Whole blood was treated directly with 40 nmol/L ZK for 30 minutes at RT.

P-Selectin Determination
P-selectin expressed on the platelet surface was detected with an anti–P-selectin mAb, EP5C7, which was directly conjugated with CY3 (Amersham Life Science). The CY3 fluorochrome is excited at 488 nm and is detected in the orange (FL2) wavelength fluorescence channel. During neutrophil aggregation, 5-µL aliquots of the 1:5 diluted blood were withdrawn and incubated with 50 µL of HEPES buffer containing a saturating concentration of EP5C7-CY3 for 3 minutes at 37°C. Samples were then diluted with 1 mL of HEPES buffer (final blood dilution of 1:1000) and analyzed by flow cytometry, as previously described.³¹ One thousand events were acquired, and EP5C7-CY3 fluorescence was measured.

Electron Microscopy
Whole blood samples containing neutrophil-platelet aggregates were prepared for electron microscopy (EM) by standard procedures. Briefly, sheared whole blood suspensions were fixed in 2% glutaraldehyde at room temperature for 30 minutes and postfixed for 1 hour in PBS containing 1% osmium tetroxide. Cells were then dehydrated in a graded series of ethanol and embedded in LX-112 (Ladd Research Industries). After polymerization, ultrathin sections were obtained on an RMC 7000 ultramicrotome equipped with a diamond knife. Sections were stained with uranyl acetate and lead citrate before being viewed on an electron microscope.

Statistical Analysis
Data are expressed as mean±SEM. Statistical significance of differences between means was determined by ANOVA, followed by the Student-Newman-Keuls test. P<0.05 was selected to be statistically significant.

	Results

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Hydrodynamic Shear Supports Formation of Neutrophil-Platelet Aggregates
Isolated neutrophils subjected to hydrodynamic shear do not form aggregates in the absence of exogenous stimulus.^{25 32} However, in undiluted whole blood samples subjected to the same shear conditions, both neutrophil-platelet and neutrophil-neutrophil aggregates were observed. Shear mixing induced a low level of homotypic platelet aggregation in blood, which was negligible in samples diluted 1:5. The addition of an anti–GP IIb/IIIa mAb (c7E3 Fab) blocked platelet aggregation in undiluted whole blood, leaving intact platelet-neutrophil adhesion and aggregation (data not shown). In the present study, we primarily used diluted whole blood specimens to monitor in real time by flow cytometry neutrophil-platelet adhesion and neutrophil aggregation in the absence of the significant artifact caused by coincident detection arising from the 1000-fold excess of erythrocytes and platelets. Moreover, we wished to study the molecular constituents that mediate adhesion in the absence of the platelet-platelet aggregation favored in blood, thus eliminating the possibility of passive neutrophil entrapment in the platelet aggregates.

We confirmed that the extents of platelet attachment and neutrophil aggregation were comparable in both undiluted and diluted blood specimens subjected to shear. A baseline level of neutrophil-platelet adherence (31±4%) was observed before shear mixing (Figures 1a and 2a). This finding is in accord with previously published reports.^{5 13 15} However, neutrophil aggregation was not detected in blood before shear (Figures 1a and 2b). Platelet adhesion to neutrophils increased rapidly on application of shear, as determined by the increase in neutrophil-bound platelet fluorescence (Figure 1). This preceded the onset of neutrophil aggregation, which followed with a 20-second lag. The rate of platelet recruitment by neutrophils was similar to that of neutrophil aggregation, with both processes plateauing at 7 minutes and being irreversible for 14 minutes (Figure 2). At this time, >85% of neutrophils had platelets adherent to their surface, and 75% of neutrophils were in aggregates of 2 cells (Figure 1b and 1d). The number of platelets bound to neutrophils increased with time of shear, as evidenced by the 5-fold increase in the mean platelet fluorescence intensity of single neutrophils associated with platelets (Figure 1c). This correlated to an average of 1 to 2 platelets adherent to a single neutrophil before shear and 4 to 6 platelets/neutrophils after application of shear for 14 minutes.

View larger version (16K): [in this window] [in a new window]   Figure 2. Kinetics of neutrophil-platelet and neutrophil aggregate formation measured in real time by flow cytometry. Diluted blood was placed at 37°C either in the absence or presence of shear (100 s-1). In some experiments, samples were sheared on addition of either anti-GP IIb/IIIa (20 µg/mL c7E3 Fab), PGI2 analogue (40 nmol/L ZK363374), or EDTA (5 mmol/L). Time course of neutrophil-platelet adhesion (a) and neutrophil aggregation (b). Data in a and b are representative of 3 independent experiments. c, Correlation between neutrophil-platelet adhesion and neutrophil aggregation over time in diluted whole blood subjected to shear. Data from the adhesion kinetics obtained from 16 separate donors are plotted. represents an experiment in which blood was sheared for 14 minutes in the presence of ZK (40 nmol/L).

Activation and Cation Dependence of Neutrophil-Platelet Adhesion
To examine the molecular requirements for adhesion in sheared blood, the effect of blocking with anti–GP IIb/IIIa mAb (c7E3 Fab), the PGI₂ analogue ZK, or EDTA was assessed (Figure 2). The addition of anti–GP IIb/IIIa mAb has been shown to block the receptor from binding fibrinogen and von Willebrand factor, thus preventing platelet aggregation. In whole blood, the addition of this mAb did not significantly alter the kinetics of neutrophil-platelet attachment or neutrophil aggregation (Figure 2). These data suggest that platelet attachment to neutrophils in this low regimen of shear did not involve GP IIb/IIIa or require platelet aggregation.

To assess the mechanism of platelet activation in the formation of neutrophil-platelet aggregates, we tested the effect of the PGI₂ analogue ZK. This compound has been shown to elevate cAMP levels in platelets and to inhibit platelet aggregation.³³ In addition, high levels of ZK (10 to 100 µmol/L) have been reported to substantially inhibit neutrophil function in vitro.³⁴ We confirmed that the addition of 40 nmol/L ZK did not alter the upregulation of ß₂-integrin in blood or the efficiency of homotypic aggregation in isolated neutrophil suspensions stimulated by the chemotactic peptide formyl-methionine-leucyl-phenylanine (fMLP; data not shown). In contrast, the pretreatment of blood with 40 nmol/L ZK immediately after collection substantially decreased the rate and extent of platelet-neutrophil adhesion and blocked neutrophil aggregation to baseline (Figure 2). At 14 minutes of shear, 60% of neutrophils bound to an average of 1 to 2 platelets. Although platelet attachment was a component of neutrophil aggregation, the attachment of small numbers of platelets to even a majority of neutrophils was not sufficient to mediate aggregation.

Experiments were performed in the presence of 5 mmol/L EDTA to assess the divalent cation requirements. These ions are necessary for selectin- and integrin-mediated adhesion.^{8 26} When EDTA was added, the baseline platelet adhesion decreased from 30% to 5% and remained at this level on application of shear. Neutrophil aggregation was totally abrogated under these conditions. The baseline level of neutrophil-platelet adhesion (5%) detected in the presence of EDTA was due to loosely attached or nonadherent platelets. These coincident platelet events depended on the extent of blood dilution, as confirmed by the elimination of baseline neutrophil-platelet adhesion in the presence of EDTA at a 1:50 dilution of blood (data not shown).

Under conditions of shear, a direct correlation was found between the fraction of neutrophils adherent to platelets and aggregation at 5 and 14 minutes (Figure 2c). Above a level of 1 to 2 bound platelets, neutrophil aggregation increased almost linearly with platelet attachment in response to shear. The addition of ZK revealed a dissociation between platelet attachment and aggregation because there was no detectable neutrophil aggregation, although 60% of neutrophils bound 1 to 2 platelets. These results suggest that in sheared suspensions of whole blood, platelet attachment is a necessary but not sufficient condition for neutrophil aggregation. Aggregation increases as a function of both the fraction of neutrophils with bound platelets and the number of attached platelets/neutrophil. We determined that activation can be attributed in part to the release of ADP, as evidenced by the addition of ADP scavengers (apyrase, CP-CPK), which were partially effective in inhibiting aggregation (30% inhibition).

Hydrodynamic Shear Increases Expression of Platelet P-Selectin
Platelet P-selectin (CD62P) has been shown to be an important molecule for platelet-neutrophil adhesion and activation.²³ We examined the role of this adhesion receptor by measuring its surface expression in response to shear (Table 1). A small fraction of platelets (<2%) expressed P-selectin before application of shear. With time of shear, an increase in the expression of P-selectin was detected; by 14 minutes, 6% of the platelets increased their mean fluorescence intensity to a level 5- to 10-fold higher than the background EP5C7-CY3 fluorescence of unactivated platelets. The increase was not attributed to platelet-platelet microaggregation because P-selectin–positive platelets spanned the entire platelet size range, including the smallest platelets. Maximal stimulation of platelets with thrombin resulted in 95% of the platelets expressing P-selectin. The mean fluorescence of these TRAP-stimulated platelets was identical to that of the maximally activated platelets under shear conditions and significantly greater than background fluorescence (data not shown).

View this table: [in this window] [in a new window]   Table 1. Flow Cytometric Determination of Platelet P-Selectin Expression

The fraction of P-selectin–positive platelets steadily increased in blood subjected to shear, but not in samples incubated under static conditions (Table 1). Treatment of blood with the PGI₂ analogue ZK before the application of shear resulted in abrogation of P-selectin upregulation of platelets over the time course of shear. The data are consistent with a mechanism in which P-selectin is released from intracellular stores onto the plasma membrane in response to venous levels of shear. This precipitated platelet attachment and neutrophil aggregation.

In isolated suspensions of neutrophils, homotypic aggregation stimulated by chemotactic factors is blocked with antibodies against ß₂-integrin.^{30 32} We assessed the role of P-selectin and ß₂-integrin in neutrophil-platelet adhesion by incubating samples with the PGI₂ analogue ZK and function-blocking mAbs to these adhesion molecules. Neutrophil-platelet adhesion and aggregation were significantly blocked at all time points under shear by treatment with ZK alone (Figure 3). The addition of anti–P-selectin (EP5C7) did not further increase the level of inhibition observed with ZK. However, the residual amount of platelet adhesion and aggregation was blocked to baseline with the addition of anti-ß₂-integrin mAb (IB4). These results indicate that ZK acted on the platelets to diminish the expression of P-selectin and partially block their adhesion to neutrophils. The residual level of adhesion, which was particularly evident at 14 minutes of shear, was apparently due to de novo neutrophil activation and adhesion via ß₂-integrin.

View larger version (19K): [in this window] [in a new window]   Figure 3. Dependence of platelet adhesion and neutrophil aggregation on binding via P-selectin and ß2-integrin. Kinetics of neutrophil-platelet adhesion (a) and neutrophil aggregation (b). Diluted blood was subjected to shear at 37°C either in the absence of any treatment or on addition of ZK (40 nmol/L) and blocking mAbs to CD62P (20 µg/mL EP5C7) or CD18 (30 µg/mL IB4). *P<0.05 with respect to no-treatment control. §P<0.05 with respect to ZK. Data are mean±SEM from 3 to 5 experiments.

Dependence of Adhesion on Selectins and Integrins
Neutrophil localization onto a surface-adherent layer of platelets in a flow chamber assay has been modeled as a multistep process involving initial capture via P-selectin and firm adhesion via ß₂-integrin.^{8 9} We examined the molecules involved in whole blood aggregation in sheared suspensions by performing mAb blocking studies against various members of the selectin (Figure 4) and integrin families (Figure 5). Blocking either P-selectin (with EP5C7) or PSGL-1 (with KPL-1) was equally effective in inhibiting neutrophil-platelet adhesion by 30% and neutrophil aggregation by 70% (Figure 4). On addition of these mAbs, both the rate and extent of platelet adhesion was decreased. Furthermore, the number of platelets bound decreased from 4 to 5 platelets to 1 to 2 platelets per neutrophil. Two other antibodies to PSGL-1, PSL 275 and 4RB, only partially inhibited platelet-neutrophil adhesion by 10%, and neutrophil aggregation by 35% (data not shown). These observations support our hypothesis that neutrophil aggregation increases not only as a function of the fraction of neutrophils with bound platelets but also with the number of adherent platelets per neutrophil. Blocking with anti–P-selectin and anti–PSGL-1 (KPL-1) did not further inhibit adhesion compared with treatment with either mAb alone, which indicates that PSGL-1 on neutrophils acts as the primary ligand for P-selectin.

View larger version (25K): [in this window] [in a new window]   Figure 4. Contribution of P-selectin and L-selectin to platelet adhesion and neutrophil aggregation. Diluted blood was incubated for 10 minutes at room temperature with a panel of blocking mAbs to selectins and PSGL-1: anti-CD62P (20 µg/mL EP5C7), anti–PSGL-1 (30 µg/mL KPL-1), and/or anti-CD62L (20 µg/mL LAM1-3). The suspension was then subjected to shear at 37°C, and adhesion kinetics were measured for neutrophil-platelet adhesion (a) and neutrophil aggregation (b). *P<0.05 vs shear alone. P<0.05 vs CD62P. §P<0.05 vs CD62L. Values are mean±SEM from 3 to 13 experiments.

View larger version (29K): [in this window] [in a new window]   Figure 5. Contribution of ß2-integrin to neutrophil-platelet adhesion and aggregation. Diluted blood from normal donors and LAD-I patients (<4% normal CD18 expression) was incubated for 10 minutes at RT with a panel of blocking mAbs: to CD62P (20 µg/mL EP5C7), to PSGL-1 (30 µg/mL KPL-1), to CD18 (30 µg/mL IB4), to LFA-1 (CD11a, 30 µg/mL R3.1Fab), or to Mac-1 (CD11b, 30 µg/mL h60.1). The suspension was then subjected to shear at 37°C, and adhesion kinetics were displayed for neutrophil-platelet adhesion (a) and neutrophil aggregation (b). *P<0.05 vs shear alone. P<0.05 vs CD62P. §P<0.05 vs CD18. Data for normal donors are mean±SEM for 3 to 13 experiments. Data for LAD-I patients are representative of blood from 2 donors.

Blocking L-selectin with mAb LAM1–3 alone did not significantly decrease either neutrophil-platelet adhesion or neutrophil aggregation (Figure 4). However, in combination with anti–P-selectin, neutrophil-platelet adhesion was inhibited by 65%. As previously shown,³⁵ L-selectin on neutrophils may bind to a yet-unidentified ligand on platelets.

Blocking ß₂-integrin with anti-CD18 (IB4) alone did not yield any inhibition (Figure 5). However, in combination with anti–P-selectin, it almost completely blocked both neutrophil-platelet attachment and neutrophil aggregation. Using function-blocking mAbs, we next determined which of the ß₂-integrin subunits, Mac-1 (mAb 60.1) or LFA-1 (mAb R3.1Fab), supported platelet-neutrophil adhesion and aggregation. A combination of anti–PSGL-1 and anti–Mac-1, but not anti–LFA-1, was more effective than anti–PSGL-1 or anti–P-selectin alone in blocking platelet-neutrophil adhesion (Figure 5a). Although there was a trend toward increased inhibition of neutrophil aggregation on combined treatment with anti–P-selectin/anti–PSGL-1 and anti–Mac-1, it was not significantly different from blocking P-selectin or its ligand on neutrophils (Figure 5b). Taken together, the data indicated that platelet P-selectin recognized PSGL-1 in mediating adhesion to the neutrophil. This interaction was sufficient to mediate most neutrophil aggregation. The subsequent activation of neutrophils led to Mac-1 binding to an undefined ligand on platelets.

We further examined the role of ß₂-integrin in supporting adhesion in blood from patients whose leukocytes expressed very low levels (4% of normal donors) of ß₂-integrin (Figure 5). These individuals suffered from a variety of immune disorders associated with neutrophil dysfunction and were clinically characterized as LAD-I patients.^{36 37} Exposure of LAD-I blood to shear resulted in levels of neutrophil-platelet and neutrophil aggregation comparable to those of normal volunteers. The addition of anti–P-selectin mAb EP5C7 drastically reduced the extent of neutrophil-platelet adhesion and completely blocked neutrophil aggregation. At 14 minutes, 90% of the neutrophils in LAD-I blood were recruited by platelets in the control sample, compared with 43% in the presence of anti–P-selectin. A nonblocking P-selectin–binding control mAb S12 did not alter either neutrophil-platelet or neutrophil-neutrophil aggregation (data not shown). These observations confirm that in the presence of low levels of ß₂-integrin, P-selectin alone was sufficient for platelet adhesion to neutrophils and requisite for neutrophil aggregation.

Morphology of Neutrophil Aggregation Assessed by EM
To visualize the cellular interactions supporting the aggregation process, blood samples were fixed over the time course of shear and examined by transmission EM. Observation of EM grids revealed only 5 to 6 leukocytes on sections containing up to 1000 red cells. This ratio is consistent with the fact that at a 1:5 dilution of blood, the leukocyte hematocrit was decreased to 0.3% and red blood cells outnumbered neutrophils by several hundred to 1. After exposure to shear for 10 minutes, few neutrophils were observed without adherent platelets, and these single neutrophils remained in a spherical shape, an indication of an unactivated state (Figure 6A). Most of the neutrophils were adherent to 2 platelets in the planar section (Figure 6B). These cells had clearly undergone shape change and were activated. Virtually all of these neutrophils were in aggregates (Figure 6C and 6D). Neutrophil aggregates were observed with platelets either forming bridges between neutrophils (Figure 6C) or attached at the periphery away from the neutrophil-neutrophil contact region (Figure 6D). Larger aggregates with multiple neutrophils and platelets were also observed at 10 minutes. Mononuclear cells adherent to platelets in the EM sections were also observed. These events were largely gated out in the flow cytometric analysis and therefore were not included in our determination. The EM observations were consistent with the aggregation data obtained by flow cytometry and lead us to conclude that platelet attachment promoted neutrophil activation and aggregation in whole blood. Although platelet bridging of neutrophils seemed to provide 1 mechanism that promoted neutrophil aggregation, direct membrane contact between neutrophils was also evident by 10 minutes.

View larger version (139K): [in this window] [in a new window]   Figure 6. Morphology of neutrophil-platelet and neutrophil-neutrophil aggregation. Diluted whole blood specimens were subjected to hydrodynamic shear (100 s-1) for 10 minutes, fixed with 2% glutaraldehyde and examined by transmission EM. A, Single neutrophils with few to no adherent platelets appear round (unactivated). B, Neutrophil bound to 4 platelets has undergone shape change (activated). Neutrophil aggregates were observed with platelets forming bridges between neutrophils (C) and on the periphery (D). Arrows mark platelets; arrowheads indicate neutrophil-neutrophil contact region. Scale bar in each panel=2 µm.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Leukocyte adhesion to activated platelets represents a key event in the sequence of thrombus formation, as demonstrated in vitro³⁸ and observed in vivo after deep arterial injury and in the propagation of venous thrombosis.^{39 40} Neutrophils are the largest leukocyte component in these thrombi³⁸ and have been reported to increase the vasoconstrictive response at the site of endothelial injury in vivo.⁴¹ In the present study, we demonstrated that venous levels of hydrodynamic shear supported platelet-mediated neutrophil aggregation in whole blood in the absence of exogenous stimuli, such as thrombin to activate platelets or chemotactic factor to stimulate neutrophils. In contrast, isolated neutrophils subjected to identical conditions of shear do not aggregate until stimulated with a chemotactic factor.^{25 32} We investigated the molecular mechanisms by which shear induces platelet activation, neutrophil-platelet adhesion, and neutrophil aggregation in blood.

Neutrophil-Platelet Adhesion Is a Prerequisite for Neutrophil Aggregation
Previous studies demonstrated that exposing isolated platelets to shear stresses 1.2 Pa (12 dyne/cm²) did not result in activation because these platelets did not aggregate or release procoagulant-containing microparticles.⁴² In the current study, exposing blood to low levels of shear (0.1 Pa) promoted activation of platelets and neutrophil aggregation. A likely mechanism for platelet activation and upregulation of P-selectin may involve hemolysis and release of ADP. This has been reported to occur in blood exposed to relatively low levels of shear stress.⁴³ However, additional sources of activation may derive from the experimental environment, such as exposure to foreign surfaces and altered O₂ levels. Nonetheless, platelet activation was associated with signal transduction because inhibition of P-selectin upregulation was achieved by treating blood with the PGI₂ analogue ZK. This compound has been shown to elevate cAMP levels in platelets and inhibit platelet aggregation induced by chemical agonists, including ADP.³³

Activated platelets appeared to capture neutrophils, as evidenced by inhibition of this heterotypic interaction on whole blood treatment with ZK. An early onset of neutrophil-platelet adhesion led to the formation of neutrophil-neutrophil aggregation, indicating that platelet capture was a prerequisite for neutrophil aggregation. In the absence of shear, we observed that only a fraction of the neutrophils (30%) were adherent to 1 to 2 platelets. With time of shear, neutrophil aggregation increased almost linearly with platelet attachment, the latter reaching a level of 4 to 6 platelets per neutrophil at maximal aggregation. The addition of either the PGI₂ analogue ZK or the anti–P-selectin mAb decreased this ratio to 1 to 2. Although 60% of the neutrophils remained bound to 1 platelet, neutrophil aggregation was reduced by 70% under these conditions. Taken together, the data suggest that the level of neutrophil aggregation is closely correlated with the onset of platelet activation and the number of adherent platelets per neutrophil. Above a threshold of 1 to 2 adherent platelets, neutrophil aggregation increased linearly with platelet adhesion to neutrophils.

Neutrophil-Platelet Adhesion Is Mediated by P-Selectin and ß₂-Integrin
Neutrophil aggregation in blood differs markedly from that of isolated suspensions stimulated with fMLP,^{30 32} in that mAbs that block L-selectin and ß₂-integrin function failed to reduce platelet-mediated neutrophil aggregation. Only the combination of anti–P-selectin (or anti–PSGL-1) and anti–ß₂-integrin resulted in abrogation of neutrophil-platelet adhesion. In agreement with previous reports,^{6 7} the functional activity of the ß₂-integrin subunit Mac-1 but not LFA-1 supported neutrophil-platelet adhesion. This was illustrated by complete inhibition after blocking with a combination of mAbs to PSGL-1 and Mac-1 but not LFA-1. Furthermore, PSGL-1 seemed to be the primary ligand for P-selectin because blocking either receptor individually or both simultaneously gave the same level of inhibition. Although PSGL-1 has been shown to bind purified P-selectin on coated surfaces or expressed P-selectin on transfected cells,^{28 44 45} the present study is the first to show that platelet P-selectin mediates adhesion to neutrophils through PSGL-1. L-Selectin also seemed to be involved in neutrophil-platelet binding, because blocking with a combination of mAbs to L- and P-selectin was twice as effective at blocking platelet adhesion as P-selectin alone. P-selectin upregulation was a necessary and sufficient condition for neutrophil aggregation in whole blood, as evidenced by mAb blocking experiments. This observation was confirmed in experiments with blood from LAD-I patients whose neutrophils were deficient in functional levels of ß₂-integrin. They exhibited normal levels of P-selectin–dependent aggregation in response to shear.

A multistep process that outlines the molecular events leading to leukocyte emigration on cytokine-stimulated endothelium also applies to neutrophil adhesion on surface-adherent platelets in a parallel flow geometry. Published data support a mechanism in which P-selectin initiates capture of neutrophils, followed by activation by platelet-mediated PAF that results in firm adhesion via Mac-1 recognition of an unknown ligand on the platelet surface.^{6 7 8 24} It seems that platelet adhesion to neutrophils and subsequent aggregation in whole blood exposed to a low shear regimen (0.1 Pa) is fundamentally different from the multistep cascade of neutrophil recruitment to surface-adherent activated platelets subjected to higher levels of shear (0.2 Pa). In this experimental system, P-selectin was necessary and sufficient for platelet capture of neutrophils. Moreover, this event was the primary factor in precipitating neutrophil aggregation. This novel function for P-selectin in mediating stable adhesion in sheared blood suspensions appears to be different from its role in tethering neutrophils to immobilized and activated platelets.

A commensurate level of platelet activation in vivo would not necessarily result in the rate and extent of aggregation measured in our experiments. The rotational streamlines in our system lead to substantially more frequent intercellular collisions than occur in a parallel flow geometry that predominates in laminar flow in the microcirculation. This phenomenon, in combination with the relatively low shear stresses prevailing in our experiments, enabled determination of the maximal adhesion efficiency between platelets and neutrophils.

Model of Neutrophil Aggregation
Taken together with the receptor blocking studies, the EM analysis supported a model for neutrophil aggregation in whole blood. Exposure of blood to shear induces platelet activation via a PGI₂-dependent pathway, as manifested by upregulation of P-selectin surface expression. Platelet P-selectin binding to PSGL-1 on neutrophils initiates signal transduction and neutrophil activation.^{6 23 46 47} This is supported by the EM observations that neutrophils with few adherent platelets remained round in shape, whereas those adherent to several platelets consistently exhibited shape changes with numerous extended pseudopodia. Platelet-mediated activation of ß₂-integrin has also been demonstrated by the increased expression of an antibody that reports on activated CD18 (mAb24).⁶ In addition to P-selectin/PSGL-1 interaction and L-selectin involvement, activated Mac-1 binds to an unidentified ligand on platelets, as demonstrated in the present and previous studies.^{6 7 8 24} This heterotypic cell interaction is a prerequisite for neutrophil aggregation. Platelets seem to support aggregation by 2 mechanisms: (1) by the formation of bridges between neutrophils and (2) through contact-mediated activation. In the latter case, we propose that sufficient numbers of bound platelets (>2) induce neutrophil activation. This may lead to neutrophil adhesion mediated through a multistep mechanism analogous to that of isolated neutrophil suspensions stimulated with a chemotactic factor in which L-selectin tethering leads to ß₂-integrin–dependent aggregation.^{30 48} In this regard, activated surface-adherent platelets have been reported to capture neutrophils in shear flow and to activate spreading and firm adhesion that is dependent on PAF.^{7 24} Alternatively, platelets bound to neutrophils may provide an adhesive surface for the capture of a second neutrophil through P-selectin tethering, thus facilitating neutrophil-neutrophil contact and subsequent engagement of activated ß₂-integrin. The implication of our data is that the formation of neutrophil-platelet aggregates under venous levels of shear, which can occur in thrombotic and inflammatory disorders, cannot be prevented unless both selectin and integrin molecules are blocked.

Study Limitations and Pathophysiological Significance
Our results suggest that exposure of whole blood to venous levels of hydrodynamic shear induces platelet activation via a PGI₂-dependent pathway. P-selectin upregulation was mainly attributed to hemolysis and release of ADP from erythrocytes in response to shear stress. In the normal circulation, release of endothelial ecto-ATP and ADPases, as well as PGI₂ and nitric oxide, from endothelial cells tends to keep platelets quiescent.^{49 50} This may provide a homeostatic control that maintains platelet-mediated neutrophil aggregation at basal levels in healthy individuals, despite the ever-present shear of flowing blood. However, in certain pathological conditions, such as stroke and AMI, enhanced platelet activation and platelet-neutrophil complexes occur.^{12 13 51} Under such conditions, venous levels of shear and release of proinflammatory mediators may be important determinants in promoting platelet-neutrophil adhesion and subsequent neutrophil aggregation, thus providing additional modes of neutrophil recruitment. Our experimental approach provides a continuous measurement of the kinetics of platelet activation and platelet-neutrophil interactions and achieves determination of the maximal efficiency of adhesion. Elucidation of the detailed molecular basis underlying neutrophil-platelet conjugate formation enables an understanding of the roles they play and the time course involved.

	Acknowledgments

 
This work was supported by National Institutes of Health grants AI31545–01, HL-18672, and NS-23327 (Dr McIntire); AI31652 (Dr Simon); HL-42550 and AI19031 (Dr Smith); HL-18584, 5P50-NS-23327 (Dr Hellums); NASA NSCORT grant NAG 54072, Robert A. Welch Foundation grant C-938, and a grant from the Butcher Fund (Dr McIntire); a Whitaker Foundation grant (Dr Simon); and a Methodist Foundation grant (Dr Burns). Dr Burns is a recipient of the Chao fellowship. Dr Simon and Dr Kansas are Established Investigators of the American Heart Association.

	Footnotes

 
¹ Drs Konstantopoulos and Neelamegham contributed equally to this article. Dr Konstantopoulos is now at the Department of Chemical Engineering, Johns Hopkins University, Baltimore, Md. Dr Neelamegham is now at the Department of Chemical Engineering, State University of New York at Buffalo.

Guest editor for this article was Dr Allan M. Lefer, Jefferson Medical College, Philadelphia, Pa.

Received December 16, 1997; revision received March 30, 1998; accepted April 22, 1998.

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