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Keywords:

  • coagulation factor X;
  • fibrinogen;
  • flow cytometry;
  • Mac-1;
  • monocytes;
  • neutrophils;
  • platelets;
  • tissue factor

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conflicts of interest disclosure
  8. References

Summary. Background: Monocytes and neutrophils form heterotypic aggregates with platelets initially via engagement of platelet surface P-selectin with leukocyte surface P-selectin glycoprotein ligand-1 (PSGL-1). The resultant intracellular signaling causes the leukocyte surface expression of tissue factor and activation of leukocyte surface Mac-1 (integrin αMβ2, CD11b/CD18). The activation-dependent conformational change in monocyte surface Mac-1 results in the binding of coagulation factor Xa (FXa) and/or fibrinogen to Mac-1. The aim of this study was to develop whole blood flow cytometry assays of these procoagulant activities and to investigate the effects of platelet binding to monocytes and neutrophils. Methods: Citrate or D-Phe-Pro-Arg-chloromethylketone (PPACK) anticoagulated whole blood was incubated with monoclonal antibodies against CD14 (PECy5), CD42a (PE), FITC-conjugated test antibody and an agonist, and then fixed with FACS lyse. Appropriate isotype negative controls were prepared in parallel. A BD FACSCalibur was used to analyze monocytes and neutrophils, which were identified based on CD14 fluorescence, forward and 90° light scatter. These populations were further gated into CD42a-positive (platelet-bound) and CD42a-negative (platelet-free). Geometric mean fluorescence and per cent positive data were collected for each subpopulation to measure the binding of test antibodies directed at CD42a, tissue factor, coagulation FXa, bound fibrinogen, activated Mac-1, and CD11b. Compensation controls were prepared on six normal donors prior to the study and these settings were used throughout the 10 donor study. Negative controls verified the lack of cross talk, particularly in the quantified FITC and PE parameters. Results: The physiologic agonists collagen and ADP increased monocyte-platelet and neutrophil-platelet aggregates and increased leukocyte surface Mac-1/CD11b and surface-bound tissue factor, FXa and fibrinogen. Whereas the increases in Mac-1/CD11b were mainly independent of leukocyte-platelet binding, the increases in surface-bound tissue factor, FXa and fibrinogen were mainly dependent on leukocyte-platelet binding. Conclusions: (i) We have developed novel whole blood flow cytometry assays to measure bound tissue factor, coagulation FXa, fibrinogen, activated Mac-1 and CD11b on the surface of monocytes and neutrophils, allowing independent analysis of monocytes and neutrophils with and without surface-adherent platelets. (ii) The monocyte and neutrophil surface binding of tissue factor, FXa and fibrinogen is mainly dependent on platelet adherence to monocytes and neutrophils, whereas the monocyte and neutrophil surface expression of CD11b and activated Mac-1 is mainly independent of platelet adherence to monocytes and neutrophils.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conflicts of interest disclosure
  8. References

Monocytes and neutrophils form heterotypic aggregates with platelets via engagement of platelet surface P-selectin (CD62P) with leukocyte surface P-selectin glycoprotein ligand-1 (PSGL-1, CD162) [1,2]. The resultant intracellular signaling causes the leukocyte surface expression of tissue factor (CD142) [3] and activation of leukocyte surface Mac-1 (CD11b/CD18, integrin αMβ2) [4,5]. The activation-dependent conformational change in monocyte surface Mac-1 [6,7] results in the binding of coagulation factor Xa (FXa) and/or fibrinogen to Mac-1 [8–10]. The platelet surface also binds coagulation factors via exposed negatively charged phospholipids such as phosphatidylserine [11,12]. Tissue factor from the vascular wall and platelets forms a complex with coagulation factor VIIa facilitating the activation of factor X [11]. Tissue factor is a key component of monocyte surface procoagulant activity [13]. Fibrinogen binds to the platelet surface upon activation of GPIIb-IIIa (integrin αIIbβ3, CD41/CD61) [14]. The aim of the present study was to develop whole blood flow cytometry assays of surface bound tissue factor, FXa and fibrinogen and to investigate the effects on these procoagulant assays of platelet binding to monocytes and neutrophils.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conflicts of interest disclosure
  8. References

Materials

After IRB-approved written informed consent, peripheral blood was drawn from healthy, non-smoking adult volunteers [five men and five women, average age 40.4 years (range 28–50 years)] who had not received any platelet inhibitory drugs in the preceding 2 weeks. After discarding the first 2 mL, blood was drawn into a 3.2% sodium citrate Vacutainer (Becton Dickinson, Franklin Lakes, NJ, USA) and syringes containing 300 μm D-Phe-Pro-Arg-chloromethylketone (final concentration, PPACK; Calbiochem, San Diego, CA, USA). All assays were carried out in endotoxin free Hanks buffered saline solution (HBSS; Gibco, Gaithersburg, MD, USA) to minimize leukocyte activation.

Monocyte-specific anti-CD14-PECy5 clone RM052 was purchased from Immunotech (Marseille, France). Platelet-specific anti-CD42a-PE (GPIX) clone ALMA 16 was purchased from BD-Pharmingen (San Diego, CA, USA). Anti-platelet CD42b (GPIb) clone 6D1 was a gift from Dr Barry S. Coller (Rockefeller University, NY, USA) [15]. Clone mAb24 which recognizes an activated conformation of Mac-1 (CD11b/CD18) was a gift from Dr Nancy Hogg (Cancer Research UK, London, UK) [7]. Antibody F26, which recognizes a receptor-induced binding site (RIBS) on fibrinogen bound to the dimeric integrin αIIbβ3 (GPIIb-IIIa CD41/CD61), was a gift from Dr Margaret Rick (NIH, Bethesda, MD, USA) [16]. The latter three antibodies were fluorescein isothiocyanate (FITC) conjugated in our laboratory as previously described [17]. Antibody F26 was used as a F(ab’)2 fragment, because we have previously demonstrated that F26 induces platelet activation via the platelet Fc receptor, CD32 [18]. Anti-CD11b-PE clone LPM19c was purchased from DAKO-Cytomation (Carpinteria, CA, USA). Anti-tissue factor-FITC (CD142, clone 4508), anti-coagulation factor X/Xa-FITC (clone 5010) and purified coagulation FX were obtained from (American Diagnostica, Stamford, CT, USA). Appropriate matched isotype control antibodies were purchased from BD-Pharmingen and DAKO-Cytomation. ADP was purchased from Bio/Data Corp (Hatboro, PA, USA), phorbol myristate acetate (PMA) from Calbiochem, and collagen from Chronolog Corp (Havertown, PA, USA).

Coagulation FXa assay

The PPACK anticoagulated whole blood was diluted 1:2 in HBSS containing 20 μg mL−1 purified human FX. Aliquots were incubated at 37°C with 2 or 20 μm ADP, 20 μg mL−1 collagen, or HBSS buffer only (no agonist) for 15 min. Incubation at 37°C is required for assembly of the tenase complex and subsequent binding of coagulation FX in its activated form (Xa). Three-antibody cocktails containing CD14-PECy5, CD42a-PE and 12 μg mL−1 anti-factor X/Xa-FITC or MIgG1-FITC were added and the samples incubated for an additional 15 min at 22°C. FACS lysing solution (Becton Dickinson) was added and samples were stored at 4°C until analysis.

Cell surface-bound fibrinogen assay

The PPACK anticoagulated whole blood was diluted 1:2 in HBSS and then incubated at either 22°C (for ADP, to minimize plasma and ectoADPase effects) or 37°C (for collagen whose action requires this temperature) for 15 min. Three-antibody cocktails containing CD14-PECy5, CD42a-PE and 10 μg mL−1 F26-FITC or MIgG1-FITC were incubated simultaneously with an agonist. FACS lysing solution was added and samples were stored at 4°C until analysis.

Constitutive Mac-1 (CD11b) expression assay

Citrate anticoagulated whole blood was diluted 1:2 in HBSS and then incubated at either 22°C (ADP samples) or 37°C (collagen and PMA samples) for 15 min. Three-antibody cocktails containing CD14-PECy5, CD42b-FITC (6D1) and 3 μg mL−1 anti-CD11b-PE (clone LPM19c) or MIgG1-PE were included during the agonist incubations. FACS lysing solution was added and samples were stored at 4°C until analysis.

Activated MAC-1 assay

The PPACK anticoagulated whole blood was diluted 1:2 in HBSS and aliquots were incubated for 15 min at 22°C with buffer or ADP and 37°C with collagen. In this same incubation three-antibody cocktails were included containing CD14-PECy5, CD42a-PE and 10 μg mL−1 mAb24-FITC or MIgG1-FITC. FACS lysing solution was added and samples were stored at 4°C until analysis.

Tissue factor assay

Citrated whole blood was incubated at 37°C for 10 min with 2 or 20 μM ADP, 20 μg mL−1 collagen (final concentrations), or HBSS buffer only (no agonist). Incubation at 37°C is required for tissue factor binding. An aliquot was then transferred to a three-antibody mixture containing 1 μg mL−1 CD14-PECy5, 1 μg mL−1 CD42a-PE and either 10 μg mL−1 anti-tissue factor FITC or MIgG1-FITC. After 20 min incubation at 22°C, 500 μL of FACS lysing solution was added and the samples were analyzed using a BD FACSCalibur flow cytometer as described below.

Flow cytometric analysis

Samples were run on a three color FACSCalibur which was calibrated daily with Spherotech RCP-30-5 rainbow beads. PMT voltages for FL1 through FL3 were adjusted daily to established target values to maintain a consistent fluorescence output. This instrument is equipped with 530/30 band pass, 585/42 band pass and 650 long pass filters for FL1, FL2, and FL3, respectively. Monocytes and neutrophils were initially gated by a combination of light scattering and CD14-PECy5 fluorescence (Fig. 1, upper panels). Dim CD14, forward and 90° light scatter define neutrophils (blue, R1 + R3). Bright CD14 and 90° light scatter define monocytes (red, R2). Because forward light scatter increases significantly upon formation of both monocyte-platelet and neutrophil-platelet aggregates, large gate regions were placed to accommodate these increases. A minimum of 1000 monocytes and 5000 neutrophils was acquired for each determination. All list mode analysis was performed using CellQuest software (Becton Dickinson).

image

Figure 1. Flow cytometry gating logic. Upper panels: monocytes and neutrophils were initially gated by a combination of light scattering and CD14 PECy5 fluorescence. Dim CD14, forward and 90° light scatter define neutrophils (blue, R1 + R3). Bright CD14 and 90° light scatter define monocytes (red, R2). Forward and 90° light scatter increase significantly upon formation of heterotypic aggregates. Lower panels: in this collagen-stimulated example, monocytes (red) and neutrophils (blue) were further gated into CD42a-negative platelet-free (R6 and R8) and CD42a-positive platelet-bound (R5 and R7) sub-populations. Note monoclonal antibody F26 (directed against surface-bound fibrinogen) (x-axis) increases with increased numbers of leukocyte-bound platelets (y-axis).

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Monocytes and neutrophils were further gated (Fig. 1, lower panels) into a CD42a-negative, platelet-free population (regions R6 and R8 used in combination with the leukocyte gates) and a CD42a-positive, platelet-bound populations (regions R5 and R7). Geometric mean fluorescence and per cent positive statistics were collected using single parameter histograms to take advantage of the 1024 channel resolution for more accurate region placement. This was particularly important for the monocytes which are relatively rare compared with neutrophils. Negative control mouse IgG fluorescence values were subtracted from the test antibody geometric mean fluorescence prior to graphic analysis (Microsoft Excel and GraphPad Prism software).

Correct electronic color compensation for fluorescence cross talk is critical in the analysis of leukocyte-platelet aggregates and was determined on six normal donors. Using voltages of 590 (FL1), 490 (FL2), and 500 (FL3) ± 10 V (adjusted daily to target mean fluorescence values), the following average compensation values were used: FL1-1% FL2, FL2 - 14.4% FL1, FL2 - 2% FL3, and FL3 - 20% FL2. Of particular importance is the PE (FL2) bleed-over to the FITC (FL1) channel because of the bright signal which develops in FL2 as leukocyte-platelet aggregates form. Anti-tissue factor and coagulation factor X/Xa signals were comparatively dim, and care was taken to assure an antigen-specific signal. Figure 2 demonstrates that with addition of increasing amounts of anti-CD42a-PE to maximally activated whole blood (10 μm PMA) FL1 - 1% FL2 was adequate to avoid an artefactual increase in mean FL1 fluorescence. As the geometric mean FL2 increased from 70 to nearly 700, there was no increase in FL1 background signal. This is also shown in the representative monocyte gated histograms below the graph in Fig. 2.

image

Figure 2. Flow cytometry color compensation. FITC test antibodies were correctly compensated to eliminate PE signal crossover. In this demonstration, all samples were activated with 10 μm PMA in the presence of increasing concentrations of anti-CD42a-PE (platelet-specific). The FL1 isotype control mean fluorescence did not change, verifying adequate FL1-FL2 color compensation. Data shown are monocytes, mean ± SEM, n = 4. The histograms below are a representative example.

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Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conflicts of interest disclosure
  8. References

Surface expression of bound tissue factor, coagulation FXa and fibrinogen on platelet-bound and platelet-free monocytes and neutrophils

Collagen and ADP resulted in increased monocyte and neutrophil surface expression of bound tissue factor (Fig. 3), coagulation FXa (Fig. 4) and fibrinogen (Fig. 5). Although platelet-free monocytes and neutrophils had a slight increase in the surface expression of bound tissue factor, FXa and fibrinogen in response to ADP (Figs 3, 4 and 5), tissue factor, FXa and fibrinogen bound to platelet-bound monocytes and neutrophils to a much greater extent than to platelet-free monocytes and neutrophils (Figs 3, 4 and 5). Under the conditions employed for the tissue factor and coagulation FXa assays, collagen results in >98% of monocytes and neutrophils to be platelet-bound; there were therefore insufficient platelet-free monocytes and neutrophils to generate data in the collagen-treated samples.

image

Figure 3. Surface expression of tissue factor on platelet-bound monocytes and neutrophils and platelet-free monocytes and neutrophils. These whole blood flow cytometric assays required pre-incubations at 37°C to allow physiological ligand binding prior to adding the test antibody – see Methods for details. Data are mean ± SEM, n = 10. Asterisks indicate P < 0.05 by paired t-test for surface tissue factor with agonist compared with no agonist. Cross symbols indicate P < 0.05 by paired t-test for platelet-free monocytes compared with platelet-bound monocytes and for platelet-free neutrophils compared with platelet-bound neutrophils. There were insufficient platelet-free monocytes or neutrophils after collagen stimulation to generate these data points.

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image

Figure 4. Surface expression of coagulation FXa on platelet-bound monocytes and neutrophils and platelet-free monocytes and neutrophils. These whole blood flow cytometric assays required pre-incubations at 37°C to allow physiological ligand binding prior to adding the test antibody – see Methods for details. Data are mean ± SEM, n = 10. Asterisks indicate P < 0.05 by paired t-test for surface FXa binding with agonist compared with no agonist. Cross symbols indicate P < 0.05 by paired t-test for platelet-free monocytes compared with platelet-bound monocytes and for platelet-free neutrophils compared with platelet-bound neutrophils. There were insufficient platelet-free monocytes or neutrophils after collagen stimulation to generate these data points.

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image

Figure 5. Surface expression of bound fibrinogen on platelet-bound monocytes and neutrophils and platelet-free monocytes and neutrophils. Surface-bound fibrinogen was reported in whole blood by monoclonal antibody F26 – see Methods for details. Data are mean ± SEM, n = 10. Asterisks indicate P < 0.05 by paired t-test for bound fibrinogen binding with agonist compared with no agonist. Cross symbols indicate P < 0.05 by paired t-test for platelet-free monocytes compared with platelet-bound monocytes and for platelet-free neutrophils compared with platelet-bound neutrophils.

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Surface Mac-1 expression on platelet-bound and platelet-free monocytes and neutrophils

Collagen and, to a lesser extent, ADP resulted in increased monocyte and neutrophil surface expression of CD11b (Fig. 6) and activated Mac-1 (integrin αMβ2, CD11b/CD18; Fig. 7). This increase in surface CD11b and activated Mac-1 was slightly, but significant statistically, lower on platelet-free monocytes and neutrophils as compared with platelet-bound monocytes and neutrophils.

image

Figure 6. Surface expression of CD11b on platelet-bound monocytes and neutrophils and platelet-free monocytes and neutrophils. Surface CD11b was reported in whole blood by monoclonal antibody LPM19c – see Methods for details. Data are mean ± SEM, n = 10. Asterisks indicate P < 0.05 by paired t-test for surface CD11b with agonist compared with no agonist. Cross symbols indicate P < 0.05 by paired t-test for platelet-free monocytes compared with platelet-bound monocytes and for platelet-free neutrophils compared with platelet-bound neutrophils.

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image

Figure 7. Surface Expression of activated Mac-1 on platelet-bound monocytes and neutrophils and platelet-free monocytes and neutrophils. Activated Mac-1 was reported in whole blood by monoclonal antibody MAb24 – see Methods for details. Data are mean ± SEM, n = 10. Asterisks indicate P < 0.05 by paired t-test for activated Mac-1 with agonist compared with no agonist. Cross symbols indicate P < 0.05 by paired t-test for platelet-free monocytes compared with platelet-bound monocytes and for platelet-free neutrophils compared with platelet-bound neutrophils.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conflicts of interest disclosure
  8. References

In summary, (i) we have developed whole blood flow cytometry assays to measure bound tissue factor, coagulation FXa, fibrinogen, activated Mac-1 (integrin αMβ2, CD11b/CD18) and CD11b on the surface of monocytes and neutrophils, allowing independent analysis of monocytes and neutrophils with and without surface-adherent platelets. These novel methods will be applicable to future in vivo and in vitro studies of pharmacological agents targeted to either coagulation and/or cellular activation processes. (ii) The monocyte and neutrophil surface binding of tissue factor, coagulation FXa and fibrinogen is mainly dependent on platelet adherence to the monocytes and neutrophils, whereas the monocyte and neutrophil surface expression of CD11b and activated Mac-1 is mainly independent of platelet adherence to the monocytes and neutrophils.

Circulating leukocyte-platelet aggregates are increased in stable coronary artery disease [19], unstable angina [20], acute myocardial infarction [21,22], and cardiopulmonary bypass [23]. Circulating leukocyte-platelet aggregates also increased after coronary angioplasty, with a greater magnitude in patients experiencing late clinical events [24]. Circulating monocyte-platelet aggregates are a more sensitive marker of in vivo platelet activation than platelet surface P-selectin in the clinical settings of stable coronary artery disease [19], percutaneous coronary intervention [25], and acute myocardial infarction [25]. In addition, circulating monocyte-platelet aggregates are an early marker of acute myocardial infarction [26]. Increased circulating monocyte-platelet and or neutrophil-platelet aggregates have also been demonstrated in peripheral venous disease [27], hemodialysis [28], sickle cell disease [29], systemic inflammatory response syndrome [30], septic multiple organ dysfunction syndrome [31,32], antiphospholipid syndrome [33], systemic lupus erythematosus [33], rheumatoid arthritis [33], myeloproliferative disorders [34,35], and Alzheimer's disease [36]. High levels of circulating monocyte-platelet aggregates can predict rejection episodes after orthotopic liver transplantation [37].

However, the function of these circulating monocyte-platelet and neutrophil-platelet aggregates is not well characterized. At in vivo sites of local injury, leukocyte-platelet aggregates are procoagulant [38]. Thus, the findings of the present study, that the monocyte and neutrophil surface binding of tissue factor, coagulation FXa and fibrinogen is mainly dependent on platelet adherence to the monocytes and neutrophils, suggests that monocyte-platelet and neutrophil-platelet aggregates are vehicles for the local delivery of surface bound tissue factor, coagulation FXa and fibrinogen – each of which is a key component of the local coagulant response, with its end result of a stable fibrin clot [11]. Although the origin of tissue factor exposure on the surface of leukocytes is controversial, our findings are consistent with the concept that tissue factor is of intraplatelet origin and that tissue factor is transferred from platelets to leukocytes [39–42].

Conflicts of interest disclosure

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conflicts of interest disclosure
  8. References

This study was funded in part by Bristol-Myers Squibb/Sanofi Aventis.

References

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  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conflicts of interest disclosure
  8. References
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