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. 2011 Jul;96(7):954-62.
doi: 10.3324/haematol.2010.029298. Epub 2011 May 5.

Loss of Expression of Neutrophil proteinase-3: A Factor Contributing to Thrombotic Risk in Paroxysmal Nocturnal Hemoglobinuria

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Free PMC article

Loss of Expression of Neutrophil proteinase-3: A Factor Contributing to Thrombotic Risk in Paroxysmal Nocturnal Hemoglobinuria

Anna M Jankowska et al. Haematologica. .
Free PMC article

Abstract

Background: A deficiency of specific glycosylphosphatidyl inositol-anchored proteins in paroxysmal nocturnal hemoglobinuria may be responsible for most of the clinical features of this disease, but some functional consequences may be indirect. For example, the absence of certain glycosylphosphatidyl inositol-anchored proteins in paroxysmal nocturnal hemoglobinuria cells may influence expression of other membrane proteins. Membrane-bound proteinase 3 co-localizes with glycosylphosphatidyl inositol-linked neutrophil antigen 2a, which is absent in patients with paroxysmal nocturnal hemoglobinuria.

Design and methods: We compared expression of proteinase 3 and neutrophil antigen 2a by flow cytometry and western blotting in normal and paroxysmal nocturnal hemoglobinuria cells and measured cytoplasmic and soluble levels of proteinase 3 by enzyme-linked immunosorbent assays in controls and patients with paroxysmal nocturnal hemoglobinuria. Finally, we studied the effects of proteinase 3 on platelet activation using an in vitro aggregometry assay and flow cytometry.

Results: We showed that membrane-bound proteinase 3 is deficient in patients' cells, but invariantly present in the cytoplasm regardless of disease phenotype. When we isolated lipid rafts from patients, both molecules were detected only in the rafts from normal cells, but not diseased ones. Membrane-bound proteinase 3 was associated with a decrease in plasma proteinase 3 levels, clone size and history of thrombosis. In addition, we found that treating platelets ex vivo with proteinase 3, but not other agonists, decreased the exposure of an epitope on protease activated receptor-1 needed for thrombin activation. Conversely, treatment of whole blood with serine protease inhibitor enhanced expression of this epitope on protease activated receptor-1 located C-terminal to the thrombin cleavage site on platelets.

Conclusions: We demonstrated that deficiency of glycosylphosphatidyl inositol-anchored proteins in paroxysmal nocturnal hemoglobinuria results in decreased membrane-bound and soluble proteinase 3 levels. This phenomenon may constitute another mechanism contributing to a prothrombotic propensity in patients with paroxysmal nocturnal hemoglobinuria.

Figures

Figure 1.
Figure 1.
NB1 and PR3 surface expression in controls and patients with PNH. Freshly obtained neutrophils were stained using monoclonal antibodies directed against NB1 (CD177), PR3 and CD16. The panels represent flow dot blots/histograms (A) from a healthy individual and (B) from a PNH patient with a PNH clone size of 67% of granulocytes determined by expression of the GPI-AP CD16. In healthy subjects, CD16 is expressed on the majority of neutrophils; the NB1 surface staining is bimodal and coincides with mPR3 expression. In patients with PNH, CD16 GPI cells expressing NB1 and mPR3 were not found. In contrast, co-expression of NB1 and mPR3 was found in GPI+ cells of the same patient.
Figure 2.
Figure 2.
PR3 neutrophil membrane expression requires expression of GPI-linked NB1. (A) The percentage of mPR3-expressing cells is plotted as a function of the percentage of NB1 expressing cells. Percentages were nearly identical in all donors, independently of the proportion of mPR3+ cells (r=0.986, n=24). (B) Loss of GPI-AP proteins after cleavage with phosphatidylinositol-specific phospholipase C (PI-PLC) (1U; 30 min at 37°C) results in decreased membrane expression of NB1 accompanied by a similar reduction of mPR3 expression. (C) Treatment of GPI-AP+ neutrophils (as determined by CD16 expression) from a PNH patient with TNF-α induced up-regulation of mPR3 and surface NB1 whereas no such increase was detected when GPI-AP cells were stimulated by TNF-α.
Figure 3.
Figure 3.
Cytoplasmic PR3 is expressed normally in patients with PNH (A) Double immunofluorescence staining of permeabilized (right group of histograms) and non-permeabilized (left group) neutrophils using antibodies to PR3 and NB1 along with GPI-linked surface molecule CD16. All neutrophils contain intracellular PR3, independent of the status of GPI-AP expression. In contrast, NB1 was only detected in GPI-AP-expressing cells. (B) Western blot analysis showing PR3 in the membrane fraction of GPI-AP+ cells, but not GPI-AP cells.
Figure 4.
Figure 4.
PR3 and NB1 localize in lipid rafts. Detergent-resistant membranes (DRM) were isolated from GPI-AP+ (A) and GPI-AP (B) neutrophils derived from a PNH patient or control and subjected to immunoblot analysis for CD55, PR3, and NB1. CD55 was seen in the DRM fractions 3 and 4 from GPI-AP+ cells, but not from GPI-AP cells and was also present in soluble form in the cellular proteome of both cell types (lane 1). Similarly, PR3 and NB1 were detected in the DRM fractions in GPI-AP+ cells, but not in GPI-AP cells (lanes 2–3). PR3 was detected in the non-DRM fractions of both cell types, while NB1 was not seen either in DRM (fractions 3 and 4) or in the cellular form (lanes 2–3). Fractions 1, 2, 3 and 4 contain DRM enriched proteins; fractions 9, 10 and 11 represent non-DRM enriched proteins. The remaining fractions (5, 6, 7 and 8) represent migration of buoyant rafts. Lipid rafts are mostly localized in fractions 3 and 4.
Figure 5.
Figure 5.
Circulating levels of PR3 are decreased in plasma from patients with PNH. Plasma PR3 levels were plotted as a function of PNH clone percentage (A) in patients with PNH and aplastic anemia (AA)/PNH syndrome. The percentage of GPI-AP neutrophils was determined at the time of plasma sampling by flow cytometry. Solid circles represent patients without a history of thrombosis (n=27). Open circles represent patients with a history of thrombotic events (n=8). A negative correlation between PNH clone size and PR3 plasma level was observed, n=36, P=0.0093 (Spearman’s non-parametric correlation coefficient was −0.42). (B) Plasma PR3 levels were plotted as a function of percentage of NB1/mPR3+ neutrophils in individuals with the NB1-null phenotype and those with unrelated conditions characterized by decreased membrane-bound NB1/PR3 expression (≤mean±2 standard deviations of 50 healthy controls) (r=−0.32, n=18, P=0.19). The solid line represents the linear regression and broken lines show 95% confidence intervals. (C) The concentration of PR3 was measured by enzyme-linked immunosorbent assay in 49 healthy donors, 18 subjects with unrelated hematologic conditions, and 40 patients with PNH or AA/PNH syndrome. Decreased levels of PR3 were seen in PNH and AA/PNH patients (mean 201 ng/mL) compared to those in both control groups (mean 256 ng/mL for normal subjects and 311 ng/mL for those with unrelated hematologic disorders). (D) Plasma PR3 levels in patients grouped by history of thrombosis. Lower levels of PR3 were seen in those who had had thrombotic events (mean 160 ng/mL) compared to other patients (mean 213 ng/mL). Student’s t test was used to determine statistical significance.
Figure 6.
Figure 6.
PR3-treated platelets show decreased activation and aggregation induced by thrombin. (A) Pretreatment of platelets with 400 nM PR3 (5 min at 37°C) resulted in significant loss of binding of a monoclonal antibody against WEDE (an epitope on PAR-1 located C-terminal to the thrombin cleavage site. (B) Representative flow cytometry histograms showing inhibition of surface expression of CD62P in PR3–treated platelets following exposure of the platelets to thrombin, ADP or TRAP. PR3 treatment (400 nM, 5 min at 37°C) led to loss of thrombin-induced CD62P expression, but did not affect ADP- or TRAP-induced expression (black line represents isotype control; blue line- PR3-treated platelets, solid line- untreated platelets). (C) Pre-incubation of platelets from seven healthy donors for 5 min with PR3 (400 nM) resulted in a 35–75% inhibition of CD62P surface expression after exposure to thrombin (0.2 U/mL). The dashed line represents the mean level of inhibition. (D) Pretreatment of washed platelets from four healthy donors with PR3, as in panel (C), inhibited thrombin-induced platelet aggregation by 10–58%. The dashed line represents the mean level of inhibition.

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