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. 2020 Jul;99(7):1531-1542.
doi: 10.1007/s00277-020-04075-6. Epub 2020 May 19.

LPS-induced expression and release of monocyte tissue factor in patients with haemophilia

Katharina Holstein #  1 , Anna Matysiak #  1 , Leonora Witt  1 , Bianca Sievers  1 , Lennart Beckmann  1 , Munif Haddad  2 , Thomas Renné  2 , Minna Voigtlaender  1 , Florian Langer  3
Affiliations

LPS-induced expression and release of monocyte tissue factor in patients with haemophilia

Katharina Holstein et al. Ann Hematol. 2020 Jul.

Abstract

In haemophilia, thrombin generation and fibrin deposition upon vascular injury critically depend on the tissue factor (TF)-driven coagulation pathway. TF expression by monocytes/macrophages and circulating microvesicles contributes to haemostasis, thrombosis and inflammation. Inflammation is a hallmark of blood-induced joint disease. The aim of this study is to correlate TF production by whole-blood monocytes with inflammatory markers and clinical parameters in patients with moderate-to-severe haemophilia A or B (n = 43) in comparison to healthy males (n = 23). Monocyte TF antigen and microvesicle-associated TF procoagulant activity (MV TF PCA) were measured immediately after blood draw (baseline) and following incubation of whole blood with buffer or lipopolysaccharide (LPS) using two-colour flow cytometry and chromogenic FXa generation assay, respectively. Patients with HIV or uncontrolled HBV/HCV infections were excluded. TF was hardly detectable and not different in baseline and buffer-treaded samples from both groups. Stimulation with LPS, however, induced monocyte TF production, with increased TF-specific mean fluorescence intensity (P = 0.08) and MV TF PCA (P < 0.05) in patients compared to controls. Patients also had elevated hs-CRP and IL-6 serum levels (P < 0.001), which correlated with LPS-induced TF parameters. Further exploratory analyses revealed that the presence of systemic (low-grade) inflammation and boosted LPS-induced monocyte TF production were mainly restricted to patients with clinically controlled HBV and/or HCV infection (n = 16), who were older and also had a significantly worse orthopaedic joint score than patients with no history of viral hepatitis (P < 0.01). Our study delineates a previously unrecognised link between systemic inflammation and inducible monocyte TF production in patients with haemophilia A or B.

Keywords: Haemophilia; Hepatitis; Microvesicles; Monocytes; Tissue factor.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Monocyte TF antigen expression and release of MV-associated TF PCA in patients and controls. a TF antigen on whole-blood monocytes was analysed by two-colour flow cytometry both at baseline and after incubation for 4 h at 37 °C with buffer (PBS) or lipopolysaccharide (LPS). AU denotes arbitrary units. b Plasma microvesicles (MVs) were isolated from baseline and PBS- or LPS-treated whole blood and analysed for TF-specific procoagulant activity (PCA) using a chromogenic FXa generation endpoint assay. Results are presented as AU per 200 μL of platelet-poor plasma. Horizontal bars indicate median MV TF PCA levels. The P value is according to Mann-Whitney U test. c MV-associated TF PCA was plotted against TF-specific mean fluorescence intensity (MFI) of monocytes. Values were obtained from LPS-treated patient samples. Correlation coefficient (r) and P value are according to the method of Spearman. Values for MV TF PCA are missing for one patient and two controls
Fig. 2
Fig. 2
hs-CRP and IL-6 serum levels in patients and controls. Antigen levels of high-sensitivity C-reactive protein (hs-CRP, a) and interleukin-6 (IL-6, b) were measured in baseline serum samples from patients and controls. Median levels and upper limits of the normal reference ranges are indicated by horizontal bars and dashed lines, respectively. P values are according to Mann-Whitney U test
Fig. 3
Fig. 3
Correlations between hs-CRP/IL-6 and LPS-induced monocyte TF in the patient cohort. Baseline serum levels of hs-CRP (a) and IL-6 (b) were plotted against monocyte TF antigen, expressed as TF-specific MFI, and release of MV-associated TF PCA. Values were obtained from LPS-treated patient samples. Correlation coefficients (r) and P values are according to the method of Spearman. AU denotes arbitrary units. The value for MV TF PCA is missing for one patient
Fig. 4
Fig. 4
Association of the HBV/HCV infection status with inflammatory markers and LPS-induced monocyte TF. Baseline serum levels of hs-CRP (a) and IL-6 (b), results for LPS-induced monocyte TF antigen, expressed as TF-specific MFI (c), and release of MV-associated TF PCA (d) are shown for healthy male controls (n = 23) and patients with (n = 16) or without positive HBV/HCV test results (n = 27) at study inclusion. P values are according to ANOVA and Tukey’s post hoc test (c) or the Kruskal-Wallis and Dunn’s post hoc test (a, b, d). AU denotes arbitrary units. Values for MV TF PCA are missing for one patient and two controls
Fig. 5
Fig. 5
Correlation of the OJS with inflammatory markers and association of haemophilic arthropathy with the HBV/HCV infection status. Baseline serum levels of hs-CRP (a) and IL-6 (b) were plotted against the orthopaedic joint score (OJS). Correlation coefficients (r) and P values are according to the method of Spearman. c The OJS was assessed in patients with (n = 16) or without positive HBV/HCV test results (n = 27) at study inclusion. Horizontal bars indicate median OJS. The P value is according to Mann-Whitney U test. d Black bars indicate proportions of patients with clinically significant haemophilic arthropathy, defined by an OJS > 4, as a function of the HBV/HCV infection status. The P value is according to Fisher’s exact test
Fig. 6
Fig. 6
Hypothetical role of monocyte TF in haemophilia-associated inflammation. In adult patients with moderate-to-severe haemophilia, clinically significant arthropathy and a positive HBV/HCV infection status are still frequently encountered complications caused by the bleeding disorder and its treatment, respectively. Both conditions contribute to systemic (low-grade) inflammation and share advanced patient age as a common determinant. Based on our current study and previous literature on the role of monocyte/macrophage TF in thrombo-inflammation, we hypothesise that pro-inflammatory pathways prime whole-blood monocytes to boost TF production in response to pathophysiological stimuli (a desirable effect in the context of deficient thrombin generation and fibrin formation), while monocyte TF may reciprocally fuel inflammation, e.g. through PAR-dependent signalling, thus potentially contributing to synovitis and hepatitis
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