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M. Brusson et al.
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Figure 3. Increased adhesion to laminin of red blood cells (RBCs) from hydroxycarbamide (HC)-treated patients. (A) Adhesion to laminin at 3 dyn/cm2 of RBCs from 11 control (CT), 17 untreated (UT), 16 HC-treated (HC) and 11 IFN-treated (IFN) polycythemia vera patients. Horizontal lines represent medians. (B) The JAK2V617F allele burden (%JAK2V617F). RBC adhesion as a function of (C) %JAK2V617F (R2=0.048) and (D) Lu/BCAM mean fluorescence intensity (MFI) (R2=0.67) for 16 HC- treated patients.
when compared to UT patients. However, we observed increased expression of CD147 in the HC group (median MFI=8472) when compared to the UT group (median MFI=5406) (P<0.0001, Mann-Whitney test) (Figure 2C) suggesting a common upregulation of Lu/BCAM and CD147 by HC. This increase was confirmed by pro- teomics in the 3 pre-post patients who showed increased CD147 expression during HC treatment (Table 2).
Increased RBC adhesion to laminin in HC-treated patients
As Lu/BCAM mediates abnormal adhesion of PV RBCs to laminin,17 we examined the effect of HC treatment on PV RBC adhesion by performing adhesion assays under flow conditions. RBCs from HC-treated patients adhered much more than those from UT patients, with respective medians of 969 and 231 RBCs/mm2 at 3 dyn/cm2 (P=0.0047, Mann-Whitney test) whereas no significant difference was found between the IFN (271 RBCs/mm2) and UT groups (P=0.8508, Mann-Whitney test) (Figure 3A). Adhesion of RBCs from 6 control donors was minor (median=37 RBCs/mm2), and significantly lower than in the UT, HC and IFN groups (P<0.01, Mann-Whitney test).
The impact of HC and IFN on cell adhesion was also analyzed by comparing the UT, HC and IFN groups in terms of RBC adhesion value distributions. We found that these values were broadly dispersed in the UT and HC groups but were less scattered in the IFN group (Figure 3A), with a significant difference between the variance of the IFN group as compared to the UT (P<0.05) or the HC
group (P<0.05) (Fisher exact test). This was probably linked to the JAK2V617F allele burden of each group. As a matter of fact, we have previously shown that PV RBC adhesion to laminin in UT patients was correlated with the JAK2V617F allele burden, defined as the percentage of circulating JAK2 alleles with the V617F mutation (%V617F).26 We determined the %V617F of all patients and, as expected and as reported in previous studies,13 the median was significantly lower in the IFN than in the UT and HC groups (Figure 3B). Moreover, we found no signif- icant difference between the UT and the HC groups, despite a lower median in the latter (Figure 3B), indicating that increased RBC adhesion in the HC group was inde- pendent of the %V617F and was most probably due to the expression level of Lu/BCAM. This was demonstrated by plotting RBC adhesion values against the percentage of JAK2V617F (Figure 3C) and the MFI of Lu/BCAM (Figure 3D).
Longitudinal study of HC treatment in 4 PV patients
As only HC had activating effects on RBC adhesion, and because of wide interindividual variability in PV, we fur- ther investigated the effects of HC treatment in the 4 pre- post HC patients of our cohort by performing adhesion and flow cytometry assays. This longitudinal analysis showed a great increase of adhesion after HC treatment for all 4 patients (Figure 4A and B), confirming the results obtained with the UT and HC groups. As seen in the UT and HC groups, we found that the increased adhesion was associated with increased Lu/BCAM expression, both in
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