Primary Myelofibrosis Is Associated with Truncation of the Plasma Chemokine SDF-1
Authors: Cho Sool Yeon , Roboz John , Xu Mingjiang , Hoffman Ronald 
Published: October 2019

Abstract
Primary myelofibrosis (PMF) is a myeloproliferative disorder (MPD) characterized by abnormal cellular trafficking leading to constitutive mobilization of CD34+ cells. This abnormal trafficking has been associated with elevation of the plasma and marrow levels of the chemokine SDF-1 and reduced expression of its receptor CXCR4 by CD34+ cells. We have reported that plasma levels of matrix metalloproteinases (MMPs) and neutrophil elastase (NE) are elevated in the plasma of patients with PMF and polycythemia vera (PV). Such proteases are capable of degrading VCAM-1 and SDF-1 both of which function to retain CD34+ cells within the marrow (Xu et al. Blood, 105:4508, 2005). The elevation of SDF-1 both in the blood and marrow of PMF patients has been difficult to reconcile with the mobilization of CD34+ cells. It has been reported that several plasma proteases truncate SDF-1 by removing two or more amino acids from the N-terminal. The degradation of SDF-1 results in the generation of truncated forms of SDF-1 which can compete with the intact SDF-1 for CXCR4, possibly leading to the release of CD34+ cells from the marrow. We studied the truncating activities of dipeptidyl peptidase IV (CD26), NE, MMP, and cathepsin G (CG). We characterized the truncated form SDF-1 in vitro upon incubation of recombinant SDF-1 with each of these individual proteases and analyzed the truncated SDF-1 forms in the plasma of patients with MPD. Electrospray ionization mass spectrometry was used
to determine characteristic multiply charged ions from the truncation products and, to search for these products directly in MPD patient plasma by monitoring up to 15 representative masses (obtained in incubation experiments).
We confirmed the expected molecular mass of SDF-1, using the masses of the +6, +7, +8 multiply charged ions. The estimated quantity of SDF-1 in normal plasma (n=3) was 6 ng/mL. The corresponding amount in plasma from patients with PV (n=5) and PMF (n=3) were 2 to 5-fold greater. We have utilized the same approach to determine the degree of SDF-1 truncation due to CD26 (removal of two amino acids (aa), NE (removal of 3 aa), MMP (removal of 4 aa), and CG (removal of 5 aa, also yielded products corresponding to the removal of 2, 3 and 4 aa). Based on these results, we have monitored these representative masses to search for the truncated SDF-1 forms in MPD. No detectable levels of the truncated forms of SDF-1 was found in normal plasma (n=3), since the peaks were below the estimated detection limit of 500 pg/mL. By contrast, each of the truncated products was detected in the MPD plasma. Intact (full length) SDF-1 was present in MPD plasma, but the total quantity of the truncated forms was 3–4 fold greater than that of intact SDF-1. In the PV samples, the estimated quantity of each truncated SDF-1 form due to MMP, NE, and CD26 activity was similar (approx. 12 ng/mL); the corresponding amounts in the PMF plasma samples were approx. two-fold greater than that present in PV plasma. The quantity of the 5 aa truncated form was 19 ng/mL in PV and 53 ng/mL in PMF samples. These data suggest that proteases present in the plasma of MPD patients digest SDF-1 leading to the generation of several truncated forms of SDF-1. We hypothesize that these truncated forms of SDF-1 likely compete with the full length form of SDF-1 for CXCR4 expression by CD34+ cells leading to the release of CD34+ cells from the marrow and their constitutive mobilization.