The endothelial specific isoform of type XVIII collagen correlates to annual bleeding rate in haemophilia patients

Abstract

Introduction: The medical need in the haemophilic (HF) field to reduce bleeding incidents requires measurement of the annual bleeding rate (ABR) in haemophiliacs. Vascular rupture is associated with damage to the vascular endothelium causing exposure of the basement membrane. Endothelial cells and matrix impairment may be associated with joint bleeds and later development of HF arthropathy. Imbalanced extracellular matrix turnover is a central pathological feature in many diseases consequent to epithelial or endothelial cell damage. Type XVIII collagen is an essential basement membrane component, with an endothelial specific isoform.

Aim: To quantify the basement membrane specifically for the endothelial cells, as that may have particular relevance to endothelial cell stability and rupture in haemophiliacs. A newly developed ELISA assay detecting endothelial type XVIII collagen (COL-18N) was used to assess the clinical relevance of endothelial basement membrane turnover in patients diagnosed with HF arthropathy and correlation to ABR.

Methods: We developed an ELISA assay for quantification of COL-18N. Serum from 35 male HF patients was investigated using the COL-18N ELISA.

Results: COL-18N correlated to the ABR of haemophiliacs, r = 0.45, P<0.006.

Conclusion: Vascular rupture and consequent bleeding are associated with joint damage and deterioration of life quality in haemophiliacs. Quantification of ABR is an important part in efficacy assessment of different interventions, and the benchmark of these. Objective biomarkers reflecting endothelial dysfunction, vascular leaks and rupture, like the COL-18N biomarker that associate with ABR, may assist in identifying the most optimal treatment and monitoring of HF patients.

Fig 1. Type XVIII collagen location and structure.

(A) Structure of the vascular basement membrane (BM). The capillary subendothelial layer is composed of a BM and an interstitial matrix (IM). The main components of vascular basement membranes include type IV collagen, laminin and nidogen. Minor components include type XV collagen and type XVIII collagen. The components of the BM self-assemble into sheet-like structures. The BM is tightly connected to the IM through interactions between collagen type I and VI and collagen type IV and XV. (B) Isoforms of Type XVIII collagen. Collagen type XVIII exists in three isoforms, which differ in their N-terminus. The COL18A1 gene encodes these variants by the use of two promoters and alternative splicing. The short isoform has a different signal peptide from the other two, and coded by promoter 1, while the others have the same signal peptide and both coded by promoter 2. All isoforms include a thrombospondin-like domain, heparin sulphate chains and a globular C-terminal containing the type XVIII collagen fragment, endostatin.

Fig 2. COL-18N antibody specific reactivity against type XVIII collagen.

Monoclonal NB632-13H11/G5 antibody reaction towards standard peptide, truncated, elongated and de-selection peptides. The antibody has high reactivity towards standard peptide and no or minimal cross-reactivity with the other peptides.

Fig 3. Biomarker COL-18N correlate with ABR.

Serum from 35 male HF patients aged 26 and over was measured with the COL-18N ELISA. Correlations between vascular endothelial type XVIII collagen concentration and ABR were analyzed using Spearman rank correlation coefficient and shown r = 0.45, p<0.006. Differences between ABR and COL-18N levels were considered statistically significant if p<0.05 and significant levels are displayed as: * = p<0.05; ** = p<0.01, and *** = p<0.001.