Characterization of anticoagulant heparinoids by immunoprofiling

Heparinoids are used in the clinic as anticoagulants. A specific pentasaccharide in heparinoids activates antithrombin III, resulting in inactivation of factor Xa and–when additional saccharides are present–inactivation of factor IIa. Structural and functional analysis of the heterogeneous heparinoids generally requires advanced equipment, is time consuming, and needs (extensive) sample preparation. In this study, a novel and fast method for the characterization of heparinoids is introduced based on reactivity with nine unique anti-heparin antibodies. Eight heparinoids were biochemically analyzed by electrophoresis and their reactivity with domain-specific anti-heparin antibodies was established by ELISA. Each heparinoid displayed a distinct immunoprofile matching its structural characteristics. The immunoprofile could also be linked to biological characteristics, such as the anti-Xa/anti-IIa ratio, which was reflected by reactivity of the heparinoids with antibodies HS4C3 (indicative for 3-O-sulfates) and HS4E4 (indicative for domains allowing anti-factor IIa activity). In addition, the immunoprofile could be indicative for heparinoid-induced side-effects, such as heparin-induced thrombocytopenia, as illustrated by reactivity with antibody NS4F5, which defines a very high sulfated domain. In conclusion, immunoprofiling provides a novel, fast, and simple methodology for the characterization of heparinoids, and allows high-throughput screening of (new) heparinoids for defined structural and biological characteristics

Differential Expression of Specific Dermatan Sulfate Domains in Renal Pathology.

Dermatan sulfate (DS), also known as chondroitin sulfate (CS)-B, is a member of the linear polysaccharides called glycosaminoglycans (GAGs). The expression of CS/DS and DS proteoglycans is increased in several fibrotic renal diseases, including interstitial fibrosis, diabetic nephropathy, mesangial sclerosis and nephrosclerosis. Little, however, is known about structural alterations in DS in renal diseases. The aim of this study was to evaluate the renal expression of two different DS domains in renal transplant rejection and glomerular pathologies. DS expression was evaluated in normal renal tissue and in kidney biopsies obtained from patients with acute interstitial or vascular renal allograft rejection, patients with interstitial fibrosis and tubular atrophy (IF/TA), and from patients with focal segmental glomerulosclerosis (FSGS), membranous glomerulopathy (MGP) or systemic lupus erythematosus (SLE), using our unique specific anti-DS antibodies LKN1 and GD3A12. Expression of the 4/2,4-di-O-sulfated DS domain recognized by antibody LKN1 was decreased in the interstitium of transplant kidneys with IF/TA, which was accompanied by an increased expression of type I collagen, decorin and transforming growth factor beta (TGF-β), while its expression was increased in the interstitium in FSGS, MGP and SLE. Importantly, all patients showed glomerular LKN1 staining in contrast to the controls. Expression of the IdoA-Gal-NAc4SDS domain recognized by GD3A12 was similar in controls and patients. Our data suggest a role for the DS domain recognized by antibody LKN1 in renal diseases with early fibrosis. Further research is required to delineate the exact role of different DS domains in renal fibrosis

Localization and functional characterization of glycosaminoglycan domains in the normal human kidney as revealed by phage display-derived single chain antibodies

Glycosaminoglycans (GAG) play an important role in renal homeostasis. They are strongly negatively charged polysaccharides that bind and modulate a myriad of proteins, including growth factors, cytokines, and enzymes. With the aid of specific phage display-derived antibodies, the distribution of heparan sulfate (HS) and chondroitin sulfate (CS) domains in the normal human kidney was studied. HS domains were specifically located in basement membranes and/or surfaces of renal cells and displayed a characteristic distribution over the nephron. A characteristic location in specific parts of the tubular system was also observed. CS showed mainly an interstitial location. Immunoelectron microscopy indicated specific ultrastructural location of domains. Only partial overlap with any of seven different proteoglycan core proteins was observed. Two HS domains, one highly sulfated (defined by antibody HS4C3) and one low sulfated (defined by antibody RB4Ea12), were studied for their cell biologic relevance with respect to the proliferative effect of FGF-2 on human mesangial cells in vitro. Fibroblast growth factor 2 (FGF-2) binding was HS dependent. Addition of purified HS4C3 antibody but not of the RB4Ea12 antibody counteracted the binding and the proliferative effect of FGF-2, indicating that the HS4C3 domain is involved in FGF-2 handling by mesangial cells. In conclusion, specific GAG domains are differentially distributed in the normal human kidney and are likely involved in binding of effector molecules such as FGF-2. The availability of tools to identify and study relevant GAG structures allows the development of glycomimetica to halt, for instance, mesangial proliferation and matrix production as seen in diabetic nephropathy

Clinical characteristics of transplanted patients with rejection.

<p>Abbreviations: SCreat, Serum creatinine; ESRD, End Stage Renal Disease; MPGN, Membranoproliferative glomerulonephritis; ANCA-vasculitis, Anti-neutrophil cytoplasmic antibodies-associated vasculitis; N.A., not available.</p><p>Clinical characteristics of transplanted patients with rejection.</p

Semi-quantitative analysis of the expression the 4/2,4-di-O-sulfated and IdoA-Gal-NAc4S DS domains defined by LKN1 and GD3A12, type I collagen, decorin and TGF-β in the interstitium (A) and glomeruli (B) of patients with FSGS, MGP and SLE.

<p>Staining intensities of the 4/2,4-di-O-sulfated and IdoA-Gal-NAc4S DS domains defined by LKN1 and GD3A12 respectively and of type I collagen, decorin and transforming growth factor beta (TGF-β) were scored using a scale of 0–4 and revealed a significantly increased expression of the 4/2,4-di-O-sulfated DS domain and type I collagen in the interstitium (A) and glomeruli (B) of patients with glomerular diseases. Glomerular expression of TGF-β also was increased in patients with glomerular diseases (B), while expression of the IdoA-Gal-NAc4S DS domain and decorin was similar in the interstitium (A) of patients and controls. *P<0.05 vs control.</p

Expression of the 4/2,4-di-O-sulfated and IdoA-Gal-NAc4S DS domains defined by the antibodies LKN1 (A-D) and GD3A12 (E-H), type I collagen (I-L), decorin (M-P) and TGF-β (Q-T) in renal allograft rejection and controls.

<p>Representative photographs showing the expression of the 4/2,4-di-O-sulfated DS domain defined by LKN1 in the control human kidneys (A). Tubular interstitial expression of this 4/2,4-di-O-sulfated DS domain defined by LKN1 was increased in acute interstitial (B) and acute vascular (C) renal allograft rejections compared to interstitial fibrosis and tubular atrophy (IF/TA) (D). Expression of the IdoA-Gal-NAc4S DS domain recognized by GD3A12 was similar in the control human kidney and the three types of renal allograft rejection (E-H), while expression of type I collagen (coll I) and decorin was increased in IF/TA (L, P) compared to the control human kidney (I, M), and acute interstitial (J, N) and acute vascular renal allograft rejections (K, O). Glomerular expression of transforming growth factor beta (TGF-β) was increased in the three types of renal allograft rejection (R-T) compared to the control human kidney (Q). Magnification A-P 100x, magnification Q-T 200x.</p

Correlation between the expression of the 4/2,4-di-O-sulfated DS domain recognized by antibody LKN1 and type I collagen (A) and the expression ratio of LKN1 and GD3A12 (B) in glomerular diseases, acute rejection and IF/TA.

<p>In acute (vascular and interstitial) rejection, the interstitial expression of the 4/2,4-di-O-sulfated DS domain recognized by antibody LKN1 is increased, while expression of collagen type I is decreased (A). Expression of collagen type I is increased in glomerular diseases and IF/TA, which is accompanied by an increased expression of the 4/2,4-di-O-sulfated DS domain in glomerular diseases and a decreased expression of the 4/2,4-di-O-sulfated DS domain in IF/TA. The expression ratio of the 4/2,4-di-O-sulfated DS domain recognized by antibody LKN1 to the IdoA-Gal-NAc4S DS domain recognized by antibody GD3A12 is decreased in the IF/TA patients in contrast to the patients with acute rejection or glomerular diseases (B).</p

Semi-quantitative analysis of the expression of type I collagen, decorin, TGF-β and the 4/2,4-di-O-sulfated and IdoA-Gal-NAc4S DS domains defined by LKN1 and GD3A12 in the interstitium (A) and glomeruli (B) in renal allograft rejection.

<p>Staining intensities of type I collagen, decorin, transforming growth factor beta (TGF-β) and the DS domains defined by LKN1 and GD3A12 were scored using a scale of 0–4 and revealed a significantly increased tubular interstitial expression of type I collagen, decorin and TGF-β and a decreased expression of the 4/2,4-di-O-sulfated DS domain recognized by LKN1 in interstitial fibrosis and tubular atrophy (IF/TA). Expression of the IdoA-Gal-NAc4S DS domain recognized by GD3A12 was similar in controls and the three types of renal allograft rejection (A). Glomerular expression of the 4/2,4-di-O-sulfated DS domain defined recognized by LKN1 and TGF-β was increased in the acute interstitial, acute vascular and chronic renal allograft rejections compared to controls (B). *P<0.05 vs control.</p

Expression of the 4/2,4-di-O-sulfated and IdoA-Gal-NAc4S DS domains defined by LKN1 (A-D) and GD3A12 (E-H), type I collagen (I-L), decorin (M-P) and TGF-β (Q-T) in renal sections obtained from patients with glomerular diseases.

<p>Representative photographs showing the immunofluorescence stainings of renal sections obtained from controls (A, E, I, M, Q), patients with focal segmental glomerulosclerosis (FSGS) (B, F, J, N, R), membranous glomerulopathy (MGP) (C, G, K, O, S) and systemic lupus erythematosus (SLE) (D, H, L, P, T) with the anti-DS antibodies LKN1 (A-D) and GD3A12 (E-H), anti-collagen type I (Coll I) (I-L), decorin (M-P) and transforming growth factor beta (TGF-β) (Q-T). Tubular interstitial and glomerular expression of the 4/2,4-di-O-sulfated DS domain defined by LKN1 and of type I collagen was increased in FSGS (B, J), MGP (C, K) and SLE (D, L) compared to control human kidney (A, I). Expression of the IdoA-Gal-NAc4S DS domain, recognized by GD3A12, and decorin was similar in the interstitium and glomeruli of patients (F-H, N-P) and controls (E, M). Glomerular expression of TGF-β was increased in FSGS (R), MGP (S) and SLE (T) compared to control (Q). Magnification A-P 100x, magnification Q-T 200x.</p