Jing Pan

Loss of Dermatan-4-Sulfotransferase 1 Function Results in Adducted Thumb-Clubfoot Syndrome

Adducted thumb-clubfoot syndrome is an autosomal-recessive disorder characterized by typical facial appearance, wasted build, thin and translucent skin, congenital contractures of thumbs and feet, joint instability, facial clefting, and coagulopathy, as well as heart, kidney, or intestinal defects. We elucidated the molecular basis of the disease by using a SNP array-based genome-wide linkage approach that identified distinct homozygous nonsense and missense mutations in CHST14 in each of four consanguineous families with this disease. The CHST14 gene encodes N-acetylgalactosamine 4-O-sulfotransferase 1 (D4ST1), which catalyzes 4-O sulfation of N-acetylgalactosamine in the repeating iduronic acid-alpha1,3-N-acetylgalactosamine disaccharide sequence to form dermatan sulfate. Mass spectrometry of glycosaminoglycans from a patients fibroblasts revealed absence of dermatan sulfate and excess of chondroitin sulfate, showing that 4-O sulfation by CHST14 is essential for dermatan sulfate formation in vivo. Our results indicate that adducted thumb-clubfoot syndrome is a disorder resulting from a defect specific to dermatan sulfate biosynthesis and emphasize roles for dermatan sulfate in human development and extracellular-matrix maintenance.

Oversulfated chondroitin sulfate is not the sole contaminant in heparin

To the Editor: Contaminated heparin was associated with at least 149 deaths in 2007 and 2008, according to the information published at the US Food and Drug Administration (FDA) website (http://www.fda.gov/ Drugs/DrugSafety/PostmarketDrug SafetyInformationforPatientsandProviders/ ucm112669.htm). Sasisekharan and his colleagues1–3 have analyzed 6 lots of heparin associated with the contamination event and 28 lots of heparin produced by a heparin manufacturer from 2004 to 2007 (ref. 2). Using NMR analysis, they report that the contaminants in heparin include an impurity, specifically dermatan sulfate, and a contaminant, oversulfated chondroitin sulfate (OSCS) that is presumed to be derived from animal cartilage1. Work in our laboratory analyzing the same 28 heparin samples reveals the presence of other contaminants in heparin—some oversulfated and some undersulfated—all of which, like dermatan sulfate and OSCS, are found in, or can be made from, heparin by-product (a highly variable waste product comprising heparan sulfate, dermatan sulfate and chondroitin sulfate generated during heparin purification from crude heparin). With the exception of dermatan sulfate and OSCS, which can be definitively detected by NMR, our results suggest these additional contaminants cannot be distinguished from heparin in samples because of their similar NMR profiles2 and similar rates of migration under capillary electrophoresis and anion exchange highperformance liquid chromatography (HPLC). Taken together, our findings suggest the need for further work to unambiguously identify and characterize additional contaminants present in different batches of heparin. Heparin is the most highly sulfated naturally occurring glycosaminoglycan (GAG). It is enriched in porcine, ovine and bovine intestines or bovine lung entrails along with less sulfated GAGs, including heparan sulfate, dermatan sulfate and chondroitin sulfate. These GAGs are made by all animal cells and are present in all tissues4. Pharmaceutical-grade heparin is prepared from crude heparin by removal of the less sulfated GAGs, the so-called heparin by-product, from heparin. As a result of its high degree of sulfation, heparin has higher anticoagulation activities than its less sulfated heparin by-product. Indeed, danaparoid (also referred to as Orgaran), an anticoagulant derived from porcine mucosa heparin byproduct containing 70–80% heparan sulfate and 20–30% chondroitin sulfate and/or dermatan sulfate5, was removed from the US market in 2002 due to its unsatisfactory anticoagulation activities and difficulties in obtaining heparin by-product that had a constant heparan sulfate to chondroitin sulfate/dermatan sulfate ratio5,6. In this context, we examined whether the OSCS observed by Sasisekharan1–3 in contaminated heparin might be derived by oversulfation of heparin by-product, rather than from animal cartilage chondroitin sulfate. Similarly, we sought to determine whether heparan sulfate, dermatan sulfate, chondroitin sulfate and oversulfated forms of these GAGs were also present in adulterated heparin samples. As heparin contains no, or vanishingly low, levels of galactosamine, we commenced our analysis by quantifying the glucosamine/ galactosamine ratio in each of the heparin lots (derived as previously reported3) (Supplementary Methods). This assay7,8 has passed a double-blind test designed by the FDA with a set of heparin and galactosamine-containing GAG mixtures. We found that the 28 heparin samples contained 0–37% galactosamine-containing GAGs, with an average value of 14.8% (Fig. 1). The most contaminated heparin lot (2007-29) contained 37% galactosamine, consistent with the 39% galactosamine found by a recently published assay9. We also analyzed 28 ‘uncontaminated’ heparin lots produced by different heparin manufacturers in the United States and found that 20 out of 28 heparin lots had no galactosaminecontaining GAGs (Fig. 1, far right). Finally, we confirmed our findings using a different assay in which the samples were digested with a mixture of heparin lyases I, II and III. These enzymes degrade heparin and heparan sulfate to component disaccharides generating a chromophore at 232 nm. The adulterated preparations did not yield the expected optical density values compared with control, unadulterated heparin (Supplementary Fig. 1). Oversulfated GAGs can inhibit these enzymes, so the decrease in optical density could result from contaminating chondroitin sulfate, other impurities or oversulfated material. In contrast to our findings, Sasisekharan and colleagues3 previously reported these same 28 heparin lots to contain 0–27% (average, 6.4%) galactosamine-containing GAGs, including the impurity, dermatan sulfate and the previously identified contaminant, OSCS (Supplementary Table 1 in ref. 3). Their calculation of the amount of dermatan sulfate and OSCS in contaminated heparin was based on NMR analysis using two assumptions: first, each of the 15 protons in heparin, dermatan sulfate and OSCS measured by NMR produces the same signal intensity by integration; and second, dermatan sulfate has 100% iduronic acid and 0% glucuronic acid residues. However, we assert that these assumptions are questionable. First, the NMR proton signal intensity by integration is not equal for each proton of OSCS and heparin1,10 (Supplementary Fig. 2). Second, two proton shifts (A1 and U5) of OSCS are invisible in the contaminated heparins tested (Supplementary Fig. 2), indicating that either the contaminants were not OSCS or OSCS had different proton shifts when mixed with heparin or other unidentified contaminants. Indeed, in Figure 4 of their Nature Biotechnology paper1, the two-dimensional (2D) NMR profiles of isolated heparin contaminant reported by c o r r e s p o n d e n c e

Chemically oversulfated glycosaminoglycans are potent modulators of contact system activation and different cell signaling pathways

Contaminated heparin was associated with adverse reactions by activating the contact system. Chemically oversulfated/modified glycosaminoglycans (GAGs) consisting of heparan sulfate, dermatan sulfate, and chondroitin sulfate have been identified as heparin contaminants. Current studies demonstrated that each component of oversulfated GAGs was comparable with oversulfated chondroitin sulfate in activating the contact system. By testing a series of unrelated negatively charged compounds, we found that the contact system recognized negative charges rather than specific chemical structures. We further tested how oversulfated GAGs and contaminated heparins affect different cell signaling pathways. Our data showed that chemically oversulfated GAGs and contaminated heparin had higher activity than the parent compounds and authentic heparin, indicative of sulfation-dominant and GAG sequence-dependent activities in BaF cell-based models of fibroblast growth factor/fibroblast growth factor receptor, glial cell line-derived neurotrophic factor/c-Ret, and hepatocyte growth factor/c-Met signaling. In summary, these data indicate that contaminated heparins intended for blood anticoagulation not only activated the contact system but also modified different GAG-dependent cell signaling pathways.

High affinity glycosaminoglycan and autoantigen interaction explains joint specificity in a mouse model of rheumatoid arthritis

In the K/BxN mouse model of rheumatoid arthritis, autoantibodies specific for glucose-6-phosphate isomerase (GPI) can transfer joint-specific inflammation to most strains of normal mice. Binding of GPI and autoantibody to the joint surface is a prerequisite for joint-specific inflammation. However, how GPI localizes to the joint remains unclear. We show that glycosaminoglycans (GAGs) are the high affinity (83 nm) joint receptors for GPI. The binding affinity and structural differences between mouse paw/ankle GAGs and elbows/knee GAGs correlated with the distal to proximal disease severity in these joints. We found that cartilage surface GPI binding was greatly reduced by either chondroitinase ABC or β-glucuronidase treatment. We also identified several inhibitors that inhibit both GPI/GAG interaction and GPI enzymatic activities, which suggests that the GPI GAG-binding domain overlaps with the active site of GPI enzyme. Our studies raise the possibility that GAGs are the receptors for other autoantigens involved in joint-specific inflammatory responses.

Identification of Chemically Sulfated/desulfated Glycosaminoglycans in Contaminated Heparins and Development of a Simple Assay for the Detection of Most Contaminants in Heparin.

Contaminated heparin was linked to at least 149 deaths and hundreds of adverse reactions. Published report indicates that heparin contaminants were a natural impurity, dermatan sulfate, and a contaminant, oversulfated chondroitin sulfate (OSCS). OSCS was assumed to derive from animal cartilage. By analyzing 26 contaminated heparin lots from different sources, our data indicate that the heparin contaminants were chemically sulfated or chemically sulfated/desulfated glycosaminoglycans (GAGs) consisting of heparan sulfate, chondroitin sulfate, and dermatan sulfate based on monosaccharide quantification, CE, heparin lyase digestion, and liquid chromatography/mass spectrometry analysis. Since currently recommended heparin quality control assays had failed to detect certain heparin contaminants, a simple method that detects most contaminants in heparin was developed. This assay detects specific heparin structures that most contaminants cannot mimic and can be performed in any laboratory equipped with an UV spectrometer.

Molecular Mechanism Underlines Heparin-Induced Thrombocytopenia and Thrombosis

Heparin-induced thrombocytopenia (HIT) with thrombosis is the most severe side effect of heparin administration. HIT patients may die or have permanent sequelae, such as a stroke or limb amputation. Contaminated heparin is associated with anaphylactic reactions and deaths by activating the contact system. It is also associated with high incidence of HIT via a yet unknown mechanism. This chapter shows that: (1) the contact system can be activated by a variety of unrelated molecules; (2) kallikrein directly cuts prothrombin to generate functional thrombin through contact system activation; and (3) while heparin contaminants, oversulfated heparin by-product (OS-HB), induce thrombin generation in both normal and HIT patient plasmas through contact system activation, authentic heparin induces thrombin activities only in HIT patient plasmas containing autoantibodies against protein/heparin complex. These data suggest that the negatively charged IgG/protein/heparin or OS-HB complex activate the contact system and produce thrombin in human plasma and thrombin partially activates the platelets allowing subsequent platelet activation through IgG/Fc receptor II signaling. The newly discovered mechanism of heparin-induced thrombin activity could explain the increased incidence of HIT in patients exposed to contaminated heparin. Furthermore, the assays used in these studies would be valuable for HIT diagnosis, prevention, and treatment.

Oversulfated heparin by-products induce thrombin generation in human plasmas through contact system activation.

Thrombin generation is thought to be mediated predominantly by the tissue factor or ‘‘extrinsic’’ coagulation pathway. An alternate pathway to thrombin generation (the ‘‘intrinsic’’ pathway or contact system) has been observed when blood or plasma comes in contact with artificial surfaces. Here we present evidence for a new route to thrombin formation that begins with the activation of the contact system protein prekallikrein by oversulfated heparin (OS-HB). Kallikrein, instead of activated factor X, cleaves prothrombin to form thrombin. Thrombin then cleaves fibrinogen to form fibrin clots. Moreover, we show that OS-HB by-products induce kallikrein- and thrombin-like activities in normal human plasma and in human plasma devoid of coagulation factor X or downstream contact system components factor IX or factor XI. Oversulfated heparin by-product-induced thrombin generation may have had a role in the adverse reactions associated with the recent clinical use of contaminated heparin.

Activated contact system and abnormal glycosaminoglycans in lupus and other auto- and non-autoimmune diseases.

Systemic lupus erythematosus (SLE), heparin-induced thrombocytopenia (HIT), rheumatoid arthritis (RA) are marked by the presence of autoantibodies against negatively changed DNA, phospholipids, heparin, and chondroitin sulfate, respectively. Heparin/protein complexes induce contact system activation in HIT patient plasmas. The activated contact system generates thrombin. Thrombin is responsible for thrombosis, a common cause of death and disabilities for both HIT and SLE. In this chapter, we analyze plasma contact system proteins, thrombin- and kallikrein-like activities, glucosamine and galactosamine content from SLE-, RA-, osteoarthritis (OA)-, and psoriasis (Ps)-patient plasmas in addition to pooled 30+ healthy patient plasmas. We found that all SLE patient plasmas exhibited abnormal contact systems marked by the absence of high molecular weight kininogen, the presence of processed C1 inhibitor (C1inh), the display of abnormal thrombin- and kallikrein-like activities, and increased levels of plasma glucosamine and galactosamine. Different patterns of contact system activation distinguish SLE, RA, and Ps whereas no contact system activation is observed in normal and OA patient plasmas. The presence of paradoxical lupus anticoagulants in certain thrombosis-prone SLE patient plasmas, marked by delayed clotting in clinical plasma test, was explained by the consumption of contact system proteins, especially high molecular weight kininogen. Finally, we discovered that mouse and human SLE autoantibodies bind to cell surface GAGs with structural selectivity. In conclusion, markers of abnormal contact system activation represent potential new targets for autoimmune disease diagnosis, prevention, and treatment. These markers might also be useful in monitoring SLE activity/severity and in pinpointing patients with SLE-associated arthritis and psoriasis.

Chondroitin sulfate and abnormal contact system in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a heterogeneous autoimmune disease that affects 1% of the population worldwide. In the K/BxN mouse model of RA, autoantibodies specific for glucose-6-phosphate isomerase (GPI) from these mice can transfer joint-specific inflammation to normal mice. The binding of GPI/autoantibody to the cartilage surface is a prerequisite for autoantibody-induced joint-specific inflammation in the mouse model. Chondroitin sulfate (CS) on cartilage surface is the long sought high-affinity receptor for GPI. The binding affinity and structural differences between mouse paw/ankle CS and knee/elbow CS correlate with the distal to proximal disease severity in these joints. The data presented in this chapter indicate that autoantigen/autoantibodies in blood circulation activate contact system to produce vasodilators to allow immune complex, protein aggregates, and other plasma proteins to get into the joints. Cartilage surface CS binds and retains autoantigen/autoantibodies. The CS/autoantigen/autoantibody complexes could induce C3a and C5a production through contact system activation. C3a and C5a trigger degranulation of mast cells, which further recruit plasma contact system and complement proteins, immune cells, and immune activation factors to facilitate joint-specific tissue destruction. Therefore, either reducing autoantibody production or inhibiting autoantibody-induced contact system activation might be effective in RA prevention.

Glycosaminoglycans and Activated Contact System in Cancer Patient Plasmas

Oncogenic mutations create cancer cells. Cancer cells require thrombin for growth, angiogenesis, and metastasis. All cancer patients display a hypercoagulable state, which includes platelet activation, blood coagulation, complement activation, vasodilatation, and inflammation. This often results in thrombosis, the second leading cause of death in cancer patients. It is established that chemically oversulfated glycosaminoglycans (GAGs) induce thrombin generation through contact system activation in human plasma. Thrombin is responsible for thrombosis. In this chapter, we show that plasmas from lung cancer patients contain activated contact systems apparent by the absence of high molecular weight kininogen and processed C1inh, by abnormal kallikrein and thrombin activities, and by increased glucosamine, galactosamine, and GAG levels. Activated contact systems were also evident in plasmas from breast, colon, and pancreatic cancer patients. These data suggest that GAGs or other molecules produced by tumors induce abnormal thrombin generation through contact system activation. Therefore, the contact system and glycans represent new targets for cancer diagnosis, prevention, and treatment.