Circulating anticoagulant glycosaminoglycans in mucopolysaccharidosis type I.

Abstract

Mucopolysaccharidosis type I (MPS I) is caused by mutations in the α‐L‐iduronidase (IDUA) gene. The protein product of this gene plays a central role in the catabolism of complex sugars, called glycosaminoglycans. Severe congenital deficiency of IDUA, or Hurler syndrome (MPS IH), leads to an accumulation of glycosaminoglycans in tissues and multi‐organ dysfunction. MPS IH is lethal unless treated with hematopoietic cell transplantation [1Boelens J.J. Wynn R.F. O’Meara A. Veys P. Bertrand Y. Souillet G. Wraith J.E. Fischer A. Cavazzana‐Calvo M. Sykora K.W. Sedlacek P. Rovelli A. Uiterwaal C.S. Wulffraat N. Outcomes of hematopoietic stem cell transplantation for Hurler’s syndrome in Europe: a risk factor analysis for graft failure.Bone Marrow Transplant. 2007; 40: 225-33Crossref PubMed Scopus (177) Google Scholar]. The beneficial effects of transplantation result from endogenous production of IDUA following stable engraftment of allogeneic cells. The efficacy of transplantation for MPS IH, however, is limited by transplantation toxicity, including anecdotal reports suggesting that MPS IH patients are at increased risk of pulmonary bleeding [2Gassas A. Sung L. Doyle J.J. Clarke J.T. Saunders E.F. Life‐threatening pulmonary hemorrhages post bone marrow transplantation in Hurler syndrome. Report of three cases and review of the literature.Bone Marrow Transplant. 2003; 32: 213-5Crossref PubMed Scopus (0) Google Scholar]. To investigate this issue further, we determined the incidence of pulmonary bleeding in children (<18 years) who received transplantation at the University of Minnesota over a period of 25 years (1983–2007). Of 106 MPS IH patients, 14 children (13.2%) experienced pulmonary bleeding after transplantation. In contrast, of 2573 children who received transplantation for a reason other than MPS IH, only 179 patients (7%) experienced pulmonary hemorrhage after transplantation. Thus, MPS IH patients were significantly more likely to develop this life‐threatening complication (P = 0.01). We hypothesized that the bleeding propensity in MPS IH is related to the accumulation of glycosaminoglycans resulting from IDUA deficiency. We evaluated five MPS IH patients (two females and three males) prior to any therapy. The median age at assessment was 1.1 years. First, urinary glycosaminoglycan levels were determined colorimetrically, and normalized to urinary creatinine values as described [3Whitley C.B. Ridnour M.D. Draper K.A. Dutton C.M. Neglia J.P. Diagnostic test for mucopolysaccharidosis. I. Direct method for quantifying excessive urinary glycosaminoglycan excretion.Clin Chem. 1989; 35: 374-9Crossref PubMed Scopus (178) Google Scholar]. Of note, the renal function (which can impact the urinary clearance of glycosaminoglycans) was normal in all five MPS IH patients. Next, platelet‐poor plasma was prepared from whole blood by centrifugation at 1500 × g for 15 min at room temperature, and the activated partial thromboplastin time (APTT) was determined using equal parts of pre‐warmed platelet‐poor plasma, Stago APTT reagent, and 30 mm CaCl2. We observed that two of five patients had an APTT elevated above the normal APTT range of 24–36 s (mean = 30 s) in one‐ to five‐year‐old unaffected children [4Andrew M. Vegh P. Johnston M. Bowker J. Ofosu F. Mitchell L. Maturation of the hemostatic system during childhood.Blood. 1992; 80: 1998-2005Crossref PubMed Google Scholar]. Remarkably, aggregate data from all five patients indicated that a dose–effect relationship existed between whole body glycosaminoglycan load (as determined by the urinary glycosaminoglycan levels) and coagulation parameters [mean ± standard deviation (SD); glycosaminoglycans: 1217 ± 469 mg glycosaminoglycans/g urinary creatinine; APTT: 34 ± 6 s; antifactor Xa (anti‐FXa), 0.14 ± 0.02 international units (IU); in three patients with elevated anti‐FXa the APTT was measured after addition of heparinase, and found to be corrected to <36 s; Fig. 1]. In the context of our finding that MPS IH patients are at an almost 2‐fold risk of pulmonary hemorrhage after transplantation (described above), it is important to note that the patient with the second highest value of urinary glycosaminoglycans (1568 mg glycosaminoglycan/g urinary creatinine) and APTT (39 s), and the highest value of anti‐FXa (0.19 IU; Fig. 1) experienced pulmonary hemorrhage on day 12 after transplantation and required intubation and ventilatory support for 25 days. Because no therapeutic heparin was used in any of these patients, and as the APTT changes were directly proportional to the whole body glycosaminoglycan load, we reasoned that glycosaminoglycan accumulation may serve as a heparin‐like anticoagulant through antithrombin III‐mediated inhibition of FXa. Thus, in order to better understand the impaired coagulation underlying the clinical observations, we assessed anti‐FXa levels in mice with IDUA deficiency, an animal model of MPS I (C57Bl/6J‐MPS I; MPS I mice), to determine whether coagulation defects associated with glycosaminoglycan accumulation were species‐specific. Concentrations of anticoagulant glycosaminoglycans in platelet‐free mouse plasma were determined by chromogenic heparin anti‐FXa assay (to increase accuracy over the clinical APTT assay), using the Coatest Heparin Kit (Chromogenix AB, Molndal, Sweden). Normal mouse plasma was used both as a control and for dilutions of the glycosaminoglycan standards. As expected, anti‐FXa values of MPS I mutant mice (n = 14) were significantly elevated when compared both to the age‐matched and sex‐matched, wild‐type (n = 3) and heterozygous (n = 6) controls: mean ± SD 0.19 ± 0.05 IU mL−1 vs. 0.11 ± 0.005 IU mL−1 vs. 0.10 ± 0.004 IU mL−1 plasma (both P < 0.001). As there is little published evidence to confirm the assumption that urinary glycosaminoglycans represent total body burden of glycosaminoglycans, we have assessed total tissue glycosaminoglycans in the parenchymal tissue most relevant to vascular tissue, in the myocardium, and compared myocardial glycosaminoglycans to urinary glycosaminoglycan clearance in MPS I mice (MPS I male mice, aged 12 ± 0.7 months; n = 8). A direct relationship existed between whole body glycosaminoglycan load (as determined by the urinary glycosaminoglycan levels) and the myocardial glycosaminoglycan levels (mean ± SD; urinary glycosaminoglycans: 5 ± 1 μg mL–1 of urine; myocardial glycosaminoglycans: 28 ± 7 μg mg–1 total protein; R2 = 0.85; Fig. S1). Critically, we observed that the endogenous anticoagulant activity was partially neutralized in representative samples by addition of heparinase: MPS I (n = 3) mean ± SD 0.16 ± 0.02 IU mL−1 vs. MPS I + heparinase (n = 3) 0.10 ± 0.003 IU mL−1 plasma vs. wild‐type 0.11 ± 0.005 IU mL−1; both P < 0.01), thereby confirming the presence of heparan sulfate. The partial correction can be explained by the presence of multiple species of glycosaminoglycans (dermatan sulfate, chondroitin sulfate and heparan sulfate) and numerous oligosaccharides resulting from partial degradation of these glycosaminoglycans, as heparinase would be expected to be specific only to a subgroup of these heterogenous glycosaminoglycan species. Thus, the major finding in this report is that IDUA deficiency is linked to a hemostatic defect in MPS I mice and humans. This observation is in agreement with the known anticoagulant activities of glycosaminoglycans, harnessed for clinical use, for example, in the low‐molecular‐weight heparinoid, danaparoid (Orgaran®; Organon, West Orange, NJ, USA). Based on extensive studies with various species of glycosaminoglycans [5Fuller M. Meikle P.J. Hopwood J.J. Glycosaminoglycan degradation fragments in mucopolysaccharidosis I.Glycobiology. 2004; 14: 443-50Crossref PubMed Scopus (46) Google Scholar], it seems likely that endogenous glycosaminoglycans in MPS I have a similar effect, and it is of interest that the heparin cofactor II–thrombin complex has been identified as a marker of MPS I [6Randall D.R. Sinclair G.B. Colobong K.E. Hetty E. Clarke L.A. Heparin cofactor II‐thrombin complex in MPS I: a biomarker of MPS disease.Mol Genet Metab. 2006; 88: 235-43Crossref PubMed Scopus (0) Google Scholar]. Coagulation abnormalities have been noted in other lysosomal storage diseases, for example, mucopolysaccharidosis type II [7Street A.M. Hunter’s syndrome with an endogenous anticoagulant.Am J Hematol. 1996; 53: 277Crossref PubMed Scopus (0) Google Scholar]; however, endogenous circulating anticoagulant activity has not been previously described in MPS IH. Only two out of five MPS IH patients had elevated APTT. This may be related to heterogeneity of the mutations within the IDUA gene, or because of the impact of other factors not yet identified [8Bunge S. Clements P.R. Byers S. Kleijer W.J. Brooks D.A. Hopwood J.J. Genotype‐phenotype correlations in mucopolysaccharidosis type I using enzyme kinetics, immunoquantification and in vitro turnover studies.Biochim Biophys Acta. 1998; 1407: 249-56Crossref PubMed Scopus (0) Google Scholar]. In addition, lung specific variables that likely impact hemostasis (e.g., the level of glycosaminoglycan deposition, infection, graft‐versus‐host disease and inflammation) are likely a part of multifactorial predisposition of MPS IH patients to bleeding complications. At the time of writing, it remains unknown whether enzyme replacement therapy prior to transplantation [9Wraith J.E. Beck M. Lane R. van der Ploeg A. Shapiro E. Xue Y. Kakkis E.D. Guffon N. Enzyme replacement therapy in patients who have mucopolysaccharidosis I and are younger than 5 years: results of a multinational study of recombinant human alpha‐L‐iduronidase (laronidase).Pediatrics. 2007; 120: e37-46Crossref PubMed Scopus (0) Google Scholar, 10Tolar J. Grewal S.S. Bjoraker K.J. Whitley C.B. Shapiro E.G. Charnas L. Orchard P.J. Combination of enzyme replacement and hematopoietic stem cell transplantation as therapy for Hurler Syndrome.Bone Marrow Transplant. 2007; 6: 531-5Google Scholar] may decrease the glycosaminoglycan burden in MPS IH patients, and thereby reduce the risk of hemorrhage. The implication of the current study is that a state of impaired hemostasis may exist in untreated patients with MPS IH, and that MPS IH patients with high urinary glycosaminoglycans may prove more likely to experience bleeding complications such as life‐threatening pulmonary hemorrhage. As such, this information may be useful in identifying MPS IH patients at higher risk for bleeding, especially in the peri‐transplant period, when glycosaminoglycan levels have not yet normalized and additional, transplantation‐related factors (e.g., thrombocytopenia and hepatic dysfunction affecting clotting factors) contribute to the risk of hemorrhage. The authors state that they have no conflict of interest. We thank T. Kivisto, E. Hanson, B. Peacock and Q. Cao for obtaining the necessary data. Fig. S1. Myocardial glycosaminoglycans are directly proportional to glycosaminoglycan load in MPS I mice. Please note: Blackwell Publishing are not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. Download .jpg (.02 MB) Help with files Supporting info item