Osmotic stress regulates the anticoagulant efficiency of dermatan sulfate Maria P. McGee a, * , William Wagner b , Lynne Li a a Section on Rheumatology, Department of Internal Medicine, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, United States b Department of Pathology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC, United States Received 14 May 2004; received in revised form 22 July 2004; accepted 23 July 2004 Abstract Glycosaminoglycans (GAGs) in pericellular and interstitial spaces help to maintain local water homeostasis and blood coagulation balance. This study explored whether dehydrating microenvironment conditions influence dermatan sulfate’s (DS) anticoagulant activity. Water transfer during antithrombin activation by dermatan sulfate was measured using osmotic stress techniques. Anticoagulant activity was determined from the change in the rate of coagulation factor Xa (fXa) inhibition. Osmotic stress accelerated reaction rates, indicating water transfer from reactants to bulk. The net volume transferred, measured using osmotic probes similar in size to the reacting proteins, was approximately 2500 mol of water per mole of fXa inhibited. The reaction efficiency, V sat /K 1/2 (rate at saturation/concentration resulting in half-maximal rates), determined in titrations with monosulfated dermatan sulfate and disulfated dermatan sulfate (DDS), were 410 4 and 210 5 M 1 s 1 under osmotic stress and in the presence of calcium, corresponding to 34- and 81-fold increases over efficiency measured under standard conditions. These results indicate that dermatan sulfate can contribute significantly to antithrombin activation, and that in dehydrating environments and depending of ionic conditions, its anticoagulant efficiency can exceed that of heparan sulfate (HS). D 2004 Elsevier B.V./International Society of Matrix Biology. All rights reserved. Keywords: Coagulation; Glycosaminoglycans; Osmotic stress 1. Introduction Glycosaminoglycans (GAGs) activate antithrombin, the principal inhibitor of coagulation proteases (Olds et al., 1994; Olson et al., 1993; Gettins, 2002). The mechanisms of antithrombin activation by pharmacological heparins have been studied in considerable detail (Craig et al., 1989; Stratikos and Gettins, 1999; Jordan et al., 1980; Rezaie, 1998; Streusand et al., 1995; Johnson and Huntington, 2004), but much less is known about the physiological activation mediated by vascular GAGs in situ. Glycosami- noglycan species with a high affinity for antithrombin are very rare in the vascular wall (Wight et al., 1987; Wight, 2003; Kejellen and Lindahl, 1991). Studies in cultured endothelial cells expressing primarily heparan sulfate (HS) containing proteoglycans (Marcum et al., 1986) suggest that high-affinity sequences comprise less than 1% of the total GAG content. In arterial tissue, particularly the intima, high- affinity sequences must be even rarer, because chondroitin and dermatan sulfate (DS) proteoglycans are more abundant than heparan sulfate proteoglycans (Wight et al., 1987; Wight, 2003; Shirk et al., 2000). The quantity, structure, and distribution of proteoglycans in interstitial tissues vary markedly during tissue develop- ment, differentiation, and the progression of chronic pathologic conditions, such as atherosclerosis, osteoarthritis, and cancer (Shirk et al., 2000; Plaas et al., 1998; Iozzo, 1998). For example, the distribution and amount of dermatan sulfate proteoglycans differ between normal arterial tissue and arteries undergoing progressive athero- sclerosis. Dermatan sulfate isolated from decorin and biglycan from lesions prone to thrombosis was previously found to activate heparin cofactor II less effectively than dermatan sulfate species from healthy arterial tissue (Shirk et al., 2000). 0945-053X/$ - see front matter D 2004 Elsevier B.V./International Society of Matrix Biology. All rights reserved. doi:10.1016/j.matbio.2004.07.006 * Corresponding author. Tel.: +1 336 716 6716; fax: +1 336 716 9821. E-mail address: mmcgee@wfubmc.edu (M.P. McGee). Matrix Biology 23 (2004) 363 – 370 www.elsevier.com/locate/matbio