Mechanisms responsible for the failure of protamine to inactivate low-molecular-weight heparin MARK A. CROWTHER, 1 L ESLIE R. BERRY, 2,3 P AUL T. MONAGLE 4 AND ANTHONY K. C. CHAN 2,3,5 1 Department of Medicine, McMaster University, 2 Hamilton Civic Hospitals Research Centre, 3 Department of Paediatrics, McMaster University, Hamilton, Canada, 4 Royal Children's Hospital, Melbourne, Australia, and 5 Hospital for Sick Children, Toronto, Ontario, Canada Received 12 February 2001; accepted for publication 20 July 2001 Summary. Protamine is unable to completely reverse the anticoagulant effect of the low-molecular-weight heparins (LMWH), a fact of clinical importance given the rapid increase in use of LMWH in clinical practice. This investi- gation sought to determine the mechanism by which LMWH were able to resist protamine-mediated inactivation. Af®nity fractionation of LMWH by passage through a pro- tamine column, with subsequent determination of mole- cular mass and sulphate charge density, demonstrated that the protamine-resistant fraction in LMWH is an ultra- low-molecular-weight fraction with low sulphate charge density. This group of molecules was not found in unfrac- tionated heparin, even when species of similar molecular mass were compared. We then determined that different commercially available LMWH varied in their ability to be neutralized by protamine, and that this variability corre- lated with the total sulphate content of the LMWH. We conclude that reduced sulphate charge, not molecular mass, is the principle reason that protamine is unable to fully inactivate LMWH. Furthermore, different LMWH vary in their ability to be neutralized by protamine, suggesting that product-speci®c recommendations for neutralization might be developed. Keywords: unfractionated heparin, low-molecular-weight heparin, protamine, haemorrhage, neutralization. Unlike unfractionated heparin (UFH), the antifactor Xa activity of the low-molecular-weight heparins (LMWH) is not neutralized by protamine (Makris et al, 2000). Even at protamine/heparin ratios of more than 5, protamine does not neutralize all the Xa-inhibiting activity of LMWH (Massonnet-Castel et al, 1986; Racanelli & Fareed, 1992). It is unknown why LMWH retains antifactor Xa activity after protamine neutralization. Some investigators have suggested that the LMWH molecules are too small to interact with protamine (Harenberg et al, 1985). However, protamine is able to completely neutralize the antifactor Xa activity of UFH, in spite of the fact that UFH contains a small population of low-molecular-mass molecules (Wolzt et al, 1995). An alternate explanation for the persistent anti- coagulant effect of LMWH after protamine neutralization is that the molecular interaction between protamine and LMWH is altered during the synthesis of LMWH. This hypothesis is supported by experiments with protamine and poly(L-lysine) which demonstrated that the degree of sulphonation (particularly N-sulphonation) of unfraction- ated heparin is critical in the interaction between protamine and UFH (Jones et al, 1986). However, to our knowledge, there is no experimental evidence supporting the hypothesis that reduced sulphate charge density is responsible for the inability of protamine to neutralize LMWH. This investiga- tion sought to explore these issues by determining whether differences in sulphate density existed between neutralizable and non-neutralizable fractions of UFH and LMWH. Fur- thermore, we attempted to determine if the protamine neutralizability of commercially available LMWH correlated with their sulphate charge density. EXPERIMENTAL PROCEDURES Chemicals. All reagents were of analytical grade. Stan- dard UFH was from Sigma (grade I-A, Na salt, 15000 average molecular weight, from porcine intestinal mucosa; Mississauga, Canada). LMWH were either: Enoxaparin (Na salt, C 90 050 99 from Rho Ãne-Poulenc Rorer, Paris, France), Clivarin (3500±4500 molecular weight range, Nordmark, Heidelberg, Germany), Tinzaparin (Leo Pharma Correspondence: Dr Anthony K. C. Chan, Hamilton Civic Hospitals Research Centre, 711 Concession St, Hamilton, L8V 1C3 Canada. E-mail: achan@thrombosis.hhscr.org British Journal of Haematology, 2002, 116, 178±186 178 Ó 2002 Blackwell Science Ltd