Fibrinolysis(I 996) 10( I ), 49-63 © Pearson Professional Ltd 1996 The role of carbohydrate in the activation of plasminogen 2 glycoforms by streptokinase S. R. Pirie-Shepherd, R. L. Serrano, N. L. Andon, M. Gonzalez-Gronow, S. V. Pizzo Department of Pathology, Duke University Medical Center, Box 3712, Durham, North Carolina, 27710, USA. Summary Plasminogen 2 has six giycoforms that differ in their sialic acid content. In this study we have examined the effect that this differential sialylation has on activation of these glycoforms by streptokinase (SK). We find that increases in sialic acid content, above a threshold level, result in a decrease in the catalytic efficiency of activation. This decrease in catalytic efficiency appears to be due to an increase in Km values and a decrease in/(ca t values. This phenomenon is most apparent in the activation of plasminogen 2¢, the most extensively sialyiated glycoform with 13 mols of sialic acid/mol protein. Gel filtration demonstrated that SK forms an activator complex with plasminogen 2t), indicating that the carbohydrate on plasminogen 2 interferes with the stability of the Michaelis complex, ra, ther than the ability of streptokinase to form activator complexes with plasminogen 2 glycoforms. For comparison we also show data pertaining to activation of plasminogen 2 giycoforms by urinary plasminogen activator and tissue-type plasminogen activator. INTRODUCTION Plasmin, the central proteinase in fibrinolysis, is generated from plasminogen, which exists in two main glycosylated variants. ]-a Plasminogen 1 contains two carbohydrate chains, one N-linked to Asn-289 and the other a mucin- type O-linked oligosaccharide at Thr-345, whilst form 2 contains only the carbohydrate chain linked to Thr-345. Further studies of the glycoforms have demonstrated 6 glycoforms of plasminogen 24 termed tx, 13, 7, 8, e and with pI values ranging from approx, pH 8.2 to pH 6.6. The microheterogeneity stems from variation in sialic acid con- tent of plasminogen, s Recent work from this laboratory4 has demonstrated that the sialic acid content affects the activation of plasminogen by tissue-type plasminogen acti- vator (t-PA) bound to a fibrin surface, as a general rule, the greater the sialic acid content, the lower the catalytic effi- ciency of activation of the plasminogen glycoforms. Received: 19 September 1995 Accepted after revision: 11 December 1995 Correspondence to: S. R. Pirie-Shepherd, Duke University Medical Center, Box 3712, Durham, North Carolina, 27710, USA. Tel: 1-919-684-8986; Fax: 1-919-684-8689. Plasminogen can also be activated by urinary-type plasminogen activator (u-PA) and streptokinase (SK). The activity of t-PA is largely confined to the vasculature owing to its marked affinity for fibrin, which facilitates its physiologic activity on clot lysis. Monocyte cells provide binding sites for plasminogen and u-PAil The u-PA recep- tor (u-PAR) is a GPI-anchored protein that serves a dual function localizing u-PA and mediating the internaliza- tion of u-PA complexed to its physiological inhibitor plas- minogen activator inhibitor 1 (PAI-1) (see review ref. 6). The activation of bound plasminogen is kinetically enhanced by cell bound u-PA, and the generated plasmin is also resistant to inhibition by ct2-antiplasmin7 (ct2-AP). SK is a 414 amino acid, 47kDa extracellular protein secreted by hemolytic streptococci. 8 SK forms an equimolar complex with plasminogen (Pg-SK) or plasmin which acts as an activator of plasminogen. 9 SK has no inherent proteolytic activity.I° The Pg-SK complex catal- yses the conversion of free plasminogen to plasmin, a functional activity that free plasmin does not possess. 9 Recent work has attempted to elucidate the regions of SK responsible for binding plasminogen. Rodriguez et al ]~ demonstrated that a 17 kDa fragment of SK, was the smallest stable plasminogen binding domain to be 49