Anti-thrombin activities of heparin. Effect of saccharide chain length on thrombin inhibition by heparin cofactor II and by antithrombin.
The interactions of two proteinase inhibitors, heparin cofactor II and antithrombin, with thrombin are potentiated by heparin. Using two methods, we have studied the potentiating effects of a series of heparin (poly)saccharides with high affinity for antithrombin and mean Mr ranging from approx. 1700 to 18,800. First, catalytic amounts of heparin (poly)saccharide were added to purified systems containing thrombin and either heparin cofactor II or antithrombin. Residual thrombin activity was determined with a chromogenic substrate. It was found that only the higher-Mr polysaccharides (Mr greater than 8000) efficiently catalysed thrombin inhibition by heparin cofactor II, there being a progressive catalytic effect with increasing Mr of the polysaccharide. Weak accelerating effects were noted with low-Mr saccharides (Mr less than 8000). This contrasted with the well-characterized interaction of heparin with antithrombin and thrombin, where heparin oligosaccharides of Mr less than 5400 had absolutely no ability to accelerate the reaction, while (poly)saccharides of Mr exceeding 5400 showed rapidly increasing catalytic activity with increasing Mr. Secondly, these and other heparin preparations were added in a wide concentration range to plasma with which 125I-labelled thrombin was then incubated for 30 s. Inhibited thrombin was determined from the distribution of labelled thrombin amongst inhibitor-thrombin complexes, predominantly antithrombin-thrombin and heparin cofactor II-thrombin complexes. In this situation, where the inhibitors competed for thrombin and for the (poly)saccharides, it was found that, provided the latter were of high affinity for antithrombin and exceeded a Mr of 5400, thrombin inhibition in plasma was mediated largely through antithrombin. Polysaccharides of Mr exceeding 8000 that were of low affinity for antithrombin accelerated thrombin inhibition in plasma through their interaction with heparin cofactor II. High concentrations of saccharides of Mr 1700-5400 exhibited a size-dependent acceleration of thrombin inhibition, not through their interaction with antithrombin, but through their interaction with heparin cofactor II.
Bray
B
B
Department of Haematology, Charing Cross and Westminster Medical School, Hammersmith, London, U.K.
Lane
D A
DA
Freyssinet
J M
JM
Pejler
G
G
Lindahl
U
U
D000991:Antithrombins / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006493:Heparin / Q000494:pharmacology*; D015844:Heparin Cofactor II / Q000494:pharmacology*; D006801:Humans; D008970:Molecular Weight; D009844:Oligosaccharides; D013917:Thrombin / Q000037:antagonists & inhibitors*
D000991:Antithrombins; D009844:Oligosaccharides; D015844:Heparin Cofactor II; D006493:Heparin; D013917:Thrombin