Heparin inhibits the intrinsic tenase complex by interacting with an exosite on factor IXa.
The specific molecular target for direct heparin inhibition of factor X activation by intrinsic tenase (factor IXa-factor VIIIa) was investigated. Comparison of size-fractionated oligosaccharides demonstrated that an octasaccharide was sufficient to inhibit intrinsic tenase. Substitution of soluble dihexanoic phosphatidylserine (C6PS) for phospholipid (PL) vesicles demonstrated that inhibition by low-molecular weight heparin (LMWH) was independent of factor IXa-factor VIIIa membrane assembly. LMWH also inhibited factor X activation by the factor IXa-PL complex via a distinct mechanism that required longer oligosaccharides and was independent of substrate concentrations. The apparent affinity of LMWH for the factor IXa-PL complex was higher in the absence of factor VIIIa, suggesting that the cofactor adversely affected the interaction of heparin with factor IXa-phospholipid. LMWH did not interact directly with the active site, as it failed to inhibit chromogenic substrate cleavage by the factor IXa-PL complex. LMWH induced a modest decrease in factor IXa-factor VIIIa affinity [K(D(app))] on PL vesicles that did not account for the inhibition. In contrast, LMWH caused a substantial reduction in factor IXa-factor VIIIa affinity in the presence of C6PS that fully accounted for the inhibition. Factor IXa bound LMWH with significantly higher affinity than factor X by competition solution affinity analysis, and the K(D(app)) for the factor IXa-LMWH complex agreed with the K(I) for inhibition of the factor IXa-PL complex by LMWH. Thus, LMWH binds to an exosite on factor IXa that antagonizes cofactor activity without disrupting factor IXa-factor VIIIa assembly on the PL surface. This exosite may contribute to the clinical efficacy of heparin and represents a novel target for antithrombotic therapy.
D001665:Binding Sites; D001667:Binding, Competitive; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015949:Factor IXa / Q000737:chemistry / Q000378:metabolism*; D015944:Factor VIIIa / Q000037:antagonists & inhibitors / Q000737:chemistry; D005170:Factor X / Q000378:metabolism; D006493:Heparin / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology*; D006495:Heparin, Low-Molecular-Weight / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry; D020349:Surface Plasmon Resonance
D006495:Heparin, Low-Molecular-Weight; D009363:Neoplasm Proteins; D010743:Phospholipids; D015944:Factor VIIIa; D005170:Factor X; D006493:Heparin; D015949:Factor IXa; D003546:Cysteine Endopeptidases; C030910:cancer procoagulant