Abstract
Human histidine-rich glycoprotein was found to interact strongly with heparin both in purified systems and in plasma, resulting in neutralization of the anti-coagulant activity of heparin. In purified systems, histidine-rich glycoprotein and heparin react with apparent 1:1 stoichiometry to form a complex with a dissociation constant of 7 nM. Covalent heparin-antithrombin complex still reacts with histidine-rich glycoprotein to form a complex with a dissociation constant of 29 nM. The interaction between a Mr = 4300-heparin fragment and histidine-rich glycoprotein appeared to be more complex. The mechanism of the interaction between histidine-rich glycoprotein and heparin appeared to be different from that between antithrombin III and heparin, since the former is abolished by EDTA and occurs both with heparin molecules having a high affinity or a low affinity for antithrombin III. In plasma, histidine-rich glycoprotein efficiently counteracts the anticoagulant activity of heparin. Both the thrombin times and the activated factor X inhibition following addition of heparin are markedly prolonged in the absence of histidine-rich glycoprotein and shortened by addition of purified histidine-rich glycoprotein. Low affinity heparin was found to efficiently compete with high affinity heparin for binding to histidine-rich glycoprotein but not to antithrombin III. This results in an increased anticoagulant activity of high affinity heparin in the presence of low affinity heparin. Since the effect of histidine-rich glycoprotein on the anticoagulant properties of heparin is clearly demonstrated in normal plasma, it may be of clinical significance.
Highlights
Human histidine-rich glycoprotein was found to in- with albumin [7], fibrinogen [7], fibronectin [8], lipoprotein teract strongly with heparin both in purified systems lipase [9], and with cY1-acidglycoprotein [10]
These findings indicate that the histidine-rich glycoprotein efficiently competes with antithrombin I11for binding of heparin, thereby neutralizing the heparin activity and decreasing the inhibition rate of factor Xa
When the apparent rate constants measured at different histidine-rich glycoprotein concentrations were converted to their corresponding apparent heparin concentrations and plotted against the concentrations of free histidine-rich glycoprotein, sigmoidal curves were obtained (Fig. 1 A ) .The shape of the curve obtained for high affiity heparin is compatible with a single association reaction with histidine-rich glycoprotein,but that obtained for the M, = 4,300 heparin is not
Summary
From the Center for Thrombosis and Vascular Research, Departmpnt of Medical Research, University of Leuuen, Belgium. The residual free affinity heparin or of the M, = 4,300 fragment of heparin and by a heparin concentration is determined from the apparent rate constant covalent heparin-antithrombin I11 complex was investigated in the in the presence of histidine-rich glycoprotein,using calibration curves presence of the synthetic substrate S-2222 [28]. FactorXa wasthen added (1.2nM final concentration) a known amount of heparin (0.08 IU/ml for clinical grade and 0.04 and the disappearance rate of the amidolyticactivity was continuously I U / d for the M, = 4,300 heparin) the residual factor Xa activity was recorded Under these conditions, heparin and the M , = 4,300heparin measured in the same way in the depleted plasma, which was reconstituted with puritied histidine-rich glycoprotein to different The abbreviations used are: Xa, activated factor X; S-2222; N- concentrations. The histidine-rich glycoprotein (1 p~ in 0.1 M Tris-HC1buffer, pH 7.60)was treated with EDTA (10m~ final concentration) and the EDTA was subsequently removed by dialysis.The influenceof the addition of different divalent cations (10 p~ final concentration) to the incubation mixture was investigated using the same kinetic system, after treatment of all buffers and solutions with Chelex 100
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