Abstract
Endothelial cells expose specific receptors for blood clotting factors and, upon perturbation, can initiate and propagate the reactions of the extrinsic pathway of blood coagulation leading to fibrin formation on the cell surface. The existence of an intrinsic mechanism of Factor IX activation on cultured human umbilical vein cells (HUVECs) was investigated by studies of the interaction between HUVECs and two proteins of the contact activation system, the cofactor high molecular weight kininogen (H-kininogen) and the zymogen Factor XI. In the presence of zinc ions (10-300 microM), 125I-labeled H-kininogen bound to HUVECs in a time-dependent, reversible, and saturable manner, with calcium ions exerting an inhibitory effect on the zinc-dependent binding. Analysis of the binding data by the LIGAND computer program indicated that HUVECs, in the presence of 2 mM CaCl2 and 100 microM ZnCl2 at 37 degrees C, bound 1.14 x 10(7) H-kininogen molecules per cell with an apparent dissociation constant of 55 nM. HUVEC-bound H-kininogen functions as the cell surface receptor for both 125I-labeled Factor XI and 125I-labeled Factor XIa, since HUVECs cultured in contact factor-depleted serum do not detectably bind either the zymogen or the enzyme in the absence of H-kininogen and zinc ions. In the presence of saturating concentrations of H-kininogen, 2 mM CaCl2 and 100 microM ZnCl2, the binding of 125I-labeled Factor XI and Factor XIa to HUVECs was time-dependent, reversible, and saturable, with apparent dissociation constants of 4.5 and 1.5 nM, respectively. HUVEC-bound complexes of H-kininogen and Factor XI generated Factor XIa activity only after the addition of purified Factor XIIa, and cell-bound Factor XIa in turn activated Factor IX, as documented by a 3H-labeled activation peptide release assay for 3H-Factor IX activation. The results indicate that cultured HUVECs provide a surface for the assembly and expression of an intrinsic Factor IX activator complex that may participate in the initiation of blood coagulation at sites of vascular injury.
Highlights
'"I-labeled F.XI/F.XIa did not bind to HUVECs in the presence of H-kininogen. All these observations suggest that HUVECs do not have H-kininogen independent bindingsites for F.XI/F.XIa and that H-kininogen is an essential requirement for the association of both F.XI and F.XIa to thesurface of endothelial cells
A illustrates the experimentpserformed with HUVECs grown in 20% fetal calf serum, while E illustratestheresults of experiments using HUVECs which had been grown in 20% contact factor-depleted human serum(see "Materials andMethods")
Activationof "H-F.IX was studied in the absence or in the presenceof 1 pg/ml F.XIIa, addedto HUVECs simultaneouslywith'H-F.IX.In controlexperiments,F.XIIaand 3H.FIXwere added to HUVECspreviously incubated with buffer,but without H-kininogen and F.XI.In separate wells, HUVECs grown in 20% fetal calf serum ( A )were preincubated with Hkininogen, F.XI, and 1 pg/ml of popcorn inhibitor, and the activation of 3H-F.IX was monitored without adding F.XIIa
Summary
Enzymes-F.XIIa was prepared by incubation of purified F.XII with pg/ml dextran sulfate at 37 “C for 90 min as previously described (29) and consisted of disulfide-linked heavy and light chains (u-F.XIIa)as judged by SDS-PAGE of reduced and nonreduced samples. This preparation had a specific activity of 16.6 /LM min” mg” when testedagainst the substrate S-2302, 0.4 mM in 0.09 M Tns-HC1, 0.09 M NaC1, 1mg/ml BSA, pH 8.3, at room temperature. 3H-F.IX was added to HUVECs in a final volume of 0.4 ml of incubation buffer, and therelease of 3H-activation peptide wasfollowedby measuring the trichloroacetic acid-soluble radioactivity in 50-pl aliquots aspirated from the wells at various times.
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