Localization of anticoagulantly active heparan sulfate proteoglycans in vascular endothelium: antithrombin binding on cultured endothelial cells and perfused rat aorta.

Abstract

Abstract. We have studied the interaction of 1251-antithrombin (125I-AT) with microvascular endothelial cells (RFPEC) to localize the cellular site of anticoagulantly active heparan sulfate proteoglycans (HSPG). The radiolabeled protease inhibitor bound specifically to the above HSPG with a Kd of ,'050 nM. Confluent monolayer RFPEC cultures exhibited a linear increase in the amount of AT bound per cell for up to 16 d, whereas suspension RFPEC cultures possessed a constant number of protease inhibitor binding sites per cell for up to 5 d. These results suggest that monolayer RFPEC cultures secrete anticoagulantly active HSPG, which then accumulate in the extracellular matrix. This hypothesis was confirmed by quantitative light and EM level autoradiography which demonstrated that the AT binding sites are predominantly 1o-cated in the extracellular matrix with only small quantities of protease inhibitor complexed to the cell surface.We have also pinpointed the in vivo position of anticoagulantly active HSPG within the blood vessel wall. Rat aortas were perfused, in situ, with 125I-AT, and bound labeled protease inhibitor was localized by light and EM autoradiography. The anticoagulantly active HSPG were concentrated immediately beneath the aortic and vasa vasorum endothelium with only a very small extent of labeling noted on the luminal surface of the endothelial cells. Based upon the above data, we propose a model whereby luminal and abluminal anticoagulantly active HSPG regulate coagulation mechanism activity.

Partial Activation of Antithrombin without Heparin through Deletion of a Unique Sequence on the Reactive Site Loop of the Serpin

“…AT, by virtue of its unique reactive site loop, can inhibit thrombin in the native conformation, but requires activation before it can effectively inhibit fXa. It follows, therefore, that fXa in vivo can be inhibited primarily by the small fraction of AT that is bound to the heparin-like molecules on the vasculature (6,26). Otherwise, in the presence of a high concentration of circulating active AT (2.3 M), maintenance of normal hemostasis would have been compromised due to rapid inactivation of fXa, which is generated at a minute concentration (at picomolar range) during the propagation phase of the coagulation cascade (27).…”

Syndecans: multifunctional cell-surface co-receptors

“…In some cases, syndecan binding appears to play an obvious role in sequestering these proteins to the appropriate physiological compartment. For example, binding of antithrombin III to syndecan molecules on the luminal surfaces of endothelial cells would contribute to the establishment of a nonthrombogenic lining for blood vessels [150]. In other cases, the main function of syndecan binding might be to mediate internalization and possible degradation of the bound proteins.…”

Identification of the Antithrombin III Heparin Binding Site

“…ATIII inhibitory activity can be potentiated, however, by heparin-like molecules (15). The physiological source of cofactor activity is heparan sulfate proteoglycans on and under the endothelium, which serve to localize and concentrate antithrombin on the vessel wall where activated coagulation enzymes are generated (16,17). Additionally, pharmaceutical heparin may be administered to boost the anticoagulant activity of circulating antithrombin.…”