Cell-surface heparan sulfate proteoglycan–mediated regulation of human neutrophil migration by the serpin antithrombin III

Abstract

The serpin antithrombin III (AT III) is reported to have hemostasis-regulating and anti-inflammatory properties. To determine its ability to influence thrombin-independent leukocyte responses, the direct effects of the AT III concentrate Kybernin P and a monoclonal antibody-purified AT III on neutrophil migration were studied. Chemotactic activity of human neutrophils isolated from the blood of healthy donors was determined in modified Boyden microchemotaxis chambers, and binding studies were performed according to standard experimental protocols. Preincubation in vitro of neutrophils with Kybernin P or immune-adsorbed AT III significantly deactivated migration toward fMet-Leu-Phe, or interleukin-8 (IL-8), in a concentration-dependent manner. In the absence of additional attractants, neutrophils exhibited a migratory response toward gradients of AT III preparations. True chemotaxis was confirmed in checkerboard assays. Analyses revealed that the AT III heparin-binding site interacts with neutrophil membrane-associated heparan sulfate proteoglycan receptors. Mechanisms of intracellular signaling differed; the deactivation of IL-8-induced chemotaxis resulted from tyrphostin-sensitive interactions of AT III-signaling with the IL-8 signal transduction pathway, whereas AT III-induced chemotaxis involved protein kinase C and phosphodiesterases. Signaling similarities between AT III and the proteoglycan syndecan-4 may suggest the binding of AT III to this novel type of membrane receptor. Under physiological conditions, AT III may prevent neutrophils from premature activation. Moreover, the systemic administration of AT III concentrate could have beneficial effects in combating systemic inflammation.

Antithrombin reduces ischemia/reperfusion-induced renal injury in rats by inhibiting leukocyte activation through promotion of prostacyclin production

“…Recently, results of in vitro experiments raised the possibility that AT might inhibit leukocyte activation directly. Dunzendorfers et al 13 reported that AT directly inhibited neutrophil chemotaxis by binding to the cell surface-heparan sulfate glycosaminoglycans. Souter et al 14 demonstrated that AT directly inhibited interleukin-6 production by monocytes stimulated with lipopolysaccharide.…”

“…11,12 In contrast to these reports, AT has been demonstrated to inhibit leukocyte activation directly. [13][14][15] These observations suggest that anti-inflammatory effects of AT may be mediated mainly by its direct inhibitory properties on leukocyte activation.…”

Attenuation of Leukocyte β2-Integrin Expression by Antithrombin-III

“…The proposed mechanism of action is reduced production of prostacyclin by endothelium upon interaction of AT-III with cell surface heparan sulfate proteoglycans (14)(15)(16). In the absence of endothelium, AT-III ligates with heparan sulfate proteoglycans of leukocytes and affects their migratory responses directly via syndecan-mediated signaling mechanisms (17)(18)(19)(20). This suggests that a different mechanism exists by which leukocyte activation may be attenuated with AT-III during cardiopulmonary bypass.…”

Expression and function of the endothelial protein C receptor in human neutrophils

“…17 In various experiments, neutrophil migration was tested toward phPC, ArhPC, AphPC (0.1 pg/mL to 10 g/mL), interleukin-8 (IL-8) (1 nM), formyl-Met-Leu-Phe (fMLP), C5a (both 10 nM), or AT (1 U/mL). For deactivation, neutrophils were incubated for 20 minutes at various concentrations of PC or APC (0.1 pg/mL to 10 g/mL).…”