Binding of recombinant annexin V to endothelial cells: effect of annexin V binding on endothelial-cell-mediated thrombin formation

Abstract

Annexin V binds with high affinity to procoagulant phospholipid vesicles and thereby inhibits the procoagulant reactions catalysed by these surfaces in vitro. In vivo, vascular endothelial cells are known to catalyse the formation of thrombin by the expression of binding sites at which procoagulant complexes can assemble. Here, we have studied the binding capacity of recombinant annexin V (rANV) to quiescent, phorbol 12-myristate 13-acetate (PMA)- and tumour necrosis factor alpha (TNF-alpha)-stimulated cultured human umbilical-vein endothelial cells (HUVEC). The dissociation constant (Kd) was 15.5 +/- 3.3 nM and the number of binding sites was 8.8 (+/- 3.9) x 10(6)/cell. These binding parameters did not change significantly during a 30 h incubation period with PMA or TNF-alpha. rANV inhibited HUVEC-mediated factor Xa formation via the extrinsic as well as the intrinsic route. Activation of factor X by the tissue factor-factor VII-factor X complex and tenase complex was inhibited with IC50 values of 43 +/- 30 nM and 33 +/- 24 nM respectively. Endothelial-cell-mediated generation of thrombin by the prothrombinase complex was inhibited by rANV with an IC50 of 16 +/- 12 nM. Preincubation of rANV with the endothelial cells did not significantly influence the IC50 values. These results show that rANV binds to the same extent to quiescent, PMA- and TNF-stimulated HUVEC, and, as a result of this binding, rANV efficiently inhibits endothelial-cell-mediated thrombin formation.

Leishmaniadisease development depends on the presence of apoptotic promastigotes in the virulent inoculum

“…AnxA5 binds in a Ca 2ϩ -dependent manner with anionic phospholipids present in membranes of apoptotic cells. AnxA5 binds with high affinity to PS (12). A recent study demonstrated that, in addition to AnxA5, serum protein S specifically binds PS.…”

Lactadherin inhibits enzyme complexes of blood coagulation by competing for phospholipid-binding sites

“…26 However, annexin V binds poorly to curved membranes, 41 requires supraphysiologic Ca ϩϩ concentrations for optimal binding, and inhibits less than 80% of procoagulant function on endothelial cell membranes unless the concentration exceeds 200 nM. 50 Likewise, annexin V is an incomplete inhibitor of the factor Xase complex on platelet membranes. 51 Our results, indicating that inhibition of the prothrombinase complex exceeds 80% at 60 nM annexin V only when the curvature of the membrane is minimal and the PS content is 15%, are in agreement with these prior studies.…”

Viable, apoptotic and necrotic monocytes expose phosphatidylserine: cooperative binding of the ligand Annexin V to dying but not viable cells and implications for PS-dependent clearance

“…The Kd has been reported to have values between 15 and 0.03 nM. [9][10][11] Cooperativity has been reported for the interaction with Ca 2 þ . 12 Freshly isolated, viable monocytes (00h-wo in Figure 1a) as shown by an intact mitochondrial membrane potential (C m ; DiO 6 C), no morphological changes of the nucleus (nSSC), G1/ 0 DNA content (PI-Triton) and no detectable caspase activity (zVAD-fitc) were analysed for their AxV binding in the presence and absence of EDTA.…”