Heeb Mary J, Prashun Duane, Griffin John H, Bouma Bonno N
Department of Molecular and Experimental Medicine, MEM276, Scripps Research Institute, 10550 N. Torrey Pines Rd, La Jolla, CA 92037, USA.
FASEB J. 2009 Jul;23(7):2244-53. doi: 10.1096/fj.08-123174. Epub 2009 Feb 24.
Protein S (PS) is a cofactor for activated protein C (APC), which inactivates coagulation factors (F) Va and VIIIa. Deficiency of protein C or PS is associated with risk of thrombosis. We found that PS also has APC-independent anticoagulant activity (PS-direct) and directly inhibits thrombin generated by FXa/FVa (prothrombinase complex). Here we report that PS contains Zn(2+) that is required for PS-direct and that is lost during certain purification procedures. Immunoaffinity-purified PS contained 1.4 +/- 0.6 Zn(2+)/mol, whereas MonoQ-purified and commercial PS contained 0.15 +/- 0.15 Zn(2+)/mol. This may explain the controversy regarding the validity of PS-direct. Zn(2+) content correlated positively with PS-direct in prothrombinase assays and clotting assays, but APC-cofactor activity of PS was independent of Zn(2+) content. PS-direct and Zn(2+) were restored to inactive PS under mildly denaturing conditions. Conversely, o-phenanthroline reversibly impaired the PS-direct of active PS. Zn(2+)-containing PS bound FXa more efficiently (K(d)(app)=9.3 nM) than Zn(2+)-deficient PS (K(d)(app)=110 nM). PS bound TFPI efficiently, independently of Zn(2+) content (K(d)(app)=21 nM). Antibodies that block PS-direct preferentially recognized Zn(2+)-containing PS, suggesting conformation differences at or near the interface of 2 laminin G-like domains near the PS C terminus. Thus, Zn(2+) is required for PS-direct and efficient FXa binding and may play a role in stabilizing PS conformation.
蛋白S（PS）是活化蛋白C（APC）的辅因子，活化蛋白C可使凝血因子（F）Va和VIIIa失活。蛋白C或PS缺乏与血栓形成风险相关。我们发现PS还具有不依赖APC的抗凝活性（PS-直接作用），并直接抑制由FXa/FVa（凝血酶原酶复合物）产生的凝血酶。在此我们报告，PS含有PS-直接作用所需的Zn(2+)，且在某些纯化过程中会丢失。免疫亲和纯化的PS含有1.4±0.6 Zn(2+)/mol，而MonoQ纯化的和市售的PS含有0.15±0.15 Zn(2+)/mol。这可能解释了关于PS-直接作用有效性的争议。在凝血酶原酶测定和凝血测定中，Zn(2+)含量与PS-直接作用呈正相关，但PS的APC辅因子活性与Zn(2+)含量无关。在轻度变性条件下，PS-直接作用和Zn(2+)可恢复到无活性的PS。相反，邻菲罗啉可逆地损害活性PS的PS-直接作用。含Zn(2+)的PS比缺乏Zn(2+)的PS更有效地结合FXa（表观解离常数K(d)(app)=9.3 nM）（K(d)(app)=110 nM）。PS有效地结合组织因子途径抑制物（TFPI），与Zn(2+)含量无关（K(d)(app)=21 nM）。阻断PS-直接作用的抗体优先识别含Zn(2+)的PS，表明在PS C末端附近的2个层粘连蛋白G样结构域界面处或其附近存在构象差异。因此，Zn(2+)是PS-直接作用和有效结合FXa所必需的，并且可能在稳定PS构象中起作用。
