Côté H C, Bajzar L, Stevens W K, Samis J A, Morser J, MacGillivray R T, Nesheim M E
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, V6T 1Z3 Canada.
J Biol Chem. 1997 Mar 7;272(10):6194-200. doi: 10.1074/jbc.272.10.6194.
Recombinant human prothrombin (rII) and two mutant forms (R155A, R271A,R284A (rMZ) and R271A,R284A (rMZdesF1)) were expressed in mammalian cells. Following activation and purification, recombinant thrombin (rIIa) and stable analogues of meizothrombin (rMZa) and meizothrombin(desF1) (rMZdesF1a) were obtained. Studies of the activation of protein C in the presence of recombinant soluble thrombomodulin (TM) show TM-dependent stimulation of protein C activation by all three enzymes and, in the presence of phosphatidylserine/phosphatidylcholine phospholipid vesicles, rMZa is 6-fold more potent than rIIa. In the presence of TM, rMZa was also shown to be an effective activator of TAFI (thrombin-activatable fibrinolysis inhibitor) (Bajzar, L., Manuel, R., and Nesheim, M. E. (1995) J. Biol. Chem. 270, 14477-14484). All three enzymes were capable of inducing platelet aggregation, but 60-fold higher concentrations of rMZa and rMZdesF1a were required to achieve the effects obtained with rIIa. Second order rate constants (M-1.min-1) for inhibition by antithrombin III (AT-III) were 2.44 x 10(5) (rIIa), 6.10 x 10(4) (rMZa), and 1.05 x 10(5) (rMZdesF1a). The inhibition of rMZa and rMZdesF1a by AT-III is not affected by heparin. All three enzymes bound similarly to hirudin. The results of this and previous studies imply that full-length meizothrombin has marginal procoagulant properties compared to thrombin. However, meizothrombin has potent anticoagulant properties, expressed through TM-dependent activation of protein C, and can contribute to down-regulation of fibrinolysis through the TM-dependent activation of TAFI.
重组人凝血酶原（rII）及其两种突变形式（R155A、R271A、R284A（rMZ）和R271A、R284A（rMZdesF1））在哺乳动物细胞中表达。经过激活和纯化后，获得了重组凝血酶（rIIa）以及中凝血酶（rMZa）和中凝血酶（去F1）（rMZdesF1a）的稳定类似物。在重组可溶性血栓调节蛋白（TM）存在下对蛋白C激活的研究表明，这三种酶均能在TM依赖的情况下刺激蛋白C的激活，并且在磷脂酰丝氨酸/磷脂酰胆碱磷脂囊泡存在时，rMZa激活蛋白C的能力比rIIa强6倍。在TM存在的情况下，rMZa还被证明是凝血酶激活的纤溶抑制物（TAFI）的有效激活剂（Bajzar，L.，Manuel，R.，和Nesheim，M. E.（1995）《生物化学杂志》270，14477 - 14484）。这三种酶均能诱导血小板聚集，但与rIIa相比，rMZa和rMZdesF1a需要高60倍的浓度才能达到相同效果。抗凝血酶III（AT - III）抑制的二级速率常数（M⁻¹·min⁻¹）分别为2.44×10⁵（rIIa）、6.10×10⁴（rMZa）和1.05×10⁵（rMZdesF1a）。AT - III对rMZa和rMZdesF1a的抑制不受肝素影响。这三种酶与水蛭素的结合情况相似。本研究及之前研究的结果表明，与凝血酶相比，全长中凝血酶的促凝血特性较弱。然而，中凝血酶具有强大的抗凝特性，通过TM依赖的蛋白C激活来体现，并且可以通过TM依赖的TAFI激活促进纤维蛋白溶解的下调。
