Analysis of the kinetics of prothrombin activation and evidence that two equilibrating forms of prothrombinase are involved in the process
Analysis of the kinetics of prothrombin activation and evidence that two equilibrating forms of prothrombinase are involved in the process
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DOI：
10.1074/jbc.m206413200
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发表时间：
2003-02-28
影响因子：
4.8
通讯作者：
Nesheim, ME
中科院分区：
文献类型：
--
作者：
Brufatto, N;Nesheim, ME
凝血酶原酶在Arg（271）和Arg（320）处切割凝血酶原以产生凝血酶。测定了五种重组凝血酶原的裂解动力学：野生型凝血酶原（WT-II）、R155 AIR284 AIR271 A凝血酶原（rMZ-II）、R155 A/R284 A/R320 A凝血酶原（rP 2-II）、荧光素标记的S525 C凝血酶原（WT-II-F*）和荧光素标记的R155 A/R284 A/R271 A/S525 C凝血酶原（rMZ-II-F*）。rMZ-II和rP 2-II分别仅在Arg（320）和Arg（271）处裂解，分别产生中间体美佐凝血酶和凝血酶前体-2。WT-II-F* 和rMZ-II-F* 在Cys（525）处用荧光素标记;通过增强的荧光监测切割。WT-II、rMZ-II和rP 2-II的活化动力学表明，辅因子Va使Arg（320）处裂解的催化效率增加了30，000倍，凝血酶原转化为凝血酶也是如此。然而，因子Va仅使Arg（271）处的切割增加34倍。尽管WT-II竞争性抑制WT-II-F* 的切割，但即使在饱和浓度下，rMZ-II或rP 2-II也不能完全抑制。然而，rMZ-II和rP 2-II一起竞争性地抑制WT-II-F* 切割。WT-II和rMZ-II都竞争性抑制rMZ-II-F* 裂解，而rP 2-II则没有。一个模型的凝血酶原激活，包括，两个平衡形式的凝血酶原酶，每个识别的裂解位点之一，是定量与所有的实验观察一致。因此，我们得出结论，凝血酶原激活的动力学可以描述的“乒乓”样机制。
Prothrombinase cleaves prothrombin at Arg(271) and Arg(320) to produce thrombin. The kinetics of cleavage of five recombinant prothrombins were measured: wildtype prothrombin (WT-II), R155AIR284AIR271A prothrombin (rMZ-II), R155A/R284A/R320A prothrombin (rP2-II), S525C prothrombin labeled with fluorescein (WT-II-F*), and R155A/R284A/R271A/S525C prothrombin labeled with fluorescein (rMZ-II-F*). rMZ-II and rP2-II are cleaved only at Arg(320) and Arg(271), respectively, to yield the intermediates meizothrombin and prethrombin-2, respectively. WT-II-F* and rMZ-II-F* were labeled at Cys(525) with fluorescein; cleavage was monitored by enhanced fluorescence. Activation kinetics of WT-II, rMZ-II, and rP2-II indicated that the catalytic efficiency of cleavage at Arg(320) was increased by 30,000-fold by the cofactor factor Va, as was the conversion of prothrombin to thrombin. However, factor Va increased cleavage at Arg(271) only by 34-fold. Although WT-II competitively inhibited cleavage of WT-II-F*, rMZ-II or rP2-II did not inhibit completely even at saturating concentrations. However, rMZ-II and rP2-II together inhibited WT-II-F* cleavage competitively. Both WT-II and rMZ-II competitively inhibited rMZ-II-F* cleavage, whereas rP2-II did not. A model of prothrombin activation that includes, two equilibrating forms of prothrombinase, each recognizing one of the cleavage sites, is quantitatively consistent with all of the experimental observations. Therefore, we conclude that the kinetics of prothrombin activation can be described by a "ping-pong"-like mechanism.