Ivanov Ivan, Matafonov Anton, Sun Mao-Fu, Cheng Qiufang, Dickeson S Kent, Verhamme Ingrid M, Emsley Jonas, Gailani David
Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN.
Department of Bioengineering and Organic Chemistry, Tomsk Polytechnic University, Tomsk, Russia; and.
Blood. 2017 Mar 16;129(11):1527-1537. doi: 10.1182/blood-2016-10-744110. Epub 2017 Jan 9.
When blood is exposed to variety of artificial surfaces and biologic substances, the plasma proteins factor XII (FXII) and prekallikrein undergo reciprocal proteolytic conversion to the proteases αFXIIa and α-kallikrein by a process called contact activation. These enzymes contribute to host-defense responses including coagulation, inflammation, and fibrinolysis. The initiating event in contact activation is debated. To test the hypothesis that single-chain FXII expresses activity that could initiate contact activation, we prepared human FXII variants lacking the Arg353 cleavage site required for conversion to αFXIIa (FXII-R353A), or lacking the 3 known cleavage sites at Arg334, Arg343, and Arg353 (FXII-T, for "triple" mutant), and compared their properties to wild-type αFXIIa. In the absence of a surface, FXII-R353A and FXII-T activate prekallikrein and cleave the tripeptide S-2302, demonstrating proteolytic activity. The activity is several orders of magnitude weaker than that of αFXIIa. Polyphosphate, an inducer of contact activation, enhances PK activation by FXII-T, and facilitates FXII-T activation of FXII and FXI. In plasma, FXII-T and FXII-R353A, but not FXII lacking the active site serine residue (FXII-S544A), shortened the clotting time of FXII-deficient plasma and enhanced thrombin generation in a surface-dependent manner. The effect was not as strong as for wild-type FXII. Our results support a model for induction of contact activation in which activity intrinsic to single-chain FXII initiates αFXIIa and α-kallikrein formation on a surface. αFXIIa, with support from α-kallikrein, subsequently accelerates contact activation and is responsible for the full procoagulant activity of FXII.
当血液暴露于各种人工表面和生物物质时，血浆蛋白因子 XII（FXII）和前激肽释放酶通过一种称为接触激活的过程相互进行蛋白水解转化，形成蛋白酶αFXIIa和α-激肽释放酶。这些酶参与宿主防御反应，包括凝血、炎症和纤维蛋白溶解。接触激活的起始事件存在争议。为了验证单链 FXII 表达的活性可引发接触激活这一假说，我们制备了缺乏转化为αFXIIa所需的 Arg353 切割位点的人 FXII 变体（FXII-R353A），或缺乏 Arg334、Arg343 和 Arg353 这 3 个已知切割位点的变体（FXII-T，即“三重”突变体），并将它们的特性与野生型αFXIIa 进行比较。在没有表面的情况下，FXII-R353A 和 FXII-T 可激活前激肽释放酶并切割三肽 S-2302，显示出蛋白水解活性。该活性比αFXIIa 的活性弱几个数量级。多磷酸盐是接触激活的诱导剂，可增强 FXII-T 对 PK 的激活，并促进 FXII-T 对 FXII 和 FXI 的激活。在血浆中，FXII-T 和 FXII-R353A，但不包括缺乏活性位点丝氨酸残基的 FXII（FXII-S544A），以表面依赖性方式缩短了 FXII 缺陷血浆的凝血时间并增强了凝血酶生成。其效果不如野生型 FXII 强。我们的结果支持一种接触激活诱导模型，其中单链 FXII 的固有活性在表面引发αFXIIa 和α-激肽释放酶的形成。在α-激肽释放酶的支持下，αFXIIa 随后加速接触激活，并负责 FXII 的全部促凝血活性。
