van Galen Josse, Freato Nadia, Przeradzka Małgorzata A, Ebberink Eduard H T M, Boon-Spijker Mariëtte, van der Zwaan Carmen, van den Biggelaar Maartje, Meijer Alexander B
Department of Molecular and Cellular Hemostasis, Sanquin Research, Amsterdam, The Netherlands.
Department of Biomolecular Mass Spectrometry and Proteomics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.
Thromb Haemost. 2021 May;121(5):594-602. doi: 10.1055/s-0040-1721422. Epub 2020 Dec 10.
Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was employed to gain insight into the changes in factor VIII (FVIII) that occur upon its activation and assembly with activated factor IX (FIXa) on phospholipid membranes. HDX-MS analysis of thrombin-activated FVIII (FVIIIa) revealed a marked increase in deuterium incorporation of amino acid residues along the A1-A2 and A2-A3 interface. Rapid dissociation of the A2 domain from FVIIIa can explain this observation. In the presence of FIXa, enhanced deuterium incorporation at the interface of FVIIIa was similar to that of FVIII. This is compatible with the previous finding that FIXa contributes to A2 domain retention in FVIIIa. A2 domain region Leu631-Tyr637, which is not part of the interface between the A domains, also showed a marked increase in deuterium incorporation in FVIIIa compared with FVIII. Deuterium uptake of this region was decreased in the presence of FIXa beyond that observed in FVIII. This implies that FIXa alters the conformation or directly interacts with this region in FVIIIa. Replacement of Val634 in FVIII by alanine using site-directed mutagenesis almost completely impaired the ability of the activated cofactor to enhance the activity of FIXa. Surface plasmon resonance analysis revealed that the rates of A2 domain dissociation from FVIIIa and FVIIIa-Val634Ala were indistinguishable. HDX-MS analysis showed, however, that FIXa was unable to retain the A2 domain in FVIIIa-Val634Ala. The combined results of this study suggest that the local structure of Leu631-Tyr637 is altered by FIXa and that this region contributes to the cofactor function of FVIII.
采用氢-氘交换质谱法（HDX-MS）来深入了解凝血因子VIII（FVIII）在激活并与磷脂膜上的活化凝血因子IX（FIXa）组装时所发生的变化。对凝血酶激活的FVIII（FVIIIa）进行HDX-MS分析显示，沿A1-A2和A2-A3界面的氨基酸残基的氘掺入量显著增加。A2结构域从FVIIIa的快速解离可以解释这一现象。在FIXa存在的情况下，FVIIIa界面处增强的氘掺入与FVIII相似。这与之前的发现一致，即FIXa有助于A2结构域保留在FVIIIa中。A2结构域区域Leu631-Tyr637并非A结构域之间界面的一部分，与FVIII相比，其在FVIIIa中的氘掺入也显著增加。在FIXa存在的情况下该区域的氘摄取量比FVIII中观察到的有所降低。这意味着FIXa改变了FVIIIa中该区域的构象或直接与其相互作用。使用定点诱变将FVIII中的Val634替换为丙氨酸几乎完全损害了活化辅因子增强FIXa活性的能力。表面等离子体共振分析表明，A2结构域从FVIIIa和FVIIIa-Val634Ala解离的速率没有区别。然而，HDX-MS分析显示，FIXa无法将A2结构域保留在FVIIIa-Val634Ala中。这项研究的综合结果表明，Leu631-Tyr637的局部结构被FIXa改变，并且该区域有助于FVIII的辅因子功能。
