Sheffield William P, Eltringham-Smith Louise J, Gataiance Sharon, Bhakta Varsha
Department of Pathology and Molecular Medicine, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada.
BMC Biotechnol. 2009 Mar 3;9:15. doi: 10.1186/1472-6750-9-15.
The plasma protein alpha2-antiplasmin (alpha2AP) is cross-linked to fibrin in blood clots by the transglutaminase factor XIIIa, and in that location retards clot lysis. Competition for this effect could be clinically useful in patients with thrombosis. We hypothesized that fusion of N-terminal portions of alpha2-antiplasmin to human serum albumin (HSA) and production of the chimeric proteins in Pichia pastoris yeast would produce a stable and effective competitor protein.
Fusion protein alpha2AP(13-42)-HSA was efficiently secreted from transformed yeast and purified preparations contained within a mixed population the full-length intact form, while fusions with longer alpha2AP moieties were inefficiently secreted and/or degraded. The alpha2AP(13-42)-HSA protein, but not recombinant HSA, was cross-linked to both chemical lysine donors and fibrin or fibrinogen by factor XIIIa, although with less rapid kinetics than native alpha2AP. Excess alpha2AP(13-42)-HSA competed with alpha2AP for cross-linking to chemical lysine donors more effectively than a synthetic alpha2AP(13-42) peptide, and reduced the alpha2AP-dependent resistance to fibrinolysis of plasma clots equally effectively as the peptide. Native alpha2AP was found in in vivo clots in rabbits to a greater extent than alpha2AP(13-42), however.
In this first report of transfer of transglutamination substrate status from one plasma protein to another, fusion protein alpha2AP(13-42)-HSA was shown to satisfy initial requirements for a long-lasting, well-tolerated competitive inhibitor of alpha2-antiplasmin predicted to act in a clot-localized manner.
血浆蛋白α2-抗纤溶酶（α2AP）通过转谷氨酰胺酶因子XIIIa与血凝块中的纤维蛋白交联，在该位置可延缓凝块溶解。对这种作用的竞争在血栓形成患者中可能具有临床应用价值。我们推测，将α2-抗纤溶酶的N端部分与人血清白蛋白（HSA）融合，并在毕赤酵母中生产嵌合蛋白，将产生一种稳定且有效的竞争蛋白。
融合蛋白α2AP(13 - 42)-HSA从转化酵母中有效分泌，纯化制剂在混合群体中包含全长完整形式，而与较长α2AP部分的融合分泌效率低和/或被降解。α2AP(13 - 42)-HSA蛋白而非重组HSA，可被因子XIIIa与化学赖氨酸供体以及纤维蛋白或纤维蛋白原交联，尽管动力学速度比天然α2AP慢。过量的α2AP(13 - 42)-HSA比合成的α2AP(13 - 42)肽更有效地与α2AP竞争与化学赖氨酸供体的交联，并且与该肽一样有效地降低了α2AP依赖的血浆凝块对纤溶的抗性。然而，在兔体内凝块中发现天然α2AP的程度高于α2AP(13 - 42)。
在这篇关于转谷氨酰胺底物状态从一种血浆蛋白转移到另一种血浆蛋白的首次报告中，融合蛋白α2AP(13 - 42)-HSA被证明满足对一种长效、耐受性良好的α2-抗纤溶酶竞争抑制剂的初步要求，预计该抑制剂以凝块局部作用方式起作用。
