Inter-α-inhibitor heavy chain 4 (ITIH4) is a poorly characterized plasma protein that is proteolytically processed in multiple pathological conditions. However, no biological function of ITIH4 has been identified. Here, we show that ITIH4 is cleaved by several human proteases within a protease-susceptible region, enabling ITIH4 to function as a protease inhibitor. This is exemplified by its inhibition of mannan-binding lectin–associated serine protease-1 (MASP-1), MASP-2, and plasma kallikrein, which are key proteases for intravascular host defense. Mechanistically, ITIH4 acts as bait that, upon cleavage, forms a noncovalent, inhibitory complex with the executing protease that depends on the ITIH4 von Willebrand factor A domain. ITIH4 inhibits the MASPs by sterically preventing larger protein substrates from accessing their active sites, which remain accessible and fully functional toward small substrates. Thus, we demonstrate that ITIH4 functions as a protease inhibitor by a previously undescribed inhibitory mechanism.

α-抑制剂间重链 4 (ITIH4) 是一种特征较差的血浆蛋白，在多种病理条件下经过蛋白水解加工。然而，尚未确定 ITIH4 的生物学功能。在这里，我们展示了 ITIH4 被蛋白酶敏感区域内的几种人类蛋白酶切割，使 ITIH4 能够作为蛋白酶抑制剂发挥作用。这可以通过抑制甘露聚糖结合凝集素相关的丝氨酸蛋白酶 1 (MASP-1)、MASP-2 和血浆激肽释放酶来证明，这些是血管内宿主防御的关键蛋白酶。从机制上讲，ITIH4 充当诱饵，在切割后与依赖于 ITIH4 von Willebrand 因子 A 结构域的执行蛋白酶形成非共价抑制复合物。ITIH4 通过在空间上阻止较大的蛋白质底物进入其活性位点来抑制 MASP，对于小型基板，它们仍然可访问且功能齐全。因此，我们通过以前未描述的抑制机制证明 ITIH4 作为蛋白酶抑制剂发挥作用。