Microlyse design and characteristics. (A) Microlyse design. Microlyse is a single polypeptide composed of the enzymatic domain of uPA, fused together with a VWF-targeting VHH through a glycine-serine linker sequence. (B) Microlyse variants (20.7 nM) gain urokinase-like activity in buffer after activation by 11.6 nM plasmin. (C) Microlyse variants (20.7 nM) activate 1.16 μM plasminogen in buffer. (D) Microlyse variants (207 nM) activate plasminogen in plasma. (E) Binding of Microlyse variants to immobilized VWF. (F) Capture of full-length VWF or a VWF variant lacking the D’D3 domain (ΔD’D3) by immobilized Microlyse variants. (G) Capture of recombinant D’D3 by immobilized Microlyse variants. (H) VWF-FVIII complexes in plasma after incubation with Microlyse variants. (I,J) Capture of full-length VWF, C-terminal VWF truncation variants, or recombinant CT/CK domain by immobilized Microlyse variants. Data represent the mean ± SD of 3 separate experiments, each performed in duplicate.