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. 2019 Apr 16;9:6125. doi: 10.1038/s41598-019-42524-z

Figure 5.

Figure 5

Critical role of residue R296 in the allosteric open-closed equilibrium and activation of prothrombin. (A) Chymotrypsin efficiently digests prothrombin R296A yielding a ~38 kDa specie after cleavage at Y277 and F281 in Lnk3, L202 in kringke-2 and W357 in the protease domain. (B) Residue R296 is located towards the N-terminal of the A-chain (yellow, PDB ID: 5EDM) of the protease domain where it is involved in the R296-E300-D306-E309 ionic cluster. (C) Activation of prothrombin R296A by the prothrombinase complex monitored by SDS-PAGE under reducing conditions indicates the generation of prethrombin-2 (P2, 38 kDa). Under the same experimental conditions, prothrombin wild-type is first cleaved at R320 producing fragment-1.2.A (40 kDa) and the B chain of thrombin (30 kDa), and then at R271 with generation of the smaller A chain (6 kDa). The R296A mutation switches the pathway of activation from meizothrombin to prethrombin-2 and significantly reduces the amount of thrombin activity generated upon interaction with the physiological activator prothrombinase. All reactions were conducted in TBS buffer at 37 °C. Chemical identities of the bands were verified by N-terminal sequencing. None of the gels were cropped and originals are available in the Supplementary Information as Fig. S5. (D) Michaelis-Menten plot of the initial rates of prothrombin cleavage by prothrombinase plotted versus the concentration of prothrombin for wild-type (WT) and mutant R296A. The rates measure the activity of thrombin toward the chromogenic substrate FPR-pNA after generation from prothrombin.