Residues 1680-1684 in the A3 Domain of Factor VIII Contain a Novel Thrombin-Interactive Site Responsible for Proteolytic Cleavage at Arg1689
Authors: Nakajima Yuto , Nogami Keiji , Minami Hiroaki , Sasai Kana , Shima Midori 
Published: November 2019

Thrombin-catalyzed activation of factor (F)VIII by proteolytic cleavages at Arg372, Arg740, and Arg1689 is essential for the propagation phase of blood coagulation cascade. Activated FVIII (FVIIIa) forms the tenase complex and markedly amplifies FX activation as a cofactor of FIXa. We previously reported that thrombin interacts with FVIII through the A2 domain (residues 392-394 and 484-509) and C2 domain, and these interactions governed the cleavages at Arg740, Arg372 and Arg1689, respectively (Nogami, JBC 2000, 2005, BJH 2008). A previous report suggested, however, that FVIII lacking the C2 domain retained >50% cofactor activity (Wakabayashi, JBC 2010), supportive of the presence of other thrombin-binding site responsible for cleavage at Arg1689 within the A3-C1 domain. Recently, we focused on similar sequence in A3 acidic region to hirugen residues 54-65, and demonstrated that two region of residues 1659-1669 and 1675-1685 might contained the thrombin-binding site(s) responsible for cleavage at Arg1689 by functional and binding experiments using synthetic peptide (Minami. ASH 2015). In this study, to identify the crucial residues, seven acidic clustered residues and two sulfated Tyr residues in these regions as a series of rFVIII mutants were converted to Ala, and 9 single mutants for D1663A, Y1664A, D1665A, D1666A, Y1680A, D1681A, E1682A, D1683A, E1684A and 2 double mutants for D1665A/D1666A, D1683A/E1684A, were prepared by using a BHK system.
Specific activities of FVIII in all of 5 mutants (D1663A, Y1664A, D1665A, D1666A, D1665/D1664) in the former region and 4 mutants (Y1680A, D1683A, E1684A, D1683A/E1684A) in the latter region, assessed by a one-stage clotting assay were 40-70% and 50-70% of wild type (WT), respectively. These mutants exhibited the assay discrepancy for FVIII activity between one-stage clotting assay and FVIIIa-dependent FXa generation assay (one-stage < FXa generation assay), guessing possible association with these mutated residues for thrombin reaction.
Next, FVIII mutants (10 nM) were examined for activation by thrombin (0.4 nM) in a one-stage clotting assay. Regards FVIII mutants in the 1663-1666 region, FVIII activation by thrombin in all mutants were not significant difference from that in WT. We further examined thrombin-catalyzed cleavage at Arg1689 of these mutants by using SDS-PAGE and Western blot using an anti-FVIII monoclonal antibody recognizing the A3 acidic region for detection. The initial velocity of thrombin cleavage at Arg1689 in these mutants showed no significant difference from that in WT. In addition, the rate of thrombin cleavage at Arg372 in them was also almost similar to that in WT. These results indicated that the 1663-1666 region appeared unlikely to participate in the functional association between FVIII and thrombin.
On the other hand, regards the 1680-1684 region, peak activity in FVIII activation by thrombin for D1683A, E1684A and D1683A/E1684A mutants were modestly reduced with an ~60% level of WT, and that for Y1680A mutant was significantly diminished with peak activity of ~30% of WT. FVIII activation by thrombin in D1681A and E1682A mutants was not significant difference from WT, however. Evaluated by SDS-PAGE, the initial velocities of cleavage at Arg1689 of D1681A and E1682A were comparable to WT, whilst those of D1683A, E1684A and D1683A/E1684A mutants were ~60% level of WT. It was note that Y1680A mutant showed a ~10% of velocity rates of WT on the cleavage at Arg1689, supportive of the results obtained from thrombin-catalyzed activation of FVIII. However, the rate of thrombin cleavage at Arg372 in these mutants was almost similar to that in WT. These results suggested that three residues were involved with the cleavage at Arg1689.
In conclusion, we for the first time identified that that 1680-1684 residues in A3 acidic region, in particular sulfated Tyr1680, played a key role in thrombin-interactive sites responsible for cleavage at Arg1689.

Disclosures
No relevant conflicts of interest to declare.