Blocking domain 6 of high molecular weight kininogen to understand intrinsic clotting mechanisms

Abstract

Background The contact system is initiated by factor (F) XII activation and the assembly of high molecular weight kininogen (HK) with either FXI or prekallikrein (PK) on a negatively charged surface. Overactivation of this system contributes to thrombosis and inflammation in numerous diseases. To develop effective therapeutics for contact system disorders, a detailed understanding of this pathway is needed. Methods We performed coagulation assays in normal human plasma and various factor‐deficient plasmas. To evaluate how HK‐mediated PK and FXI activation contributes to coagulation, we used an anti‐HK antibody to block access to domain 6 of HK, the region required for efficient activation of PK and FXI. Results FXI's binding to HK and its subsequent activation by activated FXII contributes to coagulation. We found that the 3E8 anti‐HK antibody can inhibit the binding of FXI or PK to HK, delaying clot formation in human plasma. Our data show that in the absence of FXI, however, PK can substitute for FXI in this process. Addition of activated FXI (FXIa) or activated PK (PKa) abolished the inhibitory effect of 3E8. Moreover, the requirement of HK in intrinsic coagulation can be largely bypassed by adding FXIa. Like FXIa, exogenous PKa shortened the clotting time in HK‐deficient plasma, which was not due to feedback activation of FXII. Conclusions This study improves our understanding of HK‐mediated coagulation and provides an explanation for the absence of bleeding in HK‐deficient individuals. 3E8 specifically prevented HK‐mediated FXI activation; therefore, it could be used to prevent contact activation‐mediated thrombosis without altering hemostasis.

Plasma Kallikrein as a Forgotten Clotting Factor

“…These data support a role for PKa in downstream intrinsic coagulation independent of FXII. 19 A study by Henderson et al examined protease:serpin complexes in the plasma from patients with COVID-19, to assess activation of the contact system and intrinsic pathway. 45 Levels of FXIa:serpin complexes and, to a lesser extent, PKa:C1 complexes were positively correlated with FIXa:AT level and it was thus suggested that both FXIa and PKa may contribute to FIX activation in vivo.…”

“…A recent study by Singh et al used an antibody “3E8” against domain 6 of HK, the binding site for both PK and FXI, which prevents binding of PK or FXI to HK. 19 Delayed clotting time in FXI-deficient plasma was prolonged further by the addition of 3E8, due to inhibition of PK activation, as PK-HK binding was prevented. The addition of PKa to FXI-deficient plasma in the presence of 3E8 and CTI (an inhibitor of FXIIa) significantly lessened the clotting delay, demonstrating a FXI- and FXII-independent role of PKa in clot formation.…”

“…[3][4][5] Both FXI and PK largely circulate in plasma with HK, allowing the efficient activation of both zymogens by FXIIa. 19 FXIa can cleave components of the kallikrein-kinin system; like PKa, FXIa can activate FXII, and can cleave HK to yield bradykinin, although not as efficiently as PKa. 20 PK protein is made up of four tandem repeats of 90 amino acid apple domains (A1-A4) at the N-terminus, and a catalytic domain at the C-terminus (►Fig.…”

“…3 4 5 Both FXI and PK largely circulate in plasma with HK, allowing the efficient activation of both zymogens by FXIIa. 19 FXIa can cleave components of the kallikrein–kinin system; like PKa, FXIa can activate FXII, and can cleave HK to yield bradykinin, although not as efficiently as PKa. 20…”

Factor XII Structure–Function Relationships

“…1B). [31][32][33] During contact activation, FXIIa also converts factor XI (FXI), a homolog of PK, to factor XIa (FXIa; ►Fig. 1B).…”