Contact ignition by single-chain XIIa

In this issue of Blood, Ivanov et al¹  demonstrate that factor XII (FXII) does not have to be cleaved into a 2-chain protein to initiate contact activation by creating a form of single-chain FXIIa (scXIIa) with low-level proteolytic activity.

When teaching students, it is challenging to explain how the coagulation cascade is activated in the absence of active enzymes. The contact activation system (CAS) consists of FXII, prekallikrein (PK) and high-molecular-weight kininogen (HK) that assemble on negatively charged surfaces. The holy grail of the field is to understand how the first enzyme becomes active. There are 2 old theories. First, activation is initiated by very low concentrations of enzymes, FXIIa or kallikrein (Kal), that are present in the circulation despite the presence of natural inhibitors. Second, the proenzyme, free or when bound to a negatively charged surface, has a very low level of activity. Both hypotheses were difficult to confirm or refute, because preparations of purified proteins have low levels of contaminating enzymes resulting from isolation. As described by Silverberg et al, it is the classic story of what comes first, the chicken or egg.²  Ivanov et al have resolved the technical issues for proper investigation by preparing recombinant proteins that lack the (activation) cleavage sites.

FXII is cleaved at multiple arginine residues: Arg334, Arg343, and Arg353.³  Cleavage at Arg353 is required for enzymatic FXIIa, since the defect in factor XII Locarno, in which Arg353 is mutated to proline, prevents the formation of functional 2-chain αFXIIa.^3,4  Ivanov et al prepared cleavage-resistant FXII mutant molecules wherein each arginine is replaced by alanine. A triple mutant was also prepared with 3 alanine residues at positions 334, 343, and 353. This FXII-T mutant (T for triple) is not susceptible to cleavage, and the key novel tool developed. It displays a low level of activity toward a chromogenic substrate, and it activates PK, FXII, and FXI in purified systems, especially in the presence of negatively charged surfaces such as polyphosphates. The activity was several-thousand-fold lower than normal FXII, something predicted by Silverberg and Kaplan in 1982.²  Inclusion of polyphosphates as negatively charged surface enhances the activity of FXII-T toward its substrates. The observation that noncleaved (or noncleavable) FXII has proteolytic activity is novel and demonstrates a new biochemical form of protein, one that we would like to designate scXIIa.

The concept of FXII having enzymatic activity is not unique, since Engel et al introduced the phenomenon of scXIIa, demonstrating that zymogen FXII when complexed to polyphosphate develops chromogenic activity.⁴  This event is distinguished from dextran sulfate, which forms 2-chain FXIIa under the same conditons.⁴  The importance of the Ivanov et al study is that these authors extended the findings of Engel et al and established a convincing biochemical basis for the initiation of the contact system.¹  Single-chain enzymes are not without precedent. Both urokinase and tissue-type plasminogen activator have enzymatic activity as single-chain proteins. Importantly, FXII is structurally similar to urokinase and tissue-type plasminogen activator and distinct from other proteins that contribute to thrombin formation.

The present investigation proposes scXIIa as an initiator of CAS (see figure). In the absence of polyphosphates, scXIIa has the ability to convert zymogen PK into plasma Kal, while in the presence of polyphosphates, scXIIa activates PK faster and is also able to activate FXII or FXI into 2-chain FXIIa (αFXIIa) or active factor XI (FXIa). This step is an initiation phase. Formed Kal and αFXIIa activate more zymogen FXII, PK, and FXI. This step is the propagation phase. Formed Kal and αFXIIa then reciprocally activate each other to magnify contact activation. This amplification phase leads to thrombin formation, inflammation, complement activation, and fibrinolysis.

The influence of FXII-T is demonstrated in in vitro systems with purified proteins and in plasma. FXII-T shortens the activated partial thromboplastin time and generates thrombin at reduced rates compared with wild-type FXII (FXII-WT). However, the mutant proteins have limited activity. In a murine model with FXII-deficient mice that have reduced thrombosis, reconstitution of the animals with FXII-T or FXII-R353A does not correct the delayed time to vessel occlusion as did FXII-WT. Apparently, the initiation of contact activation by scXIIa in the ferric chloride model was insufficient for thrombus formation in the absence of normal FXII.

The study by Ivanov et al makes a substantial biochemical contribution to our understanding on the initiation of the CAS. First, the identification of scXIIa highlights the surface-dependent activation that may arise in vivo. We need to know if polyphosphate length influences outcome and if other recently recognized biologic surfaces such as DNA, RNA, misfolded proteins, or collagen amplify scXIIa.⁵  It is remarkable that the authors observed that activation of PK, FXII, and FXI occurs with scXIIa in the absence and presence of polyphosphates. Is so-called zymogen FXII always a low-level enzyme? These observations are new and important. However, virtually all PK and FXI circulate in complex with HK, and the strength of this novel proposed initiation of activation of the CAS rests on the ability to show that these processes also occur in the presence of physiologic concentrations of HK. Additionally, in plasma, C1 inhibitor is the major serpin that regulates formed Kal and αFXIIa. Polyphosphates have a variable effect on C1 inhibitor’s action: polyphosphates protect αFXIIa from inhibition by C1 inhibitor but potentiate inhibition of complement enzymes.⁶  Demonstration that scXIIa (FXII-T) is functional in the presence of C1 inhibitor would be a major step forward in understanding how activation of CAS is initiated. Last, contact activation is not the only mechanism by which FXII becomes activated. Vessel wall endothelium is rich with propylcarboxypeptidase, a serine protease that activates PK to Kal in a kinetically favorable manner. This enzyme has the ability to create the first molecules of Kal that through the complex interactions shown in the figure may lead to FXII and FXI activation and the cascade of activities associated with these systems.⁷  It remains to be established which and when initiating processes for contact activation are operative under different physiologic and pathophysiologic states.