FXII was discovered in 1955 (29
), when plasma from an asymptomatic individual, John Hageman (FXII is also known as Hageman factor), was found to have a profoundly prolonged clotting time in vitro due to a deficiency in this protease. After the discovery of FXII, the components and functions of the contact system were elucidated, mostly using in vitro studies. The absence of a bleeding diathesis in FXII, HK, and PK deficiencies contradicts the key role of these proteins in coagulation via the intrinsic pathway in vitro, and this has left the physiological significance of the contact system unclear.
Here, we demonstrate that FXII-dependent kallikrein generation is initiated by misfolded protein aggregates, which unexpectedly is not associated with FXIa generation, indicating that the misfolded proteins are not procoagulant (see schematic illustration, Figure ). These observations were confirmed in vivo by the detection of elevated activation of both FXII and PK, but not FXI, in systemic amyloidosis patients. This finding implies that the kallikrein-kinin system and the intrinsic pathway of coagulation can be differentially regulated by FXII and can be activated independently of each other. However, it is in contrast to the simultaneous generation of kallikrein and FXIa when plasma is exposed to negatively charged surfaces such as kaolin that are commonly used in aPTT-based clotting assays. We show that adsorption of native proteins to surfaces such as kaolin leads to a conformational change (Supplemental Figure 5) that corresponds to the observed induction of FXII-dependent kallikrein generation (Figure ), just as misfolded protein aggregates (in the absence of a surface) do (Figure ). We propose that the kallikrein generation that is observed during aPTT-based clotting assays is the result of a misfolded protein cofactor; indeed, misfolded proteins in absence of a surface induce activation of PK in plasma (Figure and Supplemental Figure 7). However, no such misfolded protein cofactor is required for or capable of inducing FXII-dependent FXIa formation (Figure ).
Activation of FXII by misfolded protein aggregates leads to specific activation of the kallikrein-kinin system.
We have shown here that proteins adsorbed to a negatively charged surface induce FXII-dependent kallikrein generation and propose that this can also occur when plasma contacts a surface material. Adsorption of plasma proteins is of a transient nature, and changes can occur within a matter of seconds — a phenomenon referred to as the Vroman effect (27
). The sequence of adsorption and desorption events by different proteins on a surface is explained by the relative abundance and (in)stability of proteins, as has been postulated by Vroman and coworkers. The sequence is albumin, IgG, Fg/fibronectin, and HK/FXII. Based on our findings, we propose now that these transiently adsorbed proteins, such as albumin and Fg used in our studies, are responsible for the kallikrein generation that is seen within the time frame of surface-induced kallikrein formation, but not for clotting, whereas the direct adsorption of contact system proteins is responsible for the clotting response to a foreign surface.
Our findings demonstrate that FXII is able to activate either the intrinsic pathway of coagulation or the kallikrein-kinin system, based on the type activator that is present. But how can 1 protease activate these 2 proteolytic pathways separately? So far, 2 forms of activated FXII have been identified, called α-FXIIa and β-FXIIa. A recent publication elegantly demonstrates that adsorption of FXII to surfaces leads to changes in orientation and ordering of the molecules, which controls its activation (13
). This activation of FXII leads to the formation of α-FXIIa, retaining its full-length molecular weight (80 kDa). In plasma, α-FXIIa can undergo a single cleavage by kallikrein, but this form remains surface bound and can activate FXI (resulting in clotting). A second form of FXIIa, called β-FXIIa, is formed when α-FXIIa is cleaved a second time by kallikrein. This 28-kDa fragment is not surface bound and has lost its capacity to induce FXIa formation but has a strong kallikrein-generating potential (31
). Generation of different molecular forms of FXIIa could explain why FXII can control 2 distinct pathways in plasma, but this needs to be elucidated further.
What do our findings contribute to the understanding of the role of FXII in hemostasis? An upsurge in interest in the contact system and FXII took place recently when it was demonstrated that FXII has a role in pathological arterial thrombus formation in experimental mouse models (32
), which is suggested to occur via direct interaction with platelets (33
). This role of FXII in pathological thrombus formation is attributed to FXII-dependent generation of FXIa, since FXII- and FXI-knockout mice have the same phenotype (32
). However, there also seems to be a role for HK in arterial thrombosis, since both kininogen (34
) and BK receptor B2–deficient mice (35
) are protected from thrombosis. Unfortunately, the role of plasma PK has not been elucidated in a similar fashion. Taken together, these reports suggest that there is still no clear role for the contact system (i.e., FXII, PK, and HK) in normal hemostasis. Our findings here do confirm, however, that the procoagulant role for the contact system lies in its direct interaction with a surface (at least, with respect to intrinsic coagulation), and although this system may not be physiologically relevant, it seems to play a role in thrombosis.
What are the implications of these findings? We propose that FXII-dependent activation of PK by misfolded proteins reflects a conserved protective response that is meant to perceive and clear damaged proteins in the extracellular compartment. Misfolded proteins are generated during injury and are detected by FXII, which leads to kallikrein and BK formation. Several downstream events, which include activation of ECs, are put in motion. First, BK is a potent stimulator of EC prostacyclin synthesis, NO formation, and smooth muscle hyperpolarization factor formation (36
). These factors together make for a fast vasoactive and proinflammatory response. Second, the fibrinolytic system is activated by BK, which induces tissue-type plasminogen activator (tPA) release from ECs (36
), while kallikrein activates both urokinase-type plasminogen activator (uPA) and plasminogen. We reported earlier that tPA-dependent plasmin formation is also stimulated by misfolded proteins (23
) and occurs in systemic amyloidosis (38
). Activation of the fibrinolytic system has various consequences besides removal of fibrin polymers: active plasmin plays a role in inflammation (39
) and wound regeneration (40
). In addition, it has been reported that the crosstalk between the fibrinolytic and the kallikrein system (kallikrein-mediated plasmin formation) seems to be of importance for wound repair (41
). Last, more long-term cellular events are triggered through recruitment and activation of several cell types, including macrophages as well as cells involved in adaptive immunity (42
We here describe that misfolded protein aggregates, but not native, monomeric proteins, activate FXII, resulting in kallikrein formation. Considering the relatively wide variety of unrelated proteins that possess the capacity to trigger this system once they have adopted this aggregated structure, it is conceivable that they share a common conformation-dependent feature. Such features may resemble those of prefibrillar, oligomeric protein species that are currently held responsible for the toxicity seen in protein misfolding disease, for example, as has been described by Kayed et al. (43
). It is therefore tempting to speculate that FXII may play a protective role by recognizing these potentially harmful protein species, thereby aiding in their clearance. Once proteins have assembled into amyloid fibrils, they have lost the ability to activate FXII-dependent kallikrein generation. This in turn may reflect the inability of the human body to cope with this pathological structure, which is seen in many protein misfolding diseases.
Excessive FXII-dependent kallikrein generation may have adverse consequences. A recent paper by Gao et al. elegantly demonstrates that diabetic retinopathy is caused by alkalinization of the vitreous, which in turn results in activation of the contact system (44
). Generation of kallikrein and BK in turn leads to increased vascular permeability, followed by retinopathy. In similar fashion, lactic acidosis (which leads to low pH) has been described to induce activation of FXII and hypotension (45
). It is attractive to postulate that changes in pH lead to misfolding of proteins in vitreous, which would explain the consequent FXII and/or kallikrein generation and retinopathy. From a similar point of view, it would be of interest to investigate whether protein misfolding, FXII, and the kallikrein-kinin system are involved in the (unknown) etiology of inflammatory diseases. An example would be inflammatory bowel disease, which is in part mediated by BK and can be experimentally induced by oral administration of DXS to mice (47
). Additionally, 2 recent publications have reported that the presence of the contaminant oversulfated chondroitin sulfate in certain preparations of heparin is responsible for serious adverse clinical events that are specifically attributed to activation of FXII and PK (48
). Our data suggests that the anaphylactoid reactions are possibly mediated via the generation of misfolded protein intermediates on this negatively charged molecule that resembles the DXS used in our study, but this remains to be elucidated.
Despite a number of recent reports on the role of FXII in (thrombotic) pathology, a number of epidemiological studies on the contact system show that FXII has a protective role in vivo (28
), which seems contradictory at first glance. In most of these studies, there seems to be a nonlinear relationship between FXII levels (or FXIIa-C1inh complexes) and disease risk. Such a phenomenon suggests that FXII fulfills more than one role in vivo: one is at risk for developing pathological problems when there is a low concentration of FXII (suggesting a protective role) but also when there is too much FXII (suggesting a pathological role). Since the pathological role of FXII has been elucidated by its crucial contribution to thrombosis (32
), but not to normal hemostasis, we hereby propose that the protective role of FXII lies in its selective control over the kallikrein-kinin system. In this role, FXII recognizes misfolded proteins during tissue damage and infection and responds by activation of the kallikrein-kinin system.
In conclusion, FXII and the underlying kallikrein-kinin system are activated by misfolded proteins without induction of coagulation, which finally offers an explanation for the paradoxical role of FXII and the contact system.