A mechanism for hereditary angioedema caused by a lysine 311-to-glutamic acid substitution in plasminogen.

Patients with hereditary angioedema (HAE) experience episodes of bradykinin (BK)-induced swelling of skin and mucosal membranes. The most common cause is reduced plasma activity of C1 inhibitor, the main regulator of the proteases plasma kallikrein (PKa) and factor XIIa (FXIIa). Recently, patients with HAE were described with a Lys311 to glutamic acid substitution in plasminogen (Plg), the zymogen of the protease plasmin (Plm). Adding tissue plasminogen activator to plasma containing Plg-Glu311 vs plasma containing wild-type Plg (Plg-Lys311) results in greater BK generation. Similar results were obtained in plasma lacking prekallikrein or FXII (the zymogens of PKa and FXIIa) and in normal plasma treated with a PKa inhibitor, indicating Plg-Glu311 induces BK generation independently of PKa and FXIIa. Plm-Glu311 cleaves high and low molecular weight kininogens (HK and LK, respectively), releasing BK more efficiently than Plm-Lys311. Based on the plasma concentrations of HK and LK, the latter may be the source of most of the BK generated by Plm-Glu311. The lysine analog ε-aminocaproic acid blocks Plm-catalyzed BK generation. The Glu311 substitution introduces a lysine-binding site into the Plg kringle 3 domain, perhaps altering binding to kininogens. Plg residue 311 is glutamic acid in most mammals. Glu311 in patients with HAE, therefore, represents reversion to the ancestral condition. Substantial BK generation occurs during Plm-Glu311 cleavage of human HK, but not mouse HK. Furthermore, mouse Plm, which has Glu311, did not liberate BK from human kininogens more rapidly than human Plg-Lys311. This indicates Glu311 is pathogenic in the context of human Plm when human kininogens are the substrates.

Activated Coagulation FXII: A Unique Target for In Vivo Molecular Imaging.

BACKGROUND: Current clinical imaging of thromboembolic diseases often relies on indirect detection of thrombi, which may delay diagnosis and ultimately the institution of beneficial, potentially lifesaving treatment. Therefore, the development of targeting tools that facilitate the rapid, specific, and direct imaging of thrombi using molecular imaging is highly sought after. One potential molecular target is FXIIa (factor XIIa), which initiates the intrinsic coagulation pathway but also activates the kallikrein-kinin system, thereby initiating coagulation and inflammatory/immune responses. As FXII (factor XII) is dispensable for normal hemostasis, its activated form (FXIIa) represents an ideal molecular target for diagnostic and therapeutic approaches, the latter combining diagnosis/identification of thrombi and effective antithrombotic therapy. METHODS: We conjugated an FXIIa-specific antibody, 3F7, to a near-infrared (NIR) fluorophore and demonstrated binding to FeCl3-induced carotid thrombosis with 3-dimensional fluorescence emission computed tomography/computed tomography and 2-dimensional fluorescence imaging. We further demonstrated ex vivo imaging of thromboplastin-induced pulmonary embolism and detection of FXIIa in human thrombi produced in vitro. RESULTS: We demonstrated imaging of carotid thrombosis by fluorescence emission computed tomography/computed tomography and measured a significant fold increase in signal between healthy and control vessels from mice injected with 3F7-NIR compared with mice injected with nontargeted probe (P=0.002) ex vivo. In a model of pulmonary embolism, we measured increased NIR signal in lungs from mice injected with 3F7-NIR compared with mice injected with nontargeted probe (P=0.0008) and healthy lungs from mice injected with 3F7-NIR (P=0.021). CONCLUSIONS: Overall, we demonstrate that FXIIa targeting is highly suitable for the specific detection of venous and arterial thrombi. This approach will allow direct, specific, and early imaging of thrombosis in preclinical imaging modalities and may facilitate monitoring of antithrombotic treatment in vivo.

Dual Inhibition of Factor XIIa and Factor XIa Produces a Synergistic Anticoagulant Effect.

ABSTRACT: Clinical practice shows that a critical unmet need in the field of thrombosis prevention is the availability of anticoagulant therapy without bleeding risk. Inhibitors against FXIa or FXIIa have been extensively studied because of their low bleeding risk. However, whether these compounds produce synergistic effects has not yet been explored. In this study, analyses of activated partial thromboplastin time in combination with the FXIa inhibitor PN2KPI and the FXIIa inhibitor Infestin4 at different proportions were performed using the SynergyFinder tool identifying synergistic anticoagulation effects. Both an FeCl 3 -induced carotid artery thrombosis mouse model and a transient occlusion of the middle cerebral artery mouse model showed that the combination of PN2KPI and Infestin4, which are 28.57% and 6.25% of the effective dose, respectively, significantly prevents coagulation, and furthermore, dual inhibition does not cause bleeding risk.

Iron-Deficiency and Estrogen Are Associated With Ischemic Stroke by Up-Regulating Transferrin to Induce Hypercoagulability.

RATIONALE: Epidemiological studies have identified an associate between iron deficiency (ID) and the use of oral contraceptives (CC) and ischemic stroke (IS). To date, however, the underlying mechanism remains poorly understood. Both ID and CC have been demonstrated to upregulate the level and iron-binding ability of Tf (transferrin), with our recent study showing that this upregulation can induce hypercoagulability by potentiating FXIIa/thrombin and blocking antithrombin-coagulation proteases interactions. OBJECTIVE: To investigate whether Tf mediates IS associated with ID or CC and the underlying mechanisms. METHODS AND RESULTS: Tf levels were assayed in the plasma of IS patients with a history of ID anemia, ID anemia patients, venous thromboembolism patients using CC, and ID mice, and in the cerebrospinal fluid of some IS patients. Effects of ID and estrogen administration on Tf expression and coagulability and the underlying mechanisms were studied in vivo and in vitro. High levels of Tf and Tf-thrombin/FXIIa complexes were found in patients and ID mice. Both ID and estrogen upregulated Tf through hypoxia and estrogen response elements located in the Tf gene enhancer and promoter regions, respectively. In addition, ID, administration of exogenous Tf or estrogen, and Tf overexpression promoted platelet-based thrombin generation and hypercoagulability and thus aggravated IS. In contrast, anti-Tf antibodies, Tf knockdown, and peptide inhibitors of Tf-thrombin/FXIIa interaction exerted anti-IS effects in vivo. CONCLUSIONS: Our findings revealed that certain factors (ie, ID and CC) upregulating Tf are risk factors of thromboembolic diseases decipher a previously unrecognized mechanistic association among ID, CC, and IS and provide a novel strategy for the development of anti-IS medicine by interfering with Tf-thrombin/FXIIa interactions.

An Ultrapotent and Selective Cyclic Peptide Inhibitor of Human ß-Factor XIIa in a Cyclotide Scaffold.

Cyclotides are plant-derived peptides with complex structures shaped by their head-to-tail cyclic backbone and cystine knot core. These structural features underpin the native bioactivities of cyclotides, as well as their beneficial properties as pharmaceutical leads, including high proteolytic stability and cell permeability. However, their inherent structural complexity presents a challenge for cyclotide engineering, particularly for accessing libraries of sufficient chemical diversity to design potent and selective cyclotide variants. Here, we report a strategy using mRNA display enabling us to select potent cyclotide-based FXIIa inhibitors from a library comprising more than 1012 members based on the cyclotide scaffold of Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II). The most potent and selective inhibitor, cMCoFx1, has a pM inhibitory constant toward FXIIa with greater than three orders of magnitude selectivity over related serine proteases, realizing specific inhibition of the intrinsic coagulation pathway. The cocrystal structure of cMCoFx1 and FXIIa revealed interactions at several positions across the contact interface that conveyed high affinity binding, highlighting that such cyclotides are attractive cystine knot scaffolds for therapeutic development.

Development of an updated assay for prekallikrein activator in albumin and immunoglobulin therapeutics.

BACKGROUND: Prekallikrein activator (PKA) is a contaminating enzyme found in therapeutic albumin and immunoglobulin products. The level is commonly measured using methods such as that defined by the European Pharmacopoeia (Ph Eur) with traceability to the WHO International Standard for PKA. This method generally works well, but problems are sometimes observed. MATERIALS AND METHODS: A simplified one-step method has been developed to replace the existing Ph Eur two-step method which consists of kallikrein generation followed by kallikrein measurement using a chromogenic substrate. Analysis of data from the one-stage method is simplified by the use of a dedicated online app. RESULTS: The one-stage method was validated against the current Ph Eur method using batches of albumin and immunoglobulins. Problem batches of immunoglobulins were investigated using the one-stage method. Improved methodology using true initial rate determinations and use of acid-treated prekallikrein substrate (PKS) helped understand and reduce artefactual results. CONCLUSIONS: The one-stage method and associated app streamline real-time determination of PKA and promote good principles of enzyme assays to limit substrate depletion, while also conserving expensive PKS. Blanking steps and reproducibility are simplified.

Peptidoglycan induces disseminated intravascular coagulation in baboons through activation of both coagulation pathways.

Anthrax infections exhibit progressive coagulopathies that may contribute to the sepsis pathophysiology observed in fulminant disease. The hemostatic imbalance is recapitulated in primate models of late-stage disease but is uncommon in toxemic models, suggesting contribution of other bacterial pathogen-associated molecular patterns (PAMPs). Peptidoglycan (PGN) is a bacterial PAMP that engages cellular components at the cross talk between innate immunity and hemostasis. We hypothesized that PGN is critical for anthrax-induced coagulopathies and investigated the activation of blood coagulation in response to a sterile PGN infusion in primates. The PGN challenge, like the vegetative bacteria, induced a sepsis-like pathophysiology characterized by systemic inflammation, disseminated intravascular coagulation (DIC), organ dysfunction, and impaired survival. Importantly, the hemostatic impairment occurred early and in parallel with the inflammatory response, suggesting direct engagement of coagulation pathways. PGN infusion in baboons promoted early activation of contact factors evidenced by elevated protease-serpin complexes. Despite binding to contact factors, PGN did not directly activate either factor XII (FXII) or prekallikrein. PGN supported contact coagulation by enhancing enzymatic function of active FXII (FXIIa) and depressing its inhibition by antithrombin. In parallel, PGN induced de novo monocyte tissue factor expression in vitro and in vivo, promoting extrinsic clotting reactions at later stages. Activation of platelets further amplified the procoagulant state during PGN challenge, leading to DIC and subsequent ischemic damage of peripheral tissues. These data indicate that PGN may be a major cause for the pathophysiologic progression of Bacillus anthracis sepsis and is the primary PAMP behind the pathogen-induced coagulopathy in late-stage anthrax.

Proteolytic properties of single-chain factor XII: a mechanism for triggering contact activation.

When blood is exposed to variety of artificial surfaces and biologic substances, the plasma proteins factor XII (FXII) and prekallikrein undergo reciprocal proteolytic conversion to the proteases αFXIIa and α-kallikrein by a process called contact activation. These enzymes contribute to host-defense responses including coagulation, inflammation, and fibrinolysis. The initiating event in contact activation is debated. To test the hypothesis that single-chain FXII expresses activity that could initiate contact activation, we prepared human FXII variants lacking the Arg353 cleavage site required for conversion to αFXIIa (FXII-R353A), or lacking the 3 known cleavage sites at Arg334, Arg343, and Arg353 (FXII-T, for "triple" mutant), and compared their properties to wild-type αFXIIa. In the absence of a surface, FXII-R353A and FXII-T activate prekallikrein and cleave the tripeptide S-2302, demonstrating proteolytic activity. The activity is several orders of magnitude weaker than that of αFXIIa. Polyphosphate, an inducer of contact activation, enhances PK activation by FXII-T, and facilitates FXII-T activation of FXII and FXI. In plasma, FXII-T and FXII-R353A, but not FXII lacking the active site serine residue (FXII-S544A), shortened the clotting time of FXII-deficient plasma and enhanced thrombin generation in a surface-dependent manner. The effect was not as strong as for wild-type FXII. Our results support a model for induction of contact activation in which activity intrinsic to single-chain FXII initiates αFXIIa and α-kallikrein formation on a surface. αFXIIa, with support from α-kallikrein, subsequently accelerates contact activation and is responsible for the full procoagulant activity of FXII.

Establishing the Transient Mass Balance of Thrombosis: From Tissue Factor to Thrombin to Fibrin Under Venous Flow.

OBJECTIVE: We investigated the coregulation of thrombin and fibrin as blood flows over a procoagulant surface. APPROACH AND RESULTS: Using microfluidic perfusion of factor XIIa-inhibited human whole blood (200 s-1 wall shear rate) over a 250-µm long patch of collagen/TF (tissue factor; ≈1 molecule per µm2) and immunoassays of the effluent for F1.2 (prothrombin fragment 1.2), TAT (thrombin-antithrombin complex), and D-dimer (post-end point plasmin digest), we sought to establish the transient mass balance for clotting under venous flow. F1.2 (but almost no free thrombin detected via TAT assay) continually eluted from clots when fibrin was allowed to form. Low-dose fluorescein-Phe-Pro-Arg-chloromethylketone stained fibrin-bound thrombin-a staining ablated by anti-γ'-fibrinogen or the fibrin inhibitor glypro-arg-pro but highly resistant to 7-minute buffer rinse, demonstrating tight binding of thrombin to γ'-fibrin. With fibrin polymerizing for 500 seconds, 92 000 thrombin molecules and 203 000 clot-associated fibrin monomer equivalents were generated per TF molecule (or per µm2). Fibrin reached 15 mg/mL in the pore space (porosity ≈0.5) of a 15-µm-thick thrombus core by 500 seconds and 30 mg/mL by 800 seconds. For a known rate of ≈60 FPA (fibrinopeptide-A) per thrombin per second, each thrombin molecule generated only 3 fibrin monomer equivalents during 500 seconds, indicating an intraclot thrombin half-life of ≈70 ms, much shorter than its diffusional escape time (≈10 seconds). By 800 seconds, gly-pro-arg-pro allowed 4-fold more F1.2 generation, consistent with gly-pro-arg-pro ablating fibrin's antithrombin-I activity and facilitating thrombin-mediated FXIa activation. CONCLUSIONS: Under flow, fibrinogen continually penetrates the clot, and γ'-fibrin regulates thrombin.

Factor XII Activation Promotes Platelet Consumption in the Presence of Bacterial-Type Long-Chain Polyphosphate In Vitro and In Vivo.

Objective- Terminal complications of bacterial sepsis include development of disseminated intravascular consumptive coagulopathy. Bacterial constituents, including long-chain polyphosphates (polyP), have been shown to activate the contact pathway of coagulation in plasma. Recent work shows that activation of the contact pathway in flowing whole blood promotes thrombin generation and platelet activation and consumption distal to thrombus formation ex vivo and in vivo. Here, we sought to determine whether presence of long-chain polyP or bacteria in the bloodstream promotes platelet activation and consumption in a coagulation factor (F)XII-dependent manner. Approach and Results- Long-chain polyP promoted platelet P-selectin expression, microaggregate formation, and platelet consumption in flowing whole blood in a contact activation pathway-dependent manner. Moreover, long-chain polyP promoted local fibrin formation on collagen under shear flow in a FXI-dependent manner. Distal to the site of thrombus formation, platelet consumption was dramatically enhanced in the presence of long-chain polyP in the blood flow in a FXI- and FXII-dependent manner. In a murine model, long-chain polyP promoted platelet deposition and fibrin generation in lungs in a FXII-dependent manner. In a nonhuman primate model of bacterial sepsis, pre-treatment of animals with an antibody blocking FXI activation by FXIIa reduced lethal dose100 Staphylococcus aureus-induced platelet and fibrinogen consumption. Conclusions- This study demonstrates that bacterial-type long-chain polyP promotes platelet activation in a FXII-dependent manner in flowing blood, which may contribute to sepsis-associated thrombotic processes, consumptive coagulopathy, and thrombocytopenia.

[Hereditary angioedema]. / Hereditäres Angioödem.

Hereditary angioedema (HAE) encompasses a heterogeneous group of diseases with similar phenotypes but different underlying genotypes. Specific clinical signs may point to HAE as opposed to histaminergic angioedema: the typical prolonged development of angioedema over time, positive family history, a lack of response to antihistamines and steroids and response to bradykinin antagonists are typical signs of HAE. The different types of HAE may be associated with a severe clinical course. They are life-long conditions and are still potentially life-threatening. The quality of life of patients with HAE may be considerably impaired. Management plans should be individualized, which is facilitated by the variety of specific medicastions available.

Plasma contact activation by a fucosylated chondroitin sulfate and its structure-activity relationship study.

Plasma contact system is the initial part of both the intrinsic coagulation pathway and kallikrein-kinin pathway, which mainly involves three proteins: coagulation factor XII (FXII), prekallikrein (PK) and high-molecular weight kininogen. Fucosylated chondroitin sulfate (FCS) is a unique sulfated glycosaminoglycan (GAG) composed of a chondroitin sulfate-like backbone and sulfated fucose branches. The native FCS was preliminary found to cause undesired activation of the plasma contact system. How this unusual GAG functions in this process remains to be clarified. Herein, the relationship between its structure, plasma contact activation and its effects on the PK-FXII reciprocal activation loop were studied. The recalcification time assay indicated that the FCS at high concentration could be procoagulant which may be attributed to its contact activation activity. The structure-activity relationship study indicated that its high molecular weight and distinct fucose side chains are required for contact activation by FCS, although the sulfate substitution types of its side chains have less impact. In human plasma, the native FCSs potently induced FXII-dependent contact activation. However, in purified systems FCS did not significantly activate FXII per se or induce its autoactivation, whereas FCS significantly promoted the activation of PK by factor XIIa. Polysaccharide-protein interaction assays showed that FCS bound to PK with higher affinity than other contact system proteins. These data suggested that potent contact activation by FCS requires the positive feedback loop between PK and FXII. These findings contribute to better understanding of contact activation by complex GAG.

Hemostasis in Allergy.

The involvement of the hemostatic system in immune-mediated inflammation is widely reported. Many coagulation factors play a role in the pathogenesis of autoimmune diseases, such as systemic vasculitis and systemic lupus erythematosus. Hemostatic disorders are also involved in asthma and chronic spontaneous urticaria (CSU). Factor XIIa (FXIIa) was one of the first coagulation factors implicated in inducing both humoral and cellular responses and is therefore considered a prime new therapeutic target in immune-mediated inflammation. The involvement of coagulation factors, such as tissue factor and fibrinogen, in the pathogenesis of asthma has been reported. The finding of platelet activation in asthma also indicates a link between bronchial inflammation and hemostasis. The pathogenesis of mast cell degranulation and CSU was also shown to be associated with the activation of hemostatic factors such as fibrinogen and FXIIa. Increased plasma levels of D-dimer have been widely reported as a biological marker for the duration and severity of CSU. In addition, endothelial-induced cell activation by the kallikrein-high molecular weight complex and the release of heat shock protein 90 was shown to be involved in mast cell degranulation disorders. In this narrative review, the authors aim to summarize the role of hemostasis in inflammation, asthma, and CSU by focusing on the increasing information linking hemostatic factors and immune-mediated disorders.

Factor XII as a Therapeutic Target in Thromboembolic and Inflammatory Diseases.

Coagulation factor XII (FXII, Hageman factor) is a plasma protease that in its active form (FXIIa) initiates the procoagulant and proinflammatory contact system. This name arises from FXII's unique mechanism of activation that is induced by binding (contact) to negatively charged surfaces. Various substances have the capacity to trigger FXII contact-activation in vivo including mast cell-derived heparin, misfolded protein aggregates, collagen, nucleic acids, and polyphosphate. FXII deficiency is not associated with bleeding, and for decades, the factor was considered to be dispensable for coagulation in vivo. However, despite the fact that humans and animals with deficiency in FXII have a normal hemostatic capacity, animal models revealed a critical role of FXIIa-driven coagulation in thromboembolic diseases. In addition to its role in thrombosis, FXIIa contributes to inflammation through the activation of the inflammatory bradykinin-producing kallikrein-kinin system. Pharmacological inhibition of FXII/FXIIa interferes with thrombosis and inflammation in animal models. Thus, targeting the FXIIa-driven contact system seems to be a promising and safe therapeutic anticoagulation treatment strategy, with additional anti-inflammatory effects. Here, we discuss novel functions of FXIIa in cardiovascular thrombotic and inflammatory disorders.

Activation of the factor XII-driven contact system in Alzheimer's disease patient and mouse model plasma.

Alzheimer's disease (AD) is characterized by accumulation of the ß-amyloid peptide (Aß), which likely contributes to disease via multiple mechanisms. Increasing evidence implicates inflammation in AD, the origins of which are not completely understood. We investigated whether circulating Aß could initiate inflammation in AD via the plasma contact activation system. This proteolytic cascade is triggered by the activation of the plasma protein factor XII (FXII) and leads to kallikrein-mediated cleavage of high molecular-weight kininogen (HK) and release of proinflammatory bradykinin. Aß has been shown to promote FXII-dependent cleavage of HK in vitro. In addition, increased cleavage of HK has been found in the cerebrospinal fluid of patients with AD. Here, we show increased activation of FXII, kallikrein activity, and HK cleavage in AD patient plasma. Increased contact system activation is also observed in AD mouse model plasma and in plasma from wild-type mice i.v. injected with Aß42. Our results demonstrate that Aß42-mediated contact system activation can occur in the AD circulation and suggest new pathogenic mechanisms, diagnostic tests, and therapies for AD.

Single-chain factor XII: a new form of activated factor XII.

PURPOSE OF REVIEW: Exposure of blood to foreign surfaces induces reciprocal conversion of the plasma proteins factor XII (fXII) and plasma prekallikrein (PPK) to the proteases α-fXIIa and α-kallikrein. This process, called contact activation, has a range of effects on host defence mechanisms, including promoting coagulation. The nature of the triggering mechanism for contact activation is debated. One hypothesis predicts that fXII has protease activity, either intrinsically or upon surface-binding, that initiates contact activation. We tested this by assessing the proteolytic activity of a recombinant fXII variant that cannot be converted to α-fXIIa. RECENT FINDINGS: The proteolytic activity of fXII-T (for 'triple' mutant), a variant with alanine substitutions for arginine at activation cleavage sites (Arg334, Arg344, and Arg353) was tested with known α-fXIIa substrates. FXII-T activates PPK in solution, and the reaction is enhanced by polyphosphate, an inducer of contact activation released from platelets. In the presence of polyphosphate, fXII-T converts fXII to α-fXIIa, and also converts the coagulation protein factor XI to its active form. SUMMARY: The findings support the hypothesis that contact activation is initiated through activity intrinsic to single-chain fXII, and indicate that preexisting α-fXIIa is not required for induction of contact activation.

Alleviation of secondary brain injury, posttraumatic inflammation, and brain edema formation by inhibition of factor XIIa.

BACKGROUND: Traumatic brain injury (TBI) is a devastating neurological condition and a frequent cause of permanent disability. Posttraumatic inflammation and brain edema formation, two pathological key events contributing to secondary brain injury, are mediated by the contact-kinin system. Activation of this pathway in the plasma is triggered by activated factor XII. Hence, we set out to study in detail the influence of activated factor XII on the abovementioned pathophysiological features of TBI. METHODS: Using a cortical cryogenic lesion model in mice, we investigated the impact of genetic deficiency of factor XII and inhibition of activated factor XII with a single bolus injection of recombinant human albumin-fused Infestin-4 on the release of bradykinin, the brain lesion size, and contact-kinin system-dependent pathological events. We determined protein levels of bradykinin, intracellular adhesion molecule-1, CC-chemokine ligand 2, and interleukin-1ß by enzyme-linked immunosorbent assays and mRNA levels of genes related to inflammation by quantitative real-time PCR. Brain lesion size was determined by tetrazolium chloride staining. Furthermore, protein levels of the tight junction protein occludin, integrity of the blood-brain barrier, and brain water content were assessed by Western blot analysis, extravasated Evans Blue dye, and the wet weight-dry weight method, respectively. Infiltration of neutrophils and microglia/activated macrophages into the injured brain lesions was quantified by immunohistological stainings. RESULTS: We show that both genetic deficiency of factor XII and inhibition of activated factor XII in mice diminish brain injury-induced bradykinin release by the contact-kinin system and minimize brain lesion size, blood-brain barrier leakage, brain edema formation, and inflammation in our brain injury model. CONCLUSIONS: Stimulation of bradykinin release by activated factor XII probably plays a prominent role in expanding secondary brain damage by promoting brain edema formation and inflammation. Pharmacological blocking of activated factor XII could be a useful therapeutic principle in the treatment of TBI-associated pathologic processes by alleviating posttraumatic inflammation and brain edema formation.

Factor XII Contact Activation.

Contact activation is the surface-induced conversion of factor XII (FXII) zymogen to the serine protease FXIIa. Blood-circulating FXII binds to negatively charged surfaces and this contact to surfaces triggers a conformational change in the zymogen inducing autoactivation. Several surfaces that have the capacity for initiating FXII contact activation have been identified, including misfolded protein aggregates, collagen, nucleic acids, and platelet and microbial polyphosphate. Activated FXII initiates the proinflammatory kallikrein-kinin system and the intrinsic coagulation pathway, leading to formation of bradykinin and thrombin, respectively. FXII contact activation is well characterized in vitro and provides the mechanistic basis for the diagnostic clotting assay, activated partial thromboplastin time. However, only in the past decade has the critical role of FXII contact activation in pathological thrombosis been appreciated. While defective FXII contact activation provides thromboprotection, excess activation underlies the swelling disorder hereditary angioedema type III. This review provides an overview of the molecular basis of FXII contact activation and FXII contact activation-associated disease states.

Heparan sulfate proteoglycans mediate factor XIIa binding to the cell surface.

Hageman factor (FXIIa) initiates the intrinsic coagulation pathway and triggers the kallikrein-kinin and the complement systems. In addition, it functions as a growth factor by expressing promitogenic activities toward several cell types. FXIIa binds to the cell surface via a number of structurally unrelated surface receptors; however, the underlying mechanisms are not yet fully understood. Here, we demonstrate that FXIIa utilizes cell membrane-bound glycosaminoglycans to interact with the cell surface of human lung fibroblasts (HLF). The combination of enzymatic, inhibitory, and overexpression approaches identified a heparan sulfate (HS) component of proteoglycans as an important determinant of the FXIIa binding capacity of HLF. Moreover, cell-free assays and competition experiments revealed preferential binding of FXIIa to HS and heparin over dextran sulfate, dermatan sulfate, and chondroitin sulfate A and C. Finally, we demonstrate that fibroblasts isolated from the lungs of the patients suffering from idiopathic pulmonary fibrosis (IPF) exhibit enhanced FXIIa binding capacity. Increased sulfation of HS resulting from elevated HS 6-O-sulfotransferase-1 expression in IPF HLF accounted, in part, for this phenomenon. Application of RNA interference technology and inhibitors of intracellular sulfation revealed the cooperative action of cell surface-associated HS and urokinase-type plasminogen activator receptor in the accumulation of FXIIa on the cell surface of IPF HLF. Moreover, FXIIa stimulated IPF HLF migration, which was abrogated by pretreatment of cells with heparinase I. Collectively, our study uncovers a novel role of HS-type glycosaminoglycans in a local accumulation of FXIIa on the cell membrane. The enhanced association of FXIIa with IPF HLF suggests its contribution to fibrogenesis.

C1-inhibitor polymers activate the FXII-dependent kallikrein-kinin system: Implication for a role in hereditary angioedema.

BACKGROUND: The FXII-dependent kallikrein-kinin system (KKS) is tightly regulated by the serine protease inhibitor (serpin) C1-inhibitor (C1-inh). When regulation of the FXII-dependent KKS fails, which is the case in hereditary angioedema (HAE), patients consequently experience invalidating edema attacks. HAE is caused by mutations in the C1-inh encoding gene, and we recently demonstrated that some mutations give rise to the presence of polymerized C1-inh in the plasma of HAE patients. METHODS: C1-inh polymers corresponding to the size of polymers observed in vivo were produced using heat denaturation and gel filtration. The ability of these polymers to facilitate FXII activation was assessed in vitro in an FXII activation bandshift assay. After spiking of plasma with C1-inh polymers, kallikrein generation was analyzed in a global kallikrein generation method. Prekallikrein consumption in the entire Danish HAE cohort was analyzed using an ELISA method. RESULTS: C1-inh polymers mediated FXII activation, and a dose dependent kallikrein generation in plasma spiked with C1-inh polymers. An increased (pre)kallikrein consumption was observed in plasma samples from HAE patients presenting with C1-inh polymers in vivo. CONCLUSION: Polymerization of the C1-inh transforms the major inhibitor of the FXII-dependent KKS, into a potent activator of the very same system. GENERAL SIGNIFICANCE: The C1-inh polymers might play a role in the pathophysiology of HAE, but several diseases are characterized by the presence of serpin polymers. The role of serpin polymers has so far remained elusive, but our results indicate that such polymers can play a role as inflammatory mediators through the FXII-dependent KKS.