FXII

The plasma protein FXII (Hageman factor) has been shown to be linked with the plasma defence systems of coagulation, fibrinolysis, kallikrein-kinin and complement. It can be activated by surface contact activation and in solution. Surface contact activation is a complex phenomenon involving negatively charged surfaces, FXII, high molecular weight kininogen and plasma kallikrein. Fluid-phase activation can be effected by a variety of serine proteases. In both types of activation the FXII zymogen is converted to active enzymes. FXII levels in plasma are low or undetectable in both inherited deficiencies and in a variety of clinical conditions. FXII levels can also be elevated in some clinical conditions. Although discovered as a clotting protein FXII appears to play an important role in the kallikrein-kinin and fibrinolytic systems and also has effects on cells. Recent studies suggest that therapeutic blockade of activation of FXII can be of benefit in certain clinical conditions.     

Pleiotropic effects of a vibrio extracellular protease on the activation of contact system

Many proteases secreted by pathogenic bacteria can affect seriously on hemostatic system. We have reported that an extracellular zinc metalloprotease (named vEP-45) from Vibrio vulnificus ATCC29307 activates prothrombin to active thrombin, leading the formation of fibrin clot. In this study, the effects of vEP-45 on the intrinsic pathway of coagulation and the kallikrein/kinin system were examined. The protease could activate proteolytically clotting factor zymogens, including FXII, FXI, FX, and prothrombin, to their functional enzymes in vitro and plasma milieu. In addition, it could cleave plasma prekallikrein (PPK) to form an active kallikrein as well as actively digest high-molecular weight kininogen (HK), probably producing bradykinin. In fact, vEP-45 could induce a vascular permeability in a dose-dependent manner in vivo. Taken together, the results demonstrate that vEP-45 can activate plasma contact system by cleaving key zymogen molecules, participating in the intrinsic pathway of coagulation and the kallikrein/kinin system.     

Factor XII in coagulation,inflammation and beyond

Factor XII (FXII) is a protease that is mainly produced in the liver and circulates in plasma as a single chain zymogen. Following contact with negatively charged surfaces, FXII is converted into the two-chain active form, FXIIa. FXIIa initiates the intrinsic blood coagulation pathway via activation of factor XI. Furthermore, it converts plasma prekallikrein to kallikrein (PK), which reciprocally activates FXII and liberates bradykinin from high molecular weight kininogen. In addition, FXIIa initiates fibrinolysis via PK-mediated urokinase activation and activates the classical complement pathway. Even though the main function of FXII seems to relate to the activation of the intrinsic coagulation pathway and the kallikrein-kinin system, a growing body of evidence suggests that FXII may also directly regulate cellular responses. In this regard, it has been found that FXII/FXIIa induces the expression of inflammatory mediators, promotes cell proliferation, and enhances the migration of neutrophils and lung fibroblasts. In addition, it has been reported that genetic ablation of FXII protects against neuroinflammation, reduces the formation of atherosclerotic lesions in Apoe^−/− mice, improves wound healing, and inhibits postnatal angiogenesis. Although the aforementioned effects can be partially explained by the downstream products of FXII activation, the ability of FXII/FXIIa to directly regulate cellular responses has recently emerged as an alternative hypothesis. These direct cellular reactions to FXII/FXIIa will be discussed in the review.     

Activated human platelets induce factor XIIa-mediated contact activation

Earlier studies have shown that isolated platelets in buffer systems can promote activation of FXII or amplify contact activation, in the presence of a negatively charge substance or material. Still proof is lacking that FXII is activated by platelets in a more physiological environment. In this study we investigate if activated platelets can induce FXII-mediated contact activation and whether this activation affects clot formation in human blood.Human platelets were activated with a thrombin receptor-activating peptide, SFLLRN-amide, in platelet-rich plasma or in whole blood. FXIIa and FXIa in complex with preferentially antithrombin (AT) and to some extent C1-inhibitor (C1INH) were generated in response to TRAP stimulation. This contact activation was independent of surface-mediated contact activation, tissue factor pathway or thrombin. In clotting whole blood FXIIa-AT and FXIa-AT complexes were specifically formed, demonstrating that AT is a potent inhibitor of FXIIa and FXIa generated by platelet activation. Contact activation proteins were analyzed by flow cytometry and FXII, FXI, high-molecular weight kininogen, and prekallikrein were detected on activated platelets. Using chromogenic assays, enzymatic activity of platelet-associated FXIIa, FXIa, and kallikrein were demonstrated. Inhibition of FXIIa in non-anticoagulated blood also prolonged the clotting time.We conclude that platelet activation triggers FXII-mediated contact activation on the surface and in the vicinity of activated platelets. This leads specifically to generation of FXIIa-AT and FXIa-AT complexes, and contributes to clot formation. Activated platelets may thereby constitute an intravascular locus for contact activation, which may explain the recently reported importance of FXII in thrombus formation.     

Hageman factor-dependent pathways: mechanism of initiation and bradykinin formation

The concentration of bradykinin in human plasma depends on its relative rates of formation and destruction. Bradykinin is destroyed by two enzymes: a plasma carboxypeptidase (anaphylatoxin inactivator) removes the COOH-terminal arginine to yield an inactive octapeptide, and a dipeptidase (identical to the angiotensin-converting enzyme) removes the COOH-terminal Phe-Arg to yield a fragment of seven amino acids that is further fragmented to an end product of five amino acids. Formation of bradykinin is initiated on binding of Hageman factor (HF) to certain negatively charged surfaces on which it autoactivates by an autodigestion mechanism. Initiation appears to depend on a trace of intrinsic activity present in HF that is at most 1/4000 that of activated HF (HFa); alternatively traces of circulating HFa could subserve the same function. HFa then converts coagulation factor XI to activated factor XI (XIa) and prekallikrein to kallikrein. Kallikrein then digests high-molecular-weight kininogen (HMW-kininogen) to form bradykinin. Prekallikrein and factor XI circulate bound to HMW-kininogen and surface binding of these complexes is mediated via this kininogen. In the absence of HMW-kininogen, activation of prekallikrein and factor XI is much diminished; thus HMW-kininogen has a cofactor function in kinin formation and coagulation. Once a trace of kallikrein is generated, a positive feedback reaction occurs in which kallikrein rapidly activates HF. This is much faster than the HF autoactivation rate; thus most HFa is formed by a kallikrein-dependent mechanism. HMW-kininogen is also therefore a cofactor for HF activation, but its effect on HF activation is indirect because it occurs via kallikrein formation. HFa can be further digested by kallikrein to form an active fragment (HFf), which is not surface bound and acts in the fluid phase. The activity of HFf on factor XI is minimal, but it is a potent prekallikrein activator and can therefore perpetuate fluid phase bradykinin formation until it is inactivated by the C1 inhibitor. In the absence of C1 inhibitor (hereditary angioedema) HFf may also interact with C1 and activate it enzymatically. The resultant augmented bradykinin formation and complement activation may account for the pathogenesis of the swelling characteristic of hereditary angioedema and the serologic changes observed during acute attacks.     

Contact phase of blood coagulation is not activated in edema of high altitude

To examine whether bradykinin generated by the activation of the contact phase of blood coagulation is involved in the pathogenesis of edema occurring after acute exposure to high altitude, 15 mountaineers were examined at 490 m and 1, 3, and 5 days after arrival at 4,559 m. The clotting activity levels of factor XII, factor XI, plasma prekallikrein, and high-molecular-weight kininogen (HMWK) were measured, and plasma kallikrein-induced proteolytic cleavage of HMWK was assessed by ligand blotting by use of radiolabeled factor XI. After an ascent on foot from 1,170 to 4,559 m in 3 days, three subjects developed high-altitude pulmonary edema, and four subjects presented facial edema. There was no evidence for activation of the contact system in any subject as demonstrated by the lack of proteolytic cleavage of HMWK at high altitude. The absence of contact system activation was further supported by stable plasma levels of the individual factors of contact activation. Therefore, we conclude that bradykinin generated by plasma kallikrein-induced cleavage of HMWK is not involved in the pathogenesis of edema due to acute exposure to high altitude.     

The hyaluronan-binding serine protease from human plasma cleaves HMW and LMW kininogen and releases bradykinin

The influence of the hyaluronan-binding protease (PHBSP), a plasma enzyme with FVII- and pro-urokinase-activating potency, on components of the contact phase (kallikrein/kinin) system was investigated. No activation or cleavage of the proenzymes involved in the contact phase system was observed. The pro-cofactor high molecular weight kininogen (HK), however, was cleaved in vitro by PHBSP in the absence of any charged surface, releasing the activated cofactor and the vasoactive nonapeptide bradykinin. Glycosoaminoglycans strongly enhanced the reaction. The cleavage was comparable to that of plasma kallikrein, but clearly different from that of coagulation factor FXIa. Upon extended incubation with PHBSP, the light chain was further processed, partially removing about 60 amino acid residues from the N-terminus of domain D5 of the light chain. These cleavage site(s) were distinct from plasma kallikrein or FXIa cleavage sites. PHBSP and, more interestingly, also plasma kallikrein could cleave low molecular weight kininogen in vitro, indicating that domains D5H and D6H are no prerequisite for kininogen cleavage. PHBSP was also able to release bradykinin from HK in plasma where the pro-cofactor circulates predominantly in complex with plasma kallikrein or FXI. In conclusion, PHBSP represents a novel kininogen-cleaving and bradykinin-releasing enzyme in plasma that shares significant catalytic similarities with plasma kallikrein. Since they are structurally unrelated in their heavy chains (propeptide), their similar in vivo catalytic activities might be directed at distinct sites where PHBSP could induce processes that are related to the kallikrein/kinin system.     

Local bradykinin formation is controlled by glycosaminoglycans

Bradykinin is a potent inflammatory mediator that induces vasodilation, vascular leakage, and pain sensations. This short-lived peptide hormone is liberated from its large precursor protein high molecular weight kininogen (HK) through the contact system cascade involving coagulation factor XII and plasma kallikrein. Although bradykinin release is well established in vitro, the factors and mechanisms controlling bradykinin generation in vivo are still incompletely understood. In this study we demonstrate that binding of HK to glycosaminoglycans (GAGs) of the heparan and chondroitin sulfate type efficiently interferes with bradykinin release in plasma and on endothelial surfaces. Proteolytic bradykinin production on endothelial cells is restored following degradation of cell surface GAG through heparinase. Alternatively, application of HK fragments D3 or light chain, which compete with uncleaved HK for cell binding, promote kininogen proteolysis and bradykinin release. Intravital microscopy revealed that HK fragments increase bradykinin-mediated mesentery microvascular leakage. Topical application of D3 or light chain enhanced bradykinin generation and edema formation in the mouse skin. Our results demonstrate that bradykinin formation is controlled by HK binding to and detachment from GAGs. Separation of the precursor from cell surfaces is a prerequisite for its efficient proteolytic processing. By this means, fragments arising from HK processing propagate bradykinin generation, revealing a novel regulatory level for the kallikrein-kinin system.     

Stimulation of blood mononuclear cells with bacterial virulence factors leads to the release of pro-coagulant and pro-inflammatory microparticles

Severe infectious diseases remain a major and life-threatening health problem. In serious cases a systemic activation of the coagulation cascade and hypovolemic shock are critical complications that are associated with high mortality rates. Here we report that blood mononuclear cells, stimulated with M1 protein of Streptococcus pyogenes or other bacterial virulence factors, produce not only pro-coagulant, but also pro-inflammatory microparticles (MPs). Our results also show that activation of the contact system on MPs contributes to these two effects. Phosphatidylserine (PS) plays an important role in these processes as its upregulation on MPs allows an assembly and activation of the contact system. This in turn results in stabilization of the tissue factor-induced clot and a processing of high-molecular-weight kininogen by plasma kallikrein followed by the release of bradykinin, a potent vascular mediator. Pro-coagulant monocyte-derived MPs were identified in plasma samples from septic patients and further analysis of MPs from these patients revealed that their pro-coagulant activity is dependent on the tissue factor- and contact system-driven pathway.     

Assembly, activation, and signaling by kinin-forming proteins on human vascular smooth muscle cells

Cardiovascular disease is the number one cause of death in the United States. Vascular smooth muscle cells (VSMC) are an important constituent of the vessel wall that can bring about pathological changes leading to vascular disease. Depending on the environment, the function of VSMC can deviate profoundly from its normal contractile role. Despite advances in research, the underlying mechanisms that activate VSMC toward vascular disease are poorly understood. For the first time, we have observed that factor XII and high-molecular-weight kininogen, constituents of the blood plasma, can bind to VSMC in a Zn2+-dependent manner. In the presence of prekallikrein, this assembly of factor XII and high-molecular-weight kininogen on VSMC leads to the activation of prekallikrein to kallikrein with a rapid formation of bradykinin. The amount of bradykinin in the culture medium then decreases, presumably because of the presence of a kininase activity. p44/42 mitogen-activated protein kinase is rapidly phosphorylated in response to in situ-generated or in vitro-added bradykinin and is inhibited by bradykinin antagonist HOE-140. Binding of factor XII to VSMC also results in a concentration-dependent phosphorylation of p44/42 mitogen-activated protein kinase. This early mitogenic signal, which is also implicated in atherogenesis, may change the metabolic and proliferative activity of VSMC, which are key steps in the progression of atherosclerosis.     

Characterization of serine proteinases isolated from rat submaxillary gland: with special reference to the degradation of rat kininogens by these enzymes

From the homogenate of rat submaxillary gland, two kinds of serine proteinases, named tentatively proteinases A and B, were isolated and their chemical properties and activities toward rat kininogens were examined, in comparison with those of submaxillary kallikrein. Proteinase A with Mr of 28,200 rapidly cleaved high-molecular-weight (HMW) kininogen into a protein of 67 kDa, which retained thiol-proteinase inhibitory activity, but had lost the correcting activity of HMW kininogen on the prolonged clotting time of Fitzgerald trait plasma. It liberated bradykinin from HMW kininogen but did not liberate kinin from T-kininogen and did not degrade T-kininogen. On the other hand, proteinase B with Mr of 30,400 showed a very weak activity for the liberation of kinin from T-kininogen and the cleavage of T-kininogen at pH 8.0. However, the enzyme extensively degraded T-kininogen at pH 4.5. Proteinase B also degraded HMW kininogen at pH 4.5 and pH 8.0, but liberated bradykinin only at pH 8.0. Thiol-proteinase inhibitory activities of HMW kininogen and T-kininogen were inactivated after the incubation with proteinase B at pH 4.5 but not at pH 8.0, while the correcting activity of HMW kininogen on the Fitzgerald trait plasma was inactivated at pH 4.5 and 8.0. The NH2-terminal amino acid sequences of proteinases A and B were different from each other, and distinguishable with those of serine proteinases in rat submaxillary gland so far reported. These results provide evidence that in addition to the known kallikrein, there exist at least two kinds of serine proteinases in rat submaxillary gland, both of which liberate bradykinin from rat HMW kininogen at pH 8.0 and modulate the functional activities of HMW kininogen and T-kininogen, degrading these proteins at pH 8.0 or 4.5.     

Canine Plasma Kininogen: Evidence for the Presence of Two Kininogens and Purification of High Molecular Weight Kininogen and Characterization as Multi-Functional Molecule

The ratio of kininogen that is substrate of plasma kallikrein to kininogen, which is not substrate of plasma kallikrein in canine plasma, was about 1:3.6 by differential assay of kininogens. When the plasma was gel-filtered through a column of Sephacryl S-300 superfine, two fractions, which released kinin by trypsin, were obtained. These results indicate that two kininogens with different molecular weights are present in the plasma and they show different susceptibility to plasma kallikrein. One kininogen was purified by ion-exchange and zinc-chelating affinity chromatographies. Purified kininogen showed a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing condition and its molecular weight was 125 kDa. Released kinin from the kininogen by trypsin was bradykinin. The kininogen inhibited papain and ficin but did not inhibit bromelain at the concentration used. The kininogen bound to carboxymethylated-papain and this binding was dissociated by 3M NaSCN. Canine plasma shortened the abnormal clotting time of human high molecular weight kininogen-deficint plasma. The kininogen also shortened the abnormal clotting time of the plasma. From these results, the purified kininogen was high molecular weight kininogen and it was multi-functional protein.     

Identification and characterization of a new kallikrein-kinin system inhibitor from the salivary glands of the malaria vector mosquito Anopheles stephensi

A new kallikrein-kinin system inhibitor, designated anophensin, was identified in the salivary glands of the malaria vector mosquito, Anopheles stephensi. In vitro reconstitution experiments showed that anophensin inhibits activation of the kallikrein-kinin system by inhibiting the reciprocal activation of factor XII (FXII) and prekallikrein (PK), and subsequent release of bradykinin. Additionally, anophensin inhibits activation of the kallikrein-kinin system on cultured human umbilical vein endothelial cells (HUVECs). Direct binding assays show that this inhibitory effect is due to Zn(2+)-dependent specific binding of anophensin to both FXII and high molecular weight kininogen (HK). Furthermore, anophensin interacts with both the N-terminus of FXII and domain D5 of HK, which are the binding domains for biological activating surfaces. These results suggest that anophensin inhibits activation of the kallikrein-kinin system by interfering with the association of FXII and HK with biological activating surfaces, resulting in the inhibition of bradykinin release in a host animal during insect blood-feeding.     

Activation mechanism of human Hageman factor-plasma kallikrein-kinin system by Vibrio vulnificus metalloprotease.

Vivrio vulnificus, an opportunistic human pathogen, secretes a metalloprotease (VVP). The VVP inoculated into a guinea pig is known to generate bradykinin through activation of the Hageman factor-plasma kallikrein-kinin system. VVP was shown to possess the ability to activate the human system through the same mechanism as that clarified in the guinea pig system, namely, VVP converted both human zymogens (Hageman factor and plasma prekallikrein) to active enzymes (activated Hageman factor and plasma kallikrein), and the then generated kallikrein liberated bradykinin from high-molecular-weight kininogen. However, in the presence of plasma alpha 2-macroglobulin (alpha 2M), the VVP action was drastically decreased. This finding suggests that the human system might be activated only at the interstitial-tissue space which contains negligible amounts of alpha 2M or in the bloodstream of the individuals whose plasma alpha 2M level is extremely reduced.     

Cleavage of human high molecular weight kininogen by factor XIa in vitro. Effect on structure and function

We have recently demonstrated that human high molecular weight kininogen (HMWK) is a pro-cofactor that is cleaved by kallikrein to yield a two-chain cofactor (HMWKa) and the nanopeptide bradykinin. This proteolysis enhances its association with an activating surface, an event necessary for expression of its cofactor activity. We now report that factor XIa is capable of hydrolyzing HMWK and releasing bradykinin in a purified system as well as cleaving and inactivating HMWK in a plasma environment during the contact-activation process. The profile of proteolysis differs from that produced by kallikrein and by factor XIIa in that the first cleavage by factor XIa yields 75- and 45-kDa polypeptides, whereas both factor XIIa and kallikrein initially produce 65- and 56-kDa species. Further proteolysis by all three enzymes eventually produces similar heavy chains (Mr = 65,000) and light chains (Mr = 45,000). However, the amount of factor XIa generated in plasma during contact activation further degrades the light chain of HMWK, eventually destroying its coagulant activity. Furthermore, in a purified system, enhancement of the degradation of HMWK coagulant activity by factor XIa was achieved when kallikrein was included in the incubation mixture, suggesting that the preferred substrate for factor XIa is the active form of HMWK (HMWKa), and not the pro-cofactor. These data suggest that factor XIa has the potential to act as a regulator of contact-activated coagulation by virtue of its ability to destroy the cofactor function of HMWK after its generation by either kallikrein, factor XIIa, or to a lesser extent, factor XIa, itself.     

The fragments of bovine high molecular weight kininogen promote osteoblast proliferation in vitro

High molecular weight (HMW) kininogen is known to be a large plasma protein and cleaved by plasma proteinase kallikrein, then it generates four fragments in the blood coagulation cascade: heavy chain, bradykinin, fragment 1.2, and light chain. The fragment 1.2 has also been found in the basic protein fraction of bovine milk as a bioactive protein which promotes osteoblast proliferation. The milk basic protein has been shown to be a multi functional edible protein which promotes bone formation and inhibits bone resorption. In the present study, we purified the fragment 1.2 from bovine plasma and assessed it could promote osteoblast proliferation and posses the activity after pepsin digestion. Purified plasma HMW kininogen did not promote the proliferation, however, the kallikrein-cleaved HMW kininogen promoted the proliferation. The fragment 1.2, purified from the proteolysate, also promoted the proliferation. The pepsin digestion was performed according to the method of the assessment of allergenesity of genetically modified crops. After pepsin digestion, the fragment 1.2 generated resistant fragments and showed the promoting activity of osteoblast proliferation. These results suggest that the enzymatically-digested fragments of bovine HMW kininogen are able to be a naturally occurred active protein that promotes the bone formation by oral administration.     

Pathogenesis of serratial infection: activation of the Hageman factor-prekallikrein cascade by serratial protease

A serratial protease with an apparent molecular weight of 56,000 (56K protease), which had been purified from the culture supernatant of a strain of Serratia marcescens isolated from a corneal lesion of a human eye [Matsumoto, K. et al. (1984) J. Bacteriol. 157, 225-232], greatly enhanced vascular permeability when injected into guinea pig skin. The 56K protease, which requires zinc ion for activity, was found to possess plasma kallikrein-like properties in vitro as judged by (i) preferential amidolysis of carbobenzoxy-Phe-Arg-4-methylcoumaryl-7-amide and Pro-Phe-Arg-4-methylcoumaryl-7-amide, which are known substrates for plasma kallikrein; (ii) release of kinin from high-molecular-weight kininogen; and (iii) prompt activation of Hageman factor followed by generation of kallikrein from plasma prekallikrein. These results suggest that the 56K protease enhances vascular permeability through activation of a Hageman factor-kallikrein-kinin pathway in vivo, and this molecular process appears to be a rational mechanism of enhancement of permeability and serratial pathogenesis.     

Interaction of human plasma kallikrein and its light chain with alpha 2-macroglobulin

Human plasma kallikrein participates in the contact activation system of plasma. The light chain of kallikrein contains the enzymatic active site; the heavy chain is required for binding to high molecular weight kininogen and for surface-dependent activation of coagulation. This study has examined the functional contributions of the heavy chain of kallikrein and of high molecular weight kininogen in the inactivation of kallikrein and of its isolated light chain by alpha 2-macroglobulin (alpha 2M). Irreversible inhibition was observed for both kallikrein and its light chain, with the initial formation of a reversible enzyme-inhibitor complex. The second-order rate constants for these reactions were 3.5 X 10(5) and 4.8 X 10(5) M-1 min-1 for kallikrein and its light chain, respectively. When present in excess, high molecular weight kininogen decreased the rate of kallikrein inactivation by alpha 2M, whereas the rate of inactivation of the light chain was unaffected by high molecular weight kininogen. Although at a drastically reduced rate, high molecular weight kininogen was cleaved by alpha 2M-bound kallikrein. Sodium dodecyl sulfate gradient polyacrylamide gel electrophoresis was used to study complex formation between alpha 2M and kallikrein or its light chain. Under reducing conditions, four kallikrein-alpha 2M complexes were observed. Three of these complexes consisted of alpha 2M and the light chain of kallikrein (Mr 123 000, 235 000, and 330 000). Two alpha 2M-kallikrein light chain complexes incorporated [3H]diisopropyl fluorophosphate ( [3H]DFP) whereas the Mr 330 000 complex did not react with [3H]DFP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contact activation of prekallikrein and plasminogen in plasma

Activation of the Hageman factor-prekallikrein system in the whole human blood plasma is studied as affected by organic silica (aerosils) with anionic and cationic properties. Positive- and negative-charged aerosils are shown to possess the same ability to activate prekallikrein. Activity of prekallikrein was manifested in hydrolysis of the chromogenic substrate--Benz-Pro-Phen-Arg-paranitroanilide . HCl, kininogen and protamine sulphate formed by kallikrein. The data permit supposing that optimal activation of the Hageman factor requires the polar (but not ionic) groups with hydrophilic properties on activating surfaces. Plasminogen under contact activation, in contrast to prekallikrein is activated only in the diluted plasma (pH 4.8), and not completely. Possible mechanisms of the contact activation and interaction of the Hageman factor, prekallikrein and high-molecular kininogen in this process are discussed.     

A mosquito salivary protein inhibits activation of the plasma contact system by binding to factor XII and high molecular weight kininogen

The salivary glands of female mosquitoes contain a variety of bioactive substances that assist their blood-feeding behavior. Here, we report a salivary protein of the malarial vector mosquito, Anopheles stephensi, that inhibits activation of the plasma contact system. This factor, named hamadarin, is a 16-kDa protein and a major component of the saliva of this mosquito. Assays using human plasma showed that hamadarin dose-dependently inhibits activation of the plasma contact system and subsequent release of bradykinin, a primary mediator of inflammatory reactions. Reconstitution experiments showed that hamadarin inhibits activation of the plasma contact system by inhibition of the reciprocal activation of factor XII and kallikrein. Direct binding assays demonstrated that this inhibitory effect is due to hamadarin binding to both factor XII and high molecular weight kininogen and interference in their association with the activating surface. The assays also showed that hamadarin binding to these proteins depends on Zn(2+) ions, suggesting that hamadarin binds to these contact factors by recognizing their conformational change induced by Zn(2+) binding. We propose that hamadarin may attenuate the host's acute inflammatory responses to the mosquito's bites by inhibition of bradykinin release and thus enable mosquitoes to take a blood meal efficiently and safely.