Activated Hageman Factor Research Articles

Deciphering the canonical blockade of activated Hageman factor (FXIIa) by benzamidine in the coagulation cascade: A thorough dynamical perspective.

The experimental inhibitory potency of benzamidine (BEN) paved way for further design and development of inhibitors that target β-FXIIa. Structural dynamics of the loops and catalytic residues that encompass the binding pocket of β-FXIIa and all serine proteases are crucial to their overall activity. Employing molecular dynamics and post-MD analysis, this study sorts to unravel the structural and molecular events that accompany the inhibitory activity of BEN on human β-FXIIa upon selective non-covalent binding. Analysis of conformational dynamics of crucial loops revealed prominent alterations of the original conformational posture of FXIIa, evidenced by increased flexibility, decreased compactness, and an increased exposure to solvent upon binding of BEN, which could have in turn interfered with the essential roles of these loops in enhancing their procoagulation interactions with biological substrates and cofactors, altogether resulting in the consequential inactivation of FXIIa. A sustained interaction of the catalytic triad residues and key residues of the autolysis loop impeded their roles in catalysis which equally enhanced the inhibitory potency of BEN toward β-FXIIa evidenced by a favorable binding. Findings provide essential structural and molecular insights that could facilitate the structure-based design of novel antithrombotic compounds with enhanced inhibitory activities and low therapeutic risk.

Pathophysiology of Hereditary Angioedema

Laryngeal angioedema may be associated with significant morbidity and even mortality. Because of the potential severity of attacks, both allergists and otolaryngologists must be knowledgeable about the recognition and treatment of laryngeal angioedema. This study describes the clinical characteristics and pathophysiology of bradykinin-mediated angioedema. A literature review was conducted concerning the clinical characteristics and pathophysiology of types I and II hereditary angioedema (HAE), type III HAE, acquired C1 inhibitor (C1INH) deficiency, and angiotensin-converting enzyme (ACE) inhibitor-associated angioedema. The diagnosis of type I/II HAE is relatively straightforward as long as the clinician maintains a high index of suspicion. Mutations in the SERPING1 gene result in decreased secretion of functional C1INH and episodic activation of plasma kallikrein and Hageman factor (FXII) of the plasma contact system with cleavage of high molecular weight kininogen and generation of bradykinin. In contrast, there are no unequivocal criteria for making a diagnosis of type III HAE, although a minority of these patients may have a mutation in the factor XII gene. Angioedema attacks and mediator of swelling in acquired C1INH deficiency are similar to those in type I or II HAE; however, it occurs on a sporadic basis because of excessive consumption of C1INH in patients who are middle aged or older. ACE inhibitor-associated angioedema should always be considered in any patient taking an ACE inhibitor who experiences angioedema. ACE is a kininase, which when inhibited is thought to result in increased bradykinin levels. Bradykinin acts on vascular endothelial cells to enhance vascular permeability. Laryngeal swelling is not infrequently encountered in bradykinin-mediated angioedema. Novel therapies are becoming available that for the first time provide effective treatment for bradykinin-mediated angioedema. Because the characteristics and treatment of these angioedemas are quite distinct from each other and from histamine-mediated angioedema, it is crucial that the physician be able to recognize and distinguish these swelling disorders.

Bradykinin and the Pathogenesis of Hereditary Angioedema

Bradykinin and the Pathogenesis of Hereditary Angioedema

Low anticoagulant heparin targets multiple sites of inflammation, suppresses heparin-induced thrombocytopenia, and inhibits interaction of RAGE with its ligands

While heparin has been used almost exclusively as a blood anticoagulant, important literature demonstrates that it also has broad anti-inflammatory activity. Herein, using low anti-coagulant 2-O,3-O-desulfated heparin (ODSH), we demonstrate that most of the anti-inflammatory pharmacology of heparin is unrelated to anticoagulant activity. ODSH has low affinity for anti-thrombin III, low anti-Xa, and anti-IIa anticoagulant activities and does not activate Hageman factor (factor XII). Unlike heparin, ODSH does not interact with heparin-platelet factor-4 antibodies present in patients with heparin-induced thrombocytopenia and even suppresses platelet activation in the presence of activating concentrations of heparin. Like heparin, ODSH inhibits complement activation, binding to the leukocyte adhesion molecule P-selectin, and the leukocyte cationic granular proteins azurocidin, human leukocyte elastase, and cathepsin G. In addition, ODSH and heparin disrupt Mac-1 (CD11b/CD18)-mediated leukocyte adhesion to the receptor for advanced glycation end products (RAGE) and inhibit ligation of RAGE by its many proinflammatory ligands, including the advanced glycation end-product carboxymethyl lysine-bovine serum albumin, the nuclear protein high mobility group box protein-1 (HMGB-1), and S100 calgranulins. In mice, ODSH is more effective than heparin in reducing selectin-mediated lung metastasis from melanoma and inhibits RAGE-mediated airway inflammation from intratracheal HMGB-1. In humans, 50% inhibitory concentrations of ODSH for these anti-inflammatory activities can be achieved in the blood without anticoagulation. These results demonstrate that the anticoagulant activity of heparin is distinct from its anti-inflammatory actions and indicate that 2-O and 3-O sulfate groups can be removed to reduce anticoagulant activity of heparin without impairing its anti-inflammatory pharmacology.

Pathogenesis and laboratory diagnosis of hereditary angioedema

Hereditary angioedema (HAE) was first described in the 19th century. Over the past 50 years, many details of the pathophysiology and molecular biology of HAE have been elucidated. Two types of HAE, type I and type II, result from mutations in the gene for the broad-spectrum protease inhibitor C1 inhibitor (C1INH). Type I HAE is characterized by low antigenic and functional C1INH levels and type II HAE has normal antigenic but low functional C1INH levels. Type III HAE, by contrast, has normal antigenic and functional C1INH levels. In some families, type III HAE has been linked to mutations in Hageman factor. C1INH is the primary inhibitor of the complement proteases C1r and C1s as well as the contact system proteases activated Hageman factor (coagulation factor XIIa and XIIf) and plasma kallikrein. It is also an inhibitor of plasmin and coagulation factor XIa. The primary mediator of swelling in HAE has now been unequivocally shown to be bradykinin, generated from activation of the plasma contact system. The knowledge gained concerning the underlying mechanisms of the different types of HAE allow the clinician to approach the laboratory diagnosis with confidence and provides opportunities for novel therapeutic strategies.

Procoagulant activity of surface-immobilized Hageman factor

Procoagulant activity of surface-immobilized coagulation factor XIIa (activated Hageman factor) is reported. Activity of FXIIa immobilized onto the surfaces of three silanized-glass procoagulants spanning a wide range of wettability was assayed in normal and FXII-deficient plasmas. Previously published mathematical models were used to characterize the procoagulant activity of protein-immobilized materials and soluble enzymes. Results show that FXIIa activity is unrelated to underlying procoagulant surface chemistry and is similar to soluble FXIIa activity. The uninfluential role of the surface on FXIIa suggests that the solid surface activates FXII in biomaterial-induced blood coagulation but is not otherwise involved in FXIIa activity as described by the classical mechanism.

The multiple faces of the partial thromboplastin time APTT

Converting the PTT into the APTTIn his historical sketch The partial thromboplastin time:defining an era in coagulation,G.C.WhiteII[1]describesthegenesis in 1953 of the original partial thromboplastin time(PTT) [2].In 1961 animprovedversionwas publishedfrom mylaboratory [3] in which adding kaolin powder to a partialthromboplastin reagent optimally activated Hageman factor(HF) and, consequently, plasma thromboplastin antecedent[PTA, factor (F)XI] during the test’s incubation period. Todaycommercial reagents provide this function. Although stillcommonly referred to as the PTT, the correct name of theimproved test is the APTT (activated partial thromboplastintime). My memory of the events of long ago leading toconversion of the PTT into the APTT follows.In1956Ireportedthatafour-partoriginalPTTassaysystemcontainingeitheradsorbedoxplasma(sourceoffactorVIII)oraged serum (source of factor IX) as a correcting agent hadproved its worth as a simple sensitive screening test. It hadenabledmetodeterminethekindofhemophiliapresentineachof a previously unscreened large group of 162 hemophilicchildren and adolescents in Los Angeles [4]. By 1957 using thisassay system I had also identified three adults with plasmathromboplastin antecedent (PTA, FXI) deficiency.Thencameaproblem.Normal silicone testplasmafailedtoshorten the prolonged PTT of glass-activated substrate plasmafrom these three patients that had stood for 4 h at 4 Cbeforetesting.Incontrast,thesame silicone testplasmareadilydidsoif shaken with glass beads. Submitting these data to the JLabClin Med in January 1958, I postulated that plasma PTAcirculates as an inert precursor and emphasized the need tostandardize exposure to glass in constructing a quantitativeassay for PTA activity [5].In accepting my paper the editor, C. Loosli, informed me ofan abstract from the Proceedings of the Thirtieth AnnualMeeting of the Central Society for Clinical Research thatappeared in the December 1957 issue of the JClinLabMed.Itwas from Oscar Ratnoff and Jerold Rosenblum on the role inthe initiation of clotting by glass of HF [6]. A full paper fromRatnoff and Rosenblum was published in 1958 [7] and soonotherinvestigatorsconfirmedthatbothHFandPTA(FXI)arerequired for the glass activation of normal plasma.Bjarne Waaler’s 1959 monograph [8] of work carried outbetween 1955 and 1958 in Paul Owren’s Institute for Throm-bosis Research in Norway particularly interested me. Inaddition to confirming that both HF and PTA were requiredfor plasma to form an activation product after contact withglass, Waaler had found a cause for the disconcertingly widenormal range of the PTT test. Clotting times depended uponwhether new or previously used glass test tubes were used, andif the latter, how the tube had been washed. New glass tubesyielded50–60-sclottingtimeswhereasglasstubesthathadbeenwashed with a commonly used acid cleaning solution yielded80–90-s clotting times.Waaler also reported that the clotting time of plasmalengthened markedly if after exposure to glass powder, oneremoved the powder and allowed the plasma to stand forseveral hours at 37 C. He called such plasma exhaustedplasma and believed it was depleted of both HF and PTA. Iused his method to create an exhausted plasma substrate foruse in my laboratory.In late 1958 I was recruited as the full-time hematologist forthe Department of Medicine of the University of SouthernCalifornia (USC) Medical School and its clinical services at theLos Angeles (LA) County Hospital. By the fall of 1989 mytransplanted Coagulation Research Laboratory was up andrunning at USC. A young couple, Norman and SandraSchiffman, had moved to Los Angeles, he to enter medicalschool at USC and she to enter the PhD program of itsDepartment of Biochemistry. Arnold G. Ware of the Depart-ment of Biochemisty, who was the Director of the ClinicalChemistry Laboratory of the LA County Hospital, suggestedthatS. Schiffman,whilesearching fora thesis project, talk withme. Fortune smiled upon me. This wonderfully bright,energetic graduate student elected to work on a project in mylaboratory. She would separate PTA (FXI) and HF fromnormal plasma by ion-exchange chromatography using therecently available ion exchange resin diethylaminoethyl cellu-lose (DAEA).Although I had PTA-deficient, HF-deficient and exhaustedplasmas ready for use as substrates for assays of eluatefractions, I knew from the experience cited above that suchassays should not be based upon the original PTT procedure.J.Margolishadpublisheda paperin1958onanewtestthathecalled the kaolin clotting time [9]. When S. Schiffman and Iread his paper, for us, a light turned on. We would design fourcomponent assays for her project that contained equal parts ofa cephalin (partial thromobplastin)–kaolin (HF activator)suspension, substrate plasma, an eluate sample, and CaCl

Activation of the kallikrein-kinin system and release of new kinins through alternative cleavage of kininogens by microbial and human cell proteinases

Kinins are released from kininogens through the activation of the Hageman factor-prekallikrein system or by tissue kallikrein. These peptides exert various biological activities, such as vascular permeability increase, smooth muscle contraction, pain sensation and induction of hypotension. In many instances kinins are thought to be involved in the pathophysiology of various diseases. Recent studies have revealed that microbial and human cell proteinases activate Hageman factor and/or prekallikrein, or directly release kinin from kininogens. This review discusses the activation of the kinin-release system by mast-cell tryptase and microbial proteinases, including gingipains, which are cysteine proteinases from Porphyromonas gingivalis , the major pathogen of periodontal disease. Each enzyme is evaluated in the context of its association to allergy and infectious diseases, respectively. Furthermore, a novel system of kinin generation directly from kininogens by the concerted action of two proteinases is described. An interesting example of this system with implications to bacterial pathogenicity is the release of kinins from kininogens by neutrophil elastase and a synergistic action of cysteine proteinases from Staphylococcus aureus . This alternative production of kinins by proteinases present in diseased sites indicates a significant contribution of proteinases other than kallikreins in kinin generation. Therefore kinin receptor antagonists and proteinase inhibitors may be useful as therapeutic agents.

Roles of factor XI, platelets and tissue factor-initiated blood coagulation

Roles of factor XI, platelets and tissue factor-initiated blood coagulation

Oscar Ratnoff: his contributions to the golden era of coagulation research.

Oscar Ratnoff: his contributions to the golden era of coagulation research.

Availability in pooled normal blood plasma of activated Hageman factor and kallikrein for contact induced coagulation.

Availability in pooled normal blood plasma of activated Hageman factor and kallikrein for contact induced coagulation.

C1 esterase inhibitor deficiency, airway compromise, and anesthesia.

C1 esterase inhibitor deficiency, airway compromise, and anesthesia.

The corn inhibitor of activated Hageman factor: purification and properties of two recombinant forms of the protein.

A cDNA clone that encodes the 14-kDa bifunctional inhibitor from corn seeds (L. Wen et al., Plant Mol. Biol. 18, 813-814, 1992) has been expressed in Escherichia coli after being incorporated into the pT7 expression vector. This inhibitor protein, referred to as CHFI (for the corn inhibitor of activated Hageman factor) or as the popcorn inhibitor, is an important tool for specific inhibition of human activated Hageman factor (activated forms of coagulation Factor XII) and has been well characterized as isolated from corn seeds. Recombinant CHFI was expressed in E. coli in high levels but was insoluble. We solubilized the expressed protein by sonication in 5 M urea and 1% Triton X-100. Several steps of purification, culminating with reversed-phase HPLC, yielded pure, recombinant corn inhibitor in about 5% yield (about 1 mg per liter of culture). The form with which we have worked most, 7N-CHFI, contains 7 amino acid residues at its N-terminus that are encoded by the expression vector. Physical properties of this recombinant protein indicate it has the expected mass and is properly folded. Functionally, 7N-CHFI is indistinguishable from the inhibitor isolated from corn seeds in its inhibition of porcine trypsin, human beta-Factor XIIa, failure to inhibit human plasma kallikrein, and its inhibition of an insect alpha-amylase. A second recombinant form, (4N-11)-CHFI, which lacks 11 residues from the corn inhibitor's N-terminus, is indistinguishable from 7N-CHFI in its pattern of inhibition of the three test proteinases but is inactive against the insect alpha-amylase. This suggests that the N-terminal region of 7N-CHFI forms at least part of the protein's site of interaction with alpha-amylase.

Species differences in amino acid sequences of Hageman factor and prekallikrein at region around scissile bond in activation

The initial step in activation of the plasma kinin system is activation of Hageman factor (coagulation factor XII) and/or plasma prekallikrein. Two types of activation mechanisms, contact activation on a negatively charged surface and a cascade activation by exogenous proteases are known. Since these factors are serine protease zymogens, the activation of these molecules usually results from the cleavage of a particular -Arg-Ile(Val)- bond in either mechanism. Hence, these zymogens are regard to be substrates of their activator proteases. Sensitivity of the substrate for the protease basically depends on the amino acid sequence of six to eight residues around the scissile bond of the substrate. We found different activation efficiencies of these zymogens between human and guinea pig in both types of activation, and micro-heterogeneity of the sequence around the scissile bond among human, guinea pig and bovine Hageman factors, or between human and guinea pig prekallikreins. The sequence heterogeneity may explain the different activation efficiencies of these zymogens among mammalian species.

Pathogenic mechanisms induced by microbial proteases in microbial infections.

Most bacterial and fungal proteases excreted into infected hosts exhibit a wide range of pathogenic potentials ranging from pain, edema or even shock to translocation of bacteria from the site of infection into systemic circulation, thus resulting in septicemia. The basic mechanism or principle common to all these phenomena is explained by kinin generation, either directly from high- and/or low-molecular weight kininogens or indirectly via activation of the bradykinin generating cascade: i.e. Hageman factor-->activated Hageman factor-->prekallikrein-->kallikrein-->high-molecular weight kininogen-->bradykinin. Some bacterial proteases are also involved in activation of other host protease zymogens such as plasminogen, procollagenase (matrix metallo proteases) and proenzymes of the clotting system. Furthermore, most bacterial proteases are not only resistant to plasma protease inhibitors of the hosts, most of which belong to a group of serine protease inhibitors called serpins (serine protease inhibitors), but they also quickly inactivate serpins. Some bacterial proteases may also activate bacterial toxins thus rendering toxigenic pathogenesis. They are also capable of degrading immunoglobulins and components of the complement system and facilitate propagation of micro organisms. All in all, microbial proteases are very critical in enhancing pathogenesis of severe diseases. It is also noteworthy that bacterial cell wall components themselves, i.e. endotoxin (or lipopolysaccharide) of gram negative bacteria and teichoic/lipoteichoic acid of gram positive bacteria, are also able to activate the bradykinin generating cascade-involving activation of Hageman factor as mentioned above.

Primary structure of bovine Hageman factor (blood coagulation factor XII): comparison with human and guinea pig molecules

A bovine Hageman factor cDNA was cloned from a liver cDNA library. The nucleotide sequence was analyzed and the amino-acid sequence was deduced. The sequence deduced was consistent with the partial amino-acid sequences of bovine Hageman factor protein. The sequences for three portions including the amino terminal had been previously reported (Fujikawa et al. (1977) Biochemistry 16, 2270–2278). In comparison with the primary structures of human and guinea pig Hageman factors, the putative domain structures were totally conserved. Each domain possessed high sequence homology with the human molecule (66–88%) and the guinea pig one (63–81%) except for the proline-rich region (less than 10%) which connects the amino-terminal five domains with a serine proteinase portion. Significant heterogeneities were observed among the three species around the essential cleavage sites for the conversion to the activated Hageman factors. Bovine Hageman factor has no suitable amino-acid sequence as the substrate for the trypsin-type proteinases at the proline-rich region in difference from the human and guinea pig molecules. Probably this is the reason why the β-form activated Hageman factor (the proteinase moiety) is not liberated in the activation of the bovine molecule with trypsin or plasma kallikrein.

The Corn Inhibitor of Blood Coagulation Factor XIIa: Crystallization and Preliminary Crystallographic Analysis

A 13·6 kDa protein from corn seeds is known to be a highly selective inhibitor of human blood coagulation Factor XIIa (or activated Hageman factor). We have crystallized this inhibitor at 23°C and pH 7·5 from a solution of 30% polyethylene glycol 400, 0·2 M MgCl 2, and 0·1 M Hepes. The crystals diffract to at least 2·1 Å resolution. The space group is P4 22 12 with a = b = 57·15 Å and c = 80·5 Å. The crystals contain 51% solvent. Two heavy atom derivatives have been identified.

Bradykinin generation by dialysis membranes

Several recent reports have described a high incidence of anaphylactic reactions in patients being dialyzed with high-flux membranes while simultaneously using angiotensin-converting enzyme inhibitors. Many of these reports implicate polyacrylonitrile (PAN) as the membrane commonly involved in these reactions. To elucidate potential mechanisms of these anaphylactic reactions, whether dialysis membranes can activate the Hageman factor-dependent (contact) pathways as assessed by the in vitro generation of activated Hageman factor (Hfa), as well as the formation of kallikrein and subsequent bradykinin generation was examined. Both cuprophane (CUP) and PAN membranes were able to activate Hageman factor and convert prekallikrein to kallikrein as measured by an ELISA against kallikrein-C1-inactivator complexes. Subsequently, the active kallikrein was able to cleave bradykinin from its endogenous substrate, high-molecular-weight kininogen. However, it was found that the PAN membrane consistently led to an earlier and significantly higher formation of Hfa and kallikrein when compared with CUP. Importantly, there was also a pronounced but transient generation of bradykinin by the PAN membrane, in contrast to slower bradykinin formation by CUP, with both normal and uremic blood. It was proposed that the early and vigorous bradykinin generation induced by the contact of blood with PAN could explain, in part, the pathogenesis of the reported anaphylactoid reactions.

Inhibition of the activation of hageman factor (factor XII) by eosinophils and eosinophilic constituents

Several syndromes characterized by striking eosinophilia may be complicated by thrombosis. The experiments described indicate that, paradoxically, eosinophils and certain of their constituents inhibit the activation of Hageman factor (HF, factor XII). In earlier studies, suspensions of mixed types of granulocytes, other nucleated peripheral blood cells, and platelets inhibited activation of Hageman factor by ellagic acid, glass, and sulfatides. After these cells were sedimented by centrifugation, the supernatant fluids were also inhibitory. No attempt had been made earlier to distinguish among different granulocytic species. In the present study, suspensions of eosinophils and the supernatant fluid after eosinophils had been separated by centrifugation inhibited activation of Hageman factor by ellagic acid. The protein concentration of that amount of supernatant fluid that inhibited activation by about half was 16 micrograms/ml, approximately the same as had been described for suspensions of peripheral blood mononuclear cells. Activation of Hageman factor by ellagic acid was also inhibited by certain constituents of eosinophils, including eosinophil peroxidase, eosinophil major basic protein and eosinophil cationic protein. Inhibition was not specific for ellagic acid-induced activation of Hageman factor, as inhibition was also observed with sulfatide-induced activation. Inhibition was presumably related to neutralization of the negative charge of activators of Hageman factor. Thus, bismuth subgallate, a particulate activator of Hageman factor, was no longer effective after it had been exposed to eosinophil cationic protein. The observations reported here raise the question of whether in vivo eosinophils modulate certain of the defense reactions ascribed to Hageman factor.

Difference between human and guinea pig Hageman factors in activation by bacterial proteinases: cleavage site shift due to local amino acid substitutions may determine the activation efficiency of serime proteinase zymogens

Human and guinea pig Hageman factors have been subjected to the action of pseudomonal elastase and serratial E15 proteinase. The pseudomonal elastase cleaved 22–24% of the human molecule at Arg 353-Val 354, and the remainder at Gly 357-Leu 358 resulting in the generation of about 20% of potential activity as activated Hageman factor, compared with trypsin activation, while ti hydrolyzed Arg 340-Ile 341 bond in guinea pig molecule and generated about 75% of activity as activated Hageman factor. The serratial proteinase did not hydrolyze the essential cleavage site (Arg 353-Val 354) of the human zymogen but Gly 356-Gly 357 (30%) and Gly 357-Leu 358 (70%) bonds. Both products showed no activity. The guinea pig zymogen, in contrast, was cleaved mostly at Arg 340-Ile 341 (70%) and less abundantly at Gly 344-Leu 345 (30%), generating about 85% of the whole potential activity as activated Hageman factor. From the high correspondence between the proportions of activationo of hydrolysis at the essential cleavage site in activation, it was concluded that hydrolysis of the bonds different from the essential bond did not cause activation, even when the spatial separation was only 3 or 4 residues. Considering the amino acid differences between human and guinea pig Hageman factors, - Met 351- Thr -Arg- Val -Val-Gly-Gly-Leu-Val-Ala 360- and - Leu 338- Ser -Arg- Ile -Val-Gly-Gly_leu-Val-Ala 347-, respectively, it was realized that even the minor amino acid substitutions caused the cleavage site shift which resulted in significant differences in activation efficiency of the proteinase zymogens.