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PubMed1985

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.

Bradykinin↗
PubMed1991

Proteinase inhibitors in Brazilian Leguminosae.

Serine proteinase inhibitors, in the seeds of several Leguminosae from the Pantanal region (West Brazil), were studied using bovine trypsin, a digestive enzyme, Factor XIIa and human plasma kallikrein, two blood clotting factors. The inhibitors were purified from Enterolobium contortisiliquum (M(r) = 23,000), Torresea cearensis (M(r) = 13,000), Bauhinia pentandra (M(r) = 20,000) and Bauhinia bauhinioides (M(r) = 20,000). E. contortisiliquum inhibitor inactivates all three enzymes, whereas the T. cearensis inhibitor inactivates trypsin and Factor XIIa, but does not affect plasma kallikrein; both Bauhinia inhibitors, on the other hand, inactivate trypsin and plasma kallikrein but only the B. pentandra inhibitor affects Factor XIIa. Ki values were calculated between 10(-7) and 10(-8) M.

Brazil↗
PubMed1987

A new assay for high molecular weight kininogen in human plasma using a chromogenic substrate.

High molecular weight kininogen (HK), the cofactor of contact-activated plasma proteolysis, is currently assayed by coagulant or immunological methods. The former is limited by the need for rare, congenitally-deficient plasma and a high coefficient of variation (CV), and the latter, by failure to distinguish nonfunctional protein. The surface activation of factor XI requires HK as its cofactor to transport its zymogen form to a negatively-charged surface where it is converted to its enzymatic form by factor XIIa. Based on this principle, we developed an assay for HK using the chromogenic substrate pyroGlu-Pro-Arg-p-nitroanilide (S-2366, KabiVitrum), which is hydrolyzed by factor XIa. Plasma is first acidified to inactivate protease inhibitors. After neutralization and dilution, the plasma is incubated with an excess of factor XI, factor XIIa, and soybean trypsin inhibitor (to inactivate generated kallikrein), in the presence of a negatively-charged surface (kaolin) in order to form factor XIa. EDTA is included in the buffer to prevent calcium-dependent reactions. This activation process is stopped by adding corn trypsin inhibitor to inactivate the enzyme in this reaction, factor XIIa. Then, S-2366 is added and is hydrolyzed by the factor XIa that was formed. Since factor XI and factor XIIa are in excess of the concentration of HK in the diluted plasma, HK is the rate-limiting protein in this assay for the formation of factor XIa (after subtracting the small amount of factor XIa generated in the absence of HK). The assay is specific for HK, since no activity is detected in kininogen-deficient plasma, and when compared with the HK coagulant assay, r = 0.95 and slope = 0.95. The mean of 21 normal donors was 0.98 U/ml (range 0.68 - 1.28 U/ml) as compared with pooled, normal plasma. The CV for 1 U/ml HK for the chromogenic assay was 2% as compared with 9.5% for the coagulant assay. When purified reagents become commercially available, this assay could prove useful in clinical laboratories or intensive care units for monitoring the progression of various disease states in which contact activation occurs.

Colorimetry↗
PubMed1986

Purification of human factor XII from plasma using zinc chelate affinity chromatography.

Human factor XII (Hageman Factor) was isolated from human plasma to apparent homogeneity using a four step procedure with yields up to 30%. The method, which is more rapid than the current conventional procedures, consists of a 25-50% ammonium sulfate fractionation, two affinity chromatography steps using Zinc Chelate Sepharose, followed by gel filtration. The isolated zymogen factor XII was a single protein component when examined by SDS PAGE with a Mr of 80,000. Activation of zymogen factor XII to its active enzymatic forms by kallikrein resulted in factor XIIa and factor XIIf as observed with factor XII purified by other procedures.

Ammonium Sulfate↗
PubMed1983

Benzyl p-guanidinothiobenzoate hydrochloride, a new active-site titrant for trypsin and trypsin-like enzymes.

Benzyl p-guanidinothiobenzoate hydrochloride was synthesized and demonstrated to be useful for active-site titration of bovine trypsin, bovine thrombin, human lung tryptase, bovine activated protein C, human Factor XIIa fragment and bovine Factor Xa beta. The titration is based on rapid formation of a stable acyl-enzyme with a stoichiometric release of benzyl thiol. Thiol production is measured quantitatively by including 4,4'-dithiodipyridine in the reaction mixture and measuring the increase in absorbance at 324 nm. Ellman's reagent has also been successfully employed, allowing measurement at 410 nm. Unlike p-nitrophenyl p'-guanidinobenzoate, the thioester titrant reacts slowly with chymotrypsin A alpha thus eliminating interference by this enzyme in most titrations. Advantages of this reagent as a titrant include: flexibility in detection of the released thiol, selectivity between trypsin and chymotrypsin-like enzymes, minimal pH-dependence of the epsilon of the absorbing species, relative stability of the reagent under titration conditions, and high epsilon at pH 7.2 with either 4,4'-dithiodipyridine or Ellman's reagent. The reagent should prove useful as an alternative to p-nitrophenyl p'-guanidinobenzoate hydrochloride for the determination of active-site concentrations of the enzymes employed, as well as of other related enzymes.

Animals↗
PubMed2004

Identification of a binding site for glycoprotein Ibalpha in the Apple 3 domain of factor XI.

Factor XI (FXI) is a homodimeric plasma zymogen that is cleaved at two internal Arg(369)-Ile(370) bonds by thrombin, factor XIIa, or factor XIa. FXI circulates as a complex with the glycoprotein high molecular weight kininogen (HK). FXI binds to specific sites (K(d) = approximately 10 nM, B(max) = approximately 1,500/platelet) on the surface of stimulated platelets, where it is efficiently activated by thrombin. The FXI Apple 3 (A3) domain mediates binding to platelets in the presence of HK and zinc ions (Zn(2+)) or prothrombin and calcium ions. The platelet glycoprotein (GP) Ib-IX-V complex is the receptor for FXI. Using surface plasmon resonance, we determined that FXI binds specifically to glycocalicin, the extracellular domain of GPIbalpha, in a Zn(2+)-dependent fashion (K(d) = approximately 52 nM). We now show that recombinant FXI A3 domain inhibits FXI inbinding to glycocalicin in the presence of Zn(2+), whereas the recombinant FXI A1, A2, or A4 domains have no effect. Experiments with full-length recombinant FXI mutants show that, in the presence of Zn(2+), glycocalicin binds FXI at a heparin-binding site in A3 (Lys(252) and Lys(253)) and not by amino acids previously shown to be required for platelet binding (Ser(248), Arg(250), Lys(255), Phe(260), and Gln(263)). However, binding in the presence of HK and Zn(2+) requires Ser(248), Arg(250), Lys(255), Phe(260), and GLn(263) and not Lys(252) and Lys(253). Thus, binding of FXI to GPIbalpha is mediated by amino acids in the A3 domain in the presence or absence of HK. This interaction is important for the initiation of the consolidation phase of blood coagulation and the generation of thrombin at sites of platelet thrombus formation.

Binding Sites↗
PubMed2003

Kinetic characterization of factor Xa binding using a quenched fluorescent substrate based on the reactive site of factor Xa inhibitor from Bauhinia ungulata seeds.

The specific Kunitz Bauhinia ungulata factor Xa inhibitor (BuXI) and the Bauhinia variegata trypsin inhibitor (BvTI) blocked the activity of trypsin, chymotrypsin, plasmin, plasma kallikrein and factor XIIa, and factor Xa inhibition was achieved only by BuXI (K(i) 14 nM). BuXI and BvTI are highly homologous (70%). The major differences are the methionine residues at BuXI reactive site, which are involved in the inhibition, since the oxidized protein no longer inhibits factor Xa but maintains the trypsin inhibition. Quenched fluorescent substrates based on the reactive site sequence of the inhibitors were synthesized and the kinetic parameters of the hydrolysis were determined using factor Xa and trypsin. The catalytic efficiency k(cat)/K(m) 4.3 x 10(7) M(-1)sec(>-1) for Abz-VMIAALPRTMFIQ-EDDnp (lead peptide) hydrolysis by factor Xa was 10(4)-fold higher than that of Boc-Ile-Glu-Gly-Arg-AMC, widely used as factor Xa substrate. Lengthening of the substrate changed its susceptibility to factor Xa hydrolysis. Both methionine residues in the substrate influence the binding to factor Xa. Serine replacement of threonine (P(1)') decreases the catalytic efficiency by four orders of magnitude. Factor Xa did not hydrolyze the substrate containing the reactive site sequence of BvTI, that inhibits trypsin inhibitor but not factor Xa. Abz-VMIAALPRTMFIQ-EDDnp prolonged both the prothrombin time and the activated partial thromboplastin time, and the other modified substrates used in this experiment altered blood-clotting assays.

Amino Acid Sequence↗
PubMed1975

[Activation of the blood coagulation system during gram-negative infections and endotoxemias].

Blood coagulation may be activated by the extrinsic or intrinsic pathways. The extrinsic clotting system is put into action by tissue thromboplastin, originating from injured tissue cells, but also from damaged leukocytes and erythrocytes. Tissue thromboplastin is a phospholipoprotein with an enzymatic component, capable of converting the clotting factor VII to its activated form, factor VIIa, which in turn activates factor X. The factor Xa-complex (containing also factor Va, phospholipid, and calcium) is the prothrombinconverting principle. The intrinsic clotting system is based on factors which are contained in the circulating blood. Its activation requires the availability of phospholipid and of activated factor XII (factor XIIa), or factor XIa. Factor XII is activated by collagen, i.e., whenever the vascular endothelium is injured, and to a lesser extent also by "activated" blood platelets. Platelets in turn are activated primarily by thrombin, collagen, and, in a self-perpetuating process, since all these materials are released from activated platelets, also by adenosine-5-diphosphate, adrenaline, and serotonin. The activation of platelets leads to a variety of morphological and biochemical alterations, culminating in their aggregation and in the selective release from storage organelles of different substances, among them those mentioned above. Of particular importance is the fact that in the course of platelet alterations, procoagulant phospholipid also becomes available on the platelet surface. The significance of the activation of the intrinsic system is seen in the possibility of the initiation of a self-sustained process which, after a primary event, e.g. vascular or cellular injury, will continue to convert prothrombin into thrombin. The effects of endotoxin on the blood clotting system show striking species differences. In the rabbit, endotoxin, with the involvement of factors of the complement system, will directly act upon blood platelets and thus initiate intravascular, intrinsic coagulation. In man, endotoxin remains without a direct effect on platelets and alternative possibilities of initiating thrombin formation must be considered. One possibility is extrinsic activation via tissue thromboplastin from injured leukocytes. Another pathway, which is supported by several experimental findings, starts out with endotoxin-mediated endothelial damage. Endothelial cells are in fact severely affected by endotoxin and may even be removed from the vascular wall, thus making accessible the subendothelial activator of factor XII. Thrombin in turn affects the vascular endothelium: therefore, one initiated, the process of intravascular activation of coagulation will perpetuate, this the more as platelets in turn will be stimulated into activity. The possible intervention of other vasoactive factors must also be considered...

Animals↗
PubMed1988

Pathophysiology of thrombophilic states.

The coagulation system can be considered as a balance in which clotting and fibrinolysis have to be in a state of equilibrium. Increased fibrin formation or decreased fibrinolysis can predispose to thromboembolic diseases. Derailments in the clotting system leading to thrombosis center around the regulatory mechanisms, antithrombin III, protein C, protein S and possibly heparin cofactor II. Many cases of congenital or acquired deficiencies or abnormalities or antithrombin III, protein C and S have been described, all predisposing to thrombotic events. Alterations of the fibrinolytic system can also be associated with thromboembolisms. In particular, abnormalities of plasminogen, tissue plasminogen activator release and elevated tissue plasminogen activator inhibitor levels seem to be associated with thromboses. Conceivably also factor XIIa (Hageman factor) and prekallikrein deficiencies, when associated with thrombosis, exert their mechanism through the fibrinolytic system. Finally, about 50% of patients with lupus anticoagulant seem to suffer from thromboembolic disorders. The pathophysiology of this particular association is not known with certainty. Undoubtedly, there will be more disturbances discovered in the hemostasis system that are associated with increased intravascular fibrin formation. The understanding of these derailments is at this time only in its earliest stages of development.

Humans↗
PubMed1989

Amidolytic detection of prothrombin activation products after SDS-gel electrophoresis.

In this paper we report a method via which enzymatically active products formed during prothrombin activation can be detected by simple photographic means after SDS-gel electrophoresis, blotting onto a nitrocellulose membrane and visualization with the chromogenic substrate, S2238. After amidolytic detection the same nitrocellulose membrane can also be used for immunologic detection of prothrombin activation products, thus allowing a complete description of product formation during prothrombin activation. The detection limit of the so-called "amidoblot" is approximately 3 ng thrombin per gel sample which is comparable to the sensitivity of immunoblotting. It is further shown that the amidoblot technique can also be applied to other coagulation factors for which a suitable chromogenic substrate is available (factor XIIa, kallikrein, factor XIa, factor Xa, plasmin and activated protein C).

Blood Coagulation Tests↗
PubMed1983

Mammalian tissue trypsin-like enzymes. Comparative reactivities of human skin tryptase, human lung tryptase, and bovine trypsin with peptide 4-nitroanilide and thioester substrates.

The subsite specificity of human lung and skin tryptase (trypsin-like enzyme) has been studied at pH 7.5 using 17 amino acid and dipeptide thioester substrates and 14 tripeptide 4-nitroanilide substrates. The reactivity and specificity of the human tryptases were compared with bovine trypsin and other trypsin-like enzymes. Neither tryptase was similar to either kallikrein or factor XIIa (Hageman factor). The skin enzyme was the most reactive as measured by the specificity constant kcat/KM. The best substrate was benzyloxycarbonyl(Z)-Lys-Arg-S-CH2CH(CH3)2 which had a kcat/KM value of 59,000,000 M-1 S-1. Only a single substrate, Z-Glu-Phe-Arg-4-nitroanilide, was slightly more reactive with the lung tryptase. Both enzymes have extended substrate-binding sites and proline residues at P3 substantially decrease kcat/KM. Both enzymes preferred the tripeptide 4-nitroanilides with a P2 Gly residue over Phe, and both favored the substrate Z-Lys-Gly-Arg-4-nitroanilide over similar substrates containing six other representative amino acid residues at P3. The lung enzyme was inhibited over three times faster by p-amidinophenylmethanesulfonyl fluoride than the skin enzyme. The preference of the skin tryptase for substrates with two terminal basic residues indicates that this enzyme could process prohormones and proproteins which contain this structural feature at the cleavage site. The substrates reported in this paper should be useful for the further characterization of the physiologic function of tryptases.

Anilides↗
PubMed1993

Mechanisms of thrombin generation during surgery and cardiopulmonary bypass.

Although in vitro studies have been invaluable in revealing the complex biochemistry of the blood coagulation system, the mechanisms involved during the in vivo response to hypercoagulable stimuli are still unclear. We have used plasma-based enzyme-linked immunosorbent assays (ELISAs) to study the mechanisms by which the coagulation system is activated in vivo during human cardiopulmonary bypass (CPB) surgery (n = 8). A novel immunoassay for factor XIIa was used to detect activation of the contact system, factor IX activation peptide (FIXAP) was used as a marker for activation of factor IX, and prothrombin fragment F1 + 2 (F1 + 2) was used as a marker for thrombin generation. The ELISA for FIXAP is described for the first time herein. F1 + 2 levels increased early in response to surgical intervention: from a baseline of 38.7 +/- 9.7 ng/mL (mean +/-SE), levels increased rapidly during surgery and bypass to a maximum of 448.5 +/- 92.0 ng/mL. A modest yet significant increase in factor XIIa levels from 3.47 +/- 0.54 ng/mL to 4.33 +/- 0.85 ng/mL was evident during surgery before bypass, but no further significant increase was detected on establishing extracorporeal circulation. FIXAP levels demonstrated a small and late increase during surgery from 4.98 +/- 0.55 ng/mL to a maximum of 10.20 +/- 1.23 ng/mL, the increase beginning at the time of near maximal F1 + 2 levels. There was no association between activation of the contact system (factor XIIa levels) and the generation of thrombin (F1 + 2 levels). However, a strong association (r = .705) was apparent between the generation of thrombin (F1 + 2 levels) and activation of factor IX (FIXAP levels), despite the delay between the activation of prothrombin and factor IX. The data do not support the established view that contact activation resulting from exposure of blood to foreign surfaces is the major procoagulant stimulus in CPB. Instead, the results suggest that the main trigger to coagulation during CPB surgery was provided via the tissue factor-factor VIIa mechanism in response to the cutting of blood vessels, which directly activated factor X and then prothrombin. The late activation of factor IX, which presumably also contributed to maximal prothrombin activation, could have arisen due to direct tissue factor-factor VIIa action, or by secondary feedback action of thrombin on the intrinsic system.

Aged↗
PubMed1982

The contact activation mechanism in human plasma: activation induced by dextran sulfate.

Incubation of normal human plasma with dextran sulfate for 7 min at 4 degrees C generates kallikrein amidolytic activity. No kallikrein activity is generated in factor XII or prekallikrein-deficient plasma and only small amounts (8%) in high molecular weight (HMW) kininogen-deficient plasma. Addition of specific antisera directed against prekallikrein or HMW kininogen to normal plasma blocked the generation of kallikrein activity by dextran sulfate. Thus, factor XII, prekallikrein, and HMW kininogen are essential components for optimal activation of prekallikrein. The role of limited proteolysis in the activation of prekallikrein induced by dextran sulfate was studied by adding 125I-prekallikrein to plasma. The generation of kallikrein activity paralleled the proteolytic cleavage of prekallikrein as judged on SDS gels in the presence of reducing agents. The same cleavage fragments were observed as obtained by activation of purified prekallikrein by beta-factor-XIIa. Addition of 131I-HMW kininogen and 125I-factor XII or 131I-HMW kininogen and 125I-prekallikrein to normal plasma followed by activation with dextran sulfate and analysis on SDS gels indicated that the observed cleavage of prekallikrein and HMW kininogen is fast compared to the observed cleavage of factor XII, which is much slower and less extensive. During the first minutes of incubation of normal plasma with dextran sulfate, mainly alpha-factor-XIIa is formed. During prolonged incubation, beta-factor-XIIa is also formed.

Dextran Sulfate↗
PubMed1996

beta 2 glycoprotein-I inhibits factor XII activation on triglyceride rich lipoproteins: the effect of antibodies from plasma of patients with antiphospholipid syndrome.

It is now well recognised that antiphospholipid antibodies are associated with thrombosis and recurrent fetal loss. Some antiphospholipid antibodies (aPAs) have been shown to require a cofactor, beta 2 glycoprotein-I (beta 2 GPI), for binding to phospholipids, and recently beta 2 GPI has been identified as the antigenic target for some aPAs. beta 2 GPI possesses in vitro anticoagulant properties and modulation of beta 2 GPI function may therefore result in altered haemostatic regulation. In the present study, the influence of plasma derived aPAs and beta 2 GPI on factor XII activation on the surface of very low density lipoprotein (VLDL) was investigated. Factor XIIa generation was dependent on lipoprotein lipase treatment of VLDL and beta 2 GPI inhibited the factor XIIa generation in a concentration dependent manner. No consistent effects on factor XIIa generation were demonstrated with the IgG fractions from patients with aPAs. Inhibition of the beta 2 GPI activity was demonstrated by some antibodies, and study with cardiolipin affinity purified antibody indicated that antibody concentration is critical. These results suggest that perturbation of beta 2 GPI function may contribute to the pathogenic mechanism for thrombosis in some patients with aPAs.

Adult↗
PubMed1993

Surface-independent acceleration of factor XII activation by zinc ions. II. Direct binding and fluorescence studies.

To determine the role of Zn(II)-factor XII interactions in the rate-enhancing effect of Zn(II) on factor XII activation demonstrated in the preceding paper, equilibrium binding of zinc ions to factor XII, and the spectroscopic changes accompanying this binding were investigated. Equilibrium dialysis provided direct evidence for the binding of Zn(II) to factor XII. The binding data were consistent with 7.8 +/- 0.3 zinc ions binding with an indistinguishable Kd of 91 +/- 6 microM. Binding of Zn(II) was accompanied by a 10% quenching of the intrinsic protein fluorescence and a 2-nm red shift of the wavelength of maximum emission. These spectroscopic changes were specific for factor XII and were not observed with factor XIIa. The Zn(II) concentration dependence of factor XII fluorescence quenching was sigmoid and paralleled the Zn(II)-accelerating effect of factor XII activation by kallikrein and factor XIIa, indicating that the spectral change was reporting Zn(II)-factor XII interactions responsible for the enhanced activation rate. The apparent cooperativity of Zn(II) effects on factor XII fluorescence quenching and activation kinetics, and the apparent noncooperativity in Zn(II) binding to factor XII measured by equilibrium dialysis could be explained by a two-state model in which Zn(II) binding is linked to a conformational change in the protein. The Zn(II)-induced quenching of factor XII fluorescence exhibited a pH dependence consistent with the involvement of histidine residues in the binding of Zn(II). Dynamic quenching of factor XII protein fluorescence by iodide or acrylamide, in the absence and presence of Zn(II), revealed heterogeneity in the environment of the 13 tryptophan residues of factor XII that was markedly reduced by metal ion binding. Together, these results indicate that cooperative interactions of Zn(II) with factor XII induce structural changes in the zymogen that facilitate its proteolytic cleavage and activation.

Acrylamide↗
PubMed1985

Kinetic studies on surface-mediated activation of bovine factor XII and prekallikrein. Effects of kaolin and high-Mr kininogen on the activation reactions.

The kaolin-mediated reciprocal activation of bovine factor XII and prekallikrein was divided into the following two reactions: the activation of factor XII by plasma kallikrein (reaction 1) and the activation of prekallikrein by factor XIIa (reaction 2). The effects of high-Mr kininogen and kaolin surface on the kinetics of these activation reactions were studied. High-Mr kininogen markedly enhanced the rate of reactions 1 and 2 in the presence of kaolin, and the enhancements were highly dependent on the concentrations of the protein cofactor and amount of kaolin surface. For the activation of factor XII by plasma kallikrein (reaction 1), high-Mr kininogen was required when a low concentration of factor XII and kaolin was used. The molar ratio of the protein cofactor to factor XII for optimal activation was found to be approximately 1:1. The apparent Km value and the kcat/Km value for plasma kallikrein on factor XII were calculated to be 4 nM and 5.2 X 10(7) s-1 X M-1, respectively. The activation of prekallikrein by factor XIIa, (reaction 2) proceeded even in the absence of high-Mr kininogen and kaolin. The addition of the protein cofactor and surface to the reaction mixture remarkably accelerated the reaction, and the apparent Km value for factor XIIa on prekallikrein was reduced from 1 microM to 40 nM. Moreover, the kcat/Km value was altered from 7.3 X 10(4) to 1.1 X 10(6) s-1 X M-1). These results suggest that high-Mr kininogen accelerates the surface-mediated activation of factor XII and prekallikrein by enhancing the susceptibility of factor XII to plasma kallikrein, on the one hand, and the affinity of factor XIIa for prekallikrein, on the other hand. Kaolin may play an important role in the concentration and organization of these components on the negatively charged surface.

Amidohydrolases↗
PubMed2001

Factor XI assembly and activation on human umbilical vein endothelial cells in culture.

Biotin-FXI optimally bound to HUVEC in the presence of 40 nM high molecular weight kininogen (HK) and > or =7 microM Zn2+. There was little specific FXI binding in the absence of added HK and at concentrations of Zn2+ <15 microM. FXI and prekallikrein, but not prothrombin, blocked biotin-FXI binding to HUVEC in the presence of HK with an IC50 of 18 nM and 180 nM, respectively. Monoclonal antibody HKL16 and peptide SDD31 also inhibited biotin-XI binding in the presence of HK with an IC50 of 4.7 nM and 50 microM, respectively. Alternatively, peptide T249-F260 of FXI's apple domain 3 and heparin monosulfate were weak inhibitors of FXI binding to HUVEC. FXI bound to HUVEC with an apparent Kd of 6.9 +/- 3.0 nM and Bmax of 13 +/- 2.6 x 10(6) sites/cell. FXI bound to HK on HUVEC, but not prothrombin, became converted to FXIa. FXI activation on HUVEC resulted from tissue culture media bovine factor XIIa. HUVEC grown in human factor XI-deficient serum did not support FXI activation. FXI binding to HUVEC in culture was mostly mediated by HK and FXI activation on HUVEC is dependent on cell-associated factor XIIa.

Binding, Competitive↗
PubMed1985

Binding and activation properties of human factor XII, prekallikrein, and derived peptides with acidic lipid vesicles.

The binding of human factor XII and prekallikrein to vesicles of various compositions and the relationship to activation of factor XII were studied. Factor XII, factor XIIa, and the 40-kilodalton binding fragment of factor XII bound tightly to all of the negatively charged lipids investigated, including sulfatide, phosphatidylserine, and phosphatidylethanolamine, but not to the neutral lipid phosphatidylcholine. Binding could be reversed by high salt, and the dissociation constant for binding to sulfatide vesicles was in the nanomolar range at an ionic strength of 0.15 M. Prekallikrein did not bind significantly to either sulfatide or phosphatidylethanolamine vesicles under the conditions used. Stopped-flow studies showed that the association rate for the factor XII-sulfatide interaction was biphasic and very rapid; the faster rate corresponded to about 30% collisional efficiency. The kinetics of activation of factor XII was investigated and was in agreement with previous studies; sulfatide promoted activation but phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine did not. Autoactivation rates correlated closely with the packing density of factor XII and factor XIIa on the vesicle surface. In contrast, kallikrein activation of factor XII correlated with the amount of sulfatide-bound factor XII and was relatively insensitive to the density of factor XII on the vesicle surface. When the concentration of factor XII was reduced to only several molecules per vesicle, the autoactivation rate dropped very low whereas kallikrein activation held relatively constant. These results indicated that the autoactivation and the kallikrein activation of factor XII were dependent on different properties of the surface component.

Enzyme Activation↗