Zinc ions promote the binding of factor XII/factor XIIA to acidic phospholipids but have no effect on the binding of high-Mr kininogen

Binding of high-Mr kininogen and factor XII/factor XIIa to phospholipids coated on to polystyrene microtiter plates was investigated by ELISA. Both high-Mr kininogen and factor XII/factor XIIa bound specifically to the phospholipid surface. Binding was observed to negatively charged phospholipids only. The binding of high-Mr kininogen was not affected by the presence of zinc ions. At a surface concentration of 20% phosphatidylinositol phosphate in phosphatidylcholine a dissociation constant (kD) of 10 nM for the binding of high-Mr kininogen was calculated. The amount of bound purified alpha-factor XIIa could be increased 4-5-fold in the presence of zinc ions. The lowest zinc ion concentration giving maximal binding was 0.1 mM. The binding of alpha-factor XIIa was inhibited by high-Mr kininogen. Independent of the presence of zinc ions or high-Mr kininogen, a kD of 7.9 nM was calculated for alpha-factor XIIa binding. The binding of prekallikrein was dependent upon the presence and the concentration of high-Mr kininogen. In plasma containing aprotinin, the binding of high-Mr kininogen was apparently inhibited in the presence of zinc ions, which was a prerequisite for the binding of factor XII. This apparently inhibitory effect of zinc ions on the binding of high-Mr kininogen was probably due to the increased binding of factor XII, which displaced high-Mr kininogen.     

Possible basis for the apparent surface selectivity of the contact activation of human blood coagulation factor XII

The activation of factor XII by the proteases factor XIIa and kallikrein is known to be greatly enhanced by certain negatively charged surfaces. Studies that compared factor XII surface binding to factor XII activation found that binding alone was insufficient to account for surface enhancement of the activation rate. The temperature dependence of the reaction showed unusual behavior that may be related to the conformational change of factor XII following binding; the rate of factor XII activation had a relatively low temperature optimum (0-47 degrees C) that was sensitive to choice of surface and salt concentration. In temperature studies, below 47 degrees C, the decrease in the activation rate was not related to the thermal denaturation of enzyme or substrate, nor to the choice of activator enzyme (factor XIIa or kallikrein), nor to the species of factor XII (human or bovine) but to a behavior, designated a thermal transition, associated with the surface or the protein-surface interaction. The previously reported surface selectivity of contact activation is possible due to the temperature characteristics and other properties of the thermal transition; a surface that has a low-temperature thermal transition and that is highly sensitive to salt will be a "poor" contact surface under the usual choice of reaction conditions (approximately 150 mM ionic strength and 37 degrees C). However, solution conditions were identified that allowed the following negatively charged surfaces to function, in nearly equal potency, in the activation of factor XII: phosphatidylserine, phosphatidylglycerol, phosphatidic acid, phosphatidylinositol 4-phosphate, heparin, and 5-kDa dextran sulfate, as well as the previously characterized sulfatide and 500-kDa dextran sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human factor XII binding to the glycoprotein Ib-IX-V complex inhibits thrombin-induced platelet aggregation

Factor XII deficiency has been postulated to be a risk factor for thrombosis suggesting that factor XII is an antithrombotic protein. The biochemical mechanism leading to this clinical observation is unknown. We have previously reported high molecular weight kininogen (HK) inhibition of thrombin-induced platelet aggregation by binding to the platelet glycoprotein (GP) Ib-IX-V complex. Although factor XII will bind to the intact platelet through GP Ibalpha (glycocalicin) without activation, we now report that factor XIIa (0. 37 microm), but not factor XII zymogen, is required for the inhibition of thrombin-induced platelet aggregation. Factor XIIa had no significant effect on SFLLRN-induced platelet aggregation. Moreover, an antibody to the thrombin site on protease-activated receptor-1 failed to block factor XII binding to platelets. Inhibition of thrombin-induced platelet aggregation was demonstrated with factor XIIa but not with factor XII zymogen or factor XIIf, indicating that the conformational exposure of the heavy chain following proteolytic activation is required for inhibition. However, inactivation of the catalytic activity of factor XIIa did not affect the inhibition of thrombin-induced platelet aggregation. Factor XII showed displacement of biotin-labeled HK (30 nm) binding to gel-filtered platelets and, at concentrations of 50 nm, was able to block 50% of the HK binding, suggesting involvement of the GP Ib complex. Antibodies to GP Ib and GP IX, which inhibited HK binding to platelets, did not block factor XII binding. However, using a biosensor, which monitors protein-protein interactions, both HK and factor XII bind to GP Ibalpha. Factor XII may serve to regulate thrombin binding to the GP Ib receptor by co-localizing with HK, to control the extent of platelet aggregation in vivo.     

Surface-dependent activation of human factor XII (Hageman factor) by kallikrein and its light chain

In this paper we report the effect of sulfatides on the rate constants of factor XII activation by kallikrein and its isolated light chain (the domain of kallikrein that contains the active site of the enzyme). In the absence of sulfatides, kallikrein and the light chain were equally effective in factor XII activation (k1 = 1.57 X 10(3) M-1 s-1 at pH 7.0). The pH optima were the same (pH 7.0) and the reaction was not affected by variation of the ionic strength. Sulfatides strongly increased the rate constants of factor XIIa formation. In the presence of sulfatides kallikrein was, however, much more active than its light chain. At 330 microM sulfatides, pH 7.0 and 100 mM NaCl the rate constants of factor XII activation were 5.34 X 10(6) M-1 s-1 and 4.17 X 10(4) M-1 s-1 for kallikrein and its light chain, respectively. The pH optimum of factor XII activation by kallikrein in the presence of sulfatides was shifted to pH 6.3, and the reaction became highly ionic-strength-dependent. The rate constant increased considerably at decreasing NaCl concentrations. The optimum pH for light-chain-dependent factor XII activation in the presence of sulfatides remained unaltered and the reaction was not affected by the ionic strength. Binding studies revealed that both kallikrein and factor XII bind to the sulfatide surface, whereas no binding of the light chain of kallikrein was detectable. The isolated heavy chain of kallikrein had the same binding properties as kallikrein, which indicates that the heavy-chain domain contains the functional information for kallikrein binding to sulfatides. Since the effects of pH and ionic strength on the rate constants of kallikrein-dependent factor XII activation in the presence of sulfatides correlated with effects on the binding of kallikrein, it is concluded that under these conditions surface-bound factor XII is activated by surface-bound kallikrein. Our data suggest that sulfatides stimulate kallikrein-dependent factor XII activation by two distinct mechanisms: by making factor XII more susceptible to peptide bond cleavage by kallikrein and by promoting the formation of the enzyme-substrate complex through surface binding of kallikrein and factor XII.     

Activation of the contact system of coagulation by a monoclonal antibody directed against a neodeterminant in the heavy chain region of human coagulation factor XII (Hageman factor)

We studied the characteristics of two monoclonal antibodies (mAbs), F1 and F3, against human coagulation factor XII (Hageman factor). Experiments with trypsin-digested 125I-factor XII revealed that the epitope for mAb F1 is located in the NH2-terminal Mr 40,100 portion of factor XII, whereas that for mAb F3 resides in the COOH-terminal Mr 30,000 portion of this protein. Factor XII in fresh plasma (single-chain factor XII) bound approximately 190 times less to mAb F1 than factor XII in dextran sulfate-activated plasma (cleaved factor XII). However, no difference in accessibility of the epitope for mAb F1 was observed between cleaved and single-chain factor XII when bound to glass. mAb F3 appeared to bind to both single-chain and cleaved factor XII in plasma as well as when bound to glass. Neither mAb F1, nor F3 affected the amidolytic activity of factor XIIa, whereas both mAb F1 and F3 inhibited factor XII-coagulant activity to about 15 and 70%, respectively, at a molar ratio of mAb to factor XII of 20 to 1. mAb F1, as well as F(ab')2 and F(ab') fragments of this antibody induced activation of the contact system in plasma, as reflected by the generation of factor XIIa. C1 inhibitor and kallikrein. C1 inhibitor complexes. Activation was induced neither upon incubation with mAb F3, nor with that of control mAbs. mAb F1-induced contact activation required the presence of factor XII, prekallikrein, and high molecular weight kininogen and, in contrast to activation by negatively charged surfaces, was not inhibited by the presence of Polybrene. Based on these results we propose that a conformational change in factor XII is a key event in the activation process of this molecule. This conformational change can be induced by binding of factor XII to a surface as well as by proteolytic cleavage. As mAb F1 can also induce this conformational change, this antibody may provide a unique tool in studies of the activation of factor XII.     

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.     

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.     

Identification and characterization of plasma kallikrein-kinin system inhibitors from salivary glands of the blood-sucking insect Triatoma infestans

Two plasma kallikrein-kinin system inhibitors in the salivary glands of the kissing bug Triatoma infestans, designated triafestin-1 and triafestin-2, have been identified and characterized. Reconstitution experiments showed that triafestin-1 and triafestin-2 inhibit the activation of the kallikrein-kinin system by inhibiting the reciprocal activation of factor XII and prekallikrein, and subsequent release of bradykinin. Binding analyses showed that triafestin-1 and triafestin-2 specifically interact with factor XII and high molecular weight kininogen in a Zn2+-dependent manner, suggesting that they specifically recognize Zn2+-induced conformational changes in factor XII and high molecular weight kininogen. Triafestin-1 and triafestin-2 also inhibit factor XII and high molecular weight kininogen binding to negatively charged surfaces. Furthermore, they interact with both the N-terminus of factor XII and domain D5 of high molecular weight kininogen, which are the binding domains for biological activating surfaces. These results suggest that triafestin-1 and triafestin-2 inhibit activation of the kallikrein-kinin system by interfering with the association of factor XII and high molecular weight kininogen with biological activating surfaces, resulting in the inhibition of bradykinin release in an animal host during insect blood-feeding.     

Activation of factor XII and prekallikrein with polysaccharide sulfates and sulfatides: comparison with kaolin-mediated activation

The activation of Factor XII and prekallikrein by polysaccharide sulfates and sulfatides in the presence of high-molecular-weight (HMW) kininogen was studied, and compared with the kaolin-mediated activation reaction. Among a variety of artificially-sulfated polysaccharides and native polysaccharide sulfates, amylose sulfate (M.W.= 380,000 and sulfur content, 19.1%) and sulfatide were found to have the most efficient ability to trigger the activation of prekallikrein by Factor XII. The effects of these two kinds of negatively-charged surfaces on the following three activation reactions were compared; the activation of prekallikrein by Factor XII (reaction 1), the activation of Factor XII by kallikrein (reaction 2) and the activation of prekallikrein by Factor XIIa (reaction 3). All three reactions mediated by the selected surfaces were strongly accelerated by HMW kininogen and its derivatives, kinin-free protein and fragment 1.2-linked light chain, like the kaolin-mediated activation. However, this accelerating effect of HMW kininogen on the amylose sulfate- and sulfatide-mediated activations (reaction 1) was diminished after treatment with fluorescein iso-thiocyanate, whereas the effect on the kaolin-mediated activation was not influenced by fluorescein-labeling. In addition, reaction 2 mediated by amylose sulfate and sulfatide was extremely slow even in the presence of HMW kininogen, and the results also differed from those with kaolin. The sulfatide-mediated activation of reaction 1 was not inhibited by fragment 1.2 (His-rich fragment), which is released from HMW kininogen by the action of kallikrein, and is known to be a potent inhibitor of the kaolin-dependent activation. These results indicate that the mechanisms responsible for surface activation triggered by soluble amylose sulfate, sulfatide micelles and kaolin differ from each other as regards the molecular interaction with the contact factors.     

Mapping of a putative surface-binding site of human coagulation factor XII

We have localized the binding epitope(s) of two murine monoclonal antibodies (B7C9 and P5-2-1) that were shown previously to inhibit the activation of human coagulation factor XII by negatively charged surfaces. A factor XII cDNA expression library in lambda gt11 was screened with antibody B7C9, and 16 immunoreactive bacteriophage were isolated. Fusion proteins from each of the recombinant phage were reactive with both monoclonal antibodies. Two of the phage cDNA inserts were found to code for amino acid residues -6-+31 and +1-+47 of factor XII, respectively, thereby defining the limits of the antigenic peptide to amino acids +1-+31. Each of the remaining 14 recombinant phage contained longer factor XII cDNA inserts that included sequences coding for the amino-terminal 31 amino acid residues. These results were confirmed by direct binding of antibody B7C9 to synthetic peptides containing amino acids 1-14 and 1-28 of factor XII. Further experiments with a set of nested peptides also indicated that amino acid residues 1-4 were essential but not sufficient for binding of B7C9 to the peptides. Hydrophobicity analysis of the amino-terminal region of plasma factor XII revealed a highly hydrophilic region between amino acid residues 5 and 15 that contained positively charged lysine residues at positions 8, 11, and 13. We conclude that a major epitope(s) recognized by monoclonal antibodies B7C9 and P5-2-1 is present in the amino-terminal 28 amino acids of factor XII. It is proposed that binding of these antibodies to factor XII blocks interaction of the positively charged region between residues 5 and 15 with negatively charged surfaces, thereby inhibiting activation.     

Activation of human blood coagulation factor XI independent of factor XII. Factor XI is activated by thrombin and factor XIa in the presence of negatively charged surfaces

Human blood coagulation factor XI was activated by either autoactivation or thrombin. These reactions occurred only in the presence of negatively charged materials, such as dextran sulfate (approximately Mr 500,000), sulfatide, and heparin. During the activation, factor XI was cleaved at a single Arg-Ile bond by thrombin or factor XIa to produce an amino-terminal 50-kDa heavy chain and a carboxyl-terminal 35-kDa light chain. This activation pattern is identical to that produced by factor XIIa. The addition of a small amount of thrombin and sulfatide to factor XII-deficient plasma produced shorter clotting times than when these agents were added to factor XI/factor XII combined-deficient plasma. These results suggest that the activation of factor XI by thrombin and possibly the autoactivation of factor XI proceed in plasma to lead fibrin clot formation. These reactions may have a role on an appropriate negatively charged surface in normal hemostasis.     

Studies on the effect of serine protease inhibitors on activated contact factors. Application in amidolytic assays for factor XIIa, plasma kallikrein and factor XIa

Amidolytic assays have been developed to determine factor XIIa, factor XIa and plasma kallikrein in mixtures containing variable amounts of each enzyme. The commercially available chromogenic p-nitroanilide substrates Pro-Phe-Arg-NH-Np (S2302 or chromozym PK), Glp-Pro-Arg-NH-Np (S2366), Ile-Glu-(piperidyl)-Gly-Arg-NH-Np (S2337), and Ile-Glu-Gly-Arg-NH-Np (S2222) were tested for their suitability as substrates in these assays. The kinetic parameters for the conversion of S2302, S2222, S2337 and S2366 by beta factor XIIa, factor XIa and plasma kallikrein indicate that each active enzyme exhibits considerable activity towards a number of these substrates. This precludes direct quantification of the individual enzymes when large amounts of other activated contact factors are present. Several serine protease inhibitors have been tested for their ability to inhibit those contact factors selectively that may interfere with the factor tested for. Soybean trypsin inhibitor very efficiently inhibited kallikrein, inhibited factor XIa at moderate concentrations, but did not affect the amidolytic activity of factor XIIa. Therefore, this inhibitor can be used to abolish a kallikrein and factor XIa contribution in a factor XIIa assay. We also report the rate constants of inhibition of contact activation factors by three different chloromethyl ketones. D-Phe-Pro-Arg-CH2Cl was moderately active against contact factors (k = 2.2 X 10(3) M-1 s-1 at pH 8.3) but showed no differences in specifity. D-Phe-Phe-Arg-CH2Cl was a very efficient inhibitor of plasma kallikrein (k = 1.2 X 10(5) M-1 s-1 at pH 8.3) whereas it slowly inhibited factor XIIa (k = 1.4 X 10(3) M-1 s-1) and factor XIa (k = 0.11 X 10(3) M-1 s-1). Also Dns-Glu-Gly-Arg-CH2Cl was more reactive towards kallikrein (k = 1.6 X 10(4) M-1 s-1) than towards factor XIIa (k = 4.6 X 10(2) M-1 s-1) and factor XIa (k = 0.6 X 10(2) M-1 s-1). Since Phe-Phe-Arg-CH2Cl is highly specific for plasma kallikrein it can be used in a factor XIa assay selectively to inhibit kallikrein. Based on the catalytic efficiencies of chromogenic substrate conversion and the inhibition characteristics of serine protease inhibitors and chloromethyl ketones we were able to develop quantitative assays for factor XIIa, factor XIa and kallikrein in mixtures of contact activation factors.     

Binding and calcium-induced aggregation of laminin onto lipid bilayers

Direct binding of laminin in the form of its complex with nidogen to planar lipid bilayers was demonstrated with total internal reflection fluorescence microscopy. Binding occurred equally well to zwitterionic (phosphatidylcholine) and negatively charged (phosphatidylglycerol) lipids and was enhanced by sulfatides but only at nonphysiological molar ratios higher than 30 mol %. Strong interactions with lipid bilayers were also observed for bovine serum albumin. This explains a strong inhibition of laminin binding by this protein. However, binding of laminin to sulfatide-rich bilayers was not completely inhibited. Observable by the microscopic technique was the formation of laminin clusters on the surface of the bilayer which occurred concomitantly with binding. Both processes were strongly enhanced by the presence of calcium. These results show that calcium-induced laminin self-assembly is enhanced at lipid surfaces.     

Sulfated glycolipids and cell adhesion

The adhesive glycoproteins laminin, thrombospondin, and von Willebrand factor bind specifically and with high affinity to sulfatides, and it is this binding that probably accounts for their ability to agglutinate glutaraldehyde-fixed erythrocytes. The three proteins differ, however, in the inhibition of their binding to sulfatides by sulfated polysaccharides. Fucoidan strongly inhibits binding of both laminin and thrombospondin, but not of von Willebrand factor, suggesting the involvement of laminin or thrombospondin, or other unknown sulfatide-binding proteins in specific cell interactions that are also inhibited by fucoidan. Thrombospondin adsorbed on plastic promotes the attachment and spreading of some melanoma cells. Interestingly, fucoidan and an antibody against the sulfatide-binding domain of thrombospondin selectively inhibit spreading but not attachment to thrombospondin-coated surfaces. Sulfatides, but not neutral glycolipids or gangliosides, when adsorbed on plastic also promote attachment and spreading of some cultured cell lines. Direct adhesion of melanoma cells requires high densities of adsorbed sulfatide. In the presence of laminin, however, specific adhesion of some cell types to sulfatide is strongly stimulated and requires only low densities of adsorbed lipid, suggesting that laminin is mediating adhesion by crosslinking receptors on the cell surface to sulfatide adsorbed on the plastic. Although thrombospondin also binds to sulfatides and to melanoma cells, it does not enhance but rather inhibits direct and laminin-dependent melanoma cell adhesion to sulfatide, presumably because it is unable to bind simultaneously to ligands on opposing surfaces. Thus, sulfated glycolipids can participate in both laminin- and thrombospondin-mediated cell adhesion, but their mechanisms of interaction are different.     

A monoclonal antibody recognizing an icosapeptide sequence in the heavy chain of human factor XII inhibits surface-catalyzed activation

A monoclonal antibody (mAb B7C9) to human factor XII was raised in murine somatic cell using purified factor XII antigen. The purified antibody was subtyped IgG1 kappa and had a KD of 9.8 nM for antigen factor XII. Functional studies indicated that mAb B7C9 blocks surface-mediated coagulant activity of factor XII but not the amidolytic activity of factor XIIa against the small substrate H-D-Pro-Phe-Arg-p-nitroanilide (S-2302), suggesting that the mAb B7C9 epitope is located at or near the surface binding domain of the heavy chain region of factor XII. Western blot analysis indicated that the antibody reacts with factor XII and the heavy chain of factor XIIa. Affinity isolation of factor XII peptides, produced after cleavage by kallikrein, resulted in three factor XII heavy chain domain segments that were identified in the known factor XII sequence by limited N-terminal analysis. The epitope was located to a 20-amino acid sequence of 2.5 kDa in the heavy chain of factor XII which is the putative surface binding region of factor XII. The 2.5-kDa peptide was synthesized and demonstrated to react with mAb B7C9. mAb B7C9 was immobilized on an affinity resin and was successfully utilized to purify functionally active factor XII from plasma.     

The sequence HGLGHGHEQQHGLGHGH in the light chain of high molecular weight kininogen serves as a primary structural feature for zinc-dependent binding to an anionic surface.

The histidine-glycine-rich region of the light chain of cleaved high molecular weight kininogen (HK) is thought to be responsible for binding to negatively charged surfaces and initiation of the intrinsic coagulation, fibrinolytic, and kinin-forming systems. However, the specifically required amino acid sequences have not been delineated. An IgG fraction of a monoclonal antibody (MAb) C11C1 to the HK light chain was shown to inhibit by 66% the coagulant activity and by 57% the binding of HK to the anionic surface of kaolin at a concentration of 1.5 microM and 27 microM, respectively. Proteolytic fragments of HK were produced by successive digestion with human plasma kallikrein and factor XIa (FXIa). Those polypeptides that bound tightly (Kd = 0.77 nM) to a C11C1 affinity column were eluted at pH 3.0 and purified by membrane filtration. On 15% SDS polyacrylamide electrophoresis, the approximate M(r) was 7.3 kDa (range 6.2-8.1 kDa). Based on N-terminal sequencing, this polypeptide (1(2)), which extends from the histidine residue 459 to a lysine at position 505, 509, 511, 512, 515, or 520, inhibits by 50% the coagulant activity expressed by HK at a concentration of 22 microM. The synthetic peptide HGLGHGH representing the N-terminal of the 1(2)) fragment was synthesized, tested, and found at 4 mM to inhibit the procoagulant activity of HK 50%. A synthetic heptadecapeptide, HGLGHGHEQQHGLGHGH (residues 459-475) included within the 1(2) fragment, and with the ability to bind zinc, inhibited 50% of the HK coagulant activity at a concentration of 325 microM in the absence and presence of added Zn2+ (30 microM). The specific binding of 125I-HK to a negatively charged surface (kaolin) was inhibited 50% by unlabeled HK (5 microM). HGLGHGH, at a concentration of 7.0 mM, inhibited the binding to kaolin by 50%. The heptadecapeptide inhibited the specific binding of 125I-HK to kaolin by 50%, at a concentration of 2.3 mM, in the absence of Zn2+. In contrast, when Zn2+ was added, the concentration to achieve 50% inhibition decreased to 630 microM, indicating that Zn2+ was required to attain a favorable conformation for binding. Moreover, the 1(2) fragment was found to inhibit 50% of the 125I-HK binding to kaolin at a concentration of 380 microM. These results suggest that residues contained within the 1(2) fragment, notably HGLGHGHEQQHGLGHGH, serves as a primary structural feature for binding to a negatively charged surface.     

Contribution of the N-terminal and C-terminal domains of haemaphysalin to inhibition of activation of plasma kallikrein-kinin system

Haemaphysalin is a kallikrein-kinin system inhibitor from hard tick Haemaphysalis longicornis, and consists of two Kunitz type protease inhibitor domains. Each domain as well as haemaphysalin inhibited intrinsic coagulation by inhibiting activation of the kallikrein-kinin system without affecting the amidolytic activities of intrinsic coagulation factors, indicating that both domains were involved in the inhibition through a similar mechanism to that for haemaphysalin. Reconstitution experiments showed that the C-terminal domain contributed more predominantly to this inhibition. Direct binding assaying showed that the C-terminal domain could bind to the cell-binding region of high molecular weight kininogen (HK), suggesting that it also binds to the cell-binding region of factor XII. Judging from these findings, the C-terminal domain may more effectively inhibit the association of factor XII and HK with the cell surface by binding to cell-binding regions, and hence would predominantly contribute to the inhibition of activation of the kallikrein-kinin system.     

The sorbinil trap: a predicted dead-end complex confirms the mechanism of aldose reductase inhibition

Kinetic and crystallographic studies have demonstrated that negatively charged aldose reductase inhibitors act primarily by binding to the enzyme complexed with oxidized nicotinamide dinucleotide phosphate (E.NADP(+)) to form a ternary dead-end complex that prevents turnover in the steady state. A recent fluorescence study [Nakano and Petrash (1996) Biochemistry 35, 11196-11202], however, has concluded that inhibition by sorbinil, a classic negatively charged aldose reductase inhibitor, results from binding to the enzyme complexed with reduced cofactor (E.NADPH) and not binding to E.NADP(+). To resolve this controversy, we present transient kinetic data which show unequivocally that sorbinil binds to E.NADP(+) to produce a dead-end complex, the so-called sorbinil trap, which prevents steady-state turnover in the presence of a saturating concentration of aldehyde substrate. The reported fluorescence binding results, which we have confirmed independently, are further shown to be fully consistent with the proposed sorbinil trap mechanism. Our conclusions are supported by KINSIM simulations of both pre-steady-state and steady-state reaction time courses in the presence and absence of sorbinil. Thus, while sorbinil binding indeed occurs to both E.NADPH and E.NADP(+), only the latter dead-end complex shows significant inhibition of the steady-state turnover rate. The effect of tight-binding kinetics on the inhibition patterns observed for zopolrestat, another negatively charged inhibitor, is further examined both experimentally and with KINSIM, with the conclusion that all reported aldose reductase inhibition can be rationalized in terms of binding of an alrestatin-like inhibitor at the active site, with no need to postulate a second inhibitor binding site.     

Binding of coagulation factor XI to washed human platelets

The binding of human coagulation factor XI to washed human platelets was studied in the presence of zinc ions, calcium ions, and high molecular weight kininogen. Significant factor XI binding occurred at physiological levels of these metal ions when high molecular weight kininogen was present. Binding required platelet stimulation and was specific, reversible, and saturable. Scatchard analysis of the binding yielded approximately 1500 binding sites per platelet with an apparent dissociation constant of approximately 10 nM. Since the concentration of factor XI in plasma is about 25 nM, this suggests that in plasma factor XI binding sites on stimulated platelets might be saturated. Calcium ions and high molecular weight kininogen acted synergistically to enhance the ability of low concentrations of zinc ions to promote factor XI binding. The similarity between the concentrations of metal ions optimal for factor XI binding and those optimal for high molecular weight kininogen binding, as well as the ability of high molecular weight kininogen to modulate these metal ion effects, implies that factor XI and high molecular weight kininogen may form a complex on the platelet surface as they do in solution and on artificial negatively charged surfaces.     

Mechanisms of binding of recombinant extrinsic pathway inhibitor (rEPI) to cultured cell surfaces. Evidence that rEPI can bind to and inhibit factor VIIa-tissue factor complexes in the absence of factor Xa.

Extrinsic pathway inhibitor plays a key role in modulating tissue factor-dependent blood coagulation. We have studied binding of radioiodinated recombinant extrinsic pathway inhibitor (rEPI) to cultured cell surfaces. rEPI in the absence of added reactants bound to a limited extent to three cell lines studied. Binding of rEPI to two cell lines possessing surface tissue factor, but not to a cell line lacking surface tissue factor, was markedly increased in the presence of both factor VIIa and factor Xa, and calcium ions. Moreover, some increased tissue factor-dependent binding was also demonstrated with factor VIIa alone. Binding isotherms of rEPI to factor VIIa-tissue factor obtained with an ovarian carcinoma cell line were hyperbolic. Scatchard plots indicated the following: a Kd value of 4.5 +/- 1.5 nM and 335,000 +/- 84,000 sites/cell when factor Xa was present; a Kd value of 11.9 +/- 3.5 nM and 236,000 +/- 68,000 sites/cell when factor Xa was absent. In functional studies, high concentrations of rEPI, e.g. 27-67.5 nM, were found to inhibit factor VIIa-tissue factor-catalyzed release of activation peptide from tritiated factor IX in the absence of factor Xa. Whereas factor Xa was thus shown not to be required for rEPI to inhibit factor VIIa-tissue factor catalytic activity, its presence markedly enhanced rEPI's inhibitory function. Since the local concentration of extrinsic pathway inhibitor achieved at a site of tissue injury is unknown, the physiologic significance of the observation of extrinsic pathway inhibitor-induced inhibition of factor VIIa-tissue factor activity in the absence of factor Xa is not clear. However, factor Xa-independent inhibition could play a significant role when large doses of rEPI are administered in experimental studies of thrombosis.