Localization of a heparin binding site in the catalytic domain of factor XIa.

Inhibition of factor XIa by protease nexin II (K(i) approximately 450 pM) is potentiated by heparin (K(I) approximately 30 pM). The inhibition of the isolated catalytic domain of factor XIa demonstrates a similar potentiation by heparin (K(i) decreasing from 436 +/- 62 to 88 +/- 10 pM) and also binds to heparin on surface plasmon resonance (K(d) 11.2 +/- 3.2 nM vs K(d) 8.63 +/- 1.06 nM for factor XIa). The factor XIa catalytic domain contains a cysteine-constrained alpha-helix-containing loop: (527)CQKRYRGHKITHKMIC(542), identified as a heparin-binding region in other coagulation proteins. Heparin-binding studies of coagulation proteases allowed a grouping of these proteins into three categories: group A (binding within a cysteine-constrained loop or a C-terminal heparin-binding region), factors XIa, IXa, Xa, and thrombin; group B (binding by a different mechanism), factor XIIa and activated protein C; and group C (no binding), factor VIIa and kallikrein. Synthesized peptides representative of the factor XIa catalytic domain loop were used as competitors in factor XIa binding and inhibition studies. A native sequence peptide binds to heparin with a K(d) = 86 +/- 15 nM and competes with factor XIa in binding to heparin, K(i) = 241 +/- 37 nM. A peptide with alanine substitutions at (534)H, (535)K, (538)H, and (539)K binds and competes with factor XIa for heparin-binding in a manner nearly identical to that of the native peptide, whereas a scrambled peptide is approximately 10-fold less effective, and alanine substitutions at residues (529)K, (530)R, and (532)R result in loss of virtually all activity. We conclude that residues (529)K, (530)R, and (532)R comprise a high-affinity heparin-binding site in the factor XIa catalytic domain.     

Structural and mutational analyses of the molecular interactions between the catalytic domain of factor XIa and the Kunitz protease inhibitor domain of protease nexin 2

Factor XIa (FXIa) is a serine protease important for initiating the intrinsic pathway of blood coagulation. Protease nexin 2 (PN2) is a Kunitz-type protease inhibitor secreted by activated platelets and a physiologically important inhibitor of FXIa. Inhibition of FXIa by PN2 requires interactions between the two proteins that are confined to the catalytic domain of the enzyme and the Kunitz protease inhibitor (KPI) domain of PN2. Recombinant PN2KPI and a mutant form of the FXI catalytic domain (FXIac) were expressed in yeast, purified to homogeneity, co-crystallized, and the structure of the complex was solved at 2.6 angstroms (Protein Data Bank code 1ZJD). In this complex, PN2KPI has a characteristic, disulfide-stabilized double loop structure that fits into the FXIac active site. To determine the contributions of residues within PN2KPI to its inhibitory activity, selected point mutations in PN2KPI loop 1 11TGPCRAMISR20 and loop 2 34FYGGC38 were tested for their ability to inhibit FXIa. The P1 site mutation R15A completely abolished its ability to inhibit FXIa. IC50 values for the wild type protein and the remaining mutants were as follows: PN2KPI WT, 1.28 nM; P13A, 5.92 nM; M17A, 1.62 nM; S19A, 1.86 nM; R20A, 5.67 nM; F34A, 9.85 nM. The IC50 values for the M17A and S19A mutants were not significantly different from those obtained with wild type PN2KPI. These functional studies and activated partial thromboplastin time analysis validate predictions made from the PN2KPI-FXIac co-crystal structure and implicate PN2KPI residues, in descending order of importance, Arg15, Phe34, Pro13, and Arg20 in FXIa inhibition by PN2KPI.     

The role of factor XIa (FXIa) catalytic domain exosite residues in substrate catalysis and inhibition by the Kunitz protease inhibitor domain of protease nexin 2

To select residues in coagulation factor XIa (FXIa) potentially important for substrate and inhibitor interactions, we examined the crystal structure of the complex between the catalytic domain of FXIa and the Kunitz protease inhibitor (KPI) domain of a physiologically relevant FXIa inhibitor, protease nexin 2 (PN2). Six FXIa catalytic domain residues (Glu(98), Tyr(143), Ile(151), Arg(3704), Lys(192), and Tyr(5901)) were subjected to mutational analysis to investigate the molecular interactions between FXIa and the small synthetic substrate (S-2366), the macromolecular substrate (factor IX (FIX)) and inhibitor PN2KPI. Analysis of all six Ala mutants demonstrated normal K(m) values for S-2366 hydrolysis, indicating normal substrate binding compared with plasma FXIa; however, all except E98A and K192A had impaired values of k(cat) for S-2366 hydrolysis. All six Ala mutants displayed deficient k(cat) values for FIX hydrolysis, and all were inhibited by PN2KPI with normal values of K(i) except for K192A, and Y5901A, which displayed increased values of K(i). The integrity of the S1 binding site residue, Asp(189), utilizing p-aminobenzamidine, was intact for all FXIa mutants. Thus, whereas all six residues are essential for catalysis of the macromolecular substrate (FIX), only four (Tyr(143), Ile(151), Arg(3704), and Tyr(5901)) are important for S-2366 hydrolysis; Glu(98) and Lys(192) are essential for FIX but not S-2366 hydrolysis; and Lys(192) and Tyr(5901) are required for both inhibitor and macromolecular substrate interactions.     

SERPIN regulation of factor XIa. The novel observation that protease nexin 1 in the presence of heparin is a more potent inhibitor of factor XIa than C1 inhibitor

In the present studies we have made the novel observation that protease nexin 1 (PN1), a member of the serine protease inhibitor (SERPIN) superfamily, is a potent inhibitor of the blood coagulation Factor XIa (FXIa). The inhibitory complexes formed between PN1 and FXIa are stable when subjected to reducing agents, SDS, and boiling, a characteristic of the acyl linkage formed between SERPINs and their cognate proteases. Using a sensitive fluorescence-quenched peptide substrate, the K(assoc) of PN1 for FXIa was determined to be 7.9 x 10(4) m(-)(1) s(-)(1) in the absence of heparin. In the presence of heparin, this rate was accelerated to 1.7 x 10(6), M(-)(1) s(-)(1), making PN1 a far better inhibitor of FXIa than C1 inhibitor, which is the only other SERPIN known to significantly inhibit FXIa. FXIa-PN1 complexes are shown to be internalized and degraded by human fibroblasts, most likely via the low density lipoprotein receptor-related protein (LRP), since degradation was strongly inhibited by the LRP agonist, receptor-associated protein. Since FXIa proteolytically modifies the amyloid precursor protein, this observation may suggest an accessory role for PN1 in the pathobiogenesis of Alzheimer's disease.     

A low molecular weight platelet inhibitor of factor XIa: purification, characterization, and possible role in blood coagulation.

A low molecular weight platelet inhibitor of factor XIa (PIXI) has been purified 250-fold from releasates of washed and stimulated human platelets. Molecular weight estimates of 8400 and 8500 were determined by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively, although a second band of Mr 5000 was present upon electrophoresis. The inhibitor does not appear to be one of the platelet-specific, heparin-binding proteins, since it neither bound to nor was affected by heparin. An amount of PIXI which inhibited by 50% factor XIa cleavage of the chromogenic substrate S2366 (Pyr-Glu-Pro-Arg-pNA-2H2O) only slightly inhibited (5-9%) factor XIIa, plasma kallikrein, plasmin, and activated protein C and did not inhibit factor Xa, thrombin, tPA, or trypsin, suggesting specificity for factor XIa. Kinetic analyses of the effect of PIXI on factor XIa activity demonstrated mixed-type, noncompetitive inhibition of S2366 cleavage and of factor IX activation with Ki's of 7 x 10(-8) and 3.8 x 10(-9) M, respectively. Immunoblot analysis showed that PIXI is not the inhibitory domain of protease nexin II, a potent inhibitor of factor XIa also secreted from platelets. Amino acid analysis showed that PIXI has no cysteine residues and, therefore, is not a Kunitz-type inhibitor. PIXI can prevent stable complex formation between alpha 1-protease inhibitor and factor XIa light chain as demonstrated by SDS-polyacrylamide gel electrophoresis. The inhibition by PIXI of factor XIa-catalyzed activation of factor IX and its capacity to prevent factor XIa inactivation by alpha 1-protease inhibitor, combined with the specificity of PIXI for factor XIa among serine proteases found in blood, suggest a role for PIXI in the regulation of intrinsic coagulation.     

Exosite-mediated substrate recognition of factor IX by factor XIa. The factor XIa heavy chain is required for initial recognition of factor IX

Studies of the mechanisms of blood coagulation zymogen activation demonstrate that exosites (sites on the activating complex distinct from the protease active site) play key roles in macromolecular substrate recognition. We investigated the importance of exosite interactions in recognition of factor IX by the protease factor XIa. Factor XIa cleavage of the tripeptide substrate S2366 was inhibited by the active site inhibitors p-aminobenzamidine (Ki 28 +/- 2 microM) and aprotinin (Ki 1.13 +/- 0.07 microM) in a classical competitive manner, indicating that substrate and inhibitor binding to the active site was mutually exclusive. In contrast, inhibition of factor XIa cleavage of S2366 by factor IX (Ki 224 +/- 32 nM) was characterized by hyperbolic mixed-type inhibition, indicating that factor IX binds to free and S2366-bound factor XIa at exosites. Consistent with this premise, inhibition of factor XIa activation of factor IX by aprotinin (Ki 0.89 +/- 0.52 microM) was non-competitive, whereas inhibition by active site-inhibited factor IXa beta was competitive (Ki 0.33 +/- 0.05 microM). S2366 cleavage by isolated factor XIa catalytic domain was competitively inhibited by p-aminobenzamidine (Ki 38 +/- 14 microM) but was not inhibited by factor IX, consistent with loss of factor IX-binding exosites on the non-catalytic factor XI heavy chain. The results support a model in which factor IX binds initially to exosites on the factor XIa heavy chain, followed by interaction at the active site with subsequent bond cleavage, and support a growing body of evidence that exosite interactions are critical determinants of substrate affinity and specificity in blood coagulation reactions.     

Inactivation of human plasma kallikrein and factor XIa by protein C inhibitor

The inhibition of kallikrein and factor XIa by protein C inhibitor (PCI) was studied. The method of Suzuki et al. [Suzuki, K., Nishioka, J., & Hashimoto, S. (1983) J. Biol. Chem. 258, 163-168] for the purification of PCI was modified in order to avoid the generation of proteolytic activity and subsequent inactivation of PCI. With the use of soybean trypsin inhibitor, an efficient inhibitor of kallikrein and factor XIa, the generation of proteolytic activity was avoided. The kinetics for the inactivation of activated protein C (APC), kallikrein, and factor XIa by PCI were determined. In the absence of heparin, no inactivation of APC was observed, in contrast to kallikrein and factor XIa, which are inhibited with second-order rate constants of (11 +/- 4) X 10(4) and (0.94 +/- 0.07) X 10(4) M-1 s-1, respectively. Addition of heparin potentiated the inhibition of APC [(1.2 +/- 0.2) X 10(4) M-1 s-1] and factor XIa [(9.1 +/- 0.7) X 10(4) M-1 s-1] by PCI, whereas the inhibition of kallikrein by PCI was unchanged [(10 +/- 1) X 10(4) M-1 s-1]. The second-order rate constants for the inhibition of kallikrein or factor XIa by PCI were similar to the second-order rate constants for the inhibition of their isolated light chains by PCI, indicating a minor role for the heavy chains of both molecules in the inactivation reactions. With sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and immunoblotting, complex formation of APC, kallikrein, and factor XIa with PCI could be demonstrated. APC and kallikrein formed 1:1 molar complexes with PCI.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Amyloid Precursor Protein/Protease Nexin 2 Kunitz Inhibitor Domain Is a Highly Specific Substrate of Mesotrypsin

The amyloid precursor protein (APP) is a ubiquitously expressed transmembrane adhesion protein and the progenitor of amyloid-β peptides. The major splice isoforms of APP expressed by most tissues contain a Kunitz protease inhibitor domain; secreted APP containing this domain is also known as protease nexin 2 and potently inhibits serine proteases, including trypsin and coagulation factors. The atypical human trypsin isoform mesotrypsin is resistant to inhibition by most protein protease inhibitors and cleaves some inhibitors at a substantially accelerated rate. Here, in a proteomic screen to identify potential physiological substrates of mesotrypsin, we find that APP/protease nexin 2 is selectively cleaved by mesotrypsin within the Kunitz protease inhibitor domain. In studies employing the recombinant Kunitz domain of APP (APPI), we show that mesotrypsin cleaves selectively at the Arg¹⁵-Ala¹⁶ reactive site bond, with kinetic constants approaching those of other proteases toward highly specific protein substrates. Finally, we show that cleavage of APPI compromises its inhibition of other serine proteases, including cationic trypsin and factor XIa, by 2 orders of magnitude. Because APP/protease nexin 2 and mesotrypsin are coexpressed in a number of tissues, we suggest that processing by mesotrypsin may ablate the protease inhibitory function of APP/protease nexin 2 in vivo and may also modulate other activities of APP/protease nexin 2 that involve the Kunitz domain.     

Functional and structural similarities between protease nexin I and C1 inhibitor

Protease nexin I is a proteinase inhibitor that is secreted by human fibroblasts and forms stable complexes with certain serine proteinases; the complexes then bind to the fibroblasts and are rapidly internalized and degraded. In this report, we show that this inhibitor, which is present in very low concentrations in plasma, has functional and structural similarities to C1 inhibitor, an abundant proteinase inhibitor in plasma. Both inhibitors complex and inactivate certain proteinases that previously were known to rapidly react only with C1 inhibitor. Kinetic inhibition studies show that protease nexin I inhibits Factor XIIa and plasma kallikrein with second-order rate constants of 2.3 x 10(3) and 2.5 x 10(5) M-1 s-1, respectively, which are similar to the rate constants for inhibition of these proteinases by C1 inhibitor. Protease nexin I inhibits C1s about one-tenth as rapidly as does C1 inhibitor. Alignment of the amino acid sequences of protease nexin I and C1 inhibitor shows that these proteins have similarity at their reactive centers (from sites P7 to P1). The remaining regions of the two proteins share much less similarity. In contrast to protease nexin I, C1 inhibitor is not secreted by human fibroblasts. Although 125I-C1s-protease nexin I complexes readily bind to human fibroblasts, binding of 125I-C1s-C1 inhibitor complexes or other 125I-proteinase-C1-inhibitor complexes to these cells is not detectable. Thus, protease nexin I and C1 inhibitor may control some common regulatory proteinases in the extravascular and vascular compartments, respectively.     

Characterization of Novel Forms of Coagulation Factor XIa: independence of factor XIa subunits in factor IX activation

Factor XI is the zymogen of a dimeric plasma protease, factor XIa, with two active sites. In solution, and during contact activation in plasma, conversion of factor XI to factor XIa proceeds through an intermediate with one active site (1/2-FXIa). Factor XIa and 1/2-FXIa activate the substrate factor IX, with similar kinetic parameters in purified and plasma systems. During hemostasis, factor IX is activated by factors XIa or VIIa, by cleavage of the peptide bonds after Arg145 and Arg180. Factor VIIa cleaves these bonds sequentially, with accumulation of factor IX alpha, an intermediate cleaved after Arg145. Factor XIa also cleaves factor IX preferentially after Arg145, but little intermediate is detected. It has been postulated that the two factor XIa active sites cleave both factor IX peptide bonds prior to releasing factor IX abeta. To test this, we examined cleavage of factor IX by four single active site factor XIa proteases. Little intermediate formation was detected with 1/2-FXIa, factor XIa with one inhibited active site, or a recombinant factor XIa monomer. However, factor IX alpha accumulated during activation by the factor XIa catalytic domain, demonstrating the importance of the factor XIa heavy chain. Fluorescence titration of active site-labeled factor XIa revealed a binding stoichiometry of 1.9 +/- 0.4 mol of factor IX/mol of factor XIa (Kd = 70 +/- 40 nm). The results indicate that two forms of activated factor XI are generated during coagulation, and that each half of a factor XIa dimer behaves as an independent enzyme with respect to factor IX.     

Protease nexin. Properties and a modified purification procedure

The present paper describes chemical and functional properties of protease nexin, a serine protease inhibitor released from cultured human fibroblasts. It is shown that protease nexin is actually synthesized by fibroblasts and represents about 1% of their secreted protein. Analysis of the amino acid composition of purified protease nexin indicates that it is evolutionarily related to antithrombin III and heparin cofactor II. Protease nexin contains approximately 6% carbohydrate, with 2.3% amino sugar, 1.1% neutral sugar, and 3.0% sialic acid. The Mr calculated from equilibrium sedimentation analysis is 43,000. Protease nexin is a broad specificity inhibitor of trypsin-like serine proteases. It reacts rapidly with trypsin (kassoc = 4.2 +/- 0.4 X 10(6) M-1 s-1), thrombin (kassoc = 6.0 +/- 1.3 X 10(5) M-1 s-1), urokinase (kassoc = 1.5 +/- 0.1 X 10(5) M-1 s-1), and plasmin (kassoc = 1.3 +/- 0.1 X 10(5) M-1 s-1), and slowly inhibits Factor Xa and the gamma subunit of nerve growth factor but does not inhibit chymotrypsin-like proteases or leukocyte elastase. In the presence of heparin, protease nexin inhibits thrombin at a nearly diffusion-controlled rate. Two heparin affinity classes of protease nexin can be detected. The present characterization pertains to the fraction of protease nexin having the higher affinity for heparin. The low affinity material, which is the minor fraction, is lost during purification.     

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.     

Heparin is a major activator of the anticoagulant serpin, protein Z-dependent protease inhibitor

Protein Z-dependent protease inhibitor (ZPI) is a recently identified member of the serpin superfamily that functions as a cofactor-dependent regulator of blood coagulation factors Xa and XIa. Here we provide evidence that, in addition to the established cofactors, protein Z, lipid, and calcium, heparin is an important cofactor of ZPI anticoagulant function. Heparin produced 20-100-fold accelerations of ZPI reactions with factor Xa and factor XIa to yield second order rate constants approaching the physiologically significant diffusion limit (k(a) = 10(6) to 10(7) M(-1) s(-1)). The dependence of heparin accelerating effects on heparin concentration was bell-shaped for ZPI reactions with both factors Xa and XIa, consistent with a template-bridging mechanism of heparin rate enhancement. Maximal accelerations of ZPI-factor Xa reactions required calcium, which augmented the heparin acceleration by relieving Gla domain inhibition as previously shown for heparin bridging of the antithrombin-factor Xa reaction. Heparin acceleration of both ZPI-protease reactions was optimal at heparin concentrations and heparin chain lengths comparable with those that produce physiologically significant rate enhancements of other serpin-protease reactions. Protein Z binding to ZPI minimally affected heparin rate enhancements, indicating that heparin binds to a distinct site on ZPI and activates ZPI in its physiologically relevant complex with protein Z. Taken together, these results suggest that whereas protein Z, lipid, and calcium cofactors promote ZPI inhibition of membrane-associated factor Xa, heparin activates ZPI to inhibit free factor Xa as well as factor XIa and therefore may play a physiologically and pharmacologically important role in ZPI anticoagulant function.     

Glial-derived neurite-promoting factor is a slow-binding inhibitor of trypsin, thrombin, and urokinase

Glial-derived neurite-promoting factor was found to be a slow-binding inhibitor of trypsin, urokinase, and thrombin. The kinetic mechanism of the inhibition differs among the three proteases. With trypsin and urokinase, an initial protease-factor complex formed which isomerized to a tighter complex. For thrombin, however, no initial complex was kinetically observed. The dissociation constants of the equilibrium complexes of the factor with trypsin, urokinase, and thrombin were 17, 280, and 18 pM, respectively, and the apparent second-order rate constants for the interaction of the factor with these enzymes were, respectively, 4.7 X 10(6), 1.2 X 10(5), and 2.1 X 10(6) M-1S-1. Heparin increased the rate at which the factor reacted with thrombin by over 40-fold to 8.9 X 10(7) M-1S-1 and decreased the dissociation constant of the complex by over 80-fold to 0.3 pM. The values obtained for the apparent second-order rate constants when compared with the kinetics of neurite induction by the factor indicate that the neurite-promoting activity of the factor is not due to the inhibition of urokinase but could be due to the inhibition of an enzyme with a specificity similar to that of thrombin or trypsin. Comparison of the values of the apparent second-order rate constants obtained for the factor with those obtained for protease nexin suggests that these two molecules are very similar in their inhibitory properties.     

A catalytic domain exosite (Cys527-Cys542) in factor XIa mediates binding to a site on activated platelets

The zymogen, factor XI, and the enzyme, factor XIa, interact specifically with functional receptors on the surface of activated platelets. These studies were initiated to identify the molecular subdomain within factor XIa that binds to activated platelets. Both factor XIa (Ki approximately 1.4 nM) and a chimeric factor XIa containing the Apple 3 domain of prekallikrein (Ki approximately 2.7 nM) competed with [125I]factor XIa for binding sites on activated platelets, suggesting that the factor XIa binding site for platelets is not located in the Apple 3 domain which mediates factor XI binding to platelets. The recombinant catalytic domain (Ile370-Val607) inhibited the binding of [125I]factor XIa to the platelets (Ki approximately 3.5 nM), whereas the recombinant factor XI heavy chain did not, demonstrating that the platelet binding site is located in the light chain of factor XIa. A conformationally constrained cyclic peptide (Cys527-Cys542) containing a high-affinity (KD approximately 86 nM) heparin-binding site within the catalytic domain of factor XIa also displaced [125I]factor XIa from the surface of activated platelets (Ki approximately 5.8 nM), whereas a scrambled peptide of identical composition was without effect, suggesting that the binding site in factor XIa that interacts with the platelet surface resides in the catalytic domain near the heparin binding site of factor XIa. These data support the conclusion that a conformational transition accompanies conversion of factor XI to factor XIa that conceals the Apple 3 domain factor XI (zymogen) platelet binding site and exposes the factor XIa (enzyme) platelet binding site within the catalytic domain possibly comprising residues Cys527-Cys542.     

The interaction of factor XIa with activated platelets but not endothelial cells promotes the activation of factor IX in the consolidation phase of blood coagulation

We have previously shown that the zymogen factor XI (FXI) binds to activated platelets but not to human umbilical vein endothelial cells (HUVEC), a conclusion that is in conflict with previous reports stating that FXI binds to 2.7-13 x 10(6) high affinity sites per HUVEC (Berrettini, M., Schleef, R. R., Heeb, M. J., Hopmeier, P., and Griffin, J. H. (1992) J. Biol. Chem. 267, 19833-19839; Shariat-Madar, Z., Mahdi, F., and Schmaier, A. H. (2001) Thromb. Haemostasis 85, 544-551). It has also been reported that activated FXI (FXIa) binds to 1.5 x 10(6) sites per HUVEC and promotes the activation of factor IX by cell bound FXIa (Berrettini, M., Schleef, R. R., Heeb, M. J., Hopmeier, P., and Griffin, J. H. (1992) J. Biol. Chem. 267, 19833-19839). Therefore, the binding of FXIa to activated platelets was compared with FXIa binding to HUVEC and HEK293 cells immobilized on microcarrier beads. Specific and saturable zinc-dependent FXIa binding was demonstrated to 250 +/- 48 sites per activated platelet (K(D) = 1.7 +/- 0.78 nm) and 6.5 +/- 0.4 x 10(4) sites per HUVEC (K(D) = 2.4 +/- 0.5 nm), whereas no binding to HEK293 cells was detected. A titration with high molecular weight kininogen had no effect on FXIa binding to platelets, but revealed a concentration-dependent decrease in the amount of FXIa bound to HUVEC. The rate of factor IXa generation catalyzed by FXIa was unaffected by the presence of surfaces; however only the activated platelet surface protected FXIa from inhibition by protease nexin 2. The results presented here confirm the conclusion that activated platelets are procoagulant while unstimulated endothelial cells are not.     

Inhibition of factor XIa by antithrombin III

The inactivation of human factor XIa by human antithrombin III was studied under pseudo-first-order reaction conditions (excess antithrombin III) both in the absence and in the presence of heparin. The time course of inhibition was followed by using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. After electrophoresis, proteins were blotted onto nitrocellulose and stained either for glycoprotein or for antithrombin III using antibodies against antithrombin III. Concomitant with factor XIa inactivation, two new slower migrating bands, one of which represented the intermediate complex consisting of one antithrombin III complexed with factor XIa, appeared as a transient band. Complete inactivation resulted in a single band representing the complex of factor XIa with two antithrombin III molecules. Quantitative analysis of the time course of inactivation was accomplished by measurement of the disappearance of factor XIa amidolytic activity toward the chromogenic substrate S2366. Pseudo-first-order reaction kinetics were observed throughout. The rate constant of inactivation was found to be 10(3) M-1 s-1 in the absence of heparin and 26.7 X 10(3) M-1 s-1 in the presence of saturating amounts of heparin. From the kinetic data, a binding constant (Kd) of 0.14 microM was inferred for the binding of antithrombin III to heparin. The time course of inactivation and the distribution of the reaction products observed upon gel electrophoresis are best explained assuming a mechanism of inactivation in which the two active sites present in factor XIa are inhibited in random order (i.e., independent of each other) with the same rate constant of inhibition.     

Substrate-dependent modulation of the mechanism of factor XIa inhibition

Factor XIa is a serine protease which participates in both the extrinsic and intrinsic pathways of blood coagulation. In this work we used active site directed inhibitors to study the mechanism of factor IX activation by factor XIa. To this end, we developed a new sensitive method for the detection of factor IXa based on its affinity to antithrombin III. Using this assay, we found that the peptidic inhibitors, leupeptin and aprotinin, exhibited similar potencies in inhibiting factor IX activation and the cleavage of a tripeptidic chromogenic substrate by factor XIa. As expected, leupeptin and aprotinin were competitive with respect to the tripeptidic chromogenic substrate. However, the inhibition of factor IX activation was best described by mixed-type inhibition with the affinity of leupeptin and aprotinin to the factor XIa-factor IX complex only approximately 10-fold lower than their affinity toward factor XIa. These results, consistent with previous factor XI domain analyses, suggest that the active site of factor XIa does not contribute significantly to the affinity of factor XIa toward factor IX. The competitive component of the inhibition of factor IX activation suggests that binding of factor IX to factor XIa heavy chain affects the interactions of leupeptin and aprotinin with the active site.     

Role of the PH domain in regulating in vitro autophosphorylation events required for reconstitution of PDK1 catalytic activity

In addition to its catalytic domain, phosphoinsositide-dependent protein kinase-1 (PDK1) contains a C-terminal pleckstrin homology (PH) domain, which binds the membrane-bound phosphatidylinositol (3,4,5)-triphosphate [PI(3,4,5)P3] second messenger. Here, we report in vitro kinetic, phosphopeptide mapping, and oligomerization studies that address the role of the PH domain in regulating specific autophosphorylation events, which are required for PDK1 catalytic activation. First, 'inactive' unphosphorylated forms of N-terminal His6 tagged full length (His6-PDK1) and catalytic domain constructs [His6-PDK1(Delta PH)] were generated by treatment with Lambda protein phosphatase (lambda PP). Reconstitution of lambda PP-treated His6-PDK1(Delta PH) catalytic activity required activation loop Ser-241 phosphorylation, which occurred only upon trans-addition of 'active' PDK1 with an apparent bimolecular rate constant of (app)k1(S241) = 374+/-29 M(-1) s(-1). In contrast, full length lambda PP-treated His6-PDK1 catalyzed Ser-241 cis-autophosphorylation with an apparent first-order rate constant of (app)k1(S241) = (5.0+/-1.5) x 10(-4) s(-1) but remained 'inactive'. Reconstitution of lambda PP-treated His(6)-PDK1 catalytic activity occurred only when autophosphorylated in the presence of PI(3,4,5)P3 containing vesicles. PI(3,4,5)P3 binding to the PH domain activated apparent first-order Ser-241 autophosphorylation by 20-fold [(app)k1(S241) = (1.1+/-0.1) x 10(-2) s(-1)] and also promoted biphasic Thr-513 trans-autophosphorylation [(app)k2(T513) = (4.9+/-1.1) x 10(2) M(-1) s(-1) and(app)k3(T513) = (1.5+/-0.2) x 10(3) M(-1) s(-1)]. The results of mutagenesis studies suggest that Thr-513 phosphorylation may cause dissociation of autoinhibitory contacts formed between the contiguous regulatory PH and catalytic kinase domains.     

The factor IX gamma-carboxyglutamic acid (Gla) domain is involved in interactions between factor IX and factor XIa

During hemostasis, factor IX is activated to factor IXabeta by factor VIIa and factor XIa. The glutamic acid-rich gamma-carboxyglutamic acid (Gla) domain of factor IX is involved in phospholipid binding and is required for activation by factor VIIa. In contrast, activation by factor XIa is not phospholipid-dependent, raising questions about the importance of the Gla for this reaction. We examined binding of factors IX and IXabeta to factor XIa by surface plasmon resonance. Plasma factors IX and IXabeta bind to factor XIa with K(d) values of 120 +/- 11 nm and 110 +/- 8 nm, respectively. Recombinant factor IX bound to factor XIa with a K(d) of 107 nm, whereas factor IX with a factor VII Gla domain (rFIX/VII-Gla) and factor IX expressed in the presence of warfarin (rFIX-desgamma) did not bind. An anti-factor IX Gla monoclonal antibody was a potent inhibitor of factor IX binding to factor XIa (K(i) 34 nm) and activation by factor XIa (K(i) 33 nm). In activated partial thromboplastin time clotting assays, the specific activities of plasma and recombinant factor IX were comparable (200 and 150 units/mg), whereas rFIX/VII-Gla activity was low (<2 units/mg). In contrast, recombinant factor IXabeta and activated rFIX/VIIa-Gla had similar activities (80 and 60% of plasma factor IXabeta), indicating that both proteases activate factor X and that the poor activity of zymogen rFIX/VII-Gla was caused by a specific defect in activation by factor XIa. The data demonstrate that factor XIa binds with comparable affinity to factors IX and IXabeta and that the interactions are dependent on the factor IX Gla domain.