Identification of the binding site for plasma prekallikrein in human high molecular weight kininogen. A region from residues 185 to 224 of the kininogen light chain retains full binding activity

We studied the ability of fragments of the light chain of human high molecular weight kininogen to bind to plasma prekallikrein. In a competitive fluorescence polarization assay, kallikrein-cleaved light chain (light chain-2; residues 49-255), a cyanogen bromide fragment (residues 185-242), and a tryptic peptide (T-7; residues 185-224) had binding affinities of approximately 20 nM, equivalent to the value for the intact light chain (residues 1-255) of high-molecular-weight kininogen. In contrast, fragments consisting of residues 49-184 and 243-255 showed no binding activity (Kd much greater than 1,000 nM). Direct titrations of fluorescein-labeled derivatives of light chain-2 and peptide T-7 with prekallikrein confirmed that T-7 retained full binding activity for prekallikrein (Kd = 12 +/- 2 nM for labeled light chain-2; Kd = 7 +/- 1 nM for labeled T-7). These results localize the binding site of high molecular weight kininogen for prekallikrein within a region of 40 amino acids (residues 185-224) that resides in the near carboxyl terminus of the light chain of kininogen.     

Primary structure requirements for the binding of human high molecular weight kininogen to plasma prekallikrein and factor XI

We recently identified residues 185-224 of the light chain of human high molecular weight kininogen (HMWK) as the binding site for plasma prekallikrein (Tait, J.F., and Fujikawa, K. (1986) J. Biol. Chem. 261, 15396-15401). In the present study, we have further defined the primary structure requirements for binding of HMWK to factor XI and prekallikrein. In a competitive fluorescence polarization binding assay, a 31-residue synthetic peptide (residues 194-224 of the HMWK light chain) bound to prekallikrein with a Kd of 20 +/- 6 nM, indistinguishable from the previously determined value of 18 +/- 5 nM for the light chain. We also prepared three shorter synthetic peptides corresponding to different portions of the 31-residue peptide (residues 205-224, 212-224, and 194-211), but these peptides bound to prekallikrein more than 100-fold more weakly. Factor XI also bound to the same region of the HMWK light chain, but at least 58 residues (185-242) were required for optimal binding (Kd = 69 +/- 4 nM for the light chain; Kd = 130 +/- 50 nM for residues 185-242). The four synthetic peptides inhibited kaolin-activated clotting of blood plasma with potencies paralleling their affinities for prekallikrein and factor XI. Peptide 194-224 can also be used for rapid affinity purification of prekallikrein and factor XI from plasma.     

Structural Requirements for the Procoagulant Activity of Nucleic Acids

Nucleic acids, especially extracellular RNA, are exposed following tissue- or vessel damage and have previously been shown to activate the intrinsic blood coagulation pathway in vitro and in vivo. Yet, no information on structural requirements for the procoagulant activity of nucleic acids is available. A comparison of linear and hairpin-forming RNA- and DNA-oligomers revealed that all tested oligomers forming a stable hairpin structure were protected from degradation in human plasma. In contrast to linear nucleic acids, hairpin forming compounds demonstrated highest procoagulant activities based on the analysis of clotting time in human plasma and in a prekallikrein activation assay. Moreover, the procoagulant activities of the DNA-oligomers correlated well with their binding affinity to high molecular weight kininogen, whereas the binding affinity of all tested oligomers to prekallikrein was low. Furthermore, four DNA-aptamers directed against thrombin, activated protein C, vascular endothelial growth factor and nucleolin as well as the naturally occurring small nucleolar RNA U6snRNA were identified as effective cofactors for prekallikrein auto-activation. Together, we conclude that hairpin-forming nucleic acids are most effective in promoting procoagulant activities, largely mediated by their specific binding to kininogen. Thus, in vivo application of therapeutic nucleic acids like aptamers might have undesired prothrombotic or proinflammatory side effects.     

High molecular weight kininogen-binding site of prekallikrein probed by monoclonal antibodies.

A panel of monoclonal antibodies against human prekallikrein was raised in mice and characterized with respect to the major antigenic epitopes. Of 18 antibodies, nine were directed against the light chain portion performing the proteolytic function of activated kallikrein, and nine recognized the heavy chain mediating the binding of prekallikrein to high molecular weight (H-)kininogen. Among the anti-heavy chain antibodies, one (PK6) interfered with the procoagulant activity of prekallikrein, and prolonged in a concentration-dependent manner the activated partial thromboplastin time of reconstituted prekallikrein-deficient plasma (Fletcher type). Antibody PK6 was subtyped IgG1,k and had an apparent Kass of 6.8 +/- 0.44.10(8) M-1 for prekallikrein. Functional analyses revealed that PK6 does not interfere with prekallikrein activation by activated Hageman factor (beta-F XIIa), and has no effect on the kininogenase function of activated kallikrein. Monoclonal antibody PK6 but none of the other anti-heavy chain antibodies completely prevented complex formation of prekallikrein with H-kininogen, and readily dissociated preformed complexes of prekallikrein and H-kininogen. Likewise, Fab' and F(ab')2 fragments of PK6 blocked H-kininogen binding to prekallikrein. A synthetic peptide of 31 amino acid residues encompassing the entire prekallikrein binding region of H-kininogen effectively competed with PK6 for prekallikrein binding indicating that the target epitope of PK6 is juxtaposed to, if not incorporated in the H-kininogen-binding site of prekallikrein. Extensive cross-reactivity of PK6 with another H-kininogen-binding protein of human plasma, i.e. factor XI, suggested that the structure of the target epitope of PK6 is well conserved among prekallikrein and factor XI, as would be expected for the kininogen-binding site shared by the two proteins. It is anticipated that monoclonal antibody PK6 will be an important tool for the precise mapping of the hitherto unknown kininogen-binding site of prekallikrein.     

Protein-protein interactions in contact activation of blood coagulation. Characterization of fluorescein-labeled human high molecular weight kininogen-light chain as a probe

Limited proteolysis of high molecular weight kininogen by kallikrein resulted in the generation of an inactive heavy chain of Mr = 64,000 and active light chains of Mr = 64,000 and 51,000 when analyzed by sodium dodecyl sulfate (SDS)-gel electrophoresis under reducing conditions. Starting with kininogen from outdated plasma, a light chain with an apparent molecular weight of 51,000 on 7.5% SDS gels was purified and characterized. Molecular weights of 28,900 +/- 1,100 and 30,500 +/- 1,600 were obtained by gel filtration of the reduced and alkylated protein in 6 M guanidine HCl and equilibrium sedimentation under nondenaturing conditions in the air-driven ultracentrifuge, respectively. The light chain stained positively with periodic acid-Schiff reagent on SDS gels indicating that covalently attached carbohydrate may be responsible for the anomalously high molecular weight estimated by SDS-gel electrophoresis. A single light chain thiol group reacted with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) in the presence and absence of 6 M guanidine HCl. Specific fluorescent labeling of the thiol group with 5-(iodoacetamido)fluorescein (IAF) occurred without loss of clotting activity. Addition of purified human plasma prekallikrein to the IAF-light chain resulted in a maximum increase in fluorescence anisotropy of 0.041 +/- 0.001 and no change in the fluorescence intensity. Fluorescence anisotropy measurements of the equilibrium binding of prekallikrein to the IAF-light chain yielded an average Kd of 17.3 +/- 2.5 nM and stoichiometry of 1.07 +/- 0.07 mol of prekallikrein/mol of IAF-light chain. Measurements of the interaction of prekallikrein with iodoacetamide-alkylated light chain using the IAF-light chain as a probe gave an average Kd of 16 +/- 4 nM and stoichiometry of 1.0 +/- 0.2 indicating indistinguishable affinities for prekallikrein.     

Role of charged groups in factor XI/XIa activity

To elucidate the role of charged groups in expression of factor XI coagulant activity, the charged groups of purified human blood coagulation factor XI/XIa containing 125I-XI/XIa were derivatized: free amino groups by succinylation, guanido groups of arginine by reaction with phenylglyoxal hydrate, and free carboxyl groups by reaction with ethylenediamine. The modified proteins were tested for: 1) ability to adsorb to glass, 2) ability to be cleaved by trypsin or factor XII-high molecular weight kininogen, 3) coagulant activity. The amino group-modified factor XI had a significantly decreased ability to bind to glass; modification of arginine or carboxyl groups did not affect adsorption. Trypsin cleaved factor XI with modified free amino, guanido, or carboxyl groups. Factor XII-high molecular weight kininogen could cleave only the arginine-modified factor XI. Amino group-modified factor XI and carboxyl group-modified factor XI lost all their factor XI assay activity, whereas arginine-modified factor XI retained 50% of the original activity. Amino group-modified factor XI could not be activated by trypsin, but arginine-modified and carboxyl group-modified factor XI could be activated by trypsin to 50% of the original activity. Succinylation of the amino groups of factor XIa destroyed all its factor XIa activity. Arginine-modified and carboxyl group-modified factor XIa retained 50% of their factor XIa activity. We conclude that epsilon-amino groups are essential for adsorption; activation by factor XII-high molecular weight kininogen requires free amino and carboxyl but not guanido groups; free amino, carboxyl, and guanido groups in factor XIa all appear to be critical for interaction of factor XIa with factor IX.     

The role of bovine high-molecular-weight (HMW) kininogen in contact-mediated activation of bovine factor XII: interaction of HMW kininogen with kaolin and plasma prekallikrein

Previous studies from our laboratories (Sugo et al. (1980) Biochemistry 19, 3215-3220) have shown that bovine high-molecular-weight (HMW) kininogen remarkably accelerates the kaolin-mediated activation of Factor XII in the presence of prekallikrein, and that both fragment 1.2 and the light chain regions located in the COOH terminal half of the kininogen molecule are essential for the activation. In the present study, we demonstrate that the accelerating effect of HMW kininogen is mediated through its adsorption on the kaolin surface through the fragment 1.2 region and its complex formation with prekallikrein through the light chain region. The evidence is as follows: 1. HMW kininogen radio-labeled with 125I was adsorbed on kaolin and the adsorption was inhibited by the prior treatment of kaolin with fragment 1.2, fragment 1.2-light chain, kinin-free protein or HMW kininogen, but not with kinin- and fragment 1.2-free protein, light chain or low molecular-weight (LMW) kininogen. 2. The complex formation of HMW kininogen with prekallikrein in bovine plasma or in the purified system was examined by gel-filtration on a column of Sephacryl S-200 In bovine plasma, prekallikrein was eluted in the same fraction as HMW kininogen, showing an apparent molecular weight of 250,000, whereas purified prekallikrein was eluted in the fraction corresponding to an apparent molecular weight of 100,000. When purified prekallikrein was mixed with purified HMW kininogen in a mol ratio of 1 to 2, all prekallikrein was found to be associated with HMW kininogen. Furthermore, purified prekallikrein mixed with kininogen derivatives, such as kinin- and fragment 1.2-free protein, fragment 1.2-light chain or light chain, was eluted in the higher molecular weight fraction. HMW kininogen did not form a complex with prekallikrein. Using the same technique, it was shown that kinin- and fragment 1.2-free protein forms a complex not only with prekallikrein but also with kallikrein.     

Mapping of the prekallikrein-binding site of human H-kininogen by ligand screening of lambda gt11 expression libraries. Mimicking of the predicted binding site by anti-idiotypic antibodies.

High molecular weight (H-)kininogen, a non-enzymatic cofactor of the contact activation system, has on the COOH-terminal part of its light chain a unique binding site which complexes prekallikrein or factor XI with high affinity and specificity. In a conventional protein fragmentation approach, the prekallikrein-binding site was mapped to positions 556-595 of the human H-kininogen sequence (Tait, J. F., and Fujikawa, K. (1986) J. Biol. Chem. 261, 15396-15401). To gain more insight into the minimum structural requirements of the prekallikrein-binding site, we have developed an alternative strategy employing the lambda gt11 expression cloning system. A ligand assay was established which probes for the binding site in H-kininogen or recombinant fusion proteins thereof by complexation with prekallikrein, followed by a specific antibody against prekallikrein and a secondary labeled antibody. A cDNA library constructed in lambda gt11 from random fragments of a cDNA clone encoding the COOH-terminal part of the kininogen light chain was screened by the ligand assay, and 17 positive clones were identified. Analysis of their inserted cDNA sequences revealed a consensus sequence of 119 nucleotides which maps to the extreme 3' end (positions 1759-1877) of the coding part of the prekininogen mRNA. The consensus sequence encodes positions 569-607 of the kininogen light chain and overlaps by 27 residues (positions 569-595) with the binding segment identified previously by the fragment approach. Analysis of successively shortened peptides revealed that the common segment of 27 residues but not truncated versions thereof contains the essential structural elements for prekallikrein binding. This conclusion was corroborated by the finding that anti-idiotypic antibodies toward a monoclonal antibody directed to the binding segment of 27 residues bear internal image(s) of the binding site of H-kininogen. It is pointed out that the methodology described in this study may prove generally useful in the cloning and mapping of high affinity binding sites of proteins.     

Molecular weight of human high-molecular-weight kininogen light chain by equilibrium sedimentation in an air-driven ultracentrifuge

High-molecular-weight kininogen, a nonenzymatic glycoprotein of the intrinsic blood coagulation system, is proteolytically cleaved by kallikrein as an early event in the activation of this system. The light chain of cleaved kininogen retains the ability to form specific noncovalent complexes with prekallikrein and factor XI, other members of this system. We have determined the molecular weight of human kininogen light chain by equilibrium sedimentation in buffers of differing density, using an air-driven benchtop ultracentrifuge. The resulting molecular weight (30,500 +/- 800 g/mol) and partial specific volume (0.660 +/- 0.008 ml/g) are consistent with the idea that a sizeable fraction of the carbohydrate of high-molecular-weight kininogen is associated with the light chain. This level of precision is relatively easy to attain. The procedures are detailed, along with expressions for error propagation, to permit ready application of the technique.     

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.     

Human high molecular weight kininogen. Studies of structure-function relationships and of proteolysis of the molecule occurring during contact activation of plasma.

Human high Mr kininogen was purified from normal plasma in 35% yield. The purified high Mr kininogen appeared homogeneous on polyacrylamide gels in the presence of sodium dodecyl sulfate and mercaptoethanol and gave a single protein band with an apparent Mr = 110,000. Using sedimentation equilibrium techniques, the observed Mr was 108,000 +/- 2,000. Human plasma kallikrein cleaves high Mr kininogen to liberate kinin and give a kinin-free, two-chain, disulfide-linked molecule containing a heavy chain of apparent Mr = 65,000 and a light chain of apparent Mr = 44,000. The light chain is histidine-rich and exhibits a high affinity for negatively charged materials. The isolated alkylated light chain quantitatively retains the procoagulant activity of the single-chain parent molecule. 125I-Human high Mr kininogen undergoes cleavage in plasma during contact activation initiated by addition of kaolin. This cleavage, which liberates kinin and gives a two-chain, disulfide-linked molecule, is dependent upon the presence of prekallikrein and Factor XII (Hageman factor) in plasma. Addition of purified plasma kallikrein to normal plasma or to plasmas deficient in prekallikrein or Factor XII in the presence or absence of kaolin results in cleavage of high Mr kininogen and kinin formation.     

Localization of the high molecular weight kininogen binding site in the heavy chain of human factor XI to amino acids phenylalanine 56 through serine 86

We have previously demonstrated that a monoclonal antibody (5F7) directed against the heavy chain region of factor XI inhibits the binding of factor XI to high molecular weight kininogen (high Mr kininogen) and the surface-mediated proteolytic activation of factor XI by factor XIIa in the presence of high Mr kininogen. In order to identify the structural domain of factor XI that binds high Mr kininogen, CNBr-digested factor XI was passed over a 5F7 antibody affinity column. One of two CNBr peptides that bound to this 5F7 affinity column inhibited binding of 125I-factor XI to high Mr kininogen, as did intact factor XI. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of an inhibitory peptide purified by high performance liquid chromatography revealed an Mr of 10,000-15,000. Gas-phase sequencing of this peptide revealed the following amino-terminal sequence: X-X-Val-Thr-Gln-Leu-Leu-Lys-Asp-Thr. These data together with the amino acid composition of the isolated peptide indicate that both the epitope recognized by antibody 5F7 and at least a portion of the high Mr kininogen binding site are contained within the amino-terminal portion of factor XI comprising residues Glu-1 through Met-102. Further cleavage of this peptide with o-iodosobenzoic acid at a tryptophanyl peptide bond revealed that an Mr 5,000 peptide (with the amino-terminal sequence Trp-Phe-Thr-Cys-Val-Leu) bound to a high Mr kininogen affinity column and inhibited binding of 125I-factor XI to high Mr kininogen. Finally, a synthetic peptide comprising residues Phe-56 through Ser-86 inhibited 125I-factor XI binding to high Mr kininogen. These experiments strongly suggest that the high Mr kininogen binding site is contained within the domain in the heavy chain region of factor XI comprising residues Phe-56 through Ser-86.     

Isolation and characterization of bovine plasma prekallikrein (Fletcher factor)

Prekallikrein (Fletcher factor) has been purified from bovine plasma approximately 25 000-fold with an overall yield of 14%. Purification steps included ammonium sulfate fractionation and column chromatography on heparin-agarose, DEAE-Sephadex, CM-Sephadex, benzamidine-agarose, and arginine methyl ester-agarose. The purified protein was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal sequence analysis. Bovine plasma prekallikrein is a glycoprotein with a molecular weight of 82 000 as determined by sedimentation equilibrium centrifugation. It contains 12.9% carbohydrate, including 6.2% hexose, 4.5% N-acetylglucosamine, and 2.2% N-acetylneuraminic acid. Prekallikrein is a single polypeptide chain with an amino-terminal sequence of Gly-Cys-Leu-Thr-Gln-Leu-Tyr-His-Asn-Ile-Phe-Phe-Arg-Gly-Gly. This sequence is homologous to the amino-terminal sequence of human factor XI (plasma thromboplastin antecedent). Both prekallikrein and kallikrein require kaolin to correct Fletcher factor deficient plasma. Kallikrein, however, has a specific activity 3.5 times greater than prekallikrein. Prekallikrein does not correct plasma deficient in factor XII (Hageman factor), factor XI, or high molecular weight kininogen (Fitzgerald factor).     

Protein-protein interactions in contact activation of blood coagulation. Binding of high molecular weight kininogen and the 5-(iodoacetamido) fluorescein-labeled kininogen light chain to prekallikrein, kallikrein, and the separated kallikrein heavy and light chains

Binding of the 5-(iodoacetamido)fluorescein (IAF)-labeled high molecular weight (HMW) kininogen light chain to prekallikrein and D-Phe-Phe-Arg-CH2Cl-inactivated kallikrein was monitored by a 0.040 +/- 0.002 increase in fluorescence anisotropy. Indistinguishable average dissociation constants and stoichiometries of 14 +/- 3 nM and 1.1 +/- 0.1 mol of prekallikrein/mol of IAF-light chain and 17 +/- 3 nM and 0.9 +/- 0.1 mol of kallikrein/mol of IAF-light chain were determined for these interactions at pH 7.4, mu 0.14 and 22 degrees C. Prekallikrein which had been reduced and alkylated in 6 M guanidine HCl lost the ability to increase the fluorescence anisotropy of the IAF-kininogen light chain, suggesting that the native tertiary structure was required for tight binding. The kallikrein heavy and light chains were separated on the basis of the affinity of the heavy chain for HMW-kininogen-Sepharose, after mild reduction and alkylation of kallikrein under nondenaturing conditions. Under these conditions, alkylation with iodo [14C]acetamide demonstrated that only limited chemical modification had occurred. Binding of the IAF-kininogen light chain to the isolated alkylated kallikrein heavy chain, when compared to prekallikrein and kallikrein, was characterized by an indistinguishable increase in fluorescence anisotropy, average dissociation constant of 14 +/- 3 nM, and stoichiometry of 1.2 +/- 0.1 mol of kallikrein heavy chain/mol of IAF-light chain. In contrast, no binding of the D-Phe-Phe-Arg-CH2Cl-inactivated kallikrein light chain was detected at concentrations up to 500 nM. Furthermore, 300 nM kallikrein light chain did not affect IAF-kininogen light chain binding to prekallikrein, kallikrein, or the kallikrein heavy chain. The binding of monomeric single chain HMW-kininogen to prekallikrein, kallikrein, and the kallikrein heavy and light chains was studied using the IAF-kininogen light chain as a probe. Analysis of the competitive binding of HMW-kininogen gave average dissociation constants and stoichiometries of 12 +/- 2 nM and 1.2 +/- 0.1 mol of prekallikrein/mol of HMW-kininogen, 15 +/- 2 nM and 1.3 +/- 0.1 mol of kallikrein/mol of HMW-kininogen, 14 +/- 3 nM and 1.4 +/- 0.2 mol of kallikrein heavy chain/mol of HMW-kininogen, and no detectable effect of 300 nM kallikrein light chain on these interactions. We conclude that a specific, nonenzymatic interaction between sites located exclusively on the light chain of HMW-kininogen and the heavy chain of kallikrein or prekallikrein is responsible for the formation of 1:1 noncovalent complexes between these proteins.     

Mapping of the discontinuous H-kininogen binding site of plasma prekallikrein. Evidence for a critical role of apple domain-2.

Plasma prekallikrein, a zymogen of the contact phase system, circulates in plasma as heterodimeric complex with H-kininogen. The binding is mediated by the prekallikrein heavy chain consisting of four apple domains, A1 to A4, to which H-kininogen binds with high specificity and affinity (K(D) = 1.2 x 10(-8) M). Previous work had demonstrated that a discontinuous kininogen-binding site is formed by a proximal part located in A1, a distal part exposed by A4, and other yet unidentified portion(s) of the kallikrein heavy chain. To detect relevant binding segment(s) we recombinantly expressed single apple domains and found a rank order of binding affinity for kininogen of A2 > A4 approximately A1 > A3. Removal of single apple domains in prekallikrein deletion mutants reduced kininogen binding by 21 (A1), 64 (A2), and 24% (A4), respectively, whereas deletion of A3 was without effect. Transposition of homologous A2 domain from prekallikrein to factor XI conferred high-affinity kininogen binding from the former to the latter. The principal role of A2 for H-kininogen docking to the prekallikrein heavy chain was further substantiated by the finding that cleavage of a single peptide bond in A2 drastically diminished the H-kininogen binding affinity. Furthermore, the epitope of monoclonal antibody PKH6 which blocks kallikrein-kininogen complex formation with an IC(50) of 8 nM mapped to the center portion of domain A2. Our data indicate that domain A2 and two flanking sequence segments of A1 and A4 form a discontinuous binding platform for H-kininogen on the prekallikrein heavy chain. Domain-specific antibodies directed to these critical sites efficiently interfered with contact phase-induced bradykinin release from H-kininogen.     

Mapping of functional domains of human high molecular weight and low molecular weight kininogens using murine monoclonal antibodies

Thirty-four monoclonal antibodies directed against human high molecular weight (HMW) and low molecular weight (LMW) kininogens and their derivatives were obtained, and the specificities of the antibodies were assayed by enzyme-linked immunosorbent assay (ELISA). By use of HMW kininogen, kinin-free HMW kininogen, kinin-free and fragment 1.2 (fr 1.2) free HMW kininogen, fr 1.2-light chain of HMW kininogen, LMW kininogen, kinin-free LMW kininogen, heavy chain of LMW kininogen, and light chain of LMW kininogen, the monoclonal antibodies were characterized and classified into four groups: (A) 20 monoclonal antibodies reacting with only the heavy chain, a common region of HMW and LMW kininogens; each of these monoclonal antibodies possessed the specificity to domain 1 (2 monoclonal antibodies), domain 2 (2 monoclonal antibodies), domain 3 (7 monoclonal antibodies), and both domains 2 and 3 (7 monoclonal antibodies) of the heavy chain; (B) 7 monoclonal antibodies reacting with fr 1.2, a unique histidine-rich region; (C) 5 monoclonal antibodies reacting with the light chain of HMW kininogen; (D) 2 monoclonal antibodies reacting with the light chain of LMW kininogen. Two monoclonal antibodies in the first group (group A), designated HKG H7 and H12, effectively suppressed the thiol proteinase inhibitor activity of HMW kininogen to papain and calpains and of LMW kininogen to papain, but the others did not affect it. Further, all the monoclonal antibodies which recognized the fr 1.2 or light chain of HMW kininogen (groups B and C) suppressed the clotting activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activated platelets but not endothelial cells participate in the initiation of the consolidation phase of blood coagulation

To address the question of whether initiation of the consolidation phase of coagulation occurs on platelets or on endothelium, we have examined the interaction of coagulation factor XI with human umbilical vein endothelial cells (HUVEC) and with platelets. In microtiter wells factor XI binds to more sites in the absence of HUVEC (1.8 x 10(10) sites/well, K(D) = 2.6 nm) than in their presence (1.3 x 10(10) sites/well, K(D) = 12 nm) when high molecular weight kininogen (HK) and zinc are present. Binding was volume-dependent and abrogated by HUVEC or Chinese hamster ovary cells and was a function of nonspecific binding of HK to the artificial plastic surface. Factor XI did not bind to HUVEC or to HEK293 cell monolayers anchored to microcarrier beads. Activation of HUVEC resulted in von Willebrand's factor secretion, but factor XI binding was not observed. Only activated platelets supported factor XI binding in the presence of HK and zinc (K(D) = 8 nm, B(max) = 1319 sites/cell). Activation of factor XI was observed in plasma in the presence of platelets activated by the thrombin receptor activation peptide but not with activated HUVEC. These results support the concept that activated platelets, but not endothelial cells, expose a procoagulant surface for binding and activating factor XI, thereby initiating the consolidation phase of coagulation.     

Interaction of factor XII, high-molecular-weight (HMW) kininogen and prekallikrein with sulfatide: analysis by fluorescence polarization

The interaction of high-molecular-weight (HMW) kininogen, Factor XII and prekallikrein with sulfatide was studied by fluorescence polarization. Fluorescein-conjugated derivatives of HMW kininogen, Factor XII and prekallikrein were prepared by reacting the purified bovine factors with fluorescein isothiocyanate (FITC). The apparent dissociation constant (Kd) for the binding of FITC-labeled HMW kininogen (F-HMW kininogen) with sulfatide was calculated to be 3.2 (+/- 0.3) X 10(-8) M. This binding was partially inhibited by three kininogen derivatives, fragment 1 X 2, kinin-free protein and fragment 1 X 2-light chain, but not by kinin and fragment 1 X 2-free protein. In the presence of Factor XII, the binding of F-HMW kininogen with sulfatide was strongly inhibited, suggesting that the zymogen and the protein cofactor compete for the same or a closely related binding site on the sulfatide surface. In contrast, the binding of FITC-labeled Factor XII (F-Factor XII) with sulfatide was weakly inhibited by HMW kininogen but not by prekallikrein. The Kd value for binding of F-Factor XII with sulfatide was calculated to be 2.0 (+/- 0.3) X 10(-8) M. F-Prekallikrein did not interact with sulfatide. Moreover, the fluorescence polarization value of F-HMW kininogen decreased in the presence of prekallikrein, leveling off at a one-to-one molar ratio of prekallikrein to F-HMW kininogen. The Kd value for binding of F-HMW kininogen-light chain (F-light chain) with prekallikrein was calculated to be 3.8 (+/- 0.6) X 10(-8) M and the stoichiometry was estimated as 1 to 1.2 on a molar basis from the Scatchard plot.     

Comparative studies of measuring condition of activated partial thromboplastin time and contact factors in experimental animals

For establishing the optimal incubation time (OIT) for measurement of the activated partial thromboplastin time (APTT) in dogs, rabbits, guinea pigs, rats and mice, we determined the shortest clotting time of the plasma from each animal species and compared them with that of human plasma. The OIT for APTT determination was 15 to 30 sec in guinea pigs, rats and mice and 5 to 10 minutes in dogs and rabbits. The mouse APTT (about 30 sec) with the OIT thus determined was similar to human APTT, and relatively longer than APTT in other animal species (10-20 sec). To elucidate the mechanism of the species differences in OIT, we examined the plasma of each animal species for the activity of the contact factors such as factor XII, factor XI, high molecular weight kininogen (HMWK) and prekallikrein (PK) and their effect on the coagulation of contact factor-deficient plasma. The total activity of contact factors was higher in dogs and guinea pigs and lower in rabbits and mice than that in humans. Species difference with the factor XII, Factor XI and HMWK was noted in clotting time but not in OIT. These results suggest that the species difference in OIT for APTT is probably due to difference in activity of the plasma contact factors and in the mode of coagulation for each contact factor.     

Calorimetric and spectroscopic examination of the solution phase structures of prekallikrein binding domain peptides of high molecular weight kininogen

Unique sequence-binding sites are exposed on the surface of high molecular weight kininogen which complex prekallikrein or factor XI with high affinity and specificity. A sequence comprising 31 residues of the mature kininogen molecule (Asp565-Lys595) retains full binding activity for prekallikrein (K _D =20 nM) and assumes a complex folded structure in solution which is stabilized by long-range interactions between N- and C-terminal residues. The sequence Trp569-Lys595 (27 residues) shows only 28% of this binding affinity and lacks the key structural features required for protein recognition (Scarsale, J. N., and Harris, R. B.,J. Prot. Chem. 9, 647–659, 1990). We were thus able to predict that N- or C-terminal truncations of the binding-site sequence would disrupt the conformational integrity required for binding. Two new peptides of 20- and 22- residues have now been synthesized and their solution phase structures examined. These peptides are N- and C-terminal truncations, respectively, of the 27-residue sequence and correspond to the sequences Asp576-Lys595 and Trp569-Asp590 of high molecular weight kininogen. The results of fluorescence emission and circular dichroism (CD) spectroscopies in the range 25–90°C and from differential scanning calorimetry (DSC) all substantiate the idea that the C-terminal truncation peptide binds prekallikrein 35-fold poorer than the 31-residue peptide because it is relatively unoredered and possesses a less stable structure. Surprisingly, the N-terminal truncation peptide (20-mer) shows structural stability even at elevated temperatures and, like the 31-residue peptide, undergoes cold-induced denaturation observable in the DSC. 2D-NMR analysis of the 20-residue peptide revealed two distinct structures; one conformer possesses a more compact, folded structure than the other. However, the predicted structures assumed by either conformer are very different from those of either the 31- or 27-residue peptides. Hence, the binding affinity of the 20-residue peptide is 60-fold poorer than that for the 31-residue peptide because it assumes a nonproductive binding conformation(s).