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.     

Human plasma prekallikrein. Immunoaffinity purification and activation to alpha- and beta-kallikrein

Prekallikrein was purified from human plasma with a final yield of 76% using as the principal step adsorption to immobilized chicken antikallikrein IgY. When purified prekallikrein (3.4 microM) was incubated in the presence of beta-Factor XIIa (0.068 microM) for 5 min at 37 degrees C and pH 7.5, alpha-kallikrein was obtained. Upon prolonged incubation (0.5-28 h), the Mr 52,000 heavy chain of alpha-kallikrein was progressively cleaved, resulting in the formation of beta-kallikrein. The formation of beta-kallikrein was characterized as an autolytic process because it was prevented by specific inhibitors of kallikrein, including aprotinin and antikallikrein antibody but not by corn trypsin inhibitor, an inhibitor specific for beta-Factor XIIa.     

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.     

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.     

Characterization of the H-kininogen-binding site on factor XI: a comparison of factor XI and plasma prekallikrein.

Factor XI (FXI), the zymogen of the blood coagulation protease FXIa, and the structurally homologous protein plasma prekallikrein circulate in plasma in noncovalent complexes with H-kininogen (HK). HK binds to the heavy chains of FXI and of prekallikrein. Each chain contains four apple domains (F1-F4 for FXI and P1-P4 for prekallikrein). Previous studies indicated that the HK-binding site on FXI is located in F1, whereas the major HK-binding site on prekallikrein is in P2. To determine the contribution of each FXI apple domain to HK-FXI complex formation, we examined binding of recombinant single apple domain-tissue plasminogen activator fusion proteins to HK. The order of affinity from highest to lowest is F2 F4 > F1 F3. Monoclonal antibodies against F2 are superior to F4 or F1 antibodies as inhibitors of HK binding to FXI. Antibody alphaP2, raised against prekallikrein, cross-reacts with FXI F2 and inhibits FXI-HK binding with an IC(50) of 8 nm. HK binding to a platelet-specific FXI variant lacking the N-terminal half of F2 is reduced > 5-fold compared with full-length FXI. A chimeric FXI molecule in which F2 is replaced by P2 is cleaved within P2 during activation by factor XIIa, resulting in greatly reduced HK binding capacity. In contrast, wild-type FXI is not cleaved within F2, and its binding capacity for HK is unaffected by factor XIIa. Our data show that HK binding to FXI involves multiple apple domains, with F2 being most important. The findings demonstrate a similarity in mechanism for FXI and prekallikrein binding to HK.     

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.     

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.     

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.     

Identification and characterization of prolylcarboxypeptidase as an endothelial cell prekallikrein activator

Our recent investigations have postulated a human umbilical vein endothelial cell (HUVEC)-associated prekallikrein activator (PKA). When prekallikrein (PK) assembles on high molecular weight kininogen on HUVEC, PK is activated to kallikrein. PKA was found in the 15,800 x g pellet of HUVEC lysates using an assay that measures PK activation only when bound to high molecular weight kininogen linked to microtiter plates. Sequential DEAE, wheat germ lectin affinity, and hydroxyapatite chromatography resulted in four protein bands on SDS-PAGE. One protein in the 73-kDa band was identified by amino acid sequencing as prolylcarboxypeptidase (PRCP). On gel filtration, PKA activity was a single homogenous peak identical in migration to the 73-kDa immunoblot of PRCP. Anti-PRCP inhibits PKA activity and PK activation on HUVEC. Purified PKA was blocked by diisopropyl fluorophosphate (1 mm), phenylmethylsulfonyl fluoride (3 mm), leupeptin (100 microm), antipain (IC(50) = 2 microm), HgCl(2) (IC(50) = 500 microm), Z-Pro-Pro-aldehyde-dimethyl acetate (IC(50) = 1 microm), and corn trypsin inhibitor (IC(50) = 40 nm). PKA did not correct the coagulant defect in factor XII deficient plasma, was purified from HUVEC cultured in factor XII-deficient serum, was not detected by antibody to factor XII, did not activate FXI, and was not inhibited by a neutralizing antibody to FXII. Angiotensin II (IC(50) = 2 microm) or bradykinin (IC(50) = 100 microm), but not angiotensin II-(1-7) or bradykinin(1-5), and the prolyl oligopeptidase inhibitor Fmoc-Ala-Pyr-CN (IC(50) = 50 nm) also blocked purified PKA activation of PK. The K(m) of PK activation by PRCP is 6.7 nm. PRCP antigen is present on the membrane of fixed but not permeabilized HUVEC. PRCP appears to be a HUVEC-associated PK activator.     

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.     

Platelet glycoprotein Ib: a zinc-dependent binding protein for the heavy chain of high-molecular-weight kininogen.

Domains 3 and 5 of high-molecular-weight kininogen (HK) have been shown to bind to platelets in a zinc-dependent reaction. However, the platelet-binding proteins responsible for this interaction have not been identified. We have focused on the platelet-binding site for the heavy chain (domain 3), which we approached using a domain 3-derived peptide ligand and isolated binding proteins by affinity chromatography. The domain 3-derived peptide, thrombin, HK, factor XII, as well as antibody to glycocalicin (the N-terminal portion of the alpha chain of GPIb) recognized a protein at 74 kD. We also isolated the thrombin receptor (PAR 1) at 45 kD, however, none of the above-mentioned ligands bound to this protein. Isolation of platelet membrane proteins using a monoclonal anti-glycocalicin antibody column revealed the same HK binding protein at 74 kD, which was reactive with anti-GPIb and represents a GPIb fragment. By photoaffinity labeling, HK interacted with membrane GPIb, which was then isolated in native form (135 kD) along with gC1qR, a ligand for the HK light chain. Finally, (125)I-HK binding to platelets was significantly inhibited by the anti-GPIb antibody. These results suggest that the GPIb alpha chain, a known thrombin binding protein, is also one of the zinc-dependent platelet membrane binding sites for HK domain 3.     

Hageman factor substrates. Human plasma prekallikrein: mechanism of activation by Hageman factor and participation in hageman factor-dependent fibrinolysis.

Two molecular forms of prekallikrein can be isolated from pooled normal human plasma. Their approximate molecular weights by sodium dodecyl sulfate-gel electrophoresis are 88,000 and 85,000. The two bands observed are shown to represent prekallikrein by functional, immunochemical, and structural criteria. Both forms are cleaved by activated Hageman factor, they appear to share antigenic determinants, they are not interconvertible upon incubation with activated Hageman factor or kallikrein, and the ratio of kinin-generating, and plasminogen-activating activities of the preparations are independent of the relative proportion of each band. Activated Factor XII converts prekallikrein to kallikrein by limited proteolysis and two disulfide-linked chains designated kallikrein heavy chain (Mr = 52,000) and kallikrein light chains (Mr = 36,000 or 33,000) are formed. The active site is associated with the light chains as assessed by incorporation of [3H]diisopropyl fluorophosphate. No dissociable fragments were observed in the absence of reducing agents. However, kallikrein could digest prekallikrein to diminish its molecular weight by 10,000. In addition, two factors capable of activating plasminogen to plasmin have been isolated; one is identified as kallikrein. The second principle fractionates with Factor XI and is demonstrable in normal and prekallikrein-deficient plasma.     

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 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.     

Evidence that the platinum-reactive methionyl residue of the alpha 2-macroglobulin receptor recognition site is not in the carboxyl-terminal receptor binding domain

Digestion of human alpha 2-macroglobulin-methylamine (alpha 2M-CH3NH2) with papain prior to gel filtration resulted in the resolution of three distinct peaks. The material in peak I (Mr approximately 600,000) and peak II (Mr approximately 55,000) did not have any receptor binding ability as determined by in vivo clearance studies and in vitro competitive binding studies using mouse peritoneal macrophages. In contrast, the material in peak III (Mr approximately 20,000) bound to macrophage alpha 2-macroglobulin (alpha 2M) receptors with a Kd of 250 nM. This represents a 500-fold decrease in affinity relative to undigested alpha 2M-CH3NH2. Sequence analysis demonstrated that this material constituted the carboxyl-terminal fragment (COOH-terminal fragment) of alpha 2M. alpha 2M is known to possess a methionyl residue which is susceptible to modification by cis-dichlorodiammineplatinum (II) (cis-DDP) with the result being a loss of receptor binding ability by alpha 2M. For this reason, experiments were performed to determine if the platinum-reactive methionyl residue is located in the COOH-terminal receptor binding fragment of alpha 2M. The results of this investigation demonstrate that cis-DDP is not reactive with either the isolated COOH-terminal fragment or the COOH-terminal fragment isolated from alpha 2M-CH3NH2 which had been pretreated with cis-DDP. In addition, the COOH-terminal fragment did not bind to monoclonal antibody 7H11D6, a monoclonal antibody which binds to the platinum-reactive epitope of the alpha 2M-CH3NH2 receptor recognition site. In contrast, the 55-kDa fragment of alpha 2M bound approximately 1 mol platinum/mol of 55-kDa fragment and also bound to monoclonal antibody 7H11D6. Since the COOH-terminal fragment retains some receptor binding ability, the results of this investigation demonstrate that this fragment is not the complete receptor recognition site and suggest that a platinum-reactive methionyl residue located in the 55-kDa fragment of alpha 2M is another component of this site.     

Use of chromogenic substrates in the clarification of disorders in the early stages of blood coagulation

The kallikrein specific chromogenic peptide substrates S-2302 (KABI) and Chromozym PK (Boehringer) were used in the first analysis of a familial defect in the early stage of clotting. Slight to extensive prolongation of the activated partial thromboplastin time was seen in the affected persons. Using dextransulfate for activation of plasma marked deficiency in kallikrein activity was found in 3 persons. Using factor XIIa (activated Hageman factor) for activation normal prekallikrein levels were found in 2 of them whereas factor XII levels, however, were below normal. The third had a prekallikrein deficiency presumably caused by oral contraceptives. In a fourth member of the family factor XII deficiency was found with normal kallikrein activity. The application of chromogenic peptide substrates for analysing the early stage of clotting has to take into account the special mechanisms of activation.     

Monoclonal antibodies to bovine UDP-galactosyltransferase. Characterization, cross-reactivity, and utilization as structural probes

A series of mouse monoclonal antibodies has been developed against a soluble form of bovine UDP-galactose:N-acetylglucosamine galactosyltransferase purified to apparent chemical homogeneity by a combination of affinity and immunoadsorption chromatography. The purified enzyme consists of two molecular mass variants of 42 and 48 kDa. Individual monoclonal antibodies were selected for by their ability to recognize immobilized affinity-purified galactosyltransferase and were not reactive against bovine alpha-lactalbumin and bovine immunoglobulins. Based on competitive binding assays and Western blot analysis with either galactosyltransferase or lactose synthetase (covalently cross-linked alpha-lactalbumin galactosyltransferase), these monoclonal antibodies can be subdivided into four groups. Group A (3 clones) recognize an epitope at or near the alpha-lactalbumin binding site. In addition, this group is cross-reactive with soluble galactosyltransferase from human milk and pleural effusion. Group B (6 clones) and D (1 clone) appear to recognize two different epitopes on the 6-kDa fragment which is released when the 48-kDa galactosyltransferase polypeptide is converted to the 42-kDa form, apparently by proteolysis. Groups A and C (1 clone) recognize epitopes found on both the 48- and 42-kDa polypeptide. Interestingly, immunofluorescence studies indicate that only two monoclonal antibody groups (C and D) are able to decorate membrane-bound galactosyltransferase (Golgi-associated) in formalin-fixed, methanol-, or detergent-permeabilized cells. Thus, these groups of monoclonal antibodies appear to identify four separate structural/functional domains on soluble galactosyltransferase, two of which are not readily accessible for binding in situ.     

Overexpression of prolylcarboxypeptidase enhances plasma prekallikrein activation on Chinese hamster ovary cells

Plasma prekallikrein (PK) complexes with its receptor, high-molecular-weight kininogen (HK), on human umbilical vein endothelial cells (HUVEC). When assembled on endothelial cells, PK is activated to plasma kallikrein independent of factor XIIa by the serine protease prolylcarboxypeptidase (PRCP, Km= 9 nM). PRCP was shown to be a PK activator when isolated from HUVEC (J Biol Chem 277: 17962-17969, 2002) and produced as a recombinant protein (Blood 103: 4554-4561, 2004). To additionally confirm that human PRCP is a physiological PK activator, PRCP was overexpressed in Chinese hamster ovary (CHO) cells. CHO cells were transfected with full-length PRCP under the control of a cytomegalovirus promoter, and CHO recombinant PRCP was expressed as a fusion protein with COOH-terminal enhanced green fluorescence protein (EGFP). The presence of recombinant PRCP in transfected CHO cells was detected by real-time RT-PCR, immunoblot, and immunoprecipitation. PRCP mRNA and PK activation were two- to threefold higher in transfected than in control CHO cells. The increase in PRCP-induced PK activation in the transfected CHO cells paralleled the increase in PRCP antigen expression, as determined by anti-PRCP and anti-green fluorescence protein antibodies. PK activation of the transfected cells was blocked by small interfering RNA to PRCP. Anti-PRCP antibody and Z-Pro-Pro-aldehyde dimethyl acetate also blocked PK activation (IC50= 0.01 and 7.0 mM, respectively). Localization of PRCP in intact cells observed via confocal microscopy and flow cytometry also confirmed overexpression of PRCP on the external membrane. These investigations independently confirm that PRCP is expressed on cell membranes and that PRCP expression increases PK activation.     

Detection and localization of a cytoplasmic domain on the beta-subunit of Na+,K+-ATPase. A monoclonal antibody study

A monoclonal antibody directed against the beta-subunit of dog kidney Na+,K+-ATPase was generated. Immunoblots demonstrate that monoclonal antibody III 18A binds exclusively to the denaturated beta-subunit. Binding experiments with membranes and whole cells reveal that III 18A binds to membranes but not to whole cells, indicating that the antibody binds to a cytoplasmic domain on the native beta-subunit. To localize the antibody-binding epitope, purified membrane-bound enzyme was fragmented by protease treatment. Tryptic digestion yields a 30-kDa fragment of the beta-subunit, which still retains the binding capacity for the antibody. Thus III 18A probably does not bind to the NH2-terminal segment of the protein. On the other hand, fragmentation of the beta-subunit with low concentrations of papain, which is known to yield a 40-kDa NH2-terminal and a 16-kDa COOH-terminal fragment, results in a complete loss of III 18A binding. These results suggest that the antibody-binding epitope is localized at or near a papain cleavage site on the COOH-terminal part of the beta-subunit. This is inconsistent with a structure model of the beta-subunit containing only a single transmembrane hydrophobic segment with a cytoplasmic NH2-terminal portion, but agrees quite well with a hypothetical structure with four intramembrane segments.