High Molecular Weight Kininogen: A Review of the Structural Literature.

Kininogens are multidomain glycoproteins found in the blood of most vertebrates. High molecular weight kininogen demonstrate both carrier and co-factor activity as part of the intrinsic pathway of coagulation, leading to thrombin generation. Kininogens are the source of the vasoactive nonapeptide bradykinin. To date, attempts to crystallize kininogen have failed, and very little is known about the shape of kininogen at an atomic level. New advancements in the field of cryo-electron microscopy (cryoEM) have enabled researchers to crack the structure of proteins that has been refractory to traditional crystallography techniques. High molecular weight kininogen is a good candidate for structural investigation by cryoEM. The goal of this review is to summarize the findings of kininogen structural studies.

Genetic determinants of activity and antigen levels of contact system factors.

Essentials Genetic variation may provide valuable insight into the role of the contact system in thrombosis. Explored associations of genetic variants with activity, antigen, and disease in RATIO study. Two novel loci were identified: KLKB1 rs4253243 for prekallikrein; KNG1 rs5029980 for HMWK levels. Contact system variants and haplotypes were not associated with myocardial infarction or stroke. SUMMARY: Background The complex, interdependent contact activation system has been implicated in thrombotic disease, although few genetic determinants of levels of proteins from this system are known. Objectives Our primary aim was to study the influence of common F11, F12, KLKB1, and KNG1 variants on factor (F) XI activity and FXI, FXII, prekallikrein (PK) and high-molecular-weight kininogen (HMWK) antigen levels, as well as the risk of myocardial infarction and ischemic stroke. Patients/methods We analyzed samples from all 630 healthy participants, 182 ischemic stroke patients and 216 myocardial infarction patients in the RATIO case-control study of women aged < 50 years. Forty-three tagging single nucleotide variants (SNVs) were genotyped to represent common genetic variation in the contact system genes. Antigen and activity levels were measured with sandwich-ELISA-based and one-stage clotting assays. We performed single variant, age-adjusted, linear regression analyses per trait and disease phenotype, assuming additive inheritance and determined conditionally independent associations. Haplotypes based on the lead SNV and all conditionally independent SNVs were tested for association with traits and disease. Results We identified two novel associations of KLKB1 SNV rs4253243 with PK antigen (ßconditional = -12.38; 95% CI, -20.07 to -4.69) and KNG1 SNV rs5029980 with HMWK antigen (ßconditional = 5.86; 95% CI, 2.40-9.32) and replicated previously reported associations in a single study. Further analyses probed whether the observed associations were indicative of linkage, pleiotropic effects or mediation. No individual SNVs or haplotypes were associated with the disease outcomes. Conclusion This study adds to current knowledge of how genetic variation influences contact system protein levels and clarifies interdependencies.

Cleaved high-molecular-weight kininogen inhibits neointima formation following vascular injury.

Cleaved high-molecular-weight kininogen (HKa) or its peptide domain 5 (D5) alone exert anti-adhesive properties in vitro related to impeding integrin-mediated cellular interactions. However, the anti-adhesive effects of HKa in vivo remain elusive. In this study, we investigated the effects of HKa on leukocyte recruitment and neointima formation following wire-induced injury of the femoral artery in C57BL/6 mice. Local application of HKa significantly reduced the accumulation of monocytes and also reduced neointimal lesion size 14 days after injury. Moreover, C57BL/6 mice transplanted with bone marrow from transgenic mice expressing enhanced green fluorescence protein (eGFP) showed a significantly reduced accumulation of eGFP+-cells at the arterial injury site and decreased neointimal lesion size after local application of HKa or the polypeptide D5 alone. A differentiation of accumulating eGFP+-cells into highly specific smooth muscle cells (SMC) was not detected in any group. In contrast, application of HKa significantly reduced the proliferation of locally derived neointimal cells. In vitro, HKa and D5 potently inhibited the adhesion of SMC to vitronectin, thus impairing their proliferation, migration, and survival rates. In conclusion, application of HKa or D5 decreases the inflammatory response to vascular injury and exerts direct effects on SMC by impeding the binding of integrins to extracellular matrix components. Therefore, HKa and D5 may hold promise as novel therapeutic substances to prevent neointima formation.

Domain 5 of high molecular weight kininogen inhibits collagen-mediated cancer cell adhesion and invasion in association with α-actinin-4.

High molecular weight kininogen (HK) is a plasma glycoprotein with multiple functions, including the regulation of coagulation. We previously demonstrated that domain 5 (D5(H)), a functional domain of HK, and its derived peptides played an important role in the vitronectin-mediated suppression of cancer cell adhesion and invasion. However, the underlying mechanisms of the D5(H)-mediated suppressive effects remain to be elucidated. Here, we showed that D5(H) and its derivatives inhibited the collagen-mediated cell adhesion and invasion of human osteosarcoma MG63 cells. Using purified D5(H) fused to glutathione-S-transferase (GST) and D5(H)-derived peptides for column chromatography, an actin-binding protein, α-actinin-4, was identified as a binding protein of D5(H) with high-affinity for P-5m, a core octapeptide of D5(H). Immunofluorescence microscopy demonstrated that D5(H) co-localized with α-actinin-4 inside MG63 cells. In addition, exogenous GST-D5(H) added to the culture media was transported into MG63 cells, although GST alone as a control was not. As α-actinin-4 regulates actin polymerization necessary for cell adhesion and is related to the integrin-dependent attachment of cells to the extracellular matrix, our results suggest that D5(H) may modulate cell adhesion and invasion together with actinin-4.

GHGKHKNK octapeptide (P-5m) inhibits metastasis of HCCLM3 cell lines via regulation of MMP-2 expression in in vitro and in vivo studies.

P-5m, an octapeptide derived from domain 5 of HKa, was initially found to inhibit the invasion and migration of melanoma cells. The high metastatic potential of melanoma cells was prevented by the HGK motif in the P-5m peptide in vitro and in an experimental lung metastasis model, suggesting that P-5m may play an important role in the regulation of tumor metastasis. The aim of this study was to measure the effect of P-5m on tumor metastasis of human hepatocarcinoma cell line (HCCLM3) in vitro and in vivo in a nude mouse model of hepatocellular carcinoma (HCC), and detect the mechanisms involved in P-5m-induced anti-metastasis. By gelatin zymography, matrix metallo-proteinases 2 (MMP-2) activity in HCCLM3 was dramatically diminished by P-5m peptide. In addition, the migration and metastasis of HCCLM3 cells was also inhibited by the peptide in vitro. In an orthotopic model of HCC in nude mice, P-5m treatment effectively reduced the lung metastasis as well as the expression of MMP-2 in the tumor tissues. Overall, these observations indicate an important role for P-5m peptide in HCC invasion and metastasis, at least partially through modulation MMP-2 expression. These data suggests that P-5m may have therapeutic potential in metastatic human hepatocarcinoma.

Genome-wide association study for adiponectin levels in Filipino women identifies CDH13 and a novel uncommon haplotype at KNG1-ADIPOQ.

Adiponectin is an adipocyte-secreted protein involved in a variety of metabolic processes, including glucose regulation and fatty acid catabolism. We conducted a genome-wide association study to investigate the genetic loci associated with plasma adiponectin in 1776 unrelated Filipino women from the Cebu Longitudinal Health and Nutrition Survey (CLHNS). Our strongest signal for adiponectin mapped to the gene CDH13 (rs3865188, P ≤ 7.2 × 10(-16)), which encodes a receptor for high-molecular-weight forms of adiponectin. Strong association was also detected near the ADIPOQ gene (rs864265, P = 3.8 × 10(-9)) and at a novel signal 100 kb upstream near KNG1 (rs11924390, P = 7.6 × 10(-7)). All three signals were also observed in 1774 young adult CLHNS offspring and in combined analysis including all 3550 mothers and offspring samples (all P ≤ 1.6 × 10(-9)). An uncommon haplotype of rs11924390 and rs864265 (haplotype frequency = 0.050) was strongly associated with lower adiponectin compared with the most common C-G haplotype in both CLHNS mothers (P = 1.8 × 10(-25)) and offspring (P = 8.7 × 10(-32)). Comprehensive imputation of 2653 SNPs in a 2 Mb region using as reference combined CHB, JPT and CEU haplotypes from the 1000 Genomes Project revealed no variants that perfectly tagged this haplotype. Our findings provide the first genome-wide significant evidence of association with plasma adiponectin at the CDH13 locus and identify a novel uncommon KNG1-ADIPOQ haplotype strongly associated with adiponectin levels in Filipinos.

Expression of endogenous nuclear bradykinin B2 receptors mediating signaling in immediate early gene activation.

Bradykinin (BK) represents a pro-inflammatory mediator that partakes in many inflammatory diseases. The mechanism of action of BK is thought to be primarily mediated by specific cell surface membrane B2 receptors (B2Rs). Some evidence has suggested, however, the existence of an intracellular/nuclear B2R population. Whether these receptors are functional and contribute to BK signaling remains to be determined. In this study, by mean of Western blotting, 3D-confocal microscopy, receptor autoradiography and radioligand binding analysis, we showed that plasma membrane and highly purified nuclei from isolated rat hepatocytes contain specific B2R that bind BK. The results depicting B2R nuclear expression in isolated nuclear organelles were reproduced in situ on hepatic sections by immunogold labeling and transmission electron microscopy. Functional tests on single nuclei, by means of confocal microscopy and the calcium-sensitive probe fluo-4AM, showed that BK induces concentration-dependent transitory mobilization of nucleoplasmic calcium; these responses were blocked by B2R antagonist HOE 140, not by the B1R antagonist R954 and, were also found in wild-type C57/Bl6 mice, but not in B2R-KO mice. In isolated nuclei, BK elicited activation/phosphorylation of Akt, acetylation of histone H3 and ensuing pro-inflammatory iNOS gene induction as determined by Western blot and RT-PCR. ChIP assay confirmed binding of acetylated-histone H3 complexes, but not B2R, to promoter region of iNOS gene suggesting that B2R-mediated gene expression is bridged with accessory downstream effectors. This study discloses a previously undescribed mechanism in BK-induced transcriptional events, via intracrine B2R-mediated signaling, occurring in rat autologous hepatic cells.

A second expressed kininogen gene in mice.

We isolated PCR, RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE-PCR)-, and RT-PCR-generated clones from mouse kininogen family transcripts. DNA sequencing indicated that the clones were from two distinct genes. One set (K1) is from the previously reported mouse kininogen gene. The second set (K2) has an open reading frame, is 93% identical to K1 in the overlapping nucleotide sequence, and, unlike T-kininogens in the rat, encodes a bradykinin motif identical to K1. We discovered that K2 exists with two different 5' ends. We used RT-PCR to determine the distribution and relative abundance of K1 and K2 mRNA in mouse tissues. K2 is transcribed and K1 and K2 are generally both expressed in the same tissues; however, they differ in their regulation of the alternative splicing event that yields either low-molecular-weight kininogen (LMWK) or high-molecular-weight kininogen (HMWK). For example, in the liver K1 is expressed as both HMWK and LMWK, whereas K2 is only expressed as LMWK. Conversely, in the kidney K2 is strongly expressed as both HMWK and LMWK, whereas K1 is not expressed as HMWK and expressed only very weakly as LMWK.

El kininógeno de alto peso molecular: su participación en la respuesta inflamatoria y en la angiogénesis. Propiedades y posible aplicación terapéutica / High molecular weight kininogen in inflammation and angiogenesis: a review of its properties and therapeutic applications

The plasma kallikrein-kinin system (KKS) participates in the pathogenesis of inflammatory reactions involved in cellular injury, coagulation, fibrinolysis, kinin formation, complement activation, cytokine secretion and release of proteases. It has been shown that KKS activation in the systemic inflammatory response syndrome results in decrease of its component plasma proteins. Similar changes have been documented in diabetes, sepsis, children with vasculitis, allograft rejection, disseminated intravascular coagulation, patients with recurrent pregnancy losses, hereditary angioedema, adult respiratory distress syndrome and coronary artery disease. Direct involvement of the KKS in the pathogenesis of experimental acute arthritis and acute and chronic enterocolitis has been documented by previous studies from our laboratory using experimental animal models. It has been found that in HK deficient Lewis rats, experimental IBD was much less severe. We showed a genetic difference in kininogen structure between resistant Buffalo and susceptible Lewis rats, which results in accelerated cleavage of HK and it is responsible for the susceptibility to the inflammatory process in the Lewis rats. It has been demostrated that therapy with a specific plasma kallikrein inhibitor (P8720) modulated the experimental enterocolitis, arthritis and systemic inflammation. Furthermore, it has been shown that a bradykinin 2 receptor (B2R) antagonist attenuates the inflammatory changes in the same animal model. We have showed that a monoclonal antibody targeting HK decreases angiogénesis and arrests tumor growth in a syngeneic animal model. In summary, these results indicate that the plasma KKS plays a central role in the pathogenesis of chronic intestinal inflammation, arthritis and angiogenesis.

Se ha demostrado la participación del sistema plasmático de kalikreína-kininas (KKS) en el proceso inflamatorio, el cual incluye reacciones de daño celular, coagulación y fibrinólisis, formación de kininas, activación del complemento, secreción de citoquinas y liberación de proteasas. El KKS se encuentra activado en el síndrome de respuesta inflamatoria sistémica con una disminución en la concentración plasmática de las proteínas que lo constituyen. También se ha demostrado una activación similar en la diabetes, choque séptico, vasculitis en infantes, enfermedad injerto-huésped, coagulación intravascular diseminada, pacientes con abortos de repetición, angioedema hereditario, el síndrome de estrés respiratorio del adulto y enfermedad coronaria arterial. Mediante el uso de modelos animales experimentales, nuestro laboratorio ha demostrado una participación directa del KKS en la patogénesis de la artritis experimental aguda y la enterocolitis aguda y crónica. Se ha demostrado que en la rata tipo Lewis, cuando es deficiente de kininógeno de alto peso molecular (HK), la enfermedad inflamatoria intestinal es menos severa comparada con la presentada en ratas con niveles normales de HK como la Buffalo. Nosotros mostramos una diferencia entre el gene que codifica la molécula del kininógeno de la rata tipo Buffalo (resistentes) y Lewis (susceptibles), que resulta en un incremento de la actividad proteolítica de kalikreína sobre su substrato HK, lo cual predispone a las ratas Lewis al desarrollo de la enfermedad inflamatoria crónica. Se ha demostrado una disminución en las manifestaciones inflamatorias sistémicas de la enterocolitis y artritis experimental mediante el uso de un inhibidor específico de la kalikreína (P8720). Además, el antagonista del receptor 2 de la bradikinina (BR2) atenuó los cambios inflamatorios en el mismo modelo animal. Asimismo, se ha demostrado que las ratas Lewis deficientes de kininógeno desarrollaron inflamación intestinal sistémica menos severa. Mediante el uso del anticuerpo monoclonal C11C1 contra HK se logró una disminución de la angiogenesis y, consecuentemente, el crecimiento tumoral. En conclusión, los resultados demuestran que el sistema plasmático de KKS desempeña un papel preponderante en la patogénesis de la artritis reumatoide, la enfermedad intestinal crónica y en el proceso angiogénico.

[High molecular weight kininogen in inflammation and angiogenesis: a review of its properties and therapeutic applications]. / El kininógeno de alto peso molecular: su participación en la respuesta inflamatoria y en la angiogénesis. Propiedades y posible aplicación terapéutica.

The plasma kallikrein-kinin system (KKS) participates in the pathogenesis of inflammatory reactions involved in cellular injury, coagulation, fibrinolysis, kinin formation, complement activation, cytokine secretion and release of proteases. It has been shown that KKS activation in the systemic inflammatory response syndrome results in decrease of its component plasma proteins. Similar changes have been documented in diabetes, sepsis, children with vasculitis, allograft rejection, disseminated intravascular coagulation, patients with recurrent pregnancy losses, hereditary angioedema, adult respiratory distress syndrome and coronary artery disease. Direct involvement of the KKS in the pathogenesis of experimental acute arthritis and acute and chronic enterocolitis has been documented by previous studies from our laboratory using experimental animal models. It has been found that in HK deficient Lewis rats, experimental IBD was much less severe. We showed a genetic difference in kininogen structure between resistant Buffalo and susceptible Lewis rats, which results in accelerated cleavage of HK and it is responsible for the susceptibility to the inflammatory process in the Lewis rats. It has been demostrated that therapy with a specific plasma kallikrein inhibitor (P8720) modulated the experimental enterocolitis, arthritis and systemic inflammation. Furthermore, it has been shown that a bradykinin 2 receptor (B2R) antagonist attenuates the inflammatory changes in the same animal model. We have showed that a monoclonal antibody targeting HK decreases angiogenesis and arrests tumor growth in a syngeneic animal model. In summary, these results indicate that the plasma KKS plays a central role in the pathogenesis of chronic intestinal inflammation, arthritis and angiogenesis.

Primary structures of guinea pig high- and low-molecular-weight kininogens.

Guinea pig high-molecular-weight and low-molecular-weight (HMW and LMW) kininogen cDNA were amplified from liver mRNA by RT-PCR. Their nucleotide sequences were analyzed and deduced to amino acid sequences. The HMW kininogen, composed of 607 amino acid residues with a 18-residue signal sequence, possessed the cysteine protease inhibitor domains I and II, the bradykinin domain, the histidine-rich region, and the prekallikrein-binding region. The amino acid sequence preceding the bradykinin domain was found not to be -Leu-Met-Lys- but -Leu-Thr-Arg-. Therefore, kallidin (Lys-bradykinin) and Met-kallidin are not liberated from the guinea pig kininogens. We purified the HMW kininogen protein from plasma and prepared the kinin-free form using guinea pig plasma kallikrein. Although the amino-terminal of the HMW kininogen was modified, the 25 amino-terminal residues of the light chain of the kinin-free kininogen corresponded to the deduced sequence just after the bradykinin moiety of the HMW kininogen. With regard to the LMW kininogen, the nucleotide sequence down to T(1200) as well as the amino acid sequence till Thr(382) was identical to that of the HMW kininogen. We also examined the localization of the guinea pig kininogen gene on the prometaphase lymphocyte chromosomes by fluorescence in situ hybridization method. Two pair signals were observed on a pair of homologous chromosomes, each of which is composed of two chromatids. Based on these findings, we concluded that HMW and LMW kininogens are produced from the single kininogen gene in guinea pig as in the cases of the other mammalian species reported so far.

Structure and expression of two kininogen genes in mice.

Kininogens serve dual functions by forming a scaffold for the assembly of the protein complex initiating the surface-activated blood coagulation cascade and as precursors for the kinin hormones. While rats have three kininogen genes, for mice, cattle, and humans only one gene has been described. Here, we present sequence and expression data of a second mouse kininogen gene. The two genes, kininogen-I and kininogen-II, are located in close proximity on chromosome 16 in a head-to-head arrangement. In liver and kidney, both genes are expressed and for each gene three alternative splice variants are synthesized. Two of them are the expected high and low molecular weight isoforms known from all mammalian kininogens. However, for both genes also a third, hitherto unknown splice variant was detected which lacks part of the high molecular weight mRNA due to splicing from the low molecular weight donor site to alternative splice acceptor sites in exon 10. The physiological functions of the six kininogen isoforms predicted by these findings need to be determined.

Porcine endometrial expression of kininogen, factor XII, and plasma kallikrein in cyclic and pregnant gilts.

Establishment of pregnancy in the pig is accompanied by a localized uterine acute inflammatory response and increase in uterine blood flow. Following rapid trophoblast elongation on Day 12 of pregnancy there is an increase in tissue kallikrein activity and release of bradykinin into the uterine lumen, suggesting the kallikrein-kininogen-kinin system is active in the porcine uterus. The present study investigated endometrial expression and presence of the various factors of the kallikrein-kininogen-kinin system. Endometrial L- and H-kininogen gene expression as well as presence of kininogens in the uterine flushings was evaluated throughout the estrous cycle and early pregnancy in the pig. The possible involvement of plasma kallikrein and Factor XII, activators of the kallikrein-kininogen-kinin system, were evaluated through analysis of gene expression in endometrial and conceptus tissues. Gene expression for plasma kallikrein, Factor XII, and H-kininogen were detected in endometrium but not early conceptus tissues. Factor XII and H-kininogen gene expression were similar across the days of the estrous cycle and early pregnancy. Endometrial plasma kallikrein gene expression was low but increased on Day 15 of the estrous cycle, whereas expression was similar across the days of early pregnancy. In comparison to cyclic gilts, endometrial L-kininogen gene expression increased fourfold on Days 15 and 18 of pregnancy. Both L- and H-kininogen were detected in the uterine flushings of cyclic and pregnant gilts. Presence of L- and H-kininogen in the porcine uterus and endometrial gene expression of plasma kallikrein and Factor XII provide evidence that the kallikrein-kininogen-kinin system is biologically active during establishment of pregnancy in the pig.

Fine mapping of the sequences in domain 5 of high molecular weight kininogen (HK) interacting with heparin and zinc.

We previously localized the heparin binding region on high molecular weight kininogen to domain 5 (D5) by quantifying the binding using surface plasmon resonance of D5 fused at its N-terminal to glutathione-S-transferase. We further examined GST-(H475-S626) which at 100 nm was previously shown to be ineffective in reversing the heparin acceleration of antithrombin inhibition of thrombin. However, we now show that at a concentration of 400 nm, complete reversal of accelerated inhibition occurred. To characterize the interacting sequences on D5, four peptides representing surface loops of a molecular model were synthesized. Peptides H475-H485 and G440-G455, rich in histidine and low in lysine, showed weak or no detectable binding in the absence of Zn++, but tighter binding in the presence of Zn++. H483-K497 containing three histidines and six lysines showed tight binding without Zn++, and increased in avidity with Zn++. In contrast, G486-K502, low in histidine and high in lysine, showed tight binding (KD = 0.8 microm) in the absence and presence of Zn++. Both H483-K497 and G486-K502 were effective in neutralizing the accelerated inhibition by heparin of thrombin by antithrombin in the absence of Zn++. Therefore, a set of lysine residues in the sequence of K487-K502 is responsible for Zn++-independent binding of heparin. Further, a group of histidine residues in sequence range of H475-H485 contributes to Zn++-dependent binding of heparin to HK-D5.

Characterization of molecular defects of Fitzgerald trait and another novel high-molecular-weight kininogen-deficient patient: insights into structural requirements for kininogen expression.

A 6-year-old male with vertebral-basilar artery thrombosis was recognized to have high-molecular-weight kininogen (HK) deficiency. The propositus had no HK procoagulant activity and antigen (< 1%). Using monoclonal antibodies (Mabs) to kininogen domain 3, the propositus, family members, and Fitzgerald plasma were determined to have detectable low-molecular-weight kininogen. Mabs to HK domains 5 and 6 do not detect HK antigen in the propositus' plasma. The propositus has a single base pair (bp) deletion in cDNA position 1492 of exon 10 affecting amino acid 480 of the mature protein and resulting in a frameshift and a premature stop codon at position 1597 (amino acid 532). Unexpectedly, Mabs to the heavy chain and domain 5 of HK detect a 92-kDa form of HK in Fitzgerald plasma, the first HK-deficient plasma. The 92-kDa Fitzgerald HK has amino acid residues through 502, corresponding to domains 1 through 5, but lacks epitopes of domain 6 (positions 543 to 595). Fitzgerald DNA has a normal exon 10, but a 17-bp mutation in intron 9. These combined results indicate that mutations in the kininogen gene may differentially affect biosynthesis, processing, and/or secretion of HK.

Chronic intestinal inflammation and angiogenesis in genetically susceptible rats is modulated by kininogen deficiency.

Genetically susceptible Lewis rats injected in the intestinal wall with peptidoglycan-polysaccharide (PG-APS) polymers develop chronic granulomatous enterocolitis associated with activation of the kallikrein-kinin system. To elucidate the role of high-molecular-weight kininogen (HK), we backcrossed Brown Norway rats having an HK deficiency with Lewis rats for five generations. Two new strains were produced, wild-type F5 (F5WT) and HK deficient (F5HKd), each with a approximately 97% Lewis genome. The HK values of F5WT rat plasma and F5HKd rat plasma were 0.62 +/- 0.20 and 0.08 +/- 0.03 U/ml, respectively. Among the inflammatory changes, the mean gross gut, total intestinal histologic and liver granuloma score and the white blood count were significantly lower in the F5HKd than the F5WT rats. Plasma T-kininogen was significantly less in F5HKd. Angiogenesis (mean vascular density) in the cecum was decreased significantly in F5HKd compared to F5WT. These results indicate the importance of the kallikrein-kinin system in this model of chronic enterocolitis and systemic inflammation.

The antiangiogenic activity of cleaved high molecular weight kininogen is mediated through binding to endothelial cell tropomyosin.

Conformationally altered proteins and protein fragments derived from the extracellular matrix and hemostatic system may function as naturally occurring angiogenesis inhibitors. One example of such a protein is cleaved high molecular weight kininogen (HKa). HKa inhibits angiogenesis by inducing apoptosis of proliferating endothelial cells, effects mediated largely by HKa domain 5. However, the mechanisms underlying the antiangiogenic activity of HKa have not been characterized, and its binding site on proliferating endothelial cells has not been defined. Here, we report that the induction of endothelial cell apoptosis by HKa, as well as the antiangiogenic activity of HKa in the chick chorioallantoic membrane, was inhibited completely by antitropomyosin monoclonal antibody TM-311. TM-311 also blocked the high-affinity Zn2+-dependent binding of HKa to both purified tropomyosin and proliferating endothelial cells. Confocal microscopic analysis of endothelial cells stained with monoclonal antibody TM-311, as well as biotin labeling of cell surface proteins on intact endothelial cells, revealed that tropomyosin exposure was enhanced on the surface of proliferating cells. These studies demonstrate that the antiangiogenic effects of HKa depend on high-affinity binding to endothelial cell tropomyosin.

Whale high-molecular-weight and low-molecular-weight kininogens.

The expression of high-molecular-weight and low-molecular-weight kininogen mRNAs in the whale liver was examined by reverse transcription-polymerase chain reaction. The nucleotide sequences of the high-molecular-weight and low-molecular-weight kininogen cDNAs were analyzed and deduced to the amino acid sequences. The high-molecular-weight kininogen composed of 609 amino acid residues with 18 signal peptides possessed the consensus sequences of the cysteine protease inhibitor domains I and II, the bradykinin domain, the histidine-rich region, and the prekallikrein-binding region. Except for the histidine-rich region, the overall homologies with bovine, human, and rat high-molecular-weight kininogens were 81%, 76%, and 62%, respectively. The low-molecular-weight kininogen is composed of 408 amino acid residues. The nucleotide sequence down to C(1200) as well as the amino acid sequence till Ile(382) is identical to that of the high-molecular-weight kininogen. The remaining low-molecular-weight kininogen-specific carboxy-terminal portion possessed an amino acid sequence similar to that of the land mammals. The overall homologies with bovine, human, and rat low-molecular-weight kininogens were 82%, 79%, and 64%, respectively. The amino acid sequences of both whale high-molecular-weight and low-molecular-weight kininogens are most similar to those of the bovine among the land mammals analyzed so far. An incubation of dolphin/whale plasma with human plasma kallikrein, or with bovine trypsin, in the presence of carboxypeptidase inhibitors generated bradykinin antigen as well as the spasmogenic activity to the estrous rat uterus. The amount of bradykinin released by the latter enzyme was almost double of the former, indicating that the dolphin/whale plasma contained similar concentrations of low-molecular-weight and high-molecular-weight kininogens.

Studies on porcine high molecular weight kininogen. An improved method for the purification of porcine high molecular weight kininogen and cleavage of the kininogen by the action of porcine plasma kallikrein.

By introduction of stepwise DEAE Sephadex A-50 and copper-Chelating Sepharose 6B column chromatographies, about 18.5 mg of high molecular weight kininogen (HK) composed of a single polypeptide chain was obtained from 500 ml of porcine plasma. Molecular weights of reduced or non-reduced preparation were estimated to be 110 kDa and 116 kDa, respectively, by SDS-PAGE. Using the preparation, cleavage of HK by porcine plasma kallikrein (KK) was investigated. A single polypeptide HK was cleaved into two chains cross-linked by disulfide bond(s), accompanying the release of kinin. Further degradation was not observed. Molecular weights of heavy-chain (H-chain) and light-chain (L-chain) were estimated to be 61 kDa and 56 kDa, respectively, by SDS-PAGE. The amino- (N-) terminal sequences of intact HK, reduced and carboxymethylated- (RCM-) H-chain, RCM-L-chain and the peptide around the kinin moiety obtained by BrCN digestion were determined. Their sequences were highly homologous with those of bovine or human HK. These results indicate that plasma KK first cleaved the Arg-Ser bond of HK, and formed nicked HK. The second cleavage yielded bradykinin (BK) and kinin-free protein, which was apparently of equal size to the nicked HK. The structure of HK was from the N-terminus to the carboxy- (C-) terminus, H-chain-BK-L-chain.