scholarly journals Domain 2 of uPAR regulates single-chain urokinase-mediated angiogenesis through β1-integrin and VEGFR2

2013 ◽  
Vol 305 (3) ◽  
pp. H305-H320 ◽  
Author(s):  
Gretchen A. LaRusch ◽  
Alona Merkulova ◽  
Fakhri Mahdi ◽  
Zia Shariat-Madar ◽  
Robert G. Sitrin ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Kinase Inhibitors ◽  
Umbilical Vein ◽  
Endothelial Cell Proliferation ◽  
Vegf Receptor ◽  
High Molecular Weight Kininogen ◽  
Single Chain ◽  
Urokinase Plasminogen Activator Receptor ◽  
Integrin Binding Site

How single-chain urokinase (ScuPA) mediates angiogenesis is incompletely understood. ScuPA (≥4 nM) induces phosphorylated (p)ERK1/2 (MAPK44 and MAPK42) and pAkt (Ser473) in umbilical vein and dermal microvascular endothelial cells. Activation of pERK1/2 by ScuPA is blocked by PD-98059 or U-0126, and pAkt (Ser473) activation is inhibited by wortmannin or LY-294002. ScuPA (32 nM) or protease-inhibited two-chain urokinase stimulates pERK1/2 to the same extent, indicating that signaling is not dependent on enzymatic activity. ScuPA induces pERK1/2, but not pAkt (Ser473), in SIN1−/−cells, indicating that the two pathways are not identical. Peptides from domain 2 of the urokinase plasminogen activator receptor (uPAR) or domain 5 of high-molecular-weight kininogen compete with ScuPA for the induction of pERK1/2 and pAkt (Ser473). A peptide of the integrin-binding site on uPAR, a β1-integrin peptide that binds uPAR, antibody 6S6 to β1-integrin, tyrosine kinase inhibitors AG-1478 or PP3, and small interfering RNA knockdown of VEFG receptor 2, but not HER1–HER4, blocked ScuPA-induced pERK1/2 and pAkt (Ser473). ScuPA-induced endothelial cell proliferation was blocked by inhibitors of pERK1/2 and pAkt (Ser473), antibody 6S6, and uPAR or kininogen peptides. ScuPA initiated aortic sprouts and Matrigel plug angiogenesis in normal, but not uPAR-deficient, mouse aortae or mice, respectively, but these were blocked by PD-98059, LY-294002, AG-1478, or cleaved high-molecular-weight kininogen. In summary, this investigation indicates a novel, a nonproteolytic signaling pathway initiated by zymogen ScuPA and mediated by domain 2 of uPAR, β1-integrins, and VEGF receptor 2 leading to angiogenesis. Kininogens or peptides from it downregulate this pathway.

Assembly of High Molecular Weight Kininogen and Activation of Prekallikrein on Cell Matrix

2001 ◽  
Vol 86 (09) ◽  
pp. 840-847 ◽  
Author(s):  
Z. Shariat-Madar ◽  
F. Mahdi ◽  
C. A. M. Sampaio ◽  
A. H. Schmaier ◽  
G. Motta
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
High Molecular Weight ◽  
Trypsin Inhibitor ◽  
Umbilical Vein ◽  
Sodium Dodecyl ◽  
Factor Xii ◽  
High Molecular Weight Kininogen ◽  
Urokinase Plasminogen Activator Receptor ◽  
Corn Trypsin Inhibitor

SummaryInvestigations determined if extracellular matrix of endothelial cells (EC) is a platform for HK assembly and PK activation. In buffers containing bovine serum albumin, biotin-HK binding to ECV304 cells or their matrix requires ≥ 50 µM added Zn 2+. Ortho-phenanthroline or a HK domain 5 peptide blocks HK binding. Binding to umbilical vein EC or matrix, but not ECV304 cells or matrix, is mediated by cytokeratin 1. Biotin-HK binds to ECV304 cells or matrix with a Kd of 15.8 or 9.0 nM and a Bmax of 2.6 107 or 2.4 107 sites/cell, respectively. PK activation on ECV304 cells or matrix is blocked by antipain or SBTI and corn trypsin inhibitor partially inhibits kallikrein formation. PK activation occurs on ECV304 cells or matrix prepared without serum or in human factor XII deficient serum, indicating that the PK activator is not factor XIIa. EC matrix promotes plasminogen activation after the assembly of HK, PK and pro-urokinase. These studies indicate that matrix of various EC has the ability to assemble HK allowing for PK activation and subsequent activities.Abbreviations: EC: endothelial cells, FXII: factor XII, HK: high molecular weight kininogen, HKa: bradykinin-free HK, PK: plasma prekallikrein, Pro-UK: pro-urokinase, uPAR: urokinase plasminogen activator receptor, tcuPA: twochain urokinase, CK1: cytokeratin 1, SBTI: soybean trypsin inhibitor, HUVEC: human umbilical vein endothelial cell, SDS-PAGE: sodium dodecyl sulfatepolyacrylamide gel electrophoresis, CTI: corn trypsin inhibitor, p-APMSF: p-amidinophenylmethylsulfonylfluoride, EBSS: Earle’s Balanced Salt Solution

High molecular weight kininogen and factor XII binding to endothelial cells and astrocytes

2003 ◽  
Vol 90 (11) ◽  
pp. 787-795 ◽  
Author(s):  
Lawrence Fernando ◽  
Snehlatha Natesan ◽  
Kusumam Joseph ◽  
Allen Kaplan
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
High Molecular Weight ◽  
Umbilical Vein ◽  
Microvascular Endothelial Cells ◽  
Factor Xii ◽  
High Molecular Weight Kininogen ◽  
Contact Activation ◽  
Urokinase Plasminogen Activator Receptor ◽  
Microvascular Endothelial

SummaryWe have quantitated the binding of high molecular weight kininogen (HK) to human microvascular endothelial cells of lung and dermal origin as well as to astrocytes and compared the results with those reported for human umbilical vein endothelial cells (HUVEC). We also reassessed parameters of binding to HUVEC employing cells in suspension as well as cells attached to the culture plate and report similar numbers of sites varying from 6.96x105to 7.71x105per cell. The present study shows that HK binds with high specificity and affinity to microvascular endothelial cells (Kd = 1.86 to 4.5 nM) compared to HUVEC (Kd = 10.35nM) but with lower affinity to astrocytes (Kd = 23.73 nM). Human cytokeratin 1, urokinase plasminogen activator receptor and gC1qR were found to be HK binding proteins present at the surface of microvascular endothelial cells and astrocytes analogous to that seen in HUVEC, as assessed by inhibition of binding with antibody to each protein. Lung microvascular endothelial cells had approximately half the number of HK binding sites as HUVEC while dermal micro vascular endothelial cells and astrocytes had only 8-10% of the sites/cell. The affinity of binding to the microvascular endothelial cells was greater than HUVEC, the affinity of binding to astrocytes was considerably less, nevertheless binding to each cell type involves gC1qR, cytokeratin 1 and u-PAR to varying degrees. We also demonstrate, for the first time, that factor XII binds to all of these cell types in a saturable and Zn+2dependent manner. Given that factor XII accelerates the interactions among cell surfaces and proteins of the contact activation cascade to generate bradykinin, binding of factor XII (and the prekallikrein-HK complex) may serve as a mechanism by which these proteins are concentrated locally to facilitate their interactions.

scholarly journals High Molecular Weight Kininogen Peptides Inhibit the Formation of Kallikrein on Endothelial Cell Surfaces and Subsequent Urokinase-Dependent Plasmin Formation

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 690-697 ◽  
Author(s):  
Yingzhang Lin ◽  
Robert B. Harris ◽  
Wuyi Yan ◽  
Keith R. McCrae ◽  
Hong Zhang ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Structural Information ◽  
Umbilical Vein ◽  
High Molecular Weight Kininogen ◽  
Cell Surfaces ◽  
Free System ◽  
Order Of Magnitude ◽  
A Cell ◽  
Umbilical Vein Endothelial Cells

A sequence of 31 amino acids (S565-K595) in domain 6 of the light chain of high molecular weight kininogen (HK) has previously been shown to be responsible for the binding of plasma prekallikrein (PK) or kallikrein. To find effective peptides that might block binding between HK and PK on cell surfaces, a new series of synthetic peptides has now been prepared that incorporates portions of this binding domain sequence. For mapping the minimal sequence within HK, these new peptides were tested for their ability to compete with HK for binding PK in a cell-free system and on human umbilical vein endothelial cells (HUVEC). In the former, at pH 7.4, the kds for binding between kallikrein and either D567-K595, S565-P594, D567-S593, or D567-T591 were all similar to that for the binding of S565-K595 (0.2 to 0.4 μmol/L), but those for the binding of D568-K595, W569-K595, and D567-P589 were an order of magnitude greater (kd = 2 to 5 μmol/L). D567-S586, the shortest chain length of the N- and C-terminal truncation sequences tested, does not effectively compete with kininogen for kallikrein binding (kd = 100 μmol/L). These results imply that D567-T591, a 25-residue peptide (HK25c), contains sufficient structural information for binding kallikrein in solution. D567-T591 also is the minimum structural sequence to block binding of kallikrein to HUVEC-bound HK (IC50 = 50 nmol/L) and to inhibit PK activation to kallikrein on the cell surface (IC50 = 80 nmol/L). In addition, D567-T591 also inhibits the generation of kallikrein-activated urokinase, which activates plasminogen to plasmin (IC50 = 100 nmol/L). Thus, HK-derived peptides may be useful compounds for modulating excessive fibrinolysis and hypotension in sepsis and multiple trauma.

Commercial Immunodepleted Deficient Plasmas Contain Cleaved High Molecular Weight Kininogen (HK)

1988 ◽  
Vol 60 (03) ◽  
pp. 447-452
Author(s):  
C Mannhalter ◽  
H Lang
Keyword(s):  
Molecular Weight ◽  
Quality Control ◽  
Comparative Analysis ◽  
High Molecular Weight ◽  
Control Method ◽  
Immunoblot Analysis ◽  
High Molecular Weight Kininogen ◽  
Single Chain ◽  
Quality Control Method ◽  
Chain Form

SummaryComparative analysis of high molecular weight kininogen (HK) in various commercial congenital and immunodepleted deficiency plasmas was performed by immunoblotting of HK. It was found, that some artificially depleted deficiency plasmas contained proteolytically cleaved, kinin-free kininogen. In contrast, in all congenitally deficient plasmas, HK was present in the intact, single chain form. Thus, cleavage of kininogen could have been triggered by or during the immunodepletion procedure. It was seen, that the degree of proteolytic cleavage and degradation of HK in depleted plasmas differed among various manufacturers. E.g. depleted products of one company contained only trace amounts of cleaved HK, in contrast to products of another one, in which HK was completely degraded. The immunoblot analysis of HK reflects the occurrence of proteolytic events during the production of artificially deficient plasmas and can therefore serve as a quality control method.

The Murine Genome Contains Two Functional Kininogen Genes Leading to Tissue Specific Expression of High Molecular Weight Kininogen (HK) and Expression of a Novel HK Splice Variant.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3979-3979
Author(s):  
Sergei Merkoulov ◽  
Anton A. Komar ◽  
Keith R. McCrae
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Expression Patterns ◽  
Endothelial Cell Proliferation ◽  
Head Orientation ◽  
High Molecular Weight Kininogen ◽  
Specific Expression ◽  
Tissue Specific ◽  
Tissue Specific Expression ◽  
Exon 10

Abstract High molecular weight kininogen (HK) plays an important role in the assembly and activation of the kallikrein/kinin system. While the human genome contains only a single copy of the kininogen gene, three copies are present in the rat (one K-kininogen and two T-kininogen). Here, we report that the mouse genome contains two homologous kininogen genes (overall homology 91%), denoted mHK1 and mHK2. Both genes are located on chromosome 16 in a head-to-head orientation, and contain open reading frames. The size of intronic sequences between the 11 kininogen gene exons is similar (Figure). HK mRNA transcripts derived from the mHK1 and mHK2 genes differ slightly in size due to gaps of 33 and 18 nucleotides in exon 10 of mHK2. RT-PCR analysis of HK gene expression in adult and embryonic murine tissues revealed that HK mRNA was derived from mHK1 in liver, adrenal and embryo, but from mHK2 in kidney and lung. HK mRNA derived from both genes was present in testis, brain and muscle, though expression levels were low relative to those in other tissues. HK mRNA was not detected in ovary, bone marrow, heart or bladder. mHK1-derived HK mRNA was alternatively spliced, as demonstrated by the presence of an HK mRNA transcript encoding a novel HK1 isoform, ΔmD5, that lacked the portion of exon 10 encoding Thr400 - Asp582 of HK domains 5 and 6. Examination of the putative promoter regions of the two genes using the MatInspector Professional program (Genomatix) demonstrated distinct differences, perhaps explaining in part their tissue-specific expression patterns. Like domain 5 of human HK (hD5), domain 5 of murine HK (mD5), in which the histidine and lysine-rich C-terminal region of this domain previously shown to mediate the antiangiogenic activity of domain 5 is highly conserved, inhibited endothelial cell proliferation. While the function of each of the kininogen genes in the intact animal has yet to be defined, characterization of the two genes may provide new information concerning the role of high molecular weight kininogen in development, normal physiology, and pathological processes. Figure Figure

High Molecular Weight Kininogen Fragments Stimulate the Secretion of Interleukin 1β through the Urokinase-Type Plasminogen Activator Receptor (uPAR) and CD11b/CD18 (Mac-1) in Human Blood Mononuclear Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3800-3800
Author(s):  
Mohammad M. Khan ◽  
Harlan N. Bradford ◽  
Irma Isordia-Salas ◽  
Ricardo Espinola ◽  
Robert W. Colman
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Human Blood ◽  
Mononuclear Cells ◽  
Inflammatory Diseases ◽  
Interleukin 1Β ◽  
High Molecular Weight Kininogen ◽  
Single Chain ◽  
Blood Mononuclear Cells ◽  
Domain 3

Abstract High molecular weight kininogen (HK) is known to bind specifically and saturably to Mac-1 with a Kd = 9–18 nM for neutrophils and to uPAR with a Kd =30 nM for endothelial cells. However, the functional results of HK interaction with Mac-1 or uPAR on leukocytes is not fully understood. Kallikrein cleavage of single chain HK to a two chain form (HKa) with release of bradykinin (BK) occurs in sepsis, arthritis, and inflammatory bowel disease. We hypothesized that HKa stimulates secretion of inflammatory cytokines. Mononuclear cells were isolated from normal subjects by a Histopaque density gradient. We have expressed kininogen domain 3 (D3) and a fragment of domain 3, coded for by exon 7, E7P (aaG235-Q292), in E. Coli as glutathione S-transferase (GST) fusion proteins. HK and HKa were purified proteins. GST was recombinant. All proteins contained <0.01 EU/ml endotoxin. For all experiments, 2 X 106/ml mononuclear cells/ml were preincubated with monoclonal antibodies, murine IgG (both at 1.8 mM) or HANKS buffer containing 0.15 M NaCl, pH 7.4 for 30 minutes at 37°C. HK, HKa, GST-D3, GST-E7P, GST-D5 or GST all at 600 nM were added. Centrifugation allowed separation of the mononuclear cell suspension into cells and supernatant. The latter was used for assay of interleukin-1β (IL-1β) by ELISA. HK and all fragments tested stimulated secretion of IL-1β of 84.8 to 306.3 pg/ml when incubated with mononuclear cells for 30 minutes at 37°C. Anti-Mac-1 antibody inhibited IL-1β secretion by HK 100%, by HKa 89%, by GST-D3 78%, by GST-E7P 94% and by GST-D5 98%. Anti-uPAR antibody inhibited IL-1β release by HK 88%, by HKa 77%, by GST-D3 95%, by GST-E7P 85%, and by GST-D5 76%. Inhibition by both receptor antibodies is consistent with their known complex formation. A monoclonal antibody (mAb) to HK D5 (C11C1) and a mAb to HK D3 (2B5) both inhibited IL-1β release by HK, HKa, GST-D5 and GST-D3 indicate that both D3 and D5 are important in cytokine release. Murine IgG gave 0% inhibition in all studies. These results indicate that kininogen may contribute to the pathogenesis of inflammatory diseases by releasing IL-1β from human blood mononuclear cells.

scholarly journals Characterization of the procoagulant chain derived from human high molecular weight kininogen (Fitzgerald factor) by human tissue kallikrein

Blood ◽  
1983 ◽  
Vol 62 (2) ◽  
pp. 457-463 ◽  
Author(s):  
M Maier ◽  
KF Austen ◽  
J Spragg
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Broad Band ◽  
Limited Proteolysis ◽  
Procoagulant Activity ◽  
Plasma Kallikrein ◽  
High Molecular Weight Kininogen ◽  
Single Chain ◽  
Amino Terminal ◽  
A Chain

Abstract Human high molecular weight kininogen (HMWK), a single-chain protein with mol wt 120,000, is cleaved by human urinary kallikrein (HUK) to release kinin from within a disulfide loop and form a two-chain protein that retains all the procoagulant activity of the native molecule. Cleavage of HMWK by HUK is associated with a reduction in size to mol wt 115,000, as assessed by SDS-PAGE of unreduced protein, whereas the two chains of the reduced protein present together as a single broad band with mol wt 64,000. The 64,000 chain with procoagulant activity was chromatographically separated from the nonfunctional chain of similar size. The homogeneous procoagulant chain had an amino acid composition similar to that of smaller procoagulant (“light”) chains isolated by others upon cleavage of HMWK with plasma kallikrein and elicited an antiserum that was monospecific by Ouchterlony analysis and inhibited the procoagulant function of HMWK. Thus, the limited proteolysis of HMWK by HUK has permitted, for the first time, the isolation of a stable procoagulant chain that is equal in size to the nonfunctional chain. The common terminology of “heavy” and “light” chain for kinin-free kininogen obtained with plasma kallikrein reflects the continued degradation of the procoagulant carboxyterminal chain and is not appropriate for the initial two-chain product formed when kinin is released from HMWK. It is proposed that the initial cleavage products of HMWK be designated the A-chain, the B-fragment, and the C- chain, representing the amino-terminal chain, the released vasoactive peptide containing the bradykinin sequence, and the carboxy-terminal procoagulant chain, respectively. Thus, intact HMWK would contain, in sequence, A, B, and C regions.

scholarly journals Inhibition of cell adhesion by high molecular weight kininogen.

1992 ◽  
Vol 116 (2) ◽  
pp. 465-476 ◽  
Author(s):  
S Asakura ◽  
R W Hurley ◽  
K Skorstengaard ◽  
I Ohkubo ◽  
D F Mosher
Keyword(s):  
Molecular Weight ◽  
Cell Adhesion ◽  
High Molecular Weight ◽  
High Molecular Weight Kininogen ◽  
Single Chain ◽  
Adhesive Protein ◽  
Rich Domain ◽  
Growth Promoting ◽  
Heparin Affinity ◽  
Mononuclear Blood Cells

An anti-cell adhesion globulin was purified from human plasma by heparin-affinity chromatography. The purified globulin inhibited spreading of osteosarcoma and melanoma cells on vitronectin, and of endothelial cells, platelets, and mononuclear blood cells on vitronectin or fibrinogen. It did not inhibit cell spreading on fibronectin. The protein had the strongest antiadhesive effect when preadsorbed onto the otherwise adhesive surfaces. Amino acid sequence analysis revealed that the globulin is cleaved (kinin-free) high molecular weight kininogen (HKa). Globulin fractions from normal plasma immunodepleted of high molecular weight kininogen (HK) or from an individual deficient of HK lacked adhesive activity. Uncleaved single-chain HK preadsorbed at neutral pH, HKa preadsorbed at pH greater than 8.0, and HKa degraded further to release its histidine-rich domain had little anti-adhesive activity. These results indicate that the cationic histidine-rich domain is critical for anti-adhesive activity and is somehow mobilized upon cleavage. Vitronectin was not displaced from the surface by HKa. Thus, cleavage of HK by kallikrein results in both release of bradykinin, a potent vasoactive and growth-promoting peptide, and formation of a potent anti-adhesive protein.

Induction of Endothelial Cell Apoptosis by Cleaved High Molecular Weight Kininogen Is Mediated Through a Lck and p53-Dependent Pathway

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3305-3305
Author(s):  
Venkaiah Betapudi ◽  
Keith R. McCrae
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
High Molecular Weight ◽  
Cell Apoptosis ◽  
Negative Regulator ◽  
Dependent Manner ◽  
High Molecular Weight Kininogen ◽  
Endothelial Cell Apoptosis ◽  
Single Chain

Abstract Abstract 3305 Background and objective: High molecular weight kininogen (HK) is an abundant plasma protein that serves as an important component of the intrinsic pathway of coagulation. HK normally circulates as in the single chain form, but may be cleaved by plasma kallikrein to release the nonapeptide bradykinin, resulting in the formation cleaved high molecular weight kininogen (HKa) that consists of a heavy and light chain linked by a single disulfide bond. Conformational changes occurring after kallikrein cleavage result in increased exposure of histidine and glycine-rich regions with kininogen domain 5 that impart HKa with unique properties, including the ability to inhibit angiogenesis by causing selective apoptosis of proliferating endothelial cells. However, the receptors that mediate the antiangiogenic activity of HKa remain controversial, and the signaling pathways that lead to apoptosis have not been defined. Previous studies suggested possible involvement of SRC family kinases (SFK) in this process, and the purpose of this work was to further define the activation of SFKs and their downstream targets during HKa-induced endothelial cell apoptosis. Results: We first assessed the activation of SFKs in proliferating endothelial cells stimulated with bFGF before and after incubation with HKa (6–20 nM). SFKs are maintained in an inactive state through tyrosine phosphorylation of their C-terminal region mediated by the negative regulator C-terminal Src kinase (Csk). Exposure of endothelial cells to HKa caused downregulation of Csk in a dose-dependent manner within 60 minutes. In parallel, we observed a significant increase in expression of the proapoptotic SFK Lck in endothelial cells exposed to HKa, though expression of other SFKs including Lyn, Fyn, Src, Hck and Blk were not significantly altered. Increased expression of Lck was associated with activation of p53 and increased expression of the pro-apoptotic Bcl-2 family members Bax and Bak. Endothelial cell lysates prepared within 60 minutes of exposure to HKa demonstrated significant increases in the activity of caspases 3 and 7, as well as depletion of DNA fragmentation factors (DFF) 45 and 35, which cleave and inactivate DFF40, a major endonuclease involved in apoptosis. In parallel studies, endothelial cells depleted of Lck by treatment with Lck siRNA displayed loss of p53 phosphorylation, caspase 3 and 7 activity, and expression of Bax and Bad with no effects on the expression of Bad and Bid. Conclusion: These findings demonstrate a critical role for Csk in regulation of SFK activation and endothelial homeostasis, and demonstrate that downregulation of Csk by HKa leads to activation of a Lck-dependent, p53-mediated apoptotic pathway. Increasing the expression of Lck may represent a novel mechanism for regulation of aberrant angiogenesis. Disclosures: No relevant conflicts of interest to declare.

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