Cleaved high molecular weight kininogen binds directly to the integrin CD11b/CD18 (Mac-1) and blocks adhesion to fibrinogen and ICAM-1

Blood ◽  
2000 ◽  
Vol 95 (12) ◽  
pp. 3788-3795 ◽  
Author(s):  
Nijing Sheng ◽  
Michael B. Fairbanks ◽  
Robert L. Heinrikson ◽  
Gabriela Canziani ◽  
Irwin M. Chaiken ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Direct Interaction ◽  
Cellular Adhesion ◽  
Intercellular Adhesion Molecule ◽  
Dependent Manner ◽  
High Molecular Weight Kininogen ◽  
Intercellular Adhesion Molecule 1 ◽  
Free System ◽  
Transfected Cells

High molecular weight kininogen (HK) and its cleaved form (HKa) have been shown to bind to neutrophils. Based on studies using monoclonal antibodies (mAbs), we postulated that CD11b/CD18 (Mac-1) might be the receptor on the neutrophils for binding to HK/HKa. However, the direct interaction of HK/HKa and Mac-1 had not been demonstrated. We therefore transfected HEK 293 cells with human Mac-1. Cell binding assays using fluorescein isothiocyanate-labeled HKa showed increased binding to the Mac-1 transfected cells compared with the control transfected cells. The binding was specific because unlabeled HKa, Mac-1–specific antibody, and fibrinogen can inhibit the binding of biotin-HKa to Mac-1 transfected cells. HKa bound to Mac-1 transfected cells (20 000 molecules/cell) with a Kd = 62 nmol/L. To demonstrate directly the formation of a complex between HKa and Mac-1, we examined the interaction of HKa and purified Mac-1 in a cell-free system using an IAsys resonant mirror optical biosensor. The association and dissociation rate constants (kon and koff, respectively) were determined, and they yielded a dissociation constant (Kd) of 3.2×10−9mol/L. The functional significance of direct interaction of HKa to Mac-1 was investigated by examining the effect of HKa on cellular adhesion to fibrinogen and intercellular adhesion molecule-1 (ICAM-1), molecules abundant in the injured vessel wall. HKa blocked the adhesion of Mac-1 transfected cells to fibrinogen and ICAM-1 in a dose-dependent manner. Thus, HKa may interrupt Mac-1–mediated cell–extracellular matrix and cell–cell adhesive interactions and may therefore influence the recruitment of circulating neutrophils/monocytes to sites of vessel injury.

Cleaved high molecular weight kininogen binds directly to the integrin CD11b/CD18 (Mac-1) and blocks adhesion to fibrinogen and ICAM-1

Blood ◽  
2000 ◽  
Vol 95 (12) ◽  
pp. 3788-3795 ◽  
Author(s):  
Nijing Sheng ◽  
Michael B. Fairbanks ◽  
Robert L. Heinrikson ◽  
Gabriela Canziani ◽  
Irwin M. Chaiken ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Direct Interaction ◽  
Cellular Adhesion ◽  
Intercellular Adhesion Molecule ◽  
Dependent Manner ◽  
High Molecular Weight Kininogen ◽  
Intercellular Adhesion Molecule 1 ◽  
Free System ◽  
Transfected Cells

Abstract High molecular weight kininogen (HK) and its cleaved form (HKa) have been shown to bind to neutrophils. Based on studies using monoclonal antibodies (mAbs), we postulated that CD11b/CD18 (Mac-1) might be the receptor on the neutrophils for binding to HK/HKa. However, the direct interaction of HK/HKa and Mac-1 had not been demonstrated. We therefore transfected HEK 293 cells with human Mac-1. Cell binding assays using fluorescein isothiocyanate-labeled HKa showed increased binding to the Mac-1 transfected cells compared with the control transfected cells. The binding was specific because unlabeled HKa, Mac-1–specific antibody, and fibrinogen can inhibit the binding of biotin-HKa to Mac-1 transfected cells. HKa bound to Mac-1 transfected cells (20 000 molecules/cell) with a Kd = 62 nmol/L. To demonstrate directly the formation of a complex between HKa and Mac-1, we examined the interaction of HKa and purified Mac-1 in a cell-free system using an IAsys resonant mirror optical biosensor. The association and dissociation rate constants (kon and koff, respectively) were determined, and they yielded a dissociation constant (Kd) of 3.2×10−9mol/L. The functional significance of direct interaction of HKa to Mac-1 was investigated by examining the effect of HKa on cellular adhesion to fibrinogen and intercellular adhesion molecule-1 (ICAM-1), molecules abundant in the injured vessel wall. HKa blocked the adhesion of Mac-1 transfected cells to fibrinogen and ICAM-1 in a dose-dependent manner. Thus, HKa may interrupt Mac-1–mediated cell–extracellular matrix and cell–cell adhesive interactions and may therefore influence the recruitment of circulating neutrophils/monocytes to sites of vessel injury.

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.

Inhibition of tumor angiogenesis in vivo by a monoclonal antibody targeted to domain 5 of high molecular weight kininogen

Blood ◽  
2004 ◽  
Vol 104 (7) ◽  
pp. 2065-2072 ◽  
Author(s):  
James S. Song ◽  
Irma M. Sainz ◽  
Stephen C. Cosenza ◽  
Irma Isordia-Salas ◽  
Abdel Bior ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Cell Growth ◽  
Tumor Cell ◽  
High Molecular Weight ◽  
Control Group ◽  
Dependent Manner ◽  
Tumor Cell Growth ◽  
High Molecular Weight Kininogen

Abstract We have shown that human high molecular weight kininogen is proangiogenic due to release of bradykinin. We now determined the ability of a murine monoclonal antibody to the light chain of high molecular weight kininogen, C11C1, to inhibit tumor growth compared to isotype-matched murine IgG. Monoclonal antibody C11C1 efficiently blocks binding of high molecular weight kininogen to endothelial cells in a concentration-dependent manner. The antibody significantly inhibited growth of human colon carcinoma cells in a nude mouse xenograft assay and was accompanied by a significant reduction in the mean microvascular density compared to the IgG control group. We also showed that a hybridoma producing monoclonal antibody C11C1 injected intramuscularly exhibited markedly smaller tumor mass in a syngeneic host compared to a hybridoma producing a monoclonal antibody to the high molecular weight kininogen heavy chain or to an unrelated plasma protein. In addition, tumor inhibition by purified monoclonal antibody C11C1 was not due to direct antitumor effect because there was no decrease of tumor cell growth in vitro in contrast to the in vivo inhibition. Our results indicate that monoclonal antibody C11C1 inhibits angiogenesis and human tumor cell growth in vivo and has therapeutic potential for treatment of human cancer. (Blood. 2004;104:2065-2072)

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.

HIGH MOLECULAR WEIGHT KININOGEN SPECIFICALLY BLOCKS THROMBIN-INDUCED AGGREGATION BY INHIBITING PLATELET CALPAIN

1987 ◽  
Author(s):  
R N Puri ◽  
F Zhou ◽  
H Bradford ◽  
E J Gustafson ◽  
R F Colman ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Platelet Aggregation ◽  
High Molecular Weight ◽  
Metabolic Inhibitors ◽  
Dependent Manner ◽  
High Molecular Weight Kininogen ◽  
Normal Plasma ◽  
Calpain 2 ◽  
Induced Aggregation ◽  
Aggregation Of Platelets

We have previously shown that platelet-aggregation induced by alpha-thrombin (1.7 nM) involves complete cleavage of the surface membrane polypeptide, Mr = 100 kDa (MP 100) labeled by FSBA in intact platelets. The failure to cleave MP100 in membrane preparations or in platelets treated with metabolic inhibitors or leupeptin, suggested that thrombin was acting by activating platelet calpain. Since high molecular weight kininogen (HMWK) is the most potent plasma inhibitor of calpain(s), we now report that HMWK inhibited thrombin-induced aggregation in a dose-dependent manner over a range of plasma concentrations. HMWK did not inhibit aggregation induced by ADP, collagen, U46619, A23187 and/or PMA. In order to study the action of HMWK in a plasma environment we utilized Y-thrombin. The aggregation induced by γ-thrombin (25 nM) in washed human platelets was also inhibited by HMWK. A much higher concentration of γ - thrombin (200 nM) was required to induce similar aggregation of platelets suspended in normal plasma. In contrast, γ -thrombin (50nM) induced complete aggregation of platelets suspended in plasma completely deficient in total kininogen indicating that a kininogen was predominently responsible for the inhibitory effect of plasma. When platelets were suspended in plasma deficient only in HMWK, aggregation required 75 nMY-thrombin. When plasma deficient in HMWK was supplemented with a physiological concentration of HMWK (0.67 μM) the aggregation of suspended washed platelets was similar to that in normal plasma. Finally, we found that purified platelet calpain-2 not only exposed fibrinogen binding sites and induced platelet aggregation, but also completely cleaved MP 100 in both intact platelets and membrane preparations. We conclude: a) Thrombin-induced platelet aggregation involves the indirect proteolytic cleavage of MP100 by activating calpain-2, and b) Inhibition of thrombin-induced platelet aggregation by HMWK involves specific inactivation of platelet calpain.

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

Abstract 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.

Cleavage of high-molecular-weight kininogen by elastase and tryptase is inhibited by ferritin

2008 ◽  
Vol 294 (3) ◽  
pp. L505-L515 ◽  
Author(s):  
Lan G. Coffman ◽  
Julie C. Brown ◽  
David A. Johnson ◽  
Narayanan Parthasarathy ◽  
Ralph B. D'Agostino ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Solid Phase ◽  
Direct Interaction ◽  
Human Monocyte ◽  
Biologically Active ◽  
High Molecular Weight Kininogen ◽  
Binding Studies ◽  
Iron Storage

Ferritin is a protein principally known for its role in iron storage. We have previously shown that ferritin can bind high-molecular-weight kininogen (HK). Upon proteolytic cleavage by the protease kallikrein, HK releases the proinflammatory peptide bradykinin (BK) and other biologically active products, such as two-chain high-molecular-weight kininogen, HKa. At inflammatory sites, HK is oxidized, which renders it a poor substrate for kallikrein. However, oxidized HK remains a good substrate for elastase and tryptase, thereby providing an alternative cleavage mechanism for HK during inflammation. Here we report that ferritin can retard the cleavage of both native HK and oxidized HK by elastase and tryptase. Initial rates of cleavage were reduced 45–75% in the presence of ferritin. Ferritin is not a substrate for elastase or tryptase and does not interfere with the ability of either protease to digest a synthetic substrate, suggesting that ferritin may impede HK cleavage through direct interaction with HK. Immunoprecipitation and solid phase binding studies reveal that ferritin and HK bind directly with a Kdof 134 nM. To test whether ferritin regulates HK cleavage in vivo, we used THP-1 cells, a human monocyte/macrophage cell line that has been used to model pulmonary inflammatory cells. We observed that ferritin impedes the cleavage of HK by secretory proteases in stimulated macrophages. Furthermore, ferritin, HK, and elastase are all present in or on alveolar macrophages in a mouse model of pulmonary inflammation. Collectively, these results implicate ferritin in the modulation of HK cleavage at sites of inflammation.

scholarly journals The autoactivation of rabbit hageman factor

1979 ◽  
Vol 150 (5) ◽  
pp. 1122-1133 ◽  
Author(s):  
RC Wiggins ◽  
CC Cochrane
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Clotting Factor ◽  
Dependent Manner ◽  
High Molecular Weight Kininogen ◽  
Single Chain ◽  
Hageman Factor ◽  
Reducing Conditions ◽  
Factor Α

Proteolytic cleavage and activation of isolated, single chain, zymogen Hageman factor was observed in the presence of kaolin alone. The rate of cleavage of kaolin-bound Hageman factor was enhanced 50-fold by the presence of prekallikrein and high molecular weight kininogen. The two-chain 82,000 dalton form of activated Hageman factor (α-HF(a)) also cleaved kaolin- bound single-chain Hageman factor in a dose-dependent manner, yielding fragments of 28,000 and, 50,000 dahons under reducing conditions. Cleavage of kaolin-bound single-chain Hageman factor was not inhibited by preincubation with diisopropylfluorophosphate (12 mM) for 10 min, but long-term incubation of Hageman factor with diisopropylfluorophosphate (up to 48 h) resulted in inhibition of cleavage of kaolin-bound Hageman factor to an extent proportional to the inhibition of procoagulant Hageman factor activity. Hageman factor cleavage was maximal when the kaolin concentration was {approximately} 10-fold greater than the Hageman factor concentration (wt:wt), and was partially inhibited by high molecular weight kininogen. Kaolin-bound Hageman factor cleaved clotting factor XI in an amount which correlated with the extent of cleavage of the Hageman factor. These findings are compatible with the concept that single-chain Hageman factor and α- HF(a), are both capable of cleaving and activating kaolin-bound Hageman factor and that a close molecular association of kaolin-bound Hageman factor molecules is required for this reaction.

Inhibition of angiogenesis by two-chain high molecular weight kininogen (HKa) and kininogen-derived polypeptides

2002 ◽  
Vol 80 (2) ◽  
pp. 85-90 ◽  
Author(s):  
Jing-Chuan Zhang ◽  
Xiaoping Qi ◽  
Jose' Juarez ◽  
Marian Plunkett ◽  
Fernando Donaté ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
High Molecular Weight ◽  
Cell Apoptosis ◽  
Endothelial Cell Proliferation ◽  
Dependent Manner ◽  
High Molecular Weight Kininogen ◽  
Endothelial Cell Apoptosis ◽  
Domain 3

We recently reported that the two-chain form of human high molecular weight kininogen (HKa) inhibits angiogenesis by inducing endothelial cell apoptosis (Zhang et al. 2000). This property appears to be primarily conferred by HKa domain 5 (HKa D5). In this manuscript, we further characterize the activity of these polypeptides toward proliferating endothelial cells, as well as their in vivo anti-angiogenic activity in the chick chorioallantoic membrane (CAM). We also demonstrate that short peptides derived from endothelial cell binding regions in HKa domains 3 and 5 inhibit endothelial cell proliferation and induce endothelial cell apoptosis. Like HKa and HKa D5, peptides derived from the latter domain induce endothelial cell apoptosis in a Zn2+-dependent manner, while those derived from domain 3 function independently of Zn2+. The implications of these findings to the regulation of angiogenesis and development of anti-angiogenic therapeutics are discussed.Key words: angiogenesis, kininogen, endothelial cells, apoptosis, peptides.

Different mechanisms define the antiadhesive function of high molecular weight kininogen in integrin- and urokinase receptor–dependent interactions

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 514-522 ◽  
Author(s):  
Triantafyllos Chavakis ◽  
Sandip M. Kanse ◽  
Florea Lupu ◽  
Hans-Peter Hammes ◽  
Werner Müller-Esterl ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
High Molecular Weight ◽  
Urokinase Receptor ◽  
Immunogold Electron Microscopy ◽  
Dependent Manner ◽  
High Molecular Weight Kininogen ◽  
Terminal Portion ◽  
Single Chain ◽  
Amino Terminal

Abstract Proteolytic cleavage of single-chain high molecular weight kininogen (HK) by kallikrein releases the short-lived vasodilator bradykinin and leaves behind 2-chain high molecular weight kininogen (HKa) that has been previously reported to exert antiadhesive properties as well as to bind to the urokinase receptor (uPAR) on endothelial cells. In this study we defined the molecular mechanisms for the antiadhesive effects of HKa related to disruption of integrin- and uPAR-mediated cellular interactions. Vitronectin (VN) but not fibrinogen or fibronectin-dependent vβ3 integrin–mediated adhesion of endothelial cells was blocked by HKa or its isolated domain 5. In a purified system, HKa but not HK competed for the interaction of VN with vβ3 integrin, because HKa and the isolated domain 5 but not HK bound to both multimeric and native VN in a Zn2+-dependent manner. The interaction between HKa or domain 5 with VN was prevented by heparin, plasminogen activator inhibitor-1, and a recombinant glutathione-S-transferase (GST)-fusion peptide GST-VN (1-77) consisting of the amino terminal portion of VN (amino acids 1-77), but not by a cyclic arginyl-glycyl-aspartyl peptide, indicating that HKa interacts with the amino terminal portion of VN (“somatomedin B region”). Furthermore, we have confirmed that HKa but not HK bound to uPAR and to the truncated 2-domain form of uPAR lacking domain 1 in a Zn2+-dependent manner. Through these interactions, HKa or its recombinant His-Gly-Lys–rich domain 5 completely inhibited the uPAR-dependent adhesion of myelomonocytic U937 cells and uPAR-transfected BAF-3 cells to VN and thereby promoted cell detachment. By immunogold electron microscopy, both VN and HK/HKa were found to be colocalized in sections from human atherosclerotic coronary artery, indicating that the described interactions are likely to take place in vivo. Taken together, HK and HKa inhibit different VN-responsive adhesion receptor systems and may thereby influence endothelial cell- or leukocyte-related interactions in the vasculature, particularly under inflammatory conditions.

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