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.

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.

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.

scholarly journals Human high molecular weight kininogen binds to human umbilical vein endothelial cells via its heavy and light chains

Blood ◽  
1993 ◽  
Vol 81 (5) ◽  
pp. 1306-1311 ◽  
Author(s):  
SR Reddigari ◽  
P Kuna ◽  
G Miragliotta ◽  
Y Shibayama ◽  
K Nishikawa ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Light Chain ◽  
Binding Sites ◽  
Umbilical Vein ◽  
Light Chains ◽  
Dependent Manner ◽  
High Molecular Weight Kininogen ◽  
High Affinity ◽  
Umbilical Vein Endothelial Cells

Abstract High molecular weight kininogen (HK) is a multifunctional plasma glycoprotein that occupies a critical position in pathways that link inflammation and coagulation. Excision of the vasoactive peptide bradykinin by plasma kallikrein results in kinin-free HK that consists of a 65-Kd N-terminal heavy chain (HK-HC) linked to the C-terminal 45- Kd light chain (HK-LC) by a disulfide bridge. HK-HC is an inhibitor of SH-proteases and HK-LC contains the binding sites for coagulation cofactors prekallikrein and factor XI. HK has previously been shown to bind specifically to human umbilical vein endothelial cells (HUVEC) in a zinc(2+)-dependent manner by a single class of high-affinity binding sites. We have further characterized that interaction in order to determine the cell-binding regions of HK. Competition binding experiments have indicated that either HK-LC or HK-HC was able to inhibit the binding of labeled HK with a 50% inhibitory concentration (IC50) of 77 nmol/L and 89 nmol/L, respectively. Cleaved two-chain HK (HKa) had an IC50 of 73 nmol/L, whereas uncleaved HK had an IC50 of 335 nmol/L. Direct binding experiments have indicated that HUVEC bind both purified [125I]HK-HC and [125I]HK-LC in a zinc(2+)-dependent manner and that HK-LC did not displace bound HK-HC. The light chain of low molecular weight kininogen or prekallikrein-binding region of HK did not inhibit the binding of HK to HUVEC. Our results, therefore, indicate that (1) HK is capable of binding to endothelial cells via both heavy and light chain moieties, (2) HKa has a higher affinity to HUVEC, and (3) purified heavy and light chains are capable of directly binding to HUVEC. The data are consistent with the presence of a single high-affinity site for HK on endothelial cells within which are subsites that bind to heavy and light chains.

scholarly journals Composition of the von Willebrand factor storage organelle (Weibel-Palade body) isolated from cultured human umbilical vein endothelial cells.

1987 ◽  
Vol 104 (5) ◽  
pp. 1423-1433 ◽  
Author(s):  
B M Ewenstein ◽  
M J Warhol ◽  
R I Handin ◽  
J S Pober
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
High Molecular Weight ◽  
Von Willebrand Factor ◽  
Umbilical Vein ◽  
Proteolytic Processing ◽  
Storage Organelle ◽  
Umbilical Vein Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor

von Willebrand factor (VWF) is a large, adhesive glycoprotein that is biosynthesized and secreted by cultured endothelial cells (EC). Although these cells constitutively release VWF, they also contain a storage pool of this protein that can be rapidly mobilized. In this study, a dense organelle fraction was isolated from cultured umbilical vein endothelial cells by centrifugation on a self-generated Percoll gradient. Stimulation of EC by 4-phorbol 12-myristate 13-acetate (PMA) resulted in the disappearance of this organelle fraction and the synchronous loss of Weibel-Palade bodies as judged by immunoelectron microscopy. Electrophoretic and serologic analyses of biosynthetically labeled dense organelle fraction revealed that it is comprised almost exclusively of VWF and its cleaved pro sequence. These two polypeptides were similarly localized exclusively to Weibel-Palade bodies by ultrastructural immunocytochemistry. The identity of the dense organelle as the Weibel-Palade body was further established by direct morphological examination of the dense organelle fraction. The VWF derived from this organelle is distributed among unusually high molecular weight multimers composed of fully processed monomeric subunits and is rapidly and quantitatively secreted in unmodified form after PMA stimulation. These studies: establish that the Weibel-Palade body is the endothelial-specific storage organelle for regulated VWF secretion; demonstrate that in cultured EC, the VWF concentrated in secretory organelles is of unusually high molecular weight and that this material may be rapidly mobilized in unmodified form; imply that proteolytic processing of VWF involved in regulated secretion takes place after translocation to the secretory organelle; provide a basis for further studies of intracellular protein trafficking in EC.

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