Human Hageman factor (factor XII) and high molecular weight kininogen compete for the same binding site on human umbilical vein endothelial cells

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

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

Thrombosis and Haemostasis ◽

10.1160/th03-04-0231 ◽

2003 ◽

Vol 90 (11) ◽

pp. 787-795 ◽

Cited By ~ 17

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.

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Studies of the Factor Xa-Dependent Inhibitor of Factor VIIa/Tissue Factor (Extrinsic Pathway Inhibitor) from Cell Supernates of Cultured Human Umbilical Vein Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646535 ◽

1989 ◽

Vol 61 (01) ◽

pp. 101-105 ◽

Cited By ~ 27

Author(s):

Bonnie J Warn-Cramer ◽

Fanny E Almus ◽

Samuel I Rapaport

Keyword(s):

Molecular Weight ◽

Endothelial Cells ◽

Tissue Factor ◽

Phorbol Ester ◽

Umbilical Vein ◽

Primary Cultures ◽

Factor Xa ◽

Extrinsic Pathway ◽

Human Umbilical Vein ◽

Umbilical Vein Endothelial Cells

SummaryCultured human umbilical vein endothelial cells (HUVEC) have been reported to produce extrinsic pathway inhibitor (EPI), the factor Xa-dependent inhibitor of factor VHa/tissue factor (TF). We examined the release of this inhibitor from HUVEC as a function of their growth state and in response to the induction of endothelial cell TF activity. HUVEC constitutively produced significant amounts of EPI at all stages of their growth in culture including the post-confluent state. Rate of release varied over a 3-fold range for primary cultures from 12 different batches of pooled umbilical cord cells. Constitutive EPI release was unaltered during a 6 hour period of induction of TF activity with thrombin or phorbol ester but slowed during longer incubation of the cells with phorbol ester. Whereas plasma contains two molecular weight forms of EPI, only the higher of these two molecular weight forms was demonstrable by Western analysis of HUVEC supernatants with 125I-factor Xa as the ligand.

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Assembly and Activation of the Intrinsic Fibrinolytic Pathway on the Surface of Human Endothelial Cells in Culture

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649800 ◽

1995 ◽

Vol 74 (02) ◽

pp. 698-703 ◽

Cited By ~ 29

Author(s):

Catherine Lenich ◽

Ralph Pannell ◽

Victor Gurewich

Keyword(s):

Endothelial Cells ◽

Umbilical Vein ◽

Factor Xii ◽

Plasminogen Activation ◽

High Molecular Weight Kininogen ◽

Human Endothelial Cells ◽

Intrinsic Pathway ◽

Local Fibrinolysis ◽

Umbilical Vein Endothelial Cells ◽

And Function

SummaryFactor XII has long been implicated in the intrinsic pathway of fibrinolysis, but the mechanism by which it triggers plasminogen activation and targets fibrinolysis has not been established. In the present study, the assembly and function of activated Factor XII (F.XIIa), prourokinase (pro-u-PA), high molecular weight kininogen (H-kininogen), and prekallikrein on human umbilical vein endothelial cells (HUVEC) was investigated. 125I-prekallikrein was shown to bind to HUVEC via receptor-bound H-kininogen in the presence of 50 μM ZnCl2. After the addition of F.XIIa, 78% of the 125I-prekallikrein initially bound to HUVEC was converted to 125I-kallikrein. However, only 6% of the HUVEC-bound 125I-pro-u-PA was thereby activated. This discrepancy was shown to be related to rapid dissociation (>50% within 15 min) of prekallikrein/kallikrein, but not pro-u-PA, from HUVEC. Increasing the level of cell-bound kallikrein increased the portion of cell-bound pro-u-PA activated, indicating that their co-localization was important for this pathway. Finally, F.XIIa was shown to trigger plasminogen activation on HUVEC via this pathway. This assembly of reactants on the endothelium suggests a mechanism whereby local fibrinolysis may be triggered by blood coagulation.

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

Blood ◽

10.1182/blood.v90.2.690.690_690_697 ◽

1997 ◽

Vol 90 (2) ◽

pp. 690-697 ◽

Cited By ~ 1

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.

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