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Alvin H. Schmaier - One of the best experts on this subject based on the ideXlab platform.
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Why do we want to know how factor XII levels are modulated
Thrombosis research, 2009Co-Authors: Alvin H. SchmaierAbstract:New interest in factor XII has been found due to the fact that its murine knockout animal is protected from arterial thrombosis in several models (1,2). The mechanism(s) for these observations have not been precisely described. However, several investigators have shown that factor XII converting to factor XIIa presumably by autoactivation on surfaces such as platelet polysomes, expressed RNA, exposed vascular collagen, and/or aggregated protein contributes to developing thrombus (3–6). Since factor XII participates in the size of an induced-clot formation it has a role in thrombosis. This activity describes for the first time an in vivo pathophysiologic event contributed to by factor XII/XIIa. It is known from the observations of Ratnoff and others that factor XII is essential for surface activated blood coagulation reactions (7). Recognition that factor XII initiates test-tube blood coagulations was the basis for the waterfall hypothesis for the blood coagulation system (8). However, factor XII deficient patients do not have a bleeding defect even though their surface-activated blood coagulation tests are prolonged. Thus, factor XII is not a hemostatic protein. The dichotomy of factor XII being needed for normal blood coagulation tests, but not hemostasis has confused this field for decades. Now that it is generally agreed that tissue factor is the physiologic initiator of blood coagulation leading to hemostasis, the contact activation system needs to be redefined (9,10). Contact activation mechanisms explain how common blood coagulations tests like the activated partial thromboplastin time (APTT) or activated coagulation time (ACT) which are performed on millions of patients annually work. The molecular mechanism for factor XII autoactivation still is not known. Factor XII autoactivation at the molecular level occurs by imposing specific orientation and ordering on the adsorbed protein molecules on sum frequency generation vibrational spectroscopy (11). Alternatively, factor XII in vivo may be activated by contact as described above with various biologic substances in developing thrombus or secondary to formed plasma kallikrein, its only known enzyme activator. The kinetics of factor XII activation by plasma kallikrein (Km=11 µM) is similar to factor XIIa activation of prekallikrein (Km=2.4 µM). However, the Km of prekallikrein activation in vivo by its endothelial cell membrane expressed serine protease, prolylcarboxypeptidase, is 9 nM (12). These latter kinetic data suggest that physiologic factor XII activation is secondary to plasma kallikrein formation. However, it has yet to be shown if prolylcarboxypeptidase is important for in vivo prekallikrein activation for factor XII activation. Although C1 inhibitor deficient mice have angioedema, indicating higher constitutive bradykinin formation from either unregulated plasma kallikrein or factor XIIa, factor XII deficient mice have lower levels of basal plasma bradykinin, indicating factor XII’s importance in the peptide’s formation (13,14). The observation that the presence of factor XII contributes to the size of induced thrombus formation indicates that its inhibition may be a pharmacologic target to decrease the extent of thrombosis. Although factor XII deficiency was initially thought to be a risk factor for venous thrombosis, more recent studies indicate that it is not. However, human in vivo studies on the role of factor XII in arterial thrombosis also are not clear. Kanaji et al. reported that a polymorphism 46C to T substitution in the 5’-untranslated region of factor XII gene is associated with low translational efficiency and decreased plasma levels (15). This polymorphism may account for the commonly recognized fact that factor XII levels are lower in Asians when compared to Caucasians. An initial report suggested that the homozygous 46TT allele protects from acute coronary syndrome and that lower XII levels may be associated with lower thrombosis risk (16,17). Two other reports suggested that opposite, i.e., the TT genotype is associated with a higher risk of coronary artery disease (18,19). The degree of lowering of factor XII levels by the TT allele is probably insufficient to significantly reduce the amplifying effect of factor XII’s autoactivation on developing clot to really influence thrombosis risk (20). Not much is known how factor XII levels in plasma are regulated. Estrogen therapy increases plasma factor XII levels which are mainly produced in the liver as zymogens. The factor XII gene contains a sequence resembling the consensus estrogen-responsive element (20). The hepatocyte nuclear factor-4 (HNF-4) transcription factor inhibits estrogen induction of factor XII promoter in fibroblasts but not in HepG2 cells where its binding contributes to estrogen induction of the gene (21). HNF-4 null mice exhibit reduced expression of factor XII (22). In the present report of Thrombosis Research, Sabater-Lleal et al. contribute to our understanding on what regulates factor XII plasma levels (23). These authors describe two new mutations, a C/G substitution at position -8 and a C/T substitution at position -13 of the factor XII gene that result in decreased expression levels. These mutations interfere with nuclear proteins interacting with the factor XII promoter. This region is in the putative HNF-4 binding site of the factor XII gene promoter. These polymorphisms summated with the 46C/T polymorphism to lower plasma factor XII levels. What is the physiologic role of factor XII? The answer to this question also is not completely known but new interpretations are coming to this field. In addition to its possible influence on arterial thrombosis, factor XII also may be a growth factor. In the intravascular compartment, factor XII has a multiprotein receptor that consists of the urokinase plasminogen activator receptor, gC1qR, and Cytokeratin 1 (24). Factor XII has been recognized as a growth factor leading to ERK1/2 activation (25). Factor XII’s artificial and cell binding site has been localized to its fibronectin type II domain which is adjacent to its epidermal growth factor domain, suggesting its growth promoting activities may be mediated by its cell binding site. Since collagen-activated platelets provide sufficient zinc ion to promote factor XII binding to cells and developing clot and collagen exposure is sufficient for its activation in flowing blood, factor XII in the developing thrombosis may also stimulate repair for this injury (6,24–26). An appreciation for the role of factor XII as a growth factor is emerging. This activity may be important for repair after thrombosis or inflammation. Better understanding of factor XII expression and activities should lead to better characterization of thrombus formation and its repair.
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myeloperoxidase interacts with endothelial cell surface Cytokeratin 1 and modulates bradykinin production by the plasma kallikrein kinin system
American Journal of Pathology, 2007Co-Authors: Joshua M Astern, Alvin H. Schmaier, Fakhri Mahdi, William F Pendergraft, Ronald J Falk, Charles J Jennette, Gloria A PrestonAbstract:During an inflammatory state, functional myeloperoxidase (MPO) is released into the vessel as a result of intravascular neutrophil degradation. One mechanism of resulting cellular injury involves endothelial internalization of MPO, which causes oxidative damage and impairs endothelial signaling. We report the discovery of a protein that facilitates MPO internalization, Cytokeratin 1 (CK1), identified using affinity chromatography and mass spectrometry. CK1 interacts with MPO in vitro, even in the presence of 100% human plasma, thus substantiating biological relevance. Immunofluorescent microscopy confirmed that MPO added to endothelial cells can co-localize with endogenously expressed CK1. CK1 acts as a scaffolding protein for the assembly of the vasoregulatory plasma kallikrein-kinin system; thus we explored whether MPO and high molecular weight kininogen (HK) reside on CK1 together or whether they compete for binding. The data support cooperative binding of MPO and HK on cells such that MPO masked the plasma kallikrein cleavage site on HK, and MPO-generated oxidants caused inactivation of both HK and kallikrein. Collectively, interactions between MPO and the components of the plasma kallikrein-kinin system resulted in decreased bradykinin production. This study identifies CK1 as a facilitator of MPO-mediated vascular responses and thus provides a new paradigm by which MPO affects vasoregulatory systems.
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recombinant prolylcarboxypeptidase activates plasma prekallikrein
Blood, 2004Co-Authors: Zia Shariatmadar, Fakhri Mahdi, Alvin H. SchmaierAbstract:The serine protease prolylcarboxypeptidase (PRCP), isolated from human umbilical vein endothelial cells (HUVECs), is a plasma prekallikrein (PK) activator. PRCP cDNA was cloned in pMT/BIP/V5-HIS-C, transfected into Schneider insect (S2) cells, and purified from serum-free media. Full-length recombinant PRCP (rPRCP) activates PK when bound to high-molecular-weight kininogen (HK). Recombinant PRCP is inhibited by leupeptin, angiotensin II, bradykinin, anti-PRCP, diisopropyl-fluorophosphonate (DFP), phenylmethylsulfonyl fluoride (PMSF), and Z-Pro-Proaldehyde-dimethyl acetate, but not by 1 mM EDTA (ethylenediaminetetraacetic acid), bradykinin 1-5, or angiotensin 1-7. Corn trypsin inhibitor binds to prekallikrein to prevent rPRCP activation, but it does not directly inhibit the active site of either enzyme. Unlike factor XIIa, the ability of rPRCP to activate PK is blocked by angiotensin II, not by neutralizing antibody to factor XIIa. PRCP antigen is detected on HUVEC membranes using flow cytometry and laser scanning confocal microscopy. PRCP antigen does not colocalize with LAMP1 on nonpermeabilized HUVECs, but it partially colocalizes in permeabilized cells. PRCP colocalizes with all the HK receptors, gC1qR, uPAR, and Cytokeratin 1 antigen, on nonpermeabilized HUVECs. PRCP activity and antigen expression on cultured HUVECs are blocked by a morpholino antisense oligonucleotide. These investigations indicate that rPRCP is functionally identical to isolated HUVEC PRCP and is a major HUVEC membrane-expressed, PK-activating enzyme detected in the intravascular compartment. (Blood. 2004;103:4554-4561)
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the relative priority of prekallikrein and factors xi xia assembly on cultured endothelial cells
Journal of Biological Chemistry, 2003Co-Authors: Fakhri Mahdi, Zia Shariatmadar, Alvin H. SchmaierAbstract:Abstract Investigations determined the relative preference of prekallikrein (PK) or factor XI/XIa (FXI/FXIa) binding to endothelial cells (HUVECs). In microtiter plates, biotinylated high molecular weight kininogen (biotin-HK) or biotin-FXI binding to HUVEC monolayers or their matrix proteins, but not fibronectin-coated plastic microtiter plate wells, was specifically blocked by antibodies to each of the receptors of HK, uPAR, gC1qR, or Cytokeratin 1. Fluorescein isothiocyanate (FITC)-PK specifically bound to HUVEC suspensions without added Zn2+, whereas FITC-FXI or -FXIa binding to HUVEC suspensions required 10 μm added Zn2+ to support specific binding. Plasma concentrations of FXI did not block FITC-PK binding to HUVECs in the absence or presence of 10 μm Zn2+. In the absence of HK, the level of FITC-FXI or -FXIa binding was half that seen in its presence. At physiologic concentrations, PK (450 nm) abolished FITC-FXI or -FXIa binding to HUVEC suspensions in the absence or presence of HK in the presence of 10 μm Zn2+. Released Zn2+ from 2–8 × 108 collagen-activated platelets/ml supported biotin-FXI binding to HUVEC monolayers, but platelet activation was not necessary to support biotin-PK binding to HUVECs. At physiologic concentrations, PK also abolished FXI binding to HUVECs in the presence of activated platelets, but FXI did not influence PK binding. PK in the presence or absence of HK preferentially bound to HUVECs over FXI or FXIa. Elevated Zn2+ concentrations are required for FXI but not PK binding, but the presence of physiologic concentrations of PK and HK also prevented FXI binding. PK preferential binding to endothelial cells contributes to their anticoagulant nature.
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Mapping the interaction between uPAR and high molecular weight kininogen
2003Co-Authors: F. Mahdi, A. Kuo, Z. Shariat‐madar, D. B. Cines, Alvin H. SchmaierAbstract:OC124 Mapping the interaction between uPAR and high molecular weight kininogen Hall 5 09:30 15th July, 2003 Session Type: Oral communications Subject area: Contact phase Session title: Prekallikrein and kininogens Abstract: OC124 Authors: F. Mahdi*, A. Kuo+, Z. Shariat-Madar*, D. B. Cines+ & A. H. Schmaier* *University of Michigan, USA; +University of Pennsylvania, USA The urokinase plasminogen activator receptor (uPAR) is a GPI-linked receptor in membrane rafts on HUVEC that shuttles between proteins, modulating their behavior. In order to describe the regulatory functions of uPAR, its interaction with high molecular weight kininogen (HK) was mapped. Using purified HK, kallikrein- cleaved 56 kDa HK (56HKa) and 46 kDa HK (46HKa), low molecular weight kininogen (LK) and the isolated 56 kDa light chain of HK (LC), we mapped where uPAR binds to HK and where HK binds to soluble, recombinant uPAR (suPAR). The regions on HK that bind to uPAR were determined first. Biotin-HK binding to HUVEC is inhibited by HK, 56HKa or 46HKa with an IC50 of 110, 110 or 55 nM, respectively. Biotin-HK binding to suPAR also is inhibited by HK, 56HKa, or 46HKa with an IC50 of 60, 100, or 9 nM, respectively. The region on the light chain of HK and 46HKa that binds best to suPAR is H477-G496. Biotin-HK or -46HKa also binds by its heavy chain to suPAR at 10-fold lower affinity. The heavy chain HK/46HKa binding region to suPAR is C333-K345. Biotinylated peptides of these suPAR binding regions on HK specifically bind to suPAR. The HK binding site(s) on uPAR was determined next. Mab 3139 that blocks HK binding to HUVEC and suPAR immunoblots epitopes on uPAR's domains 2 and 3. Purified domain 1 (D1) of uPAR blocks biotin-HK and - 46HKa binding to suPAR 18-35%. Purified isolated domains 2 & 3 (D2D3) block biotin-HK or -46HKa binding 55-60%. Combined D1 and D2D3 completely inhibit biotin-HK or -46HKa binding to suPAR. Mutagenizing the uPA binding region on the aminoterminal portion of domain 2 of suPAR does not interfere with HK binding. Fine mapping of the HK binding site on uPAR was performed by using sequential and overlapping 20 amino acid peptides prepared from uPAR. Two regions on uPAR contribute to HK binding. One region on the carboxyterminal end of D2 (L166-T195) blocks HK binding to HUVEC or suPAR and, when biotinylated, directly binds to HK. A second region on the aminoterminal portion of D3 (Q215-N255) blocks HK binding to HUVEC or suPAR and, when biotinylated, directly binds to HK. Both suPAR and recombinant Cytokeratin 1 block biotin-HK or -46HKa binding to suPAR with equal affinity (IC50 = 1 μM). These investigations indicate that HK has the ability to intimately interact with uPAR. This activity influences vitronectin's interactions with file:///E|/working/LAXMI-PRASAD/WileyML-3G/deepak/31-Jan/Tuesday/Abstract%20OC124.html (1 of 2) [1/31/2014 3:46:19 PM]
Allen P Kaplan - One of the best experts on this subject based on the ideXlab platform.
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Pathogenic mechanisms of bradykinin mediated diseases: dysregulation of an innate inflammatory pathway
Advances in immunology, 2014Co-Authors: Allen P Kaplan, Kusumam JosephAbstract:Binding of negatively charged macromolecules to factor XII induces a conformational change such that it becomes a substrate for trace amounts of activated factor present in plasma (less than 0.01%). As activated factor XII (factor XIIa or factor XIIf) forms, it converts prekallikrein (PK) to kallikrein and kallikrein cleaves high molecular weight kininogen (HK) to release bradykinin. A far more rapid activation of the remaining unactivated factor XII occurs by enzymatic cleavage by kallikrein (kallikrein-feedback) and sequential cleavage yields two forms of activated factor XII; namely, factor XIIa followed by factor XII fragment (factor XIIf). PK circulates bound to HK and binding induces a conformational change in PK so that it acquires enzymatic activity and can stoichiometrically cleave HK to produce bradykinin. This reaction is prevented from occurring in plasma by the presence of C1 inhibitor (C1 INH). The same active site leads to autoactivation of the PK-HK complex to generate kallikrein if a phosphate containing buffer is used. Theoretically, formation of kallikrein by this factor XII-independent route can activate surface-bound factor XII to generate factor XIIa resulting in a marked increase in the rate of bradykinin formation as stoichiometric reactions are replaced by Michaelis-Menton, enzyme-substrate, kinetics. Zinc-dependent binding of the constituents of the bradykinin-forming cascade to the surface of endothelial cells is mediated by gC1qR and bimolecular complexes of gC1qR-Cytokeratin 1 and Cytokeratin 1-u-PAR (urokinase plasminogen activator receptor). Factor XII and HK compete for binding to free gC1qR (present in excess) while Cytokeratin 1-u-PAR preferentially binds factor XII and gC1qR-Cytokeratin 1 preferentially binds HK. Autoactivation of factor XII can be initiated as a result of binding to gC1qR but is prevented by C1 INH. Yet stoichiometric activation of PK-HK to yield kallikrein in the absence of factor XII can be initiated by heat shock protein 90 (HSP-90) which forms a zinc-dependent trimolecular complex by binding to HK. Thus, endothelial cell-dependent activation can be initiated by activation of factor XII or by activation of PK-HK. Hereditary angioedema (HAE), types I and II, are due to autosomal dominant mutations of the C1 INH gene. In type I disease, the level of C1 INH protein and function is proportionately low, while type II disease has a normal protein level but diminished function. There is trans-inhibition of the one normal gene so that functional levels are 30% or less and severe angioedema affecting peripheral structures, the gastrointestinal tract, and the larynx results. Prolonged incubation of plasma of HAE patients (but not normal controls) leads to bradykinin formation and conversion of PK to kallikrein which is reversed by reconstitution with C1 INH. The disorder can be treated by C1 INH replacement, inhibition of plasma kallikrein, or blockade at the bradykinin B-2 receptor. A recently described HAE with normal C1 INH (based on inhibition of activated C1s) presents similarly; the defect is not yet clear, however one-third of patients have a mutant factor XII gene. We have shown that this HAE has a defect in bradykinin overproduction whether the factor XII mutation is present or not, that patients' C1 INH is capable of inhibiting factor XIIa and kallikrein (and not just activated C1) but the functional level is approximately 40-60% of normal, and that α2 macroglobulin protein levels are normal. In vitro abnormalities can be suppressed by raising C1 INH to twice normal levels. Finally, aggregated proteins have been shown to activate the bradykinin-forming pathway by catalyzing factor XII autoactivation. Those include the amyloid β protein of Alzheimer's disease and cryoglobulins. This may represent a new avenue for kinin-dependent research in human disease. In allergy (anaphylaxis; perhaps other mast cell-dependent reactions), the oversulfated proteoglycan of mast cells, liberated along with histamine, also catalyze factor XII autoactivation.
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pathogenic mechanisms of bradykinin mediated diseases dysregulation of an innate inflammatory pathway
Advances in Immunology, 2014Co-Authors: Allen P Kaplan, Kusumam JosephAbstract:Abstract Binding of negatively charged macromolecules to factor XII induces a conformational change such that it becomes a substrate for trace amounts of activated factor present in plasma (less than 0.01%). As activated factor XII (factor XIIa or factor XIIf) forms, it converts prekallikrein (PK) to kallikrein and kallikrein cleaves high molecular weight kininogen (HK) to release bradykinin. A far more rapid activation of the remaining unactivated factor XII occurs by enzymatic cleavage by kallikrein (kallikrein-feedback) and sequential cleavage yields two forms of activated factor XII; namely, factor XIIa followed by factor XII fragment (factor XIIf). PK circulates bound to HK and binding induces a conformational change in PK so that it acquires enzymatic activity and can stoichiometrically cleave HK to produce bradykinin. This reaction is prevented from occurring in plasma by the presence of C1 inhibitor (C1 INH). The same active site leads to autoactivation of the PK–HK complex to generate kallikrein if a phosphate containing buffer is used. Theoretically, formation of kallikrein by this factor XII-independent route can activate surface-bound factor XII to generate factor XIIa resulting in a marked increase in the rate of bradykinin formation as stoichiometric reactions are replaced by Michaelis–Menton, enzyme–substrate, kinetics. Zinc-dependent binding of the constituents of the bradykinin-forming cascade to the surface of endothelial cells is mediated by gC1qR and bimolecular complexes of gC1qR-Cytokeratin 1 and Cytokeratin 1-u-PAR (urokinase plasminogen activator receptor). Factor XII and HK compete for binding to free gC1qR (present in excess) while Cytokeratin 1-u-PAR preferentially binds factor XII and gC1qR-Cytokeratin 1 preferentially binds HK. Autoactivation of factor XII can be initiated as a result of binding to gC1qR but is prevented by C1 INH. Yet stoichiometric activation of PK–HK to yield kallikrein in the absence of factor XII can be initiated by heat shock protein 90 (HSP-90) which forms a zinc-dependent trimolecular complex by binding to HK. Thus, endothelial cell-dependent activation can be initiated by activation of factor XII or by activation of PK–HK. Hereditary angioedema (HAE), types I and II, are due to autosomal dominant mutations of the C1 INH gene. In type I disease, the level of C1 INH protein and function is proportionately low, while type II disease has a normal protein level but diminished function. There is trans-inhibition of the one normal gene so that functional levels are 30% or less and severe angioedema affecting peripheral structures, the gastrointestinal tract, and the larynx results. Prolonged incubation of plasma of HAE patients (but not normal controls) leads to bradykinin formation and conversion of PK to kallikrein which is reversed by reconstitution with C1 INH. The disorder can be treated by C1 INH replacement, inhibition of plasma kallikrein, or blockade at the bradykinin B-2 receptor. A recently described HAE with normal C1 INH (based on inhibition of activated C1s) presents similarly; the defect is not yet clear, however one-third of patients have a mutant factor XII gene. We have shown that this HAE has a defect in bradykinin overproduction whether the factor XII mutation is present or not, that patients’ C1 INH is capable of inhibiting factor XIIa and kallikrein (and not just activated C1) but the functional level is approximately 40–60% of normal, and that α 2 macroglobulin protein levels are normal. In vitro abnormalities can be suppressed by raising C1 INH to twice normal levels. Finally, aggregated proteins have been shown to activate the bradykinin-forming pathway by catalyzing factor XII autoactivation. Those include the amyloid β protein of Alzheimer’s disease and cryoglobulins. This may represent a new avenue for kinin-dependent research in human disease. In allergy (anaphylaxis; perhaps other mast cell-dependent reactions), the oversulfated proteoglycan of mast cells, liberated along with histamine, also catalyze factor XII autoactivation.
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the plasma bradykinin forming pathways and its interrelationships with complement
Molecular Immunology, 2010Co-Authors: Allen P Kaplan, Berhane GhebrehiwetAbstract:Abstract The plasma bradykinin-forming cascade and the complement pathways share many elements, including cross-activation, common control mechanisms, and shared binding proteins. The C1 inhibitor (C1 INH) is not only the inhibitor of activated C1r and C1s, but it is the key control protein of the plasma bradykinin-forming cascade. It inhibits the autoactivation of Factor XII, the ability of Factor XIIa to activate prekallikrein and Factor XI, the activation of high molecular weight kininogen (HK) by kallikrein, and the feedback activation of Factor XII by kallikrein. Thus in the absence of C1 INH (hereditary angioedema or acquired C1 INH deficiency) there is unimpeded formation of bradykinin leading to angioedema. Activated Factor XII (Factor XIIa, 80,000 kDa) is further cleaved by kallikrein or plasmin to yield Factor XII fragment (Factor XIIf, 30,000 kDa) and Factor XIIf can activate the C1r subcomponent of C1, particularly when C1 INH (which inhibits Factor XIIf) is absent. Once bradykinin is formed, it causes vasodilatation and increased vascular permeability by interaction with constitutively expressed B-2 receptors. However degradation of bradykinin by carboxypeptidase N (in plasma) or carboxypeptidase M (on endothelial cells) yields des-arg-9 ( Kerbiriou and Griffin, 1979 ) bradykinin which interacts with B-1 receptors. B-1 receptors are induced in inflammatory states by cytokines such as Interleukin 1 and its interaction with bradykinin may prolong or perpetuate the vascular response until bradykinin is completely inactivated by angiotensin converting enzyme or aminopeptidase P, or neutral endopeptidase. The entire bradykinin-forming cascade is assembled and can be activated along the surface of endothelial cells in zinc dependent reactions involving gC1qR, Cytokeratin 1, and the urokinase plasminogen activated receptor (u-PAR). Although Factors XII and HK can be shown to bind to each one of these proteins, they exist in endothelial cells as two bimolecular complexes; gC1qR-Cytokeratin 1, which preferentially binds HK, and Cytokeratin 1–u-PAR which preferentially binds Factor XII. The gC1qR, which binds the globular heads of C1q is present in excess and can bind either Factor XII or HK however the binding sites for HK and C1q have been shown to reside at opposite ends of gC1qR. Activation of the bradykinin-forming pathway can be initiated at the cell surface by gC1qR-induced autoactivation of Factor XII or direct activation of the prekallikrein–HK complex by endothelial cell-derived heat-shock protein 90 (HSP 90) or prolylcarboxypeptidase with recruitment or Factor XII by the kallikrein produced.
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Formation of bradykinin: a major contributor to the innate inflammatory response.
Advances in immunology, 2005Co-Authors: Kusumam Joseph, Allen P KaplanAbstract:The plasma kinin-forming cascade can be activated by contact with negatively charged macromolecules leading to binding and autoactivation of factor XII, activation of prekallikrein to kallikrein by factor XIIa, and cleavage of high molecular weight kininogen (HK) by kallikrein to release the vasoactive peptide bradykinin. Once kallikrein formation begins, there is rapid cleavage of unactivated factor XII to factor XIIa, and this positive feedback is favored kinetically over factor XII autoactivation. Examples of surface initiators that can function in this fashion are endotoxin, sulfated mucopolysaccharides, and aggregated Abeta protein. Physiological activation appears to occur along the surface of endothelial cells both by the aforementioned contact-initiated reactions as well as bypass pathways that are independent of factor XII. Factor XII binds primarily to cell surface u-PAR (urokinase plasminogen activator receptor); HK binds to gC1qR via its light chain (domain 5) and to Cytokeratin 1 by its heavy chain (domain 3) and, to a lesser degree, by its light chain. Prekallikrein circulates bound to HK (as does coagulation factor XI), and prekallikrein is thereby brought to the surface as HK binds. All cell-binding reactions are dependent on zinc ion. Endothelial cells (HUVECs) have bimolecular complexes of u-PAR-Cytokeratin 1 and gC1qR-Cytokeratin 1 at the cell surface plus free gC1qR, which is present in substantial molar excess. Factor XII appears to interact primarily with the u-PAR-Cytokeratin 1 complex, whereas HK binds primarily to the gC1qR-Cytokeratin 1 complex and to free gC1qR. Release of endothelial cell heat shock protein 90 (Hsp90) or the enzyme prolylcarboxypeptidase leads to activation of the bradykinin-forming cascade by activating the prekallikrein-HK complex. In contrast to factor XIIa, neither will activate prekallikrein in the absence of HK, both reactions require zinc ion, and the stoichiometry suggests interaction of one molecule of Hsp90 (for example) with one molecule of prekallikrein-HK complex. The presence of factor XII, however, leads to a marked augmentation in reaction rate via the kallikrein feedback as well as to a change to classic enzyme-substrate kinetics. The circumstances in which activation is initiated by factor XII autoactivation or by these factor XII bypasses are yet to be defined. The pathologic conditions in which bradykinin generation appears important include hereditary and acquired C1 inhibitor deficiency, cough and angioedema due to ACE inhibitors, endotoxin shock, with contributions to conditions as diverse as Alzheimer's disease, stroke, control of blood pressure, and allergic diseases.
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interaction of high molecular weight kininogen binding proteins on endothelial cells
Thrombosis and Haemostasis, 2003Co-Authors: Kusumam Joseph, Berhane Ghebrehiwet, Baby G Tholanikunnel, Allen P KaplanAbstract:Cell surface proteins reported to participate in the binding and activation of the plasma kinin-forming cascade includes gC1qR, Cytokeratin 1 and u-PAR. Each of these proteins binds high molecular weight kininogen (HK) as well as Factor XII. The studies on the interaction of these proteins, using dot-blot analysis, revealed that Cytokeratin 1 binds to both gC1qR and u-PAR while gC1qR and u-PAR do not bind to each other. The binding properties of these proteins were further analyzed by gel filtration. When biotinylated Cytokeratin 1 was incubated with either gC1qR or u-PAR and gel filtered, a new, higher molecular weight peak containing biotin was observed indicating complex formation.The protein shift was also similar to the biotin shift. Further, immunoprecipitation of solubilized endo-thelial cell plasma membrane proteins with anti-gC1qR recovered both gC1qR and Cytokeratin 1, but not u-PAR. Immunoprecipitation with anti-u-PAR recovered only u-PAR and Cytokeratin 1. By competitive ELISA, gC1qR inhibits u-PAR from binding to Cytokeratin 1; u-PAR inhibits gC1qR binding to a lesser extent and requires a 10-fold molar excess. Our data suggest that formation of HK (and Factor XII) binding sites along endothelial cell membranes consists of bimolecular complexes of gC1qR-Cytokeratin 1 and u-PAR-Cytokeratin 1, with gC1qR binding being favored.
Kusumam Joseph - One of the best experts on this subject based on the ideXlab platform.
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pathogenic mechanisms of bradykinin mediated diseases dysregulation of an innate inflammatory pathway
Advances in Immunology, 2014Co-Authors: Allen P Kaplan, Kusumam JosephAbstract:Abstract Binding of negatively charged macromolecules to factor XII induces a conformational change such that it becomes a substrate for trace amounts of activated factor present in plasma (less than 0.01%). As activated factor XII (factor XIIa or factor XIIf) forms, it converts prekallikrein (PK) to kallikrein and kallikrein cleaves high molecular weight kininogen (HK) to release bradykinin. A far more rapid activation of the remaining unactivated factor XII occurs by enzymatic cleavage by kallikrein (kallikrein-feedback) and sequential cleavage yields two forms of activated factor XII; namely, factor XIIa followed by factor XII fragment (factor XIIf). PK circulates bound to HK and binding induces a conformational change in PK so that it acquires enzymatic activity and can stoichiometrically cleave HK to produce bradykinin. This reaction is prevented from occurring in plasma by the presence of C1 inhibitor (C1 INH). The same active site leads to autoactivation of the PK–HK complex to generate kallikrein if a phosphate containing buffer is used. Theoretically, formation of kallikrein by this factor XII-independent route can activate surface-bound factor XII to generate factor XIIa resulting in a marked increase in the rate of bradykinin formation as stoichiometric reactions are replaced by Michaelis–Menton, enzyme–substrate, kinetics. Zinc-dependent binding of the constituents of the bradykinin-forming cascade to the surface of endothelial cells is mediated by gC1qR and bimolecular complexes of gC1qR-Cytokeratin 1 and Cytokeratin 1-u-PAR (urokinase plasminogen activator receptor). Factor XII and HK compete for binding to free gC1qR (present in excess) while Cytokeratin 1-u-PAR preferentially binds factor XII and gC1qR-Cytokeratin 1 preferentially binds HK. Autoactivation of factor XII can be initiated as a result of binding to gC1qR but is prevented by C1 INH. Yet stoichiometric activation of PK–HK to yield kallikrein in the absence of factor XII can be initiated by heat shock protein 90 (HSP-90) which forms a zinc-dependent trimolecular complex by binding to HK. Thus, endothelial cell-dependent activation can be initiated by activation of factor XII or by activation of PK–HK. Hereditary angioedema (HAE), types I and II, are due to autosomal dominant mutations of the C1 INH gene. In type I disease, the level of C1 INH protein and function is proportionately low, while type II disease has a normal protein level but diminished function. There is trans-inhibition of the one normal gene so that functional levels are 30% or less and severe angioedema affecting peripheral structures, the gastrointestinal tract, and the larynx results. Prolonged incubation of plasma of HAE patients (but not normal controls) leads to bradykinin formation and conversion of PK to kallikrein which is reversed by reconstitution with C1 INH. The disorder can be treated by C1 INH replacement, inhibition of plasma kallikrein, or blockade at the bradykinin B-2 receptor. A recently described HAE with normal C1 INH (based on inhibition of activated C1s) presents similarly; the defect is not yet clear, however one-third of patients have a mutant factor XII gene. We have shown that this HAE has a defect in bradykinin overproduction whether the factor XII mutation is present or not, that patients’ C1 INH is capable of inhibiting factor XIIa and kallikrein (and not just activated C1) but the functional level is approximately 40–60% of normal, and that α 2 macroglobulin protein levels are normal. In vitro abnormalities can be suppressed by raising C1 INH to twice normal levels. Finally, aggregated proteins have been shown to activate the bradykinin-forming pathway by catalyzing factor XII autoactivation. Those include the amyloid β protein of Alzheimer’s disease and cryoglobulins. This may represent a new avenue for kinin-dependent research in human disease. In allergy (anaphylaxis; perhaps other mast cell-dependent reactions), the oversulfated proteoglycan of mast cells, liberated along with histamine, also catalyze factor XII autoactivation.
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Pathogenic mechanisms of bradykinin mediated diseases: dysregulation of an innate inflammatory pathway
Advances in immunology, 2014Co-Authors: Allen P Kaplan, Kusumam JosephAbstract:Binding of negatively charged macromolecules to factor XII induces a conformational change such that it becomes a substrate for trace amounts of activated factor present in plasma (less than 0.01%). As activated factor XII (factor XIIa or factor XIIf) forms, it converts prekallikrein (PK) to kallikrein and kallikrein cleaves high molecular weight kininogen (HK) to release bradykinin. A far more rapid activation of the remaining unactivated factor XII occurs by enzymatic cleavage by kallikrein (kallikrein-feedback) and sequential cleavage yields two forms of activated factor XII; namely, factor XIIa followed by factor XII fragment (factor XIIf). PK circulates bound to HK and binding induces a conformational change in PK so that it acquires enzymatic activity and can stoichiometrically cleave HK to produce bradykinin. This reaction is prevented from occurring in plasma by the presence of C1 inhibitor (C1 INH). The same active site leads to autoactivation of the PK-HK complex to generate kallikrein if a phosphate containing buffer is used. Theoretically, formation of kallikrein by this factor XII-independent route can activate surface-bound factor XII to generate factor XIIa resulting in a marked increase in the rate of bradykinin formation as stoichiometric reactions are replaced by Michaelis-Menton, enzyme-substrate, kinetics. Zinc-dependent binding of the constituents of the bradykinin-forming cascade to the surface of endothelial cells is mediated by gC1qR and bimolecular complexes of gC1qR-Cytokeratin 1 and Cytokeratin 1-u-PAR (urokinase plasminogen activator receptor). Factor XII and HK compete for binding to free gC1qR (present in excess) while Cytokeratin 1-u-PAR preferentially binds factor XII and gC1qR-Cytokeratin 1 preferentially binds HK. Autoactivation of factor XII can be initiated as a result of binding to gC1qR but is prevented by C1 INH. Yet stoichiometric activation of PK-HK to yield kallikrein in the absence of factor XII can be initiated by heat shock protein 90 (HSP-90) which forms a zinc-dependent trimolecular complex by binding to HK. Thus, endothelial cell-dependent activation can be initiated by activation of factor XII or by activation of PK-HK. Hereditary angioedema (HAE), types I and II, are due to autosomal dominant mutations of the C1 INH gene. In type I disease, the level of C1 INH protein and function is proportionately low, while type II disease has a normal protein level but diminished function. There is trans-inhibition of the one normal gene so that functional levels are 30% or less and severe angioedema affecting peripheral structures, the gastrointestinal tract, and the larynx results. Prolonged incubation of plasma of HAE patients (but not normal controls) leads to bradykinin formation and conversion of PK to kallikrein which is reversed by reconstitution with C1 INH. The disorder can be treated by C1 INH replacement, inhibition of plasma kallikrein, or blockade at the bradykinin B-2 receptor. A recently described HAE with normal C1 INH (based on inhibition of activated C1s) presents similarly; the defect is not yet clear, however one-third of patients have a mutant factor XII gene. We have shown that this HAE has a defect in bradykinin overproduction whether the factor XII mutation is present or not, that patients' C1 INH is capable of inhibiting factor XIIa and kallikrein (and not just activated C1) but the functional level is approximately 40-60% of normal, and that α2 macroglobulin protein levels are normal. In vitro abnormalities can be suppressed by raising C1 INH to twice normal levels. Finally, aggregated proteins have been shown to activate the bradykinin-forming pathway by catalyzing factor XII autoactivation. Those include the amyloid β protein of Alzheimer's disease and cryoglobulins. This may represent a new avenue for kinin-dependent research in human disease. In allergy (anaphylaxis; perhaps other mast cell-dependent reactions), the oversulfated proteoglycan of mast cells, liberated along with histamine, also catalyze factor XII autoactivation.
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interaction of high molecular weight kininogen with endothelial cell binding proteins supar gc1qr and Cytokeratin 1 determined by surface plasmon resonance biacore
Thrombosis and Haemostasis, 2011Co-Authors: Robin A Pixley, Berhane Ghebrehiwet, Kusumam Joseph, Ricardo Espinola, Alice Kao, Khalil Bdeir, Douglas B Cines, Robert W ColmanAbstract:The physiologic activation of the plasma kallikrein-kinin system requires the assembly of its constituents on a cell membrane. High- molecular-weight kininogen (HK) and cleaved HK (HKa) both interact with at least three endothelial cell binding proteins: urokinase plasminogen activator receptor (uPAR), globular C1q receptor (gC1qR,) and Cytokeratin 1 (CK1). The affinity of HK and HKa for endothelial cells are KD=7–52 nM. The contribution of each protein is unknown. We examined the direct binding of HK and HKa to the soluble extracellular form of uPAR (suPAR), gC1qR and CK1 using surface plasmon resonance. Each binding protein linked to a CM-5 chip and the association, dissociation and KD (equilibrium constant) were measured. The interaction of HK and HKa with each binding protein was zinc-dependent. The affinity for HK and HKa was gC1qR>CK1>suPAR, indicating that gC1qR is dominant for binding. The affinity for HKa compared to HK was the same for gC1qR, 2.6-fold tighter for CK1 but 53-fold tighter for suPAR. Complex between binding proteins was only observed between gC1qR and CK1 indicating that a binary CK1-gC1qR complex can form independently of kininogen. Although suPAR has the weakest affinity of the three binding proteins, it is the only one that distinguished between HK and HKa. This finding indicates that uPAR may be a key membrane binding protein for differential binding and signalling between the cleaved and uncleaved forms of kininogen. The role of CK1 and gC1qR may be to initially bind HK to the membrane surface before productive cleavage to HKa.
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Interaction of High Molecular Weight Kininogen with Endothelial Cell Receptors suPAR, gC1qR and Cytokeratin 1 by Surface Plasmon Resonance (BiaCore).
Blood, 2005Co-Authors: Ricardo Espinola, Berhane Ghebrehiwet, Kusumam Joseph, Robin A Pixley, Douglas B Cines, Alice Kuo, Robert W ColmanAbstract:The physiologic activation of the plasma kallikrein kinin system (KKS) requires the assembly of these proteins on the cell membrane. High molecular weight kininogen (HK) binds to endothelial cells through an interaction with a multiprotein receptor complex that consist of: urokinase plasminogen activator receptor (uPAR), globular C1q receptor (gC1qR) and Cytokeratin 1 (CK1). The affinity of HK and cleaved HK (Hka) for endothelial cells is K D =7–52 nM but the affinity for each of the three binding proteins is unknown. We first examined the direct binding of HK and Hka to the soluble receptor form of uPAR (suPAR), gC1qR and CK1 using surface plasmon resonance (BiaCore). We linked suPAR, gC1qR and CK1 (800–1100 pg/mm 2 ) by amine coupling to a CM-5 chip and perfused HK and Hka at a concentration ranging from 50 to 400 nM in the presence or absence of 10 μM ZnCl 2 . A Langmuir binding model with local fit (stoichiometry of 1:1) was used to analyse k on (association rate constant), k off (dissociation rate constant) and K D (equilibrium dissociation constant) for HK and Hka. The binding of HK and Hka to the three receptors was zinc dependent. The affinity for HK and Hka was gC1qR>CK1>suPAR. The high affinity for gC1qR and CK1 was similar for HK and Hka and was due to a very slow k off . The affinity for Hka compared to HK was 2.5-fold tighter for CK1 but 50-fold tighter for suPAR. A reversed immobiization was then performed. We immobilized HK, Hka and LK (800 pg/mm 2 ) and suPAR, gC1qR and CK1 were flowed over the chip at a concentration ranging from 50 to 700 nM. Only gC1qR bound to immobilized HK and Hka, with a K D of 6.31± 2.8 pM for HK and 2.86±1.5 pM for Hka while the other 2 receptors did not bind. Receptor integrity was shown by preincubating HK or Hka with the pure receptors and observing competition with the immobilized receptors. Among the HK/Hka receptors, complex formation was only observed between gC1qR and immobiized CK1 with or without Hka indicating that a CK1-gC1qR complex can form independently of kininogen. This study indicates that although suPAR has the weakest affinity of the three receptors it is the only one that distinguishes between HK and Hka. Only one binary complex was revealed by this technique between CK1 and gC1qR. These results lay the foundation for anlayzing the affinity of these receptors in a cellular environment.
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Formation of bradykinin: a major contributor to the innate inflammatory response.
Advances in immunology, 2005Co-Authors: Kusumam Joseph, Allen P KaplanAbstract:The plasma kinin-forming cascade can be activated by contact with negatively charged macromolecules leading to binding and autoactivation of factor XII, activation of prekallikrein to kallikrein by factor XIIa, and cleavage of high molecular weight kininogen (HK) by kallikrein to release the vasoactive peptide bradykinin. Once kallikrein formation begins, there is rapid cleavage of unactivated factor XII to factor XIIa, and this positive feedback is favored kinetically over factor XII autoactivation. Examples of surface initiators that can function in this fashion are endotoxin, sulfated mucopolysaccharides, and aggregated Abeta protein. Physiological activation appears to occur along the surface of endothelial cells both by the aforementioned contact-initiated reactions as well as bypass pathways that are independent of factor XII. Factor XII binds primarily to cell surface u-PAR (urokinase plasminogen activator receptor); HK binds to gC1qR via its light chain (domain 5) and to Cytokeratin 1 by its heavy chain (domain 3) and, to a lesser degree, by its light chain. Prekallikrein circulates bound to HK (as does coagulation factor XI), and prekallikrein is thereby brought to the surface as HK binds. All cell-binding reactions are dependent on zinc ion. Endothelial cells (HUVECs) have bimolecular complexes of u-PAR-Cytokeratin 1 and gC1qR-Cytokeratin 1 at the cell surface plus free gC1qR, which is present in substantial molar excess. Factor XII appears to interact primarily with the u-PAR-Cytokeratin 1 complex, whereas HK binds primarily to the gC1qR-Cytokeratin 1 complex and to free gC1qR. Release of endothelial cell heat shock protein 90 (Hsp90) or the enzyme prolylcarboxypeptidase leads to activation of the bradykinin-forming cascade by activating the prekallikrein-HK complex. In contrast to factor XIIa, neither will activate prekallikrein in the absence of HK, both reactions require zinc ion, and the stoichiometry suggests interaction of one molecule of Hsp90 (for example) with one molecule of prekallikrein-HK complex. The presence of factor XII, however, leads to a marked augmentation in reaction rate via the kallikrein feedback as well as to a change to classic enzyme-substrate kinetics. The circumstances in which activation is initiated by factor XII autoactivation or by these factor XII bypasses are yet to be defined. The pathologic conditions in which bradykinin generation appears important include hereditary and acquired C1 inhibitor deficiency, cough and angioedema due to ACE inhibitors, endotoxin shock, with contributions to conditions as diverse as Alzheimer's disease, stroke, control of blood pressure, and allergic diseases.
Berhane Ghebrehiwet - One of the best experts on this subject based on the ideXlab platform.
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Targeting gC1qR Domains for Therapy Against Infection and Inflammation
Advances in experimental medicine and biology, 2012Co-Authors: Berhane Ghebrehiwet, Jolyon Jesty, Rama Vinayagasundaram, Uma Vinayagasundaram, Alisa Valentino, Nithin Tumma, Kinga K. Hosszu, Ellinor I.b. PeerschkeAbstract:The receptor for the globular heads of C1q, gC1qR/p33, is a widely expressed cellular protein, which binds to diverse ligands including plasma proteins, cellular proteins, and microbial ligands. In addition to C1q, gC1qR also binds high molecular weight kininogen (HK), which also has two other cell surface sites, namely, Cytokeratin 1 and urokinase plasminogen activator receptor (uPAR). On endothelial cells (ECs), the three molecules form two closely associated bimolecular complexes of gC1qR/Cytokeratin 1 and uPAR/Cytokeratin 1. However, by virtue of its high affinity for HK, gC1qR plays a central role in the assembly of the kallikrein–kinin system, leading to the generation of bradykinin (BK). BK in turn is largely responsible for the vascular leakage and associated inflammation seen in angioedema patients. Therefore, blockade of gC1qR by inhibitory peptides or antibodies may not only prevent the generation of BK but also reduce C1q-induced or microbial-ligand-induced inflammatory responses. Employing synthetic peptides and gC1qR deletion mutants, we confirmed previously predicted sites for C1q (residues 75–96) and HK (residues 204–218) and identified additional sites for both C1q and HK (residues190–202), for C1q (residues 144–162), and for HIV-1 gp41 (residues 174–180). With the exception of residues 75–96, which is located in the αA coiled-coil N-terminal segment, most of the identified residues form part of the highly charged loops connecting the various β-strands in the crystal structure. Taken together, the data support the notion that gC1qR could serve as a novel molecular target for the design of antibody-based and/or peptide-based therapy to attenuate acute and/or chronic inflammation associated with vascular leakage and infection.
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interaction of high molecular weight kininogen with endothelial cell binding proteins supar gc1qr and Cytokeratin 1 determined by surface plasmon resonance biacore
Thrombosis and Haemostasis, 2011Co-Authors: Robin A Pixley, Berhane Ghebrehiwet, Kusumam Joseph, Ricardo Espinola, Alice Kao, Khalil Bdeir, Douglas B Cines, Robert W ColmanAbstract:The physiologic activation of the plasma kallikrein-kinin system requires the assembly of its constituents on a cell membrane. High- molecular-weight kininogen (HK) and cleaved HK (HKa) both interact with at least three endothelial cell binding proteins: urokinase plasminogen activator receptor (uPAR), globular C1q receptor (gC1qR,) and Cytokeratin 1 (CK1). The affinity of HK and HKa for endothelial cells are KD=7–52 nM. The contribution of each protein is unknown. We examined the direct binding of HK and HKa to the soluble extracellular form of uPAR (suPAR), gC1qR and CK1 using surface plasmon resonance. Each binding protein linked to a CM-5 chip and the association, dissociation and KD (equilibrium constant) were measured. The interaction of HK and HKa with each binding protein was zinc-dependent. The affinity for HK and HKa was gC1qR>CK1>suPAR, indicating that gC1qR is dominant for binding. The affinity for HKa compared to HK was the same for gC1qR, 2.6-fold tighter for CK1 but 53-fold tighter for suPAR. Complex between binding proteins was only observed between gC1qR and CK1 indicating that a binary CK1-gC1qR complex can form independently of kininogen. Although suPAR has the weakest affinity of the three binding proteins, it is the only one that distinguished between HK and HKa. This finding indicates that uPAR may be a key membrane binding protein for differential binding and signalling between the cleaved and uncleaved forms of kininogen. The role of CK1 and gC1qR may be to initially bind HK to the membrane surface before productive cleavage to HKa.
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the plasma bradykinin forming pathways and its interrelationships with complement
Molecular Immunology, 2010Co-Authors: Allen P Kaplan, Berhane GhebrehiwetAbstract:Abstract The plasma bradykinin-forming cascade and the complement pathways share many elements, including cross-activation, common control mechanisms, and shared binding proteins. The C1 inhibitor (C1 INH) is not only the inhibitor of activated C1r and C1s, but it is the key control protein of the plasma bradykinin-forming cascade. It inhibits the autoactivation of Factor XII, the ability of Factor XIIa to activate prekallikrein and Factor XI, the activation of high molecular weight kininogen (HK) by kallikrein, and the feedback activation of Factor XII by kallikrein. Thus in the absence of C1 INH (hereditary angioedema or acquired C1 INH deficiency) there is unimpeded formation of bradykinin leading to angioedema. Activated Factor XII (Factor XIIa, 80,000 kDa) is further cleaved by kallikrein or plasmin to yield Factor XII fragment (Factor XIIf, 30,000 kDa) and Factor XIIf can activate the C1r subcomponent of C1, particularly when C1 INH (which inhibits Factor XIIf) is absent. Once bradykinin is formed, it causes vasodilatation and increased vascular permeability by interaction with constitutively expressed B-2 receptors. However degradation of bradykinin by carboxypeptidase N (in plasma) or carboxypeptidase M (on endothelial cells) yields des-arg-9 ( Kerbiriou and Griffin, 1979 ) bradykinin which interacts with B-1 receptors. B-1 receptors are induced in inflammatory states by cytokines such as Interleukin 1 and its interaction with bradykinin may prolong or perpetuate the vascular response until bradykinin is completely inactivated by angiotensin converting enzyme or aminopeptidase P, or neutral endopeptidase. The entire bradykinin-forming cascade is assembled and can be activated along the surface of endothelial cells in zinc dependent reactions involving gC1qR, Cytokeratin 1, and the urokinase plasminogen activated receptor (u-PAR). Although Factors XII and HK can be shown to bind to each one of these proteins, they exist in endothelial cells as two bimolecular complexes; gC1qR-Cytokeratin 1, which preferentially binds HK, and Cytokeratin 1–u-PAR which preferentially binds Factor XII. The gC1qR, which binds the globular heads of C1q is present in excess and can bind either Factor XII or HK however the binding sites for HK and C1q have been shown to reside at opposite ends of gC1qR. Activation of the bradykinin-forming pathway can be initiated at the cell surface by gC1qR-induced autoactivation of Factor XII or direct activation of the prekallikrein–HK complex by endothelial cell-derived heat-shock protein 90 (HSP 90) or prolylcarboxypeptidase with recruitment or Factor XII by the kallikrein produced.
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Interaction of High Molecular Weight Kininogen with Endothelial Cell Receptors suPAR, gC1qR and Cytokeratin 1 by Surface Plasmon Resonance (BiaCore).
Blood, 2005Co-Authors: Ricardo Espinola, Berhane Ghebrehiwet, Kusumam Joseph, Robin A Pixley, Douglas B Cines, Alice Kuo, Robert W ColmanAbstract:The physiologic activation of the plasma kallikrein kinin system (KKS) requires the assembly of these proteins on the cell membrane. High molecular weight kininogen (HK) binds to endothelial cells through an interaction with a multiprotein receptor complex that consist of: urokinase plasminogen activator receptor (uPAR), globular C1q receptor (gC1qR) and Cytokeratin 1 (CK1). The affinity of HK and cleaved HK (Hka) for endothelial cells is K D =7–52 nM but the affinity for each of the three binding proteins is unknown. We first examined the direct binding of HK and Hka to the soluble receptor form of uPAR (suPAR), gC1qR and CK1 using surface plasmon resonance (BiaCore). We linked suPAR, gC1qR and CK1 (800–1100 pg/mm 2 ) by amine coupling to a CM-5 chip and perfused HK and Hka at a concentration ranging from 50 to 400 nM in the presence or absence of 10 μM ZnCl 2 . A Langmuir binding model with local fit (stoichiometry of 1:1) was used to analyse k on (association rate constant), k off (dissociation rate constant) and K D (equilibrium dissociation constant) for HK and Hka. The binding of HK and Hka to the three receptors was zinc dependent. The affinity for HK and Hka was gC1qR>CK1>suPAR. The high affinity for gC1qR and CK1 was similar for HK and Hka and was due to a very slow k off . The affinity for Hka compared to HK was 2.5-fold tighter for CK1 but 50-fold tighter for suPAR. A reversed immobiization was then performed. We immobilized HK, Hka and LK (800 pg/mm 2 ) and suPAR, gC1qR and CK1 were flowed over the chip at a concentration ranging from 50 to 700 nM. Only gC1qR bound to immobilized HK and Hka, with a K D of 6.31± 2.8 pM for HK and 2.86±1.5 pM for Hka while the other 2 receptors did not bind. Receptor integrity was shown by preincubating HK or Hka with the pure receptors and observing competition with the immobilized receptors. Among the HK/Hka receptors, complex formation was only observed between gC1qR and immobiized CK1 with or without Hka indicating that a CK1-gC1qR complex can form independently of kininogen. This study indicates that although suPAR has the weakest affinity of the three receptors it is the only one that distinguishes between HK and Hka. Only one binary complex was revealed by this technique between CK1 and gC1qR. These results lay the foundation for anlayzing the affinity of these receptors in a cellular environment.
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interaction of high molecular weight kininogen binding proteins on endothelial cells
Thrombosis and Haemostasis, 2003Co-Authors: Kusumam Joseph, Berhane Ghebrehiwet, Baby G Tholanikunnel, Allen P KaplanAbstract:Cell surface proteins reported to participate in the binding and activation of the plasma kinin-forming cascade includes gC1qR, Cytokeratin 1 and u-PAR. Each of these proteins binds high molecular weight kininogen (HK) as well as Factor XII. The studies on the interaction of these proteins, using dot-blot analysis, revealed that Cytokeratin 1 binds to both gC1qR and u-PAR while gC1qR and u-PAR do not bind to each other. The binding properties of these proteins were further analyzed by gel filtration. When biotinylated Cytokeratin 1 was incubated with either gC1qR or u-PAR and gel filtered, a new, higher molecular weight peak containing biotin was observed indicating complex formation.The protein shift was also similar to the biotin shift. Further, immunoprecipitation of solubilized endo-thelial cell plasma membrane proteins with anti-gC1qR recovered both gC1qR and Cytokeratin 1, but not u-PAR. Immunoprecipitation with anti-u-PAR recovered only u-PAR and Cytokeratin 1. By competitive ELISA, gC1qR inhibits u-PAR from binding to Cytokeratin 1; u-PAR inhibits gC1qR binding to a lesser extent and requires a 10-fold molar excess. Our data suggest that formation of HK (and Factor XII) binding sites along endothelial cell membranes consists of bimolecular complexes of gC1qR-Cytokeratin 1 and u-PAR-Cytokeratin 1, with gC1qR binding being favored.
Fakhri Mahdi - One of the best experts on this subject based on the ideXlab platform.
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myeloperoxidase interacts with endothelial cell surface Cytokeratin 1 and modulates bradykinin production by the plasma kallikrein kinin system
American Journal of Pathology, 2007Co-Authors: Joshua M Astern, Alvin H. Schmaier, Fakhri Mahdi, William F Pendergraft, Ronald J Falk, Charles J Jennette, Gloria A PrestonAbstract:During an inflammatory state, functional myeloperoxidase (MPO) is released into the vessel as a result of intravascular neutrophil degradation. One mechanism of resulting cellular injury involves endothelial internalization of MPO, which causes oxidative damage and impairs endothelial signaling. We report the discovery of a protein that facilitates MPO internalization, Cytokeratin 1 (CK1), identified using affinity chromatography and mass spectrometry. CK1 interacts with MPO in vitro, even in the presence of 100% human plasma, thus substantiating biological relevance. Immunofluorescent microscopy confirmed that MPO added to endothelial cells can co-localize with endogenously expressed CK1. CK1 acts as a scaffolding protein for the assembly of the vasoregulatory plasma kallikrein-kinin system; thus we explored whether MPO and high molecular weight kininogen (HK) reside on CK1 together or whether they compete for binding. The data support cooperative binding of MPO and HK on cells such that MPO masked the plasma kallikrein cleavage site on HK, and MPO-generated oxidants caused inactivation of both HK and kallikrein. Collectively, interactions between MPO and the components of the plasma kallikrein-kinin system resulted in decreased bradykinin production. This study identifies CK1 as a facilitator of MPO-mediated vascular responses and thus provides a new paradigm by which MPO affects vasoregulatory systems.
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recombinant prolylcarboxypeptidase activates plasma prekallikrein
Blood, 2004Co-Authors: Zia Shariatmadar, Fakhri Mahdi, Alvin H. SchmaierAbstract:The serine protease prolylcarboxypeptidase (PRCP), isolated from human umbilical vein endothelial cells (HUVECs), is a plasma prekallikrein (PK) activator. PRCP cDNA was cloned in pMT/BIP/V5-HIS-C, transfected into Schneider insect (S2) cells, and purified from serum-free media. Full-length recombinant PRCP (rPRCP) activates PK when bound to high-molecular-weight kininogen (HK). Recombinant PRCP is inhibited by leupeptin, angiotensin II, bradykinin, anti-PRCP, diisopropyl-fluorophosphonate (DFP), phenylmethylsulfonyl fluoride (PMSF), and Z-Pro-Proaldehyde-dimethyl acetate, but not by 1 mM EDTA (ethylenediaminetetraacetic acid), bradykinin 1-5, or angiotensin 1-7. Corn trypsin inhibitor binds to prekallikrein to prevent rPRCP activation, but it does not directly inhibit the active site of either enzyme. Unlike factor XIIa, the ability of rPRCP to activate PK is blocked by angiotensin II, not by neutralizing antibody to factor XIIa. PRCP antigen is detected on HUVEC membranes using flow cytometry and laser scanning confocal microscopy. PRCP antigen does not colocalize with LAMP1 on nonpermeabilized HUVECs, but it partially colocalizes in permeabilized cells. PRCP colocalizes with all the HK receptors, gC1qR, uPAR, and Cytokeratin 1 antigen, on nonpermeabilized HUVECs. PRCP activity and antigen expression on cultured HUVECs are blocked by a morpholino antisense oligonucleotide. These investigations indicate that rPRCP is functionally identical to isolated HUVEC PRCP and is a major HUVEC membrane-expressed, PK-activating enzyme detected in the intravascular compartment. (Blood. 2004;103:4554-4561)
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the relative priority of prekallikrein and factors xi xia assembly on cultured endothelial cells
Journal of Biological Chemistry, 2003Co-Authors: Fakhri Mahdi, Zia Shariatmadar, Alvin H. SchmaierAbstract:Abstract Investigations determined the relative preference of prekallikrein (PK) or factor XI/XIa (FXI/FXIa) binding to endothelial cells (HUVECs). In microtiter plates, biotinylated high molecular weight kininogen (biotin-HK) or biotin-FXI binding to HUVEC monolayers or their matrix proteins, but not fibronectin-coated plastic microtiter plate wells, was specifically blocked by antibodies to each of the receptors of HK, uPAR, gC1qR, or Cytokeratin 1. Fluorescein isothiocyanate (FITC)-PK specifically bound to HUVEC suspensions without added Zn2+, whereas FITC-FXI or -FXIa binding to HUVEC suspensions required 10 μm added Zn2+ to support specific binding. Plasma concentrations of FXI did not block FITC-PK binding to HUVECs in the absence or presence of 10 μm Zn2+. In the absence of HK, the level of FITC-FXI or -FXIa binding was half that seen in its presence. At physiologic concentrations, PK (450 nm) abolished FITC-FXI or -FXIa binding to HUVEC suspensions in the absence or presence of HK in the presence of 10 μm Zn2+. Released Zn2+ from 2–8 × 108 collagen-activated platelets/ml supported biotin-FXI binding to HUVEC monolayers, but platelet activation was not necessary to support biotin-PK binding to HUVECs. At physiologic concentrations, PK also abolished FXI binding to HUVECs in the presence of activated platelets, but FXI did not influence PK binding. PK in the presence or absence of HK preferentially bound to HUVECs over FXI or FXIa. Elevated Zn2+ concentrations are required for FXI but not PK binding, but the presence of physiologic concentrations of PK and HK also prevented FXI binding. PK preferential binding to endothelial cells contributes to their anticoagulant nature.
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assembly and activation of the plasma kallikrein kinin system a new interpretation
International Immunopharmacology, 2002Co-Authors: Zia Shariatmadar, Fakhri Mahdi, A H SchmaierAbstract:Abstract Understanding the importance and physiologic activity of the plasma kallikrein/kinin system (KKS) has been thwarted by the absence of an inclusive theory for its assembly and activation. The contact activation hypothesis describes the assembly and activation of this system in test tubes and disease states, but not under physiologic circumstances. Recent investigations have indicated a new cohesive hypothesis for understanding physiologic activation of this system. Prekallikrein (PK) and factor XI (FXI) through high molecular weight kininogen (HK) assemble on a co-localized, multiprotein receptor complex on endothelial cells that consists of at least Cytokeratin 1 (CK1), gC1qR, and urokinase plasminogen activator receptor (uPAR). When assembled on these proteins, prekallikrein becomes activated to kallikrein by the membrane-expressed enzyme prolylcarboxypeptidase (PRCP). Formed kallikrein then activates factor XII (FXII) for amplification of its activation and single chain urokinase. The plasma kallikrein/kinin system may serve as a physiologic counterbalance to the plasma renin angiotensin system (RAS) by lowering blood pressure and preventing thrombosis. Insights into the integrated role of these two systems may afford the development of novel therapeutic drugs to manage hypertension and thrombosis.
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factor xii interacts with the multiprotein assembly of urokinase plasminogen activator receptor gc1qr and Cytokeratin 1 on endothelial cell membranes
Blood, 2002Co-Authors: Fakhri Mahdi, Alvin H. Schmaier, Carlos D. Figueroa, Zia Shariat MadarAbstract:Investigations were performed to define the factor XII (FXII) binding site(s) on cultured endothelial cells (HUVECs). Biotin- or fluorescein isothiocyanate (FITC)–FXII in the presence of 10 μM Zn2+ specifically binds to HUVEC monolayers or cells in suspension. Collagen-stimulated platelets release sufficient Zn2+ to support FXII binding. On laser scanning confocal microscopy or electron microscopy, FITC-FXII or Nanogold-labeled FXII, respectively, specifically bind to HUVECs. Antibodies to gC1qR, urokinase plasminogen activator receptor (uPAR) and, to a lesser extent, Cytokeratin 1 (CK1) block FXII binding to HUVECs as determined by flow cytometry and soluble or solid phase binding assays. FITC-FXII on endothelial cells colocalizes with gC1qR, uPAR and, to a lesser extent, CK1 antigen. Combined recombinant soluble uPAR and CK1 inhibit 80% FITC-FXII binding to HUVECs. Peptide Y(39)HKCTHKGR(47) (YHK9) from the N-terminal region of FXII and peptide H(479)KHGHGHGKHKNKGKKNGKH(498) from HK's domain 5 cell-binding site block FITC-FXII binding to HUVECs. Peptide YHK9 also inhibits FXIIa's activation of prekallikrein and FXI on HUVECs. These combined investigations indicate that FXII through a region on its fibronectin type II domain binds to the same multiprotein receptor complex that comprises the HK binding site of HUVECs. However, plasma concentrations of HK and vitronectin inhibit FXII binding to HUVECs 100% and 50%, respectively, and plasma albumin and other proteins prevent a sufficient level of free Zn2+ to be available to support FXII binding to HUVECs. Thus, physiologic FXII expression on HUVECs is secondary to HK binding and highly restricted in its ability to initiate prekallikrein or FXI activation.
