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  • 1
    ISSN: 1573-4943
    Keywords: Kininogen ; cathepsin B ; cathepsin H ; cysteine protease inhibitor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Domain 3 (D3) of human kininogens, the major cysteine proteinase inhibitors in plasma, has been shown to be the tightest binding inhibitory domain for cathepsins B and H. D3 was expressed in three fragments as its exon products as follows: exon 7 (Gly235-Gln292), exon 8 (Gln292-Gly328), and exon 9 (Gln329-Met357). Exon products 7, 8, and 9 alone as well as exon product 7 + 9 each exhibited an IC50 value 5- to 30-fold higher (5–30μM) than exon products 7 + 8 and 8 + 9 (0.9–1.3μM) for cathepsins B and H, respectively. However, in turn, the exon products 7 + 8 and 8 + 9 seemed to be less potent inhibitors than the intact D3 (10, 200 nM) or HK (200, 500 nM) molecule. These results clearly indicate that an intact molecule of HK or its domain 3 as a whole is required for optimal inhibition of cathepsins B and H.
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  • 2
    Publication Date: 2007-11-16
    Description: Human prothrombinase assembled on synthetic membranes composed of phosphatidylcholine and phosphatidylserine (PCPS) catalyzes thrombin formation almost exclusively by sequential cleavage of prothrombin at Arg320 to yield the protease meizothrombin (mIIa) as an intermediate which is then further cleaved at Arg271. This cleavage pathway arises because Arg320 in intact prothrombin is cleaved ∼30-fold faster than Arg271. When prothrombin lacks γ-carboxyglutamic acid modifications (desGlaII), product formation is modestly decreased but bond cleavage largely occurs in the opposite order, yielding the zymogen, prethrombin 2 (P2), as an intermediate. This results from a reduction in the rate of cleavage at Arg320 in intact prothrombin and a partly compensating gain of function in cleavage at Arg271. Thus, membrane binding and/or other interactions mediated by γ-carboxyglutamic acids in the substrate play a major role in modulating the pathway for cleavage and whether the intermediate is a zymogen or protease. We now extend these approaches to evaluate prothrombinase function on activated platelets and human umbilical vein endothelial cells (HUVECs) at the physiologic concentration of prothrombin. Prothrombin activation was detected by measuring the appearance of product(s) with proteolytic activity and by analysis of the cleavage process by SDS-PAGE and western blot analysis quantitatively imaged by infrared fluorescence. This approach was validated by documenting equivalence in the progress curves obtained by western blot imaging or following staining of total protein in a purified system with PCPS membranes. Prothrombin cleavage was assessed using human platelets (108/ml), activated with thrombin and studied with saturating concentrations of Va and a limiting concentration of Xa. The pattern of prothrombin cleavage, intermediate accumulation and product formation was clearly distinct from that observed with PCPS membranes, indicating substantial flux towards thrombin formation via initial cleavage at Arg271 followed by cleavage at Arg320 producing the zymogen P2 as an intermediate. Transient formation of mIIa from initial cleavage at Arg320 was undetectable. Thus, prothrombinase assembled on thrombin activated platelets cleaves prothrombin in a way that is reminiscent of the cleavage of desGlaII rather than fully carboxylated prothrombin seen with PCPS membranes. In contrast, equivalent studies with prothrombinase assembled on thrombin activated HUVECs produced cleavage patterns consistent with significant flux towards thrombin formation via initial cleavage of prothrombin at Arg320 yielding mIIa as an intermediate. The use of desGlaII with HUVECs yielded lower rates and cleavage patterns similar to those obtained with fully carboxylated prothrombin in the platelet reactions. Our results document that the cleavage pathway for thrombin formation is dependent on cell type. Because mIIa is a protease with a different spectrum of activities from thrombin, its formation restricted to the vessel wall suggests an important regulatory role for the modulation of the pathway of prothrombin cleavage. Such bimodal regulation of the pathway for prothrombin cleavage on HUVECs and platelets suggests differential roles of prothrombinase assembled on platelets versus endothelium in regulating the hemostatic response to vascular injury.
    Print ISSN: 0006-4971
    Electronic ISSN: 1528-0020
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  • 3
    Publication Date: 2008-11-16
    Description: Prothrombin activation by prothrombinase is a paradigm for proteolytic activation reactions wherein product is produced following cleavage at more than one site in the substrate. Prothrombin is tethered to prothrombinase through exosite binding and is preferentially cleaved at R320. Subsequent cleavage at R271, in the resulting intermediate, yields thrombin. Selective presentation of the R320 site for active site docking and cleavage likely arises from the combined constraints of exosite-dependent tethering and the position of the cleavage site within the polypeptide sequence of prothrombin. A series of recombinant variants with substitutions in residues at and preceding the R320 cleavage site have been employed to investigate the role of geometric effects in enforcing the selective action of prothrombinase on this site in prothrombin. Replacement of the sequence D-G-R320 in wild type prothrombin (IIWT) with D-R-R320 (IIRR) or R-G-R320 (IIRGR) yielded substrate species that were indistinguishable from IIWT in their conversion to thrombin by prothrombinase. Equivalent progress curves for bands produced following initial cleavage at R320 followed by cleavage at R271 were established by SDS-PAGE for all three substrate species. Specific and quantitative cleavage at R320 rather than at R319 or R318 in the initial cleavage reaction was established by N-terminal sequencing. Substitution of R320 in IIWT with Q (IIQ320) rendered this site uncleavable and led to very slow cleavage only at the R271 site. These findings support a role for geometric constraints in precisely positioning R320 in II for preferential cleavage. Surprisingly, substitution of flanking sequences in IIQ320 to yield R-G-R-Q320 (II-1Shift) and R-G-R-G-Q320 (II-2Shift) yielded variants that were cleaved in a manner similar to IIWT. For either variant, the rate of prothrombin consumption was within 2-fold that observed with IIWT and could be quantitatively explained by cleavage at R319 (II-1Shift) or R318 (II-2Shift) determined by N-terminal sequencing. Thus, provided position 320 cannot be cleaved, geometric effects are not absolute and flexibility can be tolerated in the position of the scissile bond presented for preferential active site engagement. Variants containing RGR substitutions further shifted to the -3 and -4 positions were instead cleaved ~30-fold slower but at R271 similar to IIQ320. Dramatic loss of cleavage seen with these further N-terminal shifts establishes the limits of possible flexibility in substrate presentation to the active site. Further insights were provided by binding studies examining the ability of these variants to engage and displace 4-aminobenzamidine from the active site of the catalyst within prothrombinase assembled with XaA195. R320 in prothrombin engaged the active site with an equilibrium constant that was ~600-fold more favorable than R271. The register shift variants showed systematic decreases in their affinities for engaging the active site. Loss of cleavage at R316 in II-4Shift correlated with a markedly reduced affinity for active site docking. Thus, geometric constraints, arising from exosite binding, play an essential role in affecting the thermodynamics of active docking by prothrombin. However, very significant decreases in the equilibrium constant for active site docking of an otherwise highly favorable interaction are evident as only minor changes in rate. Large changes in rate and qualitative differences in cleavage pathway result when the equilibrium constant for cleavage at R316 in II-4Shift approaches that for R271. Our findings provide an explanation for how the action of prothrombinase on prothrombin is regulated by both precise substrate geometry and competition for active site docking between different sites within the substrate. The unexpected relationship between thermodynamics and rate reveals new mechanistic insights into the ability of prothrombinase to discriminate between sites within prothrombin and how thrombin formation is regulated.
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  • 4
    Publication Date: 1997-08-15
    Description: We and others have shown that both high and low molecular mass kininogens are able to inhibit the thrombin-induced aggregation of gel-filtered platelets, indicating that the locus for inhibition resides in the heavy chain. The inhibitory site is present in domain 3, confined to the C-terminal portion of the region encoded by exon 7 (K270-G292), and the minimal effective sequence is a heptapeptide (L271-A277; Kunapuli et al, J Biol Chem 271:11228, 1996). Kininogens inhibit thrombin binding to platelets and thus inhibit thrombin-induced aggregation. The molecular mechanism by which kininogens inhibit thrombin-induced aggregation of platelets is unknown. Thrombin has previously been shown to bind to two receptors on the platelet surface, glycoprotein (GP) Ib-IX-V complex and the hepta-spanning transmembrane receptor coupled to G protein(s). We now show that, unlike its effect on normal platelets, kininogen (2 μmol/L) did not inhibit the thrombin-induced aggregation of Bernard-Soulier platelets, which lack the GP Ib-IX-V complex, suggesting that kininogen interacts either directly or indirectly with that complex and restricts access by thrombin to this receptor. We further show that both recombinant K270-G292 polypeptide and the synthetic peptide L271-A277 derived from high molecular mass kininogen lower thrombin binding to platelets in a manner similar to monoclonal antibodies to or ligands (von Willebrand factor and echicetin) of GP Ib-IX. The anti–GP Ib-IX-V complex antibodies, TM-60 and SZ 2, can inhibit 125I-high molecular mass kininogen binding to platelets. Conversely, kininogen could block the binding of biotinylated TM-60 or of 125I-SZ 2. Kininogen inhibited the binding of biotinylated thrombin bound to a mouse fibroblast cell line transfected with the GP Ib-IX-V complex. These results indicated that kininogen binds to the GP Ib-IX-V complex modulating thrombin binding to platelets and the consequent platelet aggregation. Kininogen can thus serve as an important regulator of the early stages of platelet stimulation by thrombin.
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  • 5
    Publication Date: 2004-10-01
    Description: 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)
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  • 6
    Publication Date: 2018-11-29
    Description: Anticoagulation requires a careful balance to achieve antithrombotic efficacy without disrupting normal hemostasis. Although current generation direct oral anticoagulants (DOACs) offer advantages over traditional warfarin or heparin therapy they still carry a bleeding risk, highlighting an unmet need for novel anticoagulants with improved therapeutic indices. DOACs such as apixaban and dabigatran bind to the active sites of their target proteases thereby inhibiting proteolysis of all substrates. In contrast, the recombinant antibody JNJ-64179375 (JNJ-9375) specifically binds to the exosite I region of thrombin without inhibiting active site function. Interestingly, JNJ-9375 has demonstrated an improved therapeutic index compared to apixaban in preclinical models of venous thrombosis (Chintala, M et al. Res Pract Thromb Haemost, 2017:1 (suppl 1): P201). To understand how the mechanism of action of JNJ-9375 contributes to its improved therapeutic index in preclinical models, we evaluated the impact of JNJ-9375 on the activity of thrombin toward physiological substrates in vitro. Fibrinogen is the primary in vivo substrate of thrombin and binds to it via exosite I. In a turbidimetric assay JNJ-9375 dose-dependently inhibited fibrinogen cleavage, reflecting direct competition between fibrinogen and JNJ-9375 for exosite I binding. Factor V (FV), Factor VIII (FVIII), and Factor XI (FXI) are activated by thrombin in positive feedback reactions. Exosites I and II in thrombin have been differentially implicated in its action on Factors V and VIII. Accordingly, JNJ-9375 potently inhibited thrombin-dependent FV activation while exhibiting a more modest inhibition of FVIII activation as determined by quantitative Western blotting. Activation of FXI by thrombin, another exosite I-mediated reaction, was reduced approximately 8-fold in the presence of JNJ-9375. Thrombin also interacts with the anticoagulant regulators Protein C and antithrombin III (ATIII). Thrombin activates Protein C through an exosite I-mediated interaction with thrombomodulin (TM). In chromogenic assays JNJ-9375 reduced the TM-dependent activation of Protein C approximately 7-fold. Plasma supplemented with JNJ-9375 also demonstrated a modest resistance to TM-dependent Protein C activation measured in thrombin generation assays. ATIII inactivates thrombin by binding to the active site of thrombin in a suicide substrate reaction. Despite this reaction being independent of exosite I binding the rate of thrombin inactivation by ATIII was reduced approximately 3-fold in the presence of JNJ-9375, indicating an allosteric effect of exosite I binding on the reactivity of thrombin with ATIII. In contrast to exosite I-dependent reactions, JNJ-9375 had no apparent effect on the exosite II-dependent interaction of thrombin with heparin. Pre-incubation with JNJ-9375 did not alter the elution profile of thrombin in heparin sepharose chromatography. Furthermore, both unfractionated heparin and low molecular weight heparin dramatically accelerated inactivation of thrombin by ATIII in the presence of JNJ-9375, indicating that exosite II function was preserved. In summary these in vitro studies illustrate a global mechanism wherein JNJ-9375 attenuates the activity of thrombin toward both pro- and anti-coagulant components of the coagulation cascade through competitive binding to the exosite I region. This distinct mechanism in which JNJ-9375 modulates thrombin without inhibiting its proteolytic activity or exosite II-mediated activity differentiates JNJ-9375 from active site-specific DOACs. We hypothesize that the residual catalytic activity of JNJ-9375-bound thrombin enables antithrombotic efficacy without the concomitant bleeding risk seen with current generation DOACs, resulting in an improved therapeutic index. Disclosures Krishnaswamy: Baxalta: Consultancy; Portola: Research Funding; Janssen Research & Development: Research Funding.
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  • 7
    Publication Date: 2004-11-16
    Description: High molecular weight kininogen (HK) is known to bind specifically and saturably to Mac-1 with a Kd = 9–18 nM for neutrophils and to uPAR with a Kd =30 nM for endothelial cells. However, the functional results of HK interaction with Mac-1 or uPAR on leukocytes is not fully understood. Kallikrein cleavage of single chain HK to a two chain form (HKa) with release of bradykinin (BK) occurs in sepsis, arthritis, and inflammatory bowel disease. We hypothesized that HKa stimulates secretion of inflammatory cytokines. Mononuclear cells were isolated from normal subjects by a Histopaque density gradient. We have expressed kininogen domain 3 (D3) and a fragment of domain 3, coded for by exon 7, E7P (aaG235-Q292), in E. Coli as glutathione S-transferase (GST) fusion proteins. HK and HKa were purified proteins. GST was recombinant. All proteins contained
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  • 8
    Publication Date: 1997-08-15
    Description: We and others have shown that both high and low molecular mass kininogens are able to inhibit the thrombin-induced aggregation of gel-filtered platelets, indicating that the locus for inhibition resides in the heavy chain. The inhibitory site is present in domain 3, confined to the C-terminal portion of the region encoded by exon 7 (K270-G292), and the minimal effective sequence is a heptapeptide (L271-A277; Kunapuli et al, J Biol Chem 271:11228, 1996). Kininogens inhibit thrombin binding to platelets and thus inhibit thrombin-induced aggregation. The molecular mechanism by which kininogens inhibit thrombin-induced aggregation of platelets is unknown. Thrombin has previously been shown to bind to two receptors on the platelet surface, glycoprotein (GP) Ib-IX-V complex and the hepta-spanning transmembrane receptor coupled to G protein(s). We now show that, unlike its effect on normal platelets, kininogen (2 μmol/L) did not inhibit the thrombin-induced aggregation of Bernard-Soulier platelets, which lack the GP Ib-IX-V complex, suggesting that kininogen interacts either directly or indirectly with that complex and restricts access by thrombin to this receptor. We further show that both recombinant K270-G292 polypeptide and the synthetic peptide L271-A277 derived from high molecular mass kininogen lower thrombin binding to platelets in a manner similar to monoclonal antibodies to or ligands (von Willebrand factor and echicetin) of GP Ib-IX. The anti–GP Ib-IX-V complex antibodies, TM-60 and SZ 2, can inhibit 125I-high molecular mass kininogen binding to platelets. Conversely, kininogen could block the binding of biotinylated TM-60 or of 125I-SZ 2. Kininogen inhibited the binding of biotinylated thrombin bound to a mouse fibroblast cell line transfected with the GP Ib-IX-V complex. These results indicated that kininogen binds to the GP Ib-IX-V complex modulating thrombin binding to platelets and the consequent platelet aggregation. Kininogen can thus serve as an important regulator of the early stages of platelet stimulation by thrombin.
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  • 9
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