FIG 1 - uploaded by Raul A DeLa Cadena
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Primary structure of kininogen domain 3. Domain 3 (amino acids 235–357) is coded for by exons 7 (Gly 235 –Gln 292 ), 8 (Val 293 – Gly 328 ), and 9 (Gln 329 –Met 357 ). Boxed N is N-linked carbohydrate. The shaded area indicates the site for cysteine protease binding. Figure is modified from DeLa Cadena et al. (1994a).
Source publication
The ability of kininogens to modulate thrombin-induced aggregation of human platelets has been assigned to domain 3 (D3) in the common heavy chain coded for by exons 7, 8, and 9 of kininogen gene. We expressed each of the exons 7, 8, and 9, and various combinations as glutathione S-transferase fusion proteins in Escherichia coli. Each of the exon p...
Citations
... 35 Kunapuli et al. found that recombinant domain 3 inhibited thrombin-induced platelet aggregation. 40 Sugi and McIntyre 24 reported that certain aPE are not specific for PE per se, but are directed to PE-binding plasma proteins, kininogens. Several studies report strong association of aPE with thrombosis and recurrent pregnancy losses. ...
Factor XII, plasma prekallikrein and high-molecular-weight kininogen were first identified as coagulation proteins in the intrinsic pathway because patients deficient in these proteins had marked prolongation of in vitro surface-activated coagulation time. However, deficiencies of these proteins are not associated with clinical bleeding. Paradoxically, studies suggest that these proteins have anticoagulant and profibrinolytic activities. In fact, association between deficiencies of these proteins and thrombosis has been reported. Recently, autoantibodies to these proteins and antiphospholipid antibodies are frequent coagulation-related abnormalities found in unexplained recurrent aborters. Evidence has accumulated for the presence of the kallikrein-kininogen-kinin system in the fetoplacental unit. The contact system, or kallikrein-kininogen-kinin system, in the reproductive tract plays an essential roll in the regulation of thrombosis, hemostasis, angiogenesis and in the defense against invasive bacterial infection. Autoantibodies to these proteins may be associated with pregnancy losses due to disruption of this system. These possibilities will be reviewed, the functions of the individual components will be summarized, and their role in blood coagulation and pregnancy discussed.
... 4 Two distinct sequences from domain 3 and domain 4 are each capable of inhibiting thrombin-induced platelet activation, although the affinities and the mechanism are different. Most of the inhibitory activity of domain 3 is in the region coded for by exon of G235-Q292 (IC 50 ϭ 0.2 M). 5 This sequence competes with thrombin for binding to platelet GPIb. The second sequence in domain 4 is minimally contained in the breakdown product of bradykinin, catalyzed by ACE, bradykinin 1-5 peptide RPPGF. ...
Comment on Shariat-Madar et al, page [192][1]
In this issue, Shariat-Madar and colleagues provide evidence that BK B2 receptor knockout (BKB2R-/-) mice have a long bleeding time and delayed thrombosis that depends in part on nitric oxide and prostacyclin elevation produced by angiotensin II binding
... Moreover, do-main 5 of HK binds to the  2 -integrin Mac-1 on granulocytes and competes for fibrinogen and ICAM-1 binding in vitro and serves to regulate the Mac-1-dependent leukocyte recruitment in vivo (21). On platelets, cell-associated thrombospondin as well as GPIb mediate the binding of HK (22)(23)(24). Whereas binding to GPIb␣ has been mapped to the region Lys 270 -Gln 292 of HK domain 3, multiple binding sites for HK have been proposed within the GPIb␣ molecule, among others the region 269 -282 that contains three sulfated tyrosine residues, which is recognized by the mAb SZ2 (23). In contrast, the region 201-268 of GPIb␣ recognized by mAb AP1 mediates the Mac-1-GPIb␣ interaction (10). ...
... Biotin-labeled HK and HKa were produced as previously described (21). Glutathione S-transferase fused to domains 3, 5, and 6 of HK or to sequences derived from domain 5 were produced as previously described (24). Glutathione Stransferase was amino terminally attached to the following sequences of HK: Gly 235 -Met 357 (domain 3), Lys 420 -Ser 513 (domain 5), Thr 503 -Ser 626 (domain 6) as well as Lys 420 -Asp 474 and His 475 -Lys 502 (amino-and carboxyl-terminal domain 5 sequences). ...
... Furthermore, HK was previously described to regulate platelet aggregation. Domains 3 and 5, as well as fragments thereof inhibit thrombin-induced platelet aggregation and interfere with ligand binding to ␣ IIb  3 -integrin, respectively (24,40). However, the present work indicates that the antithrombotic properties of these domains might also be attributable to their ability to interfere with Mac-1-GPIb␣-dependent leukocyteplatelet interactions. ...
Leukocyte-platelet interaction is important in mediating leukocyte adhesion to a thrombus and leukocyte recruitment to a site of vascular injury. This interaction is mediated at least in part by the β 2-integrin Mac-1 (CD11b/CD18) and its counter-receptor on platelets, glycoprotein Ibα (GPIbα). High molecular weight kininogen (HK) was previously shown to interact with both GPIbα and Mac-1 through its domains 3 and 5, respectively. In this study we investigated the ability of HK to interfere with the leukocyte-platelet interaction. In a purified system, HK binding to GPIbα was inhibited by HK domain 3 and the monoclonal antibody (mAb) SZ2, directed against the epitope 269-282 of GPIbα, whereas mAb AP1, directed to the region 201-268 of GPIbα had no effect. In contrast, mAb AP1 inhibited the Mac-1-GPIbα interaction. Binding of GPIbα to Mac-1 was enhanced 2-fold by HK. This effect of HK was abrogated in the presence of HK domains 3 or 5 or peptides from the 475-497 region of the carboxyl terminus of domain 5 as well as in the presence of mAb SZ2 but not mAb AP1. Whereas no difference in the affinity of the Mac-1-GPIbα interaction was observed in the absence or presence of HK, maximal binding of GPIbα to Mac-1 doubled in the presence of HK. Moreover, HK/HKa increased the Mac-1-dependent adhesion of myelomonocytic U937 cells and K562 cells transfected with Mac-1 to immobilized GPIbα or to GPIbα-transfected Chinese hamster ovary cells. Finally, Mac-1-dependent adhesion of neutrophils to surface-adherent platelets was enhanced by HK. Thus, HK can bridge leukocytes with platelets by interacting via its domain 3 with GPIbα and via its domain 5 with Mac-1 thereby augmenting the Mac-1-GPIbα interaction. These distinct molecular interactions of HK with leukocytes and platelets contribute to the regulation of the adhesive behavior of vascular cells and provide novel molecular targets for reducing atherothrombotic pathologies.
... These results confirm that FXR is a permissive receptor and that it is the FXR/RXR␣ heterodimer that transactivates the kininogen promoter. DISCUSSION HKa has been shown to exert important functions in antiadhesion (26 -29), anti-inflammation (30), and anti-platelet aggregation (31). In addition, HKa has also been shown to be a mediator and modulator of vascular inflammation and local injury (9). ...
Human kininogen belongs to the plasma kallikreinkinin system. High molecular weight kininogen is the precursor for two-chain kinin-free kininogen and bradykinin. It has been shown that the two-chain kinin-free kininogen has the properties of anti-adhesion, anti-platelet aggregation, and anti-thrombosis, whereas bradykinin is a potent vasodilator and mediator of inflammation. In this study we show that the human kininogen gene is strongly up-regulated by agonists of the farnesoid X receptor (FXR), a nuclear receptor for bile acids. In primary human hepatocytes, both the endogenous FXR agonist chenodeoxycholate and synthetic FXR agonist GW4064 increased kininogen mRNA with a maximum induction of 8-10-fold. A more robust induction of kininogen expression was observed in HepG2 cells, where kininogen mRNA was increased by chenodeoxycholate or GW4064 up to 130-140-fold as shown by real time PCR. Northern blot analysis confirmed the up-regulation of kininogen expression by FXR agonists. To determine whether kininogen is a direct target of FXR, we examined the sequence of the kininogen promoter and identified a highly conserved FXR response element (inverted repeat, IR-1) in the proximity of the kininogen promoter (-66/-54). FXR/RXRalpha heterodimers specifically bind to this IR-1. A construct of a minimal promoter with the luciferase reporter containing this IR-1 was transactivated by FXR. Deletion or mutation of this IR-1 abolished FXR-mediated promoter activation, indicating that this IR-1 element is responsible for the promoter transactivation by FXR. We conclude that kininogen is a novel and direct target of FXR, and bile acids may play a role in the vasodilation and anti-coagulation processes.
... Domain 3 (D3) inhibits papain and cathepsin L [3], binds to cell receptors GPIb-IX-V on platelets [4,5], the integrin Mac-1 (a M b 2 ) [6] on neutrophils [7], and cytokeratin 1 on endothelial cells [8]. D3, by inhibiting thrombin binding to platelets [9,10], accounts for HK antithrombotic action in vivo [11]. Domain 4 contains bradykinin, which binds to B1 and B2 receptors. ...
We previously localized the heparin binding region on high molecular weight kininogen to domain 5 (D5) by quantifying the binding using surface plasmon resonance of D5 fused at its N-terminal to glutathione-S-transferase. We further examined GST-(H475-S626) which at 100 nm was previously shown to be ineffective in reversing the heparin acceleration of antithrombin inhibition of thrombin. However, we now show that at a concentration of 400 nm, complete reversal of accelerated inhibition occurred. To characterize the interacting sequences on D5, four peptides representing surface loops of a molecular model were synthesized. Peptides H475-H485 and G440-G455, rich in histidine and low in lysine, showed weak or no detectable binding in the absence of Zn++, but tighter binding in the presence of Zn++. H483-K497 containing three histidines and six lysines showed tight binding without Zn++, and increased in avidity with Zn++. In contrast, G486-K502, low in histidine and high in lysine, showed tight binding (KD = 0.8 microm) in the absence and presence of Zn++. Both H483-K497 and G486-K502 were effective in neutralizing the accelerated inhibition by heparin of thrombin by antithrombin in the absence of Zn++. Therefore, a set of lysine residues in the sequence of K487-K502 is responsible for Zn++-independent binding of heparin. Further, a group of histidine residues in sequence range of H475-H485 contributes to Zn++-dependent binding of heparin to HK-D5.
... HK contains the anti-angiogenic domain 5, kininostatin, which inhibits endothelial cell proliferation and stimulates apoptosis through an anti-adhesive mechanism that involves the displacement of endothelial cells from vitronectin (10). Other HK domains interact with platelets and inhibit calpainrelated platelet aggregation via domain 2 and prevent thrombin binding to glycoprotein Ib-IX-V (GPIb-IX-V) via domain 3 or protease activated receptor-1 activation via domain 4 (11)(12)(13). Both FXI (30 nM) and prekallikrein (PK) (490 nM) circulate in plasma in high-affinity (K D ϳ 10 Ϫ8 M) complexes with HK (670 nM) (14 -18). ...
Previous studies on the interaction of high molecular weight kininogen (HK) with endothelial cells have reported a large number of binding sites (106-107 sites/cell) with differing relative affinities (KD = 7-130 nm) and have implicated various receptors or receptor complexes. In this study, we examined the binding of HK to human umbilical vein endothelial cells (HUVEC) with a novel assay system utilizing HUVEC immobilized on microcarrier beads, which eliminates the detection of the high affinity binding sites found nonspecifically in conventional microtiter well assays. We report that HK binds to 8.5 x 104 high affinity (KD = 21 nm) sites per HUVEC, i.e. 10-100-fold fewer than previously reported. Although HK binding is unaffected by the presence of a physiological concentration of prekallikrein, factor XI abrogates HK binding to HUVEC in a concentration-dependent manner. Disruption of the naturally occurring complex between factor XI and HK by the addition of a 31-amino acid peptide mimicking the factor XI-binding site on HK restored HK binding to HUVEC. Furthermore, HK inhibited thrombin-stimulated von Willebrand factor release by HUVEC but not thrombin receptor activation peptide (SFLLRN-amide)-stimulated von Willebrand factor release. Factor XI restored the ability of thrombin to stimulate von Willebrand factor release in the presence of low HK concentrations. These results suggest that free HK, or HK in complex with prekallikrein but not in complex with factor XI, interacts with the endothelium and can maintain endothelial cell quiescence by preventing endothelial stimulation by thrombin.
... By using specific monoclonal antibodies, Jiang et al. [36] showed the D3 region to be responsible for the inhibition of thrombin binding to platelets. Kunapuli et al. [37] found that recombinant D3 inhibited thrombin-induced platelet aggregation. We thus hypothesized that the APE can bind to kininogens on the platelet. ...
... Twenty-four previously determined kininogen-dependent APEpositive RPL patients were chosen for this study. The mean age of the patients was 31 years (range [22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41], and the mean number of pregnancy losses was 2.7. The APE ELISA followed a previously described procedure [11,12,32,38]. ...
Antiphosphatidylethanolamine antibodies (APE) have been described in patients with thrombotic diseases and recurrent pregnancy loss (RPL). It has been reported that certain APE are not specific for phosphatidylethanolamine (PE) per se, but are directed to PE-binding plasma proteins, called kininogens. Our recent in vitro data suggest that APE may recognize the domain 3 (D3) region of kininogens. In this study, we have used synthetic peptides that span the D3 of kininogens in inhibition and direct binding studies to identify epitopes that are sites for binding APE. Our present data demonstrate that among 24 RPL patients who were positive for kininogen-dependent immunoglobulin (IgG) APE, 17 patients (70.8%) recognized the LDC27 peptide. We mapped the APE-binding region on D3 using plasma from a RPL patient (X) who had a high titer of IgG APE that recognized LDC27. APE of patient X recognized a 13-residue segment in LDC27, named CNA13. Leu331-Met357 (LDC27) and Cys333-Lys345 (CNA13) are located on the carboxyl-terminal portion of kininogen D3, which is known as the major kininogen heavy chain cell attachment site where it overlaps its cysteine protease inhibitory region. Because APE interferes with the balance of hemostasis in vitro, APE may therefore induce a similar condition in patients thereby causing thrombosis and RPL.
... These include the following. (i) Binding to the glycoprotein Ib-IX-V complex on platelets, which is mediated by domain 3 of HK, may lead to inhibition of thrombin-dependent platelet aggregation (48). (ii) ␣-granulederived HK can inhibit the function of platelet calpain involved in, e.g. ...
The adhesive glycoprotein vitronectin (VN) forms a function-stabilizing complex with plasminogen activator inhibitor-1 (PAI-1),
the major fibrinolysis inhibitor in both plasma and vessel wall connective tissue. VN also interacts with two-chain high molecular
weight kininogen (HKa), particularly its His-Gly-Lys-rich domain 5, and both HKa and PAI-1 are antiadhesive factors that have
been shown to compete for binding to VN. In this study the influence of HKa and domain 5 on the antifibrinolytic function
of PAI-1 was investigated. In a purified system, HKa and particularly domain 5 inhibited the binding of PAI-1 to VN and promoted
PAI-1 displacement from both isolated VN as well as subendothelial extracellular matrix-associated VN. The sequence Gly486-Lys502 of HKa domain 5 was identified as responsible for this inhibition. Although having no direct effect on PAI-1 activity itself,
HKa domain 5 or the peptide Gly486-Lys502 markedly destabilized the VN·PAI-1 complex interaction, resulting in a significant reduction of PAI-1 inhibitory function
on plasminogen activators, resembling the effect of VN antibodies that prevent stabilization of PAI-1. Furthermore, high affinity
fibrin binding of PAI-1 in the presence of VN as well as the VN-dependent fibrin clot stabilization by the inhibitor were
abrogated in the presence of the kininogen forms mentioned. Taken together, our data indicate that the peptide Gly486-Lys502 derived from domain 5 of HKa serves to interfere with PAI-1 function. Based on these observations potential low molecular
weight PAI-1 inhibitors could be designed for the use in therapeutic interventions against thromboembolic complications.
... Apart from its role as precursor of bradykinin, which serves important vasodilator functions in vascular homeostasis, high molecular weight kininogen exhibits anti-adhesive and antithrombotic properties, especially in the two chain, kinin-free form (HKa) (18,(32)(33)(34)(35)48). In the present study we continued to define the underlying mechanisms of these functions of HKa, related to disruption of αIIbß3-mediated platelet adhesion and aggregation. ...
... Moreover, domain 5 and particularly the carboxy-terminal region G486-K502 were identified as potent inhibitors of platelet aggregation by blocking the αIIbß3-VN interaction. Our results extend the previously described antithrombotic properties of kininogen (32,33,48) and indicate that novel anti-aggregatory drugs could be designed on the basis of the region G486-K502 derived from domain 5. ...
... Besides the inhibitory properties of HK for VN-dependent platelet adhesion and aggregation described in the present study, previous reports have demonstrated that HK can bind to thrombospondin and to glycoprotein Ib-IX-V complex on platelets (30,31). The latter interaction, attributable to domain 3 of HK, mediates one of the two pathways for inhibition of thrombin-dependent platelet aggregation by HK, a property that has also been ascribed to breakdown products of bradykinin (32,33). In addition to interactions of extracellular HK with platelets, α-granule-derived HK can also inhibit the function of platelet calpain, an effect mainly mediated by domain 2 of HK (55). ...
Proteolytic cleavage of single chain high molecular weight kininogen (HK) by kallikrein releases the short-lived vasodilator bradykinin and leaves behind two-chain high molecular weight kininogen (HKa). HKa and particularly its His-Gly-Lys-rich domain 5 have been previously reported to exert anti-adhesive properties by binding to the extracellular matrix protein vitronectin (VN). In this study the ability of HKa and domain 5 to interfere with platelet adhesion and aggregation was investigated. In a purified system HKa and particularly domain 5 but not HK inhibited the binding of VN to the alpha(IIb)beta(3) integrin, whereas the binding of fibrinogen to this integrin was not affected. The region Gly-486-Lys-502 from the carboxyl terminus of the domain 5 was identified as responsible for inhibition of the VN-alpha(IIb)beta(3)-integrin interaction, as this portion was also found to mediate kininogen binding to VN. Through these interactions, HKa, the isolated domain 5, and the peptide Gly-486-Lys-502 abrogated the alpha(IIb)beta(3)-integrin-dependent adhesion of human platelets to VN but not to fibrinogen. The codistribution of VN and HKa at sites of ex vivo platelet aggregation was demonstrated by transmission immune electron microscopy, indicating that the described interaction is likely to take place in vivo. Moreover, domain 5 and the peptide Gly-486-Lys-502 dose-dependently blocked platelet aggregation, resembling the inhibitory effect of monoclonal antibody 13H1 against multimeric VN. Finally, treatment of mice with isolated domain 5 resulted in a significantly prolonged tail bleeding time. Taken together, our data emphasize the inhibitory role of HK domain 5 on platelet adhesion and aggregation; new anti-thrombotic compounds may become available on the basis of peptide Gly-486-Lys-502 of HK domain 5.
... Platelet Isolation and Aggregation-Platelet isolation by gel filtration and platelet aggregation was performed as described previously (21). Briefly, an aliquot of platelets (Ͼ100,000/l) and added inhibitors in a 0.5-ml cuvette at 37°C was incubated with stirring for 2 min in the presence of 50 M free Zn 2ϩ . ...
... Briefly, platelets were washed by centrifugation, and after the addition of 1 mM EDTA and 1 M prostaglandin E 1 the resulting platelet pellet was incubated with 100 M SFLLRN peptide at room temperature for 5 min without stirring. Following incubation with the SFLLRN peptide, the resuspended platelet preparation was gel-filtered to remove any remaining inhibitors or agonists (21). The resulting platelet pool yielded concentrations between 2 and 6 ϫ 10 8 cells/ml and were further tested for response to SFLLRN-and thrombin-induced aggregation at 37°C. ...
Factor XII deficiency has been postulated to be a risk factor for thrombosis suggesting that factor XII is an antithrombotic protein. The biochemical mechanism leading to this clinical observation is unknown. We have previously reported high molecular weight kininogen (HK) inhibition of thrombin-induced platelet aggregation by binding to the platelet glycoprotein (GP) Ib-IX-V complex. Although factor XII will bind to the intact platelet through GP Ibalpha (glycocalicin) without activation, we now report that factor XIIa (0. 37 microm), but not factor XII zymogen, is required for the inhibition of thrombin-induced platelet aggregation. Factor XIIa had no significant effect on SFLLRN-induced platelet aggregation. Moreover, an antibody to the thrombin site on protease-activated receptor-1 failed to block factor XII binding to platelets. Inhibition of thrombin-induced platelet aggregation was demonstrated with factor XIIa but not with factor XII zymogen or factor XIIf, indicating that the conformational exposure of the heavy chain following proteolytic activation is required for inhibition. However, inactivation of the catalytic activity of factor XIIa did not affect the inhibition of thrombin-induced platelet aggregation. Factor XII showed displacement of biotin-labeled HK (30 nm) binding to gel-filtered platelets and, at concentrations of 50 nm, was able to block 50% of the HK binding, suggesting involvement of the GP Ib complex. Antibodies to GP Ib and GP IX, which inhibited HK binding to platelets, did not block factor XII binding. However, using a biosensor, which monitors protein-protein interactions, both HK and factor XII bind to GP Ibalpha. Factor XII may serve to regulate thrombin binding to the GP Ib receptor by co-localizing with HK, to control the extent of platelet aggregation in vivo.
