Thrombin-induced Platelet Aggregation Is Inhibited by the Heptapeptide LeuAla of Domain 3 in the Heavy Chain of High Molecular Weight Kininogen

Abstract 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 <0.01 EU/ml endotoxin. For all experiments, 2 X 106/ml mononuclear cells/ml were preincubated with monoclonal antibodies, murine IgG (both at 1.8 mM) or HANKS buffer containing 0.15 M NaCl, pH 7.4 for 30 minutes at 37°C. HK, HKa, GST-D3, GST-E7P, GST-D5 or GST all at 600 nM were added. Centrifugation allowed separation of the mononuclear cell suspension into cells and supernatant. The latter was used for assay of interleukin-1β (IL-1β) by ELISA. HK and all fragments tested stimulated secretion of IL-1β of 84.8 to 306.3 pg/ml when incubated with mononuclear cells for 30 minutes at 37°C. Anti-Mac-1 antibody inhibited IL-1β secretion by HK 100%, by HKa 89%, by GST-D3 78%, by GST-E7P 94% and by GST-D5 98%. Anti-uPAR antibody inhibited IL-1β release by HK 88%, by HKa 77%, by GST-D3 95%, by GST-E7P 85%, and by GST-D5 76%. Inhibition by both receptor antibodies is consistent with their known complex formation. A monoclonal antibody (mAb) to HK D5 (C11C1) and a mAb to HK D3 (2B5) both inhibited IL-1β release by HK, HKa, GST-D5 and GST-D3 indicate that both D3 and D5 are important in cytokine release. Murine IgG gave 0% inhibition in all studies. These results indicate that kininogen may contribute to the pathogenesis of inflammatory diseases by releasing IL-1β from human blood mononuclear cells.

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High Molecular Weight Kininogen Fragments Stimulate the Secretion of Interleukin-1β through the NFκ B, JNK and p38 MAP Kinase Signaling Pathways in Monocytes.

Blood ◽

10.1182/blood.v108.11.1644.1644 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1644-1644

Author(s):

Mohammad M. Khan ◽

Harland N. Bradford ◽

Irma Isordia-Salas ◽

Yuchuan Liu ◽

Yi Wu ◽

...

Keyword(s):

Molecular Weight ◽

Signaling Pathways ◽

High Molecular Weight ◽

Intracellular Signaling ◽

Inflammatory Diseases ◽

Specific Inhibitor ◽

P38 Map Kinase ◽

High Molecular Weight Kininogen ◽

Recombinant Peptide ◽

Domain 3

Abstract Plasma high molecular weight kininogen (HK) is cleaved in inflammatory diseases by kallikrein to cleaved HK (HKa). We have reported that HKa releases cytokines (TNFα , IL-1β , IL-6) and chemokines (IL-8 and MCP-1) from isolated human peripheral blood monocytes. At a concentration of 600nM, Glutathione -S- transferase (GST) fusion proteins of kininogen domain 3 (D3), E7P - an active fragment of domain 3 (aaG255-Q292), HK domain 5 (D5), and D5 recombinant peptide HG (aa K420-D474) each stimulated secretion of IL-1β from monocytes. Receptors on monocytes including Mac-1, LFA-1, uPAR and gC1qR are required for IL-1β secretion from monocytes. We now report the signaling pathways and evidence for synthesis of IL-1 β in monocytes stimulated by HKa. Inhibitors of signaling pathways initiated by NFkB, JNK and p38, but not ERK, decreased IL-1b release from monocytes. A specific inhibitor of NFkB, MG-132 (1, 10 and 100 μM) significantly reduced IL-1β release from monocytes by 69.7, 67.3, 88.5% respectively, when stimulated by GST-E7P. The release of IL-1β by LPS (10 μg/ml), was blocked 83.4% by MG-132 (100 μM). A specific inhibitor of JNK, SP 600125 (1, 10 and 100 μM) significantly reduced IL-1β release from monocytes by 55.7, 76.3, 78.9% respectively, when stimulated by GST-E7P. The release of IL-1β by LPS (10 μg/ml), was blocked 90.23% by SP 600125 (100 μM). A specific inhibitor of p38, SB202190 (1,10 and 100 μM) significantly reduced IL-1β release from monocytes by 27.8, 14.2, 91.0% respectively, when stimulated by E7P. The release of IL-1β by LPS (10 μg/ml), was blocked 76.8 % by SB202190 (100 μM). In contrast, the ERK activation inhibitor U0126 (1, 10 and 100 μM) failed to inhibit GST-E7P-induced release of IL-1β from monocytes but the release of IL-1β with LPS (10 μg/ml), was blocked by 77.4% at 100 μM. After monocytes (4 X 106/ml) were treated with HKa (600 nM) or LPS (10 ng/ml), total RNA was extracted and RT-PCR reactions for expression of IL-1β and gC1qR mRNA using specific primers were carried out. PCR products was separated in 4% ethidium bromide-stained agarose gels and photographed. No IL-1b mRNA was detected prior to exposure to HKa or LPS but both were detected at 1 hour. In contrast, gC1qR mRNA was present without stimulation by HKa. HKa domains 3 and 5 may contribute to the pathogenesis of inflammatory diseases by stimulating the synthesis and release of IL-1β from human monocytes using intracellular signaling pathways initiated by uPAR, β 2 integrins and gC1qR receptors.

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Relationship between structure and correcting activity of bovine high molecular weight kininogen upon the clotting time of fitzgerald-trait plasma

Journal of Experimental Medicine ◽

10.1084/jem.149.4.847 ◽

1979 ◽

Vol 149 (4) ◽

pp. 847-855 ◽

Cited By ~ 14

Author(s):

AG Scicli ◽

R Waldmann ◽

JA Guimaraes ◽

G Scicli ◽

OA Carretero ◽

...

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Heavy Chain ◽

Light Chain ◽

Plasma Kallikrein ◽

High Molecular Weight Kininogen ◽

Clotting Time ◽

Active Inhibitor ◽

Charged Surfaces ◽

Bovine Plasma

Bovine high molecular weight kininogen (bHMWK) partially corrects the activated plasma thromboplastin time (aPTT) of Fitzgerald trait plasma which is congenitally deficient in HMWK. The relationship between the structure and activity of HMWK was clarified by studying the effects of different fragments of bHMWK on the aPTT of Fitzgerald-trait plasma. The peptides studied were lys-bradykinin-free HMWK, bradykinin-fragment 1-2-free HMWK, heavy chain, fragment 1-2-light chain, and light chain. All fragments were tested in equimolar concentrations. Bradykinin-fragment 1-2-free HMWK, heavy chain, and light chain have little or no correcting activity upon Fitzgerald-trait plasma aPTr. Fragment 1-2 light chain has the same correcting activity as intact bHMWK, while that of lys-bradykinin-free HMWK appears to be higher. Both fragment 1-2 and fragment 2 inhibit the clotting time of normal human plasma. When compared on a molar basis, fragment 2 is a more active inhibitor than fragment 1-2. When the effects of bovine plasma kallikrein upon bHMWK and hHMWK were studied, it was found that it released kinins from both kininogens. However, while the correcting activity of bHMWK was completely destroyed after 60 min of incubation, that of hHMWK was fully retained. These data suggest that: (a) the active part of bHMWK is comprised of the fragment 1-2 light chain portion; (b) fragment 1-2 or fragment 2 is the binding site to negatively charged surfaces, while the light chain interacts with other components of the surface-mediated reactions; and (c) bovine plasma kallikrein releases kinins, but probably does not cause the release of fragment 1-2 from human HMWK.

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Localization of the Binding Site on Plasma Kallikrein for High-Molecular-Weight Kininogen to Both Apple 1 and Apple 4 Domains of the Heavy Chain

Archives of Biochemistry and Biophysics ◽

10.1006/abbi.1994.1424 ◽

1994 ◽

Vol 314 (1) ◽

pp. 159-164 ◽

Cited By ~ 17

Author(s):

J.D. Page ◽

J.L. You ◽

R.B. Harris ◽

R.W. Colman

Keyword(s):

Molecular Weight ◽

Binding Site ◽

High Molecular Weight ◽

Heavy Chain ◽

Plasma Kallikrein ◽

High Molecular Weight Kininogen

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HIGH MOLECULAR WEIGHT KININOGEN SPECIFICALLY BLOCKS THROMBIN-INDUCED AGGREGATION BY INHIBITING PLATELET CALPAIN

10.1055/s-0038-1643860 ◽

1987 ◽

Author(s):

R N Puri ◽

F Zhou ◽

H Bradford ◽

E J Gustafson ◽

R F Colman ◽

...

Keyword(s):

Molecular Weight ◽

Platelet Aggregation ◽

High Molecular Weight ◽

Metabolic Inhibitors ◽

Dependent Manner ◽

High Molecular Weight Kininogen ◽

Normal Plasma ◽

Calpain 2 ◽

Induced Aggregation ◽

Aggregation Of Platelets

We have previously shown that platelet-aggregation induced by alpha-thrombin (1.7 nM) involves complete cleavage of the surface membrane polypeptide, Mr = 100 kDa (MP 100) labeled by FSBA in intact platelets. The failure to cleave MP100 in membrane preparations or in platelets treated with metabolic inhibitors or leupeptin, suggested that thrombin was acting by activating platelet calpain. Since high molecular weight kininogen (HMWK) is the most potent plasma inhibitor of calpain(s), we now report that HMWK inhibited thrombin-induced aggregation in a dose-dependent manner over a range of plasma concentrations. HMWK did not inhibit aggregation induced by ADP, collagen, U46619, A23187 and/or PMA. In order to study the action of HMWK in a plasma environment we utilized Y-thrombin. The aggregation induced by γ-thrombin (25 nM) in washed human platelets was also inhibited by HMWK. A much higher concentration of γ - thrombin (200 nM) was required to induce similar aggregation of platelets suspended in normal plasma. In contrast, γ -thrombin (50nM) induced complete aggregation of platelets suspended in plasma completely deficient in total kininogen indicating that a kininogen was predominently responsible for the inhibitory effect of plasma. When platelets were suspended in plasma deficient only in HMWK, aggregation required 75 nMY-thrombin. When plasma deficient in HMWK was supplemented with a physiological concentration of HMWK (0.67 μM) the aggregation of suspended washed platelets was similar to that in normal plasma. Finally, we found that purified platelet calpain-2 not only exposed fibrinogen binding sites and induced platelet aggregation, but also completely cleaved MP 100 in both intact platelets and membrane preparations. We conclude: a) Thrombin-induced platelet aggregation involves the indirect proteolytic cleavage of MP100 by activating calpain-2, and b) Inhibition of thrombin-induced platelet aggregation by HMWK involves specific inactivation of platelet calpain.

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STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF DOMAINS IN THE HEAVY CHAIN REGION OF FACTOR XI (XIa) INVOLVED IN BINDING HIGH MOLECULAR WEIGHT KININOGEN AND FACTOR IX

10.1055/s-0038-1642804 ◽

1987 ◽

Author(s):

F A Baglia ◽

D Sinha ◽

P N Walsh

Keyword(s):

Molecular Weight ◽

Monoclonal Antibody ◽

Binding Site ◽

High Molecular Weight ◽

Heavy Chain ◽

Substrate Binding ◽

The Other ◽

High Molecular Weight Kininogen ◽

Factor Xi ◽

Substrate Binding Site

Previous studies from our laboratory (J. Biol. Chem. 260:10714,1985; J. Clin. Invest. 78:1631,1986) provide evidence that a monoclonal antibody (3C1) directed against the heavy chain region of factor XIa (FXIa) recognizes an epitope near a substrate binding site for FIX and a binding site for high molecular weight kininogen (HMWK). The present studies were carried out to determine whether these two sites are identical or different. Another heavy-chain-specific murine monoclonal antibody (5F7) was found to recognize an epitope distinct from that recognized by 3C1 since 3C1 did not compete with 5F7 for binding to FXI in a solid-phase radioimmunoassay. Antibody 3C1 was a competitive inhibitor of F-XIa-catalyzed F-IX activation, assayed by the release of a 3H-labeled activation peptide from FIX, whereas 5F7 had no effect on F—IX activation by FXIa. In contrast, 5F7 (which also inhibited F-XIIa-catalyzed F-XI activation in the presence of HMWK and kaolin) completely blocked FXI binding to immobilized HMWK at concentrations 1,000-fold lower than 3C1. Finally, HMWK had no effect on F-IX activation by FXIa. We therefore conclude that two separate and distinct domains are present in the heavy-chain region of FXI, one of which is a substrate binding site for FIX and the other a binding site for HMWK. A 15,000 Mr peptide containing the HMWK binding site was isolated using cyanogen bromide digests of factor XI which were bound to and eluted from a ,5F7 antibody affinity column and further purified using high performance liquid chromatography. Gas phase sequencing studies are in progress to characterize this peptide and place its sequence within the known structure of the heavy chain of FXIa. In conclusion, our antibodies have defined two domains within the heavy chain region of FXI: one defined by 5F7 is near the HMWK binding site, whereas the other, recognized by 3C1, is a substrate binding site for FIX. Finally, a peptide domain in the heavy chain of FXI that compriaes the HMWK binding site has been identified and isolated.

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Characterization of molecular defects of Fitzgerald trait and another novel high-molecular-weight kininogen-deficient patient: insights into structural requirements for kininogen expression

Blood ◽

10.1182/blood-2002-11-3329 ◽

2003 ◽

Vol 101 (11) ◽

pp. 4430-4436 ◽

Cited By ~ 13

Author(s):

Yelena Krijanovski ◽

Valerie Proulle ◽

Fakhri Mahdi ◽

Marie Dreyfus ◽

Werner Müller-Esterl ◽

...

Keyword(s):

Molecular Weight ◽

Amino Acid ◽

High Molecular Weight ◽

Stop Codon ◽

Premature Stop Codon ◽

Amino Acid Residues ◽

High Molecular Weight Kininogen ◽

Basilar Artery Thrombosis ◽

Domain 3 ◽

Exon 10

Abstract A 6-year-old male with vertebral-basilar artery thrombosis was recognized to have high-molecular-weight kininogen (HK) deficiency. The propositus had no HK procoagulant activity and antigen (< 1%). Using monoclonal antibodies (Mabs) to kininogen domain 3, the propositus, family members, and Fitzgerald plasma were determined to have detectable low-molecular-weight kininogen. Mabs to HK domains 5 and 6 do not detect HK antigen in the propositus' plasma. The propositus has a single base pair (bp) deletion in cDNA position 1492 of exon 10 affecting amino acid 480 of the mature protein and resulting in a frameshift and a premature stop codon at position 1597 (amino acid 532). Unexpectedly, Mabs to the heavy chain and domain 5 of HK detect a 92-kDa form of HK in Fitzgerald plasma, the first HK-deficient plasma. The 92-kDa Fitzgerald HK has amino acid residues through 502, corresponding to domains 1 through 5, but lacks epitopes of domain 6 (positions 543 to 595). Fitzgerald DNA has a normal exon 10, but a 17-bp mutation in intron 9. These combined results indicate that mutations in the kininogen gene may differentially affect biosynthesis, processing, and/or secretion of HK.

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