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1.
The activation of factor XI initiates the intrinsic coagulation pathway. Until recently it was believed that the main activator of factor XI is factor XIIa in conjunction with the cofactor high molecular weight kininogen on a negatively charged surface. Two recent reports have presented evidence that in a purified system factor XI is activatable by thrombin together with the soluble polyanion dextran sulfate. To assess the physiological relevance of these findings we studied the activation of factor XI in normal and factor XII-deficient plasma. We used either kaolin/cephalin or dextran sulfate as a surface for the intrinsic coagulation pathway, tissue factor to generate thrombin via the extrinsic pathway, or the addition of alpha-thrombin directly. 125I-factor XI, added to factor XI-deficient plasma at physiologic concentrations (35 nmol/L), is rapidly cleaved on incubation with kaolin. The kinetics appear to be exponential with half the maximum cleavage at 5 minutes. Similar kinetics of factor XI cleavage are seen when 40 nmol/L factor XIIa (equal to 10% of factor XII activation) is added to factor XII-deficient plasma if an activating surface is provided. Tissue factor (1:500) added to plasma did not induce cleavage of factor XI during a 90-minute incubation, although fibrin formation within 30 seconds indicated that thrombin was generated via the extrinsic pathway. Adding 1 mumol/L alpha-thrombin (equivalent to 50% prothrombin activation) directly to factor XII deficient or normal plasma (with or without kaolin/cephalin/Ca2+ or dextran sulfate) led to instantaneous fibrinogen cleavage, but again no cleavage of factor XI was observable. We conclude that in plasma surroundings factor XI is not activated by thrombin, and that proposals of thrombin initiation of the intrinsic coagulation cascade are not supportable. 相似文献
2.
We developed a mouse monoclonal antibody (MoAb 115-21) to human high- molecular-weight kininogen (HK) that recognizes its prekallikrein binding site (residues 565 through 595 of HK). The corresponding synthesized 31-amino acid peptide (peptide IV) was recently shown to retain native HK's prekallikrein binding property. The same peptide bound factor XI also, although less avidly. Our MoAb recognizes purified HK, peptide IV, and the light chain moiety of HK (where the peptide IV resides), as shown by enzyme-linked immunosorbent assay (ELISA) and Western blotting experiments. The apparent dissociation constant for the HK and MoAb 115-21 interaction was 2.2 nmol/L. It does not recognize low-molecular-weight kininogen (LK) with which HK shares its heavy chain moiety or any antigens in human plasma congenitally deficient in kininogens. The binding of MoAb 115-21 to purified light chain of HK was competitively inhibited by peptide IV. In addition, the antibody inhibits HK-dependent clotting activity of normal human plasma and dextran sulfate-mediated activation of prekallikrein in plasma and retards cleavage of HK in normal plasma after contact activation with dextran sulfate. Also, purified Fab fragments of MoAb 115-21 inhibited the HK-dependent coagulant activity and dextran sulfate-mediated prekallikrein activation in normal plasma. Since the kd for HK-MoAb 115- 21 interaction is ten times lower than that of HK-prekallikrein, our data suggest that binding of MoAb 115-21 to HK's peptide IV site increases the free prekallikrein concentration in plasma and thus results in the decreased efficiency of factor XIIa-mediated activation of prekallikrein. Decreased levels of kallikrein thus formed may be responsible for the inhibition of HK-dependent clotting activity and the decrease in rate and extent of HK cleavage in normal plasma on contact activation with dextran sulfate. MoAb 115-21 may thus prove very useful, especially with its high affinity for HK, in further delineation of the role of HK and prekallikrein in contact activation and kinin-related human pathology. 相似文献
3.
Relationship of one form of human histamine-releasing factor to connective tissue activating peptide-III.
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M L Baeza S R Reddigari D Kornfeld N Ramani E M Smith P A Hossler T Fischer C W Castor P G Gorevic A P Kaplan 《The Journal of clinical investigation》1990,85(5):1516-1521
We have previously reported purification of three forms of histamine-releasing factors (HRFs) from mixtures of streptokinase-streptodornase stimulated human mononuclear cells and platelets with apparent molecular masses of 10-12, 15-17, and 40-41 kD (1989. J. Clin. Invest. 83:1204-1210). We have also prepared mouse MAbs against the 10-12-kD HRF (1989. J. Allergy Clin. Immunol. 83:281). Affinity-purified 10-12-kD HRF appears as a broad band upon polyacrylamide gel electrophoresis in the presence of SDS. We determined the NH2-terminal amino acid sequence of the top and bottom halves of this broad band. Sequence analysis revealed striking homology between this HRF and connective tissue activating peptide-III (CTAP-III), a platelet-derived 8-10-kD protein known to cause mitogenesis and extracellular matrix formation in fibroblast cultures. 19 of 21 NH2-terminal residues in the top half of the HRF band were identical to the NH2-terminal sequence of CTAP-III. 20 of 21 NH2-terminal residues in the bottom half were identical to the NH2-terminal sequence of neutrophil-activating peptide-2, which is derived from CTAP-III by proteolytic cleavage between residues 15 and 16. Purified CTAP-III also released histamine from basophils. Rabbit antiserum raised against either native or recombinant CTAP-III recognized affinity-purified HRF in immunodot blot assays, and MAb against HRF recognized CTAP-III in both dot blot and microtiter plate based immunoassays. These data demonstrate the first structural, functional, and immunologic relationship between one form of human HRF and a previously described cell product. 相似文献
4.
Chemokines and the allergic response 总被引:3,自引:0,他引:3
Abstract The β subfamily of chemokines contains-cytokine-like factors which are chemotactic for human basophils and eosinophils. They also stimulate these cells to secrete pro-inflammatory substances such as histamine or eosinophil cationic protein. MCAF/MCP-1, MCP-2, MCP-3, RANTES and MIP-lα all attract and stimulate basophils; MCP-I and MCP-3 are the most potent. RANTES, MCP-3 and to a lesser degree MIP-Ia are chemotactic factors and activators of eosinophils. Cytokines such as IL3, IL5 and GM CSF can augment the responses of these cells to the various chemokines and function as primers. These substances may have particular importance as mediators of allergic inflammation, particularly the late phase component of the response. 相似文献
5.
Kaplan AP Joseph K Shibayama Y Reddigari S Ghebrehiwet B 《International archives of allergy and immunology》2001,124(1-3):339-342
Proteins of the plasma kinin-forming cascade bind to endothelial cells and activation of the cascade can be initiated along the surface. The light chain of high molecular weight kininogen (HK) (domain 5) and factor XII bind to gC1qR, the heavy chain of HK (domain 3) binds to cytokeratin 1 and the interactions are zinc dependent. Prekallikrein binds to domain 6 of HK. Antisera to gC1qR and cytokeratin 1 inhibit binding and activation. Incubation of normal plasma with endothelial cells leads to gradual conversion of prekallikrein to kallikrein, while plasma deficient in factor XII or HK are inactive within a 2-hour time frame. Thus factor XII is critical for activation to proceed. Augmentation of these reactions may occur when C1 inhibitor is functionally deficient or with ACE inhibitors which also inhibit kininases. 相似文献
6.
Purification and further characterization of human mononuclear cell histamine-releasing factor. 总被引:4,自引:2,他引:2
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M L Baeza S Reddigari M Haak-Frendscho A P Kaplan 《The Journal of clinical investigation》1989,83(4):1204-1210
We have purified and further characterized a histamine releasing factor (HRF) derived from human mononuclear cells using gel-filtration HPLC, reverse-phase HPLC, anion exchange chromatography, and elution from SDS gels after electrophoresis. Considerable heterogeneity is seen, far exceeding that published in prior reports. Gel filtration HPLC yielded a major peak at molecular weight 30,000 and minor peaks at 50,000 and 12,000. Reverse-phase HPLC gave one major fraction in the void volume and an eluted peak at 50-60% acetonitrile. Accell QMA anion exchange HPLC revealed three peaks of activity; one in the void volume similar to that published previously using QAE-Sephadex, and peaks that eluted at 0.5 and 0.8 M ammonium acetate, respectively. Electroelution following SDS-PAGE yielded peaks at MW 12,000 and 15-17,000 plus variable peaks at 25-27,000, 31-34,000, and 80-90,000 D. Using a combination of the aforementioned procedures, we have purified molecular species of HRF at 41,000 and 17,000 D to apparent homogeneity, as judged by SDS PAGE and autoradiography. Since human interleukin 3 and granulocyte-macrophage colony-stimulating factor possess histamine-releasing capability, it is clear that multiple cytokines can share this activity. However, the major HRF we isolate from human mononuclear cells appears, thus far, to be unique. 相似文献
7.
High molecular weight kininogen (HK) is a multifunctional plasma glycoprotein that occupies a critical position in pathways that link inflammation and coagulation. It is an inhibitor of sulfhydryl proteases and has procoagulant properties. It is also a source of the vasoactive peptide bradykinin (BK). It has been previously shown that HK binds to human umbilical vein endothelial cells (HUVEC) in culture. We have further characterized that interaction herein. Immunohistochemical experiments have indicated that when freshly obtained umbilical vein segments were treated with HK, washed, and probed with anti-HK antibodies, HK was localized on the endothelium. We next determined whether HUVEC-bound HK can be cleaved by plasma kallikrein to release BK. Cultured HUVEC were incubated with unlabeled HK for varying times, washed, and the kinetics of BK release by plasma kallikrein were assayed by radioimmunoassay. Results indicated that kallikrein released BK from HUVEC in proportion to the initial amount of bound HK. No release of BK occurred in the absence of kallikrein. Also, there was no BK release upon kallikrein treatment of the HUVEC not treated with exogenous HK. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of HUVEC-bound 125I-HK indicated that addition of kallikrein resulted in cleavage of HK, thus corroborating the BK release experiments. Comparison of cleavage patterns has also indicated that cell-bound HK is slightly less susceptible to digestion by kallikrein than free HK. Therefore, our data suggest that human HK can bind to vascular endothelium in situ and that plasma kallikrein can recognize endothelial-bound HK as a substrate and liberate the vasoactive peptide BK. 相似文献
8.
To study the digestion pattern of human high-molecular weight (mol wt) kininogen (HMWK) in plasma during contact activation we have prepared monoclonal antibodies (MoAbs) to the light-chain (LC) and the heavy-chain moiety of HMWK. One MoAb from each set was purified, and neither MoAb inhibited the clotting activity of HMWK. In enzyme-linked immunosorbent assay and immunoblotting experiments neither antibody bound to kininogen-deficient plasma. Digestion of purified HMWK with plasma kallikrein yielded, on reduced sodium dodecyl sulfate gels, two LC forms, at 62 and 49 kd, respectively. Digestion of HMWK with tissue kallikrein (TK) yielded mainly the 62-kd form. In immunoblot analyses of these digests, the anti-LC MoAb detected products at 62 and 49 kd respectively. With plasma kallikrein, the 62-kd species slowly shifted to 49 kd, and with TK, the 62-kd species accumulated with time. Anti-LC MoAb was also used as a probe in immunoblotting experiments to study the digestion pattern of HMWK in whole plasma activated with kaolin or dextran sulfate. In activated normal pooled plasma (NHP) and factor XI-deficient plasma, native HMWK (mol wt, 115 kd) was cleaved within five to ten minutes, and two LC forms at 62 and 49 kd were detected. In kaolin-activated prekallikrein (PK)-deficient plasma, the disappearance of the 115-kd form was relatively slow, and only the 62-kd form of LC was seen. HMWK was not cleaved when factor XII-deficient plasma was incubated with kaolin. LC-dependent coagulant activity paralleled the presence of LC bands seen in the immunoblots, and lower-mol wt fragments of LC were not identified. These data indicate that in activated NHP two forms of LC of HMWK (62 and 49 kd) are formed sequentially. Further, the LC-dependent coagulant activity remains detectable long enough to suggest that proteolytic inactivation of LC is too slow to be an important control mechanism. 相似文献
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