摘要:

Tissue factor (TF), a transmembrane protein, is the main initiator of blood clottingin vivowhich functions by complexing the enzyme, factor VIIa, which then activates its natural substrates, factors IX and X. TF functions by increasing the affinity of factor VIIa for the surface and increasing the catalytic rate of factor VIIa. TF is not proteolytically activated, but is regulated by its exposure to blood, for example by cellular expression after stimulation with endotoxin, tumour necrosis factor, or interleukin I. The function of TF is modulated by the surrounding phospholipid surface. Anionic phospholipids stimulate TF:VIIa activity by lowering the apparentKM.Since the enzyme is localized on a two-dimensional surface, the apparent enzymatic parameters are dependent on the transport of substrate to the surface. This, in turn, is a function of enzyme density and flow conditions.

ISSN:0957-5235    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

Congenital dysfibrinogenaemia was found in three non-related patients. None of them had a haemorrhagic tendency, but one gave a thrombotic history. When their fibrinogens were treated with thrombin, they released fibrinopeptides A and B at normal rates, but the resultant fibrin monomers produced exhibited abnormal polymerization curves. This abnormality was more marked in fibrinogen Villajoyosa than in Barcelonas III and IV. Plasminogen and t-PA binding to fibrin monomers from the three dysfibrinogenaemias was similar to that of normal fibrin monomers. The γ chain was purified from the three fibrinogens, treated with CNBr and the peptides produced were separated by reversed-phase HPLC. Chromatograms of digested fibrinogens showed an abnormal peak that was not present in the normal γ chain. Amino acid sequence analysis of abnormal peptides and genomic DNA sequencing revealed that the γ arginine 275 had been changed in the three fibrinogens; in two cases it was substituted by histidine, and in the third by cysteine. The altered properties observed in fibrin monomers produced from fibrinogen with the γArg 275→His or γArg 275→Cys substitution, suggests that this amino acid is important in maintaining the protein structure necessary for normal polymerization, but is not essential for the binding of t-PA or plasminogen to fibrin. It also suggests that the change Arg→Cys produces more severe alterations in the functions of fibrinogen than the substitution Arg→His.

ISSN:0957-5235    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

The interaction between the plasminogen kringle 4 module and a synthetic peptide corresponding to the tryptic heptapeptide fragment Ala-Phe-Gln-Tyr-His-Ser-Lys (AFQYHSK), segment 44–50 of the plasminogen N-terminal peptide (Wiman and Wallén,Eur J Biochem1975; 50: 489–494), has been investigated by1H-NMR spectroscopy. AFQYHSK, as well as the shorter fragments thereof, FQYHSK, QYHSK and YHSK, all bound to kringle 4 with equilibrium association constant (Ka) values ranging between 2.5 and 8.5 mM-1. The NMR evidence also indicates that binding is mediated by the canonical kringle lysine binding site and involves the C-terminal Lys residue of the ligand peptide. The results (a) support a potential interaction between plasminogen Lys-binding kringles and the N-terminal activation peptide, and (b) unambiguously demonstrate the capability of such kringles to bind polypeptides ending with C-terminal lysine.

ISSN:0957-5235    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

Cross-linked fibrin degradation products have been used to detect venous thrombosis. While the sensitivity of plasma D-dimer measured by ELISA in the diagnosis of deep vein thrombosis (DVT) is high, the utility of ELISA methods is limited in a clinical setting. This study analysed the diagnostic value of a rapid D-dimer assay performed on whole blood samples (SimpliRed D-dimer) compared with latex and ELISA in 86 patients suspected of having DVT. SimpliRed D-dimer was positive in 47/50 of patients with DVT established by Doppler ultrasound (DU; sensitivity 94%). SimpliRed D-dimer was positive in 35/37 of patients with proximal DVT, nine out of nine with popliteal DVT and three out of four with superficial thrombophlebitis. The specificity of SimpliRed D-dimer in the diagnosis of DVT was 61 %. The sensitivity of the SimpliRed D-dimer assay was at least comparable with the ELISA (87%) and superior to the latex assay (80%). The positive predictive value (77%), the negative predictive value (88%) and the overall accuracy (80%) of the SimpliRed assay were better than the ELISA and latex methods. It is concluded that SimpliRed D-dimer is a rapid useful assay for screening of patients suspected of having deep vein thrombosis.

ISSN:0957-5235    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

Several ligand blotting or immunoblotting assays for the detection of single-chain and proteolytically cleaved two-chain high molecular weight kininogen (HK) in whole plasma have been described. Since they may suffer from poor sensitivity for the light chain species of cleaved HK on reduced blots, an antiserum against the reduced and alkylated 47 kDa light chain of HK was raised in rabbits allowing improved immunodetection of HK species on blots of reduced electropherograms. This immunoblotting method is highly specific and sensitive, permitting detection of 0.2 ng single-chain HK or the light chains of 2 ng proteolytically cleaved HK in whole plasma. Thus, this immunoblotting technique is at least 50–100 times more sensitive than ligand blotting with radiolabelled factor XI overlay. A similar cleavage pattern was observed followingin vitroactivation of normal human plasma by dextran sulphate and after plasma kallikrein-induced proteolysis of purified HK. However, bands of different molecular weights were generated after HK had been cleaved by purified leukocyte elastase. During acute attack in a patient with hereditary angioedema, high levels ofin vivocleaved HK were noticed, whereas concentration of cleaved HK in plasma samples and synovial fluids from patients suffering from various inflammatory conditions were not substantially higher than those in normal plasma. Duringin vitrocold activation of plasma samples of pregnant women concomitant HK cleavage and plasma kallikrein generation were observed.

ISSN:0957-5235    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

This study investigated the effect of carbazochrome sodium sulphonate (AC-17) on the accumulation of tissue-type plasminogen activator antigen (t-PA:Ag) and plasminogen activator inhibitor-1 antigen (PAI-l:Ag) in culture medium of human umbilical vein endothelial cells (HUVEC). HUVEC were cultured in the presence of AC-17, and the concentration of t-PA:Ag and PAI-1:Ag in the medium was measured by an enzyme-linked immunoassay. AC-17, after 48 and 72 h incubation, significantly decreased the t-PA:Ag in the culture medium, while it had no significant effect on the PAI-1:Ag. The result of fibrin zymography was consistent with the result obtained by ELISA. It also revealed that all of the t-PA present in the culture medium formed complexes with PAI-1, indicating that AC-17 did not interfere with t-PA/PAI-1 complex formation. Furthermore, AC-17 did not inhibit the activities of t-PA and plasmin when measured by a fibrin plate method. These results suggest that AC-17 may modulate the fibrinolytic balance of blood through changing the function of endothelial cells, a new aspect of action of AC-17 as a haemostatic drug.

ISSN:0957-5235    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

The response of coagulation tests to heparin can be expressed as the coagulation time of plasma containing heparin divided by the coagulation time of the same plasma without heparin (CT ratio). The purpose of the present study was to assess the influence of liposomes on the response to heparin of four coagulation tests: the kaolin-induced coagulation time, the tissue factor-induced coagulation time, the factor Xa-induced coagulation time, and the thrombin-induced coagulation time. Liposomes were prepared from dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), and dioleoylphosphatidylserine (DOPS). High concentrations of DOPS/DOPE/DOPC (20:40:40) liposomes enhanced the CT ratio of the four coagulation tests, more than DOPS/DOPC (20:80) or DOPE/DOPC (40:60) or a mixture of DOPS/DOPC and DOPE/DOPC liposomes. These experiments demonstrate that there is synergism between DOPS and DOPE in promoting heparin's anticoagulant effect if both phospholipids are incorporated into the same liposome surface.

ISSN:0957-5235    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

This study compared a rapid method to detect the nucleotide mutation 1691 G→A, responsible for the factor V Arg506→Gln substitution, with a previously established denaturing gradient gel electrophoresis (DGGE) technique in 136 patients with unexplained thrombosis. The new method comprises amplification of the factor V gene exon 10 with a modified oligonucleotide, permitting the introduction of a cleavage site for the restriction endonucleaseHindIII in the fragments bearing the mutation. This simple, rapid, inexpensive and non-isotopic method gave the same results as the DGGE method in all subjects tested.

ISSN:0957-5235    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

Among six monoclonal antibodies raised against the human plasmin-α2-plasmin inhibitor complex (PPI), three antibodies were found to recognize the plasmin part (group 1) and another three the α2-plasmin inhibitor (α2-PI) part (group 2) of the complex. One of the group-1 monoclonal antibodies, designated JIPPI-3, specifically reacted with a segment of plasmin containing kringles 2 and 3. Although all three group 2 antibodies reacted with both α2-PI and PPI on immunoblotting and ELISA, one of them, JIPPI-50, was unable to react with α2-PI, when the antibody had been covalently conjugated to Sepharose 4B gels and tested for reactivity against the antigens in solution. The results indicated that the epitope for this antibody had been buried in nascent α2-PI, but had been exposed by complex formation with plasmin or by possible conformational changes induced in the α2-PI molecule on insolubilization to nitrocellulose membranes or immunoplates. By utilizing a set of JIPPI-3 and JIPPI-50, individually coated onto latex beads, PPI could be measured in plasma in the range of 0.8–100 μg/ml without interference by coexisting plasminogen (120–200 μg/ml) or α2-PI (70 μg/ml). This measurable range seems to cover the level of PPI clinically observed under hyperfibrinolytic states.

ISSN:0957-5235    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

Elastase, released by stimulated polymorphonuclear leukocytes (PMN), is thought to play an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD) especially pulmonary emphysema. A test that can detect release of elastase activity from PMN would be valuable to monitor therapy or to identify patients at risk. The authors aimed to isolate and characterize monoclonal antibodies (mAb) with a high affinity for a neo-antigenic determinant in a high-molecular weight degradation product of fibrinogen (Fbg) generated by PMN-derived elastase. Using synthetic peptides, they mimicked the new amino or carboxy terminal sequences of the Aα-, Bβ-and γ-chains of Fbg that are generated by elastase. These synthetic peptides (Aα22–36, Aα350–360, Bβ44–55, and γ295–305), unidirectionally coupled to a carrier protein, were used to generate mAb specific for elastase-degraded fibrinogen (EDF). mAb that appeared to be specific for a neo-antigenic determinant (neotope) consisting of the new amino terminal amino acid(s) of the Fbg Aα-chain that is generated by elastase activity were isolated only with the Aα22–36 synthetic peptide. One mAb, designated as EF1–4, was further characterized and had an approximately 75-fold higher affinity for EDF, as compared with Fbg, in solution. Using the other peptides, no mAb specific for elastase generated fibrinogen degradation products were obtained. Incubation of immobilized fibrin(ogen) with stimulated PMN resulted in the generation of this specific neotope recognized by mAb EF1–4; the protease inhibitors α1-proteinase inhibitor (α1-PI) and antileukoprotease (ALP) decreased the generation by PMN of this neotope on Fbg. Fibrinogen degradation products generated by plasmin or by other leukocyte proteases, i.e. cathepsin G and proteinase 3, did not react with mAb EF1–4.

ISSN:0957-5235    

出版商:OVID    

年代:1995

数据来源: OVID