ISSN:0957-5235    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

&NA;To characterize potential mechanisms for enhanced thrombolysis in the presence of thrombin inhibitors, we measured lysis of125I‐fibrin‐(ogen)‐labelled clots after incubation in rotating tubes with whole blood containing tissue‐type plasminogen activator (t‐PA, 1500 ng/ml) and either saline or increasing concentrations of recombinant desulphatohirudin (hirudin), or heparin. Thrombin activity in washed clots was negligible, but incubation of clots with t‐PA in non‐anticoagulated blood resulted in marked thrombin activity within 5 min as measured by fibrinopeptide A generation. Incubation with hirudin over a range of concentrations increased clot lysis compared with t‐PA alone (control) from 132 ± 18% of control with 0.5 μg/ml (n= 4) to 216 ± 48% of control with 10 μg/ml (n= 4). Incubation with less than 0.5 U/ml of heparin attenuated clot lysis (35 ± 22% of control with 0.08 U/ml,n= 3) while concentrations ≥ 0.5 U/ml increased lysis (178 ± 13% of control with 1.7 U/ml,n= 4). Similar results were obtained for incubations in recalcified platelet‐poor plasma indicating that platelets are not required for the enhancement of clot lysis induced by thrombin inhibitors. However, incubations in recalcified plasma from a patient with afibrinogenaemia abolished the increased lysis observed with 10 μg/ml of hirudin and addition of physiological concentrations of fibrin monomer (400‐800 nM) or fibrinogen degradation products (500 nM) to the mixture of whole blood, t‐PA and hirudin blunted the extent of clot lysis, Thus, inhibition of thrombin activity induced by exposure of clots to t‐PA prevents concomitant formation of fibrin that attenuates fibrinolysis. Conjunctive administration of potent thrombin inhibitors during pharmacological thrombolysis should be useful to accelerate recanalization and contribute to sustained vessel patency.

ISSN:0957-5235    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

&NA;The recent observation that coagulation factor XI is activated by the serine protease thrombin indicates that factor XI may play a role in sustaining the haemostatic process by activating factor IX, after coagulation has been initiated by the factor VIIa/tissue factor catalytic complex. Since negatively charged substances, such as dextran sulphate or sulphatides, have been shown to enhance the activation of factor XI by thrombin, we investigated the effect of glycosaminoglycans on this reaction. A 60‐fold enhancement in activation was observed in the presence of heparin and more modest increases were seen with dermatan sulphate and chondroitin sulphates A and C. The increase in activation was greater if Zn2+was included in the reactions with glycosaminoglycans. The combination of heparin or chondroitin sulphate C and Zn2+supported factor XI autoactivation in addition to factor XI activation by thrombin; an effect noted previously only with dextran sulphate.

ISSN:0957-5235    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

&NA;To study the long‐term overall effect of physical exercise on blood coagulation, 20 sedentary males and 15 sedentary females were trained three to four times a week with increasing intensity for 9 months. After 24 and 36 weeks all subjects ran a 15 km and a half‐marathon (21 km) race, respectively, Blood samples were drawn before the training programme, 5 days before both races and 5 days after the halfmarathon run. Plasma factor VIII coagulant activity and von Willebrand factor antigen concentration did not increase during the training programme. In both males and females plasma fibrinogen concentration was not enhanced after 24 weeks of training but increased in preparation for the 21 km race and was still raised significantly (P< 0.01) 5 days later. No significant changes in plasma thrombin‐antithrombin III concentrations were observed in either group during the training programme. The results of this study demonstrate that an exercise programme of increasing intensity induces physical stress which has significant effects on plasma fibrinogen concentration, even at rest. However, in contrast to acute post‐exercise effects, a regular physical fitness programme does not induce a long‐term activation of the haemostatic system.

ISSN:0957-5235    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

&NA;uK2t‐PA is a hybrid plasminogen activator in which the epidermal growth factor‐like domain of the urokinase‐type plasminogen activator precedes the kringle 2 and catalytic domains of tissue‐type plasminogen activator. The molecules are expressed in Chinese hamster ovary cells in two variant forms, a type II form in which only the protease domain is glycosylated, and a type I form in which both the kringle 2 and the protease domains carryN‐acetyllactosamine type glycans. The two forms differed slightly in their affinity for fibrin and fibrinogen, which allowed their separation, but the stimulation of plasminogen activation of the type II form by fibrin was up to eightfold lower than that of the type II form. The sensitivity to fibrin could be restored by treatment of the type I form withN‐glycanase or sialidase. Enzymatic activity vs low molecular weight substrates was not influenced by the glycosylation of kringle 2.

ISSN:0957-5235    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

&NA;Aprotinin, a broad spectrum serine proteinase inhibitor, is becoming increasingly used to control bleeding during surgery. Heparinized patients treated with aprotinin for cardiac surgery have a much longer activated clotting time (ACT) than expected for the dose of heparin used and a similar effect has been observed with the activated partial thromboplastin time (APTT). Since APTT reagents vary in their sensitivity to heparin we compared the effect of aprotinin on 25 commercially available products with respect to the APTT response to heparin. Aprotinin (Bayer) was added to normal, pooled, citrated plasma at concentrations of 0, 100, 200, 300 and 400 kallikrein inhibition units (KIU)/ml and mucosal heparin was then added to give concentrations of 0, 0.25, 0.5, 0.75 and 1.0 IU/ml. Duplicate APTTs were performed on each of these 25 plasma samples according to the manufacturers' instructions. As expected, different commercial reagents exhibited different sensitivities to heparin. There was also variation in the sensitivity to aprotinin but this did not correlate with the heparin sensitivity. The prolongation of the APTT by heparin was markedly increased by aprotinin. For example, APTT ratios at 0.5 IU/ml heparin increased up to eight‐fold in the presence of ‘therapeutic’ levels of aprotinin (200 KIU/ml) and this effect was even more pronounced at higher heparin levels. The activator used did not significantly influence the effect of aprotinin on the APTT although there was a trend for kaolin‐activated reagents to be less affected by the addition of aprotinin to heparinised plasma. These results suggest that therapeutic ranges for APTT ratios in heparin therapy should be re‐evaluated for each APTT reagent used if high dose aprotinin is being given concomitantly.

ISSN:0957-5235    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

&NA;In cardiopulmonary bypass (CPB) surgery high dose heparin is necessary to inhibit the clotting cascade which is activated through damage to the vessels (extrinsic pathway) as well as contact activation of the blood with the various artificial surfaces of the CPB machine (intrinsic pathway). In most European heart surgery centres, the fibrinolytic activation that always occurs in CPB due to contact activation is reduced by the proteinase inhibitor aprotinin. Monitoring of anticoagulation during CPB is performed with the activated whole blood clotting time (ACT). The two machines commonly used for this purpose, Hemotec® and Hemochron®, use different contact system activators, kaolin and celite. These activators displayed highly significant differences, in bothin vitrotests (modified APTT with whole blood in a neutral coagulometer), and ACT in both machines where aprotinin and heparin were used, as well as with parallel measurements with the two machines with blood from patients undergoing CPB with high dose aprotinin therapy (P< 0.001). The Hemotec machine with kaolin as activator was not affected by aprotinin throughout surgery, while the Hemochron clotting times almost doubled as soon as aprotinin and heparin were combined. Our studies show that for the determination of ACT in CPB where high dose aprotinin therapy is used only ACT determinations with machines using kaolin as activator yield accurate results.

ISSN:0957-5235    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

&NA;Bovine factor XIIa inhibitor was purified by an improved method employing affinity for heparin. N‐terminal amino acid sequencing revealed a unique sequence without homology to any other known protein sequences. Peptide sequencing, however, showed that a part of the bovine factor XIIa inhibitor was homologous to human CÏ‐inhibitor with a fraction of identical amino acid residues around 70%. Deglycosylation studies and carbohydrate analysis showed the presence of N‐ and O‐linked carbohydrate. Bovine factor XIIa inhibitor did not inhibit plasma kallikrein and trypsin. The reactive site comprised an Arg‐Asn bond, and represents the first example of asparagine as a P1' residue in Serpins with well documented inhibitory activity.

ISSN:0957-5235    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

&NA;Our laboratory has been using protein engineering to study the relationship of primary structure to fibrinogen function. In order to examine genetically altered domains in the context of the intact, functional fibrinogen molecule, we have expressed recombinant human fibrinogen in Chinese Hamster Ovary (CHO) cells. The cDNA for each fibrinogen chain was individually cloned into the same expression vector. Each vector was cotransfected with the selection vector pRSVneo into CHO cells. In addition, the plasmids encoding A&agr; and &ggr; were cotransfected with pRSVneo. Cells resistant to G418, a neomycin analogue, were isolated and clonal lines developed. Analysis of these lines demonstrated that CHO cells express and secrete free &ggr; chain, and an A&agr;‐&ggr; complex. To obtain recombinant fibrinogen, the A&agr;‐&ggr; G418‐resistant clones were transfected with the B&bgr; expression plasmid and a second selection vector, pMSVhis. Colonies resistant to neomycin and histidinol were selected and clonal lines obtained. These clones secreted biologically active recombinant human fibrinogen, which was purified from serum‐free culture media by protamine‐Sepharose chromatography. Analysis of the purified protein on SDS‐polyacrylamide gels demonstrated a pattern indistinguishable from plasma fibrinogen. Removal of Asn‐linked carbohydrate with glycosidase F revealed the presence of carbohydrate on the B&bgr; and &ggr; chains, as is seen for plasma fibrinogen.

ISSN:0957-5235    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

&NA;The six ploypeptide chains of normal fibrinogen are covalently linked by interchain disulphide bonds, and there are no free sulphhydryl groups. Fibrinogen Dusart is a congenital fibrinogen variant in which A&agr; 554 Arg is replaced by Cys; albumin is disulphide linked to these fibrinogen molecules, possibly at A&agr; 554 Cys. Functionally, Dusart fibrinogen displays markedly abnormal fibrin polymerization, characterized by delayed lateral fibril association and matrix fibre bundles that are thinner than normal fibrin bundles. These observations are consistent with experiments suggesting that the carboxy terminal region of the A&agr;‐chain contains a polymerization domain that participates in lateral fibril associations. In order to investigate the location and the effect of albumin binding to Dusart fibrinogen, we examined the fibrinogen by electron microscopy, and compared the polymerization and ultrastructure of fibrin prepared from normal fibrinogen containing intact A&agr;‐chains (fraction I‐2) or plasmin degraded fibrinogen molecules lacking carboxy terminal regions of A&agr;‐chains (fraction I‐9D), with fibrin prepared from Dusart fraction I‐2 and I‐9D. Most bound albumin was released from Dusart fibrinogen by plasmin degradation involving the A&agr;‐chains. Nevertheless, we were able to visualize albumin molecules remaining covalently bound to Dusart I‐9D as well as to Dusart I‐2 fibrinogen, as distinct globular domains situated near the fibrinogen D domain. The presence of albumin in these fractions was confirmed by Western blotting using anti‐albumin. Dusart fibrin polymerized much more slowly than normal I‐2, as previously reported, whereas polymerization of Dusart I‐9D fibrin was faster than Dusart I‐2 and nearly the same as normal I‐9D fibrin. The ultrastructure of the Dusart I‐9D fibrin matrix was indistinguishable from that of normal I‐9D; both contained intensely striated thick fibres. In contrast, Dusart I‐2 fibrin had thinner, more highly branched fibres with few striations. These results indicate; (1) most albumin bound to fibrinogen Dusart is removed by release of the carboxyl‐terminal region of the A&agr;‐chain, suggesting that at least 90% of the albumin is bound at A&agr; 554 Cys; (2) removal of the carboxyl‐terminal region of the fibrinogen (I‐9D) normalizes its polymerization properties relative to normal I‐9D.

ISSN:0957-5235    

出版商:OVID    

年代:1993

数据来源: OVID