摘要:

&NA;The aim of this study was to characterize soluble fibrin(ogen) species in human, arterial, in‐vivo‐formed thrombi, using the immunoblotting technique. Specimens were collected via intra‐arterial catheters in six patients scheduled for catheter‐directed thrombolysis. Unreduced and reduced samples of the supernatants from the arterial thrombi‐derived specimens were electrophoresed on polyacrylamide gels and immunoblotted, using specific mono‐ and polyclonal anti‐fibrin(ogen) antibodies. The reduced samples disclosed substantial amounts of high molecular weight material, consistent with &agr;‐chain polymers and &ggr;&ggr;‐dimers, as well as lower molecular weight material, such as &agr;‐, &bgr;‐ and &ggr;‐chains. No fibrinogen with intact fibrinopeptide A was detectable, and des‐AABB fibrin represented a major fibrin derivative in the soluble part of the arterial thrombi. The &agr;‐chains were C‐terminally degraded, most of them distal to position 259. In conclusion, we have demonstrated the presence of cross‐linked fibrin derivatives in soluble, arterial thrombus‐related material, without signs of fibrinogen‐fibrin hybrids. The fibrin derivatives were C‐terminally degraded, thus representing X‐oligomeric material, most probably originating from plasmin degradation of insoluble thrombus fibrin. The present study supports the hypothesis of a dynamic equilibrium between clotting and lysis in thrombi.

ISSN:0957-5235    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

&NA;In the search for a more acceptable route of heparin administration that can also be used for long‐term treatment, we evaluated the bioavailability and antithrombotic activity of intraduodenal administration of unfractioned heparin (UFH) in rats. A radioiodinate derivative of UFH was administered intraduodenally in rats in conjunction with unlabeled UFH. We found that radioactivity increases very rapidly in plasma, as well as on the surface of aortic and caval segments, so that peak radioactivity was already achieved within 5 min of drug administration. Subsequently, plasma radioactivity declined rapidly, although 1.5‐2.5% of the total radioactivity administered was still circulating 3 h after drug administration. The plasma anti‐Xa activity was much lower and longer lasting than expected from the radioactivity counts in both peripheral and portal blood, and its level was very similar in the two circulatory districts, never exceeding 0.23 U/ml. This suggests that extensive degradation of the drug already occurs during its gastrointestinal absorption. Nevertheless, in a stasis‐induced venous thrombosis model, intraduodenal UFH prevented thrombus formation in a dose‐dependent way (ED50, 2000 IU/kg). The maximum antithrombotic effect was observed when the drug was administered 30‐60 min before ligature of the vena cava, and a 40% reduction of thrombus weight was still present at 180 min. Since antithrombotic kinetics does not match the kinetics of either the plasma or vessel‐bound radioactivity but approaches what is found in anti‐Xa activity, the antithrombotic activity of oral heparin may be dependent on the release of unlabeled endogenous glycosaminoglycans deposited in the vessels.

ISSN:0957-5235    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

&NA;Mostin vitrostudies of human endothelial cells have relied on cells derived from human umbilical veins (HUVEC); however, heterogeneity of primary cultured endothelial cells can make critical interpretation of results difficult. Several endothelial cell lines have been produced to serve as a more constant source of endothelial cells. In this study, we characterized the endothelial cell lines EVLB3and EVLC2derived from HUVEC, and EVLK1and EVLK2derived from human iliac vein endothelial cells (HIVEC). These cell lines maintained the typical endothelial cell cobblestone morphology and appeared to be growth factor independent. They lost PECAM‐1 and von Willebrand factor, GP96 was reduced to the level of vascular smooth muscle cells (SMC), but &agr;SMC‐actin was far less than in vascular SMC. Antigen levels of tissue‐type plasminogen activator (tPA) and plasminogen activator inhibitor (PAI‐1) were comparable with young endothelial cells, and mRNA was present for tPA, PAI‐1, tissue factor (TF), tissue factor pathway inhibitor and thrombomodulin. This study revealed that mRNA and protein expression of coagulation and fibrinolytic factors was influenced by the stage of cell confluence. No differences could be detected between the endothelial cell lines derived from HUVEC and HIVEC. These cell lines may be a useful tool for studies on cellular interactions of fibrinolytic components or exploring the regulation of TF expression.

ISSN:0957-5235    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

&NA;The use of libraries of phage‐displayed human single‐chain antibody fragments (scFv) has become a new, powerful tool in rapidly obtaining therapeutically useful antibodies. Here, we describe the generation of human scFv and F(ab')2directed against the &ggr;‐carboxyglutamic acid (Gla) domain of coagulation factor IX. A large library of human scFv, displayed either on M13 phage or expressed as soluble proteins, was screened for binding to human Gla‐domain peptide (Tyr1‐Lys43). Among a panel of scFv that bound to the factor IX‐Gla domain, six scFv clones recognized full‐length factor IX and exhibited strong inhibitory activity of factor IXin vitro.After reformatting as F(ab')2, the affinity for factor IX of three selected clones was determined: 10C12Kd= 1.6 nmol/l, 13D1Kd= 2.9 nmol/l, and 13H6Kd= 0.46 nmol/l. The antibodies specifically bound to factor IX and not to other coagulation factors, as assessed by enzyme‐linked immunosorbent‐type and human plasma clotting assays. The complementarity determining region amino acid sequences of clones 10C12 and 13D1 only differed at a single residue, whereas 13H6 showed little homology, suggesting that 13H6 binds to a different epitope within the factor IX‐Gla domain. Despite the slightly lower affinity of 10C12 F(ab')2versus 13H6 F(ab')2, 10C12 was consistently more potent than 13H6 in prolonging the activated partial thromboplastin time (APTT), in inhibiting platelet‐mediated plasma clotting, and in inhibiting factor X activation by the intrinsic Xase complex. Finally, 10C12 F(ab')2also recognized and neutralized factor IX/factor IXa of different species, as demonstrated by the specific APTT prolongation of dog, mouse, baboon and rabbit plasma. In summary, the results validate the usefulness of scFv phage‐displayed libraries to rapidly generate fully human antibodies as potential new therapeutics for thrombotic disorders.

ISSN:0957-5235    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

&NA;The role of fibrinogen and other haemostatic factors in prediction of peripheral arterial disease (PAD) has not been established. We examined the associations of plasma fibrinogen, von Willebrand Factor (vWF), tissue plasminogen activator (t‐PA) antigen, fibrin D‐dimer, and factor VII with the development and clinical progression of PAD. In the Edinburgh Artery Study, 1592 men and women, aged between 55 and 74 years, were followed prospectively over 5 years to detect the onset of PAD, and the deterioration of established PAD. At baseline, 418 individuals had evidence of PAD and 60 (14.4%) subsequently deteriorated. 1080 subjects had no baseline disease, but 59 (5.5%) developed PAD during follow‐up. Median levels of fibrinogen and vWF were higher in the groupdevelopingdisease compared with the group which did not (2.78 g/l versus 2.57 g/l,P≤ 0.01; 116 IU/dl versus 104 IU/dl,P≤ 0.05; respectively). After adjusting for age and sex, fibrinogen (P≤ 0.01) and vWF (P≤ 0.05) were significantly associated with the risk of developing PAD. The association between fibrinogen and development of disease remained after adjusting for cardiovascular risk factors and baseline ischaemic heart disease (relative risk, 1.35, 95% confidence interval, 1.05, 1.73;P≤ 0.05). None of the haemostatic factors were significantly associated withprogressionof PAD. In conclusion, plasma fibrinogen levels are related to the future onset of PAD, providing further evidence of a possible role of elevated fibrinogen in the development of atherosclerotic disease.

ISSN:0957-5235    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

&NA;Procoagulant activities associated with human clots may contribute to thrombus extension. We investigate the inhibition of clot‐associated factor Xa and thrombin activities by purified human antithrombin either alone or as combination with a low molecular weight heparin (enoxaparin) as compared with unfractionated heparin (UFH). The standard clots were prepared by recalcification of frozen platelet‐poor human plasma. Clot‐associated thrombin was measured on the clot after clot incubation in recalcified buffer or recalcified prothrombin solution. The enzymatic reaction was measured using a specific substrate for thrombin (CBS 3447). The thrombin concentration was determined both on the clots and in the reaction mixtures. In parallel, prothrombin fragment 1.2 and thrombin‐antithrombin complexes (TAT) were measured using enzyme‐linked immunosorbent assay methods.We demonstrated that in the presence of purified human prothrombin and antithrombin (AT), a partial inhibition of clot associated thrombin activity correlated with an increase of TAT complexes. However, antithrombin was unable to inhibit thrombin generation induced by the clot‐associated factor Xa. Enoxaparin (low molecular weight heparin) and UFH did not enhance clot‐bound thrombin inhibition induced by AT. We conclude that clot‐bound thrombin is accessible to human antithrombin alone. AT is also able to inhibit thrombin generated by factor Xa‐associated clot. However, neither a low molecular weight heparin or UFH enhanced the effect of AT alone.

ISSN:0957-5235    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

&NA;The present study was undertaken to evaluate the effects of an intravenous infusion of angiotensin II (10 ng/kg per min) on platelet function and coagulationin vivoin 18 healthy males. The infusion increased mean arterial pressure by 23 ± 2 mmHg. Plasma &bgr;‐thromboglobulin levels, reflecting platelet secretion, increased by 66 ± 24% during the infusion, as did also platelet surface expression of P‐selectin as measured by flow cytometry. Platelet fibrinogen binding increased, whereas platelet aggregability, assessed byex vivofiltragometry, was unaltered. Angiotensin II caused mild activation of the coagulation cascade with increases in plasma levels of thrombin‐antithrombin complex and prothrombin fragment F1 + 2. In conclusion, angiotensin II has mild platelet‐activating effectsin vivoand also enhances coagulation. Markers of platelet secretion are significantly increased, whereas markers of platelet aggregability are less affected. The clinical relevance of these findings remains to be clarified.

ISSN:0957-5235    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

&NA;The association between both plasma viscosity and fibrinogen concentration with clustering of metabolic risk markers was examined within a cross‐sectional study of employed middle‐aged men. Analyses were performed on a subsample of 629 non‐smokers (46.7 ± 7.8 years) without diabetes. The effect of obesity and cardiorespiratory fitness on these haemorheological parameters and their association with the metabolic syndrome was also investigated. The cohort was grouped by the number of metabolic markers present. Metabolic markers included high‐density lipoprotein‐cholesterol (< 1.13 mmol/l), triglycerides (≥ 1.805 mmol/l), glucose (≥ 5.5 mmol/l) and diastolic blood pressure (≥ 90 mmHg). The age‐adjusted odds ratio for hyperviscosity (≥ 1.67 mPa/s) was 2.08 [95% confidence interval (CI), 1.06‐4.05;P= 0.031] for the subjects with the metabolic syndrome (three or more metabolic markers) when compared with those with no metabolic abnormalities. The comparable age‐adjusted odds ratio for hyperfibrinogenaemia (≥ 3.47 g/l) was non‐significantly higher at 1.69 (95% CI, 0.87‐3.27;P= 0.119). The mean age‐adjusted plasma viscosity level and the prevalence of hyperviscosity increased significantly from 1.629 to 1.692 mPa/s (P= 0.0005) and from 21.0 to 36.0% with accumulating metabolic markers (P= 0.006). Plasma viscosity and fibrinogen concentration both increased with higher quartiles of skinfolds (P= 0.003 andP= 0.01, respectively) following adjustment for age, lipids and leucocyte count. Plasma viscosity was also significantly lower with higher levels of predicted maximum oxygen consumption (&OV0312;o2max) (P= 0.0005). The odds ratio for hyperviscosity in subjects with the metabolic syndrome as compared with those with no metabolic markers was attenuated following adjustment for age, sum of skinfolds and predicted maximum oxygen consumption (&OV0312;o2max) (1.44; 95% CI, 0.72‐2.90;P= 0.307). These cross‐sectional results suggest that plasma viscosity is associated with increased clustering of metabolic markers in middle‐aged men of high socio‐economic status. Obesity and poor cardiorespiratory fitness may be important in the development of haemorheological abnormalities associated with the metabolic syndrome.

ISSN:0957-5235    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

&NA;To better understand the regulation of tissue plasminogen activator (t‐PA) and plasminogen activator inhibitor 1 (PAI‐1) during liver transplantation, we used a computer model of the human circulatory system to simultaneously evaluate the effect of t‐PA secretion, t‐PA inhibition by PAI‐1, hepatic clearance of t‐PA, blood loss, transfusion and hemodynamics on t‐PA and PAI‐1 levels during liver transplantation in three patients that differed in severity of liver disease, blood loss and anhepatic changes in t‐PA. Higher preoperative t‐PA levels were primarily related to underlying liver disease and reduced hepatic clearance. During the anhepatic stage, when hepatic t‐PA clearance was eliminated: (1) the expected rise in t‐PA was modulated by the extent of bleeding, which acted as an alternate t‐PA clearance mechanism; and (2) the ratio of t‐PA:PAI‐1 was increased due both to lower t‐PA clearance and reduced PAI‐1 secretion. Recirculation of the new liver was associated with renewed clearance of t‐PA, an acute phase increase in PAI‐1 and a drop in the t‐PA:PAI‐1 ratio. Understanding fibrinolytic regulation required simultaneous analysis of t‐PA secretion, inhibition and clearance. Anhepatic t‐PA levels could be predicted based on preoperative liver function and surgical blood loss, which acted as an alternate t‐PA clearance mechanism.

ISSN:0957-5235    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

&NA;Activated factor V (FVa) serves as a cofactor to activated factor X in the prothrombinase complex. FVa is homologous to activated factor VIII (FVIIIa), the light chains of both proteins being formed by similar domains (A3‐C1‐C2). Interaction of FVa and FVIIIa with negatively charged phospholipid membranes is crucial for the function of both cofactors. In both proteins, the C2 domains are important for membrane binding but a detailed understanding of the binding mechanisms is missing. Recently, knowledge has been gained into the three‐dimensional structures of the C domains facilitating studies of structure‐function relationships. Structural analysis of the C2 domain in FVa predicted a surface‐exposed loop (K2060, K2061, S2062, W2063, W2064) to be involved in membrane binding. Three double mutants were created, K2060Q‐K2061Q, W2063Y‐W2064Y and W2063A‐W2064A, and expressed in a transient expression system. In addition, a FV variant in which all four residues were mutated, K2060Q‐K2061Q‐W2063A‐W2064A, was produced. Mutagenesis of the two lysines showed no functional consequences, whereas mutagenesis of the two tryptophanes yielded FVa with impaired ability to interact with the phospholipid, as demonstrated by a poor functional activity at limiting phospholipid concentrations. A molecular model of FVa, anchored at the surface of a phospholipid membrane, was developed and used as a template for the interpretation of the mutagenesis experiments.

ISSN:0957-5235    

出版商:OVID    

年代:2000

数据来源: OVID