摘要:

&NA;Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz‐type protease inhibitor, which inhibits factor Xa directly and in a factor Xa dependent manner inhibits the factor VIIa/tissue factor catalytic complex. Altered forms of recombinant TFPI (rTFPI) were tested for their ability to inhibit human factor Xa and bovine &ggr;‐carboxyglutamate (Gla)‐domainless factor Xa in the presence and absence of calciumions, heparin, phospholipids, and factor Va. Sequential deletions of the positively charged C‐terminus of TFPI produces proteins that have decreasing inhibitory activity against factor Xa as well as decreasing affinity for heparin‐agarose. Deletion of the C‐terminus distal to Leu111,which eliminates the third Kunitz‐type domain, results in the loss of heparin‐agarose binding at physiological ionic strength. Furthermore, the entire C‐terminal polypeptide, including at least a portion of the third Kunitz‐type domain, appears to be involved in heparin binding. Residues 230‐241 probably form an alpha helix in which Lys231and Arg237within the Kunitz domain and Lys241and Lys241could provide a positively charged surface epitope capable of binding heparin. Heparin and Ca2+together, but not individually, enhance the rate of factor Xa inhibition by full‐length TFPI. The effect of heparin is concentration dependent and biphasic (maximal between 0.1 and 1.0 unit/ml) suggesting that the acceleration of factor Xa inhibition occurs at least in part through a ‘template’ mechanism. Full‐length rTFPI inhibits factor Xa in the presence of Ca2+, phospholipids and factor Va more effectively than factor Xa in the presence of Ca2+alone and is a more potent inhibitor of factor Xa under the former conditions than carboxy‐terminal truncated forms of rTFPI. Additional studies show that the acidic N‐terminus of TFPI is not required for Xa inhibition and suggest that in a non‐physiological system lacking Ca2+the cluster of basic amino acids near the C‐terminus of TFPI may interact with factor Xa in a Gla domain dependent fashion.

ISSN:0957-5235    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

&NA;Conditions for triggering factor XII to function in the autoactivation reaction in blood coagulation in the presence of different surfaces have been studied using prekallikrein‐deficient plasma. Autoactivation was recorded in a chromogenic assay by measuring the amidolytic activity of factor XIIa. The results showed that autoactivation of factor XII was achieved only after modulation of factor XII. This modulation was mediated by Zn2+and did not require an activating surface. Following modulation, the autoactivation proceeded in the presence of a negatively charged phospholipid or sulphatide, but the sulphatide‐mediated autoactivation was inhibited by Zn2+. In the presence of Zn2+(6.6 μmol/ml plasma), the rate constant for the autoactivation following modulation was calculated to be 3.1 × 104M−1s−1and 1.2 × 104M‐1s‐1for the phospholipid and the sulphatide‐mediated reactions, respectively. In the absence of Zn2+no activation was observed in the presence of a negatively charged phospholipid. After removal of Zn2+by EDTA, the rate constant for the sulphatide‐mediated autoactivation increased to 5.7 × 104M−1s−1in the presence and 1.0 × 105M−1s−1in the absence of a negatively charged phospholipid (phosphatidylinositol phosphate). Dextran sulphate was not able to mediate autoactivation, in either the presence or absence of Zn2+. Aprotinin completely blocked the modulation and/or autoactivation. Soy bean trypsin inhibitor prolonged the period required for autoactivation to start, but had no effect on the autoactivation rate. The main conclusions are that Zn2+is required to initiate contact activation, modulating factor XII for autoactivation. This modulation does not require the presence of an activating surface.

ISSN:0957-5235    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

&NA;The aim of this study was to investigate the interactions of t‐PA and plasminogen with fibrin derived from an abnormal fibrinogen detected in a 40‐year‐old male patient who had had an episode of thrombophlebitis with pulmonary embolism. An abnormal fibrinogen was diagnosed on the basis of prolonged thrombin and reptilase times also detected in two other family members. Fibrinogen purified from plasma, in the presence of protease inhibitors, by glycine precipitations, gel filtration and affinity chromatography, was devoid of plasminogen, fibronectin, and vWf. SDS‐PAGE analysis according to Laemmli under reducing conditions, showed an abnormal &ggr; chain (˜50% of the total) migrating in a more anodic position (M,48 kDa). By PCR amplification and DNA sequencing, the abnormality was identified as an Asn104Lys mutation of the &ggr; chain. Since such a mutation constitutes a new plasmin cleavage site as first reported for fibrinogen Kyoto 1, it may modify interactions of plasminogen and t‐PA with carboxy‐terminal lysine residues. Ligand‐binding studies were therefore performed using intact and plasmin‐degraded fibrin surfaces obtained from the abnormal fibrinogen. The plasminogen and t‐PA binding isotherms obtained with the abnormal fibrinogen were similar to the control. Moreover, the stimulation by fibrin of plasminogen activation by t‐PA was not different from the control. These results suggest (i) that the lysine 308 residue may not be exposed to plasmin cleavage in fibrin, and (ii) that the thrombotic accident of the propositus cannot be explained by an abnormality of the plasminogen/t‐PA binding to fibrin.

ISSN:0957-5235    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

&NA;Ioxaglate, an iodinated contrast agent, decreases the rates of fibrin clot formation induced by thrombin or reptilase. This effect is not related to an increase in the ionic strength of the medium since a specific control of equivalent composition does not induce such variation. The concentration of ioxaglate which led to a 50% decrease of the control clot turbidity induced by thrombin was 17.5 ± 2 mM. Macroscopically, clots formed with ioxaglate were larger and less turbid than the isotonic control. An increase in fibrin fibre diameters and a decrease in their densities were observed. During the fibrin polymerization process, all the fibrinogen was converted into fibrin, as for both the control and ioxaglate quantitative analysis of clots and supernatants showed (1) an identical quantity of FpA in clot supernatants, (2) the same quantities of protein incorporated into clots, and (3) no trace of fibrin monomers in the clot supernatants. Furthermore, dissolution in urea of clots formed in the presence of ioxaglate occurred more rapidly than in the control. Total incorporation of fibrinogen into clots, associated with a decrease in clot turbidity, indicated the existence of a qualitative abnormality in the construction of the three‐dimensional fibrin structure. Using differential scanning calorimetry, it was observed that the two domains (D and E) of fibrinogen were modified by ioxaglate, showing the absence of specificity in the interaction between ioxaglate and a particular domain.

ISSN:0957-5235    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

&NA;Disseminated intravascular coagulation (DIC) is a common complication in sepsis, and may result from endotoxin‐induced exposure of tissue factor on the surface of monocytes and endothelial cells. Tissue factor pathway inhibitor (TFPI) is a factor Xa‐dependent feedback inhibitor of the tissue factor‐factor VIIa complex. In the present study the effect on DIC of a two‐domain TFPI analogue (2D‐TFPI), consisting of the first two Kunitz domains of TFPI but lacking the third domain, was tested. DIC was induced in rabbits by two intravenous bolus injections of endotoxin fromEscherichia coli(10 and 50 μg/kg) 24 h apart. Simultaneously with the last endotoxin injection an infusion of 2D‐TFPI (0, 0.3, 1.0 or 3.0 mg/kg/h) was given. Blood samples were obtained at 0 h, 24 h and 31 h. At 31 h the animals were sacrificed and the kidneys were submitted to histological examination. The degree of fibrin deposition in glomeruli was scored blindly using an arbitrary scale from 0 to 3. Between 24 and 31 h the group receiving endotoxin alone showed a significant decrease in platelet count (65%), plasma fibrinogen (41%), antithrombin III (25%), and factor VIII (63%), and a significant prolongation of the aPTT (14%). Furthermore, massive fibrin deposition was detected in the renal glomeruli at 31 h. Infusions of 2D‐TFPI inhibited all the endotoxininduced changes in a dose‐dependent manner. In conclusion, the data demonstrate that inhibition of the TF/FVIIa complex by infusion of 2D‐TFPI significantly counteracts endotoxin‐induced coagulopathy in rabbits, and might thus be an attractive drug for treatment of endotoxin‐induced DIC in humans.

ISSN:0957-5235    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

&NA;The purpose of this study was to clarify differences in coagulation and fibrinolytic activation between the various subtypes and phases of ischaemic stroke. Haemostatic activation markers were measured in 52 patients with cardioembolic stroke, 32 with atherothrombotic stroke and 54 with lacunar stroke and compared with 23 age‐matched controls. Data were obtained in the acute (≤ 7 days after onset), subacute (8‐28 days) and chronic (≥ 29 days) phases of stroke. In patients with cardioembolic stroke, D‐dimer and &agr;2‐antiplasminplasmin complex levels were higher during the acute and subacute phases, while thrombin‐antithrombin III complex levels were higher during the acute phase than in patients with lacunar stroke and controls. In cardioembolic stroke, fibrinopeptide A was increased during the acute and subacute phases, thrombin‐antithrombin III complexes were higher during the subacute phase and D‐dimer levels were higher during the chronic phase. Protein C activity was lower during the acute phase than in atherothrombotic stroke, lacunar stroke and controls. Protein C antigen was lower during the acute phase than in lacunar stroke and controls and during the chronic phase than in lacunar stroke. In contrast, only D‐dimer levels were higher in atherothrombotic stroke patients than controls during the acute and chronic phases and no significant alterations in these markers were observed in the patients with lacunar stroke. These findings suggest that measurement of molecular markers of coagulation and fibrinolysis may be useful for detecting intracardiac thrombin and plasmin generation in patients with cardioembolic stroke.

ISSN:0957-5235    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

&NA;We have previously shown that the C‐terminus of TFPI is essential for its anticoagulant activity. In the present study we have assessed the role of this region in the binding of TFPI to lipoproteins. We found that full length TFPI, but not C‐terminal degraded TFPI, was capable of coeluting with the plasma lipoprotein fraction on a Superose‐6 column. The importance of the TFPI C‐terminus in lipoprotein interactions was also assessed using a microtitre plate binding assay. We found that full‐length TFPI was capable of binding to VLDL or LDL coated microtitre plates. C‐terminal degraded TFPI also bound to VLDL, but with a ten‐fold lower affinity than full length TFPI. Interestingly, removal of the C‐terminus along with the third Kunitz‐type domain resulted in a TFPI form incapable of lipoprotein binding. Since heparin shows strong binding to the C‐terminus of TFPI, we also tested its effect on the binding of full length TFPI to VLDL. We found that co‐incubation of TFPI with heparin inhibited this binding in a dose‐dependent manner. Heparin was also capable of releasing TFPI from a preformed TFPI:VLDL complex, although this reaction required unphysiological amounts of heparin. To assess the physiological function of heparin on FL‐TFPI:lipoprotein interactions we also performed gel filtration chromatography of rabbit plasma immediately following i.v. administration of FL‐TFPI with and without heparin. Previous experiments indicated that heparin has a protective effect on exogenously added FL‐TFPI, increasing its recovery by ten‐fold. We found that FL‐TFPI remaining in circulation 5 min after administration migrates solely in high molecular weight fractions, whereas the TFPI recovered after co‐injection of heparin migrates solely in the low molecular weight fractions. Collectively our results indicate that the C‐terminus of TFPI is involved in lipoprotein binding, and that binding of TFPI to lipoproteins and glycosaminoglycans may be related phenomena.

ISSN:0957-5235    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

&NA;We have previously demonstrated that therapeutic concentrations of unfractionated heparin (UFH) impair fibrin polymerization leading to the formation of clots which are more sensitive to lysis induced by tissue plasminogen activator (t‐PA). The aim of this study was to compare the effect of UFH with that of three different low molecular weight heparins (LMWHs) on clot sensitivity to t‐PA‐induced lysis. Labelled fibrin clots, prepared from plasma containing UFH, Fraxiparine®, Reviparine®, Enoxaparine®or saline, were incubated in phosphate buffer containing t‐PA (0.1 and 0.5 μg/ml) and plasminogen (20 μg/ml). The extent of clot lysis was quantified by counting the residual radioactivity of the clots and by measuring D‐dimer levels released into the medium. Fibrin polymerization and clot structure were evaluated by means of a turbidimetric assay and by electron microscopic scanning. Pre‐incubation of plasma with 0.5 or 1.0 U/ml UFH resulted in a marked dose‐dependent acceleration of lysis induced by 0.1 or 0.5 μg/ml t‐PA. In contrast, lysis rates induced by 0.5 μg/ml t‐PA were not modified by pre‐incubation of plasma with LMWHs. When exposed to 0.1 μg/ml t‐PA clots formed from plasma containing 0.5‐2 IU/ml of Fraxiparine, Reviparine and Enoxaparine showed only a minor increase in lysis rates compared to control clots. There was not a clear dose‐response curve with LMWHs. Furthermore, lysis rates obtained with UFH‐treated clots were always significantly higher than those seen with LMWHs‐treated clots. We found that UFH, in contrast to LMWHs, induced marked changes of fibrin assembly and clot structure, resulting in the formation of clots with thicker fibres and larger pores. The relatively poor effects of LMWHs on clot structure and clot sensitivity to t‐PA‐induced lysis may contribute to their lower haemorrhagic potential.

ISSN:0957-5235    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

&NA;Fluorescence flow cytometry (FC) was employed to monitor the platelet surface in humans receiving intravenous infusions of Fab fragments of a chimaeric (murine/human) construct of monoclonal antibody 7E3, which binds to the fibrinogen receptor (glycoprotein IIb‐IIIa) and inhibits platelet aggregation. Platelet surface‐bound chimaeric 7E3‐Fab (c7E3‐Fab) was measured using a fluorescein‐conjugated polyclonal anti‐7E3 antibody and residual c7E3‐Fab binding capacity was measured using fluorescein‐conjugated c7E3‐Fab. Turbidometrically measured platelet aggregation response to ADP was shown to be a linear function (r= 0.9) of the logarithm of residual free binding sites for c7E3‐Fab. The distribution of c7E3‐Fab in the platelet population was unimodal at all time points following the infusion, demonstratingin vivotransfer of antibody to newly released circulating platelets. Clearance of platelet surface‐bound antibody followed an exponential model but all circulating platelets were bearing c7E3‐Fab at time points well beyond the lifespan of the platelets exposed to c7E3‐Fab during the infusion.Ex vivoandin vitromixing experiments showed that c7E3‐Fab transfer between platelets was possible and suggested that differences in thein vivokinetics between monovalent and bivalent forms of monoclonal antibodies might be relevant in their therapeutic application.

ISSN:0957-5235    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

&NA;To determine the best way to reverse the excessive effect of regular anticoagulation in patients with INR > 5 and no bleeding complications, 23 patients with INR > 5 were randomly subdivided into two groups: group A (n= 12) discontinued warfarin for one day and group B (n= 11) received 2 mg of vitamin K1orally in addition to the usual warfarin dose. INR was determined after 24 h (day 1), after which both groups continued with their usual dose of warfarin. After 48h (day 2), warfarin dosage was changed according to the INR value. On day 9, INR values were determined again. Five out of twelve patients in group A had INR values > 5 on day 1. One patient in group A had an INR value < 5 both on days 1 and 2. All eleven patients in group B had INR values < 5 on day 1, and all but one on day 2. On day 9, INR values were acceptable (INR 2.0‐4.5) in ten group A patients and eight group B patients. These findings suggest that a low oral dose of vitamin K1is a convenient treatment for excessive anticoagulation in patients with no bleeding complications.

ISSN:0957-5235    

出版商:OVID    

年代:1993

数据来源: OVID