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T C Wun

Publications and source records attributed to T C Wun.

At least 37 records · Page 2Linked to original sources
PubMed1994

Renaturation and purification of human tissue factor pathway inhibitor expressed in recombinant E. coli.

Tissue factor pathway inhibitor is an inhibitor of the extrinsic coagulation pathway. Evaluation of the pharmacological effects of tissue factor pathway inhibitor in animal models has been limited by the high cost and low availability of mammalian tissue culture produced protein. In order to circumvent this obstacle, a 277-amino-acid nonglycosylated tissue factor pathway inhibitor variant possessing an N-terminal alanine was expressed in recombinant E. coli using the tac promoter expression system. High-level expression in recombinant E. coli resulted in the accumulation of ala-tissue factor pathway inhibitor in inclusion bodies. Active protein was produced by solubilization of the inclusion bodies in 8 M urea, purification of the full-length molecule by cation exchange chromatography, and renaturation in 6 M urea. Fractionation of crude refold mixtures using cation exchange chromatography yielded a purified nonglycosylated tissue factor pathway inhibitor possessing in vitro prothrombin time activity comparable to inhibitor purified from mammalian cell lines.

Amino Acid Sequence↗
PubMed1994

Recombinant E. coli-derived tissue factor pathway inhibitor reduces coagulopathic and lethal effects in the baboon gram-negative model of septic shock.

Excessive coagulation is a typical response to the vascular injury occurring in gram negative sepsis. This study evaluated the pharmacological effects of the use of a recombinant Escherichia coli derived form of tissue factor pathway inhibitor (ala-TFPI) in a baboon model of septic shock. Several doses of ala-TFPI were administered either 30 or 120 min after the initiation of a lethal intravenous infusion of E. coli into baboons. Treatment at 30 min with either 2.7 or 7.4 mg/kg of ala-TFPI resulted in the same survival rates and attenuation of both the coagulation response and cellular injury, as measured by clinical chemistry. When administration of ala-TFPI was delayed for 120 min, a dose of ala-TFPI protein continued to provide a benefit to survival. Ala-TFPI reduced the drop in mean systemic arterial pressure compared to control baboons in addition to partially attenuating the coagulopathic response. Baboons given ala-TFPI also maintained lower levels of plasma interleukin-6 (IL-6) and thrombin-antithrombin. These results suggest that the site of action of the protein may involve the later stage components of the coagulation and inflammatory pathways.

Animals↗
PubMed1993

Kinetics of factor Xa inhibition by tissue factor pathway inhibitor.

Tissue factor pathway inhibitor is a multivalent, Kunitz-type proteinase inhibitor. It directly inhibits factor Xa and, in a factor Xa-dependent fashion, produces feedback inhibition of the factor VIIa/tissue factor catalytic complex which is responsible for the initiation of coagulation. Human recombinant TFPI (rTFPI) produced in Escherichia coli was used to define the kinetic constants describing the human factor Xa:TFPI interaction. The inactivation of factor Xa by E. coli-rTFPI is indistinguishable from that of rTFPI produced in mammalian SK-hepatoma cells, suggesting that post-translational modifications such as glycosylation and phosphorylation do not play a major role in the inhibitory process. The slow, tight-binding inhibition of factor Xa follows the scheme: [formula: see text] Where the enzyme (E) and inhibitor (I) form an initial, immediate collision complex (EI) that then isomerizes slowly to a tightened final EI* complex. In the absence of other additions, the initial Ki (=k2/k1) and final Ki* for the inhibition of factor Xa by E. coli-rTFPI are 1.24 nM and 26.4 pM, respectively. In the presence of calcium ions (5 mM) the interaction between factor Xa and rTFPI is substantially weaker, with a Ki of 42.7 nM and Ki* of 85.2 pM. The addition of other components of the prothrombinase complex produces enhanced factor Xa inhibition predominantly through an effect on the initial Ki. In the presence of calcium ions and saturating concentrations of phospholipids and factor Va, the Ki and Ki* for factor Xa inactivation are 2.04 nM and 52.3 pM. The enhancing effect of heparin on the inhibitory process is concentration dependent and exhibits an optimum, reminiscent of the "template" model for heparin's acceleration of thrombin and factor IXa inhibition by antithrombin III. At optimal concentrations, the major mechanism of heparin action is also a reduction in the Ki of the initial encounter complex between factor Xa and rTFPI.

Amino Acid Sequence↗
PubMed1993

Prevention of thrombosis by topical application of tissue factor pathway inhibitor in a rabbit model of vascular trauma.

The extrinsic pathway of coagulation is initiated when tissue factor complexes with factor VII. A naturally occurring protein inhibitor of this complex, tissue factor pathway inhibitor (TFPI), has recently been isolated and the cDNA coding for this protein cloned. We used a rabbit ear artery model of crush/avulsion injury and microvascular repair to investigate the efficacy of TFPI as a topically applied antithrombotic agent. Traumatized arteries treated through lumenal irrigation with normal saline vehicle (controls) achieved patency rates of 8% and 0% at 1 and 7 postoperative days, respectively. Heparin irrigation (10 U/ml) resulted in patencies of 40% at both evaluation times. In contrast, TFPI at a dose of 20 micrograms/ml (0.2 ml total volume; 10 minute exposure) yielded a 91% patency rate at 1 day and 73% at 7 days postoperatively (p < 0.0005 vs controls). Prothrombin time and activated partial thromboplastin time values were not altered after topical treatment with TFPI. Scanning electron microscopy revealed dramatically inhibited thrombogenesis upon the injured surfaces of TFPI-treated vessels. These results suggest that TFPI used as a topically applied antithrombotic agent is effective for the prevention of thrombosis in microvascular anastomoses.

Administration, Topical↗
PubMed1992

Separation and characterization of nonphosphorylated and serine-phosphorylated urokinase. Catalytic properties and sensitivity to plasminogen activator inhibitor type 1.

Urokinase synthesized by human A431 epidermoid carcinoma cells is phosphorylated on serine (Mastronicola, M. R., Stoppelli, M. P., Migliaccio, A., Auricchio, F., and Blasi, F. (1990) FEBS Lett. 266, 109-114). To test the possibility that phosphorylation may have specific effects on urokinase function, the phosphorylated and nonphosphorylated forms of urokinase were separated by Fe(3+)-Sepharose chromatography. Both forms exhibit indistinguishable Km and kcat for plasminogen activation. On the other hand, their sensitivity toward the specific plasminogen activator inhibitor type 1 is different as assessed by measuring both the stability of the covalent complex and the residual enzymatic activity. Phosphorylated urokinase was 50% inhibited at a concentration of plasminogen activator inhibitor type 1 4-fold higher than nonphosphorylated urokinase (0.7 versus 0.15 nM). Furthermore about 10% of phosphorylated urokinase was resistant to plasminogen activator inhibitor type 1 at a concentration as high as 20 nM. Thus, phosphorylation affects urokinase sensitivity to plasminogen activator inhibitor type 1, therefore resulting in a net, although indirect, increase of urokinase activity. These results suggest the existence of a novel cellular regulatory mechanism of extracellular proteolysis.

Catalysis↗
PubMed1992

Comparison of recombinant tissue factor pathway inhibitors expressed in human SK hepatoma, mouse C127, baby hamster kidney, and Chinese hamster ovary cells.

Recombinant tissue factor pathway inhibitor (rTFPI) has been expressed in four mammalian expression systems using human SK hepatoma, mouse C127, baby hamster kidney (BHK), and Chinese hamster ovary (CHO) cells as hosts. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, the immunoaffinity purified rTFPIs all show broad bands and the mean molecular weight of SK hepatoma and C127 rTFPIs (M(r) approximately 38,000) appear larger than those of BHK and CHO rTFPIs (M(r) approximately 35,000). All these proteins inhibit factor Xa and appear to bind factor Xa with 1:1 stoichiometry. The ability of these proteins to inhibit tissue factor-induced coagulation in plasma was examined using a prothrombin time assay. The relative activities of SK rTFPI:C127 rTFPI:BHK rTFPI:CHO rTFPI were found to be 28:15:2.1:1. By Western blot using specific antisera against the amino- and carboxy-termini of TFPI as probes, it is found that all the immunoaffinity purified rTFPIs possess approximately equal amounts of the amino terminus, but the C127 and BHK rTFPIs are deficient in carboxy terminus and the CHO rTFPI is essentially devoid of this region of the protein. Mono S chromatography allowed separation of the full-length and the truncated molecules with high and low anticoagulant activities, respectively. The above results suggest that proteolysis of the carboxy terminus of TFPI occurs to different extent when TFPI is expressed in different cells and that the carboxy terminal region of the TFPI molecule is important for the inhibition of tissue factor-induced coagulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Clearance of recombinant tissue factor pathway inhibitor (TFPI) in rabbits.

The pharmacokinetics of recombinant tissue factor pathway inhibitor (TFPI) after an intravenous bolus injection was studied in rabbits. Clearance of TFPI was followed by measurement of the radioactivity of the 125I-labelled compound in the whole plasma or the trichloroacetic acid precipitate and by quantitation of the functional TFPI activity of the unlabelled compound using a tissue factor-induced coagulation assay. When iodinated TFPI was used, the ratios of the trichloroacetic acid precipitable counts vs. that of the whole plasma was about 1 in the first 10 min after TFPI injection, but this ratio gradually decreased to less than 0.5 after 2 h. This result suggested that the iodinated TFPI in the plasma was partially degraded after prolonged circulation in the animal. When unlabelled TFPI was used, the clearance of TFPI activity from the plasma exhibited bi-exponential elimination kinetics with a rapid alpha phase half-life (t1/2 alpha) of 2.3 min, and a terminal beta phase half-life (t1/2 beta) of 79 min. The plasma clearance was 4.2 ml kg-1 min-1. The tissue distribution of intravenously administered 125I-TFPI in the rabbit was studied using whole-body autoradiography. At 3 min after dosing, significant levels of TFPI were apparent in the liver, kidney, and other highly blood perfused tissues. Significant levels of 125I-TFPI-derived radioactivity were also apparent in the liver and kidney at 30 min after intravenous administration. The localization within the liver demonstrated a mottled appearance, suggesting regions of higher uptake within the liver. In the kidney, the outer cortex consistently revealed the highest activity.

Animals↗
PubMed1992

Internalization of the urokinase-plasminogen activator inhibitor type-1 complex is mediated by the urokinase receptor.

The role of the urokinase receptor (uPAR) in the internalization of the urokinase-plasminogen activator inhibitor type-1 (uPA.PAI-1) complex has been investigated. First, exploiting the species specificity of uPA binding, we show that mouse LB6 cells (that express a mouse uPAR) were unable to bind or degrade the human uPA.PAI-1 complex. On the other hand, LB6 clone 19 cells, which express a transfected human uPAR, degraded uPA.PAI-1 complexes with kinetics identical to the human monocytic U937 cells. We also show by immunofluorescence experiments with anti-uPA antibodies that in LB6 clone 19 cells, the uPA.PAI-1 complex is indeed internalized. While at 4 degrees C uPA fluorescence was visible at the cell surface, shift of the temperature to 37 degrees C caused a displacement of the immunoreactivity to the cytoplasmic compartment, with a pattern indicating lysosomal localization. If uPA.PAI-1 internalization/degradation is mediated by uPAR, inhibition of uPA.PAI-1 binding to uPAR should block degradation. Three different treatments, competition with the agonist amino-terminal fragment of uPA, treatment with a monoclonal antibody directed toward the binding domain of uPAR or release of uPAR from the cell surface with phosphatidylinositol-specific phospholipase C completely prevented uPA.PAI-1 degradation. The possibility that a serpin-enzyme complex receptor might be primarily or secondarily involved in the internalization process was excluded since a serpin-enzyme complex peptide failed to inhibit uPA.PAI-1 binding and degradation. Similarly, complexes of PAI-1 with low molecular mass uPA (33 kDa uPA), which lacks the uPAR binding domain, were neither bound nor degraded. Finally we also show that treatment of cells with uPA.PAI-1 complex caused a specific but partial down-regulation of uPAR. A similar result was obtained when PAI-1 was allowed to complex to uPA that had been previously bound to the receptor. The possibility therefore exists that the entire complex uPA.PAI-1-uPAR is internalized. All these data allow us to conclude that internalization of the uPA.PAI-1 complex is mediated by uPAR.

Amino Acid Sequence↗
PubMed1992

Tissue factor pathway inhibitor: the carboxy-terminus is required for optimal inhibition of factor Xa.

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type protease inhibitor that binds to and inactivates factor Xa directly, and in a factor Xa-dependent fashion inhibits the factor VIIa/tissue factor catalytic complex. TFPI is a slow, tight-binding, competitive, and reversible inhibitor of factor Xa, in which the formation of an initial encounter complex between TFPI and factor Xa is followed by slow isomerization to a final, tightened complex. Wild-type recombinant TFPI (rTFPI), expressed in mouse C127 cells, separates into two forms on heparin-agarose chromatography that elute at 0.3 mol/L and 0.6 mol/L NaCl. Western blot analysis shows that both forms contain the N-terminus of full-length TFPI, but only rTFPI(0.6) is recognized by an antibody directed against the C-terminus. rTFPI(0.3) and rTFPI(0.6) inhibit factor Xa with 1:1 stoichiometry and inhibit factor VIIa/tissue factor equally in an endpoint-type assay. However, rTFPI(0.6) is a more potent inhibitor than rTFPI(0.3) of coagulation in normal plasma induced by either factor Xa or tissue factor. The initial inhibition of factor Xa (less than 5 seconds) produced by rTFPI(0.6) is several-fold greater than that produced by rTFPI(0.3), presumably reflecting a lower Ki of the immediate encounter complex between factor Xa and TFPI. The differential effect of these forms of TFPI on tissue factor-induced coagulation in normal plasma appears to be directly related to their ability to inhibit factor Xa. To confirm the role of the C-terminal region of TFPI in optimal factor Xa inhibition, a carboxy-terminal mutant of rTFPI, which is truncated after leucine 252 and thus lacks the basic sequence K T K R K R K K Q R V K (residues 254-265), was expressed in C127 cells. This form of rTFPI elutes from heparin-agarose at 0.28 mol/L NaCl and inhibits factor Xa at a rate that is slower than rTFPI(0.3). The Ki(final)s for factor Xa inhibition by rTFPI(0.6), rTFPI(0.3), and rTFPI1-252 are 3.1 +/- 0.6, 19.6 +/- 0.8, and 19.6 +/- 3.0 pmol/L, respectively.

Amino Acid Sequence↗
PubMed1992

The effect of leukocyte elastase on tissue factor pathway inhibitor.

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type inhibitor that directly inhibits factor Xa and, in a factor Xa-dependent fashion, also inhibits the factor VIIa/tissue factor (TF) catalytic complex. The Kunitz-2 domain in TFPI is needed for the binding and inhibition of factor Xa, while the Kunitz-1 domain appears to be responsible for binding factor VIIa in a quaternary factor Xa-TFPI-factor VIIa/TF inhibitory complex. Human leukocyte elastase (HLE) proteolytically cleaves TFPI between threonine-87 and threonine-88 within the polypeptide that links the Kunitz-1 and Kunitz-2 domains in the TFPI molecule. HLE treatment not only affects the ability of TFPI to inhibit factor VIIa/TF, but also dramatically reduces its inhibition of factor Xa. Both purified HLE and stimulated neutrophils regenerate TF activity from a preformed factor Xa-TFPI-factor VIIa/TF inhibitory complex. Kinetic analysis suggests that HLE cleavage does not effect the affinity of the initial encounter interaction between factor Xa and TFPI, whereas it markedly reduces the affinity of the final factor Xa:TFPI complex with Ki (final) values for untreated and HLE-treated TFPI of 58 pmol/L and 4.4 nmol/L, respectively. Thus, an epitope in the amino-terminal region of TFPI or a conformation of the TFPI molecule that requires the presence of this region is needed in concert with the Kunitz-2 domain to produce optimal inhibition of factor Xa by TFPI.

Amino Acid Sequence↗
PubMed1992

Lipoprotein-associated coagulation inhibitor (LACI) is a cofactor for heparin: synergistic anticoagulant action between LACI and sulfated polysaccharides.

Lipoprotein-associated coagulation inhibitor (LACI) is a plasma-derived protein that inhibits tissue factor (TF)/factor VIIa-induced coagulation in a factor Xa-dependent manner. The roles of endogenous plasma LACI and exogenously added LACI and heparin, in the regulation of coagulation, initiated via the intrinsic and extrinsic pathways, were studied using the activated partial thromboplastin time (APTT) and the modified prothrombin time (PT) assays, respectively. Both LACI-depleted plasma and normal plasma have identical APTTs and similar prolongations of the APTT in response to heparin; both are fully anticoagulated (arbitrarily defined as clotting times of greater than 1 hour) at similar concentrations of heparin. These results indicate that heparin is an effective anticoagulant when coagulation is initiated by the intrinsic pathway and that endogenous LACI is not significantly involved in the regulation of this pathway. The PT of normal plasma is only marginally longer than that of LACI-depleted plasma in the absence of heparin, suggesting that endogenous plasma LACI has a very limited capacity to inhibit TF-induced clotting. However, in the presence of heparin, the PTs of LACI-depleted plasma and normal plasma are different. Prolongation of the PT occurred only moderately and linearly with increasing concentrations of heparin in LACI-depleted plasma. In contrast, normal plasma showed a greater extent of PT prolongation in response to heparin and the plasma became fully anticoagulated at a certain threshold concentration of heparin. These results suggest that LACI serves as a cofactor for heparin and thus greatly enhances the inhibition of TF-induced coagulation. LACI-depleted plasma was supplemented with purified recombinant LACI and/or heparin and the effects on TF-induced clotting were studied. A combination of LACI and heparin greatly enhanced anticoagulation compared with LACI or heparin alone. Many sulfated polysaccharides were also found to enhance the LACI-dependent inhibition of TF-induced clotting. By weight, the relative potencies of these compounds are: low molecular weight heparin (mean Mr, 5,100) greater than unfractionated heparin greater than low molecular weight heparin (mean Mr, 3,700) greater than pentosan polysulfate greater than dermatan sulfate greater than dextran sulfate greater than heparan sulfate. Based on the above results, it is concluded that LACI is a cofactor for heparin in the inhibition of TF-induced clotting and that LACI and sulfated polysaccharides act synergistically in whole plasma.

Animals↗
PubMed1992

Venous thrombosis produced in the vena cava of rabbits by vascular damage and stasis.

The main objective of these experiments was to develop and characterize a new experimental model of venous thrombosis, and determine whether a combination of vascular wall damage (crushing with hemostat clamps) and prolonged stasis produced more reproducible clots than prolonged stasis per se. Rabbits were laparotomized, and a segment of the vena cava was dissected free and looped with two silk ligatures, 2.5 cm apart. The proximal tie was ligated 5 min after release of the clamps; the distal tie applied shortly thereafter, trapping a volume of blood in the isolated segment. At 2 hr after ligation, the isolated venous sac was excised and examined for the presence of a clot. Large, well-formed clots, which could be readily transferred and weighed, were invariably observed in the "clamp" and "no clamp" groups, the latter being a sham control. Mean clot weights did not differ in the two groups (23.1 +/- 1.6 versus 30.8 +/- 5.4 mg dry weight, clamped versus no clamp, respectively, p greater than 0.05). However, the precision of the method was improved significantly (p less than 0.005) by clamping as determined by homogeneity of variance testing. Time-course studies showed that a considerable lag period (about 60 min) preceded development of a detectable clot, and that the thrombus evolved rapidly during the interval of 60-90 min postligation. The location of the small clots at 60 min in clamped segments, as well as the failure of prolonged (120 min) stasis without caval isolation to cause substantial thrombi, strongly suggests that clot formation attributed to "stasis per se" is in fact due to focal vascular lesions created at the tie-down points. The present study is also the first report of blockade of localized venous thrombosis by recombinant tissue factor pathway inhibitor (rTFPI). When given as an i.v. bolus 20 min prior to ligation, rTFPI at 400 and 800 micrograms/kg completely blocked formation of the thrombus or greatly reduced its size in five of the six animals tested.

Animals↗
PubMed1991

Effect of lipoprotein-associated coagulation inhibitor (LACI) on thromboplastin-induced coagulation of normal and hemophiliac plasmas.

Lipoprotein-associated coagulation inhibitor (LACI) is a plasma-derived protein that inhibits the tissue factor/factor VIIa/calcium/phospholipid complex in a factor Xa-dependent manner. Recombinant LACI (rLACI) added exogenously to plasma prolonged the activated partial thromboplastin time (APTT) and prothrombin time (PT) as a function of rLACI concentration in linear and curvilinear manners, respectively. Under these standard assay conditions, the amounts of rLACI required to double the APTT and PT were approximately 350- and 90-fold the plasma concentration of LACI, respectively. Likewise, addition of antibodies against LACI to pooled normal, factor VIII-deficient, or factor IX-deficient plasma had no effect on their respective APTTs and PTs, demonstrating the insensitivity of these assays to endogenous LACI. The prothrombin time assay was modified by using dilute thromboplastin. Unlike the standard prothrombin time assay, the clotting times were prolonged for factors VIII- or IX-deficient plasma relative to pooled normal plasma in this modified PT assay. Additionally, the degree of factor deficiency, as determined by the APTT assay, was correlated with that determined by the modified PT assay using dilute thromboplastin. When antibodies against LACI were added to pooled normal, factor VIII-deficient, or factor IX-deficient plasma and the prothrombin time assay initiated using dilute thromboplastin, the clotting times for antibody-treated plasma were shorter than for the corresponding plasma in the absence of antibodies. Moreover, the clotting times for factors VIII- and IX-deficient plasmas treated with antibodies raised against LACI were at least as fast as pooled normal plasma in the absence of LACI antibodies when dilute thromboplastin was used to initiate clotting. These results suggest that the prothrombin time assay using dilute thromboplastin may more accurately reflect what occurs in vivo and that LACI may play an important role in the prolonged bleeding of those with hemophilia A or B.

Antibodies, Monoclonal↗
PubMed1991

Purification and properties of heparin-releasable lipoprotein-associated coagulation inhibitor.

The lipoprotein-associated coagulation inhibitor (LACI) is present in vivo in at least three different pools: sequestered in platelets, associated with plasma lipoproteins, and released into plasma by intravenous heparin, possibly from vascular endothelium. In this study we have purified the heparin-relesable form of LACI from post-heparin plasma and show that it is structurally different from lipoprotein LACI. The purification scheme uses heparin-agarose chromatography, immunoaffinity chromatography, and size-exclusion chromatography and results in a 185,000-fold purification with a 33% yield. Heparin-releasable LACI (HRL), as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under reducing conditions, appears as a major band at 40 Kd and a minor band at 36 Kd. Immunoblot analysis suggests that the 36-Kd band arises from carboxyl-terminus proteolysis that occurs during the purification. HRL has a specific activity similar to that of HepG2 or lipoprotein LACI. HRL and lipoprotein LACI combine with lipoproteins in vitro while purified HepG2 LACI does not. I125-labeled HRL, injected into a rabbit, is cleared more slowly than I125-labeled HepG2 LACI, which may be due to attachment to lipoproteins in vivo. Preliminary evidence suggests that HRL is associated with vascular endothelium, possibly by attachment to glycosaminoglycans.

Adult↗
PubMed1991

Binding of plasminogen activator inhibitor type-1 to extracellular matrix of Hep G2 cells. Evidence that the binding protein is vitronectin.

Catabolism of plasminogen activators by Hep G2 cells is mediated by a specific receptor which recognizes complexes of these serine proteases with their physiological inhibitor, plasminogen activator inhibitor type-1 (PAI-1). This catabolic process is initiated by interaction of exogenous plasminogen activators with bioactive PAI-1, which is secreted and localizes in an active form to the extracellular matrix (ECM) of Hep G2 cells. We now report that vitronectin (VN) mediates the specific binding of PAI-1 to the ECM of these cells. Purified bovine or human VN competes for specific binding of PAI-1 to Hep G2 ECM, and ligand blotting reveals specific binding of PAI-1 to ECM-associated VN. Hep G2 cells secrete both VN and PAI-1, and pulse-chase studies strongly suggest that these proteins associate only following secretion. Although Hep G2 cell-derived VN does not significantly bind to ECM in vitro, 30-40% of endogenous PAI-1 binds to the ECM, even in the presence of human serum, suggesting that ECM-associated VN is entirely derived from bovine serum. PAI-1 was localized by indirect immunofluorescence to ECM beneath cells and at cell margins, whereas VN exhibited a uniform distribution throughout the growth substratum. VN associated with the ECM may confer retention and bioactivity to PAI-1, potentially facilitating both pericellular regulation of plasmin generation and the rapid hepatic clearance of plasminogen activators.

Carcinoma, Hepatocellular↗
PubMed1991

Prevention of arterial reocclusion after thrombolysis with recombinant lipoprotein-associated coagulation inhibitor.

BACKGROUND: This study was designed to determine whether arterial reocclusion after thrombolysis can be prevented by lipoprotein-associated coagulation inhibitor (LACI), a physiological inhibitor of tissue factor-induced coagulation mediated by the extrinsic pathway. METHODS AND RESULTS: Thrombosis was induced in femoral arteries of anesthetized dogs with the use of anodal current to elicit extensive vascular injury and formation of platelet-rich thrombi in one artery and with thrombogenic copper wire to elicit fibrin-rich thrombi without appreciable vascular injury in the contralateral artery. Recanalization of both vessels was induced with t-PA (1.7 mg/kg i.v. over 1 hour) and verified with Doppler flow probes. Reocclusion occurred within 2 hours in seven of seven arteries with electrical injury-induced thrombosis and in four of seven arteries with copper wire-induced thrombosis in the absence of LACI. In dogs given infusions of recombinant DNA-produced LACI (225 micrograms/kg over 15 minutes, followed by 4 micrograms/kg/min i.v.) after completion of the infusion of t-PA, no reocclusion occurred during the 2-hour interval of observation in any of the five arteries subjected to electrical injury (p less than 0.001), and cyclic partial occlusions were nearly abolished (0.4 +/- 0.4/hr in LACI-treated dogs compared with 13.7 +/- 5.5/hr in saline-treated dogs, p less than 0.0001). In contrast, reocclusion occurred in two of five arteries with indwelling copper wires, and cyclic partial occlusions were unaffected despite LACI. LACI prolonged the partial thromboplastin time modestly (1.7 +/- 0.2 x baseline) but did not affect platelet counts or aggregation assessed ex vivo. CONCLUSIONS: Inhibition of the extrinsic pathway of coagulation with LACI prevents thrombotic arterial reocclusion after thrombolysis in vessels subjected to extensive vascular injury. Our results demonstrate that activation of the extrinsic pathway plays a critical role in thrombotic reocclusion and that LACI provides a highly targeted approach to facilitate sustained recanalization without directly inhibiting platelets.

Animals↗
PubMed1990

Secretion of active kringle-2-serine protease in Escherichia coli.

Active human tissue plasminogen activator variant kringle-2-serine protease (K2 + SP domains; referred to as MB1004) was synthesized as a secreted protein in Escherichia coli, isolated, and characterized. MB1004 is a relatively large and complex protein, approximately 38 kDa in size and containing nine disulfide bonds. MB1004 without a pro region was secreted into the periplasm of E. coli by fusing the protein to the PhoA leader peptide expressed from the tac promoter. Approximately 1% (20 micrograms/L broth) of the secreted MB1004 was purified from E. coli homogenates as a soluble, active enzyme by using a combination of lysine and Erythrina inhibitor affinity chromatography. Purified MB1004 was monomeric and single-chain, and the N-terminus was identical with the predicted amino acid sequence. The specific activity of purified MB1004 from E. coli was compared against the equivalent recombinant material purified from mammalian cells that was naturally glycosylated (MB1004G) or deglycosylated after treatment with N-glycanase (MB1004N). Results from four different in vitro assays showed that MB1004 and MB1004N had similar activities. Both exhibited 4-12-fold higher specific activity than MB1004G in plasminogen activation assays. These results suggest that an inaccurate picture of specific activity can be obtained if the effects of glycosylation are not considered. By utilization of secretion in E. coli, nonglycosylated MB1004 was purified without in vitro refolding and was shown to be suitable for structure-function studies.

Base Sequence↗