scholarly journals Processing of Laminin-5 and Its Functional Consequences: Role of Plasmin and Tissue-type Plasminogen Activator

1998 ◽  
Vol 141 (1) ◽  
pp. 255-265 ◽  
Author(s):  
Lawrence E. Goldfinger ◽  
M. Sharon Stack ◽  
Jonathan C.R. Jones
Keyword(s):  
Extracellular Matrix ◽  
Epithelial Cell ◽  
Cell Motility ◽  
Plasminogen Activator ◽  
Tissue Type ◽  
Breast Epithelial Cell ◽  
Epithelial Cell Line ◽  
Laminin 5 ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

The laminin-5 component of the extracellular matrices of certain cultured cells such as the rat epithelial cell line 804G and the human breast epithelial cell MCF-10A is capable of nucleating assembly of cell– matrix adhesive devices called hemidesmosomes when other cells are plated upon them. These matrices also impede cell motility. In contrast, cells plated onto the laminin-5–rich matrices of pp126 epithelial cells fail to assemble hemidesmosomes and are motile. To understand these contradictory phenomena, we have compared the forms of heterotrimeric laminin-5 secreted by 804G and MCF-10A cells with those secreted by pp126 cells, using a panel of laminin-5 subunit-specific antibodies. The α3 subunit of laminin-5 secreted by pp126 cells migrates at 190 kD, whereas that secreted by 804G and MCF-10A cells migrates at 160 kD. The pp126 cell 190-kD α3 chain of laminin-5 can be specifically proteolyzed by plasmin to a 160-kD species at enzyme concentrations that do not apparently effect the laminin-5 β and γ chains. After plasmin treatment, pp126 cell laminin-5 not only impedes cell motility but also becomes competent to nucleate assembly of hemidesmosomes. The possibility that plasmin may play an important role in processing laminin-5 subunits is supported by immunofluorescence analyses that demonstrate colocalization of laminin-5 and plasminogen in the extracellular matrix of MCF-10A and pp126 cells. Whereas tissue-type plasminogen activator (tPA), which converts plasminogen to plasmin, codistributes with laminin-5 in MCF-10A matrix, tPA is not present in pp126 extracellular matrix. Treatment of pp126 laminin-5–rich extracellular matrix with exogenous tPA results in proteolysis of the laminin-5 α3 chain from 190 to 160 kD. In addition, plasminogen and tPA bind laminin-5 in vitro. In summary, we provide evidence that laminin-5 is a multifunctional protein that can act under certain circumstances as a motility and at other times as an adhesive factor. In cells in culture, this functional conversion appears dependent upon and is regulated by tPA and plasminogen.

Binding of 131I-Labeled Tissue-Type Plasminogen Activator on De-Endothelialized Lesions in Rabbits

1987 ◽  
Vol 26 (05) ◽  
pp. 224-228 ◽  
Author(s):  
Y. Isaka ◽  
H. Etani ◽  
K. Kimura ◽  
S. Yoneda ◽  
T. Kamada ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Abdominal Aorta ◽  
Half Life ◽  
Balloon Catheter ◽  
Biochemical Properties ◽  
Tissue Type ◽  
Fibrin Deposition ◽  
Tissue Type Plasminogen Activator ◽  
High Affinity ◽  
Type Plasminogen Activator

Tissue-type plasminogen activator (t-PA) which has a high affinity for fibrin in the clot, was labeled with 131I by the iodogen method, and its binding to de-endothelialized lesions in the rabbit was measured to assess the detectability of thrombi. The de-endothelialized lesion was induced in the abdominal aorta with a Fogarty 4F balloon catheter. Two hours after the de-endothelialization, 131I-labeled t-PA (125 ± 46 μCi) was injected intravenously. The initial half-life of the agent in blood (n = 12) was 2.9 ± 0.4 min. The degree of binding of 131I-labeled t-PA to the de-endothelialized lesion was evaluated at 15 min (n = 6) or at 30 min (n = 6) after injection of the agent. In spite of the retention of the biochemical properties of 131I-labeled t-PA and the presence of fibrin deposition at the de-endothelialized lesion, the binding of t-PA to the lesion was not sufficiently strong. Lesion-to-control ratios (cpm/g/cpm/g) were 1.65 ± 0.40 (at 15 min) and 1.39 ± 1.31 (at 30 min), and lesion-to-blood ratios were 1.39 ± 0.32 (at 15 min) and 1.36 ± 0.23 (at 30 min). These results suggest that radiolabeled t-PA may be inappropriate as a radiopharmaceutical for the scintigraphic detection of a pre-existing thrombotic lesion.

The Influence of Carbohydrate Structure on the Clearance of Recombinant Tissue-Type Plasminogen Activator

1988 ◽  
Vol 60 (02) ◽  
pp. 255-261 ◽  
Author(s):  
A Hotchkiss ◽  
C J Refino ◽  
C K Leonard ◽  
J V O'Connor ◽  
C Crowley ◽  
...  
Keyword(s):  
Amino Acid ◽  
Plasminogen Activator ◽  
Sodium Periodate ◽  
Glycosylation Site ◽  
Tissue Type ◽  
Carbohydrate Structure ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
High Mannose ◽  
Oligosaccharide Structures

SummaryModification of the carbohydrate structures of recombinant tissue-type plasminogen activator (rt-PA) can increase or decrease its rate of clearance in rabbits. When rt-PA was treated with sodium periodate to oxidize carbohydrate residues, the rate of clearance was decreased from 9.6 ± 1.9 ml min−1 kg−1 to 3.5 ± 0.6 ml min−1 kg−1 (mean ± SD, n = 5). A similar change in the clearance of rt-PA was introduced by the use of endo-β-N-acetyl- glucosaminidase H (Endo-H), which selectively removes high mannose asparagine-linked oligosaccharides; the clearance of Endo-H-treated rt-PA was 5.0 ± 0.5 ml min−1 kg−1. A mutant of rt-PA was produced with an amino acid substitution at position 117 (Asn replaced with Gin) to remove a potential glycosylation site that normally contains a high mannose structure. The clearance of this material was also decreased, similar to the periodate and Endo-H-treated rt-PA. Conversely, when rt-PA was produced in the CHO 15B cell line, which can produce only high mannose oligosaccharide structures on glycoproteins, the clearance was increased by a factor of 1.8. These results demonstrate that the removal of rt-PA from the blood depends significantly upon the nature of its oligosaccharide structures.

Recombinant Variants of Tissue-Type Plasminogen Activator Containing Amino Acid Substitutions in the Finger Domain

1992 ◽  
Vol 68 (06) ◽  
pp. 672-677 ◽  
Author(s):  
Hitoshi Yahara ◽  
Keiji Matsumoto ◽  
Hiroyuki Maruyama ◽  
Tetsuya Nagaoka ◽  
Yasuhiro Ikenaka ◽  
...  
Keyword(s):  
Amino Acid ◽  
Plasminogen Activator ◽  
Half Life ◽  
Tissue Type ◽  
Clot Lysis ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Plasma Clot ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

SummaryTissue-type plasminogen activator (t-PA) is a fibrin-specific agent which has been used to treat acute myocardial infarction. In an attempt to clarify the determinants for its rapid clearance in vivo and high affinity for fibrin clots, we produced five variants containing amino acid substitutions in the finger domain, at amino acid residues 7–9, 10–14, 15–19, 28–33, and 37–42. All the variants had a prolonged half-life and a decreased affinity for fibrin of various degrees. The 37–42 variant demonstrated about a 6-fold longer half-life with a lower affinity for fibrin. Human plasma clot lysis assay estimated the fibrinolytic activity of the 37–42 variant to be 1.4-fold less effective than that of the wild-type rt-PA. In a rabbit jugular vein clot lysis model, doses of 1.0 and 0.15 mg/kg were required for about 70% lysis in the wild-type and 37–42 variant, respectively. Fibrinogen was degraded only when the wild-type rt-PA was administered at a dose of 1.0 mg/kg. These findings suggest that the 37–42 variant can be employed at a lower dosage and that it is a more fibrin-specific thrombolytic agent than the wild-type rt-PA.

Enhanced Effective Fibrinolysis following the Neutralization of Heparin in Open Heart Surgery Increases the Risk of Post-Surgical Bleeding

1990 ◽  
Vol 63 (02) ◽  
pp. 241-245 ◽  
Author(s):  
Jørgen Gram ◽  
Thomas Janetzko ◽  
Jørgen Jespersen ◽  
Hans Dietrich Bruhn
Keyword(s):  
Blood Loss ◽  
Plasminogen Activator ◽  
Heart Surgery ◽  
Degradation Products ◽  
Open Heart Surgery ◽  
Tissue Type ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Chest Tube Drain ◽  
Median Blood Loss

SummaryThe tissue-type plasminogen activator related fibrinolytic system was studied in 24 patients undergoing cardiopulmonary bypass surgery. The degradation of fibrinogen and fibrin was followed during and after surgery by means of new sensitive and specific assays and the changes were related to the blood loss measured in the chest tube drain during the first 24 postoperative hours. Although tissue-type plasminogen activator was significantly released into the circulation during the period of extracor-poreal circulation (p <0.01), constantly low levels of fibrinogen degradation products indicated that a systemic generation of plasmin could be controlled by the naturally occurring inhibitors. Following extracorporeal circulation heparin was neutralized by protamine chloride, and in relation to the subsequent generation of fibrin, there was a short period with increased concentrations of fibrinogen degradation products (p <0.01) and a prolonged period of degradation of cross-linked fibrin, as detected by increased concentrations of D-Dimer until 24 h after surgery (p <0.01). Patients with a higher than the median blood loss (520 ml) in the chest tube drain had a significantly higher increase of D-Dimer than patients with a lower than the median blood loss (p <0.05).We conclude that the incorporation of tissue-type plasminogen activator into fibrin and the in situ activation of plasminogen enhance local fibrinolysis, thereby increasing the risk of bleeding in patients undergoing open heart surgery

Single-Chain and Two-Chain Tissue-Type Plasminogen Activator (t-PA) Bind Differently to Cultured Human Endothelial Cells

1989 ◽  
Vol 62 (02) ◽  
pp. 699-703 ◽  
Author(s):  
Rob J Aerts ◽  
Karin Gillis ◽  
Hans Pannekoek
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Binding Sites ◽  
Umbilical Vein ◽  
Tissue Type ◽  
Single Chain ◽  
Human Endothelial Cells ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Umbilical Vein Endothelial Cells

SummaryIt has recently been shown that the fibrinolytic components plasminogen and tissue-type plasminogen activator (t-PA) both bind to cultured human umbilical vein endothelial cells (HUVEC). After cleavage of t-PA by plasmin, “single-chain” t-PA (sct-PA) is converted into “two-chain” t-PA (tct-PA), which differs from the former in a number of respects. We compared binding of sct-PA and tct-PA to the surface of HUVEC. Removal of t-PA bound to HUVEC by a mild treatment with acid and a subsequent quantification of eluted t-PA both by activity- and immunoradiometric assays revealed that, at concentrations between 10 and 500 nM, HUVEC bind about 3-4 times more sct-PA than tct-PA. At these concentrations, both sct-PA and tct-PA remain active when bound to HUVEC. Mutual competition experiments showed that sct-PA and tct-PA can virtually fully inhibit binding of each other to HUVEC, but that an about twofold higher concentration of tct-PA is required to prevent halfmaximal binding of sct-PA than visa versa. These results demonstrate that sct-PA and tct-PA bind with different affinities to the same binding sites on HUVEC.

Pharmacokinetics and Haemostatic Status During Consecutive Infusions of Recom binant Tissue-Type Plasminogen Activator in Patients with Acute Myocardial Infarction

1989 ◽  
Vol 61 (03) ◽  
pp. 497-501 ◽  
Author(s):  
E Seifried ◽  
P Tanswell ◽  
D Ellbrück ◽  
W Haerer ◽  
A Schmidt
Keyword(s):  
Myocardial Infarction ◽  
Acute Myocardial Infarction ◽  
Plasminogen Activator ◽  
Peak Plasma ◽  
Peak Plasma Concentration ◽  
Tissue Type ◽  
Tissue Type Plasminogen Activator ◽  
Precise Control ◽  
Type Plasminogen Activator

SummaryPharmacokinetics and systemic effects of recombinant tissue type plasminogen activator (rt-PA) were determined during coronary thrombolysis in 12 acute myocardial infarction patients using a consecutive intravenous infusion regimen. Ten mg rt-PA were infused in 2 minutes resulting in a peak plasma concentration (mean ±SD) of 3310±950 ng/ml, followed by 50 mg in 1 h and 30 mg in 1.5 h yielding steady state plasma levels of. 2210±470 nglml and 930±200 ng/ml, respectively. All patients received intravenous heparin. Total clearance of rt-PA was 380±74 ml/min, t,½α was 3.6±0.9 min and t,½β was 16±5.4 min.After 90 min, in plasma samples containing anti-rt-PA-IgG to inhibit in vitro effects, fibrinogen was decreased to 54%, plasminogen to 52%, α2-antiplasmin to 25%, α2-macroglobulin to 90% and antithrombin III to 85% of initial values. Coagulation times were prolonged and fibrin D-dimer concentrations increased from 0.40 to 2.7 μg/ml. It is concluded that pharmacokinetics of rt-PA show low interpatient variability and that its short mean residence time in plasma allows precise control of therapy. Apart from its moderate effect on the haemostatic system, rt-PA appears to lyse a fibrin pool in addition to the coronary thrombus.

Effects of Moderate Alcohol Consumption on Platelet Function, Tissue-Type Plasminogen Activator and Plasminogen Activator Inhibitor

1990 ◽  
Vol 63 (03) ◽  
pp. 345-348 ◽  
Author(s):  
J Veenastra ◽  
C Kluft ◽  
Th Ockhuizen ◽  
H v d Pol ◽  
M Wedel ◽  
...  
Keyword(s):  
Alcohol Consumption ◽  
Plasminogen Activator ◽  
Platelet Function ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Moderate Alcohol Consumption ◽  
Tissue Type Plasminogen Activator ◽  
Postprandial Phase ◽  
Type Plasminogen Activator ◽  
Activator Inhibitor

SummaryShort-term effects of moderate alcohol consumption on platelet function, tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI) were studied in two age groups of volunteers (20–30 and 45–55 years), each consisting of eight healthy males. The alcohol (30 g in red port and wine) was consumed during a standard dinner. Two blood samples were drawn: one in the postprandial phase, and one the next morning after fasting overnight. Alcohol consumption tended to increase platelet aggregation and production of hydroxy fatty acids, reduced plasma t-PA activity and increased PAI activity in the postprandial phase. After the overnight fast the effects on t-PA and PAI had disappeared whereas at that time alcohol consumption tended to decrease platelet function. The effects of alcohol on t-PA and PAI activity appeared mainly in the older age group, whereas the t-PA activity in this group was already much lower, irrespective of alcohol consumption.

Sign in / Sign up

Export Citation Format

Share Document