Similarities beween single-chain tissue plasminogen activator and the cell-surface proteinase, guanidinobenzoatase

1990 ◽  
Vol 18 (4) ◽  
pp. 632-633
Author(s):  
FRANK S. STEVEN ◽  
MARGARET M. GRIFFIN
Keyword(s):  
Cell Surface ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Single Chain ◽  
Tissue Plasminogen

A Collaborative Study to Establish the 2nd International Standard for Tissue Plasminogen Activator (t-PA)

1987 ◽  
Vol 58 (04) ◽  
pp. 1085-1087 ◽  
Author(s):  
P J Gaffney ◽  
A D Curtis
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Collaborative Study ◽  
Chinese Hamster ◽  
World Health ◽  
Clot Lysis ◽  
Expert Committee ◽  
Single Chain ◽  
International Standard ◽  
Tissue Plasminogen

SummaryAn international collaborative study involving ten laboratories located in eight different countries was undertaken in order to replace the current International Standard (I.S.) for tissue plasminogen activator (t-PA). Two lyophilised candidate preparations of high purity were assessed in comparison with the current I.S. for t-PA using only a clot lysis assay. One preparation (coded 861670) was purified from a cultured melanoma cell supernatant and was about 98% single chain t-PA while the other preparation (coded 861624) was derived from Chinese hamster ovary (CHO) cells following DNA recombinant procedures and was 75% single chain t-PA.Both candidate preparations of t-PA compared in quite a satisfactory manner with the current I.S. from the viewpoint of the biometrics of parallel line bioassays and both preparations were quite stable for long periods at low temperatures and stable from up to 1 month at temperatures of 20° and 38° C. Both fultil the criteria to serve as a satisfactory Znd International Standard for t-PA. The Fibrinolysis Subcommittee of the International Committee for Thrombosis and Haemostasis recommended the melanoma source t-PA (861670) as the next I.S. in order to maintain continuity with the 1st I.S. which was also a melanomatype preparation. The data from the ten laboratories indicated that each ampoule of the new proposed standard contains 850 international units of t-PA activity by the clot lysis assay. It is planned to present the results of this study to the Expert Committee on Biological Standardization of the World Health Organization at its next meeting and to request that the preparation of t-PA, coded 861670, be established as the 2ndlnternational Standard for t-PA.

THE BINDING OF TISSUE PLASMINOGEN ACTIVATOR TO PLATELETS

1987 ◽  
Author(s):  
J Loscalzo ◽  
D E Vaughan
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Human Platelets ◽  
Tissue Type ◽  
Single Chain ◽  
Platelet Surface ◽  
Binding Domains ◽  
Tissue Plasminogen ◽  
Platelet Aggregate ◽  
A Site

Since the platelet surface has been shown to be a site for plasminogen conversion by tissue-type and other plasminogen activators, we examined the binding of tissue plasminogen activator (tPA) to human platelets. Resting, washed platelets were found to bind single chain, radioiodinated, recombinant tPA specifically and saturably with an apparent, estimated dissociation constant (KD) of 458 nM, binding approximately 570 molecules per platelet at saturation. Washed platelets activated with adenosine 5'-diphosphate in the presence of 0.1 mg/ml fibrinogen and 1 mM CaCl2 bound tPA with greater affinity, having an estimated apparent KD of 30.6 nM and binding approximately 29,000 molecules per platelet at saturation. Bound tPA could be completely displaced by an excess of unlabeled tPA. Interestingly, bound tPA could also be displaced from activated platelets with increasing concentrations of soluble fibrin with an estimated IC50 of 37.5 μg/ml of fibrin. In contrast, increasing concentrations of fibrinogen failed to reduce binding. These data show that tPA binds to the activated platelet surface by a mechanism that involves platelet-bound fibrinogen. In addition, these data suggest that on binding to the platelet surface, fibrinogen expresses domains that are similar to the tPA binding domains of fibrin. It is the presence of these domains within the platelet aggregate that likely supports tPA binding and facilitates plasminogen activation.

scholarly journals Competition between plasminogen and tissue plasminogen activator for cellular binding sites

Blood ◽  
1993 ◽  
Vol 82 (8) ◽  
pp. 2433-2441 ◽  
Author(s):  
J Felez ◽  
CJ Chanquia ◽  
P Fabregas ◽  
EF Plow ◽  
LA Miles
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Proteolytic Activity ◽  
Binding Sites ◽  
Cell Types ◽  
Receptor Expression ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Cellular Receptors ◽  
Tissue Plasminogen

Cellular receptors for plasminogen and tissue plasminogen activator (t- PA) regulate plasminogen activation and cell-associated proteolytic activity. The characteristics of the interactions of both ligands with monocytes and monocytoid cell lines bear certain similarities, including affinity (kd approximately 1 mumol/L) capacity and susceptibility to carboxypeptidase treatment. Therefore, we have undertaken the present study to determine directly whether t-PA and plasminogen share common binding sites on cells. We found that recombinant human single-chain t-PA (rt-PA) could inhibit the binding of 125I-plasminogen to the cells and, conversely, plasminogen could inhibit 125I-rt-PA binding. This relationship was observed with 9 cell types, including both adherent cells and cells in suspension. In addition, under several conditions of cell treatment, plasminogen and t- PA receptor expression was modulated in parallel. Furthermore, molecules that have been implicated as candidate plasminogen receptors, gangliosides, and an alpha-enolase--related molecule, also interacted with t-PA. These results suggest that at least a component of the binding sites for plasminogen is shared with t-PA. Occupancy of these sites by either or both ligand(s) should result in arming the cells with the proteolytic activity of plasmin.

scholarly journals Kringle glycosylation in a modified human tissue plasminogen activator improves functional properties

Blood ◽  
1993 ◽  
Vol 81 (5) ◽  
pp. 1312-1322
Author(s):  
DT Berg ◽  
PJ Burck ◽  
DH Berg ◽  
BW Grinnell
Keyword(s):  
Serine Protease ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Functional Properties ◽  
Site Directed Mutagenesis ◽  
Type I ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Tissue Plasminogen ◽  
Kringle 2

Native tissue plasminogen activator (ntPA) has a variable glycosylation site on its kringle-2 domain. We have examined the effects of kringle glycosylation on functional properties by studying the simplified tPA molecule, tPA-6. tPA-6 is composed of kringle-2 and the serine protease domains and, like ntPA, cells expressing tPA-6 process it into two glycoforms: the monoglycosylated tPA-6-primary (tPA-6P, type II) with N- linked glycosylation at Asn-448 in the serine protease domain and diglycosylated tPA-6-variant (tPA-6V, type I) with glycosylation at Asn- 448 and at Asn-184 in kringle-2. When the two glycoforms were separated, we found that purified tPA-6V had reduced fibrin-stimulated plasminogenolytic activity toward Glu-plasminogen when compared to purified tPA-6P. However, in the presence of fibrin, tPA-6V unexpectedly exhibited a sixfold increase in selectivity toward Lys- plasminogen. In addition, tPA-6V was less susceptible than tPA-6P to plasmin-mediated conversion to the two-chain form. By site-directed mutagenesis of tPA-6, we eliminated variable glycosylation at Asn-184 and engineered a new glycosylation signal at a remnant site in the kringle. This derivative, designated tPA-6D, was secreted with complete kringle glycosylation. Like the naturally occurring tPA-6V, tPA-6D had lower rates of fibrin-stimulated Glu-plasminogen activation, increased specificity toward Lys-plasminogen, and greater resistance to plasmin digestion. Although the activity of tPA-6D could be stimulated by fibrin, its activity was not stimulated significantly by fibrinogen, and in human plasma the rate of fibrinogen depletion was reduced threefold. Although fibrin binding to kringle-2 of tPA-6D was slightly improved, there was a substantial increase in the dissociation constant (kd) for lysine binding, demonstrating a lack of correlation between these ligand-binding sites. Overall, our data demonstrate the marked effect of kringle glycosylation on functional properties. In addition, we have generated a derivative with properties that potentially improve clot specificity and single-chain half-life and reduce the potential for plasminogen activation in the plasma.

scholarly journals The kinetics of plasminogen activation by thrombin-cleaved pro- urokinase and promotion of its activity by fibrin fragment E-2 and by tissue plasminogen activator

Blood ◽  
1993 ◽  
Vol 81 (4) ◽  
pp. 980-987
Author(s):  
JN Liu ◽  
V Gurewich
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Catalytic Efficiency ◽  
Clot Lysis ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Carboxypeptidase B ◽  
Thrombin Cleavage ◽  
Tissue Plasminogen ◽  
A Chain

Thrombin hydrolyzes the Arg156-Phe157 bond in pro-urokinase (pro-UK), two residues from the activation site, generating a two-chain form (thromb-UK) believed to have little activity and that is resistant to plasmin activation. The kinetic constants for thromb-UK against synthetic substrate (S2444) were found to be essentially identical to pro-UK. Against native plasminogen, thromb-UK had a lower Michaelis constant (KM) and a higher (2-fold) catalytic efficiency. However, this difference with pro-UK was nullified by carboxypeptidase B (CpB) treatment of thromb-UK to remove the C-terminal arginine on the A- chain. Plasminogen activation by thromb-UK was substantially promoted by fibrin fragment E-2 but not by other fibrin derivatives, a phenomenon previously observed with pro-UK. Similarly, clot lysis by thromb-UK was promoted by tissue plasminogen activator because their combined effect was synergistic. Fibrinogenolysis in plasma occurred at 80-fold the concentration of thromb-UK as pro-UK, reflecting the 90- fold greater plasmin resistance of thromb-UK. Addition of a CpB inhibitor to the plasma enhanced fibrinogenolysis by thromb-UK and pro- UK by approximately 16%, consistent with the promotion of both forms by certain C-terminal lysines. In conclusion, CpB-thromb-UK corresponds functionally to a plasmin resistant form of pro-UK, indicating that the catalytic site of the single-chain pro-UK is unaffected by thrombin cleavage. The effect of CpB indicates that the C-terminal Arg of thromb- UK slightly enhances its affinity for plasminogen. Thromb-UK has potential plasminogen-activating activity at surfaces where C-terminal lysines, functionally comparable to fragment E-2, are found.

Preimplantation mouse embryos express a cell surface receptor for tissue-plasminogen activator

Development ◽  
1993 ◽  
Vol 119 (1) ◽  
pp. 191-198 ◽  
Author(s):  
P.M. Carroll ◽  
W.G. Richards ◽  
A.L. Darrow ◽  
J.M. Wells ◽  
S. Strickland
Keyword(s):  
Cell Surface ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Binding Activity ◽  
Mouse Embryos ◽  
Preimplantation Mouse Embryos ◽  
Tissue Plasminogen ◽  
Preimplantation Mouse ◽  
Morula Stage ◽  
A Cell

The serine protease tissue-plasminogen activator (t-PA) has previously been shown to be intracellular in mouse secondary oocytes and extracellular in fertilized eggs. Here we demonstrate that extracellular t-PA activity is bound to the surface of the fertilized egg. The level of t-PA activity associated with preimplantation mouse embryos decreases in the 2-cell stage embryo, then increases in 4-cell and morula stage embryos. However, morulae grown in culture from fertilized eggs lack t-PA activity but are able to bind exogenously added mouse t-PA. Additionally, northern analysis indicates that preimplantation embryos do not contain detectable levels of t-PA mRNA. Therefore, the enzyme activity associated with 4-cell and morula stage embryos in vivo is derived from t-PA present in the oviduct lumen that binds the embryo, and not from protein produced from translation of embryonic mRNA. The binding activity is species and protein specific in that neither mouse urokinase-type plasminogen activator (u-PA) nor human t-PA bind to cultured morulae. Furthermore, binding activity is dose-dependent and saturable, and does not require the active site of t-PA. These data indicate that a cell surface-specific t-PA-binding activity exists in the preimplantation mouse embryo and may localize function and concentrate the proteolytic activity of t-PA in early mouse development.

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