Glycobiology: Toward Understanding the Function of Sugars.

Publisher Summary This chapter discusses the role of plasminogen activators in various biological processes. In specific, it describes two types of plasminogen activators—namely, the urokinase-type plasminogen activator (u-PA) and the tissue-type plasminogen activator (t-PA), which are essentially different gene products. The amino acid sequences of these activators and nucleotide sequences of the corresponding cDNAs have largely been determined, and the cDNAs have been cloned using recombinant techniques. A variety of enzymatic as well as immunological assay and detection methods have also been developed that allows a precise quantification of the activators, a distinction between u-PA and t-PA, determination of whether an activator is present in its active or zymogen form, analysis of the kinetics of different steps of the cascade reaction, and immunocytochemical identification of u-PA and t-PA in tissue sections. Much of the studies on plasminogen activators and cancer has been guided by the hypothesis that proteolysis of the components of extracellular matrix, initiated by the release of plasminogen activator from the cancer cells, plays a decisive role for the degradation of normal tissue, and thereby for invasive growth and metastases.

Plasminogen activators, tissue degradation, and cancer.

Endothelial Cells in Physiology and in the Pathophysiology of Vascular Disorders

On the regulation and control of fibrinolysis. Edward Kowalski Memorial Lecture.

http://www.chemie.uni-muenchen.de/ac/kluefers/homepage/L/bac/ca_2004.pdf

Synthetic analogues relevant to the structure and function of zinc enzymes.

Utilizing the immunoglobulin fraction from a goat antiserum against human uterine tissue plasminogen activator, an enzyme-linked immunoassay for tissue-type plasminogen activator in human plasma has been developed. With the new method, the concentration of t-PA in normal human acidified plasma is found to be 4.0 +/- 1.8 (SD) ng/ml. It increases to 12 ng/ml after a tourniquet test, and to 14 ng/ml after strenuous physical exercise. In a group of patients with idiopathic thromboembolic disease, the resting t-PA concentration was 5 ng/ml and the post-occlusion value 16 ng/ml. Furthermore, the patients also exhibited a normal post-occlusion rise in the concentration of plasmin-alpha 2-antiplasmin complex. However, in 37% of the post-occlusion patient plasmas, virtually no increase in t-PA could be detected by a specific activity assay. The results indicate that the reason for a defective post-occlusion fibrinolytic activity in a majority of cases may be the presence of increased concentrations of a fast-acting specific t-PA inhibitor.

An enzyme linked immunosorbent assay for determination of tissue plasminogen activator applied to patients with thromboembolic disease.

The specific interaction between plasminogen and fibrin. A physiological role of the lysine binding site in plasminogen.

A sensitive assay for tissue plasminogen activator

Activation of plasminogen was performed by perfusing a plasminogen solution through a small column loaded with insolubilized urokinase. The effluent was immediately collected in an excess of plasmin inhibitor (p-nitrophenyl-p-guanidino-benzoate). This provides a practically instantaneous inhibition of the plasmin formed and autocatalytic degradation is thus reduced to a minimum. Different degrees of activation are obtained by changing the perfusion rate.



Dodecylsulfate-polyacrylamide electrophoresis, amino acid and NH2-terminal amino acid analyses show that the activation of human plasminogen involves cleavage of at least two peptide bonds, the first event being the release of a peptide of 64 amino acid residues from the NH2-terminal end. An intermediate inactive product with a molecular weight of about 86000 and methionine as NH2-terminal amino acid is formed. Cleavage at the second site results in formation of a molecule with two chains connected by disulfide bridges. One chain which has a molecular weight of about 63000 is very sensitive to proteolytic degradation in its NH2-terminal part. However, there is evidence that methionine is the true NH2-terminal amino acid of this fragment. The other chain has a molecular weight of about 25000 and has valine as NH2-terminal amino acid.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1973.tb02880.x

Activation of human plasminogen by an insoluble derivative of urokinase. Structural changes of plasminogen in the course of activation to plasmin and demonstration of a possible intermediate compound.

Tissue plasminogen activator, separated into variants I and II (differing in Mr by 2000-3000), was reduced and [14C]carboxymethylated. Fragments from cleavages with enzymes and cyanogen bromide (CNBr) were separated by reverse-phase high-performance liquid chromatography and subjected to sequence degradations. All seven CNBr fragments were purified and found to be compatible with the cDNA-derived amino acid sequence [Pennica, D., Holmes, W. E., Kohr, W. J., Harkins, R. N., Vehar, G. A., Ward, C. A., Bennett, W. F., Ylverton, E., Seeburg, P. H., Heynecker, H. L., Goeddel, D. V., & Collen, D. (1983) Nature (London) 301, 214-221]. Chemical characterization of 93% of the 527 residues recovered in 50 peptides confirmed the indirectly deduced primary structure of the protein. The tryptic peptide patterns from the two variants were found to differ for one peptide (T15). Since carbohydrate was present in this peptide for variant I and since a marked difference in chromatographic behavior for T15 was observed in variant II, we conclude that carbohydrate differences in this peptide (i.e., Asn-184 in the numbering system of the cDNA-derived amino acid sequence) are the explanation for the size differences between variants I and II. Carbohydrate was also found at two other positions in the protein, corresponding to Asn-117 and Asn-448. However, a fourth potential glycosylation site, Asn-218, is apparently not utilized for carbohydrate attachment. The enzyme is inactivated by diisopropyl phosphorofluoridate, which covalently modifies the serine residue corresponding to position 478, identifying this as the active site serine residue.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue plasminogen activator: peptide analyses confirm an indirectly derived amino acid sequence, identify the active site serine residue, establish glycosylation sites, and localize variant differences.

Per Wallén

Papers

An enzyme linked immunosorbent assay for determination of tissue plasminogen activator applied to patients with thromboembolic disease.

The specific interaction between plasminogen and fibrin. A physiological role of the lysine binding site in plasminogen.

A sensitive assay for tissue plasminogen activator

Activation of human plasminogen by an insoluble derivative of urokinase. Structural changes of plasminogen in the course of activation to plasmin and demonstration of a possible intermediate compound.

Tissue plasminogen activator: peptide analyses confirm an indirectly derived amino acid sequence, identify the active site serine residue, establish glycosylation sites, and localize variant differences.