What is Plasminogen and Plasminogen Function Overview

What is plasminogen: Plasminogen is a 92‐kDa protein that is present in the blood as the inactive precursor of the serine protease, plasmin.391,392, therefore, it is converted to plasmin by the cleavage at the Arg561‐Val562 peptide bond by urokinase‐type or tissue‐type plasminogen activator (tPA and uPA, respectively).

Related Terms:

Tissue Plasminogen Activator
Fibrinolysin
Urokinase,
Fibrinolysis
Plasminogen Activator

Overview

Plasminogen is that the inactive precursor of plasmin, and a potent serine protease involved in the dissolution of fibrin blood clots. there are each hereditary and acquired forms of plasminogen deficiency have been described. furthermore, these are usually associated with either a thrombotic or a hyperfibrinolytic condition. therefore, this monograph, we review the plasminogen-plasmin system and describe 2 chromogenic-based assay kits for the photometric determination of plasminogen activity in human plasma.

Blood coagulation is a complex enzymatic event culminating in the formation of an insoluble threadlike protein called fibrin. therefore, together with platelets, fibrin forms a hemostatic plug to prevent excessive bleeding. thus, Fibrin blood clots are ultimately dissolved in due course in order to restore vascular patency. furthermore, the enzymes involved in this physiologically important process are part of the fibrinolytic system.

The central component in the fibrinolytic system is the glycoprotein plasminogen, which is produced by the liver and is present in plasma and most extravascular fluids. Therefore, it is a precursor enzyme that, following partial cleavage by a plasminogen activator is converted to its active and proteolytic form, plasmin. Therefore, Its primary target is fibrin, but it is also able to degrade several constituents of the extracellular matrix and to convert a number of pro-hormones and cytokine precursors to their active form. Therefore, Plasmin also appears to be involved in the metastatic spread of cancer.

The generation of plasmin occurs preferentially on the fibrin surface, which offers binding sites for plasminogen and its principal activator in blood, t-PA. This binding stimulates plasminogen activation but also localizes the action of plasmin to sites of fibrin formation which promotes efficient clot lysis. Further regulation is provided by the presence in plasma of inhibitors, primarily the plasmin inhibitor and the plasminogen activator inhibitor 1 (PAI-1).

The important role of plasminogen in fibrinolysis makes it an interesting parameter to evaluate in various diseases. Therefore, a decreased plasminogen level may in some situations compromise the body’s ability to degrade fibrin and as such predispose to thrombosis. Hereditary plasminogen deficiency, as a cause of thrombosis, has also been reported in several cases.

Thus, however, plasminogen deficiency is usually an acquired condition and since plasminogen is the inactive precursor of plasmin, most acquired defects are found in situations with increased fibrinolytic activity. furthermore, An acquired deficiency is often seen with severe liver disease and acute disseminated intravascular coagulation (DIC), or as a result of thrombolytic therapy with plasminogen activators.

Plasminogen Facts

Evolution of Fibrinolytic Enzymes

Plasminogen and its natural activators (t-PA, u-PA) belong to a large family of enzymes considered to have evolved from an ancestral protease similar to trypsin- a serine protease of broad specificity that breaks down dietary proteins. The kinship between the fibrinolytic enzymes in question and trypsin is attributed to their similar protease moiety, which cleaves proteins on the C-terminal side of arginyl and lysyl residues. Stretches of high homology are generally found in the active site pocket composed of serine, histidine, and aspartic acid.

During evolution, several types of homologous units of domain structures (generally coded by individual exons) have been added to the trypsin-like protease, enabling the fibrinolytic proteases to gradually fulfill more specialized tasks. The addition of five kringle domains gave rise to plasminogen, whereas the addition of two kringles, one finger, and one EGF (epidermal growth factor) domain gave rise to t-PA. In a similar fashion, the addition of one kringle and EGF domain gave rise to u-PA.

Schematic illustration of physiologic fibrinolysis. Plasminogen is the proenzyme of plasmin, whose primary target is the degradation of fibrin in the vasculature. t-PA is the principal activator of plasminogen in blood, while u-PA is the predominant activator outside the bloodstream in the extracellular matrix. t-PA is produced by the vascular endothelial cells and is released into the circulation after stimulation. Fibrin regulates its own destruction by providing receptors or binding sites for plasminogen and t-PA, thus localizing the action of plasmin. Therefore, inhibition of the system may occur at the level of plasminogen activation (PAI-1) or at the level of plasmin (plasmin inhibitor). Furthermore, Free t-PA as well as complex t-PA/PAI-1 is cleared from the circulation by receptors in the liver.

Abbreviations:

t-PA= tissue-type plasminogen activator,
PAI-1= plasminogen activator inhibitor 1,
FDP= fibrin degradation products.

Plasmin Function

Activation of plasminogen by its natural activators, t-PA, and u-PA, involves a bond cleavage at a specific site in the plasminogen molecule, which gives rise to a two-chain molecule linked by two disulfide bonds. The plasmin formed may degrade fibrin in a variety of ways resulting in insoluble fibrin degradation products or fragments called X, Y, D, and E. Plasmin is a relatively non-specific protease and can degrade not only fibrin but also many proteins in both plasma and extracellular spaces. In the coagulation pathway factors, V, VIII, and von Willebrand factor are known targets of plasmin.
Therefore, Plasmin activity is inhibited by binding to the plasmin inhibitor, therefore, which forms a stable complex with plasmin devoid of proteolytic activity.

Plasminogen Function

Several forms of plasminogen in plasma are called and it can be separated by affinity chromatography. Furthermore, the native form of plasminogen in plasma has glutamic acid at the N-terminal and is termed Glu-plasminogen. Therefore, the other plasminogen forms are generated by the catalytic cleavage by plasmin and containing mostly lysine at the N-terminal position, are termed Lys-plasminogen.

Human plasminogen is a single-chain glycoprotein that’s containing 791 amino acid residues and 2% carbohydrate. Its molecular mass is about 92,000 daltons. The plasminogen molecule contains a total of six structural domains, each with different properties. The N-terminal portion of the molecule consists of five kringle domains with the capacity to bind to fibrin. The kringle domain was first described by Magnussen et al (1975) who compared the structure with Danish pastry. Together with the preactivation peptide, the kringles control the ability of plasminogen to adopt different conformations. The protease domain resembles that of other serine proteases and contains the active site pocket His603, Asp646, and Ser741. This region also contains Ala601 which appears to be essential for the normal function of plasminogen, since mutation to Thr601 leads to an increased risk of thrombosis.

Glu-plasminogen exists in a closed conformation that becomes extended when binding to lysine residues on a fibrin surface. A similar conformational change is believed to take place when Glu-plasminogen is converted to Lys-plasminogen. The physiological role of these conformational changes is not well known although the general effect is believed to be an increased plasminogen activation rate catalyzed by t-PA. The opposite effect is observed in the presence of anions, in particular with Cl-, which stabilizes the closed-form of Glu-plasminogen rendering plasminogen poorly activatable.

Plasminogen activators

Plasminogen activators can be divided into two groups: endogenous activators (t-PA and u-PA), present in blood and other body fluids, and exogenous activators (e.g. streptokinase). Plasminogen activators are used as clot-dissolving (thrombolytic) agents for the treatment of pulmonary embolism and acute myocardial infarction.

Tissue Plasminogen Activator (tPA)

Tissue-type plasminogen activator (tPA) is the principal endogenous activator of plasminogen in blood. It is produced as a single-chain molecule by the vascular endothelial cells and is secreted into the plasma continuously or by an acute release reaction following stimulation of certain endothelial cell receptors. Rapid fluctuations in tPA concentration can be observed in response to exercise, venous occlusion, alcohol, and drugs, such as DDAVP and anabolic steroids. furthermore, individuals who do not show increased tPA activity

Therefore, the Plasmin cleavage of t-PA produces the more active two-chain molecule. However, unlike many other serine proteases, t-PA is active in its single-chain form, especially in the presence of fibrin or fibrin fragments.

Single-chain t-PA is a 68 kDa glycoprotein, which consisting of the 530 amino acids and the containing 7-13% carbohydrate. therefore, in the human plasma, t-PA may occur mainly as the complex together with its principal inhibitor PAI-1. The level of t-PA antigen is about 5 mg/ml, whereas the concentration of free t-PA is only about 1 mg/l or 0.5 IU/ml (specific activity range 500,000 to 700,000 U/mg). The single-chain t-PA molecule is converted by plasmin to a two-chain form by cleavage of the Arg275-Ile276 peptide bond. Binding to fibrin concentrates and correctly orientates t-PA and plasminogen, as well as inducing conformational changes in the molecules that promote efficient clot lysis.

Domain structures of t-PA and u-PA. The t-PA molecule is composed of at least five domains: a finger domain, the epidermal growth factor domain (EGF), two kringle domains, and the protease domain. The u-PA molecule consists of an EGF domain, one kringle, and the protease domain. The finger domain is homologous to structures found in fibronectin. Therefore, in the t-PA molecule, these domain is implicated with the fibrin binding. The EGF domain often confers affinity to specific receptors on cell surfaces.

Streptokinase

Streptokinase (SK) is an exogenous plasminogen activator of 47 kDa, derived from streptococci bacteria. It is not an enzyme and functions by forming a stoichiometric 1:1 complex with human plasminogen. These complex can function as an activator of the other plasminogen molecules. Therefore, the complex (difficult) formation is accompanied by the conformational change in the plasminogen molecule, exposing the active site to activate a second plasminogen molecule and that is followed by the conversion of the SK plasminogen into an SK-plasmin complex. furthermore, both types of SK-complexes are equally efficient activators of plasminogen.

Urokinase

Urokinase-type plasminogen activator (u-PA) is mainly produced in the kidneys as an inactive single-chain molecule (SCU-PA). u-PA has its major function in tissue-related proteolysis and is believed to play only a secondary role to t-PA as a physiological activator in blood.

The activation of SCU-PA by catalytic amounts of plasmin results in a two-chain structure with increased activity towards plasminogen. with this mechanism, the initial traces of plasmin can catalyze the production of active u-PA, leading to the formation of more plasmin. u-PA can only activate plasminogen in the presence of fibrin. However, it does not bind to fibrin and is not activated by fibrin. Therefore, in the human plasma, u-PA antigen concentrations range from 2 to 7 ng/ml. Higher values are often found in subjects with liver cirrhosis and hepatoma.

Mechanism of Plasminogen Activation

Full-length plasminogen comprises seven domains. additionally, to a C-terminal chymotrypsin-like serine protease domain, plasminogen contains an N-terminal Pan Apple domain together with five Kringle domains (KR1-5). Therefore, The Pan-Apple domain contains determinants for maintaining plasminogen in the closed form, and therefore, the kringle domains are liable for binding to lysine residues present in receptors and substrates.

Therefore, The X-ray crystal structure of closed plasminogen reveals that the PAp and SP domains maintain the closed conformation through interactions created throughout the kringle array. furthermore, Chloride ions further bridge the PAp / KR4 and SP / KR2 interfaces, explaining the physiological role of serum chloride in stabilizing the closed conformer. Therefore, the structural studies also reveal which differences in glycosylation alter the position of KR3. This data helps to explain the functional differences between type I and type II plasminogen glycoforms.

Thus, in the closed plasminogen, access to the activation bond (R561/V562) which targeted for cleavage by tPA and uPA is blocked through the position of the KR3/KR4 linker sequence and therefore, the O-linked sugar on T346. Here, the position of KR3 can also hinder access to the activation loop. The Inter-domain interactions block all kringle ligand-binding sites apart from that of the KR-1, which suggests that the latter domain governs pro-enzyme recruitment to targets.

Furthermore, an analysis of an intermediate plasminogen structure suggests which plasminogen conformational change to the open form is initiated through the KR-5 transiently peeling away from the PAp domain. Furthermore, These movements expose the KR5 lysine-binding site to potential binding partners and that suggests a requirement for spatially distinct lysine residues in the eliciting plasminogen recruitment and conformational change respectively.

Mechanism of Plasmin Inactivation

Plasmin is inactivated by proteins like α2-macroglobulin and α2-antiplasmin. Therefore, the mechanism of plasmin inactivation involves the cleavage of an α2-macroglobulin at the bait region by plasmin. furthermore, these initiates a conformational change like that the α2-macroglobulin collapses about the plasmin. Therefore, in the resulting α2-macroglobulin-plasmin complex, furthermore, the active site of plasmin is sterically shielded, thus substantially decreasing the plasmin’s access to protein substrates. Here, two additional events may occur as a consequence of bait region cleavage, namely
(i) an h-cysteinyl-g-glutamyl thiol ester of the α2-macroglobulin becomes the highly reactive and
(ii) The major conformational change exposes the conserved COOH-terminal receptor binding domain. Furthermore, the exposure of this receptor-binding domain also allowed the α2-macroglobulin protease complex to bind to clearance receptors and therefore, be removed from circulation.

REFERENCES

1. Plasmin and plasminogen From Wikipedia https://en.wikipedia.org/wiki/Plasmin
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5.Mouse PubMed Reference: National Center for Biotechnology Information, U.S. National Library of Medicine.
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