Physiology, Plasminogen Activation

Function

The fibrinolytic system functions to dissolve fibrin, one of the main products of thrombin activity. Within this system, plasmin serves as the major protease, utilizing fibrin contained within clots as a substrate for proteolysis, producing soluble products and thus maintaining the patency within the vascular system.[1] The precursor to plasmin, plasminogen, serves as zymogen produced by the liver circulates throughout the endovascular network, and participates in the fibrinolytic pathway, serving as both a catalytic enzyme to initiate the process of fibrinolysis, as well as a substrate, which once cleaved to become plasmin, continues on the process of fibrinolysis at an accelerated rate. This conversion from the zymogen to plasminogen is highly regulated and involves several different circulating factors and feedback mechanisms from the substrate products.[1] Regulation of the activation of plasminogen functions to control the homeostasis between fibrin deposition and fibrinolysis, particularly in the setting of hemostasis. This regulation results from the molecular structure of plasminogen, its conformational state, the interactions between plasminogen and fibrin, plasminogen activators, inhibitors of activation, and the effects of plasmin during fibrinolysis, which initiates a positive feedback mechanism.

Although plasmin’s role is largely thought to be contained in the fibrinolytic system, plasminogen activators seem to play roles beyond regulating fibrinolysis. Interestingly, deficiencies of plasminogen are not associated with increased thrombotic events but rather with the thickening of mucous membranes from an accumulation of fibrin; the hypothesis is that the lack of tendency towards thrombosis is from subsequent increases in other serine protease activity.[2]

Mechanism

Plasminogen is a zymogen that initiates the fibrinolytic cascade by binding to intact fibrin via structural domains within plasminogen called "kringle" domains. Structurally, kringle domains are large loops of amino acids stabilized by disulfide bonds. These kringle domains, present in several enzymes within the fibrinolytic system, allow for plasminogen binding to carboxy-terminal lysine residues and have been considered the first stage of fibrinolysis.[3] Plasminogen binding is regulated in hemostatic thrombi by removing the carboxy-terminal lysine groups when fibrin is formed in thrombomodulin, expressed by vascular endothelial cells. Thrombomodulin binds thrombin and generates carboxypeptidase B, which cleaves free carboxyterminal lysine residues.[3][4] This regulatory step prevents premature lysis of clots acting in a hemostatic role and limits plasminogen activation on large, actively forming thrombi.[4] However, once the plasminogen is bound to fibrin, a conformational change occurs in the plasminogen’s structure, increasing the susceptibility of the plasminogen to activation.

Studies indicate plasminogen has 3 distinct conformational forms: alpha, beta, and gamma.

• Alpha-conformation: A closed conformation and is the confirmation adapted predominantly while plasminogen circulates
• Beta-conformation: A semi-open conformation occurs when plasminogen is bound to intact fibrin via 1 carboxy-terminal lysine residue
• b A fully open conformation occurs when the plasminogen is bound to 2 carboxy-terminal lysine residues.[3]

Additionally, the literature indicates that circulating plasminogen can be modified via hydrolysis reactions, which increase plasminogen’s binding affinity to fibrin.[3] These conformational forms and modifications allow for the regulation of plasminogen activation at the molecular level.

Tissue Plasminogen Activator

The most physiologically active plasminogen activator is the tissue plasminogen activator (tPA); its production and secretion are predominantly from endothelial cells.[1] The endothelial release of tPA gets triggered by numerous local stimuli, including shear stress, thrombin activity, histamine, and bradykinin.[3]  When synthesized, tPA contains 5 structural domains, of these domains which include a fibronectin finger domain, 2 kringle domains, which are homologs to the kringle structures found in plasminogen, an epidermal growth factor analog, and a serine protease domain.[1] tPA production occurs first as a single-chain protein, and its affinity for plasminogen decreases in this single-chain form. Once plasmin has been produced, plasmin works in a positive feedback mechanism, cleaving tPA into its 2-chain form. This form has a 10-fold increase in affinity for converting plasminogen into plasmin and accelerates the conversion rate.[4] TPA remains suppressed in a normal patent lumen via a molar excess of its inhibitor, plasminogen activator inhibitor-1 (PAI-1).[4] In the presence of fibrin, plasminogen and tPA can bind, the concentration-dependent inhibitory effect of PAI-1 is lost, and tPA is brought close enough to cleave plasminogen into active plasmin. This activation occurs through the cleavage of an Arg-Val peptide bond within plasminogen, giving rise to the active protease plasmin.[1] This cleavage event is a similar activating step for all the different activators of plasminogen, of which tPA is the most ubiquitous.

Urokinase-type plasminogen activator

Urokinase-type plasminogen activator (uPA) is the second major plasminogen activator and is known to have numerous functions beyond its involvement in plasminogen activation.[5] To be proteolytically active and participate in plasminogen activation, uPA binds with a cell surface receptor on vascular endothelium. Like tPA, uPA secretion is in a single-chain form with a low affinity for plasminogen, and similar to tPA has a more active 2-chain form. When single-chain uPA binds to its cell membrane receptor, and plasminogen is bound close by via a carboxyterminal lysine residue, the 2 proenzymes can reciprocally activate one another. It is important to note that uPA-mediated plasminogen activation plays a minor role compared to tPA activation.[5] While uPA and tPA are the major plasminogen activators, the literature describes several others. These include kallikrein, as well as factor XIa and factor XIIa. The overall effect of these proteases on the total plasma plasmin production is reported in the literature to be about 15%.[1]

Once activated, mechanisms exist within the plasma to degrade the plasmin response. Inhibition of plasmin occurs by alpha-antiplasmin which is a member of the serpin protein family, alpha-antiplasmin circulates within the plasma at a relatively high concentration to inhibit the activity of plasmin.[1] Concurrently, mechanisms exist to decrease the activity of tPA and uPA, accomplished by the action of 2 other members of the serpin family, plasminogen activator inhibitor-1(PAI-1) and plasminogen activator inhibitor-2 (PAI-2).[1]

PAI-1 and PAI-2 

Numerous cell types, including endothelial cells and platelets, release PAI-1 and PAI-2 in response to cytokines involved in inflammatory cascades. PAI-1 is produced in endothelial cells. Synthesis is highly regulated, and PAI-1 produced is active, which rapidly decays in solution.[6] Thus, the conclusion is that upon release, PAI-1 and PAI-2 are structurally labile and require stabilization. Stabilization occurs via a circulating component of plasma called vitronectin; the vitronectin and PAI complex exhibits less spontaneous inactivation than PAI-1 alone, and the fibronectin and PAI complex is then further stabilized in a molecular locking mechanism by binding with ligands that restrict PAI-1's labile structural center.[7] Once stabilized, PAI-1 and PAI-2 form irreversible complexes at the cutting sites of tPA and uPA, inhibiting them within the vascular space. Of the 2, PAI-1 exists at a higher concentration and is the most physiologically active compared to PAI-2, inhibiting both uPA and tPA. PAI-2 has been shown to have a minimal inhibitory effect on tPA and no inhibition on uPA.  Genetic polymorphisms of PAI-1 were thought to contribute to the pathogenesis of atherosclerotic disease; however, recent meta-analyses do not support this contribution.[8]

More recent evidence suggests the endocrine role adipose tissue plays, and an adipose-derived plasminogen activator inhibitor has been identified in plasminogen activation. Production of adipose-derived plasminogen activator inhibitor increases as total visceral body fat increases, thus increasing the inhibiting effect on plasminogen activation and leading to dysregulation of fibrinolysis.[9] PAI-1 is known to have roles beyond inhibition of plasminogen activation, and evidence indicates that it has roles in stimulating extracellular matrix remodeling, cell adhesion, and motility. Dysregulation of these roles is thought to have implications in fibrotic disease, neoplastic metastasis, and gestational complications.[10] 

In summary, plasminogen exists in 3 distinct conformational forms, which confer different accessibility to plasminogen's activating site.  Activation can occur via several different catalytic enzymes, tPA and uPA being the most physiologically important. The activity of these plasminogen activators is regulated primarily by PAI-1 and PAI-2. In contrast, the active form of plasminogen, plasmin, is inhibited by alpha-antiplasmin, a serpin protein in the same class as PAI-1 and PAI-2.

Clinical Significance

Acute ischemic stroke is one of the most significant causes of mortality and morbidity in the United States. An acute ischemic stroke occurs when there is a sudden onset occlusion of blood supply to an area of the brain. Primary mechanisms of acute ischemic stroke include embolic events and atherothrombosis (a clot forming over an atherosclerotic plaque). Treatment involves recanalization of the affected vessel through mechanical or pharmacological mechanisms.  

One of the first pharmacological mechanisms for treating acute ischemic strokes was the IV recombinant tissue plasminogen activator (IV tPA). It was approved in 1996 in the United States for patients presenting within the first 3 hours of symptom onset. Recent data support the use of IV-rtPA between 3 to 4.5 hours.[11] Despite the benefit of IV rtPA over placebo, literature reports only a 10% to 30% reduction in adverse outcomes when patients receive treatment within the 3-hour window and lower in the 3- to 4.5-hour window.[11]  Additionally, research has identified several limitations to reperfusion therapy, including delays to reperfusion resulting in irreversible ischemic injury, incomplete recanalization, and one of the more dangerous complications from IV rtPA, hemorrhagic transformation. IV rtPA has also been noted to have therapeutic limitations, including decreased efficacy in the removal of large thrombi. Contraindications to IV rtPA include but are not limited to recent head trauma or stroke within the last 3 months, history of intracranial hemorrhage, Current use of anticoagulants such as Xa inhibitors or thrombin inhibitors and major surgery within the past 2 weeks, or GI bleeding within the last 3 weeks.[11] While the major limiting factor for IV rtPA is the window for symptom onset, numerous trials and literature reviews are ongoing, looking for ways to mitigate the adverse effects of IV rtPA and extend the window for treatment.[12] Currently, research has begun to come out proposing combination medical therapy in the treatment of acute ischemic stroke, including suggesting medications that can support the blood-brain barrier and work to facilitate the preservation of cerebral vasculature alongside IV rtPA use.[12] Other plasminogen activators have been studied in treating acute ischemic stroke and include recombinant pro-urokinase, which has been studied in patients presenting within up to 6 hours of symptom onset; however, it did not receive FDA approval.[11] 

While limited by symptom onset, IV rtPA is still considered one of the gold standards of care in acute ischemic stroke. Currently, the use of IV rtPA in the absence of contraindications is widespread within the United States, and research is ongoing into ways to enhance the medical management of acute ischemic events.