The fibrinolytic system selectively degrades cross-linked fibrin within thrombi by converting plasminogen into plasmin. Streptokinase, urokinase, and tissue-type plasminogen activator initiate this process through different mechanisms and exhibit clear differences in fibrin selectivity, duration of action, and bleeding risk.

Keywords: fibrinolytic system; plasminogen; plasmin; streptokinase; urokinase; tissue-type plasminogen activator; tPA; fibrin; thrombolysis; bleeding risk

1 Components of the Fibrinolytic System and Mechanisms of Thrombus Dissolution

1.1 Formation and Degradation of Fibrin Thrombi

Thrombin converts soluble fibrinogen into fibrin monomers, which subsequently polymerize and undergo cross-linking by coagulation factor XIIIa to form a stable fibrin network. Platelets, erythrocytes, and other blood components become embedded within this network and together constitute a thrombus. The fibrinolytic system primarily degrades cross-linked fibrin, and the strength of this process depends on the balance among plasminogen recruitment, activator localization, and inhibitory proteins.

1.2 Core Components of the Fibrinolytic System

The fibrinolytic system mainly includes plasminogen, plasmin, tissue-type plasminogen activator, urokinase-type plasminogen activator, and their inhibitory proteins. Plasminogen is an inactive serine protease precursor that is converted into plasmin after activation and subsequently cleaves fibrin. Plasminogen activator inhibitor 1 inhibits tPA and uPA, whereas α2-antiplasmin rapidly neutralizes free plasmin. Thrombin-activatable fibrinolysis inhibitor reduces the recruitment of plasminogen and tPA by removing carboxy-terminal lysine residues from the fibrin surface.

1.3 Major Stages of the Fibrinolytic Reaction

(1) Plasminogen Recruitment

Initial fibrin hydrolysis exposes additional carboxy-terminal lysine sites. Lysine-binding sites within plasminogen bind to these regions, resulting in enrichment of plasminogen on the thrombus surface.

(2) Plasmin Formation

tPA, uPA, or the streptokinase-plasminogen complex promotes the conversion of plasminogen into plasmin. Newly formed plasmin continues to cleave fibrin and generates additional plasminogen-binding sites, producing a local amplification effect.

(3) Fibrin Degradation

Plasmin cleaves the α, β, and γ chains of fibrin and progressively disrupts the thrombus network. Degradation of cross-linked fibrin produces D-dimer and related products, whereas degradation of fibrinogen and non-cross-linked fibrin generates other fibrinogen or fibrin degradation products.

(4) Reaction Termination

Free plasmin that dissociates from the fibrin surface is readily inhibited by α2-antiplasmin, whereas tPA and uPA are regulated by inhibitors such as PAI-1. This mechanism limits the spread of fibrinolysis to circulating fibrinogen and other proteins.

1.4 Dynamic Balance Between Coagulation and Fibrinolysis

Physiological hemostasis is not controlled by a simple switch between coagulation and fibrinolysis but by a dynamic temporal and spatial balance between the two systems. The coagulation system produces fibrin and stabilizes the hemostatic plug, whereas the fibrinolytic system gradually removes excess fibrin during tissue repair. Insufficient fibrinolysis can promote thrombus persistence, whereas excessive fibrinolysis can destabilize the hemostatic plug and cause bleeding.

Figure 1. Schematic diagram of the dynamic balance between coagulation and fibrinolysis

2 Plasminogen Activation and Plasmin Formation

2.1 Molecular Structure of Plasminogen

Plasminogen consists of an amino-terminal peptide, multiple kringle domains, and a carboxy-terminal serine protease domain. Lysine-binding sites within the kringle domains mediate the binding of plasminogen to fibrin, cell-surface receptors, and other proteins. Circulating plasminogen generally maintains a relatively compact conformation and can transition into a more readily activated open conformation after binding to fibrin or lysine analogs.

2.2 Proteolysis of the Activation Site

tPA and uPA cleave a specific peptide bond within plasminogen, converting the inactive single-chain precursor into plasmin with a two-chain structure. This cleavage rearranges the protease domain and forms a complete catalytic active site. The resulting plasmin contains the typical catalytic triad of a serine protease and can hydrolyze fibrin and various protein substrates.

2.3 Promotion of Plasmin Formation by Fibrin

Fibrin can simultaneously bind tPA and plasminogen, allowing the two molecules to form a catalytically favorable local complex on the thrombus surface. Fibrin binding not only increases the local substrate concentration but can also alter the conformations of plasminogen and tPA, thereby markedly accelerating plasmin formation. This mechanism is an important basis for the relative preference of tPA for activity on thrombus surfaces.

2.4 Plasmin Substrates and Inhibition

The principal physiological substrate of plasmin is fibrin, but in the absence of spatial restriction, plasmin can also degrade fibrinogen and certain coagulation factors. Binding to the fibrin surface can partially protect plasmin from inhibition by α2-antiplasmin, whereas free plasmin entering the circulation is rapidly neutralized. Detection of plasmin formation in an experiment does not necessarily indicate effective thrombolysis localized to the thrombus.

3 Nonproteolytic Activation of Plasminogen Mediated by Streptokinase

3.1 Molecular Characteristics of Streptokinase

Streptokinase (SK) is a secreted protein produced by certain streptococci and does not itself possess classical serine protease catalytic activity. Multiple structural regions of streptokinase can bind human plasminogen and form an approximately equimolar streptokinase-plasminogen complex. This complex acquires the ability to activate plasminogen.

3.2 Nonproteolytic Conformational Activation

Streptokinase first binds plasminogen and induces conformational rearrangement of the plasminogen protease domain. This process does not require prior cleavage of the plasminogen activation peptide bond and is therefore referred to as nonproteolytic activation. The conformational change forms a catalytically competent active site, enabling the streptokinase-plasminogen complex to recognize and cleave other free plasminogen molecules.

3.3 Plasmin Formation and Reaction Amplification

The activated streptokinase-plasminogen complex converts substrate plasminogen into plasmin. Newly formed plasmin further degrades fibrin and participates in the processing and amplification of streptokinase-containing complexes. Because streptokinase has relatively low dependence on fibrin, the reaction can occur not only on the thrombus surface but also continuously within the circulating fluid phase.

3.4 Fibrin Selectivity and Systemic Fibrinolysis

Streptokinase has relatively low fibrin selectivity and can cause consumption of circulating plasminogen and fibrinogen. Systemic fibrinolysis may enhance thrombolytic capacity but can also weaken the stability of hemostatic plugs at other sites. When streptokinase is used, decreases in fibrinogen, increases in fibrin degradation products, and bleeding risk should be closely monitored.

3.5 Antibodies and Immunogenicity

Streptokinase is a bacterial protein. Individuals with a history of streptococcal infection or previous streptokinase treatment may have anti-streptokinase antibodies. These antibodies can neutralize streptokinase activity, reduce efficacy, and increase the risk of allergic or infusion-related reactions. Repeated use of streptokinase generally requires comprehensive consideration of previous exposure, antibody levels, and the availability of alternative agents.

4 Catalytic Mechanisms of Urokinase and Tissue-Type Plasminogen Activator

4.1 Activation and Catalysis of Urokinase

Urokinase-type plasminogen activator (uPA) is a serine protease. The single-chain urokinase precursor can be cleaved by plasmin or other proteases to form the more active two-chain uPA. Two-chain uPA directly cleaves plasminogen and generates plasmin. Unlike streptokinase, uPA contains its own protease catalytic domain and does not need to form a nonenzymatic complex with plasminogen before acquiring catalytic activity.

4.2 uPA Localization on the Cell Surface

uPA can bind the urokinase-type plasminogen activator receptor through its amino-terminal domain, thereby localizing fibrinolytic activity to the cell surface. This mechanism participates in cell migration, tissue remodeling, and extracellular matrix degradation. In pharmacological thrombolysis, free urokinase has relatively limited fibrin selectivity, and high doses may therefore consume circulating plasminogen and fibrinogen.

4.3 Fibrin-Dependent Activation by tPA

Tissue-type plasminogen activator (tPA) is also a serine protease, but its activity is markedly enhanced by fibrin. tPA can bind fibrin through regions including its finger domain and kringle domains, while simultaneously localizing plasminogen to the thrombus surface. The local complex formed by tPA, fibrin, and plasminogen increases catalytic efficiency and allows plasmin to form preferentially within the thrombus.

4.4 Single-Chain and Two-Chain Forms of tPA

Native tPA can exist in single-chain and two-chain forms, both of which exhibit plasminogen-activating activity, although their substrate environments and sensitivities to inhibitors differ. Plasmin can cleave single-chain tPA to generate two-chain tPA, thereby contributing to local reaction amplification. After leaving the fibrin surface, tPA may still activate circulating plasminogen. Therefore, fibrin selectivity is a relative property rather than absolute thrombus specificity.

4.5 Molecular Engineering of tPA-Based Preparations

tPA-based preparations can be engineered through structural truncation, modification of glycosylation sites, or amino acid substitutions to alter half-life, fibrin affinity, and sensitivity to PAI-1. Alteplase is structurally similar to native human tPA, reteplase is a derivative lacking certain domains, and tenecteplase contains modifications at multiple sites. These preparations differ in pharmacokinetics and administration regimens, but all may cause severe bleeding.

5 Comparison of the Functional Characteristics of Streptokinase, Urokinase, and tPA-Based Preparations

5.1 Differences in Activation Mechanisms

Streptokinase acquires activation capacity by forming a complex with plasminogen and inducing a conformational change, representing an indirect activation mechanism. Urokinase and tPA are themselves serine proteases and directly cleave plasminogen. The common endpoint of all three activator classes is plasmin formation, but their initial mechanisms and spatial localization differ.

5.2 Differences in Fibrin Selectivity

Streptokinase and free urokinase have relatively low dependence on fibrin and are more likely to cause systemic fibrinolysis. The catalytic efficiency of tPA increases markedly in the presence of fibrin, giving it relatively greater local selectivity for thrombi. Engineered tPA derivatives may further alter fibrin affinity but cannot completely eliminate systemic fibrinolysis or bleeding risk.

5.3 Differences in Pharmacokinetics and Administration

Native tPA and some urokinase-related molecules are rapidly cleared from the circulation and may require continuous infusion or administration according to specific regimens. Some engineered tPA derivatives have longer half-lives and permit simplified administration. The effective duration of streptokinase is also influenced by antibody neutralization, plasminogen consumption, and the stability of protein complexes.

5.4 Immunogenicity and Repeated Use

Streptokinase is a bacterially derived protein and is markedly more immunogenic than human-derived or recombinant human plasminogen activators. Urokinase and tPA-based preparations generally do not produce the same type of antibody-related problem as streptokinase, although allergic reactions, infusion reactions, and other adverse events may still occur. Selection of a preparation should consider efficacy, previous medication history, disease type, and bleeding risk factors.

Table 1 Functional Differences Among Streptokinase, Urokinase, and tPA-Based Preparations

6 Thrombolytic Applications and Safety Evaluation of Fibrinolytic Activators

6.1 Applications in Acute Thrombotic Diseases

Fibrinolytic activators can be used for pharmacological dissolution of acute ischemic thrombi, including in selected cases of acute ischemic stroke, acute myocardial infarction, pulmonary embolism, and other thrombotic occlusive diseases. Thrombus composition, vascular location, duration of ischemia, and consequences of bleeding differ among diseases. Therefore, preparations and treatment regimens cannot be simply interchanged. Actual use should be determined according to imaging findings, time from disease onset, bleeding risk, contraindications, locally approved prescribing information, and clinical guidelines.

6.2 Systemic and Local Thrombolysis

(1) Systemic Thrombolysis

Systemic administration is relatively straightforward and allows the activator to enter the systemic circulation and act on the thrombus. Its limitation is broad drug exposure, which may increase distant bleeding and consumption of circulating fibrinogen.

(2) Catheter-Directed Thrombolysis

Catheter-directed thrombolysis delivers the drug directly near the thrombus, potentially increasing local drug concentration and reducing some systemic exposure. This method requires an interventional procedure and may still cause bleeding at the puncture site, distal embolization, and severe systemic bleeding.

(3) Device-Assisted Thrombolysis

Mechanical thrombectomy, thrombus aspiration, or other interventional procedures can be combined with pharmacological thrombolysis. Combination strategies may accelerate reperfusion, but their benefits depend on thrombus location, device availability, treatment timing, and the patient’s bleeding risk.

6.3 Mechanisms Underlying Bleeding Risk

Fibrinolytic activators can degrade pathological thrombi but may also weaken physiological hemostatic plugs. Systemic plasmin formation, decreased fibrinogen, vascular injury, and concomitant use of anticoagulant or antiplatelet drugs can all increase bleeding risk. Intracranial hemorrhage is relatively uncommon but has severe consequences and is therefore a major adverse event requiring careful assessment during thrombolytic therapy.

6.4 Safety Monitoring Indicators

Safety evaluation can incorporate clinical signs of bleeding, hemoglobin levels, platelet counts, prothrombin time, activated partial thromboplastin time, and fibrinogen levels. Increased D-dimer and fibrin degradation products may indicate enhanced fibrin degradation but cannot independently determine whether thrombolysis has succeeded or whether severe bleeding has occurred. High-risk patients also require dynamic assessment using imaging and organ-function indicators.

6.5 Antifibrinolytic and Supportive Treatment

When severe fibrinolysis-related bleeding occurs, the thrombolytic agent may be discontinued according to the specific situation, and antifibrinolytic drugs, fibrinogen replacement, blood-component support, or other hemostatic interventions may be considered. Tranexamic acid and aminocaproic acid block the lysine-binding sites of plasminogen and inhibit its binding to fibrin. Specific management should be determined according to the bleeding site, thrombolytic agent used, coagulation test results, and clinical guidelines.

7 Fibrinolytic Activity Assays and Interpretation of Experimental Results

7.1 Chromogenic Substrate Assays

Chromogenic substrate assays use artificial peptide substrates that release a chromogenic group after cleavage by plasmin or plasminogen activators, allowing enzyme activity to be calculated from absorbance changes. Direct addition of a plasmin substrate can measure plasmin activity. In contrast, assays of tPA, uPA, or streptokinase generally require plasminogen and indirectly measure activity through subsequent plasmin formation. Free plasmin, other serine proteases, or protease inhibitors in the sample may generate background signals or inhibitory effects.

7.2 Fibrin Plate Assays

In fibrin plate assays, the test sample is added to a gel containing fibrin and plasminogen, and fibrinolytic activity is evaluated by the formation of a clear lysis zone. This method visually reflects sample diffusion and fibrin-degrading capacity, but the size of the lysis zone is influenced by enzyme activity, molecular diffusion rate, sample volume, and reaction time. Plate thickness, fibrin concentration, and incubation conditions should be standardized when different samples are compared.

7.3 Clot-Lysis Curves

An in vitro fibrin clot can be constructed using fibrinogen, thrombin, plasminogen, and the activator being tested, followed by continuous monitoring of turbidity changes. Absorbance increases during clot formation and decreases after fibrinolysis begins. The onset time of lysis, half-lysis time, and maximum lysis rate can then be calculated. This method reflects initiation, amplification, and termination of fibrinolysis more effectively than a single endpoint measurement.

7.4 Detection of Fibrinogen and Degradation Products

A decrease in fibrinogen can reflect substrate consumption but may result from either effective thrombus dissolution or excessive systemic fibrinolysis. D-dimer is mainly derived from the degradation of cross-linked fibrin and indicates that fibrinolysis has occurred after clot formation. General fibrin or fibrinogen degradation products have lower specificity. Results should be interpreted according to baseline levels, dynamic changes, and thrombus burden.

7.5 Antigen Detection and Activity Measurement

ELISA can detect the antigen concentrations of tPA, uPA, PAI-1, and other proteins, but total protein concentration is not equivalent to free activity. Activators may be present as inactive precursors, inhibitor complexes, or degradation fragments. When studying fibrinolytic function, antigen concentration, enzyme activity, and inhibitor levels should be measured together whenever possible.

7.6 Inhibitors and Experimental Controls

Fibrinolytic experiments should include controls without activator, controls without plasminogen, and heat-inactivated sample controls to distinguish direct fibrin hydrolysis from plasminogen-dependent effects. Tranexamic acid, aminocaproic acid, aprotinin, or specific inhibitors can be added to verify the reaction mechanism. When streptokinase is used, the possible influence of anti-streptokinase antibodies in the sample on measured activity should also be considered.

7.7 Main Principles for Result Interpretation

Enhanced fibrinolytic activity is not equivalent to improved thrombolytic selectivity. An ideal activator should produce strong fibrinolysis in the presence of fibrin while maintaining low circulating proteolytic activity in the absence of thrombus substrate. Experimental evaluation should simultaneously compare fibrin-clot dissolution, fibrinogen consumption, plasmin formation, and sensitivity to inhibitors rather than relying on a single chromogenic substrate readout to assess thrombolytic performance.

Table 2 Common Methods for Evaluating Fibrinolytic Activity and Thrombolytic Effects

8 Fibrinolytic Activators, Regulatory Proteins, and Activity Assay Products

Table 3 Fibrinolytic Activators, Core Substrates, and Products for Functional Research

Table 4 Assay Kits for Human Fibrinolytic Activators, Inhibitors, and Related Indicators

Table 5 Products for Animal Fibrinolytic System Detection and Clot-Status Evaluation

The core of the fibrinolytic system is the conversion of plasminogen into plasmin on the thrombus surface. Streptokinase induces conformational activation to form a catalytically competent complex, urokinase directly cleaves plasminogen, and tPA uses fibrin to enhance local activation efficiency.

For more related articles, please see below:

[1] Proteinase Functional Systems and Regulatory Logic in Hemostasis, Fibrinolysis, and Vascular Homeostasis

[2] Research Methods and Applications of Fibrinolytic Proteases: Thrombolysis, Fibrin Degradation, and Anticoagulant Evaluation