The Application of Coagulation reagent substrate in IVD reagents

Product Name：Coagulation reagent substrate
Article No.： 10002603

In clinical coagulation function testing, the accuracy of coagulation reagents is directly related to the accuracy of disease diagnosis. As the core functional component of reagents, substrates are key carriers for initiating coagulation reactions and achieving signal conversion, and their performance determines the reliability of test results.

Coagulation reagent substrates refer to biological or chemically active substances that specifically interact with the coagulation factors to be tested or related enzymes in the coagulation reaction system, thereby triggering detectable signals such as color development and coagulation. Their core role is to provide specific targets, convert the activity of coagulation factors into quantifiable indicators, and achieve accurate judgment of abnormal coagulation function. Therefore, they need to have extremely high specificity and reaction activity to meet the stringent requirements of clinical diagnosis.

According to the detection items, types of reaction signals and chemical properties, coagulation reagent substrates can be divided into three categories, each with a clear specificity.

1，Chromogenic substrates：Chromogenic substrates are widely used in chromogenic substrate assays. They are mostly synthetic peptides containing specific recognition sequences for coagulation-related enzymes, with chromogenic groups such as p-nitroaniline (pNA) attached to their ends. After specific hydrolysis by the target enzyme, the chromogenic groups are released. The enzyme activity is quantified through changes in absorbance, and the level of coagulation factors is derived. Common examples include S-2238 (for thrombin detection) and S-2765 (for factor Xa detection), which ensure detection accuracy by virtue of their specific recognition sequences.

2，Coagulation substrates ：Coagulation substrates are used in coagulation assays. Their core function is to simulate the in vivo fibrinogen conversion process, and coagulation function is determined by the coagulation time. The most common one is fibrinogen (the final substrate in the coagulation cascade, which can be converted into fibrin clots by thrombin). In addition, auxiliary substrates such as cephalin and tissue thromboplastin are added to the reagents to initiate specific coagulation pathways. For example, tissue thromboplastin in PT assay reagents is the auxiliary substrate that initiates the extrinsic pathway.

3，Fluorescent substrates ：Fluorescent substrates are peptides with fluorescent groups such as 7-amino-4-methylcoumarin (AMC) attached to their ends. They are used in the fluorescent substrate method for detection. The mechanism of action is similar to that of chromogenic substrates: fluorescent groups are released through enzymatic hydrolysis, and the fluorescence intensity is measured to quantify enzyme activity. They have higher sensitivity and are suitable for the detection of low-concentration coagulation factors or precise scientific research analysis. However, due to the high cost of detection equipment, they are less commonly used in routine clinical applications.

Mechanism of action of coagulation substrates

The coagulation process is a complex cascade reaction (coagulation cascade). Substrates achieve the detection of specific coagulation factors through precise matching with different links. The typical clinical application mechanisms are as follows:

1.     Extrinsic pathway detection (such as PT): Tissue thromboplastin (cofactor substrate) activates factor VII to initiate the pathway, ultimately activating thrombin; thrombin acts on fibrinogen to form a clot, and the extrinsic coagulation function is judged by the clot formation time.

2. Endogenous pathway detection (such as APTT): Activators like kaolin activate factor XII to initiate the pathway, and the cascade reaction activates thrombin, which ultimately acts on fibrinogen to form a clot. The activity of endogenous coagulation factors is reflected through the clotting time.

3. Specific thrombin detection: Chromogenic substrates such as S-2238 are specifically hydrolyzed by thrombin to release pNA. The change in absorbance is positively correlated with thrombin activity, which can quantify its level and is used for monitoring the efficacy of anticoagulant drugs such as heparin.

In short, through the logic of "specific recognition - reaction activation - signal release", the substrate converts the biological activity of coagulation factors into quantifiable signals, serving as a key bridge connecting coagulation reactions and clinical detection.

Coagulation testing is crucial for clinical diagnosis and treatment decisions, and substrates must meet the following stringent performance requirements.

1, Extremely high specificity: It must specifically react only with the target coagulation factor/enzyme and avoid non-specific reactions with other components or impurities that could lead to result deviations. For example, the factor Xa detection substrate S-2765 must accurately recognize its active center and not cross-react with thrombin, etc.

2, Excellent stability: It directly determines the shelf life of the reagent and the repeatability of the detection. It needs to maintain stable activity for a long time under standard conditions such as refrigeration at 2-8°C, and have stable performance in reaction systems with different pH values, temperatures, etc., to ensure the consistency of results.

3, Precise reactivity: It needs to meet clinical testing requirements, being able to both capture normal coagulation factor activity and identify minor abnormal changes such as mild coagulation dysfunction, so as to avoid narrowing the detection range caused by excessively strong or weak reaction activity.

4, Extremely low impurity content: Impurities may interfere with reactions or affect signals. It is necessary to use multiple purification processes to achieve sufficient purity, ensuring that the impurity content is below the detection threshold to avoid interfering with the accuracy of the results.

Clinical application value of coagulation substrates: assisting in precise diagnosis and treatment of coagulation-related diseases

1. Diagnosis of coagulation dysfunction: Detect the activity of coagulation factors (such as factors Ⅷ and Ⅸ in hemophilia patients) to clarify the type and severity of the disease.

2. Monitoring of anticoagulant drugs: Detection of indicators such as PT and APTT, evaluation of the efficacy of drugs such as heparin and warfarin, adjustment of dosages to avoid the risk of bleeding/thrombosis.

3. Preoperative screening: Assess the patient's coagulation function and conduct early intervention to reduce the risk of surgical bleeding.

4. Thrombosis early warning: Detect the activity of coagulation factors and the level of fibrinogen, providing early warning for high-risk groups such as those at risk of deep vein thrombosis.

Substrates are the core foundation for accurate detection of coagulation function. They are characterized by clear classification, mechanisms that align with the coagulation cascade, and must meet stringent requirements such as high specificity and high stability. With the increasing demand for precision in clinical testing, the research, development, and production of substrates are moving towards greater efficiency, specificity, and stability. In the future, relying on advanced molecular design and purification processes, substrates will further enhance reagent performance, providing stronger support for the precise diagnosis and treatment of coagulation-related diseases.