Safety
Risk Assessment and safety and security measures.
Diagnostics
The diagnosis of protein S deficiency has many components including a detailed patient history, identification of symptoms, and clinical testing. Determining if a patient has the congenital form of protein S deficiency can be difficult because there are many conditions and medications that can temporarily lower protein S levels, which is also known as acquired protein S deficiency. In order to determine if an individual has a mutation in the PROS1 gene, molecular genetic testing is required and that service is only available at specialized laboratories (Lumpkins 2019). Instead, there are immunological and functional assays that are used to diagnose protein S deficiency.
Protein S and APC Coagulation Cascade
Factor IXa and VIIIa are cofactors. FXa and FVa are cofactors as well. FVIIIa activates FX into FXa, then FVa converts prothrombin (FII) into thrombin (FIIa). The natural antithrombotic process begins with thrombomodulin (TM) binding to thrombin. This complex cleaves fibrinogen (FI) to fibrin (FIa), which initiates clotting. When normal levels of Protein S are seen in plasma, protein S acts as a cofactor for Protein C to activate it, therefore inhibiting FVa and FVIIIa, which prevents the conversion of prothrombin to thrombin and fibrinogen to fibrin suppressing clot formation. When a patient's plasma is PS deficient, PS and APC can no longer inhibit FVa and FVIIIa, which means that FVa and FVIIIa are constitutively active, increasing the risk of thrombosis, shown in the diagrammatic pathway below.
Immunological Assays
- ELISA: in these assays, the wells of a microtiter plate are coated with goat or rabbit anti-human PS antibodies. The wells are then incubated with plasma, and washed, and horseradish peroxidase (HRP) that is conjugated to a second anti-PS antibody is added. Then, using a specific substrate, the amount of HRP bound antibody is measured. A calibration curve of a known concentration of protein S is compared against the sample specimen.
- Latex particle-based Agglutination Assays: in this assay, purified C4-binding protein is used (C4BP). C4BP and the latex reagent are incubated with the patient plasma sample. FPS will be adsorbed and prompt an agglutination reaction with the PS monoclonal antibody. For this assay, the amount of agglutination is proportional to the concentration of FPS contained in the patient sample.
Functional Assays
- APTT-based functional Protein S Assays, a patient plasma sample with low platelets is incubated with a plasma sample that is protein S deficient, phospholipid, a contact activator, and either activated protein C (APC) or an activator of protein C. Following incubation of about 1-4 minutes, Ca2+ is added to trigger the clotting process. The amount of time that it takes for a clot to form is measured. Based on this measurement, the PS level is established by comparing this to a reference curve that is constructed using dilutions from a plasma sample that has a known concentration of PS. The logic for this assay is that the PS and APC will inhibit factor Va and factor VIIIa. Therefore the concentration of PS will determine the clotting times.
- PT-based functional protein S assays, a plasma sample with deficient PS is mixed with the patient sample, an activator of protein C called Protac, tissue factor (TF), phospholipid and Ca2+ ions are added. The additional time it takes for a clot to form is directly proportional to the PS concentration in the patient sample (Protein S Assays).
We were able to locate the patent for Method for detecting Protein S Deficiency, in which the inventors also published a paper, New quantitative total protein S-assay system for diagnosing protein S type II deficiency clinical application of the screening system for protein S type II deficiency. The protein S Tokushima mutation (K155) seen in the Asian population, is commonly associated with protein S deficiency type II. In type II deficiency, there are normal levels of both free protein S and total protein S, but activity is decreased. These authors developed a novel assay which is able to detect total protein S activity and total protein S antigen with high accuracy. This K155 mutation is a genetic polymorphism that is found in 1.6-1.8% of healthy Japanese individuals, but in patients with VTE, the prevalence increases to 5-10%. The Tokushima mutation is described as an inconsistency between the protein S activity and the antigen levels in the plasma. The diagnosis of type II deficiency can be concluded by measuring the ratio of protein S activity to the protein S antigen level. The assays that are typically performed to detect protein S levels and activity based on coagulation timing are not sensitive enough to accurately diagnose type II deficiencies.
A recent case report was published about a case of protein S deficiency type II that presented as a cerebral venous thrombosis (CVT) in an 18 year old female. With a negative family history of VTE, the patient went to the hospital complaining of a headache for approximately five days. Other symptoms included the development of an abnormal sensation that presented on the left side of her body. No visual interruptions, seizures, loss of consciousness, fever, neck pain, or sustained trauma to the head. Following a CT scan, results showed the possibility of CVT. An MRI of the brain confirmed that the patient had a CVT of the deep cerebral veins and dural venous sinuses. The patient was immediately started on an anticoagulant regimen. Genetic testing revealed that she was negative for the FVL mutation and her functional protein C values were normal. Free protein S antigen values were normal as well. Tests revealed a decrease in the functional protein S activity. The case concluded the importance of conducting a thorough analysis of patients presenting with the same system. The creation of a more sensitive assay for the detection of type II deficiency will increase the likelihood of early detection and hopefully decrease possible CVT incidences (Agarwal et al., 2022).
Our Proposal
In a partnership with iGEM Ashei-Ghana, we designed a biosensor to directly enable detection and quantification of protein S in blood samples. The design involves engineering aptazymes that can specifically target and bind to protein S in blood samples. More details about the design of the model, specified by Ashei-Ghana, can be found in the infographic at this link:
If implemented, this biosensor could greatly improve the diagnosis of protein S deficiency.
We also worked to help streamline the diagnostic procedure for protein S deficiency. This involved establishing a comprehensive and cost-effective algorithm to help in protein S assessment. This algorithm cuts the cost of testing by more than 40%, and can be distributed to healthcare providers to implement. In order to make this guide more accessible, with the help of the Bilkent UNAM iGEM team, we also translated it into Turkish. Both versions can be seen at the links below.
References
Agarwal S, A., Santhanam, J., K, A., Degapudi, S., & K, S. (2022). A case of type 2 protein S deficiency presenting as cerebral venous thrombosis (CVT) in an 18-year-old female. Cureus, 14(8), e28221. doi:10.7759/cureus.28221
Tsuda, T., Jin, X., Tsuda, H., Ieko, M., Morishita, E., Adachi, T., & Hamasaki, N. (2012, January). New quantitative total protein S-assay system for diagnosing protein S type II deficiency clinical application of the screening system for protein S type II deficiency. Retrieved August 5, 2022, from Lww.com website: https://journals.lww.com/bloodcoagulation/Fulltext/2012/01000/New_quantitative_total_protein_S_assay_system_for.9.aspx
Lumpkins, C. (2019, April 10). Protein S deficiency - NORD (national organization for Rare disorders). Retrieved August 4, 2022, from NORD (National Organization for Rare Disorders) website: https://rarediseases.org/rare-diseases/protein-s-deficiency/
Protein S Assays. (n.d.). Retrieved August 4, 2022, from Practical-haemostasis.com website: https://practical-haemostasis.com/Thromobophilia/ps_assays.html
