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A simplified functional assay for protein C in plasma samples
Insights
A new functional protein C (PC) assay accurately identifies PC deficiency, crucial for managing thromboembolic events. This simplified test uses readily available reagents and is suitable for routine clinical use.
Area of Science:
- Biochemistry
- Hematology
- Clinical Diagnostics
Background:
- Protein C (PC) plays a vital role in regulating hemostasis.
- PC deficiency is linked to severe thromboembolic events, necessitating reliable diagnostic methods.
- Existing diagnostic approaches may lack efficiency or require complex procedures.
Purpose of the Study:
- To develop and validate a novel, efficient functional assay for assessing protein C activity in plasma.
- To establish a reliable method for early identification of protein C deficiency.
- To simplify the diagnostic process for protein C-related coagulation disorders.
Main Methods:
- A new assay utilizing the thrombin/thrombomodulin complex for rapid activated PC generation.
- Employing a chromogenic substrate to quantify the proteolytic capacity of activated PC.
- Using rabbit lung homogenate as a substitute for purified thrombomodulin.
Main Results:
- The assay demonstrated specificity and accuracy, unaffected by common proteases like plasmin, streptokinase, and urokinase.
- Heparin's inhibitory effect on PC activation was effectively neutralized by protamine sulfate.
- Unselected patients (n=50) showed normal PC activity (100 +/- 12%), while phenprocoumon-treated patients (n=103) exhibited reduced PC activity (40 +/- 11%) and antigen levels (43 +/- 10%).
Conclusions:
- The developed functional protein C assay is specific, sensitive, and time-efficient.
- This assay simplifies testing procedures without compromising accuracy, making it suitable for routine diagnostics.
- The assay aids in the early and reliable identification of protein C deficiency and monitoring of anticoagulant therapy.
Abstract:
The important role of protein C (PC) in the regulation of hemostasis has been appreciated since the description of patients who were deficient in PC and presented with severe thromboembolic events. The potentially fatal complications associated with PC-deficiency require an early and reliable identification of those patients affected with this inherited disorder. The present study introduces a test procedure for the functional assessment of PC in plasma samples. The test utilizes the thrombin/thrombomodulin complex to achieve complete and rapid formation of activated PC whose proteolytic capacity is subsequently determined with a chromogenic substrate. Homogenate obtained from rabbit lung effectively substituted the purified component thrombomodulin in the assay system. This new approach simplifies the test procedure without losing specificity and accuracy. Proteases, such as plasmin, streptokinase and urokinase did not influence the assay and the inhibitory effect of heparin on the PC-activation could easily be overcome by the addition of protamine sulphate. The PC-activity in a group of unselected patients (n = 50), who did not reveal any abnormalities in global coagulation tests, amounted to 100 +/- 12% (mean +/- SD) with a range from 54 to 143% when analyzed in comparison to a plasma pool constituted from healthy volunteers. Since the synthesis of PC depends on the availability of vitamin K, patients receiving phenprocoumon have also been analyzed. These patients (n = 103) presented 40 +/- 11% residual PC-activity accompanied by a concomitant decrease in PC-antigen levels to 43 +/- 10% (mean +/- SD). The test described is specific, sensitive, less time-consuming and can be performed on a routine basis.