Method of determination

Automatic analyzer of the parameters of the coagulation system ACL TOP, method - kinetic colorimetric.

Material under study Plasma (citrate)

One of the most important natural clotting inhibitors.

Protein C is one of the most important physiological clotting inhibitors. In its active form, it cleaves and inactivates coagulation factors VIIIa and Va (but not factor V Leiden). Protein C exhibits anticoagulant activity, indirectly activates fibrinolysis, limits the size of the thrombus. In vivo, protein C is activated by thrombin, repeatedly accelerated by a complex of thrombin and thrombomodulin (a protein on the surface of endothelial cells).

The anticoagulant activity of protein C is enhanced by its cofactor - . Protein C is synthesized in the liver and is a vitamin K-dependent protein, so its activity also depends on vitamin K deficiency and oral anticoagulant therapy. The level of protein C in newborns and young children is physiologically lower than in adults due to the immaturity of the liver. Congenital deficiency of protein C is associated with a tendency to severe thrombotic disorders. Among congenital types of physiological anticoagulant deficiency, such as antithrombin III deficiency, protein C deficiency, protein S deficiency, protein C deficiency is the most common (0.2-0.4% of the population). Homozygous states present in early childhood with fulminant purpura of newborns and are often fatal, with undetectable levels of protein C in these newborns.

Patients with protein C deficiency are usually heterozygous, in which thrombosis does not appear before the second or third decade of life. Among them, about 5% may also have a factor V mutation (factor V Leiden) in a heterozygous state. The presence of this mutation is considered a risk factor for the development of early thrombotic pathology (see genetic studies, thrombophilia, test no. 7171). Protein C deficiency is associated with an increased risk of pregnancy complications (deep vein thrombosis, preeclampsia, intrauterine growth retardation, and recurrent miscarriages). There is an increased risk of developing warfarin-induced skin necrosis. The effect of risk factors associated with bad habits is aggravated.

Congenital deficiency conditions can be diagnosed when the causes of acquired protein C deficiency have been excluded. The study of protein C for this purpose is not recommended during acute illness / acute thrombotic episodes, due to the consumption of protein C, as well as in patients receiving oral anticoagulant therapy (warfarin lowers protein C levels).

Re-testing of protein C is recommended after cessation of oral coagulant therapy (preferably one month after the end of therapy), in correlation with the examination of family members. In heterozygotes for protein C deficiency, the values ​​partially overlap with the normal reference range. Violation of protein C activation occurs in pathological conditions associated with the presence of factors such as hypoxia, endotoxin, interleukin-1, tumor necrosis factor alpha, high levels of homocysteine ​​(all of which accelerate clotting by inducing tissue factor expression and suppressing transcription of thrombomodulin by endothelial cells).

The informative value of protein C testing for prognostic purposes in septic conditions (characterized by increased consumption, destruction and impaired synthesis of protein C) has been shown. Protein C activity level< 40%, а также снижение более чем на 10% за 1 день при сепсисе коррелирует с неблагоприятным прогнозом.

Literature

Shorr A.F. R92 Protein C concentrations in severe sepsis: an early directional change in plasma levels predicts outcome Critical Care 2006,10: R92 http://ccforum.com/content/10/3/R9.

Methodological materials of the manufacturer of reagents.

The gene responsible for the synthesis of protein C is located on chromosome 2 (ql3-ql4). The main function of this physiological anticoagulant is to inactivate the main non-enzymatic coagulation factors (FVa, FVIlla).

Patients with congenital protein C deficiency are prone to recurrent thrombosis and thromboembolism. Protein C deficiency is inherited autosomal, homozygotes and double heterozygotes die in early childhood from thrombosis.

Method principle

The method is based on the assessment of the APTT mixture (diluted PTP sample, protein C deficient plasma, protein C activator, APTT reagent). Protein C of the studied PRP sample is activated with a reagent obtained from the venom of the muzzle Agkistrodon contortrix. Activated protein C destroys the coagulation factors Va and Villa contained in the mixture of the test sample and the added protein-C-deficient plasma, due to which, after the addition of calcium chloride, an increase in APTT is recorded. With a low activity of protein C, the prolongation of APTT is slightly expressed. Dilutions of the BTP calibration sample allow you to plot a curve and determine the activity of protein C.

Reagents and equipment

• Protein C activator (Protac).
• APTT reagent.
• Protein C deficient plasma.
• buffer solution.
• PRP sample with known protein C activity.
• Coagulometer.

Blood samples for research

PRP is used to determine the activity of protein C.

To construct a calibration curve, a PRP sample with known protein C activity is required. To construct a calibration curve, the clotting time in seconds obtained from the study of diluted calibration samples with known protein C activity is used.

The clinical picture in congenital protein C deficiency is dominated by relapses of venous thrombosis and thromboembolism. A number of patients experience skin necrosis, miscarriage, etc. In newborns with a deficiency of protein C, malignant purpura (purpura fulminans) is often observed.

Acquired deficiency of protein C may be due to insufficient synthesis by hepatocytes, increased consumption due to DIC, treatment with indirect anticoagulants, etc. In some patients with VA, an overestimation of its activity is observed.

Causes of errors

• Errors of the preanalytical stage of the study.
• Ingress of heparin into the test blood from a venous catheter.

Other analytical technologies

The functional activity of protein C is determined by amidolytic or coagulation methods.

ELISA is used to determine the concentration of protein C, however, when comparing the results of immunological and functional methods, there is a discrepancy between the results in patients with molecular abnormalities of protein C.

Protein S activity

Protein S is a vitamin K-dependent glycoprotein involved as a non-enzymatic cofactor of activated protein C in the proteolytic degradation of coagulation factors Va and Villa. The gene responsible for the synthesis of protein S is located on human chromosome 3 at position pll.l-qll.2. In the blood, protein S is present in two versions: in the form of a free protein (about 40%) and in the form of a complement associated with the C4b component (about 60%). A decrease in protein S activity increases the risk of thrombosis and thromboembolism.

Method principle

Functional methods are based on taking into account the severity of the prolongation of the clotting time of a mixture of protein S-deficient plasma and test plasma when activated protein C is introduced into the test system. To assess the clotting time in such a test system, manufacturers use different coagulation stimulants (Russell's viper venom, APTT reagent , activated coagulation factor IX or others). With a normal content of protein S under the influence of activated protein C, a significant lengthening of the clotting time occurs (due to the destruction of non-enzymatic coagulation factors), while with a deficiency of protein S, it is much less pronounced (due to the inefficient destruction of non-enzymatic factors by activated protein C).

Reagents and equipment

• Protein S-deficient plasma.
• Activating reagent (phospholipids, Russell's viper venom or other activators, heparin neutralizer, etc.)
• Activated protein C.
• Calcium chloride solution (0.025 M).
• buffer solution.
• PRP sample with known protein S content.
• Coagulometer.

Blood Samples for Research PRP is used to determine protein S activity.

Method of determination

The course of determining the activity of protein S varies significantly when using reagents and equipment from different manufacturers, so the sequence of actions of a laboratory doctor must strictly comply with the instructions for the reagent kit and its adaptation to the coagulometer available in the laboratory.

Evaluation of the results of the study

The study of the coagulation time of diluted BTP samples with a known concentration of protein S allows you to build a calibration curve and determine the activity of this physiological anticoagulant as a percentage of the norm.

In healthy people, protein S activity is in the range of 60-130%.

Interpretation of study results

Congenital protein S deficiency is a rare defect in the anticoagulant link of hemostasis. The first description of protein S deficiency was presented in 1984 by H.R. Schwarz at al. The clinical picture of this disease is dominated by recurrent phlebothrombosis and thromboembolism. Like many other defects in the anticoagulant link of hemostasis, this pathology is transmitted autosomal. It is customary to distinguish three types of congenital protein S deficiency.

Variants of Congenital Protein S Deficiency

• Type: I Coalition method: Reduced; Free protein S: Decreased; Total protein S: Decreased.
• Type: II Coalition method: Reduced; Free protein S: Normal; Total protein S: Normal.
• Type: III Coalition method: Reduced; Free protein S: Decreased; Total protein S: Normal.

Type I is characterized by a low content of protein S using different variants of its immunological determination, as well as a decrease in its functional activity. In type II deficiency, a decrease in functional activity is observed, however, the total and free fractions of protein S are not impaired. Type III is manifested by a combined decrease in the functional activity of protein S and its free fraction. Thus, to identify the type of protein S deficiency, it is necessary to use coagulation and immunological methods for its determination.

Acquired protein S deficiency is much more common in clinical practice. A decrease in protein S activity can be detected with nephrotic syndrome, pregnancy, treatment with estrogen, L-asparaginase, etc. Newborns with protein S deficiency have malignant purpura (purpura fulminans).

Causes of errors

• Heparin from a venous catheter.
• Hemolysis in the test blood sample.
• Incorrect dosage of citrate when taking blood.

Other Analytical Techniques A fairly common method is based on the use of ELISA for the determination of free protein S and associated with the C4b component of complement. In addition, functional methods for the determination of this anticoagulant based on the use of chromogenic substrates have been described (but not marketed).

Protein C is a protein synthesized in the liver, which is coagulation factor XIV, one of the most important anticoagulant factors that allow blood to be in a liquid state. But in order for this factor to begin to act, it is necessary to activate it - this task is performed by vitamin K.

A deficiency of protein C in the blood can lead to an increased risk of developing blood clots. However, it must be said that this pathology is quite rare. In addition to preventing blood clotting, protein C promotes the natural death of spent body cells and is an agent that relieves and prevents inflammation.

The norm of protein C in the blood. Result interpretation (table)

A blood test for protein C is performed in conjunction with an analysis for its counterpart, protein S, and often in conjunction with other blood clotting tests. It is prescribed if the patient has an unexplained tendency to form blood clots, or if thrombosis is a genetically inherited disease in this family. Moreover, such an analysis is necessary if blood clots form in a patient younger than 50 years old or in some non-traditional place, for example, in the hepatic vein or in the vessels of the brain. The level of protein C should be checked if the patient has a habitual miscarriage or if someone in the family suffered from a deficiency of this protein. In addition, a blood test for protein C helps to quickly identify liver disease and vitamin K deficiency in the body.

Blood is taken from a vein.

The norm of protein C in the blood of ordinary people and pregnant women.

If protein C is elevated, what does it mean?

As a rule, an increase in the level of protein C in the patient's blood is not of clinical interest and is not used in diagnosis. However, you need to understand that high levels of protein C can lead to difficult-to-stop bleeding.

If protein C is low, what does this mean?

Protein C deficiency in the body can be hereditary, acquired, or develop as a result of some other factors. In any case, a decrease in the level of protein C leads to pathologies that increase the risk of developing abnormal blood clots.

With a moderate protein C deficiency, the patient is at risk of developing deep vein thrombosis. These clots form in veins located far from the surface of the skin and are extremely dangerous because they can lead to pulmonary embolism. Although, in most cases, pathological blood clots do not occur, some factors can significantly increase this threat. For example, age or surgery, pregnancy or a sedentary lifestyle.

During pregnancy, the rate of protein C in the blood can decrease and protein deficiency can lead to an increased risk of thrombosis, which persists after the birth of the child. The fact is that pregnancy itself is a risk factor for the development of thrombosis. In addition, there is an opinion that a reduced level of protein C in the blood of an expectant mother may increase the risk of miscarriage, both in early and late pregnancy.

Severe deficiency of protein C can lead to an extremely life-threatening phenomenon in newborns - fulminant purpura. This is the name of a pathology in which multiple blood clots form in small blood vessels located throughout the body. These clots lead to blockage of normal blood flow, and this leads to tissue necrosis and death of the child. But even in those children who managed to survive, lightning purpura can recur in the future at any time.

Protein C deficiency can be genetically transmitted. That is, if your family has already had cases of such a deficiency, you are more likely to develop this pathology. However, such a deficiency can also occur without a link to genetics. For example, in the cases:

• deficiency of vitamin K in the body,
• severe liver disease:
• kidney disease,
• severe infectious diseases
• DIC,
• cancer and the spread of metastases.

The use of certain blood-thinning drugs, such as warfarin, can lead to a decrease in the level of protein C in the body.

When registering a woman and after the 30th week of pregnancy, it is very important to monitor the main indicators of hemostasis. The change in these indicators during pregnancy is of a compensatory-adaptive nature and is aimed at the formation of a normal feto-placental complex and at reducing blood loss during childbirth. Let's list these changes:

during pregnancy, there is an increase in coagulant potential (total activity of coagulation factors) - the level of almost all coagulation factors increases (except XI and XIII);
during pregnancy, the level of fibrinogen significantly increases and at the end of a normal pregnancy, it increases by at least two times compared with the non-pregnant state.

in increasing the overall coagulant potential;
in increasing the functional activity of platelets with a slight decrease in their number;
in a decrease in fibrinolytic activity with an increase in fibrin degradation products (PDF);
in a decrease in the activity of antithrombin-III (AT-III) with a slight decrease in its content.

To date, it is possible to determine a fairly wide range of hemostasis indicators of both tissue and plasma levels. Of course, it is not rational to carry out the determination of all indicators, both from a clinical and from an economic point of view. A number of authors recommend a phased clinical diagnostic algorithm for detecting thrombophilic conditions in pregnant women:

at the first - screening - stage, basic information about the external and internal pathways of blood coagulation can be obtained using only two tests: prothrombin time - PT (expressed as a percentage of Quick activity and / or as MHO) and activated partial thromboplastin time - APTT (expressed in seconds );
with an increase in PT and / or APTT, the next step is to diagnose antiphospholipid syndrome (APS) [ read about APS and its diagnosis];
in the absence of APS, but with an increase in PT and / or APTT values, it is necessary to proceed to the next stage - the diagnosis of genetically determined thrombophilia.

Currently, six main genetically determined forms of thrombophilia are known and well studied:

resistance to activated protein C or factor V mutation (Leiden mutation);
hyperhomocysteinemia;
deficiency or violation of the structure of AT-III;
deficiency or violation of the structure of protein C;
deficiency or violation of the structure of protein S;
mutation of the prothrombin G 20210 A gene;
to this list we can add an increase in the activity of factor VIII, which may also be a consequence of a hereditary disease, but, of course, most often a significant increase in the activity of this indicator is observed during pregnancy.

Deficiency of antithrombin III (AT-III). AT-III is a natural anticoagulant. It accounts for 75 - 80% of the total coagulant potential of the blood. Synthesized in the liver and endothelial cells. The frequency of occurrence in patients with thrombophilia is 5%. A decrease in AT-III activity of less than 60% may indicate its hereditary deficiency, however, in the diagnosis, it is necessary to exclude liver diseases, since violations of its function, along with septic diseases and acute thrombosis, are the main causes of acquired and temporary deficiency. Also, when diagnosing AT-III, it is important to use a chromogenic method based on the ability of plasma to inactivate factor X(a).

Protein C deficiency. Protein C (PC), a natural anticoagulant, vitamin K-dependent glycoprotein, is synthesized in the liver in an inactive form. The frequency of occurrence in patients with thrombophilia is 4%. PC passes into the active form (APC) by interacting with the thrombin-thrombomodulin complex. PC deficiency leads to a decrease in the concentration of aPC, which leads to a slowdown in the inactivation of factors Va and VIII(a). There are two types of deficiency states: type I - a quantitative deficiency of protein C (reduced synthesis or reduced protein lifetime) and type II - a violation of the protein structure. Described violations of the protein structure, leading to disruption of interaction with phospholipids, thrombomodulin, factors V/VIII and other substances. Protein C can be determined by various methods: chromogenic, clotting, and immunochemical. Optimal results for clinical interpretation are obtained using the chromogenic method.

Protein S deficiency. Protein S (PS) is a non-enzymatic cofactor of protein C, is involved in the inactivation of factors V and VIII, and has its own anticoagulant activity, independent of protein C. PS is a vitamin K-dependent glycoprotein synthesized in the liver. It exists in two forms: free protein S and associated with C4 (compliment protein). Normally, 60 - 70% is in a bound form. The level of PS binding to C4 determines its activity, tk. only freeform is active. Normally, the level of PS 80 - 120%, during pregnancy, its level drops and is 60 - 80%. The frequency of occurrence of hereditary deficiency in patients with thrombosis is 4%. The most accurate determination of the concentration of free PS is carried out
immunological method.

Mutation of the prothrombin gene G 20210 A. As a result of this mutation, the synthesis of prothrombin increases. The frequency of occurrence among patients with thrombophilia is 1%. Diagnosis - molecular genetic method.

Hyperhomocysteinemia (HHC) is determined in patients with thrombophilia with a frequency of 13 - 27%. HHC may be due to an enzyme defect or a deficiency in folic acid and vitamins B12 and B6. The molecular mechanisms that cause thrombogenic effects have not yet been identified. Determination of homocysteine ​​is carried out by ELISA (enzymatic immunoassay).

Increased activity of factor VIII. A persistent increase in factor VIII activity of more than 150% without a simultaneous increase in the concentration of C-reactive protein as an indicator of the acute phase reaction is observed in approximately 20% of patients with thrombophilia. The hereditary cause of increased plasma levels of factor VIII has not yet been identified. Determination of the activity of factor VIII is most appropriate to carry out using the chromogenic method.