RU2234857C1 - Method for quantitative estimation of effectiveness of shift regulation in platelet function - Google Patents

Abstract

FIELD: medicine, clinical laboratory diagnosis.

SUBSTANCE: method involves carrying out an average size of aggregates and number of objects adhesized on rigid backing and adhesion and aggregation of platelets are assayed in blood sample taken from one patient with low (200 c^-1) and high (1300 c^-1) rates of shift and index of shift regulation of aggregation is calculated as the ratio of value of an average size of aggregates at high shift rate to its value at low shift rate, and index of shift regulation of adhesion is calculated as the ratio of an average number of adhesized objects at high shift rate to value of this index at low shift rate. Method provides accelerating and simplicity in carrying out of investigation and quantitative estimation of effectiveness of shift regulation in platelets function.

EFFECT: improved method for estimation.

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Description

The invention relates to medicine, in particular to clinical laboratory diagnostics, and can be used in various fields of experimental and clinical medicine to assess the state of platelets in various forms of pathology and the effects of various physical and medical factors. The proposed method allows to evaluate the sensitivity of platelets to a changing shear rate, provided by the viscous interaction of elements of a moving medium.

Evaluation of platelet function in various pathological conditions is an important link in the diagnosis and serves as the basis for the development of effective methods of pathogenetic therapy of many diseases. It is known that one of the regulatory circuits that provide the optimal adhesive and aggregation function of platelets is the so-called “shear regulation”, which is carried out due to the emergence of a membrane cell signal with a change in the rate of displacement of viscous elements of the medium during the movement of uniform elements in the blood stream [1, 3, 5]. Due to the local features of vasoarchitectonics, differences in the diameter of blood vessels and the value of perfusion pressure, real fluctuations in the blood flow velocity occur in different parts of the vascular bed and, therefore, different values of the shear rate occur. An important factor in the perception and implementation of a shear signal is the state of the blood platelets themselves [3, 5, 6].

Under conditions of pathology, for example, with the development of the atherosclerotic process, the rheological properties of blood change, laminar disturbances of blood flow occur due to the formation of plaques in the lumen of the vessel, and the velocity regimes of the blood flow change, which naturally affects the nature of the shift regulation of the function of the blood platelets [4, 7]. In this case, the state of the platelets themselves changes, which leads to a change in the efficiency of conjugation of the membrane shear signal and the final cellular response in the form of adhesion and aggregation.

If the fact of the presence and significance of shear regulation of platelet function is not currently in doubt, then the literature does not describe methods that allow a quantitative assessment of the state of shear regulation in normal and pathological conditions.

We are the first to propose a method for quantifying the effectiveness of shear regulation of platelet function, including determining the average size of aggregates and the number of objects adhered to a rigid substrate in a blood sample of one patient at low (200 s ^-1 ) and high (1300 s ^-1 ) shear rates and calculation of the index of shear regulation of aggregation (ISRagr) as the ratio of the average aggregate size at high shear rate to its value at low shear rate, and the index of shear regulation of adhesion (ISRag) as the ratio of he number of adhered objects at a high shear rate to the value of the indicator at low shear rate. The method is implemented in vitro and is as follows.

A sample of citrated blood is taken from a patient by a standard technique (blood-citrate ratio 9: 1). Blood is maintained at room temperature for 40 minutes and is divided into 2 aliquots (for testing at low and high shear rates). Then, a study is made of the behavior of platelets on a rigid substrate - extracellular matrix (ECM), which is a complex of products produced by bovine corneal epithelium and containing various types of collagen, fibrinogen, fibronectin, vitronectin, von Willebrand factor, thrombospondin, laminin and glycoproteins [2] on polystyrene. Standard 4-cell polystyrene plates are used with ECM applied to the bottom of cells, or cells without ECM. 200 μl of whole citrated blood are placed in the well. Studies are performed using the Cone and Plate Analyzer (CPA) instrument, which allows the study of platelet behavior at various shear rates. The device uses the principle of rotational viscometry. A cylinder with an end conical configuration is lowered into the cells with biomaterial, due to which a gap is formed between it and the bottom of the cell. The rotation of the cylinder at a given speed creates the necessary shear rate parameters. Two shear rates are used: low - 200 s ^-1 (which is the subthreshold for triggering shear activation mechanisms) and high - 1300 s ^-1 (activating platelet functions). The study on the device of each aliquot takes 2 minutes.

Then the cells with settled platelets for 2 min are filled with dye May-Grunwald. Then the paint is sucked off, the preparation dries in air and is analyzed on an image analyzer. In this case, the following parameters are calculated - the number of adhered objects (an indicator characterizing the adhesive ability of platelets) and the average size of aggregates (an indicator characterizing the aggregation ability of platelets).

Since, in the presence of shear rate, an additional factor stimulating blood platelets is included, differences in recorded parameters at low and high shear rates will reflect the effectiveness (severity) of shear regulation in stimulating platelet function. For its quantitative expression, we propose for each indicator to calculate the shear regulation index (SRI), which is the ratio of the value of the studied indicator at a high shear rate to its value at a low shear rate. So, the index of shear regulation of platelet aggregation (ISRagr) is calculated by the formula

ISRagr = CPAH / CPA

where CPA is the average size of aggregates at a high shear rate, CPA is the average size of aggregates at a low shear rate.

The platelet adhesion shift index (ISDadg) is calculated using the formula

ISRadg = KAOvys / KAOizn,

where KAOysh is the number of adhesive objects at a high shear rate, KAOysh is the number of adhesive objects at a low shear rate.

Platelet activation at a high shear rate depends on the reactivity of the blood platelets themselves, as well as on the content of von Willebrand factor in the blood plasma and ECM [3, 5]. However, in the proposed method, the concentration of von Willebrand factor in the two compared blood samples, as well as on an ECM, is constant and, therefore, the change in SRI reflects a change in the reactivity of blood plates in terms of their sensitivity to shear regulation (activation).

To verify this method, we conducted studies of the shear regulation of platelet function in a group of healthy male donors (n = 15). The age of the subjects is from 18 to 34 years. The research results are presented in the table.

Example. Donor M., 28 years old. 5 ml of citrated blood were obtained from the ulnar vein. After 40 minutes at room temperature, 200 μl of blood was placed in an ECM cell, which was installed in a Cone and Plate Analyzer and the study was conducted at a low (200 s ^-1 ) shear rate. Next, the drug is painted with May-Grunwald paint for 2 minutes. Then, the next portion (200 μl) of blood was placed in another ECM cell and its study was conducted at a high (1300 s ^-1 ) shear rate. The drug is painted May-Grunwald 2 min. After drying, both drugs were analyzed on an image analyzer. The number of adhesive objects (CAO) and the average size of adhesive objects (SRO) were calculated. The following results were obtained:

KAOish = 6412; KAOniz = 4287;

SRW = 42.8 μm ² ; SROS = 22.9 μm ² ;

Given these indicators, the calculated ISRadg and ISRagr:

ISRadg = KAOv / KAOiz = 6412: 4287 = 1.50

ISRagr = SROvys / SROiz = 42.8: 22.9 = 1.87

This test, due to its simplicity and speed of execution, can be an important additional criterion in assessing the functional state of platelets in various diseases. In addition, it can be used to study the effect of various pharmacological preparations and physical factors on the state of shear regulation of platelet function. With an increase in SRI, a conclusion is made about strengthening (increasing the efficiency) of shear regulation, which is the basis for the use of agents that inhibit platelet function. A decrease in ISI indicates hyporeactivity (lower regulation efficiency) of blood platelets, which requires the use of drugs that activate their function.

Sources of information

1. Levtov V.A., Regirer S.A., Shadrina N.Kh. Blood rheology. M., Medicine, 1982.

2. Gospodarowich D., Vlodavsky I., Savion N. The extracellular matrix and the control of proliferation of vascular endothelial and smooth muscle cells // J. Supermol. Struct. - 1980. - Vol.13. - P.339-372.

3. Kroll M.H., Hellums J.D., McIntire L.V., Schafer A.I., Moake J.L. Platelets and shear stress // J. Amer. Soc. Hematol. - 1996. - Vol. 88. - N.5. - P. 1525-1541.

4. Ross J.M., McIntire L.V. Molecular mechanisms of mural thrombosis under dynamic flow conditions // News in Physiological Sciences. - 1995 .-- Vol.10. - P.117-122.

5. Ruggeri Z.M. Mechanisms of shear-induced platelet adhesion and aggregation // Thrombosis and Haemostasis. - 1993. - Vol.70, N1. - P.119-123.

6. Shenkman B., Linder N., Savion N., Tamarin I., Dardik R., Kennet G., German B., Varon D. Increased neonatal platelet deposition on subendothelium under flow conditions: the role of plasma von Willebrand factor // Pediatric Research. - 1999. - Vol. 45, N 2. - P.270-275.

7. Slack S.M., Cui Y., Turitto V.T. The effect of flow on blood coagulation and thrombosis // Thrombosis and Haemostasis. - 1993. - Vol.70, N1. - P.129-134.

Claims (1)

A method for quantifying the effectiveness of shear regulation of platelet function, including determining the average size of the aggregates and the number of objects adhered to a rigid substrate, characterized in that the adhesion and aggregation of platelets is determined in a blood sample of one patient at low (200 s ^-1 ) and high (1300 s ^-1) shear rates and shear index calculated aggregation regulation (ISRagr) the ratio of the average size of the aggregates at a high shear rate to its value at low shear rate and shear index p gulyatsii adhesion (ISRadg) as the ratio of the average number of adhered objects at a high shear rate to the value of the indicator at low shear rate.