The Effect of Ceruloplasmin on the Functional State of Platelets under Experimental Hemostasis Disorders

The dissertation is devoted to the study of the effect of ceruloplasmin — an acute-phase protein — on the number and functional state of platelets under experimental disorders of hemostasis. The research was conducted using materials from the intact organism and experimental models of exogenous and endogenous thrombocytemias, acute and chronic blood loss. In the work, the adhesiveness, aggregatability of platelets, the release reaction, and the intensity of free radical oxidation processes in platelets and plasma were comprehensively evaluated. It is demonstrated that ceruloplasmin exerts a dual effect on platelets depending on the duration of contact and the dose of the preparation, and its action is mediated through a change in the activity of free radical oxidation processes. The obtained data expand the understanding of the role of acute-phase proteins in the regulation of platelet hemostasis and substantiate the possibility of using ceruloplasmin as an antiaggregant for the prevention and correction of hemostasis disorders.

Research Relevance. The study of the functional state of the platelet component of hemostasis has attracted the attention of many research groups. There is a sufficient number of publications in the literature on the role of various factors that alter the functional properties of platelets. Among such substances actively affecting platelet hemostasis are acute-phase proteins. These include fibrinogen, prothrombin, thrombin, thrombopoietin, von Willebrand factor, and many other proteins [53, 78].

Ceruloplasmin (CP) belongs to acute-phase proteins of the "third echelon"; its polyfunctionality and involvement in various types of metabolism do not allow a definitive assessment of its true biological and pathophysiological role in the organism [4].

CP is a carrier of copper and iron, an immunomodulator, an inhibitor of adrenaline, noradrenaline, serotonin, dopamine, and histamine; anti-inflammatory activity of the protein is also mentioned [3, 44, 74, 88, 100, 169]. CP possesses hematoprotective action, and there is evidence of its stimulatory influence on erythropoiesis and leukopoiesis [58, 65, 66]. There are data in the literature on the positive influence of CP on rheological properties of blood, but these reports are few in number and do not reveal the mechanisms of action of the protein [49]. Many researchers regard CP as one of the plasma antioxidants, and there are also references to its prooxidant properties [45, 110].

The concentration of CP increases in many pathological conditions accompanied by general or local disturbances of hemostasis, of which platelets are a functional component.

In platelets, the process of free radical oxidation (FRO) is actively represented; it begins with the activation of membrane phospholipases and initiates the phosphatidylinositol cycle, as well as leads to the formation from arachidonic acid of a series of active prostaglandin endoperoxides and the final product of their metabolism, thromboxane A2 [113, 130, 228]. Platelet activation is accompanied by a change in the intensity of FRO processes [112]. According to Byshevsky A.Sh., Galyan Ch.L. and co-authors, the activation of FRO processes in plasma, secondarily, leads to a change in the functional state of platelets [24].

Thus, it can be hypothesized that CP, being one of the main plasma antioxidants, may influence FRO processes in plasma and platelets, and therefore directly or indirectly alter the functional state of blood cells. At present, the indications for the use of CP do not include conditions requiring correction of the platelet component of hemostasis. All of the above determined the choice of the research topic.

Objective of the Research: To determine the nature and mechanism of the effect of CP on platelets under experimental disorders of hemostasis.

Tasks of the Research

1. To investigate the effect of CP on the number of platelets in the intact organism and in experimental pathology: exogenous thrombocytemias, acute and chronic blood loss (endogenous thrombocytemias).

2. To determine the presence, specificity, and nature of the dose-dependent effect of CP on the functional properties of platelets.

3. To investigate the effect of CP on the adhesiveness, aggregatability of platelets, and release reaction under conditions of the intact organism.

4. To study the effect of CP on the adhesiveness and aggregatability of platelets against the background of experimental thrombocytemias.

5. To investigate certain mechanisms of the effect of CP on the functional state of platelets under thrombocytemias of different genesis:

- intensity of FRO in plasma;

- intensity of FRO in platelets.

Scientific Novelty. For the first time, it is demonstrated that the acute-phase protein CP influences the number and functional state of platelets both under normal conditions and under experimental disorders of hemostasis.

Under in vitro conditions, it is shown that the effect of CP depends on the duration of contact with platelets. During a short contact of 1–2 minutes, the aggregatability of platelets increases, FRO is activated, final products of lipid peroxidation (LPO) accumulate, and the total antioxidant activity decreases. During a more prolonged contact of 30 minutes, the adhesiveness-aggregatability of platelets decreases, while the total antioxidant activity and the activity of a number of enzymes of this group — superoxide dismutase and catalase — increase in platelets, and the content of total, primary, and intermediate LPO products decreases. It is shown that during a short-term temporal contact of CP with platelets, small doses of the preparation (5% of its physiological level in serum) exert the greatest effect, whereas during prolonged contact, medium and high doses (25%, 50%) are most effective.

Under in vivo conditions, CP reduces the functional capacity of platelets both in the intact organism and under experimental exogenous and endogenous thrombocytemias. The effect of CP on the aggregatability of platelets is associated predominantly with the second wave of aggregation. CP alters the intensity of FRO processes in platelets and plasma. Under the influence of CP, new strong and moderate correlational links are formed between the intensity of FRO processes in plasma and the functional properties of platelets.

A new research method has been developed, which was confirmed by a positive decision of the formal examination dated July 12, 2002, on the patent application "Method for Detecting Platelet Luminescence" (No. 200210555291/20(0055267)).

Theoretical and Practical Significance

The results of the dissertation research on the effect of the acute-phase protein CP on the number and functional state of platelets complement the existing data on the regulation of the platelet component of hemostasis and also clarify the pathophysiological significance of acute-phase proteins in the development of many pathological processes.

The use of CP as an antiaggregant for the purpose of prevention and correction of hemostasis disorders has been experimentally substantiated. The obtained data allow the expansion of indications for the use of the medicinal form of CP in hematological practice.

Implementation of the Results

The results of the dissertation work have been introduced into the teaching of pathophysiology and biochemistry in the sections "Pathophysiology of the Blood System" and "Protein Metabolism" at the Chelyabinsk State Medical Academy; as well as into the department of physiology at the Ural State Academy of Physical Culture in the section "Hematopoiesis, General Principles."

The developed "Method for Detecting Platelet Luminescence" has been introduced into the department of pathophysiology of the Chelyabinsk State Medical Academy for the conduct of further scientific research.

The obtained data served as the basis for the use of CP to normalize hematological parameters in patients with chronic renal insufficiency at City Clinical Hospital No. 8 (Chelyabinsk).

Main Propositions to Be Defended

1. CP increases the number of platelets in the intact organism and against the background of chronic blood loss.

2. Under in vitro conditions, CP has a dual effect on the functional state of platelets. At a short contact (1–2 min), it increases the aggregatability of platelets; at a prolonged contact (30 min), it decreases their adhesiveness-aggregatability activity.

3. CP possesses a specific dose-dependent effect on the functional properties of platelets.

4. Under experimental exogenous and endogenous thrombocytemias, CP decreases the adhesiveness-aggregatability of platelets.

5. The effect of CP on the functional state of platelets under normal conditions and experimental disorders of hemostasis is mediated through a change in the intensity of FRO processes in plasma blood and platelets.

Presentation of the Work

The main provisions of the work were presented at the Second Russian Congress on Pathophysiology with international participation (Moscow, 2000), the intercity conference of young scientists "Actual Problems of Pathophysiology" (Saint Petersburg, 2000), the Russian scientific conference "Fundamental Sciences — Practice of Healthcare" (Omsk, 2001), and the scientific-practical conference "New Technologies and Fundamental Research in Medicine" (Chelyabinsk, 2002).

Publications. Ten printed works have been published on the dissertation topic.

Structure and Volume of the Dissertation. The dissertation is presented on 181 pages of typed text and consists of an introduction, a literature review, a description of the materials and methods of research, three chapters of the results of original research, a discussion of the results, conclusions, and a list of references. The work is illustrated with 94 tables and 18 figures. The bibliographic reference includes 252 sources, of which 137 are by foreign authors.