Chapter 3 PATHOGENETIC MECHANISMS OF ACTION OF AUTOLOGOUS BLOOD PLASMA

This chapter provides a more detailed description of the pathogenetic mechanisms of autologous blood plasma action.

The pathological mechanisms are similar to those of arterial thrombus ("white clot") formation, as suggested by the biological aspects which lie at the heart of the Plasmolifting method. In both cases, platelets and leukocytes are the morphological substrates. However, the Plasmolifting method implies a minimal presence of white blood cells, especially neutrophils. The following information, in my opinion, provides new insights into the mechanisms underlying the action of autologous plasma in the body tissues.

The use of autologous blood plasma has now become common in many fields of medicine. Most often, it is used for the treatment of conditions characterized by long-term chronic inflammation that follows a course of alternating exacerbations and remissions. Such diseases include, for example, acne, chronic generalized or localized periodontitis, gingivitis, peri-implantitis, endocervicitis, etc. That is, we are talking about congested tissues derived from either the ectoderm or endoderm (epidermal and connective tissues) that show diffuse or focal mononuclear cell infiltration.

Since the reserves are reduced, and the local immune response is impaired in such tissues, instead of active cell proliferation in the site of inflammation and resulting tissue repair, secondary tissue damage (alteration), caused by the inflammatory cells, occurs. This creates a vicious circle.

3.1. The first step: the vascular wall response.

Inflamed tissues are usually characterized by a damaged endothelium, which in turn causes circulatory disturbances. The response of the blood vessels to injury is vasoconstriction, which, in large vessels, is mediated by neural reflex mechanisms. The capillary constriction occurs through a contraction in the endothelial cell myofibrils. Vascular wall contraction can be stimulated by vasoactive substances released from platelets that are adhered to the site of injury, more specifically by serotonin, adrenaline, and thromboxane A2. In turn, bradykinin, activating factor XII, can contribute to increasing the microvascular permeability and constricting capillaries from the outside with the filtered fluid.

The endothelium itself also plays an essential role in regulating interactive cascades occurring within the vascular wall in response to injury, being capable of producing both anti-thrombogenic and thrombogenic factors.

3.2. The second step: platelet adhesion.

Platelets adhere to the collagen fibers in the disrupted vessel wall within the first seconds following damage. Platelet adhesion is primarily carried out by the physiological mechanisms mediating the platelet response to vascular injury via receptors that act as an intermediary between platelets and various environmental factors.

Most receptors anchored within the platelet cell plasma membrane are glycoproteins (GP) (Table 3.1).

Table 3.1

Platelet receptors and their agonists

 

 One end of the GP receptor molecule is located in the extracellular space, while the other "permeates" the plasma membrane and contacts platelet structures embedded in its inner leaflet. The outer parts of the GP molecules host receptor loci that are specific for different substances (ligands). After the coupling of the ligands to specific receptor loci, an activation signal is transduced to the internal parts of the platelets.

Ligands are substances that bind specific receptors, causing its conformational changes and thus modulating the functional activity of the platelet. Each receptor has one or more physiological agonists and can bind to them with high or low affinity (Fig. 3.1).

Fig. 3.1. Surface platelet glycoprotein receptors

	Platelet
	Lysosomes
	Granules

 The direct adhesion of platelets to subendothelial collagen fibers occurs due to the presence of a receptor for collagen on platelets, i.e., GP Ia-IIa, belonging to the integrin family. The resulting complex is stabilized by the von Willebrand factor, which serves as a bridge between the subendothelial collagen fibers and the platelet glycoprotein Ib-IX receptor (Fig. 3.2.). 

 

Fig. 3.2. Scheme of platelet adhesion to the vessel wall

	Collagen fibers
	"Bridges" (Von Willebrand factor and other proteins)
	Activated platelet
	Ib platelet receptors

 

3.3. The third step: activation and degranulation of platelets.

As mentioned above, platelet activation occurs through subendothelial structures of the vessel wall (collagen and microfibrils) due to adhesion, which leads to a change in the disk-shaped form of platelets that turn into spiculated spheres with protruding filopodia. Collagen-induced platelet aggregation has a fairly pronounced latent phase and can last up to 5–7 minutes.

The release reaction is the selective secretion of compounds stored in aggregated platelets. During this release, cellular integrity is maintained. Specifically, the platelet granule exocytosis into the plasma is not accompanied by cell lysis, in which the membranes are destroyed, so the organelles, together with their cargo, are released to the extracellular environment. This quick release of secreted substances is facilitated by contracting of the surface-connected microtubular system. At the same time, the platelets maintain their integrity or, in any case, the ability to perform their functions (Fig. 3.3).

 

Fig. 3.3. Activation and degranulation of platelets

The release reaction is conditionally subdivided into two consecutive steps: induction and transmission. During induction, the release inducers (such as collagen, thrombin, and other factors) interact with the membrane receptors, by which calcium ions (Ca²⁺) are liberated from the membrane.  Transmission is the penetration of Ca²⁺ into the cell.

In recent years, it has been established that Ca²⁺ plays a crucial role in the functional activity of platelets. This assertion is supported by some evidence, primarily by appealing to the analogous situation with other cells, for which it is known that Ca²⁺ is the causative agent of secretion and contraction. Indirect evidence includes the well-known fact that adhesion and secretion of platelet granule contents are induced by the cation ionophore A23187, and the response to the action of this compound is the same as with other stimuli. Finally, as direct proof, one may add the blocking of the functional activity of blood plaques with drugs (with several local anesthetics) along with inhibiting the release of Ca²⁺ from the sarcoplasmic reticulum. Although conclusive experimental evidence supporting this assertion is lacking, there are suggestions that intracellular Ca²⁺ resources play the primary role in the regulation of platelet functions.

An increase in cytoplasmic Ca²⁺ also triggers Ca²⁺ secretion from the membrane, mediating rapid platelet shape changes, then vesicle store organelles discharge Ca²⁺ into the cytoplasm and induce the release reaction. At the same time, Ca²⁺ is secreted into the environment, and changes in the cell plasma membrane occur, increasing its permeability for Ca²⁺. When Ca²⁺ is released from the dense granules, the membrane of these organelles attaches to the cell plasma membrane or to that of the surface-connected canalicular system, which, by contracting, pushes Ca²⁺ and some other substances into the cytoplasm.

Since the regulation of the Ca²⁺ level ensures the required cell contractility, the activity of the contractile mechanism necessary for aggregation, the release reaction, and the retraction of the blood clot, many studies have been carried out to identify the platelet receptors for Ca²⁺. One of the most striking results of these studies is the isolation of four proteins (labeled with 32P-ATP) from platelet lysates. These proteins bind Ca²⁺ (molecular weight 50,000, 28,000, 15,000 and 11,000 Daltons). The greatest incorporation of the label was found in the protein fraction with a molecular weight of 11,000 Daltons, and only this protein of the four receptors was tagged as 32P in intact platelets. It is localized on the cell surface and binds 1 mole of Ca²⁺ per 1 mole of phosphorylated protein.

As a result of activation, several substances that serve as powerful platelet stimulants (ADP, serotonin, adrenaline, unstable prostaglandins, thromboxane A2, platelet-activating factor) are released.

Adrenaline, collagen, and thrombin, binding to membrane receptors, activate two membrane enzymes: phospholipase C and phospholipase A2. These enzymes catalyze the breakdown of two membrane phospholipids, phosphatidylinositol-4,5-diphosphate, and lecithin, with the formation of arachidonic acid. First, a small amount of arachidonic acid is converted to thromboxane A2, which, in turn, activates phospholipase C. The formation of thromboxane A2 from arachidonic acid is catalyzed by cyclooxygenase.

Phospholipase C catalyzes the hydrolysis of phosphatidylinositol-4,5-diphosphate to DAG (diacylglycerol) and IF₃ (inositol 1,4,5-trisphosphate). IF₃ causes a release of calcium into the cytosol, thereby resulting in phosphorylation of myosin light chains. The interaction of myosin with actin provides the movement of granules and the change in the platelet shape. 

DAG activates protein kinase C, which phosphorylates some proteins, including myosin light-chain kinase and pleckstrin (a protein with a molecular weight of 47,000). It is supposed that phosphorylation of these or other proteins also regulates platelet degranulation.

Thromboxane A2 synthesized from arachidonic acid by platelets stimulates their activation, while prostacyclin (prostaglandin I2) produced from the same acid by vascular endothelium, inhibits platelet activation (by increasing the level of cAMP).

The final steps of the release reaction are the discharge of dense granule cargo (mainly serotonin, ADP, Ca²⁺) and the secretion of several substances from platelet α-granules, i.e., growth factors that can trigger regeneration processes, as well as ATP, factor 4, able to attach and neutralize heparin, factor III, catalyzing the formation of fibrin as the final stage of coagulation, and also the secretion of Ca²⁺, lipids and some hydrolases in trace amounts. Enzymes located in the cytoplasm, mitochondria, and membrane are retained by the cell.

Therefore, it becomes evident that an injection of autologous blood plasma into a tissue induces the same normal processes of platelet adhesion and release of the corresponding growth factors from α-granules.3

3.4. Growth factors and mechanisms underlying their effects on tissues.

 

Growth factors are protein molecules containing specific sequences of amino acids. The main growth factors found in platelets and their functions are shown in Table 3.2.

Table 3.2

 

EGF (epidermal growth factor) is a protein compound, a polypeptide with a molecular weight of 6000, consisting of 53 amino acid residues. It was first isolated from mouse salivary glands (Fig. 3.4). In 1962, EGF was accidentally discovered by American biochemist Cohen Stanley while studying nerve growth factor. It is a low-molecular-weight polypeptide that is present in many body tissues. The proven and hypothetical functions of EGF can be classified as endocrine and paracrine. EGF is found in blood, urine, cerebrospinal fluid, milk, saliva, gastric and pancreatic juice.

 The epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases, which also includes HER2/erbB2 and HER3/erbB3. All of them are attractive targets for cancer therapy. EGFR is a complex protein molecule that is encoded in one of the genes. Its mechanism of action is as follows: the factor binds to specific extracellular receptors, the two extracellular parts combine, tyrosine kinase is activated, signaling molecules interact with tyrosine kinase, prompting a signal for cell division (Fig. 3.5).

	Growth factor binds to the receptor
	Activating cell receptor
	Activation of tyrosine kinase
	Mitotic signal
	Nucleus
	Receptors

 

It is this mechanism that suppresses the gene responsible for aging and stimulating the activity and growth of skin cells. EGF induces the formation of young skin cells by accelerating the regeneration process, resulting in a significant reduction in the depth of wrinkles; age spots disappear, and the skin looks young and fresh.

It helps combat age-related changes:

• Helps get rid of dead skin cells and reduce wrinkles, makes skin soft and smooth, and has a rejuvenating effect on the skin.

• Protects skin from damage and irritation.

• Enhances the action of the BCL-2 gene, which delays cell aging, has a wound healing property, reduces the harmful effects of UV radiation, low temperatures, and the medication side effects.

• Relieves symptoms of dryness, including skin feeling tight or rough, and enhances the ability of the epidermis to retain moisture.

• Improves skin complexion.

• Promotes cellular metabolism, and helps prevent hyperpigmentation, or to stop it becoming more prominent after exposure to the sun.

• Boosts and improves blood circulation, makes skin look more natural and healthier.

• A source of skin health and beauty, as it deeply moisturizes it and enhances the synthesis of macromolecular proteins that provides elasticity to the skin.

Usually, chronically inflamed tissue is characterized by impaired angiogenesis associated with insufficient production of vascular growth factors, or with increased secretion of angiogenesis inhibitors (thrombospondin, matrix metalloproteinases, and plasminogen activators, endostatin, etc.) Angiogenesis is driven by growth factors and strictly regulated, both in time and space. Low oxygen in tissues (hypoxia or ischemia) is the central stimulus for angiogenesis in numerous physiological and pathological conditions. This lack of oxygen, through an activator of angiogenesis transcription factors, hypoxia-inducible factor-1 (HIF-1), mediates the expression of many angiogenic factors and, above all, the primary regulator of angiogenesis, i.e., vascular endothelial growth factor (VEGF) and its receptors. VEGF selectively induces proliferation and migration of endothelial cells, their precursors and monocytes expressing receptors to it, increases vessel permeability, promoting leakage of plasma proteins into the perivascular space required for the migration of endothelial cells; induces expression of endothelial NO-synthase and NO formation, which enhances vasodilation and stimulates the expression of proteases that break down the bonds between endothelial cells and the extracellular matrix necessary for the targeted cell migration.

The following growth factors are responsible for the maturation and stabilization of the newly formed immature vessels: 1) angiopoietin-1, which suppresses the proliferation of endothelial cells, reduces vascular permeability and attracts pericytes, 2) platelet-derived GF (PDGF), which attracts pericytes and smooth muscle cells, and 3) transforming GF-beta 1 (TGF- beta 1), stimulating the synthesis of matrix proteins.

Fibroblast growth factors (FGF) make up a family of about 20 homologous polypeptide growth factors with similar properties. The main growth factor (FGF, FGF-2, 18 kD) is most studied. FGF stimulates the proliferation and differentiation of mesoderm-derived stromal cells: fibroblasts, osteoblasts, chondroblasts, myeloblasts, and endothelial cells.

3.5. Proliferation inhibitors and their effect on tissue cells.

The required extent of cell proliferation is determined not only by stimulators but also by proliferation inhibitors. The latter include chalones. The mechanism of action of these substances is well understood through the study of the epithelium. These substances are synthesized and deposited in mature desquamating cells. A decrease in the number of these cells results in a decrease in chalone concentration, which means that the inhibition effect is also decreased, and cell division is accelerated. The site of inflammation is characterized by a small number of mature cells and, consequently, by a low level of inhibitors of mitosis, i.e., chalones. One of the mechanisms of inhibition of cell proliferation is tapped into the alpha-granules themselves and is mediated via alpha-2-macroglobulin. This protein has a broad spectrum of action; it is the primary inhibitor of quinine-forming blood enzymes eliminating their influence, which results in dilatation and increased permeability of the vessels. Besides, it inhibits the majority of leukocyte proteinases, including collagenase and elastase, and thereby protects the connective tissue from destruction. Finally, macroglobulin can bind to neutrophil membranes and thus inhibit their response to C3 and C5a (chemotaxis).

Consequently, the administration of autologous blood plasma into damaged and inflamed tissues may increase the platelet concentration in this area up to several hundred-fold compared to a natural baseline, which allows one to break the vicious cycle of chronic inflammation due to artificial stimulation of chemotaxis, proliferation, and differentiation of cells, and therefore of tissue regeneration.