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Platelets and Exosomes

By June 21, 2024No Comments

Platelets, also called thrombocytes, are disc shaped components of blood are essential for many physiological processes. They do not have a nucleus and therefore cannot reproduce themselves, yet they exhibit active RNA metabolism during their 7-day life span.

Platelets are derived from megakaryocytes in the bone marrow. The primary function of platelets is hemostasis but also contain and transport molecules implicated in numerous regenerative processes. These processes include wound healing, cell activation and proliferation, angiogenesis, immune cell recruitment and inflammation, bone regeneration and cartilage repair.

Once platelets are activated, they secrete large amounts of growth factors, cytokines and bioactive proteins that contribute and support the tissue repair process.

The highest amount of growth factors is the following:

  • Platelet derived growth factor
  • Transforming growth factor
  • Epidermal growth factor
  • Insulin like growth factor
  • Fibroblast growth factor
  • Vascular endothelial growth factor

These growth factors are contained on the alpha granules of the platelets and are released upon activation to stimulate cell proliferation, migration, chemotaxis, differentiation and angiogenesis. They also promote the formation of the extracellular matrix.

Role of Extracellular Vesicles (EVs)

Besides the growth factors, these blood products contain a heterogenous group of cell membrane derived vesicles. These are called extracellular vesicles which may be the main contributors to PRP effects. These blood derived extracellular vesicles in the plasma may originate from different cell types such as leukocytes, erythrocytes, dendritic cells, platelets, mast cells, epithelial cells and endothelial cells. Most originate from the megakaryocytes in the bone marrow and thought to originate from platelet precursor cells. The extracellular vesicles are classified as to their size. The larger are the extracellular vesicles and the smaller are the exosomes. However, both classes are called EV’s.

EV’s, including exosomes and micro-vesicles, sometimes called microparticles or ectosomes exist in almost all biological fluids carrying molecules such as proteins, lipids, and RNA either on their surface or within the lumen. They are generated and released from nearly all cell types into the extracellular space. The biomolecules carried by EV’s are reflective of the cell of origin.

These EVs were observed in 1967 for the first time by Wolf et al and he named them “platelet dust”. Recent studies shows that platelet derived-EVs act as a cargo of several molecules including signaling molecules, growth factors, lipids, proteins., nucleic acids (mRNA) which mediate cell to cell communication, immune reaction, inflammatory response, and reparation.

Platelet Rich Plasma is defined as a higher concentration of autologous human platelets exceeding physiological concentration in a small volume of plasma.

Numerous publications show that PRP is effective in the treatment of multiple disorders including musculoskeletal pathologies such as chronic sports related injuries of the muscles and tendons and degenerative joint disorders. Various uses in basically all fields of medicine have proven the efficacy of platelet rich plasma treatments with no reported significant side effects.

With all the studies that have been done to date and all the published reports, the exact mode of action of platelet rich plasma is still up for debate. Perhaps, it is the extracellular vesicles that act to ultimately direct the action of platelet rich plasma.

Activation is a critical step in the action of PRP. Activation can be accomplished by tissue damage at the site or by extraneous means, but inactivated PRP does not achieve the same regeneration as activated PRP.

After activation, the outer membrane of macrovesicles is invaded and bioproteins, cytokines and growth factors are released out of the platelets through extracellular secretion. The dynamic content of platelet-EVs including proteins from the platelet membrane, cytosol, organelles, adhesion receptors, coagulation factors, transcription factors, growth factors, active enzymes, cytokines, chemokines and their receptors is mainly dependent on the mechanism of platelet activation, the agonist used and the time of stimulation.

Different modes of platelet activation may lead to the heterogeneous PL-EV populations with different surface marker expression and protein profiles. This can affect their role in intercellular communication. Studies have proven that EVs from activated platelets by ADP contain different protein cargo in comparison to those activated by collagen or thrombin. This certainly implies that EVs can have different functions based on the mode of activation.

Isolation of Platelet Derived Exosomes

There is no standard method to isolate PRP-EVs and there is no single detection technique with high sensitivity to detect EVs from platelets. It is often impossible to compare results of studies as various isolation methods produce EVs with different yields and purities. The isolation process of EVs from plasma is complicated because the plasma contains other non EVs with similar size and densities and can contaminate the sample.

The typical protocol to obtain EVs from platelets involved centrifugation, separation of the PRP, activation and a second centrifugation. These steps are unwieldy, and the platelets can be prematurely activated to release the exosomes. The blood collection process, the handling, timing, choice of anti-coagulant, storage are all variables causing inaccurate results in treatment and outcomes.

It is well known that EV’s are present in a wide range of body fluids including blood, urine, saliva, CSF, amniotic fluid, breast milk and ascites.

Do Platelet Derived Exosomes Regenerate?

PL-EVs may play a duel role in inflammation stimulating either pro or anti- inflammatory effects depending on the expression of surface markers or microenvironment. Data has shown that

EV’s exert strong immunomodulatory activity on smooth muscle cells and stimulates vascular remodeling.

Platelet-EV’s induce angiogenesis both in vitro and in vivo. This is important to revascularization of the ischemic myocardium.

Platelet -EV’s may be utilized for treatment following brain injury according to results describing their positive role In neuronal cell proliferation, survival and differentiation to form glia cells and neurons.

In a diabetic rat model, encapsulated PL-EV’s increased re-epithelialization and collagen synthesis which led to faster wound healing and closure compared to untreated wounds.

In an osteoarthritic model, it was demonstrated that PL-EVs had a better effect in promoting chondrocyte proliferation and migration and attenuating apoptosis than PRP alone in vivo and in vitro rat model.

PL-EV’s positively modulate the growth, migration and differentiation potential of stem cells from different sources. This enhanced potency of stem cell populations resident in tissues is important in treating and preventing degenerative diseases.

PRP has long been known to be effective in accelerating tissue repair and regeneration, but the underlying mechanism is still not fully understood. The result of clinical research is encouraging as PL-EV’s might be the key part of the mechanism in tissue regeneration induced by platelet rich plasma.

Platelet Rich Plasma and Exosomes

To date, studies have shown the great potential of PRP-EV’s in the field of tissue repair and regeneration. Recent findings suggest that PRP-EV’s may be a superior alternative in regenerative medicine when compared to PRP alone. PRP-EV’s may have more significant advantages over PRP in regenerative medicine for the following reasons:

  • Some studies have demonstrated a higher concentration of growth factors.
  • The smaller size is beneficial for the transfer across biological barriers.
  • Lower immunogenicity
  • Provide a more sufficient protection from loaded cargo due to the phospholipid layer.
  • Contain abundant information molecules such as proteins, lipids and RNA for intercellular communication.

As previously noted, there are many barriers to the manufacturing process and technical challenges to the formation and harvesting of platelet rich plasma extracellular vesicles. Nonetheless, the therapeutic applications and clinical outcomes to date, exemplifies that PRP-EV’s may be the regenerative treatment of the future.

What is the Best Regenerative Product Currently?

The advantage of Platelet Rich Plasma is well documented in the literature. There are currently 20,000 articles or studies on the PubMed website concerning the applications and merits of

PRP. However, this article has outlined the advantages of extracellular vesicles in regenerative medicine.

Exosomes sourced from mesenchymal stem cells offers a superior regenerative product. The exosomes from this natural source contain proteins, messenger RNA and other bioactive proteins that augment regenerative capabilities when used with PRP.

More and more studies have revealed that platelets and exosomes induced activation of intracellular signaling pathways, alteration of vascular reactivity and induction of angiogenesis. This can restore endothelial integrity after vascular injury and trigger neurogenesis in a stroke model.

Torreggiani et al demonstrated that bone marrow stromal cells treated with the PRP-exosome combination showed a significant increase in cell proliferation, migration and osteogenesis. Torreggiani implied that the combination might be one of the effectors of the actions of PRP.

Although exosomes derived from platelets are difficult and costly to extract and concentrate, the exosomes from mesenchymal stem cells are just the opposite. They are readily obtainable, and the manufacturing and concentration process has been virtually perfected. These products have shown superior regenerative capabilities.

Perhaps the exosomes obtained from platelet rich plasma may be a superior product when used in regenerative medicine but until they can be reliably obtained, in guaranteed concentrations at a reasonable cost, the best exosomes are sourced from mesenchymal stem cells.

Consider the combination of PRP and Exosomes extracted from mesenchymal stem cells as the ultimate treatment in regenerative therapy today.

Studies

  • Platelet Derived Extracellular Vesicles: An Emerging Therapeutic Approach Int J Biol Sci 2017 Jul 6;13(7):828-834 PMID 28808416
  • Characterization and Therapeutic Use of Extracellular Vesicles Derived From Platelets Int J Mol Sci 2021 Sep 8;22(18):9701 PMID 34575865
  • Platelet Rich Plasma Derived Extracellular Vesicles: A Superior Alternative in Regenerative Medicine? Cell Prolif 2021 Dec;54(12):e13123 PMID 34609779
  • Advances in Platelet Rich Plasma Derived Extracellular Vesicles for Regenerative Medicine: A Systematic Narrative Review Int J Mol Sci 2023 Aug 22;24(17):13043 PMID 37685849
  • The Value of Platelet Rich Plasma Derived Extracellular Vesicles in Modern Medicine Ann Med 2023 Dec ;55(2):2287705 PMID 38065677

Why Juventix?

Juventix Regenerative Medical is an industry leader in the regenerative medical field. Our Platelet Rich Plasma Kits are FDA cleared and designed for safety, sterility and effectiveness. Our kits are scientifically manufactured to provide a platelet concentrate, devoid of red blood cells with a minimum number of leukocytes that are critical to the regenerative process.

Juventix Regenerative Medical offers a LED Activator to activate the platelets and begin the regenerative process. The activation is a critical step in the release of cytokines, growth factors and bioactive proteins from the alpha granules on the platelets and is accomplished with LED light. This negates the use of chemical additives such as Calcium Chloride, Thrombin or Collagen. This mode of activation by LED light provides sustained growth factor release versus older methods of activation while adhering to the minimal manipulation guidelines of the FDA.

Juventix Regenerative Medical supplies a bio-incubator to transform the Platelet Rich Plasma into an Injectable Platelet Rich Fibrin. The Platelet Rich Fibrin, commonly called the “second generation of platelet products” has different cytokines and growth factors than the original PRP. These different cytokines provide an anti-inflammatory microenvironment and can be used confidently in inflammatory conditions.

Juventix Regenerative Medical is proud to collaborate with Evolutionary Biologics to offer exosomes and other related biologics. This collaboration together provides the ultimate regenerative treatments for the regenerative professional today.

RESTORE, REVIVE, REGENERATE- JUVENTIX REGENERATIVE MEDICAL

 

Regenerative Regards,

Dr. Robert McGrath

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