Platelet Generation Research Articles

PEGylated thrombopoietin mimetic, JNJ‑26366821 a novel prophylactic radiation countermeasure for acute radiation injury

Thrombopoietin (TPO) is the primary regulator of platelet generation and a stimulator of multilineage hematopoietic recovery following exposure to total body irradiation (TBI). JNJ‑26366821, a novel PEGylated TPO mimetic peptide, stimulates platelet production without developing neutralizing antibodies or causing any adverse effects. Administration of a single dose of JNJ‑26366821 demonstrated its efficacy as a prophylactic countermeasure in various mouse strains (males CD2F1, C3H/HeN, and male and female C57BL/6J) exposed to Co-60 gamma TBI. A dose dependent survival efficacy of JNJ‑26366821 (− 24 h) was identified in male CD2F1 mice exposed to a supralethal dose of radiation. A single dose of JNJ‑26366821 administered 24, 12, or 2 h pre-radiation resulted in 100% survival from a lethal dose of TBI with a dose reduction factor of 1.36. There was significantly accelerated recovery from radiation-induced peripheral blood neutropenia and thrombocytopenia in animals pre-treated with JNJ‑26366821. The drug also increased bone marrow cellularity and megakaryocytes, accelerated multi-lineage hematopoietic recovery, and alleviated radiation-induced soluble markers of bone marrow aplasia and endothelial damage. These results indicate that JNJ‑26366821 is a promising prophylactic radiation countermeasure for hematopoietic acute radiation syndrome with a broad window for medical management in a radiological or nuclear event.

Donor-related EDTA dependent pseudothrombocytopenia after allogeneic stem cell transplantation. Can it be real?

Hematopoietic stem cell transplantation (HSCT) may take place in the form of an autologous or allogeneic transplant depending on the indication for transplantation. Because of the myeloablative conditioning regimens preceding HSCT, deep thrombocytopenia is experienced by most of the stem cell recipients in whom replenishment of leukocytes and platelets is expected within the first month following the transplantation. Prolonged thrombocytopenia, on the other hand, usually develops as a delayed complication of allogeneic stem cell transplantation (allo-SCT) and is associated to the source of stem cells, quantity of the infused CD34+ cells, graft-versus-host-disease (GVHD), insufficient engraftment, relapse of the malignancy, microangiopathy, alloimmunisation, medications, or viral infections. In an attempt to explain pathogenesis leading to post-HSCT thrombocytopenia, two main theories have been proposed. First one is the peripheral destruction caused by anti-platelet antibodies, splenic sequestration, or other factors. The latter blames insufficient platelet generation due to impaired thrombopoiesis. Nevertheless, most of the clinical conditions arise with overlapping of both mechanisms.Here we present a pseudothrombocytopenia case induced by donor-related ethylene-diamine-tetra-acetic acid (EDTA) as an unanticipated cause of thrombocytopenia to which most recipients of allo-SCT are prone to.

Dynamic actin/septin network in megakaryocytes coordinates proplatelet elaboration.

Megakaryocytes (MK) undergo extensive cytoskeletal rearrangements as they give rise to platelets. While cortical microtubule sliding has been implicated in proplatelet formation, the role of the actin cytoskeleton in proplatelet elongation is less understood. It is assumed that actin filament reorganization is important for platelet generation given that mouse models with mutations in actin-associated proteins exhibit thrombocytopenia. However, due to the essential role of the actin network during MK development, a differential understanding of the contribution of the actin cytoskeleton on proplatelet release is lacking. Here, we reveal that inhibition of actin polymerization impairs the formation of elaborate proplatelets by hampering proplatelet extension and bead formation along the proplatelet shaft, which was mostly independent of changes in cortical microtubule sliding. We identify Cdc42 and its downstream effectors, septins, as critical regulators of intracellular actin dynamics in MK, inhibition of which, similarly to inhibition of actin polymerization, impairs proplatelet movement and beading. Super-resolution microscopy revealed a differential association of distinctive septins with the actin and microtubule cytoskeleton, respectively, which was disrupted upon septin inhibition and diminished intracellular filamentous actin dynamics. In vivo, septins, similarly to F-actin, were subject to changes in expression upon enforcing proplatelet formation through prior platelet depletion. In summary, we demonstrate that a Cdc42/septin axis is not only important for MK maturation and polarization, but is further required for intracellular actin dynamics during proplatelet formation.

IN VITRO PROPAGATION OF PLATELETS FROM EMBRYONIC STEM CELLS WITH C. PAPAYA LEAF EXTRACT

Objective: The depletion of platelets in patients under different clinical conditions like thrombocytopenia is often associated with a significant mortality factor. Alternative modes of platelet propagation from other resources, including stem cells, are evaluated under in vitro conditions. Embryonic stem (ES) cells can proliferate and differentiate into various cell types. This study evaluated the generation of functional platelets from embryonic stem cells with C. papaya leaf extract through a controlled sequential pathway to meet the clinical demand.
 Methods: In vitro culture of embryonic stem cells was carried out with the help of growth factors and a suitable medium. The differentiated megakaryocytes and platelets were analyzed by staining, flow cytometer and functional studies. The normal megakaryocytes were compared with the megakaryocytes differentiated from Embryonic Stem cells.
 Results: The platelets derived from embryonic stem cells are morphologically identical to normal platelets isolated from peripheral blood, and the number of viable cells was analyzed by flow cytometer. The distinctive two waves of platelet production represented normal primitive and definitive hematopoiesis.
 Conclusion: This study is a preliminary milestone in platelet propagation from embryonic stem cells with C. papaya leaf extract. The differentiation of ESCs into megakaryocyte-mediated platelets with C. papaya leaf extract has shown valuable information on the positive effects that embryonic stem cells can be used for large-scale platelet production.

Abstract 123: Protein Kinase C Theta (PKCθ) Signaling Associated With The Generation Of Procoagulant Platelets Revealed By Phosphoproteomics And Systems Analysis

Protein kinase C (PKC) family members integrate platelet activation programs to support hemostasis, and, also drive inflammatory and vascular diseases. While some PKC isoforms have established roles in platelets, roles for PKCθ are less specified. Here, we aim to determine how PKCθ signaling orchestrates platelet function. We prepared washed platelets from human donors for biochemical, physiological and omics studies. Platelet signaling and physiology were studied following stimulation with agonists, including the glycoprotein receptor GPVI agonist crosslinked collagen-related peptide (CRP-XL), and protease activated receptor PAR1 and PAR4 agonists, TRAP6 and AYPGKF-NH 2 . Platelets were treated with inhibitors of PKC family members to study PKC activation mechanisms by Western blot using antisera against specific phosphorylation sites on PKC isoforms and PKC substrates. Phosphoproteomics studies of GPVI and PAR agonist-stimulated platelets quantified significant, site-specific changes in the phosphorylation of PKC isoforms as well as PKC substrates. In addition to established phosphorylation events (e.g., PKCδ Tyr311), we noted a novel, prominent phosphorylation on the C-terminus PKCθ at Ser685. After generating and characterizing antisera specifically reactive to PKCθ phospho-Ser685, Western blot analysis determined that PKCθ Ser685 phosphorylation occurred later following GPVI platelet activation (>5 min) relative to PKCδ Tyr311 phosphorylation and PKC substrate phosphorylation driven by calcium-dependent PKCs. Treatment of platelets with specific PKC inhibitors revealed that PKCα/β, PKCθ, and, to a lesser extent, PKCδ activities all determine PKCθ Ser685 phosphorylation. In parallel, platelet function studies found that inhibition of PKCθ had only minor effects on platelet secretion and aggregation; however, PKCθ inhibition significantly reduced platelet phosphatidylserine exposure - a marker of procoagulant platelet activity - in response to GPVI and PAR agonists. Together, our results suggest that PKCδ and PKCα/β activities drive the phosphorylation of PKCθ Ser685 to support procoagulant platelet generation in a manner that may be blunted by therapeutically relevant small molecule kinase inhibitors.

Transcription factors in megakaryocytes and platelets.

Transcription factors bind promoter or regulatory sequences of a gene to regulate its rate of transcription. However, they are also detected in anucleated platelets. The transcription factors RUNX1, GATA1, STAT3, NFκB, and PPAR have been widely reported to play key roles in the pathophysiology of platelet hyper-reactivity, thrombosis, and atherosclerosis. These non-transcriptional activities are independent of gene transcription or protein synthesis but their underlying mechanisms of action remain poorly defined. Genetic and acquired defects in these transcription factors are associated with the production of platelet microvesicles that are known to initiate and propagate coagulation and to promote thrombosis. In this review, we summarize recent developments in the study of transcription factors in platelet generation, reactivity, and production of microvesicles, with a focus on non-transcriptional activities of selected transcription factors.

Apoptosis is one cause of thrombocytopenia in patients with high-altitude polycythemia

High-altitude polycythemia (HAPC) can occur in individuals who are intolerant to high-altitude hypoxia. In patients with HAPC, erythrocytosis is often accompanied by a decrease in platelet count. Chronic hypoxia can increase the incidence of arteriovenous thrombosis and the risk of bleeding during antithrombotic treatment due to thrombocytopenia; therefore, understanding the cause of thrombocytopenia can reduce the risk of treatment-related bleeding. In this study, we examined platelet production and apoptosis to understand the cause of thrombocytopenia in patients with HAPC. The classification of myeloid-derived megakaryocytes (MKs) in HAPC patients was mainly granular MKs rather than mature MKs, suggesting impaired differentiation and maturation. However, the total number of MKs and newly generated reticulated platelets in the peripheral blood increased, indicating sufficient platelet generation in HAPC thrombocytopenia. Increased platelet apoptosis may be one of the causes of thrombocytopenia. Platelet activation and GP1bα pathway activation induced by thrombin and von Willebrand factor can lead to platelet apoptosis. Platelet production was not reduced in patients with HAPC, whereas platelet apoptosis was associated with thrombocytopenia. These findings provide a rationale for considering the bleeding risk in HAPC patient while treating thrombotic diseases.

Progress of concentrated growth factors in the treatment of jaw cysts

The activation of platelets can release multiple growth factors associated with wound healing and bone regeneration. With the development of autologous platelet concentrate preparation techniques, significant progress has been made in the repair of bone defects in oral and jaw cysts. Concentrated growth factors (CGF)are the latest generation of platelet concentrates obtained by differential centrifugation, rich in growth factors without the addition of anticoagulants, and with no risk of cross-infection and immune rejection. CGF is convenient for material preparation, simple preparation, high efficiency, and has a certain anti-inflammatory effect, and the surrounding soft tissue structure is better recovered. This paper briefly reviews the promoting effect of CGF in the treatment of jaw cysts on soft and hard tissue healing and its related clinical applications, and aims to provide some theoretical basis for the further application of CGF in promoting osteogenesis, which can be better applied in clinical practice and play its maximum value.

Ex vivo manufacturing of platelets: beyond the first-in-human clinical trial using autologous iPSC-platelets.

Platelet transfusion is a common clinical approach to providing platelets to patients suffering from thrombocytopenia or other ailments that require an additional platelet source. However, a stable supply of platelet products is challenged by aging societies, pandemics, and other factors. Many groups have made extensive efforts toward the in vitro generation of platelets for clinical application. We established immortalized megakaryocyte progenitor cell lines (imMKCLs) from human induced pluripotent stem cells (iPSCs) and achieved clinical-scale manufacturing of iPSC-derived platelets (iPSC-PLTs) from them by identifying turbulent flow as a key physical condition. We later completed the iPLAT1 study, the first-in-human clinical trial using autologous iPSC-PLTs. This review summarizes current findings on the ex vivo generation of iPSC-PLTs that led to the iPLAT1 study and beyond. We also discuss new insights regarding the heterogeneity of megakaryocytes and the implications for the ex vivo generation of iPSC-PLTs.

Bifunctional effect of the inflammatory cytokine tumor necrosis factor α on megakaryopoiesis and platelet production

Background and ObjectivesPlatelets are affected by many factors, such as infectious or aseptic inflammation, and different inflammatory states may induce either thrombocytopenia or thrombocytosis. Tumor necrosis factor α (TNFα) is an important inflammatory cytokine that has been shown to affect the activity of hematopoietic stem cells. However, its role in megakaryocyte (MK) development and platelet production remains largely unknown. This study aimed to investigate the effects of TNFα on MK and platelet generation. Methods and ResultsThe ex vivo study with human CD34+ cells demonstrated that TNFα differentially modulated commitment toward the MK lineage. Specifically, a low concentration of 0.5 ng/ml TNFα promoted MK maturation, proplatelet formation, and platelet production, whereas a high concentration of 10 ng/ml or more TNFα exhibited a substantial inhibitory effect on MK and platelet production. The distinct effect of TNFα on MKs was mainly dependent on TNFα receptor 1. TNFα differentially regulated the MAPK–ERK1/2 signaling pathway and the cytoskeletal proteins cofilin and MLC2. The in vivo study with Balb/c mice indicated that low‐dose or high‐dose TNFα administration differentially affected short‐term platelet recovery after bone marrow transplantation. ConclusionsOur study revealed distinct roles for TNFα in megakaryopoiesis and thrombopoiesis and may provide new insights regarding the treatment for platelet disorders.

Inflammatory platelet production stimulated by tyrosyl-tRNA synthetase mimicking viral infection

Platelets play a role not only in hemostasis and thrombosis, but also in inflammation and innate immunity. We previously reported that an activated form of tyrosyl-tRNA synthetase (YRSACT) has an extratranslational activity that enhances megakaryopoiesis and platelet production in mice. Here, we report that YRSACT mimics inflammatory stress inducing a unique megakaryocyte (MK) population with stem cell (Sca1) and myeloid (F4/80) markers through a mechanism dependent on Toll-like receptor (TLR) activation and type I interferon (IFN-I) signaling. This mimicry of inflammatory stress by YRSACT was studied in mice infected by lymphocytic choriomeningitis virus (LCMV). Using Sca1/EGFP transgenic mice, we demonstrated that IFN-I induced by YRSACT or LCMV infection suppressed normal hematopoiesis while activating an alternative pathway of thrombopoiesis. Platelets of inflammatory origin (Sca1/EGFP+) were a relevant proportion of those circulating during recovery from thrombocytopenia. Analysis of these "inflammatory" MKs and platelets suggested their origin in myeloid/MK-biased hematopoietic stem cells (HSCs) that bypassed the classical MK-erythroid progenitor (MEP) pathway to replenish platelets and promote recovery from thrombocytopenia. Notably, inflammatory platelets displayed enhanced agonist-induced activation and procoagulant activities. Moreover, myeloid/MK-biased progenitors and MKs were mobilized from the bone marrow, as evidenced by their presence in the lung microvasculature within fibrin-containing microthrombi. Our results define the function of YRSACT in platelet generation and contribute to elucidate platelet alterations in number and function during viral infection.

Deciphering transcriptome alterations in bone marrow hematopoiesis at single-cell resolution in immune thrombocytopenia

Immune thrombocytopenia (ITP) is an autoimmune disorder, in which megakaryocyte dysfunction caused by an autoimmune reaction can lead to thrombocytopenia, although the underlying mechanisms remain unclear. Here, we performed single-cell transcriptome profiling of bone marrow CD34+ hematopoietic stem and progenitor cells (HSPCs) to determine defects in megakaryopoiesis in ITP. Gene expression, cell-cell interactions, and transcriptional regulatory networks varied in HSPCs of ITP, particularly in immune cell progenitors. Differentially expressed gene (DEG) analysis indicated that there was an impaired megakaryopoiesis of ITP. Flow cytometry confirmed that the number of CD9+ and HES1+ cells from Lin−CD34+CD45RA− HSPCs decreased in ITP. Liquid culture assays demonstrated that CD9+Lin−CD34+CD45RA− HSPCs tended to differentiate into megakaryocytes; however, this tendency was not observed in ITP patients and more erythrocytes were produced. The percentage of megakaryocytes differentiated from CD9+Lin−CD34+CD45RA− HSPCs was 3-fold higher than that of the CD9− counterparts from healthy controls (HCs), whereas, in ITP patients, the percentage decreased to only 1/4th of that in the HCs and was comparable to that from the CD9− HSPCs. Additionally, when co-cultured with pre-B cells from ITP patients, the differentiation of CD9+Lin−CD34+CD45RA− HSPCs toward the megakaryopoietic lineage was impaired. Further analysis revealed that megakaryocytic progenitors (MkP) can be divided into seven subclusters with different gene expression patterns and functions. The ITP-associated DEGs were MkP subtype-specific, with most DEGs concentrated in the subcluster possessing dual functions of immunomodulation and platelet generation. This study comprehensively dissects defective hematopoiesis and provides novel insights regarding the pathogenesis of ITP.

Augmented Production of Platelets From Cord Blood With Euchromatic Histone Lysine Methyltransferase Inhibition.

Cord blood hematopoietic stem/progenitor cells (CB-HSPCs) have emerged as a promising supply for functional platelets to potentially alleviate the increasing demand for platelet transfusions, but the clinical application has been limited by the undefined molecular mechanism and insufficient platelet production. Here, we performed single-cell profiling of more than 16 160 cells to construct a dynamic molecular landscape of human megakaryopoiesis from CB-HSPCs, enabling us to uncover, for the first time, cellular heterogeneity and unique features of neonatal megakaryocytes (MKs) and to also offer unique resources for the scientific community. By using this model, we defined the genetic programs underlying the differentiation process from megakaryocyte-erythroid progenitors (MEPs) to MKs via megakaryocyte progenitors (MKPs) and identified inhibitors of euchromatic histone lysine methyltransferase (EHMT), which, when applied at the early stage of differentiation, significantly increase the final platelet production. At the mechanistic level, we found that EHMT inhibitors act to selectively induce the expansion of MEPs and MKPs. Together, we uncover new mechanistic insights into human megakaryopoiesis and provide a novel chemical strategy for future large-scale generation and clinical applications of platelets.

Use of platelet-rich fibrin a backbone in facial plastic surgery and wound healing: A review of literature

Platelet aggregates has been used in surgery for many years. The primary goal of these ‘Autologous Preparation’ was to aggregate platelets and their growth factors and to convey it to a surgical site, to improve healing only. The latest research work shows that the PRF also can be used for Protection of Graft, neo-angiogenesis, vascularization locally and release of various growth factors apart from healing. Platelet rich fibrin (PRF) is considered as the new generation of platelet concentrate. “Choukroun’s PRF” or Leucocyte PRF (L-PRF) is the new development, which was first developed by Choukroun’s et al. in 2001 as an ‘Autologous biomaterial.’ A systematic literature search was performed to identify relevant articles that describes the use of PRF, through PubMed and Scopus databases. The focus of this article is to explore the role of PRF in esthetic facial surgery.

A Comprehensive Review on Efficacy of Leukocyte and Platelet-rich Fibrin (L-PRF) Administration in Reconstruction of Oral and Maxillofacial Structures

Several studies have shown that growth factors are effective in soothing inflammatory processes involved in the initial hemostasis phase of wound healing. Leukocyte and platelet-rich fibrin (L-PRF) is the second generation of concentrated platelets that have been shown to stimulate the proliferation of osteoblasts, gingival fibroblasts, and periodontal ligament cells. Rich platelet components have an immune function and antibacterial properties, which play a critical role in the wound healing process. The present study aimed to review the reported documents on L-PRF administration in regeneration and wound healing of oral and maxillofacial surgeries. The research was performed using well-known medical databases to find controlled clinical trials that used L-PRF as a cotreatment strategy during oral and maxillofacial surgeries. Eligible articles published between 2000 and 2022 in PubMed, Web of Science, Science Direct, and Scopus databases were selected for this review. According to the results of this study, L-PRF has significant effectiveness as a maxillofacial regenerative therapy by improving para-clinical parameters after surgery. However, more clinical practice and additional research are essential for the development of this complementary treatment method

Neutrophil extracellular traps participate in the development of cancer-associated thrombosis in patients with gastric cancer.

BACKGROUNDThe development of venous thromboembolism (VTE) is associated with high mortality among gastric cancer (GC) patients. Neutrophil extracellular traps (NETs) have been reported to correlate with the prothrombotic state in some diseases, but are rarely reported in GC patients.AIMTo investigate the effect of NETs on the development of cancer-associated thrombosis in GC patients.METHODSThe levels of NETs in blood and tissue samples of patients were analyzed by ELISA, flow cytometry, and immunofluorescence staining. NET generation and hypercoagulation of platelets and endothelial cells (ECs) in vitro were observed by immunofluorescence staining. NET procoagulant activity (PCA) was determined by fibrin formation and thrombin–antithrombin complex (TAT) assays. Thrombosis in vivo was measured in a murine model induced by flow stenosis in the inferior vena cava (IVC).RESULTSNETs were likely to form in blood and tissue samples of GC patients compared with healthy individuals. In vitro studies showed that GC cells and their conditioned medium, but not gastric mucosal epithelial cells, stimulated NET release from neutrophils. In addition, NETs induced a hypercoagulable state of platelets by upregulating the expression of phosphatidylserine and P-selectin on the cells. Furthermore, NETs stimulated the adhesion of normal platelets on glass surfaces. Similarly, NETs triggered the conversion of ECs to hypercoagulable phenotypes by downregulating the expression of their intercellular tight junctions but upregulating that of tissue factor. Treatment of normal platelets or ECs with NETs augmented the level of plasma fibrin formation and the TAT complex. In the models of IVC stenosis, tumor-bearing mice showed a stronger ability to form thrombi, and NETs abundantly accumulated in the thrombi of tumor-bearing mice compared with control mice. Notably, the combination of deoxyribonuclease I, activated protein C, and sivelestat markedly abolished the PCA of NETs.CONCLUSIONGC-induced NETs strongly increased the risk of VTE development both in vitro and in vivo. NETs are potential therapeutic targets in the prevention and treatment of VTE in GC patients.

DNA Origami-Platelet Adducts: Nanoconstruct Binding without Platelet Activation.

Objective. Platelets are small, mechanosensitive blood cells responsible for maintaining vascular integrity and activatable on demand to limit bleeding and facilitate thrombosis. While circulating in the blood, platelets are exposed to a range of mechanical and chemical stimuli, with the platelet membrane being the primary interface and transducer of outside-in signaling. Sensing and modulating these interface signals would be useful to study mechanochemical interactions; yet, to date, no methods have been defined to attach adducts for sensor fabrication to platelets without triggering platelet activation. We hypothesized that DNA origami, and methods for its attachment, could be optimized to enable nonactivating instrumentation of the platelet membrane. Approach and Results. We designed and fabricated multivalent DNA origami nanotile constructs to investigate nanotile hybridization to membrane-embedded single-stranded DNA-tetraethylene glycol cholesteryl linkers. Two hybridization protocols were developed and validated (Methods I and II) for rendering high-density binding of DNA origami nanotiles to human platelets. Using quantitative flow cytometry, we showed that DNA origami binding efficacy was significantly improved when the number of binding overhangs was increased from two to six. However, no additional binding benefit was observed when increasing the number of nanotile overhangs further to 12. Using flow cytometry and transmission electron microscopy, we verified that hybridization with DNA origami constructs did not cause alterations in the platelet morphology, activation, aggregation, or generation of platelet-derived microparticles. Conclusions. Herein, we demonstrate that platelets can be successfully instrumented with DNA origami constructs with no or minimal effect on the platelet morphology and function. Our protocol allows for efficient high-density binding of DNA origami to platelets using low quantities of the DNA material to label a large number of platelets in a timely manner. Nonactivating platelet-nanotile adducts afford a path for advancing the development of DNA origami nanoconstructs for cell-adherent mechanosensing and therapeutic agent delivery.

Hemolytic uremic syndrome: An updated review

Hemolytic uremic syndrome (HUS) is a microangiopathic thrombotic disease. HUS is classified into typical, secondary, and atypical types. All types are characterized by thrombocytopenia, acute kidney impairment, and hemolysis. Infection with Shiga toxin from Escherichia coli causes typical HUS. Atypical HUS is frequently caused by abnormal complement activation via the alternative pathway because of gene mutations or autoantibodies synthesis. Secondary HUS accompanies post-transplantation, autoimmunity, cancer, etc. Endothelial cell injury initiates cells destruction stimulates procoagulant state, which activates platelet and thrombus generation, producing ischemic tissue injury.
 HUS pathogenesis is the result of an ineffective complement activation cycle that causes endothelial cells damage, activating platelet and thrombus formation. In some atypical HUS cases, interrupting the pathogenesis cycle of HUS by inhibiting complement activation might be beneficial, but not in those with gene mutations and patients with typical or secondary HUS. Hence, the pathogenesis of HUS types understanding is required; therefore, a comprehensive review of the differences and resemblances between the HUS types will be discussed and updated.

Low COAT platelets are frequent in patients with bleeding disorders of unknown cause (BDUC) and can be enhanced by DDAVP

Low COAT platelets are frequent in patients with bleeding disorders of unknown cause (BDUC) and can be enhanced by DDAVP

Production Protocol Standardisation, Macroscopic and Histological Evaluation, and Growth Factor Quantification of Canine Leukocyte-and Platelet-Rich Fibrin Membranes.

Leukocyte-Platelet-Rich Fibrin (L-PRF) is a second generation of platelet concentrates; it was widely used, as an autologous platelet-based wound sealant and hemostatic agent in surgical wound healing. L-PRF clot or membrane is a solid fibrin-based biomaterial, with a specific 3D distribution of the leukocytes and platelet aggregates. This biological scaffold releases growth factors (i.e., TGF- β1, PDGF-AB, VEGF) and matrix proteins (fibronectin, vitronectin and thrombospondin-1) during the healing process after the application. To the Authors' knowledge both in human and veterinary medicine a single standardised protocol was not reported. This prospective study aimed to apply Crisci's L-PRF protocol (which is characterised by 30” of acceleration, 2' at 2,700 rpm, 4' at 2,400 rpm, 3' at 3,000 rpm, and 36” of deceleration and arrest) sin canine species, evaluate macroscopically and histologically the L-PRF membranes obtained by using Wound Box to standardise the L-PRF protocol in dogs and to evaluate the clinical feasibility of using L-PRF membranes by quantitative in vitro analysis of growth factors over 7 days. One hundred twenty-eight dogs in good general condition with no history of recent NSAIDs intake (15 days of washout) and/or any medication or disease related to coagulation process met inclusion criteria and therefore were enrolled. We obtained 172 membrane L-PRF membranes by 86 dogs: half of them underwent macroscopic and histological analysis, the other 86 underwent ELISA analysis. The Wound Box gave a membrane of mean (±SD) length (cm), width (cm) and weight (g) of 1.97 (±0.89), 0.95 (±0.36), 0.46 (±0.20) respectively. Histology analysis confirmed a well-defined histoarchitecture with five layers reproducing density and distribution of blood cells in this biomaterial. Finally, the ELISA assay performed with 22 L-PRF membranes showed a peak in growth factors at 6 h after membrane production, followed by a decrease in release at 24 and 72 h and a second peak in release at 168 h after production. Statistical analysis of demographic variables (age, sex, and body condition score BCS) and the average of growth factors determined by the ELISA assay did not reveal statistical significance, except for the BCS factor compared with the production of VEGF. Our data confirm the effectiveness of this protocol and of Wound Box to produce L-PRF membranes in dogs.