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Background: Platelet-rich plasma (PRP) has the potential to be used for bone regeneration. However, its effect on osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and its effect on cell autophagy of hPDLSCs remain unknown. In this study, we investigated the effects of PRP on cell viability and osteogenic differentiation of hPDLSCs and the underlying molecular mechanisms., Methods: hPDLSCs were isolated and identified by morphology and flow cytometry analysis. Next, thrombin-activated PRP was used to stimulate hPDLSCs. The MTT assay was used to analyze cell viability. Osteogenic differentiation was investigated using alkaline phosphatase (ALP) activity assay, alizarin red S (ARS) staining, and gene expression analysis of osteogenic markers. Expression of the autophagic proteins was determined using western blotting., Results: Thrombin-activated PRP significantly enhanced cell viability, ALP activity, osteogenic-related mRNA levels and alizarin red-mineralization activity in hPDLSCs in a dose-dependent manner. Furthermore, activated PRP dose-dependently increased LC3-II/I ratio and the expression of SIRT1 and Beclin-1. PRP treatment also enhanced the autophagic flux. It was also demonstrated that the inhibition of SIRT1 using sirtinol or suppression of autophagy by 3-methyladenine (3-MA) abrogated PRP-induced viability and osteogenic differentiation of hPDLSCs., Conclusion: Our study suggested that thrombin-activated PRP accelerated the viability and osteogenic differentiation of hPDLSCs via SIRT1-mediated autophagy induction., (© 2021. The Author(s).)

Platelets mediate circulating endothelial progenitor cell (EPC) recruitment and maturation, participating in vascular repair, however the underlying mechanism(s) remain unclear. We investigated the effect of platelet-rich plasma (PRP) on the functionality of CD34 + -derived late-outgrowth endothelial cells (OECs) in culture. Confluent OECs were coincubated with PRP under platelet aggregation (with adenosine diphosphate; ADP) and nonaggregation conditions, in the presence/absence of the reversible P2Y12 platelet receptor antagonist ticagrelor. Outgrowth endothelial cell activation was evaluated by determining prostacyclin (PGI 2 ) and monocyte chemoattractant protein-1 (MCP-1) release and intercellular adhesion molecule-1 (ICAM-1) membrane expression. Similar experiments were performed using human umbilical vein endothelial cells (HUVECs). Platelet-rich plasma increased ICAM-1 expression and PGI 2 and MCP-1 secretion compared with autologous platelet-poor plasma, whereas ADP-aggregated platelets in PRP did not exhibit any effect. Platelet-rich plasma pretreated with ticagrelor prior to activation with ADP increased all markers to a similar extent as PRP. Similar results were obtained using HUVECs. In conclusion, PRP induces OEC activation, a phenomenon not observed when platelets are aggregated with ADP. Platelet inhibition with ticagrelor restores the PRP capability to activate OECs. Since EPC activation is important for endothelial regeneration and angiogenesis, we suggest that agents inhibiting platelet aggregation, such as ticagrelor, may promote platelet-EPC interaction and EPC function.

To be faced with an infected bone defect and the need to accelerate bone union while controlling infection is a welcome challenge for orthopedists. Platelet-rich plasma (PRP) has been applied in tissue defects given their composition of growth factors however the weak antibacterial effects have limited the use of PRP in the clinical setting. Therefore, the aim of this study was to explore the feasibility of using PRP in a local antibiotic delivery system (PADS) with the characteristics of promoting wound healing of bone infection. PADS was prepared with the addition of antibiotics or no antibiotics as control after PRP was prepared by a two-step centrifugation procedure. Antibacterial tests showed zones of inhibition produced by antibiotics were not significantly different with antibiotics combined with PRP. HPLC analysis demonstrated that about 60% of the total vancomycin (VAN) and ceftazidime (CAZ) dose were released within 10 min, then the release rate gradually decreased. However, 90% clindamycin was released within 10 min. Interestingly, above 10 times the minimum inhibitory concentration was presented after 72 h. Additionally, ELISA and morphology studies of PADS indicated that loaded antibiotics could reduce the PRP-released growth factor concentration and disturb the structure of platelet-fibrin beams and fibrin network in a dose-dependent manner. Fortunately, the lower dose of antibiotics maintained their anti-microbial effect, meanwhile growth factors released from PADS, the structure of platelet-fibrin beams, fibrin network remained unaffected. In addition, a patient experiencing infected bone defect receiving this PADS treatment achieved union within the 15-month follow-up. Therefore, this novel PADS approach might represent a potential therapy for patients who have sustained infected bone defects.

PRP-Exos are nanoscale cup-shaped vesicles that carry a variety of proteins, mRNAs, microRNAs, and other bioactive substances. PRP-Exos can be formed through several induction pathways, which determine their molecular profiles and facilitate their tailormade participation in intercellular communication. Currently, little is known on how the PRP-Exos activation method influences the quality and quantity of PRP-Exos. The present study aims to observe and analyze the number, profile, and growth factors of PRP-Exos through TEM, Nanoflow, and WB after PRP activation and compare the difference in function of PRP-Exos on HUVECs, with different stimuli (calcium gluconate, thrombin, or both). We found that PRP activated with both thrombin and calcium gluconate harvested the highest concentration of exosomes [(7.16 ± 0.46) × 10 10 particles/ml], compared to thrombin group [(4.87 ± 0.15) × 10 10 particles/ml], calcium gluconate group [(5.85 ± 0.43) × 10 10 particles/ml], or saline group [(7.52 ± 0.19) × 10 9 particles/ml], respectively ( P < 0.05) via Nanoflow analysis. The WB analysis showed that cytokines (VEGF, PDGFBB, bFGF, TGF-β) are differentially encapsulated in PRP-Exos, depending on the PRP stimulus, in which the mixture-PRP-Exos yielded the highest concentration of cytokines. In the function assay of PRP-Exos on HUVECs, the mixture-PRP-Exos promoted HUVECs proliferation, increased HUVECs migration, promoted the formation of vessel-like by HUVECs via the AKT ERK signal pathway more dramatically, compared with other groups. In summary, our studies showed that PRP activated by the mixture of calcium gluconate and thrombin harvested the best quality of exosomes which had the top biological functions. This study provides a protocol for selecting appropriate PRP activators to obtain high-quality exosomes for future applications.

The expansion of nanotechnology for drug delivery applications has raised questions regarding the safety of nanoparticles (NPs) due to their potential for interacting at molecular and cellular levels. Although polymeric NPs for drug delivery are formulated using FDA-approved polymers such as lactide- and glycolide-based polymers, their interactions with blood constituents, remain to be identified. The aim of this study was to determine the impact of size-selected Poly-lactide-co-glycolide-polyethylene glycol (PLGA-PEG) NPs on platelet activity. The NPs of 113, 321, and 585 nm sizes, were formulated and their effects at concentrations of 0-2.2 mg/mL on the activation and aggregation of platelet-rich plasma (PRP) were investigated. The results showed that NPs of 113 nm did not affect adenosine diphosphate (ADP)-induced platelet aggregation at any NP concentration studied. The NPs of 321 and 585 nm, at concentrations ≥0.25 mg/mL, reduced ADP-activated platelet aggregation. The platelet activation profile remained unchanged in the presence of investigated NPs. Confocal microscopy revealed that NPs were attached to or internalised by platelets in both resting and activated states, with no influence on platelet reactivity. The results indicate minimal risks of interference with platelet function for PLGA-PEG NPs and that these NPs can be explored as nanocarriers for targeted drug delivery to platelets.

Mesenchymal stem cells (MSCs) have been recently introduced in veterinary medicine as a potential therapeutic tool for several pathologies. The large-scale in vitro expansion needed to ensure the preparation of a suitable number of MSCs for clinical application usually requires the use of xenogeneic supplements like the fetal bovine serum (FBS). The substitution of FBS with species-specific supplements would improve the safety of implanted cells, reducing the risk of undesired immune responses following cell therapy. We have evaluated the effectiveness of canine adipose tissue-derived stromal vascular fraction (SVF) and MSCs (ADMSCs) expansion in the presence of canine blood-derived supplements. Cells were cultured on traditional plastic surface and inside a 3D environment derived from the jellification of different blood-derived products, i.e., platelet-poor plasma (PPP), platelet-rich plasma (PRP), or platelet lysate (PL). PPP, PRP, and PL can contribute to canine ADMSCs in vitro expansion. Both allogeneic and autologous PPP and PL can replace FBS for ADMSCs culture on a plastic surface, exhibiting either a similar (PPP) or a more effective (PL) stimulus to cell replication. Furthermore, the 3D environment based on homospecific blood-derived products polymerization provides a strong stimulus to ADMSCs replication, producing a higher number of cells in comparison to the plastic surface environment. Allogeneic or autologous blood products behave similarly. The work suggests that canine ADMSCs can be expanded in the absence of xenogeneic supplements, thus increasing the safety of cellular preparations. Furthermore, the 3D fibrin-based matrices could represent a simple, readily available environments for effective in vitro expansion of ADMSCs using allogeneic or autologous blood-products.

Our objective in this study was to determine the biocompatibility and hemocompatibility of thin film metallic glass (TFMG) and its potential use in hypodermic needles for intramuscular or intravenous injection. Mouse and rabbit models were employed under approval from the Institutional Animal Care and Use Committee (n = 5/group, two groups in total for both animal models). Platelet-rich plasma (PRP) was collected from the whole blood of rabbits (ear vein) without anti-coagulant for use in in vitro coagulation tests. Histological analysis and optical microscopy were used to assess the endothelial structure of the inner lining of veins after being punctured with needles and detained for 3 days. Histological analysis of ear vein sections revealed that the extent of endothelial damage after puncturing with a TFMG-coated needle was 33% less than that produced by bare needles. Our results confirm that the deposition of a thin TFMG layer (e.g., Zr 53 Cu 33 Al 9 Ta 5 ) on the surface of hypodermic needle can have remarkably clinical benefits, including anti-adhesion, reduced invasion, and minimal endothelial damage. Our results also confirm the good biocompatibility and hemocompatibility of the TFMG coatings.

Rhizoma Bletillae, the tubes of Bletilla striata, has been traditionally used in China as a hemostatic agent. In preliminary studies, the major active fraction responsible for its hemostatic effect have been confirmed to be Rhizoma Bletillae polysaccharide (RBp), but the hemostatic mechanism of action of RBp is still unknown.The main aim of this study was to clarify its mechanism of hemostatic effect. RBp was prepared by 80 % ethanol precipitation of the water extract of Rhizoma Bletillae followed by the Sevag method to remove proteins. The average molecular weight (Mw) of the crude RBp maintained at a range of 30.06-200 KDa. The hemostatic effects of RBp were evaluated by testing its effect on the platelet aggregation of rat platelet-rich plasma (PRP). PRP was dealt with different concentrations of RBp and platelet aggregation was measured by the turbidimetric method. The hemostatic mechanism of RBp was investigated by examining its effect on platelet shape, platelet secretion, and activation of related receptors (P2Y 1 , P2Y 12 and TXA2) by electron microscopy and the turbidimetric method. RBp significantly enhanced the platelet aggregations at concentrations of 50-200 mg/L in a concentration-dependent manner. The inhibitory rate of platelet aggregation was significantly increased by apyrase and Ro31-8220 in a concentration-dependent manner, while RBp-induced platelet aggregation was completely inhibited by P2Y 1 , P2Y 12 and the PKC receptor antagonists. However, the aggregation was not sensitive to TXA2. RBp, the active ingredients of Rhizoma Bletillae responsible for its hemostatic effect, could significantly accelerate the platelet aggregation and shape change. The hemostatic mechanism may involve activation of the P2Y 1 , P2Y 12 , and PKC receptors in the adenosine diphosphate (ADP) receptor signaling pathway., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: This study evaluated blood components processed by the platelet rich plasma (PRP) method from fresh whole blood (FWB) treated with a pathogen reduction technology (PRT). The effects of storage temperature on PRT treated platelet concentrates (PCs) were also examined., Study Design and Methods: PRT was performed using riboflavin and ultraviolet light on FWB in citrate phosphate dextrose anticoagulant. Following PRT, red blood cells (RBCs), PCs, and plasma for fresh frozen plasma (FFP), were isolated by sequential centrifugation. RBCs were stored at 4°C, FFP at -80°C, and PC at 22°C or at 4°C. Components were assayed throughout their storage times for blood gases, chemistry and CBC, hemostatic function as well as platelet (PLT) and RBC integrity., Results: Component processing following PRT resulted in a significant drop in platelet recovery. Most PRT-PC bags fell below AABB guidelines for platelet count. PRT-PC also showed a decrease in clot strength and decreased aggregometry response. Platelet caspases were activated by PRT. Storage at 4°C improved platelet function. In PRT-FFP, prothrombin time and partial thromboplastin time (PT and aPTT) were prolonged; factors V, VII, VIII, and XI, protein C, and fibrinogen were significantly decreased. Free hemoglobin was elevated two-fold in PRT-RBC., Conclusion: Blood components isolated by the PRP method from PRT-treated WB result in a high percentage of PC that fail to meet AABB guidelines. FFP also shows diminished coagulation capacity. However, PRT-RBC are comparable to control-RBC. PRT-WB retains acceptable hemostatic function but alternatives to the PRP method of component separation may be more suitable., (Published 2020. This article is a U.S. Government work and is in the public domain in the USA.)

Previous in vitro studies suggest a direct relevance for the peptide-free lipid fraction (LF) of platelet-rich plasma (PRP) in biological mechanisms related to wound healing. However, there are no scientific reports to date on the wound healing activities of this lipid component in vivo . Thus, the present study provides a scientific evaluation for the wound healing potential of the lipid portion of the activated PRP. For the wound healing activity assessment, in vivo full-thickness excisional wounds were created on the dorsal skin of Sprague-Dawley rats. Lipid extract from pooled PRP was applied topically to the wounds on 0, 3, and 7 days after injury. Histological assessment of epidermal and dermal regeneration, granulation tissue thickness and angiogenesis by Sirius red and Masson's trichrome staining, in addition to immunohistochemical staining for transforming growth factor beta-1 (TGF-β1), collagen type I (COL I), and collagen type III (COL III) were performed on skin biopsies at 3, 7 and 14 days. The total histological scores of the LF group were significantly higher than the 25% dimethylsulfoxide-control group. According to the immunohistochemical staining, the observed expression changes for TGF-β1, COL I and III at 3, 7, and 14 days after wounding were significantly better in the study group than the control group. Furthermore, COL I/III ratio in the lipid extract-treated group at day 14 was much higher than that of the control group. Meanwhile, analysis of the data also indicated that the LF has less positive effects on all evaluated parameters than PRP. From the present data, it could be concluded that the peptide-free LF of PRP has potent wound healing capacity in vivo for cutaneous wounds, although not as much as that of PRP. Strengthening our understanding of the wound healing potential of lipid components of PRP and platelet-derived lipid factors may provide new avenues for improving the healing process of a wound with elevated protease activity.

Losartan and honokiol are small molecules which have been described to inhibit aggregation of platelets by collagen. Losartan has been proposed to block clustering of GPVI but not to affect binding of collagen. Honokiol has been reported to bind directly to GPVI but only at a concentration that is three orders of magnitude higher than that needed for inhibition of aggregation. The mechanism of action of both inhibitors is so far unclear. In the present study, we confirm the inhibitory effects of both agents on platelet aggregation by collagen and show that both also block the aggregation induced by the activation of CLEC-2 or the low affinity immune receptor FcγRIIa at similar concentrations. For GPVI and CLEC-2, this inhibition is associated with a reduction in protein tyrosine phosphorylation of multiple proteins including Syk. In contrast, on a collagen surface, spreading of platelets and clustering of GPVI (measured by single molecule localisation microscopy) was not altered by losartan or honokiol. Furthermore, in flow whole-blood, both inhibitors suppressed the formation of multi-layered platelet thrombi at arteriolar shear rates at concentrations that hardly affect collagen-induced platelet aggregation in platelet rich plasma. Together, these results demonstrate that losartan and honokiol have multiple effects on platelets which should be considered in the use of these compounds as anti-platelet agents.

Background . Studies of platelet aggregation (PA) in essential thrombocythemia (ET) reported contrasting results, likely due to differences in analytical conditions. Objective . We investigated platelet aggregation using different techniques and analytical conditions. Patients and Methods . PA was studied by light-transmission aggregometry (LTA) in platelet-rich plasma (PRP) and impedance aggregometry in PRP and whole blood (WB). ADP, collagen, thrombin receptor activating peptide (TRAP-14) and adrenaline were used as agonists. Since ET patients (n = 41) were on treatment with aspirin (100 mg/d), healthy controls (n = 29) were given aspirin (100 mg/d) for 5 days before testing: therefore, thromboxane A 2 -independent PA was tested in all subjects. Blood samples were collected in citrate (C) [low Ca 2+ ] or lepirudin (L) [physiological Ca 2+ ]; platelet count was adjusted to 250 x 10 9 /L in a set of C-PRP (adjusted C-PRP) and left unmodified in the other samples. Results . Results of PA in 17 ET patients who were poor responders to aspirin (high serum thromboxane B2 levels) were not included in the analysis. With LTA, PA in ET was lower than in controls in adjusted C-PRP and normal in native C-PRP and L-PRP. With impedance aggregometry, PA in L-PRP and L-WB tended to be higher in ET than in controls. Platelet serotonin and ADP contents were reduced in ET. The percentages of circulating platelets expressing P-selectin and platelet-leukocyte hetero-aggregates were higher in ET. Conclusions . Analytical conditions dramatically affect in vitro PA of ET patients, which appears defective under the least physiological conditions and normal/supranormal under conditions that are closer to the physiological.

Background: Light transmission aggregometry (LTA) is the gold standard for platelet function assessment. The automated coagulation analyzer from Sysmex that performs LTA offers the advantage of being a walk-away technology. Recently, a new parameter "ADP-induced platelet aggregation level (APAL)" was developed to support the interpretation of results. APAL is calculated as a score from 0.0 to 10.0 based on platelet aggregation patterns with 1 and 10 µM adenosine diphosphate (ADP). Here, the basic performance of the newly developed APAL system and comparison with the maximum aggregation rate of ADP (ADP-MA) was evaluated., Methods: The within-run precision was calculated by conducting five replicate analyses of the platelet-rich plasma (PRP) from healthy volunteers and 0.05 µM of cangrelor-spiked PRP. Cangrelor is a P2Y12 inhibitor that does not require liver CYP activation. The reference interval was calculated from the results of 67 healthy volunteers. The effect of the antiplatelet P2Y12 agent was evaluated using several concentrations of cangrelor. A comparative study was performed using 103 PRP samples with different levels of aggregation. Each test was analyzed with both APAL and ADP-MA., Results: The percentage coefficient of variation in within-run precision was within 7% for APAL and 10 µM ADP-MA. Reference interval of APAL and 10 µM ADP-MA was 7.1 - 10.0 and 80.0 - 99.2%, respectively. APAL signifi-cantly decreased with the addition of 0.02 µM cangrelor, while 10 µM ADP-MA was barely affected. A significant correlation was observed between APAL and 10 µM ADP-MA (r = 0.94; p < 0.0001)., Conclusions: The newly developed APAL system exhibited an acceptable performance. APAL score showed a good correlation with ADP-MA and was adequate to detect the weak effect of P2Y12 inhibitors. APAL is a new platelet aggregation scoring system with the potential to monitor the effects of P2Y12 inhibitor over a wide range.

Background: The aim of this study was to assess the possible association between different factors such as age, sex, antibiotic consumption duration, angiogenesis and pain and "acceleration of wound healing" in pilonidal sinus patients after treating with platelet-rich plasma (PRP)., Methods: In this clinical trial, 110 patients were randomly divided into treatment arm and control group. After surgery, control group underwent classic wound dressing and the treatment arm experienced PRP gel therapy. Before achieving complete healing, wound incisional biopsy was performed in order to evaluate angiogenesis. During the study, other data such as pain and antibiotic consumption duration were also collected. Wound healing time of pilonidal sinus disease was analyzed using Extended and Stratify Cox model. Data were analyzed using R and STATA software. p<0.05 were considered statistically significant., Results: The average wound volume was calculated 41.9 ± 8.01 cc in the controls and 42.35 ± 10.81 in the treatment arm group. The mean of healing time was 8.7 ± 1.18, 4.8 ± 0.87 weeks for control and treatment arm, respectively. There was a significant and strong negative association between healing time and wound volume (p<0.01). Moreover, a significant negative association was found between pain duration and angiogenesis (p<0.001), a strong positive significant association was found between healing time of the treatment arms (p<0.01), and the rate of wound healing for participants treated with PRP gel was 37.2 times more than that of controls., Conclusion: Authors hope for these finding to help the future researches to more thoroughly focus on the mentioned factors in order to find a suitable strategy for wound healing using PRP., (Copyright © 2019 Chang Gung University. Published by Elsevier B.V. All rights reserved.)

Background: Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating antibodies that recognize platelet factor 4/heparin (PF4/hep)-complexes. The in vitro demonstration of PF4/hep antibodies using functional assays is essential for an optimal management of patients suspected to have HIT. However, conventional functional assays are technically challenging and limited to specialized laboratories. In contrast, flow cytometers are commonly used in routine laboratories. The aim of this study is to investigate the performance characteristics of a commercially available, flow cytometer based assay in the diagnosis of HIT., Study Design: Sera of consecutive patients with suspected HIT were investigated using the Emo-test HIT Confirm® assay and compared to the standard method consisting of an IgG-specific enzyme immunoassay (EIA) for anti-PF4/hep antibodies and the heparin induced platelet aggregation (HIPA) test., Results: 390 sera were included in the study, 164 sera tested IgG EIA-positive, of which 33 also tested HIPA-positive. No HIPA-positive samples were EIA-negative. In the Emo-test HIT Confirm® assay, 112 sera revealed positive results (%Hepla > 13); however, 51 (45.5%) were EIA-negative. Of the 33 HIPA-positive/EIA-positive HIT sera, 23 tested positive in the Emo-test HIT Confirm® assay, 2 gave ambiguous results, and 8 sera yielded false-negative results. Accordingly, the HIT Confirm® assay showed a sensitivity of 69.7% with a slightly better specificity of 75.4% compared to the EIA (sensitivity 100%, specificity 63.3%). An increase in diagnostic specificity for HIT to 85% was found when positive results were obtained in both the Emo-test HIT Confirm® assay and EIA., Conclusion: The Emo-Test HIT Confirm® assay may improve the specificity of laboratory investigations of HIT. However, the assay can only be recommended in combination with an immunoassay due to the high rate of false negativity. Our observation indicates a need to establish external quality assessment for functional assays to avoid such clinically relevant pitfalls., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: The benefits of platelet-rich plasma (PRP) are believed to be in part dependent on growth factor release after platelet activation. Platelet activation is complex and involves multiple mechanisms. One important mechanism is driven by cyclooxygenase 1 (COX-1)-mediated conversion of arachidonic acid (AA) to precursor prostaglandins that then mediate proinflammatory responses that trigger growth factor release. Acetylsalicylic acid (ASA; also known as aspirin) is known to irreversibly inhibit COX-1, thereby blocking AA-mediated signaling; however, it is unclear whether ASA use alters growth factor release from freshly isolated PRP., Purpose: To assess the effects of low-dose ASA use on activation of growth factor release from freshly isolated human PRP via AA and thrombin (TBN)., Study Design: Controlled laboratory study., Methods: Twelve healthy men underwent blood collection and leukocyte-rich PRP (LR-PRP) preparation through a double-spin protocol to obtain baseline whole blood and PRP counts the same day. PRP was aliquoted into 3 groups: nonactivated, AA activated, and TBN activated. Immediately after activation, the concentrations of transforming growth factor β1 (TGF-β1), vascular endothelial growth factor (VEGF), and platelet-derived growth factor AB (PDGF-AB) were measured using enzyme-linked immunosorbent assays (ELISAs). The same 12 participants were then placed on an 81-mg daily dose of oral ASA for 14 days. Repeat characterization of whole blood and PRP analyses was done on day 14, followed by repeat ELISAs of growth factors under the same nonactivated and activated settings as previously stated., Results: Fourteen days of daily ASA had no effect on the number of platelets and leukocytes measured in whole blood and LR-PRP. Compared with nonactivated LR-PRP, AA- and TBN-mediated activation led to significant release of VEGF and PDGF-AB. In contrast, release of TGF-β1 from LR-PRP was observed only with activation by AA, not with TBN. Consistent with its inhibitory role in AA signaling, ASA significantly inhibited AA-mediated release of all 3 growth factors measured in this study. Although ASA had no effect on TBN-mediated release of VEGF and TGF-β1 from LR-PRP, ASA did partially block TBN-mediated release of PDGF-AB, although the mechanism remains unclear., Conclusion: Daily use of low-dose ASA reduces VEGF, PDGF-AB, and TGF-β1 expression in freshly isolated human LR-PRP when activated with AA., Clinical Relevance: Reduction in growth factor release attributed to daily use of low-dose ASA or other COX inhibitors can be mitigated when PRP samples are activated with TBN. Clinical studies are needed to determine whether activation before PRP injection is needed in all applications where ASA is in use and to what extent ASA may inhibit growth factor release in vivo at the site of injury.

: Freeze-drying is an effective means of storing platelets. In this study, we investigated the effects of a protective agent on freeze-dried platelet-rich plasma (FD-PRP) after a 12-week preservation period. Platelet structure was measured by transmission electron microscopy (TEM), and the expression levels of procaspase activating compound (PAC)-1 and CD62P were measured by flow cytometry. The levels of transforming growth factor-beta (TGF-β), platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) were determined by ELISA. The effect of FD-PRP on cell proliferation was measured by cell counting. TEM revealed that most platelets were intact, and their internal structure was evident. The expression levels of the platelet activation marker CD62P in FD-PRP and fresh PRP were 36.83% ± 8.21 and 35.47% ± 4.11, respectively, without a significant difference (P > 0.05). The expression levels of PAC-1 in FD-PRP and fresh PRP were 3.23% ± 0.49 and 2.83% ± 0.44, respectively, without a significant difference (P > 0.05). Upon activation of FD-PRP and fresh PRP by thrombin, the levels of TGF-β, PDGF and VEGF were not significantly decreased in FD-PRP. Moreover, FD-PRP promoted cell proliferation in a manner similar to that of fresh PRP. The protective agent maintained the biological activity of FD-PRP after a 12-week preservation period.

The aim of this study was to investigate the influence of different incubation methods on the growth factor content of lysates of platelet-rich fibrin (PRF), advanced-platelet-rich fibrin (A-PRF) and platelet-rich plasma (PRP) products. A comparison of related studies suggests that the method of sample preparation has a significant influence on growth factor content. There are few reports on the comparison of non-Ca 2+ -activated PRP, Ca 2+ -activated PRP, A-PRF, and PRF, along with a lack of information on the release of PDGF-BB, TGF-β1, and VEGF among the different incubation methods. The lysate preparation was made of non-Ca 2+ -activated PRP, Ca 2+ -activated PRP, PRF, and A-PRF, using a room-temperature, 37 °C, or freeze-thaw-freeze incubation method. Afterwards the VEGF, PDGF-BB, and TGF-β1 content was investigated by running ELISA tests. Growth factor levels were significantly increased in the non-Ca 2+ -activated PRP with freeze-thaw-freeze incubation, and in the PRF preparation there was a significant disadvantage to using room temperature incubation for releasing growth factors. In conclusion, the freeze-thaw-freeze method is sufficient for releasing growth factors, and calcium activation is not necessary. Finally, the study demonstrates the possibility of preparing PRP products from platelet concentrates, so that preoperative blood sampling might not be required., (Copyright © 2018 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

Marine and salmon polar lipids (PLs) extracted by conventional extractions with non-food-grade solvents (CE-salmon-PLs) possess antithrombotic bioactivities against platelet-activating factor (PAF) and thrombin. Similar effects of food-grade-extracted (FGE) marine PLs have not yet been reported. In this study, food-grade solvents were used to extract PLs from Irish organic farmed salmon ( Salmo salar ) fillets (FGE-salmon-PLs), while their antithrombotic bioactivities were assessed in human platelets induced by platelet aggregation agonists (PAF/thrombin). FGE-salmon-PLs were further separated by thin layer chromatography (TLC) into lipid subclasses, and the antithrombotic bioactivities of each subclass were also assessed. LC-MS was utilized to elucidate the structure-activity relationships. FGE-salmon-PLs strongly inhibited PAF-induced platelet aggregation, while their relevant anti-thrombin effects were at least three times more potent than the previously reported activities of CE-salmon-PLs. TLC-derived lipid fractions corresponding to phosphatidylcholines (PC) and phosphatidylethanolamines (PE) were the most bioactive lipid subclasses obtained, especially against thrombin. Their LC-MS analysis elucidated that they are diacyl- or alkyl-acyl- PC and PE moieties baring ω3 polyunsaturated fatty acids (PUFA) at their sn -2 position, such as eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA). Our results concerning the potent antithrombotic effects of FGE-salmon-PLs against both PAF and thrombin pathways strongly suggest that such food-grade extracts are putative candidates for the development of novel cardioprotective supplements and nutraceuticals.

As the aged population continues to markedly increase worldwide, the incidences of diabetes mellitus (DM) and cardiovascular disease (CVD) are increasing. In this study, we investigated the effects of aging, DM, and antiplatelet drugs on growth factors and anti-aging proteins in platelet-rich plasma (PRP). The study participants were classified into the following four groups: Group A, healthy individuals aged ≤45 years; Group B, healthy individuals aged >45 years; Group C, DM patients aged >45 years; and Group D, CVD patients aged >45 years taking antiplatelet drugs. The concentrations of epidermal growth factor (EGF), fibroblast growth factor (FGF)-2, platelet-derived growth factor (PDGF)-AA, PDGF-AB/BB, vascular endothelial growth factor (VEGF)-A, tissue inhibitor of metalloproteinase 2 (TIMP2), insulin-like growth factor 1 (IGF-1), growth differentiation factor (GDF)11, and clusterin in PRP samples were determined to analyze the effects of aging, DM, and antiplatelet drugs. Overall, the concentrations of IGF-1, TIMP2, and clusterin did not vary significantly between the four groups. The concentrations of PDGF-AB/BB ( P  = 0.010), VEGF-A ( P  = 0.000), and GDF11 ( P  = 0.026) were significantly different between Group A and Group B. Further, the concentrations of EGF ( P  = 0.000) and GDF11 ( P  = 0.000) were significantly different between Groups B and C. The concentrations of EGF ( P  = 0.001), VEGF-A ( P  = 0.000), and GDF11 ( P  = 0.002) significantly differed between Groups A and C. The concentrations of FGF-2 ( P  = 0.048), PDGF-AA ( P  = 0.03), and GDF11 ( P  = 0.001) were significantly different between Groups B and D. The concentrations of PDGF-AB/BB ( P  = 0.032), VEGF-A ( P  = 0.010), and GDF11 ( P  = 0.02) significantly differed between Groups A and D. We found that PRP contains high concentrations of the growth factors, TIMP2 and GDF11. Aging, DM, and antiplatelet drugs can decrease the concentration of some growth factors and GDF11, which weakens the regenerative capacity and anti-aging effects of PRP and reduces the quality of PRP.

Lithium (Li) is used for the treatment and prophylaxis of mental disorders, associated with many serious hazards. Resveratrol (RSV) has various beneficial therapeutic effects. Platelet-rich plasma (PRP) is a new promising curative tool. This study aimed to assess the impacts of RSV versus PRP on lithium-induced thyroid follicular cell toxicity in adult male rats. Forty-nine adult male rats were divided into four groups: group I: control rats; group II: lithium-treated rats; group III: lithium- and resveratrol-treated rats; group IV: lithium and PRP-treated rats. Thyroid specimens were taken and processed for histological and immunohistochemical methods. Morphometrical studies and statistical analysis were done. Group II showed distorted thyroid follicles, significantly increased collagen fibers, and highly positive P53 immunostaining (P < 0.01). Ultrastructural examination showed dilated rough endoplasmic reticulum and damaged mitochondria. Groups III and IV exhibited significant amelioration of the histological and electron microscopic changes mentioned previously. PRP remedy was more effective than RSV for treatment of Li-induced thyroid follicular cell toxicity.

Proteostasis, which includes the repair and disposal of misfolded proteins, depends, in part, on the activity of heat shock proteins (HSPs), a well-known class of chaperone molecules. When this process fails, abnormally folded proteins may accumulate in cells, tissues, and blood. These species are a hallmark of protein aggregation diseases, but also amass during aging, often in the absence of an identified clinical disorder. We report that a neuroprotective cyclic heptapeptide, CHEC-7, which has been applied systemically as a therapeutic in animal neurodegeneration models, disrupts such aggregates and inhibits amyloidogenesis when added in nanomolar concentrations to human plasma. This effect includes aggregates of amyloid beta (Aβ1-40, 1-42), prominent features of Alzheimer's disease pathology. The activity of endogenous HSP70, a recently discovered target of the peptide, is required as demonstrated by both antibody blocking and application of pifithrin-μ, an HSP70 inhibitor. CHEC-7 is the first high-affinity compound to stimulate HSP70's disaggregase activity and therefore enable this endogenous mechanism in a human systemic environment, increasing the likelihood of a convenient therapy for protein aggregate disease, including age-related failures of protein repair.

Objective: To compare and analyze the effects of different activators on the release curve of TGF-β(1) and PDGF-AB in platelet rich plasma(PRP). Methods: A total of 36 ml peripheral venous blood was obtained from 10 healthy adult volunteers, and the PRP was made by secondary centrifugation. The platelet activator was made by bovine thrombin 1 000 U in 1 ml 10% calcium chloride solution. The Thrombin-PRP group was made by PRP and the activator in a ratio of 10∶1.The Calcium chloride-PRP group was made in a ratio of 10∶1 by PRP and 10% calcium chloride solution instead. The fresh whole blood(whole blood group) and inactived PRP(PRP group) were used as the control groups. The 4 groups were incubated in warm water of 37 ℃ for 0, 1, 8, 24,72 and 168 h. A quantitative sandwich enzyme-linked immunosorbent assays(ELISA) was used to examine the amount of TGF-β(1) and PDGF-AB in different time points of each group. The release curves of TGF-β(1) and PDGF-AB were based on afore-mentioned data, and then comparisons of the release curves of TGF-β(1) and PDGF-AB in different groups were performed by repeated measurement variance analysis. Results: (1)The levels of TGF-β(1) and PDGF-AB in the whole blood group and the PRP group continued to increase within 168 h. PRP immediately formed into a gel after mixture with thrombin combined and calcium chloride, and the concentrations of TGF-β(1) and PDGF-AB reached the peak in 1 h after activation; increased from (42±21)ng/ml and (77±18)ng/ml to (84±21)ng/ml and (124±35)ng/ml, respectively, and then decreased gradually. The release curve was direct and rapid. The PRP became a gel state in approximate 1 h after mixture with calcium chloride, and the concentrations of TGF-β(1) and PDGF-AB were slowly rising and remained high at 168 h. (2)The AUC(0-168h) of TGF-β(1) and PDGF-AB in the PRP group was higher than that in the whole blood group (all P< 0.05) , and the AUC(0-168h) of TGF-β(1) in the Calcium chloride-PRP group was higher than that in the Thrombin-PRP group( Z= -2.26, P< 0.05).However, there was no significant difference in the AUC(0-168h) of PDGF-AB between the Calcium chloride-PRP group and the Thrombin-PRP group( Z= -1.512, P= 0.131). Conclusion: Using calcium chloride as activator can get a higher release concentration of TGF-β(1) and PDGF-AB and a longer release time, with the largest area under the curve.

Vascular endothelial dysfunction and platelet activation play a key role in tumor metastasis, and therefore, both of these processes are considered important therapeutic targets in cancer. The aim of our studies was to analyze antimetastatic activity of combination therapy using nitric oxide donor DETA/NO and antiplatelet drug clopidogrel. Nitric oxide acts as a vasoprotective mediator, while clopidogrel inhibits ADP-mediated platelet aggregation. 4T1-luc2-tdTomato cell line transplanted intravenously (i.v.) and 4T1 cell line transplanted orthotopically were used as metastatic mammary gland cancer models. Moreover, antiaggregation action of compounds was tested ex vivo on the blood samples taken from breast cancer patients. We have shown that in selected dosage regimes, DETA/NO combined with clopidogrel significantly reduced lung metastatic foci formation in an i.v. model, and such inhibition was transiently observed also in an orthotopic model. The antimetastatic effect was correlated with a significant increase of prostacyclin (PGI2) metabolite and reduction of endothelin-1, sE-selectin, sI-CAM, and TGF-β plasma levels as well as decreased V-CAM expression on the endothelium. Combination therapy decreased fibrinogen binding to the resting platelets at the early stage of tumor progression (day 14). However, at the later stages (days 21 and 28), the markers of platelet activation were detected (increased JON/A antibody bound, P-selectin level, binding of fibrinogen, and vWf). Decreased aggregation as well as a lower release of TGF-β were detected in platelets incubated ex vivo with compounds tested from metastatic breast cancer patients. Although combination therapy increases E-cadherin, the increase of N-cadherin and α-SMA in tumor tissue was also observed. The results showed that at the early stages of tumor progression, combined therapy with DETA/NO and clopidogrel improves vasoprotective and antiplatelet activity. However, in advanced tumors, some adverse effects toward platelet activation can be observed.

Background: Knee osteoarthritis (KOA) is a progressive joint disease involving intraarticular and periarticular structures. In recent years, there has been increasing interest in the use of autologous growth factors, such as intraarticular injections of platelet-rich plasma (PRP), to treat KOA. It is necessary to update the research and reevaluate the efficacy and safety of PRP to provide up-to-date evidence for KOA management. Therefore, we provide a protocol for a systematic review of PRP for KOA., Methods: The aim of this study was to retrieve papers on the topic of PRP treatment for KOA in electronic databases including PubMed, Embase, and the Cochrane Library. The search will include studies that were published from the time the databases were established until April 2018. The entire process will include study selection, data extraction, risk of bias assessment, and meta-analyses., Results: The literature will provide a high-quality analysis of the current evidence supporting PRP for KOA based on various comprehensive assessments including the Western Ontario and McMaster Universities Osteoarthritis Index, visual analog scale scores, International Knee Documentation Committee scores, Lequesne index scores, and adverse events., Conclusion: This proposed systematic review will provide up-to-date evidence to assess the effect of PRP treatment for patients with KOA., Prospero Registration Number: CRD42018108825.

Background: The aim of this study was to report the short-term clinical and imaging outcomes of lateral meniscus allograft transplantations (LMAT) combined with intra-articular platelet-rich plasma (PRP) injection., Methods: Thirty-three patients who had undergone LMAT combined with intra-articular PRP injection were evaluated. The Lysholm, International Knee Documentation Committee (IKDC), Western Ontario and McMaster Universities Osteoarthritis Index, Tegner activity level scale and visual analog scale for pain scores were used to evaluate the outcomes. Magnetic resonance imaging scans were performed postoperatively to assess graft position and chondral degeneration/damage., Results: A total of 31 of the original 33 patients were evaluated over a mean follow-up period of 37.0months. Patients demonstrated statistically significant improvements in all scoring data from the pre-operative to two-year follow-up period. The mean postoperative extrusion was 1.59±1.20mm (range 0-3.9mm). There were no significant differences in the distribution of the grade of chondral damage between the pre-operative and two-year follow-up periods. Three patients (9.7%) showed no improvements or had lower evaluation scores. One patient underwent matrix-induced autologous chondrocyte implantation at one year after LMAT., Conclusion: Lateral meniscus allograft transplantation combined with intra-articular PRP injection resulted in statistically significant improvements in all functions and pain scores, and clinical improvements in Tegner, IKDC, and Lysholm values during short-term follow-up. A further case-control study with a larger sample size and longer follow-up is required to obtain an overall assessment of the benefits of PRP on MAT patients. Level of evidence IV., (Copyright © 2018. Published by Elsevier B.V.)

Essentials Successful outcome of platelet transfusion depends on specific antiplatelet therapy in use. We assessed if ticagrelor, clopidogrel or prasugrel impacts on donor platelet activity ex vivo. Ticagrelor and/or its active metabolite in plasma or bound to platelets can inhibit donor platelets. This might compromise the effectiveness of platelet transfusion therapy., Summary: Background Platelet transfusion is the conventional approach to restore platelet function during acute bleeds or surgery, but successful outcome depends on the specific antiplatelet therapy. Notably ticagrelor is associated with inadequate recovery of platelet function after platelet transfusion. We examined whether plasma and/or platelets from ticagrelor-treated patients influence donor platelet function, in comparison with clopidogrel and prasugrel. Methods Platelet transfusion was mimicked ex vivo by mixing naïve donor platelet-rich plasma (PRP) or gel-filtered platelets (GFP) in defined proportions with PRP, plasma or GFP from cardiovascular patients receiving standard care including medication with prasugrel, clopidogrel or ticagrelor (n = 20 each). Blood was taken 4 h after the previous dose. HLA2/HLA28 haplotyping let us distinguish net (all platelet) and individual patient/donor platelet reactivity in mixtures of patient/donor platelets, measured by flow cytometry analysis of ADP-induced fibrinogen binding and CD62P expression. Results ADP responsiveness of donor platelets was dramatically reduced by even low (10%) concentrations of PRP or plasma from ticagrelor-treated patients. Clopidogrel and prasugrel were associated with more modest donor platelet inhibition. GFP from ticagrelor-treated patients but not patients receiving clopidogrel or prasugrel also suppressed donor GFP function upon mixing, suggesting the transfer of ticagrelor from patient platelets to donor platelets. This transfer did not lead to recovery of ADP responsiveness of patient's platelets. Conclusion Collectively, these observations support the concept that ticagrelor and/or its active metabolite in plasma or bound to platelets can inhibit donor platelets, which might compromise the effectiveness of platelet transfusion therapy., (© 2018 International Society on Thrombosis and Haemostasis.)

The current study aimed to enhance the efficacy of peripheral nerve regeneration using a biodegradable porous neural guidance conduit as a carrier to transplant allogeneic Schwann cells (SCs). The conduit was prepared from polyurethane (PU) and gelatin nanofibrils (GNFs) using thermally induced phase separation technique and filled with melatonin (MLT) and platelet-rich plasma (PRP). The prepared conduit had the porosity of 87.17 ± 1.89%, the contact angle of 78.17 ± 5.30° and the ultimate tensile strength and Young's modulus of 5.40 ± 0.98 MPa and 3.13 ± 0.65 GPa, respectively. The conduit lost about 14% of its weight after 60 days in distilled water. The produced conduit enhanced the proliferation of SCs demonstrated by a tetrazolium salt-based assay. For functional analysis, the conduit was seeded with 1.50 × 10 4 SCs (PU/GNFs/PRP/MLT/SCs) and implanted into a 10-mm sciatic nerve defect of Wistar rat. Three control groups were used: (1) PU/GNFs/SCs, (2) PU/GNFs/PRP/SCs, and (3) Autograft. The results of sciatic functional index, hot plate latency, compound muscle action potential amplitude and latency, weight-loss percentage of wet gastrocnemius muscle and histopathological examination using hematoxylin-eosin and Luxol fast blue staining, demonstrated that using the PU/GNFs/PRP/MLT conduit to transplant SCs to the sciatic nerve defect resulted in a higher regenerative outcome than the PU/GNFs and PU/GNFs/PRP conduits.

The 2013 ISTH-SSC guidelines for the standardization of light transmission aggregometry (LTA) were largely based on expert consensus, as studies directly comparing LTA methodologies were lacking. We experimentally tested the cogency of ISTH-SSC recommendations pertaining to use of anticoagulant, in particular whether: (1) buffered citrate (BC) is preferable to unbuffered citrate (C); (2) the two recommended concentrations of sodium citrate (109 and 129 mM) are equivalent in terms of platelet aggregation (PA). Blood from 16 healthy volunteers was collected into BC and C (109 and 129 mM). PA was measured by LTA in platelet-rich plasma (PRP) stimulated by adenosine diphosphate (ADP) (2 μM) immediately after PRP preparation and up to 4 hr after blood collection; pH and platelet counts in PRP were measured in parallel. pH in PRP increased with time up to about 8 for all anticoagulants, although it was lower in BC than in C at all times. In BC, PA was lower at 45 min, but equivalent at all other times. PA was higher and more stable in sodium citrate 109 mM than in 129 mM at all times. The extent of PA did not change for up to 2 hr after blood collection, and subsequently dramatically decreased. In contrast with ISTH-SSC recommendations, (1) BC does not show advantages compared to C; (2) 109 mM citrate is preferable to 129 mM, because it better supports PA; and (3) LTA studies should be completed within 2 hr of blood collection, instead of the recommended 4 hr.

The objective of this network meta-analysis (NMA) was to assess the pain relief performance of eight different plantar fasciitis therapies, including nonsteroidal anti-inflammatory medications, corticosteroid injections (CSs), autologous whole blood, platelet-rich plasma (PRP), extracorporeal shockwave therapy (ESWT), ultrasound therapy (US), botulinum toxin A (BTX-A), and dry needling (DN). Published prospective or randomized controlled trials (RCTs) as for the above eight therapies were identified by searching CNKI, PubMed, and Embase. Mean difference (MD) and 95% credible intervals (CrIs) of visual analogue scale (VAS) were used to evaluate multiaspect comparisons. The ranking result was obtained by utilizing surface under cumulative ranking curve (SUCRA). Node-splitting plots were conducted to assess the consistency between direct and indirect evidence. Egger's test and funnel plots were performed to examine publication bias. Forty-one trials with a total of 2,889 cases were involved in this NMA. In terms of 1-month VAS, only ESWT turned out to be of better efficacy than placebo (MD = -3.3; CrI: [-5.3, -1.1]). No statistically significant difference was found between pair-wise comparisons concerning 2-month VAS. ESWT also demonstrated better efficacy as for 3-month results (MD = -2.7; CrI: [-4.2, -1.3]). Besides, CSs was significantly better than placebo as well in 3-month results (MD = -2.1; CrI: [-4.1, -0.19]). With regard to 6-month VAS results, ESWT performed better than placebo (MD = -3.0; CrI: [-5.0, -0.51]). According to the SUCRA, ESWT ranked the first as for all seven outcomes. ESWT might be the optimal treatment. In addition, BTX-A and PRP were considered as suboptimal., (© 2018 Wiley Periodicals, Inc.)

Cell-based therapy and regenerative medicine offer a paradigm shift in regard to various diseases causing loss of substance or volume and tissue or organ damage. Recently, many authors have focused their attention on mesenchymal stem cells for their capacity to differentiate into many cell lineages. The most widely studied types are bone marrow mesenchymal stem cells and adipose derived stem cells (ADSCs), which display similar results. Based on the literature, we believe that the ADSCs offer advantages because of lower morbidity during the harvesting procedure. Additionally, platelet-rich plasma can be used in this field for its ability to stimulate tissue regeneration. The aim of this chapter is to describe ADSC preparation and isolation procedures, preparation of platelet-rich plasma, and the application of ADSCs in regenerative plastic surgery. We also discuss the mechanisms and future role of ADSCs in cell-based therapy and tissue engineering.

Under ischemic conditions, tissues are exposed to hypoxia. Although human physiology, to a certain extent, can adapt to hypoxic conditions, the impact of low oxygen levels on platelet function is unresolved. Therefore, we explored how reduction of atmospheric oxygen levels to 1% might affect agonist-induced aggregation and static adhesion of isolated human platelets. We uncovered that isolated, washed human platelets exposed to hypoxic conditions show reduced thrombin receptor-activating peptide-6 (TRAP-6) and convulxin-induced aggregation. Of note, this hypoxia-triggered effect was not observed in platelet-rich plasma. Independent of the agonist used (TRAP-6, ADP), activation of the platelet fibrinogen receptor integrin α IIb β 3 (GPIIbIIIa, CD41/CD61) was strongly reduced at 1% and 8% oxygen. The difference in agonist-induced integrin α IIb β 3 activation was apparent within 5 minutes of stimulation. Following hypoxia, re-oxygenation resulted in the recovery of integrin α IIb β 3 activation. Importantly, platelet secretion was not impaired by hypoxia. Static adhesion experiments revealed decreased platelet deposition to fibrinogen coatings, but not to collagen or vitronectin coatings, indicating that specifically the function of the integrin subunit α IIb is impaired by exposure of platelets to reduced oxygen levels. Our results reveal an unexpected effect of oxygen deprivation on platelet aggregation mediated by the fibrinogen receptor integrin α IIb β 3 .

Electric pulses can induce various changes in cell dynamics and properties depending upon pulse parameters; however, pulsed power generators for in vitro and ex vivo applications may have little to no flexibility in changing the pulse duration, rise- and fall-times, or pulse shape. We outline a compact pulsed power architecture that operates from hundreds of nanoseconds (with the potential for modification to tens of nanoseconds) to tens of microseconds by modifying a Marx topology via controlling switch sequences and voltages into each capacitor stage. We demonstrate that this device can deliver pulses to both low conductivity buffers, like standard pulsed power supplies used for electroporation, and higher conductivity solutions, such as blood and platelet rich plasma. We further test the effectiveness of this pulse generator for biomedical applications by successfully activating platelets ex vivo with 400 ns and 600 ns electric pulses. This novel bioelectrics platform may provide researchers with unprecedented flexibility to explore a wide range of pulse parameters that may induce phenomena ranging from intracellular to plasma membrane manipulation.

Purpose: To determine whether platelet-rich plasma (PRP) injections are associated with improved pain and function scores when compared with corticosteroid injections for plantar fasciopathy., Methods: A systematic review of published literature was performed for studies comparing PRP injections and corticosteroid injections for plantar fasciopathy. Studies were assessed using the Cochrane Risk of Bias Tool and the Newcastle Ottawa Scale (NOS). The primary endpoint was pain and function score at three and six month follow-up. Sensitivity analysis was performed for high quality studies and randomised studies., Results: Ten studies totalling 517 patients were included. Seven studies were randomised. All studies included patients who had failed conservative measures and excluded patients with systemic illness and other causes of foot pain. Studies reported outcomes using the visual analogue score (VAS) and American Orthopaedic Foot and Ankle Score (AOFAS). At 3-month follow-up, PRP injections were associated with improved VAS scores (standard mean difference [SMD], -0.66; 95% CI, -1.3 to -0.02; p = 0.04) and AOFAS scores (SMD, 1.87; 95% CI, 0.16-3.58; p = 0.03). At 6-month follow-up, there was no difference in VAS score (SMD, -0.66; 95% CI, -1.65 to 0.3; p = 0.17) or AOFAS scores (SMD, 1.69; 95% CI, -1.06 to 4.45; p = 0.23). No studies reported adverse event rates or cost analysis. There was no difference in pain or function score at one, six- or 12-month follow-up. Sensitivity analyses of high-quality studies showed no differences between the PRP and steroid group at any of the follow-up points., Conclusions: PRP injections are associated with improved pain and function scores at three month follow-up when compared with corticosteroid injections. Information regarding relative adverse event rates and cost implications is lacking. Further, large-scale, high-quality, randomised controlled trials with blinding of outcome assessment and longer follow-up are required.

Background/aim: Platelet-rich plasma (PRP) application has gained widespread interest for musculoskeletal injuries. Nonsteroidal antiinflammatory drugs are frequently used in sports medicine before and/or after PRP application. Our study seeks to determine whether serum levels of platelet-derived growth factor-AB (PDGF-AB) and vascular endothelial growth factor (VEGF) levels of PRP would be affected by nonsteroidal antiinflammatory drugs., Materials and Methods: Two different final concentrations of diclofenac (0.5 μg mL -1 and 2.5 μg mL -1 ), meloxicam (0.8 μg mL -1 and 2.0 μg mL -1 ), and acetylsalicylic acid (final concentration 450 μm) were obtained in separate tubes with PRPs prepared from 20 healthy male volunteers. Medicine-free PRP was the control group. Growth factors were measured using ELISA., Results: PDGF-AB and VEGF serum levels did not change with diclofenac, meloxicam, or acetylsalicylic acid addition. PDGF-AB and VEGF serum levels correlated with each other., Conclusion: Diclofenac, meloxicam, and acetylsalicylic acid did not affect PDGF-AB and VEGF serum levels.

Purpose: Early stage adult acquired flatfoot deformity (AAFD) is traditionally treated with osteotomy and tendon transfer. Despite a high success rate, the long recovery time and associated morbidity are not sufficient. This study aims to evaluate the functional and radiological outcomes following the use of the arthroereisis screw with tendoscopic delivered PRP for early stage AAFD., Methods: Patients with stage IIa AAFD who underwent the use of the arthroereisis screw with tendoscopic delivered PRP with a minimum follow-up time of 24 months were retrospectively evaluated. Clinical outcomes for pain were evaluated with the Foot and Ankle Outcomes Score (FAOS) and Visual Analog Score (VAS). Radiographic deformity correction was assessed using weight-bearing imaging., Results: Thirteen patients (13 feet) with mean follow-up of 29.5 months were included. The mean age was 37.3 years (range, 28-65 years). FAOS-reported symptoms, pain, daily activities, sports activities, and quality of life significantly improved from 52.1, 42.6, 57.6, 35.7, and 15.4 pre-operatively to 78.5, 68.2, 83.3, 65.0, and 49.6 post-operatively, respectively (p < 0.05). Statistically significant radiographic improvements (lateral talus first metatarsal angle, calcaneal pitch, and cuneiform to ground distance) were also observed between the pre- and post-operative images., Conclusions: This study elucidates the successful implementation of a less invasive approach to stage IIa AAFD. Through the use of a subtalar arthroereisis screw, PTT tendoscopy, and PRP injection, clinical and radiographic outcomes were improved.

Purpose: The treatment of large bone defects represents a significant challenge for orthopaedic surgeons. In recent years, biologic agents have also been used to further improve bone healing. Among these, platelet-rich plasma (PRP) is the most exploited strategy. The aim of the present study was to systematically review the available literature to identify: 1) preclinical in-vivo results supporting the rational of PRP use for bone healing; 2) evidence from the clinical practice on the actual clinical benefit of PRP for the treatment of fractures and complications such as delayed unions and non-unions., Methods: A systematic review of the literature was performed on the application of PRP in bone healing, using the following inclusion criteria: pre-clinical and clinical reports of any level of evidence, written in English language, published in the last 20 years (1996-2016), on the use of PRP to stimulate long-bone defect treatment, with focus on fracture and delayed/non-unions healing., Results: The search in the Pubmed database identified 64 articles eligible for inclusion: 45 were preclinical in-vivo studies and 19 were clinical studies. Despite the fact that the overall pre-clinical results seem to support the benefit of PRP in 91.1 % of the studies, a more in depth analysis underlined a lower success rate, with a positive outcome of 84.4 % in terms of histological analysis, and even lower values considering radiological and biomechanical results (75.0 % and 72.7 % positive outcome respectively). This was also mirrored in the clinical literature, where the real benefit of PRP use to treat fractures and non-unions is still under debate., Conclusion: Overall, the available literature presents major limitations in terms of low quality and extreme heterogeneity, which hamper the possibility to optimize PRP treatment and translate it into a real clinical benefit despite positive preclinical findings on its biological potential to favour bone healing.

Objective: To explore the effects of calcium gluconate and thrombin on the formation of platelet-rich gel (PRG) and the release of bioactive substances in human platelet-rich plasma (PRP) and the clinical significance. Methods: Six healthy blood donors who met the inclusion criteria were recruited in our unit from May to August in 2016. Platelet samples of each donor were collected for preparation of PRP. (1) PRP in the volume of 10 mL was collected from each donor and divided into thrombin activation group (TA, added with 0.5 mL thrombin solution in dose of 100 U/mL) and calcium gluconate activation group (CGA, added with 0.5 mL calcium gluconate solution in dose of 100 g/L) according to the random number table, with 5 mL PRP in each group. Then the PRP of the two groups was activated in water bath at 37 ℃ for 1 h. The formation time of PRG was recorded, and the formation situation of PRG was observed within 1 hour of activation. After being activated for 1 h, one part of PRG was collected to observe the distribution of fibrous protein with HE staining, and another part of PRG was collected to observe platelet ultrastructure under transmission electron microscope (TEM). After being activated for 1 h, the supernatant was collected to determine the content of transforming growth factor β(1, )platelet-derived growth factor BB (PDGF-BB), vascular endothelial growth factor, basic fibroblast growth factor (bFGF), epidermal growth factor, and insulin-like growth factorⅠby enzyme-linked immunosorbent assay. (2) Another 10 mL PRP from each donor was collected and grouped as above, and the platelet suspension was obtained after two times of centrifugation and resuspension with phosphate buffered saline, respectively. And then they were treated with corresponding activator for 1 h as that in experiment (1). Nanoparticle tracking analyzer was used to detect the concentrations of microvesicles with different diameters and total microvesicles derived from platelet. Data were processed with t test. Results: (1) The formation time of PRG in group TA was (228±40) s, and the PRG volume reached the maximum at this moment. The PRG volume shrunk to the minimum after 30 minutes of activation. The formation time of PRG in group CGA was (690±71) s, and the PRG volume reached the maximum at this moment. After 55 minutes of activation, the PRG volume shrunk to the minimum. The formation time of PRG in group TA was obviously shorter than that in group CGA ( t =15.17, P <0.01). (2) HE staining showed that after 1 hour of activation, the red-stained area of fibrous protein in PRG of group TA was large and densely distributed, while that of group CGA was small and loosely distributed. TEM revealed that after 1 hour of activation, the platelets in PRG of group TA were fragmented, while lysing platelet structure, lysing α granule structure, intact α granule structure, and intact dense body structure were observed in PRG of group CGA. (3) The content of PDGF-BB released by PRP in group TA was (7.4±0.8) ng/mL, which was obviously higher than that in group CGA [(4.9±0.5) ng/mL, t =5.41, P <0.01]. The content of bFGF released by PRP in group CGA was (960±151) pg/mL, which was significantly higher than that in group TA [(384±56) pg/mL, t =8.75, P <0.01]. The content of the other 4 growth factors released by PRP in the two groups was close (with t values from 0.11 to 1.97, P values above 0.05). (4) The concentrations of total microvesicles, microvesicles with diameter more than 100 nm, and exosomes with diameter less than or equal to 100 nm derived from platelet in group CGA were (165.8±15.1)×10(8)/mL, (142.4±12.3)×10(8)/mL, and (23.4±2.9)×10(8)/mL respectively, which were significantly higher than those in group TA [(24.7±4.6)×10(8)/mL, (22.6±4.0)×10(8)/mL, and (2.1±0.7)×10(8)/mL, with t values from 17.36 to 22.66, P values below 0.01]. Conclusions: Calcium gluconate can slowly activate PRP, resulting in slowly shrunk PRG with high content of bFGF and high concentration of microvesicles, which is suitable for repairing articular cavity and sinus tract wound. Thrombin can rapidly activate PRP, resulting in quickly shrunk PRG with high content of PDGF-BB and a certain concentration of microvesicles, which is suitable for repairing acute trauma.

Objective: To compare five activation methods in equine platelet-rich plasma (PRP) by determination of platelet-derived growth factor BB (PDGF-BB) and transforming growth factor beta 1 (TGF-β1) concentrations in platelet-rich gel (PRG) supernatants., Methods: Platelet-rich plasma from 20 horses was activated by calcium chloride (CC), calcium gluconate (CG), bovine thrombin (BT), and their combinations, BTCC and BTCG. Both growth factor concentrations in PRG supernatants were measured by ELISA and compared with plasma and platelet lysates (PL) over time., Results: Growth factor concentrations were significantly lower in plasma and higher for all PRG supernatants. Platelet lysates contained a significantly lower concentration of PDGF-BB than PRG supernatants and a significantly higher concentration of TGF-β1 than PRG supernatants. Clots from PRP activated with sodium salts were more stable over time and had significant growth factor release, whereas CC produced gross salt deposition. Significant correlations were noticed for platelet with leukocyte concentrations in PRP (r s : 0.76), platelet counts in PRP with TGF-β1 concentrations in PRG supernatants (r s : 0.86), platelet counts in PRP with PDGF-BB concentrations in PRG supernatants (r s : 0.78), leukocyte counts in PRP with TGF-β1 concentrations in PRG supernatants (r s : 0.76), and PDGF-BB concentrations with activating substances (r s : 0.72)., Clinical Significance: Calcium gluconate was the better substance to induce PRP activation. It induced growth factor release free from calcium precipitates in the clots. Use of BT alone or combined with calcium salts was not advantageous for growth factor release.

Background: Diabetic foot ulcer is considered as a major health problem that predisposes to limb amputation. Among the different methods to achieve ulcer healing, platelet-rich plasma (PRP) gel is gaining popularity. It is thought to stimulate wound closure by providing essential growth factors for healing. This study aims to evaluate the value of autologous PRP gel in the treatment of diabetic ulcers., Methods: The study included 56 patients of both sex from 18 to 80 years, with clean chronic diabetic foot ulcers divided into 2 equal groups. The first group was treated by antiseptic ointment dressing, and the second group was treated by autologous platelet gel. PRP together with thrombin were prepared by centrifugation at each dressing session. Thrombin and calcium chloride were used to activate the PRP. The formed platelet gel was applied to the wound twice weekly., Results: Statically significant increase in healing rate was found in the PRP-treated group, and complete healing was achieved in 86% of them in comparison to 68% of the control group. In the study group, rate of healing per week was greater during the first 8 weeks and starts to decline afterward. The use of platelet gel showed a lower rate of wound infection., Conclusions: Autologous platelet gel is more effective than the local antiseptic dressing in terms of healing rate and prevention of infection in clean diabetic ulcers., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: High on-aspirin treatment platelets reactivity (HPR) is a significant problem in long-term secondary prevention of cardiovascular events. We hypothesize that imbalance between platelets MMPs/TIMPs results in cardiovascular disorders. We also explored whether chronically elevated blood glucose affects MMP-2/TIMP-4 release from platelets., Materials and Methods: Seventy patients with stable coronary artery disease, supplemented with aspirin, participated in this pilot study. The presence of HPR and/or diabetes mellitus was considered as the differentiating factor. Light aggregometry, impedance aggregometry, and ELISA tests for TXB2, MMP-2, MMP-9, and TIMP-4 were performed in serum, plasma, platelet-rich plasma, and platelets-poor plasma, as appropriate., Results: Aspirin-HPR did not affect plasma MMP-2, MMP-9, and TIMP-4. Arachidonic acid-induced aggregation of platelets from aspirin-HPR patients did not lead to increased release of MMP-2, MMP-9, and TIMP-4. Studying patients at the lowest TXB2 serum concentration quartile revealed that high concentration of plasma TIMP-4 and TIMP-4 negatively correlated with TXB2 and platelet aggregation. Diabetics showed an increased plasma MMP-2 as well as an increased MMP-2 in supernatants after platelet aggregation. However, diabetes mellitus did not affect MMP-9 and TIMP-4., Conclusion: Aspirin-HPR did not affect the translocation and release of MMPs and TIMP-4 from platelets. TIMP-4 may serve as a marker of TXA2-mediated platelet aggregation. Chronically elevated plasma glucose increases plasma MMP-2, and HPR potentiates this phenomenon.

Background: Benzimidazoles are privileged biomolecules which form an integral part of vitamin B12 and have been attracting numerous researchers all over the world to assess their potential therapeutic significance., Objectives: The comparative in vitro antiplatelet activity of newly synthesized benzimidazole derivatives, M3BIM, C2BIM, and L2BIM in thrombin, adenosine diphosphate (ADP) and epinephrineinduced washed human platelets was investigated., Method: Reversed-phase silica gel column chromatography, Aggregometry, Flow cytometry and Immunoblotting were used in this study., Results: M3BIM exhibited a concentration (25-100 µM) dependent inhibitory effect on platelet aggregation induced by thrombin (0.01 U/mL) in washed human platelets and by epinephrine (10 µM) only at a maximum concentration of 500 µM in platelet-rich plasma (PRP); however, C2BIM and L2BIM had no response even at 500 µM against thrombin and 1mM against epinephrine-induced platelet aggregation. Moreover, all these three compounds were not inhibited platelet aggregation induced by ADP (20 µM). Additionally, these compounds showed no effects in thrombin-induced P-selectin expression and αIIbβ3 activation, as evidenced by flow cytometry and clot reaction assays, respectively. Besides, M3BIM (100 µM) significantly abolished thrombin-induced Akt and mitogen-activated protein kinases (MAPKs) phosphorylation; whereas 200 µM C2BIM and L2BIM were not effective on these proteins., Conclusion: This study affords confirmation for the inhibitory effect of M3BIM in a low dose thrombin and epinephrine-induced platelet aggregation in vitro compared to other imidazole derivatives, C2BIM and L2BIM. These outcomes may recommend that M3BIM can be appraised as a prospective benzeimidazole compound for the treatment of thrombin -induced platelet defect and its related diseases., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Introduction: The Toll-like receptor 9 (TLR9) pathway can activate platelets but its role in acute coronary syndromes (ACS) is unknown. This study examined TLR9 expression and platelet activation in response to ODN2006, a TLR9 agonist, in healthy subjects and in ACS subjects treated with dual anti-platelet therapy (DAPT)., Materials and Methods: TLR9 expression was examined in both resting and thrombin receptor activator peptide (TRAP)-activated platelets (1 and 10μM) from healthy and ACS subjects by flow cytometry. In both cohorts, ODN2006-mediated platelet activation (5μM) was examined in whole blood (WB) and platelet-rich plasma (PRP) using cell-surface CD62p and CD63 expression by flow cytometry., Results: Baseline TLR9 expression was significantly greater in ACS subjects compared to healthy subjects (p<0.01). Following TRAP activation, TLR9 expression increased dose-dependently in healthy subjects. However, no difference in TLR9 expression was seen in ACS platelets following TRAP activation. ODN2006 treatment resulted in significant increases in cell-surface expression of CD62p and CD63 in both WB (all p<0.001) and PRP (all p<0.001) in comparison to unstimulated platelets in healthy subjects. Despite DAPT, ODN2006 treatment produced significant increases in both activation markers in the ACS cohort across WB and PRP (all p<0.0001). Elevated baseline expression of TLR9 in ACS platelets may indicate increased sensitivity to TLR9 agonists and contribute to increased platelet activation in these patients. Furthermore, ODN2006 stimulation can activate platelets in ACS subjects despite treatment with DAPT., Conclusion: This study demonstrates TLR9 expression and activation to be of potential therapeutic importance in ASC patients., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective: Our aim was to investigate the biological effects of bone morphogenic protein 2 (BMP2) on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into chondrocyte-like cells in platelet-rich plasma (PRP) gel in vitro. In addition, the effectiveness of BMP2-transduced BMSCs in combined with PRP gel to repair the degenerated intervertebral disc in a rabbit model was also evaluated. Previous studies have shown that tissue engineering provides many promising advantages to treating disc degeneration and may reverse the pathological process of disc degeneration., Methods: The expressions of types I, II and X collagen, aggrecan and Sox9 of the BMP2-transduced BMSCs in monolayer or PRP gel were examined by reverse transcriptase polymerase chain reaction (RT-PCR). Sixty New Zealand white rabbits were divided into five groups: 12 normal controls; 12 puncture operated with only disc degeneration being induced; 12 PRP transplantation animals; 12 BMSC and PRP-transplantation animals; 12 BMP2-transduced BMSCs and PRP-transplantation animals. The effect of BMP2-transduced BMSCs on degenerated discs were evaluated by magnetic resonance image (MRI) scan, histology, immunohistochemistry and Western blot analysis., Results: BMP2 could facilitate chondrogenic differentiation of BMSCs in monolayer or PRP gel. The discs treated with BMP2-transduced BMSCs exhibited relatively well-preserved nucleus pulposus (NP) structure. Significantly higher T2-weighted signal intensity and a greater amount of extracellular matrix were observed in the BMP2-transduced BMSC group compared with other groups. In addition, the presences of BMP2-transduced BMSCs were identified at week 12 postoperatively in vivo., Conclusions: BMP2-transduced BMSCs can maintain the chondrocyte-like phenotype in PRP gel in vitro, and the combined use of these two agents can significantly promote repair of the degenerated discs in vivo.

Purpose: The aim of this article is to provide an overview of clinical and translational research on intradiscal platelet-rich plasma (PRP) as a minimally invasive treatment for discogenic low back pain., Methods: A literature review of in vitro, in vivo, and clinical studies was performed., Results: There is strong in vitro evidence that supports the use of intradiscal PRP for discogenic low back pain. There are also promising findings in select preclinical animal studies. A clinical study of 29 participants who underwent intradiscal PRP injections for discogenic low back pain found statistically and clinically significant improvements in pain and function through two years of follow-up., Conclusions: Intradiscal PRP is a safe and a possibly effective treatment for discogenic low back pain. Future studies are warranted to determine the best candidates for this treatment, what the optimal injectate is and what relationships exist between patient-reported outcomes and radiological findings.

Purpose: There is evidence showing that mesenchymal stromal cells (MSC) may constitute a potential therapeutic strategy to induce bone regeneration. In this work, we investigate the capacity of autologous bone marrow (BM) MSC loaded on collagen microspheres (CM) and included into autologous platelet-rich plasma (PRP) clots (MSC/CM/PRP) to induce bone formation in patients with nonunion lesions., Methods: MSC were isolated from BM cells of patients with nonunion lesions. Phenotypical (marker expression) and functional studies (osteogenic differentiation) were performed. MSC were seeded on CM and included into autologous PRP clot (MSC/CM/PRP). The capacity of MSC/CM/PRP to induce bone formation was evaluated in three patients diagnosed with nonunion., Results: MSC loaded on CM/PRP clots maintain their biological functions, in vitro. After three months, post-MSC transplantation, all patients showed evidence of osteogenesis at the site of nonunion. After one year, all patients showed a complete healing of the nonunion., Conclusions: Our results support the use of autologous MSC transplanted as MSC/CM/PRP for the treatment of nonunion fractures. Future studies incorporating a larger number of patients may confirm the results obtained in this work.

Purpose: The aim of this study is evaluation of the efficacy of the use of platelet rich plasma (PRP) in management of femoral neck fractures., Materials and Methods: This is a prospective study that was conducted between February 2010 and March 2013. A total of 60 patients were included in this study, categorized randomly into two groups. Group A included fracture neck femur treated by closed reduction and internal fixation with three cannulated screws and group B by addition of PRP to internal fixation. We planned to compare time of healing, need for revision and incidence of complications between the two groups., Results: Union occurred in 53 patients (88.33 %) in both groups, 25 cases (83.3 %) in group A and 28 cases (93.3 %) in group B, including three cases (5 %) with avascular necrosis (AVN): two in group A (6.7 %) and one case in group B (3.3 %).Revision surgery was done for six cases (20 %) in group A and for two cases (6.7 %) in group B. In both groups, all united cases had good to excellent clinical outcome as regards Harris hip score (HHS) at the end of the follow up., Conclusion: Despite advances in surgical techniques and medical care, the risk of nonunion and avascular necrosis (AVN) after treatment of femoral neck fractures have not been changed appreciably in the last 50 years. Results of this study generally showed that both the median clinical and radiographic healing time were lower in group B compared to group A.

Background: The authors have developed a tendon hydrogel that may be injected into the site of tendon injury to improve speed and strength of repair. The aim of this study was to compare the biological and physical properties of fresh, hydrated tendon hydrogel with its reconstituted lyophilized counterpart with the goal of increasing clinical feasibility., Materials: Hydrogel was prepared from fresh human cadaveric flexor tendon. Fresh gel was compared to gel aliquots that were lyophilized and reconstituted with sterile deionized water. Scanning electron microscopy was used to examine the microarchitecture of gelated samples. Rat adipose-derived stem cells were seeded in hydrogel, and cell viability was assessed after 7 days. MTS colorimetric assay was used to evaluate both the effect of prolonged storage on gel and the ability of reconstituted lyophilized hydrogel to activate platelet-rich plasma. The viability and proliferation of luciferase-transfected adipose-derived stem cells embedded within hydrogel in vivo was assessed by a bioluminescence in vivo imaging system., Results: Reconstituted lyophilized hydrogel demonstrated similar handling properties compared to fresh gel. Adipose-derived stem cells remained viable 7 days after reseeding in both conditions. Lyophilized hydrogel retained its ability to activate platelet-rich plasma and retained 95 percent of its maximal proliferative capacity at 30 days. The in vivo imaging system demonstrated similar cell proliferation, with signal persisting through day 13., Conclusions: Reconstitution of lyophilized hydrogel stimulated cell proliferation and platelet-rich plasma activation to a greater degree than did fresh hydrogel. Efficacy after prolonged storage was also shown to be superior. Therefore, this lyophilized formulation of tendon hydrogel may have wider clinical applicability.

Objectives: Our objectives were as follows: 1) to validate a protocol for producing rabbit platelet-rich plasma (PRP); 2) to determine the influence of two anticoagulants, sodium citrate and acid-citrate-dextrose solution A, and gender on cell count in PRP and growth factor concentration in pure platelet-rich gel supernatants; 3) to correlate the variables evaluated., Methods: Whole blood from 18 New Zealand rabbits (9 males and 9 females) was obtained with sodium citrate and acid-citrate-dextrose solution A for processing PRP fractions (A and B), which were evaluated for haematology. The PRP fractions were either activated with calcium gluconate or lysated with a detergent. The concentrations of transforming growth factor beta 1 and platelet-derived growth factor BB were assayed by ELISA., Results: The sodium citrate PRP-B had significantly higher counts of platelets in comparison to PRP-A and whole blood obtained with the same anticoagulant and the homologous acid-citrate-dextrose solution A PRP fraction. The sodium citrate PRP-A had a significantly higher count of leukocytes compared to the homologous acid-citrate-dextrose solution A fraction. All the PRP fractions had a significant leuko-reduction when compared to whole blood. The sodium citrate PRP-A fraction from female rabbits had significantly lower platelet counts and significantly higher leukocyte counts than the same acid-citrate-dextrose solution A fraction. Growth factor concentration was not affected by the type of anticoagulant or gender., Clinical Significance: The type of anticoagulant and gender affected the cell counts in PRP, but they did not influence the growth factor concentration. More complete rabbit PRP studies should be performed before evaluating this type of substance in models of disease.

Objective: The role of nitrates and nitric oxide on platelet functions has obtained an increasing attention with respect to their potential effects on cardiovascular disorders. In this study we aimed to analyze the effect of sodium nitrite on platelet functions in human platelets., Patients and Methods: This in vitro study was designed to show the effect of sodium nitrite on platelet functions in seven healthy volunteers. Blood samples were centrifuged to prepare platelet rich plasma and platelet poor plasma. Platelet rich plasma was diluted with the platelet poor plasma to have a final count of 300,000 ± 25,000 platelets. Platelet rich plasma was incubated with six different increasing doses (from 10 μM to 5 mM) of sodium nitrite for 1 hour at 37°C. Then stimulating agents including collagen (3 μg ml-1), adenosine diphosphate (10 μM), and epinephrine (10 μM) were added to the cuvette. Changes in light transmission were observed for 10 minutes. In addition spontaneous aggregation were performed in control group with all aggregating agents separately., Results: Effect of sodium nitrite on agonist-induced platelet aggregation depends on the concentration of sodium nitrite. Compared with control group, agonist-induced platelet aggregations were significantly suppressed by sodium nitrite at the concentration of 5, 1.0 and 0.5 mM., Conclusions: Our results suggested that sodium nitrite has inhibitory effects in vitro on platelet aggregation in a dose-dependent manner.