Large Number Of Platelets Research Articles

A Comparison of Efficacy of Autologous Platelet-rich Plasma and Conventional Sutures in Anchoring Split Skin Graft on Wounds

Abstract Context: Split thickness skin grafting (STSG) restores cutaneous cover over wounds thus protecting the underlying surface from contamination, fluid loss and stimulates healing. Autologous platelet rich plasma (PRP) provides a large number of platelets and high concentration of growth factors which promote overall uptake of STSG. Aims: The aim of this study was to assess the immediate, subsequent adhesion and final take of STSG with application of PRP over recipient site. Methods and Material: 1 year Randomised Control Trial where 80 wounds of various aetiologies were randomised into intervention group (n=40) which received PRP before placing STSG on recipient site and control group (n=40) in which the graft was fixed in place with sutures alone. Immediate graft adhesion and subsequent graft uptake were compared between the two groups and statistical analysis was done with: SPSS Version 20. Results: Irrespective of aetiology, and size, among a total of 80 wounds 87.5% grafts had adhered by 1st minute of application in the intervention group compared to nil in control group (p< 0.0001). Graft uptake was assessed on first three consecutive dressings. There was significantly better graft uptake in intervention group compared to control group [third dressing uptake (98.29%, 93%, p< 0.0001) respectively].Difference in seroma and haematoma formation were also compared between the two groups and found to be not significant. (p>0.05) Conclusions: Application of topical PRP facilitates STSG uptake. It decreases operative time by decreasing mobility of graft over the wound bed. Thus, use of PRP improves outcome of split thickness skin graft in wounds of various aetiologies and we recommend use of the same at recipient site of STSG. CTRI/2021/06/034401

Locally Injected Autologous Platelet-Rich Plasma Improves Cutaneous Wound Healing in Cats.

Simple SummaryWound healing in cats is a complex process that can be accomplished by first or second intention. Autologous platelet-rich plasma (PRP) is a product from patients’ own blood that has been found to promote wound healing in both humans and animals. The aim of this study was to investigate the efficacy of PRP infiltration in open wounds in cats. It was found that experimentally created wounds infiltrated with PRP showed a more rapid total wound healing and a better perfusion, supporting the use of PRP in open wounds in laboratory cats. In conclusion, PRP is considered a cost-effective and simple method to accelerate open-wound healing in cats.Cutaneous defects in cats are commonly encountered in clinical practice, and healing can be accomplished by first or second intention. Platelet-rich plasma (PRP) is characterized by a plasma concentration containing a large number of platelets in a small volume of plasma. The objective of the present study was to record the efficacy of PRP infiltration in open wounds in laboratory cats. Six wounds were created in the dorsal midline of eight laboratory cats, with the wounds of one side designated as the PRP group and the wounds of the other side as the control group. Wound healing was evaluated by daily clinical examination, planimetry, laser Doppler flowmetry, and histologic examination on days 0, 7, 14, and 25, and by measurement of metalloproteinases (MMPs)-2 and -9 and tissue inhibitor metalloproteinase (TIMP)-1 on days 0, 14, and 25. Based on the results of the present study, the mean time for full coverage with granulation tissue was shorter in the PRP group, the mean contraction and total wound healing percentage were increased compared to the control group, and finally, the perfusion measured with laser Doppler flowmetry was higher in the PRP group during all examination days. In conclusion, this is the first study focusing on the topical application of PRP in the treatment of open wounds in laboratory cats, and our results are encouraging—showing a more rapid healing in the PRP group.

Preliminary study on the involvement of platelets in mouse experimental periodontitis

Background/purposeAlthough some studies have taken an interest in the participation of platelets in periodontitis, so far, we know very little about the roles of platelets in periodontitis. The objective of this study is to explore the involvement of platelets in the development of experimental periodontitis in mice. Materials and methodsTwenty C57BL/6 male mice were used for this study. Experimental periodontitis models of mice were constructed by ligating for 1, 3, 7, and 14 days, respectively. Morphological changes in the alveolar bone were assessed by micro-computed tomography (Micro-CT). The gingival crevicular fluid samples of ligation sites were collected and stained by immunocytochemistry. Immunohistochemistry was used to detect platelets infiltration in gingival tissues of mice. ResultsThe results of Micro-CT showed that with the extension of ligation time, alveolar bone resorption increased, suggesting that the experimental periodontitis models were established. Immunochemical staining showed that there were almost no platelets in the gingival crevicular fluid of mice ligated for 1 and 3 days. And at 7 and 14 days of ligation, a large number of platelets were present in the gingival crevicular fluid and formed complexes with neutrophils. And with the extension of ligation time, the extent of platelet infiltration increased in mice gingival tissues. ConclusionPlatelets were infiltrated increasedly in the gingival sulcus and gingival tissues following the experimental time, and may participate in the development of mouse experimental periodontitis.

Correlation of laboratory markers of hemostatic system activation with concentration and size of plasma extracellular microparticles in patients with COVID-19

Introduction. In recent years, much attention has been paid to the study of extracellular microparticles (microvesicles and exosomes) and their role in the pathogenesis of human diseases.The objective of this study was to determine the number and size of plasma extracellular microparticles (PEMP) in patients with severe and extremely severe COVID-19 and correlate these data with the markers of hemostasis activation, inflammation, and tissue damage.Methods and Materials. The study included 29 patients with severe and extremely severe COVID-19. Concentration and size of PEMP were determined by nanoparticle trajectory analysis (NTA). All patients underwent the complete blood count and the thromboelastometry (TEM). Hemostatic, biochemical, and immunological parameters were assessed including fibrinogen, prothrombin time, activated partial thromboplastin time, D-dimer, C-reactive protein, lactate dehydrogenase, procalcitonin, von Willebrand factor antigen, interleukin 6, and interleukin 18.Results. There were 14 patients (48.3 %) discharged from the ICU with improvement (group 1— survived patients), and 15 patients (51.7 %) with lethal outcomes (group 2 — lethal outcome); the PEMP concentration did not differ between these groups. In group 2, there were heterogeneity of PEMP population, and a tendency to the larger PEMP size (p=0.074). In all patients, the PEMP concentration correlated negatively with both prothrombin time and the number of large platelets; the size of PEMP correlated negatively with the level of von Willebrand factor antigen, and positively with the fibrinogen. In group 1, the PEMP concentration had a direct correlation with both the level of interleukin 18 and maximum clot lysis in TEM; the PEMP size had a direct correlation with the maximum clot lysis in TEM and an inverse correlation with both the level of procalcitonin and maximum clot density in TEM.Conclusion. Our study confirms the importance of the process of extracellular microparticles formation in the COVID-19 pathogenesis. Our findings are consistent with the hypothesis that the parameters of PEMP population can be predictive biomarkers of the COVID-19 severity.

Investigation of platelet measurands in dogs with hematologic neoplasia.

Thrombocytopenia has been associated with some neoplastic processes, including hematologic neoplasia. There is no information regarding specific changes in platelet measurands in dogs with hematologic neoplasia compared with healthy dogs. The objectives of our study were to establish RIs, evaluate platelet measurands in dogs with hematologic neoplasia, and compare these measurands in patients with hematologic malignancies with or without thrombocytopenia. This was a retrospective study. Platelet measurands were determined using the ADVIA 120 Hematology analyzer when a CBC was performed and included the platelet count, MPV, platelet distribution width (PDW), plateletcrit (PCT), mean platelet component (MPC), platelet component distribution width (PCDW), mean platelet mass (MPM), platelet mass distribution width (PMDW), and number of large platelets. Reference intervals were determined retrospectively using data from 129 healthy dogs. Patients with hematologic neoplasia (n=50) were identified through retrospective evaluation of medical records from the Auburn University Veterinary Teaching Hospital and separated into thrombocytopenic (n=20) and nonthrombocytopenic groups (n=30). Platelet count and PCT were significantly higher in older healthy dogs compared with younger dogs. Significant differences were identified when comparing healthy dogs with those with hematologic neoplasia without thrombocytopenia for PDW, PCDW, PMDW, and the number of large platelets, indicating the presence of more heterogeneous platelets. Thrombocytopenic dogs with hematologic neoplasia had significantly decreased MPCs and increased MPVs, MPMs, and PCDWs compared with nonthrombocytopenic dogs with neoplasia. Dogs with hematologic neoplasia had more heterogeneous platelets, whereas thrombocytopenic patients with neoplasia had more activated platelets.

Platlet-Rich Plasma - Review of Literature.

Wound healing is a dynamic and physiological process for restoring the normal architecture and functionality of damaged tissue. Platelet-rich plasma (PRP) is an autologous whole blood product that contains a large number of platelets in a small volume of plasma with complete set of coagulation factors, which are in physiological concentrations. PRP has haemostatic, adhesive properties and acts supraphysiologically in the process of wound healing and osteogenesis. Platelets play a very important role in the wound healing process by providing growth factors that enhance the rate and quality of wound healing by many different mechanisms. The aim of this review is to describe: the biology of platelets and their role in the wound healing process, the terminology of platelet rich products, PRP preparation, activation and concentration of PRP, as well as the use of PRP in plastic surgery.

The effect of platelet-rich fibrin on normal dermal fibroblast proliferation after mitomycin-c treatment: An in vitro study

BackgroundDisturbance in the wound healing can cause the wound turn into a chronic wound, which histologically shows fibroblast senescence with weak proliferation ability. Mitomycin-C could block cell proliferation that causes cell senescence which is similar to the chronic wound morphology. Platelet-Rich Fibrin (PRF) contains a large number of platelets, leukocytes, cytokines and growth factors. This study aims to determine whether PRF could improve the fibroblast proliferation after treatment with Mitomycin-C. MethodsCultured normal human skin fibroblasts forth passage divided into five groups. The first group was treated with culture medium, and the second group with 10 μg/mL mitomycin-C for 2 h. The third, 4th and 5th group were treated with mitomycin-C for the same dose and period, then adding it with 100%, 50%, and 25% of PRF. The fibroblast proliferation was measured by MTT assay. ResultsThe fibroblast proliferation in the group with culture medium is 11.366,56 ± 4.073,32, meanwhile in the group with mitomycin-C treatment is 5.690,41 ± 2.834,22. The fibroblast proliferation in group with 100% PRF is 7.909,8 ± 3.392,19; group with 50% PRF 15.347,91 ± 8.413,02; and group with 25% PRF 13.449,56 ± 7.523,83. All of the PRF groups increased significantly compared to the group with Mitomycin-C treatment. ConclusionsPlatelet-Rich Fibrin can improve normal dermal fibroblast proliferation after treatment with mitomycin-C in vitro.

Abstract 5172: Deposition of platelets in sinusoids and its contribution to hepatocarcinogenesis

Abstract Platelets can activate hepatocyte proliferation not only via releasing the platelet factors, but also promoting for liver sinusoidal endothelial cells (LSEC) and Kupffer cells to produce the factors such as IL-6 and TGF-α that are indispensable for hepatocyte proliferation. We previously reported that in the transgenic (TG) mice with liver specific human BRAFV600E mutation, which corresponds to the mouse BrafV637E that is highly prevalent in mouse DEN-induced hepatic tumors, exhibit increased liver weight five times larger than that of normal mice. Furthermore, hepatocytes in these mice overexpress thrombopoietin (TPO) in association with megakaryocytosis/thrombocytosis, and large numbers of platelets were deposited in the liver sinusoids. In the present study, we investigated whether platelets are deposited in liver sinusoids in hepatic tumors in human and mice. CD61 immunohistochemical staining revealed the increased numbers of platelets were deposited in the sinusoids in foci, adenomas and hepatocellular carcinomas (HCC) induced by neonatal treatment with DEN compared to surrounding normal hepatic tissues. Moreover, increased numbers of platelets were deposited in sinusoids of dysplastic nodules and well-differentiated HCC compared to normal liver tissue and cirrhotic nodules in human. In addition, we evaluated the correlation between the tumor diameters and peripheral platelets numbers in the patients who were primarily diagnosed as HCC in our institute. The result indicated that the numbers of peripheral platelets are positively correlated with the tumor diameters. These results indicate that platelets contribute to development of HCC via interacting sinusoidal cells. Citation Format: Hiroki Tanaka, Kie Horioka, Masahiro Yamamoto, Koji Sawada, Takumu Hasebe, Kosuke Yamazaki, Katsuhiro Ogawa. Deposition of platelets in sinusoids and its contribution to hepatocarcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5172.

Platelet and Red Blood Cell Counts, as well as the Concentrations of Uric Acid, but Not Homocysteinaemia or Oxidative Stress, Contribute Mostly to Platelet Reactivity in Older Adults.

Purpose The goal of this study was to estimate the hierarchical contribution of the most commonly recognized cardiovascular risk factors associated with atherogenesis to activation and reactivity of blood platelets in a group of men and women at ages 60-65. Methods Socioeconomic and anthropometric data were taken from questionnaires. Blood morphology and biochemistry were measured with standard diagnostic methods. Plasma serum homocysteine was measured by immunochemical method. Plasma concentrations of VCAM, ICAM, total antioxidant status, and total oxidant status were estimated with commercial ELISA kits. Markers of oxidative stress of plasma and platelet proteins (concentrations of protein free thiol and amino groups) and lipids (concentrations of lipid peroxides) and generation of superoxide anion by platelets were measured with colorimetric methods. Platelet reactivity was estimated by impedance aggregometry with arachidonate, collagen, and ADP as agonists. Expression of selectin-P and GPIIb/IIIa on blood platelets was tested by flow cytometry. Results Platelet aggregation associated significantly negatively with HGB and age and significantly positively with PLT, MPV, PCT, PDW, and P-LCR. When platelet reactivity (“cumulative platelet reactivity_aggregation”) was analyzed in a cumulated manner, the negative association with serum concentration of uric acid (Rs = −0.169, p = 0.003) was confirmed. Multivariate analysis revealed that amongst blood morphological parameters, platelet count, plateletcrit, and number of large platelets and uric acid are the most predictive variables for platelet reactivity. Conclusions The most significant contributors to platelet reactivity in older subjects are platelet morphology, plasma uricaemia, and erythrocyte morphology.

In vitro large scale production of megakaryocytes to functional platelets from human hematopoietic stem cells

Megakaryocytopoiesis results in the formation of platelets, which are essential for hemostasis. Decreased production or increased destruction of platelets can cause thrombocytopenia, in which platelet transfusion is the mode of treatment. The present study is aimed in generation of megakaryocytes (MKs) and platelet from human hematopoietic stem cells (HSCs). The purity of HSCs was assessed through Flow cytometry and immunocytochemistry (ICC) studies. These pure HSCs were induced with thrombopoietin (TPO), similarly with Andrographis paniculata extract (APE) for 21 days to generate MKs. The APE is mainly composed of andrographolide which stimulates TPO from the liver, and this binds to CD110 present on the surface of HSCs and triggers the proliferation of HSCs and initiate higher MKs population subsequently, a large number of platelets. The results of the present study showed increased proliferation of HSCs grown in the presence of APE and revealed a high population of CD41a and CD42b positive MKs as enumerated by Flow cytometry compared with TPO induced MKs. These results also concurred with qRT-PCR and western blot analysis. The scanning electron microscopy (SEM) revealed the morphology of differentiated MKs and platelets were similar to human blood platelets. The differentiated MKs in APE exhibited polyploidy up to 32 N while TPO induced MKs showed polyploidy of 8 N, these results corroborated with colony forming unit assay. On thrombin stimulation, high expression of P-selectin (CD62p) and fibrinogen binding were detected in APE induced platelets. Autologous transplantation of platelets generated from APE may be a useful option in thrombocytopenia condition.

Optimization of the spectra optia for leukapheresis of very small subjects

![Graphic][1] Leukapheresis procedures in very small volume subjects require procedure modifications related to anticoagulant, venous access, and volume. Historically, we accomplished this procedure in 4-5 kg rhesus macaques using a modified CS3000Plus cell separator, but this instrument is no longer available. The Spectra Optia apheresis system and its mononuclear cell (MNC) collection set were evaluated to develop leukapheresis procedures for such subjects. Five rhesus macaques, weight 7-9 kg, blood volume 60 mL/kg, were studied. They pre-donated 100-120 mL of autologous blood (25-30 mL/wk x 4 wks). Aspirin 81 mg was given daily for 3 days prior to 5 of 8 procedures. AMD3100 1 mg/kg SQ 2-3 hours prior to leukapheresis was given as a mobilizing agent. Autologous blood was pooled, diluted 1:1 with saline, and washed to remove citrate and plasma. The Optia was primed first with saline, and then with a customized albumin option in which 30-35 mL of the 40-50 mL of packed RBCs were pumped into the disposable apheresis kit at 10 mL/min. The albumin option shortens procedure ramp-up time. A closed circuit was created between the return line and the packed RBC bag and the flow rate increased to 40 mL/min until 400 mL was processed. To minimize citrate toxicity and avoid volume overload, animals received heparin 50 U/kg bolus IV followed by an ACDA solution containing heparin 5U/mL at an inlet:AC ratio of 25:1. A Collection Preference (CP) setting of 20 was selected using the automatic interface management mode. In the last procedure a semi-automatic mode was utilized so that collection deeper than CP of 20 was possible. Procedures were terminated at 120 min. Animals were anesthetized and catheters placed in the saphenous and cephalic as draw and return lines. Blood counts, chemistries, and ionized calcium (iCa) levels were monitored every 30 min. The elutriation chamber, which concentrates MNCs and allows platelets to return to the donor, was filled 1-2 times per procedure resulting in a product volume of 17-34 mL. A total of eight leukapheresis procedures were performed. Inlet flow rates were typically 10.5 mL/min. In one animal, an inlet rate of 12.0 mL/min was achieved; however, at 120 minutes severe citrate toxicity developed with bradycardia and an iCa level of 0.62 mmol/L, requiring IV calcium gluconate. Excluding this case, a mean(SD) baseline iCa value of 1.17(0.2) dropped to 0.84(0.03) mmol/L at120 min. Mean hematocrit (HCT) decreased from 40.4(1.7)% to a nadir of 36.8(2.6)% at 90 min. Mean platelets declined to a greater degree, from 342(42) x103/mcL to 109(33) x103/mcL at 60 min and 121(43) x103/mcL at 120 min (p<0.001). This drop was reflected in the large number of platelets in the product, 2.49(1.09)x1010 total. Occasional platelet aggregate formation was observed in animals both receiving and not receiving aspirin. Product WBC differential was 65.5(11.1)% MNC and 34.5(10)% granulocytes. Total product MNC content was 1.95(0.54)x109 . MNC collection efficiency (CE) was 26.2(8.5)% using CP=20 and improved to 38.1% using the semi-automatic mode. Product MNC composition consisted of 5.1(2.0)x108 CD4+ , 5.6(2.1)x108 CD8+, and 3.0(2.0)x108 CD20+ lymphocytes, and 9.8(7.0)x108 CD14+ monocytes. As a measure of product contamination, the CE for granulocytes was 5.8(3.0)% and for platelets 11.2(4.4)%. In 3 procedures, two-chamber collections were obtained, the remaining 5 procedures had one-chamber collections (all within 120 min). The HCT of the product was low, 3.7(1.2)%. The product also was enriched for reticulocytes (10.57(2.44)%) and CD4+CD8+ cells(3.4(2.2)x107). Relatively low numbers of CD34+ cells were collected, 5.2(1.9)x106; however, the mobilization protocol was not optimized for CD34+ collection in that AMD3100 alone and older monkeys were used. We are currently optimizing the procedure for even smaller rhesus and combination cytokine mobilization. MNC collections through the process of leukapheresis have proven to be effective in isolating cells for transplantation and cellular therapeutics. The use of leukapheresis in a pediatric setting is difficult due to small blood volumes and a high risk of adverse events. Here we have identified a novel means to perform leukapheresis procedures safely and effectively in small volume subjects. This methodology will have numerous therapeutic applications in both human and veterinary medicine. Disclosures Parker: Terumo BCT, Inc.: Employment. Lienesch: Terumo BCT, Inc.: Employment. [1]: /embed/inline-graphic-2.gif

P-Selectin-Mediated Adhesion between Platelets and Tumor Cells Promotes Intestinal Tumorigenesis in ApcMin/+ Mice

Studies have indicated that platelets play an important role in tumorigenesis, and an abundance of platelets accumulate in the ovarian tumor microenvironment outside the vasculature. However, whether cancer cells recruit platelets within intestinal tumors and how they signal adherent platelets to enter intestinal tumor tissues remain unknown. Here, we unexpectedly found that large numbers of platelets were deposited within human colorectal tumor specimens using immunohistochemical staining, and these platelets were fully associated with tumor development. We further report the robust adhesion of platelet aggregates to tumor cells within intestinal tumors, which occurs via a mechanism that is dependent on P-selectin (CD62P), a cell adhesion molecule that is abundantly expressed on activated platelets. Using spontaneous intestinal tumor mouse models, we determined that the genetic deletion of P-selectin suppressed intestinal tumor growth, which was rescued by the infusion of wild-type platelets but not P-selectin-/- platelets. Mechanistically, platelet adhesion to tumor cells induced the secretion of vascular endothelial growth factor (VEGF) to promote angiogenesis and accelerate intestinal tumor cell proliferation. Our results indicate that the adherence of platelets to tumor cells could promote tumor growth and metastasis. By targeting this platelet-tumor cell interaction, recombinant soluble P-selectin may have therapeutic value for the treatment of intestinal tumors.

Optimization of the Spectra Optia for Leukapheresis of Very Small Subjects

▪Leukapheresis procedures in very small volume subjects require procedure modifications related to anticoagulant, venous access, and volume. Historically, we accomplished this procedure in 4-5 kg rhesus macaques using a modified CS3000Plus cell separator, but this instrument is no longer available. The Spectra Optia apheresis system and its mononuclear cell (MNC) collection set were evaluated to develop leukapheresis procedures for such subjects.Five rhesus macaques, weight 7-9 kg, blood volume 60 mL/kg, were studied. They pre-donated 100-120 mL of autologous blood (25-30 mL/wk x 4 wks). Aspirin 81 mg was given daily for 3 days prior to 5 of 8 procedures. AMD3100 1 mg/kg SQ 2-3 hours prior to leukapheresis was given as a mobilizing agent. Autologous blood was pooled, diluted 1:1 with saline, and washed to remove citrate and plasma. The Optia was primed first with saline, and then with a customized albumin option in which 30-35 mL of the 40-50 mL of packed RBCs were pumped into the disposable apheresis kit at 10 mL/min. The albumin option shortens procedure ramp-up time. A closed circuit was created between the return line and the packed RBC bag and the flow rate increased to 40 mL/min until 400 mL was processed. To minimize citrate toxicity and avoid volume overload, animals received heparin 50 U/kg bolus IV followed by an ACDA solution containing heparin 5U/mL at an inlet:AC ratio of 25:1. A Collection Preference (CP) setting of 20 was selected using the automatic interface management mode. In the last procedure a semi-automatic mode was utilized so that collection deeper than CP of 20 was possible. Procedures were terminated at 120 min. Animals were anesthetized and catheters placed in the saphenous and cephalic as draw and return lines. Blood counts, chemistries, and ionized calcium (iCa) levels were monitored every 30 min. The elutriation chamber, which concentrates MNCs and allows platelets to return to the donor, was filled 1-2 times per procedure resulting in a product volume of 17-34 mL.A total of eight leukapheresis procedures were performed. Inlet flow rates were typically 10.5 mL/min. In one animal, an inlet rate of 12.0 mL/min was achieved; however, at 120 minutes severe citrate toxicity developed with bradycardia and an iCa level of 0.62 mmol/L, requiring IV calcium gluconate. Excluding this case, a mean(SD) baseline iCa value of 1.17(0.2) dropped to 0.84(0.03) mmol/L at120 min. Mean hematocrit (HCT) decreased from 40.4(1.7)% to a nadir of 36.8(2.6)% at 90 min. Mean platelets declined to a greater degree, from 342(42) x103/mcL to 109(33) x103/mcL at 60 min and 121(43) x103/mcL at 120 min (p<0.001). This drop was reflected in the large number of platelets in the product, 2.49(1.09)x1010 total. Occasional platelet aggregate formation was observed in animals both receiving and not receiving aspirin. Product WBC differential was 65.5(11.1)% MNC and 34.5(10)% granulocytes. Total product MNC content was 1.95(0.54)x109 . MNC collection efficiency (CE) was 26.2(8.5)% using CP=20 and improved to 38.1% using the semi-automatic mode. Product MNC composition consisted of 5.1(2.0)x108 CD4+ , 5.6(2.1)x108 CD8+, and 3.0(2.0)x108 CD20+ lymphocytes, and 9.8(7.0)x108 CD14+ monocytes. As a measure of product contamination, the CE for granulocytes was 5.8(3.0)% and for platelets 11.2(4.4)%. In 3 procedures, two-chamber collections were obtained, the remaining 5 procedures had one-chamber collections (all within 120 min). The HCT of the product was low, 3.7(1.2)%. The product also was enriched for reticulocytes (10.57(2.44)%) and CD4+CD8+ cells(3.4(2.2)x107). Relatively low numbers of CD34+ cells were collected, 5.2(1.9)x106; however, the mobilization protocol was not optimized for CD34+ collection in that AMD3100 alone and older monkeys were used. We are currently optimizing the procedure for even smaller rhesus and combination cytokine mobilization.MNC collections through the process of leukapheresis have proven to be effective in isolating cells for transplantation and cellular therapeutics. The use of leukapheresis in a pediatric setting is difficult due to small blood volumes and a high risk of adverse events. Here we have identified a novel means to perform leukapheresis procedures safely and effectively in small volume subjects. This methodology will have numerous therapeutic applications in both human and veterinary medicine. DisclosuresParker:Terumo BCT, Inc.: Employment. Lienesch:Terumo BCT, Inc.: Employment.

Scalable Generation of Universal Platelets from Human Induced Pluripotent Stem Cells

SummaryHuman induced pluripotent stem cells (iPSCs) provide a potentially replenishable source for the production of transfusable platelets. Here, we describe a method to generate megakaryocytes (MKs) and functional platelets from iPSCs in a scalable manner under serum/feeder-free conditions. The method also permits the cryopreservation of MK progenitors, enabling a rapid “surge” capacity when large numbers of platelets are needed. Ultrastructural/morphological analyses show no major differences between iPSC platelets and human blood platelets. iPSC platelets form aggregates, lamellipodia, and filopodia after activation and circulate in macrophage-depleted animals and incorporate into developing mouse thrombi in a manner identical to human platelets. By knocking out the β2-microglobulin gene, we have generated platelets that are negative for the major histocompatibility antigens. The scalable generation of HLA-ABC-negative platelets from a renewable cell source represents an important step toward generating universal platelets for transfusion as well as a potential strategy for the management of platelet refractoriness.

Platelet parameters from an automated hematology analyzer in dogs with inflammatory clinical diseases

The mean platelet component (MPC) is a proprietary algorithm of an automated laser-based hematology analyzer system which measures the refractive index of platelets. The MPC is related linearly to platelet density and is an indirect index of platelet activation status. Previous investigations of canine inflammatory conditions and models of endotoxemia demonstrated a significant decrease in the MPC, consistent with platelet activation. The purpose of this study was to evaluate the MPC and other platelet parameters in dogs with different diseases to determine if they could show differential platelet activation with different pathologies. The hypothesis was that the MPC would decrease in clinical conditions associated with systemic inflammation or platelet activation. Complete blood counts run on the analyzer from dogs with different inflammatory conditions (primary immune-mediated hemolytic anemia (IMHA) or thrombocytopenia (ITP), pituitary-dependent hyperadrenocorticism, intra-abdominal sepsis, pancreatitis, intravascular thrombus or thromboembolus and hemangiosarcoma) were reviewed retrospectively and compared with those of control dogs presenting for orthopedic evaluation.Dogs with ITP had a decreased plateletcrit and MPC, with an increased platelet volume and number of large platelets (P < 0.001). Dogs with IMHA had an increased plateletcrit and mass, and more numerous large platelets (P < 0.001).With the exception of the ITP group, there was no difference in MPC in the diseased groups when compared with the controls. The results of this study suggest the MPC does not change in certain canine diseases associated with systemic inflammation.

Platelet Activation in Acute Pancreatitis

The aim of this study was to assess the functional state of platelets in patients with mild acute pancreatitis and severe acute pancreatitis (S-AP). The number of platelets and their morphological parameters were measured with Advia 2120. β-Thromboglobulin and platelet factor 4 concentrations were determined by enzyme-linked immunosorbent assay method. To evaluate the expression of platelet glycoproteins, flow cytometry method was used. At the time of admission, a multiparameter evaluation of the platelets' function in AP patients showed enhanced platelet activation, which was reflected by an increase in the number of large platelets, concentration of degranulation markers (platelet factor 4 and β-thromboglobulin), expression of glycoprotein (Gp) IIb/IIIa, and decreased mean platelet component. Only in S-AP patients at day 1 a decreased number of platelets and high expression of P-selectin and GpIa were observed, which may suggest their prognostic value. At day 30, the procoagulation state was still present in S-AP patients, because of increased platelets and number of large platelets as well as high GpIIb/IIIa expression. These results may indicate an important role of platelet activation in the pathogenesis of acute pancreatitis and the development of complications in S-AP.

Effect of submaximal aerobic exercise on platelet function, platelet activation, and secondary and tertiary hemostasis in dogs

To investigate whether submaximal aerobic exercise in dogs is followed by activation of all phases of coagulation as has been reported for humans. 9 healthy Beagles. 30 minutes before dogs were exercised, a 16-gauge central venous catheter was placed in a jugular vein of each dog by use of the catheter-through-the-needle technique. Samples were collected before exercise, after running on a treadmill (6 km/h for 13 minutes), and at 60 minutes. Platelet activation was evaluated with platelet morphology indices (mean platelet component, mean platelet volume, and number of large platelets) provided by a laser-based hematology system. Platelet function was assessed in hirudin-anticoagulated whole blood with an impedance-based aggregometer with collagen as the agonist (final concentrations, 0, 1.6, 3.2, 5, and 10 μg/mL). Prothrombin time, activated partial thromboplastin time, and concentrations of fibrinogen, factor VIII, antithrombin, protein C, protein S, and fibrin D-dimer were determined automatically. Kaolin-activated thromboelastography variables R (reaction time), K (clot formation time), angle α, maximal amplitude, and G (clot stability) were measured in recalcified citrated whole blood. Exercise resulted in a significant decrease in mean platelet volume and the number of large platelets but did not change the mean platelet component, which reflected platelet activation as well as platelet function. Secondary and tertiary coagulation did not change significantly, nor did thromboelastography variables. Aerobic exercise resulted in a decrease in the number of large and thus most likely activated platelets but otherwise had no major impact on coagulation in dogs.

Surface Modification of Polycarbonateurethane by Grafting Phosphorylcholine Glyceraldehydes for Improving Hemocompatibility

ABSTRACTPhosphorylcholine glyceraldehyde (PCGA) was used as a phosphorylcholine (PC) group containing compound to graft onto the surface of polycarbonateurethane (PCU) film using 1,6-hexanediamine (HDA) or α,ω-diamino-poly(ethylene glycol) (APEG, Mn = 200) as a spacer, in order to introduce biomimetic structure onto the polymer surface. X-ray photoelectron spectroscopy (XPS) analysis shows that PCGA has been covalently linked to the PCU surface. Water contact angle test suggests that the surface hydrophilicity has been improved after PCGA is grafted onto the surface of PCU film. Scanning electron microscope (SEM) observation of the modified PCU films after contacting with plasma-rich plasma demonstrates that platelets rarely adhere but a large number of platelets adhere to the original PCU surface. The hemocompatibility of the PC modified PCU film has been improved obviously after grafting with PCGA with PEG spacer.

Polypropylene modified with 2-hydroxyethyl acrylate-g-2-methacryloyloxyethyl phosphorycholine and its hemocompatibility

Polypropylene (PP) was modified with 2-hydroxyethyl acrylate (HEA) by solution radical grafting to introduce active hydroxyl groups on polypropylene backbone (PP-g-HEA). Then the biomimic monomer, 2-methacryloyloxyethyl phosphorycholine (MPC), was grafted onto the surface of PP-g-HEA film (PP-g-HEA-g-MPC) by redox graft polymerizations with ceric(IV) ammonium nitrate as an initiator. Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) showed that the HEA and MPC were introduced onto PP molecular chains and the copolymer, PP-g-HEA-g-MPC were formed. The water contact angle measurements demonstrated that the final modified PP film exhibited a better hydrophilic surface compared to the neat PP film. The platelets adhesion on the neat PP, PP-g-HEA and PP-g-HEA-g-MPC film was examined by scanning electron microscopy (SEM). It was found that a large number of platelets were adhered and activated on the surface of neat PP and PP-g-HEA films, while the number of platelets on PP-g-HEA-g-MPC surface was decreased remarkably. The result revealed that the introduction of poly(MPC) onto the PP surface improved the hemocompatibility of PP substantially.

Escape of intraluminal platelets into brain parenchyma after subarachnoid hemorrhage

Platelet aggregates are present in parenchymal vessels as early as 10 min after experimental subarachnoid hemorrhage (SAH). Structural injury to parenchymal vessel walls and depletion of collagen-IV (the major protein of basal lamina) occur in a similar time frame. Since platelets upon activation release enzymes which can digest collagen-IV, we investigated the topographic relationship between platelet aggregates, endothelium, and basal lamina after SAH produced by endovascular perforation, using triple immunofluorescence and confocal microscopy with deconvolution. The location of platelet aggregates in relation to zymography-detected active collagenase was also examined. As reported previously, most cerebral vessels profiles contained platelets aggregates at 10 min after SAH. High-resolution three-dimensional image analysis placed many platelets at the ab-luminal (basal) side of endothelium at 10 min, and others either within the vascular basal lamina or in nearby parenchyma. By 24 h post hemorrhage, large numbers of platelets had entered the brain parenchyma. The vascular sites of platelet movement were devoid of endothelium and collagen-IV. Collagenase activity colocalized with vascular platelet aggregates. Our data demonstrate that parenchymal entry of platelets into brain parenchyma begins within minutes after hemorrhage. Three-dimensional analysis suggests that platelet aggregates initiate or stimulate local disruption of endothelium and destruction of adjacent basal lamina after SAH.