PLT-rich Plasma Research Articles

Gamma irradiation induces a pro-apoptotic state in longer stored platelets, without progressing to an overt apoptosis by day 7 of storage.

Although gamma-irradiation to platelet products is a standard method to prevent the risk of TA-GVHD in vulnerable recipients, it induces some proteomic and redox changes, of which irradiation-induced ROS increments may potentiate platelet mitochondrial dysfunction. However, whether these changes cause platelet apoptosis, or affect their viability during storage, is the main subject of this study. PLT-rich plasma PC was split into two bags, one kept as control while other was subjected to gamma-irradiation. Within 7-days storage, cytosolic and mitochondrial levels of cytochrome c and pro-apoptotic molecules of Bak and Bax were evaluated by western-blotting. Intraplatelet active caspase (using FAM-DEVD-FMK) and PS-exposure were detected by flowcytometry. Caspase activity in platelet lysate was also confirmed by immunofluorescence detection of Caspase-3/7 Substrate N-Ac-DEVD-N'-MC-R110 while platelet viability was evaluated with MTT assays. Cytosolic cytochrome c gradually increased while its mitochondrial content steadily declined during 7 days of storage. In a contrary trend, reverse patterns were observed for Bak and Bax expressions. Gamma-irradiated platelets showed higher release of mitochondrial cytochrome c that reflected by higher cytosolic cytochrome c levels on day 7 of storage. Concurrently mitochondrial pro-apoptotic Bak and Bax proteins increased on day 7 in irradiated products. However, gamma-irradiation didn't significantly increase caspase activity or PS-exposure, nor did it decrease platelet viability. Here, consistent with studies on "gamma-irradiation-induced oxidative stress", we showed that gamma-ray also increases platelet pro-apoptotic signals during storage, although not strongly enough to affect platelet viability by overt apoptosis induction. Conclusively, whether supplementing ROS scavengers or antioxidants to irradiated platelets can improve their quality during storage may be of interest for future research.

The Relationship between Premature Adrenarche and Platelet Aggregation.

Premature adrenarche has been associated with an increase in adrenal androgens, and the hyperandrogenic hormonal environment is known to lead to increased platelet aggregation. Here, we evaluated the effects of premature adrenarche (PA) on platelet aggregation in platelet-rich plasma samples from female patients. The study included 40 female patients diagnosed with PA between February 2014 and June 2018 and 30 healthy female individuals in the control group. Adenosine diphosphate (ADP) and collagen-induced platelet aggregation were studied with the photometric aggregometry method. There was no significant difference in the platelet count or volume values between the participants with PA and the control group. Additionally, the ADP-induced maximum aggregation time, value, and slope values did not significantly differ between the patient and control groups (p>0.05). However, the collagen-induced maximum aggregation time, value, and slope values were significantly higher in the patient group (p < 0.001). Increased collagen-induced platelet aggregation was detected in female patients with PA. As PA is associated with a higher risk of cardiovascular events later in life, close follow-up of PA in this respect may be beneficial.

Neutrophil extracellular traps promote thrombogenicity in cerebral venous sinus thrombosis

BackgroundNeutrophil extracellular traps (NETs) contribute to the creation of a coagulation state in various diseases. Currently, it is not clear whether NETs are present in the thrombi and plasma of patients with cerebral venous sinus thrombosis (CVST). This study aimed to investigate the presence of NETs in thrombi and blood samples from CVST patients and the procoagulant activity (PCA) of NETs during the progression of CVST.ResultsThrombi obtained from CVST patients undergoing thrombectomy were examined by immunochemistry using neutrophil elastase (NE), CD66b and citrullinated histone H3(citH3). The presence of NET markers in samples from 37 CVST patients and 32 healthy people was evaluated by ELISA. NET-producing neutrophils and neutrophil-platelet (PLT) aggregates were examined in samples obtained from CVST patients and healthy people by flow cytometry. The TAT complex in plasma sample from each group was detected by ELISA to evaluate the procoagulant activity of NETs in CVST patients. Neutrophils from healthy subjects were treated with PLT-rich plasma in the presence of anti-PF4 antibodies or an autophagy inhibitor and analyzed by flow cytometry and confocal microscopy. After treatment with NETs, the expression of von Willebrand factor (VWF), tissue factor (TF) and CD31 in human brain microvascular endothelial cells (HBMECs) was measured by confocal microscopy and western blotting. Our results showed that NETs were abundant in the plasma and thrombi from CVST patients. Platelet factor 4 (PF4) from CVST PLTs induced NET generation through autophagy. NETs could induce PCA by modulating TF and phosphatidylserine (PS) in CVST. NETs also disrupted the endothelial barrier and transformed ECs into a procoagulant phenotype to exacerbate thrombogenicity.ConclusionsNET generation was mediated by PF4 from PLTs through autophagy and contribute to thrombosis in CVST patients.

Antioxidant prevents clearance of hemostatically competent platelets after long-term cold storage.

Cold storage of platelets (PLTs) has the potential advantage of prolonging storage time while reducing posttransfusion infection given the decreased likelihood of bacterial outgrowth during storage and possibly beneficial effects in treating bleeding patients. However, cold storage reduces PLT survival through the induction of complex storage lesions, which are more accentuated when storage is prolonged. Whole blood-derived PLT-rich plasma concentrates from seven PLT pools (n = 5 donors per pool). PLT additive solution was added (67%/33% plasma) and the product was split into 50-mL bags. Split units were stored in the presence or absence of 1 mM of N-acetylcysteine (NAC) under agitation for up to 14 days at room temperature or in the cold and were analyzed for PLT activation, fibrinogen-dependent spreading, microparticle formation, mitochondrial respiratory activity, reactive oxygen species (ROS) generation, as well as in vivo survival and bleeding time correction in immunodeficient mice. Cold storage of PLTs for 7 days or longer induces significant PLT activation, cytoskeletal damage, impaired fibrinogen spreading, enhances mitochondrial metabolic decoupling and ROS generation, and increases macrophage-dependent phagocytosis and macrophage-independent clearance. Addition of NAC prevents PLT clearance and allows a correction of the prolonged bleeding time in thrombocytopenic, aspirin-treated, immunodeficient mice. Long-term cold storage induces mitochondrial uncoupling and increased proton leak and ROS generation. The resulting ROS is a crucial contributor to the increased macrophage-dependent and -independent clearance of functional PLTs and can be prevented by the antioxidant NAC in a magnesium-containing additive solution.

Buffy coat platelets coming to America: Are we ready?

Buffy coat (BC) platelets (PLTs) have been used globally for many years. In 2004 Canadian Blood Services (CBS) made the decision to transition from PLT-rich plasma (PRP) to BC PLTs. We reviewed the benefits and manufacture process of BC and the implementation challenges involved. A literature review was performed in the following areas: BC efficacy, donor population shifts, production and good stewardship of PLTs, logistic considerations with overnight holds, advantages of the overnight hold, the CBS experience, licensure and standards, and changes needed to produce BC PLTs in the United States. The aim was to analyze current practice and identify possible actions for blood centers and hospitals. Implementation of BC would offer an additional source of PLTs to address the growing elderly population and the declining apheresis donor base. Substantial logistic, operational, and financial benefits were seen when CBS transitioned to BC with overnight hold. Buffy coat blood products are widely used throughout the world. Recent conversion from PRP to BC by CBS showed that conversion can be accomplished with planning, communication, and partnership from all stakeholders. In conclusion, BC PLTs are worth serious consideration in the United States, but regulatory barriers in the United States will need to be addressed.

Platelet transfusion: The effects of a fluid warmer on platelet function.

Thirty-one units of hospital blood bank apheresis PLTs were obtained. PLT-rich plasma (PRP) aggregometry and thromboelastography (TEG) were performed on the unit samples before and after the units were infused through a Ranger blood/fluid warming device. There were no differences in any of the aggregometry results before and after infusion of the PLTs through the blood warmer (all P > .32). There was a significant reduction in the TEG maximum amplitude (MA) of 69.8 ± 7.9 mm before and 66.0 ± 8.8 mm after (P < .001) infusion of the PLTs through the blood warmer and α angle 61.8 ± 9.4° before and 59.3 ± 8.2° after (P = .044) infusion of the PLTs through the blood warmer, although both mean values were within normal range for the TEG and not clinically significant. There were very good correlations of aggregometry and TEG results before and after infusion of the PLTs through the blood warmer device. This study did not demonstrate significant deleterious effect on PLT function from infusing apheresis PLT units through a blood warming device by PRP aggregometry. We did detect a statistically significant-but not clinically significant-reduction in TEG MA and α angle. The prohibition of transfusing PLT units though the Ranger blood warming device is not indicated.

Assessment of platelet function utilizing viscoelastic testing.

Assessment of platelet function utilizing viscoelastic testing.

Analysis of platelet parameters and activation markers in hematologic metastases of lung cancer.

Analysis of platelet parameters and activation markers in hematologic metastases of lung cancer.

Platelets directly regulate DNA damage and division of Staphylococcus aureus.

Platelets (PLTs) are classically used in the clinical setting to maintain hemostasis. Recent evidence supports important roles for PLTs in host inflammatory and immune responses, and PLT-rich plasma has been demonstrated to inhibit the growth of bacteria in vitro and in vivo; however, few studies have examined whether PLTs can inhibit bacterial growth directly, and related mechanisms have not been elucidated further. Accordingly, in this study, we evaluated the effects of PLTs on bacterial growth. We washed and purified PLTs from peripheral blood, then confirmed that PLTs significantly inhibited the growth of Staphylococcus aureus when cocultured in vitro. Moreover, PLTs damaged DNA and blocked cell division in S. aureus. During coculture, PLT-derived TGF-β1 was dramatically down-regulated compared with that in PLT culture alone, and the addition of TGF-β1 to the coculture system promoted the inhibition of PLTs on S. aureus. Analysis of a murine S. aureus infection model demonstrated that the depletion of PLTs exacerbated the severity of infection, whereas the transfusion of PLTs alleviated this infection. Our observations demonstrate that PLTs could directly inhibit the growth of S. aureus by damaging DNA and blockage cell division, and that PLT-derived TGF-β1 may play an important role in this machinery.-Xu, J., Yi, J., Zhang, H., Feng, F., Gu, S., Weng, L., Zhang, J., Chen, Y., An, N., Liu, Z., An, Q., Yin, W., Hu, X. Platelets directly regulate DNA damage and division of Staphylococcus aureus.

Reduced alloimmunization in mice following repeated transfusion with pathogen-reduced platelets.

Allogeneic transfusion can result in alloimmunization, leading to platelet (PLT) refractoriness and rejection of solid organ transplants. Previously we demonstrated that pathogen reduction using UV light and riboflavin (UV + R) eliminates the immunogenicity of white blood cells (WBCs) in vitro, blocks alloimmunization from transfusion in mice, and results in reduced ex vivo cytokine responses to subsequent untreated transfusions. We sought to determine if repeated transfusion with pathogen-reduced PLT-rich plasma (PRP) would eventually cause breakthrough alloimmunization or enhanced tolerance. BALB/cJ mice were transfused weekly for 2, 4, or 8 weeks with C57Bl/6J PRP that was either untreated or pathogen reduced with UV + R and leukoreduced or not. Alloimmunization was determined by measuring donor antibody levels, ex vivo cytokine responses, and 24-hour donor PLT recovery. The role of donor antibodies in PLT refractoriness was also assessed by transfer of diluted immune sera into naïve recipients. Donor antibody levels increased with the number of transfusions, but levels were significantly reduced using either UV + R or leukoreduction, and combining UV + R and leukoreduction gave the best protection. Priming of ex vivo cytokine responses required WBCs and remained suppressed with repeated UV + R-treated transfusion. PLT recovery was reduced with UV + R in naïve mice, and multiply transfused mice had poor PLT recovery even when antibody levels were relatively low. Approximately 1/100 dose of serum from a multiply transfused mouse was sufficient for complete rejection of donor PLTs. Pathogen reduction significantly reduces alloimmunization in repeatedly transfused mice and combined with leukoreduction provides a high level of protection from alloimmunization.

The association between platelet transfusion and idiopathic pneumonia syndrome is unaffected by platelet product type.

Methods used to produce platelet (PLT) components, pooling of PLT-rich plasma (PRP-PLT) and apheresis (AP-PLT), may variably contribute to the pathogenesis and severity of idiopathic pneumonia syndrome (IPS). We performed a retrospective cohort study of 906 allogeneic hematopoietic cell transplant recipients to examine associations between PLT product type and risks of developing IPS and dying after IPS onset. Proportional hazards models included separate terms for the sum of all PLT transfusions and the sum of PRP-PLT units received in the 3 or 7 days before IPS onset. Similarly constructed models analyzed the outcome of time to death after IPS onset. All analyses were adjusted for known IPS risk factors. Patients received a median of three PRP-PLT transfusions (interquartile range [IQR], 0-6) and five AP-PLT transfusions (IQR, 1-13) while at risk for IPS. Seventy-five patients (8%) developed IPS by Posttransplant Day 120. The proportion of PRP-PLT transfusions was not associated with risk of developing IPS (3-day hazard ratio [HR] 0.98, 95% CI 0.74-1.29, p = 0.86; 7-day HR 1.00, 95% CI 0.86-1.15, p = 0.95) or dying after IPS onset (3-day HR 0.99, 95% CI 0.75-1.31, p = 0.97; 7-day HR 0.98, 95% CI 0.78-1.12, p = 0.47). The association between PLT transfusions and risk of developing IPS or dying after IPS onset does not differ according to PLT product type. Further research is required to identify potentially modifiable steps in PLT component production that contribute to IPS.

The transfusion‐related acute lung injury controversy: lessons from heparin‐induced thrombocytopenia

The transfusion‐related acute lung injury controversy: lessons from heparin‐induced thrombocytopenia

Use of a cytokine-release assay to demonstrate loss of platelet secretory capacity during blood bank processing and storage.

The extent to which changes in secretory function contribute to the storage lesion of platelets (PLTs) prepared for transfusion is not well described. To develop a cytokine-release assay for the assessment of PLT secretory capacity during the preparation and storage of PLTs. Small volumes of PLT-rich plasma and PLT concentrate (PC) were prepared from whole blood (WB; N = 4 donors). Aliquots of WB, PLT-rich plasma, and PC were treated with 20 μM adenosine diphosphate or saline (control). Samples of WB-derived PCs obtained from a regional blood center were similarly stimulated at various storage times (N = 10 units). Plasma levels of RANTES (chemokine ligand 5; regulated on activation, normal T cell expressed and secreted) and PLT aggregation were measured following agonist addition. Adenosine diphosphate stimulated RANTES release from PLTs in fresh WB on average by 4.1-fold (P < .001), in PLT-rich plasma by 4.7-fold (P = .002), and in PC by 1.3-fold (P < .001). For blood center PCs, adenosine diphosphate failed to stimulate RANTES release at day 2 of storage or later (P ≥ .31). Baseline RANTES levels in the plasma/supernatant increased 660% during PC preparation (P = .02) and an additional 30% during subsequent storage (P < .001). Mean PLT aggregation decreased during processing from WB (95.6%) to PC (60.5%; P = .04). For blood center PCs, mean PLT aggregation decreased substantially from days 2 (41.0%) to 7 (2.3%; P < .001). A cytokine-release assay revealed a diminution in PLT secretory capacity during PC processing and storage, with complete elimination by day 2 of storage. Loss of PLT aggregability occurred more slowly. The cytokine-release assay may be a useful endpoint for optimizing PLT preparation and storage.

Treatment with fibrinogen γ-chain peptide-coated, adenosine 5'-diphosphate-encapsulated liposomes as an infusible hemostatic agent against active liver bleeding in rabbits with acute thrombocytopenia.

We evaluated the hemostatic efficacy of H12-(adenosine 5'-diphosphate [ADP])-liposomes in the setting of active liver bleeding in rabbits with dilutional thrombocytopenia after massive transfusion. Acute thrombocytopenia (platelet [PLT] count < 50 × 10(9) /L) was induced in rabbits by repeated blood withdrawal and isovolemic transfusion of autologous washed red blood cells. Liver hemorrhage was initiated by a penetrating liver injury. Subsequently, the animals received tamponade treatment for the liver hemorrhage for 5 minutes and were intravenously administered H12-(ADP)-liposomes with PLT-poor plasma (PPP), PLT-rich plasma (PRP), PPP alone, H12-(phosphate-buffered saline [PBS])-liposome/PPP, or H12-(ADP)-liposomes/PPP plus fibrinogen concentrate during the tamponade. Administration of H12-(ADP)-liposomes/PPP rescued 60% of the rabbits from the liver hemorrhage; PRP administration rescued 50%. In contrast, rabbits receiving PPP or H12-(PBS)-liposome/PPP achieved only 10 or 17% survival, respectively, for the first 24 hours. H12-(ADP)-liposomes/PPP as well as PRP consistently reduced bleeding volumes and shortened clotting times (CTs) in comparison to PPP administration. Specifically, bleeding volumes in the initial 5 minutes averaged 11 mL (H12-(ADP)-liposomes/PPP) and 17 mL (PRP) versus 30 mL (PPP; p < 0.05); CTs averaged 270 and 306 seconds versus 401 seconds (p < 0.05). H12-(ADP)-liposomes were observed at the bleeding site with thrombus formation, suggesting an induction of thrombi. Neither macro- nor microthrombi were detected in the lung, kidney, spleen, or liver in rabbits treated with H12-(ADP)-liposomes. Supplementation of fibrinogen to H12-(ADP)-liposomes/PPP did not significantly improve rabbit survival. H12-(ADP)-liposomes might be a safe and effective therapeutic tool during damage control surgery for trauma patients with acute thrombocytopenia and massive bleeding.

Extended storage of buffy coat platelet concentrates in plasma or a platelet additive solution

Platelet (PLT) concentrates (PCs) prepared from whole blood in the United States are made using the PLT-rich plasma method. The PCs must be made within 8 hours of blood collection and stored for only 5 days. In Europe and Canada, PCs are made using the buffy coat (BC) method from whole blood held overnight at 22 °C and storage times may be up to 7 days. Our studies were designed to determine how long BC PLTs can be stored in plasma or Plasmalyte while meeting the FDA's poststorage viability criteria. Normal subjects donated whole blood that was stored at 22 °C for 22 ± 2 hours before preparation of BC PLTs. PLTs were stored for 5 to 8 days in either plasma or Plasmalyte concentrations of 65 or 80%. Radiolabeled autologous stored versus fresh PLT recoveries and survivals were assessed as well as poststorage in vitro assays. BC PLTs stored in either plasma or 65% Plasmalyte met FDA poststorage PLT recovery criteria for 7 days but survivals for only 6 days, while storage in 80% Plasmalyte gave very poor results. Both stored PLT recoveries and survivals correlated with the same donor's fresh results, but the correlation was much stronger between recoveries than survivals. In vitro measures of extent of shape change, morphology score, and pH best predicted poststorage PLT recoveries, while annexin V binding best predicted PLT survivals. BC PLTs stored in either plasma or 65% Plasmalyte meet FDA's poststorage viability criteria for 6 days.

Decreasing phosphodiesterase 5A activity contributes to platelet cGMP accumulation during storage of apheresis‐derived platelet concentrates

Platelet storage lesion (PSL) considerably decreases the quality of platelets (PLTs) in concentrates characterized by a loss of signaling responses to agonists and impaired PLT activation, secretion, and aggregation. To understand the role of inhibitory signaling pathways in the mechanism of PSL, the basal state of the cyclic nucleotide (CN)-dependent signaling systems in stored PLTs was investigated. Whole blood samples (WB) and apheresis-derived PLT concentrates (APCs) were obtained from healthy volunteers. Washed PLTs were prepared from WB on Day 0 and from APCs on Days 0, 2, and 5. The basal phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) and phosphodiesterase 5A (PDE5A) levels were quantified by Western blot. CN and PDE5A activity were measured by enzyme-linked immunoassay kits. Fibrinogen binding and aggregation were measured in PLT-rich plasma of WB or APC samples. Unpaired t test was used for statistical analysis. Basal VASP phosphorylation levels were comparable in WB and APCs on Day 0. VASP phosphorylation increased significantly during storage of APCs, more pronounced at Ser(239) than at Ser(157) . Similarly, intracellular cGMP, but not cAMP, concentration continuously increased in stored PLTs, whereas PDE5A levels and activity significantly decreased accompanied by diminished thrombin receptor activator peptide 6-induced fibrinogen binding and aggregation. Storage of APCs leads to intracellular cGMP accumulation that could be caused by degradation of PDE5A. Enhanced cGMP level supports subsequent cGMP-dependent protein kinase-mediated increase of VASP phosphorylation resulting in reduced fibrinogen binding and aggregation.

In reply

In reply: We read with interest the article by Feys and colleagues1 comparing nitrite/nitrate (NO2−/NO3−) levels during storage and preparation of platelet concentrate (PC) from pooled buffy coats. Although they did not measure nitrite separately, their results suggest no substantial production of nitric oxide (NO) in stored PCs since NO produced in large quantities would likely affect the nitrite/nitrate levels. It is also consistent with our report2 that apheresis PCs did not produce NO by endogenous nitric oxide synthase (NOS) during storage. However, based on our electron paramagnetic resonance results, NO level in PCs was higher than that in plasma. Furthermore, we also showed that there was a slight decrease in nitrite levels during storage. We believe that our results suggest a possibility of a nitrite reduction system in PCs or nitrite reaction with residual hemoglobin, cell free or in red blood cells (RBCs), possibly indicative of some NO metabolic processes. Interestingly, Feys and colleagues observed an increase in nitrite/nitrate levels immediately after the procedures of PC preparation from buffy coats in which centrifugations and dilution with additive solution are used. As they suggested, it is possible that RBCs and white blood cells in buffy coats contribute to this increase in nitrite/nitrate levels as a native mechanism to prevent overt platelet (PLT) activation during preparation. Some authors have suggested the presence of active NOS in RBCs, although the definitive proof of functional NOS in RBCs is still under debate.3 In addition, it is still not clear if PLTs themselves express NOS.4 Considering the fact that buffy coat PCs show the lowest level of PLT activation by in vitro assays compared with apheresis PCs or PLT-rich plasma (PRP)-derived PCs,5 it would be interesting to see if the procedures of apheresis or PRP PC preparations also increase nitrite and nitrate levels. As Feys and colleagues note, production of NO in these preparations may be important for the maintenance of PLT function during in vitro storage, allowing clinical utility of PLT transfusions. ANS is a co-inventor of a patent issued to NIH for the use of sodium nitrite in the treatment of cardiovascular diseases. The authors declare that they have no conflicts of interest relevant to the manuscript submitted to TRANSFUSION.

Alteration of candidate circulating microRNA cancer biomarkers by blood sample processing.

8 Background: Circulating, cell-free microRNAs (miRNAs) are promising candidates as cancer biomarkers due to their association with cancer, stability in plasma and serum, and easy and sensitive detection using standard PCR-based methods. However, little is known about optimal conditions for collecting and processing blood specimens for miRNA biomarker measurements, or what effects blood processing has on miRNA levels. We systematically evaluated the effect of specimen processing differences on miRNA expression levels, an essential step in developing circulating miRNAs as robust cancer biomarkers. Methods: Matched plasma and serum samples drawn at a single timepoint from 3 healthy individuals were processed with increasing stringency to remove platelets (PLTs) and cell debris by differential centrifugation or 0.22µM filtration to produce: PLT-rich plasma, standard plasma, PLT-poor plasma, filtered plasma, serum, and filtered serum samples. PLTs were quantified by Coulter Counter. MiRNA expression profiling of differently processed samples was performed using Exiqon miRCury LNA qPCR panels with TaqMan qPCR confirmation. Results: Of 369 miRNAs profiled for expression after increasingly stringent sample processing, 150 (41%) showed a difference in cycle threshold (ΔCt) of ≥5 (≥30-fold), and 23 miRNAs showed a ΔCt of ≥10 (≥1,000-fold). For example, expression of miR-142-3p, a proposed biomarker for lung and esophageal cancers, ranged from a Ct of 19.9±2.4 to 30.8±0.8 (1,800-fold difference, p&lt;10-5). A minority of miRNAs (34/369) showed a ΔCt ≤2 (≤4-fold) between differently processed samples, suggesting expression of this subset is relatively unaffected by processing differences. Differences attributable to processing were reproducible, with a majority of miRNAs with ΔCt ≥5 overlapping between the 3 individuals. Conclusions: Blood-sample processing substantially and reproducibly affects about half of circulating miRNAs, while a minority of miRNAs are relatively unaffected. In pursuing circulating miRNAs as cancer biomarkers, focusing efforts on miRNAs less affected by processing and ensuring adherence to standardized processing protocols will minimize confounding effects.

Enhanced shear‐induced platelet aggregation due to low‐temperature storage

Refrigeration of platelets (PLTs) offers an attractive alternative to the currently practiced storage at room temperature since it may mitigate problems associated with bacterial contamination and extend storage lifetime. Refrigeration causes a number of biophysical and biochemical changes in PLTs and decreases PLT circulation time in vivo. However, the effect of refrigeration on PLT hemostatic functions under physiologic and pathophysiologic shear conditions has not been adequately characterized. Washed PLTs prepared from either fresh PLT-rich plasma (PRP) or PRP stored at 4°C for 2 days was mixed with exogenous von Willebrand factor (VWF) and fibrinogen and sheared in a cone-and-plate viscometer. PLT aggregation, activation, and VWF binding after shear and glycoprotein (GP) Ibα receptor expression and ristocetin-induced PLT agglutination were measured. PLTs stored at 4°C for 2 days aggregated significantly more than fresh PLTs particularly at high shear rates (10,000/sec), and this increase was independent of PLT concentration or suspension viscosity. Further, refrigerated PLTs showed a greater increase in GP Ibα-dependent PLT activation under shear and also bound more VWF than fresh PLTs. However, the GP Ibα expression levels as measured by three different antibodies were significantly lower in refrigerated PLTs than in fresh PLTs, and refrigeration resulted in a modest decrease in ristocetin-induced PLT agglutination. The combined results demonstrate that refrigeration increases PLT aggregation under high shear, but not static, conditions and also increases shear-induced VWF binding and PLT activation. Clinically, enhanced shear-induced PLT aggregation due to low temperature storage may be a beneficial strategy to prevent severe bleeding in trauma.

Antimicrobial activity of platelet (PLT)‐poor plasma, PLT‐rich plasma, PLT gel, and solvent/detergent‐treated PLT lysate biomaterials against wound bacteria

Platelet (PLT) gels exhibit antimicrobial activity useful for wound healing. The nature of the antibacterial component(s) is unknown. PLT-poor plasma (PPP), PLT-rich plasma (PRP), PLT gel (PG), and solvent/detergent-treated PLT lysate (S/D-PL) from two donors were evaluated either native or after complement heat inactivation. Materials were spiked at a 10% ratio (vol/vol) with approximately 10(7-8) colony-forming units/mL with four Gram-positive and four Gram-negative bacteria of the wound flora. Bacterial count was determined by plate assays at time of spiking and after 3 and 48 hours at 31°C. Bacteria growth inhibition tests were also performed. There was no viable Escherichia coli colony for 48 hours after spiking to the plasma and PLT materials from both donors, corresponding to greater than 7.51 to greater than 9.05 log inactivation. Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus were inactivated (approx. 4.7, 7, and 2 log, respectively) 3 hours after spiking to PRP, PPP, or S/D-PL from the first donor but less (1.1, 4.6, and 0.2 log, respectively) in PG, before a regrowth at 48 hours in all materials. Similar data were obtained with the second donor. No plasma and PLT material had antimicrobial activity against Enterobacter cloacae, Bacillus cereus, Bacillus subtilis, and Staphylococcus epidermidis. Complement-inactivated samples had no antimicrobial activity. Plasma complement is mostly responsible for the activity of plasma and PLT biomaterials against E. coli, P. aeruginosa, K. pneumoniae, and S. aureus. Activation of the coagulation to prepare PG may reduce antimicrobial activity. These findings may help optimize the control of wound infections by blood biomaterials.