Inactivated Platelets Research Articles

Comparative assessment of hemocompatibility of the suture material modified with heparin using different techniques

Postoperative complications in surgery, including thrombosis, are often associated with the suture material. The search for new types of suture materials that reduce the risk of vascular anastomotic complications remains an urgent task. The aim of the study was to compare and evaluate the effectiveness of two types of suture materials modified with heparin in terms of the hemocompatibility. Material and methods. The study involved a thread based on the Prolene 3.0 Polypropylene Suture (Ethicon, USA) that had undergone subsequent modification. The suture was modified with either heparin with polydimethylsiloxane or heparin with polyhydroxybutyrate/oxyvalerate. The consistency and hemocompatibility of the modifying layer were evaluated by scanning electron microscopy, diffuse scattering spectroscopy, hemolysis, platelet adhesion and aggregation. Results and discussion. The modifying layer based on heparin and polydimethylsiloxane was nonhomogeneous, with either thick or completely absent coating layer. The modifying layer based on heparin and polyhydroxybutyrate/oxyvalerate was homogeneous, without signs of thickening. Diffuse reflectance spectroscopy confirmed its presence on the thread surface. Evaluation of the hemocompatibility of the modified thread revealed the absence of hemolysis in all the studied groups. The modifying layer based on heparin and polyhydroxybutyrate/ oxyvalerate significantly reduced the maximum platelet aggregation (46.28 %) compared with the unmodified thread (82.64 %), the thread modification with heparin and polydimethylsiloxane did not influence on the indicator (77.72 %). The number of platelets adhered on the surface of the thread modified with heparin and polyhydroxybutyrate/oxyvalerate was insignificant, consisting of single inactivated platelets, in contrast to a thread modified with polydimethylsiloxane rubber and heparin, on the surface of which a large number of adherent activated platelets were found. Conclusions. The results obtained indicate the promising approach for the prevention of postoperative thrombosis by layer-by-layer modification of thread with heparin and polyhydroxybutyrate/oxyvalerate.

SCUBE-1 as a novel predictor of thromboembolic event

SCUBE-1 is a newly identified, secreted cell surface protein that is determined during early embryogenesis. This protein consists of cascading EGF-like repeats following the N-terminal signal peptide sequence, a spacer region, cysteine-rich repeat motifs, and a CUB domain at the C-terminus. These molecules are stored in alpha granules in inactivated platelets, translocated to the platelet surface after activation by thrombin, secreted as small soluble fragments, and incorporated into the thrombus.

Mouse Model for Platelet Aggregation using Flow Cytometry

Abstract Platelets play crucial roles in hemostasis, bleeding, and thrombosis. Within the United States, an estimated 7,000 platelet units are transfused daily. The gold standard to measure platelet survival in humans is to determine post-transfusion recovery after an autologous transfusion of radiolabeled platelet units, however, the ability to detect platelets circulating after transfusion does not provide information on how well these platelets function in hemostasis. Clinically, platelet unit function is routinely measured using aggregometry, which requires large volumes of platelet concentrates and normal platelet counts. While the clinical demand for platelets continues to increase with advances in medical care, there remains a lack of in vitro measures to properly assess platelet function in animal models. Here we outline an in vitro method for characterizing platelet function using flow cytometry to assess platelet activation and aggregation simultaneously in a mouse model. Using two commercially available transgenic mouse lines, one with platelets expressing red fluorescent protein (RFP) and the other with platelets expressing green fluorescent protein (GFP), whole blood is collected from each transgenic mouse line by aseptic cardiac puncture, and leukocyte-reduced platelet rich plasma (LRPRP) is isolated after two consecutive centrifuge spins and leukocyte reduction. RFP- and GFP-labeled platelets can be visually separated by flow cytometry. Platelets are activated after incubation with 4 mM GPRP peptide followed by exposure to 0.5U/mL high activity bovine thrombin for 2 minutes. Platelets are then stained with anti-CD41a, labelling all platelets, and anti-CD62P, labelling activated platelets; these platelet populations can be analyzed by flow cytometry. When RFP- and GFP-LRPRP are combined prior to activation with thrombin, RFP- and GFP-labeled platelet aggregation can be measured as the number of cells doubly expressing RFP and GFP by flow cytometry. Thrombin stimulation activated 93.31% of the total number of RFP- and GFP-platelets. Of the activated, CD62P-positive platelets, 11.03% expressed both RFP and GFP, indicating that these platelets were aggregated. Of the inactivated, CD62P-negative platelets, 0.81% expressed both RFP and GFP. These results suggest that thrombin exposure activates platelets and causes platelet aggregation, while <1% of inactivated platelets aggregate. Additionally, platelet aggregation is easily measured by flow without differentially labeling the two separate platelet populations. These data indicate that our model is a viable measure of platelet function in vitro. Our model of platelet activation and aggregation provides a baseline measure of in vitro platelet function in RFP and GFP mice. This in vitro method of assessing platelet function can be applied to various experimental populations and used to quantify the effects of experimental conditions on platelet function in mice. Using this model of platelet function, factors influencing platelet storage can be characterized and ultimately used to improve platelet transfusion therapies.

Facile fabrication of Bi-layered perfusable hydrogel tubes as biomimetic 3D arterial construct

Small-diameter arterial conduits with native physiological and biological equivalence continues to be a constant global demand posing critical challenges in fabrication. Advent of various strategies towards mimicking the structural hierarchy of a native blood vessel, often involve complex instrumentation and template-assistance with post-processing complications eventually compromising structural fidelity. In the present research, we report a template-free, facile strategy- ‘3D wet writing’ by peripheral-core differential ionic gelation to fabricate perfusable customizable constructs of any dimension, thickness and length in <5 mins. Dual-crosslinking using di-diol complexation of borax with Alginate- poly (vinyl alcohol) was performed to enhance the stability of fabricated bi-layered tubular constructs (BLT). These fabricated BLTs demonstrated non-linear mechanical characteristics of native blood vessels in withstanding physiological (120/80 mmHg) hemodynamic loading conditions with cyclic strain (5.82 ± 0.88%). The BLTs also ensured adequate longitudinal (0.176 ± 0.03 MPa) & circumferential (0.29 ± 0.012 MPa) tensile strength and burst pressure strength of 353.875 ± 22.69 mmHg. Hemocompatible characteristics of BLT were clearly evident with lower hemolytic index (0.21 ± 0.03%) and maintenance of erythrocyte structural integrity under dynamic conditions. Further, non-thrombogenic and non-inflammatory characteristics of BLTs were confirmed by in-activated platelets and monocytes under dynamic conditions. The developed wet-writing technique exhibited facile integration of layer-specific cells concurrently with the BLT fabrication. The spatial cell-specific expressions of smooth muscle (α-SMA) and endothelial (CD-31) cells in BLT were comparable to native hierarchical cellular organization with the multi-layered medial and mono-layered intimal layers. Further, ex-vivo dynamic studies on anastomotic interface between BLT and rat abdominal aorta clearly evidenced the functional efficacy of fabricated BLTs as physiologically relevant small-diameter vascular construct.

Effects of material interfaces on orientation and function of fibrinogen

Fibrinogen (Fg) in human plasma plays an important role in hemostasis, vascular repair and tissue integrity. The surface chemistry of extracellular matrix or biological materials affects the orientation and distribution of Fg, and changes the exposure of integrin binding sites, thereby affecting its adhesion function to platelets. Here, the quantity, morphology and side chain exposure of Fg adsorbed on hydrophilic, hydrophobic and avidin surfaces were measured by atomic force microscopy (AFM) and flow cytometry (FCM), then the rolling behavior of platelets on Fg was observed through a parallel plate flow chamber system. Our results show that the hydrophobic surface leads to a large amount of cross-linking and aggregation of Fg, while the hydrophilic surface reduces the adsorption and accumulation of Fg while causing the exposure and spreading of the α chain on Fg and further mediating the adhesion of platelets. Fg immobilized by avidin / biotin on hydrophilic surface can maintain the monomer state, avoid over exposure and stretching of α chain, and bind to the platelets activated by the A1 domain of von Willebrand factor instead of inactivated platelets. This study would be helpful for improving the blood compatibility of implant biomaterials and reasonable experimental design of coagulation in vitro.

Sulfenylome analysis of pathogen‐inactivated platelets reveals the presence of cysteine oxidation in integrin signaling pathway and cytoskeleton regulation

Unlabelled Box AbstractBackgroundCysteine‐containing protein modifications are involved in numerous biological processes such redox regulation or signal transduction. During the preparation and storage of platelet concentrates, cell functions and protein regulations are impacted. In spite of several proteomic investigations, the platelet sulfenylome, ie, the proteins containing cysteine residues (R‐SH) oxidized to sulfenic acid (R‐SOH), has not been characterized. MethodsA dimedone‐based sulfenic acid tagging and enrichment coupled to a mass spectrometry identification workflow was developed to identify and quantify the sulfenic acid‐containing proteins in platelet concentrates treated or not with an amotosalen/ultraviolet A (UVA) pathogen inactivation technique. ResultsOne hundred and seventy‐four sulfenylated proteins were identified belonging mainly to the integrin signal pathway and cytoskeletal regulation by Rho GTPase. The impact on pathogen inactivated platelet concentrates was weak compared to untreated ones where three sulfenylated proteins (myosin heavy chain 9, integrin βIII, and transgelin 2) were significantly affected by amotosalen/UVA treatment. Of particular interest, the reported oxidation of cysteine residues in integrin βIII is known to activate the receptor αIIbβIII. Following the pathogen inactivation, it might trigger the phosphorylation of p38MAPK and explain the lesions reported in the literature. Moreover, procaspase activating compound‐1 (PAC‐1) binding assays on platelet activation showed an increased response to adenosine diphosphate exacerbated by the tagging of proteins with dimedone. This result corroborates the hypothesis of an oxidation‐triggered activation of αIIbβIII by the pathogen inactivation treatment. ConclusionsThe present work completes missing information on the platelet proteome and provides new insights on the effect of pathogen inactivation linked to integrin signaling and cytoskeleton regulation.

Effects of amotosalen treatment on human platelet lysate bioactivity: A proof-of-concept study.

Clinical application of mesenchymal stromal cells (MSCs) usually requires an in vitro expansion step to reach clinically relevant numbers. In vitro cell expansion necessitates supplementation of basal mammalian cell culture medium with growth factors. To avoid using supplements containing animal substances, human platelet lysates (hPL) produced from expired and pathogen inactivated platelet concentrates can be used in place of fetal bovine serum. However, globally, most transfusion units are currently not pathogen inactivated. As blood banks are the sole source of platelet concentrates for hPL production, it is important to ensure product safety and standardized production methods. In this proof-of-concept study we assessed the feasibility of producing hPL from expired platelet concentrates with pathogen inactivation applied after platelet lysis by evaluating the retention of growth factors, cytokines, and the ability to support MSC proliferation and tri-lineage differentiation. Bone marrow-derived MSCs (BM-MSCs) were expanded and differentiated using hPL derived from pathogen inactivated platelet lysates (hPL-PIPL), with pathogen inactivation by amotosalen/ultraviolet A treatment applied after lysis of expired platelets. Results were compared to those using hPL produced from conventional expired pathogen inactivated platelet concentrates (hPL-PIPC), with pathogen inactivation applied after blood donation. hPL-PIPL treatment had lower concentrations of soluble growth factors and cytokines than hPL-PIPC treatment. When used as supplementation in cell culture, BM-MSCs proliferated at a reduced rate, but more consistently, in hPL-PIPL than in hPL-PIPC. The ability to support tri-lineage differentiation was comparable between lysates. These results suggest that functional hPL can be produced from expired and untreated platelet lysates by applying pathogen inactivation after platelet lysis. When carried out post-expiration, pathogen inactivation may provide a valuable solution for further standardizing global hPL production methods, increasing the pool of starting material, and meeting future demand for animal-free supplements in human cell culturing.

Neonatal and pediatric platelet transfusions: current concepts and controversies.

In this review, we focus on three specific concepts related to platelet transfusion in the neonatal and pediatric population: choice of transfusion threshold; use of ABO-mismatched platelets; transfusion of pathogen-reduced or inactivated platelets. Recent trials support the use of lower platelet transfusion thresholds (25 000/μl) in preterm neonates, although data is limited to guide transfusion among more mature neonates. In children, there is low-level evidence as to what the prophylactic platelet transfusion threshold should be in many situations of thrombocytopenia, revealing major variability in platelet transfusion practices. Most pediatric guidelines are extrapolated from adult studies with the most evidence in treatment-associated hypoproliferative thrombocytopenia varying between a platelet transfusion threshold of 10 000/μl to 20 000/μl. Although pathogen-reduced platelets may lower the risks of transfusion-transmitted infection, the effects on platelet refractoriness and transfusion burden in this population warrant additional study. Our review highlights recent advances in neonatal and pediatric platelet transfusion and also emphasizes the urgent need for better evidence to guide practice given recent studies showing the potential harms of platelet transfusion, particularly with liberal use.

In vitro evaluation of pathogen inactivated platelet quality: An 8 year experience of routine use in Galicia, Spain

BackgroundPlatelet concentrates (PCs) treated by the pathogen inactivation technology (PI) using amotosalen and UVA illumination (PI-PCs) can be manufactured in additive solutions (PAS-III and PAS-IIIM) or in 100% Plasma. Quality control (QC) is an integral part of the production. We capitalized on our ongoing QC program to capture 8 years-worth of data on parameters related to the quality of 116,214 PI-PCs produced under different manufacturing methods. Materials and methodsSelected in vitro parameters of metabolism, activation, and storage were analyzed for the different manufacturing periods to compare PI-PCs versus conventional PCs (C-PCs) resuspended in different PAS. Results and discussionAll BC-PCs met quality standards for pH and dose and residual leucocytes. As expected, storage time correlated with increased lactate, LDH, Annexin V, CD62, sCD40 L levels and decreased glucose and pH. With PAS-IIIM, higher levels of glucose were observed toward the end of shelf life (p < 0.0001) with lower platelet activation markers Annexin V (p = 0.038) and CD62 (p = 0.0006). Following PI implementation, a low expire rate of <0.5% was observed. While a 2.3% mean increase in the production of PCs occurred from 2011 to 2015, the distribution of red blood cell concentrates dropped by 4.4%. A mean incidence of 0.14% for transfusion-related adverse reaction was observed while PI-PCs were distributed, similar to the one observed with C-PCs. Overall, PI-PCs prepared in additive solutions consistently met quality standards. Those prepared in PAS-IIIM appeared to have better retention of in vitro characteristics compared to PAS-III though all demonstrated functionality and clinical effectiveness.

Biosynthesis, property comparison, and hemocompatibility of bacterial and haloarchaeal poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

Polyhydroxyalkanoates (PHAs) are a class of natural biopolyesters accumulated intracellularly by many microorganisms. These polymers have attracted particular attention as green plastic in biomedical and industrial applications due to their good biodegradability and biocompatibility. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is one of the most common members of PHAs. However, there is no report comparing the properties of PHBV from different groups of producers, e.g., bacteria and haloarchaea. In this study, two types of PHBV copolymers were synthesized in Halogranum amylolyticum and Ralstonia eutropha, respectively, by feeding different carbon sources. They possessed a similar concentration of 3HV monomers (21mol%) and were named PHBV-H (produced by H. amylolyticum) and PHBV-B (produced by R. eutropha) based on their source. Interestingly, they exhibited different behaviors especially in thermal stability, melting temperature, crystallinity percentage, and mechanical properties. Furthermore, the films of PHBV-H and PHBV-B possessed different surface properties, such as surface roughness, wettability, and surface free energy. The value of hemolysis on the PHBV-H film was lower in comparison with the PHBV-B film, although both values were within the limit of 5% permissible for biomaterials. Notably, few inactivated platelets adhered to the surface of the PHBV-H film, whereas numerous activated platelets were seen on film PHBV-B. These results indicated that PHBV-H was a better potential component of blood-contact biomaterials than PHBV-B. Our study clearly revealed that the properties of PHAs are source dependent and haloarchaeal species provide a new opportunity for the production of desired PHAs.

Effect of acute plateletpheresis and back-transfusion on platelet activation in patients undergoing open heart surgery with CPB

Objective To evaluate the effect of acute plateletpheresis(APP) and back-transfusion on platelet activation in the patients undergoing open heart surgery with cardiopulmonary bypass(CPB). Methods Forty patients, aged 35-64 yr, with body mass index within the normal range, of ASA physical status Ⅱ or Ⅲ(NYHA Ⅱ or Ⅲ), scheduled for elective cardiac valve replacement under CPB, were randomly divided into 2 groups(n=20 each) using a random number table: control group(group C)and APP group.In group APP, after induction of anesthesia, APP was performed, packed red blood cells and platelet-poor plasma were transfused back to the patient after termination of the previous cycle, and another cycle was started simultaneously.Platelet-rich plasma(PRP)was harvested and transfused back to the patient after termination of CPB and neutralization of heparin with protamine.Before induction of anesthesia(baseline), after APP and before heparinization, after neutralization of heparin with protamine and before back-transfusion of PRP, at the end of operation, and at 24 h after operation, venous blood samples were collected for determination of the expression of CD62p and PAC-1 in inactivated platelets and activated platelets by adenosine diphosphate(ADP). After APP and before heparinization, and after neutralization of heparin with protamine and before back-transfusion of PRP, the expression of CD62p and PAC-1 in inactivated platelets and ADP-activated platelets was detected in the whole blood and PRP in group APP. Results Compared with C group, the expression of CD62p and PAC-1 in inactivated platelets was down-regulated at the end of operation, and the expression of CD62p and PAC-1 in ADP-activated platelets was up-regulated in APP group.The expression of CD62p and PAC-1 in inactivated platelets was down-regulated in PRP, and the expression of CD62p and PAC-1 in ADP-activated platelets was up-regulated in PRP as compared with those in the whole blood. Conclusion APP can not induce platelet activation, however, platelet back-transfusion can enhance platelet activation in the patients undergoing open heart surgery with CPB. Key words: Plateletpheresis; Platelet activation; Cardiopulmonary bypass; Cardiac surgical procedures

Evaluation of relationship between endothelial function and vascular-platelet hemostasis in patients with a good compensation of type 2 diabetes mellitus and hypertension

The aim of research. The aim of this study is to determine the relationships between endothelial dysfunction, the degree of renal function damage, lipidemic profile and morphological changes of vascular-platelet hemostasis in patients with good compensation of type 2 diabetes mellitus and arterial hypertension. Material s and m ethods . In 69 patients with good glycemic compensation using clinical, laboratory, morphological methods and correlational analysis association between endothelial dysfunction, degree of renal function damage, lipidemic profile and morphological changes of vascular-platelet hemostasis have been identified. Results. Results of correlation analysis showed the presence of credible negative relation between the content of inactivated platelets and concentrations of endothelin-1 (r=-0.53; p<0.05), von Willebrand factor (r=-0.68; p<0.05) and thrombomodulin (r=-0.55; p<0.05). The largest number and strength of correlations between morphological indicators of intravascular platelet activation and studied clinical and laboratory parameters were observed with admiration to the activated and aggregated forms of cells. In particular, elevated levels of activated platelets in patients of this contingent had reliable high degree of direct correlation with the content of glycated hemoglobin (r=+0.79), systolic blood pressure (r=+0.63), the concentration of endothelial dysfunction markers, including the level of albuminuria (r=+0.73), as well as with the values of total cholesterol (r=+0.64) and low density lipoprotein cholesterol (r=+0.79). Content of aggregated platelets statistically significantly positively correlated with the level of glycated hemoglobin (r=+0.73), all markers of endothelial dysfunction, urea concentration (r=+0.51) and creatinine (r=+0.58). In contrast, the relative content of degranulated platelet establish a reliable correlation only with the levels of endothelin-1 (r=+0.75) and thrombomodulin (r=+0.62). On the level of endothelin-1 and thrombomodulin concentration positive linear correlation was found, but alpha-granules located in shut inverse correlation not only with endothelin-1 (r=-0.57; p<0.05), as well as with the concentration of glycated hemoglobin (r=-0.76; p<0.05) and with dyslipidemic changes. Increased platelet aggregation activity in patients with good glycemic compensation, which was manifested in a twofold increase in the content of circulating small units was significantly correlated with the level of glycated hemoglobin (r=+0.66) and the values of systolic blood pressure (r=+0.58), as well as concentrations of markers of endothelial dysfunction and renal function, but did not have any significant relationships with blood lipids in patients of this contingent. Conclusion. It has been established that in the good glycemic control morphological criteria of platelet hemostasis damage clearly correlated with markers of endothelial dysfunction, but did not show significant relationships with indicators of renal function and blood lipid profile.

Pathogen inactivation – defining ‘adequate’ bacterial protection

Background and ObjectivesClinical acceptance of pathogen inactivation technologies depends on the demonstration of product safety, a minimal effect on product efficacy and the range and degree of pathogen inactivation. The major rationale for pathogen inactivation of platelets is the prevention of transfusion‐associated bacterial sepsis, a persistent problem despite multiple innovations to minimize and detect contamination.Materials and MethodsRecent reports and an empirical model are used to describe our current understanding of the efficacy of pathogen inactivation in improving platelet transfusion safety.ResultsOptimized in vitro testing evaluated the efficacy of riboflavin/UV light (Mirasol™), amotosalen/UVA (Intercept™) and UVC irradiation (THERAFLEX™ UV platelets) systems to effect a 102‐ &gt;106 fold reduction of a variety of bacterial species. All systems are less effective at inactivating bacterial spores. Reviewing reports on the level of pathogen inactivation at very low and high bacterial concentrations identifies areas for further scrutiny.ConclusionsClinical trials of pathogen inactivated platelets have focused on safety and clinical efficacy in terms of count increments and the prevention and treatment of bleeding. Little attention has been paid to efficacy in reducing the risk of bacterial transmission. Pathogen inactivation is expected to significantly reduce transfusion of contaminated platelet units, especially ones that are the result of low initial bacterial titres that are a systemic issue with the current testing methodology. Nevertheless, available data suggest that breakthrough cases of sepsis are likely and that their rates will vary substantially by technology.

Qualitative analysis of platelet rich plasma prepared by acute plateletpheresis in patients undergoing heart surgery

This study was aimed to evaluate the efficiency and effectiveness of platelet-rich plasma(PRP) prepared by acute plateletpheresis in patients undergoing open heart surgery, and to analyze the quality of prepared platelet-rich plasma. Whole blood from 20 patients with ASAII-III was collected and PRP was harvested by machine after induction of anesthesia. Platelet count (Plt), mean platelet volume (MPV), platelet distribution width (PDW), plasma pH, plasma lactic acid (LA) concentration, and lactic dehydrogenase (LDH) concentration, germiculture result, CD62p and PAC-1 positive rate of inactivated and activated platelets by ADP in the whole blood before plateletpheresis (T1) , in the PRP after plateletpheresis (T2) and PRP before back-transfusion (T3) were determinated. The results showed that as compared with whole blood the platelet count in the PRP at T2 was (783 ± 184) ×10(9)/L, MPV, PDW and pH significantly decreased (P < 0.01) , while the plasma LDH, LA concentration, CD62p and PAC-1 positive rate of inactivated platelets were not significantly different from the whole blood at T1. In the PRP at T3, the platelet count, MPV, PDW and pH significantly decreased (P < 0.01) , while plasma LDH concentration, CD62p and PAC-1 positive rate of inactivated platelet significantly increased (P < 0.05 or P < 0.01) compared with the whole blood at T1. There were no significant difference among the CD62p and PAC-1 positive rate of activated platelets in the whole blood and PRP. It is concluded that PRP can be efficiently obtained from the patients undergoing open heart surgery by acute plateletpheresis, and the platelets in PRP are not activated during the preparing process. Some platelets in PRP are activated during the preserving process, but the whole activating function of platelets keeps normal.

Ultralarge von Willebrand Factor Fibers Mediate Luminal Staphylococcus aureus Adhesion to an Intact Endothelial Cell Layer Under Shear Stress

During pathogenesis of infective endocarditis, Staphylococcus aureus adherence often occurs without identifiable preexisting heart disease. However, molecular mechanisms mediating initial bacterial adhesion to morphologically intact endocardium are largely unknown. Perfusion of activated human endothelial cells with fluorescent bacteria under high-shear-rate conditions revealed 95% attachment of the S aureus by ultralarge von Willebrand factor (ULVWF). Flow experiments with VWF deletion mutants and heparin indicate a contribution of the A-type domains of VWF to bacterial binding. In this context, analyses of different bacterial deletion mutants suggest the involvement of wall teichoic acid but not of staphylococcal protein A. The presence of inactivated platelets and serum increased significantly ULVWF-mediated bacterial adherence. ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin motifs 13) caused a dose-dependent reduction of bacterial binding and a reduced length of ULVWF, but single cocci were still tethered by ULVWF at physiological levels of ADAMTS13. To further prove the role of VWF in vivo, we compared wild-type mice with VWF knockout mice. Binding of fluorescent bacteria was followed in tumor necrosis factor-α-stimulated tissue by intravital microscopy applying the dorsal skinfold chamber model. Compared with wild-type mice (n=6), we found less bacteria in postcapillary (60±6 versus 32±5 bacteria) and collecting venules (48±5 versus 18±4 bacteria; P<0.05) of VWF knockout mice (n=5). Our data provide the first evidence that ULVWF contributes to the initial pathogenic step of S aureus-induced endocarditis in patients with an apparently intact endothelium. An intervention reducing the ULVWF formation with heparin or ADAMTS13 suggests novel therapeutic options to prevent infective endocarditis.

Evaluation of amotosalem treated platelets over 7 days of storage with an automated cytometry assay panel

Pathogen Inactivation allows to overcome microbial contamination and growth related to storage of platelets concentrates (PC) at room temperature. The aim of our study was to evaluate the platelet storage lesion extending the storage period of pathogen inactivated platelet concentrates over 7days using an automated cytometry assay panel. We analyzed 43 concentrates subjected to pathogen inactivation (CPPI) at 3, 5 and 7days evaluating: platelet count, mean platelet volume, platelets at low optical density, platelets at high density, GPIIb-IIIa glycoprotein, platelet microparticles, lactate dehydrogenase. The collection bags (Fenwal) and the IBS kit made in PL2410/PL2411 are approved for the conservation of PC up to 7days. Data analysis was performed with anova test. All the parameters except small platelets and PMP were statistically different among day 7 vs. 3 and day 7 vs. 5. Our study showed a progressive modification of pathogen inactivated platelet concentrates observed up to 7days. The persistence of the secretory pool and the presence of the platelet membrane fibrinogen receptor suggest the persistence of a potential hemostatic efficacy. Clinical studies are necessary to directly correlate this type of analysis to 24h recovery or survival of transfused platelets in humans.

Therapeutic efficacy of platelet components treated with amotosalen and ultraviolet A pathogen inactivation method: results of a meta‐analysis of randomized controlled trials

There are conflicting data regarding the therapeutic efficacy of platelets inactivated using amotosalen and ultraviolet A light. We have performed a meta-analysis to summarize the results of different randomized controlled trials (RCT). Five RCTs reported through March 2011 met the criteria for meta-analysis. Weighted mean difference (WMD) in corrected count increment (CCI) at 1 h, CCI-24 h, and transfusion interval (days) and summary odds ratio (OR) of bleeding in inactivated platelet (I-P) group vs. noninactivated platelet (C-P) group were calculated across studies. Randomized controlled trials were statistically homogeneous when we analysed CCI-24 h, and the transfusion of C-P was associated with a higher CCI-24 h when compared with the transfusion of I-P (WMD, 3×10(3); 95% CI, 2·32×10(3)-3·69×10(3); P<0·00001). RCTs were statistically heterogeneous when we analysed CCI-1 h, transfusion interval and OR of bleeding. Regarding the OR of bleeding in the I-P and C-P groups, it varied by as much as a multiple of four among the trials, from 0·66 to 2·66. When we combined double-blinded and high methodologic quality score RCTs, the use of I-P was not statistically associated with an increase in the OR of bleeding when compared with the use of C-P (OR, 0·97; 95% CI, 0·75-1·27; P=0·84). Although the transfusion of I-P was associated with lower CCI-24 h when compared with the transfusion of C-P, this was not associated with differences in the OR of bleeding between I-P and C-P.

Determination of acute lung injury after repeated platelet transfusions

AbstractAcute lung injury (ALI) during hematopoietic stem cell transplant (HSCT) is associated with substantial morbidity; however, the frequency of ALI in HSCT patients is poorly characterized. Platelets are postulated to play a critical role in the pathogenesis of ALI. Using a transfusion trial of pathogen inactivated platelet components (PC-Test) compared with conventional PC (Reference) populated with HSCT patients, data were reviewed by an adjudication panel to determine the frequency of ALI overall, by treatment groups, and key outcomes: PC exposure, ventilator-free days, and mortality. The diagnosis of ALI was based on American European Consensus Criteria. Of 645 patients who received PC over 28 days, 100 (15.5%) had clinically serious pulmonary adverse events, and 35 (5.4%) met criteria for ALI. Days of platelet support and number of platelet transfusions for patients with ALI were not significantly different from patients without ALI (P > .05). Mortality was greater for patients with ALI (57%) than those without (17%, P < .001) but not significantly different between treatment groups. For patients with ALI, the distributions of time to onset of mechanical ventilation were significantly different (P = .04). Patients supported with Reference PC were more likely to be ventilated sooner than patients receiving Test PC.

Hemocompatibility of soft hydrophobic poly(n-butyl acrylate) networks with elastic moduli adapted to the elasticity of human arteries

Small calibre vascular prostheses (<6 mm) still lack medium and long term patency. Inelasticity of the prosthesis is one of the characteristics, which is involved in the mechanisms of failure (e.g. the development of neointimal hyperplasia at the distal anastomosis). Here we report about covalently crosslinked poly(n-butyl acrylate) networks (cPnBA) with adjustable elastic moduli, which can be tailored to values of human arteries (between 100 and 1000 kPa). Motivated by the potential application of such polymer networks as cardiovascular prosthesis, adhesion, activation and thrombus formation of human platelets on cPnBA networks were evaluated. All cPnBA-samples displayed a high thrombogenicity compared to the control (silicone). Significantly less platelets adhered on the surface of the soft cPnBA04 than on cPnBA73. All cPnBA samples displayed a higher number of platelet aggregates and a lower number of inactivated platelets in comparison to the control. While the elastic modulus of cPnBA networks could be successfully adjusted to that of human arteries, the tested polymers did not show an optimal hemocompatibility. Future studies aim at improving the biofunctionality by surface modification of these polymer networks.

Shear Induced Reversible Conglomerates of Von Willebrand Factor Networks with Aggregates of Inactivated Platelets - a Fast Switchable Plugging Mechanism

AbstractAbstract 2104Thrombus growth rate has been reported to be a linear function of very high shear rates beyond 40,000 s-1 (Ku DN, Bioreology. 2007;44(4):273-84), which are thought to occur in severe arterial stenosis. We therefore applied shear rates continuously increasing from 2,000 s-1 to 40,000 s-1 and examined platelet and von Willebrand factor (VWF) interaction in a parallel plate flow chamber under direct visualization. Above a critical threshold of 10,000 to 15,000 s-1 aggregates of inactivated platelets formed when VWF was present in solution and immobilized on the perfused surface (Ruggeri, … Reininger; Blood, 108: 1903–1910, 2006). A new finding was the shear dependent simultaneous formation of VWF strands in and around those platelet aggregates, growing to strands up to several microns thick and up to several tens of microns long, thus forming large VWF networks. Platelets appeared to be enmeshed in the VWF networks but were nevertheless a prerequsite for their formation, mutually fostering each other. Platelet-VWF conglomerates formed at the perfused VWF surface, were constantly rolling in the flow direction, grew in size with increasing shear rate, and were completely reversible below the critical shear rate threshold of 10,000 s-1. VWF networks assembled in whole blood as well as in washed blood cell suspensions reconstituted with VWF. Recombinant full length VWF generated larger VWF networks than a commercial VWF preparation from pooled human plasma. Perfusion of collagen coated surfaces caused assembly of VWF networks anchored to the collagen fibrils and capture of activation-blocked platelets within them at shear rates of 2,000 s-1, leading to residence times of platelet-VWF conglomerates of more than a minute. Newly formed platelet conglomerates could be visualized rolling on the surfaces of already attached conglomerates. When we used a gain of function mutation of VWF, rolling platelet aggregates could be detected at a shear rate as low as 1,500 s-1. We conclude that shear rate activation of VWF leads to reversible platelet-VWF network generation, which may be a crucial mechanism of fast platelet accrual and prolonged arrest times at sites of thrombosis as well as physiological hemostasis. Disclosures:No relevant conflicts of interest to declare.