Platelet Surface Research Articles

Targeting transmembrane-domain-less MOG expression to platelets prevents disease development in experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system with no cure yet. Here, we report genetic engineering of hematopoietic stem cells (HSCs) to express myelin oligodendrocyte glycoprotein (MOG), specifically in platelets, as a means of intervention to induce immune tolerance in experimental autoimmune encephalomyelitis (EAE), the mouse model of MS. The platelet-specific αIIb promoter was used to drive either a full-length or truncated MOG expression cassette. Platelet-MOG expression was introduced by lentivirus transduction of HSCs followed by transplantation. MOG protein was detected on the cell surface of platelets only in full-length MOG-transduced recipients, but MOG was detected in transmembrane-domain-less MOG1-157-transduced platelets intracellularly. We found that targeting MOG expression to platelets could prevent EAE development and attenuate disease severity, including the loss of bladder control in transduced recipients. Elimination of the transmembrane domains of MOG significantly enhanced the clinical efficacy in preventing the onset and development of the disease and induced CD4+Foxp3+ Treg cells in the EAE model. Together, our data demonstrated that targeting transmembrane domain-deleted MOG expression to platelets is an effective strategy to induce immune tolerance in EAE, which could be a promising approach for the treatment of patients with MS autoimmune disease.

Inhibition of platelet activation suppresses reactive enteric glia and mitigates intestinal barrier dysfunction during sepsis

BackgroundIntestinal barrier dysfunction, which is associated with reactive enteric glia cells (EGCs), is not only a result of early sepsis but also a cause of multiple organ dysfunction syndrome. Inhibition of platelet activation has been proposed as a potential treatment for septic patients because of its efficacy in ameliorating the organ damage and barrier dysfunction. During platelet activation, CD40L is translocated from α granules to the platelet surface, serving as a biomarker of platelet activation a reliable predictor of sepsis prognosis. Given that more than 95% of the circulating CD40L originate from activated platelets, the present study aimed to investigate if inhibiting platelet activation mitigates intestinal barrier dysfunction is associated with suppressing reactive EGCs and its underlying mechanism.MethodsCecal ligation and puncture (CLP) was performed to establish the sepsis model. 24 h after CLP, the proportion of activated platelets, the level of sCD40L, the expression of tight-junction proteins, the intestinal barrier function and histological damage of septic mice were analyzed. In vitro, primary cultured EGCs were stimulated by CD40L and LPS for 24 h and EGCs-conditioned medium were collected for Caco-2 cells treatment. The expression of tight-junction proteins and transepithelial electrical resistance of Caco-2 cell were evaluated.ResultsIn vivo, inhibiting platelet activation with cilostazol mitigated the intestinal barrier dysfunction, increased the expression of ZO-1 and occludin and improved the survival rate of septic mice. The efficacy was associated with reduced CD40L+ platelets proportion, decreased sCD40L concentration, and suppressed the activation of EGCs. Comparable results were observed upon treatment with compound 6,877,002, a blocker of CD40L–CD40–TRAF6 signaling pathway. Also, S-nitrosoglutathione supplement reduced intestinal damage both in vivo and in vitro. In addition, CD40L increased release of TNF-α and IL-1β while suppressed the release of S-nitrosoglutathione from EGCs. These EGCs-conditioned medium reduced the expression of ZO-1 and occludin on Caco-2 cells and their transepithelial electrical resistance, which could be reversed by CD40-siRNA and TRAF6-siRNA transfection on EGCs.ConclusionsThe inhibition of platelet activation is related to the suppression of CD40L-CD40-TRAF6 signaling pathway and the reduction of EGCs activation, which promotes intestinal barrier function and survival in sepsis mice. These results might provide a potential therapeutic strategy and a promising target for sepsis.

Stable binding to phosphatidylserine-containing membranes requires conserved arginine residues in tandem C domains of blood coagulation factor VIII.

At sites of vascular damage, factor VIII (fVIII) is proteolytically activated by thrombin and binds to activated platelet surfaces with activated factor IX (fIXa) to form the intrinsic "tenase" complex. Previous structural and mutational studies of fVIII have identified the C1 and C2 domains in binding to negatively charged membrane surfaces through β-hairpin loops with solvent-exposed hydrophobic residues and a ring of positively charged basic residues. Several hemophilia A-associated mutations within the C domains are suggested to disrupt lipid binding, preventing formation of the intrinsic tenase complex. In this study, we devised a novel platform for generating recombinant C1, C2, and C1C2 domain constructs and performed mutagenesis of several charged residues proximal to the putative membrane binding region of each C domain. Binding measurements between phosphatidylserine (PS)-containing lipid membrane surfaces and fVIII C domains demonstrated an ionic strength dependence on membrane binding affinity. Mutations to basic residues adjacent to the surface-exposed hydrophobic regions of C1 and C2 differentially disrupted membrane binding, with abrogation of binding occurring for mutations to conserved arginine residues in the C1 (R2163) and C2 (R2320) domains. Lastly, we determined the X-ray crystal structure of the porcine fVIII C2 domain bound to o-phospho-L-serine, the polar headgroup of PS, which binds to a basic cleft and makes charge-charge contact with R2320. We conclude that basic clefts in the fVIII C domains bind to PS-containing membranes through conserved arginine residues via a C domain modularity, where each C domain possesses modest electrostatic-dependent affinity and tandem C domains are required for high affinity binding.

Surface-anchored tumor microenvironment-responsive protein nanogel-platelet system for cytosolic delivery of therapeutic protein in the post-surgical cancer treatment

Nanoparticle-anchored platelet systems hold great potential to act as drug carriers in post-surgical cancer treatment due to their intrinsic ability to target the bleeding sites. However, rational design is still needed to further improve its cargo release profiles to meet the cytosolic delivery of therapeutic proteins with intracellular targets. Herein, we developed a tumor microenvironment (TME)-responsive backpack-conjugated platelet system to enhance intracellular protein delivery, thereby significantly inhibiting tumor recurrence after surgery. Specifically, protein nanogels encapsulating GALA and Granzyme B (GrB) are conjugated on the platelet surface via an acid-sensitive benzoic-imine linker through a biorthogonal reaction (GALA-GNGs-P). Taking advantage of wound-tropism of platelets, GALA-GNGs-P could actively accumulate at the surgical trauma and release nanogels in response to acidic TME for promoting deep penetration. Following cellular uptake, the pore-forming peptide GALA helps nanogels escape from lysosome. Subsequently, high glutathione (GSH) concentration in tumor cytoplasm facilitates GrB release from NGs, leading to intense cell apoptosis. GALA-GNGs-P shows remarkable tumor-targeting capability, high cellular uptake, and outstanding lysosomal escaping ability, which can significantly inhibit tumor recurrence in mice models with incomplete tumor resection. Our findings indicate that platelets bioengineered with TME-responsive protein nanogels provide an option to intracellularly deliver therapeutic proteins for the post-surgical treatment of cancer. Statement of significancePlatelet-based drug delivery systems (DDSs) have gained considerable achievements in post-surgical cancer treatment. However, there is no research exploring their potential in realizing the controllable release of cargoes in the acidic tumor microenvironment (TME). Herein, we developed a TME-responsive bioengineered platelet delivery platform (GALA-GNGs-P) for achieving controllable and effective protein intracellular delivery to overcome post-surgical tumor recurrence. Our surface-anchored nanogel-platelet system has the following advantages: (i) improving the loading efficiency of therapeutic proteins, (ii) affecting no physiological function of platelets, (iii) realizing on-demand cargo release in the acidic TME, and (iv) helping proteins escape from endosomal entrapment. Our findings further explored the prospect of cellular backpack system and realized the controllable release of cargoes in the acidic TME.

Potential of Mean Force for Face-Face Interactions between Pairs of 2:1 Clay Mineral Platelets.

Bottom-up modeling of clay behavior from the molecular scale requires a detailed understanding of the free energy between pairs of clay platelets. We investigate the potential of mean force (PMF) for hydrated clays in face-to-face interactions with free energy perturbation (FEP) methods through molecular dynamics simulations using simple overlap sampling (SOS). We show that PMF results for open systems with one finite in-plane dimension are affected by migration of counterions from within the interlayer space compared with fully confined closed system conditions. We compare PMFs for two common 2:1 clay sheet minerals Illite (IMt-1) and Na-smectite. The PMFs for the open illite systems exhibit a strong attractive energy well at a basal layer separation, d = 11 Å and interlayer water content, wIL = ∼0.4% while the attractive minimum for the closed system occurs at d = 12 Å, wIL = 3.5%. In contrast, net repulsion occurs between pairs of Na-smectite platelets for both open and closed systems (for d < 15-16 Å). The free energy is closely related to the distribution of counterions; while K+ ions are bound closely to the surfaces of the illite platelets, Na+ ions are more spatially disperse. This PMF results contradict prior findings for Na-smectite and prompted further comparisons with other published results. We find that most of the published results do not represent accurately the free energy for face-face interactions between pairs of clay platelets that are effectively infinite (with width/thickness O[104]). The PMF results presented in this paper form a reliable basis for mesoscale, coarse-grained modeling of illite and smectite particle assemblies. We show that the Gay-Berne potential provides a reasonable first-order model for upscaling, while the solvation potential proposed by Masoumi enables a more accurate representation of the computed PMFs.

Preparation and action mechanism of temperature sensitive N-isopropylacrylamide/nanosilica hybrid as rheological modifier for water-based drilling fluid

A temperature-sensitive silica nanohybrid with a temperature response at 35 °C was prepared by copolymerization of temperature-sensitive monomer N-isopropylacrylamide (NIPAM) with reactive nanosilica (RNS-D).The rheological properties of the as-prepared NIPAM/nano-SiO2 nanohybrid as the rheological modifier of water-based drilling fluid were tested. Findings demonstrate that NIPAM/nano-SiO2 nanohybrid contributes to increasing the viscosity of the drilling fluid upon elevating temperature, and the drilling fluid fits to the Herschel-Bulkley model. The drilling fluid containing 1.0% (mass fraction) of NIPAM/nano-SiO2 nanohybrid exhibits significant thermally thickening performance at the critical association temperature (Tass) of NIPAM (Tass; 35 °C). In terms of the action mechanism of NIPAM/nano-SiO2 nanohybrid in drilling fluid, the amide group of NIPAM/nano-SiO2 nanohybrid can form hydrogen bond with bentonite, thereby being well adsorbed on the surface and edge of bentonite platelets or even entrapped into the interlay of the bentonite platelets. The resultant cross-linked structure can function to prevent the flocculation of bentonite platelets and increase the viscosity of the drilling fluid. After heating to Tass, the temperature-sensitive polymer molecular chains undergo hydrophilic to hydrophobic transformation and hydrophobic association to form cross-linked three-dimensional network structure and increase the viscosity of the drilling fluid. More importantly, the water-based drilling fluid with 1.0% NIPAM/nano-SiO2 nanohybrid still retains thermally thickening behavior and flat rheology even after being aged at 150 °C. Therefore, the temperature-sensitive NIPAM/nano-SiO2 nanohybrid could be a potential rheological modifier for flat rheology water-based drilling fluid.

The High Expression of Adhering and Circulating Integrin Serves as a Diagnostic Marker in Venous Thromboembolism.

Objective To investigate the diagnostic value of circulating integrins β1, 2, and 3 in venous thrombosis (VTE). Materials and Methods A total of 474 VTE patients and 306 patients with nonhigh risk for VTE as the control group were studied. Levels of adhering integrins β1, 2, and 3 were detected by flow cytometry. Levels of circulating integrins β1, 2, and 3 in serum were measured by enzyme-linked immunosorbent assay. Results We found that integrins β1, 2, and 3 were expressed highly both in serum and on the surface of leukocytes and platelets in venous thromboembolism. The levels of circulating integrins β1, 2, and 3 are positively correlated with adhering integrins. It showed excellent clinical diagnostic performance of circulating integrins β1, 2, and 3 in venous thromboembolism. Conclusions Integrin subunit β can be used as a diagnostic marker with high sensitivity and specificity for venous thromboembolism.

Proprotein convertase subtilisin/kexin type 9 (PCSK9), platelet activation and interaction with the vascular endothelium: the impact of PCSK9 inhibition with evolocumab in acute coronary syndrome

Abstract Background/Introduction Platelet activation and endothelial dysfunction may contribute to adverse outcomes in patients with acute myocardial infarction. Pre-clinical studies indicate PCSK9 enhances platelet activation. Purpose Our goals were (1) to examine the role of PCSK9 as a potential mediator of platelet activation in patients with acute coronary syndrome (ACS) and (2) to perform immunohistochemical studies of left internal mammary artery (LIMA) specimens to examine interactions among PCSK9, platelets, and endothelial cells in specimens obtained during coronary bypass surgery. Methods Participants from the Evolocumab in Acute Coronary Syndrome trials (EVACS; NCT03515304, NCT04082442), with a NSTEMI and a troponin-I of &amp;gt;5 ng/ml or a STEMI were randomized to placebo or to 420 mg SC of evolocumab, a monoclonal anti-PCSK9 antibody, within 24 hours of hospitalization. We performed serial ex vivo analysis of PF4, a marker of platelet activation, in serum samples obtained at baseline (prior to study drug administration) and at day 30. PF4 values were normalized to total platelet count. All of the participants were on guideline-directed therapies for ACS. Additionally, LIMA samples from patients undergoing coronary bypass surgery were immunostained with PCSK9, the endothelial cell transmembrane glycoprotein CD31 (platelet endothelial cell adhesion molecule-1) and the platelet surface marker CD61 (integrin beta-3). Outcome data were summarized using medians and interquartile ranges. Results Forty-six participants were randomized in a 1:1 fashion to placebo or to evolocumab. Mean (±SD) age of the cohort was 60±13 years, 48% were women and 22% were African American. Baseline PF4 levels (expressed as ng/1k platelets) were similar between the two groups (placebo: 9.3 [4–12] vs evolocumab 8.0 [4–12], p=0.8). In the placebo group, there was a significant increase in PF4 at 30 days to 13.1 [11–14], p&amp;lt;0.01 (baseline vs 30 days). In contrast, there was no significant change from baseline in the evolocumab group at 30 days (10.7 [6–13]), which was significantly lower than the placebo group (p=0.04). Furthermore, immunostaining of LIMA specimens obtained from patients during coronary artery bypass surgery revealed colocalization of PCSK9, CD31, and CD61 on the vascular endothelial cell surface (see Figure). Conclusion PCSK9 inhibition with evolocumab decreases platelet activation in ACS patients on dual anti-platelet therapy. PCSK9 is associated with platelets and endothelial cells at the vascular endothelium. PCSK9 is a potential mediator of the interaction between platelets and vascular endothelial cells in patients with coronary artery disease. Funding Acknowledgement Type of funding sources: Other. Main funding source(s): Amgen

D121 Located within the DRY Motif of P2Y12 Is Essential for P2Y12-Mediated Platelet Function.

Platelets are anucleate cells that mediate hemostasis. This occurs via a primary signal that is reinforced by secreted products such as ADP that bind purinergic receptors (P2Y1 and P2Y12) on the platelet surface. We recently identified a human subject, whom we termed platelet defect subject 25 (PDS25) with a platelet functional disorder associated with the P2Y12 receptor. PDS25 has normal blood cell counts and no history of bleeding diathesis. However, platelets from PDS25 have virtually no response to 2-MeSADP (a stable analogue of ADP). Genetic analysis of P2Y12 from PDS25 revealed a heterozygous mutation of D121N within the DRY motif. Rap1b activity was reduced in platelets from PDS25, while VASP phosphorylation was enhanced, suggesting that signaling from the P2Y12 receptor was interrupted by the heterozygous mutation. To explore this further, we produced knock-in mice that mimic our subject. Bleeding failed to cease in homozygous KI mice during tail bleeding assays, while tail bleeding times did not differ between WT and heterozygous KI mice. Furthermore, occlusions failed to form in most homozygous KI mice following carotid artery injury via FeCl3. These data indicate that the aspartic acid residue found in the DRY motif of P2Y12 is essential for P2Y12 function.

Provisional Matrix Formation at Implant Surfaces-The Bridging Role of Calcium Ions.

The success of dental implants lies in their strong and lasting integration into the patient’s receiving bone. The first biological interactions at the implant surface determine the subsequent evolution of the integration process. In this study we set our objective to analyze the mechanistic interaction of the early regenerative matrix at implant surfaces modified with calcium ions (Ca) as compared to standard implant surfaces (NoCa). We put the surfaces in a Quartz Crystal Microbalance with Dissipation (QCM-D) to monitor the frequency shift (f) and the viscoelastic properties of the adsorbed biofilms and used Scanning Electron Microscopy (SEM) to visualize the resulting interfaces. Upon the addition of human blood plasma, Ca surfaces formed an adsorbed three-dimensional film attached to the surface (∆f = −40 Hz), while with NoCa, the biofilm formed but was not attached to the surface (∆f = 0 Hz). After 20 min in blood, two representative commercial implants with Ca and NoCa surfaces showed also distinct interfaces: Ca implants formed a visible clot attached to the implant which was composed mainly of platelets (Surface Coverage: 40 ± 20%) and some red blood cells (SC: 9 ± 3%) entrapped within a fibrin network (SC: 93 ± 5%). The NoCa implants were largely populated by red blood cells (SC: 67 ± 12%) with scarce fibrin remnants (SC: 3 ± 2%), and the implants showed no clot on their surfaces macroscopically. The pre-clinical and clinical results discussed in this work encourage the modification of titanium implant surfaces with calcium ions to improve the bone regenerative process. Taken together, these results add more information about the roles of Ca ions in bridging the formation of the provisional matrix at implant surfaces and their effects on implant osseointegration.

Role of thrombin to non-physiological shear stress induced platelet activation and function alternation

ObjectiveNon-physiological shear stress (NPSS) and thrombin have two distinct mechanisms for activating platelets. NPSS in mechanically assisted circulation (MAC) devices can cause platelet dysfunction, e.g., by shedding its key receptors. In addition, patients with heart failure have increased levels of thrombin generation, which may further affect the NPSS-induced platelet dysfunction, resulting in device-associated complications. This study aimed to assess the combined effect of NPSS and thrombin in platelet activation, expression of adhesion receptors on the platelet surface, and alterations of platelet aggregation. MethodsFresh human blood from healthy donors was divided into two groups; one group was treated by adding 0.01 U/mL thrombin, and another group not treated with thrombin served as a control comparison. They were then pumped through a novel blood shearing device which produces similar shear stress conditions to those in the MAC devices. Three levels of NPSS (i.e., 75, 125, and 175 Pa) with a 1.0 s exposure time were selected for the shearing conditions. Expression of platelet activation markers (PAC-1, activated GPIIb/IIIa and CD62P, platelet surface P-selectin) were investigated along with the shedding of platelet receptors (GPIb, GPIIb/IIIa, and GPVI), generation of platelet microparticles, and Phosphatidylserine (PS)-positive platelets detected by flow cytometry. Platelet aggregation (induced by collagen/ristocetin) was measured by Lumi-aggregometry. ResultsPlatelet receptors were shed after exposure to NPSS showing a positive correlation with the level of shear stress. The generation of platelet microparticles and PS-positive platelets also increased with greater NPSS. Elevated NPSS decreased the platelet aggregation capacity. Platelet activation level increased with greater NPSS. Being treated by thrombin can further exacerbate these characteristics under same level of NPSS, except that platelet activation level drastically dropped after the exposure to 175 Pa NPSS in the thrombin-treated blood. ConclusionAfter being treated by thrombin, platelets became more susceptible to NPSS, resulting in more receptor shedding, platelet microparticles, and PS-positive platelets, thus limiting platelet aggregation capacity after exposure to NPSS. Platelet activation, in terms of PAC-1 and P-selectin, is an interim status competing between the expression and shedding of these makers/receptors. When platelets have reached a saturation level of activation, exposure to excessive NPSS can potentially impair activation.

Recent changes in paradigms in the management of adulthood chronic idiopathic thrombocytopenic purpura

The scenario of immune pathomechanism-based first- and second-line therapies of immune thrombocytopenia (i.e., immunosuppression, splenectomy) substantially changed approximately more than 20 years ago, giving place and important role for thrombopoietin analogs in steroid refractory cases, positioning splenectomy as third-line intervention. Although this approach became dominant and powerful, refractory cases urged the search for further medications. More recently, the traditional immunological approach received more attention again, and new targets were determined: (1) shortening selectively IgG life span and clearance, (2) modifying complement system in an effort to reduce antibody binding to platelet surface, (3) cell signaling pathway modification efforts to reduce antiplatelet phagocytic events, mainly spleen tyrosine kinase and Bruton tyrosine kinase inhibitory compounds. These new agents seem to be capable alone and probably in different combinations and sequence of increasing platelet count even in thrombopoietin analog refractory adult chronic thrombocytopenic purpura cases. These promising approaches probably will bring new treatment schedules in the adult chronic thrombocytopenic purpura field quite soon, and the scenario moves back to the refreshed immunological basis rewriting many elements of the therapeutic algorithm of thrombocytopenic purpura.

Increased platelet activation and platelet-inflammasome engagement during chikungunya infection

Chikungunya fever is a viral disease transmitted by mosquitoes of the genus Aedes. The infection is usually symptomatic and most common symptoms are fever accompanied by joint pain and swelling. In most cases symptoms subside within a week. However, severe prolonged and disabling joint pain, that may persist for several months, even years, are reported. Although the pathogenesis of Chikungunya infection is not fully understood, the evolution to severe disease seems to be associated with the activation of immune mechanisms and the action of inflammatory mediators. Platelets are recognized as inflammatory cells with fundamental activities in the immune response, maintenance of vascular stability and pathogenicity of several inflammatory and infectious diseases. Although the involvement of platelets in the pathogenesis of viral diseases has gained attention in recent years, their activation in Chikungunya has not been explored. The aim of this study was to analyze platelet activation and the possible role of platelets in the amplification of the inflammatory response during Chikungunya infection. We prospectively included 132 patients attended at the Quinta D’Or hospital and 25 healthy volunteers during the 2016 epidemic in Rio de Janeiro, Brazil. We observed increased expression of CD62P on the surface of platelets, as well as increased plasma levels of CD62P and platelet-derived inflammatory mediators indicating that the Chikungunya infection leads to platelet activation. In addition, platelets from chikungunya patients exhibit increased expression of NLRP3, caspase 4, and cleaved IL-1β, suggestive of platelet-inflammasome engagement during chikungunya infection. In vitro experiments confirmed that the Chikungunya virus directly activates platelets. Moreover, we observed that platelet activation and soluble p-selectin at the onset of symptoms were associated with development of chronic forms of the disease. Collectively, our data suggest platelet involvement in the immune processes and inflammatory amplification triggered by the infection.

Mobilisation of HLA-F on the surface of bronchial epithelial cells and platelets in asthmatic patients.

Uncontrolled inflammation of the airways in chronic obstructive lung diseases leads to exacerbation, accelerated lung dysfunction and respiratory insufficiency. Among these diseases, asthma affects 358million people worldwide. Human bronchial epithelium cells (HBEC) express both anti-inflammatory and activating molecules, and their deregulated expression contribute to immune cell recruitment and activation, especially platelets (PLT) particularly involved in lung tissue inflammation in asthma context. Previous results supported that HLA-G dysregulation in lung tissue is associated with immune cell activation. We investigated here HLA-F expression, reported to be mobilised on immune cell surface upon activation and displaying its highest affinity for the KIR3DS1-activating NK receptor. We explored HLA-F transcriptional expression in HBEC; HLA-F total expression in PBMC and HBEC collected from healthy individuals at rest and upon chemical activation and HLA-F membrane expression in PBMC, HBEC and PLT collected from healthy individuals at rest and upon chemical activation. We compared HLA-F transcriptional expression in HBEC from healthy individuals and asthmatic patients and its surface expression in HBEC and PLT from healthy individuals and asthmatic patients. Our results support that HLA-F is expressed by HBEC and PLT under healthy physiological conditions and is retained in cytoplasm, barely expressed on the surface, as previously reported in immune cells. In both cell types, HLA-F reaches the surface in the inflammatory asthma context whereas no effect is observed at the transcriptional level. Our study suggests that HLA-F surface expression is a ubiquitous post-transcriptional process in activated cells. It may be of therapeutic interest in controlling lung inflammation.

Lyophilized platelets inhibit platelet aggregation with simultaneous paradoxical promotion of platelet adhesion.

Lyophilized platelets have been explored as a potential hemostatic agent due to their long-term ambient storage capabilities that make them readily available in various scenarios. Additionally, their high biocompatibility and the key role of platelet interactions in various clinical conditions make them a promising platform for drug delivery. To explore these applications and for wider clinical deployment, the interactions between lyophilized platelets and fresh platelets must be examined. This project characterized receptor expression on the lyophilized platelet surface and their ability to bind fibrinogen using flow cytometry. The effect of lyophilized platelets on aggregation of unaltered platelets was assessed using light transmission aggregometry while the effect on adhesion was evaluated using static and microfluidic assays. Lyophilized platelets maintained significant levels of GPIIb and GPVI receptors on their surface, though the expression was reduced from fresh platelets. Additionally, lyophilized platelets maintained GPIb expression similar to fresh platelets. Furthermore, 15.8% of the lyophilized platelets exhibited the active conformation of the GPIIb/IIIa receptor, indicating a significant increase over fresh platelets. Lyophilized platelets also exhibited an increase in exposed phosphatidylserine and fibrinogen binding. Despite the effect of lyophilized platelets in promoting the adhesion of fresh platelets on a collagen-coated surface, their net effect was inhibitory on platelet aggregation. This study demonstrates that lyophilized platelets can have paradoxical effects on platelet adhesion and aggregation, which could have an impact for clinical applications. Detailed characterization and engineering of these effects will be important for their continued development as a drug delivery platform.

Aspirin Mediated Platelet Reactivity Following Vascular Procedures: The Path of Least Resistance is to Ignore it

Dehne et al. have examined aspirin resistance after vascular intervention in this issue of the European Journal of Vascular and Endovascular Surgery.1 Antiplatelet resistance comes in several forms. It can be genetic, such as a platelet surface receptor mutation, which decreases the ability of a drug to bind and subsequently inhibits platelet function.2 More commonly it can be due to changes in metabolism, such as those seen during the response to surgical trauma, as examined by Dehne et al.1 Patients will also have normal platelet function if they do not take their medication.

Prophylactic administration of HPA-1a–specific antibodies prevents fetal/neonatal alloimmune thrombocytopenia in mice

AbstractFetal/neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder caused by maternal alloantibodies directed against paternally inherited human platelet alloantigens (HPAs) present on the surface of fetal and neonatal platelets. There are currently no approved therapies for the prevention of FNAIT. We report herein the ability of 2 human HPA-1a–specific therapeutic candidates, one a polyclonal, and the other a monoclonal antibody, to prevent alloimmunization in a novel preclinical mouse model of FNAIT. Both antibody preparations effected the rapid and complete elimination of HPA-1a+ platelets from circulation and prevented the development of HPA-1a alloantibodies. HPA-1a− female mice treated prophylactically with anti–HPA-1a antibody prior to exposure to HPA-1a+ platelets gave birth to HPA-1a+/− pups with significantly improved platelet counts and no bleeding symptoms. These preclinical data establish both the potential and threshold exposure targets for prophylactic treatment with HPA-1a–specific antibodies for the prevention of FNAIT in humans.

O079 Predictive value for elevated platelet-derived SGPVI in venous in-stent stenosis in patients with post-thrombotic syndrome

Abstract Introduction In-stent stenosis following intervention for post-thrombotic syndrome (PTS) occurs in ∼30% of cases, despite therapeutic anticoagulation. Our aim was to investigate whether platelets are involved. Methods Blood was taken from case-matched patients before and after venous stenting. Flow-cytometry was used to assess platelet activation (P-selectin and/or phosphatidylserine exposure) while= functional activity was measured by plate-based aggregation and through a flow-cell model of thrombus formation. Soluble glycoprotein VI (sGPVI) was measured in plasma. Patients with in-stent stenosis requiring reintervention (&amp;gt;50% diameter reduction) were compared with those who did not. Results Forty-five patients were recruited (median age: 43yrs (33–55yrs); 65% female), and 19/45 (42%) required reintervention (median time: 3wks (1day-3mths)). There was no difference in platelet activation or reactivity after stenting, but P-selectin exposure pre-stent was significantly higher in patients who developed in-stent stenosis (2.7%±0.4 vs 1.7%±0.2; P&amp;lt;0.05). Pre-stent sGPVI levels were increased in patients who developed in-stent stenosis (18.9±3.6ng/mL vs. 7.4±0.9ng/mL; P&amp;lt;0.01). Platelet reactivity to collagen-related peptide, a GPVI-specific platelet agonist, was reduced in patients who developed in-stent stenosis (logEC50 = -6.5M±0.3 vs -7.2M±0.2; P&amp;lt;0.05; n=33). Pre-stent platelet accumulation (26.6AU±0.5 vs 19.0AU±8.1; P=0.04) was significantly increased in PTS patients compared with healthy controls. Conclusion Venous stenting does not activate platelets, but patients who developed in-stent stenosis exhibited greater levels of pre-stent platelet activation, greater loss of platelet surface GPVI in the form of sGPVI and reduction in reactivity to GPVI activation. sGPVI may have potential to risk stratify patients undergoing venous stenting and predict who requires closer surveillance. Take-home message Soluble GPVI may have potential to risk-stratify patients undergoing deep venous reconstruction and predict who requires closer surveillance.

Platelet Priming and Activation in Naturally Occurring Thermal Burn Injuries and Wildfire Smoke Exposure Is Associated With Intracardiac Thrombosis and Spontaneous Echocardiographic Contrast in Feline Survivors.

Wildfires pose a major health risk for humans, wildlife, and domestic animals. We previously discovered pathophysiologic parallels between domestic cats with naturally occurring smoke inhalation and thermal burn injuries and human beings with similar injuries; these were characterized by transient myocardial thickening, cardiac troponin I elevation and formation of intracardiac thrombosis. While the underlying mechanisms remain unclear, results from murine models suggest that platelet priming and activation may contribute to a global hypercoagulable state and thrombosis. Herein, we evaluated and compared the degree of platelet activation, platelet response to physiologic agonists and levels of platelet-derived microvesicles (PDMV) in 29 cats with naturally occurring wildfire thermal injuries (WF), 21 clinically healthy cats with subclinical hypertrophic cardiomyopathy (HCM) and 11 healthy cats without HCM (CC). We also quantified and compared circulating PDMVs in WF cats to CC cats. In addition, we examined the association between thrombotic events, severity of burn injuries, myocardial changes, and the degree of platelet activation in cats exposed to wildfires. Flow cytometric detection of platelet surface P-selectin expression showed that WF cats had increased platelet response to adenosine diphosphate (ADP) and thrombin compared to the two control groups indicating the presence of primed platelets in circulation. In addition, cats in the WF group had increased circulating levels of PDMV, characterized by increased phosphatidylserine on the external leaflet. Cats in the WF group with documented intracardiac thrombosis had elevated platelet activation and platelet priming in the presence of ADP. While high dose arachidonic acid (AA) mostly resulted in platelet inhibition, persistent response to AA was noted among cats in the WF group with intracardiac thrombosis. Univariate and multiple logistic regression analyses demonstrated that increased platelet response to AA was independently associated with thrombotic events. This is the first study reporting the significant association between platelet priming and intracardiac thrombosis in domestic cats with naturally occurring wildfire-related injuries and smoke inhalation. Further studies are required to delineate additional mechanisms between inflammation and thrombosis, especially regarding platelet primers and the cyclooxygenase pathway.One Sentence SummaryPlatelet activation and shedding of platelet-derived microvesicles due to platelet priming is present following naturally occurring wildfire smoke exposure and thermal burn injuries in a population of domestic cats.

Supramolecularly functionalized platelets for rapid control of hemorrhage

Excessive bleeding has always been of great medical challenge, particularly in trauma and surgery. Due to the fast clearance of medicine and complex hemodynamics during hemorrhage, it is often difficult to achieve rapid and effective hemostasis on irregularly shaped, noncompressible visceral bleeding wounds. Herein, we report a hemostatic derived from supramolecularly functionalized platelets (SPLTs), showing rapid hemorrhage controlling effects via efficiently targeting injured vessels and in-situ aggregation. Von Willebrand factor-binding peptide (VBP) modified hyaluronic acid (HA-VBP) decorated platelets (PLTs) were fabricated via supramolecular host-guest interactions between cucurbit[7]uril (CB[7], a host molecule) modified on HA-VBP (HA-CB[7]-VBP) and adamantane (ADA, a guest molecule) anchored on the surface of PLTs (ADA-PLTs). The SPLTs demonstrated approximately 10-fold improvements than the native PLTs in the targeting efficiency into the injured vessels in mice upon intravenous injection. More significantly, the total bleeding time and bleeding volume were dramatically reduced down to less than 1/4 and 1/10 of the control group, respectively, in both external and internal major bleeding mice models. This SPLTs provide a facile yet effective approach for rapid control of major hemorrhage and offers important new insights to the design and development PLTs-based hemostatics. Statement of significanceHemorrhage is one of the greatest threats to humans in trauma and surgery. To reduce bleeding volume and time, transfusion of hematological products such as platelets (PLTs)-rich plasma is one of the most commonly used therapeutics, but with low targeting and hemostatic efficiency. Thus, engineered PLTs with expanded structural repertoire and functionalities are in urgent clinical needs. Herein, we developed supramolecularly functionalized PLTs (SPLTs), prepared with a mild and facile approach, for rapid control of hemorrhage with significantly enhanced targeting efficiency. The SPLTs not only provide a facile approach for rapid control of major hemorrhage, but also offer important new insights into the development PLTs-based hemostatics.