GPIb Expression Research Articles

Platelet changes and bleeding symptoms in children, adolescents, and adults with 22q11.2 deletion syndrome.

The deletion region of 22q11.2 deletion syndrome (22q11.2DS) contains a gene encoding glycoprotein Ibβ (GPIbβ), which is required to express the GPIb/IX/V complex on the platelet surface. Therefore, patients with 22q11.2DS may have congenital platelet disorders. However, information is limited on platelets and bleeding symptoms. In this study, we investigated clinical information, including bleeding symptoms, platelet counts, and GPIb expression levels in children and adolescents/adults with 22q11.2DS. Thirty-two patients with 22q11.2DS were enrolled in a prospective cohort study between 2022 and 2023 at outpatient clinics within our institute. The median platelet counts in adolescents/adults with 22q11.2DS were significantly lower than those in children (p<.0001). A gradual decrease was found along with increasing age (p=.0006). Values of median GPIb expression on platelet surfaces (66% in children and 70% in adolescents/adults) were significantly lower than those in healthy controls (p<.0001 and p=.0002). Bleeding symptoms included surgery-related bleeding (52%), purpura (31%), and epistaxis (22%); most of them were minor. The median International Society on Thrombosis and Hemostasis bleeding assessment tool score was not significantly different between children and adolescents/adults (p=.2311). Although there was an age-related decrease in platelet count and a disease-related decrease in GPIb expression, no difference in bleeding symptoms was found between children and adolescents/adults. 22q11.2DS overall had minor bleeding symptoms in daily life, and the disease had little effect on spontaneous bleeding. However, some patients had major bleeding events; further accumulation of data on hemostasis during surgery and trauma is required.

Western Diet Modifies Platelet Activation Profiles in Male Mice.

The correlation between obesity and cardiovascular disease has long been understood, yet scant investigations endeavored to determine the impact of an obesogenic diet on platelet activation or function. As platelets drive clot formation, the terminus of cardiovascular events, we aimed to elucidate the longitudinal effect of an obesogenic diet on platelet phenotype by assessing markers of platelet activation using flow cytometry. Male, weanling mice were fed either a Western diet (30% kcal sucrose, 40% kcal fat, 8.0% sodium) or Control diet (7% kcal sucrose, 10% kcal fat, 0.24% sodium). At 12, 16 and 20 weeks on diets, platelets were collected and stained to visualize glycoprotein Ibα (GPIbα), P-selectin and the conformationally active state of αIIbβ3 (a platelet specific integrin) after collagen stimulation. At all time points, a Western diet reduced GPIbα and αIIbβ3 expression in platelets broadly while P-selectin levels were unaffected. However, P-selectin was diminished by a Western diet in the GPIbα- subpopulation. Thus, a Western diet persistently primed platelets towards a blunted activation response as indicated by reduced active αIIbβ3 and P-selectin surface expression. This study provides a first look at the influence of diet on platelet activation and revealed that platelet activation is susceptible to dietary intervention.

Pedigree Analysis and Molecular Mechanism Study of Hereditary Glanzmann Thrombasthenia Caused by Compound Heterozygous Mutation of the ITGA2B Gene

Objective: The phenotype and genotype of a pedigree with Glanzmann thrombasthenia caused by compound heterozygous mutation in the ITGA2B gene and its molecular pathogenesis were explored. Methods: The platelet aggregation rate of the proband and his family was detected by using a platelet aggregation test with adenosine diphosphate, collagen, epinephrine, arachidonic acid, and ristocetin. The expression levels of CD41 (αⅡb), CD61 (β3), and CD42b (GPⅠb) on the platelet surface was detected by flow cytometry. Gene sequencing technology was used for the genetic identification of the family. RT-PCR was used in the detection of mRNA splicing, and qRT-PCR was used in detecting the relative mRNA level of the ITGA2B gene. Bioinformatics analysis was used to evaluate the pathogenicity of mutation sites and their effects on protein structure and function. The expressions of total αⅡb and β3 in platelets were analyzed by Western blot. Results: Except ristocetin, the other four inducers could not induce platelet aggregation in the proband. Flow cytometry showed that the expression levels of αⅡb and β3 were only 0.25% and 9.76%, respectively, on the platelet surface of the proband, whereas GPⅠb expression was relatively normal. The expression levels of glycoproteins in the other family members were almost normal. c.480C>G and c.2929C>T mutations were detected in the proband through gene sequencing. The c.480C>G mutation was inherited from his mother, and the c.2929C>T mutation was inherited from his father. The RT-PCR and sequencing results showed that the c.480C>G mutation caused mRNA splicing in the proband and his mother, resulting in the deletion of 99 bases in c.476G-574A (p.S160-S192). qRT-PCR showed that the c.2929C>T variant reduced the mRNA level of the ITGA2B gene in the proband and his father. Bioinformatics analysis suggested that the c.480C>G mutation might form a binding sequence with hnRNP A1 protein and generate the 5'SS splice site. The three-dimensional structural model of the αⅡb subunit showed that the β-propeller domain of the p.S160-S192 deletion lost two β-strands and one α-helix in blade 2. The c.2929C>T nonsense mutation caused premature translation termination and produced a truncated protein with the deletion of p.R977-E1039, including the cytoplasmic domain, transmembrane domain, and a β chain of the extracellular Calf-2 domain. The total αⅡb expression of the proband was absent, and the relative expression of β3 was 11.36% of the normal level. Conclusion: The compound heterozygous mutation c.480C>G in exon 4 and c.2929C>T in exon 28 of the ITGA2B gene probably underlies Glanzmann thrombasthenia in this pedigree.

Modulation of Staphylococcus aureus gene expression during proliferation in platelet concentrates with focus on virulence and platelet functionality.

Staphylococcus aureus is a well-documented bacterial contaminant in platelet concentrates (PCs), a blood component used to treat patients with platelet deficiencies. This bacterium can evade routine PC culture screening and cause septic transfusion reactions. Here, we investigated the gene expression modulation within the PC niche versus trypticase soy media (TSB) of S. aureus CBS2016-05, a strain isolated from a septic reaction, in comparison to PS/BAC/317/16/W, a strain identified during PC screening. RNA-seq analysis revealed upregulation of the capsule biosynthesis operon (capA-H), surface adhesion factors (sasADF), clumping factor A (clfA), protein A (spa), and anaerobic metabolism genes (pflAB, nrdDG) in CBS2016-05 when grown in PCs versus TSB, implying its enhanced pathogenicity in this milieu, in contrast to the PS/BAC/317/16/W strain. Furthermore, we investigated the impact of S. aureus CBS2016-05 on platelet functionality in spiked PCs versus non-spiked PC units. Flow cytometry analyses revealed a significant decrease in glycoprotein (GP) IIb (CD41) and GPIbα (CD42b) expression, alongside increased P-selectin (CD62P) and phosphatidylserine (annexin V) expression in spiked PCs compared to non-spiked PCs (p = 0.01). Moreover, spiked PCs exhibited a drastic reduction in MitoTrack Red FM and Calcein AM positive platelets (87.3% vs. 29.4%, p = 0.0001 and 95.4% vs. 24.7%, p = 0.0001) in a bacterial cell density manner. These results indicated that S. aureus CBS2016-05 triggers platelet activation and apoptosis, and compromises mitochondrial functionality and platelet viability, in contaminated PCs. Furthermore, this study enhanced our understanding of the effects of platelet-bacteria interactions in the unique PC niche, highlighting S. aureus increased pathogenicity and deleterious effect on platelet functionality in a strain specific manner. Our novel insights serve as a platform to improve PC transfusion safety.

Turbulence Enhances Fine-Tuning of Mitochondria Delivery in Megakaryocyte Maturation Contributing to Biogenesis of High Quality Platelets

Platelet (PLT) products have a short shelf life of 4-5 days but blood donations are limited. As a solution, we established induced pluripotent stem cell (iPSC)-derived immortalized megakaryocyte cell lines (imMKCLs) (Nakamura, Cell Stem Cell, 2014), which generated platelet-like particles (iPSC-PLTs) under static conditions, albeit with low yield and function. Subsequently, by developing turbulence-flow bioreactors, imMKCLs generated high yield of competent iPSC-PLTs (Ito, Cell, 2018). Meanwhile, it is still unclear the reason why turbulence stimulation into imMKCLs allow higher production efficiency and improved function. Herein we found turbulence increases CD42b (GPIbα) expression in iPSC-PLTs, which was inversely correlated with phosphatidylserine (PS) expression levels (as represented by Annexin V binding). Only CD42b + population of iPSC-PLTs responded to agonist stimulation, detected by P-selectin expression or PAC1 binding. We demonstrated functional CD42b + iPSC-PLTs are determined by mitochondria activity in iPSC-PLTs, but not in imMKCLs. Regarding CD42b expression, it is known that ADAM17 causes CD42b shedding upon exposure of phosphatidylserine on the cell surface (Sommer, Nature communications, 2016). Therefore, we examined whether mitochondria activity influence CD42b shedding. To test this, we added FCCP, a mitochondrial uncoupler depleting mitochondria membrane potential, in the absence or presence of shedding inhibitors in imMKCL culture. Depleting mitochondria activity decreased CD42b expression levels on iPSC-PLTs, independently of culture conditions. Pre-incubation with ADAM17 inhibitor or different type shedding inhibitors prior to FCCP treatment allowed to retain CD42b levels, suggesting that mitochondria function might regulate downstream ADAM17 activity or shedding mechanisms. This was confirmed by GPIX deficiency in imMKCLs, as no difference in mitochondria activity was found compared to the control. Differences in CD42b levels were not seen in MKs during maturation, independent of culture conditions, therefore we suspected turbulence promote intact mitochondria delivery in imMKCLs. To examine this hypothesis, we used Mdivi-1, which inhibit Drp1 activity and thereby suppress mitochondria fission, to mimic impaired mitochondria delivery. Administration of Mdivi-1 to turbulence-exposed imMKCLs decreased CD42b expression in subsequently generated iPSC-PLTs. Using confocal microscopy, we also visualized fused mitochondria in imMKCLs upon addition of Mdivi-1, strongly suggesting that negative CD42b expression in iPSC-PLTs is due to impaired mitochondria delivery. Next, to gain mechanistic insight at molecular levels, we differentiated imMKCLs in static or turbulence conditions and swapped the culture conditions on day 3 of the total 6 days of imMKCL maturation. It revealed that turbulence is required only after day 3 onwards, but not before. scRNA analysis and pharmacological examination results strongly suggested that increased HDAC5, 6, and 9 levels by turbulence stimulation are crucial for efficient mitochondria delivery into iPSC-PLTs. We conclude that turbulence increases ‘fine-tuning’ of mitochondria distribution in imMKCLs and efficient delivery to PLTs with relatively sufficient activity via an HDAC mediated mechanism, where mitochondria activity is required for inhibition of phosphatidylserine exposure on the cell surface and of ADAM17 activity.

Non-Activating Integrin β3(R734C) Mutation Associated with Macrothrombocytopenia and Impaired Platelet Function

Introduction We and others identified several mutations in ITGA2B or ITGB3 associated with congenital macrothrombocytopenia. These mutations are located around transmembrane domains of αIIb or β3, and basically induce constitutive activation of αIIbβ3. Recently, we identified a mutation, β3(R734C), which is located in cytoplasmic tail of β3 without inducing αIIbβ3 activation in a Japanese family with macrothrombocytopenia. We analyzed the effect of the mutation on platelet production and function using knock-in (KI) mice. Cases and Transfection assay The proband was a 14-year-old Japanese girl, who showed macrothrombocytopenia with 60-90 x 10 9/L platelet counts and mild bleeding tendency. Her mother, her maternal aunt and a cousin also showed macrothrombocytopenia. The expression of αIIbβ3 on their platelets was decreased to ~50% of healthy control. GPVI expression was decreased, whereas GPIb expression was increased probably due to the increase in platelet size. Genetic analysis revealed that all affected subjects were heterozygous of β3(R734C) mutation. No PAC-1 binding was observed to the platelets of the affected subjects or αIIbβ3(R734C)-transfected 293T cells, indicating that the mutation does not cause αIIbβ3 activation. Methods and Results of mouse studies We generated β3(R734C) KI mouse (C57BL6/J background) by CRISPR/Cas9-mediated genome editing. The β3(R734C) KI mice were viable with no apparent bleeding tendency. Platelet counts of heterozygous (Hetero), and homozygous (Homo) KI mice were decreased relative to those of wild-type (WT) mice [WT: 1095 ± 52 (x10 9/L), Hetero: 834 ± 93**, Homo: 445 ± 30** (mean±SD, **P&amp;lt;0.01 compared with WT, n=6 respectively)], with an increase in platelet size, indicating that β3(R734C) leads to macrothrombocytopenia. Moreover, the expression of αIIbβ3 and GPVI in platelets was decrease in KI mice like human subjects. Platelet aggregations were impaired in KI mice. JON/A binding was not detected on non-stimulated platelets and agonist-induced P-selectin and JON/A binding were impaired in homo KI mice, particularly in PAR4-AP stimulation. Platelet spreading on fibrinogen (Fgn) was impaired in KI mice with or without ADP and thrombin stimulation. Next, we analyzed megakaryocytes (Mgk) derived from bone marrow after 5 days incubation with murine thrombopoietin. Interestingly, the expression of αIIbβ3 in Mgk of KI mice was compatible with WT and the rate of filopodia and/or lamellipodia formation of Fgn-adhered Mgk was decreased in KI mice [WT: 78(%), Homo: 32(%) (P&amp;lt;0.01)]. RhoA activation was assessed by ELISA and it was significantly decreased in Fgn-adhered Mgk of KI mice [WT: 0.33±0.039, Homo: 0.16±0.029 (mean OD 490nm Δblank±SD, P&amp;lt;0.01)]. Finally, we observed impaired proplatelet formation of fetal liver derived-Mgk in KI mice [WT: 26.2±2.7(%), Homo: 15.2±1.9(%) (P&amp;lt;0.01)] with abnormal morphology (Figure 1). These results suggest that impaired αIIbβ3 outside-in signaling and cytoskeletal remodeling in KI mice lead to macrothrombocytopenia. Conclusions We revealed that a non-activating β3(R734C) is a causal mutation of macrothrombocytopenia in human and mouse. This mutation leads to impaired inside-out and outside-in signaling of αIIbβ3, which results in abnormal platelet production with impaired cytoskeletal reorganization. Our results also suggest that abnormal signaling induced by β3(R734C) causes reduced expression of GPVI, which has not been reported in other αIIbβ3-related macrothrombocytopenia.

Shear Forces Induced Platelet Clearance Is a New Mechanism of Thrombocytopenia.

Thrombocytopenia has been consistently described in patients with extracorporeal membrane oxygenation (ECMO) and associated with poor outcome. However, the prevalence and underlying mechanisms remain largely unknown, and a device-related role of ECMO in thrombocytopenia has been hypothesized. This study aims to investigate the mechanisms underlying thrombocytopenia in ECMO patients. In a prospective cohort of 107 ECMO patients, we investigated platelet count, functions, and glycoprotein shedding. In an ex vivo mock circulatory ECMO loop, we assessed platelet responses and VWF (von Willebrand factor)-GP Ibα (glycoprotein Ibα) interactions at low- and high-flow rates, in the presence or absence of red blood cells. The clearance of human platelets subjected or not to ex vivo perfusion was studied using an in vivo transfusion model in NOD/SCID (nonobese diabetic/severe combined Immunodeficient) mice. In ECMO patients, we observed a time-dependent decrease in platelet count starting 1 hour after device onset, with a mean drop of 7%, 35%, and 41% at 1, 24, and 48 hours post-ECMO initiation (P=0.00013, P<0.0001, and P<0.0001, respectively), regardless of the type of ECMO. This drop in platelet count was associated with a decrease in platelet GP Ibα expression (before: 47.8±9.1 versus 24 hours post-ECMO: 42.3±8.9 mean fluorescence intensity; P=0.002) and an increase in soluble GP Ibα plasma levels (before: 5.6±3.3 versus 24 hours post-ECMO: 10.8±4.1 µg/mL; P<0.0001). GP Ibα shedding was also observed ex vivo and was unaffected by (1) red blood cells, (2) the coagulation potential, (3) an antibody blocking VWF-GP Ibα interaction, (4) an antibody limiting VWF degradation, and (5) supraphysiological VWF plasma concentrations. In contrast, GP Ibα shedding was dependent on rheological conditions, with a 2.8-fold increase at high- versus low-flow rates. Platelets perfused at high-flow rates before being transfused to immunodeficient mice were eliminated faster in vivo with an accelerated clearance of GP Ibα-negative versus GP Ibα-positive platelets. ECMO-associated shear forces induce GP Ibα shedding and thrombocytopenia due to faster clearance of GP Ibα-negative platelets. Inhibiting GP Ibα shedding could represent an approach to reduce thrombocytopenia during ECMO.

A Western Diet Modifies the Activation Profile of Circulating Platelets

Background: While it is known that obesity increases the risk for cardiovascular disease (CVD), there has been no clear investigation into the effect an obesogenic diet exerts onto circulating platelets. As platelets are the critical actors in the formation of occlusive clots that manifest as a myocardial infarction or stroke, it is pertinent to establish any potential link between obesity and platelet activity. Methods: To examine the effect of a Western diet on baseline platelet activity, male C57BL6/J mice were fed either a control (10% fat, 7% sucrose, 0.3% salt Research Diets Inc. D12450J) or Western (40% fat, 30% sucrose, 8% salt Research Diets Inc. D06111701) diet ad libitum for 20 weeks beginning at the time of weaning. At 12, 16 and 20 weeks after beginning diet administration whole blood was collected via the retro-orbital sinus. Blood was used to generate platelet rich plasma (PRP) which was stimulated with collagen (20mg/ml) prior to staining for flow cytometry analysis. Flow cytometry staining utilized antibodies to recognize the platelet receptors GPIbα, P-Selectin and the activated form of integrin α IIb β 3 (JON/A). Additionally, body composition was likewise determined at these time points using Echo-MRI while bodyweight was measured weekly. Results: Western diet fed mice gained slightly more weight than control mice and had a higher percentage of body fat (measured by EchoMRI). Surprisingly, the two groups differed in GPIbα and activated α IIb β 3 expression on circulating platelets at all three observed time points. Specifically, platelets from control-fed mice displayed significantly higher GPIbα expression throughout the study while Western diet fed animals had higher levels of activated α IIb β 3 . There was no difference in platelet expression of P-Selectin between the groups at any time point. Conclusions: Platelet activation is not a binary process where platelets display a definitive “active” or “inactive” phenotype. Though terminal platelet activation elicits de-granulation and an increase in P-Selectin expression, the spectrum of platelet stimulation preceding this is marked by variable incidences of integrin activation and GPIbα shedding. Here we demonstrate proof of principle that dietary factors, such as the consumption of a Western diet, can facilitate early changes to the stimulation pattern of circulating platelets which is then maintained long-term. While the consequence of this altered platelet stimulation profile is unknown with regards to the pathways of hemostasis and thrombosis, further investigations of downstream signaling and amplification pathways are needed. Funding: Arkansas Children's Research Institute/Arkansas Bioscience Institute Postgraduate Fellowship: AWD-037176 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sepsis-induced coagulopathy in preterm neonates with Gram-positive sepsis presents with hypercoagulation and reduced platelet activation compared with healthy preterm neonates

BackgroundNeonatal sepsis is frequently accompanied by coagulopathy and thrombocytopenia attributed to the cross-link between inflammation and coagulation. However, sepsis-induced coagulopathy and platelet function in septic preterm neonates remain to be elucidated. In addition, there is no robust evidence for a causal relationship between thrombocytopenia and bleeding in preterm neonates with sepsis. ObjectiveThis single-center prospective cohort study aimed to assess sepsis-induced coagulopathy and platelet function in preterm neonates during sepsis. MethodsWe included 25 preterm neonates with Gram-positive sepsis born at gestational age 24 + 1 to 34 + 3 and studied in comparison to 30 healthy counterparts. Coagulation was assessed using conventional coagulation tests (CCTs) and rotational thromboelastometry (ROTEM). Platelet function was evaluated by flow cytometry. The study was conducted at 3-time points, at 1st, at 2nd to 3rd, and at 5th to 7th day of sepsis, respectively. ResultsCompared with healthy controls, neonates with Gram-positive sepsis present in ROTEM a hypercoagulable state; a higher maximum clot firmness (MCF) and higher amplitudes of intrinsic rotational thromboelastometry (INTEM) (INTEM MCF: median, 71; P .004 and INTEM A10: median, 67; P .005, respectively), extrinsic rotational thromboelastometry (EXTEM) (EXTEM MCF: median, 70; P .02 and EXTEM A10: median, 67; P .02, respectively), and rotational thromboelastometry assay for fibrin formation (FIBTEM) (FIBTEM MCF: median, 25; P < .001 and FIBTEM A10: median, 23; P .002, respectively). Conversely, CCTs exhibited hypocoagulation. Thrombocytopenia in preterm neonates with Gram-positive sepsis is not associated with an increased bleeding risk. In Gram-positive sepsis, platelets display increased glycoprotein (GP) surface receptors' expression (GPIb: median, 2.8; P .03, GPIIb: median, 3.1; P .004, and GPIIIa: median, 3.9; P .008, respectively) and reduced activation (P-selectin: median, 1; P < .001). A higher expression of platelets GP and improved degranulation capacity were recorded in patients in higher gestational age groups of >32 weeks of gestation. Platelet GPIb expression is age-dependent in healthy neonates. ConclusionNeonatal Gram-positive sepsis is characterized by a progressive hypercoagulation along with increased GP expression, reduced platelet activation, and thrombocytopenia without bleeding. Platelet GP expression and degranulation capacity are age-dependent among neonates with sepsis. Platelet GP expression is age-dependent among healthy counterparts.

Platelet dysfunction and thrombus instability in flow conditions in patients with severe COVID-19

Severe COVID-19 has been associated with a high rate of thrombotic events but also of bleeding events, particularly when the level of prophylactic anticoagulation was increased. Data on the contribution of platelets to these thrombotic events are discordant between reports, while the involvement of platelets in bleeding events has never been investigated. The objective of the present study was to assess platelet function during the first week of ICU hospitalization in patients with severe COVID-19 pneumonia. A total of 35 patients were prospectively included and blood samples were drawn on day (D) 0, D2 and D7. COVID-19 pneumonia was severe with a median PaO2/FiO2 ratio of 91 [68-119] on D0. Platelets from these patients showed evidence of pre-activation and exhaustion with a significant reduction in the surface expression of GPVI, GPIb and GPIIbIIIa, together with a decrease in serotonin content. Platelets from patients with severe COVID-19 were hyporesponsive with a reduced maximal aggregation response to several platelet agonists and decreased adhesion to immobilized fibrinogen. Aggregation of washed platelets and plasma substitution experiments indicated that a plasma factor was at least partially responsible for this hyporeactivity of platelets. Blood flow experiments showed that severe COVID-19 platelets formed smaller, less stable aggregates on a collagen-coated surface, which could explain why some patients develop bleeding events. These findings should prompt us to carefully evaluate the risks and benefits of high-dose prophylactic anticoagulation, and to decrease the level of anticoagulation once the initial phase of the disease has resolved. Trial registrationClinicalTrials.gov identifier: NCT04359992.

Platelet findings in 22q11.2 deletion syndrome correlate with disease manifestations but do not correlate with GPIb surface expression.

Prior studies have demonstrated that patients with chromosome 22q11.2 deletion syndrome (22q11.2DS) have lower platelet counts (PC) compared to non-deleted populations. They also have an increased mean platelet volume. The mechanism for this has been postulated to be haploinsufficiency of the GPIBB gene. We examined platelet parameters, deletion size and factors known to influence counts, including status of thyroid hormone and congenital heart disease (CHD), in a population of 825 patients with 22q11.2DS. We also measured surface expression of GPIB-IX complex by flow cytometry. The major determinant of PC was deletion status of GP1BB, regardless of surface expression or other factors. Patients with nested distal chromosome 22q11.2 deletions (those with GP1BB present) had higher PCs than those with proximal deletions where GP1BB is deleted. Patients with 22q11.2DS also demonstrated an accelerated PC decrease with age, occurring in childhood. These data demonstrate that genes within the proximal deletion segment drive PC differences in 22q11.2DS and suggest that PC reference ranges may need to be adjusted for age and deletion size in 22q11.2DS populations. Bleeding did not correlate with either platelet count or GPIb expression. Further studies into drivers of expression of GPIb and associations with severe thrombocytopenia and immune thrombocytopenia are needed to inform clinical care.

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.

Ibrutinib Inhibits BMX-Dependent Endothelial VCAM-1 Expression In Vitro and Pro-Atherosclerotic Endothelial Activation and Platelet Adhesion In Vivo.

Inflammatoryactivation of the vascular endothelium leads to overexpression of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), contributing to the pro-thromboticstate underpinning atherogenesis. While the role of TEC family kinases (TFKs) in mediating inflammatory cell and platelet activation is well defined, the role of TFKs in vascular endothelial activation remains unclear. We investigated the role of TFKs in endothelial cell activation in vitro and in a nonhuman primate model of diet-induced atherosclerosis in vivo. In vitro, we found that ibrutinib blocked activation of the TFK member, BMX, by vascular endothelial growth factors (VEGF)-A in human aortic endothelial cells (HAECs). Blockade of BMX activation with ibrutinib or pharmacologically distinct BMX inhibitors eliminated the ability of VEGF-A to stimulate VCAM-1 expression in HAECs. We validated that treatment with ibrutinib inhibited TFK-mediated platelet activation and aggregation in both human and primate samples as measured using flow cytometry and light transmission aggregometry. We utilized contrast-enhancedultrasound molecular imaging to measure platelet GPIbα and endothelialVCAM-1 expression in atherosclerosis-prone carotid arteries of obese nonhuman primates. We observed that the TFK inhibitor, ibrutinib, inhibited platelet deposition and endothelial cell activation in vivo. Herein we found that VEGF-A signals through BMX to induce VCAM-1 expression in endothelial cells, and that VCAM-1 expression is sensitive to ibrutinib in vitro and in atherosclerosis-prone carotid arteries in vivo. These findings suggest that TFKs may contribute to the pathogenesis of atherosclerosis and could represent a novel therapeutic target.

Specialized Proresolving Mediators (SPMs) Help to Alleviate the Cold Platelet Storage Lesion

Introduction: Refrigeration of platelets in plasma is a promising alternative to conventional room temperature storage, but the cold temperature leads to increased levels of activation (i.e. P-Selectin and phosphatidylserine [PS]). Resolvins (Rv) are a class of specialized proresolving mediators (SPMs) that promote the resolution of inflammation and, more specifically, have been shown to regulate ADP activation of human platelets. In this study, we tested the hypothesis that the addition of Rv would help to reduce platelet activation during cold (4°C) storage and better preserve platelet function.Methods: Apheresis platelets were collected from healthy donors (n=3-4) using a Trima Accel collection system. On the day of collection (Day 0) platelets were aliquotted into 4 mini-bags (10 mL). Three of the bags received either RvE1 (100μM), RvD2 (100 μM), or Maresin 1 (MaR1; 100 μM), while the final bag received 0.1% EtOH (Vehicle control). Treated mini-bags were incubated for 15 minutes with compound or vehicle prior to long-term storage at 4°C. The following assays were performed on Day 0 and Day 7 of storage: platelet count, Multiplate aggregation (ADP, Collagen, TRAP), and flow cytometry (GPIb expression, P-Selectin, lactadherin [PS], and PAC-1 [activated GPIIb/IIIa]). Data were analyzed using paired t-tests. Significance was determined as P <0.05.Results: P-Selectin and PAC-1 expression in Day 7 Vehicle and compound-treated samples were significantly higher than Day 0 Vehicle levels (Fig. 1A, C). Compared to Vehicle at Day 7, RvD2- and MaR1-treated samples showed a significant reduction in PS expression (RvD2 vs Vehicle, P=0.031; MaR1 vs Vehicle, P=0.018) and PAC-I binding (RvD2 vs Vehicle, P=0.012; MaR1 vs Vehicle, P=0.036) (Fig. 1B, C). No differences in P-Selectin expression were observed for any of the compounds tested (Fig. 1A). TRAP response was higher in RvD2-treated samples compared to Day 7 Vehicle (P=0.047). ADP response was higher in MaR1-treated samples compared to Day 7 Vehicle (P=0.03). No differences in collagen aggregation were observed between samples. All SPM-treated bags resulted in higher platelet counts than Day 7 Vehicle.Conclusions: Cold storage of platelets at 4°C has gained increased attention as an alternative to room temperature storage due to the lower risk of bacterial contamination, better preservation of platelet function, and the potential for a prolonged shelf life; however, increased levels of platelet activation and platelet clumping in cold-stored platelets limit shelf life. Results from our study suggest that the addition of SPMs (RvE1, RvD2, and MaR1) to cold-stored platelets helps minimize platelet activation and even preserve platelet function (RvD2, MarR1) during long-term storage. A platelet product with an extended shelf life would improve platelet inventories and expand access to under-served patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

β1,4-Galactosyltransferase 1 Is a Key Regulator of Thrombopoiesis

Sialylated derivatives of N-Acetyl Lactosamine (LacNAc) regulate platelet lifespan, hepatic thrombopoietin (TPO) production, and thus thrombopoiesis. A major enzyme that synthesizes LacNAc is β1,4-galactosyltransferase 1 (β4GalT1), the gene of which is highly conserved through evolution. Long considered a housekeeping gene, the transcriptional regulation of B 4GALT1 remained widely understudied, and recent observations have shown that itspromoter region is rich in enhancer sequences.Both TPO and the chemokine CXCL12 regulate thrombopoiesis. Here we report that TPO and CXCL12 regulate megakaryocyte (MK) function by increasing β 4galt1 transcription . TPO and CXCL12 induced a two-wave expression of LacNAc in MKs. TPO was required for LacNAc production in wild type (WT) MKs, and β4GalT1 deletion in mice (β4GalT1-/-) resulted in severe thrombocytopenia due to increased integrin activity in MKs, and defective MK localization within the bone marrow (BM).Detailed analysis of β4GalT1-/- MKs and platelets showed that β4GalT1-/- MKs are almost completely devoid of LacNAc expression. Complete lack of galactosyltransferase activity in β4GalT1-/- MKs and platelets was demonstrated using in vitro enzymatic assays, showing that LacNAc synthesis in MKs and platelets depends primarily on β4GalT1. In WT MKs cultured in the presence of TPO alone, and in isolated circulating platelets, LacNAc expression was particularly evident in polypeptides with molecular weight ranging between 100-150 kDa, which were identified as αIIbβ3, GPIbα and β1 integrin. Addition of CXCL12 in vitro increased LacNAc expression specifically on a 130-kDa polypeptide, identified as the β1 integrin subunit. Further detailed investigation of receptor expression and function showed that WT MKs expressed two β1 integrin glycoforms of 130 and 100 kDa, and WT platelets expressed only the 130-kDa glycoform. β4GalT1-/- MKs and platelets expressed only the immature 100-kDa β1 integrin glycoform. While the subcellular distribution β1 integrin was indistinguishable between WT and β4GalT1-/- MKs and platelets, lack of LacNAc synthesis resulted in increased of β1 integrin surface expression, as determined by flow cytometry. β4GalT1-/- platelet adhesion to collagen and laminin was significantly increased and was unaffected by addition of RGDS. By contrast, GPIbα and αIIbβ3 expression and function were not affected by β4GalT1 loss.Together, the results suggest that TPO and CXCL12 upregulate β4GalT1-dependent LacNAc synthesis to promote thrombopoiesis, likely regulating MK β1 integrin expression, glycosylation and subsequent interaction with components of the extracellular matrix. Investigating the mechanisms regulating posttranslational modifications induced by various hematopoietic stimulating chemokines and cytokines will contribute to better understand thrombopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Platelet Function in Thrombocytopenic Patients with Chronic Liver Disease

Background: Thrombocytopenia, the most common hematologic abnormality encountered in patients with chronic liver disease (CLD), results from multiple factors including splenic sequestration, relatively reduced production of thrombopoietin for the degree of thrombocytopenia, consumption of platelets secondary to autoantibodies directed against platelets, and bone marrow suppression. Elevated plasma levels of von Willebrand factor (VWF) in CLD patients may partly compensate for the reduced platelet count via interaction of VWF with the platelet receptors glycoprotein (GP) Ib-IX-V and GPIIb-IIIa (integrin αIIβ3), to promote platelet adhesion and aggregation following vessel injury. Platelets in patients with CLD are reported to have reduced function as measured by light transmission platelet aggregation (LTA) and other assays. However, LTA and most other platelet function tests are unreliable in thrombocytopenic samples. In contrast, platelet function evaluated by whole blood flow cytometry is accurate in samples with low platelet counts. Therefore, in the present study we used whole blood flow cytometry to determine whether platelet function as measured by platelet surface P-selectin, activated GPIIb-IIIa and GPIb on in vivo -circulating and in vitro -stimulated platelets is altered in thrombocytopenic CLD patients compared to healthy controls.Methods: Platelet function end points were evaluated in untreated CLD patients with thrombocytopenia (n = 21) participating at U.S. sites in the Phase 3 E5501-G000-310 or E5501-G000-311 studies of a novel thrombopoietin receptor agonist, avatrombopag, compared to healthy controls (n=10). IRB approval was obtained at each study site and written informed consent was obtained from each subject. CLD patient samples analyzed in the present study were collected prior to the administration of any study drug. No patient received a platelet transfusion within 10 days of the blood draw for this study. Platelet surface P-selectin, activated GPIIb-IIIa and GPIb, without or with in vitro stimulation (0.5 μM adenosine diphosphate [ADP], 20 μM ADP, 1.5 μM thrombin receptor activating peptide [TRAP] or 20 μM TRAP) were measured by whole blood flow cytometry. To explore whether the differences in these markers between CLD patients and healthy subjects were related to the difference in platelet count rather than to differences in platelet activation or reactivity, whole blood from healthy controls was diluted with platelet-poor plasma to adjust the platelet counts to those of the thrombocytopenic CLD patients.Results: Circulating platelets (i.e., no in vitro stimulation) from thrombocytopenic CLD patients showed low levels of platelet surface P-selectin and activated GPIIb-IIIa, similar to the healthy subject controls, demonstrating minimal in vivo platelet activation in thrombocytopenic CLD patients. However, the platelets of thrombocytopenic CLD patients had higher platelet surface GPIb mean fluorescence intensity (MFI) than either diluted or undiluted circulating platelets from healthy controls (Figure). In vitro stimulation of CLD patient platelets by ADP or TRAP resulted in lower levels of platelet surface P-selectin and activated GPIIb-IIIa compared to healthy controls, except for the levels of surface P-selectin after stimulation with 20 μM TRAP. The results for the diluted healthy subject controls were unaffected by the platelet count, except for the samples activated with 1.5 μM TRAP in which dilution resulted in reduced platelet surface P-selectin and activated GPIIb-IIIa.Conclusions: 1) Circulating platelets in thrombocytopenic CLD patients are not activated. 2) Agonist-stimulated platelet surface expression of P-selectin and activated GPIIb-IIIa is reduced in thrombocytopenic CLD patients compared to healthy controls with the same degree of thrombocytopenia, which likely contributes to the excessive bleeding in thrombocytopenic CLD patients. 3) Thrombocytopenic CLD patients have a higher expression of GPIb on circulating platelets compared with healthy controls with the same degree of thrombocytopenia. This finding is consistent with the presence of an increased proportion of large immature platelets. The high levels of platelet surface GPIb, together with the previously reported high levels of circulating VWF in these patients, may contribute to vascular integrity at lower platelet counts. [Display omitted] DisclosuresFrelinger:Sysmex: Research Funding; Pfizer: Research Funding; Ironwood: Research Funding; Baxalta: Research Funding; GLSynthesis: Research Funding; Ionis: Research Funding. Michelson:GLSynthesis: Research Funding; Eisai: Research Funding; Ironwood: Research Funding; Sysmex: Research Funding; Ionis: Research Funding; Pfizer: Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Elsevier: Patents & Royalties; Baxalta: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Instrumentation Laboratory: Consultancy.

To Bleed or Not to Bleed: MCS-Related Shear Stress Promotes Redistribution of GPIb and αIIbβ3 from Platelets to Microparticles Thus Impairing Platelet Hemostatic Function

Purpose Bleeding is a leading cause of readmission and 1-year mortality for mechanical circulatory support (MCS) patients. Mechanical damage of platelets and coagulation proteins induced by MCS-generated high shear stress is the main driver of bleeding. We previously demonstrated that hypershear promotes platelet pro-apoptotic behavior resulting in membrane reorganization and generation of platelet-derived microparticles (PDMP). Here, we test the hypothesis that hypershear alters surface expression and shedding of adhesion receptors GPIb and αIIbβ3 from platelets and PDMPs thus affecting platelet aggregability - all potential bleeding contributors. Methods Human platelets were exposed to shear stress in a hemodynamic shearing device (30-70 dyn/cm2, 10 min). Integrin GPIb and αIIbβ3 surface expression and PDMP generation were quantified by flow cytometry. Single platelets and PDMPs were distinguished by FS/SS characteristics. Shedding of soluble GPIb and αIIbβ3 fragments was quantified by ELISA. Platelet aggregation was measured by optical aggregometry. Results Platelet exposure to hypershear led to a reduction of platelet GPIb surface expression, with a significant drop of GPIb+ platelet number and fluorescence (73.5 ± 2.9% and 953.1 ± 101.0 AU vs. 94.3 ± 0.7% and 1620 ± 72.4 AU for intact platelets; M ± SEM, ANOVA, p ≤ 0.05). Yet, no shear-mediated shedding of soluble GPIb fragment or β3 subunit was found. Downregulation of platelet GPIb expression coincided with a progressive increase of GPIb- & αIIbβ3-positive PDMPs after shear (up to 3.3 ± 0.6% & 12.5 ± 2.8%, correspondingly). Surface density of GPIb and αIIbβ3 on PDMPs was 2-4x higher than on platelets, post-shear. Platelet aggregation to increasing concentrations of ADP and TRAP-6 was significantly impaired by 70 dyn/cm2 shear stress. Conclusion MCS-mediated hypershear and platelet stress accumulation 1) induces downregulation of platelet adhesion receptors via ejection of receptor-enriched PDMPs; 2) does not induce shedding of soluble fragments of GPIb and β3, and; 3) mechanistically limits platelet aggregatory and hemostatic responses. Shear-mediated redistribution of primary adhesion receptors from platelets to PDMPs coupled with impaired aggregability may mechanistically contribute to MCS-related bleeding.

Blood cell adhesion to arterial filters analysis by scanning electron microscopy and real-time PCR assay: observational clinical study in cardiac surgery patients.

Arterial filter is the part of the cardiopulmonary bypass circuit where blood cells are exposed to high mechanical stress and where cellular aggregates may fasten in large quantities. The aim of this study was to analyse blood cell adhesiveness in the arterial filter through scanning electron microscopy and real-time PCR assay. Prospective, clinical and observational study performed on 28 patients undergoing cardiac surgery with cardiopulmonary bypass. Arterial filters were analysed by scanning electron microscopy. Real-time PCR assay was performed in extracted material from the arterial filters for analysis of platelet GPIb and CD45 leucocyte gene expression. Blood coagulation was analysed during cardiopulmonary bypass. Patients were followed until hospital discharge or 28 days after surgery. All studied arterial filters used in the subject patients showed a degree of adhesion from blood elements at scanning electron microscopy. All studied filters were positive for platelets GPIb gene expression and 15% had CD45 leucocyte gene expression. The GPIb platelet gene expression in blood lowered at the end of cardiopulmonary bypass (p = 0.019). There was negative correlation between blood GPIb platelet gene expression and Clot SR (HEPSCREEN2 ReoRox®) (rho = 0.635; p = 0.027). The filter fields count was correlated to the D-dimer dosage (rho = 0.828; p < 0.001). There was adhesion of blood elements, especially nucleated platelets, on all arterial filters studied. Although the arterial filter worked as a safety device, that possibly prevented arterial embolisation, it may also have caused greater hyperfibrinolysis during cardiopulmonary bypass.

Assessing bleeding risk in 18 children with Osteogenesis imperfecta

Assessing bleeding risk in 18 children with <i>Osteogenesis imperfecta</i>

Influence of long-term proteasome inhibition on platelet responsiveness mediated by bortezomib

Introduction: Although platelets contain a full proteasome system, its role in platelet function is not completely understood yet. Since the proteasome system may be involved in time-delayed processes, platelet responsiveness was investigated after long-term, bortezomib-mediated proteasome inhibition.Materials and Methods: Citrate-anticoagulated whole blood was stored with 5 nM and 1 μM bortezomib for 24 h. Consecutively, aggregation was measured by light transmission in platelet-rich-plasma (PRP). Flow cytometry was performed to determine phosphorylation levels of the vasodilator-stimulated phosphoprotein (VASP), fibrinogen binding, PAC1-antibody binding and purinergic receptor expression in PRP, P2Y12 activity or glycoprotein (GP) Ib and IIb expression in whole blood. P2Y1 and P2X1 activities were assessed by calcium flux-induced fluorescence in washed platelets. Using PRP, adherent platelets on fibrinogen-, collagen- and ristocetin-coated surfaces were visualized and quantified by immunostaining.Results: Under bortezomib, VASP phosphorylation was less inducible and nitric oxide-induced inhibition of fibrinogen binding was slightly reduced. Proteasome inhibition did not tamper adenosine diphosphate-mediated aggregation or purinergic receptor expression and activity. Induced expression of activated fibrinogen receptors and fibrinogen binding were not significantly influenced by incubation with bortezomib for 24 h. Aggregation values with threshold agonist concentrations were increased under bortezomib. Despite unchanged GPIb expression, bortezomib-treated platelets showed enhanced adhesion on coated surfaces.Conclusions: In platelets incubated for 24 h, bortezomib mediates a slight attenuation of inhibitory signaling, associated with facilitated platelet aggregation using threshold agonist concentrations and enhanced adhesion on agonist-coated surfaces.