Influence of 4-pyridone-3-carboxamide-1Β-D-ribonucleoside (4PYR) on activities of extracellular enzymes in endothelial human cells

ABSTRACT

Previous studies demonstrated that human endothelial cells were capable to phosphorylate 4-pyridone-3-carboxamide-1β-D-ribonucleoside (4PYR) to monophosphate (4PYMP) and formed another metabolite—an analog of NAD (4PYRAD). Elevated levels of 4PYMP and 4PYRAD had an adverse effect on energy balance—depressed adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD) concentration in human endothelial cells. Ecto-enzymes such as ecto-nucleoside triphosphate diphosphohydrolase (eNTPD); ecto-5′-nucleotidase (e5’NT); and ecto-adenosine deaminase (eADA) are involved in controlling of inflammation and platelet aggregation. This study aimed to evaluate influence of 4PYR and its metabolites on activities of extracellular enzymes in human endothelial cells. Endothelial cells (endothelial cell line HMEC-1) were treated with 100 uM 4PYR for 0, 24, 48, or 72 hours. After incubation, intact HMEC-1 cells were incubated with suitable substrate. Simultaneously, in another path of experiments intracellular concentration of 4PYMP and 4PYRAD had been analyzed. Conversion of extracellular nucleotides into their products and intracellular concentration of 4PYMP and 4PYRAD were measured by high performance liquid chromatography (HPLC). We demonstrated that eNTPD and e5’NT activities increase after 72 hours of cell treatment with 4PYR as compared to control (0.40 ± 0.02 versus 0.29 ± 0.02 nmol/min/mg protein; 13.3 ± 0.6 versus 8.30 ± 0.34 nmol/min/mg protein, respectively, mean ± SEM). eADA activity decreases after 24 hours of cells treatment with 4PYR as compared to control (1.55 ± 0.06 versus 1.92 ± 0.13 nmol/min/mg protein, respectively, mean ± SEM). 4PYR and its derivatives have positive effect on ecto-enzymes related with ATP degradation pathway. We conclude that these increases in extracellular enzyme activities are an adaptive response to decreased intracellular ATP and NAD arising from 4PYR uptake. These changes may protect the cells from the inflammatory result of external ATP degradation.     

Increased plasma concentration of 4-pyridone-3-carboxamide-1-ß-D-ribonucleoside (4PYR) in lung cancer. Preliminary studies

Abstract

4-pyridone-3-carboxamide-1-β-D-ribonucleoside (4PYR) is a new nicotinamide derivative, which is potentially toxic to the endothelium. Dysfunction of the endothelium promotes cancer cell proliferation, invasiveness, and inflammatory signaling. The aim of this study was to analyze 4PYR concentration in the plasma of lung cancer patients and its relationship to other known biochemical parameters associated with the endothelium function.

The concentration of 4PYR, nicotinamide, 1-methylnicotinamide (MNA), amino acids, and their derivatives were measured in samples obtained from patients with primary squamous cell carcinoma (n?=?48) and control group (n?=?100).

The concentration of 4PYR and 4PYR/MNA ratio were significantly higher in lung cancer patients as compared to controls (0.099?±?0.009 vs. 0.066?±?0.006?µmol/L and 1.10?±?0.08 vs. 1.97?±?0.15, respectively). The plasma arginine/asymmetric dimethylarginine (Arg/ADMA) ratio was considerably lower in lung cancer patients (253?±?17 vs. 369?±?19) as well as plasma MNA (0.057?±?0.004 vs. 0.069?±?0.003?µmol/L). There was no difference in the plasma concentrations of nicotinamide and nicotinamide riboside in both groups (0.116?±?0.019 vs. 0.131?±?0.014 and 0.102?±?0.006 vs. 0.113?±?0.011, respectively).

In this study, a higher 4PYR concentration was observed for the first time in patients with squamous cell carcinoma. This change may be related to the endothelial dysfunction that promote cancer progression since 4PYR and its derivatives are known to disrupt glycolytic pathway.     

Metabolic pathway of 4-pyridone-3-carboxamide-1β-d-ribonucleoside and its effects on cellular energetics

4-pirydone-3-carboxamide-1β-d-ribonucleoside (4PYR) is an endogenous nucleoside that could be converted to triphosphates, diphosphates, monophosphates and an analogue of NAD − 4PYRAD. Elevated level of these compounds have been reported in chronic renal failure, cancer and active HIV infection. However, little is known about the effect on cell functionality and the metabolic pathways. This study tested effects of 4PYR in different cell types on nucleotide, energy metabolism and clarified enzymes that are involved in conversions of 4PYR.We have found that human neuroblastoma cells, human malignant melanoma cells, human adipose-derived stem cells, human bone marrow-derived stem cells, human dermal microvascular endothelial cells and human embryonic kidney cells, were capable to convert 4PYR into its derivatives. This was associated with deterioration of cellular energetics. Incubation with 4PYR did not affect mitochondrial function, but decreased glycolytic rate (as measured by extracellular acidification) in endothelial cells. Silencing of adenosine kinase, cytosolic 5′-nucleotidase II and nicotinamide nucleotide adenylyltransferase 3, blocked metabolism of 4PYR. Incubation of endothelial cells with 4PYR decreased AMP deaminase activity by 40%.The main finding of this paper is that human cells (including cancer type) are capable of metabolizing 4PYR that lead to deterioration of energy metabolism, possibly as the consequence of inhibition of glycolysis. This study, it was also found that several enzymes of nucleotide metabolism could also contribute to the 4PYRconversions     

Effects of oxysterols on the blood–brain barrier: Implications for Alzheimer’s disease

Altered brain cholesterol homeostasis plays a key role in neurodegenerative diseases such as Alzheimer’s disease (AD). For a long time, the blood–brain barrier (BBB) was basically considered as a barrier isolating the brain from circulating cholesterol, however, several lines of evidence now suggest that the BBB strictly regulates the exchanges of sterol between the brain and the peripheral circulation. Oxysterols, synthesized by neurons or by peripheral cells, cross the BBB easily and modulate the expression of several enzymes, receptors and transporters which are involved not only in cholesterol metabolism but also in other brain functions. This review article deals with the way oxysterols impact BBB cells. These perspectives open new routes for designing certain therapeutical approaches that target the BBB so that the onset and/or progression of brain diseases such as AD may be modulated.     

4-Pyridone-3-Carboxamide-1β-D-Ribonucleoside Metabolism in Endothelial Cells and Its Impact on Cellular Energetic Balance

4-Pyridone-3-carboxamide-1β-D-ribonucleoside (4PYR) is a naturally occurring compound related to nicotinamide that could be metabolized to mono-, di-, and triphosphates of 4PYR (4PYMP, 4PYDP, 4PYTP) and nicotinamide adenine dinucleotide (NAD) analogue (4PYRAD) in all types of cells. Previous studies demonstrated that formation of 4PYMP and 4PYTP was dependent on adenosine kinase activity. Pathway of 4PYRAD production is not yet identified, but most likely this process involves production of 4PYMP. This study aimed to evaluate influence of 4PYR on metabolism of endothelial cells and to test effect of nucleoside transport inhibitors. 4PYR was obtained by chemical synthesis. Endothelial cell line (HMEC-1) was incubated for 24 or 48 hours with 100 μM 4PYR. After incubation, cells were separated from medium and analyzed for concentrations of ATP, NAD, and 4PYR metabolites by using reversed-phase high performance liquid chromatography. We demonstrated progressive accumulation of 4PYR metabolites in endothelium that reached 33.2 ± 0.8 nmol/mg protein for 4PYMP and 5.25 ± 0.17 nmol/mg protein for 4PYRAD after 48-hour incubation with 4PYR. Dipyridamole protected from accumulation of 4PYR metabolites in endothelial cells. We conclude that endothelium is capable to convert 4PYR into intracellular metabolites and this causes disruption of cell energy balance. Nucleoside transport inhibition with dipyridamole could protect endothelium from this effect. This finding could be of clinical relevance in conditions associated with accumulation of 4PYR such as chronic renal disease.     

Metabolism of 4-Pyridone-3-Carboxamide-1-ß-D- Ribonucleoside Triphosphate and Its Nucleoside Precursor in the Erythrocytes

We recently discovered new nucleotides (4-pyridone-3-carboxamide-1-β -D-ribonucleoside phosphates) in human erythrocytes. To establish the precursor compound and pathways of nucleotide derivative formation and breakdown, human erythrocytes were incubated for 3 hours with 0.3 mM 4-pyridone-3-carboxamide-1-β -D-ribonucleoside (4PYR) and erythrocyte concentrations of 4PYR and adenine nucleotides were followed. 4PYR triphosphate increased from 16.1 ± 0.6 μ M to 74.9 ± 9.17 and 4PYR monophosphate increased from 5 μ M to 254.7 ± 13.9 μ M. Conversely, incubation with 0.3 mM 4-pyridone-3-carboxamide (4PY) did not lead to additional 4PYR nucleotide formation. 4PYR nucleotides were catabolized to 4PYR. We conclude that 4PYR nucleotides are formed in erythrocytes by nucleoside kinase-mediated 4PYR phosphorylation and catabolized by 5′nucleotidase-mediated dephosphorylation.     

Influence of thrombophlebitis on TGF-β1 and its signaling pathway in the vein wall

Extensive extracellular matrix remodeling of the vein wall is involved in varicose veins pathogenesis. This process is controlled by numerous factors, including peptide growth factors. The aim of the study was to evaluate influence of thrombophlebitis on TGF-β1 and its signaling pathway in the vein wall. TGF-β1 mRNAlevels, growth factor content and its expression were evaluated by RT-PCR, ELISA, and western blot methods, respectively, in the walls of normal veins, varicose veins and varicose veins complicated by thrombophlebitis. Western blot analysis was used to assess TGF-β receptor type II (TGF-β RII) and p-Smad2/3 protein expression in the investigated material. Unchanged mRNA levels of TGF-β1, decreased TGF-β1 content, as well as decreased expression of latent and active forms of TGF-β1 were found in varicose veins. Increased expression of TGF-β RII and p-Smad2/3 were found in varicose veins. Thrombophlebitis led to increased protein expression of the TGF-β1 active form and p-Smad2/3 in the vein wall compared to varicose veins. TGF-β1 may play a role in the disease pathogenesis because of increased expression and activation of its receptor in the wall of varicose veins. Thrombophlebitis accelerates activation of TGF-β1 and activity of its receptor in the varicose vein wall.     

5-Aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) enhances the efficacy of rapamycin in human cancer cells

mTOR – the mammalian/mechanistic target of rapamycin – has been implicated as a key signaling node for promoting survival of cancer cells. However, clinical trials that have targeted mTOR with rapamycin or rapamycin analogs have had minimal impact. In spite of the high specificity of rapamycin for mTOR, the doses needed to suppress key mTOR substrates have proved toxic. We report here that rapamycin when combined with AICAR – a compound that activates AMP-activated protein kinase makes rapamycin cytotoxic rather than cytostatic at doses that are tolerated clinically. AICAR by itself is able to suppress mTOR complex 1 (mTORC1), but also stimulates a feedback activation of mTORC2, which activates the survival kinase Akt. However, AICAR also suppresses production of phosphatidic acid (PA), which interacts with mTOR in a manner that is competitive with rapamycin. The reduced level of PA sensitizes mTORC2 to rapamycin at tolerable nano-molar doses leading reduced Akt phosphorylation and apoptosis. This study reveals how the use of AICAR enhances the efficacy of rapamycin such that rapamycin at low nano-molar doses can suppress mTORC2 and induce apoptosis in human cancer cells at doses that are clinically tolerable.     

Metabolism of 4-pyridone-3-carboxamide-1-??-d-ribonucleoside (4PYR) in rodent tissues and in vivo

Our previous studies identified 4-pyridone-3-carboxamide-1-β-D-ribonucleoside (4PYR) phosphates in human erythrocytes. We demonstrated formation of these nucleotides by phosphorylation of 4PYR and potential toxicity due to disruption of erythrocyte energy balance. This study aimed to evaluate the ability of the other cell types to phosphorylate 4PYR to characterize function and toxicity of these compounds. Homogenates of rat heart, kidneys, and liver were used to study the rate of 4PYR phosphorylation in the presence of ATP. In another experiment, 4PYR was administered into mouse as repeated subcutaneous injections and into rats as intraperitoneal infusion. After 7 days, heart, liver, kidney, lungs, and skeletal muscle were collected, and the concentration of 4PYR nucleotides was evaluated. HPLC was used to measure 4PYR and 4PYR nucleotides in homogenate and specimens from in vivo experiments. 4PYR was rapidly phosphorylated by the liver homogenate (390 ± 27 nmol/min/g wet wt). Significant rates were reported in the heart and kidneys' homogenates: 34.3 ± 4.3 nmol/min/g and 33.2 ± 9.2 nmol/min/g, respectively. Phosphorylation of 4PYR was almost completely inhibited by adenosine kinase inhibitor 5'-iodotubercidin. Administration of 4PYR in vivo resulted in accumulation of 4PYR monophosphate in the liver, heart, skeletal muscle, and lung (20-220 nmol/g dry wt) except kidney (<1 nmol/g). In contrast to erythrocytes, no 4PYR triphosphate formation (<1 nmol/g) was observed in any of the organs studied. We conclude that not only the erythrocytes but also other cell types are capable of phosphorylating 4PYR to form 4PYR monophosphate. Potential toxicity or physiological role of 4PYR in peripheral organs could be considered, but mechanisms will be different from that in erythrocytes.     

Effects of the 3β-hydroxysteroid dehydrogenase inhibitor trilostane on luteal progesterone production in the dog

Interference with the pregnancy-maintaining influence of progesterone is the basis of most methods for termination of unwanted pregnancy in dogs. The currently available methods are based on induction of luteolysis or blocking of the progesterone receptor. Inhibition of progesterone synthesis using a competitive inhibitor of 3β-hydroxysteroid dehydrogenase (3β-HSD) could be another strategy to terminate unwanted pregnancies.In this study we investigated the effects of the 3β-HSD inhibitor trilostane on corpus luteum function in non-pregnant bitches. Trilostane was administered orally for seven consecutive days in either the pituitary-independent part of the luteal phase (PIP, start of treatment on D11 after ovulation, n = 6) or the pituitary-dependent part (PDP, start of treatment on D31 after ovulation, n = 6), in an oral dose of about 4.5 mg/kg bw, twice daily. Results were compared with those obtained in control bitches (n = 6). ACTH stimulation tests were performed to assess adrenocortical reserve capacity.Trilostane caused no apparent side effects and ACTH stimulation tests revealed good suppression of cortisol secretion. Trilostane also caused a significant decrease in plasma progesterone concentration. When it was stopped during PIP, progesterone secretion was completely restored and there was no difference in the length of the luteal phase between those dogs and control dogs (99 days, range 70-138 d and 99 d, range 60-112 d, respectively). When trilostane was stopped during PDP there was no post-treatment recovery of progesterone secretion and although the luteal phase tended to be shorter (66 d, range 41-101 d) the difference was not significant (P = 0.09). Plasma prolactin concentration did not increase after the trilostane-induced decrease in plasma progesterone. The interoestrous interval in dogs treated during PIP (234 d, range 175-269 d) or PDP (198 d, range 120-287 d) was not significantly shorter than the control interval (247 d, range 176-313 d).In conclusion, trilostane treatment was effective in decreasing plasma progesterone concentration in bitches during the luteal phase, but the dose regimen used in this study produced less clear-cut inhibition of ovarian steroidogenesis than have other strategies to decrease plasma progesterone concentration. Further studies are warranted to determine whether trilostane can be used to terminate unwanted pregnancy in the bitch without inducing adrenocortical insufficiency.     

Structural Basis Underlying the Binding Preference of Human Galectins-1, -3 and -7 for Galβ1-3/4GlcNAc

Galectins represent β-galactoside-binding proteins and are known to bind Galβ1-3/4GlcNAc disaccharides (abbreviated as LN1 and LN2, respectively). Despite high sequence and structural homology shared by the carbohydrate recognition domain (CRD) of all galectin members, how each galectin displays different sugar-binding specificity still remains ambiguous. Herein we provided the first structural evidence of human galectins-1, 3-CRD and 7 in complex with LN1. Galectins-1 and 3 were shown to have higher affinity for LN2 than for LN1, while galectin-7 displayed the reversed specificity. In comparison with the previous LN2-complexed structures, the results indicated that the average glycosidic torsion angle of galectin-bound LN1 (ψ^LN1 ≈ 135°) was significantly differed from that of galectin-bound LN2 (ψ^LN2 ≈ -108°), i.e. the GlcNAc moiety adopted a different orientation to maintain essential interactions. Furthermore, we also identified an Arg-Asp/Glu-Glu-Arg salt-bridge network and the corresponding loop (to position the second Asp/Glu residue) critical for the LN1/2-binding preference.     

The influence of Congo red on the cell wall and (1 → 3)-β-d-glucan microfibril biogenesis in Saccharomyces cerevisiae

Congo red was applied to growing yeast cells and regenerating protoplasts in order to study its effects on wall biogenesis and cell morphogenesis. In the presence of the dye, the whole yeast cells grew and divided to form chains of connected cells showing aberrant wall structures on both sides of the septum. The wall-less protoplasts in solid medium with the dye exhibited an abnormal increase in volume, regeneration of aberrant cell walls and inability to carry out cytokinesis or protoplast reversion to cells. In liquid medium, the protoplasts synthesized glucan nets composed mainly of thin fibrils orientated at random, whereas normally, in the absence of dye, the nets consist of rather thick fibrils, 10 to 20 nm in width, assembled into broad ribbons. These fibrils are known to consist of triple 6/1 helical strands of (1 » 3)--d-glucan aggregated laterally in crystalline packing. The thin fibrils (c. 4 to 8 nm wide) can contain only a few triple helical strands (c. 1.6 nm wide) and are supposed to be prevented from further aggregation and crystallization by complexing with Congo red on their surfaces. Some loose triple 6/1 helical strands (native elementary fibrils) are also discernible. They represent the first native (1 » 3)--d-glucan elementary fibrils depicted by electron microscopy.The effects of Congo red on growth and the wall structure in normal cells and regenerating protoplasts in solid medium can be explained by the presence of a complex which the dye forms with (helical) chain parts of the glucan network and which results in a loss of rigidity by a blocked lateral interaction between the helices.In memory of Dr. D. R. Kreger of the University of Groningen, The Netherlands, who died on 7 January 1992     

Effects of 3 weeks GMP oral administration on glutamatergic parameters in mice neocortex

Overstimulation of the glutamatergic system (excitotoxicity) is involved in various acute and chronic brain diseases. Several studies support the hypothesis that guanosine-5′-monophosphate (GMP) can modulate glutamatergic neurotransmission. The aim of this study was to evaluate the effects of chronically administered GMP on brain cortical glutamatergic parameters in mice. Additionally, we investigated the neuroprotective potential of the GMP treatment submitting cortical brain slices to oxygen and glucose deprivation (OGD). Moreover, measurements of the cerebrospinal fluid (CSF) purine levels were performed after the treatment. Mice received an oral administration of saline or GMP during 3 weeks. GMP significantly decreases the cortical brain glutamate binding and uptake. Accordingly, GMP reduced the immunocontent of the glutamate receptors subunits, NR2A/B and GluR1 (NMDA and AMPA receptors, respectively) and glutamate transporters EAAC1 and GLT1. GMP treatment significantly reduced the immunocontent of PSD-95 while did not affect the content of Snap 25, GLAST and GFAP. Moreover, GMP treatment increased the resistance of neocortex to OGD insult. The chronic GMP administration increased the CSF levels of GMP and its metabolites. Altogether, these findings suggest a potential modulatory role of GMP on neocortex glutamatergic system by promoting functional and plastic changes associated to more resistance of mice neocortex against an in vitro excitotoxicity event.     

Construction of SH-EP1-α4β2-hAPP695 Cell Line and Effects of Nicotinic Agonists on β-amyloid in the Cells

(1) Nicotinic acetylcholine receptors in central nervous system are thought to be new targets for Alzheimer’s disease. However, the most involved nicotinic receptor subtype in Alzheimer’s disease is unclear. α4β2 receptor is the most widely spread subtype in brain, involving in several important aspects of cognitive and other functions. We constructed cell line by transfecting human amyloid precursor protein (695) gene into SH-EP1 cells which have been transfected with human nicotinic receptor α4 subunit and β2 subunit gene, to observe effects of α4β2 receptors activation on β-amyloid, expecting to provide a new cell line for drug screening and research purpose. (2) Liposome transfection was used to express human amyloid precursor protein (695) gene in SH-EP1-α4β2 cells. Function of the transfected α4β2 receptors was tested by patch clamp. Effects of nicotine and epibatidine (selective α4β2 nicotinic receptor agonist) on β-amyloid were detected by Western blot and ELISA. Effects of nicotine and epibatidine on amyloid precursor protein (695) mRNA level were measured using real-time PCR. (3) Human amyloid precursor protein (695) gene was stably expressed in SH-EP1-α4β2 cells; Nicotine (1 μM) and epibatidine (0.1 μM) decreased intracellular and secreted β-amyloid in the cells; and activation of α4β2 receptors did not affect amyloid precursor protein (695) mRNA level. (4) These results suggest that the constructed cell line, expressing both amyloid precursor protein (695) gene and human nicotinic receptor α4 subunit and β2 subunit gene, might be useful for screening specific nicotinic receptor agonists against Alzheimer’s disease. Alteration of Aβ level induced by activation of α4β2 nAChR in our study might occur at a post-translational level.     

Effects of 17β-estradiol on the distribution and activity of some oxydative enzymes of uterine and cervical epithelium in neonatal mice

Summary The following enzymes were studied histochemically in uterine and cervical epithelium from neonatal mice treated with 17-estradiol for the first four days after birth: NADH-, NADPH-, succinate-, -glycerophosphate-, lactate-, glucose-6-phosphate-, and 17-OH-steroid dehydrogenases.It was demonstrated that estradiol administration had a marked influence on distribution and activity of several of the enzymes compared with the control animals. In cervix there was an increase of activity for most of the enzymes, especially in the apical parts of the epithelium cells. The uterine epithelium was also estradiol sensitive as regards most enzymes, and in the case of glucose-6-phosphate dehydrogenase there was a dramatic enhancement of reaction in the uterus of the experimental animals. The differences obtained between cervical and uterine epithelium are described.17-OH-steroid dehydrogenase could not be detected histochemically in the present material.Supported by the Norwegian Research Council for Science and the Humanities.     

Disruption of Nucleotide Homeostasis by the Antiproliferative Drug 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside Monophosphate (AICAR)

5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside monophosphate (AICAR) is a natural metabolite with potent anti-proliferative and low energy mimetic properties. At high concentration, AICAR is toxic for yeast and mammalian cells, but the molecular basis of this toxicity is poorly understood. Here, we report the identification of yeast purine salvage pathway mutants that are synthetically lethal with AICAR accumulation. Genetic suppression revealed that this synthetic lethality is in part due to low expression of adenine phosphoribosyl transferase under high AICAR conditions. In addition, metabolite profiling points to the AICAR/NTP balance as crucial for optimal utilization of glucose as a carbon source. Indeed, we found that AICAR toxicity in yeast and human cells is alleviated when glucose is replaced by an alternative carbon source. Together, our metabolic analyses unveil the AICAR/NTP balance as a major factor of AICAR antiproliferative effects.     

Modeling and Molecular Dynamics of HPA-1a and -1b Polymorphisms: Effects on the Structure of the β3 Subunit of the αIIbβ3 Integrin

Background

The HPA-1 alloimmune system carried by the platelet integrin αIIbβ3 is the primary cause of alloimmune thrombocytopenia in Caucasians and the HPA-1b allele might be a risk factor for thrombosis. HPA-1a and -1b alleles are defined by a leucine and a proline, respectively, at position 33 in the β3 subunit. Although the structure of αIIbβ3 is available, little is known about structural effects of the L33P substitution and its consequences on immune response and integrin functions.

Methodology/Principal Findings

A complete 3D model of the L33-β3 extracellular domain was built and a P33 model was obtained by in silico mutagenesis. We then performed molecular dynamics simulations. Analyses focused on the PSI, I-EGF-1, and I-EGF-2 domains and confirmed higher exposure of residue 33 in the L33 β3 form. These analyses also showed major structural flexibility of all three domains in both forms, but increased flexibility in the P33 β3 form. The L33P substitution does not alter the local structure (residues 33 to 35) of the PSI domain, but modifies the structural equilibrium of the three domains.

Conclusions

These results provide a better understanding of HPA-1 epitopes complexity and alloimmunization prevalence of HPA-1a. P33 gain of structure flexibility in the β3 knee may explain the increased adhesion capacity of HPA-1b platelets and the associated thrombotic risk. Our study provides important new insights into the relationship between HPA-1 variants and β3 structure that suggest possible effects on the alloimmune response and platelet function.