Platelet Spreading Research Articles

Noncanonical Sonic Hedgehog signaling amplifies platelet reactivity and thrombogenicity

AbstractSonic Hedgehog (Shh) is a morphogen in vertebrate embryos that is also associated with organ homeostasis in adults. We report here that human platelets, though enucleate, synthesize Shh from preexisting mRNAs upon agonist stimulation, and mobilize it for surface expression and release on extracellular vesicles, thus alluding to its putative role in platelet activation. Shh, in turn, induced a wave of noncanonical signaling in platelets leading to activation of small GTPase Ras homolog family member A and phosphorylation of myosin light chain in activated protein kinase-dependent manner. Remarkably, agonist-induced thrombogenic responses in platelets, which include platelet aggregation, granule secretion, and spreading on immobilized fibrinogen, were significantly attenuated by inhibition of Hedgehog signaling, thus, implicating inputs from Shh in potentiation of agonist-mediated platelet activation. In consistence, inhibition of the Shh pathway significantly impaired arterial thrombosis in mice. Taken together, the above observations strongly support a feed-forward loop of platelet stimulation triggered locally by Shh, similar to ADP and thromboxane A2, that contributes significantly to the stability of occlusive arterial thrombus and that can be investigated as a potential therapeutic target in thrombotic disorders.

Main Factors Influencing the Platelet Spreading

To explore the main factors of platelet spreading and provide the foundation for related research. Platelets (2×107/ml) were draw from C57BL/6J mouse and kept at 22 ℃ for 1-2 hours. Platelets (2×107/ml) were were allowed to adhere and spread on the fibrinogen-coated slides, after staining F-actin in platelets, the platelets were observed with the confocal microscopy. The effects of different concentrations of fibrinogen (10 μg/ml, 30 μg/ml, 100 μg/ml) and kinds of agonists ［thrombin(0.01,0.05,0.1 U/ml), ADP（5,10,20 μmol/L）, U46619（0.125,0.25,0.5 μmol/L）］ on platelets were analyzed. The platelet spreading was successful if the spreading rate was higher after treated with agonists. Compared to the group which coated with 10 μg/ml and 100 μg/ml fibrinogen, the platelet density is optimal when coated with 30 μg/ml fibrinogen. In addition, under the stimulation of thrombin, ADP and U46619, the spreading rate of platelets showed a certain concentration-dependent increasing. The platelet spreading is easily influenced by various factors, the platelet spreading can be induced successfully at 0.1 U/ml thrombin, 20 μmol/L ADP and 0.5 μmol/L U46619 on the slide coated with 30 μg/ml fibrinogen.

Nanofiber Topographies Enhance Platelet-Fibrinogen Scaffold Interactions.

The initial contact with blood and its components, including plasma proteins and platelets, directs the body's response to foreign materials. Natural scaffolds of extracellular matrix or fibrin contain fibrils with nanoscale dimensions, but how platelets specifically respond to the topography and architecture of fibrous materials is still incompletely understood. Here, planar and nanofiber scaffolds are fabricated from native fibrinogen to characterize the morphology of adherent platelets and activation markers for phosphatidylserine exposure and α-granule secretion by confocal fluorescence microscopy and scanning electron microscopy. Different fibrinogen topographies equally support the spreading and α-granule secretion of washed platelets. In contrast, preincubation of the scaffolds with plasma diminishes platelet spreading on planar fibrinogen surfaces but not on nanofibers. The data show that the enhanced interactions of platelets with nanofibers result from a higher locally accessible surface area, effectively increasing the ligand density for integrin-mediated responses. Overall, fibrinogen nanofibers direct platelets toward robust adhesion formation and α-granule secretion while minimizing their procoagulant activity. Similar results on fibrinogen-coated polydimethylsiloxane substrates with micrometer-sized 3D features suggest that surface topography could be used more generally to steer blood-materials interactions on different length scales for enhancing the initial wound healing steps.

Cytoskeleton Dependent Mobility Dynamics of FcγRIIA Facilitates Platelet Haptotaxis and Capture of Opsonized Bacteria.

Platelet adhesion and spreading at the sites of vascular injury is vital to hemostasis. As an integral part of the innate immune system, platelets interact with opsonized bacterial pathogens through FcγRIIA and contribute to host defense. As mechanoscavangers, platelets actively migrate and capture bacteria via cytoskeleton-rich, dynamic structures, such as filopodia and lamellipodia. However, the role of human platelet FcγRIIA in cytoskeleton-dependent interaction with opsonized bacteria is not well understood. To decipher this, we used a reductionist approach with well-defined micropatterns functionalized with immunoglobulins mimicking immune complexes at planar interfaces and bacteriamimetic microbeads. By specifically blocking of FcγRIIA and selective disruption of the platelet cytoskeleton, we show that both functional FcγRIIA and cytoskeleton are necessary for human platelet adhesion and haptotaxis. The direct link between FcγRIIA and the cytoskeleton is further explored by single-particle tracking. We then demonstrate the relevance of cytoskeleton-dependent differential mobilities of FcγRIIA on bacteria opsonized with the chemokine platelet factor 4 (PF4) and patient-derived anti-PF4/polyanion IgG. Our data suggest that efficient capture of opsonized bacteria during host-defense is governed by mobility dynamics of FcγRIIA on filopodia and lamellipodia, and the cytoskeleton plays an essential role in platelet morphodynamics at biological interfaces that display immune complexes.

A novel nonsense variant in TPM4 caused dominant macrothrombocytopenia, mild bleeding tendency and disrupted cytoskeleton remodeling

BackgroundRare inherited thrombocytopenias are caused by alterations in genes involved in megakaryopoiesis, thrombopoiesis and/or platelet release. Diagnosis is challenging due to poor specificity of platelet laboratory assays, large numbers of culprit genes, and difficult assessment of the pathogenicity of novel variants. ObjectivesTo characterize the clinical and laboratory phenotype, and identifying the underlying molecular alteration, in a pedigree with thrombocytopenia of uncertain etiology. Patients/MethodsIndex case was enrolled in our Spanish multicentric project of inherited platelet disorders due to lifelong thrombocytopenia and bleeding. Bleeding score was recorded by ISTH‐BAT. Laboratory phenotyping consisted of blood cells count, blood film, platelet aggregation and flow cytometric analysis. Genotyping was made by whole‐exome sequencing (WES). Cytoskeleton proteins were analyzed in resting/spreading platelets by immunofluorescence and immunoblotting. ResultsFive family members displayed lifelong mild thrombocytopenia with a high number of enlarged platelets in blood film, and mild bleeding tendency. Patient's platelets showed normal aggregation and granule secretion response to several agonists. WES revealed a novel nonsense variant (c.322C>T; p.Gln108*) in TPM4 (NM_003290.3), the gene encoding for tropomyosin‐4 (TPM4). This variant led to impairment of platelet spreading capacity after stimulation with TRAP‐6 and CRP, delocalization of TPM4 in activated platelets, and significantly reduced TPM4 levels in platelet lysates. Moreover, the index case displayed up‐regulation of TPM2 and TPM3 mRNA levels. ConclusionsThis study identifies a novel TPM4 nonsense variant segregating with macrothrombocytopenia and impaired platelet cytoskeletal remodeling and spreading. These findings support the relevant role of TPM4 in thrombopoiesis and further expand our knowledge of TPM4‐related thrombocytopenia.

Rac Inhibition Causes Impaired GPVI Signalling in Human Platelets through GPVI Shedding and Reduction in PLCγ2 Phosphorylation.

Rac1 is a small Rho GTPase that is activated in platelets upon stimulation with various ligands, including collagen and thrombin, which are ligands for the glycoprotein VI (GPVI) receptor and the protease-activated receptors, respectively. Rac1-deficient murine platelets have impaired lamellipodia formation, aggregation, and reduced PLCγ2 activation, but not phosphorylation. The objective of our study is to investigate the role of Rac1 in GPVI-dependent human platelet activation and downstream signalling. Therefore, we used human platelets stimulated using GPVI agonists (collagen and collagen-related peptide) in the presence of the Rac1-specific inhibitor EHT1864 and analysed platelet activation, aggregation, spreading, protein phosphorylation, and GPVI clustering and shedding. We observed that in human platelets, the inhibition of Rac1 by EHT1864 had no significant effect on GPVI clustering on collagen fibres but decreased the ability of platelets to spread or aggregate in response to GPVI agonists. Additionally, in contrast to what was observed in murine Rac1-deficient platelets, EHT1864 enhanced GPVI shedding in platelets and reduced the phosphorylation levels of PLCγ2 following GPVI activation. In conclusion, Rac1 activity is required for both human and murine platelet activation in response to GPVI-ligands, but Rac1’s mode of action differs between the two species.

Saccharides from Arctium lappa L. root reduce platelet activation and thrombus formation in a laser injury thrombosis mouse model.

Arctium lappa L., also known as burdock, is a popular medicinal plant in traditional Chinese medicine due to its potential therapeutic properties. Saccharides from Arctium lappa L. root (ALR-S) have been extensively studied for their anti-inflammatory and anti-diabetes effects. Platelets play a pivotal role in thrombosis. The present study describes the effects of ALR-S on platelet activation and thrombosis using a laser injury thrombosis in vivo model. The study also measured the effects of ALR-S on platelet activation by analysing aggregation, ATP release, platelet spreading, adhesion and clot retraction in vitro. Specifically, the effects were ALR-S concentration-dependent inhibition of platelet aggregation and ATP release. Activated platelets pretreated with ALR-S showed diminished CD62P expression levels and fibrinogen binding, as measured by flow cytometry. ALR-S inhibited platelet spreading on fibrinogen and adhesion on collagen under shear. ALR-S attenuated platelet activation by decreasing oxidative stress and thrombus formation. These results demonstrated the antiplatelet effects of ALR-S, suggesting the antithrombotic and cardiovascular protective activities of ALR-S as a functional food.

Fruitflow inhibits platelet function by suppressing Akt/GSK3\u03b2, Syk/PLC\u03b32 and p38 MAPK phosphorylation in collagen-stimulated platelets

BackgroundPlatelets play an important role in the progression of atherosclerosis and cardiovascular events. The inhibition of platelet function is a main strategy to reduce risk of cardiovascular events. Some studies have shown that tomato extracts inhibit platelet function, but the molecular mechanisms remain unclear. Fruitflow is a water-solute tomato extract and the main ingredients including flavonoids, adenosine, chlorogenic acid, phytosterols, naringenin, and carotenoids. The present study investigated the effects of fruitflow on adenosine diphosphate (ADP)- and collagen- stimulated platelet aggregation, platelet adhesion, and levels of thromboxane B2 (TXB2), 6-keto-prostaglandin F1α (PGF1α), and platelet factor 4 (PF4) and explored the underlying molecular mechanisms.MethodsPlatelet-rich plasma (PRP) was used for measurement of platelet aggregation, TXB2, 6-keto- PGF1α, and PF4 levels. Platelet aggregation was analyzed using a Chrono-Log aggregometer. TXB2, 6-keto- PGF1α, and PF4 levels were determined using enzyme-linked immunosorbent assay kits. Immunoblotting was used to detect protein expression and phosphorylation on washed platelets. Platelet adhesion and spreading were determined by immunofluorescence.ResultsFruitflow (1, 3, 10 and 100 μg/ml) dose-dependently inhibited platelet aggregation that was induced by ADP and collagen. Fruitflow (100 μg/ml) treatment completely suppressed ADP- and collagen-stimulated platelet aggregation. Fruitflow (100 μg/ml) significantly decreased TXB2 and 6-keto-PGF1α generation and PF4 release in ADP- and collagen-stimulated platelets. Treatment with fruitflow effectively blocked collagen-induced platelet spreading. To determine the potential molecule mechanism of action of fruitflow, we investigated the protein expression and phosphorylation of several signaling molecules in collagen-activated platelets. Fruitflow dose-dependently suppressed Akt, Glycogen synthase kinase-3β (GSK-3β), spleen tyrosine kinase (Syk) and phospholipase Cγ2 (PLCγ2) and p38 MAPK phosphorylation that was induced by collagen.ConclusionFruitflow inhibited platelet aggregation and reduced TXB2, 6-keto-PGF1α, and PF4 levels in ADP- and collagen-stimulated platelets. The mechanism of action of fruitflow may be associated with the suppression of Akt/GSK3β, Syk/PLCγ2, and p38 MAPK phosphorylation in collagen-activated platelets. Fruitflow is a natural product derived from tomato and can be used as a health food for decreasing platelet activity.

Fully automated platelet differential interference contrast image analysis via deep learning

Platelets mediate arterial thrombosis, a leading cause of myocardial infarction and stroke. During injury, platelets adhere and spread over exposed subendothelial matrix substrates of the damaged blood vessel wall. The mechanisms which govern platelet activation and their interaction with a range of substrates are therefore regularly investigated using platelet spreading assays. These assays often use differential interference contrast (DIC) microscopy to assess platelet morphology and analysis performed using manual annotation. Here, a convolutional neural network (CNN) allowed fully automated analysis of platelet spreading assays captured by DIC microscopy. The CNN was trained using 120 generalised training images. Increasing the number of training images increases the mean average precision of the CNN. The CNN performance was compared to six manual annotators. Significant variation was observed between annotators, highlighting bias when manual analysis is performed. The CNN effectively analysed platelet morphology when platelets spread over a range of substrates (CRP-XL, vWF and fibrinogen), in the presence and absence of inhibitors (dasatinib, ibrutinib and PRT-060318) and agonist (thrombin), with results consistent in quantifying spread platelet area which is comparable to published literature. The application of a CNN enables, for the first time, automated analysis of platelet spreading assays captured by DIC microscopy.

Force-enhanced biophysical connectivity of platelet \u03b23 integrin signaling through Talin is predicted by steered molecular dynamics simulations

Platelet β3-integrin signaling through Talin is crucial in platelet transmembrane signaling, activation, adhesion, spreading and aggregation, and remains unclear in mechano-microenvironments. In order to examine Talin-β3 integrin biophysical connectivity, a series of “ramp-clamp” steered molecular dynamics (SMD) simulations were performed on complex of F3 domain of Talin and cytoplasmic tail of β3 integrin to imitate different force-loads in platelet. Pull-induced allostery of the hydrophobic pocket in F3 domain might markedly enhance complex rupture-force (> 150pN) and slow down breakage of the complex; the complex should mechano-stable for its conformational conservation under loads (≤ 80pN); increasing force below 60pN would decrease the complex dissociation probability, and force-induced extension of β5 strand on Talin and binding site residues, ASP740 and ALA742 as well as Asn744, on β3-integrin were responsible for the force-enhanced linkage of the Talin-β3 integrin. Force might enhance biophysical connectivity of β3-integrin signaling through Talin by a catch bond mechanism, which be mediated by the force-induced allostery of complex at clamped stage. This work provides a novel insight into the force-regulated transmembrane β3-integrin signaling and its molecular basis for platelet activation, and exhibited a potential power of the present computer strategy in predicting mechanical regulation on ligand-receptor interaction under loads.

Deletion of Grin1 in mouse megakaryocytes reveals NMDA receptor role in platelet function and proplatelet formation

AbstractThe process of proplatelet formation (PPF) requires coordinated interaction between megakaryocytes (MKs) and the extracellular matrix (ECM), followed by a dynamic reorganization of the actin and microtubule cytoskeleton. Localized fluxes of intracellular calcium ions (Ca2+) facilitate MK-ECM interaction and PPF. Glutamate-gated N-methyl-D-aspartate receptor (NMDAR) is highly permeable to Ca2+. NMDAR antagonists inhibit MK maturation ex vivo; however, there are no in vivo data. Using the Cre-loxP system, we generated a platelet lineage–specific knockout mouse model of reduced NMDAR function in MKs and platelets (Pf4-Grin1−/− mice). Effects of NMDAR deletion were examined using well-established assays of platelet function and production in vivo and ex vivo. We found that Pf4-Grin1−/− mice had defects in megakaryopoiesis, thrombopoiesis, and platelet function, which manifested as reduced platelet counts, lower rates of platelet production in the immune model of thrombocytopenia, and prolonged tail bleeding time. Platelet activation was impaired to a range of agonists associated with reduced Ca2+ responses, including metabotropic like, and defective platelet spreading. MKs showed reduced colony and proplatelet formation. Impaired reorganization of intracellular F-actin and α-tubulin was identified as the main cause of reduced platelet function and production. Pf4-Grin1−/− MKs also had lower levels of transcripts encoding crucial ECM elements and enzymes, suggesting NMDAR signaling is involved in ECM remodeling. In summary, we provide the first genetic evidence that NMDAR plays an active role in platelet function and production. NMDAR regulates PPF through a mechanism that involves MK-ECM interaction and cytoskeletal reorganization. Our results suggest that NMDAR helps guide PPF in vivo.

Rare missense variants in Tropomyosin‐4 (TPM4) are associated with platelet dysfunction, cytoskeletal defects, and excessive bleeding

BackgroundA significant challenge is faced for the genetic diagnosis of inherited platelet disorders in which candidate genetic variants can be found in more than 100 bleeding, thrombotic, and platelet disorder genes, especially within families in which there are both normal and low platelet counts. Genetic variants of unknown clinical significance (VUS) are found in a significant proportion of such patients in which functional studies are required to prove pathogenicity. ObjectiveTo identify the genetic cause in patients with a suspected platelet disorder and subsequently perform a detailed functional analysis of the candidate genetic variants found. MethodsGenetic and functional studies were undertaken in three patients in two unrelated families with a suspected platelet disorder and excessive bleeding. A targeted gene panel of previously known bleeding and platelet genes was used to identify plausible genetic variants. Deep platelet phenotyping was performed using platelet spreading analysis, transmission electron microscopy, immunofluorescence, and platelet function testing using lumiaggregometry and flow cytometry. ResultsWe report rare conserved missense variants (p.R182C and p.A183V) in TPM4 encoding tromomyosin‐4 in 3 patients. Deep platelet phenotyping studies revealed similar platelet function defects across the 3 patients including reduced platelet secretion, and aggregation and spreading defects suggesting that TPM4 missense variants impact platelet function and show a disordered pattern of tropomyosin staining. ConclusionsGenetic and functional TPM4 defects are reported making TPM4 a diagnostic grade tier 1 gene and highlights the importance of including TPM4 in diagnostic genetic screening for patients with significant bleeding and undiagnosed platelet disorders, particularly for those with a normal platelet count.

Platelets and Antiplatelet Medication in COVID-19-Related Thrombotic Complications.

Coronavirus disease 2019 (COVID-19) induces a hypercoagulatory state that frequently leads to thromboembolic complications. Whereas anticoagulation is associated with reduced mortality, the role of antiplatelet therapy in COVID-19 is less clear. We retrospectively analyzed the effect of anticoagulation and antiplatelet therapy in 578 hospitalized patients with COVID-19 and prospectively monitored 110 patients for circulating microthrombi and plasma markers of coagulation in the first week of admission. Moreover, we determined platelet shape change and also thrombi in postmortem lung biopsies in a subset of patients with COVID-19. We observed no association of antiplatelet therapy with COVID-19 survival. Adverse outcome in COVID-19 was associated with increased activation of the coagulation cascade, whereas circulating microthrombi did not increase in aggravated disease. This was in line with analysis of postmortem lung biopsies of patients with COVID-19, which revealed generally fibrin(ogen)-rich and platelet-low thrombi. Platelet spreading was normal in severe COVID-19 cases; however, plasma from patients with COVID-19 mediated an outcome-dependent inhibitory effect on naïve platelets. Antiplatelet medication disproportionally exacerbated this platelet impairment in plasma of patients with fatal outcome. Taken together, this study shows that unfavorable outcome in COVID-19 is associated with a profound dysregulation of the coagulation system, whereas the contribution of platelets to thrombotic complications is less clear. Adverse outcome may be associated with impaired platelet function or platelet exhaustion. In line, antiplatelet therapy was not associated with beneficial outcome.

Pharmacological actions of dieckol on modulation of platelet functions and thrombus formation via integrin αIIbβ3 and cAMP signaling

Background and purposeDieckol is a phlorotannin that can be found in seaweeds, particularly in Eisenia bicyclis (brown algae) and is known to have anti-oxidant, anti-inflammatory, and anti-microbial properties. It also possesses anti-thrombotic and pro-fibrinolytic activities; however, the mechanistic aspects of anti-platelet and anti-thrombotic activity are yet to be explored. Study design and methodologyWe investigated the pharmacological effects of dieckol on the modulation of platelet functions using human, rat, and mice models. Inhibitory effects of dieckol on platelet aggregation were assessed using platelet-rich plasma and washed platelets, followed by measurement of dense granule secretions, fibrinogen binding to integrin αIIbβ3, fibronectin adhesion assay, platelet spreading on immobilized fibrinogen, and clot retraction. Cyclic nucleotide signaling events were evaluated, such as cyclic-AMP production followed by vasodilator-stimulated phosphoprotein (VASP) stimulation. The in vivo anti-thrombotic potential was evaluated in mice using an acute pulmonary thromboembolism model and tail bleeding assay. ResultsDieckol markedly inhibited platelet aggregation and granule secretion; furthermore, it down-regulated integrin αIIbβ3–mediated inside-out and outside-in signaling events, including platelet adhesion, spreading, and clot retraction, whereas it upregulated the cAMP–PKA–VASP pathway. Dieckol-treated mice significantly survived the thrombosis than vehicle treated mice, without affecting hemostasis. Histological examinations of lungs revealed minimum occluded vasculature in dieckol-treated mice. ConclusionDieckol possesses strong anti-platelet and anti-thrombotic properties and is a potential therapeutic drug candidate to treat and prevent platelet-related cardiovascular disorders.

Three-dimensional analysis of blood platelet spreading using digital holographic microscopy: a statistical study of the differential effect of coatings in healthy volunteers and dialyzed patients.

In cardiovascular disorders, the study of thrombocytes, commonly known as platelets, is highly important since they are involved in blood clotting, essential in hemostasis, and they can in pathological situations affect the blood circulation. In this paper, single deposited platelets are measured using interferometric digital holographic microscopy. We have shown that the average optical height of platelets is significantly lower in healthy volunteers than in dialyzed patients, meaning a better spreading. It demonstrates the great interest for assessing this parameter in any patients, and therefore the high potential of analyzing single spread platelets using digital holographic microscopy in fundamental research as well as a diagnostic tool in routine laboratories, for usual blood tests.

Practical Considerations of Dissolved Oxygen Levels for Platelet Function under Hypoxia.

Investigating human platelet function in low-oxygen environments is important in multiple settings, including hypobaric hypoxia (e.g., high altitude), sea level hypoxia-related disease, and thrombus stability. These studies often involve drawing blood from which platelets are isolated and analysed at atmospheric conditions or re-exposed to low oxygen levels in hypoxia chambers before testing. However, it remains unknown how the in vitro handling of the samples itself changes their dissolved oxygen concentration, which might affect platelet function and experimental results. Here, we prepared healthy donor platelet-rich plasma and washed platelet (WP) suspensions and exposed them to 2% oxygen. We found that the use of hypoxia pre-equilibrated tubes, higher platelet concentrations (>2 × 108/mL versus 2 × 107/mL), smaller volumes (600 µL versus 3 mL), and presence of plasma reduced the time for samples to reach 2% oxygen. Notably, oxygen levels decreased below 2% in most suspensions, but also in WP maintained at atmospheric 21% oxygen. Additionally, platelet spreading on fibrinogen was decreased when using hypoxic fibrinogen-coated culture plates regardless of the oxygen percentage (2% or 21%) in which platelet incubation took place. Thus, sample handling and experimental conditions should be carefully monitored in platelet-hypoxia studies as they might compromise results interpretation and comparison across studies.

Novel disintegrin-like peptides derived from an amphibian skin cDNA sequence of Hypsiboas punctatus.

Disintegrins comprise a family of small proteins that bind to and alter the physiological function of integrins, especially integrins that mediate platelet aggregation in blood. Here, we report a lysine-glycine-aspartic acid (KGD) disintegrin-like motif present in a 15-amino acid residue peptide identified in a cDNA library of the amphibian Hypsiboas punctatus skin. The original peptide sequence was used as a template from which five new analogs were designed, chemically synthesized by solid phase, and tested for disintegrin activity and tridimensional structural studies using NMR spectroscopy. The original amphibian peptide had no effect on integrin-mediated responses. Nevertheless, derived peptide analogs inhibited integrin-mediated platelet function, including platelet spreading on fibrinogen.

Paxillin binding to the PH domain of kindlin‐3 in platelets is required to support integrin αIIbβ3 outside‐in signaling

BackgroundKindlin‐3 is essential for supporting the bidirectional signaling of integrin αIIbβ3 in platelets by bridging the crosstalk between integrin αIIbβ3 and the cytoplasmic signaling adaptors. ObjectiveIn this study, we identified a previously unrecognized paxillin binding site in the pleckstrin homology (PH) domain of kindlin‐3 and verified its functional significance. MethodsStructure‐based approaches were employed to identify the paxillin binding site in the PH domain of kindlin‐3. In addition, the bidirectional signaling of integrin αIIbβ3 were evaluated in both human and mouse platelets. ResultsIn brief, we found that a β1‐β2 loop in the PH domain of kindlin‐3, an important part of the canonical membrane phospholipid binding pocket, was also involved in mediating paxillin interaction. Interestingly, the binding sites of paxillin and membrane phospholipids in the PH domain of kindlin‐3 were mutually exclusive. Specific disruption of paxillin binding to the PH domain by point mutations inhibited platelet spreading on immobilized fibrinogen while having no inhibition on soluble fibrinogen binding to stimulated platelets. In addition, a membrane‐permeable peptide derived from the β1‐β2 loop in the PH domain of kindlin‐3 was capable of inhibiting platelet spreading and clot retraction, but it had no effect on soluble fibrinogen binding to platelets and platelet aggregation. Treatment with this peptide significantly reduced thrombus formation in mice. ConclusionTaken together, these findings suggest that interaction between paxillin and the PH domain of kindlin‐3 plays an important role in supporting integrin αIIbβ3 outside‐in signaling in platelets, thus providing a novel antithrombotic target.

A novel role for endoplasmic reticulum protein 46 (ERp46) in platelet function and arterial thrombosis in mice

AbstractAlthough several members of protein disulfide isomerase (PDI) family support thrombosis, other PDI family members with the CXYC motif remain uninvestigated. ERp46 has 3 CGHC redox-active sites and a radically different molecular architecture than other PDIs. Expression of ERp46 on the platelet surface increased with thrombin stimulation. An anti-ERp46 antibody inhibited platelet aggregation, adenosine triphosphate (ATP) release, and αIIbβ3 activation. ERp46 protein potentiated αIIbβ3 activation, platelet aggregation, and ATP release, whereas inactive ERp46 inhibited these processes. ERp46 knockout mice had prolonged tail-bleeding times and decreased platelet accumulation in thrombosis models that was rescued by infusion of ERp46. ERp46-deficient platelets had decreased αIIbβ3 activation, platelet aggregation, ATP release, and P-selectin expression. The defects were reversed by wild-type ERp46 and partially reversed by ERp46 containing any of the 3 active sites. Platelet aggregation stimulated by an αIIbβ3-activating peptide was inhibited by the anti-ERp46 antibody and was decreased in ERp46-deficient platelets. ERp46 bound tightly to αIIbβ3 by surface plasmon resonance but poorly to platelets lacking αIIbβ3 and physically associated with αIIbβ3 upon platelet activation. ERp46 mediated clot retraction and platelet spreading. ERp46 more strongly reduced disulfide bonds in the β3 subunit than other PDIs and in contrast to PDI, generated thiols in β3 independently of fibrinogen. ERp46 cleaved the Cys473-Cys503 disulfide bond in β3, implicating a target for ERp46. Finally, ERp46-deficient platelets have decreased thiols in β3, implying that ERp46 cleaves disulfide bonds in platelets. In conclusion, ERp46 is critical for platelet function and thrombosis and facilitates αIIbβ3 activation by targeting disulfide bonds.

Introducing a Novel Biophysical Platelet Function Panel to Investigate Disorders of Primary Hemostasis and Bleeding of Unknown Cause

A subset of patients with chronic bleeding remain undiagnosed even after extensive diagnostic evaluation are labeled as “bleeding of unknown cause” (BUC). The key barrier to treating these patients is that they have a clinical bleeding tendency in the presence of normal diagnostic tests, and optimal methods for monitoring and treating patients with BUC remain unknown. While patients with BUC have symptoms of a primary hemostatic disorder, there is no diagnostic test or biomarker that can accurately identify which patients are at risk for bleeding such as those with mild Von Willebrand Disease (VWD) which comprise a broad spectrum of patients with varying degrees of bleeding. In order to fill this diagnostic gap in disorders of primary hemostasis, there is a clinical need for more assays of platelet function.To that end, we have engineered multiple new biophysical assays to assess disorders of primary hemostasis and apply this panel of platelet function testing to potentially define new bleeding disorders, characterize platelet phenotypes in patients with BUC, and refine the definition of mild VWD. Our panel of platelet function tests (Fig 1) collectively enables us to simultaneously assess different facets of primary hemostasis from the microscopic level of single-platelet physiology to hemostatic plug formation, thereby capturing various aspects of platelet function with a single blood sample. Our platelet function panel ranges from platelet adhesion and bulk clot contraction assays to spatially-regulated platelet granule secretion assay, single-platelet contraction cytometry, microfluidics, and a microengineered vascularized bleeding model. As such, we are leveraging these biophysical assays to correlate platelet function with bleeding phenotype severity and establish the dynamic range of this diagnostic panel. We have now established that our assays can be utilized to study blood samples from patients with disorders including hemophilia A, Hermansky-Pudlak Syndrome, FLI-1 mutation, and sickle cell disease among others (Fig 2), demonstrating the clinical utility of our platelet function panel.Our panel can also be used to assess the effects of novel therapeutics on different aspects of platelet function simultaneously. To investigate how crizanlizumab (p-selectin inhibitor) affects hemostatic plug formation, healthy human blood was treated with crizanlizumab. Platelet α-granule secretion enables exposure of P-selectin, and with crizanlizumab we observed restricted platelet filopodial extension and diminished α-granule exocytosis, and an overall decrease in adhered platelets to the fibrinogen micropattern (Fig 3A). The adhesion assay demonstrated a decrease in spreading and adhesion of platelets to collagen and fibrinogen with treatment (Fig 3B). Using the bleeding model, hemostasis was achieved within the normal established range and platelets contracted normally. This suggests that p-selectin has a limited role in the setting of minor injury. Utilizing the bulk contraction assay, we exhibited increased contraction early in clot formation, however over time the treated platelets contracted similarly to the control (Fig 3C). Interestingly, the effect of crizanlizumab-induced restriction of filopodial extension did not correlate with impaired bulk clot contraction or time to form hemostatic plug. Our work suggests crizanlizumab affects platelet spreading at the single-cell level but does not impair platelet function in achieving primary hemostasis at the whole blood level.Here we demonstrate the translational utility of our platelet function panel in providing a deeper understanding of platelet biophysics as it relates to hematologic conditions, with implications for investigation to include pharmaceutical applications. The versatility of this novel panel in capturing platelet function from single-platelet contraction to providing in vitro models with the bleeding device provides multiple dimensions to platelet investigation for primary hemostatic disorders and BUC that have not yet been elucidated. Ongoing research is being conducted using our comprehensive platelet function panel to investigate platelet properties in BUC and mild VWD and correlate these biophysics with bleeding phenotypes. Using this approach we aim to provide novel diagnostic testing with clinical relevance for disorders that have been incompletely characterized until now. [Display omitted] DisclosuresMeeks: National Institutes of Health: Research Funding; Hemophilia of Georgia: Research Funding; National Hemophilia Foundation: Research Funding; Spark Therapeutics: Consultancy; Sangamo Therapeutics: Consultancy; Pfizer: Consultancy; Sanofi: Consultancy; CSL Behring: Consultancy; Genentech: Consultancy; Takeda: Consultancy. Lam: Sanguina, Inc.: Current holder of individual stocks in a privately-held company.