Integrin αIIbβ3 Activation Research Articles

MAGT1 deficiency in XMEN disease is associated with severe platelet dysfunction and impaired platelet glycoprotein N-glycosylation

BackgroundX-linked immunodeficiency with magnesium defect, Epstein–Barr virus infection, and neoplasia (XMEN) disease is a primary immunodeficiency due to loss-of-function mutations in the gene encoding for magnesium transporter 1 (MAGT1). Furthermore, as MAGT1 is involved in the N-glycosylation process, XMEN disease is classified as a congenital disorder of glycosylation. Although XMEN-associated immunodeficiency is well described, the mechanisms underlying platelet dysfunction and those responsible for life-threatening bleeding events have never been investigated. ObjectivesTo assess platelet functions in patients with XMEN disease. MethodsTwo unrelated young boys, including one before and after hematopoietic stem cell transplantation, were investigated for their platelet functions, glycoprotein expression, and serum and platelet-derived N-glycans. ResultsPlatelet analysis highlighted abnormal elongated cells and unusual barbell-shaped proplatelets. Platelet aggregation, integrin αIIbβ3 activation, calcium mobilization, and protein kinase C activity were impaired between both patients. Strikingly, platelet responses to protease-activated receptor 1 activating peptide were absent at both low and high concentrations. These defects were also associated with decreased molecular weights of glycoprotein Ibα, glycoprotein VI, and integrin αIIb due to partial impairment of N-glycosylation. All these defects were corrected after hematopoietic stem cell transplantation. ConclusionOur results highlight prominent platelet dysfunction related to MAGT1 deficiency and defective N-glycosylation in several platelet proteins that could explain the hemorrhages reported in patients with XMEN disease.

Small-Molecule Cyclophilin Inhibitors Potently Reduce Platelet Procoagulant Activity.

Procoagulant platelets are associated with an increased risk for thrombosis. Procoagulant platelet formation is mediated via Cyclophilin D (CypD) mediated opening of the mitochondrial permeability transition pore. Inhibiting CypD activity could therefore be an interesting approach to limiting thrombosis. In this study, we investigated the potential of two novel, non-immunosuppressive, non-peptidic small-molecule cyclophilin inhibitors (SMCypIs) to limit thrombosis in vitro, in comparison with the cyclophilin inhibitor and immunosuppressant Cyclosporin A (CsA). Both cyclophilin inhibitors significantly decreased procoagulant platelet formation upon dual-agonist stimulation, shown by a decreased phosphatidylserine (PS) exposure, as well as a reduction in the loss of mitochondrial membrane potential. Furthermore, the SMCypIs potently reduced procoagulant platelet-dependent clotting time, as well as fibrin formation under flow, comparable to CsA. No effect was observed on agonist-induced platelet activation measured by P-selectin expression, as well as CypA-mediated integrin αIIbβ3 activation. Importantly, whereas CsA increased Adenosine 5'-diphosphate (ADP)-induced platelet aggregation, this was unaffected in the presence of the SMCypIs. We here demonstrate specific cyclophilin inhibition does not affect normal platelet function, while a clear reduction in procoagulant platelets is observed. Reducing platelet procoagulant activity by inhibiting cyclophilins with SMCypIs forms a promising strategy to limit thrombosis.

IL-37 Attenuates Platelet Activation and Thrombosis Through IL-1R8 Pathway.

IL-37 (interleukin-37), a natural suppressor of innate inflammatory and immune responses, is increased in patients with myocardial infarction. Platelets play an important role in the progress of myocardial infarction, but the direct effects of IL-37 on platelet activation and thrombosis, as well as the underlying mechanisms, still remain unclear. We evaluated the direct effects of IL-37 on agonists-induced platelet activation and thrombus formation, as well as revealed the underlying mechanisms using platelet-specific IL-1R8 (IL-1 receptor 8)-deficient mice. Using myocardial infarct model, we explored the effects of IL-37 on microvascular obstruction and myocardial injury. IL-37 directly inhibited agonists-induced platelet aggregation, dense granule ATP release, P-selectin exposure, integrin αIIbβ3 activation, platelet spreading, and clot retraction. IL-37 inhibited thrombus formation in vivo in a FeCl3-injured mesenteric arteriole thrombosis mouse model and ex vivo in a microfluidic whole-blood perfusion assay. Mechanistic studies using platelet-specific IL-1R8-deficient mice revealed that IL-37 bound to platelet IL-1R8 and IL-18Rα, and IL-1R8 deficiency impaired the inhibitory effects of IL-37 on platelet activation. Using PTEN (phosphatase and tensin homolog)-specific inhibitor and PTEN-deficient platelets, we found that IL-37 combined with IL-1R8 to enhance PTEN activity, inhibit Akt (protein kinase B), mitogen-activated protein kinases, and spleen tyrosine kinase pathways, as well as decrease the generation of reactive oxygen species to regulate platelet activation. Exogenous IL-37 injection suppressed microvascular thrombosis to protect against myocardial injury in wild-type mice but not in platelet-specific IL-1R8-deficient mice after permanent ligation of the left anterior descending coronary. Finally, a negative correlation between plasma IL-37 concentration and platelet aggregation was observed in patients with myocardial infarction. IL-37 directly attenuated platelet activation, thrombus formation, and myocardial injury via IL-1R8 receptor. Accumulated IL-37 in plasma inhibited platelet activation to ameliorate atherothrombosis and infarction expansion, and thus may have therapeutic advantages as potential antiplatelet drugs.

Platelet αIIbβ3 integrin binds to SARS-CoV-2 spike protein of alpha strain but not wild type and omicron strains

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 causes a pandemic infectious disease, Coronavirus disease 2019 (COVID-19). It causes respiratory infection. Then, it progresses into a systemic infection by involving other organs. This progression mechanism remains to be elucidated, although thrombus formation plays an important role in its progression. Platelets is involved in the thrombus formation by aggregating each other through association of activated αIIbβ3 integrin with the Arg-Gly-Asp (RGD) motif-containing its ligands such as fibrinogen and von Willebrand factor. SARS-CoV-2 enters host cells through association of the spike protein (S-protein) with its receptor, angiotensin-converting enzyme 2 (ACE-2), on the host cells. While presence of ACE2 in platelets is suspicious, S-protein harbors the RGD sequences within its receptor binding domain. Therefore, it could be possible SARS-CoV-2 enter platelets through association of S-protein with αIIbβ3. In this study, we found that receptor binding domain of S-protein of WT SARS-CoV-2 strain barely bound to isolated healthy human platelets. In contrast, highly toxic alpha-strain-based N501Y substitution strongly bound to platelets in a RGD dependent manner, although binding of S protein did not induce platelet aggregation or activation. This binding may serve for transferring the infection to systemic organs.

Activation of Hippo Pathway Proteins in Thrombin-Stimulated Platelets and Regutates Platelets Activation in Vitro

To investigate the phosphorylation levels of Hippo pathway proteins in thrombin stimulated platelets and to explore its effects on platelet activation. The phosphorylation levels of Hippo pathway proteins - Mammalian STE20-like kinase 1/2 (MST1/2), Nuclear Dbf2 related kinase 1/2 (NDR1/2) and Mps one binder 1(MOB1) in human thrombin stimulated platelets was detected by Western blot. The effect of MST1/2 inhibitor XMU-MP-1 on platelet aggregation was detected by Platelet Aggregometer. The effect of XMU-MP-1 on platelet integrin αIIbβ3 activation and CD62p expression was detected by flow cytometry. The effect of XMU-MP-1 on the "outside-in" signal of platelet integrin was detected by blood clot retraction test. The effects of XMU-MP-1 on platelet Hippo pathway proteins and p38 phosphorylation levels was detected by Western blot. The phosphorylation levels of MST1/2, NDR1/2 and MOB1 were significantly increased in thrombin activated platelets; XMU-MP-1 inhibited thrombin-induced platelet aggregation and αIIbβ3 activation, but did not affect α-granules release and clot retraction. In addition, thrombin induced phosphorylation of the Hippo proteins were decreased in XMU-MP-1 treated platelets, while the phosphorylation of p38 was not affected. In thrombin stimulated platelets, Hippo pathway proteins were activated and contributed to platelets activation.

Alterations in platelet proteome signature and impaired platelet integrin αIIbβ3 activation in patients with COVID-19

BackgroundPatients with COVID-19 are at increased risk of thrombosis, which is associated with altered platelet function and coagulopathy, contributing to excess mortality. ObjectivesTo characterize the mechanism of altered platelet function in COVID-19 patients. MethodsThe platelet proteome, platelet functional responses, and platelet-neutrophil aggregates were compared between patients hospitalized with COVID-19 and healthy control subjects using tandem mass tag proteomic analysis, Western blotting, and flow cytometry. ResultsCOVID-19 patients showed a different profile of platelet protein expression (858 altered of the 5773 quantified). Levels of COVID-19 plasma markers were enhanced in the platelets of COVID-19 patients. Gene ontology pathway analysis demonstrated that the levels of granule secretory proteins were raised, whereas those of platelet activation proteins, such as the thrombopoietin receptor and protein kinase Cα, were lowered. Basally, platelets of COVID-19 patients showed enhanced phosphatidylserine exposure, with unaltered integrin αIIbβ3 activation and P-selectin expression. Agonist–stimulated integrin αIIbβ3 activation and phosphatidylserine exposure, but not P-selectin expression, were decreased in COVID-19 patients. COVID-19 patients had high levels of platelet-neutrophil aggregates, even under basal conditions, compared to controls. This association was disrupted by blocking P-selectin, demonstrating that platelet P-selectin is critical for the interaction. ConclusionsOverall, our data suggest the presence of 2 platelet populations in patients with COVID-19: one of circulating platelets with an altered proteome and reduced functional responses and another of P-selectin-expressing neutrophil–associated platelets. Platelet–driven thromboinflammation may therefore be one of the key factors enhancing the risk of thrombosis in COVID-19 patients.

Fatty acid oxidation fuels agonist-induced platelet activation and thrombus formation: Targeting β-oxidation of fatty acids as an effective anti-platelet strategy.

Platelet mitochondria possess remarkable plasticity for oxidation of energy substrates, where metabolic dependency on glucose or fatty acids is higher than glutamine. Since platelets metabolize nearly the entire pool of glucose to lactate rather than fluxing through mitochondrial tricarboxylic acid cycle, we posit that majority of mitochondrial ATP, which is essential for platelet granule secretion and thrombus formation, is sourced from oxidation of fatty acids. We performed a comprehensive analysis of bioenergetics and function of stimulated platelets in the presence of etomoxir, trimetazidine and oxfenicine, three pharmacologically distinct inhibitors of β-oxidation. Each of them significantly impaired oxidative phosphorylation in unstimulated as well as thrombin-stimulated platelets leading to a small but consistent drop in ATP level in activated cells due to a lack of compensation from glycolytic ATP. Trimetazidine and oxfenicine attenuated platelet aggregation, P-selectin externalization and integrin αIIb β3 activation. Both etomoxir and trimetazidine impeded agonist-induced dense granule release and platelet thrombus formation on collagen under arterial shear. The effect of inhibitors on platelet aggregation and dense granule release was dose- and incubation time- dependent with significant inhibition at higher doses and prolonged incubation times. Neither of the inhibitors could protect mice from collagen-epinephrine-induced pulmonary embolism or prolong mouse tail bleeding times. However, mice pre-administered with etomoxir, trimetazidine and oxfenicine were protected from ferric chloride-induced mesenteric thrombosis. In conclusion, β-oxidation of fatty acids sustains ATP level in stimulated platelets and is therefore essential for energy-intensive agonist-induced platelet responses. Thus, fatty acid oxidation may constitute an attractive therapeutic target for novel antiplatelet agents.

Artificial cerebrospinal fluid restores aspirin-inhibited physiological hemostasis through recovery of platelet aggregation function.

Optimal hemostasis provides safety and reliability during neurosurgery which improves surgical outcomes. Previously, artificial cerebrospinal fluid (aCSF) and its component sodium bicarbonate were found to facilitate physiological hemostasis by amplifying platelet aggregation. This study aimed to verify whether aCSF amplifies platelet-dependent hemostasis in the presence of antiplatelet agents. We prepared platelet-rich plasma (PRP) or washed platelets using aspirin (acetylsalicylic acid, (ASA)) or normal saline (NS). We evaluated samples treated with a commercially available aCSF solution or NS for amplification of aggregation, activation of integrin αIIbβ3, phosphatidylserine (PS) exposure, P-selectin (CD62P) expression, and formation of microparticles (MPs). We assessed the effect of aCSF on in vivo hemostasis in the presence of ASA by measuring the tail bleeding time in ASA-or NS-injected C57BL/6N mice. Compared with NS, aCSF amplified ASA-inhibited platelet aggregation by recovering platelet activation including PS exposure, MP release, CD62P expression, and integrin αIIbβ3 activation. When using washed platelets, aCSF almost completely counteracted the inhibition of platelet aggregation by ASA. Prolonged bleeding time from the amputated tail of ASA-injected mice was significantly shortened by the treatment with aCSF compared to NS. Sodium bicarbonate also directly amplified ASA-inhibited platelet aggregation. aCSF and sodium bicarbonate facilitate physiological hemostasis through the recovery of inhibited platelet aggregation even in the presence of ASA. The utilization of aCSF in the operative field may be advantageous for facilitating hemostasis in patients with impaired platelet function and contribute to improving outcomes of neurosurgery.

Heparin and heparin proteoglycan-mimetics activate platelets via PEAR1 and PI3Kβ

BackgroundPlatelet endothelial aggregation receptor 1 (PEAR1) is a single-transmembrane orphan receptor primarily expressed on platelets and endothelial cells. Genetic variants of PEAR1 have repeatedly and independently been identified to be associated with cardiovascular diseases, including coronary artery disease. ObjectivesWe have identified sulfated fucoidans and their mimetics as ligands for PEAR1 and proposed that its endogenous ligand is a sulfated proteoglycan. The aim of this study was to test this hypothesis. MethodsA heparin proteoglycan-mimetic (HPGM) was created by linking unfractionated heparin (UFH) to albumin. The ability of the HPGM, UFH and selectively desulfated heparins to stimulate platelet aggregation and protein phosphorylation was investigated. Nanobodies against the 12th to 13th epidermal growth factor-like repeat of PEAR1 and phosphoinositide 3-kinase (PI3K) isoform-selective inhibitors were tested for the inhibition of platelet activation. ResultsWe show that HPGM, heparin conjugated to an albumin protein core, stimulates aggregation and phosphorylation of PEAR1 in washed platelets. Platelet aggregation was abolished by an anti-PEAR1 nanobody, Nb138. UFH stimulated platelet aggregation in washed platelets, but desulfated UFH did not. Furthermore, HPGM, but not UFH, stimulated maximal aggregation in platelet-rich plasma. However, both HPGM and UFH increased integrin αIIbβ3 activation in whole blood. By using PI3K isoform-selective inhibitors, we show that PEAR1 activates PI3Kβ, leading to Akt phosphorylation. ConclusionOur findings reveal that PEAR1 is a receptor for heparin and HPGM and that PI3Kβ is a key signaling molecule downstream of PEAR1 in platelets. These findings may have important implications for our understanding of the role of PEAR1 in cardiovascular disease.

Mammalian STE20-like kinase-1/2 are activated in human platelets stimulated by collagen or thrombin and play a vital role in collagen-activated platelets

IntroductionMammalian ste20-like kinases-1/2 (MST1/2), the core kinases of the Hippo pathway, play critical roles in the biology of hematopoietic cells via noncanonical mechanisms and contributes to megakaryocyte differentiation, polyploidization, and maturation to produce platelets. However, the role of MST1/2 in platelet functions remains unclear. Materials and methodsIn this study, we investigated this topic by determining platelet aggregation and through flow cytometry, ATP release assay, clot retraction assay, and immunoblotting analysis. ResultsWe found that MST1/2 were rapidly phosphorylated and activated upon platelet stimulation by thrombin and collagen. XMU-MP-1, a specific inhibitor of MST1/2, blocks the activation of MST1/2 in platelets. Inhibitor-pretreated platelets showed impaired platelet aggregation and dense-granule secretion mediated by collagen, thrombin, and U46619, whereas ristocetin or ADP mediated platelet aggregation was unaffected by XMU-MP-1. Although platelet-mediated clot retraction was not affected by MST1/2 inhibitors, integrin αIIbβ3 activation was significantly attenuated in XMU-MP-1-treated platelets. Moreover, MST1/2 inhibition significantly attenuated the mobilization of platelet calcium ions and the secretion of α-granules induced by convulxin. ConclusionsThis study is the first to demonstrate that MST1/2 play vital roles in human platelets and contributes to collagen-induced platelet activation and aggregation.

A familial case of MYH9 gene mutation associated with multiple functional and structural platelet abnormalities

Mutations in the MYH9 gene result in macrothrombocytopenia often associated with hemorrhages. Here, we studied the function and structure of platelets in three family members with a heterozygous mutation R1933X in the MYH9 gene, characteristic of closely related disorders known as the May-Hegglin anomaly and Sebastian syndrome. The examination included complete blood count, blood smear microscopy, platelet flow cytometry (expression of P-selectin and active integrin αIIbβ3 before and after activation), the kinetics of platelet-driven contraction (retraction) of blood clots, as well as scanning/transmission electron microscopy of platelets. Despite severe thrombocytopenia ranging (36–86) × 109/l, none of the patients had hemorrhages at the time of examination, although they had a history of heavy menstruation, spontaneous ecchymosis, and postpartum hemorrhage. Flow cytometry showed background platelet activation, revealed by overexpression of P-selectin and active αIIbβ3 integrin above normal levels. After TRAP-induced stimulation, the fractions of platelets expressing P-selectin in the proband and her sister were below normal response, indicating partial platelet refractoriness. The initiation of clot contraction was delayed. Electron microscopy revealed giant platelets with multiple filopodia and fusion of α-granules with dilated open canalicular system, containing filamentous and vesicular inclusions. The novel concept implies that the R1933X mutation in the MYH9 gene is associated not only with thrombocytopenia, but also with qualitative structural and functional defects in platelets. Platelet dysfunction includes impaired contractility, which can disrupt the compaction of hemostatic clots, making the clots weak and permeable, therefore predisposing patients with MYH9 gene mutations to the hemorrhagic phenotype.

Deletion of 12-Lipoxygenase Improves Platelet Function after Storage and Transfusion in Thrombocytopenic Mice

Introduction Human platelets are stored for up to 7 days for transfusion. During storage, platelets undergo numerous detrimental functional changes. Results from two recent randomized clinical trials challenge the hemostatic efficacy of stored platelets for transfusion. In a recent study in healthy humans, increased 12-HETE levels were associated with reduced platelet performance in vivo. In the current study, we sought to understand how genetic deletion of 12-LOX (the enzyme that generates 12-HETE) affects platelets during storage, before, and after transfusion in vitro and in mouse models. Methods and Results: We obtained platelets from wild-type (WT) and 12-LOX-/- mice and performed storage studies for 24 and 48 hours. Using LC-MS/MS-MRM, we showed that ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) increased significantly in stored platelets from 12-LOX-/- mice, while oxylipins were significantly lower in 12-LOX-/- platelets than in WT platelets. The ω-6/ω-3 PUFA ratio decreased significantly in stored 12-LOX-/- compared to stored WT platelets, highlighting a pronounced increase in ω-3 PUFAs. The circulation time of stored 12-LOX-/- platelets did not differ from stored WT platelets. Baseline, pre-transfusion αIIbβ3 integrin activation was significantly lower before and after 24 hours of storage in 12-LOX-/- platelets than in WT platelets. Surprisingly, once stored platelets were transfused, we observed significantly increased baseline αIIbβ3 integrin activation in 12-LOX-/- platelets than in WT platelets. To test whether this translates into better in vivo function, we exposed 24-hour stored platelets to an in vivo model of thrombosis and hemostasis. In brief, hIL4R-TG mice express the receptor for human interleukin 4 instead of GPIbα on the platelet surface. hIL4R-TG mice were rendered thrombocytopenic with an anti-human IL4R antibody. We transfused 24-hour stored WT, or 12-LOX-/- platelets, to thrombocytopenic hIL4R-TG mice, injured the carotid artery by FeCl3, and recorded the arterial blood flow. Indeed, stored 12-LOX-/- platelets had better in vivo function, as evidenced by more frequent and significantly faster vessel occlusions with stored and transfused 12-LOX-/- platelets than transfusion of stored WT platelets. Importantly, stored and transfused 12-LOX-/- occluded vessels in a similar time as normocytic WT mice. Untransfused, thrombocytopenic hIL4R-TG mice and untransfused, normocytic hIL4R-TG mice did not show vessel occlusion. Conclusion Deleting 12-LOX improves the post-transfusion function of stored murine platelets. Pharmacologic inhibition of 12-LOX or dietary alterations of ω-3 and ω-6 PUFAs could significantly enhance human platelet quality and function after storage. Future studies must determine the feasibility and safety of 12-LOX inhibition in stored and transfused human platelets.

Platelet ERO1α Is Crucial for Ca2+ Mobilization and Platelet Activation in Atherothrombosis

Endoplasmic reticulum oxidoreductase 1α (ERO1α) is a key oxidase of protein disulfide isomerase (PDI) during protein folding in the ER. Platelet ERO1α was reported to interact with PDI and αIIbβ3 integrin and regulate platelet function. However, the regulatory mechanism and pathophysiological role remain unknown. In the present study, we demonstrate that platelet ERO1α orchestrates intracellular Ca2+ signaling during platelet activation and contributes to thrombogenesis. Intravital microscopy using megakaryocyte-specific Ero1α conditional knockout (CKO) and KO mice reveals that platelet ERO1α is crucial for platelet thrombus formation in mouse models of laser-induced cremaster arteriolar thrombosis and FeCl3-induced carotid arterial thrombosis. Deletion of Ero1α does not affect bleeding times and blood loss at the site of tail amputation, implying that ERO1α is not required for hemostasis. Studies using Ero1α-null platelets, novel function-blocking antibodies, and small-molecule inhibitors reveal that intracellular, but not extracellular, ERO1α promotes P-selectin exposure, αIIbβ3 integrin activation, and platelet aggregation induced by multiple agonists, such as thrombin and collagen-related peptide. Importantly, the regulatory function of ERO1α is independent of PDI activity. Mass spectrometry and immunoprecipitation assays identify stromal interaction molecule 1 (STIM1) and sarco/endoplasmic reticulum Ca2+-ATPase 2 (SERCA2) as ERO1α binding partners. We have found that ERO1α-STIM1 binding is enhanced during platelet activation, whereas ERO1α-SERCA2 binding is reduced. Intriguingly, platelet ERO1α interacts with STIM1 through Cys49 and Cys56 residues and promotes its function by oxidizing the Cys49-Cys56 disulfide bond, contributing to Ca2+ store content and enhancing Ca2+ mobilization during platelet activation. Using a high throughput screen, we identify a small-molecule ERO1α inhibitor, B20, which inhibits ERO1α activity with an IC50 value of 10.0 μM but does not affect the activities of H2O2 and PDI. Treatment of platelets with B20 recapitulates the functional defects as seen in Ero1α-null platelets. Intravenous injection of B20 and eptifibatide, an αIIbβ3 integrin antagonist, into WT mice significantly inhibits platelet thrombus formation following laser- or FeCl3-induced vascular injury. While treatment of mice with eptifibatide markedly prolongs tail bleeding times and causes blood loss, B20 does not impair hemostatic function. Furthermore, treatment with B20 reduces the infarct volume in ischemic stroke. These results indicate that platelet ERO1α is a novel regulator of Ca2+ signaling and promotes platelet activation and aggregation in a PDI-independent manner, contributing to the pathogenesis of arterial thrombosis and thromboinflammation. Targeting ERO1α may be an attractive therapeutic strategy for treating patients with thrombotic disease.

Coagulation Factor XII Is Activated Via Noncanonical Binding to Platelet Integrin αIIbβ3

Coagulation factor XII (FXII) has attracted significant interest as a potentially transformative antithrombotic drug target, with preclinical models consistently showing that FXII deletion or blockade protects against thrombosis without causing hemorrhage. A long-standing observation has been that stimulated platelets can support activation of FXII, thereby linking platelet function to FXIIa generation and thrombus formation. However, the mechanism by which FXII is recruited and activated at the platelet surface remains unclear and a source of considerable controversy. In order to explore the FXII-platelet interaction, we performed a mass spectrometry-based proteomic screen of potential FXII binding partners in the solubilized membrane fraction of SFLLRN-activated platelets. FXII co-immunoprecipitated with integrins αIIb and β3, with strong signals also observed for several integrin-associated proteins. These results were corroborated by Western blot of FXII co-immunoprecipitation with washed stimulated platelets and pulldown of FXII from dilute human plasma by beads coated with activated integrin αIIbβ3. Using inactive integrin from resting platelet lysate, we found that only by inducing the activated conformation of αIIbβ3 with MnCl2 could the receptor bind FXII. When flow cytometry was performed on K562 cell lines stably transfected with integrin αIIbβ3, αvβ3, or αvβ1, only those expressing αIIbβ3 in the presence of MnCl2 supported binding of FXII-FITC (Figure 1). We next evaluated the need for αIIbβ3 to support platelet-dependent FXIIa generation. Platelets from integrin β3 knockout mice (ITGB3-/-) demonstrated significantly decreased binding to FXII-FITC by flow cytometry, and ITGB3-/- platelet-rich plasma (PRP) stimulated with PAR4 agonist peptide failed to generate FXIIa in a chromogenic assay compared to PRP from wild-type littermate controls. Similarly, platelets from a patient with congenital biallelic loss of integrin αIIb (Glanzmann's thrombasthenia) also failed to bind or activate FXII compared to platelets from healthy donors. To further interrogate the role of αIIbβ3 in FXII activation, we recombinantly generated the soluble αIIbβ3 ectodomain (rED). Bio-layer interferometry (Octet) steady state analysis showed that FXII bound the rED with KD=16 nM. We found that rED greatly enhanced the rate of FXIIa generation in solution (Figure 2), with an increase of 133-fold in Kcat/Km and 203-fold in Vmax measured for FXII catalytic activity. Further, when rED was added to FXII in the presence of factor XI (FXI), we found a significant enhancement in FXIIa-dependent activation of FXI. All known physiologic ligands of αIIbβ3 contain an RGD motif, which is required for binding to the integrin. However, FXII does not have an RGD or equivalent sequence. We sought to better understand the potentially non-canonical nature of FXII-αIIbβ3 binding. We found that PAC-1 antibody, which blocks RGD-dependent macromolecular ligand binding to αIIbβ3, inhibited binding of fibrinogen-FITC to activated human platelets but failed to block FXII-FITC binding. We next studied the impact of two αIIbβ3 inhibitors on the FXII-integrin interaction. Eptifibatide blocks RGD-dependent ligand binding to αIIbβ3 and induces a large structural change in the receptor, causing it to assume an "extended open" conformation. By contrast, antibody 10E5 blocks ligand binding without inducing a conformational change in αIIbβ3. In a solid-phase assay, 10E5 did not inhibit binding of FXII to αIIbβ3, whereas eptifibatide partially blocked binding, consistent with an allosteric effect mediated by the transition to the extended open conformation. Likewise, eptifibatide partially inhibited FXIIa generation by stimulated PRP and by purified αIIbβ3, while 10E5 had no effect in either assay. Our data are consistent with a model in which non-canonical binding of FXII to integrin αIIbβ3 is responsible for FXII activation at the platelet surface. Future studies will be directed at dissecting the molecular mechanism of FXII activation by αIIbβ3 and determining whether non-RGD dependent binding may generalize to other ligands of the integrin. Taken together, our work contributes to a novel understanding of pathologic FXIIa generation during platelet activation and thrombus formation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Identification of Receptors and Signaling Pathways Mediating the Platelet Contribution to Clotting Parameters in Thromboelastography (TEG)

Viscoelastic testing is used for real-time determination of hemostatic status in patients with acute risk of bleeding. Clot formation and contraction in thromboelastography (TEG) are assumed to be driven by thrombin through its two main actions, the conversion of fibrinogen to fibrin and activation of platelets through protease activated receptors (PARs). Platelet stimulation leads to inside-out activation of αIIbβ3 integrin, a process regulated by the small GTPase Rap1, and fibrin binding to activated αIIbβ3 triggers outside-in signaling and platelet contraction. However, studies have shown no impact of PAR inhibitors on viscoelastic clotting parameters, and the impact of defects in Rap1 signaling on TEG have not been determined. Furthermore, the role of novel ligand-receptor interactions such as fibrin-GPVI on TEG parameters have not been studied. Here we investigated the specific receptors and signaling pathways required for platelet function in standard (kaolin-triggered) TEG using genetic or pharmacological inhibition of platelet proteins in murine and human blood samples. We confirmed the requirement of platelets and αIIbβ3 integrin for mouse whole blood clot formation/contraction in TEG. Loss of the integrin adaptor Talin1 in mouse platelets impaired clotting parameters, but to a lesser extent than αIIbβ3 blockade. We observed no changes in clotting parameters using blood from mice lacking Rap1b, or with combined deficiency of CalDAG-GEFI and P2Y12, the two main pathways mediating Rap1 activation. Deficiency of PAR4, which fully mediates the platelet response to thrombin in mice, only partially reduced the speed of clot formation (α-angle) with no impact on maximal clot strength (maximal amplitude, MA). However, TEG parameters similar to samples lacking platelets were observed for blood from PAR4-deficient mice treated with inhibitors of the GPVI receptor or a key downstream signaling protein Syk. These results were reproduced in human blood samples, where inhibition of both PAR1 and PAR4 combined with Syk inhibition robustly reduced α-angle and MA. In summary, our results suggest that global hemostasis assays such as TEG, which are driven by high concentrations of thrombin, are not sensitive to platelet signaling pathways critical for integrin inside-out activation. Furthermore, we provide evidence that platelet-mediated clot contraction in TEG is dependent on both the thrombin-PAR interaction and GPVI, likely interacting with fibrin. Our findings suggest that current interpretation of platelet function in TEG may not reflect platelet function in vivo, and that novel interactions need to be integrated into clinical assays.

A gain‐of‐function filamin A mutation in mouse platelets induces thrombus instability

BackgroundFilaminopathies A are rare disorders affecting the brain, intestine, or skeleton, characterized by dominant X‐linked filamin A (FLNA) gene mutations. Macrothrombocytopenia with functionally defective platelets is frequent. We have described a filaminopathy A male patient, exhibiting a C‐terminal frame‐shift FLNa mutation (Berrou et al., Arterioscler Thromb Vasc Biol. 2017;37:1087–1097). Contrasting with female patients, this male patient exhibited gain of platelet functions, including increased platelet aggregation, integrin αIIbβ3 activation, and secretion at low agonist concentration, raising the issue of thrombosis risk. ObjectivesOur goal is to assess the thrombotic potential of the patient FLNa mutation in an in vivo model. MethodsWe have established a mutant FlnA knock‐in mouse model. ResultsThe mutant FlnA mouse platelets phenocopied patient platelets, showing normal platelet count, lower expression level of mutant FlnA, and gain of platelet functions: increased platelet aggregation, secretion, and αIIbβ3 activation, as well as increased spreading and clot retraction. Surprisingly, mutant FlnA mice exhibited a normal bleeding time, but with increased re‐bleeding (77%) compared to wild type (WT) FlnA mice (27%), reflecting hemostatic plug instability. Again, in an in vivo thrombosis model, the occlusion time was not altered by the FlnA mutation, but arteriolar embolies were increased (7‐fold more frequent in mutant FlnA mice versus WT mice), confirming thrombus instability. ConclusionsThis study shows that the FlnA mutation found in the male patient induced gain of platelet functions in vitro, but thrombus instability in vivo. Implications for the role of FLNa in physiology of thrombus formation are discussed.

Clot‐targeted magnetic hyperthermia permeabilizes blood clots to make them more susceptible to thrombolysis

BackgroundThrombolysis is a frontline treatment for stroke, which involves the application of tissue plasminogen activator (tPA) to trigger endogenous clot‐degradation pathways. However, it is only effective within 4.5 h of symptom onset because of clot contraction preventing tPA permeation into the clot. Magnetic hyperthermia (MH) mediated by tumor‐targeted magnetic nanoparticles is used to treat cancer by using local heat generation to trigger apoptosis of cancer cells. ObjectivesTo develop clot‐targeting magnetic nanoparticles to deliver MH to the surface of human blood clots, and to assess whether this can improve the efficacy of thrombolysis of contracted blood clots. MethodsClot‐targeting magnetic nanoparticles were developed by functionalizing iron oxide nanoparticles with an antibody recognizing activated integrin αIIbβ3 (PAC‐1). The magnetic properties of the PAC‐1‐tagged magnetic nanoparticles were characterized and optimized to deliver clot‐targeted MH. ResultsClot‐targeted MH increases the efficacy of tPA‐mediated thrombolysis in contracted human blood clots, leading to a reduction in clot weight. MH increases the permeability of the clots to tPA, facilitating their breakdown. Scanning electron microscopy reveals that this effect is elicited through enhanced fibrin breakdown and triggering the disruption of red blood cells on the surface of the clot. Importantly, endothelial cells viability in a three‐dimensional blood vessel model is unaffected by exposure to MH. ConclusionsThis study demonstrates that clot‐targeted MH can enhance the thrombolysis of contracted human blood clots and can be safely applied to enhance the timeframe in which thrombolysis is effective.

Reversible Platelet Integrin αIIbβ3 Activation and Thrombus Instability.

Integrin αIIbβ3 activation is essential for platelet aggregation and, accordingly, for hemostasis and arterial thrombosis. The αIIbβ3 integrin is highly expressed on platelets and requires an activation step for binding to fibrinogen, fibrin or von Willebrand factor (VWF). A current model assumes that the process of integrin activation relies on actomyosin force-dependent molecular changes from a bent-closed and extended-closed to an extended-open conformation. In this paper we review the pathways that point to a functional reversibility of platelet αIIbβ3 activation and transient aggregation. Furthermore, we refer to mouse models indicating that genetic defects that lead to reversible platelet aggregation can also cause instable thrombus formation. We discuss the platelet agonists and signaling pathways that lead to a transient binding of ligands to integrin αIIbβ3. Our analysis points to the (autocrine) ADP P2Y1 and P2Y12 receptor signaling via phosphoinositide 3-kinases and Akt as principal pathways linked to reversible integrin activation. Downstream signaling events by protein kinase C, CalDAG-GEFI and Rap1b have not been linked to transient integrin activation. Insight into the functional reversibility of integrin activation pathways will help to better understand the effects of antiplatelet agents.

A Novel GATA1 Variant in the C-Terminal Zinc Finger Compared with the Platelet Phenotype of Patients with A Likely Pathogenic Variant in the N-Terminal Zinc Finger.

The GATA1 transcription factor is essential for normal erythropoiesis and megakaryocytic differentiation. Germline GATA1 pathogenic variants in the N-terminal zinc finger (N-ZF) are typically associated with X-linked thrombocytopenia, platelet dysfunction, and dyserythropoietic anemia. A few variants in the C-terminal ZF (C-ZF) domain are described with normal platelet count but altered platelet function as the main characteristic. Independently performed molecular genetic analysis identified a novel hemizygous variant (c.865C>T, p.H289Y) in the C-ZF region of GATA1 in a German patient and in a Spanish patient. We characterized the bleeding and platelet phenotype of these patients and compared these findings with the parameters of two German siblings carrying the likely pathogenic variant p.D218N in the GATA1 N-ZF domain. The main difference was profound thrombocytopenia in the brothers carrying the p.D218N variant compared to a normal platelet count in patients carrying the p.H289Y variant; only the Spanish patient occasionally developed mild thrombocytopenia. A functional platelet defect affecting αIIbβ3 integrin activation and α-granule secretion was present in all patients. Additionally, mild anemia, anisocytosis, and poikilocytosis were observed in the patients with the C-ZF variant. Our data support the concept that GATA1 variants located in the different ZF regions can lead to clinically diverse manifestations.

Circadian nuclear receptor Rev-erbalpha is expressed by platelets and potentiates platelet activation and thrombus formation

Abstract Background Adverse cardiovascular events have day/night patterns with peaks in the morning, potentially related to endogenous circadian clock control of platelet activation. Circadian nuclear receptor Rev-erbα is an essential and negative component of the circadian clock. Purpose We aim to investigate the expression profile and biological function of Rev-erbα in platelets. Methods and results Here we report the presence and functions of circadian nuclear receptor Rev-erbα in human and mouse platelets. Both human and mouse platelet Rev-erbα showed a circadian rhythm that positively correlated with platelet aggregation. Global Rev-erbα knockout and platelet-specific Rev-erbα knockout mice exhibited defective in hemostasis as assessed by prolonged tail-bleeding times. Rev-erbα deletion also reduced ferric chloride-induced carotid arterial occlusive thrombosis, prevented collagen/epinephrine-induced pulmonary thromboembolism, and protected against microvascular microthrombi obstruction and infarct expansion in an acute myocardial infarction model. In vitro thrombus formation assessed by CD41-labeled platelet fluorescence intensity was significantly reduced in Rev-erbα knockout mouse blood. Platelets from Rev-erbα knockout mice exhibited impaired agonist-induced aggregation responses, integrin αIIbβ3 activation and α-granule release. Consistently, pharmacological inhibition of Rev-erbα by specific antagonists decreased platelet activation markers in both mouse and human platelets. Mechanistically, mass spectrometry and co-immunoprecipitation analyses revealed that Rev-erbα potentiated platelet activation via oligophrenin-1-mediated RhoA/ERM (ezrin/radixin/moesin) pathway. Conclusion We provide the first evidence that circadian protein Rev-erbα is functionally expressed in platelets and potentiates platelet activation and thrombus formation. Rev-erbα may serve as a novel therapeutic target for managing thrombosis-based cardiovascular disease. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): This work was supported by grants from the National Science Fund for Distinguished Young Scholars (81625002), the National Natural Science Foundation of China (81930007).