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Platelets are essential for hemostasis and vascular integrity. Platelets recognize dengue virus through the DC-SIGN receptor. Upon pathogen recognition, platelets rapidly modulate the expression of adhesion molecules to trigger immune cell interactions and regulate the immune response. In this study, we aimed to examine the expression levels of three molecules, CD151, CD154, and CD148 on platelets in dengue patients. A significantly increased expression of CD154 and reduced expression of CD148 was observed in dengue patients compared to healthy subjects (p < 0.0001). Moreover, a strong positive correlation between CD148 and CD41/CD61 (p < 0.0001) was noted in dengue patients. In summary, we demonstrated the altered expressions of CD154 and CD148 on platelets in dengue patients that may play a crucial role in dengue pathogenesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Objective: Intrahepatic cholangiocarcinoma (iCCA) is a highly lethal hepatobiliary malignancy with an increasing incidence annually. Extensive research has elucidated the existence of a reciprocal interaction between platelets and cancer cells, which promotes tumor proliferation and metastasis. This study aims to investigate the function and mechanism underlying iCCA progression driven by the interplay between platelets and tumor cells, aiming to provide novel therapeutic strategies for iCCA., Methods: The associations between platelets and cancer development were investigated by analyzing the peripheral blood platelet count, degree of platelet activation and infiltration in the microenvironment of patients with iCCA. By co-culturing tumor cells with platelets, the influence of platelet stimulation on the epithelial-mesenchymal transition (EMT), proliferation, and metastasis of iCCA cells was assessed through in vitro and in vivo experiments. Quantitative proteomic profiling was conducted to identify key downstream targets that were altered in tumor cells following platelet stimulation. The RNA interference technique was utilized to investigate the impacts of gene silencing on the malignant biological behaviors of tumor cells., Results: Compared with healthy adults, patients with iCCA presented significantly higher levels of peripheral blood platelet counts, platelet activation and infiltration degrees, which were also found to be correlated with patient prognosis. Platelet stimulation greatly facilitated the EMT of iCCA cells, leading to enhanced proliferative and metastatic capabilities. Mechanistically, proteomic profiling identified a total of 67 up-regulated and 40 down-regulated proteins in iCCA cells co-cultured with platelets. Among these proteins, two elevated targets ILK and ITGB3, were further demonstrated to be partially responsible for platelet-induced iCCA progression, which might depend on their regulatory effects on FAK/PI3K/AKT signaling transduction., Conclusions: Our data revealed that platelet-related indices were abnormally ascendant in iCCA patients compared to healthy adults. Co-culturing with platelets enhanced the progression of EMT, and the motility and viability of iCCA cells in vitro and in vivo. Proteomic profiling discovered that platelets promoted the development of iCCA through FAK/PI3K/AKT pathway by means of elevating the expression of ILK and ITGB3, indicating that both proteins are promising therapeutic targets for iCCA with the guidance of platelet-related indices., Competing Interests: Declarations. Ethics approval and consent to participate: Human iCCA tissues were procured from patients who underwent surgical resection of the bile duct at the Department of Biliary-Pancreatic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China). All patients received prior informed consent. Human sample research involved in this study was approved by the Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. All animal studies in this work were approved by the Laboratory Animal Welfare and Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (Ethical number: TJH-202206004). And the maximal subcutaneous tumor size was not exceeded the standard permitted by the ethics committee. Consent for publication: The authors declare that they give consent for publication with the contents of this article. Competing interests: The authors declare no conflict of interest., (© 2024. The Author(s).)

Background: Platelets prevent bleeding in a variety of inflammatory settings, the adhesion receptors and activation pathways involved being highly context-dependent and functionally redundant. In some situations, platelets recruited to inflammatory sites act independently of aggregation. The mechanisms underlying stable platelet adhesion in inflamed microvessels remain incompletely understood, in particular, whether and if so, how β1 and β3 integrins are involved., Methods: The impact of isolated or combined platelet deficiency in β1 and β3 integrins on inflammation-associated hemostasis was investigated in 3 models of acute inflammation: immune complex-based cutaneous reverse passive Arthus reaction, intranasal lipopolysaccharide-induced lung inflammation, and cerebral ischemia-reperfusion following transient (2-hour) occlusion of the middle cerebral artery., Results: Mice with platelet-directed inactivation of Itgb1 (PF4Cre-β1 -/- ) displayed no bleeding in any of the inflammation models, while mice defective in platelet Itgb3 (PF4Cre-β3 -/- ) exhibited bleeding in all 3 models. Remarkably, the bleeding phenotype of PF4Cre-β3 -/- mice was exacerbated in the reverse passive Arthus model by the concomitant deletion of β1 integrins, PF4Cre-β1 -/- /β3 -/- animals presenting increased bleeding. Intravital microscopy in reverse passive Arthus experiments highlighted a major defect in the adhesion of PF4Cre-β1 -/- /β3 -/- platelets to inflamed microvessels. Unlike PF4Cre-β1 -/- and PF4Cre-β3 -/- mice, PF4Cre-β1 -/- /β3 -/- animals developed early hemorrhagic transformation 6 hours after transient middle cerebral artery occlusion. PF4Cre-β1 -/- /β3 -/- mice displayed no more bleeding in lipopolysaccharide-induced lung inflammation than PF4Cre-β3 -/- animals., Conclusions: Altogether, these results show that the requirement for and degree of functional redundancy between platelet β1 and β3 integrins in inflammation-associated hemostasis vary with the inflammatory situation., Competing Interests: None.

Several investigations are being done to increase the short lifetime of mesenchymal stem cells (MSCs). One of the crucial genes involved in the immortalization of MSCs, hTERT (human telomerase reverse transcriptase), is activated in most publications using viral-based techniques. In this work, we investigated the use of platelet-derived (PMPs) and B cell precursor leukemia-derived microparticles as a nonviral method to trigger and compare the expression of the hTERT gene in MSCs. MSCs were extracted from the umbilical cord for the current investigation and identified using a flow cytometry approach and an inverted microscope. The Nalm-6 cell line and platelet concentrate were used to isolate microparticles (MPs). MSCs and MPs were cocultured for 14 days at 25-, 50-, and 100 μg/ml concentrations. qRT-PCR was used to research the expression of the hTERT gene. SPSS 26.0's t test was used to compare the outcomes. After coculture with platelet MPs, MSCs had higher levels of hTERT gene expression than the control group. In contrast, this gene's expression was concurrently decreased in MSCs exposed to MPs generated from Nalm-6. We demonstrated that following 14-day treatment, PMP significantly boosted the hTERT gene expression in MSCs, while the Nalm-6 MPs lowered the gene expression. However, additional studies are necessary due to the stability of hTERT gene expression and the immortalization of MSCs following exposure., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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Background: Macrothrombocytopenia (MTP) is a rare but enigmatic complication of Glanzmann thrombasthenia (GT), an inherited bleeding disorder caused by the absence of platelet aggregation due to deficiencies of the αIIbβ3 integrin., Objectives: We report a family with type I GT and a prolonged bleeding time but unusually associated with congenital mild thrombocytopenia and platelet size heterogeneity with giant forms., Methods and Results: Sanger sequencing of DNA from the propositus identified 2 heterozygous ITGB3 gene mutations: p.P189S and p.C210S both of which prevent αIIbβ3 expression and are causative of GT but without explaining the presence of enlarged platelets. High-throughput screening led to the detection of a predicted disease-causing heterozygous mutation in the TUBB1 gene: p.G146R, encoding β1-tubulin, a component of the platelet cytoskeleton and a gene where mutations are a known cause of MTP., Conclusions: Family screening confirmed that this rare phenotype results from oligogenic inheritance while suggesting that the GT phenotype dominates clinically., (© 2019 International Society on Thrombosis and Haemostasis.)

Antiplatelet therapy resistance against acetylsalicylic acid (ASA) and/or clopidogrel in coronary heart disease (CHD) is common with diabetes mellitus. One factor might involve platelet receptor ITGB3 gene polymorphism. We aimed to assess resistance together with platelet reactivity parameters, the polymorphism, plus diabetes type 2 coexistence. The study included 185 patients with CHD, including 58 diabetics, aged 62.3 ± 8.2 years. Patients were treated long-term with ASA, plus clopidogrel, both 75 mg/d. Platelet aggregation was measured with arachidonic acid (ASPI test; ASA-response assessment) or ADP (ADP test; clopidogrel-response assessment). Thromboxane B2 (TXB2) and fibrinogen concentrations were measured and ITGB3 PIA1>A2 variants identified. Increases in PLT, glucose and SBP were demonstrated with dual resistance or to clopidogrel. Regardless of response, diabetics (versus non-diabetics) had elevated platelet aggregation with the ADP test (P = 0.0198), higher TXB2 (P = 0.0501), BMI (P = 0.0003) and SBP (P = 0.0627). ITGB3 PIA1/A1 homozygotes had higher platelet aggregation with the ASPI test (P = 0.0513), and fibrinogen concentrations (P = 0.0133), relative to A2 allele carriers. Significant associations of diabetes with clopidogrel resistance (P = 0.0011) and PIA1/A1 homozygotes with ASA resistance (P = 0.0518) were found. Higher concentrations of TXB2 (P = 0.0223) and higher SBP (P = 0.0063) were found with diabetes (versus non-diabetic) in PIA1/A1 homozygotes. We concluded that diabetes with CHD weakens response to antiplatelet drugs, especially to clopidogrel; and hyperglycaemia, hypertension and obesity might also play an important role. Diabetics' resistance to ASA is associated with increased platelet reactivity, perhaps related to the more frequent ITGB3 PIA1 allele and increased TXB2 generation. The PIA1 allele may be a potential factor for aspirin resistance with elevated fibrinogen concentration.

Background and Objective: Glycoprotein IIIa and GPIIb constitutes are the fibrinogen receptor (integrin αIIbβ3). This glycoprotein has a fundamental role in atherothrombosis. This study aimed to detect the association of αIIbβ3 polymorphisms with atherosclerotic coronary heart disease in Sudanese patients and the association between the risk factors and platelet integrin αIIbβ3 polymorphisms., Materials and Methods: This is a case-control hospital-based study contain 50 atherosclerotic patients (>18 years) with coronary heart disease that admitted to Khartoum hospital and were compared to apparently 50 healthy Sudanese subjects at the same ages. About 5 mL of venous blood sample was collected from each patient and control. The laboratory analyses were done for HbA1c, lipid profile and for DNA genotyping., Results: LDL, HDL, HbA1c and body mass index have shown highly significant influence on patients. No significant differences were observed for triglycerides and total cholesterol levels. The risks of coronary heart disease were higher with A/B genotype in HPA3, but no association detected with coronary heart disease patients in HPA1 polymorphism., Conclusion: In conclusion, HPA3 polymorphism was associated with atherosclerotic in Sudanese patients, while HPA1 polymorphism has not.

Objectives: Shear stress can activate platelet integrin-mediated signaling that leads to shear-induced platelet aggregation (SIPA) and eventually contribute to acute myocardial infarction. The major platelet integrin αIIbβ3 is polymorphic at residue 33 [Leu33Pro (PlA) polymorphism]. The Pro33 isoform has been shown to have a prothrombotic phenotype. In this work, we studied the impact of Leu33/Pro33 polymorphism on the shear-induced integrin-mediated Src and FAK activation in platelets., Methods: Platelets of both genotypes were placed on immobilized fibrinogen or heat activated BSA and were exposed to physiological (500/s) or abnormally high (5000/s) shear rates for 2-10 min. Platelets after exposure to shear were analysed for Src pY418 and FAK pY397 activities., Results: Whereas physiological shear stress does not affect platelet signaling, abnormally high-shear stress considerably elevates Src and FAK phosphorylation in both Pro33 and Leu33 platelets. Both under static and flow conditions, Pro33 platelets exhibited a significantly higher Src and FAK activities than Leu33 platelets. Interestingly, even in the absence of the αIIbβ3-fibrinogen interaction, we could detect a shear-induced integrin-mediated signaling of Src and FAK in platelets. In parallel experiments in which platelets were pretreated with abciximab, an integrin αIIbβ3 antagonist, activation of both kinases by shear was inhibited., Conclusion: Taken together, our data indicates an important role of αIIbβ3 and shows that Leu33Pro polymorphism modulates the integrin-mediated Src and FAK signaling in platelets in response to shear stress.

Glycoprotein IIb/IIIa (GPIIb/IIIa) is the most abundant platelet surface receptor for fibrinogen and von Willebrand factor. Polymorphism PlA1/A2 in the gene of GPIIb/IIIa is among the risk factors for the development of arterial and venous thrombosis. The aim of this study is to evaluate the effect of the carriage of PlA1/A2 on the size, topographic features, and membrane stiffness of platelets from healthy controls and patients with deep venous thrombosis (DVT). Atomic force microscopy (AFM) imaging and nanoindentation (force-distance curves) were applied to investigate the morphological and nanomechanical properties (Young's modulus) of platelets immobilized on glass surface. The surface roughness ( R a ) and height ( h) of platelets from patients with DVT, carriers of mutant allele PlA2 ( R a = 30.2 ± 6 nm; h = 766 ± 182 nm) and noncarriers ( R a = 28.6 ± 6 nm; h = 865 ± 290 nm), were lower than those of healthy carriers of allele PlA2 ( R a = 48.1 ± 12 nm; h = 1072 ± 338 nm) and healthy noncarriers ( R a = 49.7 ± 14 nm; h = 1021 ± 433 nm), respectively. Platelets isolated from patients with DVT, both carriers and noncarriers, exhibit much higher degree of stiffness at the stage of spreading ( E = 327 ± 85 kPa and 341 ± 102 kPa, respectively) compared to healthy noncarriers ( E = 198 ± 50 kPa). In addition, more pronounced level of platelet activation was found in polymorphism carriers. In conclusion, the carriage of PlA2 allele modulates the activation state, morphology, and membrane elasticity of platelets.

Hemangioendothelioma (HE) is a type of angiomatous lesions that features endothelial cell proliferation. Understanding the mechanisms orchestrating HE angiogenesis can provide therapeutic insights. It has been shown that platelets can support normal and malignant endothelial cells during angiogenesis. Using the mouse endothelial-derived EOMA cell line as a model of HE, we explored the regulatory effect of platelets. We found that platelets stimulated EOMA proliferation but did not mitigate apoptosis. Furthermore, direct platelet-EOMA cell contact was required and the proliferation was mediated via integrin β3/Akt signaling in EOMA cells. SiRNA knockdown of integrin β3 and inhibition of Akt activity significantly abolished platelet-induced EOMA cell proliferation in vitro and tumor development in vivo. These results provide a new mechanism by which platelets support HE progression and suggest integrin β3 as a potential target to treat HE.

Previous studies in Chinese hamster ovary cells showed that truncational mutations of β3 at sites of F754 and Y759 mimicking calpain cleavage regulate integrin signaling. The roles of the sequence from F754 to C-terminus and the conservative N756ITY759 motif in platelet function have yet to be elaborated. Mice expressing β3 with F754 and Y759 truncations, or NITY deletion (β3-ΔTNITYRGT, β3-ΔRGT, or β3-ΔNITY) were established through transplanting the homozygous β3-deficient mouse bone marrow cells infected by the GFP tagged MSCV MigR1 retroviral vector encoding different β3 mutants into lethally radiated wild-type mice. The platelets were harvested for soluble fibrinogen binding and platelet spreading on immobilized fibrinogen. Platelet adhesion on fibrinogen- and collagen-coated surface under flow was also tested to assess the ability of the platelets to resist hydrodynamic drag forces. Data showed a drastic inhibition of the β3-ΔTNITYRGT platelets to bind soluble fibrinogen and spread on immobilized fibrinogen in contrast to a partially impaired fibrinogen binding and an almost unaffected spreading exhibited in the β3-ΔNITY platelets. Behaviors of the β3-ΔRGT platelets were consistent with the previous observations in the β3-ΔRGT knock-in platelets. The adhesion impairment of platelets with the β3 mutants under flow was in different orders of magnitude shown as: β3-ΔTNITYRGT>β3-ΔRGT>β3-ΔNITY to fibrinogen-coated surface, and β3-ΔTNITYRGT>β3-ΔNITY>β3-ΔRGT to collagen-coated surface. To evaluate the interaction of the β3 mutants with signaling molecules, GST pull-down and immunofluorescent assays were performed. Results showed that β3-ΔRGT interacted with kindlin but not c-Src, β3-ΔNITY interacted with c-Src but not kindlin, while β3-ΔTNITYRGT did not interact with both proteins. This study provided evidence in platelets at both static and flow conditions that the calpain cleavage-related sequences of integrin β3, i.e. T755NITYRGT762, R760GT762, and N756ITY759 participate in bidirectional, outside-in, and inside-out signaling, respectively and the association of c-Src or kindlin with β3 integrin may regulate these processes., Competing Interests: The authors have declared that no competing interests exist.

For many years, splenectomy has been used to treat immune thrombocytopenia (ITP), and this procedure benefits approximately two-thirds of the treated patients. Although splenectomy may raise platelet counts, antibody-coated platelets and cytotoxic T lymphocytes appear to persist or can change over time. To better understand how the spleen may affect anti-platelet immune responses, we used a murine model of ITP demonstrating both antibody-mediated and T lymphocyte-mediated thrombocytopenia. Mice with severe combined immunodeficiency (SCID) were either splenectomized or not and transfused with splenocytes from CD61 (GPIIIa) knockout mice immunized against CD61(+) platelets. Platelet counts and anti-platelet antibody levels were performed weekly. After 4 weeks, the mice were sacrificed, and lymphoid organs were harvested and examined by flow cytometry to quantify CD4(+)CD25(+)FoxP3(+) Tregs and conventional cross-presenting XCR1(+) and tolerizing SIRPα+ dendritic cells. The results indicate that compared with control non-splenectomized mice, thrombocytopenia was improved and anti-platelet antibody production was significantly diminished in all splenectomized mice that received immune splenocytes. Splenectomized SCID mice also had a marked reduction in Tregs in the thymus together with an increased proportion of both thymic dendritic cell subsets that correlated with increased platelet counts. Of interest, although splenectomy diminished anti-platelet antibody production and raised platelet counts, marrow megakaryocyte densities were still significantly reduced in mice that received immune splenocytes. These results suggest that the spleen in murine ITP not only is the primary site responsible for platelet destruction, but it also controls, to a significant extent, the antibody response against platelets and the migration patterns of lymphocyte subsets., (Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Background: Pathogen inactivation (PI) techniques use ultraviolet (UV) illumination with or without a photosensitizer to destroy pathogen RNA and DNA. Although lacking a nucleus and innate DNA transcription, platelets (PLTs) contain RNA and can synthesize proteins. The impact of PI on PLT protein synthesis and function is unknown; altered synthesis may affect overall PLT quality. In this study we determine to what extent PLT RNA is affected by PI., Study Design and Methods: In a pool-and-split design, paired apheresis PLT concentrates were treated with riboflavin and UV illumination or were left untreated. PLT total RNA and mRNA amounts specific for glycoproteins (GP)IIIa, GPIIb, and GPIb; α-granule proteins PLT factor (PF)4; osteonectin and thrombospondin (TSP); and housekeeping protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined using absorbance and quantitative polymerase chain reaction., Results: After treatment, amounts of all analyzed mRNAs were significantly reduced (p < 0.05), but to different degrees. For GAPDH and PF4, transcripts appeared less susceptible to the treatment, with 70% remaining 1 hour after UV illumination. For GPIIIa and TSP, less than 15% remained after treatment. There was a correlation (R(2)  = 0.85) between transcript length and amount of mRNA remaining 1 hour after treatment. Total RNA demonstrated a life span equal to the PLT life span of 10 to 11 days., Conclusion: This is the first report of the impact of riboflavin and UV illumination on PLT mRNA. Results suggest that all mRNA present in PLTs is affected by the treatment although the degree of the effect varies among transcripts., (© 2016 AABB.)

In addition to mutations in ITG2B or ITGB3 genes that cause defective αIIbβ3 expression and/or function in Glanzmann's thrombasthenia patients, platelet dysfunction can be a result of genetic variability in proteins that mediate inside-out activation of αIIbβ3 The RASGRP2 gene is strongly expressed in platelets and neutrophils, where its encoded protein CalDAG-GEFI facilitates the activation of Rap1 and subsequent activation of integrins. We used next-generation sequencing (NGS) and whole-exome sequencing (WES) to identify 2 novel function-disrupting mutations in RASGRP2 that account for bleeding diathesis and platelet dysfunction in 2 unrelated families. By using a panel of 71 genes, we identified a homozygous change (c.1142C>T) in exon 10 of RASGRP2 in a 9-year-old child of Chinese origin (family 1). This variant led to a p.Ser381Phe substitution in the CDC25 catalytic domain of CalDAG-GEFI. In 2 Spanish siblings from family 2, WES identified a nonsense homozygous variation (c.337C>T) (p.Arg113X) in exon 5 of RASGRP2 CalDAG-GEFI expression was markedly reduced in platelets from all patients, and by using a novel in vitro assay, we found that the nucleotide exchange activity was dramatically reduced in CalDAG-GEFI p.Ser381Phe. Platelets from homozygous patients exhibited agonist-specific defects in αIIbβ3 integrin activation and aggregation. In contrast, α- and δ-granule secretion, platelet spreading, and clot retraction were not markedly affected. Integrin activation in the patients' neutrophils was also impaired. These patients are the first cases of a CalDAG-GEFI deficiency due to homozygous RASGRP2 mutations that are linked to defects in both leukocyte and platelet integrin activation., (© 2016 by The American Society of Hematology.)

Direct effects of circulating blood microparticles on fibrin formation and structure were studied. Clots made from platelet-free plasma and from microparticle-depleted plasma obtained by filtration was studied in parallel, including clots from the microparticle-depleted plasma replenished with phospholipids. Fibrin formation was induced by exogenous thrombin without Ca2+ to prevent formation of endogenous thrombin and exclude indirect kinetic effects of microparticles related to thrombin generation. In the presence of natural microparticles or exogenous phospholipids the maximal turbidity of fibrin clots was significantly smaller, indicating structural distinctions from the clots formed in the absence of microparticles. Scanning electron microscopy and confocal microscopy showed that clots formed from platelet-free plasma, i. e. in the presence of microparticles, unlike clots from the microparticle-depleted plasma, contained 0.1-0.5-μm-large CD61-positive granules associated with fibrin fibers that were identical to the particles found on the surface of filters used for microparticle removal. The results show that platelet-derived microparticles bind to fibrin and affect its structure. The revealed interactions of cell-derived microparticles with fibrin highlight a previously unknown role of microparticles in hemostasis and thrombosis as constituents and modulators of a fibrin clot structure.

Human platelets contain microRNAs (miRNAs) and miRNA processing machinery, but their contribution to platelet function remains incompletely understood. Here, we show that murine megakaryocyte (MK)-specific knockdown of Dicer1, the ribonuclease that cleaves miRNA precursors into mature miRNAs, reduces the level of the majority of miRNAs in platelets. This leads to altered platelet messenger RNA (mRNA) expression profiles and mild thrombocytopenia. Fibrinogen receptor subunits Itga2b (αIIb) and Itgb3 (β3) mRNAs were among the differentially expressed transcripts that are increased in platelets lacking Dicer1. Argonaute 2 (Ago2), a member of the miRNA silencing complex, co-immunoprecipitated with αIIband β3mRNAs in wild-type platelets. Furthermore, co-immunoprecipitation experiments suggested reduced αIIb/β3/Ago2 complexes in miRNA-deficient platelets. These results suggested that miRNAs regulate both integrin subunits. Subsequent 3' untranslated region luciferase reporter assays confirmed that the translation of both αIIband β3mRNAs can be regulated by miRNAs miR-326, miR-128, miR-331, and miR-500. Consistent with these molecular changes, the deletion ofDicer1resulted in increased surface expression of integrins αIIband β3, and enhanced platelet binding to fibrinogen in vivo and in vitro. Heightened platelet reactivity, shortened tail-bleeding time, and reduced survival following collagen/epinephrine-induced pulmonary embolism were also observed in Dicer1-deficient animals. CombinedPf4-cre-mediated deletion of Drosha and Dicer1 did not significantly exacerbate phenotypes observed in single Dicer1 knockout mice. In summary, these findings indicate that Dicer1-dependent generation of mature miRNAs in late-stage MKs and platelets modulates the expression of target mRNAs important for the hemostatic and thrombotic function of platelets., (© 2016 by The American Society of Hematology.)

Objective: To explore the function of a novel nonsense mutation c.1476G>A of ITGB3 gene using an in vitro expression system., Methods: An eukaryotic expression vector containing ITGB3 c.1476G>A cDNA was generated by site-directed mutagenesis and transformed into E.coli. Plasmid DNA was extracted and sequenced to confirm the target mutations. Wild-type and mutant recombination plasmids were transfected into Chinese hamster ovarian cancer (CHO) cells by nonliposome method, and the stable expression cells were harvested by G418 screening. The ITGB3 gene mRNA transcription and GPIIIa expression level in CHO cells were detected with real-time quantitative PCR, Western blotting and flow cytometry, respectively., Results: The eukaryotic expression vectors of wild ITGB3 cDNA and c.1476G>A mutant were successfully constructed. CHO cells with stable expression were obtained after transfection and screening. Compared with the wild-type transfected cells, the amount of CD61 antigen expression was 37% and mRNA transcription level was only 6% in the mutant-transfected cells. Full length GPIIIa protein was found only in the stably wild-type-transfected cells, but not in mutant-transfected cells by Western blotting analysis., Conclusion: The ITGB3 c.1476G>A mutation can decrease the transcription level and further affect GPIIIa synthesis and CD61 antigen expression.

Several patients have been reported to have variant dominant forms of Glanzmann thrombasthenia, associated with macrothrombocytopenia and caused by gain-of-function mutations of ITGB3 or ITGA2B leading to reduced surface expression and constitutive activation of integrin αIIbβ3. The mechanisms leading to a bleeding phenotype of these patients have never been addressed. The aim of this study was to unravel the mechanism by which ITGB3 mutations causing activation of αIIbβ3 lead to platelet dysfunction and macrothrombocytopenia. Using platelets from two patients carrying the β3 del647-686 mutation and Chinese hamster ovary cells expressing different αIIbβ3-activating mutations, we showed that reduced surface expression of αIIbβ3 is due to receptor internalization. Moreover, we demonstrated that permanent triggering of αIIbβ3-mediated outside-in signaling causes an impairment of cytoskeletal reorganization arresting actin turnover at the stage of polymerization. The induction of actin polymerization by jasplakinolide, a natural toxin that promotes actin nucleation and prevents depolymerization of stress fibers, in control platelets produced an impairment of platelet function similar to that of patients with variant forms of dominant Glanzmann thrombasthenia. del647-686β3-transduced murine megakaryocytes generated proplatelets with a reduced number of large tips and asymmetric barbell-proplatelets, suggesting that impaired cytoskeletal rearrangement is the cause of macrothrombocytopenia. These data show that impaired cytoskeletal remodeling caused by a constitutively activated αIIbβ3 is the main effector of platelet dysfunction and macrothrombocytopenia, and thus of bleeding, in variant forms of dominant Glanzmann thrombasthenia., (Copyright© Ferrata Storti Foundation.)

The aim of the study was to investigate ITGA2B and ITGB3 genetic polymorphisms and to evaluate the variability in the platelet function in healthy Chinese subjects. The genetic sequence of the entire coding region of the ITGA2B and ITGB3 genes was investigated. Adenosine diphosphate-induced platelet aggregation, glycoprotein IIb/IIIa content, bleeding time, and coagulation indexes were detected. Thirteen variants in the ITGA2B locus and 29 variants in the ITGB3 locus were identified in the Chinese population. The rs1009312 and rs2015049 were associated with the mean platelet volume. The rs70940817 was significantly correlated with the prothrombin time. The rs70940817 and rs112188890 were related with the activated partial thromboplastin time, and ITGB3 rs4642 was correlated with the thrombin time and fibrinogen. The minor alleles of rs56197296 and rs5919 were associated with decreased ADP-induced platelet aggregation, and rs55827077 was related with decreased GPIIb/IIIa per platelet. The rs1009312, rs2015049, rs3760364, rs567581451, rs7208170, and rs117052258 were related with bleeding time. Further studies are needed to explore the clinical importance of ITGA2B and ITGB3 SNPs in the platelet function., Competing Interests: The authors declare that there is no conflict of interests regarding the publication of this paper.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening disease in which intracranial hemorrhage (ICH) is the major risk. Although thrombocytopenia, which is caused by maternal antibodies against β3 integrin and occasionally by maternal antibodies against other platelet antigens, such as glycoprotein GPIbα, has long been assumed to be the cause of bleeding, the mechanism of ICH has not been adequately explored. Utilizing murine models of FNAIT and a high-frequency ultrasound imaging system, we found that ICH only occurred in fetuses and neonates with anti-β3 integrin-mediated, but not anti-GPIbα-mediated, FNAIT, despite similar thrombocytopenia in both groups. Only anti-β3 integrin-mediated FNAIT reduced brain and retina vessel density, impaired angiogenic signaling, and increased endothelial cell apoptosis, all of which were abrogated by maternal administration of intravenous immunoglobulin (IVIG). ICH and impairment of retinal angiogenesis were further reproduced in neonates by injection of anti-β3 integrin, but not anti-GPIbα antisera. Utilizing cultured human endothelial cells, we found that cell proliferation, network formation, and AKT phosphorylation were inhibited only by murine anti-β3 integrin antisera and human anti-HPA-1a IgG purified from mothers with FNAIT children. Our data suggest that fetal hemostasis is distinct and that impairment of angiogenesis rather than thrombocytopenia likely causes FNAIT-associated ICH. Additionally, our results indicate that maternal IVIG therapy can effectively prevent this devastating disorder.

Objective: Neointima formation is associated with stenosis and subsequent thrombosis in arteriovenous grafts (AVGs). A role of integrin β3 in the neointima formation of AVGs remains poorly understood., Approach and Results: In integrin β3(-/-) mice, we found significantly accelerated occlusion of AVGs compared with the wild-type mice. This is caused by the development of neointima and lack of endothelial regeneration. The latter is a direct consequence of impaired functions of circulating angiogenic cells (CACs) and platelets in integrin β3(-/-) mice. Evidence suggests the involvement of platelet regulating CAC homing to and differentiation at graft sites via transforming growth factor-β1 and Notch signaling pathway. First, CACs deficient of integrin β3 impaired adhesion activity toward exposed subendothelium. Second, platelets from integrin β3(-/-) mice failed to sufficiently stimulate CACs to differentiate into mature endothelial cells. Finally, we found that transforming growth factor-β1 level was increased in platelets from integrin β3(-/-) mice and resulted in enhanced Notch1 activation in CACs in AVGs. These results demonstrate that integrin β3 is critical for endothelial cell homing and differentiation. The increased transforming growth factor-β1 and Notch1 signaling mediates integrin β3(-/-)-induced AVG occlusion. This accelerated occlusion of AVGs was reversed in integrin β3(-/-) mice transplanted with the bone marrow from wild-type mice., Conclusions: Our results suggest that boosting integrin β3 function in the endothelial cells and platelets could prevent neointima and thrombosis in AVGs., (© 2015 American Heart Association, Inc.)

Activated platelets have been recognized as an accessory character in the cascade of tumor hematogenous metastasis, and intervention of tumor cell attachment to the activated platelets or microemboli formation might be a leading strategy to prevent tumor cells surviving in the blood vessels and sequential metastasis. Recently, we have demonstrated that holothurian glycosaminoglycan (hGAG), a sulfated polysaccharide with potent anticoagulant activity extracted from the sea cucumber Holothuria leucospilota Brandt, was highly efficacious against tumor metastasis. In this study, we identified the potential effects of hGAG on the disruption of interactions of cancer cells and platelets and the underlying mechanisms, which were supported by the following evidence: hGAG (1) inhibited thrombin-induced platelet activation and aggregation, (2) reduced adhesion between platelet and breast cancer cells, and abrogated platelets/cancer cells adhering to fibrinogen, (3) attenuated platelet-cancer cell complex formation (the number and size of aggregates) and (4) suppressed both mRNA and protein levels of β1 and β3 integrins, matrix metalloproteinase (MMP)-2 and MMP-9, while increasing the expression of the MMP inhibitor, tissue inhibitor of metalloproteinase (TIMP)-1 in MDA-MB-231 cells. These results suggested that both the antiplatelet properties and mitigation of the levels of cellular adhesion molecules contributed to the anticancer effects of hGAG, and might thus be exploited for clinical adjuvant therapy to attenuate tumor hematogenous metastasis., (© 2015 S. Karger AG, Basel.)

It has been identified that platelet glycoprotein IIIa PIA1/A2 polymorphism plays an important role in atherothrombotic disease such as myocardial infarction and stroke, but results remain controversial. Here, we investigated whether the PIA2 allele is associated with ST myocardial infarction or idiopathic ischemic stroke in young individuals in two independent studies. In a case-control study 275 patients with ST elevation myocardial infarction ≤45 years of age and 278 controls were recruited. In a second study, 200 patients with idiopathic ischemic stroke ≤45 years of age and 200 controls were enrolled. In both studies cases and controls were matched by age and gender. The PIA1/A2 polymorphism was determined in all participants by a polymerase chain reaction-restriction fragment length polymorphism assay. There was a significant difference in the PIA1/A2 genotype distribution (P = 0.001) and allele frequency (P = 0.001), between ST elevation myocardial infarction and control groups, but not in the PIA1/A2 genotype distribution (P = 0.61) and allele frequency (P = 0.80), between idiopathic ischemic stroke. The allele PIA2 represented an independent risk for ST elevation myocardial infarction but not for idiopathic ischemic stroke. Hypertension, smoking, and family history of atherothrombotic disease were also associated with ST elevation myocardial infarction and idiopathic ischemic stroke. Our results suggest that PA2 allele represents a risk factor for ST elevation myocardial infarction in young Mexican individuals but not for idiopathic ischemic stroke.

Human alloimmune thrombocytopenic conditions caused by exposure to a platelet-specific alloantigen include neonatal alloimmune thrombocytopenia, posttransfusion purpura, and platelet transfusion refractoriness. More than 30 platelet-specific alloantigens have been defined in the human platelet antigen (HPA) system; however, there is no previous information on canine platelet-specific alloantigens. Using the HPA system as a model, we evaluated the canine ITGB3, ITGA2B, and GP1BB genes encoding GPIIIa (β3), GPIIb (αIIb), and GPIbβ, respectively, which account for 21 of 27 HPA, to determine whether amino acid polymorphisms are present in the orthologous canine genes. A secondary objective was to perform a pilot study to assess possible association between specific alleles of these proteins and a diagnosis of idiopathic thrombocytopenic purpura (ITP) in dogs. By using genomic DNA from dogs of various breeds with and without ITP, sequencing of PCR products encompassing all coding regions and exon-intron boundaries for these 3 genes revealed 4 single-nucleotide polymorphisms in ITGA2B resulting in amino acid polymorphisms in the canine genome, 3 previously reported and 1 newly identified (Gly[GGG]/Arg[AGG] at amino acid position 576 of ITGA2B. Of 16 possible ITGA2B protein alleles resulting from unique combinations of the 4 polymorphic amino acids, 5 different protein isoforms were present in homozygous dogs and explain all of the genotype combinations in heterozygous dogs. There was no amino acid polymorphism or protein isoform that was specific for a particular breed or for the diagnosis of ITP.

Background and Objectives: β(3)-Deficient megakaryocytes were modified by human β(3)-lentivirus transduction and transplantation to express sufficient levels of a C560Rβ(3) amino acid substitution, for investigation of how an activated αII b β(3) conformation affects platelets in vivo in mice., Patient/methods: As in our previous report of an R560β(3) mutation in a patient with Glanzmann thrombasthenia, R560β(3) murine platelets spontaneously bound antibody that only recognizes activated αII b β3 bound to its ligand, fibrinogen., Results: With this murine model, we showed that αII b -R560β3 mutation-mediated continuous binding of fibrinogen occurred in the absence of P-selectin surface expression, indicating that the integrin was in an active conformation, although the platelets circulated in a quiescent manner. Remarkably, only 35% of R560β(3) 'mutant' mice survived for 6 months after transplantation, whereas 87% of C560β(3) 'wild-type' mice remained alive. Pathologic examination revealed that R560β(3) mice had enlarged spleens with extramedullary hematopoiesis and increased hemosiderin, indicating hemorrhage. R560β(3) megakaryocytes and platelets showed abnormal morphology and irregular granule distribution. Interestingly, R560β(3) washed platelets could aggregate upon simultaneous addition of fibrinogen and physiologic agonists, but aggregation failed when platelets were exposed to fibrinogen before activation in vitro and in vivo., Conclusions: The results demonstrate that continuous occupancy of αIIb β3 with fibrinogen disrupts platelet structure and function, leading to hemorrhagic death consistent with Glanzmann thrombasthenia rather than a thrombotic state., (© 2013 International Society on Thrombosis and Haemostasis.)

Introduction: Peptide LSARLAF (LSA) can bind and activate integrin αIIbβ3 in the absence of 'inside-out' signal. The active αIIbβ3 mediates 'outside-in' signaling that elicits platelet aggregation, granule secretion and TxA2 production. Here we identify the membrane glycoproteins which mediate LSA-induced platelet activation other than αIIbβ3, and determine the roles of Src, PLCγ2, FcRγ-chain, and SLP-76 in LSA-induced platelet activation., Method: Ligand-receptor binding assay was performed to study the effect of peptide LSA or its control peptide FRALASL (FRA) on integrins binding to their ligands. Spreading of CHO cells expressing αIIbβ3 or αVβ3 on immobilized fibrinogen was measured in the presence of LSA or FRA. Washed β3, Src, FcRγ-chain, LAT and SLP-76 deficient platelets aggregation and secretion were tested in response to LSA., Results: Ligand-receptor binding assay indicated that LSA promoted the binding of multiple ligands to αIIbβ3 or αVβ3. LSA also enhanced CHO cells with αIIbβ3 or αVβ3 expression spreading on immobilized fibrinogen. β3 deficient platelets failed to aggregate and secrete in response to LSA. The phosphorylation of PLCγ2 and Syk was also β3 dependent. Src, FcRγ-chain, LAT and SLP-76 deficient platelets did not aggregate, secrete ATP or produce TxA2 in response to LSA., Conclusion: LSA-induced platelet activation is β3 dependent, and signaling molecules Src, FcRγ-chain, SLP-76 and LAT play crucial roles in LSA-induced β3 mediated signaling., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: During and shortly after coronary artery bypass graft (CABG) surgery, there is an increase in thromboembolic events. CABG, a strong inflammatory stimulus, is associated with a hypercoaguable state. Platelets might contribute to this hypercoaguable state because they have a pivotal role in thrombosis. In the days following surgery there is augmented platelet regeneration in response to the inflammatory stimulus., Objectives: The aim of this study was to investigate any changes in platelet mRNA profiles to test the hypothesis that post-CABG surgery platelets are associated with a prothrombotic state., Methods: Blood was sampled and platelets purified from 11 patients before and 3-6 days after CABG. Gene expression profiling was performed using low density array (LDA) plates for seven of the patients., Results: Forty-five genes were examined and those significantly up-regulated were glycoprotein (GP)IIb, GPIIIa and cyclooxygenase-1 (COX-1). These findings were confirmed in four more patients, including flow cytometry analysis of the GPIIb/IIIa receptor., Conclusions: CABG surgery up-regulates mRNA and protein levels of proteins that are key players in platelet aggregation. Marked elevation of GPIIb/IIIa mRNA levels results in significantly increased GPIIb/IIIa expression in platelets post-CABG surgery, which may be a reason for increased thrombus formation and myocardial infarction after CABG., (© 2012 International Society on Thrombosis and Haemostasis.)

Maternal thrombophilia was recently discussed as possible cause for pregnancy complication, although the roles of some coagulation factors have not been clarified. Carrier status for platelet integrin beta3 polymorphism A1/A2 (PL A1/A2) was considered as possible risk factor for pregnancy complication. Seventy women with one or more stillbirth (intrauterine fetal death after 20 week of gestation) and 100 healthy control subjects were evaluated for PL A1/A2 to assess the impact of polymorphism for late pregnancy loss. The prevalence for PL A1/A2 in women with stillbirth was higher but not significantly differs from carrier status in control subjects (respectively 28.3% and 17%, OR = 1.93; 95% CI: 0.84 - 4.45). After adjustment for carrier status for Factor V Leiden (FVL) and Prothrombin (FII) gene mutation 20210 G > A the prevalence of PL 1/A2 remains a similar (28.2% O R = 1.92; 95% Il: 0.78 - 4.75). Combined carriers status for PL A1/ A2 with FVL or III 20210 G > A have had significantly higher prevalence in investigated group comparing with control subjects (respectively 20% and 2%, p < 0.0001). An independent impact of PL A1/A2 on risk of stillbirth development is not be yet established but additive role of the polymorphism in combination with other thrombophilic factors should be considered.

Objectives: To investigate potential associations between the PlA1/A2 polymorphism of the platelet glycoprotein receptor IIIA (GpIIIa) gene and venous thrombosis and other clinical manifestations in Italian patients with Behçet's disease (BD)., Methods: Two hundred consecutive Italian patients satisfying the International Study Group criteria for BD who were followed up for seven years and 241 healthy Italian age- and gender-matched blood donors were molecularly genotyped for the PlA1/A2 polymorphism of the platelet GpIIIa gene; 118 and 117 of the 200 BD patients were also respectively genotyped for factor V Leiden and prothrombin gene G20210A polymorphisms. A standard microlymphocytotoxicity technique was used to type serological HLA class B51. The patients were grouped on the basis of the presence or absence of clinical manifestations. The diagnoses of deep vein thrombosis (DVT) and superficial thrombophlebitis were initially made clinically, and then confirmed by means of ultrasonography or contrast venography. The distribution of the PlA1/A2 genotype was investigated, and odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated., Results: The allele and genotype frequency of the PlA1/A2 polymorphism were not significantly different in the BD patients and controls, but the PlA2 allele was significantly more frequent in the BD patients with DVT than the controls (p=0.023; Pcorr=0.046; OR 2.0, 95% CI 1.1-3.7). There were no associations between thrombotic events and the PlA1/A2 polymorphism in the BD patients carrying factor V Leiden or prothrombin gene G20210A mutations. The PlA2 allele was significantly less frequent in the BD patients with genital genital ulcers than in those without (26.9% vs. 43.2%; p=0.022; P corr 0.044; OR 0.48, CI 0.27-0.88)., Conclusions: The PlA1/A2 polymorphism of the GpIIIa gene was associated with DVT in our Italian BD patients, but does not seem to increase the risk of DVT due to factor V Leiden or prothrombin gene G20210A mutations. There was a negative association between the A2 allele and genital ulcers.

Angiogenesis alleviates hypoxic stress in ischemic tissues or during tumor progression. In addition to endothelial cell proliferation and migration, the angiogenic process requires bone marrow-derived cell (BMDC) recruitment to sites of neovascularization. However, the mechanism of communication between hypoxic tissues and the BM remains unknown. Using 2 models of hypoxia-induced angiogenesis (ischemic hindlimb surgery and subcutaneous tumor growth), we show that platelet infusion promotes BMDC mobilization into the circulation, BMDC recruitment into growing neovasculature, tumor vascularization, and blood flow restoration in ischemic limbs, whereas platelet depletion inhibits these effects. Thus, platelets are required for BMDC recruitment into ischemia-induced vasculature. Secretion of platelet α-granules, but neither dense granules nor platelet aggregation is crucial for BMDC homing and subsequent angiogenesis, as determined using VAMP-8(-/-), Pearl, and integrin Beta 3(-/-) platelets. Finally, platelets sequester tumor-derived promoters of angiogenesis and BMDC mobilization, which are counterbalanced by the antiangiogenic factor thrombospondin-1. A lack of thrombospondin-1 in platelets leads to an imbalance in proangiogenic and antiangiogenic factors and accelerates tumor growth and vascularization. Our data demonstrate that platelets stimulate BMDC homing in a VAMP-8-dependent manner, revealing a previously unknown role for platelets as key mediators between hypoxic tissues and the bone marrow during angiogenesis.

Cardiac vegetations result from bacterium-platelet adherence, activation and aggregation, and are associated with increased morbidity and mortality in infective endocarditis. The GPIIb/IIIa and FcγRIIa platelet receptors play a central role in platelet adhesion, activation and aggregation induced by endocarditis pathogens such as Staphylococcus aureus, but the influence of known polymorphisms of these receptors on the pathogenesis of infective endocarditis is unknown. We determined the GPIIIa platelet antigen Pl(A1/A2) and FcγRIIa H131R genotype of healthy volunteers (n = 160) and patients with infective endocarditis (n = 40), and investigated the influence of these polymorphisms on clinical outcome in infective endocarditis and S. aureus-platelet interactions in vitro. Platelet receptor genotype did not correlate with development of infective endocarditis, vegetation characteristics on echocardiogram or the composite clinical end-point of embolism, heart failure, need for surgery or mortality (P > 0.05 for all), even though patients with the GPIIIa Pl(A1/A1) genotype had increased in vivo platelet activation (P = 0.001). Furthermore, neither GPIIIa Pl(A1/A2) nor FcγRIIa H131R genotype influenced S. aureus-induced platelet adhesion, activation or aggregation in vitro (P > 0.05). Taken together, our data suggest that the GPIIIa and FcγRIIa platelet receptor polymorphisms do not influence S. aureus-platelet interactions in vitro or the clinical course of infective endocarditis., (Copyright © 2010 Institut Pasteur. All rights reserved.)

Background: Various genetic polymorphisms have been proposed to explain the persistent platelet hyperactivity (HPR) under aspirin treatment. PlA polymorphism of platelet GPIIIa receptor has been largely studied. However, its influence on platelet sensitivity to aspirin remains controversial., Objectives: The aim of this prospective study is to investigate whether this PlA polymorphism is associated with a greater prevalence of HPR in stable coronary artery disease patients Material and Methods: 188 stable coronary artery disease patients were included. Platelet aspirin inhibitory effect was determined with PFA-100 using Collagen/Epinephrine closure time (CEPI-CT). A CEPI-CT<160sec was defining the HPR status. GPIIIa PlA polymorphism was established using polymerase chain reaction and classical restriction fragments length polymorphism., Results: The observed frequencies of different genotypes were not different from those predicted by the Hardy-Weinberg equilibrium: PlA1/lA1 (55.3%), PlA1/PlA2 (39.4%) and PlA2/PlA2 (5.3%). HPR patients with inadequate aspirin inhibition were significantly more often homozygous PlA1/A1 (65.4% vs. 47.7%, p=0.015). After multivariate analysis, PlA1/A1 genotype was the only independent risk factor for persistent HPR (OR: 2.07; 95% CI [1.14 to 3.76; p=0.016)., Conclusion: In CAD patients receiving daily low dose of aspirin, there is a significant and independent association between the expression of GPIIIa PlA1 allele and the occurrence of persistent HPR detected with PFA-100., (Copyright 2009 Elsevier Ltd. All rights reserved.)

The purpose of this work was to determine platelet and myeloid cell-specific requirements for beta3-containing integrins in hemostasis, bone resorption, and tumor growth. LoxP-flanked mice were generated to study the conditional deletion of beta3-integrin in platelets [knockout in platelets (KOP)] and myeloid cells [knockout in myeloid (KOM)]. Using the beta3KOP and beta3KOM strains of mice, we studied the role of beta3-integrin in hemostasis, bone resorption, and subcutaneous tumor growth. Tissue-specific deletion of platelet beta3-integrins in beta3KOP mice did not affect bone mass but resulted in a severe bleeding phenotype. No growth difference of tumor xenografts or in neoangiogenesis were found in beta3KOP mice, in contrast to the defects observed in germline beta3(-/-) mice. Conditional deletion of myeloid beta3-integrins in beta3KOM mice resulted in osteopetrosis but had no effect on hemostasis or mortality. Tumor growth in beta3KOM mice was increased and accompanied by decreased macrophage infiltration, without increase in blood vessel number. Platelet beta3-integrin deficiency was sufficient to disrupt hemostasis but had no effect on bone mass or tumor growth. Myeloid-specific beta3-integrin deletion was sufficient to perturb bone mass and enhance tumor growth due to reduced macrophage infiltration in the tumors. These results suggest that beta3-integrins have cell-specific roles in complex biological processes.-Morgan, E. A., Schneider, J. G., Baroni, T. E., Uluçkan, O., Heller, E., Hurchla, M. A., Deng, H., Floyd, D., Berdy, A., Prior, J. L., Piwnica-Worms, D., Teitelbaum, S. L., Ross, F. P., Weilbaecher, K. N. Dissection of platelet and myeloid cell defects by conditional targeting of the beta3-integrin subunit.

Background: Defects of integrin alpha(IIb)beta(3) are typical of Glanzmann's thrombasthenia, an inherited autosomal recessive bleeding disorder characterized by the failure of platelets to aggregate in response to all physiological agonists, but with no abnormalities in the number or size of platelets. Although large heterogeneity has been described for Glanzmann's thrombasthenia, no family has so far been described as having an autosomal dominant form of this disease., Design and Methods: We describe two Italian families with moderate thrombocytopenia with large platelets, defective platelet function and moderate/severe mucocutaneous bleeding, transmitted as an autosomal dominant trait and associated with a novel integrin beta(3)-gene (ITGB3) mutation., Results: The characteristics of our families are moderate macrothrombocytopenia and defective platelet function associated with a mild reduction of surface alpha(Ib) beta(3), impaired platelet aggregation to physiological agonists but not to ristocetin, normal clot retraction, reduced fibrinogen binding and expression of activated alpha(IIb)beta(3) upon stimulation, normal platelet adhesion to immobilized fibrinogen but reduced platelet spreading and tyrosine phosphorylation, indicating defective alpha(IIb)beta(3)-mediated outside-in signaling. Molecular analysis revealed a novel mutation of ITGB3 that determines an in-frame deletion producing the loss of amino acids 647-686 of the betaTD ectodomain of integrin beta(3). Haplotype analysis indicated that the two families inherited the mutation from a common ancestral chromosome., Conclusions: This novel autosomal dominant macrothrombocytopenia associated with platelet dysfunction raises interesting questions about the role of integrin beta(3), and its betaTD domain, in platelet formation and function.

We report a 3-generation pedigree with 5 individuals affected with a dominantly inherited macrothrombocytopenia. All 5 carry 2 nonsynonymous mutations resulting in a D723H mutation in the beta3 integrin and a P53L mutation in glycoprotein (GP) Ibalpha. We show that GPIbalpha-L53 is phenotypically silent, being also present in 3 unaffected pedigree members and in 7 of 1639 healthy controls. The beta3-H723 causes constitutive, albeit partial, activation of the alphaIIbbeta3 complex by disruption of the highly conserved cytoplasmic salt bridge with arginine 995 in the alphaIIb integrin as evidenced by increased PAC-1 but not fibrinogen binding to the patients' resting platelets. This was confirmed in CHO alphaIIbbeta3-H723 transfectants, which also exhibited increased PAC-1 binding, increased adhesion to von Willebrand factor (VWF) in static conditions and to fibrinogen under shear stress. Crucially, we show that in the presence of fibrinogen, alphaIIbbeta3-H723, but not wild-type alphaIIbbeta3, generates a signal that leads to the formation of proplatelet-like protrusions in transfected CHO cells. Abnormal proplatelet formation was confirmed in the propositus's CD34+ stem cell-derived megakaryocytes. We conclude that the constitutive activation of the alphaIIbbeta3-H723 receptor causes abnormal proplatelet formation, leading to incorrect sizing of platelets and the thrombocytopenia observed in the pedigree.

Platelet glycoprotein (GP) IIb/IIIa has an important role in platelet aggregation. A polymorphism of platelet GPIIIa (PlA2, also called HPA1b) has been associated with a higher risk of thrombosis, but its implication in heparin-induced thrombocytopenia (HIT) is unclear. To investigate the hypothesis that the PlA2 polymorphism influences the prothrombotic effects of HIT, we conducted a prospective study of 66 consecutive patients with a laboratory diagnosis of HIT. The end point of the study was the diagnosis of a thrombus within 30 days of the positive HIT test result. The Diagnostica Stago (Asnières, France) enzyme-linked immunosorbent assay was used to detect HIT antibodies, and a polymerase chain reaction assay was used to detect the PlA2 polymorphism. Of the 66 patients, thrombotic complications developed in 27 (41%). Patients with the PlA2 allele demonstrated a significantly higher thrombosis risk than did patients without (69% vs 32%; P = .0088; odds ratio, 4.68; 95% confidence interval, 1.39-15.72). The risk was stronger for arterial thrombosis and for patients 60 years or older. There was a significant association between the PlA2 polymorphism of GPIIIa and the risk of thrombosis in patients with HIT antibodies.

Introduction: In South Africa coronary artery disease (CAD) is less common in African than Indian or white subjects. Although the association between CAD and metabolic factors have been well documented, the role of genetic factors is as yet poorly understood. Specific polymorphisms in the platelet membrane glycoprotein (GP) IIIa gene Pl(A1/A2), have been implicated in the development of CAD., Methods: The prevalence of platelet GPIIIa (Pl(A1/A2)) polymorphisms and their effect on platelet function was determined in 313 Indian, 267 white and 227 African subjects with and without a history of CAD., Results: In subjects without a history of CAD the frequency of the unfavourable Pl(A2) allele was 8.0%, 14.8% and 8.7% in the Indian, white and African populations respectively, with the frequency being significantly higher (p<0.05) in the white than both other groups. The frequency of the Pl(A2) allele was higher in subjects with (23.0%) than without (10.0%; p<0.0001) a history of CAD. Aggregation studies showed that platelets carrying the Pl(A2) allele were hypersensitive to the platelet aggregating agonists ADP and collagen and produced a higher amount of TXA(2) when stimulated with low concentrations of both these agonists., Conclusions: The positive association observed between the platelet GPIIIa Pl(A1/A2) polymorphism and platelet function suggests that the GPIIIa Pl(A2) allele may be a genetic factor that contributes to the risk of sudden death from myocardial infarction in the absence of known risk factors but it does not explain ethnic differences in the prevalence of CAD.

Background: Platelet (PLT) products have a short shelf life (5 days) owing in part to the deterioration of the quality of PLTs stored at 22 degrees C. This creates significant inventory challenges, and blood banks may suffer shortages and high wastage as a result. The precise biochemical pathways involved in the PLT storage lesion are unknown and must be understood before storage time can be extended., Study Design and Methods: Informed by previous proteomics analysis, specific PLT glycoprotein (GP) concentration and surface expression were examined by Western blot and flow cytometry. mRNA concentration was determined by Northern blot and real-time polymerase chain reaction. Protein synthesis was confirmed by [(35)S]methionine labeling., Results: Western blots of GPIIIa revealed a twofold increase in concentration on Day 7 of storage and a fourfold increase on Day 10. By flow cytometry, surface expression of the GPIIb/IIIa increased by 13.4 percent on Day 7 and 41.9 percent on Day 10. Full-length GPIIIa mRNA was present throughout this storage period and was shown to have a half-life of approximately 2.9 days. Translation of GPIIb and IIIa during storage was confirmed by [(35)S]methionine labeling., Conclusion: This article confirms that PLTs are capable of synthesizing biologically relevant proteins ex vivo throughout a 10-day storage period with particularly long-lived mRNA and provides a framework through which the biochemical mechanisms involved in the translational regulation of proteins thought to be involved in the initiation or exacerbation of the PLT storage lesion can be investigated.

A panel of commercially available anti-human mab was screened for cross-reactivity on chicken cells. All mab were screened at least twice on PBL collected from two different chicken lines. Out of the 377 mab tested, only two consistently reacted with subpopulations of PBL. The mab HUH73A reactive with CD11a was detected on all lymphocytes. In contrast, the mab 23C6 obtained from Serotec and reactive with an epitope formed by humanVbeta3 integrin chains (CD51/CD61) reacted with all thrombocytes, but not with other cells. In double immunofluorescence analyses, the 23C6+ cells were found to coexpress the CD45 antigen and the chicken thrombocyte marker K1. The chicken genes encoding CD51 and CD61 were analyzed by database mining. The CD51 gene is encoded by a 43 kb region containing 30 exons on chicken chromosome 7, whereas the 16 kb CD61 gene consisted of 15 exons and was localized to chromosome 27. In conclusion, the mab 23C6 is a useful reagent to identify chicken thrombocytes.

Autism is a neurodevelopmental disorder of unclear etiology. The consistent finding of platelet hyperserotonemia in a proportion of patients and its heritability within affected families suggest that genes involved in the serotonin system play a role in this disorder. The role in autism etiology of seven candidate genes in the serotonin metabolic and neurotransmission pathways and mapping to autism linkage regions (SLC6A4, HTR1A, HTR1D, HTR2A, HTR5A, TPH1 and ITGB3) was analyzed in a sample of 186 nuclear families. The impact of interactions among these genes in autism was assessed using the multifactor-dimensionality reduction (MDR) method in 186 patients and 181 controls. We further evaluated whether the effect of specific gene variants or gene interactions associated with autism etiology might be mediated by their influence on serotonin levels, using the quantitative transmission disequilibrium test (QTDT) and the restricted partition method (RPM), in a sample of 109 autistic children. We report a significant main effect of the HTR5A gene in autism (P = 0.0088), and a significant three-locus model comprising a synergistic interaction between the ITGB3 and SLC6A4 genes with an additive effect of HTR5A (P < 0.0010). In addition to the previously reported contribution of SLC6A4, we found significant associations of ITGB3 haplotypes with serotonin level distribution (P = 0.0163). The most significant models contributing to serotonin distribution were found for interactions between TPH1 rs4537731 and SLC6A4 haplotypes (P = 0.002) and between HTR1D rs6300 and SLC6A4 haplotypes (P = 0.013). In addition to the significant independent effects, evidence for interaction between SLC6A4 and ITGB3 markers was also found. The overall results implicate SLC6A4 and ITGB3 gene interactions in autism etiology and in serotonin level determination, providing evidence for a common underlying genetic mechanism and a molecular explanation for the association of platelet hyperserotonemia with autism.

Background: Ultra-large von Willebrand factor (ULVWF) and the receptor P-selectin are released from endothelial Weibel-Palade bodies during injury or inflammation. VWF mediates platelet adhesion and P-selectin promotes leukocyte rolling. ADAMTS-13 limits the duration of platelet adhesion by cleaving the ULVWF. In the absence of ADAMTS-13, long VWF filaments decorated with platelets form. Recent in vitro studies suggested that P-selectin might anchor these platelet strings to endothelium, but whether the same mechanism exists in vivo remains to be elucidated., Methods: We address the role of P-selectin and beta(3) integrin in platelet string formation in vivo using intravital microscopy by infusing inhibitory ADAMTS-13 antibody in P-selectin-/- and beta(3)-deficient mice and activating the endothelium by injecting histamine., Results: We show that inhibition of ADAMTS-13 combined with endothelial activation leads to similar extents of platelet string formation in wild-type, P-selectin- and integrin beta(3)-deficient mice. Further, in venules the platelet strings can coalesce into VWF-platelet aggregates. This process utilizes neither the platelet beta(3) integrin nor P-selectin. We also show in vitro that platelets can act as a bridge between the VWF fibers and that VWF can self-associate even in areas devoid of platelets., Conclusions: The formation or retention of the platelet strings does not require P-selectin or the endothelial VWF receptor alpha(v)beta(3). Furthermore, in the presence of low ADAMTS-13 activity, VWF-dependent and alpha(IIb)beta(3)-independent platelet clustering occurs in veins, as has been shown at high arterial shear rates. Our study further supports the importance of regulation of VWF multimer size upon secretion from Weibel-Palade bodies.

With the aim to detect genetic factors of risk of development of early myocardial infarction (MI) we studied 29 allele variants of 19 genes in 206 men who had survived MI in the age before 45 years and in 195 men of similar age without cardiovascular diseases. All subjects were inhabitants of North-West region of Russia. The following factors were associated with history of myocardial infarction: genotype RR191 of paraoxonase-1 (PON1) gene (RR 2.8 [95% CI: 1.24 - 6.30]), P1A2 allele of glycoprotein (GP) IIIa subunit of platelet fibrinogen receptor GPIIb/IIIa (RR 1.8 [95% CI: 1.11 - 2.93]), and Met145 allele of GPIbalpha platelet von Willebrand factor receptor gene. Genotype CC ( - 108) PON1 was associated with lowered risk of MI development (RR 0.6 [95% CI: 0.40 - 0.91]). During 7 years of follow-up 30 men from MI group died of recurrent acute coronary syndromes. In the group of those who died we noted increased prevalence of P1A2 GPIIIa allele compared with those who survived (p < 0.03). The results allow to suggest that contribute to development of MI in young men factors associated with elevation of functional state of platelets and levels of oxidized lipids in blood plasma.

In recent years inherited variations in platelet proteins have emerged as potential risk factors that could predispose individuals to arterial thrombosis. Although many studies have examined the association of platelet gene polymorphisms with particular disease states, the underlying mechanisms by which most of these polymorphisms contribute to the pathophysiology of thrombosis have remained largely unexplored. This review will focus on the cellular and molecular features by which these genetic changes affect platelet physiology. Although many genes have been investigated in this regard, only the genes encoding integrins beta3 and alpha2, and the platelet Fc receptor, Fc(gamma)RIIA, have been studied in any depth. In some cases (such as integrin alpha2), evidence supports a quantitative trait locus. For other genes, nonsynonymous nucleotide substitutions lead to structural and functional consequences. A large portion of this review will focus on the widely studied Leu33Pro (Pl(A)) polymorphism of integrin beta3, and will consider the potential mechanisms by which the Pro33 polymorphism could induce a prothrombotic risk. A detailed understanding of how polymorphisms modulate platelet physiology will be important for understanding individual differences in response to antiplatelet therapy.

Some studies have suggested that genetic variability in the glycoprotein (GP) IIIa gene modulates expression of platelet GPIIb/IIIa (alpha(2b)beta(3)). We sought to determine as to whether combinations of genetic variants within the GPIIIa gene (haplotypes) influenced the expression of GPIIIa RNA and protein levels in human platelets. Three promoter polymorphisms, Pl(A1/A2) genotype and platelet receptor densities were determined in 207 acute coronary syndrome (ACS) patients. Allele-specific quantitative reverse transcription-polymerase chain reaction of platelet RNA from Pl(A1/A2) heterozygotes identified a greater expression of Pl(A2) bearing transcripts among heterozygotes. Among the patients studied, the ratio of Pl(A1)/Pl(A2) RNA expression was significantly influenced by promoter haplotype (P < 0.01). However, this effect reflected carriership of rare not common haplotypes (P = 0.2). There was a threefold variation between subjects in the number of GPIIb/IIIa receptors expressed per platelet, although no association between receptor density and the Pl(A2) (P = 0.93) or promoter polymorphisms was demonstrated (-468A, P = 0.52; -425C, P = 0.59; -400A, P = 0.52). Among common haplotypes, Pl(A1)/Pl(A2) RNA expression was negatively correlated with adjusted GPIIb/IIIa receptor density (P = 0.04). The overall trend towards higher expression of Pl(A2) bearing message in Pl(A1/A2) heterozygotes, and the existence of rare haplotypes with more pronounced changes indicate the existence of cis-acting genetic factors that remain to be identified.

Toll-like receptors (TLRs) play a critical role in stimulating innate immunity by recognizing pathogen-associated molecular patterns (PAMPs) on invading microorganisms. Platelets also play a role in innate immunity, and we studied whether they express TLR. Results show that human and murine platelets variably expressed TLR2, TLR4, and TLR9 by flow cytometry and Western blotting. TLR4 expression was confirmed by demonstrating murine platelet binding to lipopolysaccharide (LPS). Thrombin activation of the platelets significantly enhanced the expression of TLR9, suggesting that at least some TLRs may derive from intracellular compartments. When LPS was administered to LPS-sensitive C3H/HeN and LPS-resistant C3H/HeJ mice, functional TLR4 expression in vivo was shown to be responsible for LPS-induced thrombocytopenia. However, when the C3H/HeN mice were first rendered thrombocytopenic by an antiplatelet antibody and then administered LPS, a significant reduction occurred in their ability to produce TNF-alpha. The decreased cytokine production in the thrombocytopenic mice was restored with platelet transfusion. These results suggest that platelets express various TLRs and that the functional significance of one of these, TLR4, appears to be a role in the modulation of LPS-induced thrombocytopenia and TNF-alpha production. This work implicates platelets as important mediators of innate immune responses against invading microorganisms.

Individual variability in response to clopidogrel is known but its mechanism is poorly understood. We examined the relationship between glycoprotein IIIa polymorphism P1(A1/A2) and anti-thrombotic actions of clopidogrel. Clopidogrel (75 mg/d; 2 weeks) was administered to 48 normolipemic patients with coronary artery disease. Bleeding time, thrombin generation at the site of microvascular injury, platelet function under high shear, using PFA-100 with ADP cartridge, and platelet surface activation markers (P-selectin and fibrinogen binding sites on GPIIb/IIIa complex detected by PAC-1 antibody), were studied both before and after clopidogrel treatment. Both unstimulated and low-dose (0.02 microM and 1 microM) in vitro ADP-stimulated platelets were examined. GP IIIa polymorphism was assessed by polymerase chain reaction and restriction fragment length polymorphism analysis. We identified 32 P1(A1/A1) homozygotes, 15 P1(A1/A2 heterozygotes and one P1(A2/A2) homozygote. Clopidogrel significantly prolonged bleeding time in all subjects, but this effect was greater in P1(A2 carriers (p < 0.01). Furthermore, clopidogrel only depressed thrombin generation at the site of microvascular injury (p < 0.01) in P1(A2) patients and prolonged closure time measured in vitro by PFA-100 (p < 0.05). At baseline spontaneous expression of PAC-1 and P-selectin was higher in P1(A2) subjects as compared to P1(A1) homozygotes (p < 0.05 for both antigens). Clopidogrel lowered the expression of both markers affecting more P1(A2) carriers, so that the difference in binding PAC-1 antibody between platelets from P1(A1) and P1(A2) carriers disappeared, while the difference in P-selectin expression slightly diminished. Anti-thrombotic effects of clopidogrel are more pronounced in CAD patients carrying the P1(A2) allele than in P1(A1) homozygotes.

A quantitative detection assay for analysis of platelet glycoprotein GPIIIa gene expression is presented. The assay uses two fluorescently labeled TaqMan MGB probes to detect the polymorphic site in GPIIIa nucleotide sequence, leading to antigens HPA-1a and HPA-1b. In order to avoid the influence of DNA contamination on RNA quantification, a forward primer was constructed to span an exon-exon junction. The assay is therefore applicable to expression studies also in samples containing only a small amount of contaminating DNA. To standardize the amount of sample cDNA added to the reaction, amplification of endogenous control 18SrRNA was included in a separate well. The amplification validation experiment showed a high real-time PCR efficiency for HPA-1a, HPA-1b and 18SrRNA. Relative quantification was therefore performed using the comparative C(T) method. The assay was optimized on a reversely transcribed total RNA from platelets, and the specificity rate was determined by sequencing. The amount of cDNA at which amplification was still clearly detectable was 5 ng. This newly developed real-time quantitative PCR assay is a sensitive, reproducible and reliable method. It is suitable for studying different stages of megakaryopoiesis, monitoring molecular alteration in defective platelets and determining differences in the GPIIIa expression level between normal and pathological megakaryocytic differentiation pathways.