Platelet Fibrinogen Receptor Research Articles

Myricetin as a promising inhibitor of platelet fibrinogen receptor in humans

Platelets play a vital role in the formation of dangerous arterial thrombosis. Platelets are activated by adhesive proteins or soluble agonists through their specific receptors. The receptor-mediated signaling pathways lead to common signaling events, which result in shape changes and inside–out signaling, leading fibrinogen binding to glycoprotein IIb/IIIa complex (integrin αIIbβ3). This interaction initiates integrin αIIbβ3-mediated outside-in signaling, subsequently culminating in granule secretion and aggregation. Myricetin is a flavonoid that occurs in a variety of plants. Although myricetin has been demonstrated to have several bioactive properties, its role in platelet activation has not been extensively studied. The present study demonstrated the ability of myricetin to inhibit platelet aggregation stimulated by collagen, thrombin, and U46619. Myricetin reduced the ATP-release, cytosolic Ca2+ mobilization, and P-selectin expression and the activation of PLCγ2/PKC, PI3K/Akt/GSK3β, and MAPK. Myricetin exerted a direct inhibitory effect on the activation of integrin αIIbβ3 by disrupting the binding between FITC-PAC-1 and the integrin. Moreover, myricetin suppressed integrin αIIbβ3-mediated outside–in signaling, such as integrin β3, Src, and Syk phosphorylation on immobilized fibrinogen. In animal studies, myricetin significantly prolonged the occlusion time of thrombotic platelet plug formation in mesenteric microvessels without extending bleeding time. This study concludes that myricetin is a natural integrin αIIbβ3 inhibitor and a novel antithrombotic agent.

A picogram BA-ELISA quantification assay for rLj-RGD3, a platelet fibrinogen receptor antagonist, in the rat plasma and its application to a pharmacokinetic study.

rLj-RGD3, a new member of the RGD (Arginine-Glycine-Aspartate)-motif toxin protein family obtained from Lampetra japonica by means of recombinant DNA techniques, has been demonstrated to be a platelet fibrinogen receptor antagonist and holds potential as a drug candidate for a specific indication. The present article reports an innovative validated highly sensitive and specific biotin-avidin enzyme linked immunosorbent assay (BA-ELISA) to provide a bio-analytical method for pharmacokinetic (PK) studies of rLj-RGD3. The concentration of picogram level rLj-RGD3 in rat plasma was measured using the developed double sandwich BA-ELISA assay, which used two mouse anti-rLj-RGD3 monoclonal antibodies that recognize different epitopes for capture and detection. This method was verified to be highly specific (blank plasma did not interfere with detection), precise (RSD <15%), and accurate (86%-113%). Absolute recovery was in the 94%-119% range. The calibration curve showed good linearity within the 50 to 1600 pg/mL range. The LOQ was as low as 50 pg/mL. The above validated assay was successfully employed to assess PK of rLj-RGD3 in rats. After i.v. and s.c. dosing with 30 μg/kg, the rLj-RGD3 plasma concentration declined bi-exponentially with time. This decay was best fitted to a two-compartment model. In conclusion, the BA-ELISA method described here meets all requirements for PK studies of rLj-RGD3 with an effective pharmacological dose in the μg/kg BW range.

Letter: Nationwide Readmission Rates and Hospital Charges for Patients With Surgical Evacuation of Nontraumatic Subdural Hematomas: Part 1-Craniotomy.

Letter: Nationwide Readmission Rates and Hospital Charges for Patients With Surgical Evacuation of Nontraumatic Subdural Hematomas: Part 1-Craniotomy.

Preferential Use of Coagulation Pathways Differs between Classical Myeloproliferative Neoplasms: Results of Global Coagulation Assays with Respect to Clinical and Genetic Features

Introduction Classical Myeloproliferative Neoplasms (MPN) are hematopoietic stem cell diseases characterized by inflammation, promotion of atherosclerosis, hypercoagulability, fibrosis, and clonal evolution. Our previous studies led us to the construction of a model, called "the circulating wound", for the likely exploitation of tissue factor (TF)-dependent thrombin generation and the consequent amplification of pro-fibrotic mechanisms mediated by Protease Activated Receptors (PARs). The indirect demonstration of PARs hyperactivation is the marked reduction of platelet fibrinogen receptors (PFRs) in patients with MPNs, following the rapid conversion of functional fibrinogen to fibrin. Acetylsalicylic acid (ASA), endowed with hypoprothrombinemic and hyperfibrinolytic properties, could restore the expression of PFRs. The aim of the present study is to verify the existence of imbalances in hemostatic function in rotational thromboelastometry (ROTEM), with respect to the different classical forms of MPN, their cell counts, the presence of genetic mutations in Next Generation Sequencing (NGS), PFR expression, the use of ASA and the history of vascular events. Methods We enrolled 53 patients affected by MPNs, naïve from any therapy, with the only exception of ASA (taken by 50% of patients). We used the samples to run assays of genetics (30-gene panel in NGS - SOPHiA Myeloid Solution™, SOPHiA Genetics, Switzerland), cytofluorimetric determination of PFRs, global coagulation by rotational tromboelastometry (ROTEM® Delta, Werfen, Spain) for INTEM, EXTEM, FIBTEM parameters. Results We found that ROTEM parameters show significant divergence depending on disease type, cell number and some mutations. The results should be read in terms of "efficiency" or "preferential use" of one pathway over another, as the instrument (as expected) mostly yields values in the ranges. Essential thrombocythemia (ET) and CALR mutation exhibit preserved efficiency of both classical coagulation pathways, with significantly more contracted clot formation times (CFTs). In contrast, primary myelofibrosis (PMF) and polycythemia vera (PV) show greater imbalances in the hemostatic system. PV patients have longer CFTs (EXTEM 114 sec, p= 0.008, INTEM 82 sec, p=0.027) and selective contraction of parameters in INTEM for higher counts of platelets (Plt), white blood cells (WBC) and hemoglobin (Hb). PMF - in contrast - seems to exploit the extrinsic pathway (EXTEM) more - see figure. Multivariate analysis gives Hb levels the largest contribution to the alterations, while only Plt retain statistical significance in PMF and ET. Selected mutations are associated with changes in ROTEM parameters. Among the DTA mutations, the presence of DNMT3A shows a significant reduction in clotting time (CT) in EXTEM, while ASXL1 is associated with reduced maximum lysis (ML). EZH2 could be responsible for CFT elongations in the INTEM assay. In addition, increased expression of PFRs is associated with bleeding history and sustained FIBTEM CT under ASA prophylaxis. Conclusions In more indolent MPNs (such as ET, especially if CALR mutated) the balance and efficiency of the classical pathways are preserved. In PMFs, the hemostatic system is unbalanced toward the extrinsic pathway, confirming the link between increased fibrosis and plasma-dependent hypercoagulable states. The experiment also provides further evidence of the robustness of the "circulating wound" model. Rheological characteristics and endothelial stress in PV appear to result in increased involvement of the intrinsic pathway. Interestingly, mutations involving genes that regulate DNA methylation are confirmed to interfere with hemostatic balance. Finally, excessive restitution of PFR expression by ASA appears to be related to the history of bleeding events, making their cytofluorimetric determination a potential tool for monitoring antiplatelet prophylaxis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Molecular-genetic substantiation of combined therapy in the early postoperative period in diffuse peritonitis

The OBJECTIVE was to give a molecular genetic foundation of combination therapy in the early postoperative period for peritonitis.METHODS AND MATERIALS. 70 patients with diffuse peritonitis were studied: the first (n=35) – patients underwent basic treatment, the second (n=35) – combined treatment with the inclusion of Remaxol and laser irradiation. The severity of endotoxemia and oxidative stress, the state of the hemostasis system and the functional status of the liver were determined. Molecular genotyping of the following genes was performed: beta subunits of platelet fibrinogen receptor (T1565C, ITGB3), fibrinogen (G(-455)A, FGB), catalase (-262C/T, CAT), superoxide dismutase (C47T, SOD2).RESULTS. It was found that the early postoperative period in patients with peritonitis is accompanied by endotoxemia, oxidative stress, impaired functional status of the liver, hypercoagulation and hypofibrinolysis. In patients with conditionally «mutant» genotypes of the studied genes, peritonitis is more severe, the severity of changes in the studied indicators of homeostasis is greater. At the same time, the effectiveness of therapy decreased, the number of complications increased, and the stay of patients in the hospital increased. The early inclusion of Remaxol and laser radiation in basic therapy increases the overall effectiveness of treatment: the severity of endotoxemia and oxidative stress decreases, the functional state of the liver and hemostasis system is restored relatively quickly, and as a result, an improvement in clinical results has been registered (a 37.5 % decrease in the frequency of complications (χ2=3.360, p=0.047) and a reduction in the length of stay of patients in the hospital by 16.6 % (p&lt;0.05)).CONCLUSION. The inclusion of Remaxol and laser therapy in the early postoperative period in the treatment regimen of patients with peritonitis makes it possible to influence pathogenetically important links of the disease relatively quickly, which improves treatment results. This type of therapy is particularly effective in patients with polymorphic genotypes T1565C and C1565C of the ITGB3 gene.

Platelet Phenotype and Function Changes With Increasing Duration of Extracorporeal Membrane Oxygenation.

To investigate platelet pathophysiology associated with pediatric extracorporeal membrane oxygenation (ECMO). Prospective observational study of neonatal and pediatric ECMO patients from September 1, 2016, to December 31, 2019. The PICU in a large tertiary referral pediatric ECMO center. Eighty-seven neonates and children (< 18 yr) supported by ECMO. None. Arterial blood samples were collected on days 1, 2, and 5 of ECMO and were analyzed by whole blood flow cytometry. Corresponding clinical data for each patient was also recorded. A total of 87 patients were recruited (median age, 65 d; interquartile range [IQR], 7 d to 4 yr). The median duration of ECMO was 5 days (IQR, 3-8 d) with a median length of stay in PICU and hospital of 18 days (IQR, 10-29 d) and 35 days (IQR, 19-75 d), respectively. Forty-two patients (48%) had at least one major bleed according to a priori determined definitions, and 12 patients (14%) had at least one thrombotic event during ECMO. Platelet fibrinogen receptor expression decreased (median fluorescence intensity [MFI], 29,256 vs 26,544; p = 0.0005), while von Willebrand Factor expression increased (MFI: 7,620 vs 8,829; p = 0.0459) from day 2 to day 5 of ECMO. Platelet response to agonist, Thrombin Receptor Activator Peptide 6, also decreased from day 2 to day 5 of ECMO, as measured by binding with anti-P-selectin, PAC-1 (binds activated GPIIb/IIIa), and anti-CD63 monoclonal antibodies (P-selectin area under the curve [AUC]: 63.46 vs 42.82, respectively, p = 0.0022; PAC-1 AUC: 93.75 vs 74.46, p = 0.0191; CD63 AUC: 55.69 vs 41.76, p = 0.0020). The loss of platelet response over time may contribute to bleeding during ECMO. These novel insights may be useful in understanding mechanisms of bleeding in pediatric ECMO and monitoring platelet markers clinically could allow for prediction or early detection of bleeding and thrombosis.

TLT-1, a Potential Regulator of Inflammatory Pathogenesis in Obesity and Non-Alcoholic Fatty Liver Disease (NAFLD)

Background:Obesity, a nationwide health issue, has related medical costs ranging between $147-210 billion per year in United States and has been associated with a 3.5-fold increased risk of developing NAFLD. In obesity, platelets work in a pleotropic manner with vascular and immune cells to amplify the chronic inflammatory process. Interestingly, studies have demonstrated that platelet numbers and reactivity are increased in obese individuals. The emerging role of activated platelets during obesity induced inflammation introduces the novel concept of platelet targeted therapeutic interventions.Kopec et al, further supports the idea that the mechanism underlying the progression of obesity lies in a platelet mediated pro-inflammatory state, illustrating that there is extravascular fibrin(ogen) deposition, macrophages and inflammatory cytokines within white adipose tissue and liver of mice on western diet. Kopec et al uses a fibrinogen mutant mouse (Fiby390-396 ) which lacks the binding motif for Mac-1 and inhibits the ligand interaction with leukocytes, diminishing inflammation, reducing macrophage counts, reducing weight, protects mice from NAFLD and glucose dysmetabolism. Taken together, all evidence points towards a platelet/fibrinogen/leukocyte pathophysiological mechanism underlying the development of obesity.TREM-Like Transcript-1 (TLT-1) is a platelet specific receptor found in the a-granules of platelets and released to the surface upon platelet activation. TLT-1 is a type 1 receptor that, like the integrin a2bb3, binds fibrinogen and facilitates platelet aggregation . However, although TLT-1 may assist in clot formation and hemostasis to arrest bleeding in a non-inflammatory/nonimmune mediated setting, TLT-1's main association is with regulating inflammatory-derived bleeding. This is demonstrated by increased hemorrhage after inflammatory treatments such as lipopolysaccharide LPS in the treml1 -/- mice as compared to controls.Considering the emerging evidence in support of a platelet-fibrinogen receptor ligand interaction as a key mechanism underlying the development of obesity and that TLT-1, a platelet specific receptor binds fibrinogen and mediates leukocyte trafficking, our laboratory set out to determine whether TLT-1 could be implicated as the main culprit underlying this mechanism. When placed on a western diet, treml1 -/- mice are more prone to weight gain, based on these finding we hypothesize that: The TLT-1/Fibrinogen molecular interaction regulates metabolic inflammation in obesityAims:Evaluate the effects of western diet on obesity and NAFLD in the treml1 -/- mouse modelMethods:TLT-1 (treml1 -/-) - apolipoprotein E (apoe -/-) double null (AT-DKO;n=11) mice and control apoe +/-/treml1 +/- littermate controls (AT-Hets;n=20) were fed western diet for 20 weeks. Plasma samples were collected for adipokine, glucose, insulin, liver enzyme and lipid profiling. Mouse were perfused, liver and adipose tissue were collected for histological analysis.Results:Overall AT-DKO mice gained more weight compared to AT-Hets (12.94±1.90 vs 8.51±1.70 grams p=0.02). Plasma analysis demonstrates that the AT-DKO have higher levels of TNF-a (0.54±0.60 vs 0.118±0.17 pg/ml p=0.03), and IL-10 (2.50±1.40 vs 1.50±2.10 pg/ml p=0.004) compared to littermate controls. Histological analysis of livers illustrates increased lipid vacuoles and inflammatory foci in the AT-DKO mice as compared to controls, while preliminary data is not significant for these differences, liver damage in the AT-DKO was significantly greater as demonstrated by increased AST levels (166.21±91.00 vs 102±68.10 U/L p=0.02). Moreover, the AT-DKO mice had higher levels of ALT, direct bilirubin, cholesterol, pai-1 , triglycerides and lower IL-6 and Adiponectin (Table 1 data not significant). These findings suggest that in the absence of TLT-1 these mice are more prone to liver disfunction , hyperlipidemia and inflammatory alterations.Conclusions:Mutant AT-DKO mice are more prone to obesity and NAFLD compared to littermate controls, suggesting that TLT-1, a platelet gene, plays a surprising role in metabolism. Further investigation could adjudicate TLT-1 administration as a potential therapeutic intervention for prevention and amelioration of Obesity and related pathologies. The current state of this project will be reported here. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clinical and genetic factors associated with the risk of recurrent ischemic events in patients with stable stenocardia

The aimof the study was to assess the clinical and genetic factors associated with the risk of recurrent ischemic events in patients with stable stenocardia (SS).Materials and methods.A total of 100 patients with SS were examined and followed-up for 15.3±8.3 months. The patients were divided into subgroups (SG): SG1 (n=51) – persons without events, SG2 (n=49) persons with recurrent ischemic events (hospitalization due to the development of pain syndrome, re-stenting due to stent restenosis, myocardial infarction, cerebral infarction and death from cardiovascular causes), SGB (n=11) – persons with «major» recurrent ischemic events (re-stenting due to stent restenosis, myocardial infarction, cerebral infarction and death from cardiovascular causes) , SGG (n=89) – persons without «major» events. The obtained survey data (general clinical, aggregometry, polymor phism of genes of platelet fibrinogen receptor ITGB3 (T1565C), platelet collagen receptor ITGA2 (C807T), ADP platelet receptor P2RY12, H1/H2 (T744C)) were analyzed using the STATISTICA 10.0 software.Results.In SG2, men predominated (χ2=9.2; p&lt;0.01), past MI was more common (χ2=4.8; p&lt;0.05), more stents were implanted (2.4±1.9 versus 1.7±1.1, p&lt;0.05), TRAP-test values were higher (p&lt;0.05) compared to SG1. In SGB, greater number of stents were implanted (3.1±2.2 versus 1.61±1.57, p&lt;0.05), the carriage of the TC genotype of the ITGB3 gene was more common, (p&lt;0.05), a combination of gene mutations ITGB3 and P2RY12 was more common, (p&lt;0.05) compared to SGG. A logistic regression equation was constructed, including the presence of diabetes mellitus, the number of platelets in the blood test, the ASPI-test values, the carriage of the 1565C allele of the ITGB3 gene, the number of stents implanted, which makes it possible to determine the likelihood of developing «major» recurrent ischemic events with a cut-off threshold LP₀=0.0965, with sensitivity – 81.82 %, specificity – 78.48 %, overall accuracy – 78.89 %.Conclusions.The factors associated with the development of recurrent ischemic events are: male sex, previous MI, a greater number of implanted stents, and high TRAP-test values. The factors associated with the development of recurrent «major» ischemic events are: a greater number of implanted stents, carriage of the TC genotype of the ITGB3 gene, carriage of a combination of mutations of the H1/H2 polymorphic locus of the P2RY12 gene and the T1565C polymorphic locus of the ITGB3 gene, diabetes mellitus, the number of platelets in blood test, ASPI-test values.

Assessment of platelet function utilizing viscoelastic testing.

Assessment of platelet function utilizing viscoelastic testing.

Glycoprotein VI is not a Functional Platelet Receptor for Fibrin Formed in Plasma or Blood.

Glycoprotein VI (GPVI), a platelet collagen receptor, is crucial in mediating atherothrombosis. Besides collagen, injured plaques expose tissue factor (TF) that triggers fibrin formation. Previous studies reported that GPVI also is a platelet receptor for fibrinogen and fibrin. We studied the effect of anti-GPVI antibodies and inhibitors of GPVI signaling kinases (Syk and Btk) on platelet adhesion and aggregate formation onto immobilized fibrinogen and different types of fibrin under arterial flow conditions. Fibrin was prepared from isolated fibrinogen ("pure fibrin"), recombinant fibrinogen ("recombinant fibrin"), or generated more physiologically from endogenous fibrinogen in plasma ("plasma fibrin") or by exposing TF-coated surfaces to flowing blood ("blood fibrin"). Inhibition of GPVI and Syk did not inhibit platelet adhesion and aggregate formation onto fibrinogen. In contrast anti-GPVI antibodies, inhibitors of Syk and Btk and the anti-GPIb antibody 6B4 inhibited platelet aggregate formation onto pure and recombinant fibrin. However, inhibition of GPVI and GPVI signaling did not significantly reduce platelet coverage of plasma fibrin and blood fibrin. Plasma fibrin contained many proteins incorporated during clot formation. Advanced optical imaging revealed plasma fibrin as a spongiform cushion with thicker, knotty, and long fibers and little activation of adhering platelets. Albumin intercalated in plasma fibrin fibers left only little space for platelet attachment. Pure fibrin was different showing a dense mesh of thin fibers with strongly activated platelets. We conclude that fibrin formed in plasma and blood contains plasma proteins shielding GPVI-activating epitopes. Our findings do not support a role of GPVI for platelet activation by physiologic fibrin.

Role of arrestin1 in regulation of GPCR signaling in platelet activation

The arrestins are a class of soluble proteins that function in concert with the GPCR kinases to stop or arrest GPCR‐mediated intracellular signaling. Although β‐arrestins function in the desensitization of most GPCRs, the functional differences of β‐arrestin1 (arrestin2, BARR1) versus β‐arrestin2 (arrestin1, BARR2) in the regulation of GPCR signaling in platelets and the mechanisms of desensitization of platelets to GPCR agonists by β‐arrestins have not been understood. In this study, we identify the role of BARR2 in the regulation of platelet function and GPCR signaling in platelets. We used mice lacking BARR1 and BARR2 to evaluate the functional role of BARR2 in platelet activation. Platelet aggregation, dense‐granule secretion, and fibrinogen receptor activation induced by 2‐MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in BARR2 −/− platelets compared to WT platelets. In contrast to BARR2 −/− platelets, AYPGKF‐, thrombin‐, and U46619‐induced platelet aggregation and secretion were significantly inhibited in BARR1 −/− platelets compared to WT platelets, while 2‐MeSADP‐induced platelet aggregation and secretion were minimally affected in BARR1 −/−platelets. However, CRP‐induced platelet aggregation and secretion were not affected in both BARR1 −/− and BARR2 −/− platelets. These results suggest that BARR2 functions as the predominant regulator in the termination of GPCR signaling in platelets. In addition, platelet aggregation was restored in response to second challenge of 2‐MeSADP and AYPGKF in BARR2 −/− platelets whereas re‐stimulation of agonist failed to induce aggregation in WT and b‐arrestin1 −/− platelets, indicating BARR2 contributes to P2Y1, P2Y12, and PAR receptor desensitization. Furthermore, 2MeSADP‐ and AYPGKF‐induced Akt and ERK phosphorylations were significantly potentiated in BARR2 −/− platelets. Finally, BARR2 −/− mice show an enhanced and stable thrombus formation after FeCl3 injury to the carotid artery, indicating BARR2 is critical for thrombus formation in vivo. We conclude that BARR2 plays an important role in regulation of platelet function through selective GPCR desensitization.Support or Funding InformationThis work was supported by the National Research Foundation of Korea (NRF) Grant of the Korean government (NRF‐2016R1D1A1B01010310) and the Global Research and Development Center (GRDC) Program through the NRF funded by the Ministry of Education, Science and Technology (2017K1A4A3014959).

Unexpected low expression of platelet fibrinogen receptor in patients with chronic myeloproliferative neoplasms: how does it change with aspirin?

SummaryThis study was conducted to evaluate the expression of fibrinogen receptors on platelets of Philadelphia‐negative chronic myeloproliferative neoplasm (MPN) patients. We collected blood samples from 40 consecutive MPN patients and healthy volunteers. We performed flow cytometry analysis of P‐selectin expression and integrin beta‐3, activation of glycoprotein (GP) IIb/IIIa and fibrinogen receptor exposure (PAC‐1 binding). Surprisingly, we found a very low PAC‐1 binding capacity in MPN patients; however, the expression of PAC‐1 was almost completely recovered with aspirin intake. We hypothesize that the hypercoagulable states observed in MPN patients could depend on a primarily plasma‐driven impairment of fibrin turnover and thrombin generation.

The Critical Role of Kindlin-3 in Intiation of Physiological Thrombus Formation ~ Analysis of Kindlin-3 Deficient Patient

Background: Kindlin-3 which is expressed mainly in hematopoietic cells, is essential for platelet fibrinogen receptor integrin αIIbβ3 (GPIIb-IIIa) activation and kindlin-3 deficiency causes severe bleeding problems. αIIbβ3 activation is tightly regulated by inside-out signaling, and the direct interaction of talin and kindlin-3 with β3-cytoplasmic tail following CalDAG-GEFI-induced Rap1 activation is critical for αIIbβ3 activation. We have reported that immediate and sustained αIIbβ3 activation by inside-out signaling is important for thrombus formation under physiological conditions (Blood 2016). However, the details of inside-out signaling are not still fully understood. Recently we identified patient with kindlin-3 deficiency as the first Japanese patient with kindlin-3 deficiency caused by novel p.W277X nonsense mutation. To clarify the role of kindlin-3 and the molecular mechanism of inside-out signaling, we analyzed single platelet behavior adhered on collagen under physiological flow conditions, and established kindlin-3 deficient human erythroleukemia HEL cell line. Case: The patient was 8-month old female, born to Japanese consanguineous parents. She has been suffering from bleeding tendency shortly after birth. Her peripheral blood showed slightly decreased platelet count (95x103/L), anemia (Hb 6.9 g/L), and elevated leukocyte count of 37.2x106/L with 1.0% blast. Although the surface expressions of glycoproteins were comparable to healthy control, her platelet aggregations and αIIbβ3 activation were strongly impaired in all agonist stimulations due to defective kindlin-3 expression. The sequencing analysis revealed the homozygous novel nonsense mutation, p.W277X (c.918G&gt;A) in kindlin-3. Methods: Human platelets were obtained from healthy control subjects (CT) and patients with kindlin-3 deficiency and Glanzmann thrombasthenia (GT). Whole blood was perfused at a shear rate 1,250s-1 on collagen surface and shear-induced in vitro thrombus formation during 10 minutes was observed. To evaluate the single platelet behavior after the initial attachment on collagen, each single platelet was analyzed by CellTracker software (Pccinini F. et al. Bioinformatics 2015). To further determine the role of kindlin-3 in inside-out signaling, Kindlin-3 was knocked out by CRISPR-Cas9 system and established kindlin-3 deficient HEL cell line. Results: First, we compared shear-induced thrombus formation between CT, GT, and kindlin-3 deficiency. In contrast to the stable and large platelet aggregate formation in CT after 10 minutes blood perfusion, almost no aggregate was observed in both GT and Kindlin-3 deficiency. In kindlin-3 deficiency, the initial platelet attachment on collagen seemed comparable to that of CT and GT. However, the single adherent platelets looked unstable. Next, we determined the behavior of initially attached platelets. Between CT, GT, and kindlin-3 deficiency, the numbers of platelets attached on collagen during 10 seconds were comparable. Between the initially attached platelets, 31.25% of CT platelets formed stable adhesion followed by platelet aggregation. Similar to CT, 33% of GT platelets adhered stably. However, these GT platelets did not proceed to aggregate formation due to αIIbβ3 deficiency. In contrast to GT, kindlin-3 deficient platelets showed increased detachment, only 9% of initially attached platelets stably adhered. These results suggest that kindlin-3 is indispensable for platelet initial adhesion to collagen by integrin α2β1 activation and explains severe bleeding symptoms in kindlin-3 deficiency than GT. To further investigate how kindlin-3 contributes in initial platelet adhesion to collagen, we established kindlin-3 deficient HEL cell line. In contrast to parental HEL cells, PMA stimulation did not induce αIIbβ3 activation in kindlin-3 deficient HEL cells suggesting impaired inside-out signaling. The introduction of wild type kindlin-3 cDNA, but not W227X mutant, rescued αIIbβ3 activation. Conclusion: The detailed analyses by tracking single adherent platelet on collagen confirmed the role of kindlin-3 in initial step of physiological thrombus formation. Newly established kindlin-3 deficient HEL cell line is useful for further exploration of the role of kindlin-3 and inside-out signaling in platelets and expected contribution for development of new antiplatelet therapy. Disclosures Tomiyama: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Chugai: Honoraria; Kyowa-Kirin: Honoraria.

Prevalence of the Mutations Responsible for Glanzmann Thrombasthenia in Horses in Brazil.

Simple SummaryHereditary bleeding disorders occur in different species due to mutations in genes coding specific hemostatic proteins leading to alterations in their synthesis, or to the production of non-functional proteins which leads to impairment of hemostasis. Some of these disorders have been described in horses, i.e., Von Willebrand disease (VWD), hemophilia A, and Glanzmann’s thrombasthenia (GT). GT is an inherited disease characterized by hemorrhage and has been described in different species including horses of varied breeds (Thoroughbred, Standardbred, Oldenburg, Peruvian Paso, and Quarter Horse). There are two different mutations described in horses a single guanine to cytosine substitution (CGG for CCG) and a 10 base pair deletion in the ITGA2B gene.Glanzmann’s thrombasthenia (GT) is an autosomal recessive inherited disorder characterized by changes in platelet aggregation, leading to hemorrhage and epistaxis. To date, two independent mutations have been described in horses and associated with this disorder, a point mutation (c.122G > C) and a 10-base-pair deletion (g.1456_1466del) in the Integrin subunit alpha2β gene (ITGA2B) of horses of different breeds (Quarter Horse, Thoroughbred, Oldenburg, and Peruvian Paso). ITGA2B codifies the αIIb subunit of the αIIbβ3 integrin, also termed platelet fibrinogen receptor. Horses with GT have been diagnosed in the USA, Canada, Japan, and Australia. However, there are no studies on the prevalence of GT in horses. The aim of this study is to evaluate the prevalence of the mutations responsible for GT in horses in Brazil. A total of 1053 DNA samples of clinically healthy Quarter Horse (n = 679) and Warmblood horses (n = 374) were used. DNA fragments were amplified by PCR and sequenced. The genotype of each animal was analyzed and compared to the nucleotide sequence of the ITGA2B gene found on GenBankTM. There were no carriers in the analyzed samples, that is, all animals tested were wild type. Therefore, under the conditions in which this study was carried out, it can be inferred that GT seems to be extremely rare in the population of Quarter Horses and Warmbloods in Brazil, although it is not possible to affirm that there are no horses carrying mutated alleles in Brazil.

Molecular underpinnings of integrin binding to collagen-mimetic peptides containing vascular Ehlers–Danlos syndrome–associated substitutions

Collagens carry out critical extracellular matrix (ECM) functions by interacting with numerous cell receptors and ECM components. Single glycine substitutions in collagen III, which predominates in vascular walls, result in vascular Ehlers-Danlos syndrome (vEDS), leading to arterial, uterine, and intestinal rupture and an average life expectancy of <50 years. Collagen interactions with integrin α2β1 are vital for platelet adhesion and activation; however, how these interactions are impacted by vEDS-associated mutations and by specific amino acid substitutions is unclear. Here, we designed collagen-mimetic peptides (CMPs) with previously reported Gly → Xaa (Xaa = Ala, Arg, or Val) vEDS substitutions within a high-affinity integrin α2β1-binding motif, GROGER. We used these peptides to investigate, at atomic-level resolution, how these amino acid substitutions affect the collagen III-integrin α2β1 interaction. Using a multitiered approach combining biological adhesion assays, CD, NMR, and molecular dynamics (MD) simulations, we found that these substitutions differentially impede human mesenchymal stem cell spreading and integrin α2-inserted (α2I) domain binding to the CMPs and were associated with triple-helix destabilization. Although an Ala substitution locally destabilized hydrogen bonding and enhanced mobility, it did not significantly reduce the CMP-integrin interactions. MD simulations suggested that bulkier Gly → Xaa substitutions differentially disrupt the CMP-α2I interaction. The Gly → Arg substitution destabilized CMP-α2I side-chain interactions, and the Gly → Val change broke the essential Mg2+ coordination. The relationship between the loss of functional binding and the type of vEDS substitution provides a foundation for developing potential therapies for managing collagen disorders.

Molecular genetic diagnosis of Glanzmann syndrome in Iranian population; reporting novel and recurrent mutations

BackgroundGlanzmann thrombasthenia (GT) is a rare autosomal recessive abnormality of platelet aggregation with quantitative and/or qualitative abnormality of αIIbβ3 integrin. The αIIbβ3 is a platelet fibrinogen receptor, which is required for platelet aggregation, firm adhesion, and also spreading.The disease is more prevalent in the populations with a higher rate of consanguineous marriages as in some Middle Eastern populations including Iraq, Jordan, and Iran. Different types of mutations in ITGA2B and ITGB3 genes have been previously reported to cause the disease.ResultIn this study, 16 patients with the clinical diagnosis of GT were studied. Direct sequencing of the exons and exon-intron boundaries of the above genes revealed mutations in 14 patients (detection rate: 87.5%). Briefly, out of fifteen types of identified mutations, 14 were novel. Seven mutations in the ITGB3 gene included 4 missense [c.2T > C, c.155 G > T, c. 538 G > A, c.1990 G > T], one nonsense mutation [c.1303 G > T], a small deletion [c.1656_1658delCTC] and a deletion of one nucleotide [c.401delA]. Mutations in the ITGA2B were 8 different mutations consisting 2 missense [c.286 T > A, c.842 C > T], 2 deletions [c.1899 del T, c.189-319_236del], an insertion [c.1071_1072insG] and one splice site mutations [c.409–3 C > G], one synonymous mutation that might alter the normal splicing process [c.1392 A > G] and a nonsense mutation [c.1555 C > T].The causative mutation in 2 patients remained unknown. Using long-range PCR and sequencing, we found a rather large deletion. The break point of this deletion covers 319 nt from the last part of the first intron and 48 nt from the beginning of the second exon of ITGA2B gene. The deletion was also detected in two unrelated patients with the same ethnicity. In addition, in silico analyses of novel mutations were performed.ConclusionThere was no recurrent mutation in the studied population. This may be due to either small sample size or the heterogeneity of the studied population.

Unexpected Low Expression of Platelet Fibrinogen Receptor in Patients with Chronic Myeloproliferative Neoplasms: How Does It Change with Aspirin?

INTRODUCTIONPatients affected by Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) are considered at high risk of thrombo-haemorrhagic events, but the role of the platelet count in the assessment of the risk of vascular events is still controversial. A tight correlation was found between the platelet count and plasma sCD40L, which appears to be required for thrombus formation in vivo. However sCD40L is increased both in MPNs and reactive thrombocytosis. Intravascular aggregates of platelets and leukocytes, mediated by P-selectin and CD11b on the former and the latter, respectively, have been observed.Both of this processes should imply a perpetual - and measurable - platelet activation. Several studies on platelet function have been already proposed, nevertheless the mechanisms through which platelets are able to trigger vascular events, are not yet adequately clarified.A refined method for the determination of platelet activation appears to be the use of platelet PAC-1 antibody, able to identify the expression of the fibrinogen receptor of platelet glycoprotein IIb/IIIa. This expression is indeed unique in the process of platelet activation, and yet rarely analyzed. Moreover, since the platelet fibrinogen receptor exposure seems to be influenced by turbulence in blood flow, we have thought that its evaluation could provide important biological evidences to explain some clinical manifestations, such as microvascular disturbances.METHODSBlood samples from 40 consecutive MPNs patients who never received cytoreductive agents, were obtained. 28/40 patients were receiving a continuative antiplatelet prophylaxis with low dose aspirin (ASA, 75-100 mg) at the time of collection, while 12/40 of them were not on such therapy. Our aim was to verify the expression of platelet fibrinogen receptors (PFRs) in the two different groups of patients compared to healthy volunteers, using whole blood flow cytometry. In each experiment sodium citrate and heparin (positive control of platelet activation) tubes were collected from the same patient. Within 10 minutes from blood sampling, 5 ml of whole blood from each tube was incubated for 20 minutes at room temperature in the dark with saturating concentration of CD61 PerCP, CD62P PE and PAC-1 FITC. Positive control was also incubated with PAC-1 in the presence of Arg-Gly-Asp-Ser (RGDS) in order to test the specific antibody binding. Samples were fixed with paraformaldehyde 1% for 30 minutes at 4°C in the dark and analyzed on a flow cytometer.RESULTSSurprisingly, we have been able to verify a very low PAC-1 binding to platelets in patients with MPNs not receiving cytoreduction nor antiplatelet agents (33%) if compared to that observed in healthy subjects (61%; p<0.0001). The use of aspirin seems conversely to restore the expression of platelet fibrinogen receptor, as PAC-1 binding capacity is comparable to that of healthy volunteers (56%). No difference was found with respect to the JAK2 Val617Phe mutation and its allele burden. Interestingly, by focusing on the group of patients under antiplatelet prophylaxis and with no history of thrombosis, it was found that subjects with persistent microcirculatory disorders show a higher PAC-1 binding capacity if compared to the asymptomatic ones (67% vs 52%, p= 0.04).CONCLUSIONIn untreated MPNs, a large amount of platelets are resting in a conformation that is unable to bind fibrinogen, as demonstrated by the low PAC-1 expression in cytofluorimetry. This lack of activity can be reversed by administering ASA, which is also known for its fibrinolytic and hypoprothrombinemic effects. We hypotesize that the hypercoagulable states observed in these patient could depend on a primarly plasma-driven impairment of fibrin turnover and thrombin generation. Further investigations are going to be promoted by our Group. PAC-1 could be at the same time a good marker of aspirin resistance in patients experiencing microcirculatory disorders. [Display omitted] DisclosuresMartinelli:Janssen: Consultancy; Jazz Pharmaceuticals: Consultancy; Ariad/Incyte: Consultancy; Pfizer: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy; Abbvie: Consultancy; Roche: Consultancy; Novartis: Speakers Bureau.

Platelet Integrin αIIbβ3 Activation Kinetics in Inherited Platelet Functional Disorders ∼ the Role of ADP Receptor P2Y12, Caldag-GEFI and Kindlin-3 αIIbβ3 Activation By inside-out Signaling

Background and objectivesActivation of platelet fibrinogen receptor integrin αIIbβ3 (GPIIb-IIIa) by inside-out signaling is essential for platelet aggregate formation. In αIIbβ3 activation, it has been established that CalDAG-GEFI-induced Rap1 activation is necessary to induce the direct interaction of β3 cytoplasmic tail with talin and kindlin-3. Agonist induced platelet and αIIbβ3 activation are generally assessed using platelet aggregation assay and flow cytometric analysis of monoclonal antibody specific for activated αIIbβ3, PAC-1, binding. However, we found that the results of PAC-1 binding assay were not associated with severity of bleeding symptoms in patient with CalDAG-GEFI (Blood 2016) or ADP receptor P2Y12 (J Thromb Haemost 2005) deficiency.To further determine the role of molecules in inside-out signaling on αIIbβ3 activation and the impact on hemostasis, we studied the αIIbβ3 activation kinetics in patient's platelets deficient for αIIbβ3, P2Y12, CalDAG-GEFI, or kindlin-3 using the αIIbβ3 velocity assay (Exp Hematol 2013).MethodsHuman platelets were obtained from healthy control subjects and patients with inherited platelet disorders (Glanzmann thrombasthenia, P2Y12 deficiency, CalDAG-GEFI deficiency, and kindlin-3 deficiency). Agonist induced platelet and αIIbβ3 activation were assessed by light transmission aggregometer and PAC-1 binding on flow cytometry. In addition to the conventional PAC-1 binding assay which is simultaneous incubation of stimulated platelets with FITC-PAC-1 for 20 minutes, the time course of αIIbβ3 activation was determined by αIIbβ3 velocity assay. In brief, to measure the activation state of αIIbβ3 at the time of interest (0, 30, 60, 120 and 300 seconds), FITC-labeled PAC-1 was added after the PAR1-AP stimulation. After 30 seconds incubation with FITC-PAC-1, the bound FITC-PAC-1 was immediately assessed by flow cytometry. To determine the thrombus formation under the physiological condition, shear-induced thrombus formation on collagen coated surface was observed.ResultsIn the patient with kindlin-3 deficiency, strongly impaired platelet aggregation and αIIbβ3 activation determined by conventional PAC-1 binding were correlated to the patients severe bleeding problems appeared from early infancy. The αIIbβ3 activation kinetics of kindling-3 deficient platelets showed no PAC-1 binding during 300 seconds after the thrombin receptor agonist PAR1-AP stimulation like platelets of Granzmann thrombasthenia, which suggests the essential role of Kindlin-3 in αIIbβ3 activation. In contrast, in spite of the strongly impaired PAC-1 binding in conventional assay for P2Y12 deficient platelets, the 67 year-old patient showed only minor bleeding symptoms and no transfusion was required during delivery. The αIIbβ3 activation observed in P2Y12 deficiency was transient and the level of initial activation at 30 seconds was the same as healthy control platelets. Under the flow conditions, P2Y12 deficient platelets could form thrombus albeit unstable and fragile. In the 16-yaer-old patient with CalDAG-GEFI deficiency who has been suffering from severe nose bleed and menorrhagia, her platelet aggregation and conventional PAC-1 binding were only modestly affected. The time course of αIIbβ3 activation showed delayed activation. Although αIIbβ3 activation in CalDAG-GEFI deficient platelets reached to similar to that of control at 300 seconds, the initial activation at 30 seconds was significantly decreased to only 19% of control. Her platelets could adhere on collagen under the flow condition. However, almost no aggregate formation was observed which explains her severe bleeding symptoms.ConclusionThe analyses of αIIbβ3 activation kinetics in platelets with inherited platelet disorder revealed the importance of immediate and sustained αIIbβ3 activation for hemostasis in physiological conditions. In addition to well established platelet functional assay, the analysis of αIIbβ3 activation kinetics contributes to understand the patient's bleeding symptoms and also clarifies the role of each molecule in inside-out αIIbβ3 activation, CalDAG-GEFI for the immediate activation, P2Y12 for the maintenance of activation, and Kindlin-3 at the last step of αIIbβ3 activation. DisclosuresKanakura:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding. Tomiyama:Kyowa Hakko Kirin Co., Ltd.: Honoraria; Novartis Pharma Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sysmex Corporation: Consultancy; Chugai Pharmaceutical Co., Ltd.: Honoraria.

Abstract WP56: Imaging Intra-Carotid Thrombosis Using Near InfraRed Fluorescent-NanoDiamond Particles Bio-engineered With the Disintegrin Bitistatin

Background: Thromboembolism is a major cause of events in carotid artery (CA) disease and predisposes the dissemination of clots to upstream ischemic strokes. Although diagnostic methods are available to define thromboembolic strokes (CT, MRI, etc.), there is no technology to provide ambulatory assessment to ‘content image’ thrombi within the CA bifurcation free of the hospital setting. To address this unmet medical need we bioengineered the joining of the disintegrin bitistatin (Bit) to fluorescent NanoDiamond Particles (F-NDP) in order to bind to and non-invasively image vascular clots. F-NDP-Bit preferentially binds to the human platelet fibrinogen receptor (GPIIB/IIIA). F-NDP-Bit was systematically evaluated for NIR emission capacity in various tissues and demonstrated penetration of NIR fluorescence across distances similar to the distance from the human skin surface to the CA bifurcation. Methods: In vitro studies were conducted to evaluate binding of F-NDP-Bit to purified human GPIIB/IIIA. Also, the ability of the F-NDP-Bit to bind to vascular clots In Vivo was investigated in rat FeCl3-induced internal CA coagulation. Results: Using the specific GPIIB/IIIA antagonist lotrafiban, In Vitro specific and concentration-related F-NDP-Bit binding was demonstrated. In Vivo , intravenous administration of F-NDP-Bit during the evolution of FeCl3-induced internal CA thrombosis resulted in the dose-related clot accumulation of F-NDP-Bit that was cross-validated by three independent methods (all measures were significant as described; p &lt; 0.05): ( 1 ) In Situ whole rat imaging via In Vivo Imaging System (IVIS: using Ex 54 nm; Em 695-770 nm); ( 2 ) Ex Vivo whole vessel/clot imaging (Scanning Confocal Microscopy), and ( 3 ) Extract, isolate and quantitate F-NDP-Bit following internal CA clot dissolution (12N HNO3) and direct counting (hemocytometer). Conclusions: These data indicate that F-NDP-Bit might address the need for fast, safe and highly affordable ambulatory imaging of vascular clot burden. Functionalization of F-NDP with selective ligands that can be pathognomonic for many disease situations also could facilitate risk assessment, earlier intervention and preventative medicine.

Hypoxia impairs agonist-induced integrin \u03b1IIb\u03b23 activation and platelet aggregation

Under ischemic conditions, tissues are exposed to hypoxia. Although human physiology, to a certain extent, can adapt to hypoxic conditions, the impact of low oxygen levels on platelet function is unresolved. Therefore, we explored how reduction of atmospheric oxygen levels to 1% might affect agonist-induced aggregation and static adhesion of isolated human platelets. We uncovered that isolated, washed human platelets exposed to hypoxic conditions show reduced thrombin receptor-activating peptide-6 (TRAP-6) and convulxin-induced aggregation. Of note, this hypoxia-triggered effect was not observed in platelet-rich plasma. Independent of the agonist used (TRAP-6, ADP), activation of the platelet fibrinogen receptor integrin αIIbβ3 (GPIIbIIIa, CD41/CD61) was strongly reduced at 1% and 8% oxygen. The difference in agonist-induced integrin αIIbβ3 activation was apparent within 5 minutes of stimulation. Following hypoxia, re-oxygenation resulted in the recovery of integrin αIIbβ3 activation. Importantly, platelet secretion was not impaired by hypoxia. Static adhesion experiments revealed decreased platelet deposition to fibrinogen coatings, but not to collagen or vitronectin coatings, indicating that specifically the function of the integrin subunit αIIb is impaired by exposure of platelets to reduced oxygen levels. Our results reveal an unexpected effect of oxygen deprivation on platelet aggregation mediated by the fibrinogen receptor integrin αIIbβ3.