Platelet Defects Research Articles

Comparative study of hemostatic profile between conventional methods and “Sonoclot Coagulation and Platelet Function Analyzer” in patients with septicemia

Abstract: BACKGROUND: Sonoclot Coagulation and Platelet Functional Analyzer (Sonoclot) operating on the principle of micro-viscometry is known to provide comprehensive information on the entire hemostatic process. We hypothesized that performing global hemostasis assessment in patients with sepsis would identify many more patients with altered hemostasis than the conventional tests. OBJECTIVES: We compared the hemostatic status determined by Sonoclot and that by conventional hemostatic tests. METHODS: It was a prospective analytical study conducted for 18 months in a tertiary care hospital. Sixty-one cases of septicemia admitted in the intensive care units were included in the study. RESULTS: Conventional hemostatic tests (platelet count, prothrombin time [PT], international normalized ratio, and activated partial thromboplastin time [APTT]) were compared with the Sonoclot parameters. Conventional hemostatic parameter PT was prolonged in 60.7% and APTT in 31.1%. Overall, 65.6% of the patients had prolonged coagulation on assessment by conventional hemostatic parameters. Sonoclot-derived parameter-activated clotting time was prolonged in 31.1%, indicating 31.1% of cases to have a hypocoagulable state. The clot rate was increased in 93.4% of subjects, indicating 93.4% to have a hypercoagulable state. Thrombocytopenia was seen in 34.4% and thrombocytosis in 10%. Defective platelet function was observed in 92% of the cases in Sonoclot, and all of them had normal platelet counts. This finding was statistically significant. CONCLUSION: Information about hemostatic status identified by Sonoclot is significantly higher and more complete than that identified by conventional coagulation tests in septicemic patients.

Suppressed ORAI1-STIM1-dependent Ca2+ entry by protein kinase C isoforms regulating platelet procoagulant activity

Agonist-induced rises in cytosolic Ca2+ control most platelet responses in thrombosis and hemostasis. In human platelets we earlier demonstrated that the ORAI1-STIM1 pathway is a major component of extracellular Ca2+ entry, in particular when induced via the ITAM-linked collagen receptor, glycoprotein VI (GPVI). In the present paper, using functionally defective platelets from patients with a loss-of-function mutation in ORAI1 or STIM1, we show that Ca2+ entry induced by the endoplasmic reticulum ATPase inhibitor, thapsigargin, fully relies on this pathway. We demonstrate that both the GPVI-induced and thapsigargin-induced Ca2+ entry is strongly suppressed by protein kinase C (PKC) activation, while leaving intracellular Ca2+ mobilization unchanged. Comparing effects of a PKC inhibitory panel pointed to redundant roles of beta and theta PKC isoforms in Ca2+-entry suppression. In contrast, tyrosine kinases positively regulated GPVI-induced Ca2+ entry and mobilization. Label-free and stable isotope phosphoproteome analysis of GPVI-stimulated platelets suggested a regulatory role of bridging integrator-2 (BIN2), known as important mediator of the ORAI1-STIM1 pathway in mouse platelets. Identified were 25-45 regulated phospho- sites in BIN2 and 16-18 in STIM1. Five of these were characterized as direct substrates of the expressed PKC isoforms alpha, beta delta and theta. Functional platelet testing indicated that the downregulation of Ca2+ entry by PKC resulted in suppressed phosphatidylserine exposure and plasmatic thrombin generation. Conclusively, our results indicate that in platelets multiple PKC isoforms constrain the store-regulated Ca2+ entry via ORAI1-BIN2-STIM1, and hence downregulate platelet-dependent coagulation.

Glanzmann Thrombasthenia – A Rare Case Report

Glanzmann thrombasthenia is a rare inherited bleeding disorder resulting from mutation in platelet membrane glycoprotein (GP) IIb or IIIa leading to impaired platelet function which is characterized by defective platelet aggregation and diminished clot retraction. Glanzmann thrombasthenia patients commonly visit to physician with features of bleeding. Here we discuss about the case of a 32-years-old young female presented with menorrhagia, ecchymosis and occasional gum bleeding. Her coagulation profile was in favor of Glanzmann thrombasthenia. This rare disease has a good outcome if early diagnosis and proper management can be ensured. Delta Med Col J. Jan 2021;9(2): 101-103

A circle of life: platelet and megakaryocyte cytoskeleton dynamics in health and disease.

Platelets are blood cells derived from megakaryocytes that play a central role in regulating haemostasis and vascular integrity. The microtubule cytoskeleton of megakaryocytes undergoes a critical dynamic reorganization during cycles of endomitosis and platelet biogenesis. Quiescent platelets have a discoid shape maintained by a marginal band composed of microtubule bundles, which undergoes remarkable remodelling during platelet activation, driving shape change and platelet function. Disrupting or enhancing this process can cause platelet dysfunction such as bleeding disorders or thrombosis. However, little is known about the molecular mechanisms underlying the reorganization of the cytoskeleton in the platelet lineage. Recent studies indicate that the emergence of a unique platelet tubulin code and specific pathogenic tubulin mutations cause platelet defects and bleeding disorders. Frequently, these mutations exhibit dominant negative effects, offering valuable insights into both platelet disease mechanisms and the functioning of tubulins. This review will highlight our current understanding of the role of the microtubule cytoskeleton in the life and death of platelets, along with its relevance to platelet disorders.

Linking Computational Fluid Dynamics Modeling to Device-Induced Platelet Defects in Mechanically Assisted Circulation.

Thrombotic and bleeding events are the most common hematologic complications in patients with mechanically assisted circulation and are closely related to device-induced platelet dysfunction. In this study, we sought to link computational fluid dynamics (CFD) modeling of blood pumps with device-induced platelet defects. Fresh human blood was circulated in circulatory loops with four pumps (CentriMag, HVAD, HeartMate II, and CH-VAD) operated under a total of six clinically representative conditions. Blood samples were collected and analyzed for glycoprotein (GP) IIb/IIIa activation and receptor shedding of GPIbα and GPVI. In parallel, CFD modeling was performed to characterize the blood flow in these pumps. Numerical indices of platelet defects were derived from CFD modeling incorporating previously derived power-law models under constant shear conditions. Numerical results were correlated with experimental results by regression analysis. The results suggested that a scalar shear stress of less than 75 Pa may have limited contribution to platelet damage. The platelet defect indices predicted by the CFD power-law models after excluding shear stress <75 Pa correlated excellently with experimentally measured indices. Although numerical prediction based on the power-law model cannot directly reproduce the experimental data. The power-law model has proven its effectiveness, especially for quantitative comparisons.

Abstract 119: LRRC8 Complex Is An ATP Release Channel Regulating Platelet Activation And Thrombosis

Platelet shape and volume changes are early mechanical events in platelet activation proposed to contribute to arterial thrombosis. Here, we identify single-nucleotide polymorphisms (SNPs) in four leucine-rich repeat containing protein subunits (LRRC8A, B, C, and D) expressed in human platelets that are independently associated with increased mean platelet volume in human genetic studies - implicating the volume-sensing LRRC8 channel complex as regulating platelet function in humans. We show LRRC8A is required for functional LRRC8/VRAC in megakaryocytes (MKs). MK-specific LRRC8A conditional knockout mice give rise to LRRC8A-null platelets with larger mean volumes. LRRC8A-null platelets and CRISPR-edited human LRRC8A KO MKs (hiPSC MKs) have reduced agonist -stimulated P-selectin exposure, and αIIbβ3 integrin activation. LRRC8A-null platelets exhibit impaired adhesion to collagen-coated surfaces, agonist-induced platelet aggregation and ATP secretion. MK-specific LRRC8A conditional knockout mice have reduced platelet thrombus formation in laser-induced cremaster arteriolar thrombosis and prolonged occlusion times in FeCl 3 -induced carotid arterial thrombosis, compared to control mice, without affecting tail bleeding times. Mechanistically, LRRC8A mediates swelling-induced ATP release to amplify agonist-stimulated calcium influx and aggregation in platelets. Treatment of platelets with small-molecule LRRC8 channel inhibitors recapitulate the defects in LRRC8A-null platelets. These studies identify the mechanoresponsive LRRC8 channel complex as a regulator of platelet function and thrombosis - providing a proof-of-concept for a novel anti-thrombotic drug target.

Surface immobilization mechanisms of cobalt ions on hydroxyapatite catalyst supports

Cobalt deposition in an excess of solution was used to design Co-modified hydroxyapatite materials with various Co loadings and controlled dispersion. It is rationalized how the properties of hydroxyapatite supports (more or less stoichiometric compositions, nanorod or platelet morphologies and crystalline (100) zig-zag termination or non-apatitic hydrated external layer), influence the immobilization processes of Co by operating either with slightly acidic (natural) or basic pH of the suspension media. Four cobalt immobilization mechanisms impacting the final dispersion of Co on hydroxyapatites were identified by combining structural (XRD, 1H and 31P solid-state NMR, UV-Vis, Raman and X-ray fluorescence spectroscopies), surface (XPS) characterizations of Co-modified hydroxyapatites after drying and thermal treatment at 500 °C, and monitoring of the pH and the composition of the supernatant solutions during the Co deposition step. On dried Co-modified crystalline stoichiometric hydroxyapatite nanorods, cobalt is highly dispersed through cationic exchange or strong electrostatic adsorption (SEA) at slightly acidic or basic pH, respectively. After thermal treatment at 500 °C, only cation exchange preserved atomic dispersion of Co(II) ions since Co3O4 nanoparticles were observed on samples for which Co deposition occurred via SEA. On defective hydroxyapatite platelets, cobalt deposited at acidic natural pH could diffuse in an external non-apatitic layer, whereas under basic pH media, this surface layer was hydrolysed, resulting in the formation of a cobalt-substituted hydroxyapatite layer in which only a limited fraction of the surface cobalt species could be probed by XPS.

Altered platelet-megakaryocyte endocytosis and trafficking of albumin and fibrinogen in RUNX1 haplodeficiency

AbstractPlatelet α-granules have numerous proteins, some synthesized by megakaryocytes (MK) and others not synthesized but incorporated by endocytosis, an incompletely understood process in platelets/MK. Germ line RUNX1 haplodeficiency, referred to as familial platelet defect with predisposition to myeloid malignancies (FPDMMs), is associated with thrombocytopenia, platelet dysfunction, and granule deficiencies. In previous studies, we found that platelet albumin, fibrinogen, and immunoglobulin G (IgG) were decreased in a patient with FPDMM. We now show that platelet endocytosis of fluorescent-labeled albumin, fibrinogen, and IgG is decreased in the patient and his daughter with FPDMM. In megakaryocytic human erythroleukemia (HEL) cells, small interfering RNA RUNX1 knockdown (KD) increased uptake of these proteins over 24 hours compared with control cells, with increases in caveolin-1 and flotillin-1 (2 independent regulators of clathrin-independent endocytosis), LAMP2 (a lysosomal marker), RAB11 (a marker of recycling endosomes), and IFITM3. Caveolin-1 downregulation in RUNX1-deficient HEL cells abrogated the increased uptake of albumin, but not fibrinogen. Albumin, but not fibrinogen, partially colocalized with caveolin-1. RUNX1 KD resulted in increased colocalization of albumin with flotillin and fibrinogen with RAB11, suggesting altered trafficking of both proteins. The increased uptake of albumin and fibrinogen, as well as levels of caveolin-1, flotillin-1, LAMP2, and IFITM3, were recapitulated by short hairpin RNA RUNX1 KD in CD34+-derived MK. To our knowledge, these studies provide first evidence that platelet endocytosis of albumin and fibrinogen is impaired in some patients with RUNX1-haplodeficiency and suggest that megakaryocytes have enhanced endocytosis with defective trafficking, leading to loss of these proteins by distinct mechanisms. This study provides new insights into mechanisms governing endocytosis and α-granule deficiencies in RUNX1-haplodeficiency.

A novel GATA1 variant p.G229D causing the defect of procoagulant platelet formation

IntroductionGATA1 is one of the master transcription factors in hematopoietic lineages development which is crucial for megakaryocytic differentiation and maturation. Previous studies have shown that distinct GATA1 variants are associated with varying severities of macrothrombocytopenia and platelet dysfunction. ObjectiveTo determine the underlying pathological mechanisms of a novel GATA1 variant (c. 686G > A, p. G229D) in a patient with recurrent traumatic muscle hematomas. MethodsComprehensive phenotypic analysis of the patient platelets was performed. Procoagulant platelet formation and function were detected using flow cytometry assay and thrombin generation test (TGT), respectively. The ANO6 expression was measured by qPCR and western blot. The intracellular supramaximal calcium flux was detected by Fluo-5N fluorescent assay. ResultsThe patient displayed mild macrothrombocytopenia with defects of platelet granules, aggregation, and integrin αIIbβ3 activation. The percentage of the procoagulant platelet formation of the patient upon the stimulation of thrombin plus collagen was lower than that of the healthy controls (40.9 % vs 49.0 % ± 5.1 %). The patient platelets exhibited a marked reduction of thrombin generation in platelet rich plasma TGT compared to the healthy controls (peak value: ∼70 % of the healthy controls; the endogenous thrombin potential: ∼40 % of the healthy controls). The expression of ANO6 and intracellular calcium flux were impaired, which together with abnormal granules of the patient platelets might contribute to defect of procoagulant platelet function. ConclusionsThe G229D variant could lead to a novel platelet phenotype characterized by defective procoagulant platelet formation and function, which extended the range of GATA1 variants associated platelet disorders.

PFA-100 System: A New Method for Assessment of Platelet Dysfunction.

This is a celebratory reprint of a historical paper published in STH in 1998. The original Abstract follows.The PFA-100 system is a platelet function analyzer designed to measure platelet-related primary hemostasis. The instrument uses two disposable cartridges: a collagen/epinephrine (CEPI) and a collagen/ADP (CADP) cartridge. Previous experience has shown that CEPI cartridges detect qualitative platelet defects, including acetylsalicylic acid (ASA)-induced abnormalities, while CADP cartridges detect only thrombocytopathies and not ASA use. In this seven-center trial, 206 healthy subjects and 176 persons with various platelet-related defects, including 127 ASA users, were studied. The platelet function status was determined by a platelet function test panel. Comparisons were made as to how well the defects were identified by the PFA-100 system and by platelet aggregometry. The reference intervals for both cartridges, testing the 206 healthy subjects, were similar to values described in smaller studies in the literature (mean closure time [CT] of 132 seconds for CEPI and 93 seconds for CADP). The use of different lot numbers of cartridges or duplicate versus singleton testing revealed no differences. Compared with the platelet function status, the PFA-100 system had a clinical sensitivity of 94.9% and a specificity of 88.8%. For aggregometry, a sensitivity of 94.3% and a specificity of 88.3% were obtained. These values are based on all 382 specimens. A separate analysis of sensitivity by type of platelet defect, ASA use versus congenital thrombocytopathies, revealed for the PFA-100 system a 94.5% sensitivity in identifying ASA users and a 95.9% sensitivity in identifying the other defects. For aggregometry, the values were 100% for ASA users and 79.6% for congenital defects. Analysis of concordance between the PFA-100 system and aggregometry revealed no difference in clinical sensitivity and specificity between the systems (p > 0.9999). The overall agreement was 87.5%, with a Kappa index of 0.751. The two tests are thus equivalent in their ability to identify normal and abnormal platelet defects. Testing 126 subjects who took 325 mg ASA revealed that the PFA-100 system (CEPI) was able to detect 71.7% of ASA-induced defects with a positive predictive value of 97.8%. The overall clinical accuracy of the system, calculated from the area under the receiver operating characteristic curve, was 0.977. The data suggest that the PFA-100 system is highly accurate in discriminating normal from abnormal platelet function. The ease of operation of the instrument makes it a useful tool to use in screening patients for platelet-related hemostasis defects.

Management of pregnant women who have bleeding disorders.

Bleeding disorders, including von Willebrand disease (VWD), hemophilia, other coagulation factor deficiencies, platelet disorders, defects of fibrinolysis, and connective tissue disorders, have both maternal and fetal implications. Successful management of bleeding disorders in pregnant women requires not only an understanding of bleeding disorders but also an understanding of when and how bleeding occurs in pregnancy. Bleeding does not occur during a normal pregnancy with a healthy placenta. Bleeding occurs during pregnancy when there is an interruption of the normal utero-placental interface, during miscarriage, during an ectopic pregnancy, or at the time of placental separation at the conclusion of pregnancy. Although mild platelet defects may be more prevalent, the most commonly diagnosed bleeding disorder among women is VWD. Other bleeding disorders are less common, but hemophilia carriers are unique in that they are at risk of bleeding themselves and of giving birth to an affected male infant. General guidance for maternal management of a woman who is moderately or severely affected includes obtaining coagulation factor levels at a minimum in the third trimester; planning for delivery at a center with hemostasis expertise; and anticipating the need for hemostatic agents. General guidance for fetal management includes pre-pregnancy counseling; the option of preimplantation genetic testing for hemophilia; delivery at a tertiary care center with pediatric hematology and newborn intensive care; consideration of cesarean delivery of a potentially severely affected infant; and avoidance of invasive procedures such as scalp electrodes and operative vaginal delivery in any potentially affected infant.

Functional Classification of Germline RUNX1 Variants Identified in Patients Suspected of Familial Platelet Disorder with Associated Myeloid Malignancies

RUNX1 is a master regulator of hematopoiesis. A highly conserved runt-homology domain (RHD) located near the N-terminus is responsible for DNA binding and interaction with its heterodimeric partner CBFβ, whereas the C-terminus contains the transactivation domain (TAD). Somatic variants and chromosomal abnormalities affecting RUNX1 are commonly observed in hematological malignancies, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Heterozygous germline RUNX1 variants are responsible for familial platelet disorder with associated myeloid malignancies (FPDMM), an autosomal dominant disorder with incomplete penetrance and a broad spectrum of clinical phenotypes, including thrombocytopenia, functional platelet defects, and an increased risk (35-45%) of developing MDS and AML. In 2019, our group launched a longitudinal natural history study of patients with germline RUNX1 variants to increase our understanding of FPDMM, which will hopefully lead to better clinical management and potentially new therapies. The majority of RUNX1 variants are private with limited segregation data available, making variant interpretation challenging. A significant number of cases have variants of unknown significance (VUS) in RUNX1. Therefore, it is important to determine if these VUS are pathogenic (or benign) in order to improve the diagnosis and clinical management for these individuals. To functionally characterize each variant, we used well-established in vitro functional assays to assess the damaging effect of each RUNX1 VUS, including transactivation reporter assays, electrophoretic mobility shift assays, co-immunoprecipitation, immunofluorescence, and western blot with cell fractionation. All assays include RUNX1 wildtype and known benign and pathogenic controls, as well as co-expression with its binding partner CBFβ. The VUS Ser94Arg located in RHD of RUNX1 has showed impaired CSF1R transactivation (&amp;lt; 20% of wildtype), reduced subcellular co-localization of RUNX1 with CBFβ, and reduced heterodimerization with CBFβ. These results together with population frequency data, in silico prediction, phenotype and segregation data support the reclassification of this variant into the likely pathogenic category. On the other hand, the VUS Asp123His shows no damaging effect in vitro, and could be reclassified to likely benign. Using these assays, we have confirmed or reclassified several RUNX1 variants using the criteria set by the ACGM Myeloid Malignancy VCEP (Luo et al, Blood Adv. 2019; 3:2962). Together, these results highlight the importance of using in vitro functional assays for RUNX1 variant classification. Moreover, we sought to determine the potential of using platelet transcriptomics for RUNX1 variant classification and to gain a deeper understanding of FPDMM pathogenesis. Clariom™ D microarrays identified 218 differentially expressed genes (DEG; absolute fold change ≥ 2; q-value &amp;lt; 0.05) between FPDMM patients and healthy controls, with 90 genes (41%) being upregulated and 128 genes (59%) being downregulated in patients. Hierarchical clustering showed that the selected FPDMM patients with pathogenic and likely pathogenic RUNX1 variants clustered together and separately from healthy controls, with well-established pathogenic variants, such as Arg201Ter and Arg201Gln, at the opposite end of the dendrogram, and likely pathogenic variants such as Tyr403Cysfs153 closer to healthy controls. Our results show that FPDMM patients exhibit reduced expression of important genes related to thrombocytopenia and platelet dysfunction. Furthermore, several top upregulated genes identified in FPDMM patient's platelets are associated with poorer outcomes in AML patients, according to the TCGA dataset. Overall, our study shows the importance of performing functional assays to aid RUNX1 variant classification and to facilitate the study of FPDMM pathogenesis. We are currently analyzing the platelet proteome data of FPDMM patients by LC-MS/MS, which will be reported at the annual meeting.

Qualitative platelet alterations in May — Hegglin anomaly

Introduction: May — Hegglin anomaly is an autosomal dominant mutation in the MYH9 gene that disrupts the dynamics of the cytoskeleton in megakaryocytes. Megakaryocyte dysfunction entails a violation of thrombocytopoiesis, which is manifested by macrothrombocytopenia, sometimes associated with bleeding. Aim: to study the structure and function of platelets in the family members with documented May — Hegglin anomaly. Patients and methods. The proband, her sister and their mother who all had a heterozygous mutation R1933X in the MYH9 gene were examined. The examination included complete blood count, peripheral blood microscopy, platelet flow cytometry, blood clot contraction kinetics, scanning and transmission electron microscopy of platelets. The control group included10 healthy donors. Results. In the proband and her sister the degree of contraction of blood clots was reduced. Unstimulated platelets displayed overexpression of P-selectin and active αIIbβ3 integrin. After TRAP-induced stimulation the proportion of platelets expressing P-selectin in the patients was below normal, indicating partial platelet refractoriness. Electron microscopy of the unstimulated platelets revealed macrothrombocytosis and multiple filopodia, as well as enlargement of the open canalicular system. Conclusion. The May — Hegglin anomaly caused by a heterozygous mutation R1933X in the MYH9 gene is accompanied by thrombocytopenia associated with structural and functional platelet defects. Partial platelet refractoriness and contractile dysfunction can impair the retraction of hemostatic clots, predisposing patients with MYH9-related disorders to bleeding.

Pregnancy and Delivery Outcome Among Ladies with Glanzmann Thrombasthenia: A Report from Glanzmann Thrombasthenia Registry of Al-Madinah, Saudi Arabia

Introduction Glanzmann's thrombasthenia (GT) is an autosomal recessive bleeding disorder due to functional platelet defects impairing platelet aggregation. AlMadinah has reported the highest prevalence of GT globally, with a prevalence of 1:10,000. Pregnancy had been reported among ladies with GT with different outcomes. As a bleeding disorder, Pregnancy among ladies with GT carries a higher risk for bleeding either during pregnancy or intra and postpartum. Neonates have an increased risk for hemorrhage, either due to alloimmunization or they may carry the gene of GT. Many GT ladies got pregnant earlier when GT diagnosis was difficult, and they did not know their diagnosis; even after the diagnosis was possible, ceasing the menstrual cycle was not culturally acceptable. In this study, we are describing the pregnancies among GT ladies and their outcomes. Methods This is a report from the AlMadinah GT registry in the Madinah Hemophilia Comprehensive Care Center. The registry covers a period of 20 years and includes all patients with a confirmed diagnosis of GT. 136 GT patients were identified. 11 patients had no data available. A spreadsheet designed to collect detailed data about pregnancy, delivery, and neonates. Electronic and hard copy medical records have been accessed to collect data. Data was analyzed by the statistical package of social sciences (SPSS) version 23. The research and ethical committee approved the study. Results Twenty-eight pregnancies have been identified. The median age of mothers at the time of pregnancy was 30 ± 5.7 years (20 - 42 years). Mothers' Gravidity was 1-7 (median 2.5 ± 1.6), and parity was 1-6 (median 2 ±1.4). Five miscarriages (18%) identified occurred at the 17 th, 11 th, 13 th and 15 th gestational weeks. In contrast, 5 th one is not on record. Three miscarriages took place after moderate to severe vaginal bleeding. Mild to severe post-abortion bleeding was controlled with platelets transfusion, activated human recombinant factor 7 (rFVIIa), and tranexamic acid. Thirty-two bleeding episodes occurred during 16 pregnancies, with 1-5 bleeding episodes per pregnancy (Median of 2 episodes). Seven (22%) bleeding episodes were vaginal bleeds, while 25 (78%) were GT-related bleeds such as gum bleeding or epistaxis. Two patients must undergo plasma exchange and be on steroids due to alloimmunization development during pregnancy. Delivery occurred at the gestational age of 34-40 weeks (median 38 ± 2 weeks). Sixteen (70%) deliveries were vaginal, and 7(30%) were cesarean sections. The indications for cesarean sections were family request in 4 cases, fetal distress in two instances, and precautional section in one case. Prophylactic platelets transfusion had given pre-delivery on four occasions, rFVIIa on 4, and a combination of rFVIIa and platelets transfusion on six occasions. Tranexamic acid was used in all cases. Ten deliveries were conducted with no prophylaxis. Post-partum hemorrhage (PPH) occurred in 18 (64%) cases, 13 (72%) cases of primary PPH, and 5 (28%) cases of secondary PPH. We could not find a correlation between pre-delivery prophylaxes and PPH (P=0.2). PPH had controlled successfully with platelets transfusion and rFVIIa though red blood cell transfusion was needed on eight occasions. Twenty-eight pregnancies produced 24 babies (13 girls and 11 boys). Four babies had bleeding (2 intracranial hemorrhages, one subdural hematoma, and one gum oozing), and 9 had alloimmunization with mild to severe thrombocytopenia. Of all babies tested for GT, 6 of them were GT. Conclusions Pregnancy among ladies with GT carries a higher risk of bleeding. Management of pregnancy in women with GT has to be in a Multidisciplinary approach. The high rate of PPH among GT ladies makes it essential to treat it promptly. Pregnancy among ladies with GT should be studied in multi-centers sitting to learn more about clinical courses and management.

Blood Coagulation and Platelet Function Are Impaired By the ARP2 p.R258G Variant in Mice

Introduction: Venous thromboembolism (VTE) displays significant heritability, yet the genes explaining this heritability are not completely characterized. We previously identified that tissue factor pathway inhibitor heterozygosity ( Tfpi+/−) on the homozygous F5L background ( F5L/L) produces a lethal perinatal thrombotic phenotype. Using a sensitized ENU mutagenesis screen, we discovered an autosomal dominant missense mutation in the Actr2 gene (p.R258G, Actr2G) that suppresses thrombosis and restores survival to F5L/LTfpi+/− mice. Inheritance of the Actr2Gmutation is associated with nearly complete suppression of the lethal F5L/LTfpi+/- phenotype. Actr2 codes for the ARP2 protein, an essential component of the ARP2/3 complex, which mediates actin cytoskeletal rearrangements during platelet activation. We hypothesized that the Actr2G variant affects blood coagulation and platelet function to reduce thrombosis. Methods: To investigate potential effects of the Actr2G mutation on the coagulation cascade, prothrombin time (PT), activated partial thromboplastin time (aPTT), and plasma-dependent thrombin generation assays (TGA) were used to evaluate clot formation. A mouse tail clip assay was used to measure bleeding time and quantify blood loss. Finally, platelets were assessed for functional defects using platelet spreading and aggregometry assays. Results: Actr2 +/Gplasma had significantly prolonged PT and aPTT compared to wild type controls (N&amp;gt;17, p&amp;lt;0.03). There was also a significant reduction in Actr2+/G thrombin peak height compared to wildtypes (N=10; p=0.03). Despite the delayed time to clot formation, Actr2+/G mice did not exhibit a bleeding phenotype and showed no significant differences in blood loss from tail clip assays. Activated Actr2+/G platelets exhibited reduced surface area and adhesion (N=5; p&amp;lt;0.02), as well as significant defects in collagen induced whole blood aggregation (N=10; p&amp;lt;0.02). Discussion: The results of the PT, aPTT and TGA indicate reduced thrombin generation potential, suggesting a defect in the common pathway of coagulation. This hypocoaguability may be due to a decrease in circulating procoagulant factors, or an increase in endogenous thrombin inhibitors. Both the significantly decreased aggregation and defective platelet spreading in the Actr2+/G mice indicate that Actr2+/G platelets are less responsive to agonists, a protective mechanism that contributes to overall thrombosis suppression. Conclusion: In summary, our studies demonstrate the pleiotropic effects that the Actr2G mutation exerts in suppressing thrombosis. The combination of altered plasma coagulation and platelet reactivity likely contribute to suppression of the F5L/LTfpi+/− phenotype. Defects in these actin-mediated processes precipitated by Actr2+/G are the likely root of the F5L/LTfpi+/- phenotype rescue, demonstrating the power of this gene in maintaining hemostatic balance. However, the combination of these thrombosis suppressing phenotypes is not significant enough to prolong the bleeding time in mice with the Actr2G mutation. Thus, our results suggest that targeting actin proteins such as ARP2 for novel antithrombotic drug therapies may have clinical applicability with limited effects on bleeding.

Adding to the evidence of gene-disease association of RAP1B and syndromic thrombocytopenia.

Syndromic constitutive thrombocytopenia encompasses a heterogeneous group of disorders characterised by quantitative and qualitative defects of platelets while featuring other malformations. Recently, heterozygous, de novo variants in RAP1B were reported in three cases of syndromic thrombocytopenia. Here, we report two additional, unrelated individuals identified retrospectively in our data repository with heterozygous variants in RAP1B: NM_001010942.2(RAP1B):c.35G>A, p.(Gly12Glu) (de novo) and NM_001010942.2(RAP1B):c.178G>A, p.(Gly60Arg). Both individuals had thrombocytopenia, as well as congenital malformations, and neurological, behavioural, and dysmorphic features, in line with previous reports. Our data supports the causal role of monoallelic RAP1B variants that disrupt RAP1B GTPase activity in syndromic congenital thrombocytopenia.

Novel hemizygous CORO1A variant leads to combined immunodeficiency with defective platelet calcium signaling and cell mobility

To date, fewer than 20 patients have been identified as having germline biallelic mutations in the coronin-1A gene (CORO1A) and its protein with clinical features of combined immunodeficiency characterized by T-cell lymphopenia ranging from the severe phenotype to the mild phenotype, recurrent infections, and lymphoproliferative disorders. However, the effects of CORO1A protein disruption on actin-dependent functions in primary cells have not been fully delineated. We sought to characterize the underlying defects of actin-dependent cellular functions in a female patient with combined immunodeficiency caused by a novel missense variant in the CORO1A gene in combination with a de novo heterozygous microdeletion of chromosome 16p11.2 and also to provide evidence of the pathogenicity of this gene mutation. To identify the genetic defect, next-generation sequencing followed by Sanger confirmation and array comparative genomic hybridization were performed. Western blot and quantitative PCR tests were used to assess the effects on the protein. Flow cytometry and live microscopy were performed to investigate cellular motility and immune cell counts and function. We demonstrated that the CORO1A hemizygous variant c.19C>T, p. A7C induces significant decreases in cellular levels of the CORO1A protein while leaving mRNA concentrations unaffected. The observed mutation resulted in impaired natural killer cell cytotoxicity and platelet calcium signaling. In addition, primary granulocytes and mesenchymal cells showed significant defects in motility. Collectively, we added new data about the CORO1A gene as a key player in actin cytoskeleton dynamics and cell signaling. Our findings expand the clinical spectrum regarding CORO1A protein deficiency and confirm the importance of a personalized therapeutic approach for each patient.

Clopidogrel Responsiveness in Patients Undergoing Percutaneous Coronary Intervention using Multiplate Analyzer: Frequency and Outcomes

Objectives: Over and under response to dual antiplatelet therapy (DAPT) can lead to bleeding and thrombotic events in patients undergoing coronary stent placement. The present study aimed to assess the platelet response to clopidogrel in patient undergoing percutaneous coronary intervention (PCI) using Multiplate Analyzer. The primary outcome in the present study was the short-term incidence of stent thrombosis and bleeding events. Background: Multiple electrode aggregometry is a rapid and standardized tool to for diagnosis of platelet defects and monitoring response to DAPT. Methods: A hospital-based, prospective study was conducted on 431 patients who underwent PCI from September 2016 to November 2017 and received clopidogrel therapy. The platelet aggregometry was done using a Multiplate analyzer (Dynabyte, Munich, Germany). Patients were followed for 30 days to assess the incidence of stent thrombosis and bleeding. Results: The patients’ mean age was 58 ± 6.7 years. A total of 40% of the patients were diabetic and 7.7% had chronic renal failure. The rate of clopidogrel non-responders was 10.7%, while clopidogrel over-responders were 18.3%. Patients with diabetes and chronic renal failure had significantly lower platelet responsiveness (40.1% with p &lt; 0.05 and 7.7% with p &lt;0.005, respectively). Smoking was significantly associated with platelet over-responsiveness (39.4%, p &lt; 0.001). Patients with low platelet responsiveness to clopidogrel were associated with an increased risk of definite stent thrombosis (p &lt; 0.005), while increasing bleeding risk was significantly associated with over-responsiveness to patients to clopidogrel (p &lt;0.001). Conclusions: Antiplatelet responsiveness showing individual variability with increased risk of stent thrombosis among the cases with no response to the effect of clopidogrel and high risk of bleeding with the over-responsiveness group.

SINE-VNTR-Alu retrotransposon insertion as a novel mutational event underlying Glanzmann thrombasthenia

BackgroundGlanzmann thrombasthenia (GT) is an autosomal recessive platelet aggregation disorder caused by mutations in ITGA2B or ITGB3. ObjectivesWe aimed to assess the phenotype and investigate the genetic etiology of a GT pedigree. MethodsA patient with bleeding manifestations and mild mental retardation was enrolled. Complete blood count, coagulation, and platelet aggregation tests were performed. Causal mutations were identified via whole exome and genome sequencing and subsequently confirmed through polymerase chain reaction and Sanger sequencing. The transcription of ITGB3 was characterized using RNA sequencing and reverse transcription polymerase chain reaction. The αⅡb and β3 biosynthesis was investigated via whole blood flow cytometry and in vitro studies. ResultsGT was diagnosed in a patient with defective platelet aggregation. Novel compound heterozygous ITGB3 variants were identified, with a maternal nonsense mutation (c.2222G>A, p.Trp741∗) and a paternal SINE-VNTR-Alu (SVA) retrotransposon insertion. The 5' truncated SVA element was inserted in a sense orientation in intron 11 of ITGB3, resulting in aberrant splicing of ITGB3 and significantly reducing β3 protein content. Meanwhile, both the expression and transportation of β3 were damaged by the ITGB3 c.2222G>A. Almost no αⅡb and β3 expressions were detected on the patient's platelets surface. ConclusionNovel compound heterozygous ITGB3 mutations were identified in the GT pedigree, resulting in defects of αⅡbβ3 biosynthesis. This is the first report of SVA retrotransposon insertion in the genetic pathogenesis of GT. Our study highlights the importance of combining multiple high-throughput sequencing technologies for the molecular diagnosis of genetic disorders.

Combined immunodeficiency and impaired PI3K signaling in a patient with biallelic LCP2 variants

Inborn errors affecting components of the T-cell receptor signaling cascade cause combined immunodeficiency with various degrees of severity. Recently, homozygous variants in LCP2 were reported to cause pediatric onset of severe combined immunodeficiency with neutrophil, platelet, and T-and B-cell defects. We sought to unravel the genetic cause of combined immunodeficiency and early-onset immune dysregulation in a 26-year-old man who presented with specific antibody deficiency, autoimmunity, and inflammatory bowel disease since early childhood. The patient was subjected to whole-exome sequencing of genomic DNA and examination of blood neutrophils, platelets, and T and B cells. Expression levels of the Src homology domain 2-containing leukocyte protein of 76 kDa (SLP76) and tonic and ligand-induced PI3K signaling were evaluated by flow-cytometric detection of phosphorylated ribosomal protein S6 in B and T cells. Compound heterozygous missense variants were identified in LCP2, affecting the proline-rich repeat domain of SLP76 (p.P190R and p.R204W). The patient's total B- and T-cell numbers were within the normal range, as was platelet function. However, neutrophil function, numbers of unswitched and class-switched memory B cells, and serum IgA were decreased. Moreover, intracellular SLP76 protein levels were reduced in the patient's B cells, CD4+ and CD8+ T cells, and natural killer cells. Tonic and ligand-induced levels of phosphorylated ribosomal protein S6 and ligand-induced phosphorylated PLCγ1 were decreased in the patient's B cells and CD4+ and CD8+ T cells. Biallelic variants in LCP2 impair neutrophil function and T-cell and B-cell antigen-receptor signaling and can cause combined immunodeficiency with early-onset immune dysregulation, even in the absence of platelet defects.