Research

10 pages, 439 KiB

Open AccessArticle

Glanzmann Thrombasthenia in Pakistani Patients: Identification of 7 Novel Pathogenic Variants in the Fibrinogen Receptor αIIbβ3

by Muhammad Younus Jamal Siddiqi, Doris Boeckelmann, Arshi Naz, Ayisha Imran, Shariq Ahmed, Akbar Najmuddin and Barbara Zieger

Cells 2023, 12(2), 213; https://doi.org/10.3390/cells12020213 - 04 Jan 2023

Cited by 2 | Viewed by 2097

Abstract

Glanzmann thrombasthenia (GT) is a rare autosomal recessive inherited platelet disorder occurring frequently in populations with high incidence of consanguineous marriages. GT is characterized by quantitative and/or qualitative defect of the platelet αIIbβ3 (GPIIb/IIIa) receptor caused by pathogenic variants of the encoding genes: [...] Read more.

Glanzmann thrombasthenia (GT) is a rare autosomal recessive inherited platelet disorder occurring frequently in populations with high incidence of consanguineous marriages. GT is characterized by quantitative and/or qualitative defect of the platelet αIIbβ3 (GPIIb/IIIa) receptor caused by pathogenic variants of the encoding genes: ITGA2B and ITGB3. Patients present with a moderate to severe bleeding tendency with normal platelet count. Platelets show reduced/absent aggregation for all agonists except ristocetin in light transmission aggregometry and reduced/absent αIIbβ3 expression in flow cytometry (FC). In this study, we investigated a cohort of 20 Pakistani patients and 2 families collected from the National Institute of Blood Disease, Karachi and Chughtai’s Lab, Lahore. Platelet aggregation studies, FC (platelet CD41, CD61, CD42a, CD42b) and direct sequencing of the candidate genes were performed. All patients showed altered platelet aggregation, but normal agglutination after stimulation with ristocetin. Absent/reduced αIIbβ3 receptor expression was present in the platelets of 16 patients, in 4 patients expression was borderline/normal. Candidate gene sequencing identified pathogenic/likely pathogenic variants in 15 patients. Seven variants are novel. One patient with absent receptor expression remained without genetic finding. 13 (86.7%) of 15 patients stated consanguinity reflected by homozygosity finding in 14 (93.3%) patients. Full article

(This article belongs to the Special Issue Impact of Platelet Defects on Pathophysiological Processes)

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10 pages, 1514 KiB

Open AccessArticle

Pervasive Platelet Secretion Defects in Patients with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)

by Johannes Kalbhenn, Jan-Steffen Pooth, Georg Trummer, David Kranzhöfer, Axel Schlagenhauf and Barbara Zieger

Cells 2023, 12(1), 193; https://doi.org/10.3390/cells12010193 - 03 Jan 2023

Cited by 3 | Viewed by 1960

Abstract

Critically ill COVID-19 patients suffer from thromboembolic as well as bleeding events. Endothelial dysfunction, spiking of von Willebrand factor (vWF), and excessive cytokine signaling result in coagulopathy associated with substantial activation of plasmatic clotting factors. Thrombocytopenia secondary to extensive platelet activation is a [...] Read more.

Critically ill COVID-19 patients suffer from thromboembolic as well as bleeding events. Endothelial dysfunction, spiking of von Willebrand factor (vWF), and excessive cytokine signaling result in coagulopathy associated with substantial activation of plasmatic clotting factors. Thrombocytopenia secondary to extensive platelet activation is a frequent finding, but abnormal platelet dysfunction may also exist in patients with normal platelet counts. In this study, we performed analyses of platelet function and of von Willebrand factor in critically ill COVID-19 patients (n = 13). Platelet aggregometry was performed using ADP, collagen, epinephrin, and ristocetin. VWF and fibrinogen binding of platelets and CD62 and CD63 expression after thrombin stimulation were analyzed via flow cytometry. In addition, VWF antigen (VWF:Ag), collagen binding capacity (VWF:CB), and multimer analysis were performed next to routine coagulation parameters. All patients exhibited reduced platelet aggregation and decreased CD62 and CD63 expression. VWF binding of platelets was reduced in 12/13 patients. VWF:CB/VWF:Ag ratios were pathologically decreased in 2/13 patients and elevated in 2/13 patients. Critically ill COVID-19 patients exhibit platelet secretion defects independent of thrombocytopenia. Platelet exhaustion and VWF dysfunction may result in impaired primary hemostasis and should be considered when treating coagulopathy in these patients. Full article

(This article belongs to the Special Issue Impact of Platelet Defects on Pathophysiological Processes)

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22 pages, 80706 KiB

Open AccessArticle

Only Acute but Not Chronic Thrombocytopenia Protects Mice against Left Ventricular Dysfunction after Acute Myocardial Infarction

by Friedrich Reusswig, Amin Polzin, Meike Klier, Matthias Achim Dille, Aysel Ayhan, Marcel Benkhoff, Celina Lersch, Anika Prinz, Simone Gorressen, Jens Walter Fischer, Malte Kelm and Margitta Elvers

Cells 2022, 11(21), 3500; https://doi.org/10.3390/cells11213500 - 04 Nov 2022

Cited by 6 | Viewed by 1550

Abstract

Background: Platelets are major players of thrombosis and inflammation after acute myocardial infarction (AMI). The impact of thrombocytopenia on platelet-induced cellular processes post AMI is not well defined. Methods: The left anterior descending artery was ligated in C57/Bl6 mice and in two thrombocytopenic [...] Read more.

Background: Platelets are major players of thrombosis and inflammation after acute myocardial infarction (AMI). The impact of thrombocytopenia on platelet-induced cellular processes post AMI is not well defined. Methods: The left anterior descending artery was ligated in C57/Bl6 mice and in two thrombocytopenic mouse models to induce AMI. Results: Platelets from STEMI patients and from C57/Bl6 mice displayed enhanced platelet activation after AMI. This allows platelets to migrate into the infarct but not into the remote zone of the left ventricle. Acute thrombocytopenia by antibody-induced platelet depletion resulted in reduced infarct size and improved cardiac function 24 h and 21 days post AMI. This was due to reduced platelet-mediated inflammation after 24 h and reduced scar formation after 21 days post AMI. The collagen composition and interstitial collagen content in the left ventricle were altered due to platelet interaction with cardiac fibroblasts. Acute inflammation was also significantly reduced in Mpl^−/− mice with chronic thrombocytopenia, but cardiac remodeling was unaltered. Consequently, left ventricular function, infarct size and scar formation in Mpl^−/− mice were comparable to controls. Conclusion: This study discovers a novel role for platelets in cardiac remodeling and reveals that acute but not chronic thrombocytopenia protects left ventricular function post AMI. Full article

(This article belongs to the Special Issue Impact of Platelet Defects on Pathophysiological Processes)

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18 pages, 2218 KiB

Open AccessArticle

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

by José M. Bastida, Stefano Malvestiti, Doris Boeckelmann, Verónica Palma-Barqueros, Mira Wolter, María L. Lozano, Hannah Glonnegger, Rocío Benito, Carlo Zaninetti, Felix Sobotta, Freimut H. Schilling, Neil V. Morgan, Kathleen Freson, José Rivera and Barbara Zieger

Cells 2022, 11(20), 3223; https://doi.org/10.3390/cells11203223 - 14 Oct 2022

Cited by 5 | Viewed by 2002

Abstract

The GATA1 transcription factor is essential for normal erythropoiesis and megakaryocytic differentiation. Germline GATA1 pathogenic variants in the N-terminal zinc finger (N-ZF) are typically associated with X-linked thrombocytopenia, platelet dysfunction, and dyserythropoietic anemia. A few variants in the C-terminal ZF (C-ZF) domain are [...] Read more.

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

(This article belongs to the Special Issue Impact of Platelet Defects on Pathophysiological Processes)

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23 pages, 6978 KiB

Open AccessArticle

Novel GATA1 Variant Causing a Bleeding Phenotype Associated with Combined Platelet α-/δ-Storage Pool Deficiency and Mild Dyserythropoiesis Modified by a SLC4A1 Variant

by Kerstin Jurk, Anke Adenaeuer, Stefanie Sollfrank, Kathrin Groß, Friederike Häuser, Andreas Czwalinna, Josef Erkel, Nele Fritsch, Dana Marandiuc, Martin Schaller, Karl J. Lackner, Heidi Rossmann and Frauke Bergmann

Cells 2022, 11(19), 3071; https://doi.org/10.3390/cells11193071 - 29 Sep 2022

Cited by 3 | Viewed by 1877

Abstract

Germline defects in the transcription factor GATA1 are known to cause dyserythropoiesis with(out) anemia and variable abnormalities in platelet count and function. However, damaging variants closely located to the C-terminal zinc finger domain of GATA1 are nearly unknown. In this study, a 36-year-old [...] Read more.

Germline defects in the transcription factor GATA1 are known to cause dyserythropoiesis with(out) anemia and variable abnormalities in platelet count and function. However, damaging variants closely located to the C-terminal zinc finger domain of GATA1 are nearly unknown. In this study, a 36-year-old male index patient and his 4-year-old daughter suffered from moderate mucocutaneous bleeding diathesis since birth. Whole exome sequencing detected a novel hemizygous GATA1 missense variant, c.886A>C p.T296P, located between the C-terminal zinc finger and the nuclear localization sequence with non-random X-chromosome inactivation in the heterozygous daughter. Blood smears from both patients demonstrated large platelet fractions and moderate thrombocytopenia in the index. Flow cytometry and electron microscopy analysis supported a combined α-/δ (AN-subtype)-storage pool deficiency as cause for impaired agonist-induced platelet aggregation (light transmission aggregometry) and granule exocytosis (flow cytometry). The absence of BCAM in the index (Lu(a-b-)) and its low expression in the daughter (Lu(a-b+)) confirmed a less obvious effect of defective GATA1 also on erythrocytes. Borderline anemia, elevated HbF levels, and differential transcription of GATA1-regulated genes indicated mild dyserythropoiesis in both patients. Furthermore, a mild SLC4A1 defect associated with a heterozygous SLC4A1 c.2210C>T p.A737V variant maternally transmitted in the daughter may modify the disease to mild spherocytosis and hemolysis. Full article

(This article belongs to the Special Issue Impact of Platelet Defects on Pathophysiological Processes)

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9 pages, 947 KiB

Open AccessArticle

Neonatal Platelets: Lower G_12/13 Expression Contributes to Reduced Secretion of Dense Granules

by Axel Schlagenhauf, Sheila Bohler, Mirjam Kunze, Tanja Strini, Harald Haidl, Miriam Erlacher and Barbara Zieger

Cells 2022, 11(16), 2563; https://doi.org/10.3390/cells11162563 - 17 Aug 2022

Cited by 1 | Viewed by 1117

Abstract

Despite fully functional primary hemostasis, platelets of healthy neonates exhibit hypoaggregability and secretion defects, which may be adaptations to specific requirements in this developmental stage. The etiologies for reduced signal transduction vary with the type of agonist. The discovered peculiarities are lower receptor [...] Read more.

Despite fully functional primary hemostasis, platelets of healthy neonates exhibit hypoaggregability and secretion defects, which may be adaptations to specific requirements in this developmental stage. The etiologies for reduced signal transduction vary with the type of agonist. The discovered peculiarities are lower receptor densities, reduced calcium mobilization, and functional impairments of G proteins. Reduced secretion of dense granules has been attributed to lower numbers of granules. Signaling studies with adult platelets have shown a regulating effect of the G_12/13 signaling pathway on dense granule secretion via RhoA. We comparatively analyzed secretion profiles using flow cytometry and expression levels of G_q, G_i, and G_12/13 using Western blot analysis in platelets from cord blood and adults. Furthermore, we evaluated Rho activation after in vitro platelet stimulation with thrombin using a pulldown assay. We observed a markedly reduced expression of the dense granule marker CD63 on neonatal platelets after thrombin stimulation. G_α12/13 expression was significantly decreased in neonatal platelets and correlated with lower Rho activation after thrombin stimulation. We conclude that lower expression of G_12/13 in neonatal platelets results in attenuated activation of Rho and may contribute to reduced secretion of dense granules after exposure to thrombin. Full article

(This article belongs to the Special Issue Impact of Platelet Defects on Pathophysiological Processes)

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20 pages, 4688 KiB

Open AccessArticle

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

by Raghavendra Palankar, Laura Sachs, Jan Wesche and Andreas Greinacher

Cells 2022, 11(10), 1615; https://doi.org/10.3390/cells11101615 - 11 May 2022

Cited by 4 | Viewed by 1718

Abstract

Platelet adhesion and spreading at the sites of vascular injury is vital to hemostasis. As an integral part of the innate immune system, platelets interact with opsonized bacterial pathogens through FcγRIIA and contribute to host defense. As mechanoscavangers, platelets actively migrate and capture [...] Read more.

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

(This article belongs to the Special Issue Impact of Platelet Defects on Pathophysiological Processes)

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18 pages, 2534 KiB

Open AccessArticle

Adipose Triglyceride Lipase Deficiency Attenuates In Vitro Thrombus Formation without Affecting Platelet Activation and Bleeding In Vivo

by Madeleine Goeritzer, Stefanie Schlager, Katharina B. Kuentzel, Nemanja Vujić, Melanie Korbelius, Silvia Rainer, Dagmar Kolb, Marion Mussbacher, Manuel Salzmann, Waltraud C. Schrottmaier, Alice Assinger, Axel Schlagenhauf, Corina T. Madreiter-Sokolowski, Sandra Blass, Thomas O. Eichmann, Wolfgang F. Graier and Dagmar Kratky

Cells 2022, 11(5), 850; https://doi.org/10.3390/cells11050850 - 01 Mar 2022

Cited by 3 | Viewed by 2652

Abstract

According to genome-wide RNA sequencing data from human and mouse platelets, adipose triglyceride lipase (ATGL), the main lipase catalyzing triglyceride (TG) hydrolysis in cytosolic lipid droplets (LD) at neutral pH, is expressed in platelets. Currently, it is elusive to whether common lipolytic enzymes [...] Read more.

According to genome-wide RNA sequencing data from human and mouse platelets, adipose triglyceride lipase (ATGL), the main lipase catalyzing triglyceride (TG) hydrolysis in cytosolic lipid droplets (LD) at neutral pH, is expressed in platelets. Currently, it is elusive to whether common lipolytic enzymes are involved in the degradation of TG in platelets. Since the consequences of ATGL deficiency in platelets are unknown, we used whole-body and platelet-specific (plat)Atgl-deficient (−/−) mice to investigate the loss of ATGL on platelet function. Our results showed that platelets accumulate only a few LD due to lack of ATGL. Stimulation with platelet-activating agonists resulted in comparable platelet activation in Atgl−/−, platAtgl−/−, and wild-type mice. Measurement of mitochondrial respiration revealed a decreased oxygen consumption rate in platelets from Atgl−/− but not from platAtgl−/− mice. Of note, global loss of ATGL was associated with an anti-thrombogenic phenotype, which was evident by reduced thrombus formation in collagen-coated channels in vitro despite unchanged bleeding and occlusion times in vivo. We conclude that genetic deletion of ATGL affects collagen-induced thrombosis without pathological bleeding and platelet activation. Full article

(This article belongs to the Special Issue Impact of Platelet Defects on Pathophysiological Processes)

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