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

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.

Type 2B von Willebrand Disease: Early Manifestation as Neonatal Thrombocytopenia.

Here, we report about a preterm female newborn with a prolonged course of severe thrombocytopenia and hematomas. The family history was positive for von Willebrand disease type 2B (VWD 2B). Diagnosis of VWD 2B was identified analyzing von Willebrand factor (VWF) parameters (VWF:antigen, VWF:activity, VWF multimer analyses) and performing light transmission aggregometry (with half concentration of ristocetin). In addition, the diagnosis was confirmed by molecular genetic analysis: identification of a disease-causing missense mutation (Val1316Met) in the VWF gene associated with a severe course of VWD 2B, which had been previously reported. Treatment with a VWF-containing plasma concentrate was initiated. Because the combination of prematurity and very low platelet count is often associated with intracranial bleeding, at the beginning platelet concentrates were transfused. Fortunately, the patient did not develop serious bleeding episodes. Interestingly, the patient had a mutation in the VWF gene, which had been described to be associated with aggravation of thrombocytopenia especially in stressful situations. Therefore, we replaced venous blood withdrawals by capillary blood samplings when possible and, consequently, we observed an increase of the platelet count after this change in management. At the age of 2 months, the patient was discharged after stabilization of the platelet count without any bleeding signs and without a need of long-term medication.

Distinct epigenetic programs regulate cardiac myocyte development and disease in the human heart in vivo.

Epigenetic mechanisms and transcription factor networks essential for differentiation of cardiac myocytes have been uncovered. However, reshaping of the epigenome of these terminally differentiated cells during fetal development, postnatal maturation, and in disease remains unknown. Here, we investigate the dynamics of the cardiac myocyte epigenome during development and in chronic heart failure. We find that prenatal development and postnatal maturation are characterized by a cooperation of active CpG methylation and histone marks at cis-regulatory and genic regions to shape the cardiac myocyte transcriptome. In contrast, pathological gene expression in terminal heart failure is accompanied by changes in active histone marks without major alterations in CpG methylation and repressive chromatin marks. Notably, cis-regulatory regions in cardiac myocytes are significantly enriched for cardiovascular disease-associated variants. This study uncovers distinct layers of epigenetic regulation not only during prenatal development and postnatal maturation but also in diseased human cardiac myocytes.

5'-Hydroxymethylcytosine Precedes Loss of CpG Methylation in Enhancers and Genes Undergoing Activation in Cardiomyocyte Maturation.

BACKGROUND: Cardiomyocytes undergo major changes in DNA methylation during maturation and transition to a non-proliferative state after birth. 5'-hydroxylation of methylated cytosines (5hmC) is not only involved in DNA loss of CpG methylation but is also thought to be an epigenetic mark with unique distribution and functions. Here, we sought to get insight into the dynamics of 5'-hydroxymethylcytosine in newborn and adult cardiomyocytes. METHODS: Cardiomyocyte nuclei from newborn and adult C57BL/6 mice were purified by flow cytometric sorting. 5hmC-containing DNA was captured by selective chemical labeling, followed by deep sequencing. Sequencing reads of library replicates were mapped independently (n = 3 for newborn, n = 2 for adult mice) and merged for further analysis steps. 5hmC coverage was normalized to read length and the total number of mapped reads (RPKM). MethylC-Seq, ChIP-Seq and RNA-Seq data sets of newborn and adult cardiomyocytes served to elucidate specific features of 5hmC at gene bodies and around low methylated regions (LMRs) representing regulatory genomic regions with enhancer function. RESULTS: 163,544 and 315,220 5hmC peaks were identified in P1 and adult cardiomyocytes, respectively. Of these peaks, 66,641 were common between P1 and adult cardiomyocytes with more than 50% reciprocal overlap. P1 and adult 5hmC peaks were overrepresented in genic features such as exons, introns, 3'- and 5'-untranslated regions (UTRs), promotors and transcription end sites (TES). During cardiomyocyte maturation, 5hmC was found to be enriched at sites of subsequent DNA loss of CpG methylation such as gene bodies of upregulated genes (i.e. Atp2a2, Tnni3, Mb, Pdk4). Additionally, centers of postnatally established enhancers were premarked by 5hmC before DNA loss of CpG methylation. CONCLUSIONS: Simultaneous analysis of 5hmC-Seq, MethylC-Seq, RNA-Seq and ChIP-Seq data at two defined time points of cardiomyocyte maturation demonstrates that 5hmC is positively associated with gene expression and decorates sites of subsequent DNA loss of CpG methylation.