Reciprocal stabilization of coagulation factor XIII-A and -B subunits is a determinant of plasma FXIII concentration.

ABSTRACT: Transglutaminase factor XIII (FXIII) is essential for hemostasis, wound healing, and pregnancy maintenance. Plasma FXIII is composed of A and B subunit dimers synthesized in cells of hematopoietic origin and hepatocytes, respectively. The subunits associate tightly in circulation as FXIII-A2B2. FXIII-B2 stabilizes the (pro)active site-containing FXIII-A subunits. Interestingly, people with genetic FXIII-A deficiency have decreased FXIII-B2, and therapeutic infusion of recombinant FXIII-A2 (rFXIII-A2) increases FXIII-B2, suggesting FXIII-A regulates FXIII-B secretion, production, and/or clearance. We analyzed humans and mice with genetic FXIII-A deficiency and developed a mouse model of rFXIII-A2 infusion to define mechanisms mediating plasma FXIII-B levels. Like humans with FXIII-A deficiency, mice with genetic FXIII-A deficiency had reduced circulating FXIII-B2, and infusion of FXIII-A2 increased FXIII-B2. FXIII-A-deficient mice had normal hepatic function and did not store FXIII-B in liver, indicating FXIII-A does not mediate FXIII-B secretion. Transcriptional analysis and polysome profiling indicated similar F13b levels and ribosome occupancy in FXIII-A-sufficient and -deficient mice and in FXIII-A-deficient mice infused with rFXIII-A2, indicating FXIII-A does not induce de novo FXIII-B synthesis. Unexpectedly, pharmacokinetic/pharmacodynamic modeling of FXIII-B antigen after rFXIII-A2 infusion in humans and mice suggested FXIII-A2 slows FXIII-B2 loss from plasma. Accordingly, comparison of free FXIII-B2 vs FXIII-A2-complexed FXIII-B2 (FXIII-A2B2) infused into mice revealed faster clearance of free FXIII-B2. These data show FXIII-A2 prevents FXIII-B2 loss from circulation and establish the mechanism underlying FXIII-B2 behavior in FXIII-A deficiency and during rFXIII-A2 therapy. Our findings reveal a unique, reciprocal relationship between independently synthesized subunits that mediate an essential hemostatic protein in circulation. This trial was registered at www.ClinicalTrials.com as #NCT00978380.

Antithrombin, Protein C, and Protein S: Genome and Transcriptome-Wide Association Studies Identify 7 Novel Loci Regulating Plasma Levels.

BACKGROUND: Antithrombin, PC (protein C), and PS (protein S) are circulating natural anticoagulant proteins that regulate hemostasis and of which partial deficiencies are causes of venous thromboembolism. Previous genetic association studies involving antithrombin, PC, and PS were limited by modest sample sizes or by being restricted to candidate genes. In the setting of the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium, we meta-analyzed across ancestries the results from 10 genome-wide association studies of plasma levels of antithrombin, PC, PS free, and PS total. METHODS: Study participants were of European and African ancestries, and genotype data were imputed to TOPMed, a dense multiancestry reference panel. Each of the 10 studies conducted a genome-wide association studies for each phenotype and summary results were meta-analyzed, stratified by ancestry. Analysis of antithrombin included 25 243 European ancestry and 2688 African ancestry participants, PC analysis included 16 597 European ancestry and 2688 African ancestry participants, PSF and PST analysis included 4113 and 6409 European ancestry participants. We also conducted transcriptome-wide association analyses and multiphenotype analysis to discover additional associations. Novel genome-wide association studies and transcriptome-wide association analyses findings were validated by in vitro functional experiments. Mendelian randomization was performed to assess the causal relationship between these proteins and cardiovascular outcomes. RESULTS: Genome-wide association studies meta-analyses identified 4 newly associated loci: 3 with antithrombin levels (GCKR, BAZ1B, and HP-TXNL4B) and 1 with PS levels (ORM1-ORM2). transcriptome-wide association analyses identified 3 newly associated genes: 1 with antithrombin level (FCGRT), 1 with PC (GOLM2), and 1 with PS (MYL7). In addition, we replicated 7 independent loci reported in previous studies. Functional experiments provided evidence for the involvement of GCKR, SNX17, and HP genes in antithrombin regulation. CONCLUSIONS: The use of larger sample sizes, diverse populations, and a denser imputation reference panel allowed the detection of 7 novel genomic loci associated with plasma antithrombin, PC, and PS levels.

Novel genetic regulators of fibrinogen synthesis identified by an in vitro experimental platform.

BACKGROUND: Fibrinogen has an established, essential role in both coagulation and inflammatory pathways, and these processes are deeply intertwined in the development of thrombotic and atherosclerotic diseases. Previous studies aimed to better understand the (patho) physiological actions of fibrinogen by characterizing the genomic contribution to circulating fibrinogen levels. OBJECTIVES: Establish an in vitro approach to define functional roles between genes within these loci and fibrinogen synthesis. METHODS: Candidate genes were selected on the basis of their proximity to genetic variants associated with fibrinogen levels and expression in hepatocytes and HepG2 cells. HepG2 cells were transfected with small interfering RNAs targeting candidate genes and cultured in the absence or presence of the proinflammatory cytokine interleukin-6. Effects on fibrinogen protein production, gene expression, and cell growth were assessed by immunoblotting, real-time polymerase chain reaction, and cell counts, respectively. RESULTS: HepG2 cells secreted fibrinogen, and stimulation with interleukin-6 increased fibrinogen production by 3.4 ± 1.2 fold. In the absence of interleukin-6, small interfering RNA knockdown of FGA, IL6R, or EEPD1 decreased fibrinogen production, and knockdown of LEPR, PDIA5, PLEC, SHANK3, or CPS1 increased production. In the presence of interleukin-6, knockdown of FGA, IL6R, or ATXN2L decreased fibrinogen production. Knockdown of FGA, IL6R, EEPD1, LEPR, PDIA5, PLEC, or CPS1 altered transcription of one or more fibrinogen genes. Knocking down ATXN2L suppressed inducible but not basal fibrinogen production via a post-transcriptional mechanism. CONCLUSIONS: We established an in vitro platform to define the impact of select gene products on fibrinogen production. Genes identified in our screen may reveal cellular mechanisms that drive fibrinogen production as well as fibrin(ogen)-mediated (patho)physiological mechanisms.

DNA methylation analysis is used to identify novel genetic loci associated with circulating fibrinogen levels in blood.

BACKGROUND: Fibrinogen plays an essential role in blood coagulation and inflammation. Circulating fibrinogen levels may be determined based on interindividual differences in DNA methylation at cytosine-phosphate-guanine (CpG) sites and vice versa. OBJECTIVES: To perform an EWAS to examine an association between blood DNA methylation levels and circulating fibrinogen levels to better understand its biological and pathophysiological actions. METHODS: We performed an epigenome-wide association study of circulating fibrinogen levels in 18 037 White, Black, American Indian, and Hispanic participants, representing 14 studies from the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium. Circulating leukocyte DNA methylation was measured using the Illumina 450K array in 12 904 participants and using the EPIC array in 5133 participants. In each study, an epigenome-wide association study of fibrinogen was performed using linear mixed models adjusted for potential confounders. Study-specific results were combined using array-specific meta-analysis, followed by cross-replication of epigenome-wide significant associations. We compared models with and without CRP adjustment to examine the role of inflammation. RESULTS: We identified 208 and 87 significant CpG sites associated with fibrinogen levels from the 450K (p < 1.03 × 10-7) and EPIC arrays (p < 5.78 × 10-8), respectively. There were 78 associations from the 450K array that replicated in the EPIC array and 26 vice versa. After accounting for overlapping sites, there were 83 replicated CpG sites located in 61 loci, of which only 4 have been previously reported for fibrinogen. The examples of genes located near these CpG sites were SOCS3 and AIM2, which are involved in inflammatory pathways. The associations of all 83 replicated CpG sites were attenuated after CRP adjustment, although many remained significant. CONCLUSION: We identified 83 CpG sites associated with circulating fibrinogen levels. These associations are partially driven by inflammatory pathways shared by both fibrinogen and CRP.

COVID-19 pandemic perspectives: A scientific silver lining?

Accounts of the numerous negative effects caused by COVID-19 are pervasive, but few perspectives have identified any positive impacts of this massive societal shift. This forum examines potentially positive changes that have occurred within the scientific community amid the chaotic pandemic. Among these positives are the formation of virtual supergroups and an interdisciplinary brain trust. In forcing scientists away from their lab benches, COVID-19 has created time and space for more conversations about science and experimental design. Being away from the lab in this time of social unrest has also given scientists time to directly address institutional racism and its suppression of diversity in science. Although COVID-19 has been an unforeseen disaster of epic proportions, some of the resulting changes in our scientific community should remain in place after the pandemic is over. By leveraging these small wins, we will undoubtedly return to our laboratories stronger, smarter, and more efficient.