Extreme γ' fibrinogen levels in COVID-19 patients.

COVID-19 disease progression can be accompanied by a "cytokine storm" that leads to secondary sequelae such as acute respiratory distress syndrome. Several inflammatory cytokines have been associated with COVID-19 disease progression, but have high daily intra-individual variability. In contrast, we have shown that the inflammatory biomarker γ' fibrinogen (GPF) has a 6-fold lower coefficient of variability compared to other inflammatory markers such as hs-CRP. The aims of the study were to measure GPF in serial blood samples from COVID-19 patients at a tertiary care medical center in order to investigate its association with clinical measures of disease progression. COVID-19 patients were retrospectively enrolled between 3/16/2020 and 8/1/2020. GPF was measured using a commercial ELISA. We found that COVID-19 patients can develop extraordinarily high levels of GPF. Our results showed that ten out of the eighteen patients with COVID-19 had the highest levels of GPF ever recorded. The previous highest GPF level of 80.3 mg/dL was found in a study of 10,601 participants in the ARIC study. GPF levels were significantly associated with the need for ECMO and mortality. These findings have potential implications regarding prophylactic anticoagulation of COVID-19 patients.

γ' fibrinogen levels as a biomarker of COVID-19 respiratory disease severity.

Coronavirus disease 2019 (COVID-19) is characterized by a pro-inflammatory state associated with organ failure, thrombosis, and death. We investigated a novel inflammatory biomarker, γ' fibrinogen (GPF), in 103 hospitalized patients with COVID-19 and 19 healthy controls. We found significant associations between GPF levels and the severity of COVID-19 as judged by blood oxygen saturation (SpO2). The mean level of GPF in the patients with COVID-19 was significantly higher than in controls (69.8 (95 % CI 64.8-74.8) mg/dL compared with 36.9 (95 % CI 31.4-42.4) mg/dL, p < 0.0001), whereas C-reactive protein (CRP), lactate dehydrogenase (LDH), and total fibrinogen levels were not significantly different between groups. Mean GPF levels were significantly highest in patients with severe COVID-19 (SpO2 ≤ 93 %, GPF 75.2 (95 % CI 68.7-81.8) mg/dL), compared to mild/moderate COVID-19 (SpO2 > 93 %, GPF 62.5 (95 % CI 55.0-70.0) mg/dL, p = 0.01, AUC of 0.68, 95 % CI 0.57-0.78; Youden's index cutpoint 62.9 mg/dL, sensitivity 0.64, specificity 0.63). In contrast, CRP, interleukin-6, ferritin, LDH, D-dimers, and total fibrinogen had weaker associations with COVID-19 disease severity (all ROC curves with lower AUCs). Thus, GPF may be a useful inflammatory marker of COVID-19 respiratory disease severity.

Gamma' fibrinogen levels as a biomarker of COVID-19 respiratory disease severity.

Coronavirus disease 2019 (COVID-19) is characterized by a pro-inflammatory state associated with organ failure, thrombosis, and death. We investigated a novel inflammatory biomarker, γ' fibrinogen (GPF), in 103 hospitalized patients with COVID-19 and 19 healthy controls. We found significant associations between GPF levels and the severity of COVID-19 as judged by blood oxygen saturation (SpO 2 ). The mean level of GPF in the patients with COVID-19 was significantly higher than in controls (69.8 (95% CI 64.8-74.8) mg/dL compared with 36.9 (95% CI 31.4-42.4) mg/dL, p < 0.0001), whereas C-reactive protein (CRP), lactate dehydrogenase (LDH), and total fibrinogen levels were not significantly different between groups. Mean GPF levels were significantly highest in patients with severe COVID-19 (SpO 2 ≤ 93%, GPF 75.2 (95% CI 68.7-81.8) mg/dL), compared to mild/moderate COVID-19 (SpO 2 > 93%, GPF 62.5 (95% CI 55.0-70.0) mg/dL, p = 0.01, AUC of 0.68, 95% CI 0.57-0.78; Youden's index cutpoint 62.9 mg/dL, sensitivity 0.64, specificity 0.63). In contrast, CRP, interleukin-6, ferritin, LDH, D-dimers, and total fibrinogen had weaker associations with COVID-19 disease severity (all ROC curves with lower AUCs). Thus, GPF may be a useful inflammatory marker of COVID-19 respiratory disease severity.

Fibrinolysis in Traumatic Brain Injury: Diagnosis, Management, and Clinical Considerations.

Posttraumatic coagulopathy involves disruption of both the coagulation and fibrinolytic pathways secondary to tissue damage, hypotension, and inflammatory upregulation. This phenomenon contributes to delayed complications after traumatic brain injury (TBI), including intracranial hemorrhage progression and systemic disseminated intravascular coagulopathy. Development of an early hyperfibrinolytic state may result in uncontrolled bleeding and is associated with increased mortality in patients with TBI. Although fibrinolytic assays are not routinely performed in the assessment of posttraumatic coagulopathy, circulating biomarkers such as D-dimer and fibrin degradation products have demonstrated potential utility in outcome prediction. Unfortunately, the relatively delayed nature of these tests limits their clinical utility. In contrast, viscoelastic tests are able to provide a rapid global assessment of coagulopathy, although their ability to reliably identify disruptions in the fibrinolytic cascade remains unclear. Limited evidence supports the use of hypertonic saline, cryoprecipitate, and plasma to correct fibrinolytic disruption; however, some studies suggest more harm than benefit. Recently, early use of tranexamic acid in patients with TBI and confirmed hyperfibrinolysis has been proposed as a strategy to further improve clinical outcomes. Moving forward, further delineation of TBI phenotypes and the clinical implications of fibrinolysis based on phenotypic variation is needed. In this review, we summarize the clinical aspects of fibrinolysis in TBI, including diagnosis, treatment, and clinical correlates, with identification of targeted areas for future research efforts.

Fibrinolytic Activation in Patients with Progressive Intracranial Hemorrhage after Traumatic Brain Injury.

Progression of intracranial hemorrhage (PICH) is a significant cause of secondary brain injury in patients with traumatic brain injury (TBI). Previous studies have implicated a variety of mediators that contribute to PICH. We hypothesized that patients with PICH would display either a hypocoagulable state, hyperfibrinolysis, or both. We conducted a prospective study of adult trauma patients with isolated TBI. Blood was obtained for routine coagulation assays, platelet count, fibrinogen, thrombelastography, markers of thrombin generation, and markers of fibrinolysis at admission and 6, 12, 24, and 48 h. Univariate analyses were performed to compare baseline characteristics between groups. Linear regression models were created, adjusting for baseline differences, to determine the relationship between individual assays and PICH. One hundred forty-one patients met entry criteria, of whom 71 had hemorrhage progression. Patients with PICH had a higher Injury Severity Score and Abbreviated Injury Scale score (head), a lower Glasgow Coma Scale score, and lower plasma sodium on admission. Patients with PICH had higher D-dimers on admission. After adjusting for baseline differences, elevated D-dimers remained significantly associated with PICH compared to patients without PICH at admission. Hypocoagulation was not significantly associated with PICH in these patients. The association between PICH and elevated D-dimers early after injury suggests that fibrinolytic activation may contribute to PICH in patients with TBI.

Tranexamic acid administration in the field does not affect admission thromboelastography after traumatic brain injury.

BACKGROUND: No Food and Drug Administration-approved medication improves outcomes following traumatic brain injury (TBI). A forthcoming clinical trial that evaluated the effects of two prehospital tranexamic acid (TXA) dosing strategies compared with placebo demonstrated no differences in thromboelastography (TEG) values. We proposed to explore the impact of TXA on markers of coagulation and fibrinolysis in patients with moderate to severe TBI. METHODS: Data were extracted from a placebo-controlled clinical trial in which patients 15 years or older with TBI (Glasgow Coma Scale, 3-12) and systolic blood pressure of ≥90 mm Hg were randomized prehospital to receive placebo bolus/placebo infusion (placebo), 1 g of TXA bolus/1 g of TXA infusion (bolus maintenance), or 2 g of TXA bolus/placebo infusion (bolus only). Thromboelastography was performed, and coagulation measures including prothrombin time, activated partial thromboplastin time, international ratio, fibrinogen, D-dimer, plasmin-antiplasmin (PAP), thrombin antithrombin, tissue plasminogen activator, and plasminogen activator inhibitor 1 were quantified at admission and 6 hours later. RESULTS: Of 966 patients receiving study drug, 700 had laboratory tests drawn at admission and 6 hours later. There were no statistically significant differences in TEG values, including LY30, between groups (p > 0.05). No differences between prothrombin time, activated partial thromboplastin time, international ratio, fibrinogen, thrombin antithrombin, tissue plasminogen activator, and plasminogen activator inhibitor 1 were demonstrated across treatment groups. Concentrations of D-dimer in TXA treatment groups were less than placebo at 6 hours (p < 0.001). Concentrations of PAP in TXA treatment groups were less than placebo on admission (p < 0.001) and 6 hours (p = 0.02). No differences in D-dimer and PAP were observed between bolus maintenance and bolus only. CONCLUSION: While D-dimer and PAP levels reflect a lower degree of fibrinolysis following prehospital administration of TXA when compared with placebo in a large prehospital trial of patients with TBI, TEG obtained on admission and 6 hours later did not demonstrate any differences in fibrinolysis between the two TXA dosing regimens and placebo. LEVEL OF EVIDENCE: Diagnostic test, level III.

γ' fibrinogen levels are associated with blood clot strength in traumatic brain injury patients.

BACKGROUND: γ' fibrinogen is an alternatively-spliced fibrinogen variant that displays different coagulation parameters in vitro than the major form of fibrinogen. Purified γ' fibrinogen has slower clotting kinetics than unfractionated fibrinogen, but forms clots that are stronger and resistant to fibrinolysis. However, these properties have only been investigated in human populations in a limited number of studies. We therefore performed a retrospective analysis to test the hypothesis that γ' fibrinogen levels influence coagulation in vivo. METHODS: In the present study, we utilized blood samples that were collected from traumatic brain injury patients to probe the relationship between γ' fibrinogen levels and traditional coagulation parameters. RESULTS: The results show that the levels of γ' fibrinogen were inversely associated with clotting kinetics, indicated by a shortened INR. In addition, the levels of γ' fibrinogen were associated with stronger clots by thrombelastography. However, these changes were not associated with significant changes in hemorrhage progression. CONCLUSIONS: These findings verify that γ' fibrinogen properties observed in purified systems result in similar properties in a clinical setting, and may affect coagulation.

The 95RGD97 sequence on the Aα chain of fibrinogen is essential for binding to its erythrocyte receptor.

BACKGROUND: Erythrocyte aggregation, a cardiovascular risk factor, is increased by high plasma fibrinogen levels. Here, the effect of different fibrinogen mutations on binding to its human erythrocyte receptor was assessed in order to identify the interaction sites. METHODS: Three fibrinogen variants were tested, specifically mutated in their putative integrin recognition sites on the Aα chain (mutants D97E, D574E and D97E/D574E) and compared with wild-type fibrinogen. RESULTS: Atomic force microscopy-based force spectroscopy measurements showed a significant decrease both on the fibrinogen-erythrocyte binding force and on its frequency for fibrinogen with the D97E mutation, indicating that the corresponding arginine-glycine-aspartate sequence (residues 95-97) is involved in this interaction, and supporting that the fibrinogen receptor on erythrocytes has a ß3 subunit. Changes in the fibrin clot network structure obtained with the D97E mutant were observed by scanning electron microscopy. CONCLUSION: These findings may lead to innovative perspectives on the development of new therapeutic approaches to overcome the risks of fibrinogen-driven erythrocyte hyperaggregation.

The Platelet Integrin αIIbß3 Differentially Interacts with Fibrin Versus Fibrinogen.

Fibrinogen binding to the integrin αIIbß3 mediates platelet aggregation and spreading on fibrinogen-coated surfaces. However,in vivoαIIbß3 activation and fibrinogen conversion to fibrin occur simultaneously, although the relative contributions of fibrinogenversusfibrin to αIIbß3-mediated platelet functions are unknown. Here, we compared the interaction of αIIbß3 with fibrin and fibrinogen to explore their differential effects. A microscopic bead coated with fibrinogen or monomeric fibrin produced by treating the immobilized fibrinogen with thrombin was captured by a laser beam and repeatedly brought into contact with surface-attached purified αIIbß3. When αIIbß3-ligand complexes were detected, the rupture forces were measured and displayed as force histograms. Monomeric fibrin displayed a higher probability of interacting with αIIbß3 and a greater binding strength. αIIbß3-fibrin interactions were also less sensitive to inhibition by abciximab and eptifibatide. Both fibrinogen- and fibrin-αIIbß3 interactions were partially inhibited by RGD peptides, suggesting the existence of common RGD-containing binding motifs. This assumption was supported using the fibrin variants αD97E or αD574E with mutated RGD motifs. Fibrin made from a fibrinogen γ'/γ' variant lacking the γC αIIbß3-binding motif was more reactive with αIIbß3 than the parent fibrinogen. These results demonstrate that fibrin is more reactive with αIIbß3 than fibrinogen. Fibrin is also less sensitive to αIIbß3 inhibitors, suggesting that fibrin and fibrinogen have distinct binding requirements. In particular, the maintenance of αIIbß3 binding activity in the absence of the γC-dodecapeptide and the α-chain RGD sequences suggests that the αIIbß3-binding sites in fibrin are not confined to its known γ-chain and RGD motifs.

Prognostic utility of biochemical markers of cardiovascular risk: impact of biological variability.

BACKGROUND: Although a variety of biochemical markers are used to help predict the risk of cardiovascular disease, the prognostic utility of any marker used as a risk assessment tool is dependent on the long- and short-term biological variability that the marker shows in different individuals. METHODS: We measured total, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) cholesterol; triglycerides; high-sensitivity C-reactive protein (hsCRP); total fibrinogen; and γ' fibrinogen in blood samples collected from 15 apparently healthy individuals over the course of 1 year. Repeated measures variation estimates were used to calculate short- and long-term intraclass correlation coefficients (ICC), within- and between-subject coefficients of variation (CVI and CVG, respectively), validity coefficients, and indices of individuality for each marker. RESULTS: HDL cholesterol demonstrated the lowest variability profile, with an ICC of 0.84 and CVI of 11.1 (95% CI: 8.3, 17.0). hsCRP showed the highest levels of short- and long-term within-subject variability [CVI (95% CI): 54.8 (32.8, 196.3) and 77.1 (53.3, 141.3), respectively]. Stated differently, it would require five separate measurements of hsCRP, performed on samples collected over multiple days, to provide the risk assessment information provided by a single measurement of HDL cholesterol. γ' Fibrinogen demonstrated an ICC of 0.79 and CVI of 14.3 (95% CI: 10.6, 21.9). CONCLUSIONS: hsCRP showed very high biological variability, such that a single measurement of hsCRP lacks sufficient clinical utility to justify routine measurement. The variability profile of γ' fibrinogen was not markedly different than HDL cholesterol, necessitating only a limited number of measurements to establish an individual's risk of cardiovascular disease.

Obesity-related increased γ' fibrinogen concentration in children and its reduction by a physical activity-based lifestyle intervention: a randomized controlled study.

OBJECTIVE: To determine if elevated plasma γ'-fibrinogen, typically involved in the formation of fibrinolysis-resistant clots, confers an increased risk for cardiovascular disease (CVD) and thrombosis in children as it does in adults. Although obesity-related hyperfibrinogenemia is frequently reported in children, the role of γ' fibrinogen and its response to physical activity-based lifestyle are less clear in this population. STUDY DESIGN: In a randomized controlled 3-month physical activity-based lifestyle intervention, γ' fibrinogen concentration was measured in 21 children (aged 14-18 years; Tanner stage > IV), including 15 in the obese group and 6 in the normal weight group, with body mass index percentiles for age and sex of >95 and <85, respectively. RESULTS: The relationships between γ' fibrinogen and other risk factors for CVD, such as markers of insulin resistance and subclinical inflammation, along with body composition (as measured by dual-energy X-ray absortiometry), were assessed before and after the intervention. γ' fibrinogen concentration was higher in the obese group compared with the normal weight group (P < .05) and was correlated with other risk factors for CVD (adjusted R(2) = 0.9; P < .05), and insulin emerged as the major predictor of γ' fibrinogen. The intervention reduced γ'-fibrinogen concentration (P < .05). CONCLUSION: Our data reveal: (1) elevated γ' fibrinogen concentrations in obese insulin-resistant children compared with normal lean controls; (2) a relationship between γ' fibrinogen and other CVD risk factors; and (3) physical activity-induced reduction in γ' fibrinogen in obese children.

Differential regulation of fibrinogen γ chain splice isoforms by interleukin-6.

INTRODUCTION: Fibrinogen is a major structural protein in blood clots, and is also a well-known acute phase reactant. The γ chain gene of fibrinogen has two alternative splice variants, γA and γ' chains. γ' fibrinogen constitutes about 7% of total fibrinogen. Total fibrinogen levels and γ' fibrinogen levels have been associated with cardiovascular disease, but the mechanisms regulating the production of the two isoforms are unknown. Several inflammatory cytokines are known to influence the production of total fibrinogen, but the role of cytokines in the production of γ' fibrinogen has not been examined. However, epidemiologic studies have shown an association between γ' fibrinogen levels and inflammatory markers in humans. MATERIALS AND METHODS: The expression of γ' fibrinogen and total fibrinogen by HepG2 liver cells was quantitated after treatment with interleukin-1ß, transforming growth factor-ß, tumor necrosis factor-α, and interleukin-6. RESULTS: Interleukin-1ß, transforming growth factor-ß, and tumor necrosis factor-α, known down-regulators of total fibrinogen synthesis, also downregulate γ' fibrinogen synthesis in HepG2 cells. However, interleukin-6 differentially up-regulates the production of total and γ' fibrinogen, leading to a 3.6-fold increase in γA mRNA, but an 8.3-fold increase in γ' mRNA. CONCLUSIONS: These findings indicate that γ' fibrinogen is disproportionately up-regulated by inflammatory responses induced by interleukin-6.

γ' Fibrinogen as a novel marker of thrombotic disease.

γ' Fibrinogen is an isoform of fibrinogen that normally constitutes about 7% of total plasma fibrinogen, and arises from an alternative processing event in the γ chain mRNA. γ' Fibrinogen is a newly-emerging cardiovascular disease (CVD) risk factor that appears to have an independent association with CVD from that of total fibrinogen, which is itself a well-established CVD risk factor. γ' Fibrinogen shows a significant association with coronary artery disease and myocardial infarction in at least four case-control studies, including the Stockholm Coronary Artery Risk Factor study and the Framingham Heart Study. γ' Fibrinogen is also significantly associated with stroke, as shown in the Erasmus Stroke Study and others. The role of genetic polymorphisms in the association between γ' fibrinogen and CVD is under active investigation. γ' Fibrinogen increases during inflammation, and is differentially regulated from total fibrinogen under pathologic conditions, as demonstrated in the Periodontitis and Vascular Events study. The association between γ' fibrinogen and venous thromboembolism remains unclear, however, with some studies showing an inverse association with γ' fibrinogen levels and other studies showing the opposite.

Designing allosteric regulators of thrombin. Monosulfated benzofuran dimers selectively interact with Arg173 of exosite 2 to induce inhibition.

Earlier, we reported on the design of sulfated benzofuran dimers (SBDs) as allosteric inhibitors of thrombin (Sidhu et al. J. Med. Chem.201154 5522-5531). To identify the site of binding of SBDs, we studied thrombin inhibition in the presence of exosite 1 and 2 ligands. Whereas hirudin peptide and heparin octasaccharide did not affect the IC(50) of thrombin inhibition by a high affinity SBD, the presence of full-length heparin reduced inhibition potency by 4-fold. The presence of γ' fibrinogen peptide, which recognizes Arg93, Arg97, Arg173, Arg175, and other residues, resulted in a loss of affinity that correlated with the ideal Dixon-Webb competitive profile. Replacement of several arginines and lysines of exosite 2 with alanine did not affect thrombin inhibition potency, except for Arg173, which displayed a 22-fold reduction in IC(50). Docking studies suggested a hydrophobic patch around Arg173 as a plausible site of SBD binding to thrombin. The absence of the Arg173-like residue in factor Xa supported the observed selectivity of inhibition by SBDs. Cellular toxicity studies indicated that SBDs are essentially nontoxic to cells at concentrations as high as 250 mg/kg. Overall, the work presents the localization of the SBD binding site, which could lead to allosteric modulators of thrombin that are completely different from all clinically used anticoagulants.

Role of electrostatic interactions in binding of thrombin to the fibrinogen γ' chain.

Thrombin binds to the highly anionic fibrinogen γ' chain through anion-binding exosite II. This binding profoundly alters thrombin's ability to cleave substrates, including fibrinogen, factor VIII, and PAR1. However, it is unknown whether this interaction is due mainly to general electrostatic complementarity between the γ' chain and exosite II or if there are critical charged γ' chain residues involved. We therefore systematically determined the contribution of negatively charged amino acids in the γ' chain, both individually and collectively, to thrombin binding affinity. Surface plasmon resonance binding experiments were performed using immobilized γ' chain peptides with charged-to-uncharged amino acid substitutions, i.e., Asp to Asn, Glu to Gln, and pTyr to Tyr. Individually, the substitution of uncharged for charged amino acids resulted in only minor changes in binding affinity, with a maximal change in K(d) from 0.440 to 0.705 µM for the Asp419Asn substitution. However, substitution of all three charged amino acids in a conserved ß-turn that is predicted to contact thrombin, pTyr418Tyr, Asp419Asn, and pTyr422Tyr, resulted in the loss of measurable binding, as did substitution of all the flanking charged amino acids. In addition, the binding of the γ' chain to thrombin was weakened in a dose-dependent manner with increasing NaCl concentration, resulting in a net loss of three or four ion pairs between thrombin and the γ' chain. Therefore, although each of the individual charges in the γ' chain contributes only incrementally to the overall binding affinity, the ensemble of the combined charges plays a profound role in the thrombin-γ' chain interactions.

Assessment of genetic determinants of the association of γ' fibrinogen in relation to cardiovascular disease.

OBJECTIVE: γ' fibrinogen is a newly emerging biomarker that is associated with cardiovascular disease (CVD). However, the genetic determinants of γ' fibrinogen levels are unknown. We therefore conducted a genome-wide association study on 3042 participants from the Framingham Heart Study Offspring Cohort. METHODS AND RESULTS: A genome-wide association study with 2.5 million single-nucleotide polymorphisms (SNPs) was carried out for γ' fibrinogen levels from the cycle 7 examination. Fifty-four SNPs in or near the fibrinogen gene locus demonstrated genome-wide significance (P<5.0×10(-8)) for association with γ' fibrinogen levels. The top-signal SNP was rs7681423 (P=9.97×10(-110)) in the fibrinogen gene locus near FGG, which encodes the γ chain. Conditional on the top SNP, the only other SNP that remained genome-wide significant was rs1049636. Associations between SNPs, γ' fibrinogen levels, and prevalent CVD events were examined using multiple logistic regression. γ' fibrinogen levels were associated with prevalent CVD (P=0.02), although the top 2 SNPs associated with γ' fibrinogen levels were not associated with CVD. These findings contrast those for total fibrinogen levels, which are associated with different genetic loci, particularly FGB, which encodes the Bß chain. CONCLUSIONS: γ' fibrinogen is associated with prevalent CVD and with SNPs exclusively in and near the fibrinogen gene locus.

Association between γ' fibrinogen levels and inflammation.

The γ' fibrinogen isoform produces clots that are stiffer and more resistant to breakdown than the more common fibrinogen isoform, γA. Increased levels of γ' fibrinogen are associated with several forms of cardiovascular disease. The purpose of this cross-sectional study was to investigate the relationship between γ' fibrinogen, an emerging risk factor for cardiovascular disease, and inflammatory markers in subjects with a chronic inflammatory state. The 284 subjects for this study came from the Periodontitis And Vascular Events (PAVE) study, and γ' fibrinogen and total fibrinogen in plasma were measured by ELISA. Information on patient demographics and health status, as well as levels of C-reactive protein (CRP), an inflammatory marker, have previously been collected for this study. The mean (SE) γ' fibrinogen level in the subjects was 0.622 (0.017) mg/ml. Levels of γ' fibrinogen were correlated with CRP (p = 0.006), with a one unit increase in CRP associated with a 1.9% increase in γ' fibrinogen, after adjustment for potential confounders. Total fibrinogen was not correlated with γ' fibrinogen in these subjects. The number of dental sites with evidence of tissue inflammation was also significantly associated with γ' fibrinogen levels. These results provide an important step in the evolution of γ' fibrinogen not only as a general risk factor for cardiovascular disease, but as a potentially useful biomarker for assessing a patient's inflammatory state and associated cardiovascular disease risk.