Does Fibrin Structure Contribute to the Increased Risk of Thrombosis in COVID-19 ICU Patients?

Introduction: SARS-CoV-2 is responsible for a global pandemic, with almost 200 million confirmed cases. SARS-CoV-2 infection can lead to various disease states, from only mild symptoms in the majority of cases to severe disease, which is associated with an increased incidence of venous thromboembolism (VTE). We hypothesized that an altered fibrin network structure contributes to VTE in COVID-19 patients by affecting thrombus stability and fibrinolysis sensitivity. By studying the fibrin network of COVID-19 patients, we aimed to unravel the mechanisms that contribute to the increased risk of VTE in COVID-19 patients. Methods: Between April 2020 and December 2020, we collected plasma samples from patients with COVID-19 admitted to the intensive care unit (ICU) of the Erasmus Medical Center. We included patients with confirmed VTE diagnosed on CT-angiography, and COVID-19 patients without confirmed VTE during ICU admission. Samples were collected on admission to the ICU and after confirmed VTE or at similar time points in ICU patients without confirmed VTE. In addition, we collected plasma from COVID-19 patients at admission to general wards without confirmed VTE and from healthy controls. Clots were formed by mixing citrated plasma with thrombin (final concentration 1 U/ml) and calcium (17 mM). We imaged the clots using stimulated emission depletion (STED) microscopy, a super-resolution technique in which a depletion laser is used to selectively switch off fluorophores surrounding the focal point, thereby increasing the resolution. In these images, fibrin fiber diameters were measured using the Local Thickness plugin of ImageJ. Fiber density was quantified as percentage of area in Z-stacks of confocal microscopy images. Finally, a clot lysis assay based on turbidity was used to determine sensitivity to fibrinolysis (clot lysis time) and clot density (difference between maximum and baseline absorbance). Differences in fibrin network properties between groups were tested using One-Way ANOVA with Bonferroni post-hoc tests and linear regression with and without adjustment for fibrinogen levels. Results: We included 21 COVID-19 ICU patients with confirmed VTE, 20 COVID-19 ICU patients without confirmed VTE, 10 COVID-19 ward patients and 7 healthy controls. Mean age was comparable between the groups, while BMI was higher in COVID-19 patients than in healthy controls (Table 1). Levels of fibrinogen, D-dimer and anti-Xa were significantly higher in COVID-19 ICU patients than in COVID-19 ward patients and healthy controls. FVIII levels were significantly higher in COVID-19 ICU patients than in healthy controls, while FXIII levels were significantly lower. On admission to the ICU, clot density was significantly higher in COVID-19 ICU patients with and without confirmed VTE than in healthy controls (Figure 1 and Table 2). However, after adjustment for fibrinogen levels, this difference disappears. Clot lysis time was significantly longer in clots from COVID-19 ICU patients than in clots from healthy controls, regardless of fibrinogen levels (Table 2). COVID-19 ICU patients with confirmed VTE also showed a significant longer clot lysis time than COVID-19 ward patients. Interestingly, in the clot lysis assay, fibrinolysis did not occur in 25% of COVID-19 ICU patients with VTE versus 9.5% of COVID-19 ICU patients without VTE (Figure 2). This fibrinolysis shutdown was never observed in clots from healthy controls and COVID-19 ward patients. Fibrin fiber diameters were comparable between the groups. In the clots from plasma samples collected at admission to the ICU, there were no differences between COVID-19 ICU patients with and without VTE (Figure 2). However, when comparing clots prepared from plasma collected at the second time point (after VTE or at a similar time point for patients without VTE), we observed significant longer clot lysis times in patients with confirmed VTE (97.4 [88.5-158.8] min) than in patients without confirmed VTE (80.0 [76.0-97.8] min) (p=0.03). Finally, there were no significant changes between clots from plasma before and after VTE or between the two time points in patients without VTE, except for a decreased clot lysis time over time for COVID-19 ICU patients without confirmed VTE. Conclusion: Our results suggest that SARS-CoV-2 infection increases clot density and decreases clot susceptibility to fibrinolysis, and that these changes relate to the severity of the disease. Figure 1 Figure 1. Disclosures Kruip: Daiichi Sankyo: Research Funding; Bayer: Honoraria, Research Funding.

Labeling fibrin fibers with microparticle sized beads alter single fibrin fiber lysis, external clot lysis, and produce large fibrin aggregates upon lysis

Background Fluorescent beads are often used as a tool for visualizing fibrin fibers and can mimic the size of microparticles in the blood. Studies showed microparticles alter the appearance and behavior of whole blood clot systems. Objectives Here we investigate the effect of beads on fibrin fiber lysis and extensibility to enhance understanding of this common research technique and as a biomimetic system for fibrin-microparticle interaction. Methods We used fluorescence microscopy, atomic force microscopy (AFM), and scanning electron microscopy (SEM) to quantify changes in lysis, extensibility, and clot structure of fibrin fibers and clots in the presence and absence of beads. Results and Conclusions Fibrin clot structure and lysis were altered in the presence of beads. Fibrin clots formed with beads had a higher fiber density, smaller fibers, and smaller pores. The rate of lysis for clots was reduced when beads were present. Lysis of bead-labeled individual fibers showed that beads, at concentrations similar to those reported for microparticles in the blood, cause a subset of fibers to resist lysis. In the absence of beads, all fibers lyse. These results demonstrate that beads alter fiber lysis through both a change in fibrin clot structure as well as changes to individual fiber lysis behavior. Additionally, the lysis of clots with beads produced large fibrin aggregates. This data encourages researchers to use careful consideration when labeling fibrin fibers with fluorescent beads and suggests that particles binding fibrin(ogen) in the bloodstream may be an underappreciated mechanism increasing the risk of thrombosis.

Dysfibrinogenemia—Potential Impact of Genotype on Thrombosis or Bleeding

The congenital dysfibrinogenemias, most often associated with bleeding disorders, encompass mutations in the amino-terminal end of fibrinogen α-chain consisting of Gly17-Pro18-Arg19-Val20, known as knob A, which is a critical site for fibrin polymerization. Here we review the studies reporting dysfibrinogenemia due to mutations affecting fibrinogen knob A and identified 29 papers. The number of reports on dysfibrinogenemias related to residues Gly17, Pro18, Arg19, and Val20 is 5, 4, 18, and 2, respectively. Dysfibrinogenemias related to residues Gly17, Pro18, and Val20 are exclusively associated with bleeding tendency. However, the clinical picture associated with dysfibrinogenemia related to residue Arg19 varies, with most patients suffering from bleeding tendencies, but also transitory ischemic attacks and retinal thrombosis may occur. The reason for this variation is unclear. To elaborate the genotype–phenotype associations further, we studied a Danish family with knob A-related dysfibrinogenemia caused by the Aα Arg19Gly (p.Arg19Gly) mutation using whole-exome sequencing and fibrin structure analysis. Our family is the first reported carrying the p.Arg19Gly mutation combined with one or more single nucleotide polymorphisms (SNP)s in FGA, FGB, and/or FGG and increased fibrin fiber thickness and fibrin mass-to-length ratio suffering from pulmonary emboli, suggesting that compound genotypes may contribute to the thrombogenic phenotype of these patients. Our review, accordingly, focuses on significance of SNPs, compound genotypes, and fibrin structure measures affecting the genotype–phenotype associations in fibrinogen knob A mutations.

Reduced Plasma Magnesium Levels in Type-1 Diabetes Associate with Prothrombotic Changes in Fibrin Clotting and Fibrinolysis

Individuals with type-1 diabetes mellitus (T1DM) have a higher risk of thrombosis and low plasma magnesium concentrations. As magnesium is a known regulator of fibrin network formation, we investigated potential associations between fibrin clot properties and plasma magnesium concentrations in 45 individuals with T1DM and 47 age- and sex-matched controls without diabetes. Fibrin clot characteristics were assessed using a validated turbidimetric assay and associations with plasma magnesium concentration were examined. Plasma concentrations of fibrinogen, plasminogen activator inhibitor-1 (PAI-1), and lipids were measured and fibrin fiber diameters assessed using scanning electron microscopy. Fibrin clot maximum absorbance was unchanged in subjects with T1DM compared with controls, while lysis time was prolonged (p = 0.0273). No differences in fibrin fiber diameters or in lipid profile were observed between T1DM and controls. PAI-1 concentration was lower in the T1DM group compared with the controls (p = 0.0232) and positively correlated with lysis time (p = 0.0023). Plasma magnesium concentration was lower in the T1DM group compared with controls (p < 0.0001). Magnesium concentration negatively correlated with clot maximum absorbance (p = 0.0215) and lysis time (p = 0.0464). A turbidimetric fibrin clot lysis assay performed in a purified system that included PAI-1 and 0 to 3.2 mM Mg2+ showed a shortening of lysis time with increasing Mg2+ concentrations (p = 0.0004). Our findings reveal that plasma magnesium concentration is associated with changes in fibrin clot and lysis parameters.

In vitro examination of the thrombolytic efficacy of tenecteplase and therapeutic ultrasound compared to rt-PA

Background: Optimizing thrombolytic therapy is vital for improving stroke outcomes. We aimed to develop standardized thrombolysis conditions to evaluate the efficacy of tenecteplase (TNK) compared to the current gold standard rt-PA (alteplase), with and without additional ultrasound treatment. We also wanted to introduce a new analytical approach to quantify fibrin fiber density in transmission electron microscopy (TEM). Methods: In vitro clots that are similar to ex vivo clots concerning their histological condition and their durability were generated from whole blood. For five treatment groups we compared relative clot weight loss (each n=60) and fibrin fiber density in TEM (each n=5). The control group (A) was treated only with plasma. Two groups were designated for each rt-PA (B+C) and TNK (D+E). Groups C and E were additionally treated with ultrasound. Dosages were 50µg/ml for rt-PA and 30µg/ml for TNK. Results were evaluated by using analyses of variance (ANOVA) and post-hoc t-tests. Results: Weight loss was increased significantly for all groups compared to the control group. Both TNK groups showed significantly increased weight loss compared to their counterpart rt-PA group (p≤0.001). For TEM only group D showed significantly decreased fibrin fiber density (p<0.05) compared to both rt-PA groups. Ultrasound did not significantly increase dissolution of clots with either method (best p=0.16). Conclusions: Tenecteplase dissolved clots more effectively than rt-PA with and without ultrasound. A higher sample size could provide more convincing results for TEM.

In vitro examination of the thrombolytic efficacy of tenecteplase and therapeutic ultrasound compared to rt-PA

Background: Optimizing thrombolytic therapy is vital for improving stroke outcomes. We aimed to develop standardized thrombolysis conditions to evaluate the efficacy of tenecteplase (TNK) compared to the current gold standard rt-PA (alteplase), with and without additional ultrasound treatment. Also we wanted to introduce a new analytical approach to quantify fibrin fiber density in transmission electron microscopy (TEM). Methods: In vitro clots that are similar to ex vivo clots concerning their histological condition and their durability were generated from whole blood. For five treatment groups we compared relative clot weight loss (each n=60) and fibrin fiber density in TEM (each n=5). The control group (A) was treated only with plasma. Two groups were designated for each rt-PA (B+C) and TNK (D+E). Groups C and E were additionally treated with ultrasound. Dosages were 50µg/ml for rt-PA and 30µg/ml for TNK. Results were evaluated by using analyses of variance (ANOVA) and post-hoc t-tests. Results: Weight loss was increased significantly for all groups compared to the control group. Both TNK groups showed significantly increased weight loss compared to their counterpart rt-PA group (p≤0.001). For TEM only group D showed significantly decreased fibrin fiber density (p<0.05) compared to both rt-PA groups. Ultrasound did not significantly increase dissolution of clots with either method (best p=0.16). Conclusions: Tenecteplase dissolved clots more effectively than rt-PA with and without ultrasound. A higher sample size could provide more convincing results for TEM.

In vitro examination of the thrombolytic efficacy of tenecteplase and therapeutic ultrasound compared to rt-PA

Background: Optimizing thrombolytic therapy is vital for improving stroke outcomes. We aimed to evaluate the efficacy of tenecteplase (TNK) compared to the current gold standard rt-PA (alteplase), with and without additional ultrasound treatment. Methods: In vitro clots that are similar to ex vivo clots concerning their histological condition and their durability were generated from whole blood. For five treatment groups we compared relative clot weight loss (each n=60) and fibrin fiber density in transmission electron microscopy (TEM) (each n=5). The control group (A) was treated only with plasma. Two groups were designated for each rt-PA (B+C) and TNK (D+E). Groups C and E were additionally treated with ultrasound. Dosages were 50µg/ml for rt-PA and 30µg/ml for TNK. Results were evaluated by using analyses of variance (ANOVA) and post-hoc t-tests. Results: Weight loss was increased significantly for all groups compared to the control group. Both TNK groups showed significantly increased weight loss compared to their counterpart rt-PA group (p≤0.001). For TEM only group D showed significantly decreased fibrin fiber density (p<0.05) compared to both rt-PA groups. Ultrasound did not significantly increase dissolution of clots with either method (best p=0.16). Conclusions: Tenecteplase dissolved clots more effectively than rt-PA with and without ultrasound. A higher sample size could provide more convincing results for TEM. Keywords: Stroke, thrombolysis, tenecteplase, ultrasound, transmission electron microscopy.