Comprehensive Analysis of the Role of Fibrinogen and Thrombin in Clot Formation and Structure for Plasma and Purified Fibrinogen

Abstract

Altered properties of fibrin clots have been associated with bleeding and thrombotic disorders, including hemophilia or trauma and heart attack or stroke. Clotting factors, such as thrombin and tissue factor, or blood plasma proteins, such as fibrinogen, play critical roles in fibrin network polymerization. The concentrations and combinations of these proteins affect the structure and stability of clots, which can lead to downstream complications. The present work includes clots made from plasma and purified fibrinogen and shows how varying fibrinogen and activation factor concentrations affect the fibrin properties under both conditions. We used a combination of scanning electron microscopy, confocal microscopy, and turbidimetry to analyze clot/fiber structure and polymerization. We quantified the structural and polymerization features and found similar trends with increasing/decreasing fibrinogen and thrombin concentrations for both purified fibrinogen and plasma clots. Using our compiled results, we were able to generate multiple linear regressions that predict structural and polymerization features using various fibrinogen and clotting agent concentrations. This study provides an analysis of structural and polymerization features of clots made with purified fibrinogen or plasma at various fibrinogen and clotting agent concentrations. Our results could be utilized to aid in interpreting results, designing future experiments, or developing relevant mathematical models.

Keywords: fibrin; fibrinogen; thrombin; thrombosis; tissue factor.

Figure 1

Structural analysis of clots made from purified fibrinogen with varying fibrinogen concentrations. (A–D) SEM images obtained on a Zeiss EVO10 (Zeiss, Oberkochen, Germany) at ~20,000× magnification on clots made with (A) 1 mg/mL, (B) 2.7 mg/mL, (C) 5 mg/mL, and (D) 10 mg/mL fibrinogen. (E–H) Confocal images obtained on a Zeiss LSM800 (Zeiss, Oberkochen, Germany) using a 63× oil immersion objective on clots made with (E) 1 mg/mL, (F) 2.7 mg/mL, (G) 5 mg/mL, and (H) 10 mg/mL fibrinogen. (I) Diameters acquired from SEM images. (J) Pore sizes acquired from confocal images. (K) Percent area covered obtained from confocal images. (L) Fiber lengths obtained from confocal images. (All clots contain 0.1 U/mL thrombin and 25 L-U/mL FXIIIa in a buffer of 150 mM NaCl, 20 mM HEPES, 5 mM CaCl₂, pH 7.4; scale bars 10 µm;, ** p < 0.01, **** p < 0.0001.) Kruskal–Wallis with Dunn’s multiple comparison tests were used due to non-normal distributions present for each parameter. Horizontal number inside bar is the mean; vertical number above bar is sample size.

Figure 2

Plasma clot structural analysis with varying fibrinogen concentrations. (A–D) SEM images obtained on a Zeiss SIGMA (Zeiss, Oberkochen, Germany) at 20,000× magnification on clots made with (A) 1 mg/mL, (B) 2.7 mg/mL, (C) 5 mg/mL, and (D) 10 mg/mL fibrinogen. (E–H) Confocal images obtained on a Zeiss LSM800 using a 63× oil immersion objective on clots made with (E) 1 mg/mL (adjusted in FIJI for visualization), (F) 2.7 mg/mL (adjusted in FIJI for visualization), (G) 5 mg/mL, and (H) 10 mg/mL fibrinogen. (I) Diameters acquired from SEM images. (J) Pore sizes acquired from confocal images. (K) Percent area covered obtained from confocal images. (L) Fiber lengths obtained from confocal images. (All clots contain 1:80 volume ratio of tissue factor; scale bars 10 µm; ns not significant, * p < 0.05, ** p < 0.01, **** p < 0.0001.) Brown–Forsythe and Welch ANOVA tests and Dunnett multiple comparisons test (I,K), Kruskal–Wallis test and Dunn multiple comparison test (J,L). Horizontal number inside bar is the mean; vertical number above bar is sample size of individual measurements from 2 to 6 images.

Figure 3

Diameter values obtained from the Carr–Hermans and Yeromonahos turbidimetric approaches as well as from SEM imaging for (A) purified fibrinogen and (B) plasma clots. Mean ± standard deviation displayed for scanning electron microscopy. Mean ± uncertainty due to approximation displayed for Carr–Hermans and Yeromonahos. n = 3.

Figure 4

Structural analysis of clots from purified fibrinogen with varying thrombin concentrations (2.7 mg/mL fibrinogen). (A–D) SEM images obtained on a Zeiss EVO10 at ~20,000× magnification on clots made with (A) 0.1 U/mL, (B) 0.25 U/mL, (C) 0.5 U/mL, and (D) 1 U/mL thrombin. (E–H) Confocal images obtained on a Zeiss LSM800 using a 63x oil immersion objective on clots made with (E) 0.1 U/mL, (F) 0.25 U/mL, (G) 0.5 U/mL, and (H) 1 U/mL thrombin. (I) Diameters acquired from SEM images. (J) Pore sizes acquired from confocal images. (K) Percent fluorescent density obtained from confocal images. (L) Fiber lengths obtained from confocal images. (All clots contain 2.7 mg/mL fibrinogen and 25 L-U/mL FXIIIa in a buffer of 150 mM NaCl, 20 mM HEPES, 5 mM CaCl₂, pH 7.4; scale bars 10 µm; ns not significant * p < 0.05, ** p < 0.01, **** p < 0.0001.) Kruskal–Wallis with Dunn’s multiple comparison tests were used to account for non-normal distributions present for each parameter. Horizontal number inside bar is the mean; vertical number above bar is sample size of individual measurements from at least 6 images.

Figure 5

Plasma clot structural analysis with varying thrombin concentrations (2.9 mg/mL fibrinogen). (A–D) SEM images obtained on a Zeiss SIGMA at 20,000× magnification on clots made with (A) 0.1 U/mL, (B) 0.25 U/mL, (C) 0.5 U/mL, and (D) 1 U/mL thrombin. (E–H) Confocal images obtained on a Zeiss LSM800 using a 40x water immersion objective on clots made with (E) 0.1 U/mL, (F) 0.25 U/mL, (G) 0.5 U/mL, and (H) 1 U/mL thrombin. (I) Diameters acquired from SEM images. (J) Pore sizes acquired from confocal images. (K) Percent fluorescent density obtained from confocal images. (L) Fiber lengths obtained from confocal images. (Scale bars 10 µm; ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.) Brown–Forsythe and Welch ANOVA tests, Dunnett multiple comparisons test (I–L). Horizontal number inside bar is the mean; vertical number above bar is sample size of individual measurements from at least 6 images.

Figure 6

Diameters obtained from the Carr–Hermans and Yeromonahos turbidimetric approaches as well as from SEM imaging for (A) clots from purified fibrinogen and (B) plasma clots. Mean ± standard deviation displayed for scanning electron microscopy. Mean ± uncertainty due to approximation displayed for Carr–Hermans and Yeromonahos. n = 3.