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. Author manuscript; available in PMC: 2025 Jun 1.
Published in final edited form as: J Thromb Haemost. 2024 Mar 15;22(6):1675–1688. doi: 10.1016/j.jtha.2024.02.020

Time-dependent ultrastructural changes during venous thrombogenesis and thrombus resolution

Irina N Chernysh 1,*, Subhradip Mukhopadhyay 2,3,*, Tierra A Johnson 2, Jacob A Brooks 2, Rajabrata Sarkar 2,3, John W Weisel 1, Toni M Antalis 2,4,5,#
PMCID: PMC11139557  NIHMSID: NIHMS1979036  PMID: 38492853

Abstract

Background:

Deep venous thrombosis (DVT) is a common vascular event that can result in debilitating morbidity and even death due to pulmonary embolism. Clinically, patients with faster resolution of a venous thrombus have improved prognosis, but the detailed structural information regarding changes that occur in a resolving thrombus over time is lacking.

Objective:

To define the spatial-morphological characteristics of venous thrombus formation, propagation, and resolution at the submicron level over time.

Methods:

Using murine model of stasis-induced DVT along with scanning electron microscopy and immunohistology, we determined the specific structural, compositional and morphological characteristics of venous thrombi formed after 4 days and identify the changes that take place during resolution by day 7. Comparison is made with the structure and composition of venous thrombi formed in mice genetically deficient in plasminogen activator inhibitor type-1 (PAI-1).

Results:

As venous thrombus resolution progresses, fibrin exists in different structural forms and there are dynamic cellular changes in the compositions of leukocytes, platelet aggregates and red blood cells. Intra-thrombus microvesicles are present that are not evident by histology, and red blood cells in the form of polyhedrocytes are an indicator of clot contraction. Structural evidence of fibrinolysis is observed early, during thrombogenesis, and is accelerated by PAI-1 deficiency.

Conclusion:

The results reveal unique, detailed ultrastructural and compositional insights along with documentation of the dynamic changes that occur during accelerated resolution that are not evident by standard pathological procedures, and can be applied to inform diagnosis and effectiveness of thrombolytic treatments to improve patient outcomes.

Keywords: venous thrombosis, fibrinolysis, scanning electron microscopy, fibrin, PAI-1

1. INTRODUCTION

Deep vein thrombosis (DVT) is a serious clinical health problem, with significant morbidity and mortality, characterized by the formation of blood clots primarily within the deep veins of the leg. DVT affects approximately 1 in 900,000 individuals per year in the USA [1]. An often-fatal consequence of DVT is pulmonary embolism, where a part of a thrombus ruptures and enters the pulmonary circulation. Despite standard care with anticoagulants, approximately 20–50% of patients with DVT eventually will develop a condition called post-thrombotic syndrome, an intractable vascular disorder associated with chronic leg swelling, edema and foot ulcers [2]. Clinical studies have shown that thrombus resolution is central to the pathogenesis of this condition; the longer the thrombus is in contact with the vein wall, the greater the eventual damage [3]. Patients with faster venous thrombus resolution have better prognosis than those with slower resolution [4][5]. Therapeutic approaches aimed at accelerating the process of venous thrombus resolution therefore should reduce post-thrombotic complications, by decreasing valvular damage, excessive scarring, and residual obstruction.

Venous thrombosis can be initiated by stasis, increased blood hypercoagulability or vessel wall damage [6]. The activated vascular endothelium triggers the recruitment of platelets and innate immune cells that initiate the formation of a thrombus, including fibrin. Thrombus resolution involves the recruitment of inflammatory cells, specifically neutrophils and macrophages, and proteases to degrade fibrin and remodel the injured tissue to restore venous blood flow. Lacking is detailed structural information on the nature of the initial thrombus formed and the time-dependent structural changes that occur as thrombus resolution progresses, which will be important to advance therapies to alleviate post-thrombotic vein wall damage.

Previously, we characterized the structure and composition of human venous thrombi extracted by thrombectomy using high resolution scanning electron microscopy (SEM) and compared venous thrombus structures and composition to human pulmonary emboli and arterial thrombi [7]. Although this analysis identified unique structural and compositional features that are linked to both vascular origin and thrombus age, logistical difficulties in investigating DVT in humans makes it impractical to directly analyze the formation and progression of the clot, which is missed by static studies. To address this deficiency, here we have applied SEM imaging and quantitative analysis to the well-established and reproducible preclinical mouse stasis-induced DVT model that accurately mimics many of the clinical and pathophysiological features observed in human DVT [8] and allows for analysis of time-dependent changes in structure and composition during thrombus resolution. DVT is induced by inferior vena cava (IVC) ligation immediately below the renal veins [8][9], and the forming thrombus reaches a maximal size at approximately 4 days. The thrombus resolves gradually over time, and resolution can be monitored by the decrease in thrombus weight/size at 7 days. While the stasis model of DVT does not reproduce the clinical scenario where a thrombus is nonocclusive, it reproducibly mimics complete occlusion, which is pathologically significant, since human DVTs are initially occlusive in 88% of cases [10].

The degradation of fibrin, or fibrinolysis, is a fundamental element of venous thrombus resolution [11], and PAI-1 is a central regulative factor, playing a critical role in hemostatic clot stabilization and vein wall remodeling [12]. PAI-1 modulates the proteolytic activities of plasminogen activators (PAs) by forming inhibitory complexes with PAs, thereby inhibiting plasmin-catalyzed fibrin degradation [13]. PAI-1 also interacts with ligands such as vitronectin and cell-surface receptors, which extend the functions of PAI-1 to pericellular proteolysis, cell migration, and tissue remodeling [13]. We and others have found that mice genetically deficient in PAI-1 (PAI-1KO) that have undergone stasis-induced DVT surgery develop thrombi that are smaller and exhibit accelerated thrombus resolution compared to littermate control mice [14][15][16]. Conversely, mice overexpressing PAI-1 exhibit impaired thrombus resolution that leads to larger thrombi after IVC ligation [17]. These findings underscore the inhibitory activities of PAI-1 during DVT formation and resolution.

To better understand mechanisms involved in thrombogenesis and the process of venous thrombus resolution, we have applied scanning electron microscopy (SEM) to characterize the structure and composition of venous thrombi formed after IVC ligation in stasis induced DVT and the time dependent changes that occur during thrombus resolution. We provide qualitative and quantitative evaluation of the proportions of different forms of fibrin, the presence of biconcave, compressed polyhedral and intermediate forms of red blood cells (RBCs) [18], as well as volume fractions of the thrombi comprised of other components, including leukocytes, platelets, and cellular microvesicles present during venous thrombogenesis and changes that occur as the thrombus resolves. These data are compared with those that occur in PAI-1KO mice, where thrombus resolution is accelerated. The data reveal unique, detailed structural and compositional insights into experimental venous thrombi and for the most part, compare well with data from human venous thrombi. Moreover, the results demonstrate that fibrinolysis unexpectedly occurs very early in thrombogenesis, via a process accelerated by PAI-1 deficiency.

2. MATERIAL AND METHODS

2.1. Stasis model of venous thrombosis

The murine stasis-induced venous thrombosis model has been previously described [9][14][19][20] and detailed methods are provided in Supplemental Information. Wild-type (WT) C57BL/6 mice and mice genetically deficient in PAI-1 obtained from the Jackson Laboratory (JAX#002507) and backcrossed a total of 9 times to the C57BL/6 strain were used for this study. IVC ligation was performed on 12–16 week old male mice as described [14]. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Maryland Baltimore and the Veterans Health Administration Chief Medical Veterinary Officer.

2.2. Histopathology and immunohistochemistry

Venous thrombi were prepared for histological analyses and analyzed by immunostaining. Signals were quantified in 4 random high-power (×200) images which were combined for each sample. For additional details, see Supplemental Information.

2.3. Scanning electron microscopy

SEM was used to examine the structure and composition of the exterior and interior of the venous thrombi from WT and PAI-1KO mice (n=3 for both strains and n=3 for each time point). All dissected thrombi were placed in saline solution immediately after their removal. Specimens were rinsed with saline and fixed in 2% glutaraldehyde in 50mM sodium cacodylate buffer with 150mM NaCl (pH 7.4) and processed as described [21]. High-definition micrographs were obtained from 7 different randomly chosen areas of each section to eliminate selection bias and examined in a FEI Quanta 250FEG scanning electron microscope (FEI, Hillsboro, OR). The representative images were evaluated as previously described [22].

2.4. Image Quantification and analysis

Quantitative assessment of thrombi composition by SEM was carried out using previously described procedures [7]. The specimens were sectioned into 3–6 pieces and 5–6 micrographs from 7 randomly selected areas were taken at 2,000× magnification. The images were transposed onto a computer screen and a fine grid (1.5μm×1.5μm) was overlaid using Image J 1.48 software (National Institutes of Health, Bethesda, MD, USA). The size of these grid squares was chosen such that usually only one predefined structural element was present in each. If the square was occupied by two structures in the same ratio, each would be counted as ½ structure, or similarly for other types of multiple occupancy.

2.5. Statistical Analyses

Details of the statistical analyses are provided in the Supplementary Information.

3. RESULTS

3.1. PAI-1 deficiency leads to reduced thrombus size and enhanced thrombus resolution

Stasis-induced DVT was created by IVC ligation in WT and PAI-1KO mice (Fig. 1). As measured by both the thrombus weight (Fig. 1A), and length of the thrombosed IVC (Fig. 1B), PAI-1−/− mice developed significantly reduced thrombi than WT mice at post-ligation day 4 as we have reported previously [14], and which has been attributed to contributions of PAI-1 to clot stabilization during thrombus formation [23]. When compared 7 days after IVC ligation (Fig. 1A–B), the thrombus weight and length were again significantly decreased in PAI-1KO animals compared to WT thrombi. This phenotype is consistent with past studies by ourselves and other investigators [14][15][16].

Fig. 1. PAI-1 deficiency enhances venous thrombus resolution.

Fig. 1.

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Thrombus weights (A) and thrombus lengths (B) of wild-type (WT) animals compared with PAI-1 knockout (PAI-1KO) animals at day 4 and day 7 after IVC ligation (n = 6 per group). Statistical analysis of data sets was performed using the unpaired two-tailed Student’s t test with the exception of the Day 7 PAI-1KO thrombus length which was not normally distributed and was analyzed using the Wilcoxon Rank Sum test. * P < 0.05.

3.2. Characterization of thrombi from WT and PAI-1KO animals by light microscopy

Histological characterization of venous thrombi from WT and PAI-1KO animals at days 4 and 7 carried out by H&E staining (Fig. 2A) showed no gross morphological differences in composition. Martius Scarlet Blue (MSB) staining of venous thrombi for detection of fibrin (red), RBCs (yellow) and platelets (gray)(Fig. 2B) also indicated no differences between WT and PAI-1KO thrombi at either day 4 or 7.

Fig. 2. Morphological and intra-thrombus immune cell analysis.

Fig. 2.

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Histochemical analyses of WT and PAI-1KO thrombi at day 4 and day 7 after IVC ligation. (A) Representative images of H&E staining showing normal architecture of venous thrombi. RBC areas are highlighted in red, platelet areas in pink, and inflammatory cell areas in purple. Upper row shows images of entire thrombi. Magnification bar = 1000 μm. Lower row shows higher magnification images. Magnification bar = 100 μm. (n=3 for each group). (B) Representative images of MSB staining showing intrathrombus fibrin content. Fibrin stains red, RBCs stain yellow and platelets stain gray. Upper row shows images of entire thrombi. Magnification bar = 1000 μm. Lower row shows higher magnification images. Magnification bar = 100 μm. (n=3 for each group). Representative images of immunohistochemical analysis of intra-thrombus (C) Ly6G positive neutrophils and (D) CD68 positive macrophages in venous thrombi sections from WT and PAI-1KO mice at 4 and 7 days after IVC ligation. Magnification bar = 100 μm. (n=3 for each group). (E) Quantification of intra-thrombotic Ly6G positive neutrophils and CD68 positive macrophages. Cells were counted in 4 random high-power fields (hpf, 200x) and combined for each sample. Statistical analysis was performed using the unpaired two-tailed Student’s t test, * P < 0.05.

Inflammation is central to both the initiation and resolution of venous thrombi [24]. Immunostaining of WT and PAI-1KO thrombus sections for leukocyte infiltrates showed the presence of neutrophils (Lys-6G/C positive)(Fig. 2C) and a few macrophages (CD68 positive) within venous thrombi at day 4 (Fig. 2D), consistent with the early infiltration of neutrophils [25]. By day 7 post-ligation, the intra-thrombotic accumulation of CD68 positive macrophages increased significantly (Fig. 2D,E) reflecting the extravasation of monocytes and macrophages into the thrombus during thrombus resolution [25]. The cellular recruitment was similar between both genotypes (Fig. 2E).

3.3. Overall structural elements and composition of WT and PAI-1KO thrombi

SEM analysis of venous thrombi harvested at post-ligation days 4 and 7 from WT (Fig. 3) and PAI-1KO (Fig. 4) mice revealed structural elements that were present in all thrombi. These included: fibrin fibers, straight thin structures that make up a branched, space-filling network; fibrin fibers with ends, fibers that have undergone fibrinolytic cleavage; fibrin sponge, network of fibrin that appears as very fine fibers often with associated platelets and microvesicles; fibrin bundles, laterally aggregated fibrin fibers formed due to cleavage of fibrin fibers; fibrin mesh, which appear when fibers are partially lysed but continue to be attached to the network, forming stubs if the cut end of a fiber is short, or bundles if the cut end is long; four shapes of RBCs (biconcave, intermediate-shaped, polyhedrocytes and echinocytes), two types of platelets (small and balloon-like); and fibroblasts, macrophages, neutrophils and collagen-like structures (Examples in Fig. S1).

Fig. 3. Selected SEM images of structures identified in venous thrombi from WT mice at days 4 and 7 after IVC ligation.

Fig. 3.

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Panels (A, C, E and G) are images of thrombi from WT mice on day 4. Panels (B, D, F and H) are images of thrombi from WT mice on day 7. (A) Fibrin sponge structures are indicated by black arrows; white arrowheads point to fibrin bundles. (B) Fibroblasts present in the thrombus are indicated by black arrows. (C) Fibrin fibers with ends were present in WT thrombi at day 4, indicated by black arrows. (D) Black arrows point to collagen-like structures. (E) Thrombi from WT mice were contracted with tightly packed RBCs in the form of polyhedrocytes (black arrows) and (F) intermediate-shaped RBCs (black arrows). (G) Neutrophils with protrusions (black arrows) were mostly observed in thrombi at day 4. (H) Neutrophils with holes (black arrows) were mostly present at day 7. Magnification bar = 10 μm.

Fig. 4. Selected SEM images of structures identified in thrombi from PAI-1KO mice at days 4 and 7 after IVC ligation.

Fig. 4.

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Panels (A, C, E and G) are images of thrombi from PAI-1KO mice at day 4. Panels (B, D, F and H) are images of thrombi from PAI-1KO mice at day 7. (A) Abundant fibrin fibers with ends (black arrows) in thrombi at day 4. (B) Fibrin fibers with ends (black arrows) at day 7. White arrows point to fibrin bundles. (C and (D) Fibrin mesh at day 4 and day 7, respectively. Black arrows point to pore-like structures within the fibrin mesh, while white arrows point to fibrin bundles. (E) Image shows an area where there were no fibrin structures present and most of the RBCs were biconcave (black arrows) or intermediate-shaped (white arrows). (F) Black arrows point to macrophages at day 7. (G) Balloon-like platelets (black arrows) at day 4. (H) Activated platelets, most degranulated (black arrows) at day 7. Magnification bar = 10 μm.

The relative volume fractions of each component present in WT and PAI-1KO thrombi for both days 4 and 7 analyzed as mean values over all quantified micrographs are presented in Table 1, and the proportions of total volume occupied by each structural component, calculated as average weighted volume fractions, are listed in Table S1 and represented in pie charts in Fig. 5. These analyses showed that as the thrombus matures from day 4 to resolution at day 7, the overall composition of WT thrombi between the days differed significantly (P<0.0001) (Fig. 5). Similarly, the overall composition of PAI-1KO thrombi differed significantly between days 4 and 7 (P<0.0001) (Fig. 5). The overall composition of PAI-1KO thrombi was significantly different than the composition of WT thrombi both at day 4 (P=0.0196) and day 7 (P=0.0008)(Fig. 5). The relative volume fractions for each structural component calculated across all quantified electron micrographs at each of the two time points (Fig. 6) show similar significant compositional changes.

Table 1.

The averaged volume fraction (%) of structures identified in all quantified electron micrographs obtained for all samples at the two time points expressed as the mean ± standard error of the mean.

Day 4 Day 7
WT PAI-1KO WT PAI-1KO
Major Components 91.0 ± 2.5 82.8 ± 3.0 76.8 ± 4.1 56.6 ± 5.3
Fibrin total 42.9 ± 3.7 41.2 ±3.3 30.9 ±3.85 20.0 ± 3.0
 Single fibers  7.2 ± 1.5  5.6 ± 1.1  3.1 ± 0.7  1.2 ± 0.1
 Bundles  13.7 ± 2.0  12.2 ± 2.2  11.5 ± 1.9  6.7 ± 1.1
 Sponge  21.9 ± 2.9  14.9 ± 2.1  6.2 ± 1.5  4.5 ± 2.0
 Fibers with ends  2.3 ± 0.9  7.7 ± 1.5  5.8 ± 1.1  3.2 ± 0.6
 Fibrin mesh  ND  2.3 ± 0.4  4.2 ± 0.9  5.3 ± 1.1
RBC total 19.9 ± 3.1 21.2 ± 2.70 23.1 ± 2.5 10.0 ± 1.9
 Polyhedrocytes  8.9 ± 1.3  2.2 ± 0.2  3.3 ± 0.7  0.0 ± 0.0
 Intermediate  10.9 ± 1.2  18.5 ± 2.6  21.0 ± 3.2  4.8 ± 1.3
 Biconcave  0.1 ± 0.03  1.1 ± 0.4  0.8 ± 0.3  4.1 ± 1.0
 Echinocytes  ND  ND  1.4 ± 0.9  0.6 ± 0.03
Platelets total 21.2 ± 2.3 14.6 ± 2.5 14.0 ± 2.3 16.6 ± 2.6
 Balloon like  13.7 ± 1.8  7.8 ± 1.4  9.0 ± 1.6  2.0 ± 0.5
 Activated platelets  7.5 ± 1.6  6.8 ± 1.6  4.9 ± 1.1  14.6 ± 2.6
Other Components 9.4 ± 1.3 16.7 ± 2.0 21.7 ± 3.1 44.7 ± 4.2
Microvesicles 7.1 ± 1.3 15.6 ± 1.7 6.2 ± 1.3 12.0 ± 1.1
Neutrophils 1.0 ± 0.4 1.3 ± 0.7 0.3 ± 0.1 1.4 ± 0.2
Macrophages ND ND 5.6 ± 1.4 15.1 ± 2.1
Fibroblasts ND ND 4.6 ± 0.1 9.2 ± 0.2
Collagen-like structures ND ND 2.2 ± 0.1 4.5 ± 0.2
Cells debris  0.03 ± 0.01  0.02 ± 0.07 0.8 ± 0.03 1.2 ± 0.1
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Fig. 5. Pie charts representing the proportions of total volumes (%) occupied by each structural component identified in venous thrombi from WT and PAI-1KO mice.

Fig. 5.

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Thrombi were obtained from mice on day 4 and 7 after IVC ligation. The average volume fractions are weighted relative to the total structural components in each sample as described in the Materials and Methods. The types of fibrin structures, RBCs, platelets, and other structures are colored and listed in the key at the bottom. Differences in composition were compared statistically by using the Chi-square test. P values for differences between WT and PAI-1KO thrombi at day 4 are indicated by * P=0.0196; differences between WT and PAI-1KO thrombi at day 7 are indicated by ** P=0.0008. P values for differences between WT at day 4 vs WT at day 7 are indicated by *** P<0.0001 and PAI-1KO at day 4 vs PAI-1KO at day 7 are indicated by ****P<0.0001. RBC = red blood cell.

Fig. 6. Quantitative analysis of relative volume fractions (%) differing significantly between WT and PAI-1KO venous thrombi.

Fig. 6.

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Structures in thrombi visualized by SEM were quantified as described in the Materials and Methods section. The various structural components identified in thrombi are compared as a percentage of the total thrombus volume. Quantitative differences in the relative volume fractions were compared statistically by an ANOVA with Dunn’s test for multiple comparisons, P values for differences between WT and PAI–1 KO thrombi are indicated by *. (A) Total fibrin structures; * P<0.0001; (B) Single fibrin fibers; *P<0.0001; (C) Fibrin fibers with ends; *P<0.0001; (D) Fibrin sponge; *P=0.0008; (E) Total RBCs; *P=0.0033; (F) Polyhedrocytes; *P=0.0213; (G) Intermediate-shaped RBCs; *P<0.0001. (H) Biconcave RBCs; *P<0.0001; (I) Activated platelets; *P=0.0107; (J) Balloon–like platelets; *P=0.007; (K) Microvesicles; *P=0.004; (L) Macrophages; *P<0.0001. ND = not detected. See Supplementary Table S1 for the results of the relative volume fractions averaged over all quantified electron micrographs obtained for all samples at two time points expressed as a mean ± standard error of the mean.

3.4. Ultrastructural changes during venous thrombogenesis and resolution

3.4.1. Fibrin

Fibrin structures were mostly present as fibrin fibers, fibrin bundles and fibrin sponge in both WT (Fig. 3) and PAI-1KO thrombi (Fig. 4). Thrombi harvested at day 4 from WT mice had a higher total fibrin content compared to day 7 (42.9% vs 30.9%)(Table 1), and there were also significant differences in the composition of fibrin structures between days 4 and 7 (Fig. 5). The content of single fibers was higher at day 4 compared to day 7 (7.2% vs 3.1%, P<0.0001)(Table 1, Fig. 6B and Table S1). Fibrin sponge (Fig. 3A) was the major fibrin component of WT thrombi at day 4, and the relative proportion of this structure decreased significantly as the thrombus resolved by day 7 (21.9% vs 6.2%, P= 0.0008)(Table 1, Fig. 6D and Table S1), at which time fibrin bundles (11.5%) and fibrin fibers with ends (5.8%) predominated. Unexpectedly, 2.3% of fibrin fibers had ends on day 4 in WT thrombi (Fig. 3C and Table 1), indicating ongoing fibrinolysis, even at this very early stage of thrombogenesis. Thus, even when thrombi are still forming, natural resolution has already initiated. The relative content of fibrin fibers with ends increased significantly by day 7 (2.3% vs 5.8%, P=0.0008) (Table 1, Fig. 6C and Table S1), indicating increased fibrinolysis. In addition, by day 7, most of fibrin structures were replaced with other, less highly branched fibers, likely to be collagen, as they were infiltrated with fibroblasts and were not apparent at day 4 (Fig. 3B,D).

Comparing WT and PAI-1KO thrombi at day 4, the total fibrin content of PAI-1KO thrombi was not significantly different (42.9% vs 41.2%)(Table 1). Between days 4 and 7 however, the total fibrin content in PAI-KO thrombi significantly decreased (41.2% vs 20%, P< 0.0001)(Table 1, Fig. 6A), and this was greater than the decrease in total fibrin content seen with WT thrombi (42.9% vs 30.9%)(Table 1), indicating accelerated fibrinolysis in the thrombi lacking PAI-1. The composition of fibrin types between WT and PAI-1KO thrombi was significantly different at both days (Fig. 5). PAI-1KO thrombi had much lower single fiber content compared to the WT thrombi at day 4 (5.6% vs 7.2%, P<0.0001)(Table 1, Fig. 6B). There was also reduced volume occupied by fibrin sponge in the PAI-1KO thrombi at day 4 compared to WT thrombi (14.9% vs 21.9%), which decreased significantly in both WT and PAI-1KO genotypes by day 7 (6.2% and 4.5%, respectively, P=0.0008)(Table 1, Fig. 6D). Corresponding to this decrease, the proportion of fibrin fibers with ends (Fig. 4A, B) at day 4 was significantly higher in PAI-1KO thrombi as compared to WT thrombi at day 4 (7.7% vs 2.3%, P<0.0001) (Table 1 and Fig. 6C), reflecting enhanced fibrinolysis in the thrombi lacking PAI-1. Interestingly, fibrin mesh was observed at day 4 in PAI-1KO thrombi (Fig. 4C, D), but not in the WT thrombi (Table 1), further signifying early enhanced fibrinolysis in PAI-1KO thrombi.

Ongoing fibrinolysis will result in a fibrin structure with big pores and fibrin bundles, as observed in PAI-1KO thrombi on day 7 (Fig. 4D). The content of fibrin bundles at day 4 was similar between PAI-1KO and WT thrombi (12.2% vs 13.7%) (Table 1). From day 4 to day 7, there was a reduction in fibrin bundles in PAI-1KO thrombi (12.2% to 6.7%) (Table 1) and the proportion of fibrin fibers with ends decreased significantly from day 4 to day 7 (7.7% vs 3.2%, P<0.0001) (Table 1 and Fig. 6C). This decrease was accompanied by infiltration of fibroblasts into the thrombi (Table 1, Fig. S2) that were observed in multiple areas (Fig. 3B), replacing the lysed fibrin network with less branched collagen-like structures (Fig. 3D) at day 7 in PAI-1KO thrombi.

3.4.2. RBCs

At day 4, the majority of RBCs in WT thrombi were present in the compressed forms, polyhedrocytes (8.9%) or intermediate shape (10.9%)(Figs. 3E,F; Table 1) and very few in the normal biconcave shape (0.1%), indicating clot contraction at this early-stage [18]. Although the difference in overall proportions of thrombus volume occupied by RBCs between days 4 and 7 was not significant (Fig. 6E), the individual component composition differed significantly. Polyhedrocyte forms of RBCs were reduced significantly between days 4 and 7 (8.9% vs 3.3%, P=0.0213)(Table 1 and Fig. 6F), whereas there was a significant increase in RBCs present in the intermediate shape (10.9% vs 21.0%, P=0.001)(Table 1 and Fig. 6G), as well as the biconcave shape (0.1% vs 0.8%), p<0.0001 (Table 1 and Fig. 6H), indicating less clot contraction at day 7 as the thrombus undergoes resolution. Another notable difference was that echinocytes, a form of RBC with short blunt projections (Fig. S2), appeared at day 7 but were not observed at day 4.

Comparing PAI-1KO thrombi between days 4 and 7, the overall proportion of thrombus volume occupied by RBCs was greatly reduced (21.2% vs 10.0%)(Table 1, Fig. 5). RBCs in PAI-1KO thrombi at day 4 were present mostly in intermediate (18.5%) and biconcave (1.1%) shapes (Fig. 4E, Table 1). The content of polyhedrocytes was significantly lower compared to the WT thrombi at day 4 (2.2% vs 8.9%, P=0.0213)(Table 1, Fig. 6F and Table S1), indicating less contraction and increased fibrinolysis even at the early stage of thrombogenesis in the absence of PAI-1. At day 7, surprisingly, no polyhedrocytes were found in PAI-1KO thrombi (Table 1) and the majority of the RBCs were present in intermediate and biconcave shapes. While the proportion of intermediate-shape RBCs was significantly lower compared to the WT thrombi at day 7 (4.8% vs 21.0%, P<0.0001)(Table 1, Fig. 6G and Table S1), the proportion of biconcave RBCs was significantly higher compared to the WT thrombi at day 7 (4.1% vs 0.8%, P<0.0001)(Table 1 and Fig. 6H). Moreover, at day 7, the proportion of biconcave RBCs present in the PAI-1KO thrombi was significantly greater compared with WT thrombi at day 4 (4.1% vs 0.1%, P<0.0001)(Table 1, Fig. 6H and Table S1), indicative of less clot contraction and increased fibrin network dissolution in PAI-1-deficient thrombi. Indeed, measurement of clot contraction using the ruler method [26] also showed that clot contraction in PAI-1KO thrombi at Day 7 was significantly decreased (Fig. S4). These data show that in thrombi lacking PAI-1, there was less constraint on RBCs, leading them to return to the biconcave shape and possibly better able to escape from the thrombus compared to the more restricted WT thrombus, explaining the reduced proportion of thrombus volume occupied by RBCs at day 7.

3.4.3. Platelets

Two types of platelets were observed in thrombi: balloon-like platelets and smaller activated platelets. Balloon–like platelets were large, about 3–4 μm in size, and smaller activated platelets were less than 2 μm in size and were also distinguished by the presence of holes due to degranulation. At day 4, platelets comprised 21.2% of the volume in WT thrombi, and most were present as large balloon-like platelets (13.7%), whereas 7.5% were activated platelets (Table 1). As the thrombus undergoes resolution, the volume occupied by platelets decreased from 21.2% to 14.0% at day 7 (Table 1). In contrast to the WT thrombi, platelets occupied only 14.6% of the volume in PAI-1KO thrombi at day 4, and this remained relatively unchanged at day 7 (Table 1), indicating early, enhanced resolution. Interestingly, compared to day 4, the proportion of activated platelets significantly increased in PAI-1KO thrombi at day 7 (6.8% vs 14.6% (Table 1), (P=0.0107) (Fig. 4H, Fig. 6I and Table S1) and the proportion of balloon-like platelets decreased, (7.8% vs 2.0% (Table 1) and those differences were significant as well (P=0.007) (Fig. 6J, Table S1).

3.4.4. Other components

At day 4, only 9.4% of the volume of the WT thrombi was composed of other components, including microvesicles (7.1%), neutrophils (1.0%) and cellular debris (0.03%) (Table 1). There was a more than 2-fold increase in the volume of other components in WT thrombi at day 7 (21.7%) (Table 1). This increase can mostly be attributed to the appearance of new structures on day 7, e.g., macrophages (5.6%), fibroblasts (4.6%) and collagen-like structures (2.2%) (Table 1), which were not observed at day 4.

Compared to WT thrombi, the volume occupied by other components e.g., microvesicles, neutrophils, macrophages, collagen, and cell debris at days 4 and 7 was higher in the PAI-1KO thrombi (16.7% vs 9.4% and 44.7% vs 21.7%, respectively) (Table 1). Interestingly, the portion occupied by microvesicles was significantly higher in thrombi of PAI-1KO mice as compared to WT thrombi both at day 4 (15.6% vs 7.1%) and day 7 (12.0% vs 6.2%) (Table 1, Fig. 6K). Large areas packed with microvesicles, which might originate from multiple cell types, including platelets, neutrophils, macrophages and fibroblasts, were observed in PAI-1KO thrombi and those differences were significant (P=0.004)(Fig. 6K and Table S1).

The substantial increase of other components observed at day 7 in PAI-1KO thrombi was mostly due to the increased infiltration of both macrophages (Fig. 4F) and fibroblasts (Fig. S3), compared to WT thrombi (macrophages, 15.1% vs 5.6% (Table 1) and those differences were significant (P<0.0001) (Fig. 6L and Table S1); fibroblasts 4.6% vs 9.2%, respectively). Since macrophages are a type of scavenger cell, they are likely phagocytosing cell debris within the resolving thrombus. Fibroblasts were observed in large quantities on day 7 in both WT and PAI-1KO thrombi and some areas of thrombi were packed with fibroblasts surrounded by unbranched fibrous structures, likely to be collagen (Fig. S3), since fibrin forms branched networks. Histochemical staining of thrombus sections with picrosirius red for collagen content (red, Fig. S3) also showed staining for collagen at day 7 within the thrombus, which was absent at day 4. During the process of thrombus resolution, fibrin is replaced by collagen via a remodeling process resembling wound healing [24]. The substantial increase in these components on day 7 provides structural evidence that not only fibrinolysis but other processes of thrombus remodeling are occurring.

4. DISCUSSION

While venous thrombosis has been studied extensively in experimental animal models by monitoring thrombus weight and analysis of morphological changes by histology, immunohistochemistry or in vivo microfluidic models, these studies are limited in providing detailed structural information. This study has revealed the high-resolution structure of the cellular composition of venous thrombi and time-dependent structural changes that occur during venous thrombus resolution. Using SEM, we were able to visualize detailed structural features at the sub-micron spatial scale of thrombus formation and document the dynamic morphological changes that occur during dissolution that were not apparent by light microscopy. We identified different forms of fibrin reflecting thrombus resolution, platelet fragmentation and death, and the prevalence of polyhedrocytes as a measure of clot contraction and an indicator of reduced susceptibility to fibrinolysis, as well as structural evidence for fibrinolysis as an early event in venous thrombogenesis. Moreover, the direct effects of PAI-1 deficiency on the increase in fibrin fiber ends and the decrease in fibrin sponge provide microscale structural evidence that the absence of PAI-1 promotes thrombus resolution and as a result accelerates tissue remodeling.

In the stasis DVT model, venous thrombi are formed beginning from 3 h, reaching a maximal size by 3–4 days after IVC ligation [9]. While single fibrin fibers and fibrin sponge structures were abundant, even at this early stage, the presence of fibers with ends, fibrin bundles and fibrin mesh provide evidence for fibrinolysis. Thus, natural thrombus resolution has already initiated even when thrombi are forming. As thrombus resolution progresses from Day 4 to Day 7, there is a substantial increase in these indicators of fibrinolysis, and the additional appearance of fibrin mesh, a structure characterized by fine fibers and big pores that is associated with strong fibrinolysis [27]. The genetic deletion of PAI-1, a potent and highly effective attenuator of plasmin-dependent fibrinolysis [12][13], accelerates the entire fibrinolytic process, as shown by an increase in fibrin fiber ends and a decrease in fibrin sponge, with some areas of the thrombus completely devoid of fibrin by day 7.

Clot contraction, which is altered in prothrombotic conditions [18], influences the efficiency of fibrinolysis [28] and therefore is a major effector of thrombus resolution. Clot contraction also causes redistribution of platelets and fibrin to the clot periphery and RBCs to the interior, where they are compressed from biconcave to polyhedral, or intermediate forms, forming an array of very tightly packed cells [29]. As thrombus resolution progresses to day 7, increased biconcave forms were observed, an indication of reduced RBC packing and increased thrombus permeability. Polyhedrocytes were found to be the major component of WT thrombi at day 4, a feature that has been observed in extracted human venous thrombi [7]. Biconcave RBCs were found to be significantly more pronounced in PAI-1KO thrombi at both days 4 and 7 compared to WT thrombi, likely reflecting two separate mechanisms. There is less compaction due to accelerated fibrinolysis, such that fibers are severed, relieving the compression of RBCs and allowing them to return to their normal biconcave shape. At the same time, new biconcave RBCs may enter the thrombus from the circulation. The proportion of volume occupied by RBCs in day 7 PAI-1KO thrombi was significantly lower than that observed at day 4. These data suggest less constraint on RBCs in PAI-1KO thrombi, leading them to be more biconcave-shaped and potentially better able to escape from the thrombus, compared to the tightly packed polyhedrocytes.

Prior research using a vascular injury model suggests that suboptimal levels of plasmin activity can promote clot retraction within two hours by rendering the fibrin network more amenable to platelet contractile forces [30]. Our results are consistent with this finding since we observe early fibrinolysis and strong clot contraction. Here we also observed that as thrombus resolution progresses between day 4 and day 7, PAI-1KO thrombi undergo increased fibrinolysis, relieving the compression of RBCs by lysing fibrin fibers, and as a result, clot contraction (as measured by compressed RBCs (Fig. S4) decreases, resulting in increased thrombus permeability. There is a significant increase in intrathrombotic plasmin-generating uPA activity in both WT and PAI-1KO thrombi between days 4 and 7 [14]. These differences in the outcome of the effect of plasmin on clot contraction vs fibrinolysis are likely regulated by the amount, location and timing of plasmin activity.

Platelets were a major cell type found in venous thrombi early in the process of DVT formation at day 4. Two types of platelets were observed: normal-sized activated platelets and large balloon-like platelets. Activated platelets are irregular structures with filopodia that are necessary for clot contraction. Mostly all activated platelets observed in this study had large cavities from degranulation, indicating that they are highly activated. Balloon-like procoagulant platelets are larger, attaining their shape due to calcium influx and high phosphatidylserine surface exposure, allowing for coagulation factors to bind and thrombin to be generated [31]. These procoagulant platelets were the majority platelet structures in WT thrombi at day 4, while PAI-1KO thrombi had a considerably lower platelet content. After platelets have performed their clotting functions, they expire via a calpain-dependent cell death [32]. The lower platelet content indicates that this process is accelerated in PAI-1KO thrombi.

The later stages of venous thrombus resolution involve infiltration by monocytes, macrophages, and fibroblasts, which synthesize collagen [24]. Inflammatory cells play crucial roles in thrombus recanalization and restoration of blood flow via fibrinolysis and vascular remodeling. Very few macrophages were identified at Day 4 by both CD68 staining and by SEM, but macrophages, which contribute significantly to fibrinolysis and tissue remodeling by the release of plasminogen activators, were abundantly present at Day 7 [33]. By enhancing uPA-mediated plasminogen activation, PAI-1 deficiency may augment macrophage pericellular proteolysis associated with tissue remodeling and cell migration [13], reflected in the significant increase in macrophages at day 7 in PAI-1KO thrombi compared to WT thrombi.

An increased proportion of microvesicles were detected in the thrombi of PAI-1KO mice as compared to WT thrombi at both Days 4 and 7. Microvesicles may originate from multiple cell types present in the thrombus and are believed to function as mediators of cell-to-cell communication [34]. Large areas packed with microvesicles were observed, which may correlate with accelerated fibrinolysis. In addition, infiltrating fibroblasts appear at day 7 in both WT and PAI-1KO thrombi, along with the presence of collagen-like structures, providing a scaffold for migrating inflammatory cells.

The structures and content identified in mouse venous thrombi at day 7 are similar to those identified in human venous thrombi, but there are some distinct differences. The average amount of fibrin in human thrombi, 35%, is remarkably similar to the 33% present in WT thrombi at day 7. In both human and mouse venous thrombi, the vast majority of RBCs were present in compressed forms, polyhedrocytes or intermediate forms [26], with very few biconcave cells, although there was a difference in relative amount of RBCs present, 63.4% in human venous thrombi versus 28.0% in WT mice at day 7. In addition, the fraction of platelets in human venous thrombi was only 0.4%, compared to 13.6% in WT mouse thrombi, which can probably be accounted for by the much greater age of the human thrombi, which results in the fragmentation and death of platelets over time.

Therapeutic strategies aimed at accelerating the process of thrombus resolution should prevent post-thrombotic complications, by reducing valvular damage and residual obstruction. PAI-1 deficiency accelerates the temporal progression of venous thrombus resolution, suggesting that blocking or inhibiting PAI-1 activities could promote faster thrombus resolution and lead to better prognosis for patients. However, fibrinolysis is a delicate balance between proteases and inhibitors. Individuals with complete PAI-1 gene deficiency are highly susceptible to bleeding disorders, including intracranial hemorrhage [35]. The use of PAI-1 inhibitors in animal models of DVT to decrease circulating PAI-1 levels and achieve faster venous thrombus resolution has been investigated, but the outcomes have been mixed [36][37][38][39].

The preclinical stasis-induced mouse model of DVT provides valuable insight into the formation, propagation, and resolution of venous thrombi as well as the composition of key components of these processes. Genetically defined animals are used, undergoing the same surgical procedure, resulting in consistent thrombosis in each case and a defined timeline of disease. The structures and content identified in mouse venous thrombi are similar to those identified in our previous work on human venous thrombi [7], but human DVT samples only provide static snapshots of structural data, with the age of the thrombus usually unknown. Further, patient-derived samples show high variability due to patient genetics, environment, clinical histories, comorbidities leading up to the DVT event, and thrombus evolution due to dynamic changes in inflammatory vascular remodeling over time that can result in mature thrombi that are resistant to thrombolysis [40][41]. On the other hand, the interpretation of time-dependent changes in thrombus structure obtained from mouse models are more defined and much more meaningful. The mechanistic insights from the mouse structural data obtained here along with the documentation of the dynamic changes that occur with accelerated fibrinolysis, as exemplified by PAI-1 deficiency, critically inform our understanding of clot dynamics and can be applied to determining the effectiveness of thrombolytic treatments as well as help identify specific targets within thrombi that may be vulnerable to thrombolytic drugs [42].

Supplementary Material

MMC1

Acknowledgements

This work was supported by Merit Review Award I01 BX001921 from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service, the UMGCCC Cancer Center Support Grant NIH P30CA134274 and by funds through the Maryland Department of Health’s Cigarette Restitution Fund Program to T.M.A., and by NIH grants R01 HL148227, R01 HL148014, R01 HL159256, P01 HL146373 to J.W.W. T.J. was supported by a NIH R25 IMSD Fellowship (R25 GM055036) and by NIH T32 Training Grant (T32HL007698). J.A.B. was supported by NIH R25M113262. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health, the Department of Veterans Affairs or the United States Government.

Footnotes

Declaration of competing interests

There are no competing interests to disclose.

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