The role of platelets in thrombus fibrosis and vessel wall remodeling after venous thrombosis

Abstract

Purpose: Platelets are known to play an important role in venous thrombogenesis, but their role in thrombus maturation, resolution, and postthrombotic vein wall remodeling is unclear. The purpose of this study was to determine the role that circulating platelets play in the later phases of venous thrombosis.

Methods: We used a murine inferior vena cava (IVC) stenosis model. Baseline studies in untreated mice were performed to determine an optimal postthrombotic time point for tissue harvest that would capture both thrombus maturation/resolution and postthrombotic vein wall remodeling. This time point was found to be postoperative day 10. After undergoing IVC ultrasound on day 2 to confirm venous thrombus formation, mice were treated with a daily injection of platelet-depleting antibody (anti-GP1bα) to maintain thrombocytopenia or with control IgG until postoperative day 10, at which time IVC and thrombi were harvested and thrombus length, volume, fibrosis, neovascularization, and smooth muscle cell invasion analyzed. Vein wall fibrosis and intimal thickening were also determined.

Results: Mice that were made thrombocytopenic after venous thrombogenesis had thrombi that were less fibrotic, with fewer invading smooth muscle cells. Furthermore, thrombocytopenia in the setting of venous thrombosis resulted in less postthrombotic vein wall intimal thickening. Thrombus volume did not differ between thrombocytopenic mice and their control peers.

Conclusions: This work suggests that circulating platelets contribute to venous thrombus maturation, fibrosis, and adverse vein wall remodeling, and that that inhibition of platelet recruitment may decrease thrombus and vein wall fibrosis, thus helping thrombolysis and preventing postthrombotic syndrome.

Keywords: fibrosis; platelets; post-thrombotic syndrome; vascular remodeling; venous thrombosis.

FIGURE 1

Thrombus length and volume decrease over time in the IVC stenosis model of DVT. A, Pre- and postoperative platelet counts were measured in all mice 24 h before and 24 h after IVC stenosis surgery (n = 30). B, Thrombus incidence in mice that underwent tissue harvest on postoperative day 2 (n = 10), 8 (n = 10), or 16 (n = 10). C, Thrombus length and (D) thrombus volume on postoperative days 2, 8, and 16. Data are reported as mean ± SEM. Unpaired, Student’s t test and χ²test used for statistical analysis. (P < .05*, P < .01**, P < .001***, P < .0001****)

FIGURE 2

Thrombus and vein wall fibrosis evolution over time in the IVC stenosis model of DVT. Cross sections (CS) of vein wall and thrombi harvested from mice 2 (n = 4), 8 (n = 5), or 16 (n = 4) days after IVC stenosis were stained with Masson’s Trichrome for analysis of collagen content and distribution. A-C, Representative low magnification (bar 500 μm) and D-F, high magnification (bar 50 μm) images of vein wall and thrombus collagen staining (white dotted line indicates vein wall thickness). G, Quantification at 2, 8, and 16 d post-stenosis of average vein wall collagen area, (H) average vein wall cross-sectional area, and (I) percent vein wall occupied by collagen. J, Quantification at 2, 8, and 16 d poststenosis of average thrombus collagen area, (K) average thrombus cross-sectional area, and (L) percent thrombus occupied by collagen. Data are reported as mean ± SEM. Unpaired, Student’s t test used for statistical analysis. (P < .05*, P < .01**, P < .001***, P < .0001****)

FIGURE 3

Thrombus neovascularization over time in the IVC stenosis model of DVT. Cross sections of vein wall/thrombi harvested from mice 2 (n = 3), 8 (n = 3), or 16 (n = 4) days after IVC stenosis were stained with immunofluorescent antibodies against PECAM/CD31 (488λ) and VWF (568λ). A-C, Representative low magnification (bar 200 μm) intrathrombus PECAM and VWF staining on postoperative day 2, 8, or 16 (thrombus/vein wall interface indicated by red arrows, dotted white box indicates area magnified in panel below). D-F, High magnification (bar 50 μm) PECAM and VWF staining at postoperative day 2, 8, or 16 in the peripheral aspect of the thrombus. G, Quantification of average number of PECAM/VWF dual-positive cells/high-powered field (HPF). Data are displayed as mean number of cells/HPF averaged across 6 HPF at 400x magnification. H, Quantification of PECAM/VWF dual-positive cells/HPF in the peripheral (-p) vs central (-c) aspects of thrombi. Data are reported as mean ± SEM. Unpaired, Student’s t test used for statistical analysis. (P < .05*, P < .01**, P < .001***, P < .0001****)

FIGURE 4

Sustained platelet depletion after thrombogenesis does not alter thrombus length or volume. A and B, Characteristics of thrombi before antibody treatment. Thrombus length and volume 48 hours after IVC stenosis, as determined by ultrasound. C, Platelet counts in control (n = 14) and platelet-depleted (n = 16) mice. Surgery occurred on day 0 and platelet depletion was initiated on postoperative day 2. D, Thrombus length was measured at the time of harvest. E, Change in thrombus length over time from postoperative days 2 through 10 in control-treated and platelet-depleted mice. F, Thrombus volume at postoperative day 10. Data are reported as mean ± SEM. Unpaired, Student’s t test used for statistical analysis. (P < .05*, P < .01**, P < .001***, P < .0001****)

FIGURE 5

Platelet depletion for 8 d after 2 d of thrombogenesis decreases intrathrombus collagen content, vein wall intimal thickening, and intrathrombus smooth muscle cells. Thrombus/vein wall samples from control (n = 7) or platelet-depleted (n = 8) mice stained for (A-D) collagen with Masson’s Trichrome, (I,J) Elastic Van Gieson for elastin, and (L,M) anti-αSMC actin for smooth muscle cells. A-D, Representative low-magnification (bar 500 μm) and high-magnification (bar 50 μm) images of trichrome-stained tissue shown above. White arrow indicates region of intrathrombus collagen deposition, and dotted line indicates vein wall thickness. E and F, Quantification of average vein wall collagen area and percent vein wall occupied by collagen over time. G-H, Quantification of average thrombus collagen area and percent thrombus occupied by collagen. I and J, Images of Van Gieson stained thrombus/vein wall cross sections at high magnification (bar 50 μm). Images shown above are representative of cohort (white dotted line indicates intimal thickness). K, Quantification of intimal thickness in mice treated with platelet-depleting antibody or control IgG. L and M, Representative low-magnification (bar 200 μm) images of smooth muscle (red) and DAPI (blue) staining in thrombi from control and platelet-depleted mice. White arrows indicate the approximate borders of the thrombus/vein wall interface, coming from the vein wall pointing toward the thrombus. N, Quantification of intrathrombus smooth muscle cell staining, performed across 6 HPF at 400×. Data are shown as mean ± SEM (E-H) or median with IQR (K-N). Unpaired, Student’s t test (E-H) or Mann-WhitneyUtest (K-N) were used for statistical analysis. (P < .05*, P < .01**, P < .001***, P < .0001****)