Cellular procoagulant activity dictates clot structure and stability as a function of distance from the cell surface

Abstract

Background: Thrombin concentration modulates fibrin structure and fibrin structure modulates clot stability; however, the impact of localized, cell surface-driven in situ thrombin generation on fibrin structure and stability has not previously been evaluated.

Methods and results: Human fibroblasts were incubated with factors Xa, Va, prothrombin and fibrinogen, or plasma. Fibrin formation, structure, and lysis were examined using laser scanning confocal microscopy and transmission electron microscopy. In situ thrombin generation on the cell surface produced clots with a significantly denser fiber network in a 10-microm region proximal versus distal to (40 to 50 microm) the cell surface. This morphology was not altered by addition of integrin-blocking RGDS peptide and was not apparent in clots made by exogenous thrombin addition, suggesting that spatial morphology was dictated predominantly by localized thrombin generation on the fibroblast surface. The fibrin network lysed more rapidly distal versus proximal to the cell surface, suggesting that the structural heterogeneity of the clot affected its fibrinolytic stability.

Conclusions: In situ thrombin generation on the cell surface modulates the three-dimensional structure and stability of the clot. Thrombus formation in vivo may reflect the ability of the local cell population to support thrombin generation and, therefore, the three-dimensional structure and stability of the fibrin network.

Figure 1. In situ thrombin generation on the cell surface modulates clot structure in three dimensions

Clots were formed as described in the Methods in the absence (a–f) or presence (g–i) of RGDS peptide. Three-dimensional projections show clot architecture in 10-μm sections at (0–10 μm) and above (40–50 μm) the cell surface. Each image is from one experiment, representative of 3–6 independent experiments. Darker areas show increased fibrin density. Bar = 10 μm.

Figure 2. Fibrin structure is determined by the distance from the cell surface

A) Images from one experiment in which clots were formed by combining factors Xa, Va, II, fibrinogen, and CaCl₂ (1 nM, 5 nM, 1.4 μM, 2 mg/mL, and 5 mM, respectively) as described in the Methods. a) Differential interference contrast image. b – k) Individuals slices every 1.08 μm in the z-plane: b) 0, c) 1.08, d) 2.16, e) 3.24, f) 4.32, g) 5.4, h) 6.48, i) 7.56, j) 8.64, and k) 9.72 μm from the cell surface. Darker areas show increased fibrin density. Bar = 10 μm. B) Fibrin network density (± standard error) was determined as described in Methods in the presence of cells with 0.014 μM prothrombin (circles), 1.4 μM prothrombin (squares), 2 nM thrombin (diamonds), or in the absence of cells with 2 nM thrombin (triangles).

Figure 2. Fibrin structure is determined by the distance from the cell surface

A) Images from one experiment in which clots were formed by combining factors Xa, Va, II, fibrinogen, and CaCl₂ (1 nM, 5 nM, 1.4 μM, 2 mg/mL, and 5 mM, respectively) as described in the Methods. a) Differential interference contrast image. b – k) Individuals slices every 1.08 μm in the z-plane: b) 0, c) 1.08, d) 2.16, e) 3.24, f) 4.32, g) 5.4, h) 6.48, i) 7.56, j) 8.64, and k) 9.72 μm from the cell surface. Darker areas show increased fibrin density. Bar = 10 μm. B) Fibrin network density (± standard error) was determined as described in Methods in the presence of cells with 0.014 μM prothrombin (circles), 1.4 μM prothrombin (squares), 2 nM thrombin (diamonds), or in the absence of cells with 2 nM thrombin (triangles).

Figure 3. Clot formation and lysis are determined by the distance from the cell surface

Factors Xa, Va, prothrombin, fibrinogen, and CaCl₂ were combined in the presence of plasmin, as described in Methods. A) Images are from one experiment, representative of four independent experiments. Images are the same size as in figure 1. The circular lysis pattern reflects the distribution of light power across the lens (30% greater in the center than at the periphery at 63× oil objective). B) Change in the fiber number over time (± standard error, n = 4 – 6) in clots formed over cells with 0.014 μM prothrombin (circles), 1.4 μM prothrombin (squares), or 2 nM thrombin (diamonds), at (open symbols) and above (closed symbols) the cell surface. C) Fiber formation was defined as the first appearance of fibers in the field of view. D) Fiber lysis was defined as the disappearance of all fibers in the field of view.

Figure 3. Clot formation and lysis are determined by the distance from the cell surface

Factors Xa, Va, prothrombin, fibrinogen, and CaCl₂ were combined in the presence of plasmin, as described in Methods. A) Images are from one experiment, representative of four independent experiments. Images are the same size as in figure 1. The circular lysis pattern reflects the distribution of light power across the lens (30% greater in the center than at the periphery at 63× oil objective). B) Change in the fiber number over time (± standard error, n = 4 – 6) in clots formed over cells with 0.014 μM prothrombin (circles), 1.4 μM prothrombin (squares), or 2 nM thrombin (diamonds), at (open symbols) and above (closed symbols) the cell surface. C) Fiber formation was defined as the first appearance of fibers in the field of view. D) Fiber lysis was defined as the disappearance of all fibers in the field of view.

Figure 3. Clot formation and lysis are determined by the distance from the cell surface

Factors Xa, Va, prothrombin, fibrinogen, and CaCl₂ were combined in the presence of plasmin, as described in Methods. A) Images are from one experiment, representative of four independent experiments. Images are the same size as in figure 1. The circular lysis pattern reflects the distribution of light power across the lens (30% greater in the center than at the periphery at 63× oil objective). B) Change in the fiber number over time (± standard error, n = 4 – 6) in clots formed over cells with 0.014 μM prothrombin (circles), 1.4 μM prothrombin (squares), or 2 nM thrombin (diamonds), at (open symbols) and above (closed symbols) the cell surface. C) Fiber formation was defined as the first appearance of fibers in the field of view. D) Fiber lysis was defined as the disappearance of all fibers in the field of view.

Figure 3. Clot formation and lysis are determined by the distance from the cell surface

Factors Xa, Va, prothrombin, fibrinogen, and CaCl₂ were combined in the presence of plasmin, as described in Methods. A) Images are from one experiment, representative of four independent experiments. Images are the same size as in figure 1. The circular lysis pattern reflects the distribution of light power across the lens (30% greater in the center than at the periphery at 63× oil objective). B) Change in the fiber number over time (± standard error, n = 4 – 6) in clots formed over cells with 0.014 μM prothrombin (circles), 1.4 μM prothrombin (squares), or 2 nM thrombin (diamonds), at (open symbols) and above (closed symbols) the cell surface. C) Fiber formation was defined as the first appearance of fibers in the field of view. D) Fiber lysis was defined as the disappearance of all fibers in the field of view.

Figure 4. Cells modulate the network density of plasma clots

Re-calcified PFP was incubated with or without fibroblasts, as described in the Methods. A) Three-dimensional projections show clot architecture in 10 μm sections at (0–10 μm) and above (40–50 μm) the cell surface. Each image is from one experiment, representative of 2 independent experiments. Bar = 10 μm. B) Fibrin network density (± standard deviation) in the absence and presence (open and closed symbols, respectively) of 500 μM RGDS. C) TEMs of fibrin fibers (black dots) in clots formed in the presence or absence of fibroblasts. In both images, the bottom of the reaction chamber is oriented at the bottom of the image. Bar = 1 μm. D) Fibrin fibers were counted on micrographs recorded at 6300X by placing a 1-μm thick box parallel to the cell surface 0.25 μm and 5 μm from the cell surface and counting fibers inside the box.

Figure 4. Cells modulate the network density of plasma clots

Re-calcified PFP was incubated with or without fibroblasts, as described in the Methods. A) Three-dimensional projections show clot architecture in 10 μm sections at (0–10 μm) and above (40–50 μm) the cell surface. Each image is from one experiment, representative of 2 independent experiments. Bar = 10 μm. B) Fibrin network density (± standard deviation) in the absence and presence (open and closed symbols, respectively) of 500 μM RGDS. C) TEMs of fibrin fibers (black dots) in clots formed in the presence or absence of fibroblasts. In both images, the bottom of the reaction chamber is oriented at the bottom of the image. Bar = 1 μm. D) Fibrin fibers were counted on micrographs recorded at 6300X by placing a 1-μm thick box parallel to the cell surface 0.25 μm and 5 μm from the cell surface and counting fibers inside the box.

Figure 4. Cells modulate the network density of plasma clots

Re-calcified PFP was incubated with or without fibroblasts, as described in the Methods. A) Three-dimensional projections show clot architecture in 10 μm sections at (0–10 μm) and above (40–50 μm) the cell surface. Each image is from one experiment, representative of 2 independent experiments. Bar = 10 μm. B) Fibrin network density (± standard deviation) in the absence and presence (open and closed symbols, respectively) of 500 μM RGDS. C) TEMs of fibrin fibers (black dots) in clots formed in the presence or absence of fibroblasts. In both images, the bottom of the reaction chamber is oriented at the bottom of the image. Bar = 1 μm. D) Fibrin fibers were counted on micrographs recorded at 6300X by placing a 1-μm thick box parallel to the cell surface 0.25 μm and 5 μm from the cell surface and counting fibers inside the box.

Figure 4. Cells modulate the network density of plasma clots

Re-calcified PFP was incubated with or without fibroblasts, as described in the Methods. A) Three-dimensional projections show clot architecture in 10 μm sections at (0–10 μm) and above (40–50 μm) the cell surface. Each image is from one experiment, representative of 2 independent experiments. Bar = 10 μm. B) Fibrin network density (± standard deviation) in the absence and presence (open and closed symbols, respectively) of 500 μM RGDS. C) TEMs of fibrin fibers (black dots) in clots formed in the presence or absence of fibroblasts. In both images, the bottom of the reaction chamber is oriented at the bottom of the image. Bar = 1 μm. D) Fibrin fibers were counted on micrographs recorded at 6300X by placing a 1-μm thick box parallel to the cell surface 0.25 μm and 5 μm from the cell surface and counting fibers inside the box.