Spatial distribution of factor Xa, thrombin, and fibrin(ogen) on thrombi at venous shear

Abstract

Background: The generation of thrombin is a critical process in the formation of venous thrombi. In isolated plasma under static conditions, phosphatidylserine (PS)-exposing platelets support coagulation factor activation and thrombin generation; however, their role in supporting coagulation factor binding under shear conditions remains unclear. We sought to determine where activated factor X (FXa), (pro)thrombin, and fibrin(ogen) are localized in thrombi formed under venous shear.

Methodology/principal findings: Fluorescence microscopy was used to study the accumulation of platelets, FXa, (pro)thrombin, and fibrin(ogen) in thrombi formed in vitro and in vivo. Co-perfusion of human blood with tissue factor resulted in formation of visible fibrin at low, but not at high shear rate. At low shear, platelets demonstrated increased Ca(2+) signaling and PS exposure, and supported binding of FXa and prothrombin. However, once cleaved, (pro)thrombin was observed on fibrin fibers, covering the whole thrombus. In vivo, wild-type mice were injected with fluorescently labeled coagulation factors and venous thrombus formation was monitored in mesenteric veins treated with FeCl(3). Thrombi formed in vivo consisted of platelet aggregates, focal spots of platelets binding FXa, and large areas binding (pro)thrombin and fibrin(ogen).

Conclusions/significance: FXa bound in a punctate manner to thrombi under shear, while thrombin and fibrin(ogen) distributed ubiquitously over platelet-fibrin thrombi. During thrombus formation under venous shear, thrombin may relocate from focal sites of formation (on FXa-binding platelets) to dispersed sites of action (on fibrin fibers).

Figure 1. Thrombus formation and fibrin deposition on fibrinogen under shear.

Human whole blood was co-perfused with CaCl₂/TF over fibrinogen for 15 minutes at a shear rate of 200 s⁻¹, 500 s⁻¹, or 1000 s⁻¹. Experiments were performed in the presence of vehicle or hirudin (2.9 µmol/L). A, Phase-contrast micrographs of platelet adhesion and fibrin formation. B, Following perfusion, flow chambers were washed and sequentially treated with lysis buffer and plasmin. Samples were analyzed for fibrin formation by western blot analysis, as measured by the fibrin degradation product, D-dimer. Lysis of fibrinogen-coated capillaries prior to blood perfusion served as a control (Blank).

Figure 2. Thrombin generation and platelet activation under shear.

Human whole blood was co-perfused with CaCl₂/TF over fibrinogen in the presence of vehicle or hirudin (2.9 µmol/L). A-B, The fluorogenic thrombin substrate, Z-GGR-AMC (420 µM) was added to whole blood prior to perfusion. The accumulation of fluorescence from cleaved Z-GGR-AMC after 10 minutes of flow at 200 s⁻¹ is shown. A, Increase in fluorescence intensity from thrombus area; B, first-derivative curves of transient thrombin generation. C-E, Platelets loaded with the calcium indicator Fluo-4 were added to whole blood before perfusion. Platelet calcium responses under flow are shown. C, Traces of F/F_o values of platelets after adhesion representing [Ca²⁺]_i; D, average F/F_o traces; E, fold increase of Ca²⁺ responses during 1 minute of adhesion. F, Following blood perfusion, adherent cells were labeled with OG488-annexin A5. The graph indicates accumulation of PS-exposing platelets (fraction of platelets staining with OG488-annexin A5 as a percentage of 200 s⁻¹ control). Mean ± SEM (n = 3–5). *P<0.05.

Figure 3. Localization of factor Xa (FXa) and (pro)thrombin on thrombi formed under shear.

Human whole blood was co-perfused with CaCl₂/TF at 200 s⁻¹ over fibrinogen. Thrombi were dual-labeled with AF647-annexin A5 (14 nmol/L, red) and active-site OG488-labeled coagulation factors (1 µmol/L, green) or an antibody to the integrin β₃ (α-CD61, 20 µg/ml, green). Where indicated, serine proteases were inhibited during labeling with PPACK (40 µmol/L). A, Confocal images in green are shown for OG488-labeled FXa, prothrombin (Prothr), or thrombin (Thr) fluorescence; images in red indicate annexin A5 binding. B, Fluorescence surface area coverage for each green label and annexin A5. C, Pearson's correlation coefficient, showing overlap between PS-exposing platelets and green labels. Mean ± SEM (n = 3–5).

Figure 4. Spatial localization of fibrin(ogen) and procoagulant platelets during thrombus formation.

Thrombi were formed by a 15 minute co-perfusion of whole blood and CaCl₂/TF at 200 s⁻¹ over fibrinogen. Dual-labeling was with AF647-annexin A5 (14 nmol/L, red) and the indicated fibrin(ogen) labels (green). A, Confocal fluorescence images in green are shown for OG488-fibrinogen (FG, 0.3 µmol/L), anti-fibrinogen antibody (α-FG, 20 µg/mL), or anti-fibrin antibody (α-Fibrin, 20 µg/mL). Images in red indicate staining of PS-exposing platelets. B, Fluorescence surface area coverage for each OG488 label and annexin A5. C, Pearson's correlation coefficient (R_r), between annexin A5 and each green label. Mean ± SEM (n = 3–5 experiments).

Figure 5. Role of PS-exposing platelets in fibrin formation during thrombus formation.

Thrombi were formed on fibrinogen by co-perfusion of whole blood and CaCl₂/TF at a shear rate of 200 s⁻¹. Blood samples were incubated with vehicle, annexin A5, or mutant M1234 annexin A5 (each 0.28 µmol/L) before perfusion. Thrombi were labeled with OG488-fibrinogen (0.3 µmol/L). A, Representative phase-contrast micrographs of platelets and fibrin after 15 minutes of perfusion. B, Relative accumulation of labeled fibrin(ogen). Data are presented as integrated fluorescence intensity per microscopic field. Results of one representative experiment are shown.

Figure 6. Incorporation of labeled coagulation factors during venous thrombus formation in vivo.

Mice were injected with OG488-labeled fibrinogen (FG), prothrombin (Prothr), factor Xa (FXa), or anti-CD41 mAb (anti-platelet integrin α_IIb). Thrombus formation in exteriorized mesenteric vessels was induced by 30 µL of 500 mM FeCl₃ (n = 3–5). A, Fluorescence images of venous thrombi (arrows) and adjacent arteries (arrowheads). B, Increase in fluorescence surface area coverage over time. C, Distribution of fluorescence in smaller or larger features.