Factor XIII activity mediates red blood cell retention in venous thrombi

Abstract

Venous thrombi, fibrin- and rbc-rich clots triggered by inflammation and blood stasis, underlie devastating, and sometimes fatal, occlusive events. During intravascular fibrin deposition, rbc are thought to become passively trapped in thrombi and therefore have not been considered a modifiable thrombus component. In the present study, we determined that activity of the transglutaminase factor XIII (FXIII) is critical for rbc retention within clots and directly affects thrombus size. Compared with WT mice, mice carrying a homozygous mutation in the fibrinogen γ chain (Fibγ390-396A) had a striking 50% reduction in thrombus weight due to reduced rbc content. Fibrinogen from mice harboring the Fibγ390-396A mutation exhibited reduced binding to FXIII, and plasma from these mice exhibited delayed FXIII activation and fibrin crosslinking, indicating these residues mediate FXIII binding and activation. FXIII-deficient mice phenocopied mice carrying Fibγ390-396A and produced smaller thrombi with fewer rbc than WT mice. Importantly, FXIII-deficient human clots also exhibited reduced rbc retention. The addition of FXIII to FXIII-deficient clots increased rbc retention, while inhibition of FXIII activity in normal blood reduced rbc retention and produced smaller clots. These findings establish the FXIII-fibrinogen axis as a central determinant in venous thrombogenesis and identify FXIII as a potential therapeutic target for limiting venous thrombosis.

Figure 7. FXIII-deficient human clots retain fewer rbc after clot retraction, and FXIII inhibition prevents rbc retention in clots.

(A) Serum rbc content following tissue factor–initiated clot retraction of human FXIII-deficient patient plasma reconstituted with washed platelets and washed rbc from normal donors and treated with increasing doses of FXIII (0–1 U/ml, n = 6). Data represent mean ± SEM. *P < 0.02 versus FXIII-deficient plasma with no additional FXIII by ANOVA. (B) Serum rbc content following tissue factor–initiated clot retraction of normal human whole blood in the presence of the irreversible FXIIIa inhibitor, T101 (0.1–5 μM, n = 7). Data represent normalized mean ± SEM. *P < 0.02 versus absence of T101 by ANOVA.

Figure 6. FXIII-deficient mice produce smaller thrombi with reduced rbc content.

(A) Thrombus weights 1 day after ligation (stasis model). (B) Total neutrophil content of thrombus lysates, measured by Ly6G antigen. (C) Total platelet content of thrombus lysates, measured by CD41 antigen. (D) Total fibrin (β-chain) antigen of thrombus lysates. (E) Total rbc content of stasis thrombus lysates, measured as hemoglobin absorbance at 575 nm. Each dot represents an individual mouse. Lines are means. (F and G) Serum rbc content following thrombin-initiated clot retraction of recalcified blood from (F) F13a^–/– (n = 4) and (G) Fibγ^390–396A mice (n = 7). Data represent mean ± SEM.

Figure 5. Compared with WT, plasma from Fibγ^390–396A mice exhibits delayed FXIII activation and, consequently, delayed fibrin crosslinking during tissue factor–initiated coagulation.

(A) Representative Western blots for FXIII-A during time course of tissue factor–initiated clotting in WT and Fibγ^390–396A plasma. Activated FXIII (FXIII-A*) appears over time as a lower MW band. (B) Quantitation of FXIII-A* appearance over time in blots from A. Data represent mean ± SEM (n = 5). (C) Representative Western blots for fibrin(ogen) during time course of tissue factor–initiated clotting in WT and Fibγ^390–396A plasma showing γ-γ dimer and high MW crosslinked species (α-α and α-γ polymers). (D) Quantitation of γ-γ dimer formation over time in Western blots. (E) Quantitation of high MW crosslinked species over time in Western blots. Data represent mean ± SEM (n = 6).

Figure 4. FXIII-A₂B₂ does not coprecipitate with Fibγ^390–396A fibrinogen.

(A) Blue silver–stained 10% Tris-glycine gel containing purified fibrinogens from WT and Fibγ^390–396A mice. Bands indicated with arrows were analyzed by mass spectrometry. (B) Western blot detection of FXIII-A subunit in purified fibrinogen (Fgn) samples shown in A and commercial murine FXIII as a positive control. (C) Relative level of FXIII-A in WT and Fibγ^390–396A plasmas. Data represent means ± SEM (n = 4). (D) Relative binding curves of plasma-purified FXIII-A₂B₂ (Fibrogammin-P) to purified fibrinogen from WT or Fibγ^390–396A mice. Data represent normalized means ± SEM (n = 4).

Figure 3. rbc adhere to fibrin(ogen) from WT and Fibγ^390–396A mice.

(A) Adherence of rbc to BSA, WT fibrin(ogen), or Fibγ^390–396A fibrin(ogen) before and after application of flow with a shear rate of 1000 s^–1. Scale bar: 30 μm. (B) Quantitation of rbc adhesion after application of flow. Data represent mean ± SEM (n = 3). *P < 0.009, versus BSA by ANOVA.

Figure 2. rbc are extruded from Fibγ^390–396A clots during clot retraction, resulting in decreased clot weight.

After clot formation, platelet contractile force retracts clots away from siliconized tube walls, leaving the clots surrounded by serum. (A) Image of fully retracted WT and Fibγ^390–396A clots 90 minutes after initiation of clot formation by tissue factor and CaCl₂. (B) Number of rbc present in the serum following clot retraction (n = 3). (C) Wet clot weights following clot retraction (n = 3). (D) Image of fully retracted PRP clots 90 minutes after initiation of clot formation. (E) Percentage retraction of PRP clots (n = 3). (F) Serum rbc content of retracted clots containing WT PRP with Fibγ^390–396A rbc and vice versa (n = 3). Data represent mean ± SEM.

Figure 1. Compared with thrombi from WT mice, thrombi from Fibγ^390–396A mice are smaller and have reduced rbc content.

(A) Thrombus weights 1 day after ligation (stasis model). (B) Total rbc content of stasis thrombus lysates, measured as hemoglobin absorbance at 575 nm. (C) Total neutrophil content of thrombus lysates, measured by Ly6G antigen. (D) Total platelet content of thrombus lysates, measured by CD41 antigen. (E) Total fibrin (β-chain) antigen of thrombus lysates. (F) Thrombus weights 1 day after ligation (stenosis model). (G) Total rbc content of stenosis thrombus lysates, measured as hemoglobin absorbance at 575 nm. Each dot represents an individual mouse. Lines represent means.