Thrombin in complex with dabigatran can still interact with PAR‐1 via exosite‐I and instigate loss of vascular integrity

Figures

• 

  FIGURE 1. Tissue factor (TF)‐mediated thrombin generation in PFP incubated on ECs. A, HUVECs stimulated with TNF‐α (0–10 ng/ml, 5 h) show enhanced TF‐mRNA expression. (B, HUVECs stimulated with TNF‐α (0–10 ng/ml, 5 h) generate FXa due to expression of active TF on their surface. Data represent two independent experiments; means were compared using one‐way analysis of variance with Tukey’s multiple comparisons test. *P < .05, **P < .01; ***P < .001. C, Immunofluorescence images of permeabilized HUVECs stimulated with TNF‐α (5 ng/ml for 5 h) show TF‐protein expression stained with 10H10 and VWF–protein secretion. D‐E, HUVECs stimulated with TNF‐α (0–10 ng/mL, 5 hrs) show an enhanced surface expression of (D) TF and TFPI and a decreased expression of (E) EPCR and TM as measured by flow cytometry. F, Thrombin generation assays were performed on monolayers of confluent HUVECs, expressed as thrombin peak (nM) or lag time (min) measured in separate experiments: ± TNF‐α (1 µg/ml, 5 h); ± CTI to inhibit the intrinsic pathway component FXIIa; ± function‐blocking antibody directed against TF (5G9, 50 μg/ml) or an IgG control (50 μg/mL); ± ECs as source of TF and phospholipids; ± function‐blocking…

• 

  FIGURE 2. Rivaroxaban maintains EC integrity, while dabigatran does not. A, Schematic of ECIS experimental setup. The barrier‐recovery capacity was expressed as percentage of maximal recovery measured before the washing procedure (barrier reference). B and H, Examples of ECIS barrier recovery curves over time on TNF‐α–stimulated HUVECs from one PFP donor and an EC donor (mean ± SD values of triplicate wells in dotted lines) measured from −20 to 120 min of PFP, PFP + dabigatran (Dabi), PFP + rivaroxaban (Riva), PFP + hirudin (Hirudin), and PFP + PPACK (PPACK). Recovery phase = 0–40/60 min; maintenance phase = 40/60–120 min, varying per experiment. C and I, Barrier resistance values of the recovery phase (three wells per condition at 40/60 min) and (D and J) maintenance phase (at 120 min). E and K, Examples of thrombin generation curves over time from the same plasma‐ and EC donors used in (B) and (H), respectively. Quantification of thrombin generation expressed as (F and L) thrombin peak (mean ± SD in nM) and (G and M) lag time (mean ± SD in min). Means were compared using one‐way analysis of variance with Tukey’s multiple comparisons test. *P < .05, **P < .01, ***P < .001. ECs,…

• 

  FIGURE 3. Role of TF and APC on EC barrier function. A, Representation of ECIS barrier recovery curves over time on TNF‐α–stimulated HUVECs with PFP in the absence or presence of function‐blocking TF antibody (5G9, 50 µg/ml) or an isotype IgG (IT 50 µg/ml). B, Quantification of the barrier resistance at the recovery phase (at 40 min) and C, maintenance phase (at 120 min) shows that blockage of TF supports barrier recovery significantly (two‐tailed Student’s t‐test, P < .001). D, Barrier recovery curves with the quantification for the (E) recovery phase at 50 min and the (F) maintenance phase at 120 min showed that inhibition of PC activation with antibody HPC4 (αPC, 100 μg/ml) reduced the barrier in the recovery and maintenance phase in the absence of dabigatran compared to isotype IgG (IT, 100 μg/ml). In the presence of dabigatran, HPC4 had no effect on the (E) recovery phase nor the (F) maintenance phase compared to IT. G, Thrombin generation curves with quantification expressed as (H) thrombin peak (nM) and (I) lag time (min) showed no effect of HPC4 versus IT in PFP and no effect of HPC4 versus IT in PFP + dabigatran. Means were compared using one‐way analysis of variance with Tukey’s…

• 

  FIGURE 4. Thrombin binding capacity of peptide D50‐S64. A, Schematic illustration of free thrombin with its active site and exosite‐I and ‐II and in complex with different direct thrombin inhibitors. Dabigatran is a non‐peptidic, reversible inhibitor interacting with thrombin’s active site only, leaving exosite‐I and ‐II available. Hirudin binding is irreversible and will interact with both the active site and exosite‐I. PPACK is a peptide derivative, which forms an irreversible interaction with the active site, leaving exosite‐I available. Peptide D50‐S64 was designed, which mimics the PAR‐1 sequence that will interact with exosite‐I of thrombin. Incubation of thrombin with both peptide and dabigatran will reflect the situation of hirudin‐blocked thrombin. B, Light transmission aggregometry was performed with purified platelets (200.106/ml) incubated with thrombin (0–1 U/ml). C, Thrombin‐induced (0.1 U/ml) platelet aggregation was dose‐dependently inhibited with peptide D50‐S64 (0–100 µM). Individual points of three separate experiments with three different platelet donors are shown. A standardized CAT assay was performed using pooled PFP, to which purified TF and phospholipids were…

• 

  FIGURE 5. The effect of peptide D50‐S64 on thrombin generation and barrier function. A, Representation from one plasma‐donor and an endothelial cell donor of thrombin generation (nM) over time in platelet‐free plasma (PFP), PFP supplemented with dabigatran (D or dabi), peptide D50‐S64 (1–1000 µM), or dabigatran plus peptide D50‐S64 (1–1000 µM) incubated on tumor necrosis factor‐‐stimulated human umbilical vein endothelial cells as source of tissue factor and phospholipids. B, Quantification of thrombin peak values (mean ± standard deviation [SD] in nM) and (C) lag time (min) showed no effect of peptide D50‐S64 compared to PFP alone and no extra effect in combination with dabigatran. D, An example of electric cell‐substrate impedance sensor (ECIS) recovery curves over time (mean values of triplicate wells with SD in dotted lines) in the absence and (G) presence of dabigatran (PFP + dabi) in combination with increasing dose of peptide D50‐S64 (1–1000 µM). E, Quantification for the recovery phase (at 50 min) and (F) the maintenance phase (at 120 min) showed no effect of peptide D50‐S64 on barrier recovery in the absence of dabigatran. H, In the presence of dabigatran, peptide D50‐S64 had no…