The Effect of Factor VIII Deficiencies and Replacement and Bypass Therapies on Thrombus Formation under Venous Flow Conditions in Microfluidic and Computational Models

<div><p>Clinical evidence suggests that individuals with factor VIII (FVIII) deficiency (hemophilia A) are protected against venous thrombosis, but treatment with recombinant proteins can increase their risk for thrombosis. In this study we examined the dynamics of thrombus formation in individuals with hemophilia A and their response to replacement and bypass therapies under venous flow conditions. Fibrin and platelet accumulation were measured in microfluidic flow assays on a TF-rich surface at a shear rate of 100 s⁻¹. Thrombin generation was calculated with a computational spatial-temporal model of thrombus formation. Mild FVIII deficiencies (5–30% normal levels) could support fibrin fiber formation, while severe (<1%) and moderate (1–5%) deficiencies could not. Based on these experimental observations, computational calculations estimate an average thrombin concentration of ∼10 nM is necessary to support fibrin formation under flow. There was no difference in fibrin formation between severe and moderate deficiencies, but platelet aggregate size was significantly larger for moderate deficiencies. Computational calculations estimate that the local thrombin concentration in moderate deficiencies is high enough to induce platelet activation (>1 nM), but too low to support fibrin formation (<10 nM). In the absence of platelets, fibrin formation was not supported even at normal FVIII levels, suggesting platelet adhesion is necessary for fibrin formation. Individuals treated by replacement therapy, recombinant FVIII, showed normalized fibrin formation. Individuals treated with bypass therapy, recombinant FVIIa, had a reduced lag time in fibrin formation, as well as elevated fibrin accumulation compared to healthy controls. Treatment of rFVIIa, but not rFVIII, resulted in significant changes in fibrin dynamics that could lead to a prothrombotic state.</p></div

Fibrin deposition dynamics in response to rFVIIa treatment.

<p>Two patients (5–6) with severe FVIII deficiency with high inhibitor titer were treated with 90 µg/mL rFVIIa. Recalcified whole blood was perfused over glass slides coated with 2.3 fmol TF/cm² and type 1 fibrillar collagen at 100 s⁻¹ for 5 min before and 30 min after treatment with rFVIIa. Platelets (<i>blue</i>, anti-CD41), fibrin(ogen) (<i>green</i>, Alexa488-fibrinogen) and their overlay immediately before (A–C) and 30 min. after (D–F) rFVIIa injection. (G) Transient fibrin density pre-treatment (•) and post-treatment (○) in comparison to a normal control (◊). The dynamics of fibrin deposition before (black bars) and after (white bars) was characterized by (H) maximum fibrin density, (I) the lag time, and (J) the velocity. Error bars represent standard deviations of n = 3. Lines indicate comparisons between pairs according to the Mann-Whitney U-test.</p

Fibrin deposition dynamics from individuals with FVIII deficiencies.

<p>(A) Fibrin density as a function of time for a normal control (◊) and hemophilia samples with plasma FVIII levels of 11.1% (▵), 3.1% (□), and 0.4% (○). The dynamics of fibrin deposition were quantified by three metrics: (B) Maximum fibrin density, which is the integrated fluorescence of the fibrino(gen) signal at the end of the 5 min. assay. (C) The lag time, which is the time to 10% of the maximum fibrin density for normal controls (4 RFU). (D) The velocity, which is the slope of the fibrin density curve from the lag time to the end of the assay. Each data point (•) represents a single individual with either normal or deficient FVIII levels.</p

Clinical characteristics of hemophilia A patients and controls.

<p>FVIII∶C was measured by a one-stage clotting assay and expressed as percent of normal pooled plasma (NPP). Fibrin density and platelet aggregate size were measured at the end of a 5 min. flow assay on type I collagen and 2.3 fmol TF/cm² at 100 s⁻¹. Data is presented as the average and standard deviation of n = 3.</p>†<p>Categories are based on FVIII∶C where control >50%, mild 5–30%, moderate 1–5%, and severe <1%.</p

Thrombin generation under flow for rFVIIa treatment.

<p>The computationally calculated cumulative thrombin production (A), average thrombin concentration (B), maximum thrombin concentration (C), velocity of thrombin production (D) and lag time (E) for 1% FVIII levels at 0.1, 1, and 10 nM FVIIa plasma concentration compared to 100% FVIII levels and 0.1 nM FVIIa. The cumulative thrombin production is normalized by the maximum for 100% FVIII, 0.1 nM FVIIa.</p

Thrombin generation under flow in FVIII deficiencies.

<p>The average thrombin concentration within a thrombus was calculated using a spatial-temporal computational model of thrombus formation on 2.3 fmol TF/cm². (A) Average thrombin concentration as a function of time for FVIII levels of 1, 5, 10, 20, and 100%. The dynamics of thrombin generation were quantified by three metrics: (B) Maximum thrombin concentration, which is the thrombin concentration at the end of the 5 min. simulation. (C) The lag time, which is the time to 1 nM thrombin. (D) The velocity, which is the slope of the average thrombin curve from the lag time to the end of the simulation. Each data point (•) represents a single simulation. The lines are extrapolations between simulation data points.</p