The investigators plan to measure routine laboratory parameters of coagulation and
fibrinolysis, the procoagulant or profibrinolytic activity of microparticles (MPs), and
explore the role of the procoagulant and profibrinolytic activating factor of MPs in the
pathogenesis of coagulopathy in patients with APL.
i. Dynamic turbidimetry of plasma clot formation. The effects of MPs on the kinetics of
fibrin formation and on the optical properties of clots are studied using dynamic
turbidimetry of re-calcified plasma samples (platelet-free plasma and microparticle-depleted
plasma) without adding any clotting activator. Clotting of plasma samples induced by Ca2+ is
followed by monitoring the optical density at λ = 405 nm at 37 °C.
ii. Thrombin generation assay. The amount of thrombin formed in plasma upon re-calcification
is measured directly using a modified thrombin generation test . Because fibrin interferes
with colorimetric measurements, plasma samples are first defibrinated by adding reptilase
followed by incubation at 37 °C. The clots are removed. Then a chromogenic substrate for
thrombin is added to the plasma samples. Thrombin generation is started by adding CaCl2 with
simultaneous recording of the absorbance at λ = 405 nm.
iii. Thrombin generating capacity of the MPs. MPs are reconstituted in defibrinated
(reptilase treated), normal pooled microparticle-depleted plasma. Then a chromogenic
substrate for thrombin is added to the samples. Thrombin generation is started by adding
CaCl2 with simultaneous recording of the absorbance at λ = 405 nm.
iv. Thrombin generation inhibitory experiments. The following inhibitors are pre-incubated
with the microparticles: Annexin V, anti-human tissue factor (TF) and irrelevant control
immunoglobulin G (IgG). Then repeats the experiment iii.
v. Fibrinolytic activity. Incubate a fixed concentration of plasminogen with the plasma
samples in the presence of a chromogenic substrate selective for plasmin. Plasmin formed from
plasminogen bound at the surface of microparticles cleaves the chromogenic substrate and the
released p-nitroaniline is detected by measuring A405nm as a function of time.
vi. Determination of fibrinolytic activity on microparticles. The capacity of microparticles
to activate plasminogen is determined by incubating a fixed concentration of plasminogen
(1mM) with the microparticles with or without t-PA and/or u-PA in the presence of a
chromogenic substrate selective for plasmin. Plasmin formed from plasminogen bound at the
surface of microparticles cleaves the chromogenic substrate and the released p-nitroaniline
is detected by measuring A405nm.
vii. Fibrinolytic activity inhibitory experiments. The following inhibitors are pre-incubated
with the microparticles: anti-human tissue type plasminogen activator (tPA) , anti-human
urokinase type plasminogen activator (uPA), and respective irrelevant control IgGs;
ε-aminocaproic acid and plasminogen activator inhibitor-1 (PAI-1).Then repeat the experiment
vi.