Brief Title
Role of Microparticles in the Coagulopathy of Acute Promyelocytic Leukemia
Official Title
Role of Microparticles in the Coagulopathy of Acute Promyelocytic Leukemia
Brief Summary
Although the clinical application of differentiation therapy has made great success in the
treatment of acute promyelocytic leukemia (APL), early fatal bleeding remains an unsolved
problem which accounts for the main reason of induction failure in APL patients. The clinical
manifestation of both serious bleeding and thrombosis illustrate the complexity of the
pathogenesis of coagulopathy in APL. Despite extensive research, the pathogenesis of
coagulopathy in APL is still unclear. Microparticles, 0.11μm in diameter, are small membrane
vesicles released to circulation by blood cells and vascular endothelial cells during
activation or apoptosis. Microparticles (MPs) derived from different cells types all exert
procoagulant activity mediated by phosphatidylserine (PS) and carry some basic substances
derived from their origin cells. Also, the biological activity of microparticles is often
significantly higher than that of the cells they come from. According to these problems and
background knowledge, our project aims to observe the roles of microparticles derived from
APL cells and the procoagulant or profibrinolytic activating factors resided on these
microparticles in the pathogenesis of coagulopathy in APL, and the effects of different
induction therapies, chemotherapeutic drugs or differentiation agents on these microparticles
and their procoagulant or profibrinolytic activating factors. To carry out this study,
microparticles are obtained from patients who undergo different induction therapies at
different time points or from primary bone marrow APL cells which are treated by different
drugs in vitro at different time points, the expressions and activities of five procoagulant
or profibrinolytic activating factors, which are highly expressed in APL cells, PS exposure
and the functional state of these microparticles, will be dynamically monitored. Further
study of the pathogenesis of coagulopathy in APL can provide clues and help for deep
understanding of clinical manifestations, guiding clinical treatment as well as judging
prognosis, and establishing theoretical basis for exploring new treatment.
Detailed Description
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.
Study Type
Observational
Primary Outcome
Change From Baseline in the Levels and Cellular Origin of MPs at 5 Weeks
Condition
Acute Promyelocytic Leukemia
Study Arms / Comparison Groups
Patients
Description: patients with de novo acute promyelocytic leukemia with hemorrhage.
Publications
* Includes publications given by the data provider as well as publications identified by National Clinical Trials Identifier (NCT ID) in Medline.
Recruitment Information
Estimated Enrollment
20
Start Date
October 2014
Completion Date
December 2020
Primary Completion Date
December 2019
Eligibility Criteria
Inclusion Criteria:
- Patients with de novo APL accompanied by hemorrhage.
- The diagnosis was confirmed by the presence of t(15;17) and/or the PML (promyelocytic
leukemia)/RARa(retinoic acid receptor alpha) fusion gene.
- Patients should receive single-agent arsenic trioxide (ATO) for induction therapy.
Exclusion Criteria:
- Patients with relapsed acute promyelocytic leukemia.
- Patients without evidence of bleeding.
- Patients younger than 18 years.
Gender
All
Ages
18 Years - N/A
Accepts Healthy Volunteers
Accepts Healthy Volunteers
Contacts
Jin Zhou, MD, PhD, 008645185555951, [email protected]
Location Countries
China
Location Countries
China
Administrative Informations
NCT ID
NCT02991066
Organization ID
1309
Responsible Party
Sponsor
Study Sponsor
First Affiliated Hospital of Harbin Medical University
Study Sponsor
Jin Zhou, MD, PhD, Study Chair, First Affiliated Hospital of Harbin Medical University
Verification Date
April 2018