Role of Microparticles in the Coagulopathy of Acute Promyelocytic Leukemia

Acute Promyelocytic Leukemia Clinical Trial

Official title:

Role of Microparticles in the Coagulopathy of Acute Promyelocytic Leukemia

Clinical Trial Details — Status: Recruiting

Administrative data

• NCT number: NCT02991066
• Other study ID #: 1309
• Status: Recruiting
• First received: March 2, 2016
• Last updated: April 22, 2018
• Start date: October 2014
• Est. completion date: December 2020

Study information

• Verified date: April 2018
• Source: First Affiliated Hospital of Harbin Medical University
• Contact: Jin Zhou, MD, PhD
• Phone: 008645185555951
• Email: zhoujin1111[@]126.com
• Is FDA regulated: No
• Study type: Observational

Clinical Trial 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.

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.

Recruitment information / eligibility

• Status: Recruiting
• Enrollment: 20
• Est. completion date: December 2020
• Est. primary completion date: December 2019
• Accepts healthy volunteers: Accepts Healthy Volunteers
• Gender: All
• Age group: 18 Years and older

Eligibility

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.

Related Conditions & MeSH terms

• Acute Promyelocytic Leukemia
• Blood Coagulation Disorders
• Hemostatic Disorders
• Leukemia
• Leukemia, Promyelocytic, Acute

Locations

Country	Name	City	State
China	the First Affiliated Hospital of Harbin Medical University	Harbin	Heilongjiang

Sponsors (1)

Lead Sponsor	Collaborator
First Affiliated Hospital of Harbin Medical University

Country where clinical trial is conducted

China, 

References & Publications (14)

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Pereira J, Alfaro G, Goycoolea M, Quiroga T, Ocqueteau M, Massardo L, Pérez C, Sáez C, Panes O, Matus V, Mezzano D. Circulating platelet-derived microparticles in systemic lupus erythematosus. Association with increased thrombin generation and procoagulant state. Thromb Haemost. 2006 Jan;95(1):94-9. — View Citation

Sinauridze EI, Kireev DA, Popenko NY, Pichugin AV, Panteleev MA, Krymskaya OV, Ataullakhanov FI. Platelet microparticle membranes have 50- to 100-fold higher specific procoagulant activity than activated platelets. Thromb Haemost. 2007 Mar;97(3):425-34. — View Citation

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* Note: There are 14 references in all — Click here to view all references

Outcome

Type	Measure	Description	Time frame	Safety issue
Primary	Change From Baseline in the Levels and Cellular Origin of MPs at 5 Weeks	Demonstration that the some procoagulant or profibrinolytic activating factors expressed on MP in APL patients' plasma associate with the thrombin generating capacity and fibrinolytic activity of patients' plasma.	5 weeks