Evaluation of a New Approach of the Diagnosis of Constitutional Functional Disorders of Platelets

NCT ID: NCT01957345

Last Updated: 2025-12-10

Basic Information

• Recruitment Status: COMPLETED
• Clinical Phase: NA
• Total Enrollment: 322 participants
• Study Classification: INTERVENTIONAL
• Study Start Date: 2013-02-28
• Study Completion Date: 2017-06-29

Brief Summary

The primary purpose of the study is to evaluate a standardized method of screening for platelet signalling defects in patients with constitutional disorders of platelet function of unknown origin. We hypothesize that such defects are under-diagnosed in patients, due to heavy workup and requirement of relatively large blood sample by conventional biochemical methods. We propose to analyse kinase signalling downstream platelet membrane receptors using multiplex flow cytometry quantification and fluorescent platelet barcoding.

Detailed Description

Little is known of the molecular basis of disorders of signalling pathways potentially responsible of constitutional defects of platelet functions (adhesion, aggregation or secretion) (1-5). Indeed, in routine practice, this investigation is limited by the complexity of the analyse using biochemical methods (western blotting), and the requirement of large amount of platelets.

We have designed a new approach for the quantification of platelet cytoplasmic phosphoproteins by flow cytometry. Fresh platelets in platelet rich plasma are analysed at baseline or after stimulation by major agonists (ADP, TRAP, thromboxane analogue, or collagen-related peptide), with and without relevant inhibitors of each pathway. Multiplex barcoding is used to identify each condition, allowing a high throughput analysis (6, 7). Platelet signalling profiling of Akt, Slp76, P38 MAPK and LIMK can be obtained from a blood sample of less than 10 ml, and within 6h The main objective of the study is to standardize the method between clinical laboratories with a standard expertise in flow cytometry. The study will be performed in 4 academic hospitals members of the French reference network of rare platelet diseases. Three groups of patients referred for mild or severe bleeding disorders will be included: 1) a control group of patients (30 per centre) with a bleeding disorder definitely other than of platelet origin (e.g. "low" von Willebrand); 2) a group of 10 patients per centre with definite constitutional platelet disorder (e.g. Glanzmann thrombasthenia) and 3) a group of 10 patients per centre with a defect of platelet function of unknown origin, potentially defective in signalling pathway.

The control group will serve to standardize the method between centres and to establish the reference values. A quality control will be set up by using frozen platelet preparations. The patients with definite platelet disorder will be useful for detecting potential signalling defects still not described in these pathologies. Platelet signalling defects which could be evidenced in these groups will be further identified by conventional biochemical and molecular methods after confirmation on a new sample.

If this new approach can be proposed to clinical laboratories working on rare platelet diseases, we expect an advance in our knowledge in the field. In addition the method has a potential in pharmaceutical innovation, for identifying (8) or monitoring new antiplatelet agents (9, 10), or identifying platelet defects induced by new "target therapies" designed for other diseases such as cancer or immune pathologies (10).

Conditions

Platelet Dysfunction

Study Design

• Allocation Method: NON_RANDOMIZED
• Intervention Model: PARALLEL
• Primary Study Purpose: DIAGNOSTIC
• Blinding Strategy: NONE

Study Groups

bleeding disorder

Blood punction at patients with bleeding disorder definitely other than of platelet origin (e.g. "low" von Willebrand)

Group Type: OTHER

Blood punction

Intervention Type: OTHER

T1 and T2: one blood punction with signalisation test (T1 for each arm, T2 at +6 months only if an anomaly at signalisation test is detected)

signalisation test is made by flow cytometry

Stage 1: for arm "bleeding disorder" : flow cytometry analysis to establish reference values observed in subjects without thrombopathy for each marker after agonist stimulation and corresponding search of an effect if a center-center effect is observed on the reference values its origin will be sought and corrected.

Stage 2: for the two others arms: Several markers will be analyzed, corresponding to routes or levels of different signaling. For each of these markers, the test will evaluate quantitatively the phosphorylation activity of the protein tested.

constitutional platelet disorder

Blood punction at patients with constitutional platelet disorder (e.g. Glanzmann thrombasthenia)

Group Type: OTHER

Blood punction

Intervention Type: OTHER

T1 and T2: one blood punction with signalisation test (T1 for each arm, T2 at +6 months only if an anomaly at signalisation test is detected)

signalisation test is made by flow cytometry

Stage 1: for arm "bleeding disorder" : flow cytometry analysis to establish reference values observed in subjects without thrombopathy for each marker after agonist stimulation and corresponding search of an effect if a center-center effect is observed on the reference values its origin will be sought and corrected.

Stage 2: for the two others arms: Several markers will be analyzed, corresponding to routes or levels of different signaling. For each of these markers, the test will evaluate quantitatively the phosphorylation activity of the protein tested.

defect of platelet function

Blood punction at patients with defect of platelet function of unknown origin, potentially defective in signalling pathway.

Group Type: OTHER

Blood punction

Intervention Type: OTHER

T1 and T2: one blood punction with signalisation test (T1 for each arm, T2 at +6 months only if an anomaly at signalisation test is detected)

signalisation test is made by flow cytometry

Stage 1: for arm "bleeding disorder" : flow cytometry analysis to establish reference values observed in subjects without thrombopathy for each marker after agonist stimulation and corresponding search of an effect if a center-center effect is observed on the reference values its origin will be sought and corrected.

Stage 2: for the two others arms: Several markers will be analyzed, corresponding to routes or levels of different signaling. For each of these markers, the test will evaluate quantitatively the phosphorylation activity of the protein tested.

Interventions

Blood punction

T1 and T2: one blood punction with signalisation test (T1 for each arm, T2 at +6 months only if an anomaly at signalisation test is detected)

signalisation test is made by flow cytometry

Stage 1: for arm "bleeding disorder" : flow cytometry analysis to establish reference values observed in subjects without thrombopathy for each marker after agonist stimulation and corresponding search of an effect if a center-center effect is observed on the reference values its origin will be sought and corrected.

Stage 2: for the two others arms: Several markers will be analyzed, corresponding to routes or levels of different signaling. For each of these markers, the test will evaluate quantitatively the phosphorylation activity of the protein tested.

Intervention Type: OTHER

Eligibility Criteria

Inclusion Criteria

• patient consulting for hemorrhagic symptomatology with a bleeding disorder definitely other than of platelet origin (e.g. "low" von Willebrand) / or constitutional platelet disorder (e.g. Glanzmann thrombasthenia) / or with a defect of platelet function of unknown origin, potentially defective in signalling pathway.
• Informed consent form
• patient with social security insurance or equivalent

Exclusion Criteria

• treatment interfering with platelets function within 7 days prior to enrollment
• thrombocytopenia <100G/L
• pregnant or lactating females
• subjects under juridical protection guardianship or tutelage measure
• subjects involved in another clinical trial

• Minimum Eligible Age: 18 Years
• Eligible Sex: ALL
• Accepts Healthy Volunteers: No

Sponsors

University Hospital, Toulouse

OTHER

Sponsor Role: lead

Responsible Party

Responsibility Role: SPONSOR

Principal Investigators

Pierre SIE, MD PhD

Role: PRINCIPAL_INVESTIGATOR

University Hospital, Toulouse

Locations

University Hospital Toulouse

Toulouse, , France

Countries

France

References

Garcia C, Dejean S, Savy N, Bordet JC, Series J, Cadot S, Ribes A, Voisin S, Rugeri L, Payrastre B, Sie P. Multicolor flow cytometry in clinical samples for platelet signaling assessment. Res Pract Thromb Haemost. 2023 May 16;7(4):100180. doi: 10.1016/j.rpth.2023.100180. eCollection 2023 May.

Reference Type: RESULT

PMID: 37538502 (View on PubMed)

Other Identifiers

PHRCI 2012

Identifier Type: OTHER_GRANT

Identifier Source: secondary_id

12 068 08

Identifier Type: -

Identifier Source: org_study_id