Saliva of Persons with Hemophilia A Triggers Coagulation via Extrinsic Tenase Complexes

Medicine Hematology
Hemophilia A Extrinsic Tenase Complex Saliva Coagulation FVII Deficiency

Extrinsic Tenase Complexes in Human Saliva Trigger Coagulation in Patients with Severe Hemophilia A

Background Introduction

Hemophilia A is a hereditary bleeding disorder caused by a deficiency or defect in clotting factor VIII (FVIII), inherited in an X-linked recessive pattern. Patients with severe hemophilia A often experience recurrent joint bleeding; however, compared to joint bleeding, oropharyngeal mucosal bleeding is significantly less common in these individuals. This phenomenon lacks a clear biological explanation. By contrast, patients with a rare coagulation disorder, factor VII (FVII) deficiency, show a higher prevalence of oropharyngeal bleeding. Understanding the mechanisms behind these contrasting bleeding patterns is crucial for optimizing clinical management and patient education.

Recent studies revealed that human body fluids, such as amniotic fluid, breast milk, and saliva, contain extracellular vesicles (EVs) that carry tissue factor (TF) and activated factor VII (FVIIa). These form extrinsic tenase complexes that can trigger coagulation. This study hypothesizes that the extrinsic tenase complexes present in saliva might provide protection against oropharyngeal bleeding in severe hemophilia A patients. Conversely, the absence of functional extrinsic tenase complexes in the saliva of patients with FVII deficiency might explain their susceptibility to oropharyngeal bleeding.

Source and Authors

This research was led by Johannes Thaler and his team, with contributions from internationally renowned institutions, including the Medical University of Vienna in Austria, Amsterdam University Medical Centers in the Netherlands, and the University of Mainz in Germany. Published on December 19, 2024, in the journal Blood, the study received funding from the German Research Foundation and the Medical Scientific Fund of the Mayor of Vienna.

Methods and Approach

Study Design and Experimental Workflow

The study involved collecting blood and saliva samples from two groups: 1. Severe Hemophilia A Patients (FVIII activity IU/dL): 5 adult males. 2. Severe FVII Deficiency Patients (FVII activity IU/dL): 3 adult males.

The samples were collected both before and after coagulation factor replacement therapy. Key steps and techniques included:

  1. Sample Preparation:

    • Saliva was rendered cell-free through differential centrifugation.
    • EVs were separated from non-vesicle components using ultracentrifugation and size exclusion chromatography (SEC).

  2. Coagulation Function Tests:

    • Whole Blood and Plasma Clotting Tests: Saliva was added to FVIII-deficient whole blood or plasma, and clotting times, fibrin formation, and FXa activity were monitored.
    • Thrombin Generation Assay: The ability of saliva to trigger thrombin generation was tested.
    • Factor Xa (FXa) Generation Assay: A chromogenic assay was used to measure the direct activation of factor X to FXa by saliva.

  3. Characterization of EVs:

    • Transmission electron microscopy (TEM) was used to observe EV morphology.
    • Flow cytometry and Western blot analyses identified EV markers (e.g., CD63, CD9) and evaluated their association with TF/FVIIa activity.

  4. Bleeding Pattern Analysis:

    • A retrospective review of bleeding events over 12 months was conducted in 6 patients with severe hemophilia A who were not on prophylactic FVIII replacement therapy.

Data and Statistical Analysis

GraphPad Prism was used for paired t-tests, with continuous variables presented as mean ± standard deviation. Statistical significance was defined as p < 0.05.

Key Findings

Role of Saliva in Coagulation for Severe Hemophilia A Patients

  1. Saliva Triggers FVIII-Independent Coagulation in Whole Blood and Plasma:

    • Saliva significantly shortened the clotting time of FVIII-deficient whole blood (from 1125 ± 139 seconds to 270 ± 37 seconds; p = 0.0006).
    • Saliva induced fibrin formation and FXa activity in FVIII-deficient plasma.

  2. Presence of Extrinsic Tenase Complexes in Saliva:

    • EVs in saliva induced FXa generation, mediated by TF/FVIIa complexes. This activity was confirmed through antibody inhibition experiments.

  3. Calcium’s Role in Stabilizing Complexes:

    • Chelation of calcium ions in saliva caused dissociation of TF/FVIIa complexes, indicating the importance of calcium in maintaining their activity.

  4. Bleeding Pattern Survey:

    • Among 105 bleeding episodes reported by 6 severe hemophilia A patients, only 19 (18%) were oropharyngeal events. All these episodes stopped spontaneously without FVIII replacement. In contrast, joint and muscle bleeds (66% and 14%, respectively) required FVIII therapy.

Observations in FVII Deficiency Patients

Unlike hemophilia A patients, saliva from FVII-deficient patients showed no functional extrinsic tenase activity. However, the addition of recombinant FVIIa restored FXa generation, suggesting a high affinity between TF in saliva and FVIIa.

Conclusions and Significance

This study reveals for the first time that EVs in the saliva of severe hemophilia A patients can trigger coagulation via extrinsic tenase complexes. These findings explain the rarity of oropharyngeal bleeding in hemophilia A patients and highlight the absence of similar activity in saliva from FVII deficiency patients. The study’s implications include:

  1. Scientific Insights:

    • Provides a molecular basis for the differing bleeding phenotypes in hemophilia A and FVII deficiency.
    • Demonstrates a potential high-affinity interaction between TF and FVIIa in saliva.

  2. Clinical Relevance:

    • Offers a potential educational tool for hemophilia A patients, emphasizing their body’s compensatory mechanisms in certain bleeding scenarios.
    • Could help refine bleeding management strategies and enable more personalized treatment for patients with heterogeneous bleeding patterns.

  3. Future Directions:

    • Suggests that similar mechanisms might exist in other EV-containing body fluids, such as breast milk, urine, and semen.

Study Innovations and Highlights

  1. This study is the first to link the coagulation potential of human saliva to the bleeding phenotype in hemophilia A patients, addressing a longstanding clinical question.
  2. A combination of advanced experimental techniques, including thrombin generation and high-efficiency EV separation, ensured robust and reproducible results.

Overall, these findings deepen our understanding of EV-mediated coagulation in saliva and their role in explaining the divergent bleeding patterns between hemophilia A and FVII deficiency. The study paves the way for further research and clinical applications in hemostasis-related disorders.