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    Oral Communication

    Diagnostics and OMICs

    High Throughput Methods for Improved Understanding of Coagulation and Bleeding

    OC 57.1 - Peptidomics reveals expected and unexpected proteolytic events in plasma upon activation of the coagulation cascade

    Tuesday, June 27, 2023
    10:45 – 11:00 ET
    Room: 518
    Session Available As: On-Demand

      Presenting Author(s)

    • Jessica Del Castillo Alferez, Msc (she/her/hers)

      PhD student
      Department of Molecular Hematology, Sanquin Research, Amsterdam, The Netherlands.
      Amsterdam, Noord-Holland, Netherlands

      • Co-Author(s)

    • FV

      Floris Van Alphen

      Technician
      Department of Molecular Hematology, Sanquin Research, 1066 CX Amsterdam, the Netherlands
      Amsterdam, Noord-Holland, Netherlands

    • Cv

      Carmen van der Zwaan

      Technician
      Department of Molecular Hematology, Sanquin Research, Amsterdam, The Netherlands.
      Amsterdam, Noord-Holland, Netherlands

    • MZ

      Marleen Zuurveld

      senior technician
      Department of Molecular Hematology, Sanquin Research, Amsterdam, The Netherlands
      Amsterdam, Noord-Holland, Netherlands

    • KB

      Kamran Bakhtiari

      Technician
      Department of Molecular Hematology, Sanquin Research, Amsterdam, The Netherlands.
      Amsterdam, Noord-Holland, Netherlands

    • Arie J. Hoogendijk, PhD

      PI
      Department of Molecular Hematology, Sanquin Research, Amsterdam, The Netherlands.
      Amsterdam, Noord-Holland, Netherlands

    • Herm-Jan M. Brinkman, PhD (he/him/his)

      Biochemist
      Department of Molecular Hematology, Sanquin Research, Amsterdam, The Netherlands
      Amsterdam, Noord-Holland, Netherlands

    • JM

      Joost CM Meijers

      Biochemist
      Department of Molecular Hematology, Sanquin Research, Amsterdam, The Netherlands
      Amsterdam, Noord-Holland, Netherlands

    • AM

      Alexander B. Meijer

      PI
      Department of Molecular Hematology, Sanquin Research, 1066 CX Amsterdam, the Netherlands
      Amsterdam, Noord-Holland, Netherlands

    Background: Hemostasis is driven by several proteolytic events resulting in the formation and dissolution of a fibrin clot. The proteolytic processing of procoagulant and anticoagulant plasma proteins has been well characterized. However the impact on other plasma proteins is not fully understood. Insight therein may increase our understanding in the putative link between coagulation and other biological processes in plasma.

    Aims: To assess proteolytic events in plasma that are associated with hemostasis.

    Methods: Citrated plasma from healthy individuals was recalcified and the extrinsic pathway of coagulation was initiated with tissue factor in the absence or presence of platelets. Resulting endogenous peptides were extracted and analyzed by LC-MS/MS. Spectra were interpreted with PEAKSX De Novo algorithm. Identified peptides were mapped in silico against the Human UniProt database. This is a novel approach that allows making snapshots at distinct time points of the proteolytic events in and around the coagulation cascade.

    Results: Upon activation of coagulation by tissue factor, peptides were generated including (truncated) variants of activation peptides of amongst others fibrinogen and factor XIII. In addition, we identified fibrinogen-derived peptides that could be mapped to cleavages by thrombin and plasmin. Also, protease inhibitors were processed such as alpha1-antitrypsin, antithrombin and alpha-2-macroglobulin. Surprisingly, In the presence of platelets, an increased number of proteolytic peptides were observed of prothrombin, vitronectin and proteins of the complement system including C4a, C4b, and C7.

    Conclusion(s): Our data reveal the general impact of coagulation on plasma proteins. We found expected and novel proteolytic events upon activation of coagulation. Our results demonstrate that peptidomics can be leveraged to obtain functional insights of proteolysis during coagulation and fibrinolysis, and may be a useful approach to identify novel modulators of hemostasis.

    Learning Objectives:

    • Recognize the medical need to identify novel modulators of hemostasis and define how limited proteolysis of proteins regulates hemostasis.
    • Distinguish peptidomics as a leverage to obtain functional insights of hemostatic processes where proteolysis is involved and illustrate what proteolytic signatures are.
    • Name examples of proteolytic events in plasma upon activation of the coagulation cascade. Describe proteolytic signatures in plasma that are characteristic of different modulators of hemostasis.