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Thromb Haemost
DOI: 10.1055/a-2747-9083
Original Article: Cellular Haemostasis and Platelets

Trypsin Activates Human Platelets via Proteolytic Cleavage of Protease-Activated Receptor 4

Authors

  • Emma M. Webb 

    1   Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, United States

  • Kendal Seymour

    2   Department of Neuroscience, Vanderbilt University, Nashville, Tennessee, United States

  • Heidi E. Hamm

    1   Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, United States
    3   Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, United States

Funding Information This work was supported by grants from the NIH (1R01AG068623).
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Graphical Abstract

Abstract

Background

Human platelets express protease-activated receptor (PAR) 1 and 4 on the platelet membrane. PAR1 activation is crucial for hemostasis, while PAR4 activation produces a prolonged pro-inflammatory platelet response. Once activated, the PAR receptors cause platelet activation and aggregation. The PARs are activated by proteolytic cleavage of their N-terminus, revealing a new N-terminus called the tethered ligand. This tethered ligand folds back and activates the receptor. PAR1 and PAR4 differ in their tethered ligand sequence as well as their signaling cascades; however, they are both activated by various serine proteases, specifically thrombin.

Objective

Much is known about PAR4 activation via thrombin activation; however, little is known about activation by other proteases. Thrombin cleavage of PAR4 and subsequent activation leads to G proteins, Gαq and Gα12/13, and β-arrestin signaling. The objective of this study is to characterize trypsin activation of PAR4 on human platelets.

Methods

Platelets were isolated from the whole blood of healthy human volunteers by centrifugation. Platelets were utilized in flow cytometry assays, calcium mobilization assays, and western blot experiments to characterize trypsin activation of human platelets.

Results

Here, we demonstrate that trypsin activates human platelets solely through PAR4. Trypsin activation of PAR4 signaling through both Gαq and Gα12/13 is as effective as α-thrombin activation of PAR4.

Conclusion

Thrombin may be the main protease to activate PAR4 under typical conditions; however, under pathological conditions such as acute pancreatitis (AP), trypsin activation of PAR4 may be a main driver of platelet activation and disease progression.

Keywords

PAR - proteases - platelet pharmacology

Contributors' Statement

Participated in research design: E.M.W., K.S., and H.E.H. Conducted experiments: E.M.W. and K.S. Performed data analysis: E.M.W. Wrote or contributed to the writing of the manuscript: E.M.W. and H.E.H.


Informed Consent

Informed consent was obtained from all healthy volunteers, in accordance with IRB #130814.


Supplementary Material



Publication History

Received: 28 July 2025

Accepted after revision: 13 November 2025

Accepted Manuscript online:
17 November 2025

Article published online:
04 December 2025

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