Dematin Regulates Calcium Mobilization, Thrombosis, and Early Akt Activation in Platelets

Abstract

The complex intrinsic and extrinsic pathways contributing to platelet activation profoundly impact hemostasis and thrombosis. Detailed cellular mechanisms that regulate calcium mobilization, Akt activation, and integrin signaling in platelets remain incompletely understood. Dematin is a broadly expressed actin binding and bundling cytoskeletal adaptor protein regulated by phosphorylation via cAMP-dependent protein kinase. Here, we report the development of a conditional mouse model specifically lacking dematin in platelets. Using the new mouse model termed PDKO, we provide direct evidence that dematin is a major regulator of calcium mobilization, and its genetic deletion inhibits the early phase of Akt activation in response to collagen and thrombin agonists in platelets. The aberrant platelet shape change, clot retraction, and in vivo thrombosis observed in PDKO mice will enable future characterization of dematin-mediated integrin activation mechanisms in thrombogenic as well as nonvascular pathologies.

Keywords:

• platelets
• calcium signaling

SUPPLEMENTAL MATERIAL

Supplemental data for this article can be accessed online at https://doi.org/10.1080/10985549.2023.2210033.

ACKNOWLEDGEMENTS

We thank Dr. Peter Newman of Blood Research Institute, Milwaukee, Wisconsin, for generously sharing the collagen-related peptide (CRP) for platelet activation studies. We also thank Dr. Lidija Covic of Tufts Medical Center, Boston, MA, for sharing the TRAP4 agonist for initial studies of platelet activation. Dr. Covic’s technical guidance for the calcium mobilization experiments was invaluable. Anti-kindlin-3 antibodies were kindly provided by Dr. Edward Plow (Cleveland Clinic) and Dr. Stephen C. Bunnell (Tufts University). We thank Stephen Kwok (Tufts Core Flow Cytometry Facility) for invaluable guidance with the flow cytometry measurements. Finally, we thank Donna-Marie Mironchuk for her many contributions to the administrative organization of the project, proofreading, and improvements of figures. Figure 9 was created with BioRender.com.

AUTHORS’ CONTRIBUTIONS

Contributions: DF, TH, and AC conceived and designed the study. TH generated the PDKO mice. DF performed all the major experiments reported in this study. The in vivo thrombosis experiments (Fig. 8) were performed by GM-S using the resources and advice from RF. RF provided critical feedback during the course of this study. YD played a pivotal role in the characterization and genotyping of PDKO mice and data reported in Fig. 1 to Fig. 3 and Fig. 7 as well as kindlin-3 expression analysis (Supplemental Fig. 3). DF wrote the first draft of the manuscript. AC edited the manuscript and guided the project from its inception to completion. All authors edited various versions of the manuscript and approved the final manuscript.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author, (AC), upon reasonable request.

Additional information

Funding

This work was supported in part by grants from the National Heart, Lung, and Blood Institute (NHLBI) [RO1-HL060961 (AC), R01-HL095050 (AC), R35HL135775 (RF)], Tufts Collaborates Seed Grant Program (AC), American Heart Association Grant-in-Aid [15GRNT25710346 (AC)], and American Heart Association Predoctoral Fellowship to Daniel Fritz [ 20PRE35210897; 2020-2021].