OC 61.4 - Manipulating platelet secretion to affect hemostasis

Tuesday, June 27, 2023

15:30 – 15:45 ET

Room: 710A

Session Available As: On-Demand

Presenting Author(s)

Smita Joshi, PhD (she/her/hers)

Scientist
Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY, USA
Lexington KY, Kentucky, United States

Co-Author(s)

Alexis N. Smith (she/her/hers)

Graduate Research Assistant
Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY, USA
Lexington, Kentucky, United States
HA

Hammodah R. Alfar

Graduate Student
Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY, USA
Lexington, Kentucky, United States
Kanakanagavalli Shravani Prakhya, PhD (she/her/hers)

Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY, USA
Lexington, Kentucky, United States
IP

Irina Pokrovskaya

Research Associate
University of Arkansas for Medical Sciences
Little Rock, Arkansas, United States
HC

Harry Chanzu

Sr. Technical Account Manager
GenScript
Lexington, Kentucky, United States
BS

Brian Storrie

Professor
University of Arkansas for Medical Sciences
Little Rock, Arkansas, United States
SW

Sidney W. Whiteheart

University Research Professor
Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY, USA
Lexington, Kentucky, United States

Background: Platelet granule secretion is crucial for maintaining vascular microenvironments. The members of soluble N ethylmaleimide sensitive Attachment Receptor family (SNARE) are critical to secretion. Vesicle SNAREs/Vesicle-Associated Membrane Proteins (VAMPs)/v-SNAREs on granules and t-SNAREs on the plasma membrane mediate granule fusion and cargo release. How platelet SNAREs are used and what role(s) secretion plays in hemostasis are still unclear.

Aims: To study the contributions of individual VAMPs in secretion and hemostasis by genetically manipulating VAMP-2, -3, -7, and -8, either alone or in combinations.

Methods: We generated global V7-/-8-/-, V3-/-7-/- and platelet-specific V2Δ3Δ7-/-8-/- mice. Ex vivo assays were used to monitor the kinetics and extent of cargo release (serotonin from dense, PF4 from alpha, and β-hexosaminidase from lysosomes). In vivo assays (FeCl3-mediated arterial injury, venous puncture injury, and tail bleeding) were used to monitor hemostasis.

Results: Secretion analysis demonstrated that V3-/- and V7-/- platelets show no defect, but V3-/-7-/- platelets show mild defect in alpha and lysosomal secretion.  The combined deletion of VAMP-7 and -8 had minimal defect in secretion beyond that of V8-/-, partially due to increased VAMP-2 levels. V2Δ3Δ7-/-8-/- platelets exhibited the most decrease in secretion, although not completely abolished. V7-/-8-/-, and V2Δ3Δ7-/-8-/- mice had the most robust bleeding. Loss of VAMP-8 alone delayed occlusion upon FeCl3 injury without causing bleeding.

Conclusion(s): VAMP-8 is the major platelet VAMP isoform. When it is present, deletion of others (i.e., 2,3,7) alone or in combination minimally affected secretion. Residual secretion when VAMP-2,3,7,8 are deleted may suggest a role for YKT6; only remaining v-SNARE at significant concentrations. Hemostatic phenotypes show that by manipulating the types and levels of platelet VAMPs, we can modulate bleeding, however, the degree of defect is context-specific. These models will be instrumental in probing physiological processes in which platelet contribution is critical, including wound healing and inflammation.
Supported by the NIH, NHLBI (HL56652, HL138179, and HL150818) and the VA.

Learning Objectives:

Appreciate how residual Vesicle-Associated membrane Proteins/ VAMPs levels are co-ordinated in platelets.
Describe which Vesicle-Associated membrane Proteins/ VAMPs are important for platelet granule secretion.
Recognize how platelet granule secretion influences arterial vs venous thrombosis.