2017 Cardiovascular Research Day Abstract Book

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Fine Tuning Platelet Secretion to Modulate Hemostasis
Smita Joshi, MS 1 • Irina Pokrovskaya, MS 2 • Brian Storrie, PhD 2 • Sidney W. Whiteheart, PhD 1
1Molecular and Cellular Biochemistry, University of Kentucky • 2 Department of Physiology and
Biophysics, University of Arkansas for Medical Sciences
Graduate Student
Globally, occlusive thrombotic events: e.g., heart attacks and cerebral strokes, cause > 50% of total
deaths attributed to the noninfectious disease. However, aggressive attempts to limit thrombosis
cause bleeding, which can be equally catastrophic. What is needed is a strategy to limit clot
formation, but not prevent it. Platelets play a critical role in controlling bleeding by sensing vascular
damage and releasing a host of components to seal breaches. This secretion process is mediated by
Soluble N-ethylmaleimide Sensitive Factor Attachment Protein Receptors (SNAREs) and their
regulators. To drive secretion, vesicle (v)-SNARE on granules and target (t)-SNARE on the plasma
membrane (PM) form a trans-bilayer complex that mediates membrane fusion. Syntaxin 11 and
SNAP-23 form the functionally relevant t-SNARE heterodimer. For v-SNAREs, platelets contain
Vesicle-Associated Membrane Protein (VAMP)-2, -3, -4, -5, -7, and -8. We focused on how the
platelet VAMPs influence secretion and whether modulating secretion can modulate clot formation.
To address this goal, we genetically titrated the different VAMPs to define their roles in exocytosis
and hemostasis.
We gathered global VAMP-3-/- and VAMP-8-/- animals. To overcome embryonic lethality of global
VAMP-2 deletion, we generated platelet-specific VAMP-2/3-/- mice by using a tissue-specific
promoter that facilitates expression of tetanus toxin that cleaves VAMP-2 and 3. We crossed these
with VAMP-8-/- mice to create platelet-specific VAMP-2/3/8-/- mice. Structural analysis of wildtype
and VAMP-deficient platelets showed that the α granule cargo solubilization/decondensation
follows granule fusion. To define the structure of secretion, activation intermediates were fixed at
various time points, post stimulation, and electron microscopy was performed. The data indicate
that granule decondensation is time- and agonist concentration-dependent. Moreover,
decondensation of granule cargo was VAMP dependent. Three dimensional EM analyses indicate
that VAMP-8 plays a major role in compound, intra-granule fusion and also contributes to single,
granule-PM fusion. Our structural data elucidate how platelet secretion occurs at the cellular level
and explains the complex secretion kinetics previously reported in activated platelets.
To further measure the functional importance of the VAMPs, ex vivo secretion assays were used to
monitor the kinetics and the extent of release from all three platelet granules (dense, α, and
lysosomes). Only VAMP-2/3/8-/- platelets showed a robust defect in secretion (~70% decrease),
more than observed for VAMP-8-/- platelets (~50%). When we studied the effects of secretion on
hemostasis, only VAMP-2/3/8-/- mice showed significantly increased tail-bleeding times and
delayed arterial thrombosis. VAMP-8-/- animals did show a delay in thrombus formation but no
overt bleeding diathesis. Our data show that small differences in secretion kinetics alter hemostasis,
thus by modulating platelet secretion, we can control thrombus formation without inducing
pathological bleeding. These data identify the secretory machinery as a viable target to control
occlusive cardiovascular diseases.
Our work is the first comprehensive study showing how by targeting secretion we can achieve the
long-sought balance between occlusive thrombosis and spurious hemostasis. Additionally, by
titrating amounts and types of VAMPs in platelets we have created a valuable set of animals to
precisely analyze the role of platelet secretion in other vascular processes.
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