UNC. US: Researchers at the UNC School of Medicine have found that the blood
platelet protein Rasa3 is critical to the success of the common
anti-platelet drug Plavix, which breaks up blood clots during heart
attacks and other arterial diseases.
The findings could lead to more personalized approaches to controlling
platelet activity during heart attacks and other vascular emergencies
and diseases.
The discovery, published in the Journal of Clinical Investigation,
details how Rasa3 is part of a cellular pathway crucial for platelet
activity during clot formation. Understanding the protein’s role could
also prove vital in the development of new compounds aimed at altering
platelet function.
“We believe these findings could lead to improved strategies for
treatment following a heart attack and a better understanding of why
people respond differently to anti-platelet drugs, such as aspirin and
Plavix,” said Wolfgang Bergmeier, PhD, professor of biochemistry and
biophysics, member of the McAllister Heart Institute at UNC, and senior
author of the paper.
The research, which was conducted in mice, may also open the door to
developing antidotes to Plavix, which was the second-best selling drug
in the world prior to its patent expiring in 2012. It is still
prescribed under its generic name clopidogrel to millions of people with
heart disease, peripheral vascular disease, and cerebrovascular
disease. However, the drug’s anti-platelet effect increases the risk of
bleeding in patients and makes emergency surgery too risky because
Plavix affects the ability of platelets to prevent blood loss after
vascular injury. An antidote would bypass the need to wait until the
kidneys eliminate the drug from circulation.
Since the 1970s, scientists knew that clopidogrel had an
anti-clotting effect on platelets. In 2001, they found the compound’s
target – a cell receptor called P2Y12. As Plavix was developed into a
multi-billion-dollar drug, scientists still didn’t know how this
receptor communicated with other proteins in the cell pathways important
for platelet activation. This also meant they didn’t know why people
responded differently to the drug.
Researchers have since learned that the receptor P2Y12 communicates
with a small protein called Rap1, which is like a cellular switch. In
platelets, this switch is typically off, which keeps platelets in a
non-sticky state.
In this quiet state, the 2.5 trillion platelets can patrol blood vessels
and arteries without sticking to the endothelium – or inside wall – of,
say, a coronary artery. If there’s a problem in the endothelium, the
Rap1 switch is flipped and platelets morph into super sticky cells that
clot fast to keep blood from gushing into tissue.
This is crucial when we have a severe injury or even a cut.
But this clotting also happens during a heart attack, when a massive clot is the last thing a person with heart disease needs.
In the arteries that feed blood to the heart, plaque builds over
time. But this buildup isn’t typically the cause of heart attacks; they
occur when the plaque ruptures and platelets rush in to plug the
rupture. This clotting blocks the artery, which blocks oxygen from
entering the heart. And that causes the heart attack.
To counteract the effects of the clot, Plavix hits its P2Y12 target
to flip the Rap1 switch back to the off position so the platelets return
to their quiet, non-sticky state. Aspirin also helps keep platelets
from sticking.
Until now, no one knew how hitting the P2Y12 receptor triggered the
Rap1 protein to switch off. The experiments conducted by the Bergmeier
lab show that the Rasa3 protein is a crucial player in this process.
“Platelets live in unique environment and they need to be very
sensitive to changes in that environment,” Bergmeier said. “They are
ready to jump into action almost without anything happening. You could
say they’re in a preloaded state. But for that to be possible, they need
a breaking system that keeps the platelets in the off state so that
they don’t do anything until they absolutely have to.”
Rasa3 is a key part of that breaking system, and Plavix makes sure that the break stays on.
Think of a platelet like a circuit with Rap1-GDP representing the off
state and Rap1-GTP representing the on state. In between, there are
proteins called exchange factors (GEFs), which flip on the platelet’s
Rap1 machinery. The proteins needed to switch off Rap1 are called GAPs.
(see illustration)
Using deep sequencing techniques, Bergmeier’s team found that Rasa3
was the only highly expressed GAP gene for Rap1 in platelets. He thought
that a malfunctioning Rasa3 protein would lead to platelet activation
and clearance from circulation.
His team knocked out Rasa3 in mice to show that the offspring had no
platelets and could not survive. The researchers then used mice from The
Jackson Laboratory to study platelets in mice with a Rasa3 mutation.
These mice had 3 to 5 percent of the typical platelet count. Bergmeier’s
team found that the rest of the platelets were being activated and
cleared from circulation.
When the researchers disabled the major GEF proteins, the platelet
counts rose to normal amounts in the mice. This showed that a tightly
controlled balance between GEF and GAP proteins, especially Rasa3, is
vital for platelet activity.
At the sites of vascular injury there’s a shift in this balance
inside a platelet that makes the cells very sticky. Plavix ensures that
Rasa3 cannot be turned off in platelets; the drug irreversibly limits
the cell’s ability to stick. It keeps the cell’s breaking system
perpetually on.
“These experiments show that this Rap1 GEF-GAP pathway is crucial for
platelets to jump into action to plug a hole in the endothelium,”
Bergmeier said. “And now we know that Rasa3 is a critical negative
regulator, a break, on the process.”
Bergmeier added, “We have good reason to believe that the Rap1
switch, controlled by the same GEF and GAP proteins, also regulates the
active state of human platelets. We expect this research will provide
critical information for improving anti-platelet therapies, possibly
including approaches that eliminate some of the patient-to-patient
variability and the increased bleeding risk associated with current
anti-platelet drugs.”
Co-first authors of the study are David Paul, PhD, and Lucia Stefanini, PhD, both postdoctoral fellows in the Bergmeier lab
when this research was conducted. Stefanini is now member of the
Institute for Cardiovascular and Metabolic Research at the University of
Reading in the United Kingdom. The co-corresponding author, along with
Bergmeier, is Luanne Peters, PhD, professor at The Jackson Laboratory.
Other UNC authors include Kathleen Caron, PhD, professor and chair
of the department of cell biology and physiology; Nigel Mackman, PhD,
the John C. Parker Professor of Medicine and director of the McAllister
Heart Institute; Matthew Parrott, PhD, assistant professor of radiology
and member of the UNC Biomedical Research Imaging Center; Todd Getz,
PhD, former UNC graduate student and current ORISE research fellow the
U.S. Army Institute of Surgical Research; Yacine Boulaftali, PhD, and
Caterina Casari, PhD, both postdoctoral fellows in the Bergmeier lab;
and graduate student Dan Kechele.
This research was supported through grants from the National
Institutes of Health, The American Heart Association, the European
Hematology Association, and the International Society of Thrombosis and
Hemostasis.