Have you ever wondered how those cuts on your hands stop bleeding? What is involved in this process? How can we control this process using science? I hope to address these questions and more in this blog. But I’m getting ahead of myself. Do you even know who I am?
My name is Jeff and I am a 4th year Biology/Chemistry student at the University of New Brunswick. In my spare time I compete for UNB as a member of the Varsity REDS track and field team, where I run indoors as fast as my scrawny legs will carry me. My secret to running fast also happens to be my two favourite foods: hummus, and bagels with cream cheese. My interest in going fast sometimes leads to taking a spill going at high speeds. Let’s just say that I know what it’s like being on the bicycle side of a bicycle-car collision. I am still here to talk about science for two reasons:
I was wearing a helmet (it’s a good idea - do it, even if you think you look silly.)
One of my favourite biochemical pathways: blood clotting or coagulation.
If you weren’t already able to guess from my degree, I am very interested in the chemical reactions that happen inside the body. Our bodies are made up of cells, which communicate using proteins and other molecules to facilitate a series of chemical reactions. These chemical reactions are the reason that you can eat, think, breathe, and anything else you can imagine. Each group of reactions happen one after another in a controlled fashion to perform a specific task, also known as a pathway. These pathways are amongst the coolest things to study in science (in my completely unbiased opinion). Oddly enough, I’ve already mentioned one of these pathways in this post, which will be the topic of my blog. Aren’t I a sneaky one?
In this blog, I am going to explore the coagulation pathway. This occurs in damaged blood vessels, and the end result of this pathway is the cessation of bleeding thanks to a blood clot. You might think that this only happens when you get a wound, like a cut on your hand, and that cut becomes a scab that eventually heals. Since that doesn’t happen all too often, your body doesn’t need to form clots on a daily basis, right? The truth is, your body forms blood clots all of the time! Your arteries that carry your blood are always under a high amount of pressure, and so small arteries in your body can rupture on a daily basis. If we didn’t have this pathway in our bodies, we’d be leaking blood into different parts of our body all of the time. Thank goodness for coagulation!
Although this pathway is important, it can sometimes be a treacherous beast. Blood clots can form in your blood vessels when they are not needed and this can cause strokes, deep venous thrombosis (DVT), and other unfortunate things like heart attacks (myocardial infarctions) and pulmonary emboli (clots lodged in the lung). It is also possible for someone to fail to produce blood clots, which is equally unfortunate and can lead to haemorrhaging. Luckily, drugs that control the coagulation process have been developed in order to maintain this fine line of optimal blood clotting in humans. I can’t wait to explore with you the kinds of drugs used in the past and present in order to get a better understanding on how this pathway gets controlled to save people’s lives.
Also, feel free to compliment me on the sweet graphics that I make. Hold on to your life juice!
But hold on… plasmin inhibitors already exist and are being used today, so why would we want new ones? Well, the inhibitors that exist now are mostly large molecules that bind to the active site of plasmin. The issues with the current molecules are that they have limited toxicity and selectivity, requiring a higher dose and yielding more side-effects. A smaller inhibitor molecule that is currently being used, tranexamic acid (TXA), requires a whopping dose of 1 to 20 grams to achieve the desired inhibition. The active site of plasmin is very similar to those of thrombin, factor Xa, and factor XIa. This means that that there is a potential for this drug to slow down the clotting cascade and increase blood loss; the opposite of the desired effect. Other side effects of the low specificity of the drug can include: seizures, renal dysfunction, and chest tube “drainage” (although I think they mean clogging).
So we could definitely use some new drugs that are more specific and also more toxic. The specificity would yield less side effects, and a more toxic drug requires a lower dosage to be effective. But how does one go about designing a new drug? Well, you start with what you know first.
Heparin is a sulphated glycosaminoglycan (carbohydrate monomer repeats with amide functional groups) which is known to allosterically control plasmin activity with an IC50 in the nanomolar range, among other interactions. Al-Horani et al. reasoned that small sulphated drugs would have interactions similar to the sulphur groups in heparin, while the specificity of the interaction would increase due to two things: 1. a smaller molecule will have fewer functional groups, leading to a statistically lower chance of having non-specific interactions; 2. the highly charged sulphur groups prevents the molecule from entering cells and crossing the blood brain barrier, keeping the molecule from ending up in places where it is not useful and can even cause undesirable reactions. Supported by the results of others that have been able to make active site plasmin inhibitors, they began synthesis of allosteric inhibitors in hopes of finding the next big plasmin inhibitor.
After using a variety of molecular backbones, a library of molecules was built. By using a chromogenic substrate hydrolysis assay they were able to identify 55 molecules with promise as inhibitors for heparin (the top four compounds being 31, 32, 52, and 54 from scheme 1 of the paper). Inhibition of plasmin with 31 and 32 yielded IC50 values of around 50 uM while 52 and 54 had IC50 values of around 75 uM. The compounds were tested for selectivity by comparing their ability to inhibit closely related serine proteases: thrombin and factor Xa. It was found that compared to plasmin, the compounds inhibited thrombin 7 to 10-fold less (IC50 >500uM) and factor Xa was inhibited about 5-fold less (IC50 >250 uM), indicating that selectivity was achieved with these compounds.
In summary, this paper is mostly a proof-of-concept that we can allosterically inhibit plasmin in order to reduce blood loss in people undergoing surgery. Allosteric inhibition will increase specificity and will have fewer side effects than drugs currently used to inhibit plasmin, like tranexamic acid. All this so that you can be more comfortable while you keep holding on to your life juice.
Sometimes I find it hard to imagine how it can be that everybody in the world is different. Out of 7 billion people, there isn’t anyone who has the same fingerprint, which essentially blows my mind. Likewise, everybody has a different affinity to form clots in the body: for some, clots form easily… while others do not form clots well at all. This affinity is a kind of spectrum, where being on either extreme of the spectrum can be dangerous. Medication can be used to assist high risk individuals to reduce risk of haemorrhage or stroke. These medicines influence enzymes in the coagulation cascade, but in order to learn about how that works, it is important to have a firm grasp on how the cascade works.
For the next few paragraphs, I am going to go into some depth about the blood clotting pathway - known as the coagulation cascade - and try to explain how it works. If you want the short version, here it is: when a blood vessel is damaged, enzymes in the body produce fibrin which will ‘glue’ platelets together, forming a stable plug over the damaged area (see figure 1). If you want a longer version of how this works, read on:
Figure 1: Repairing a damaged blood vessel.
The Cascade (see figure 2): There are 13 compounds involved known as clotting factors which are normally present in the body. The clotting factors are assigned to roman numerals (I to XIII), although there is nothing assigned to factor VI for some reason. These factors are mostly proenzymes, meaning that they are in their inactive form. When they are activated (denoted by an “a” next to their name i.e. factor VIIa), they will perform their task in the cascade. Specialized blood cells called “platelets” also play an important role. I will now try to provide a clearer picture of how this cascade works.
I enjoy beginning with the end in mind because it helps me understand why certain things are happening, so keep in mind that the end goal of the coagulation cascade is to produce fibrin. This fibrin will bind to platelets and other blood components, producing a stable blood clot and stopping blood loss. But how does this cascade start? Lets take a look:
Once a blood vessel is damaged, a few things happen at the wound site: tissue factor is released, tissue phospholipids are released, and collagen becomes exposed. The exposure of these molecules mark the start of the cascade, which is comprised of two different pathways: the extrinsic pathway and the intrinsic pathway. I will now talk about each pathway individually.
The released tissue factor will start the extrinsic pathway by forming a complex with blood coagulation factor VII. This complex, with the help of the tissue phospholipids, will activate factor X to factor Xa. Xa will complex with factor V and tissue phospholipids to form the prothrombin activator. Prothrombin activator will split prothrombin (blood clotting factor II) into pieces, one of those pieces being thrombin (factor IIa). Thrombin will then interact with fibrinogen (factor I) and cleave off two small pieces. The result is the active monomer form of fibrin (factor Ia) that will polymerize at the wound site in the form of fibrin threads with the help of factor XIII. These threads will adhere together, trapping platelets, blood cells, and plasma to form the blood clot. The clot will then adhere to the damaged opening of the vessel and stop bleeding.
Meanwhile, platelets in the bloodstream as well as Factor XII will bind to collagen and become active, initiating the intrinsic pathway. XIIa then activates XI, and XIa subsequently activates IX. IXa will act with factor VIII and platelet phospholipids in order to activate factor X. The rest of this pathway proceeds the same way as the extrinsic pathway, except that the phospholipids involved are from platelets, not the tissue. The following diagram gives a visual on how this process works.
Figure 2: The clotting cascade in all its glory.
Now, one might ask why two different pathways are needed to form a blood clot. Surely one would be enough? The extrinsic pathway is more of a “first responder” in this cascade. It can take as little as 15 seconds to form a clot via the extrinsic pathway, where it can take around 2 to 6 minutes for the intrinsic pathway alone to form a clot. Since the extrinsic pathway happens so quickly, it uses up the tissue factors and phospholipids from the damaged site quickly and then slows down due to lack of nearby resources. The intrinsic pathway is a more sustainable pathway that uses compounds from the platelets to drive the cascade, which are constantly flowing through the bloodstream. Having two pathways allow for a quick response while being able to keep driving the cascade if necessary.
Coagulation is a very helpful thing, although it can be very dangerous. Thrombin can have a direct influence on prothrombin, cleaving it into more thrombin and causing a potentially infinite feedback loop, but this is usually prevented by the removal of thrombin and other clotting factors from the clot site via the flow of blood. Since humans all have a varying affinity to form blood clots, some can have a higher chance of forming unnecessary blood clots in their bodies. Because the flow of blood is so important to the cessation of clot formation, people with conditions such as atrial fibrillation (irregularly irregular rhythm in the left atrium, causing blood flow to slow) can have a high risk of undesirable clot formation in their bodies. These clots can get stuck in places like the capillaries of the lung or in the brain and block the flow of blood, causing serious conditions like a pulmonary embolism or ischemic stroke. Let’s start looking at some drugs that influence this pathway in order to prevent these undesirable conditions.
Warfarin: When we talk about influencing the coagulation cascade with medications, it is often useful to consider what the two pathways have in common. Somebody who is prone to clotting will see more of an effect on their coagulation if a drug that slows down both pathways is used.
Let’s start by talking about one of the oldest and most prescribed oral anticoagulants, Warfarin. An interesting fact about warfarin is that it was first used as a rat poison in the 1940s, and still is today to an extent. It works by inhibiting the rats’ ability to form blood clots, causing them to bleed to death. I first thought that the cause of blood loss would be from the rats rubbing against something and getting a scratch, but it is primarily caused by gastrointestinal bleeding. This makes sense, considering the harsh conditions inside the stomach and bowel. Warfarin works by inhibiting the vitamin K dependent synthesis of clotting factors. When warfarin is present, it binds to the protein vitamin K epoxide reductase (VKOR), preventing it from recycling vitamin K. Vitamin K is essential for the synthesis of the clotting factors (II, VII, IX, and X; see figure 2). When vitamin K is not present (which is essentially the case when it cannot be recycled), the clotting factors are synthesized in a biologically inactive form, rendering them unable to contribute to the clotting cascade. Warfarin is a very effective drug, however its use does have drawbacks.
When somebody is taking warfarin, they must monitor their vitamin K intake. If too much vitamin K is consumed, the effects of warfarin are diminished and the risk of clot formation increases. The consumption of vitamin K rich foods (leafy greens) has to remain consistent in order for warfarin to work properly.
Since warfarin works by causing the synthesis of biologically inactive clotting factors, the anticoagulant effects of warfarin are only noticeable once the biologically active clotting factors are degraded by the body, which takes up to 5 days. Frequent blood testing is required to ensure that the body is forming clots at an appropriate rate, indicating correct dosage of warfarin. If the blood test indicates that clots are forming too easily or too slowly, the dosage must be adjusted. The results of the adjusted dosage can only be observed another 2 to 5 days later, making it often difficult to maintain appropriate levels of drug in the body.
The shortcomings of the old “blood thinners” is what has driven the discovery of newer drugs. Let’s take a look a more recently discovered drug.
Dabigatran: In recent years, more novel anticoagulants have been discovered with their own sets of advantages and disadvantages. Let’s take a look at Dabigatran (sold as Pradaxa), an alternative to warfarin that was brought into the market in 2010.
Pradaxa is a direct thrombin inhibitor (clotting factor IIa; see figure 2). It works by binding to the active site of thrombin, preventing it from cleaving fibrinogen. This step is shared by both clotting pathways, so inhibition will prevent clot formation. The advantage of dabigatran is that it will have its full effect on the body in as little as two hours. It has a half life of around 12 hours, which is much easier to work with when compared to warfarin’s half life that is multiple days long. The FDA studied Pradaxa versus warfarin and in 2014 concluded that Pradaxa had a lower mortality rate, as well as lower risks of cerebral bleeding and ischemic stroke. One of the main reasons people like taking dabigatran is that regular blood testing is not required.
Although dabigatran is in many cases preferable to warfarin, it still has its drawbacks. The instances of gastrointestinal bleeding are higher when taking dabigatran. While warfarin treatment costs a few cents a day, treatment with dabigatran is about 100 times more expensive, costing multiple dollars a day.
While the short half life is a good feature to have in dabigatran, 12 hours could still be too long for somebody to wait if they need to have emergency surgery. Research is being conducted on potential antidotes for dabigatran which I am excited to share with you in my next major post. For now, be sure to hold on to your life juice.
-Jeff
Sources: -Barash PG, Cullen BF, Stoeling RK (1992). “Clinical Anesthesia” 2nd ed. East Washing Square, Philidalphia: J. B. Lippincott Company. Print. -Blommel ML et al. (2011). “Dabigatran etexilate: A novel oral direct thrombin inhibitor”. Am J Health Syst Pharm 68 (16): 1506–19. PMID 21817082. -Di Nisio M, Middeldorp S, Büller H (2005). “Direct thrombin inhibitors.”. N Engl J Med 353 (10): 1028–40. PMID 16148288. -Guyton AC (1981). “Textbook of Medical Physiology” 6th ed. 1 Goldthorne Avenue, Toronto: W. B. Sunders Company. Print. -Link KP (1 January 1959). “The discovery of dicumarol and its sequels”. Circulation 19 (1): 97–107. PMID 13619027. -Whitlon DS, Sadowski JA, Suttie JW (1978). “Mechanism of coumarin action: significance of vitamin K epoxide reductase inhibition”. Biochemistry 17 (8): 1371–7. PMID 646989.
Poetry was not my first idea for my post this week. Here’s a haiku hint of what i was planning on doing:
To test the styptic I was to cut my hand twice Then decided: nope
I wanted to test how well a styptic pen would slow down bleeding, but it would have involved causing self-harm and I decided that may not be the best course of action. If you really want to see a styptic pen used, Weston Summers has an interesting demonstration that you can watch (warning: blood).
Here is a lovely poem instead:
In animals large to small, short to tall, Something is common in all. Blood, the juice of life, circulates through the veins, They flow though the arteries just the same. You don’t want that juice to come flowing out,
That’s what the clotting cascade is about.
So just in case something were to happen,
Like a bite from a komodo dragon,
These clotting factors thirteen all work together,
To produce fibrin, which will weave and tether
Platelets and other compounds in the blood alike,
Staunching the flow of blood and leaving a sense of delight,
Now that you know how the bleeding will stop,
Keep holding on to your life juice, every single drop.
My blog will explore what is known about blood clotting. I will be looking at drugs that control the enzymatic pathways involved in forming blood clots and how these help prevent death due to blood loss, undesirable blood clotting, etc.
This article gives a very apt description about treating strokes using blood thinners and the advantages/disadvantages associated with this treatment. I like it particularly because it gives specific examples of different issues that can be encountered when taking these medications. Many different drugs are addressed in this article, and I want you to focus on Warfarin and Pradaxa during reading.
Warfarin has been used on humans since the 1950‘s, making it one of the oldest drugs in the blood thinner class. Warfarin has been used to effectively treat people at risk for thrombosis (undesirable blood clotting), although it has its disadvantages. Pradaxa is a newer drug that has entered the scene in 2010 with its own set of benefits and drawbacks. I am excited to explore the impact of these two drugs on the coagulation pathway in my first of two major blog posts.
One of my favourite sections from this article talks about how “some clinicians and patients are concerned the newer drugs do not have a “reversal agent"”. I can’t wait to address this topic in a future post!
Some references:
Holbrook AM, Pereira JA, Labiris R, McDonald H, Douketis JD, Crowther M, Wells PS (May 2005). "Systematic overview of warfarin and its drug and food interactions”. Arch. Intern. Med.165 (10): 1095–106. http://www.ncbi.nlm.nih.gov/pubmed/15911722
A thrombus, or blood clot, is the final product of the blood coagulation step in hemostasis.
Blood clotting is an important mechanism to help the body repair injured blood vessels.
Blood consists of: -red blood cells containing hemoglobin that carry oxygen to cells and remove carbon dioxide (the waste product of metabolism) -white blood cells that fight infection -platelets that are part of the clotting process of the body -blood plasma, which contains fluid, chemicals and proteins that are important for bodily functions.
Complex mechanisms exist in the bloodstream to form clots where they are needed. If the lining of the blood vessels becomes damaged, platelets are recruited to the injured area to form an initial plug. These activated platelets release chemicals that start the clotting cascade, using a series of clotting factors produced by the body. Ultimately, fibrin is formed, the protein that crosslinks with itself to form a mesh that makes up the final blood clot.
Blood clots are healthy and lifesaving when they stop bleeding. However, blood clots can also form abnormally, causing a heart attack, stroke, or other serious medical problems.
Most heart attacks and strokes result from the sudden formation of a blood clot on a waxy cholesterol plaque inside an artery in the heart or brain. When the plaque ruptures suddenly, thrombogenic substances inside the plaque are exposed to blood, triggering the blood clotting process.
Modern
medicine wouldn’t have gotten as far as it has without the development of medicinal
imaging. This technology is used every day for the diagnosis of illness,
cancers, and diseases for millions of patients around the world, and gives us
the amazing ability to detect and study diseases as they progress right down to
the molecular level - and it even helps to save lives.
So
how can we make this technology better, cheaper, and more accessible? Bioluminescent
imaging might just be the answer we’re looking for.
The birth of the GFP:
Using
molecules that have a natural “glow” has been a staple in biological studies
for decades. This all began in the 1960s (almost 60 years ago) when a marine
biologist discovered a natural fluorescent protein derived from a crystal
jellyfish off the coast of Japan. With its fluorescent properties, this protein
was able to absorb ultraviolet light and, in return, release its own light as a
green glow. It wasn’t long before researchers discovered that this protein
could be attached to cellular components, such as proteins and DNA, and used to
observe cellular processes at a molecular level.
This
marked the discovery of the infamous Green Fluorescent Protein (GFP) - a protein
that has become a key asset in the study of biological systems, even to this
day.
Figure 1: Structure and
activation of the Green Fluorescent Protein.
This
was only the beginning of “harnessing nature’s glow” to help further biological
and medicinal research.
The power of medicinal imaging:
In
order to begin looking at the use of bioluminescence in medicinal imaging, we
first need to understand what medicinal imaging is. Medicinal imaging is the
use of non-invasive technology to produce images of anatomical structures that
are usually hidden from our sight. Imaging can be used for observing internal systems
and tissues in real time, and to detect abnormalities within these systems, thus
allowing for fast and easy diagnosis of diseases.
Over
the years there have been many different methods for observing internal
processes. Some of the more well-known methods of imaging include X-ray radiography, CAT scans, Magnetic
resonance imaging (MRI), and ultrasound - and these are just to
name a few. Although these imaging methods are non-invasive procedures, many of
them are way too expensive for practical use and can also be potentially harmful
to patients (for example x-ray radiation).
Fluorescent vs bioluminescent imaging: what’s the difference?
Another
highly used imaging method is fluorescent imaging. Fluorescent imaging is an
ideal examination technique that uses compounds called fluorophores. These
compounds are capable of emitting light after being excited by an external light
source. When these compounds enter an excited state they become unstable, and as
the excited material stabilizes within the excited state, energy is released as
heat. When the material relaxes back down to a ground state, photons of energy
are re-released from the material in the form of light.
Figure 2: Fluorescence
at its finest. This figure shows the energy levels involved in light release of
a fluorophore. A is the energy from the
external light source, B is the energy lost through heat, and C is the light
energy released by the fluorophore. The E’s represent the different excitation
states, with E0 being the ground state.
Different
fluorescent stains and markers can be used to tag molecules within cells for observational
studies. One of the more commonly used fluorescent markers is the infamous GFP
that I mentioned earlier.
Unfortunately
fluorescent imaging has many limitations, the first being that fluorophores lose
their ability to give off light over time. This is due to an event called photobleaching
- which refers to damage that accumulates within the fluorophore from the
constant excitement of its electrons –this causes the fluorophore to slowly break
down and also reduces the time allotted for observation.
Although
the use of fluorescent imaging allows us to observe living cells in their
natural habitat, it also leaves our cells open to phototoxicity. This is a toxic
response that occurs when light travelling from an external light source (needed
to excite the fluorophore) comes in contact with our cells. Phototoxicity can
be compared to UV damage from sun rays. (And we definitely know that UV damage
is not good for our skin!) Fluorophores are
also known to generate reactive chemical species which can enhance this
phototoxic effect.
To
bypass the downfalls of fluorescent imaging, researchers have been looking into
the possibilities of using bioluminescent imaging instead.
The Bioluminescent difference:
Bioluminescent imaging (BLI) is a sensitive,
and relatively new, tool that is used to detect light energy given off from bioluminescently
tagged cells and tissues to study in-vivo pocesses. Unlike fluorescent imaging,
BLI does not need an external light source, since it produces its own light.
BLI
works by adding bioluminescent reporter proteins to the tissues, cells, or
molecules are being studied, and these natural reporter molecules are derived from
bioluminescent organisms such as fireflies (D-luciferin), cnidarians (Coelenterazine
and Renilla luciferin), and bacteria (the LUX operon). These bioluminescent
markers can be added to cells in different ways. A couple of examples include: using
modified DNA (ex: transgenic mice) or using antigens and antibodies specific to
the target cells to attach the reporters molecules.
The
light produced through BLI is too small to be seen with the naked eye. Imaging technology
like charge coupled cameras are used to detect the light through the tissue. This
camera device works by converting the incoming photons into electrical charges.
These electrical charges can then be converted into an image.
Figure 3: An example of
how BLI works. 1. Bioluminescent reporter tags are added to the cell to be
studied, in this case cancer cells. 2. Luciferin is then added via injection to
activate the luminescent enzymes and produce light. 3. The light from the
reporter molecules is then visualized using CCD cameras allowing for observation
of the cancer cells.
Although
BLI has only been used in small in-vivo animal studies so far, it still shows a
lot of promise in someday becoming a mainstream imaging technique.
The
main problem with BLI is that it depends on the presence of oxygen as well as
the addition of luciferin in order to work. It is also not yet powerful enough
for use in larger animal testing, mostly
due to the fact that BLI signals do not travel very far through tissue, so
visualization is restricted to only a few centimeters deep.
But
beside these few issues, there are plenty of reasons to use bioluminescent
imaging!
The
benefits to using BLI include the fact that it does not need an external light source,
so cells involved in the screening are not exposed to any phototoxicity and
remain damage free. BLI is also much cheaper compared to other imaging methods
(for example x-ray machinery), it is non-invasive, and it is an easy way to
visualize a variety of in vivo cellular events as they are happening in real
time. BLI can be used to study and diagnose a very wide range of molecular
functions and diseases, including: gene function, cell trafficking, tumor
development in cancers, disease progression, protein-protein interactions,
bacterial and viral infection progression. It can also be used to test the
effectiveness of newly developed drugs and antibiotics.
Future Prospects for this technology:
So
what can we do with this technology?
The
medicinal potential of bioluminescent imaging is enormous. With this technology
we might someday be able to map brain activity in a new level of detail, or track
electrical impulses translating into muscular actions. It could be used as an
alternate process for diagnosis of diseases, replacing radioactive or other
harmful techniques that had been previously used, or it could be merged with
other imaging techniques to look into deeper tissues. BLI could also be used as
an aid in drug discovery in pharmaceuticals by acting as a visual aid to
determine the effectiveness of drugs.
And
with millions of blood tests and scans and operations being performed every
day, using bioluminescent imaging could revolutionize the way we see things both
medicine and in science.
Since
this technology is still developing, there is a ways to go before it’s ready
for human use, but who knows where this technology could take us next? Bioluminescent
imaging might someday even become as famous as the GFP.
illuminated-shrooms
