Understanding the role of vitamin K-dependent proteins in vascular calcification
What if the process of arterial calcification was regulated from within the cells of the blood vessels, and that it had nothing directly to do with what you ate and what circulated in the bloodstream because calcification takes place not anywhere near the surface but inside the blood vessel wall?
What if the process of arterial calcification was actually a process by which muscle is transformed into bone, a process by which vascular smooth muscle cells transform themselves into bone cells which then actually build bone tissue within the blood vessel wall?
And what if apoptosis preceded calcification, what if cell death was what triggered the process of calcification, and it was the apoptotic bodies of dead vascular smooth muscle cells within the blood vessel wall that served as the nodes around which calcium crystals formed?
Would you not find this shocking? Find it incredible that any of these could be true, let alone all of them? It’s entirely not at all what we’ve been told by “health experts” and “health authorities” for more than half a century!
All of these statements are hard to believe. It is especially unbelievable that muscle cells can change into bone-building cells, and begin to grow bone tissue within the artery wall. It sounds surreal, kind of like science fiction. But it isn’t. All of it is true. All of this has been observed.
Interesting, you may think, but what does any of this have to do vitamin K? Everything! It has everything to do with vitamin K.
How clever we are
The sophistication and precision of biochemical reactions and processes in animals and humans are mind blowing. Understanding how they work is a wonderfully noble endeavour that is certainly very fulfilling in its own right. In some cases though, it can be a matter of life and death. And in the case of the processes related to and regulated by vitamin K dependent proteins it definitely is.
This is not an exaggeration to push you to read on. It’s a statement of fact. And you’ll see how this is true by the time we finish. I believe it is essential, for each one of us to understand the details of how things in our body work and how they are related and connected in order to appreciate their significance and their importance.
We are so clever. We can figure out such complicated things when we put our minds to it. Things like complex biochemical pathways, or long chains of enzymatic reactions that, one step at a time, transform molecules from one form into another. And it is this kind of cleverness that has enabled us to develop the hundreds of different types of medications we can find today in drug stores.
We have designed medications to address basically every symptom we can think of. If it’s a symptom we’ve had, it’s most likely a symptom that many others have or have had. And if many have the same or a similar symptom, we can be sure that at least one pharmaceutical company will have made a drug for it.
Warfarin was developed in the 1950s to prevent or at least suppress coagulation, and in so doing help prevent or at least reduce the number of strokes and heart attacks. Because so many people either suffer from, are susceptible to, or are at risk of cardiovascular disease, many people take warfarin.
And what I mean by many in this case is between 20 and 30 million prescriptions per year in the United States alone. The number went up to 35 million in 2010 and dropped back to 20 million in 2015. That’s a lot of warfarin pills! You can see the stats here (http://clincalc.com/DrugStats/Drugs/Warfarin). Warfarin is in the top 50 drugs. It’s 42nd down the list. Just below aspirin at 39, insulin at 36, and ibuprofen at 34, as you can see here (http://clincalc.com/DrugStats/Top200Drugs.aspx).
Surely close to every household in the western world will have somewhere in a bathroom cupboard or drawer a bottle of aspirin or ibuprofen. Given how close to warfarin they are in popularity of usage, there’s clearly no need to even say that this anti-coagulant drug is in broad and widespread use.
Isn’t this great, though? Millions of people at risk of having blood clots that would possibly cause them a stroke or heart attack, protected by taking a little warfarin every day? Yes, I suppose in some ways, it is, if these people are actually at risk. But, unfortunately, with a drug like that, we can be pretty sure that most are taking it preventatively, as in, just in case. And this is a problem.
Warfarin works by disrupting the process that leads to the activation of coagulation factors. The blood’s ability to form clots quickly is one of its most vital functions, because without it we would just bleed to death from a flesh wound. Evolutionarily, we simply would not have made it to here without this protection mechanism that ensured that when we were wounded, the blood would immediately thicken to stop it bleeding out of our body by forming clots at the surface of the open wound as fast as possible. The special proteins responsible for regulating coagulation are vitamin K-dependent proteins (VKDPs).
It has taken a long time to understand, first of all, that there wasn’t just vitamin K, but in fact two different kinds of vitamin K. It is also true that it has taken a long time to identify the major vitamin K-dependent proteins and figure out how they work. We are talking about 40 years from the 1950s to the 1990s. So, you really shouldn’t be surprised if you haven’t read or heard about this before.
But today, a lot has been understood through in vitro and in vivo observations, trials and studies both in animal models and in humans. And even though we will inevitably continue to deepen our understanding of the subtleties of the molecular mechanisms, the species, and the interactions involved in the life of cells and proteins in how they affect the state of our blood vessels and organs, this is a sketch of the picture we have at this stage.
Vitamin K dependent proteins
There are about twenty identified VKDPs belonging to two classes: hepatic—those produced by the liver, and extra-hepatic—those produced in other tissues. Those from the first class are the most well-known and well-studied. They are the coagulation factors (II, VII, IX, and X) manufactured by the liver and activated within it before being pushed into the bloodstream and circulated throughout the body to maintain a healthy coagulation response in case it is ever needed. These are the ones targeted by warfarin. Naturally, since that drug has been around since the 1950s, the role and function of these vitamin-K dependent coagulation factors have also been known at least since that time.
The second class is less known and less studied but has—luckily for us—gained much more attention in the last two decades. It includes three very important proteins whose functions are essential in maintaining healthy blood vessels. But unlike the coagulation factors produced in the liver, these proteins are instead produced by the vascular smooth muscle cells and activated there locally in the vasculature. These vascular health factors, we call them that in analogy to but to distinguish them from the coagulation factors, were identified much more recently in the 1980s and 1990s. All are proteins that contain gamma-carboxyglutamic acid abbreviated Gla.
Some important ones for us here are osteocalcin, for which it took 30 years to be identified as an inhibitor of calcification when it was discovered in vitro to prevent the precipitation of crystals in a supersaturated calcium solution. This means that without it, calcium crystals would have inevitably formed spontaneously in the solution. Osteocalcin is also called bone Gla protein. Growth arrest specific protein 6 is involved in the regulation of cell proliferation, and seems to inhibit premature cell death. And the most important one in relation to soft tissue calcification, matrix Gla protein abbreviated MGP.
Matrix Gla protein was originally isolated from bone, but it has been found to be expressed in several other tissues including kidney, lung, heart, and—most critically—vascular smooth muscle cells or VSMCs. It is now known to be the most potent inhibitor of calcification of blood vessels, and even though the liver does produce and secrete MGP into the bloodstream, only the MGP produced in the vasculature inhibits calcification.
Besides being produced in different tissues, another important difference between the two classes of VKDPs is that the liver-produced coagulation factors are phylloquinone—or vitamin K1-dependent, whereas the vascular smooth muscle cell-produced proteins are menaquinone—or vitamin K2-dependent. In light of the fact that it is rather hard to find vitamin K1 insufficiency with a diet that contains at least some green plant foods, while the exact opposite is true for vitamin K2 of which the western diet is practically devoid, this difference is highly significant.
Both vitamin K1 and K2 are absorbed in the second and third portions of the small intestine, the jejunum and ileum, K1 is delivered to the liver, whereas K2 is transported via LDL and HDL to other organs. K1 is mainly found in the liver, whereas K2 is preferentially stored in peripheral tissues, with the highest levels in the brain, aorta, pancreas, and fat tissues. This obviously attests to the importance of these essential vitamins.
While vitamin K1 and K2 are really two different vitamins with different functions, transport mechanisms, and distribution in the tissues, and while there are several differences between the vitamin K1-dependent and the vitamin K2-dependent proteins, these have one essential thing in common. This is, as their name says, that they are vitamin K-dependent. What this means is that all these proteins share the same enzymatic chain of activation—whether it mediated by K1 or K2—that transforms them into their biologically active form, the form they need to have in order to do the things they are meant to do.
All VKDPs must be carboxylated in order to be activated. The process is complicated and not yet completely understood. We know that it is targeted to the glutamic acid (Glu) residues on the protein that must be made into gamma-carboxylglutamic acid (Gla). We also know that the process is mediated by the enzyme gamma-glutanyl carboxylase (GGC), and that vitamin K is the main co-factor that enables the enzyme to perform the activation. In the end, the process leads to the addition of a carbon dioxide molecule to the gamma-carbon of Glu, which transforms it into Gla. However, it is the reduced form of vitamin K that is required.
Vitamin K, whether it is the plant-based phylloquinone (K1) or the animal-based menaquinone (K2), enters the body through the diet in its non-reduced form. Reduction involves the addition of hydrogen in molecular form, H2, to make KH2. Transformations of this kind are generally always done by enzymes, and so is this one. In this case the enzyme is vitamin K epoxide reductase (VKOR). Its action is essential because it is the reduced form KH2 that acts as the co-factor in the process of carboxylation.
The energy released by the oxidation of KH2 drives the addition of the carboxyl group unto the glutamic acid residues. But the oxidised form of vitamin K, KO, can subsequently be reduced again to KH2. Thus vitamin K is first reduced, then oxidised to help push the carboxyl group unto the glutamic residue, and then reduced once more to start the whole cycle again. This cycle is called the vitamin K epoxide reductase or VKOR cycle.
For this class of proteins, the VKDPs, activation through carboxylation means for them to acquire the structure and properties needed to bind calcium in order to transport it. You may recall from a previous chapter in the story of vitamin K2, matrix Gla protein generally transports calcium out of soft tissues in order to prevent calcification, and bone Gla protein generally transports it into bones and teeth to prevent osteopenia, osteoporosis, and tooth decay.
The big red flag
Now you understand why it is that when, in our remarkable cleverness, we understood that the main coagulation factors depended on the action of these enzymes to be activated and rendered functional, we naturally concluded that the best way to prevent clot formation would be to prevent coagulation, and that this could be achieved by blocking these enzymes from doing what they are intended to in a healthy organism. This is precisely what warfarin does.
And it does it well. Otherwise it wouldn’t have become as commonly used as it is. And we can be certain it has saved a lot of people much of the pain and possibly life-threatening conditions that a blood clot could have caused them. The problem is that the vascular health factors so critical for maintaining healthy blood vessels, depend on the same enzymes for activation as do the coagulation factors. Preventing the carboxylation of coagulation factors, prevents, in exactly the same way, the carboxylation of the vascular health factors.
This was only understood to be a major problem relatively recently. We first had to understand that there isn’t just one kind of vitamin K, but that there are two, and that they are very different in their functions. We had to understand that both vitamin K1 and vitamin K2-dependent proteins rely on the same enzymes to get activated. We had to understand the carboxylation process by which they are activated. And we had to understand that MGP, BGP, and Gas 6 are vitamin K-dependent proteins, that they are specifically vitamin K2-dependent, how they are activated, what they actually do in our veins and arteries, and what happens if they can’t do what they are designed to do.
A major red flag about anticoagulants and warfarin came up from what was seen in mice. The first part of the study was with MGP-knockout mice, (mice in which the MGP-encoding gene was deactivated). They were observed to have stunted growth from the premature calcification of the epiphysis—the part at the end of bones and at the junction with the cartilage of the joint which allows the bone to grow longer. As as soon as the epiphysis calcifies, longitudinal growth stops. But this was the least severe of the problems that were observed.
The MGP-knockout mice very quickly developed severe arterial calcification, and died highly prematurely, within 6 to 8 weeks, of strokes, heart attacks, and rupture of the aorta. Normal lab mice live 2 to 3 years and some even up to 4 years. So, in the least extreme case, a MGP-knockout mouse dying from aortic rupture at 2 months instead of living a relatively short normal life of only 2 years, would be equivalent for a human that would normally live to the age of 72 to die at the age of 6!
Here is what severe coronary calcification looks like in humans:
It was also observed that although the liver did produce and release MGP into the bloodstream, it had no effect on the arteries. Only the tissue-specific, locally-produced MGP within the vascular smooth muscle cells was able to inhibit calcification.
To check these conclusions, a similar study was done on normal mice that were given vitamin K1 to ensure proper liver function and healthy coagulation, and warfarin to block all extra-hepatic MGP action in tissues. The result? Stunted growth, pervasive arterial calcification, and premature death from stroke, heart attack, and aortic rupture.
The conclusions were solid: matrix Gla protein is the organism’s primary protection against soft tissue and arterial calcification; liver MGP has no protective effect on arteries, and only VSMCs-produced MGP can inhibit calcification in the arteries; both vitamin K deficiency and disruptions of the action of the enzymes that activate MGP cause extensive soft tissue calcification; and only vitamin K2, not vitamin K1, can inhibit warfarin-induced calcification.
When this was understood, more attention began to be paid to matrix Gla protein. Many other details were elucidated through further investigations. It was found that MGP is an 84-amino acid protein with five Gla residues. That all of these Gla residues are produced by gamma-carboxylation, which is mediated by the enzyme gamma-carboxylase that requires vitamin K2 as a cofactor, and that until now, the only known function of Gla residues is to bind calcium ions and crystals (calcium apatite). It was discovered that the concentration of calcium and phosphate in extracellular fluids is high enough to trigger and sustain growth of crystals, but that MGP and BGP prevent this from happening. That MGP is required by VSMCs to maintain their elastic and contractile nature. And not just that.
MGP actually inhibits the transformation of VSMCs into bone cells by antagonising the action of Bone Morphogenic Protein 2 (BMP2). It turns out that the muscle cells of the blood vessels have in them the potential to either stay smooth elastic contractile muscle cells, or turn into osteoblast-like bone building cells. BMP2 triggers that osteogenetic gene expression in the VSMCs: it tells muscle cells of the blood vessels to transform into bone-building cells. And as if this wasn’t enough, BMP2 also induces apoptosis: it tells blood vessel muscle cells to commit suicide, which is certainly to help in the process given that once dead, they can be used as seeds for calcium crystal formation, and thus promote a faster and more efficient calcification.
What induces expression of BMP2 in cells? Probably several things that we haven’t yet identified. But for now we know that BMP2 is stimulated by oxidative stress, chronic inflammation, and high blood sugar levels. The good news is that MGP protects against all of these effects by antagonising BMP2. So if there is enough MGP and enough vitamin K2, if there are no disruptions to the action of the vitamin K dependent enzymes by anticoagulants like warfarin, and if oxidative stress, inflammation, and blood sugar are kept low, then there is protection against calcification of the arteries and other soft tissues like the liver, kidneys, and heart.
Here we have it. We have now understood the role of vitamin K dependent proteins in vascular calcification. And although it was a little long and maybe somewhat arduous, all the details are clear. It is complicated. I won’t deny that. But I have strived to make it all as accessible as I could without diluting the mechanisms of action and relationship between the different players. Let’s recap to make sure you are left with the essential elements in mind.
Vitamin K dependent proteins can either be vitamin K1 or vitamin K2 dependent. The dependence comes from the fact that vitamin K is required to activate the protein. This activation is the carboxylation in which a carbon dioxide is added to the glutamic acid residues along the protein. Carboxylation is mediated by carboxylase (GGC) that requires the reduced form of vitamin K in order to oxidise it and get the energy to push the carbon dioxide molecule onto the glutamic acid residue. Vitamin K is reduced by reductase (VKOR) which can do it over and over again in what is called the VKOR cycle.
Vitamin K1 dependent proteins are mostly liver based coagulation factors. Vitamin K2 dependent proteins are mostly outside the liver and generally involved in inhibiting soft tissue calcification. The most important calcification-inhibiting VKDP is matrix Gla (MGP), which performs a wide range of tasks to maintain elastic, flexible, calcium-free blood vessel walls.
Calcification is triggered by the death of vascular smooth muscle cells. These dead muscle cells act at seeds for calcium apatite crystals to form. VSMCs can be induced to become osteoblast bone-building like cells that then go on to stimulate the growth of bone tissue within the artery walls. This process is stimulated by bone morphogenic protein 2 (BMP2), which is expressed under conditions of oxidative stress, inflammation, and hyperglycaemia.
To prevent and reverse calcification the most important is to provide a good supply of vitamin K2 through diet and supplementation. Because it is essential in the activation of Gla proteins but only through its role in the VKOR cycle, the amount of K2 is the rate limiting factor. Hence more is better than less, and excess will simply remain unused but will not cause harm.
Naturally, matrix Gla protein needs to be available. Cells of tissues where calcification occurs (kidney, liver, heart, and blood vessels) secrete MGP. An interesting evolutionary self-protection adaptation mechanism. And here’s another: the amount of MGP that is produced by a cell depends on at least two factors that have been identified. One is the amount of calcium; the other is the amount of vitamin D3. In both cases, the more there is, the more MGP is produced.
So, vitamin D3 has the role of making calcium available but at the same time stimulates the production of MGP in order for the calcium to be available to the bones and not to the soft tissues. But for this, it relies on vitamin K2. This is why vitamin D3 without vitamin K2 leads to calcification: because MGP and BGP remain inactive and incapable of binding to the calcium ions to move them into bones and out of tissues. On the other hand, plenty of vitamin K2 would indeed activate the available MGP, but without enough vitamin D3 there might not be enough MGP to confer proper protection against calcification. This is a perfect example of the complementarity of action and function in essential micronutrients. There are certainly many more, but this one is particularly remarkable.
I want to close on a final consideration. It is so easy and seems so natural for us to think in terms of this and that, good and bad, for and against, that our tendency is to look at everything in these terms. This is also true when we look at biochemical processes like the ones we have described and explored here. We naturally lean towards looking at the calcification inhibiting mechanisms as protective, and those that promote calcification and apoptosis as destructive.
But the reality is that cells, proteins, and enzymes don’t behave in these terms, they don’t think in these terms simply because they don’t think. They react biochemically to what they are exposed to, to the molecules and chemical messengers they encounter, to the quality of the liquids in which they bathe, to the characteristics of the environment in which they live, microsecond after microsecond, without any forethought or concern for the microsecond that will follow. The only guiding principle that can be used to lead us to understand why things happen the way they do is evolutionary adaptation to survive.
Having recognised this, we immediately see that the mechanisms that promote apoptosis of VSMCs, their subsequent transformation to osteoblast-like cells, and the growth of bone tissue within the artery walls that we refer to as arterial calcification, can only be a protection mechanism. A mechanism to protect the tissues and cells from the damaging effects of exposure to free radicals, inflammatory molecules, and glucose. Because, as we have seen, the process is reversible, it would be perfectly natural to undergo periods of calcification followed by periods during which the bone tissue is broken down and removed from our arteries and other soft tissues and organs when the circumstances allow it. Actually, we should say when the circumstances dictate it, because no matter what happens, it is always the circumstances—the environment—that dictate what is to happen.
What we can do, with the knowledge of what we have understood, is make choices about what we eat and drink, when and how much we eat, and how we live, sleep, and exercise. Choices that will shape or reshape, define or redefine the makeup of this internal environment of the body to always move us in the direction of optimal biochemistry, optimal physiology, optimal metabolism, and optimal health.
Everything that we explore together is always about just this. But sometimes the corrective action requires effort, sometimes even a lot of effort. In this case, however, it is as simple as can be, because it only requires us to supplement with vitamin K2 and possibly also D3. Of course, the last thing we want is a lifestyle that promotes the expression of BMP2 and the growth of bone tissue within our arteries. But supplementing with K2 and D3 together will in general bring only benefits. I know it was a very long-winded way to get to this, but now you understand why. That was—and is—the whole point of this blog, after all. I hope you enjoyed reading.
The information in this article comes primarily from the following papers: Molecular Mechanisms Mediating Vascular Calcification by Proudfoot and Shanahan (2006); Vitamin K-dependent Proteins, Warfarin, and Vascular Calcification by Danziger (2008); The Role of Vitamin K in Soft Tissue Calcification by Theuwissen, Smit, and Vermeer (2012).
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