Coagulation on negatively charged surfaces: The effects of vitamin K
Welcome to Chalk Talk! In our previous episode, we looked at coagulation in vivo and presented
the cell-based model of coagulation. As seen, a central element of this model is that clotting
factors don’t just interact with each other loosely in plasma, but mostly bind to the
cell surface. This is clinically relevant, because this mechanism can be selectively
inhibited with vitamin K antagonists to reduce blood coagulability. In this Chalk Talk episode,
we’ll be taking a closer look at how this works.
Let’s begin with the binding of clotting factors to the platelet surface. Calcium is
a decisive mediator in the binding of clotting factors to the platelet membrane. In contrast
to all the other clotting factors, calcium isn’t a protein, but is present in blood
as a doubly charged positive ion. Using this positive charge, calcium can adhere to the
negative surface of activated platelets. In addition, several clotting factors also have
negative charges on their surface, which are capable of binding to calcium. So, why is
this favorable for the coagulation process? Let’s take a quick look:
Most clotting factors are proteases, that is, they activate downstream factors by cleaving
specific segments of the molecule. This is only possible if the proteases and their substrates
meet. Compared to the many options for interaction in three-dimensional space, such as in a blood
vessel, these options are reduced in two-dimensional space, such as on a surface. This dimensionality
reduction is achieved via calcium-mediated binding of clotting factors to the platelet
membrane. As a result, clotting factors can be rapidly activated and push coagulation
forward. So, as you can see, calcium is an important
mediator of coagulation. It’s so important that it’s even designated as a clotting
factor, namely factor four. To understand when calcium binds to platelets,
let’s take a look at the platelet surface. As seen in the second Chalk Talk episode on
primary hemostasis, platelets change shape as a result of platelet activation, as schematically
shown here. This activation causes changes in the molecular
structure of the platelet membrane, which consists of a lipid bilayer. In an inactivated
state, the outer layer mainly contains uncharged lipids, whereas negatively charged lipids
are present in the inner layer. Upon platelet activation, an increasing number of negatively
charged lipids are transferred from the inner to the outer layer of the plasma membrane.
This results in the creation of a negative charge on the outer platelet surface, which
calcium is able to bind to. This transverse diffusion of some membrane lipids from one
side of the membrane to the other is called the flip-flop mechanism.
The other negative charge that binds to calcium is present on the surface of the clotting
factors ten, nine, seven, and two. Why not try remembering this combination as a year?
Nineteen seventy-two, the year that saw the beginning of the Watergate scandal. An important
element for further understanding concerns the origin of the negative charge of these
four clotting factors. The negative charge originates from carboxyl groups. The general
chemical formula of a carboxyl group is C O O H. In blood, which has a pH of 7.4, carboxyl
groups are deprotonated. In other words, a proton is released resulting in a negatively
charged C O O minus group. Things are now going to become a bit more
complicated. We’d like to answer two important questions: Where are these carboxyl groups
located, and, how do they attach to clotting factors?
The carboxyl groups are located on the glutamate side chains of clotting factors. The correct
chemical term for these groups is gamma-carboxyglutamic acid, which implies that it has a glutamate
side chain with an additional carboxyl group at the gamma carbon atom of the side chain.
So, on to the second question: How do the additional carboxyl groups attach to the clotting
factors? This carboxyl group is only attached to the protein after translation. The process
is, therefore, termed post-translational modification. So, why does all of this matter? This post-translational
modification is only made possible through the action of vitamin K, which means that
blood coagulability can be positively or negatively influenced by its effect. The reason for this
is that in the absence of the gamma carboxyl group, clotting factors cannot bind well to
membrane lipids via calcium. This importance is also reflected in the name
vitamin K, referring to its essential role in coagulation. In 1929, the Danish scientist
Henrik Dam fed chickens on a vitamin K-free diet. The chickens subsequently developed
bleeding. The results were published in a German journal
and the substance was named Koagulationsvitamin or vitamin K. In 1943, Henrik Dam received
the Nobel prize in medicine for his discovery. From this important discovery, drugs were
later developed that are now vitally important in daily clinical practice. These are the
vitamin K antagonists such as warfarin. They prevent the carboxylation of clotting factors
and, therefore, work precisely on the mechanism previously described.
In the next slide, we’ll be taking a closer look at vitamin K-dependent clotting factors.
There are four clotting factors that use gamma-carboxyl groups to bind to the cell membrane. As previously
mentioned, these factors are ten, nine, seven, and two.
In addition to these four factors, there are other vitamin K-dependent proteins. Examples
include proteins C and S. Both of these proteins are inhibitors of coagulation.
OK, let's now take a look at the positions of factors ten, nine, seven, and two during
coagulation. At this stage, it would be beneficial to have watched the previous Chalk Talk episode
on the cell-based model of coagulation. But don’t worry if you haven’t; here’s
the link. Factor seven is part of the extrinsic tenase
complex, which is formed at the beginning of coagulation. Factor nine is part of the
intrinsic tenase complex, whereas factor ten is part of prothrombinase. Therefore, all
three factors are components of complexes that play a central role in coagulation.
The fourth and final factor, prothrombin, is also bound to the membrane.
This is quite useful, because prothrombin is directly delivered to prothrombinase. Prothrombinase
is a complex comprising activated factors ten and five, which is also membrane-bound.
This interaction results in rapid activation of prothrombin on the cell surface.
By the way, recent studies have shown that other factors are also capable of binding
to the platelet membrane, such as factors five and eight. However, the exact mechanism
by which these factors bind is still not completely understood, but is under investigation.
OK. In this episode, we’ve shown you how clotting factors bind to the cell membrane.
In some textbooks, you may find the following alternative notation for the complexes formed
during the process. This notation may be initially quite puzzling, but it should be easier to
understand at the end of this episode. These are complexes of activated clotting
factors; in this example, of the factors ten and five. The second part indicates that the
complex is bound to the phospholipids in the cell membrane by calcium.
The older term platelet factor 3 is sometimes used for phospholipids. The term was established
after discovering the contribution of platelets to the coagulation process. Today, we know
that platelet factor 3 is the membrane of activated platelets, more precisely, the negatively
charged phospholipids that are exposed through the flip-flop mechanism.
So, that just about wraps it up for this Chalk Talk episode on secondary hemostasis.