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Imitating Rube
Does the human clotting system lend itself to the
same kind of analysis? At its core, the actual mechanism of clotting is
remarkably simple. A fibrous, soluble protein called fibrinogen
("clot-maker") comprises about 3% of the protein in blood plasma.
Fibrinogen has a sticky portion near the center of the molecule, but the
sticky region is covered by little amino acid chains with negative
charges. Because like charges repel, these chains keep fibrinogen
molecules apart.
When a clot forms, a protease (protein-cutting)
enzyme clips off the charged chains. This exposes the sticky parts of the
molecule, and suddenly, fibrinogens (which are now called fibrins) start
to stick together, beginning the formation of a clot.
The protease that cuts off the charged chains is
called thrombin. So, just like the lobster clotting system, the heart of
the reaction involves just two molecules: fibrinogen and thrombin. But,
unlike the lobster, there's a lot more to this machine. It turns out that
thrombin itself exists in an inactive form called prothrombin. So it, just
like fibrinogen, has to be activated before it can start the clotting
process. What activates prothrombin? Here's where life gets really
interesting. Prothrombin, a protease itself, is activated by
another protease called Factor X which clips of part of the
inactive protein to produce active, clot-forming thrombin. OK, so what
activates Factor X? Believe it or not, there are still more proteases, two
of them, actually, called Factor VII and Factor IX, that can switch on
Factor X. What switches them on? Here's where a good teacher goes the the
blackboard, and so will I:
Now, "beauty" is a word that most people wouldn't
think to put in the same sentence with "biochemistry," but the
biochemistry of this pathway is beautiful indeed. Start at the bottom,
with the conversion of soluble fibrinogen into clot-forming fibrin. As you
look up, you can trace this process to one of two different external
stimuli, both of which make good sense. At the upper left, the pathway can
be started with damage to a cell surface, something that happens whenever
blood is exposed to the air or a foreign object at the surface of a wound.
At the upper right, tissue factor, a soluble protein found in most tissues
but not in the bloodstream, activates the pathway. This is where clotting
starts from an internal hemorrage, a broken vessel within the tissues of
the body. So, both of these ultimate stimuli lead to the same set of
clot-forming proteins (Factor X, thrombin, and fibrin), but neither does
it directly. Instead, each activates a "cascade" of intermediate factors,
nearly all of them proteases, which eventually activate clot
formation.
It sure does look pretty, but why a cascade? Why
couldn't we have a simpler pathway, like the lobster, where something like
tissue factor activated clotting directly? Well, we could, but a complex
pathway, even if it drives biochemistry students to distraction, has
advantages of its own. For one thing, the multiple steps of the cascade
amplify the signal from that first stimulus. If a single active
molecule of Factor XII could activate, say, 20 or 30 molecules of Factor
XI, then each level of the cascade would multiply the effects of a
starting signal. Put 5 or 6 steps in the cascade, and you've amplified a
biochemical signal more than a million times. Clotting with fewer steps
would still work, but it would take longer to produce a substantial clot,
and would be much less responsive to smaller injuries.
Michael Behe is in awe of the the intricate
complexity of this system, and so am I. And he is also correct in pointing
out that if we take away part of this system, we're in trouble.
Hemophiliacs, for example, are unable to synthesize the active form of
Factor VIII. This means that they are unable to complete the final step of
one of the pathways, and that's why hemophilia is sometimes known as the
bleeder's disease. Defects or deficiencies in any of the other factors are
equally serious. No doubt about it - clotting is an essential function and
it's not something to be messed with. But does this also mean that it
could not have evolved? Not at all. The key to understanding the evolution
of this intricate system, as Russell Doolittle has pointed out, is the
fact that the clotting factors share an exquisite and revealing
similarity.
Building the Machine
To paraphrase Darwin, the notion that evolution
could have produced a system as intricate as the blood clotting cascade
seems, we might freely confess, "absurd in the highest possible degree."
This is especially true if you believe, as Behe seems to, that clotting is
not possible until the entire cascade of factors is assembled.
But we already know that evolution doesn't start
from scratch, and it doesn't need fully-assembled systems to work.
Remember the lobster system as an example. Blood clotting evolved there
from two pre-existing proteins, normally found in separate compartments of
the body, that had a fortuituous interaction when damage to a blood vessel
brought them together. Once that interaction was established, natural
selection did the rest.
Could something like this have happened
here?
Remember, we're not starting from nothing. We're
starting about 600 million years ago in a small pre-vertebrate. with a
low-volume low-pressure circulatory system. Just like any small
inverterbate with a circulatory system, our ancestral organism would have
had a full compliment of sticky white cells to help plug leaks. In
addition, that ancestral system would have had something else. Most of the
time, hemorrage starts with cell injury, meaning that cells are broken in
the vicinity of a wound and their contents are dumped out. That means,
among other things, that all of a cell's internal signalling molecules are
suddenly spilled out into the damaged vascular system. Included among the
contents are a whole slew of internal signalling molecules, including
prominent ones like cyclic adenosine monophosphate (abbreviated:
cAMP), all dumped into the tissue surrounding a wound.
Why would a sudden gusher of cAMP in a wound be
significant? Well, it turns out that vertebrates use cAMP as a signalling
molecule to control the contractions of smooth muscle cells, the very sort
of muscle cells that surround blood vessels. Therefore, the release of
internal cAMP from broken cells would automatically cause smooth muscles
around a broken vessel to contract, limiting blood flow and making it more
likely that the blood's own sticky white cells would be able to plug the
leak. That means that we already have some ability to limit damage and
plug leaks in a primitive, low-pressure system. Not a bad place to
begin.
Our next step is to consider the nature of blood
itself. For reasons relating to osmotic pressure, the tendency of water to
move across cell membranes, blood plasma is a viscous, protein-laden
solution. And it's also important to note that the extracellular
environment of ordinary tissue is very different from blood. These spaces
are laden with protein signals, insoluble matrix molecules, and
extracellular proteases that cut and trim these molecules to their final
shapes and sizes. In fact, such proteases constitute one of the major
forms of extracellular signalling. So the tissues of our ancestral
vertebrate would be laden with protein-cutting enzymes for reasons
completely unrelated to clotting.
Keeping all of this in mind, what would happen
when a blood vessel broke in such an organism?
Well, protein-rich plasma flows into an unfamiliar
environment, and sticky white cells quickly "glom" up against the fibers
of the extracellular matrix. Tissue proteases, quite accidentally, are now
exposed to a new range of proteins, and they cut many of them to pieces.
The solubility of these new fragments vary. Some are more soluble than the
plasma proteins from which they were trimmed, but many are much less
soluble. The result is that clumps of newly-insoluble protein fragments
begin to assumulate at the tissue-plasma interface, helping to seal the
break and forming a very primitive clot. (Could one object that this is
too primitive and too nonspecific to work? That it wouldn't be sufficient
to seal breaks? Well, it turns out that you can't make this objection for
the very simple reason that this is pretty much the clotting mechanism
used today by a large number of invertebrates. Works for them, and
therefore there is no reason why it wouldn't have worked for the ancestors
of today vertebrates, either!)
Now we get down to business. A mutation duplicates
an existing gene for a serine protease, a digestive enzyme produced in the
pancreas. Gene duplications happen all the time, and they are generally of
such little importance that they are known as "neutral" mutations, having
no effect on an organism's fittness. However, the original gene had a
control region that switched it on only in the pancreas. During the
duplication, the control region of the duplicate is damaged so that the
new gene is switched on in both the pancreas and the liver. As a
result, the inactive form of the enzyme, a zymogen, is relesased into the
bloodstream.
This causes no problem for the organism - most
pancreatic proteases are inactive until a small piece near their active
sites can be cut away by another protease. However, when damage to a blood
vessel allows plasma to seep into tissue, suddenly our previously inactive
plasma serine protease is activated by tissue proteases, increasing
the overall protein-cutting activity at the site of the hemorrage. Blood
clotting is enhanced, so our duplicate gene (with the mistargeted protein)
is now favored by natural selection.
That plasma protease gene is now subject to the
same witches' brew of copying errors, rearrangements, and genetic
reshuffling that affect the genes for every other cellular protein. Over
time, bits and pieces of other genes are accidentally spliced into the
plasma protease sequence. Because the selective value of the plasma
protease is pretty low (it doesn't help clotting all that much), most of
these changes make very little difference. But one day, through a
well-understood process called "exon shuffling," a DNA sequence known as
an "EGF domain" is spliced into one end of the protease gene. EGF stands
for epidermal growth factor, a small protein used by cells throughout the
body to signal other cells. EGF is so common that just about every tissue
cell has "receptors" for it. These receptors are cell surface proteins
shaped in such a way that they bind EGF tightly.
The fortuitious combination of a EGF sequence with
the plasma protease changes everything.
In a flash, the tissue surrouding a broken blood
vessel is now teeming with receptors that bind to the new EGF sequence on
our serum protease. As a result, high concentrations of the circulating
protease bind directly to the surfaces of cells near a wound. The
proteases are activated in the same way, but now their proteolytic
activities are highly localized. The production of a clot of insoluble
protein fragments is now faster and more specific than ever. Organisms
with the new EGF-protease can clot their blood much more quickly than
before, and therefore are favored by natural selection. To emphasize its
role in the clotting process, that cell surface protein with the EGF
receptor is called Tissue Factor.
What happens next? Well, remember the case of the
lobster in which a duplicate of a circulating protein (vitellogenin)
became specialized to produce a clot-forming protein (lobster fibrinogen)?
Once we have a situation in which every hemorrage activates a protease
bound to tissue receptors, a gene duplicate of one of the major plasma
proteins would then be under strong selective pressure to increase its
ability to interact with the bound protease. Fibrinogen, the soluble
protein that now is now the primary target of proteolysis in the clotting
cascade, clearly arose in this way. Natural selection would favor each and
every mutation or rearrangement that increased the sensitivity of
fibrinogen to the plasma protease, dramatically enhancing the ability of
the new protease to form specific clots of insoluble protein.
There is no doubt that these three steps, each one
supported by classic Darwinian mechanisms, would have been sufficient to
fashion a rudimentary clotting system. This would leave us with system in
which circulating plasma contains both an inactive serine protease and its
fibrinogen target. The protease would activated by contact with tissue
factor, and the active protease, in turn, would cleave sensitive sites in
fibrinogen to form a clot. This system wouldn't be nearly as quick, as
responsive, or as sensitive as the current system of vertebrate clotting,
but it would work a little better than the system that preceeded it, and
that's all that evolution requires.
Adding Complexity
Could evolution take this rudimentary system and
produce a multilayered cascade of factors? Just watch. Most serine
proteases, including trypsin and thrombin, are auto-catalytic. That means
that some extent they can activate themselves, in many cases by cleaving a
few amino acids to switch on their active sites. So, we could diagram the
actual functions of our ancestral plasma protease (which we'll call
protease A) like this:
As we have seen, the inactive form of the protease
(A) is changed into the active form (A*) when two things happen: it is
bound to tissue factor (TF) and it is activated by tissue proteases,
including our protease itself (that's the autocatalytic part). This means
- and this is important - that our protease is actually involved in
cutting two things: Fibrinogen, and also itself, converting A's
inactive precursor protein into A*.
Now, let's suppose that a gene duplication occurs
in the gene for our protease, producing a new (B) version of the
gene:
At first, just like most gene duplications, this
is no big deal. Proteins A and B are identical. Each can bind to TF, each
can cleave fibrinogen into fibrin, and each can activate itself or its
sister serum protease. So nothing has really changed - we've just got two
copies of the same gene. But now let's suppose that a mutation in the
active site of B changes its behavior, making it a little less likely to
cut fibrinogen and a little more likely to activate protease A. In
essence, this would change the relationship between these previously
duplicate genes to something like this:
Suddenly, the ability of A to bind to TF becomes
much less important. If B can saturate all of the available TF-binding
sites itself (by virtue of its EGF domain), then the TF-mediated
activation of B, combined with B's affinity for A, will result in a rapid
activation of A, producing plenty of activated A to convert fibrinogen
into clottable fibrin. Sounds good. But why would natural selection favor
a mutation like this in B's active site? Simple: it would increase the
efficiency of the clotting process by producing a 2-level cascade. Look
closely, and you'll see that our 1-step clotting system required a direct
interaction with TF to activate each protease. The new 2-step system
allows each TF to activate a protease B, each of which in turn can
activate scores or hundreds of A's. With so many more active proteases in
the neighborhood of the injury, clotting can now occur more quickly,
increasing the chances of surviving a hemorrage. Exactly the sort of stuff
that natural selection favors.
Step back for a second and think about what we've
just seen. A simple gene duplication sets the stage for the selection of
active site mutations that would dramatically improve the clotting
process. Gene duplications are neutral mutations, the sort that occur all
the time and therefore, given enough time, are highly probable. Once the
duplication has taken place, any mutation in the active site that shifts
the preferences of the active site in the direction I have mentioned will
be strongly favored. And that means that a true 2-step system will evolve
very quickly.
Two additional points have to be mentioned. The
first one is obvious. If gene duplication and subsequent mutation of the
duplicate protease can change a 1-step system into a 2-step one, they
could certainly change a 2-step system into a 3-step one. This means that
increases in biochemical complexity are not only accomodated by
evolutionary theory, they are actually predicted by it. The second point
is a little more subtle. Early stages in the evolution of a clot
forming-system are bound not to work very well. But as the system starts
to work better, as it increases in complexity and efficiency, it begins to
present a danger to the organism. That danger, simply put, is that
clotting might get out of hand. As the clot-forming cascade evolves larger
and larger, there is a chance that a small stimulus will start a reaction
that might cause all of an organism's blood to clot, or at least
enough of it to cause serious problems. Does evolution have an answer to
that, too?
Well, it turns out that it does. First, keep in
mind that a primitive clotting system, adequate for an animal with low
blood pressure and minimal blood flow, doesn't have the clotting capacity
to present this kind of a threat. But just as soon as the occasional clot
becomes large enough to present health risks, natural selection would
favor the evolution of systems to keep clot formation in check. And where
would these systems come from? From pre-existing proteins, of course,
duplicated and modified. The tissues of the body produce a protein known
as a1-antitrypsin which binds to the active site of serine
proteases found in tissues and keeps them in check. So, just as soon as
clotting systems became strong enough, gene duplication would have
presented natural selection with a working protease inhibitor that could
then evolve into antithrombin, a similar inhibitor that today
blocks the action of the primary fibrinogen-cleaving protease,
thrombin.
In similar fashion, plasminogen, the
precursor to a powerful clot-dissolving protein now found in plasma, would
have been generated from duplicates of existing protease genes, just as
soon as it became advantageous to develop clot-dissolving
capability.
In short, the key to understanding the evolution
of blood clotting is to appreciate that the current system did not evolve
all at once. Like all biochemical systems, it evolved from genes and
proteins that originally served different purposes. The powerful
opportunistic pressures of natural selection progressively recruited one
gene duplication after another, gradually fashioning a system in which
high efficiences of controlled blood clotting made the modern vertebrate
circulatory system possible.
Mining the Biochemical Past
Can we know for sure that this is how blood
clotting (or any other biochemical system) evolved? The strict answer, of
course, is we cannot. The best we can hope from our vertebrate ancestors
are fossils that preserve bits and pieces of their form and structure, and
it might seem that their biochemistry would be lost forever. But that's
not quite true. Today's organisms are the descendents of that biological
(and biochemical) past, and they provide a perfect opportunity to test
these ideas.
Even a general scheme, like the one I've just
presented, leads to a number of very specific predictions, each of which
can be tested. First, the scheme itself is based on the use of well-known
biochemical clues. For example, most of the enzymes involved in clotting
are serine proteases, protein-cutting enzymes so-named because of the
presence of a highly reactive serine in their active sites, the business
ends of the protein. Now, what organ produces lots of serine
proteases? The pancreas, of course, which releases serine proteases to
help digest food. The pancreas, as it turns out, shares a common embryonic
origin with another organ: the liver. And, not surprisingly, all of the
clotting proteases are made in the liver. So, to "get" a masked protease
into the serum all we'd need is a gene duplication that is turned on in
the pancreas' "sister" organ. Simple, reasonable, and supported by the
evidence.
Next, if the clotting cascade really evolved the
way I have suggested, the the clotting enzymes would have to be
near-duplicates of a pancreatic enzyme and of each other. As it turns out,
they are. Not only is thrombin homologous to trypsin, a pancreatic serine
protease, but the 5 clotting proteases (prothrombin and Factors X, IX, XI,
and VII) share extensive homology as well. This is consistent, of course,
with the notion that they were formed by gene duplication, just as
suggested. But there is more to it than that. We could take one organism,
humans for example, and construct a branching "tree" based on the relative
degrees of similarity and difference between each of the five clotting
proteases. Now, if the gene duplications that produced the clotting
cascade occurred long ago in an ancestral vertebrate, we should be able to
take any other vertebrate and construct a similar tree in which the
relationships between the five clotting proteases match the relationships
between the human proteases. This is a powerful test for our little scheme
because it requires that sequences still undiscovered should match a
particular pattern. And, as anyone knows who has followed the work in
Doolittle's lab over the years, it is also a test that evolution passes in
one organism after another.
There are many other tests and predictions that
can be imposed on the scheme as well, but one of the boldest was made by
Doolittle himself more than a decade ago. If the modern fibrinogen gene
really was recruited from a duplicated ancestral gene, one that had
nothing to do with blood clotting, then we ought to be able to find a
fibrinogen-like gene in an animal that does not possess the vertebrate
clotting pathway. In other words, we ought to be able to find a
non-clotting fibrinogen protein in an invertebrate. That's a mighty bold
prediction, because if it could not be found, it would cast Doolittle's
whole evolutionary scheme into doubt.
Not to worry. In 1990, Xun Yu and Doolittle won
their own bet, finding a fibrinogen-like sequence in the sea cucumber, an
echinoderm. The vertebrate fibrinogen gene, just like genes for the other
proteins of the clotting sequence, was formed by the duplication and
modification of pre-existing genes.
Now, it would not be fair, just because we have
presented a realistic evolutionary scheme, supported by gene sequences
from modern organisms, to suggest that we now know exactly how the
clotting system has evolved. That would be making far too much of our
limited ability to reconstruct the details of the past. But nonetheless,
there is little doubt that we do know enough to develop a plausible and
scientifically valid scenario for how it might have evolved. And that
scenario makes specific predictions that can be tested and verified
against the evidence.
References:
Doolittle, R. F., (1993) "The evolution of
vertebrate blood coagulation: A case of yin and yang," Thrombosis and
Haemostasis 70: 24-28.
Doolittle, R. F., and Feng, D. F., (1987)
"Reconstructing the evolution of vertebrate blood coagulation from a
consideration of the amino acid sequences of clotting proteins," Cold
Spring Harbor Symposia on Quantitative Biology 52: 869-874.
Doolittle, R. F., and Riley, M. (1990) "The
amino-acid sequence of lobster fibriongen reveals common ancestry with
vitellogenin." Biochemical and Biophysical Research Communications. 167:
16-19.
Xu, X., and Doolittle, R. F., (1990) "Presence of
a vertebrate fibrinogen-like sequence in an echinoderm." Proceedings of
the National Academy of Sciences (USA) 87: 2097-2101.
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