Tell us how it was established that the blood clotting cascade came about naturally.
If you had simply gone to The Ultimate Creation vs. Evolution Resource (available right here on FR), you wouldn't be making such requests. Indeed, Is the Blood Clotting Cascade Irreducibly Complex? is right there, and it specifically addresses Behe's works. The relevant information is below:
As I wrote in my 1999 book Finding Darwin's God, Russell Doolittle's pioneering work on protein evolution has indeed shown that the blood clotting cascade could, and indeed was, produced by Darwinian evolution. Dr. Behe's defense of his position to the contrary requires him to explain why my description of the system's evolution (pp. 152-161) is not valid. In his web-published defense he writes that my description is "a just-so story that doesn't deal with any of the difficulties the evolution of such an intricate system would face."
Curiously, Behe all but ignores my description of the evolution of blood clotting in the lobster, and provides no rebuttal to my scenario for its evolution (based, of course, on Doolittle's research work). I described the lobster system in my book for two reasons: (1) It, like the vertebrate system is (by Behe's standards) irreducibly complex, and (2) It is a simpler system whose step-by-step evolution is relatively easy to account for.
Behe quite properly notes that my own description of the evolution of the verterbate clotting system is rather brief... a decision, alas, of my Editor at Harper-Collins (the book's publisher). Nonetheless, I still have my original draft of this edited-out section, which the reader may wish to consult. (Click here for my ideas on the evolution of the vertebrate clotting system). However, which elements of my description, besides its sketchiness, does he take issue with?
Behe asserts that the targeting of a protease, a digestive enzyme, to the bloodstream is a "potentially deadly situation," and tells the readers of his web document that we can tell how deadly this might be by looking at situations "where regulatory proteins are missing from modern organisms." In other words, Behe wants us to look at what happens when the highly-regulated current versions of clotting proteases are missing their regulatory factors. Despite this bluster, however, Behe has no evidence that the mistargeting of an inactive protease to the bloodstream would cause harm. Indeed, the recent discovery that antifreeze protein genes in fish arose from exactly such a mistargeting of proteases into the bloodstream (Chen, L., DeVries, A. L. & Cheng, C.- H. C. Proc. Natl Acad. Sci. USA 94, 38113816 (1997); and Chen, L., DeVries, A. L. & Cheng, C.-H. C. Proc. Natl Acad. Sci. USA 94, 38173822 (1997)) suggests that exactly the opposite is true.
Having made unsupported claims about the "danger" of such a mutation, Behe says that it would be difficult to see what "advantage" this would present to the organism. The answer, of course, is that it would provide a slight improvement in the organism's ability to clot blood - and that's the point. The clotting system doesn't have to work full-blast right away. In a primitive vertebrate with a low-pressure circulatory system, a very slight improvement in clotting would be advantageous, and would be favored by natural selection.
Behe then wonders how the circulating protease could become localized at the site of a clot, as if this were an insurmountable difficulty. It's not. As I suggested in my original draft on the evolution of clotting, a well-understood process called exon shuffling could have placed an "EGF domain" onto the protease sequence, and the "problem" that Behe puzzles over is solved in a flash.
Finally, Behe emphasizes that the real problem is not to generate a clot - it is to "regulate" that clot by means of an inhibitor of the protease so that it doesn't become destructive. But that's not a problem for evolution, either. As usual, Behe envisions a clotting protease that is just as powerful as the fully-evolved proteases in modern vertebrates. However, remember that this is the same guy who fretted a moment or two ago that the protease would not be strong enough to clot effectively. He wants to have it both ways. The answer to his objection is just what I wrote in the draft:
" ... 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 alpha-1-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 short, none of the points raised by Behe are adequate to explain why the vertebrate clotting system could not have evolved. Furthermore, as Doolittle's work has shown clearly, the hypothesis of evolution makes testable predictions with respect to the DNA sequences of clotting proteins, and these predictions have turned out to be correct time and time again.
Why has Behe's "Biochemical Challenge to Evolution" met with so little support within the scientific community? I would suggest that the reason is simple. His hypothesis is wrong. The complex biochemical systems of living organisms, including the vertebrate clotting cascade, are fully understandable in terms of Darwinian evolution.
Next in line, we have The Evolution of Vertebrate Blood Clotting, which contains additional information. For instance:
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.
