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Summary. Coagulation evolved as a means to stem the loss of blood and to defend against pathogens. The complexity of the clotting cascade has been cited as evidence for the existence of divine intervention. The objective of this review is to draw on the debate between creationists and evolutionary biologists to highlight important evolutionary principles that underlie the hemostatic mechanism. I propose the following: (a) as with all biological systems, the hemostatic mechanism displays non-linear complexity; (b) the cellular response represents primary hemostasis owing to its place in the evolutionary time scale and functional importance; and (c) the rapid evolution of the hemostatic mechanism in vertebrates is testimony to the power and versatility of gene duplications and exon shuffling.
Many health care providers are confused and intimidated by the clotting cascade. They should not feel alone. The complexity of the coagulation mechanism has been cited as proof for the existence of divine intelligence and the clotting cascade has found itself of all places at center stage in a riveting debate between Christian fundamentalists and evolutionary biologists.
A consideration of this debate provides a refreshing perspective on the subject of blood clotting, not because it has scientific merit but because it encourages us to think about hemostasis in evolutionary terms. Complex design refers to an object whose constituent parts are arranged in a way that is unlikely to have arisen by chance alone. Commonly cited examples include the pocket watch or airplane . Complex design also exists in nature. In the early 19th century, William Paley noted that ‘every manifestation of design, which existed in the watch, exists in the works of nature with the difference on the side of nature of being greater or more…’. In supporting his argument, Paley drew heavily on descriptions of the human eye. For purposes of this discussion, we will focus on the coagulation system as an example of biological complexity.
The question has never been whether complexity exists, but rather how did it arise? In the case of the pocket watch or airplane, the answer is unambiguous. These are the products of purposeful design. In the case of biological complexity, the answer, while it may seem obvious to most of us today, has been hotly debated in some circles for the last 140 years.
The nature of this debate is perhaps best framed by the landscape metaphor, as originally described by Richard Dawkins in his book, Climbing Mount Improbable . According to this metaphor, complex design rests at the peak of a mountain. Dawkins called the mountain Mount Improbable because the species or organ could not have reached the summit by chance alone.
There are two sides to the mountain. For centuries, mankind recognized only the side with the cliff. Reaching the summit depended on giant leaps through divine intervention or single generation macromutations, a process referred to as saltation. In 1859, Charles Darwin exposed the other side of the mountain. He proposed that the gradual incline was surmountable by the cumulative selection of chance mutations, a mechanism that came to be known as natural selection . Once at the summit, the product carries the illusion of design.
As discussed by Dawkins, there are several rules to climbing this side of the mountain . First, each step represents an improvement. Every change must confer a survival or reproductive advantage. Second, the path is one way up. A species or organ system cannot get worse as a means towards eventual improvement. Third, there are no sudden leaps, or precipitous increases in ordered complexity. Finally, there is more than one peak or more than one way to solve a given problem.
So sure of his hypothesis was Darwin that he proclaimed: ‘If it could be demonstrated that any complex organ existed which could not possibly have been formed by numerous successive slight modifications my theory would absolutely break down’.
This of course opened an eternal door for the creationists and set the stage for decades of debate. Which brings us back to the clotting cascade. The coagulation mechanism, metaphorically resting at the peak of a mountain, does indeed impress us with improbable perfection, so much so that creationists have argued that it could not possibly have arisen from small beginnings by a gradual series of step-by-step changes.
How can we test this hypothesis? Traditionally, there are two approaches for studying gradual transformation. One is to identify fossil forms of the intermediates. The other is to guess at intermediates by looking at modern animals. Since the coagulation mechanism does not fossilize, we are forced to examine other living creatures with the assumption that what works for them should have worked for ancestral forms.
In lower invertebrates, such as the echinoderm or insect, which have an open circulation and low blood pressures, coagulation is mediated primarily by a cellular response. Upon injury to a vessel, cells quickly aggregate, retract and seal the wound. This mechanism is the rudimentary form of the platelet plug.
Coagulation in higher invertebrates is generally mediated by a combination of cells and clottable protein. The best-characterized system is that of the horseshoe crab [5–9]. These creatures, often referred to as ‘living fossils’, belong to a subclass that spans over 500 million years of evolution. Coagulation in horseshoe crabs is initiated by the aggregation of circulating cells, termed hemocytes. The cellular plug is quickly reinforced by a protein gel. In this reaction, a clottable protein (coagulogen) is cleaved and the resulting products cross-linked to one another to form a coagulin clot . Cleavage of coagulogen is mediated by the serine protease, coagulase. The reaction takes place very quickly and therefore the enzyme and substrate cannot circulate together. Rather, they are sequestered in circulating cells and are released only upon stimulation, resulting in clot formation. Thus, even in this ‘ancient’ hemostatic system, the cellular and protein components of the hemostatic response are critically coupled to one another.
Bacterial endotoxin is a potent stimulator of hemocytes. Indeed, an important function of coagulin is to immobilize pathogens on the cell surface of the cell, thereby promoting engulfment and disposal of the pathogen. Interestingly, when the inactive proteases are cleaved, the clipped domain has antimicrobial activity. Taken together, these observations suggest that the protease cascade plays a dual role in higher invertebrates: defense against exanguination and protection against pathogens.
Now let us jump a few hundred million years in the evolutionary time scale and consider the coagulation response in humans. The first line of defense is a cellular one, involving platelet adhesion, shape change, aggregation and release. The platelet, like its invertebrate counterpart the hemocyte, sets in motion a cascade of enzymes that ultimately leads to a protein clot. In humans (and other vertebrates), the clottable protein is fibrinogen and its product, fibrin. The conversion of fibrinogen to fibrin is mediated by the serine protease, thrombin. While the invertebrate coagulase is sequestered in the granules of circulating hemocytes, thrombin circulates as an inactive precursor, prothrombin. The conversion of prothrombin to thrombin is mediated by factor (F)Xa, which in turn is activated by either FVIIa of the extrinsic pathway or FIXa of the intrinsic pathway. Indeed, the coagulation cascade consists of a series of linked reactions in which a serine protease, once activated, is free to cleave its downstream substrate [11,12]. Blood coagulation is initiated by membrane bound tissue factor, and is amplified thought the intrinsic pathway by mechanisms that involve cross-talk and feedback. Virtually all of the coagulation reactions take place on activated cell surfaces, and is some cases are accelerated by the presence of a cofactor (FVa and FVIIIa). Finally, for every procoagulant response, there is a natural anticoagulant mechanism. Tissue factor pathway inhibitor neutralizes the extrinsic pathway; heparan is a cofactor for antithrombin III, which inhibits all of the serine proteases of the clotting cascade; protein C is converted to its active form by endothelial bound thrombomodulin and one activated, works with protein S to inactivate the cofactors, FVa and FVIIIa; plasmin, which is the end product of the fibrinolytic system, serves to degrade fibrin. In the final analysis, hemostasis can be considered a finely tuned balance between procoagulant and anticoagulant forces. Clinical phenotypes, namely bleeding or thrombosis, arise when the scale is tipped towards one side or the other.
Why have creationists embraced the coagulation pathway as a rallying point? First, they claim that the pathway is irreducibly complex, that is to say it is ‘composed of several well-matched, interacting parts that contribute to the basic function, wherein the removal of any one of the parts causes the system to effectively cease functioning’. Implicit in this definition, according to the creationist camp, is the notion that such a system ‘cannot be produced by continuously improving the initial function or by slight successive modifications of a precursor system, because any precursor that is missing is by definition non-functional’. A mousetrap is a fitting example of irreducible complexity . It has five basic components, all of which must be present and properly assembled in order to function.
As biologists, we should not have a problem with the concept of irreducible complexity. After all, there are many examples in which the removal of a single component would be predicted to short-circuit an entire system. However, the logic that follows, that irreducible complexity is proof for intelligent design, is seriously flawed. Surely, the interlocking nature of complex systems is as much a product of evolution as the single components themselves. Indeed, an inherent property of biological systems is that they display non-linear dynamics . Such systems consist of a large number of components, which display marked variability over time, and are characterized by a high degree of connectivity or interdependence. A key feature of non-linear systems is the relationship or coupling between the variables, not just the isolated variables themselves. The emergent properties of such a system cannot be understood by studying the individual parts in isolation.
While there is no question that the coagulation system is a complex non-linear dynamic, does it meet the definition of irreducible complexity ? If irreducibly complex, then one would predict that the loss of a serine protease or cofactor from the cascade would lead to a non-functional state, specifically a severe/lethal bleeding phenotype. Congenital deficiencies of FVIII or FIX are relatively common and give rise to hemophilia A and B, respectively, each of which confers a high risk of spontaneous deep tissue hemorrhage . Deficiencies in FVII and in components of the common pathway are less common, yet they too lead to hemorrhagic diatheses . While these relative deficiency states are associated with significant morbidity and even mortality, they are compatible with life and reproduction.
What we would really like to know is what happens when hemostasis is completely disrupted. We can answer that question by examining genetic mouse models in which the fibrinogen A α-chain gene is deleted . These animals have an absolute deficiency of secondary hemostasis. They cannot form fibrin plugs. Although null females develop fatal uterine bleeding during pregnancy, fibrogen-deficient animals are viable, and may lead a full healthy life. On the other hand, mouse models of severe thrombocytopenia are at higher risk for hemorrhagic death . Taken together, these observations suggest that the clotting cascade has evolved on a solid foundation of primary hemostasis (or cellular clot) and was therefore afforded the luxury of small incremental changes. From a functional standpoint, this hardly meets the definition of irreducible complexity.
A second, more interesting aspect of the coagulation mechanism that has attracted the attention of the creationists relates to the time frame of evolution. Studies of the most primitive extant vertebrate, the jawless fish, have revealed a well-developed coagulation cascade . In contrast, there is no ancestral form of the vertebrate clotting cascade in invertebrate animals (the serine protease cascade of the horseshoe crab evolved independently and is an example of convergent evolution). These observations suggest that the coagulation mechanism was assembled over a period of only 50 million years and then remained relatively unchanged for the next 450 million years (Fig. 1) . That is to say, the clotting cascade took a giant leap during evolution and plateaued early. How do we explain this apparent departure from the rules of the mountain?
Figure 1. Evolutionary timescale. The earth formed approximately 4600 million years ago. The first prokaryotic fossils date back to 3500 million years ago, while the first eukaryotic fossils appear 1800 million years ago. The first multicellular organisms appeared approximately 600 million years ago. The vertebrates and invertebrates diverged from a common ancestor 500 million years ago. The time elapsed between the earliest vertebrate and today's oldest extant vertebrate, the jawless fish, is estimated to be approximately 50 million years. All living vertebrates have platelets (or their nucleate counterpart, the thrombocyte) and a well-developed coagulation cascade; to date many, but not all, of the components have been cloned from the jawless fish. In contrast, the invertebrates do not possess even a vestige of the vertebrate clotting cascade.
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This is no easy task. In 1993, Russell Doolittle from the University of California, San Diego, an authority on protein evolution wrote a state-of-the-art review about the evolution of blood clotting . In the absence of a fossil record and without the benefit of existing intermediates, Doolittle proposed a sequence of step-by-step changes that may have given rise to the modern cascade, based on a series of gene duplications, exon shuffling, mutations and the development of highly refined regulatory processes. The thesis was well reasoned, speculative and necessarily simplified for the clinical readership.
Michael Behe, a Professor of Biochemistry at Lehigh University was quick to criticize Doolittle's paper and devoted an entire chapter in his book ‘Darwin's Black Box' to the clotting cascade . In the book, he states that: ‘blood coagulation is a paradigm of staggering complexity that underlies even apparently simple bodily processes. Faced with such complexity beneath even simple phenomena, Darwinian theory falls silent.’ Behe went on to write that: ‘Doolittle's scenario implicitly acknowledges that the clotting cascade is irreducibly complex, but it tries to paper over the dilemma with a hail of metaphorical references to yin and yang. The bottom line is that clusters of proteins have to be inserted all at once into the cascade. This can be done by postulating… the guidance of an intelligent agent' . How does one respond to such assertions? Doolittle wrote a letter to the Boston Review, sardonically concluding that Behe's brilliant revelation had rendered his career obsolete . Richard Dawkins would have characterized Behe's position as the Argument from Personal Incredulity , only this time wrapped in the cloak of biochemistry.
The humor and futility of this debate notwithstanding, the above considerations help to capture the essence of a quotation of a famous biologist from the early 20th century: ‘nothing makes sense except in the light of evolution’. According to proponents of a new branch in medicine, called Darwinian medicine, evolutionary perspectives provide an ‘integrating framework on which to hang a million otherwise arbitrary facts’[22,23]. A consideration of the evolutionary principles of hemostasis does indeed teach us important lessons. First, hemostasis does not exist in a vacuum. It evolved for a reason, namely as a mechanism to protect increasingly pressurized cardiovascular systems from leaking (and to protect multicellular organisms from pathogenic invasion). Second, the most important response is the cellular clot. Traditionally, we refer to the platelet response as primary hemostasis because it precedes thrombin generation at the site of injury. I would argue that it should also be considered primary owing to its place in the evolutionary time scale and its relative functional importance in mediating hemostasis. Third, the coagulation cascade was assembled over a very short period of time. This observation speaks to the power and versatility of gene duplications and exon shuffling and explains the high degree of homology between the various components of the clotting cascade. Finally, the cascade has weathered many storms and remained relatively unchanged for 450 million years. In other words, for all its complexity, the coagulation mechanism is extraordinarily durable.