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Has Russell Doolittle Provided an Evolutionary Explanation of the Blood Clotting Cascade?

Photo: A hagfish, by Peter Southwood, CC BY-SA 4.0 , via Wikimedia Commons.

In two previous articles (here and here), I reviewed the process of vertebrate blood clotting and summarized why it cannot readily be explained by naturalistic unguided processes, such as those proposed by neo-Darwinian evolutionary theory. In this final installment, with the foregoing challenges in mind, let us inspect Russell Doolittle’s paper on the evolution of vertebrate blood clotting1 to determine to what extent his analysis assuages these concerns. Since Doolittle has also published a book dealing with this subject2, in which he elaborates on the arguments expressed in the paper in more detail, I occasionally will refer to things said in the book as well.

Do Gene Duplications Explain Vertebrate Blood Clotting?

Doolittle contends that “Many of the proteins involved [in coagulation] are clearly related to one another by gene duplications, and in the past, sequence-based phylogenies have offered insights into the relative order in which certain factors appeared.”3 But sequence similarity does not necessarily imply common ancestry (due to the possibility of common design) and common ancestry does not necessarily imply a stepwise evolutionary pathway. Michael Behe explains this point:

Although useful for determining lines of descent… comparing sequences cannot show how a complex biochemical system achieved its function—the question that most concerns us in this book. By way of analogy, the instruction manuals for two different models of computer put out by the same company might have many identical words, sentences, and even paragraphs, suggesting a common ancestry (perhaps the same author wrote both manuals), but comparing the sequences of letters in the instruction manuals will never tell us if a computer can be produced step-by-step starting from a typewriter… Like the sequence analysts, I believe the evidence strongly supports common descent. But the root question remains unanswered: What has caused complex systems to form?4

This aside, however, Doolittle’s proposed mechanism runs into the problem enumerated above — namely, that duplicating a gene coding for one of the blood clotting factors would lead to that factor becoming over-expressed, disrupting the cascade’s delicate balance and leading to excessive clotting. This difficulty is nowhere even acknowledged in Doolittle’s paper, let alone addressed.

Doolittle raises a valid concern: “The question may be asked, how can new factors be introduced into an existing pathway?” Good question. How does Doolittle respond? He writes, “It was long ago suggested that in the case of clotting pathways, new factors that are the products of gene duplications could easily be sandwiched into the middle of pathways where they initially were only performing the same operation as the original gene product. Only a few amino acid replacements were likely needed to broaden the proteolytic specificity to the point where the duplicon could itself activate the other surviving gene product.”5 In support of this thesis, Doolittle notes that “all of the vitamin-K dependent proteases (prothrombin, factors VII, IX, and X, and protein C) cleave after arginine residues in the same general regions of their homologous substrates.”6 This, again, however, runs into the problem described above — namely, that gene duplication events would be likely to upset the delicate balance of the system, increasing the risk of thrombosis. Such duplicate genes are thus unlikely to be preserved by selection. Moreover, a few specific amino acid replacements in animals such as vertebrates (assuming none of them are beneficial until all have arisen) are unlikely to happen on a realistic timescale. As has been much discussed at Evolution News in the past, and in the academic literature, evolution depends on prohibitively long times to attain and fix multiple co-dependent mutations, where none of them confer a fitness benefit until all have arisen.7,8,9,10,11,12 Doolittle gives a time window for the emergence of the coagulation cascade of approximately fifty to a hundred million years, since fibrin clots have never been detected in any protochordate (i.e., organisms lacking a backbone but possessing a notochord at some stage during development) though it has been identified in the earliest vertebrates — that is, jawless fish. Thus, he argues, blood clotting must have arisen between the emergence of protochordates and jawless fish. As he writes in his book, 

Because fibrin clots have never been observed in our nearest non-vertebrate relatives, the protochordates, we must accept that the clotting system was assembled in the relatively brief interval since protochordates diverged from the lineage leading to vertebrates and the appearance of creatures like the hagfish and lamprey. In years, the available time is estimated to have been 50 to 100 million.13

Given that it is highly probable that each step in the evolution of coagulation would require multiple co-dependent mutations (since each proenzyme would have to evolve in a coordinated way with its activating enzyme), this time window appears to be quite brief. Compounding this is the fact that the mutations in the evolving gene duplicate would need to occur in a coordinated way with its own activating enzyme to ensure that the new factor is active only when needed.

Simpler Blood Clotting Systems in Jawless Fish

There are two extant genera of jawless fish — hagfish (pictured above) and lamprey. Doolittle predicts, from an evolutionary framework, that jawless vertebrates would have a simpler blood clotting cascade than the human system described above. Doolittle limits the scope of his analysis to the lamprey, for which there was better genomic data than for hagfish. Doolittle’s research has revealed that lampreys lack both factor IX and factor VIII.14,15 It may thus be concluded that factors IX and VIII are not essential, at least in the jawless vertebrates. Why are jawless vertebrates not hemophilic, unlike humans who lack these factors? Without knowing more about how coagulation works in jawless vertebrates, it is impossible to say for sure. Clearly, there are other differences as well — since apparently there are two factor X genes in lamprey. Is it possible that one of those is functioning as a factor IX gene? There are also three factors VII. Unfortunately, the precise mechanisms of coagulation in lampreys have yet to be elucidated. Given that humans who are deficient in factors IX or VIII suffer from hemophilia, this provides an indirect justification for believing that there are, quite probably, compensating mechanisms in lampreys that have yet to be uncovered.

Failing to Address Behe’s Argument

A crucial point is that, even if Doolittle’s entirely evolutionary scheme were correct, it would not even address — much less refute — Behe’s original thesis about the blood clotting cascade. In Darwin’s Black Box, Behe only argued that the common pathway is irreducibly complex — he does not give an assessment of whether the intrinsic pathway (to which these factors IX and VIII, missing in jawless fish, belong) or the extrinsic pathway are irreducibly complex as well.16 To summarize, in Darwin’s Black Box, Michael Behe only argued for irreducible complexity of the common pathway — i.e., the pathway after the convergence of the extrinsic and intrinsic initiation pathways, or what Behe calls the components “after the fork.” Behe made this very explicit in his book, where he wrote:

Leaving aside the system before the fork in the pathway, where some details are less well known, the blood-clotting system fits the definition of irreducible complexity.17

The system “before the fork in the pathway” is the intrinsic and extrinsic initiation pathways highlighted by Doolittle. But Behe explicitly leaves this part of the system “aside” and does not argue it is irreducibly complex. Thus, even if Doolittle’s arguments held merit, they would still not refute, even address, the portion of the pathway that Behe argues is irreducibly complex.

Regarding the systems “after the fork,” to my knowledge, there are no vertebrates with a functional coagulation system that lack thrombin, fibrinogen, factor X, or factor V. Clearly, there also has to be some way of activating factor X in response to tissue damage. Thus, to be conservative, blood coagulation must require a minimum of five parts (and probably more) – the very components which Behe argues comprise the irreducibly compelx portion of the blood clotting cascade. Behe’s argument has not been refuted. This same mistake was made in response to Behe by Kenneth Miller, as has been previously noted at Evolution News by Casey Luskin.

Co-Option of Thrombin / Fibrinogen

Doolittle concedes that “it seems unlikely that thrombin and fibrinogen would appear simultaneously,” and posits that “one already existed with an alternative function.”18 He suggests that “fibrinogen may have had a role in cell-cell interactions, a property of many proteins with fibrinogen-related domains.”19 Doolittle postulates that “a more likely scenario, however, is that thrombin had an early role in agglutinating thrombocytes by proteolyzing cell surface proteins, something it is known to do today, attacking a set of G-protein-coupled receptors called PAR proteins.”20 On this hypothesis, “a tissue factor would become exposed during the course of injury, activating prothrombin that would then clump cells which were the ancient ancestors of mammalian platelets. A GLA domain could have helped to keep thrombin localized on the surface of the thrombocytes.”21 The emergence of fibrinogen “would allow thrombin to broaden its attack, generating a more durable clot composed of fibrin. Duplications of the prothrombin gene would lead to the appearance of factors VII, X, and eventually IX.”22

Such a scenario, however, presents a number of problems. One is acknowledged by Doolittle himself: “Besides the GLA domain, thrombin has two kringle domains, usually thought to have an affinity for fibrin. The kinds of domains that interact with tissue factor, however, are the EGF domains found in factors VII, X, and IX.”23 He, therefore, has to postulate that “there was much domain shuffling in the early stages and that thrombin originally had EGF domains, or no peripheral domains at all.”24 Second, he makes no attempt to estimate how durable a clot composed of nothing more than clumped cells would be — though, if the initial flow rate was relatively low, this issue could be less of a concern. Finally, a duplication of a prothrombin gene would result in a second prothrombin gene. Doolittle makes no attempt to determine how difficult it would be for factors VII, X and IX to arise from a duplicated prothrombin gene (and evolve in a coordinated way with their corresponding activating enzymes), nor how the overexpression problem described above may be overcome.

Doolittle’s Four Stage Scenario

In his book, Doolittle proposes four stages in the evolution of vertebrate blood clotting, based on the presence or absence of coagulation factors in various animals. The timepoints along evolutionary history at which each of his four stages takes place is illustrated in the diagram below, reprinted under fair use from figure 13.1 of his book.25

The first stage, according to Doolittle, “existed in the last common ancestor of jawless and jawed vertebrates and was characterized by the presence of only six different proteins, three of which are vitamin K-dependent proteases.”26 These six proteins included tissue factor, factor VII, factor X, factor V, prothrombin and fibrinogen. The second stage, Doolittle suggests, involved the emergence of factors VIII and IX prior to the evolution of jawed fish. The third stage was characterized by the acquisition of prekallikrein and factor XII. A duplication of the gene coding for prekallikrein, resulting in the origins of factor XI, led, according to Doolittle’s scenario, to the fourth stage.

Notice that even the primitive system that constitutes stage one contains all of the four components that Behe claimed to comprise the irreducibly complex system — that is, thrombin, fibrinogen, factor X and factor V — in addition to tissue factor and factor VII (key components of the extrinsic pathway, required for initiating coagulation in response to tissue damage). Behe’s hypothesis predicts that every coagulation system will contain these four proteins (or equivalents), in addition to there being some way of activating factor X in response to tissue damage. This is precisely what the data show. The only aspect of the coagulation cascade that emerged subsequent to the origins of coagulation itself, then, so far as can be told by the data, are the components that make up the intrinsic pathway. That the intrinsic pathway is redundant is not particularly surprising, since there only needs to be one mechanism by which factor V is activated. Adding the intrinsic pathway is certainly helpful, though not necessarily essential. The intrinsic pathway serves to amplify the coagulation response initiated by the extrinsic pathway, and provides additional layers of activation and enzyme generation. Again, Behe’s fundamental thesis about irreducible complexity of the blood clotting cascade has not been refuted or even touched by Doolittle’s arguments.

But perhaps the most damning problem confronting Doolittle’s proposed scenario is that his analysis entirely ignores all of the clotting inhibitors (such as antithrombin, protein C, and protein S) that prevent excessive clot formation, as well as those factors that dismantle clots (as he himself acknowledges27). And this even though he correctly notes elsewhere that “this suppression of activity is very important; there is enough prothrombin in one milliliter of plasma to clot all the fibrinogen in the whole body if the prothrombin were all converted to thrombin.”28 Thus, the emergence of the pathway for coagulation along the lines suggested by Doolittle would likely result in runaway thrombosis without the simultaneous advent of inhibitors.

A Formidable Challenge 

In summary, the coagulation cascade presents a formidable challenge to evolutionary theory. While Doolittle is to be commended for his work in attempting to provide an evolutionary account for coagulation, the failure of those attempts underscores the difficulty of the problem. They simply do not address the issues at the heart of Behe’s argument in Darwin’s Black Box, which remains untouched by Doolittle’s thesis.


  1. Doolittle RF. Step-by-step evolution of vertebrate blood coagulation. Cold Spring Harb Symp Quant Biol. 2009;74:35-40.
  2. Doolittle RF. The Evolution of Vertebrate Blood Clotting. University Science Books 2013.
  3. Doolittle RF. Step-by-step evolution of vertebrate blood coagulation. Cold Spring Harb Symp Quant Biol. 2009;74:35-40.
  4. Behe, Michael J. (1996). Darwin’s Black Box: The Biochemical Challenge to Evolution. Free Press, kindle.
  5. Doolittle RF. Step-by-step evolution of vertebrate blood coagulation. Cold Spring Harb Symp Quant Biol. 2009;74:35-40.
  6. Ibid.
  7. Hössjer O, Bechly G, Gauger A. On the waiting time until coordinated mutations get fixed in regulatory sequences. J Theor Biol. 2021 Sep 7;524:110657.
  8. Sanford J, Brewer W, Smith F, Baumgardner J. The waiting time problem in a model hominin population. Theor Biol Med Model. 2015 Sep 17;12:18.
  9. Durrett R, Schmidt D. Waiting for two mutations: with applications to regulatory sequence evolution and the limits of Darwinian evolution. Genetics. 2008 Nov;180(3):1501-9. Erratum in: Genetics. 2009 Feb;181(2):819-20; author reply 821-2.
  10. Behe MJ, The Edge of Evolution: The Search for the Limits of Darwinism. Free Press 2007.
  11. Behe MJ, Snoke DW. Simulating evolution by gene duplication of protein features that require multiple amino acid residues. Protein Sci.2004 Oct;13(10):2651-64. doi: 10.1110/ps.04802904. Epub 2004 Aug 31. PMID: 15340163; PMCID: PMC2286568.
  12. Axe DD. The Limits of Complex Adaptation: An Analysis Based on a Simple Model of Structured Bacterial Populations. Bio-Complexity2010.
  13. Doolittle RF. The Evolution of Vertebrate Blood Clotting. University Science Books 2013, 184.
  14. Doolittle RF, Jiang Y, Nand J. Genomic evidence for a simpler clotting scheme in jawless vertebrates. J Mol Evol. 2008 Feb;66(2):185-96. doi: 10.1007/s00239-008-9074-8.
  15. Doolittle RF. Bioinformatic Characterization of Genes and Proteins Involved in Blood Clotting in Lampreys. J Mol Evol. 2015 Oct;81(3-4):121-30.
  16. Behe, Michael J. Darwin’s Black Box: The Biochemical Challenge to Evolution. Free Press 1996, kindle.
  17. Ibid.
  18. Doolittle RF. Step-by-step evolution of vertebrate blood coagulation. Cold Spring Harb Symp Quant Biol. 2009;74:35-40.
  19. Ibid.
  20. Ibid.
  21. Ibid.
  22. Ibid.
  23. Ibid.
  24. Ibid.
  25. Doolittle RF. The Evolution of Vertebrate Blood Clotting. University Science Books 2013, 185.
  26. Ibid.
  27. Doolittle RF. The Evolution of Vertebrate Blood Clotting. University Science Books 2013, 196.
  28. Ibid., 17.