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EEAVEEETGDGLDEDSD--RAIEGRTATSEYQTFFNPRTFGSGEADCGLR 339

EEAVEEETGDGLDEDPD--RAIEGRTATSEYQPFFNPRTFGSGEADCGLR 339

EEPVDGDLGDRLGEDPDPDAAIEGRTSEDHFQPFFNEKTFGAGEADCGLR 342

EEAVGEEN-----YDVD--ESIAGRTTDAEFHTFFNEKTFGLGEADCGLR 336

DEAVGEEN-----HDGD--ESIAGRTTDAEFHTFFDERTFGLGEADCGLR 335

DSSLEDEN--------EQVEEIAGRTIFQEFKTFFDEKTFGEGEADCGTR 326

------------------------RTTLDQRKAFFNPRSFGNGELDCGER 238

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PLFEKKSLEDKTERELLESYIDGRIVEGSDAEIGMSPWQVMLFRKSPQEL 389

PLFEKKSLEDKTERELLESYIDGRIVEGSDAEIGMSPWQVMLFRKSPQEL 389

PLFEKKQVQDQTEKELFESYIEGRIVEGQDAEVGLSPWQVMLFRKSPQEL 392

PLFEKKSLKDTTEKELLDSYIDGRIVEGWDAEKGIAPWQVMLFRKSPQEL 386

PLFEKKSLTDKTEKELLDSYIDGRIVEGWDAEKGIAPWQVMLFRKSPQEL 385

PLFEKKQITDQSEKELMDSYMGGRVVHGNDAEVGSAPWQVMLYKKSPQEL 376

PLFEKINKADKNEKELLMSYTGSRIVGGDEAEVASAPWQVMLYKRSPQEL 288

***** .  * .*:**: **  .*:* * :** . :******:::*****

LCGASLISDRWVLTAAHCLLYPPWDKNFTENDLLVRIGKHSRTRYERNIE 439

LCGASLISDRWVLTAAHCLLYPPWDKNFTENDLLVRIGKHSRTRYERNIE 439

LCGASLISDRWVLTAAHCLLYPPWDKNFTVDDLLVRIGKHSRTRYERKVE 442

LCGASLISDRWVLTAAHCILYPPWDKNFTENDLLVRIGKHSRTRYERNVE 436

LCGASLISDRWVLTAAHCILYPPWDKNFTENDLLVRIGKHSRTRYERNVE 435

LCGASLISNSWILTAAHCLLYPPWDKNLTTNDILVRMGLHFRAKYERNKE 426

LCGASLISDEWILTAAHCILYPPWNKNFTINDIIVRLGKHSRTKYERGIE 338

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KISMLEKIYIHPRYNWRENLDRDIALMKLKKPVAFSDYIHPVCLPDRETA 489

KISMLEKIYIHPRYNWRENLDRDIALMKLKKPVAFSDYIHPVCLPDRETA 489

KISMLDKIYIHPRYNWKENLDRDIALLKLKRPIELSDYIHPVCLPDKQTA 492

KISMLEKIYVHPRYNWRENLDRDIALLKLKKPVPFSDYIHPVCLPDKQTV 486

KISMLEKIYIHPRYNWRENLDRDIALLKLKKPVPFSDYIHPVCLPDKQTV 485

KIVLLDKVIIHPKYNWKENMDRDIALLHLKRPVIFSDYIHPVCLPTKELV 476

KIVAIDEIIVHPKYNWKENLNRDIALLHMKKPVVFTSEIHPVCLPTKSIA 388

**  :::: :**:***:**::*****:::*:*: ::. ******* :. .

ASLLQAGYKGRVTGWGNLKETWTANVGKGQPSVLQVVNLPIVERPVCKDS 539

ASLLQAGYKGRVTGWGNLKETWTANVGKGQPSVLQVVNLPIVERPVCKDS 539

AKLLHAGFKGRVTGWGNRRETWTTSVAEVQPSVLQVVNLPLVERPVCKAS 542

TSLLRAGYKGRVTGWGNLRETWTTNINEIQPSVLQVVNLPIVERPVCKAS 536

TSLLQAGYKGRVTGWGNLRETWTTNINEIQPSVLQVVNLPIVERPVCKAS 535

QRLMLAGFKGRVTGWGNLKETWATTP-ENLPTVLQQLNLPIVDQNTCKAS 525

KNLMFAGYKGRVTGWGNLRESWTSNP-SNLPAVLQQIHLPIVDQSICRNS 437

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TRIRITDNMFCAGYKPDEGKRGDACEGDSGGPFVMKSPFNNRWYQMGIVS 589

TRIRITDNMFCAGYKPDEGKRGDACEGDSGGPFVMKSPFNNRWYQMGIVS 589

TRIRITDNMFCAGYKPGEGKRGDACEGDSGGPFVMKSPYNNRWYQMGIVS 592

TRIRITDNMFCAGFKVNDTKRGDACEGDSGGPFVMKSPFNNRWYQMGIVS 586

TRIRITDNMFCAGFKVNDTKRGDACEGDSGGPFVMKSPYNHRWYQMGIVS 585

TRVKVTDNMFCAGYSPEDSKRGDACEGDSGGPFVMKNPDDNRWYQVGIVS 575

TSVIITDNMFCAGYQPDDSKRGDACEGDSGGPFVMKSPSDNRWYQIGIVS 487

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WGEGCDRDGKYGFYTHVFRLKKWIQKVIDQFGE---- 622

WGEGCDRDGKYGFYTHVFRLKKWIQKVIDQFGE---- 622

WGEGCDRDGKYGFYTHVFRLKKWIQKVIDRLGS---- 625

WGEGCDRKGKYGFYTHVFRLKRWIQKVIDQFG----- 618

WGEGCDRNGKYGFYTHVFRLKRWMQKVIDQHR----- 617

WGEGCDRDGKYGFYTHVFRLKKWMRKTIEKQG----- 607

WGEGCDRDGKYGFYTHLFRMRRWMKKVIEKTDSGDDE 524

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    Before prothrombin becomes thrombin, it forms the prothrombinase complex. This is comprised of Coagulation factors Va and Xa and calcium, which all bind to the prothrombin protein (shown in Figure 3). The thrombin active site is located in the B chain and possibly associated with the light A chain. The first two Kringle domains and the Vitamin K dependent Domain are all involved with the binding of these other coagulation factors (Mann et al, 1982).

    Factor Xa is the proteolytic component of the prothrombinase complex. By itself, it can produce alpha-thrombin, but at a vastly decreased rate when compared to its abilities as part of the complex. It cuts after two Arg residues, one right after the other. Depending on which Arginine it cleaves first, it will either produce D or E in Figure 2.

    Possibly, the loss of fragment II (as depicted in Figure 2, also shown as the second kringle domain in Figure 1), might have some affect upon the formation of the prothrombinase complex in zebrafish. This would also suggest that zebrafish do not form the intermediate proteins shown in Figure 2 (D and E), or that their intermediate forms are different than those of the mammals’ proteins that are shown in the alignment. It is possible that this loss causes an alternative form.

    Studies have shown, however, that the active site of bovine thrombin is sufficiently similar to zebrafish thrombin to induce thrombosis (formation of clots) in zebrafish embryos. This suggests that there is a high level of conservation between the active site within the heavy B chain in both zebrafish and mammals (shown also in Figure 4), which allows the enzyme to work in both native systems (Kalish et al, 2009).

Figure 1: Alignment of prothrombin protein in the following vertebrates:  Homo sapiens (Human), Pan troglodytes (Chimpanzee), Bon taurus (Cow), Mus musculus (Mouse), Rattus norvegicus (Rat), Gallus gallus   (Chicken), Danio rerio  (Zebrafish). The domains labelled are the thrombin light chain (orange), Vitamin K dependent domain (blue), Kringle domain (green), Trypsin-like serine protease (purple). (Figure made using Hologene Search).

    The trypsin-like serine protease is the most highly conserved segment of the thrombin protein. The specificity of thrombin (discussed on the Protein page) is controlled mainly by the active site found in this protease section of the thrombin protein. Divergence between species is found mainly in the kringle domains and the Vitamin K dependent domain (Figure 4). In order for thrombin to perform it’s specific, intended cleavage of the fibrinogen peptide, this sequence containing the active site (Heavy B chain/serine protease domain) must be conserved.

    It has been shown that F2‘s first Kringle domain is receptive to certain F2 repressors--specifically an anticoagulant caller warfarin. Human and cow thrombin is unaffected by the presence of warfarin, but rat thrombin bound to warfarin in vivo is degraded within the cell, as opposed to being secreted in order to perform its coagulative duties. Chimeric rat f2 containing the first human Kringle domain in place of the rat version is not susceptible to warfarin interference (Wu et al, 1997). Although the differences between human and rat prothrombin are minimal, they convey immunities to different repressors, yet are similar enough to make viable chimeric proteins.

    As you can see in Figure 4 of the thrombin proteins, zebrafish lack the second Kringle domain found in six other species. The two kringle domains and the Vitamin K-dependent domain are part of the prothrombin protein but not the final acive thrombin. They are necessary to form the active form of thrombin by cleavage between the second Kringle domain and the thrombin light chain (Bode, et al, 1989). Cleavage also occurs between the light chain (A) and protease domain (Heavy chain B); these two segments are only held together by a single disulfide bond as shown in Figure 2.

    Thrombin mRNA has been found in the liver, stomach and brain of mammalian embryos, as well as zebrafish embryos. It’s catalytic abilities are tied strongly but not limited to the coagulation cascade and the cleavage of fibrinogen to form fibrin and it acts as an activator for several other enzymes. In other words, thrombin’s effects upon the body are far-reaching and numerous. Mutations that affect thrombin are the cause of several serious illnesses.

    High levels of thrombin have been tied to inflammatory brain disease (Chapman, 2006), coronary artery disease(Small et al, 1988) and cardiovascular disease (Russell, 2003). In many of these cases, it is predicted that a hypercoagulative state is at least partly responsible for the illness. Inflammation can cause narrowing of the blood vessels, which would lead to any of the above-mentioned diseases. However, it has been hypothesized that high levels of thrombin would lead to increased fibrin production as well as activation of other coagulation factors. These factors would then activate platelets that would add to the fibrin clot and encourage an inflammatory response in the surrounding tissue, as if a breach of the primary immune defense system had been discovered.

Figure 2: Figure showing The different forms of thrombin and all the intermediate steps between prothrombin and thrombin activation. A shows the complete prothrombin protein. B shows the cleavage of the light and heavy thrombin chains while the disulfide bridge between the two fragments is maintained. C, D and E show other intermediate molecules. F, G and H show variations of the thrombin molecule. Each involve the heavy and light thrombin chains, but with additional modifications and divisions in the final thrombin molecule(Evidence That Meizothrombin Is an Intermediate Product in the Clotting of Whole Blood).

A

B

C

D

E

F

G

H

Figure 3: A) This shows the association of Factor Xa and Factor Va to form the prothrombinase complex. By associating with the Tissue Factor, the prothrombinase complex remains fixed to phospholipid layers on the surface of specific cells and organelles. B) Here we can see the formation of thrombin from prothrombin by Factor Xa in association with Factor Va and thrombin’s further catalytic role as it cleaves fibrinogen to form the thrombus.

A

B

Figure 4: This figure shows a multiple alignment of prothrombin proteins from different species, shown in the following order: Homo sapiens (Human), Pan troglodytes (Chimpanzee), Bon taurus (Cow), Mus musculus (Mouse), Rattus norvegicus (Rat), Gallus gallus   (Chicken), Dani rerio  (Zebrafish). Domains are highlighted by colored boxes and labelled as follows: thrombin light chain (orange), Vitamin K dependent domain (blue), Kringle domain (green), Trypsin-like serine protease (purple). Notice the missing segment in the second Kringle Domain of the Zebrafish prothrombin sequence. (Made using ClustalW multiple alignment)

    The original Thrombin crystal structure was determined with partial assistance by the 3D structure of bovine chymotrypsin (Bode et al 1989). Thrombin’s trypsin-like serine protease domain (surprisingly?) is very similar to trypsin and chymotrypsin’s active site domain.  As shown in Figure 5, trypsin or chymotrypsin proteins from our six simlar species are highly conserved in prothrombin’s trypsin-like serine protease domain.

   The fact that this secondary protein structure has lasted over vast amounts of evolutionary time is not surprising, given its importance within the coagulation cascade. The differences in the active domain of both trypsin and thrombin are minor, considering trypsin’s ability to cleave any serine residue, and thrombin’s much more specific abilities (As discussed in Protein).

Bode, W., Turk, D., Karshikov A. The refined 1.9-A X-ray crystal structure of D-Phe-Pro-Arg chloromethylketone-inhibited human a-thrombin: Structure analysis, overall structure, electrostatic properties, detailed active-site geometry, and structure-function relationships. 1992. Protein Science. 426-471.

Bode, W., Mayr, I., Baumann, U., Stone, S. R., Hofsteenge, J. 1989. The Refined A crystal structure of human a-thrombin: interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment. The EMBO Journal. 8(11): 3467-3475.

Chapman, Joab. Thrombin in inflammatory brain diseases. 2006. Autoimmunity Reviews. 5:8 528-531. <http://www.sciencedirect.com/science/article/B6W8V-4JHW9RY-2/2/5d719318e4f1388e1ada011d9d898b6b> Accessed March 8 2010.

Kalish, Yosef MD, Arunima Ghosh, MBBS, PhD, Jordan A. Shavit, MD, PhD and David Ginsburg, MD. Conservation of Hemostatic System Component Function Between Zebrafish and Mammals. 2009. American Society of Hematology. <http://ash.confex.com/ash/2009/webprogram/Paper25113.html>Accessed March 8 2010.

Mann, K.G., Nesheim, P.B., Tracy, L. S., Hibbard, L.S., Bloom, J.W. ASSEMBLY OF THE PROTHROMBINASE COMPLEX. 1982. BIOPHYS.J.37:106-107

Russell, T. P. Thrombin, Inflammation, and Cardiovasular Disease. 2003. CHEST. 124: (3) 49S-57S . <http://chestjournal.chestpubs.org/content/124/3_suppl/49S.full>. Accessed March 8 2010.

Small, M., Lowe, G. D., Douglas, J. T., Hutton, I., Lorimer, A. R., and Forbes, C. D. Thrombin and plasmin activity in coronary artery disease. 1988. Br Heart J. 60: 201-3.

Wu, W., Bancroft, J. D., Suttie, J. W. Structural features of the kringle domain determine the intracellular degradation of under-γ-carboxylated prothrombin: Studies of chimeric rat/human prothrombin. 1997. 94: (25) 13654-13660. <http://www.pnas.org/content/94/25/13654.abstract> Accessed March 7 2010.

*All Figures are from Wikimedia Commons unless otherwise noted.

    Thrombin (also known as prothrombin or Coagulation Factor II) is a highly conserved protein in many eukaryotes. Since it functions within a coagulation cascade that is unique to Animalia, similar proteins are not found in prokaryotes.  There are seven species in which the thrombin protein (gene symbol F2), is very highly conserved: Homo sapiens (Human), Pan troglodytes (Chimpanzee), Bon taurus (Cow), Mus musculus (Mouse), Rattus norvegicus (Rat), Gallus gallus   (Chicken), Dani rerio  (Zebrafish) (Figure 1 from Hologene).

    There several domains that are highly conserved. These are the thrombin light chain, kringle domain, Vitamin K-dependent domain and a segment that resembles a trypsin-like serine protease. As is shown in Figure 1, several species have all four domains in the same order, but some (like the zebrafish) have one fewer kringle domains than the other six.