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Lee et al., (2010). A revisit to the one form kinetic model of prothrombinase.

December 2011, model of the month by Massimo Lai
Original model: BIOMD0000000364

Blood clotting, along with platelet activation, is a key mechanism of hemostasis, i.e. the mechanism that stops the bleeding of a damaged blood vessel by promoting the formation of a clot, that is made of platelets and blood cells in a network of fibrin. Fibrin filaments are produced when their soluble precursor, fibrinogen, polymerises after being appropriately cleaved by thrombin. Production of thrombin is tightly regulated, and unsurprisingly so. The formation of fibrin must be sufficiently fast and efficient to allow fast clotting and prevent excessive bleeding. On the other hand the activation of the pathway, despite involving components that are ubiquitous in blood, must be localised to the damage area and must not spread (as thrombosis would be obviously lethal). Any pathology that severely affects the coagulation cascade makes individuals vulnerable to accidental death or disability even after minor injuries.

Thrombin is finally produced when its precursor, prothrombin, is cleaved by prothrombinase. However, the cleavage process has a peculiarity, whose elucidation is the main point of Lee's model [[1], BIOMD0000000364]. Prothrombin has two cleavage points, that can produce two intermediate forms, called meizothrombin (if the first cleavage happens at Arg320) and prethrombin-2 (if the first cleavage happens at Arg271). A second cleavage of either form produces thrombin. The outstanding question is weather prothrombinase can flip-flop between two distinct structural conformations (called E1 and E2), that are responsible for the two different first cleavages, or if both cleavages are carried forward by a single form of the enzyme (called E). The numerous published papers on the subject are nearly equally split in support of either view. Further hypothesis about the thrombin production mechanism were also introduced in order to fit the experimental results: in particular channelling (direct production of thrombin without intermediate states) and ratcheting (after the first cleavage, the intermediate product changes conformation and makes the second site also available to protease) (Figure 1a). The leading models in the field of the two-form and one-form supporters were produced by the Nesheim group and the Krishnaswamy group respectively [2,3].

Figure 1

Figure 1: (a) One-form model proposed by Nesheim and coworkers: both cleavages of prothrombin are performed by the same form of prothrombinase. (b) Simplified one-form model (without ratcheting nor channelling) by Lee et al. Figure taken from [1].

Figure 1

Figure 2: Averaged datasets by the Nesheim and Krishnaswamy groups, and concentration profiles predicted by Lee's model. Figure taken from [1].

Figure 1

Figure 3: Structure of the equilibrated prothrombin complex, showing the two cleavage residues in the middle of long, flexible loops. Figure taken from [1].

Lee's paper argues that an, improved one-form model (Figure 1b) can produce correct concentration profiles of the four main observable chemical species: prothrombin, thrombin, meizothrombin, and prethrombin-2. Moreover, for such a model, no ratcheting or channelling is required in order to reproduce consistent concentration profiles (Figure 2).

Molecular dynamics simulation of a prothrombin complex in explicit solvent further showed that the two cleavage sites Arg320 and Arg271 are situated in the middle of two long, flexible loops of about 40 aminoacids each (Figure 3), that are likely to be very mobile. Therefore, the authors suggest that the differential cleavage is due to competition between the two sites for the active site of the protease. In summary, this paper supports the one-form view of prothrombinase and produces a model that is both consistent with available datasets, and much simpler that its predecessors.

Bibliographic References

  1. Lee CJ, Wu S, Eun C, Pedersen LG. A revisit to the one form kinetic model of prothrombinase. Biophys Chem Jun;149(1-2):28-33, 2010. [CiteXplore]
  2. Kim PY, Nesheim ME. Further evidence for two functional forms of prothrombinase each specific for either of the two prothrombin activation cleavages. J Biol Chem. Nov 9;282(45):32568-81, 2007. [CiteXplore]
  3. Bianchini EP, Orcutt SJ, Panizzi P, Bock PE, Krishnaswamy S. Ratcheting of the substrate from the zymogen to proteinase conformations directs the sequential cleavage of prothrombin by prothrombinase. Proc Natl Acad Sci U S A. Jul 19;102(29):10099-104. 2005. [CiteXplore]