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Figure 1: Thrombin and Its Two Anion-binding Exosites
extensive investigation. Apart from the active site of thrombin, there are
mixed positive charges and hydrophobic patches on both sides of the
molecules known as anion-binding exosites I and II (ABE I and ABE II)
that confer thrombin substrate specificity (see Figure 1). ABE I is
Exosite II
Exosite I
responsible for fibrinogen, factors V, VIII, XI, PAR and TM contacts, while
the ABE II binds platelet GPIb and chondroitin sulphate part of TM.
18
Thrombin inhibitors may target the ABE I (hirudin) exosite II (heparin and
hemadin) or both exosites (Bothrojaracin, a C-type lectin from the snake
Bothrops jararaca).
Thrombin-like enzymes are named according to the ability of venom
Active site cleft
proteases to clot fibrinogen in vitro. Notably, these enzymes do not
contain the full effects of thrombin. They most commonly release only
fibrinopeptide A, termed venombin A, for example ancrod from the
Malayan pit viper, Calloselasma rhodostoma,
19
batroxobin from B. atrox
20
and GPV-TLs from the green pit viper, C. albolabris;
21
or only
fibrinopeptide B, termed venombin B, for example contortrixobin from
Agkistrodon contortrix,
22
or both fibrinopeptides, termed venombin AB,
for example bilineobin from A. bilineatus.
23
However, bilineobin
the tetramers can cluster platelet receptors, resulting in stronger platelet preferentially cleaves fibrinopeptide B. In addition, factor XIII is not
activation.
16
Disulphide bonds also linked two peptides, either in the same activated, resulting in friable clots subjected to fibrinolysis. Some SVSPs
family, e.g. a dimeric disintegrin, or different families of proteins, e.g. SVMP show other activities of thrombin. For example, platelet PAR is activated
and CLPs. These create novel and/or composite functions. Although cysteine by PA-BJ from B. jararaca.
24
A TM-independent protein C activator, ACC-
bonds may be beneficial for snake venom evolution, they make these C, was isolated from A. contortrix contortrix,
25
and a factor V activator,
proteins difficult to recombinantly express, even in eukaryotic systems. RVV-V, was purified from Russell’s viper, D. russelii.
26
Other SVSPs contain
fibrinogenolytic
27
and/or plasminogen-activating activities.
28
Snake Venom Serine Proteases
Almost all of the enzymes in coagulation cascade and fibrinolysis are Sequence analysis of thrombin-like SVSPs could not define ABE I and II
serine proteases characterised by the catalytic triad: serine (S), aspartate homologous to thrombin.
21
This is not surprising because their functions
(D) and histidine (H). However, most SVSPs are originate from the do not have the same multiplicity and their evolutionary origin is different
glandular kallikrein that later evolved to acquire coagulation and from that of thrombin. However, parts of molecules, which may be called
fibrinolytic/fibrinogenolytic activities. This may explain hypotensive effects ‘exosites’, away from the active site are likely to play roles in substrate
of some enzymes due to the kallikrin-like activity producing kinin. The recognition. Unfortunately, there is no reported high-resolution crystal
largest group of venom enzymes in this family is termed ‘thrombin-like’ structure of a fibrinogen-clotting enzyme; only crystallography data of
enzymes due to their activities, not the evolutionary origin. plasminogen activator
28
and protein C activator
29
are available.
Interestingly, the overall structures of these two are similar. Structure–
Thrombin plays pivotal roles in haemostasis.
17
The inactive precursor, function correlations of SVSPs require further study.
prothrombin, is composed of the N-terminal domain that is cleaved from
the C-terminal catalytic domain or thrombin upon activation. In contrast The properties of thrombin-like enzymes have been employed for
to factors VII, IX, X, which require their N-terminal domains and co- diagnostic and investigational uses. For example, venombin A and
factors (tissue factor, factor VIII and factor V, respectively) to form venombin B were used to define the contributions of fibrinopeptide A or
proteinase complexes on membrane, thrombin is released freely in B releases in fibrin polymerisation.
30
In addition, SVSP from B. atrox
solution to perform its myriad functions. The primary function of (reptilase time) can determine the causes of prolonged thrombin time
thrombin is the conversion of fibrinogen to fibrin by cleaving because the test is unaffected by heparin. The protein C and factor V
fibrinopeptide A and, subsequently, fibrinopeptide B from Aα and Bβ activators are used for activated protein C resistance and lupus
chains of fibrinogen, respectively. On the fibrin surface, thrombin anticoagulant tests, respectively. Furthermore, thrombin-like enzymes
activates factor XIII, resulting in fibrin cross-link. It also amplifies the have the potential to be therapeutic anticoagulants.
31
However, a large
positive feedback loops of coagulation by activating factors V, VIII and randomised trial revealed that ancrod is not better than placebo in the
XI, as well as platelet protease-activated receptor (PAR). The activation treatment of ischaemic stroke.
32
of PAR not only stimulates platelet aggregation, but also provides a
negatively charged surface, supporting blood coagulation. The Snake Venom Metalloproteases/Disintegrin
activation of factor XI and PAR is dependent on the ability of thrombin SVMPs are zinc-dependent multidomain proteins. The nascent peptides
to bind platelet glycoprotein (GP) Ib. Furthermore, in the presence of the contain a signal peptide and a pro-sequence that keeps the enzyme
co-factor thrombomodulin (TM), thrombin activates protein C and inactive. The mature secreted proteins from class P-I comprise only a
thrombin-activatable fibrinolysis inhibitor (TAFI) to inhibit coagulation metalloproteinase domain, while class P-II contains additional disintegrins
(via degradation of factors Va and VIIIa) and fibrinolysis (via digestion of and P-III class possesses disintegrin-like and CR domains. The disintegrin
plasminogen activator-binding sites on fibrin), respectively. Molecular domains are usually released from the mature proteins. Free disintegrins
mechanisms of the multifunctionality of thrombin have been under interfere with cell–matrix interaction via integrin binding, while attached
8 EUROPEAN HAEMATOLOGY
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