C. Anastasopoulos et al.
Introduction
prothrombin to thrombin, which acts on fibrinogen to
generate a fibrin monomer, polymerized rapidly to form
fibrin clot.
Anticoagulant therapy is a mainstay in medical practice to
cope with the cardiovascular diseases (CVDs), such as
deep vein thrombosis, myocardial infarction, unstable
angina, pulmonary embolism and ischemic stroke. The
current anticoagulant therapies cover a range of Vitamin K
antagonists, heparins, direct thrombin or Factor Xa inhib-
itors. Although these drugs are effective, they have
numerous limitations. Most of them have a slow onset of
action, a variable dose requirement due to common genetic
polymorphisms (e.g., warfarin), food-drug or drug–drug
interactions, etc. Because of these issues, routine patient
monitoring is essential to ensure that a therapeutic anti-
coagulation response is obtained. Newer anticoagulants
have many potential advantages; however, there are some
potential disadvantages, particularly the lack of established
monitoring tests, therapeutic ranges and reversal agents
(Witt and Clark 2013; Maan et al. 2012). In addition, at
least some of the extended research studies in orthopedic
knee or hip surgery suggest that there will continue to be a
trade-off (Garcia et al. 2010). After all, it is clear that there
is a continuous requirement in targeting new directions to
succeed further development of new and safer antithrom-
botic drugs without the above limitations. These include
inhibitors of the factors VIIa, Xa, IXa, VIIIa and XII (Lin
et al. 2006; Minors 2007; Howard et al. 2007; Franchini
and Mannucci 2011; Baeriswyl et al. 2013).
Factor VIII is a multidomain glycoprotein responsible
for blood coagulation pathway. This is constituted of six
domains, which can be schematically represented as fol-
lowed: A1–A2–B–A3–C1–C2 (Vehar et al. 1984). FVIII
circulates in blood mainly as a heterodimer composed of a
heavy chain (A1–A2–B) and a light chain (A3–C1–C2)
complexed to Von Willebrand factor (vWF) associated
with a lipoprotein receptor-related protein (LRP) (Fay
2004). The main role of vWF is the engagement in other
proteins, particularly in factor VIII and consequently it is
important in the adherence of platelets at the area of
wounded vessels (Terraube et al. 2010). Factor VIII is
actually inactive as a cofactor in blood coagulation, but is
converted into its active cofactor form by proteolytic
cleavage (Fang et al. 2007). Among the FVIII six domains,
the A2 one is a key element that regulates its overall
function. In addition, the A2 domain is of primary impor-
tance in the cofactor function of FVIII, because it contains
FIXa binding sites (Fay et al. 1994; O’Brien et al. 1995;
Bajaj et al. 2001). The A2 subunit includes an epitope,
residues 558–565, which interacts with FIXa, residues
330–338, and it is likely the major catalytic interaction
between the FVIIIa and FIXa (Shen et al. 2008; Ngo et al.
2008; Jagannathan et al. 2009). Recently, Plantier et al.
(2012) provided a detailed map of the FVIII A2 domain
between residues 371 and 649, identifying residues crucial
for maintaining FVIII function and residues that can be
mutated without jeopardizing the coagulant activity of the
factor. Moreover, Griffiths et al. (2013) demonstrated a
previously unidentified high affinity interaction site
between the FVIIIa A2 subunit and FIXa, region 707–714.
We published recently (Anastasopoulos et al. 2013) that
small synthetic linear peptides, analogs of the sequence
558–565 of the A2 subunit, might be used as lead com-
pounds for novel anticoagulant drugs by inhibiting the
FVIIIa–FIXa interaction. Linear peptides that contain 2–10
residues, as in the case of 558–565 loop, are especially
flexible in solution. Their geometry and especially their
flexibility can be modified by the synthesis of cyclic pep-
tides using a wide variety of classical and novel chemical
methods (Perlman et al. 2005; Thakkar et al. 2013). Usu-
ally, their constrained structure results in higher receptor-
binding affinities as compared to their linear analogs.
Moreover, cyclization of peptides usually enhances their
resistance to enzymatic degradation by proteases, so that
could prolong their biological activity. Considering all the
above-mentioned remarks, we synthesized a series of
cyclic peptides (head to tail), analogs of the above
sequence of A2 aiming at preventing the interaction of
FVIIIa with FIXa, to suspend the process of blood
Haemostasis basics Upon vessel injury, the platelets
adhere to macromolecules in subendothelial tissues at the
site of injury and then aggregate to form the primary he-
mostatic plug. Coagulation, one of the most important host
defense mechanism, is a complex protease cascade
involving about 30 interacting proteins. The result is the
formation of thrombin, which catalyzes the conversion of
fibrinogen, a soluble protein, to insoluble fibrin strands,
which forms a stable clot in conjunction with platelets.
This cascade follows two biochemical pathways initiated
by either the exposure of tissue factor (TF) on the vessel
wall at the site of injury, ‘‘extrinsic pathway’’, or by the
activation of blood-borne components (FXII) by biologic
or foreign negatively charged surfaces, ‘‘intrinsic path-
way’’. Recently, new evidences (Gailani and Renne 2007;
Woodruff et al. 2010) indicate the important participation
of the intrinsic pathway in sustaining thrombus develop-
ment in vivo. A heterotrimer complex, called tenase, is
formed among FVIIIa, FIXa, Ca2? (which come from
activated platelets) and negatively charged phospholipids
of the cells membrane. Tenase is responsible for the con-
version of Factor X to the activated form FXa, another
protease that binds to cofactor Va to form a complex
known as prothrombinase. The latter complex converts
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