The organs and tissues that make up the human body rely on a continual

supply of blood, which is circulated throughout the body via a complex series of
arteries, veins, and capillaries. This blood not only carries vital oxygen and
nutrients to the tissues, as well as removing carbon dioxide and other waste
products, but it is also a vehicle for various blood cells, including important
clotting cells known as platelets .
Platelets are small blood cells produced by megakaryocytes. In the event of
blood vessel injury, these tiny blood cells are rapidly recruited to the area of
damage, where they effectively seal off the injured site to prevent blood loss.
This is achieved through the execution of a series of functional events
beginning with adhesion, followed by spreading and aggregation, leading
to thrombus (clot) formation.

Figure 1. Disruption of the endothelial lining of a blood vessel, due to injury (

), leads to exposure of subendothelial proteins (
). Platelets adhere
and spread over these subendothelial proteins, in an effort to seal off the site of
injury and prevent blood loss.
All platelet functional responses must be tightly regulated to ensure that the
formation a blood clot is of sufficient size to seal off the damaged area, whilst
not disrupting blood flow to vital organs by causing vessel occlusion (blockage).
Unfortunately, the consequences of abnormal platelet regulation are seen all
too frequently in the clinical setting, with the incidence of cardiovascular
related diseases such as heart attack and stroke, remaining some of the major
causes of death in the western world today.
Therefore, it is important to gain a
comprehensive understanding of the functional
responses executed by platelets that participate
in vessel wall maintenance, as this knowledge
may have important implications not only for our
understanding of existing disease states, but
also for the future development of novel
antithrombotic strategies.
Figure 2 . Excessive adhesion of platelets to a
diseased vessel leads to the blockage or
occlusion of the vessel, preventing blood flow
from travelling to vital organs and tissues.
Within the Platelet Signalling Unit, current research interests include:
Investigation of signalling enzymes that regulate platelet adhesive

function
Investigation of signalling factors that regulate blood clot consolidation
or "clot retraction"
Investigation of Signalling Enzymes thatRegulate Platelet Adhesive Function.

Many normal and pathological processes in the human body depend on the
ability of cells to attach firmly to a biological surface (adhesion). A classic
example of this is the adhesion of platelets to an injured blood vessel, in order
to prevent blood loss. Our laboratory is investigating the regulation of an
important attachment protein (or receptor), called integrin (Figure 1, ) which
is critical for platelet adhesion. This receptor is fundamental for the
physiological process of haemostasis, as evidenced by patients who suffer from
severe bleeding complications due to a lack of this receptor. Integrin aIIbb3 is
also implicated in the development of pathological thrombosis, and many
pharmaceutical companies have used this receptor as a candidate drug target
for treating thrombosis.
The adhesive properties of this major platelet integrin aIIbb3 must be tightly
regulated to ensure efficient haemostasis and avoid pathological thrombosis.
Recent evidence from our laboratory and others has implicated members of the
Ras family of small G-proteins, Rap1b and RhoA, as critical enzymes controlling
integrin adhesion. Our studies have demonstrated for the first time that the
activation of RhoA downstream of integrin aIIbb3 is responsible for maintaining
stable adhesion of platelets, particularly under conditions of blood flow. This
effect is achieved through modulation of integrin stability itself, rather than
regulation of the platelet interaction with vWf, or initial activation of the
integrin (Schoenwaelder et al, 2002). In further studies that are ongoing, we
are investigating preliminary evidence demonstrating the existence of distinct
yet cooperative roles for both RhoA, and another Ras family member, Rap1b, in
regulating integrin aIIbb3 adhesive function (Chrzanowska-Wodnicka et al, 2003;
manuscript submitted )
Related Publications

Schoenwaelder SM , Hughan SC, Boniface K, Fernando S, Holdsworth M,

Thompson PE, Salem HH, Jackson SP. RhoA sustains integrin aIIbb3 adhesion
contacts under high shear. J Biol Chem. 2002 277 (17):14738-46
http://www.jbc.org/cgi/content/abstract/277/17/14738
Chrzanowska-Wodnicka M, Schoenwaelder SM and White GC (2003) Rap1b
regulates Integrin aIIbb3 activity and platelet function lessons from a knockout.
Blood, 102(11):773a.

Investigation of signalling factors that regulateblood clot consolidation or

clot retraction.

One of the mechanisms utilised by platelets to regulate the size of the blood
clot is known as platelet-mediated clot retraction. This process describes the
ability of platelets to consolidate or shrink the size of the blood clot once it has
formed, through actomyosin contractile forces. This process is thought to be
important for both maintenance of the vasculature and also the subsequent
manner in which the blood clot is removed once wound healing has finished.
Although this process is of critical importance to the over maintenance of blood
vessels, our understanding of the signalling events regulating this process is
limited.
Our previous studies have highlighted important roles for several signalling
proteins in platelet mediated clot retraction, inlcuidng the Src family tyrosine
kinases (Schoenwaelder et al, 1994), and the protease calpain (Schoenwaelder
et al, 1997a, 1997b). We have recently set up a novel confocal-based imaging
assay in the laboratory to study the process of clot retraction, that allows us to
directly monitor platelet dynamics within a retracting blood clot, through the
use of fluorescence markers, monitoring both biochemical and physical
changes. By gaining more insight into the signalling mechanisms regulating
clot retraction and ultimately thrombus consolidation, it is hoped that these
studies will identify new targets for therapeutic intervention.
Figure 2. Following clot
formation, platelet within the
clot can bind to the fibrin
meshwork. Build-up of
contractile forces within the
cytoskeleton of the platelet are
then transmitted outside to
shrink the mass of the clot.

[Frontiers in Bioscience 10, 2504-2517, September 1, 2005]

PLATELET AGGREGATION: INVOLVEMENT OF THROMBIN AND

FIBRIN(OGEN)
Ton Lisman, Cees Weeterings, and Philip G. de Groot
Thrombosis and Haemostasis Laboratory, Department of Haematology, University
Medical Centre Utrecht, Utrecht, and Institute of Biomembranes, Utrecht University,
Utrecht, The Netherlands
FIGURES

Figure 1. Schematic representation of platelet adhesion and aggregation under

flow conditions. A) Rolling of platelets over collagen-bound vWF mediated by GPIb.
B) Firm attachment mediated by alpha(2)beta(1) and glycoprotein VI (GP VI)
binding to collagen, and by alpha(IIb)beta(3) binding to collagen-bound vWF. C)
Platelet activation, secretion, and spreading. D) Aggregate formation.

Figure 2. Thrombin-mediated activation of PARs in human platelets. Human

platelets express both PAR-1 and -4. Thrombin cleaves at the N-terminal
extracellular part of the receptor, thereby exposing a new N-terminus (SFLLRN and
GYPGQV, respectively), which binds to the body of the receptor, leading to Gprotein-coupled signal transduction.

Figure 3. Thrombin-mediated activation of PARs in mouse platelets. Mouse

platelets express PAR-3 and -4. PAR-3, however, is not cleaved by thrombin, but
rather binds thrombin with high affinity and serves as a docking molecule to present

thrombin to PAR-4, which is cleaved by thrombin. PAR-3 is necessary for PAR-4

activation at low concentrations of thrombin. At higher thrombin levels, PAR-4 can
be activated without the requirement for PAR-3.

Figure 4. Schematic representation of thrombin binding to GPIb(alpha). (A)

Thrombin binds to the tyrosine-sulphated region of GPIb(alpha) via its exosite II.
After binding to GPIb(alpha) thrombin is able to cleave GPV, resulting in the ability
to bind via exosite I to another GPIb molecule. The cleavage of GPV is necessary
for this clustering as the intact GPV most likely sterically hinders the interaction
between the two GPIb(alpha) molecules. From this point on the two crystal
structures of Celikel et al. and Dumas et al. suggest opposite consequences. (B)
Celikel et al. suggest clustering of GPIb(alpha) on the same platelet via thrombin,
which could subsequently lead to intracellular signaling. (C) Dumas et al. suggest
an adhesion mechanism, which involves the interaction of thrombin between two
GPIb(alpha) molecules on two different platelets, eventually resulting in possible
platelet aggregation.

Figure 5. Platelet aggregation independent of alpha(IIb)beta(3) via polymerizing

fibrin. In this experiment, the adhesion of platelets treated with an alpha(IIb)beta(3)
inhibitor to collagen was studied under flow conditions in absence (panel A) or
presence (B) of rFVIIa. In absence of thrombin generation via rFVIIa, perfusion of
alpha(IIb)beta(3)-inhibited blood over collagen resulted in adhesion of platelets, but
not platelet-platelet contacts or generation of fibrin was observed. In contrast, after
addition of rFVIIa, extensive fibrin deposition and the formation of platelet
aggregates was observed. This research was originally published in Blood. Ton
Lisman, Jelle Adelmeijer, Harry F. G. Heijnen, Philip G. de Groot, Recombinant
factor VIIa restores aggregation of alpha(IIb)beta(3)-deficient platelets via tissue
factor-independent fibrin generation (98) with permission form the American
Society of Hematology