NEUTROPHIL-PLATELET INTERACTION
Johnny Nijm, MD; PhD
Specialist in Internal medicine, Cardiology & Clinical Physiology
Department of Medicine Diagnostic, Division of Clinical Physiology,
County Hospital Ryhov, Jönköping
&
Department of Medical and Health Sciences, Division of Cardiovascular
Medicine, Linköping University, Linköping, SWEDEN
NEUTROPHIL-PLATELET INTERACTION
Sanofi-aventis AB & Bristol-Myers Squibb

Neutrophils develop from the same early precursors
as Monocytes and Macrophages but in contrast to
these cells, they have a very short life time (1-2 days)

Neutrophils are the first inflammatory cells that
appear in intimal lesions

Neutrophils represent more than 50 % of the total
circulating Leukocytes and play a pivotal role in
innate immunity
Hematopoiesis
WHAT ARE WE DEALING WITH....
Thrombocyte
activation
Aktiverade
trombocyter
Skumceller
Monocyter
T-celler
Aktiverat
endotel
I nt im
a
M ed ia
Aktiverade
T-celler
Glatta muskelceller
AUTOIMMUNITY
AUTOANTIGEN
INFLAMMATION
TNF-α INF-γ
IL-4
Th1
IL-6
Th2
IL-10
IL-1
IL-2
ANTI INFLAMMATION

Neutrophils may contribute to plaque instability
and rupture by mediating their effects through:
 The
production of Proteases such as Cathapsins,
MMPs and Elastase
 Cytokines
like IL-1 beta, TNF-alpha, IL-8
 Generation
of Reactive Oxygen Species (ROS)
FURTHERMORE:

Neutrophil activation in peripheral blood may be
assessed by:
CD11b up-regulation
 Neutrophil-platelet aggregates
 Elastase, and Myeloperoxidase (MPO) release

Sanofi-aventis AB & Bristol-Myers Squibb
FACS plot showing the definition of neutrophil-platelet aggregates
CELL POPULATIONS AND NEUTROPHIL-PLATELET AGGREGATES
antal i medelvärde
Hundratal
diff,2
CD41a+CD11b+CD45+
** p < 0,01
50
45
* p = 0,5
* p < 0,05
**
600
**
** p < 0,001
40
*
500
35
400
30
Kontroller
25
300
SA
UA
20
200
15
100
*
10
376
541
483
0
5
0
Kontroller
Neutrofiler
Lymfocyter Monocyter
Eosinofiler
Basofiler
S1
SA
UA
Expression of CD11b and CD41a (MFI)
390
380
370
360
350
340
330
320
310
300
290
MFI CD41a
p = 0,001
**
500
325
331
379
Kontroll
SA
UA
Antal i medelvärde
Antal i medelvärde
MFI CD11b
400
300
200
100
0
370
Kontroll
383
249
SA
UA
Nijm J et al Am J Cardiol, 2005
NEUTROPHIL-PLATELET AGGREGATES CORRELATE TO
INFLAMMATORY MARKERS.
CRP
mg/ml
IL-6
pg/ml
IL-1Ra
pg/ml
IL-6
pg/ml
IL-1Ra
pg/ml
CD41a+CD11b+CD45+
cells/mm³
p<0,001
(r = 0,6)
p<0,001
(r = 0,4)
p<0,01
(r = 0,3)
p<0,0001
(r = 0,5)
p<0,01
(r = 0,3)
p<0,05
(r = 0,2)

The number of neutrophils was significantly higher
in the ACS patients compared to patients with
stable disease

However, the number of circulating neutrophilplatelet aggregates tended to be lower in ACS
patients compared to stable CAD patients

The expression of CD41a was significantly lower in
the ACS group compared to the stable group,
(probably due to the clopidogrel therapy).

Jochen Graff et al,
Clinical Pharmacology & Therapeutics, 2005
showed that Clopidogrel inhibits the expression of
platelet activation markers and leukocyte-platelets
interaction
Sanofi-aventis AB & Bristol-Myers Squibb
THE INTERACTION BETWEEN NEUTROPHILS AND
PLATELETS IS MAINLY MEDIATED BY:

P-selectin: on the surface of activated endothelial
cells and platelets

E-selectin: on the surface proteins of certain
leukocytes (monocytes, granulocytes and Tlymphocytes)

L-selectin: on leukocytes, acting as “homing
receptor” for leukocytes to enter secondary
lymphoid tissues

Beta I (CD11b/CD18) and beta II (CD41/CD61)
Integrins regulate neutrophil transmigration
across the vascular endothelium.

Integrins exists in different conformations, from a
low-affinity to a high-affinity state.
THE AFFINITY STATE OF THE BETA2-INTEGRINS BEFORE
AND AFTER STIMULATION EX VIVO
Takagi J and Springer TA, Immunol. Rev. 2002

There was no activation of neutrophils in patients with
stable CAD, expressed as CD18 or high affinity CD11b.
Särndahl E, Nijm J et al, PLoS ONE 2007
The ROS production was reduced in neutrophils from patients
with stable CAD.
Särndahl E, Nijm J et al, PLoS ONE 2007
In conclusion:

The number of neutrophil-platelet aggregates was higher in patients with stable CAD
compared to healthy controls, suggesting a “primed” state of circulating neutrophils
and increased thrombus susceptibility

Neutrophils were not more activated in vivo than were cells in healthy controls,
neither were the neutrophils in patients more prone to activation ex vivo

In contrast, we obtained evidence for an impaired activation state in the patients, as
assessed by decreased ROS production which may rather indicate an “exhausted” or
“frustrated” neutrophils

The clinical relevance of neutrophil dysfunction in CAD remains to be elucidated. Is it
a marker of chronic inflammatory disease or a factor with impact on the progress of
CAD ? Ongoing studies on neutrophils from ACS patients might bring more light on
their role in atherosclerosis
However , a recent study, by Jönsson S et al, using IL-8 as a physiological activator,
showed that MMP-9 release by neutrophils was significantly increased in stable CAD
patients compared with controls, a result that might indicate neutrophil activation. At
the same time the release of MMP-9, after a maximal stimulation with PMA, tended
to be lower, a fact that might suggest an “exhausted” state of neutrophils
Neutrophil release of MMP-9
400
300
200
100
IL
-8
C
on
tr
ol
nt
s
IL
-8
P
at
ie
on
tr
ol
C
at
ie
nt
s
0
P
MMP-9 (ng/ml)

Jönsson S et al, ESC 2010
Thank you for your attention
ADP-Stimulated trombocyts.
Sanofi-aventis AB & Bristol-Myers Squibb

The expression of annexin-1 was increased in
neutrophils from patients with stable CAD.
Särndahl E, Nijm J et al, Metabolism 2010
Matrixmetaloprotases, MMPs
Group
Name
MMP
Substrate
Collagenases
Fibroblast
Neutrophil
Collagenase-3
Collagenase-4
MMP-1
MMP-8
MMP-13
MMP-18
fibrillar collagen
”
”
”
Gelatinases
Gelatinase A
Gelatinase B
MMP-2
MMP-9
Gelatin, collagen IV, fibronectin, elastin, laminin
Gelatin, elastin, fibronectin, vitronectin
stromelysins
Stromelysin 1
Stromelysin 2
Stromelysin 3
MMP-3
MMP-10
MMP-11
Gelatine, fibronectin, casein, laminin, elastin, MMP2/TIMP-2
“
Fibronectin, laminin, gelatine, aggrecan
Matrilysins
Matrilysin 1
Matrilysin 2
MMP-7
MMP-26
Fibronectin, vitronectin, laminin, gelatine, aggrecan
Collagen IV, gelatine,
fibronectin, fibrin, fibrinogen, type 1 gelatine
Elastase
Metalloelastase
MMP-12
Elastin, gelatine, collagen IV, fibronectin, laminin,
vitronectin, Proteoglycan
Membrane Type
MT1- MMP
MT2- MMP
MT3- MMP
MT4 – MMP
MT5- MMP
MT6- MMP
MMP-14
MMP-15
MMP-16
MMP-17
MMP-24
MMP-25
Pro MMP-2, procollagenase 3
Pro MMP-2
“
Gelatine, TNF-α precursor, fibrin
Collagen IV, gelatine, laminin
MMP-19
MMP-20
MMP-22
MMP-23
MMP-27
MMP-28
Collagen IV, gelatine, laminin, nidogen, tenacin,
fibronectin, aggrecan
Amelogenin
Synthetic MMP substrate
Other MMPs
Enamelysin
Cluster of differentiation (CD)
CD antigen
Alternate name
Function
Cellular expression in blood
CD3
Signalling component of T cells, part of a bigger
complex which includes the T cell receptor.
All T cells
CD4
Co-receptor for MHC class II molecules.
T helper cells,
monocytes (weak)
CD8
Co-receptor for MHC class I molecules.
Cytotoxic T cells,
subset of NK cells
CD14
High-affinity receptor for lipopolysacharide.
Monocytes
CD19
B cell surface antigen Involved in the regulation of B cells. Forms complex B cells
B4
with complement receptor 2.
CD25
IL-2R a-chain
In conjunction with IL-2Rb- and IL-2Rg, the CD25
antigen forms the high-affinity IL-2R complex.
Activated T and B cells.
CD45
Leukocyte common
antigen
Tyrosine phosphate, mediating signalling through B
and T cell receptors
All differentiated haematopoietic cells
except erythrocytes and plasma cells
CD62L
L-selectin, LAM1
Mediates rolling interactions between leukocytes and
endothelium
B cells, T cells, monocytes, NK cells
and granulocytes
CD89
CD11b
FcaR
Mac-1 a chain,
complement receptor
3
Neutrophil Fc receptor for IgA
Subunit of b2-integrin, associated with CD18.
Neutrophils, monocytes
Granulocytes, monocytes, NK cells,
subsets of T and B cells
Conformation to the high-affinity state of b2-integrin
As above (activated)
CD11b
I-domain
CD18
Mac-1 b chain
The second subunit of the b2-integrin, associated
with CD11b
Granulocytes, monocytes, NK cells,
subsets of T and B cells
CD41a
Glycoprotein IIb
Part of the glycoproteinIIb/IIIa complex which is the
soluble receptor for fibrinogen
Platelets

Platelets plays a major role in activating
neutrophils and endothelial cells via cytokines,
MMPs, TNF acting by CD40/CD40L

Platelets secrete neutrophil and endothelial
activators inducing production of the
inflammatory cytokines found on APC and is
required for their activation

The binding of CD40L on Th cells to CD40
activates APC and induces a variety of
downstream effects
Monocytes-Platelet interaction befor and after stimulation with TRA
Lindmark et al, Arterioscler Thromb Vasc Biol 2000
INFECTION AS AN ACTIVATOR OF THE IMMUNE SYSTEM, BOTH
IN A CHRONIC OR IN AN ACUTE MATTER

CMV, EB, Chlamydia

There is some data that links Adenovirus infection (subgroup) and
the bacterial flora found in the colon with obesities (as a chronic
inflammation in fat tissue)

Other authors speculate of the possibility of a chronic Coxaccii
virus B4 and the autoimmunity found in patients with type I
diabetes

There is documented data that showed a relationship between
helicobacter pylori infection (after a non successful eradication)
and a new onset of ACS during 6-12 months
WHAT ABOUT MEDICATION…!

There is data indicating that neutrophil-activating peptid-2 (NAP-2) activates
both EC and leukocytes and could result in plaque rupture

ASA-treatment results in decreased NAP-2 activity but do not influence CRP
levels, whereas ACE-I does. Statin-treatment increase NAP-2 activity

A study by Jochen Graff et al, Clinical Pharmacology & Therapeutics, 2005
showed that Clopedogrel inhibits the expression of platelet activation markers
and the leukocyte-platelets interaction

A possible explanation of these results is that the synthesis of vascular disease
markers and inflammatory products such as sCD40L and MMP-9 has been
inhibited, and anti-inflammatory properties of Clopedogrel are likely to be a
result of decreasing platelet activation