1 Platelets role in the interaction between coagulation and inflammation
after myocardial infarction.
Felipe Donoso. Medicine Programme, Uppsala University.
Introduction
Even though major advances have been made in treatment and the secondary prophylaxis of cardiovascular disease
1; the illness is still the predominant cause of mortality and morbidity worldwide
2and coronary atherosclerosis is its major cause
3.
The development of atherosclerotic plaques is a long process that is driven by inflammation and culminates with plaque rupture, activation of the coagulation system and thrombus formation. It can be divided in 3 stages:
-initiation: in susceptible areas, as bifurcations and branching points, due to hemodynamic forces there is a rearrangement of the intima with subendothelial smooth muscle accumulation and endothelial dysfunction. This lead to deposition of lipids by low-density lipoproteins (LDL) into the intima and extravasation of monocytes/macrophages to the lesion; which in turn stimulate to further lipid deposition, accumulation of smooth muscle and rearrangement of the extracellular matrix. Oxidized LDL induces tissue damage, macrophage extravasation and inflammatory response. As the lesion growths necrosis is developed in the deepest part of the intima and the plaque is formed with a fibrous cap and a necrotic lipid core.
- adaptation: As the plaque growths remodeling occurs in the vessel to maintain the lumen size, but when the plaque occludes 50% of the lumen or more, no further remodeling can compensate for the loss of lumen size.
- clinical: the plaque has developed ulcerations, calcifications, a great inflammatory response and maybe aneurysm formation. All of these can lead to stenosis or plaque rupture with activation of the coagulation system, thrombus formation and occlusion of the vessel lumen with subsequent myocardial infarction
4.
The process of thrombus formation involves the coagulation system and platelets. Upon exposure of subendothelial collagen, platelets activate and degranulate. Subendothelial cells express tissue factor (TF). The exposed TF form complexes with circulating activated factor VII (FVIIa) and cleaves factor X to Xa and IX to IXa. These activated factors bind to factor Va and VIIIa respectively on the surface of activated platelets. The FIXa/VIIIa complex generates more Xa. And the Xa/Va complex cleaves prothrombin to thrombin which in turn activates platelets and the factors VII, XI and V. Thrombin participates directly in the coagulation cascade by cleaving fibrinogen to fibrin which stabilizes the plug of platelet aggregates
5.
Platelet activation
Activation of platelets is crucial in the process of thrombus formation. Platelets contribute to coagulation by providing an appropriate surface for the coagulation cascade to take place.
They amplify the coagulation process trough activation of other platelets, stimulation of the
inflammatory response and stimulation of intravascular TF expression by de novo synthesis
2
and complex formation with leukocytes. Platelets are also directly involved in thrombus formation trough interactions with other platelets and fibrinogen
5-7.
There are several “pathways” for platelet activation. The following is an illustrative model of the events leading to vessel occlusion and myocardial infarction, in conditions of shear stress due to diminished lumen size.
After plaque rupture the subendothelial tissue is exposed. Von Willebrand factor (VWF) binds to collagen and mediates a reversible binding to circulating platelets by the platelet receptor glycoprotein (GP) Ib which enables recruitment of platelets, rolling and interactions between collagen and platelet’s GPVI, the major activating platelet collagen receptor. By intracellular signaling through the PKC-pathway, the content of dense and alpha granulae (i.e. adenosine diphosphate (ADP), calcium, adenosine triphosphate (ATP) and coagulation/inflammatory factors respectively) are released. Negatively charged phospholipids are exposed in the
cellular surface, which provides a platform for the coagulation cascade. Furthermore, platelets release thromboxane A2 (TXA2).
ADP, TXA2 (released by platelets) and thrombin (formed by the coagulation cascade) induce up-regulation and activation of GPIIb/IIIa- in platelets- via G-protein coupled receptors P2Y1, P2Y12 and PAR-1, PAR-2 respectively. GPIIb/IIIa mediates the irreversible binding of platelets to collagen and to other platelets trough fibrinogen
8.
The activated platelet undergoes conformational changes and acquires several new functions due to the expression of different receptors. The pathway by which platelets are activated influence their expression of surface proteins and the release of different factors. For instance, it has been shown that platelets stimulated with thrombin and collagen respond with high levels of procoagulant proteins on their surface
7.
Platelet-leukocyte aggregates
In addition to homotypic cell-cell interactions between platelets, as describes above, the activated platelets can establish heterotypic cell-cell interaction with monocytes and granulocytes through P-selectin (CD62P). P-selectin becomes up-regulated from alpha
granules after platelet activation and is expressed for a prolonged period of time. Another way to establish these interactions is through CD40-CD40-ligand interactions and GPIIb/IIIa- fibrinogen-MAC1- interactions. CD40-ligand is expressed on platelet surfaces upon activation and CD40 is expressed by B-lymphocytes, monocytes and endothelial cells
9.
Of particular interest is the formation of platelet-monocyte aggregates (PMA) which has been shown to be elevated in patients with cardiovascular morbidity
7,9,10. Upon p-selectin exposure the activated platelet secretes RANTES, a chemoattractant for monocytes. The combination of stimuli from RANTES and the interaction with platelets trough p-selectin/PSGL-1 (P-selectin glycoprotein ligand-1 on monocytes) activates monocytes, induces the production of
proinflammatory mediators (such as IL-8, IL-1β, matrix metalloproteinases, MIP1β, MCP-1), induces TF expression in monocytes, enhances phagocytosis, superoxide anion production and PAF secretion
7,11-14.
PMA can be reinforced by GPIIb/IIIa-fibrinogen-MAC1- interactions
7.
3
Intracellular signaling pathways
Even though the effects of p-selectin/PSGL-1 interactions in monocytes are well understood, the intracellular pathways leading to these effects are not.
Evangelista et al showed that PSGL-1 functioned as a signaling molecule
15and that Src- family kinases (SFK) are involved in intracellular signaling in granulocytes
16. Christersson et al were able to present evidence that links the phosphorylation of Lyn (a SFK member) to IL8 and TF production
13. Further steps in the pathway are not elucidated but it is presumed that mitogen-activated protein (MAP) kinases are involved as in endothelial cells
5.
Blood borne TF
TF is the main initiator of coagulation in vivo. TF is constitutively expressed in cells outside the vascular system. However, intravascular TF-expression with coagulatory activity have been described and investigated for more than a decade.
The source of this blood-borne TF has long been attributed exclusively to monocytes that synthesize and express an inactive or “encrypted” form of TF that becomes active upon interactions with platelets or neutrophils. This idea has been reconsidered due to the
presentation of evidence that support TF expression by endothelial cells after activation, TF synthesis and expression by activated platelets and TF expression on monocyte and platelet derived microparticles
6,17.
Granulocytes also express TF on their surface but it has been shown that this TF is not
synthesized by the granulocytes instead it has been acquired from circulating monocytes
6,17. The role of blood borne TF in cardiovascular disease is debatable, but the main stream of thoughts is that it contributes to the expansion of the coagulation process
5,6.
Treatment of myocardial infarction
Because of platelets central role in thrombus formation they have been the main target for treatment and prophylaxis of myocardial infarction. The acute anti-thrombotic treatment with effects on platelets consists of acetylsalicylic acid (cyklooxygenase inhibition), clopidogrel (P2Y
12antagonist) and tirofiban or eptifibatide (glycoprotein IIb/IIIa-inhibitors).
The coagulation cascade is also a target for anti-thrombotic treatment with low molecular weight heparin (as dalteparin).
Other aspects of myocardial infarction are also treated in the acute situation, as:
- Pain and anxiety relief with morphine and diazepam.
- Anti-ischemic treatment with oxygen, nitroglycerin and β-blockers (as metoprolol) or calcium channel blockers (as diltiazem).
- Reperfusion treatment with percutaneous coronary intervention, thrombolysis (tenecteplase/alteplase) or coronary arterial bypass grafting
18.
The secondary prophylaxis against myocardial infarction consists of: acetylsalicylic acid,
clopidogrel, β-blockers, angiotensin-converting enzyme inhibitors, lipid-lowering with statins
and nitroglycerin. This treatment is initiated prior patient discharge from hospital
18.
4
The aim of this thesis was to:
- Understand the importance of platelets and their interaction with other cell types in acute myocardial infarction, i.e. the interaction between coagulation and inflammation. This was performed by analyzing the interactions between platelets and monocytes/granulocytes and their TF expression through flow cytometry methods.
- Perform experiments to distinguish intracellular signal pathways in monocytes activated by p-selectin.
Materials and methods
The analysis of platelets and monocytes/granulocytes interactions and their TF expression was performed by flow cytometry methods in which leukocytes was distinguished from other blood components by isotope-labeled monoclonal antibodies against CD45. By plotting the CD45+ population and cellular complexity (obtained by side scattering) in a 2D dot plot different cell populations emerged (Fig.1). By gating the monocytes or granulocytes other parameters were analyzed with other isotope-labeled monoclonal antibodies, as their cell surface expression of TF, p-selectin (CD62P) and CD41 (Fig.2). The last two are specific for platelets and were interpreted as PMA or PGA (platelet-granulocyte aggregates).
Figure 1 Differentiation of leukocytes (CD45+ cells) by cellular complexity.
Modified from http://probes.invitrogen.com/resources/education/tutorials/4Intro_Flow/player.html Granulocytes
s
Monocytes
Lymphocytes CD45+ cells
Cellular complexity
5
Figure 2 Example of an analysis of CD45+ cells regarding their expression of TF and CD62P on their cell surface.
Modified from http://probes.invitrogen.com/resources/education/tutorials/5Data_Analysis/player.html
Groups analyzed
I performed a series of analyses in a group of healthy individuals which consisted on a total of 5 people, 3 men and 2 women. A single sample was collected from those individuals.
The patient group consisted of 16 subjects included in REBUS. 10 of these subjects were followed up with 3 serial samples, 5 with 2 serial samples and for 1 subject only 1 sample was collected. REBUS is an ongoing observational study performed at the Department of
Cardiology, Uppsala University Hospital, in which an unselected patient population with resent myocardial infarction is followed during 2 years. These individuals have been treated for myocardial infarction as described above and were taking medication included in
secondary prophylaxis at sampling collection.
Flow cytometry analyses
Whole blood was collected one time from healthy individuals and three times for patients; 3-5 days (visit 0), 2-3 weeks (visit 1) and 3 months (visit 2) after the cardiac event. The collection was performed with a 21G needle at no stasis to BD Vacutainer® 3.8% citrate tubes.
Collected blood was analyzed within 30 minutes. The analysis was performed by
spectrophotometry using a Cytomics FC500 (Beckman Coulter, Fullerton, CA), anti-CD45
ECD(Beckman Coulter, Fullerton, CA) antibodies (Ab), anti-CD62P
PEAb(AbD serotec), anti- CD41
PC7Ab (Beckman Coulter, Fullerton, CA), anti-TF
FITCAb (American diagnostic Inc., Stanford) and anti-IgG1 labeled with PE, FITC and PC7.
For each sample a control was establish using irrelevant Ab labeled with FITC, PE and PC7 and the biomarkers were analyzed in unstimulated whole blood, whole blood stimulated with 20µM ADP and whole blood stimulated with 20µmM thrombin receptor activator peptide (TRAP). The stimulated or unstimulated blood was incubated with the Ab and Hepes for 20 minutes at room temperature prior flow cytometry analysis.
CD62P+: 27,6% CD62P+/TF+: 2%
TF+: 68%
CD62P-/TF-: 2,4%
Flourescence TF
Flourescence CD62P
CD45 CD62P TF
6
A minimum of 5000 leukocytes were analyzed and 2% of the control was considered positive.
The leukocytes were gated by CD45 expression and then divided in subpopulations of lymphocytes, monocytes and granulocytes by side scattering.
In the healthy group, the amount of platelet-leukocyte aggregates was established by the presence of CD41 (GpIIb) on the surface of the subpopulations of leukocytes.
In the patient group, TF expression was analyzed using the appropriate Ab. The amount of platelet-leukocyte aggregates was established by the presence of CD62P (p-selectin) and CD41 (GpIIb) on the surface of the subpopulations of leukocytes.
Experiment on monocytes
36 ml of whole blood was collected from three individuals using the same methodology as described above into Terumo Venoject
®Lithium-Heparin tubes. In order to purify monocytes platelet rich plasma was extracted from the blood, centrifuged and then the platelet poor plasma (PPP) was reincorporated to the blood. The PPP-blood was incubated for 20 min at room temperature with RosetteSep® Human Monocyte Enrichment Cocktail (Stemcell technologies). RosetteSep® consists on antibodies that link erythrocytes to all the cellular components of blood but monocytes. The monocytes were separated from the erythrocytes with Ficoll-Paque® (GE Healthcare) and then washed a couple of times. The purified
monocytes were resuspended in RPMI 1640 with 5% FBS and 1% glutamine and incubated at 37ºC for one hour. The cells were then divided in 3 propropylene tubes. The monocytes in two of the tubes were stimulated with rh-P-selectin at 150 ng/ml or 250 ng/ml. All the tubes containing monocytes were incubated at 37ºC for 30 min. then the cells were lysed and the amount of ERK, p38 and jnk in both native and phosphorylated state was analyzed by western blotting. The antibodies used for this purposes are anti erk, anti phosphorylated erk, anti p38, anti phosphorylated p38 and anti phosphorylated jnk.
Statistics
In the in vitro whole blood experiments the amount of cells expressing the different
parameters were normalized to a 2000-cells population (amount of cells/2000). Descriptive statistical analyses were made on this values and the results are presented as median (25
thpercentile- 75
thpercentile) if not state otherwise. To determine statistical significance within and between groups the Kruskal-Wallis test was used. For more specific analysis Wilcoxon matched-pairs signed rank test was performed. The statistics software used was GraphPad Prism 5
®. P-values are summarized as: ns (p>0,05), S* (0,01-0,05), S**(0,001-0,01) and S***
(p<0,001).
For the monocytes experiments, no statistical analysis was performed.
Results
Healthy group
In the group of healthy individuals I found that PMA and PGA increased after ADP and
TRAP stimulation from 136/2000 monocytes (72-285) to 515/2000 monocytes (226-1329)
and 407/2000 monocytes (335-1151) for PMA; and from 952/2000 granulocytes (824-1245)
to 1396/2000 granulocytes (1204-1623) and 1628/2000 granulocytes (1478-1717) for PGA.
7
The amount of PGA in healthy individuals was higher both in unstimulated blood and in blood stimulated with either ADP or TRAP compared to PMA.
No difference in the amount of PMA after TRAP stimulation was observed compared to ADP stimulated blood (Fig.3-4).
None of these observations were statistical significant.
Figure 3 Amount of PMA (CD41)/ 2000 monocytes in unstimulated blood and stimulated with ADP or TRAP
Figure 4 Amount of PMA (CD41)/ 2000 granulocytes in unstimulated blood and stimulated with ADP or TRAP
Patient’s group
As described above, the patient group consisted of 16 subjects treated for acute myocardial infarction at the Department of Cardiology, Uppsala University Hospital. 3 of these
individuals were women and 13 were men. 8 had suffered a ST elevation myocardial infarction and 8 have had a non ST elevation myocardial infarction. The mean age for the group was 66,6 (54-79) and the median 64,5 (62,75-71,25) years.
For one individual under visit 0 no parameters were analyzed due to technical problems with the flow cytometer.
Monocytes and PMA
The amount of monocytes expressing TF in unstimulated blood from patients that had
suffered a myocardial infarction the last 3-5 days was 313/2000 monocytes (269-470) and did not change over time. After ADP stimulation the amount of monocytes expressing TF
increased to 869/2000 monocytes (707-1194). This increase was statistical significant (S**) but no variation was seen over time. After TRAP stimulation the amount of monocytes expressing TF further increased to 1304/2000 monocytes (955-1436). This increase was S***
compared to the unstimulated blood but ns to ADP stimulated blood. No variation over time was seen (Fig.5).
The amount of PMA expressing CD62P in the unstimulated blood at visit 0 was 95/2000 monocytes (78-100), no variation over time was seen. After ADP-stimulation the amount of PMA CD62P increased to 258/2000 monocytes (189-626). This increase was S**. Even thou
0 500 1000 1500 2000
ctrl unstim ADP TRAP
PMA CD41
Mean
0 500 1000 1500 2000
ctrl unstim ADP TRAP
PGA CD41
Mean
8
it seemed that the amount of PMA after ADP-stimulation decreased in visit 1 and 2, these differences were ns (Fig.6).
Upon TRAP-stimulation the amount of PMA CD62P increased to 1036/2000 monocytes (811-1358). This increase was S*** compared to the unstimulated blood but ns to ADP stimulated blood. No variation over time was seen (Fig.6).
The amount of PMA expressing CD41 in the unstimulated blood at visit 0 was 136/2000 monocytes (101-145), no variation over time was seen. After ADP-stimulation the amount of PMA CD41 increased to 672/2000 monocytes (326-1018). This increase was S**. Even thou it seemed that the amount of PMA CD41 after ADP-stimulation decreased in visit 1 and 2, these differences were ns (Fig.7).
Upon TRAP-stimulation the amount of PMA CD41 increased to 1167/2000 monocytes (862- 1396). This increase was S*** compared to the unstimulated blood but ns to ADP stimulated blood. No variation over time was seen (Fig.7).
It seemed that there was an important part of the monocytes that expressed TF that did not form complexes with platelets, particularly in the unstimulated blood (Fig.5-8).
Figure 5 Amount of TF expressing monocytes/ 2000
monocytes in unstimulated blood and stimulated with ADP or TRAP at visit 0, 1 and 2.
Figure 6 Amount of PMA (CD62P)/ 2000 monocytes in unstimulated blood and stimulated with ADP or TRAP at visit 0, 1 and 2.
0 500 1000 1500 2000
unstim 0 ADP 0 TRAP 0 unstim 1 ADP 1 TRAP 1 unstim 2 ADP 2 TRAP 2
/2000 cells
Monocytes expressing TF
Mean
0 500 1000 1500 2000
unstim 0 ADP 0 TRAP 0 unstim 1 ADP 1 TRAP 1 unstim 2 ADP 2 TRAP 2
/2000 cells
PMA (CD62P)
Mean
9
Figure 7 Amount of PMA (CD41)/ 2000 monocytes in unstimulated blood and stimulated with ADP or TRAP at visit 0, 1 and 2.
Figure 8 Percentage of monocytes in PMA calculated by CD62P.
The proportion of PMA CD62P, in unstimulated blood, that expressed TF was 81% (78-88).
Upon stimulation with ADP 94% (89-97) of PMA CD62P expressed TF. This increase was S* in visit 0 and ns in visit 1 and 2.
After TRAP-stimulation 95% (90-97) of PMA CD62P expressed TF. This increase was S**
in all visits compared to unstimulated blood but ns compared to ADP-stimulated blood (Fig.9).
When the same analysis was performed in PMA CD41 50% (43-55) expressed TF in
unstimulated blood (fig.10). The difference in TF expression between PMA CD41 and PMA CD62P in unstimulated blood was S*** (Fig.9-10).
Upon stimulation with ADP 87% (77-93) of PMA CD41 expressed TF. This increase was S**
in visit 0 and 1, but ns in visit 2.
After TRAP-stimulation 91% (88-94) of PMA CD41 expressed TF. This increase was S** or S*** in all visits compared to unstimulated blood but ns compared to ADP-stimulated blood (Fig.10).
0 500 1000 1500 2000
unstim 0 ADP 0 TRAP 0 unstim 1 ADP 1 TRAP 1 unstim 2 ADP 2 TRAP 2
/2000 cells
PMA (CD41)
Mean
0%
20%
40%
60%
80%
100%
% Monocytes in PMA (CD62P)
Mean
10
Figure 9 Percentage PMA (CD62P) that express TF Figure 10 Percentage PMA (CD41) that express TF
Granulocytes and PGA
The amount of granulocytes that expressed TF in unstimulated blood from patients that had suffered a myocardial infarction was 201/2000 granulocytes (160-224), 3-5 days after the cardiac event.
Upon ADP-stimulation the amount increased to 269/2000 granulocytes (233-370). After TRAP-stimulation the amount of granulocytes expressing TF was 400/2000 granulocytes (250-489). In the measurements performed 2 weeks and 3 months after the myocardial infarction, the increment in TF-expressing granulocytes was more discrete. None of these comparisons were statistical significant (Fig.11).
The amount of PGA expressing CD62P in the unstimulated blood at visit 0 was 84/2000 granulocytes (68-112), no variation over time was seen. After ADP-stimulation the amount of PGA increased to 245/2000 granulocytes (194-444). This increase was S***. In visit 1 the increase was seen (S*) but in visit 2, even thou it seemed to be an increase in PGA CD62P, it was ns.
Upon TRAP-stimulation the amount of PGA CD62P increased to 477/2000 granulocytes (398-722). This increase was S*** compared to the unstimulated blood but ns to ADP stimulated blood. No variation over time was seen (Fig.12).
About 5% of granulocytes that expressed TF were not in complexes with platelets in the unstimulated blood (Fig.11-14).
0%
20%
40%
60%
80%
100%
% PMA (CD62P) expressing TF
Mean
0%
20%
40%
60%
80%
100%
% PMA (CD41) expressing TF
Mean
11
Figure 11 Amount of TF expressing granulocytes/ 2000 granulocytes in unstimulated blood and stimulated with ADP or TRAP at visit 0, 1 and 2.
Figure 13 Amount of PGA (CD41)/ 2000 granulocytes in unstimulated blood and blood stimulated with ADP or TRAP at visit 0, 1 and 2.
Figure 12 Amount of PGA (CD62P)/ 2000 granulocytes in unstimulated blood and stimulated with ADP or TRAP at visit 0, 1 and 2.
Figure 14 Percentage of granulocytes in PGA calculated by CD62P.
The proportion of PGA CD62P that expressed TF in unstimulated blood was 65% (51-71).
Upon ADP-stimulation 43% (35-47) of PGA CD62P expressed TF (S**). No variation over time was observed.
After TRAP-stimulation 38% (27-50) of PGA CD62P expressed TF. This is S*** compared to unstimulated blood but ns to ADP-stimulated blood. No variation was observed over time (Fig.15).
0 500 1000 1500 2000
unstim 0 ADP 0 TRAP 0 unstim 1 ADP 1 TRAP 1 unstim 2 ADP 2 TRAP 2
/2000 cells
Granulocytes expressing TF
Mean
0 500 1000 1500 2000
unstim 0 ADP 0 TRAP 0 unstim 1 ADP 1 TRAP 1 unstim 2 ADP 2 TRAP 2
/2000 cells
PGA (CD41)
Mean
0 500 1000 1500 2000
unstim 0 ADP 0 TRAP 0 unstim 1 ADP 1 TRAP 1 unstim 2 ADP 2 TRAP 2
/2000 cells
PGA (CD62P)
Mean
0%
20%
40%
60%
80%
100%
% Granulocytes in PGA (CD62)
Mean
12
When PGA CD41 was analyzed in unstimulated blood, 34% (30-38) of PGA CD41 expressed TF. No variation in the percentage of PGA CD41 expressing TF was observed upon
stimulation with neither ADP nor TRAP. No variation was observed over time (Fig.16).
Figure 15 Percentage PGA (CD62P) that express TF Figure 16 Percentage PGA (CD41) that express TF
PMA vs PGA
The amount of monocytes that expressed TF was nearly twice the amount of granulocytes.
The granulocytes expressed TF at first while the monocytes expressed TF over the 3 month period (Fig.5 and 11).
The amount of PMA and PGA was similar in unstimulated blood, but upon stimulation about twice as much PMA was formed. The amount of PMA expressing TF was higher than PGA expressing TF (Fig.6-8 and 12-14).
Cell signaling
For the monocyte experiment, blood samples were taken from 3 healthy individuals. The results presented correspond to one of those individuals. The analysis of the other two faild. In one case no flourescens were detected probably due to lack of cellular material, in the other case the purification of monocytes failed (they were contaminated with erytrocytes).
The control sample was considered as 100% flourescens. Upon stimulation with 150 ng/ml rh p-selectin p-erk flouresced 75% of control, p-p38 43% and p-jnk 24%. After stimulation with 250 ng/ml rh p-selectin p-erk flouresced 75%, p-p38 28% and p-jnk 16% (Fig.17).
0%
20%
40%
60%
80%
100%
% PGA (CD62P) expressing TF
Mean
0%
20%
40%
60%
80%
100%
unstim 0 ADP 0 TRAP 0 unstim 1 ADP 1 TRAP 1 unstim 2 ADP 2 TRAP 2
% PGA (CD41) expressing TF
Mean
13
Figure 17 Percentage of phosphorylated erk, p38 and jnk in relation to total erk.
Discussion
Platelets have a central role in thrombus formation. They contribute to coagulation by providing an appropriate surface for the coagulation cascade to take place, amplifying the coagulation process trough activation of other platelets, stimulation of the inflammatory response and stimulation of intravascular TF expression by de novo synthesis and complex formation with leukocytes. Platelets are also directly involved in thrombus formation trough interactions with other platelets and fibrinogen.
All this together imply that platelets are able to sustain the growth of a thrombus
6, creating a favorable microenvironment for the coagulation process and linking it to inflammation at the site of injury. This key role has made the platelets a perfect target for pharmacological
treatment of acute coronary syndrome. Several agents, as acetylsalicylic acid, clopidogrel and tirofiban or eptifibatide, are used to prevent platelet activation or interaction with other cells.
Despite these major advances myocardial infarction is still the predominant cause of mortality and morbidity worldwide
2. This means that more understanding of the pathophysiological process and new therapeutical approaches that consider interpersonal variation are needed to be able to manage this disease.
The results presented in this thesis have to be interpreted as indications of the made
observation regardless of their statistical significance. Mainly because of the small groups that were analyzed.
The responses to ADP and TRAP stimulation between the healthy and patient group are very different in the type of platelet-leukocyte aggregates that are preferentially formed. In the healthy group PGA is formed in a wider extent than PMA while the opposite is true for the patient group (Fig.3, 4, 6, 7, 12, 13). This observation is interesting but may be a result of methodological errors due to the learning process during the analysis of the healthy group.
The groups are different regarding to age. A proper control group has to be analyzed to establish whether there is a difference in the type of platelet-leukocyte aggregates formed in a healthy population compared to the patients.
In the patients group, monocytes express more TF than granulocytes. Moreover, monocytes are able to express TF after stimulation over time, while granulocytes increase in TF
0%
20%
40%
60%
80%
100%
control 150 ng/ml rh p-selectin
250 ng/ml rh p-selectin
% p-erk
%p-p38
%p-jnk
14
expression 3-5 days after myocardial infarction but after 2 weeks the ability to express TF after stimulation is practically absent. The fact that granulocytes do not synthesize TF is an obvious explanation but the difference could also be a consequence of the natural course of inflammation with accumulation of granulocytes at the site of injury in the acute period.
It was expected to see an increment in the amount of PMA and PGA after stimulation due to the platelet activation potential of both ADP and TRAP. It is clear, however, that PMA formation is prioritized over PGA formation. This priority order is clearly procoagulatory since the formation of PMA implicates TF expression (Fig.9, 10) that does not seems to be affected by time after myocardial infarction or medication. The procoagulatory potential of PGA is constant or diminished by platelet activation (Fig.15, 16) since TF expression in granulocytes is not p-selectin dependent and TF is not transmitted to granulocytes after ADP or TRAP stimulation.
Despite the lack of statistical significance, it is of interest to further investigate the eventual decrease in PMA formation after ADP stimulation that occurs over time; and if it is a consequence of resistance to monocytes/platelet activation, decreased ability to recruit monocytes, diminished platelet activity due to natural course of the disease or medication?
The intracellular p-selectin/PSGL-1 dependent pathway that leads to TF expression in
monocytes is highly interesting. An adequate understanding of this pathway could elucidate a pharmacological approach to regulate the procoagulatory activity of PMA which is not possible with current treatment. This would mean a significant advance in secondary prophylaxis and possibly in primary as well. The observation of MAP-kinase inhibition presented here adds questions about this pathway that need further investigation to be elucidate.
Acknowledgment
I want to thank C. Christersson and A. Siegbahn and the members of the coagulation and
inflammation research group for their support and assistance.
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