Series on Biomechanics, Vol.27, No. 1-2 (2012), 51- 58
Interaction of Blood Components and Blood Cells with Body Foreign
Surfaces
R.P. Franke1, F. Jung2
1
2
University of Ulm, ZBMT, Dept. of Biomaterials, Ulm, Germany,
Institute for Clinical Hemostasiology and Transfusion Medicine, University of Saarland,
Homburg/Saar, Germany, Email: Friedrich.Jung@gkss.de
1. Introduction
Surfaces of biomaterials not derived from biological tissues activate the coagulation cascade as well
as the complement system when they are exposed to blood e.g. after implantation of cardiovascular implants
(e.g. stents, heart valves, occluder systems, vascular grafts) or during procedures like renal haemodialysis.
These biomaterials, however, are assumed to be less immunologically active than tissue derived biomaterials
[15].
Most proteins involved in the coagulation cascade as well as in the complement system are in precursor forms
present in the plasma. For their specific functions, precursor forms have to be activated by either specific
cleavage, or by conformational changes, or by both. Blood in contact with artificial surfaces can alterate or
even activate the coagulation and complement glycoproteins which is often followed by activation especially
of platelets and of leukocytes [20]. The activation of complement was shown to critically raise the
susceptibility to infection, pulmonary dysfunction, morbidity, and to effect survival rates of patients with
renal failure [12].
Endothelial cells are the key holders of non-thrombogenicity in the blood vasculature. Endothelial cells not
activated will generate or present no or only sparse amounts of pro-coagulant proteins like e.g. P-selectin or
coagulation-activating factors, at the same time maintaining a sufficiently high level of anti-fibrinolytic
activity. Under certain conditions the functionality of endothelial cells can be diminished so that activation
especially of thrombocytes and other blood cells, of the coagulation cascade (fibrin generation and
polymerization) and of the complement system can occur. One or more of these activating conditions were
regularly described in atherosclerotically diseased parts of blood vessels where the endothelial function was
demonstrated to be disturbed. So far, in most cases with diseased endothelial cells it could be shown that the
endothelial tissue factor pathway inhibitors (TFPI) production was disturbed together with other unbalanced
activities of endothelial cells.
After application of vascular prostheses the balancing of tissue factor is drastically disturbed
primarily because there are no endothelial cells inhabiting the prosthesis, but, secondly because
monocytes/macrophages will inhabit the prosthesis wall instead which can release massive amounts of TF. TF
is acting both on the activation directly of platelets and the coagulation cascade and at least indirectly on
leukocytes.
Beside the imbalance of TF due to the lack of EC and the additional local accumulation of
monocytes/macrophages there are further influences of body foreign and polymer surfaces especially on
thrombogenicity of the blood. Thrombogenicity is still not clearly defined, but at least comprises the
activation of platelets and of the coagulation cascade and the generation of thrombi or emboli, respectively.
In a very short period of time platelet activation and the so called “contact activation” occur where high
molecular weight kininogen (HMWK) and the activated factor XII interact and generate bradykinin. This will
further enhance the activation of the coagulation cascade also implying the further activation of the
complement system. Several polymer surfaces were reported to have a strong influence on contact activation
and in consequence also on the activation of complement and coagulation.
A more or less generalized complement activation can have serious consequences due to the reduced
removal of antibody complexes which generally need complement decoration for their elimination. And there
are more mechanisms to burden the immune system beside the lack of complement. Degradation particles
from polymers can become coupled to haptens and lead to immune reactions.
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R.P. Franke et al./ Interaction of Blood Components and Blood Cells with Body Foreign Surfaces
Thrombogenesis with the activation of platelets and of the coagulation cascade can lead to thrombi
adhering to the vascular wall in regions with deteriorated endothelial cell function or to emboli which are
reaction products of platelets, erythrocytes and the coagulation cascade, which are carried in the blood stream
and can lead to the obstruction of supplied blood vessels. So one of the fatal consequences of the generation
of thrombi/emboli could be the complete obstruction either of vessels of the heart (myocardial infarction), or
of the brain (stroke), or of the lungs (pulmonary embolism).
Under certain conditions the vasculature is able to lyse these thrombemboli. This again implies a
plasmatic component, plasmin. For the lysis of clots the activation of the plasmin system from plasminogen
also needs to be initialized by endothelial cells, via plasminogen activators mainly of the tissue type
plasminogen activator (tPA). This is another element of imbalance especially in cases when endothelial cells
are lacking. So, not only the generation of thrombemboli can be enhanced (lack of endothelial cell tissue
factor inhibitors) but also the lysis of emboli can be strongly decreased (lack of endothelial cell plasminogen
activators) in the absence of endothelial cells. And there are not just problems with reduced fibrinolysis, some
of the fibrinolytic products can lead to further activation of the plasmatic coagulation. Adding to that, some of
these fibrin degradation products where described to be toxic or impeding for other cell species e.g.
endothelial cells.
Kallikrein-Kinine
system
Thrombogenicity
and coagulation
Complement
activation
Cytotoxicity
Opsonisation:
Activation of monocytes/
granulocytes (PMN)
Fig. 1. Activation sequences and situation in vivo
2. Contact phase activation
Within seconds body foreign surfaces will be covered by blood products to varying extent depending
on physico-chemical characteristics of the biomaterial surface. Among these are components indicating the
activation of the Kallikrein-Kinine system which is also called the contact phase activation [27]. High
molecular weight Kininogen (HMWK) is the starting element of Kallikrein-Kinine activity and of
vasodilatory effects as well as of the activation of the plasmatic coagulation.
It is important to note that negatively charged biomaterial surfaces generally were described to exhibit a
strong tendency to activate the contact phase.
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R.P. Franke et al./ Interaction of Blood Components and Blood Cells with Body Foreign Surfaces
Vasodilation
Coagulation
KK
KK
PK
-
HMWK
Surface
Fig. 2. Contact activation via Kallikrein-Kinine system
3. Complement activation
Proteins adsorbing to artificial surfaces usually will be altered in their structure and can be activated
as was shown for the two major plasma protein systems, namely the complement system and the coagulation
cascade. While it is possible, to control coagulation using anticoagulants, there are no clinical agents available
to prevent activation of complement on the contact of blood with biomaterial surfaces.
Of the three described pathways of complement activation, the classical pathway of complement
activation (CP) is usually started by binding of antigen-antibody complexes to C1q (complement-fixing
antibodies), leading to formation of C1r, C1s, C4, C2, C3 and C5. An antibody independent activation, called
lectin pathway (LP), was found in which the C-type lectin domain present in mannose binding lectin (MBL)
together with MBL-associated serine-protease (MASP) bound to mannose or N-acetylglycosamine structures.
The alternative pathway (AP) usually will become activated slowly and spontaneously by a change of C3conformation upon adsorption and hydrolysis of the internal C3 thioester bond [29, 16] and will further be
enhanced by C3 contact with body foreign surfaces [34]. In AP activation on cell surfaces, C3b was found to
bind covalently to hydroxyl or amino groups [16], whereas in AP activation on body foreign surfaces C3b
binding occurred when no hydroxyl or amino groups were available [15].
Body foreign polymeric surfaces will initially trigger limited CP activation followed by C3b binding
and AP amplification where most of the effect is determined by AP amplification [3, 16]. Nascent C3b will
bind covalently with the thioesterbond to surface components forming CP clusters, then will bind factor B and
factor D and properdin, resulting in stabilisation of C3 convertases and in additional amplification [11]. In
plasma there is a spontaneous hydrolysis of C3 occurring continuously as part of AP, denominated as
“tickover”.
The three pathways meet together in the formation of two C3 convertases (C3Bb and C4bC2a) and
two C5 convertases (C3bBbC3b and C4bC2aC3b) implying cleavage of C2 and C4 (CP and LP) or the serine
proteases factor B and factor D (AP). Subsequently, the anaphylatoxins C3a and C5a, the opsonins C3b and
C4b and the membrane attack complex (MAC; C5b-C6-C7-C8-C9 abbreviated as C5b-C9) are generated.
C5b was shown to induce P-selectin expression on platelets [36]. The C5b-C9 complex can also play a role in
the activation of platelets [17]. The blockade of the C5b-C9 complex was demonstrated to prevent platelet
activation [31].
Deposition of C3b/C4b on pathogens or body foreign material lead either to ingestion by phagocytic
cells (opsonisation) or to their lysis by interaction with the MAC, termed as lytic mechanism. Factor H is a
potent soluble inhibitor of AP serving as cofactor for factor I in conversion of active C3b to inactive iC3b.
With a broad variety of complement activation stimulators or inhibitors existing [11] there can be many and
very different reasons why a body foreign surface could appear as compatible or incompatible. E.g. Factor H
could specifically bind to such a surface and be available for the down regulation of C3 activation or,
conversely, factor B could bind to such a surface and be available for further AP activation.
The complement system is assumed to act as an instructor of the humoral immune response in
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R.P. Franke et al./ Interaction of Blood Components and Blood Cells with Body Foreign Surfaces
Cell lysis
Cell
Cell stimulation
stimulation
TH2
(Mo,
(Mo, PMN,
PMN,TTH1
H,1,
TH, 2,
regT)
regT)
TCC
C5a
C3a
C5
C9 C5
Alternative
pathway (AP)
on cell surfaces
C3
OH
NH2
cell surface
Fig. 2a. Complement activation on cell surfaces
Cell lysis
Cell
Cell stimulation
stimulation
(Mo,
TH2
H,1,
TH, 2,
regT)
(Mo, PMN,
PMN,TTH1
regT)
TCC
C5a
C3a
C5
C9 C5
C3
OH
C1q
2: Alternative pathway
amplification
1: Limited
Classical pathway
Body foreign surface
Fig. 2b. Complement activation on body foreign surfaces
lowering the threshold for B-cell activation and promoting optimal B-cell memory [8]. Complement can also
modulate T-cell responses [9] in all phases of immunological activity either through direct modulation of Tcells themselves or indirectly through alteration of antigen presenting cells (APC) [21]. Some of the
complement activation products act as mediators of inflammation. They can bind to specific receptors on
polymorphonuclear leukocytes (PMN), monocytes, macrophages, and e.g. mast cells. Binding in already
adherent cells can induce e.g. release of oxidative products and/or lysosomal enzymes. Monocytes can be
induced by C3a to generate cytokines.
Activation of complement in the presence of polymer based biomaterials is assumed to proceed most
likely through limited CP activation followed by massive alternative pathway amplification. However, contact
of blood with negatively charged surfaces seems to activate complement via the classical pathway.
4. Activation of platelets and of the plasmatic coagulation
The main function of platelets is the formation of mechanical plugs at vessel wall injuries, following
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R.P. Franke et al./ Interaction of Blood Components and Blood Cells with Body Foreign Surfaces
platelet activation. Therefore, the platelet surface has various receptors and there are different stimuli, which
activate platelets, with diverse platelet responses to these stimuli, mediated by the binding of various
stimulants to specific platelet receptors.
Fig. 3. Platelet thrombus growing on a stent strut in a closed loop in vitro system perfused with platelet rich plasma
During the early phase of adhesion (tethering phase) the platelet responses are reversible [32], which
include adhesion, shape change and reversible aggregation. When platelets adhere, they change their shape
from discoid to spherical with the extrusion of the pseudopods [18, 37].
There is a suggestion that the procoagulant activity and subsequent production of thrombin increases on the
platelet surface due to this platelet shape change [10]. Platelet adhesion occurs in flowing blood and it has to
withstand the shear stress exerted by the haemodynamics of the blood flow [19].
Platelets not only adhere to injured vessels walls but also to particulate matter in the blood
stream, bacteria and other microorganisms, macrophages and to the artificial surfaces of
prosthetic devices [28] Apparently, the only surface at which platelets do not adhere seems to be
the surface of non-activated endothelial cells.
The release reaction, which augments the platelet aggregation, is regulated by two positive feedback loops.
Firstly, endoperoxides like thromboxane A2 and ADP, which are released during the reaction provoke by
intracellular mechanisms further expression of the fibrinogen receptors on the platelet surface, thus inducing
further platelet aggregation [14]. Secondly, the synergism between the different platelet agonists augments
platelet aggregation [2, 4, 6]. Full platelet aggregation can also be induced by the simultaneous addition of
subthreshold levels of platelet stimuli, which fail to induce platelet aggregation on their own merit. The
linking of the platelets via fibrinogen brings about platelet aggregation. A great number of agents (including
ADP, epinephrine, collagen and thrombin) can induce platelet aggregation [22]. Simplistically, vWF and
fibrinogen bind to receptors on one platelet and create crosslinks to another platelet by binding to receptors on
the latter [25].
Activated platelets contribute to haemostasis and it is generally believed that these activities are
relevant to thrombus growth, where the bulk of the thrombus often seems to be a mass of platelets.
Platelet membrane phospholipids potentiate the intrinsic pathway of coagulation, which eventually forms
thrombin from prothrombin by activated factor X [13]. The platelet surface also protects active coagulation
factors from inactivation by their natural inhibitors [35]. The release products of platelets play an important
role in the formation of platelet thrombi. For example, Platelet Factor 4 seems to possess antiheparin activity,
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R.P. Franke et al./ Interaction of Blood Components and Blood Cells with Body Foreign Surfaces
and release of fibrinogen could potentially contribute further to the formation of the thrombus [38]. As
mentioned previously, P-selectin expression could result in platelet–leukocyte interaction leading to fibrin
deposition to stabilize the thrombus [30].
In addition, there has been more evidence on the role of procoagulant activity of platelets. For
example, coagulation Factor V located in the alpha granules of the platelets becomes membrane bound when
activated simultaneously with two agonists, thrombin and convulsin, an activator of the collagen receptor
glycoprotein VI [1]. These platelets are referred to as convulsin and thrombin induced Factor V platelets.
These convulsin and thrombin activated platelets were capable of generating more prothrombinase activity
than any other physiological agent and thus exhibited much stronger procoagulant activity. Factor V could
also be expressed on the platelet membrane by simultaneous activation with thrombin and most of the
subtypes of collagen, as would be expected at sites of endothelial damage.
A spontaneously formed primary platelet plug is unstable. But the formation of a platelet thrombus is
accompanied by the generation of thrombin, which results in the generation of fibrin required for the
stabilization of the thrombus [26]. Platelet plug formation and coagulation are closely linked processes. The
activation of the plasmatic coagulation can be triggered by different mechanisms:
1: thrombogenic body foreign surfaces induce the contact activation (via FXI, FXII, pre-kallikrein and
HMW-kininogen).
2: the vessel wall injury during an implantation procedure leads to the liberation of unbalanced tissue
factor that together with FVIIa activates the extrinsic coagulation cascade. In addition, sub-endothelial
structures - like collagen, laminin or vWF – activate the so-called surface-sensible coagulation factors
(e. g. FXI und FXII). Both mechanisms lead to the activation of the prothrombinase complex and to the
transformation of fibrinogen to fibrin and subsequent polymerisation.
3: in consequence of platelet aggregation (activation via FXIIa and FXII-subunits) as well as of
fibrinolysis – effected by plasmin - a complement activation can occur (with a feedback loop into the
contact activation).
4: Activated platelets release platelet factor 4. This factor can neutralize heparin and acts prothrombotic
via the inactivation of heparin.
The thrombus stabilisation by the polymerisation and subsequent cross-linking of fibrin under the
influence of FXIII is a very complex process with numerous feed-back loops.
5. Determinants of thrombogenicity of cardiovascular implants
It has been shown repeatedly that cell adhesion to an artificial material depends strongly on the
physico-chemical properties of the material surface, e.g. its roughness, chemical composition, energy, polarity
and wettability. The chemical composition of the material surface is an important factor determining the
surface energy, polarity, wettability and zeta potential, and consequently the character of the cell–material
interaction [5].
Surface roughness influences the platelet adhesion, not only but also, because an increase in surface
roughness means also an increase in surface area, where surfaces with greater roughness induce platelet
adherence much stronger then smoother surfaces [7]. Surface wettability, described as hydrophilic
(“wettable”) or hydrophobic (“non wettable”) is also discussed to be essential. Very hydrophilic surfaces
either prevent the adsorption of proteins, or bind these molecules very weakly, prevent also cell attachment
and spreading partially or completely. Such surfaces are known to adsorb cell adhesion-mediating molecules
relatively weakly, which could lead to the detachment of these molecules especially at later culture periods,
when they contain larger numbers of adherent cells. However, optimal cell adhesion was described for
moderately hydrophilic surfaces. Tamada et al. [33] claimed that a polymer surface with a water contact angle
of 70 o seemed to be the most appropriate surface for cell adhesion, while Lee [24] found a contact angle of 55
o
as most appropriate for another polymer. Synthetic polymers used in biotechnologies and in medicine are
usually too hydrophobic in their pristine and unmodified state (a water drop contact angle of about 100° or
more), so they cannot grant sufficient cellular colonization.
The intimal blood vessel layer (vessel intima) is mostly negatively charged against the external blood
vessel layer (adventitia of the blood vessel; 1-5 mV) due to the negative charge of polysaccharides of the
glykocalyx of the vessel intima. Since blood cells and platelets are also negatively charged, they are usually
repelled from the vessel intima. The electrical charge of the material surface is also an important factor for its
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R.P. Franke et al./ Interaction of Blood Components and Blood Cells with Body Foreign Surfaces
colonization with cells. It has been shown repeatedly that there is better cell adhesion to positively charged
surfaces than to negatively charged surfaces. A result that possibly depends on the fact, that negatively
charged surfaces were shown to activate the complement system.
At present, there is no theory which allows to predict the haemocompatibility of a new material.
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