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Another effect of Eap on the immune system is that it modulates the proliferation of distinct T-cell-populations in the way that the balance between cellular and humoral immune response is shifted towards the humoral defence.

In this work it is shown that Eap can modulate platelet functions. It activates platelets, which can be characterized by fibrinogen binding. This is more intense after incubation with Eap compared to the binding intensity after incubation with thrombin, collagen or ADP. In contrast to that, secretion of the α-granules turned out to be rather poor after incubation of human thrombocytes with Eap and secretion of the dense bodies could not be detected. Therefore, Eap-induced activation was not secondarily enhanced by agonists of the dense bodies. Eap also aggregated human platelets.

Hence, different platelet activation parameters were influenced by Eap unequally.

In addition to the modulation of the innate immune response via the p-selectin-PSGL-1-connection and the inhibition of leukocytic transmigration via ICAM-1, there is still another factor making Eap an immune modulator. Incubated with Eap, thrombocytes express CD40L.

CD40L on the platelet surface stimulates the immune and endothelial cells, which express CD40 on their cell surfaces. Therefore, the CD40L-expression on platelets might also stimulate the T-cell-proliferation as it was described by Haggar et al. (2005).

It is quite possible that Eap induces the formation of associates between thrombocytes and leukocytes via the CD40L-CD40-connection and the P-selectin-PSGL-1-connection.

The evidence of an Eap-induced procoagulant surface on thrombocytes detected by binding of FITC-conjugated coagulation factors and Annexin-V-FITC, leads to the conclusion that Eap induces the binding of procoagulant complexes to the cell surface and owing to this to a sufficient synthesis of thrombin on the platelet surface.

But surprisingly, the experiments with anticoagulants show that the activation of thrombocytes - here detected by fibrinogen binding, takes place independently from thrombin or Ca2+. This can be concluded from the fact that neither thrombin inhibitors nor Ca2+ -chelators can prevent platelet activation detected by fibrinogen binding. Just the group of heparines can block the Eap-induced platelet activation completely.

In this work, also the effect of thiol reactive substances was assayed, which had shown an inhibition of platelet activation induced by known agonists like thrombin. So it is of great interest which effect they have on Eap-induced platelet activation.

It was reported before that in case of platelet activation by known agonists thiol blocking substances like pCMPS or DTNB are only able to inhibit the binding of fibrinogen, but not granule secretion. That means the degranulation of platelets induced by known agonists occurs independently of free thiol groups on the receptors, whereas free thiol groups are necessary for the binding of fibrinogen.

VII. Summary 129

In contrast, the results of this work show that after incubation with Eap the thiol blocking substances inhibited the binding of fibrinogen to platelets as well as platelet degranulation.

So there is a definitive dependency on free thiol groups on the platelet surface, not only for fibrinogen binding, but also for granule secretion.

Additionally, the activation of platelets by Eap seems to be dependent on the protein disulfide isomerase (PDI) concluded from the experiments that have shown an inhibition of Eap-induced fibrinogen binding after incubation with the PDI-inhibitor bacitracin.

Based on the knowledge that Eap reacts with free thiol groups there could be a possible explanation for the effect of the heparines. Probably it is not the antithrombotic effect of the heparines inhibiting the platelet activation induced by Eap, but much more their molecular structure with lots of sulphate groups that Eap possibly interacts with forming complexes with the heparine molecules.

In addition to the effects of Eap on human thrombocytes, the effect of Eap on thrombocytes of different animal species was studied. Further experiments with blood samples from animals have shown that there is a great variation in the platelet reactions in response to Eap. This could possibly be explained by the fact that there is a species dependent different reactivity of platelets to the different agonists. Horses for example show a high tendency to thrombo-embolic complications, especially in connection with inflammation processes as found in case of colic or laminitis.

In contrast, ruminants have been reported to have a very low tendency to those complications, which has also been reflected by a very low reactivity of ruminant platelets to different agonists in this work.

Besides, the exact mechanism of platelet activation induced by Eap is not known yet. This could possibly imply further details to understand these differences in reactivity. We suppose that similar to other adhesion proteins of S. aureus immune globulins and the Fc-receptor could play a key role. It is therefore conceivable that a more intense production of antibodies in the rat compared to other species could induce a stronger activation of thrombocytes; or may be on the surface of rat platelets there are more receptors or structures expressed that Eap could interact with compared to platelets of other species. Further research is necessary to find out more about the mechanism of platelet activation induced by Eap. The results of this work indicate that Eap activates platelets via structures or receptors that possess accessible thiol groups on their surface.

In addition it is evident that MSCRAMMs like the fibrinogen- and fibronectin binding proteins (FnBPs), the clumping factors A and B (Clf A and B) and the staphylococcal protein A (SPA) activate platelets via the FcγRIIA-receptor.

Beside the staphylococcal bacteria several other bacterial species are known to activate platelets via this receptor, for example Sc. pyogenes or Sc. sanguis which also express adhesion proteins on their surface. So, obviously, in further experiments it should be checked out if Eap activates platelets via this receptor as well. In the present literature it is not mentioned if there are thiol groups on the Fc-receptor, which are accessible on its surface.

Additionally, in case of activation via this receptor, immune globulins could have a mediating role indeed, since the Fc-receptor is the receptor for the immune globulins as well.

In case of a sepsis, Eap is distributed over the whole circulation system. With its capability to activate platelets it can cause clot forming, so that the bacteria are sequestered in the clots being protected against the immune cells and medications; in worst case a disseminated intravasal coagulation (DIC) could be initiated. The situation seems to be still more precarious taking into account that S. aureus develops continuously more resistances to antibiotics and how heavy the course of a sepsis induced by S. aureus can be, for example by developing an infectious endocarditis that can result in a permanent heart deficiency.

Based on these facts it becomes clear how important it is to find new therapeutic ways to fight septicaemia more successfully. In general, it is important not only to fight the bacteria, but also to do a prophylaxis, respectively a therapy against platelet activation and DIC.

Further research on the effects of Eap on platelet function could lead to a better understanding of the intracellular processes of platelet activation caused by Eap. Gaining more detailed information about these mechanisms and the pathogenesis of complications in connection with a sepsis could yield to possible new therapeutic approaches.