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Platelets have a key physiological role in haemostasis however, inappropriate thrombus formation can lead to cardiovascular diseases such as myocardial infarction or stroke. Although, such diseases are common worldwide there are comparatively few anti-platelet drugs, and these are associated with an increased risk of bleeding. Platelets also have roles in thrombo-inflammation, immuno-thrombosis and cancer, in part via C-type lectin-like receptor 2 (CLEC-2) and its ligand podoplanin. Although CLEC-2 contributes to these diseases in mice, as well as to thrombus stability, it is unclear whether CLEC-2 has similar roles in humans, particularly as human CLEC-2 (hCLEC-2) cannot be investigated experimentally in vivo.
To investigate hCLEC-2 in vivo, we generated a humanised CLEC-2 mouse (hCLEC-2KI) model, as well as a novel monoclonal antibody, HEL1, that binds to a different site than an existing antibody, AYP1. Using these antibodies, we have provided proof of principle for the use of hCLEC-2KI mice to test potential therapeutics targeting hCLEC-2, and shown for the first time that hCLEC-2 can be immunodepleted, with little effect on haemostasis. However, our results have also suggested that there are species differences in the role of CLEC-2 in arterial thrombosis. We further confirmed this using human blood where blocking CLEC-2 ligand binding had no effect on thrombosis, whereas we confirmed a minor role for mouse CLEC-2 in thrombus stability. We also investigated the effect of blocking CLEC-2 signalling using the Bruton’s tyrosine kinase inhibitor PRN473 on CLEC-2 mediated immuno-thrombosis in a Salmonella typhimurium infection model. However, no effect on thrombosis was observed suggesting that CLEC-2 signalling is not involved.
Overall, our results suggest that there may be differences in the role of human and mouse CLEC-2, at least in arterial thrombosis, which could limit the potential of CLEC-2 as an anti-thrombotic target. However, it appears that the interaction between CLEC-2 and podoplanin is conserved and therefore CLEC-2 could still be a therapeutic target in immuno-thrombosis, thrombo-inflammation and cancer. Furthermore, any potential human specific therapeutics could be investigated in vivo using hCLEC-2KI mice.
Every year, stroke affects over 100 million people worldwide and the number of cases continues to grow. Ischemic stroke is the most prevalent form of stroke and rapid restoration of blood flow is the primary therapeutic aim. However, recanalization might fail or reperfusion itself induces detrimental processes leading to infarct progression. Previous studies identified platelets and immune cells as drivers of this so-called ischemia/reperfusion (I/R) injury, establishing the concept of ischemic stroke as thrombo-inflammatory disease. Reduced cerebral blood flow despite recanalization promoted the hypothesis that thrombus formation within the cerebral microcirculation induces further tissue damage. The results presented in this thesis refute this: using complementary methodologies, it was shown that infarct growth precedes the occurrence of thrombi excluding them as I/R injury-underlying cause. Blood brain barrier disruption is one of the hallmarks of ischemic stroke pathology and was confirmed as early event during reperfusion injury in the second part of this study. Abolished platelet α-granule release protects mice from vascular leakage in the early reperfusion phase resulting in smaller infarcts. Using in vitro assays, platelet α-granule-derived PDGF-AB was identified as one factor contributing to blood-brain barrier disruption.
In vivo visualization of platelet activation would provide important insights in the spatio-temporal context of platelet activation in stroke pathology. As platelet signaling results in elevated intracellular Ca2+ levels, this is an ideal readout. To overcome the limitations of chemical calcium indicators, a mouse line expressing an endogenous calcium reporter specifically in platelets and megakaryocytes was generated. Presence of the reporter did not interfere with platelet function, consequently these mice were characterized in in vivo and ex vivo models.
Upon ischemic stroke, neutrophils are among the first cells that are recruited to the brain. Since for neutrophils both, beneficial and detrimental effects are described, their role was investigated within this thesis. Neither neutrophil depletion nor absence of NADPH-dependent ROS production (Ncf-/- mice) affected stroke outcome. In contrast, abolished NET-formation in Pad4-/- mice resulted in reduced infarct sizes, revealing detrimental effects of NETosis in the context of ischemic stroke, which might become a potential therapeutic target.
Cerebral venous (sinus) thrombosis, CV(S)T is a rare type of stroke with mainly idiopathic onset. Whereas for arterial thrombosis a critical contribution of platelets is known and widely accepted, for venous thrombosis this is less clear but considered more and more. In the last part of this thesis, it was shown that fab-fragments of the anti-CLEC-2 antibody INU1 trigger pathological platelet activation in vivo, resulting in foudroyant CVT accompanied by heavy neurological symptoms. Using this novel animal model for CVT, cooperative signaling of the two platelet receptors CLEC-2 and GPIIb/IIIa was revealed as major trigger of CVT and potential target for treatment.