In the Western world, thrombotic disease as an underlying cause in cardiovascular disease, is a major cause of mortality. The clinical definition of thrombosis is that of the pathological presence of a clot (thrombus) in a blood vessel or in the heart that causes the obstruction of blood flow through the circulatory system. Depending on the location where thrombus formation takes place (i.e. in the venous or arterial part of the vessel tree), thrombotic disease can be classified into venous and arterial thrombosis. One of the most frequent forms of arterial thrombosis is in heart attacks, where one of the coronary arteries, is occluded, which leads to hypoxia of the heart tissue that is nourished by the artery that is occluded. Another well-known and commonly seen form of thrombosis is deep vein thrombosis (DVT) which is a form of venous thromboembolism, caused by one or more thrombi that occlude the normal blood flow in the lower leg (usually the calf).
Cause of death in the Netherlands for the years 1980-2004.
Source: Hart- en Vaatziekten in Nederland 2006, Nederlandse Hartstichting
Arterial and venous thromboses are considered as distinct disease states that are characterized by different pathogenic mechanisms and underlying risk factors. However, central to the pathogenesis of both venous and arterial thrombosis is the perturbation of the normal haemostatic balance. A commonly accepted view of homeostasis is that in healthy individuals, haemostasis is carefully balanced by several anticoagulant mechanisms that counteract the procoagulant forces and thus prevent inappropriate vascular blood clotting. In other words, under physiological conditions, homeostasis is in fact a dynamic equilibrium between the pro- and anticoagulant forces that are ongoing at a low level. Several observations are even in favor of a slight dominance of the anticoagulant forces during homeostasis. The equilibrium between pro- and anticoagulant factors can be rapidly shifted in favor of coagulation in case of a physiological need for cessation of blood loss. However, this proneness towards clot formation, necessary to prevent excessive blood loss by a swift formation of a blood clot also implies a risk to develop thrombosis.
While venous disease remains to be important, arterial thrombotic disease appears as the clinically most prevalent form, even though both have been coined to represent two aspects of a same disease. The relative ease with which venous thrombotic disease can be studied however has made that this has been the topic of many studies in the home institute, and remains to do so being valuable in itself but also with respect to its implications for arterial thrombotic disease. In arterial thrombotic disease however, it appears as if more pathological factors come together and the onset of the disease appears intrinsically more complicated. Atherosclerosis is a progressive chronic inflammatory disease that is characterized by the formation of lesions in the vessel walls of large and medium-sized arteries. Already in children the earliest lesions, called fatty streaks, start to develop. These small lesions are localized just beneath the endothelial layer of the arterial wall and consist of lipid-containing macrophage-derived foam cells and T lymphocytes. Thus, lipid metabolism and inflammation are processes intrisically involved in formation of plaques, that may weaken vessel walls, leading to rupture and sudden blockades of blood supply. The involvement of the human immune system and the clear contributions from both the vessel wall and haematopoietic cells have become a focuss of our current work. One very important condition in which many of the factors come into play that are equally important for haemostasis and immune regulation is sepsis.
Sepsis arises when the body responds to an infection, of either bacteria, fungi or viruses and this response culminates into a systemic inflammatory response that inflicts damage to own tissues and organs. Based on extrapolation of the number of treated sepsis patients in the United States, the estimated worldwide incidence rate of sepsis amounts to a staggering 19 million cases per year. According to official criteria sepsis is diagnosed by a proven or suspected clinical infection in combination with at least two systemic inflammatory response syndrome (SIRS) criteria:
- 1) a body temperature greater than 38 °C or less than 36 °C
- 2) a heart rate greater than 90 beats/minute
- 3) tachypnea, manifested by a respiratory rate greater than 20 breaths/minute or hyperventilation, as indicated by a PaCO2 of less than 32 mmHg
- 4) an alteration in the white blood cell count, such as a count greater than 12.000/mm3, a count less than 4.000/mm3, or the presence of more than 10% immature neutrophils.
Severe sepsis is diagnosed when patients meet the aforementioned criteria and have developed sepsis-induced organ dysfunction, hypoperfusion abnormality (lactic acidosis, oliguria, acute alteration of mental status) or hypotension. Patients are diagnosed with septic shock when they meet the severe sepsis criteria and have hypotension despite adequate fluid resuscitation. Although advances are made in the treatment of sepsis patients by the improved availability of potent antibiotics and the more advanced supportive therapy for failing organs, mortality rates are still as high as 20-30% for sepsis patients and even higher for severe sepsis and septic shock patients.
Increased concentrations of histones and nucleosomes were found in circulation of patients with trauma, systemic inflammation and sepsis. Histones are cationic nuclear proteins that package and order DNA into structural units called nucleosomes. Nucleosomes consist of a superhelix of 146 base pairs DNA wrapped around an octamer containing two copies of histones H2A, H2B, H3 and H4. These extracellular histones and nucleosomes could originate from either excessive apoptotic and necrotic cell death or neutrophil extracellular trap (NET) formation. NETs are intricate fibrous networks that contain DNA as their major structural component, as well as antimicrobial proteins such as histones and neutrophil elastase. Extracellular traps are not exclusively released by activated neutrophils. Recently it was discovered that other immune cells like macrophages, eosinophils and mast cells can also release extracellular traps to disarm and kill bacteria. Extracellular histones possess bactericidal activity, but unfortunately also exhibit cytotoxic activity towards host cells, including endothelial cells. Thus, release of histones into circulation may trigger a positive feedback loop, resulting in more cell death and additional histone release.
The mechanism of histone-mediated cytotoxicity is not exactly known yet. One hypothesis is that histones induce channel formation in phospholipid membranes of cells and platelets, resulting in calcium influx, plasma-membrane depolarization and finally cell death. Another hypothesis is that extracellular histones act as pathogen-associated molecular patterns (PAMPs) through activation of Toll-like receptors (TLR) 2 and TLR4 in vivo, leading to production of cytokines. These cytokines cause severe sterile inflammation and tissue injury and can ultimately lead to development of organ failure and death in mice.
In collaboration with the research group of Prof. Chris Reutelingsperger from our department, we focus on the role of histones in cellular activation and in particular in sepsis. In 2014 we published our joint finding that non-anticoagulant heparins are able to safely neutralize the cell toxicity ofextracellular histones, not only in in vitro systems, but also in in vivo models of sepsis (PMID:24264231). Treatment of septic mice resulted in a much increased survival rate during sepsis. This study has been the direct motivation for us to engage in a university spin-off called Matisse Pharmaceuticals B.V. (Matisse), which was created in order to support the further development of non-anticoagulant heparins for use in humans.