Variations in fibrinogen: effect on fibrin matrix and its interaction with cells
The fibrin matrix is an important component of the extracellular matrix and as such an important factor in tissue repair. It is involved in determining and modulating the reorganization and behaviour of a wide variety of cells, amongst which endothelial cells (EC) and smooth muscle cells (SMC). Importantly, previous studies, including our own experiments, show that modification of the fibrin matrix directly modifies its characteristics, which may affect the outcome of tissue repair.
During wound healing, a new vascular bed needs to be developed in the wound area and the fibrin network has an active role in this process. EC adhere to fibrin molecules and thus migrate and form tubular structures, the starting point for new vessels. SMC are then involved in the next stage of the formation of the new vessels. Fibrin networks specifically bind numerous cells and proteins that are resident in normal blood or released into it in response to wounding.

Figure 1. Endothelial cell (red) in fibrin matrix (confocal microscopy)

Plasma fibrinogen shows a high degree of heterogeneity, which is due to alternative processing of fibrinogen mRNAs, post-translational modifications and proteolytic degradation of the protein. The molecular variants of fibrinogen and fibrin demonstrate different functional properties with respect to matrix structure, porosity of the polymer, adhesiveness to tissue, interaction with cells, clotting properties, stability etc. We recently showed that fibrinogen heterogeneity can have a very large effect on the fibrin matrix support of angiogenesis. The HMW fibrinogen form strongly stimulates angiogenesis in vivo and in vitro, while the LMW fibrinogen forms strongly inhibits this process. However, our understanding of the role of the fibrin matrix in these processes is still limited. In our research group we aim to increase our understanding of the characteristics of the fibrin matrix and we will study the possibilities of modulating these fibrin matrix characteristics in order to modify its interaction with various cell types and thereby improve tissue repair in vascular diseases.

Figure 2. Tube-like for mation of EC in matrices of unfractionated fibrin (left), HMW fibrin (middle) and LMW fibrin (right).

Hemostasis in arterial thrombosis
Hemostasis is a main process in arterial thrombosis, and because genetic variation explains a large part of the risk of arterial thrombosis and also of the levels of hemostasis factors, our research group studies how (genetic variation in) hemostasis factors are associated with arterial thrombosis. Since genetic factors are expected to play a larger role in young people then in the elderly, a unique study group of patients with arterial thrombosis at a young age (men < 45 years and women < 55 years) is being recruited in which the (genetic) role of hemostasis will be studied in more detail. In addition, there is collaboration with a number of epidemiological and clinical studies. In addition to association studies, also more mechanistic and fundamental research questions will be addressed to determine the functionality of genetic variants. Hemostasis factors that have our specific interest are:

• Fibrinogen
We study the association between genetic variation in the three fibrinogen genes, with the emphasis is on haplotype analysis, with a selection of cardiovascular disease endpoints (myocardial infarction, ischemic stroke, dementia, atherosclerosis). Fibrinogen polymorphisms may have a direct effect on the function or level of fibrinogen and thus on the association between fibrinogen levels and risk of cardiovascular disease. We study this in a number of population studies. Since fibrinogen is an acute phase protein, we study its role in cardiovascular disease in association with that of other inflammatory markers, such as C-reactive protein.

• Platelet receptors
Platelet aggregation plays a pivotal role in the pathogenesis of arterial thrombosis, such as ischemic stroke (IS) and acute myocardial infarction (AMI). Platelet aggregation is initiated by several agonists that act via different platelet receptors. Polymorphisms in these platelet receptor genes may influence the extent of platelet aggregation and therefore these polymorphisms may be associated with the risk of arterial thrombosis.
One of the agonists is ADP, which gives platelet aggregation and stabilization of existing aggregates by activating two G-protein coupled receptors (P2Y1 and P2Y12). We study how polymorphisms in the gene encoding the ADP receptor P2Y12 influence the function of the receptor and the efficacy of medication, for example the platelet-aggregation inhibitor clopidogrel.
The aim of this research line is to study the relationship of haplotypes in the P2Y12 ADP receptor with platelet aggregation and with the risk of arterial thrombosis. This will lead to new insights on treatment of patients suffering from arterial thrombosis with clopidogrel and related drugs.

• Von Willebrand factor (vWF)
A high circulating vWF has been identified as an independent risk factor for coronary heart disease (CHD). Studies on the association between vWF and CHD are complicated by the fact that multiple factors influence the plasma levels of vWF, such as blood group, the acute phase reaction, and endothelial damage. It is therefore unclear whether vWF has a causal effect on coronary heart disease (CHD) and/or whether it is a marker of endothelial damage. In our research we want to study this further.
vWF levels are to a large and significant extent genetically determined and we have already studied a number of polymorphisms. Furthermore, plasma levels of vWF are also influenced by ADAMTS13, a recently identified protease that is involved in the proteolysis of vWF. Our research also addresses the role of plasma levels and genetic haplotypes of ADAMTS13 in arterial thrombosis.

• Fibrinolysis
In recent years the role of Thrombin Activatable Fibrinolysis Inhibitor (TAFI) in arterial thrombotic disease has been studied. TAFI is a recently described fibrinolysis inhibitor, which attenuates fibrinolysis by binding to lysine binding sites of fibrin. We have studied levels and genetic polymorphisms of TAFI in arterial and venous thrombotic disease.  In patients with unstable angina pectoris, TAFI levels seem to predict with the severity of the disease.
Both these research projects are carried out in close co-operation with the research laboratories of Dick C. Rijken, PhD and Frank Leebeek, M.D., PhD.