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Frank Leebeek

Role of coagulation factors and platelets in the pathogenesis of arterial thrombosis

Hemostasis in arterial thrombosis

Leebeek Lab:
Principal investigator: Frank W.G. Leebeek, MD PhD

Our research is focussed on the role of coagulation factors and platelets in the pathogenesis of arterial thrombosis (see also introduction). In recent years several case-control studies have been initiated to investigate if genetically determined coagulation disorders are a risk factor for the development of ischemic stroke or acute myocardial infarction. This study was performed in close collaboration with the departments of Neurology (Dr. D.W.J. Dippel and Prof.  P. Koudstaal),  Cardiology (Prof. M.L. Simoons) and Vascular Medicine (Dr. A.H. van den Meiracker). We have recently recruited a unique study group of patients with arterial thrombosis at a young age (men < 45 years and women <55 years) in which the role of hemostasis disorders will be studied in more detail. Also more mechanistic and fundamental research questions will be addressed. Our focus will be on platelet receptors, including the ADP receptor, fibrin formation and fibrin degradation (fibrinolysis). This includes work on (functional) polymorphisms in coagulation factor genes or in the promoter regions (TAFI, P2Y12).

I. Hemostasis in arterial thrombosis
Fibrin and fibrinolysis and arterial thrombosis
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 partially degraded fibrin. We have implemented and developed plasma TAFI assays (antigen and activity assays) to study levels of TAFI in arterial thrombotic disease. We have recently described a new polymorphism in the TAFI gene that influences both TAFI activity and antigen levels. At least 14 polymorphisms in the TAFI gene have been described. In patients with unstable angina pectoris TAFI levels seem to predict severity of the disease. We also found that increased TAFI levels may also be associated with an increased risk of ischemic stroke. We are planning to extend our research on TAFI in several patients groups with arterial or venous thrombosis (including hepatic vein thrombosis). This research is funded by the Revolving Fund, Erasmus MC Rotterdam, and in part by the Netherlands Organisation for Scientific Research (NWO).

Primary hemostasis and arterial thrombosis
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.

Drugs inhibiting platelet aggregation are widely used as secondary prophylaxis in patients with AMI and IS. A new class of anti-platelet drugs are the ADP receptor blockers, the thienopyridines, including clopidogrel and ticlopidin. Normally the activation of platelets by ADP results in platelet aggregation and stabilization of existing aggregates. ADP-dependent platelet aggregation requires activation of two G-protein coupled receptors. One of these, the P2Y12 receptor, is the target of thienopyridines. Recently the P2Y12 gene has been cloned. Because polymorphisms in the gene encoding the ADP receptor may influence the function of the receptor, it is of extreme interest to identify polymorphisms in the P2Y12 gene. It is likely that genetic mutations or polymorphisms in the ADP receptor gene affect the function of this receptor and possibly also the efficacy of the thienopyridines. The aim of this research project is to identify polymorphisms in the recently cloned gene of the P2Y12 ADP receptor, to investigate whether these result in enhanced platelet aggregability and to study the association of the polymorphisms with arterial thrombosis. This may lead to new insights on treatment of patients suffering from arterial thrombosis with thienopyridines. This study is supported by the Netherlands Organisation for Scientific Research (NWO).

In addition we will extend our research on von Willebrand factor (vWF) and arterial thrombosis. Several epidemiological studies have shown that a high circulating vWF is an independent risk factor for coronary heart disease (CHD), although the results are not consistent. Studies on the association between vWF and CHD are complicated by the fact that multiple factors, such as blood group, the acute phase reaction, and endothelial damage influence levels of vWF. vWF may therefore be causally associated with coronary heart disease (CHD) or merely be a marker of endothelial damage. It has recently been shown that vWF levels are to a large and significant extent genetically determined. The G allele of the –1793C/G promoter polymorphism in the vWF gene has been associated with higher plasma levels of vWF. We have recently shown that strongly elevated levels of vWF are seen in patients with a first ischemic stroke. In the Rotterdam study we have established that carriers of the G-allele of the –1793 C/G promoter polymorphism of vWF, associated with a higher vWF level, have an increased risk of  myocardial infarction.. These carriers did not have a higher risk of developing atheroscleross, which indicates that high vWF levels may primarily be involved in thrombus formation, rather than in atherogenesis. Other factors may influence vWF plasma levels, such as ADAMTS13, a recently identified protease that is involved in the proteolysis of vWF. This will be addressed in future studies (funded by the Erasmus MC, Translational Research Grant 2004).
Both these research projects are carried out in close co-operation with the research laboratories of Dick C. Rijken, PhD and Moniek P.M. de Maat, PhD.

II. Clinical studies on Haemophilia, von Willebrand disease and other bleeding disorders
The clinical research is focussed on haemophilia and other bleeding disorders, including von Willebrand disease. Our department has been appointed as a Haemophilia Treatment Center in 2000. Three hematologists of our department are members of the Dutch Society for Haemophilia treaters (NVHB).  In recent years we have performed several clinical studies in patients with haemophilia and von Willebrand disease (Mies C. Kappers and Huub H.D.M. van Vliet). We are also involved in several research projects initiated by other Haemophilia Treatment Centers. We have performed clinical, pharmacokinetic and in vitro studies on coagulation factor products that are used for von Willebrand disease. This research area may be extended in the next few years. In addition clinical studies are performed on TTP and ITP (Mies C. Kappers and Huub H.D.M. van Vliet).
Our department has a longstanding tradition of performing hemostasis-oriented research in patients with liver disease. In the past we have studied hemostatic disturbances in patients with liver cirrhosis and in patients undergoing liver transplantation. This research has recently been extended, in close collaboration with the Dept of Hematology of UMC Utrecht (Ton Lisman, PhD). We are studying also the role of coagulation disorders in the development of hepatic vein and portal vein thrombosis. This work is carried out in collaboration with the department of  Hepatology of the Erasmus MC (Harry L.A. Janssen, MD PhD). Our department has recently participated in a large nation-wide diagnostic management study on pulmonary embolism (Frank.W.G. Leebeek and Marieke J.H.A Kruip). More studies on venous thromboembolism, both diagnostic and therapeutic will be performed in the near future.