Cardiovascular & Hematological Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry - Cardiovascular & Hematological Agents) - Volume 6, Issue 3, 2008

Cardiovascular and cerebrovascular diseases continue to be a major public health burden worldwide. According to the WHO data the mortality related to atherothrombosis is the leading cause of death responsible for 22.3% of the total deaths in the world preceding infectious diseases (19.1%) and neoplasms (12.5%) [1]. Some of the improvements in death rates seen in many developed countries may be partly attributable to the application of thrombolytic therapy for myocardial infarction or ischemic stroke [2, 3], designed to lyse the clots blocking the arteries and restore blood flow as quickly as possible. This therapeutic approach is based on the administration of plasminogen activators (urokinase, streptokinase, tissue-type plasminogen activator and its recombinant variants), which convert the plasminogen in blood plasma, on the surface of or inside the thrombi to plasmin which then dissolves fibrin, the solid matrix of thrombi. However, persistent recanalization of the occluded blood vessels often fails (in 15 to 40% of patients) and efficient doses of current fibrinolytic agents have significant bleeding side-effects [4]. These limitations of fibrinolytic therapy maintain a continuous interest in the basic research of the molecular mechanisms underlying fibrin dissolution, which could help the development of more efficient and safe clot busting agents. The present collection of reviews explores recent advances in understanding the interconnections between the structure of the fibrin clots and the action of the enzymes destroying it. In their review entitled “The biochemical and physical process of fibrinolysis and effects of clot structure and stability on the lysis rate” Weisel and Litvinov (University of Pennsylvania, Philadelphia) present a comprehensive overview of all participants in the fibrinolytic process, which helps the reader to unify the more specialized aspects of the molecular mechanisms discussed in the accompanying papers [5]. A special focus of this review is the microscopic structure of the fibrin mesh and its impact on the enzymatic steps of the dissolution process. The minireview entitled “Searching for differences between fibrinogen and fibrin that affect the initiation of fibrinolysis” contributed by Doolittle (University of California, San Diego) summarizes the ultrastructural changes accompanying the conversion of fibrinogen to fibrin [6]. This minireview addresses the structural determinants of the self-destructing nature of fibrin as opposed to fibrinogen, which co-exists in peace with all participants in fibrinolysis. Muszbek, Bagoly, Bereczky and Katona (University of Debrecen Medical and Health Science Center, Debrecen) explain the basic biochemical function of factor XIII in their minireview entitled “The involvement of blood coagulation factor XIII in fibrinolysis and thrombosis” [7]. The stability of fibrin conferred by factor XIIIa is discussed in the context of clinical states of thrombosis related to variations in factor XIII level and its genetic polymorphisms. In the minireview entitled “Alterations of fibrinogen structure in human disease” Hoffman (Duke University Medical Center, Durham) explores the consequences of post-translational modification of fibrinogen through oxidation, nitration, homocysteinylation and glycation [8]. This minireview helps the understanding of the altered fibrinolysis in disease states with increased rate of covalent modification of proteins (e.g. diabetes). Longstaff, Williams and Thelwell (National Institute for Biological Standards and Control, South Mimms) approach the structure-function relationships in fibrinolysis from the side of plasminogen activators in their minireview entitled “Fibrin binding and the regulation of plasminogen activators during thrombolytic therapy” [9]. A special focus in this minireview is the methodology for evaluating and modeling fibrinolysis in vitro as a tool for assessing new thrombolytic agents. In the last review of the series, entitled “Role of cellular elements in thrombus formation and dissolution” Wohner (Semmelweis University, Budapest) presents a cellular view of fibrinolysis [10]. This minireview outlines the contribution of platelets, leukocytes and red blood cells to the modification of fibrin structure as well as their direct effects on discrete steps of fibrinolysis........

The effectiveness of fibrinolysis results from the combination of regulated enzymatic activity and the physical properties of the fibrin scaffold. Physiologically, clots or thrombi are dissolved from within via internal lysis. In contrast, with therapeutic thrombolysis, lytic agents are introduced at one surface and lysis proceeds across the thrombus. In the latter case, there are complex changes that take place at the lysis front in a narrow zone. However, at the microscopic level the mechanisms for either general type of fibrinolysis appear to be similar. Fibrinolysis proceeds by fibers being transected laterally, rather than digestion of fibers by surface erosion from the outside. A molecular model to account for these observations together with what is known from the biochemical characterization of fibrinolysis involves the movement of plasmin laterally across fibers, binding to sites created by its own proteolytic activity. Fibrin clots can have a great diversity of structural, biological, physical, and chemical properties depending on the conditions of formation, and the rate and nature of fibrinolysis is related to these properties. In general, the rate of lysis appears to be faster for clots made up of thicker fibers than for clots made up of thinner fibers, but the lysis rate is not simply a function of fiber diameter and also depends on other physical properties of the clot. Platelet aggregation and clot retraction have a dramatic effect on the structure of fibrin and hence on fibrinolysis.

Although in a gross sense fibrin is merely a collection of fibrinogen molecules packed together in bundles, numerous small structural differences can arise as a result of the conversion of the soluble precursor into the gelled product. Some of the consequences are obvious, others more subtle. In one way or another, all these changes are the result of a sequence of events that includes the release of the fibrinopeptides A and B, the formation of protofibrils, the cross-linking of γ chains, the assembly into mature fibers and the cross-linking of α chains. Numerous immunologic differences between fibrinogen and fibrin have been cataloged, and putative sites for fibrin enhancing the activity of plasminogen activators have been identified. Although some conformational changes have been found by X-ray crystallography, the structural changes leading to the exposure of sites thought to bind t-PA and/or plasminogen remain to be demonstrated.

It has been known for a long time that blood coagulation factor XIII (FXIII) is essential for maintaining haemostasis, its deficiency leads to severe bleeding complication. Biochemical studies have revealed that FXIII is a key regulator of fibrinolysis and, in addition to its role in haemostasis, it has also been implicated in the pathology of arterial and venous thrombosis. Most recently, the polymorphisms in the FXIII subunit genes and their influence on the risk of thrombotic diseases have stirred a lot of interest. This review, besides including the basic biochemistry of FXIII, mainly concentrates on the biochemical and clinical aspects of the involvement of FXIII in fibrinolysis and thrombosis. Biochemical aspects: Basics on the structure and activation of plasma and cellular FXIII. The enzymological features of activated FXIII and its main substrates. The interaction of FXIIIa with fibrinogen/fibrin and with components of the fibrinolytic system. The impact of cross-linked fibrin clot formation on the fibrinolytic processes. The down-regulation of FXIIIa within the fibrin clot. FXIII polymorphisms and their biochemical consequences. Clinical Aspects: FXIII level and the risk of arterial thrombosis (coronary artery disease, peripheral artery disease, ischemic stroke). The effect of FXIII subunit polymorphisms on the risk of arterial thrombotic diseases. The interplay between FXIII polymorphisms and other factors influencing the risk of arterial thrombosis. FXIII and venous thromboembolism.

Products of normal and pathologic metabolism can react with proteins to cause covalent modification. When such modifications affect fibrinogen they can potentially alter fibrinogen function. Those that have been best studied are oxidation, nitration, homocysteinylation and glycation. It appears that the clottability of fibrinogen is maintained unless the degree of modification is extensive. However, modest degrees of fibrinogen modification can alter the rate of assembly of fibrin monomers into a fibrin clot and the fiber structure and packing. In addition, some types of modification affect lysine residues that are critical to binding, activation and activity of fibrinolytic enzymes. Any of these alterations could potentially affect the susceptibility of fibrin clots to fibrinolysis, and have been shown to do so in vitro. In the case of homocysteinylation and glycation, good evidence exists that fibrinogen modification affects clot stability in vivo. However, direct evidence is still lacking that these modifications contribute to the increased atherothrombotic risk associated with hyperhomocysteinemia and diabetes.

First generation thrombolytics (streptokinase and urokinase) had no fibrin binding capabilities and caused systemic plasminogen activation with concomitant destruction of haemostatic proteins. A primary driving force behind the development of the second generation plasminogen activator tissue plasminogen activator (tPA or alteplase) was its ability to bind to fibrin and target thrombolysis. Although in vitro assays highlighted advantages of fibrin binding, clinical trials were disappointing, showing only small benefits in mortality with tPA versus streptokinase, but also with some increase in haemorrhagic stroke. Third generation thrombolytic agents (reteplase, tenecteplase and pamiteplase) are variants of tPA engineered to have improved structure/function, such as longer half life and resistance to inhibitors. However, clear therapeutic advantages of third generation thrombolytics in clinical trials have also been difficult to demonstrate. Although fibrin binding is critical in regulating the activity of tPA, it is not clear how important it is for thrombolytic treatment. Advances are needed in our understanding of the relationship between structure/binding and activity of PAs in vivo under normal conditions and when administered in pharmacological doses. Clearly the impact of fibrin structure and the other components in fibrin clots must also be considered. Ultimately these studies may lead to better engineered therapeutics or optimised mixtures of molecules. With a more detailed understanding of the regulation of plasminogen activation and fibrinolysis it might be possible to tailor thrombolytic therapy to different situations such as myocardial or cerebrovascular treatment or to the patient's age and sex and other characteristics.

Although fibrin forms the core matrix of thrombi, their structure depends also on the cellular elements embedded in its meshwork. Platelets are essential in the initial stages of thrombus formation, because they adhere and aggregate at sites of blood vessel wall injury and then serve as a surface for coagulation reactions, the overall rate of which determines the final structure of fibrin. In addition, platelets affect fibrinolysis through their proteins and phospholipids, which modulate plasmin activity. Leukocytes form mixed aggregates with platelets and thus influence the structure of thrombi. After activation they secrete different proteases (elastase, cathepsin G, matrix metalloproteinases) that enhance the von Willebrand factor-dependent platelet adhesion. Leukocyte-derived enzymes, first of all elastase, effect fibrinolysis by direct digestion of fibrin or indirectly modulate it by partial degradation of zymogens and inhibitors of coagulation and fibrinolytic proteases.