Platelet Activation: Pathophysiology, Methodology and Clinical Application
ACVIM 2008
Annemarie T. Kristensen, DVM, PhD, DACVIM-SA, DECVIM (CA & Oncology); Inge Tarnow, DVM, PhD
Copenhagen, Denmark

Introduction

Activated platelets have a well-defined critical role in many human conditions incl. cardiovascular disease, immunologic and inflammatory conditions. In veterinary medicine, activated platelets have been associated with disease, but considering the overwhelming evidence of involvement of platelets in human disease, we are just scratching the surface. In this presentation, the pathophysiology of platelet activation, detection methods and potential clinical applications will be reviewed and evidence of platelet activation in disease states incl. heart failure discussed.

Background

Physiological hemostasis is essential for maintaining the integrity of the vessel wall. It halts bleeding and participates in the initiation of wound healing if trauma occurs. Under these instances the components of the hemostatic system: platelets, coagulation factors, fibrinolytic factors, their regulators and components of the vessel wall are important players participating in a perfectly orchestrated but complex beneficial process. In many disease states, the hemostatic factors including the platelets function as in physiological hemostasis, but in addition through their activation and imbalances in the system may become key players in hemostatic complications, activation of inflammatory processes, but also play an important part in the innate immune response, contributing to control, progression and/or exacerbation of the disease state.

During this presentation, physiological hemostasis focusing on the role of activated platelets is presented as well as the pathophysiological role platelets and the mediators they release play in hemostatic complications, inflammation, infection and innate immunity. Methodologies for measuring platelet activation including PFA100, thromboelastography, Multiplate/aggregometry, flow cytometry and confocal microscopy as well as the results obtained in selected disease states including heart disease will be presented as well.

The Role of Activated Platelets in Physiologic and Pathological Hemostasis

The cell based model of coagulation is a dynamic model that involves cellular regulation of coagulation in three phases--initiation, amplification, and propagation.In this model tissue factor bearing cells initiate hemostasis.Recent research facilitated by the availability of specific reagents and cell based model systems has confirmed the key role of tissue factor and activated platelets in the key processes of the hemostatic process. Tissue factor (TF) is a transmembrane glycoprotein receptor found in extravascular tissues including organ capsules and the adventitia of blood vessel walls. It is constitutively expressed on fibroblasts and upon cellular activation on vascular smooth muscle cells, monocytes and neutrophils. Recently evidence has surfaced that activated platelets can express TF. TF-bearing cells and especially the membrane surfaces formed by activated platelets act as the main cellular surfaces for assembly of the procoagulant complexes.

The tissue factor pathway is the essential step in initiation of the coagulation cascade. Any vessel injury or inflammatory process leads to TF expression or exposure. An initial small amount of thrombin close to the cell is formed. The initially generated thrombin is responsible for activation of platelets, release of FV from the platelets, activation of FVIII and release of FVIII from von Willebrand factor and activation of FXI and later amplification of the process and finally fibrin formation.But platelets may also be activated by other mechanisms including 1) exposed vessel wall collagen leading to adhesion and aggregation at the injured site; 2) infectious agents such as bacteria and viruses, as well as 3) local or systemic inflammatory processes, or 4) tumor cells. It has recently been shown that a subpopulation of highly procoagulant platelets named coated platelets form after simultaneous stimulation by thrombin and a GPVI collagen receptor agonist. Dual stimulation with potent agonists are needed to form these platelets and it is believed that they are formed locally where an efficient control of bleeding is essential. Coated platelets are reported to expose phosphatidylserine (PS) and α-granulae proteins including fibrinogen, factor V, von Willebrand Factor (vWF), and fibronectin on their surface, forming a highly procoagulant surface.

Activated platelets interact with key cells involved in inflammation, including endothelial cells and inflammatory cells such as monocytes and neutrophils. Activated platelets secrete a multitude of factors involved in inflammation, angiogenesis and wound healing. These factors can promote disease exacerbation and cause activation of e.g., the inflammatory and complement systems, as part of the body's first line of defense. Activated platelets form heterotypic cell aggregates with leukocytes incl. neutrophils and monocytes, and these leukocyte-platelet aggregates play an essential role in leukocyte recruitment to inflamed sites.

Activated platelets are also important in cancer. It is known that tumor emboli are often surrounded by platelets and it has been shown that activated platelets promote tumor metastasis in mice models. In addition, platelet function may be abnormal in cancer, this includes both decreased function, but platelets may also be more easily activated and this hyperreactivity is known to occur in both human and canine cancer patients. The mechanism by which platelets are activated by the tumor cells are several, but primarily believed to be caused by tumor procoagulant factor, the majority of which has been shown to be tissue factor. Thrombin is formed, an extremely potent promoters of both cell growth and angiogenesis.

Measuring Activated Platelets--Methods and Conditions

There are numerous methods to measure platelet function. They provide valuable results for research or patient studies. The platelet function analyzer, PFA-100, is a relatively new method which has been developed as a quantitative, simple and rapid in vitro tool of assessing primary platelet-related hemostasis at high shear stress. In response to the high shear rates and the agonists, a platelet aggregate forms that blocks blood flow through the aperture; the time taken to occlude the aperture is reported as the closure time (CT). Platelet aggregation is still one of the main stays of evaluating platelet function incl the traditional aggregometers as well as newer technologies such as the MultiPlate. Thromboelastography (TEG) allows rapid and global assessment of hemostatic function in whole blood. TEG may also be used to evaluate platelet activation. During the past few years increasing numbers of studies have evaluated platelet activation using flow cytometry in several disease states incl. heart disease and immune-mediated hemolytic anemia. Furthermore confocal microscopy may be used as a research tool to detect the localization of activated platelets in the forming clot.

References

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9.  Høier L, Kristensen AT, Haggstrom J, Jensen AL, Klitgaard B, Hanson H, Pedersen HD. Increased platelet aggregation response in Cavalier King Charles Spaniels with mitral valve prolapse. J Vet Int Med, 2001; 15:209-216.

10. Tarnow I, Falk T, Tidholm A, Martinussen T, Jensen AL, Olsen LH, Pedersen HD, Kristensen AT. Hemostatic biomarkers in dogs with chronic congestive heart failure. J Vet Intern Med. 2007 May-Jun;21(3):451-7.

11. Kristensen AT, Wiinberg B, Jessen LR, Jensen AL. Evaluation of human recombinant tissue factor activated thromboelastography (TEG) in 49 dogs with neoplasia. J Vet Int Med, 2007, (in press).

Speaker Information
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Annemarie Kristensen, DVM, PhD, DACVIM-SA, DECVIM-CA & ONC
Royal Veterinary & Agricultural University
Frederiksberg C, DK, Denmark


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