Endothelial Function and Platelet Activation

Platelets are anucleate cells produced by megakaryocytes in the bone marrow. Once they have traversed from the bone marrow to the general circulation, their lifespan is approximately 10 days. They function mainly to limit hemorrhage after trauma resulting in vascular injury. Normally in the vasculature, platelets are in a resting state and only become activated after exposure to a stimulus leads to a shape change and release reaction that causes the platelet to export many of its biologically important proteins. Some of the agonists that can initiate this response include thromboxane A₂, adenosine diphosphate (ADP), thrombin, and serotonin. In areas of vascular injury, platelets are attracted to the impaired site by collagen through binding with von Willebrand factor (vWF) via the glycoprotein (GP) Ib/V/IX complex. This initial binding results in platelet activation, with a subsequent feedback mechanism in which ADP, thrombin, and thromboxane A₂ further activate the platelets and recruit additional platelets to the area. The complex firmly binds the platelet to the area of injury so there is no disruption by the high shear forces of turbulent blood flow that occur with vessel disruption. This amplification of the response is essential to form a hemostatic plug and represents the first stage in the hemostatic process. When vWF is not present, hemostatic abnormalities result, with deficiencies leading to von Willebrand’s disease, which can be associated with severe bleeding. Hemostasis issues also arise when the platelet receptor complex GPIb/V/IX is mutated, resulting in inability of vWF to bind, a disorder termed Bernard-Soulier’s syndrome.^1,2

Additional platelet aggregation occurs through activation of G protein–coupled receptors (GPCRs), with the final pathway relying on the GP IIb/IIIa complex, the main receptor for platelet aggregation and adhesion.^3,4 Fibrinogen tethers GP IIb/IIIa complexes on different platelets, stabilizing the clot. The integral role of this receptor is manifest in Glanzmann thrombasthenia, a disorder in which fibrinogen binding is impaired, leading to spontaneously occurring mucocutaneous bleeding episodes.⁵

Vascular endothelium is essential to this hemostatic process; this is the cellular site where regulation and initiation of coagulation begins. Endothelial cells (ECs) modulate vascular tone, generate mediators of inflammation, and provide a resistant surface that allows for platelets to experience laminar flow with minimal shear. Endothelial cells regulate hemostasis by releasing a number of inhibitors of platelets and inflammation. Vascular endothelium is essential for regulating uncontrolled platelet activity through mechanisms of inhibition including the arachidonic acid–prostacyclin pathway, L-arginine–nitric oxide pathway, and endothelial ectoadenosine diphosphatase (ecto-ADPase) pathway⁶ (Table 5-1).

^{Table 5-1} Factors Involved in Fibrinolysis

Nitric oxide (NO) is produced constitutively by (ECs) via an endothelial isoform of nitric oxide synthase (eNOS) in a process dependent on conversion of L-arginine to L-citrulline. Vascular tone is regulated by NO as it controls smooth muscle cell (SMC) contraction. It also inhibits platelets directly, blocking platelet aggregation through stimulation of guanylyl cyclase and cyclic guanosine monophosphate (cGMP) and inhibition of platelet phosphoinositol3-kinase (PI-3 kinase). Nitric oxide functions by decreasing the intracellular Ca^2 + level through cGMP, which inhibits the conformational change in GP IIb/IIIa suppressing fibrinogen’s ability to bind to the receptor, thereby attenuating platelet aggregation.⁷

Prostacyclin, which is synthesized in the ECs from arachidonic acid through cyclooxygenase-1 or -2 (COX-1, COX-2)-dependent pathways, inhibits platelet function by increasing cyclic adenosine monophosphate (cAMP). This is essential for aspirin’s ability to diminish platelet function through acetylation of platelet COX1 at serine 529.

The last pathway important in modulating vascular endothelium’s interaction with platelets is the endothelial ecto-ADPase pathway, which impairs ADP-mediated platelet activation. By hydrolyzing ADP, this enzyme inhibits the critical state of platelet recruitment to a growing aggregate, thereby limiting thrombus formation. Once the platelet aggregate has been stabilized by fibrin with red cells to the vessel wall, the next stage of hemostasis involves activation of the highly regulated coagulation cascade (Fig. 5-1).

Figure 5-1 Coagulation cascade.

Coagulation Cascade Leading to Fibrin Formation

Disruption in the endothelium not only recruits platelets for plug formation, it also stimulates activation of the coagulation cascade, which is essential for secondary clot formation through fibrin generation. The coagulation cascade is a dynamic integrated process in which each step is dependent on another step for activation of proenzymes or zymogens to their active forms through proteolytic cleavage. This process is dependent upon calcium and the phospholipid bilayer allowing inactive clotting factors to be converted to active enzymes through serine protease activity. These coagulation proteins function in a step-by-step fashion to activate downstream members of the cascade, leading to production of the penultimate clotting factor, thrombin. Thrombin is versatile, playing a role in many of the essential stages of hemostasis. Not only is it important for platelet activation, it is also necessary for the cross-linking of fibrin. Recently there have been attempts to limit thrombus formation by directly inhibiting thrombin activity through anticoagulants such as ximelagatran and the oral medication, dabigatran, which is now available for clinical use.⁸

The clotting cascade is divided into two main pathways, the intrinsic and extrinsic pathways. The extrinsic pathway begins with establishment of a complex between tissue factor, found on the cell surface or on microparticles, and factor VIIa. This complex leads to activation of factor X to Xa, which can then further the response by looping back and converting factor VII to VIIa in a feedback mechanism. When factor Xa is present, it binds to factor Va on the membrane surface and again generates prothrombinase, which converts prothrombin to thrombin and then generates fibrin as detailed earlier. The activity of factor Xa is accelerated by the presence of factor Va through calcium and formation of a noncovalent association γ-carboxyglutamate residues of factor Xa and the phospholipid surface of activated platelets.⁹ The extrinsic pathway is measured by prothrombin time (PT), which is determined by adding an extrinsic substance such as tissue factor or thromboplastin.¹⁰

The extrinsic pathway, which is dependent on tissue factor, appears to be the main pathway responsible for hemostasis, with the intrinsic pathway playing a supporting role. Tissue factor is a membrane-bound GP that is constitutively expressed by SMCs and fibroblasts but selectively expressed by ECs when there is vessel wall injury. The “encrypted” activated form of factor VIIa is made functional through a conformational change that occurs at cysteines 186 and 209, leading to disulfide bond formation upon vessel wall injury. Protein disulfide isomerase, glutathione, and NO all may have a role in these allosteric changes; however, recent studies have questioned the importance of “de-encryption” in this process.^11–14 Tissue factor functions through activation of factors X and IX after interactions with factor VII as a complex. Factor VII, although at low levels in an active state (factor VIIa) in the circulation, only becomes biologically important after it is bound to tissue factor in complex with factors X and IX. This complex formation is essential for activation of thrombin.⁹

The role of tissue factor has recently been expanded. It circulates in the blood in association with microvesicles that are derived from cellular membranes produced from lipid rafts on monocytes and macrophages.¹⁵ These tissue factor–bearing microvesicles can directly initiate the coagulation cascade on activated platelets in a process that may be important for understanding the hypercoagulable state.^16,17