USMLE – Hemostasis
With the evolution of the circulation as a transport system, an efficient mechanism developed, not only to prevent blood loss from a damaged vessel to secure hemostasis but also to prevent the inappropriate cessation of flow. Hemostasis depends upon interactions between the vessel wall, platelets and clotting factors. Two phases of hemostasis can be recognized: primary and secondary. In the initial primary phase, the damaged vessel contracts and activated platelets aggregate at the site of damage to form a plug to arrest hemorrhage. This occurs over a number of minutes and is subsequently followed by the secondary deposition of a fibrin mesh to secure the platelet plug. These two processes are interlinked; damaged endothelium activates platelets, which then provide the optimal surface for the enzymatic generation of insoluble fibrin.
Structure, function and development
Resting platelets are discoid-shaped, with a diameter of 2-4 um. The surface membrane invaginates to form a tubular network, the canalicular system. This provides a large surface area of phospholipid on to which clotting factors can absorb. Three types of granule are present in the cytoplasm:
-alpha granules contain fibrinogen and von Willebrand factor
-dense (delta) granules store adenosine diphosphate (ADP) and 5-hydroxytryptamine (5-HT, serotonin)
-lysosomes contain acid hydrolases.
When platelets are activated by ADP, thrombin or collagen they contract to become spherical and extend pseudopodia which adhere to the subendothelium and other platelets. Upon activation, platelet granules discharge their contents which encourage further platelet aggregation and fibrin formation. At the same time, arachidonic acid is released from the platelet membrane and convened by cycle-oxygenase to endoperoxides and the powerful platelet aggregating agent, thromboxane A2. Aspirin and non-steroidal anti-inflammatory drugs irreversibly inhibit platelet cycle-oxygenase and impair platelet function. Platelet-binding to subendothelium is dependent on high molecular weight von Willebrand factor released from endothelial cells, which bridges the gap between platelet membrane glycoproteins and subendothelial collagen. Interplatelet aggregation is dependent upon fibrinogen binding to platelet glycoproteins.
Platelets are derived from megakaryocytes, each of which sheds several thousand platelets directly into the circulation in the marrow sinusoids. Megakaryocytes are derived from lineage-committed stem cells, the CFU-Meg and their proliferation and ploidy are controlled by a newly described growth factor termed thrombopoietin (Tpo). Platelets circulate for 8-14 days before they are destroyed in the reticulo-endothelial system. Some 30% of peripheral platelets are normally pooled in the spleen and not circulating.
The clotting system consists of a series of soluble inactive zymogens designated by roman numerals. When proteo-lytically cleaved and activated, each is capable of activating one or more components of the cascade. Activated factors are designated by the suffix ‘a’. These reactions usually require a source of appropriate phospholipid and calcium. Two pathways of activation are recognized. The ‘extrinsic’ pathway is the principal physiological hemostatic mechanism in vivo. When vessels are damaged, subendothelial cells are exposed. All cells not exposed to the circulation express a 100 kDa transmembrane protein termed tissue factor (TF); the brain, uterus and tumor tissues are particularly rich in this factor. Tissue factor can also be expressed by damaged endothelial cells and activated monocytes. Tissue factor activates factor VII, which in turn activates factor X. In the ‘intrinsic’ pathway, the negatively charged subendothelial surface and collagen exposed by vessel damage activate the ‘contact system’, which in turn activates the complex of factors VIII and IX. This complex then activates factor X. Activated factor X forms a complex with factor V on the surface of activated platelets and this complex converts prothrombin to thrombin. Thrombin converts fibrinogen to fibrin monomer, which polymerizes and is cross-linked by factor XIII to form stable clot. Thrombin plays a crucial role in this ‘final common pathway’; platelets, factors V, VIII and XI are activated by thrombin, which generates a positive feedback loop. Congenital deficiencies of any of these factors will result in a bleeding diathesis.
The extrinsic pathway is assessed by the ‘prothrombin time’ (PT). The intrinsic pathway is assessed by the ‘activated partial thromboplastin time’ (APTT).
Clotting factors are synthesized by the liver but factor V is also produced in platelets and endothelial cells. Factors II, VII, IX and X are produced as inactive proteins. These factors are rich in glutamic acid (Gla) residues, which must be carboxylated to permit calcium-binding and association with phospholipid to generate an active catalytic site. The carboxylase enzyme responsible for this in the liver uses vitamin K as a cofactor. Factors II, VII, IX and X are therefore termed vitamin K-dependent. Vitamin K is converted to an epoxide in this reaction and must be regenerated to its active form by a reductase enzyme. This reductase is inhibited by warfarin and this mechanism forms the basis of the anticoagulant effect of coumarins.
To prevent inappropriate activation of the clotting cascade, natural inhibitors of the clotting systems are present. Antithrombin III is a protein produced by the liver which has weak inhibitory activity against thrombin and factor Xa. When antithrombin binds to heparin, however, this inhibitory activity is markedly accelerated and this forms the basis of the anticoagulant action of heparin. Protein C is a vitamin K-dependent factor produced by the liver; when activated by interaction with protein S, it inhibits factor Va. These natural inhibitors are powerful control points in the positive feedback cascade of clotting and abnormalities in their function result in a tendency to thrombosis.