Lawrence L. K. Leung M.D.1
1Professor of Medicine and Chief, Division of Hematology, Department of Medicine, Stanford University School of Medicine
The author has no commercial relationships with manufacturers of products or providers of services discussed in this subsection.
Hemostasis, the process of blood clot formation, is a coordinated series of responses to vessel injury. It requires complex interactions between platelets, the clotting cascade, blood flow and shear, endothelial cells, and fibrinolysis.
Platelet Plug Formation
Platelets are activated at the site of vascular injury to form a plug to stop bleeding. Physiologic platelet stimuli include adenosine diphosphate (ADP), epinephrine, thrombin, and collagen. ADP and epinephrine are relatively weak platelet stimulators; thrombin and collagen are strong agonists. Thrombin activation is mediated by G protein-coupled protease-activated receptors (PAR),1 specifically PAR-1 and PAR-4. Thrombin cleaves the external domain of the PAR to initiate transmembrane signaling [see Figure 1].2 Platelet responses to ADP require the coordinated activation of two G-protein-coupled receptors, P2Y1 and P2Y12, which lead to activation of phospholipase C and suppression of cyclic adenosine monophosphate (cAMP) formation, respectively. Antiplatelet drugs such as ticlopidine and clopidogrel block activation of P2Y12.3 There are also specific receptors for epinephrine, thromboxane A2, and collagen.
Figure 1. Thrombin Activation
Thrombin activation is mediated by G protein-coupled protease-activated receptor (PAR). Thrombin cleaves the NH2-terminal exodomain of the PAR, exposing a new NH2 terminus, which then serves as a tethered ligand to bind intramolecularly to the body of the receptor to initiate transmembrane signaling.
Platelet activation involves four distinct processes: adhesion (deposition of platelets on subendothelial matrix); aggregation (cohesion of platelets); secretion (release of platelet granule proteins); and procoagulant activity (enhancement of thrombin generation) [see Figure 2].
Figure 2. Response of Activated Platelets
After platelets are activated, they undergo significant morphologic changes, producing elongated pseudopods. They also become extremely adhesive. The functional response of activated platelets involves four distinct processes: adhesion (deposition of platelets on subendothelial matrix); aggregation (cohesion of platelets); secretion (release of platelet granule proteins); and procoagulant activity (enhancement of thrombin generation).
Platelet adhesion is primarily mediated by the binding of platelet surface receptor glycoprotein (GP) Ib-IX-V complex to the adhesive protein von Willebrand factor (vWF) in the subendothelial matrix.4 Deficiency of GPIb-IX-V complex or vWF leads to two congenital bleeding disorders, Bernard-Soulier disease and von Willebrand disease, respectively [see 5:XIII Hemorrhagic Disorders]. Other adhesive interactions (e.g., binding of platelet collagen receptor GPIa-IIa to collagen fibrils in the matrix) also contribute to platelet adhesion.5
Platelet aggregation involves binding of fibrinogen to the platelet fibrinogen receptor (i.e., the GPIIb-IIIa complex). GPIIb-IIIa (also termed aIIbb3) is a member of a superfamily of adhesive protein receptors, called integrins, which are found in many different cell types. It is the most abundant receptor on the platelet surface. GPIIb-IIIa does not bind fibrinogen on nonstimulated platelets. After platelet stimulation, GPIIb-IIIa undergoes a conformational change and is converted from a low-affinity fibrinogen receptor to a high-affinity receptor in a process termed inside-out signaling. Fibrinogen, a divalent molecule, serves to bridge the activated platelets [see Figure 3]. The cytosolic portion of the activated GPIIb-IIIa complex binds to the platelet cytoskeleton and can mediate platelet spreading and clot retraction (in a process termed outside-in signaling).6 Congenital deficiency of GPIIb-IIIa or fibrinogen leads to Glanzmann thrombasthenia and afibrinogenemia. The GPIIb-IIIa-fibrinogen pathway is the final common course for platelet aggregation. Blockade of this pathway is the basis of an important class of antiplatelet drugs.
Figure 3. Platelet Aggregation
Platelet aggregation involves binding of the divalent molecule fibrinogen to the platelet fibrinogen receptor (the GPIIb-IIIa complex). After platelet stimulation, GPIIb-IIIa is converted from a low-affinity fibrinogen receptor to a high-affinity receptor (inside-out signaling). The cytosolic portion of the activated GPIIb-IIIa complex can mediate platelet spreading and clot retraction (outside-in signaling).
After stimulation, platelet granules release ADP and serotonin, which stimulate and recruit additional platelets; adhesive proteins such as fibronectin and thrombospondin, which reinforce and stabilize platelet aggregates; factor V, a component of the clotting cascade; thromboxane, which stimulates vasoconstriction; and growth factors such as platelet-derived growth factor (PDGF), which stimulate proliferation of smooth muscle cells and mediate tissue repair. PDGF may also contribute to the development of atherosclerosis and reocclusion after coronary angioplasty.
Platelet procoagulation involves the assembly of the enzyme complexes of the clotting cascade on the platelet surface. It is an important example of the close interrelationship between platelet activation and the activation of the clotting cascade.
The central feature of the clotting cascade is the sequential activation of a series of proenzymes (zymogens) to enzymes, ultimately generating fibrin and reinforcing the platelet plug. Another key feature, amplification, ensures rapid response for effective hemostasis but demands tight regulation to prevent untoward thrombosis.
The clotting cascade is usually depicted as comprising intrinsic and extrinsic pathways [see Figure 4]. The intrinsic pathway is initiated by the exposure of blood to a negatively charged surface (e.g., glass), whereas the extrinsic pathway is activated by tissue factor or thromboplastin. Both pathways converge on the activation of factor X, which then activates prothrombin (factor II) to thrombin, the final enzyme of the clotting cascade.
Figure 4. Clotting Cascade
In the classic view of the clotting cascade (left), the intrinsic pathway is initiated by the exposure of blood to a negatively charged surface (e.g., glass) and the extrinsic pathway is activated by tissue factor or thromboplastin. In the modified view (right), generation or exposure of tissue factor at the wound site is the primary physiologic event that initiates clotting.
Although this classic view of the clotting cascade has been useful in the interpretation of clotting times, it is not completely accurate. Patients who are severely deficient in factor XII—as well as many patients deficient in factor XII—do not bleed clinically, which indicates that the initiation part of the intrinsic pathway (the contact phase) is not important in vivo. It is now established that generation or exposure of tissue factor at the wound site is the primary physiologic event that initiates clotting [see Figure 4].7 Tissue factor functions as a cofactor that is absolutely required by factor VII/factor VIIa to initiate clotting. Factor VIIa activates factor X directly and indirectly via the activation of factor IX. This dual pathway of factor X activation is necessary apparently because of the limited amount of tissue factor generated in vivo and the presence of the tissue factor pathway inhibitor (see below), which, when complexed with factor Xa, inhibits the tissue factor/factor VIIa complex.
All of the procoagulants are synthesized in the liver except vWF, which is synthesized in megakaryocytes and endothelial cells. The vitamin K-dependent procoagulants are prothrombin, factor VII, factor IX, and factor X; the vitamin K-dependent anticoagulants are protein C and protein S. For these factors, the formation of α-carboxyglutamic acid residues by vitamin K-dependent carboxylation of glutamic acid residues endows them with calcium-binding properties and the ability to interact with phospholipid membrane surfaces, which are required for biologic activity.8
INTERACTION BETWEEN ACTIVATED PLATELETS AND THE CLOTTING CASCADE
There is an extremely close interaction between the clotting cascade and activated platelet surface in vivo. When platelets are activated, anionic lipids become exposed on the platelet surface, and factor V (stored in platelet granules) is released and bound on the anionic lipids. The factor V on the platelet surface is activated to factor Va and acts as an assembly site for the binding of factor Xa (enzyme) and prothrombin (substrate) known as the prothrombinase complex. At the assembly site, thrombin generation by the prothrombinase complex is approximately 300,000 times more efficient than thrombin generation by fluid-phase factor Xa and prothrombin alone, and the platelet plug keeps the thrombin localized. Factor Xa bound on factor Va is also relatively protected from inhibition by circulating inhibitors such as antithrombin III (AT-III) (see below). Similar enzyme complex assembly applies to the activation of factor X by factor VIIIa (cofactor) and factor IXa (the intrinsic tenase). The result of these processes is efficient amplification and localization of clotting.
Coagulation is modulated by a number of mechanisms: dilution of procoagulants in flowing blood; removal of activated factors through the reticuloendothelial system, especially in the liver; and control by natural antithrombotic pathways. At least seven separate and distinct control systems modulate each phase of hemostasis and protect against thrombosis, vascular inflammation, and tissue damage [see Table 1]. Antithrombin III, protein C, protein S, and tissue factor pathway inhibitor (TFPI) collectively regulate the clotting cascade; prostacyclin and nitric oxide modulate vascular and platelet reactivity; ecto-ADPase inhibits platelet recruitment; and fibrinolysis removes the fibrin clot.
Table 1 Natural Antithrombotic Mechanisms of Endothelial Cells
ANTITHROMBIN III-HEPARAN SULFATE SYSTEM
Antithrombin III is a circulating plasma protease inhibitor. It inhibits thrombin and factor Xa, the two key enzymes in the clotting cascade. AT-III also inhibits activated factor XII and factor XI. In the absence of the glycosaminoglycan heparin, AT-III inhibits thrombin and factor Xa relatively slowly (complete inhibition requires a few minutes). When present, heparin binds to a discrete binding site on AT-III that causes a conformational change in AT-III, which then inhibits thrombin instantaneously and irreversibly. This augmentation of the inhibition of thrombin and factor Xa is the basis for the therapeutic use of heparin as an anticoagulant. Heparan sulfate proteoglycans on the luminal surface of endothelial cells appear to activate AT-III in a manner similar to that of heparin [see Figure 5].9
Figure 5. Binding of Heparan Sulfate
In the absence of heparan sulfate (HS), antithrombin III (AT-III) inhibits thrombin slowly. When HS is present, it binds to a specific site on AT-III that causes a conformational change in AT-III, allowing it to reach the active site of thrombin and inhibit the enzyme instantaneously. HS also binds to a specific site on thrombin, positioning it for optimal inhibition by AT-III.
Thus, the endothelial surface is normally coated with a layer of AT-III that is already activated by the endogenous heparan sulfate. Because 1 ml of blood can be exposed to as much as 5,000 cm2 of endothelial surface, the AT-III-heparan sulfate system is poised to rapidly inactivate any thrombin in the general circulation.
PROTEIN C AND PROTEIN S-THROMBOMODULIN SYSTEM
Thrombomodulin is an integral membrane protein found on the luminal surface of the vascular endothelium in the microcirculation. The binding of thrombin to thrombomodulin results in a remarkable switch in thrombin's substrate specificities: it no longer clots fibrinogen or activates platelets [see Figure 6]. On the other hand, it acquires the ability to activate protein C in plasma.10 A distinct endothelial receptor for protein C has been found that enhances the activation of protein C by the thrombin-thrombomodulin complex.11 Activated protein C degrades factor Va and factor VIIIa, the two cofactors responsible for the assembly of the prothrombinase and intrinsic tenase complex in the clotting cascade. Protein S serves as a cofactor for activated protein C. Deficiencies of AT-III, protein C, and protein S are important causes of a hypercoagulable state.
Figure 6. Protein C/Protein S Pathway
The protein C/protein S pathway is complementary to the AT-III pathway. When thrombin binds to thrombomodulin, thrombin undergoes a conformational change and no longer clots fibrinogen or activates platelets. However, it acquires the ability to activate protein C in plasma. Protein S serves as a cofactor for activated protein C. Activated protein C degrades activated factors V and VIII, the two cofactors in the clotting cascade.
Protein C and protein S both show some structural similarity to the vitamin K-dependent clotting factors (prothrombin, factor VII, factor IX, and factor X). Protein S circulates in two forms: a free form, in which it is active as an anticoagulant, and a bound, inactive form, in which it is complexed to C4b-binding protein of the complement system. C4b-binding protein acts as an acute-phase reactant. The resultant increase in inflammatory states reduces the activity of free protein S, enhancing the likelihood of thrombosis.
TISSUE FACTOR PATHWAY INHIBITOR
Tissue factor pathway inhibitor is a circulating plasma protease inhibitor that is synthesized by the microvascular endothelium. Unlike AT-III, TFPI has a very low plasma concentration. TFPI inhibits factor Xa. The TFPI/factor Xa complex becomes an effective inhibitor of tissue factor/factor VIIa, thus mediating feedback inhibition of tissue factor/factor VIIa [see Figure 7]. Animal studies have shown that depletion of the endogenous TFPI sensitizes the animals to the development of disseminated intravascular coagulation induced by tissue factor or endotoxin.12
Figure 7. Tissue Factor Pathway Inhibitor
Tissue factor pathway inhibitor (TFPI) binds to and inhibits factor Xa. After binding to factor Xa, TFPI undergoes a conformational change. The TFPI/factor Xa complex then mediates feedback inhibition of tissue factor/factor VIIa.
TFPI is primarily synthesized by the microvascular endothelium. Approximately 20% of TFPI circulates in plasma associated with lipoproteins; the majority remains associated with the endothelial surface, apparently bound to the cell-surface glycosaminoglycans. The plasma level of TFPI is greatly increased after intravenous administration of heparin. This release of endothelial TFPI may contribute to the antithrombotic efficacy of heparin and low-molecular-weight heparin. Recombinant TFPI is now in early clinical trials.13
Upon cell perturbation, the fatty acid arachidonic acid is released from cell membrane phospholipids by phospholipase A2. The enzyme prostaglandin endoperoxide H synthase-1 (PGHS-1) converts arachidonic acid into prostaglandin endoperoxides and finally to thromboxane A2 (TXA2) in platelets and prostacyclin (PGI2) in endothelial cells. TXA2 and PGI2 have opposite functions. TXA2 is a potent stimulator of platelet aggregation and causes vasoconstriction, whereas PGI2 inhibits platelet aggregation and induces vasodilatation. PGI2 functions by activating adenylate cyclase, which leads to an increase in intracellular cAMP [see Figure 8].
Figure 8. Action of Nitric Oxide and Prostacyclin
Significant synergism exists between nitric oxide (NO) and prostacyclin (PGI2), leading to platelet inactivation and vasodilatation. The enzyme prostaglandin endoperoxide H synthase-1 (PGHS-1) converts arachidonic acid into PGI2 in endothelial cells. PGI2 activates adenylate cyclase, which leads to an increase in intracellular cyclic adenosine monophosphate (cAMP), inhibiting platelet aggregation and inducing vasodilatation. NO, formed from l-arginine, stimulates production of cyclic guanosine monophosphate (cGMP). Cyclooxygenase-2 (COX-2) is the induced isoform of PGHS; its formation presumably results from hemodynamic shear in the circulation. NO formation is catalyzed by NO synthases (NOS).
Cyclooxygenase-1 and Cyclooxygenase-2
Cyclooxygenase-1 (COX-1) is the constitutive isoform of PGHS. Cyclooxygenase-2 (COX-2) is an inducible isoform of PGHS. COX-2 is undetectable in most tissues. However, it can be rapidly induced in response to growth factors, endotoxins, and cytokines in endothelial cells and monocytes (although not in platelets).14 Recent evidence indicates that endothelial COX-2 is a major source of PGI2 under physiologic conditions in humans, perhaps because of continual COX-2 induction by hemodynamic shear in the circulation.15 Aspirin acetylates and irreversibly inhibits both COX-1 and COX-2. Other nonsteroidal anti-inflammatory drugs (NSAIDs) also inhibit COX-1 and COX-2, although not permanently. Selective COX-2 inhibitors are now available as a new generation of NSAIDs.16
Because aspirin irreversibly inhibits COX-1 and because platelets cannot make new COX-1, brief exposure to aspirin will permanently inhibit TXA2 production for the life span of affected platelets.
Nitric oxide (NO) is formed from l-arginine in endothelial cells. NO stimulates guanylate cyclase, leading to an increase in cyclic guanosine monophosphate (cGMP) in target cells; causes vasodilatation; and inhibits platelet adhesion and aggregation [see Figure 8].17 NO is rapidly destroyed by hemoglobin and thus functions as a local (i.e., paracrine) hormone. Intravenous infusion of an arginine analogue that blocks NO production leads to an immediate and substantial rise in blood pressure. This phenomenon suggests that NO is released continually and basally to regulate vascular tone (in contrast to the production of PGI2, which is more stimulus-responsive). There is significant synergism between NO and PGI2. Formation of NO is catalyzed by NO synthases, which exist in different isoforms in various tissues. In addition to regulating vascular events, NO has a wide range of biologic effects (e.g., neurotransmittal function in the central nervous system).
CD39 is an integral membrane protein found on the endothelial cell surface. It is an active enzyme that rapidly hydrolyzes ADP to AMP, thus functioning as a cell-bound ecto-ADPase. It limits the recruitment of additional platelets into the growing platelet plug by removing ADP released from the dense granules of activated platelets and from damaged erythrocytes and endothelial cells.18
Tissue plasminogen activator (t-PA) is released from perturbed endothelial cells near the site of vascular injury. t-PA converts plasminogen to plasmin. Like the AT-III interaction with thrombin, which is accelerated in the presence of endothelial cell surface heparan sulfate, generation of plasmin takes place optimally on a surface (in this case, the fibrin clot). Both t-PA and plasminogen bind to fibrin (via recognition of lysine residues), which facilitates plasmin generation and localized fibrinolysis [see Figure 9].
Figure 9. Action of t-PA
Tissue-type plasminogen activator (t-PA), released from perturbed endothelial cells near an injured blood vessel, converts plasminogen to plasmin. Free plasmin is rapidly inactivated by plasma α2-antiplasmin; plasmin bound to the fibrin clot is protected from inactivation.
Plasmin cleaves the polymerized fibrin strand at multiple sites, releasing fibrin degradation products. One of the major fibrin degradation products is D-dimer, which consists of two D domains from adjacent fibrin monomers that have been cross-linked by activated factor XIII [see Figure 10]. Plasmin has a broad substrate specificity and, in addition to fibrin, cleaves fibrinogen and a variety of plasma proteins and clotting factors. Plasmin bound on the fibrin clot is protected from inactivation, whereas plasmin released into the circulation is rapidly inactivated by plasma α2-antiplasmin. Thus, localized fibrinolysis is achieved, but nonspecific plasmin degradation of plasma proteins is prevented. In rare cases, patients have bleeding problems caused by a congenital deficiency in α2-antiplasmin.
Figure 10. Transformation of Fibrinogen to Fibrin
The transformation of fibrinogen to fibrin is initiated by thrombin cleavage of fibrinopeptides A and B from the E domains of fibrinogen to form fibrin monomer. The cleavage apparently changes the overall negative charge of the E domain to a positive charge. This change in charge permits the spontaneous polymerization of fibrin monomers, because the positively charged E domain assembles with the negatively charged D domains of other monomers. The polymer is initially joined by hydrogen bonds. Thrombin activates factor XIII, which catalyzes the formation of covalent bonds between adjacent D domains in the fibrin polymer. Plasmin cleaves the polymerized fibrin strand at multiple sites and releases fibrin degradation products, including D-dimer.
Urokinase is the second physiologic plasminogen activator. It is present in high concentration in the urine. Although t-PA is largely responsible for initiating intravascular fibrinolysis, urokinase is the major activator of fibrinolysis in the extravascular compartment. Urokinase is secreted by many cell types in the form of prourokinase, also termed single-chain urokinase-type plasminogen activator (scu-PA). Prourokinase is converted to urokinase by plasmin. Urokinase lacks fibrin specificity in converting plasminogen to plasmin, whereas prourokinase displays such specificity.
The major physiologic inhibitor of t-PA and urokinase plasminogen activator (u-PA) is plasminogen activator inhibitor-1 (PAI-1).19Substantial amounts of PAI-1 are found in platelets. PAI-1 is also released from endothelial cells. PAI-1 deficiency is associated with bleeding diathesis, usually related to trauma or surgery.20 A second inhibitor, PAI-2, is normally secreted by monocytes. During pregnancy, PAI-2 levels are greatly increased because of synthesis by the placenta. The biologic importance of PAI-2 remains to be established.
Thrombin-Activatable Fibrinolysis Inhibitor
Plasma carboxypeptidase is a newly recognized thrombin-activatable fibrinolysis inhibitor (TAFI) [see Figure 11].21,22 TAFI is the second known physiologic substrate for the thrombin-thrombomodulin complex. One may envisage that after the initial fibrin clot is formed by thrombin at the site of a vascular wound, thrombin binds to thrombomodulin on the nearby intact endothelial surface. The thrombomodulin-bound thrombin leads to the generation of activated protein C, which dampens the clotting cascade and prevents excessive thrombin generation. At the same time, the thrombomodulin-bound thrombin activates TAFI, thus slowing down the lysis of the existing clot. In hemophilia, the decreased generation of thrombin may lead to suboptimal activation of TAFI and result in premature clot lysis, which contributes to the delayed bleeding observed in these patients.22 Whether excessive TAFI activity leads to thrombosis is unknown at present.
Figure 11. Plasma Carboxypeptidase
Plasma carboxypeptidase is a thrombin-activatable fibrinolysis inhibitor (TAFI). When fibrin is degraded by plasmin, new carboxyl-terminal lysines are exposed in the partially digested clot. These lysines provide additional sites for plasminogen incorporation and activation in the clot, setting up a positive feedback loop in clot lysis. Thrombin activates carboxypeptidase-B in plasma, which removes the exposed carboxyl-terminal lysines and prevents further plasminogen incorporation into the clot.
Overview of Blood Coagulation
The clotting cascade is initiated by the exposure of tissue factor at a vascular wound, which leads to the generation of thrombin and the deposition of a fibrin clot [see Figure 12]. Simultaneously, the damaged endothelium releases t-PA, which converts plasminogen to plasmin, which then lyses the clot. Both pathways are regulated: TF/factor VIIa is regulated by the TFPI/factor Xa complex, and thrombin is regulated by protein C and protein S. Similarly, the activity of t-PA is regulated by PAI-1. Thrombin and plasmin are under the control of their respective inhibitors, AT-III and α2-antiplasmin. When these two pathways work in coordinated symmetry, a clot is laid down to stop bleeding, and clot lysis and tissue remodeling follow. Diminished thrombin generation (as in factor VIII deficiency) or enhanced plasmin production (as in α2-antiplasmin deficiency) causes hemorrhage [see 5:XIII Hemorrhagic Disorders]. Conversely, excessive production of thrombin (as in AT-III or protein C deficiency) leads to thrombosis [see 5:XIV Thrombotic Disorders].
Figure 12. Initation of Clotting Cascade
Exposure of tissue factor at a vascular wound initiates the clotting cascade. Generation of thrombin and deposition of a fibrin clot occur simultaneously with release of t-PA from the damaged epithelium and conversion of plasminogen to plasmin. Plasmin then lyses the clot. When these two pathways work in coordinated symmetry, a clot is laid down to stop bleeding, and clot lysis and remodeling follow. (α2-AP—α2-antiplasmin; AT-III—antithrombin III; PAI-1—plasminogen activator inhibitor-1; PC/PS—protein C/protein S; TF—tissue factor; TFPI—tissue factor pathway inhibitor; t-PA—tissue-type plasminogen activator)
Heterogeneity of Endothelial Cells and Vascular Bed-Specific Hemostasis
Although the endothelium is generally considered to be a distinct, homogeneous organ system, there are significant differences between arterial, venous, and capillary endothelial cells in terms of morphology and disease susceptibility. Recent studies have shown distinct sets of proteins that mark the arterial and venous endothelial cells from the earliest stages of angiogenesis. Ephrin-B2, an Eph family transmembrane ligand, marks arterial but not venous endothelial cells. Conversely, Eph-B4, a receptor tyrosine kinase for ephrin-B2, marks veins but not arteries.23
It is also likely that endothelia from different vascular beds are not identical.24 For example, the high endothelium in the postcapillary venules of lymph nodes and Peyer patches regulates the circulation of lymphocytes from blood to lymphatics and peripheral tissues. Specific adhesive protein receptors and matrix proteins are highly expressed in these high endothelial venules. The specialized endothelium representing the blood-brain barrier is another example.
These differences between arterial and venous endothelial cells and the vascular bed-specific endothelium may partly account for their different susceptibilities to thrombosis. For example, whereas AT-III and protein C deficiencies are usually associated with deep vein thrombosis of the lower extremities, thrombosis of portal and hepatic veins is frequently associated with myeloproliferative diseases.25 In both conditions, the underlying defect is a systemic hypercoagulable state, and yet there is a clear predisposition of thrombosis to specific vascular beds. Thus, clinical thrombosis is attributable to an imbalance between systemic prothrombotic stimuli and local antithrombotic mechanisms [see 5:XIV Thrombotic Disorders].
Platelet Production and Thrombopoietin
Platelets are derived from megakaryocytes, which arise from pluripotent myeloid stem cells. Platelet production is controlled by a thrombopoietin that is involved in the final maturation of the megakaryocyte. Thrombopoietin has multiple actions in megakaryocyte development.26 It shares some structural homology with erythropoietin and is produced principally by the liver. It increases the size and number of megakaryocytes, stimulates the expression of platelet-specific markers, and is a potent megakaryocyte colony-stimulating factor. Although thrombopoietin is clearly a key factor, stem cell factor (also called kit ligand), interleukin-3 (IL-3), IL-6, and IL-11 all play contributory roles in controlling megakaryocytopoiesis.
Megakaryocytes undergo endomitosis, in which nuclear divisions occur without cell division and are followed by nuclear fusion, to yield a cell with a chromosomal content of 8n, 16n, or 32n. The megakaryocyte cytoplasm then changes into a series of thin, cylindrical strands that eventually fragment into small pieces of megakaryocytes, called proplatelets, that are released into the circulation. Megakaryocyte volume correlates with ploidy and cytoplasmic maturity; the largest megakaryocytes produce the greatest number of platelets. Large platelets called megathrombocytes are seen in the peripheral blood in thrombocytopenic states, especially in idiopathic thrombocytopenic purpura [see5:XIII Hemorrhagic Disorders]. These megathrombocytes probably are young proplatelets and account for the increase in mean platelet volume that occurs during response to or recovery from acute thrombocytopenia.
Platelets entering the circulation survive about 8.5 to 10 days and have a half-life of about 4 days. Approximately 30% to 40% of the platelets are present in a splenic pool that can freely exchange with the circulation. When the need for platelets arises, production can increase sevenfold to eightfold. Because there is no marrow pool of platelets waiting to be released, meeting increased requirements for platelets may require a few days. Platelets have receptors for thrombopoietin and remove it from plasma, and the platelet mass functions as a major thrombopoietin regulator.27 In states of megakaryocyte hypoplasia and thrombocytopenia, little thrombopoietin is metabolized and the plasma thrombopoietin level rises, leading to increased production of megakaryocytes and platelets. In the setting of thrombocytosis, thrombopoietin metabolism increases, lowering the plasma thrombopoietin level and decreasing platelet production.
Coagulation Tests and Their Use
TESTS OF COAGULATION CASCADE
Most coagulation tests measure the time required for fibrinogen from plasma to form fibrin strands, which can be detected by either optical or electrical devices. Prolongation may represent a low factor concentration, inactive factor or factors, or the presence of inhibitors.
Partial Thromboplastin Time
The partial thromboplastin time (PTT), sometimes termed the activated PTT (aPTT), tests the intrinsic coagulation system. A negatively charged surface (e.g., kaolin or silica), followed by cephalin, is added to whole plasma to activate factors XII and XI. The PTT is most sensitive to abnormalities and deficiencies in the sequence of the coagulation cascade before factor X activation. The PTT is also quite sensitive to the action of heparin. It is used to monitor and adjust anticoagulant therapy with regular heparin but not with low-molecular-weight heparins.
The prothrombin time (PT) is a test of the extrinsic system. It detects deficiencies in fibrinogen, factor II (prothrombin), factor V, factor VII, and factor X. Tissue factor is added to whole plasma, leading to fibrin formation, normally in 9 to 12 seconds. Results are usually reported using the international normalized ratio (INR). The INR is calculated by using the following equation:
where C represents the international sensitivity index (ISI). In this way, the thromboplastin used in an individual laboratory, with its specific ISI, is calibrated against a standard reference thromboplastin, and the PT is reported as an INR.28 The presence of a lupus anticoagulant may also interfere with the PT.29
Dilute Russell Viper Venom Time
Russell viper venom contains an enzyme that activates factor X; therefore, the dilute Russell viper venom time (DRVVT) measures the common pathway of the clotting cascade. It is sensitive to the presence of a lupuslike anticoagulant that inhibits the phospholipid-dependent prothrombinase complex.
The thrombin time (TT) is used to test abnormalities of the conversion of fibrinogen to fibrin. It can be prolonged because of hypofibrinogenemia, abnormal fibrinogen (dysfibrinogen), or the presence of inhibitors (e.g., fibrin degradation products) that interfere with fibrin polymerization. The clinical factors commonly associated with prolonged TT are severe liver disease, disseminated intravascular coagulation, and heparin therapy.
Reptilase is a thrombinlike enzyme that converts fibrinogen to fibrin. The reptilase time (RT) is prolonged under conditions similar to those for prolonged TT, with one significant difference: reptilase is not inhibited by the AT-III-heparin complex. Therefore, RT is not prolonged by heparin. A long thrombin time and normal RT suggest a heparin effect.
Thrombin activates fibrinogen by splitting off two peptides, fibrinopeptide A (FPA) and FPB, from the Aα and Bβ chain of fibrinogen and converting fibrinogen to fibrin monomer. Measurement of FPA in the blood can be used as an index of thrombin activity in vivo. Because the clotting cascade can be activated during the blood-sample collection, however, precautions are required in the measurement and interpretation of FPA levels.
The fibrinogen level in plasma can be measured either antigenically or more commonly by clotting assays. The results are reported in mg/dl.
D-Dimer and Fibrin-Fibrinogen Degradation Products
Fibrinogen degradation products (FDP) and fibrin-fibrinogen split products (FSP) result from plasmin degradation of fibrinogen and fibrin clot [see Figure 9]. D-dimer is released by the plasmin-mediated degradation of fully polymerized fibrin. Plasmin cleavage of fibrinogen or soluble fibrin monomer does not yield the D-dimer. Thus, elevated D-dimer is a specific measure of intravascular fibrin deposition and plasmin degradation characteristic of disseminated intravascular coagulation. The D-dimer test has largely replaced the FSP test.
Factor XIII is the only clotting factor whose activity is not assessed in PT or PTT because the end point for both tests is the formation of fibrin polymers, irrespective of whether these polymers are cross-linked covalently by activated factor XIII. Factor XIII deficiency may be suspected in an infant who has significant bleeding after circumcision or, more rarely, in an adult patient who has unexplained bleeding.
Plasminogen and a2-Antiplasmin
The activation of the plasminogen-plasmin system can be inferred from the findings of a long TT, a low plasma fibrinogen level, and an elevated D-dimer level. Another crude test used to measure plasminogen-plasmin activation is the euglobulin lysis time. The sensitivity and specificity of this test is not well defined, however. During extensive thrombosis and fibrinolysis, both plasminogen and α2-antiplasmin (the physiologic inhibitor of plasmin) are consumed. The direct measurement of plasma levels of plasminogen and α2-antiplasmin is sometimes useful to assess the extent of fibrinolysis and the requirement for replenishment of these plasma proteins using fresh frozen plasma.
TESTS OF PLATELETS AND OF PLATELET FUNCTION
Peripheral Blood Smear Evaluation
This examination provides quick, definitive information to confirm or question a platelet count. Normally, there are eight to 12 platelets per high-power field (1,000 × magnification), corresponding to a normal platelet count of 150,000 to 300,000/ml. The smear also shows platelet granularity and whether megathrombocytes are present.
This test primarily measures platelet function. A spring-loaded device is used to make a standard skin incision on the forearm. A prolonged bleeding time with platelets greater than 100,000/ml suggests impaired function. The bleeding time is difficult to standardize, and a normal bleeding time does not predict the safety of surgical procedures or accurately predict hemorrhage.30 It should not be used as a general screening test in a preoperative setting. Although once used in the screening of patients for von Willebrand disease or certain platelet function disorders, for these purposes bleeding time has been largely replaced by the platelet function-100 assay (PFA-100).
Platelet Function Assay-100
PFA-100 is a newly developed automated test for platelet function. Citrated whole blood is aspirated through a capillary tube under high shear onto a membrane coated with collagen and epinephrine or collagen and ADP in which a central aperture is made. The time it takes for blood flow through the membrane to stop is denoted as closure time and is a measure of platelet function. The closure time is prolonged in patients with von Willebrand disease or other platelet functional defects.31 PFA-100 should be considered the first-line test for platelet function disorders.
Platelet aggregometers are photometric devices for recording the transmission of light through a suspension of platelets. When platelets aggregate, light passes through the suspension more readily. To test aggregation, dilute concentrations of platelet agonists (e.g., ADP, epinephrine, collagen, and ristocetin) are added to citrated platelet-rich plasma. With the weak agonists, such as ADP and epinephrine, the initial primary wave of aggregation is followed by a secondary wave. The secondary wave reflects the induction of the platelet release reaction, in which platelet granule contents are released to augment further platelet aggregation. A suboptimal secondary wave is seen with platelet storage pool defects in which either platelet granule content is diminished or its release activity is impaired. The latter is commonly associated with aspirin intake or uremia-related thrombocytopathy. Patients with von Willebrand disease will have a suboptimal platelet aggregation response to ristocetin but a normal response to the other agonists. Platelet aggregation testing is labor intensive and expensive and should be performed only in clinical coagulation laboratories that do this test regularly.
TESTS OF INHIBITORS OF HEMOSTASIS
A prolonged clotting time (e.g., PTT of 60 seconds [normal, 28 to 30 seconds]) can be caused by either a clotting factor deficiency or an inhibitor. An inhibitor is generally an antibody directed against a specific clotting factor or against a phospholipid-protein complex, the so-called lupus anticoagulant [see 5:XIV Thrombotic Disorders]. In a mixing study, one volume of a patient's plasma is mixed with an equal volume of normal plasma. The resulting mixture will provide at least 50% of a deficient factor and correct the abnormality. If the problem is caused by an inhibitor, the resulting plasma mixture still has a prolonged clotting time. A mixing study should always be done when a prolonged clotting time is noted.
Bioassays and immunoassays are available for assessing AT-III activity. A functional assay is preferable to an antigenic assay.
Protein C and Protein S
Functional and immunologic methods are available. Because protein C and protein S are vitamin K dependent, their measurement can be problematic in patients taking warfarin. It is best to measure protein C or protein S when the patient has been off warfarin for 3 to 4 weeks.
Figures 1, 2, 3, 5, 6, and 8 through 11 Seward Hung.
Figures 4, 7, and 12 Marcia Kammerer.
Editors: Dale, David C.; Federman, Daniel D.