HEMOSTASIS AND FIBRINOLYSIS

We have already noted that two essential requirements of the circulatory system are that the blood must be a liquid and that it cannot leak through the walls of the blood vessels. Meeting these two requirements is the job of the fibrinolytic and hemostatic machinery. Hemostasis (from the Greek hemos [blood] + stasis [standing]), or the prevention of hemorrhage, can be achieved by four methods: (1) vasoconstriction, (2) increased tissue pressure, (3) the formation of a platelet plug in the case of capillary bleeding, and (4) coagulation or clot formation. Vasoconstriction contributes to hemostasis because it raises the critical closing pressure - as we discuss on page 455 - and thus collapses vessels that have an intravascu-lar pressure below the critical closing pressure. Vessel constriction is also promoted by chemical byproducts of platelet-plug formation and of coagulation. For example, activated platelets release the vasoconstrictors thromboxane A2 (TXA2) (p. 106) and serotonin (5-HT) (p. 305). Moreover, thrombin, a major product of the clotting machinery, triggers the endothelium to generate endothelin-1 (ET-1) (p. 480), the most powerful physiological vasocon-strictor.
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Increased tissue pressure contributes to hemostasis because it decreases transmural pressure (p. 428), which is the difference between intravascular pressure and tissue pressure. Transmural pressure is the main determinant of blood-vessel radius. Given the fourth-power relationship between flow and blood-vessel radius (p. 429), an increase in tissue pressure that causes radius to decrease by a factor of 2 would diminish flow by a factor of 16. We all take advantage of this principle when we press a finger against a small cut to stop the bleeding. A tourniquet increases extravascular pressure and can thus halt an arterial hemorrhage in a limb. Finally, surgeons routinely make use of this principle when applying hemostatic clamps to close off "bleeders." In this section, we discuss the third and fourth methods of hemostasis, platelet-plug formation and coagulation.
Platelets Can Plug Holes in Small Vessels

Platelets form in the bone marrow by budding off from large cells called megakaryocytes, each of which can produce up to a few thousand platelets. In their unactivated state, these nucleus-free fragments are disk shaped and 2 to 3 m in diameter. Normal blood contains 150,000 to 450,000 platelets per microliter. The lifespan of these platelets is about ten days. In a highly controlled fashion, platelets plug small breaches in the vascular endothelium. Plug formation is a process that includes adhesion, activation, and aggregation. ADHESION. Normally platelets do not adhere to themselves, to other blood cells, or to endothelial membranes. One preventive factor may be the negative surface charge on both platelets and endothelial cells. In the case of endothelial cells, the negative surface charge reflects the presence of proteoglycans, mainly heparan sulfate. Platelet adhesion occurs in response to an increase in the shearing force (p. 429) at the surface of platelets or endothelial cells and in response to vessel injury or humoral signals. Platelet adhesion - the binding of platelets to themselves or to other components - is mediated by "platelet receptors," which are glycoproteins in the platelet membrane. These platelet receptors are integral membrane proteins belonging to a class of matrix receptors known as integrins (p. 19). They are usually heterodimers linked by disulfide bonds. One ligand naturally present in the blood plasma is von Willebrand factor (vWf), a glyco-protein made by endothelial cells and megakaryocytes (and carried by the product of megakaryoctyes - platelets). High shear, certain cytokines, and hypoxia all trigger the release of vWf from endothelial cells. vWf binds to the platelet receptor known as glycoprotein Ib/Ia (Gp Ib/Ia), which is a dimer of Gp Ib linked to Gp Ia. A breach of the endothelium exposes platelet receptors to ligands that are components of the subendothelial matrix. These ligands include collagen, which binds to Gp Ia/IIa, and fibronectin and laminin (p. 19), both of which bind to Gp Ic/IIa. ACTIVATION.

The binding of the above ligands - or of certain other agents (e.g., thrombin) that we will discuss later triggers a conformational change in the platelet receptors that initiates an intracellular signaling cascade that leads to an exocytotic event known as the "release reaction" or platelet activation. The signal transduction cascade involves the activation of phospholipase C (p. 100) and an influx of Ca2+. Activated platelets exocytose the contents of their dense storage granules, which include ADP, serotonin, and Ca2+. Activated platelets also exocytose the contents of their granules, which contain several proteins, including a host of growth factors and three hemostatic factors: von Willebrand factor (see above) and two clotting factors that we will discuss below, clotting factor V and fibrinogen. Activated platelets also use cyclooxygenase (p. 104) to initiate the breakdown of arachidonic acid to thromboxane A2, which they release. Platelet activation is also associated with marked cytoskeletal and morphological changes as the platelet extends first a broad lamellipodium and then many finger-like filopodia. AGGREGATION. Signaling molecules released by activated platelets amplify the platelet-activation response. ADP (which binds to P2Y12 receptors on platelets), serotonin, and TXA2 all activate additional platelets, and this recruitment promotes platelet aggregation. Aspirin, an inhibitor of cyclooxygenase, inhibits clotting by reducing the release of TXA2. As noted above, the von Willebrand factor released by activated platelets binds to the platelet receptor Gp Ib/Ia, thereby activating even more platelets and forming molecular bridges between platelets. Platelet activation also induces a conformational change in Gp IIb/IIIa, another platelet receptor, endowing it with the capacity to bind fibrinogen. Thus, as a result of the conformational change in Gp IIb/IIIa, the fibrinogen that is always present in blood forms bridges between platelets and thus participates in the formation of a platelet plug. As we will see later, when cleaved by thrombin, fibrino-gen also plays a critical role in clotting.
A Controlled Cascade of Proteolysis Creates a Blood Clot

A blood clot is a semisolid mass composed of both platelets, fibrin, and - entrapped in the mesh of fibrin -erythrocytes, leukocytes, and serum. A thrombus is also a blood clot, but the term is usually reserved for an intra-vascular clot. Thus, the blood clot formed at the site of a skin wound would usually not be called a thrombus. The relative composition of thrombi varies with the site of thrombosis (i.e., thrombus formation). A higher proportion of platelets is present in clots of the arterial circulation, whereas a higher proportion of fibrin is present in clots of the venous circulation. Platelet-plug formation and blood clotting are related but distinct events that may occur in parallel or in the absence of the other. As we will see later, activated platelets can release small amounts of some of the factors (e.g., Ca2+) that play a role in blood clotting. Conversely, as we have already noted, some clotting factors (e.g., thrombin and fibrinogen) play a role in platelet-plug formation. Thus, molecular crosstalk between the machinery involved in platelet-plug formation and clot formation helps coordinate hemostasis.
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The cardiovascular system normally maintains a precarious balance between two pathological states. On the one hand, inadequate clotting would lead to the leakage of blood from the vascular system and, ultimately, to hypo-volemia. On the other hand, overactive clotting would lead to thrombosis and, ultimately, to cessation of blood flow. The cardiovascular system achieves this balance between an antithrombotic (anticoagulant) and a prothrom-botic (procoagulant) state by a variety of components of the vascular wall and blood. Promoting an "antithrombotic" state is a normal layer of endothelial cells, which line all luminal surfaces of the vascular system. Promoting a "pro-thrombotic" state are events associated with vascular damage: (1) the failure of endothelial cells to produce the proper antithrombotic factors or (2) the physical removal or injury of endothelial cells, which permits the blood to come into contact with thrombogenic factors that lie beneath the endothelium. Also promoting a "prothrombotic" state is the activation of platelets by any of the ligands that bind to platelet receptors, as discussed earlier. For instance, as platelets flow past artificial mechanical heart valves, the shearing forces can activate the platelets. Table 17-1. PROCOAGULANT AND ANTICOAGULANT FACTORS NAME ALTERNATE NAMES Procoagulant Factors Factor I Factor Ia Fibrinogen Fibrin PROPERTIES A plasma globulin

Factor II Factor IIa Factor III (cofactor)

Prothrombin Thrombin Tissue factor Tissue thromboplastin

A plasma 2 globulin Synthesis in liver requires Vitamin K* A serine protease An integral membrane glycoprotein; member of Type II cytokine receptor family Receptor for Factor VIIa Must be present in a phospholipid membrane for procoagulant activity

Factor IV Factor V

Ca2+ Labile factor Proaccelerin Accelerator globulin A plasma protein synthesized by liver and stored in platelets Single-chain protein Heterodimer held together by a single Ca2+ ion Highly homologous to Factor VIIIa Stable factor Serum prothrombin conversion accelerator (SPCA) Proconvertin Antihemophilic factor (AHF) Factor VIII procoagulant component (FVIII:C) A plasma protein Synthesis in liver requires Vitamin K*

Factor Va (cofactor)

Factor VII

Factor VIIa Factor VIII

A serine protease A plasma protein with phospholipid binding domain Highly homologous to Factor Va Christmas factor Plasma thromboplastin component (PTC) A plasma protein Synthesis in liver requires Vitamin K* A protease A disulfide-linked heterodimer Stuart factor A plasma glycoprotein Synthesis in liver requires Vitamin K* A protease Plasma thromboplastin antecedent (PTA) A plasma protein produced by megakaryocytes and stored in platelets A protease A disulfide-linked homodimer Hageman factor (HAF) A plasma glycoprotein A protease Fibrin stabilizing factor (FSF) A plasma protein stored in platelets A transglutaminase A tetramer of two A chains and two B chains High molecular weight kininogen Fitzgerald factor A plasma protein stored in platelets Kallikrein clips bradykinin from HMWK A plasma protein A serine protease Kallikrein clips bradykinin from HMWK

Factor VIIIa (cofactor) Factor IX

Factor IXa Factor X Factor Xa Factor XI Factor XIa Factor XII Factor XIIa Factor XIII Factor XIIIa

HMWK

Plasma prekallikrein Fletcher factor Plasma kallikrein precursor Plasma kallikrein

von Willebrand factor

vWf

A plasma glycoprotein made by endothelial cells and mega-karyocytes Stabilizes Factor VIIIa Promotes platelet adhesion and aggregation Protease inhibitor produced by endothelial cells GPI-linked to the cell membrane A plasma protein Serine protease inhibitor, member of serpin family Inhibits Factor Xa and thrombin, and probably also Factors XIIa, XIa, and IXa Heparan and heparin enhance the inhibitory action Glycosaminoglycan on surface of endothelial cell Binds thrombin and promotes activation of protein C

Anticoagulant Factors TFPI Tissue factor pathway inhibitor AT III

Antithrombin III

Thrombomodulin (cofactor)

Protein C Protein Ca Protein S (cofactor)

Anticoagulant protein C Autoprothrombin IIA

A plasma protein Synthesis in liver requires Vitamin K* A serine protease Disulfide-linked heterodimer A plasma protein Synthesis in liver requires Vitamin K* Cofactor for protein C

*See page 1226 for a discussion of vitamin K.

According to the classical view, two distinct sequences can precipitate coagulation: the intrinsic pathway and the extrinsic pathway. It is the intrinsic pathway that becomes activated when blood comes into contact with a negatively charged surface - in the laboratory we can mimic this process by putting blood into a glass test tube. The extrinsic pathway is activated when blood comes in contact with material from damaged cell membranes. In both cases, the precipitating event triggers a chain reaction that converts precursors into activated factors, which in turn catalyze the conversion of other precursors into other activated factors, and so on. Most of these "precursors" are zymogens that give rise to "activated factors" that are serine proteases. Thus, controlled proteolysis plays a central role in amplifying the clotting signals. However, the cascades do not occur in the fluid phase of the blood, where the concentration of each of these factors is low. In the case of the intrinsic pathway, the chain reaction occurs mainly at the membrane of activated platelets. In the case of the extrinsic pathway, the reactions occur mainly at a "tissue factor" that is membrane bound. Both pathways converge on a common pathway that culminates in generating thrombin and, ultimately, "stable" fi-brin. Table 17-1 summarizes the names, synonyms, and properties of the procoagulant and anticoagulant factors in various parts of the clotting scheme. INTRINSIC PATHWAY (SURFACE CONTACT-ACTIVATION). The left branch of Figure 17-23 shows the intrinsic pathway, a cascade of protease reactions initiated by factors that are all present within blood. When in contact with a negatively charged surface such as glass or the membrane of an activated platelet, a plasma protein called Factor XII (Hageman factor) can become Factor XIIa -the suffix "a" indicates that this is the activated form of Factor XII. A molecule called high molecular weight kininogen (HMWK), a product of platelets that may in fact be attached to the platelet membrane, helps to anchor Factor XII to the charged surface, and thus serves as a cofactor. However, this HMWK-assisted conversion of Factor XII to Factor XIIa is limited in speed. Once a small amount of Factor XIIa accumulates, this protease converts prekallikrein to kallikrein, with HMWK as an anchor. In turn, the newly produced kallikrein accelerates the conversion of Factor XII to Factor XIIa-an example of positive feedback. On page 555, we see another example of an interaction between kallikreins and kininogens (e.g., HMWK), in which the proteolytic activity of kallikreins on kininogens leads to the release of small vasodilatory peptides called kinins.

page 446D

Figure 17-23 The coagulation cascade, showing only the procoagulant factors. The abbreviations are listed in Table 17-1.

In addition to amplifying its own generation by forming kallikrein, Factor XIIa (together with HMWK) also proteolytically cleaves Factor XI to Factor XIa. In turn, Factor XIa (also bound to the charged surface by HMWK) proteolytically cleaves Factor IX (Christmas factor) to Factor IXa, which is a protease. Factor IXa and two downstream products of the cascade-Factors Xa and, most importantly, thrombin-proteolytically cleave Factor VIII to Factor VIIIa, a cofactor in the next reaction. Finally, Factors IXa and VIIIa, together with Ca2+ (which may come largely from activated platelets) and negatively charged phospholipids form a trimolecular complex called "tenase." Tenase then converts Factor X (Stuart factor) to Factor Xa, yet another protease. EXTRINSIC PATHWAY (TISSUE FACTOR ACTIVATION). The right branch of Figure 17-23 shows the extrinsic pathway, a cascade of protease reactions initiated by factors that are outside the vascular system. Nonvascu-lar cells constitutively express an integral membrane protein called tissue factor (tissue thromboplastin, or Factor III), which is a receptor for a plasma protein called Factor VII. When an injury to the endothelium allows Factor VII to come into contact with tissue factor, the tissue factor nonproteolytically activates Factor VII to Factor VIIa. Subsequently, tissue factor, Factor VIIa, and Ca2+ form a trimolecular complex analogous to tenase. Like tenase, the trimolecular complex of [tissue factor + Factor VIIa + Ca 2+] proteolytically cleaves the proenzyme Factor X to Factor Xa. An interesting feature is that when Factor X binds to the trimolecular complex, Factor VIIa undergoes a conformational change that prevents it from dissociating from tissue factor. Regardless of whether Factor Xa arises by the intrinsic or extrinsic pathway, the cascade proceeds along the "common pathway." COMMON PATHWAY.
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Figure 17-24 An abbreviated version of the coagulation cascade, showing the anticoagulant factors. The anticoagulant pathways are indicated in red. The abbreviations are listed in Table 17-1.

Factor Xa from either the intrinsic or extrinsic pathway is the first protease of the common pathway (center of Figure 17-23). Reminiscent of the conversion of Factor VIII to the cofactor VIIIa in the intrinsic pathway, the downstream product thrombin clips Factor V to form the cofactor Va. Factor V is highly homologous to Factor VIII, and in both cases the proteo-lytic activation clips a single protein into two peptides that remain attached to one another. Factors Xa and Va, together with Ca2+ and phospholipids, form yet another trimolecular complex called prothrombinase. Prothrombi-nase acts on a plasma protein called prothrombin to form thrombin. Thrombin is the central protease of the coagulation cascade, responsible for three major kinds of actions: 1. Activation of downstream components in the clotting cascade. The main action of thrombin is to catalyze the proteolysis of a soluble plasma protein called fibrinogen (p. 432) to form fibrin monomers that are still soluble. Fibrin monomers then polymerize to form a gel of fibrin polymers that traps blood cells. Thrombin also activates Factor XIII to Factor XIIIa, which mediates the covalent crosslinking of the fibrin polymers to form a mesh called stable fibrin that is even less soluble than fibrin polymers.

2. Positive feedback at several upstream levels of the cascade. Thrombin can catalyze the formation of new thrombin from prothrombin and can also catalyze the formation of the co-factors Va and VIIIa. 3. Paracrine actions that influence hemostasis. First, thrombin causes endothelial cells to release NO, PGI2, ADP, von Willebrand factor, and tissue plasminogen activator (see below). Second, thrombin can activate platelets through PAR-1, a protease-activated receptor that belongs to the family of G-protein - coupled receptors (p. 92). Thus, thrombin is a key part of the molecular crosstalk introduced earlier between platelet activation and blood clotting, both of which are required for optimal clot formation. On the one hand, thrombin is a strong catalyst for platelet activation, and on the other hand, activated platelets offer the optimal surface for the intrinsic pathway leading to additional thrombin generation. COAGULATION AS A CONNECTED DIAGRAM.
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The concept of independent intrinsic and extrinsic branches converging on a common pathway is becoming obsolete. In such a "branching tree" (see p. 574), multiple branches converge to form larger downstream branches, eventually converging on a single "trunk" - with no crosstalk between branches. However, coagulation is best conceptualized as a "connected diagram" (p. 574) in which the branches may interconnect in both the upstream and downstream directions. One example of interconnections is thrombin's multiple actions just discussed. Another example is the trimolecular complex of [tissue factor + Factor VIIa + Ca2+] of the extrinsic pathway, which activates Factors IX and XI of the intrinsic pathway. In the other direction, Factors IXa and Xa of the intrinsic pathway can activate Factor VII of the extrinsic pathway. Thus, the intrinsic pathway and extrinsic pathway are strongly interconnected to form a network. What parts of this network are most important for coagulation in vivo? Clinical evidence suggests that coagulation depends largely on the extrinsic pathway. Although tissue factor is normally absent from intravascular cells, inflammation can trigger peripheral blood monocytes and endothelial cells to express tissue factor, increasing the risk of coagulation. Indeed, during sepsis, the tissue factor produced by circulating monocytes initiates intravascular thrombosis.
Anticoagulants Keep the Clotting Network in Check

Thus far, our discussion has focused on the coagulation cascade and attendant positive feedback. Just as important are the mechanisms that prevent hemostasis from running out of control. Endothelial cells are the main sources of the agents that help maintain normal blood fluidity. These agents are of two general types, paracrine factors and anticoagulant factors. PARACRINE FACTORS. Endothelial cells generate prostacyclin (PGI2 ; see p. 106), which promotes vasodilation (p. 479) and thus blood flow, and also inhibits platelet activation and thus clotting. Stimulated by thrombin, endothelial cells also produce nitric oxide (NO; see p. 110). Via cGMP, NO inhibits platelet adhesion and aggregation. ANTICOAGULANT FACTORS. As summarized in Figure 17-24, endothelial cells also generate anticoagulant factors that interfere with the clotting cascade that generates fibrin. Table 17-1 lists these factors: 1. Tissue factor pathway inhibitor (TFPI). TFPI is a plasma protein that binds to the tri-molecular complex [tissue factor + Factor VIIa + Ca 2+] in the extrinsic pathway and blocks the protease activity of Factor VIIa. TFPI is GPI linked (p. 34) to the endothelial cell membrane, where it maintains an antithrombotic surface. 2. Antithrombin III (AT III). AT III binds to and inhibits Factor Xa and thrombin. The sulfated glycosamino-glycans (p. 40) heparan sulfate and heparin enhance the binding of AT III to Factor Xa or to thrombin, thus inhibiting coagulation. Heparan sulfate is present on the external surface of most cells, including endo-thelial surfaces. Mast cells and basophils release heparin. 3. Thrombomodulin. A glycosaminoglycan product of endothelial cells, thrombomodulin can form a complex with thrombin, thereby removing thrombin from the circulation and inhibiting coagulation. In addition, thrombomodulin also binds protein C. 4. Protein C. After protein C binds to the thrombomod-ulin portion of the thrombin-thrombomodulin

complex, the thrombin activates protein C. Activated protein C (Ca) is a protease. Together with its cofactor protein S, Ca inactivates the cofactors Va and VIIIa, thus inhibiting coagulation. 5. Protein S. This is the cofactor of protein C and is thus an anticoagulant.
Fibrinolysis Breaks Up Clots

As noted on page 425, cross-linked stable fibrin traps red and white blood cells as well as platelets in a freshly formed thrombus. Through the interaction of actin and myosin in the platelets, the clot shrinks to a plug and thereby expels serum. After plug formation, fibrinoly-sis- the breakdown of stable fibrin - breaks up the clot in a more general process known as thrombolysis. As shown in Figure 17-25, the process of fibrinolysis begins with the conversion of plasminogen to plasmin, catalyzed by one of two activators: tissue-type plasminogen activator or urokinase-type plasminogen activator. Table 17-2 summarizes the properties of fibrinolytic factors. The source of tissue plasminogen activator (t-PA), a serine protease, is endothelial cells. t-PA consists of a single peptide chain with two "kringles" at the N-terminal portion of the molecule and a protease motif in the C-terminal portion. Kringles are loop structures created by three disulfide bonds and serve to anchor the molecule to its substrate. t-PA converts the plasma zymogen plasmino-gen to the active fibrinolytic protease plasmin. The presence of fibrin greatly accelerates the conversion of plas-minogen to plasmin. Besides t-PA, the other plasminogen activator, uroki-nase-type plasminogen activator (u-PA), is present in plasma either as a single-chain protein or as the two-chain product of a proteolytic cleavage. Like t-PA, u-PA converts plasminogen to the active protease plasmin. However, this proteolysis requires that u-PA attaches to a receptor on the cell surface called urokinase plasminogen activator receptor (u-PAR). Plasminogen is a large, single-chain glycoprotein that is composed of an N-terminal heavy chain (A chain) and a C-terminal light chain (B chain). The N-terminal heavy chain contains five kringles, and the C-terminal light chain contains the protease domain. t-PA cleaves plasmin-ogen at the junction between the heavy and light chains, yielding plasmin. However, the two chains in plasmin remain connected by disulfide bonds. Plasmin is a serine protease that can break down both fibrin and fibrinogen. The five kringles of the heavy chain of plasminogen are still present in plasmin. These anchors attach to lysine residues on fibrin, holding the protease portion of the molecule in place to promote hydrolysis. Plasmin proteolytically cleaves stable fibrin to fibrin breakdown products. Plasmin can also cleave t-PA between the kringle and protease motifis of t-PA. The C terminus of single-chain t-PA nonetheless retains its protease activity.

Figure 17-25 The fibrinolytic cascade. The abbreviations are listed in Table 17-2 on the following page.
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Table 17-2. FIBRINOLYTIC FACTORS NAME ALTERNATE NAMES Tissue-type plasminogen t-PA activator PROPERTIES A serine protease that catalyzes hydrolysis of plasminogen at the junction between the N-terminal heavy chain and C-terminal light chain N terminus contains two loop structures called kringles A serine protease Binds to and required for the activity of u-PA

Urokinase-type plasminogen activator Urokinase-type plasminogen activator receptor Plasminogen

u-PA u-PAR

Single-chain plasma glycoprotein with large N-terminal and small C-terminal domain. N terminus contains five kringles

Plasmin Plasminogen activator inhibitor-1 Plasminogen activator inhibitor-2 2-Antiplasmin

Fibrinolysin PAI-1

A serine protease A serpin (serine protease inhibitor) In plasma and platelets Forms 1:1 complex with t-PA in blood A serpin (serine protease inhibitor) Detected only in pregnancy A serpin (serine protease inhibitor) Forms 1:1 complex with plasmin in blood

PAI-2 2-AP

The cardiovascular system regulates fibrinolysis at several levels, using both enhancing and inhibitory mechanisms. Catecholamines and bradykinin increase the levels of circulating t-PA. Two ser ine p rotease inhibitors ("ser-pins") reduce the activity of the plasminogen activators: plasminogen activator inhibitor-1 (PAI-1) and plasmino-gen activator inhibitor-2 (PAI-2). PAI-1 complexes with and inhibits both single-chain and two-chain t-PA. PAI-2 mainly inhibits u-PA. It is of interest that activated protein C, which inhibits coagulation as shown in Figure 17-24, also inhibits PAI-1 and PAI-2, thereby facilitating fibrinolysis. Only one serpin targets plasmin, 2-antiplasmin (2-AP). When plasmin is not bound to fibrin (i.e., when the plasmin is in free solution), 2-AP complexes with and thereby readily inactivates plasmin. However, when plasmin is attached to lysine residues on fibrin, this inhibition is greatly reduced. In other words, the very presence of a clot (i.e., fibrin) promotes the breakdown of the clot (i.e., fibrinolysis).
References REFERENCES Books and Reviews
Caro CG, Pedley TJ, Schroter RC, Seed WA: The Mechanics of the Circulation. Oxford, Oxford University Press, 1978. Haynes RH: Physical basis of the dependence of blood viscosity on tube radius. Am J Physiol 198:1193-1200, 1960. Lassen NA, Henriksen O, Sejrsen P: Indicator methods for measurement of organ and tissue blood flow. In Handbook of Physiology, Section 2: The Cardiovascular System, Vol III. Bethesda, MD, American Physiological Society, 1979, pp. 21-63. Levine RA, Gillam LD, Weyman AE: Echocardiography in cardiac research. In Fozzard HA, Haber E, Jennings RB, et al (eds): The Heart and Cardiovascular System. New York, Raven Press, 1986, pp 369-452. Maeda N, Shiga T: Velocity of oxygen transfer and erythrocyte rheology. News Physiol Sci 9:22-27, 1994.

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Coulter NA Jr, Pappenheimer JR: Development of turbulence in flowing blood. Am J Physiol 159:401-408, 1949. Cournand A, Ranges HA: Catheterization of the right auricle. Proc Soc Exp Biol Med 46:462-466, 1941. Fhraeus R, Lindqvist T: The viscosity of the blood in narrow capillary tubes. Am J Physiol 96:562-568, 1931. Hamilton WF, Moore JW, Kinsman JM, Spurling RG: Studies on the circulation. IV. Further analysis of the injection method and of changes in hemodynamics under physiological and pathological conditions. Am J Physiol 99:534-551, 1932. Poiseuille JLM: Recherches exprimentales sur le mouvement des liquides dans les tubes de trs petits diamtres. Mm Savant Etrangers Paris 9:433-544, 1846. Reynolds O: An experimental investigation of the circumstances which determine whether the motion of water shall be direct or sinusoid, and of the law of resistance in parallel channels. Philos Trans R Soc Lond B Biol Sci 174:935-982, 1883.
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