Chapter 36: Hemostasis and Blood Coagulation PDF

CHAPTER 36 UNIT VI Hemostasis and Blood Coagulation Physical...

CHAPTER 36 UNIT VI Hemostasis and Blood Coagulation Physical and Chemical Characteristics of Platelets Events in Hemostasis Platelets (also called thrombocytes) are minute discs 1 to 4 micrometers in diameter. They are formed in the bone The term hemostasis means marrow from megakaryocytes, which are extremely large prevention of blood loss. cells of the hematopoietic series in the marrow; the mega- Whenever a vessel is severed karyocytes fragment into the minute platelets either in the or ruptured, hemostasis is achieved by several mechanisms: bone marrow or soon after entering the blood, especially (1) vascular constriction, (2) formation of a platelet plug, (3) as they squeeze through capillaries. The normal concen- formation of a blood clot as a result of blood coagulation, tration of platelets in the blood is between 150,000 and and (4) eventual growth of fibrous tissue into the blood clot 300,000 per microliter. to close the hole in the vessel permanently. Platelets have many functional characteristics of whole cells, even though they do not have nuclei and cannot reproduce. In their cytoplasm are such active Vascular Constriction factors as (1) actin and myosin molecules, which are con- tractile proteins similar to those found in muscle cells, Immediately after a blood vessel has been cut or ruptured, and still another contractile protein, thrombosthenin, the trauma to the vessel wall causes the smooth muscle in that can cause the platelets to contract; (2) residuals of the wall to contract; this instantaneously reduces the flow both the endoplasmic reticulum and the Golgi apparatus of blood from the ruptured vessel. The contraction results that synthesize various enzymes and especially store from (1) local myogenic spasm, (2) local autacoid factors large quantities of calcium ions; (3) mitochondria and from the traumatized tissues and blood platelets, and (3) enzyme systems that are capable of forming adenosine nervous reflexes. The nervous reflexes are initiated by pain triphosphate (ATP) and adenosine diphosphate (ADP); nerve impulses or other sensory impulses that originate (4) enzyme systems that synthesize prostaglandins, from the traumatized vessel or nearby tissues. However, which are local hormones that cause many vascular and even more vasoconstriction probably results from local other local tissue reactions; (5) an important protein myogenic contraction of the blood vessels initiated by called fibrin-stabilizing factor, which we discuss later in direct damage to the vascular wall. And, for the smaller relation to blood coagulation; and (6) a growth factor that vessels, the platelets are responsible for much of the vaso- causes vascular endothelial cells, vascular smooth mus- constriction by releasing a vasoconstrictor substance, cle cells, and fibroblasts to multiply and grow, thus caus- thromboxane A2. ing cellular growth that eventually helps repair damaged The more severely a vessel is traumatized, the greater vascular walls. the degree of vascular spasm. The spasm can last for many The cell membrane of the platelets is also important. minutes or even hours, during which time the processes On its surface is a coat of glycoproteins that repulses of platelet plugging and blood coagulation can take place. adherence to normal endothelium and yet causes adher- ence to injured areas of the vessel wall, especially to injured endothelial cells and even more so to any exposed Formation of the Platelet Plug collagen from deep within the vessel wall. In addition, the If the cut in the blood vessel is very small—indeed, platelet membrane contains large amounts of phospho- many very small vascular holes do develop throughout lipids that activate multiple stages in the blood-clotting the body each day—the cut is often sealed by a platelet process, as we discuss later. plug, rather than by a blood clot. To understand this, it Thus, the platelet is an active structure. It has a half- is important that we first discuss the nature of platelets life in the blood of 8 to 12 days, so over several weeks its themselves. functional processes run out. Then it is eliminated from 451 Unit VI Blood Cells, Immunity, and Blood Coagulation the circulation mainly by the tissue macrophage system. More than one half of the platelets are removed by mac- rophages in the spleen, where the blood passes through a latticework of tight trabeculae. 1. Severed vessel 2. Platelets agglutinate Mechanism of the Platelet Plug Platelet repair of vascular openings is based on sev- eral important functions of the platelet. When platelets come in contact with a damaged vascular surface, espe- cially with collagen fibers in the vascular wall, the platelets 3. Fibrin appears 4. Fibrin clot forms immediately change their own characteristics drastically. They begin to swell; they assume irregular forms with numerous irradiating pseudopods protruding from their surfaces; their contractile proteins contract forcefully and cause the release of granules that contain multiple active 5. Clot retraction occurs factors; they become sticky so that they adhere to collagen Figure 36-1 Clotting process in a traumatized blood vessel. in the tissues and to a protein called von Willebrand fac- (Modified from Seegers WH: Hemostatic Agents, 1948. Courtesy tor that leaks into the traumatized tissue from the plasma; of Charles C Thomas, Publisher, Ltd., Springfield, Ill.) they secrete large quantities of ADP; and their enzymes form thromboxane A2. The ADP and thromboxane in Within 3 to 6 minutes after rupture of a vessel, if the ves- turn act on nearby platelets to activate them as well, and sel opening is not too large, the entire opening or broken the stickiness of these additional platelets causes them to end of the vessel is filled with clot. After 20 minutes to an adhere to the original activated platelets. hour, the clot retracts; this closes the vessel still further. Therefore, at the site of any opening in a blood Platelets also play an important role in this clot retraction, vessel wall, the damaged vascular wall activates succes- as is discussed later. sively increasing numbers of platelets that themselves Table 36-1 Clotting Factors in Blood and Their Synonyms attract more and more additional platelets, thus form- ing a platelet plug. This is at first a loose plug, but it is usually successful in blocking blood loss if the vascular Clotting Factor Synonyms opening is small. Then, during the subsequent process Fibrinogen Factor I of blood coagulation, fibrin threads form. These attach Prothrombin Factor II tightly to the platelets, thus constructing an unyield- ing plug. Tissue factor Factor III; tissue thromboplastin Importance of the Platelet Mechanism for Closing Calcium Factor IV Vascular Holes. The platelet-plugging mechanism is Factor V Proaccelerin; labile factor; extremely important for closing minute ruptures in very Ac-globulin (Ac-G) small blood vessels that occur many thousands of times Factor VII Serum prothrombin conversion daily. Indeed, multiple small holes through the endothe- accelerator (SPCA); proconvertin; lial cells themselves are often closed by platelets actu- stable factor ally fusing with the endothelial cells to form additional Factor VIII Antihemophilic factor (AHF); endothelial cell membrane. A person who has few blood antihemophilic globulin (AHG); platelets develops each day literally thousands of small antihemophilic factor A hemorrhagic areas under the skin and throughout the internal tissues, but this does not occur in the normal Factor IX Plasma thromboplastin component (PTC); Christmas factor; person. antihemophilic factor B Factor X Stuart factor; Stuart-Prower factor Blood Coagulation in the Ruptured Vessel Factor XI Plasma thromboplastin antecedent The third mechanism for hemostasis is formation of the (PTA); antihemophilic factor C blood clot. The clot begins to develop in 15 to 20 seconds if the trauma to the vascular wall has been severe, and in Factor XII Hageman factor 1 to 2 minutes if the trauma has been minor. Activator Factor XIII Fibrin-stabilizing factor substances from the traumatized vascular wall, from Prekallikrein Fletcher factor platelets, and from blood proteins adhering to the trau- High-molecular-weight Fitzgerald factor; HMWK matized vascular wall initiate the clotting process. The kininogen (high-molecular-weight kininogen) physical events of this process are shown in Figure 36-1, Platelets and Table 36-1 lists the most important of the clotting factors. 452  Chapter 36 Hemostasis and Blood Coagulation Fibrous Organization or Dissolution Prothrombin of the Blood Clot Prothrombin Ca++ Once a blood clot has formed, it can follow one of two activator courses: (1) It can become invaded by fibroblasts, which UNIT VI Thrombin subsequently form connective tissue all through the clot, or (2) it can dissolve. The usual course for a clot that forms in a small hole of a vessel wall is invasion by Fibrinogen Fibrinogen monomer fibroblasts, beginning within a few hours after the clot is Ca++ formed (which is promoted at least partially by growth factor secreted by platelets). This continues to complete Fibrin fibers organization of the clot into fibrous tissue within about Thrombin activated fibrin-stabilizing 1 to 2 weeks. factor Conversely, when excess blood has leaked into the tis- sues and tissue clots have occurred where they are not Cross-linked fibrin fibers needed, special substances within the clot itself usually Figure 36-2 Schema for conversion of prothrombin to thrombin become activated. These function as enzymes to dissolve and polymerization of fibrinogen to form fibrin fibers. the clot, as discussed later in the chapter. the thrombin causes polymerization of fibrinogen mol- ecules into fibrin fibers within another 10 to 15 seconds. Mechanism of Blood Coagulation Thus, the rate-limiting factor in causing blood coagulation is usually the formation of prothrombin activator and not Basic Theory. More than 50 important substances the subsequent reactions beyond that point, because these that cause or affect blood coagulation have been found in terminal steps normally occur rapidly to form the clot. the blood and in the tissues—some that promote coag- Platelets also play an important role in the conversion ulation, called procoagulants, and others that inhibit of prothrombin to thrombin because much of the pro- coagulation, called anticoagulants. Whether blood will thrombin first attaches to prothrombin receptors on the coagulate depends on the balance between these two platelets already bound to the damaged tissue. groups of substances. In the blood stream, the anticoagu- lants normally predominate, so the blood does not coag- Prothrombin and Thrombin. Prothrombin is a ulate while it is circulating in the blood vessels. But when plasma protein, an alpha2-globulin, having a molecular a vessel is ruptured, procoagulants from the area of tissue weight of 68,700. It is present in normal plasma in a con- damage become “activated” and override the anticoagu- centration of about 15 mg/dl. It is an unstable protein that lants, and then a clot does develop. can split easily into smaller compounds, one of which is thrombin, which has a molecular weight of 33,700, almost General Mechanism. Clotting takes place in three exactly one half that of prothrombin. essential steps: (1) In response to rupture of the vessel or Prothrombin is formed continually by the liver, and it damage to the blood itself, a complex cascade of chemi- is continually being used throughout the body for blood cal reactions occurs in the blood involving more than a clotting. If the liver fails to produce prothrombin, in a day dozen blood coagulation factors. The net result is for- or so prothrombin concentration in the plasma falls too mation of a complex of activated substances collectively low to provide normal blood coagulation. called prothrombin activator. (2) The prothrombin acti- Vitamin K is required by the liver for normal activa- vator catalyzes conversion of prothrombin into thrombin. tion of prothrombin, as well as a few other clotting fac- (3) The thrombin acts as an enzyme to convert fibrinogen tors. Therefore, either lack of vitamin K or the presence of into fibrin fibers that enmesh platelets, blood cells, and liver disease that prevents normal prothrombin formation plasma to form the clot. can decrease the prothrombin level so low that a bleeding Let us discuss first the mechanism by which the blood tendency results. clot itself is formed, beginning with conversion of pro- thrombin to thrombin; then we will come back to the initi- Conversion of Fibrinogen to Fibrin—Formation ating stages in the clotting process by which prothrombin of the Clot activator is formed. Fibrinogen. Fibrinogen is a high-molecular-weight protein (MW 340,000) that occurs in the plasma in Conversion of Prothrombin to Thrombin quantities of 100 to 700 mg/dl. Fibrinogen is formed in the First, prothrombin activator is formed as a result of rupture liver, and liver disease can decrease the concentration of of a blood vessel or as a result of damage to special sub- circulating fibrinogen, as it does the concentration of pro- stances in the blood. Second, the prothrombin activator, in thrombin, pointed out earlier. the presence of sufficient amounts of ionic Ca++, causes con- Because of its large molecular size, little fibrinogen version of prothrombin to thrombin (Figure 36-2). Third, normally leaks from the blood vessels into the interstitial 453 Unit VI Blood Cells, Immunity, and Blood Coagulation fluids, and because fibrinogen is one of the essential fac- platelets entrapped in the clot continue to release proco- tors in the coagulation process, interstitial fluids ordinar- agulant substances, one of the most important of which ily do not coagulate. Yet, when the permeability of the is fibrin-stabilizing factor, which causes more and more capillaries becomes pathologically increased, fibrinogen cross-linking bonds between adjacent fibrin fibers. In does then leak into the tissue fluids in sufficient quantities addition, the platelets themselves contribute directly to to allow clotting of these fluids in much the same way that clot contraction by activating platelet thrombosthenin, plasma and whole blood can clot. actin, and myosin molecules, which are all contractile proteins in the platelets and cause strong contraction of Action of Thrombin on Fibrinogen to Form Fibrin. the platelet spicules attached to the fibrin. This also helps Thrombin is a protein enzyme with weak proteolytic compress the fibrin meshwork into a smaller mass. The capabilities. It acts on fibrinogen to remove four low- contraction is activated and accelerated by thrombin, molecular-weight peptides from each molecule of fibrin- as well as by calcium ions released from calcium stores ogen, forming one molecule of fibrin monomer that has in the mitochondria, endoplasmic reticulum, and Golgi the automatic capability to polymerize with other fibrin apparatus of the platelets. monomer molecules to form fibrin fibers. Therefore, As the clot retracts, the edges of the broken blood ves- many fibrin monomer molecules polymerize within sec- sel are pulled together, thus contributing still further to onds into long fibrin fibers that constitute the reticulum of hemostasis. the blood clot. In the early stages of polymerization, the fibrin mono- Positive Feedback of Clot Formation mer molecules are held together by weak noncovalent Once a blood clot has started to develop, it normally hydrogen bonding, and the newly forming fibers are extends within minutes into the surrounding blood. That not cross-linked with one another; therefore, the resul- is, the clot itself initiates a positive feedback to promote tant clot is weak and can be broken apart with ease. But more clotting. One of the most important causes of this is another process occurs during the next few minutes that the fact that the proteolytic action of thrombin allows it to greatly strengthens the fibrin reticulum. This involves act on many of the other blood-clotting factors in addition a substance called fibrin-stabilizing factor that is pres- to fibrinogen. For instance, thrombin has a direct prote- ent in small amounts in normal plasma globulins but is olytic effect on prothrombin itself, tending to convert this also released from platelets entrapped in the clot. Before into still more thrombin, and it acts on some of the blood- fibrin-stabilizing factor can have an effect on the fibrin clotting factors responsible for formation of prothrombin fibers, it must itself be activated. The same thrombin that activator. (These effects, discussed in subsequent para- causes fibrin formation also activates the fibrin-stabiliz- graphs, include acceleration of the actions of Factors VIII, ing factor. Then this activated substance operates as an IX, X, XI, and XII and aggregation of platelets.) Once a enzyme to cause covalent bonds between more and more critical amount of thrombin is formed, a positive feedback of the fibrin monomer molecules, as well as multiple develops that causes still more blood clotting and more cross-linkages between adjacent fibrin fibers, thus adding and more thrombin to be formed; thus, the blood clot tremendously to the three-dimensional strength of the continues to grow until blood leakage ceases. fibrin meshwork. Blood Clot. The clot is composed of a meshwork Initiation of Coagulation: Formation of fibrin fibers running in all directions and entrapping of Prothrombin Activator blood cells, platelets, and plasma. The fibrin fibers also Now that we have discussed the clotting process, we turn adhere to damaged surfaces of blood vessels; therefore, to the more complex mechanisms that initiate clotting the blood clot becomes adherent to any vascular opening in the first place. These mechanisms are set into play by and thereby prevents further blood loss. (1) trauma to the vascular wall and adjacent tissues, (2) trauma to the blood, or (3) contact of the blood with dam- Clot Retraction—Serum. Within a few minutes aged endothelial cells or with collagen and other tissue after a clot is formed, it begins to contract and usually elements outside the blood vessel. In each instance, this expresses most of the fluid from the clot within 20 to 60 leads to the formation of prothrombin activator, which minutes. The fluid expressed is called serum because all then causes prothrombin conversion to thrombin and all its fibrinogen and most of the other clotting factors have the subsequent clotting steps. been removed; in this way, serum differs from plasma. Prothrombin activator is generally considered to be Serum cannot clot because it lacks these factors. formed in two ways, although, in reality, the two ways Platelets are necessary for clot retraction to occur. interact constantly with each other: (1) by the extrinsic Therefore, failure of clot retraction is an indication that the pathway that begins with trauma to the vascular wall and number of platelets in the circulating blood might be low. surrounding tissues and (2) by the intrinsic pathway that Electron micrographs of platelets in blood clots show that begins in the blood itself. they become attached to the fibrin fibers in such a way that In both the extrinsic and the intrinsic pathways, a they actually bond different fibers together. Furthermore, series of different plasma proteins called blood-clotting 454  Chapter 36 Hemostasis and Blood Coagulation factors plays a major role. Most of these proteins are inac- prothrombin to form thrombin, and the clotting process tive forms of proteolytic enzymes. When converted to the proceeds as already explained. At first, the Factor V active forms, their enzymatic actions cause the successive, in the prothrombin activator complex is inactive, but cascading reactions of the clotting process. once clotting begins and thrombin begins to form, Most of the clotting factors, which are listed in Table the proteolytic action of thrombin activates Factor V. UNIT VI 36-1, are designated by Roman numerals. To indicate the This then becomes an additional strong accelerator of activated form of the factor, a small letter “a” is added prothrombin activation. Thus, in the final prothrom- after the Roman numeral, such as Factor VIIIa to indicate bin activator complex, activated Factor X is the actual the activated state of Factor VIII. protease that causes splitting of prothrombin to form thrombin; activated Factor V greatly accelerates this Extrinsic Pathway for Initiating Clotting protease activity, and platelet phospholipids act as a The extrinsic pathway for initiating the formation of pro- vehicle that further accelerates the process. Note espe- thrombin activator begins with a traumatized vascular cially the positive feedback effect of thrombin, acting wall or traumatized extravascular tissues that come in through Factor V, to accelerate the entire process once contact with the blood. This leads to the following steps, it begins. as shown in Figure 36-3: Intrinsic Pathway for Initiating Clotting 1. Release of tissue factor. Traumatized tissue releases a complex of several factors called tissue factor or tis- The second mechanism for initiating formation of pro- sue thromboplastin. This factor is composed especially thrombin activator, and therefore for initiating clotting, of phospholipids from the membranes of the tissue begins with trauma to the blood or exposure of the blood plus a lipoprotein complex that functions mainly as to collagen from a traumatized blood vessel wall. Then the a proteolytic enzyme. process continues through the series of cascading reac- 2. Activation of Factor X—role of Factor VII and tissue tions shown in Figure 36-4. factor. The lipoprotein complex of tissue factor fur- 1. Blood trauma causes (1) activation of Factor XII and (2) ther complexes with blood coagulation Factor VII and, release of platelet phospholipids. Trauma to the blood in the presence of calcium ions, acts enzymatically on or exposure of the blood to vascular wall collagen alters Factor X to form activated Factor X (Xa). two important clotting factors in the blood: Factor XII 3. Effect of Xa to form prothrombin activator—role of and the platelets. When Factor XII is disturbed, such Factor V. The activated Factor X combines immedi- as by coming into contact with collagen or with a wet- ately with tissue phospholipids that are part of tissue table surface such as glass, it takes on a new molec- factors or with additional phospholipids released from ular configuration that converts it into a proteolytic platelets, as well as with Factor V to form the complex enzyme called “activated Factor XII.” Simultaneously, called prothrombin activator. Within a few seconds, the blood trauma also damages the platelets because in the presence of calcium ions (Ca++), this splits of adherence to either collagen or a wettable surface (or by damage in other ways), and this releases plate- let phospholipids that contain the lipoprotein called (1) Tissue trauma platelet factor 3, which also plays a role in subsequent clotting reactions. 2. Activation of Factor XI. The activated Factor XII acts Tissue factor enzymatically on Factor XI to activate this factor as well, which is the second step in the intrinsic pathway. This reaction also requires HMW (high-molecular- (2) Vll VIIa weight) kininogen and is accelerated by prekallikrein. 3. Activation of Factor IX by activated Factor XI. The acti- X Activated X (Xa) vated Factor XI then acts enzymatically on Factor IX to activate this factor as well. Ca++ 4. Activation of Factor X—role of Factor VIII. The acti- V Ca++ vated Factor IX, acting in concert with activated Factor Prothrombin VIII and with the platelet phospholipids and factor 3 (3) activator from the traumatized platelets, activates Factor X. It Platelet phospholipids is clear that when either Factor VIII or platelets are in short supply, this step is deficient. Factor VIII is the Prothrombin Thrombin factor that is missing in a person who has classic hemo- philia, for which reason it is called antihemophilic fac- Ca++ tor. Platelets are the clotting factor that is lacking in the Figure 36-3 Extrinsic pathway for initiating blood clotting. bleeding disease called thrombocytopenia. 455 Unit VI Blood Cells, Immunity, and Blood Coagulation Blood trauma or contact with collagen (1) XII Activated XII (XIIa) (HMW kininogen, prekallikrein) (2) XI Activated XI (XIa) Ca++ (3) IX Activated IX (IXa) VIII Thrombin VIIIa Ca++ (4) X Activated X (Xa) (5) Platelet phospholipids Thrombin Ca++ V Prothrombin activator Platelet phospholipids Prothrombin Thrombin Ca++ Figure 36-4 Intrinsic pathway for initiating blood clotting. 5. Action of activated Factor X to form prothrombin acti- Interaction Between the Extrinsic and Intrinsic vator—role of Factor V. This step in the intrinsic path- Pathways—Summary of Blood-Clotting Initiation way is the same as the last step in the extrinsic pathway. It is clear from the schemas of the intrinsic and extrinsic sys- That is, activated Factor X combines with Factor V and tems that after blood vessels rupture, clotting occurs by both platelet or tissue phospholipids to form the complex pathways simultaneously. Tissue factor initiates the extrin- called prothrombin activator. The prothrombin acti- sic pathway, whereas contact of Factor XII and platelets with vator in turn initiates within seconds the cleavage of collagen in the vascular wall initiates the intrinsic pathway. prothrombin to form thrombin, thereby setting into An especially important difference between the extrin- motion the final clotting process, as described earlier. sic and intrinsic pathways is that the extrinsic pathway can be explosive; once initiated, its speed of completion to the Role of Calcium Ions in the Intrinsic final clot is limited only by the amount of tissue factor and Extrinsic Pathways released from the traumatized tissues and by the quanti- Except for the first two steps in the intrinsic pathway, cal- ties of Factors X, VII, and V in the blood. With severe tis- cium ions are required for promotion or acceleration of sue trauma, clotting can occur in as little as 15 seconds. all the blood-clotting reactions. Therefore, in the absence The intrinsic pathway is much slower to proceed, usually of calcium ions, blood clotting by either pathway does not requiring 1 to 6 minutes to cause clotting. occur. In the living body, the calcium ion concentration sel- Prevention of Blood Clotting in the Normal dom falls low enough to significantly affect the kinetics of Vascular System—Intravascular Anticoagulants blood clotting. But, when blood is removed from a per- Endothelial Surface Factors. Probably the most son, it can be prevented from clotting by reducing the important factors for preventing clotting in the normal calcium ion concentration below the threshold level for vascular system are (1) the smoothness of the endothe- clotting, either by deionizing the calcium by causing it to lial cell surface, which prevents contact activation of the react with substances such as citrate ion or by precipitat- intrinsic clotting system; (2) a layer of glycocalyx on the ing the calcium with substances such as oxalate ion. endothelium (glycocalyx is a mucopolysaccharide adsorbed 456  Chapter 36 Hemostasis and Blood Coagulation to the surfaces of the endothelial cells), which repels clot- quantities of heparin that diffuse into the circulatory sys- ting factors and platelets, thereby preventing activation tem. The basophil cells of the blood, which are functionally of clotting; and (3) a protein bound with the endothelial almost identical to the mast cells, release small quantities membrane, thrombomodulin, which binds thrombin. Not of heparin into the plasma. only does the binding of thrombin with thrombomodu- Mast cells are abundant in tissue surrounding the cap- UNIT VI lin slow the clotting process by removing thrombin, but illaries of the lungs and, to a lesser extent, capillaries of the the thrombomodulin-thrombin complex also activates a liver. It is easy to understand why large quantities of hepa- plasma protein, protein C, that acts as an anticoagulant by rin might be needed in these areas because the capillaries inactivating activated Factors V and VIII. of the lungs and liver receive many embolic clots formed When the endothelial wall is damaged, its smoothness in slowly flowing venous blood; sufficient formation of and its glycocalyx-thrombomodulin layer are lost, which heparin prevents further growth of the clots. activates both Factor XII and the platelets, thus setting off the intrinsic pathway of clotting. If Factor XII and plate- Lysis of Blood Clots—Plasmin lets come in contact with the subendothelial collagen, the The plasma proteins contain a euglobulin called plas- activation is even more powerful. minogen (or profibrinolysin) that, when activated, becomes a substance called plasmin (or fibrinolysin). Plasmin is Antithrombin Action of Fibrin and Antithrombin a proteolytic enzyme that resembles trypsin, the most III. Among the most important anticoagulants in the important proteolytic digestive enzyme of pancreatic blood are those that remove thrombin from the blood. secretion. Plasmin digests fibrin fibers and some other The most powerful of these are (1) the fibrin fibers that protein coagulants such as fibrinogen, Factor V, Factor are formed during the process of clotting and (2) an alpha- VIII, prothrombin, and Factor XII. Therefore, whenever globulin called antithrombin III or antithrombin-heparin plasmin is formed, it can cause lysis of a clot by destroy- cofactor. ing many of the clotting factors, thereby sometimes even While a clot is forming, about 85 to 90 percent of causing hypocoagulability of the blood. the thrombin formed from the prothrombin becomes adsorbed to the fibrin fibers as they develop. This helps Activation of Plasminogen to Form Plasmin, Then prevent the spread of thrombin into the remaining blood Lysis of Clots. When a clot is formed, a large amount of and, therefore, prevents excessive spread of the clot. plasminogen is trapped in the clot along with other plasma The thrombin that does not adsorb to the fibrin fibers proteins. This will not become plasmin or cause lysis of the soon combines with antithrombin III, which further clot until it is activated. The injured tissues and vascular blocks the effect of the thrombin on the fibrinogen and endothelium very slowly release a powerful activator called then also inactivates the thrombin itself during the next tissue plasminogen activator (t-PA) that a few days later, after 12 to 20 minutes. the clot has stopped the bleeding, eventually converts plas- minogen to plasmin, which in turn removes the remaining Heparin. Heparin is another powerful anticoagulant, unnecessary blood clot. In fact, many small blood vessels in but its concentration in the blood is normally low, so only which blood flow has been blocked by clots are reopened under special physiologic conditions does it have signifi- by this mechanism. Thus, an especially important func- cant anticoagulant effects. However, heparin is used widely tion of the plasmin system is to remove minute clots from as a pharmacological agent in medical practice in much millions of tiny peripheral vessels that eventually would higher concentrations to prevent intravascular clotting. become occluded were there no way to clear them. The heparin molecule is a highly negatively charged conjugated polysaccharide. By itself, it has little or no anticoagulant properties, but when it combines with anti- thrombin III, the effectiveness of antithrombin III for Conditions That Cause Excessive removing thrombin increases by a hundredfold to a thou- Bleeding in Humans sandfold, and thus it acts as an anticoagulant. Therefore, Excessive bleeding can result from deficiency of any one of in the presence of excess heparin, removal of free throm- the many blood-clotting factors. Three particular types of bin from the circulating blood by antithrombin III is bleeding tendencies that have been studied to the greatest almost instantaneous. extent are discussed here: bleeding caused by (1) vitamin The complex of heparin and antithrombin III removes K deficiency, (2) hemophilia, and (3) thrombocytopenia several other activated coagulation factors in addition to (platelet deficiency). thrombin, further enhancing the effectiveness of antico- agulation. The others include activated Factors XII, XI, X, and IX. Decreased Prothrombin, Factor VII, Factor IX, Heparin is produced by many different cells of the body, and Factor X Caused by Vitamin K Deficiency but especially large quantities are formed by the basophilic With few exceptions, almost all the blood-clotting factors mast cells located in the pericapillary connective tissue are formed by the liver. Therefore, diseases of the liver throughout the body. These cells continually secrete small such as hepatitis, cirrhosis, and acute yellow atrophy can 457 Unit VI Blood Cells, Immunity, and Blood Coagulation sometimes depress the clotting system so greatly that the The bleeding trait in hemophilia can have various patient develops a severe tendency to bleed. degrees of severity, depending on the character of the Another cause of depressed formation of clotting fac- genetic deficiency. Bleeding usually does not occur except tors by the liver is vitamin K deficiency. Vitamin K is an after trauma, but in some patients, the degree of trauma essential factor to a liver carboxylase that adds a carboxyl required to cause severe and prolonged bleeding may be group to glutamic acid residues on five of the important so mild that it is hardly noticeable. For instance, bleeding clotting factors: prothrombin, Factor VII, Factor IX, Factor can often last for days after extraction of a tooth. X, and protein C. In adding the carboxyl group to glutamic Factor VIII has two active components, a large compo- acid residues on the immature clotting factors, vitamin K nent with a molecular weight in the millions and a smaller is oxidized and becomes inactive. Another enzyme, vita- component with a molecular weight of about 230,000. The min K epoxide reductase complex 1 (VKOR c1), reduces smaller component is most important in the intrinsic path- vitamin K back to its active form. way for clotting, and it is deficiency of this part of Factor VIII In the absence of active vitamin K, subsequent insuf- that causes classic hemophilia. Another bleeding disease with ficiency of these coagulation factors in the blood can lead somewhat different characteristics, called von Willebrand’s to serious bleeding tendencies. disease, results from loss of the large component. Vitamin K is continually synthesized in the intestinal When a person with classic hemophilia experiences tract by bacteria, so vitamin K deficiency seldom occurs severe prolonged bleeding, almost the only therapy that is in the normal person as a result of vitamin K absence truly effective is injection of purified Factor VIII. The cost of from the diet (except in neonates before they establish Factor VIII is high, because it is gathered from human blood their intestinal bacterial flora). However, in gastrointesti- and only in extremely small quantities. However, increasing nal disease, vitamin K deficiency often occurs as a result production and use of recombinant Factor VIII will make this of poor absorption of fats from the gastrointestinal tract. treatment available to more patients with classic hemophilia. The reason is that vitamin K is fat soluble and ordinarily absorbed into the blood along with the fats. One of the most prevalent causes of vitamin K defi- Thrombocytopenia ciency is failure of the liver to secrete bile into the gastroin- Thrombocytopenia means the presence of very low num- testinal tract (which occurs either as a result of obstruction bers of platelets in the circulating blood. People with of the bile ducts or as a result of liver disease). Lack of bile thrombocytopenia have a tendency to bleed, as do hemo- prevents adequate fat digestion and absorption and, there- philiacs, except that the bleeding is usually from many fore, depresses vitamin K absorption as well. Thus, liver small venules or capillaries, rather than from larger ves- disease often causes decreased production of prothrombin sels, as in hemophilia. As a result, small punctate hemor- and some other clotting factors both because of poor vita- rhages occur throughout all the body tissues. The skin of min K absorption and because of the diseased liver cells. such a person displays many small, purplish blotches, giv- Because of this, vitamin K is injected into surgical patients ing the disease the name thrombocytopenic purpura. As with liver disease or with obstructed bile ducts before per- stated earlier, platelets are especially important for repair forming the surgical procedure. Ordinarily, if vitamin K is of minute breaks in capillaries and other small vessels. given to a deficient patient 4 to 8 hours before the opera- Ordinarily, bleeding will not occur until the number tion and the liver parenchymal cells are at least one-half of platelets in the blood falls below 50,000/ l, rather than normal in function, sufficient clotting factors will be pro- the normal 150,000 to 300,000. Levels as low as 10,000/ l duced to prevent excessive bleeding during the operation. are frequently lethal. Even without making specific platelet counts in the blood, sometimes one can suspect the existence of throm- Hemophilia bocytopenia if the person’s blood fails to retract, because, as Hemophilia is a bleeding disease that occurs almost pointed out earlier, clot retraction is normally dependent on exclusively in males. In 85 percent of cases, it is caused release of multiple coagulation factors from the large num- by an abnormality or deficiency of Factor VIII; this type bers of platelets entrapped in the fibrin mesh of the clot. of hemophilia is called hemophilia A or classic hemo- Most people with thrombocytopenia have the disease philia. About 1 of every 10,000 males in the United States known as idiopathic thrombocytopenia, which means has classic hemophilia. In the other 15 percent of hemo- thrombocytopenia of unknown cause. In most of these philia patients, the bleeding tendency is caused by defi- people, it has been discovered that, for unknown reasons, ciency of Factor IX. Both of these factors are transmitted specific antibodies have formed and react against the genetically by way of the female chromosome. Therefore, platelets themselves to destroy them. Relief from bleed- almost never will a woman have hemophilia because at ing for 1 to 4 days can often be effected in a patient with least one of her two X chromosomes will have the appro- thrombocytopenia by giving fresh whole blood transfu- priate genes. If one of her X chromosomes is deficient, sions that contain large numbers of platelets. Also, sple- she will be a hemophilia carrier, transmitting the disease nectomy is often helpful, sometimes effecting almost to half of her male offspring and transmitting the carrier complete cure because the spleen normally removes large state to half of her female offspring. numbers of platelets from the blood. 458  Chapter 36 Hemostasis and Blood Coagulation Disseminated Intravascular Coagulation Thromboembolic Conditions in the Human Being Occasionally the clotting mechanism becomes activated in widespread areas of the circulation, giving rise to the Thrombi and Emboli. An abnormal clot that devel- condition called disseminated intravascular coagulation. UNIT VI ops in a blood vessel is called a thrombus. Once a clot has This often results from the presence of large amounts of developed, continued flow of blood past the clot is likely traumatized or dying tissue in the body that releases great to break it away from its attachment and cause the clot to quantities of tissue factor into the blood. Frequently, the flow with the blood; such freely flowing clots are known clots are small but numerous, and they plug a large share as emboli. Also, emboli that originate in large arteries or of the small peripheral blood vessels. This occurs espe- in the left side of the heart can flow peripherally and plug cially in patients with widespread septicemia, in which arteries or arterioles in the brain, kidneys, or elsewhere. either circulating bacteria or bacterial toxins—especially Emboli that originate in the venous system or in the right endotoxins—activate the clotting mechanisms. Plugging side of the heart generally flow into the lungs to cause pul- of small peripheral vessels greatly diminishes delivery of monary arterial embolism. oxygen and other nutrients to the tissues—a situation that leads to or exacerbates circulatory shock. It is partly for Cause of Thromboembolic Conditions. The causes this reason that septicemic shock is lethal in 85 percent or of thromboembolic conditions in the human being are more of patients. usually twofold: (1) Any roughened endothelial surface of a A peculiar effect of disseminated intravascular coag- vessel—as may be caused by arteriosclerosis, infection, or ulation is that the patient on occasion begins to bleed. trauma—is likely to initiate the clotting process. (2) Blood The reason for this is that so many of the clotting fac- often clots when it flows very slowly through blood vessels, tors are removed by the widespread clotting that too few where small quantities of thrombin and other procoagulants procoagulants remain to allow normal hemostasis of the are always being formed. remaining blood. Use of t-PA in Treating Intravascular Clots. Genetically engineered t-PA (tissue plasminogen activa- Anticoagulants for Clinical Use tor) is available. When delivered directly to a thrombosed area through a catheter, it is effective in activating plas- In some thromboembolic conditions, it is desirable to minogen to plasmin, which in turn can dissolve some intra- delay the coagulation process. Various anticoagulants vascular clots. For instance, if used within the first hour have been developed for this purpose. The ones most or so after thrombotic occlusion of a coronary artery, the useful clinically are heparin and the coumarins. heart is often spared serious damage. Heparin as an Intravenous Anticoagulant Femoral Venous Thrombosis and Massive Commercial heparin is extracted from several different Pulmonary Embolism animal tissues and prepared in almost pure form. Injection Because clotting almost always occurs when blood flow of relatively small quantities, about 0.5 to 1 mg/kg of is blocked for many hours in any vessel of the body, the body weight, causes the blood-clotting time to increase immobility of patients confined to bed plus the practice from a normal of about 6 minutes to 30 or more minutes. of propping the knees with pillows often causes intravas- Furthermore, this change in clotting time occurs instan- cular clotting because of blood stasis in one or more of taneously, thereby immediately preventing or slowing the leg veins for hours at a time. Then the clot grows, further development of a thromboembolic condition. mainly in the direction of the slowly moving venous The action of heparin lasts about 1.5 to 4 hours. The blood, sometimes growing the entire length of the leg injected heparin is destroyed by an enzyme in the blood veins and occasionally even up into the common iliac known as heparinase. vein and inferior vena cava. Then, about 1 time out of every 10, a large part of the clot disengages from its Coumarins as Anticoagulants attachments to the vessel wall and flows freely with the When a coumarin, such as warfarin, is given to a patient, venous blood through the right side of the heart and into the amounts of active prothrombin and Factors VII, IX, the pulmonary arteries to cause massive blockage of the and X, all formed by the liver, begin to fall. Warfarin causes pulmonary arteries, called massive pulmonary embolism. this effect by inhibiting the enzyme, vitamin K epoxide If the clot is large enough to occlude both of the pulmo- reductase complex 1 (VKOR c1). As discussed previously, nary arteries at the same time, immediate death ensues. this enzyme converts the inactive, oxidized form of vita- If only one pulmonary artery is blocked, death may not min K to its active, reduced form. By inhibiting VKOR occur, or the embolism may lead to death a few hours to c1, warfarin decreases the available active form of vita- several days later because of further growth of the clot min K in the tissues. When this occurs, the coagulation within the pulmonary vessels. But, again, t-PA therapy factors are no longer carboxylated and are biologically can be a lifesaver. inactive. Over several days the body stores of the active 459 Unit VI Blood Cells, Immunity, and Blood Coagulation coagulation factors degrade and are replaced by inactive the level of calcium ion in the blood, which can result in factors. Although the coagulation factors continue to be tetany and convulsive death. produced, they have greatly decreased coagulant activity. After administration of an effective dose of warfarin, the coagulant activity of the blood decreases to about 50 Blood Coagulation Tests percent of normal by the end of 12 hours and to about 20 percent of normal by the end of 24 hours. In other words, Bleeding Time the coagulation process is not blocked immediately but When a sharp-pointed knife is used to pierce the tip of the must await the degradation of the active prothrombin finger or lobe of the ear, bleeding ordinarily lasts for 1 to and the other affected coagulation factors already present 6 minutes. The time depends largely on the depth of the in the plasma. Normal coagulation usually returns 1 to 3 wound and the degree of hyperemia in the finger or ear days after discontinuing coumarin therapy. lobe at the time of the test. Lack of any one of several of the clotting factors can prolong the bleeding time, but it is Prevention of Blood Coagulation especially prolonged by lack of platelets. Outside the Body Clotting Time Although blood removed from the body and held in a glass test tube normally clots in about 6 minutes, blood Many methods have been devised for determining blood collected in siliconized containers often does not clot for 1 clotting times. The one most widely used is to collect hour or more. The reason for this delay is that preparing blood in a chemically clean glass test tube and then to tip the surfaces of the containers with silicone prevents con- the tube back and forth about every 30 seconds until the tact activation of platelets and Factor XII, the two princi- blood has clotted. By this method, the normal clotting pal factors that initiate the intrinsic clotting mechanism. time is 6 to 10 minutes. Procedures using multiple test Conversely, untreated glass containers allow contact tubes have also been devised for determining clotting time activation of the platelets and Factor XII, with rapid more accurately. development of clots. Unfortunately, the clotting time varies widely, depend- Heparin can be used for preventing coagulation of ing on the method used for measuring it, so it is no lon- blood outside the body, as well as in the body. Heparin is ger used in many clinics. Instead, measurements of the especially used in surgical procedures in which the blood clotting factors themselves are made, using sophisticated must be passed through a heart-lung machine or artificial chemical procedures. kidney machine and then back into the person. Various substances that decrease the concentration of Prothrombin Time and International calcium ions in the blood can also be used for preventing Normalized Ratio blood coagulation outside the body. For instance, a solu- Prothrombin time gives an indication of the concentra- ble oxalate compound mixed in a very small quantity with tion of prothrombin in the blood. Figure 36-5 shows the a sample of blood causes precipitation of calcium oxalate relation of prothrombin concentration to prothrombin from the plasma and thereby decreases the ionic calcium level so much that blood coagulation is blocked. Any substance that deionizes the blood calcium will 100 prevent coagulation. The negatively charged citrate ion is especially valuable for this purpose, mixed with blood Concentration (percent of normal) usually in the form of sodium, ammonium, or potassium citrate. The citrate ion combines with calcium in the blood to cause an un-ionized calcium compound, and the lack of ionic calcium prevents coagulation. Citrate anticoagulants have an important advantage over the oxalate anticoagu- 50.0 lants because oxalate is toxic to the body, whereas mod- erate quantities of citrate can be injected intravenously. After injection, the citrate ion is removed from the blood within a few minutes by the liver and is polymerized into 25.0 glucose or metabolized directly for energy. Consequently, 500 milliliters of blood that has been rendered incoagula- 12.5 6.25 ble by citrate can ordinarily be transfused into a recipient 0 within a few minutes without dire consequences. But if 0 10 20 30 40 50 60 the liver is damaged or if large quantities of citrated blood Prothrombin time or plasma are given too rapidly (within fractions of a min- (seconds) ute), the citrate ion may not be removed quickly enough, Figure 36-5 Relation of prothrombin concentration in the blood and the citrate can, under these conditions, greatly depress to “prothrombin time.” 460  Chapter 36 Hemostasis and Blood Coagulation time. The method for determining prothrombin time is blood clotting factors. In each of these tests, excesses of the following. calcium ions and all the other factors besides the one being Blood removed from the patient is immediately tested are added to oxalated blood all at once. Then the oxalated so that none of the prothrombin can change time required for coagulation is determined in the same into thrombin. Then, a large excess of calcium ion and manner as for prothrombin time. If the factor being tested UNIT VI tissue factor is quickly mixed with the oxalated blood. is deficient, the coagulation time is prolonged. The time The excess calcium nullifies the effect of the oxalate, and itself can then be used to quantitate the concentration of the tissue factor activates the prothrombin-to-thrombin the factor. reaction by means of the extrinsic clotting pathway. The time required for coagulation to take place is known as the prothrombin time. The shortness of the time is deter- Bibliography mined mainly by prothrombin concentration. The normal Andrews RK, Berndt MC: Platelet adhesion: a game of catch and release, prothrombin time is about 12 seconds. In each laboratory, J Clin Invest 118:3009, 2008. a curve relating prothrombin concentration to prothrom- Brass LF, Zhu L, Stalker TJ: Minding the gaps to promote thrombus growth bin time, such as that shown in Figure 36-5, is drawn for and stability, J Clin Invest 115:3385, 2005. the method used so that the prothrombin in the blood can Crawley JT, Lane DA: The haemostatic role of tissue factor pathway inhibi- tor, Arterioscler Thromb Vasc Biol 28:233, 2008. be quantified. Furie B, Furie BC: Mechanisms of thrombus formation, N Engl J Med The results obtained for prothrombin time may vary 359:938, 2008. considerably even in the same individual if there are dif- Gailani D, Renné T: Intrinsic pathway of coagulation and arterial thrombo- ferences in activity of the tissue factor and the analytical sis, Arterioscler Thromb Vasc Biol 27:2507, 2007. system used to perform the test. Tissue factor is isolated Jennings LK: Role of platelets in atherothrombosis, Am J Cardiol 103(3 Suppl):4A, 2009. from human tissues, such as placental tissue, and differ- Koreth R, Weinert C, Weisdorf DJ, et al: Measurement of bleeding severity: ent batches may have different activity. The international a critical review, Transfusion 44:605, 2004. normalized ratio (INR) was devised as a way to standard- Nachman RL, Rafii S: Platelets, petechiae, and preservation of the vascular ize measurements of prothrombin time. For each batch wall, N Engl J Med 359:1261, 2008. of tissue factor, the manufacturer assigns an international Pabinger I, Ay C: Biomarkers and venous thromboembolism, Arterioscler Thromb Vasc Biol 29:332, 2009. sensitivity index (ISI), which indicates the activity of the Rijken DC, Lijnen HR: New insights into the molecular mechanisms of the tissue factor with a standardized sample. The ISI usually fibrinolytic system, J Thromb Haemost 7:4, 2009. varies between 1.0 and 2.0. The INR is the ratio of the per- Schmaier AH: The elusive physiologic role of Factor XII, J Clin Invest son’s prothrombin time to a normal control sample raised 118:3006, 2008. to the power of the ISI: Smyth SS, Woulfe DS, Weitz JI, et al: 2008 Platelet Colloquium Participants. G-protein-coupled receptors as signaling targets for antiplatelet ther- PTtest ISI apy, Arterioscler Thromb Vasc Biol. 29:449, 2009. INR = Tapson VF: Acute pulmonary embolism, N Engl J Med 358:1037, 2008. PTnormal Toh CH, Dennis M: Disseminated intravascular coagulation: old disease, new hope, BMJ 327:974, 2003. The normal range for INR in a healthy person is 0.9 to Tsai HM: Advances in the pathogenesis, diagnosis, and treatment of 1.3. A high INR level (e.g., 4 or 5) indicates a high risk of thrombotic thrombocytopenic purpura, J Am Soc Nephrol 14:1072, bleeding, whereas a low INR (e.g., 0.5) suggests that there 2003. Tsai HM: Platelet activation and the formation of the platelet plug: defi- is a chance of having a clot. Patients on warfarin therapy ciency of ADAMTS13 causes thrombotic thrombocytopenic purpura, usually have an INR of 2.0 to 3.0. Arterioscler Thromb Vasc Biol 23:388, 2003. Tests similar to that for prothrombin time and INR VandenDriessche T, Collen D, Chuah MK: Gene therapy for the hemophilias, have been devised to determine the quantities of other J Thromb Haemost 1:1550, 2003. 461