an overview of is

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An Overview of Hemostasis*

*Address correspondence to: Dr. G. Daniel Boon, DepartmentofPathology, College ofVeterinary Medicine, University ofMis-souri, Columbia, Missouri 65205.

G. DANIEL BOON

Department ofPathology, College of Veterinary Medicine,University of Missouri, Columbia, Missouri 65205

ABSTRACT

Hemostasis is a remarkable and a remarkably complex mechanism. It can maintain blood in a fluid state

intravascularly but very quickly changes blood to a jellylike mass upon disruption of the vasculature. Thisreview will give a synopsis of the 3 phases of hemostasis: platelet, vascular, and coagulation. Fibrinolysisand control mechanisms of hemostasis will also be covered. In addition, brief descriptions of the clinicaland laboratory evaluation of patients and the diagnosis ofbleeding disorders will be presented.

Keywords. Platelet function; coagulation; bleeding diatheses; coagulation testing; thrombocytopenia

INTRODUCTION

The hemostatic process is remarkable in its abilityto stop the flow of blood from a breach in any rea-

sonably sized vessel within minutes. Yet blood ismaintained in a fluid state within the vascular sys-tem. This is accomplishedby the coordinated effortsof 3 distinct but intimately related mechanisms: thevascular, the platelet, and the coagulation phases ofhemostasis (90). These same mechanisms, when

overactive or inappropriately activated, can resultin thrombosis, embolism, or disseminated intra-vascular coagulation. In this article, I will providean overview of the hemostatic mechanism and an

approach to the diagnosis of hemorrhagic disorders.Because of space limitations and the expertise ofother authors within this issue, less attention to

platelet function and fibrinolysis will be given here.

HEMOSTATIC MECHANISMS

Platelets

Platelets perform functions in all phases of he-mostasis. Most apparently, they provide primaryhemostasisby producing the platelet plug that formsalmost immediately after a small vessel has been

disrupted (88, 90).As illustrated in Fig. 1, plateletsbegin adhering to the subendothelium within sec-onds after it has been exposed (84).After 30-60 secthe first fibrin strands can be seen interspersedamongthe platelets, and after several minutes the plateletplug is completely formed and stabilized by fibrin

(88). Within a few hours, the platelets lose their

integrity, and the plug appears as a mass of fibrinstrands (36, 84). This familiar sequence occurs manytimes every day and involves all phases of hemo-stasis, but platelets are central and all of their basicfunctions are involved. The fibrin formation occurs

because the platelets, in addition to adhering to thesubendothelium (adhesion) (88) and each other (ag-gregation) ( 11 ), provide a surface for the assembly

of coagulation factors that lead to the generation ofthrombin and ultimately fibrin (83, 100). Secretionis of course involved in the recruitment of more

platelets into this activity, but it also supplies a largenumber of other factors with a wide variety of ac-tivities (11, 93). Thus, the 4 primary platelet func-tions are adhesion, aggregation (cohesion), secre-tion, and procoagulant activity.Adhesion. When vessels are damaged, platelets

will adhere to different components of the suben-

dothelium, depending on the flow rate ofthe blood.In low flow areas, platelets will adhere to and spread

upon collagen and fibronectin. In higher flow areas,such as arterioles, platelet adhesion depends on the

presence ofvon Willebrand factor (vWF) in the sub-endothelium (1, 2, 33, 73, 103). Von Willebrandfactor is a large multimeric protein (27) synthesizedby megakaryocytes (91) and endothelial cells (42).Megakaryocytes, in humans, package vWF into thealpha granules of platelets (14, 91 ), whereas endo-thelial cells secrete it into both the plasma and thesubendothelium (94). In the dog, platelets containlittle vWF (72). In the subendothelium, vWF bindsto matrix and awaits vascular injury and the arrival

ofplatelets. In the plasma, vWF complexes with thecoagulation cofactor factor VIII, resulting in vWF


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FIG. I. -Formation ofthe platelet plug in primary hemostasis.

serving as a carrier protein for factor VIII and pro-longing its half-life (37, 67, 106).Aggregation. After a layer of platelets has accu-mulated on the injured subendothelium, additional

platelets participate in the formation of a complete

plug in a process known as platelet aggregation (90,102). This occurs through active platelet metabo-lism (11, 38) and stimulation by specific agonists(11, 30, 102). Platelet aggregation can be studied invitro with instruments called aggregometers (30).Aggregometers measure the transmission of lightthrough a suspension of stirred platelets.Aggrega-tion is initiated by the addition of agonists and, as

clumps ofplatelets form, light passes more freely asthe turbidity of the suspension decreases (30, 102).Aggregation can be divided into primary and sec-

ondary phases. Primary aggregation occurs as the

direct consequence of the agonist, is reversible, andis not accompanied by secretion. Secondary aggre-gation occurs with secretion and is manifested bythe formation of large irreversibly aggregated plate-lets.At high agonist concentrations, primary and

secondary aggregation may occur simultaneously (30,102).

Platelet agonists can be classified according towhether or not they stimulate secretion indepen-dently ofsecondary aggregation. Strong agonists canand weak agonists cannot stimulate secretion in-

dependently of aggregation. That is, strong agonists

can stimulate platelets to secrete their granule con-tents even in the presence of aspirin and other in-hibitors of cyclooxygenase; weak agonists cannot.

Obviously there is another pathway to release be-sides cyclooxygenase. Strong agonists include

thrombin and collagen. Weak agonists includeADPand epinephrine (11). Miscellaneous agonists are

platelet-activating factor, serotonin, and vasopres-sin.

The signal produced by the agonists and the re-sponse of the platelets, especially aggregation andsecretion, are coupled by complex biochemical re-actions that are not all completely clear.

Briefly, phospholipase C hydrolyzes membranephosphatidylinositol 4,5-biphosphate to diacylglyc-erol (DAG) and inositol 1,4,5-triphosphate(IP3) (seeFig. 2). Both of these are second messengers. DAG

activates protein kinase C, which is active in bothaggregation and secretion, although its mechanismis not entirely clear. IP3 causes the release of calciumfrom the dense tubular system, one of the intracel-

lular storage depots for calcium. The elevated cy-toplasmic calcium, in turn, activates phospholipaseA2, which then hydrolyzes the membrane lipids andreleases arachidonic acid (50, 56).

Secretion. Secretion is mediated by many of thesame second messengers discussed under aggrega-tion. The increased concentration of cytoplasmicCa++ is involved in the assembly of microtubules


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FIG. 2.-A schematic diagram of platelet metabolism. (See text for discussion.) lib and Illa = membrane proteins that

form the fibrinogen receptor; Va=

activated coagulation factor V; VaR=

activated coagulation factor V receptor;A=

agonist; Ca++ = ionized calcium; DG = diacylglyceride; G = guanine nucleotide-binding protein; IP3 = inositol 1,4,5-triphosphate ; PIP2 = phosphatidylinositol 4,5-biphosphate; R = receptor.

that are involved in the central movement of the

granules (50). This brings them into close proximityto the open canalicular system. There granule and

canalicular membranes fuse and granular contentsare emptied. The open canalicular system com-municates to the exterior, allowing granular con-tents access to the plasma.

There are 4 types of granules in platelets: alphagranules, dense or 6 granules, lysosomes, and mi-croperoxisomes (93). The alpha granules contain a

large number of constituents.A few examples areplatelet factor 4 (26, 87), thromboglobulin (87),platelet-derived growth factor (44), and thrombo-

spondin (107, 108). Dense granules also contain a

variety of components includingADP,ATP, cal-cium, pyrophosphate, and serotonin (32, 93). The

ADP andATP are present in a ratio of 3:2 and

exchange very slowly if at all with the metabolic

pool of nucleotides.ADP is the agent responsiblefor recruitment of additional platelets into the plugformed during primary hemostasis (32, 93).ProcoagulantActivity. Platelets contain coagu-

lation factors including fibrinogen, factor V, andfactor VIII (45, 63, 71, 87, 98). In fact, 20% of wholeblood factor V is contained in platelets (98), sug-gesting some importance. However, their most im-

portant contribution to coagulation is surfaces and

specific receptors upon which complexes of coagu-lation factors form (89, 99, 100). These complexeswill be discussed later.

Coagulation CascadeThe coagulation cascade has classically been con-

sidered to be a series of sequential zymogen acti-

vation steps (Fig. 3). Each factor is the substrate forthe previous enzyme and the activator of the sub-

sequent proenzyme (15, 5 5).Although this conceptis basically correct, it has been enlarged a great deal.Surprisingly, the mechanism ofinitiation ofthe co-

agulation sequence is still uncertain.The coagulation cascade can be intimidating. It

is complex, and the roman numeral system of no-menclature can be confusing. The numerals seem-ingly were assigned at random. However, ifthe cas-cade is subdivided into some functional areas, each

can be addressed with much less trepidation.Contact System. The intrinsic system (so named

because all factors are found in blood) has been

thought to start with the contact activation system.Factor XII is activated (factor XIIa) by contact withnegatively charged surfaces. This probably occurs

through autoactivation after contact with negativelycharged surfaces, collagen, or other contact activa-tors (80). Factor XIIa activates factor XI to factorXIa and prekallikrein to kallikrein. Kallikrein sub-

sequently activates more factor XII.All of thesereactions require the presence of high molecular

weight kininogen (13, 31, 54, 60, 66, 80, 85, 96).The factor XII-prekallikrein amplification systemresults in a great deal of factor XIIa to activate factor

XI and initiate the intrinsic system. However, theclinical relevance of this sequence is in serious doubt.

Deficiency of any or all of the factors involved inthe contact system is not accompanied by clinical

bleeding (13, 23, 31, 34, 54). This has always casta shadow on the importance of contact activationand suggested that there must be another pathwayfor the activation of factor XI. Indeed, as will be


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FIG. 3.-A schematic diagram ofthe coagulation cascade. (See text for a more complete discussion.) The dotted arrowfrom unactivated factor VII to the conversion of X to Xa indicates a small degree ofenzyme activity. This may representthe initial activation of the coagulation cascade in vivo.

discussed, another pathway for the activation of fac-tor XI has been recently described (24).

Intrinsic System. Factor Xla activates factor IXto factor IXain the presence of ionized calcium (21 ).However, the most prominent reaction of the in-trinsic system is the activation of factor X by factorIXa (23, 37, 39-41, 61, 69, 82, 104) and its cofac-tors. This reaction occurs on a surface that is prob-ably provided by platelets (23, 41). The enzyme(factor IXa) and the substrate (factor X) are broughtinto close proximity on the surface by binding toCa++. Factor V also enters the reaction and accel-

erates it 500-fold (61). This is 1 of 3 complexes

formed by coagulation factors, and it illustrates sev-eral important points. First, a cofactor is very im-

portant in allowing the reaction to take place at amaximum rate. Loss or inactivation ofthe cofactor

is a very effective way of controlling the reaction

(see discussion on protein in the later section Con-trol of Coagulation). Second, each of the complexesformed involves at least 1 ofthe factors ofthe &dquo;pro-thrombin complex.&dquo; The prothrombin complex isa group of coagulation factors (factors II, VII, IX,and X) that have been modified by a reaction in-

volving vitamin K so that they will bind calcium

(22, 68, 95).The

bindingof calcium is

important

in the formation of the complexes (64). This par-ticular complex of factors VIII, IX, and X, as wellas Ca++ and membrane (probably platelet mem-

brane), is sometimes called ten-ase because it cleavesfactor X (ten). These complexes are very importantto the understanding and control of coagulation.Common Pathway. The common pathway con-

tains the second of the 3 complexes. FactorXa formsa complex with its substrate prothrombin (factor II),its cofactor factor V, a membrane surface (platelet),and Ca++ (99, 100) to form a complex sometimesreferred to as prothrombinase. The prothrombin iscleaved and thrombin is released when the reaction

is complete. The primary action of thrombin is, ofcourse, to convert fibrinogen to fibrin. However,thrombin has a wide range of other actions (40, 49,52, 75), one of which is the activation of factor XI

(24). This casts a whole new light on the importanceof the extrinsic system for in vivo coagulation (seelater).

Extrinsic System. The extrinsic system consistsalmost entirely ofthe third enzyme-coenzyme-sub-strate complex. The extrinsic system is also whereone of the more important additions to knowledgeof the coagulation cascade has been made. FactorVII forms a

complexwith tissue factor and Ca-+


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that enzymatically activates factor X to factor Xa

by limited proteolysis (7, 9, 23, 25, 65, 69, 109).Factor VII is the only coagulation enzyme that pos-sesses activity without undergoing activation (7, 75,76, 113). It does indeed do that. It also activatesfactor IX (70, 112), providing further amplificationof factor Xa production and perhaps explaining whyhemophiliacs have clinical bleeding problems. Iffactor IX activation were not initiated by the ex-trinsic system and the extrinsic system was a pri-mary initiator of in vivo coagulation, there wouldbe no reason for a hemophiliac to have bleedingproblems.

Coagulation Cascade Rearrangements. The 2

relatively recent discoveries of thrombin activationof factor XI and factor Vlla activation offactor IX

have resulted in some new ways to think about the

coagulation cascade. In addition, some old enigmasmay have cleared up. It has

longbeen a mystery

why individuals deficient in contact activation fac-tors do not have a clinical bleeding problem. It maybe that the contact activation system is not impor-tant in coagulation in vivo. The extrinsic system maybe the primary initiator of coagulation. This makessense in that injury would expose tissue factor, whichis an absolute requirement for the activation of fac-tor X and factor IX by factor VII (66). It may alsobe that the thrombin activation of factor XI is an-

other amplification system for the production ofthrombin. Certainly, the importance this places on

factors IX, VIII, X, and Ca++-phospholipidcom-

plexes makes sense of the clinical picture presented

by hemophiliacs. The contact activation systemshould not be dismissed too quickly, however. Thereis still more to be learned. Passovoy factor is some-how involved in the contact system, and patientsdeficient in it do have a bleeding diathesis (34,35, 58).

Control of Coagulation. With all the amplifica-tion that takes place in the coagulation cascade, itwould seem that once coagulation was initiated itwould continue out of control. There are several

mechanisms to prevent this. The complexes thatoccur in the coagulation cascade not only allow the

enzymes involved to act more efficiently, but theyalso result in the localization of the coagulant ac-

tivity (23, 57). Some of this complexing takes placeon endothelial cell membranes but most probablyoccurs on platelet membranes (23, 83, 99). Regard-less, the receptors involved are exposed by injuryor activation and are localized to the area where

hemostasis is needed.All of these complexes in-volve the vitamin K-dependent coagulation factors.These are prothrombin and factors VII, IX, and X.

Vitamin K mediates a posttranslational modifica-tion of these factors that involves the addition of

gamma-carboxyl groups to a series of glutamylgroups. This gamma-glutamyl carboxylation allowsthe factors involved to bind calcium and therefore

participate in complex formation (64, 68, 95).Protein C and its cofactor protein S are also im-

portant controls of coagulation. Both are also vi-tamin K-dependent. Protein C is activated bythrombin in a reaction that requires the binding ofthrombin by a membrane protein, thrombomodu-lin, of endothelial cells.Activated protein C enzy-matically cleaves factor Va and factor Villa intoforms that will no longer support coagulation. Thisis a very effective inhibition of the coagulation cas-cade. Interestingly, while thrombin bound to throm-bomodulin will activate protein C, it will no longeractivate platelets or factor V or convert fibrinogento fibrin. Thrombomodulin transforms it from a

coagulant to an anticoagulant intermediate. ProteinC also promotes

fibrinolysis by protecting plasmin-ogen activator from inhibition (12, 18, 19, 23, 47,59, 105).A variety ofother control mechanisms also affectthe coagulation cascade. These include antithrom-bin III (10, 101), heparin cofactor II (5, 6, 29, 97),and (X2-macroglobulin (51).

Vascular Phase

The blood vessel has traditionally been thoughtto be an inert conduit through which blood flowsbut which did not participate in hemostasis. Grad-

ually, discussions of hemostasis have come to in-clude comments on endothelial contributions. Manyofthe interactions are detailed in the discussions of

the various phases of hemostasis.As can be seen,endothelial cells participate extensively. Some he-mostatic contributions are vasoconstriction (43, 53),production of vWF (42, 103), and tissue factor pro-duction (23).Antithrombotic contributions include

prostacyclin production (62), thrombomodulin pre-sentation (23, 59), and tissue plasminogen activator

production (48).

FibrinolysisOnce hemostasis is complete and healing has oc-

curred, clots must be disposed of and vascular lu-mina maintained. This is accomplished by the fi-

brinolytic system. The operative enzyme of thefibrinolytic system is plasmin. Plasmin is a rathernonspecific protease and is generated by the actionof plasminogen activators on plasminogen. Plas-

minogen activators present in normal tissues aretissue-type plasminogen activator and urokinase-type plasminogen activator. Plasminogen is incor-

porated into fibrin clots. Plasminogen activator isreleased from endothelium in response to, amongother things, high concentrations ofthrombin. This


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mechanism serves to localize the fibrinolytic actionof plasmin and results in the production of the fa-miliar fibrin split products (8, 20, 48, 52, 77, 78,86, 92, 110).

CLINICALAPPROACH TO THE PATIENT

Perhaps the most important rule to keep in mind

when evaluating the bleeding patient is that bloodcan be lost from vessels for reasons other than hem-

orrhagic tendencies. Because the most common causeof bleeding is trauma, every bleeding animal doesnot require a hemostatic workup. When a bleedingdiathesis is suspected clinically, a great deal can belearned by performing a thorough physical exami-nation and obtaining a good history.

History

History can, in clinical situations, be an effective

way of diagnosing hemostatic disorders. It is less

importantin the controlled conditions of drug safetyassessment, but it should be mentioned that con-

genital disorders are occasionally seen in laboratoryanimals. In those situations, age of onset, pedigree,and other pertinent clinical historical information

may be of benefit.As noted, because trauma is themost likely cause of any bleeding episode, the ap-propriateness of the degree of hemorrhage can be

very helpful in determining whether or not a bleed-

ing tendency is present.

Physical Examination

The type of bleeding can be a clue as to the un-

derlying disorder. Petechiae are usually associatedwith thrombocytopenia but can also be seen withvascular defects. Ecchymoses are larger than pete-chiae and are often thought to be confluent pete-chiae. This is usually true, but they can also be seenin coagulation disorders. Prolonged bleeding fromminor trauma (venepuncture, scratches, etc.) is as-sociated with defective primary hemostasis-

thrombocytopenia. Rebleeding after a phase of ap-parently normal hemostasis is characteristic of co-

agulation factor deficiencies (4, 79).Although information about types of bleeding is

the primary goal of physical examination, additionalimportant information should not be overlooked.Underlying diseases may be detected that can con-tribute to or result in hemorrhage. Such abnormal-ities including splenomegaly, lymphadenopathy, and

jaundice may be associated with diseases that cause

bleeding diatheses.

LABORATORYAPPROACH TO THE PATIENT

General Concepts of Abnormal Bleeding

Levels of Platelets. Thrombocytopenia is the mostcommon cause of abnormal bleeding in both hu-

mans and animals. The degree of bleeding is quitevariable and often does not correlate with the degreeof thrombocytopenia. The reasons are probably re-lated to the underlying cause of the disorder. Con-ditions involving peripheral destruction cause a

compensatory increase in marrow production of

platelets. This results in a population of relatively

young platelets that may function more efficientlythan older platelets and prevent bleeding at lowerconcentrations. Conversely, in situations wheremarrow production is impaired, the platelets re-

maining in the circulation are relatively old and maynot prevent bleeding at higher concentrations.A

good rule ofthumb for the platelet concentration atwhich to expect bleeding is the familiar 40,000-50,000/ ~l, although this will vary depending on thehemostatic challenges the animal encounters. Per-

haps better rules are (a) spontaneous bleeding is

probably below platelet concentrations of 20,000/wl, (b) spontaneous bleeding is unlikely more than

platelet concentrations of 50,000/jnl, and (c) bleed-

ing after challenge (e.g., surgery) is probably at plate-let concentrations of 20,000-100,000/~1 (4, 79).

Levels of Coagulation Factors. The associationbetween levels ofcoagulation factors and the clinical

tendency to bleed is better than that between plate-lets and bleeding. There is, however, considerablevariation from factor to factor. In general, the con-centration needed to prevent bleeding is no morethan 25% of the normal, and some factors can beeven lower in concentration and not result in a prob-lem. This is in contrast to the 50% concentrations

needed to maintain most coagulation screening testswithin normal limits. This means that, in general,if the concentrations of factors are high enough forthe screening tests ofcoagulation to be normal, theyare high enough that clinical bleeding should not bea problem (17, 46, 74, 81, 111).

Principles of Laboratory Examination

Platelets. Because thrombocytopenia is the mostcommon cause of abnormal bleeding, they shouldbe routinely evaluated in any abnormally bleedinganimal. In clinical practice, this evaluation is quick-

ly and easily done by examining a blood smear. Inmost safety assessment laboratories, platelet countsare routinely performed. This makes examinationof smears unnecessary for diagnosis, but it is in-valuable in distinguishing true thrombocytopeniafrom artificial thrombocytopenia due to aggrega-tion.A thorough discussion of thrombocytopeniaand thrombopathy cannot be accomplished in thisshort article.

Coagulation Factors.A large variety of tests ofthe coagulation cascade are available. Because ofthe complexity of the cascade and the number of


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factors involved, a thorough test of coagulation in-

volving each factor would necessarily be cumber-some and impractical. In spite of the mentioneddiscoveries that have changed our view of the ap-parent importance ofthe intrinsic and extrinsic sys-tems for in vivo hemostasis, the old familiar schemeis still useful for sorting out abnormalities. Forscreening purposes, 2 simple tests still serve quitewell. These tests are the activated partial throm-

boplastin time (APTT) and the prothrombin time

(PT), sometimes called the 1-stage PT. Using these

tests, coagulation abnormalities (if severe enough)can be detected as well as localized to a relativelysmall area of cascade. TheAPTT tests the intrinsic

system and the common pathway by providing con-tact activation, Ca++, and phospholipids for the in-trinsic system reactions. The PT tests the extrinsic

system and common pathway by providing tissue

factor, Ca++, and phospholipid. Note that the phos-pholipid portion of theAPTT and PT reagents re-

places the platelet contribution. Thrombocytopeniawill therefore have no effect on these tests. Three

abnormal patterns are possible. NormalAPTT and

prolonged PT localize the problem to factor VII.Normal PT and prolongedAPTT localize the prob-lem to the intrinsic system. If the patient has noclinical bleeding problem with this pattern, the ab-

normality is most likely in the contact activation

system. If the patient exhibits bleeding problems,the abnormality most likely involves factor VIII,

IX,or XI.

Prolongationsofboth theAPTT and the

PT indicate either an abnormality in the common

pathway or multiple abnormalities involving the in-trinsic and extrinsic systems.Abnormalities of the

common pathway are exceedingly rare, and this sce-nario is almost invariably due to multiple abnor-malities. In clinical medicine warfarin intoxication

is a common culprit (3, 4, 16, 28, 79).

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