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57. Godbout JP, Glaser R. Stress-induced immune dysregulation: implications for wound healing,

infectious disease and cancer. J Neuroimmune Pharmacol 2006;1(4):421–427.

58. Peyroux J, Sternberg M. Advanced glycation endproducts (AGEs): pharmacological inhibition in

diabetes. Pathol Biol (Paris) 2006;54(7):405–419.

59. Godbout JP, Glaser R. Stress-induced immune dysregulation: implications for wound healing,

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60. Siana JE, Rex S, Gottrup F. The effect of cigarette smoking on wound healing. Scand J Plast Reconstr

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61. Ahn C, Mulligan P, Salcido RS. Smoking-the bane of wound healing: biomedical interventions and

social influences. Adv Skin Wound Care 2008; 21(5):227-236; quiz 237–238.

62. Levin L, Schwartz-Arad D. The effect of cigarette smoking on dental implants and related surgery.

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64. Scolaro JA, Schenker ML, Yannascoli S, et al. Cigarette smoking increases complications following

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65. S⊘rensen LT, J⊘rgensen S, Petersen LJ, et al. Acute effects of nicotine and smoking on blood flow,

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66. Siasos G, Tsigkou V, Kokkou E, et al. Smoking and atherosclerosis: mechanisms of disease and new

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67. McMaster SK, Paul-Clark MJ, Walters M, et al. Cigarette smoke inhibits macrophage sensing of

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68. S⊘rensen LT, Zillmer R, Agren M, et al. Effect of smoking, abstention, and nicotine patch on

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70. S⊘rensen LT, Jorgensen LN, Zillmer R, et al. Transdermal nicotine patch enhances type I collagen

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complications. Curr Probl Surg 2007;44(11):691–763.

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Chapter 6

Hemostasis

Peter K. Henke and Thomas W. Wakefield

Key Points

1 At the same time that thrombin forms, natural anticoagulant mechanisms oppose further thrombin

formation and help to localize thrombin activity to areas of vascular injury. Just as thrombin

generation is key to coagulation, antithrombin is the central anticoagulant protein.

2 The endothelial cell acts as a nonthrombogenic surface, and inflammation tips the balance to

procoagulant state.

3 Thrombosis and inflammation are closely linked, and may perpetuate each other. Leukocytes and

chemokines are involved with normal DVT resolution. These essential inflammatory mechanisms

may drive vein wall injury.

4 Both acquired and inherited factors contribute to pathologic thrombosis; often occurring together.

5 Heparin-induced thrombocytopenia (HIT) occurs in 0.6% to 30% of patients in whom heparin is

given; severe thrombocytopenia associated with thrombosis (HITTS) is much less frequent. Cessation

of heparin is critical.

6 Factor VIII and IX deficiency states are involved in hemophilia A and B and von Willebrand disease.

BASIC CONSIDERATIONS

Coagulation is an essential homeostatic mechanism for survival, and involves tightly controlled

processes to maintain vascular integrity including thrombosis localization, amplification, and

neutralization. These coordinated steps occur at the vessel, cellular and subcellular levels. Thrombosis,

directly or indirectly, is the underlying leading cause of death in the world, and is an essential part of

surgery.

Platelets form the initial hemostatic plug after vascular injury, and are locally activated and

aggregation induced (Fig. 6-1). Platelet aggregation is mediated by receptors that are part of the

mammalian integrin family. This family includes the β1

family, mediating platelet interaction with cells,

collagen, fibronectin, and laminin; the β2

family (LeuCAM), present on leukocytes mediating

interactions between leukocytes and other cells important in inflammation; and the β3

family

(cytoadhesion), including the megakaryocyte-specific glycoprotein (Gp) IIb/IIIa receptor and the

vitronectin (Vn) receptor present on platelets and other cells.1 Platelet aggregation is mediated by

GpIIb/IIIa, which binds fibrinogen, von Willebrand factor (vWF), fibronectin, Vn, and thrombospondin

to activated platelets. These high-density receptors are hidden on inactivated platelets and become

exposed on the surface of activated platelets.

Two platelet activation routes are thought to occur physiologically.2 Without direct vessel damage,

platelet activation may occur via tissue factor (TF) de-encryption and activation by protein disulfide

isomerase (PDI), with factor VIIa generation and activation of platelets. Alternatively, subendothelial

collagen may directly bind to GpVI and vWF, leading to platelet capture and activation. Of note, PDI

inhibition can directly block experimental thrombus formation.3

Once stimulated, activated platelets contract, with externalization of negatively charged procoagulant

phospholipids, including phosphatidylserine and phosphatidylinositol (termed platelet factor 3). This

allows the coagulation proteins to assemble on the surface membrane of platelets, accelerating the

coagulation reaction.4 During platelet activation, granules release their contents of calcium, serotonin,

and membranes are exposed that are rich in receptors for factors Va and VIIIa,5,6 as well as fibrinogen,

vWF, and ADP, a potent activator of other platelets. vWF is responsible for platelet adhesion through

binding to GpIb,7 whereas fibrinogen forms bridges between activated platelets by binding to GpIIb/IIIa

on adjacent stimulated platelets.8 Platelets also release polyphosphates that activate factor XII and the

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intrinsic pathway.9

While long thought to be a bystander in venous thrombosis (VT) as compared to the arterial system,

the role of the platelet is now thought to play a critical role, as well as directing later inflammatory cell

actions.10,11 First, recent clinical trials suggest antiplatelet therapy may reduce recurrent VTE.12 Second,

in stasis and nonstasis experimental murine VT, genetic deletion of vWF was associated with

significantly reduced VT size and not restored with recombinant factor rVIII.13 Intravital microscopy

also showed direct association of leukocytes and platelets in a growing acute thrombus. Consistently,

platelets via GpIb2

, may promote VT by colocalizing leukocytes and coagulation factors at the site of

injury or stasis in the vein.14

Once the platelet plug has formed, the stage is set for coagulation protein assembly (Fig. 6-2).

Initiating agents for coagulation include subendothelial collagen and TF, usually from vascular injury.2

There is also growing evidence that blood-borne TF associated with leukocytes, or circulating in soluble

form, is also involved with venous thrombogenesis.15,16 Leukocyte adhesion to platelets may trigger

leukocyte activation, causing recruitment of blood-borne TF onto the surface of leukocytes associated

with thrombus, or recruitment of TF-positive leukocytes onto the growing thrombus.17 TF, both blood

borne and local, activates the extrinsic pathway of coagulation by complexing with activated factor VII

(VIIa), activating factors IX and X to factors IXa and Xa.16,18 Factor Xa, activated factor V (Va), ionized

calcium, and factor II (prothrombin) form on the platelet phospholipid surface to initiate the

prothrombinase complex, which catalyzes the formation of thrombin faster than can be achieved with

factor Xa alone.

Thrombin is central to coagulation and acts to cleave fibrinopeptide A (FPA) from the α chain of

fibrinogen and fibrinopeptide B (FPB) from the β chain. This leads to the release of fibrinopeptides and

the formation of new fibrin monomers, which then cross-link, resulting in fibrin polymerization.

Thrombin also activates factor XIII, which catalyzes the cross-linking of fibrin to make the clot firm,

activates platelets, and activates factors V and VIII, two nonenzymatic cofactors, to Va and VIIIa. This is

important because only activated factors Va and VIIIa are involved in coagulation. Factor XIIIa also

cross-links other plasma proteins, such as fibronectin and α2

-antitrypsin, resulting in their incorporation

into clot.

Figure 6-1. Primary hemostasis is achieved initially with a platelet aggregation as illustrated. Note that platelet adhesion, shape

change, granule release, followed by recruitment and the hemostatic plug at the area of subendothelial collagen (binds to GpVI)

exposure are the initial events for thrombus formation. Platelets can also be activated by PDI with de-encryption of TF.

The intrinsic pathway of blood coagulation requires activation of factor XI to XIa. This may occur by

both the contact activation system through activation of factor XII, plasma prekallikrein, and high–

molecular-weight kininogen and, more important, through thrombin with negatively charged surfaces.19

Factor XIa activates factor XI autocatalytically and also catalyzes the conversion of factor IX to IXa.

After activation, factor VIIIa dissociates from vWF and assembles with factors IXa and X. Factor IXa,

factor X, ionized calcium, and thrombin-activated factor VIII (VIIIa) then assemble on the platelet

surface in a complex called the Xase complex to catalyze the activation of factor X to Xa. Factor Xa then

shunts into the prothrombinase complex for further amplification of thrombin formation.

The importance of a mechanism of factor XI activation independent of the contact activation system is

apparent because patients deficient in those factors of the contact activation system, including factor XI,

bleed, whereas patients deficient in factor XII, prekallikrein, and high–molecular-weight kininogen do

not usually bleed.20 The contact activation system is the most important coagulation process involved in

extracorporeal bypass circuits, including cardiopulmonary bypass and extracorporeal membrane

oxygenation.

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Figure 6-2. The classical pathway showing the interface between the intrinsic pathway, extrinsic pathway, and the common

pathway is illustrated with the ultimate production of thrombin. This catalyzes fibrin from fibrinogen, and then cross-linking the

fibrin to form a stable clot.

NATURAL ANTICOAGULANT MECHANISMS

1 At the same time that thrombin forms, natural anticoagulant mechanisms oppose further thrombin

formation and help to localize thrombin activity to areas of vascular injury. Just as thrombin generation

is key to coagulation, antithrombin (AT) is the central anticoagulant protein (Fig. 6-3). This

glycoprotein of 70-kD molecular weight binds to thrombin, preventing the removal of FPA and FPB

from fibrinogen, prevents the activation of factors V and VIII, and inhibits the activation and

aggregation of platelets. In addition, AT directly inhibits factors IXa, Xa, and XIa.

A second natural anticoagulant is activated protein C (APC), which inactivates factors Va21,22 and

VIIIa, thus reducing the Xase and prothrombinase complex acceleration of the rate of thrombin

formation. In the circulation, protein C is activated on endothelial cell surfaces by thrombin complexed

with one of its receptors, thrombomodulin (TM).23–25 The formation of this thrombin–TM complex

accelerates the activation of protein C compared with thrombin alone. Thrombin, at the same time, by

binding to TM, loses its platelet-activating activity as well as its enzymatic activity for fibrinogen and

factor V. Protein S is a cofactor for APC.

Another innate anticoagulant is tissue factor pathway inhibitor (TFPI). The protein binds to TF–VIIa

complex, inhibiting the activation of factor X to Xa and the formation of the prothrombinase complex.26

A fourth natural anticoagulant is heparin cofactor II.27 Its concentration in plasma is estimated to be

significantly less than that of AT, and its action is implicated primarily in the regulation of thrombin

formation in extravascular tissues. Finally, thrombin is inactivated when it becomes incorporated into

the clot.

FIBRINOLYSIS

In addition to natural anticoagulants such as protein C and S, physiologic clot formation is balanced by a

contained process of clot lysis, which prevents thrombus formation from proceeding outside of the

injured area (Fig. 6-4). The central fibrinolytic enzyme is plasmin, a serine protease generated by the

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proteolytic cleavage of the proenzyme, plasminogen. Its main substrates include fibrin, fibrinogen, and

other coagulation factors. Plasminogen, tissue plasminogen activator (tPA), and α2

-antiplasmin (α2

-AP)

become incorporated into the fibrin clot as it forms. Plasminogen activators are serine proteases that

activate plasminogen, by cleavage of a single arginine–valine peptide bond, to the enzyme plasmin.

Plasminogen activation provides localized proteolytic activity.28–30 In fact, thrombin promotes tPA

release from endothelial cells as well as the production of plasminogen activator inhibitor (PAI-1) from

endothelial cells.31,32

Figure 6-3. Antithrombin is a primary anticoagulant. Note antithrombin complexes with IIa to inhibit fibrin polymerization, as

well as factor Xa, and an inactivating factor Va and VIIIa.

Figure 6-4. Hemostasis with thrombus production is a tight and intricate process that is locally confined. Balancing thrombus

production is tissue plasmin activator and urokinase plasminogen activator which activates plasmin and causes thrombolysis. These

are balanced by plasminogen activator inhibitor-1 and alpha-2-antiplasmin. Free plasmin is complexed rapidly. Fibrin degradation

products, such as D-dimer are produced.

The major endogenous plasminogen activators include tPA and urokinase, and intrinsic factors, such

as factor XII, prekallikrein, and high–molecular-weight kininogen. These later factors of the contact

system are more important in clot lysis than thrombus formation. These enzymes may also liberate

bradykinin from high–molecular-weight kininogen, resulting in an increase in vascular permeability,

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prostacyclin (PGI2

) liberation, and tPA secretion. Finally, APC has been found to proteolytically

inactivate the inhibitor to tPA, thus promoting tPA activity and fibrinolysis.33

Fibrin, when digested by plasmin, yields one molecule of fragment E and two molecules of fragment

D. In physiologic clot formation, fragment D is released in dimeric form (D-dimer),20,34 and is a marker

for fibrinolysis of formed clot. An elevated D-dimer level after treatment of DVT is one biomarker that

has been found to accurately predict an ongoing risk of recurrent VTE.35

Two primary inhibitors of plasmin are important. First, α2

-AP is released by endothelial cells and

complexes with plasmin. In physiologic fibrinolysis, α2

-AP is bound to fibrin and excess plasmin is

readily inactivated. In plasma, PAI-1 is the primary inhibitor of plasminogen activators. It is secreted in

an active form from liver and endothelial cells and stabilized by binding to Vn. PAI-1 levels are elevated

by hyperlipidemia, and PAI-1 elevation appears to synergize with factor V Leiden genetic

abnormalities.36

In summary, coagulation is an ongoing process of thrombus formation, inhibition of thrombus

formation, and thrombus dissolution. The central mediators are TF, platelets, thrombin, and plasmin.

Abnormalities in coagulation occur when one process – thrombus formation, thrombus inhibition, or

fibrinolysis – overcomes the others and dominates the delicate balance.

ENDOTHELIUM AND HEMOSTASIS

2 Through its ability to express procoagulants and anticoagulant factors, vasoconstrictors and

vasodilators, as well as key cell adhesion molecules and cytokines, the endothelial cell is a key regulator

of hemostasis (Fig. 6-5).37 Vascular endothelium maintains a vasodilatory and local fibrinolytic state in

which coagulation, platelet adhesion and activation, and leukocyte activation are suppressed.

Vasodilatory endothelial products include adenosine, nitric oxide (NO), and PGI2

. A nonthrombogenic

endothelial surface is maintained by four main mechanisms including; endothelial production of TM and

subsequent activation of protein C, endothelial expression of surface heparan- and dermatan sulfate,

constitutive expression of TFPI by endothelium (which is markedly accelerated in response to heparin),

and local production of tPA and urokinase plasminogen activator (uPA).37,38 Finally, the elaboration of

NO and interleukin (IL)-10 by endothelium inhibits leukocyte adhesion and activation.

During states of endothelial disturbances such as injury, a prothrombotic and proinflammatory state

of vasoconstriction is driven by the endothelial surface. Endothelial release of platelet-activating factor

(PAF) and endothelin-1 promotes vasoconstriction.38 Endothelial cells increase production of vWF, TF,

PAI-1, and factor V to augment thrombosis with exposure to prothrombotic stimuli. Finally, in response

to endothelial injury, endothelial cells are activated, resulting in increased surface expression of cell

adhesion molecules, promoting leukocyte adhesion and activation.

THROMBOSIS, INFLAMMATION, AND RESOLUTION

3 After VT, an acute to chronic inflammatory response occurs in the vein wall and thrombus progressing

from thrombus amplification, to organization, and vein recanalization (often at the expense of vein wall

fibrosis and vein valvular damage). Initially, there is an increase in neutrophils (PMN) in the vein wall

followed by monocytes/macrophages. Cytokines, chemokines, and inflammatory factors (e.g., tumor

necrosis factor [TNF]) facilitate inflammation. The ultimate response of the vein wall depends on

proinflammatory and anti-inflammatory mediator balance at the interface between the leukocyte,

activated platelet, and endothelium.39

Cell Adhesion Molecules

Selectins (P- and E-selectin) have been found to be intimately involved in this process (Fig. 6-6).40

Selectins are the first upregulated glycoproteins on activated platelets (alpha granules) and endothelial

cells (Weibel–Palade bodies). They mediate the adhesion of leukocytes, platelets, and even cancer cells

in inflammation and thrombosis. The P-selectin receptor is P-selectin glycoprotein ligand-1 (PSGL-1). Pselectin:PSGL-1 interactions trigger the release of procoagulant microparticles (MPs), that support fibrin

formation, thrombus growth, and increase monocyte TF expression.

MPs <1 micron fragments may be central for P-selectin’s effect. It is believed that with initial

thrombosis, P-selectin upregulation leads to MP formation. These procoagulant MPs, which may express

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