A New Understanding of the Coagulation Process

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Ruseva A., Dimitrova A. A new understanding of the coagulation process...

Review

A NEW UNDERSTANDING OF THE COAGULATION PROCESS –THE CELL-BASED MODEL

Adelaida L. Ruseva,1

Anelia A. Dimitrova

Clinical Laboratory,University Hospital, Pleven1 Department of Physiology and Pathophysiology, Faculty of Medicine, Medical University-Pleven

Corresponding Author: Adelaida L. RusevaClinical Laboratory,University Hospital8a “G. Kochev” str.Pleven, 5800Bulgariae-mail:

Received: April 15, 2011Revision received: August 09, 2011 Accepted: October 25, 2011

[email protected]

Summary

The cell-based model of coagulation replaces thetraditional “cascade” hypothesis. The incorporation of therole of cells in coagulation allows for an integratedunderstanding of the mechanism by which coagulationoccur in the dynamic vascular system in vivo. The newmodel proposes that coagulation takes place on differentcell surfaces in three overlapping steps: initiation,amplification, and propagation.Key words: cell-based model, coagulation cascade,hemostasis, tissie factor, platelet, haemophilia

The hemostatic system is a vital protectivemechanism that is responsible for preventing bloodloss by sealing sites of injury in the vascular system.However, hemostasis must be controlled so that

blood does not coagulate within the vasculature andrestrict normal blood flow. Understanding ofhemostatic mechanisms has progressed substantiallyover the last century, with the majority ofinvestigations occurring in static, cell-free in vitro

systems [1].

Previous models of coagulation

The classic theory of coagulation is described byPaul Morawitz in 1905. He assembled 4“coagulation factors” in his scheme of coagulation. In the presence of calcium and thromboplastin,

prothrombin was believed to be converted tothrombin. In turn, the thrombin converted fibrinogento fibrin, enabling the formation of a fibrin clot.Morawitz posited that all the ingredients of clotting

were present in circulating blood and that the factthat such blood did not normally clot was due to thelack of a wettable surface in the blood vessels. Thisclassic theory persisted for 40 years.

The modern understanding of the biochemical processes of coagulation began in the 1940s, whenPaul Owren (1947) recognized that a bleedingdiathesis in a young woman could not be explained

by the 4-factor concept, positing that she lacked afifth coagulation factor in her plasma. Throughoutthe 1940s and 1950s, several more coagulationfactors were discovered. Coagulation factors were

designated by roman numerals. Importantly, the


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numeric system that was adopted assigned thenumber to the factor according to the sequence ofdiscovery and not to the point of interaction in thecascade [2].

The cascade model of coagulation

In the 1960s, 2 independent groups of biochemists introduced a model of coagulation asa series of steps in which activation of eachclotting factor led to the activation of another,culminating in a burst of thrombin generation.(Figure 1). Each clotting factor was believed toexist as a proenzyme that cold be converted to anactive enzyme. The article proposing the cascademodel by Macfarlane (1964) appeared in the

journal Nature and was shortly followed by thewaterfall model reported by Davie and Ratnoff

(1964) in the journal Science. The originalcascade models were subsequently modified toinclude the observation that some procoagulantswere cofactors and did not possess enzymaticactivity. The coagulation process is now oftenoutlined in a Y-shaped scheme, with distinctintrinsic and extrinsic pathways. The extrinsicsystem consisted of factor VIIa and tissue factor(TF), the latter being viewed as occurring onlyextrinsic to the circulating blood. By contrast, thefactors in the intrinsic system were all thought to

be intravascular. Both extrinsic and intrinsic

pathways could activate factor X which incomplex with its cofactor Va, could convert

prothrombin to thrombin. This sequencecontinued until sufficient thrombin wasgenerated to convert F1 to fibrin [3].

Utility of the cascade model

The cascade model was extremely useful inadvancing the understanding of how coagulationenzymatic steps occur in plasma-based in vitrocoagulation. The description of the cascade

model has also allowed for clinically useful interpretation of laboratory tests for plasmacoagulation abnormalities. Specifically,deficiencies in the extrinsic or common pathwaysare identified using the prothrombin time (PT),while deficiencies in the intrinsic or common

pathways are reflected with prolongation of theactivated partial thromboplastin time (aPTT). The cascade model, functions reasonably well toexplain the way coagulation occurs in plasmawhere the fluid is static and does not interact withvascular wall or cell surfaces [4].

Deficiencies in the cascade model

While separating the various enzymatic processes of coagulation into this Y-shapedcascade was useful in further understanding ofhow coagulation processes occur in plasma-

based in vitro coagulation, it is obvious that thismodel does not adequately explain thehemostatic process as it occurs in vivo.

Deficiencies in the initial components of theintrinsic pathway (FXII, HMWK, or PK) cause

marked prolongation of the aPTT, but they are notassociated with a tendency for bleeding. Deficiency of the next downstream enzyme FXI(hemophilia C) is associated with variable hemostatic deficits, with some individuals experiencing bleeding. In contrast, deficiency ineither of the next downstream components of theintrinsic pathway (FVIII and FIX) results in theserious bleeding tendencies seen with hemophiliaA and B, despite the fact that these patients havean intact extrinsic pathway. If the coagulationmechanism was represented by a step-by-step

activating cascade, a deficiency in a factor higherin the cascade should result in increased bleedingtendencies compared with a deficiency in a factorlower in the cascade. Similarly, deficiency of the

primary enzyme of the extrinsic pathway (FVII)can be associated with bleeding, despite the

presence of an intact intrinsic pathway [3, 5-10].

The Cell-Based Model of FibrinFormation

A major development over the past 15 years was

Figure 1. The cascade model of fibrin formationModified after :www.frca.co.uk/article.aspx?articleid=100805 [21].


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the discovery that exposure of blood to cells thatexpress TF on their surface is both necessary andsufficient to initiate blood coagulation in vivo.This hypothesis and the experimental evidence tosupport it were collected by several investigatorsand presented in a series of articles authored by a

group from the Department of Pathology at DukeUniversity and the University of North Carolina(Allen, Monroe, Roberts, Hoffman, Kjalke, etal.) [2].

This group hypothesized that the key tounderstanding the hemostatic process was thecorrect incorporation of the roles of cells. They

began developing a new conceptual model ofhemostasis on the basis of a cell-basedexperimental model using monocytes culturedwith agents to induce tissue factor (TF), unactivated platelets and also the factors II, V,

VIII, IX, X, XI as well as the inhibitors: Tissuefactor pathway inhibitor (TFPI) andAntithrombin III (AT III). Data obtained in thismodel system strongly suggested that the processof coagulation requires the joint participation of2 cell types: a TF-bearing cell and platelets andthat it occurs in distinct overlapping steps: 1.initiation, 2. amplification, and 3. propagation.The process of coagulation is prevented, at leastin part, by keeping the 2 cell types apart until aninjury makes activation of the coagulationsystem desirable. At that time and within the

limited region of the injury, these intra- andextravascular cells can come into contact [2, 3].

Initiation of Coagulation on TF-bearing Cells It is now generally accepted that coagulation isinitiated by TF in vivo. Cells expressing TF arenormally found outside the vasculature and so

preventing initiation of coagulation undernormal flow circumstances. Some circulatingcells (eg, monocytes or tumor cells) may expressTF on their membrane surface, but this TF is

thought to be inactive unter normal conditionsand its exact role is not clear.Once an injury occurs and the flow-in blood is

exposed to a TF-bearing cell, F VIIa rapidly binds to the exposed TF. FVIIa is the onlycoagulation protein that routinely circulates inthe blood in its inactive and active forms(approximately 1% of total FVII is F VIIa). Thefactor VIIa/TF complex activates additionalFVII to F VIIa and small amounts of factors IXand X. This factor Xa associates with its cofactor, factor Va, to form prothrombinase complexes on the surface of the TF-bearing cell. Factor V can

be activated by factor Xa or by non-coagulation proteases (Figure 2). Any factor Xa thatdissociates from the TF-bearing cell is rapidlyinhibited in the fluid phase by TFPI and ATIII.Thus, the presence of inhibitors effectivelylocalizes factor Xa activity to the surface on

which it was formed. It cannot move from onecell surface to another through the fluid phase. Bycontrast, factor IXa can move from the TF-

bearing cell on which it was formed to a nearby platelet or to another cell surface, since it is notinhibited by TFPI and is inhibited very slowly byATIII.

Low-level activity of the TF pathway occursat all times within the extravascular space. Thus,factor VII is probably bound to extravascular TFeven in the absence of an injury; and theextravascular factors X and IX can be activated asthey pass through the tissues. This idea isconsistent with the finding that low levels of theactivation peptides of coagulation factors are

present in the blood of normal individuals. This phenomenon has been called “basal coagulation” or “idling” . This ongoing basal process does notlead to clot formation under normalcircumstances because the large components ofthe coagulation pro ce ss (pl atelets andFVIII/vWF) are kept sequestered within the vascular space.

Coagulation progresses beyond thegeneration of the small amount of thrombin thatoccurs with initiation only when the injury allows

platelets and larger proteins to leave the vascular

Figure 2. Initiation occurs on the TF-bearing cell

Modified after : M. Hoffman In: Remodeling theBlood Coagulation Cascade. Journal of Thrombosisand Thrombolysis 16(1/2), 17–20, 2003. [3].



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space and adhere to the TF-bearing cells in theextravascular area [3, 5, 11-13].

Amplification of the Procoagulant Signal As a result of vessel damage, componenets of the

haemostatic system that are normally unable toleave the vasculature due to their large size arenow able to do so. As they leave the vascularsystem, they come into contact with the smallamount of thrombin, generated during theinitiation of the coagulation process.

The small amount of thrombin generated onthe TF-bearing cell has several importantfunctions (Figure 3). One major function isactivation of platelets. They stick to the site ofinjury, forming a plug at the damaged vessel walland become fully activated by the thrombin.

Another function of the thrombin formed duringthe initiation phase is the activation of thecofactors V and VIII on the activated platelet surface. In the process the factor VIII/vWFcomplex is dissociated, permitting vWF tomediate additional platelet adhesion andaggregation at the site of injury. Small amounts ofthrombin also activate factor XI to XIa on the

platelet surface during this phase. This mayexplain why FXII and other contact factors arenot always necessary for coagulation, as initially

postulated by the original cascade hypothesis. At

the end of the amplification phase, plateletsactivated by the limited amount of thrombinduring the initiation phase, are clad in activatedcofactors and FXIa, and the process ofhaemostasis moves into the propagation phase [3,8, 9, 12, 14-7 ].

Propagation of Thrombin Generationon the Platelet SurfaceThe propagation phase occurs on the surface ofac t iva ted p la te le ts where the tenase(FVIIIa/FIXa) and prothrombinase (FVa/FXa)complexes are formed (Figure 4). Factor IXa

activated during the initiation phase migrate tothe platelet surface. Factor IXa com bines with itscofactor VIIIa on the surface of activated

platelets. Additional factor IXa can be supplied by platelet-bound factor XIa. Since factor Xacannot move effectively from the TF-Bearing cell to the activated platelet, factor Xa must be

provided directly on the platelet surface by the factor VIIIa/IXa complex. The factor Xa rapidly associates with its cofactor Va bound to the

platelet during the amplification phase. Thecompletion of platelet prothrombinase assembly

leads to a burst of thrombin generation ofsufficient magnitude to clot fibrinogen.

The platelet is probably the only cell type onwhich the propagation phase of coagulation canoccur effectively. The platelet surface is

specialized to coordinate assembly of the tenase(FVIIIa/IXa) and prothrombinase (FVa/Xa)complexes. This scheme also addresses the roleof factor XI in coagulation. The role of factor XIais to enhance the amount of platelet surface factorIXa. This increases the supply of platelet surfacefactor Xa, and thereby enhances the amount andrate of thrombin generation leading to cleavage offibrinopeptide A from fibrinogen. When enoughthrombin is generated with enough speed to resultin a critical mass of fibrin, these soluble fibrinmolecules will spontaneously polymerize into

Figure 3. The small amount of thrombin generatedon TF-bearing cell amplifies the procoagulantresponceModified after : M. Hoffman In: Remodeling theBlood Coagulation Cascade. Journal of Thrombosisand Thrombolysis 16(1/2), 17–20, 2003. [3].

Figure 4. The large burst of thrombin required foreffective hemostasis is formed on the plateletsurface during the propagation phase.Modified after : M. Hoffman In: Remodeling theBlood Coagulation Cascade. Journal of Thrombosisand Thrombolysis 16(1/2), 17–20, 2003. [3].


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References

1. Furie B, Furie BC. In vivo thrombus formation. JThromb Haemost. 2007;5(Suppl. 1):12-17.

2. Riddel JP, Aouizerat BE, Miaskowski C, Lillicrap

DP. Theories of Blood Coagulation. J PediatrOncol Nurs. 2007;24(3):123-31.3. Hoffman M. Remodeling the Blood Coagulation

C a s c a d e . J T h r o m b T h r o m b o l y s i s .2003;16(1/2):17-20.

4. Mann KG, Brummel K, Butenas S. What is all thatt h r o m b i n f o r ? J T h r o m b H a e m o s t . 2003;1(7):1504-14.

5. Roberts HR, Hoffman M, Monroe DM. A cell- based model of thrombin generation. SeminThromb Hemost .2006;32(Suppl 1):32-8.

6. Becker RC. Cell-based models of coagulation: a paradigm in evolution. J Thromb Thrombolysis. 2005;20(1):65-8.

7. Gailani D, Renne T. The intrinsic pathway ofcoagulation: a target for treating thromboembolicdisease? J Thromb Haemost. 2007;5(6):1106-12.

8. Gregory Romney BA, Michael Glick. An updatedconcept of coagulation with clinical implications.JADA. 2009,140(5):567-74

9. Hoffman M. A cell-based model of coagulationand the role of factor VIIa. Blood Rev.2003;17:51-5.

10. Hoffman M. Mechanism of action of NovoSevenusing a cell-based model. Bloodline Reviews2002;1:5-6.

11. Morrissey JH, Mutch NJ. Tissue factor structure

fibrin strands, resulting in an insoluble fibrinmatrix [3, 8, 13-15, 18].

Hemophilia and t he cell-based modelof coagulation The cell-based model of coagulation explains

why the FXa generated by the extrinsic pathwayis insufficient to compensate for a deficiency inFVIII or FIX, resulting in hemophilia. Accordingto the cell-based model, in normal coagulation itis important that both the tenase complex(FVIIIa/FIXa) and the prothrombinase complex(FVa/FXa) be functional. In hemophilia, thetenase complex is deficient due to to the lack ofFVIIIa or FIXa. Without an adequate tenasecomplex, FX is not activated on the plateletsurface, effectively impairing the pro-

thrombinase complex. The prothrombinase

complex is responsible for converting prothrombin to thrombin.

Logic would suggest that hemophilia could betreated by providing FXa, thus bypassing thetenase complex. However, similar to FXa

produced in the initiation phase, FXa used to treata patient is not able to migrate to the plateletsurface because it is inhibited by TFPI and ATIIIin plasma. FXa activated in plasma by TF/FVIIaor provided as a treatment is not as effective atgenerating thrombin as is FXa on a plateletsurface. FX activated on the platelet is able to

remain localized to the platelet's surface and is protected from plasma inhibition. According tothe cell-based model, the end result ofnonfunctional complexeson the platelet surfacesis the insufficient generation of thrombin, whichcauses the clinical bleeding tendencies seen inhemophilia [8, 15, 19].

The cell-based model also may provide ahypothesis to explain the role of FXI incoagulation. FXI can be activated by the smallamount of thrombin that is generated during theinitiationphase, which in turn activates additional

FIX. Additional FXa may then be manufactured by the tenase complex, leading to increasedthrombin generation. FXIa has been described asa possible thrombin mechanism booster. Theclinical representation of FXI deficiency isvariable because even in the absence of FXI, thetenase (FVIIIa/IXa) and prothrombinase(FXa/FVa) complexes are formed on the plateletsurface and are functional. For this reason, thelack of FXI results in a mild or absent bleedingtendency, because the coagulation mechanismstill may produce sufficient thrombin on thecellular surface. FXI thus is not essential for

platelet-surface thrombin generation, as are FIXand FVIII, and its deficiency does notcompromise hemostasis to the degree seen in FIXand FVIII deficiency [8, 20].

Conclusions

This review is an attempt to provide a comprehensive summary of the newunderstanding of the coagulation process. Thecell-based model represents a significantimprovement in our understanding of thehemostatic process. This model suggests that theextrinsic and intrinsic systems are in fact parallelgenerators of FXa that occur on different cellsurfaces, rather than redundant pathways. Inorder for coagulation to occur effectively,thrombin must be generated directly on the

activated platelet surface, not just on the surfaceof the TF-bearing cell.



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and function. In: Colman RW, Marder VJ, ClowesAW, George JN, Goldhaber SZ , editors.Hemostasis and Thrombosis: Basic Principles andClinical Practice. 5th ed. Philadelphia: LippincottWilliams and Wilkins; 2006, pp. 91-106.

12. Hoffman M, Monroe DM. Coagulation 2006: AModern View of Hemostasis. Hematol Oncol Clin N Am. 2007;21(1):1-11.

13. Tanaka KA , Key NS, Levy JH. BloodCoagulation: hemostasis and thrombin regulation.Anesth Analg. 2009;108(5):1433-46.

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15. Hoffman M, Monroe DM 3rd. A cell-based modelof hemostasis. Thromb Haemost. 2001;85(6):958-65

16. Crawley JT, Zanardelli S, Chion CK, Lane DA.The central role of thrombin in hemostasis. JThromb Haemost. 2007;5(Suppl 1):95-101.

17.Baglia FA, Badellino KO, Li CQ, Lopez JA, Walsh PN. Factor XI binding to the platelet glycoproteinIb-IX-V complex promotes factor XI activation bythrombin. J Biol Chem. 2002;277:1662-8.

18. Schenone M, Furie BC, Furie B. The bloodcoagulation cascade. Curr Opin Hematol. 2004;11(4):272-7.

19. Butenas S, Orfeo T, Brummel-Ziedins KE, MannKG. Tissue factor in thrombosis and hemorrhage.Surgery 2007;142(Suppl 4):S2–S14.

20. Monroe DM, Hoffman M. What does it take tomake the perfect clot? Arterioscler Thromb VascBiol. 2006;26(1):41-8.

21. AnaesthesiaUK [Internet]. The Royal College ofAnaesthetists, The Irish College of Anaesthetistsand The Intensive Care [updated 2007 march 15;cited 2011 April 5] . Available from:www.frca.co.uk/article.aspx?articleid=100805.

A New Understanding of the Coagulation Process

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