Blood Coagulation

BLOOD COAGULATION

A. "Intrinsic" and "extrinsic" coagulation pathways
B. Identification of distinct coagulation factors
C. Sequence of coagulation reactions

II. Coagulation in vivo

A. Exposure of plasma to tissue factor initiates coagulation
B. Coagulation can be initiated via the "intrinsic" pathway in vitro
C. Concentrations of coagulation factors required for normal hemostasis

III. Biochemistry of coagulation

A. Structure of coagulation protease zymogens
B. Non-enzymatic protein cofactors
C. Prothrombin activation
D. Fibrinogen
E. Factor XIII
F. Amplification and localization of coagulation reactions

Blood coagulation is part of an important host defense mechanism termed hemostasis (the cessation of blood loss from a damaged vessel). Upon vessel injury, platelets adhere to macromolecules in the subendothelial tissues and then aggregate to form the primary hemostatic plug. The platelets stimulate local activation of plasma coagulation factors, leading to generation of a fibrin clot that reinforces the platelet aggregate. Later, as wound healing occurs, the platelet aggregate and fibrin clot are broken down. Mechanisms that restrict formation of platelet aggregates and fibrin clots to sites of injury are necessary to maintain the fluidity of the blood.

Hemorrhage can result from trauma, vascular defects (e.g., esophageal varices, peptic ulcer disease), platelet abnormalities, or deficiencies of one or more of the plasma coagulation factors.

Thrombosis is a pathologic process in which a platelet aggregate and/or a fibrin clot forms in the lumen of an intact blood vessel or in a chamber of the heart. If thrombosis occurs in an artery, the tissue supplied by the artery may undergo ischemic necrosis (e.g., myocardial infarction due to thrombosis of a coronary artery). If thrombosis occurs in a vein, the tissues drained by the vein may become edematous and inflamed. Thrombosis of a deep vein in the lower extremity may be complicated by pulmonary embolism, in which all or a portion of the thrombus breaks loose, is carried in the bloodstream through the vena cava and the right side of the heart, and becomes lodged in a pulmonary artery. Massive pulmonary embolism can cause hypoxemia, shock, and death.

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I. Coagulation in vitro

When blood is removed from the body and placed in a glass test tube, it clots fairly quickly (Table 1). Calcium ions are required for this process. Addition of EDTA or citrate prevents clotting by binding calcium. Clotting can be initiated in vitro at a later time by adding back an excess of calcium ions. As shown in Table 1, recalcified plasma (minus platelets and other blood cells) will clot in 2-4 min. The clotting time after recalcification can be shortened by adding an emulsion of negatively-charged phospholipids (PL). The clotting time is further shortened to 21-32 sec by preincubation of the plasma with particulate substances such as kaolin (insoluble aluminum silicate). The reaction initiated by kaolin, PL, and calcium is termed the activated partial thromboplastin time (aPTT) test. Alternatively, the clotting time of recalcified plasma can be shortened to 11-12 sec by adding "thromboplastin" (a saline brain extract containing tissue factor, a lipoprotein described below). The reaction initiated by thromboplastin and calcium is termed the prothrombin time (PT) test.

Table 1
Coagulation in vitro

	Clotting time
Whole blood	4-8 min
Whole blood + EDTA or citrate	infinite
Citrated platelet-poor plasma + Ca++	2-4 min
Citrated platelet-poor plasma + PL + Ca++	60-85 sec
Citrated platelet-poor plasma + kaolin + PL + Ca++	21-32 sec (aPTT)
Citrated platelet-poor plasma + thromboplastin + Ca++	11-12 sec (PT)

A. "Intrinsic" and "extrinsic" coagulation pathways. Many patients with inherited bleeding disorders have prolongation of the aPTT, the PT, or both. Thus, two pathways for coagulation were proposed (Fig. 1). A patient with a prolonged aPTT and a normal PT is considered to have a defect in the "intrinsic" coagulation pathway. The name indicates that all of the components of the aPTT test, except kaolin, are "intrinsic" to the plasma. On the other hand, a patient with a prolonged PT and a normal aPTT has a defect in the "extrinsic" coagulation pathway (tissue factor is "extrinsic" to the plasma). Prolongation of both the aPTT and the PT suggests that the defect lies in a common pathway.

Fig. 1

B. Identification of distinct coagulation factors. Early investigators discovered that mixing plasma from two patients with defects in the same coagulation pathway would sometimes correct the clotting time to normal. This type of result suggests that the two patients have different coagulation factor deficiencies (e.g., the hypothetical orange and blue factors in Fig. 2). In this manner, 11 plasma coagulation factors were discovered: 6 in the "intrinsic" pathway (factors VIII, IX, XI, XII, prekallikrein, and high-molecular weight kininogen), 1 in the "extrinsic" pathway (factor VII), and 4 in the common pathway (factors II, V, X, and fibrinogen).

Fig. 2
Differentiation of Congenital Bleeding Disorders

C. Sequence of coagulation reactions. The classical model of blood coagulation involves a series (or "cascade") of zymogen activation reactions as shown in Fig. 3. At each stage a precursor protein (zymogen) is converted to an active protease by cleavage of one or more peptide bonds in the precursor molecule. The types of components that can be involved at each stage include the following:

(a) a protease (from the preceding stage)
(b) a zymogen
(c) a non-enzymatic protein cofactor
(d) calcium ions
(e) an organizing surface (provided by a phospholipid emulsion in vitro or by platelets in vivo)

The final protease generated is thrombin (factor IIa). Thrombin converts the soluble protein fibrinogen into an insoluble fibrin gel, which is strengthened further by covalent cross-linking catalyzed by factor XIIIa.

Fig. 3
Coagulation Cascade

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II. Coagulation in vivo

Although the concept of "intrinsic" and "extrinsic" pathways served for many years as a useful model for coagulation, more recent evidence has shown that the pathways are not, in fact, redundant but are highly interconnected. For example, the tissue factor/VIIa complex activates not only factor X (as shown in Fig. 3) but also factor IX of the intrinsic pathway. Furthermore, patients with severe factor VII deficiency may bleed even though the intrinsic pathway is intact. Likewise, the severe bleeding associated with deficiencies of factors VIII or IX would not be expected if the extrinsic pathway alone were sufficient to achieve normal hemostasis.

A. Exposure of plasma to tissue factor initiates coagulation. Tissue factor is a non-enzymatic lipoprotein constitutively expressed on the surface of cells that are not normally in contact with plasma (e.g., fibroblasts and macrophages). Exposure of plasma to these cells initiates coagulation outside a broken blood vessel. Endothelial cells also express tissue factor when stimulated by endotoxin, tumor necrosis factor, or interleukin-1, and may be involved in thrombus formation under pathologic conditions.

Tissue factor binds factor VIIa and accelerates factor X activation about 30,000-fold. Although factor VII is activated by its product, factor Xa, a trace amount of factor VIIa appears to be available in plasma at all times to interact with tissue factor. Factor VIIa also activates factor IX in the presence of tissue factor, providing a connection between the "extrinsic" and "intrinsic" pathways (cf. Figs. 3 & 4). Factors IXa and Xa assemble with their non-enzymatic protein cofactors (VIIIa and Va, respectively) on the surface of aggregated platelets. This leads to local generation of large amounts of Xa and thrombin (IIa), followed by conversion of fibrinogen to fibrin.

Fig. 4
Initiation of Coagulation In Vivo

Tissue factor pathway inhibitor (TFPI) is a 34-kDa protein associated with plasma lipoproteins and with the vascular endothelium. It binds to and inhibits factor Xa. The Xa-TFPI complex then interacts with VIIa/tissue factor and inhibits activation of factors X and IX. TFPI may prevent coagulation unless the VIIa/tissue factor initially present generates a sufficient amount of factor IXa to sustain factor X activation via the "intrinsic" pathway. Thus, VIIa/tissue factor may provide the initial stimulus to clot (in the form of relatively small amounts of IXa and Xa) and then be rapidly turned off, while IXa and VIIIa may be responsible for generating the larger amounts of Xa and thrombin required for clot formation.

B. Coagulation can be initiated via the "intrinsic" pathway in vitro when factor XII, prekallikrein, and high-molecular weight kininogen (HMWK) bind to kaolin, glass, or another artificial surface. Once bound, reciprocal activation of XII and prekallikrein occurs (Fig. 3). Factor XIIa triggers clotting via the sequential activation of factors XI, IX, X, and II (prothrombin).

Activation of factor XII is not required for hemostasis, since patients with deficiency of factor XII, prekallikrein, or HMWK do not bleed even though their aPTT values are prolonged. Patients with factor XI deficiency tend to have a mild bleeding disorder, however, implying that XI is involved in hemostasis. The mechanism for activation of factor XI in vivo is unknown, although thrombin has been shown to activate XI in vitro.

C. Concentrations of coagulation factors required for normal hemostasis are summarized in Table 2. With the exception of fibrinogen, factor levels are usually reported as percentages of the concentrations present in plasma pooled from normal individuals. Tissue factor is not present in plasma and cannot be quantified in patients.

Table 2
Plasma Coagulation Factors

Factor	Molecular Weight	Plasma Concentration (µg/ml)	Required for Hemostasis (% of normal concentration)
Fibrinogen	330,000	3000	30
Prothrombin	72,000	100	40
Factor V	300,000	10	10-15
Factor VII	50,000	0.5	5-10
Factor VIII	300,000	0.1	10-40
Factor IX	56,000	5	10-40
Factor X	56,000	10	10-15
Factor XI	160,000	5	20-30
Factor XIII	320,000	30	1-5
Factor XII	76,000	30	0
Prekallikrein	82,000	40	0
HMWK	108,000	100	0

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III. Biochemistry of coagulation

A. Structure of coagulation protease zymogens. The structures of the protease zymogens involved in coagulation (i.e., factors II (prothrombin), VII, IX, X, XI, XII, and kallikrein) are shown in Fig. 5. Each protein is secreted by hepatocytes into the bloodstream and contains a signal peptide that is removed during transit into the endoplasmic reticulum. About 200 amino acid residues at the C-terminal end of each zymogen are homologous to trypsin and contain the active site Ser, Asp, and His residues of the protease (catalytic domain). The proteins also contain a variety of domains that are homologous to portions of other proteins such as epidermal growth factor (EGF) and fibronectin. These domains appear to be involved in specific interactions between the proteases and their substrates, cofactors, and/or inhibitors.

Fig. 5
Structures of Coagulation Factors

Factors II, VII, IX, and X are homologous to each other at their N-terminal ends. After removal of the signal peptide, a carboxylase residing in the endoplasmic reticulum or Golgi binds to the propeptide region of each of these proteins and converts ~10-12 glutamate (Glu) residues to g-carboxyglutamate (Gla) in the adjacent "Gla domain" (Fig. 6). The propeptide is removed from the carboxylated polypeptide prior to secretion. The Gla residues bind calcium ions and are necessary for the activity of these coagulation factors. Synthesis of Gla requires vitamin K. During g-carboxylation, vitamin K becomes oxidized and must be reduced subsequently in order for the cycle to continue (Fig. 7). The anticoagulant drug warfarin (Coumadin®) inhibits reduction of vitamin K and thereby prevents synthesis of active factors II, VII, IX, and X.

Fig. 6
g-Carboxylation of Prothrombin (Factor II)

Fig. 7
Role of Vitamin K in Biosynthesis of Factors II, VII, IX, and X

B. Non-enzymatic protein cofactors include factors V and VIII, tissue factor, and high-molecular weight kininogen (HMWK) (Table 3). Factors V and VIII are large plasma proteins that contain repeated sequences homologous to the copper-binding protein ceruloplasmin (A1, A2 & A3 domains in Fig. 8). Thrombin cleaves V and VIII to yield activated factors (Va and VIIIa) that have at least 50 times the coagulant activity of the precursor forms (Fig. 8). Va and VIIIa have no known enzymatic activity. Instead, they serve as cofactors that increase the proteolytic efficiency of Xa and IXa, respectively. Factor VIII circulates in plasma bound to von Willebrand factor; thus, a patient with von Willebrand factor deficiency will generally have a concomitant decrease in the plasma concentration of factor VIII.

Tissue factor is an integral membrane protein that is expressed on the surface of "activated" monocytes, endothelial cells exposed to various cytokines (e.g., tumor necrosis factor), and other cells. It is not found in plasma. Tissue factor greatly increases the proteolytic efficiency of VIIa.

Table 3
Non-enzymatic Protein Cofactors

Cofactor	Location	Mol Wt	Activated by	Cofactor for
Factor V	Plasma	300,000	Thrombin	Factor Xa
Factor VIII	Plasma (bound to vWF)	300,000	Thrombin	Factor IXa
Tissue factor	Cell membranes	40,000		Factor VIIa
HMWK	Plasma	110,000		Factor XIIa

Fig. 8
Activation of Factor VIII by Thrombin

C. Prothrombin activation. Factor Xa converts prothrombin (factor II) to thrombin (factor IIa) by cleaving two peptide bonds in the zymogen (indicated by arrows in Fig. 5). Activation of prothrombin by Xa is accelerated by Va, platelets (or phospholipids), and calcium ions (Table 4). The complete system activates prothrombin at a rate about 300,000 times greater than that of Xa and calcium alone. Phospholipids and Va can accelerate prothrombin activation independently (cf. lines 1, 2, and 3 in Table 4), but they act synergistically in the complete system (i.e., 50 x 350 ª 19,000). Rapid activation occurs only when prothrombin and Xa both contain Gla residues and, therefore, have the ability to bind calcium (cf. lines 4 and 5 in Table 4). Binding of calcium alters the conformation the Gla domains of these factors, enabling them to interact with a membrane surface provided by phospholipids in vitro or platelets in vivo (note that platelets are more efficient in this regard). Interactions among the components of the "prothrombinase" complex are shown schematically in Fig. 9. Aggregated platelets are thought to provide the surface upon which prothrombin activation occurs at a site of hemostasis. Activation of factor X by IXa and its cofactor VIIIa appears to occur by a mechanism similar to that of prothrombin activation and may also be accelerated by platelets in vivo.

Table 4
Acceleration of Prothrombin Activation by Factor Va and Platelets

	Purified Components	Relative Rate of Thrombin Generation
(1)	Prothrombin, Xa, Ca⁺⁺	1	(35 days)
(2)	Prothrombin, Xa, PL, Ca⁺⁺	50	(17 hours)
(3)	Prothrombin, Xa, Va, Ca⁺⁺	350	(2.4 hours)
(4)	Prothrombin, Xa, Va, PL, Ca⁺⁺	<1000	(>50 min)
(5)	Prothrombin, Xa, Va, PL, Ca⁺⁺	19,000	(2.5 min)
(6)	Prothrombin, Xa, Va, platelets, Ca⁺⁺	300,000	(10 sec)

PL = phospholipids
Prothrombin* and Xa* are Gla-less forms of prothrombin and Xa, synthesized in the absence of vitamin K.

Fig. 9
Prothrombin Activation Complex

D. Fibrinogen consists of three pairs of polypeptide chains covalently linked by disulfide bonds (Fig. 10) and has a molecular weight of approximately 330,000. Thrombin converts fibrinogen to fibrin monomers by cleaving fibrinopeptides A (16 amino acid residues) and B (14 amino acid residues) from the N-terminal ends of the Aa and Bb chains, respectively. Removal of the fibrinopeptides allows the fibrin monomers to form a gel consisting of long polymers as shown in Fig. 11. Formation of the fibrin gel constitutes the end point of the aPTT and PT tests. At this stage, the fibrin monomers are bound to each other non-covalently.

Fig. 10
Structure of Fibrinogen

Fig. 11
Fibrin Polymerization

E. Factor XIII. Covalent cross-linking of fibrin polymers by activated factor XIII (XIIIa) is required for adequate clot strength and normal wound healing in vivo. The zymogen form of factor XIII is converted to an active enzyme (XIIIa) by thrombin (Fig. 12). XIIIa catalyzes a transglutamination reaction that initially cross-links the C-terminal ends of the g chains on adjacent fibrin monomers. Several other sites become cross-linked more slowly and give the clot additional strength. Because cross-linking of fibrin occurs after formation of a visible clot in vitro, the aPTT and PT tests do not depend on the activity of factor XIII. However, plasma factor XIII can be assayed by determining the solubility of a clot in the presence of urea, which cannot dissolve cross-linked fibrin.

Fig. 12
Transglutaminase Activity of Factor XIIIa

F. Amplification and localization of coagulation reactions. The catalytic nature of coagulation reactions allows tremendous amplification of the initial stimulus. Each VIIa/tissue factor complex can produce many Xa molecules, which subsequently produce an even greater amount of thrombin. Amplification also results from positive feedback reactions (see Fig. 13). These include activation of V, VIII, and possibly XI by thrombin, as well as activation of VII by Xa. Assembly of activation complexes on cell membranes normally serves to localize coagulation to sites of vessel injury. Membrane-associated reactions include

(a) activation of X and IX by the VIIa/tissue factor complex on the surface of smooth muscle cells or other cells located beneath the vascular endothelium,
(b) activation of X by the IXa/VIIIa complex on the surface of platelets that have become activated at the site of injury, and
(c) activation of prothrombin by the Xa/Va complex on the surface of activated platelets.

Fig. 13
Feedback Reactions

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