Fibrin Meshwork Formation

FIBRIN MESHWORK FORMATION

The formation of the fibrin meshwork involves two unique pathways that converge to form a common pathway (Figure 34.2). In each pathway, the major components are proteins (called factors) designated by Roman numerals. The factors are glycoproteins that are synthesized and secreted by the liver, primarily. [Note: Several factors are also denoted by alternative names. For example, active factor X (FXa), the point of pathway convergence, is also known as Stuart factor.]

Figure 34.2 Three pathways involved in formation of the fibrin meshwork.

A. Protolytic cascade

Within the pathways, a cascade is set up in which proteins are converted from an inactive form, or zymogen, to an active form by proteolytic cleavage in which the protein product of one activation reaction initiates another. The active form of a factor is denoted by a lower case “a” after the numeral. The active proteins FIIa, FVIIa, FIXa, FXa, FXIa, and FXIIa are enzymes that function as serine proteases with trypsin-like specificity and, therefore, cleave a peptide bond on the carboxyl side of an arginine or lysine residue in a polypeptide. For example, FIX (Christmas factor) is activated through cleavage at arginine 145 and arginine 180 by FXIa (Figure 34.3). The proteolytic cascade results in enormous rate acceleration, because one active protease can produce many molecules of active product each of which, in turn, can activate many molecules of the next protein in the cascade. In some cases, activation can be caused by a conformational change in the protein in the absence of proteolysis. [Note: Nonproteolytic proteins play a role as accessory proteins (cofactors) in the pathways. FIII, FV, and FVIII are the accessory proteins.]

Figure 34.3 Activation of FIX (Christmas factor) via proteolysis by the serine protease FXIa. [Note: Activation can occur by conformational change for some of the factors.] a = active; Arg = arginine.

B. Role of phosphatidylserine and calcium

The presence of the negatively charged phospholipid phosphatidylserine (PS) and positively charged calcium ions (Ca²⁺) accelerates the rate of some steps in the cascade.

1. Negatively charged phosphatidylserine: PS is located primarily on the intracellular (cytosolic) face of the plasma membrane. Its exposure signals injury to the endothelial cells that line blood vessels. PS is also exposed on the surface of activated platelets.

2. Calcium ions: Ca²⁺ binds the negatively charged γ-carboxyglutamate (Gla) residues present in certain clotting serine proteases (FII, FVII, FIX, and FX), facilitating the binding of these proteins to exposed phospholipids (Figure 34.4). The Gla residues are good chelators of Ca²⁺ because of their two adjacent negatively charged carboxylate groups (Figure 34.5). [Note: The use of chelating agents such as sodium citrate to bind Ca²⁺ in blood-collecting tubes or bags prevents the blood from clotting.]

Figure 34.4 Ca²⁺ facilitates the binding of γ-carboxyglutamate (Gla)- containing factors to membrane phospholipids. F = factor.

Figure 34.5 Gla residue.

C. Formation of γ-carboxyglutamate residues

γ-Carboxylation is a posttranslational modification in which 9–12 glutamate residues (at the amino or N terminus of the target protein) get carboxylated at the γ carbon, thereby forming γ-carboxyglutamate (Gla) residues. The process occurs in the rough endoplasmic reticulum (RER) of the liver.

1. γ-Carboxylation: This carboxylation reaction requires a protein substrate, O₂, CO₂, γ-glutamyl carboxylase, and the hydroquinone form of vitamin K as a coenzyme (Figure 34.6). In the reaction, the hydroquinone form of vitamin K gets oxidized to its epoxide form as O₂ is reduced to water. [Note: Vitamin K, a fat-soluble vitamin, is reduced from the quinone form to the hydroquinone coenzyme form by vitamin K reductase (Figure 34.7).]

Figure 34.6 γ-Carboxylation of a glutamate (Glu) residue to γ-carboxyglutamate (Gla) by vitamin K-requiring γ-glutamyl carboxylase. The γ carbon is shown in blue.

Figure 34.7 The vitamin K cycle. VKOR = vitamin K epoxide reductase.

2. Inhibition by warfarin: The formation of Gla residues is sensitive to inhibition by warfarin, a synthetic analog of vitamin K that inhibits the enzyme vitamin K epoxide reductase (VKOR). The reductase, an integral protein complex of the RER membrane, is required to regenerate the functional hydroquinone form of vitamin K from the epoxide form generated in the γ-carboxylation reaction. Thus, warfarin is an anticoagulant that inhibits clotting by functioning as a vitamin K antagonist. Warfarin salts are used therapeutically to limit clot formation. [Note: Warfarin is used commercially as a pest-control agent such as in rat poison. It was developed by the Wisconsin Alumni Research Foundation, hence the name.]

Genetic differences (genotypes) in the gene for subunit 1 of the VKOR complex (VKORC1) influence patient response to warfarin. For example, a polymorphism in the promoter region of the gene decreases gene expression, resulting in less VKOR being made, thereby necessitating a lower dose of warfarin to achieve a therapeutic level. Polymorphisms in the cytochrome P450 enzyme (CYP2C9) that metabolizes warfarin are also known. In 2010, the U.S. Food and Drug Administration added a genotype-based dose table to the warfarin label (package insert). The influence of genetics on an individual’s response to drugs is known as pharmacogenetics.

D. Pathways

Three distinct pathways are involved in formation of the fibrin meshwork: the extrinsic pathway, the intrinsic pathway, and the common pathway. Production of FXa by the extrinsic and intrinsic pathways initiates the common pathway (see Figure 34.2).

1. Extrinsic pathway: This pathway involves a protein, tissue factor (TF), that is not in the blood but becomes exposed when blood vessels get injured. TF (FIII) is a transmembrane glycoprotein abundant in vascular subendothelium. It is an extravascular accessory protein and not a protease. Any injury that exposes FIII to blood rapidly (within seconds) initiates the extrinsic (or TF) pathway. Once exposed, TF binds a circulating Gla-containing protein, FVII, activating it through conformational change. [Note: FVII can also be activated proteolytically by thrombin (see Section 3. below).] Binding of FVII to TF requires the presence of Ca²⁺ and phospholipids. The TF–FVIIa complex then binds and activates FX by proteolysis (Figure 34.8). Therefore, activation of FX by the extrinsic pathway occurs in association with the membrane. The extrinsic pathway is quickly inactivated by tissue factor pathway inhibitor (TFPI) that, in a FXa-dependent process, binds to the TF–FVIIa complex and prevents further production of FXa. [Note: TF and FVII are unique to the extrinsic pathway.]

Figure 34.8 The extrinsic or tissue factor (TF) pathway. Binding of FVII to exposed TF (FIII) activates FVII. [Note: The pathway is quickly inhibited by tissue factor pathway inhibitor (TFPI).] F = factor; Gla = γ-carboxyglutamate; PL = phospholipid; a = active.

2. Intrinsic pathway: All of the protein factors involved in the intrinsic pathway are present in the blood and are, therefore, intravascular. The intrinsic pathway involves two phases: the contact phase and the FX-activation phase, each with known deficiencies.

a. Contact phase: This phase results in the activation of FXII (Hageman factor) to FXIIa by conformational change through binding to a negative surface. Deficiencies in FXII (or in the other proteins of this phase, high-molecular-weight kininogen and prekallikrein) do not result in bleeding problems, calling into question the importance of this phase in coagulation. However, the contact phase does play a role in inflammation. [Note: FXII can be activated proteolytically by thrombin (see Section 3. below)].

b. Factor X–activation phase: The sequence of events leading to the activation of Factor X to FXa by the intrinsic pathway is initiated by FXIIa (Figure 34.9). FXIIa activates FXI, and FXIa activates FIX, a Gla-containing protein. FIXa combines with FVIIIa (a bloodborne accessory [nonenzymatic] protein), and the complex activates FX, a Gla-containing serine protease. [Note: The complex containing FIXa, FVIIIa, and FX forms on exposed negatively charged membrane regions, and FX gets activated to FXa. This complex is sometimes referred to as Xase. Binding of the complex to membrane phospholipids requires Ca²⁺.]

Figure 34.9 FX activation phase of the intrinsic pathway. [Note: von Willebrand factor (VWF) stabilizes FVIII in the circulation.] Gla = γ-carboxyglutamate; PL = phospholipid; a = active; F = factor.

c. Factor XII deficiency: A deficiency in FXII does not lead to a bleeding disorder. This is because FXI, the next protein in the cascade, can be activated proteolytically by thrombin (see Section 3. below).

d. Hemophilia: Hemophilia is a coagulopathy, a defect in the ability to clot. Hemophilia A (the most common form of hemophilia) results from deficiency of FVIII, whereas deficiency of FIX results in hemophilia B. Each deficiency is characterized by decreased and delayed ability to clot and/or formation of abnormally friable (easily disrupted) clots. This can be manifested, for example, by bleeding into the joints (Figure 34.10). The extent of the factor deficiency determines the severity of the disease. Current treatment for hemophilia is factor replacement therapy using factors obtained from pooled human blood or from recombinant DNA technology. Gene replacement therapy is a goal. Because the genes for both proteins are on the X chromosome, hemophilia is an X-linked disorder. [Note: Deficiency of FXI results in a bleeding disorder that sometimes is referred to as hemophilia C.]

Figure 34.10 Acute bleeding into joint spaces (hemarthrosis) in an individual with hemophilia.

The inactivation of the extrinsic pathway by TFPI results in dependence on the intrinsic pathway for continued production of FXa. This explains why individuals with hemophilia bleed even though they have an intact extrinsic pathway.

3. Common pathway: FXa produced by both the intrinsic and the extrinsic paths initiates the common pathway, a sequence of events that results in the generation of fibrin (FIa) (Figure 34.11) . FXa associates with FVa (a bloodborne accessory [nonenzymic] protein) and, in the presence of Ca²⁺ and phospholipids, forms a membrane-bound complex referred to as prothrombinase. The complex cleaves prothrombin (FII) to thrombin (FIIa). [Note: FVa potentiates the proteolytic activity of FXa.] The binding of Ca²⁺ to the Gla residues in FII facilitates the binding of FII to the membrane and to the prothrombinase complex, with subsequent cleavage to thrombin. Cleavage excises the Gla-containing region, releasing thrombin from the membrane and, thereby, freeing it to activate fibrinogen (FI) in the blood. [Note: This is the only example of cleavage of a Gla protein that results in the release of a Gla-containing peptide. The peptide travels to the liver where it is thought to act as a signal for increased production of clotting proteins.] Oral, direct inhibitors of FXa have been approved for limited clinical use as anticoagulants.

A common point mutation (G20210A) in which an adenine (A) replaces a guanine (G) at nucleotide 20210 in the 3′ untranslated region of the gene for prothrombin leads to increased levels of prothrombin in the blood. This results in thrombophilia, a condition characterized by an increased tendency to clot.

Figure 34.11 Generation of fibrin by FXa and the common pathway. F = factor; Gla = γ-carboxyglutamate; PL = phospholipid; a = active.

a. Conversion of fibrinogen to fibrin by thrombin: Fibrinogen is a soluble glycoprotein made by the liver. It consists of dimers of three different polypeptide chains [(Aα)2(Bβ)2(γ)2] held together at the N termini by disulfide bonds. [Note: Aα and Bβ each represent a single polypeptide.] The N termini of the Aα and Bβ chains form “tufts” on the central of three globular domains (Figure 34.12). The tufts are negatively charged and result in repulsion between fibrinogen molecules. Thrombin cleaves the charged tufts (releasing fibrinopeptides A and B), and fibrinogen becomes fibrin. As a result of the loss of charge, the fibrin monomers are able to noncovalently associate in a staggered array, and a soft (soluble) fibrin clot is formed.

Figure 34.12 Conversion of fibrinogen to fibrin and formation of the soft fibrin clot. [Note: D and E refer to domains on fibrin.]

b. Cross-linking of fibrin: The associated linked. This converts the soft clot to fibrin molecules get covalently cross-a hard (insoluble) clot. FXIIIa, a transglutaminase, covalently links the γ-carboxamide of a glutamine residue in one fibrin molecule to the ε-amino of a lysine residue in another through formation of an isopeptide bond and release of ammonia (Figure 34.13). [Note: FXIII is also activated by thrombin.]

Figure 34.13 Cross-linking of fibrin. FXIIIa forms a covalent isopeptide bond between a lysine residue and a glutamine residue. F = factor.

c. Importance of thrombin: The activation of FX by the extrinsic path provides the “spark” of FXa that results in the initial activation of thrombin. Active thrombin then activates factors of the common (FV, FI, FXIII), intrinsic (FXI, FVIII), and extrinsic (FVII) pathways (Figure 34.14). It also activates FXII of the contact phase. The extrinsic pathway, then, initiates clotting by the generation of FXa, and the intrinsic pathway amplifies and sustains clotting after the extrinsic pathway has been inhibited by TFPI. [Note: Hirudin, a peptide secreted from the salivary gland of medicinal leeches, is a potent, direct, oral thrombin inhibitor. Recombinant hirudin has been approved for limited clinical use.] Additional crosstalk between the pathways of clotting is achieved by the FVIIA^-TF-mediated activation of the intrinsic pathway and the FXIIa-mediated activation of the extrinsic pathway. The complete picture of physiologic blood clotting via the formation of a hard fibrin clot is shown in Figure 34.15. The factors of the clotting cascade are shown organized by function in Figure 34.16.

Figure 34.14 The importance of thrombin in formation of the fibrin clot. a = active; F = factor.

Figure 34.15 The complete picture of physiologic blood clotting via the formation of a cross-linked (hard) fibrin clot. a = active; F = factor; TF = tissue factor; TFPI = tissue factor pathway inhibitor; PL = phospholipid; Gla = γ-carboxyglutamate.

Clinical laboratory tests are available to evaluate the extrinsic through common pathways (prothrombin time [PT] using thromboplastin and expressed as the International Normalized Ratio [INR]) and the intrinsic through common pathways (activated partial thromboplastin time [aPTT]). Thromboplastin is a combination of phospholipids + FIII. A derivative, partial thromboplastin, contains just the phospholipid portion because FIII isn’t needed to activate the intrinsic pathway.

Figure 34.16 Protein factors of the clotting cascade organized by function. The activated form would be denoted by an a after the numeral. [Note: Ca²⁺ is IV. There is no VI. I (fibrin) is neither a protease nor an accesory protein. XIII is a transglutaminase.] Gla = γ-carboxyglutamate.