Normal Coagulation - Clinical Tree

Introduction

Vascular endothelial cells, blood, and extravascular tissue maintain blood flow fluidity or produce an integrated response to attenuate blood leakage by localized clotting at the site of vascular injury. The processes of blood coagulation and fibrinolysis are the primary defense systems of the vasculature. The opposing forces of fibrin clot formation and dissolution maintain hemostasis and preserve vascular function and integrity. Procoagulant events that culminate in the generation of thrombin and the formation of a fibrin clot protect the vasculature from perforating injury and excessive blood loss. Fibrinolysis removes the clot and initiates mechanisms involved in tissue repair and regeneration. Therefore, hemostasis refers to multiple discrete processes that center on thrombin generation, fibrin clot formation, and fibrin clot dissolution.

Hemostasis is not a passive state, but instead it is actively maintained by the vascular system. Specific cellular and protein interactions are required to maintain a state of equilibrium. When the system is perturbed, an integrated series of processes are required to initiate procoagulant events and to promote fibrinolysis and tissue repair. Each individual process that contributes to hemostasis must operate properly or the entire system is compromised. A balance among procoagulant, anticoagulant, and fibrinolytic factors is required to prevent uncontrolled bleeding or, conversely, excessive clot formation.

Procoagulant, Anticoagulant, Fibrinolytic Proteins, Inhibitors, And Receptors

History and Nomenclature

Current knowledge of the components involved in the complex process of blood coagulation ( Fig. 38.1 ) is the result of observations that date back to the 2nd century. Many hypotheses were envisioned about the transformation of fluid blood to a gel-like mass as it escaped the body, but the realization that clots stem blood loss did not occur until the beginning of the 18th century, ^, and it was not until the 19th century that the existence of thrombin, the key enzyme in blood coagulation, was recognized. ^, Four clotting factors were initially identified : factor I, fibrinogen; factor II, prothrombin; factor III, thromboplastin; and factor IV, calcium ions (or Ca ²⁺ ). As more coagulation factors were identified, initially by bleeding pathology and subsequently by laboratory clotting tests, ^, they were assigned consecutive Roman numerals, with activated forms distinguished by an a after the Roman numeral designation.

To describe the multiple simultaneous processes involved in generation of a hemostatic response, we identify an inventory of the procoagulant, anticoagulant, and fibrinolytic participants in blood coagulation. Subsequently, we describe the connections between these components and the dynamics of this process.

Vitamin-K-Dependent Proteins

The vitamin-K-dependent proteins, synthesized in the liver, play central roles in both the procoagulant or anticoagulant pathways. The family includes the procoagulant factors VII, IX, X, and prothrombin and the anticoagulants protein C, protein S, and protein Z ( Fig. 38.2 and Tables 38.1 and 38.2 ). Except for proteins S and Z, these proteins in their active forms are serine proteases. The cleavage of specific peptide bonds converts the vitamin-K-dependent zymogens to their active serine protease forms. All share noncatalytic domains, each of which is characterized by highly conserved regions that provide specific binding characteristics essential for their function. The domain organizations of the vitamin-K-dependent proteins are illustrated in Figure 38.2 .

TABLE 38.1

Procoagulant Proteins

Protein	Molecular Weight (kD)	Plasma Concentration		Plasma t _1/2 (Days)	Clinical Phenotype Associated with Deficiency	Functional Classification
Protein	Molecular Weight (kD)	(nmol/L)	(μg/mL)	Plasma t _1/2 (Days)	Clinical Phenotype Associated with Deficiency	Functional Classification
Factor XII	80	500	40	2–3	None	Protease zymogen
HMW kininogen	120	670	80		None	Cofactor
LMW kininogen	66	1300	90			Cofactor
Prekallikrein	85/88	486	42			Protease zymogen
Factor XI	160	30	4.8	2.5–3.3	Sometimes bleeding	Protease zymogen
Tissue factor	44			N/A		Cell-associated cofactor
Factor VII	50	10	0.5	0.25	Bleeding (occasionally thrombotic)	VKD protease zymogen
Factor X	59	170	10	1.5	Bleeding	VKD protease zymogen
Factor IX	55	90	5	1	Bleeding	VKD protease zymogen
Factor V	330	20	6.6	0.5	Bleeding ^a	Soluble procofactor
Factor VIII	285	1.1–1.5 ^d	0.3–0.4	0.3–0.5	Bleeding	Soluble procofactor
vWF	255	Varies	10		Bleeding	Carrier for factor VIII
Factor II	72	1400	100	2.5	Bleeding ^b	VKD protease zymogen
Fibrinogen	340	7400	2500	3–5	Bleeding ^c	Structural clot protein
Factor XIII	320	94	30	9–10	Bleeding	Transglutaminase zymogen

HMW, high-molecular-weight; LMW, low-molecular-weight; VKD, vitamin-K-dependent; vWF, von Willebrand factor.

a Factor V Leiden mutation associated with thrombosis.

b Prothrombin 20210A mutation associated with thrombosis.

c Some fibrinogen mutations associated with thrombosis.

d Butenas S, Parhami-Seren B, Mann KG. The influence of von Willebrand factor on factor VIII activity measurements. J Thromb Haemost . 2009;7:132–137; Butenas S, Parhami-Seren B, Undas A, Fass DN, Mann KG. The “normal” factor VIII concentration in plasma. Thromb Res . 2010;126:119–123.

TABLE 38.2

Anticoagulant Proteins, Inhibitors, and Receptors

Protein	M _r (kD)	Plasma Concentration		Plasma t _1/2 (Days)	Clinical Phenotype Associated with Deficiency	Functional Classification
Protein	M _r (kD)	(nmol/L)	(μg/mL)	Plasma t _1/2 (Days)	Clinical Phenotype Associated with Deficiency	Functional Classification
Protein C	62	65	4	0.33	Thrombotic	Proteinase zymogen
Protein S	69	300	20	1.75	Thrombotic	Inhibitory cofactor
Protein Z	62	47	2.9	2.5	Sometimes thrombotic	Inhibitory cofactor
Thrombomodulin	100	N/A	N/A	N/A		Cofactor/modulator
Tissue factor pathway inhibitor	40	1–4	0.1	minutes		Proteinase inhibitor
Antithrombin	58	2400	140	2.5–3	Thrombotic	Proteinase inhibitor
Heparin cofactor II	66	500–1400	33–90	2.5	Often thrombotic	Proteinase inhibitor
α ₂ -Macroglobulin	735	2700–4000	2–3000	<1 h		Proteinase inhibitor
α ₁ -Proteinase inhibitor	53	28,000–65,000	1500–3500	6		Proteinase inhibitor
Endothelial protein C receptor						Receptor

Vitamin K is essential for the biosynthesis of these clotting factors by participating in a cyclic oxidation and reduction process that converts 9 to 13 amino-terminal glutamate residues into γ-carboxyglutamate (Gla) residues. ^, ^, This posttranslational modification enables the vitamin-K-dependent proteins to interact with Ca ²⁺ (or calcium ions) and appropriate membranes. Inhibition of the Gla residue modification is the basis for “blood-thinning” anticoagulant therapy with coumarin derivatives (e.g., warfarin) that interfere with the redox cycle. The level of anticoagulation achieved with vitamin K antagonist therapy in individuals taking the same dose regimen is variable. ^, For instance, altered sensitivity to warfarin has been identified in patients when it is prescribed after surgery. Factors affecting the efficacy of treatment include liver function in the synthesis of clotting factors, the influence of other medications, and the dietary intake/absorption of vitamin K ; therefore, monitoring of warfarin therapy is essential (see Ch. 41 , Anticoagulant Therapy). ^,

NH ₂ -terminal Gla domains are followed by either a Kringle domain or an epidermal growth factor-like domain (see Fig. 38.2 ). Protein S is not a serine protease precursor but instead contains a thrombin-sensitive region before the epidermal growth factor domain and a sex hormone–binding globulin-like domain in the COOH terminus. Protein Z contains a “pseudo-catalytic domain” in the COOH terminus and is not a zymogen.

Cofactor Proteins

Cofactor proteins ( Fig. 38.3A ,B) either circulate in plasma (factor V and factor VIII) or are the cell-bound tissue factor (TF) and thrombomodulin (TM).

Soluble Plasma Profactors

Factor V

Factor V (see Fig. 38.3A and Table 38.1 ) is a large single-chain glycoprotein found in plasma or platelet alpha granules (18%–25% of the total factor V pool) and is secreted upon platelet activation. It is proteolytically activated by thrombin to form the cofactor factor Va ( Fig. 38.3A ). Factor Va functions as positive modulator of factor Xa’s catalytic potential in the prothrombinase complex in the presence of Ca ²⁺ and an appropriate membrane surface. It is proteolytically inactivated by activated protein C (APC) (see Fig. 38.3A ). ^, Cleavage at Arg ⁵⁰⁶ by APC reduces factor Va activity, while the slower cleavage at Arg ³⁰⁶ eliminates activity. The importance of this regulatory mechanism is illustrated by the factor V Leiden, characterized by an Arg ⁵⁰⁶ Gln mutation. Therefore, despite normal activity in the prothrombinase complex, factor Va Leiden is more slowly inactivated resulting in a prothrombotic phenotype.

Factor VIII

The procofactor factor VIII circulates in plasma in complex with the large multimeric protein von Willebrand factor (vWF; see Fig. 38.3A ). It is homologous (40% identity) with factor V, ^, but circulates in plasma as a two-chain molecule. Factor VIII is activated by thrombin cleavage at three sites to generate the heterotrimeric cofactor factor VIIIa that lacks a vWF binding site. Factor VIIIa enhances the enzymatic activity of the factor IXa in the presence of Ca ²⁺ and an appropriate membrane surface, forming intrinsic tenase complex. Its function is downregulated by the rapid and spontaneous dissociation of the noncovalently bound A ₂ subunit or proteolysis of the factor VIIIa light chain by APC. Deficiency of factor VIII (hemophilia A) is a well-characterized bleeding disorder linked to the X chromosome.

Cell-Bound Cofactors

Tissue factor

TF is a transmembrane protein that functions as a nonenzymatic cofactor with factor VIIa in the extrinsic tenase complex that activates both factors IX and X ( Fig. 38.4 ). TF is not expressed in blood in the absence of injury or inflammatory stimulus, and its presentation triggers the extrinsic pathway for hemorrhage control (see Fig. 38.1 ). Functional TF is presented following vascular damage that exposes the sub-endothelium or cytokine stimulation of monocytes. There are no known deficiencies of human TF, and the absence of TF in mice is lethal during embryonic development.

Figure 38.4, Vitamin-K-Dependent Complexes.

Thrombomodulin

TM is a high-affinity receptor for α-thrombin and acts as a cofactor for the activation of protein C (see Fig. 38.3B ). The endothelial cell protein C receptor provides cell-specific binding sites for both protein C and APC. ^, Once thrombin is bound to TM, its procoagulant activities are neutralized and the rate of inactivation of α-thrombin by antithrombin (AT) is increased. Protein C activation by the thrombin-TM complex (protein Case) leads to inactivation of the cofactors factor Va and factor VIIIa, suppressing thrombin formation (see Fig. 38.2 ). The protein Case complex also has an antifibrinolytic role through activation of thrombin-activatable fibrinolysis inhibitor (TAFI). TM activity on the surface of endothelial cells is decreased by inflammatory cytokines ; this decrease may contribute to hypercoagulation in inflammatory states.

Complexes

Vitamin-K-dependent protein complexes are essential for establishing hemostatic balance ( Fig. 38.4 ). Each complex is composed of a serine protease (factor VIIa, factor IXa, factor Xa, or thrombin [factor IIa]), a cofactor that functions as a receptor/enhancer for the enzyme (factor VIIIa, factor Va, TF, TM), Ca ²⁺ , and an appropriate anionic phospholipid membrane of cellular origin. There are four vitamin-K-dependent enzyme complexes (see Fig. 38.4 ): the extrinsic tenase complex (factor VIIa–TF), the intrinsic tenase complex (factor IXa–factor VIIIa), the prothrombinase complex (factor Xa–factor Va), and the anticoagulant protein Case complex (thrombin–TM). These membrane-bound complexes serve to localize enzymatic activity to the appropriate regional site for their required enzymatic functions and result in the only biologically relevant enzymatic activity for factor VIIa, factor IXa, and factor Xa – 10 ⁴ -fold to 10 ⁹ -fold faster reaction rates than the enzyme in solution.

Intrinsic (Accessory) Pathway Proteins

Deficiencies of proteins associated with the intrinsic pathway (factor XII, prekallikrein, and high-molecular-weight kininogen [HMWK]) are not associated with abnormal bleeding, even after surgical challenge. In contrast, deficiencies of the protein components of the extrinsic or primary pathway (prothrombin and factors V, VII, VIII, IX, and X) can lead to severe bleeding diatheses. The physiologic significance of the intrinsic pathway is still debated, but mounting evidence suggests that it serves as the link between inflammation and innate immunity.

Factor XI represents an intersection point for the two pathways. Individuals with factor XI deficiency (hemophilia C) express variable bleeding with surgical challenge, ^, thus establishing a role for factor XI in hemostasis. During the hemostatic process, formation of factor XIa appears to be catalyzed by thrombin as part of a positive feedback loop. Factor XIa then functions in the propagation phase of thrombin generation in association with the primary pathway by activation of factor IX.

Three proteins, factor XII, prekallikrein, and HMWK, are required for activity of the intrinsic pathway. Factor XII and prekallikrein are zymogens that are activated to generate serine proteases, and HMWK is a nonenzymatic procofactor ( Fig. 38.5 ). Activation of this pathway in vitro is accomplished when factor XII autoactivates to factor XIIa with exposure to a foreign surface (e.g., glass, kaolin, dextran sulfate, sulfatides). The substrates for factor XIIa, prekallikrein and factor XI, exist in a noncovalent complex with HMWK and become activated to kallikrein and factor XIa, respectively. This pathway is positively and negatively regulated via cleavage of HMWK by kallikrein and FXIa, respectively. ^, It has also been reported that the intrinsic pathway is activated by inorganic polyphosphates ^, or the assembly of these proteins on endothelial cell membranes. ^, Activation of the intrinsic pathway is important in cardiopulmonary bypass because of contact between blood components and synthetic surfaces.

Figure 38.5, Schematic Representation of the Intrinsic Pathway Proteins.

Stoichiometric Inhibitors

An array of inhibitors is present in blood with both specific and broad-spectrum actions. Inhibitors of clot formation are AT, tissue factor pathway inhibitor (TFPI), heparin cofactor II, and protein C inhibitor ( Fig. 38.6 ).

Antithrombin

AT is a member of the serpin proteinase family and circulates in blood as a single-chain glycoprotein (see Fig. 38.6 ). ^, Congenital AT deficiency exhibits an autosomal dominant pattern of inheritance, with an incidence of 1 in 2000 to 5000. Individuals with this deficiency have partial expression of AT and are prone to thromboembolic disease (see Ch. 40 , Disorders of Coagulation: Hypercoagulable States). The complete absence of AT is lethal. AT is a broad-spectrum anticoagulant, interacting with most proteases participating in the coagulation cascade (see Fig. 38.1 ), including thrombin, factor Xa, factor IXa, factor VIIa–TF, factor XIa, factor XIIa, plasma kallikrein, and HMWK. Heparins and heparan sulfates potentiate these reactions and are used in the treatment of thrombosis. When AT is complexed with heparin, its rate of inhibition of several coagulation proteases is accelerated by up to 10,000-fold as heparin induces structural changes that expose its reactive center loop. Antithrombin also displays antiproliferative and anti-inflammatory properties that primarily derive from its ability to inhibit thrombin. In addition, latent or cleaved forms of AT have antiangiogenic activity.

Tissue Factor Pathway Inhibitor

TFPI (also called “extrinsic pathway inhibitor” or “lipoprotein-associated coagulation inhibitor”) is a multivalent, Kunitz-type inhibitor that circulates in plasma as a heterogeneous collection of partially proteolyzed forms (see Fig. 38.6 ). Ninety percent of circulating TFPI is found associated with lipoproteins. ^, ^, Circulating TFPI is cleared principally by the liver and has an unusually short half-life compared with other proteinase inhibitors.

Complete deficiency of TFPI is incompatible with birth and survival in transgenic mice; however, this lethality can be rescued by heterozygous or homozygous factor VII deficiency. Mice bred to have a combined heterozygous TFPI deficiency and homozygous apolipoprotein E deficiency exhibit an increased atherosclerotic burden, suggesting a role for TFPI in protection from atherosclerosis and as a regulator of thrombosis.

TFPI is the principal stoichiometric inhibitor of the extrinsic tenase complex (factor VIIa–TF). Effective TFPI inhibition of the factor VIIa–TF complex depends on the presence of factor Xa; thus, inhibition of the extrinsic factor tenase by TFPI occurs only after significant generation of factor Xa. The TFPIα splice variant can also bind to some forms of factor Va formed early in the coagulation process and inhibit the prothrombinase complex. This interaction is the basis of the east Texas bleeding disorder, which is associated with life-threatening bleeding after trauma or surgery due to the production of a factor V splice variant that binds tightly to TFPIα. ^,

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