blood coagulation

Introduction
Platelet Activation and von Willebrand Factor (vWF)
Description of Clotting Factors
Image of the Clotting Cascade
Kallikrein-Kinin System in Coagulation
Intrinsic (contact activation) Clotting Cascade
Extrinsic (tissue factor) Clotting Cascade
Activation of Thrombin
Regulation of Thrombin Levels
Activation of Fibrin
Dissolution of Fibrin Clots
Clinical Significances of Hemostasis: The Bleeding Disorders
Pharmacological Intervention in Bleeding
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Introduction

The ability of the body to control the flow of blood following vascular injury is paramount to continued survival. The process of blood clotting and then the subsequent dissolution of the clot, following repair of the injured tissue, is termed hemostasis. Hemostasis, composed of 4 major events that occur in a set order following the loss of vascular integrity:

1. The initial phase of the process is vascular constriction. This limits the flow of blood to the area of injury.

2. Next, platelets become activated by thrombin and aggregate at the site of injury, forming a temporary, loose platelet plug. The protein fibrinogen is primarily responsible for stimulating platelet clumping. Platelets clump by binding to collagen that becomes exposed following rupture of the endothelial lining of vessels. Upon activation, platelets release the nucleotide, ADP and the eicosanoid, TXA2 (both of which activate additional platelets), serotonin, phospholipids, lipoproteins, and other proteins important for the coagulation cascade. In addition to induced secretion, activated platelets change their shape to accommodate the formation of the plug.

3. To insure stability of the initially loose platelet plug, a fibrin mesh (also called the clot) forms and entraps the plug. If the plug contains only platelets it is termed a white thrombus; if red blood cells are present it is called a red thrombus

4. Finally, the clot must be dissolved in order for normal blood flow to resume following tissue repair. The dissolution of the clot occurs through the action of plasmin

Two pathways lead to the formation of a fibrin clot: the intrinsic and extrinsic pathway. Although they are initiated by distinct mechanisms, the two converge on a common pathway that leads to clot formation. Both pathways are complex and involve numerous different proteins termed clotting factors. Fibrin clot formation in response to tissue injury is the most clinically relevant event of hemostasis under normal physiological conditions. This process is the result of the activation of the extrinsic pathway. The formation of a red thrombus or a clot in response to an abnormal vessel wall in the absence of tissue injury is the result of the intrinsic pathway. The intrinsic pathway has low significance under normal physiological conditions. Most significant clinically is the activation of the intrinsic pathway by contact of the vessel wall with lipoprotein particles, VLDLs and chylomicrons. This process clearly demonstrates the role of hyperlipidemia in the generation of atherosclerosis. The intrinsic pathway can also be activated by vessel wall contact with bacteria.
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Platelet Activation and von Willebrand Factor (vWF)

In order for hemostasis to occur, platelets must adhere to exposed collagen, release the contents of their granules, and aggregate. The adhesion of platelets to the collagen exposed on endothelial cell surfaces is mediated by von Willebrand factor (vWF). Inherited deficiencies of vWF are the causes of von Willebrand disease, (vWD) (also see below for more details). The function of vWF is to act as a bridge between a specific glycoprotein complex on the surface of platelets (GPIb-GPIX-GPV) and collagen fibrils. The importance of this interaction between vWF and the GPIb-GPIX-GPV complex of platelets is demonstrated by the inheritance of bleeding disorders caused by defects in any one of the four the proteins of the complex, the most common of which is Bernard-Soulier syndrome (also called giant platelet syndrome). The GPIb part of the complex is composed of two proteins, GPIbα and GPIbβ encoded by separate genes.

In addition to its role as a bridge between platelets and exposed collagen on endothelial surfaces, vWF binds to and stabilizes coagulation factor VIII. Binding of factor VIII by vWF is required for normal survival of factor VIII in the circulation.

von Willebrand factor is a complex multimeric glycoprotein that is produced by and stored in the α-granules of platelets. It is also synthesized by megakaryocytes and found associated with subendothelial connective tissue.

The initial activation of platelets is induced by thrombin binding to specific receptors on the surface of platelets, thereby initiating a signal transduction cascade. The thrombin receptor is coupled to a G-protein that, in turn, activates phospholipase C-γ (PLC-γ). PLC-γ hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP2) leading to the formation of inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 induces the release of intracellular Ca2+ stores, and DAG activates protein kinase C (PKC).

The collagen to which platelets adhere as well as the release of intracellular Ca2+ leads to the activation of phospholipase A2 (PLA2), which then hydrolyzes membrane phospholipids, leading to liberation of arachidonic acid. The arachidonic acid release leads to an increase in the production and subsequent release of thromboxane A2 (TXA2). TXA2 is a potent vasoconstrictor and inducer of platelet aggregation that functions by binding to receptors that function through the PLC-γ pathway.

Another enzyme activated by the released intracellular Ca2+ stores is myosin light chain kinase (MLCK). Activated MLCK phosphorylates the light chain of myosin which then interacts with actin, resulting in altered platelet morphology and motility.

One of the many effects of PKC is the phosphorylation and activation of a specific 47,000-Dalton platelet protein. This activated protein induces the release of platelet granule contents; one of which is ADP. ADP further stimulates platelets increasing the overall activation cascade. The important role of ADP in platelet activation can be appreciated from the use of the ADP receptor antagonist, Plavix® (clopidogrel), in the control of thrombosis (see below). ADP also modifies the platelet membranes leading to exposure platelet glycoprotein receptor complex: GPIIb-GPIIIa. GPIIb-GPIIIa constitutes a receptor for vWF and fibrinogen, resulting in fibrinogen-induced platelet aggregation. The GPIIb-GPIIIa complex is a member of the integrin family of cell-surface receptors that interact with the extracellular matrix. The GPIIb-GPIIIa complex is also called integrin αIIb-β3. The importance of the GPIIb-GPIIIa in platelet activation and coagulation is demonstrated by the fact that bleeding disorders result from inherited defects in this glycoprotein complex. The most commonly inherited platelet dysfunction is Glanzmann thrombasthenia which results from defects in the GPIIb protein of this complex. In addition, the importance of this complex in overall hemostasis is demonstrated by the use of antibodies that block this receptor as anti-coagulants (e.g. ReoPro®, abciximab: see below).

Activation of platelets is required for their consequent aggregation to a platelet plug. However, equally significant is the role of activated platelet surface phospholipids in the activation of the coagulation cascade.
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Primary Factors
Factor

Trivial Name(s)

Pathway

Characteristic
Prekallikrein (PK) Fletcher factor Intrinsic Functions with HMWK and factor XII
High molecular weight kininogen (HMWK) contact activation cofactor; Fitzgerald, Flaujeac Williams factor Intrinsic Co-factor in kallikrein and factor XII activation, necessary in factor XIIa activation of XI, precursor for bradykinin (a potent vasodilator and inducer of smooth muscle contraction
I Fibrinogen Both
II Prothrombin Both Contains N-term. gla segment
III Tissue Factor Extrinsic
IV Calcium Both
V Proaccelerin, labile factor, accelerator (Ac-) globulin Both Protein cofactor
VI (same as Va) Accelerin Both This is Va, redundant to Factor V
VII Proconvertin, serum prothrombin conversion accelerator (SPCA), cothromboplastin Extrinsic Endopeptidase with gla residues
VIII Antihemophiliac factor A, antihemophilic globulin (AHG) Intrinsic Protein cofactor
IX Christmas Factor, antihemophilic factor B,plasma thromboplastin component (PTC) Intrinsic Endopeptidase with gla residues
X Stuart-Prower Factor Both Endopeptidase with gla residues
XI Plasma thromboplastin antecedent (PTA) Intrinsic Endopeptidase
XII Hageman Factor Intrinsic Endopeptidase
XIII Protransglutaminase, fibrin stabilizing factor (FSF), fibrinoligase Both Transpeptidase
Functional Classification of Clotting Factors
Zymogens of Serine Proteases

Activities
Factor XII binds to exposed collagen at site of vessel wall injury, activated by high-MW kininogen and kallikrein
Factor XI activated by factor XIIa
Factor IX activated by factor XIa in presence of Ca2+
Factor VII activated by thrombin in presence of Ca2+
Factor X activated on surface of activated platelets by tenase complex and by factor VIIa in presence of tissue factor and Ca2+
Factor II activated on surface of activated platelets by prothrombinase complex
Cofactors

Activities
Factor VIII activated by thrombin; factor VIIIa is a cofactor in the activation of factor X by factor IXa
Factor V activated by thrombin; factor Va is a cofactor in the activation of prothrombin by factor Xa
Factor III (tissue factor) a subendothelial cell-surface glycoprotein that acts as a cofactor for factor VII
Fibrinogen

Activity
Factor I cleaved by thrombin to form fibrin clot
Transglutaminase

Activity
Factor XIII activated by thrombin in presence of Ca2+; stabilizes fibrin clot by covalent cross-linking
Regulatory/Other Proteins

Activities
von Willebrand factor associated with subendothelial connective tissue; serves as a bridge between platelet glycoprotein GPIb/IX and collagen
Protein C activated to protein Ca by thrombin bound to thrombomodulin; then degrades factors VIIIa and Va
Protein S acts as a cofactor of protein C; both proteins contain gla residues
Thrombomodulin protein on the surface of endothelial cells; binds thrombin, which then activates protein C
Antithrombin III most important coagulation inhibitor, controls activities of thrombin, and factors IXa, Xa, XIa and XIIa

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The Clotting Cascades

The clotting cascades: The intrinsic cascade (which has less in vivo significance in normal physiological circumstances than the extrinsic cascade) is initiated when contact is made between blood and exposed negatively charged surfaces. The extrinsic pathway is initiated upon vascular injury which leads to exposure of tissue factor, TF (also identified as factor III), a subendothelial cell-surface glycoprotein that binds phospholipid. The green dotted arrow represents a point of cross-over between the extrinsic and intrinsic pathways. The two pathways converge at the activation of factor X to Xa. Factor Xa has a role in the further activation of factor VII to VIIa as depicted by the green arrow. Active factor Xa hydrolyzes and activates prothrombin to thrombin. Thrombin can then activate factors XI, VIII and V furthering the cascade. Ultimately the role of thrombin is to convert fribrinogen to fibrin and to activate factor XIII to XIIIa. Factor XIIIa (also termed transglutaminase) cross-links fibrin polymers solidifying the clot. HMWK = high molecular weight kininogen. PK = prekallikrein. PL = phospholipid.

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The Kallikrein-Kinin System in Coagulation

The kallikrein-kinin system comprises a complex of proteins that when activated leads to the release of vasoactive kinins. The kinins are released from both high molecular weight kininogen (HMWK) and low molecular weight kininogen (LMWK) as a result of activation of either tissue kallikrein or plasma kallikrein. The kallekreins themselves exist in inactive pre-forms. The kinins are involved in many physiological and pathological processes including regulation of blood pressure and flow (via modulation of the renin-angiotensin pathway), blood coagulation, cellular proliferation and growth, angiogenesis, apoptosis, and inflammation. Kinin action on endothelial cells leads to vasodilation, increased vascular permeability, release of tissue plasminogen activator (tPA), production of nitric oxide (NO), and the mobilization of arachidonic acid, primarily resulting in prostacyclin (PGI2) production by endothelial cells. Although the activities of the kallikrein-kinin system are involved in numerous processes, this section will deal only with their function in blood coagulation. With respect to hemostasis the most important kinin is bradykinin which is released from HMWK.

The two forms of prekallikrein, plasma and tissue, are derived from distinct genes on different chromosomes. The plasma kallikrein gene (symbol KLKB1) is on chromosome 4q34-q35 and the tissue kallikrein gene (symbol KLK1) located on chromosome 19q13.2-q13.4. These two kallikreins are serine proteases whose major substrates are HMWK (plasma kallikrein) and LMWK (tissue kallikrein). When plasma prekallikrein is activated to kallikrein it cleaves HMWK in a two-step process that ultimately releases bradykinin. Bradykinin is a 9-amino acid vasoactive peptide that induces vasodilation and increases vascular permeability. Activated tissue kallikrein cleaves lysyl-bradykinin (also called kallidin) from LMWK. Lysyl-bradykinin is bradykinin with a lysine residue at the N-terminus making it a 10-amino acid vasoactive peptide. Its activities are essentially identical to those of bradykinin.

Both HMWK and LMWK are derived from the same gene on chromosome 3q26-qter which is composed of 11 exons. Exons 1 to 9 encode the heavy chain of both kininogens. Exon 10 encodes bradykinin as well as the light chain of HMWK. Exon 11 encodes the light chain of LMWK. The heavy and light chain nomenclature refers to the disulfide-bonded structure of each kininogen after their activation by through kallikrein cleavage.

HMWK is considered an α-globulin and is composed of six functional domains. The protein circulates in the plasma as single-chain polypeptide with a molecular weight of 88 - 120 kDa dependent upon the level of glycosylation. The heavy chain is 64 kDa and contains domains 1, 2, and 3 whereas the light chain is 45 - 56 kDa and comprises domains 5 and 6. The heavy and light chains are linked together through domain 4 which also contains the bradykinin sequence. Domain 1 contains a low affinity calcium-binding site. Domains 2 and 3 contain amino acid sequences (QVVAG) that inhibit cysteine proteases. Domain 3 also has platelet and endothelial cell-binding activity. Domain 5 has sequences for heparin binding, cell-binding sites, and antiangiogenic properties. The binding of HMWK to negatively charged surfaces occurs through a histidine region of the light chain which is in domain 5. Domain 6 contains the prekallikrein and factor XI-binding sites. By being able to bind to charged surfaces via domain 5 and simultaneously bind factor XI and prekallikrein via domain 6, HMWK can serve as the cofactor in contact activation of plasma. LMWK is considered a β-globulin and has a molecular weight of 50 - 68 kDa. The structure of LMWK is similar to that of HMWK, however, the light chain is only 4 - 5 kDa and has no contact activation nor prekallikrein-binding sites.

The plasma kinin forming system is called the contact system of plasma and is composed of factor XII, factor XI, prekallikrein and HMWK. Factor XII, prekallikrein, and HMWK saturably and reversibly bind to endothelial cells, platelets, and granulocytes in a zinc-dependent reaction. When plasma makes contact with a negatively charged surface factor XII binds and is autoactivated to factor XIIa (the "a" signifies the activated factor). Factor XIIa then activates prekallikrein to kallikrein and kallikrein cleaves HMWK releasing bradykinin. There is also reciprocal activation of factor XII by kallikrein resulting in amplification of the system. The actual surface that leads to factor XII autoactivation is unknown however, several physiologic substances support the process. These substances include hematin, skin, fatty acids, sodium urate crystals, protoporphyrin, sulfatides, heparins, chondroitin sulfates, articular cartilage, endotoxin, L-homocysteine, and amyloid β-protein. Once the contact system is activated the intrinsic pathway (described below) is initiated.
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The Intrinsic Clotting Cascade

The intrinsic pathway (also called the contact activation pathway) is much less significant to hemostasis under normal physiological conditions than is the extrinsic pathway. However, abnormal physiology such as hyperlipidemic states or bacterial infiltration can lead to activation of thrombosis via the intrinsic clotting cascade.

The intrinsic pathway requires the clotting factors VIII, IX, X, XI, and XII. Also required are the proteins prekallikrein (PK) and high-molecular-weight kininogen (HK or HMWK), as well as calcium ions and phospholipids secreted from platelets. The role of PK and HMWK is described in the above section. Each of these pathway constituents leads to the conversion of factor X (inactive) to factor Xa. Initiation of the intrinsic pathway occurs when prekallikrein, high-molecular-weight kininogen, factor XI and factor XII are exposed to a negatively charged surface. This is termed the contact phase and can occur as a result of interaction with the phospholipids (primarily phosphatidylethanolamine, PE) of circulating lipoprotein particles such as chylomicrons and VLDLs. This is the basis of the role of hyperlipidemia in the promotion of a pro-thrombotic state and the development of atherosclerosis. Contact activation of the intrinsic pathway can also occur on the surface of bacteria.

The assemblage of contact phase components results in conversion of prekallikrein to kallikrein, which in turn activates factor XII to factor XIIa. Factor XIIa then activates factor XI to factor XIa. Factor XIIa will also hydrolyze more prekallikrein to kallikrein, establishing a reciprocal activation cascade. Kallikrein acts upon HMWK leading to the release of bradykinin, a potent vasodilator.

In the presence of Ca2+, factor XIa activates factor IX to factor IXa. Factor IX is a proenzyme that contains vitamin K-dependent γ-carboxyglutamate (gla) residues, whose serine protease activity is activated following Ca2+ binding to these gla residues. Several of the serine proteases of the cascade (II, VII, IX, and X) are gla-containing proenzymes. Active factor IXa cleaves factor X at an internal arg-ile (R-I) bond leading to its activation to factor Xa.

The activation of factor Xa requires assemblage of the tenase complex (Ca2+ and factors VIIIa, IXa and X) on the surface of activated platelets. One of the responses of platelets to activation is the presentation of phosphatidylserine (PS) and phosphatidylinositol (PI) on their surfaces. The exposure of these phospholipids allows the tenase complex to form. The role of factor VIII in this process is to act as a receptor, in the form of factor VIIIa, for factors IXa and X. Factor VIIIa is termed a cofactor in the clotting cascade. The activation of factor VIII to factor VIIIa (the actual receptor) occurs in the presence of minute quantities of thrombin. As the concentration of thrombin increases, factor VIIIa is ultimately cleaved by thrombin and inactivated. This dual action of thrombin, upon factor VIII, acts to limit the extent of tenase complex formation and thus the extent of the coagulation cascade.
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Extrinsic Clotting Cascade

Activated factor Xa is the site at which the intrinsic and extrinsic coagulation cascades converge. The extrinsic pathway is initiated at the site of injury in response to the release of tissue factor (factor III) and thus, is also known as the tissue factor pathway. Tissue factor is a cofactor in the factor VIIa-catalyzed activation of factor X. Factor VIIa, a gla residue containing serine protease, cleaves factor X to factor Xa in a manner identical to that of factor IXa of the intrinsic pathway. The activation of factor VII occurs through the action of thrombin or factor Xa. The ability of factor Xa to activate factor VII creates a link between the intrinsic and extrinsic pathways. An additional link between the two pathways exists through the ability of tissue factor and factor VIIa to activate factor IX. The formation of complex between factor VIIa and tissue factor is believed to be a principal step in the overall clotting cascade. Evidence for this stems from the fact that persons with hereditary deficiencies in the components of the contact phase of the intrinsic pathway do not exhibit clotting problems. A major mechanism for the inhibition of the extrinsic pathway occurs at the tissue factor-factor VIIa-Ca2+-Xa complex. The protein, lipoprotein-associated coagulation inhibitor, LACI specifically binds to this complex. LACI is also referred to as extrinsic pathway inhibitor, EPI or tissue factor pathway inhibitor, TFPI and was formerly named anticonvertin. LACI is composed of 3 tandem protease inhibitor domains. Domain 1 binds to factor Xa and domain 2 binds to factor VIIa only in the presence of factor Xa.
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Activation of Prothrombin to Thrombin

The common point in both pathways is the activation of factor X to factor Xa. Factor Xa activates prothrombin (factor II) to thrombin (factor IIa). Thrombin, in turn, converts fibrinogen to fibrin. The activation of thrombin occurs on the surface of activated platelets and requires formation of a prothrombinase complex. This complex is composed of the platelet phospholipids, phosphatidylinositol and phosphatidylserine, Ca2+, factors Va and Xa, and prothrombin. Factor V is a cofactor in the formation of the prothrombinase complex, similar to the role of factor VIII in tenase complex formation. Like factor VIII activation, factor V is activated to factor Va by means of minute amounts and is inactivated by increased levels of thrombin. Factor Va binds to specific receptors on the surfaces of activated platelets and forms a complex with prothrombin and factor Xa.

Prothrombin is a 72,000-Dalton, single-chain protein containing ten gla residues in its N-terminal region. Within the prothrombinase complex, prothrombin is cleaved at 2 sites by factor Xa. This cleavage generates a 2-chain active thrombin molecule containing an A and a B chain which are held together by a single disulfide bond.

In addition to its role in activation of fibrin clot formation, thrombin plays an important regulatory role in coagulation. Thrombin combines with thrombomodulin present on endothelial cell surfaces forming a complex that converts protein C to protein Ca. The cofactor protein S and protein Ca degrade factors Va and VIIIa, thereby limiting the activity of these 2 factors in the coagulation cascade.

Thrombin also binds to a class of G-protein-coupled receptors called protease activated receptors (PARs), specifically PAR-1, -3 and -4. PARs utilize a unique mechanism to convert the result of extracellular proteolytic cleavage into an intracellular signaling event. PARs carry their own ligand which remains inactive until protease cleavage, such as by thrombin, "unmasks" the ligand. Following thrombin cleavage the unmasked ligand is still a part of the intact PAR but is now capable of interacting with the ligand-binding domain of the PAR resulting in the activation of numerous signaling cascades. Because the activation of PARs requires proteolytic cleavage the activation process is irreversible. The signaling cascades activated by thrombin-activated PARs include release of the interleukins, (ILs), IL-1 and IL-6, increased secretion of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). The thrombin-induced signaling also leads to increased platelet activation and leukocyte adhesion.

Thrombin also activates thrombin-activatable fibrinolysis inhibitor (TAFI) thus modulating fibrinolysis (degradation of fibrin clots). TAFI is also known as carboxypeptidase U (CPU) whose activity leads to removal of C-terminal lysines from partially degraded fibrin. This leads to an impairment of plasminogen activation, thereby reducing the rate of fibrin clot dissolution (i.e. fibrinolysis).
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Control of Thrombin Levels

The inability of the body to control the circulating level of active thrombin would lead to dire consequences. There are 2 principal mechanisms by which thrombin activity is regulated. The predominant form of thrombin in the circulation is the inactive prothrombin, whose activation requires the pathways of proenzyme activation described above for the coagulation cascade. At each step in the cascade, feedback mechanisms regulate the balance between active and inactive enzymes.

The activation of thrombin is also regulated by 4 specific thrombin inhibitors Antithrombin III is the most important since it can also inhibit the activities of factors IXa, Xa, XIa and XIIa. The activity of antithrombin III is potentiated in the presence of heparin by the following means: heparin binds to a specific site on antithrombin III, producing an altered conformation of the protein, and the new conformation has a higher affinity for thrombin as well as its other substrates. This effect of heparin is the basis for its clinical use as an anticoagulant. The naturally occurring heparin activator of antithrombin III is present as heparan and heparan sulfate on the surface of vessel endothelial cells. It is this feature that controls the activation of the intrinsic coagulation cascade.

However, thrombin activity is also inhibited by α2-macroglobulin, heparin cofactor II and α1-antitrypsin. Although a minor player in thrombin regulation α1-antitrypsin is the primary serine protease inhibitor of human plasma. Its physiological significance is demonstrated by the fact that lack of this protein plays a causative role in the development of emphysema.
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Activation of Fibrinogen to Fibrin

Fibrinogen (factor I) consists of 3 pairs of polypeptides ([Aα][Bβ][γ])2. The 6 chains are covalently linked near their N-terminals through disulfide bonds. The A and B portions of the Aα and Bβ chains comprise the fibrinopeptides, A and B, respectively. The fibrinopeptide regions of fibrinogen contain several glutamate and aspartate residues imparting a high negative charge to this region and aid in the solubility of fibrinogen in plasma. Active thrombin is a serine protease that hydrolyses fibrinogen at four arg-gly (R-G) bonds between the fibrinopeptide and the a and b portions of the protein.

Thrombin-mediated release of the fibrinopeptides generates fibrin monomers with a subunit structure (αβγ)2. These monomers spontaneously aggregate in a regular array, forming a somewhat weak fibrin clot. In addition to fibrin activation, thrombin converts factor XIII to factor XIIIa, a highly specific transglutaminase that introduces cross-links composed of covalent bonds between the amide nitrogen of glutamines and e-amino group of lysines in the fibrin monomers.
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Dissolution of Fibrin Clots

Degradation of fibrin clots is the function of plasmin, a serine protease that circulates as the inactive proenzyme, plasminogen. Any free circulating plasmin is rapidly inhibited by α2-antiplasmin. Plasminogen binds to both fibrinogen and fibrin, thereby being incorporated into a clot as it is formed. Tissue plasminogen activator (tPA) and, to a lesser degree, urokinase are serine proteases which convert plasminogen to plasmin. Inactive tPA is released from vascular endothelial cells following injury; it binds to fibrin and is consequently activated. Urokinase is produced as the precursor, prourokinase by epithelial cells lining excretory ducts. The role of urokinase is to activate the dissolution of fibrin clots that may be deposited in these ducts.

Active tPA cleaves plasminogen to plasmin which then digests the fibrin; the result is soluble degradation product to which neither plasmin nor plasminogen can bind. Following the release of plasminogen and plasmin they are rapidly inactivated by their respective inhibitors. The inhibition of tPA activity results from binding to specific inhibitory proteins. At least 4 distinct inhibitors have been identified, of which 2: plasminogen activator-inhibitors type 1 (PAI-1) and type 2 (PAI-2) are of greatest physiological significance.
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Clinical Significances of Hemostasis
The Bleeding Disorders

Defects in the process of hemostasis, leading to bleeding disorders, have been identified at the level of the proteins of the clotting cascades, platelet activation and function, contact activation and antithrombin function This list is not all inclusive and for more details please visit the Inborn Errors: Clotting Factors page.
Hemophilia A

Hemophilia A is classic hemophilia (a disease referring to the inability to clot blood). It is an X-linked disorder resulting from a deficiency in factor VIII, a key component of the coagulation cascade. There are severe, moderate and mild forms of hemophilia A that reflect the level of active factor VIII in the plasma.

Hemophilia A arises from a variety of mutations. Some 150 different point mutations have been characterized in the factor VIII gene in hemophilia A. Inheritence of the disorder occurs with a frequency of 1:5,000 to 1:10,000 males in all populations. Factor VIII is a cofactor in the activation of factor X to factor Xa in a reaction catalyzed by factor IXa. Activation of factor VIII occurs via proteolytic cleavage by thrombin and factor Xa. Inactivaqtion of factor VIIIa occurs by limited proteolysis by factor Xa or activated protein C.

Individuals with deficiencies in factor VIII suffer joint and muscle hemorrhage, easy bruising and prolonged bleeding from wounds. Treatment of hemophilia A is accomplished by infusion of factor VIII concentrates prepared from either human plasma or by recombinant DNA technology.
Hemophilia B

Hemophilia B results from deficiencies in factor IX. The prevalence of hemophilia B is approximately one-tenth that of hemophilia A. All patients with hemophilia B have prolonged coagulation time and decreased factor IX clotting activity. Like hemophilia A, there are severe, moderate and mild forms of hemophilia B and reflect the factor IX activity in plasma.

At least 300 unique factor IX mutations have been identified, 85% are point mutations, 3% are short nucleotide deletions or insertions and 12% are gross gene alterations.
Disorders of Fibrinogen and Factor XIII

Several cardivascular risk factors are associated with abnormalities in fibrinogen. As a result of the acute-phase response or through other poorly understood mechanisms, elevated plasma fibrinogen levels have been observed in patients with coronary artery disease, diabetes, hypertension, peripheral artery disease, hyperlipoproteinemia and hypertriglyceridemia. In addition, pregnancy, menopause, hypercholesterolemia, use of oral contraceptives and smoking lead to increased plasma fibrinogen levels.

Although rare, there are inherited disorders in fibrinogen. These disorders include afibrinogenemia (a complete lack of fibrinogen), hypofibrinogenemia (reduced levels of fibrinogen) and dysfibrinogenemia (presence of dysfunctional fibrinogen). Afibrinogenemia is characterized by neonatal umbilical cord hemorrhage, ecchymoses, mucosal hemorrhage, internal hemorrhage, and recurrent abortion. The disorder is inherited in an autosomal recessive manner. Hypofibrinogenemia is characterized by fibrinogen levels below 100mg/dL (normal is 250-350mg/dL) and can be either aquired or inherited. Symptoms of hypofibrinogememia are similar to, but less severe than, afibrinogenemia. Dysfibrinogenemias are extremely heterogeneous affecting any of the functional properties of fibrinogen. Clinical consequences of dysfibrinogenemias include hemorrhage, spontaneous abortion and thromboembolism.

Factor XIII is the proenzyme form of plasma transglutaminase and is activated by thrombin in the presence of calcium ions. Active factor XIII catalyzes the cross-linking of fibrin monomers. Factor XIII is a tetramer of two different peptides, A and B (forming A2B2). Hereditary deficiencies (autosomal recessive) occur resulting in the absence of either subunit. Clinical manifestation of factor XIII deficiency is delayed bleeding although primary hemostasis is normal. Deficiency leads to neonatal umbilical cord bleeding, intracranial hemorrhage and soft tissue hematomas.
von Willebrand Disease

von Willebrand disease (vWD) is due to inherited deficiency in von Willebrand factor (vWF). vWD is the most common inherited bleeding disorder of humans. Using sensitive laboratory testing, abnormalities in vWF can be detected in approximately 8000 people per million. Clinically significant vWD occurs in approximatley 125 people per million. This is a frequency at least twice that of hemophilia A.

Deficiency of vWF results in defective platelet adhesion and causes a secondary deficiency in factor VIII. The result is that vWF deficiency can cause bleeding that appears similar to that caused by platelet dysfunction or hemophilia. vWD is an extremely heterogeneous disorder that has been classified into several major subtypes. Type I vWD is the most common and is inherited as an autosomal dominant trait. This variant is due to simple quantitative deficiency of all vWF multimers. Type 2 vWD is also subdivided further dependent upon whether the dysfunctional protein has decreased or paradoxically increased function in certain laboratory tests of binding to platelets. Type 3 vWD is clinically severe and is characterized by recessive inheritance and virtual absence of vWF.
Factor XI and Contact Activation

When blood makes contact with negatively charged surfaces it triggers a series of interactions that involve factor XI, prekallikrein and high molecular weight kininogen leading to blood coagulation. This process is referred to as contact activation. Deficiency in factor XI confers an injury-related bleeding tendency. This deficiency was identified in 1953 and originally termed hemophilia C. Factor XI deficiency is very common in Ashkenazic Jews and is inherited as an autosomal disorder with either homozygosity or compound heterozygosity. Three independent point mutations in factor XI have been identified.
Antithrombin Deficiency

Antithrombin functions to inhibit several activated coagulation factors including thrombin, factor IXa and factor Xa, by forming a stable complex with the various factors. Heparin and heparan sulfates increase the activity of antithrombin at least 1000 fold.

Deficiency in antithrombin is seen in approximately 2% of patients with venous thromboembolic disease. Inheritance occurs as an autosomal dominant trait. The prevalence of symptomatic antithrombin deficiency ranges from 1 per 2000 to 1 per 5000 in the general population. Deficiencies results from mutations that affect synthesis or stability of antithrombin or from mutations that affect the protease and/or heparin binding sites of antithrombin.

Clinical manifestations of antithrombin deficiency include deep vein thrombosis and pulmonary embolism. Arterial thrombosis is rare in anththrombin deficiency. Thrombosis may occur spontaneously or in association with surgery, trauma or pregnancy. Treatment of acute episodes of thrombosis is by infusion of heparin (for 5-7 days) followed by oral anticoagulant therapy.
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Pharmacological Intervention in Bleeding

Coumarin drugs (based on the chemical benzopyrone), such as warfarin (trade name Coumadin®) as well as the glycosaminoglycans, heparin and heparan sulfate, are useful as anticoagulants. Heparin is useful as an anticoagulant because it binds to, and activates, antithrombin III which then inhibits the serine proteases of the coagulation cascade. Heparin is abundant in granules of the mast cells that line the vasculature. In response to injury, the heparin is released and inhibits coagulation. The coumarin drugs inhibit coagulation by inhibiting the vitamin K-dependent γ-carboxylation reactions necessary to the function of thrombin, and factors VII, IX, and X as well as proteins C and S. These drugs act by inhibiting the reduction of the quinone derivatives of vitamin K to their active hydroquinone forms. Because of the mode of action of coumarin drugs, it takes several days for their maximum effect to be realized. For this reason, heparin is normally administered first followed by warfarin or warfarin-related drugs.

The plasminogen activators also are useful for controlling coagulation. Because tPA is highly selective for the degradation of fibrin in clots, it is extremely useful in restoring the patency of the coronary arteries following thrombosis, in particular during the short period following myocardial infarct. Streptokinase (an enzyme from the Streptococci bacterium) is another plasminogen activator useful from a therapeutic standpoint. However, it is less selective than tPA, being able to activate circulating plasminogen as well as that bound to a fibrin clot.

Aspirin is an important inhibitor of platelet activation. By virtue of inhibiting the activity of cyclooxygenase, aspirin reduces the production of TXA2 by platelets. Aspirin also reduces endothelial cell production of prostacyclin (PGI2), an inhibitor of platelet aggregation and a vasodilator. Localized to the site of coagulation is a balance between the levels of platelet derived TXA2 and endothelial cell derived PGI2. This allows for platelet aggregation and clot formation but preventing excessive accumulation of the clot, thus maintaining blood flow around the site of the clot. Endothelial cells regenerate active cyclooxygenase faster than platelets because mature platelets cannot synthesize the enzyme, requiring new platelets to enter the circulation (platelet half-life is approximately 4 days). Therefore, PGI2 synthesis is greater than that of TXA2. The net effect of aspirin is more in favor of endothelial cell-mediated inhibition of the coagulation cascade. This reflects one of the the cardiovascular benefits to low dose administration of aspirin. Aspirin also has important effects on inflammatory processes that impact cardiovascular systems (see the Aspirin page for more details).

Newer classes of anticoagulation drugs are being developed that function by inhibiting the activation of platelets and their subsequent aggregation. The drug clopidogrel: Plavix® (Bristol-Myers Squibb) is an irreversible inhibitor of the ADP receptor on platelet membranes. When ADP binds to platelets they are activated and aggregate leading to amplification of the coagulation response, thus Plavix interferes with this process. Plavix is prescribed for the treatment of peripheral vascular and cerebrovascular disease as well as coronary artery disease to prevent the formation of thrombotic plaques.

Another target of pharmacologic intervention in coagulation involving platelets is the role of GPIIb-GPIIIa in fibrinogen-induced platelet aggregation. Glanzmann thrombasthenia is an inherited platelet function defect characterized by a lack of platelet aggregation in response to all physiological agonists. This disorder is due to a lack of the GPIIb-GPIIIa receptor complex on platelets. Patients with this disorder present with significantly increased bleeding times. Although a rare disorder, study of the pathophysiology of the disease led to the development of anticoagulant drugs that inhibit platelet aggregation regardless of the agonist. Therefore, the GPIIb-GPIIIa antagonists more completely inhibit platelet aggregation than do aspirin or Plavix. The current family of these drugs includes ReoPro® (abciximab: a human monoclonal antibody), Integrilin® (eptifibatide: a cyclic hexapeptide derived from a protein found in the venom of the southeastern pygmy rattlesnake) and Aggrastat® (tirofiban: a synthetic organic non-peptide molecule).