Blood Clotting

Blood Clotting http://ntri.tamuk.edu/homepage-ntri/lectures/clotting.html The circulatory system must be self-sealing to prevent life threatening injury. Bleeding is rapidly stopped by a process known as hemostasis that involves three processes. 1. First, platelets adhere to damaged blood vessels and then to each other forming a plug that can stop minor bleeding. This process is mediated by von Willebrandt factor, a large (104 kD) multimeric plasma glycoprotein of subunit mass 225 kD. This protein binds to both a specific receptor on the platelet membrane and to the collagen and possibly other components of the subendothelial membrane. 2. As the platelets aggregate, they release several physiologically active substances, including serotonin (5-hydroxytryptamine) and thromboxane A2, which stimulates vasoconstriction, thereby reducing the blood flow at the injury. 3. Finally, the aggregating platelets and damaged tissue initiate blood clotting, which is the major defense against bleeding. Platelet Plug Until platelets are needed for hemostasis, circulating platelets are maintained in a nonreactive state for the following reasons: 1. Partly by inhibitory compounds secreted by endothelial cells , such as nitric oxide and prostacyclin  [a prostaglandin (any of various oxygenated unsaturated cyclic fatty acids of animals that perform a variety of hormonelike actions (as in controlling blood pressure or smooth muscle contraction)that is a metabolite of arachidonic acid, inhibits platelet aggregation, and dilates blood vessels]. 2. The negative surface charge limits platelet interaction with circulating erythrocytes, leukocytes, or the vascular endothelium. 3. The intact endothelium also represents a barrier separating platelets form adhesive substrates in the subendothelial connective tissue matrix such as collagen. Disruption of the endothelium by trauma, or by a disease such as atherosclerosis, allows platelets to come in contact with and adhere to sub-endothelial matrix. Adhesion is the first stage of platelet function. Platelets tend to stick to the exposed ends of injured blood vessels. Actually, they adhere to any rough surface, particularly to the collagen (the chief fibrous protein found in skin, bone, cartilage, tendon, and other connective tissue that yields gelatin when boiled in water) in connective tissue that underlies the endothelial lining of blood vessel. At

lower shear rates, the integrins α2 Β1, α5 Β1 , and α6 Β3 bind to exposed collagen, fibronectin [any of a group of glycoproteins  (a complex protein in which the nonprotein part is a carbohydrate ) of cell surfaces, blood plasma, and connective tissue that promote cellular adhesion and migration], and laminin , respectively (Helmler et. al., 1988). At higher shear rates, platelet

adhesion requires sub-endothelial von Willebrand factor (vWf) (Turrito et al. 1985). VWf binds to the GPIb-IX-V complex on un-activated platelets (Coller et al. 1983) and to the αIIb Β3 complex on activated platelets (Ruggeri et al. 1983).

Collagen is not only an important substrate for platelet adhesion but also a binding site for vWf in the sub-endothelium ( Savage et al 1998). A number of platelet proteins interact with collagen besides α2 Β1. Other that interact include αIIb Β3, GPIV, GPVI and two other proteins with a molecular weight of 65,00 and 85,000 molecular weight protein (Clemetson et. Al 1999). Clinical reports suggest that α2 Β1 and BPVI are the physiological platelet receptors for collagen (Moroi et al. 1989, Mieuwenhuis et al. 1985, and Arai et al. 1995)). When platelets contact collagen, their shapes change drastically, and numerous spiny processes begin to protrude from their membranes. At the same time, they tend to stick to each other with fibrinogen forming a platelet plug in the vascular break. This starts the second phase of platelet function aggregation. In contrast to adhesion, platelet aggregation is an active metabolic process in which platelet agonist binding to specific membrane receptor initiates signaling pathways that enable the integrins αIIbΒ3 to bind soluble macromolecular ligands, such as fibrinogen or vWf (Bennett 1996). The fibrinogen or vWf bound to a αIIbΒ3 cross-links platelets into a hemostatic plug or thrombus.

Platelets are important for blood clotting and provide a good example of how cell-cell interactions are modulated by controlling integrin activity. The αIIb Β3 integrin normally is present on the plasma membrane of platelets but is unable to bind the blood protein fibrinogen or the other protein ligands. Only after binding collagen or thrombin in a forming clot activates the platelet can αIIb Β3 integrin bind fibrinogen. This interaction accelerates the formation of the clot. Platelet activation is accompanied by a conformational change in the αIIb Β3 integrin. The nature of this change is unknown, but as platelet activation also involves a major change in the platelet cytoskeleton. This change probably involves binding of a cytoskeletal protein to the integrin cytosolic domain. Patients with genetic defects in the β3 integrin subunit are prone to excessive bleeding. Unstimulated platelets do not aggregate in circulation since they do not bind fibrinogen. Antagonists such as ADP, epinephrine, thrombin, or prostaglandin endoperoxides stimulate platelets exposing fibrinogen receptors to associate with αIIb Β3 integrin, which results in fibrinogen binding and subsequent platelet aggregation. Platelets are the smallest corpuscular components of human blood (diameter 2-4µm) - the physiological number varies from 150,000 to 300,000/mm3 blood. Despite their appearance on the face of it platelets are not cells, as they are not provided with a nucleus. The origin of platelets is the bone marrow. Megakaryocytes as the results of mitotic proliferation of a committed progenitor cells liberate platelets as the end product of protrusions of their membrane and cytoplasm. The typical shape of resting platelets is discoid (Figure 2 & 3), upon activation they undergo a shape change to a globular form with pseudopodia (up to 5µm long). Figure 2: Scanning electromicrograph of activated platelets.

Figure 3: Scanning electromicrograph of platelets.

The membrane consists of a typical phospholipid bilayer (Figure 4). Embedded in membrane are different kinds of glycoproteins (GP), the receptors for activation and interaction with other cells (figure 4). The integrin α1β1 mediates platelet adhesion to subendothelial collagen via von Willebrand factor (vWf) and activate the αIIb Β3 integrin.

Figure 4: Capillary with platelet plug

and receptors of platelet aggregation. Collagen will induce formation of aggregates as well as secretion of granule contents. Platelet responses that are well recognized include development of stickiness, adhesion, change in shape, irreversible aggregation and secretion of granule contents.

Blood Clotting Cascade A blood clot (thrombus) forms through the action of a cascade of proteolytic reactions involving the participation of nearly 20 different substances, most of which are liversynthesized plasma glycoproteins. The cascade is diagrammed below. All but two of these factors are designated by both a roman numeral and a common name. Seven of the clotting factors are zymogens of serine proteases [any of numerous enzymes that hydrolyze proteins and are classified according to the most prominent functional group(i.e serine)] that are proteolytically activated by serine proteases further up the cascade. Other clotting proteins, termed accessory factors, which are also activated by these serine proteases, enhance the rate of activation of some of the zymogens. In both cases the active forms of a factor is designated by subscript a.

The above figure is the blood clotting cascade in humans showing the division of its primary stages into the so-called intrinsic and extrinsic pathways. The clotting factors are named in black. The active clotting factors, with the exception of fibrin, are serine proteases indicated in red. Red arrows represent their proteolytic activation of other factors in the cascade. Accessory factors including Ca2+ and phospholipid membrane (PL) are indicated in green. There are many feedback reactions that accelerate the clotting process.

The blood clotting system is in all vertebrates, contains a number of homologous serine proteases and therefore appears to have developed through a series of gene duplications. The C-terminal ~250 residues of these proteases, which comprise their catalytically active domains, are also homologous to the pancreatic serine proteases trypsin, chymotrypsin, and elastase. The blood clotting proteases and digestive proteases are activated by proteolytic cleavages that precede their C-terminal segments. The clotting proteases differ from the digestive enzymes in the presence of Ca 2+ on an appropriate phospholipid membrane. The resulting N-terminal fragments are quite large (150-582) residues and, with the exception of prothrombin are linked to their C-terminal segments via disulfide bonds so that these segments do not separate upon activation. The Nterminal segments are thought to be responsible, at least in part, for the exquisite specificities of the proteolytic blood clotting factors. Fibrinogen to Fibrin Blood clots consist of arrays of cross-linked fibrin that forms an insoluble fibrous network. See the scanning electron micrograph of a blood clot showing a red cell enmeshed in a fibrin network. Fibrin is made from the soluble plasma protein fibrinogen (factor I) through a proteolytic reaction catalyzed by the serine protease thrombin (factor II). Fibrinogen comprises 2 to 3% of plasma protein. A molecule of fibrinogen consists of three pairs of chains Aα(610 residues), Bβ (461 residues) and γ (411 residues) and two pairs of N-linked oligosaccharides of ~2.5 kD each. Here A and B represent the 16- and 14-residue N-terminal fibrinopeptides that thrombin cleaves from fibrinogen, so a fibrin monomer designated α2β2γ2.

Electron microscopic and low-resolution X-ray crystallographic studies indicate that fibrinogen is a two fold symmetric elongated molecule ~450 A in length, that has two nodules at each end and one in the middle. Its 6 polypeptide chains are joined by 17 disulfide bonds, 7 within each half of the dimer and 3 linking these two protomers. The central region of each protomer consists mainly of a three-stranded coiled coil of a

helices in which the α, β, and γ chains each contribute a strand. The peripheral nodules diagrammed are formed by the C-terminal domains of the β and γ chains. The C-terminal segment of the α chain apparently lacks a defined conformation and is therefore not represented. Fibrin Polymerization Thrombin specifically cleaves the Arg-X peptide bond (in most animals X is Gly) joining each fibrinopeptide to fibrin. Fibrin then spontaneously aggregates to form fibers that have a banded structure that repeats every 225 A . This is one half repeat length of fibrin monomer 450 A, which suggests that fibrin monomers associate as a half-staggered array as shown below. The main reason the fibrin aggregates is that the loss of the fibrinopeptides exposes otherwise masked sites that mediate intermolecular association. In addition, fibrinogen aggregation is inhibited by charge repulsions. The fibrinopeptides are highly anionic, and the fibrinogen has a charge of -8 at fibrinopeptide site whereas that of fibrin is +5.

The diameters of fibrin fibers, which are fairly uniform (~50 nm) are important determinants of a clot's physical properties. Through electron microscope studies the fibrin is uniformly twisted. Consequently, the in-register molecules near the periphery of a twisted fiber must traverse a longer path than molecules near the fiber's center. The degree to which a molecule can stretch therefore limits the diameter of the fiber. Molecules add to the outside of a growing fiber until the energy to stretch an added molecule exceeds its energy of binding. Fibrin-Stabilizing Factor A soft clot is rather fragile. It is rapidly converted to a more stable hard clot by the covalent cross-linking of neighboring fibrin molecules in a reaction catalyzed by fibrinstabilizing factors (FSF or XIIa). This transamidase initially joins the C-terminal segments of adjacent γ chains by forming isopeptide bonds between the side chains of a Gln residue on on γ chain and a Lys residue on another. Two such symmetrically equivalent bonds are rapidly formed between each neighboring pair of γ chains as shown below. The α chains are similarly cross linked to one another, but at slower rate.

The transamidation reaction forming the isopeptide bonds cross-linking fibrin monomers in "hard" clots as catalyzed by activate fibrin-stabilizing factor (FSF, XIIIa).

FSF is present in both platelets and plasma. Platelet FSF consists of two 75 kD α chains, whereas plasma FSF additionally has two 88 kD β chains. Both species of FSF occur as zymogens that undergo thrombin-catalyzed cleavage of a specific Arg-Gly bond near the N-terminus of each α chain with the consequent release of a 37 residue propeptide.

Thrombin Activation Thrombin is a serine protease that consists of two disulfide-linked polypeptide chains. In humans there are 36 residues for A chain and a 256 residues for B chain. The thrombin B chain is homologous to trypsin and has similar specificity but is far more selective. It cleaves only certain Arg-S and less frequently cleaves Lys-X bonds with a clear preference for a Pro preceding the Arg or Lys. Human thrombin is synthesized as a 579 residue zymogen, prothrombin (II), which is activated by two proteolytic cleavages by activated Stuart factor (Xa), the product of the preceding steps of the clotting cascade. The cleavage of prothrombin's Arg 271-Thr 272

and Arg 320-Ile 321 bonds releases its N-terminal propeptide and separates the A and B chains. The latter cleavage, which yields active enzyme, results in the formation of an ion pair between Ile 321 and Asp 524, much like that formed between chymotrypsinogen's homologous Ile 16 and Asp194 in the activation of this zymogen. Thrombin then autolytically cleaves its Arg 285-Thr 286 bond, thereby trimming away the N-terminal 13 residues of the A chain to yield thrombin. Prothrombin's propeptide consists of three domains. An N-terminal 40 residue Gla domain followed by two 40% identical ~115 residue kringle domains. Kringles are crosslinked by three characteristically located disulfide bonds that gives three triple looped sequence motifs. Gla domains and kringles occur in several of the proteins involved in the formation and the breakdown of blood clots.

Vitamin K is an Essential Cofactor in the Synthesis of γ-Carboxyglutamate Prothrombin, as well as the Factors VII, IX and X, is synthesized in the liver in a process that requires and adequate dietary intake of vitamin K. Lack of vitamin K or the presence of a competitive inhibitor such as dicoumarol or warfarin causes the production of an abnormal prothrombin that is activated by factor Xa (Stuart factor) at only 1 to 2% of the normal rate. This was puzzling because normal and abnormal prothrombins seemed to have identical amino acid compositions. NMR studies eventually established however, that normal prothrombin contains γ-Carboxyglutamate (Gla) residue, 10 of which occur between residues 6 and 32 in human prothrombin. Abnormal prothrombin, in contrast, contains Glu in the place of Gla residues. Vitamin K must therefore be a cofactor in the post-translational conversion of Glu to Gla. The reason why prothrombin's Gla residues were not initially detected is because they decarboxylate to Glu under the condition of acid hydrolysis normally used in amino acid composition determinations.

Prothrombin Activation

Factor Xa (Stuart factor) by itself, is an extremely sluggish prothrombin activator. In the presence of activated proaccelerin (Factor Va), Ca2+, and phospholipid membrane, its activity is enhanced 20,000-fold. The membrane surface in contact with the activation complex must contain negatively charged phospholipids such as phosphatidylserine in order to stimulate this rate enhancement. Such phospholipids occur almost exclusively on the cytoplasmic side of cell membranes which of course are normally not in contact with the blood plasma. In addition, 20% of factor V (proaccelerin) in the blood is stored in the platelets and released only upon platelet activation. Consequently, physiological prothrombin activation normally takes place at a significant rate only in the vicinity of an injury. Ca2+ is required for either prothrombin or factor Xa to bind phospholipid membranes. These proteins are anchored to the membrane via Ca2+ bridges. Prothrombin (II) and factor Xa (Stuart factor) from vitamin K-deficient animals have greatly reduce membrane binding affinities compared to the corresponding normal proteins. The Gla side chains, which are much stronger Ca2+ binding sites. Prothrombin (II), proconvertin (VII), Christmas (XI), and Stuart (X) have homologous N-terminal segments which contain the 9 to 12 conserved Gla residues. The excision of prothrombin's N-terminal propeptide releases the resulting thrombin from the phospholipid membrane so that it can activate fibrinogen in plasma. The other vitamin K-dependent zymogen (proconvertin (VII), Christmas (XI), and Stuart (X) remain bound to the phospholipid membrane after their activation Activated thrombin specifically cleaves prothrombin's propeptide at it's Arg 155- Ser 156 bond to yield its 155 residue N-terminal segment, the so called fragment 1, which consists of prothrombin's Gla domain and its kringle 1. Activated proaccelerin (VA), the accessory factor in prothrombin, is activated by a thrombin-catalyzed proteolytic cleavage. Prothrombin activation, in this indirect way, is thereby autocatalytic. Thrombin, in vitro can also directly activate prothrombin by cleaving its Arg 283-Thr 284 bond, but this reaction has been shown to be physiologically insignificant. VA is subject to further thrombin-catalyzed proteolysis, which inactivates it. Thrombin can proteolytically inactivate thrombin molecules. Clot formation is therefore self limiting, a safeguard that helps prevent blood clots from propagating away from the site of an injury. Thrombin structurally resembles trypsin. The structure of the B-chain closely resembles that of pancreatic serine proteases as had been expected from the high degree of sequence homology. The thrombin's substrate-binding cleft is much deeper than those of the pancreatic serine proteases. Steric hindrance by loops greatly restrict access to the active site and presumably contributes to thrombin's high specificity and its poor binding of most natural serine protease inhibitors. The specificity of thrombin for fibrinogen is largely attributable to its so called anion-binding site, and extension of thrombin's substrate binding cleft which is lined with positively charged side chains and which bind the highly anionic fibrinopeptides. Intrinsic Pathway Factor X (Stuart factor) may be activated by two different proteases. By factor IXa (Christmas factor) a product of the intrinsic pathway and factor VIIa (proconvertin) the

product of the extrinsic pathway. It has long been known that bringing blood into contact with negatively charged surfaces, such as those of glass, or kaolin (clay) initiates clotting. In vivo, collagen and platelet membranes are though to have the same effect. The contact system consist of four glycoproteins: 1. 2. 3. 4.

Hageman factor (XII), Prekallikrein, Plasma thromboplastin antecedent or PTA (factor XII) High molecular weigh kininogen (HMK)

Adsorption to a suitable surface is thought to somehow activate the Hageman factor which, in the presence of high molecular weight kininogen (HMK), hydrolyzes to the active protease kallikrein. Kallikrein in turn proteolytically activates Hageman factor do that these two proteins reciprocally activate each other. The nature of contact-activated Hageman factor is enigmatic; it is by no means certain that physical adsorption to a surface cleaves the same bond as does kallikrein or, for that matter, cleaves any bond at all. Much of the experimental difficulty in resolving this issue is a consequence of the contact-activation process's autocatalytic nature. Prekallikrein, contact activated Hageman factor's substrate is the zymogen of the protease that activate Hageman factor. Consequently, in any measurement of its activity, the nature of contact activated Hageman factor is immediately obscured by large amounts of rapidly generated kalikrein activated Hageman factor. The final reaction mediated by the contact system is the proteolytic activation of factor XI by activate Hageman factor ((XIIa) in the presence of HMK. Although the contact system is clearly effective in initiating in vitro clot formation, its in vivo importance is in doubt because individuals deficient in Hageman factor, prokallikrein, or HMK do not suffer from bleeding problems. Factor XIa (plasma thromboplastin antecedent PTA) catalyzes the proteolytic activation of Christmas factor (IX), a Gla-containing glycoprotein, in a Ca2+ requiring reaction that takes place on a phospholipid membrane surface. No accessory factor are known for this reaction. Christmas factor may also be activated by activated proconvertin (VIIa), a product of the extrinsic pathway. Factor X is proteolytically cleaved by activated Christmas factor (IXa) on a phospholipid membrane in a reaction requiring Ca2+ and the accessory factor activated antihemophilic factor (VIIIa). Antihemophilic factor, as is proaccelerin (V), is proteolytically activated by thrombin in a second autocatalytic process leading to prothrombin activation. Not surprising, proaccelerin and antihemophilic factor are homologous proteins. Antihemophilic factor circulates in the plasma in complex with von Willebrandt factor; in fact, the activities of these two substances were initially attributed to a single protein. Extrinsic Pathway

The extrinsic pathway, the alternative arm of the clotting cascade, is initiated by the proteolysis of proconvertin (VII), a process that can be catalyzed by activated Hageman factor (XIIa) as well as by thrombin. Activated proconvertin (VIIa) mediates the activation of Stuart factor (X). In the presence of phospholipid membrane, Ca2+ and tissue factor thromboplastin (III), the rate is enhanced 16,000-fold. Tissue factor thromboplastin (III) is an integral membrane glycoprotein that occurs in many tissues and is particularly abundant in brain, lung, blood vessel walls and placenta. Consequently, an injury that exposes blood to tissue rapidly initiates the extrinsic pathway. In fact, the addition of tissue factor thromboplastin to the extrinsic system causes clot formation in 12 s, where as the intrinsic system requires several minute to do so. These observations suggest that the intrinsic pathway is normally of little significance. However, the severity of the hemophilias resulting from intrinsic pathway clotting factor deficiencies clearly establishes the importance of the intrinsic pathway in blood clotting. The two pathways are not really independent since they are coupled through a number of reactions. Control of Clotting The multilevel cascade of the blood clotting system permits enormous amplification of it triggering signals. Proconvertin (VII), Stuart Factor (X) prothrombin (II) and fibrinogen (I) are present in plasma in concentration of 1, 8, 150, and 4000 µg/ml respectively. Clotting must be regulated since even one clot can have fatal consequences. Indeed, blood clots are the leading cause of strokes and heart attacks, which are the two major causes of human death in developed countries. A variety of factors limit clot growth. There are several interactions among the various clotting factors that inhibit blood coagulation. The dilution of active clotting factors by blood flow reduces the risk of clotting. Clots are selectively removed from the circulation by the liver. Plasma contains several serine protease inhibitors whose presence prevents clots from spreading beyond the vicinity of an injury. For example, antithrombin (58 kD) inhibits all active proteases of the clotting system except Proconvertin (VIIa) by binding to them in a1:1 complex. The presence of heparin, a sulfated glycoaminoglycan, enhances the activity of antithrombin by several hundred folds. Heparin occurs almost exclusively in the intercellular granules of the mast cells that line certain blood vessels. Protein C is another plasma protein that limits clotting. This Gla residue containing 62 kD zymogen is activated by thrombin to proteolytically inactivate proaccelerin (V) and antihemophilic factor (VII). Activated protein C attacks the active forms of these accessory factors more readily than their non-active forms. The importance of protein C is demonstrated by the observation that individuals who lack it often die in infancy of massive thrombotic complications. Thrombomodulin is a 74 kD glycoprotein that projects from the cell surface membranes of the vascular endothelium. Thrombomodulin specifically binds thrombin so as to

convert it to a form with decreased ability to catalyze clot formation and 1000 fold increased capacity to activate protein C. Clot lysis Blood clots are only temporary patches; they must be eliminated as wound repair progresses. This is a particularly urgent need when a clot has inappropriately formed or has broken free into the general circulation. Fibrin is easily dismantled in a process termed fibrinolysis. The demolition agent is a plasma serine protease named plasmin. This enzyme specifically cleaves fibrin that is a triple coiled protein. Plasmin cuts away a covalently linked α chain which is not in the triple coiled region. The rather open meshlike structure of a blood clot gives plasmin relatively free access to polymerized fibrin molecules thereby facilitating clot lysis. Plasmin is formed through the proteolytic cleavage of 82 kD zymogen plasminogen, a protein that is homologous to the zymogen of the blood-clotting cascade. There are several serine proteases that activate plasminogen, most notably the 54 kD enzyme urokinase, which is synthesized by the kidney and occurs as its name implies in the urine. Tissue type plasminogen activator (t-PA), a 70 kD protein, occurs in vascular tissues and activates plasminogen. In addition, activated Hageman factor, in the presence of prekallikrein and HMK activates plasminogen. Fibrinolysis is not as simple as just a zymogen and its activators. There are several inhibitors. The 70 kD glycoprotein α2antiplasmin forms an irreversible equal molar complex with plasmin and prevents it from binding to fibrin. The α2-antiplasmin cross-links to fibrin α chains through the action of activated FSF (XIIIa) thereby making hard clots less susceptible to fibrinolysis than soft clots. Plasminogen activators have received considerable medical attention aimed at rapidly dissolving the blood clots responsible for heart attacks and strokes. Streptokinase, a 45 kD protein produced by certain streptococci, has shown considerable utility in this regard particularly when administered together with aspirin. Despite its name streptokinase exhibits no enzymatic activity. Rather, it acts by forming a tight 1:1 complex with plasminogen that proteolytically activates other plasminogen molecules. The use of streptokinase to dissolve clots has the apparent disadvantage that it activates plasmin to degrade fibrinogen as well as fibrin thereby increasing the risk of bleeding problems, particularly strokes. The therapeutic use of t-PA, which has been synthesized by recombinant DNA techniques, is thought to eliminate these problems because this enzyme activates plasminogen only in the presence of a blood clot. Clinical Laboratory Procedures Clinical laboratory procedure used in hematology. View an animation of eurythrocyte clearance View an animation of how the sonoclot works

View an animation of how the Chronolog works Strokes