Physiology and Pathophysiology

Regulation of Hemostasis

July 23, 2017 Anticoagulant Therapy, Cardiology, Hematology, Physiology and Pathophysiology No comments , , , , , , , , , , , , , , ,

Key events that initiate and propagate coagulation are the redistribution of negatively charged phospholipids to the cell surface and the exposure of tissue factor to the blood. The appropriate negatively charged phospholipids, primarily phosphatidylserine, can arise as a result of either cellular activation with strong agonists like thrombin together with collagen in the case of platelets or tissue damage or death. The negatively charged phospholipids promote the activation of factor X and IX by the tissue factor-factor VIIa pathway, the activation of prothrombin by factors Va and Xa, and the activation of factor X by factors VIIIa and IXa. In addition, it has been suggested that oxidation of a specific disulfide in tissue factor is important for expression of its procoagulant activity, a process that may be regulated by protein disulfide isomerase, although this concept remains controversial.

Normally tissue factor is not present on cells in contact with blood. Tissue factor is found on extravascular cells surrounding the blood vessel so that a potent procoagulant surface is exposed when the endothelium is breached. This helps to seal the breach. Intravascularly, inflammatory stimuli can induce tissue factor synthesis and expression on leukocytes, particularly monocytes, providing a mechanism for the initiation of coagulation. This response likely contributes to disseminated intravascular coagulation (DIC). Animal studies suggest that this coagulation response plays a role in innate immunity and prevents the dissemination of infectious agents.

Other key events that can play a major role in the pathogenesis of thrombosis, at least in animal models, involve the release of intracellular components, including ribonucleic acid (RNA) and polyphosphates. These can trigger activation of factor XII, which initiates coagulation via the contact pathway. Although factor XII does not appear to contribute to hemostasis, it drives several models of thrombosis, including pulmonary embolism and myocardial infarction.

Polyphosphates are stored in the dense granules of platelets and when released, contribute to the procoagulant potential of the platelet. In regions of cellular death or severe inflammation, histones released from the tissue can bind to these polyphosphates, increasing the procoagulant activity more than 20-fold. Heparin analogues can block polyphosphate stimulation of coagulation by histones. In addition, neutrophils in hyperinflammatory environments can release their nuclear contents to form neutrophil extracellular traps (NETs). These NETs are effective in killing bacteria and other pathogens and also provide a potent agonist for the activation of platelets and the development of thrombi. Interestingly, heparin, an anticoagulant often used in the setting of inflammation, can disrupt the NETs and diminish the activation of platelets by histones and NETs.

Inhibition of Coagulation

There are three major natural anticoagulant mechanisms that serve to limit the coagulation process. These are tissue factor pathway inhibitor (TFPI), which blocks the initiation of coagulation by tissue factor-factor VIIa; the antithrombin-heparin mechanism, which inhibits thrombin and factors VIIa, Xa, and IXa; and the protein C pathway, which inactivates factors Va and VIIIa.

  1. Tissue factor pathway inhibitor (TFPI)
  2. Antithrombin-heparin mechanism
  3. Protein C pathway

Tissue Factor Pathway Inhibitor

TFPI is a complex molecule composed of three similar domains related to a protease inhibitor type known as Kunitz inhibitor. To inhibitor the tissue factor-factor VIIa complex, the Kunitz-1 domain of TFPI binds factor VIIa, whereas the Kunitz-2 domain binds factor Xa, either because the factor X is activated on tissue factor or (probably less effectively) by first reacting with factor Xa in solution and then inhibiting factor VIIa bound to tissue factor, thus blocking the initiation of coagulation. The carboxy terminal portion of TFPI is very basic, potentially facilitating interaction with the endothelium. Protein S can augment the activity of TFPI by increasing the rate of TFPI inactivation of factor Xa. The physiologic importance of TFPI is highlighted by the fact that gene deletion results in embryonic lethality apparently because of thrombosis and subsequent hemorrhage.

  1. Inhibiting Xa (Xa-TFPI complex)
  2. Xa-TFPI complex Inhibits FVIIa-TF complex
  3. TFPI is necessary for life

Antithrombin and Heparin

Antithrombin is the major inhibitor of the coagulation proteases thrombin, factor VIIa, factor Xa, and factor IXa. Antithrombin is a member of a large class of protease inhibitors referred to as serine protease inhibitors and abbreviated sermons. Antithrombin forms a very tight complex with these proteases. This reaction is rather slow with a half-life in the order of 30 seconds in plasma. Heparin markedly accelerates the reaction, thus accounting for most of its anticoagulant activity. There is a “bait” region in antithrombin that is involved in its interaction with the proteases. In the absence of heparin, this bait region is only partially available to the protease, resulting in the slow inhibition. Heparin binding to antithrombin induces a conformational change in antithrombin that enhances protease access to the bait loop.

In addition to the conformational change in antithrombin, heparin has additional roles in the inhibition of coagulation proteases. High-molecular-weight heparin can bind to both antithrombin and the protease, creating a situation where both reactants are brought into close proximity, thus increasing the reaction rate. This function is of variable importance with different proteases of the cascade. It is essential for thrombin inhibition, but plays a less important role in the inhibition of factor Xa. High-molecular-weight heparins accelerate factor Xa inhibition somewhat better than low-molecular-weight forms, but in contrast to most of the other heparin-mediated inhibitory functions, the additional acceleration gained by the high-molecular-weight forms is dependent on calcium ions. It is of interest that as heparins become processed to generate lower-molecular-weight fractions, such as enoxaparin, the capacity to promote thrombin inhibition versus factor Xa inhibition decreases. With the smallest functional form of heparin, the synthetic pentasaccharide fondaparinux, the ability to augment thrombin inhibition is almost completely lost, while good inhibition of factor Xa is maintained. Although heparin is a potent antithrombin-dependent anticoagulant, it is not effective against clot-bound thrombin or the factor Va-factor Xa complex assembled on membrane surfaces.

The importance of antithrombin is documented by the clinical observation that antithrombin deficiency, even to 50% of normal, is associated with significant thrombotic problems in humans and mice. Deletion of the gene in mice causes embryonic lethality, apparently in a mechanism that involves thrombosis followed by hemorrhage. Although heparin-like proteoglycans have been proposed to be important in modulating antithrombin function, immunohistochemical analysis indicates that most of these are localized to the basolateral side of the endothelium. No definitive deletion of heparin sulfate proteoglycans has been reported, possibly because there are alternative mechanisms for its biosynthesis. However, deletion of ryudocan, another heparin-like proteoglycan, is associated with a thrombotic phenotype.

  1. Antithrombin is ineffective against clot-bound thrombin or the factor Va-factor Xa complex assembled on membrane surfaces
  2. Heparin itself has no anticoagulation effect
  3. Heparin accelerates the anticoagulation capacity of antithrombin
  4. Deficiency of heparin-like proteoglycan potentiates patients at risk of thrombosis

The Thrombomodulin and Protein C Anticoagulation Pathway

The protein C pathway serves many roles, probably the primary role being to work to generate activated protein C (APC) that in turn inactivates factors Va and VIIIa to inhibit coagulation. The importance of this anticoagulant activity is readily apparent because patients born without protein C die in infancy of massive thrombotic complication (purport fulminans) unless provided a protein concentrate.

The protein C anticoagulant pathway serves to alter the function of thrombin, converting it from a procoagulant enzyme into the initiator of an anticoagulant response. This occurs when thrombin binds to thrombomodulin, a proteoglycan receptor primarily on the surface of the endothelium. Thrombomodulin binds thrombin with high affinity, depending on the posttranslational modifications of thrombomodulin. Binding thrombin to thrombomodulin blocks most of thrombin’s procoagulant activity such as the ability to clot fibrinogen, activate platelets, and activate factor V but does not prevent thrombin inhibition by antithrombin. The thrombin-thrombomodulin complex gains the ability to rapidly activate protein C. In the microcirculation, where there is high ratio of endothelial surface to blood volume, it has been estimated that the thrombomodulin concentration is in the range of 100 to 500 nM. Thus a single pass through the microcirculation effectively strips thrombin from the blood, initiates protein C activation, and holds a coagulantly inactive thrombin molecule in place for inhibition by antithrombin or protein C inhibitor. Activation of protein C is augmented approximately 20-fold in vivo by the endothelial cell protein C receptor (EPCR). EPCR binds both protein C and APC with similar affinity. The EPCR-APC complex is capable of cytoprotective functions, but at least with soluble EPCR, this complex is not an effective anticoagulant. Instead, when APC dissociates from EPCR, it can interact with protein S to inactivate factors Va and VIIIa and thus inhibit coagulation. Factor V increases the rate at which the APC-protein S complex inactivates factor VIIIa.

Thrombomodulin also increases the rate at which thrombin activates thrombin-activatable fibrinolysis inhibitor (TAFI) to a similar extent as it dose protein C. Once activated, TAFI releases C-terminal lysine and arginine residues of fibrin chains. C-terminal lysine residues on fibrin enhance fibrin degradation by serving as plasminogen- and tissue plasminogen activator-binding sites. Consequently, their removal by activated TAFI (TAFIa) attenuates clot lysis.

More than any other regulatory pathway except tissue factor, the protein C pathway is sensitive to regulation by inflammatory mediators. Tumor necrosis factor-alpha and IL-1beta downregulate thrombomodulin both in cell culture and in at least some patients with sepsis. Downregulation of thrombomodulin has been observed in animal models of diabetes, inflammatory bowel disease, reperfusion injury in the heart, over human atheroma, and in villitis, in addition to sepsis.

  1. Protein C, thrombomodulin, and EPCR are essential for life
  2. Protein C targets against factors Va and VIIIa
  3. EPCR increases the protein C activation by thrombin-thormbomodulin complex by about 20-fold
  4. Protein C’s anticoagulation function needs the help of protein S
  5. Thrombin-thrombomodulin complex removes the C-terminal lysine residues of fibrin, which attenuates clot fibrinolysis.

Regulation of Fibrinolysis

Fibrinolysis is the process by which fibrin clots are dissolved, either through natural mechanisms or with the aid of pharmaceutical interventions. Plasmin is the serine protease that solubilizes fibrin. Plasmin is generated by the activation of its precursor plasminogen either by natural activators, tissue plasminogen activator (tPA), the major activator in the circulation, or urokinase plasminogen activator (uPA), or by administration of recombinant tPA or streptokinase, a bacterial protein that promotes plasmin generation and aids in dissemination of the bacteria. Staphylokinase, a protein from Staphylococcus aureus, is similar to streptokinase and also has been examined as a potential therapeutic.

Fibrin plays an active role in its own degradation because it binds plasminogen and tPA, thereby concentrating them on the fibrin surface and promoting their interaction. The resultant plasmin cleaves fibrin and exposes C-terminal lysine residues that serve as additional plasminogen and tPA binding sites. TAFIa attenuates fibrinolysis by releasing these C-terminal lysine residues. Furthermore, lysine analogues, such as ε-aminocaproic acid or tranexamic acid, bind to tPA and plasminogen. Consequently, these lysine analogues can be used to treat patients with hyperfibrinolysis and can reduce bleeding complications.

Plasmin is not specific for fibrin and degrades a variety of other proteins. Consequently, plasmin is tightly controlled. There are two major inhibitors of fibrinolysis: α2-antiplasmin, and serpin that inactivates plasmin, and plasminogen activator inhibitor 1 (PAI-1), which inhibits tPA and uPA. α2-Antiplasmin is cross-linked to fibrin by activator factor XIII. PAI-1 is an intrinsically unstable serpin that can be stabilized by interaction with vitronectin. Because of its instability, the antigen and functional levels of PAI-1 are often quite different and can vary among individuals. PAI-1 is upregulated by lipopolysaccharide, tumor necrosis factor, and other inflammatory cytokines. PAI-1 levels are increased in vascular diseases such as atherosclerosis and in metabolic syndrome, sepsis, and obesity. Increased levels of PAI-1 result in decreased fibrinolytic activity, which increases the risk for thrombosis. PAI-1 is also stored in platelets and is released with platelet activation. Platelet-derived PAI-1 may limit the degradation of platelet-rich thrombi, such as those that trigger acute coronary syndromes.

Overall the fibrinolytic system is dynamic and responsive to local phenomena, such as platelet-derived PAI-1, as well as to systemic alterations like obesity and inflammation. In this sense, the fibrinolytic control mechanisms share many similarities with the control of coagulation, in which similar considerations affect their function.

Renal Handling of Urea

July 22, 2017 Laboratory Medicine, Nephrology, Physiology and Pathophysiology, Urology No comments , , , , , , ,

Renal Handling of Urate

Urate, an anion that is the base form of uric acid, provides a fascinating example of the renal handling of organic anions that is particularly important for clinical medicine and is illustrative of renal pathology. An increase in the plasma concentration of urate can cause gout and is thought to be involved in some forms of heart disease and renal disease; therefore, its removal from the blood is important. However, instead of excreting all the urate it can, the kidneys actually reabsorb most of the filtered urate. Urate is freely filterable. Almost all the filtered rate is reabsorbed early in the proximal tubule, primarily via antiporters (URAT1) that exchange urate for another organic anion. Further on the proximal tubule urate undergoes active tubular secretion. Then, in the straight portion, some of the urate is once again reabsorbed. Because the total rate of reabsorption is normally much greater than the rate of secretion, only a small fraction of the filtered load is excreted.

Although urate reabsorption is greater than secretion, the secretory process is controlled to maintain relative constancy of plasma urate. In other words, if plasma urate begins to increase because of increased urate production, the active proximal secretion of urate is stimulated, thereby increasing urate excretion.

Given these mechanisms of renal urate handling, the reader should be able to deduce the 3 ways by which altered renal function can lead to decreased urate excretion and hence increased plasma urate, as in gout: 1) decreased filtration of urate secondary to decreased GFR, 2) excessive reabsorption of urate, and 3) diminished secretion of urate.

Urate, and some other organic solutes, although more membrane permeable in the neutral form, are less soluble in aqueous solution and tend to precipitate. The combination of excess plasma urate and low urinary pH, which converts urate to the neutral uric acid, often leads to the formation of uric acid kidney stones.

Renal Handling of Urea

Urea is a very special substance for the kidney. It is an end product of protein metabolism, waste to be excreted, and also an important component for the regulation of water excretion. Urea differs from all the other organic solutes in several significant ways. 1) There are no membrane transport mechanisms in the proximal tubule; instead, it easily permeates the tight junctions of the proximal tubule where it is reabsorbed paracellularly. 2) Tubular elements beyond the proximal tubule express urea transporters and handle urea in a complex, regulated manner.

Urea is derived from proteins, which form much of the functional and structural substance of body tissues. Proteins are also a source of metabolic fuel. Dietary protein is first digested into its constituent amino acids. These are then used as building blocks for tissue protein, converted to fat or oxidized immediately. During fasting, the body breaks down proteins into amino acids that are used as fuel, in essence consuming itself. The metabolism of amino acids yields a nitrogen moiety (ammonium) and a carbohydrate moiety. The carbohydrate goes on to further metabolic processing, but the ammonium cannot be further oxidized and is a waste product. Ammonium per se is rather toxic to most tissues and the liver immediately converts most ammonium to urea and a smaller, but crucial amount to glutamine. While normal levels of urea are not toxic, the large amounts produced on a daily basis, particularly on a high protein diet, represent a large osmotic load that must be excreted. Whether a person is well fed or fasting, urea production proceeds continuously and constitutes about half of the usual solute content of urine.

The normal level of urea in the blood is quite variable, reflecting variations in both protein intake and renal handling of urea. Over days to weeks, renal urea excretion must match hepatic production; otherwise plasma levels would increase into the pathological range producing a condition called uremia. On a short-term basis (hours to days), urea excretion rate may not exactly match production rate because urea excretion is also regulated for purposes other than keeping a stable plasma level.

The gist of the renal handling of urea is the following: it is freely filtered. About half is reabsorbed passively in the proximal tubule. Then an amount equal to that reabsorbed is secreted back into the loop of Henle. Finally, about half is reabsorbed a second time in the medullary collecting duct. The net result is that about half the filtered load is excreted.

pH Dependence of Passive Reabsorption or Secretion

Many of the organic solutes handled by the kidney are weak acids or bases and exist in both, neutral and ionized forms. The state of ionization affects both the aqueous solubility and membrane permeability of the substance. Neutral solutes are more permeable than ionized solutes. As water is reabsorbed from the tubule, any substance remaining in the tubule becomes progressively more concentrated. And the luminal pH may change substantially during flow through the tubules. Therefore, both the progressive concentration of organic solutes and change in pH strongly influence the degree to which they are reabsorbed by passive diffusion through regions of tubule beyond the proximal tubule.

At low pH weak acids are predominantly neutral, while at high pH they dissociate into an anion and a proton. Imagine the case in which the tubular fluid becomes acidified relative to the plasma, which it does on a typical Western diet. For a weak acid in the tubular fluid, acidification converts much of the acid to the neutral form and therefore, increases its permeability. This favors diffusion out of the lumen (reabsorption). Highly acidic urine tends to increase passive reabsorption of weak acids (and promote less excretion). For many weak bases, the pH dependence is just opposite. At low pH they are protonated cations. As the urine becomes acidified, more is converted to the impermeable charged form and is trapped in the lumen. Less is reabsorbed passively, and more is excreted.

Ammonia and Urea Cycle

July 20, 2017 Gastroenterology, Medicinal Chemistry, Nephrology, Physiology and Pathophysiology No comments , , , , ,

Ammonia (NH3) is a small metabolite that results predominantly from protein and amino acid degradation. It is highly membrane-permeant and readily crosses epithelial barriers in its nonionized form.

Ammonia does not have a physiologic function. However, it is important clinically because it is highly toxic to the nervous system. Because ammonia is being formed constantly from the deamination of amino acids derived from proteins, it is important that mechanisms exist to provide for the timely and efficient disposal fo this molecule. The liver is critical for ammonia catabolism because it is the only tissue in which all elements of the urea cycle, also known as the Krebs-Henseleit cycle, are expressed, providing for the conversion of ammonia to urea. Ammonia is also consumed in the synthesis of nonessential amino acids, and in various facets of intermediary metabolism.

Ammonia Formation and Disposition

Ammonia in the circulation originates in a number of different sites. A diagram showing the major contributors to ammonia levels is shown in 14-1. Note that the liver is efficient in taking up ammonia from the portal blood in health, leaving only approximately 15% to spill over into the systemic circulation.

Intestinal Production

The major contributor to plasma ammonia is the intestine, supplying about 50% of the plasma load. Intestinal ammonia is derived via two major mechanisms. First, ammonia is liberated from urea in the intestinal lumen by enzymes known as ureases. Ureases are not expressed by mammalian cells, but are products of many bacteria, and convert urea to ammonia and carbon dioxide. Indeed, this provides the basis for a common diagnostic test, since H. pylori, which colonizes the gastric lumen and has been identified as a cause of peptic ulcer disease, has a potent urease. Therefore, if patients are given a dose of urea labeled with carbon-13, rapid production of labeled carbon dioxide in the breath is suggestive of infection with this microorganism.

Second, after proteins are digested by either host or bacterial proteases, further breakdown of amino acids generates free ammonia. Ammonia in its unionized form crosses the intestinal epithelium freely, and enters the portal circulation to travel to the liver; however, depending on the pH of the colonic contents, a portion of the ammonia will be protonated to ammonium ion. Because the colonic pH is usually slightly acidic, secondary to the production of short-chain fatty acids, the ammonium is thereby trapped in the lumen and can be eliminated in the stool.

Extraintestinal Production

The second largest contributor to plasma ammonia levels is the kidney. Ammonia is also produced in the liver itself during the deamination of amino acids. Minor additional components of plasma ammonia derive from adenylic acid metabolism in muscle cells, as well as glutamine released from senescent red blood cells.

Urea Cycle

The most important site for ammonia catabolism is the liver, where the elements of the urea cycle are expressed in hepatocytes. Ammonia derived from the sources described earlier is converted in the mitochondria to carbamoyl phosphate, which in turn reacts with ornithine to generate citrulline. Citrulline, in turn, reacts in the cytosol with aspartate, produced by the deamination of glutarate, to yield sequentially arginine succinate then arginine itself. The enzyme arginase then dehydrates arginine to yield urea and ornithine, which returns to the mitochondria and can reenter the cycle to generate additional urea. The net reaction is the combination of two molecules of ammonia with one of carbon dioxide, yielding urea and water.

Urea Disposition

A “mass balance” for the disposition of ammonia and urea is presented in Figure 14-2. As a small molecule, urea can cross cell membranes readily. Likewise, it is filtered at the glomerulus and enters the urine. While urea can be passively reabsorbed across the renal tubule as the urine is concentrated, its permeability is less than that of water such that only approximately half of the filtered load can be reabsorbed. Because of this, the kidney serves as the site where the majority of the urea produced by the liver is excreted. However, some circulating urea may passively back diffuse into the gut, where it is acted on by bacterial ureases to again yield ammonia and (CO2?). Some of the ammonia generated is excreted in the form of ammonium ion; the remainder is again reabsorbed to the handled by the liver once more.

Overview of the Hemostasis System

March 15, 2017 Hematology, Physiology and Pathophysiology No comments , , , , , , , , , , , , , , , , , , , , ,

Procoagulant Pathways

Two procoagulant pathway have been identified which converge at the “intrinsic” (accessory) fXase (fIXa*fVIIIa) complex. The contact or “intrinsic” pathway is activated by the interaction of blood with a foreign surface. This pathway is activated by the factor XIIa-high-molecular-weight kininogen (HMWK)-prekallikrein complex in association with foreign surfaces including glass, dextran sulfate, or kaolin. The complex catalyst activates factor XI leading to the factor XIa-HMWK complex which activates fIX to fIXa. The “intrinsic” fXase rapidly cleaves fX to fXa.

Factor Xa is directly but less effectively produced by the “extrinsic” fXase, which is composed of plasma-derived fVIIa and tissue factor and expressed when the latter is exposed to blood. The “extrinsic” fXase also activates fIX to fIXa as the reaction progresses, with suppression of the quaternary complex occurring by TFPI. The “intrinsic” fXase complex is kinetically superior and ultimately produces the majority of fXa.

Since bleeding pathology is not ordinarily associated with defects of the initiation protein complexes of the contact pathway, most investigations conclude that the “intrinsic” pathway is not the primary provider for hemostasis. The “intrinsic” pathway, however, has been implicated in some forms of thrombosis. The primary (“extrinsic”) and accessory (“intrinsic”) pathways, initiated by independent routes, both lead to the activation of factor IX and converge at the “intrinsic” fXase complex. Regardless of the path, the outcome is the formation of the prothrombinase complex and thrombin generation. Each reaction of the primary pathway of coagulation involves the vitamin K-dependent zymogens and serine proteases, cofactor proteins, and Ca2+ ions assembled on membranes. The complexes display reaction rates 10^5 to 10^9 times greater than the respective serine proteases alone.

Clinical laboratory tests differentiate between the pathways. The activated partial thromboplastin time (aPTT) initiates coagulation through the accessory pathway, whereas the prothrombin time (PT) assay initiates coagulation through the primary pathway. The designations of primary and accessory pathways are based on clinical evidence of bleeding diseases. Deficiencies of proteins associated with the “intrinsic” or accessory pathway (factor XII, prekallikrein, and HMWK) exist but are not associated with abnormal bleeding events, even after surgical challenge. However, 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. Factor XI deficiency may also result in bleeding episodes subsequent to trauma or surgery. The role of the accessory pathway is therefore not clearly understood.

Factor XII, prekallikrein, and HMWK are required for activity of the contact or accessory pathway and deficiencies are reported by the aPTT. Factor XII and prekallirein are zymogens that are activated to sarin proteases, while HMWK is a cofactor. The accessory pathway factors are hypothesized to play a role in disseminated intravascular coagulation (DIC) associated with the systemic inflammatory response syndrome, and may also be involved in the promotion of thrombus stability. The accessory pathway may also be important in cardiopulmonary bypass because of contact between blood components and synthetic surfaces.

The importance of the membrane component in coagulation was initially identified by kinetic studies of the prothrombinase complex. In the absence of the membrane surface, the cofactor (factor Va)-enzyme (factor Xa) interaction is relatively weak, with dissociation constant (Kd) of 800 nmol/L. The factor Va-lipid interaction (Kd = 3 nmol/L) and factor Xa-lipid interaction (Kd = 110 nmol/L) show higher affinity. However, all of the components must be present to generate the high-affinity factor Va-factor Xa-Ca2+ membrane complex, with a Kd of 1 nmol/L. The fully assembled complex is stabilized through factor Va-factor Xa, factor Va-lipid, and factor Xa-lipid interactions. Similar properties have been observed for the fIXa/fVIIIa, TF/fVIIa, and Tm/fIIa complexes.

The primary pathway of coagulation is initiated or triggered by the interaction of circulating factor VIIa with its cofactor tissue factor (TF). In general, the serine proteases associated with hemostasis circulate in their zymogen or inactive forms; however, low levels of circulate in their zymogen or inactive forms; however, low levels of circulating factor VIIa are present in blood. This factor VIIa binds to tissue factor expressed by pathology and initiates the procoagulant response. Free factor VIIa is a poor enzyme with virtually no proteolytic activity, but as a consequence is protected from interacting with the circulating inhibitors in the absence of tissue factor. Tissue factor, an integral membrane protein not normally expressed on vascular cell surfaces, is constitutively expressed on extravascular cellular surfaces and thus becomes exposed upon damage to the endothelial cell layer. Tissue factor is also expressed on peripheral blood cells and endothelial cells stimulated by inflammatory cytokines.

Upon interaction of plasma factor VIIa and the injury/pathology present, tissue factor, the “extrinsic” fXase complex (factor VIIa-tissue factor) is formed and initiates coagulation by activating factor IX and X. Factor IXa forms a complex with its cofactor, factor VIIIa, to generate the “intrinsic” fXase complex, and factor Xa combines with factor Va to form the prothrombinase complex. The factor VIIIa-factor IXa complex more efficiently activates factor X to factor Xa, providing a robust source of the enzyme component (factor Xa) of the prothrombinase complex.

Deficiencies of the “intrinsic” fXase components factor VIIIa (hemophilia A) and factor IXa (hemophilia B) illustrate the significance of the factor IX activation by the fVIIa/TF complex and the enhanced rate of factor X activation by the intrinsic fXase and the inhibition of the extrinsic fXase by TFPI. Hemophilia A and B are detected using the aPTT. It would appear activation of factor X by the extrinsic fXase should compensate for the lack of factor activation by the intrinsic fXase in hemophilias A and B. However, this compensatory mechanism only occurs during clinical administration of supraphysiologic concentrations of recombinant factor VIIa during replacement therapy for hemophilia with inhibitors. The natural physiologic levels of factor VIIa are not able to provide sufficient levels of factor Xa to support normal coagulation. Factor Xa generation is suppressed to approximately one-half the level observed when factor X is the only substrate presented.

Factor IX, not factor X, appears to be the preferred substrate of the extrinsic fXase. In addition, factor IXalpha, the intermediate species in factor IX activation, is generated more rapidly in the presence of factor X. Factor IXalpha activation to the final product factor IXa occurs at a higher rate than factor IX activation, thereby providing a burst of factor IXa to form the intrinsic fXase complex. The low level of factor Xa generated by the tissue factor-factor VIIa complex most likely functions in the activation of factor IX. A model of extrinsic fXase behavior suggests that factor IX is converted to factor IXalpha by the extrinsic fXase or factor Xa-phospholipid complex. Factor IXalpha is then rapidly converted to factor IXa by the extrinsic fXase. The factor VIIIa-factor IXa complex subsequently activates the major fraction of factor X to factor Xa and provides the enzyme component for the prothrombinase complex. Measurements of second-order rate constants for factor Xa generation by the intrinsic and extrinsic fXase complexes also support this model. The rate of factor Xa generation by the tissue factor-factor VIIa complex is 1/50th the rate of factor Xa generation by the factor VIIIa-factor IXa complex. Both complexes thus have distinct roles in the procoagulant response.

Screen Shot 2017 03 13 at 9 34 32 PM

Accessory Pathway

The procoagulant proteins that make up the intrinsic or accessory pathway consist of factor XII, plasma prekallikrein, HMWK, and factor XI. These proteins are responsible for the contact activation of blood coagulation. The physiologic role of the intrinsic pathway is not clearly understood, but does not appear to be essential for hemostasis because individuals deficient in factor XII, plasma prekallirein, or HMWK do not manifest abnormal bleeding. Factor XI appears to play a more prominent role, unrelated to its activities in the intrinsic pathway, after activation by thrombin.

Update on March 15 2017

Hemostatic System

The major components of the hemostatic system are the vascular endothelium, platelets, and the coagulation and fibrinolytic systems.

Vascular Endothelium

A monolayer of endothelial cells lines the intimal surface of the circulatory tree and separates the blood from the prothrombotic sub endothelial components of the vessel wall. As such, the vascular endothelium encompasses about 10^13 cells and covers a vast surface area. Rather than serving as a static barrier, the healthy vascular endothelium is a dynamic organ that actively regulates hemostasis by inhibiting platelets, suppressing coagulation, promoting fibrinolysis, and modulating vascular tone and permeability.

Platelet Inhibition

Endothelial cells synthesize prostacyclin and nitric oxide and release them into the blood.These agents not only serve as potent vasodilators but also inhibit platelet activation and subsequent aggregation by stimulating adenylate cyclase and increasing intracellular levels of cyclic adenosine monophosphate (cAMP). In addition, endothelial cells express (CD39) on their surface, a membrane-associated ecto-adenosine diphosphatase (ADPase). By degrading ADP, which is a platelet agonist, CD39 attenuates platelet activation.

  • Prostacyclin
  • Nitric oxide
  • CD39

Anticoagulant Activity

Intact endothelial cells play an essential part in the regulation of thrombin generation through a variety of mechanisms. Endothelial cells produce heparan sulfate proteoglycans, which bind circulating antithrombin and accelerate the rate at which it inhibits thrombin and other coagulation enzymes. Tissue factor pathway inhibitor (TFPI), a naturally occurring inhibitor of coagulation, binds heparan sulfate on the endothelia cell surface. Administration of heparin or low-molecular-weight heparin (LMWH) displaces glycosaminoglycan-bound TFPI from the vascular endothelium, and released TFPI may contribute to the antithrombotic activity of these drugs.

Endothelial cells regulate thrombin generation by expressing thrombomodulin and endothelial cell protein C receptor (EPCR) on their surfaces. Thrombomodulin binds thrombin and alters this enzyme’s substrate specificity such that it no longer acts as a procoagulant but becomes a potent activator of protein C. Activated protein C serves as an anticoagulant by degrading and inactivating activated factor V (FVa) and factor VIII (FVIIIa), key cofactors involved in thrombin generation. Protein S acts as a cofactor in this reaction, and EPCR enhances this pathway by binding protein C and presenting it to the thrombin-thrombomodulin complex for activation. In addition to its role as an anticoagulant, activated protein C also regulates inflammation and preserves the barrier function of the endothelium.

  • Heparan
  • TFPI
  • Thrombomodulin
  • EPCR

Fibrinolytic Activity

The vascular endothelium promotes fibrinolysis by synthesizing and releasing tissue-type and urokinase-type plasminogen activator (t-PA and u-PA, respectively), which initiate fibrinolysis by converting plasminogen to plasmin. Endothelial cells in most vascular beds synthesize t-PA constitutively. In contrast, perturbed endothelial cells produce u-PA in the settings of inflammation and would repair.

Endothelial cell also produce type 1 plasminogen activator inhibitor 1 (PAI-1), the major regulator of both t-PA and u-PA. Therefore, net fibrinolytic activity depends on the dynamic balance between the release of plasminogen activators and PAI-1.  Fibrinolysis localizes to the endothelial cell surface because these cells express annexin II, a coreceptor for plasminogen and t-PA that promotes their interaction. Therefore, healthy vessels actively resist thrombosis and help maintain platelets in quiescent state.

  • t-PA
  • u-PA
  • PAI-1
  • Annexin II

Vascular Tone and Permeability

In addition to synthesizing potent vasodilators, such as prostacyclin and nitric oxide, endothelial cells also produce a group of counter-regulatory peptides known as endothelins that induce vasoconstriction. Endothelial cell permeability is influenced by the connections that join endothelial cells to their neighbors. Macromolecules traverse the endothelium via patent intercellular junctions, by endocytosis, or through transendothelial pores. Vasodilatation, servers thrombocytopenia, and high doses of heparin can increase endothelial permeability, which may contribute to bleeding. Activated protein C may also contribute to the barrier function of the endothelium.


Platelets are anucleate particles released into the circulation after fragmentation of bone marrow megakaryocytes. Because they are anucleate, platelets have limited capacity to synthesize proteins. Thrombopoietin, a glycoprotein synthesized in the liver and kidneys, regulates megakaryocytic proliferation and maturation as well as platelet production. After they enter the circulation, platelet have a life span of 7 to 10 days.

Screen Shot 2017 03 14 at 3 10 37 PM

Damage to the intimal lining of the vessel exposes the underlying subendothelial matrix. Platelets home to sites of vascular disruption and adhere to the exposed matrix proteins. Adherent platelets undergo activation and not only release substances that recruit additional platelets to the site of injury but also promote thrombin generation and subsequent fibrin formation. A potent platelet agonist, thrombin amplifies platelet recruitment and activation. Activated platelets then aggregate to form a plug that seals the leak in the vasculature. An understanding of the steps in these highly integrated processes helps pinpoint the sites of action of the antiplatelet drugs and rationalizes the utility of anticoagulants for the treatment of arterial thrombosis and venous thrombosis.


Platelets adhere to exposed collagen and von Willebrand factor (vWF) and form a monolayer that supports and promotes thrombin generation and subsequent fibrin formation. These events depend on constitutively expressed receptors on the platelet surface, alpha2beta1 and glycoprotein (GP) VI, which bind collagen, and GPIbalpha and GPIIb/IIIa (alphaIIbbeta3), which bind vWF. The platelet surface is crowded with receptors, but those involved in adhesion are the most abundant: every platelet has about 40,000 to 80,000 copies of GPIIb/IIIa and 25,000 copies of GPIbalpha. Receptors cluster in cholesterol-enriched subdomains, which render them more mobile, thereby increasing the efficiency of platelet adhesion and subsequent activation.

  • Platelet receptors: alpha2beta1, GP VI, GPIbalpha, GPIIb/IIIa
  • Platelet receptors’ ligands: collagen (alpha2beta1, GP VI), vWF (GPIbalpha, GPIIb/IIIa)

Under low shear conditions, collagen can capture and activate platelets on its own. Captured platelets undergo cytoskeletal reorganization that causes them to flatten and to adhere more closely to the damaged vessel wall. Under high shear conditions, however, collagen and vWF must act in concert to support optimal platelet adhesion and activation. vWF synthesized by endothelial cells and megakaryocytes assembles into multimers that range from 550 to more than 10,000 kDa. When released from storage in the Weibel-Palade bodies of endothelial cells or the alpha-granules of platelets, most of the vWF enters the circulation, but the vWF released from the abluminal surface of endothelial cells accumulates in the subendothelial matrix, where it binds collagen via its A3 domain. This surface-immobilized vWF can simultaneously bind platelets via its A1 domain. In contrast, circulating vWF does not react with unstimulated platelets. This difference in reactivity likely reflects vWF conformation; whereas circulating vWF is in a coiled conformation that prevents access of its platelet-binding domain to vWF receptors on the platelet surface, immobilized vWF assumes an elongated shape that exposes its A1 domain. In this extended conformation, large vWF multimers serve as the molecular glue that tethers platelets to the damaged vessel wall with sufficient strength to withstand higher shear forces. Large vWF multimers provide additional binding sites for collagen and heighten platelet adhesion because platelets have more vWF results in platelet activation, the next step in platelet plug formation.

  • Low shear conditions
  • High shear conditions
  • Free vWF
  • Surface-immobilized vWF
  • Interaction: vWF and collagen

Activation and Secretion

Adhesion to collagen and vWF initiates signaling pathways that result in platelet activation. These pathway induce cyclooxyrgenase-1 (COX-1)-dependent synthesis and release of thromboxane A2, and trigger the release of adenosine diphosphate (ADP) from storage granules. Thromboxane A2 is a potent vasoconstrictor, and similar to ADP, locally activates ambient platelets and recruits them to the site of injury. This process results in expansion of the platelet plug. To activate platelets, thromboxane A2 and ADP must bind to their respective receptors on the platelet membrane. The thromboxane receptor (TP) is a G-protein coupled receptor that is found on platelets and on the endothelium, which explains why thromboxane A2 induces vasoconstriction as well as platelet activation. ADP interacts with a family of G protein-coupled receptors on the platelet membrane. Most important of these is P2Y12, which is the target of the theienopyridines, but P2Y1 also contributes to ADP-induced platelet activation, and maximal ADP-induced platelet activation requires activation of both receptors. A third ADP receptor, P2X1, is an adenosine triphosphate (ATP)-gated calcium channel. Platelet storage granules contain ATP as well as ADP; ATP released during the platelet activation process may contribute to the platelet recruitment process in a P2X1-dependent fashion.

  • TXA2
  • Thromboxane receptor
  • ADP
  • ADP receptors (P2Y12, P2Y1, P2X1)
  • Pathway: collagen and/or vWF > thromboxane A2 release, and ADP release

Although TP and the various ADP receptors signal through different pathways, they all trigger an increase in the intracellular calcium concentration in platelets. This in turn induces shape change via cytoskeletal rearrangement, granule mobilization and release, and subsequent platelet aggregation. Activated platelets promote coagulation by expressing phosphatidylserine on their surfaces, and anionic phospholipid that supports assembly of coagulation factor complexes. After being assembled, these clotting factor complexes trigger a burst of thrombin generation and subsequent fibrin formation. In addition to converting fibrinogen to fibrin, thrombin amplifies platelet recruitment and activation and promotes expansion of the platelet plug. Thrombin binds to protease-activated receptors types 1 and 4 (PAR1 and PAR4, respectively) on the platelet surface and cleaves their extended amino-termini, thereby generating new amino-termini that serve as tethered ligands that bind and activate the receptors. Whereas low concentrations of thrombin cleave PAR1, PAR4 cleavage requires higher thrombin concentrations. Cleavage of either receptor triggers platelet activation.

  • Pathway: thrombin (ligand), and PAR1 and PAR4 (receptors)

In addition to providing a surface on which clotting factors assemble, activated platelets also promote fibrin formation and subsequent stabilization by releasing factor V, factor XI, fibrinogen, and factor XIII. Thus, there is coordinated activation of platelets and coagulation, and the fibrin network that results from thrombin action helps anchor the platelet aggregates at the site of injury. Activated platelets also release adhesive proteins, such as vWF, thrombospondin, and fibronectin, which may augment platelet adhesion at sites of injury, as well as growth factors, such as platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGFbeta), which promote wound healing. Platelet aggregation is the final step in the formation of the platelet plug.

  • Secretion: platelet derived factor V, factor XI, fibrinogen, and factor XIII, vWF


Platelet aggregation links platelets to each other to form clumps. GPIIb/IIIa mediates these platelet-to-platelet linkages. On inactivated platelets, GPIIb/IIIa exhibits minimal affinity for its ligands. Upon platelet activation, GPIIb/IIIa undergoes a conformational transformation, which reflects transmission of inside-out signals from its cytoplasmic domain to its extracellular domain. This transformation enhances the affinity of GPIIb/IIIa for its ligands; fibrinogen; and, under high shear conditions, vWF. Arginine-glycine-aspartic acid (RGD) sequences located on fibrinogen and vWF, as well as platelet-binding lycine-glycine-aspartic acid (KGD) sequence on fibrinogen, mediate their interaction with GPIIb/IIIa. When subjected to high shear, circulating vWF elongates and exposes its platelet-binding domain, which enables its interaction with the conformationally activated GPIIb/IIIa. Divalent fibrinogen and multivalent vWF molecules serve as bridges and bind adjacent platelets together. After being bound to GPIIb/IIIa, fibrinogen and vWF induce outside-inside signals that augment platelet activation and result in the activation of additional GPIIb/IIIa receptors, creating a positive feedback loop. Because GPIIb/IIIa acts as the final effector in platelet aggregation, it is a logical target for potent antiplatelet drugs. Fibrin, the ultimate product of the coagulation system, tethers the platelet aggregates together and anchors them to the site of injury.

  • Platelet (activated) receptors: GPIIb/IIIb (fibrinogen, vWF)


Coagulation results in the generation of thrombin, which converts soluble fibrinogen to fibrin. Coagulation occurs through the action of discrete enzyme complexes, which are composed of a vitamin K-dependent enzyme and a non-enzyme cofactor, and assemble on anionic phospholipid membranes in a calcium-dependent fashion. Each enzyme complex activates a vitamin K-dependent substrate that becomes the enzyme component of the subsequent complex. Together, these complexes generate a small amount of thrombin, which amplifies its own generation by activating the non-enzyme cofactors and platelets, which then provide an anionic surface on which the complexes assemble. The three enzyme complexes involved in thrombin generation are extrinsic tenase, intrinsic tenase, and prothrombinase. Although extrinsic tenase initiates the system under most circumstances, the contact system also plays a role in some situations.

Extrinsic Tenase (FVIIa-TF complex)

This complex forms upon exposure of tissue factor-expressing cells to the blood. Tissue factor exposure occurs after atherosclerotic plaque rupture because the core of the plaque is rich in cells that express tissue factor. Denuding injury to the vessel wall also exposes tissue factor constitutively expressed by subendothelial fibroblasts and smooth muscle cells. In addition to cells in the vessel wall, circulating monocytes and monocyte-derived microparticles (small membrane fragments) also provide a source of tissue factor. When tissue factor-bearing monocytes or microparticles bind to platelets or other leukocytes and their plasma membranes fuse, tissue factor transfer occurs. By binding to adhesion molecules expressed on activated endothelial cells or to P-selectin on activated platelets, these tissue factor-bearing cells or microparticles can initiate or augment coagulation. This phenomenon likely explains how venous thrombi develop in the absence of obvious vessel wall injury.

Tissue factor is an integral membrane protein that serves as a receptor for factor VIIa. The blood contains trace amounts of factor VIIa, which has negligible activity in the absence of tissue factor. With tissue factor exposure on anionic cell surfaces, factor VIIa binds in a calcium-dependent fashion to form the extrinsic tenase complex, which is a potent activator of factor IX and X. After being activated, factor IXa and factor Xa serve as the enzyme components of intrinsic tenase and prothrombinase, respectively.

Intrinsic Tenase (FVIIIa-FIXa complex)

Factor IXa binds to factor VIIIa on anionic cell surfaces to form the intrinsic tenase complex. Factor VIII circulates in blood in complex with vWF. Thrombin cleaves factor VIII and releases it from vWF, converting it to its activated form. Activated platelets express binding sites for factor VIIIa. After being bound, factor VIIIa binds factor IXa in a calcium-dependent fashion to form the intrinsic tenase complex, which then activates factor X. The change in catalytic efficiency of factor IXa-mediated activation of factor X that occurs with deletion of individual components of the intrinsic tenase complex highlights their importance. Absence of the membrane or factor VIIIa almost completely abolishes enzymatic activity, and the catalytic efficiency of the complete complex is 10^9-fold greater than that of factor IX alone. Because intrinsic tenase activates factor X at a rate 50- to 100-fold faster than extrinsic tenase, it plays a critical role in the amplification of factor Xa and subsequent thrombin generation.

Prothrombinase (FXa-FVa complex)

Factor Xa binds to factor Va, its activated cofactor, on anionic phospholipid membrane surfaces to form the prothrombinase complex. Activated platelets release factor V from their alpha-granules, and this platelet-derived factor V may play a more important role in hemostasis than its plasma counterpart. Whereas plasma factor V requires thrombin activation to exert its cofactor activity, the partially activated factor V released from platelets already exhibits substantial cofactor activity. Activated platelets express specific factor Va binding sites on their surface, and bound factor Va serves as a receptor for factor Xa. The catalytic efficiency of factor Xa activation of prothrombin increase by 10^9-fold when factor Xa incorporates into the prothrombinase complex. Prothrombin binds to the prothrombinase complex, where it undergoes conversion to thrombin in a reaction that release prothrombin fragment 1.2. Plasma levels of F1.2, therefore, provide a marker of prothrombin activation.

Fibrin Formation

The final effector in coagulation is thrombin. Thrombin converts soluble fibrinogen into insoluble fibrin. Fibrinogen is a dimeric molecule, each half of which is composed of three polypeptide chains – the Aalpha, Bbeta, and gamma chains. Numerous disulfide bonds covalently link the chains together and join the two halves of the fibrinogen molecule. Electron micrographic studies of fibrinogen reveal a trinodular structure with a central E domain flanked by two D domains. Crystal structures show symmetry of design with the central E domain, which contains the amino termini of the fibrinogen chains, joined to the lateral D domains by coiled-coil regions.

Fibrinogen circulates in an inactive form. Thrombin binds to the amino termini of the Aalpha and Bbeta chains of fibrinogen, where it cleaves specific peptide bonds to release fibrinopeptide A and fibrinopeptide B and generates fibrin monomer. Because they are products of thrombin action on fibrinogen, plasma levels of these fibrinopeptides provide an index of thrombin activity. Fibrinopeptide release creates new amino termini that extend as knobs from the E domain of one fibrin monomer and insert into preformed holes in the D domains of other fibrin monomers. This creates long strands known as protofibrils, consisting of fibrin monomers noncovalently linked together in a half-staggered overlapping fashion.

Noncovalently linked fibrin protofibrils are unstable. The stability of the fibrin network is enhanced by platelets and procoagulant cells. Platelets not only bind fibrin via GPIIb/IIIa and promote the formation of a dense fibrin network, but the also release factor XIII. By covalently cross-linking the alpha and gamma chains of adjacent fibrin monomers, factor XIIIa stabilizes the fibrin in a calcium-dependent fashion and renders it relatively resistant to degradation. Factor XIII circulates in blood as a heterodimer consisting of pairs of A and B subunits. The active and calcium binding sites on factor XIII are localized to the A subunit. Platelets contain large amounts of factor XIII in their cytoplasms, but platelet-derived factor XIII consists only of the A subunits. Both plasma and platelet factor XIII are activated by thrombin.

Contact Pathway

Current thinking is that tissue factor exposure represents the sole pathway for activation of coagulation and that the contact system – which includes factor XII, prekallikrein, and high-molecular-weight kininogen – is unimportant for hemostasis because patients deficient in these factors do not have bleeding problems. The physiologic role of factor XI is more difficult to assess because the plasma level of factor XI does not predict the propensity for bleeding. Although the capacity of thrombin to feed back and activate platelet-bound factor XI may explain this phenomenon, platelet-derived factor XI may be more important for hemostasis than circulating factor XI.

We cannot ignore the contact pathway, however, because coronary catheters and other blood-contacting medical devices, such as stents or mechanical valves, likely trigger clotting through this mechanism. Factor XII bound to the surface of catheters or devices undergoes a conformational change that results in it activation. Factor XIIa converts prekallikrein to kallikrein in a reaction accelerated by high-molecular-weight kininogen, and factor XIIa and kallikrein then feedback to activate additional factor XII. Factor XIIa propagates coagulation by activating factor XI.

In addition to its role in device-related thrombosis, the contact pathway may also contribute to the stability of arterial and venous thrombi. DNA and RNA released from damaged cells in atherosclerotic plaques activates factor XII, and mice DNA- or RNA-degrading enzymes exhibit attenuated thrombosis at site of arterial injury. Polyphosphates released from activated platelets also activate factor XII and may provide another stimulus for contact pathway activation. Mice deficient in factor XII or factor XI form small unstable thrombi at sites of arterial or venous damage, suggesting that factor XII and factor XI contribute to thrombogenesis. It is unknown whether the same is true in humans. Patients with unstable angina have increased plasma levels of factor XIa, but it is unclear whether this reflects activation by factor XIIa or by thrombin. Although the contribution of the contact pathway to thrombin generation remains uncertain, the final product of coagulation is fibrin. Hemostasis depends on the dynamic balance between the formation of fibrin and its degradation.

Interactions Between Platelet and Coagulation System (Summary)

Platelet and coagulation system act in synergism to maintain hemostasis.

Platelet for coagulation system

  • Platelet provides membrane where clotting factors reactions could take place quickly
  • Platelet secrets FV (partially activated), FXI, fibrinogen, FXIII, and vWF
  • Polyphosphates released from activated platelets activate FXII

Coagulation system for platelet

  • Thrombin activates platelet via PAR-1 and PAR-4
  • Fibrinogen helps platelet binds adjacent platelet together

Update on Mar 25 2017

Fibrinolytic System

Fibrinolysis initiates when plasminogen activators convert plasminogen to plasmin, which then degrades fibrin into soluble fragments. Blood contains two immunologically and functionally distinct plasminogen activators, t-PA and u-PA. Whereas t-PA mediates intravascular fibrin degradation, u-PA binds to a specific u-PA receptor (u-PAR) on the surface of cells, where it activates cell-bound plasminogen. Consequently, pericellular proteolysis during cell migration and tissue remodeling and repair are the major functions of u-PA.

Regulation of fibrinolysis occurs at two levels. PAI-1, and to a lesser extent, PAI-2, inhibit the plasminogen activators, and alpha2-antiplasmin inhibits plasmin. Endothelial cells synthesize PAI-1, which inhibits both t-PA and u-PA, whereas monocytes and the placenta synthesize PAI-2, which specifically inhibits u-PA. Thrombin-activated fibrinolysis inhibitor (TAFI) also modulates fibrinolysis and provides a link between fibrinolysis and coagulation. Whereas thrombosis can occur if there is impaired activation of the fibrinolytic system, excessive activation leads to bleeding.

  • Plasminogen
  • t-PA
  • u-PA
  • PAI-1
  • PAI-2
  • TAFI

Mechanism of Action of Tissue Plasminogen Activator

Tissue plasminogen activator, a serine protease, contains five discrete domains: a fibronectin-like finger domain, and epidermal growth factor (EGF) domain, two kringle domains, and a protease domain. Synthesized as a single-chain polypeptide, plasmin readily converts t-PA into a two-chain form. Single- and two-chain forms of t-PA convert plasminogen to plasmin. Native Glu-plasminogen is a single-chain polypeptide with a Glue residue at its amino-terminus. Plasmin cleavage at the amino-terminus generates lysine-plasminogen, a truncated form with a Lys residue at its new amino terminus. t-PA cleaves a single peptide bond to convert single-chian glutamine- or lysine-plasminogen into two-chain plasmin composed of a heavy chain containing five kringle domains and a light chain containing the catalytic domain. Because its open conformation exposes the t-PA cleavage site, lysine-plasminogen is a better substrate than glutamic acid-plasminogen, which assumes a circular conformation that renders this bond less accessible.

Tissue plasminogen activator has little enzymatic activity in the absence of fibrin, but its activity increases by at least three orders of magnitude when fibrin is present. This increase in activity reflects the capacity of fibrin to serve as a template that binds t-PA and plasminogen and promotes their interaction. Whereas t-PA binds fibrin via its finger and second kringle domains, plasminogen binds fibrin via its kringle domains. Kringle domains are loop-like structures that bind Lys residues on fibrin. As fibrin undergoes degradation, more Lys residues are exposed, which provide additional binding sites for t-PA and plasminogen. Consequently, degraded fibrin stimulates t-PA activation of plasminogen more than intact fibrin.

alpha2-antiplasmin rapidly inhibits circulating plasmin by docking to its first kringle domain and then inhibiting the active site. Because plasmin binds to fibrin via its kringle domains, plasmin generated on the fibrin surface resists inhibition by alpha2-antiplasmin. This phenomenon endows fibrin-bound plasmin with the capacity to degrade fibrin. Factor XIIIa cross-links small amounts of alpha2-antiplasmin onto fibrin, which prevents premature fibrinolysis.

Similar to fibrin, annexin II on endothelial cells binds t-PA and plasminogen and promotes their interaction. Cell-surface gangliosides and alpha-enolase also may bind plasminogen and promote its activation by altering its conformation into the more readily activated open form. Plasminogen binds to endothelial cells via its kringle domains. Lipoprotein a, which also possesses kringle domains, impairs cell-based fibrinolysis by competing with plasminogen for cell-surface binding. This phenomenon may explain the association between elevated lipoprotein a levels and atherosclerosis.

Mechanism of Action of Urokinase-Type Plasminogen Activator

Synthesized as a single-chain polypeptide, single-chain u-PA (scu-PA) has minimal enzymatic activity. Plasmin converts scu-PA into a two-chain form that is enzymatically active and capable of binding u-PAR, the u-PA receptor on cell surfaces. Further cleavage at the amino terminus of two-chain u-PA yields a truncated, lower-molecular-weight form that lacks the u-PAR binding domain.

Two-chain forms of u-PA readily convert plasminogen to plasmin in the absence or presence of fibrin. In contrast, scu-PA does not activate plasminogen in the absence of fibrin, but it can activate fibrin-bound plasminogen because plasminogen adopts a more open and readily activatable conformation when immobilized on fibrin. Similar to the higher-molecular-weight form of two-chain u-PA, scu-PA binds to cell surface u-PAR, where plasmin can activate it. Many tumor cells elaborate u-PA and express u-PAR on their surfaces. Plasmin generated on these cells endows them with the capacity for metastasis.

Mechanism of Action of TAFI

Thrombin-activated fibrinolysis inhibitor, a procarboxpeptidase B-like molecule synthesized in the liver, circulates in blood in a latent form while thrombin bound to thrombomodulin can activate it. Unless bound to thrombomodulin, thrombin activates TAFI inefficiently. TAFIa attenuates fibrinolysis by cleaving lysine residues form the carboxy termini of chains of degrading fibrin, thereby removing binding sites for plasminogen, plasmin, and t-PA. TAFI links fibrinolysis to coagulation because the thrombin-thrombomodulin complex not only activates TAFI, which attenuates fibrinolysis, but also activates protein C, which mutes thrombi in generation.

Activated TAFI (TAFIa) has a short half-life in plasma because the enzyme is unstable. Genetic polymorphisms can result in synthesis of more stable forms of TAFIa. Persistent attenuation of fibrinolysis by these variant forms of TAFIa may render patients susceptible to thrombosis.

[Physiology][Hematology] General Concepts in Hemolytic Anemias

November 18, 2016 Hematology, Physiology and Pathophysiology No comments , , , , , , , , , , , , , , , , , , , , ,

Hemolysis is the accelerated destruction of red blood cells (RBCs), leading to decreased RBC survival. The bone marrow's response to hemolysis is increased erythropoiesis, reflected by reticulocytosis. If the rate of hemolysis is modest and the bone marrow is able to completely compensate for the decreased RBC life span, the hemoglobin concentration may be normal; this is called fully compensated hemolysis. If the bone marrow is unable to completely compensate for hemolysis, the anemia occurs. This is called incompletely compensated hemolysis.

PS (from wikipedia): The Reticulocyte production index (RPI) is a calculated value used in the diagnosis of anemia. This calculation is necessary because the raw reticulocyte count is misleading in anemic patients. The problem arises because the reticulocyte count is not really a count but rather a percentage: it reports the number of reticulocytes as a percentage of the number of red blood cells. In anemia, the patient's red blood cells are depleted, creating an erroneously elevated reticulocyte count.

Calculation of RPI

Step 1 – Reticulocyte Index is calculated using the formla on the left

Step 2 – The next step is to correct for the longer life span of prematurely released reticulocytes in the blood—a phenomenon of increased red blood cell production. This relies on a table:

Hematocrit (%) Retic survival (days) = maturation correction
36-45 1.0
26-35 1.5
16-25 2.0
15 and below 2.5

Step 3 – The Reticulocyte Production Index is calcualted using the formla below:

Hemolysis can be classified as extravascular or intravascular. Extravascular hemolysis, in which erythrocyte desstruction occurs by macrophages in the liver and spleen, is more common. Intravascular hemolysis refers to RBC destruction occurring primarily within blood vessels. The distinction between intravascular and extravascular hemolysis is not absolute because both occur simultaneously, at least to some degree, in the same patient, and the manifestations of both can overlap. The site of RBC destruction in different conditions can be conceptualized to occur in a spectrum between pure intravascular and pure extravascular hemolysis. Some hemolytic anemias are predominantly intravascular, and some are predominantly extravascular. Others have substantial components of both.

To understand better, the hemolytic anemias can be classified according to whether the cause of hemolysis is intrinsic or extrinsic to the RBC.Intrinsic causes of hemolysis include abnormalities in hemoglobin structure or function, the RBC membrane, or RBC metabolism (cytosolic enzymes). Extrinsic causes may be due to a RBC-directed antibody, a disordered vasculature, or the presence of infecting organisms or toxins. In general, intrinsic causes of hemolysis are inherited and extrinsic causes are acquired, but there are notable exceptions.

Hemolysis Due to Intrinsic Abnormalities of the RBC

screen-shot-2016-11-14-at-9-19-43-pmIntrinsic causes of hemolysis include abnormalities of hemoglobin structure or function, the RBC membrane, or RBC metabolism (cytosolic enzymes). Hemoglobin is the oxygen-carrying protein within RBCs. It is composed of four globular protein subunits, called globins, each with an oxygen-binding heme group. The two main types of globins are the alpha-globins and the beta-globins, which are made in essentially equivalent amount in precursors of RBCs. Normal adult hemoglobin (Hb A) has two alpha-globins and two beta-globins (alpha2beta2).

Abnormalities of Hemoglobin

Disorders of hemoglobin can be classified as qualitative or quantitative disorders. Qualitative abnormalities of hemoglobin arise from mutations that change the amino acid sequence of the globin, thereby producing structrual and functional changes in hemoglobin. There are four ways in which hemoglobin can be qualitatively abnormal: (i) decreased solubility, (ii) instability, (iii) altered oxygen affinity, (iv) altered maintenance of the oxidation state of the heme-coordinated iron. Hemolytic anemia (qualitative abnormalities) result from decreased solubility and instability of hemoglobin. Qualitative hemoglobin disorders often are referred to as hemoglobinopathies, even though the term technicially can apply to both qualitative and quantitative disorders.

Quantitative hemoglobin disorders result from the decreased and imbalanced production of generally structurally normal globins. For example, if beta-globin production is diminished by a mutation, there will be a relative excess of alpha-globins. Such imbalanced production of alpha- and beta-globins damages RBCs and their precursors in the bone marrow. These quantitative hemoglobin disorders are called thalassemias.

Abnormalities of the RBC Membrane

Some hemolytic diseases is characterized by abnormal shape and flexibility of RBCs because of a deficiency or dysfunction of one or more of the membrane proteins, which leads to shortened RBC survival (hemolysis).

The RBC membrane consists of a phospholipid-cholesterol lipid bilayer intercalated by integral membrane proteins such as band 3 (the anion transport channel) and the glycophorins. This relatively fluid layer is stabilized by attachment to a membrane skeleton. Spectrin is the major protein of the skeleton, accounting for approximately 75% of its mass. The skeleton is organized into a hexagonal lattice. The hexagon arms are formed by fiber-like spectrin tetramers, whereas the hexagon corners are composed of small oligomers of actin that, with the aid of other proteins (4.1 and adducin), connect the spectrin tetramers into a two-dimensional lattice. The membrane cytoskeleton and its fixation to the lipid-protein bilayer are the major determinants of the shape, strength, flexibility, and survival of RBCs. When any of these constituents are altered, RBC survival may be shortened.

Abnormalities of RBC Metabolism (cytosolic enzymes)

Normal metabolism of the mature RBC involves two principal pathways of glucose catabolism: the glycolytic pathway and the hexose-monophosphate shunt. The three major functions of the products of glucose catabolism in the erythrocyte are (i) maintenance of protein integrity, cellular deformability, and RBC shape; (ii) preservation of hemoglobin iron in the ferrous form; and (iii) modulation of the oxygen affinity of hemoglobin. These functions are served by the regulation of appropriate production of five specific molecules: ATP, reduced glutathione, reduced NADH, reduced NADPH, and 2,3-BPG. Maintenance of the biochemical and structural integrity of the RBC depends on the normal function of >20 enzymes involved in these pathways as well as the availability of five essential RBC substrates: glucose, glutathione, NAD, NAD phosphate (NADP), and adenosine diphosphate (ADP).

  • ATP

The primary function of the glycolytic pathway is the generation of ATP, which is necessary for the ATPase-linked sodium-potassium and calcium membrane pumps essential for cation homeostasis and the maintenance of erythrocyte deformability.

  • 2,3-BPG

The production of 2,3-BPG is regulated by the Rapoport-Luebering shunt, which is controlled by bisphosphoglyceromutase, the enzyme that converts 1,3-BPG to 2,3-BPG. Concentration of 2,3-BPG in the RBC in the RBC in turn regulates hemoglobin oxygen affinity, thus facilitating the transfer of oxygen from hemoglobin to tissue-binding sites.

  • Reduced gluthione

The major function of the hexose-monophosphate shunt is preservation and regeneration of reduced gluthione, which protects hemoglobin and other intracellular and membrane proteins from oxidant injury.

Abnormalities of the glycolytic pathway

Defects in the glycolytic pathway lead to a decrease in the production of ATP or a change in the concentration of 2,3-BPG.

Deficiencies of erythrocyte hexokinase, glucose phosphate isomerase, phosphofructokinase, and pyruvate kinase (PK) all lead to a decrease in ATP concentration. Although genetic disorders involving nearly all of the enzymes of the glycolytic pathway have been described, PK accounts for >80% of the clinically significant hemolytic anemias from defects in this pathway. With the exception of phosphoglycerate kinase deficiency, which is X-linked, all other glycolytic enzyme defects are autosomal recessive.

PK deficiency is the most common congenital nonspherocytic hemolytic anemia caused by a defect in glycolytic RBC metabolism. The syndrome is both genetically and clinically heterogeneous. Both glucose phosphate isomerase and hexokinase deficiencies produce nonspherocytic hemolytic anemia associated with decreased ATP and 2,3-BPG content.

Abnormalities of the hexose-monophosphate shunt

G6PD deficiency is the most frequently encountered abnormality of RBC metabolism, affecting >200 million people worldwide. The gene for G6PD is carried on the X chromosome and exhibits extensive polymorphism. Enzyme deficiency is observed in males carrying a variant gene. Hemolysis in G6PD-deficient RBCs is due to a failure to generate adequate NADPH, leading to insufficient levels of reduced glutathione. This renders erythrocytes susceptible to oxidation of hemoglobin by oxidant radicals, such as hydrogen peroxide. The resulting denatured hemoglobin aggregates and forms intraerythrocytic Heinz bodies, which bind to membrane cytoskeletal proteins. Membrane proteins are also subject to oxidation, leading to decreased cellular deformability. Cells containing Heinz bodies are entrapped or partially destroyed in the spleen, resulting in loss of cell membranes through pitting of Heinz bodies and leading to hemolysis.

Abnormalities of nucleotide metabolism

Pyrimidine-5'-nucleotidase deficiency is an enzymatic abnormality of pyrimidine metabolism associated with hemolytic anemia. The peripheral blood smear in patients with this defect often shows RBCs containing coarse basophilic stippling. Lead intoxication also inactivates the enzyme, leading to an acquired variant of pyrimidine-5'-nucleotidase deficiency.

Adenosine deaminase (ADA) excess is an unusual abnormality. It is caused by a genetically determined increase in the activity of a normal erythrocyte enzyme. The excessive deaminase activity prevents normal salvage of adenosine and causes subsequent depletion of ATP and hemolysis.