blood and lymph3 Flashcards
Immunomodulators.
immunomodulation is the use of drugs, alone or in combination with other maneuvers, to change the function of all, or part, of the immune system. hey are a diverse array of recombinant, synthetic and natural preparations, often cytokines. Some of these substances, such as granulocyte colony-stimulating factor (G-CSF), interferons, imiquimod and cellular membrane fractions from bacteria are already licensed for use in patients. Others including IL-2, IL-7, IL-12, various chemokines, synthetic cytosine phosphate-guanosine (CpG) oligodeoxynucleotides and glucans are currently being investigated extensively in clinical and preclinical studies. Immunomodulatory regimens offer an attractive approach as they often have fewer side effects than existing drugs, including less potential for creating resistance in microbial diseases.
Categories of immunomodulation drugs
Many of the drugs that are used to alter immune responses are also used in other conditions; this is most true of the older drugs. Some are true immunomodulators, and other drugs that don’t really affect the immune system but are commonly used in the treatment of immune diseases. These are some of the main categories: Non-steroidal anti-inflammatory drugs (NSAIDs), Disease-modifying antirheumatic drugs (DMARDs), glucocorticoids, biological response modifiers, Tumor-specific monoclonal antibodies, other antibodies, and miscellaneous drugs.
Biological response modifiers
These are a loose class of substances targeted mostly at cytokines or their receptors, or at cellular communication and signaling molecules. They can be antagonists or agonists. They can be genetically-engineered receptor antagonists. And they can be cloned, mass-produced normal gene products. Many of these agents are antibodies to various components of the immune or inflammatory system (which stimulate, inhibit, or opsonize, depending on the designer’s intentions), including monoclonal antibodies.
Monoclonal antibodies (mAb or moAb)
are monospecific antibodies that are made by identical immune cells that are all clones of a unique parent cell, in contrast to polyclonal antibodies which are made from several different immune cells. Monoclonal antibodies have monovalent affinity, in that they bind to the same epitope. Monoclonal antibodies (mAb) are a revolution in therapeutics; they can be manufactured under ideal conditions, and any quantity desired can be made, with complete uniformity of the product. The main problem is cost. Production costs are currently about $1,000/g (most small molecules cost drug companies $5/g to produce). The typical monoclonal antibody derives from the progeny of a single B cell, that has been fused with a multiple myeloma tumor cell; the resultant hybrid line can grow forever in culture like its tumor parent, but make the specific antibody of its B cell parent. They are truly monoclonal. Thousands are used in labs around the planet, and 33 are already drugs.
Monoclonal antibody production
Monoclonal antibodies are typically made by fusing myeloma cells with the spleen cells from a mouse that has been immunized with the desired antigen. This mixture of cells is then diluted and clones are grown from single parent cells on microtitre wells. The antibodies secreted by the different clones are then assayed for their ability to bind to the antigen (with a test such as ELISA or Antigen Microarray Assay) or immuno-dot blot. The most productive and stable clone is then selected for future use.
Chimeric antibody
Early on, a major problem for the therapeutic use of monoclonal antibodies in medicine was that initial methods used to produce them yielded mouse, not human antibodies. While structurally similar, differences between the two were sufficient to invoke an immune response when murine monoclonal antibodies were injected into humans, resulting in their rapid removal from the blood, as well as systemic inflammatory effects, and the production of human anti-mouse antibodies (HAMA). In an effort to overcome this obstacle, mouse DNA encoding the binding portion of a monoclonal antibody was merged with human antibody-producing DNA in living cells. The expression of this chimeric DNA through cell culture yielded partially mouse, partially human monoclonal antibodies. For this product, the descriptive terms “chimeric” and “humanised” monoclonal antibody have been used to reflect the combination of mouse and human DNA sources used in the recombinant process.
Human monoclonal antibodies
Transgenic mice technology is by far the most successful approach to making “fully” human monoclonal antibody therapeutics: 7 of the 9 “fully” human monoclonal antibody therapeutics on the market were derived in this manner.
Compare and contrast murine, chimeric, humanized, and human monoclonal antibodies. Discuss which might have disadvantages when used in human patients, and the reason for that.
The first monoclonals were made using B cells directly derived from immunized mice; such antibodies are murine [-omab] (e.g., ibritumomab). Some mAbs have been engineered at the DNA level to have the mouse VL and VH domains, but human C domains; these are chimeric, [-ximab] and less likely to be recognized by your patient’s own immune system. Going further, there are monoclonals which are humanized [-zumab]; only the CDR’s of the V domains are from the mouse. Finally, fully human [-umab] monoclonals are now becoming common.
NK (natural killer) cells
are large granular lymphocytes (LGL) which make up 5-10% of blood lymphocytic cells. They are killers with mechanisms available similar to those of CTL, but they do not have rearranged V(D)J genes and are not thymic-derived. They have a few NK receptors which recognize molecules on the surface of ‘stressed’ or dysregulated cells, such as virally- infected cells or many tumors, which they then kill; therefore, they are part of the innate immune system. They have a second cytotoxic trick available called antibody-dependent cell-mediated cytotoxicity, or ADCC. Not all tumor cells express the markers that NK cells recognize via NK receptors (tumors would gradually be selected to downregulate such markers). For example, with tumors, antibody against some specific protein on the tumor cells is added to them in culture; the antibody binds but has no observable effect. Normal blood leukocytes (which include LGL) are now added; the tumor cells are killed by induced apoptosis. If you hadn’t added both antibody and the LGL, nothing would have happened. Anyone’s leukocytes can be used; the phenomenon is not MHC-restricted the way CTL-mediated killing is.
How ADCC works
NK cells also have receptors for the Fc end of IgG (FcγR), and so they have a second, antibody-dependent, way to interact with target cells. The mechanism of ADCC is this: IgG antibody binds to the target cell, then the NK cell binds to the Fc end of the antibody. Just like a killer T cell, the NK cell is now triggered and delivers lethal signals to the target, which dies by apoptosis. We know that many of the new therapeutic monoclonal antibodies (used to modulate the immune response, or treat cancer) work by triggering ADCC. The normal role of ADCC has been hard to define; recently it has been reported that some HIV elite controllers have particularly strong early ADCC that destroys their HIV-infected cells.
Passive antibody therapy in Cancer
Antibody to tumor-associated antigens should be useful, and quite a few monoclonal antibodies (mAb) are already available. A few activate complement, and the tumor is lysed or phagocytosed; more often they invoke ADCC. Antibodies can also be tagged with a poison such as calicheamicin, or diphtheria toxin, or a radioisotope (such modified antibodies are called immunotoxins). They provide highly-targeted delivery of the toxic moiety. At least one mAb is available for use as both an imaging and a therapeutic drug, depending on which radioisotope is attached.
BiTE for Bispecific T-cell Engager
A group coupled together two single-chain engineered antibodies, one against CD19 and one against CD3 (remember, CD3 is the signaling component of the T cell receptor). This construct can bind T cells via their CD3 to CD19+ B cell lymphoma cells. [VL1-linker-VH1]-big linker-[VH2-linker-VL2]. Small doses given to lymphoma patients resulted in some cases in complete clearance of the tumor cells. It’s for use in Philadelphia-chromosome negative acute lymphoblastic leukemia (ALL). The drug is called blinatumomab [Blincyto, Amgen.]
Chimeric antigen receptor, CAR
In trials now are several systems of amazing complexity and daring, in which T cells are removed from a cancer patient and transformed using lentivirus vectors with a chimeric antigen receptor, CAR. The constructs usually involve the CDRs of a high-affinity antibody (some use natural or engineered T-cell receptor CDRs) linked to a transmembrane region and a T-cell intracellular signaling molecule, one of the components of CD3. This allows a transformed CTL to bind a tumor target with high affinity and chosen specificity, and, like an antibody, no MHC- restriction, and then be triggered via its normal TCR-associated pathway to become a fully- cytotoxic cell. Some of the preliminary reports are startling. Cost will be a huge factor in determining the future of this approach.
Solid organ transplants
Remember that the better the match the better the result. This is less true nowadays, because surgeons rely on highly effective immunosuppressive regimes. The purpose of treatment is to prevent organ rejection. The drug regimens that are available are very effective at doing this; organ failure on a purely immunological basis is rare. Problems arise from three currently unavoidable consequences of using these drugs. They are all inherently toxic, some severely so; they can increase risk of cancer; and they commonly increase risk of opportunistic infections. So you may one day be faced with the very difficult decision: Is this patient suffering from infection, or organ failure, or a transplant rejection crisis? Or all three?
Azathioprine (related to 6-mecaptopurine)
Major Drugs Used in Organ Transplantation. This agent decreases DNA synthesis and mRNA transcription. It is gradually being replaced by Mycophenolate mofetil
Mycophenolate mofetil
Major Drugs Used in Organ Transplantation. This drug is less toxic and has the same mode of action as azathioprine.
Glucocorticoids
Major Drugs Used in Organ Transplantation. Essential anti-inflammatories in transplantation. Usually start with very high dose, taper as soon as possible; discontinue if possible. High doses can also be used briefly for threatened rejection episodes.
Cyclosporine-A
Major Drugs Used in Organ Transplantation. Its primary function is to decrease IL-2 production. Thus it is synergistic with glucocorticoids, which, by down-regulating macrophage function as APCs, lessen stimulation of T cells.
Tacrolimus
Major Drugs Used in Organ Transplantation. Can synergize with cyclosporine-A. The combination decreases both synthesis and response to IL-2.
Sirolimus
Major Drugs Used in Organ Transplantation. (rapamycin), a new relative of cyclosporine. It binds FKBP-12 as does tacrolimus, but the complex has no effect on calcineurin; instead, it inhibits a kinase called Target of Rapamycin (mTOR) which is needed for T cell activation. Approved in kidney transplantation.
Anti-thymocyte globulin (ATGAM)
Major Drugs Used in Organ Transplantation. Made in horses, and now rabbits, immunized with human thymocytes. Useful as a part of the regimen to prepare recipients for bone marrow transplantation and in acute organ rejection.
mAbs are available to CD3 and the IL-2 receptor.
Major Drugs Used in Organ Transplantation. Both can be gotten humanized, though the most common one, Muromomab, is simply a mouse monoclonal against CD3, the same as the well-known diagnostic monoclonal OKT3. They are replacing anti-thymocyte globulin but are expensive. Too much anti-CD3 can destroy so many T cells at once that the patient undergoes a “cytokine storm,” something like the flu but 1000 times worse.
Hemostasis
The term “hemostasis” refers to the ability of the body to stop bleeding from a damaged blood vessel when it occurs and to eventually repair the defect in the vessel wall so that normal blood flow to and from the involved area can be maintained. There are several aspects occurring at the same time including the coagulation cascade, anticoagulation regulatory pathways, fibrinolytic system (breaks down formed clots), endothelial cell lining of blood vessels that work to prevent clots in the resting state and promotes clot formation following injury, and platelets.
Thromboplastin
is a plasma protein aiding blood coagulation through catalyzing the conversion of prothrombin to thrombin. It is a complex enzyme that is found in brain, lung, and other tissues and especially in blood platelets and that functions in the conversion of prothrombin to thrombin in the clotting of blood—called also thrombokinase. Although sometimes used as a synonym for the protein tissue factor (with its official name “Coagulation factor III [thromboplastin, tissue factor]”), this is a misconception. Historically, thromboplastin was a lab reagent, usually derived from placental sources, used to assay prothrombin times (PT time). Thromboplastin, by itself, could activate the extrinsic coagulation pathway. When manipulated in the laboratory, a derivative could be created called partial thromboplastin. Partial thromboplastin was used to measure the intrinsic pathway. This test is called the aPTT, or activated partial thromboplastin time. It was not until much later that the subcomponents of thromboplastin and partial thromboplastin were identified. Thromboplastin is the combination of both phospholipids and tissue factor, both needed in the activation of the extrinsic pathway. However, partial thromboplastin is just phospholipids, and not tissue factor.
Identify which coagulation factors are serine proteases
Serine proteases in the coagulation pathways factor II, factor VII, factor IX, factor X, Factor XI, Factor XII, and prekallikrein.
Cofactors in the coagulation pathways
factor III, factor V, factor VIII, and high molecular weight kininogen.
Serine proteases
almost all of the enzymes in the coagulation cascade are serine proteases, related to trypsin and/or chymotrypsin, which function by cleaving their targets at arginyl residues. The inactive precursor proteins that are activated through cleavage into active enzymes are called zymogens. Zymogens that become active serine proteases in the cascade include factor XII, Prekallikrein, factor XI, factor IX, factor X, factor VII, and factor II (prothrombin).
Classic blood coagulation pathways
there are two main pathways leading to the activation of factor X, the extrinsic pathway and the intrinsic pathway. The extrinsic pathway, so called because it requires a factor extrinsic to the plasma (tissue factor) to function, is initiated by tissue factor binding to factor VIIa, leading to activation of factor X. Xa, with its cofactor Va, activates factor II to IIa, which then converts fibrinogen to fibrin. The separate intrinsic pathway, so called because all of the necessary components are contained in plasma, begins with activation of the contact factors (factor XII, prekallikrein, HMWK), that leads to activation of factor XI, followed by activation of factor IX which, with its cofactor VIIIa, then activates factor X, which, with its cofactor Va, activates factor II, which converts fibrinogen to fibrin. Both pathways converge at the level of activation of factor X, so the events occurring following activation of factor X are sometimes referred to as the common pathway. While the modern conception of coagulation has changed somewhat from this model, it is still useful to keep in mind when interpreting the PT and aPTT coagulation screening tests, which test the function of the extrinsic and intrinsic pathways, respectively. Two-step process of coagulation: Prothrombin + Thromboplastin + Calcium = Thrombin. Fibrinogen + Thrombin = Fibrin
Synthesis of coagulation factors
Nearly all of the proteins in the coagulation cascade are synthesized primarily in the liver. Thus, one of the major problems observed in patients with severe liver disease is a bleeding diathesis (bleeding tendency or predisposition). Two exceptions are tissue factor, expressed on the surface of many cell types, and VWF, produced in endothelial cells and megakaryocytes. Also, factor VIII, while produced by the liver, can also be produced in other organs such as the spleen, lung, and kidney, explaining why factor VIII levels can be normal or even increased in patients with liver failure and coagulopathy. Biliary obstruction can lead to vitamin K deficiencies due to malabsorption (vit K is fat soluble)
The half-lives (t1/2) of the various factors
factor VII has the shortest plasma t1/2 of 4-6 hours, making it one of the first factors to be depleted in some disease states. Once activated, the factors generally have a much shorter t1/2 and are rapidly inactivated, sometimes within a couple of minutes, as part of a tightly regulated control mechanism.
The role of vitamin K in coagulation
A special subset of this group of proteases are the vitamin-K dependent factors, including factors II, VII, IX, and X, along with the anticoagulant protein C. Protein S is also vitamin K dependent, though it functions in coagulation as a cofactor for protein C and not as a serine protease. These proteins share significant homology and are likely derived evolutionarily from a common ancestor. A special property that they all share is that they contain a “Gla” domain. This domain contains several (9 to 13) glutamic acid residues that undergo post-translational modification to γ- carboxy glutamic acid residues (“Gla” residues). These Gla residues are able to bind calcium, which leads to shape change of the protein, allowing binding to an anionic phospholipid surface, which is necessary for normal protein function. This γ-carboxylation is carried out by the enzyme γ- glutamyl carboxylase in the liver. Vitamin K is required for generation of the precursor for the reaction, so vitamin K deficiency leads to inability to make the Gla residues, resulting in non-functional protein and making vitamin K deficiency an important cause of bleeding to consider.
Hemorrhagic disease of the newborn
due to vitamin K deficiency. Some reasons for this are decreased stores, low levels in breast milk, and developing gut flora.
Factor XIII
An enzyme in the coagulation cascade that is not a serine protease. One other enzyme involved in the coagulation cascade is factor XIII, which covalently links fibrin molecules together to form a stable clot. Factor XIII is a transglutaminase that can form cross-linked amide bonds between specific lysine and glutamine residues.
Cofactors in the coagulation cascade
In addition to the enzymes in the cascade, there are several cofactors that are essential for initiating or accelerating enzymatic reactions but lack intrinsic enzyme activity themselves. The cofactors are thought to work by bringing the components together and orienting them properly to make the reaction more efficient. Cofactors in the coagulation cascade include tissue factor, factor VIII, factor V, and high molecular weight kininogen (HMWK).
Tissue factor
is not ordinarily expressed on cells in direct contact with the blood (endothelial cells, leukocytes). It is, however, expressed on the fibroblasts and smooth muscle cells surrounding blood vessels. With blood vessel injury, factor VIIa from the plasma will become exposed to tissue factor, leading to activation of the extrinsic pathway. Tissue factor can also become expressed on endothelial cells and monocytic blood cells under conditions of stress or injury, or when stimulated by LPS or other proinflammatory agents.
Factors VIII and V
are both non-enzymatic procofactors with significant sequence homology to each other. Both are cleaved by thrombin to their active form, allowing them to participate in the tenase and prothrombinase complexes, respectively.
High molecular weight kininogen (HMWK)
participates as a cofactor with the contact factors, factor XIIa, kallikrein, and factor XI.
Fibrinogen
is the soluble plasma protein that is cleaved by thrombin into its insoluble form, fibrin, which then participates in forming the actual blood clot. Fibrinogen is made up of 3 pairs of polypeptide chains (2 Aα-chains, 2 Bβ-chains, and 2 γ-chains) arranged into identical half molecules in an elongated polypeptide composed of three globules: a central globule containing the N-terminal domain of all of the polypeptides, and globules on each end of the molecule. With activation, thrombin cleaves off two small peptides, fibrinopeptide A and fibrinopeptide B. The release of fibrinopeptide A leads to exposure of a site on the middle domain that aligns non-covalently with a complementary site in the side domain of another fibrin molecule to form overlapping fibrils. The subsequent cleavage and release of fibrinopeptide B allows increasing aggregation of the growing fibrils. Factor XIIIa then covalently cross-links adjacent side domains, stabilizing and strengthening the developing clot.
Von Willebrand factor (VWF)
plays a critical role in platelet adhesion and aggregation and also plays an important role in the coagulation cascade by serving as the carrier protein for factor VIII in the plasma. VWF is a large, multimeric protein that is produced and stored in what are known as Weibel-Palade bodies in endothelial cells and in α-granules of platelets. In the circulating plasma, it binds to and protects factor VIII, significantly prolonging its half-life (12 hrs vs 2 hrs). Patients with severe deficiency of VWF have low levels of circulating factor VIII, leading to a bleeding disorder similar to classical hemophilia A (factor VIII deficiency). They have a coexistent platelet function defect, leading to a very severe bleeding problem.
protease + cofactor + phospholipid surface + calcium
A common theme in the coagulation cascade is a situation where an activated enzyme will combine with a cofactor, on a negatively charged phospholipid surface and in the presence of calcium, forming an enzyme complex that accelerates the speed of the reaction several hundred- to several thousand-fold. These complexes are often referred to as “_____ase.” Examples of these complexes in the coagulation system include: extrinsic tenase, intrinsic tenase, and prothrombinase complex.
Extrinsic tenase (Xase )T
issue factor (a cofactor) combines with factor VIIa (a serine protease) on a phospholipid surface in the presence of calcium to bind to and activate either factor IX or factor X. This is called “extrinsic tenase” (or Xase), since it is part of the extrinsic pathway.
Intrinsic tenase
Alternatively, two members of the intrinsic pathway, Factor IXa (a serine protease) and factor VIIIa (a cofactor), can combine with phospholipid and calcium to bind and activate factor X. This complex is called intrinsic tenase.
Prothrombinase complex
Activated factor X (Xa), a serine protease, can then combine with factor Va (a cofactor), on a phospholipid surface in the presence of calcium, to bind to and activate prothrombin (factor II) to thrombin (factor IIa). This is known as the prothrombinase complex.
Our current understanding of coagulation
While it is useful to learn the classic coagulation cascade, our conception of how coagulation actually works has evolved. The current concept of coagulation stresses the physiologic importance of the extrinsic pathway in the initiation of coagulation. Also, instead of the intrinsic and extrinsic pathways working independently and converging at the level of factor X, there is crosstalk between them. And, the role of the contact factors is de-emphasized. A current model of coagulation divides it into three phases: an initiation phase, an amplification phase, and a propagation phase.
Initiation phase
Coagulation is initiated with vascular disruption that leads to exposure of plasma to tissue factor (TF). In the plasma, a small amount (~1 %) of circulating factor VII can normally be found in its activated (VIIa) form. Free factor VIIa that is not bound to TF is inactive, which is why the small amount of circulating VIIa doesn’t initiate clotting. With exposure of tissue factor on the cell surface, it can bind factor VIIa in the presence of calcium, and the TF-VIIa (extrinsic tenase) complex can then bind factor X, leading to production of minute amounts of factor Xa. TF-VIIa also converts factor IX to factor IXa. The TF-VIIa complex can also generate more VIIa to amplify the process even more. This process, termed the initiation phase of coagulation, produces sufficient quantities of Xa to start the process of clot generation. Factor V can be slowly activated by factor Xa. Factor Xa then binds to factor Va and forms the prothrombinase complex, which generates small amounts of thrombin.
Amplification phase
The amplification phase takes place on the surface of platelets which have adhered to the exposed subendothelium and been activated. In this phase, the initial procoagulant signal is amplified by small amounts of thrombin generated on TF-bearing cells. Thrombin activates Factors V and VIII to Va and VIIIa, respectively. Upon platelet activation, factors Va and VIIIa bind to the platelet surface. Xa can then form the prothrombinase complex with Va to generate small amounts of thrombin from prothrombin.
Propagation phase
The propagation phase of coagulation begins with assembly of procoagulant complexes on the cell surface. The intrinsic tenase complex (factor VIIIa and factor IXa) activates more factor X on the platelet surface. The rate of activation of factor X by the intrinsic tenase complex is about 50-100 times faster than the extrinsic tenase complex, making formation of the intrinsic tenase complex critical for producing a sufficient quantity of Xa to initiate coagulation. Factor Xa then rapidly binds to factor Va (generated by thrombin in the amplification phase), forming the prothrombinase complex, which initiates conversion of prothrombin to thrombin, producing the thrombin burst necessary for fibrin-clot formation. Thrombin cleaves fibrinogen to form fibrin, leading to formation of the fibrin clot, and activates factor XIII to XIIIa, which covalently cross-links and stabilizes the forming clot. This fibrin latticework develops between and around the activated platelets, which have adhered and aggregated at the site of injury. The activated platelets provide a surface for the coagulation reactions to continue to occur, generating even more thrombin. Platelets also secrete the contents of their granules, which contain many of the components of coagulation to provide more substrate for the reactions to take place. Simultaneously, anticoagulation and fibrinolytic reactions are occurring, which will rapidly quench this process and prevent its spread beyond the site of injury.
The Central role of thrombin in coagulation
At this point, it is important to emphasize the central role that thrombin plays in blood coagulation. Not only does it cleave fibrinogen to fibrin to form the actual clot. It also cleaves and activates the procofactors V and VIII to Va and VIIIa, which can then participate in the prothrombinase and intrinsic tenase complexes, respectively. It also activates factor XI to XIa, which can then go on to generate more factor IXa to participate in the intrinsic tenase complex. This all has the end result of amplifying the coagulation response and producing a burst of thrombin sufficient to form the clot. It also cleaves and activates factor XIII, which covalently links the fibrin to form a more stable clot. In addition to its role in the coagulation cascade, thrombin is the most potent known activator of platelets. One of the results of platelet activation is the translocation of the anionic phospholipid phosphatidylserine from the inner leaflet of the platelet cell membrane to the outer leaflet. Exposure provides a surface for the coagulation reactions to take place, greatly increasing their efficiency. These multiple roles of thrombin emphasize its central importance to all aspects of coagulation.
The role of the contact factors in coagulation
One could question the real role of contact factors (factor XII, prekallikrein, HMWK, and, to a lesser extent, factor XI) in this whole process. Deficiencies of factor XII, prekallikrein and HMWK exist and are not associated with a bleeding tendency. Patients with factor XI deficiency can have a mild bleeding disorder, likely related to its role in the amplification phase of coagulation through activation of factor IX. One role of factor XII may be clot stabilization, since patients with factor XII deficiency may be more prone to development of thrombotic emboli. Factor XII may also contribute to regulation of fibrinolysis and may play a role in activation of coagulation during extracorporeal membrane oxygenation (i.e., cardiac bypass), which involves exposure of the blood to foreign surfaces. HMWK is a source for bradykinin, a vasoactive peptide with multiple physiologic effects. In the end, the contact factors may play their most important role in linking coagulation and inflammation, the details of which are still being worked out. Their most important feature is that their (very rare) deficiency will lead to abnormal prolongation of the aPTT, placing them in the differential for a prolonged aPTT.
Describe how antithrombin functions as a regulator of coagulation and explain how heparin affects its function.
Fibrinolysis mechanisms to break down a clot. Both this and repair mechanisms are activated when clotting is initiated. The endothelial cells lining the blood vessels, while helping to promote clotting with damage or injury, also play a key role in minimizing risk of intravascular clotting in the resting state. And, of course, there are mechanisms to regulate and keep in check these systems, leading to a complex system of checks and balances. mechanisms to break down a clot. Both this and repair mechanisms are activated when clotting is initiated. The endothelial cells lining the blood vessels, while helping to promote clotting with damage or injury, also play a key role in minimizing risk of intravascular clotting in the resting state. And, of course, there are mechanisms to regulate and keep in check these systems, leading to a complex system of checks and balances.
Serine protease inhibitors (serpins)
a large and diverse group of proteins which share the feature of being able to bind to chymotrypsin-like serine proteases at their active-site serine, generally for purposes of protease inactivation. They can also perform other functions as well, such as storage or transport of the target protease. A large number of the proteins in the coagulation cascade are serine proteases and require the presence of a serine residue in their catalytic domain. Serpins have a domain called the variable reactive site loop, which is able to bind with specificity to the catalytic groove of their target serine protease. With this interaction, and sometimes the participation of a cofactor, structural changes lead to the exposure of the reactive site loop to the serine residue of the protease, which then forms a covalent bond between the two and significant structural changes in both proteins. This causes complete inactivation of both (sometimes serpins are called suicide protease inhibitors).
Antithrombin
(formerly known as antithrombin-III) is a serpin that plays a critical role in anticoagulation. It targets multiple serine proteases in the coagulation cascade, probably most importantly thrombin and factor Xa, though it can also bind to and inactivate factors IXa, the VIIa-tissue factor complex, factor XIa, factor XIIa, and kallikrein. It inactivates its target by binding of an arginyl residue in its reactive site loop to the serine in the catalytic site of the protease, leading to 1:1 complex formation of serpin and protease, both of which are inactivated and cleared. Antithrombin deficiency is a well-known risk for venous thromboembolism.
Heparin
acts as a key cofactor for antithrombin, accelerating its rate of protease inactivation by several hundred to several thousand-fold. Heparin is a polysaccharide that is a highly sulfated glycosaminoglycan. It works as a cofactor by two mechanisms: 1. A specific pentasaccharide sequence contained within the heparin polymer induces an allosteric conformational change in antithrombin that permits more efficient binding to and inhibition of the target protease. This shortened version of heparin is able to accelerate inactivation of factor Xa but not of thrombin. 2. A second mechanism in which heparin acts as a cofactor for antithrombin requires a longer form of the heparin molecule that is able to bind not only antithrombin but also its serine protease target, bringing the two molecules into close proximity to allow inactivation of the protease to occur. The longer form of heparin is required for acceleration of thrombin inactivation by antithrombin.
Heparin as a drug
Unfractionated heparin and its derivatives (low-molecular weight heparin, fondaparinux) are important drugs used therapeutically to treat people at an increased risk of clotting. Heparin is derived from the mucosal tissues of pigs and cattle.
Heparin sulfate
a closely related polysaccharide to heparin, is expressed on the surface of multiple cell types, including endothelial cells, and is likely the cofactor for antithrombin under normal conditions.
Heparin cofactor II
another in the serpin family. The only serine protease heparin cofactor II inhibits is thrombin. It is a cofactor for heparin and dermatan sulfate (“minor antithrombin”)., which is synthesized primarily by fibroblasts and vascular smooth muscle cells. It also may play a role as an anticoagulant during pregnancy, when elevated levels of both heparin cofactor II and dermatan sulfate are seen.
C1 esterase inhibitor
another serpin that plays a small role in anticoagulation. It is primarily an important regulator of the classic complement pathway. Also regulates the contact factors of the intrinsic pathway (kallikrein, factor XIIa, and factor XIa).
Protein C inhibitor
a non-specific protease inhibitor, it is the most physiologically important inhibitor of activated protein C.
Protein C
is a vitamin K-dependent serine protease. It circulates as a zymogen. It circulates as a zymogen. When thrombin is generated from prothrombin, in addition to its role as a procoagulant, it binds to thrombomodulin. Once bound to thrombomodulin, thrombin’s procoagulant activity is neutralized, but the thrombin- thrombomodulin complex on the cell surface can bind to and activate protein C.
Thrombomodulin
a transmembrane protein constitutively expressed on endothelial cells and serves as a cofactor for thrombin. It reduces blood coagulation by converting thrombin to an anticoagulant enzyme from a procoagulant enzyme, accelerating the reaction about 1000-fold. It is analogous to the tenase and prothrombinase complexes that are part of the coagulation cascade and is sometimes referred to as the ‘protein Case” complex. Thrombomodulin expression can be down-regulated with exposure of the endothelial cells to proinflammatory agents, likely contributing to the hypercoagulability that can be seen with inflammation.
Activated protein C (APC)
protein C is activated when cleaved by thrombin. Once generated, APC goes on to cleave and inactivate the cofactors Va and VIIIa, leading to decreased generation of thrombin. This reaction is enhanced by interaction of protein C with its cofactor, protein S, on an anionic phospholipid surface (i.e., the activated platelet surface). Interaction of protein S with APC alters the structure of APC alters the structure of APC and moves the APC active site closer to the membrane surface.
Protein S
protein C’s cofactor. It is the only vitamin K-dependent factor that is not a serine protease. Only about 40% of protein S circulates in the blood in the free form, whereas the remaining 60% circulates bound to C4b-binding protein (C4bBP), a regulator of the complement pathway. This becomes important when measuring protein S levels in plasma, since protein S bound to C4bBP is inactive. Protein S probably has other roles in inhibiting coagulation outside of its role as a cofactor for protein C. Though its precise function still isn’t fully clear, it undoubtedly plays an important role as an anticoagulant as evidenced by the pro- thrombotic risk associated with deficiency.
Neonatal purpura fulminans
protein C deficiency, which leads to increased thrombotic risk, particularly severe in the case of homozygous deficiency and can result in fatal neonatal thrombotic events.
Half life of protein C
it has a short half life of only 8-10 hours. Warfarin is a commonly used oral anticoagulant that functions by blocking the action of vitamin K on post-translational modification of vitamin K-dependent factors. Because of protein C’s short half-life, at onset of treatment with Coumadin it will be one of the first vitamin K-dependent coagulation factor to be depleted, leading to a transient hypercoagulable state. This becomes an important consideration when starting anticoagulation therapy with Coumadin.
Facot V leiden
another inherited pro-thrombotic condition related to protein C. A mutation in one of protein C’s target, factor V. It is caused by a point mutation that changes amino acid 506 of the protein from glutamine to arginine, which results in factor Va resistant to the proteolytic activation of activated protein C, so that factor Va remains activated longer than it should. Also known as APC resistance, since factor Va is resistant to inactivation by activated protein C. 3-8% of the Caucasian population is heterozygous for the factor V Leiden defect, making it one of the most common inherited risk factors for venous thromboembolism.
Tissue factor pathway inhibitor (TFPI)
expressed constitutively by endothelial cells, is an important inhibitor of the extrinsic arm of the coagulation pathway and thus the initiation phase of thrombin generation. As factor Xa is generated by the extrinsic Xase complex (factor VIIa-tissue factor), TFPI can bind to Xa’s active site. Once surface bound, the factor Xa-TFPI complex rapidly binds and inactivates tissue factor-factor VIIa, forming a stable quaternary complex of tissue factor, factor VIIa, TFPI, and factor Xa. Factor IXa-TFPI can also bind to and inhibit factor VIIa-tissue factor, though TFPI binds to factor IXa with significantly less affinity, making this less likely to be of physiologic relevance. Ninety percent of circulating TFPI is found in association with lipoproteins, and TFPI has been implicated, in addition to its role in coagulation, to play a role in protection from atherosclerosis. Recombinant TFPI is currently being studied to see if may play a therapeutic role in the treatment of hypercoagulable states. The combination of TFPI inhibition of the initiation phase of thrombin generation and protein C pathway inhibition of the amplification phase of thrombin generation leads to a situation where, under physiologic conditions, a sufficient signal needs to be generated to reach the threshold necessary for thrombin generation to proceed to the propagation phase, serving as a mechanism to protect the body from excessive clot formation.
α2-macroglobulin
a nonspecific proteinase inhibitor that inhibits a broad range of proteinases, including thrombin, kallikrein, and plasmin, thus affecting both the coagulation and fibrinolytic pathways. It has an interesting mechanism for inactivating proteases through presentation of a “bait region” for the protease. With proteolysis of this bait region, the α2-macroglobulin molecule undergoes a rapid conformational change that traps the proteinase inside the molecule. The complex is then rapidly cleared through a receptor-mediated process.
The Fibrinolytic System
Fibrinolysis is the process of clot breakdown that occurs following clot formation, allowing eventual repair of the damaged blood vessel following injury. It begins as soon as the clot begins forming. The key enzyme is plasmin,
Plasmin
a serine protease which is cleaved from its zymogen precursor, plasminogen, to its active form. Plasmin can cleave both fibrinogen (fibrinogenolysis) and fibrin (fibrinolysis) to form innumerable different types and sizes of fragments, which can be detected in the blood (collectively called fibrin degradation products or FDP). Plasmin can also break down extracellular matrix proteins, aiding in the remodeling process involved with repair of the damaged vessel. Plasminogen is synthesized in the liver and circulates in the plasma as well as being present in a wide variety of extravascular tissues and body fluids.
Tissue plasminogen activator (t-PA)
The primary activator of plasminogen in vivo. t-PA is a serine protease produced predominantly in endothelial cells. Its secretion from the endothelium is regulated by numerous important mediators of blood clotting and inflammation, such as thrombin, histamine, bradykinin, epinephrine, serotonin, and interleukins. Once released, t-PA has a very short half-life of about 2.5 minutes. It is rapidly cleared in a receptor-mediated fashion by the liver and by endothelial cells, as well as inactivated by various inhibitors, the most important being plasminogen activator inhibitor-1 (PAI-1). t-PA is a poor activator of plasmin in the absence of fibrin but efficiently activates plasminogen to plasmin upon binding to fibrin, with catalytic efficiency enhanced several hundredfold. t-PA itself can be cleaved by plasmin or other enzymes into a more active form. As a clot forms, t-PA and plasminogen bind to the fibrin being generated, localizing plasmin generation to the site of the clot. Initial plasmin degradation of fibrin increases the number of plasminogen-binding sites in the clot, further amplifying plasmin generation. Recombinant t-PA is extensively used today therapeutically for clot lysis.
Urokinase plasminogen activator (u-PA)
The other main activator of plasminogen. It is synthesized by kidney cells as well as endothelial cells. It also can be secreted by tumors and is thought to contribute to metastasis by breaking down extracellular matrix to facilitate tumor invasion. It is a serine protease that is synthesized and released as prourokinase or single-chain u-PA (scu-PA). Similar to t-PA, it has a very short half-life of about 5 minutes. The prourokinase becomes bound to the clot and is then cleaved, primarily by already generated plasmin, into its active form, which can then contribute to further clot lysis. A small amount of plasmin can also be generated by cleavage of plasminogen to plasmin by the contact factors of the intrinsic pathway (factor XIIa, kallikrein, factor XIa).
Inhibitors of fibrinolysis
Several mechanism exist to inhibit fibrinolysis through inhibition of plasmin itself or through inhibition of the conversion of plasminogen to plasmin. Some mechanisms include thrombin- activatable fibrinolysis inhibitor (TAFI), plasminogen activator inhibitors 1 and 2, and α2-antiplasmin.
Thrombin-activatable fibrinolysis inhibitor (TAFI)
is a zymogen that is synthesized in the liver and circulates in the blood in complex with plasminogen. Factor XIIIa can covalently attach it to fibrin. Like protein C, it is cleaved to its active form through binding to the thrombin-thrombomodulin complex. Once activated, it is an exopeptidase that removes basic amino acids (arginine, lysine) from the C- terminal of proteins. It targets the C-terminal of fibrin molecules and FDPs. Removal of these amino acids reduces the number of plasminogen-binding sites on fibrin, which decreases the amount of plasminogen available to t-PA or u-PA to cleave to its active form, thus down-regulating plasmin generation and slowing clot lysis.
plasminogen activator inhibitor-1 (PAI-1)
The primary physiologic inhibitor of plasminogen activation, targeting t-PA and u-PA. PAI-1 is produced in multiple different cell types and has a half-life of less than 10 minutes in the blood. A major fraction of PAI-1 in blood is present in the α-granules of platelets. PAI-1 is a serpin that binds to and inactivates t-PA. Circulating levels of PAI-1 are in several-fold excess to levels of t-PA. Thus, any t-PA circulating in the blood is rapidly bound to and inactivated by PAI-1. For u-PA, PAI-1 only binds to the activated form. Deficiency of PAI-1 leads to excessive bleeding, and higher levels of PAI-1 lead to slower clot breakdown and a more thrombotic state.
PAI-2
another member of the serpin family, initially identified in human placenta. High levels are observed in the blood of pregnant women. Its role in clot lysis is not clear.
α2-Antiplasmin
the primary plasmin inhibitor in blood. It is another member of the serpin family. It acts by binding to and inactivating plasmin in a 1:1 fashion. α2-Antiplasmin rapidly inhibits plasmin free in the circulation, preventing systemic fibrinogen degradation. By contrast, plasmin bound to fibrin is somewhat protected from inactivation by circulating α2-antiplasmin, leading to localization of fibrinolysis to the site of the clot. As with TAFI, factor XIIIa can covalently link α2-antiplasmin to fibrin, leading to stabilization of the fibrin scaffold in the developing clot. Individuals with deficiency show a bleeding disorder, indicating its important physiologic role in preventing fibrinolysis.
The Endothelial Cell Lining and prevention of clot formation
Under resting conditions, the endothelial cell lining employs several mechanisms to prevent thrombosis from occurring. Endothelial cells have mechanism including anticoagulation, fibrinolytic, and antiplatelet.
Anticoagulation mechanisms of endothelial cells
include: 1. Expression of heparan sulfate and dermatan sulfate, which act as cofactors for antithrombin and heparin cofactor II, respectively. 2. Expression of thrombomodulin, which acts as a cofactor with thrombin for activation of protein C. 3. Expression of TFPI to inhibit the extrinsic Xase complex.
Fibrinolytic mechanisms of endothelial cells
include synthesis and release of t-PA and u-PA.
Antiplatelet mechanisms of endothelial cells
include: 1. Synthesis and secretion of prostacyclin (PGI2) and nitric oxide (NO), which prevent adhesion of activated platelets and cause vasodilation. 2. Expression of an enzyme that rapidly metabolizes ADP (a potent platelet agonist) to AMP and adenosine (a potent inhibitor of platelet function).
Endothelial cells promotion of clot formation
With damage or injury to the endothelial cell line through a host of mechanisms, several changes occur that promote clot formation, including: 1. Exposure and/or expression of tissue factor to initiate the extrinsic pathway. 2. Exposure of the subendothelium, leading to binding and activation of platelets, which then release substances such as ADP and thromboxane A2 which cause vasoconstriction, release procoagulant factors from their α-granules, and provide an anionic phospholipid surface for the coagulation reactions to take place. 3. Release of von Willebrand factor from the Weibel-Palade bodies of the endothelial cells.
Primary hemostasis
With exposure of the subendothelium, von Willebrand factor (VWF) binds to the subendothelial matrix. Circulating platelets adhere to the subendothelium through platelet integrin receptor interactions with VWF, collagen, and other subendothelial components. Binding leads to platelet shape change and activation as well as platelet aggregation. This leads to release of platelet granules containing many of the coagulation proteins as well as platelet agonists and vasoactive substances that cause vasoconstriction to help stem blood loss. In addition, phosphatidylserine, normally kept on inner leaflet of the cell membrane, is translocated to the outer leaflet, providing a surface for the coagulation reactions to take place. This part of the hemostatic process, involving platelet adhesion, aggregation, and activation, is sometimes referred to as “primary hemostasis.”
secondary hemostasis
At the same time, tissue factor, either constitutively expressed on cells in the extravascular space or exposed on damaged endothelial cells in the area of the injury, becomes exposed to the small amount of factor VIIa circulating in the plasma, leading to initiation of the extrinsic coagulation pathway. This leads to activation of factors IX and X. A prothrombinase complex made up of factor Xa and factor Va, on the anionic phospholipid surface of the platelet and in the presence of calcium, is able to bind and activate prothrombin to thrombin. The generated thrombin is able to activate multiple factors, including factor XI, factor VIII, and factor V, leading to amplification of the coagulation process. This then leads to the propagation phase of coagulation, with generation of a burst of thrombin. Thrombin cleaves fibrinogen to fibrin to form a fibrin network around the activated platelets in the area of injury. Thrombin also cleaves and activates factor XIII, which covalently cross-links the fibrin, stabilizing the clot. This process, involving activation of the coagulation cascade and formation of the fibrin clot, is sometimes referred to as “secondary hemostasis.”
The functions of thrombin
Thrombin simultaneously carries out several other functions. It is a potent activator of platelets, supporting development of the platelet plug and generating more phospholipid surface for coagulation to take place. It also initiates the anticoagulation process through activation of protein C, which goes on to inactivate factors V and VIII, and contributes to regulation of the fibrinolytic process through activation of TAFI. Thrombin itself, along with several of the other serine proteases that participate in coagulation, is rapidly inactivated and cleared by antithrombin, a reaction which is greatly accelerated by heparin or heparin-like molecules expressed on endothelial cells. At the same time, the extrinsic pathway is inhibited by TFPI.
t-PA and u-PA activation and inactivation
t-PA is being released by the injured endothelial lining. t-PA (primarily), as well as u-PA, binds to the forming fibrin network and cleaves plasminogen to plasmin. Plasmin begins the process of breaking down the forming clot (fibrinolysis). Uncontrolled fibrinolysis is avoided by rapid inactivation of plasmin (particularly circulating plasmin) by α2-antiplasmin. Also, t-PA and u-PA are being inactivated by PAI-1.
Discuss events occurring during hemostasis, comparing primary and secondary hemostasis.
Adhesion, activation, and aggregation of platelets to form a platelet plug constitute the first events in formation of a clot (primary hemostasis). The platelet plug is stabilized by formation of a fibrin network generated through the coagulation cascade (secondary hemostasis). Optimal numbers and function of platelets are key to cessation of bleeding from small vascular injuries. Disorders of platelet number or function can lead to bleeding from the skin, mucous membranes, brain, or other sites.
Platelet Structure
The circulating platelet is a small anuclear discoid cell ~2-3 microns in diameter that arises from megakaryocytes, with a maturation time of 4-5 days and a circulating life span of 9-10 days. In patients with normal spleen size, 80% of platelets are circulating and 20% are in the spleen. In some pathologic states (e.g., hypersplenism), the spleen may contain up to 90% of platelets. The bone marrow reserve of platelets is limited and can be rapidly depleted after sudden platelet loss or destruction. Newly formed platelets are larger in size and termed megathrombocytes. Platelets do not have a nucleus, but they do contain mitochondria. They have three kinds of functional granules: dense, alpha, and lysosomal granules. Dense granules contain ATP, ADP, serotonin, and calcium. α-granules contains a number of proteins essential for platelet function, including procoagulant proteins (fibrinogen, factor V, von Willebrand factor, etc), platelet-specific factors for platelet activation, and growth factors such as platelet-derived growth factor. Lysosomal granules contain acid hydrolases. Platelets have an extensive system of internal membrane tunnels, called the surface-connected canalicular system, through which the contents of the platelet granules are extruded during platelet aggregation and secretion. Platelets also have a cytoplasmic framework of monomers, filaments, and tubules that constitute the cytoskeleton and allow shape change with activation.
Platelet Function
Platelets play several important roles in hemostasis, including adhesion to the vascular subendothelium at sites of injury to begin the hemostatic process, activation of intracellular signaling pathways leading to cytoskeletal changes and release of intracellular granules to enhance platelet plug formation, aggregation to form the platelet plug, and support of thrombin generation by providing a phospholipid surface for the coagulation cascade to take place. These processes are a continuous and dynamic interaction of vessel, platelet, and plasma components. The endothelial cells of intact vessels prevent blood coagulation by secretion of a heparin- like molecule and through expression of thrombomodulin, which when bound to thrombin activates protein C and S. Intact endothelial cells prevent platelet aggregation by the secretion of nitric oxide and prostacyclin, inhibitors of platelet activation.
Platelet Adhesion
With vessel injury, subendothelial components are exposed. Circulating von Willebrand factor (vWF) adheres to the damaged, exposed subendothelium. Under conditions of high shear flow, circulating platelets then contact the exposed subendothelium in a rolling fashion and adhere by interaction between glycoprotein Ib (GP1b)on the platelet surface and vWF. With exposure to soluble agonists such as thrombin, ADP, epinephrine, and thromboxane A2, or to adhesive proteins in the subendothelial matrix such as collagen and vWF, the platelet integrin GPIIb-IIIa (αIIbβ3) increases its affinity for vWF, leading to tighter binding. GPVI also interacts directly with collagen in the subendothelium. Numerous ligands in the subendothelium, such as collagen, laminin, and fibronectin, also interact with β1 integrins on the platelet surface. All of these interactions lead to firm adherence of the platelet to the subendothelial surface.
Platelet Activation
With adherence to the injured vessel wall, platelets undergo shape change through cytoskeletal activation, becoming more spherical with extended pseudopods and spreading over the exposed subendothelium. The contents of platelet granules are released. Soluble agonists, including thrombin, thromboxane A2, epinephrine, and ADP, interact with their respective G protein coupled platelet membrane receptors, leading to intracellular signaling and calcium mobilization. Calcium activates phospholipase A2, which releases arachidonic acid from phospholipids. Cyclooxygenase (COX-1) then converts arachidonic acid to prostaglandin H2, which in turn is converted to thromboxane A2 by thromboxane synthetase. Thromboxane A2, along with other agonists, is released, acting to further amplify platelet activation. With platelet activation, membrane reorganization also occurs, with switching of the phospholipid phosphatidylserine from the inner to the outer membrane leaflet, making it available to interact with clotting factors that then lead to thrombin generation.
Platelet Aggregation
With platelet adhesion and with binding of soluble agonists to receptors to amplify platelet activation, GPIIb-IIIa is converted to a high-affinity state where it can bind fibrinogen and vWF. Binding of the membrane protein talin to GPIIb-IIIa is the last step to mediate the change from a low-affinity to a high-affinity state. GPIIb-IIIa can then bind fibrinogen, which acts as a bridge to lace platelets together into an aggregate. Thombin generated through activation of the coagulation cascade then converts fibrinogen to fibrin to stabilize the platelet plug.
Tests evaluating platelets
Several tests are used to evaluate platelet function. A CBC with peripheral blood smear provides the platelet count and allows evaluation of platelet size and granularity. A bleeding time (or, more commonly used now, a platelet function analyzer or PFA-100 test) is a diagnostic test for platelet dysfunction. To do a bleeding time, a small incision in the skin is made using a standardized template and the time until cessation of bleeding is measured. A normal bleeding time is less than 9 minutes. A hemophiliac with a normal platelet count and normal platelet function will have a normal bleeding time. A platelet count below 100,000/uL will lead to a prolonged bleeding time, as will a qualitative platelet disorder. Platelet aggregation studies are done to evaluate platelet aggregation in response to a set of agonists, including thrombin, ADP, epinephrine, collagen, arachidonic acid, and ristocetin (an antibiotic which causes vWF to bind to GP1b, inducing platelet aggregation).
Drugs that inhibit platelet function
Major classes of drugs which inhibit platelet function include cyclooxygenase inhibitors such as Aspirin and nonsteroidal anti-inflammatory drugs such as Ibuprofen, ADP receptor inhibitors such as Ticlopidine (Ticlid) and Clopidogrel (Plavix), and GPIIb-IIIa receptor antagonists such as Abciximab.
Thrombocytopenia
(a low platelet count) can be due to decreased platelet production, increased platelet destruction or consumption, or sequestration of platelets in the spleen. A normal platelet count is between 150,000 and 400,000/uL. Spontaneous hemorrhage and increased risk of hemorrhage with trauma or surgery may be seen with platelet counts <50,000/uL, and with platelet counts less than 10- 20,000/uL, life-threatening spontaneous hemorrhage, such as spontaneous intracranial hemorrhage, can be seen. Thrombocytopenia due to decreased platelet production can occur with primary bone marrow disorders such as aplastic anemia, myelodysplasia, and leukemia. It can also occur with bone marrow invasion by metastatic cancer, myelofibrosis, or infections such as tuberculosis. Toxins such as chemotherapeutic drugs, chemicals, and exposure to radiation can injure the bone marrow and lead to thrombocytopenia. And, severe nutritional disorders such as B12 or folate deficiency can affect megakaryopoiesis. Finally, rare congenital disorders can lead to a decreased platelet count. The most common cause of thrombocytopenia due to increased destruction is immune thrombocytopenic purpura (ITP), formerly known as idiopathic thrombocytopenic purpura.
immune thrombocytopenic purpura (ITP)
In patients with ITP, autoantibodies develop which are directed against platelet antigens, leading to their removal by macrophages of the reticuloendothelial system of the liver and spleen, a similar mechanism to that seen with autoimmune hemolytic anemia. There are two forms of ITP: acute and chronic. Acute ITP is usually seen in children or young adults, often preceded by a viral infection. Onset of the thrombocytopenia is sudden and can be severe. Patients present with petechiae and nosebleeds. Recovery is generally within 2 to 6 weeks without treatment or after treatment with steroids. Chronic ITP is more common in adults and is often associated with concurrent autoimmune disorders (e.g., SLE or rheumatoid arthritis), lymphoma, or HIV, although most cases remain idiopathic. Spontaneous remissions are infrequent, and most patients require treatment. The most commonly used treatment options include corticosteroids, intravenous immunoglobulin (IVIG), and splenectomy. Steroids work by dampening proliferation of the B cell clone making the autoantibody. An effect is usually seen within 7 to 10 days of starting treatment. IVIG acts by blocking splenic Fc receptors to prevent their binding to antibody-coated platelets, with an effect being seen within 1 to 2 days. Splenectomy works by removing the site of autoantibody-induced platelet removal and leads to lasting responses in 60 to 70% of patients.
Alloimmune thrombocytopenia
occurs when a patient develops antibodies to platelet antigens not present on the patient’s own platelets. It can occur in the setting of a patient receiving platelet transfusions, or it can occur in the neonate through passive transfer of maternal IgG alloantibodies across the placenta. Drug-induced immune thrombocytopenia can occur when an antibody recognizes a neoepitope created by the binding of a drug to a platelet surface glycoprotein. Heparin-induced thrombocytopenia (HIT) can occur in patients on heparin therapy and can be associated with development of thromboemboli due to platelet activation.
Other non- immune-mediated causes of thrombocytopenia
include DIC, sepsis, thrombotic thrombocytopenic purpura (TTP), and hemolytic uremic syndrome (HUS). Thrombocytopenia with these disorders is due to increased platelet consumption.
thrombotic thrombocytopenic purpura (TTP)
TTP is characterized by fever, renal insufficiency, microangiopathic hemolytic anemia, mental status changes, and thrombocytopenia. It occurs when endothelial damage occurs from a variety of mechanisms (e.g., infection, immune complexes, HIV, pregnancy, cancer), leading to abnormal release of unusually large vWF molecules from storage sites. These large vWF multimers mediate platelet adhesion and aggregation, forming diffuse platelet plugs in small arterioles. The large multimers are present because of the absence of a metalloprotease called ADAMTS13 that normally digests the vWF into smaller multimers. While a congenital form of the disease exists, most of the time it is acquired through development of autoantibodies to ADAMTS13. Treatment is with plasmapheresis to remove the large vWF multimers and replace the missing ADAMTS13.
Hemolytic uremic syndrome (HUS)
HUS has a similar presentation but tends to more often be associated with renal failure and tends to occur more often in children. It is due to damage to the endothelial lining, usually by a bacterial toxin, leading to platelet adhesion and activation and microthrombi formation. It is often a self-limited process and is generally treated with supportive care alone
Von Willebrand disease (vWD)
the most common congenital bleeding disorder. It can also be an acquired problem if antibodies develop against the vWF molecule. It can be due to an inadequate amount of vWF or it can be due to mutations in the vWF gene leading to abnormal protein function. vWF plays a key role in adhesion of platelets to injured vascular endothelium. Lack of vWF leads to abnormal platelet/endothelial interaction, leading to a primary hemostatic bleeding disorder characterized by mucosal and skin bleeding. VWF also serves as a carrier protein for factor VIII. So, with severe deficiency, defects in secondary hemostasis can be seen as well, due to a functional factor VIII deficiency. Lab tests for diagnosis of vWD include the bleeding time or PFA-100, which will be abnormally prolonged with vWD, a factor VIII level, a von Willebrand antigen test to measure the amount of vW protein, and a test of von Willebrand activity, also known as the ristocetin cofactor activity, which measures function of a patient’s von Willebrand protein using donor platelets. vWF multimer assays are occasionally obtained when evaluating for a qualitative defect in vWF function. A commonly used treatment for vWD is DDAVP (arginine vasopressin), which enhances release of vWF from endothelial stores. It is effective for treatment of type 1 vWD (partial quantitative deficiency) but not type 2 (qualitative defects) or type 3 (near-complete absence of vWF). Factor replacement can also be used is some situations. Patients should be told to avoid aspirin and other platelet inhibiting agents.
Bernard-Soulier syndrome
a rare autosomal recessive disorder where expression of GP1b on the platelet surface is reduced, leading to a defect in platelet adhesion. Platelet aggregation studies only show abnormal aggregation with ristocetin.
Platelet disorders of activation
Storage pool deficiencies can occur, with a deficiency of either dense granules or α-granules. Deficiency of α-granules is known as gray platelet syndrome. Several syndromes can be associated with a lack of dense granules. These disorders can also be acquired when platelets pass across abnormal vascular surfaces (such as cardiopulmonary bypass apparatus) leading to partially degranulated platelets. Disorders can also be due to rare defects in signal transduction pathways within the platelets.
Platelet disorders of aggregation
Rare inherited defects include afibrogenemia, which leads to both primary (platelet plug formation) and secondary (formation of cross-linked fibrin) hemostatic defects. The defect in primary hemostasis is due to lack of fibrinogen for binding to GPIIb-IIIa to allow platelet aggregation. The defect in secondary hemostasis is due to lack of fibrinogen for formation of cross-linked fibrin. Patients have platelet-type mucosal and cutaneous bleeding as well as deep muscle hematomas more characteristic of coagulation defects. Glanzmann thrombasthenia is a rare autosomal recessive bleeding disorder caused by absent or defective GPIIb-IIIa. Platelets can adhere but are unable to aggregate in response to normal agonist stimuli. Patients have petechiae and easy bruising.
List important questions to ask when obtaining a bleeding history in a patient with excessive bleeding.
Brisk bleeding from obvious trauma suggests a local vascular defect. Prolonged or recurrent bleeding is more likely a generalized hemostatic disorder. Sudden resumption of bleeding from an injured site raises the possibility of excessive fibrinolysis or abnormal crosslinking of fibrin. Multiple site bleeding suggests a more severe, generalized hemostatic disorder. Mucocutaneous bleeding (bruising, petechiae, epistaxis, menorrhagia, prolonged oozing after tooth extraction, increased bleeding after aspirin intake) is indicative of a defect in primary hemostasis (platelet disorder or von Willebrand disease) while soft tissue/joint/deep bleeding is more consistent with a defect in secondary hemostasis (coagulopathy). Findings on physical exam may suggest an underlying disorder, such as petechiae with thrombocytopenia, an enlarged spleen and lymph nodes with chronic infections or malignancies, signs of liver disease such as jaundice or edema with liver coagulopathy, or musculoskeletal abnormalities and joint disease with the hemophilias. Complaints or signs of easy bruising are common in children and many elderly people without an underlying bleeding disorder. It is rare, however, for children < 1 year of age to show bruising. Trauma (accidental or non-accidental) should be considered as a cause of multiple or unusual bruises at any age. Large (>2 inches in diameter) or indurated purpuric lesions should lead to an evaluation for a bleeding problem. Recurrent, brief nosebleeds are frequently seen in children. Nosebleeds that occur every 1-2 months, last longer than 10 minutes, involve both nares, and require medical attention or transfusion are suspicious of a bleeding defect.
Basic screening tests when evaluating excessive bleeding can include
Platelet count and blood smear to evaluate for thrombocytopenia or other hematologic abnormalities. Bleeding time or platelet function analyzer (PFA-100) to evaluate primary hemostasis. APTT as a screening test for the intrinsic coagulation pathway. PT/INR as a screening test for the extrinsic coagulation pathway. Thrombin clotting time (TCT) to evaluate for fibrinogen defects, the presence of fibrin split products, or heparin effects. Fibrinogen level. Further testing is based on results of the basic screen and clinical suspicion. If the initial studies are negative in a patient with a definitive history of bleeding, further diagnostic studies should be done (under the guidance of a hematologist) to evaluate for such things as mild hemophilia, factor XIII deficiency, and fibrinolytic defects. Mild cases of von Willebrand disease may require repeated testing to establish a diagnosis if clinical suspicion remains. Occasionally, screening tests are obtained prior to a surgical procedure and abnormalities, such as an isolated prolonged APTT, are seen in an otherwise asymptomatic patient. Such a patient may have a factor XII deficiency (not associated with bleeding) or a lupus anticoagulant (associated with thrombosis, not bleeding). Or, they may have a mild form of hemophilia (factors VIII, IX, or XI deficiencies) or von Willebrand disease. Appropriate factor levels to rule out factor deficiency and a 1:1 mixing of normal plasma with patient plasma to evaluate for a lupus anticoagulant or factor inhibitor can then be done.
General principles of immunohematology
Red cells do no carry MHC antigens in humans, and the antigens they do carry are much less polymorphic in the population (many fewer alleles). The white cells that come along in transfusion are recognized and destroyed. Platelets do bear HLA (class I) and when people repeatedly need platelets, they may develop an alloimmunization problem, in which case HLA typing becomes necessary.
Blood group antigens
are glycolipids found on the surface of all body cells, including red cells. The lipid backbone spans the plasma membrane and the terminal sugars confer the antigenic specificity A, B, or O. people who are O are somewhat protected from pancreatic cancer and much less likely to develop venous thromboembolic disorders. Antigens are assembled by a set of glycosyl transferases that first builds the H antigen, the basic core sugar chain which almost everybody has. Then a final glycosyl transferase, of which there are three alleles, can act. The O allele is an amorph (it does not code for a working transferase and so group O only have the H antigen). People who are group A have a glycosyl transferase allele, which puts an additional sugar on the H antigen and people who are B have a different allelic form of this enzyme, which adds a different sugar. Group AB individuals have both the A and B antigens on their red cells, because they have both the A and B transferases from their parents. There are some people who lack the transferase gene that puts the final sugar on the core and thus do not express even the H antigen, so there is no substrate for the A or B glycosyltransferases to modify. This is the Bombay phenotype (Oh) and it is rare. All blood, even type O, is foreign to such people.
Blood group substances
glycoproteins with the same sugar, found in the body fluids of people who have the secretor (Se) phenotype.
Antibodies to blood antigens
the antigens that are not the same as your own are foreign to you and you will become immunized to them. Thus a person who is group A will make antibody foreign to B but not A. a person who is O will have antibodies to both A and B.
Isohemagglutinins
An isoantibody that causes agglutination of cells of genetically different members of the same species. Measuring their titer can be use in the diagnosis of B cell immunodeficiency, since they should begin to appear in the blood between 3 and 6 months of age, as antigen exposure occurs. Isohemagglutinins are of the IgM class. There are variant A and B types (A2, A3, Ax, Bx, etc.) in which the A or B antigen is expressed rather weakly; such people may be typed incorrectly or with difficulty in the blood bank. Suppression of ABO antigens can be seen in some diseases such as leukemia. In addition, titers of isohemagglutinin can be low in the elderly and in hypogammaglobulinemia. Any of these conditions can lead to an “ABO discrepancy” (lack of correlation between ABO phenotype as determined by cell and serum typing) which must be resolved. DNA typing is possible. AB is the rarest blood group, O the most useful as blood donors, and group B people are the best looking.
Rh
the second most important blood group system. Rh antigens are on proteins coded for at two loci; one is for the alleles d/D and the other for c/C and e/E. The most important allele is D. D is dominant over d (the d allele, another amorph, is heavily mutated and does not make a protein). Ninety-two percent of U.S. blacks are Rh+; 85% of whites. Rh(D)- is rare in sub-Saharan Africa. There are no “naturally occurring” isohemagglutinins for Rh; it’s a protein, and not ubiquitous in nature, so you don’t make antibody to it unless you’re Rh(D)- and become immunized with Rh(D)+ red cells.
