|Hemostasis Basics - Programmed Learner|
|Table of Contents
When you cut yourself,the process of coagulation begins by the formation of a blood clot. This is followed shortly after by digestion or breakdown of the clot. Patients clot and/or bleed because of a variety of identifiable hemostatic abnormalities. Logical and effective treatment depends upon the proper identification of the abnormality. The coagulation or hemostasis laboratory performs tests to determine the cause and to monitor the proper treatment of the defect.
This educational module will teach you a systematic and practical approach to understanding the laboratory's role in diagnosis and therapy of bleeding and clotting disorders.
Platelets and Hemostasis:
There is a sequence of events which occurs at the site of vascular injury. First, the platelet is attracted to the exposed sub-endothelial layer of collagen and adheres to it. To accomplish this, the platelet undergoes a shape change. Secondly, the platelets release intrinsic adenosine diphosphate (ADP), among other substances. The released ADP stimulates other platelets to stick together at the wound site, and, thirdly, aggregation occurs. In this process, platelets adhere to each other to form a beginning plug. Finally, coagulation occurs and fibrin forms around the platelet aggregate to initiate repair.(See figure 1)
The model generally used to describe the mechanism of coagulation is the cascade system. The cascade is separated into three areas: the intrinsic system, commonly measured by the aPTT test, which is activated by surface contact; the extrinsic system, commonly measured by the PT test, which is activated by vascular injury, and, the common pathway, which is set into motion by activation from the intrinsic and/or the extrinsic pathway. Because of the variety of constituents involved with the common pathway, there are several different tests that could be used to monitor activity. The systems and tests are described in later sections of this module.
These coagulation factors, which are proteins,with the exception of Calcium and Thromboplastin, can conveniently be divided into three families: the fibrinogen, prothrombin, and contact family. The fibrinogen family includes fibrinogen, Factors V, VIII, and XIII. The prothrombin family includes Factors II, VII, IX, X, Protein C and Protein S. The contact family of plasma coagulation proteins include: Factor XII or Hageman factor, Factor XI, Fletcher factor or Prekallikrein (PK), Fitzgerald factor or High Molecular Weight Kininogen (HMWK) and possibly the Passovoy factor. They are all involved in the mechanism that generates insoluble fibrin as a final product, by means of the coagulation cascade. Disorders of secondary hemostasis many times involve a change in the coagulation proteins. These changes can be a decreased level of a particular factor or a defect in the way the factor functions.
Warfarin is a drug used in patient therapy to prevent thrombosis. It inhibits the synthesis of the vitamin K dependent factors, factors II, VII, IX and X by blocking the regeneration of vitamin K and shows a dose dependent effect. As more warfarin is ingested orally, the greater the reduction in the functional levels of vitamin K dependent factors. See Figure 4 for the effect of warfarin on the synthesis of clotting factors. Because 3 of the 4 factors affected by warfarin are evaluated by the PT test, it is commonly used to monitor therapy.
The PT test is performed by adding tissue thromboplastin and calcium to plasma and measuring the time for clot formation. It can be performed either manually by tilt tube method or mechanically by use of a fibrometer or a photo-optical instrument. The PT reagent used in the testing provides the tissue thromboplastin and calcium. The sources of thromboplastin can be human or rabbit brain, lung, placental, brain/lung combination, or produced by recombinant technology. The necessary calcium is added to the reagent either at the time of manufacture or prior to testing.
The PT can be done as either a one-stage or a two-stage assay, although the one-stage procedure is the most widely used and preferred. Thromboplastin reagent (0.2 ml) is warmed at 37C then forcibly added to plasma (0.1ml) which also has been heated to 37C and a timer is started. As soon as the clot forms indicating fibrin formation, the timing stops and the time is recorded to the nearest tenth of a second. The expected normal range for a PT is 10-14 seconds depending on the type of reagent used.
Variation in the composition and responsiveness of PT reagents have necessitated the use for standardization. The International Normalized Ratio or INR was developed for the purpose of standardizing the monitoring of warfarin therapy.
Several factors may contribute to the differing degrees of responsiveness observed for various thromboplastin reagents. Some of these include the species and tissue source, the relative concentrations of other components of the reagent formula etc. The responsiveness of the thromboplastin reagent needs to be considered to make the PT an effective way of monitoring warfarin treatment. The responsiveness of a thromboplastin reagent toward plasma samples from patients receiving warfarin is described by a value called the International Sensitivity Index (ISI).
The calculation of the INR is obtained by using the following calculation:
In summary, defects in the normal hemostatic mechanism can be listed as two types. One is the failure of any of the processes that lead to the hemostatic plug formation which may lead to a bleeding disorder and inappropriate activation of the hemostatic mechanism which may cause thrombosis. Laboratory investigations and determinations are needed to identify the eXact nature of the underlying bleeding disorder. Screening tests such as the PT are initially performed. Based on these results, further, more complex testing may be needed leading to follow-up corrective action and treatment.
The intrinsic pathway of Coagulation is activated when circulating Factor XII comes in contact with and is bound to a negatively charged surface. This causes a change in the molecular configuration of Factor XII and in concert with HMWK and prekallikrein it becomes an active enzyme, XIIa. This activated enzyme is then able to bring about a similar change in Factor XI. After activation, Factor XIa, in a calcium dependent reaction, converts Factor IX to its active form, Factor IXa. A phospholipid surface is also needed for Factor IXa conversion and is provided by activated platelets, as Platelet Factor Three (PF3). Factor IX can also be activated by the tissue factor, Factor VII complex; the initiating complex of the extrinsic pathway. Factor X can be activated to Factor Xa by either the Factor VIIa complex or by the complex of Factor IXa and Factor VIII. Factor Xa in the presence of Factor V, calcium and phospholipid surface converts Factor II (prothrombin) to Factor IIa (thrombin) which converts Factor I (fibrinogen) to fibrin (see Figure 5).
Activated partial thromboplastin time (aPTT) is an assay used to screen for abnormalities of the intrinsic clotting system. It is also used to monitor the anticoagulant effect of circulating heparin.
An aPTT assay is performed by adding to platelet poor plasma a Factor XII activator, a phospholipid, and calcium ions. Factors I, II, V, VIII, IX, X, XI, XII, prekallikrein (Fletcher Factor) and high molecular weight kininogen (HMWK) are measured. An abnormal aPTT result might indicate the presence of an acquired inhibitor or a deficiency in any of the coagulation factors except Factors VII and XIII.
For in vitro analysis, some commonly used activators are glass, ellagic acid, kaolin, silica and celite. All of these except glass, are used in aPTT reagents and serve the same function of activating the clotting mechanism. Phospholipids are platelet substitutes and accelerate the reactions involved. Sources of phospholipids are rabbit brain, cephalin (dehydrated rabbit brain), bovine brain, and soy bean.
When adequate levels of all the coagulation factors are present in plasma, the aPTT test result is normal. Normal ranges of the factors vary from approximately 50-150% of normal. In general an aPTT reagent should be able to detect factor levels of 30% or less. If aPTT results are prolonged and there is no indication of a factor deficiency, an acquired inhibitor may be present.
Heparin will also cause a prolonged aPTT. This commercial product is prepared from beef lung or porcine intestinal mucosa and is administered via intravenous or subcutaneous injection. Heparin with its plasma co-factor Antithrombin III, inhibits coagulation immediately after being administered. It is the drug of choice for treating venous thrombosis by preventing fibrin formation.
The aPTT, although useful in monitoring high level heparin therapy, has had variable effectiveness in monitoring low dose heparin therapy and low molecular weight forms of heparin.
Once Coagulation is initiated, the body has mechanisms for avoiding massive thrombus formation. Physiologic balancing of the Hemostatic mechanism to limit uncontrolled bleeding and clotting is an important aspect in the Hemostatic response. There are a variety of biological control mechanisms which aid in the control of blood coagulation. These include the ability of the liver and the reticulo-endothelial system to clear activated clotting factors from the circulation, the prevention of the high concentrations of activated factors at a given location within the circulation by a constant blood flow, and natural inhibitors in the plasma such as Antithrombin III and the Protein C-S System.
Antithrombin III (AT-III) is the most important inhibitor of the coagulation enzymes. AT-III binds to activated factors rendering them inactive (Figure 6). The primary function is to inactivate thrombin. Inactive factors and cofactors are not neutralized by AT-III, since it only binds to the enzymatic factors. The process of binding the active forms of the clotting factors (XIIa, XIa, Xa, IXa) and thrombin to AT-III is greatly accelerated by heparin to an almost instant neutralization. AT-III inhibits not only coagulation enzymes but also plasmin and kallikrein.
Patients with decreased AT-III levels are subject to an increased risk of thromboembolism even in cases of slightly reduced AT-III levels, therefore the Antithrombin III assay is an important part of a prethrombotic workup.
Antithrombin III levels are affected by several other disease states. Individuals suffering from severe hepatic disorders such as cirrhosis or acute hepatitis have significantly depressed AT-III levels, while disease accompanied by inflammation may show elevations.
Protein C is an inhibitor of the activated Factors Va
The last stage of coagulation is fibrinolysis, which is the dissolution and localization of a fibrin clot. These functions are carried out by enzymes and their inhibitors. A disruption or breach of the fine balance of this fibrinolytic system can result in bleeding or thrombosis.
The components of the fibrinolytic system are schematically shown in Figure 7. Fibrinolysis is mediated by activation of plasminogen to plasmin. This is accomplished by:
Activators of plasminogen convert it to the active enzyme plasmin. Plasmin, in turn, acts to split the fibrin clot into fibrin degradation products. To balance this activity there are inhibitors. The most important inhibitor of plasminogen activators is PAI-1, which is fast acting. Alpha2-antiplasmin, another principal inhibitor of fibrinolysis, inhibits plasmin (See Figure 8).
Soluble fibrinogen is cleaved by thrombin to form fibrin monomers. The fibrin monomers aggregate to form fibrin polymers, unstable fibrin clots. Thrombin also activates factor XIII to an activated enzyme, factor XIIIa, which in the presence of calcium converts fibrin polymers to a stable fibrin clot. Plasmin can degrade or split both fibrinogen and fibrin into fragments, X, Y, D and E. Fibrinogen degradation products (FDP) are the products of fibrinogenolysis and are detected by the FDP assay. Fibrin degradation products (fdp) are the product of fibrinolysis. The only time D-dimers (cross linked D-domains) are present is after the degradation of a stable fibrin clot (See Figure 9). These tests (FDP and D-Dimer) will be described in future modules.
There are many conditions that can affect the fibrinolytic system resulting in an increased or decreased activity of fibrinolysis. Samples of such conditions are Disseminated Intravascular Coagulation (DIC), trauma from surgical procedures or accidents, deficiencies in or consumption of the various inhibitors and activators of the fibrinolytic system.
Continued study of the fibrinolytic system unlocks it's complexities . Always on the horizons are newer and more sensitive and specific methods of evaluating this system, thus providing better diagnostic tools.