HEMATOLOGY EBOOK

D-dimer is a fibrin degradation product, a small protein fragment present in the blood after a blood clot is degraded by fibrinolysis. It is so named because it contains two crosslinked D fragments of the fibrinogen protein. D-dimer concentration may be determined by a blood test to help to help diagnose thrombosis. Since its introduction in the 1990s, it has become an important test performed in patients suspected of thrombotic disorders. While a negative result practically rules out thrombosis, particularly in young and healthy patients, a positive result can indicate thrombosis but does not rule out other potential causes. Its main use, therefore, is to exclude thromboembolic disease where the probability is low. In addition, it is used in the diagnosis of the blood disorder disseminated intravascular coagulation

Principles

Principles of D-dimer testing

Coagulation, the formation of ablood clot of thrombin, occurs when the proteins of the “coagulation cascade” are activated, either by contact with damaged blood vessel wall (extrinsic pathway) or by activation of high-molecular-weight kininogen by a number of stimuli. Both pathways lead to the generation of thrombin, an enzyme that turns the soluble blood protein fibrinogen into fibrin, which aggregates into proteofibrils. Another thrombin-generated enxyme, factor XIII, then crosslinks the fibrin ptoteofibrils at the D fragment site, leading to the formation of an insoluble gel which serves as a scaffold for blood clot formation. The circulating enzyme plasmin, the main enzyme of fibrinolysis, cleaves the fibrin gel in a number of places. The resultant fragments, “high molecular weight polymers”, are digested several times more by plasmin to lead to intermediate and then to small polymers (fibrin degradation product or FDPs). The crosslink between two D fragments remains intact, however, and these are exposed on the surface when the fibrin fragments are sufficiently digested. The typical D-dimer containing fragment cotains two D domains and one E domain of the fibrinogen molecule. D-dimer are not normally present in human blood plasma, except when the coagulation system has been activated, for instance because of the presence of thrombosis or disseminated intravascular coagulation. The D-dimer assay depends in the binding of a monoclonal antibody to a particular epitope on the D-dimer fragment. Several detection kits are commerically available; all of them rely on a different monoclonal antibody against D-dimer. Of some of these it is known to which area on the D-dimer the antibody binds. The binding of the antibody is then measured quantitatively by one of various laboratory methods.

Indications

D-dimer testing is of clinical use when there is a suspicion of deep venous thrombosis (DVT) or pulmonary embolism (PE). In patients suspected of disseminated intravascular coagulation (DIC), D-dimers may aid in the diagnosis.

For DVT and PE, there are various scoring systems that are used to determine the a priori clinical probability of these diseases; the best-known were introduced by Wells et al. (2003).

• For a very high score, or pretest probability, a D-dimer will make little difference and anticoagulant therapy will be initiated regardless of test results, and additional testing for DVT or pulmonary embolism may be performed.
• For a moderate or low score, or pretest probability:
  • A negative D-dimer test will virtually rule out thromboembolism: the degree to which the D-dimer reduces the probability of thrombotic disease is dependent on the test properties of the specific test used in your clinical setting: most available D-dimer tests with a negative result will reduce the probability of thromboembolic disease to less than 1% if the pretest probability is less than 15-20%
  • If the D-dimer reads high, then further testing (ultrasound of the leg veins or lung scintigraphy or CT scanning) is required to confirm the presence of thrombus. Anticoagulant therapy may be started at this point or withheld until further tests confirm the diagnosis, depending on the clinical situation.

In some hospitals, they are measured by laboratories after a form is completed showing the probability score and only if the probability score is low or intermediate. This would reduce the need for unnecessary tests in those who are high-probability.

Test properties

Various kits have a 93-95% sensitivity and about 50% specificity in the diagnosis of thrombotic disease.

• False positive readings can be due to various causes: liver disease, high rheumatoid factor, inflammation, malignancy, trauma, pregnancy, recent surgery as well as advanced age
• False negative readings can occur if the sample is taken either too early after thrombus formation or if testing is delayed for several days. Additionally, the presence of anti-coagulation can render the test negative because it prevents thrombus extension.
• Likelihood ratios are derived from sensitivity and specificity to adjust pretest probability.

History

D-dimer was originally described in the 1970s, and found its diagnostic application in the 1990s.

The euglobulin lysis time (ELT) is a test that measures overall fibrinolysis. The test is performed by mixing citrated platelet-poor plasma with acid in a glass test tube. This acidification causes the precipitation of certain clotting factors in a complex called the euglobulin fraction. The euglobulin fraction contains the important fibrinolytic factors fibrinogen, PAI-1, tPA, plasminogen, and to a lesser extent alpha 2-antiplasmin. The euglobulin fraction also contains factor VIII. After precipitation, the euglobulin fraction is resuspended in a borate solution. Clotting is then activated by the addition of calcium shloride at 37 C. Historically, subsequent amount of fibrinolysis was determined by eye, by observing the clot within the test tube at ten minute intervals until complete lysis had occured. Newer automated methods have also been developed. These methods use the same principle as the older technique, but use a spectrophotometer to track clot lysis as a function of optical density.

Fibrinolysis is the process wherein a fibrin clot, the product of coagulation, is broken down. Its main enzyme plasmin cuts the fibrin mesh at various places, leading to the production of circulating fragments that are cleared by other proteases or by the kidney and liver.

Physiology

Plasmin is produced in an inactive form, plasminogen, in the liver. Although plasminogen cannot cleave fibrin, it still has an affinity for it, and is incorporated into the clot when it is formed. Plasminogen contains secondary structure motifs known as kringles, which bind specifically to lysine and arginine residues on fibrin(ogen). When converted from plasminogen into plasmin, it functions as a serine protease, cutting C-terminal to these lysine and arginine residue. Fibrin monomers, when polymerized, form protofibrils. These protofibrils contain two strandm the fibrin monomers are covalently. Within a single strand, the fibrin monomers are covalently linked through the actions of coagulation factor XIII. Thus, plasmin action on a clot initially creates nicks in the fibrin, further digestion leads to solubilization. Tissue plasminogen activator (t-PA) and urokinase are the agents that convert plasminogen to the active plasmin, thus allowing fibrinolysis to occur. t-PA is released into the blood very slowly by the damaged endothelium of the blood vessels, such that, after several days (when the bleeding has stopped), the clot is broken down. This occurs because plasminogen became entrapped within the clot when it formed; as it is slowly activated, it breaks down the fibrin mesh. t-PA and urokinase are themselves inhibited by plasminogen activator inhibitor-1 and plasminogen activator inhibitor-2 (PAI-A and PAI-2). In contrast, plasmin further stimulates plasmin generation by producing more active forms of both tPA and urokinase. Alpha 2-antiplasmin and alpha 2-macroglobulin inactivate plasmin. Plasmin activity is also reduced by thrombin-activatable fibrinolysis inhibitor (TAFI), which modifies fibrin to make a less potent cofactor for the tPA-mediated plasminogen.

Measurement

When plasmin breaks down fibrin, a number of soluble parts are produced. These are called fibrin degradation products (FDPs). FDPs compete with thrombin, and so slow down the conversion of fibrinogen to fibrin (and thus slows down clot formation). This effect can be seen in the similar results are also seen after administration of DDAVP or after severe stress. A more rapid detection of fibrinolytic activity, especially hyperfibrinolysis, is possible with thromboelastometry (TEM) in whole blood, even in patients on heparin. With various assays an enhanced fibrinolysis becomes visible in the curve signature and from the calsulated values e.g. the maximum lysis parameter. A spcial test for the identification of increased fibrinolysis (APTEM) compares the TEM profile in the absence or presence of the fibrinolysis inhibiotr aprotinin. In severe cases of activated fibrinolysis, this assay confirms the syndrome already in less thn 15 min during the early phases of clot formation.

Role in disease

Few congenital disorders of the fibrinolytic system have been documented. Nevertheless, excess levels of PAI and alpha 2-antiplasmin have been implicated in the metabolic syndrome and various other disease states. However, acquired disturbance of fibrinolysis (Hyperfibrinolysis), is not uncommon. Many trauma patients suffer from an overwhelming activation  of tissue factor and thus massive hyperfibrinolysis. Also in other disease states hyperfibrinolysis may occur. It could lead to massive bleeding if not dianosed and treates early enough. The fibrinolytic system is closely linked to control of inflammation, and plays a role in disease states associated with inflammation. Plasmin, in addition to lysing fibrin clots, also cleaves the complement system component C3, and fibrin degradation products have some vascular permeability inducing effects.

Pharmacology

Fibrinolytic drugs are given after a heart attack to dissolve the thrombus blocking the coronary artery, experimentally in stroke to reperfuse the affected part of the brain, and in massive pulmonary embolism. The process is called thrombolysis. Antifibrinolytics, such as aminocaproic acid (ε-aminocaproic acid) and tranexamic acid are used as inhibitors of fibrinolysis. Their application may be benefecial in patients with hyperfibrinolysis because they arrest bleeding rapidly if the other components of the haemostatic system are not severely affected. This may help to avoid the use of blood products such as fresh frozen plasma with its associated risks of infections or anaphylactic reactions. The antifibrinolytic drug aprotinin was abandoned after identification of major side effects, especially on kidney.

Bleeding time is a medical test done on someone to assess their platelet function

The term “template bleeding time” is used when the test is performed to standardized parameters.^[1] This makes it easier to compare data collected at different facilities.

 Process

It involves cutting the underside of the subject’s forearm, in an area where there is no hair or visible veins. The cut is of a standardised width and depth, and is done quickly by an automatic device.

A blood pressure cuff is used above the wound, to maintain venous pressure at a special value. The time it takes for bleeding to stop (as thus the time it takes for a platelet plug to form) is measured. Cessation of bleeding can be determined by blotting away the blood every several seconds until the site looks ‘glassy’.

 Ivy method

The Ivy method is the traditional format for this test. While both the Ivy and the Duke method require the use of a sphygmomanometer, or blood pressure cuff, the Ivy method is more invasive than the Duke method, utilizing an incision on the ventral side of the forearm, whereas the Duke method involves puncture with a lancet or special needle. In the Ivy method, the blood pressure cuff is placed on the upper arm and inflated to 40 mmHg. A lancet or scalpel blade is used to make a shallow incision that is 1 millimeter deep on the underside of the forearm.

A standard-sized incision is made around 10 mm long and 1 mm deep. The time from when the incision is made until all bleeding has stopped is measured and is called the bleeding time. Every 30 seconds, filter paper or a paper towel is used to draw off the blood.

The test is finished when bleeding has stopped completely.

A normal value is less than 9 and a half minutes.

A prolonged bleeding time may be a result from decreased number of thrombocytes or impaired blood vessels. However, it should also be noted that the depth of the puncture or incision may be the source of error.

Normal values fall between 2 – 9 minutes depending on the method used.

 Duke Method

With the Duke method, the patient is pricked with a special needle or lancet, preferably on the earlobe or fingertip, after having been swabbed with alcohol. The prick is about 3-4 mm deep. The patient then wipes the blood every 30 seconds with a filter paper. The test ceases when bleeding ceases. The usual time is about 1-3 minutes.

 Interpretation

Bleeding time is affected by platelet function, certain vascular disorders and von Willebrand Disease–not by other coagulation factors such as haemophilia. Diseases that cause prolonged bleeding time include thrombocytopenia, disseminated intravascular coagulation (DIC), Bernard-Soulier disease, and Glanzmann’s thrombasthenia.

Aspirin and other cyclooxygenase inhibitors can prolong bleeding time significantly. While warfarin and heparin have their major effects on coagulation factors, an increased bleeding time is sometimes seen with use of these medications as well.

People with von Willebrand disease usually experience increased bleeding time, as von Willebrand factor is a platelet agglutination protein, but this is not considered an effective diagnostic test for this condition.

It is also prolonged in hypofibrinogenemia.

 Representation in Media

Apart from mentions in Medical Dramas such as House, Bleeding Times most famous appearance came in the film “Doctor in the House”, when Sir Lancelot Spratt was teaching a group of Junior Doctors and was talking about the bleeding time. He noticed that Doctor Simon Sparrow wasn’t listening and in the series’ most famous line went “Doctor Sparrow! Whats the Bleeding Time”. Sparrow looks flustered and then suddenly pipes up “10 past Two sir”. (Bleeding in UK Slang is a very mild form of Expletive.)

The Thrombin Time (TT), is a blood test which measures the time it takes for a clot to form in the plasma from a blood sample in anticoagulant which had added an excess of thrombin,. This test is repeated with pooled plasma from normal patients. The difference in time between the test and the ‘normal’ indicates an abnormality in the conversion of fibrinogen(a soluble protein) to fibrin an insoluble protein. This test is also known as the Thrombin Clotting Time (TCT).

Thrombin time compares a patient’s rate of clot formation to that of a sample of normal pooled plasma. Thrombin is added to the samples of plasma. If the plasma does not clot immediately, a fibrinogen deficiency is present. If a patient is receiving heparin, a substance derived from snake venom called reptilase is used instead of thrombin. Reptilase has a similar action to thrombin but unlike thrombin it is not inhibited by heparin.

The thrombin time is used to diagnose bleeding disorders and to assess the effectiveness of fibrinolytic therapy. Reference values for thrombin time are 10 to 15 seconds or within 5 seconds of the control. If reptilase is used, the reptilase time should be between 15 and 20 seconds. Thrombin time can be prolonged by: heparin, fibrin degradation products, lupus anticoag

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 Procedure

Within the realm of coagulation assays, the Thrombin Clotting Time is one of the most procedurally simple. After liberating the plasma from the whole blood by centrifugation, bovine Thrombin is added to the sample of plasma. The clot is formed and is detected optically or mechanically by a coagulation instrument. The time between the addition of the thrombin and the clot formation is recorded as the thrombin clotting time

 Specimen Requirements

Whole blood is taken with either citrate or oxalate additive (if using the vacutainer system, this is a light blue top tube). As with other coagulation assays, the tube must not be over- or under-filled in order to ensure the correct anticoagulant-to-blood ratio: 1 part anticoagulant per 9 parts blood.

 Reference Interval

The reference interval of the Thrombin Clotting time is generally <22 seconds, depending on the method and the endemic patient population. Results outside of reference interval indicate heparin therapy, Hypofibrinogenemia, hyperfibrinogenemia fibrinogen abnormality, or Lupus anticoagulant.

 Causes for specimen rejection

Causes for Rejection of the specimen include QNS, severe hemolysis, improper storage or delay in processing, error in labeling.

The partial thromboplastin time (PTT) or activated partial thromboplastin time (aPTT or APTT) is a performance indicator measuring the efficacy of both the “intrinsic” (now referred to as the contact activation pathway) and the common coagulation pathways. Apart from detecting abnormalities in blood clotting, it is also used to monitor the treatment effects with heparin, a major anticoagulant. It is used in conjunction with the prothrombin time (PT) which measures the extrinsic pathway.

Method

A phlebotomist collects blood samples in vacu-tubes with oxalate or citrate to arrest coagulation by binding calcium. The specimen is then delivered to the laboratory. In order to activate the intrinsic pathway, phospholipid, an activator (such as silica, celite, kaolin, ellagic acid), and calcium (to reverse the anticoagulant effect of the oxalate) are mixed into the plasma sample . The time is measured until a thrombus (clot) forms. This testing is performed by a medical technologist.

The test is termed “partial” due to the absence of tissue factor from the reaction mixture.

 Interpretation

Values below 25 seconds or over 39 s (depending on local normal ranges) are generally abnormal. Shortening of the PTT has little clinical relevance. Prolonged APTT may indicate:

• use of heparin (or contamination of the sample)
• antiphospholipid antibody (especially lupus anticoagulant, which paradoxically increases propensity to thrombosis)
• coagulation factor deficiency (e.g. hemophilia)

To distinguish the above causes, mixing tests are performed, in which the patient’s plasma is mixed (initially at a 50:50 dilution) with normal plasma. If the abnormality does not disappear, the sample is said to contain an “inhibitor” (either heparin, antiphospholipid antibodies or coagulation factor specific inhibitors), while if it does correct a factor deficiency is more likely. Deficiencies of factors VIII, IX, XI and XII and rarely von Willebrand factor (if causing a low factor VIII level) may lead to a prolonged aPTT correcting on mixing studies.

 History

The aPTT was first described in 1953 by researchers at the University of North Carolina at Chapel Hill.

The prothrombin time (PT) and its derived measures of prothrombin ratio (PR) and international normalized ratio (INR) are measures of the extrinsic pathway of coagulation. They are used to determine the clotting tendency of blood, in the measure of warfarin dosage, liver damage, and vitamin K status. The reference range for prothrombin time is usually around 12-15 seconds; the normal range for the INR is 0.8-1.2. PT measures factors II, V,VII,X and fibrinogen. It is used in conjunction with the activted partial thromboplastin time (aPTT) which measures the intrinsic pathway.

Laboratory measurement

Methodology

The prothrombin time is most commonly measured using blood plasma. Blood is drawn into a test tube containing liquid citrate, which acts as an anticoagulant by binding the calsium in a sample. The blood is mixed, then centrifuged to separate blood cells from plasma. In newborns, whole blood is used. The plasma is analyzed by a biomedical scientist on an automated instrument at 37°C, which takes a sample of the plasma. An excess of calcium is added (thereby reversing the effects if citrate), which enables the blood to clot again. For an accurate measurement the proportion of blood to citrate needs to be fixed; many laboratories will not perform the assay if the tube is underfied and contains a relatively high concentration of citrate. If the tube is underfilled or overfilled with blood, the standardized dilution of 1 part antocoagulant to 9 parts whole blood is no longer valid. For the prothrombin time test the appropriate sample is the blue top tube, or sodium citrate tube, which is a liquid anticoagulant. Tissue factor (also known as factor III) is added, and the time the sample takes to clot is measured optically. Some laboratories use a mechanical measurement, which eliminates interferences from lipemic and icteris samples. The prothrombin ratio is the prothrombin time for a patient, divided by the result for control plasma.

International normalized ratio

The result (in seconds) for a prothrombin time performed on a normal individual will vary depending or what type of analytical system it is performed. This is due to the differences between different batches of manufacturer’s tissue factor used in the reagent to perform the test. The INR was devised to standardize the results. Each manufacturer assigns an ISI value (International Sensitive Index) for any tissue factor they manufacture. The ISI value indicates how a particular batch of tissue factor compres to an internally standardized sample. The ISI is usually between 1.0 and 2.0. The INR is the ratio of a patient’s prothrombin time to a normal (control) sample, raised to the power of the ISI value for the analytical system used.

Interpratation

The prothrombin time is the time it takes plasma to clot after addition of tissue factor (obtained from animals). This measures the quality of the extrinsic pathway (as well as the common pathway) of coagulation. The speed of the extrinsic pathway is greatly affected by evels of factor VII in the body. Factor VII has ashort half-life and its snthesis requires vitamin K. The prothrombin time can be prolonged as a result of deficiency in vitamin K, which can be caused by warfarin, malabsorption, or lack of intestinal colonization by bacteria (such as in newborns). In addition, poor factor VII synthesis (due to liver disease) or increased consumption (in disseminated intravascular coagulation) may prolong the PT. A high INR level such as INR=0.5 then there is a high chance of having a clot. Normal range for a healthy person is 0.9-1.3, and for people on warfarin therapy, 2.0-3.0, although the target INR may be higher in particular situations, such as for those with a mechanical heart valve,  or bridging warfarin with a low-molecular weight heparin (such as anoxaparin) perioperatively.

Factors determining accuracy

Lupus anticoagulant, a circulating inhibitor predisposing for thrombosis, may skew PT results, depending on the assay used. Variations between various thromboplastin preparations have in the past led to decreased accuracy of INR readings, and a 2005 study suggested that despite international calibration efforts (by INR) there were still statistically significant differences between various kits, casting doubt on the long-term tenability of PT/INR as a measure for anticoagulant therapy.

Statistic

An estimated 800 million PT/INR assays are performed annually worldwide.

Near-patient testing

In addition to the laborator method outlined above, near patient testing (NPT) or home INR monitoring is becoming increasingly common is some countris. In the United Kingdom, for example, near-patient testing is used both by patients at home. and by some anticoagulant clinics (often hospital-based) as a fast and convenient alternative to the lab method. After a period of doubt about the accuracy of NPT results, a new generation of machines and reagents seems to be gaining acceptance for its ability to deliver results close in accuracy to those of the lab. In a typical NPT setup a small table-top device is used; for example the Roche Coaguchek S, or the more recently (2005) introduced HemoSense INRatio. A drop of capillary blood is obtained with an automated finger-prick, which is almost painless. This drop is placed on a disposable test strip with which the machine has been prepared. The resulting INR comes up on the display a few seconds later. Similar testing methods are used by diabetics on insulin, and are easily taught and practiced. Local policy determines whether the patient or a coagulation specialist (nurse, general practitioner or hospital doctor) interprets the result and determines the dose of medication. In Germany, patients may adjust the medication dose themselves, while in the UK and the USA this remains in the hands of a health care professional. For example, patients using services such as Philips INR@Home  will phone in their INR results on a weekly basis and this information is transmitted to their doctor, who is also alerted if out-of-range levels should require an immediate intervention or adjustment to medications. A significant advantage of home testing is the evidence that patient self-testing with medical support and patient self-management (where patients adjust their own anticoagulant dose) improves anticoagulant control. A meta analysis which reviewed 14 trials showed that home testing led to a reduced incidence of complications (bleeding and thrombosis) and improved the time in the therapeutic range, which is an indirect measure of anticoagulant control. Other advantages of the NPT approach are that it is fast and convenient, usually less painful, and offers, in home use, the ability for patients to measure their own INRs when required. Among its problems are that quite a steady hand is needed to deliver the blood to the exact spot, that some patients find the finger-pricking difficult, and that the cost of the test strips must also be taken into account. In the UK these are available on prescription so that elderly and unwaged people will not pay for them and others will pay only a standard prescription charge, which at the moment represents only about 20% of the retail price of the strips. In the USA, NPT in the home is currently reimbursed by Medicare for patients with mechanical heart valves, while private insurers may cover for other indications.Medicare is now covering home testing for patients with chronic atrial fibrillation. Requires a doctor’s prescription. There is some evidence to suggest that NPT may be less accurate for certain patients, for example those who have the lupus anticoagulant.

Guidelines

International guidelines were published in 2005 to govern home monitoring of oral anticoagulation by the International Self-Monitoring Association for Oral Anticoagulation.The international guidelines study stated, “The consensus agrees that patient self-testing and patient self-management are effective methods of monitoring oral anticoagulation therapy, providing outcomes at least as good as, and possibly better than, those achieved with an anticoagulation clinic. All patients must be appropriately selected and trained. Currently available self-testing/self-management devices give INR results which are comparable with those obtained in laboratory testing.” Medicare coverage for home testing of INR has been expanded in order to allow more people access to home testing of INR in the USA. The release on the 19th March 2008 said, “[t]he Centers for Medicare & Medicaid Services (CMS) expanded Medicare coverage for home blood testing of prothrombin time (PT) International Normalized Ratio (INR) to include beneficiaries who are using the drug warfarin, an anticoagulant (blood thinner) medication, for chronic atrial fibrillation or venous thromboembolism.” In addition, “[t]hose Medicare beneficiaries and their physicians managing conditions related to chronic atrial fibrillation or venous thromboembolism will benefit greatly through the use of the home test.”

History

The prothrombin time was discovered by Dr Armand Quick and colleagues in 1935 , and a second method was published by Dr Paul Owren, also called the “p and p” or “prothrombin and proconvertin” method. It aided in the identification of the anticoagulants dicumarol and warfarin, and was used subsequently as a measure of activity for warfarin when used therapeutically. The INR was introduced in the early 1980s when it turned out that there was a large degree of variation between the various prothrombin time assays, a discrepancy mainly due to problems with the purity of the thromboplastin (tissue factor) concentrate. The INR became widely accepted worldwide, especially after endorsement by the World Health Organisation.

The ristocetin induced platelet aggregation (RIPA) is an in vitro assay for von Willebrand factor activity used to diagnose von Willebrand disease. It has the benefit over the ristocetin cofactor activity in that it can diagnose type 2B vWD and Bernard-Soulier syndrome.

In an unknown fashion, the antibiotic ristocetin causes von Willebrand factor to bind the platelet receptor glycoprotein Ib (GpIb), so when ristocetin is added to normal blood, it causes agglutination. In von Willebrand disease, where von Willebrand factor is absent or defective, abnormal agglutination occurs:

• In type 1 vWD: no agglutination occurs
• In type 2A vWD: no agglutination occurs
• In type 2B vWD: hyperactive agglutination occurs
• In type 2N vWD: normal agglutination occurs
• In type 3 vWD: no agglutination occurs

Von Willebrand factor (vWF) is a blood glycoprotein involved in hemostasis. It is deficient or defective in von Willebrand disease and is involved in a large number of other diseases, including thrombotic thrombocytopenic purpura, Heyde’s syndrome, and possibly hemolytic-uremic syndrome.

 Biochemistry

Synthesis

vWF is a large multimeric glycoprotein present in blood plasma and produced constitutively in endothelium (in the Weibel-Palade bodies), megakaryocytes (α-granules of platelets), and subendothelial connective tissue.^[1]

 Structure

The basic vWF monomer is a 2050 amino acid protein. Every monomer contains a number of specific domains with a specific function; elements of note are:^[1]

• the D’/D3 domain, which binds to Factor VIII
• the A1 domain, which binds to:
  • platelet GPIb-receptor
  • heparin
  • possibly collagen
• the A3 domain, which binds to collagen
• the C1 domain, in which the RGD domain binds to platelet integrin α_IIbβ₃ when this is activated
• the “cysteine knot” domain (at the C-terminal end of the protein), which vWF shares with platelet-derived growth factor (PDGF), transforming growth factor-β (TGFβ) and β-human chorionic gonadotropin (βHCG, of pregnancy test fame).

Monomers are subsequently N-glycosylated, arranged into dimers in the endoplasmic reticulum and into multimers in the Golgi apparatus by crosslinking of cysteine residues via disulfide bonds. With respect to the glycosylation, vWF is one of the few proteins that carry ABO blood group system antigens.

Multimers of vWF can be extremely large, >20,000 kDa, and consist of over 80 subunits of 250 kDa each. Only the large multimers are functional. Some cleavage products that result from vWF production are also secreted but probably serve no function.

 Function

Von Willebrand factor is not an enzyme and therefore has no catalytic activity. Its primary function is binding to other proteins, particularly Factor VIII and it is important in platelet adhesion to wound sites.

vWF binds to a number of cells and molecules. The most important ones are:

• Factor VIII is bound to vWF while inactive in circulation; Factor VIII degrades rapidly when not bound to vWF. Factor VIII is released from vWF by the action of thrombin.
• vWF binds to collagen, e.g., when it is exposed in endothelial cells due to damage occurring to the blood vessel.
• vWF binds to platelet gpIb when it forms a complex with gpIX and gpV; this binding occurs under all circumstances, but is most efficient under high shear stress (i.e., rapid blood flow in narrow blood vessels, see below).
• vWF binds to other platelet receptors when they are activated, e.g., by thrombin (i.e., when coagulation has been stimulated).

vWF appears to play a major role in blood coagulation. vWF deficiency or dysfunction (von Willebrand disease) therefore leads to a bleeding tendency, which is most apparent in tissues having high blood flow shear in narrow vessels. From studies it appears that vWF uncoils under these circumstances, decelerating passing platelets.^[1]

 Catabolism

The biological breakdown (catabolism) of vWF is largely mediated by the protein ADAMTS13 (acronym of “a disintegrin-like and metalloprotease with thrombospondin type 1 motif no. 13“). It is a metalloproteinase which cleaves vWF between tyrosine at position 842 and methionine at position 843 (or 1605–1606 of the gene) in the A2 domain. This breaks down the multimers into smaller units, which are degraded by other peptidases.

 Role in disease

In thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS), ADAMTS13 either is deficient or has been inhibited by antibodies directed at the enzyme. This leads to decreased breakdown of the ultra-large multimers of vWF and microangiopathic hemolytic anemia with deposition of fibrin and platelets in small vessels, and capillary necrosis. In TTP, the organ most obviously affected is the brain; in HUS, the kidney.

Higher levels of vWF are more common among people that have had ischaemic stroke (from blood-clotting) for the first time. Occurrence is not affected by ADAMTS13, and the only significant genetic factor is the person’s blood group.

 History

vWF is named after Dr. Erik von Willebrand (1870–1949), a Finnish doctor who in 1924 first described a hereditary bleeding disorder in families from the Åland islands, who had a tendency for cutaneous and mucosal bleeding, including menorrhagia. Although von Willebrand could not identify the definite cause, he distinguished von Willebrand disease (vWD) from haemophilia and other forms of bleeding diathesis.

In the 1950s, vWD was shown to be caused by a plasma factor deficiency (instead of being caused by platelet disorders), and, in the 1970s, the vWF protein was purified.

 Interactions

Von Willebrand factor has been shown to interact with Collagen, type I, alpha 1.

Coagulation is a complex process by which blood forms clots. It is an important part of hemostasis (the cessation of blood loss from a damaged vessel), wherein a damaged blood vessel wall is covered by a platelet and fibrin-containing clot to stop bleeding and begin repair of the damaged vessel. Disorders of coagulation can lead to an increased risk of bleeding (hemorrhage) or clotting (thrombosis). Coagulation is highly conserved throughout biology; in all mammals, coagulation involves both a cellular (platelet) and a protein (coagulation factor) component. The system in humans has been the most extensively researched and, therefore, the best-understood. Coagulation begins almost instantly after an injury to the blood vessel has damaged the endothelium (lining of the vessel), this releases phospholipid components called tissue factor and fibrinogen that initiate a chain reaction. Platelets immediately form a plug at the site of injury; this is called primary hemostasis. Secondary hemostasis occurs simultaneously: Proteins in the blood plasma, called coagulation factors or clotting factors, respons in a complex cascade to form fibrin strands, which strengthen the platelet plug.

Physiology

Platelet activation

Damage to blood vessel walls exposes subendothelium proteins, most notably von willebrand factor (VWF), present under the endothelium. VWF is a protein secreted by healthy endothelium, forming a layer between the endothelium and underlying basement membrane. When the endothelium is damaged, the normally-isolated, underlying vWF is exposed to blood and recruits Factor VII, collagen, and other clotting factors. Circulating platelets bind to collagen with surface collagen-specific glycoprotein Ia/IIa receptors. This adhesion is strengthtened further by additional circulating proteins vWF), which forms additional links between the platelets glycoprotein Ib/IX/V and the collagen fibrils. These adhesions activate the platelets. Activated platelets release the contents of stored granules into the blood plasma. The granules include ADP, serotonin, platelet-activating factor (PAF), vWF, platelet factor 4, and thromboxane A₂ (TXA₂), which, in turn, activate additional platelets. The granules’ contents activte a  G_q-linked protein receptor cascade, resulting in increased calcium concentration in the platelets’ cytosol. The calcium activates protein kinase C, which, in turn, activates phospholipase A₂ (PLA₂). PLA₂ then modifies the integrin membrane glycoprotein IIb/IIIa, increasing its affinity to bind fibrinogen. The activated platelets change shape from spherical to stellate, and the fibrinogen cross-links with glycoprotein IIb/IIIa aid in aggregation of adjacent platelets.

The coagulation cascade