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Introduction to Haemostasis and Haemostatic Disorders
Chapter Outline
• Haemostasis (Stoppage of Bleeding)
▪ Vascular response
▪ Role of platelets
▪ Role of coagulation factors
• Mechanism of Blood Coagulation
▪ Stage 1: Generation of plasma thromboplastin
▪ Stage 2: Formation of thrombin from prothrombin
▪ Stage 3: Formation of brin from brinogen
• Fibrinolysis
• Disorders of Haemostasis
▪ Hereditary haemostatic disorders
▪ Hereditary platelet disorder
▪ Acquired disorders of haemostasis
• Control Mechanisms of Haemostasis
• Laboratory Tests for Haemostatic Function
• Review Questions
13
Ritwik Bhatia
Bleeding occurs when the blood vessel is injured and bleeding stops by a process called
haemostasis (stoppage of bleeding). This process consists of biochemical and physical reactions
that help to plug the leak in the blood vessel. Any interruption in this process leads to abnormal
bleeding. This calls for clinical as well as laboratory investigations. For example, patients
prepared for surgical procedures must be routinely tested to ensure normal haemostasis and
appropriate care must be taken in case of any abnormality in the haemostatic process.
Haemostatic disorders can be inherited or acquired. Classic haemophilia (Haemophilia A)
is inherited and runs in the family while vitamin К deciency is acquired (see “Disorders of
Haemostasis” below). Vitamin К deciency can be triggered by the use of warfarin (Coumadin),
intestinal obstruction or malabsorption, or more rarely, poor nutritional intake. Similarly, use
of aspirin, a blood thinner, might lead to bleeding problems, and other drugs can accelerate
clot formation. These alterations of haemostasis can prove to be fatal if neglected. They
aect patients’ lives and require a combination of clinical examination, laboratory tests and
investigation into family medical history for diagnosis. The coagulation laboratory (a part of the
haematology laboratory) helps in the identication of the bleeding defect. Before we proceed to
learn the laboratory tests used in the diagnosis of bleeding disorders, we need to understand
the normal mechanism of haemostasis and the clinical approach to diagnose abnormalities.
Introduction to Haemostasis and Haemostatic Disorders
339
Haemostasis (stoppage of Bleeding)
Haemostasis is a complex process that involves four major steps in sequence (Figure 13.1)—
vascular response, platelet plug formation, coagulation (clot formation) and nally, brinolysis
(dissolving of the clot after healing). The last step is important in order to keep the wall of
the blood vessel smooth for the ow of blood (Figure 13.1). The defence system of the body
towards vascular injury is illustrated in Figure 13.3. It will be appropriate here to evaluate
the roles of blood vessels (vascular response), platelets, coagulation factors and brinolysis in
this complex mechanism that leads to the stoppage of bleeding.
Figure 13.1 Interaction of four processes in haemostasis
Vascular Response
Vasoconstriction is the immediate response of the blood vessels to the injury. This constriction
reduces the blood ow which assists in the process of platelet plug formation. Muscular
contraction and release of serotonin (and other chemical mediators) by the damaged platelets
are contributory factors in the formation of the plug. The vascular response cannot be clearly
separated from the platelet response, and hence, the basic screening tests—capillary fragility
test and bleeding time—measure both.
Role of Platelets
This occurs when platelets adhere to the vessel wall at the site of injury. The steps of plug
formation include aggregation of platelets followed by deposition of brin around the platelet
aggregate. The brin binds platelets together and anchors the platelet plug to the vessel wall
as an impermeable seal over the injury site in order to arrest bleeding. The eectiveness of
platelets in haemostasis and platelet-related defects can be assessed routinely by bleeding
time-determination, platelet count and clot retraction test.
Role of Coagulation Factors
The circulating plasma and tissues surrounding blood vessels contain a total of twelve factors,
though numbered from I-XIII. This is because factor VI is now found to be the same as factor
V and hence, it is missing from the list. The coagulation factors are numbered I through
XIII in the order in which they were discovered, not in the order of their action. Factor IV
(calcium) is also not used in the modern nomenclature. Instead, it is represented as ionized
calcium (Ca++). Other than calcium, an inorganic element, all other coagulation factors are
proteins. These coagulation factors are present in the circulation under normal conditions,
in an inactive form, with the readiness to be converted into active enzymes to initiate and
complete the coagulation process whenever there is a bleeding episode. These coagulation
factors participate in three sequential stages in order to form the clot (brin). The list of the
coagulation factors is presented in Table 13.1.
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Medical Laboratory Technology: Volume 1
Table 13.1 International Roman numerical nomenclature for blood coagulation factors
Factor Synonym
1 Fibrinogen
II Prothrombin
III Thromboplastin, tissue factor
Ionized Calcium (Ca
V Prothrombin accelerator (Proaccelerin)
VII Proconvertin or stable factor
VIII Anti-haemophilic factor (AHF) or anti-haemophilic factor A
IX Plasma thromboplastin component (PTC) or Christmas factor or anti-haemophilic
factor В
X Stuart–Prower factor
XI Plasma thromboplastin antecedent (PTA) or anti-haemophilic factor С
XII Hageman factor or contact factor
XIII Fibrin-stabilizing factor
No number assigned Figerald factor
No number assigned Fletcher factor
++
)
Platelets are the cells and are not included in the list of coagulation factors. Platelets,
however, release several factors that help in the cloing process. In addition, thromboplastin
and thrombin are also not included in the list. These are intermediates of the coagulation
process, and are not normally present in the body unless bleeding occurs.
Although coagulation factor interaction involves very complex reactions but it can be
summarized in three general sequential reactions:
1. Activation of tissue factors (vascular response).
2. The conversion of prothrombin to thrombin in the presence of thrombokinase and
calcium.
3. The conversion of brinogen to brin due to the action of thrombin.
Introduction to Haemostasis and Haemostatic Disorders
341
Normal fresh plasma contains all factors listed in Table 13.2 except factor IV (calcium),
which is removed by the anticoagulant. Thus, addition of calcium makes normal plasma
clot within a few seconds. Fresh normal plasma can be processed in a way that a group of
factors can be isolated in various plasma preparations shown in Table 13.2. These reagent–
plasma components can be used in the replacement of decient factors for the laboratory
investigation of missing coagulation factors in order to conrm ndings. The procedure for
the preparation of reagent-plasma will be presented later.
Table 13.2 Preparation of reagent plasma for coagulation studies
Reagent-plasma Factors missing Factors present
Aged plasma or serum I*, V, VIII VII, IX, X, XI, XII
Adsorbed plasma II, VII, IX, X I, V, VIII
Normal plasma
* In aged plasma, factor I is present and in aged serum, it is missing.
†
In anticoagulated plasma, calcium (Factor V) is removed by the anticoagulant in order to prevent cloing.
†
None All factors present
mecHanism of Blood coagulation
An understanding of the mechanism of blood coagulation is necessary in order to comprehend
the procedure and signicance of routine coagulation tests.
Blood coagulation occurs in three sequential stages (Figure 13.2)—(1) generation of plasma
thromboplastin, (2) formation of thrombin from prothrombin, and (3) formation of brin clot
from brinogen.
Stage1 : Generation of Plasma Thromboplastin
The tissue extract or tissue thromboplastin (factor III) enters the blood vessel through the
site of injury and combines with factor VII and calcium (factor IV). This is designated as the
extrinsic system that originates from outside the blood vessel and includes factors such as
III, VII and IV (calcium). The extrinsic system then joins the intrinsic system. The factors that
participate in the intrinsic system are all present in the blood circulation (XII, XI, IX, VIII and
calcium). The extrinsic and intrinsic systems after joining together within the blood vessel
follow a common path involving factors X and V. Finally, the end product of Stage 1— plasma
thromboplastin—is formed. The thromboplastin then triggers the Stage 2, which otherwise
remains dormant in the blood with the inactive prothrombin (factor II).
Any defect in the intrinsic system of Stage 1 is recognized by activated partial thromboplastin
time (APTT).
Stage 2: Formation of Thrombin from Prothrombin
In Stage 2, prothrombin (factor II) is activated by the thromboplastin, the end product of
Stage 1. This results in the formation of thrombin. Any defect in Stage 2 (factor II deciency)
will be recognized by prolonged prothrombin time (PT). As stated earlier, the APTT will also
be prolonged as a secondary eect. The cascade system of coagulation steps further continues
to Stage 3.
Stage 3: Formation of Fibrin from Fibrinogen
This is the nal stage of the coagulation process. Inactive brinogen (factor I) is activated by
the end product of Stage 2, thrombin. The brinogen is rst converted into soluble brin form
(brin monomers). Soluble brin polymerizes with the help of activated factor XIII to form an
insoluble and mechanically strong brin clot (Figure 13.2).
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Medical Laboratory Technology: Volume 1
Figure 13.2 Cascade hypothesis of haemostasis—MacFarlane (1964); Davie and Ratno (1964)
Fibrinolysis
Fibrinolysis is a process whereby the brin clot is degraded into soluble brin degradation
products (FDP) around the site of injury, while the brin beyond this point is normally removed
(Figure 13.3). Activated intermediates of the coagulation process, which escape from the site
of injury, are diluted and dispersed in the blood stream. If these defences are insucient and
the brin is deposited within the blood vessel, at a distance from the haemostatic plug, it will
be removed by brinolysis (Figure 13.4).
Introduction to Haemostasis and Haemostatic Disorders
Figure 13.3 Platelet activation and the process of healing of blood vessel injury
343
Disorders of Haemostasis
Haemostatic disorders have a variety of causes. They are broadly divided as hereditary or
acquired causes. Diagnosis of these bleeding disorders requires a combination of clinical
examination, laboratory tests and investigation into family medical history for diagnosis. The
following account provides a general idea of the clinical approach and its laboratory support
for the diagnosis.
Hereditary Haemostatic Disorders
The haemophilias are inherited diseases in which there is a deciency or functional disorder
in one or more of the coagulation factors listed in Table 13.1. Classic haemophilia, called
haemophilia A,, is caused by a functional deciency of coagulation factor VIII. Haemophilia
B, also known as Christmas disease, is named after the rst patient in whom it was discovered.
The defect in Christmas disease is a functional deciency of coagulation factor IX.
Haemophilia A and В are inherited as sex-linked recessive genes carried on the X
chromosome. Since males inherit one X and one Y, if the inherited X chromosome carries
the recessive haemophilia gene, the gene will be expressed. Since females have two X
chromosomes, those who carry the recessive haemophilia gene on one X chromosome will
not manifest the disease because the normal X chromosome will be dominant. These females
are called carriers and can pass the recessive gene to their ospring. Therefore, diseases are
limited almost exclusively to males, although there have been rare cases documented in
females.
344
Figure 13.4 Fibrinolytic pathway and formation of brinogen and brin degradation products
The clinical symptoms of haemophilias A and В are identical. Aected infants do not have
symptoms unless they undergo circumcision or other surgery. However, as the child grows
and is subject to bumps and falls, large bruises appear. Abnormal bleeding occurs into the
joints, causing swelling and pain. In addition, bleeding occurs in the mouth, muscles, renal
tract, and gut and after dental extractions.
The most common inherited factor deciency is von Willebrand’s disease (vWD). It is not
really a disease, but is a condition caused by the deciency or functional abnormality of von
Willebrand factor (vWF). The vWF is a portion of factor VIII molecule, the VIII:vWF segment.
Although the deciency is in a coagulation factor, it results in alteration of platelet adhesion.
Most cases of von Willebrand’s disease are mild and display symptoms such as nose bleeds,
bleeding of the gums and easy bruising. Unlike the classic haemophilias, von Willebrand’s
disease is inherited as an autosomal trait and so can occur in both males and females.
Medical Laboratory Technology: Volume 1
Hereditary Platelet Disorder
Several inherited disorders of platelets cause prolonged bleeding in the patient. BernardSoulier syndrome is a disorder in which the platelets are larger than normal and are present
in normal or decreased numbers. A defect on the surface of the platelet membrane causes a
decrease in the adherence of platelets. The aected patients develop small, purplish spots
on the skin called petechiae. They also suer gastrointestinal bleeding, nose bleeding and
abnormal menstrual bleeding or intracranial bleeding. The disease can be severe and even
fatal.
In case of Glanzmann’s thrombasthenia, symptoms are similar but here platelets are normal
in number with the ability to adhere to each other but they do not have normal aggregation. If
one performs the cloing test, the clot does not form the characteristic clump.
As discussed earlier, in von Willebrand’s disease, the factor deciency of VIII is combined
with the poor adhesion function of platelets, although the number of platelets remains normal.
Introduction to Haemostasis and Haemostatic Disorders
345
Acquired Disorders of Haemostasis
Some of the disorders of haemostasis may not be hereditary. In these cases, abnormal bleeding
may be acquired by an individual that can aect one or more of the coagulation factors or
platelets.
The acquired factor disorders can be due to four common causes:
1. Vitamin К deciency
2. Disseminated intravascular coagulation
3. Circulating inhibitors to coagulation factors
4. Platelet disorder
Several coagulation factors are vitamin K-dependent. Thus a deciency of vitamin К leads
to their decreased production—II, VII, IX and X—and their deciency in circulation. The
deciency of vitamin К is rarely due to dietary insuciency. It may, however, be related to its
malabsorption from the intestine due to prolonged antibiotic treatment. Some drugs like oral
anticoagulant Coumadin, which is prescribed to reduce unwanted cloing, acts as vitamin
К antagonist. This results in a decrease in vitamin K-dependent factors and thus works as a
therapeutic measure against intravascular cloing.
Disseminated intravascular coagulation (DIC) is a serious condition in which a pathological
process initiates coagulation and secondary brinolysis. Depending on the balance between
the two systems, cloing and brinolysis, there may be thrombosis or bleeding, or a combination
of the two. Some conditions that contribute to DIC are crush injuries; certain bacterial, viral
or rickesial infections; and drug reactions. Disseminated intravascular coagulation occurs
when intravascular coagulation is triggered. This causes brin deposition in blood vessels,
which in turn uses up the available supply of coagulation factors. Microthrombi forms in
vital organs, including kidneys, lungs and brain. The brinolytic system is then activated,
releasing brin fragments that can act as coagulation inhibitors.
Circulating inhibitors to coagulation factors is another acquired haemostatic disorder.
These inhibitors are also called circulating anticoagulants. Usually they are autoantibodies,
produced within the patient’s circulatory system and directed at one or more of the cloing
factors, causing factor(s) to be removed from the blood, creating a factor deciency.
Acquired platelet disorder can arise from several sources. This is grouped under the
general name of idiopathic thrombocytopenic purpura (ITP). One such clinical situation
is when the immune system makes antibodies against the patient’s own platelets making
them non-functional. It may also be caused by an enlarged spleen that traps many platelets
(sequestration) and thus reduces the number of platelets in circulation. Infection by certain
viruses can decrease the bone marrow production of megakaryocytes, cells that produce
platelets. Ingestion of aspirin can also aect the aggregation of platelets by inhibiting the
platelet-release reaction. The eect of aspirin persists for about 8–10 days.
Control Mechanisms of Haemostasis
Under normal circumstances, a clot forms only at the site of injury. Regulating systems
prevent an isolated injury from initiating the cloing mechanism throughout the body. The
body’s defence mechanism prevents the formation of unwanted clots.
Unfortunately, sometimes the haemostatic mechanism goes amiss and intravascular blood
clots can form and cause problems. In atherosclerosis, arterial blood vessel walls become
rough and irregular. This can cause platelets to become activated and initiate the formation
of clots within the blood vessels, known as intravascular cloing. A blood clot aached
to a vessel wall is called a thrombus; however, when it breaks o and travels through the
circulatory system, it is called an embolus. Emboli are very dangerous because they become
lodged in small vessels in brain, lungs and other organs. They cause serious, irreversible
346
damage and sometimes death unless they are dissolved. Heart patients are often prescribed
aspirin at a low dosage in order to keep their blood circulating without any interruption.
Aspirin acts as a blood thinner and prevents lodging of the emboli. Patients being prepared
for any invasive procedure or surgery are asked to stop aspirin prior to the procedure so as to
allow the desirable natural healing process.
Anticoagulant therapy is recommended in order to prevent and treat thrombosis (clot
formation in blood vessels). Two types of anticoagulants are commonly used to treat this
condition—heparin and Coumadin (sodium warfarin). Heparin administered intravenously
is eective immediately but is usually used only for short term. Coumadin and heparin can
be administered orally and used for long term. Patients who have undergone major surgical
procedures or joint replacement (hip or knee) are at risk for blood clot formation. The oral
anticoagulants allow them to return home and be treated as outpatients. Patients with
certain conditions such as atherosclerosis or phlebitis may have oral anticoagulants (blood
thinners) prescribed for long term to prevent the formation of thrombi. These patients must
be monitored by having their coagulation times checked at regular intervals.
Medical Laboratory Technology: Volume 1
Laboratory Tests for Haemostatic Function
The physician depends on the laboratory report in his assessment of haemostatic function.
The testing involves an evaluation of the circulating platelets, response of blood vessels or
the absence of any coagulation factor. The commonly employed laboratory tests are: platelet
count, bleeding time, cloing time, prothrombin time and APTT. Expected laboratory results
for some disorders of haemostasis are shown in Table 13.2. Some of the tests are done on the
patient (e.g., bleeding time) while others can be carried out in the laboratory with the blood
specimen collected from the patient. Whole blood is used for platelet count. Plasma is the
specimen of choice in most coagulation tests (e.g., PT and APTT) where citrate is used as
an anticoagulant. Modern equipment can perform coagulation tests using whole blood with
results being available within minutes. They are ideal in the physician’s oce seing and
very useful in guiding heparin therapy.
Platelet count, discussed in the earlier chapter (Chapter 10) is a quantitative test for
platelets. However, even if the platelet count is in the normal range, a bleeding problem
can still exist if platelets do not function normally. Bleeding time and cloing time tests
are performed to assess quantitative and qualitative abnormalities in platelets, and also for
vascular integrity of capillaries. When bleeding time is prolonged, more denitive tests, such
as platelet adhesion and platelet aggregation, must be performed.
Prothrombin time is a measure of the extrinsic pathway while the APTT tells us regarding
intrinsic pathway function. More elaborate tests are performed in search of specic missing
factor which is beyond the scope of this book. In the following chapter (Chapter 14) we will
discuss the procedure of some of the aforementioned basic tests requested by the physician
for the diagnosis of bleeding problems.
Review Questions
1. Explain the following terms.
Haemostasis, Coagulation factors, Vascular defect, Bleeder and Platelet plug
2. Explain the mechanism of haemostasis. How is the coagulation process dierent from
that of the vascular response?
3. How would you determine defects in Stages 1, 2 and 3?
4. Which coagulation factors are missing in aged plasma and which are present in adsorbed
plasma?
5. How are the extrinsic and intrinsic systems of coagulation dierent?
Introduction to Haemostasis and Haemostatic Disorders
347
6. What is brinolysis? How does it occur in the body? What is the clinical signicance of
FDP determination?
7. Name two acquired and two inherited disorders of platelets.
8. What is the eect of aspirin on platelets?
9. List the vitamin K-dependent coagulation factors. What are the common causes of
vitamin К deciency?
10. Name two hereditary disorders of coagulation factors.
11. Explain the dierences between haemophilia A and haemophilia B.
12. Explain why haemophilia A and haemophilia В are almost exclusively limited to males.
13. Discuss conditions that contribute to DIC.
14. What causes von Willebrand’s disease?
15. Give some of the laboratory ndings in the diagnosis of haemophilia A, haemophilia В
and von Willebrand’s disease.
16. Dene petechiae and describe under what conditions they develop in patients.
17. Explain the dierences between haemophilia A and haemophilia B. Why are they almost
exclusively limited to males?
18. What cause DIC? How is this recognized?