Bleeding disorders is caused by the abnormalities of hemostasis and / or coagulation, characterized by local or extensive skin or mucocutaneous hemorrhage derived from capillary. The bleeding is usually spontaneous or from slight trauma .
Blood vessel injury triggers the following sequence:
1. vessel constricts
Circulating platelets adhere to the vessel
an intricate series of enzymatic reactions
involving coagulation proteins, produces
fibrin to form a stable haemostatic plug
Vascular damage results in initiation of clotting with the goal of producing a localized platelet or fibrin plug to prevent blood loss; this action is followed by processes that lead to clot containment, would healing, clot dissolution, and tissue regeneration and remodeling. introduction
Haemostasis or normal blood clotting is essential for survival
---platelet plug formation at sites of injury
---plasma coagulation system reaction resulting in fibrin formation
Primary and secondary hemostasis are closely linked
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 VIII , collagen , and other clotting factors. Circulating platelets bind to collagen with surface collagen-specific glycoprotein Ia/IIa receptors. Platelet activation
This adhesion is strengthened 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. Platelet activation
The activated platelets change shape from spherical to stellate, and the fibrinogen cross-links with glycoprotein IIb/IIIa aid in aggregation of adjacent platelets. Platelet activation
Coagulation involves a complex set of protease reactions involving roughly 30 different proteins. The final result of these reactions is to convert fibrinogen, a soluble protein, to insoluble strands of fibrin . Together with platelets, the fibrin strands form a stable blood clot. Coagulation cascade
After the formation of a stable vascular “plug”, The subsequent process of clot dissolution that occurs during the healing phase is known as “fibrinolysis”. Coagulation cascade
the contact activation pathway ( formerly known as the intrinsic pathway), the tissue factor pathway ( formerly known as the extrinsic pathway), It was previously thought that the coagulation cascade consisted of two pathways of equal importance joined to a common pathway. Coagulation cascade 14
The intrinsic /extrinsic pathway model X Xa IX and XII extrinsic pathway intrinsic pathway coagulation cascade IIV 15
the primary pathway : tissue factor pathway. The pathways are a series of reactions: a zymogen active components catalyze the next reaction resulting in cross-linked fibrin. Coagulation cascade 16
Coagulation factors are generally indicated by Roman numerals , with a lowercase a appended to indicate an active form. Coagulation cascade X Xa 17
The coagulation cascade is classically divided into three pathways. The tissue factor and contact activation pathways both activate the "final common pathway" of factor X , thrombin and fibrin . Coagulation cascade
The main role of the tissue factor pathway is to generate a "thrombin burst," a process by which thrombin , the most important constituent of the coagulation cascade in terms of its feedback activation roles, is released instantaneously. FVIIa circulates in a higher amount than any other activated coagulation factor. Tissue factor pathway (extrinsic) 19
FVII is itself activated by thrombin, FXIa, plasmin , FXII and FXa. FXa and its co-factor FVa form the prothrombinase complex, which activates prothrombin to thrombin. The activation of FXa by TF-FVIIa is almost immediately inhibited by tissue factor pathway inhibitor (TFPI). Tissue factor pathway (extrinsic) 21
Thrombin then activates other components of the coagulation cascade, including FV and FVIII (which activates FXI, which, in turn, activates FIX), and activates and releases FVIII from being bound to vWF. Tissue factor pathway (extrinsic) 22
FVIIIa is the co-factor of FIXa, and together they form the " tenase " complex, which activates FX; the cycle continues. ("Tenase" is a contraction of "ten" and the suffix "-ase" used for enzymes.) Tissue factor pathway (extrinsic) 23
Contact activation pathway (intrinsic) The contact activation pathway begins with formation of the primary complex on collagen by high-molecular-weight kininogen (HMWK), prekallikrein , and FXII. Prekallikrein is converted to kallikrein and FXII becomes FXIIa. 24
Final common pathway Thrombin has a large array of functions. Its primary role is the conversion of fibrinogen to fibrin, the building block of a hemostatic plug. Prothrombin Thrombin (FIIa) fibrinogen fibrin (F I) (F Ia) FXa FVa
Fibrinolysis: restoring blood flow Fibrinolysis, as the term implies, is the process that dissolves fibrin . It leads to clot dissolution. Plasminogen is the precursor of plasmin , which breaks up fibrin clots. During initial clot formation, plasminogen activators are inhibited. 27
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 . Fibrinolysis 28
Over time, endothelial cells begin to secrete tissue plasminogen activators to start dissolving the clot as the structural integrity of the blood vessel wall is restored. Fibrinolysis
While haemostasis is necessary for survival, the pathological formation of a blood clot, or thrombosis, poses significant health risks. Thrombosis plays a role in heart attack, ischaemic stroke , cardioembolic stroke in patients with atrial fibrillation (AF), and venous thromboembolism (VTE).
Types of hemorrhage Petechia: a small (1-2mm) red or purple spot on the body, caused by a minor hemorrhage (broken capillary blood vessels ) ,pinpoint hemorrhage, <2mm in diameter. 33
Petechiae are characteristic of an abnormality of the vessels or the platelets, such as thrombocytopenia, and are exceedingly rare in the coagulation disorders. These lesions are small capillary hemorrhages ranging from the size of a pinhead to much larger. Petechiae 35
the appearance of red or purple discolorations on the skin that do not blanch on applying pressure. They are caused by bleeding underneath the skin. Purpura measure 0.3-1 cm, whereas petechiae measure less than 3 mm, and ecchymoses greater than 1 cm Purpura Types of hemorrhage
Petechial spots over the back
Ecchymosis: the medical term for a subcutaneous hematoma larger than 1 centimeter, commonly called a bruise . It can be located both in the skin as well as in a mucous membrane . Types of hemorrhage
In the purpuric disorders, petechiae commonly are associated with multiple superficial ecchymoses, which usually develop without perceptible trauma but seldom spread into deeper tissues. Small isolated ecchymoses are commonly noted in apparently normal women, especially on the legs, and in small children. ecchymoses
Hemarthrosis: a bleeding into joint spaces. most seen in hemophilia A or hemophilia B, rare in disorders of the vessels and platelets or in acquired coagulation disorders. Types of hemorrhage
Hemarthrosis in knee
Bleeding from a platelet disorder
Bleeding sites localized to superficial sites such as the skin and mucous membranes
Immediately after trauma or surgery
Readily controlled by local measures
Bleeding from coagulation defects
Bleeding sites: in deep subcutaneous tissues, muscles, joints, or body cavities
time: hours or days after injury
Unaffected by local therapy
Testing of coagulation Numerous tests are used to assess the function of the coagulation system: Common: aPTT , PT (also used to determine INR ), fibrinogen testing, platelet count, platelet function testing .
a performance indicator measuring the efficacy of both the "intrinsic" 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 . activated partial thromboplastin time ( aPTT or APTT )
A phlebotomist collects blood samples in vacu-tubes with oxalate or citrate to arrest coagulation by binding calcium. In order to activate the intrinsic pathway, phospholipid , an activator (such as silica , celite , kaolin ), 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. Method
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.
Interpretation Prolonged APTT may indicate: 1. use of heparin (or contamination of the sample) 2. antiphospholipid antibody (especially lupus anticoagulant , which paradoxically increases propensity to thrombosis ) 3. coagulation factor deficiency (e.g. hemophilia )
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 . Prothrombin time The reference range for prothrombin time is usually around 12–15 seconds; the normal range for the INR is 0.8–1.2.
They are used to determine the clotting tendency of blood, in the measure of warfarin dosage, liver damage, and vitamin K status. PT measures factors II , V, VII, X and fibrinogen . It is used in conjunction with the activated partial thromboplastin time (aPTT) which measures the intrinsic pathway . Prothrombin time
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 calcium in a sample. The blood is mixed, then centrifuged to separate blood cells from plasma. Methodology
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 of citrate), which enables the blood to clot again. the standardized dilution of 1 part anticoagulant to 9 parts whole blood is no longer valid. Methodology
Tissue factor (also known as factor III)is added, and the time the sample takes to clot is measured optically. The prothrombin ratio is the prothrombin time for a patient, divided by the result for control plasma. Methodology
International normalized ratio The result (in seconds) for a prothrombin time performed on a normal individual will vary depending on 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 Sensitivity Index) for any tissue factor they manufacture. The ISI value indicates how a particular batch of tissue factor compares to an internationally 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. INR=PT （ test ） /PT(normal)
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 . Interpretation
The speed of the extrinsic pathway is greatly affected by levels of factor VII in the body. Factor VII has a short half-life and its synthesis requires vitamin K . The prothrombin time can be prolonged as a result of deficiencies in vitamin K, which can be caused by warfarin , malabsorption , or lack of intestinal colonization by bacteria (such as in newborns ). Interpretation
In addition, poor factor VII synthesis (due to liver disease ) or increased consumption (in disseminated intravascular coagulation ) may prolong the PT. Interpretation
A high INR level such as INR=5 indicates that there is a high chance of bleeding, whereas if the 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 enoxaparin ) perioperatively.
Fibrinogen (also called factor I) is a 340 KDa glycoprotein synthesised in the liver by hepatocytes and megakaryocytes. The concentration in blood plasma is 1.5-4.0 g/L (normally measured using the Clauss method) or about 7 µM. Fibrinogen
In its natural form, fibrinogen can form bridges between platelets, by binding to their GpIIb/IIIa surface membrane proteins; however its major function is as the precursor to fibrin. Fibrinogen levels can be measured in venous blood. Fibrinogen
Higher levels are, amongst others, associated with cardiovascular disease (>4.6 g/L). It may be elevated in any form of inflammation , as it is an acute phase protein Fibrinogen
Low levels of fibrinogen can indicate a systemic activation of the clotting system, with consumption of clotting factors faster than synthesis. This excessive clotting factor consumption condition is known as Disseminated Intravascular Coagulation or "DIC." Fibrinogen
Fibrin is made from fibrinogen, a soluble plasma glycoprotein that is synthesised by the liver . Processes in the coagulation cascade activate the zymogen prothrombin to the serine protease thrombin , which is responsible for converting fibrinogen into fibrin. Fibrin
Fibrin (also called Factor Ia) is a fibrous protein involved in the clotting of blood, and is non globular. It is a fibrillar protein that is polymerised to form a "mesh" that forms a hemostatic plug or clot (in conjunction with platelets ) over a wound site. Fibrin
Fibrin and ligand
Platelet 1.Platelets are produced in blood cell formation ( thrombopoiesis ) in bone marrow, by budding off from megakaryocyte. 2.The physiological range for platelets is 150- 400 x 10 9 per litre. 3.Around 1 x 10 11 platelets are produced each day by an average healthy adult. 4.The lifespan of circulating platelets is 7 to 10 days.
Platelets play a fundamental role in hemostasis and are a natural source of growth factors . They circulate in the blood of mammals and are involved in hemostasis , leading to the formation of blood clots .
Both thrombocytopenia and thrombocytosis may present with coagulation problems. In general, low platelet counts increase bleeding risks; thrombocytosis (high counts) may lead to thrombosis, although this is mainly when the elevated count is due to myeloproliferative disorder .
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
D-dimer concentration may be determined by a blood test to help diagnose thrombosis . While a negative result practically rules out thrombosis, 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.
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 to fibrin an insoluble protein. Thrombin timec
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.
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. Thrombin time can be prolonged by: heparin, fibrin degradation products, lupus anticoagulant.
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. Procedure
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 Thrombin timec