HEMOSTATIC FUNCTION

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Hemostasis

Hemostasis is the process of arresting bleeding in which 3 important events take place Reaction of the bloopd vessels to the site of injury which is VASOCONSTRICTION Formation of platelet plug in the site of injury and release of active substances from the aggregated platelets Coagulation of Blood

It is also a process of (1) retains the blood within the vascular system during periods of injury (2) localizes the reactions involved in the site of injury (3) repairs/ re-establishes the blood flow through injured vessel

Importance of Balance in Hemostasis

Hemostasis Intricate system maintaining blood in fluid state reacts to vascular injury to stop blood loss and seal vessel wall Tightly Regulated System There should be a balance as to the formation and dissolution of hemostatic plug. If there is tilting of the balance it would result to bleeding or excessive clot formation/coagulation.

It involves 3 elements to form the Hemostatic Plug: Intact Vascular System Cellular Elements (mainly: Platelets but granulocytes, monocytes play some minor role) Coagulation Factors

Under normal conditions, the formation and dissolution of thrombi is maintained in a delicate balance. (fig ). Without this balance, the individual may experience either excessive bleeding (poor clot formation or excessive Fibrinolysis) Vaso-occlusion (uncontrolled formation of thrombin in vascular system, occluding vessels and depriving organs of blood).

Process of Hemostasis: There are substances released by the endothelial and subendothelial cells that either prevent clot formation or initiate platelet formation depending on the situation When there is blood vessel (BV) injury, there is a neural mechanism that causes transient vasoconstriction and at the same time there is endothelin (a potent endothelium-derived vasoconstrictor) release.

The effect is transient and bleeding would resume if not for activation of the platelet and coagulation systems

Events that take place are the interaction of the platelets and the vascular response to injury Initial response in primary hemostasis is Platelet adhesion, whereby in requires vWF for the platelet to adhere in the injured area of the endothelium There is a change in the shape of the platelet membrane There is also release of granules and promote platelet aggregation Release of platelet factor V, in the activated plelets, will lead toactivation of fibrin function, hence having a stable fibrin clot In a normal individual, coagulation is initiated within 20 seconds after an injury occurs to the blood vessel damaging the endothelial cells. PLATELETS immediately form a hemostatic plug at the site of injury. This is called primary hemostasis. BV injury exposes collagen which will attract platelets. Platelets will adhere to that site of injury thru membrane receptors (glycoprotein Ib (GpIb) receptors) which will link with von Willebrand factor (vWF) on exposed extracellular matrix (ECM). The platelets will then change shape from round or discoid to very irregular and release granules that will recruit additional platelets to form the platelet plug. The end result is the formation of platelet plug

Plasma components called coagulation factors respond (in a complex cascade) to form fibrin strands which strengthen the platelet plug. Plasma proteins, tissue factor and calcium interacts on the surface of the platelets to form a fibrin clot.

During primary hemostasis there is constriction of damged blood vessels, which decreases the blood flow through the injured vessels. Platelets clump together and adhere to injured vessels to form a platelet plug and further inhibit bleeding.During secondary hemostais the coagulation factor present in the blood (and tissue factor) interact, forming a fibrin meshwork (clot) to more efficiently stop the bleeding.

Coagulation Cascade

Two pathways Intrinsic already found within the blood Amplifies coagulation cascade Extrinsic found outside the blood vessel, more important in response to injured blood vessels Initiates coagulation cascade Tissue factor adjacent to the site of injury activates the inactive factor VII. The activated factor VII, together with tissue factor, activates the inactive factor X. the activated factor X, together with factor V will cleave prothrombin to thrombin. The thrombin will then cleave fibrinogen to fibrin. At the same time, the thrombin will cause platelet activation. the coagulation cascade of secondary hemostasis has two pathways, the Contact Activation pathway (formerly known as the Intrinsic Pathway) and the Tissue Factor pathway (formerly known as the Extrinsic pathway) that lead to fibrin formation. it is now known that the main pathway for initiationof coagulation is the extrinsic pathway (factor VII and tissue factor), whilst the intrinsic pathway acts toamplify(but not initiate) the coagulation cascade. Two parts: intrinsicsystem - activated by coagulation factors that are already present in the blood, acts toamplify(but not initiate) the coagulation cascade.

extrinsic system - initiated outside of blood vessels in the presence of injury to a vessel. factor VII, which is present in the blood, is converted to its activated form, factor VIIa, by binding to tissue factor (TF). TF/factor VIIa complex formed in this way converts factor X into its activated form factor Xa. This in turn forms a complex with factor Va and thus brings about cleavage of prothrombin to form thrombin. In turn, thrombin cleaves fibrinogen to form fibrin, which polymerises to form fibrin threadsIn the past, most people believed that either the intrinsic or extrinsic pathway could initiate coagulation. it is now known that the main pathway for initiationof coagulation is the extrinsic pathway (factor VII and tissue factor), whilst the intrinsic pathway acts toamplify(but not initiate) the coagulation cascade.The four phases of coagulation(1) Contact phase; small amounts of factor XII automatically activate when coming in contact with (-) charged surface like collagen(2) Activation of factor X via intrinsic and extrinsic pathway; to summarize these two systems, the intrinsic coagulation mechanism requires a contact phase, followed by activation of factor IX and X. The extrinsic mechanisms primary action is the activation of factor X. From this point on, two systems will combine into common pathway. It is most likely the systems operate simultaneously in vivo.(3) Conversion of prothrombin to thrombin; marks the beginning of common pathway, which adheres to platelet surfaces along with factor Xa and factor V (cofactor). This complex converts prothrombin to thrombin(4) Formation of fibrin clot; fibrin formation occurs by the action of thrombin on fibrinogen.

Tertiary phase of hemostasis

o Defined as the formation of plasmin, which is the main enzyme responsible for fibrinolysis (breakdown of the clot). o Controls/regulates formation of clot by way of tissue plasminogen activator (t-PA). o It occurs so that the clot does not grow enough to obstruct the pathway of blood. o t-PA will activate Plasminogen to form plasmin. - While the coagulation cascade is happening, the endothelial cells at the same time releases tPA, which will activate plasminogen giving rise to plasmin, giving rise to breakdown of platelets in the clot which will produce fibrin degradation product (FDP)

PRE-ANALYTICAL VARIABLES ON HEMOSTATIC TESTS A coagulation test result is only good as the sample received. you would want to a have a result that is accurate, precise and reproducible type of sample must be strictly monitored in order to get the real actual result

Pre-analytical variables 1. Sample specimen volume 9:1 ratio of blood sample to anticoagulant-assuming with normal Hct reject clotted, hemolyse, lipemic samples - If with clotted specimen and you removed the clot result becomes prolonged

2. Specimens will be rejected in the following circumstances: presence of clots visible hemolysis, lipemic samples incorrect anticoagulant tubes filled to less than or more than the recommended volume

3. Anticoagulant- blue top ; 3.2 % buffered sodium citrate which binds with calcium (in the blood) to arrest the clotting stage

4. Manner of phlebotomy Tourniquet should be released once there is backflow of blood in the syringe in order not to activate extrinsic pathway Tourniquet should not stay > 2 min If needle punctures the blood vessel, mere puncturing already activates the clotting mechanism When the blood sample is to be used in coagulation studies, as much as possible trauma is minimize thus probing is avoided Once blood is seen just beyond the needle, tourniquet may be released

5. Storage- samples frozen at -20C, stable 2 weeks; samples frozen at -70C, stable 6 months Should be done right away Once settled, should be centrifuged Separate RBC and plasma Controls are test specimens to check if machine is working or will give you an accurate result if it falls within the control then it is good if it falls beyond, then it means that there is a problem

USES: 1. Pre- operative assessment of hemostasis2. Investigate hemostatic / bleeding disorders 3. Monitor anticoagulant therapy such as cardiac patients who underwent bypass surgery and valvular replacements

TEST OF HEMOSTASIS Short Screening tests: Bleeding.T - to test Platelet & BV function, test for primary hemostasis Prothrombin.T Extrinsic, common pathway APTT Intrinsic , common pathway Thrombin.T common pathway D-dimer test Specialized (Extended) diagnostic tests: Factor assays Tests of thrombosis TT, FDP, DDA, Platelet function studies: Adhesion, Aggregation, Release & PG pathway tests Bone Marrow study

SHORT SCREENING TESTS/PANEL

BLEEDING TIME Measures interaction between platelets and (injured) vascular endothelium. Test for adequacy of primary hemostasis or platelet function and capillary integrity Need adequate number of platelets for a normal bleeding time 3 methods: Duke crude way Meilke has a standard depth and length of puncture Ivy - uses BP cuff and lancet

Recommended criteria prior to testing 1. platelet count: >75,000/mm3 2. only 1 bleeding time/24hrs unless patient has received therapy ex. platelet infusion 3. discontinue heparin 6 hrs prior to testing

Limitations: If thrombocytopenia is present (