Katelyn Fong 20 1

Haemostasis & Blood Clotting Mechanism

Haemostasis Introduction = Maintenance of blood volume [stoppage of bleeding to prevent blood loss] Normal hemostatic response to vascular damage involves platelets, endothelial cells and coagulation factors Blood clotting: a positive feedback process Involved when a vessel is severe/ ruptured: Small blood vessels (arterioles, capillaries, venules): Hemostatic mechanisms Medium to large arteries bleeding: CANNOT control Vein bleeding: Raise body part above heart level pressure Stop bleeding Accumulation of blood in tissues pressure Eliminate pressure gradient needed for continued blood loss Haematoma = accumulation of blood in tissues due to bleeding from any vessel Usually when blood vessels bleed Changes in local vasodilator & constrictor substances released by endothelial cells & blood cells Vessel contricts flow rate of blood & glue the opposed endothelial surfaces of the vessel together When blood is taken out from the body, it forms a gel called clot and finally separates from the liquid serum 4 Steps to blood formation:(1) Vascular constriction(2) Formation of a temporary platelet plug (primary phase)(3) Formation of a blood clot due to blood coagulation (secondary phase)(4) Clot retraction & fibrinolysis (Growth of fibrous tissue into blood clot to close the hole in vessel permanently)

Vascular Constriction After a blood vessel has been ruptured, the vessel walls smooth muscle will contract due to:: Local myogenic contraction of blood vessels initiated by the direct damage to the vascular wall Can last for a long time: During this period, formation of platelet plug & blood clotting can occur Local autacoid factors from injured tissues & blood platelets E.g. thromboxane A, ADP, tissue factor, prostacyclin & endothelins, peptide hormones Stimulates: Smooth muscle contraction & promotes vascular spasms Division of endothelial cells, smooth muscle cells & fibroblasts to accelerate repair process Nervous reflexes initiated by pain/sensory nerve impulses from that area Sympathetic nerves send messages Smooth muscles in the wall contract Vessel walls smooth muscle contracts

Reduce blood flow to ruptured vessel Reduce blood loss Permanent closure by constriction & contact stickiness only occur in the smallest vessels Stoppage of bleeding in other vessels requires: Platelet plug formation Blood clotting

Formation of a Temporary Platelet Plug (/Primary Hemostatic Plug) Platelets (Thrombocytes): Disc shaped cells: 2-4 m in diameter Formed in bone marrow from megakaryocytes Do not have nuclei & cannot reproduce Cellular characteristics: Actin/myosin molecules: allow contraction Endoplasmic reticulum, Golgi apparatus, mitochondria, etc. Factors (growth factor, fibrin-stabilizing factor): help repair damaged vascular walls Glycoproteins on surface: aids adherence to injured areas of vessel walls by providing receptors for agents activating platelets Phospholipid membrane: Provide a catalytic surface for blood clotting and yield arachidonic acid for synthesis of prostaglandins Release clotting factors Main functions [Extremely important for closing tiny ruptures in very small blood vessels that occur thousands of times daily]: Closure of leak by formation of temporary patch Release of chemical mediators to regulate blood clotting Contraction of blood clot Mechanism of the platelet plug: Occurs in the first minute or so following injury Injury to vessel disrupts endothelium & expose underlying connective-tissue collagen fibres Platelets come into contact with the damaged vessel surface The platelets are activated & immediately change their own characteristics drastically: Swell Assume irregular forms with numerous pseudopods protruding from their surfaces To increase surface area Become sticky glycoproteins on membrane (GPIb, GPIIb, GPIa, etc.) adhere to von Willebrand factor (a plasma protein secreted by endothelial cells & platelets that leaks into the traumatized tissue from plasma) Secrete large quantities of ADP & thromboxane A2 These (also secreted by other ruptured cells) act on the nearby platelets to activate them Stickiness of these additional platelets causes them to adhere to the original activated platelets Their contractile proteins contract forcefully Platelets release granules that contain multiple active factors: clotting factors & platelet factors (also for platelet aggregation/repair) Platelet aggregation occurs: successively increasing number of platelets are activated & attract more & more additional platelets Platelet plug forms At first a loose plug, but is usually successful in blocking blood loss at small vascular opening During the subsequent process of blood coagulation: fibrinogen forms fibrin bridges between the aggregating platelets Fibrin threads attach tightly to the platelets, constructing an unyielding platelet plug

Positive feedback cascade: Platelets stick platelets release contents more platelets stick Platelet plug Platelet plug wont spread to undamaged endothelium synthesis of prostacyclin (a prostaglandin) & nitric oxide from undamaged endothelial cells are both vasodilator & inhibitor of platelet adhesion, activation & aggregationThey inhibit platelet aggregation

Formation of Blood Clot Due to Blood Coagulation Blood clotting = Transformation of blood into a solid gel: clot/ thrombus, with fibrin as the main component Begins to develop in 1520 seconds after the trauma if severe; 12 minutes if trauma is minor Within 3-6 minutes the whole broken end of the vessel will be covered by the clot Occurs locally around the original platelet plug Dominant hemostatic defense Supports & reinforce the platelet plug & solidifies blood that remains in the wound channel Clotting factors play an important role in the reactions: Most of these proteins are inactive forms of proteolytic enzymes. When converted to the active forms, their enzymatic actions will cause the successive, cascading reactions of the clotting process.Clotting FactorsNameFunctionsSynonyms

IFibrinogenConversion to fibrin

IIProthrombinConversion to thrombin

IIITissue ThromboplastinActivation of factor X Tissue factor

IVCalcium ionCofactor for several reactions

VProaccelerinThrombin formation (not an enzyme) Labile factor Ac-globulin (Ac-G)

VIIProconvertinFactor X activation Serum prothrombin conversion accelerator (SPCA) Stable fator

VIIIAntihemophilic globulin [AHG]Factor X activation Antihemophilic factor (AHF) Antihemophilic factor A

IXPlasma thromboplastin (component) [PTC]Formation of factor VIII Chirstmas factor Antihemophilic factor B

XStuart factorThrombin formation Stuart-Prower factor

XIPlasma thromboplastin ancedent [PTA) Factor IX activation Abtihemophilic factor C

XIIHageman factorFactor XI activation

XIIITransglutaminaseCross-linking of fibrin Fibrin-stabilizing factor

-Prekallikrein Fletcher factor

-High-molecular-weight kininogen [HMWK] Fitzgerald factor

Platelets

Initiation of coagulation: Factors that could initiate the clotting process Trauma to vascular wall & adjacent tissues Trauma to the blood Contact of the blood with damaged endothelial cells, collagen or other tissue elements outside vessel Each case leads to the formation of prothrombin activator (catalyzes conversion of prothrombin to thrombin in the presence of sufficient Ca2+) with the use of blood clotting factors (e.g. Ca2+) Theres positive feedback: thrombin later produced help increase activity & production of factor VIII (antihemophilic globulin; responsible for factor X activation and hence thrombin foramtion) & V (Proaccelerin; responsible for thrombin formation) Clotting defects due to too low level of Ca2+: NOT POSSIBLE muscles paralysis/ cardiac arrhythmias would already cause death 2 pathways [can occur simultaneously]: Extrinsic & Intrinsic Extrinsic Pathway Extrinsic: A cellular tissue protein tissue factor outside blood vessel is needed Begins with (i) traumatized vascular wall or (ii) damaged extravascular tissues come into contact with blood (1) Traumatized tissue releases factor III (tissue factor) Factor III is composed especially of phospholipids from the membranes of the tissue & a lipoprotein complex that functions mainly as proteolytic enzyme(2) Factor III & factor VII (proconvertin) leads to the activation of factor X (Stuart factor) Activation of Factor X requires calcium ions & lipoprotein complex of Factor III(3) Activated factor X combines with factor V (proaccelerin) & phospholipids to form prothrombin activator Within a few seconds, in the presence of Ca++, prothrombin activator splits prothrombin to form thrombin, and the clotting process proceeds At first, the Factor V in the prothrombin activator complex is inactive, but once clotting begins & thrombin begins to form, the proteoclytic action of thrombin activates Factor V Factor V becomes an additional strong accelerator of prothrombin activation In the final prothrombin activator complex, activated factor X is the actual protease that causes splitting of prothrombin to form thrombin, a process thats greatly accelerated by factor V & platelet phospholipids= positive feedback effect of thrombin (acting through Factor V) accelerates the entire process once it begins Intrinsic Pathway Intrinsic: Everything occurs in blood for this path Begins with (i) trauma to the blood itself or (ii) exposure of blood to collagen from traumatized vessel wall (1) Blood trauma causes activation of Factor XII (Hageman factor) & release of platelet phospholipids Factor XII is activated when it comes into contact with collagen fibres underlying damaged endothelium Platelet phospholipid contains the lipidprotein called platelet factor 3(2) Activated factor XII enzymatically activates factor XI (antihemophilic factor C) Activation of Factor XI requires HMWK (high-molecular-weight kininogen) & is accelerated by prekallikrein(3) Activated factor XI enzymatically activates factor IX (antihemophilic factor B) (4) Activated factor IX combines with activated factor VIII (antihemophilic factor A) to activates factor X (Stuart factor) Activation of Factor X requires platelet phospholipids & Ca++ Haemophilia (excessive bleeding) is due to absence of VIII(5) Activated factor X combines with factor V (proaccelerin) & phospholipids to form prothrombin activator Same as the last step in the extrinsic pathway Prothrombin activator initiates within seconds the cleavage of prothrombin to form thrombin, thereby setting into motion the final clotting process Extrinsic pathway usually start first to produce thrombin, but the amount is always too small, so there is positive feedback from thrombin on the activation of factors V, VIII, XI of the intrinsic pathway, then there would be enough amount of thrombin for blood clotting Difference between intrinsic & extrinsic pathways:PathwayIntrinsicExtrinsic

Initiator Contact of Factor XII & platelets with collagen in traumatized vascular vessel wall Tissue factor (factor III) released from traumatized tissues

Speed of completion Slow [1-6 minutes] Fast [15 Seconds](explosive once initiated)

Amplification Enzyme cascade No

Thrombin produced Much Little

3 main steps to coagulation 1. Prothrombin activator is formed Formed due to complex chemical reaction in response to vessel wall damage2. Prothrombin activator catalyzes conversion of prothrombin to thrombin Prothrombin activator in presence of enough Ca2+ can do the conversion Prothrombin is a plasma protein produced in the liver3. Thrombin coverts fibrinogen to fibrin Fibrinogen is a plasma protein produced in the liver Thrombin is a protein enzyme with proteolytic capabilities Acts on fibrinogen to remove peptides Forms molecules of fibrin monomers Fibrin monomers can polymerize to form fibrin fibres Thrombin whilst catalyzing the formation of fibrin, activates fibrin-stabilizing factor (factor XIII) Enhances the fibrin meshwork Fibrin fibres adhere to damaged surface of blood vessels Fibrin enmeshes platelets, blood cells & plasma to form clot Clot formation normally remains localized at the site of injury because: Fibrin absorbs thrombin into the clot Stops the spread of thrombin into blood Inhibit blood clotting in other places Although theres dispersal of some clotting factors to other places, their concentrations are too low to bring about clotting Thrombins functions in blood clotting: Facilitate formation of loose fibrin Activate factor XIII (stabilization of fibrin mesh) Activate factor XI Activate factor VIII Activate factor V Further activate its own production from prothrombin (positive feedback) Stimulate platelet activation platelets display receptors that bind clotting factors & platelet factor (a cofactor) Release of tissue factor (activation of extrinsic pathway) Livers functions in blood clotting: Site of production of plasma clotting factors Produce bile salts to help absorption of vitamin K Useful bacteria in the colon produce vitamin K & it can also be ingested Essential in the maturation of prothrombin and other coagulation factors (VII, IX, X, etc.) in the liver Antibiotics can have an effect on the production of vitamin In the absence of prothrombin, thrombin cant be made & fibrin fibres cant be formed Anti-coagulants (limit clotting): Tissue factor pathway inhibitor Inhibits the tissue factor-activated factor VII complex Protein C Activated by thrombin & thrombobondulin Inhibit activated factors VIII & V Antithrombin III Activated by heparin Deactivates thrombin & several other clotting factors

Clot Retraction = Stabilization of clot by squeezing serum from the fibrin strands Within a few minutes after a clot is formed, it begins to contract & express most of the fluid (serum) out within 20-60 minutes This serum has all its fibrinogen & most other clotting factors removed Platelets contribute to clot contraction by activating actin & myosin molecules (contractile proteins) As clot retracts, edges of broken blood vessel are pulled together Functions: Further closure of leak Pulls torn edges of the vessels closer together, reducing residual bleeding & stabilizing injury size Reduces the size of the damaged area, making easier for fibrocytes, smooth muscle cells & endothelial cells to complete repairs Platelet-derived growth factor stimulates vascular smooth muscle cells to build new vessel wall Stimulated by VEH, endothelial cells multiply & restore their inner endothelial lining

Fibrinolysis Plasma proteins contains plasminogen (profibrinolysin) When a clot is formed, plasminogen is trapped in the clot Plasminogen (plasmo proenzyme) is activated by plasminogen activators (secreted by endothelial cells & is activated by fibrin in a clot) after the clot has stopped bleeding Plasminogen becomes plasmin (fibrinolysin) Plasmin (a protease) digests fibrin fibres & other protein coagulants & clotting factors into soluble fragments (inactive substance like fibrinogen & prothrombin)

Blood Clotting Disorders that Leads to Abnormal Bleeding Thrombocytopenia (low platelet count): Failure of platelet production This may be due to selective megakaryocyte depression (induced by drugs, chemicals or viral infections) or as part of general bone marrow failure Increased destruction of platelets Immune (idiopathic, drug-induced, infections and other causes) Disseminated intravascular coagulation DIC (undesirable blood clotting) Abnormal distribution of platelets Dilutional loss Massive transfusion of stored blood to bleeding patients Platelet function defects Hereditary disorders eg. Glanzmanns disease when platelet aggregation fails due to deficiency in some glycoproteins Acquired disorders Antiplatelet drugs (eg. aspirin) Uraemia (kidney failure) Hyperglobulinaemia (liver disease) Disorders of coagulation Hereditary deficiencies of clotting factors Haemophilia A - factor VIII deficiency Haemophilia B (Christmas disease) - factor IX deficiency Acquired disorders Vitamin K deficiency (needed for clotting factors) Liver diseases Over dosage of anticoagulants Vascular bleeding disorders These disorders are a heterogeneous group of conditions characterized by easy bruising an spontaneous bleeding from small vessels the problem rests in the vessels or their connective tissues Hereditary hemorrhagic telangiectasia Dilated microvascular swellings develop in the skin mucous membranes and internal organs Acquired vascular defects Eg. purpura (purple spots on skin due to internal bleeding of vessels) associated with infection Vitamin K deficiency Vitamin K is essential for synthesis of several clotting factors Half of vit K from gut bacteria, half from diet Long-teraminm antibiotic use results in vitamin K deficiency and bleeding disorders