Physio Hema 3

SAN BEDA COLLEGE OF MEDICINE – PHYSIOLOGY HEMATOLOGY & IMMUNOLOGY 3: BLOOD GROUPS, HEMOSTASIS & COAGULATION ABO Blood Group System LEGEND: Video, Audio/Notes,Book

EMPHASIS/ IMPORTANT CONCEPTS from: Lecture/ Audio, Mnemonics EDUCATIONAL OBJECTIVES At the end of the 4-hour lecture, the future Bedan Doctor must be able to:  Explain the basis for classifying blood into groups. o Enumerate the blood groups of the ABO system. o Give the agglutinogens (antigen) and agglutinins (antibodies) associated with each group. o Discuss the principle involved in blood typing and cross matching. o Give the basis for the Rh blood groups. o Differentiate Rh blood group from the ABO system.  Give the functions and normal values of platelets.  Describe the underlying physiologic mechanisms of hemostasis and coagulation. o Define hemostasis. o Discuss the steps and mechanisms in hemostasis.  Reflex vasoconstriction  Primary hemostasis  Secondary hemostasis o Discuss the coagulation cascade.  Name the 12 coagulation factors.  Trace the intrinsic and extrinsic pathways of the coagulation cascade. BLOOD GROUPS Red blood cell surface antigens: glycolipids or glycoproteins A-B-O System Agglutinogens surface antigens (A, B) genes or alleles (A, B, O- are not represented by any surface antigens) inherited (two surface chromosomes) OO OA OB AA BB AB – co-dominance or 2 alleles, 1 from each parent will come together to form these genotypes Different from Mendelian inheritance, wherein there is dominant & recessive gene. Homozygous- AA, BB, OO; Heterozygous- OA, OB, AB also present on all cells in the body are called agglutinogens because they often cause blood cell agglutination. Agglutinins gamma globulins or antibodies, anti-A, anti-B, IgM, IgG basis for blood typing ABO Analogy: “Doughnut & Sprinkles” RBCs are like donuts without a hole in it Sprinkles are like the antigens

ABO Phenotypes: Type A has antigens A as its sprinkles

Type B would have B sprinkles on it Type AB would have two surface antigens  Type O is like a plain donut, w/o any sprinkles on it

 Antibodies are pentamer structures, thus, these are IgM. Type A has an A antigen & anti-B antibody (meaning that if you transfuse a type A patient w/ a type B blood, this anti- B antibody will react/attack with the B antigen/ sprinkles. Likewise, with transfusion of type A blood to a type B patient. (Remember the basis of immune system is its ability to recognize self vs non-self. Type AB has both A & B antigens but has no antibody, hence, it is called the “universal recipient” because even when you transfuse this blood with either A or B, there will be no reaction that will go against it. Type O has no surface antigens but has both anti- A & B antibodies, so, it will react/ attack with any transfused blood cells from either A or B type. But because it does not have any surface antigens, technically, it can be a donor to any blood types (“universal donor”)

Remember! There are SIX possible combinations of alleles (or genotypes): OO, OA, OB, AA. BB, and BB Type O allele is either functionless or almost functionless, so it causes no significant type O agglutinogen on the cells. Type A & B do cause strong agglutinogens on the cells.  Thus, O allele is recessive to both A & B alleles, which show co-dominance  Prevalence of blood types among one group of persons was approximately: O (47%) | A (41%) | B (9%) | AB (3%)  Procedure of blood typing & blood matching are performed in the ff. way: 1. RBCs are separated from the plasma & diluted with saline solution. 2. One portion is then mixed w/ anti-A agglutinin & the other w/ anti-B. 3. After several mins, mixtures are observed under a microscope. NOTE: - If RBCs have become clumped- “agglutinated” - Type O RBCs have no agglutinogens & therefore do not react w/ either anti-A or anti-B agglutinins. Page 1 of 10

PHYSIOLOGY

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HEMATOLOGY & IMMUNOLOGY 3: BLOOD GROUPS, HEMOSTASIS & COAGULATION

Titer of Agglutinins After birth – almost zero 2-8 months after birth – begin production 8-10 years old – maximum titer Gradually declines remaining years of life

When we were born & all the time during our neonatal period up to four months of our lives, we are dependent to the antibodies given by our mothers, mainly because we are only starting to build our own immune system & create our own immunoglobulins. IgG is usually what is given by our mothers either through breast milk or birth through the placenta.

Hemolytic Disease of the Newborn or Erythroblastosis Fetalis There is a mismatched between an Rh + mother carrying an Rh – fetus. Immune system will not attack the first fetus but the next time that mother gets pregnant& another different Rh fetus from that of hers, then that will be time immune response attack. In most instances, baby has inherited the Rh-positive antigen from the father and the mother develops anti-Rh agglutinins from exposure to the fetus’s Rh antigen. In turn, the mother’s agglutinins diffuse through the placenta into the fetus & cause RBC agglutination of the fetus’s blood. This does not develop sufficient anti-Rh agglutinins to cause any harm to the first child. But the incidence of this disease rises progressively with subsequent pregnancies. • • • •

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Rhesus System Major difference b/w A-B-O system & Rh system: A-B-O system: plasma agglutinins responsible for causing transfusion reaction to develop spontaneously Rh system: spontaneous agglutinins almost never occur. Person must first be massively exposed to an Rh antigen to cause a significant reaction to develop. Agglutinogens - 6 rhesus factors (C, D, E, c, d, e) - inherited as triplets but in various combination CDE, CDe, Cde, CdE, cDE, cDe, cde o antigen D  Rhesus positive (widely prevalent & more antigenic can induce immunologic responses)  widely prevalent in the population & considerably more antigenic than the other Rh antigens. Anyone who has this type is said to be Rh positive (where a person who does not have it is said to be Rh negative. o Follows Mendelian inheritance o C, D, E are the dominant genes; c, d, e are the recessive ones o Upon first exposure to a different RH type, you will not get an immediate reaction from the antibodies, it takes time to mount an immune response against foreign Rh antigen  E.g., when we transfuse Rh positive blood to an Rh-negative person, nothing will happen to that person but immune system will start to initiate an immune response next time we transfuse Rh + into that same Rh – patient

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Agglutinins - do not occur spontaneously, only after exposure to Rh antigens - anti-Rh agglutinins develop slowly, reaching maximum concentration of agglutinins about 2 to 4 months later. - Rh+ blood into Rh negative person: sensitization to further Rh+ transfusion

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Disease of the fetus and newborn child Fetal blood enters maternal circulation Agglutination & phagocytosis of fetus’ RBCs ABO incompatibility O mother and A or B fetus IgG anti-A and anti-B cross placenta very mild effects Rh incompatibility Rh positive fetus and a Rh negative mother Anti-D agglutinins form in mother from exposure to fetus’ Rh Ag  diffusion thru the placenta into the fetus and cause agglutination Maternal blood with anti-D usually circulate in infant’s blood for another 1-2 months after birth  destroys more RBC More critical with 2nd and succeeding Rh positive child Signs and Symptoms Jaundice (since agglutinated RBCs hemolyzed, hemoglobin is released into the blood, in which the macrophages then convert Hb into bilirubin, causing jaundice) Pallor or anemia at birth Hepatosplenomegaly – forming nucleated blastic RBCs Kernicterus (bilirubin-induced brain dysfunction) Treatment Exchange Transfusion Replace neonate’s blood with Rh (-) blood > 6 weeks - transfused Rh (-) blood is replaced by infant’s own Rh (+) blood and mother’s anti-D is destroyed Injection of IgG anti-D or Rh Ig or Rhogam to expectant mother starting at 28-30 weeks AOG` Prevention  Administer Rhogam to Rh (-) mother who delivered Rh (+) babies to prevent sensitization of mother to the D Ag, thus, reducing the risk of developing large amounts of anti-D during 2nd pregnancy  Mechanism of Rhogam  inhibits Ag-induced B lymphocytic Ab production in expectant mothers  Attaches to D-Ag sites on Rh (+) fetal RBC that may cross the placenta Transfusion Reaction Typical transfusion reactions are immediate or delayed agglutination and hemolysis of RBCs, which frequently lead to death. RBCs agglutinate as a result of agglutinins attaching themselves to the RBCs. Because agglutinins have 2 binding sites (IgG type) or 10 binding sites (IgM type), a single agglutinin can attach to 2 or more RBCs at the same time, thereby causing the cells to be bound together by agglutinin, causing it to clump. Then, these Page 2 of 10

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HEMATOLOGY & IMMUNOLOGY 3: BLOOD GROUPS, HEMOSTASIS & COAGULATION

clumps plug small blood vessels throughout circulatory system. Hemolysis: either physical distortion of the cells or attack by phagocytic WBCs destroy the membrane of the agglutinated cells, causing a release of hemoglobin into the plasma. Acute Hemolysis: recipient & donor bloods are mismatched  occurrence of immediate hemolysis of RBCs by activation of the complement system that releases proteolytic enzymes  rupture the cell membranes Immediate intravascular hemolysis is far less common than agglutination followed by delayed hemolysis because (1) there have to be a high titer of antibodies for lysis to occur and (2) IgM antibodies (hemolysins), a diff. type of antibody seems to be required.

Hemolysins IgG agglutinins o dimeric or monomeric structure o contain 2 binding sites IgM agglutinins o a pentameric structure o contain 10 binding sites Agglutination “clumping” of cells by activation of complement system (AgAb reaction) ↓ release proteolytic enzymes ↓ destroys membrane of agglutinated cells (either by physical distortion of cells or phagocytosis by WBC) ↓ release of Hb ↓ hemolysis -

Hemoglobin in blood is very toxic. It can cause hyperviscosity of blood. Transfusion reaction due to agglutination of donor blood and rarely recipient’s blood due to – plasma portion of donor blood immediately becomes diluted by plasma of recipient  decreasing titer of transfused agglutinins o Ab of recipient attacks Ag of donor o DONOR’S BLOOD undergoes hemolysis

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In a normal blood smear, notice that the RBCs are evenly well- distributed with some basophilic cells that are possibly monocytes/macrophages or neutrophils. With agglutination, notice that there is clumping of RBCs in the blood smear. Signs and Symptoms 1. Fever and chills 2. Shortness of breath • due to lack of oxygen delivery because of the massive agglutination going on 3. Jaundice • because of the release of Hb in the blood 4. Shock 5. Renal shutdown

Renal Shutdown or Renal Failure 1. Renal Vasoconstrictor– toxic substance released by hemolyzed blood 2. Circulatory Shock– due to loss of circulating RBC, the produced toxic substances and from immune reaction (Ag-Ab reaction) – hypotension, decrease renal blood flow, decrease urine output 3. Renal Tubular Blockage– total amount of free Hb released into the circulating blood >Hb bound to haptoglobin  excess leaks thru the glomerular membrane into kidney tubules, which precipitates and blocks renal tubules (worst case) HEMOSTASIS AND BLOOD COAGULATION

A. a type A recipient with a type A blood donor ↓ There is no B surface antigen ↓ no agglutination ↓ blood will be accepted B. a type A recipient given a type B blood ↓ Anti-B agglutinins binds to the antigen ↓ agglutination or clumping of RBCs REMEMBER: No foreign antigens allowed!

Platelet or Thrombocyte Characteristics • Shape Biconvex disk like (as opposed to RBCs that are biconcave) diameter about 2~4 µm, 1~4 µm average cubage 8 µm3. • Complicated structure under the electronic microscope, there are αgranule, dense body, lysin peroxide enzyme, opening tubular system, dense tubular system, canaliculus,etc. do not have nuclei • Dense body It contains ADP, ATP, 5-HT (aka Serotonin), Ca2+ (important for coagulation cascade), epinephrine (important for vasoconstriction), Page 3 of 10

PHYSIOLOGY

HEMATOLOGY & IMMUNOLOGY 3: BLOOD GROUPS, HEMOSTASIS & COAGULATION

etc(thromboxane a2, platelet derived-growth factor (pdgf) The body of the platelet itself contains a lot of chemicals. It can be argued that platelets are actually not a complete cell because they don’t contain organelles & they are technically fragments of megakaryocyte. •

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Platelet comes from megakaryocyte fractionlet eitherin the release in the bone marrow or after entering the blood

FUNCTIONAL CHARACTERISTICS 1. Cannot reproduce 2. Their cytoplasm has actin & myosin (contractile proteins similar to muscle cells) and thromosthenin(causes platelet to contract) 3. Residuals of both ER & Golgi apparatus (synthesizes various enzymes & stores large quantities of Calcium ions) 4. Have mitochondria systems (capable of forming ATP & ADP) 5. Have enzyme systems (synthesizes prostaglandins  local hormone that cause many vascular & tissue reactions) 6. Contains fibrin stabilizing factor (FSH) 7. Contains growth factor (causes vascular endothelial cells, vascular smooth muscles & fibroblasts to multiply & grow that promotes cellular growth which helps repair damaged vascular walls) 8. Cell membrane surface contains a. a coat of glycoproteins >> repulses adherence to normal endothelium & yet causes adherence to injured areas of the vessel specifically to injured endothelial cells & exposed collagen b. large amount of phospholipids >> activates multiple stages in bloodclotting process. Normal Value 150, 000 – 400, 000; 150, 000 – 300, 000 per microliter (GUTYON!!!) Nice to know: Dengue pathogens attack the platelets, wherein dropping of the platelet count increases morbidity which eventually will lead to a lot of bleeding Functions 1. It maintains capillary endothelial cells smooth and integrated (repairing endothelium and providing nutrition). 2. It is involved in physiological hemostasis,particularly, primary hemostasis, wherein a platelet plug is formed. Clinical Correlation DECREASE of platelet ↓ abnormal immune reaction ↓ results in hemorrhage or bleeding, purpuric symptoms.

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Platelet Formation and Regulation  Formation: o Birth place is bone marrow, originating from hemopoietic stem cells, and differentiating into burst forming unit- megakaryocyte, o BFU-MK, then continuously into CFU-MK, and into megakaryocyte, demarcation membrane system, DMS, into fractionlet release to the blood requiring 8~10 days. (one megakaryocyte can produce 200~7700 platelets).  Regulation: o Protein, Mpl, expressed by c-mpl (oncogene)exists in CD34+located at hemopoietic stem cells/ committed progenitors, megakaryocyte and platelet, found by Methin in 1993, and its ligand named thrombopoietin. o Thromopoietin promotes hemopoietic stem cells differentiation into megakaryocyte as a hemopoietic stem cells positive regulating factor. Life Span & Destruction of Platelets  Life-span: o Average of 7~14 days in the blood. o 8-12 days (GUYTON!!!)  Destruction: o Aged platelets are processed by phagocytosis in liver, spleen and lymphatic node because their functional processes run out as they age. Physiological Characteristics of Platelets Activation of platelet:Stimulus brings about thrombocyte adhesion, aggregation, release and contraction. 

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Thrombocyte adhesion: its membrane glycoprotein (GP, GPIb/IX and GPIIa/IIIb), collagen (underendothelial structure), vWF (plasma component; very important in platelet adhesion), fibrinogen are involved in adhesion. Thrombocyte aggregation: induced by physiological factors such as ADP, thromboxane A2 (TXA2), epinephrine, 5-HT, histamine, collagen, thrombin, prostacyclin,etc and by pathological factors like bacteria, virus, immune complex, drugs, etc. TXA2 & Epinephrine: for vasoconstriction Thrombocyte release: ADP, ATP, 5-HT, Ca2+ released from dense body, and β-platelet globin, PF4, vWF, fibrinogen, PFV, PDGF, thrombin sensitive protein from α-granule, and acid protein hydrolyzed enzyme, tissue hydrolyzed enzyme from lysosome. All of these will come out from platelet to call more platelets in to start to clump into each other, therefore plugging the leak in the blood vessels. Thrombocyte contraction: Loose platelet thrombus could turn into compact platelet thrombus by Ca2+ release and cytoskeleton movement (filament/canaliculus) within platelet. Once an injury occurs in blood vessels, exposed collagen will attract platelets or thrombocytes, therefore it starts to stick into it through membrane glycoprotein, trapping the thrombocytes causing the thrombocytes/platelets to aggregate which Page 4 of 10

PHYSIOLOGY

HEMATOLOGY & IMMUNOLOGY 3: BLOOD GROUPS, HEMOSTASIS & COAGULATION

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forms a clot that prevents more blood from being loss.

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Hemostasis “Hemo” blood | “Stasis” stop Definition: o The process from blood vessel damage to hemostasis o Prevention of blood loss Bleeding time: o The time from blood vessel damage to automatic hemostasis. o Normal time is 1~3 min and is longer when platelets are decreased. Longer time for patients with dengue.

Mechanisms/Process of Hemostasis

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BLOOD COAGULATION IN RUPTURED VESSELS  Blood clot begins to develop in  15-20 seconds if the trauma to the vascular wall has been sever  1-2 minutes for minor trauma  After 20 minutes, the clot retracts which closes the vessel still further  Platelets play an important part in this retraction

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Explanation of the picture: Blood Vessel injury stimulates the platelets to become activated (which in turn will cause platelet fusion), reflex vasoconstriction to be present (which will reduce blood flow to prevent more blood loss), & coagulation system activation (thrombin & fibrin comes into play mainly for secondary hemostatic response). The ultimate goal of hemostasis, whether primary or secondary, is to form stable hemostatic plug, in which after its formation, there will be some contraction that goes on that will eventually close the injury. 1.

Platelet thrombosisformation (Primary Hemostasis) made by platelet adhesion, aggregation, release and contraction

FORMATION OF PLATELET PLUG  for very small cut in the blood vessel  It is sealed by a platelet plug instead of a blood clot  important fir closing minute ruptures in very small blood vessels that occur many times daily 3.

FIBROUS ORGANIZATION OR DISSOLUTION OF BLOOD CLOT  2 courses after blood clot has formed A. It can become invaded by fibroblasts It is the usual course Subsequently forms connective tissue all through the clot Begins within few hours after the clot is formed  Promoted by GROWTH FACTOR secreted by platelets Continues to complete organization of clot into the fibrous tissues within ~1-2 weeks B. It can dissolve When… o excess blood has leaked to the tissues o tissue clots have occurred where they are not needed Special substances are activated and act as an enzyme that dissolves the clot

Blood vessel contraction(Reflex Vasoconstriction) induced by neuroreflex; 5-hydroxytryptamine,5HT; thromboxane A2, TXA2; endothelin, ET It contracts to prevent more blood from being loss

VASCULAR CONSTRICTION  Immediately after cut/trauma, smooth muscles in the wall contract  results from  Local myogenic spasm – initiated by direct vascular damage to vascular wall  Local autocoid factors from traumatized tissues & platelets – for smaller vessels  release of Thrombaxane A2 by the platelets  Nervous reflexes – initiated by 1) pain nerve impulse or 2) other sensory sensory impulses (from traumatized vessel or nearby tissues) 2.

“blood coagulation activation” Conversion of Fibrinogen  Fibrin will create a mesh to stabilize the clot

Fibrin, clot formation and maintenance(Secondary Hemostasis)

Explanation of the picture above: Collagen fiber will cause platelet adhesion which will then form a platelet plug to prevent more blood from going out the injury (PAULIT ULIT NO? HAHAHAHHA) Roles of Platelet in Hemostasis 1. Activated platelets supply lecithoid (phospholipid) surface for blood clotting factor and involve in activating factor X and prothrombin. 2. Surface of platelet membrane combine with many blood clotting factor, such as fibrinogen, FV, FXI, FXIII (for stabilizing fibrin meshwork that has been formed in the injury site) to speed up coagulation. 3. Activated platelets release α-granule which contains fibrinogen to intensify fibrin forming and blood coagulation. 4. Activated platelets contract clot with its contractive protein to solidify blood coagulation.

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PHYSIOLOGY

HEMATOLOGY & IMMUNOLOGY 3: BLOOD GROUPS, HEMOSTASIS & COAGULATION 

Mechanisms of Platelet in Hemostasis Mechanism #1

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These chemicals will call in other platelets, cause them to stick together, & cause vasoconstriction.

Blood Coagulation/ Coagulatory Hemostasis Stages/Steps of Coagulatory Hemostasis Basic steps of blood coagulation [typical positive feedback]:

Why do the platelets (which normally plays around the blood) stick to endothelium? Note that a healthy endothelium produces prostacyclin, that in turn, signals platelets not to adhere to the endothelium surface (similar to action of lubricant). Once an injury occurs, exposed collagen will bind and activate platelets (phospholipids will also come into the mix and bind as well) causing the release of platelet factors, w/c will then attract more platelets to come into the cycle and also bind to the other platelets, thereby will aggregate into platelet plug, thus preventing blood from going out from the blood vessel (mgailanulit kaya to? Umayhahaha sorry friends! =)))) Mechanism #2

Platelet plug may seem competent but sometimes it’s not enough. (SAD! ☹) Thus, you got to have formation of stable clot through the coagulation cascade. At the same time, the exposed collagen for every occurrence of damage to the wall of the vessel, tissue factor is also being exposed to initiate coagulation cascade, which will ultimately form thrombin, which will then catalyze conversion of fibrinogen to fibrin with the help of Ca2+. What happens next is that you will have a reinforced platelet plug because fibrins have little strand structures that will interconnect on top of the platelet plug. As clot stabilizes, you will notice that there is a tissue repair, endothelium repair, and the like, which will eventually dissolve fibrin by plasmin (derived from plasminogen) causing the clot to dissolve too. A clot is fully dissolved only when the blood vessel wall is intact. Platelet Clotting Factors • Cellular clotting factors are present in the granules of thrombocytes. • These factors are released when the platelets undergo Degranulation (like Mast cells). • They are: ADP and ATP Ionized calcium Serotonin Epinephine Thrombaxane A2

1. Activation of prothrombin activator  are set inyo play by 1) trauma to the vascular wall & adjacent tissues, 2) trauma to the blood, 3) contact of the blood with damaged endothelial cells or with collagen and other tissue elements outside the blood vessel which will lead to the formation of prothrombin activator.  formed in 2 ways that interact with each other constantly  Extrinsic pathway – begins with trauma to vascular wall and surrounding tissues  Intrinsic pathway – begins in the blood. 2. Prothrombin → Thrombin  this is the rate limiting factor of blood coagulation because these terminal steps normally occur rapidly to form clot 1. Prothrombin activator is formed 2. Prothrombin activator causes conversion of prothrombin to thrombin 3. Thrombin causes polymerization of fibrinogen molecules into fibrin fibers  Platelets play an important role in the conversion of prothrombin to thrombin because much of the prothrombin first attaches to the prothrombin receptors of the platelets already bound to the damaged tissues Prothrombin >> plasma protein, an α2-globulin. >> Normal plasma conc.: ~15 mg/dL >> unstable protein that can easily split into smaller compounds (Ex. Thrombin) >> continually produced by the liver & used throughout the body for blood clotting >> Vitamin K is required by the liver for the normal activation of prothrombin Lack of Vit. K or liver disease  prevents normal prothrombin formation  decrease prothrombin  bleeding tendencies 3.

Conversion of Fibrinogen to Fibrin – Formation of the Clot “Fibrinogen → Fibrin-monomer - fibrin-polymer – crosslinked fibrin-polymer” Significance!! Stops blood loss from the large vesselsthat have higher blood pressure by the formation of a blood clot.

 Fibrinogen formed in the liver is essential for clot formation Fibrinogen >> high molecular weight protein found in the plasma (100700 mg/dL) >> formed in the liver (so kapag may liver disease ka, magdedecrease ‘to) >>only little fibrinogen leaks from blood vessels into the interstitial fluid because of its large size >> it is one of the essential factors in the coagulation process (kaya usually hindinagcocoagulateyung interstitial kasinga di namansiyamakalabas) Leakage due to pathology  increase the amount of fibrinogen to the tissue fluids  clotting of these fluids

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HEMATOLOGY & IMMUNOLOGY 3: BLOOD GROUPS, HEMOSTASIS & COAGULATION

Thrombin acts on the fibrinogen to remove four-low-molecular weight peptides from each molecule of fibrinogen  Many fibrin monomer molecules polymerize within seconds into long fibrin monomers In the early stages of polymerization, fibrin monomer molecules are held by weak noncovalent hydrogen bonding >> the newly forming forming are not crosslinked with one another >> resultant clot is weak & can be broken apart with ease Another process occurs during the next few minutes which involves the substance FSF (present in small amounts in normal plasma globulins & released from platelets entrapped in the clot) Thrombin >> protein enzyme with weak proteolytic capabilities FSF >> must be activated by the same thrombin that causes fibrin formation >> operate as an enzyme to cause covalent bonds between more and more of the fibrin monomer molecules & multiple cross-linkages between adjacent fibrin fibers which will add to the 3D strength of fibrin meshwork  Clot forms and fibrin fibers also adhere to the damaged surfaces of blood vessel. This causes the clot to be adherent to any vascular opening and threby prevent further blood loss. Clot >> It is composed of meshwork of fibrin fibers running in all directions & entrapping blood cells, platelets and plasma.  Within a few minutes after the clot is formed, it begins to contract & usually expresses most of the fluid from the clot within 20-60 minutes Serum >> expressed fluid >> called “serum” because clotting factors and fibrinogen have been removed  Clot retraction will occur if there are enough platelets in the circulating blood Platelets (P) >> Entrapped P in the clot continue to release procoagulant substances like:  FSF – which causes more cross linking bonds between adjacent fibers >> contributes directly to clot contraction by activating thrombsthenin, actin and myosin molecules  These are all contractile proteins that causes contraction of platelet spicules attached to fibrin and helps compress the fibrin meshwork into smaller mass.  It is activated and stimulated by thrombin & Calcium ions

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Blood Coagulation Factors Number

Names

I II

Fibrinogen Prothrombin

III

Tissue Factor (Thromboplastin)

IV

Calcium ions (Ca2+)

V VII

VIII

IX X XI

XII XIII

Proaccelerin, Labile Factor, Accelerator Globulin (AcG) Serum prothrombin conversion accelerator (SPCA), stable factor, proconvertin Antihemophilic fator (AHF), antihemophilic factor A, antihemophilic globulin (AHG) Christmas Factor, plasma thromboplastin component, antihemophilic factor B Stuart Factor, thrombokinase, Stuart-Prower Factor Plasma thromboplastin antecedent (PTA), antihemophilic factor C Hageman factor, glass factor, contact factor, antihemophilic factor D Fibrin-stabilizing factor (FSF)

Source Liver Liver Damaged tissues and activated Platelets Diet, bones, platelets Liver and platelets

Pathways of Activation Common Common

Extrinsic

All Extrinsic & Intrinsic

Liver

Extrinsic

Liver

Intrinsic

Liver

Intrinsic

Liver

Extrinsic & Intrinsic

Liver

Intrinsic

Liver

Intrinsic

Liver & platelets

Common

Mnemonics!!! "Fit Pants, Tight Collars, Loose American Shirts Are Cool Says Pretty Heroine Farah" -

Deficiency of factor VIII  hemophilia A Deficiency of factor IX  hemophilia B Deficiency of factor XI  hemophilia C Deficiency of factor XII  may affect bleeding but not as much as deficiency of 8, 9, 11 Fibrin-stabilizing factor links the fibrin fibers to create the mesh NO FACTOR VI! Prothrombinase (prothrombin activator) is a combination of FVa and FXa

Blood Coagulation

 As the clot retracts, the edges of the broken blood vessel are pulled together which will lead to further hemostasis. 4. Positive Feedback of Clot formation  Once a clot starts to develop, it extends within minutes into the surrounding blood.  The clot initiates the positive feedback to promote more clotting and more thrombin to be formed. Thus, through this, the blood clot continues to grow until blood leakage ceases. >>>>>>> Kasi nga di ba yung proteolytic action ng thrombin mag act sa iba pang clotting factors and sa fibrinogen. So si thrombin, directly niyang naapektuhan si prothrombin kaya mas macoconvert ito into thrombin so madami na si thrombin. Tapos si prothrombin nag aact din sa iba pang clotting factors kaya naman mafoform si prothrombin activator. So kapag na reach na ni thrombin yung critical amount niya, magpopositive feedback na. Gets ba? LOL sorry na. 

REMEMBER!!! Intrinsic pathway is activated by subendothelial collagen. Extrinsic pathway is activated by tissue thromboplastin. •

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Intrinsic pathway of blood coagulation: • All clotting factors involved in blood coagulation comes from blood. Negative charge (collagenin) on the endothelium of blood vessel activates blood FXII as beginning of coagulation this is called surface activation. • Any contact with any other substance will start the intrinsic pathway. Extrinsic pathway of blood coagulation: • Stimulus activates tissue factor (FIII)as beginning of coagulation. Page 7 of 10

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HEMATOLOGY & IMMUNOLOGY 3: BLOOD GROUPS, HEMOSTASIS & COAGULATION

Extrinsic pathway of blood coagulation is faster than intrinsic pathwayof blood coagulation because its steps are simpler.

Hemophilia A, B, C in the clinic results from deficiency of FVIII, FIX, FXI in the blood, respectively.

Helpful ba ‘tong mnemonic na ‘to? HAHAHAH parang hindinaman gusto kolangilagaykasinatawakopiliitnapilit. Hahahahaburahinniyonalang ‘tong side comment ko di nakakatulonghahaha

The main purpose of the whole hemostatic system is to form Fibrin clot. As the blood vessel/tissue starts to heal, there will be time that the clot is no longer needed. So, Fibrinolysis will then come into play. Mechanism of Blood Coagulation REMEMBER! FX is common for both intrinsic and extrinsic pathways Both pathways will ultimately form fibrin that will then be a fibrin clot through factor XIII Tissue factor initiates Intrinsic Pathway whereas contact of FXII & platelets with collagen initiates extrinsic pathway  Extrinsic pathway can be explosive because it is limited only by the amount of tissue factor released. Thus, clotting occur within 15 seconds in a severe trauma.  Intrinsic pathway is slower to proceed because it requires 1-6 minutes before clotting occurs

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Extrinsic Pathway usually the spark initiatesactivator tissue thromboplastin - FIII (due to exposure of thromboplastin to the outside environment of the blood vessel) catalyzes the conversion of FVII to FVIIa forms VII complex that will then catalyze the conversion of FX to FXaProthrombin to Thrombin (FIIa)  Fibrinogen to Fibrin (FIa) will form a fibrin polymer (a meshlike structure) through FXIII Traumatized vascular wall or extravascular tissue ↓ Release of Tissue Factor or Tissue Thromboplastin >> composed of phospholipids (from the membranes of tissue) & lipoprotein complex (functions mainly as proteolytic enzyme) ↓ Activation of Factor X >> FIII catalyzes activation of FVII >> lipoprotein complex of FIII further complexes with FVIIa (in the presence of Ca2+) acts enzymatically on FX to form FXa ↓ Formation of Prothrombin activator >>FXa combines immediately with tissue phospholipids of FIII & additional phospholipids from platelets, as well as with FV to form the prothrombin complex ↓ Within few seconds, in the presence of Ca2+, prothrombin is split into thrombin ↓ Clotting process proceeds: 1. The beginning of clotting & formation thrombin activates FV which becomes an accelerator of the prothrombin activation. 2. In the final prothrombin activator… a. FXa is the actual protease that causes splitting of prothrombin to form thrombin b. FVa accelerates FXa activity c. Platelet phospholipids act as a vehicle that further accelerates this process Note: Positive feedback effect of thrombin (Acting through FV)  accelerate the entire process. Ulit para maalala! Extrinsic P: tissue factor III will catalyze the activation of VII  together they will form complex of III &VIIa activates X with the aid of Ca2+ & Phospholipids Xa, with the action of V, Ca2+, Phospholipids,will then catalyze conversion of prothrombin to thrombin, that will, in turn, catalyzes conversion of fibrinogen to fibrinXIIIa will then form stable fibrin clot (adhesion of fibrin)

Intrinsic Pathway is the workhorse, which is why it is very essential in forming a very stable plug 12  11  9  8  10 Page 8 of 10

PHYSIOLOGY -

HEMATOLOGY & IMMUNOLOGY 3: BLOOD GROUPS, HEMOSTASIS & COAGULATION

activators (exposed collagen of the blood vessel, glass (e.g., blood on a glass slide), & others) catalyzes the conversion of FXII to FXIIa FXI to FXIa FIX to FIXa forms VIII complex that will then activate FX

Trauma to blood or exposure of blood to collagen ↓ Activation of FXII & release of platelet phospholipids >> when FXII is disturbed (eposed to collgen or wettable surfaces), it undergoes new molecular configuration that converts it into a proteolytic enzyme (FXIIa) >> Blood trauma damages platelets because of adherence to collagen or wettable surface which releases platelet phospholipids that contain platelet factor 3 (role in subsequent clotting reaction) ↓ Activation of FXI >>FXIIa acts enzymatically on FXI >> this requires High-Molecular-Weight Kininogen (HMW kininogen) and Prekallikrein ↓ Activation of FIX by FXIa >> aided by Calcium ions ↓ Activation of FX >> Thrombin activates FVIII >>FIXa, acting in concert with FVIIIa, together with platelet phospholipids, Calcium ions & FIII (from the traumatized tissues), activates FX ↓ Action of FXa to form Prothrombin activator >> same as the last step in extrinsic pathway >>FXa combines with FV and platelet phospholipids to form prothrombin activator ↓ Prothrombin activator initiates, within seconds, the cleavage of prothrombin to form thrombin, thereby setting into motion the final clotting process. Ulit para maalala! Intrinsic P: Collagen, glass, air, or other activators will catalyze activation of XII XIIa, with the action of Ca2+, will activate XI  Xia, with Ca2+, will then activate IX IXa, with the help of Ca2+ & Phospholipids, will then catalyze the conversion of X to Xa goes to the common pathway

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Anticoagulative System in Blood Provides negative feedback to coagulation system 1-Serine protease inhibitors: inhibit the coagulation cascade. 2-Neutralizers of activated coagulation factors (components of protein C system) 1-Serine protease inhibitors: 1-Antithrombin (III). 2-Heparin and heparin like substance. 3-Alpha 1 antitypsin. 4-Alpha 2 macroglobulin 2-Neutralizers of activated coagulation factors: 1-Protein C: synthesized in the liver, vit. K dependent, activated by thrombin. o Thrombin is actually the one who regulates the whole thing. It catalyzes the breakdown of fibrinogen to fibrin and it also forms anticoagulant in order to prevent over-clotting. 2-Thrombomodulin. 3-Protein S and C4b-binding Specific Inhibitors of Clotting Factors 1. Antithrombin III • It is a plasma protein that inactivates thrombin by forming an irreversible complex with it. • It resembles alpha 1-antitrypsin except that it inhibits thrombin much more strongly than it inhibits elastase. • Also, it blocks other serine proteases in the clotting cascade namely, factors XIIa, XIa, IXa, and Xa. 2.Heparin • The inhibitory action of antithrombin III is enhanced by heparin • It is a negatively charged polysaccharide found in mast cells (contains histamine & heparin) near the walls of blood vessels and on the surfaces of endothelial cells • Heparin acts as an anticoagulant by increasing the rate of formation of irreversible complexes between antithrombin III and the serine protease clotting factors. • Antitrypsin and antithrombin are serpins, a family of serine protease inhibitors.

3. Alpha 1-antitrypsin • which normally inhibits elastase • alpha 1-Antitrypsin activity normally increases markedly after injury to counteract excess elastase arising from stimulated neutrophils. • The mutant a 1-antitrypsin causes the patient's thrombin activity to drop to such a low level that hemorrhage ensues. Nice to know: COPD (ex. Emphysema)  decrease of elastase inhibits the recoil action of the lungs

Role of Ca2+ in Intrinsic and Extrinsic Pathway >> Except for the 2 steps in the intrinsic pathway, Ca 2+ ions are required for promotion and acceleration of all blood clotting reactions. ABSENCE OF Ca2+  NO CLOTTING OF BLOOD (Both pathways) >> when blood is removed from a person, clotting is prevented through lowering the Calcium level below the threshold either: 1) by deionizing calcium to react with substances (ex. citrate ion) or 2) by precipitating calcium with substances (ex. oxalate ion)

Fibrinolysis is the process wherein a fibrin clot, the product of coagulation, is broken down (clot retraction). 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

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PHYSIOLOGY

HEMATOLOGY & IMMUNOLOGY 3: BLOOD GROUPS, HEMOSTASIS & COAGULATION

PLASMIN >> Activated form of plasminogen (profibrinolysin)  When clot is formed, a large amount of plasminogen & other plasma proteins are trapped in the clot  The injured tissues and vascular endothelium very slowly release a powerful activator called tissue plasminogen activator (t-PA)  A few days later after the clot has stopped bleeding, t-PA converts plasminogen to plasmin  Plasmin will now remove the remaining unnecessary clot. >> A proteolytic enzyme that resembles trypsin >> It digests fibrin fibers and some other protein coagulants like fibrinogen, FV, FVIII, prothrombin, FXII. >> Whenever it is formed, it causes lysis of clot by destroying many clotting factors & sometimes hypercoagulability of the blood. >> IMPORTANT FUNCTION! To remove minute clots from millions of tiny peripheral vessels that eventually would become occluded and there would be no way to clear them. It only causes lysis kapag ACTIVE!!!!

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age, cigarette smoking, hormonal contraceptives, and obesity.

 If there is any disruption in any one of these three components of the triad, it will result to clotting or thrombosis. Polycythemia- excess number of RBCs, making the blood hypercoagulate, thereby creating a clot inside the blood vessels REFERENCES

4.

Guyton & Hall Textbook of Medical Physiology 12th Edition by Hall, John &, Guyton, Arthur C. , , Published in Philadelphia, Pensylvania: Saunders/Elsevier, 2011 Berne & Levy Physiology 6th Edition bby Berne, Robert M., 1918-2001., Koeppen, Bruce M., Published: Philadelphia : Mosby/Elsevier, 2008 Ganong Review of Medical Physiology, 23rd Edition, by Barrett, Kim , Barrett, Kim E., Barman, Susan, Boitano, Scott, Brooks, Heddwen, Published: New York : McGraw-Hill Medical, 2010 BRS Physiology 5th Edition by Linda Constanzo, 2011,

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Published: Lippincott and Williams & Wilkins Kaplan Medical Step 1 Lecture Notes (Physiology) 2010

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REVIEW!!

Medical Physiology: Big Picture by By (author) Jonathan Kibble, Colby Halsey, Published: Lange 7. Harper’s Illustrated Biochemistry 27th Edition by Murray, Robert K. by Lange 8. Basic and Clinical Pharmacology 11th Edition by byKatzung, Bertram G. , Published: New York : McGraw-Hill Medical, 2009 9. SBCM Physiology Lectures 10. Various Internet Websites

Hemostasis: to prevent blood loss Breaking the integrity of the blood vessel  blood loss, decrease in blood pressure >>In atherosclerosis, there is a formation of cholesterol plaque underneath the endothelium, which eventually damage it, causing platelets to aggregate leading to formation of a big clot that will, in turn, occlude the vessels of the heart or coronary arteries leading to the condition myocardial infarction (parts of the muscles of the heart does not receive any blood supply due to the formation of clot inside the coronary vessels) Thrombosis Formation Virchow’s Triad describes the three broad categories of factors that are thought to contribute to thrombosis: 1. Stasis - These include venous stasis, mitral stenosis, prolonged immobility (as on a long plane or car ride, bed bound during hospitalization) and varicose veins. 2.

Endothelial Injury - injuries and/or trauma to endothelium includes vessel piercings and damages arising from shear stress or hypertension.

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Hypercoagulability - alterations in the constitution of blood has numerous possible risk factors such as hyperviscosity, deficiency of antithrombin III, protein C or S deficiency, Leiden V factor, nephrotic syndrome, changes after severe trauma or burn, disseminated cancer, late pregnancy and delivery, race, advanced Page 10 of 10