10/16/2018 1(G3) HEMOSTASIS AND BLOOD COAGULATION MRS, PICHT ARCILIE E. MALINTAD, RMT REFERENCE BOOK: CLINAL HEMATOLOGY BY: CHERYL A, LOTSPEICH-STEINIGER, MS,MBA, MT(ASCP), CLS(NCA) 2 3 fe) sila) HEMOSTASIS + Derived from Greek meaning "the stoppage of bled flow” Study dates back to the time of Aristotle and Piato “Hemophilia- the 1* coagulation disorder recognized during the 2»! century AD 112" century ~ “Moses Maimonides described 2 male children who died from excessive bleeding after Greumeision ‘Hemophilia ~ means” love of hemorrhage” by Schonlein “First described as such by Hopff in 1828 Functions of Blood vessels in Hemostasis: ‘After the injury, the damaged vessels initiate hemostasis, ‘Their first response to injury is constriction or narrowing of the lumen of the arterioles to minimize the flow of blood into the wound area and the escape of blood from the wound site Vasoconstriction also brings the hemostatic components of the blood{ the platelets. and the plasma proteins) closer to the vessel wall, facilitating their interactions. «Vasoconstriction occurs immediately and lasts a short time 9(63) Vasoconstriction Its caused in part by neurogenic factors and in part by several regulatory substances that interact with receptors on the surface of the cells of the blood vessel wall, * These include serotonin and thromboxane A2(TXA2), both products of activated Dlatelets, and endothelin-1 produced by endothelial cells 10(3) HEMOSTASIS + 3 Basic Components 2 1. Extravascular 2. Vascular 3. Intravascular 11(©3) HEMOSTASIS and BLOOD COAGULATION » + Extravascular Component > sTnvolves tissues surrounding the blood vessel "Mechanism: provides back pressure on the injured vessel thru swelling and the ‘rapping of escaped blood *Factors involved:10/16/2018 1. Bulk of the surrounding tissue 2. Type of tissue surrounding the injured vessel 3, Tone of the surrounding tissue 12{E3] HEMOSTASIS and BLOOD COAGULATION + Vascular Components 2 «Involves the vessels thru which blood flows *Role played by the vessels depends on their 1, Size 2. Amount of smooth muscie within their walls 3. Integrity of the endothelial cll ning 13([) HEMOSTASIS and BLOOD COAGULATION * +Intravascular Components sey components: Platelets and biochemicals (procoaguiants) in the plasma “Involved in 2 essential processes of hemostasis: 1. Coagulation (dot oF thrombus formation) 2. Fibrinolysis (dot or thrombus dissolution) 14{Gi] HEMOSTASIS and BLOOD COAGULATION 15|C)) HEMOSTASIS and BLOOD COAGULATION * «Normal Hemostatic Balance 2 sHemostasis *A.complex interaction between blood vessels, platelets and. biochemical reactants or factors in the plasma «These interactions not only create cots that stop bleeding thru the coagulation [process but also dissolve cots thru the fibrinolytic process as injured vessels are healed 6 ¥7(Cl] PROCOAGULANTS vs ANTICOAGULANTS: “FAILURE OF HEMOSTASIS. - HEMORRHAGE *FAILURE TO MAINTAIN _ FLUIDITY OF BLOOD - THROMBOSIS 18(Gi] HEMOSTASIS and BLOOD COAGULATION Hypocoagulation » sInherited or acquired conditions with abnormal bleeding Several conditions are readily diagnosed by lab tests particularly coagulation factor assays ‘Hemophilia ‘Example of inherited hypocoagulable disorder ‘Classic hemophilia (type A) +Factor VII deficiency "DIC, liver and kidney diseases ‘may cause acquired hypocoagulation10/16/2018 21[5) HEMOSTASIS and BLOOD COAGULATION Hypercoagulation ® Associated with the inappropriate formation of thrombi in the vasculature that: ‘occlude normal blood flow Thrombi usually consists of leukocytes, piatelets and RBC held together by fbrin *Thrombi can be palnful and life threatening if the blood supply to a vital organ is cut off ‘2[B) HEMOSTASIS and BLOOD COAGULATION Hypercoagulation * + Coused by a defect in or lack of activation of the fibrinolytic system "Most are associated with acquired diseases or altered physiologic states ‘Most often, a malignancy or surgical procedure is the stimulus for hypercoagulation 25|B)) HEMOSTASIS and BLOOD COAGULATION 2s|E3| HEMOSTASIS and BLOOD COAGULATION ‘Primary and Secondary Hemostasis 2 sHemostasis occurs in 2 phases: Primary and Secondary = Primary hemostasis sinvolves the vascular and platelet response to vessel injury Secondary hemostasis ‘includes the response of the coagulation process to such injury 27(G) HEMOSTASIS and BLOOD COAGULATION * Stages of hemostasis after vascular injury + Bleeding occurs after an injury to blood vessel “The hemostatic system is activated to prevent excessive blood loss + Hemostsis occurs in two stages 2) primary hemostasis when the platelets aggregate at the site of injury and form "platelet plug and b) secondary hemostasis when fibrin developes to strengthen the platelet plug eq TING he forn-latlt plat secondary hemestate plug 2B) “What happens is, the endothelial cells secrete these proteins and send a signal to the Platelets to come to that sight of injury. Then, a sequence of events happens with the platelets. I won't go into details here, but it allows the platelets to stick to that Sight of injury. They get activated. They change shape. They release wht they have10/16/2018 Inside, and then they start clumping together and call more platelets, and cause ‘more platelets to clump there as well. What we end up with is what we calla platelet plug. This isthe first step of stopping any bleeding, This is stil kind of weak, and we ‘need to make this stronger. That's where secondary hemostasis comes into play. In secondary hemostasis, we make this platelet plug stronger with a protein called fibrin. What fibrin does is it ays over and links up on top of ths platelet plug, and ‘makes it tighter and sturdier 23[G]| HEMOSTASIS and BLOOD COAGULATION. + «Primary and Secondary Hemostasis > = These processes ultimately lead to the formation of a stable fibrin-patelet plug at the site of injury, which permit vessel healing “At the same time, fibrinolysis i initiated, allowing for gradual cot dissolution 20 (G3) Basic Sequence of Events in Primary and Secondary Hemostasis After Vessel Injury 3215) Vasoconstriction “Iti caused in part by neurogenic factors and in part by several regulatory ‘substances that interact with receptors on the surface of the cells of the blood vessel wall, *Tese include serotonin and thromboxane AZCTXA2), both products of activated «any ,Platelets, and endothelin-1 produced by endothelial cells 2) HEMOSTASIS and BLOOD COAGULATION 34(Gl) HEMOSTASIS and BLOOD COAGULATION * Role of Blood vessels in Hemostasis: "Intact Vessels: CAPILLARIES » =Where metabolic exchange between the blood and tissues take place “Lined by as single continuous endothelial cell layer that is attached to a supportive basement membrane ‘The capilary lumen is large enough for a single RBC or WAC to pass through 33(G) HEMOSTASIS and BLOOD COAGULATION Intact Vessels: CAPILLARIES 2 “There are openings called "junctions" along the capillary wall that allow passage of ‘WBC, 02, and nutrients into and out of the blood as necessary RBC and platelets usually do not leave the intravascular system *Pericytes- cells that lie beneath the endothelium of capilaries, arteries and veins that ‘may differentiate into vessel related cells when needed 36(E]] HEMOSTASIS and BLOOD COAGULATION * slIntact Vessels:10/16/2018 CAPILLARIES 2 Pericytes/Rouget cells contractile cells that wrap around the endothelial ces of capilaries and venules throughout the body Embedded in the BM where they communicate with endothelial cells through direct, physical contact and paracrine signaling s7[E3] CAPILLARY Intact Vessels: ‘Arteries and Veins 263 layers 1. Tunica Intima (inner endothelial fining) * Endothelial cells deposit von Willebrand factor (VWF) In the subendothelial matrix where VWF binds to collagen ‘Separates the blood cells from a subendothelium (BM, elastic conn tisssue and collagen fibers) HEMOSTASIS and BLOOD COAGULATION + Intact Vessels:Arteries and Veins 2 3 layers 2. Tunica media ~ composed of smooth muscle cells and connective tissue 3. Tunica adventitia (outer part) + Composed of connective tissue fibroblast and collagen fibers 0(G] Artery and vein pie ‘:|B) HEMOSTASIS and BLOOD COAGULATION + Intact Vessels: Functions 2 =Vasoconstriction and vasodilation provide the means of control of blood flow rate and. blood presssure *Controlied by the smooth muscles of tunica media Endothelial cels secrete several important substances that influence coagulation, flbrinolysis and platelets 2G) Antithrombotic, Fibrinolytic, and Coagulant Substances Released from or found on the Surface of Intact Endothelial Cells ‘0(@]) HEMOSTASIS and BLOOD COAGULATION “Intact Vessels 2 +The intact endothelial tining of blood vessels is antithrombotic ‘It does not activate platelets or promote coagulation10/16/2018 “Itwll provide a smooth surface that facilitates blood flow and reduces turbulence (promotes thrombosis) (Gi HEMOSTASIS and BLOOD COAGULATION Damage _ vessels 2 =Vasocontricton occurs as a neurogenic response(nerve reflex) "Breaks the smooth endothelial ning, exposing collagen which causes adherence of platelets to the area of injury +s(G) HEMOSTASIS and BLOOD COAGULATION + =Damage Vessels * sCollagen exposure also initiates the contact phase of coagulation, which begins a series of biochemical reactions known as the intrinsic coagulation pathway + Tissue thromboplastin is released from the injured vessel, which promotes ‘coagulation thru a different series of reactions known as extrinsic coagulation pathway HISTORY 2 +1842: Platelets were first described Later, It was discovered that platelets originated from megakaryocytes in the bone marrow +1940 — platelet structure was studied using the E/M *+1950- a method for counting platelets was described that utilized phase contrast microscopy Today, platelets are routinely counted using automated methods HEMOSTASIS and BLOOD COAGULATION HISTORY ‘Qualitative (functional) platelet evaluation was first avaliable with the introduction of the bleeding time by Duke in the early 1900 ‘A prolonged BT can indicate either a thrombocytopenia or thrombocytopathy “BT Is stil the best screening test for platelet function “More specific tests such as platelet aggregation studies are now available 5:(G) HEMOSTASIS and BLOOD COAGULATION Platelet Morphology and Function in Hemostasis10/46/2018” 2 =Platelets play a central and immediate role in the response to vessel injury + Smallest microscopically visible element in the peripheral blood film at 2-4ym and have a discoid shape = Wright stain: purple granular appearance and look like specks of dust Small fragments of megakaryocyte cytoplasm «Life span: 9-10 days 3(@)) HEMOSTASIS and BLOOD COAGULATION * Platelets play the following important roles in hemostasi They adhere to injured vessels They aggregate at the injury site They promote coagulation on thelr phospholipid surface “They release important chemicals for hemostasis ‘They induced clot retraction #@) ‘+. Now in order for platelets to find the site of injury, we have to talk about normal Platelets first and their interaction with endothelial cells. Normally platelets don't adhere or stick = to endothelial cells, and this has to do with nitric oxide and prostacyclin. The two ‘Substances that we had already mentioned that are secreted by healthy endothelial cells. And what they do in addition to causing vasodilation to the smooth muscle cells, is they kind of block the platelets from sticking to the endothelial cells, ‘Like we mentioned, when there's injury to the endothelial cell, then there's less nitric ‘oxide and less prostacyclin that will block platelets from getting closer to the ‘endothelial cell. So now that there's less nitric oxide and prostacyclin, platelets are going to get closer to the site of injury because there's nothing blocking it from going there. In addition to that, we also have this glue, this molecule that provides the link to the site of injury and to the platelet. This glue is called von Willebrand factor. 1 wish there was a better way to remember that. It's a long name. And welll just refer toit as VWF. And this VWF is normally floating around in our blood, but it also gets secreted from endothelial cells at the site of injury specifically. And when von. Willebrand factor comes into contact with the site of injury, it binds to. Platelet Adhesion: “If vascular injury exposes the endothelial surface and underiying collagen, platelets ‘adhere to the subendothelal collagen fibers, spread pseudopods along the surface, ‘and clump together{aggregate). + Platelet adhesion to subendothelial connective tissues, especially collagen, occurs in 1-2 minutes after a break in endothelium + Epinephrine and serotonin promote vasoconstriction s5(E5] Platelet Aggregation + Platelet aggregation isthe attachment of platelets to one another "Newly ariving platelets flowing into the area become activated by contact with ‘agonists such as ADP and TXA2, products from the damage tissue and endothelial cells and thrombin(a procoaguiant enzyme generated by Tissue Factor or/FVIa ‘acting on prothrombin “With activation the new platelets undergo shape change10/16/2018 (G) Platelet Shape Change “The result is cohesive platelet mass that rapidly increases to form platelet plug. «The transformation of platelet from a disc to a sphere with pseudopods produce surface membrane reorganization. “Internal contraction of the platelet result inthe release of the alpha and dense (granules and the lysosomal contents Active Factors in Platelet Cytoplasm ‘Actin & myosin molecules + Residuals of endoplasmic reticulum and Golgi apparatus Mitochondria & enzyme systems Enzyme systems that synthesize prostaglandins + Fibrin stabilizing factor * Growth Factor ~ causes vascular endothelial ces, vascular «_mooth muscle cells, and fibroblast to multiply and grow = helps repair damaged vascular walls ©o(G) Active Factors in Platelet Cytoplasm * Glycoproteins on platelet cell membrane ~ repulses adherence to normal endothelium, but adherence to injured areas "Membrane contains large amounts of Phospholipid that activate multiple stages in blood dotting process. “Half life in blood = 8-12 days Eliminated form circulation by tissue macrophage system * unique cytoplasmic structures: alpha and dense granules * important membrane glycoprotein receptors: ‘Membrane Receptors: > GPIb/IX ~ Glycoprotein Ib(GPIb)is the major platelet receptor for VWF Its found in the platelet membrane as a complex with GPDX and GPV > GPIIb/IIla. Glycoprotein Hb and Ifa associate in the membrane forming 2 ‘noncovalently associated glycoprotein IIb/TITa complex, which is major plasma ‘membrane receptor for fibrinogen >-also binds other subendothelial adhesive proteins such as VWF, thrombospondin, vitronectin, and fibronectin 2(E) Important substances secreted by platelets [5 Important substances secreted by platelets «4[&) HEMOSTASIS and BLOOD COAGULATION. Platelet Morphology and Function in Hemostasis + =Platelet Induction of Clot Retraction “The last act of platelets within a platelet-brin clot is contraction of the clot10/46/2018 Requires calclum and lots of energy (ATP) Observed in vitro when blood clots in a test tube (Indicative of normal platelet function) +The retraction process requires stabilization of platelets and plateletfbrin elements «(EJ HEMOSTASIS and BLOOD COAGULATION Platelet Morphology and Function in Hemostas 2 =Platelet Induction of Clot Retraction “The puling forces are provided by contractile platelet elements in a process similar to muscle contraction Exact purpose of dt retraction is unclear + Rypothests: «Participation in the vascular constrictive response to injury The stabilization of the fibrin clot network «Debulking of the clot to help reestablish blood flow “8 HEMOSTASIS and BLOOD COAGULATION: ©7[2l) HEMOSTASIS and BLOOD COAGULATION Coagulation » sProcess whereby on vessel injury, plasma proteins, tissue factors, and calcium interact on the surface of platelets to form a clot +Platelets not only provides surface for the coagulation reaction but also interact with fibrin to form a stable platelet-fibrin clot 3(5)) Coagulant Factor Nomenclature (3) HEMOSTASIS and BLOOD COAGULATION Coagulation ® «These coagulation factors (except calcium and tissue thromboplastin) normally reulate in the plasma as inactive proteins On activation, some factors form enzymatic proteins known as "serine proteases” that activate other specific factors in the coagulation sequence 7() HEMOSTASIS and BLOOD COAGULATION ‘Coagulation 2 «Zymogens are substrates that have no biologic activity until converted by enzymes to active enzymes called serine proteases, which have exposed, serine-rich, active enzymes sites *Serine preteases selectively hydrolyzed arginine or lysine-containing peptide bonds of other zymogens, thus converting them to serine proteases10/16/2018 71(Gi] HEMOSTASIS and BLOOD COAGULATION Coagulation 2 =The zymogens are factors II, VII, IX, X, XII and prekallkrein “The co-factors are factors V, VIT, tissue factor and HMWK 7G) HEMOSTASIS and BLOOD COAGULATION Coagulation 2 +3 interrelated pathways of coagulation, each representing a unique series of biochemical reactions “Intrinsic Pathway Extrinsic Pathway Common Pathway nl a 73{C3) HEMOSTASIS and BLOOD COAGULATION activated by: “the release of tissue thromboplastin into the plasma from the injured tissue calls Tissue thromboplastin activates factor VII to the serine protease factor VIIa Factor VIT with calcium and platelet phospholipid (PL) activates factor X to factor Xa in the common pathway 79(3] 77(G HEMOSTASIS and BLOOD COAGULATION 2 +Actvated by exposure of the subendathelial BM and collagen “When the subendothelial surface is contacted by the coagulation contact factors XILXIHMWK and prekallkrein, the contact activation of the pathway is begun + Factors XII and X1 are converted to the serine proteases XIla and Xia begins with trauma to the blood or exposure of the blood to collagen from a traumatized blood vessel wall. Then the process continues through the series of ‘cascading reactions10/16/2018, 78(&3) HEMOSTASIS and BLOOD COAGULATION ‘The Intrinsic Pathway 2 +Factor Xla with calcium in turn converts factor IX to the serine protease Dxa “Factor Dia with platelet PL, calcium, and a cofactor, factor Villa converts factor X to the serine protease factor Xa In the common pathway ay HEMOSTASIS and BLOOD COAGULATION ‘The Common Pathway 2 Begins with activation of factor X to factor Xa by either the intrinsic or extrinsic pathway “Factor Xa with co factor, Factor V converts factor II, prothrombin to factor Ta, thrombin “Factor Ha converts factor I, fibrinogen to fibrin + Factor XIII then stabilizes the clot #s|53) HEMOSTASIS and BLOOD COAGULATION FIBRINOLYSIS IN HEMOSTASIS. «(3 FIBRINOLYSIS IN HEMOSTASIS History * +1700'S- John Hunter reported the unexplained finding that blood from people who had died in accidents did not clot + 1937- MacFarlane reported that damaged tissues release a substance (plasminogen activator) that activates the inert precursor called plasminogen to its active form, plasmin *Plasmin- a nonspecific proteolytic enzyme capable of degrading fibrin as well as fibrinogen and factors V and VEIT #7{G) HEMOSTASIS and BLOOD COAGULATION Function of Fibrinolysis in Hemostasis 2 Fibrinolysis- ‘system where the temporary fibrin dot is systematically and gradually dissolved as the vessel heals in order to restore normal blood flow *Plasmin aa10/16/2018 proteolytic enzyme that Is the primary substance responsible for fibrinolysis =Tissue plasminogen activator released from injured vessel walls convert plasminogen to plasmin e=(G] Plasmin causes Lysis of Blood Clots + Plasminogen / Profibrinolysin +Euglobulin contained in plasma proteins *When activated become Plasmin/ Fibrinolysin Plasmin *Resembles trypsin = Digests fibrin fibers and some other protein coaguiants (Fibrinogen, Factor V, Factor VIII, Prothrombin, Factor X11) 2 *Can cause lysis of a clot by destroying many of the clotting factors Sometimes causes Hypocoagulabilty of blood Remove minute clot from millions of tiny Peripheral Vessels, #2(Gi) HEMOSTASIS and BLOOD COAGULATION Function of Fibrinolysis in Hemostasis 2 =Plasmin Is trapped within the clot, and clot lysis begins slowly as soon as the clot is formed, with fibrin degradation (split) products (FDP or FSP) being released in the plasma “Lysis is slow because of fibrin clot stabilization by factor XIII, fibrin stabilizing factor *Protein C and S are 2 hemostatic substances that further fibrinolysis and inactivate {PA inhibitors 0 A eo = s2[) BLOOD COAGULATION TESTS : “BLEEDING TIME > +When a sharp- pointed knife is used to pierce the tip of the finger or lobe of the ear, bleeding ordinarily lasts for 1 to 6 minutes. ‘The time depends largely on the depth of the wound and the degree of hyperemia in the finger or ear lobe at the time of the test. + Lack of any one of several of the clotting factors can prolong the bleeding time, but itis especially prolonged by lack of platelets. BLOOD COAGULATION TESTS CLOTTING TIME 12