Coagulation and Hemostasis PDF

‫‪Coagulation and Hemostasis‬‬ ‫‪Part 1 – Introduction‬‬ ‫ﺗﻔﺮﻳﻎ ﻣﻼك ﻋﺒﺪ اﻟﺮازق‬...

‫‪Coagulation and Hemostasis‬‬ ‫‪Part 1 – Introduction‬‬ ‫ﺗﻔﺮﻳﻎ ﻣﻼك ﻋﺒﺪ اﻟﺮازق‬ ‫ﲢﺎﻟﻴﻞ ﻃﺒﻴﺔ‬ ‫اﻟﻠﻬﻢ ﺑﺎرك ﻟﻲ ﰲ وﻗﺘﻲ ‪ ،‬وﻻ ﲢﺮﻣﻨﻲ ﺟﻬﺪي ‪ ،‬وﻻ ﺗﺮد دﻋﻮﺗﻲ ‪ ،‬وﻋﺎﻓﻨﻲ ﰲ ﺑﺪﻧﻲ ‪ ،‬وأﺻﻠﺢ ﻟﻲ ﺷﺄﻧﻲ ‪ ،‬وأﺷﺮح ﻟﻲ ﺻﺪري ‪ ،‬وﻳﺴﺮ ﻟﻲ أﻣﺮي ‪ ،‬وأﺣﻠﻞ ﻋﻘﺪة ﻣﻦ ﻟﺴﺎﻧﻲ ﻳﻔﻘﻪ‬ ‫ﻗﻮﻟﻲ ‪ ،‬وأﺷﺪد ﻣﻦ أزري ‪ ،‬وﺑﺎرك ﻟﻲ ﰲ ﻋﻠﻤﻲ‬ ‫‪A‬‬  ↑ -10.1 · is " Hemostasis It is the process of maintaining blood as a fluid within the D blood vessels under normal circumstances and preventing - excessive blood loss upon injury. -. & - homoestasis Bleeding state ofBalance ! s tight regulation · 85554. 1 - -151 hemostasis i I Procoagulant Anticoagulant -50 lis - $1.Bleedings Thrombosis Bleeding - - 2 ’ - Blood flows through a closed system of vessels called the circulatory system. ’ - The hemostatic system is activated when and where it is needed. ’ - Blood coagulation (“clotting”) is the mechanism that transforms the fluid plasma into a gel by converting the soluble protein fibrinogen to the insoluble form, fibrin. 3d115 is Hemostasis Process Requires the interaction of three major compartments : · 96351 - Vascular integrity -> Platelets iative. -> factor I # Plasma compartment (i.e. Coagulation and Fibrinolytic 353D8 - systems) - ↳ ↳ Loading… Sass active. activator inhibitor I idlisinactive. ⑤ is$ ↳ active 15589;8 558 5 Includes four overlapping processes or stages: di & 1 5. I Bleeding 8. 1.5 i Vasoconstriction Primary hemostasis Secondary hemostasis Fibrinolysis 3 ’ - Maintaining blood fluidity requires an intact vascular endothelium, quiescent (inactive) blood platelets, and inactive plasma procoagulant proteins. On the other hand, control of bleeding requires rapid activation of platelets and plasma proteins to prevent exsanguination.  They have three major compartment: - - 25s 8 Procoagulant) & active" 8) - lds actives in ed!:. I's 85:e.sig"Platelets -g:8 lds: activator Plasmen: - · * 355.81 is active isa Ids:inhibitor *coagulation" ~ Fibrinolytic. 2 ciai,81880s ass # * '84 PH Clis ge (Bleeding) -: 2s () se s :181, · 15.3228 active a inactive - Platelets Plasma; - active inactive 65 activator coagulation. 1 -ises inactives - - & active 65s a inhibitor or 58 is sat inactive, active a 15"35 Fibrinolytic S III"** ** us * - Bleeding, 8;'y · - 8. : ggl834se Jasoconstraction ·I primary hemostasis · 2 secondary hemostasis 3 · Fibrinolysis. 2  ss Vasoconstriction After an injury, the initial response of the damaged blood vessels is constriction or narrowing to minimize the blood flow into the wound area and blood loss. In addition, vasoconstriction brings the haemostatic 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. 5,8858d · =. Process ofJasoconstructions s ( Antiplatlet effect (PGI2, NO, and ADPase) - ↑ >Anticoagulant effect (heparin sulfate, thrombomodulin tissue factor pathway inhibitor [TFPI]) >Fibrinolytic properties (t-PA)  is -> Is bi di lis · Antithrombotic activity of blood vessels " · Antiplatlet effect : dilated isit. Basel,51:8. Prostacyclin (PGI2) ->inhibition activation. Nitric oxide (NO) ADPase -> Vasodilatation and inhibiting plts recruitment and activation. Anticoagulant effect : sysl, & expression Heparin sulfate (Inhibits fibrin formation (cofactor for antithrombin III) ·(3) Thrombomodulin ( Protein C activation) Tissue factor pathway inhibitor [TFPI] (inhibits extrinsic pathway of coagulation) A - Fibrinolytic property : - clot 31.55 is "Sk6s - DD Tissue plasminogen activator (t-PA) ( Activates fibrinolytic system ) P 1115. 81,88(f- PA)J15s - 0-11 i ’ - Fibrinogen is converted into fibrin by thrombin. ’ _ Activated protein C and its cofactor protein S - inhibits coagulation by degrading F-VIIIa and F-Va. ’ - Plasmin is a serine protease that dissolves fibrin clot.  ↓ - ↳ = - - E. S S - To E E j. in SY ↳ = I 1 E ↓ P e B - S E - h 0 I -rains U. - e t I - E ③ e. - e! i. E ↓- gr # - B Se -> - = I ! I ! E * - M u - - > I so - ⑥ 3 · - E s * S E 8 H & A - S · i ⑤ E ⑨ ·- e - - = ⑤ ⑤ - :Es 7 O - =j  Thrombotic activity of blood vessels is thrombin II Damaged endothelial cells are thrombogenic and inhibit fibrinolysis : · 8S- I 1 Intitating hemostasis and the secrete endothelin-1 which acts as vasoconstructor.9ABLood - flow). facto 2.11 Loading… (2) & 2. Produce and secrete von Willebrand factor (VWF) which aids platelets in the initial stage of primary hemostasis factor, 3. Produce tissue factor that is released during injury and initiates the formation of fibrin for secondary hemostasis - Expose collagen and other adhesive proteins in the subendothelium and secrete platelet-activating factor (PAF), which activate platelets PHA d activation $ Release plasminogen activator inhibitor (PAI-1), which inhibits fibrinolysis. I 13 ’ - von Willebrand factor (VWF) is a large adhesive glycoprotein required for platelet adhesion to subendothelium at the site of vessel injury, platelet aggregation to form the platelet plug, and stabilization of factor VIII (FVIII) in the circulation. ’ - TF (also known as tissue thromboplastin or coagulation factor III) is the high-affinity receptor and cofactor for factor (F)VII/VIIa.  10 & - - 15 ’ - Antithrombotic characteristics of resting endothelium versus the prothrombotic effects of damaged or activated endothelium. ’ Resting endothelium provides an environment that inhibits activation of hemostasis. This includes secretion of substances that (1) inhibit platelet activation (PGI2,NO, ADPase); (2) inhibit coagulation (heparan sulfate as a cofactor for AT, TM for activation of protein C, which inactivates activated FVa and FVIIIa; and TFPI); and (3) activate fibrinolysis (tPA, uPA). When endothelium is damaged, it secretes substances that (1) activate platelets (TXA2,PAF, ET) and bind them to the vessel wall (VWF), (2) activate coagulation (TF, which initiates formation of fibrin), and (3) inhibit fibrinolysis (PAI- 1).  aged inactive is pH-1B removal $. I Liver, spleen 1i fibrolytic / Blood 5% sss. activePHII system (thrombocyte) - Platelets Also known as thrombocytes. fragment from megakaryocyte ~not cell- a They are small cellular fragments appear with reddish purple granules when they are examined in a stained blood film. from thrombopoiesis ~> Produced by the process of megakaryopoiesis in the bone marrow and released to the circulation where they have a life span of 7-9 - days. & They are approximately 2-3 µm in diameter. site 1 The normal concentration of platelets in the blood is 140-440 X 103/µl (or 109/L). I W reduction 1.581- oxygen carrying & capicity Strange - (I isigil -PH I1 =Od = PHsige s,i-1; & So "Big 28 thrombocytopenia 5s. $1 - 158e5isis di ⑮; 17 ’ - Platelets are the smallest of the circulating hematologic elements. They are not truly “cells” but are membrane-bound anucleate fragments of cytoplasm derived from precursor cells in the bone marrow called megakaryocytes. ’ - Platelets circulate in the peripheral blood for 7–10 days; nonviable or aged platelets are removed by the spleen and liver. ’ - Megakaryopoiesis is the proliferation, differentiation and maturation of megakaryocyte in BM under hormonal and growth factors influence. megakaryaiueel Platelets Production They are produced in the bone marrow from the same progenitor cells as the erythroid and the myeloid series (CFU- GEMM). > grythrocyte As megakaryocyte The bi-potential (CFU-E/Mk) gives rise to the precursor cells committed to the megakaryocytic development. CFU megas18 => 3 - The morphologically recognizable platelets precursor cell is the megakaryocyte. Platelets are fragments of the cytoplasm of mature megakaryocytes. 19 · is 91.5b &S SIIsisbell b ↑ · - &II, U - & 9 9 581,5 big - 1 ⑰ ↑ $158 - ⑪S & * ⑰ -> j -> megakarysaghr 20  Slide notes Slide 20 - Hierarchical pathways in hematopoietic development. Schematic diagram depicting the relationship between distinct identifiable progenitor populations during hematopoiesis. Non- - > megakaryocytic differentiation pathways are shaded. The dashed line represents an alternate pathway of MEP derivation ( 9, 10 ). = LT-HSC long-term hematopoietic stem cell, ST-HSC short-term hematopoietic stem cell, MPP multipotent progenitor, CMP common myeloid progenitor, GMP granulocyte- macrophage progenitor, CLP common lymphoid progenitor, MEP megakaryocyte–erythroid progenitor, MkP megakaryocyte progenitor, CFU colony-forming unit 21  key regulator is Regulation of Platelets Production Interleukin-3 (IL-3), IL-6, IL-11 and stem cell factor (SCF) affect megakaryocyte development. But the- major factor regulating platelets production is G thrombopoietin (TPO) which regulates all stages of platelets production from the first recognizable stage until release of mature platelets to the circulation. = 1 1.PHS85-219 S T 51 -> TPO has receptors in the circulating platelets, bone marrow megakaryocytes and the progenitors cells. -B TPO produced mainly in the liver and to a limited level in the spleen and kidneys. 22 22  Slide notes Slide 22 -$9,55. 01:s DHC2151;TPOS1 5. - TPO is thought to maintain a constant baseline number& ↑ of platelets in the peripheral blood via a unique mechanism called the sponge model. Cellular production of TPO is relatively consistent rom day to day. TPO binds to its receptor on circulating platelets and bone marrow megakaryocytes and 1 2. progenitors. TPO bound to circulating platelets is internalized and degraded - and is not available to stimulate proliferation of bone marrow progenitor cells. Therefore, the higher the peripheral platelet count, the more TPO is bound with less free TPO remaining in the plasma. This reduces both the stimulation of megakaryocyte progenitor cells in the bone marrow and platelet production. When the platelet count decreases, more TPO is free to bind to megakaryocyte progenitor cells in the bone marrow, increasing plateletTPO production.(1251: TPOsI 1S PH IPH le TPO CIb "il receptor. IPH S 35 is play, riggins sa91 8159.9-4s - TPO,PHSI 54. SSidler liver Iss. 1; s TPO(I- Ier(1,2) -siPH died 18 -TPO 1188is *d i9 Plt slid. sla.ITPOI 81, TPOC1d9ipHd - / I PL/ 5 =. -189581,-_48 major 23 23  IO IL-3 2- -> liver [Production of TPO] pH s1 > multipotenthematopoietic growth factor --g." +POSSI ↓ & 5,0 cell line 959.51.939s - circulation [have PH] FLII"is differentiation, regulation, growth 11. b mast cell, activated / compined PH+TPO ↓ IL-6 + IL-11 amount ofTPO d IL-6 act with IL-3 to shorter 60 period ↓ of early progenitor and support megakaryocytic TPo go to the B.M colony formation and maturation. ↓ I naturation). So IL-3 1 S dis- IL-6 s amount ofPI production A ·cellline I IL-11, 12-6 ds cell line / $.59. IL-3 -di s Why?! megakaryocyte &,5%.ss regulation (1,5. 9's cell line X Because in circulation when the pit compind I Stem cell factor with TPO, the mount of TPO is reduced essential cytokines interact with other cytokines So, the released that amount to the to preserve siability of ASC and progentor. B.M the B.M cell. to stamiulating to produced PHis little, that it II 1$ $56 cell cycle;15s$* if. We have alittle amount in the of pH differentiation, proliferation circulation;the amount ofTPO is not reduced, and released the BoM a lot 1 & 5 = that is mean the amount of pl - $10 455 - - will production alot X. = & -,dl's 85 3>8 -  PAI1258.3 TPO 1126, - ↓19-s 11 & Ac CFU - receptor i 11,8 i s dlc da > S ·PH 24  Slide notes Slide 24 The regulation of stem cell (HSC) and progenitor cell - (HPC) differentiation and expansion is critical because it determines the concentration of the various lineages in the marrow and eventually in the peripheral blood. - Specific glycoproteins called hematopoietic growth factors, or cytokines), govern hematopoietic precursor cell survival, self-renewal, proliferation, and differentiation. & 25  -> maturation of megakaryocyte.) Als. 1g TPO 11 major regulator. 26  Bignucleous synthesis synthesis of cells Megakaryopoiesis -> with Bignuclue. The precursor cells begin a unique nuclear maturation process consists of a series of endomitosis. During endomitosis, the cell's DNA content - - double but cell division do not take place. The resulting cells become polyploid, meaning that the ploidy level (normally 2N) range from 4 – 64N - or higher. TPO simulates platelets production by -> note increasing the number of megakaryocytes in the bone marrow, increasing the ploidy level, and increasing the maturation rate so decreasing the C maturation time. 27 I It was reported that administration of TPO can increase platelets production from 3-7 times. -  cell cycle - &. 198. Isinterphase is s -> -g? ") DNA188-89.2: M-phase (1 5I -98 [mitsin - · - M-Phase - 159 tId83 - M-Phase - -> 251- 1 25 (ssissi rsil isnucluous., cytoplasm Iyl is. 51,5! 4,5 sl 5 5s g di s4 8 - 99.5. dis -> I mitosis (581551) cytokinesis. (15_dy5t) ismiss - is 9-134 I,balbrsidibles it in- anaphase a big, sr anaphase (15598.-sdl-jed d megakaryocyte II. I ~ anaphase Bs -endomitosis & - & 11ssift 1991:anaphase-ais s yit is asis. - - (,954, a(15] · Ib - E cytoplasm II. olis11s=IK ydl l s i'd b11.sss. 95018's $8 5 megakarycyte (1 "ild is Yanaphase j.j. %"isstissanapuasebs · ssss. & A) II, s,. 16850K..i Se s s yzulnessessi ↑ I "*ncnssgt,Ess mslid listed and is sendmitosisisthe 2N 5.cellcychell's ics- 2N 8se11s b1 - II. t b & 55 t.js anaphase - all is jigs (4N) bis Promegakaryrcyte. ⑭ b -: endocytosis $55 megakaryocytes asweiss 11-4 (5, cell cycle 1! I, 111 8 r anaphase - a StIs cytokinesis, anaphase B I skip is - nucluer, cytoplasmic 8-9,.a. divison division  - Is1 +. (33) wis81-1 & note 1 S5 pIt (13885U Ploidy level 1158158% tx ploidy is Italy s - Level Sip I1 *t PH 2100033000. 851 Ploidy kual 1108 did is it Production ofJolj&& TPO Level II yi;d 5- Pl · 8i[3-1) PH is Production is,is ↓ Ploidy Level (IGN) 9-1, 81- so 5.89I...ON & =1, 8.5."YN 195, slide 37 - 1.? Why s - $.dd?s anaphase-13 g $ $28,/ anaphase - A i5"Ik s1:8 ↳ ,. , 51_18!s6 q. i s I round - D Is - round cells see is socellcycle 1? 85, 5.751 & · irregular sell 9 J5 Bigo volume 115 ig.s maturation() 59 megakaryocyte megakaryocyte. 200-300 - IPH$, 91 (10003000) - 1 megakaryocyte Isis granule => 5s.granule islarges Protein & granule 185 Als content1) , DNA i from megakaryoblast -> to the mature megakaryocy) PH 1225 -,-19297 as , - 9112-3 215."PH 1155t jggdTPO II ↓d. 11is Pluidy Level 5.S, = maturation 8,Il cell (I -- 5 · Socosoro. 5 - 1000-3000, 51 slide. 40- (notes). PH is Blood Dis. I? B.M (IPHsid &'megakaryocyte 8581/PH - st B.M is B.M 118, s s PH (1*' 111 s & release megakaryocyte is - Blood - strem 1 PH1jg 5s B.M 5.5. s -825. S Sinus  Platelets Release One mature megakaryocyte produces between 1000-3000 platelets. - B ↓j's Process 39 content PH811.. ! Jessel 31 ’ - The primary site of megakaryocyte development (megakaryopoiesis) and platelet production (thrombopoiesis) is in the bone marrow. Mature megakaryocytes are typically situated near the abluminal surface of the marrow sinus endothelial cells and shed platelets directly into the marrow sinuses. Each megakaryocyte is estimated to give rise to 1000– 3000 platelets, depending on the ploidy of the parent megakaryocyte. ’ - Platelets form by fragmentation of megakaryocyte cytoplasm. They appear to be released from membrane extensions of megakaryocytes in groups called Proplatelets which are long slender protrusions of megakaryocyte cytoplasm. Each megakaryocyte extrudes multiple cytoplasmic extensions between endothelial cells into the marrow sinuses as proplatelets, which then break up into individual platelets.  Meta Megakaryocyte megakaryocytel- i 1release PH II. ’ Also known as bare megakaryocyte nucleus. => ’ Refers to megakaryocyte nucleus remaining after platelets have been delivered to the venous sinus. ’ Nucleus is phagocytized by macrophages and broken down. -Mega * 12- -ll) g r PH 11 B. M 1155 4s oligi as - Blood (1.5 As Phagocytosis and degredation & megakaryocytelli'sDJ, s ↑Demarcation membrane system (DMS) ↑ ↑ I ’ Is an internal membrane system of channels that invade from the plasma membrane and grow over the course of terminal differentiation to subdivide the entire cytoplasm. ’ Platelets are released by fragmentation in the cytoplasm of megakaryocytes. ’ During platelets release, they are appear to be released in groups surrounding by the DMS (called pro-platelets). ’ Then, pro-platelets become individual platelets and released to the circulation. ’ Two third of the platelets that are released in the peripheral blood circulate in the blood stream, and the remaining third is stored in the spleen and is in equilibrium with those platelets in the circulation.  ~ Demarcation membrane systems - - "ber piasaemissPlasmamembranei , gr'yyte";s megakaryrcytel 1. -9 8 Siz's & s --, i. bes-ald ⑰ I granules,its cytoplasms s.-Ad PH Jk2, (1000-3000) olt's cytoplasm (I ij) g1 ss. "I ar(41-3000) 5! 5; 5) 38 cytoplasm, it "old S · () rgk bx,lisisi s(stil cytoplasmo I granules$55 11581,25881 membrane is$ 9y61,513 I demarcation sys - ↳ - 185-55e.19134.5dIPH(I1 5d / s ↑ line of Cleavage demarcation is, membranesgranuls s. bis, diss is pads d Proplatelet 11 I :Proplatelets 1 b1 igee-s.--(bcs · 68PH $1, 3d demarcationcyt1915. demarcation Zone & &Nagel s e.:glcr megakaryocyte s,s 551- - is Il sd ls- is I158u demarcation sys. I i.l Proplatelet (1,8. sei i ej -> iesi proplateletslar-K198169 I · Demarcation membrane system (DMS) 8 08&& feltgles 80 ⑧ ’ - (a) Mature megakaryocyte showing future platelets. ’ (b) Platelet release from mature megakaryocyte. Outward extrusion of cytoplasm, eventually breaking up into individual platelets delineated by demarcation membrane system (arrow). Expelled - nucleus is phagocytized by marrow macrophages.  Platelets structure &, -> unactivated 413 wI (is 'smembrane (1) 3s5t ; - ’ The platelet ultrastructure is divided into four zones: peripheral zone : Glycocalyx , Phospholipid bilayer is  - - -  structural zone : microtubules and microfilaments ( plts shape and contraction ) 1) organelle zone : Dense bodies, Alpha granules 56 - I (2)  membrane systems : open canalicular system, Dense tubular - system - Peripheral blood (1. s:2/3-PH11855, - · spleen Sis.5.)1/3 I - circulating “resting” (unactivated) platelets are disc- ’ shaped anucleate cell fragments with smooth surfaces. 2.’ - the surface membranes of platelets have several openings or pits resembling holes in a sponge. The pits - are the openings to membranous channels that extend deep into the interior of them.  Phospholipidbilayer, glyco?Y : integral protein, receptor (glycoprotein) [D 2. Platelets structure (Glycocalyx) pHIs, 1 , PHC1, RBCs -50 1,2,3,4 - ~. aj8 WBL> s ·$ 3 I ’ It is the outer coat, consists of glycolipids, 2 3 glycoproteins, mucopolysaccharides. & ’ It is responsible for the platelet's surface negative - charge which repels platelets from each other and from the endothelial cells of the vessels. "layer of glycocalyx. 1. -:21sess, glycocalyx (slide 4al - The glycocalyx, is thicker on platelets than on most other cells. It consists of 2. Phospholipid bilayer (slide 50 +6152) I glycolipids, membrane glycoproteins, 2 3. receptor [glycoprotein] slide 53 S4) + proteins, mucopolysaccharides, and 3 4. Integral protein ⑭ adsorbed plasma proteins, including $ gas s coagulation factor V (FV), VWF, and liver e ⑤ - fibrinogen. C megakaryoll]55 , - 11 enothelial is cell  Platelets structure (Outer membrane) membrane. ’ The asymmetrical aarrangement of the phospholipid bilayer is an important factor in the function of platelets. [I] x membrane pH (1.8,0865 ’ The phosphatidyl-choline (PC), which is neutral in charge, is concentrated in the outer leaflet. While negatively charged phospholipids (phosphatidylserine {PS}, phsphatidylinositol {PI} and phosphatidylethanolamine {PE}) are concentrated in the inner half of the bilayer. ’ The negatively charged phospholipids are located in the inner leaflet, so they are separated from interaction with plasma coagulation proteins. However, during platelets activation, these phospholipids become exposed in the outer leaflet facilitating their interactions with plasma coagulation factors.  Platelets 2 [WF] factor 11. 8881 receptor (1318 Glycoprotein (GP-Ib/IX) complex - Fibrinogen 88) receptor (188 Glycoprotein (GP-IIb/IIIa) complex I?Hemostasis II & 3.99)9s. 82 receptors 69 : 61; · Site of injury (2 PH (Is So adhesion - I-9 sgbssIbs' receptor (1.1 - · 8ss 7"*s PItaggregation is. A sil receptor 11.2 Platelets structure [organells] - slide 56- - electronst -:[0. ],03ps s dense gis-The dense Bodies & microscope light L [].11s :61,54PH ·electron microscopelli'sjs structured, I - 1 PHbostorage II, screation (1 - J6, PH 115.5granules (1 - s 5 component s r dense Bodies [3-8].sesad-391 - dense Body coagulation factors activations & a calciumions 1 pinal - glands - his :, PH [adliss] neurotrasmitter D&, Plasma III is a serotonin. 27-PH (13,555 activation - =50/2ADD - 8s PIs, energy 19 & ADP 3 - & , dense II s-see-I'scell I Bodies ↑ metabolic [51 = Apry non - -8 -, pool 2 9 storage 1 15 (115. (ADP 110, 395;/ - activated PIF $ 5 nonmetabo.IIl metabolic - -SIPH Ds dlaPIt II ismostprominent granules (1659- Alpha granules (2-2) - 5.... SY11's factors, (3) fibrinogen, (2) IWF 21] 8cl, b.sw [50-80]s - -IFI I1 b vwF(lessendoth.II or endothelial's S, [Mega] II'sS IWS PHsgigs ismega. S1,rweibel Paladebodies 1113 - 57. 55- ’ - The negatively charged phospholipids (PS, PE) accelerate several steps in the plasma protein coagulation sequence. The placement of these phospholipids on the platelet’s inner membrane separates them from the plasma coagulation proteins and prevents inappropriate coagulation. However, during platelet activation, activation of scramblase causes the movement of these phospholipids to the platelet surface, facilitating their interactions with plasma procoagulant proteins.  Platelets structure (Organells) ’ The Dense bodies (DB): they appear dense under the electron microscope because they contain large amount of calcium ions. They also contain serotonin and ADP. is(3) Plasma ( $1595 · -] (2) ’ Alpha granules (αG): contain VWF, fibrinogen, FV and (2) other substances (such as adhesive molecules like - - slide fibronectin and thrombospondin). PHfactor (4) A PHdrived 57 * growth factor ** VWF is synthesized in endothelial cells and megakaryocytes during platelets development, while fibrinogen is absorbed from the plasma by the megakaryocytes and stored in these granules. p  : · ↳ de. E. - - E ⑤ E a E I E. - = - · - E stee 3 SiEs r e de 8a 3 - e E e E i - - i i = -> I. -. O 8 S I E i- : i I i - : - - E ⑧ - 3 o I = 8 - t : So - E & O n - & E B I · E #. zi ↳ A I fg - S - E ↓ - - Ei - ↑ - ⑦ e E aS E i A - & ↳ E ? - - Gi - & ↳ o B  535s 20: ’ - four types of granules dispersed within the cytoplasm: [I] dense granules, alpha-granules, lysosomes, and peroxisomes[D [] ’ - The ADP in the DG is known as the non metabolic or storage pool ADP to distinguish it from metabolic ADP - => > found in the cytoplasm. ’ - The metabolic pool of ATP/ADP provides energy for normal platelet metabolism, whereas the storage pool is important in platelet activation reactions. ’ - Serotonin is taken up from the plasma and stored in the DGs. - ’ - Alpha granules also contains platelets factor 4 (neutralizer of heparin, wound healing) and plts derived - growth factor. - - -561* * " is - is18. & Platelets structure (Membrane system) granular a content opteface 5 secreational, absorbtions.I system 1 ’ Open canalicular system (OCS) which is composed of 9 interconnected series of channels leading from the platelet surface to its interior. mal Responsible for the entry of external substances into the interior of platelets and a route for the release of granule contents to the outside. 2 ’ Dense tubular system which are channels that are not connected to the surface of platelets. Storage sites of ionized calcium. calciumlis:S S-5. 8:Dense i! sU9It II gigs. 895 II'ss,s wi PIF III's calcium 185; Sysseds ↳ dense s ·thromboxane as 2.3 x55  O I ⑧ - - - S S j = ⑧8 I - sS. I S I : ⑧ S - * S·. = - je I Es ↳ S I. set bya gas 8.8. i : - S S - - g 5 8 G I 1 ⑱ & · S 1 P i : A S - i 18 · I s 9 A  Platelets Functions ’ Platelets role in hemostasis: 1.  Maintaining vascular integrity. 2.  Platelet-platelet interaction (primary hemostatic plug). 3.  Platelet-coagulation factors interaction (secondary hemostatic plug). 4.  Aid in healing injured tissue.  ~ Platelets functions = hemostasis IIs role II 25 11s)- heparingneutralizer, chemotractant 1955, 550 factorysix maintance vascular I site of injury I'dIs. integrity ↳S, infection,jest,"1. vascular endothelial (111 -4591 cell · (Bestbe 11asg · is Il.ssi mantaines. I'di sig & intercelluar adhesion&b75.2, s. factor PH II. 11s - l-al P(t(18559855518lag 2 jellies. Isit is. ;9 I ·  ’ - platelets granules contain proangiogenic cytokines and growth factors, which are released upon platelet activation. These 1 cytokines function to stabilize an intercellular adhesion protein ④ - complex between the vascular endothelial cells. When the * circulating platelets are low (thrombocytopenia), the adjacent - intercellular junctions disassemble, resulting in leakage of cells and fluid into the surrounding tissue. (1 iss ’ - Platelets granules contain mitogenic factors such as- PDGF (platelet derived growth factor) and Vascular endothelial growth - factor (Angiogenic factor). -> factor 4. ’ - platelets can support the resting vascular endothelium by at ~ i wine least four mechanisms: 1) platelets physically block potential gaps in the vascular lining 2) platelets and platelet components promote the growth of endothelial cells 3) - platelets help maintain the endothelium ultrastructure, and 4) platelets release soluble factors that enhance the barrier function of the endothelium. Importantly, it has been implied that the loss of these endothelium-supporting functions of platelets could be the cause of bleeding events, presumed to be spontaneous, that occur during thrombocytopenia.  Formation of the primary hemostatic plug ’ The major function of platelets is to seal openings in the vascular system. ’ Platelets circulating in blood vessels do not interact with other platelets or other cell types ( resting plts). 2, 1.s_ s ei ’ A Injury to the blood vessels causes a change in the normal environment and causing platelets activation. (1) (2) ’ Formation of platelets plug requires several steps: - Platelets adhesion and activation I   Platelets contraction or shape change  Platelets release  Platelets aggregation ’ - Circulating platelets are disc shaped and inert in the environment of normal endothelium. This is largely (i) the result of endothelial release of prostacyclin (PGI2)ande nitric oxide (NO), which inhibit platelet 3. activation, and the presence of an ADPase, which degrades ADP, a potent platelet activator. 958,99PH)1 21,5459555115469  1. Platelets Adhesion ’ The initial stimulus of platelets activation is the exposure to sub- endothelial components (collagen fibers) of the vessels wall that are normally hidden from the circulating platelets. ’ Platelets have two collagen receptors (GP-Ia/IIa and GP-VI). ’ However, this adhesion is weak and requires another supportive attachments especially at high shear rates. ’ The presence of Von Willebrand factor (VWF) and platelets VWF receptor (GP-Ib/IX) strengths this attachment. VWF becomes a bridge connecting platelets to collagen fibers. ~’ VWF is synthesized by endothelial cells and megakaryocytes. ’ Mutations in the gene encoding for GP-Ib/IX causes Bernard- Soulier disease while mutations in the gene encoding VWF causes Von Willebrand Disease (VWD). Both diseases result in bleeding disorders.  PIE So 11 "yu highly Collagen fiber) - thrombogenic Platelets adhesion - layer (I.s;dioLayer (18899199) ·8:15 It!, injury (15 pis dlers initial stimulate (1.8 Collagen (1,35 8 two collagen receptor ilPIt II. GP-11 s GP-la /11a - :5*jDia bd!, 5iIgg Preceptor (12 Collagen II bigt- -WF I ,5150!gU agii! Collagen (12 GP-1b 1 Collagen Cp_lalla's - 31 is 1 receptor pir j1e8/ receptor (I) - /1e-8555561. s (1,88c-TWP PHse receptor, PHt (1jS, I Fore &GP. VI Pit -8 5! iS, site ofIIs 15bill, a injury & , ls 5biststgeid, · Qualitive 8s Quntitive is a mutation - Bernard-soulier syndrome - two receptor & Ijii/ You will brand disease is JWF & jgt (s - Glanzmann thrombasthenia aisl ~ Fibrinogen I1j5 receptor ’ - vWF is stored in intracellular organelles, the Weibel- Palade bodies of endothelial cells and alpha granules of platelets. ’ - VWF is a multimeric protein made up of a series of identical subunits, each containing binding sites for GPIb/IX on the platelet surface and for collagen in the subendothelium. ’ - Platelets contain two collagen receptors: GPIa/IIa and GPVI, which mediate direct collagen binding at low shear rates. Platelet adhesion to collagen at high shear rates requires the presence of VWF and the platelet VWF receptor, GPIb/IX  Platelets Adhesion Collagen, 11ab9 PH - i 6 -. 55dig Platelets agonists - · si, il id ja Physical response ’ Are the substances that stimulate platelets and activate them. Each agonist binds to the platelet surface at its specific platelet receptor. string poise deak pos ’ Some of them are strong agonists that can activate the full range of - platelet functions themselves and some are weak that induce plts aggregation without inducing secretion. PHS 5 288s. deak - 1 I aggregation(1$ 5,85.,,320 I strong ’ Platelet-derived agonists: (weak)  ADP -> 555859511819 ldl.s 5.)  Thromboxane A2 (TXA2) -> PH 1115154 5 $53954.38 activation  Serotonin 5". 88"1s,99.9 injury ’ Other (non-platelet derived) agonists: (strong) y1 -> & 39 = -  Thrombin -> inactivated in normal/injury (13,50 injury (1  Collagen - Thrombin is the most potent -> subendothelial layer 4) activator of platelets in vivo. injury 39 rate 3.3 < Platelets activation ’ Adhesion of platelets to collagen triggers a series of changes (platelet activation) which is irreversible and localized at the site of injury. ’ Platelets activation include changes in :  Metabolic biochemistry.  Platelet morphology (shape).  Surface receptors. - Adhesion of platelets to  Release of platelets granules. subendothelial  Membrane phospholipids orientation. components triggers a series of morphologic and functional changes known as platelet activation.  [Slide 72] -> Platelets activation o step rate limit Plactivation 18 II.Si splatelets (1.1 rate limitstep s release, change shape, $.S siPathway 11.3 -- PH activation. , (s) - - I resting I-d31-5. 0-,/ lis · is 1isirreversible activation (-d51-55 is - & bij,es collagen I adhesion ofAlt Sis Localized, inversible ↓,,, u collagen I B sway siteof is sit"8age" 518 -- 19 5 l irreversible (1 - 61 sigtl'ssd5iEIsd 5 biochemistry 11. I => 5Ij disk shape i5-ss."- PIE II, morphology. 2 - 91,! -158 receptor / expression - -a surface receptor. 3 · fibrinogen, vwf (15911.55 receptor receptor · Is -5db5 vs. release of PIt 4 granules resting state &is. Its Phospholipid / asymmetrical is a membrane phospholipid. distribution orientation · 1 ↑ isISIS'sI.1SPS, PI, PE 10's PC II geds slide 73. Platelets shape change 1.115- spiny with long projection, PIE she shape 11 s S active, myosin & Polymerization, microtubules & rearganization - I I PIt A shape 15is X project en =]sI's resting, is disk is disas ·mini ti) as; membrane disk & Isgranules (1$ljs-spiny =skbsd:8155 spiny OCS & effusion of granules I s (5.851 I:8ts S 8 1. Shape change I & granules 18 dis contactwas PH (155,Sr release of content , GP-lb/ia(1 ; fodlis Sr. & other molecule (120 1581 DIE (1& aggregation () fibrinogen I1.S -.  Platelets Shape Change ’ Upon activation, platelets change their shape from flattened disc-shaped cell to spiny with long projections (pseudopods or filopodia). ’ Shape change involves reorganization of the microtubules (sub-membranous cytoskeletal proteins) as well as polymerization of actin and myosin cytoplasmic proteins. [D] ’ Shape change brings platelets granules in close proximity to Open canalicular system (OCS) , facilitating their E - secretion. Also, it provides a larger surface area for contact with other platelets or other molecules. In addition, - ③ activation of GP-IIb/IIIa (fibrinogen receptor) occurs, facilitating platelets aggregation.  2. Platelet secretion ’ Following the shape change, platelets begin to release their granules content to the surrounding area and this process requires ATP. A ’ The open canalicular system fuses with the membrane of granules so their content will be released outside the platelets. [ Alternatively, secretion also can occur by direct fusion with the plasma membrane. ’ Serotonin, ADP and thromboxane A2 (TXA2)are released and function as agonists that stimulate membrane receptors on other platelets resulting in the recruitment of additional platelets. ’ Platelet factor 4 (PF4) is released which has heparin-neutralizing activity as well being a chemoattractant for neutrophil, monocyte and fibroblast to promote wound healing. ’ Platelets contain PDGF (platelet derived growth factor) which is a mitogen, thus contributes to the healing of injured tissue. ’ Alpha granules secrete VWF, F-V and fibrinogen.  slide - 76 Platelets secreation OCs & granules (ly-s1, gs. Secreations (19 bill,I ATP 8s 1182 membrane PIC& - granules I?Other PH SidPI s. (1. Ilal s stimulate other plt & agonists's/,s/s thromboxane A, Serotonin, ADD S10 granules (10,0 chemoattractant is, heparin neutralizing $.5x, factory activity I release 19,S, · (65.s.) wound healing,fibroblast, monocyte, neutrophill I · fibrinogen, J.V, uwp si is alpha granuless) is, ⑲ng', which mitogesdeeasierareaggregateregi granules PIt s 11. j PItto subendothelial attachment 119 PI adhesion Layer -8 PIt aggregation / Step 11 3Jsll, recepter() 91 - 6,Is fibrinogen PIt slbs $.187 GP-lIb/la (18). 5- expression of fibrinogen receptor (10 surface receptor asIS. changings! 5.8) -.. Blood - I 8.1 fibrinogen 1/2 bsss Low affinity receptors55a. High Low a receptor (165-2) 7,5818, ca is secreation 3SEUPIt activation slgis affinity affinity ·Pl aggregated with 11.5 Esso active a receptor (1584%s each other 11 -) singling. Collagen (12 GP. 6 (115: PH (1-6:;11 Creceptor (1) GP-IIb/Illa -> EPI adhesion Eplain Why Plt doesn't requar ca. But - PH ca?! aggregation requar ps. we as big, 916 express, as we (161985. pissed singl Pit adhesion is injury.s, ls is & clotting factor II's fibrinogen is by inactive $950 fibrinogensreceptor (10 receptor / activated. so (a 108. Idd-in de S. Jos strong ⑦ O 11 * membranerule s · pH)1 $18.5.4 -  Platelets Aggregation ’ It is the attachment of platelets to each other. ’ GP-IIb/IIIa is a platelet membrane receptor that binds fibrinogen. ’ The active GP-IIb/IIIa complex appears soon after platelets activation. Resting platelets do not express functioning GP- IIb/IIIa, and unable to bind fibrinogen. ’ Fibrinogen serves as a bridge, cross-linking GP-IIb/IIIa molecules on two adjacent activated platelets. ’ Calcium (Ca+2) is needed for platelets aggregation, whereas platelets adhesion does not require calcium. Calcium is required to change GP-IIb/IIIa from low affinity to high affinity receptor. Calcium is supplied from both plasma and the internal platelet storage sites. ’ Patients who have decreased levels of fibrinogen have abnormal platelets aggregation. ’ Lack functional GP-IIb/IIIa receptor is also demonstrates abnormal platelets aggregation (Glanzmann thrombasthenia) - - O & - -  - Biochemistry of platelets activation ’ Platelets become activated after agonists bind to receptors - on the platelet surface, initiating signaling events within the platelets. These signaling events eventually lead to the reorganization of the platelet cytoskeleton, granule secretion, and aggregation. activating, resting (1-1).s PH II,2, 1 513 as s - ’ Role of Calcium in platelets activation v ’ Arachidonic acid pathway ~ ’ Cyclic AMP pathway /  Role of Calcium (Ca+2) ’ Resting platelets contain very low levels of calcium in the cytoplasm. Many cellular enzymes are inactive at this concentration, however, when platelets become activated, an increase in calcium concentration occurs. ’ The increase in calcium is due to both the release from internal stores and the influx from outside the cell. ’ These enzymes include phospholipase A2 (PLA2) and myosin light chain kinase (MLCK). The last enzyme plays a role in shape change of platelets during activation.  82 axes - Role of Calcium ((a +2) dense Body (Ca 1555 Sy 34 Six tubular I dense storage (1.3 it! (385. III is Low resting PH amounts I15 - o F PIt Ca* I Low concentration's & jisu - inactive cellular s ss, Li. resting I-db. 7e enzymes · 19(a 1.51555 956 iss receptor &1s. 4 extracellular signing molecule 82, b.g, Primary messenger - - intracellular activity within the 15-$12 cell o primary & II. 9915150 intracellular molecules, sojs secondary messenger specific area & 1995. 11is primary 1983. a activation recepts Physical responses. Joy, receptors &is Stadl;dbldgl 50 $1515 - - 561 -- 8";- 8618 secondary. 5 CaII's & agonist II bijlor-