LEC 25

new topic is coagulation

coagulation a complex system; make sure if cut yourself it stops right away

  • make sure system is into so sensory clots in other parts of body
  • hemorrhagevs.thrombus (if have unwanted clots in body)
  • has to be carefully regulated (don’t know all yet)
  • give basics

The final exam is mostly on coagulation, some leukemias, some other problems encountered, draw on other info still required.

Hemostasis is the medical term forcoagulation

We’ll talk about normal function of blood vessels, coagulation factors inv

then switch to disorders that occur in hemostasis; talk coagulation problems, platelets, hereditary problems, acquired problems that occur

  • look at outline carefully and read text

cartoon “my fingers stopped bleeding”

  • Why does blood clot?
  • Hemostasis is a physio process in which spontaneous or induced hemorrhageis stopped
  • anytime get breakage in blood vessels, this process automatically goes into order that will stop blood flow from the site
  • this is a rapid process
  • It’s a localizedprocess, only want clotting at this site, do not want at any other part of body
  • ensure rest of blood remains fluid—thatis essential

To get this process to occur, need interactions between blood vessels, platelets, and coagulation of proteins

These are 3 critical elements:

1.platelets

2. bloodvessels

3. coagulation proteins (plasma factors)  fibrinolysis (clot broken down, reestablishnormal blood flow) [triangle thing]

Lets look at endothelium, the blood vessels 1st

Endotheliumis the lining of the cells that are the 1st in contact w plateletsand plasmaclotting factors

The smallest blood vessels may consist ofonlyone layer of endothelium, outside it is the basement membrane

  • diapodesis movement throughendothelialcells, then basementmembrane
  • Endothelialis carpet, cells trample over it; that is what provides support
  • Basement membrane is made up of fibrin, differentkinds of connectivetissuesproteins, somecollagen

Function ofendothelial cells

  • don’t maintain shape
  • good barrier, retain RCs and all plasma w/in blood vessels
  • throughendothelium, cell transfers waste and nutrients
  • Theendothelialcells also involvedin system of factors that regulate interaction betweenbloodcomponentsand vessel wall
  • endothelialcellsare not passive; they are actively making differentproteins:
  • factor VIII
  • von Willebrant’s factor (VWF)
  • heparin (on surfacemembrane to ensure normal layer ofendothelialis non-rxn surface thatwill not be involvedin clotting. endothelial helps makefibrin, collagen, other proteins,
  • prostaglandins
  • plasminogen plasmin
  • angiogenesisfactors that promote vascular repair, regrowth of new endothelium to heal
  • Thrombin receptors (binds to thrombomodulin, which breaks down thrombin)
  • endothelial mighthave role in Ag process, lots differentthings

Underendothelium, have basementmembrane, depending on size ofblood vessels, have several layers underneath endothelial(tunica intima) layer.

  • larger blood vessels: FIG, cross-section
  • beneath basementmembrane elastic tissues (tunica media, has smooth muscle cells in it), arteries and arterioles, blood vesselscapable of contraction, importantmechanism to slow blood vessels, also vasodilatation to  blood flow there
  • Might alsohave layers where nerves and lymph vessels, your aorta is the size of garden hose, has layers, lots of pressure in there.
  • aortic aneurism is a walking time bomb

Other mechanismsthat endothelium contributes to: thrombo resistance

Wheneverhave unwanted blood clot: thrombus (pl thrombi)

To discourage thrombus,don’twant clots to form, interior of lining is thrombo-resistant

  • Heparin on surface ofendothelialcells
  • Prostacyclin, PGI2, preventsplateletactivation withinendothelial wall
  • formed fromarachidonic acid pathway
  • heard aboutVioxx, hope you don’thave stock in there
  • non-steroidal aspirin, interaction with components ofarachidonic cycle to prevent inflammation, also involvedin coagulation, in thrombosis process
  • plasminogen activatorsfrom endothelium (for fibrinolysis)
  • endothelium activelydegradesplatelet aggregating agents ADP, thrombinthrombo-modulation (endothelialcellshavereceptors called thrombo-modulin, for thrombin)
  • get binding ofthrombin on thrombo-modulin, active protein C (an anticoagulant, fibrinolytic)
  • Fibrin is made in plasma
  • fibrin formationwheredon’twant it, enhance thrombo resistance

BREAKAGE OF A BLOOD VESSEL (starts the haemostatic mechanism)

1. Vasoconstriction: contraction of blood vessels,reduced blood flow

  • allows contact activation of plateletsand plasmaclotting proteins
  • Petechia: spots of hemorrhage where platelets don’t do their job

2. Platelets adhere(They startthis whole clottingprocess)

  • Adhere onto exposed subendothelial components, first the connective tissue
  • Shape change to spiny
  • Release reaction: release contents of platelets.
  • Platelets further enhance blood vessel constriction by releasingthromboxone A2, serotonin, epinephrine(vasoconstrictors)

3. Activation of clotting factors  fibrin network

  • Boom! got clot

4. Fibrinolysis: tobreak down clot, release ofplasminogen

PLATELETS (thrombocytes)

platelets arecell fragments, unique to adhere to injured blood vesselsand activate fibrin formationto get clot

  • bleeding disorder if too few platelets or malfunction
  • 1. quantitativeproblem
  • 2. qualitativeproblem

SC CFU-C  megaloblast  megakariocyte  Platelets

  • megaloblast undergoes mitosis
  • nucleus dividesand cell gets bigger, but celldoesn’t divide, 2-4 nuclei, more cytoplasm; mature megakaryocytes have 8-64 nuclei
  • endomitosis occurs becauseplatelets are derived fromcytoplasm, want to ensure plenty ofcytoplasm, that’s why it undergoesmitosis
  • When mature, from outer membrane, have demarcations, little canals throughout whole cytoplasm, thing fragments off; megakaryocyte saddles up next tocells that line sinusoids, extends pseudo podsand flakes off asplatelets.
  • 500-1000 plateletscan be derived from single megakaryocte
  • production of plateletsunder control ofthrombopoeitin(a hormone like EPO)
  • when platelets leave marrow, 2/3 total plateletsfound in circulation, remaining 3rd pulled into spleen backand forth so maintain 1/3 pool in spleen
  • 200-400,000/mm3, normal # ofcirculationplatelets
  • turn over all platelets in 8-10 days, gets pulled out by spleen breaks down somewhere
  • If hypersplenism (spleen enlarges), expectmore platelets sequestered in spleen, could bereasonforthrombocytopenia(too few)
  • thrombocytes or thrombocythemia

Light microscopy

Morphology ofplateletsincirculationis not impressive (little bits ofcytoplasm, have full membrane, havesome stainable granulesin interior

Electron microscopy

3 distinct structuralzones in platelets, each related to specificplateletfunction

1. Peripheral zone, there are glycoproteins (surface on cellmembrane)

  • plasmamembrane, canalicular system (derivedfrom invagination ofcellmembrane, sameas in megakaryocyte, resultis flaking)
  • The glycoproteins on surface ofplasmamembraneincreases thesurface area of plasmamembrane itself, open canalicular system, thissurface involvedin adherence
  • peripheral zone involvedin adhesion (1stinteraction of platelets withendothelialcell)
  • Peripheral zone also involved with interaction: binds to thrombin, ADP, epinephrine, VWF.

2. beneath it is sol-gel zone, thisis wherecytoplasm oftheplateletsis located, alsowherehavevariousmicrofilaments with actin and myosin, microtubules that maintain shape- (little flat discs), called plateletsbecause saucers in blood in inactivation state, when activated, change shape; instead of flat saucer, becomes sea urchin, spiny thing with projections on surface; this sol-gel zone, cytoplasm, MFs, contractile proteins that enable them to change shape. Shape change increases surface area for reactions.

3. In center, organelle zone involvedin metabolicspecializations ofplatelets, increased bunch of differentgranules, plateletshavemitochondria (metabolites, cell fragments, alsohave glycogen to keep mitochondria going; also have stainable granules). Platelets make granules and absorb and store certain proteins.

  • SECRETES: PF4, βTG, PDGF, TSP, chemotactic factors, permeability factors, and bactericidal factors.
  • ABSORBED FROM PLASMA: Fibrinogen, VWF, F5, albumin, serotonin, fibrinonectin.
  • You need electron microscopetodistinguish less dense and dense granules.
  • dense granules contain Ca, ADP, serotonin
  • whenplatelets adhere, contribute tovasoconstriction, becausereleasesgranuleswhenplateletsactivated
  • less dense granules contain platelet factor 4 that has heparin neutralizing activity; factor 4fibrinogen fibrin
  • VWFproteinis presentin less densegranules
  • platelets also contain PDGF (in book), plateletderived Growth Factor, stimulate healing viamitochondria of muscle cellsand fibroticcells
  • has lysosomal granules to break down itself
  • Some plateletspecializationproteinsare made by plateletsand are carriedaround with it; plateletsantibodies other factors fromplasma
  • Platelets are also sponges
  • capable of soaking up plasmaproteins and increase into granules
  • once sponge gets activatedand adheres,contractile proteins under cell membrane squeeze sponge on itself to release all internal granulesand substances necessary for healingand coagulationprocess
  • Calledrelease rxn, shape change, release rxn FIG [end fig lookslike starfish with dots inside]. Allows more platelets to come (aggregation).

Platelets contribute toclottingprocess

plateletsin circulation looks like flat disks

When it encounters damaged endothelial, basement membraneand collagenare exposed to them, which triggers adhesion.

1. Adhesion

  • processrequires glycoproteins onsurface ofplateletsand VWF thatplateletsantibodies
  • VWF serves as bridge tohelpplatelets to adhere
  • fibrinogen also helps adhesion

2. Aggregation – platelets now undergo shape change and release dense granulesand granules so somethrombin formed and also get thomboxin A2 (TA2) released and will have vasoconstriction to slow blood flow, attract moreplatelets to site, form aggregate; because change shape, means stick together better

  • Seen bristle blocks? If stick one on top ofanother, don’t pull apart easily
  • You can test plateletsaggregation in vitro, haveplatelet aggregonmeter, it s a spectrophotomer that has a heating block in it thatwillmaintain 37C, always go on at body temp; add anticoagulation blood sample, spin down, take off plasma, low speed to maintain platelets in plasma, makePRP (platelets rich plasma)  put into test tube, has somereagent to keep plateletsin suspension put into aggregonmeter (a spectrophotometer), shine a light, see how much light reflected off, establish a baseline of %transmissionthrough suspension ofplatelets
  • then add differentaggregation agents
  • An in vitro test to see if platelets aggregate asshould; ADP, collagen, fibrin (any componentscauseplatelets adhere and undergo shape change  a plateletsfunction test)
  • also add thrombin (plasmaclotting factor), to PRP, measure a small blip; these thingsundergoinitial shape change, add higher concentration, get initial 1st wave of shape change and initial aggregation, because not much thrombin, fall apart
  • higher concentration, furtherrecruitment of platelets, 2nd wave, add amountthrombin, 2 wave, in % transmission, platelets aggregate,  light, so measure plateletsaggregation withdifferent agents; an in vitro test to test to see if aggregation occurs

Whenplateletsundergo shape change, furtherprovide glycoproteinsurfaceforplasmacoagulationfactorsto act on, platelet factor III aidsin formation of eventual fibrin becauseactivation ofplasmaclotting factors

coagulation factors

There are at least13 distinctplasmaglycoproteins thatinteractin series of rxns that lead tobloodclotting. Table in notes lists all differentclotting factors, have roman numerals, have names attached todifferent factors, unfortunately, roman numerals designated not in order ofinteraction, but by order of discovery.

Coagulation is a cascade; the initial factors are activated by exposure to a substrate.

  • plasmacoagulation factors are proenzyme or enzymes precursors that are normallypresentin plasmain inactivation state, so you have all variousclotting factors circulationin blood but circulatein inactive state
  • once get activation ofclottingprocess, then these interactin sequential order
  • Initial stages of hemostasis, proteins undergo conformational changes. Later, proteolytic cleavage off next co-enzyme, then gains force and amplification and there is modulation of these factors.
  • Hemostasis is clotting cascade and fibrinolytic mechanism.
  • enzymes all gong to beactivated in sequential mode, next stages structuralchanges, asfurther along process, also coagulationcascadebecauselike a water fall, once get thisclottingfactor, this one, this one, this one
  • early stages, configurationchangesin enzyme, later ones, proteolytic changes that occur
  • initial stages occur on surface, that’swhereplatelets activated and where damage toblood vessels effects (causes abnormalsurface)
  • watch your cholesterol, servesassurface for plateletsand clottingproteins
  • Thisis a linked multiple stagesystem, along cascade, get amplification and modulation of initial stimulus.

Plasmaclottingmechanismhasclottingproteins, and fibrinolyticmechanism

  • clotting leads tofibrinolysis, 1 an enzymaticprocess (both)
  • The final product is fibrin and a fibrin network.
  • in order for clotting to occur, 2 distinctpathways that exist for conversion of factor 10, midway in cascade, have factor 10 thatmust beactivated, then common pwy leads toformation of fibrin (final product)
  • Start withinitialproductionfactors, final is fibrin (form fibers of clot), also need to break down fibrin later.
  • needthisfor DIAGNOSIS; dark line separates intrinsic and extrinsic system; two mechanisms by which to activate fibrin formation
  • EXTRINSIC PATHWAY
  • INTRINSIC PATHWAY starts at factor 12
  • F11 binds to platelets in the presence of calcium  binds to platelets and activates factor 9
  • factor 10 midway factor, initial factor, that overlaps between intrinsic and extrinsic system; final step is fibrinogen to fibrin
  • usefulbecausethisdiagram shows you factors necessaryto be tested, two mechanisms to test whethernormalamounts ofvarious factors and whether factors arefunctional, two plasmaclotting tests done: 1st: most commonPTT (activates partial thromboplastin time, procedureis in lab manual and extensiveinstructionsin back oftext, be sure like at theses tests), PT
  • intrinsic system, PTT test used
  • PT (prothrombin time; test integrity of extrinsic system
  • in intrinsicsystem, initialstimulusis change in surface, any abnormalsurfacein blood vesselswillactivate factor 12 whichstarts intrinsic system
  • extrinsic system, initial agent is tissues factor, released whenever havetissues damage, ill defined, problem isdifferentproteins released when havetissues injury
  • tissue damage  release tissues factor
  • big cut  breakage ofblood vessels, surface contact release  activate intrinsic system
  • tissues factor releasebecause damage totissuesstimulates extrinsic system
  • this ensures that you stop bleeding right away because activated by twomechanisms, intrinsicand extrinsic

Hemophilia is sex-linked, caused by a block in F8 or problem in VWF (VWF deficiency is in men and women). F8 is a large protein. VWF carries and protects F8 and helps platelets to adhere and forms a bridge.

Lets start with intrinsic system:

Breakage in blood vesselsand endothelialand cholesterol plaques, abnormalsurfacerevealed, initialplasma factor 12 12aundergoes conformational change, to 12A

  • 12A activated leads to several differentprocesses that occur: withactivation of factor 12, undergoclottingprocess, get activation of factor 11 and activation of prekallikrein to kallikrein to bradykinin (inflammation mediator, one of the factors that leads to pain, part ofinflammationprocess)
  • also, activation factor 12 plasminogen plasmin  fibrinolysis
  • in initial stages ofclottingsystem, startprocess ofthis clot (occurs at slightlydifferent rate)

LECTURE 26

Review

Know where platelets come from, and what mechanisms are for platelets maturation, normal numbers. Start w CFU-GM, then megakaryoblast.

Cell gets bigger and bigger, more cytoplasm

  • Averageendomitosisdivisions? up to 32-64 nuclei, cell matures, saddles up next to sinusoids, flakes off platelets
  • # Platelets circulation? 200,000-400,000
  • Lifespan? 8-10 days
  • anything below 5000 per mm3, have thrombocytopenia, risk different bleeding disorders

know about intrinsic system for coagulation; completed structure of platelets, function o platelets

  • How does a clot form?
  • Adheres, then shape change, becomes secretory cell fragment (know what stuff secreted by platelets); platelets then recruit other platelets; get aggregation, surface membrane of platelets important because that’s how clottingprocessis localized. Surface membrane itself because presents subendothelial components, platelets adhere there, undergo activation, their surfaces are sites where plasma coagulation occurs; main mechanism where clotting is localized (don’t want clotting anywhere else)

At same time ofplatelet activation going on, gonna get plasma clotting factors activated.

2 mechanisms plasma clotting factors activated: extrinsic and intrinsic system

  • two systems overlap; actually different 2 systemsfor getting plasma clotting
  • intrinsic system, have activation mechanism: surface change, disruption ofendothelial, subendothelial components exposed, platelets come in, between collagenand fibrinand other components of basement membrane, serve as activation sites
  • What factor activated 1st in intrinsic system? 12
  • useful to pull out diagram (keep handy)

FACTOR 12

  • 1st factor activated in intrinsic system is factor 12 (12 is an important factor for coagulationand also capable of activating other systems (kallikreinbradykinin = vasoconstriction,  BP, pain,  capillary permeability, promotesmigration of WCs, important mediator for inflammation)
  • At same time, activation of plasminogen plasmin, eventually leads to dissolution of clot
  • plasminogenand plasmin will be locked into clot as it’s being formed

FACTORS 11 AND 9

Then factor 12a will activate factor 11, which undergoes proteolytic cleavage to get activation of factor 9

  • 12, 11, 9
  • 9a binds to phospholipid surface (e.g. plasma membrane), requires Ca (most common anticoagulation in hematology lab is EDTA, it’s a chelator of Ca). That’s how coagulation works; ties up calcium.
  • So, 9 activated by 11; in presence of Ca, binding onto platelet membrane (lipid surface), also requires Ca
  • this rxn alsorequires activated factor 8 because this is most common factor missing or not formed correctly; contributes to hemophilia

Factor 8

This is a huge molecule, made up of subunits of 200,000 molecule wt; 8 related to VWF

  • VWF is a bridge betweenplatelets and its ability to bind tocollagen
  • VWF also binds to factor 8 and heparin
  • normal endothelial cells remain non-reactive because has heparin at surface
  • VWF also binds factor 8, bring clotting factors together
  • can consider VWF as carrier for factor 8 and protects factor8
  • VWF synthesized by endothelial cells

FACTOR 10

This is where intrinsic system and extrinsic system overlap

  • EXTRINSIC COAGULATION
  • factor 10 can be activated by intrinsic system and also be activated by extrinsic system
  • extrinsic system needs factor 7 and tissue factor, TF alsoknown as tissue thromboplastin (glycoproteins released by damaged cell; if cut, disruptsendothelial surface and causessurrounding tissue damage, get release of tissues factor)
  • TF reacts with factor 7; factor 7 circulatingin blood, part ofclotting mechanism, once have tissues factor, get activation of factor 7
  • factor 7 + Ca + platelet surface activates factor 10  10a
  • now formed complex that activates 10; F10 reacts with factor 5
  • factor 5 and 10 join on platelets surface to form prothrombinase
  • Final step; prothrombinthrombin(which converts fibrinogen to fibrin)

Prothrombinis a circulating glycoprotein, 65,000 molecule wt; factor 10 converts prothrombin to thrombin by cleavage of peptides, prothrombin is a vitamin K dependentclotting factor—need vit K to make prothrombin)

  • prothrombinis theclotting factor present in highest concentration
  • start with very small amounts of 12 or 7; because prothrombin hi quantities, magnify clottingprocess
  • higher plasma concentration ofprothrombin allowsfor amplification (even if only few molecules of initiators—get large burst ofthrombin activity, thrombin able to modify factor 5 and factor 8 so get further magnification)
  • when prothrombin becomes thrombin, converts fibrinogen to fibrin (the final product)

FIG fibrinogen: a plasma protein, fairly large, 40,000 molecule wt; composed of 3 pairs of polypeptide chains, A, B, ; 6 chains all held by disulfide bonds