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1、Hemostasis/CoagulationGregory S.Travlos,DVM,DACVPNational Institute of Environmental Health SciencesResearch Triangle Park,NC 27709919-541-0653Travlosniehs.nih.govHemostasisThe process by which bleeding is arrested.It is a series of physiological and biochemical events which terminate in the formati
2、on of an insoluble fibrin clotHemostatic Sequence:Interaction between vessel wall and plateletsBlood coagulationFibrinolysisHemostatic Component InteractionsThompson&Harker,1983Blood VesselsIntact endothelium forms a thromboresistant surface Required for the free flow of blood;does not promote plate
3、let adherence or activate coagulation Passive mechanisms:Endothelial glycocalyx(negative charge-repels like-charged particles,e.g.,platelets).Presence of a a2-macroglobulin at cell surface(protease inhibitor).Active mechanisms:Endothelial cells remove platelet aggregation promoters from circulation(
4、e.g.,PGF1,bradykinin,serotonin,adenine nucleotides).Secretion of PGI2-potent inhibitor of platelet aggregation,induces vasodilation.Proteoglycan matrix of the vessel wall influences thrombogenicity.Heparin,heparan sulfate and dermatan sulfate have anticoagulant activity;other glycosaminoglycans and
5、hyaluronic acid do not.Veins have the highest concentration.EndotheliumBesides their role in thromboresistance,endothelial cells have additional synthetic functions.Produce Von Willebrands factorAbsorbed by platelets;needed for adherence to collagenProduce plasminogen activator(tPA)Mediates fibinoly
6、sisInjured cells release thromboplastin(factor III)Activates the“extrinsic”coagulation cascadeOthers(e.g.,type III and IV collagens,elastin,fibronectin,etc.)Blood Vessel StructureThompson&Harker,1983PlateletsAdhere to exposed collagen(platelet plug)Occurs in seconds;can control hemorrhage of minute
7、injuriesSecretory functions;mediators of coagulation and fibrinolysisReleases ADP;sticky and promotes platelet adherenceADP activates phospholipase A2 which stimulates thromboxane A2 synthesisRelease of membrane fibrinogen,factor V,factor VIII and calciumRelease of membrane platelet phospholipid.Pla
8、telet-TEMmitochodrionmicrotublulesOCSgranulesUltrastructural and Functional Platelet AnatomyPlatelets-cont.The role of platelets in hemostasis is as important as the coagulation mechanism.Thrombocytopenia,thrombasthenia or thromobopathia-impair hemostasisThrombocytosis or thrombocythemia-may impair,
9、but usually promotes clotting(predisposes to thrombosis).Platelets promote hemostasis by:Release of ADP and other agonists;promotes adherence.ADP activates phospholipase A2 which stimulates thromboxane A2 synthesisThromboxane A2-stimulates vasoconstriction and platelet aggregationRelease of membrane
10、 fibrinogen,factor V,factor VIII and calciumComponents of coagulation localized at site of injuryRelease of membrane platelet phospholipid.Accelerates the“intrinsic”and“common”pathways of coagulationProstaglandin MetabolismHarlan&Harker,1981Hemostatic Platelet FunctionsThompson&Harker,1983Platelet R
11、esponseWhen a vessel is injured or severed a brief,local,reflex vasoconstriction occurs.Reduces blood flow at site.Maintained by vasoactive compounds(platelets,surrounding tissues).Passing platelets adhere to exposed collagen.Occurs in seconds;initially adhere in a single layer and become activated.
12、Severe injury-collagen serves as a potent platelet activator.Less severe injury-vWF and fibrinogen become the major activators.The adhered platelets undergo a conformational change.From discoid to development of long filopodia.Activation of GP receptors for fibrinogen and/or vWF(GPIIb/IIIa and GPIb/
13、IX/V).Structure of the GPIb-IX-V receptorTablin,2000Platelet Response to AgonistsPlatelets-unstimulatedAddition of ADP(mild stimulation)Addition of thrombin(strong stimulation)Characteristic discoid shapeShape change(elongation and crescents)and filaform process formation(arrows)Increased spreading,
14、filaform process extension(arrows)and aggregate formation(stars)SEM plates;Gentry,2000Platelet Response cont.Activated platelets release their a a-granule and dense body contents inducing additional platelet recruitment.Dense granules-ADP,serotonin and epinephrine.alpha-granules-fibrinogen(and vWF i
15、n human and pig).Synthesis and release of PAF and TxA2.The agonists accelerate the development of an irreversible platelet aggregate(platelet plug).Reversible v.irreversible responses.Thrombocytes of birds and reptiles do not respond to ADP.Serotonin and epinephrine:Serotonin-shape change(rat,g.pig
16、and dog);aggregation(human,rabbit,cow,horse,pig,sheep and cat).Epinephrine-only human,primate,cat and horse platelets appear responsive.Either serotonin or epinephrine combined with another agonist-strong response in all species.Platelet Response cont.More about agonists.Platelet Activating Factor(P
17、AF).Cow,horse,sheep,primate,dog,g.pig and rabbit respond to PAF.Human less sensitive and rat and mouse are insensitive to this agonist.Thromboxane A2(TxA2).Strong agonist-human,g.pig and rabbit.Weak agonist-horse.Insensitive-rat,cow,pig.In real life,however,platelets are exposed to multiple agonists
18、 from platelets and other cells(e.g.,red cells,ADP;white cells,PAF).Platelet Aggregation to ThrombinHarlan&Harker,1981Hemostatic Plug FormationBaumgartner&Muggli,1980Coagulation SystemConsists of a cascading system of proteinsPrimarily originating from liver(except factor III)Circulate in inactive f
19、orm(except,possibly,factor VII)System includes:Enzymatic factorsNon-enzymatic factorsTissue thromboplastin(factor III)Calcium(factor IV)Platelet phospholipid(PF 3)-structural component;accelerates factor activationAnticoagulant factorsThe coagulation system consists of three pathways(intrinsic,extri
20、nsic and common)Procoagulant FactorsCoagulation Systems-cont.Enzymatic factorsCirculate as non-active zymogens-must be activated to functionActivated enzymatic factors are not consumed during clotting(except factors II and XIII)Partial deficiency results in partial loss of clotting abilityActivated
21、enzymatic factors inhibited by antithrombin III(complexed with heparin)and some alpha-2-glycoproteinsEnzymatic factors:XI and XII(contact factors)II,VII,IX and X(vitamin K-dependent factors)XIII(clot stabilizing factor or fibrin-stabilizing factor)Coagulation Systems-cont.Non-enzymatic factorsOrigin
22、ate from liver but associate with platelet membranes(also found in plasma)Normal clotting with partial deficiency;almost total absence needed to affect hemostasis or clottingClotting consumes these factors-absent in serumNo known natural inhibitorsConsidered reactive proteins-increased during inflam
23、matory and neoplastic processes(except factor III)Non-enzymatic factors:Fibrinogen(factor I)Factor VFactor VIII:C(associated with Von Willebrands factor)Coagulation Cascade InteractionsDoes this turkey have factor XII?Of course,he doesBut,his feathered companion does notCoagulation Systems-cont.Clot
24、 stabilizationFibrin stabilizing factor(factor XIII)forms fibrin strand cross-links.Synthesized by monocytes and hepatocytes.Zymogen is activated by thrombin(plus calcium).A very small amount of factor XIII(2-10%)is adequate for hemostasis.Converts soluble fibrin monomers(unstable)to a fibrin polyme
25、r(stable).Lead,silver,zinc and snake venoms are known inhibitors.Coagulation InhibitorsThe activity of coagulation system must be attenuated.Numerous inhibitors are found in blood.Coagulation is controlled by three types of actions.Inhibition of converting enzymes(e.g.,AT III,C1 esterase inhibitor,a
26、 a2-macroglobulin,a a2-antiplasmin,a a1-antitrypsin,HC-II).Act on one or more of the converting enzymes(activated factors).AT III-heparin pathway:major system-80%of the thrombin inhibitory action in plasma.Destruction of protein cofactors(e.g.,TM-PC-PS system).TM-PC-PS system degrades cofactors V&VI
27、II:C,inhibiting prothrombinase and tenase complexes,respectively.Blocking receptor availability needed for complex formation(e.g.,Tissue factor pathway inhibitor(TFPI)and annexin V).Proposed Mechanism of AT III-Heparin SystemHeparinThrombinAntithrombin IIILysine sitesSerine siteArgininesiteHThHAT II
28、IAT IIIThProposed Mechanism of Thrombomodulin,Protein C and Protein S(TM-PC-PS)SystemThrombinProthrombinProtein CThrombomodulinThrombinF-XaActivatedplateletPSF-VaxCa+Ca+ActivatedProtein CProposed Mechanism of Tissue Factor Pathway Inhibitor(TFPI)ActivityF-XaEndotheliumTissue factorF-VIIaTFPIF-XaTFPI
29、TFPIF-XaAnticoagulant FactorsFibrinolytic SystemMethod for removing clots and maintenance of a patent vascular system and fibrin deposited during inflammation and tissue injury must be removed.Plasmin(serine protease)primarily responsible for fibrinolysis.Produced in the liver and kidney,it circulat
30、es in an inactive form(plasminogen).Activators:tissue plasminogen activator(tPA),cytokinases-urokinases(urine,CSF,tears,saliva,milk,bile,synovial,prostatic and amniotic fluids),erythrocyte erythrokinase,neutropil activator and factor XII-dependent activator(XII-prekallikrien-hageman factor cofactor
31、complex).In addition to fibrin and fibrinogen,plasmin will hydrolyse a variety of proteins.While plasminogen is normally found in blood and body fluids,plasmin is usually absent due to numerous antiplasmins.Inactivators:antithrombin III,a a2-macroglobulin,a a1-antitrypsin and C1 inactivator.Fibrinol
32、ytic System and Factors Regulating Fibrinolysis(Fibrinogenolysis)PlasminogenActivationInhibitionDamagedendotheliumKallikreinPlasminogen activator inhibitore-aminocaproic acidPrekallikreinStreptokinaseUrokinaseFHIIatPAPlasmina2-Antiplasmina2-MacroglobulinComplement activationFibrin/fibrinogenDegradat
33、ion productsBiodegradation of FV,FVIII,FIX,FXI fibrinogenFirbrinogen/fibrinDegradation of Fibrin/FibrinogenFibrinogen or FibrinFragment XSmall PeptidesFragment YFragment DSmall PeptidesFragment EFragment DSmall PeptidesPlasminPlasminPlasminEvaluation of HemostasisFundamental physiology and pathophys
34、iology of hemostasis is similar in mammalian species.Variables identical for laboratory animals and human patients PlateletsPlatelet count-detection of thrombocytopeniaClot retraction-non-anticoagulated bloodFailure to separate-platelet function defect or thrombocytopeniaBleeding time(BT)-in vivo te
35、st;simple;low sensitivityUsed to evaluate platelet function defects Thrombocytopenia -prolongs BTClotting factor deficiency does not alter BTVascular disease(eg.,scurvy)can prolong BT(humans,guinea pigs)Considerations for Blood CollectionClean/smooth surfacesWant to avoid platelet clumping or activa
36、tion of factor XII Use plastic or siliconized glass for sample collectionAnimal blood clots faster than human blood-prime needle with anticoagulantCollect sample from an endothelial-lined vessel and careful venipunctureWant avoid contamination with tissue juice(factor III)Small clot activates coagul
37、ation system invalidating results Samples from indwelling catheters are usually unacceptableSample Handling/AnticoagulantsPlasma samples separated from cells within 30 minutesPerform analyses immediatelyPlasma samples may be quickly frozen(dry ice/alcohol or liquid nitrogen)and stored at-70o for ana
38、lysis at a later dateActivity of factors V and VIII is lost rapidly in samples held at room temperatureCitrate(trisodium salt)is the anticoagulant of choice.Oxalate anticoagulants are acceptable -not commonly usedHeparin-unacceptableEDTA-unacceptable(except for indirect evaluation of fibrinogen conc
39、entration by heat precipitation and refractometry)Evaluation-cont.Activated Coagulation Time(ACT)-in vivo testMeasures(seconds)time to clot formation in fresh whole bloodCareful attention to sample collection/handlingPlatelet counts 10,000 cause slight increase in ACTResults from lack of platelet ph
40、ospholipid for testIncreased ACT suggests factor deficiency in intrinsic or common pathwaysDeficiency must be 5%of normal to prolong ACTActivated Partial Thromboplastin Time(APTT)Measures(seconds)time to clot formation in citrated plasmaIncreased APTT-factor deficiency in intrinsic or common pathway
41、sDeficiency must be 30%of normal to prolong APTTFibrinogen 50 mg/dL will prolong APTT;inflammation may shorten APTTSensitivity increased with saline-diluted plasmaHeparin therapy prolongs APTT-differentiate using a 1:1 dilution with normal plasmaEvaluation-cont.One-Stage Prothrombin Time(OSPT,PT)Mea
42、sures(seconds)time to clot formation in citrated plasmaRabbit or synthetic tissue thromboplastin preferred;human origin reagent gives longer PT timesIncreased PT-factor deficiency in factor VII or common pathwayDeficiency must be 30%of normal to prolong PTFibrinogen 50 mg/dL will prolong PTSensitivi
43、ty increased with saline-diluted plasmaRussels Viper Venom Time(RVVT)Measures(seconds)time to clot formation in citrated plasmaIncreased RVVT-in or common pathway but insensitive to factor VII deficiencyDeficiency must be 30%of normal to prolong RVVTFibrinogen 50 mg/dL will prolong RVVTSensitivity i
44、ncreased with saline-diluted plasmaEvaluation-cont.Thrombin Clotting Time(TCT)Measures(seconds)time to clot formation in citrated plasmaIncreased TCT-decreased fibrinogen concentration(100 mg/dL),dysfibrinogenemia,increased FDP concentration,heparin therapyFibrinogen Concentration(factor I)In most s
45、pecies,fibrinogen is 100-400 mg/dLFibrinogen decreases in DIC,severe liver insufficiency and hereditary hypofibrinogenemiaInflammation can increase fibrinogen concentrationEvaluation-cont.Fibrin-Fibrinogen Degradation Products(FDP)Measures,by latex agglutination,the concentration of products of fibr
46、inolysis;D-dimer assay is another method for measuring FDPIncreased FDP-occurs with disseminated intravascular coagulation or severe internal bleedingIn most species,normal FDP is 10 micrograms/mLExampleAcute oral study in dogsAnimals given 3 X LD50 in food BrodifacoumBromadioloneDiphacinoneCoagulation studies ACTRVVTPT谢谢观看/欢迎下载BY FAITH I MEAN A VISION OF GOOD ONE CHERISHES AND THE ENTHUSIASM THAT PUSHES ONE TO SEEK ITS FULFILLMENT REGARDLESS OF OBSTACLES.BY FAITH I BY FAITH