1、Hemostasis/CoagulationGregory S. Travlos, DVM, DACVPNational Institute of Environmental Health SciencesResearch Triangle Park, NC 27709919-541-0653T銷售信 bilantianHemostasisThe process by which bleeding is arrested.It is a series of physiological and biochemical events which termina

2、te in the formation of an insoluble fibrin clotHemostatic Sequence:Interaction between vessel wall and plateletsBlood coagulationFibrinolysis.Hemostatic Component InteractionsThompson &Harker, 1983.Blood VesselsIntact endothelium forms a thromboresistant surface Required for the free flow of blood;

3、does not promote platelet adherence or activate coagulation Passive mechanisms:Endothelial glycocalyx (negative charge - repels like-charged particles, e.g.,platelets).Presence of a2-macroglobulin at cell surface (protease inhibitor).Active mechanisms:Endothelial cells remove platelet aggregation pr

4、omoters from circulation (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 acti

5、vity; other glycosaminoglycans and 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 plasm

6、inogen activator (tPA)Mediates fibinolysisInjured cells release thromboplastin (factor III)Activates the “extrinsic coagulation cascadeOthers (e.g., type III and IV collagens, elastin, fibronectin, etc.).Blood Vessel StructureThompson &Harker, 1983.PlateletsAdhere to exposed collagen (platelet plug)

7、Occurs in seconds; can control hemorrhage of minute 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

8、and calciumRelease of membrane platelet phospholipid.Platelet - TEMmitochodrionmicrotublulesOCSgranules.Ultrastructural and Functional Platelet Anatomy.Platelets - cont.The role of platelets in hemostasis is as important as the coagulation mechanism.Thrombocytopenia, thrombasthenia or thromobopathia

9、 - impair hemostasisThrombocytosis or thrombocythemia - may impair, 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 -

10、 stimulates vasoconstriction and platelet aggregationRelease of membrane fibrinogen, factor V, factor VIII and calciumComponents of coagulation localized at site of injuryRelease of membrane platelet phospholipid.Accelerates the “intrinsic and “commonpathways of coagulation.Prostaglandin MetabolismH

11、arlan &Harker, 1981.Hemostatic Platelet FunctionsThompson & Harker, 1983.Platelet ResponseWhen 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 ex

12、posed collagen.Occurs in seconds; initially adhere in a single layer and become activated.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 developme

13、nt of long filopodia.Activation of GP receptors for fibrinogen and/or vWF (GPIIb/IIIa and GPIb/IX/V).Structure of the GPIb-IX-V receptorTablin, 2000.Platelet Response to AgonistsPlatelets - unstimulatedAddition of ADP(mild stimulation)Addition of thrombin(strong stimulation )Characteristic discoid s

14、hapeShape change (elongation and crescents) and filaform process formation (arrows)Increased spreading, filaform process extension (arrows) and aggregate formation (stars)SEM plates;Gentry, 2000.Platelet Response cont.Activated platelets release their a-granule and dense body contents inducing addit

15、ional platelet recruitment. Dense granules - ADP, serotonin and epinephrine.alpha-granules - fibrinogen (and vWF in 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.

16、Thrombocytes of birds and reptiles do not respond to ADP.Serotonin and epinephrine:Serotonin - shape change (rat, g. pig 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

17、combined with another agonist - strong response in all species.Platelet Response cont.More about agonists. Platelet Activating Factor (PAF).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).S

18、trong agonist - human, g. pig and rabbit.Weak agonist - horse.Insensitive - rat, cow, pig.In real life, however, platelets are exposed to multiple agonists from platelets and other cells (e.g., red cells, ADP; white cells, PAF).Platelet Aggregation to ThrombinHarlan &Harker, 1981.Hemostatic Plug For

19、mationBaumgartner & Muggli, 1980.Coagulation SystemConsists of a cascading system of proteinsPrimarily originating from liver (except factor III)Circulate in inactive form (except, possibly, factor VII)System includes:Enzymatic factorsNon-enzymatic factorsTissue thromboplastin (factor III)Calcium (f

20、actor IV)Platelet phospholipid (PF 3) - structural component; accelerates factor activationAnticoagulant factorsThe coagulation system consists of three pathways (intrinsic, extrinsic and common).Procoagulant Factors.Coagulation Systems - cont.Enzymatic factorsCirculate as non-active zymogens - must

21、 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 enzymatic factors inhibited by antithrombin III (complexed with heparin) and some alpha-2-glycoproteinsEnzymatic f

22、actors: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 factorsOriginate from liver but associate with platelet membranes (also found in plasma)Normal clotting with partial

23、deficiency; almost total absence needed to affect hemostasis or clottingClotting consumes these factors - absent in serumNo known natural inhibitorsConsidered reactive proteins - increased during inflammatory and neoplastic processes (except factor III)Non-enzymatic factors:Fibrinogen (factor I)Fact

24、or VFactor VIII:C (associated with Von Willebrands factor).Coagulation Cascade Interactions.Does this turkey have factor XII?Of course, he doesBut, his feathered companion does not.Coagulation Systems - cont.Clot stabilizationFibrin stabilizing factor (factor XIII) forms fibrin strand cross-links.Sy

25、nthesized 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 polymer (stable).Lead, silver, zinc and snake venoms are known inhibitors.Coagulation

26、 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, a2-macroglobulin, a2-antiplasmin, a1-antitrypsin, HC-II).Act on one or m

27、ore 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 & VIII:C, inhibiting prothrombinase and tenase complexes, respective

28、ly.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 IIIAT IIITh.Proposed Mechanism of Thrombomodulin, Protein C

29、and Protein S (TM-PC-PS) SystemThrombinProthrombinProtein CThrombomodulinThrombinF-XaActivatedplateletPSF-VaxCa+Ca+ActivatedProtein C.Proposed Mechanism of Tissue Factor Pathway Inhibitor (TFPI) ActivityF-XaEndotheliumTissue factorF-VIIaTFPIF-XaTFPITFPIF-Xa.Anticoagulant Factors.Fibrinolytic SystemM

30、ethod 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 circulates in an inactive form (plasminogen).Activator

31、s: 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 complex).In addition to fibrin

32、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, a2-macroglobulin, a1-antitrypsin and C1 inactivator.Fibrinolytic System and Factors Regula

33、ting Fibrinolysis (Fibrinogenolysis)PlasminogenActivationInhibitionDamagedendotheliumKallikreinPlasminogen activator inhibitore-aminocaproic acidPrekallikreinStreptokinaseUrokinaseFHIIatPAPlasmina2-Antiplasmina2-MacroglobulinComplement activationFibrin/fibrinogenDegradation productsBiodegradation of

34、 FV, FVIII, FIX, FXI fibrinogenFirbrinogen/fibrin.Degradation of Fibrin/FibrinogenFibrinogen or FibrinFragment XSmall PeptidesFragment YFragment DSmall PeptidesFragment EFragment DSmall PeptidesPlasminPlasminPlasmin.Evaluation of HemostasisFundamental physiology and pathophysiology of hemostasis is

35、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 test; simple; low

36、 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 activation o

37、f 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 coagulati

38、on system invalidating results Samples from indwelling catheters are usually unacceptable.Sample 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 an

39、alysis 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 fibrin

40、ogen concentration 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 lac

41、k of platelet phospholipid 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 intri

42、nsic or common pathwaysDeficiency 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 plasma.Evaluation -cont.One-Stage Prothrombin Time (OSPT, PT)Measures (seconds) time to clot formation in citrated plasmaRabbit or synthetic tissue thromboplastin preferred; human origin reagent gives longe