The Normal Hemostatic Process

HEMOSTASIS

1HEMOSTASIS COMPONENTSVessel wallPlateletsCoagulation enzymesFibrinolytic systemControl mechanisms, including inhibitors

NOTE: Normal hemostasis involves the complex interaction of the vessel wall, circulating proteins and biochemical mediators, cells, promoters and inhibitors. Activation of hemostasis usually begins with damage to the vessel wall, exposing the subendothelium. Conversely, the intact vessel wall helps to maintain fluidity of blood, not simply through being a passive container wall, but also by synthesizing chemicals and proteins that actively contribute to the process. When the vessel wall is damaged, platelets are at the forefront of defense by sticking to the damaged area. The clotting enzymes contribute by developing a fibrin mesh that holds the platelets in place. Control mechanisms come into play to limit hemostatic process to the are of injury. Otherwise the whole body would clot up at the slightest stimulus.

2Normal hemostasis in involves the complex interaction of the vessel wall, circulating proteins and biochemical mediators, cells, promoters and inhibitors.Activation of hemostasis usually begins with damage to the vessel wall, exposing the subendothelium. Conversely, the intact vessel wall helps to maintain fluidity of blood, not simply through being a passive container wall, but also by synthesizing chemicals and proteins that actively contribute to the process. When the vessel wall is damaged, platelets are at the forefront of defense by sticking to the damaged area. The clotting enzymes contribute by developing a fibrin mesh that holds the platelets in place. Control mechanisms come into play to limit hemostatic process to the are of injury. Otherwise the whole body would clot up at the slightest stimulus.

Sequence of Changes With Vascular Injury Injury to vessel wall (endothelium) with resultant exposure of subendotheliumPlatelet adhesion mediated by HMW vWFSimultaneous activation of clotting enzymesPlatelet aggregation via fibrinogen receptors on plateletsAnchoring of platelet plug by cross-linked fibrin

NOTE: Lets expand on the sequence of events that occur when a vessel wall is injured and define a few basic terms. Injury exposes the subendothelial collagen and the soluble substances that are normally between the subendothelium and collagen. 3Lets expand on the sequence of events that occur when a vessel wall is injured and define a few basic terms. Injury exposes the subendothelial collagen and the soluble substances that are normally between the subendothelium and collagen. Normal HemostasisVessel WallExposed collagenContractionvFWlarge multimersTissue TPLPlateletadhesionActivation ofCoagulationPlateletAggregationThrombin1 HemostaticPlugDefinitiveHemostatic PlugLimiting Reactions

This slide gives a diagrammatic representation of the whole process and the functional interrelationships. We will now go on to dissect the various componentsof this process in more detail.4This slide gives a diagrammatic representation of the whole process and the functional interrelationships. We will now go on to dissect the various componentsof this process in more detail.Platelet ComponentsRECEPTORSvWFFibrinogen Clotting FactorsAlpha granulesDense GranulesActinCanaliculus

5Platelet GranulesAlpha granulesvWFFibrinogenPF4Beta thromboglobulinPDGFDense granules (delta)ADPSerotonin

Platelet Role in HemostasisvFW binding sites- platelet adhesionFibrinogen binding sites- platelet aggregationMultiple binding sites for coagulation factors - enhances appropriate steric relationships Production of multiple chemical mediators Binding sites for chemical mediators

First, lets look more closely at the role of platelets. They play multiple roles in the hemostatic process, and, contrary to the view held 30 years ago, when they were thought to a small part of the process, many would now consider them to have a central role. We already have alluded to this by indicating that the first step after injury is platelet adhesion to the subendothelium. This is mediated by subendothelial HMW vWF binding to specific receptor sites on the platelet membrane. In, addition there binding sites that play a role in virtually every step of the process. Also. chemical mediators are synthesized by internal organelles, and they contain a contractile protein that is responsible for clot retraction. The coagulation enzymes are present in relative low concentrations in plasma. Binding of certain of these factors to specific receptors on the surface of activated platelets allows them to line up in appropriate steric configuration, catalyzing the process and limiting the reactions to the area of injury.7First, lets look more closely at the role of platelets. They play multiple roles in the hemostatic process, and, contrary to the view held 30 years ago, when they were thought to a small part of the process, many would now consider them to have a central role. We already have alluded to this by indicating that the first step afterinjury is platelet adhesion to the subendothelium. This is mediated by subendothelial HMW vWF binding to specific receptor sites on the platelet membrane. In, addition there binding sites that play a role in virtually every step of the process. Also. chemical mediators are synthesized by internal organelles, and they contain a contractile protein that is responsible for clot retraction. The coagulation enzymes are present in relative low concentrations in plasma. Binding of certain of these factors to specific receptors on the surface of activated plateletsallows them to line up in appropriate steric configuration,catalyzing the process and limiting the reactions to the area of injury.

von Willebrands FactorSynthesized in megakaryocytes and endothelial cells - approx. 230,000 M.W.Macromolecular multimer plasma: M.W. 1 x 106 - 10 X106. Plasma carrier of Factor VIII, stabilizes itLarge molecular forms: a. Most effective in platelet adhesionb. Predominate in endothelial cells and subendothelium

Coagulation Cascade TISSUE FACTOR

FACTOR VII VIIa (ACTIVATED)

( XI?) IX IXa TFPI VIII

FACTOR X Xa

V, Ca

FACTOR II IIa

FIBRINOGEN FIBRIN MONOMERS

FACTOR XIII

IN VITRO CROSS-LINKED FIBRIN PT (STABLE FIBRIN CLOT)APTT

APTTCONTACT ACTIVATIONFXII, FXI, FLETCHER, FITZGERALD FACTORSVIIIIX IXaPTPHOSPHOLIPIDPLATELETACTIVATIONINHIBITION OFCLOTTINGTHROMBOMODULINPROTEIN C

Coagulation Cascade- PT TISSUE FACTOR

FACTOR VII VIIa (ACTIVATED)

( XI?) IX IXa TFPI VIII

FACTOR X Xa

V, Ca

FACTOR II IIa

FIBRINOGEN FIBRIN MONOMERS

FACTOR XIII

IN VITRO CROSS-LINKED FIBRIN PT (STABLE FIBRIN CLOT)APTT

APTTCONTACT ACTIVATORFXII, FXI, FLETCHER, FITZGERALD FACTORSVIIIIX IXaPTPHOSPHOLIPIDPLATELETACTIVATIONINHIBITION OFCLOTTINGTHROMBOMODULINPROTEIN C(ENDPOINT)PHOSPHOLIPID

Coagulation Cascade- APTT TISSUE FACTOR

FACTOR VII VIIa (ACTIVATED)

( XI?) IX IXa TFPI VIII

FACTOR X Xa

V, Ca

FACTOR II IIa

FIBRINOGEN FIBRIN MONOMERS

FACTOR XIII

IN VITRO CROSS-LINKED FIBRIN PT (STABLE FIBRIN CLOT)APTT

APTTCONTACT ACTIVATORFXII, FXI, FLETCHER, FITZGERALD FACTORSVIIIIX IXaPTPHOSPHOLIPIDPLATELETACTIVATIONINHIBITION OFCLOTTINGTHROMBOMODULINPROTEIN C(ENDPOINT)

Control MechanismsIntact endothelial cells Chemical mediatorsMembrane bound receptorsSynthesizes activators of fibrinolysisCirculating inhibitorsProtein C systemFibrinolytic systemEndothelial componentplasma protein componentNOPGI2ADPaseINHIBITS PLATELET ACTIVATIONTHROMBOMODULINPROTEIN C PROTEIN CaPROTEIN SINACTIVATES Va + VIIIaATIIIINHIBITS Xa + THROMBINPLASMINOGENACTIVATORS (tPA) (uPA)PLASMINOGEN PLASMIN FIBRINOLYSISHEPARAN+Intact Endothelium Limits HemostasisTHROMBIN INACTIVATES PAIvFW multimersSubendothelium

NOPGI2ADPaseINHIBITS PLATELET ACTIVATIONTHROMBOMODULINPROTEIN C PROTEIN CaPROTEIN SINACTIVATES Va + VIIIaATIIIINHIBITS Xa + THROMBINPLASMINOGENACTIVATORS (tPA) (uPA)PLASMINOGEN PLASMIN FIBRINOLYSISHEPARAN+Intact Endothelium Limits Hemostasis FibrinolysisTHROMBIN INACTIVATES PAIvFW multimersSubendothelium

NOPGI2ADPaseINHIBITS PLATELET ACTIVATIONTHROMBOMODULINPROTEIN C PROTEIN CaPROTEIN SINACTIVATES Va + VIIIaATIIIINHIBITS Xa + THROMBINPLASMINOGENACTIVATORS (tPA) (uPA)PLASMINOGEN PLASMIN FIBRINOLYSISHEPARAN+Intact Endothelium Limits HemostasisProtein C SystemTHROMBIN INACTIVATES PAIvFW multimersSubendothelium

PGI2ADPaseINHIBITS PLATELET ACTIVATIONTHROMBOMODULINPROTEIN C PROTEIN CaPROTEIN SINACTIVATES Va + VIIIaATIIIINHIBITS Xa + THROMBINPLASMINOGENACTIVATORS (tPA) (uPA)PLASMINOGEN PLASMIN FIBRINOLYSISHEPARAN+Intact Endothelium Limits Hemostasis - Chemical MediatorsTHROMBIN INACTIVATES PAIvFW multimersSubendotheliumNO

Production of Coagulation FactorsSynthesized in the liver-All except Factor VIIIVitamin K dependentII, VII, IX, and XProtein C, Protein SFactor VIII- unknown

Synthesized in liverSerine proteases, inactivated by AT3Activation on surface of biologic membranesHave an affinity for binding calciumVitamin K Dependent Enzymes: Factors II, VII, IX, X

Coagulation Cascade- PT TISSUE FACTOR

FACTOR VII VIIa (ACTIVATED)

( XI?) IX IXa TFPI VIII

FACTOR X Xa

V, Ca

FACTOR II IIa

FIBRINOGEN FIBRIN MONOMERS

FACTOR XIII

IN VITRO CROSS-LINKED FIBRIN PT (STABLE FIBRIN CLOT)APTT

APTTCONTACT ACTIVATORFXII, FXI, FLETCHER, FITZGERALD FACTORSVIIIIX IXaPTPHOSPHOLIPIDPLATELETACTIVATIONINHIBITION OFCLOTTINGTHROMBOMODULINPROTEIN C(ENDPOINT)PHOSPHOLIPID

Coagulation Cascade- APTT TISSUE FACTOR

FACTOR VII VIIa (ACTIVATED)

( XI?) IX IXa TFPI VIII

FACTOR X Xa

V, Ca

FACTOR II IIa

FIBRINOGEN FIBRIN MONOMERS

FACTOR XIII

IN VITRO CROSS-LINKED FIBRIN PT (STABLE FIBRIN CLOT)APTT

APTTCONTACT ACTIVATORFXII, FXI, FLETCHER, FITZGERALD FACTORSVIIIIX IXaPTPHOSPHOLIPIDPLATELETACTIVATIONINHIBITION OFCLOTTINGTHROMBOMODULINPROTEIN C(ENDPOINT)

Prothrombin TimePoor reproducibility from lab to lab in USNo good assayed standardsMany manufacturersMany chemically different reagentsMany different types of instrumentsPoor lot-to-lot reproducibility even from same manufacturer

Reporting Protime ResultsEach laboratory must establish its own normal range using the instrument and reagents that it is usingIt may have to be redone with each new lot of reagents, certainly, at least rechecked and verified- insist on it from the laboratory you useResults should be expressed in seconds, not INRResults should be compared to the normal range. The true Control value is meaningless for clinical use.

Prothrombin TimeThe INR is the answer to our prayers- Hallelujah- NOT!!Poor calibration by reagent manufacturers is the weak link in the chainIntended only for inter laboratory comparisons in patients who are on steady state anticoagulation with coumadin: at least two weeks of therapy, ambulatory, normal activity and diet Widely misapplied to express protime results in all other situations

Bleeding TimeWidely misused as a screening test for platelet function abnormalitiesCan predict trends when used to study large populationsCannot predict bleeding risk in individual patients - use for this purpose has been discredited

Screening for Hemostatic DefectsPT, APPT- sensitivity is too poor to pick up mild defectsBleeding time- poorly reproducible, too many false positives and false negatives, Most common cause of a prolonged bleeding time- improperly performed Best screen: good history

Screen for Platelet AbnormalitiesNo good tests, historyImmediate bleedingMucous membrane bleedingEasy bruisingPetechiae

Screen for Clotting Factor DeficienciesDelayed onset of bleedingLarge ecchymoses or hematomasBleeding into joints

Screening History for Bleeding ProblemsDo you think you have a bleeding problem? Does anyone in your family have a bleeding problem?Easy bruising?Previous hemostatic challenges:Major surgeryTraumaExtraction of impacted teeth

Bleeding Problems Pre-operative screeningPatient with suspicious historyActively bleeding patient

Pre-operative ScreeningMost common hereditary bleeding problems?Acquired bleeding problems?Sensitivity of screening tests?

Hereditary Bleeding Disordersvon Willebrands disease - platelet functionStorage pool disease (delta granule deficiency) - platelet functionFactor VIII deficiency (Hemophilia A) Factor IX deficiency (Christmas disease)Factor XI deficiency

Patient with Suspicious HistoryRefer to laboratory or specialist that specializes in bleeding disorders.

Actively Bleeding PatientFocal bleeding - catgut insufficiencyGeneralized bleeding - ThrombocytopeniaVitamin K deficiencies - commonDIC - most over-diagnosed cause of bleeding in the acute care/ICU setting. Primary fibrinolysis - rare

Acquired Bleeding Problemsdrug-induced platelet function defectsThrombocytopeniavitamin K deficiencyLiver diseaseCoagulation inhibitorsPost viralMisc. othersIdiopathic

Vitamin K DeficiencyAppropriate clinical setting: a. Poor or no oral intake b. Broad spectrum antibioticsProlonged PT, PTT with a normal platelet count and fibrinogen - presumptive diagnoses of Vitamin K deficiency

Acute DIC: A clinical-pathologic DxSeverely, acutely ill patient (not clinically stable).Decreasing platelet count and/or fibrinogen.

36Acute DIC PrinciplesMost over-diagnosed cause of bleeding in a hospital/ICU setting.Should be approached as a diagnosis of exclusionIf it is the only diagnosis you can think of, you are over your head. GET HELPVitamin K deficiency is much more common.Many other factors are more likely to be the cause of thrombocytopenia.

Possible DICRun all tests on the same specimen: PT, PTT, Fibrinogen, FDP Platelets (Factor V, Factor VIII).It may take sequential testing to establish diagnosis.Sources of Vitamin KDiet- Fresh, green leafy vegetablesSynthesis by bacteria in the intestinal trackTypical ICU/acutely ill, hospitalized patientNo or poor oral intakeBroad spectrum antibiotic therapyIncreased vitamin K requirement because of illnessResult: Acquired vitamin K deficiency within two to three days of admission

Vitamin K Dependent Factors II, VII, IX, X PT - II, VII, X APTT - II, IX, X

Vitamin K Deficiency vs. Acute DICVitamin KDICPTAPTFDPFibrinogen*PlateletsN or prolongedProlongedNormalNormalNormalProlongedN or prolongedUsually elevatedN or decreasedUsually decreased

41Elevated Levels of FDPRecent surgeryAcute thromboembolic eventRenal failureHepatic failureAcute myocardial infarctionDICTTP/HUSPrimary fibrinolysis

Suggested Approach to the Bleeding Hospitalized PatientDraw PT, APTT, FDP, fibrinogen and platelet count on same specimen as a panel. Do not try to use values drawn at different times.Immediately give Vitamin KRedraw panel in 4-6 hours. K deficiency should show some degree of correction of PT and APTT within this time periodDIC should manifest itself by a decreasing fibrinogen without any significant correction of PT< APTT

Diagnostic Approach to ThrombocytopeniaGood history; medication - dont forget heparin; Acuteness of onset; Underlying diseasesPhysical - splenomegalyExamination of blood smear by an experienced individual; platelet morphology, Other hematologic abnormalitiesBone marrow examination - almost never helpful in the absence of other hematologic abnormalities