Clin Path - Hemostasis

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Light Blue top tube

Back 1

-citrate (binds Ca2+)

-plasma (anticoagulant)
-used for most coagulation assays

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Primary Hemostasis vs. Secondary Hemostasis vs. Fibrinolysis

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-Primary = formation of platelet plug
-Secondary = formation of fibrin clot
-Fibrinolysis = removal of fibrin clot

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Assays for assessing Primary Hemostasis

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-Platelet function assessment
-von Willebrand Factor concentration

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Assays for Assessing Secondary Hemostasis

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Assays for assessing blood clotting:
-PT (Prothrombin time)
-PTT (Partial Thromboplastin Time)
-Fibrinogen Concentration

Assay for assessing inhibition of clotting:
-AT (antithrombin activity)

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Assays for Assessing Fibrinolysis

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-FDP (Fibrin/Fibrinogen Degredation Products)
-D-dimer (Fibrin Fragment D-dimer)

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General hemostasis after an injury

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-vasoconstriction reducing blood flow to the area
-platelet aggregation to plug the hole
-Fibrin formation to solidify the plug
-Fibrinolysis to control fibrin formation and dissolve clots

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Effect of a defect in platelet aggregation to plug the hole

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-Petechiae
-Ecchymosis

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Effect in the ability to coagulate and have fibrin solidify the plug

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-Gross bleeding

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Effect of defective fibrinolysis

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-Thrombosis

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Assays for measuring Platelet Function

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-Buccal mucosal bleeding time
-clotting time

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Difference between buccal mucosal bleeding time and clotting time

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-Buccal mucosal bleeding time (in vivo)
-Clotting time (in vitro)

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What does a prolonged Buccal Mucosal Bleeding Time mean?

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-decreased platelet function (too few, not functional)
-vWF deficiency

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Types of Clotting Times

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-PT
-PTT
-ACT

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What are PT & PTT assessing vs. ACT?

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-PT, PTT = time for fibrin clot
-ACT = time for blood clot

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Reasons for increased time to form a fibrin or blood clot

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-dec. [clotting factor]
-inc. [clotting inhibitor]

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Clot Retraction assay
-tube use

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-red top

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Clot retraction Assay
-how is the blood collected

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-into cold saline

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Clot Retraction Assay
-what does delayed retraction mean

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-dec. platelet conc.
-dec. platelet function
-cold sample --> dec. enzyme activity

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Clot retraction assay
-how long is it supposed to take to clot

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-30 mins

-60 min in horses

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von Willebrand Factor
-composition

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-plasma glycoprotein

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von Willebrand Factor
-circulates with what clotting factor

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-Factor VIII

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von Willebrand Factor
-produced by

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-endothelial cells
-platelets and megakaryocytes (cats, people)

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von Willebrand Factor
-function

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-bridge platelets to injured vessel wall
-platelet-platelet bridging

(part of primary hemostasis)

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Blood samples to test for vWF
-how to collect
-how to ship

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-Plasma

-EDTA
-citrated blood

-ship frozen plasma overnight with cold packs

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Why may vWF be falsely low

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-poor sample collection --> platelet clumps/clots

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vWF assays

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-ELISA

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vWF disease types

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-Type 1: All types of vWF multimers present but dec. [vWF]

-Type 2: Large vWF multimers are deficient (uncommon)

-Type 3: No vWF present

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Type 1 vWF deficiency
-dog breeds

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-Dobermans

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Type 2 vWF deficiency
-dog breeds

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-GSH
-GWH

-Horses

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Type 3 vWF deficiency
-dog breeds

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-Chesapeake Bay Retrievers
-Dutch Kooikers
-Scottish terriers
-Shetland sheepdogs
-Border collies

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Signs of dogs having vWF < 35%

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Defective primary hemostasis
-spontaneous mucosal bleeding
-prolonged iatrogenic bleeding time (surgical, venipuncture)
-prolonged buccal mucosal bleeding time (BMBT)

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Extrinsic Coagulation
-aka
-enzymes
-evaluated by

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-aka: Tissue factor

-enzymes: VII

-evaluated by: PT

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Intrinsic Coagulation
-aka
-enzymes
-evaluated by

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-aka: Surface-induced

-enzymes: XII, XI, IX, VIII

-evaluated by: PTT & ACT

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Common Coagulation Pathway
-enzymes

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-X
-V
-II
-I

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What does TT assess?

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-conversion of fibrinogen to fibrin with the addition of thrombin

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How is the Tissue Factor Pathway (Extrinsic) activated?

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TF released from:
-damaged tissue
-monocytes, macrophages, endothelial cells activated by endotoxin and some inflammatory cytokines

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Coagulation factors in vivo
-typically circulate as...

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-circulate as plasma zymogens (proenzymes) that act on other factors when activated

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Plasma zymogen half-life

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-short (hours)

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Coagulation factor IV
-aka

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-free Ca2+

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Coagulation factor II
-aka

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-Prothrombin

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Coagulation factor I
-aka

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-Fibrinogen

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Coagulation Factors produced by hepatocytes

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-XII
-XI
-X
-IX
-VIII
-VII
-V
-Prothrombin (II)
-Fibrinogen (I)

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How is the surface-induced (intrinsic) coagulation pathway initiated?

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-when contact coagulation factors HMWK, PK, and Factor XII contact negatively charged surfaces

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Negatively charged surfaces that will induce intrinsic coagulation
-in vivo
-in vitro

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in vivo
-collagen

in vitro
-Kaolin
-Diatomes
-Glass

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Ca2+ is needed as a cofactor for which coagulation enzymes

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-VII
-X
-IX
-II

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Blood tubes to use to remove Ca2+ from coagulation cascade

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-Purple
-Light blue
-Grey

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Phospholipids are needed as cofactors in coagulation. Where do the phospholipids come from in vivo and in vitro?

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-in vivo: platelets

-in vitro: reagents

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Citrate to blood ratio in a collection tube
-effect of too much citrate
-effect of too little citrate

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-1 : 9

-too much: increased PTT &/or PT
-too little: blood sample clots

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Proper plasma sample collection technique for coagulation assay

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-mix blood with citrate in a 1:9 ratio (citrate:blood)
-centrifuge citrated blood within 1 hr
-analyze citrated plasma within 4 hrs or freeze
-if shipping, ship frozen overnight with ice

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Why should centrifuged citrated blood be analyzed within 1 hr?

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-platelet poor plasma may begin to form clots by then

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Thromboplastin
-function

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-activator of coagulation activity

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Partial thromboplastin
-function

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-Procoagulant that is devoid of tissue factor

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How does PTT work?

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-add Partial Thromboplastin to Citrated plasma
-activation of factors XII and XI
-incubated to prime the process and get it ready to clot with the addition of excess Ca2+
-Excess Ca2+ added
-Fibrin forms --> (PTT)

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How does PT work?

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-Thromboplastin with excess Ca2+ is added to citrated plasma
-Time until Fibrin is detected (PT)

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Rules of Thumb for prolonged plasma clotting times

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-PTT prolonged if < 30% activity from 1 or more factors (XII, XI, IX, VIII, X, V, II)

PT prolonged if < 30% activity from 1 or more factors (VII, X, V, II)

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Increased Thrombin Time generally means

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-decreased fibrinogen

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Prolonged PTT due to

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Deficiencies in the surface-induced pathway (intrinsic) or common pathway
-acquired ( > 1 factor)
-hereditary (1 factor)

Inhibitors of surface-induced (intrinsic) or common pathway

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Acquired defects in the intrinsic/common pathway

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-hepatic disease (decreased production)
-Decreased Vit K recycling in hepatocytes
-decreased Vit K absorbtion (biliary)
-DIC
-Pathway inhibition

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What can cause decreased Vit K recycling by hepatocytes

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-rodenticide

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Inhibition of the intrinsic or common pathway can be caused by...

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-heparin
-FDPs
-antibody to phospholipid
-antibody to coagulation factors

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Prolonged PT due to

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Deficiencies in the tissue factor (extrinsic) or common pathway
-acquired ( > 1 factor)
-hereditary (1 factor)

Inhibitors of tissue factor (extrinsic) or common pathway

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Acquired deficiencies of the intrinsic/common pathway are:

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-dec. production (hepatic, Vit K)
-inc. activation/consumption (DIC)

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Vit K dependent coagulation factors

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-II
-VII
-IX
-X

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PIVKAs
-define

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-Proteins Induced by Vit K Antagonism, Absence or deficiency

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PIVKA
-importance

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-coagulation factors (II, VII, IX, X) cannot bind to Ca2+

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Prolonged PTT & PT due to Warfarin toxicity
-pathogenesis

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Ingestion & absorption of warfarin
-inhibition of the recycling of Vit. K by hepatocytes
--less reduced Vit K for the carboxylation of Factors II, XII, IX, X
---Production of defective factors II, VII, IX, X (PIVKAs) that can't bind Ca2+
----Reduced plasma VII activity (shortest half-life)
-----Prolonged PT
----Reduced plasma factor II, IX, X activity
-----Prolonged PT & PTT

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Prolonged PT & PTT due to warfarin toxicity has a similar pathogenesis to

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-prolonged cholestasis
-malabsorption of Vit K

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How Does the PIVKA test work?

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Thrombotest
-add special thromboplastin with TF, fibrinogen, factor V and excess Ca2+ to citrated plasma
-time until fibrin is detected

-has a greater diagnositic sensitivity for samples with decreased factors II, X, VII, IX

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Thrombin Time
-definition

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-thrombin-induced conversion of fibrinogen to fibrin clot

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Thrombin Time
-dependent on

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-[Fibrinogen]

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Diseases causing hypofibrinogenemia

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Increased fibrinogen consumption
-Intravascular coagulation
-Increased fibrinogenolysis

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Physiologic inhibitor of coagulation

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-Antithrombin III (AT)

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Antithrombin binds to what factors?

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-IIa (thrombin)
-IXa
-Xa

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Coagulation factor used in the Antithrombin assay

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-Xa

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Tube to collect blood in for Antithrombin assay

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-green top (heparin)

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Describe how antithrombin works

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-AT is produced by hepatocytes
-AT binds with heparin sulfate, creating a receptor for Thrombin
-AT-hep. complex binds Thrombin
-Thrombin-AT complex removed by hepatocytes

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Decreased Antithrombin activity can be due to:

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-Dec. production (dec. functional mass)
-Inc. loss (PLN)
-Inc. consumption (DIC, Sepsis, Heparin administration)

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What can happen if there is decreased inhibition of clotting?

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-more prone to forming thrombi in vessels

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Describe the 10ase assay

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-heparin binds to antithrombin
-the more antithrombin that gets bound, the more enzyme Xa will be inhibited

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FDP
-defintion

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-Fibrinogen Degredation product

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What cleaves fibrin and fibrinogen to form FDPs

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-Plasmin

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Where does Plasmin come from?

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Tissue injury-->tissue releases t-PA --> plasminoen converted to plasmin

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Fibrinolysis
-pathogenesis

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Tissue injury (hypoxia, heat, neoplasia, infection....)
-release of T-PA
--conversion of plasminogen to plasmin
---plasmin cleaves plasma fibrinogen to FDPs
----Inc. [FDPs] if the formation is greater than liver removal
-activation of coagulation pathways
--formation of fibrin in vessels
-Release of T-PA
--conversion of plasminogen to plasmin
---plasmin cleaves fibrin to FDPs and D-dimers
----inc. [FDPs] & [D-dimers]

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FDP assay
-best blood sample to use

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-serum

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Increased FDP can be due to:

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-inc. fibrinolysis (DIC)
-inc. fibrinogenolysis (inc. plasmin activity)
-dec. FDP clearance (liver, kidney, macrophages)

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How does FDP act as an inhibitor?
-what are the effects

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Competes for fibrinogen-binding sites

-On thrombin: prolonged PT, PTT, TT, ACT, WBCT
-On platelets: dec. platelet function

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Consequence of high FDP in vivo

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-diffuse bleeding in DiC

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Causes of defects in Primary hemostasis

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-Thrombocytopenia
-vWF deficiency

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Causes of defects in secondary hemostasis related to bleeding

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Coagulation factor deficiency:
-acquired (dec. production, inc. consumption)
-hereditary

Inhibitors (heparin, FDPs)

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Causes of defects in secondary hemostasis related to thrombosis

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Decreased inhibitors (AT):
-dec. production
-inc. loss
-Inc. consumption

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Causes of excessive fibrinolysis

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-Marked tissue damage --> Inc. Plasmin