3ry Hemostasis & Coag Regulators

which steps require Ca2+ and phospholipid (PL) in coag cascade?

11, 9, 10, 2 (XI, IX, X, II)

how is thrombin the MVP?

forms fibrin (Ia)

activates factor 13, 11, 8, 5 (XIII, XI, VIII, V)

activates protein C on endothelium → inhibits coag

activates PAR-1 & PAR-4 on plts → plt activation

IIa (thrombin) acts on which coag cascade factors?

VIII

V

I

XIII

XI

(1, 5, 8, 11, 13)

inhibitors of coag

protein C & S

• thrombin activates thrombomodulin (on endothelial cells) → PC becomes APC (activated protein C) → APC + PS form complex inhibits factor 8 & 5

antithrombin

• inhibits IIa and Xa

• AT is enhanced by heparin

TFPI (tissue factor pw inhibitor)

• inhibits TF:VII complex (extrinsic pw) & factor X (start of common pw)

fibrinolysis: how to remove clot?

tPA (tissue plasminogen activator) + plasminogen → plasmin degrades fibrin polymers’ covalent bonds

stable fibrin clots require ___

covalent crosslinking by factor XIIIa which crosslinks at outer D domains and central E domains

ie: D—E

D-dimer vs FDP (fibrinolysis degradation products)

D-dimer

• latex immunoassay to assess fibrin degradation

FDP assay: also latex immunoassay

all D-dimers are FDPs, but not all FDPs are D-dimers

DIC panel results would look like:

• what score is required for dx?

prolonged PT, aPTT

dec fibrinogen

dec plt

inc D-dimer and FDPs (inc bc in DIC both xs coag and fibrinolysis are occuring)

each of the above contribute to DIC score

• DIC score >=5 = compatible w overt DIC

Protein C system

• what is required of PS?

thrombin activates thrombomodulin (on endothelial cells) → PC becomes APC (activated protein C) → APC + PS form complex inhibits factor 8 & 5

free PS is required for PC to work!

• lab measures free PS first

• PS in dynamic equilibrium w protein C4b binding protein

• PS+C4b binding protein = inactive PS → cannot bind to APC

antithrombin (ATIII) function?

• how does heparin affect it?

inhibits IIa (thrombin) and Xa (Stuart Prower factor)

heparin binds ATIII via pentasaccharide sequence → accelerates inhibition of IIa & Xa

• AT is enhanced by heparin → why thrombin time (TT) is sensitive to heparin → TT prolonged

thrombophilia: venous vs arterial

tendency towards recurrent thromboembolism (pathogenic blood clots)

• venous: deep vein thrombosis (DVT), pulmonary embolus (PE)

• arterial: myocardial infarction (MI), cerebrovascular accident (CVA)

factors that predispose pts to thrombosis

factor gene mutations

• factor V Leiden

• prothrombin variatn 20210A

defic of naturally found inhibitor

• ATIII

• protein C or S

presence of acquired inhibitor (anti-PL Ab)

• Lupus anticoagulant

• anti-cardiolipin Ab

Factor V Leiden leads to _?

• how is it tested?

abnormal factor V → resistant to APC inhibition

(m/c in Caucasians, Netherlands hence name)

tests:

• function assay (APC resistance)

• PCR for mutation

Prothrombin Variant 20210A leads to __?

inc levels (2-3X) prothrombin (II) in plasma → inc risk of thrombosis

• 1-2% in Caucasians

• tested via PCR

ATIII deficiency

• how is it acquired?

• can lead to?

• what test is used?

can be acquired or inherited (auto dominant)

• acquired: DIC, acute thrombosis, liver dz, nephrotic syndrome, extracorporeal membrane oxygenation (ECMO), asparaginase therapy, heparin therapy

→ thromboembolism, reduced efficacy of heparin

tests: chromogenic assay

• measures ability to inhibit factor IIa & Xa

  • more color → less ATIII, more factor Xa cleaving chromogenic substrate

protein C defic

• homo vs heterozygous risks?

• test?

• homozygous → severe thrombosis w necrosis at birth (purpura fulminans)

• heterozygous → inc risk of thrombosis

test: chromogenic assay

protein S defic

• inc risk of __

• how to test?

inc risk of thrombosis

• in severe cases → infants get purpura fulminans soon after birth

test: free protein S assay

Lupus anticoagulant (LA)

• what is it? why is it a misnomer?

• what dz states are assoc’d w/it?

LA = antiphospholipid → inc thrombosis

• misnomer bc it’s Ab interfere w coag in vitro (prolong PTT), but activates coag in vivo (→ thrombosis)

• found in pt w/ or w/o SLE

• assoc’d w APS

APS: antiphospholipid syndrome

• autoimmune disorder char by xs blood clot formation, organ failure, pregnancy complications 2ry to anti-PL Ab

SLE: systemic lupus erythrematosus

• systemic autoimmune dz which may accompany APS

what steps does LA interfere w/in the coag cascade?

11, 9, 10, 2 (XI, IX, X, II)

how to screen for Lupus anticoagulant?

• Lupus Anticoagulant

• Beta-2 GP1 Ab (IgG/IgM > IgA)

• Cardiolipin Ab (IgG/IgM > IgA)

  must be medium or high titer

how to confirm/dx Lupus anticoag? 3 tests:

• what do they have in common?

• dilute russell viper venom time (DRVVT)

• silica clotting time (SCT)

• Lupus anticoagulant sensitive aPTT method (hexagonal phase)

all have a screen and confirmation, where the confirmation has added PL to neutralize LA if present → expect corrected

dilute russell viper venom time (DRVVT)

• what is the function of the venom?

• how is the screen performed?

• how is the confirmation done?

LA will prolong clot time

venom + Ca2+ → activates factor X

• → triggers coag cascade, thus don’t need upstream factors

  • not affected by contact factor anomalies or factor VIII & IX defic or inhibitors

screen: DRVVT performed at low conc of PL

• if LA present, clot time prolonged

confirm: DRVVT performed at high conc of PL → neutralize the LA present in plasma (trying to bind all free LA’s)

• clot time is shorter than screen bc neutralized all free LA → should get normal clot time

positive result = % correction above cutoff

silica clotting time (SCT)

• what test is this identical to?

• how is the screen done?

• confirmation?

LA will prolong clot time

identical to PTT assay

• silica = activator of factor XII (intrinsic pw)

• more sensitive to effects of LA vs regular PTT rgts

screen: use low conc of PL

• if LA, clot time prolonged

confirm: use high conc of PL → neutralize LA in plasma

• clot time normalized, shorter than screen

positive result: ratio above cutoff

Lupus sensitive-PTT w hexagonal phase

• what test is this based on?

• screen?

• confirmation?

LA will prolong clot time

• aPTT based, PL-dependent coag assay (ie activates intrinsic pw)

screen: run aPTT-based, PL-dep test on plasma

confirm: adding rgt that contains xs PL (hexagonal phase PL), w/1:1 mix correcting for factor defic

pos result: time difference b/t screen & confirm (usually >8 sec)

why is glass avoided for coag testing?

glass is neg charged → can activate contact pw (intrinsic pw) → inaccurate results

to avoid false pos/neg for pt care, what has been recommended for dx’ing APS?

• incorporate mixing studies into screen/confirm steps

• beware anticoag medication: heparin, DOACs, warfarin

• at least 2 LA assays (eg DRVVT + SCT)

• anti-cardiolipin & beta2 GPI IgM alone → can no longer dx

UCI thrombophilia panel testing

• protein C activity

• free protein S Ag

• antithrombin III (ATIII)

• Lupus anticoagulant assays

  • DRVVT

  • SCT

  • PTT-LA/hexagonal phase = sendout

  • anti-B2 GP and anti-cardiolipin Ab

• factor V Leiden (molecular)

• prothrombin 20210A (molecular)

caveats of thrombophilia testing

• most adults will test neg or mildly dec

• acquired causes > > inherited causes

• following acute thrombosis, PC/PS and ATIII levels may dec

• 1 lab tests is not enough to dx defic or thrombophilia → must RPT testing at least 12 weeks