Coag Part 2/3

FII, FVII, FIX, FX, FXI, FXII, FXIII
Prekalikrein (Fletcher)
(F2,F7,F9,F10,F11,F12,F13)

All serine proteases except FXIII

Stabilize and accelerate proteolytic reactions

high MW kininogen
Tissue factor
FV
FVIII
Protein S
Thrombomodulin
Protein Z

HMWK, Fitzgerald
cofactors for FXIIa and PK

FIII, tissue thromboplastin
cofactor for FVIIa

Labile factor, proaccelerin
cofactor for FXa

Labile factor, AHF A
cofactor for FIXa

Cofactor for protein C

Cofactor for thrombin in protein C activation

Cofactor for protein Z-dependent protease inhibitor ZPI

Anti-thrombin
Protein C
Heparin cofactor II
TFPI (tissue pathway inhib)

FI (fibrinogen), vWF, Ca+ (FIV), phospholipids (PF3)

Active or activated

Based on the order of discovery or description of factors

Fibrinogen

Prothrombin

In liver except vWF

By ECs and MKs
EC: store in Weibel-Palade granules
MKs: store in plt alpha granules

VWF (500-20000 kd)

Shortest: VII (3-6 hrs)
Longest: HMWK

Carried in plasma as bound to vWF to maintain 12 hr half life

Let's coag complexes to bind to plt or cell membrane phospholipids

Coag proteins act on each other in cascade reaction (each factor activates the next)
outcome: conversion of fibrinogen into stable fibrin clot

Vitamin K dependent coag protein
AKA prothrombin group

Require vit K to become y-carboxylated
(to become functional)

All have 10-12 glutamic acid residues at N terminal

Important for assembly of coag complexes to generate thrombin

Complex 1: extrinsic tenase
complex 2: intrinsic tenase
complex 3: prothrombinase

Tissue factor + VIIa + Ca2+ + Phospholipid

activates X to Xa and
IX to IXa

IXa + VIIIa + phospholipid (PLT surface) + Ca2+

X to Xa

Xa+ Va + phospholipid + Ca2+

Activates prothrombin to thrombin

FVIII and vWF
(severe bleeding in hemophilia A and vWD)

By genes encoded on X chromosome

Gets degraded faster than other factors

vWF

Multimeric subunits of 240kd polymerized to 500-2000kd in MK and ECs
degraded to smaller multimers (ADAMTS13)in blood vessels with higher shear rate

Receptors for plts and collagen
(bridges plts to sub endothelium during plt adhesion)

With ABO groups
O group has lower level

Acute phase reactants (APR)

FXII, PK, HMWK
(contact factors, contact factor complex)

Contact system

By negatively charged surfaces

PK (bound to HMWK) to K
K further activates FXII

HMWK+PK+FXIIa

FXI (with then activates FIX)

Thrombin (generated by extrinsic tenase)

Does not cause bleeding but prolonged aPTT

Thrombin

Cleaving fibrinopeptides A and B (FPA and FPB) from fibrinogen
triggers spontaneous polymerization, clot initiation

FV, FVIII, FXI (positive feedback)
protein C
TAFI
FXIII

Thrombin bound to thrombomodulin

Aggregation of plt

Activates TAFI (thrombin activatable fibrinolysis inhibitor)

Thrombin activates FXIII

Fibrinogen

200-400 mg/dL
an APR

Plt aggregation through binding the IIa, IIIa receptor ?

Plts absorb it and store in alpha granules

Plasma transglutaminase

Deficiency does not affect PT or PTT but leads to clinical bleeding

TF:FVII is the primary initiator

VII, X, V, prothrombin, and fibrinogen

Prothrombin time (PT) test

XII, pre-K, HMWK, XI, IX, VIII, X, V, prothrombin, and fibrinogen

By activated partial thromboplastin time (aPTT) test

Includes X, V, prothrombin, and fibrinogen

Plus thrombin time (TT)

Extrinsic tenase complex

FVII:TF complex activators low level of FX and FIX and small amt of thrombin
(ignites more thrombin generation through positive feedback, max3%)

>95% of thrombin becomes generated

On plt membrane

Large plt aggregates, intrinsic tenase activates FX 50-100X higher
prothrombinase complex generates burst of thrombin

Serine protease inhibitor

With Xa, binds TF:VIIa

EC surface receptor for thrombin

Serine protease

Cofactor

Serpin
(serine protease inhibitor)

Fibrinolysis

As it is forming

Liver

ECs secrete TPA and UPA
(TPA more effective)

TPA-tissue plasminogen activator
UPA-urokinase plasminogen activator

Degrades fibrin-bound plasminogen to generate plasmin

Degrades fibrin (and fibrinogen) into several fragments (X, Y, D, E, and D-D)

D dimer
fibrin-derived and diagnostic test for thrombosis and fibrinolysis

PAI-1
alpha2-AP
TAFI

Binds to and inactivates TPA and UPA
(PAI-1 is serpin produced by ECs and MKs)

Inactivates free plasmin (plasmin not bound to fibrin)

Thrombin-thrombomodulin complex

Blocks binding of TPA and plasminogen to fibrin and formation of plasmin

Trauma-induced &
Liver disease

Systemic shock leading to acute reduction in ADAMTS13, coag factor activation, TF release, and hyperfibrinolysis

Reduction in plasma level of SCZ27910

FVII reduced, PT Is sensitive to low FVII; first PT is a prolonged in mild liver disease
sensitive early marker

Vit K deficiency has same effect on PT

FV level (whereas liver disease is FVII)

Thrombocytopenia due to high sequestration of enlarged spleen;
abnormal plt function (low aggregation in aggregometry)

Due to low production of anti thrombin or protein C and S
may be compensated or acute uncompensated

Prolonged PT, PTT, TT, low fibrinogen, increased D-dimmer test

Only D-diner is increased

>400 mg/dL-early mild liver dx
<200 mg/dL mod-severe liver dx (which causes dysfibrinogenemia and hypofibrinogenemia)

Prolonged in dysfibrinogenemia, hypofibrinogenemia, elevated fibrin degradation production, and unfractionated heparin therapy

Prolonged in hypofibrinogenemia, significantly prolonged in dysfibrinogenemia;
unaffected by heparin

Prolonged, even in mild liver dx, because of does-y-carbonyl factors replacing normal factor II (prothrombin), VII, and X

Mildly prolonged in severe liver dx bc of DIC or des-y-carbonyl prothrombin, IX, and X

Reduced in liver dx but is unaffected by vit K def, so factor V lvl helps distinguish liver dx from vit K def

Mild thrombocytopenia plt ct <150,000L

Mild suppression of plt aggregation and secretion in response to most agonists

>240 ng/ml or >500 ng/ml fibrinogen equiv units (FEUs)

Older pts may develop auto ab against FVIIa which causes acquired hemophilia (sudden bleeding into soft tissue or GI bleeding)

RA, IBD, SLE, or lymphoproliferative disease (CLL)

FVIII inhibitors
PT, PTT, TT for any pt experiencing sudden hemorrhage like acquired hemophilia
Mixing studies to confirm presence of inhibitor

PTT is prolonged but PT and TT are normal
FVIII <40 units/dL should be established by factor assay

pts plasma mixed 1:1 with normal plasma,
prolonged PTT will be corrected initially but prolonged after incubation 37C (temp sensitive)

indicates presence of lupus anticoagulants or patient is under heparin therapy
15% of inhibitors (high avidity) immediately prolong the PTT's mixture

autosomal dominant
affecting both sexes (females>males)
qual or quant changes in vWF due to gene mutations

inherited mucocutaneous bleeding disorder causing decreased plt adhesion and impaired primary hemostasis

FVIII deficiency

vWF plasma levels 30-50%, maintain sufficient FVIII

Type 1 (quant, 40-70%, mild to mod)
Type 2 (qualitative)
Type 2A, Type 2B, Type 2M, Type 2N,
Type 3 (null, severe vWF def, very low FVIII)

history of mucocutaneous bleeding and decreased vWF concentration or activity (function)

do CBC to rule out thrombocytopenia

rule out factor deficiency other than vWF

vWF: Rco, vWF:Ag, vWF activity to vWF:Ag ratio, RIPA, FVIII activity, vWF multimers

vWF:Rco

Subtype 2B
Subtype 2M

Subtype 2B

Type 3

Type 3

Type 1
Subtype 2M
Subtype 2N

Type 3

congenital, single factor deficiency
X-linked recessive (males have disease while females are carriers)

anatomic soft tissue bleeding

FVIII deficiency (85% pts)

FIX def (14% pts)

FXI def (1% of pts)

FVIII protein translated from X chromosome
various deletions, stop codons, nonsense/missense mutations result in quant def of FVIII

FVIII def slows coagulation pathway production of thrombin
leads to bleeding

anatomic bleeds
deep muscle and jt hemorrhages
hematomas
wound oozing after trauma/surgery
bleeding in CNS, peritoneum, GI tract, and kidneys

PT prolonged
PTT prolonged
TT prolonged

fibrinogen assay

PT prolonged
PTT prolonged
TT normal

PT prolonged
PTT prolonged
TT normal

PT prolonged
PTT normal
TT normal

PT normal
PTT prolonged
TT normal

PT normal
PTT prolonged
TT normal

PT prolonged
PTT prolonged
TT normal

PT normal
PTT prolonged
TT normal

PT normal
PTT normal
TT normal
What test do you test for when:
PT prolonged
PTT prolonged

fibrinogen assay
What test do you test for when:
PT prolonged
PTT prolonged

Prothrombin, V, VII, and X
What test do you test for when:
PT prolonged
PTT normal

VII assay
What test do you test for when:
PT normal
PTT prolonged

VIII, XI, and XI
What test do you test for when:
PT normal
PTT normal

XIII quant assay

on plasma used to distinguish factor deficiencies from factor inhibitors

factor deficiencies OR specific factor ab OR non-specific antibodies/inhibitors

req 50% of normal level of factors in circulation

next steps to take to diagnose cause of abnormal APTT and/or PT results

1. repeat PT or APTT using pt PPP
2. run normal pooled plasma allow to ensure normal results or 100% factor lvl
3. run pt PPP mixed 1:1 with NPP
4. compare

inhibitor
run inhibitor assay (DRVVT)

factor def or AB to factor

1:1 mix + 1-2 hr incubation at 37C

factor deficiency

factor antibody
do factor antibody assay

no correction occurred, circulating inhibitor such as lupus anticoag is suspected

correct occurred
factor deficiency or specific factor antibody may be suspected as the cause of originally prolonged test

FXIII deficiency or inhibition

FXIII insoluble in urea

fibrin clot will dissolve rapidly in presence of urea

by FXIII assay
quantitative activity assay

bovine thrombin reagent transforms plasma fibrinogen to detectable fibrin polymer

1. thrombin reagent and pt plasma prewarmed at 37C 3 mins
2. 200uL thrombin added to pt plasma and chronometer is started, clot formation recorded
RI: 15-20s

hypofibrinogenemia (<100 mg/dL) or afibrinogenemia or dysfibrinogenemia

repitlase time test

1. pt reagent warmed to 37C; PPP warmed to 37C 3-10 min
2. reagent added to plasma aliquot. Start timer
3. as clot forms, stop timer and record elapsed time
4. fibrin clot detectable visually or by optical or electromechanical sensors

becomes prolonged
coag factors begin to deteriorate or are affected by evaporation and pH change

INR standardizes PT results and reporting to allow for monitoring of vit K antagonist therapy (Coumadin/Warfarin) across diff labs
reported along with PT result

(pt PT/mean normal PT)^ISI

geometric mean normal PT derived from 20 fresh plasma specimens obtained from healthy individuals

how sensitive reagent is to def in vit K dependent factors compared to WHO reference standard

10.6-14.6 s

could be slightly diff
e.g., 9.5-13.5 s

0.9-1.2

clot based routine coag test

deficiencies or inhibitors of the factors in the intrinsic and common pathways

Factor XII, XI, IX, VIII, X, V, II, I
all factors except FVII

standard (unfractionated) heparin anticoag therapy

1. equal PPP and 1st reagent (contact activator) and PL combined and incubated
2. 2nd reagent (CaCl2) added, sample mixed at 37C
3. timing stopped when fibrin strands seen
4. fibrin clot detectable visually or optical

25-35 s, could vary lab to lab

3.2% buffered sodium citrate
(leaves all clotting factors available except calcium; citrate binds to calcium in plasma and therefore prevents coag)
not as strong as EDTA

plasma (contains coag factors) is separated from plt (phospholipid source) by centrifuged
plt poor plasma
PPP plt <10x109/L
centrifuge 3000 RPM for 15 mins at RT

9:1
9 parts whole blood to 1 part sodium citrate

high hematocrit (>55%)

c=(1.85x10-3)(100-H)xV
C=sodium citrate (mL)
V=volume whole blood (mL)
H=hematocrit(%)