Thrombophilia- Lab Assessment CH 14 & CH 9&19 (algorithmic) Flashcards
What are some common things to
know about thrombophilia?
- hereditary thrombophilias only marginally increase the risk of VTE and recurrence
- there is no evidence that a negative lab thrombophilia panel is associated with lower risk of recurrence
- clinical risk factors typically outweigh genetic ones in management
- recurrent VTE is common after the initial unprovoaked VTE
What recurrence risk of VTE is
used to weight the benefits vs. risks of anticoagulation?
- recurrence risk of >5% in the first year would outweigh the risk of long term anticoagulation
What are the 2 main lab findings
that could portend a clinically significant increased
risk of recurrent VTE if anticoagulation is stopped ?
- antiphospholipid antibodies
- antithrombin III deficiency
What is associated with VTE formation
in family members ?
- unprovoked VTE at a young age (<45 years)
- assocaited with increased risk of VTE in family members
- the risk is further increased if it occurs in multiple family members
Note: Factor V Leiden and PT gene mutations are rather weak predictors
What is the relationship between
estrogen supplementation and thrombosis ?
- dose dependent increased risk of thrombosis
- thought to be related to altered levels of clotting factors
- synergistic effect with hereditary causes, particularly Factor V leiden
What is key to know about Antithrombin III ?
- also called Heparin cofactor I
- synthesized in the liver
- half life is 2.8-4.8 days
- Inhibits
- thrombin (factor II)
- IXa, Xa, XIa, and XIIa
- accelerated 1000x by Heparin
What is the inheritance pattern of
Antithrombin III ?
- autosomal dominant with variable penetrance
- Type I deficiency
- quantitative
- homozygous deficiency is lethal
- Type II deficiency
- qualitative
- Type I deficiency
What are the three types of
type II defects in Antithrombin III ?
- Type II reactive site (RS), defect in the thrombin binding site
- Type II heparin binding site (HBS)
- most common
- least thrombophilic mutation
- exception is the homozygous type that develops thrombosis early on in life
- Type II PE
- produces a conformational change that affects binding to heparin and thrombin
How is testing for antithrombin III deficiency performed ?
- first line testing
- functional assay - Heparin cofactor activity
- chromogenic assay
- detects all types of antithrombin III defects
- performed by adding heparin to patient plasma which will bind to patient’s antithrombin III
- then exogenous thrombin and/or factor Xa is added
- once the antithrombin III activity is exceded there is light emittance
- functional assay - Heparin cofactor activity
What is a pitfall in treatment with heparin
derivatives when evaluating Antithrombin III ?
- derivative of heparin reduce antithrombin III levels
- other anticoagulants may cause increases in levels
- lead to false positive
- direct factor Xa inhibitors may lead to an overestimation of ATIII IF using a Xa based assay
- direct thrombin inhibitors may lead to overestimation of ATIII in thrombin based assays
What are causes of acquired antithrombin III
deficiencies ?
- acute thrombosis
- transiently reduces Antithrombin III
- cirrhosis
- leads to reduced synthesis of both procoagulant and anticoagulant proteins
- nephrotic syndrome
- reduces ATIII levels in the absence of clotting
- protein losing enteropathies
- ECMO
- due to heparin, consumption, and dilutional defects
- may lead to heparin resistance
- heparin therapy
- reduces ATIII by 30%
- likely due to increased clearance of heparin:ATIII complexes
- burns- levels correlate with the extent of thermal injury
How does aspariginase therapy (frequently seen in
treatment for B-ALL) affect ATIII levels ?
- asparaginase impairs the synthesis of proteins that contain asparagine
- ATIII is one of them
What is the odds ratio and clinical
presentation of ATIII deficiency ?
- first VTE OR is 16
- at risk of recurrent VTEs if they stop anticoagulation
- low levels of ATIII
- lead to heparin resistance, where you need higher levels to reach anticoagulation
Which medications do not require
antithrombin III levels in order to work ?
- direct Xa inhibitors and direct thrombin inhibitors
- their actions are not affected by low ATIII levels
Can people with ATIII deficiency develop
arterial thrombosis ?
- arterial thrombosis has been documented but is uncomon and may not actually be associated with this
What is the mechanism of action of
Protein C ?
- synthesized in the liver and is vitamin K dependent
- Protein C when activated is a serine protease
- primary function is to prevent clot extension into areas of intact endothelium
- thrombomodulin
- found on intact endothelium
- thrombomodulin binds thrombin and alters it’s function
- promotes cleavage of protein C into a functional protein C
Where is protein C located and
what is it’s function ?
- localized to the area of thrombomodulin
- connected by endothelial cell C protein
- activated C
- cleaves factor Va and VIIIa
- thus slowing clot formation
What are the protein C deficiencies?
- Type I deficiency (most common type)
- quantitative
- decreased protein with full function
- Type II deficiency
- qualitative
- dysfunctional
- Protein C deficiency is autosomal dominant
- rare homozygotes may present with neonatal purpura fulminans
Note: acquired protein C deficiency is much more common
What testing is performed to evaluate for
Protein C deficiency ?
- functional assays detect most types of Protein C deficiency
- 2 types of assays: clot based and chromogenic
- both use venom from the southern copperhead snake which activates protein C
- clot based tests (use either a PTT or Russell viper venom clotting time) suffer from lots of interference
- DOACs or lupus anticoagulants can cause false increases in protein C
- elevations of VIII and factor V leiden can falsely decrease protein C activity
- clot based tests (use either a PTT or Russell viper venom clotting time) suffer from lots of interference
- chromogenic assays are preferred due to the interferences
How does the chromogenic assay work
for the Protein C functional assay ?
- southern copperhead snake venom is added to the patient’s plasma
- this activates protein C
- a chromogenic protein C substrate is added and when cleaved emits a light detected by spectrophotometry
How do protein C levels vary in
certain normal conditions ?
- levels are significantly lower in neonates
- levels increase throughout childhood and may not reach final levels until age 30
- increase in normal pregnancy and postpartum
- in contrast to protein S which decreases
If low levels of protein C are found
on the chromogenic assay, what subsequent
test is performed ?
- immunoassay to determine levels
What can cause an
acquired protein C deficiency ?
- acute thrombosis
- cirrhosis cause reduced production
- DIC, with severe purpura fulminans in meningococcal disease
- Vitamin K or Warfarin
- Nephrotic syndrome
- L-asparaginase therapy
What types of VTE can
occur in protein C deficiency ?
- cerebral venous thrombosis
- splanchnic vein thrombosis
What are some important facts
about protein S ?
- synthesized in the liver, vitamin K dependent
- does not have direct enzymatic activity
- is a cofactor for protein C
- approximately 70% is bound in a 1:1 ratio to the complement regulatory protein C4b-binding protein
- 30% free protein S is responsible for most of the activity
- 3 types of deficiencies
- Type I- quantitative
- Type II- qualitative
- Type III- decreased free protein S antigen but normal total protein levels
How is protein S deficiency inherited ?
- autosomal dominant
- rare homozygotes get neonatal purpura fulminans
How is testing for protein S deficiency conducted?
- functional assay NOT recommended up front
- technically difficult
- functional protein S deficiencies are rare (type II)
- clot based assay technique
- DOACs and Lupus anticoagulants falsely increase protein S levels
- increased Factor VIIIa and Factor V leiden falsely decrease protein S levels
- also SUPER sensitive to pre-analytical variables (~10% of cases)
- preferred test is the quantitative test
How is the quantitative protein S test performed ?
- preferred initial evaluation
- both total protein S levels and free protein S levels are important
- total protein S levels should not be ordered in isolation because there is poor correlation with thrombosis
- IMP
- free protein S levels of < 33% correlate with VTE
- many patients have mutations in protein S
- free protein S levels of < 33% correlate with VTE
What are causes of acquired protein S
deficiency ?
- acute thrombosis
- cirrhosis
- pregnancy
- oral contraceptives
- DIC
- Vitamin K deficiency or Warfarin therapy
- nephrotic syndrome
- newborns (healthy) have 15-30% of adult levels of total protein S
- but C4b binding is also less so free protein levels are about the same
What are the most common presentations
of patients with protein S deficiency ?
- DVT and PE
- splanchnic vein thrombosis
What is the cause of Factor V Leiden?
- caused by a single nucleotide polymorphism in the factor V gene that changes arginine 506 to glutamine
- arginine 506 is the normal cleavage site for activated protein C
- mutation here leads to slower inactivation of Va
- thus there is more thrombin generation
What is the testing for Factor V leiden ?
- genetic testing for the mutation is standard
- can do a functional assay but not necessary because most cases are caused by the single mutation
What ethnicity is factor V Leiden most common ?
- European ancestry
- 4-5% of white individuals
- many are heterozygous for the mutation
- most patients never experience a VTE and of those that do fatal VTE is very rare
What clinical manifestations are seen
with Factor V leiden ?
- DVT and PE
- cerebral vein thrombosis
- portal/hepatic vein thrombosis
What is the prothrombotic mechanism
of the prothrombin mutation ?
what is the mutation ?
- mutation: G20210
- there is an increase in the prothrombin plasma levels
- increases PT mRNA and ultimately protein
- leads to increased thrombin formation
- enhanced tendency to clot
- test for by genetics
Why is testing for plasma PT
levels not recommended ?
- becuase the levels fluctuate throughout the day normally
Are patients with the PT gene mutation
at risk for recurrent VTE ?
- No
Which coagulation test do Lupus
anticoagulants affect more ?
- PTT more often affected
- PT less often affected because there is a higher amount of excess phospholipid in the test
What is the source of extra phospholipid
for determining lupus anticoagulantS?
- frozen washed platelets
- aka platelet neutralization procedure (PNP)
- purified hexagonal phase array phospholipids
- LA-insensitive phospholipid
Note: all of these are clot based assays
What factors can confound
LA testing ?
- anticoagulant medication
- factor deficiency
- specific factor inhibitor
What is the association between
increased factor VIII levels and VTE ?
- appear to increase VTE risk in a dose-dependent manner
- no definite cutoff that defines the disorder for how high the levels need to be
- independent risk factor for first unprovoaked DVT
What are common things that can
elevate factor VIII?
- aging
- acute phsyiologic stress (epinephrine)
- liver disease
- pregnancy
- possibly warfarin
Where is factor VIII produced ?
- made in specialized hepatic sinusoids
- IMP:
- most other factors are made in the hepatocytes themselves
- most factor VIII circulates noncovalently bound to von Willebrand factor (vWF)
- lots of variability in levels in different people
- 50% of variability may be related to blood type
- type O blood has lower levels of FVIII and vWF (due to faster clearance)
What are acquired causes of elevated
factor VIII ?
- acute thrombosis
- levels may remain high for up to 6 months
- Warfarin therapy
- cirrhosis
- normal aging
- pregnancy
- African American race
please see pg. 254 for a list of
minimal or null increase in thrombophilia
When should testing for protein C
deficiency occur ?
- delay testing for 4 weeks after discontinuing Warfarin
- delay testing for several days after factor Xa inhibitors
Note: elevated protein C levels are of no significance
How does heparin affect the clot-based
functional assay for Protein C activity ?
- it will overestimate protein C activity
- also seen with direct thrombin inhibitors or factor Xa anticoagulants
- a specific factor inhibitor
- or lupus anticoagulant
What is the type I protein S
deficiency?
- accounts for 80% of inherited forms
- concordant reductions of total protein S antigen, free protein S antigen and protein S activity
What is the type II protein S
deficiency ?
- due to protein S mutations that lead to a dysfunctional protein
- decreased protein S activity
- normal free protein
- normal total protein S antigen
What is type III protein S
deficiency ?
- debateable if this even exists
- low activity
- normal total S protein
- low free protein S antigen
Note:
- attributed to an increased binding affinity of protein S for C4bBP
- but it could just be seen due to age related increases in C4bBP
IMP: acquired protein S deficiency is much more common
Why do protein S activity and free protein S
antigen decrease with some combined
hormone OCPs and/or pregnancy ?
- usually do to increased hepatic synthesis of C4b binding protein
Where is antithrombin produced
and what is it’s function ?
- produced in the liver
- inactivates factor Xa and thrombin (factor IIa)
- accelarated by Heparin
- Type I mutation
- hyposynthesis
- antigen=activity
- Type II mutation
- dysnfunctional protein
- antigen > activity
- there are 3 subtypes
How is antithrombin affected by
hemodilution ?
- hemodilution from cardiopulmonary bypass reduces the response of antithrombin to heparin
How is antithrombin measured ?
- chromogenic assay
- in the presence of heparin (heparin cofactor activity)
- or absence of heparin (progressive antithrombin activity)
- use of bovine thrombin or human factor Xa is needed
- because they are not inactivated by Heparin cofactor II, which is another plasma protein with inhibitor activity
What are the advantages of the
heparin cofactor activity antithrombin assay ?
- sensitive for all types of antithrombin deficiency
- superior precision and accuracy
- lack of interference from all types of coagulation factor activities, lupus anticoagulants, or heparin
Because it is a measure of optical density things that interfere include:
- high concentrations of hemoglobin, bilirubin, and triglycerides
What has homocysteine been implicated as a
risk factor for?
- implicated as a risk factor for venous thrombosis, coronary artery disease, MI and stroke
- no mitigation in thrombosis risk by treating elevated homocysteine levels with vitamin B12 and B6
