Coagulation Flashcards
Why is FFP a poor choice for correcting mild INR abnormalities?
FFP itself has an INR > 1.0; mild INRs also do not predict significant bleeding.
What is the preferred choice to rapidly reverse warfarin effect?
4-factor prothrombin complex concentrate. Also add vitamin K.
What is a normal fibrinogen level? In a pregnant patient?
2-4g/dL»_space; 6g/dL in pregnancy.
Fibrin sealants
Topical treatments mostly for intraoperative use.
Downsides include antibody formation, viral transmission, and excessive clotting.
What drives coagulopathy in bypass circuits?
Contact with non-endothelial surfaces causes release of tissue factor (prothrombotic). Patients also acquire deficiencies of platelets, fibrinogen, fXIII and vWF (pro-bleeding) and must be on systemic anticoagulation.
What is the mainstay of anticoagulation in bypass, and how is it monitored?
Heparin; monitor with aPTT or anti-factor-Xa (note: need to ensure no acquired antithrombin deficiency!)
Thrombin-activatable fibrinolysis inhibitor (TAFI)
Endothelial protein that is activated by thrombin-thrombomodulin complex to cleave lysine residues from fibrin, making it less recognizable as a plasmin substrate.
PAI-1
Endothelial prothrombotic protein which inhibits plasminogen activator’s ability to activate plasmin.
Tissue factor pathway inhibitor (TFPI)
Endothelial antithrombotic protein. Partially secreted. Inhibits the extrinsic Tenase and prothrombinase but can be displaced by heparin.
Endothelial protein C receptor
Endothelial antithrombotic protein that facilitates the activation of protein C by thrombin.
PAR-1
Endothelial antithrombotic receptor that upregulates PGI2 and NO synthesis following cleavage activation by thrombin. Also releases tPA from Weibel-Palade bodies?
What complications are associated with use of topical bovine thrombin products?
Antibodies to thrombin but also factor V.
What coagulation factors are affected by use of oral contraceptives?
Factor VIII and von willebrand factor levels may increase wiht OCPs.
What isolated factor deficiencies may be ween with the lupus anticoagulant? With myelodysplastic or myeloproliferative disorders? Amyloidosis? Gaucher disease?
Lupus anticoagulant - Factor II
MDS, MPN - Factor V
Amyloidosis - Factor X
Gaucher disease - Factor XIT
Why do hemophilia A/B carriers often have lower levels of those factors?
Lyonization of X chromosomes.
Is PNH associated with bleeding or thrombotic complications?
Thrombotic; usually venous thrombi of visceral organs.
What pathophysiologic mechanisms cause acquired von willebrand disease?
Autoantibodies against vWF (as in lymphoma/myeloma), decreased production (as in hypothyroidism), or increased destruction (as in cardiac valve abnormalities)
What lab assays can (maybe) distinguish acquired from congenital von willebrand disease?
Von willebrand propeptide (decreased in congenital forms).
Ristocetin cofactor mixing studies (no correction upon mixing if inhibitor is present).
Pre-test risk assessments for HIT
HIT Expert Probability (HEP)
4T: Thrombocytopenia, Timing, Thrombosis, oTher causes
How can EIA specificity of HIT be improved?
Use IgG-specific test (IgA, IgM do not cause significant HIT).
Use confirmatory high-dose heparin second step (inhibition is confirmatory)
Use high OD cutoffs.
Rapid HIT immunoassays
“HemosIL” latex particle agglutination or chemiluminescence assays
“PaGIA” Particle gel immunoassay
Akers particle immunofiltration assay
Heparin-induced platelet aggregation
A disfavored confirmatory assay for diagnosis of HIT. Essentially platelet light transmission aggregometry; very labor intensive, but requires donor platelets and
How are argatroban and bivalirudin monitored?
aPTT
ACT
*Prone to underdosing because of nonlinearity of these tests.
What coagulation factors are merely cofactors? What do they do?
Factor V, VIII
Serve as serine protease binding sites to coordinate substrate docking.
What genetic polymorphisms can affect the metabolism and effect of warfarin?
CYP2C9 - Metabolism
Mutation in VKORC1 can affect response
What drive neonatal vitamin K deficiency, and how is it treated?
Lack of gut flora to produce vitamin K
Treat with prophylactic IM supplementation
What processes contribute to feedback amplification of the coagulation cascade?
Thrombin-based feedback activation of factor V, VIII, and XI. Polyphosphate secreted from activated platelets.
What is the structure of fibrinogen?
Dimer composed of three pairs of protein chains (Aa, Bb, and gamma). Can be divided into a central E globular domain flanked by two D globular domains.
What is the mechanism of action of factor XIIIa?
Crosslinks adjacent fibrin monomers in a covalent fashion through glutaminase function. Links E+D domains, but also D+D.
How does thrombin interact with platelets and endothelial cells?
Platelets: Activates PAR-1, PAR-4
Endothelium: Activates PAR-1, TAFI, and protein C (when bound to thrombomodulin).
What is EPCR?
Endothelial protein C receptor; an endothelial surface marker that concentrates protein C enough to be activated by the thrombin-thrombomodulin complex.
In what form is circulating protein S found?
40% unbound
60% bound to C4b
How does antithrombin prevent clotting?
Binds serine proteases (thrombin, but also many others) to form an inactive complex. It is potentiated by a heparin pentasaccharide.
What is heparin cofactor II?
Another serpin similar to antithrombin but more specific to inhibition of thrombin.
What are usual causes of pre-analytical errors in coag testing?
Specimen underfilling Hemolyzed/clotted specimen Old or mistimed specimen Improper transport Thrombophilia testing in acute setting or on anticoagulation...
What are the relevant binding domains on VWF?
A1 (binds platelets, collagen) and A3 (binds collagen), D’ (fVIII)
What is the ratio of vWF to fVIII binding?
50:1. Most fVIII is bound to vWF, but vWF is hardly saturated.
What platelet interactions dominate at high and low shear?
High-shear: Gp1b-VWF
Low-shear: Gp1a/IIa-Collagen
What compounds are in alpha granules?
VWF, fV, fVIII, fXIII, fibrinogen, fibronectin, P-selectin, GpIIb-IIIIa, PDGF
What compounds are in dense granules
ADP/ATP, Ca2+, Mg2+, polyphosphate, histamine, serotonin
How do red cells facilitate hemostasis?
Improve plt-endothelial interactions, contribute to hemostatic plug, increase blood viscosity (and by extension, shear force), secrete ADP
What is the cutoff value of VWF antigen used to diagnose VWD?
<30%; although normal range is generally 50-200%, some group O patients will naturally fall under 50%.
What conditions can increase von willebrand levels?
Pregnancy
Acute phase reaction
Non-white race
How is VWF RCO testing performed?
Mix PPP with REAGENT plts and add ristocetin to mimic shear force function (binding of Gp1b to vWF)
What is the “VWF functional antibody”?
Antibody against A1 domain. Note that the antigen tested in VWF antigen levels is a structural, not binding domain.
How is VWF Collagen binding testing performed?
Benchtop ELISA using microtiter wells coated with collagen. Note, same method applies to Gp1b bnding.
What is the benefit of a VWF propeptide test?
Propeptide is a cleaved byproduct of mature vWF. It is cleared more quickly than vWF. In patients with Type 1 VWD due to increased clearance (1C, Vicenza) the pp:vWF ratio will be increased.
What VWF test is useful to detect Type 2N vWF?
VWF:FVIII binding assay
Describe the utility of genetic von willebrand disease testing?
Challenging to sequence due to size and presence of a pseudogene.
Large deletions predict a higher rate of inhibitors or analphylactic reactions.
To what conditions is the PFA-100 sensitive?
Anemia (affected by hematocrit)
Thrombocytopenia (need >100k/uL to use PFA)
Qualitative defects (duh)
VWD (normal study rules out VWD?)
Compare the lumiaggregometry profiles of BS and GT.
BS: Responds to all agonists except Ristocetin.
GT: Responds only to ristocetin agonists.
How does the VerifyNow work? What testing cartridges exist?
Fibrinogen coated beads are aggregated by platelets that are activated by either AA (to detect ASA), ADP (to detect P2Y12 inhibs), or TRAP (to detect IIb/IIIa inhibs).
What is the use of electron microscopy in coag testing?
For assessing granule (particularly dense granule) structure.
What NON-COAGULATION-FACTOR targets are activated by thrombin?
Protein C, platelet activating receptor (PAR), TAFI. tPA release?
What are the longest and shortest lived coagulation factors?
Longest: Factor XIII (144hrs)
Shortest: Factors VII (6hrs), VIII (12hrs)
What is the function of these domains in coagulation factors: Kringle, Discoidin, GLA, Kunitz
Kringle: Bind positively-charged residues (especially in fibrin[ogen])
GLA: Binds calcium, requires VKORC
Discoidin: Found on cofactors V/VIII
Kunitz: Protease inhibitors, for feedback inhibition and found on SERPINs
How do the extrinsic and intrinsic pathways actually probably contribute to clotting in vivo?
Extrinsic is usually the spark (note that VIIa is rapidly shut down by TFPI), then the intrinsic feeds forward the cascade.
What is the “Josso loop”?
The extrinsic Tenase can also activate factor IX, promoting the intrinsic Tenase.
What are the five components of the tenase or prothrombinase?
Enzyme Cofactor Calcium Substrate zymogen Negatively-charged surface
What is the relative affinity of antithrombin for activated coagulation factors?
IIa, Xa > XIIa, XIa, IXa
How are different detection methods of PT/aPTT affected by H/I/L?
Optical detection methods are affected by all 3 in the usual fashion.
Mechanical detection methods are affected by hemolysis (releases phospholipid) and lipemia (activates factor VII?).
What are the Quick and Owren methods?
Methods of PT; Quick method is common and uses a 1:3 dilution of plasma to buffer. Owren method is a 1:21 dilution which uses bovine reagent and is insensitive to factor V and I levels.
Batroxobin time
AKA reptilase time, directly cleaves fibrinogen and so is unaffected by direct thrombin inhibitors. Note, only generates fibrinopeptide A.
For what factors do chromogenic assays exist?
8, 9, 10
What is a Bethesda unit? How is it calculated from the residual activity level?
The amount of inhibitor needed to reduce activity level by half. BU = (2-log[residual]/.301)
How are protein C levels measured?
Activate plasma with protac venom, mix with C-deficient plasma, and compare to a standard curve. A chomogenic protac venom assay also exists.
How are protein S levels measured?
Add APC to patient plasma (and S-deficient plasma) and compare the rate of prolongation of aPTT to a standard curve.
How are antithrombin levels measured?
Add heparin and activated Xa or IIa to PPP with a chromogenic substrate. Compare rate to a standard curve.
What is the function of factor XIII and where is it found?
It crosslinks fibrin D-domains and also incorporates alpha-2 antiplasmin into the clot. It is found half-bound to fibrinogen, and half in platelet alpha granules (and cytoplasm?)
What is the structure of factor XIII?
Composed of subunit A (the transglutaminase) which is made in megakaryocytes, and subunit B (the zymogen) which is made by hepatocytes
How does t-PA promote degradation of fibrin(ogen)? How do most antifibrinolytics work?
Binds fibrin surface using kringle domains (at lysine residues) to attract and activate plasminogen. Most antifibrinolytics work by mimicing soluble lysine analogues.
What proteins regulate plasmin activity?
Alpha-2 antiplasmin (inhibits plasmin at clot)
PAI-1 (inhibits tPA)
TAFI (cleaved by thrombin»_space; cleaves lysine domains)
Urea clot lysis
Form clot, try to dissolve in 5M urea. A surrogate marker of factor XIII activity which is not recommended by ISTH.
How are factor XIII levels detected?
Urea clot lysis (old, irrelevant)
Antigenic assays
Functional assays (detect ammonia release)
What is a D-dimer? What are the units?
Soluble fibrin fragment which was cross-linked by fXIIIa. A sensitive but nonspecific assay. Can be reported as 1 FEU (1 of which equals 0.5 DDU).
Euglobulin clot lysis
Form euglobulin fraction (which lacks serpins) by using acetate. If clot lyses in <120min, suggests hyperfibrinolysis.
How is alpha-2 antiplasmin measured?
Add pt plasma and plasmin to a normal sample, measure degree of inhibition and compare to standard curve.
What is the effect of anemia on VET?
Actually improves clotting MA/MCF on TEG/ROTEM (RBCs disrupt plt-fibrinogen interactions)
How can clot retraction be ruled in on ROTEM?
Use aprotinin (stops fibrinolysis)
Activated Clotting Time
Whole blood POC test using a contact activator (kaolin, celite). Variable and unreliable but reportable over high levels of UFH»_space; used in bypass
Tachyphylaxis
Rapid resistance to treatment, applicable to von willebrand disease response to DDAVP
How should Type 1 VWD ideally respond to DDAVP challenge?
Expect threefold increase in VWF levels.
How is von willebrand factor dosed?
No agreed upon dosing level. Aim for a minimum activity level of 30%, up to 80% for major surgeries. Example loading dose 30-60 U/kg.
Why is it not recommended to prophylactically transfused qualitative platelet disorders?
Many will be prone to developing antibodies against missing antigens (eg, BSS»_space; Gp1b antibodies).
What are the lab features of BSS?
Macrothrombocytopenia
Lack of activation with ristocetin
Absent Gp1b on flow
What are the lab features of Glanzmann’s thrombasthenia?
Lack of activation with all agonists except ristocetin
Resembles afibrinogenemia, but fails to correct with PNP mix?
Grey platelet syndrome
No alpha granules (compare with dense granule deficiency).
Genetic thrombocytopenias
MYH9
MPL/MECOM
RUNX1/ANKRD26/ETV6
What are some causes of acquired von willebrand disease?
Inhibition by paraprotein Clearance by autoantibody Neoplastic clearance (ie, essential thrombocythemia) Shear stress (defects, cardiac hardware) Hypothyroidism (decreased synthesis)
How does uremia cause bleeding? How can it be treated?
Causes abnormalities of the von willebrand factor multimers. Treat with cryo/desmopressin, estrogens, and maintaining hematocrit above 30%. IE, not platelet transfusion.
What severity of hemophilia is most common?
Severe (<1% factor activity)
How many cases of hemophilia are de novo?
1/3
What is the goal activity level desired with factor repletion in hemophilia?
30%
How are factors VIII and IX dosed?
1U fVIII per kilogram»_space; +2% activity level
1U fIX per kilogram»_space; +1% activity level
How are severe hemophilias with inhibitors treated?
Bypass agents (FEIBA, NovoSeven) Emicizumab (do not mix with FEIBA) (porcine factors may be less cross-reactive)
How do hemophilia A and B differ clinically?
A is more common, and is generally more severe (arthropathy).
DDAVP is not an option in hemophilia B.
Factor IX dosing is 2x that of factor VIII.
Hemophilia B has fewer inhibitors, but patients may have anaphylactic reactions.
How is RiaSTAP dosed?
Divide (difference in desired level) by 1.7 mg/dL per mg/kg bodyweight. Just, divide by 1.7
What treatments exist for deficiencies of…
Factor V
Factor XI
Factor XIII
fV: FFP, plts (contain fV)
fXI: FFP only (EU has concentrate available)
fXIII: Plasma-derived (Corifact) or recombinant (novo13)
How does liver disease cause thrombocytopenia?
Splenism due to portal hypertension
Decreased liver production of TPO
Direct bone marrow suppression by EtOH
How is coagulopathy of liver disease managed perioperatively?
Correct vitamin K deficiency pre-op.
Transfuse to TEG, not PT/aPTT.
Consider antifibrinolytics.
What are some general transfusion goals in DIC?
Plt: 30k/uL
PT: 150mg/dL
How does hypothermia affect coag factor function?
-10% factor activity per -1 degree C
How does acquired factor VIII inhibitor present?
Usually elderly patient with severe subcutaneous, muscle, or GI bleeding. Not hemearthrosis!
How rapidly is heparin cleared?
During the initial phase, it is cleared rapidly by endothelium with a half-life of 1.5hrs.
Once endothelium is saturated, half-life extends to 4-6hrs depending on renal function
How do LMWH and fondaparinux compare to UFH?
More Xa activity, less IIa activity. Cannot be monitored with aPTT (use anti-Xa for LMWH; no consensus for fondaparinux).
What is the purpose of the heparin bridge when starting warfarin therapy?
To cover for prothrombotic state caused by loss of protein C and S (VKORC-dependent, first to go)
How are DTIs metabolized?
Argatroban is hepatically metabolized
Dabigatran and bivalirudin are renally excreted.
How are DTIs monitored?
Can follow aPTT, ACT, or DTT. Ecarin clotting time can be used for Dabigatran.
What are the antidotes for DOACs and DTIs?
DOACs: Adnexanet-alfa, 4F-PCCs
Dabigatran: Idaricizumab, FEIBA
What isoforms of COX does aspirin block?
At low dose, blocks COX-1 (in plts)
At high dose, blocks COX-2 (inflammatory/pain)
Compare the thienopyridines antiplatelet drugs
Plavix: Slow, irreversible, prone to polymorphisms in metabolism
Prasugrel: Faster, no resistances from cytochromes
Ticagrelor, cangrelor: Fast and reversible.
