Module 1 (1st week) Flashcards
Compare FV and FVIII structure
Both have A domains (A1, A2 then A3 after B dom.) (protein - protein interactions, 40% homology) Both have B domains (Similar in size but low sequence identity between FV & FVIII, both heavily glycosylated, inessential for FVIII function but not FV) C Domains (C1, C2) (used in PL binding) F8 has a domains,which are small acidic peptides, sulphated tyrosines, contains protein binding site for VWF (a3) and thrombin cleavage
FVIII binding points
A2 binds to Factor IXa
B domain for FX
FVIII and FV function
FV - PROTHROMBINASE complex
FVIII - INTRINSIC TENASE complex
Both on PL surface, use Ca2+
FV vs FVIII synthesis
FV- hepatocytes
F8 - EC + others
FV vs FVIII (any other comparisons e.g. plasma concn, binding partners, presence in locations)
FV: - Taken up by platelets (murine Mgk) - Plasma Concn 20nM, - 20% of total is in platelets - Some bound to TFPI in circulation FVIII: - Plasma concen of 0.4nM, - not present in platelets - Most bound to vwf in circulation
Role of vwf with F8
VWF protects VIII from degradation and prevents premature association with Factor X
Free FVIII half life ≈ 2 hours
VWF-bound FVIII half life≈ 12 hours
FVIII half-life is determined by VWF - implications
50x excess of FVIII binding sites in plasma D, D3
Half time for complex ≈ 2 seconds
FVIII activation releases it from VWF
FV and TFPI
A small (~20%) fraction of FV arises from an alternative splicing event, deleting the BR (B region) This ‘short’ FV binds TFPI with higher affinity than full length FV Excess ‘short FV’ arising from a point mutation elevates TFPI and causes bleeding. Coagulation factor VA2440G causes east Texas bleeding disorder via TFPIα (Vincent . J Clin Invest. 2013;123)
Km
is the dissociation constant of the enzyme-substrate complex, a measure of affinity
Kcat
the rate constant for the ES to EP reaction often referred to as the turnover number (the number of times the enzyme turns over/sec)
Specificity constant
kcat/Km, is known as the specificity constant and is an absolute measure of catalytic efficiency
Assumptions of the Michaelis-Menten model
1) steady state: [ES] constant, i.e. constant velocity (Briggs and Haldane, 1925)
2) [S]»_space; [E]
3) rate LIMITING step is disassociation of ES to E + P
What affects Km and Kcat
Surfaces bring reagents together.
• This reduces the substrate concentration for the reaction to occur (less reaction-by-chance)
•This reduces Km = increases affinity
•Shifts reaction curve left but same Vmax is reached
Cofactors cause conformational changes in substrates which make them more reactive
•This incr Kcat = incre Vmax
•This may also reduce Km = increased affinity
AN INCREASE IN ENZYME CONCENTRATION DOES NOT CHANGE Km- This change only increases Vmax hence the 1/2Vmax value is faster, but occurs at the same substrate concentration as before
Explain how cofactors act as enhancers using kcat/km terminology
Cofactors and surfaces act as enhancers and regulators making sure reactions happen in the right location.
5a is te cofactor for 10a and increases Kcat. Plt PL is the surface and reduced Km. (This reaction wouldn’t otherwise occur as Km would be too high sine PT conc is low in plasma)
Or, fibrinogen is cofactor for tPA and increases Kcat, and surface reduces Km.
For thrombin, heparan reduced Km but also increases Kcat slightly.
Relationship between vwf and abo?
- anti - A and anti -H can precipitate VWF
- ABH reactivity removed by endoglycosidase F
- ~13% of the N-sugar chains have H attached
- Platelet VWF does not have ABH antigens
- ABO blood groups determine vwf level (blood group O is lowest and AB highest) (Franchini, 2007)
FVL
Mutation in blood coagulation factor V associated with
resistance to activated protein C (Bertina et al. 1994)
APCR mostly caused by a single polymorphism in the factor V gene,
FV 1691 G to A, coding for 506Arg to Gln (factor V Leiden).
Population prevalence ~4% in the Dutch.
Other risks for thrombophilia
Standard thrombophilia testing identifies an important contributory factor in only approx. 50% of idiopathic thromboses How are the rest explained?
GWAS for thrombosis genome wide association analyses using genotype data imputed to ~2.5 million single nucleotide polymorphisms (SNPs) from adults with diagnosed VTE identifies F5 rs6025, ABO rs8176719 and rs2519093, and F2 rs1799963 polymorphisms. Heit 2012
Also there is a hyper-coagulability in NR (Butenas 1999)
Inactivation of FV vs FVL
Inactivation occurs on the surface of anionic phospholipid
vesicles, activated platelets and endothelial cell. Biphasic - Arg 506 is rapid cleavage (results in FVa with intermediate activity) and Arg 306 is slow cleavage and completely abolishes
FVL: The rapid cleavage at Arg506 is lost. Factor Va is still inactivated by the slow cleavage at Arg306
Secondary haemostasis
Initiation: Tf : FVIIa complex = Factors Xa, IXa & thrombin are formed Amplification: Small amounts of thrombin activate platelets and factors V, VIII and XI Propagation: Activated factor complexes form on platelet surface = Thrombin Burst (A Cell-based Model of Hemostasis, 2001)
PT
Extrinsic & Common Pathway factors Used to monitor Warfarin therapy (INR) Reference range: 9.0 – 12.0 seconds (INR is PT with reagent sensitivity corrected for) Massive RBC transfusion prolongs PT
APTT
Intrinsic & Common Pathway factors
Used to monitor heparin therapy APTT ratio
Reference range: 23.0 – 31.0 seconds
Severe Prekallikrein / HMWK deficiency prolongs APTT
Mixing studies
Patient plasma + Normal plasma (50:50)
Deficient factors replaced
Correction with mixing = Factor deficiency
Incomplete correction of PT/APTT = Inhibitors
How do the D Dimer Assay works?
Immunoturbidimetric method
Latex beads coated with antibody bind D-dimer
Cross-linking leads to increase in optical density
Increase in oD proportional to D-dimer concentration
28 y/o male – Polytrauma. RTC Bleeding evident at venepuncture sites PLT count: 38 x 109/L (Ref: 150 – 400) PT (s) 19.5. 9.0 – 12.0 s APTT (s) 50.2; 23.0 – 32.0 s Thrombin Time (s) 39.4; 15.0 – 19.0 s Fibrinogen (g/L) 0.7; 1.8 – 4.0 g/L
Prolonged PT and APTT
TT likely prolonged due to apparent hypofibrinogenaemia (Fib 0.7 g/L)
D-dimer raised due to increased fibrinolysis
Interpretation: Disseminated Intravascular Coagulation (DIC)
68 y/o female presents to A&E w/ suspected GI bleeding PT (s) 98.5; 9.0 – 12.0 s APTT (s) 45.7; 23.0 – 32.0 s Thrombin Time (s) 18.0; 15.0 – 19.0 s Fibrinogen (g/L) 2.4; 1.8 – 4.0 g/L INR 9.3s
warfarin overdose
8 y/o male presents with haemarthrosis
History of recurrent joint bleeds
APTT is only increased parameter
Haemophilia A or Type 3 VWD!!! don’t forget
A because its more common, but could be B
