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.
