Module 1 - 2/3/4th week mixed Flashcards
Definition and classification of APLS
APS is an AI disease, where Abs react with phospholipids/ CSM glycoproteins, leading to in-vivo increased risk of arterial & venous thrombosis
•primary APS occurs in the abscence of other AI disease
•secondary APS occurs in conjunction with diseases eg SLE, RA, scleroderma etc
•catastrophic APS is a very rare variant which causes multi organ thrombosis and is always fatal
What antibodies are present in APLS?
Lupus anticoagulant antibodies, anti-cardiolipin antibodies and other APS Abs target lipoprotein surface antigens and components of the coagulation/fibrinolytic systems.
APLS criteria
The revised Sapporo classification diagnoses APS when one clinical and one laboratory finding are met, between 12weeks and 5years of each other.
Clinical criteria for APL
Vascular events – a history of one or more arterial/venous thrombosis episodes, objectively diagnosed by imagining, with no evidence of vessel wall inflammation
Obstetric events – requires only one of the following:
o >3 spontaneous and unexplained abortions prior to
10weeks gestation
o >1 miscarriage beyond 10weeks gestation
o >1 premature birth due to pre-eclampsia, eclampsia
or placental insufficiency
Lab criteria for APL
- ELISA to detect anti-cardiolipin antibodies at medium or high titres, at least twice in more than a 12 weeks period
o illness etc can induce transient APS Abs production
in normal people - Prolonged coagulation in dilute Russell’s viper venom time demonstrates lupus anticoagulant only when
o normal plasma addition does not correct the
prolongation
o addition of phospholipids (dilution of antibody) does
correct the prolongation
Causes of placental insufficiency?
- placental insufficiency can be due to HTN/ T2DM
* in ACS, placental insufficiency leads to intra-uterine growth restriction
Common clinical presentation of placental insufficiency
•vascular
o VTE prevalence is roughly 30%
o recurrence rate of untreated patients is 10%; this
carries a higher mortality rate
o cerebral thrombosis is most common arterial
thrombosis event
o digital necrosis and gangrene may also be seen
• cardiac – valvular heart disease
• neuro – TIA/stroke, vascular dementia
• skin – livedo reticularis (rash that appears reticular) (purple pearls)
• renal – vasculopathies → chronic renal ischaemia
rare clinical findings in APLS
Thrombocytopenia (splenic sequestration following Abs binding to Plt PL)
pulmonary HTN
peripheral thrombosis
catastrophic APS
APLS treatment
- heparin and low dose aspirin are being used as prophylaxis to prevent (recurrent) miscarriages
- steroids should only be used in treatment of coexisting AI disease, not to treat APS (side effects).
What is APLS associated with?
intra-uterine growth restriction, premature births, HELLP syndrome (haemolysis, eleveated liver enzymes, low platelets)
VTE & PE similarities
- Identical pathophysiology
- incr thrombus size leads to destruction of venous valves
- destruction of venous valves gives rise to post-phlebitic
syndrome
- predisposing Rf leads to thrombus formation in veins
thrombi form legs/pelvis embolise to pulmon. circulation
- 90% of PEs attributable to leg thrombi; 10% pelvic
about 50% of people with DVT develop a subsequent PE - Similar risk factors
pregnancy, HRT, orthopaedic surgery, thrombophilia, flights etc (Basically, Virchow’s triad) - Identical therapeutic goals
- Similar treatment strategies
Symptoms and signs of DVT /PE
symptoms of inflammation
pain, swelling, redness, warmth
unilateral signs
leg – pitting oedema, tenderness, muscle induration
lung – pleuritic chest pain, haemoptysis
systemic upset – pyrexia, tachycardia
Simplified wells score for DVT
ONE POINT: active cancer, paralysis, bed>3d, surgery within 4wk, vein tendereness, swollen leg, calf swollen>3cm, pitting oedema, collateral veins, previous DVT
Alternative diagnosis -2
Low risk/unlikely <1 Moderate 1-2 High/likely >2
Protocol for wells score showing high risk pnt
if someone has a high risk from history, negative d-dimer does not mean no DVT. high risk patients should always have an ultrasound
Protocol for wells score showing low risk pnt
If -ve d-dimer, probably don’t have a DVT because d-dimers have a high negative predictive value
If +ve d dimer but -ve US, probably confirms the diagnosis; but ultrasound is only sensitive 97% of the time – they may still require venogram/serial ultrasound within 1 week if the history points to very high risk.
Why are venograms are rarely used these days in DVT?
invasive, expensive, dangerous contrast mediated effects on kidney (especially in HTN, T2DM)
reserved for people with high risk from history and normal USS – this may suggest recurrent DVT (which USS is poor at picking up)
o recurrence is diagnosed if vessel diameter has enlarged by 2mm or more from previous venogram
Simplified wells score for PE
DVT, tachy >100, immobility or surgery 4 weeks ago, previous VTE, haemoptysis, Ca, most likely than alternative diagnoses = 1 point each. PE is deemed unlikely if score is <1.
What is the diagnostic ability of a v/q scan?
- V/Q is only diagnostic in 30% of cases, but it should be used before CTPA in the interest of decreasing radiation exposure
- V/Q may be influenced by any coexisting lung disease
What is the test of choice in a massive PE?
TTE
• you are looking for signs of RV stress/dilation in the emergency situation with TTE
• then confirm diagnosis with VQ of spiral CT
• avoid CTPA because it increases risk of major bleeding in massive PE patients
Other investigations for PE (explain each one)
ABG
ECG
Potential targets for new anticoagulants
o TFPI/PC/APC analogues
o f9, f10a, thrombin, tafi inhibitors
o rThrombomodulin
Requirements for new anticoagulants
efficacy – proven in arterial/venous thrombosis
safety – wide therapeutic window, low bleeding risk,
predictable response, antidote presence
convenience – oral, fixed dosage, few interactions, rapid on-&offset of action, no need for monitoring
Comparin warfarin, UH and LMWH
UFH and LMWH have rapid onset (warfarin does not)
UFH has rapid offset
LMWH has the most predictable response and limited interactions.
However, UFH and warfarin have antidotes but LMWH doesn’t.
The only unique advantage of warfarin is that its oral.
They all cost alot.
UH vs LMWH
UH - IV, higher incidence of HIT. LMWH - SC,
Give some examples of new anticoagulant agents in clinical practice
Synthetic pentasaccharides,
F10a inhibtors
Thrombin inhibitos
PC analogues
Give example of anticoagulants that shares the same pentasaccharide sequence as UFH and LMWH for the binding to antithrombin?
Fondaparinux
• vte prevention
• but requires parenteral (SC) admin
• excreted by kidney so cant be used in renal failure
• not reversible by protamine, but does NOT cause HIT (as with UFH / LMWH)
idraparinux
• longer half life than fondsparinux (designed to be injected weekly instead of daily)
• Its chemically related to heparin. Its a factor Xa inhibitor & binds antithrombin and accelerates its inhibition of factor Xa.
Give an example of novel f10inhibitor and thrombin inhibitor
rivaroxaban
• licensed for orthopaedic vte prophylaxis, stroke prophylaxis in AF •no antidote, but annexa 4 trials have shown promising for andexanet
dabigatran
• licensed for orthopaedic vte prophylaxis, stroke prophylaxis in AF
Give an example of a novel protein C analogues
drotrecogin-a is rAPC – used in sepsis that causes multiple organ failure but very expensive
Has been taken off market as DrotAA did not significantly reduce mortality at 28 or 90 days, as compared with placebo, in patients with septic shock. (PROWESS SHOCK study).
Compare time to peak, half life and bioavail of dabigatran, rivaroxaban, warfarin and enoxaparin
Time to peak + Half life dabig - 2-3 hrs, 8-17hrs riv - 3, 7-11 hrs enox 3-5hrs, 4.5 warf is 120, 36-48hrs apix - 3, 7-11
Bioavail (riv 80-100%, warf 100%, apix 50% enox is 92%, dabig is 6.5%)
Which novel anticoagulants are affected by renal dysfunction? Hepatic dysfunction?
renal dysfunction - dabg, riv and enox
affected hepatic - riv and warfarin
Protein binding abiity of the anticoagulants?
riv and warfarin high (95 and 99%)
Enox is 80%
Dabig is low at 25-30%
Dabigatran and warfarin reversal
Idarucizumab (Reverse-AD trial)
Humanized monoclonal antibody fragment (Fab) that binds to dabigatran and its acylglucuronide metabolites
Potent binding affinity ~350x higher than binding of dabigatran to thrombin
No direct procoagulant effect
IV administration with immediate onset of action
Warfarin:
Either wait it out (72 hours)
Or FFP (all factors) PCC (2,7, 9, 10) + vitamin K
Current reversal options for direct factor Xa inhibitors
PCC/activated PCC, 50u/kg, and andexanet reverses rivaroxaban’s & appix (not yet approved in EU)
Andexanet alfa moa
It acts as a decoy and binds to factor Xa inhibitors, thus neutralizing the anticoagulant effects of factor Xa inhibitors by preventing the inhibitors from binding to endogenous factor Xa
Effective haemostasis achieved in 83% of patients (Connolly SJ et al)
ANNEXA-4 clinical trial
On the basis of a preliminary analysis, an initial bolus and subsequent 2-hour infusion of andexanet substantially reduced anti–factor Xa activity in patients with acute major bleeding associated with factor Xa inhibitors, with effective hemostasis occurring in 79%.
Which antidotes are in development
Ciraparantag (formerly aripazine)
Small molecule that may reverse anticoagulants
Binds to UFH, LMWH, fondaparinux and DOACs
Snake venom phospholipases
A2 (PLA2)
Specific, non-competitive blood coagulation inhibitors that bind to human FXa
Why are the traditional ‘reductive’ methods of assessing haemostatic function limited?
about 50% of patients with thrombosis do not have a specific deficit
•The pros of the reductive approach are specificity of diagnosis and treatment that is directly targeted to a deficiency
•traditional methods are less attractive for complex diseases – leading to increased popularity of global assessments of haemostasis
•global assessments take into account that coagulation is an integrated system
What are the issues with global assessments of haemostatic function?
Some global assessments of haemostasis are not specific = high false positive rate
Antibodies to measure enzyme-activation markers are not in general use
•Activation peptides [these are released on zymogen activation] and enzyme inhibitor complexes [the intermediate] can both be measured
o it is a difficult process, has limited sensitivity in
thrombophilia and is largely ineffective in
predicting thrombosis – not in general use
What can be used to measure coagulation potential
thromboelastogram/ROTEM →
o involves measuring changes in clot strength on a blood sample exposed to celite
o does not give a specific diagnosis but gives idea of coagulation and fibrinolyutic ‘strength’
endogenous thrombin potential
o Uses TF to give real time thrombin generation using a fluorescent substrate on platelet rich/poor plasma
o poorly standardised so it is primarily a research tool at the moment
o may have future roles in assessing responses to therapies
What measures of primary haemostasis only?
PFA/bleeding time
VTE risk in cancer patients?
• There is a 4-7x increased risk of VTE in cancer patients
• VTE is the second biggest killer in cancer patients; 1 in 7 cancer px die of a PE
o the longer the time between cancer and VTE diagnosis (presumably because of undetected, asymptomatic disease) –t he greater the risk of dying
(Idiopathic VTE may be a predictor of occult malignancy)
Other VTE risk factor
Other VTE risk factors are age, stasis/surgery, family history of VTE, varicose veins, CHF/MI/Stroke, lower leg fractures, OCP/HRT/pregnancy
Which cancers have highest, intermediate and baseline risk of vte?
Highest (>25x RR) pancreas, lymphoma, brain
Intermediate (>17x) leukaemia, liver, cervical/uterus
baseline (lung, colon, breast, ovary, prostate)
What else increases the vte risk in cancer specifically?
Anti-tumour therapy may play a role in increasing VTE risk too
o Chemo (eg thalidomide and Lenolidomide in MM)
o hormones
o surgery/immobility
What is the pathogenesis of vte in cancer?
The pathogenesis of VTE in cancer is complex and probably poorly understood:
• It is possible that malignant cells induce monocytes/macrophages to produce TF
• Worth noting that cancer patients can often have other co-exisiting thrombophilias eg F5 leiden
• some tumours eg adenocarcinoma produce pro-thrombotic mucin
What is the management/secondary prophylaxis of VTE in cancer?
- Usual management of VTE in cancer involved 5-7days LMWH until INR reaches 2 and then warfarin for 3-6months as secondary prophylaxis
- The CLOT study 2003 suggested long term LMWH is associated with decreased risk of VTE recurrence with no change in bleeding or mortality
Thrombosis phenotypic autoimmune diseases
TTP (ADAMTS13)
HITT (heparin-pF4)
APS (PT)
APS (beta2GP1)
TTP
ADAMTS13 antibody, so vwf isn’t cleaved, vwf multimers remain huge, platelets pile on top of each other and aren’t restricted -> microthrombi & platelet depletion (the fibrin mesh of these platelets cause MAHA)
What is secondary TTP?
secondary TTP is a thrombotic microangiopathy with a predisposing condition; eg:
o cancer, sepsis/ HIV infection, pregnancy, pill, eclampsia
o drugs eg Ticlopidine, clopidogrel, quinine, ciclosporin, mitomycin C, pentostatin
HUS
thrombotic microangiopathy with acute loiguric renal failure and sparing of the other organs – shiga toxin from E-coli seems to be causative
Characterised by low RBC, AKI, and low platelets.
Bleeding phenotypic autoimmune diseases
ITP (platelet targetted) acq Haem A (F8c) acq VWD (vwf) acq F13 def (f13) acq factor def (f9/10/11c) acq Glanzmanns (GP2b3a)
Thrombosis phenotypic autoimmune diseases
TTP (ADAMTS13)
HITT (heparin-pF4)
APS (PT)
APS (beta2GP1)
TTP
ADAMTS13 antibody, so vwf isn’t cleaved, higher proporation of big vwf multimers, platelet over @, lots of clots platelets depleted -> microthrombi
What is secondary TTP?
secondary TTP is a thrombotic microangiopathy with a predisposing condition; eg:
o cancer, sepsis/ HIV infection, pregnancy, pill, eclampsia
o drugs eg Ticlopidine, clopidogrel, quinine, ciclosporin, mitomycin C, pentostatin
HUS
thrombotic microangiopathy with acute loiguric renal failure and sparing of the other organs – shiga toxin from E-coli seems to be causative
Characterised by low RBC, AKI, and low platelets.
Antiphospholipid reference
(Cervera R, 2015)
Prospective study of 1000 APS pts investigating morbi-mortality during a 10-year-follow-up period and to compare the frequency of early manifestations with those that appeared later.
The most common thrombotic events were strokes, TIAs, DVT and PE.
The most common obstetric complication in pregnant APS pts was early pregnancy loss (16.5% of the pregnancies). IUGR (26.3% of the total live births) and prematurity (48.2%) were the most frequent fetal morbidities.
9.3% patients died and the most frequent causes of death were severe thrombosis (36.5%) and infections (26.9%).
0.9% cases of catastrophic APS occurred and (55.6% of those died.
The survival probability at 10 years was 90.7%.
HELLP and APL syndrome associated? What is HELLP
(HELLP) syndrome is a thrombotic microangiopathy complicating pregnancy and shares many clinical and biological features with thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS). Thrombotic microangiopathy is also a pathological feature of catastrophic antiphospholipid syndrome (CAPS). An association between refractory HELLP syndrome and antiphospholipid syndrome (APS) has been reported in a few cases
WPB, what are they, what do they do, what do they contain?
WPB found only in endothelial cells
VWF is required for WPB formation (Specifically the propeptide)
WPB contain HMW VWF
Also contain P-selectin, CD63, IL-8, Ang2, OPG, Endothelin, FVIII
Remember this is the PRIMARY SOURCE of plasma vwf. Platelet vwf does not contribute to plasma vwf levels, but it can contribute
Describe how thrombin/other substances can cause decreases barrier integrity of the endothelium
Thrombin, TNFα, histamine and hypoxia > activate RhoA GTPases > form actin via Formin enzyme and activates myosin II via ROCK > increase assembly and contraction of actomyosin filaments > barrier dysfunction
The risk of VTE and other pathologies when using COCP
Compare this to HRT
~3 fold increase risk of VTE in women <30
•3/10,000 vs 1/10,000
o~5 fold increase risk in women 30-40
•10/10,000 vs 2/10,000
This trend continues with age due to additive/synergistic effects of age + OCP
o 5 fold increase risk of MI
o 4 fold increase risk of PVD
o 3 fold increase risk of stroke
thus PREVIOUS VTE = ABSOLUTE CONTRAINDICATION
Fort HRT: Increases: CHD x 7, Stroke x 8, PE x 8 & Invasive breast carcinoma x8.
When is the VTE risk greatest whe using COCP?
The increased risk of VTE is apparent after 4 months of taking the OCP and is greatest in the first 6-12months of use, particularly among first time users Poulter 1996 and Suissa 2000
VTE risk in pregnancy
VTE risk in pregnancy however is 29/10,000 during the pregnancy and 300/10,000 post-partum. This is far greater than any VTE risk on the COCP.
Compare HRT to COCP
Lower RR, but higher baseline, so higher absolute
Baseline: 10 in 10K
RR: 2-3x
Absolute risk: 20-30 in 10K
(OCP Baseline R: 1 in 10K, RR: 3x)
But OCP has more of a synergistic effect with FVL
RR of VTE is 3.2 on HRT, 3.9 with FVL and 15.5 on both. (Rosendaal et al, 2002)
RR of VTE is 3.7 on OC and 34.7 on OC & FVL. (Rosendaal et al, 2002
Activation of FV vs FVIII
FV
Thrombin cleaves 3 Arginine (Arg 709, 1018 and 1545) residues removing the B-domain as two separate polypeptides producing active FVa Mann 2002
o This occurs in a hierarchical order with cleavage of
Arg 709 occurring first
o Cleavage of Arg 1545 is required for full functionality as shown by tests involving Russell’s Viper Venom whereby cleavage of Arg 1545 alone was sufficient in producing full FV activity, Kane 1981
FVIII
Thrombin cleaves 3 Arg residues within the acidic peptides forming FVIIIa (Arg 372 within a1, 740 within a2 and 1689 within a3) Eaton 1986
o Removal of a3 reduces affinity for VWF causing dissociation of FVIIIa Hamer 1987
o Conformational change allows C2 to bind phospholipid and A2 to bind FIXa to form Intrinsic Xase
Similarities in the role of B domain in FV and FVIII
- Heavily glycosylated and crucial for export of both FV and FVIII. Heavily NLG and OLG in the B-domain which is crucial for export. It binds export proteins LMAN 1 and 2 required for transport from the ER to the Golgi for secretion
- Rare mutation in LMAN 1 or 2 causes FV AND FVIII
deficiency - Inhibition of NLG by tunicamycin dramatically reduces
secretion Pittman 1994 - FV & FVIII lacking B-domain are transcripted and
translated more efficiently but secreted less efficiently
o Also studies have reported than the mannose-containing NLG of the B-domain in FVIII interact with calnexin and calreticulin and are thus important in ensuring correct protein folding, Pipe 1998
o Carbohydrate moieties on FV protect it from APC degradation, Fernandez 1997
- Rare mutation in LMAN 1 or 2 causes FV AND FVIII
Dissimilarities in the role of B domain in FV and FVIII
Necessary for FV function NOT FVIII function
• Newly synthesised FV circulates, as a full-length protein which cannot interact with FXa until cleavage by thrombin. Cleavage by thrombin removes the B-domain. This allows for FXa interaction because the B domain blocks FV interaction with FXa. This ensures that circulating FV is not constantly stimulating @ of FXa and therefore thrombin generation.
FVIII: B-domain for enhanced cellular transport and inhibition of transcription only:
• Newly synthesised FVIII is immediately broken down into a heterodimer. This is cleaved by thrombin forming a heterotrimer, FVIIIa. The heterotrimer is not stable and it either spontaenously dissociates or is cleaved by APC.
• B-domain deleted FVIII has full cofactor function, indicating that the B-domain is not important for cofactor function in FVIII; instead, it is only for cellular transport and inhibiting transcription.
FV vs FVIII synthesis
FVIII:
• encoded on X chromosome
• synthesised in EC of liver
• almost entirely bound to vWF in circulation through a3.
oVWF thus determines the HL of circulating FVIII
(2hrs without, 12hrs with VWF)
oBlood group O patients have 25% less VWF and thus lower levels of FVIII- Increased bleeding risk but lower thrombotic risk
FV:
•encoded on chr1
•synthesised in hepatocytes
•Circulates with around 20% bound to TFPI (as a short form).
o People with Factor V deficiency also have lower TFPI and thus the coagulant and anti-coagulant properties tend to balance out Duckers et al 2008
