Module 1- 2nd week Flashcards
Hereditary platelets Protein C Haemophilia
Triggers for platelet synthesis
IL3, IL6 and thrombopoetin stimulate megakaryocytes in BM to release plt
In inflammatory thrombopoiesis, IL6 stimulates thrombopoiesis through TPO (Kaser et al. 2001)
IL3 and IL6 alone has a small effect on platelet production but together they have a synergistic effect (Carrington 1991)
Platelet facts (life span, count, what is contains, what it looks like)
Smallest cells in peripheral blood
Discus shaped
Membrane contains openings connecting with canalicular system
Contain granules and coagulation proteins essential for haemostasis
Normal lifespan = 7-10 days
Normal count = 150-450 x 109/l
On blood film, they’re small, anuclear and have azurophillic granules.
Plt granule contents
Alpha - vwf, glp, coag factors
Delta - ADP, serotonin, calcium etc
Plt receptors + their substrates
GPIIb-IIIa complex, is present only on the plts and is most abundant plts adhesion receptor- binds to fibrinogen.
Upon plt @, αIIbβ3 is transformed from low to high affinity state for attachment with its extra cellular ligands and promotes platelet aggregation (Inside out signalling)
GPIb-IX-Vcomplex is the 2nd most common platelet receptor, vwf and thrombin binds to it
PAR1 and PAR4- thrombin binds to this and @plt
α2β1 receptor or GPIa-IIa receptor for collagen
P2Y12 - ADP bind to these receptors
TP- TXA2
(Saboor et al., 2013) (Rivera 2009)
Clinical picture of platelet disorders
Muco-cutaneous bleeding* in the history is suggestive of platelet disorder
• spontaneous skin petechiae/ purpura
• bleeding from mucous membranes, epistaxes
• prolonged bleeding after trauma/surgery
• bad menorrhagia at menarche for girls
• family history
TAR vs CAMT
- CAMT - Congen amegakaryocytic thrombocytopenia
TAR have normal TPO and TPO receptor
CAMT has mutations in TPO receptor genes and as a result has a v high plasma TPO - After the first year of life patients with TAR often improve heamatologically, but those with CAMT often develop bi- or trilineage marrow failure
- CAMT has mixed inheritance pattern, TAR is AR
- CAMT 30-40% have orthopaedic or neuro anomalies, TAR 100% have absent radii and no neuro issues, 15-22% have cardiac abnormalities (usually septal defect) (Hedberg and Lipton 1988)
- Both have absent megakaryocytes in BM
- CAMT Platelets < 20 from birth, TAR is more variable tends to be under <50
- CAMT - a majority progress to aplasia / leukaemia (c-mpl req for stem cell proliferation). TAR - leukaemia isn’t usually associated, but some cases have been reported w/WBC > 35,000 cells/mm3. These leukemoid reactions are generally transient [Klopocki et al 2007].
TAR (how, diagonsis, management, genetics)
Normal expression in TPO and TPO receptor but issue with post-receptor binding of TPO receptor
Diagnosis: Thrombocytopenia absent radius (TAR) syndrome is characterized by bilateral absence of the radii with the presence of both thumbs and thrombocytopenia (<50 platelets/nL) that is generally transient. BM has absent megakaryocytes, defective CFU-Meg.
Mx: Platelet transfusion for thrombocytopenia as needed; central venous catheter as an alternative to repeated venipuncture; orthopedic intervention as needed to maximize function of limbs
Other: Autosomal Recessive
MYH9 disorders
features, diagnosis management
Macrothrombocytopenia, variable but can be <20 present from birth.
Features: progressive sensorineural hearing loss, presenile cataract, incr LFTs, and renal disease manifesting initially as glomerular nephropathy.
Diagnosis: MYH9 protein aggregates in neutrophils (due to mutant IIa cytoskeleton protein) detected through immunofluorescence analysis of a peripheral blood smear and/or by the identification of a heterozygous pathogenic variant in MYH9.
For active hemorrhage, DDAVP, and antifibrinolytic agents are used; platelet transfusion is necessary for: hemorrhages not controlled by the above treatments, life-threatening bleeding, or hemorrhages at critical sites.
Why are they known as “MYH9 disorders”
Before identification of the gene in which mutation is causative, MYH9, individuals with MYH9RD were diagnosed as having Epstein syndrome, Fechtner syndrome, May-Hegglin anomaly, or Sebastian syndrome based on the combination of different clinical findings at the time of diagnosis. However, the realization that they all are due to heterozygous pathogenic variants in MYH9 (22q12-13) and that the clinical findings often worsen throughout life as a result of late onset of non-haematologic manifestations has led the four conditions to be regarded as one disorder, now known as MYH9RD.
Congenital platelet dysfunction conditions
Gray syndrome (no a-granules)
Storage Pool Disease (no d-granules)
Secretion defects (granules are present but secretion is inhibited)
Glycoprotein abnormalities
Disorders of platelet production
All very rare
Almost all part of a wider problem e.g. TAR, MYH9
Some are pre-leukaemic e.g Fanconi, WAS
Variable inheritance
Categories in disorders of platelet function
Plasma membrane defects
Intracellular defects
Platelet plasma membrane dysfunction disorders
Bernard Soulier Glanzmanns thrombasthenia Scott syndrome Collagen receptor deficiencies Other receptor deficiencies - ADP, adrenaline Platelet type vWD
compare Bernard soulier vs Glanzmann
- Bernard - absence of GP1b (inital vwf receptor to A1) (1975 - Nurden and Caen)
Glanzmann - Glp2b3a (main vwf receptor to C4/fibrinogen binds to) absence/dysfunction “11234” - Both receptor complexes are heterodimers
- GT - normal plt count, morphology (no plt agg with n agonists) BT- macrothrombocytopenia
- Both are an AR disease
- absent/reduced agglutination with risto - bernard, there is agglutination with glanzmann
- Both have v high BT/PFA
- treat with ddavp both, bs is harder to mx for haemmorhage so try ddavp but also cortico and splenectomy is option
Glanzmann types
oType 1 = Absent GP2b3a / no fibrinogen in a granules
oType 2 = partial deficiency in GP2b3a / fibrinogen in a granules
oVariant Glanzmanns is due to a functionally defective GP2b3a
Pseudo vwd
AD / mild thrombocytopenia / deficient HMW multimers
Mutation within vWF-binding domain of GP1b spontaneous binding of vWF
Increased risto-induced agglutination (RIPA)
Clinical picture is similar to type 2B vWD
Scott Syndrome
Failure to generate platelet micro-particles when platelets activated & express binding sites on membrane for FVa & FXa due to abnormal expression of phosphatidylserine
Can’t facilitate fibrin polymerisation
V rare defect of platelet procoagulant activity
Moderate-severe bleeding
Normal platelet adhesion, activation & aggregation
Can use MoAb to detect microparticles and bound FVa - absence suggests Scott syndrome
Describe collagen receptor dysfunction disorders
collagen receptor defects often only causes mild bleeding because platelets have a number of membrane proteins which can bind collagen
What are the intracellular dysfunctional disorders?
intracellular disorders are more common but only give mild bleeding. platelet count and lifespan are usually normal and ATP:ADP ratio is an important diagnostic tool
1. Deficiency of granules / storage pool (SPD)
Dense-body deficiency (d-SPD)
Idiopathic (non-albino); Hermansky-Pudlak syndrome; Chediak-Higashi; Wiskott-Aldrich syndrome / XLT
2. Alpha granule deficiency
Grey platelet syndrome; Quebec platelet disorder; Combined SPD
idiopathic delta-SPD (genetics, inheritance, diagnosis)
Heterogeneous, often v mild, AD or AR
Aggregation variable but typically N with AA but abnormal with other agonists
Definitive diagnosis requires
1.EM - absent d bodies though membrane may be present
2.flow cytometry / mepacrine labelling
3.Increased ATP:ADP ratio towards that of metabolic pool
Why is there Hermansky Pudlak
The bleeding diathesis of HPS involves defective platelet aggregation due to absence of dense granules, apparent on whole mount electron microscopy. Specifically, the secondary aggregation response of platelets to exogenous stimuli is absent. Bleeding manifestations include spontaneous bruising, epistaxis, menorrhagia, and prolonged oozing after trauma or minor surgery such as a tooth extraction (Gahl, 1998)
Hermansky pudlak features
AR
mild bleeding disorder
can lead to pulmonary fibrosis in adult life and GI symptoms
due to membrane and content abnormalities for dense granules
oculocutaneous albinism and very bad nystagmus are features – treat with tranexamic acid
chediak higashi syndrome
results from mutations in the CHS1 gene
Decreased pigmentation, giant intracellular granules that are pathognomonic of the disease (Figure 7b), pigment clumping in hair shafts, and a bleeding diathesis related to platelet dense bodies that are absent or reduced in number. The granules, which are azurophilic and contain acid hydrolases and myeloperoxidase, are also present in CHS eosinophils, basophils, and monocytes. Children with CHS have life-threatening infections, primarily of the skin and respiratory systems. (Gahl, 1999)
Wiskott-Aldrich Syndrome
X-linked microthrombocytopenia associated with immunodeficiency and eczema, caused by mutations in the WASP gene, coding for a protein that regulates signal-mediated actin cytoskeleton rearrangement. WAS platelets have markedly reduced delta granules, alpha-granules and mitochondria. (Villa, 1995)
Gray platelet vs Quebec platelet disorder comparison
- Gray is AR, Quebec is AD (PM Hayward, 1997)
- Gray - Defects in a granule packaging absent / empty a granules (so platelets appear grey on blood film) but Quebec has a defect in a granule proteolysis and a deficiency of a granule multimerin (PM Hayward, 1997)
- The low levels of platelet fibrinogen, thrombospondin, vwf, and β-thromboglobulin in gray platelet syndrome suggest a defect in targeting or storing these proteins in α-granules, whereas qualitative, α-granular protein abnormalities predominate in the Quebec platelet disorder (PM Hayward, 1997)
- In contrast to the abnormal proteolysis of platelet P-selectin in the Quebec platelet disorder, P-selectin is not degraded in gray platelet syndrome (Rosa J-P, 1987)
- Although absent epinephrine aggregation is a consistent finding in the Quebec platelet disorder, this defect is not seen in gray platelet syndrome (PM Hayward, 1997)
Hereditary secretory /release defects in platelet disorders?
defects in thromboxane generation - impaired liberation of arachidonic acid - cyclo-oxygenase deficiency - thromboxane synthetase deficiency defects in signal transduction - defects of response to weak agonists - defective response to thromboxane A2 - defective response to adrenaline
Management of platelet disorders
Register at a CCC, Bleeding states card and Genetic testing if appropriate
• Supportive measures (avoid aspirin, NSAIDs, give tranexamic acid)
• DDAVP (but not in kids as it gives hyponatraemia)
• platelet transfusion – HLA match if they will need lifelong transfusion to avoid Ab synthesis
• rF8 for Haemophil/glanzmanns patients who have developed Abs
• BMT – usually just for Bernard soullier and glanzmanns; 10% mortality at least
What is ristocetin?
Ristocetin is used in in vitro agglutination (requires vWF & GP1b) - similar to adhesion in 1° haemostasis
What are the different anticoagulants and their mechanism?
- TFPI - TFPI (tissue factor pathway inhibitor) – inhibits procoagulant stimulus after initial TF-dependent FXa generation. TFPI inhibits TF-FVIIa and FXa by forming an inactive quaternary complex. Primarily influences initiation coagulation
- PC/PS - PC is @ by thrombin-TM complex on EC. APC proteolytically in@ procoagulant cofactors FVa and FVIIIa
PC is the major regulatory mechanism employed to inhibit thrombin generation. APC acts primarily upon the propagation phase of coagulation at the edge of a developing thrombus. - Antithrombin – AT is a serine protease inhibitor (serpin) that circulates in plasma. AT inactivates many activated coagulation serine proteases (FXa, thrombin (FIIa), FIXa, FXIa and FXIIa). AT inhibits free serine proteases. AT is enhanced by GAGs/heparin. (Propagation phase too)
Summarise PC (its @, de@ and synthesis)
• Produced in liver and circulates in plasma ~65nM
• Activated by T-TM complex on EC to in@ F5a & F8a
o Inactivation of 5a is by FAST (but not completely in@)
cleavage at 506 and SLOW (but completely in@)
cleavage at 306
o Inactivation of F8a is via cleavage of Arg 336/562
• APC is generated adjacent to the site of vessel injury (when thrombin fills the plug, thrombin binds to EPCR, now PC can bind and is @. It then ; hence its physiological function relates to regulating coagulation, & in particular thrombin generation, to the site of injury.
• APC has a half life of about 30mins and is inactivated (slowly) by PC inhibitor, alpha1 antitrypsin and alpha2 macroglobulin
Circulates at ~65nM
PC structure + function of each part of the structure
Serpin domain -> PCEGF2 > PCEGF1 > Gla Gla -contains 9x gamma carboxylic acid residues and can bind 7 Ca2+ ions causing a conformational change of a hydrophobic omega loop. Gla domain can now bind to hydrophobic tails in PL. It also binds to EPCR & protein S. Serpin - contains active site cleaved by TM-thrombin (Arg169) (Towie Arg) EGF1 is beta-hydroxylated to bind to Calcium ions too. binding of Ca2+ to the site in the EGF module locks the Gla module in a position relative to EGF module, a position that is commensurate with the expression of biological activity (Stenflo, 2000) EGF2 is glycosylated. The surface of contact between EGF2 and serpin can be considered a functional unit. Attempts to separate from EGF2. have been fraught with difficulty and resulted in aggregation of the serine proteinase module. Moreover, to express the serine proteinase module in a native conformation, it has to be linked to the C-terminal EGF module. (C. Valcarce, 1994) (Stenflo, 2000)
Usefulness of TM?
Thrombomodulin ‘alters’ the function of thrombin
• TM is a transmembrane protein (105kDa) expressed on all endothelial cells
• Thrombin binds to it with high affinity Kd= 1-10nM
• Binding to TM means that thrombin loses all its pro-coagulant functionality and acquires anti-coagulant (APC) and fibrinolytic (TAFI) properties
TM aligns the active site of thrombin to allow protein C activation
TM concentrates thrombin on the surface of endothelial cells adjacent to site of injury (i.e. at sites distinct from TF)
EPCR function
• EPCR is similar to MHC I and binds to protein C with high affinity
oPC-EPCR affinity is considerably more than
PC[Gla]-PL affinity
TM structure + function
Expressed on the surface of EC through the vasculature
Lectin-like domain > Hydrophobic region > 6x EGF domains > Ser/Thr rich domain + chondroitin sulphate > Transmembrane region
EGF - protein-protein interactions. EGF 4, 5, 6 - ESSENTIAL FOR FUNCTION + sufficient for @PC. 4 binds to serpin of PC (Low affinity) 5&6 binds to exosite I on the surface of thrombin.
Chondroitin sulfate - is a negative moiety and allows binding thrombin ~10 times better (Kd ~0.5nM) (due to additional interaction of chondroitin sulphate with exosite II on thrombin)
PS structure + function
SHBG- 4egf - thrombin sensitive - gla
Gla - 11 gamma carb residues (compare to PC’s9) binds to 7 Ca ions and PL. Part of the APC site
TSR - essential for its cofactor function, might be part of APC binding site
EGF domains - each EGF has a beta hydroxylated aspartic acid and so each binds to Ca2+ ions. First EGF domain is essential for cofactor function and this is probably part of the APC binding site on protein S.
SHBG - Contains the binding site for C4b-binding protein
Importance of C4b binding protein
60% of plasma protein S is tightly bound to C4BP
C4BP binding inhibits protein S co-factor activity
Therefore only 40% plasma protein S has APC cofactor activity
Bound via SHBG
How does PS function + steps?
Protein S has a high affinity for anionic phospholipids due to its Gla domain - KD ~10nM. PC/APC has a ~100-fold weaker affinity for anionic phospholipids due to subtle Gla domain differences - KD ~1ym
PS only binds APC on phospholipid surfaces
In binding APC, Protein S increases the affinity of
APC for phospholipids – i.e. the surfaces upon which FVa and FVIIIa function
Protein S enhances rate of cleavage at Arg306 only.
APC active site is optimally oriented to favour Arg506 cleavage But…APC has relatively weak affinity for membranes And…FVa is protected from APC inactivation by FXa in the prothrombinase complex.
1. PS increases the affinity of APC for membrane surface
2. PS relocates the active site of APC from 94Å- 84Å from the membrane surface Arg306 cleavage enhanced ~20 fold
3. PS enhances APC in@ of FVa-FXa complex, which is more resistant to APC inactivation than “free” FVa
In@ f5
Inactivation occurs on an anionic surface e.g. @plt surface at the edge of an injured area.
Rapid cleave at 506 (results in FVa with intermediate cofactor activity 25-40%)
Slow cleavage at 306 - completely abolishes FVa activity
o PS relocates APC active site - particularly important because this means the usually slow 306 cleavage for F5a becomes fast
o PS may enhance inactivation of the prothrombinase complex, which otherwise confers F5a ‘resistance’ to APC by keeping the factor bound. It results in a ~20-30fold enhancement.
Which 5 deficiencies in the Protein C pathway are associated with thrombophilia
PC 3/1k Purpura fulminans in homozygotes, VTE in heteros
PS 1/20k Purpura fulminans in homozygotes, VTE in heteros
EPCR, Unknown; lethal in mice
TM. V rare. PEs; MIs
F5 leiden, ~10% European caucasians, Only increases thrombotic risk slightly because usually only one allele is affected meaning that protein C mediated inactivation is only slowed, not terminated. Does increase thrombotic risk in context of other genetic/environmental risk factors
Factor 5 activation
When activated, the B domain from FV is removed by specific proteolytic cleavages catalysed by thrombin. The two fragments of FV are held together by Ca2+ ions.
Factor 5 function
FVa is the co-factor to FXa in the prothrombinase complex
Enhances activity by ~300,000x
Prothrombinase converts prothrombin to thrombin
In@ of F8 and role of PS for this
Due to the instability of FVIIIa, the A2 domain can spontaneously dissociate to yield inactivates FVIIIai, in the absence of FIXa. (FIXa binding stabilises FVIIIa). APC in@F8 at 336 >562
o PS enhances APC inactivation of F8a (3fold) by enhancing the 562 cleavage and enhancing the affinity of APC for PL surfaces making the inactivation of F8a less dependent on its spontaneous dissociation
Enhances by 2-3fold
FVIII @
Cleavage of B domain by thrombin AND additional cleavage site between A1 and A2 domains which is essential for full activation
APC inhibition
The half-life of APC in plasma is ~15-30mins (thrombin ~15 secs, FXa ~30 secs – AT & a2-macroglobulin)
The major inhibitors of APC in plasma include broad range inhibitors:-
a1-antitrypsin, a2-macroglobulin, protein C inhibitor (PCI)
Inactivation of APC is relatively slow
Slow inactivation enables therapeutic use of APC
FVL (mutation site, epi, mechanism
Factor V Leiden (Arg506Gln) is a mutation/polymorhism present in ~5% of white Caucasians associated with “APC-resistance”. It is the most prevalent genetic thrombophilia in people of European descent.
APC generally cleaves after specific Arg residues
Arg->Gln substitution prevents proteolysis by APC at 506 site i.e. it inhibits “fast” partial inactivation of FVa. Factor Va is still inactivated by the slow cleavage at Arg306
Why is FVL compatible with life?
Generally only 1 allele affected (i.e. 50% normal FV)
Inactivation of FVL by APC isn’t completely inhibited, but slowed
APC resistance of FV Leiden is diminished by function of PS
Other reg. mechanisms still exist (FVIIIa in@, AT, TFPI etc.)
HOWEVER…. in combination with other risk factors/ mutations, FVL mutation significantly enhances the likelihood of a variety of thrombotic disorders
F8 and F9 location
FVIII X chromosome (Xq28)
factor IX is located on the X chromosome (Xq27.1-q27.2)
X LINKED INHERTIANCE
Compare bleeding patterns between primary and secondary haemostatic disorders
1 haemostatic defects cover plt disorders & vWD
• Vessel wall & collagen disorders are also in this bracket, but are comparatively rare
• Bleeding is from small vessels, involves no delay and doesn’t stop
2 haemostatic defects are usually due to procoag def.
• Haemophilia is the commonest
• Heaprin therapy may produce a similar type of bleeding
• Bleeding is from deep structures, involves a delay (the time in which the plug is stable; before it breaks apart) and doesn’t stop from then on
Levels of severity in haemophilia + their symptoms
Severe <1% Moderate 2-5% Mild 6-40% Normal 40-150%
Severe - ‘spontaneous’ bleeds, Joints and muscle
Easy bruising, Bleeding often delayed, but prolonged
Bleeding time normal
Moderate - Prolonged or excessive bleeding after minor trauma. Rarely bleeds into joints
Mild - Excessive bleeding only after major trauma or surgery
Can you explain the bleeding phenotype of haemophilia?
Phenotype -> not superficial, deep ; delayed
not superficial - Primary haemostasis is intact and sufficient for small vessels
delayed - Primary haemostasis works initially
Inadequate thrombin generation leads to
Poor fibrin mesh &Increased susceptibility to fibrinolysis
deep - Tissues with low TF expression are more dependent on intrinsic pathway feedback
(high Tf tissues - brian lung uterus placenta, low tf muscles joints)
Describe the symptom of haemarthrosis
Begins at age approx 1 year, apparently spontaneous buy may be preceded by ‘tingling’
blood fills joint cavity and the rise in pressure is excruciatingly painful. The pressure eventually stop bleeding. Bleeding causes synovial hypertrophy which causes the synovium to expand and become friable which predisposes it to bleeding (vicious cycle). Also, bleeding causes inflammation and cartilage damage leading to arthritis (haemophilic arthropathy) and pseudotumours.
Haemarthrosis research
- Congenital haemophilia experiences haemarthrosis more than acquired (for unknown reasons)
- TAFI as an important modifier of haemophilic joint bleeding in aHA by inhibiting uPA-mediated fibrinolysis.
- bleed protection by TAFI was absent in congenital FVIII−/− mice because of severely defective TAFI @, underscoring the importance of clot protection in addition to clot formation when considering prohemostatic strategies for hemophilic joint bleeding
Wyseure et al., 2018
What are pseudotumours?
Results from multiple episodes of hemorrhage into bones or soft tissue spaces. Seen in severe cases of hemophilia only 1-2% of the time. Cx arise due to pain and/or compression of surrounding structures. (Lewis, 2005)
Psoas Bleeds
Often apparently spontaneous, may result from exertion. Blood fills muscle capsule or compartment ‘compartment sydrome’ may result. pressure eventually stops bleeding Psoas bleed is typical example
Causes of death recorded in haemophilia
Operations (25/113, of which 15 were circumcision)
Trivial injuries 23/113 (cut lip, bitten tongue etc)
Internal bleeding 21/113
Carol Birch, 1937
Factor replacement half lives
F8 - 8-12 hrs
Factor 9 - 18-24 hours
Treating haemophilia on alt. day dosing at 30 U/kg results in troughs of 4 IU/dl
Advantage and disadvantages of prophylactic therpay
Advantages Fewer number of haemorrhages esp per month when compared to episodic therapy. (Manco Johnson, 2007) 0 joint, spontaneous or traumatic bleeds when given prophylaxis (P<0.001) (Collins JTH 2009) no painful bleeds treat when well & not in pain no haemophilic arthropathy less concern re-activity less need to carry concentrate around life approaches 'normal' Disadvantages: regular injections need to start at early age difficult in young children may need portacath relatively expensive (?)
Prophylactic regimen
Factor VIII 3 times per week Factor IX 2 times per week Self infusion (iv) Home delivery of product New longer half life products now available
What are the other haemophilia treatments other than factor replacement?
Tranexamic acid - Lysine derivative. Binds to plasminogen , Blocks binding to fibrin, Clot lysis is reduced
Widely distributed, Oral 1.5gtds
DDAVP - vasopressin derivative, acts via v2 receptors and results in a 2-5 fold rise in VWF-VIII
Cx of Haemophilia Rx
1. Infection – historical problem with plasma products Hepatitis B Hepatitis C HIV Prions? 2. Immune Antibody production (inhibitors)
What is inhibitor development?
antibodies to factor VIII/FIX ‘foreign’ molecule to severe pts
40-50% of severe Haemophillia
~10-15% are persistent
High titre render FVIII/FIX treatment useless
Require by-pass therapy, high dosee F8 or combination therapy – partially effective
Certain mutations (e.g. large deletions of multiple or single exons) confer a high risk of inhibitors
ITT regiment?
Begin asap
Increase regimen to control bleeds and according to tire
Contine with previous F8 concentrate
Mutations in F8 and F9
in F8 a majority are nonsene, a small minority is missense with a slither being splicing intronic
In f9 3/4 are missense, 1/8 nonsense and 1/8 splicing inotronic
Factor IX deficiency is one-fifth as common as FVIII. There are No inversions. Otherwise the same range of mutations is seen
new research for preventing haemarthrosis
Blood-induced joint damage is fully prevented by blocking IL-1β with a monoclonal antibody or receptor antagonist, not by TNFα blockade.
IL-1β blockade prevents release of IL-6 but not TNFα from monocyte/macrophages, whereas TNFα blockade does not affect IL-1β or IL-6 release (Van vulpen 2015)
Inherited bleeding disorders listening in terms of commoness
VWD ~1:1000 Factor VIII 1:10,000 Factor IX 1:50,000 F XII 1:500,000 Rest are 1 : 1000000
Which assays correspond to which levels of severity?
- An assay reading of less than 1 IU/dL is deemed as an absence
- Heterozygotes (>20) may still have a bleeding problem
- <20IU = homozygote/compound heterozygotye
- <10IU = homozygote
Which rare bleeding disorders are AD?
Factor 11
Fibrinogen can be AR or AD
Which bleeding disorders can have thrombosis as a symptoms?
Fibrinogen deficiency
F13 deficiency? (inheritnce, symptoms, causes, epi and diagnosis)
AR
Symptoms: bleeding, poor wound healing and miscarraiges.
Ecchymoses, intramuscular/post op bleeding,
Umbilical bleeding (80%) suggestive, but not diagnostic
Can have acquired f13 through autoantibodies, decreased synthesis or increased consumption (DIC, IBD, liver disease)
1 in 2 million
Diagnose: N PT, APTT so you need to do a clot solubility test ,
Which factor deficiencies are fatal and what are their symptoms?
PT/F2 (Rarest of them all) Nose, periods, oral, muscle/joint
F5 (Type 1> type 2) Nose, periods, oral
F10 (joints gi urinary)
All the above would need FFP + PCC to treat
Describe fibrinogen deficiency
Types, treatment, phenotype
Type 2> 1; majority of Type 2 are acquired eg Liver dx
Phenotype: Umbilical cord, oral, muscle/joint, nose, periods. Thrombosis if mutation near to thrombin cleavage site
Rx: Fibrinogen concentrate
F11 deficiency
Described in all racial groups,
but particularly common in Ashkenazi jews (f7 levels low in liver dx, preg) Genitourinary, oral, nose, tonsils (injury related; mild)
Rx: Factor replacement, rFactor replace and tranexamic acid
F7 deficiency (mutations, symptoms and rx)
Commonest of them all, (Arg353G mutation & environmental influences cited)
Nose, periods, oral
Rx: FVII concentrate, rVIIa, PCCs, FFP
Causes of f7 deficiency
(David Perry, 2002)
Dimorphism at Ser 333 in F7 gene has a high frequency
Among environmental factors, dietary fat intake and the levels of plasma triglycerides are positively correlated with factor VII:C levels, but other factors such as age, obesity, diabetes (Heywood et al, 1996) and, in women, the use of sex hormones can all affect FVII levels (Meade, 1988; Habiba et al, 1996)
F13 function
Multifunctional
Stabilises fibrin clot
Implicated in atherothrombotic and VTE
Angiogenesis: embryo implantation, wound healing
Vascular permeability, stabilisation and mineralisation of extracellular matrix in bone.
Why is our understanding of developmental haemostasis deficient?
Multiple reference ranges needed due to a rapidly evolving system
Blood sampling difficult
Only small samples can be obtained
Microtechniques needed
Large patient numbers needed to establish normative data
Why is it important to understand developmental haemostasis?
Inherited disorders can present at birth with catastrophic haemorrhage
Acquired disorders of coagulation are common in preterm babies
Knowledge of developmental changes in haemostasis is essential for the correct interpretation of results & management
Where and when are coagulation proteins synthesised?
Inherited disorders can present at birth with catastrophic haemorrhage
Acquired disorders of coagulation are common in preterm babies
Knowledge of developmental changes in haemostasis is essential for the correct interpretation of results & management
Compare the amount of vit k and non vit k dependent coagulation factors at birth
F7 - 53% at term, F9 - 32%, F10 - 40%, F2- 44%
However, plasma levels of fibrinogen, FV, FVIII, FXIII & VWF are not decreased at birth. FVIII levels are skewed towards high measurements & fibrinogen continues to rise after birth. (F8 is 94% and fibrinogen is within normal range)
Results of coag tests in fetal life?
PT APTT and TT start high, decrease throughout fetal life and decrease more in adult life.
Why is haemostasis is imparied in the newborn?
Decreased synthesis of coagulation proteins
Accelerated clearance of coagulation proteins
Synthesis of proteins with reduced functional activity
Consumption of coagulation proteins at birth
What is the issue with impaired haemostasis in the new born?
NONE
Despite changes in coagulation proteins / tests of coagulation neonate does not appear to be disadvantaged
No increased bleeding or thrombotic risk for any given stimulus in HEALTH.
Acquired disorders of haemostasis
VKDB (HDN)
DIC
Thrombocytopenia (can be acquired too)
Causes of VKDB
Poor placental transfer of vitamin K
Low fetal vitamin K stores
Low vitamin K content of breast milk (1-2 μg/L)
Absent bacterial vitamin K synthesis in neonatal gut
Functional immaturity of the liver
Reference for VKDB
Ohishi, 2016 CASE REPORT
It has been reported that 46% patients with Crohn’s disease have vitamin K deficiency. Mother had crohn’s and this is believed to contirbuted to the infant’s VDKB as she had diarrohea, abdominal fullness led to little intake. Also, in order for vitamin K to be absorbed in the gastric tract, it needs to be ingested simultaneously with bile acid and fat. But CD pts can be on a fat-restricted diet»_space;> VK malabsorption and VKD. As fetal VK is supplied from the mother, it is possible that maternal vitamin K deficiency led to fetal vitamin K deficiency.
VKDB clinical presentation
Early 0-24 h
Maternal drugs e.g. anti-convulsants interfering with Vitamin K
Classic
1 – 7 days ‘Physiological’
Late, GI Haemorrhage
2 – 12 weeks. Liver Disease, exclusive breast feeding. Intracranial haemorrhage
VKDB Ix and Mx
Establish the diagnosis
Prolonged (PT)
Vitamin K 1 mg (IV)
Fresh frozen plasma if severe bleeding
VKDB controversy
IM vitamin K One dose of 1mg at birth 100% effective W/out proph. 1 in 400 neonate W/ I.M. proph 1 in 400,000 neonates Golding, Greenwood et al 1992: Link between vitamin K at birth & childhood leukaemia Change in practice IM>PO. 1999 WHO Statement: Not enough evidence 2 Meta analyses (2002 & 2003) -> No evidence
DIC reference
(Gando et al. 2016)
Main mechanisms of DIC are:
1) inflammatory cytokine-initiated activation of TF-dependent coagulation
2) insufficient control of anticoagulant pathways
3) plasminogen activator inhibitor 1-mediated suppression of fibrinolysis.
Together, these changes give rise to endothelial dysfunction and microvascular thrombosis, which can cause organ dysfunction and seriously affect patient prognosis.
Causes of DIC in neonates
Infection Inflammation NEC (causing inflammation) Meconium aspiration (cause infection and inflammation) Placental abruption (Ananth, 2018) Asphyxia (Schawmeis et al., 2015) Retained dead second twin (Jain, 2014)
DIC Ix and Mx
Dx Prolonged PT, APTT, TT Thrombocytopenia Low fibrinogen Elevated Fibrin Degradation Products (FDP) Mx Treat the underlying cause Further Mx Depends on how sick the baby is, gestational age, severity of coagulopathy Bleeding phenotype: Give FFP & Platelets
Commonest haematological abnormality in newborn babies? & Its causes
Neonatal thrombocytopenia
caused by increased destruction
reduced producton
Causes of reduced production neonatal thrombocyto[enia
Transient: maternal hypertension or diabetes,
fetal intrauterine growth restriction
Rare: BM stem cell disorders, e.g. inherited BM failure, CAMT
Causes of thrombocytopenia due to increased destruction
Immune: Maternal ITP, NAITP
Non immune: DIC, Congenital infections TORCH
NAITP steps
- Mother is negative for a common human
platelet antigen, e.g. she is HPA1a-negative - Fetus inherits this antigen from the father
i.e. the fetus is HPA1a+ positive - Fetal cells enter maternal circulation & mother generates antibodies against fetal platelet antigens, e.g. anti-HPA1a
- These antibodies cross the placenta and
cause fetal/neonatal thrombocytopenia: occurs in 1 in 1500 pregnancies
NAITP what is it manifestation? mx? severity?
Commonest cause of severe thrombocytopenia in newborn infants
May cause intracranial haemorrhage, chronic disability or death
Management after birth is to transfuse with HPA-1a-negative platelets; NAITP is transient and most cases resolve in 3-4 wks as the antibodies disappear
Inherited Disorders of Haemostasis that may present at birth?
Most common is haemophilia
Presentation of haemophilia + Ix
20% with severe haemophilia may present at birth Circumcision Cephalohaematoma ICH Bleeding/bruising following blood sampling Isolated prolonged APTT Can be normal Low FVIII or FIX Severe haemophilia level <1%
Mx of suspected/known haemophilic babies
No IM injections - ORAL vitamin K No antiplatelet drugs Great care with venepunctures Treat bleeding episodes promptly Surgical procedures need careful planning!
Hodkgins lymhpoa references
McNally -> EBV in 40-50% + 3-4 fold increase
Spinar -> HIV has high proportion of RS and 10fold risk of HL
Siddon -> HHV6 in 49% of RS cells
Strus et al. 2001 - > ALPS Fas
Joos 2002 - > REL is usually amplified and expressed in up to 55% of cases.
Joos 2000 - CGH of 50% loss of gain in 2p 7q 16 q
Franke 2001 - CGH of 50% loss of gain of 1q 3p 5q Xq
Bauger et al. -> REL targets anti apoptic genes and control growth promoting CKs eg IL13
Hodgkin lymphoma FMN
FMN: Tumour cells (Hodgkin and RS cells) are a minor component of the tumour mass, the bulk of which is mixed cellular infiltrate
FMN: BCL6 and 3q translocations occur in Nodular
Peripheral Lns and enarlged in NODULAR LP HL, and it can relapse as large B cell as well. Nodular is GC B cell but classical is heterogeneous (can be GC or nonGC)
Brentuximab anti cd30 for CHL - 22% CR (Rothe et al.)
Rituximab anti cd20 in NLPH
FMN: LMP1 upregreulated ahesio moecules eg ICAM1 and protect against cell death by upregulating antiapoptici genes eg bcl2 and @nfkb.
REL explanation too
Risk factors for first VTE
Risk factors for first VTE - asymptomatic thrombophilic patients
•>50% of thrombophilic people get their first VTE because of a transient risk factor
oSo the key might be preventing exposure to these factors
•Cancer also predisposes to thrombus formation
•Common polymorphisms can combine to increase risk of developing spontaneous VTE
•Warfarin does more harm than good in people with just pure AT deficiency
•F5 leiden is a less strong risk factor for thrombosis than PC/PC/AT deficiency (FVL 0.3% VTE p/a, PC/PS 1%)
Risk factors for VTE recurrence
•Previous PE has higher risk of recurrence than DVT
•Idiopathic VTE has higher risk of recurrence than VTE due to transient risk factor (surgery)
•Male
•APS
•Some thrombophilias
•Increased levels of d-dimer and ETP
•Presence of Residual vein thrombosis
Most likely to re-present at the site of the first VTE Kyrle 2016
Why and when do we assess thrombotic risk?
When? After VTE, Asymptomatic (eg family study) , Prior to risk (eg: OCP, HRT, pregnancy)
Why? Duration of a/c, A/c and prophylaxis, Assess risk and prophylaxis (e.g. after vte, 6 months is better than6 weeks)
Why do we assess a/c risk? Because we need to balance the risk of anticoagulation:
To anticoagulate -> Death from VTE/Haemorrhage, If you anticoaugulate -> Major/minor bleeding and inconvenience vs post phlebitic syndrome and recurrence and anxiety about not anticoagulating
HASBLED SCORE!
Acquired risk factors for thrombosis
Age
Inflammatory disorders (RA, IBD, lupus; Cancer)
Mobility and structural: Paraplegia .Sequelae of surgery/trauma, muscular
Therapy COCP and HRT
