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
