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Platelet Disorders Platelet Function
Following injury to the blood vessel, platelets
adhere to exposed subendothelium by a process
(adhesion) that involves the interaction of a plasma protein, von Willebrand factor (vWF), and a
specific protein on the platelet surface, glycoprotein
Ib (GPIb). Adhesion is followed by recruitment of
additional platelets that form clumps, a process called aggregation
(cohesion). This platelet-platelet
interaction involves binding of fibrinogen to specific platelet surface
receptors - a complex comprising glycoproteins IIb-IIIa (GPIIb-IIIa). In the resting state, platelets do not bind
fibrinogen; platelet activation induces a conformational change in the GPIIb-IIIa complex, leading to fibrinogen binding and
aggregation.
Activated
platelets release contents of their granules (secretion or release reaction),
such as adenosine diphosphate (ADP) and serotonin from the dense granules, which causes
recruitment of additional platelets.
Platelets also play a major role in hemostasis
by contributing to coagulation mechanisms; several key enzymatic reactions in
blood coagulation occur on the platelet membrane lipoprotein surface.
Together, the fibrin generated, the platelets, and red cells lead to clot
formation and restoration of hemostasis.
Under the
electron microscope a number of organelles can be discerned, including
mitochondria (Mt) and three types of granules: the dense granules (DG), the alpha
granules, and vesicles containing acid hydrolases. Platelets have an active machinery to
produce and use ATP. They have very
limited capacity to synthesize new proteins.
Thus, when aspirin irreversibly inactivates cyclooxygenase,
platelets cannot replenish the enzyme with new synthesis.
Alpha
granules all proteins Dense
small molecules
The
platelet surface has glycoproteins that are
associated with specific interactions or function. The GPIIb-IIIa
complex binds fibrinogen and mediates aggregation. GPIb binds vWF and mediates platelet adhesion to subendothelium. Platelet
Statistics Lifespan
= 9 days Thrombopoietin
In
contrast to the near-universal success of thrombopoietin
in ameliorating the thrombocytopenia and, in many cases,
the pancytopenia associated with myelosuppressive therapy, its effectiveness in
accelerating platelet recovery after myeloablative
therapy and stem-cell transplantation has been less impressive. In two
studies, the administration of thrombopoietin to
lethally irradiated mice receiving 1 million bone marrow cells accelerated platelet recovery by two to four days, but it
did not augment hematopoietic recovery when fewer
bone marrow cells were given. Stimulates
megakaryocyte ploidy Serum
levels correlates with megakaryocyte mass Increased
in hypo or amegakaryocytic thrombocytopenia Normal in
ITP and other thrombocytopenic states
On
platelet activation a number of responses can be shown to occur, including a change
in shape from disk to sphere, platelet aggregation, granule release or
secretion, and thromboxane (TxA2) production. Some of these products induce further
platelet activation as a positive feedback.
These include secreted ADP and TxA2.
Cyclooxygenase AC, adenylyl cyclase; cAMP, cyclic adenosine monophosphate;
CO, cyclooxygenase; DG, diacylglycerol;
IP3, inositol 1,4,5 trisphosphate;
MLC, myosin light-chain; PAF, platelet activating factor; PIP2, phosphatidylinositol bisphosphate;
PKC, protein kinase C; PLA2, phospholipase
A2; PLC, phospholipase C; R, receptor; vWD, von Willebrand Factor A number
of physiologic agonists interact with specific receptors on the platelet
surface to induce a series of responses. The agonists include ADP, epinephrine,
TxA2, thrombin, PAF, collagen, vasopressin, and serotonin.
Platelet activation initiates the production or release of several
intracellular messenger molecules, including Ca2+ ions, products of phosphoinositide (PI) hydrolysis, DG, IP3, TxA2, and cyclic
nucleotides [cAMP].
These modulate the various discernable platelet responses of Ca2+
mobilization, protein phosphorylation, aggregation,
secretion, and liberation of arachidonic acid. The interaction between the agonist
receptors on the platelet surface and the key intracellular effector enzymes (e.g. PLA2,PLC,
and AC) are mediated by a group of GTP-binding proteins that are modulated by
GTP. On platelet stimulation, phosphoinositides are
hydrolyzed by PLC to DG and various inositol
phosphates, including IP3. IP3 functions as a messenger to mobilize Ca2+ from
an intracellular
source. DG activates PKC and this results in the phosphorylation
of a 47-kD protein, pleckstrin. On activation, platelets release arachidonic acid from phospholipids,
which is mediated by PLA2. The free arachidonic acid is converted by COX to prostaglandins G2 and H2, and subsequently by thromboxane synthetase to TxA2.
Together, these events lead to aggregation and secretion.
Schematic
representation of the mechanism of action of antiplatelet
agents. When vascular cells are damaged, platelets bind to exposed collagen
via glycoprotein (GP) Ib/IX
receptors complexed to von Willebrand
factor. These bound platelets undergo degranulation,
releasing adenosine diphosphate (ADP) and numerous
other substances, including thromboxane A2, serotonin, and epinephrine, that
play a role in the recruitment and aggregation process. The released ADP
binds to two types of receptors, a low-affinity type 2 purinergic
receptor (P2Y12) and a high-affinity purinergic
receptor (P2Y1). Ticlopidine and clopidogrel
block the binding of ADP to the type 2 purinergic
receptor and prevent activation of the GP IIb/IIIa
receptor complex and the subsequent aggregation of platelets. The GP IIb/IIIa receptor antagonists prevent platelet
aggregation by blocking the binding of the GP IIb/IIIa
receptor to fibrinogen, thereby inhibiting fibrinogen-platelet bridging. Aspirin and Platelet Function Aspirin
is an irreversible inhibitor of cyclooxygenase.
That is why the PFA-100 is prolonged for Epinepherine
(CEPI) and normal for ADP (CADP) because ADP does not require TXA2 for
aggregation.
Most
platelet agonists activate platelets via G protein-coupled receptors on the
platelet surface. Thrombin activates human platelets through the protease
activated receptors (PAR), PAR-1 and PAR-4, which are coupled to Gq-, G12-, and Gi-mediated effector pathways. Cleavage of PAR-1 by thrombin between arginine 41 and serine 42 exposes a new N-terminus that
serves as a tethered ligand.
Thrombin Prothrombin
Test of platelet function 1. Bleeding time (not now done) 2. Platelet Function Analyzer- 100
(PFA-100) 3. Platelet aggregation testing Platelet
Function Analyzer - PFA 100 More accurate screen than bleeding time The
PFA-100 test is a new in vitro test of platelet function. Blood flows
through a titanium tube at high shear rate (mimics circulation). Aperture at
end of tube lined with collagen +/- Epinephrine or ADP. Platelet thrombus
blocks aperture and causes blood to stop flowing. The test measures the time
taken for blood to stop flowing through the membrane coated with collagen and
epinephrine (CEPI) or collagen and ADP (CADP). This is referred to as the
Closure Time (CT) and is measured in seconds. The test is therefore a
combined measure of platelet adhesion and aggregation. Interpretation: The
PFA test result is dependent on platelet function, plasma von Willebrand Factor level, platelet number and (to some
extent) hematocrit. CEPI
is <180 s - Normal Platelet Function CEPI
>180 s; CADP <116 s - "Aspirin Effect" CEPI
>180 s; CADP >116 s - Abnormal Platelet Function Comparison
to Bleeding Time:
Published
studies indicate that the PFA-100 and Bleeding time give equivalent results
in about 75% of cases. Most discrepancies between Bleeding time and PFA-100
are a result of aspirin ingestion since the PFA-100 is much more sensitive to
this effect. Several
studies have now shown that the PFA-100 has excellent (near-100%) sensitivity
to the presence of von Willebrand's Disease.
In contrast, the BT may be normal in as many as one third of cases. Specimen: 2.8 mL buffered sodium
citrate whole blood (blue-top tube). Store and transport at room temperature.
Sample must be received within 3 hours. Collagen/Epinephrine 81-180 sec
Caveats: Few data in childen Effect of drug unknown (except aspirin) Correlation with risk of bleeding unknown Significance of reduced closure time unknown (e.g.
Newborn have short closure) Platelet Aggregation testing
VWD exactly
like Bernard Soulier except that the Ristocetin test corrects with added VWF Platelet Disorders and Location of Mutations
Von Willebrand Factor Produced by endothelial cells and megakaryocytes Two functions: 1.
Adhesion of platelets to damaged endothelium. 2.
Binds to F8 protecting from premature destruction. VWd types von Willebrand disease (VWD),
the most common inherited bleeding disorder, is due to reduced (VWD types 1
and 3) or defective (VWD type 2) von Willebrand
factor (VWF) VWD can be classified into 3 main types, of which 70-80% are considered to be type 1. Type 1: ·
70-80% ·
Mild autosomal dominant variable penetrance ·
VWD partial quantitative decrease of qualitatively normal VWF and
FVIII. Type 1C: ·
Autosomal dominant ·
Increased Clearance of VWF ·
At the present time, all mutations for this phenotype have been found
in exons 26, 27 and 37 of the VWF gene. ·
Increased VWF propeptide/VWF antigen ratio ·
Laboratory findings include low VWF antigen and proportionately low
VWF ristocetin cofactor activity; Factor VIII
levels may be low. ·
Persistence of larger than normal size VWF multimers. ·
An increased rate of VWF clearance can be documented by DDAVP trial. ·
This observation is of clinical importance because DDAVP may not be
the treatment of choice in this subset of patients because of the short
residence time in plasma. In these patients, VWF replacement therapy may be a
more appropriate treatment choice. Type 2: ·
15-20% have type 2 disease. Can be either autosomal dominant or recessive. ·
Of the 5 known type 2 VWD subtypes (ie, 2A,
2B, 2C, 2M, 2N), type 2A VWD is by far the most common. Type 2A ·
autosomal dominant trait ·
normal-to-reduced plasma levels of factor VIIIc
(FVIIIc) and VWF. ·
reduction in intermediate and high molecular weight VWF multimer complexes. The multimeric
abnormalities are commonly the result of in vivo proteolytic
degradation of the VWF. ·
Ristocetin cofactor activity is greatly reduced, and the
platelet VWF reveals multimeric abnormalities
similar to those found in plasma. ·
Typically, type 2A mutations cause either defective assembly or
secretion of large multimers or increase the
susceptibility of large multimers to proteolysis by
ADAMTS13. (A
for ADAMTS13) While the hallmark of
type 2A VWD is plasma deficiency of high molecular weight VWF multimers, several other conditions are associated with
this finding including type 2B VWD, platelet-type VWD, and acquired VWD. ·
In patients with a clinical phenotype of type 2A VWD, molecular
testing is useful in confirming diagnosis and identifying affected family
members. ·
The majority of type 2A mutations (70-80%) are found in exon 28 of VWF. The remaining type 2A mutations that have
been described are found in exons 11-16, 26, 51 and
52. Type 2B ·
autosomal dominant trait. ·
gain-of-function mutation vWD high
molecular-weight multimers to bind more tightly to
their receptors on platelets (the alpha chains of glycoprotein
Ib (GPIb) receptors). ·
Hence increased low dose-RIPA test ·
This increased binding causes vWD because the high-molecular weight multimers
are removed from circulation in plasma since they remain attached to the
patient's platelets. ·
reduction in the proportion of high molecular weight VWF multimers, while the proportion of low molecular weight
fragments are increased. ·
hemostatic defect caused by a qualitatively
abnormal VWF and intermittent thrombocytopenia. ·
The abnormal VWF has an increased affinity for platelet glycoprotein Ib. Hence gain of
function mutation (see below). So increased sticking of the VWF multimers to platelets which are then removed from the
circulation. ·
The platelet count may fall further during pregnancy, in association
with surgical procedures, or after the administration of desmopressin
acetate (DDAVP). Although some investigators found DDAVP to be clinically
useful in persons with type 2B VWD, studies directed at excluding the 2B
variant should be completed before DDAVP is used therapeutically.
Measurements of FVIIIc and VWF in plasma are
variable; however, studies involving the use of titered
doses of ristocetin reveal that aggregation of
normal platelets is enhanced and induced by unusually small amounts of the
drug. ·
Types 2B (and platelet-type) have lower than normal amounts of ristocetin cause platelet aggregation when the patient's
platelet-rich plasma is used. This paradox is explained by these types having
gain-of-function mutations which cause the vWD high
molecular-weight multimers to bind more tightly to
their receptors on platelets (the alpha chains of glycoprotein
Ib (GPIb)
receptors). In the case of type 2B vWD, the
gain-of-function mutation involves von Willebrand's
factor (VWF gene), and in platelet-type vWD, the
receptor is the object of the mutation (GPIb). This
increased binding causes vWD
because the high-molecular weight multimers are
removed from circulation in plasma since they remain attached to the
patient's platelets. Thus, if the patient's platelet-poor plasma is used, the
ristocetin cofactor assay will not agglutinate
"standardized (ie., pooled platelets from normal donors which are fixed in formalin)" platelets, similar to the other types of vWD. ·
Mutations of exon 28 of VWF Type 2M ·
Rare type, laboratory results are similar to those of certain patients
with type 2A VWD. ·
Decreased binding to VWF to GP1b ·
A decreased platelet-directed function that is not due to a decrease
of high–molecular weight multimers. ·
decreased VWF activity, but VWF antigen, FVIII, and multimer analysis are found within reference range. ·
VWF:RCoF: VWFag = 1:2 ·
Exon 28 mutation Type 2N ·
autosomal recessive ·
markedly decreased affinity of VWF for FVIII, resulting in FVIII
levels reduced to usually around 5% of the reference range. ·
Other VWF laboratory parameters (ie, VWF
antigen [VWF:Ag], ristocetin cofactor activity) are usually normal. ·
Evaluate patients with FVIII deficiency and a bleeding disorder that
is not clearly transmitted as an X-linked disorder or those who respond
incompletely to hemophilia A therapy for type 2N
VWD. ·
Confirmatory test for type 2N VWD is not routinely available, likely
resulting in an underestimate of the true frequency of this subtype. Type 3 ·
autosomal recessive trait. ·
most severe form of VWD. ·
In the homozygous patient, marked deficiencies of both VWF and FVIIIc in the plasma ·
the absence of VWF from both platelets and endothelial cells, and a lack
of response to DDAVP. ·
severe clinical bleeding ·
consanguinity is common in kindreds with this
variant. ·
Multimeric analysis of the small amount of VWF present yields
variable results, in some cases revealing only small multimers. Diagnosis of VWD
Can start with
Bleeding time (BT) or PFA-100 The BT test is a nonspecific test and is fraught with
operational variation. It has been argued that it was a population—based test
that was never developed to test individuals.Variables
that may affect results include a crying or wiggling child, differences in
the application of the blood pressure cuff, and the location, direction, and
depth of the cut made by the device. This test also has a potential for causing keloid formation and scarring, particularly in non—Caucasian individuals. The PFA—100® result has been
demonstrated to be abnormal in the majority of persons who have VWD, other than
those who have type 2N, but its use for population screening for
VWD has not been established.Persons who have
severe type 1 VWD or who have type 3 VWD usually have abnormal PFA—100®
values, whereas persons who have mild or moderate
type 1 VWD and some who have type 2 VWD may not have abnormal results. When using the PTT in the diagnosis of VWD, results of
this test are abnormal only if the FVIII is sufficiently reduced. Because the
FVIII gene is normal in VWD, the FVIII deficiency is secondary to the
deficiency of VWF, its carrier protein. In normal individuals, the levels of
FVIII and VWF:RCo are
approximately equal, with both averaging 100 IU/dL.
In type 3 VWD, the plasma FVIII level is usually less than 10 IU/dL and represents the steady state of FVIII in the
absence of its carrier protein. In persons who have type 1 VWD, the FVIII
level is often slightly higher than the VWF level and may fall within the
normal range. In persons who have type 2 VWD (except for type 2N VWD in which
it is decreased), the FVIII is often 2–3 times higher than the VWF activity (VWF:RCo). Therefore, the PTT is
often within the normal range. If VWF clearance is the cause of low VWF, the
FVIII reduction parallels that of VWF, probably because both proteins are
cleared together as a complex. Initial Tests for
VWD ·
VWF:Ag (Antigen photometric assay) ·
VWF:RCo (agglutination assay) ·
FVIII These three tests, readily available in most larger
hospitals, measure the amount of VWF protein present in plasma (VWF:Ag), the function of the VWF
protein that is present as ristocetin cofactor
activity (VWF:RCo), and the ability of the VWF to
serve as the carrier protein to maintain normal FVIII survival, respectively.
If any of the above tests is abnormally low, the next steps should be
discussed with a coagulation specialist, who may recommend referral to a
specialized center, and/or repeating the laboratory tests plus performing
additional tests. VWF:Ag is an immunoassay that measures
the concentration of VWF protein in plasma. Commonly used methods are based
on enzyme—linked immunosorbent assay (ELISA) or
automated latex immunoassay (LIA). VWF:RCo is a functional/activity assay of
VWF that measures its ability to interact with normal platelets. The
antibiotic, ristocetin, causes VWF to bind to
platelet receptor GP1b. Serial dilutions of the patient’s serum are mixed
with formalin fixed platelets. When ristocetin is added, platelet aggregation will be visible
if WF activity is present in patient’s plasma.
Several methods are used to assess the platelet
agglutination and aggregation that result from the binding of VWF to platelet
GPIb induced by ristocetin
(ristocetin cofactor activity, or VWF:RCo). The methods include: (1)
time to visible platelet clumping using ristocetin,
washed normal platelets (fresh or formalinized),
and dilutions of patient plasma; (2)
slope of aggregation during platelet aggregometry
using ristocetin, washed normal platelets, and
dilutions of the person’s plasma; (3)
automated turbidometric tests that detect
platelet clumping, using the same reagents noted above; (4)
ELISA assays that assess direct binding of the person’s plasma VWF to
platelet GPIb (the GPIb
may be derived from plasma glycocalicin) in the
presence of ristocetin; (5)
the binding of a monoclonal antibody to a conformation epitope of the VWF A1 loop. Method 5 can be performed in
an ELISA format or in an automated latex immunoassay. It is not based on ristocetin binding. The first three assays may use platelet membrane fragments
containing GPIb rather than whole platelets. The
sensitivity varies for each laboratory and each assay; in general, however,
Methods 1 and 2, which measure platelet clumping by using several dilutions
of the person’s plasma, are quantitative to approximately 6–12 IU/dL levels. Method 3 is quantitative to about 10–20 IU/dL. Method 4 can measure VWF:RCo to <1 IU/dL, and
a variation of it can detect the increased VWF binding to GPIb
seen in type 2B VWD.173 Some automated methods are less sensitive and require
modification of the assay to detect <10 IU/dL. FVIII coagulant assay is a measure of the cofactor
function of the clotting factor, FVIII, in plasma. In the context of VWD,
FVIII activity measures the ability of VWF to bind and maintain the level of
FVIII in the circulation. In the United States, the assay is usually
performed as a one—stage clotting assay based on the PTT, although some
laboratories use a chromogenic assay. The clotting
assay, commonly done using an automated or semiautomated
instrument, measures the ability of plasma FVIII to shorten the clotting time
of FVIII—deficient plasma. Because this test is important in the diagnosis of
hemophilia, the efforts to standardize this assay have been greater than for
other hemostasis assays. FVIII activity is labile,
with the potential for spuriously low assay results if blood specimen
collection, transport, or processing is suboptimal.
Like those tests discussed above, it should be expressed in international
units per deciliter (IU/dL) based on the WHO plasma
standard. Expected patterns of laboratory results in different subtypes
of VWD, depicted in Figure 5, include results of the three initial VWD tests
(VWF:Ag, VWF:RCo, FVIII) and results of other assays for defining
and classifying VWD subtypes. The three initial tests (or at least the VWF:RCo and FVIII assays) are
also used for monitoring therapy. The VWF multimer test uses a
previously unthawed portion of the same sample or in association with a
repeated VWD test panel (VWF:Ag,
VWF:RCo, FVIII) using a fresh plasma sample. VWF multimer analysis is a qualitative assay that depicts the
variable concentrations of the different—sized VWF multimers
by using sodium dodecyl sulfate (SDS)—protein
electrophoresis followed by detection of the VWF multimers
in the gel, using a radiolabeled polyclonal antibody or a combination of monoclonal
antibodies. Alternatively, the protein is transferred to a membrane (Western
blot), and the multimers are identified by immunofluorescence or other staining techniques.
RIPA = Ristocetin Induced
Platelet Aggregation (RIPA) patients own platelets, whereas the VWF:RCo measures formalin fixed platelets from a normal person.
Thrombasthenia (Glanzmann’s Syndrome; GPllb/llla)
GP IIb/IIIa receptors
play an important role in platelets' adherence to the endothelium as well as
platelet aggregation. Acts as fibrinogen
receptor. GP IIb/IIIa complex binds
fibrinogen. Adjacent platelets are cross-linked through GP IIb/IIIa-fibrinogen-GP IIb/IIIa
complexes. When GP IIb/IIIa complex functions
abnormally, platelet aggregation is impaired and bleeding occurs. GP IIb/IIIa complex is a heterodimer that requires calcium for the GP IIb and GP IIIa to associate
normally. Both GP IIb and GP IIIa
are required for normal platelet function. A defect in either can lead to a
bleeding disorder. Autosomal recessive (Chr
17) Gene is INTEGRIN,
ALPHA-2B; ITGA2B for GP llb and INTEGRIN, BETA-3;
ITGB3 for GP llla Presents in neonatal period with mucocutaneous bleeding. Platelet counts and platelet functions that do not
depend on GP IIb/IIIa are normal in patients with thrombasthenia. Diagnosis: Complete blood count, prothrombin
time, and activated partial thromboplastin time
normal Bleeding times prolonged. PFA-100 prolonged Platelet aggregation studies SDS gel electrophoresis of platelet membrane glycoprotein Flow cytometry CD41B (GP llb) or CD61 (GP llla) The primary platelet aggregation response
to platelet agonists such as adenosine diphosphate
(ADP), epinephrine, and collagen are decreased, while the response
to ristocetin is normal. If the secondary platelet
aggregation response is abnormal, suspect a platelet storage pool defect or
an abnormality in platelet signal transduction. Ristocetin is an antibiotic, obtained from Amycolatopsis lurida,
previously used to treat staphylococcal infections. It is no longer used
clinically because of its toxicity. It causes platelet agglutination and
blood coagulation and is used to assay those functions in vitro, e.g. to
diagnose von Willebrand disease (vWD) or the Bernard-Soulier
syndrome. Platelet agglutination caused by ristocetin can occur only in the presence of large multimers of von Willebrand
factor, so if ristocetin is added to blood lacking
the factor (or its receptor -- see below), it will not coagulate. In some types of vWD
(types 2B and platelet-type), lower than normal amounts of ristocetin cause platelet aggregation when the patient's
platelet-rich plasma is used. This paradox is
explained by these types having gain-of-function mutations which cause the vWD high molecular-weight multimers
to bind more tightly to their receptors on platelets (the alpha chains of glycoprotein Ib (GPIb) receptors). In the case of type 2B vWD, the gain-of-function mutation involves von Willebrand's factor (VWF gene), and in platelet-type vWD, the receptor is the object of the mutation (GPIb). This increased binding causes vWD because the
high-molecular weight multimers are removed from
circulation in plasma since they remain attached to the patient's platelets.
Thus, if the patient's platelet-poor plasma is used, the ristocetin
cofactor assay will not agglutinate "standardized (ie., pooled platelets from
normal donors which are fixed in formalin)"
platelets, similar to the other types of vWD. ristocetin cofactor is also called also von Willebrand factor. Therefore
ristocetin enhances binding of to VWF and Gpla. So when we have VWD the ristocetin
test is abnormal but corrects with exogenous VWF whereas in Bernard Soulier’s disease (mutations of Gpla)
ristocetin test is abnormal but does not correct
with exogenous VWD. In all forms of the ristocetin
assay, the platelets are fixed in formalin prior to
the assay to prevent von Willebrand's factor stored
in platelet granules from being released and participating in platelet
aggregation. Thus, the ristocetin cofactor activity
depends only upon high-molecular multimers of the
factor present in circulating plasma. Treatment Local measures/topical agents/anti-fibrinolytic agents/hormonal) Platelet transfusion avoid due to alloimmunization Recombinant Vlla for
bleeds Stem cell transplant Bernard-Soulier Syndrome Autosomal recessive Bernard-Soulier syndrome
(BSS) has been found to be caused by mutation in the GP1BA gene;
Chr17pter-p12 (606672),
the GP1BB Chr22q11.2 gene (138720),
or the GP9; 3q21gene (173515);
the forms of BSS caused by mutations in these genes are here referred to as
types A, B, and C, respectively. Diagnosis: CBC count: Thrombocytopenia is a frequent finding
but is variable. Giant platelets are seen on the peripheral smear, possibly
exceeding the size of a red blood cell. Bleeding time: Bleeding time is usually prolonged.
The template bleeding time has largely been replaced by automatic platelet
function analyzers, such as the PFA-100. Platelet aggregation studies: Platelets do not aggregate in response to ristocetin. This is not corrected by the addition
of normal plasma, as seen in von Willebrand
disease. Platelets have normal aggregation in response to adenosine diphosphate (ADP), epinephrine, and collagen. ristocetin cofactor is also called also von Willebrand factor. Therefore
ristocetin enhances binding of to VWF and Gp lb. So when we have VWD the ristocetin
test is abnormal but corrects with exogenous VWF whereas in Bernard Soulier’s disease (mutations of Gp
lb) ristocetin test is abnormal but does not
correct with exogenous VWD.
Flow cytometry: Flow cytometry can demonstrate abnormalities of platelet
membrane glycoprotein.
Treatment local measures and platelet transfusion Neonatal
Platelets Number
lifespan same as older children Bleeding
time normal Aggregation
normal/- (epi and ADP) Adhesion
increased (high MW vWF Short PFA
100 (enhanced platelet function and high Hb) Thrombocytopenia 1. Reduced production 2. Short life span 3. Sequestration 4. loss/dilution Reduced
production Marrow
infiltration, failure, ineffective thrombopoesis Megaloblastic states Shortened
life span Immune ·
ITP ·
Neonatal alloimmune ·
Infection Non
immune ·
DIC ·
HUS ·
TTP ·
Infection Ethylenediaminetetraacetic Acid (EDTA)-Dependent Pseudothrombocytopenia
Correct
platelet count when blood collected with citrate or oxalate Drug Induced Immune Thrombocytopenias Rare Heparin/Quinine/Quninidine/sulfa/Gold/Valporic acid Drug
binds to platelets then ab binds, or drug-ab binds to platelets Heparin Induced Thrombocytopenia
(HIT)
Platelet factor-4
is a 70-amino acid protein that is released from the alpha-granules of
activated platelets and binds with high affinity to heparin. Its major
physiologic role appears to be neutralization of heparin-like molecules on
the endothelial surface of blood vessels, thereby inhibiting local antithrombin III activity and promoting coagulation. As a
strong chemo attractant for neutrophils and fibroblasts, PF4 probably has a
role in inflammation and wound repair HIT on
platelet surface Reduced
platelets, venous and arterial
thrombosis 5-10 days
after starting Heparin 1-2% More
common in high molecular wt heparin\Diagnosis ELISA Stop
heparin, use alternative Lepirudin: An anticoagulant which functions as a direct thrombin
inhibitor. Danaparoid: that works by inhibiting activated factor X (factor Xa) and is considered a "low molecular weight
heparin" by some sources, but is chemically distinct from heparin and
thus has little cross-reactivity in heparin-intolerant patients. It consists
of a mixture of heparan sulfate, dermatan sulfate and chondroitin
sulfate. Argatroban: An anticoagulant that is a small molecule direct thrombin
inhibitor. Used for prophylaxis or treatment of thrombosis in patients with
heparin-induced thrombocytopenia (HIT). Argatroban
is given intravenously. It is monitored by PTT. Thrombotic Thrombocytopenic Purpura
Clinical Manifestation Abrupt
onset, older children/ Focal or generalized CNS deficits. Need urgent
treatment. Pentad: 1. Microangiopathic hemolytic anemia 2. Thrombocytopenia 3. Neurological manifestation 4. Renal manifestation 5. Fever Differential Diagnosis HUS ITP Sepsis DIC Evan’s
Syndrome Clinical Associations ·
Idiopathic ·
Familial ·
Auto immune (SLE) ·
Drug induced o
Ticlopidine (is an antiplatelet drug
in the thienopyridine family. It is an adenosine diphosphate (ADP) receptor inhibitor. o
Clopidrogrel (ADP receptor inhibitor) o
Cyclosporin o
Tacrolimus ·
Pregnancy ·
Malignancy ·
Infection Diagnosis Exclude
above Increased
retics because of hemolysis Normal
PT/PTT/fibrinogen Reduced ADAMTS-13
metalloprotease level Antibody
against ADAMTS13 in some cases.
ULVWF multimers are normally
digested by von Willebrand cleaving protease,
ADAMTS13. In TTP, the absence of ADAMTS13 allows release of
ULVWF, triggering platelet activation. TTP, thrombotic
thrombocytopenic purpura; ULVW, unusually large von
Willebrand factor. ‘A Disintegrin-like And Metallprotease with ThromboSpondin’
(A) In normal individuals, ADAMTS-13 enzyme molecules from the
plasma attach to, and then cleave, ULVWF multimers
that are secreted in long "strings" from stimulated endothelial
cells. (B) The ULVWF multimeric "strings"
may be anchored to the endothelial cell by P-selectin
molecules. ADAMTS-13 molecules attach to exposed domains and proteolytically cleave ULVWF multimers.
The smaller VWF forms that circulate after cleavage do not induce adhesion
and aggregation of platelets during normal blood flow. (C) Absent or severely
reduced activity of ADAMTS-13 in patients with TTP. Non-cleaved ULVWF multimers induce platelet adhesion and aggregation.
Either congenital deficiencies or acquired defects of ADAMTS-13 result in
TTP. Outcome 80%
mortality untreated Plasmapheresis/Plasma
exchange daily until control of HE anemia and normal platelet count FFP vs Cryoprecipitate poor plasma Relapse
rate up to 30% Hemolytic Uremic Syndrome Infants
and young Children Bloody
diarrhea EColi O157:H7 Toxin damage
to renal endothelia results in release of ULVWF Similar
to TTP but normal ADAMTS13 Dialysis
usually no plasmapheresis required Idiopathic Thrombocytopenic
Purpura ~4.8
cases per 100,000 children (3500 per yr in US) (leukemia about 3.5/100,000 Peak
incidence 2-5 yrs Occasional
mild splenomegaly Evaluation of suspected ITP History
(recent infection otherwise normal)/Exam normal except bleeding CBC and smear
(normal except low platelets (large)) Occasional
BM needed Auto
immune test for older children (ANA, Coombs, Ig)
HIV, EBV Mechanism IgG against platelet membrane antigen usually GPllb/llla Forms ITP:
Indications for BM Planned
steroids Underlying
disease Uncertain
follow up Atypical
features (anemia, increased MCV. Neutropenia, hepatosplenomegaly) Treatment: Reassure. Avoid
aspirin Limit activates
and contact sports Weekly
counts Some
cases IVIG, Anti-D or corticosteroids No
evidence that intracranial hemorrhage (ICH)
prevented by therapy. Steroid dosage Pred 2-4mg/kg/day 1-3 wks Methylpred 30mg/kg/day iv 3 days Dexamethasone 25-30mg/m2/d po 4 days q28 days. IVIG 1g/kg/day iv 2 days Side
effects of IVIG include headache, hemolysis,
thrombosis, viral transmission, aseptic meningitis, anaphylaxis in IgA deficient patients. Anti-D therapy Binds to
D-positive RBC saturates splenic Fc receptors. Ineffective
in Rh negative patients 1/800
children develop ICH with ITP 2 deaths
from ICH with ITP/yr in the US Only a
minority of children with major bleeding respond IVIG +/- steroids Risk
factors of ICH Pl<20,
head trauma, aspirin, Cerebral AV malformation 50%
develop ICH 1 month after diagnosis Mortality
from ICH ~40% Chronic ITP Differential ITP Marrow
failure Hypersplenism Hereditary
thrombocytopenia VWD type 2B Drugs Treatment of Chronic ITP Many
drugs including rituximab Laproscopic Splenectomy definitive
60-80% respond only indicated in severe life threatening
thrombocytopenia Hereditary thrombocytopenia Thrombocytopenia
Resulting from Impaired Platelet Production Congenital
Thrombocytopenia Resulting from Impaired Platelet Production MYH9-Related
Thrombocytopenia Syndromes Genetics May-Hegglin anomaly, Fechtner
syndrome, Sebastian syndrome, and Epstein syndrome are autosomal
dominant macrothrombocytopenias with mutations in
the MYH9 gene, located on chromosome 22q12-13. This gene encodes nonmuscle myosin heavy chain (NMMHC)-IIA, which is
expressed in platelets, kidney, leukocytes and the cochlea. Although these
syndromes result from mutations in the MYH9 gene, a clear phenotype-genotype
relationship within each has not been determined. A suggestion is that they
represent a class of allelic disorders with variable phenotypic expression
leading to diversity among the group.
Autosomal recessive: Bernard Soulier X-linked Wiskott-Aldrich Syndrome. Wiskott-Aldrich Syndrome Thrombocytopenia,
eczema and frequent infections Mutation
of WAS gene (X-chromosome) Also
associated with defect in surface glycoprotein CD43 Platelet
size small and poorly functioning (storage granule deficiency) Eczema
and immunodeficiency (variable) Low IgM, high IgA Absent NK
cells Impaired
response to polysaccharide antigen Reduced
or absent isohemagglutinin levels (anti-A and
anti-B) High
mortality Median age of death ~11yrs Hemorrhage
(internal and mucosal) Infection
(post-splenetomy, viral) Lymphoma,
ALL Supportive
therapy, splenectomy, stem cell transplant. Neonatal Thrombocytopenia
TAR Syndrome Autosomal
recessive Severe
thrombocytopenia Abnormalities
of radius Normal
thumbs Leukemoid reaction Cows mild
intolerance Treated
with platelet transfusions Rise in
platelets by 12-24months Maternal ITP: Maternal
usually gestational Pl
75-150K/ul usually no clinical significance. Baby nadir
3-4 days Cord
blood then daily counts till nadir IVIG if
bleeding (steroid or platelet transfusion no clinical benefit) Neonatal Alloimmune
Thrombocytopenia Pateral HPA-1a (Human platelet antigen part of GPllb/llla complex) negative transfers across placenta
and causes antibody production Maternal
pl normal Platelet
response random donor poor, maternal donor good Antiplatelet antibody in mother Fetal intracranial hemorrhage ~ 10-20% 1-5% life
threatening bleeds Management Percuataneous umbilical venous sampling at 22 weeks Pl <50
or empirically give IVIG to mother Fetal
transfusion using compatible platelets. Management of Delivery and
neonatal period Caesarian
Section if fetus known to be at risk Maternal
platelets at delivery Bleeding
or platelets <20-30K/ul give transfusion IVIG
1g/kg /day for 2 days Resolution
2-10 weeks. Paradox of Thrombocytopenia and
thrombosis DIC HIT Antiphospholipid antibody syndrome (lupus anticoagulant) Thrombocytosis Pl>450K/ul Can be primary
or secondary (reactive) Reactive or secondary : Inflammation Connective
tissue/Malignancy/Kawasaki Iron
Deficiency Marrow
recovery Sickle
cell Post
splenectomy Young
premature No risk
treat underlying cause Exception
post splenectomy (venous and arterial thrombosis) in thalassemia
and chronic hemolytic anemia Essential Thrombocythemia Primary myeloproliferative disorder Rare in
childhood Slenomegaly/bleeding/thrombosis Pl
1000-4000K/ul Platelet
aggregation abnormal No underlying
reactive condition Management
aspirn/cytotoxics e.g. hydroxyurea/anagrelide
Anagrelide is a phosphodiesterase
inhibitor Qualitative Disorders of
Platelets. Storage Pool Diseases
A
representative tracing from a patient with dense granule SPD or a secretion
defect is shown. The primary wave of aggregation with ADP or epinephrine is
generally present with a loss of the secondary wave. The response to ristocetin is generally normal. Alpha granule deficiency = Gray
Platelet Syndrome ž
Alpha granule deficiency ž
Clinical: mild bleeding ž
Laboratory: •
Thrombocytopenia: 60 - 100 x 103/mL •
Platelets are large and appear “gray” on Wright’s stained smear (hence
the name) •
Marked deficiency of alpha granules •
Dense granules are normal •
Platelet aggregation studies are fairly normal since 2o
aggregation wave due primarily to dense granule release
Dense Granule Storage Pool Disease Seen only
by Electron Microscopy Impaired
secondary aggregation Dense granules contain serotonin,
calcium, ATP and ADP Primary
inherited disorder or secondary e.g. to Hermansky-Pudlak
or Chediak-Higashi Syndrome
Alternative
titles; symbols ALBINISM
WITH HEMORRHAGIC DIATHESIS AND PIGMENTED RETICULOENDOTHELIAL CELLS Gene map
locus 19q13,
11p15-p13, 10q24.32, 10q23.1, 6p22.3, 3q24, 22q11.2-q12.2 TEXT A number
sign (#) is used with this entry because Hermansky-Pudlak
syndrome (HPS) can be caused by mutation in several genes: HPS1 (604982),
HPS3 (606118),
HPS4 (606682),
HPS5 (607521),
and HPS6 (607522).
HPS2 (608233),
which includes immunodeficiency in its phenotype, is caused by mutation in
the AP3B1 gene (603401).
HPS7 is caused by mutation in the DTNBP1 gene (607145).
HPS8 is caused by mutation in the BLOC1S3 gene (609762).
DESCRIPTION Hermansky-Pudlak syndrome (HPS) is a rare autosomal
recessive disorder in which oculocutaneous albinism,
bleeding, and lysosomal ceroid
storage result from defects of multiple cytoplasmic
organelles: melanosomes, platelet-dense granules,
and lysosomes (Oh et al., 1998). Gain of function mutations of vWF and Gp1b In some types of vWD
(types 2B and platelet-type), lower than normal amounts of ristocetin cause platelet aggregation when the patient's
platelet-rich plasma is used. Increased platelet induced ristocetin
aggregation Reduced platelets This paradox is explained by these types having
gain-of-function mutations which cause the vWD high
molecular-weight multimers to bind more tightly to
their receptors on platelets (the alpha chains of glycoprotein
Ib (GPIb) receptors). In the case of type 2B vWD,
the gain-of-function mutation involves von Willebrand's
factor (VWF gene), In platelet-type vWD, the
receptor is the object of the mutation (GPIb). This
increased binding causes vWD
because the high-molecular weight multimers are
removed from circulation in plasma since they remain attached to the
patient's platelets. Thus, if the patient's platelet-poor plasma is
used, the ristocetin cofactor assay will not
agglutinate "standardized (ie., pooled platelets from normal donors which are fixed in formalin)" platelets, similar to the other types of vWD. However if other plasma is used there is increased
aggregation because of increased sticking to VWF. Acquired platelet dysfunction. Liver/renal/cardiac
disease/malignancy/drugs Drugs and platelet dysfunction: Aspirin Other
NSAID Valporic acid Semi
synthetic penicillins Psychotropic
drugs Treatment of Qualitative Platelet
Disorders Treat
underlying cause Local
measures (pressure, gelfoam, desiccated collagen
etc.) Desmopressin (DDAVP) Recombinant
Vlla for bleeding DDAVP
Antiplatelet drugs: Aspirin Glycoprotein llb/lla (fibrinogen receptor)
inhibitor includes abciximab and epifibatide Clopidogrel Ticlopidine and clopidogrel block the binding of ADP to the type
2 purinergic receptor and prevent activation of the
GP IIb/IIIa receptor complex and the subsequent
aggregation of platelets. The GP IIb/IIIa receptor
antagonists prevent platelet aggregation by blocking the binding of the GP IIb/IIIa receptor to fibrinogen, thereby inhibiting
fibrinogen-platelet bridging. Dipyridamole Dipyridamole inhibits the phosphodiesterase
enzymes which normally break down cAMP (increasing
cellular cAMP levels and blocking the platelet
response to ADP) and/or cGMP (resulting in added
benefit when given together with NO or statins). |
