Keywords
diagnosis management - inherited coagulation disorders - inherited/acquired platelet
disorders - platelet pathology/inherited
Introduction
Platelets play a crucial role in primary hemostasis and in the amplification of thrombin
generation during secondary hemostasis. They are produced by megakaryocytes in the
bone marrow and lungs[1] and circulate as small, granular, anucleate cytoplasmic fragments. Under physiological
conditions, their concentration ranges between 150,000 and 450,000/µL in children
and adults. When endothelial damage occurs, subendothelial structures like collagen
become exposed, recruiting the plasmatic von Willebrand factor (VWF). Platelets adhere
to these structures and form a primary hemostatic plug, wherein platelet-to-platelet
interaction is achieved through cross-linking by fibrinogen and VWF. Concurrent platelet
activation is characterized by a change in shape, exposure of anionic phospholipids,
and secretion of granule cargo. These mechanisms lead to the formation of a dense
clot pattern, providing a surface for the cell-based coagulation cascade to proceed,
allowing for fibrin formation and further clot stabilization.[2]
[3]
Inherited platelet function disorders (iPFDs) can affect platelet production, morphology,
and function, with clinical presentations varying in severity.[4] Typical clinical manifestations are mucocutaneous and gastrointestinal bleeding,
menorrhagia, epistaxis, easy bruising, and bleeding from the oral cavity.[5] The bleeding type and severity of functional platelet defects can be assessed using
the ISTH-BAT bleeding score.[6] In general, it is difficult to predict the bleeding tendency from genetic findings
only[7] since most mutations are private.[8] The application of next-generation sequencing, including whole exome sequencing,
will be able to identify rare defects and benefit the diagnostic procedures but national
and international networks with bioinformatics will be important to analyze the results.[9]
This review article summarizes the current knowledge on iPFDs ([Supplementary Table S1] (available in the online version only) with a focus on genetics, clinical presentation,
and laboratory platelet function analysis to support physicians and laboratory staff
in the differential diagnosis of platelet function defects.
Glanzmann Thrombasthenia
Glanzmann thrombasthenia (GT) is a rare autosomal recessive inherited platelet function
disorder caused by deficiency or dysfunction of the integrin αIIbβ3 (glycoprotein
[GP] IIb/IIIa).[10] αIIbβ3 allows binding of fibrin(ogen) and VWF upon platelet activation, which is
essential for aggregation.[11]
[12] Lack of functional αIIbβ3 in GT becomes clinically relevant in homozygous or compound
heterozygous states. While there are also rare cases of acquired GT, due to autoantibody
formation, most cases of GT are inherited and common, seeking medical attention for
the first time during childhood.[13] Clinical presentations of GT range in severity, and life-threatening or fatal bleedings
can also occur spontaneously and as early as during birth. Clinically relevant bleeding
is treated with antifibrinolytics, platelet concentrates, and/or recombinant-activated
factor VII concentrates.[12]
[13]
As in other bleeding disorders, the bleeding history is the starting point for the
diagnostic workup, preferably using a standardized bleeding assessment tool.[14]
[15] However, GT may also remain undiagnosed until invasive procedures are performed.[10]
[13]
[16]
Platelets of patients with GT usually appear normal regarding phenotype and number
in microscopic blood smears, except for autosomal dominant ITBG3 variants with thrombocytopenia, enlarged platelets, and anisocytosis.[12]
[17] The platelet function analyzer (PFA), as a tool for the global assessment of primary
hemostasis, is considered to have a high sensitivity for severe iPFDs such as GT,
but low sensitivity for mild iPFDs. Similar findings have been reported for whole-blood
impedance aggregometry.[18]
[19]
[20] In contrast, light transmission aggregometry (LTA) remains the gold standard for
platelet function analyses, revealing GT by reduced aggregation pattern upon stimulation
with all agonists except ristocetin. The latter is because ristocetin-mediated binding
of VWF to platelet GPIbα directly results in platelet clumping due to major passive
agglutination and not to fibrinogen-dependent aggregation. In addition, Glanzmann
platelets present with disturbed clot retraction and adhesion on fibrinogen due to
loss or impaired αIIbβ3 integrin outside-in signaling. Nevertheless, in the case of
a typical aggregation pattern of Glanzmann's disease (no/impaired response to all
agonists except ristocetin), other diseases (e.g., myeloproliferative disorders with
activation defects in αIIbβ3 or antiplatelet αIIbβ3 therapy with abciximab, eptifibatide,
tirofiban) and a preanalytical artifact (low temperature during sample transport)
should also be considered in the differential diagnosis. For instance, autoantibodies
or paraproteins that bind to the receptor can lead to loss of function without platelet
destruction. However, this is an acquired Glanzmann, which must be distinguished from
the congenital variant.[21] Distinguishing between these two forms involves several clinical and laboratory
investigations. Family history and onset of symptoms can help differentiate between
congenital and acquired disease. In addition, Glanzmann platelets present with disturbed
adhesion on fibrinogen and clot retraction.
Flow cytometric analysis (FCA) of platelet GP expression should be performed when
GT is suspected. In FCA, the binding of an antibody that recognizes the extracellular
domain of αIIbβ3 is notably reduced or missing. Furthermore, platelet activation analysis
detecting the binding of an antibody specific for activated αIIbβ3 (PAC-1) should
be applied.[22] The advantages of FCA include low sample volume, feasibility even in pronounced
thrombocytopenia, and the ability to identify heterozygous GT carriers. However, this
requires a careful gating strategy and examination of first-degree relatives should
be included. It is essential to acknowledge the importance of appropriate reference
interval validation of the method employed, especially to classify GT subtypes based
on residual expression (I: <5–10%; II: >5–10%; III: normal expression of nonfunctional
protein).[13]
[23] However, the severity of bleeding in patients with GT varies markedly. In addition
to FCA, immunofluorescence microscopy can be applied. This involves treating fixed
blood smears with antibodies of corresponding specificity to αIIbβ3 integrin or its
subunits, then analyzing their binding using fluorochrome-conjugated secondary antibodies.[24] Molecular genetics of targeting mutations in the coding of ITGA2B and ITGB3 genes
should be performed to confirm the inherited platelet disorder.[10]
Upon establishment of the diagnosis of GT, all patients should be human leukocyte
antigen (HLA)-typed and have a baseline human platelet antibody (HPA) and human leucocyte
antigen antibody screen, as the risk of isoantibody formation after platelet transfusions
against the missing αIIbβ3 integrin is high. This is of clinical importance especially
in pregnant patients, since anti-αIIbβ3 antibodies may cross the placenta and cause
fetal/neonatal thrombocytopenia.[25]
[26]
Bernard–Soulier Syndrome
In 1948, Jean Bernard and Jean-Pierre Soulier published the first description of a
patient with inherited thrombocytopenia, prolonged in vivo bleeding time, and enlarged
platelets.[27] Bernard–Soulier syndrome (BSS) is a rare hereditary, most likely underdiagnosed
bleeding disorder with variable macrothrombocytopenia, petechiae, epistaxis, gingival
bleeding, menorrhagia, rare gastrointestinal bleedings, and severe bleedings associated
with trauma or surgery.[7]
[28]
[29] Almost 50% of the patients with BSS have been misdiagnosed and treated as immune
thrombocytopenia in the past.[8]
BSS patients may have quantitative and/or qualitative defects of the platelet receptor
GPIb/IX/V, which is composed of four subunits: glycosylated GPIbα is attached by a
disulfide bond to GPIbβ, and noncovalently associated with GPIX and GPV.[9]
[30]
[31] Each platelet expresses 20,000 to 25,000 copies of GPIb/IX and ∼11,000 molecules
of GPV.[32]
The cytoplasmic tail portion of GPIbα interacts with actin-associated proteins, supporting
the maintenance of the platelet shape, size, and adhesive function. The interaction
of GPIbα with VWF mediates platelet adherence to the vascular subendothelium, which
can be investigated in ristocetin-induced platelet agglutination (RIPA).[33]
[34] Different mutations in the GP1BA gene, encoding the GPIbα subunit or in GP1BB (GPIbβ
subunit) or in GP9 (GPIX), prevent or reduce the surface expression of the whole GPIb/IX/V
complex and are typically inherited in an autosomal-recessive mode (biallelic BSS).
Few cases with single dominant mutations mainly impair the function (monoallelic BSS).[7] GPV deficiency does not cause BSS, but modulates thrombin-mediated platelet activation
and fibrin formation.[35]
The first step in laboratory testing includes the measurement of the blood cell count
ranging between 20 and 100 × 109/L in BSS patients.[28] The mean platelet volume (MPV) varies from 9.3 to 27 fL (mean 14.8 fL).[8] Of note, automated impedance-based measurement of the platelet count does not recognize
very large platelets and should be verified by microscope technique. Examination of
the stained blood smear includes the assessment of platelet size, and platelet morphology
typically showing very large or even giant platelets.[7]
[28]
[29] Classical preparation of platelet-rich plasma (PRP) by centrifugation leads to the
loss of enlarged platelets. As for other platelet disorders combined with dysfunction
and reduced platelet count, the WHO bleeding score does not always correlate with
platelet count in BSS.[8]
In the second step, platelet function assessment with the RIPA assay and/or flow cytometry
for the examination of GPIb/X/V platelet surface expression are useful.[36]
[37] Typically, RIPA is absent or reduced in BSS, while the responses to adenosine-diphosphate
(ADP), collagen, and arachidonic acid in the LTA are normal if the platelet mass is
sufficient for LTA. Depending on the underlying mutation, the expression of this receptor
is reduced.[29]
[36] Importantly, RIPA cannot be corrected by addition of normal plasma to exclude differential
diagnosis of von Willebrand disease.[7] In some cases, RIPA assay in patients with Bolzano mutation may be not sufficiently
sensitive for diagnosis.[28]
Defects in prothrombin consumption in serum from patients with BSS have been described.
GPIbα carries several binding sites for different coagulation factors, and GPIbα is
essential for thrombin generation on platelets (thrombin burst).[38]
Immunofluorescence microscopy of blood smears, using antibodies against specific proteins
affected in different hereditary platelet disorders including BSS, can be an alternative,
requiring, like flow cytometry, only a small volume of blood.[24]
The final step in laboratory testing includes molecular diagnostics. Usually, BSS
is characterized by autosomal-recessive inheritance with mutations in GP1BA, GP1BB,
or GP9 genes, including missense (54%), frameshifts (38%), and nonsense (8%) mutations;
however, none has been described for GPV.[7]
[8]
[33] In a large cohort of 211 families with biallelic BSS, 112 mutations in the GP1BA
(28%), GP1BB (28%), or GP9 (44%) have been described, while 85% were homozygous and
13% were compound heterozygous.[8] However, mutations can also be monoallelic (i.e., only one allele is affected with
autosomal dominant inheritance). The best-known mutation is the Bolzano variant (p.Ala172Val
of GPIbα) with usually only mild thrombocytopenia.[39]
Platelet-Type von Willebrand Disease
Platelet-Type von Willebrand Disease
The platelet-type von Willebrand disease (PT-VWD) is a rare, autosomal-dominant bleeding
disorder with dysfunctional platelets.[26] Bleeding becomes more severe in situations like childbirth and surgery. Laboratory
testing shows low VWF:ristocetin cofactor and low or normal VWF:antigen and characteristically
an enhanced RIPA. This disease can be identified by LTA, when a subthreshold concentration
of ristocetin (0.5 mg/mL) does induce platelet agglutination. Of note, in neonates
VWF binding induced by 0.5 mg/mL of ristocetin measured by LTA or by flow cytometry
is normal.[40] Differentiating between von Willebrand type IIB and platelet-type VWD by plasma
exchange experiments requires experience and diagnostic options. Here, fresh platelets
and plasma from healthy volunteers are required to perform cross-comparisons by incubation
in the plasma of a healthy donor with platelets from the patient and vice versa. In
the case of PT-VWD, a RIPA is normal when using platelets from healthy volunteers
and patient plasma, whereas when using patient platelets, a pathologically increased
agglutination is observed when using a low concentration of ristocetin (e.g., 0.5 mg/mL).
This makes it possible to differentiate whether the platelets or the plasma is the
cause of the phenotype.[41]
[42] However, thrombocytopenia due to increased platelet binding to VWF does not fully
explain the bleeding tendency. It has recently been described that in this disease,
a lack of signaling due to the malfunction of the GPIb receptor can also increase
the bleeding tendency in these patients.[26]
[43]
Platelet Granule Defects
Platelets contain three types of granules that can be readily distinguished by their
content, structure, release, and function: (1) α-granules, (2) δ-granules (dense bodies),
and (3) lysosomal granules.
During megakaryopoiesis, a common immature precursor of α- and δ-granules is formed
and the immature granules in megakaryocytes are referred to “multivesicular bodies”
(MVB I and II). These structures are initially formed by clathrin-dependent endosomes,
but also by endocytosis from the plasma membrane. Early MVB I contains only internal
vesicles; MVB II additionally has an electron-dense matrix. Sorting into α- and δ-granules
occurs before finally mature granules are formed.
α-Granule Defects
α-Granules are the most abundant ones with ∼50 to 80 per platelet.[44] They contain numerous proteins, including platelet-derived growth factor (PDGF),
platelet factor 4 (PF4), or VWF, most of which are synthesized in megakaryocytes,
while some, like fibrinogen or factor V, are taken up from plasma. α-Granules are
still modified in circulating platelets due to a continuous shuttling between membrane
receptors and the MVB and to further differentiate into distinct α- and δ-granules.
Several transmembrane proteins are incorporated into the α-granule membrane, including
P-selectin (CD62P), which is virtually absent from the plasma membrane of resting
platelets. CD62P is a well-established marker for platelet activation in response
to stimulation. It is also expressed on Weibel-Palade bodies in endothelial cells,
where it can be exocytosed to the plasma membrane upon endothelial cell activation.
CD62P can be cleaved from the cell surface by sheddases, resulting in a cleaved transmembrane
protein and a soluble ectodomain (sCD62P), which can be detected in plasma and whose
expression level has been assayed in distinct disease conditions, such as sickle cell
anemia.[45] Triggering receptor expressed on myeloid cells (TREM)-like transcript-1 (TLT-1)
has been considered to be a more sensitive marker for α-granule release with a comparable
specificity,[46] but not yet been broadly established.
The most common inherited disorder affecting α-granule number and content is the gray
platelet syndrome (GPS; OMIM #139090)[47] which is associated with a mild to moderate bleeding tendency, macrothrombocytopenia,
and elevated vitamin B12 levels. Bone marrow fibrosis and splenomegaly may occur during
disease progression.[47]
[48] In May–Gruenwald–Giemsa-stained blood smears, platelets appear larger and greyish
rather than purple as dyes are not incorporated into granules. GPS is caused by homozygous
or compound heterozygous mutations in NBEAL2 which encodes neurobeachin-like 2, a
protein that is essential for the formation of α-granules and deficiency thus resulting
in loss of α-granules.[49]
[50]
[51]
In addition, NBEAL-2 is required for neutrophil- and natural killer-cell function
and pathogen defense[52]
[53] and recently Delage et al described a low CTA4 expression in activated T cells from
NBEEAL-2-deficient patients.
Certain mutations in the transcription factors GFI1B and GATA1 also lead to gray platelets,[54] as many genes are not adequately expressed. The genes VPS33B and VIPAS39 are involved
in MVB and granule maturation and variants in these genes cause the arthrogryposis–renal
dysfunction–cholestasis (ARC) syndrome (OMIM #208085).[55] These disorders share several platelet features with GPS, but are not referred to
as GPS.[54]
Blood smears are often a primary hint, but the same staining pattern can be attributed
to several gene defects. This holds also true for immunofluorescence of platelet proteins
performed on blood smears, but these approaches can clearly help with the diagnosis.[24] Defects in α-granules are best detected by flow cytometry using antibodies against
CD62P or TLT-1, which should be absent on resting platelets, but becomes readily detectable
after adding a high dose of a strong agonist like thrombin. Transmission electron
microscopy (TEM) is often considered the gold standard to verify a lack of α-granules,
but typically time- and labor-intensive and restricted to specialized laboratories.
There is at the moment no curative approach for patients with α-granule defects: GPS
is associated with an increased, but overall mild bleeding risk that can be treated
with platelet transfusions, tranexamic acid, or desmopressin.[5]
[47] Patients with ARC and GPS syndrome require detailed clinical observations. Patients
harboring variants in transcription factors GATA1 should also be followed up carefully
over time; both transcription factors are associated with red blood cell defects,
and a transition into a pre-leukemic condition may occur depending on the causative
mutation.[56]
An altered composition of α-granules is found in the Quebec platelet disorder (QPD),
a disease with autosomal dominant inheritance, in which a gain-of-function defect
in fibrinolysis leads to a more than 100-fold increase in platelet stores of urokinase
plasminogen activator. This leads to increased plasmin-mediated degradation of α-granule
proteins.[57]
δ-Granule Defects
Dense bodies (δ-granules) contain ADP, adenosine triphosphate (ATP), serotonin, Ca2+ cations, pyrophosphate, and polyphosphate and express lysosomal membrane proteins
(LAMP1/2; CD107a/b), which can be detected on the platelet surface upon stimulation.
Granulophysin (CD63), another marker for lysosomes, is also expressed in δ-granules
and can be used to detect the release of both granule types. δ-Granules appear often
as “eyed” granules in TEM micrographs, but are best visualized by whole-mount TEM
where they appear as electron-dense dots on the micrographs.
Upon platelet activation, small molecules are released from the δ-granules, which
act as important autocrine platelet agonists, such as ADP, ATP, and serotonin. Released
polyphosphates and Ca2+ cations essentially contribute to the platelet-based amplification of thrombin generation
during the coagulation process. Disorders of platelet δ-granule formation are characterized
by quantitative or qualitative defects manifested as isolated or syndromic forms.[4]
[58] The bleeding severity of δ-storage pool disorders (δ-SPDs) is variable, ranging
from mild to severe when assessed by the ISTH-BAT bleeding score.[6] However, a mild spontaneous bleeding diathesis can be life-threatening after trauma
or surgery.
Congenital δ-SPDs are typically caused by pathogenic variants in genes either responsible
for the biogenesis and trafficking of lysosome-related organelles (melanosomes, lysosomes)
including platelet δ-granules during late megakaryopoiesis, or encoding distinct transcription
factors (e.g., GATA1, RUNX1, FLI1) essentially involved in early megakaryopoiesis.
The syndromic δ-SPDs, Hermansky-Pudlak syndrome (HPS), Chediak–Higashi syndrome (CHS),
and Griscelli's syndrome (GS) with autosomal recessive inheritance are predominantly
recognized rather by clinical manifestations. Currently, 11 different HPS subtypes
are known based on the mutated gene (ISTH TIER1 genes: HPS1, AP3B1 [HPS2], HPS3-HPS6,
AP3D1, BLOC1S3, BLOC1S5, BLOC1S6, DTNBP1). Prominent clinical associations of HPS
are oculocutaneous albinism, nystagmus, immunodeficiency (i.e., HPS-2), granulomatous
colitis (i.e., HPS-1, HPS-4), hemophagocytic lymphohistiocytosis (i.e., HPS-2), and
pulmonary fibrosis. Patients with CHS caused by pathogenic variants in LYST present
with partial albinism, immunodeficiency, hemophagocytic lymphohistiocytosis, neurological
defects, and hepatosplenomegaly. For all these syndromes (HPS, CHS, GS), platelet
δ-granules are absent or the number is strongly reduced, despite normal platelet count.
In comparison, GS is caused by pathogenic variants in MYO5A, RAB27A, or MLPH and patients
present with normal or reduced platelet count and partial albinism, silver hair, neurological
defects, and immunodeficiency. SLFN14 gene mutation causes a combined defect with
mild thrombocytopenia, enlarged platelets, and reduced ATP secretion. Patients show
a disproportionately high bleeding tendency compared with the rather mild thrombocytopenia.[59]
For the laboratory diagnosis of quantitative δ-SPD with reduced or absent δ-granules,
the whole mount TEM represents the “gold standard” method.[60] The sensitive and specific quantification of ADP, ATP (HPLC, luminescence), and
serotonin (HPLC, ELISA) in platelet lysates allows the evaluation of the major δ-granule
cargo.[61] However, these technologies and assays are not available in routine laboratories.
Alternatively, a combination of different platelet function assays allows the characterization
of intrinsic or secretory platelet δ-granule defects. LTA may indicate a δ-SPD when
the second aggregation wave is missing, reduced, and/or characterized by a clear disaggregation
in response to the weak agonists ADP and epinephrine or to the strong agonist collagen
in a dose-dependent manner. However, normal aggregation responses especially with
high agonist concentrations do not exclude a mild or moderate δ-SPD.[62] Therefore, a combination of LTA with lumiaggregometry, flow cytometry (δ-granule
and lysosome membrane marker CD63, mepacrine assay), and immunofluorescence (CD63,
LAMP1, and LAMP-2) is recommended to prevent missing and misdiagnosis of this platelet
function disorder.[63]
[64]
Combined α-Granule and δ-Granule Defects
Pathogenic variants in the transcription factor genes GATA1, RUNX1, and FLI1 can also
cause combined α-granule and δ-granule defects combined with thrombocytopenia with
or without enlarged platelets.[65]
[66]
[67]
Defects of Platelet Signaling Pathways
Defects of Platelet Signaling Pathways
Inherited disorders of platelet receptors, namely, fibrinogen receptor αIIbβ3 integrin
deficiency/dysfunction (GT), von Willebrand factor receptor GPIb–V–IX deficiency/dysfunction
(BSS, PT-VWD), as well as deficiencies of the collagen receptor GPVI, ADP receptor
P2Y12, and thromboxane A2 (TxA2) receptor TPα, are well known to cause dysfunctional signal transduction in platelets.
In comparison, intrinsic defects of platelet signaling affect specific or common receptor-mediated
pathways, including TxA2-, guanosine-triphosphate (GTP)-binding protein and αIIbβ3 integrin inside-out signaling.[68]
[69] Defects of the GPVI receptor are gaining increasing attention. The receptor plays
a significant role in thrombus formation and growth. Especially the procoagulant activity
of platelets is triggered via GPVI.[70] Alterations in GPVI signaling can be manifold. For instance, the protooncogenic tyrosine-protein kinase SRC (SRC) plays a crucial role in the
signaling by GPVI. SRC is also involved in the signaling processes of the integrin and GPIb–V–IX receptors
on platelets. A mutation in the PTPRJ gene is associated with absent CD148 and reduced SRC activation in platelets. The
PTPRJ gene encodes a receptor-like protein tyrosine phosphatase, known as PTPRJ or CD148,
which is abundantly expressed in platelets and megakaryocytes. Clinically, the platelets
of these patients have a reduced response to convulxin via the GPVI receptor, are
morphologically reduced in number and size, and patients have a clear clinical tendency
to bleed.[71] A mutation in the EPHB2 gene leads to reduced crosstalk between GPVI and G protein-coupled
receptor signaling. This results in reduced responsiveness of the GPVI receptor, the
consequences of which are not yet fully understood.[72]
The ADP receptor P2Y12 is predominantly expressed on platelets and enables the amplification of platelet
activation leading to irreversible platelet aggregation. Disorders associated with P2Y12 receptor include conditions like bleeding disorders
and certain types of thromboembolic diseases. The bleeding tendency is variable and often exacerbated by comedication. In particular,
bleeding during surgical interventions and trauma has been described.[73] TXA2 receptor defects are rare and not easy to distinguish from cyclooxygenase inhibitor/aspirin
intake or cyclooxygenase defects. Here, the receptor itself is affected. Patients
with this defect show a complete lack of response to thromboxane analogs such as U46619.
Although rare, defects in the common pathway of TxA2 generation are most frequent within the group of congenital platelet signal transduction
disorders. They are caused by pathogenic variants in PLA2G4A, encoding the phospholipase
A2 (group IV); in PTGS1, encoding the cyclooxygenase-1 (COX-1)/prostaglandin-endoperoxide
synthase 1; or in TBXAS1, encoding the TxA synthase 1 (Ghosal syndrome), with autosomal
recessive or dominant inheritance. Usually, the platelet count is normal, and patients
present with mild to moderate bleeding diathesis, which could be life-threatening
under hemostatic challenges. Similar to aspirin treatment, resulting in irreversible
inhibition of COX-1, platelets from patients with deficient COX-1 or TxA2 synthase showed absent or reduced arachidonic acid-mediated aggregation in the LTA
(“aspirin-like defect”).[62]
[74] However, platelet cytosolic phospholipase A2 (PLA2) deficiency cannot be identified by arachidonic acid-induced platelet aggregation
tests because PLA2 itself liberates arachidonic acid through hydrolysis of membrane phospholipids.[75] Inherited and acquired platelet disorders of TxA2 synthesis can be accompanied by impaired aggregation induced by weak agonists, such
as ADP and epinephrine, and by low concentrations of strong agonists, such as collagen
and the thrombin receptor PAR-1 agonist activating peptide TRAP-6, but the inter-individual
responses are variable. Platelet aggregation with the TxA2 analog U46619 is normal due to the direct activation of the TP receptor. The quantification
of serum TxB2, a stable metabolite of TxA2, by liquid chromatography-mass-spectrometry or ELISA-based assays is recommended
as a reference test, since it specifically detects all enzymatic dysfunctions involved
in platelet TxA2 formation.[76]
Soluble platelet agonists such as ADP, TxA2, epinephrine, serotonin (5-HT), and thrombin act as important platelet feedback agonists
via guanine nucleotide-binding protein-coupled receptors. Upon receptor ligation,
the α-subunit of the heterotrimeric G proteins converts from the GDP- to the active
GTP-bound form, dissociates from the receptor-complexed β- and γ-subunits, and transmits
downstream signaling.[77] Congenital platelet function disorders with pathogenic variants in genes of distinct
subunits from different G protein subfamilies have been described. Patients with defects
of platelet-activating G proteins frequently present with mild to moderate bleeding
symptoms, predominantly upon hemostatic challenges. Platelet deficiency in Gαq, encoded
by GNAQ, impairs platelet activation via the thrombin receptors PAR-1 and PAR-4, ADP
receptor P2Y1, TxA2 receptor TPα, and the serotonin receptor 5HT2A and can be functionally analyzed by
LTA and flow cytometry in response to intermediate concentrations of PAR-1 and PAR-4-activating
peptides and the TxA2 analog U46619. In contrast, normal platelet function is expected
in response to a high concentration of a GPVI agonist, Ca2+ ionophore, and phorbol ester, as these pathways are insensitive to ADP- and TxA2-mediated signaling.[62] However, a similar dysfunctional platelet phenotype is expected when the expression
of phospholipase-β2 (PLCβ2), encoded by PLCB2, is absent or reduced as PLCβ is a central
downstream target of Gq protein-coupled receptors.[78] Here, only the application of Western analysis, GTPase activity assay, and phospho-proteomics,
in combination with genetic screening, can specify the Gq protein-coupled receptor
signaling defect.
Activating signaling via the Gi- and Gz-coupled receptors P2Y12 for ADP and α2 adrenergic receptor for epinephrine, respectively, interact with the
inhibitory Gs-coupled IP receptor pathway by regulating the cytoplasmic level of the
inhibitory second messenger cyclic adenosine-monophosphate (cAMP). While signaling
via Gαi/z-coupled receptors leads to the inhibition of the adenylate cyclase and its
cAMP synthesis, prostacyclin-mediated activation of the Gαs-coupled receptor IP results
in adenylate cyclase activation, increase in cytoplasmic cAMP-level, activation to
protein kinase A and platelet inhibition.[79] Gi-like platelet defects are functionally detected by absent or reduced platelet
aggregation in response to epinephrine and no second wave of aggregation in response
to ADP. In comparison, maximal aggregation responses induced by arachidonic acid and
high concentration of the PAR-1 agonist TRAP-6 and collagen are within the reference
range.[62] Although dysfunction of the Gαi subunit is more frequent than that of the αq subunit,
cases of congenital Gi deficiency (i.e., pathogenic variants in GNAI1–3) are very
rare.
Inherited platelet defects affecting αIIbβ3 integrin inside-out signaling are characterized
by GT-like platelet dysfunction and bleeding symptoms.[80] Leukocyte adhesion deficiency syndrome type 3 (LAD-III) is an autosomal recessive
disease caused by pathogenic variants in FERMT3, encoding the focal adhesion protein
kindlin-3. Kindlin-3 binds to the cytoplasmic tails of the integrin β-subunit and
cooperates with talin in integrin activation. Infections, impaired wound healing,
and osteopetrosis are frequently observed in patients with LAD-III. Kindlin-3 deficiency
does not affect integrin expression, but agonist-induced activation of integrins in
leukocytes and especially of the platelet αIIbβ3 integrin.[81] Another GT-like platelet disorder is associated with mutations in RASGRP2 encoding
diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), a Ca2+-dependent activator of Rap-1, which is essentially involved in αIIbβ3 integrin inside-out
and outside-in signaling.[82]
[83]
[84] Therefore, patients with a CalDAG-GEFI defect showed reduced platelet spreading
on fibrinogen and impaired platelet aggregation in response to weak agonists and lower
concentrations of strong agonists with the exception of protein kinase C (PKC)-stimulating
phorbol esters. Specialized assays for the measurement of Rap-1 activation are established
only in research laboratories.
Defects of Platelet Procoagulant Activity
Defects of Platelet Procoagulant Activity
Dysregulation of Ca2+-dependent platelet functions primarily leads to alterations of procoagulant activity
and the surface presentation of negatively charged phospholipids such as phosphatidylserine
(PS). Platelet PS exposure is important for Ca2+-dependent binding of the Gla-domain containing coagulation factors (e.g., FX, FII
of the final prothrombinase complex) to enable platelet-dependent amplification of
thrombin generation.[85]
[86] Loss-of-function variants in ANO6/TMEM16F encoding anoctamin-6, a transmembrane
ion channel protein, scrambling Ca2+-dependent phospholipids from the inner to the outer leaflet of the cell membrane,
cause the very rare autosomal recessive Scott syndrome. Scott patients have a normal
platelet count, morphology, and aggregation responses, but platelets fail to expose
PS and subsequently to generate thrombin, fibrin, and extracellular vesicles. These
dysfunctional platelets do not promote the stabilization of the thrombus and therefore,
Scott patients present with moderate to severe bleeding diathesis during and after
surgery.[87]
[88] A bleeding tendency can also be associated with spontaneously enhanced platelet
PS exposure, caused by inherited defects of Ca2+-release-activated Ca2+ channels as observed for the rare Stormorken's syndrome, which is also linked to
immune deficiencies.[89] Autosomal dominant gain-of-function variants in sensor stromal interaction molecule
1 (STIM1), encoding the Ca2+ STIM1 within the dense tubular system, or in ORAI1, encoding Orai1 within the plasma
membrane, cause impaired store-operated Ca2+ entry accompanied by elevated cytosolic Ca2+ levels and preactivated platelets in vivo. Thus, patients with Stormorken's syndrome
showed moderate thrombocytopenia associated with increased basal platelet surface
expression of PS and P-selectin but reduced agonist-induced αIIbβ3 integrin activation
capacity, assessed by flow cytometry, and impaired thrombus formation under flow in
a multiparameter-based flow chamber assay.[90] Flow cytometric detection of fluorochrome-conjugated annexin-V binding to platelets
stimulated by strong agonists such as thrombin, thrombin plus a GPVI agonist (COAT,
coated platelets), and Ca2+ ionophore in the presence of Ca2+ is recommended to analyze platelet PS translocation.[85] Semiautomated thrombin generation assays in PRP provide functional readouts for
altered platelet coagulation capacity.
Acquired Platelet Defects
Acquired Platelet Defects
Patients can present with acquired platelet defects due to external factors or conditions
that develop during a person's lifetime.
Medications and Nutritional Deficiencies
Besides platelet function inhibitors, there are a considerable number of drugs that
can affect platelet function, as outlined in [Table 1].[91] Among the most frequently encountered types are nonsteroidal anti-inflammatory drugs,
antihypertensive drugs, antibiotics, and psychotropic medications. Furthermore, certain
diets, herbs, alcohol, and nutritional supplements or fasting itself have been shown
or are suspected to impair platelet function and laboratory tests ([Table 2]).[92] Certain nutritional deficiencies like vitamin B12 deficiency have also been shown
to affect platelet count and function.[93]
[94]
Table 1
Overview of some medications altering platelet function, modified after Scharf[91]
|
Substance class
|
Drugs
|
Suspected mechanism
|
|
NSAIDs
|
Diclofenac, ibuprofen, indomethacin, naproxen
|
Reversible COX-1-inhibition
|
|
Beta-lactam antibiotics
|
Ampicillin, penicillin G, carbenicillin, piperacillin
|
Platelet receptor a/o VWF receptor interaction
|
|
Cephalosporins
|
Cefotaxime, cefoperazone, cephalothin
|
platelet membrane constituents interaction
|
|
Other antibiotics
|
nitrofurantoin, miconazole
|
Inhibition of platelet cox-1
|
|
PDE inhibitors
|
Sildenafil, caffeine, theophylline, aminophylline
|
Inhibition of PDE5; activation defect of integrin αIIbβ3
|
|
Nitrates
|
Nitroprusside, nitroglycerin, isosorbide dinitrate
|
Nitric oxide increases with the increase in platelet cAMP/cGMP
|
|
Beta-adrenergic receptor blockers
|
Propranolol, nebivolol, pindolol
|
Independent of β-adrenoreceptor blockade
|
|
Calcium channel blockers
|
Verapamil, nifedipine, diltiazem
|
Inhibition of epinephrine to α2-adrenergic receptors and of platelet response to TXA2
and serotonin
|
|
Angiotensin-converting enzyme inhibitors
|
Ramipril, captopril, enalapril, lisinopril
|
Downregulation of αIIbβ3?
NO-mediated drug effects
|
|
Angiotensin receptor blockers
|
Valsartan, losartan, olmesartan
|
Downregulation of αIIbβ3
Interference with TXA2 receptor
|
|
Antiarrhythmic drugs
|
Quinidine
|
Blockade of α2-adrenergic receptors
|
|
HMG-CoA inhibitors (statins)
|
Atorvastatin, cerivastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin,
simvastatin
|
Changes in lipid composition of the platelet plasma membrane; inhibition of protein
prenylation with subsequent inhibition of GTP-binding proteins
|
|
Plasma expanders
|
Dextrans, hydroxyethyl starch
|
Interaction with platelet membrane constituents
|
|
Tricyclic antidepressants
|
Imipramine, amitriptyline, nortriptyline
|
Unclear
|
|
Phenothiazines
|
Chlorpromazine, promethazine, trifluoperazine
|
Inhibition of calmodulin-dependent pathways
|
|
Selective serotonin reuptake inhibitors
|
Fluoxetine, paroxetine, sertraline
|
Multifactorial mechanisms suggested
|
Abbreviations: cAMP, cyclic adenosine-monophosphate; cGMP, cyclic guanosine-monophosphate;
COX-1, cyclooxygenase-1; GTP, guanosine-triphosphate; NSAIDs, nonsteroidal anti-inflammatory
drugs; PDE5, phosphodiesterase type 5; TXA2, thromboxane A2.
Table 2
Overview of some foods and diets altering platelet function, modified after McEwen[92]
|
- Berries (especially raspberry extract)
|
|
- Coffee and caffeine
|
|
- Cocoa and dark chocolate
|
|
- Energy drink
|
|
- Garlic
|
|
- Ginger
|
|
- Kiwi fruit
|
|
- Omega-3 polyunsaturated fatty acids
|
|
- Onion
|
|
- Purple grape juice
|
|
- Red wine
|
|
- Tomato
|
|
- Turmeric/Curcumin
|
|
- Vegetarian diet
|
|
- White wine
|
|
- Bromelain
|
|
- Ginkgo biloba extract
|
Medical Conditions
In uremic patients, platelets have been shown to have similar defects as a δ-SPD (reduced
ADP and serotonin content in platelet granule; reduced δ-granule; impaired ATP release).[95]
Hematologic diseases such as myelodysplastic syndrome, myeloproliferative neoplasms
(e.g., essential thrombocythemia), or acute leukemia are associated with acquired
δ-SPD and can inherently lead to a decreased platelet function.[96]
Moreover, autoantibodies against platelet receptors result in acquired forms of Glanzmann's
disease or delta storage pool disease which should be especially considered in elderly
patients with new bleeding symptoms. Apart from bleeding control, the therapy consists
of immunosuppression.[97]
Platelet aggregation has been shown to be impaired in patients with severe sepsis
and is associated with increased mortality.[98] δ-SPD can secondarily occur due to partial or complete granule exocytosis of activated
platelets in thromboinflammatory states such as acute ischemic stroke[99] and severe COVID-19.[100]
Platelet Dysfunction Ex Vivo Due to Platelet Pathological Platelet Activation In Vivo
Certain conditions can lead to a prolonged activation of platelets in vivo and consecutively
to a release of platelet granules (also called “exhausted platelet syndrome”). Reasons
include prolonged bleeding situations in which platelets are constantly activated
and consumed.[101] Limited adhesion and activation of the platelets as well as limited maximum aggregation
are described.[102] This is also true for activation through interaction with nonphysiological surfaces
such as extracorporeal devices, especially systems with high blood flow rates such
as ventricular assist devices, extracorporeal membrane oxygenation, and cardiopulmonary
bypass leading to impaired platelet granule release ex vivo.[103]
[104]
[105]
[106]
Conclusion
iPFDs are a heterogeneous group of rare bleeding diseases in terms of frequency, bleeding
severity, platelet dysfunction, and platelet count/volume. They are caused by different
and multiple gene defects, which result in altered megakaryopoiesis, platelet production,
and platelet function. If a reduced platelet count does not explain the bleeding phenotype,
a combined iPFD should be considered. Although iPFDs are rare, their prevalence is
underestimated due to a limited diagnostic potential and challenging platelet function
tests. The complex laboratory diagnosis is usually possible only in specialized laboratories.
In case of a positive family history, platelet dysfunction should be considered after
the exclusion of a plasmatic coagulation disorder. However, in some cases, iPFDs are
also associated with VWD or other coagulation factor disorders. Especially the diagnosis
of inherited platelet function defects with mild to moderate bleeding diathesis, determined
by a standardized bleeding assessment tool, requires a combination of platelet phenotyping,
different platelet function assays, and genotyping. Therefore, a specific diagnosis
of iPFDs is not only essential for preventive bleeding treatment and advice of affected
family members but also for the therapy of bleeding. Despite the upcoming automation
of classical platelet function tests to improve standardization, the establishment
of additional specialized platelet function assays in combination with whole genome
sequencing will help identify and phenotype new iPFDs, so far classified as a bleeding
disorder of unknown cause.
