Добавил:

Sekretar kiopkiopkiop18@yandex.ru t.me/Prokururor I Вовсе не секретарь, но почту проверяю Опубликованный материал нарушает ваши авторские права? Сообщите нам.

Вуз:

Ростовский Государственный Медицинский Университет

Предмет:

Медицина общая

Файл:

Книга_104_библиотеки_им_акад_М_И_Перельмана

.pdf

Скачиваний:

Добавлен:

30.08.2026

Размер:

42 Мб

Скачать

☆

<<< < Предыдущая 16 17 18 19 20 21 22 23 24 25 26 2728 / 4528 29 30 31 32 33 34 35 36 37 38 39 40 > Следующая >>>

GPlb complex

Bernard-Soulier syndrome

7 Transmembrane

e.g. TXA

α6β

Actin laments

Myosin heavy chain IIA

-glycosylated

https://t.me/med1917

Platelet-type von Willebrand disease

Platelet disorders 253

Collagen-receptor defects

α2β1

α5β1

receptor

αllbβ3

CLEC2

Glanzmann thrombasthenia

receptor defects

Figure18.2 Diagram of a resting platelet indicating the major receptors implicated in inherited bleeding disorders.

GPVl

Figure18.3 Structure of the GPlb- V- IX complex. GPlbα is linked via disulfide bridges to two GPlbβ molecules and this complex is non- covalently associated with GPIX and GPV in the ratio of 2 : 4 : 2 : 1. Mutations and deletions leading to Bernard- Soulier syndrome and platelet- type von Willebrand disease are described in the text. Mutations affecting cytoskeletal molecules such as filamin- 1 and myosin heavy chain IIA, which interact directly or indirectly with GPlb complex, can also lead to giant platelet syndromes.

本书版权归John Wiley & Sons Inc.所有

GPIb–V–IX complex

VWF-binding site

GPIX

GPIbα

2GPIbβ

Filamin

Thrombin-binding sites

GPV

Highly O domain

Calmodulin

14-3-3ζ

254 Molecular Hematology

https://t.me/med1917

structural and signaling roles, the function of GPV remains controversial; it has been suggested that it has a role in the thrombin response (it contains a thrombin- binding and cleavage site) as well as in platelet collagen binding. It was recently shown that thrombin- mediated shedding of GPV does not mainly regulate platelet activation in thrombus formation but instead has a distinct function after platelet deposition and specifically limits thrombin- dependent generation of fibrin, a crucial mediator of vascular thromboinflammation. Genetic or pharmacologic defects in hemostatic platelet function are unexpectedly lowered by specific blockade of GPV shedding, indicating that this control of thrombin- dependent fibrin generation could also be a potential therapeutic target to improve hemostasis.

Mutations and other genetic defects causing BSS have now been collected on a website (www.bernardsoulier.org) and will therefore not be listed in detail here. Many of the mutations found to cause BSS lie in the LRR domains and appear to destabilize their folding. The subunits also contain typical disulfide bridge patterns and thus mutation of the constituent cysteines or of other amino acids close to cysteine have deleterious effects on folding, leading to a lack of expression of the affected subunit and hence decrease in expression of the rest of the complex.

Most mutations in GPIbα either cause folding problems or are nonsense mutations leading to premature termination. Mutations in GPIbβ generally have the same effect as those in GPIbα concerning expression. Mutations in GPIX, while causing many of the classic symptoms of BSS, generally do not produce such a severe phenotype because trace amounts (up to 10%) of functional GPIb are often still expressed, enough to support basic levels of hemostasis if other aspects, such as coagulation factors, are normal. The Asn45Ser mutation in GPIX is the most common cause of BSS among northern Europeans, accounting for the majority of cases among these populations. Because this mutation generally does not cause such a marked phenotype as many of the GPIbα and GPIbβ mutations, patients have not been easily detected and diagnosis is only now catching up. This mutation has also been found in patients of Turkish origin. Other mutations in GPIX, leading to its failure to express, produce similar phenotypes.

Mice models of BSS have been produced through targeted knockout of GPIbα or GPIbβ but so far not GPIX (most likely because the rest of the complex is partially expressed). The phenotype is essentially the same as in humans. Research on megakaryocytes from these mice indicates that proplatelet formation and microtubule coil assembly are defective.

GPV

No cases of BSS due to defects in GPV have been described and mice lacking GPV express GPIb–IX normally. On the other hand, expression of GPIb–IX appears to be essential

for stable expression of GPV on platelets, possibly by protecting it from proteolysis or via membrane complex structure. GPV knockout mice do not show major differences in phenotype using the present selection criteria and, in particular, do not have characteristics of BSS but do influence hemostasis positively.

Genetic and pharmacological approaches show that thrombin- mediated shedding of GPV does not primarily regulate platelet activation in thrombus formation, but has a distinct function after platelet deposition and limits thrombin- dependent generation of fibrin, a crucial mediator of vascular thrombo- inflammation. Genetic or pharmacologic defects in hemostatic platelet function are unexpectedly attenuated by specific blockade of GPV shedding, indicating that the control of thrombin- dependent fibrin generation also represents a possible therapeutic target to improve hemostasis.

GPIbα and β monoallelic mutations leading to thrombocytopenia and large platelets

Rare BSS variants have been described as having dominant inheritance and lead to expression of non- functional GPIb, although only one allele is affected. The molecular mechanism involved is not understood, although it has been suggested that the defective GPIbα molecule prevents the normal one from functioning. This class of mutation includes p(Cys20Gly), p(Val31Leu), p(Cys33Arg), p(Cys33Tyr), p(Tyr54Asp), p(Leu57Phe), p(Asn57His), p(Leu59Arg), p(Tyr70Asp), p(Leu83Phe). P(Arg127Gln), p(Asn150Ser), p(Leu1155Pro), p(Ala172Val) (known as BSS Bolzano), and Leu179del (known as BSS Nancy). Monoallelic mutations in GPIbβ have also been shown to lead to thrombocytopenia. These include p(Met1*), p(Leu16Pro), p(27Ser), p(Arg42Cys), p(Gly43Trp), p(Trp46*), p(Leu60Pro), p(Thr68Met), p(Pro79_Leu81del), p(Pro79_Leu81dup), p(Arg110fs), p(Tyr113Cys), p(Leu132Gln), p(Leu137Pro), p(Cys141*), p(Ala150Argfs*43), p(Leu168dup), and Exon2 deletion. Again, these mutations in GPIbβ are thought to prevent the GPIbnormally.

These types of mutation- causing folding problems illustrate the current limits of artificial intelligence programs such as DeepFold to indicate changes in folding caused by mutations because they try to establish “correct” folding based on comparison with the appropriate peptide sequences.

V- IX complex from functioning

Platelet- type von Willebrand disease

Platelet- type von Willebrand disease (VWD) is an inherited dominant bleeding disorder resembling VWD type IIB but caused by mutations in GPIbα rather than in the A1 domain of VWF (see Figures18.2 and18.3). Unlike BSS, platelet- type VWD shows a gain of function, with spontaneous binding of VWF to platelets. In platelet- type VWD, platelets are often

本书版权归John Wiley & Sons Inc.所有

Platelet disorders 255

Metalloprotease cleavage sit

R38C

https://t.me/med1917

enlarged like those in BSS, although the reasons for this are not yet clear. It may be due to enhanced binding of VWF to GPIb, which blocks other GPIb functions. The platelet- type VWD mutations that have been described include Gly233Val/ Ser and Met239Val, and a 27- bp deletion in the region of the gene coding for the macroglycopeptide domain of GPIbα has been reported to produce a similar effect. Recently, two related patients were diagnosed with platelet- type VWD due to a heterozygous 27 bp inframe deletion in GP1BA that removes amino acids 459–467 and therefore overlaps the deletion mentioned above. Studies on mutant proteins suggest that mutations in other amino acids of the flexible β- loop structure of GPIbα can also produce this phenotype. Recently, a p.Arg127Gln variant in GPIbα LRR5was shown to enhances affinity for VWF allosterically in a novel form of platelet- type VWD.

Collagen receptor defects

α2β1 integrin

A few patients have been reported with bleeding problems related to an α2β1 integrin deficiency, showing defective platelet adhesion to collagen while responses to other agonists were normal. Both patients were female and became normal following the menopause, suggesting a hormonal role in the disorder. Neither α2 hemostatic problems, but defects were reported in aggregation

−/−

nor β1

−/−

mice have major

to fibrillar collagen and adhesion to soluble collagen. Differences in occlusion times in thrombosis models in α2

−/−

or β1

mice seem to be dependent on the model used and

−/−

remain controversial.

GPVI

Several patients have been described with mild bleeding disorders related to low levels or deficiency of GPVI (Figure18.4). Most of the defects have not been diagnosed on a molecular level. There are two recent reports of bleeding disorders caused by compound heterozygous mutations in GPVI. One patient with a lifelong history of bleeding problems had structurally normal platelets but a functional platelet defect. Platelet aggregation was normal except for an absent response to collagen, convulxin, and the collagenrelated peptide. ATP- dense granule secretion was normal with ADP but defective with collagen. Thrombus formation on a collagen surface in flowing blood was reduced, but more single platelets are attached. PFA- 100 analysis showed a shortened collagen/ADP closure time. Flow cytometry showed absence of GPVI expression, while immunoblotting showed strongly reduced levels of GPVI. The patient is compound heterozygous for an out- of- frame 16- bp deletion and a missense mutation S175N in a highly conserved residue of the second immunoglobulin- like GPVI domain. The parents, who do not have clinical bleeding problems, are heterozygous carriers. The mother carries the S175N mutation and has a mild platelet functional defect. In vitro studies

Collagen

GPVI

Polymorphism sites

Figure18.4 Structure of the GPVI complex. GPVI is linked via a salt bridge to FcyR, which provides most of the signaling to the platelet cytoplasm. The positions of mutations leading to decreased expression and of polymorphisms thought to affect function are indicated. Activation of GPVI by autoantibodies can lead to coactivation of matrix metalloproteases and loss of GPVI by cleavage.

C2-2

C2-1

N-glycosylation

S175N

O-glycosylation

Salt bridge

ITAM-domains

本书版权归John Wiley & Sons Inc.所有

256 Molecular Hematology

Ca sites

https://t.me/med1917

showed reduced membrane expression and convulxin binding in the S175N mutant compared with the wild- type GPVI receptor.

The other patient, a 10- year- old girl, had a tendency to bruising since infancy, a prolonged bleeding time despite a normal platelet count and no antiplatelet antibodies. Collagen- induced platelet activation was null, although there was an incomplete deficiency of GPVI detected by flow cytometry. Immunoblotting showed abnormal residual GPVI, and no FcγR defect. DNA sequencing revealed an R38C mutation in exon 3 of one allele of GPVI and an insertion of five nucleotides in exon 4 of the other allele, leading to a premature nonsense codon and absence of the corresponding mRNA. Expression of the R38C mutation gave an abnormal protein migration and loss of collagen binding. This composite genetic GPVI defect leads to an absence of platelet responses to collagen and a mild bleeding phenotype.

Platelets from GPVI

−/−

mice do not adhere to collagen under static conditions, probably because the α2β1integrin is not activated. GPVI is probably important for platelet activation on subendothelium under low- shear conditions. Under high- shear conditions in GPVI

−/−

mice, α2β1 is probably activated via GPIb/VWF interactions, with VWF bound to collagen. Stable adhesion and spreading are strongly affected in GPVI

−/−

platelets, indicating that GPVI has an important role in downstream signaling to molecules critical for thrombus formation. Tail bleeding times in

−/−

GPVI wild type. Thrombosis models using both GPVI FcγR

mice are only slightly prolonged compared with

−/−

(the GPVI signaling subunit) mice are controversial

and

because the type of vascular wall damage involved is variable. Thus, laser- induced injuries did not implicate GPVI as a major factor in thrombosis, whereas FeCl3- induced injuries suggested a more substantial role. Other research pointed to thrombin production as a major factor in overcoming GPVI/ FcγR deficiencies in mice thrombosis models. There is a clear need for research involving mouse thrombosis models that are more closely related to human pathology, such as in the presence of fragile vascular plaque, before any definitive conclusions can be reached about collagen receptors in human thrombosis.

Increased amounts of soluble GPVI in plasma seem to be indicative of raised platelet activation in vivo for whatever reason. These may include vascular problems, inflammation, cirrhosis, or latent cancer.

Much recent research points to GPVI playing a role in interactions involved in fibrin formation and, possibly, in procoagulant platelet development, though there remains a lot of controversy about how this happens and how this might be affected in GPVI mutants and expression variants.

Platelet aggregation defects

Glanzmann thrombasthenia

Glanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder caused by defects in the αIIbβ3integrin or its signaling (see Figures18.2 and18.5). If this receptor is absent or defective, it is unable to interact with its ligands, including fibrinogen and VWF, but also fibronectin and vitronectin. Thus, platelet aggregates are not formed or are limited in extent, preventing efficient recruitment of platelets to a damaged vessel site. In addition, αIIbβ3–fibrinogen binding has an important role in clot retraction, the process by which wound edges are drawn together to help seal the wound. Thus, GT patients form more scar tissue than normal because wounds tend to reopen. Patients with platelets lacking, or with a severe deficiency (<5% of normal) in, αIIbβ3 are traditionally designated as type I, those with a moderate deficiency (10–50% of normal) as type II, and others as variants. The latter includes patients who express non- functional αIIbβ3 on their platelets. There is a very wide range of molecular defects in αIIb or β3 and as a consequence, the clinical symptoms are highly variable. However, even the same defect may show some variability in symptoms

Ligand-

αIIb

-binding

Thigh

Calf-1

Calf-2

Membrane

Talin Kindlin-3

Figure18.5 Structure of the αIIbβ3 complex. This major platelet integrin is shown in the activated state. The various structural domains are labeled. Cytoskeletal proteins associated with the cytoplasmic domain of β3in the activated state are shown.

β3

binding site

βAβ-Propeller

Disulde-rich domains

Hybrid PSI

EGF-1

EGF-2

EGF-3

EGF-4

βTD

本书版权归John Wiley & Sons Inc.所有

Platelet disorders 257

https://t.me/med1917

in different patients depending on other factors such as levels of coagulation factors. Typical symptoms include purpura, epistaxis, gum bleeding, and menorrhagia, but gastrointestinal and interjoint bleeding and hematuria are rare. Problems generally follow trauma and are rarely spontaneous. Diagnostic criteria are prolonged bleeding time and defective clot retraction. Platelet aggregation to all agonists except ristocetin is absent or defective. Molecular diagnosis of GT includes flow cytometry and/or Western blotting to establish whether both subunits are absent or present in reduced amounts.

Mutations and other genetic defects causing GT have now been collected on a database (http://sinaicentral. mssm.edu/intranet/research/glanzmann) and are therefore not listed in detail here. GT can be caused by mutations in either subunit αIIb or β3 and may show some distinct differences. Since fibrinogen is not synthesized in the megakaryocyte, but in the liver and transported to the α- granules from the plasma by αIIbβ3- dependent endocytosis, GT patients often lack or are deficient in platelet α- granule fibrinogen, a contributory factor to their poor hemostatic function. In patients lacking β3, who as a consequence lack the vitronectin receptor αvβ3 as well as αIIbβ3, the platelet α- granules, in addition to fibrinogen deficiency, also contain up to five times normal amounts of vitronectin. Vitronectin is synthesized in megakaryocytes, so these results suggest a possible role for αvβ3in transporting vitronectin from α- granules out of platelets. Both αIIb and β3 have a complex gene structure, with the αIIb gene (ITGA2B) composed of 30 exons and spanning 17 kb, while the β3 gene (ITGB3) consists of 15 exons and spans 46 kb. Both are located on chromosome 17q21–23. Genetic defects are distributed over the entire region.

Mutations/deletions may prevent subunit biosynthesis or

the Golgi or plasma membrane. When mutations do not affect folding too seriously, expression of lower levels of the complex may occur with some functions retained. Several GT mutations lead to expressed dysfunctional receptor. Thus, an Asp119Tyr mutation in β3 affects the key RGD binding site, while Ser752Pro removes a key phosphorylation site involved in outsidecating β3 (Arg724tTer), so that only eight of the normal 47 amino acids of the cytoplasmic domain are present, removes binding sites for the cytoskeletal proteins talin and kindlin- 2. Cys560Arg and Cys598Tyr mutations in β3 produce platelets that spontaneously bind fibrinogen because the β3in their αIIbβ3has its disulfide bridges blocked in the activated pattern and because the disulfide isomerase activity that normally switches between resting and activated and vice versa is unable to do so. A heterozygous cytoplasmic domain mutation in the αIIb subunit Arg995Gln within the

in signaling. A stop codon trun-

critical GFFKR sequence reduces expression of the complex and produces mild “thrombasthenia- like” symptoms. There is some evidence for cases of GT caused by replacement of DNA segments lying outside coding regions and affecting mRNA stability. A more general syndrome affecting insideout activation of several classes of integrins present in platelets, neutrophils, and lymphocytes has been shown to be due to mutations in the CalDAG- GEFI gene, preventing signaling to the small GTPase Rap1.

Recent studies on the linkage between ITGA2B and ITGB3 gene variants in patients with GT show disequilibrium, indicating that most disease- causing mutations are recent.

Agonist receptor defects

ADP and ATP receptor defects

Platelets interact with ADP via the purinergic seventransmembrane receptors P2Y1 and P2Y12. P2Y1 signals to release Ca2+ from the dense tubular system and cytoskeletal changes leading to shape change. P2Y12 signals to αIIbβ3lead- ing to platelet aggregation. Thus, platelet defects in P2Y12 were first thought to be GT variants. Platelets from rare patients in both France and Italy showed decreased and reversible platelet aggregation to ADP but normal shape change and calcium mobilization. This was due to an autosomal recessive hereditary disease affecting one allele of the P2Y12 gene. No patients with P2Y1 defects have been described so far, but knockout mice lacking this receptor have been prepared. P2X1 is the platelet receptor for ATP. Three molecules form a calcium channel that is regulated when platelets are activated, to control intracellular calcium levels. One case of a young girl with a bleeding syndrome caused by deletion of a single amino acid in P2X1 has been described. That this is a dominant inherited disease is presumably due to the defective molecule preventing the active calcium channel from forming.

Other primary agonist receptor defects

Rare examples of an Arg60Leu mutation in the thromboxane (TX)A2 receptor have been reported in some Japanese families that result in defective signaling. Mutations in other agonist receptors have been reported, but it is not clear that these affect platelet function. Common variants in the PAR4 gene (F2RL3) have been described, but only recently were effects of these on platelet activation observed, particularly in the presence of clinically used PAR1 inhibitors. So far, there havebeen no reports of bleeding disorders linked to defects in the PAR1 thrombin receptors.

本书版权归John Wiley & Sons Inc.所有

258 Molecular Hematology

https://t.me/med1917

Defects inintracellular signaling pathways

A wide range of defects in signaling molecules are known, often affecting other cells as well as platelets (Figure18.2). These include defects in phospholipase C (PLC) activation, calcium mobilization, and plekstrin phosphorylation. Studies in one of these patients with impaired PLC activation indicated a decrease only in PLC- β2, while other isoforms were normal. Coding sequence was normal, but mRNA for PLCβ2 was decreased, suggesting sequence changes in the gene outside the coding region affecting mRNA stability. Eight patients were described with bleeding problems and abnormal aggregation and secretion in response to several different agonists. Receptor- mediated calcium mobilization and/or plekstrin phosphorylation were abnormal in seven of these patients. Several patients have been reported with platelet cyclooxygenase deficiency, a milder bleeding disorder, and impaired platelet aggregation responses. Thromboxane synthase deficiency has been described in two patients. In other patients, a deficiency of the PLC- γ2 iso- form or a specific decrease in platelet Gαq has been reported.

Defects in signaling molecules probably account for many of the undiagnosed patients with mild to moderate bleeding problems. Techniques to diagnose these on a molecular basis are only starting to be developed and even proteomics by itself cannot be expected to detect all of these. There is still a large unmet need here.

production in megakaryocytes is defective and the proteins are directly released from the megakaryocytes. The platelets areoften larger than normal and the patients are somewhat thrombocytopenic. Platelet aggregation responses are variously affected and differ between patients. Tissue inhibitors of metalloproteinases (TIMPs) are still present in gray platelet syndrome and may account for some of this variability. In 2011, three groups applied nextstudy the genetic defect underlying gray platelet syndrome and showed mutations in NBEAL2 in all patients. NBEAL2 is a member of the LYS T (mutated in Chesiak- Higashi syndrome) gene family. Following these results, the production of NBEAL2 platelet syndrome. After injury, tissue regeneration was affected. Megakaryocyte maturation was delayed and few α- granules observed. Unusual direct secretion of VWF was also seen.

−/−

mice has extended phenotype studies in gray

generation sequencing to

Quebec platelet disorder

As the name implies, this autosomal dominant bleeding disorder was described in this region of Canada. Many α- granule proteins are degraded proteolytically, including membrane receptors such as P- selectin. Defective platelet aggregation is particularly strong with epinephrine as an agonist. The problem is caused by overexpression of urokinase plasminogen activator, a protease normally stored in α- granules and released on platelet activation.

Platelet secretion defects (storage pool disease)

This is a large and heterogeneous group of inherited disorders caused by intracellular defects of platelets (Figure18.2). Since secretion is a function of many cell types, it is clear that the phenotype may extend beyond hemostasis depending on which molecule is affected.

Defects ofα- granules

Proteins in α- granules are either synthesized in megakaryocytes or endocytosed from plasma. Membranes of α- granules also contain specific receptors, such as P- selectin and CD63, synthesized in megakaryocytes that translocate to the plasma membrane during platelet activation and secretion and are good markers for these processes. Inherited deficiencies of plasma proteins that are taken up into α- granules will be reflected in their content.

Gray platelet syndrome

Gray platelet syndrome is characterized by the absence of platelet contents and has a mostly autosomal recessive inheritance. Packaging or storage of proteins during platelet

Dense (δ) granule defects

Platelet- dense granules are storage sites for a number of small molecules including ADP, ATP, serotonin, and calcium. Defects in these granules therefore affect platelet aggregation responses (Figure18.2). Granule deficiencies are variable and disorders are known in which α- granules arealso affected, in which common pathway molecules are defective (αδorganelles exist in other cells, these are often disorders that affect pigmentation of the skin and hair, such as Hermansky– Pudlak, Chédiak–Higashi, and Griscelli syndromes. Mouse models exist for several of these.

storage pool deficiency). Since similar types of

Hermansky–Pudlak syndrome

Hermansky–Pudlak syndrome manifests as albinism and absence of ceroid- lipofuscin storage in the reticuloendothelial system. The syndrome is common in Puerto Rico and is caused by a frameshift due to a 16- bp duplication in exon 15 of the HPS- 1 gene, which normally encodes a 79- kDa protein with two membrane- spanning domains. Defects in at least eight genes cause distinct subtypes of Hermansky–Pudlak syndrome in humans. Recently. novel variants were found in the HPS3, HPS5, and DTNBP1 (HPS- 7) genes.

本书版权归John Wiley & Sons Inc.所有

Platelet disorders 259

https://t.me/med1917

Chédiak–Higashi syndrome

Severe immunological defects and progressive neurological dysfunction accompany bleeding problems. The major diagnostic criterion is the presence of giant inclusion bodies in a variety of cells with granules, including platelets. The gene responsible (LYST) has been cloned and a series of frameshift and nonsense mutations identified that produce a truncated LYST protein. A milder form of the disease is thought to be due to rare missense mutations. LYST protein is large and complex with domains suggesting that it regulates organelle protein trafficking and membrane–membrane interactions.

Griscelli syndrome

Patients with Griscelli syndrome are partially albino with silver hair. Neurological defects and/or immunodeficiency are linked to lymphocyte cytotoxicity defects. Clinical manifestations include fatal complications caused by lymphoid cell activation and cytokine release. Griscelli syndrome is associated with mutations in the genes for myosin Va, Rab27a (asmall GTPase), or melanophilin. A mouse model for this disease has recently been characterized with a mutation in Rab27a, a variant of the ashen mouse with a phenotype resembling Hermansky–Pudlak syndrome. Rab27a deficiency in humans was identified in a young patient with psychomotor retardation. The patient’s platelets lacked dense granules but had normal α- granules; epinephrine- induced aggregation was defective.

Wiskott–Aldrich syndrome

Platelets in Wiskott–Aldrich syndrome (WAS) aggregate poorly and have few granules. This is an X- linked recessive disease characterized by thrombocytopenia, small platelets, eczema, immunodeficiency, and increased autoimmunity and malignancy problems. Hereditary X- linked thrombocytopenia is a milder form lacking the immune problems. T lymphocytes are the other major blood cells affected. The

amino- acid WAS protein is encoded by the large WAS

502gene, which consists of 12 exons. Mutations in exons 1 and 2mostly lead to the mild form. WAS protein regulates actin polymerization in hematopoietic cells and its deficiency leads to premature proplatelet formation in the bone marrow. In mice, profilin- 1 deficiency in megakaryocytes was shown to lead to microtubule instability and a large increase in acetylated tubulin as well as affecting platelet production and causing a WAS- like platelet defect.

Procoagulant regulation defects

Scott syndrome

Scott syndrome is a very rare disorder found in a few humans and some dogs. So far, there is no mouse model.

When normal platelets are activated, one of the changes produced is the exposure of negatively charged phospholipids on the surface, critical for procoagulant activity. This is absent in Scott syndrome platelets and as a consequence, less thrombin and fibrin are formed in the platelet plug, leading to bleeding complications. This defect also affects membrane vesiculation, suggesting a common mechanism. Under invitro conditions, this phenomenon requires platelet activation by thrombin and collagen (or a GPVI agonist such as convulxin or collagen-

related peptide). The molecular defect in Scott syndrome was recently attributed to mutations in the gene for anoctamin 6 (TMEM16F), a Ca

activated Cl−- channel.

Surface- exposed protein Labeling using PeroxidaSe, H2O2, and Tyramide- derivative (SLAPSHOT), has been used to label extracellularly exposed proteins in a rapid, sensitive, and specific manner, while preserving cellular integrity. Soluble APEX2 peroxidase is applied to cells, allowing a wide spectrum of experimental setups. SLAPSHOT followed by quantitative mass spectrometry- based proteomics analysis was used to examine the immediate and extensive cell surface expansion and membrane shedding upon the activation of Scott syndrome- linked TMEM16F, a ubiquitously expressed calcium- dependent phospholipid scramblase and ion channel. Time- course calcium stimulation using wild- type and TMEM16F deficient cells revealed intricate co- regulation of known protein families, including those in the integrin and ICAM families. Proteins were identified that reside in intracellular organelles, including ER, in the freshly deposited membrane, and microvesicles as an abundant component and contributor to the extracellularly exposed proteome.

Stormorken syndrome

Stormorken syndrome is an even rarer disorder confined so far to one family, implying a dominant inheritance. Unlike Scott syndrome, the platelets are constitutively activated with surface exposure of negatively charged phospholipids and raised levels of microvesicles even under resting conditions. The molecular basis of Stormorken syndrome was recently shown to be a dominant mutation in STIM1.

Gain of function mutations in STIM1 is responsible for a CRAC channel defect in York platelet syndrome characterized by thrombocytopenia, ultrastructural granule abnormalities including giant dense granules and massive, multilayered target bodies, and lack of Ca

- storage in

δ- granules.

The most frequently occurring mutation in patients with Stormorken syndrome is R304W, which destabilizes and extends the STIM1 C terminus independently of ER Ca2+ store depletion, causing constitutive binding to Orai1 and CRAC channel activation. Cis deletion of one amino acid residue, Glu

296

(called E296del), reversed the pathological

effects of R304W. Homozygous Stim1 E296del + R304W

本书版权归John Wiley & Sons Inc.所有

260 Molecular Hematology

Giant platelet syndromes

Thromboxane synthase

Griscelli syndrome

https://t.me/med1917

mice were viable and phenotypically indistinguishable from wild- type mice. NMR spectroscopy, molecular dynamics simulations, and cellular experiments revealed that although the R304W mutation prevented CC1 from interacting with CC3, the additional deletion of Glu

296

opposed this effect by enabling CC1- CC3 binding and restoring the CC domain interactions within STIM1 that are critical for proper CRAC channel function.

Giant platelet syndromes andcytoskeletal defects

Although this group is diverse, many of the patients have mutations or other defects in the MYH9 gene coding for non- muscle myosin heavy chain IIA (see Figures 18.3 and18.6). Since this is expressed in a variety of cells, not only platelets are affected. Macrothrombocytopenia (variable, up to leukocyte size) is accompanied by variable phenotypes in other cells/tissues. Döhle bodies in leukocytes with a distinctive distribution of myosin IIA detected by immunofluorescence is a major diagnostic criterion. Hearing loss caused by

defective cilia, nephritis, and cataracts are all possible symptoms in later life. Characteristic mutation zones in the protein seem linked to particular symptoms: mutations in the head region can affect folding of the entire molecule and can yield more serious symptoms, whereas mutations toward the C- terminus have less- dramatic effects, possibly because function is partly maintained and there is less tendency to form denatured protein clusters. While non- muscle myosin has an important role in many cells/tissues, the presence of the IIB and IIC forms may compensate for some of these.

Giant platelets have also been reported in patients with mutations in the FLNA gene coding for filamin A. As well as platelets, neuronal migration was abnormal in these patients. In platelets, filamin A is a major attachment site for the GPIb complex, stabilizing the connection between the membrane and the cytoskeleton, and anchoring GPIb in platelets when they attach during adhesion under high shear. Myosin IIA may play a role in this process as well providing a rationale for the giant platelets in these disorders as well as BSS. Recently, a number of patients have been described with mutations in the β- tubulin gene also leading to macrothrombocytopenia and similar defects were also found in dogs. It is

Metabolic defects

ATP synthesis

Enzyme defects

Cyclooxygenase

Lipoxygenase

Myosin heavy chain llA

Filamin 1

Signaling defects

Kinases/phosphatases

G-proteins, Ca

Phospholipases/PKC

uxes

α-granule syndromes

Gray platelet syndrome

α, δ-deciency

Quebec syndrome

Dense granule syndromes

Chédiak–Higashi syndrome

Hermansky–Pudlak syndrome

Figure18.6 Diagram of a resting platelet indicating the major internal organelles and other structures implicated in inherited bleeding disorders.

本书版权归John Wiley & Sons Inc.所有

Platelet disorders 261

https://t.me/med1917

important to distinguish these large platelet syndromes (including BSS), particularly when accompanied by thrombocytopenia, from idiopathic thrombocytopenic purpura and to avoid treatments such as splenectomy, which are not useful in these disorders.

Medich giant platelet syndrome

To date, five cases of Medich giant platelet syndrome have been reported. The cases are unified by the ultrastructural findings of abnormal alpha granules and unusual cytoplasmic scrolls. All patients experienced mucosal bleeding; however, many clinical, biologic, and genetic characteristics of this rare disorder remain to be determined.

Transcription factor defects

Autosomal dominant Paris–Trousseau syndrome is a typical example of this class of disorder, with decreased platelet production and a mild bleeding tendency associated with a deletion at 11q23. Platelets are often larger than normal with giant α- granules. The defect leads to a hemizygous loss of the FLI1 gene coding for Fli1 transcription factor. A subpopulation of megakaryocyte precursors fails to mature adequately to produce platelets. Other disorders including thrombocytopenia are caused by mutations in the GATA- 1 gene. Platelets may be larger than normal with poor response to collagen. The phenotype depends on whether the mutations affect GATA- 1interactions with DNA or FOG- 1. GATA- 1dependent platelet molecules include members of the GPIb complex and α- granule components. Defects in RUNX1lead to platelets with a deficiency in protein kinase C- θ and lack of phosphorylation of its downstream targets but also an increased tendency to develop leukemia.

Glycosylation defects

X- Linked immunodeficiency with magnesium defect, Epstein–Barr virus infection and neoplasia (XMEN) disease is a primary immunodeficiency due to lossmutations in the gene encoding for the magnesium transporter 1 (MAGT1). As MAGT1 is involved in the N- glycosylation process, XMEN disease is classified as a Congenital Disorder of Glycosylation. Platelet analysis showed abnormal elongated cells and unusual barbellshaped proplatelets. Platelet aggregation, integrin α activation, calcium mobilization, and protein kinase C (PKC) activity were impaired in both patients examined. Platelet responses to protease- activated receptor 1 activating peptide (PAR1- AP) were absent at both low and high concentrations. These defects were also associated with a decreased molecular mass of glycoprotein (GP)Ibα, GPVI,

of- function

IIbβ3

and integrin α

due to a partial reduction in N- glycosylation.

IIb

All these defects were corrected after hematopoietic stemcell transplantation.

Platelet protein polymorphisms andtendency tothrombosis

Platelet activation responses vary widely in the population but are reproducible for individuals and show a strong genetic component. Following the demonstration of major polymorphisms in coagulation factors affecting thrombotic tendencies, there has been a major effort to identify polymorphisms in platelet proteins such as receptors that could explain the interindividual variation. There is also considerable interest in polymorphisms that affect patient responses to antithrombotic treatments because this could be an important way to improve treatment and reduce mortality.

Common polymorphisms in αIIbβ3were among the first targets for statistical investigation. Each of the subunits contains a common amino acid dimorphism: β3 has two isoforms, Leu33/Pro33, that lead to conformational differences in the proteins; the Ile843/Ser843 dimorphism in αIIb has also been intensively investigated. Most of the studies were controversial, with only minor effects detected. The situation in GPIbα is more complicated, with two major polymorphic systems present: (i) a Thr145/Met145 dimorphism and (ii)several versions of a tandem repeat structure of 13 amino acids within the macroglycopeptide domain; these have the nomenclature VLTR- A, - B, - C, and - D with four, three, two and one copies, respectively, of the tandem repeat. The incidence varies widely depending on ethnic origins between Europe and East Asia. Again, there has been no consensus reached about a role in thrombosis susceptibility and many studies have given contradictory results. Polymorphisms in collagen receptors α2β1 and GPVI have also been implicated in thrombosis susceptibility, but these studies still remain controversial. Perhaps if one considers that many individual genetic differences may contribute to the overall thrombosis susceptibility, it is not so surprising that these single differences do not have more pronounced effects. However, newly discovered polymorphisms, particularly in signaling molecules, are still under investigation. One area that is less controversial is the effect of cytochrome P450 polymorphisms on activation of clopidogrel and therefore on its ability to inhibit the ADP receptor P2Y12. Among persons treated with clopidogrel, carriers of a reduced- function CYP2C19 allele had significantly lower levels of the active metabolite of clopidogrel, diminished platelet inhibition, and a higher rate of major adverse cardiovascular events, including stent thrombosis, than did non- carriers.

several

本书版权归John Wiley & Sons Inc.所有

262 Molecular Hematology

https://t.me/med1917