Page 1907 - Williams Hematology ( PDFDrive )

1882 Part XII: Hemostasis and Thrombosis Chapter 112: Platelet Morphology, Biochemistry, and Function 1883

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Na /Ca antiporter, whereas the DTS/sarcoplasmic reticulum contains Platelets from mice lacking PI3K p85α aggregate normally to ADP,
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a more slowly exchanging pool of Ca regulated by a Ca /Mg ATPase thrombin, U46619 and phorbol esters, but display impaired responses
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(sarco-/endoplasmic reticulum Ca -ATPase 3 [SERCA3]), a pump that to collagen and CRP and diminished tyrosine phosphorylation of the
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also appears to be located in the plasma membrane. 1645 During ago- PI3K effectors Btk, Tec, Akt, and PLCγ . 2 1661 FcγRIIA-induced platelet
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nist stimulation, most Ca enters the platelet cytosolic compartment aggregation requires PI3K activity, which is upstream of PLCγ in the
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through receptor-operated calcium channels (reviewed in Ref. 1646) pathway. 1662 Genetic deletion of PI3Kβ in mice results in embryonic
in the plasma membrane. Collagen, for example, causes Na entry lethality, but mice possessing a kinase dead form of the enzyme have
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into platelets, which reverses the Na /Ca antiporter to promote Ca been generated. Platelets from these mice have defects in G-protein–
entry, thus contributing to platelet aggregation. 1647 Release of intracel- coupled receptor (GPCR)-, collagen-, and integrin-mediated signaling
lular Ca from the DTS/sarcoplasmic reticulum also occurs rapidly in pathways. 1663 Platelets from mice lacking PI3Kγ isoform aggregate nor-
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response to agonist stimulation, in large a result of the IP generated as mally to thrombin and collagen but have impaired responses to ADP,
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part of the phosphoinositide cycle. 1646,1648 The release of internal stores and PI3Kγ-deficient mice are protected from ADP-induced throm-
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of Ca results in translocation of STIM1 from the DTS/sarcoplasmic boembolism. 1664 Platelets from mice expressing a kinase dead form of
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reticulum, followed by activation of the plasma membrane Ca channel PI3Kγ have defective GPCR-induced activation of Rap1 and aggrega-
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OraiI, leading to store-operated Ca entry. Calcium entry is also sup- tion, but normal responses through GPVI-activated pathways. 1663 A
ported by TRPC6 mediating non–store-operated mechanisms, induced working model to explain the observations is that PI3Kβ serves as a
by DAG. 93,119,1646 The role of TRPC1 remains to be defined. 93,119,1646 Integ- common intermediary of signals elicited by GPCR, collagen, and integ-
rin α β may also participate in Ca entry. 1649 rin ligation, whereas PI3Kγ primarily affects GPCR-initiated pathways.
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An intracellular rise of Ca levels induces numerous downstream Many of the biologic actions of PI3K are mediated by their phospholipid
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events, including activation of Ca -sensitive forms of PLA 2 1650 and products, which bind to specific sequences in proteins. The pleckstrin
PKC 1651 ; calmodulin-dependent enzymes such as myosin light-chain homology (PH) domains (approximately 100-amino-acids long) present
kinase, which phosphorylates myosin light chain 1652 and promotes in pleckstrin and other platelet proteins involved in signal transduction,
cytoskeletal rearrangements required for platelet shape change; and recognize either PI(3,4)P or PI(3,4,5)P . Binding of PI(3,4,5)P to the
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gelsolin, which facilitates actin severing and rearrangement, secretion, aminoterminal PH domain in PLCγ enhances its activity. 1666 PI(3,4,5)
and aggregation. In addition, Ca probably plays a direct role in con- P binding to PH domains in BTK 1667 targets BTK to the plasma mem-
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trolling the secretory machinery, which mediates the membrane fusion brane, where it is further phosphorylated and activated. 1668 PI(3,4)P or
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events that result in degranulation and the release reaction. Calcium- PI(3,4,5)P binding to the PH domains in the serine/threonine kinase
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dependent proteases or calpains also become activated and play an Akt (or protein kinase B) changes the conformation of Akt, permitting
important role in postaggregation events. The Ca binding protein CIB it to become activated by phosphorylation on Ser and Thr by Akt-ki-
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(calcium and integrin binding protein) 1653 binds to the membrane prox- nase (PDK1). 1669,1670 Akt activation is biphasic, occurring before and
imal region of α IIb 1654 and contributes to platelet spreading. 31 after platelet aggregation. 1659 Two isoforms of Akt are present in human
platelets (Akt1 and Akt2). 1671 The Akt isoforms have multiple substrates
Phosphoinositide 3′-Kinases in platelets. One prominent substrate is glycogen synthase kinase
PI3Ks are a family of lipid kinases that phosphorylate the D-3 hydroxyl (GSK)-3β, which is inactivated by Akt-mediated phosphorylation.
group of the myoinositol ring of phosphoinositides. 1655,1656 Class I PI3Ks GSK-3β suppresses platelet function and thrombosis in mice. 1672 Akt
are heterodimeric protein complexes containing both adaptor and cat- activation also stimulates NO production and resultant protein kinase
alytic subunits that use PI, PI(4)P, and PI(4,5)P as substrates to form G (PKG)-dependent degranulation. 1673 Finally, Akt has been implicated
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PI(3)P, PI(3,4)P , and PI(3,4,5)P , respectively. Class Ia (PI3Kα, PI3Kβ, in activation of a cAMP-dependent phosphodiesterase (PDE3A), which
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and PI3Kδ) and class Ib (PI3Kγ) have distinct subunits and regulatory plays a role in reducing platelet cAMP levels after thrombin stimula-
features. The catalytic subunit of class Ia PI3K is a Mr 110,000 to 120,000 tion. 1674 Each of these Akt-mediated events is expected to contribute to
protein; the adaptor subunit, p85 (PI3K p85α), has two SH2 domains, platelet activation. Deficiency of Akt2 in mice impairs platelet aggre-
a breakpoint cluster region homology domain, a Pro-rich region, and gation, secretion, and fibrinogen binding in response to low doses of
a single SH3 domain. Members of this class of PI3K possess intrinsic thrombin and U46619, but has minimal effects on collagen signaling. 1675
serine-threonine protein kinase activity in addition to lipid kinase activ- Akt2 null mice have normal bleeding times, but are protected from
ity, and they appear to be regulated, at least in part, by binding of the p85 experimental thrombosis, as are mice with a deficiency of Akt1. 1675,1676
subunit to tyrosine-phosphorylated proteins. Although platelets possess Interestingly, in platelets containing either kinase dead PI3Kβ or PI3Kγ,
PI3Kα and PI3Kδ, the main class Ia member that is thought to contrib- activation of Akt by ADP was abolished, and yet under the same condi-
ute to platelet function is PI3Kβ. PI3Kγ has been isolated from plate- tion, aggregation was only modestly affected, 1663 which raises questions
lets and neutrophils and contains both regulatory (p101) and (p110γ) about the role of Akt in these events.
subunits; the latter is activated by the β/γ subunit of heterodimeric G
proteins. Both isoforms of PI3K appear to associate with the platelet Small G Proteins
cytoskeleton after agonist activation. Overview The Ras superfamily of small GTPases are intracellular
In platelets, 3-phosphorylated phosphoinositides are produced in transducers that act as “on–off” switches to facilitate the response to
response to a variety of agonists, including thrombin, TXA , LPA, ADP, extracellular stimuli. Platelets contain members of the Ras subfamily
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and collagen, and may mediate early signaling events that precede integ- (Ras, Ral, and Rap), the Rho subfamily (Rho, Rac, and Cdc42), the Rab
rin α β activation, as well as late events involved in stabilizing fibrino- subfamily (Rab 1, 3, 4, 6, 8, 11, 27, 31, 32), 1677–1680 and the Arf subfamily
IIb 3
gen binding and platelet aggregation. 1655–1657 Thrombin stimulates rapid (Arf1 or 3 and 6). 1681
accumulation of PI(3,4,5)P and PI(3,4)P , 2 1658 and late production of Rho family GTPases are regulators of cytoskeletal remodeling:
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Ptdlns(3,4)P ; the latter requires fibrinogen binding to α β and calpain Cdc42 for filopodia formation, Rac for lamellipodia and membrane
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activity. 1659 Collagen promotes the association of class Ia P13K via the ruffling, and Rho for focal adhesion and stress fiber formation. 1682,1683
SH2 domains with tyrosine-phosphorylated forms of FcRγ-chain and Platelets have Cdc42, 1684 Rac1, 1685 and RhoA. 1679 Resting platelets have
the regulatory protein, linker-for-activator T cells, to modulate PI3K. 1660 very low levels of the GTP-bound forms of these GTPases, 1686–1688 but all
Kaushansky_chapter 112_p1829-1914.indd 1882 17/09/15 3:30 pm