Page 1908 - Williams Hematology ( PDFDrive )

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

are converted to their GTP-bound states upon platelet activation. 1689,1690 complex termed “exocyst” for targeting secretory vesicles to specific
Thus, receptor-mediated signaling activates Rho family GTPases. Cdc42 plasma membrane domains. 1716 Both RalA and RalB in platelets are
and Rac1 are activated at a very early phase of stimulation (approxi- associated with platelet dense granules, 1717 and become rapidly activated
mately 10 sec) and reach maximal activation 30 seconds after stimu- in a Ca -dependent manner upon platelet activation. 1718 A recombinant
2+
lation with collagen, thrombin, or ADP. 1687,1688,1690,1691 This temporal Ral-interacting domain of Sec5, a downstream effector of RalA in exo-
response is consistent with an early role for these GTPases in filopodia cyst complex, inhibits serotonin release from platelet dense bodies, sug-
and lamellipodia formation. Integrin-dependent secondary signaling is gesting a role for Ral exocyst in platelet granule release. 1719
required for full activation of RhoA, 1686 but not Cdc42 or Rac1, 1687,1690 Rab Rab GTPases are the largest family of small GTPases; 63
suggesting a role for RhoA in both early (adhesion/aggregation) and late members are detected in the human genome. 1720 They are highly com-
(clot retraction) stages of platelet activation. More detailed descriptions partmentalized to different organelle membranes and function by coor-
of each subfamily follows. dinating vesicle transport, including vesicle formation and tethering to
Ras Platelets contain at least one Ras isoform (H-Ras). 1692 Despite their target compartments. 1720 Rab proteins have been shown to play
its intensively studied functions in proliferation, differentiation, and cell roles in both granule biogenesis and secretion.
survival in nucleated cells, 1693,1694 the exact role of Ras and its signal- Arf Arf family GTPases, in nucleated cells, function in secretory
ing in platelets is unclear. Platelets do contain most of the downstream and cytoskeletal processes. Platelets contain Arf1 or 3 and Arf6. Func-
Ras effectors: Raf-1, MEK (MAPK/ERK kinase), and ERK (extracellular tional studies of Arf6 show that unlike other platelet GTPases, it is in
signal-regulated kinase). 1695 Ras and ERK are both known to be acti- the GTP-bound state in resting platelets and there is a conversion to the
vated upon platelet stimulation. 1696 GDP-bound state upon platelet activation. 1681 Inhibitors of this transi-
Rho Inactivation of RhoA with C3 exoenzyme treatment inhibits tion disrupt aggregation, secretion, and clot retraction. Further analysis
agonist induced shape change, 1697–1699 adhesion/aggregation, 1686,1698,1700,1701 suggests that the Arf6-GTP to Arf6-GDP transition is required for acti-
and formation of focal adhesions. 1701 Platelets treated with the exoen- vation of Rho family proteins in platelets.
zyme also show decreased stress fibers formation, a process medi-
ated by Rho kinase-dependent phosphorylation of myosin light chain Calcium-Dependent Proteases (Calpains)
(MLC). 1686,1697,1700 After ligand binding, integrin clustering, and platelet aggregation, neu-
Rac In nucleated cells, Rac1 functions in actin remodeling via tral, cysteine proteases termed calpains become activated by a rise in
2+ 229
activation of three downstream effectors; phosphatidylinositol 4- intracellular Ca . The most important and best-studied calpains in
phosphate 5-kinase type Iα, p21-Cdc42/Rac-activated kinase, and sup- platelets are: μ-calpain (calpain-1), which is activated by micromolar
2+
pressor of cyclic AMP receptor/WASP-family verprolin-homologous concentrations of Ca and accounts for 80 percent of the Cys protease
protein. 1702 The roles of Rac1 in lamellipodia formation and aggregation activity in platelets, and m-calpain (calpain-2), which requires millimo-
2+
have been examined using platelets from mice lacking Rac1. Rac1 dele- lar levels of Ca for activation. 1721 Each calpain consists of a common
tion does not affect platelet production 1703–1705 or filopodia formation, Mr 30,000 regulatory subunit paired with a unique catalytic subunit of
but does affect lamellipodia formation upon stimulation with thrombin Mr 80,000. Activated μ-calpain cleaves numerous proteins, including
229
–/–
and collagen. 1704 Aggregation was diminished in Rac1 platelets when cytoskeletal proteins (e.g., filamin [actin binding protein], talin, WASP,
stimulated with low-dose of thrombin or collagen, or when subjected to and cortactin), tyrosine kinases (e.g., BTK, Src, Syk, and FAK), tyrosine
shear stress under flow condition. 1704,1705 phosphatases (e.g., protein tyrosine phosphatase 1B [PTP1B also called
Cdc42 The assessment of Cdc42 function in platelets is less clear. PTPN1], SHP-1, and PTPMEG), as well as other important platelet
Wiskott-Aldrich syndrome is caused by a defect in WASP, which is a proteins (e.g., integrin β , SNAP-23, Vav, PLC-β), and certain isoforms
3
downstream effector of Cdc42. However, the platelets from affected of PKC. 1721 Cleavage of talin by calpain in vitro enables talin to acti-
148
individuals have normal shape change, including filopodia formation vate integrin α β , but the role of calpain in activation of integrin
IIb 3
and Arp2/3 activation (Chap. 121). 1706 One study suggested that Cdc42 α β by talin in intact platelets is uncertain. Calpain also appears to be
IIb 3
might function in GPVI-mediated integrin α β activation and subse- upstream of, and able to affect, the activation of the small G proteins
2 1
quent platelet adhesion on collagen-coated surfaces. 969 Rac and RhoA. Calpain’s role in platelet secretion has not been defined,
Rap Rap GTPases participate in cell adhesion, cell–cell junction although it is clear that the t-SNARE, SNAP-23 is inactivated by calpain-
formation, and the development of cell polarity in nucleated cells. 1707 In mediate cleavage. 1722 Thus calpains, through their effects on structural
platelets, Rap1a, Rap1b, and Rap2 are all activated upon platelet stimu- and signaling molecules, appear to affect multiple aspects of platelet
lation. 1708,1709 Platelets from Rap1b mice have a defect in platelet aggre- function. Mice deficient in μ-calpain demonstrate abnormal platelet
–/–
gation and decreased integrin α β activation upon platelet stimulation aggregation, decreased clot retraction, and reduced tyrosine phospho-
IIb 3
with ADP or PAR-4 peptide. 1710 With the discovery of the important rylation of several platelet proteins, including the β subunit of integrin
role of CalDAG-GEF1, an exchange factor for Rap1 in platelet function, α β . These abnormalities in platelet function can be reversed by inhi-
IIb 3
Rap1’s role in integrin signaling has been a major focus of research. 1711 bition of tyrosine phosphatases or by deletion of PTP1B, suggesting that
Platelets lacking CalDAG-GEF1 have decreased Rap1B activation and μ-calpain’s effects on platelet kinases and phosphatases may be central
integrin α β activation. 1712 Integrin α β activation could be com- to its role in platelet function. 1723
IIb 3
IIb 3
pletely blocked by treating CalDAG-GEF1 platelets with a PKC inhib-
–/–
itor, suggesting that CalDAG-GEF1 and PKC function independently Inside-Out Activation of Integrin α β and Outside-in
IIb 3
to activate integrin α β . 1713 Studies using CHO cells reconstituted Signaling By Activated Integrin α β
IIb 3
IIb 3
with integrin α β , talin, and Rap1GTP-interacting adapter molecule The active state of integrin α β is defined as the conformation that
IIb 3
IIb 3
(RIAM) showed that Rap1GTP-dependent talin recruitment to integrin is competent to bind large, soluble, adhesive proteins, such as fibrin-
β by RIAM is required for integrin α β activation. 1714 The function of ogen and VWF, with relatively high affinity. Precise regulation of the
3
IIb 3
Rap2 remains to be determined; CalDAG-GEF1 does not interact with activation state of integrin α β is essential for maintenance of nor-
IIb 3
it. 1715 mal hemostasis, such that integrin α β activation only occurs upon
IIb 3
Ral In nucleated cells, Ral GTPases (RalA and RalB) are thought vascular injury. Crystallographic and electron microscope studies
to function in regulated exocytosis by recruiting a multisubunit suggest that the extracellular portion of both integrin α β and the
IIb 3
Kaushansky_chapter 112_p1829-1914.indd 1883 17/09/15 3:30 pm