Page 1867 - Williams Hematology ( PDFDrive )

1842 Part XII: Hemostasis and Thrombosis Chapter 112: Platelet Morphology, Biochemistry, and Function 1843

changes, in particular, the binding of talin to the integrin β cytoplasmic extruding a very large fraction of the serum. This process is thought
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domain. 243–246 Tyrosine kinases, including FAK, 33,247 and Src, may play to mimic in vivo phenomena that result in consolidation of thrombi
a role in this process, along with cortactin, a protein of Mr 85 kDa that and perhaps enhancement of wound healing. Clot retraction has also
is phosphorylated on tyrosine, and small GTP binding proteins such as been implicated in decreasing porosity and solute transport so as to
Rho, Rac, and Cdc42. 216,229,248,249 When the attachment of integrin α β concentrate intrathrombus thrombin, as well as decreasing the effi-
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to the cytoskeleton includes actin and myosin, the force produced by ciency of thrombolysis, which may partially account for the resistance
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the cytoskeleton on the integrin may supply the energy to produce the of platelet-rich thrombi to fibrinolytic agents. The platelet require-
conformational changes that lead to higher ligand binding affinity. ment for clot retraction is indisputable as is a requirement for integrin
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After activation, more integrin α β molecules become associated with α β and a contractile mechanism involving actin and myosin. 274,275 In
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the cytoskeleton, and this presumably reflects the interaction with talin fact, nearly complete selective disruption of the myosin Myh9 gene in
and other cytoskeletal proteins and ligand-induced integrin clustering, murine megakaryocytes gives rise to a phenotype characterized by mac-
resulting in the development of protein complexes, including cytoskel- rothrombocytopenia; absence of clot retraction; reduced secretion in
etal proteins, on the cytoplasmic surface of the receptor. 237,245,251 When response to low concentrations of agonists, but not high concentrations;
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ligand-coated beads are added to adherent platelets and bind to integrin prolonged bleeding time; and protection from thrombus formation.
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α β receptors, the beads are transported to the center of the platelets, The mice do not, however, spontaneously bleed. Myosin activation
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indicating that the cytoskeleton can move integrin receptors that have involves phosphorylation of the myosin light chain, a process that is
bound ligand. 252,253 governed by calcium-regulated myosin light-chain kinase activity and
Platelets contain calpains, which are calcium-dependent, sulfhydryl- Rho kinase–regulated myosin phosphatase activity. Calpain-cleavage
containing, neutral proteases composed of two subunits that preferen- of the cytoplasmic tail of integrin β may promote RhoA activity and
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tially cleave cytoskeletal proteins, in particular filamins and talin, 229,254 serve a molecular switch to convert platelet spreading to clot retrac-
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but have also been reported to cleave the cytoplasmic domain of integrin tion. Other signaling molecules appear to contribute to clot retrac-
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β , and a number of molecules involved in signaling, including kinases tion, including the Eph kinase EphB2, protein phosphatase 2B,
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and phosphatases (see “Calcium-Dependent Proteases [Calpains]” and PI3K. Despite these data, no model describing the details of the
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below). μ-Calpain requires micromolar calcium and m-calpain requires clot retraction process has gained acceptance. Proposed mechanisms
millimolar calcium for activation. It has been proposed that calpains include movement of platelet filopodia along fibrin strands, tugging of
are involved in cytoskeletal reorganization upon platelet activation, spe- fibrin strands by filopodia, and internalization of fibrin by the action of
cifically via cleavage of the integrin β cytoplasmic tail and talin upon the membrane skeleton. 274,275,279–282
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ligand engagement. 245,255–257 Calpain cleavage of the integrin β cytoplas- Platelet integrin α β is required for clot retraction, as demon-
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mic tail may switch the function of the integrin from promoting plate- strated by studies of patients with Glanzmann thrombasthenia
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let spreading to mediating clot retraction. Calpains have also been (Chap. 121) and studies of normal platelets in the presence of agents
implicated in platelet spreading, microparticle formation, and the gen- that block either the integrin α β receptor 280,283–288 or the fibrinogen
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eration of platelet coagulant activity. 229,256,259 Mice lacking μ-calpain have γ-chain C-terminal sequence that mediates interactions with the inte-
reduced platelet aggregation and clot retraction, but normal bleeding grin. It also requires disulfide bond exchange and the tyrosine res-
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time. 260 idues on the integrin β subunit that are phosphorylated upon platelet
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activation and contribute to outside-in signaling. Clot retraction cor-
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PLATELET CONTRACTION AND CLOT relates temporally with an integrin α β -dependent decrease in protein
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RETRACTION tyrosine phosphorylation, presumably via activation of one or more
phosphatases, and may require both integrin-mediated mitogen-ac-
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The contractile mechanism involving actin and myosin is thought to tivated protein kinase (MAPK) activation and translation of proteins
facilitate granule secretion, but the details remain obscure. 261,262 In fact, such as Bcl-3, with the latter facilitated by ligand binding to integrin
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mice with nearly complete disruption of the platelet heavy-chain myo- α β . Results with integrin α β antagonists demonstrate, however,
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sin gene, Myh9, have a defect in secretion, but only in response to low differences in their ability to inhibit clot retraction that do not cor-
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concentrations of select agonists. The cytoskeleton of resting plate- relate with their ability to block fibrinogen binding to platelets, 280,287 and
lets consists of the membrane skeleton described above, which lies just patients with Glanzmann thrombasthenia differ in the extent of their
beneath the membrane, and a lacy cytoplasmic actin filament network defect in clot retraction. Some integrin α β mutations, such as integ-
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composed of 2000 to 5000 linear actin polymers, which also contains rin β L262P, interfere with interactions with fibrinogen but do not pre-
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α-actin, filamins (actin binding proteins) A and B, tropomyosin, vin- vent interactions with fibrin and clot retraction. Of particular note,
culin, and caldesmon. 176,177,248,249,264–268 The contractile response is also fibrinogen lacking the γ-chain C-terminal sequence (amino acids 400 to
thought to be initiated by an increase in cytosolic calcium, which results 411) that mediates binding to platelet integrin α β , as well as the two
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in the formation of a calcium-calmodulin complex that then activates Arg-Gly-Asp (RGD)-containing regions in fibrinogen, is still capable of
myosin light-chain kinase; phosphatases and cyclic adenosine mono- supporting clot retraction. 296,297 It is well established that when fibrino-
phosphate (cAMP) kinase can modulate this response. After the ini- gen converts to fibrin, new sites become exposed on the surface of the
tial platelet shape change, actin becomes organized centrally into thick molecule. Therefore, one possible explanation for this paradox is that
filamentous masses, where it probably associates with phosphorylated additional or alternative integrin binding sequences in the fibrinogen
myosin filaments. 269,270 The centralization of organelles within a contrac- γ-chain (e.g., 316 to 322, 370 to 383, or other regions) may be able to
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tile ring correlates with secretion. There is controversy, however, as to mediate clot retraction. 298,299 Potential binding sites for the γ370 to 381
whether platelets secrete their granular contents by fusion with the open sequence, which is better expressed on fibrin than fibrinogen, on the
canalicular system in the center of the platelet or by direct fusion with integrin α β-propeller region, were identified and peptides from these
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the plasma membrane, or both. 95,100 regions inhibit clot retraction. Factor XIII also plays an important
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When blood initially clots in vitro, the fibrin mesh extends role in clot retraction; it has been proposed to mediate the translocation
throughout, trapping virtually all of the serum in a gel-like state. of the fibrinogen/fibrin–integrin α β complex to sphingomyelin-rich
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If platelets are present, within minutes to hours, the clot retracts, lipid rafts in the platelet membrane as well as crosslink the complex to
Kaushansky_chapter 112_p1829-1914.indd 1842 17/09/15 3:26 pm