Heat shock protein 70 regulates platelet integrin activation, granule secretion and aggregation

Abstract

Rigg RA, Healy LD, Nowak MS, Mallet J, Thierheimer ML, Pang J, McCarty OJ, Aslan JE. Heat shock protein 70 regulates platelet integrin activation, granule secretion and aggregation. Molecular chaperones that support protein quality control, including heat shock protein 70 (Hsp70), participate in diverse aspects of cellular and physiological function. Recent studies have reported roles for specific chaperone activities in blood platelets in maintaining hemostasis; however, the functions of Hsp70 in platelet physiology remain uninvestigated. Here we characterize roles for Hsp70 activity in platelet activation and function. In vitro biochemical, microscopy, flow cytometry, and aggregometry assays of platelet function, as well as ex vivo analyses of platelet aggregate formation in whole blood under shear, were carried out under Hsp70-inhibited conditions. Inhibition of platelet Hsp70 blocked platelet aggregation and granule secretion in response to collagen-related peptide (CRP), which engages the immunoreceptor tyrosine-based activation motif-bearing collagen receptor glycoprotein VI (GPVI)-Fc receptor-␥ chain complex. Hsp70 inhibition also reduced platelet integrin-␣IIb␤3 activation downstream of GPVI, as Hsp70-inhibited platelets showed reduced PAC-1 and fibrinogen binding. Ex vivo, pharmacological inhibition of Hsp70 in human whole blood prevented the formation of platelet aggregates on collagen under shear. Biochemical studies supported a role for Hsp70 in maintaining the assembly of the linker for activation of T cells signalosome, which couples GPVI-initiated signaling to integrin activation, secretion, and platelet function. Together, our results suggest that Hsp70 regulates platelet activation and function by supporting linker for activation of T cells-associated signaling events downstream of platelet GPVI engagement, suggesting a role for Hsp70 in the intracellular organization of signaling systems that mediate platelet secretion, "inside-out" activation of platelet integrin-␣IIb␤3, platelet-platelet aggregation, and, ultimately, hemostatic plug and thrombus formation. chaperones; hemostasis; integrin; platelets; thrombosis HEAT SHOCK PROTEIN (Hsp) 70 is an ATP-powered, 70-kDa molecular chaperone that regulates a myriad of protein qualitycontrol processes, including folding of nascent polypeptides, trafficking of proteins across membranes, prevention of protein aggregation, and protein complex assembly and disassembly (15,34,46). Hsp70 is an abundantly expressed and highly conserved protein with 50% sequence homology between mammals and prokaryotes, highlighting the importance of Hsp family members in protein homeostasis throughout evolution (10). In human cells, the significance and complexity of Hsp70 function are evident in the functions of the 13 members of the human Hsp70 family, including the cytosolic, stress-inducible Hsp70 (or Hsp72) and its constitutively expressed heat shock cognate 71-kDa protein (Hsc70), the endoplasmic reticulum (ER)-localized 78-kDa glucose-regulated protein (Grp78, or bindi...

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