The Pro33 isoform of integrin beta3 enhances outside-in signaling in human platelets by regulating the activation of serine/threonine phosphatases.
Integrin beta(3) is polymorphic at residue 33 (Leu(33) or Pro(33)), and the Pro(33)-positive platelets display enhanced aggregation, P-selectin secretion, and shorter bleeding times. Because outside-in signaling is critical for platelet function, we hypothesized that the Pro(33) variant provides a more efficient signaling than the Leu(33) isoform. When compared with Pro(33)-negative platelets, Pro(33)-positive platelets demonstrated significantly greater serine/threonine phosphorylation of extracellular signal-regulated kinase (ERK2) and myosin light chain (MLC) but not cytoplasmic phospholipase A2 upon thrombin-induced aggregation. Tyrosine phosphorylation of integrin beta(3) and the adaptor protein Shc was no different in the fibrinogen-engaged platelets from both genotypes. The addition of Integrilin (alpha(IIb)beta(3)-fibrinogen blocker) or okadaic acid (serine/threonine phosphatase inhibitor) dramatically enhanced ERK2 and MLC phosphorylation in the Pro(33)-negative platelets when compared with Pro(33)-positive platelets, suggesting that integrin engagement during platelet aggregation activates serine/threonine phosphatases. The phosphatase activity of myosin phosphatase (MP) that dephosphorylates MLC is inactivated by phosphorylation of the myosin binding subunit of MP at Thr(696), and aggregating Pro(33)-positive platelets exhibited an increased Thr(696) phosphorylation of MP. These studies highlight a role for the dephosphorylation events via the serine/threonine phosphatases during the integrin outside-in signaling mechanism, and the Leu(33) --> Pro polymorphism regulates this process. Furthermore, these findings support a mechanism whereby the reported enhanced alpha granule secretion in the Pro(33)-positive platelets could be mediated by an increased phosphorylation of MLC, which in turn is caused by an increased phosphorylation and subsequent inactivation of myosin phosphatase.
Vijayan
K Vinod
KV
Department of Medicine, Baylor College of Medicine, One Baylor Plaza, BCM 286, N1319, Houston, TX 77030, USA. vvijayan@bcm.tmc.edu
Liu
Yan
Y
Sun
Wensheng
W
Ito
Masaaki
M
Bray
Paul F
PF
D001792:Blood Platelets / Q000378:metabolism*; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D039661:Integrin beta3 / Q000737:chemistry*; D007930:Leucine / Q000737:chemistry; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D008954:Models, Biological; D018994:Myosin Light Chains / Q000737:chemistry; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism; D010766:Phosphorylation; D010973:Platelet Adhesiveness; D011110:Polymorphism, Genetic; D011392:Proline / Q000737:chemistry*; D020033:Protein Isoforms; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000502:physiology*; D015398:Signal Transduction; D013912:Threonine / Q000737:chemistry; D014443:Tyrosine / Q000378:metabolism
C061248:ITGB3 protein, human; D039661:Integrin beta3; D018994:Myosin Light Chains; D020033:Protein Isoforms; D013912:Threonine; D014443:Tyrosine; D005340:Fibrinogen; D011392:Proline; D017346:Protein Serine-Threonine Kinases; D019950:Mitogen-Activated Protein Kinase 1; D043263:Myosin-Light-Chain Phosphatase; D007930:Leucine