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S7.1. Molecular mechanism of shear-induced platelet aggregation
Vol. 32, Nos. 2-3
Symposia
131
SYMPOSIUM 7; BIORHEQLQGY OF CELL ADHESION; AGGREGATION I N SUSPENSIONS
$7.1.
MOLECULAR MECHANISM PLATEI.ET AGGREGATION
OF
SHEAR-INDUCED
Y. IKEDA Department of Hematology, Keio University School of Medicine, Tokyo, Japan Shear-induced platelet aggregation (SIPA) which requires the binding of vWF to both GPIb and GPIIb/IIIa, is now considered to have an important clinical significance in the pathogenesis of arterial thrombosis. Thus, it is crucial to understand the molecular mechanisms to initiate and support SIPA. In this study, we have investigated the changes in intracellular calcium ion c o n c e n t r a t i o n ([Ca++]i) and protein tyrosine phosphorylation in h u m a n platelets during SIPA. We found that the binding of multimeric vWF to GPIb u n d e r high shear force (108 dynes/cm 2) resulted in the transmembrane calcium influx. The blockage of this event suppressed aggregation, while the blockage of vWF binding to GPIIb/IIIa inhibited aggregation, but not [Ca++]i increase. Increased tyrosine phosphorylation of proteins of 40, 58, 64, 70, 74, 85, 100, and 130 KDa was observed within 30 seconds after shearing. Monoclonal antibodies against vWF-binding domain of GPIbcz, GPIb-binding domain of vWF and GPIIb/IIIa completely abolished both SSIPA and tyrosine phosphorylation. Cytochalasin D or staurosporin did not affect SIPA, but inhibited tyrosine phosphorylation. These results indicate that the increase in [Ca++] i may act as an intracellular message to cause activation of GPIIb/IIIa and that tyrosine phosphorylation of platelet proteins is a post-aggregatory event. S7.2. FIBRINOGEN AND VON WILLEBRAND FACTORMEDIATED AGGREGATION OF ACTIVATED PLATEIJETS AT MODERATE SHEAR STRESS M. F R O J M O V I C , H. GOLDSMITH, F. M C I N T O S H , AND E. B R O W N
Q.
LIU,
T.
WONG,
Departments of Physiology and Medicine, McGill University, Montreal, Canada Platelet aggregation at moderate shear rates in Poiseuille flow (<1355 s1, equivalent to shear stresses of <1.5 Nm -2) of ADP-activated platelets occurs efficiently with fibrinogen (Fg) (Xia and Frojmovic, Biophys.J., 64: 2190, 1994), but persists in the absence of plasma proteins, independent of any secreted Fg (Goldsmith et al., Thromb. Haemost. 71, 78, 1994). The present work tests the hypothesis that von Willebrand Factor (vWF) expressed on activated platelets can mediate aggregation at modest shear rates, and reports adhesive molecular domains of Fg and vWF mediating aggregation. Activated platelets were incubated with FITC-Fg to assess occupied GPIIb-IIIa receptors or with FITCmAb's against 0tRGDF, 0tRGDS, ~,400--411 or T373-385 on platelet-bound Fg; and mAb's 152B6 and NMC-4, respectively blocking the GPIIb-IIIa and GPIb binding domains on vWF, and aurin tricarboxylic acid (ATA) which blocks the vWF-binding domain on GPIb. Platelets were stirred or sheared in a
Symposia
131
SYMPOSIUM 7; BIORHEQLQGY OF CELL ADHESION; AGGREGATION I N SUSPENSIONS
$7.1.
MOLECULAR MECHANISM PLATEI.ET AGGREGATION
OF
SHEAR-INDUCED
Y. IKEDA Department of Hematology, Keio University School of Medicine, Tokyo, Japan Shear-induced platelet aggregation (SIPA) which requires the binding of vWF to both GPIb and GPIIb/IIIa, is now considered to have an important clinical significance in the pathogenesis of arterial thrombosis. Thus, it is crucial to understand the molecular mechanisms to initiate and support SIPA. In this study, we have investigated the changes in intracellular calcium ion c o n c e n t r a t i o n ([Ca++]i) and protein tyrosine phosphorylation in h u m a n platelets during SIPA. We found that the binding of multimeric vWF to GPIb u n d e r high shear force (108 dynes/cm 2) resulted in the transmembrane calcium influx. The blockage of this event suppressed aggregation, while the blockage of vWF binding to GPIIb/IIIa inhibited aggregation, but not [Ca++]i increase. Increased tyrosine phosphorylation of proteins of 40, 58, 64, 70, 74, 85, 100, and 130 KDa was observed within 30 seconds after shearing. Monoclonal antibodies against vWF-binding domain of GPIbcz, GPIb-binding domain of vWF and GPIIb/IIIa completely abolished both SSIPA and tyrosine phosphorylation. Cytochalasin D or staurosporin did not affect SIPA, but inhibited tyrosine phosphorylation. These results indicate that the increase in [Ca++] i may act as an intracellular message to cause activation of GPIIb/IIIa and that tyrosine phosphorylation of platelet proteins is a post-aggregatory event. S7.2. FIBRINOGEN AND VON WILLEBRAND FACTORMEDIATED AGGREGATION OF ACTIVATED PLATEIJETS AT MODERATE SHEAR STRESS M. F R O J M O V I C , H. GOLDSMITH, F. M C I N T O S H , AND E. B R O W N
Q.
LIU,
T.
WONG,
Departments of Physiology and Medicine, McGill University, Montreal, Canada Platelet aggregation at moderate shear rates in Poiseuille flow (<1355 s1, equivalent to shear stresses of <1.5 Nm -2) of ADP-activated platelets occurs efficiently with fibrinogen (Fg) (Xia and Frojmovic, Biophys.J., 64: 2190, 1994), but persists in the absence of plasma proteins, independent of any secreted Fg (Goldsmith et al., Thromb. Haemost. 71, 78, 1994). The present work tests the hypothesis that von Willebrand Factor (vWF) expressed on activated platelets can mediate aggregation at modest shear rates, and reports adhesive molecular domains of Fg and vWF mediating aggregation. Activated platelets were incubated with FITC-Fg to assess occupied GPIIb-IIIa receptors or with FITCmAb's against 0tRGDF, 0tRGDS, ~,400--411 or T373-385 on platelet-bound Fg; and mAb's 152B6 and NMC-4, respectively blocking the GPIIb-IIIa and GPIb binding domains on vWF, and aurin tricarboxylic acid (ATA) which blocks the vWF-binding domain on GPIb. Platelets were stirred or sheared in a