The roles of ADP and TXA in botrocetin/VWF-induced aggregation of washed platelets.
BACKGROUND
Binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein (GP) Ib-IX-V complex initiates a cascade of events leading to alphaIIbbeta3 activation and platelet aggregation. The roles of ADP and thromboxane A2 (TXA2) in agglutination-induced GPIbalpha-mediated platelet activation have not been fully described.


METHODS
Botrocetin and human VWF were used to stimulate washed mouse platelets. Platelets deficient in TXA2 receptors, Galphaq, or alphaIIbbeta3, and inhibitors and chelating agents were used to investigate the roles of TXA2, ADP, alphaIIbbeta3 and Ca2+ in botrocetin/VWF-induced signaling.


RESULTS
Our data demonstrate that botrocetin/VWF/GPIbalpha-mediated agglutination results in calcium-independent protein kinase C (PKC) and phospholipase A2 (PLA2) activities required for GPIbalpha-elicited TXA2 production that in turn causes dense granule secretion. Aggregation of washed platelets requires TXA2-induced alphaIIbbeta3 activation and ADP signaling. TXA2 or ADP can activate alphaIIbbeta3, but both are required for alpha-granule secretion and aggregation. Botrocetin/VWF-induced dense granule secretion is Galphaq-dependent. alpha-Granule secretion requires initial ADP signaling through P2Y1 and subsequent signaling through P2Y12. Signaling initiated by agglutination is propagated and amplified in an alphaIIbbeta3-dependent manner.


CONCLUSIONS
In contrast to adhesion or shear stress-induced GPIb-elicited signaling, agglutination-elicited GPIb signaling that activates alphaIIbbeta3 requires TXA2. Agglutination-elicited TXA2 production is independent of Ca2+ influx and mobilization of internal Ca2+ stores. Therefore, our results demonstrate that agglutination-elicited GPIb signaling causes alphaIIbbeta3 activation by a mechanism that is distinct from those used by adhesion, or shear stress-induced GPIb signaling.
Liu
J
J
Division of Experimental Hematology, St Jude Children's Research Hospital, Memphis, TN 38152, USA. jliu1@memphis.edu
Pestina
T I
TI
Berndt
M C
MC
Steward
S A
SA
Jackson
C W
CW
Gartner
T K
TK
D000244:Adenosine Diphosphate / Q000378:metabolism / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D001792:Blood Platelets / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism; D002448:Cell Adhesion; D003435:Crotalid Venoms / Q000494:pharmacology*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008954:Models, Biological; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D015539:Platelet Activation; D010974:Platelet Aggregation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D011796:Quinacrine / Q000494:pharmacology; D044244:Receptors, Thromboxane A2, Prostaglandin H2 / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D015398:Signal Transduction; D013928:Thromboxane A2 / Q000502:physiology*; D013997:Time Factors; D014841:von Willebrand Factor / Q000494:pharmacology*
D003435:Crotalid Venoms; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D044244:Receptors, Thromboxane A2, Prostaglandin H2; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D013928:Thromboxane A2; D000244:Adenosine Diphosphate; C030961:botrocetin; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D010741:Phospholipases A; D054467:Phospholipases A2; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D011796:Quinacrine; D002118:Calcium