von Willebrand factor bound to glycoprotein Ib is cleared from the platelet surface after platelet activation by thrombin.
We recently reported that after activation of human platelets by thrombin, glycoprotein (GP) Ib-IX complexes are translocated to the surface-connected canalicular system (SCCS) (Blood 76:1503, 1990). As GPIb is a major receptor for von Willebrand factor (vWF) in platelet adhesion, we have now examined the consequences of thrombin activation on the organization of vWF bound to GPIb on the platelet surface. Studies were performed using monoclonal or polyclonal antibodies in either immunogold staining and electron microscopy (Au-EM) or in flow cytometry. When unstirred platelet-rich plasma was incubated with ristocetin, bound vWF was located by Au-EM as discrete masses regularly distributed over the cell surface. Platelets from a patient with Glanzmann's thrombasthenia, lacking GPIIb-IIIa complexes, gave a similar pattern, confirming that this represented binding to GPIb. That ristocetin was not precipitating vWF before their binding to the platelets was shown by the detection of similar masses on the surface of platelets of a patient with type IIB von Willebrand disease. Experiments were continued using washed normal platelets incubated in Tyrode-EDTA, the purpose of the EDTA being to limit the surface expression of endogenous vWF after platelet stimulation. Under these conditions, platelets were treated with ristocetin for 5 minutes at 37 degrees C in the presence of increasing amounts of purified vWF. This was followed by incubation with thrombin (0.5 U/mL) for periods of up to 10 minutes. Flow cytometry showed a time-dependent loss in the surface expression of vWF bound to GPIb and these changes were confirmed by Au-EM. In particular, immunogold staining performed on ultrathin sections showed that the bulk of the vWF was being cleared to internal membrane systems. Surface clearance of vWF during thrombin-induced platelet activation is a potential mechanism for regulating platelet adhesivity.
Hourdillé
P
P
URA 1464 CNRS, Université de Bordeaux II, Hôpital Cardiologique, Pessac, France.
Gralnick
H R
HR
Heilmann
E
E
Derlon
A
A
Ferrer
A M
AM
Vezon
G
G
Nurden
A T
AT
D000906:Antibodies; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D006801:Humans; D066298:In Vitro Techniques; D008854:Microscopy, Electron; D016253:Microscopy, Immunoelectron; D015539:Platelet Activation; D010980:Platelet Membrane Glycoproteins / Q000187:drug effects / Q000378:metabolism*; D012310:Ristocetin / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*; D014841:von Willebrand Factor / Q000378:metabolism*
D000906:Antibodies; D010980:Platelet Membrane Glycoproteins; D014841:von Willebrand Factor; D012310:Ristocetin; D013917:Thrombin