CD9 (p24) and PECAM1 (
CD31) antigens are well-defined components of the platelet plasma membrane. Both are integral
glycoproteins (GPs) implicated in the adhesive and aggregative properties of human platelets. In the present report, we have investigated their subcellular localization using immunoelectron microscopy. The monospecificity of the two polyclonal
antibodies used was confirmed by immunoblotting. On normal resting platelets, immunolabeling for CD9 and PECAM1 was found lining the plasma membrane and the
luminal face of the open canalicular system. Some labeling was also consistently found on the alpha-granule limiting membrane. This was confirmed by double labeling experiments in which
fibrinogen and
von Willebrand factor (vWF) were used as alpha-granule markers. CD9 and
PECAM-1 were found lining the membrane of the same granules that contained
fibrinogen and vWF in their matrix. CD9 and
PECAM-1 thus appear to have an intracellular distribution identical to GPIIb-IIIa, a major aggregation platelet receptor. To rule out a cross-reactivity of the two polyclonal
antibodies with GPIIb/IIIa, we studied PECAM1 and CD9 expression on the platelets from a patient with type I Glanzmann's
thrombasthenia whose platelets are devoid of GPIIb/IIIa. The same pattern of labeling was observed for both
antigens as for normal platelets. Normal platelets were further observed after stimulation by agonists that either fail to induce (
ADP) or induce granule secretion (
thrombin).
After treatment with
ADP, platelets changed shape and centralized their granules; the plasma membrane immunolabeling remained unchanged; and
gold particles were still found decorating the periphery of the centralized alpha-granules. After
thrombin treatment, alpha-granules fused with the platelet membrane and secretion occurred. A significant increase of labeling was then observed on the platelet surface. From these results we conclude that the alpha-granule membrane contains two additional receptors in common with the plasma membrane. This suggests that alpha-granule membrane receptors may originate from a dual mechanism: direct targeting from the Golgi complex in megakaryocytes (for alpha-granule-specific receptors such as
P-selectin) or by endocytosis from the plasma membrane (for
proteins distributed in the two compartments).