Ultrastructural analysis of the distribution of von Willebrand factor and fibrinogen in platelet aggregates formed in the PFA-100.

The PFA-100 is a new apparatus used to detect platelet dysfunction in vitro . Anticoagulated blood flows under constant pressure through a capillary, and across an aperture that pierces a membrane coated with collagen and either epinephrine or ADP. Through their ability to adhere and aggregate, platelets occlude the orifice and the closure time is a test of platelet function. Using electron microscopy and immunogold staining, we have analyzed the ultrastructure of platelet aggregates formed within the aperture and that are responsible for the occlusion. Standard electron microscopy showed that the aggregates formed on both collagen-epinephrine and collagen-ADP cartridges presented the same morphological features. The aggregates were exclusively composed of platelets, some of which were degranulated. Degranulation was particularly intense at the periphery of the aggregate where platelets were often totally devoid of secretory organelles. Immunogold staining on ultrathin frozen sections with polyclonal antibodies, allowed us to evaluate the distribution of adhesive proteins such as fibrinogen and von Willebrand factor (vWF) within the aggregate. The latter was found to be abundant in the intercellular spaces between adjoining platelets. Although fibrinogen was also present, its labeling was less intense suggesting that vWF is the major protein implicated in the platelet-platelet interactions in the aggregates formed in the PFA-100 system. This may be because of the high shear rate that occurs across the aperture which suggests that the PFA-100 is particularly sensitive for detecting abnormalities of vWF-platelet interactions.

Confirmation that GP Ib-IX complexes have a reduced surface distribution on platelets activated by thrombin and TRAP-14-mer peptide.

In 1990 we reported that GP Ib-IX complexes accumulated within the surface-connected canalicular system (SCCS) of thrombin-stimulated platelets. This conclusion was reached following investigations using monoclonal antibodies (MAbs) in flow cytometry and a polyclonal antibody to GP Ib alpha in electron microscopy with immunogold staining performed on ultrathin sections of resin-embedded platelets. Recent controversy concerning these results has prompted us to perform further studies using 14 anti-GP Ib-IX MAbs obtained from the 1993 Boston Workshop on Leukocyte Antigens. Features were the use of the MAbs in mixtures and the fact that immunogold staining was performed on frozen thin sections. Platelets were stimulated with either alpha-thrombin or TRAP-14-mer peptide. In all cases a decreased density of GP Ib-IX complexes on exposed areas of the activated platelet surface was accompanied by an increased expression within the SCCS. At the same time we noted that when platelets were stimulated with TRAP-14-mer they progressively exhibited a different internal morphology in comparison to that seen with thrombin. In particular, the dense central mass disappeared and large vacuoles were present throughout the cytoplasm. Overall, these studies confirm that changes in the distribution of GP Ib-IX complexes which follow thrombin-induced platelet activation (i) are indeed observed when antibody mixtures are used to detect them, and (ii) are mediated through the receptor recognized by the TRAP-14-mer peptide.

Thrombin-induced platelet aggregates have a dynamic structure. Time-dependent redistribution of glycoprotein IIb-IIIa complexes and secreted adhesive proteins.

The role of glycoprotein (GP) IIb-IIIa complexes and of adhesive proteins in mediating platelet aggregation is now well defined. However, less is known of the changes that occur once aggregation has begun. We report immunogold staining of thin sections of platelets or platelet aggregates, embedded in Lowicryl K4M, after the use of polyclonal antibodies to GP IIb or GP IIIa, fibrinogen (Fg), von Willebrand factor (vWF), and thrombospondin (TSP). Bound immunoglobulin G (IgG) was located by species-specific anti-IgG coupled to 5-nm gold particles and by electron microscopy. Initial experiments with platelet-rich plasma confirmed the feasibility of visualizing adhesive proteins between platelets in aggregates. Experiments then continued, using stirred suspensions of washed platelets incubated with alpha-thrombin. After 20 seconds, platelets were in contact without detectable release, although giant secretory vesicles containing adhesive proteins were seen. Internal pools of GP IIb-IIIa were progressively externalized within the aggregate. Secreted Fg was readily detected between platelets at 40 seconds. After 3 minutes, when most of the secretion had occurred, Fg had a patchwork-like distribution within the aggregate. After 6 minutes, zones with closely interspaced surface membranes, usually representing pseudopods, were dominant and Fg free. Results for vWF and TSP were similar to those for Fg. Nonetheless, GP IIb-IIIa complexes continued to be located between adjacent surface membranes throughout the aggregate. Thrombin-induced platelet aggregates were isolated, and sodium dodecyl sulfate-soluble extracts were obtained. Western blot experiments showed that, although fibrinopeptide A had been cleaved, degradation of adhesive proteins by platelet proteases had not occurred. These results emphasize that a platelet aggregate is a dynamic structure and suggest that not all surface-contact interactions are mediated by Fg or the other adhesive proteins tested in this study.