CB, an agent that inhibits assembly of cytoplasmic actin filaments, was found in previous studies to make platelets more deformable to aspiration into micropipettes 35 and to prevent cold-induced shape change

CB, an agent that inhibits assembly of cytoplasmic actin filaments, was found in previous studies to make platelets more deformable to aspiration into micropipettes 35 and to prevent cold-induced shape change. external membranes of giant platelets does not differ significantly (0.93 0.20), provided the OCS is sufficiently dilated. Platelets exposed to cytochalasin B (1.01 0.31) or to hypertonic conditions (0.96 0.20) revealed comparable ratios for immunogold particles on external and internal membranes. Results of our study show that membranes of the uncovered surface and lining OCS channels of resting platelets are continuous, identical structures and GPIb is usually homogeneously distributed on external and internal membranes. Platelet membrane glycoproteins play HPGDS inhibitor 1 a critical role in the regulation of adhesive and cohesive platelet functions. The platelet surface membrane and the contiguous surface-connected open canalicular system (OCS) contain the major glycoproteins (GP), GPIb and GPIIb-IIIa. GPIb plays a key role in hemostasis by mediating the adhesion of platelets to von Willebrand factor (VWF) bound to the subendothelium. 1,2 Conversation of GPIIb-IIIa with the arginine-glycine-aspartic acid (RGD) sequence in VWF facilitates platelet distributing onto subendothelium. 3 Subsequent binding of fibrinogen to GPIIb-IIIa promotes platelet aggregation and formation of an effective hemostatic plug at intermediate HPGDS inhibitor 1 shear rates. 4 Ultrastructural studies using immunocytochemical techniques have exhibited that GPIIb-IIIa receptors HPGDS inhibitor 1 are distributed on external and internal platelet membranes and on the membranes of -granules. 5 Although detailed morphometric studies are not available, it seems that GPIIb-IIIa receptors are homogeneously distributed on external internal membranes of platelets. 6 The overall impression from ultrastructural and circulation cytometric studies is usually that the internal pools of GPIIb-IIIa located in the OCS and -granules could supply additional receptors during the process of activation. 7-9 The amount of GPIIb-IIIa stored in internal pools could account for about 100% of the surface levels. 10 Immunocytochemical techniques have also exhibited that this GPIb complexes are distributed on external and internal platelet membranes, 11,12 membranes of -granules, 13 and dense bodies. 14 Differences in opinion exist in the literature regarding the quantitative distribution of GPIb on external and internal membranes in resting platelets. Some authors have found that the density of labeling for GPIb within the OCS is usually less than that observed around HPGDS inhibitor 1 the platelet surface. 15,16 Other workers have failed to note significant differences, 12,17 whereas some using enzyme-linked immunsorbent assay techniques with monoclonal antibodies have predicted a large internal pool of GPIb that could replenish any amount of GPIb lost from the external membrane. 18 The latter authors estimated the number of internal copies of GPIb was 3 to 4 4 times greater than the number of GPIb molecules present around the platelet surface, figures relatively higher than those estimated for GPIIb-IIIa. Immunocytochemical techniques have inherent limitations to access antigens located in external or internal membranes. 19,20 The difficulty for monoclonal antibodies to gain access to GPIIb-IIIa located on internal membranes of the platelet OCS has been known for a long time. 21 Better access to these antigens can be gained when Fab fragments of the antibodies or smaller probes are used. 10,21 In the present study, we have explored the distribution of GPIb on external and internal membranes of platelets in their resting state. Several approaches were used to facilitate the convenience of antibodies to antigens located in very narrow portions of the OCS. Ultrathin cryosections of platelets from patients with giant platelet disorders, including the May-Hegglin anomaly and Epsteins syndrome, were exposed to polyclonal antibodies or to mixtures of monoclonal antibodies (AP1 and 6D1). The same techniques were applied to normal platelets where OCS of normal platelets was artificially dilated by exposure to cytochalasin-B or to hypertonic buffers. Differences in labeling were morphometrically quantified. Materials and Methods Antibodies Polyclonal antibodies to glycocalicin or a mixture of monoclonal antibodies Rabbit Polyclonal to RPC3 specific for GPIb (AP1 and 6D1) were utilized HPGDS inhibitor 1 for immunolabeling. The polyclonal antibody against GPIb, generously provided.