Prolonged bleeding time with defective platelet filopodia formation in the Wistar Furth rat.

Hereditary macrothrombocytopenia is a hallmark of Wistar Furth (WF) rats. In addition, a platelet/megakaryocyte alpha granule defect, similar to that of patients with gray platelet syndrome, is present. Several observations indicate cytoskeletal abnormalities in WF platelets and megakaryocytes, suggesting the potential for functional defects in hemostatic processes requiring cytoskeletal reorganization, such as platelet adhesion and spreading. However, no bleeding abnormality has been noted. Here, we report a prolonged bleeding time (>30 minutes in 10 of 11 rats tested) with defective clot formation in the WF strain. Prolonged bleeding time can result from defects in platelet adhesion, aggregation, or the release reaction. Because aggregation to collagen and adenosine diphosphate were reported to be normal, we determined whether WF rat platelets are defective in their ability to adhere to substrates. Platelet adherence and spreading was evaluated from 30 seconds to 30 minutes on Formvar-coated, carbon-stabilized grids or poly-L-lysine-coated glass coverslips by transmission electron microscopy or immunofluorescence, respectively, and scanning electron microscopy. We classified the adhered platelets according to their pattern of spreading, ie, rounded, rounded or spreading with short filopodia, spindle-shaped, spreading with long filopodia, spreading with lamellipodia, and fully spread. Adherent normal rat platelets displayed all stages of spreading within 30 seconds to 2 minutes, including many spindle-shaped forms, and forms with multiple, long filopodia. In contrast, adhered WF platelets at these early time points rarely developed long filopodia or were spindle shaped. The majority of adherent WF platelets at these early time points were either round, spread with a few short filopodia, or extensively spread with wide lamellipodial skirts. By 15 to 30 minutes, most platelets in both Wistar and WF samples were fully spread. These data show abnormal WF platelet spreading. The paucity of spindle-shaped forms and forms with long filopodia may reflect an inability of WF platelets to undergo the early stages of spreading, or, alternatively, their more rapid than normal progression through these stages. We hypothesize that this failure to spread normally may relate to prolonged bleeding times in vivo and defective clot formation in WF rats.

Identification of the Src family kinases, Lck and Fgr in platelets. Their tyrosine phosphorylation status and subcellular distribution compared with other Src family members.

We have identified the Src family members, Lck and Fgr in resting human and rodent platelets and compared their subcellular distributions and tyrosine phosphorylation status to those of the other Src family kinases to gain insights into the signal transduction pathways active in maintaining platelets in the circulation. Like Fyn, Lyn, and Yes, most of Fgr and Lck was detergent-insoluble in human and rat platelets. In comparison, Src showed higher detergent solubility than the Src-related kinases. Most all human platelet Src was detergent-soluble, while that of rodent platelets was present in all detergent fractions. We also compared the tyrosine-phosphorylation status of Lck and Fgr to other Src family members in resting platelets using immunoprecipitation and immunoblotting. All of these Src family members except Fgr exhibited substantial phosphotyrosine antibody labeling. The partitioning of these kinases, with the exception of Src, with the detergent-insoluble fraction, their tyrosine-phosphorylation status, and co-localization with endocytotic vesicles lead us to hypothesize that the Src family kinases are involved in signaling events that drive cytoskeletal reorganization and active endocytosis of plasma proteins by circulating platelets.

The Src family kinases, Fgr, Fyn, Lck, and Lyn, colocalize with coated membranes in platelets.

Nonreceptor protein tyrosine kinases phosphorylate proteins, thereby activating many intracellular signaling pathways and mediating protein-protein interactions. Protein phosphorylation is regulated in large part by the subcellular localization of these kinases and their respective substrates. Src is the most studied of these kinases, although other members of the Src family have been shown to be important in the differentiation of specific cell types. Src and Src family members are reported to be membrane-associated, but detergent-extraction studies have demonstrated a major difference in the solubility of Src compared with other members of the Src family (Fgr, Fyn, Lck, Lyn, and Yes), suggesting that their subcellular distributions may be different. By immunoelectron microscopy, we demonstrate that, unlike Src, the Src-related kinases are associated with electron-dense cytoplasmic domains and plasma membrane domains that correspond in size and frequency to endocytotic vesicles and coated pits. Clusters of labeling for these kinases also were seen adjacent to granule membranes. These kinases colocalize with the coated vesicle protein, clathrin, confirming their association with this class of endocytotic vesicle. We hypothesize that this vesicular association of Src-related kinases indicates a role for them in the endocytotic vesicle-mediated uptake and trafficking of plasma proteins into platelet granules.

Acute gastric pH changes alter intraluminal but not plasma peptide levels.

Gastric acidity is influenced by systemic and local peptide effects. Previous work by others has shown that intraluminally secreted peptides may have a role in local control of gastric acidity; however, the response of these peptides to acute changes in gastric pH is unknown. To determine the effects of acute changes in pH on systemic and intraluminal peptide levels, 14 normal volunteers underwent placement of a nasogastric tube after an overnight fast. Blood and gastric fluid were analyzed on a control day, 2 hours after completion of 24 hours of aluminum-magnesium antacid therapy and after 24 hours of H2 blockade. Plasma and acid-alcohol-extracted gastric peptide levels were measured with specific radioimmunoassays. Specimens were subdivided into two groups: 28 gastric fluid specimens with a pH less than 4 and 10 specimens with a pH greater than 4. In the patients with a pH greater than 4, the luminal peptides, motilin, neurotensin, pancreatic polypeptide, somatostatin, substance P, and gastrin, were decreased by 50% to 90% and gastrin-releasing peptide was decreased by 36% compared with specimens with a pH less than 4. Conversely, intraluminal vasoactive intestinal polypeptide and calcitonin levels were elevated by 60% and 27%, respectively, in the samples with a pH greater than 4. Intraluminal peptide concentrations are responsive to changes in intragastric pH; however, this response was not seen in plasma peptide levels.