The role of Rab38 in platelet dense granule defects.

BACKGROUND: The fawn-hooded hypertensive (FHH) rat has a mutation in the Rab38 gene that is associated with a platelet dense granule storage pool disease. OBJECTIVE: To better characterize the expression and function of Rab38 in FHH rat and human megakaryocytes and platelets. PATIENTS AND METHODS: Rab38 expression in FHH rat and normal tissues was demonstrated by western blotting. Platelet and megakaryocyte morphology and Rab38 expression were examined by transmission electron microscopy and by immunofluorescence confocal microscopy. Platelet surface glycoprotein and P-selectin expression and total serotonin content were assessed by flow cytometry. RESULTS: Rab38 was not expressed in FHH rat tissues, and FHH rat platelets and megakaryocytes lacked dense granules. FHH rat platelets had normal expression of surface glycoproteins and of surface P-selectin in response to thrombin. The total serotonin content in FHH rat platelets was similar to that in Brown Norway rat platelets. In a megakaryocyte cell line, Rab38 was expressed in a granular perinuclear and cytoplasmic pattern. There was partial colocalization with serotonin, and minimal colocalization with von Willebrand factor and lysosomal proteins. CONCLUSIONS: The lack of Rab38 expression in the FHH rat results in the absence of normal dense granules in the megakaryocytes and platelets, which have otherwise normal structure and function. Rab38 may play a role in the development of dense granules in the megakaryocytes and platelets.

Regulated secretion in endothelial cells: biology and clinical implications.

Vascular endothelial cells are critical participants in maintaining blood flow, with the ability to respond rapidly to injury. We have outlined above how the regulated secretion of a variety of hemostatic and inflammatory mediators contributes to these nearly instantaneous responses. The WPB are the most prominent of these regulated secretory granules, and there is growing evidence of additional granules that release their contents under a variety of conditions. The mechanisms responsible for the targeting of proteins to regulated secretory granules, and of exocytosis of these granules are being elucidated. EC appear to share some characteristics with other secretory cell types, but also are likely to have unique properties related to the storage and secretion of large multimeric proteins such as VWF and multimerin. Understanding these mechanisms may lead to new strategies for treating coronary artery disease, stroke, sickle cell disease, and hemophilia through drugs that modulate sorting and secretion, or by gene transfer approaches that introduce therapeutic molecules into the WPB for regulated release.

Targeting of a heterologous protein to a regulated secretion pathway in cultured endothelial cells.

The stimulation of regulated exocytosis in vascular endothelial cells (EC) by a variety of naturally occurring agonists contributes to the interrelated processes of inflammation, thrombosis, and fibrinolysis. The Weibel-Palade body (WPB) is a well-described secretory granule in EC that contains both von Willebrand factor (vWF) and P-selectin, but the mechanisms responsible for the targeting of these proteins into this organelle remain poorly understood. Through adenoviral transduction, we have expressed human growth hormone (GH) as a model of regulated secretory protein sorting in EC. Immunofluorescence microscopy of EC infected with GH-containing recombinant adenovirus (GHrAd) demonstrated a granular distribution of GH that colocalized with vWF. In contrast, EC infected with an rAd expressing the IgG(1) heavy chain (IG), a constitutively secreted protein, did not demonstrate colocalization of IG and vWF. In response to phorbol ester, GH as well as endogenously synthesized vWF were rapidly released from GHrAd-infected EC. By immunofluorescence microscopy, granular colocalization of GH with endogenous tissue-type plasminogen activator (tPA) was also demonstrated, and most of the tPA colocalized with vWF. These data indicate that EC are capable of selectively targeting heterologous proteins, such as GH, to the regulated secretory pathway, which suggests that EC and neuroendocrine cells share common protein targeting recognition signals or receptors.

Peptido-leukotrienes are potent agonists of von Willebrand factor secretion and P-selectin surface expression in human umbilical vein endothelial cells.

BACKGROUND: The peptido-leukotrienes (LTs) and lipoxins (LX) are produced by platelets through the transcellular conversion of leukocyte-derived LTA4 at sites of vascular inflammation and injury, such as during coronary artery balloon angioplasty. We studied the actions of these eicosanoids on vascular endothelium. METHODS AND RESULTS: We found that stimulation of cultured human umbilical vein endothelial cells (EC) with LTC4 and LTD4 resulted in the release of high-molecular-weight multimers of von Willebrand factor (vWF) in a concentration- and time-dependent fashion, as measured by ELISA. Neither LXA4 nor LXB4 stimulated vWF release. LTC4 and LTD4 also stimulated a rapid increase in the surface expression of P-selectin indicated by increased binding of anti-P-selectin monoclonal antibody-coated beads. Fluorescence cytometry detected prolonged peaks of [Ca2+]i in EC in response to concentrations of thrombin and LTD4 that induce near-maximal vWF secretion. In contrast, concentrations of LTC4 that induce similar levels of vWF secretion produced only asynchronous oscillations of [Ca2+]i in most EC and rarely induced prolonged peaks of [Ca2+]i. Depletion of external Ca2+ had no apparent impact on LT-stimulated [Ca2+]i transients and vWF secretion, implicating an intracellular pool as the source of this response. Staurosporine, sphingosine, and H-7 each had only modest effects on peptido-LT-induced vWF secretion, suggesting that protein kinase C is not a primary mediator of peptido-LT-induced exocytosis. Inhibitors of cyclooxygenase and platelet-activating factor had no effect on peptido-LT-mediated vWF secretion. CONCLUSIONS: Through the induction of vWF secretion and P-selectin surface expression, peptido-LTs are likely to play an important role in the interrelated processes of hemostasis and inflammation.

Sensitive detection of occult breast cancer by the reverse-transcriptase polymerase chain reaction.

PURPOSE: Detection of occult carcinoma in patients with breast cancer may aid the establishment of prognosis and development of new therapeutic approaches. To improve on existing methods of detection, we have developed a reverse-transcriptase polymerase chain reaction (RT-PCR) assay for keratin 19 (K19) transcripts to identify mammary carcinoma cells in the peripheral blood and bone marrow of patients with breast cancer. PATIENTS AND METHODS: Peripheral-blood or bone marrow samples obtained from 34 patients with stages I to IV breast cancer and 39 control subjects without breast cancer were screened for K19 mRNA by nested primer PCR. RESULTS: In reconstitution experiments, K19 RT-PCR reliably detected 10 mammary carcinoma cells in 1 million normal peripheral-blood mononuclear (PBMN) cells. Four of 19 patients with stage IV breast cancer had detectable K19 transcript in peripheral blood. Five of six patients with histologically negative bone marrow biopsies following preablative chemotherapy and before autologous bone marrow transplant (BMT) were positive by this assay. Stem-cell apheresis harvests obtained from one of these patients and three additional patients immediately before BMT were all K19-negative. K19 RT-PCR analysis of CSF from a breast cancer patient with known carcinomatous meningitis was also positive. Thirty-eight of 39 non-breast cancer patients had negative K19 RT-PCR assays. The one exception was a patient with chronic myelogenous leukemia. CONCLUSION: RT-PCR of K19 is a sensitive, specific, and rapid method for detection of occult mammary carcinoma cells in the peripheral blood and bone marrow of patients with breast cancer. The presence of residual breast cancer cells in histologically normal bone marrow aspirates but not in stem-cell apheresis harvests is a frequent finding. This assay may be useful in diagnosing metastatic disease, as well as in monitoring the effectiveness of systemic therapy.