Platelet Protein Expression Research Articles

Antithrombotic thrombocytes: ectopic expression of urokinase-type plasminogen activator in platelets

AbstractArterial occlusive disorders are a leading cause of human morbidity. We hypothesized that ectopic expression of fibrinolytic proteins in platelets could be used to favorably alter the hemostatic balance at sites of thrombosis. To test our hypothesis, we directed murine urokinase-type plasminogen activator transgene expression to platelets using a platelet factor 4 promoter. Urokinase was selectively expressed and stored in the platelets of these mice. These transgenic mice had altered platelet biology and a bleeding diathesis similar to that seen in patients with Quebec platelet disorder, affirming the role of ectopic urokinase expression as the etiology of this inherited disease. These mice were resistant to the development of occlusive carotid artery thrombosis in the absence of systemic fibrinolysis and displayed rapid resolution of pulmonary emboli. Moreover, transfusion of urokinase-expressing platelets into wild-type mice prevented formation of occlusive arterial thrombi. These studies show the feasibility of delivering fibrinolytic agents to sites of incipient thrombus formation through selective storage in platelets and offer a new strategy to prevent thrombosis and hemorrhage.

Enhancement of Platelet Aggregation and Thrombus Formation by Arsenic in Drinking Water: A Contributing Factor to Cardiovascular Disease

Arsenic in drinking water is a worldwide health problem that is associated with cardiovascular disease, but the cause is currently unknown. Arsenic effects on platelets, which are important in development of cardiovascular disease, were examined in vitro and in a drinking water study using a rat animal model. Trivalent inorganic arsenic (arsenite) induced in vitro aggregation when platelets were exposed to subthreshold challenge by thrombin and several other agonists in a concentration-dependent manner, with arsenite being the most potent form tested. Arsenite also induced significant increases in serotonin secretion, thromboxane A2 formation, and adhesion protein expression in platelets. Consistent with the in vitro studies, 4-week ingestion of arsenite-contaminated drinking water resulted in enhanced arterial thrombosis. Human platelets showed similar responses, suggesting that the effects seen in animal experiments are applicable to humans. These results will provide new insights into the mechanism of arsenic-induced cardiovascular disease. They will also allow regulatory agencies to estimate risk from arsenic-induced cardiovascular disease and to determine if drinking water regulatory levels based on human cancer studies will protect against noncancer effects associated with cardiovascular disease.

Bruton's tyrosine kinase is present in normal platelets and its absence identifies patients with X‐linked agammaglobulinaemia and carrier females

X-linked agammaglobulinaemia (XLA) is a primary immunodeficiency caused by mutations in the gene coding for Bruton's tyrosine kinase (Btk) and is characterized by an arrest of B-cell development. We analysed Btk protein expression in platelets using flow cytometry and found that normal platelets express large amounts of Btk. Assessment of affected males from 45 unrelated XLA families revealed that platelets of the majority of the patients (37 out of 45 families) had decreased or absent Btk expression, and that platelets from carrier females of these families had both normal and mutated Btk expression, indicating that megakaryocytes in XLA carriers undergo random X-chromosome inactivation. These observations demonstrate that Btk is not crucial for maturation of megakaryocytes and the production of platelets. No correlation between Btk expression in platelets and clinical phenotype was observed in this study. Flow cytometric evaluation using platelets is a simple and rapid method to test Btk expression. It may be used as a screening test for XLA and for carrier detection, followed, if necessary, by more expensive mutation analyses.

Changes in dietary zinc and copper affect zinc-status indicators of postmenopausal women, notably, extracellular superoxide dismutase and amyloid precursor proteins

Zinc is an essential trace element for human health and well-being; however, methods currently available for the assessment of zinc status in humans are unsatisfactory. The objective was to critically evaluate the use of various indicators of zinc status in humans in a controlled metabolic ward study. Indicators of zinc status were measured in 25 healthy postmenopausal women aged 64.9 +/- 6.7 y. After a 10-d equilibration period, volunteers consumed a diet with either a low (1 mg/d; n = 12) or a high (3 mg/d; n = 13) copper content based on a total energy content of 8.4 MJ. They received the same amount of copper throughout the study. Both groups were fed the basal diet (3 mg Zn/d) with no zinc supplement for one 90-d period, and the diet supplemented with 50 mg Zn/d for another 90-d period. Zinc supplementation significantly increased (P < 0.0001) extracellular but not erythrocyte superoxide dismutase activity. This increase was more apparent when subjects were fed the low-copper diet. Zinc supplementation in combination with the low-copper diet significantly decreased (P < 0.01) amyloid precursor protein expression in platelets. Other indicators of zinc status that were significantly elevated after zinc supplementation were as follows: plasma zinc and free thyroxine concentrations and mononuclear 5'-nucleotidase activity. The measurement of serum extracellular superoxide dismutase activity may be useful as a marker for the functional assessment of zinc status in humans.

Protease-Activated Receptors and Platelet Function

IntroductionPlatelet activation is critical for normal hemostasis, and platelet-dependent arterial thrombosis underlies most myocardial infarctions. Thrombin is the most potent activator of platelets.1,2 For this reason, understanding the process by which thrombin activates platelets is necessary for understanding hemostasis and thrombosis and may yield novel anti-platelet therapies. This chapter focuses on our recent studies of the receptors that mediate activation of human platelets by thrombin.3,4 Thrombin signaling is mediated, at least in part, by a family of G protein-coupled protease-activated receptors (PARs), for which PAR1 is the prototype.5,6 PAR1 is activated when thrombin binds to and cleaves its amino terminal exodomain to unmask a new receptor amino terminus.5 This new amino terminus then serves as a tethered peptide ligand, binding intramolecularly to the body of the receptor to effect transmembrane signaling.5,7,8 The synthetic peptide SFLLRN, which mimics the first six amino acids of the new amino terminus unmasked by receptor cleavage, functions as a PAR1 agonist and activates the receptor independent of thrombin and proteolysis.5,9,10 Such peptides have been used as pharmacological probes of PAR function in various cell types.Our understanding of the role of PARs in platelet activation is evolving rapidly. PAR1 mRNA and protein were detected in human platelets,5,11-13 SFLLRN-activated human platelets,5,9,10 and PAR1-blocking antibodies inhibited human platelet activation by low, but not high, concentrations of thrombin.11,12 These data suggested a role for PAR1 in activation of human platelets by thrombin but left open the possibility that other receptors contribute.Curiously, PAR1 appeared to play no role in mouse platelets.14-16 PAR1-activating peptides did not activate rodent platelets, and platelets from PAR1-deficient mice responded like wild-type platelets to thrombin.16 The latter observation prompted a search for additional thrombin receptors and led to the identification of PAR3.17 PAR3 is activated by thrombin and is expressed in mouse platelets. PAR3 blocking antibodies inhibited mouse platelet activation by low, but not high, concentrations of thrombin,18 and knockout of PAR3 abolished mouse platelet responses to low, but not high, concentrations of thrombin.3 These results established that PAR3 is necessary for normal thrombin signaling in mouse platelets but also pointed to the existence of another mouse platelet thrombin receptor. Such a receptor, PAR4, was recently identified.3,19 PAR4 appears to function in both mouse and human platelets.3 The role of PAR3 in human platelets, if any, remains to be determined, and whether still unidentified receptors contribute to thrombin activation of platelets is unknown. Nonetheless, available data suggest a testable, working model in which PAR3 and PAR4 mediate thrombin activation of mouse platelets and PAR1 and PAR4 mediate activation of human platelets.To determine the roles of PAR1, PAR3, and PAR4 in activation of human platelets by thrombin, we examined PAR mRNA and protein expression in platelets and probed PAR function using specific peptide agonists. We also examined the effect of receptor desensitization, receptor blocking antibodies, and a PAR1 antagonist, used alone and in combination, on platelet activation.4

Cyclooxygenase inhibition decreases nitric oxide synthase activity in human platelets

Activity of both nitric oxide (NO) synthase (NOS) and cyclooxygenase (COX) plays an important role in the regulation of platelet function. NO has been shown to directly activate COX. This study was designed to determine whether products of the COX pathway in turn regulate NOS activity. Human platelets were incubated with aspirin, indomethacin, the selective thromboxane A2 synthase inhibitor U-63557A, or the prostaglandin H2-thromboxane A2-receptor blocker SQ-29548 for 1 h at 37 degrees C. Multiple indexes of the activity of the L-arginine-NO pathway and changes in cytosolic Ca2+ concentration ([Ca2+]i) were measured in platelets. Both aspirin and indomethacin decreased NOS activity, measured as the conversion of L-arginine to L-citrulline and nitrite (+nitrate) formation, in platelets in a concentration-dependent fashion. Aspirin also decreased guanosine 3',5'-cyclic monophosphate accumulation in platelets. The NOS inhibitory effects of these aspirin and indomethacin effects were reversed by coincubation with the thromboxane A2 analog U-46619 or an excess of CaCl2. Incubation of COX inhibitors with platelets was associated with significant reductions in basal as well as thrombin-stimulated [Ca2+]i, and the reduction in [Ca2+]i was reversed by U-46619. Incubation of platelets with U-63557A and SQ-29548 resulted in inhibitory effects on NOS activity qualitatively similar to those of COX inhibitors. The effects of COX inhibitors or U-63557A were not associated with a change in NOS protein expression in platelets. These data suggest that NOS activity in human platelets is inhibited by COX inhibitors, mediated, at least in part, via suppression of thromboxane A2 and [Ca2+]i mobilization in platelets.

Human megakaryocytes. II. Expression of platelet proteins in early marrow megakaryocytes.

Analysis of various platelet proteins by immunofluorescence demonstrated that platelet glycoproteins Ib, IIb, and IIIa, as well as plasma factor VIII antigen (factor VIII:AGN), platelet factor 4, and fibronectin are present in the vast majority of morphologically recognizable megakaryocytes. In addition, a small number of lymphoid-like mononuclear marrow cells, representing approximately 1.4--2.9/10(4) marrow cells, was found to express the same platelet proteins. This population of early marrow megakaryocytes is analogous to small acetylcholinesterase-positive rat and mouse marrow cells. Fc receptors for IgG were expressed in all megakaryocytes and megakaryocyte precursors, whereas the Ia antigen was detected only on a proportion of mature megakaryocytes and not on only early or precursor megakaryocytes. Platelet glycoproteins Ib, IIb, and IIIa, as well as factor VIII:AGN, and platelet factor 4 were established as distinct markers for marrow megakaryocytes and may be helpful for identifying megakaryocytic cells as well as for monitoring events of megakaryocyte differentiation.