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D V Devine

Publications and source records attributed to D V Devine.

At least 55 records · Page 3Linked to original sources
PubMed1994

Liposome-complement interactions in rat serum: implications for liposome survival studies.

Serum complement opsonizes particles such as bacteria for clearance by the reticuloendothelial system. Complement has been reported to interact with liposomes and therefore may mediate the reticuloendothelial system clearance of liposomes. This study has used a rat serum model to define some of the characteristics of liposomes which modulate their ability to activate complement. Using functional hemolytic assays and C3/C3b crossed immunoelectrophoresis, we have demonstrated that liposomes activated rat complement in a dose-dependent manner with higher concentrations of liposomes activating higher levels of complement. The detection of complement activation required the inclusion of phospholipids bearing a net charge. Complement activation occurred via the classical pathway; no alternative pathway activation was detected. The presence of cholesterol contributed to complement activation in a dose-dependent manner. Phospholipid fatty acyl chain length did not influence complement activation while the introduction of unsaturated acyl chains markedly decreased levels of complement activation. Liposome size also influenced complement activation with 400 nm unilamellar vesicles more effectively activating complement than 50 nm vesicles for equivalent amounts of exposed lipid. These studies demonstrate that the composition of the liposome greatly affects the in vitro activation of rat serum complement and suggest that the biological half-life of liposomes in the circulation of rats may be altered by changing the liposome composition to reduce complement activation.

Animals↗
PubMed1994

Factor XIIIa binding to activated platelets is mediated through activation of glycoprotein IIb-IIIa.

Stabilization of a clot is dependent on fibrin cross-linking mediated by the transglutaminase, factor XIIIa (FXIIIa). In addition to fibrin stabilization, FXIIIa acts on a number of platelet-reactive proteins, including fibronectin and vitronectin, as well as the platelet proteins, glycoprotein (GP) IIb-IIIa, myosin, and actin. However, conditions inducing the platelet-activation dependent binding of FXIIIa have not been characterized nor have the sites mediating FXIIIa binding been identified. The generation of FXIIIa and consequent detection of FXIIIa on the platelet surface were compared with other thrombin-induced activation events; the rate at which FXIIIa bound to activated platelets was much slower than platelet degranulation or fibrin(ogen) binding. Whereas platelets could be rapidly induced to express a functional receptor for FXIIIa, the rate of FXIIIa binding to platelets is limited by the rate of conversion of FXIII to FXIIIa. Immunoprecipitation of radiolabeled platelets using polyclonal anti-FXIII A-chain antibody identified two proteins corresponding to GPIIb and GPIIIa. Preincubation of intact platelets with 7E3, a monoclonal antibody that blocks the fibrinogen binding site, or GRGDSP peptide inhibited FXIIIa binding by about 95% when measured by flow cytometry; FXIIIa binding to purified GPIIb-IIIa was also inhibited by 7E3. The binding of FXIIIa to purified GPIIb-IIIa was enhanced by the addition of fibrinogen, but not by that of fibronectin or thrombospondin, suggesting that FXIIIa also binds to fibrinogen associated with the complex. These observations suggest that activated platelets bearing FXIIIa may enhance stabilization of platelet-rich thrombi through surface-localized cross-linking events.

Adenosine Diphosphate↗
PubMed1994

Characteristics of a novel low affinity complement C3dg-binding protein of human platelets.

The breakdown products of the complement protein C3 function in receptor-mediated immune clearance. The catabolism of the C3 molecule, mediated by factors I and H, results in the generation of the fragments C3c and C3dg. C3dg binds to human platelets in a specific and saturable manner. The direct interaction of platelets with soluble C3dg may contribute to immune-mediated platelet destruction. More importantly, platelets may interact with opsonized pathogens or complement-activating immune complexes via C3dg. In this report, we investigated the interaction of C3dg with platelets and calculated the Ka to be 3.2 x 10(6) M-1 with 1100 to 2200 specific binding sites/platelet. In the presence of 5 mM calcium, both the Ka and the number of specific binding sites were modestly decreased to Ka = 2.8 x 10(6) M-1 with 1400 to 2400 specific binding sites/platelet. The Scatchard plots demonstrated a curvilinear character. On labeling C3dg with peroxidase and visualizing platelet-bound C3dg by electron microscopy, it was shown that binding sites for C3dg were restricted to the platelet plasma membrane. Using a cell attachment assay, platelet adhesion to C3dg was readily detectable; attachment to C3dg-coated plates was not blocked by fibrinogen or fibronectin. We have characterized the C3dg-binding protein of platelets using the chemical cross-linkers, SASD and DSS, to cross-link C3dg to thrombin-stimulated platelets. Gel filtration of the 125I-labeled C3dg-platelet complex revealed the presence of a large protein complex that was absent when 125I-labeled C3dg alone was analyzed. SDS-PAGE of the radiolabeled cross-linked protein, followed by autoradiography, identified a 95-kDa membrane protein. The relationship of this C3dg-binding protein to other platelet membrane proteins has yet to be determined.

Blood Platelets↗
PubMed1994

Evidence of mitochondrial DNA diversity in South American aboriginals.

The absence in South American aboriginals of an Asian-specific marker, a 9-bp deletion between the genes for the second subunit of cytochrome oxidase II and lysine transfer RNA in region V, has been interpreted as a bottlenecking effect at the Isthmus of Panama during the peopling of the Americas. We screened mitochondrial DNA (mtDNA) for this 9-bp tandem repeat and for polymorphisms in specific regions of the mtDNA in 2 ancient and 31 contemporary samples from South American aboriginals. We found additional (mtDNA) diversity in South American aboriginals in three ways. First, an Asian-specific marker not previously reported in South American aboriginals was identified by a sequencing analysis in both the contemporary Andean and Amazonian aboriginal peoples. Second, two new haplotypes so far unique to South American aboriginals were found. Additionally, we show that South American aboriginals fall into discrete populations. These results suggest that the prehistoric colonization of South America is the outcome of multiple migrations; the data do not support a bottlenecking effect at the Isthmus of Panama.

Adult↗
PubMed1994

Complement activation in platelet concentrates is surface-dependent and modulated by the platelets.

Activation of platelets during storage may contribute to the loss of function and viability known as the platelet storage lesion. We investigated the role of complement activation as a possible mediator of the platelet storage lesion. We studied platelet bags stored up to 5 days under standard blood bank conditions and monitored the generation of several complement activation fragments from both the classical and alternative pathways. To assess the role of noncellular artificial surfaces in the activation of complement, parallel bags were prepared containing platelet-poor plasma. The levels of C4d and C3a increased steadily over time in storage, as did the level of the inactivated membrane attack complex SC5b-9. Generation of C4d, C5a, and SC5b-9 was greater in the absence of platelets than when platelets were present in the container. C5a levels in both groups were low and remained so during storage, suggesting that the C5a generated became surface associated. Using flow cytometry we detected C3 and C3a, but not C9, on the platelet surface. The percentage of C3-positive platelets peaked at the third day of storage; by day five platelet-associated C3 had declined. Decay accelerating factor expression on the platelet surface increased with time in storage and in parallel with CD63 expression. Based on the C4d levels, complement activation proceeded via the classical pathway; minimal generation of the alternative pathway activation fragment. Bb was seen in either the presence or absence of platelets. As an indirect measure of the activation of C1, the functional level of C1 esterase inhibitor (C1INH) was determined. C1INH levels declined over time in storage in bags containing only plasma; however, in the presence of platelets, the levels remained constant presumably because of release of C1INH from the alpha-granules of activated platelets.

Blood Platelets↗
PubMed1993

Investigations into the clinical utility of latex D-dimer in the diagnosis of deep venous thrombosis.

The serial use of non-invasive tests has been shown to be a safe method of managing outpatients who are suspected of having lower limb deep venous thrombosis (DVT). Objective testing has shown that the majority of these outpatients do not have venous thrombosis. A rapid test to exclude DVT in these patients, without the need for expensive and inconvenient serial noninvasive vascular testing, would have practical and economic advantages. Studies measuring the fibrin degradation product D-dimer using enzyme-linked immunoassays (EIA) in patients with venographically proven DVT suggest that it should be possible to exclude this condition by the use of one of the rapid latex bead D-dimer tests. We have examined 190 patients with suspected DVT using both a latex and an EIA D-dimer assay. The latex D-dimer test used in this study was negative in 7 of the 36 proven cases of DVT. This sensitivity of only 80% is not sufficient to allow this type of assay, in its current form, to be used as an exclusion test for DVT. The same plasma samples were tested with an EIA assay. This information was used to mathematically model the effects of selecting a range of D-dimer discriminant cut off points for the diagnosis of DVT. These results indicate that 62% of suspected clinically significant DVT could have this diagnosis excluded, with a 98% sensitivity, if the rapid latex or equivalent D-dimer test could be reformulated to measure less than 185 ng/ml of D-dimer.

Adolescent↗
PubMed1993

Characterization of kinetics and target proteins for binding of human complement component C3 to the surface-exposed outer membrane of Chlamydia trachomatis serovar L2.

In order to characterize the interaction of human complement with Chlamydia trachomatis, flow cytometry was used to quantitate binding of complement component C3 to elementary bodies of C. trachomatis serovar L2 preincubated in fresh serum in the presence or absence of human polyclonal chlamydial antibody. Isolation of each of the complement activation pathways revealed that C3 was activated most effectively by the alternative pathway. The degree of binding by the classical pathway was proportional to the concentration of antibody, but dual-pathway-mediated binding was not greater than antibody-independent alternative pathway binding. Electrophoresis and immunoblotting of detergent-extracted outer membrane protein-C3b complexes indicated that the chlamydial major outer membrane protein was the primary cell surface moiety binding C3b in both the presence and absence of specific antibody. Hydroxylamine cleavage of outer membrane protein-C3b complexes provided evidence that the majority of C3b is bound to the major outer membrane protein by hydroxyl ester bonds. This result was also unchanged by the presence of specific antibody. An unexpected finding was the apparent binding of anti-C3 antibody to a 40-kDa protein of the chlamydial outer membrane complex, perhaps indicating C3 mimicry on the part of the chlamydial major outer membrane protein.

Amino Acid Sequence↗
PubMed1993

Platelet-associated factor XIII as a marker of platelet activation in patients with peripheral vascular disease.

In the past several years, monoclonal antibodies have been developed that distinguish between resting and activated platelets in vitro. These antibodies recognize epitopes expressed on membrane proteins or soluble proteins, such as factor XIIIa and thrombospondin, that bind only to activated platelets. We have used fluorescence flow cytometry to determine whether three such antibodies can detect platelet activation in patients with severe peripheral vascular disease (PVD). Using two activation-specific monoclonal antibodies and a polyclonal antiserum to factor XIII a-chain, we have examined the platelets from PVD patients, age-matched control subjects who were free of detectable PVD, and unmatched control subjects. Cells analyzed as platelets were identified by their light-scatter profile and their reactivity with monoclonal anti-glycoprotein Ib. The platelets of patients with PVD showed no increase in binding of activation-dependent 1B3 (directed against a 180-kD membrane protein) compared with age-matched control subjects (p = 0.780). Similarly, there was no difference between PVD patients and control subjects in activation-dependent CD63 expression. Conversely, the binding of anti-factor XIII a-chain was significantly higher than in the control groups (p < 0.001). These data suggest that the detection of soluble factors that bind to activated but not resting platelets may be of use in the detection of pathological in vivo platelet activation.

Adult↗
PubMed1992

Kinetic studies of the mechanism of thrombocytopenia in patients with human immunodeficiency virus infection.

BACKGROUND: Isolated thrombocytopenia accompanied by increased amounts of platelet-associated antibody is a common manifestation of human immunodeficiency virus (HIV) infection, and the thrombocytopenia often improves with zidovudine. It is not clear whether the mechanism of HIV-related thrombocytopenia primarily involves autoimmune destruction of platelets or reduced platelet production by megakaryocytes. METHODS: We studied the survival of 111In-labeled autologous platelets and performed platelet imaging in 24 men with isolated HIV-related thrombocytopenia (16 who received no treatment and 8 who received zidovudine). We also studied 20 HIV-infected men with normal platelet counts (10 who received no treatment and 10 who received zidovudine) and studied 12 healthy seronegative men as controls. RESULTS: Mean (+/- SD) platelet survival was significantly decreased in both the untreated and the zidovudine-treated patients with HIV-related thrombocytopenia (to 92 +/- 33 and 129 +/- 44 hours, respectively; both P < 0.001), as compared with the normal controls (198 +/- 15 hours). Mean platelet survival was also significantly decreased in the HIV-infected patients with normal platelet counts (untreated, 162 +/- 23 hours, P < 0.01; zidovudine-treated, 166 +/- 35 hours, P < 0.05). Imaging studies, however, revealed no evidence of increased clearance of autologous platelets in the liver or spleen in any of these groups. Mean platelet production was significantly depressed in the untreated patients with thrombocytopenia (23,000 +/- 11,000 platelets per cubic millimeter per day, P < 0.001) as compared with the healthy controls (45,000 +/- 6,000 per cubic millimeter per day). Mean platelet production was significantly increased, however, in the men treated with zidovudine, both in those with thrombocytopenia (60,000 +/- 31,000 platelets per cubic millimeter per day, P < 0.01 vs. controls) and in those without thrombocytopenia (68,000 +/- 22,000 per cubic millimeter per day, P < 0.01). CONCLUSIONS: Although there was a moderate reduction in platelet survival in HIV-infected persons, these patients, regardless of platelet counts, also had decreased production of platelets, possibly due to viral infection of the megakaryocytes. Zidovudine appears to improve platelet production.

Blood Platelets↗
PubMed1991

The role of surface charge in the activation of the classical and alternative pathways of complement by liposomes.

We have studied the complement-activating properties of liposomes. We show that surface charge is a key determinant of complement-activating liposomes. The nature of the charge, whether negative or positive, appears to dictate which pathway of the complement system is activated. Phosphatidylcholine:cholesterol (PC:CHOL, 55:45 mol/mol) liposomes were made to exhibit a positive or negative surface charge by the addition of cationic or anionic lipids, respectively. Normal human or guinea pig serum was incubated with liposomes, followed by determining the residual hemolytic activity of the serum as a measure of complement activation. Negatively charged liposomes containing phosphatidyl-glycerol, phosphatidic acid, cardiolipin, phosphatidylinositol, or phosphatidylserine activated complement in a Ca(2+)-dependent manner suggesting activation occurred via the classical pathway. Positively charged liposomes containing stearylamine or 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane activated complement via the alternative pathway. Neutral liposomes, PC:CHOL (55:45) and PC:CHOL:dipalmitoylphosphatidylethanolamine (35:45:20), failed to activate complement as measured by the hemolytic assays. We show that unsaturated liposomes are more potent complement activators than saturated liposomes and that 45 mol% cholesterol promotes complement protein-liposome interactions. Immunoblot analysis of phosphatidylglycerol-containing liposomes showed that C3b and C9 were associated with these liposomes. Thus, the complement consumption measured in the hemolytic assays represents active cleavage of the complement components and not passive adsorption to the liposome surface. These studies suggest that membranes composed of net charged phospholipids can activate the complement system. This observation underlines the importance in biologic membranes of complement regulatory proteins that protect normal cells from complement attack.

Animals↗
PubMed1991

The regulation of complement on cell surfaces.

The regulation of complement at the surface of cells is mediated by both plasma and membrane proteins. These molecules act in concert to prevent the accelerated catabolism of complement proteins and the concomitant lysis of homologous blood cells. The deficiency of either a plasma or membrane protein predisposes to the development of disease.

Angioedema↗
PubMed1990

Steroid-responsive type II anti-factor VIII:C autoantibody in a nonhemophiliac child.

Acquired antibodies to factor VIII:C in nonhemophiliac patients are uncommon in adulthood and exceedingly rare in childhood. We report a girl, 9 years of age, with no personal or familial bleeding history who presented with hematuria and bruising 2 weeks after an upper respiratory infection. The activated partial thromboplastin time was 71.9 s (normal, 25-40 s) and did not correct by mixing 1:1 with normal plasma, suggesting the presence of an inhibitor. Factor VIII:C levels were detectable at 0.03 U/ml, but inhibition experiments demonstrated the presence of an inhibitor with an activity of 24 Bethesda U/ml. This inhibitor was localized to the immunoglobulin (IgG) fraction of the patient's plasma. Incubation of the patient's IgG with normal pooled plasma resulted in a 66% decrease in factor VIII:C activity. Unlike the antibodies found in most hemophilia patients, the autoantibody produced by this patient demonstrated type II kinetics and did not inhibit all factor VIII:C activity even at very high concentrations. In addition, the rate of factor VIII:C inactivation by this autoantibody was much slower than that seen with type I inhibitors. The treatment of the patient with prednisone, 2.5 mg/kg/day, resulted in the rapid disappearance of detectable inhibitor and a rise in factor VIII:C levels to 0.70 U/ml. Normal factor VIII:C levels persisted after the discontinuation of steroids. This case is most unusual in that it occurred in a child without any evidence of an underlying autoimmune disorder, and unlike classical hemophiliac factor VIII:C inhibitors, there was a rapid response to steroids.

Autoantibodies↗
PubMed1988

Cytofluorometric identification of plasmin-sensitive factor XIIIa binding to platelets.

We have investigated the binding of blood coagulation factor XIIIa to thrombin-stimulated platelets using cytofluorometric analysis. Washed thrombin-stimulated platelets bound exogenously added factor XIIIa in a calcium-dependent reaction. The expression of endogenous platelet factor XIII was also detected on the surface of thrombin-stimulated platelets. When fluorescence analysis was performed based on particle size, factor XIIIa bound to the surface of greater than 95% of particles which contained more than one platelet, but only 50% of single platelets. The binding of factor XIIIa to thrombin-stimulated platelets was inhibited by plasmin. Plasmin also inhibited thrombin-dependent expression of the factor XIIIa binding site on platelets. Experiments in which thrombin-stimulated platelets were incubated with factor XIIIa in the presence of 125I-dimethylcasein or 3H-putrescine demonstrated that platelets bear both glutamyl and lysyl substrates for factor XIIIa. Thrombin increased the expression of factor XIIIa substrates by platelets. Plasmin inhibited both the expression of factor XIIIa substrates and degraded them. The binding of factor XIIIa to thrombin-stimulated platelets and the availability of factor XIIIa substrates on the platelet surface could provide a mechanism by which factor XIIIa stabilizes the hemostatic plug by promoting crosslinking reactions between platelet membrane proteins and adhesive glycoproteins. In contrast, plasmin inhibition of factor XIIIa binding and crosslinking could disrupt hemostasis.

Blood Platelets↗
PubMed1988

Monoclonal antibody to fibrin D-dimer (DD-3B6) recognizes an epitope on the gamma-chain of fragment D.

Laboratory determination of fibrinolysis has been facilitated by diagnostic tests that use monoclonal antibody DD-3B6 to measure fibrin D-dimer levels in plasma. When DD-3B6 is reacted with soluble fibrin fragments, it is specific for fragment D-dimer. The authors have used immunoblot analysis of DD-3B6 binding to purified fragment D and fragment D-dimer to localize the binding site of the antibody. Although DD-3B6 recognizes only fragment D-dimer in solution, it binds to both immobilized fragment D-dimer and fragment D in immunoblots. When immunoblots were performed using protein which was electrophoresed under reducing conditions, DD-3B6 bound to the gamma-chain of fragments D1, D2, and D3. Therefore, the epitope recognized by DD-3B6 resides between amino acids 86 and 302 in the gamma-chain of fragment D. This epitope is masked in soluble non-cross-linked or nondegraded fibrin, but becomes expressed after cross-linked fibrin has been cleaved by plasmin.

Antibodies, Monoclonal↗
PubMed1988

The population of paroxysmal nocturnal hemoglobinuria neutrophils deficient in decay-accelerating factor is also deficient in alkaline phosphatase.

In patients with paroxysmal nocturnal hemoglobinuria (PNH) the RBCs, neutrophils (PMNs), monocytes, and platelets derived from the abnormal clone are deficient in the complement-regulatory protein decay-accelerating factor (DAF). RBC acetylcholinesterase (AChE) and leukocyte alkaline phosphatase (LAP) activities are also characteristically low. DAF, AChE, and LAP are known to be anchored within cell membranes to glycophospholipid-containing phosphatidylinositol (PI). Because PNH progenitors contain DAF that appears to be lost with maturation, it has been proposed that this disorder results from abnormal tethering of these and possibly other proteins to membrane PI. We were puzzled, therefore, that our two PNH patients consistently had normal LAP levels. Consequently, we studied their isolated PMNs to compare DAF and LAP activities in individual cells. PMNs were separated by flow cytometry into DAF-positive and -negative populations by using rabbit anti-DAF antiserum and fluorescein-conjugated goat antirabbit IgG. In both patients the majority of PMNs were DAF deficient, and these cells contained very little alkaline phosphatase activity. In contrast, the smaller, DAF-positive cell populations were phosphatase replete. This is the first demonstration that abnormalities in DAF and LAP activity occur in the same PNH PMN population and strengthens the hypothesis that defective anchoring of proteins to membrane glycophospholipid underlies the pathophysiology of this disorder.

Aged↗