Cellular mechanisms by which proinsulin C-peptide prevents insulin-induced neointima formation in human saphenous vein.

AIMS/HYPOTHESIS: Endothelial cells (ECs) and smooth muscle cells (SMCs) play key roles in the development of intimal hyperplasia in saphenous vein (SV) bypass grafts. In diabetic patients, insulin administration controls hyperglycaemia but cardiovascular complications remain. Insulin is synthesised as a pro-peptide, from which C-peptide is cleaved and released into the circulation with insulin; exogenous insulin lacks C-peptide. Here we investigate modulation of human SV neointima formation and SV-EC and SV-SMC function by insulin and C-peptide. METHODS: Effects of insulin and C-peptide on neointima formation (organ cultures), EC and SMC proliferation (cell counting), EC migration (scratch wound), SMC migration (Boyden chamber) and signalling (immunoblotting) were examined. A real-time RT-PCR array identified insulin-responsive genes, and results were confirmed by real-time RT-PCR. Targeted gene silencing (siRNA) was used to assess functional relevance. RESULTS: Insulin (100 nmol/l) augmented SV neointimal thickening (70% increase, 14 days), SMC proliferation (55% increase, 7 days) and migration (150% increase, 6 h); effects were abrogated by 10 nmol/l C-peptide. C-peptide did not affect insulin-induced Akt or extracellular signal-regulated kinase signalling (15 min), but array data and gene silencing implicated sterol regulatory element binding transcription factor 1 (SREBF1). Insulin (1-100 nmol/l) did not modify EC proliferation or migration, whereas 10 nmol/l C-peptide stimulated EC proliferation by 40% (5 days). CONCLUSIONS/INTERPRETATION: Our data support a causative role for insulin in human SV neointima formation with a novel counter-regulatory effect of proinsulin C-peptide. Thus, C-peptide can limit the detrimental effects of insulin on SMC function. Co-supplementing insulin therapy with C-peptide could improve therapy in insulin-treated patients.

Positive follow-up blood cultures identify high mortality risk among patients with Gram-negative bacteraemia.

OBJECTIVES: The role of follow-up blood cultures (FUBCs) in the management of Gram-negative bacteraemia (GNB) is poorly understood. We aimed to determine the utility of FUBCs in identifying patients with increased mortality risk. METHODS: An observational study with a prospectively enrolled cohort of adult inpatients with GNB was conducted at Duke University Health System from 2002 to 2015. FUBCs were defined as blood cultures performed from 24 hours to 7 days from initial positive blood culture. RESULTS: Among 1702 patients with GNB, 1164 (68%) had FUBCs performed. When performed, FUBCs were positive in 20% (228/1113) of cases. FUBC acquisition was associated with lower all-cause in-hospital mortality (108/538, 20%, vs. 176/1164, 15%; p 0.01) and attributable in-hospital mortality (78/538, 15%, vs. 98/1164, 8%; p < 0.0001). Propensity score-weighted Cox proportional hazards models revealed that obtaining FUBCs was associated with reductions in all-cause (hazard ratio (HR) 0.629; 95% confidence interval (CI), 0.511-0.772; p < 0.0001) and attributable mortality (HR 0.628; 95% CI, 0.480-0.820; p 0.0007). Positive FUBCs were associated with increased all-cause mortality (49/228, 21%, vs. 110/885, 11%; p 0.0005) and attributable mortality (27/228, 12%, vs. 61/885, 7%; p 0.01) relative to negative FUBCs. Propensity score-weighted Cox proportional hazards models revealed that positive FUBCs were associated with increased all-cause (HR 2.099; 95% CI, 1.567-2.811; p < 0.0001) and attributable mortality (HR 1.800; 95% CI, 1.245-2.603; p 0.002). In a calibration analysis, a scoring system accurately identified patients at high risk of positive FUBCs. CONCLUSIONS: Rates of positive FUBCs were high and identified patients at increased risk for mortality. Clinical variables can identify patients at high risk for positive FUBCs. FUBCs should be considered in the management of GNB.

Involvement of large plasma von Willebrand factor (vWF) multimers and unusually large vWF forms derived from endothelial cells in shear stress-induced platelet aggregation.

A fluid shear stress of 180 dyn/cm2 was applied for 0.5 and 5 min to platelets in citrated plasma or blood in a cone and plate viscometer with minimal platelet-surface interactions. Platelets aggregated in the shear field if large von Willebrand Factor (vWF) multimers were present. Aggregation did not require ristocetin, other exogenous agents, or desialation of vWF. Unusually large vWF multimers produced by human endothelial cells were functionally more effective than the largest plasma vWF forms in supporting shear-induced aggregation. Shear-induced aggregation was inhibited by monoclonal antibodies to platelet glycoprotein Ib or the IIb/IIIa complex, but was little affected by the absence of fibrinogen. vWF-dependent platelet aggregation under elevated shear stress in partially occluded vessels of the arterial microcirculation may contribute to thrombosis, especially if unusually large vWF multimers are released locally from stimulated or disrupted endothelial cells.

Platelet lipoxygenase-dependent oxygen burst. Evidence for differential activation of lipoxygenase in intact and disrupted human platelets.

The metabolism of arachidonic acid in platelets by both cyclooxygenase and lipoxygenase involves the rapid consumption of molecular oxygen. However, selective inhibition of cyclooxygenase completely abolishes the arachidonate-induced oxygen burst in intact platelets. This is in contrast to platelet lysates, in which approximately 50% of the arachidonate-induced oxygen burst remains detectable following inhibition of cyclooxygenase with acetylsalicylic acid. This lipoxygenase oxygen burst is blocked by preincubation of the platelets with ETYA, which inhibits both cyclooxygenase and lipoxygenase. In cell-free 100000 x g supernatants of platelet lysates, which contain only lipoxygenase activity, arachidonate induces an oxygen burst which is not blunted by preincubation with aspirin but is completely abolished by preincubation with ETYA. The finding of a lipoxygenase-dependent oxygen burst in platelet lysates but not in intact platelet suspensions suggests differential activation or differential availability of platelet lipoxygenase in intact and disrupted platelets. This was confirmed by a 5 min lag in the generation of [14C]HETE (the major lipoxygenase product) from [14C]arachidonic acid in intact platelets, but an almost immediate initiation of [14C]HETE production in platelet lysates. In contrast, the synthesis of [14C]thromboxane B2 (the major cyclooxygenase product) from [14C]arachidonic acid began immediately in both intact and disrupted platelet preparations and peaked within 5 min. These observations provide new insight into factors controlling platelet hydroxy acid production and help to explain the nature of the platelet oxygen burst.

Effects of abciximab, ticlopidine, and combined Abciximab/Ticlopidine therapy on platelet and leukocyte function in patients undergoing coronary angioplasty.

BACKGROUND: Abciximab and ticlopidine reduce adverse cardiovascular events after percutaneous transluminal coronary angioplasty (PTCA). The goal of the current study was to determine if combined abciximab/ticlopidine therapy inhibits arterial thrombosis more effectively than either treatment alone. The effect of each therapy on platelet-leukocyte interactions was also investigated. METHODS AND RESULTS: Whole blood samples from 14 patients undergoing PTCA who received abciximab therapy, ticlopidine therapy, or both treatments were evaluated using dynamic experimental systems. Mural thrombus formation under arterial shear conditions (1500 s(-1)) was determined in a parallel plate flow chamber. Shear-induced platelet aggregation was evaluated using a cone-and-plate viscometer at a shear rate of 3000 s(-1). Of the 3 treatments, combined abciximab/ticlopidine therapy produced the most consistent reduction in shear-induced platelet aggregation and the most prolonged inhibition of mural thrombosis. Three days after PTCA, abciximab/ticlopidine treatment decreased mural thrombus formation to approximately 50% of baseline values. Abciximab treatment alone inhibited mural thrombosis for only 1 day after PTCA, whereas ticlopidine treatment alone had no significant effect. Two hours after PTCA, abciximab therapy significantly decreased the number of circulating platelet-neutrophil aggregates but significantly enhanced P-selectin-mediated leukocyte adhesion on the collagen/von Willebrand factor-platelet surface. CONCLUSIONS: Combined therapy with abciximab and ticlopidine has a prolonged inhibitory effect on mural thrombosis formation relative to either treatment alone. Further, we demonstrated an unexpected effect of abciximab in enhancing P-selectin-mediated leukocyte adhesion.

The mechanism of angiotensin II-induced extracellular signal-regulated kinase-1/2 activation is independent of angiotensin AT(1A) receptor internalisation.

The aim of this study was to determine whether internalisation of the angiotensin II (Ang II) AT(1A) receptor (AT(1A)R) was a prerequisite for Ang II-induced activation of the extracellular signal-regulated kinases, ERK-1/2. The human embryonic kidney (HEK293) cell line stably transfected with either the wild-type rat AT(1A)R or an internalisation-deficient C-terminal truncated mutant of the AT(1A)R (AT(1A)T318R) was used as a model for these studies. Inhibition of AT(1A)R internalisation by treatment with an inhibitor of clathrin-mediated endocytosis, Concanavalin A (Con A), did not inhibit Ang II-induced ERK-1/2 activation. Furthermore, cells transfected with the internalisation-deficient AT(1A)T318R mutant readily activated ERK-1/2 in response to Ang II. Ang II activated ERK-1/2 via two distinct signalling pathways in HEK-AT(1A)R cells. Approximately half of Ang II-induced ERK-1/2 activation was protein kinase C (PKC)-dependent, and the remainder was calcium- and c-Src-dependent and involved transactivation of the epidermal growth factor receptor (EGFR). In summary, Ang II-induced activation of ERK-1/2 occurs via two distinct pathways in HEK293 cells, neither of which requires AT(1A)R internalisation.

A novel flow cytometric analysis for platelet activation on immobilized von Willebrand factor or fibrillar collagen.

Under flow conditions, platelets adhere singly or in small aggregates on von Willebrand factor (VWF)-coated surfaces, but form large aggregates on immobilized fibrillar collagen. We developed a novel flow cytometric analysis to study the mechanisms underlying these distinct platelet deposition patterns. Flow cytometry was used to measure platelet activation after platelet adherence onto microspheres coated with either VWF or collagen fibrils. Two representative indices were calculated to quantify activated GpIIb-IIIa and P-selectin expression on adherent platelets. The signaling pathways responsible for platelet activation after interacting with fibrillar collagen were elucidated using various inhibitors. An in vitro endothelial cell wound model was also used to study the roles of VWF and fibrillar collagen in platelet deposition onto subendothelial matrixes. The adherent platelets on fibrillar collagen express more activated GpIIb-IIIa and P-selectin than those on VWF. Activation of GpIIb-IIIa and expression of P-selectin after platelet interaction with collagen occur via different intracellular signaling pathways; however, Ca2+ released from intracellular pools is common to both phenomena. Platelets were deposited singly or formed small aggregates on the endothelial cell wounded area, and this deposition pattern was dependent on VWF molecules secreted by endothelial cells and the absence of subendothelial collagen fibrils. As less activated GpIIb-IIIa and P-selectin are expressed after platelets interact with immobilized VWF alone, subsequent flowing platelet recruitment is minimal. Collagen fibrils, however, can activate adherent platelets sufficiently to promote the formation of large platelet aggregates.

Dependence of the induction of cytochrome P-450 by phenobarbital in primary cultures of adult rat hepatocytes on the composition of the culture medium.

A chemically defined medium developed for the maintenance of differentiated adult rat hepatocytes (T1) was compared with two commercially available media (Waymouth 752/1 and Leibovitz L-15) for maintenance of cytochrome P-450 metabolic activity in cultured hepatocytes. Specific metabolic activities of initially isolated cells and 72-hr control and phenobarbital-treated cultures were determined with 7-ethoxycoumarin, 7-ethoxyresorufin, and 7-pentoxyresorufin as substrates. Control and phenobarbital-treated cultures in T1 medium had a higher metabolic activity towards each of the three substrates than comparable cultures in the other media. These studies indicated that the metabolic activity and the response to phenobarbital of the major isozyme of the phenobarbital-inducible family of cytochrome P-450 were maintained in hepatocytes in T1 medium. However, there was anomalous expression and induction by phenobarbital of the major 3-methylcholanthrene-inducible isozyme, cytochrome P-450c, in cultured hepatocytes in each of the three media tested, but this response was more pronounced in T1 medium. In conclusion, the regulation of cytochrome P-450 metabolic activity in cultured hepatocytes was shown to be dependent on the composition of the culture medium.

In vitro and in vivo activity of a recombinant toxin, OLX-209, which targets the erbB-2 oncoprotein.

OLX-209 has readily measurable activity, is safe in experimental animals, and is efficacious in model systems. These results support the concept of OLX-209 and provide groundwork for further development of this oncoprotein targeted agent.

Down-regulation or long-term inhibition of protein kinase C (PKC) reduces noradrenaline release evoked via either PKC-dependent or PKC-independent pathways in human SH-SY5Y neuroblastoma cells.

Long-term (8-48 h) treatment of SH-SY5Y neuroblastoma cells with phorbol-12,13-dibutyrate (PDBu; 100 nM) promotes the down-regulation of protein kinase C (PKC) subtypes alpha and epsilon and reduces by up to 60% noradrenaline (NA) release evoked via both PKC-dependent (M3-muscarinic receptor activation) and PKC-independent (depolarization) pathways, over similar time courses. A similar effect on release is observed following long-term (16-48 h) incubation with the PKC inhibitor Ro 31-7549 (10 microM), even after removal of the inhibitor, indicating a mechanism which is not rapidly reversible. Evidence is presented which suggests that long-term treatment with PDBu does not (1) affect calcium entry, (2) modulate levels of proteins important in the secretory mechanism or (3) reduce the number of secretory vesicles. Thus, the decrease in NA release in SH-SYSY cells following down-regulation of PKC appears to be the result of a sustained reduction in PKC activity acting on a component of the secretory pathway not involved in the regulation of calcium entry or vesicle number.

Shear stress-induced binding of von Willebrand factor to platelets.

Shear stress-induced platelet aggregation requires von Willebrand factor (vWF), platelet glycoprotein (GP) Ib, GPIIb-IIIa, Ca2+, and adenosine diphosphate (ADP). Recent reports using vWF labeled with either 125I or fluorescein isothiocyanate (FITC) have demonstrated that in shear-fields, vWF binds to both GPIb and GPIIb-IIIa. The sequence of the vWF finding to the two platelet receptors has not been precisely determined in these reports. In this study, a flow cytometry technique using a primary anti-vWF antibody and a secondary FITC IgG antibody was used to measure shear stress-induced vWF binding to platelets. Washed normal platelets suspended at 50,000/microliters with purified large vWF multimers were exposed to laminar shear stresses of 15 to 120 dynes/cm2 for 30 sec. At this low platelet count, little or no aggregation occurred in the shear fields. A significant increase in post-shear vWF-positive platelets was consistently observed. Experiments with platelets from normal and severe von Willebrand's disease (vWD) (which lack plasma and platelet alpha-granule vWF) demonstrated that exogenous vWF predominately contributed to the platelet-vWF binding. Blockade of platelet GPIb with the monoclonal anti-GPIb antibody, 6D1, completely inhibited shear stress-induced platelet-vWF attachment. In contrast, blockade of GPIIb-IIIa with monoclonal anti-GPIIb-IIIa antibodies, 10E5, or c7E3, or with the GPIIb-IIIa-blocking tetrapeptide, RGDS had little or no inhibitory effect on platelet-vWF binding. These data demonstrate that the binding of vWF to GPIb is likely to be the initial shear-induced platelet-ligand binding event.

Protein phosphatase 2B inhibition promotes the secretion of von Willebrand factor from endothelial cells.

BACKGROUND: Secretion of Weibel-Palade body (WPB) contents is regulated, in part, by the phosphorylation of proteins that constitute the endothelial exocytotic machinery. In comparison to protein kinases, a role for protein phosphatases in regulating endothelial exocytosis is undefined. OBJECTIVE AND METHOD: In this study, we investigated the role of protein phosphatase 2B (PP2B) in the process of endothelial exocytosis using pharmacological and gene knockdown approaches. RESULTS: We show that inhibition of protein phosphatase 2B (PP2B) activity by cyclosporine A (CsA), tacrolimus or a cell-permeable PP2B autoinhibitory peptide promotes the secretion of ultralarge von Willebrand factor (ULVWF) from human umbilical vein endothelial cells (HUVECs) in the absence of any other endothelial cell-stimulating agent. PP2B inhibitor-induced secretion and anchorage of ULVWF strings from HUVECs mediate platelet tethering. In support of a role for PP2B in von Willebrand factor (VWF) secretion, the catalytic subunit of PP2B interacts with the vesicle trafficking protein, Munc18c. Serine phosphorylation of Munc18c, which promotes granule exocytosis in other secretory cells, is increased in CsA-treated HUVECs, suggesting that this process may be involved in CsA-mediated WPB exocytosis. Furthermore, the plasma VWF antigen level is also enhanced in CsA-treated mice, and small interfering RNA-mediated knockdown of the alpha and beta isoforms of the PP2B-A subunit in HUVECs enhanced VWF secretion. CONCLUSIONS: These observations suggest that CsA promotes VWF release, in part by inhibition of PP2B activity, and are compatible with the clinically observed association of CsA treatment and increased plasma VWF levels in humans.

Entamoeba invadens: the requirement for galactose ligands during encystment.

During periods of stress, trophozoites of Entamoeba invadens (strain IP-1) undergo a process of differentiation (encystment) that results in a dormant cyst with a chitin-containing cyst wall. Encystment can be induced by resuspension of trophozoites from growth medium into a diluted glucose-free medium (47% LG) containing 5% adult bovine serum (ABS). ABS is thought to be a source of gal-terminated ligands that are required for high levels of encystment. After resuspension of trophozoites in 47% LG, encystment cultures were examined every 2h for responses to the (i) addition of 10mM free-galactose, (ii) resuspension of cells to serum-free medium, (iii) and dilution of encysting cultures to cell densities below that known to support full encystment (from 5 x 10(5) to 1 x 10(4)cells/ml). The role of serum components (and the gal-terminated ligand asialofetuin; ASF) adsorbed onto the surface upon which encystment proceeds, and their effect on the multi-cellular aggregation patterns formed during encystment, were also investigated. The addition of free-galactose reduced the levels of encystment (compared with the control) even when added at 10h after resuspension of trophozoites in 47% LG. The requirement for the presence of ABS during encystment was lost within 6h, with levels of encystment of cells washed free of serum reaching 80% of the control. The ability of cells to encyst when diluted to a cell density below that normally thought to support encystment reached over 50% by 8h. Efficient encystment could be obtained in 47% LG in the absence of ABS or ASF using pre-treated glass culture tubes. Encystment (47% LG; 5% ABS) using ultra low attachment plates was poor, suggesting attachment of cells to a surface via gal-terminated ligands was important for efficient encystment. The results suggest that ABS is probably not the only source of gal-terminated ligands necessary for high levels of encystment in 47% LG. While serum may provide a source of ligands which enhance the levels of encystment initially, other gal-terminated ligands possibly released by the encysting cells are still required for the completion of the encystment process and the formation of mature cysts. In addition, the gal-terminated ligands necessary for encystment efficiency may be adsorbed onto the glass surface of culture tubes and aid the initial aggregation process, as well as be involved in cell signaling during the encystment process.

Statins for the prevention of vein graft stenosis: a role for inhibition of matrix metalloproteinase-9.

Saphenous vein (SV) grafts are commonly used to bypass coronary arteries that are diseased due to atherosclerosis. However, the development of intimal hyperplasia in such grafts can lead to patency-threatening stenosis and re-occlusion of the vessel. The proliferation and migration of smooth muscle cells (SMC) play key roles in the development of intimal hyperplasia, and an agent that inhibits both of these processes therefore has therapeutic potential. A prerequisite for SMC proliferation and migration in vivo is degradation of the basement membrane, achieved by secretion of the matrix-degrading gelatinases matrix metalloproteinase-2 (MMP-2) and MMP-9. Statins are cholesterol-lowering drugs that also have direct effects on SMC function. Here we report that neointima formation in organ-cultured human SV segments is inhibited by simvastatin, an effect that is associated with reduced MMP-9 activity. Additionally, our work shows that simvastatin not only inhibits proliferation, but importantly also inhibits invasion (migration through a matrix barrier), of cultured human SV SMC. Thus simvastatin treatment appears to inhibit neointima formation as a result of combined inhibition of SMC proliferation and invasion. The potential intracellular mechanisms by which statins affect SMC proliferation and migration, and thus attenuate intimal hyperplasia, are discussed, with particular emphasis on the role of MMP-9.

Blockade of adenosine diphosphate receptors P2Y(12) and P2Y(1) is required to inhibit platelet aggregation in whole blood under flow.

Using heparinized whole blood and flow conditions, it was shown that adenosine 5'-diphosphate (ADP) receptors P2Y(12) and P2Y(1) are both important in direct shear-induced platelet aggregation and platelet aggregation subsequent to initial adhesion onto von Willebrand factor (vWf)-collagen. In the viscometer, whole blood was subjected to shear rates of 750, 1500, and 3000 s(-1) for 30 seconds at room temperature. The extent of aggregation was determined by flow cytometry. The P2Y(12) antagonist AR-C69 931MX (ARMX) reduced shear-induced aggregation at these rates by 56%, 54%, and 16%, respectively, compared to control samples. Adenosine 3',5'-diphosphate (A3P5P; P2Y(1) antagonist) inhibited shear-induced aggregation by 40%, 30% and 29%, respectively, compared to control samples. Blockade of both ADP receptors at 3000 s(-1) with ARMX plus A3P5P further reduced the platelet aggregation by 41% compared to the addition of ARMX alone (57% compared to control samples). Using a parallel-plate flow chamber, whole blood was perfused over bovine collagen type 1 at a wall shear rate of 3000 s(-1) for 60 seconds. Platelet deposition was quantified with epifluorescence video microscopy and digital image processing. Blockade of P2Y(12) alone or blockade of P2Y(1) alone did not reduce thrombus formation on vWf-collagen. In contrast, blockade of both P2Y(12) and P2Y(1) reduced platelet deposition by 72%. These results indicate that combinations of antagonists of the ADP receptors P2Y(12) and P2Y(1) are effective inhibitors of direct shear-induced platelet aggregation and of platelet aggregation subsequent to initial adhesion under flow conditions. Inhibitors of these pathways are potentially useful as antiarterial thrombotic agents.

Urea as sole source of nitrogen for plant growth : I. The development of urease activity in Spirodela oligorrhiza.

Spirodela oligorrhiza grown in sterile culture was able to use urea as sole source of nitrogen but only when the pH of the culture medium was below 4.3. Plants inoculated into urea media at pH 6.4 initially made little growth and became nitrogen-deficient in appearance and composition although they contained about 100 µgrams of urea per gram fresh weight of tissue. After a period the pH of the medium usually fell below 4.3 and growth commenced. Growth with other compounds, e.g. ammonium, nitrate or allantoin, as sources of nitrogen was not similarly affected by the pH of the culture medium.Urease activity could always be detected in the tissues of Spirodela oligorrhiza growing on urea. Plants with little or no urease activity soon developed significant activity when inoculated into urea media at pH 4.0. When the pH of the medium was higher there was no increase in urease activity and no growth ensued. Plants growing on urea possessed an activity of about 50 milliunits per gram fresh weight of tissue, but if the pH of the medium fell to 3.5 or lower, the activity present rose to 10 times this level.Urease activity also appeared, in the absence of supplied urea, as plants became increasingly nitrogen-deficient.

Information from e.x.a.f.s. spectroscopy on the structures of different forms of molybdenum in xanthine oxidase and the catalytic mechanism of the enzyme.

X-ray spectroscopy was used to provide further information on the structure of the molybdenum centre of xanthine oxidase. Earlier work was confirmed and two states of the enzyme, not reported on by previous workers, were studied. One of these was the complex of the enzyme with pyridine-3-carboxaldehyde, in which most of the metal is in the Mo(V) state, giving the e.p.r. signal known as Inhibited. The other was the complex with the inhibitor alloxanthine, with the metal as Mo(IV). For both complexes clear evidence was obtained that an oxo ligand of molybdenum was present, but not a sulphido ligand. This information complements structural information on these complexes already available from e.p.r. spectroscopy, and has permitted us to revise and refine the structures previously proposed. The mechanism of action of the enzyme is discussed in the light of the present findings on the persistence of the oxo group in the reduced enzyme complexes, as well as of related evidence [George & Bray (1988) Biochemistry 27, 3603-3609] for an oxo group in the catalytic intermediate that gives the Mo(V) e.p.r. signal known as Very Rapid.

The Bernard-Soulier platelet: I. Correlation of adhesion defects with abnormalities of surface glycoproteins.

Bernard-Soulier disease, an inherited human platelet disorder, is characterized by decreased adhesion of platelets to the subendothelium. A review of the clinical features, laboratory diagnostic features and glycoprotein abnormalities is presented. The glycoprotein composition of platelets from patients with Bernard-Soulier disease was compared with normal using 3H-labeling of surface glycoproteins followed by two-dimensional gel electrophoresis. Decreased amounts of 3H-labeling of glycoprotein IIa as well as absence of the specific surface glycoprotein Ib was observed in Bernard-Soulier platelets. Studies of Bernard-Soulier platelets affords an opportunity to understand the mechanisms of platelet adhesion responses in the interactions of platelets with blood vessels.

The synthesis of sub-micron magnetic particles and their use for preparative purification of proteins.

A novel one-step protocol for the preparation of sub-micron magnetic particles as small as 30 nm in diameter has been developed. The surface of the particles was functionalized with carboxyl groups ( approximately 1 COOH per 15A2) to facilitate the attachment of affinity ligands. The high surface area of the resulting beads, combined with the high density of functional groups on the surface, makes them ideally suited for the preparative purification of proteins as was demonstrated by the efficient isolation of trypsin (36 mg per gram of particles) from pancreatic extract.