Alarmingly high prevalence of obesity in haemophilia in the state of Mississippi.

The state of Mississippi has consistently been ranked as the state with most number of obese people in the United States with prevalence rates of >30%. Our aims in this study were to estimate the prevalence of overweight and obesity in children and adults diagnosed with haemophilia in Mississippi, and to assess whether race/ethnicity and the severity of haemophilia are important risk factors. A retrospective chart review was performed for all haemophilic patients seen at the Mississippi Hemophilia Treatment Center. Patients were classified into two major age groups: age 2-19.9 years and > or =20 years. Body mass index (BMI) was calculated from the height and weight in kg m(-2) from the last clinic visit. Out of a total of 132 haemophilic patients, 61% were white and 37% were African American. Overall, 51% of the haemophilic patients were either obese or overweight. The prevalence of obesity in the adult (> or =20 years old) haemophilic patients was 36% and an additional 32% were overweight. A significantly greater proportion of patients >20 years old were overweight or obese as compared with the patients in the 2-19.9 year age range (P < 0.002). However, race/ethnicity and severity of haemophilia were not significant risk factors for overweight and obesity. There is a very high prevalence of obesity in the Mississippi haemophilic population, especially in adults. Particular attention at clinic visits should be paid to the BMI in order to identify patients that are overweight or obese to allow for early and appropriate intervention.

Marked temperature dependence of the platelet calcium signal induced by human von Willebrand factor.

Interaction of von Willebrand factor (vWF) with the platelet is essential to hemostasis when vascular injury occurs. This interaction elevates the intracellular free calcium concentration ([Ca2+]i) and promotes platelet activation. The present study investigated the temperature dependence of vWF-induced [Ca2+]i signaling in human platelets. The influence of temperature can provide invaluable insight into the underlying mechanism. Platelet [Ca2+]i was monitored with Fura-PE3. Ristocetin-mediated binding of vWF induced a transient platelet [Ca2+]i increase at 37 degrees C, but no response at lower temperatures (20 degrees C to 25 degrees C). This temperature dependence could not be attributed to a reduction in vWF binding, as ristocetin-mediated platelet aggregation and agglutination were essentially unaffected by temperature. Most other platelet agonists (U-46619, alpha-thrombin, and adenosine 5'-diphosphate [ADP]) induced a [Ca2+]i signal whose amplitude did not diminish at lower temperatures. The [Ca2+]i signal in response to arachidonic acid, however, showed similar temperature dependence to that seen with vWF. Assessment of thromboxane A2 production showed a strong temperature dependence for metabolism of arachidonic acid by the cyclo-oxygenase pathway. vWF induced thromboxane A2 production in the platelet. Aspirin treatment abolished the vWF-induced [Ca2+]i signal. These observations suggest that release of arachidonic acid and its conversion to thromboxane A2 play a central role in vWF-mediated [Ca2+]i signaling in the platelet at physiological temperatures.

Ristocetin-mediated interaction of human von Willebrand factor with platelet glycoprotein lb evokes a transient calcium signal: observations with Fura-PE3.

High shear stress in narrowed arteries causes von Willebrand factor (vWf) to bind to its platelet receptor, glycoprotein Ib (GpIb). This binding is reported to promote an increase in intracellular free calcium concentration ((Ca2+)i), which may be responsible for platelet activation. The present study examined the platelet (Ca2+)i signal that arises when ristocetin mediates vWf-GpIb binding. Platelet (Ca2+)i was monitored with Fura-PE3 (Vorndran C, Minta A, Poenie M. Biophys J 1995;69:2112-24), a new ratiometric calcium indicator. Fura-PE3 has calcium-binding characteristics (Kd = 146 nmol/L) and fluorescent properties similar to those of Fura-2. However, its zwitterionic nature ensured much slower extrusion from the platelet (0.2% per minute) than that for Fura-2. This eliminated one of the technical problems that seriously distorted previous measurements of vWf-induced changes in platelets (Ca2+)i. Design of a novel stirring arrangement avoided the other major problem, which is the tendency of platelet aggregates to settle to the bottom of the cuvette, beneath the detection zone of the spectrofluorometer. With Fura-PE3 and the new stirrer used in the present study, vWf-induced changes in (Ca2+)i could be measured reliably in aggregating platelets. Ristocetin-mediated vWf-GpIb binding induced a transient increase in platelet (Ca2+)i. This increase occurred after a significant lag phase; platelet (Ca2+)i rose gradually, followed by a decline to almost the resting level. Binding of vWf to platelet Gplb was responsible for the (Ca2+)i signal. A similar signal was found in the absence of extracellular calcium. These characteristics differ substantially from those described in previous reports, in which the vWf-induced rise in (Ca2+)i was attributed to calcium influx through channels in the plasma membrane. Data from those earlier studies, however, were severely distorted by indicator extrusion and loss of platelet aggregates. The present findings are a more accurate representation of the vWf-induced platelet (Ca2+]i signal.

Direct evidence for functional coupling of the vasoactive intestinal peptide receptor to Gi3 in native lung membranes.

Although vasoactive intestinal peptide (VIP) exerts many of its effects through stimulation of adenylyl cyclase, there is increasing evidence that other signaling pathways may contribute to its action. The role of inhibitory G proteins (Gi) in VIP-mediated signaling in the lung was assessed by a combination of equilibrium-binding and covalent cross-linking studies. Pertussis toxin treatment of rat lung membranes reduced the high affinity binding of 125I-VIP, implicating a member of the Gi family in signaling from the VIP receptor. The particular G protein involved was identified as Gi3 through capture of a VIP/receptor/ Gi3 ternary complex by covalent cross-linking. There was a progressive rise with increasing VIP concentration in formation of the complex reported by the cross-linking strategy. Guanine nucleotides and an anti-G alpha i3 antiserum suppressed formation of the VIP/receptor/Gi3 ternary complex, demonstrating its functional nature in native lung membranes. Inhibition of high affinity 125I-VIP binding by the anti-G alpha i3 antiserum verified this functionality. Taken together, these data suggest that receptor/ Gi3 coupling makes a significant contribution to VIP-mediated signaling in the lung and illustrate the value of covalent cross-linking as a strategy to define receptor/G protein complexes that arise under conditions in which the stoichiometry and microdomains of the native cell membrane are preserved.

Fluorescent indicators give biased estimates of intracellular free calcium change in aggregating platelets: implication for studies with human von Willebrand factor.

The ratiometric fluorescent indicators Fura-2 and Indo-1 are considered optimal probes for monitoring intracellular free calcium concentration ([Ca2+]i). Unique problems arise, however, in studying [Ca2+]i changes induced in platelets by von Willebrand factor (vWF). Binding of native multimeric vWF causes extensive platelet aggregation, and is reported to evoke a gradual [Ca2+]i increase. the present investigation examined the reliability of platelet [Ca2+]i measurements in these circumstances. Ristocetin-mediated binding of vWF to human platelets promoted a slow rise in Fura-2 fluorescence ratio. Fura-2 extrusion contributed substantially to this rise, unless blocked by probenecid. Despite this precaution, the platelets were invariably contaminated slightly with extracellular indicator. As aggregation progressively reduced the number of platelets in the spectrofluorometer beam, through settling of the larger aggregates, such extracellular Fura-2 contributed proportionately more to the observed fluorescence. This extraneous signal accounted completely for the fluorescence ratio increase, and apparent [Ca2+]i rise, in response to native multimeric vWF. The same problem arose with Indo-1, whereas the single wavelength indicator Fluo-3 showed the opposite pattern of apparent [Ca2+]i changes. Thus, none of these indicators provides reliable data on [Ca2+]i signals in aggregating platelets. Use of a dimeric form of vWF eliminated the problem of platelet aggregates settling out of suspension, but also virtually abolished the [Ca2+]i increase. These observations may explain some of the inconsistencies among previous investigations of vWF-induced calcium signaling. Moreover, similar problems may arise in studies with other adhesive proteins.

Structural characterization of heparin's binding domain for human platelets.

The structural features of heparin that are involved in binding to human platelets were investigated by a competitive binding approach. A range of heparin-derived glycosaminoglycans (GAGs) with relatively defined structure were prepared by different methods of depolymerization of pharmaceutical heparin, followed by fractionation according to molecular weight and net charge. Competitive binding to platelets was dependent on molecular weight but not on the net charge of the GAGs. The method for depolymerization significantly affected the binding activity of the resulting GAG. Heparinase I and nitrous acid depolymerization produced GAGs with lower binding affinity for platelets than those GAGs derived from the treatment with periodate followed by alkali. The IC20 (concentration producing 20% inhibition of binding) was 0.05 microM for unfractionated heparin, 0.11 microM for a periodate treated GAG, and 2 microM for comparably sized GAGs (M(r) approximately 6,000-8,000) derived by heparinase I or nitrous acid treatment. Thus, the disaccharide units GlcNSO3-6S--IdoA-2S or GlcNSO3--IdoA-2S [(2-deoxy-2-sulfoamido-6-O-sulfo-alpha-D-glycopyranosyl)-(1- 4)-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid) or (2-deoxy-2-sulfoamido-alpha-D-glycopyranosyl)-(1-4)-O-(2-O-s ulfo-alpha-L-idopyranosyluronic acid)] may be crucial elements for binding to the platelet, because these are known to be preserved during periodate/alkali treatment, but readily decomposed by heparinase I and nitrous acid. Understanding this structural specificity for platelet binding may be useful for the development of heparins with high or low platelet reactivity.

Molecular properties of the vasoactive intestinal peptide receptor in aorta and other tissues.

The molecular weight of the vasoactive intestinal peptide (VIP) receptor was assessed in bovine aorta, and rat liver, lung, and brain by covalent cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The receptor in all four tissues was found to be a single polypeptide of approximate M(r) 54,000, contradicting previous claims for substantial heterogeneity in the molecular weight of this receptor. Guanine nucleotides inhibit cross-linking of 125I-VIP to its receptor, and cross-linking with ethylene glycolbis(succinimidylsuccinate) provides further evidence for complex formation between VIP, its receptor and a guanine nucleotide-binding regulatory protein (G-protein). The precise mechanism of receptor-G-protein coupling may differ between the aorta and other tissues.

Localization and characterization of a heparin binding domain peptide of human von Willebrand factor.

Human von Willebrand factor, a plasma glycoprotein which plays a critical role in regulating hemostasis, binds heparin, but the physiological importance and mode of this interaction is poorly understood. Using the motif of an amino acid sequence of a consensus heparin binding synthetic peptide, a 23-residue sequence (Tyr565-Ala587) of human von Willebrand factor was identified that retains the consensus motif and binds heparin with affinity comparable with native von Willebrand factor and the consensus peptide. In a fluid phase binding assay, the Tyr565-Ala587 peptide competed effectively with von Willebrand factor for binding heparin. Synthesis and testing of peptides overlapping Tyr565-Ala587, as well as adjacent cationic regions, showed this core sequence to be the optimal linear binding domain. Far ultraviolet circular dichroism spectrometry of the Tyr565-Ala587 peptide suggested that the peptide undergoes conformational change upon binding heparin. The Tyr565-Ala587 peptide thus encompasses part (or all) of a functionally important heparin binding domain of von Willebrand factor. Further study of this and related peptides may be useful for exploring how heparin may influence von Willebrand factor-mediated platelet hemostasis.

The thrombin high-affinity binding site on platelets is a negative regulator of thrombin-induced platelet activation. Structure-function studies using two mutant thrombins, Quick I and Quick II.

To elucidate the thrombin domains required for high-affinity binding and platelet activation, the platelet binding properties of thrombin and two mutant thrombins, thrombin Quick I and Quick II, were compared to their agonist effects in elevating intraplatelet [Ca2+]. In Quick I, a mutation within the fibrinogen binding groove results in decreased clotting and platelet aggregating activities, whereas in Quick II, a mutation in the primary substrate binding pocket abolishes both activities. Dysthrombin binding was decreased compared to thrombin. The fibrinogen binding groove appeared more important than the primary substrate pocket for high-affinity binding since Quick I showed drastically reduced, and Quick II only slightly reduced, binding affinity (Kd approximately 200 and approximately 10 nM, respectively). The deduced interaction of thrombin with its high-affinity binding site indicated that the thrombin catalytic site is directed toward the platelet surface and therefore, when bound, is proteolytically inactive. Quick I (0.5-5 nM) elicited intraplatelet [Ca2+] fluxes at concentrations where high-affinity binding was undetectable. Saturation of high-affinity binding sites with active-site-modified thrombin did not affect thrombin-induced (0.5 nM) or Quick I-induced (5 nM) responses. In contrast, addition of D-Phe-Pro-Arg chloromethyl ketone (FPRCK) subsequent to thrombin or Quick I stimulation of platelets abolished agonist-induced responses. Since Quick I was only 10-17% as effective as thrombin in increasing intraplatelet [Ca2+], our data support a model in which thrombin acts enzymatically on a platelet membrane "substrate", through an interaction mediated in part by the fibrinogen binding groove of thrombin. This conclusion is consistent with the inhibition observed with high concentrations (greater than 100 nM) of Quick II and FPRCK-modified thrombin (FPR-thrombin) in platelets stimulated with low concentrations of thrombin (less than 0.5 nM) or Quick I (less than 2 nM), consistent with inhibition by substrate depletion. In contrast, concentrations of FPR-thrombin or Quick II (less than 100 nM), which saturated predominantly the high-affinity binding sites, enhanced the platelet responses induced by thrombin (less than 0.5 nM). Thus, occupation of the high-affinity sites with inactive thrombin increased the concentration of active thrombin available for substrate interaction. Quick I-induced responses were not enhanced, consistent with its inability to interact with the high-affinity site. Since thrombin bound to the high-affinity site is proteolytically inactive, we hypothesize that the thrombin high-affinity binding site on platelets functions to alter thrombin activity and platelet activation.

Evidence for the formation of a functional complex between vasoactive intestinal peptide, its receptor, and Gs in lung membranes.

The molecular weight of the vasoactive intestinal peptide (VIP) receptor in rat lung and its interaction with the stimulatory guanine nucleotide-binding protein (Gs) were assessed by covalent cross-linking, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunological techniques. Studies with two cross-linking agents indicated that the VIP receptor in this tissue is a single polypeptide of Mr = 54,000. The VIP-occupied receptor could be cross-linked to neighboring proteins after detergent solubilization; higher molecular weight complexes of Mr = 114,000 and 184,000 were formed. Immunoblotting with antisera against G-protein subunits demonstrated that both complexes contained the alpha-subunit of Gs as well as the 125I-VIP cross-linked receptor whereas only the Mr = 184,000 complex contained the beta-subunit. Pretreatment with GTP reduced the prominence of these complexes, verifying the functional nature of this receptor-Gs association. Studies with a third cross-linking agent, ethylene glycol bis(succinimidyl succinate), provided direct evidence of physically associated, ternary VIP-receptor-Gs complexes actually in the membrane milieu. That these complexes were functionally associated with shown by their inhibition by anti-Gs alpha anti-serum. Since treatment of membranes with guanosine 5'-O-(3-thiotriphosphate) resulted in the separation of the VIP-cross-linked receptor from Gs such that no cross-linking could occur, we conclude that the binding of GTP analogs induces a conformational change in Gs in the membrane milieu.

The significance of functional receptor heterogeneity in the biological responses of the rabbit neutrophil to stimulation by chemotactic formyl peptides.

The characteristics of binding to the chemotactic receptors on rabbit peritoneal neutrophils were examined for seven formyl peptide analogues. These receptor-binding characteristics were compared with the abilities of the analogues to induce the biological responses of degranulation and chemotaxis. Five of the analogues showed distinct functional heterogeneity in their receptor-binding patterns, whereas the two most potent compounds displayed homogeneous binding patterns. The relative potencies of the formyl peptide analogues for stimulation of degranulation correlated well with their relative potencies for high-affinity, but not low-affinity, binding. The biphasic patterns for stimulation of chemotactic migration were similar for the less potent analogues, and their potencies paralleled those for both degranulation and receptor binding. In contrast, the most potent analogues induced a greater maximal extent of chemotactic migration than the other compounds, but displayed a lower than expected potency (i.e. they required higher than expected concentrations). These anomalies in the patterns of the chemotactic response cannot be reconciled with a simple receptor model comprising two independent classes of receptors. Instead, a model comprising interconvertible states of different affinities is proposed. The state of higher affinity appears to play a central role in initiation of both degranulation and chemotaxis. The more potent formyl peptide analogues are thought to stabilize an activated, higher-affinity, state of the receptor; this can explain their greater efficacy in stimulating chemotaxis. The proposed model may also be applicable to other receptors that are coupled by a guanine-nucleotide-binding regulatory protein to their associated effector.

Heparin inhibition of von Willebrand factor-dependent platelet function in vitro and in vivo.

The intravenous administration of heparin to patients before open heart surgery reduced ristocetin cofactor activity by 58% (P less than 0.01, t test), and this impairment of von Willebrand factor-dependent platelet function was closely related to plasma heparin levels (r2 = 0.9), but not to plasma von Willebrand factor (vWF) levels. We hypothesized that heparin may inhibit vWF-dependent platelet hemostatic functions by directly binding vWF in solution and interfering with vWF-GpIb binding. Using the in vitro techniques of ristocetin-induced platelet agglutination, fluorescent flow cytometric measurement of vWF-platelet binding, and conventional radioligand binding assays we observed that heparin inhibited both vWF-dependent platelet function and vWF-platelet binding in a parallel and dose-dependent manner. Heparin also inhibited platelet agglutination induced by bovine vWF and inhibited the binding of human asialo-vWF to platelets in ristocetin-free systems. The inhibitory potency of heparin was not dependent upon its affinity for antithrombin III, but was molecular weight dependent: homogeneous preparations of lower molecular weight were less inhibitory. Heparin impairment of vWF function may explain why some hemorrhagic complications of heparin therapy are not predictable based on techniques for monitoring the conventional anticoagulant effects of heparin.

Comparison of stimulation by chemotactic formyl peptide analogs between GTPase activity in neutrophil plasma membranes and granule enzyme release from intact neutrophils.

Stimulation by fMet-Leu-Phe analogs of GTPase activity in plasma membranes from rabbit neutrophils was compared with the stimulation of degranulation in intact neutrophils. All four formyl peptides examined (fMet-Leu-Phe-Phe, fMet-Leu-Phe, fNle-Leu-Phe, and fVal-Leu-Phe) were full agonists for both responses. Their ED50 values for the two responses correlated well, although those for GTPase stimulation were uniformly about tenfold greater. The specific antagonist tBoc-Phe-Leu-Phe-Leu-Phe competitively inhibited both GTPase activity and degranulation stimulated by fMet-Leu-Phe; its Ki values were similar for the two responses. Pertussis toxin treatment, in contrast, inhibited the maximal stimulation of both responses by fMet-Leu-Phe with minimal shift in ED50. The inhibitory actions of tBoc-Phe-Leu-Phe-Leu-Phe and pertussis toxin on GTPase activity thus paralleled the effects on degranulation. These observations substantiate the hypothesis that a guanine nucleotide-binding protein that is a pertussis toxin substrate couples the formyl peptide receptors to physiological function in the neutrophil.

Receptor-purified, Bolton-Hunter radioiodinated, recombinant, human epidermal growth factor: an improved radioligand for receptor studies.

We report an assessment of the applicability of the Bolton-Hunter method to the radioiodination of epidermal growth factor (EGF). Recombinant human EGF (hEGF) could be radioiodinated successfully by this method, whereas murine EGF could not. Bolton-Hunter 125I-labeled hEGF was compared with commercial 125I-labeled hEGF prepared by the chloramine-T radioiodination method. Neither radioligand was sufficiently pure for a detailed characterization of the purportedly heterogeneous pattern of binding of EGF to its receptors. A procedure based on receptor adsorption was thus developed for repurification of the Bolton-Hunter 125I-labeled hEGF. This provided a much purer radioligand suitable for detailed studies of receptor-binding heterogeneity.

Means for the assessment of radioligand quality and its importance in receptor-binding studies. Observations with radiolabelled formylmethionyl-leucyl-phenylalanine.

Various methods for testing the quality of radioligands were applied to two different radiolabelled forms of formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe). The purpose of the study was both to examine the value of these methods for assessing radioligand quality and to determine the suitability of these particular radioligands for studying the chemotactic formylpeptide receptors on the rabbit neutrophil. It is useful in this context to distinguish two different aspects of radioligand quality: these are purity and equivalence to the native ligand. The two methods described for measuring receptor-reactivity (or 'bindability'), by measuring binding to an increasing excess of receptors and by a re-incubation procedure, provide a reliable measure of purity that should readily be applicable to other radioligands. Equivalence to the native ligand is more difficult to establish, and any uncertainty about the specific radioactivity of the radioligand can pose serious problems with this assessment. Commercial preparations of both tritiated and 35S-labelled fMet-Leu-Phe were found to be inadequately pure for detailed receptor studies. Repurification by t.l.c., however, consistently yielded radioligand preparations of high purity and close equivalence to the native ligand. Other radioligands may often also require a suitable repurification step before use for detailed receptor studies; this is especially important whenever a complex receptor-binding pattern is envisaged.