摘要:

von Willebrand Factor (vWF) is a multifunctional glycoprotein in plasma and vascular subendothelial matrix which plays a major role in cellular adhesion. vWFdependent adhesion of platelets to the subendothelium at high shear rates involves a specific platelet membrane receptor, the glycoprotein (GP) Ib-IX complex. We have previously purified a 39/34-kiloDalton (kDa) dispase fragment of vWF (Leu-480/Val-481 to Gly-718) and demonstrated that this fragment contains the binding site for the GP Ib-IX complex [Andrews R K, et al. Biochemistry 1989; 28: 8326-83361. vWF also mediates agglutination of erythrocytes by a mechanism that appears to involve binding to membrane sulfatides. In this study, we demonstrate that the 39/34-kDa vWF fragment also contains an exclusive discrete binding domain for membrane sulfatides and that the sulfatide-binding sequence also mediates binding of vWF to equine tendon collagen.Specific binding of125I-vWF to sulfatides immobilized on microtiter wells was completely inhibited by unlabeled vWF (IC50∼0.02μ;M) and by the isolated 39/34-kDa vWF fragment (IC50∼0.8μ;M). A specific anti-39/34-kDa fragment rabbit polyclonal antibody, but not nonimmune immunoglobulin, also strongly inhibited the vWF-sulfatide interaction in this assay. Using synthetic peptides corresponding to hydrophilic sequences from within the 39/34-kDa vWF fragment, a positively-charged sequence, Gln-628 to Val-646, was identified as mediating specific binding of vWF to sulfatides, since it competitively inhibited this interaction (IC50∼0.6μ;M) comparable on a molar basis to the 39/34-kDa vWF fragment (IC,−0.8μ;M). The inhibition by the Gln-626 to Val-646 peptide was specific since neither other peptides from the 39/34-kDa domain of vWF nor another highly basic peptide, polylysine, at comparable concentrations to the Gln-628 to Val-646 peptide blocked vWF binding to sulfatides. Similarly, the Gln-628 to Val-646 peptide blocked binding of vWF to equine tendon type I collagen (IC50of 0.6μ;M) suggesting that this interaction probably involves recognition of a sulfatide-like impurity in the collagen preparation. The specific binding of vWF to sulfatides via a discrete peptide sequence, Gln-628 to Val-646, within the A1 repeat domain suggests the potential for involvement of sulfatides as a class of receptors for vWF in cellular adhesion.

ISSN:0953-7104    

DOI:10.3109/09537109509023562

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor

摘要:

The interaction of the multimeric glycoprotein von Willebrand Factor (vWF) with its platelet membrane receptor, the glycoprotein (GP) Ib-IX complex plays a key role in the initial adhesion of platelets to the vascular subendothelium at high shear blood flow. The GP Ib-IX-binding site is only expressed following activation of vWF, a process that regulates vWF-mediated platelet adhesion. Binding of vWF to the GP Ib-IX complex involves the vWF A1 internal repeat domain, which also contains distinct binding sites for sulfatides, heparin, and the non-physiological modulators of the vWF-GP Ib-IX interaction, ristocetin and botrocetin. With the ultimate aim of further defining the mechanism of vWF modulation, we have analyzed the ability of various polyanionic compounds, including aurintricarboxylic acid, Evans blue, fucoidan, and a range of sulfated and phosphorylated sugars, to inhibit specific binding of purified vWF to immobilized sulfatides and heparin, and the ristocetin- and botrocetindependent binding of vWF to the platelet GP Ib-IX complex.Firstly, it was confirmed using a solid-phase binding assay that, like sulfatides, heparin specifically bound to a purified 39/WkiloDalton fragment of vWF (Leu-480 to Gly-718) that encompasses the A1 domain. Secondly, the ability of a number of polyanionic compounds to inhibit binding of vWF to heparin, but not to immobilized sulfatides, supported previous data suggesting that heparin and sulfatides bind to distinct sites on vWF. In addition, aurintricarboxylic acid, Evans blue and fucoidan all inhibited binding of vWF to both heparin and sulfatides with similar ICso values. Thirdly, many of the compounds tested that inhibited binding of vWF to heparin also effectively inhibited both ristocetin- and botrocetin-dependent binding of vWF to the GP Ib-IX complex on platelets, whereas none of the compounds tested blocked vWF binding to sulfatides and GP Ib-IX but not heparin. The majority of compounds tested inhibited the vWF-platelet interaction to a comparable degree in the presence of ristocetin or botrocetin, suggesting a similar mechanism for inhibition irrespective of the modulator used. These combined experiments provide evidence for an electrostatic model of vWF modulation, and suggest that the heparin-binding domain of vWF may be an important regulatory site involved in the adhesion of vWF to the platelet GP Ib-IX complex.

ISSN:0953-7104    

DOI:10.3109/09537109509023563

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor

摘要:

Platelet aggregation induced by microbubbles (simulating microbubbles developing during deep sea diving or clinical situations such as extracorporeal circulation) in platelet rich plasma was measured in 11 male volunteers before and after intake of 15 ml seal oil(Pagophilus groenlandica)per day for 2 weeks. The relative content of arachidonic acid (AA) decreased in platelets from all individuals, whereas the content of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) increased. Also in plasma, the relative content of EPA and DHA increased, while the change in AA content was small but variable. Generally, the platelet content of oleic acid increased while the linoleic acid decreased. Intake of seal oil decreased platelet aggregation induced by microbubbles. A significant correlation between aggregation in platelet-rich plasma (PRP) and the AA content in platelets was shown, while there was a significant negative correlation between oleic acid content and platelet aggregation. In whole blood, however, seal oil intake did not result in less platelet aggregation using ADP and U-46619 as agonists.

ISSN:0953-7104    

DOI:10.3109/09537109509023564

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor

摘要:

The cytoplasmic domain of platelet glycoprotein IIIa contains the structural information for the recruitment of integrin GPIIb/IIIa into focal contacts as well as the integration of extracellular ligand-binding signals into the intracellular pathway leading to kinase ppl25FAK phosphorylation. These events could be mediated by covalent modification of the GPIIIa intracellular tail. Controversial reports have been published regarding palmitoylation and phosphorylation of GPIIIa in activated platelets. Here, we have analyzed the structure of the cytoplasmic domain of GPIIIa isolated from either the RGD-binding and non-RGD-binding conformers of the Triton X-100 soluble fraction or from the insoluble fraction of plasma membrane lysates of platelets (free and cytoskeleton-attached GPIIb/IIIa, respectively). Only the unaltered GPIIIa cytoplasmic tail was found. Therefore, we conclude that the intracellular domain of GPIIIa is neither covalently modified constitutively nor is its modification required for either RGD-peptide-binding or cytoskeleton attachment.

ISSN:0953-7104    

DOI:10.3109/09537109509023565

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor

摘要:

It has been widely questioned as to whether the observed binding of a-thrombin to intact platelets defines receptors coupled to signal transduction or merely thrombin binding sites. We have now shown that atα-thrombin concentrations sufficient to induce a full shape change response without aggregation (0.1 nM), PPACK-thrombin (that is,α-thrombin treated with the irreversible active site inhibitor D-phenylalanyl-L-prolyl-L-arginine chloromethylketone) dose-dependently inhibits platelet shape change (IC50∼70 nM), the concomitant increases in [Ca2+Ii (IC50∼75 nM) and ATP secretion (IC50∼50 nM). Since PPACK-thrombin competes fully in the binding of a-thrombin to high, moderate and low affinity sites on intact platelets, these results show that this binding defines functional receptors coupled to platelet activation.

ISSN:0953-7104    

DOI:10.3109/09537109509023566

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor

摘要:

The anti-inflammatory effects of fish oil may partly be due to the inhibition of platelet activation induced by platelet-activating factor (PAF) and other agonists. To investigate this hypothesis, the diets of 12 healthy volunteers were supplemented with 12 fish oil capsules or 12 olive oil capsules daily for 4 weeks in a double blind crossover study. Aggregation induced by PAF (18 and 12.5 nM) and collagen (20μg/ml)tended to be reduced after fish oil but the effect was statistically significant only in subjects receiving fish oil in the 6rst 4 weeks of the study (P0.05, n=6). The effect of fish oil supplementation on platelet ATP release was more marked with significant inhibition of ATP release induced by PAF (1200 and 36 nM,P0.01, n = 12), collagen (20μg/ml,P0.005, n = 12) and ADP (15,10 and 5μM,P0.05, n = 12). Olive oil supplementation appeared to inhibit ATP release induced by collagen (45 and 30μg/ml, P>0.025, n = 12), while aggregation and ATP release induced by arachidonic acid and adrenaline were unaffected by the supplements. Plasma fibrinogen was significantly reduced after olive oil (P0.01, n = 12) while prothrombin time was reduced after fish oil (P0.001, n = 12) and olive oil (P0.0025). Reduced platelet aggregation and more importantly, inhibition of platelet release induced by PAF and other agonists may contribute to the anti-inflammatory effects of fish oil supplementation in a number of disease states but olive oil may also independently affect platelet function and influence the effect offish oil.

ISSN:0953-7104    

DOI:10.3109/09537109509023567

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor

摘要:

The antithrombotic effect of a new platelet membrane GPIIb/IIIa antagonist, SC-52012A together with the 5-HT2receptor antagonist, ketanserin was studied in two guinea-pig thrombosis models. A segment of the femoral artery was occluded by a platelet-rich thrombus following photochemical reaction between rose bengal and green light which causes endothelial injury followed by platelet adhesion and aggregation at the site of photochemical reaction. SC-52012A, (1-10μg/kg/min, intravenously (i.v.), dose-dependently prolonged the occlusion time of the artery. Ketanserin, (1 mg/kg, i.v.) or the cyclooxygenase inhibitor, aspirin (100 mg/kg, p.o.) produced no marked effect, but when ketanserin was combined with 3μg/kg/min SC-52012A, the femoral artery occlusion time was strikingly greater than that produced by the same dose of SC-52012A. In the second model, an AV shunt was established between the carotid artery and the jugular vein and the platelet thrombus formed on a copper wire within the shunt was confirmed and quantified. SC-52012A (1-10μg/kg/min) and ketanserin (1 mg/kg, i.v.), both produced a significant inhibitory effect on platelet thrombus formation in this model. As the expression of platelet membrane GPIIb/IIIa complex is the final step in the mechanism of platelet aggregation induced by most platelet agonists, inhibitors of this receptor complex may prove to be effective for the control of arterial thrombosis.

ISSN:0953-7104    

DOI:10.3109/09537109509023568

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor

摘要:

In order to compare binding of small peptide mimetics on activated vs resting platelets and with fibrinogen (fgn) on activated platelets, the binding of [3H]-SC-52012, a low molecular weight (483) mimetic of the RGDF sequence present in fgn, was evaluated. This compound is a potent inhibitor of fgn binding to activated platelets, IC, 9.0±0.6 nM (mean±SEM), and inhibits ADP induced human platelet aggregation (IC, 44±5 nM). The dissociation constant (Kd) of [3H]-SC-52012 was 21.6±4.7 nM (n = 13) in ADP-induced human washed platelets while the Kd for resting platelets was 156±8.3 nM (n = 3). The maximum number of binding sites on ADP-activated and resting platelets were 60846±7158 and 59464±5898 molecules/platelet, respectively. By comparison, results with [125I]-fgn binding to activated platelets gave values of 363±73 nM and 58046±6386 molecules/platelet (n = 8) for the Kd and receptor number, respectively. These data suggest that the small molecule binds regardless of activation state of the platelet with only a change in affinity. [3H]-SC-52012 could be displaced by unlabelled SC-52012 with an IC50of 135±20 nM.

ISSN:0953-7104    

DOI:10.3109/09537109509023569

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor