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Biomedical subjects

P A Castaldi

Publications and source records attributed to P A Castaldi.

At least 37 records · Page 2Linked to original sources
PubMed1985

Purification and preliminary characterization of the glycoprotein Ib complex in the human platelet membrane.

Human platelet glycoprotein Ib (GP Ib) is a major integral membrane protein that has been identified as the platelet-binding site mediating the factor VIII/von Willebrand-factor-dependent adhesion of platelets to vascular subendothelium. Recent evidence suggests that GP Ib is normally complexed with another platelet membrane protein, GP IX. In this study, human platelet plasma membranes were selectively solubilized with a buffer containing 0.1% (v/v) Triton X-100. The GP Ib complex (GP Ib plus GP IX) was purified to homogeneity in approximately 30% yield by immunoaffinity chromatography of the membrane extract using the anti-(glycoprotein Ib complex) murine monoclonal antibody, WM 23, coupled to agarose. GP Ib and GP IX were subsequently isolated as purified components by immunoaffinity chromatography of the GP Ib complex using a second anti-(glycoprotein Ib complex) monoclonal antibody, FMC 25, coupled to agarose. As assessed by dodecyl sulphate/polyacrylamide gel electrophoresis, purified GP Ib was identical to the molecule on intact platelets and had an apparent relative molecular mass of 170 000 under nonreducing conditions and 135 000 (alpha subunit) and 25 000 (beta subunit) under reducing conditions. GP IX had an apparent Mr of 22 000 under both nonreducing and reducing conditions. Purified Gb Ib complex and GP Ib inhibited the ristocetin-mediated, human factor VIII/von Willebrand-factor-dependent and bovine factor VIII/von Willebrand-factor-dependent agglutination of washed human platelets suggesting the proteins had been isolated in functionally active form. GP Ib alpha had a similar amino acid composition to that previously reported for its proteolytic degradation product, glycocalicin. The amino acid compositions of GP Ib beta and GP IX were similar but showed marked differences in the levels of glutamic acid, alanine, histidine and arginine. The N-termini of GP Ib alpha and GP IX were blocked; GP Ib beta had the N-terminal sequence, Ile-Pro-Ala-Pro-. On crossed immunoelectrophoresis, both GP Ib and GP IX were found to occur in the same immunoprecipitin arc(s) whether the platelets had been solubilized in the absence or presence of the calcium-dependent protease inhibitor, leupeptin. Binding studies in platelet-rich plasma indicated a similar number of binding sites (means +/- SD) for three anti-(glycoprotein Ib complex) monoclonal antibodies: AN 51, epitope on GP Ib alpha (22 000 +/- 2700, n = 3), WM 23, epitope on GP Ib alpha (21 000 +/- 3400, n = 3), FMC 25, epitope on GP IX (20 100 +/- 2700, n = 3), and FMC 25 (Fab')2 (27 100 +/- 800, n = 2).(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1985

Platelet thrombospondin haemagglutinin activity is due to aggregate formation.

Thrombospondin released from human blood platelets by thrombin activation formed high molecular weight aggregates which co-eluted with haemagglutinin activity on Sepharose 4B gel filtration. Thrombospondin aggregation was mediated by intermolecular disulphide bridges. The aggregates consisted of a series of oligomers ranging from a dimer to polymeric forms with Mr congruent to 40 X 10(6). Native monomeric thrombospondin obtained by a modified procedure was deficient in haemagglutination activity but inhibited haemagglutination induced by aggregated thrombospondin.

Blood Platelets↗
PubMed1985

Preliminary functional characterization of a 24,000 dalton platelet surface protein involved in platelet activation.

We have recently investigated three monoclonal antibodies, FMC 8, FMC 48 and FMC 56, directed against a 24,000 dalton (24K) cell surface protein on human platelets and pre-B progenitor cells. These monoclonal antibodies (at 0.1-10 micrograms/ml) were found to be potent inducers of platelet aggregation with normal and with aspirin-treated platelets. FMC 8 Fab' and FMC 56 (Fab')2 fragments blocked aggregation induced by intact antibody. The Fab' and (Fab')2 fragments, however, were ineffective by themselves (at 100 micrograms/ml) as platelet agonists but augmented (at 10-25 micrograms/ml) the aggregation response of normal and aspirin-treated platelets to threshold concentrations of ADP. It has been suggested that the 24K cell surface protein may be identical to a 24K cAMP-dependent phosphoprotein involved in regulating transmembrane calcium flux. To examine this, 32P-phosphate-loaded platelets were treated with prostaglandin E1 to raise intracellular cAMP levels and then analysed on two-dimensional O'Farrell gels. No labelled phosphoprotein corresponded in mobility to the 24K cell surface protein. Further, FMC 56 failed to immunoprecipitate a 24K phosphoprotein. The combined results suggest that the three monoclonal antibodies activate platelets via a prostaglandin-independent and as yet undefined mechanism which requires the intact bivalent antibody for full platelet response. Fab' fragments (monovalent) and (Fab')2 fragments (altered Fab-Fab configuration) are ineffective as platelet agonists but can augment the response of other stimuli. Thus, further studies with this system should allow delineation of those intracellular events necessary for partial and full platelet responses.

Antibodies, Monoclonal↗
PubMed1985

Molecular characterization of quinine/quinidine drug-dependent antibody platelet interaction using monoclonal antibodies.

Two murine monoclonal antibodies, FMC 25 and AN 51, directed against distinct epitopes on the glycoprotein Ib complex, have been used to further define the mechanism of quinine/quinidine drug-dependent antibody interaction with platelets. FMC 25, directed against an epitope on glycoprotein IX, had no effect on platelet aggregation induced by collagen or adenosine diphosphate and little, if any, effect on ristocetin-induced platelet agglutination. FMC 25 and its (Fab)2 fragment, however, were potent inhibitors of drug-dependent antibody-induced platelet aggregation and blocked binding of drug-dependent antibody to platelets as assessed by indirect platelet immunofluorescence. In contrast, AN 51, directed against an epitope on the alpha-subunit of glycoprotein Ib, blocked ristocetin-induced, factor VIII/von Willebrand factor (FVIII/vWF)-dependent platelet agglutination but not drug-dependent antibody-induced platelet aggregation or binding of drug-dependent antibody to platelets. Selective proteolytic removal of the majority of the alpha-subunit of glycoprotein Ib (glycocalicin) from platelets by treatment with calcium-dependent protease did not affect binding of drug-dependent antibody. In addition, a quinidine-dependent antiplatelet antibody immunoprecipitated glycoprotein Ib complex from normal platelets and the membrane-associated proteolytic remnant of the glycoprotein Ib complex from calcium-dependent protease-treated platelets. Preincubation of drug-dependent antibody with purified glycoprotein Ib complex inhibited subsequent binding of antibody to platelets, but the separated components, glycoprotein Ib and glycoprotein IX, were both ineffective, suggesting that the normal interaction between glycoprotein Ib and glycoprotein IX in the intact complex was necessary for drug-dependent antibody recognition. The functional response of platelets to drug-dependent antibody was not mediated by way of platelet Fc receptor, since aggregation of washed platelets by acetone-aggregated IgG was not inhibited by FMC 25 (Fab)2. FVIII/vWF was not required for drug-dependent antibody-induced platelet aggregation. The combined evidence is consistent with quinine/quinidine-dependent antibody-platelet interaction occurring by way of a FVIII/vWF-independent, Fc receptor-independent mechanism that probably involves binding of antibody to glycoprotein IX or the beta-subunit of glycoprotein Ib or both.

Antibodies, Monoclonal↗
PubMed1984

Characterization of calcium-dependent binding of endogenous factor VIII/von Willebrand factor to surface activated platelets.

Activation of washed platelets in the presence of EDTA with either 1 U/ml of alpha-thrombin or 2 microM calcium ionophore (A23187) caused the release of one-third to one-half of the platelet factor VIII/von Willebrand factor (FVIII/vWF) into the supernatant. When calcium was present in excess, only 10% of the platelet FVIII/vWF was detected free in the supernatant, regardless of whether calcium was present before stimulation or added to the platelets after thrombin activation. Release of [14C]serotonin and beta-thromboglobulin were not affected by divalent cations indicating that reduced supernatant levels of FVIII/vWF in the presence of calcium were not due to differential release, but were probably due to a calcium-dependent association of released FVIII/vWF with the platelet surface. The presence or absence of intact glycoprotein Ib on the platelet surface made no significant difference to the observed FVIII/vWF partition. Platelets from a patient with Glanzmann's thrombasthenia, however, failed to show a calcium effect with respect to released FVIII/vWF. The combined results suggest that as well as the ristocetin-dependent, divalent cation-independent binding of FVIII/vWF to glycoprotein Ib, there is a divalent cation-dependent binding of FVIII/vWF to the activated platelet surface which is mediated via the glycoprotein IIb/IIIa complex.

Blood Coagulation Factors↗
PubMed1984

Factor VIII/von Willebrand factor has potent lectin activity.

Highly-purified plasma and platelet Factor VIII/von Willebrand Factor had potent lectin activity when measured in a haemagglutination assay. This lectin activity was inhibited by monoclonal and heterologous antibodies to Factor VIII/von Willebrand Factor as well as by hexosamines, mannose and net-positively charged amino acids.

Blood Coagulation Factors↗
PubMed1984

An altered platelet granule glycoprotein in patients with essential thrombocythemia.

The protein profiles of washed platelets from nine patients with essential thrombocythemia were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In four patients, an additional protein band (reduced Mr of 170,000) was clearly identified in both unstimulated platelet preparations and thrombin-released supernatant fractions. This band was also evident, though to a lesser extent, in three more patients, but it could not be located in the two remaining patients nor in any of ten controls. Subsequent characterization of the 170,000 reduced protein in one patient indicated that (a) it was glycosylated, as judged by periodic acid-Schiff staining, and (b) that native protein was a disulfide-linked multimer (possibly trimeric), which (c) partially bound to the activated platelet plasma membrane in the presence of calcium, and (d) was immune precipitated by anti-glycoprotein G antisera. The combined evidence is consistent with the 170,000 reduced protein being a modified form of the normal subunit of the platelet alpha-granule constituent, glycoprotein G (also termed thrombospondin and thrombin-sensitive protein).

Aged↗
PubMed1983

Morphological and biochemical confirmation of gray platelet syndrome in two siblings.

A case report is presented of gray platelet syndrome in siblings. The absence of platelet alpha-granules in these patients was confirmed by electron microscopy and by analysis of the platelet protein profile using SDS-polyacrylamide gel electrophoresis. This was further confirmed by quantitation of individual alpha-granule protein constituents. The results in these two patients are compared with those of the four reported patients with this syndrome.

Adult↗
PubMed1983

Qualitative platelet defect in a case of heparin-induced thrombocytopenia.

A qualitative platelet defect was demonstrated in a patient with heparin-induced thrombocytopenia. His platelets showed impaired aggregation to collagen and arachidonate and absent second wave aggregation to adenosine diphosphate and epinephrine but normal response to ristocetin. These aggregation abnormalities were similar to those reported in idiopathic thrombocytopenia and systemic lupus erythematosus. This platelet function defect may contribute to haemorrhagic complications which appear to be common in heparin-induced thrombocytopenia.

Adenosine Diphosphate↗
PubMed1983

Quinidine-induced thrombocytopenia and leukopenia: demonstration and characterization of distinct antiplatelet and antileukocyte antibodies.

A patient with the rare syndrome of simultaneous quinidine-induced thrombocytopenia and leukopenia was studied. A quinidine-dependent antiplatelet antibody was detected in her serum by platelet aggregometry and by indirect platelet-suspension immunofluorescence. A drug-dependent antileukocyte antibody was demonstrated by leukoagglutination and by granulocyte immunofluorescence. Both antibodies were found to belong to the IgG class by immunofluorescence using monospecific antisera. There appeared to be two distinct antibodies, since the antibody eluted off sensitized platelets reacted only with platelets and that eluted off sensitized granulocytes reacted only with granulocytes. In addition, the patient's serum and quinidine gave a negative reaction on immunofluorescence with platelets from a patient with Bernard-Soulier syndrome (BSS), which lack glycoprotein lb (GPlb) complex, but gave a positive reaction with BSS granulocytes. The quinidine-dependent antiplatelet antibody immunoprecipitated GPlb and a glycoprotein of molecular weight 22,000 (GPlb complex) from Triton-solubilized, periodate-labeled platelets. Similar attempts to identify the granulocyte surface receptor/antigen were unsuccessful. Factor VIII/von Willebrand factor was not required for binding of the drug-dependent antibody to platelets.

Antibody Specificity↗
PubMed1983

Additional glycoprotein defects in Bernard-Soulier's syndrome: confirmation of genetic basis by parental analysis.

The glycoprotein profile of Bernard-Soulier platelets was examined by labeling washed platelets with periodate 3H-sodium borohydride, a procedure that labels greater than 30 glycoproteins on the membrane surface of normal platelets. Three Bernard-Soulier patients were studied; two were siblings and the third was unrelated. The platelet protein and glycoprotein profiles were evaluated under nonreduced and reduced conditions using 5%-15% exponential SDS-polyacrylamide gel electrophoresis. The two siblings completely lacked glycoprotein Ib (GPIb). The unrelated patient had congruent to 7% of the normal level. This was confirmed by two-dimensional nonreduced-reduced SDS-polyacrylamide gel electrophoresis, a procedure that allows clear separation of the disulfide-linked subunits of GPIb, GPIb alpha (mol wt 145,000), and GPIb beta (mol wt 25,000) from other membrane glycoproteins. On one-dimensional analysis, Bernard-Soulier's syndrome (BSS) platelets also lacked the peripheral membrane glycoprotein, GPV (mol wt 82,000) and a low molecular weight glycoprotein, GPIX, (nonreduced or reduced, mol wt congruent to 22,000). The two-dimensional gel system also revealed the absence of a minor glycoprotein with a molecular weight of congruent to 100,000 (GP 100). Quantitation of these proteins solubilized from electrophoretograms showed that the siblings' parents had congruent to 50% levels of GPIb, GPIX, and GP 100. A monoclonal antibody against glycoprotein Ib, FMC 25, was negative by immunofluorescence against Bernard-Soulier platelets and immuneprecipitated both GP Ib and GPIX from Triton X100 solubilized, labeled platelets. The combined results suggest that the apparent genetic absence of multiple proteins in Bernard-Soulier platelets is due, in part, to the presence in normal platelets of a tight membrane complex between glycoprotein Ib and at least one of the other absent glycoproteins.

Adolescent↗
PubMed1982

Heparin-induced thrombocytopenia: association of thrombotic complications with heparin-dependent IgG antibody that induces thromboxane synthesis in platelet aggregation.

Eleven patients in whom thrombocytopenia developed during heparin therapy were studied. Six patient (group 1) had severe thrombocytopenia with delayed onset and five of these patients had thromboembolic complications. A serum factor which induced heparin-dependent thromboxane B2 synthesis, 14C-serotonin release, and platelet aggregation was found in all patients in group 1. The serum factor was shown to be IgG. These findings suggest that the mechanism of the severe thrombocytopenia secondary to heparin therapy is immunological and the associated thromboembolic complications may be attributed to in-vivo activation of the platelet prostaglandin pathway and platelet aggregation induced by the heparin-dependent antibody. The five patients in group 2 had mild symptomless thrombocytopenia with early onset. In this group, the heparin-dependent antibody was not found and the mechanism of the thrombocytopenia is probably a direct action of heparin on platelets.

Adult↗
PubMed1982

Evidence for a platelet membrane defect in the myeloproliferative syndromes.

Bleeding and abnormal platelet aggregation occur in patients with myeloproliferative disorders. In this study, twenty patients were examined, some sequentially, and a proportion found to have defective aggregation toward adrenaline, adenosine diphosphate (ADP), and collagen. In these seven patients, the abnormality in platelet response with defective collagen-induced [14C]serotonin release correlated with poor collagen-stimulated thromboxane B2 (TXB2) production. In contrast, five of these patients showed a normal threshold aggregation response to arachidonic acid. The combined results suggest that in these patients, there is a defect between receptor-stimulus coupling and the mobilization of arachidonic acid from membrane phospholipid.

Blood Platelets↗
PubMed1982

The role of platelet surface proteins reacting with heterologous antibodies.

Platelets were labeled with surface-labelling agents which identified up to six platelet membrane proteins. Three of these labelled proteins were recognised by a specific anti-platelet membrane serum by immunoprecipitation. Affinity fractionation of platelets with insolubilised IgG obtained from the anti-platelet membrane serum indicated a greater reaction between the antiserum and the platelet surface. Fab fragments from the antiserum inhibited ristocetin-induced aggregation of platelets and partially inhibited collagen-induced aggregation but did not affect ADP aggregation. Crossed immunoelectrophoresis of platelets using the anti-platelet membrane serum showed that the antiserum was specific for platelet membranes.

Animals↗