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PubMed1992

Automated processing of leucocyte-poor platelet concentrates.

In view of transfusion reactions and alloimmunization associated with leucocyte contamination of platelet concentrates (PC), there is a general move towards the production of leuco-poor PC. This goal is currently pursued by the production of various PC using buffy coat and apheresis techniques. Although there is no overall consensus on the meaning of 'leuco-poor', by assuming that this refers to a level of 5-50 x 10(7) leucocytes per PC, we were able to make comparisons with available systems used in Europe. In addition to platelet and white cell counts, other markers of PC quality were assessed in some cases. These included traditional markers (such as hypotonic stress response, pH, and lactate and beta-thromboglobulin levels) and newer markers (such as glycocalicin and plasma von Willebrand factor levels). Our preliminary results showed appreciable differences in platelet and white cell content of PC prepared by various types of apheresis equipment. Appreciable differences in the quality of stored PC were also observed between routine PC (non-leuco-poor and buffy coat and apheresed PC (leuco-poor).

Cell Count↗
PubMed1992

Glycoproteins Ib and IIb/IIIa in the quality assessment of platelet concentrates during storage.

We have previously shown that levels of soluble glycocalicin (GC) in plasma supernatants derived from units of platelet concentrates (PC) increase progressively during storage. We now report further studies which show that the levels of both microparticle-bound and soluble GC in PC during storage are influenced by exposure of PC samples to EDTA and treatment of PC packs with ultraviolet B (UVB) irradiation. EDTA leads to a significant increase in the release of microvesicle-bound and soluble GC, while UVB irradiation leads to a dose- and rate-dependent increase in GC release. Paradoxically, UVB leads to an unexpected decrease in supernatant levels of von Willebrand factor (vWf) during storage which contrasts with its increase in untreated, stored PC. Moreover, an increase in GC release during storage is associated with a corresponding decrease in platelet size as determined by measurement of mean platelet volume (MPV) in citrated PC. The GC release is significantly correlated with standard platelet functional tests and other new generation tests such as dMPV and supernatant levels of vWf. In addition, preliminary results show the presence of microparticle-bound and soluble glycoprotein (Gp) IIb/IIIa in the supernatant plasma of stored PC. Our results suggest that supernatant levels of GpIb, GpIIb/IIIa, and vWf, together with alteration in MPV, provide essential new informative parameters for quality assessment of PC.

Blood Platelets↗
PubMed1992

Adherence of oral streptococci to salivary glycoproteins.

We used an overlay method to study the ability of human salivary glycoproteins to serve as receptors for several strains of streptococci that colonize the oral cavity. Parotid and submandibular-sublingual salivas were collected as ductal secretions, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. The resulting blots were overlaid with [35S]methionine-labeled bacteria, and salivary components to which the bacteria bound were detected by autoradiography. Potential glycoprotein receptors were identified for 8 of the 16 strains tested. In three cases (Streptococcus sanguis 72-40 and 804 and Streptococcus sobrinus OMZ176), highly specific interactions with a single salivary component were detected. Removal of sialic acid residues from the low-molecular-weight salivary mucin prevented adherence of one of these strains (S. sanguis 72-40), suggesting that this saccharide either mediates binding or is a critical component of the receptor site. In the remaining five strains (Streptococcus gordonii G9B and 10558, S. sanguis 10556, and Streptococcus oralis 10557 and 72-41), interactions with multiple salivary components, including the low-molecular-weight salivary mucin, highly glycosylated proline-rich glycoproteins, and alpha-amylase, were detected. These results suggest that some oral streptococci can bind specifically to certain of the salivary glycoproteins. The interactions identified may play an important role in governing bacterial adherence and clearance within the oral cavity.

Bacterial Adhesion↗
PubMed1992

Thrombin interaction with platelet glycoprotein Ib: effect of glycocalicin on thrombin specificity.

We describe here the alteration of thrombin specificity induced by its interaction with glycocalicin. Glycocalicin is the external part of platelet glycoprotein Ib alpha (GPIb alpha) and contains binding sites for von Willebrand factor and thrombin. Taking advantage of its solubility, we have used glycocalicin in competition assays on various thrombin activities. Glycocalicin did not inhibit chromogenic substrate hydrolysis nor diisopropylfluorophosphate iPr2 (PF) incorporation, indicating that thrombin binding to GPIb does not alter access to or the conformation of the thrombin catalytic site. Glycocalicin competitively inhibited thrombin binding to fibrin (Ki = 0.1 mumol/L) and blocked fibrinogen clotting activity of thrombin. Glycocalicin also inhibited thrombin binding to thrombomodulin in a competitive manner (Ki = 3 to 5 mumol/L), but failed to prevent thrombin interaction with protein C in the absence of thrombomodulin. Previous results have indicated that GPIb binds to thrombin within the anion binding exosite masked by the carboxy-terminal hirudin peptide 54-65. The present results confirm the implication of the anion binding exosite in GPIb recognition, and further indicate that the thrombin binding site for GPIb overlaps with the thrombin binding sites for fibrin and thrombomodulin, whereas it is distinct from the thrombin binding site for protein C. Some of the structural requirements for thrombin binding to GPIb appear to be very similar to those reported for binding to its platelet receptor. However, thrombin-GPIb interaction does not appear to compete with receptor hydrolysis but rather increases the sensitivity and the rate of platelet responses elicited by the receptor.

Amino Acid Sequence↗
PubMed1992

Coexpression of type I and type II human macrophage scavenger receptors in macrophages of various organs and foam cells in atherosclerotic lesions.

Macrophage scavenger receptors are trimeric membrane glycoproteins implicated in the pathologic deposition of cholesterol in arterial walls during atherogenesis. Two types of cDNAs for functional human receptors have been cloned, but their physiologic roles remain obscure. To study the expression of these receptors, the authors generated antibodies against scavenger receptor type-specific synthetic peptide. Immunohistochemical examination using these antibodies and other anti-human receptor antibodies shows that type I and type II receptor proteins can be detected in foam cells in various stages of atherosclerosis, most evidently in fatty streaks. Co-expression of the two types of receptor protein was also detected in macrophages of various organs. Both types of the protein were detected on the surface and the membrane of endosomes in macrophages. These results indicate that both type I and type II scavenger receptors are expressed and functionally active in physiologic and pathologic conditions.

Arteriosclerosis↗
PubMed1991

HIV-seropositive thrombocytopenia: the action of zidovudine.

The action of zidovudine when administered to individuals with severe HIV thrombocytopenia was investigated. Four individuals with platelets less than 50 x 10(9)/l and CD4 cells greater than 200 x 10(6)/l were treated with 600 mg zidovudine per day for 6 weeks, no drug for 6 weeks, 1200 mg zidovudine per day for 6 weeks, then no drug for 6 weeks. Glycocalicin, a platelet protein which correlates inversely with platelet survival, was assayed before and after treatment. Glycocalicin indices were also measured in four additional individuals with HIV thrombocytopenia. Platelet counts rose 2.5-fold [95% confidence interval (Cl), 2.0-3.0)] for four subjects who received 600 mg zidovudine per day and 4.9-fold (95% Cl, 4.0-5.8) for three subjects receiving 1200 mg zidovudine per day. Platelet counts declined during drug-free intervals. Plasma glycocalicin indices were elevated in all with untreated HIV thrombocytopenia. Indices fell after zidovudine treatment in six of seven individuals, suggesting that zidovudine prolonged platelet survival. Analysis of 170 HIV-seropositive asymptomatic individuals [mean CD4 count 474 x x 10(6)/l, standard deviation (s.d.) 245 x 10(6)/l] revealed that 14 (8%) had less than 125 x 10(9)/l platelets but only 2 (1%) had less than 50 x 10(9)/l platelets. Platelet counts increased spontaneously in eight individuals with mild HIV thrombocytopenia among the 10 for whom repeat counts were available.

Adult↗
PubMed1991

Differential redistribution of platelet glycoproteins Ib and IIb-IIIa after plasmin stimulation.

The subcellular localization of the platelet membrane receptors glycoproteins (GP) Ib and IIb/IIIa [corrected] has been studied within resting platelets by a combination of biochemical and cytochemical techniques. While both GPIb and GPIIb/IIIa are localized within the plasma membrane and surface-connected canalicular system (SCCS) membranes, only GPIIb/IIIa is present within the internal face of alpha-granular membranes. Previous studies demonstrated that plasmin can induce platelet stimulation and also decrease ristocetin-induced platelet aggregation; it was suggested that this was because of GPIb degradation by plasmin. In this study, the respective localizations of both GPIb and GPIIb/IIIa were visualized during in vitro plasmin stimulation of platelets. Generally, plasmin induced shape change, pseudopod formation, organelle centralization either with or without alpha-granule release depending on the conditions of stimulation. Plasmin treatment of platelets at 37 degrees C resulted in the disappearance of GPIb from the cell surface and its subsequent redistribution into the channels and vesicles of the SCCS with no significant modification of GPIIb/IIIa remaining on the plasma membrane. Within degranulated platelets, GPIIb/IIIa was expressed on the plasma membrane and within membranes of large vacuoles containing the alpha-granule proteins. GPIb was virtually absent from these structures and mainly restricted to the SCCS. Addition of cytochalasin D inhibited the migration of GPIb to the SCCS. Biochemical measurements confirmed that no important hydrolysis of GPIb had occurred because only very little amounts of glycocalicin were generated during the reaction. In conclusion, in plasmin-treated platelets GPIIb/IIIa is externalized to the plasma membrane while GPIb is internalized into the SCCS. Although previous studies have suggested that plasmin degrades GPIb, the reduction in ristocetin-induced aggregation may be explained by its apparent redistribution within the membranes of the SCCS.

Blood Platelets↗
PubMed1991

New enzyme-linked immunosorbent assay for glycocalicin in plasma.

A new sandwich-type enzyme-linked immunosorbent assay for quantifying glycocalicin, a proteolytic fragment of platelet membrane glycoprotein Ib, is described. The assay is based on the use of two monoclonal antibodies raised against glycoprotein Ib and involves the avidin-biotin technique. The detection limit is 7 micrograms/L and the range of glycocalicin determined in plasma is 0.01 to 1 mg/L. Assay time is 2 h. The intra-assay CV ranged from 3.6% to 5.2%, the interassay CV from 5.4% to 8.0%. Analytical recovery of purified glycocalicin added to a plasma pool averaged 96%. In 36 healthy subjects, the mean glycocalicin concentration in plasma was 0.36 (SD 0.07) mg/L (2.7 nmol/L). We conclude that this assay is suitable for measuring glycocalicin in plasma and is also more sensitive and precise than the previously published immunoassays based on competitive binding assay.

Adolescent↗
PubMed1991

Fetal platelet consumption: a feature of placental insufficiency.

The involvement of fetal platelets in placental insufficiency was investigated. Platelet life span and the rate of platelet turnover were measured by a novel technique in fetuses with moderate and severe placental insufficiency and in controls. This procedure involves the measurement of platelet count and concentration of free glycocalicin in plasma. Placental insufficiency was defined by antenatal Doppler ultrasound analysis of the umbilical artery waveform, which was used to categorize the subjects according to whether their systolic-diastolic ratios were normal, moderately raised, or severely raised. Mean platelet life span in fetuses affected by placental insufficiency was only 60% of the normal fetal platelet life span (P less than .001). In addition, affected fetuses had a higher rate of platelet turnover, reflected in an elevated plasma glycocalicin concentration (4.06 +/- 0.19 versus 3.28 +/- 0.15 microgram/mL for severe placental insufficiency and control fetuses, respectively; P less than .01). Platelet life span did not differ between the moderate and severe placental insufficiency groups. We hypothesize that the observed changes in platelet life span and turnover result from increased platelet activation, which plays a role in the development of placental insufficiency.

Female↗
PubMed1991

Measurement of receptor concentration and forward-binding rate constant via radiopharmacokinetic modeling of technetium-99m-galactosyl-neoglycoalbumin.

Technetium-99m-galactosyl-neoglycoalbumin (99mTc-NGA) is a synthetic ligand to the hepatocyte receptor, hepatic binding protein (HBP). A five-state mathematical model containing a bimolecular chemical reaction was utilized for quantitative estimation of the following physiologic and biochemical parameters: extrahepatic plasma volume Ve; hepatic plasma flow F and volume Vh; receptor-ligand forward-binding rate constant kb and reaction volume Vr; and receptor concentration [R]o. Nine normal subjects were studied. Given (a) liver and heart time-activity data, (b) the patient's weight, height, and hematocrit, (c) the fraction of injected dose in a 3-min blood sample, and (d) the amount and galactose density of the NGA dose, a computer program executed a curve-fit to the kinetic model. Systematic error, as measured by reduced chi-square, ranged from 1.43 to 2.56. Based on the nine imaging studies, the mean and relative error of each parameter were: [R]o, 0.813 +/- (0.11) microM; kb, 2.25 +/- (0.15) microM-1 min-1; F, 0.896 +/- (0.20) liter/min; Ve, 1.67 +/- (0.27) liter; and Vh, 0.228 +/- (0.22) liter. Two unique features of 99mTc-NGA radiopharmacokinetic systems permit the simultaneous estimates of receptor quantity, ligand affinity, and hepatic plasma flow. The first is the ability to administer a quantity of ligand capable of occupying a significant fraction of receptor; and the second is a simple model structure that conserves mass.

Adult↗
PubMed1991

Validation of in vivo receptor measurements via in vitro radioassay: technetium-99m-galactosyl-neoglycoalbumin as prototype model.

Hepatic binding protein (HBP) is a hepatocyte-specific receptor for serum asialoglycoproteins. The receptor also recognizes a synthetic glycoprotein that has been developed as a radiopharmaceutical, technetium-99m-galactosyl-neoglycoalbumin (99mTc-NGA). This report describes the correlation between receptor parameters measured in vivo via kinetic modeling of 99mTc-NGA and those measured by in vitro radioassay of biopsied liver tissue. Eleven patients with diffuse hepatic disease underwent percutaneous liver biopsy followed by a 99mTc-NGA functional imaging study. In vivo measurements of HBP quantity Ro and forward binding rate constant kb obtained from the kinetic analysis of 99mTc-NGA liver and blood time-activity data were compared to total receptor quantity and the HBP-99mTc-NGA association constant KA as measured by Scatchard binding assay of the biopsied tissue. The correlation coefficients between in vivo and in vitro measurements were 0.73 (df = 8, p = 0.015) and 0.98 (df = 8, p less than 0.01) for Ro and kb, respectively. The in vivo measurements of HBP biochemistry via kinetic analysis of the radiopharmaceutical time-activity data reflect the average concentration and affinity of the receptor. This study further substantiates the validity of 99mTc-NGA as a quantitative probe for the HBP receptor.

Adult↗
PubMed2026

Inherited Platelet GPIV Deficiency: First Description of a Series of Unrelated Patients with Bleeding Diathesis.

GPIV (CD36) is a multifunctional membrane protein expressed on various cells, including platelets, where it plays a role in adhesion and activation through the interaction with its ligands, including collagen types I and III and thrombospondin 1. Inherited GPIV deficiency, historically recognized in anti-Naka alloimmunized East Asian donors, is considered asymptomatic and associated with normal platelet aggregation, although impaired adhesion under high-flow conditions has been reported. Here, we reconsider the molecular basis, epidemiology and functional consequences of GPIV deficiency and report four unrelated patients in whom heterozygous CD36 variants are associated with markedly reduced platelet GPIV expression and a clinically relevant mucocutaneous bleeding diathesis. Patients suffered lifelong bleeding symptoms despite normal light-transmission aggregometry and platelet granule content and release and displayed decreased GPIV expression. Three of them showed slightly decreased VWF. Platelet adhesion to Type I collagen was reduced at high shear. These cases suggest for the first time an association between CD36 gene variants and bleeding and underscore the importance of including GPIV in the diagnostic workup of inherited platelet disorders, particularly when conventional assays do not reveal abnormalities.

Humans↗
PubMed1992

Glycoproteins V and Ib-IX form a noncovalent complex in the platelet membrane.

Platelet glycoprotein (GP) V is a Mr 82,000 plasma membrane protein of unknown function that is cleaved by the potent platelet agonist, thrombin, to yield a Mr 69,500 fragment (GPVf1). Platelet GPIb, a disulfide-linked alpha beta heterodimer (Mr 160,000) that forms a noncovalent complex with GPIX (Mr 22,000), functions as the platelet adhesion receptor for surface-bound von Willebrand factor. Association between GPV and GPIb-IX has been suggested by the finding that both proteins are deficient in the Bernard-Soulier syndrome, a bleeding disorder characterized by giant platelets and defective interaction with von Willebrand factor. Here we report that GPV and GPIb-IX are coprecipitated by monoclonal antibodies (mAbs) against GPV, GPIb, or GPIX when platelets are solubilized in the mild detergent, digitonin. Treatment of digitonin immunopreciptates with the nonionic detergent, Nonidet P-40, released GPV from anti-GPIb and anti-GPIX mAb precipitates and GPIb-IX from the anti-GPV mAb precipitate. Removal of the Mr 45,000 amino-terminal part of GPIb alpha by treatment with elastase did not abrogate association of GPV with GPIb-IX, showing that the leucine-rich repeat sequences in GPIb alpha are not required for complex formation. Binding studies with 125I-labeled mAbs showed the presence of 24,370 GPIb-IX complexes and 11,170 molecules of GPV/platelet (n = 5). These data show that the leucine-rich glycoproteins GPV and GPIb-IX form a noncovalent complex in the platelet membrane. GPV may play a role in the interaction of platelets with von Willebrand factor.

Animals↗
PubMed1991

Characterization of the binding domains on platelet glycoproteins Ib-IX and IIb/IIIa complexes for the quinine/quinidine-dependent antibodies.

Sera of 12 patients with quinine/quinidine-induced thrombocytopenia showed drug-dependent antibody binding to glycoprotein (GP) Ib-IX complex. The reaction with GPIb-IX complex of 11 of these 12 sera was strongly inhibited by the complex-specific monoclonal antibodies (MoAbs) AK1 and SZ1. The exception was a quinine-induced serum designated BU. The reaction of the six quinidine-induced sera was also partially blocked by an anti-GPIX MoAb, FMC25. Only 3 of the 12 patient sera showed drug-dependent antibody binding to GPIIb/IIIa, which was strongly inhibited by the anti-GPIIIa MoAb 22C4, and the anti-GPIIb alpha MoAb SZ22. With detergent-solubilized Serratia metalloprotease-treated platelets, quinine/quinidine-induced sera, except BU, immunoprecipitated a membrane-bound proteolytic fragment of GPIb-IX complex. In contrast, BU immunoprecipitated glycocalicin and a 40-Kd peptide tail fragment of GPIb alpha from the cell supernatant. Using purified GPIb-IX complex or its components as the target antigen, all the quinine-induced sera, except BU, immunoprecipitated GPIb-IX complex but failed to immunoprecipitate GPIb, GPIX, or the complex reformed from GPIb and GPIX. The quinidine-induced sera strongly immunoprecipitated purified GPIb-IX complex, weakly immunoprecipitated purified GPIX and the recombined complex, but did not immunoprecipitate purified GPIb. The combined data suggest that one quinine-dependent antibody (BU) recognizes an epitope in the peptide tail region of GPIb alpha and the other five quinine-dependent antibodies react with a complex-specific epitope on the membrane-associated region of GPIb-IX complex, whereas each of the six quinidine-induced sera contain two drug-dependent antibodies, one reactive with the GPIb-IX complex-specific epitope and the other reactive with GPIX. The binding domain(s) on GPIIb/IIIa for the quinine/quinidine-dependent antibodies appear to be sterically close to the epitopes for 22C4 and SZ22.

Antibodies↗