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Biomedical subjects

H J Weiss

Publications and source records attributed to H J Weiss.

At least 19 recordsLinked to original sources
PubMed1992

Defective Ca(2+)-induced microvesiculation and deficient expression of procoagulant activity in erythrocytes from a patient with a bleeding disorder: a study of the red blood cells of Scott syndrome.

The erythrocytes from a patient with Scott syndrome, a bleeding disorder characterized by an isolated defect in expression of platelet procoagulant activity, have been studied. When incubated with the calcium ionophore A23187, Scott syndrome red blood cells (RBCs) expressed less than 10% of the prothrombinase (enzyme complex of coagulation factors Va and Xa) activity of A23187-treated RBCs obtained from normal controls. Consistent with the results from enzyme assay, the ionophore-treated Scott syndrome erythrocytes exhibited diminished membrane vesiculation and decreased exposure of membrane binding sites for factor Va compared with identically treated controls. When examined by scanning electron microscopy, untreated Scott syndrome RBCs were indistinguishable from normal cells. After incubation with A23187, however, the morphology of Scott syndrome RBCs contrasted markedly from normal erythrocytes. Whereas the Ca2+ ionophore induced marked echinocytosis and spiculation of normal RBCs, Scott syndrome RBCs remained mostly discoid under these conditions, with only an occasional echinocyte-like cell observed. These aberrant responses to intracellular Ca2+ were also observed for resealed ghosts prepared from Scott syndrome erythrocytes, indicating that they are related to a defect in the membrane or membrane-associated cytoskeleton. The finding that the erythrocytes of this patient share many of the membrane abnormalities reported previously for Scott syndrome platelets suggests that this defect is common to both cell lines and involves a membrane component required for vesicle formation and for expression of prothrombinase sites.

Adult↗
PubMed1992

Human platelets deficient in dense granules contain normal amounts of pp60c-src.

We have determined that pp60c-src, a protein tyrosine kinase abundant in normal platelets, is present at comparable levels in platelets that are deficient in dense granules (Hermansky-Pudlak syndrome). Relative quantitation of pp60c-src was performed by immunoblot analysis after protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our data suggest that human platelet dense granules, unlike chromaffin cell secretory granules, are not the major intracellular site of localization of pp60c-src.

Albinism, Oculocutaneous↗
PubMed1991

Upstream thrombus growth impairs downstream thrombogenesis in non-anticoagulated blood: effect of procoagulant artery subendothelium and non-procoagulant collagen.

In the present experiments we have investigated the influence of wall shear rate and axial position on platelet and fibrin deposition which results when flowing human non-anticoagulated blood is exposed to either non-procoagulant fibrillar collagen (human type III) or procoagulant subendothelium (rabbit aorta). Platelet adhesion, thrombus volume and fibrin deposition were morphometrically evaluated at axial positions of 1 and 13 mm following perfusions for 5 min at shear rates of 100, 650 and 2,600 s-1. An axially-dependent decrease of platelet adhesion (34-57%, p less than 0.01-0.05) and thrombus volume (57-80%, p less than 0.05) was observed on collagen at all shear rates. On subendothelium, an axially-dependent decrease was observed for platelet adhesion only at 100 s-1 (29%; p less than 0.01) and for thrombus volume at shear rates of 650 s-1 and above (49-58%, p less than 0.01). Deposition of fibrin on subendothelium was axially decreased (16-42%, p less than 0.05) at all shear rates, while no significant axial differences were seen on collagen. However, substantially more fibrin was deposited on the subendothelium (p less than 0.05), and the upstream platelet adhesion and thrombus volume were lower than on collagen (p less than 0.05) at 100 s-1 and 650 s-1. The axially-dependent phenomena on the two surfaces are consistent with the concept of rapid-growing upstream thrombi which deplete the blood layer streaming adjacent ot the surface of platelets, leading to decreased platelet deposition further downstream.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Coagulation↗
PubMed1991

Further evidence that glycoprotein IIb-IIIa mediates platelet spreading on subendothelium.

In order to explore further the mechanism by which glycoprotein GPIIb-IIIa promotes platelet vessel wall interaction, platelet adhesion to subendothelium was studied in an annular chamber in which subendothelium from rabbit aorta was exposed at a shear rate of 2,600 s-1 to blood from patients with thrombasthenia. Perfusions were conducted for each of 5 exposure times (1, 2, 3, 5 and 10 min), and the percent surface coverage of the vessel segment with platelets in the contact (C) and spread (S) stage was determined. Increased values of platelet contact (C) were obtained in thrombasthenia at all exposure times; this finding is consistent with a defect in platelet spreading, based on a previously described kinetic model of platelet attachment to subendothelium. According to this model of attachment, increased values of platelet contact (C) at a single exposure time may be indicative of either a defect in spreading (S) or initial contact (C), but multiple exposures will result in increased contact only for defects which are related to defective platelet spreading (S). The results obtained over a broad range of exposure times provide more conclusive evidence that GPIIb-IIIa mediates platelet spreading than those previously obtained at single exposure times.

Animals↗
PubMed1991

Decreased content and surface expression of alpha-granule membrane protein GMP-140 in one of two types of platelet alpha delta storage pool deficiency.

To determine whether alpha-granule membranes are present in platelets of patients with storage pool deficiencies of both alpha and dense granules (alpha delta-SPD), we examined the content and surface expression of the alpha-granule membrane protein GMP-140 in one patient (J.C.) with a severe alpha-granule deficiency and in three members of a family (family C) with milder alpha-granule deficiencies. Surface expression of GMP-140 in stimulated platelets, assessed by flow cytometric measurements of the binding of two anti-GMP-140 monoclonal antibodies, was 24-38% of normal values in platelets from patient J.C., vs. 60-95% of normal values in family C. Total platelet content of GMP-140, determined in platelet lysates by antigen-capture ELISA, was 49% of normal in patient J.C., but normal in the members of family C. Platelets of patient J.C. were found to be heterogeneous with respect to GMP-140 content and surface expression by both flow cytometry and immunogold electron microscopy. Approximately 80% of her platelets expressed little or no GMP-140 after stimulation, whereas the remaining 20% expressed normal amounts of GMP-140 and showed extensive immunogold labeling of typical alpha-granules and clear vacuoles. No such heterogeneity was found in platelets from family C. These findings in the severe alpha delta-SPD patient are in clear contrast to the observations of normal GMP-140 content in the three other alpha delta-SPD patients, and in patients with the gray platelet syndrome, reported previously by others. These results illustrate the phenotypic heterogeneity of alpha-granule deficiencies in human platelets, and suggest that a defect in granule formation in the megakaryocytes may account for the alpha-granule defect in at least one form of alpha delta-SPD.

Adult↗
PubMed1990

Studies on platelet protein phosphorylation in patients with impaired responses to platelet agonists.

The phosphorylation responses of platelet proteins after platelet stimulation with agonists were studied in patients with clinical bleeding disorders and various types of impaired platelet functional responses. Impaired collagen-induced phosphorylation, particularly of the 47 kd substrate (P47) for protein kinase C, was observed in one patient whose platelet defect appears to be an impaired initial response to weak platelet agonists but whose platelet secretory mechanism is normal. Reduced phosphorylation of a 31 kd polypeptide was also observed. The phosphorylation defect in this patient differs from that seen in another patient in whom impaired P47 and myosin light chain phosphorylation was observed but whose functional defect may be more closely related to secretion. The results provide further evidence that phosphorylation of P47 may play a role in platelet activation mechanisms preceding secretion and that abnormalities of phosphorylation of both P47 and myosin light chain may be associated with platelet functional defects in some patients with bleeding disorders.

Adenosine Diphosphate↗
PubMed1989

Time dependence of aequorin-indicated calcium levels in stimulated and unstimulated platelets: evidence for multiple aequorin environments in platelets.

Aequorin-induced calcium signals were examined in human unstimulated platelets and platelets stimulated with various agonists as a function of time. The total aequorin response in unstimulated platelets, obtained by Triton x-100 lysis in the presence of 1 mM Ca, decreased in a distinctly non-linear manner over 20-60 min. This decrease was slightly, but significantly, greater in platelets maintained in the continuous presence of 1 mM Ca than in platelets maintained without external Ca, and could not be accounted for completely by leakage of aequorin from the cells. Basal Ca levels in unstimulated platelets also decreased in a non-linear manner, with a similar sensitivity to the continuous presence or absence of external Ca. These observed changes in aequorin response thus appear to be at least partially due to an intracellular discharge of aequorin, and are therefore consistent with the view that aequorin in platelets is heterogeneously distributed among localized environments differing in Ca concentration. The aequorin signals observed initially in platelets stimulated by ADP or epinephrine were lost completely over a period of 30-60 min in almost all cases studied, while initial rates of aggregation were either unchanged (epinephrine) or only partially decreased (ADP) over this same time period. In contrast, thrombin- and A23187-induced aequorin signals were virtually unchanged over periods up to 90 min. Minimal changes with time also occurred in the aequorin signals induced by phorbol ester or by collagen in the presence of indomethacin. These differences in time dependence suggest that the signals generated by ADP and epinephrine may derive from different sources of aequorin than those associated with the signals induced by other agonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Aequorin↗
PubMed1989

Assembly of the platelet prothrombinase complex is linked to vesiculation of the platelet plasma membrane. Studies in Scott syndrome: an isolated defect in platelet procoagulant activity.

Activation of human platelets by complement proteins C5b-9 is accompanied by the release of small plasma membrane vesicles (microparticles) that are highly enriched in binding sites for coagulation factor Va and exhibit prothrombinase activity. We have now examined whether assembly of the prothrombinase enzyme complex (factors VaXa) is directly linked to the process of microparticle formation. Gel-filtered platelets were incubated without stirring with various agonists at 37 degrees C, and the functional expression of cell surface receptors on platelets and on shed microparticles was analyzed using specific monoclonal antibodies and fluorescence-gated flow cytometry. In addition to the C5b-9 proteins, thrombin, collagen, and the calcium ionophore A23187 were each found to induce formation of platelet microparticles that incorporated plasma membrane glycoproteins GP Ib, IIb, and IIIa. These microparticles were enriched in binding sites for factor Va, and their formation paralleled the expression of catalytic surface for the prothrombinase enzyme complex. Little or no microparticle release or prothrombinase activity were observed when platelets were stimulated with epinephrine and ADP, despite exposure of platelet fibrinogen receptors by these agonists. When platelets were exposed to thrombin plus collagen, the shed microparticles contained activated GP IIb-IIIa complexes that bound fibrinogen. By contrast, GP IIb-IIIa incorporated into C5b-9 induced microparticles did not express fibrinogen receptor function. Platelets from a patient with an isolated defect in inducible procoagulant activity (Scott syndrome) were found to be markedly impaired in their capacity to generate microparticles in response to all platelet activators, and this was accompanied by a comparable decrease in the number and function of inducible factor Va receptors. Taken together, these data indicate that the exposure of the platelet factor Va receptor is directly coupled to plasma membrane vesiculation and that this event can be dissociated from other activation-dependent platelet responses. Since a catalytic membrane surface is required for optimal thrombin generation, platelet microparticle formation may play a role in the normal hemostatic response to vascular injury.

Antibodies, Monoclonal↗
PubMed1989

Inhibition of human platelet function in vitro and ex vivo by acetaminophen.

The effects of acetaminophen (APAP) in vitro, or ex vivo following APAP ingestion, on human platelet aggregation, 14C-5HT secretion, and thromboxane B2 (TxB2) formation were assessed. APAP added in vitro to citrated platelet-rich plasma (PRP) inhibited aggregation, secretion, and TxB2 formation induced by collagen, epinephrine, arachidonate, and the ionophore A23187, but had no effect on the responses induced by the endoperoxide analog U44069. Arachidonate-induced responses were inhibited by lower concentrations of APAP than were the responses to the other agonists. In PRP obtained 1 hour after ingestion of 650 mg or 1000 mg APAP, arachidonate-induced TxB2 formation was inhibited by 40-99% in five subjects tested, whereas inhibition of collagen- or epinephrine-induced TxB2 formation was less consistent. Aggregation and secretion responses were not altered by APAP ingestion in 4 of the 5 subjects, but were inhibited in the remaining subject, who had the highest plasma APAP levels. In contrast to aspirin and indomethacin, APAP-induced inhibition of collagen-stimulated TxB2 formation could be partially overcome with increasing collagen concentrations. No such partial correction occurred with epinephrine, however. In washed platelet suspensions labeled with 3H-arachidonate, both APAP and aspirin inhibited the formation of labeled PGD2 and PGE2, as well as TxB2. These results suggest that APAP acts in human platelets as a reversible inhibitor of cyclo-oxygenase, as found previously in other tissues, and that recent APAP ingestion can, on occasion, produce inhibition of platelet functional responses measured in vitro.

Acetaminophen↗
PubMed1989

Fibrinogen-independent platelet adhesion and thrombus formation on subendothelium mediated by glycoprotein IIb-IIIa complex at high shear rate.

Platelet adhesion and thrombus formation on subendothelium, studied at a shear rate of 2,600 s-1, were inhibited by two synthetic peptides known to interact with GPIIb-IIIa. One peptide (HHLGGAKQAGDV) corresponds to the carboxyl terminal segment of the fibrinogen gamma-chain (gamma 400-411) and the other (RGDS) contains the amino acid sequence Arg-Gly-Asp (RGD) common to fibronectin, von Willebrand factor, vitronectin and the alpha-chain of fibrinogen. Neither platelet adhesion nor thrombus formation were decreased in a patient with severe congenital fibrinogen deficiency and this was equally true when his blood was further depleted of the small amounts of fibrinogen present utilizing an anti-fibrinogen antibody. In normal subjects, adhesion and thrombus formation were inhibited by the Fab' fragments of a monoclonal anti-GPIIb-IIIa antibody (LJ-CP8), which interferes with the interaction of platelets with all four adhesive proteins in both the fluid and solid phase. However, another anti-GPIIb-IIIa antibody (LJ-P5) that had minimal effects on the interaction of platelets with fibrinogen, but inhibited to varying degrees platelet interaction with other adhesive proteins, was equally effective. The findings demonstrate that, at a shear rate of 2,600 s-1, adhesive proteins other than fibrinogen are involved in GPIIb-IIIa-mediated platelet adhesion and thrombus formation on subendothelium. In addition, since LJ-P5 inhibited the binding of soluble von Willebrand factor and vitronectin, these adhesive proteins may be involved in platelet thrombus formation. In contrast to the results obtained at a shear rate of 2,600 s-1, fibrinogen could play a role in mediating platelet-platelet interactions with weak agonists or lower shear rates.

Afibrinogenemia↗
PubMed1989

Evidence for the presence of tissue factor activity on subendothelium.

By a variety of methods, tissue factor activity was demonstrated in the subendothelium of rabbit aorta and human umbilical artery. In one method, everted segments of de-endothelialized vessels were mounted in an annular perfusion chamber and the subendothelial surface was exposed to nonanticoagulated human blood under controlled flow. Procoagulant activity was assessed by measuring fibrin deposition on subendothelium and fibrinopeptide A (FPA) levels in post chamber blood. Both fibrin deposition and FPA were decreased with rabbit vessel segments exposed (at a shear rate of 650 seconds-1) to blood from patients with factor VII deficiency and with umbilical artery segments (at shear rates of 90 to 180 seconds-1) that had been pretreated with a monoclonal antibody to human tissue factor. In a second method, everted umbilical artery segments were mounted on a stir bar and the subendothelial surface was exposed, with stirring, to plasma or purified coagulation factors. The capacity of the surface to clot plasma on addition of calcium was inhibited by the antibody to tissue factor. The surface also activated purified 3H-factor X in the presence of factor VIIa, but not in its absence, and this surface property was almost entirely eliminated by pretreating the vessel segments with antitissue factor. Tissue factor activity in subendothelium could play a role in both the arrest of bleeding and in promoting the formation of thrombi at sites of vascular injury.

Animals↗
PubMed1988

Impairment of phosphatidylinositol metabolism in a patient with a bleeding disorder associated with defects of initial platelet responses.

Phosphoinositide/polyphosphoinositide (PI/PPI) metabolism, measured by the increase of 3H-phosphatidic acid (PA) and the decrease of 3H-phosphatidylinositol (PI) in 3H-arachidonate-labeled platelet suspensions, was assessed in five patients whose platelet functional defects included impaired initial rates of ADP, epinephrine and U44069 aggregation in platelet-rich plasma (PRP). In one patient, 3H-PA formation induced by collagen and thrombin was reduced or absent on two of three occasions, and the decrease in 3H-PI was reduced on one of these two occasions in response to collagen and A23187, and on all 3 occasions in response to thrombin. The variations in the formation of 3H-PA in this patient on different occasions broadly paralleled the variations in the initial rates of ADP and U44069 aggregation and in epinephrine aggregation seen in PRP. No such abnormalities of PI metabolism were found in four other patients with similar, but not identical, functional defects. These results suggest an impairment affecting metabolism of PI/PPI via the PI/PPI cycle in this patient's platelets. The association of abnormalities of PI metabolism with defects of initial platelet responses provides further support for a physiological role of phosphoinositide metabolism in the early activation mechanism of platelets.

Arachidonic Acid↗
PubMed1988

Heterogeneous defects of platelet secretion and responses to weak agonists in patients with bleeding disorders.

Eleven patients with mild bleeding disorders had as a common abnormality, impaired platelet aggregation and secretion with low concentrations (0.5-1.0 micrograms/ml) of collagen and, in most cases, an absence of second phase aggregation with epinephrine. Platelet granule contents were normal, ruling out storage pool deficiency. To characterize further the platelet abnormalities, we measured aggregation, 14C-5HT secretion, and TxB2 formation induced by a variety of platelet agonists. In eight of the 11 patients we observed decreased initial rates as well as extents of aggregation with one or more weak agonists (ADP, epinephrine, thromboxane A2 and the endoperoxide analogue U44069), i.e. agonists which induced secretion only as a result of aggregation, but normal responses to strong agonists such as arachidonate and high (10 micrograms/ml) concentrations of collagen, which can induce secretion in the presence or absence of aggregation. In all of these patients, TxB2 formation with arachidonate and all concentrations of collagen was normal. The platelet defects in these eight patients have been designated as weak agonist response defects (WARDs). In contrast, the initial aggregation responses to all weak agonists were normal in the three other patients, while secretion and TxB2 formation induced by strong agonists were impaired. Thus, in contrast to the eight patients above, the platelet defects in these three patients were characteristic of defects in the secretion response per se. The results obtained in the 11 patients studied indicate that these types of platelet disorders, previously referred to as primary secretion defects, include defects in the initial platelet responses which precede secretion (WARD) as well as defects in the secretory mechanism per se. Both groups of defects appear to be heterogeneous in nature.

Adult↗
PubMed1988

Specific correction of impaired acid hydrolase secretion in storage pool-deficient platelets by adenosine diphosphate.

Storage pool-deficient (SPD) platelets, which have decreased amounts of dense-granule and/or alpha-granule constituents, contain normal amounts of lysosomal acid hydrolases, but in some cases exhibit impaired secretion of these enzymes. We examined this impaired secretion response in SPD patients with varying extents of granule deficiencies, and determined the effects of added dense-granule constituents. Acid hydrolase secretion was impaired in patients with severe dense-granule deficiencies, but not in patients with lesser dense-granule deficiencies, including those with alpha-granule deficiencies as well. When dense-granule constituents (ADP, ATP, serotonin, Ca+2, pyrophosphate) were added to gel-filtered platelets, ADP, but none of the other constituents, completely corrected the impairment of thrombin and A23187-induced secretion in SPD platelets. The concentration of ADP required to normalize thrombin-induced secretion varied markedly, from 0.01 to 10 microM, among the individual patients. Fixation of platelets with formaldehyde before centrifugation did not prevent the enhancement of secretion by ADP. Excess ATP, which acts as a specific antagonist of ADP-mediated responses, completely blocked this enhancement of secretion in SPD platelets by ADP, and partially inhibited acid hydrolase secretion induced by low, but not high, concentrations of thrombin in normal platelets as well. Treatment of normal platelets with acetylsalicylic acid in vivo, but not in vitro, produced an impairment of acid hydrolase secretion similar in extent to that in SPD platelets, but which could not be completely corrected by added ADP. One possible explanation of these results is that the impairment of acid hydrolase secretion may be secondary to the dense-granule deficiency in SPD platelets, and that secreted ADP may potentiate the lysosomal secretion response in normal platelets as well.

Adenosine Diphosphate↗
PubMed1988

The response of platelets to epinephrine in storage pool deficiency--evidence pertaining to the role of adenosine diphosphate in mediating primary and secondary aggregation.

Aggregation responses and thromboxane (Tx) formation in ten patients with storage pool deficiency (SPD) specific to the dense granules (delta-SPD) were studied to assess further the role of dense granule adenosine diphosphate (ADP) in mediating platelet aggregation by epinephrine. The ability of epinephrine to elicit secondary aggregation (SA) responses was highly variable in delta-SPD when tested at 5 mumol/L epinephrine, but was consistently abnormal when tested over a range of concentrations. The occurrence of SA in both delta-SPD patients and normal subjects was correlated with the magnitude of the rate of primary aggregation (PA). This PA rate was normal, on average, for the entire patient group but was greater in patients with more consistent SA responses. The PA findings were related to the Kd value obtained in binding studies with 3H-yohimbine, but not with the number of alpha 2-receptor sites. Studies on Tx production (assessed by radioimmunoassay of TxB2) showed that the ability to synthesize Tx from arachidonate was not impaired in delta-SPD, and that there was an absolute positive correlation between epinephrine-induced SA and Tx production. Aggregation in delta-SPD platelets in response to the Tx receptor agonist U44069 was consistently decreased, but could be corrected by addition of ADP. The results of the study suggest that dense granule-derived ADP is not required for PA by epinephrine, but mediates SA as a synergistic agonist with TxA2. This role of ADP in SA may be elucidated more precisely by further studies on platelet activation processes in delta-SPD.

Adenosine Diphosphate↗
PubMed1988

Evidence for tissue factor-dependent activation of the classic extrinsic coagulation mechanism in blood obtained from bleeding time wounds.

The activation of platelets and the coagulation mechanism was studied by collecting blood from a standard bleeding time incision at 30-second intervals and measuring the plasma concentrations of fibrinopeptide A (FPA), platelet factor 4 (PF4), and thromboxane B2 (TxB2). FPA was observed in the first samples (30 to 60 seconds) obtained, increased progressively until cessation of bleeding, and was markedly diminished after heparin administration, thus indicating that thrombin formation occurs early in incisional blood. PF4 increased monotonically throughout blood sampling, whereas the major increase in TxB2 appeared near the cessation of bleeding. The initial increase in FPA content occurred normally in patients with deficiencies of either factor IX or VIII, was markedly diminished in patients with factor X or V deficiency, and was delayed in patients with factor VII deficiency. These studies suggest that tissue factor activation of the classic (activation of factor X) extrinsic coagulation mechanism occurs as an early event during the arrest of bleeding from bleeding time incisions. The relation of the aforementioned to platelet activation is less clear because there was no consistent correlation between decreased FPA formation and impaired PF4 secretion or TxB2 production. In fact, the latter were normal in some subjects with the most impaired FPA formation, which suggests that both collagen and thrombin, perhaps synergistically, may contribute to platelet activation during the primary arrest of bleeding.

Biomechanical Phenomena↗