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M H Kroll

Publications and source records attributed to M H Kroll.

At least 19 recordsLinked to original sources
PubMed1992

Monoclonal antibody AG-1 initiates platelet activation by a pathway dependent on glycoprotein IIb-IIIa and extracellular calcium.

The biochemical responses of intact human platelets to the monoclonal antibody (mAb) AG-1 were investigated. AG-1 is a murine IgG mAb that recognizes a series of platelet membrane glycoproteins (Gp) from M(r) 21,000 to 29,000, one of which is the M(r) 24,000 (p24) receptor for anti-CD9 mAbs. AG-1 causes platelet aggregation and secretion. Platelets binding AG-1 demonstrate a dose- and time-dependent breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2), production of diacylglycerol, and generation of phosphatidic acid (PA). These events are associated with the activation of protein kinase C (PKC), an increase in intracellular calcium, and fibrinogen binding. Platelet PA generation and PKC activation in response to AG-1 are inhibited by mAbs to platelet GpIIb-IIIa or by extracellular EGTA, but not by a mAb to platelet GpIb or by inhibiting platelet Na+/H+ exchange with 5-(N-ethyl-N-isopropyl)amiloride. Platelet cytoplasmic free calcium ([Ca2+]i) is elevated in response to AG-1, and this elevation is inhibited by mAbs to GpIIb-IIIa, an RGDS peptide or by chelating extracellular calcium. These results suggest that AG-1 binding to a unique platelet-surface glycoprotein initiates platelet responses through the activation of PIP2-specific phospholipase C, and that this occurs through a signal pathway that is dependent on GpIIb-IIIa and extracellular calcium.

Amino Acid Sequence↗
PubMed1992

Shear stress-induced von Willebrand factor binding to platelet glycoprotein Ib initiates calcium influx associated with aggregation.

Platelets subjected to elevated levels of fluid shear stress in the absence of exogenous agonists will aggregate. Shear stress-induced aggregation requires von Willebrand factor (vWF) multimers, extracellular calcium (Ca2+), adenosine diphosphate (ADP), and platelet membrane glycoprotein (GP)Ib and GPIIb-IIIa. The sequence of interaction of vWF multimers with platelet surface receptors and the effect of these interactions on platelet activation have not been determined. To elucidate the mechanism of shear stress-induced platelet aggregation, suspensions of washed platelets were subjected to different levels of uniform shear stress (15 to 120 dyne/cm2) in an optically modified cone and plate viscometer. Cytoplasmic ionized calcium ([Ca2+]i) and aggregation of platelets were monitored simultaneously during the application of shear stress; [Ca2+]i was measured using indo-1 loaded platelets and aggregation was measured as changes in light transmission. Basal [Ca2+]i was approximately 60 to 100 nmol/L. An increase of [Ca2+]i (up to greater than 1,000 nmol/L) was accompanied by synchronous aggregation, and both responses were dependent on the shear force and the presence of vWF multimers. EGTA chelation of extracellular Ca2+ completely inhibited vWF-mediated [Ca2+]i and aggregation responses to shear stress. Aurin tricarboxylic acid, which blocks the GPIb recognition site on the vWF monomer, and 6D1, a monoclonal antibody to GPIb, also completely inhibited platelet responses to shear stress. The tetrapeptide RGDS and the monoclonal antibody 10E5, which inhibit vWF binding to GPIIb-IIIa, partially inhibited shear stress-induced [Ca2+]i and aggregation responses. The combination of creatine phosphate/creatine phosphokinase, which converts ADP to adenosine triphosphate and blocks the effect of ADP released from stimulated platelets, inhibited shear stress-induced platelet aggregation without affecting the increase of [Ca2+]i. Neither the [Ca2+]i nor aggregation response to shear stress was inhibited by blocking platelet cyclooxygenase metabolism with acetylsalicylic acid. These results indicate that GPIb and extracellular Ca2+ are absolutely required for vWF-mediated [Ca2+]i and aggregation responses to imposed shear stress, and that the interaction of vWF multimers with GPIIb-IIIa potentiates these responses. Shear stress-induced elevation of platelet [Ca2+]i, but not aggregation, is independent of the effects of release ADP, and both responses occur independently of platelet cyclooxygenase metabolism. These results suggest that shear stress induces the binding of vWF multimers to platelet GPIb and this vWF-GPIb interaction causes an increase of [Ca2+]i and platelet aggregation, both of which are potentiated by vWF binding to the platelet GPIIb-IIIa complex.

Adenosine Diphosphate↗
PubMed1992

Endothelium-derived relaxing factor inhibits thrombin-induced platelet aggregation by inhibiting platelet phospholipase C.

Endothelium-derived relaxing factor (EDRF) inhibits platelet function, but the mechanism underlying this inhibitory effect is not known. To examine this, cultured acetylsalicylic acid (ASA)-treated endothelial cells (EC) from bovine aorta (BAEC) or from human umbilical vein (HUVEC) were incubated with washed, ASA-treated human platelets. Incubation of platelets with either BAEC or HUVEC resulted in inhibition of thrombin-induced platelet aggregation that was dependent on the number of EC added. This effect was potentiated by superoxide dismutase and reversed by treating EC with NG-nitro-L-arginine or by treating platelets with methylene blue, indicating that the inhibition of platelet aggregation was due to the release of EDRF by EC. EC significantly blocked the thrombin stimulated breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) and the production of phosphatidic acid in [32P]orthophosphate-labeled platelets and of inositol trisphosphate in [3H]myoinositol-labeled platelets. In addition, the thrombin-mediated activation of protein kinase C (PKC) and phosphorylation of myosin light chain were inhibited in the presence of EC. Finally, thrombin stimulated an increase in cytosolic ionized calcium concentration ([Ca2+]i) in fura2-loaded platelets that was abolished by concentrations of EC which also blocked thrombin-induced aggregation. These data indicate that EDRF blocks thrombin-induced platelet aggregation by inhibiting the activation of PIP2-specific phospholipase C and thereby suppressing the consequent activation of PKC and the mobilization of [Ca2+]i.

Animals↗
PubMed1992

Rationale for using multiple regression analysis with complex interferences.

Non-specificities and interferences may become complex when they involve the analyte as well as other interfering substances. These non-specificities and interferences are known as analyte-dependent and multi-interferent interferences. Multiple regression analysis has proven valuable in analysing this type of interference, but the theoretical foundation for using multiple regression analysis to study the basic mechanisms of interference has not been explicitly demonstrated. Graph theory can depict and model the basic mechanisms of interferences and the possible interactions. The relationship between the analyte, the interferents, and the response of the instrument to these entities can be approximated by a polymial of order three, which includes partial derivatives and cross-terms. The partial derivatives relate to the different interactions found with the graph theory model. Further, the partial derivatives can be associated with the coefficients in the multiple regression analysis when the respective values of the three variables (analyte, interferent one, and interferent two) are multiplied by one another. One can decide to retain or discard the coefficient of a variable, based on the statistical significance of the coefficient. The respective interactions in the graphic model can then be assembled and the framework of the interference mechanism established.

Bilirubin↗
PubMed1991

Regulation of the phosphoinositide cycle by Na+/H+ exchange and intracellular pH in human platelets.

We have found that thrombin-induced activation of protein kinase C (PKC) in platelets, measured by phosphorylation of the 47 kDa protein, is synergistically enhanced by the amiloride analogue ethylisopropylamiloride (EIA), a specific inhibitor of Na+/H+ exchange. This EIA effect was further synergistically enhanced by lowering intracellular pH (pHi) with either nigericin or sodium propionate, and reversed by raising pHi with monensin or ammonium chloride. The synergistic enhancement of thrombin-activated PKC by EIA plus nigericin was not observed when PKC was directly activated by phorbol esters. EIA and EIA plus nigericin caused a 3- to 6-fold increase in thrombin-induced diacylglycerol (DAG), but not phosphatidic acid (PA), production. EIA and nigericin also caused a marked increase in thrombin-induced breakdown and inhibition of resynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2). In summary, we have presented evidence that inhibition of Na+/H+ exchange causes primarily a H(+)-mediated interruption of the phosphoinositide cycle in activated platelets, including the accumulation of DAG associated with the enhancement of PKC activation, the inhibition of conversion of DAG to PA, and increased PIP2 breakdown. These data suggest a model in which Na+/H+ and pHi play an important regulatory role in permitting the phosphoinositide cycle to proceed in thrombin-activated platelets.

Amiloride↗
PubMed1991

Platelet interaction with vascular smooth muscle in synthesis of prostacyclin.

Because vascular smooth muscle cells (SMC) can be exposed to platelets at sites of significant arterial injury, we studied whether cultured rat aorta SMC can utilize platelet-derived arachidonate and prostaglandin (PG) endoperoxides (PGG2/PGH2) in the synthesis of prostacyclin (PGI2). SMC converted exogenous PGH2 to PGI2, measured by radioimmunoassay (RIA) of 6-keto-PGF1 alpha, despite cyclooxygenase inhibition or PGH2-receptor blockade. SMC produced increasing amounts of PGI2 in the presence of an increasing number of platelets when the two cell types were coincubated with arachidonate. Furthermore, aspirin-pretreated SMC produced PGI2 in response to arachidonate, ionophore A23187, or thrombin in the presence of platelets but not in their absence. SMC, by themselves unresponsive to thrombin, produced PGI2 during coincubation with thrombin-stimulated aspirin-pretreated platelets. Separation of the SMC monolayer and platelets with a filter did not prevent platelet-dependent PGI2 formation by the SMC. Finally, aspirin-pretreated SMC, in cosuspension with platelets, inhibited platelet aggregation in association with PGI2 production. These data indicate that 1) SMC can synthesize PGI2 from exogenously added PGH2 and from platelet-derived arachidonate or endoperoxides, 2) direct cell-cell contact is not required for intercellular endoperoxide transfer, and 3) SMC can inhibit platelet aggregation possibly through PGI2 production from platelet-derived endoperoxides.

Animals↗
PubMed1991

Platelet inhibition by an L-arginine-derived substance released by IL-1 beta-treated vascular smooth muscle cells.

Experiments were performed to examine whether stimulation of cultured vascular smooth muscle cells by interleukin (IL)-1 beta would induce platelet inhibitory properties of these cells. Incubation of platelets with untreated rat aortic smooth muscle cells had no effect on thrombin-induced platelet aggregation. In contrast, incubation of platelets with IL-1 beta-pretreated smooth muscle cells or the perfusate from such cells resulted in the inhibition of thrombin-induced platelet aggregation. This effect was potentiated by superoxide dismutase and reversed by incubating the IL-1 beta-treated smooth muscle cells with NG-nitro-L-arginine (L-NNA) or by treating the platelets with methylene blue. Cytokine-treated smooth muscle cells inhibited thrombin-stimulated changes in platelet cytosolic ionized calcium, whereas untreated cells were without effect. Incubating platelets with IL-1 beta-treated smooth muscle cells resulted in a 10-fold increase in platelet guanosine 3',5'-cyclic monophosphate (cGMP) levels, whereas untreated smooth muscle cells had no effect. The elevation of platelet cGMP induced by the IL-1 beta-treated smooth muscle cells was prevented by exposing the cytokine-treated cells to L-NNA or by treating platelets with methylene blue. Treatment of smooth muscle cells with IL-1 beta also resulted in an eightfold increase in nitrite production, which was blocked when the cells were incubated with L-NNA. The addition of cycloheximide to smooth muscle cells during their incubation with IL-1 beta completely inhibited smooth muscle cell nitrite production, the effects of the smooth muscle cells on platelet cGMP levels, and platelet responses to thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

von Willebrand factor binding to platelet GpIb initiates signals for platelet activation.

The hypothesis that von Willebrand factor (vWF) binding to platelet membrane glycoprotein Ib (GpIb) initiates intracellular pathways of platelet activation was studied. We measured the biochemical responses of intact human platelets treated with ristocetin plus vWF multimers purified from human cryoprecipitate. vWF plus ristocetin causes the breakdown of phosphatidylinositol 4,5-bisphosphate, the production of phosphatidic acid (PA), the activation of protein kinase C (PKC), increase of ionized cytoplasmic calcium ([Ca2+]i), and the synthesis of thromboxane A2. PA production, PKC activation, and the rise of [Ca2+]i stimulated by the ristocetin-induced binding of vWF multimers to platelets are inhibited by an anti-GpIb monoclonal antibody, but are unaffected by anti-GpIIb-IIIa monoclonal antibodies. Indomethacin also inhibits these responses without impairing platelet aggregation induced by vWF plus ristocetin. These results indicate that vWF binding to platelets initiates specific intraplatelet signaling pathways. The mechanism by which this occurs involves an arachidonic acid metabolite-dependent activation of phospholipase C after vWF binding to platelet membrane GpIb. This signal then causes PKC activation and increases of [Ca2+]i, which promote platelet secretion and potentiate aggregation.

Calcium↗
PubMed1990

The monoclonal antibody AG-1, a potent stimulator of human platelets, interacts with a low molecular weight GTP-binding protein.

AG-1 is a monoclonal antibody that binds to human platelets and causes aggregation and secretion. Previous work has established that these responses result from phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2). To determine the mechanism by which this ligand induces signals for platelet activation, we performed a series of experiments examining the platelet binding site for AG-1. AG-1 immunoprecipitates from radioiodinated human platelet plasma membranes a protein of Mr 21,000. AG-1 immunoprecipitated proteins separated by SDS-PAGE, transferred to nitrocellulose, and incubated with [alpha 32P]GTP demonstrate binding of the radiolabeled GTP to the Mr 21,000 protein. A 100-fold molar excess of unlabeled GTP inhibits completely this binding of [alpha 32P]GTP. These results indicate that AG-1 interacts with a low Mr GTP-binding protein on the surface of platelets and suggests that either the protein recognized by AG-1 or a coprecipitating molecule of similar Mr is a low Mr GTP-binding protein that may function in platelet extracellular signal transduction.

Antibodies, Monoclonal↗
PubMed1990

Effect of serum lyophilization on the rate constants of enzymatic methods for measuring cholesterol.

We determined the equilibrium absorbances and rate constants for two enzymatic methods, aca (DuPont) and RA-1000 (Technicon), used in determining cholesterol in reconstituted lyophilized serum. The lyophilized materials included two serum pools, three control materials, a College of American Pathologists' survey material, and Standard Reference Material no. 909. We calibrated the reagents with aca standards for cholesterol (DuPont). The difference in the mean concentrations of cholesterol (aca - RA-1000) was -0.09 g/L overall and was not statistically significant by analysis of variance. The mean rate constant for all materials was 0.23 min-1 for the aca and 1.42 min-1 for the RA-1000, significantly different (P less than 0.001). Lyophilization causes lower results for the aca method than for the Ra-1000, because the reaction rate for the aca method is slower and has not reached equilibrium when the final absorbance reading is made.

Cholesterol↗
PubMed1990

Bromide interference: is less really better?

Increased serum bromide may result from the ingestion of various drugs and from environmental exposures. Chloride methods that are less susceptible to bromide interference have a clinical disadvantage in that they are less likely to arouse suspicion in cases of bromide toxicity. When there is a clinical suspicion of bromide toxicity, differences among various analyzers in the amount of interference caused by bromide may be exploited to confirm this suspicion and to estimate the concentration of bromide in serum.

Bromides↗
PubMed1989

Turnover of eicosanoid precursor fatty acids among phospholipid classes and subclasses of cultured human umbilical vein endothelial cells.

Using cultured human umbilical vein endothelial cells, in which phosphatidylcholine (PC) is equally pulse-labelled by various eicosanoid precursor fatty acids (EPFAs), we have studied the remodelling of EPFAs among the phospholipid classes and subclasses with and without activation, and the relationship of this remodelling process to the selective release of arachidonic acid (AA) by phospholipase A2-mediated cell stimulation. When endothelial cells are pulse-incubated with radiolabelled EPFA for 15 min, greater than 80% of cell-associated radioactivity is present in phospholipids, among which greater than 60% is found in 1,2-diacyl-sn-glycero-3-phosphocholine (diacyl PC). After removing unincorporated radioactivity, reincubation of the pulse-labelled cells for up to 6 h results in progressive decrease in EPFA-labelled diacyl PC, increase in AA- or eicosapentaenoic acid (EPA)-labelled 1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (plasmalogen PE) and increase only in AA-labelled 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl PC). This redistribution of radiolabelled phospholipids is not altered by the presence of excess non-radiolabelled EPFAs. When aspirin-treated EPFA-labelled endothelial cells are stimulated with ionophore A23187, a very selective release of AA is noted in comparison with eicosatrienoate (ETA) or EPA, accompanied by an equivalent decrease in AA-labelled diacyl PC and specific increase in AA-labelled plasmalogen PE and alkyl PC. These selective changes in AA radioactivity induced by A23187 are enhanced 2-fold by pretreating the AA-labelled cells with phorbol 12-myristate 13-acetate, which by itself induces no changes. The changes in radioactivity induced by A23187 without and with phorbol ester among the released AA, the diacyl PC and the plasmalogen PE are significantly correlated with each other. These results indicate that human endothelial cells incorporate EPFAs (AA, ETA, EPA) equally into diacyl PC but selectively release AA esterified into diacyl PC with specific remodelling into plasmalogen PE and alkyl PC.

Arachidonic Acid↗
PubMed1989

Second messenger function of phosphatidic acid in platelet activation.

Phosphatidic acid (PA) is synthesized as the result of the receptor-mediated response of platelets to physiologic agonists. The role of PA in platelet signal transduction, however, is largely unknown. We have examined the responses of platelets to 1-stearoyl-2-arachidonoyl phosphatidic acid (SAPA), the predominant molecular species of human platelet PA. SAPA alone causes platelet aggregation, and pretreatment of platelets with SAPA markedly enhances thrombin-induced aggregation and secretion. Addition of SAPA to intact human platelets causes rapid breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) and the generation of diacylglycerol and endogenous PA. These reactions are associated with mobilization of intracellular calcium and activation of protein kinase C. SAPA also stimulates the release of endogenous arachidonic acid and its conversion to thromboxane A2. Furthermore, platelet activation by SAPA is blocked by indomethacin, indicating that the actions of SAPA are mediated by cyclooxygenase products. These findings suggest that SAPA may play an important role as an endogenous positive feedback signal to amplify receptor-mediated activation of PIP2-specific phospholipase C in human platelets.

Adenosine Triphosphate↗
PubMed1989

Effect of lyophilization on results of five enzymatic methods for cholesterol.

We studied the effect of lyophilization of serum pools on the determination of cholesterol with the enzymatic methods used in five automated analyzers: SMAC (Technicon), the RA-1000 (Technicon), the aca (Du Pont), the TDx (Abbott), and the Ektachem 700P (Kodak). We prepared two serum pools: pool A (2.2 g/L) and pool B (1.9 g/L). We separated each pool into three groups for treatment by lyophilizing or freezing at -20 and -70 degrees C. We determined cholesterol by the above methods at regular intervals during the next 270 days. For all methods, the measured concentration of cholesterol was less for the lyophilized serum than for frozen (P less than 0.007 by the paired-sample t-test). After adjusting for the dilutional effects of reconstitution, the decrease in original value was 0.7% for the SMAC, 1.7% for the RA-1000, 5.7% for the Ektachem, 9.4% for the TDx, and 14.3% for the aca. Lyophilization of cholesterol standards or serum samples may hamper the effective standardization of enzymatic methods and may be a source of apparent inaccuracy (bias) among enzymatic methods.

Autoanalysis↗
PubMed1988

Control of platelet protein kinase C activation by cyclic AMP.

Experiments were performed to elucidate the role of adenosine 3': 5'-cyclic monophosphate (cAMP) in the control of platelet protein kinase C (PKC) activation. Platelet aggregation and secretion in response to 4 beta-phorbol 12-myristate 13-acetate (PMA) or 1-oleoyl-2-acetylglycerol (OAG) were inhibited by dibutyryl cAMP in a dose-dependent manner. Inhibition of these functional activities paralleled a decrease in the PMA-induced phosphorylation of the Mr 47,000 substrate (p47) of PKC by pre-incubation of platelets with dibutyryl cAMP. These changes were also observed when platelet cAMP was increased by prostacyclin (PGI2), forskolin, or theophylline. The ADP scavenger creatine phosphate/creatine phosphokinase (CP/CPK) and the cyclooxygenase inhibitor indomethacin also diminished the aggregation and p47 phosphorylation responses to PMA or OAG. Pre-incubation of platelets with dibutyryl cAMP significantly potentiated the inhibition of aggregation and p47 phosphorylation effected by CP/CPK and indomethacin. These results are consistent with the model that PMA- or OAG-induced activation of platelets is amplified by secreted ADP and that the response to secreted ADP is inhibited by cAMP. Furthermore, the findings that increased intracellular cAMP inhibits PMA- or OAG-induced p47 phosphorylation in excess of that due solely to CP/CPK, and that cAMP significantly potentiates the effects of ADP removal and inhibition of cyclooxygenase in blocking p47 phosphorylation suggest that cAMP also exerts non-ADP-mediated inhibitory effects on PKC in intact platelets.

Adenosine Diphosphate↗
PubMed1988

Analytical bias for cholesterol and the percent of the population deemed at risk for coronary heart disease.

The effect of methodological bias on the population at risk is dependent on the location of the reference value in the distribution of the population. We fitted the cumulative distribution for cholesterol to a rational function and calculated the apparent reference values for four biased methods: Technicon SMAC (2.6%), DuPont aca (4.0 to 4.8%), Kodak DT-60 (-2.0 to -5.5%), and BMD Reflotron (-7.4 to -7.8%). With the true and apparent reference values for cholesterol and the rational function, we determined the percentage increase or decrease in the population deemed at risk for coronary heart disease. The population at risk increased by as much as 48% for methods with positive bias, and decreased by as much as 54% for methods with negative bias. If we restrict the percentage of the population incorrectly diagnosed to 3% and use reference values (cut points) recommended by the National Cholesterol Education Program, the maximum allowable methodological bias would be 1.6% for positive bias and -1.55% for negative bias. Therefore, an absolute methodological bias of 3% (as recommended by the Laboratory Standardization Panel) may be too liberal.

Adult↗