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Biomedical subjects

M H Kroll

Publications and source records attributed to M H Kroll.

At least 37 records · Page 2Linked to original sources
PubMed1996

Binding of a novel 50-kilodalton alboaggregin from Trimeresurus albolabris and related viper venom proteins to the platelet membrane glycoprotein Ib-IX-V complex. Effect on platelet aggregation and glycoprotein Ib-mediated platelet activation.

Binding of the multimeric adhesive glycoprotein, von Willebrand Factor (vWF), to the platelet membrane glycoprotein (GP) Ib-IX-V complex mediates platelet adhesion and initiates signal transduction leading to platelet activation. Recently described viper venom proteins that bind to the GP Ib alpha-chain and inhibit vWF binding provide novel probes for studying receptor function. We have purified a 50-kDa form of alboaggregin from the white-lipped tree viper (Trimeresurus albolabris) and two 25-kDa proteins, CHH-A and CHH-B, from the timber rattlesnake (Crotalus horridus horridus) in addition to a previously described 25-kDa alboaggregin and echicetin. Complete or partial amino acid sequencing of CHH-A, CHH-B, and 50-kDa alboaggregin and cross-reactivity of these proteins with an anti-botrocetin antiserum confirmed that they were disulfide-linked heterodimers or higher multimers of the C-type lectin protein family. These proteins, together with 25-kDa alboaggregin and echicetin, specifically bound to GP Ib alpha within the N-terminal peptide domain, His-1-Glu-282, and inhibited vWF binding with comparable IC50 values (approximately 0.2 microgram/mL). However, cross-blocking studies between these structurally related proteins and anti-GP Ib alpha monoclonal antibodies demonstrated that the venom protein binding sites were not congruent. Further, the 50-kDa alboaggregin, but not the other venom proteins, potently induced platelet activation as assessed by dense granule serotonin release or elevation of cytosolic ionized calcium. Treatment of platelets with the 50-kDa alboaggregin was associated with activation of protein kinase C and tyrosine kinase(s), resulting in a platelet protein phosphorylation profile similar to that seen on shear-stress-induced vWF binding to platelets. These results suggest that the 50-kDa alboaggregin induces cytoplasmic signaling coincident with its binding to the GP Ib-IX-V complex and provides a potentially useful probe for studying the mechanism of vWF-dependent platelet activation.

Amino Acid Sequence↗
PubMed1996

Regulation of interleukin-1-beta-stimulated inducible nitric oxide synthase expression in cultured vascular smooth muscle cells by hemostatic proteins.

Experiments were performed to examine the mechanism by which specific hemostatic proteins regulate the release of nitric oxide (NO) from interleukin-1 beta (IL-1beta) stimulated cultured rate aortic smooth muscle cells. Treatment of smooth muscle cells with IL-Beta stimulated inducible nitric oxide synthase (iNOS) mRNA expression, which preceded the release of NO (as measured by the accumulation of nitrite in the culture media). The cytokine-stimulated production of nitrite was blocked by the protein synthesis inhibitor cycloheximide, the transcriptional inhibitor actinomycin D, and the competitive inhibitor of NOS nitro-L-arginine. However, only actinomycin D inhibited IL-1beta-stimulated iNOS mRNA expression, Treatment of smooth muscle cells with IL-1beta in the presence of platelet derived growth factor or thrombin resulted in the inhibition of cytokine-stimulated expression of iNOS mRNA and NO release. The inhibitory effect of thrombin was reversed by hirudin and was mimicked by a 14 amino acid thrombin receptor activating peptide. In contract, the concomitant exposure of smooth muscle cells to IL-1beta-and plasmin resulted resulted in the potentiation of both IL-1beta-stimulated iNOS expression and NO generation. Finally, treatment of smooth muscle cells with IL-1beta in the presence of the hemostatic proteins did not affect the half-life of iNOS mRNA. These results demonstrate that specific protein components of the hemostatic system regulate IL- 1beta-stimulated iNOS mRNA expression in vascular smooth muscle cells. The capacity of hemostatic proteins to modulate the induction of vascular iNOS activity may play an important role in governing the release of NO and regulating thrombogenesis in vivo.

Amino Acid Sequence↗
PubMed1996

Molecular cloning of a novel platelet protein showing homology to the angiotensin II receptor C-terminal domain.

Oligoscreening of a cDNA library obtained from 4 beta-phorbol 12-myristate 13-acetate-stimulated human erythroleukemia (HEL) cells resulted in the isolation of a novel clone coding for a protein with a calculated molecular mass of 8110 Da. This protein of 71 amino acids shows significant homology to the carboxyl-terminal regulatory domain of angiotensin II type 1 receptors. The homology encompasses four regions of amino acid residues thought to serve as consensus sequences for phosphorylation by serine/threonine kinases such as protein kinase C, which are key mediators of intracellular signaling. Reverse transcription-polymerase chain reaction identified the transcript in human platelets, human megakaryocytic DAMI cells, and HEL cells. High stringency Northern blotting revealed a tissue-specific distribution of three transcript species, with predominant expression in skeletal muscle and pancreas. Rabbit anti-peptide antiserum was used to immunoblot protein lysates from washed resting platelets and from 4 beta-phorbol 12-myristate 13-acetate-stimulated DAMI and HEL cells. These immunoblots revealed the presence of an intense approximately 8-kDa protein band in platelets and HEL cells and a faint band of identical size in DAMI cells.

Amino Acid Sequence↗
PubMed1996

Thrombin receptors activate potassium and chloride channels.

We used DAMI human megakaryocytic leukemia cells to study transmembrane ion currents activated through the G-protein-coupled thrombin receptor pathway. When the cells were stimulated by thrombin receptor-activating peptide, an increase in cytosolic Ca2+ ([Ca2+]i) developed as predicted by the known effect that thrombin exerts in the platelet. We then monitored the membrane potentials of individual DAMI cells during this response and observed complex, triphasic changes that could not be accounted for by Ca2+ fluxes alone. These consisted of rapid hyperpolarization, followed by depolarization to values more positive than the resting potential and then by slow repolarization. For the purpose of this study, we focused on the hyperpolarizing current that developed immediately after thrombin receptor activation. This proved to be composed of (1) a Ca(2+)-independent, outwardly rectifying Cl- current and (2) a strongly hyperpolarizing, inwardly rectifying, Ba(2+)-sensitive K+ current that required an increase of [Ca2+]i for activation. By analogy with their functions in other cell systems, it is logical to conclude that these prominent K+ and Cl- conductances may serve to regulate the complex volume changes that accompany thrombin receptor activation and/or to increase the electromotive drive that supports Ca2+ influx under these conditions through hyperpolarization of the cell membrane.

Amino Acid Sequence↗
PubMed1995

Fibrinolysis inhibits shear stress-induced platelet aggregation.

BACKGROUND: Shear stress-induced platelet aggregation may initiate arterial thrombosis at sites of pathological blood flow. Shear stress-induced platelet aggregation is mediated by von Willebrand factor (vWf) binding to platelet membrane glycoprotein (GP) Ib and GP IIb/IIIa. Tissue-type plasminogen activator (TPA) induces thrombolysis in coronary arteries through the local generation of plasmin. Plasmin also proteolyses GP Ib and plasma vWf. METHODS AND RESULTS: Because these effects could mitigate shear stress-induced platelet aggregation, we investigated the effect of fibrinolytic agents on platelet aggregation in response to a pathological shear stress of 120 dynes/cm2 generated by a cone-and-platen rotational viscometer. Plasmin inhibited shear stress-induced aggregation of washed platelets, and this was associated with a decrease in GP Ib. TPA, at concentrations > or = 2000 IU/mL, significantly inhibited shear stress-induced platelet aggregation of platelet-rich plasma without a decrease in platelet GP Ib. In plasma-platelet mixing experiments, we determined that the TPA effect was localized to plasma. Purified vWf multimer degradation by TPA (in the presence of exogenous plasminogen) was associated with the loss of the capacity of vWf to support shear stress-induced platelet aggregation. CONCLUSIONS: These results demonstrate that TPA inhibits platelet aggregation in response to pathological shear stress by altering the multimeric composition of vWf. This effect of TPA on shear stress-induced platelet aggregation may contribute, along with fibrinolysis, to the therapeutic effect of TPA in restoring blood flow during acute coronary artery thrombosis.

Fibrinolysin↗
PubMed1995

Vascular smooth muscle cell heme oxygenases generate guanylyl cyclase-stimulatory carbon monoxide.

BACKGROUND: Carbon monoxide (CO), like nitric oxide (NO), stimulates soluble guanylyl cyclase and thereby raises intracellular levels of cGMP. We examined the endogenous capacity of vascular smooth muscle cells (SMCs) to produce CO from heme through the activity of heme oxygenases. METHODS AND RESULTS: Cultured SMCs from rat aorta (RASMCs) expressed immunoreactive inducible heme oxygenase-1 (HO-1) and constitutive HO-2. Treatment of RASMCs with hemin and sodium arsenite, which are inducers of HO-1, stimulated RASMC cGMP without stimulating nitrite release or inducible NO synthase expression, and the induced elevations of cGMP were not inhibited by the NO synthase inhibitor NG-methyl-L-arginine. Induced CO from RASMCs likewise caused elevation of cGMP levels in platelets coincubated with the vascular cells. Zinc protoporphyrin IX, an inhibitor of HO, reversed the inducible increases in platelet cGMP. CONCLUSIONS: These results indicate that vascular SMCs have both constitutive and inducible HO activity, and they respond to specific stimuli to generate guanylyl cyclase-stimulatory CO in the same SMCs and in coincubated platelets.

Animals↗
PubMed1994

Shear-stress-induced von Willebrand factor binding to platelets causes the activation of tyrosine kinase(s).

Pathological arterial blood flow generates fluid shear stresses that directly cause platelet aggregation. The mechanism of shear-induced platelet aggregation is incompletely understood, but involves von Willebrand factor (vWF) binding to platelet glycoprotein (GP) Ib and GP IIb-IIIa, leading to the transmembrane influx of Ca2+ and the activation of protein kinase C. To investigate this further, shear-stress-induced protein tyrosine phosphorylation (PTP) of washed platelets was studied in a cone-plate viscometer. A time- and shear-stress-dependent tyrosine phosphorylation of substrates with approx. M(r) 29,000-31,000, 36,000, 50,000, 58,000, 64,000, 76,000, 85,000 and 105,000 was observed. PTP in response to a threshold shear stress of 0.3 mN/cm2 (30 dyn/cm2) was enhanced in most cases by exogenous purified human vWF, and PTP in response to a pathological shear stress of 0.9 mN/cm2 (90 dyn/cm2) was inhibited in some cases by inhibiting vWF binding to GP Ib or GP IIb-IIIa, or by inhibiting Ca2+ responses with extracellular EGTA. Shear-induced PTP of a substrate of M(r) approximately 31,000 appeared to be independent of GP Ib, and PTP of a substrate(s) of M(r) approximately 29,000 was shear-stress-dependent but independent of extracellular Ca2+. Cytochalasin D, which inhibits GP Ib-cytoskeleton interactions, inhibits the PTP of a substrate of M(r) approximately 76,000. These results suggest that tyrosine phosphorylation may be involved in transmembrane signalling that mediates platelet adhesion and aggregation in response to pathological shear stresses generated at sites of arterial vaso-occlusion.

Blood Platelets↗
PubMed1994

The activation-induced decrease in the platelet surface expression of the glycoprotein Ib-IX complex is reversible.

Thrombin decreases the platelet surface expression of the glycoprotein (GP) Ib-IX complex. To determine whether this effect is reversible, flow cytometric studies were performed with GPIb-IX-specific monoclonal antibodies. In both whole blood and washed platelet systems, incubation of platelets with thrombin or a combination of adenosine diphosphate and epinephrine resulted in a maximal decrease of the platelet surface expression of GPIb-IX within 5 minutes, after which there was a time-dependent return of the platelet surface GPIb-IX complex, which was maximal by 60 minutes. Exposure of the same platelets to additional exogenous thrombin resulted in a second decrease in platelet surface GPIb-IX, followed by a second reconstitution of platelet surface GPIb-IX. Throughout these experiments there was no measurable release from the platelets of glycocalicin (a proteolytic fragment of GPIb). Experiments in which platelets were preincubated with a biotinylated GPIb-specific MoAb showed that the GPIb molecules that returned to the platelet surface were the same molecules that had been translocated to the intraplatelet pool. The GPIb molecules that returned to the platelet surface were functionally competent to bind von Willebrand factor, as determined by ristocetin-induced platelet agglutination and ristocetin-induced binding of exogenous von Willebrand factor. Inhibitors of protein kinase C and myosin light-chain kinase enhanced the reexpression of platelet surface GPIb. In summary, the activation-induced decrease in the platelet surface expression of the GPIb-IX complex is reversible. Inactivation of protein kinase C and myosin light-chain kinase are important mechanisms in the reexpression of the platelet surface GPIb-IX complex.

Adult↗
PubMed1994

Platelets inhibit the induction of nitric oxide synthesis by interleukin-1 beta in vascular smooth muscle cells.

We have investigated the role of platelets in regulating the hemostatic and vasomotor properties of vascular smooth muscle. Experiments were performed to examine the effect of the releasate from activated platelets on the production of nitric oxide from interleukin-1 beta (IL-1 beta)-treated cultured rat aortic smooth muscle cells. Treatment of vascular smooth muscle cells with IL-1 beta resulted in significant accumulation of nitrite in the culture media and in marked elevation of intracellular cyclic guanosine monophosphate (GMP) levels. The releasate from collagen-aggregated platelets blocked the IL-1 beta-mediated production of nitrite and the accumulation of cyclic GMP in smooth muscle cells in a platelet number-dependent manner. In functional assays, the perfusates from columns containing IL-1 beta-treated smooth muscle cells relaxed detector blood vessels without endothelium and the addition of IL-1 beta-treated smooth muscle cells to suspensions of platelets inhibited their thrombin-induced aggregation. The simultaneous treatment of smooth muscle cells with IL-1 beta and the platelet releasate abolished both the vasorelaxing activities of the perfusates and the inhibition of platelet aggregation. Platelet releasates treated with a neutralizing antibody to platelet-derived growth factor (PDGF) failed to block IL-1 beta-induced nitric oxide production by the smooth muscle cells, as measured by both biochemical and functional assays. The platelet releasate from a patient with gray platelet syndrome likewise failed to block IL-1 beta-induced nitrite release by smooth muscle cells. These results demonstrate that platelets downregulate the production of nitric oxide by IL-1 beta-treated vascular smooth muscle cells through the release of PDGF. This effect may represent a novel mechanism by which platelets regulate vasomotor tone and thrombus formation at sites of vascular injury.

Animals↗
PubMed1994

Interference with clinical laboratory analyses.

Interference by endogenous and exogenous substances with assays for clinical analytes is a common problem in laboratory medicine. For this review, we defined interference as "the effect of a substance present in the sample that alters the correct value of the result, usually expressed as concentration or activity, for an analyte." There are four major endogenous compounds that consistently interfere with laboratory results: hemoglobin, bilirubin, lipids, and paraproteins. The major exogenous sources of interference are drugs prescribed for the patient; and there are several excellent compendia of the effect of drugs on clinical laboratory tests. We recommend determining whether the interference is dependent or independent of the analyte for the assay. Further, we propose an approach to the identification and resolution of an interference problem for the clinical laboratory and make recommendations to manufacturers.

Bilirubin↗
PubMed1994

Nonlinearity of high-density lipoprotein cholesterol determinations is matrix dependent.

The majority of methods for determining high-density lipoprotein (HDL) cholesterol failed the 1991 College of American Pathologists (CAP) linearity survey (sets LN2-A, B, C). We hypothesized that they failed because of the survey material matrix. We evaluated linearity with dextran sulfate or phosphotungstate as the precipitating reagents for several methods: Ektachem; TDx; Sigma; Dimension; Cobas Fara, with Roche reagents; and the Hitachi 736, with precipitating reagents from Boehringer Mannheim Diagnostics. We tested CAP survey material, Sigma HDL-cholesterol control material, and a fresh serum pool for linearity, using the polynomial method. All of the methods were nonlinear for the CAP material and for the controls precipitated after dilution. Five of eight methods were linear for the control materials precipitated before dilution. All methods but one were linear for the serum pool. These results demonstrate that the source of the nonlinearity is located in the precipitation step and depends on the sample matrix.

Chemical Precipitation↗
PubMed1993

M(r) 6,400 aurin tricarboxylic acid directly activates platelets.

ATA is a novel anticoagulant polymeric anionic aromatic compound that inhibits von Willebrand factor binding to platelet glycoprotein Ib and thereby prevents ristocetin- and shear stress-induced platelet aggregation. To investigate its mechanism of action, ATA fractions of homogeneous M(r) have been prepared by size exclusion chromatography. ATA fractions of M(r) > or = 2,500 are most effective at inhibiting vWF-mediated platelet aggregation, and ATA of M(r) = 2,500 also inhibits thrombin-induced platelet activation. Paradoxical results were observed in studies of ATA with M(r) = 6,400. This fraction of ATA stimulates aggregation of washed platelets or platelet-rich-plasma. The dose/response of aggregation shows a bell-shaped curve with maximal aggregation at approximately 2 micrograms/ml. Platelet aggregation is associated with phosphoinositide turnover and protein kinase C- and calcium-dependent protein phosphorylation. Platelet signalling responses to ATA are inhibited by platelet pretreatment with PGI2 or dibutyryl-cyclic AMP, but are unaffected by inhibiting platelet cyclooxygenase with aspirin. These results suggest that M(r) 6,400 ATA directly activates platelet phospholipase C to initiate platelet aggregation. This effect, unique to M(r) 6,400 ATA, could potentially mitigate ATA's beneficial anti-thrombotic effect on vWF-mediated platelet responses, and should be considered when analyzing results of experiments that utilize unfractionated ATA.

Aurintricarboxylic Acid↗
PubMed1993

Protein kinase C is activated in platelets subjected to pathological shear stress.

High levels of fluid shear stress at the blood vessel wall directly stimulate von Willebrand factor (vWF)-mediated platelet adhesion and aggregation and thereby contribute to the pathogenesis of arterial thrombosis. We have found that a pathological level of arterial wall shear stress (90 dynes/cm2) induces platelet aggregation that is associated with the phosphorylation of pleckstrin, a M(r) 47,000 protein kinase C substrate (p47). Shear-induced p47 phosphorylation depends entirely on vWF binding to platelet glycoprotein (Gp) Ib and GpIIb-IIIa, and the specific inhibition of protein kinase C with the staurosporine analogue Ro 31-7549 inhibits the full aggregation response to shear. Shear stress-induced platelet p47 phosphorylation occurs independent of any measurable change in diacylglycerol mass or hydrolysis of phosphatidylinositol 4,5-bisphosphate. These results indicate that mechanical shear stress induces vWF to bind to platelet GpIb and GpIIb-IIIa, stimulating a diacylglycerol-independent pathway of protein kinase C activation that contributes to platelet aggregation in response to shear.

Blood Platelets↗
PubMed1993

Nonatheromatous arterial thrombosis.

In contrast to venous thrombi, which are caused primarily by reduced blood flow and are composed predominantly of fibrin and red cells, arterial thrombi characteristically result from elevated shear stress at sites of vascular injury and are composed predominantly of platelets. Arterial thrombosis is therefore typically caused by abnormalities of platelets and/or the vessel wall. Disorders characterized by a propensity to arterial thrombosis due primarily to abnormalities of platelets include the myeloproliferative disorders, those due to vessel wall abnormalities include homocystinuria and the inflammatory vasculopathies, while those probably due to a combination of platelet and vessel wall abnormalities include heparin-associated thrombocytopenia and thrombosis and the antiphospholipid antibody syndrome.

Antiphospholipid Syndrome↗
PubMed1993

Plasmin potentiates induction of nitric oxide synthesis by interleukin-1 beta in vascular smooth muscle cells.

Experiments were performed to examine the effect of the major fibrinolytic protease, plasmin, on the production of nitric oxide from interleukin-1 beta (IL-1 beta)-treated cultured human and rat aortic smooth muscle cells. Incubation of vascular smooth muscle cells with IL-1 beta resulted in significant accumulation of nitrite and nitrate in the culture media. Plasmin, either added exogenously or generated by the reaction of tissue plasminogen activator with plasminogen, potentiated the IL-1 beta-mediated release of nitrite and nitrate from smooth muscle cells in a concentration-dependent manner, without affecting the production of nitrite and nitrate from cells untreated with IL-1 beta. This potentiating effect was abolished when plasmin was incubated with the protease inhibitor, alpha 2-antiplasmin. The perfusates from columns containing IL-1 beta-treated smooth muscle cells relaxed detector blood vessels without endothelium, and the addition of IL-1 beta-treated smooth muscle cells to suspensions of indomethacin-treated platelets inhibited their aggregation. Untreated smooth muscle cells or cells treated with plasmin alone did not have such effects. However, the simultaneous treatment of smooth muscle cells with IL-1 beta and plasmin markedly enhanced both the relaxing activities of the perfusates and the inhibition of platelet aggregation. Treatment of smooth muscle cells with NG-nitro-L-arginine inhibited the cytokine-mediated effects as well as the potentiating effect of plasmin. These results demonstrate that the plasmin can enhance the production of nitric oxide by IL-1 beta-treated vascular smooth muscle cells.

Animals↗
PubMed1993

Complex analyte-dependent and analyte-independent interferences with conjugated bilirubin in the enzymatic phenol-aminophenazone peroxidase (PAP) method for creatinine determination.

Although bilirubin interferes with the enzymatic assays for creatinine, neither a consensus of the degree of interference nor the mechanism has been established. Using multiple regression analysis, we demonstrate that the interference is negative and caused by both analyte-dependent and analyte-independent mechanisms. Furthermore, the correlative model includes terms non-linear with respect to creatinine. In the kinetic creatinine phenol-aminophenazone peroxidase method, there are analyte-dependent and analyte-independent mechanisms at work. The multivariate equation is: Crea' = 0.9879 Crea - 0.4524 Bili - 0.000828 Crea x Bili + 2.094 x 10(-7) Crea2 x Bili + 5.0 (Crea' = measured creatinine (mumol/l), Crea = true creatinine (mumol/l), and Bili = conjugated bilirubin (mumol/l)). The endpoint mode was affected less than the kinetic mode and exhibited different relationships in which two models describe the interference equally well. One is strictly analyte-dependent: Crea' = 0.9991 Crea - 0.00203 Crea x Bili + 2.390 x 10(-6) Crea2 x Bili - 1.464 x 10(-9) Crea3 x Bili + 3.261 x Bili + 3.261 x 10(-13) Crea4 x Bili - 9.9. The other is a complex combined analyte-dependent and analyte-independent: Crea' = 0.9834 Crea - 0.00680 Crea x Bili + 2.477 x 10(-7) Crea2 x Bili - 3.233 x 10(-7) Crea x Bili2 + 0.4652 Bili - 0.000458 Bili2 + 12.2. These models are valid for creatinine concentrations up to 2200 mumol/l (24.9 mg/dl) and bilirubin up to 660 mumol/l (38.6 mg/dl). The interference increases with increments of either bilirubin or creatinine. In addition, we found that unconjugated bilirubin interferes differently from conjugated bilirubin in degree and mechanism. Model building, contour plots, surface plots, and possible mechanisms are discussed. We propose multiple regression analysis as the proper way to evaluate interferences because analyte-dependence can be easily missed by simple regression analysis. True creatinine concentrations can be estimated despite the interference from conjugated bilirubin. Other phenol-aminophenazone peroxidase methods may be similarly affected.

Aminopyrine↗