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M M Frojmovic

Publications and source records attributed to M M Frojmovic.

At least 37 records · Page 2Linked to original sources
PubMed1994

Dynamics of platelet glycoprotein IIb-IIIa receptor expression and fibrinogen binding. II. Quantal activation parallels platelet capture in stir-associated microaggregation.

There is broad agreement that platelet aggregation is generally dependent on fibrinogen (Fg) binding to the glycoprotein (GP) IIb-IIIa receptor expressed on the activated platelet surface. We therefore compared rates and extents of aggregation and of fibrinogen receptor expression and specific Fg binding to activated platelets, as a function of ADP concentration. Human citrated platelet-rich plasma (diluted 10-fold) was stirred with adenosine diphosphate (ADP) for 10 s or 2 min to measure rates and extent of aggregation, respectively, determined from the decrease in the total number of particles. The number of fibrinogen receptors and bound Fg were measured from mean fluorescence values obtained with FITC-labeled IgM monoclonal antibody PAC1 and the IgG monoclonal antibody, 9F9, respectively, using flow cytometry as presented in part I (Frojmovic et al., 1994). Because flow cytometric and aggregation measurements were routinely determined at room temperature and 37 degrees C, respectively, we also compared and found temperature-independent initial rates of aggregation. The fraction of platelets with fluorescence values above one critical threshold value, corresponding to maximally "activated" platelets (P*), increased with increasing activator concentration and correlated linearly with the fraction of platelets recruited into aggregates for ADP (r > 0.9). Aggregation was not rate-limited by fibrinogen receptor expression or by Fg binding. It appears that each platelet expresses its maximal Fg receptors at a critical ADP concentration, i.e., occupancy of ADP receptors. This, in turn, leads to rapid Fg occupancy and capture of such "quantally activated" platelets into aggregates.

Adenosine Diphosphate↗
PubMed1994

Aggregation efficiency of activated normal or fixed platelets in a simple shear field: effect of shear and fibrinogen occupancy.

Shear rate can affect protein adsorption and platelet aggregation by regulating both the collision frequency and the capture efficiency (alpha). These effects were evaluated in well defined shear field in a micro-couette for shear rate G = 10 - 1000 s-1. The rate of protein binding was independent of G, shown for adsorption of albumin to latex beads and PAC1 to activated platelets. The initial aggregation rate for ADP-activated platelets in citrated platelet-rich plasma followed second order kinetics at the initial platelet concentrations between 20,000 and 60,000/microliters. alpha values, which dropped nearly fivefold for a 10-fold increase in G, were approximately proportional to G-1, contrary to a minor drop predicted by the theory that includes protein cross-bridging. Varying ADP concentration did not change alpha of maximally activated platelet subpopulations, suggesting that aggregation between unactivated and activated platelets is negligible. Directly blocking the unoccupied but activated GPIIb-IIIa receptors without affecting pre-bound Fg on "RGD"-activated, fixed platelets (AFP) by GRGDSP or Ro 43-5054 eliminated aggregation, suggesting that cross-bridging of GPIIb-IIIa on adjacent platelets by fibrinogen mediates aggregation. Alpha for AFP remained maximal (approximately 0.24) over 25-75% Fg occupancy, otherwise decreasing rapidly, with a half-maximum occurring at around 2% occupancy, suggesting that very few bound Fg were required to cause significant aggregation.

Blood Platelets↗
PubMed1994

Adenosine diphosphate-induced aggregation of human platelets in flow through tubes: III. Shear and extrinsic fibrinogen-dependent effects.

The effect of shear rate and fibrinogen concentration on adenosine diphosphate-induced aggregation of suspensions of washed human platelets in Poiseuille flow at 23 degrees C was studied using a previously described double infusion technique and resistive particle counter size analysis. Using suspensions of multiple-centrifuged and -washed cells in Tyrodes-albumin [3 x 10(5) microliters-1; (17)] with [fibrinogen] from 0 to 1.2 microM, the rate and extent of aggregation with 0.7 microM ADP in Tyrodes-albumin were measured over a range of mean transit times from 0.2 to 43 s, and at mean tube shear rates, G, = 41.9, 335 and 1,335 s-1. As measured by the decrease in singlet concentration, aggregation at 1.2 microM fibrinogen increased with increasing G up to 1,335 s-1, in contrast to that previously reported in citrated plasma, in which aggregation reached a maximum at G = 335 s-1. Without added fibrinogen, there was no aggregation at G = 41.9 s-1; at G = 335 s-1, there was significant aggregation but with an initial lag time, aggregation increasing further at G = 1,335 s-1. Without added fibrinogen, aggregation was abolished at all G upon incubation with the hexapeptide GRGDSP, but was almost unaffected by addition of an F(ab')2 fragment of an antibody to human fibrinogen. Aggregation in the absence of added fibrinogen was also observed at 37 degrees C. The activation of the multiple-washed platelets was tested using flow cytometry with the fluorescently labelled monoclonal antibodies FITC-PAC1 and FITC-9F9. It was shown that 57% of single cells in unactivated PRT expressed maximal GPIIb-IIIa fibrinogen receptors (MoAb PAC1) and 54% expressed pre-bound fibrinogen (MoAb 9F9), with further increases on ADP activation. However, incubation with GRGDSP and the F(ab')2 fragment did not inhibit the prebound fibrinogen. Moreover, relatively unactivated cells (8% expressing receptor, 14% prebound fibrinogen), prepared from acidified cPRP by single centrifugation with 50 nM of the stable prostacyclin derivative, ZK 36,374, and resuspension in Tyrodes-albumin at 5 x 10(4) microliters-1, aggregated with 2 and 5 microM ADP at G = 335 and 1,335 s-1 in the absence of added fibrinogen. We therefore postulate that a protein such as von Willebrand factor, secreted during platelet isolation or in flow at sufficiently high shear rates, may yield the observed shear-rate dependent aggregation without fibrinogen.

Adenosine Diphosphate↗
PubMed1994

Platelets from bleeding Simmental cattle mobilize calcium, phosphorylate myosin light chain and bind normal numbers of fibrinogen molecules but have abnormal cytoskeletal assembly and aggregation in response to ADP.

We have evaluated platelet function in normal Simmental cattle and in those with a congenital, inherited bleeding disorder previously attributed to impaired platelet aggregation. Affected platelets failed to aggregate and secrete in response to ADP and the ionophore A23187, and showed impaired aggregation responses to collagen and ionomycin. Aggregation and secretion of normal and affected platelets was similar in response to thrombin and PMA. Resting cytosolic calcium levels and calcium mobilization in response to ADP and ionomycin were similar in control and four affected animals. Normal and affected bovine platelets phosphorylated myosin light chain and pleckstrin in response to ADP and A23187. Transmission electron microscopy of affected platelets following stimulation with ADP, showed shape change and some degree of centralization of the actomyosin gel. Affected platelets had comparable numbers of GPIIb/IIIa complexes and expressed comparable numbers of fibrinogen receptors as normal platelets in response to ADP. Cytoskeletal assembly in affected platelets was normal in response to PMA but incomplete in response to ADP and A23187. Failure of platelet aggregation in bleeding Simmental cattle is predicted to arise from abnormal cytoskeletal assembly following calcium mobilization and phosphorylation of myosin light chain in response to ADP.

Adenosine Diphosphate↗
PubMed1993

Regulation of human neutrophil aggregation: comparable latent times, activator sensitivities, and exponential decay in aggregability for FMLP, platelet-activating factor, and leukotriene B4.

We have recently described a flow cytometry technique, whose sensitivity allows direct measurements of latent times before the onset of aggregation, and of rates, maximal extents, and reversibility of aggregation (J Leuk Biol 50:434, 1991). We report here that activators which stimulate sustained cellular signaling associated with increases in intracellular calcium (ionomycin) or protein kinase C activation (phorbol myristate acetate, PMA) cause complete (> or = 98%) and irreversible neutrophil aggregation, with latent times for the onset of aggregation inversely proportional to the activator concentration. In contrast, the receptor-specific activators leukotriene B4 (LTB4), formyl peptide FMLP, and platelet-activating factor (PAF) gave only partial and reversible aggregatory responses, limited by the following similar properties: latent times of 4.5 seconds +/- 1.5 seconds, independent of activator concentration; similar concentrations for onset of aggregation (approximately 1 nmol/L) that increased over a similar broad range of activator concentration, with one-half maximal rates of aggregation at 10 nmol/L to 30 nmol/L, corresponding to reported dissociation constant values; comparable limited recruitment and spontaneous reversibility of aggregation; absence of interactivator synergism; and similar exponential decays in activated cell stickiness (refractoriness), with t1/2 = 15 to 30 seconds. Variable cross-desensitization was seen between LTB4 and FMLP depending on donor and activator concentrations. In vivo, these properties are expected to provide localization of the aggregatory response, minimizing the otherwise detrimental effects of circulating activated neutrophils.

Adult↗
PubMed1993

Determination of adhesion force between single cell pairs generated by activated GpIIb-IIIa receptors.

A biophysical approach was used to directly determine the avidity of the junction between two Chinese hamster ovary (CHO) cells bearing recombinant GpIIb-IIIa in the presence and absence of fibrinogen. Micromanipulation was used to induce conjugation of the cell pairs with or without activating the GpIIb-IIIa molecules with monoclonal antibody (MoAb) 62. Activation of GpIIb-IIIa caused an increase in the force required to separate the conjugates. The molecular bonding force between cells bearing activated GpIIb-IIIa and fibrinogen molecules was found to be 2.1 x 10(-7) dyne, which is 3.7 times higher than that between nonactivated GpIIb-IIIa and fibrinogen (5.7 x 10(-8) dyne). The results provide a quantitative assessment of the molecular bonding force between fibrinogen and the GpIIb-IIIa expressed on cell surface. The findings indicate that the activation of GpIIb-IIIa leads to an increase in the adhesive force in CHO cell aggregation by increasing the strength of the GpIIb-IIIa-fibrinogen bonds rather than the number of these bonds.

Animals↗
PubMed1992

A model for the recruitment of neutrophils at sites of inflammation. Physiological relevance of in vivo neutrophil aggregation.

Adhesive glycoproteins on both neutrophils and vascular endothelial cells are known to mediate adhesive interaction between the two cell types. We propose that activation of endothelial cells will lead to the capture of unactivated neutrophils, localizing them at inflammatory sites. The interaction between activated endothelium and captured neutrophils will result in the stimulation of adherent neutrophils. Adherent activated neutrophils are then able to recruit incoming unactivated neutrophils by capturing them, further increasing the number of neutrophils at the inflammatory site. The formation of an adherent neutrophil aggregate will reduce plasma leakage from the vasculature as neutrophils migrate into tissue and will protect migrating neutrophils from shear stress of blood flow.

Animals↗
PubMed1992

Platelet plasma membrane is equally distributed between surface and osmotically-evaginable surface-connecting membrane, independent of size, subpopulation or species.

Osmotic swelling can double the external plasma membrane surface area of normal human platelets. The surface-connected open canalicular system (SCCS) has been proposed as the major source for this additional membrane. As bovine (B) platelets have been reported to lack SCCS, we compared osmotic swelling for B and human (H) cells. Addition of water to platelet-rich-plasma (10-90% v/v) caused sequential shape change and osmotic spherocyte (OS) formation, analyzed for size and surface area changes from time-dependent phase-contrast videomicroscopic images. Selected samples were fixed and stained with tannic acid prior to osmic acid fixation for visualization of open SCCS by transmission electron microscopy. Bovine platelets required 3-4x less water dilution of PRP than human platelets, with significant OS forming at 20% water addition. Continued water dilution converted 50% of platelets to OS, with maximally stable swelling and no significant lysis for bovine OS up to 60% dilution. Electron micrographs of unactivated discocytes (D) and of optimally-swollen OS showed open SCCS in human D not detectable in any of the swollen platelets, though granules, mitochondria and a small number of vesicles and vacuoles persisted. No evidence for any open SCCS was found for bovine D or OS, though the OS otherwise appeared similar to human OS. Geometric measurements of D and nonlysed OS showed a stable, maximal 2.1 +/- 0.1 fold increase in external plasma membrane surface area with osmotic swelling, identical for different-sized normal human platelets (mean volume V = 2.8-6.8 fl), for abnormally-large platelets (V = 8.7-11.6 fl), or for bovine or rabbit platelets (V = 3-4 fl). As osmotic swelling appears to primarily externalize SCCS in human platelets, the identical relative amounts of internal membrane externalized for bovine platelets is hypothesized to arise from an osmotically more labile, "closed", and structurally simpler SCCS or from a distinct membrane source than in human platelets. It appears that the surface to invaginated ("SCCS") plasma membrane is kept constant for human, bovine and rabbit platelets, independently of platelet production, size-dependent subpopulations, or of platelet ageing.

Animals↗
PubMed1991

Platelet glycoprotein IIb-IIIa (alpha IIb beta 3 integrin) confers fibrinogen- and activation-dependent aggregation on heterologous cells.

To analyze molecular mechanisms of platelet aggregation, we have studied the aggregation of Chinese hamster ovary (CHO) cells expressing between 1 and 4 x 10(5) recombinant human glycoprotein (GP) IIb-IIIa molecules per cell (A5 cells). These cells aggregated as measured by the disappearance of single cells during rotary agitation. Aggregation was dependent on the presence of extracellular fibrinogen (approximately 500 nmol/L) and divalent cations, and required prior activation of the GPIIb-IIIa. A synthetic peptide (GRGDSP) and monoclonal anti-GPIIb-IIIa antibody (2G12) that block platelet aggregation also blocked aggregation of these cells. Parent CHO cells or those expressing recombinant GPIIb-IIIa containing a point mutation that causes variant thrombasthenia both failed to aggregate when stimulated in the presence of fibrinogen. These data show that GPIIb-IIIa is the only unique platelet surface component required for aggregation.

Animals↗
PubMed1991

Dynamics of human neutrophil aggregation evaluated by flow cytometry.

In order to directly monitor neutrophil aggregation, we have developed a simple particle counting technique using flow cytometry. Flow times were used to determine aggregation from changes in the total number of particles per unit volume (%PA(T)), while fluorescent signals emitted from glutaraldehyde-fixed neutrophils were used to measure changes in the total number of singlet neutrophils (%PA(S)). Flow cytometrically-determined %PA values were found to be virtually identical to values determined from microscopy. We show that aggregation parameters can be evaluated and compared simply from measures of changes in total particle count without a need for distinguishing singlets from multiplets. A number of aggregation parameters are introduced and related to the initial cell concentration (N(o)) and stir speed (shear). Aggregation of neutrophils, following stimulation with 0.5 microM FMLP (N-formyl-methionyl-leucyl-phenylalanine), showed a latent time (tl) of 4 +/- 1.5 sec independent of N(o) or stir speed; had a forward rate of aggregation (vf) which was proportional to the initial cell concentration and the stir speed; plateaued for greater than or equal to 60 s; and showed partial to complete reversal by 7.5 min. Above a critical stir speed, the extent and duration of aggregation varied inversely with the stir speed. The stir and N(o) dependence of the aggregation parameters studied suggest the existence of subpopulations of neutrophils with distinct efficiencies of aggregation.

Blood Cell Count↗
PubMed1991

Ligand bridging mediates integrin alpha IIb beta 3 (platelet GPIIB-IIIA) dependent homotypic and heterotypic cell-cell interactions.

The aggregation of cells bearing recombinant integrin alpha IIb beta 3 (platelet GPIIb-IIIa) has been analyzed by two-color flow cytometry. As in normal platelets, aggregation requires functional alpha IIb beta 3, "activation" of alpha IIb beta 3, and fibrinogen (fg) binding to alpha IIb beta 3. Cellular aggregation required that both interacting cells express functional alpha IIb beta 3, because a binding defective mutant, alpha IIb beta 3 (D119----Y), failed to support interaction with wild type alpha IIb beta 3-bearing cells. In addition, cells bearing resting alpha IIb beta 3 were incorporated into aggregates formed by cells bearing a constitutively active mutant, alpha IIb beta 3 (beta 1-2), indicating that only one of the cells in an interacting pair must be activated. Finally, heterotypic interactions occurred between cells bearing activated alpha IIb beta 3 and cells bearing alpha V beta 3, a fg-binding integrin present on endothelial and tumor cells. Thus, ligand bridging between fg-binding integrins represents a mechanism of cell-cell interaction, cells bearing resting alpha IIb beta 3 (e.g., resting platelets) may be incorporated into aggregates formed by cells bearing activated alpha IIb beta 3, and alpha IIb beta 3 mediates heterotypic interactions with cells bearing other fg receptors.

Animals↗
PubMed1990

The platelet extinction coefficient measured by aggregometry is dependent on platelet composition rather than size: implications for studies of platelet heterogeneity and abnormalities.

Extinction (E) or light transmission measurements of platelet suspensions have been widely used to evaluate platelet structure and aggregation for platelet populations of varying mean platelet size (V). However, useful comparisons of platelet suspensions from donors having abnormally-sized platelets with those from healthy controls require a knowledge of the dependence of E on V. We have analyzed the extinction per platelet (e) as a function of the geometric scattering cross-section of equivalent spheres, V2/3, related to the apparent mean platelet optical efficiency, k. Such an analysis using data previously reported by Holme et al., for healthy controls and a variety of platelet-associated disorders showed e/V2/3 or k to be constant over an eight-fold variation in V. The outstanding exception was platelet suspensions from myeloproliferative disorder patients (MPD) which showed approximately 25% reduction in k values. When we analyzed e/V2/3 data for different-sized platelet subpopulations which were isolated by elutriation from healthy donors, we found that k doubled with a doubling in V from small to large platelets. It appears that the use of aggregometry devices with wide light acceptance angles yields k values for platelets which are insensitive to variations in V, but rather are sensitive to the expected variations in refractive index associated with distinct platelet internal composition and organization expected for different-sized normal subpopulations and "diseased" platelets as found in MPD. We relate these observations to photometric studies of platelet heterogeneity and of aggregation of platelets with different V and k values.

Adult↗
PubMed1990

Comparative studies of microscopically determined aggregation, degranulation, and light transmission after chemotactic activation of adult and newborn neutrophils.

Changes in the light transmission of suspensions of activated neutrophils are widely used to measure the dynamics of neutrophil aggregation. Such studies have suggested, for example, that aggregation is irreversible for human newborn neutrophils but fully reversible for adult cells. We have evaluated aggregation directly by microscopic particle counting and compared it with changes in light transmission (delta T) and with release from three granule subsets for neutrophils activated with N-formyl-methionyl-leucyl-phenylalanine (FMLP). Maximal increases in %T in response to 0.5 micromol/L FMLP were approximately 25% larger for newborn than for adult neutrophils, and were only partially reversible by 8 minutes, while %T increases for adult neutrophils were fully reversible. However, measurements of neutrophil aggregation using light microscopy showed that both newborn and adult neutrophils fully deaggregated. A further independence of delta T from aggregation was found by pretreating adult neutrophils with cytochalasin B (5 micrograms/mL) in the presence of 0.5% gelatin, a pretreatment that blocked FMLP-induced neutrophil aggregation while allowing large increases in %T and degranulation. In response to FMLP, newborn neutrophils released more enzyme from each granule subset than did adult neutrophils. Our results suggest that cellular events associated with neutrophil activation (other than aggregation) are implicated in light transmission responses and that these differ for adults and newborns. These results also suggest that reports of neutrophil aggregation should be based on direct particle counting methods rather than on %T responses.

Adult↗
PubMed1989

Montreal platelet syndrome: a defect in calcium-activated neutral proteinase (calpain).

Platelets from patients with Montreal platelet syndrome (MPS) consistently display a defect in the mechanisms that regulate platelet size during shape change and undergo spontaneous aggregation and stir-induced microaggregate formation. We now provide data that the surface glycoprotein composition of MPS platelets is indistinguishable from that of normal platelets. However, a defect in calcium-activated neutral proteinase (calpain) was detected in MPS platelets. The specific activity of calpain in the cytosolic fraction of platelets from four MPS patients was found to be only 30% of that in platelets from normal control donors (n = 18, P less than .001). Additionally, platelets from MPS patients (n = 3) contained only 50% (P less than .001) of the calpain I catalytic subunit antigen found in platelets from normal control donors (n = 9). Platelets from the asymptomatic father/grandfather of the MPS patients had normal amounts of both total calpain proteolytic activity and calpain I catalytic subunit antigen. This represents the first report of a defect in calpain in human cells. The abnormally low calpain activity in MPS platelets may account for the platelet defects characteristic of this disorder.

Blood Platelet Disorders↗
PubMed1988

Differential inhibition of the platelet activation sequence: shape change, micro- and macro-aggregation, by a stable prostacyclin analogue (Iloprost).

The relative sensitivities of adenosine diphosphate (ADP)-induced activation, and of prostaglandin-mediated inhibition, were determined for rates of platelet shape change (SC [Vs]), early platelet recruitment measured by electronic platelet counting (PA [PA3]), and turbidometrically-measured aggregation (TA [Va]). Studies were performed on stirred citrated platelet-rich plasma from 9 healthy human donors. The [ADP]1/2, [( ADP] giving half maximal rate) was determined for the sequence of activation steps: unactivated platelets----SC----PA----TA. Distinct ADP sensitivities were obtained from log dose-response studies, with a relative dose dependency for rates of change in the order of [ADP]1/2 TA greater than [ADP]1/2 PA greater than [ADP]1/2 SC of approximately 4:3:1. Differential inhibition of the above activation scheme was evaluated from log dose-response curves for Iloprost (ZK 36374), a stable carbacyclin analogue of prostacyclin (PGI2), with greater potency than PGI2 for the same platelet receptors. IC50 values corresponding to Iloprost concentrations causing 50% inhibition of rates of TA (Va), PA (PA3) and SC (Vs) were found in the relative ratios of 1: approximately 3: approximately 5, when measured at a common ADP concentration for all three parameters, or 1: approximately 2: approximately 3 when determined at respective [ADP]1/2 values for each parameter. Thus, about 3-5 times more Iloprost is required to respectively inhibit the rates of shape change (Vs) and early platelet recruitment (PA3), than that needed to inhibit the rate of turbidometrically-measured aggregation (Va).

Adenosine Diphosphate↗
PubMed1987

Unusual properties of platelet shape in coronary and cerebral artery disease.

Platelet shape was examined in freshly fixed whole blood, using phase contrast microscopy, from 7 patients post-myocardial infarction, 9 patients undergoing elective angiography for the investigation of angina, and 17 patients with stroke. Platelet shape was abnormal in 29/33 patients and in 20 of them the distribution of platelet shape did not resemble that produced by known platelet activators, by venipuncture techniques known to produce platelet activation, nor by shearing platelet-rich plasma. In particular, the proportion of spherical platelets is too high relative to the proportion of smooth discoid shaped platelets. Addition of platelet-poor plasma from a patient post-myocardial infarction to normal platelet-rich plasma did not reproduce the abnormality in platelet shape. In contrast, for patients post-myocardial infarction platelet shape was normal in platelet-rich plasma, but the platelets were hypersensitive to ADP-induced aggregation. Incubation of citrated whole blood at 37 degrees C resulted in an incomplete disappearance of the spherical platelets suggesting that the abnormal distribution of platelet shape consists of both a reversible and a more persistent abnormality. The reversible portion of the platelet shape abnormality seen in patients with stroke and coronary artery disease may be related to venipuncture-induced platelet activation; however, the nature of the persistent abnormality in platelet shape is presently unknown.

Angina Pectoris↗