Biomedical subjects

M M Frojmovic

Publications and source records attributed to M M Frojmovic.

At least 19 recordsLinked to original sources

PubMed2001

Platelets in suspension require preactivation to adhere to immobilized fibrinogen.

Previous studies using whole blood perfusion through flow chambers have suggested that unactivated platelets can adhere to surface immobilized fibrinogen (Fg). However, the red blood cells needed for surface delivery of the platelets may activate platelets by released adenosine diphosphate (ADP). Our studies of coaggregation of unactivated or ADP-activated platelets with Fg-coated latex beads in flowing suspensions show that only preactivated platelets can adhere to Fg-coated surfaces.

Blood Platelets↗

PubMed2001

Complementary roles for fibrin(ogen), thrombospondin and vWF in mediating shear-dependent aggregation of platelets stimulated at threshold thrombin concentrations.

We have evaluated the relative contribution of the adhesive ligands, von Willebrand factor (vWF), fibrinogen (Fg) and thrombospondin (TSP), all surface-expressed on washed platelets (WP) activated with a threshold thrombin concentration (approximately 0.04 U/ml), to platelet microaggregation (PA) at shear rates (G) from 300-2000 s(-1). In suspensions of thrombin-activated WP sheared immediately (tau0), all three ligands were required for optimal aggregation at all G, as shown by a 50-70% inhibition of capture efficiencies of PA (measured from initial rates of PA), by antibodies (Abs) directed against each protein. This aggregation involved both GPIb and GPIIbIIIa, as indicated by approximately 80% and 100% inhibition by Ab 6D1 and Ab 10E5, respectively. For WP preexposed to thrombin for 10 min to ensure maximal surface expression of secreted ligands and activated GPIIbIIIa (tau0), vWF was predominantly required at all G (63-75% inhibition by anti-vWF Ab), together with TSP (35-50% inhibition by anti-TSP Ab). Under these conditions, Fg was extensively converted to fibrin, so that fibrin, rather than Fg, could participate in microaggregation, with GPIb less required than GPIIbIIIa as indicated by a 30-60% inhibition by Ab 6D1 as compared to 100% inhibition by Ab 10E5. Our results show that interactions between multiple ligands and receptors favour microaggregation depending on shear and thrombin activation conditions.

Antibodies, Monoclonal↗

PubMed2000

A model of platelet aggregation involving multiple interactions of thrombospondin-1, fibrinogen, and GPIIbIIIa receptor.

Thrombospondin-1 (TSP) may, after secretion from platelet alpha granules, participate in platelet aggregation, but its mode of action is poorly understood. We evaluated the capacity of TSP to form inter-platelet cross-bridges through its interaction with fibrinogen (Fg), using either Fg-coated beads or Fg bound to the activated GPIIbIIIa integrin (GPIIbIIIa*) immobilized on beads or on activated fixed platelets (AFP), i.e. in a system free of platelet signaling and secretion mechanisms. Aggregation at physiological shear rates (100-2000 s(-1)) was studied in a microcouette device and monitored by flow cytometry. Soluble TSP bound to and induced aggregation of Fg-coated beads dose-dependently, which could be blocked by the amino-terminal heparin-binding domain of TSP, TSP18. Soluble TSP did not bind to GPIIbIIIa*-coated beads or AFP, unless they were preincubated with Fg. The interaction of soluble TSP with Fg-GPIIbIIIa*-coated beads or Fg-AFP resulted in the formation of aggregates via Fg-TSP-Fg cross-bridges, as demonstrated in a system where direct cross-bridges mediated by GPIIbIIIa*-Fg on one particle and free GPIIbIIIa* on a second particle were blocked by the RGD mimetic Ro 44-9883. Soluble TSP increased the efficiency of Fg-mediated aggregation of AFP by 30-110% over all shear rates and GPIIbIIIa* occupancies evaluated. Surprisingly, TSP binding to Fg already bound to its GPIIbIIIa* receptor appears to block the ability of this occupied Fg to recognize another GPIIbIIIa* receptor, but this TSP can indeed cross-bridge to another Fg molecule on a second platelet. Finally, TSP-coated beads could directly coaggregate at shear rates from 100 to 2000 s(-1). Our studies provide a model for the contribution of secreted TSP in reinforcing inter-platelet interactions in flowing blood, through direct Fg-TSP-Fg and TSP-TSP cross-bridges.

Acetates↗

PubMed2000

Time and force dependence of the rupture of glycoprotein IIb-IIIa-fibrinogen bonds between latex spheres.

We studied the shear-induced breakup of doublets of aldehyde/sulfate (A/S) latex spheres covalently linked with purified platelet GPIIb-IIIa receptor, and cross-linked by fibrinogen. Flow cytometry with fluorescein isothiocyanate-fibrinogen showed than an average of 22,500 molecules of active GPIIb-IIIa were captured per sphere, with a mean K(d) = 56 nM for fibrinogen binding. The spheres, suspended in buffered 19% Ficoll 400 containing 120 or 240 pM fibrinogen, were subjected to Couette flow in a counter-rotating cone-plate rheoscope. Doublets, formed by two-body collisions at low shear rate (G = 8 s(-1)) for < or =15 min, were subjected to shear stress from 0.6 to 2.9 Nm(-2), their rotations recorded until they broke up or were lost to view. Although breakup was time dependent, occurring mostly in the first 2 rotations after the onset of shear, the percentage of doublets broken up after 10 rotations were almost independent of normal hydrodynamic force, F(n): at 240 pN, 15.6, 16.0, and 17.0% broke up in the force range 70-150 pN, 150-230 pN, and 230-310 pN. Unexpectedly, at both [fibrinogen], the initial rate of breakup was highest in the lowest force range, and computer simulation using a stochastic model of breakup was unable to simulate the time course of breakup. When pre-sheared at low G for >15 min, no doublets broke up within 10 rotations at 70 < F(n) < 310 pN; it required >3 min shear (>1110 rotations) at F(n) = 210 pN for significant breakup to occur. Other published work has shown that binding of fibrinogen to GPIIb-IIIa immobilized on plane surfaces exhibits an initial fast reversible process with relative low affinity succeeded by transformation of GPIIb-IIIa to a stable high-affinity complex. We postulate that most doublet breakups observed within 10 rotations were from a population of young doublets having low numbers of bonds, by dissociation of the initial receptor complex relatively unresponsive to force. The remaining, older doublets with GPIIb-IIIa in the high-affinity complex were not broken up in the time or range of forces studied.

Binding Sites↗

PubMed2000

Efficiency of platelet adhesion to fibrinogen depends on both cell activation and flow.

The kinetics of adhesion of platelets to fibrinogen (Fg) immobilized on polystyrene latex beads (Fg-beads) was determined in suspensions undergoing Couette flow at well-defined homogeneous shear rates. The efficiency of platelet adhesion to Fg-beads was compared for ADP-activated versus "resting" platelets. The effects of the shear rate (100-2000 s(-1)), Fg density on the beads (24-2882 Fg/microm(2)), the concentration of ADP used to activate the platelets, and the presence of soluble fibrinogen were assessed. "Resting" platelets did not specifically adhere to Fg-beads at levels detectable with our methodology. The apparent efficiency of platelet adhesion to Fg-beads readily correlated with the proportion of platelets "quantally" activated by doses of ADP, i.e., only ADP-activated platelets appeared to adhere to Fg-beads, with a maximal adhesion efficiency of 6-10% at shear rates of 100-300 s(-1), decreasing with increasing shear rates up to 2000 s(-1). The adhesion efficiency was found to decrease by only threefold when decreasing the density of Fg at the surface of the beads by 100-fold, with only moderate decreases in the presence of physiologic concentrations of soluble Fg. These adhesive interactions were also compared using activated GPIIbIIIa-coated beads. Our studies provide novel model particles for studying platelet adhesion relevant to hemostasis and thrombosis, and show how the state of activation of the platelet and the local flow conditions regulate Fg-dependent adhesion.

Adenosine Diphosphate↗

PubMed2000

Glycoprotein IIb-IIIa-liposomes bind fibrinogen but do not undergo fibrinogen-mediated aggregation.

Although platelet cross-bridging is mediated primarily by the binding of fibrinogen to its activated membrane receptor, glycoprotein (GP) IIb-IIIa*, such an interaction may not be sufficient to support the aggregation process. As this question could potentially be answered by reconstituting GPIIb-IIIa* into a non-platelet environment such as liposomes, a protocol was developed for the generation of large lipid vesicles containing purified GPIIb-IIIa*. Flow cytometric techniques confirmed that the receptor was present in the lipid bilayer and were used to evaluate the characteristics of fibrinogen binding to the liposomes, which like fibrinogen-platelet interactions exhibited specificity, saturability, time dependence and calcium dependence. No fibrinogen-specific aggregation of GPIIb-IIIa* liposomes with stir or shear was observed, as determined by flow cytometric cell counting and microscopic examination of particles. In contrast, activated platelets rapidly bound Fg and rapidly formed large aggregates. The Fg associated with GPIIb-IIIa* in liposomes was 'normally' recognized by two fluorescently labelled antibodies: 4A5, which interacts with the Fg gamma chain C-terminus (residues 400-411) required for Fg-mediated cross-bridging of activated platelets in platelet aggregation (Shiba E, Lindon JN, Kushner L, Matsueda GR, Hawiger J, Kloczewiak M, Kudryk B, Salzman EW. Antibody-detectable changes in fibrinogen adsorption affecting platelet activation on polymer surfaces. Am J Physiol 1991; 260: C965-74), and anti-Fg-RIBS-I, which associates with an epitope on Fg (residues 373-385) expressed upon binding to GPIIb-IIIa. These data suggest that the Fg gamma-terminus is sterically accessible for particle cross-bridging and that an identical conformational change occurs for receptor-bound Fg on both liposomes and platelets. It thus appears that cellular elements aside from GPIIb-IIIa, such as cytoskeletal proteins proposed to be necessary for receptor 'anchoring', play a necessary role in flow-associated platelet aggregation.

Acetates↗

PubMed1999

ATP does not affect fibrinogen binding to platelet GPIIbIIIa in systems free of signal transduction.

Recent studies have suggested that the platelet fibrinogen (Fg) receptor, platelet membrane glycoprotein IIbIIIa (GPIIbIIIa, or integrin alpha(IIb)beta(3)) is also an adenosine triphosphate (ATP) binding site, and that the binding of ATP can directly inhibit the Fg-binding function of GPIIbIIIa. However, any direct effect of ATP on GPIIbIIIa function in intact fresh platelets is difficult to distinguish from indirect inhibitory effects via competition with ADP or elevation of platelet cyclic AMP levels. We therefore studied effects of ATP on Fg binding to activated GPIIbIIIa on the following model particles: externally activated and fixed platelets, as well as latex particles and liposomes containing functionally competent activated GPIIbIIIa receptors for Fg. These particles have 'normal', activated GPIIbIIIa in terms of: (1) binding affinity, (2) specificity to Fg, and (3) conformational change(s) after Fg binding. These particles neither require nor respond to further activation in order to bind Fg. With these model particles, we showed that ATP does not have any direct effect on the binding of Fg to platelet GPIIbIIIa and platelet aggregation. These simplified model particles are useful tools in the mechanistic study of platelet GPIIbIIIa function and the interaction between platelet GPIIbIIIa and its ligands.

Journal Article↗

PubMed1999

Thrombin receptor occupancy modulates aggregation efficiency and platelet surface expression of vWF and thrombospondin at low thrombin concentrations.

Previous studies evaluating requirements for occupancy of thrombin receptors in normal platelet secretion and aggregation, using the thrombin antagonists hirudin and PPACK (D-Phe-Pro-Arg-chloromethylketone), have suggested that at low thrombin activating concentrations (0.025-0.13 U/ml), occupancy was required only in the first 45-60 s following activation. In our study, we differentiate between thrombin receptor occupancy requirements for surface expression of secreted adhesive proteins, for activation of GPIIb-IIIa receptors, and for aggregation of washed platelets (WP) in laminar shear flow. Platelets activated with 0.05 U/ml thrombin for 10 min to allow maximal secretion (hereafter referred to as "pre-activated platelets"), then sheared, showed a 50-70% decrease in platelet counts after 60 s of shear. Treatment of pre-activated platelets with hirudin or PPACK produced a 65% reduction of capture efficiencies, alphaG (reflecting experimental/theoretical initial rates of aggregation), as well as a 30-40% decrease in the surface expression of von Willebrand factor (vWF) and thrombospondin (TSP). However, alpha-granule membrane P-selectin expression and numbers of activated GPIIb-IIIa receptors were comparable for treated and non-treated platelets. No significant difference in any of the parameters tested was observed when platelets were similarly pre-activated with 0.2 U/ml thrombin, due to treatment with thrombin antagonists. Binding of soluble FITC-vWF (GRGDSP-sensitive) to pre-activated, thrombin antagonist treated platelets, was greatly reduced (> or =80%). Soluble Fg was shown to bind to antagonist-treated pre-activated platelets, but could not significantly enhance platelet aggregation. Although occupancy of thrombin receptors by catalytically active thrombin is required transiently for secretion and activation of platelets, there is a further requirement for thrombin occupancy at low thrombin concentrations, for optimizing initial rates of platelet aggregation, surface expression of vWF and TSP, and activated GPIIb-IIIa ligand recognition.

Blood Platelets↗

PubMed1998

The fibrinogen RIBS-I epitope (gamma373-385) appears proximate to the gamma408-411 adhesive domain but is not involved in interaction between receptor-bound or surface-adsorbed fibrinogen and platelet GPIIbIIIa.

The carboxyl terminus of the fibrinogen (Fg) gamma chain (gamma400-411) is necessary and sufficient to support platelet aggregation and adhesion. However, a monoclonal antibody (mAb) to the Fg RIBS-I epitope (gamma373-385), the anti-Fg-RIBS-I, which binds only to platelet-bound or surface-adsorbed Fg but not soluble Fg, inhibits platelet aggregation. In this study, we showed that this same antibody also inhibits the adhesion of platelets to Fg-coated polystyrene beads. We then investigated the mechanisms by which the anti-Fg-RIBS-I antibody inhibits platelet aggregation and adhesion. The Fg RIBS-I epitope does not interact with platelet GPIIbIIIa, since recombinant Fg missing the last four amino acids, the Ala-Gly-Asp-Val, on the carboxyl terminus of its gamma chains supports neither platelet aggregation nor adhesion to surfaces, nor GPIIbIIIa binding, while it binds anti-Fg-RIBS-I normally. Purified, soluble GPIIbIIIa (265 kDa) inhibits the binding of both the anti-Fg-RIBS-I and 4A5 (a mAb specific to gamma408-411 of Fg), however, peptide G13 (1.5 kDa), corresponding to the Fg gamma chain binding domain on GPIIba (GPIIb300-312), only inhibits the binding of 4A5, and does not affect the binding of the anti-Fg-RIBS-I to Fg. The anti-Fg-RIBS-I reduces the on-rate of the 4A5 binding to Fg with no measurable changes in the dissociation of the Fg-bound 4A5. These data indicate that the inhibition of platelet aggregation and adhesion by the anti-Fg-RIBS-I antibody is due to the steric hindrance of the Fg gamma400-411 to platelet GPIIbIIIa. Thus the Fg RIBS-I epitope (gamma373-385) does not appear to be involved in direct interaction with platelet GPIIbIIIa, leaving the gamma408-411 of Fg as the sole domain mediating platelet aggregation and adhesion.

Antibodies, Monoclonal↗

PubMed1998

Role of the gamma chain Ala-Gly-Asp-Val and Aalpha chain Arg-Gly-Asp-Ser sites of fibrinogen in coaggregation of platelets and fibrinogen-coated beads.

Fibrinogen (Fg) mediates platelet aggregation and adhesion to artificial surfaces. The carboxyl terminus of the gamma chain of Fg (residues AGDV at gamma408-411) is known to play an exclusive role in platelet aggregation, while there is no known role for the consensus RGD sites in the Aalpha chain. In this study, we used flow cytometry to measure the coaggregation (CA) of platelets with Fg-coated beads, and investigated which domains in surface-immobilized Fg support platelet adhesion. CA of platelets with Fg-beads was nearly abolished in the presence of 4A5, a monoclonal antibody (mAb) whose epitope includes AGDV, while Z69/8, a mAb that also binds to the gamma chain carboxyl terminus but does not cover AGDV, had little effect. When beads were coated with recombinant Fg (rFg) lacking AGDV, CA was similarly abolished. In contrast, beads coated with Fg that lacked the RGDS site, supported platelet CA as did intact Fg. These results were confirmed in experiments that measured the binding of activated soluble glycoprotein IIb and IIIa (GPIIbIIIa), the platelet membrane glycoprotein complex known to be the Fg receptor, to immobilized Fg. This binding was inhibited by mAb 4A5, but not by mAb Z69/8. Binding was totally retained when beads were coated with Fg lacking RGDS, but was completely lost when beads were coated with Fg lacking AGDV. These results demonstrated that the AGDV sequence on the carboxyl terminus of the gamma chain of Fg plays an exclusive role in platelet adhesion to surface-immobilized Fg, while the carboxyl terminus of the Aalpha chain, including a consensus RGD site, is not required.

Antibodies, Monoclonal↗

PubMed1998

Platelet aggregation in flow: differential roles for adhesive receptors and ligands.

This article addresses the flow-dependent differential roles of the platelet receptors, glycoprotein (GP) GPIb and GPIIb-IIIa, in platelet aggregation mediated by ristocetin and soluble von Willebrand factor (vWF), by adenosine diphosphate (ADP) and soluble fibrinogen (Fg), and by thrombin and ADP in absence of exogenous ligands. Platelet-rich plasma or "activated" washed platelets were sheared in a coaxial cylinder at 100 to 1000 sec(-1) or in tubular flow, with surface ligands monitored by flow cytometry, with fluorescently labeled soluble ligands or monoclonal antibodies against specific adhesive domains on receptors or ligands. Aggregation was quantitated by monitoring the change in particle concentration with time by particle counting, and expressed as Capture efficiencies (CE) = Experimental/calculated initial rates of aggregation. The contributions of adhesive domains on putative ligands or receptors mediating aggregation at any given flow condition were evaluated with monoclonal antibodies or peptides known to block these adhesive sites. Surprisingly, ristocetin, which "chemically activates" GPIb/vWF to mediate spontaneous binding of the ligand to its receptor, at low concentrations yielding <2000 platelet-bound vWF monomers, gave efficient aggregation even at 1000 sec(-1) (CE = 0.34 +/- 0.02, n = 11) with only GPIb required. The physiologic activators ADP and thrombin both supported efficient aggregation of washed platelets with no exogenous ligands at 1000 sec(-1) by surface-secreted vWF (CE = 0.08 +/- 0.01, n = 6), in contrast to poorer ADP and soluble Fg-mediated aggregation in the absence of secretion (CE = 0.05). The secreted, platelet-bound, vWF-mediated aggregation completely depends on GPIIb-IIIa but partially and increasingly requires GPIb with increasing shear. Molecular models for these interactions are presented in terms of "rolling" and "firm" capture. Flow conditions will be critically important in designing and selecting anti-thrombotic drugs directed against the appropriate adhesive domains on receptors and ligands, which likely include other members such as thrombospondin and P-selectin.

Animals↗

PubMed1998

Ristocetin- and thrombin-induced platelet aggregation at physiological shear rates: differential roles for GPIb and GPIIb-IIIa receptor.

We recently reported that washed platelets (WP) activated with ADP and expressing surface-bound vWF aggregated in flow through small tubes or in a cylindrical couette device at physiological shear rates of G = 300 s(-1)-1000 s(-1) in the absence of exogenous ligands, with GPIb-vWF partially, and activated GPIIb-IIIa totally required for the aggregation. We have now extended these studies to aggregation of platelets "activated" with ristocetin or thrombin. Washed platelet suspensions with added soluble vWF and ristocetin (0.3-0.75 mg/ml), or activated with thrombin (0.01-0.5 U/ml) but no added ligand, were sheared in a coaxial cylinder device at uniform shear rate, G = 1000 s(-1). The collision capture efficiency (alphaG) with which small aggregates form (= experimental/calculated initial rates of aggregation) was correlated with vWF platelet binding assessed by flow cytometry. The vWF-GPIb interaction was exclusively able to support ristocetin-mediated shear aggregation of metabolically active platelets, with very few vWF monomer equivalents bound per platelet (representing < or = 10 molecules of 10 million Da) required to yield high capture efficiencies (alphaG = 0.38+/-.02; n = 11), suggesting rapid and stable bond formations between vWF and GPIb. However, platelet surface-expressed vWF, generated by addition of thrombin to washed platelets, was found to mediate platelet aggregation with alphaG = 0.08+/-.01 (n = 6), surprisingly comparable to that previously reported for WP and ADP activation. Blocking the GPIIb-Illa receptor decreased alphaG by 95+/-3% (n =3), while a monoclonal antibody to the vWF site on GPIb caused a 49+/-7% (n = 8) decrease in alphaG. The partial role for GPIb thus appears to reflect a facilitative function for increasing contact time between flowing platelets, and allowing engagement of the GPIIb-IIa receptor to yield stable attachment.

Anti-Bacterial Agents↗

PubMed1997

Surface-secreted von Willebrand factor mediates aggregation of ADP-activated platelets at moderate shear stress: facilitated by GPIb but controlled by GPIIb-IIIa.

We previously showed that ADP activation of washed human platelets in plasma-free suspensions supports aggregation at moderate shear stress (0.4-1.6 Nm-2) in Poiseuille flow. Although most activated platelets expressed maximal fibrinogen-occupied GPIIb-IIIa receptors, aggregation appeared to be independent of bound fibrinogen, but blocked by the hexapeptide GRGDSP. Here, we tested the hypothesis that von Willebrand factor (vWF) secreted and expressed on activated platelets mediates aggregation at moderate shear rates from 300 to 1000 s-1 corresponding to shear stresses from 0.3 to 1.1 Nm-2. Relatively unactivated platelets (< 15% expressing prebound fibrinogen) were prepared from acidified citrated platelet rich plasma (cPRP) by single centrifugation with 50 nM stable prostacyclin derivative ZK 36374 and resuspended in Tyrodes-albumin at 5 x 10(4) cells microliter-1. Flow cytometric measurements with monoclonal antibody (mAb) 2.2.9 reporting on surface-bound vWF, and with mAb S12 reporting on alpha-granule secreted P-selectin, showed that 65% and 80%, respectively, of all platelets were maximally activated with respect to maximal secretion and surface expression of these proteins. "Resting" washed platelets exhibited both surface-bound vWF and significant P-selectin secretion. We showed that mAbs 6D1 and NMC4, respectively blocking the adhesive domains on the GPIb receptor recognizing vWF, and on the vWF molecule recognizing the GPIb receptor, partially inhibited ADP-induced aggregation under shear in Couette flow, the degree of inhibition increasing with increasing shear stress. In contrast, mAb 10E5, blocking the vWF binding domain on GPIIb-IIIa, essentially blocked all aggregation at the shear rates tested. We conclude that vWF, expressed on ADP-activated platelets, is at least the predominant cross-bridging molecule mediating aggregation at moderate shear stress. There is an absolute requirement for free activated GPIIb-IIIa receptors, postulated to interact with platelet-secreted, surface bound vWF. The GPIb-vWF cross-bridging reaction plays a facilitative role becoming increasingly important with increasing shear stress. Since aurin tricarboxylic acid, which blocks the GPIb binding domain on vWF, was also found to completely block aggregation in Poiseuille flow, we conclude that it too affects the GPIIb-IIIa interaction.

Adenosine Diphosphate↗

PubMed1996

Optimally functional fluorescein isothiocyanate-labelled fibrinogen for quantitative studies of binding to activated platelets and platelet aggregation.

Dynamic and quantitative studies of the binding of fibrinogen (Fg) to its receptor, GPIIb-IIIa, on activated platelets, leading to platelet aggregation, are best studied with fluorescently-labelled Fg by flow cytometry. Due to conflicting reports on the functionality of FITC-labelled Fg, we have developed a reproducible and 'mild' labelling of fibrinogen with FITC-celite at pH 7.4-8.5 for direct and dynamic studies of specific Fg binding to activated platelets evaluated for native platelet-rich plasma, for washed platelets, and for activated, fixed platelets. We have demonstrated the equivalence of FITC-labelled and unlabelled Fg for binding to activated GPIIb-IIIa receptors, and in the rate and extent of mediating platelet aggregation. We found that FITC-Fg labelled at pH > or = 9 had reduced to absent specific binding to activated platelets, whether using soluble FITC or FITC-celite. The FITC-labelled Fg must be diluted 3-fold with unlabelled Fg when evaluating maximal Fg binding to activated platelets in order to prevent autoquenching of the FITC-Fg which leads to underestimation of Fg levels. The dissociation constant (KD) of Fg on stable preparations of activated, fixed platelets, determined with FITC-Fg binding to platelets by flow cytometry, was in the range reported for 125I-labelled Fg, 70-255 nm with Bmax = 10000-25000 Fg per platelet (n = 20). The FITC-Fg was used to monitor Fg binding to activated platelets directly by plasma, as well as to evaluate platelet subpopulations which maximally bind Fg according to the concentration of ADP used as activator. It is expected that this 'mildly' labelled FITC-Fg will stimulate further studies of platelet activation directly in native anticoagulated blood/plasma, for both basic and clinical research.

Blood Platelets↗

PubMed1996

Platelets from bleeding Simmental cattle have a long delay in both ADP-activated expression of GpIIB-IIIA receptors and fibrinogen-dependent platelet aggregation.

We have previously reported that platelets from bleeding Simmental cattle do not aggregate in vitro in response to ADP, collagen and calcium ionophore A23187, though calcium mobilization and myosin light chain phosphorylation do occur. The aggregation abnormality, measured by aggregometry, was ascribed to abnormal cytoskeletal expression, with the maximal numbers of activated GpIIb-IIIa receptors per platelet no different from that seen in normal bovine platelets activated with ADP. We have therefore sought to compare the kinetics of micro-aggregation with the rate of expression of GpIIb-IIIa receptors required for mediating fibrinogen (Fg)-dependent platelet aggregation, to provide a more direct molecular explanation for the aggregation abnormality. We compared aggregation kinetics of ADP-activated platelets using both aggregometry and particle counting to monitor micro-aggregation. Fibrinogen receptor expression was monitored with FITC-labelled human Fg and with the reporting antibody for activated GpIIb-IIIa, FITC-PAC1, using flow cytometry. The affected platelets show a marked delay in onset of microaggregation for ADP-activated platelets stirred with human Fg, paralleded by an unusual delay in activated GpIIb-IIIa receptor expression (DARE) for otherwise competent Fg binding. The on-rates for Fg binding to platelets maximally pre-activated with PMA are identical for normal and affected platelets, whether comparing the binding of human or bovine Fg. The unique DARE syndrome explains the observed delay in aggregation of platelets from affected Simmental cattle and predicts the bleeding problems due to delayed binding of adhesive proteins.

Adenosine Diphosphate↗

PubMed1995

p32, a platelet autoantigen recognized by an SLE-derived autoantibody that inhibits platelet aggregation.

We have previously described an SLE-derived human hybridoma autoantibody, 9604, which binds to activated but not to resting platelets, inhibits platelet aggregation, and immunoprecipitates a surface-labelled poly-peptide of 32,000 molecular weight (MW) (p32). In the present study, using a murine monoclonal anti-p32 antibody (8E8), we show that p32 is responsible for the binding of 9604 to activated platelets. First, 8E8 bound to the surface of activated but not resting platelets, as detected by flow cytometry, showing saturation with 1900 antibody molecules bound/platelet and a Kd of 72 nM. Second, complete inhibition of the binding of 9604 to ADP-activated platelets by 8E8 and inhibition of the reactivity of 8E8 with p32 by 9604 demonstrated that 8E8 and 9604 recognize the same 32,000 MW platelet polypeptide. Isolation of platelet proteins, using an 8E8-affinity column, showed high MW bands corresponding to multimers of p32 under non-reducing conditions, and isolated p32 tended to self-associate even under reducing conditions. High performance liquid chromatography (HPLC) demonstrated that the native p32-containing protein has an approximate MW of 450,000. A comparison of the functional properties of murine monoclonal 8E8 and human monoclonal 9604 revealed that both antibodies are cytotoxic to platelets in 51Cr-release assays. In contrast, 8E8, unlike 9604, did not affect platelet aggregation, suggesting that different epitopes may be recognized by the two antibodies. These findings demonstrate that p32 is a subunit of an activation marker that is expressed on the surface of activated platelets and recognized by an SLE-derived anti-platelet autoantibody. Our data also indicate that murine monoclonal antibodies produced against platelet autoantigens may not mimic the functional activities of spontaneously occurring autoantibodies, probably due to the recognition of different epitopes.

Animals↗

PubMed1995

Inhibition of platelet aggregation by an SLE-derived human hybridoma autoantibody against an activation-dependent antigen.

Anti-platelet autoantibodies may be responsible for hematological complications in patients with systemic lupus erythematosus (SLE), but the mechanisms by which these antibodies cause abnormal hemostasis remain unknown. In the present study, using fluorescence activated cell sorter (FACS) analysis, we demonstrate that an SLE-derived human hybridoma autoantibody, 9604, recognizes a surface antigen expressed on platelets activated by ADP, calcium ionophore A23187, or phorbol myristate acetate (PMA), showing saturation with approximately 2,000 antibody molecules bound per platelet and a Kd of 41 nM. The binding of 9604 to activated platelets was significantly inhibited by EDTA, indicating partial dependence on divalent cations. It did not appear to be dependent on platelet secretion, nor did it directly affect alpha-granule or dense granule secretion. The protein antigen responsible for the binding of 9604 to activated platelets was characterized by Western blot and immunoprecipitation and shown to have a native molecular weight (M. W.) of greater than 400,000, with a 32,000 M. W. subunit (p 32). Antibody 9604 had little or no effect on the shape change and the initial rate of primary aggregation of normal platelets. In contrast, 9604 inhibited secondary aggregation of stirred platelet suspensions (IC50 < or = 1 nM) following activation by ADP, thromboxane A2 mimetic U46619, or calcium ionophore A23187, but not PMA or thrombin. The inhibition of large platelet aggregate formation (secondary aggregation), with a major shift to smaller microaggregates and singlets, was confirmed by direct particle count and sizing studies. The functional inhibition of platelet aggregation by an SLE-derived human hybridoma autoantibody in vitro suggests one potential mechanism that may play a role in the hemostatic disorders found in SLE.

Adenosine Diphosphate↗

PubMed1994

Dynamics of platelet glycoprotein IIb-IIIa receptor expression and fibrinogen binding. I. Quantal activation of platelet subpopulations varies with adenosine diphosphate concentration.

We have previously reported that maximal platelet activation with adenosine diphosphate (100 microM ADP) causes rapid expression of all GPIIb-IIIa receptors for fibrinogen (FgR) (< 1-3 s), measured with FITC-labeled PAC1 by flow cytometry. We have extended these studies to examine the effects of ADP concentration on the graded expression and Fg occupancy of GPIIb-IIIa receptors. Human citrated platelet-rich plasma, diluted 10-fold with Walsh-albumin-Mg+2 (2 mM), was treated with ADP (0.1-100 microM). The rates of GPIIb-IIIa receptor expression or Fg binding were measured in unstirred samples by flow cytometry, using FITC-labeled monoclonal antibodies (mAb) PAC1 and 9F9, respectively, from on-rates, using increasing times between mAb and ADP additions. Fibrinogen receptors were all expressed rapidly at low (1 microM) or high (100 microM) ADP (few seconds), whereas Fg occupancy was 50% of maximal by about 2 min. The maximal extent of GPIIb-IIIa receptor expression and Fg occupancy was determined from maximal binding (Flmax) at 30 min incubation with PAC1 or 9F9. On-rates and maximal extents of binding for either PAC1 or 9F9 probes showed identical [ADP]-response profiles ("KD" approximately 1.4 +/- 0.1 microM). However, Flmax studies showed bimodal histograms consisting of "resting" (Po) and maximally "activated" (P*) platelets for both PAC1 and 9F9 binding, with the fraction of "activated" platelets increasing with ADP concentration. The data best fit a model where platelet subpopulations are "quantally" transformed from Po to P*, expressing all GPIIb-IIIa receptors, rapidly filled by Fg, but "triggered" at critical ADP concentrations. Larger, but not the largest, platelets appear to be the most sensitive subpopulation. The implications for clinical studies are discussed, and the relationship to dynamics of aggregation are described in a companion paper.

Adenosine Diphosphate↗