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.     

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.     

Antioxidants with carcinostatic activity (resveratrol, vitamin E and selenium) in modulation of blood platelet adhesion.

Compounds with potential antiplatelet activity can be used in the therapy of cardiovascular disorders. We investigated the effects of three different antioxidants with carcinostatic property: trans-resveratrol, Trolox a water-soluble analog of vitamin E, and inorganic selenocompounds (sodium selenite and selenate) on blood platelet adhesion to fibrinogen (Fg). Adhesion, the initial step of platelet activation, was estimated by the colorimetric method with BCA (bicinchoninic acid) solution in 96-well Fg-coated microtiter dishes. It was shown that resveratrol significantly inhibited adhesion of both thrombin- and ADP-activated platelets to Fg. After incubation of platelets for 30 min. at 37 degrees C with resveratrol at the concentration of 100 microg/ml above 40% inhibition of adhesion was achieved. The inhibition of platelet adhesion of Fg caused by Trolox was lower than by resveratrol and at higher concentration (1 mM) reached maximum 12%. We also demonstrated that neither sodium selenite nor selenate significantly altered platelet adhesion to Fg. We conclude that changed adhesion of blood platelets to Fg in the presence of resveratrol and Trolox, but not selenium may be the result of different antioxidative activities of tested compounds.     

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.     

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.(ABSTRACT TRUNCATED AT 250 WORDS)     

Integrin alpha IIb beta 3-dependent calcium signals regulate platelet-fibrinogen interactions under flow. Involvement of phospholipase C gamma 2

Platelet adhesion to fibrinogen is important for platelet aggregation and thrombus growth. In this study we have examined the mechanisms regulating platelet adhesion on immobilized fibrinogen under static and shear conditions. We demonstrate that integrin alpha IIb beta 3 engagement of immobilized fibrinogen is sufficient to induce an oscillatory calcium response, necessary for lamellipodial formation and platelet spreading. Released ADP increases the proportion of platelets exhibiting a cytosolic calcium response but is not essential for calcium signaling or lamellipodial extension. Pretreating platelets with the Src kinase inhibitor PP2, the inositol 1,4,5-trisphosphate (IP3) receptor antagonist 2-aminoethoxydiphenyl borate (APB-2), or the phospholipase C (PLC) inhibitor U73122 abolished calcium signaling and platelet spreading, suggesting a major role for Src kinase-regulated PLC isoforms in these processes. Analysis of PLC gamma 2-/- mouse platelets revealed a major role for this isoform in regulating cytosolic calcium flux and platelet spreading on fibrinogen. Under flow conditions, platelets derived from PLC gamma 2-/- mice formed less stable adhesive interactions with fibrinogen, particularly in the presence of ADP antagonists. Our studies define an important role for PLC gamma 2 in integrin alpha IIb beta 3-dependent calcium flux, necessary for stable platelet adhesion and spreading on fibrinogen. Furthermore, they establish an important cooperative signaling role for PLC gamma 2 and ADP in regulating platelet adhesion efficiency on fibrinogen.     

Blood platelet surface interactions on fibrinogen under flow as viewed with fluorescent video-microscopy

The interaction of fluorescently labeled blood platelets with fibrinogen-coated glass was studied in Poiseuille flow at 3 wall shear rates, 40, 80 and 944 s-1. Observations were made via video-microscopy at a distance of 0.5 cm from a tube's entrance over a 1370 microns 2 portion of luminal area. The rates of arrival and detachment, and the net rate of adhesion of cells increased nonlinearly with flow rate. The fraction of arriving cells, first contacts, which adhered without subsequent movement and the fraction of arriving cells which adhered, moved to new positions and then remained adherent, were maximal at 80 s-1. For platelets which adhere and then move to a number of new positions, the likelihood of permanent adhesion is greater than 85 percent. The adhesion process is one in which 40-60 percent of cells permanently adhere on first contact with an additional 30 percent adhering after several moves along the surface. Cells contacting where a platelet was previously adherent had a greater chance of adhering than they would on an unaltered fibrinogen surface. The efficiency of platelet adhesion is greater for second contacts than for first contacts on unaltered fibrinogen coated surface.     

Fluid shear regulates the kinetics and receptor specificity of Staphylococcus aureus binding to activated platelets

The interaction between surface components on the invading pathogen and host cells such as platelets plays a key role in the regulation of endovascular infections. However, the mechanisms mediating Staphylococcus aureus binding to platelets under shear remain largely unknown. This study was designed to investigate the kinetics and molecular requirements of platelet-S. aureus interactions in bulk suspensions subjected to a uniform shear field. Hydrodynamic shear-induced collisions augment platelet-S. aureus binding, which is further potentiated by platelet activation with stromal derived factor-1beta. Peak adhesion efficiency occurs at low shear (100 s(-1)) and decreases with increasing shear. The molecular interaction of platelet alpha(IIb)beta(3) with bacterial clumping factor A through fibrinogen bridging is necessary for stable bacterial binding to activated platelets under shear. Although this pathway is sufficient at low shear (相似文献   

Selective recognition of adhesive sites in surface-bound fibrinogen by glycoprotein IIb-IIIa on nonactivated platelets

We demonstrate that unstimulated platelets attach to immobilized fibrinogen in a selective process mediated by the membrane glycoprotein (GP) complex IIb-IIIa (alpha IIb beta 3). The initial attachment, independent of platelet activation, is followed by spreading and irreversible adhesion even in the presence of activation inhibitors. Using fibrinogen fragments derived from plasmin digestion, we found that unstimulated platelets do not attach to immobilized fragment E, which contains an Arg-Gly-Asp sequence at A alpha 95-97, and adhere to fragments X and D, both containing the gamma 400-411 dodecapeptide adhesion sequence, less efficiently than to intact fibrinogen. Thus, the carboxyl terminus of the A alpha chain, missing in the "early" fragment X used in these studies, appears to be involved in the interaction of fibrinogen with unstimulated platelets. In contrast, activated platelets adhere to immobilized fibrinogen and fragments X, D, and E in a time-dependent and equivalent manner. Although activated platelets adhere to immobilized vitronectin, fibronectin, and von Willebrand factor through GP IIb-IIIa, unstimulated platelets fail to adhere to vitronectin and have only a limited capacity to adhere to fibronectin and von Willebrand factor. These results demonstrate that GP IIb-IIIa on unstimulated platelets displays a recognition specificity for attachment to immobilized adhesive proteins that is distinct from that seen following platelet activation. Thus, unstimulated platelets selectively interact with fibrinogen, and the initial attachment is followed by spreading and irreversible adhesion in the absence of exogenous agonists. This process may be regulated by plasmin cleavage of the fibrinogen A alpha chain and may play an important role during normal hemostasis and during the pathological development of thrombotic vascular occlusions.     

A peptide corresponding to GPIIb alpha 300-312, a presumptive fibrinogen gamma-chain binding site on the platelet integrin GPIIb/IIIa, inhibits the adhesion of platelets to at least four adhesive ligands.

The platelet fibrinogen (Fg) receptor (GPIIb/IIIa) is an integrin which plays a critical role in hemostasis by recognizing at least the four adhesive ligands: Fg, fibronectin (Fn), vitronectin (Vn), and von Willebrand factor (vWf). We reported that residues 309-312 of GPIIb alpha appear to comprise at least part of a Fg binding site on the Fg receptor (Gartner, T. K., and Taylor, D. B. (1990) Thromb. Res. 60, 291-309). Here we report that the peptide GPIIb alpha 300-312 (G13) inhibits platelet aggregation and binds Fg and Vn. Significantly, this peptide inhibits the adhesion of stimulated platelets to Fg, Fn, Vn, and vWf, but not the adhesion of resting platelets to Fn. Thus, GPIIb 300-312 may constitute a specific but common recognition site on GPIIb/IIIa for both LGGAKQAGDV- and RGD-containing ligands.     

Hydrodynamic effects and receptor interactions of platelets and their aggregates in linear shear flow.

We have modeled platelet aggregation in a linear shear flow by accounting for two body collision hydrodynamics, platelet activation and receptor biology. Considering platelets and their aggregates as unequal-sized spheres with DLVO interactions (psi(platelet) = -15 mV, Hamaker constant = 10(-19) J), detailed hydrodynamics provided the flow field around the colliding platelets. Trajectory calculations were performed to obtain the far upstream cross-sectional area and the particle flux through this area provided the collision frequency. Only a fraction of platelets brought together by a shearing fluid flow were held together if successfully bound by fibrinogen cross-bridging GPIIb/IIIa receptors on the platelet surfaces. This fraction was calculated by modeling receptor-mediated aggregation using the formalism of Bell (Bell, G. I. 1979. A theoretical model for adhesion between cells mediated by multivalent ligands. Cell Biophys. 1:133-147) where the forward rate of bond formation dictated aggregation during collision and was estimated from the diffusional limited rate of lateral association of receptors multiplied by an effectiveness factor, eta, to give an apparent rate. For a value of eta = 0.0178, we calculated the overall efficiency (including both receptor binding and hydrodynamics effects) for equal-sized platelets with 50,000 receptors/platelet to be 0.206 for G = 41.9 s(-1), 0.05 for G = 335 s(-1), and 0.0086 for G = 1920 s(-1), values which are in agreement with efficiencies determined from initial platelet singlet consumption rates in flow through a tube. From our analysis, we predict that bond formation proceeds at a rate of approximately 0.1925 bonds/microm2 per ms, which is approximately 50-fold slower than the diffusion limited rate of association. This value of eta is also consistent with a colloidal stability of unactivated platelets at low shear rates. Fibrinogen was calculated to mediate aggregation quite efficiently at low shear rates but not at high shear rates. Although secondary collisions (an orbitlike trajectory) form only a small fraction of the total number of collisions, they become important at high shear rates (>750 s(-1)), as these are the only collisions that provide enough time to result in successful aggregate formation mediated by fibrinogen. The overall method provides a hydrodynamic and receptor correction of the Smoluchowski collision kernel and gives a first estimate of eta for the fibrinogen-GPIIb/IIIa cross-bridging of platelets. We also predict that secondary collisions extend the shear rate range at which fibrinogen can mediate successful aggregation.     

Localization of fibrinogen during ADP- or thrombin-induced aggregation of washed rabbit platelets

In contrast to human platelets, which aggregate poorly in response to ADP unless fibrinogen is present in the external medium, washed rabbit platelets form large aggregates in response to ADP without fibrinogen in the suspending medium. Addition of fibrinogen to the suspending medium of rabbit platelets frequently has little or no effect on the extent of ADP-induced platelet aggregation. We examined washed rabbit platelets by immunocytochemistry during ADP-induced aggregation and deaggregation and during thrombin-induced aggregation when the external medium did not contain added fibrinogen to determine if (a) fibrinogen was expressed on the surface of rabbit platelets that could support aggregation when the platelets were stimulated, or (b) fibrinogen secreted from the alpha granules supports platelet aggregation. Glutaraldehyde-fixed samples were prepared at different times after addition of ADP or thrombin, embedded in Lowicryl K4M, sectioned, incubated with sheep anti-rabbit fibrinogen, washed, reacted with gold-labeled anti-sheep IgG, and prepared for electron microscopy. The alpha granules of rabbit platelets were heavily labeled with immunogold; the platelet membrane was not labeled. During platelet aggregation and deaggregation in response to ADP, fibrinogen was not detectable on the platelet surface. In response to thrombin, large aggregates formed before fibrinogen was secreted from the alpha granules; fibrinogen was detectable focally at sites of granule discharge by 30-60 sec and fibrin formed by 3 min. Therefore, stimulated washed rabbit platelets can adhere to each other without large amounts of fibrinogen taking part in the close platelet-to-platelet contact, since aggregation occurs before detectable secretion, and large areas where the platelets are in contact are devoid of fibrinogen between the adherent membranes. Adhesion mechanisms not involving fibrinogen may support the aggregation of washed rabbit platelets.     

Platelet deposition onto fibrin-coated surfaces under flow conditions

Platelet deposition on fibrin-coated surfaces and release from these adherent platelets were studied in an in vitro flow system. When a mixed suspension of washed platelets and red cells flowed through a fibrin-coated glass tube, only platelets were deposited onto the fibrin-coated surfaces. The density of adhered platelets increased with flow time and decreased with distance from the tube inlet. The adhesion rate increased with increasing shear rates from 45 s-1 to 180 s-1. This adhesion process appears to fit a diffusion-limited mathematical model. Comparing with glass and other protein-coated surfaces such as collagen, fibrinogen, or albumin coated surfaces, the number of adhered platelet per unit area decreased in the following descending order: collagen, fibrin, fibrinogen, glass, albumin. On the other hand, the degree of release reaction from these platelets decreased by another order: collagen, glass, fibrinogen, fibrin. We observed little release from platelets that were in contact with a fibrin-coated surface. Our results suggest that platelets specifically adhere to fibrin-coated surface and that this interaction does not induce platelet release.     

RhoA sustains integrin alpha IIbbeta 3 adhesion contacts under high shear

The small GTPase RhoA modulates the adhesive nature of many cell types; however, despite high levels of expression in platelets, there is currently limited evidence for an important role for this small GTPase in regulating platelet adhesion processes. In this study, we have examined the role of RhoA in regulating the adhesive function of the major platelet integrin, alpha(IIb)beta(3). Our studies demonstrate that activation of RhoA occurs as a general feature of platelet activation in response to soluble agonists (thrombin, ADP, collagen), immobilized matrices (von Willebrand factor (vWf), fibrinogen) and high shear stress. Blocking the ligand binding function of integrin alpha(IIb)beta(3), by pretreating platelets with c7E3 Fab, demonstrated the existence of integrin alpha(IIb)beta(3)-dependent and -independent mechanisms regulating RhoA activation. Inhibition of RhoA (C3 exoenzyme) or its downstream effector Rho kinase had no effect on integrin alpha(IIb)beta(3) activation induced by soluble agonists or adhesive substrates, however, both inhibitors reduced shear-dependent platelet adhesion on immobilized vWf and shear-induced platelet aggregation in suspension. Detailed analysis of the sequential adhesive steps required for stable platelet adhesion on a vWf matrix under shear conditions revealed that RhoA did not regulate platelet tethering to vWf or the initial formation of integrin alpha(IIb)beta(3) adhesion contacts but played a major role in sustaining stable platelet-matrix interactions. These studies define a critical role for RhoA in regulating the stability of integrin alpha(IIb)beta(3) adhesion contacts under conditions of high shear stress.     

Glycoprotein VI but not alpha2beta1 integrin is essential for platelet interaction with collagen

Platelet adhesion on and activation by components of the extracellular matrix are crucial to arrest post-traumatic bleeding, but can also harm tissue by occluding diseased vessels. Integrin alpha2beta1 is thought to be essential for platelet adhesion to subendothelial collagens, facilitating subsequent interactions with the activating platelet collagen receptor, glycoprotein VI (GPVI). Here we show that Cre/loxP-mediated loss of beta1 integrin on platelets has no significant effect on the bleeding time in mice. Aggregation of beta1-null platelets to native fibrillar collagen is delayed, but not reduced, whereas aggregation to enzymatically digested soluble collagen is abolished. Furthermore, beta1-null platelets adhere to fibrillar, but not soluble collagen under static as well as low (150 s(-1)) and high (1000 s(-1)) shear flow conditions, probably through binding of alphaIIbbeta3 to von Willebrand factor. On the other hand, we show that platelets lacking GPVI can not activate integrins and consequently fail to adhere to and aggregate on fibrillar as well as soluble collagen. These data show that GPVI plays the central role in platelet-collagen interactions by activating different adhesive receptors, including alpha2beta1 integrin, which strengthens adhesion without being essential.     

Fibrin but not adsorbed fibrinogen supports fibronectin assembly by spread platelets. Effects of the interaction of alphaIIb beta3 with the C terminus of the fibrinogen gamma-chain

We investigated the assembly of soluble fibronectin by lysophosphatidic acid-activated platelets adherent to fibrinogen or fibrin. More fibronectin was assembled by activated platelets spread on fibrin matrices than by platelets spread on adsorbed fibrinogen. The difference between platelets adherent to fibrinogen and fibrin occurred under both static and flow conditions. Similar differences were seen in binding of the 70-kDa N-terminal fragment of fibronectin that recognizes fibronectin assembly sites on adherent cells. Antibody and peptide blocking studies demonstrated that alphaIIb beta3 integrin mediates platelet adhesion to fibrinogen, whereas both alphav beta3 and alphaIIb beta3 mediate platelet adhesion to fibrin. The hypothesis that engagement of the C-terminal QAGDV sequence of the fibrinogen gamma-chain by alphaIIb beta3 inhibits the ability of the platelet to assemble fibronectin was tested by several experiments. Activated platelets adherent to adsorbed mutant fibrinogen lacking the QAGDV sequence (gammadelta5FG) were assembly-competent, as were platelets adherent to adsorbed normal fibrinogen that had been pretreated with the 7E9 antibody to the C terminus of the gamma-chain. Moreover, adsorbed normal fibrinogen but not gammadelta5FG suppressed the ability of co-adsorbed fibronectin to direct assembly of soluble fibronectin by spread platelets. The suppressive effect was lost when a surface of co-adsorbed fibronectin and fibrinogen was pretreated with 7E9. These results support a model in which the engagement of alphaIIb beta3 by the C-terminal sequence of the fibrinogen gamma-chain initiates signals that suppress subsequent fibronectin assembly by spread platelets. This interaction is less dominant when platelets adhere to fibrin, resulting in enhanced fibronectin assembly.     

Quantitative analysis of platelet alpha v beta 3 binding to osteopontin using laser tweezers

To determine whether platelet adhesion to surfaces coated with the matrix protein osteopontin requires an agonist-induced increase in the affinity of the integrin alpha v beta 3 for this ligand, we used laser tweezers to measure the rupture force between single alpha v beta 3 molecules on the platelet surface and osteopontin-coated beads. Virtually all platelets stimulated with 10 microM ADP bound strongly to osteopontin, producing rupture forces as great as 100 piconewtons (pN) with a peak at 45-50 pN. By contrast, 90% of unstimulated, resting non-reactive platelets bound weakly to osteopontin, with rupture forces rarely exceeding 30-35 pN. However, approximately 10% of unstimulated platelets, resting reactive platelets, exhibited rupture force distributions similar to stimulated platelets. Moreover, ADP stimulation resulted in a 12-fold increase in the probability of detecting rupture forces >30 pN compared with resting non-reactive platelets. Pre-incubating stimulated platelets with the inhibitory prostaglandin E1, a cyclic RGD peptide, the monoclonal antibody abciximab, or the alpha v beta 3-specific cyclic peptide XJ735 returned force histograms to those of non-reactive platelets. These experiments demonstrate that ADP stimulation increases the strength of the interaction between platelet alpha v beta 3 and osteopontin. Furthermore, they indicate that platelet adhesion to osteopontin-coated surfaces requires an agonist-induced exposure of alpha v beta 3-binding sites for this ligand.     

Immobilised echistatin promotes platelet adhesion and protein tyrosine phosphorylation

Echistatin, a 5000-Da disintegrin, is a strong competitive inhibitor of platelet alpha(IIb)beta(3) binding to fibrinogen. In addition to its antiplatelet activity, echistatin also exhibits activating properties by inducing a switch of alpha(IIb)beta(3) conformation towards an active state. However, soluble echistatin, which is a monomeric ligand, provides only receptor affinity modulation, but it is unable to activate integrin-dependent intracellular signals. Since proteins may exhibit a multivalent functionality as a result of their absorption to a substrate, in this study we evaluated whether immobilised echistatin is able to stimulate platelet adhesion and signalling. The immobilisation process led to an increase of echistatin affinity for integrin(s) expressed on resting platelets. Unlike the soluble form, immobilised echistatin bound at comparable extent either unstimulated or ADP-activated platelets. Furthermore, echistatin presented in this manner was effective in stimulating integrin-dependent protein tyrosine phosphorylation. Platelets adhering to immobilised echistatin showed a pattern of total tyrosine phosphorylated proteins resembling that of fibrinogen-attached platelets. In particular, solid-phase echistatin induced a strong phosphorylation of tyrosine kinases pp72(syk) and pp125(FAK). Inhibitors of platelet signalling, such as apyrase, prostaglandin E(1), cytochalasin D and bisindolylmaleimide, while not affecting platelet adhesion to immobilised echistatin, abolished pp125(FAK) phosphorylation. This suggests that signals activating protein kinase C function, dense granule secretion and cytoskeleton assembly might be involved in echistatin-induced pp125(FAK) phosphorylation.     

Inhibition of fibrinogen binding to human platelets by blockage of Na+/H+ exchange

Binding of ADP to platelets enhances the binding of fibrinogen to Gp IIb-IIIa, the specific platelet receptor for adhesive proteins. The linkage between ADP and fibrinogen binding is indirect since ADP does not bind to the same receptor as fibrinogen. We have recently proposed that a third component, once affected by ADP binding, induces a conformational transition of the fibrinogen receptor from the low to the high affinity state, which is responsible for platelet aggregation [De Cristofaro, R., Landolfi, R., Castagnola, M., De Candia, E., Di Cera, E., & Wyman, J. (1988) Proc. Natl. Acad. Sci. USA 85, 8473-8476]. In the present study we provide evidence that this component should be identified with the platelet Na+/H+ antiport. Inhibition of the antiport by pharmacological agents such as amiloride, or else by decreasing extracellular Na+, results in a marked decrease of fibrinogen binding to platelets.     

Pomolic acid, triterpenoid isolated from Licania pittieri, as competitive antagonist of ADP-induced aggregation of human platelets

Pomolic acid (PA), triterpenoid isolated from Licania pittieri, has previously shown a potent ability to inhibit adenosine diphosphate (ADP)- and epinephrine-induced human platelet aggregation. To investigate whether PA could be an antagonist of ADP-activated receptors of human platelets (P2Y(1) and P2Y(12)), pharmacological studies were conducted to examining its ability to modulate the platelet shape change induced by a selective P2Y(1) receptor agonist MRS2365 and also the nature of its possible interaction with ADP receptors by analyzing the characteristics of log concentration-response curves of ADP constructed in the absence and in the presence of fixed concentrations of PA, using in vitro platelet aggregation assays. PA did not interfere with the activation of P2Y(1) receptor by MRS2365 to induce platelet shape change and displayed a competitive antagonism of ADP-induced platelet aggregation, which most probably involves competition for a single binding site in platelets. The estimated equilibrium dissociate constant (K(b)) of PA as ADP receptor antagonist was 15.4±0.06nM. Together, these findings give indirect evidence for the idea that PA could be a potent competitive antagonist of P2Y(12) receptor, and open the possibility to consider it as new member of the non-nucleotide generation of antiplatelet drugs.