Cytoskeletal changes in platelets induced by thrombin and phorbol myristate acetate (PMA).

Changes in shape, and aggregation that accompanies platelet activation, are dependent on the assembly and reorganization of the cytoskeleton. To assess the changes in cytoskeleton induced by thrombin and PMA, suspensions of aspirin-treated,32P-prelabeled, washed pig platelets in Hepes buffer containing ADP scavengers were activated with thrombin, and with PMA, an activator of protein kinase C. The cytoskeletal fraction was prepared by adding Triton extraction buffer. The Triton-insoluble (cytoskeletal) fraction isolated by centrifugation was analysed by SDS-PAGE and autoradiography. Incorporation of actin into the Triton-insoluble fraction was used to quantify the formation of F-actin. Thrombin-stimulated platelet cytoskeletal composition was different from PMA-stimulated cytoskeletal composition. Thrombin-stimulated platelets contained not only the three major proteins: actin (43 kDa), myosin (200 kDa) and an actin-binding protein (250 kDa), but three additional proteins of Mr56 kDa, 80 kDa and 85 kDa in the cytoskeleton, which were induced in by thrombin dose-response relationship. In contrast, PMA-stimulated platelets only induced actin assembly, and the 56 kDa, 80 kDa and 85 kDa proteins were not found in the cytoskeletal fraction. Exposure of platelets to thrombin or PMA induced phosphorylation of pleckstrin parallel to actin assembly. Staurosporine, an inhibitor of protein kinase C, inhibited actin assembly and platelet aggregation induced by thrombin or PMA, but did not inhibit the incorporation of 56 kDa, 80 kDa and 85 kDa into the cytoskeletal fraction induced by thrombin. These three extra proteins seem to be unrelated to the induction of protein kinase C. We conclude that actin polymerization and platelet aggregation were induced by a mechanism dependent on protein kinase C, and suggest that thrombin-activated platelets aggregation could involve additional cytoskeletal components (56 kDa, 80 kDa, 85 kDa) of the cytoskeleton, which made stronger actin polymerization and platelet aggregation more.     

Association of vinculin to the platelet cytoskeleton during thrombin-induced aggregation

Vinculin is a protein generally believed to be involved in membrane-cytoskeleton interaction, and its presence in platelets has been verified earlier. Here we show that in resting bovine platelets, vinculin is not associated with the Triton-insoluble cytoskeletal fraction but becomes incorporated into it during the thrombin-induced activation process. The incorporation starts around the same time as the release reaction and only after the shape change and the first phase of aggregation have taken place. Its time course parallels the cytoskeletal association of actin and certain other contractile proteins. Vinculin is a minor component of platelet cytoskeleton and only about 10% of the total platelet vinculin becomes incorporated into the Triton X-100 residue.     

The effect of glycoprotein IIb-IIIa receptor occupancy on the cytoskeleton of resting and activated platelets

The platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa), serves as the receptor for fibrinogen. This study examined what effect GPIIb-IIIa receptor occupancy had on the cytoskeleton of resting and activated platelets. Triton X-100-insoluble residues (cytoskeletons) were isolated from resting washed platelets incubated with either 500 microM RGDS or 500 microM RGES and examined for protein content. RGDS did not increase the amount of GPIIb-IIIa associated with the cytoskeletal residues which sedimented at either 15,800 x g or 100,000 x g. To determine the effect of receptor occupancy on the formation of the activated platelet cytoskeleton, stirred and nonstirred RGDS-treated platelets in plasma were activated with ADP. Triton X-100-insoluble residues were isolated and examined for both protein content and retention of GPIIb-IIIa. Further, morphological studies were performed on the RGDS-ADP-stimulated platelets. The results of this study suggest that 1) RGDS peptide receptor occupancy does not lead to GPIIb-IIIa linkage to the cytoskeleton, 2) ADP-stimulated platelet shape change, polymerization of actin, and association of myosin with the cytoskeleton are unaffected by RGDS peptide receptor occupancy. 3) RGDS inhibits an aggregation-dependent incorporation of ABP, alpha-actinin, talin, and GPIIb-IIIa into the Triton-insoluble residue.     

A conformation-dependent epitope of human platelet glycoprotein IIIa.

This study explores conformational states of human platelet glycoprotein IIIa (GP IIIa) and possible mechanisms of fibrinogen receptor exposure. D3GP3 is an IgG1, kappa monoclonal antibody generated against purified GP IIIa and found to be specific for GP IIIa by immunoprecipitation and Western blot analysis. The binding of D3GP3 to resting platelets caused fibrinogen binding (approximately 5,000 molecules/platelet) and platelet aggregation but not secretion. Platelets express 40,000-50,000 GP IIb-IIIa molecules in their surface membranes. However, resting platelets only bound approximately 5,000 D3GP3 molecules/platelet. D3GP3 binding to platelets could be increased 2-3-fold by dissociation of the GP IIb-IIIa complex with 5 mM EDTA or by occupying the fibrinogen receptor with either RGDS peptides or fibrinogen. Platelet stimulation with ADP in the absence of fibrinogen did not cause increased D3GP3 binding above control levels. These data suggest that 1) GP IIb-IIIa can exist in multiple conformations in the platelet membrane, 2) D3GP3 binding to GP IIIa can expose the fibrinogen receptor, 3) the binding of either RGDS peptides or fibrinogen causes exposure of the D3GP3 epitope, and 4) platelet activation in the absence of ligand does not induce the same conformational changes in GP IIb-IIIa as does receptor occupancy by RGDS peptides or fibrinogen.     

Identification of two structurally and functionally distinct sites on human platelet membrane glycoprotein IIb-IIIa using monoclonal antibodies

Platelet membrane glycoprotein IIb-IIIa exists as a calcium-dependent complex of two large peptides (designated IIb and IIIa) in Triton X-100 solutions, but it remains unknown if these peptides are subunits of one glycoprotein or are actually two individual glycoproteins in the intact platelet membrane. We used crossed immunoelectrophoresis to define the epitopes of two monoclonal antibodies to IIb-IIIa, then used these antibodies to study the structural and functional organization of IIb and IIIa in the platelet membrane. Human platelets solubilized in Triton X-100 were electrophoresed through an intermediate gel containing 125I-monoclonal IgG, then into an upper gel containing rabbit anti-human platelet antibodies. Our previously characterized antibody. Tab, and a new monoclonal antibody, T10, both bound to the immunoprecipitate corresponding to the IIb-IIIa complex. When platelets were electrophoresed after solubilization in 5 mM EDTA, 125I-Tab bound to the dissociated IIb polypeptide, but not to IIIa. In contrast, 125-I-T10 did not react with either IIb or IIIa. Thus, Tab recognizes a determinant on IIb, while T10 recognizes a determinant created only after the association of IIb and IIIa. Gel-filtered platelets from six normal donors bound 50,600 +/- 5,600 125I-T10 molecules/platelet and 47,800 +/- 11,200 125I-Tab molecules/platelet, consistent with IIb-IIIa being a heterodimer. 125I-T10 binding was identical in unactivated platelets and platelets stimulated with 10 microM ADP. However, platelets did not aggregate or bind 125I-fibrinogen until ADP was added. T10, but not Tab or nonimmune mouse antibody, inhibited ADP-induced platelet aggregation and 125I-fibrinogen binding. Our findings suggest that IIb and IIIa exist as subunits of a single membrane glycoprotein in unstimulated platelets. Fibrinogen binding appears to require not only the interaction of IIb and IIIa, but also some additional change occurring after platelet activation.     

Variations of glycoprotein IIb–IIIa (αIIb/β3-integrin) content in healthy donors. The influence on platelet aggregation activity and efficacy of aspirin action

The effects of content of a fibrinogen receptor, glycoprotein (GP) IIb–IIIa (αIIb/β3-integrin), GP IIIa genetic polymorphism (substitution Leu33Pro), and fibrinogen concentration in blood plasma on platelet aggregation activity have been investigated in a group of healthy volunteers. In 35 examined donors the GP IIb–IIIa content on platelet surface varied from 40 to 71 × 10³ per platelet. Repeated measurements revealed that the GP IIb–IIIa content coefficient of variation was 9.5%, and deviations from mean levels did not exceed 20%. The level and the rate of platelet aggregation induced by ADP (1.25–20 μM) correlated with GP IIb–IIIa number (r from 0.315 to 0.591) and were higher in the group of donors with high in comparison with low GP IIb–IIIa content (>60 and (40–50) × 10^?3 per platelet, respectively). Aspirin, the inhibitor of thromboxane A₂ synthesis, partially suppressed ADP-induced platelet aggregation. The level of residual aggregation in the presence of aspirin also correlated with GP IIb–IIIa content and increased in subjects with high receptor content. Parameters of ADP-induced aggregation did not differ in donors with genotypes GP IIIa Pro33(?) (Leu33Leu33, n = 20) and Pro33(+) (Leu33Pro33, n = 13, and Pro33Pro33, n = 2) genotype. GP IIb–IIIa content was also not affected by GP IIIa polymorphism. No significant correlations were found between the level and rate of platelet aggregation and fibrinogen concentration in blood plasma. The data obtained indicate that the effects of variations of GP IIb–IIIa content on platelet aggregation are higher than GP IIIa Leu33Pro polymorphism and variations of fibrinogen concentration. High GP IIb–IIIa content is associated with increased platelet aggregation activity and decreased efficacy of aggregation inhibition by aspirin.     

Inhibition of ADP-induced platelet activation by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole: Covalent modification of aggregin,a putative ADP receptor

ADP-induced platelet responses play an important role in the maintenance of hemostasis. There has been disagreement concerning the identity of an ADP receptor on the platelet surface. The chemical structure of 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) shows considerable resemblance to that of the adenine moiety of adenine-based nucleotides. The reagent has been previously used by other investigators as an affinity label for adenine nucleotide-requiring enzymes, such as mitochondrial ATPase and the catalytic subunit of cAMP-dependent protein kinase. Since ADP-induced platelet responses depend on the binding of ADP to its receptor, we investigated the effect on ADP-induced platelet responses and the nature of ADP-binding protein modified by NBD-Cl. NBD-Cl inhibited ADP-induced shape change and aggregation of platelets in platelet-rich plasma in a concentration- and time-dependent manner. NBD-Cl also inhibited ADP-induced shape change, aggregation, exposure of fibrinogen binding sites, secretion, and calcium mobilization in washed platelets. NBD-Cl did not act as an agonist for platelet shape change and aggregation. Covalent modification of platelets by NBD-Cl blocked the ability of ADP to antagonize the increase in intracellular levels of cAMP mediated by iloprost (a stable analogue of prostaglandin I₂). NBD-Cl was quite specific in inhibiting platelet aggregation by those agonists, e.g., ADP, collagen, and U44619 (a thromboxane mimetic), that completely or partially depend on the binding of ADP to its receptor. Autoradiogram of the gel obtained by SDS-PAGE of solubilized platelets modified by [¹⁴C]-NBD-Cl showed the presence of a predominant radiolabeled protein band at 100 kDa corresponding to aggregin, a putative ADP receptor. The intensity of this band was considerably decreased when platelets were either preincubated with ADP and ATP or covalently modified by a sulfhydryl group modifying reagent before modification by [¹⁴C]-NBD-Cl. These results (1) indicate that covalent modification of aggregin by NBD-Cl contributed to loss of the ADP-induced platelet responses, and (2) suggest that there is a sulfhydryl group in the ADP-binding domain of aggregin. © 1996 Wiley-Liss, Inc.     

Differences between platelet and microparticle glycoprotein IIb/IIIa.

Glycoprotein (GP) IIb and IIIa are major constituents of the platelet membrane which are involved in forming the fibrinogen receptor on activated platelets. We used flow cytometry to study the effects of ethylene-diamine tetraacetic acid (EDTA) on the membrane GPIIb/IIIa complexes of platelets and microparticles, and to study the effects of cations on dissociated GP complexes. Microparticles were detected by both the volume signal and by fluorescence using an FITC-conjugated anti-GPIb antibody (NNKY5-5). When platelets were stimulated with ADP, calcium ionophore A23187, or thrombin, fibrinogen binding to the platelet surface increased markedly. However, fibrinogen binding to microparticles showed little increase in response to such agonists. Microparticle GPIIb/IIIa complexes were dissociated by incubation with EDTA at 37 degrees C but did not reassociate after treatment with divalent cations (Ca2+, Mg2+, and Mn2+) in contrast to platelet GPIIb/IIIa complexes. These results suggest that some interaction of GPIIb/IIIa and linked structures like the platelet cytoskeleton may be involved in the reassociation of dissociated GPIIb and GPIIIa, perhaps explaining the failure of reassociation of microparticle GPIIb/IIIa (i.e., the fibrinogen binding to microparticles).     

Affinity labeling of a human platelet membrane protein with 5'-p-fluorosulfonylbenzoyl adenosine. Concomitant inhibition of ADP-induced platelet aggregation and fibrinogen receptor exposure

Incubation of washed human blood platelets with 5'-p-fluorosulfonylbenzoyl [3H]adenosine (FSBA) covalently labels a single polypeptide of Mr = 100,000. Protection by ADP has suggested that an ADP receptor on the platelet surface membrane was modified. The modified cells, unlike native platelets, failed to aggregate in response to ADP (100 microM) and fibrinogen (1 mg/ml). The extent of binding of 125I-fibrinogen and aggregation was inhibited to a degree related to the incorporation of 5'-p-sulfonylbenzoyl adenosine (SBA) into platelets, indicating FSBA could inhibit the exposure of fibrinogen receptors by ADP necessary for aggregation. Incubation of SBA platelets with alpha-chymotrypsin cleaved the covalently labeled polypeptide and concomitantly reversed the inhibition of aggregation and fibrinogen binding. Platelets proteolytically digested by chymotrypsin prior to exposure to FSBA did not require ADP for aggregation and fibrinogen binding. Moreover, subsequent exposure to FSBA did not inhibit aggregation or fibrinogen binding. The affinity reagent FSBA can displace fibrinogen bound to platelets in the presence of ADP, as well as promote the rapid disaggregation of the platelets. The apparent initial pseudo-first order rate constant of dissociation of fibrinogen was linearly proportional to FSBA concentrations. These studies suggest that a single polypeptide can be altered either by ADP-induced conformational changes or proteolysis by chymotrypsin to reveal latent fibrinogen receptors and promote aggregation of platelets after fibrinogen binding.     

N-Ethylmaleimide induces disaggregation of platelets preaggregated by ADP and thrombin and decreases cytoplasmic calcium level

The effect of N-ethylmaleimide (NEM, 1-200 microM) on ADP- and thrombin-induced platelet aggregation and thrombin-induced increase in intracellular Ca2+ concentration was studied. Addition of NEM to platelets preaggregated with ADP or thrombin induces platelet disaggregation. The anti-aggregant activity of NEM was different for ADP- and thrombin-induced aggregations. At 200 microM concentration, NEM completely disaggregated ADP-induced aggregates and only partially disaggregated thrombin-aggregated platelets. NEM did not influence the thrombin-induced increase in cytoplasmic Ca2+ and had no effect on the basal level of Ca2+ in the cytosol of non-activated platelets. However, NEM decreased the level of thrombin-mobilized Ca2+ in the cytosol of activated platelets. Thus, NEM can induce disaggregation of ADP- and thrombin-preaggregated platelets by activating a system which removes Ca2+ from the platelet cytosol.     

Comparison of in vitro platelet aggregation and its inhibition by three antithrombotic drugs between human and guinea pig

Platelets of guinea pigs are frequently used to evaluate the effect of new antiplatelet agents. Although several studies have compared the platelet aggregation between humans and guinea pigs, but so far the information is still limited. In this study, we compare the inhibitory effect of aspirin, dipyridamole and pentoxifylline on the platelet aggregation induced by adenosine diphosphate (ADP), collagen, arachidonic acid and thrombin between humans and guinea pigs. The results for humans and guinea pigs were compared and analysed by two-way analysis of variance (ANOVA). Our results showed: 1. The trends wherein these three drugs suppressed collagen-induced platelet aggregation was very similar in humans and guinea pigs. 2. In ADP-induced aggregation, the trend of inhibition caused by the three drugs was also similar in humans and guinea pigs except that a difference in platelet disaggregation at a late phase of platelet aggregation was noted. 3. In arachidonic acid- and thrombin-induced aggregations, the trend of inhibition caused by the three drugs was somewhat different in humans and guinea pigs. 4. Considering all activators as a whole, it was found that the status of platelet disaggregation at the late phase of platelet aggregation was different in humans and guinea pigs. Therefore, we concluded that: 1. Collagen was the most appropriate platelet activator when we used platelets of guinea pigs to study the effect of new antiplatelet agents. 2. When platelets of guinea pigs were used to study platelet aggregation, no matter which activator was used, we should avoid using the late phase of aggregation as the control index for comparison, because the results thus obtained might not be applicable to human platelets.     

Proteolytic degradation of vimentin and glial fibrillary acidic protein in rat astrocytes in primary culture

The receptor for ADP on the platelet membrane, which triggers exposure of fibrinogen-binding sites and platelet aggregation, has not yet been identified. Two enzymes with which ADP interacts on the platelet surface, an ecto-ATPase and nucleosidediphosphate kinase, have been proposed as possible receptors for ADP in ADP-induced platelet aggregation. In the present study, experiments were conducted with washed human platelets to examine if a relationship existed between platelet aggregation, fibrinogen binding and the enzymatic degradation of ADP. With 12 different platelet suspensions, a good correlation (P less than 0.01) was found between the extent of platelet aggregation and the amount of 125I-fibrinogen bound to platelets after ADP stimulation. No correlation was found between these parameters and the rate or extent of transformation of [14C]ADP to [14C]ATP or [14C]AMP. The binding of fibrinogen to platelets was inhibited in parallel with aggregation when ADP stimulation was impaired by the enzymatic degradation of ADP by the system creatine phosphate/creatine phosphokinase, or by the use of specific antagonists, such as ATP and AMP. These antagonists also influenced the enzymatic degradation of ADP. This effect occurred at lower concentrations of ATP or AMP than those required to inhibit ADP-induced platelet aggregation and fibrinogen binding. Our results demonstrate that ATP and AMP may be used as specific antagonists of the ADP-induced fibrinogen binding to platelets. They do not provide evidence to suggest that enzymes which metabolize ADP on the platelet surface are involved in the mechanism of ADP-induced platelet aggregation.     

Characterization of ATP-diphosphohydrolase activities in the intima and media of the bovine aorta: evidence for a regulatory role in platelet activation in vitro.

The inner layer of the aorta contains the enzyme ATP diphosphohydrolase (ATPDase: EC 3.6.1.5) which catalyzes the sequential phosphorolysis of ATP----ADP----AMP. Two zones of the inner layer, the intima and media, were separated and both were shown to contain ATPDase activity of similar specific activity (0.08 and 0.10 U/mg protein, respectively). However, the media exhibited about 100-times more enzyme activity than the intima. Both preparations were virtually identical with respect to pH optima (7.5), migration patterns after electrophoresis under non-denaturing conditions, relative rates of ATP and ADP hydrolysis and potency to inhibit ADP-induced platelet aggregation in both human platelet-rich plasma and whole blood. The IC50 values for ADP (2 microM)-induced aggregation were 6.8 and 12.9 mU/ml in platelet-rich plasma and whole blood, respectively. Addition of ATPDase to platelets pre-aggregated with ADP resulted in a dose-dependent disaggregation in platelet-rich plasma (IC50 4.9 mU/ml), but not in whole blood. When both ATPDase (5.6-58.7 mU/ml) and ATP (0.5-10 microM) were added to platelet-rich plasma, there was an immediate dose-dependent aggregation of platelets followed by a slowly developing disaggregation. These data show that ATPDase is present in both the intima and media layers of bovine aorta and suggest a dual role for this enzyme in platelet activation. By converting ATP released from damaged cells into ADP, the enzyme could facilitate platelet aggregation at the site of vascular injury, whereas the subsequent conversion of ADP to AMP could inhibit or reverse platelet aggregation. The consequence of these activities would be to control the growth of a platelet thrombus.     

Purification and characterization of a new RGD/KGD-containing dimeric disintegrin, piscivostatin, from the venom of Agkistrodon piscivorus piscivorus: the unique effect of piscivostatin on platelet aggregation

Piscivostatin, a novel dimeric disintegrin containing Arg-Gly-Asp (RGD) and Lys-Gly-Asp (KGD) sequences, was isolated from the venom of Agkistrodon piscivorus piscivorus. The molecule consisted of two chains designated as the alpha and beta chains, comprising 65 and 68 amino acid residues, respectively. Piscivostatin had two binding motifs recognized by platelet glycoprotein IIb/IIIa (GPIIb/IIIa), and the biological activity of dimeric disintegrin piscivostatin toward platelet aggregation differed from those of other monomeric disintegrins such as trimestatin and echistatin. We measured platelet aggregation by the laser light scattering method during the process of ADP-induced platelet aggregation. Both dimeric and monomeric disintegrins inhibited the formation of small (9 to 25 microm in diameter), medium-sized and large aggregates (25 to 70 microm in diameter) in a dose-dependent manner. The platelet aggregates disaggregated after reaching a maximal number on either treatment with ADP alone or monomeric disintegrin/ADP. However, the small aggregates did not disaggregate on treatment with piscivostatin/ADP even when applied over time. When washed platelets were incubated with an anti-GPIIb/IIIa monoclonal antibody, PT25-2, which induces conformational changes of GPIIb/IIIa to a form accessible to fibrinogen and other adhesion proteins without platelet activation, piscivostatin induced a platelet shape change alone with no aggregate formation. The present study indicated that piscivostatin has two unique contradictory activities; acting as a double inhibitor of platelet aggregation and platelet aggregate dissociation.     

Preparation of monoclonal antibodies against glycoprotein IIIa of human platelets. Their effect on platelet aggregation

Monoclonal antibodies against purified glycoprotein IIIa (GPIIIa) of human platelet membranes have been obtained. These antibodies, except one, are able to bind to intact platelets; the exception is M108/p98 antibody which recognizes a new epitope, unmasked after proteolysis of GPIIIa in vitro. Several antigenic areas can be delineated on the molecule, by testing the ability of different antibodies to compete in their simultaneous binding to GPIIIa. One of the monoclonal antibodies inhibits ADP-induced platelet aggregation while others do not have an effect or induce agglutination of platelets independent of ADP. Conventional antiserum raised against purified GPIIIa also blocks the aggregation induced by ADP. These results favour the hypothesis that GPIIIa plays a direct role in the mechanism of platelet aggregation.     

Glycoprotein Ib in the triton-insoluble (cytoskeletal) fraction of blood platelets

Glycoprotein Ib could be demonstrated in the Triton-insoluble (cytoskeletal) fraction of platelets prepared with EGTA by SDS-polyacrylamide gel electrophoresis and staining with the periodic acid Schiff's reagent. Crossed immunoelectrophoresis showed that glycoprotein Ib could be extracted from such Triton-insoluble residues when the extraction solution contained 1% Triton X-100 plus 5 mM CaCl₂, but not if it also contained leupeptin. This indicates that glycoprotein Ib was associated to structures in the cytoskeletal fraction in such a way that it could be extracted only after activation of a calcium-dependent protease, and degradation of the actin-binding protein was demonstrated. After crossed immunoelectrophoresis of platelet extracts prepared in the presence of leupeptin or EDTA, a glycoprotein Ib-related, rocket-shaped immunoprecipitate was seen originating from the application well. This was interpreted as being related to glycoprotein Ib associated to actin polymers which did not sediment at low-speed centrifugation. Incubation of platelets with ³²P as sodium phosphate led to incorporation of phosphatase-sensitive ³²P in all of the glycoprotein Ib-related immunoprecipitates except for that of glycocalicin. This support the idea that glycoprotein Ib traverses the plasma membrane and can be phosphorylated at the inner surface whereas glycocalicin represents the terminal part of the glycoprotein Ibα-chain exposed at the outer surface.     

Activation of bovine platelets induced by long-chain unsaturated fatty acids at just below their lytic concentrations,and its mechanism

The effects of long chain unsaturated fatty acids such as linoleic acid on bovine platelets were examined. Not only linoleic acid, but also oleic and linolenic acid, at just below the concentrations causing marked cell lysis, induced an absorbance decrease of the platelet suspension in the presence of Ca²⁺. Since this absorbance decrease was reversed by the addition of EDTA and moreover aggregate formation was found by macroscopic and microscopic observation, it was concluded that unsaturated fatty acids at just below their lytic concentrations caused platelet aggregation. Unsaturated fatty acids also caused release of adenine nucleotides, but there was a lag time between the release and the aggregation, just as with ADP-induced release, suggesting that the aggregation was independent of the release of ADP. It was revealed that this activation of platelets by unsaturated fatty acids was caused by marked Ca²⁺ uptake into the cytoplasm, resulting from significant membrane perturbation.     

Aggregation of platelets by muscle actin. A multivalent interaction model of platelet aggregation by ADP

Filamentous muscle actin (F-actin) aggregated blood platelets while G-actin was ineffective. This aggregation could be blocked by ATP suggesting a possible role of actin-bound ADP in this process. Actin-bound ADP caused platelet aggregation at concentrations significantly lower than equivalent concentrations of free ADP. Thus, actin potentiates the aggregating action of ADP. An actin antibody or DNase I inhibited this aggregation showing the requirement of actin in this process. Like other physiological agents, Ca⁺⁺ was necessary for platelet aggregation by actin. Platelets fixed in formaldehyde were not aggregated by actin showing the need for viable platelets. Since F-actin contains 1 mole of bound ADP/mole protein, it is postulated that actin potentiates ADP-induced aggregation by providing multiple interaction sites for platelets.     

Comparison of Aggregometry with Flow Cytometry for the Assessment of Agonists′-Induced Platelet Reactivity in Patients on Dual Antiplatelet Therapy

Data on the agreement between aggregometry and platelet activation by flow cytometry regarding the measurement of on-treatment platelet reactivity to arachidonic acid (AA) and adenosine diphosphate (ADP) are scarce. We therefore sought to compare three platelet aggregation tests with flow cytometry for the assessment of the response to antiplatelet therapy. Platelet aggregation in response to AA and ADP was determined by light transmission aggregometry (LTA), the VerifyNow assays, and multiple electrode aggregometry (MEA) in 316 patients receiving aspirin and clopidogrel therapy after angioplasty with stent implantation. AA- and ADP-induced P-selectin expression and activated glycoprotein (GP) IIb/IIIa were determined by flow cytometry. LTA, the VerifyNow P2Y₁₂ assay and MEA in response to ADP correlated significantly (all p<0.001), and the best correlation was observed between LTA and the VerifyNow P2Y₁₂ assay (r = 0.63). ADP-induced platelet reactivity by all aggregation tests correlated significantly with ADP-induced P-selectin expression and activated GPIIb/IIIa (all p<0.001). The best correlation was seen between the VerifyNow P2Y₁₂ assay and activated GPIIb/IIIa (r = 0.68). The platelet surface expressions of P-selectin and activated GPIIb/IIIa in response to ADP were significantly higher in patients with high on-treatment residual platelet reactivity (HRPR) to ADP by all test systems (all p<0.001). A rather poor correlation was observed between AA-induced platelet reactivity by LTA and the VerifyNow aspirin assay (r = 0.15, p = 0.007), while both methods did not correlate with MEA. AA-induced platelet reactivity by all aggregation tests correlated significantly, but rather poorly with AA-induced P-selectin expression (all p<0.05), while only AA-induced platelet reactivity by LTA correlated significantly with AA-induced activated GPIIb/IIIa (r = 0.21, p<0.001). The platelet surface expression of P-selectin in response to AA was significantly higher in patients with HRPR by LTA AA and MEA AA (both p<0.02). In contrast, P-selectin expression in response to AA was similar in patients without and with HRPR by the VerifyNow aspirin assay (p = 0.5), and platelet surface activated GPIIb/IIIa in response to AA did not differ significantly between patients without and with HRPR to AA by all test systems (all p>0.1). In conclusion, ADP-induced platelet reactivity by aggregometry translates partly into flow cytometry. In contrast, AA-induced platelet reactivity correlates poorly between different platelet aggregation tests, and between aggregometry and flow cytometry. Overall, both approaches capture different aspects of platelet function and are therefore not interchangeable in the assessment of agonists´-induced platelet reactivity. Clinical outcome data are needed to determine which test systems and settings are associated with different in vivo consequences.     

Disaggregation of aggregated platelets by apyrase from the tick, Ornithodoros savignyi

Apyrase, secreted by ticks during feeding, is a platelet aggregation inhibitor that functions as a regulator of the host's hemostatic system. This present study concerns the disaggregation effect of salivary gland apyrase from the tick Ornithodoros savignyi. Secondarily aggregated platelets, disaggregated by apyrase, exhibited a reversal of shape from a spherical (aggregated) form to a discoid form, reminiscent of reversible aggregation at low ADP concentrations in citrated platelet-rich plasma. However, they showed a dilatory open canaliculary system and an absence of granules indicating disaggregation after degranulation had taken place. In contrast, disaggregation by the fibrin(ogen)olytic enzyme, plasmin, showed that platelets degranulated, but retained a spherical form with numerous extended pseudopods. While thrombin had no effect on aggregation or clotting of platelets disaggregated with plasmin, it did activate those platelets disaggregated with apyrase and clotted the plasma. This is the first study to describe the disaggregating effects of tick derived apyrase on aggregated platelets. It also shows that apyrase can disaggregate platelets even after secondary aggregation and degranulation of platelets has taken place. Platelet aggregation is one of the main barriers encountered by ticks during feeding and counteraction of this process by ticks is an important factor for successful feeding.