ABSTRACT
We monitored the intracellular distribution of ionized free Ca2+ concentration ([Ca2+]i) in individual human platelets by digital imaging fluorescence microscopy with fura 2 during platelet activation induced by surface contact or a soluble platelet agonist (thrombin). Contact of platelets with glass resulted in pseudopod formation and spreading, accompanied by a nonuniform rise in [Ca2+]i. The rise in [Ca2+]i was maximal during pseudopod formation. Locally elevated [Ca2+]i was frequently found in pseudopodia and at the edge and core of spread platelets. This pattern was faithfully duplicated by the local pattern of distribution of the cytoskeletal components F-actin, gelsolin, and surface glycoproteins (GP) IIb-IIIa but not by calmodulin. Platelets stimulated by thrombin also showed an inhomogeneous rise in [Ca2+]i, which was well correlated with the staining of F-actin and GPIIb-IIIa. Cytochalasin D, an inhibitor of actin polymerization, inhibited the inhomogeneous increase or redistribution of F-actin and GPIIb-IIIa but did not inhibit the rise in mean [Ca2+]i. These observations suggest that a localized change in [Ca2+]i may be associated with cytoskeletal reorganization and redistribution of GPIIb-IIIa in activated platelets.
Subject(s)
Actins/blood , Blood Platelets/metabolism , Calcium/blood , Platelet Activation , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Calmodulin/blood , Gelsolin/blood , Humans , Image Processing, Computer-Assisted , Microscopy, Fluorescence , Tissue DistributionABSTRACT
Dibucaine and 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), which are local anesthetics, affect diverse functions of many cell types. For example, platelet aggregation is inhibited by both, and both cause changes in platelet morphology and structure. Little is known of the mechanisms. We found that both dibucaine (0.125-0.5 mM) and TMB-8 (0.25-1.0 mM) induced rapid tyrosine phosphorylation of several platelet proteins (160, 70-75, and 40 kDa) and dephosphorylation of a 62- to 64-kDa protein detectable by a specific antiphosphotyrosine monoclonal antibody (4G10). Platelet aggregation induced by alpha-thrombin (10 nM) was inhibited by the local anesthetics in approximately the same dose range. Neither dibucaine nor TMB-8 induced activation of protein kinase C (PKC) or myosin light-chain kinase. Their activation was not essential for tyrosine phosphorylation induced by local anesthetics. However, an increase in tyrosine phosphorylation of several proteins (95-130 kDa) induced by alpha-thrombin (10 nM) was inhibited by dibucaine (0.5 mM) or TMB-8 (0.5 mM). Furthermore, when dibucaine (0.5 mM) was added 1 min after addition of alpha-thrombin (10 nM), disaggregation was paralleled to dephosphorylation of many proteins, including those mentioned. Tyrosine phosphorylation and dephosphorylation of specific proteins may account for some of the diverse effects of local anesthetics on platelets and other cells. Addition of TMB-8 (0.5 mM) or dibucaine (0.5 mM) also inhibited activation of PKC, induced by alpha-thrombin (10nM), suggesting that some of the inhibitory effects of dibucaine or TMB-8 may be due to inhibitory effects of local anesthetics on PKC.
Subject(s)
Blood Platelets/drug effects , Blood Platelets/metabolism , Blood Proteins/metabolism , Dibucaine/pharmacology , Gallic Acid/analogs & derivatives , Tyrosine/metabolism , Enzyme Activation/drug effects , Gallic Acid/pharmacology , Humans , Myosin-Light-Chain Kinase/metabolism , Phosphorylation/drug effects , Protein Kinase C/metabolismABSTRACT
In human platelets, a rapid rise in cytoplasmic Ca2+ and slower rise in cytoplasmic pH follow stimulation by thrombin. With the Ca2+ probe Fura-2 and the pH probe SNARF-1 for digitized fluorescence microscopy, we studied simultaneously the distribution and changes with time of [pH]i and [Ca2+]i in individual human platelets. In platelets coloaded with both probes, the probes had no detectable fluorescence at each other's excitation wavelength. The monovalent cation ionophore, nigericin (2 microM), produced a homogeneous rise in pH but no change in [Ca2+]. Platelets, in contact with glass, spread and developed an irregular, apparently mutually independent rise in both [Ca2+] and pH. Stimulation of platelets by thrombin 1.0 U/ml elevated [Ca2+]i and produced slow alkalinization without initial acidification. Replacement of extracellular Na+ by choline abolished thrombin-induced alkalinization, but had no effect on thrombin-induced [Ca2+]i elevation. ADP 10 microM caused a rapid rise of [Ca2+]i and transient alkalinization. Most stimulated platelets developed a gradient in pH, that was highest in the center. ADP and thrombin caused oscillation of [Ca2+]i but not of [pH]i. We conclude that alkalinization in stimulated platelets, presumably involving Na+/H+ antiport, is not essential for the rise of [Ca2+]i that may accompany it.
Subject(s)
Blood Platelets/metabolism , Calcium/metabolism , Platelet Activation , Adenosine Diphosphate/pharmacology , Benzopyrans , Blood Platelets/drug effects , Cations , Cytosol/metabolism , Extracellular Matrix/chemistry , Extracellular Matrix/metabolism , Fluorescent Dyes , Fura-2 , Humans , Hydrogen-Ion Concentration , Naphthols , Nigericin/pharmacology , Rhodamines , Sodium/metabolism , Spectrometry, Fluorescence/methods , Thrombin/pharmacologyABSTRACT
The possible role of calpains in protein-tyrosine phosphorylation in platelets was examined by the use of the cell-permeant calpain inhibitor calpeptin. In platelets stimulated by 1 U/mL thrombin, protein-tyrosine phosphorylation was maximal after 2 minutes and was followed by protein-tyrosine dephosphorylation. Calpeptin (30 mumol/L) or vanadate (2 mmol/L) enhanced protein-tyrosine phosphorylation and delayed protein-tyrosine dephosphorylation. The effects of these two compounds were not additive. We also observed proteolysis of pp60src and autoproteolysis of mu-calpain. Cleavage of the former was significantly slower than that of the latter and slower than protein-tyrosine dephosphorylation. The activity of protein-tyrosine phosphatase in the platelet lysate was transiently increased to 190% by addition of Ca2+. Ca(2+)-dependent activation of protein-tyrosine phosphatase was not observed in the presence of leupeptin. Those observations suggest that platelet calpains may be involved in modulation of protein-tyrosine phosphorylation through activation of protein-tyrosine phosphatase rather than through the inactivation of pp60src, a mechanism that was previously suggested.
Subject(s)
Blood Platelets/metabolism , Calpain/metabolism , Protein Tyrosine Phosphatases/metabolism , Dipeptides/pharmacology , Humans , Phosphorylation/drug effects , Vanadates/pharmacologyABSTRACT
The protooncogene c-myb is responsible for elevating intracellular calcium concentration ([Ca2+]i) at the G1/S interface in vascular smooth muscle cells (VSMC). However, the molecular components of this pathway are undefined, and the biological effects of increased levels of divalent cation are unknown. We have demonstrated that growth-arrested c-myb-transfected VSMC, compared with wild type VSMC, exhibit a fourfold increased number of insulin-like growth factor I (IGF-I) receptors, increased amount of secreted IGF-I activity, and a twofold increased level of [Ca2+]. The c-myb transfected cells, compared with wild type cells, also possess a twofold increased rate of calcium influx and a twofold decreased rate of calcium efflux. The elevated calcium influx rate of transfected cells is decreased to that of wild type cells with IGF-I neutralizing antibody, whereas the decreased calcium efflux rate of transfected cells is increased to that of wild type cells with antisense c-myb oligonucleotides. Proliferating wild type VSMC exhibit an increased calcium influx rate in late G1, which is dependent on production of augmented amounts of IGF-I activity but not increased levels of IGF-I receptors. The wild type VSMC also show a decreased calcium efflux rate at the same point in the cell cycle, which is dependent on expression of c-myb. The treatment of wild type cells with antisense c-myb or IGF-I receptor oligonucleotides induces a late G1 block in cell proliferation, which can be overcome by exposure to the calcium ionophore, 4-bromo-A-27318, in amounts sufficient to raise [Ca2+]i to levels observed at the G1/S interface. We conclude that IGF-I/IGF-I receptors and c-myb are involved in control of [Ca2+]i at the G1/S interface by separately regulating the rates of calcium influx and efflux and that elevated levels of divalent cation are necessary for progression of VSMC into the S phase of the cell cycle.
Subject(s)
Calcium/metabolism , Muscle, Smooth, Vascular/metabolism , Proto-Oncogene Proteins/genetics , Animals , Base Sequence , Cell Cycle , Cell Line , Insulin-Like Growth Factor I/metabolism , Intracellular Membranes/metabolism , Molecular Sequence Data , Muscle, Smooth, Vascular/cytology , Oligonucleotide Probes/genetics , Proto-Oncogene Proteins c-myb , Rats , Receptors, Somatomedin/metabolism , TransfectionABSTRACT
The non-transmembrane phosphotyrosine phosphatase 1B (PTP-1B) is an abundant enzyme, normally localized to the cytosolic face of the endoplasmic reticulum via a C-terminal targeting sequence. We have found that agonist-induced platelet activation results in proteolytic cleavage of PTP-1B at a site upstream from this targeting sequence, causing subcellular relocation of its catalytic domain from membranes to the cytosol. PTP-1B cleavage is catalyzed by the calcium-dependent neutral protease calpain and is a general feature of platelet agonist-induced aggregation. Moreover, PTP-1B cleavage correlates with the transition from reversible to irreversible platelet aggregation in platelet-rich plasma. Engagement of gpIIb-IIIa is necessary for inducing PTP-1B cleavage, suggesting that integrins regulate tyrosine phosphatases as well as tyrosine kinases. PTP-1B cleavage is accompanied by a 2-fold stimulation of its enzymatic activity, as measured by immune complex phosphatase assay, and correlates with discrete changes in the pattern of tyrosyl phosphorylation. Cleavage and subcellular relocation of PTP-1B represents a novel mechanism for altering tyrosyl phosphorylation that may have important physiological implications in cell types other than platelets.
Subject(s)
Blood Platelets/enzymology , Calpain/metabolism , Protein Tyrosine Phosphatases/metabolism , Catalysis , Electrophoresis, Polyacrylamide Gel , Humans , Integrins/metabolism , Kinetics , Phosphorylation , Platelet Aggregation , Signal TransductionABSTRACT
Calpain is distributed ubiquitously in virtually every tissue (Croall, D. E., and DeMartino, G. N. (1991) Physiol. Rev. 71, 813-846), but its physiological role remains to be determined. The identification of its natural endogenous substrates would be of great interest. Since pp60src, a major tyrosine kinase in platelets, is known to be easily cleaved during purification from cells (Feder, D., and Bishop, J. M. (1990) J. Biol. Chem. 265, 8205-8211), we examined the possibility that it is an endogenous substrate of calpain. In the whole cell lysate from resting platelets, which was analyzed by Western blotting with monoclonal antibody 327, we found pp60src almost exclusively in a 60-kDa form, with a trace of 52-kDa form. Addition of A23187 (a calcium ionophore) or dibucaine, which are known to be activators of platelet calpain (Croall and DeMartino, 1991; Fox, J. E., Reynolds, C., Morrow, J. S., and Phillips, D. R. (1987) Blood 76, 2510-2519; Fox, J. E., Austin, C. D., Boyles, J. K., and Steffen, P. K. (1990b) J. Cell Biol. 111, 483-493), caused dose- and time-dependent cleavage of actin-binding protein and p235 protein (talin). At the same time, loss of the 60-kDa species of pp60src and generation of the 52-kDa (occasionally seen as doublets) and 47-kDa species were detected by the Western blotting. In platelets aggregated by 1 unit/ml thrombin, apparently identical cleavage products were found. The cleavage of pp60src was inhibited by calpeptin (20 microM), an inhibitor of calpain (Tsujinaka, T., Kajiwara, Y., Kambayashi, J., Sakon, M., Higuchi, N., Tanaka, T., and Mori, T. (1988) Biochem. Biophys. Res. Commun. 153, 1201-1208; Tsujinaka, T., Ariyoshi, H., Uemura, Y., Sakon, M., Kambayashi, J., and Mori, T. (1990) Life Sci. 46, 1059-1066; Fox, J. E., Clifford, C. C., and Austin, C. D. (1990) Blood 76, 2510-2519; Fox, J. E., Austin, C. D., Boyles, J. K., and Steffen, P. K. (1990) J. Cell. Biol. 111, 483-493; Fox, J. E., Austin, C. D., Clifford, C. C., and Steffen, P. K. (1991) J. Biol. Chem. 266, 13289-13295). Addition of EGTA (3 mM) to the extracellular media completely inhibited the cleavage of actin-binding protein, talin, and pp60src in response to A23187 (1 microM). Intact pp60src was distributed in both cytosolic and particulate (membrane) fractions. Cleaved species were found exclusively in the cytosolic fraction. pp60src-associated enolase kinase activity was reduced. Thus, pp60src is an endogenous substrate for calpain, the cleavage of which may have regulatory effects on the kinase.
Subject(s)
Blood Platelets/enzymology , Calpain/blood , Proto-Oncogene Proteins pp60(c-src)/blood , Antibodies, Monoclonal , Blotting, Western , Calcimycin/pharmacology , Calcium/pharmacology , Dimethyl Sulfoxide/pharmacology , Dipeptides/pharmacology , Egtazic Acid/pharmacology , Electrophoresis, Polyacrylamide Gel , Humans , Microfilament Proteins/metabolism , Molecular Weight , Platelet Aggregation , Proto-Oncogene Proteins pp60(c-src)/isolation & purification , Substrate Specificity , Talin/metabolismABSTRACT
To clarify the mechanism of platelet activation by immune complexes and the possible involvement of surface glycoproteins (GPs), we studied platelet activation induced by heat-aggregated IgG (HAG). We examined the effects of monoclonal antibodies (MoAbs) against GPIb, GPIIb/IIIa, and the Fc receptor on resting platelets and on platelets stimulated by HAG. HAG increased the cytosolic ionized calcium concentration ([Ca2+]i) and stimulated protein (P47 and P20) phosphorylation, phosphatidic acid (PA) synthesis, serotonin secretion, and platelet aggregation. IV.3, an anti-Fc gamma RII receptor MoAb, inhibited HAG binding to platelets and all subsequent platelet responses. Like IV.3, MoAbs against GPIIb/IIIa (Tab, 10E5, AP-3) or GPIb (AP-1, 6D1) strongly inhibited platelet activation by HAG. However, while anti-GPIIb/IIIa MoAbs inhibited binding of IV.3 and HAG to platelets, anti-GPIb MoAbs had little effect on platelet binding of IV.3 or HAG. These observations suggest a close topographical and functional association of GPIIb/IIIa with Fc gamma RII in the platelet response to HAG. Cytochalasin B, an inhibitor of actin polymerization, also inhibited platelet activation but not HAG or IV.3 binding. Measurement of the fluorescence of 7-nitrobenz-2-oxa-1,3-(NBD)-phallacidin, a specific marker for filamentous actin (F-actin), showed that both cytochalasin B and AP-1 blocked the increase of F-actin induced by HAG. The common effects of anti-GPIb MoAbs and of cytochalasin B suggest that unlike the activity of GPIIb/IIIa, the ability of anti-GPIb to inhibit the activation of platelets by immune complexes is associated with perturbation of the cytoskeleton.
Subject(s)
Antigen-Antibody Complex/immunology , Cytoskeleton/physiology , Platelet Activation , Platelet Membrane Glycoproteins/physiology , Antibodies, Monoclonal/immunology , Calcium/metabolism , Humans , Immunoglobulin G/immunology , Platelet Aggregation , Thromboxane A2/physiologyABSTRACT
Desmopressin acetate (1-deamino-8-D-arginine vasopressin [DDAVP]) improves hemostasis in hemophilia A and von Willebrand's disease and in some platelet disorders. In complex cardiac operations, excluding simple coronary artery bypass graft procedures, we found that desmopressin reduced blood loss by 40% and the need for transfusion by 34%. Conflicting reports followed. Future trials should emphasize patients with excessive bleeding. A possible post-desmopressin prothrombotic state was studied after hip replacement surgery. The incidence of deep vein thrombosis associated with warfarin sodium therapy was the same as that associated with desmopressin plus warfarin therapy. No desmopressin-induced thrombotic tendency was detected. A trend toward reduced blood loss with desmopressin was not significant. During cardiac catheterization, the plasma von Willebrand factor level was correlated with hemodynamic variables, including pulmonary vascular resistance, pulmonary arterial pressure, and (inversely) with cardiac index. von Willebrand factor concentration was highest in mitral stenosis. The relationship of these factors to the response to desmopressin remains to be defined.
Subject(s)
Cardiac Surgical Procedures , Deamino Arginine Vasopressin/therapeutic use , Hemostasis, Surgical , Hemostatics/therapeutic use , Aged , Blood Loss, Surgical/prevention & control , Cardiopulmonary Bypass , Female , Hip Prosthesis , Humans , Male , Placebos , Platelet Count/drug effects , Reoperation , Thrombophlebitis/chemically induced , von Willebrand Factor/analysisSubject(s)
Aspirin/administration & dosage , Myocardial Infarction/prevention & control , Thrombosis/prevention & control , Ticlopidine/therapeutic use , Anticoagulants/therapeutic use , Aspirin/adverse effects , Aspirin/pharmacology , Cerebrovascular Disorders/chemically induced , Cerebrovascular Disorders/prevention & control , Dipyridamole/therapeutic use , Dose-Response Relationship, Drug , Drug Synergism , Drug Therapy, Combination , Humans , Ticlopidine/adverse effectsSubject(s)
Anticoagulants/therapeutic use , Postoperative Complications/prevention & control , Thrombophlebitis/prevention & control , Wounds and Injuries/complications , Aspirin/therapeutic use , Bandages , Cerebrovascular Disorders/complications , Gravity Suits , Humans , Myocardial Infarction/complications , Thrombophlebitis/etiologySubject(s)
Anticoagulants/therapeutic use , Arterial Occlusive Diseases/drug therapy , Fibrinolytic Agents/therapeutic use , Peripheral Vascular Diseases/drug therapy , Platelet Aggregation Inhibitors/therapeutic use , Thrombolytic Therapy , Vascular Surgical Procedures , Arterial Occlusive Diseases/surgery , Humans , Peripheral Vascular Diseases/surgeryABSTRACT
Protein-tyrosine phosphorylation during platelet activation is inhibited under conditions that inhibit platelet binding of fibrinogen and aggregation. We suggested that pp60src, a major platelet tyrosine kinase, or its protein substrates might become associated with the cytoskeleton upon platelet stimulation, and that this might be related to aggregation. By Western blotting with an anti-Src monoclonal antibody, we found time-dependent association of pp60src with the cytoskeleton (10,000 x g Triton X-100-insoluble matrix) but not the "membrane" cytoskeleton (100,000 x g Triton X-100-insoluble matrix) in platelets activated by U46619 (PGH2 analog). Cytoskeletal association and platelet aggregation were inhibited by the peptide Arg-Gly-Asp-Ser (RGDS) (but not by Arg-Gly-Glu-Ser (RGES)), by 10E5 antibody against glycoprotein (Gp) IIb/IIIa, and by EGTA. U46619-induced association of pp60src with cytoskeleton but not secretion or aggregation was inhibited by cytochalasin D (2 microM). Both cytochalasin D and RGDS inhibited "slow" tyrosine phosphorylation of platelet proteins. Association of pp60src with cytoskeleton induced by U46619 or ADP was not blocked by aspirin. Aspirin blocked epinephrine-induced association of pp60src with the cytoskeleton during a second phase of aggregation when an initial phase had occurred without shape change or secretion. Association of GpIIb/IIIa with the cytoskeleton also accompanied platelet aggregation, shape change, and actin polymerization; this was shown with anti-GpIIb and anti-GpIIIa antibodies. Association of pp60src and GpIIb/IIIa with the cytoskeleton and slow tyrosine phosphorylation are related phenomena.
Subject(s)
Actins/metabolism , Blood Platelets/metabolism , Oncogene Protein pp60(v-src)/metabolism , Platelet Aggregation , Amino Acid Sequence , Blotting, Western , Cells, Cultured , Cytoskeleton/drug effects , Cytoskeleton/metabolism , Detergents , Humans , Immunohistochemistry , Molecular Sequence Data , Octoxynol , Oligopeptides/pharmacology , Phosphorylation , Platelet Aggregation Inhibitors/pharmacology , Platelet Membrane Glycoproteins/metabolism , Polyethylene Glycols , Polymers , Prostaglandin Endoperoxides, Synthetic/pharmacologyABSTRACT
Upon platelet activation, numerous proteins are known to be tyrosine phosphorylated. To investigate the mechanisms of the regulation of tyrosine phosphorylation and its physiological significance, the effects on tyrosine phosphorylation of agents that elevate the platelet level of the cyclic nucleotides cAMP and cGMP were examined in aspirin-treated gel-filtered platelets by Western blotting with a specific antiphosphotyrosine antibody. The effects of these agents on other aspects of platelet activation, i.e., aggregation, secretion, and elevation of the concentration of cytosolic ionized calcium ([Ca2+]i), were also examined in parallel experiments. Tyrosine phosphorylation in platelets activated by alpha-thrombin (1 nM) was inhibited by prostaglandin (PG) E1 (2 microM) or by sodium nitroprusside (100 microM). Elevation of [Ca2+]i, aggregation, and serotonin secretion was also strongly inhibited. On the other hand, a higher concentration of alpha-thrombin (10 nM) induced tyrosine phosphorylation of the same proteins, elevation of [Ca2+]i, platelet aggregation, and serotonin secretion, irrespective of pretreatment of platelets by either PGE1 or sodium nitroprusside. Inhibition by sodium nitroprusside of tyrosine phosphorylation induced by alpha-thrombin (1 nM) was accompanied by an increased concentration of cGMP. 8-BrcGMP (2 mM) also inhibited tyrosine phosphorylation and aggregation, although less than sodium nitroprusside. ADP (20 microM) induced platelet shape change and tyrosine phosphorylation of only a few proteins; these effects were also inhibited by either PGE1 or sodium nitroprusside. Thus tyrosine phosphorylation in platelets can be inhibited by elevation of either cAMP or cGMP, an effect that is overcome by a high concentration of thrombin, resulting in granule secretion and aggregation. Some of the proteins that are tyrosine phosphorylated may be important in the regulation of platelet functions.
Subject(s)
Adenosine Diphosphate/pharmacology , Alprostadil/pharmacology , Blood Platelets/metabolism , Nitroprusside/pharmacology , Thrombin/pharmacology , Adenosine Diphosphate/antagonists & inhibitors , Blood Platelets/drug effects , Blotting, Western , Cyclic GMP/metabolism , Cytosol/drug effects , Cytosol/metabolism , Electrophoresis, Polyacrylamide Gel , Humans , In Vitro Techniques , Kinetics , Phosphoproteins/blood , Phosphorylation/drug effects , Phosphotyrosine , Platelet Aggregation/drug effects , Thrombin/antagonists & inhibitors , Tyrosine/analogs & derivatives , Tyrosine/bloodABSTRACT
The content of filamentous actin in individual platelets was measured by flow cytometry, using a fluorescent probe specific for filamentous actin (F-actin), 7-nitrobenz-2-oxa-1,3-phallacidin (NBD-phallacidin). NBD-phallacidin binding to fixed platelets was specific in that either pretreatment of platelets with unlabeled phallacidin or absorption of NBD-phallacidin by rabbit skeletal F-actin, but not globular actin (G-actin), resulted in a significant loss in the bound fluorescent probe. Mean NBD-phallacidin binding to fixed platelets varied with the agonist and paralleled the changes in F-actin reported with the DNAse I inhibition assay. (1) NBD-phallacidin binding increased with stimulation by ADP, U46619 (a prostaglandin H2 analogue), or collagen and paralleled shape change. (2) Epinephrine did not increase NBD-phallacidin binding. (3) Platelets treated at 4 degrees C contained more F-actin than did platelets kept at 37 degrees C. (4) Cytochalasin D (10 mumol/L) inhibited the increase of phallacidin binding to individual platelets stimulated by either ADP or U46619. In measurements of cytosolic free calcium concentration ([Ca2+]i) by flow cytometry in Indo-1-loaded platelets, ADP's dose-response for actin polymerization was similar to that for calcium mobilization. As shown by flow cytometry, a tail population that had a minimal increase in F-actin upon stimulation with ADP or U46619 also contained the platelets with the least forward and right angle light scattering, which are functions of platelet size and shape. When platelets treated with NBD-phallacidin were incubated with S12-murine monoclonal antibody (a marker of alpha-granule secretion detected by phycoerythrin-conjugated antimouse IgG second antibody), phallacidin fluorescence paralleled S12 binding. Thus, human blood platelets are heterogeneous in regard to actin polymerization at rest and in association with platelet activation; different degrees of phallacidin binding may identify functionally different platelet populations.