Epinephrine induces a late thromboxane-dependent platelet shape change and enhances synergistically the shape change induced by other platelet agonists

Epinephrine is the only physiological platelet activator which induces platelet aggregation without a preceding change in platelet shape. The reason why epinephrine cannot induce this shape change is not known. Electron microscopically, we could show that during the first phase of epinephrine-induced platelet aggregation, the platelet aggregate is composed of discoid platelets, lying in rather loose contact with neighbouring platelets. During the second wave of epinephrine-induced aggregation (this is when thromboxane (TX)A₂ production has taken place), platelets have completely lost their discoid shape and are very tightly bound. In EDTA-platelet rich plasma (PRP), we could demonstrate a clear synergistic action of epinephrine 10–20 μM on the first phase of shape change (disc-to-sphere transformation), induced by low concentrations of arachidonic acid (AA), collagen, adenosine diphosphate (ADP) and platelet activating factor (PAF). In combination with moderate concentrations of AA or collagen, epinephrine induced a clear aggregation-independent secretion of platelet granules, which in the absence of epinephrine, only takes place with higher inducer concentrations. All these synergistic actions could be demonstrated in the aggregometer and electron microscopically. To explain these findings, we hypothesize that the inability of epinephrine to induce a shape change that precedes aggregation is due to slow generation of TXA₂ which is only formed as a positive feedback mechanism of aggregation. This TXA₂ will bind to its own receptor and produce a shape change coinciding with the second wave of epinephrine-induced aggregation. Collagen, in contrast, induces very rapid TXA₂ generation, causing Ca²⁺ mobilization and myosin light chain-phosphorylation, leading to shape change, clearly before aggregation starts.     

Epinephrine induces platelet fibrinogen receptor expression, fibrinogen binding, and aggregation in whole blood in the absence of other excitatory agonists

The exposure of fibrinogen receptors is an early event in agonist-induced platelet activation. Previous measurements of fibrinogen binding or aggregation in platelet-rich plasma or washed platelets have failed to define whether the initial response to epinephrine results solely from a direct effect of this agonist. To address this problem, we have measured fibrinogen receptor exposure on platelets in whole blood by using flow cytometry and a fluorescein isothiocyanate-labeled monoclonal antibody specific for the activated fibrinogen receptor (FITC-PAC1). We also measured platelet-bound fibrinogen with an antifibrinogen monoclonal antibody (FITC-9F9) as well as platelet aggregation in whole blood. In blood anticoagulated with citrate and in the presence of a cyclooxygenase inhibitor, epinephrine (0.1 to 100 mumol/L) caused significant FITC-PAC1 binding (P less than .001) that was maximal at 10 mumol/L epinephrine. The maximal epinephrine response was one third of that observed with 10 mumol/L adenosine diphosphate (ADP) and was eliminated by yohimbine, an alpha 2-adrenergic antagonist. Incubation of the blood with apyrase or phosphoenolpyruvate plus pyruvate kinase to remove extracellular ADP resulted in a 40% to 50% reduction in the epinephrine response. Despite this, FITC-PAC1 binding was still significant at epinephrine greater than or equal to 1 mumol/L (P less than .05). No reduction in epinephrine-induced FITC-PAC1 binding was observed in the presence of ATP alpha S, an ADP receptor antagonist; cinanserin, a serotonin antagonist; or WEB-2086, a platelet activating factor antagonist. Furthermore, addition of the thrombin inhibitors hirudin or leupeptin to citrated blood had no effect on the extent of the epinephrine response. Blood anticoagulated with hirudin also demonstrated an epinephrine response, even in the presence of apyrase. Similar results were obtained when FITC-9F9 was used to detect fibrinogen binding or when aggregation was assessed by a decrease in the number of single platelets. We conclude that epinephrine itself can induce fibrinogen receptor exposure, fibrinogen binding, and aggregation. This primary response is independent of synergistic interaction of epinephrine with traces of ADP, serotonin, platelet activating factor, or thrombin. However, such synergistic interaction with ADP present in whole blood may enhance the responses induced by epinephrine.     

Generation of platelet-derived microparticles and procoagulant activity by heparin-induced thrombocytopenia IgG/serum and other IgG platelet agonists: a comparison with standard platelet agonists

Heparin-induced thrombocytopenia (HIT) is a relatively common, immunoglobulin-mediated adverse drug reaction associated with in vivo thrombin generation and both venous and arterial thrombosis. Serum and purified IgG from patients with HIT induce normal platelets to generate procoagulant platelet-derived microparticles, but the magnitude of this response in comparison with other IgG and standard platelet agonists is unknown. We describe a comparison of IgG platelet agonists, including HIT-IgG/serum, heat-aggregated IgG, and platelet-activating murine monoclonal antibodies, with standard 'strong' and 'weak' platelet agonists, and have determined their relative ability to generate platelet procoagulant activity. Using washed normal platelets as targets, we observed that HIT sera as well as other IgG agonists produced similar or even greater numbers of microparticles and procoagulant activity than the standard strong platelet agonists (thrombin, collagen, and thrombin receptor agonist peptide). The only exception was the non-physiological platelet agonist, calcium ionophore, which consistently produced a platelet procoagulant response even greater than the IgG agonists. We conclude that the IgG class of platelet agonists (including pathogenic HIT antibodies) is an effective trigger of the platelet procoagulant response comparable at least to strong physiological platelet agonists. These results help to explain the association between HIT, in vivo thrombin generation, and thrombosis.     

ADP induces partial platelet aggregation without shape change and potentiates collagen-induced aggregation in the absence of Galphaq

Platelets from Galphaq knockout mice are unable to aggregate in response to physiological agonists like adenosine 5'-diphosphate (ADP), thromboxane A(2), thrombin, or collagen, although shape change still occurs in response to all of these agonists except ADP. ADP-induced platelet aggregation results from simultaneous activation of the purinergic P2Y(1) receptor coupled to calcium mobilization and shape change and of a distinct P2 receptor, P2cyc, coupled through Gi to adenylyl cyclase inhibition, which is responsible for completion and amplification of the response. P2cyc could be the molecular target of the antithrombotic drug clopidogrel and the adenosine triphosphate (ATP) analogs AR-C69931MX, AR-C67085, and AR-C66096. The aim of the present study was to determine whether externally added ADP could still act through the Gi pathway in Galphaq-deficient mouse platelets and thereby amplify the residual responses to agonists such as thrombin or collagen. It was found that (1) ADP and adrenaline still inhibited cyclic AMP accumulation in Galphaq-deficient platelets; (2) both agonists restored collagen- but not thrombin-induced aggregation in these platelets; (3) the effects of ADP were selectively inhibited in vitro by the ATP analog AR-C69931MX and ex vivo by clopidogrel and hence were apparently mediated by the P2cyc receptor; and (4) high concentrations of ADP (100 micromol/L) induced aggregation without shape change in Galphaq-deficient platelets through activation of P2cyc. Since adrenaline was not able to induce platelet aggregation even at high concentrations, we conclude that the effects of ADP mediated by P2cyc are not restricted to the inhibition of adenylyl cyclase through Gi(2).     

Generation of platelet-derived microparticles and procoagulant activity by heparin-induced thrombocytopenia IgG/serum and other IgG platelet agonists: a comparison with standard platelet agonists

Heparin-induced thrombocytopenia (HIT) is a relatively common, immunoglobulin-mediated adverse drug reaction associated with in vivo thrombin generation and both venous and arterial thrombosis. Serum and purified IgG from patients with HIT induce normal platelets to generate procoagulant platelet-derived microparticles, but the magnitude of this response in comparison with other IgG and standard platelet agonists is unknown. We describe a comparison of IgG platelet agonists, including HIT-IgG/serum, heat-aggregated IgG, and platelet-activating murine monoclonal antibodies, with standard 'strong' and 'weak' platelet agonists, and have determined their relative ability to generate platelet procoagulant activity. Using washed normal platelets as targets, we observed that HIT sera as well as other IgG agonists produced similar or even greater numbers of microparticles and procoagulant activity than the standard strong platelet agonists (thrombin, collagen, and thrombin receptor agonist peptide). The only exception was the non-physiological platelet agonist, calcium ionophore, which consistently produced a platelet procoagulant response even greater than the IgG agonists. We conclude that the IgG class of platelet agonists (including pathogenic HIT antibodies) is an effective trigger of the platelet procoagulant response comparable at least to strong physiological platelet agonists. These results help to explain the association between HIT, in vivo thrombin generation, and thrombosis.     

Thrombopoietin primes human platelet aggregation induced by shear stress and by multiple agonists

Recombinant thrombopoietin has been reported to stimulate megakaryocytopoiesis and thrombopoiesis and it may be quite useful to treat patients with low platelet counts after chemotherapy. As little is known regarding the possible activation of platelets by thrombopoietin, we examined the effects of thrombopoietin on platelet aggregation induced by shear stress and various agonists in native plasma. Using hirudin as an anticoagulant, thrombopoietin (1 to 100 ng/mL) enhanced platelet aggregation induced by 2 micromol/L adenosine- diphosphate (ADP) in a dose dependent fashion. The enhancement was not affected by treatment of platelets with 1 mmol/L aspirin plus SQ-29548 (a thromboxane antagonist, 1 micromol/L) but was inhibited by a soluble form of the thrombopoietin receptor, suggesting that the enhancement was mediated by the specific receptors and does not require thromboxane production. Epinephrine (1 micromol/L), which does not induce platelet aggregation in hirudin platelet rich plasma (PRP), did so in the presence of thrombopoietin (10 ng/mL). Thrombopoietin (10 ng/mL) also enhanced or primed platelet aggregation induced by collagen (0.5 micron.mL),. thrombin, serotonin, and vasopressin. Thrombopoietin does not induce any rise in cytosolic ionized calcium concentration nor activation of protein kinase C, as estimated by phosphorylation of preckstrin, indicating that the priming effects of thrombopoietin does not require those processes. The ADP- or thrombin-induced rise in cytosolic ionized calcium concentration was not enhanced by thrombopoietin (100 ng/mL). Further, shear (ca. 90 dyn/cm2)-induced platelet aggregation was also potentiated by thrombopoietin. The priming effect on epinephrine-induced platelet aggregation in hirudin PRP was unique to thrombopoietin, with no effects seen using interleukin-6 (IL-6), IL-11, IL-3, erythropoietin, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, or c-kit ligand. These data indicate that monitoring of platelet functions may be necessary in the clinical trials of thrombopoietin.     

Blood irradiation by He-Ne laser induces a decrease in platelet responses to physiological agonists and an increase in platelet cyclic GMP

The effect of He-Ne laser irradiation on platelet adhesion, activation and aggregation was investigated. Citrated whole blood was irradiated in vitro by He-Ne laser (632.8 nm, 7 mW) and then subjected to shear stress (1300 s-1) on subendothelial extracellular matrix (ECM)-coated plates. Laser irradiation was followed by a decrease in platelet adhesion and aggregation on ECM under flow conditions in a time exposure-dependent manner (by 30-40%). The inhibiting effect of laser light on platelets was detectable up to 1 h after the termination of irradiation. Laser irradiation of either platelet-rich plasma, gel-filtered platelets, platelet-poor plasma, or packed blood cells followed by whole blood reconstitution revealed a marked decrease in platelet deposition on ECM only in the cases of platelet-rich plasma or gel filtered platelets. In conventional aggregometry, laser-treated platelet-rich plasma demonstrated a diminished platelet response to both thrombin receptor-activating peptide (TRAP), converting a two-wave aggregation curve to reversible, and to the protein kinase C activator PMA (by 45%). In flow cytometry analysis, irradiated platelets presented lower fibrinogen binding and P-selectin expression in response to TRAP. Laser irradiation had no additional inhibitory effect on dibutyryl cGMP- and dibutyryl cAMP-pretreated platelets. A 50% increase in cGMP level was observed in laser-treated gel filtered platelets, both in the presence and in absence of the phosphodiesterase inhibitor, isobuthylmethylxanthine. The results suggest that guanylate cyclase is one of the primary mediators of the laser effect on platelet function.     

Blood irradiation by He-Ne laser induces a decrease in platelet responses to physiological agonists and an increase in platelet cyclic GMP

The effect of He-Ne laser irradiation on platelet adhesion, activation and aggregation was investigated. Citrated whole blood was irradiated in vitro by He-Ne laser (632.8 nm, 7 mW) and then subjected to shear stress (1300 s -1 ) on subendothelial extracellular matrix (ECM)-coated plates. Laser irradiation was followed by a decrease in platelet adhesion and aggregation on ECM under flow conditions in a time exposure-dependent manner (by 30-40%). The inhibiting effect of laser light on platelets was detectable up to 1 h after the termination of irradiation. Laser irradiation of either platelet-rich plasma, gel-filtered platelets, platelet-poor plasma, or packed blood cells followed by whole blood reconstitution revealed a marked decrease in platelet deposition on ECM only in the cases of platelet-rich plasma or gel filtered platelets. In conventional aggregometry, laser-treated platelet-rich plasma demonstrated a diminished platelet response to both thrombin receptor-activating peptide (TRAP), converting a two-wave aggregation curve to reversible, and to the protein kinase C activator PMA (by 45%). In flow cytometry analysis, irradiated platelets presented lower fibrinogen binding and P-selectin expression in response to TRAP. Laser irradiation had no additional inhibitory effect on dibutyryl cGMP- and dibutyryl cAMP-pretreated platelets. A 50% increase in cGMP level was observed in laser-treated gel filtered platelets, both in the presence and in absence of the phosphodiesterase inhibitor, isobuthylmethylxanthine. The results suggest that guanylate cyclase is one of the primary mediators of the laser effect on platelet function.     

Actin polymerization induces a shape change in actin-containing vesicles.

We have encapsulated actin filaments in the presence and absence of various actin-binding proteins into lipid vesicles. These vesicles are approximately the same size as animal cells and can be characterized by the same optical microscopic and mechanical techniques used to study cells. We demonstrate that the initially spherical vesicles can be forced into asymmetric, irregular shapes by polymerization of the actin that they contain. Deformation of the vesicles requires that the actin filaments be on average at least approximately 0.5 micron long as shown by the effects of gelsolin, an actin filament-nucleating protein. Filamin, a filament-crosslinking protein, caused the surfaces of the vesicles to have a smoother appearance. Heterogeneous distribution of actin filaments within the vesicles is caused by interfilament interactions and modulated by gelsolin and filamin. The vesicles provide a model system to study control of cell shape and cytoskeletal organization, membrane-cytoskeleton interactions, and cytomechanics.     

Factors affecting the observation of a synergistic response in platelet aggregation induced by pairs of excitatory agonists

When aggregation is measured as the disappearance of single platelets synergistic interaction between excitatory agonist pairs can be observed using washed platelets in a modified Tyrode's medium or platelet-rich plasma anticoagulated with hirudin; but not using citrated platelet-rich plasma. For aggregation induced by the ADP/adrenaline agonist pair, both the observation of synergistic interaction and the sensitivity of the platelets to these agonists, is a function of extracellular [Ca(2+)]. Synergistic interaction and reduced sensitivity to the individual agonists, especially adrenaline, is observed when extracellular [Ca(2+)] > 100 μM. The data suggest that lower affinity binding of Ca(2+) to the glycoprotein IIb/IIIa complex may modulate platelet sensitivity to these excitatory agonists. The conditions used to resuspend the platelets also influences the nature of the response to the ADP/adrenaline agonist pair and the sensitivity of the platelets to these agonists. A synergistic response and/or reduced sensitivity to ADP is observed on resuspension in modified Tyrode's medium but does not occur on resuspension in citrated plasma or in plasma anticoagulated with hirudin. The factor responsible for enhancing sensitivity, and hence abolishing the synergistic response, is a species of low molecular weight (M(r) less than 25 KDa). It is neither citrate nor Ca(2+).     

The platelet shape change: biophysical basis and physiological consequences

The well-known platelet shape change is the universal hallmark of activation. This review uncovers the biophysics underlying this rapid and dramatic transformation. We aim to give a broad vision of the interplay between different cytoskeletal subsystems, which is based on physical considerations and recent advances in mathematics and computational biology. These novel findings lead to the understanding that the ring of microtubules counterbalances cortical tension in the resting platelet, making it a “mechanically charged” system. Platelet activation breaks the balance via several mechanisms, triggering rapid ring buckling and cell rounding. Based on the review of known data concerning the relations between platelet shape and function, we hypothesize that disk-to-sphere transformation facilitates platelet adhesion under flow. Conclusions of the paper may be useful for the development of novel, cytoskeletal-based strategies of antiplatelet therapy.     

Protein kinase A mediates inhibition of the thrombin-induced platelet shape change by nitric oxide

The thrombin-induced platelet shape change was blocked by nitric oxide (NO), as revealed by scanning electron microscopy, light transmission, and resistive-particle volume determination. The inhibitory effect of NO was accompanied by an increase in levels of both cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) and phosphorylation of the vasodilator-stimulated phosphoprotein (VASP). However, the inhibition of the shape change was only mimicked by cAMP analogs (Sp-5,6-DClcBIMPS, 8-AHA-cAMP, and 8-CPT-cAMP) and not by cGMP analogs (8-Br-PET-cGMP, 8-Br-cGMP, and 8-pCPT-cGMP). The effect of NO on the thrombin-induced shape change was prevented by the protein kinase A (PKA) antagonists Rp-8-Br-cAMPS and Rp-cAMPS. The protein kinase G (PKG) antagonist Rp-8-CPT-cGMPS strongly inhibited PKG-mediated 46-kDa VASP Ser239 phosphorylation, but did not inhibit the thrombin-induced shape change or the PKA-mediated VASP Ser157 phosphorylation. Whereas an inhibitor of cyclic nucleotide phosphodiesterase (PDE) 3A (milrinone) mimicked the effect of NO, inhibitors of PDE2 (erythro-9-(2-hydroxy-3-nonyl)adenine) and PDE5 (dipyridamole) were poorly effective. We concluded that (1) NO was a potent and reversible inhibitor of the platelet shape change, (2) the shape change was reversible, (3) the inhibitory effect of NO was mediated through activation of PKA, (4) the onset of the NO effect coincided with VASP Ser157 phosphorylation, and (5) removal of NO and platelet shape change coincided with VASP Ser157 dephosphorylation. These findings are compatible with elevation of cGMP by NO in a compartment close to PDE3A, PKA, and VASP, leading to a local increase of cAMP able to block thrombin-induced shape change.     

Platelet aggregation through prothrombinase activation induced by non-aggregant doses of platelet agonists.

Activation of the prothrombinase complex, which catalyzes the formation of thrombin from prothrombin, is crucial for the (patho)physiological processes of hemostasis and thrombosis. We here report that washed platelets supplemented with prothrombin can be irreversibly aggregated with otherwise non-aggregant doses of adenosine diphosphate (10 micromol/l), thrombin (0.06 U/ml), or collagen (1 microg/ml). Prothrombinase-catalyzed prothrombin to thrombin conversion most probably supports this aggregation response, since inhibitors of thrombin (hirudin or heparin) and an inhibitor of activated factor X (DX-9065a) impair the response. A certain degree of agonist-induced platelet activation seems to be required for this prothrombin-supported aggregation response, since prothrombin alone does not induce aggregation, and blockade of glycoprotein Ia/IIa with a specific antibody inhibits the platelet aggregation response to collagen and prothrombin. These results may suggest that activation of the prothrombinase complex could be a common step of the platelet response to distinct agonists, which may be achieved at low levels of platelet stimulation.     

New possibilities for platelet shape change evaluation using the laser rheoaggregometer

The study of aggregation functions in blood platelets can at present be undertaken in vitro by measuring variations in intensity of transmitted light or electrical impedance change. These two methods allow only a general approach to the aggregation process. The laser rheoaggregometer was constructed to study early morphological modifications induced when the platelets are activated before aggregation. The results obtained are in keeping with the laws of light scattering and allow the measurement of platelet shape change.     

Evidence that the purinergic receptor P2Y12 potentiates platelet shape change by a Rho kinase-dependent mechanism

ADP activates human platelets through two G-protein coupled receptors, P2Y1 and P2Y12, to induce a range of functional responses. Here we have addressed the role and mechanism of P2Y12 in modulating ADP-induced platelet shape change. Although the response depended upon activation of P2Y1, it was potentiated by P2Y12 as the P2Y12-selective antagonists AR-C69931MX and 2MeSAMP partially inhibited shape change in the later phase of the response. This was paralleled by inhibition of pseudopod formation, platelet spheration, actin polymerisation and myosin light chain phosphorylation. P2Y12 is known to couple to activation of PI3 kinase and inhibition of adenylate cyclase, but we showed that neither of these signalling events couples to regulation of shape change by this receptor. However, by assessment of phosphorylation of its major substrate myosin light chain phosphatase, we provide direct evidence for activation of Rho kinase by ADP, and that although P2Y1 is required for activation of Rho kinase, P2Y12 is able to potentiate its activity. We conclude that P2Y12 plays a potentiatory role in ADP-induced shape change through regulation of the Rho kinase pathway, potentiating both myosin phosphorylation and actin polymerisation, and this forms part of an important signalling pathway additional to its well-established Gi-coupled pathways.     

Mild and moderate hypothermia increases platelet aggregation induced by various agonists: a whole blood in vitro study

The mechanisms causing temperature-dependent bleeding, especially in hypothermic patients, warrant clarification. Therefore the aim of this study was to investigate platelet aggregation at the clinically important temperature range of 30–34°C. After obtaining informed consent citrated whole blood was drawn from 12 healthy adult male volunteers, who had not taken any medication in the previous 14 days. After venipuncture blood samples were incubated at 37°C until platelet testing. Platelet aggregation was performed in whole blood using the impedance aggregometer Multiplate® at five different test temperatures between 30°C and 34°C. Aggregation responses at 37°C served as controls. At temperatures of mild and moderate hypothermia (30–34°C), overall platelet aggregation was increased compared to 37°C. Increases were recorded in response to collagen, thrombin receptor activating peptide and ristocetin between 31°C and 34°C and in response to adenosine diphosphate between 30°C and 34°C. Overall platelet aggregation is increased at mild and moderate hypothermia down to 30°C. These results indicate that bleeding complications reported in mildly hypothermic patients are not due to hypothermia-induced platelet inhibition. The pathomechanism of the overall increased platelet aggregation between 30°C and 34°C requires further detailed study.     

PPACK-thrombin inhibits thrombin-induced platelet aggregation and cytoplasmic acidification but does not inhibit platelet shape change

We have re-evaluated the previously reported ability of TLCK-thrombin (N alpha-tosyl-L-lysine chloromethyl ketone-treated alpha-thrombin) and PPACK-thrombin (D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone-treated alpha-thrombin) to inhibit alpha-thrombin-induced platelet activation (Harmon JT, Jamieson GA: J Biol Chem 261:15928, 1986; and Harmon JT, Jamieson GA: Biochemistry 27:2151, 1988). Despite several cycles of derivatization with TLCK (10,000-fold molar excess), preparations of TLCK-thrombin have been found to contain about 4% residual alpha-thrombin activity, suggesting that these preparations are an equilibrium mixture of TLCK-thrombin and alpha-thrombin and cannot be used for evaluating competition between these two agents. In contrast, alpha-thrombin activity was completely inhibited by PPACK at 15-fold molar excess. PPACK-thrombin, free of unreacted PPACK and devoid of residual alpha-thrombin activity, did not markedly affect platelet shape change at concentrations as high as 1 mumol/L, but inhibited aggregation and secretion in intact platelets activated with the minimal concentration of alpha-thrombin causing a full response (0.3 to 0.5 nmol/L) and yielded a 50% inhibition constant (IC50) for inhibition of aggregation by PPACK-thrombin of 110 nmol/L. This inhibition was specific for alpha-thrombin-induced platelet activation, and no inhibition was seen with activation induced by ADP, collagen, epinephrine, ristocetin, or arachidonate. At these low alpha-thrombin concentrations (approximately 0.4 nmol/L), a persistent cytoplasmic acidification was observed of -0.062 +/- 0.016 pH units, although alkalinization was observed at higher alpha-thrombin concentrations (greater than 1 nmol/L). While inhibition of aggregation and secretion occurred when alpha-thrombin and PPACK-thrombin were added simultaneously, inhibition of cytoplasmic acidification and of the elevation of cytoplasmic [Ca2+] induced by low concentrations of alpha-thrombin (0.4 nmol/L) occurred only if platelets were preincubated with PPACK-thrombin for 5 minutes before the addition of alpha-thrombin. In platelets treated with Serratia marcescens protease to remove glycoprotein lb (GPlb), alpha-thrombin-induced shape change was attenuated but persisted in the presence of a high concentration (2 mumol/L) of PPACK-thrombin, although aggregation and secretion were inhibited, as seen in intact platelets. The IC50 value for inhibition of aggregation by PPACK-thrombin was approximately 1 mumol/L at the higher alpha-thrombin concentrations (5 nmol/L) required for full activation in this case. These results suggest that PPACK-thrombin may be a useful probe of platelet function since it specifically blocks platelet aggregation and secretion induced by alpha-thrombin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thrombopoietin is synergistic with other hematopoietic growth factors and physiologic platelet agonists for platelet activation in vitro

Thrombopoietin (TPO) is the primary physiologic regulator of platelet production. The effect of TPO on platelet function, both alone and in combination with other hematopoietic growth factors, adenosine diphosphate (ADP), and epinephrine, was investigated using fluorescent-labeled antibodies to the activation-dependent antigen CD62 (P-selectin) and flow cytometry. TPO stimulated CD62 expression on normal human platelets, and this expression was completely inhibited by the soluble extracellular domain of the TPO receptor, MPL. The growth factors granulocyte colony-stimulating factor (G-CSF) and erythropoietin (EPO), but not interleukin-3 (IL-3) or stem-cell factor (SCF), also stimulated platelet activation. The combination of EPO, SCF, ADP, and epinephrine with TPO were synergistic for platelet CD62 expression. These data further support a role for TPO in modulating platelet function. Am. J. Hematol. 54:225–232, 1997 © 1997 Wiley-Liss, Inc.