The central role of the P(2T) receptor in amplification of human platelet activation, aggregation, secretion and procoagulant activity.

Adenosine diphosphate (ADP) is an important platelet agonist and ADP released from platelet dense granules amplifies responses to other agonists. There are three known subtypes of ADP receptor on platelets: P2X(1), P2Y(1) and P(2T) receptors. Sustained ADP-induced aggregation requires co-activation of P2Y(1) and P(2T) receptors. AR-C69931MX, a selective P(2T) receptor antagonist and novel antithrombotic agent, was studied to characterize further the function of the P(2T) receptor. The roles of the P2Y(1) receptor and thromboxane A(2) were assessed using the selective P2Y(1) antagonist A2P5P and aspirin respectively. Aggregation was measured by whole blood single-platelet counting and platelet-rich plasma turbidimetry, using hirudin anticoagulation. Dense granule release was estimated using ([14)C]-5-hydroxytryptamine (HT)-labelled platelets. Ca(2+) mobilization, P-selectin expression, Annexin V binding and microparticle formation were determined by flow cytometry. P(2T) receptor activation amplified ADP-induced aggregation initiated by the P2Y(1) receptor, as well as amplifying aggregation, secretion and procoagulant responses induced by other agonists, including U46619, thrombin receptor-activating peptide (TRAP) and collagen, independent of thromboxane A(2) synthesis, which played a more peripheral role. P(2T) receptor activation sustained elevated cytosolic Ca(2+) induced by other pathways. These studies indicate that the P(2T) receptor plays a central role in amplifying platelet responses and demonstrate the clinical potential of P(2T) receptor antagonists.

Effects of cytochalasin H, a potent inhibitor of cytoskeletal reorganisation, on platelet function.

Platelets contain a well-developed and dynamic cytoskeleton composed mainly of actin and actin-associated proteins. Upon platelet activation there is rapid polymerisation of actin and a marked reorganisation of the platelet cytoskeleton. Cytochalasins are agents that interfere with the polymerisation of actin, and it has recently been discovered that cytochalasin H (CyH) is particularly effective as an inhibitor of the cytoskeletal reorganisation that occurs in platelets following activation by adenosine diphosphate (ADP). Here we have used CyH to inhibit platelet cytoskeletal reorganisation and to determine its effects on various aspects of platelet function. Experiments were performed in hirudinized platelet-rich plasma (PRP) or whole blood obtained from human volunteers. PRP was treated with 10 microM CyH or vehicle, then activated by ADP. The effect of CyH on cytoskeletal reorganisation was determined by SDS-PAGE of the Triton X-100 insoluble cytoskeletons and quantitated by densitometry. Platelet aggregation and aggregate stability in PRP were measured by monitoring changes in light absorbance; aggregation was measured in whole blood via platelet counting. Shape change, P-selectin expression and changes in intracellular calcium were measured using flow cytometry. CyH prevented the normal incorporation of actin, alpha-actinin and actin-binding protein into the cytoskeleton that occurred following ADP activation, and incorporation of myosin was markedly reduced. Aggregation was only partially inhibited but, more dramatically, the rate of disaggregation following addition of certain agents that interfere with fibrinogen binding to glycoprotein IIb/IIIa on the surface of platelets was markedly increased. The ADP-induced shape change was also inhibited. CyH had no effect on calcium mobilisation. Curiously, expression of P-selectin was potentiated by CyH, suggesting a modulatory role of the cytoskeleton in platelet secretory activity. The results suggest that cytoskeletal reorganisation plays an important role in platelet shape change and aggregation and contributes in a major way to the stability of the aggregates that form.

Differential effects of three radiographic contrast media on platelet aggregation and degranulation: implications for clinical practice?

We have determined the effects of three radiographic contrast media on platelet aggregation and degranulation in vitro. Aggregation was measured as loss of single platelets, and degranulation was measured as P-selectin expression using flow cytometry. Iopamidol added to hirudinized blood induced aggregation directly and also potentiated that induced by weak platelet agonists such as adenosine diphosphate (ADP). Iodixanol also potentiated platelet aggregation, but ioxaglate inhibited it. Iopamidol also caused marked platelet degranulation. The pro-aggregatory effect of iopamidol was evident in non-anticoagulated blood as well as in hirudinized blood, but not in citrated blood. In platelet-rich plasma (PRP) prepared from hirudinized blood neither iopamidol nor iodixanol directly induced platelet aggregation, but they rendered platelets hypersensitive to ADP. ADP antagonists inhibited the platelet aggregation and degranulation induced by iopamidol in whole blood, whereas aspirin, an inhibitor of thromborane A2 synthesis, did not. These data are consistent with clinical reports of increased thromboembolic risk with non-ionic low-osmolar media, and raise concerns about the routine use of these contrast media during diagnostic and interventional arteriographic procedures. Routine use of citrate in previous experiments may have masked a pro-aggregatory effect of some contrast media.

Role of GPIIb-IIIa in platelet-monocyte and platelet-neutrophil conjugate formation in whole blood.

Platelets in stirred whole blood can be induced to form aggregates and also to form heterotypic platelet-monocyte (P/M) and platelet-neutrophil (P/N) conjugates. Here we have investigated the effects of three GPIIb-IIIa antagonists (GR144053F, MK-852 and Reopro, a CD62P-blocking antibody, GA6, and EDTA on the conjugate formation that occurs on stirring whole blood and in response to adding ADP and PAF. We have confirmed the identities of the conjugates by light microscopy after cell sorting. Platelet aggregation was measured by platelet counting. Monocytes, neutrophils, P/M and P/N were detected and quantitated using immunofluorescence and flow cytometry. Stirring whole blood resulted in both platelet aggregation and formation of P/M but not P/N. Adding ADP or PAF to whole blood caused rapid platelet aggregation and generation of both P/M and P/N. All of the GPIIb-IIIa antagonists studied had similar effects: inhibition of stirring-induced platelet aggregation and P/M formation, and inhibition of ADP-induced platelet aggregation and P/N formation. In contrast, they accelerated ADP induced-P/M conjugate formation and PAF-induced formation of both P/M and P/N. Both EDTA and GA6 completely inhibited P/M and P/N, which is commensurate with CD62P being involved in platelet-leucocyte conjugate formation. The results of these investigations suggest that GPIIb-IIIa has a dual role in determining the interaction between platelets and leukocytes.

Studies on the effects of agonists and antagonists on platelet shape change and platelet aggregation in whole blood.

The shape change that occurs when platelets are stimulated with an agonist can be quantitated by monitoring changes in their forward-scatter/side-scatter profile using a flow cytometer. Here we have stimulated platelets in citrated whole blood with several agonists and determined the time-course and extent of the shape change that occurs. In some experiments parallel investigations of shape change and aggregation were performed. Aggregation was measured by monitoring the fall in number of single platelets using a Whole Blood Platelet Counter. Some agents (ADP, PAF, U46619 and 5HT) produced a strong and rapid change in platelet forward-scatter/side-scatter that was maximal within 10 s. Others (A23187 and collagen) produced a strong but slower response. Adrenaline produced only a weak response that was also slow to develop, and PMA did not produce any response. The concentrations of each of ADP, PAF, U46619 and 5HT needed to induce a shape change were lower than those required for aggregation. Selective PAF, TXA2 and 5HT antagonists (WEB 2086, sulotroban and MCI-9042) clearly inhibited both the shape change and the aggregation induced by the appropriate agonist; in each case the effect of the antagonist was to move the dose-response curve to the right. These results are consistent with the shape change and aggregation brought about by each of these agonists being mediated via a single receptor. In contrast, a selective P2T purinoceptor antagonist (ARL 66096) markedly inhibited the aggregation induced by ADP but was found to have little or no effect on shape change. This is consistent with these platelet responses to ADP being mediated by different receptors, with P2T receptors mediating only the aggregation response.

Effects of ticlopidine administered to healthy volunteers on platelet function in whole blood.

Ticlopidine is thought to be a selective inhibitor of ADP-induced platelet function. Here we have investigated the effects of ticlopidine on platelet function in whole blood induced by ADP and by other platelet agonists. Whole blood was used because it was considered that ADP derived from red cells might act synergistically with other platelet agonists to enhance platelet responses, and that ticlopidine might interfere with this process. Measurements were performed using blood from 16 healthy volunteers before ticlopidine administration, after taking ticlopidine 250 mg daily for 10 days, after taking ticlopidine 250 mg twice daily for a further 10 days, and after 14 days off treatment. Ticlopidine proved to be a very effective inhibitor of the platelet aggregation induced by ADP; it was most effective in enhancing the reversibility of the aggregation response. The drug modestly but significantly reduced streptokinase, adrenaline, collagen, sodium arachidonate, PAF and U46619 - induced platelet aggregation. The drug significantly reduced the extent of the release reaction (14C-5HT release) induced by ADP, streptokinase, PAF, ristocetin and sodium arachidonate, and also reduced the extent of the synergistic 14C-5HT release induced by combinations of ADP and PAF, ADP and adrenaline and PAF and adrenaline. The various inhibitory effects of ticlopidine were evident after treatment with 250 mg daily but were more pronounced after 250 mg twice daily. All values had returned to normal after 14 days off treatment. Ticlopidine had no effect on serum thromboxane B2 production nor on several parameters of coagulation and fibrinolysis. We conclude that ticlopidine is an effective inhibitor of ADP-induced platelet aggregation and also the platelet aggregation and 14C-5HT release induced in whole blood by a number of platelet agonists and combinations of agonists. These latter effects are probably mainly via a selective effect on ADP. The inhibitory effects of the drug are dose-related.

Investigations on spontaneous aggregation and platelet responses to streptokinase and to adrenaline during pregnancy.

Platelets have been shown to be hyperresponsive during pregnancy. Studies in whole blood have revealed increased 'spontaneous' platelet aggregation (SPA) and increased aggregation and (14)C-5HT release in response to adrenaline. Here we have extended previous studies. We have explored the possibility that the nature of the agent used to anticoagulate the blood may have influenced the results obtained, and, for the first time, have investigated the effects of streptokinase (SK) on platelets in whole blood during pregnancy. We found that increased SPA is present during pregnancy irrespective of the anticoagulant used. Also, platelets in blood taken during pregnancy aggregate more extensively in response to adrenaline, even though the type of anticoagulant influences the extent of (14)C-5HT release that accompanies the aggregation response. SK was found to induce platelet aggregation in whole blood and this was also independent of the anticoagulant used. Further, SK induced aggregation at a lower concentration than in blood from non-pregnant female volunteers (NFV). Increased platelet responses had always returned to values similar to those for NFV by 12 weeks post-natal. These studies confirm the existance of a generalized increase in platelet reactivity during pregnancy, indicate that this is not an artefact consequent to the use of a particular anticoagulant, and provide new information on the effects of SK on platelets during pregnancy.