The use of laser-light scattering and controlled shear in platelet aggregometry

Laser-light scattering was used to observe and quantify the dynamics of human blood platelet aggregation in platelet-rich plasma (PRP). Aggregation was performed in a controlled shear environment by placing the PRP in the annular space between a rotating cylindrical rod and a stationary cylindrical tube. The instrument was capable of very sensitive continuous semi-quantitative measurements of chemically-induced microaggregation. As a demonstration of the technique, results are presented for ADP-induced aggregation at doses of 10, 1, and 0.1 microM and collagen-induced aggregation at a dose of 5 micrograms/ml, each at shear rates of 1,000 s-1 and 500 s-1. Extensive aggregation was observed in response to ADP at even the low dose of 0.1 microM, indicating a high sensitivity to microaggregates. The sensitivity of the ultimate size of the ADP-induced aggregates to ADP concentration was shear dependent. The formation of microaggregates by collagen stimulation was shown to be almost immediate, as contrasted with a 10-20 s typical lag when observed turbidometrically. Disaggregation was observed with 1 microM ADP, but this was only partial, as contrasted with the complete recovery of transmittance observed in the turbidometric technique. Electronic particle sizing and counting was employed to semiquantitatively verify the aggregate size distributions found from mathematical conversion of the laser-light scattering data.     

Rapid change of platelet aggregability in acute hyperglycemia. Detection by a novel laser-light scattering method

We examined the alteration of platelet aggregability in acute hyperglycemia during 75-gram oral glucose tolerance tests (OGTT). Twenty subjects underwent 75-gram OGTT and venous blood samples were obtained before (0 min), 60, 120 and 180 min postload. Platelet aggregability shown as the number of small platelet aggregates was measured with a novel laser-light scattering (LS) method. Platelet aggregability increased in parallel with both glucose and immunoreactive insulin (IRI) levels. The number of mean small aggregates at 60 min (12.30 +/- 1.10 X 10(4)) was significantly higher than the one at 0 min (8.32 +/- 0.88 x 10(4), p <0.001), 120 min (10.63 +/- 0.98 x 10(4), p <0.05) and 180 min (8.28 +/- 0.84 x 104, p <0.001) (mean +/- SEM). Small aggregates correlated positively with plasma glucose levels at 60 min postload (r = 0.67, p = 0.001) while not with IRI. It might be important to suppress transient hyperglycemia for preventing the onset of acute coronary syndromes that could be closely related to platelet hyperaggregability.     

Spin-labelled human platelets.

Human platelets were labelled with a variety of lipophilic and thiol reagent spin probes. Aggregation was not affected by the lipid probes but was blocked by the covalent protein-directed probes. ESR spectra before and after thrombin treatment indicated that lipid structure of the platelet membrane was not affected to any measurable extent by the thrombin interaction, although there was a change in the partition coefficient of the probe between solution and the membrane. From the fact that four different probes for different parts of the membrane lipid showed the same result, we conclude that thrombin interaction and resultant changes in the platelet membrane do not involve any substantial change in lipid organization.     

Measurement of fibrinogen concentrations in suspensions of washed rabbit and human platelets by radioimmunoassays

Although fibrinogen is a cofactor in platelet aggregation, washed rabbit platelets aggregate when stimulated with ADP even when no fibrinogen is added to the platelet suspension. Washed human platelets usually do not aggregate to a significant extent when stimulated with ADP unless fibrinogen is added. To study this phenomenon, radioimmunoassays for rabbit and human fibrinogens have been developed and used to measure fibrinogen concentrations in suspensions of washed platelets. The fibrinogen concentration in the suspending medium of rabbit platelets was 2.5 +/- 0.9 micrograms/10(9) platelets, and upon stimulation with 9 microM ADP it increased to 10.7 +/- 2.9 micrograms/10(9) platelets. The loss of fibrinogen from the platelets was significantly greater than the loss of 14C-serotonin (11% vs 2%). The presence of prostaglandin E1 reduced the fibrinogen concentration to approximately 1 micrograms/10(9) platelets and prevented aggregation and loss of fibrinogen when the platelets were stimulated with ADP. With human platelets, the extracellular concentrations of fibrinogen and beta-thromboglobulin, expressed as percentages of the amount in the platelets, were similar, and the increase in fibrinogen concentration upon ADP stimulation (approximately 2%) was much lower than with rabbit platelets. We conclude that rabbit platelets may release fibrinogen from their alpha-granules when stimulated with ADP, and that a portion of the released fibrinogen becomes available to support aggregation. Smaller amounts of fibrinogen would become available in the case of human platelets.     

The prognostic value of small-sized platelet aggregates in unstable angina: detection by a novel laser-light scattering method

Platelet activation plays a pivotal role in the pathogenesis of acute coronary syndromes. This study was designed to evaluate the platelet aggregability in patients with unstable angina using a new aggregometer with laser-light scattering. We also examined whether there was a relationship between these platelet aggregabilities and unfavorable outcome during in-hospital stay. We measured platelet aggregability, in particular small-sized platelet aggregates in 31 patients with unstable angina, 31 patients with stable exertional angina, and 30 patients with chest pain syndrome. The patients with unstable angina were divided into two groups by their cardiac events during in-hospital stay, cardiac events (+)(n=11) group and cardiac events (-)(n=20) group. On admission, the number of small-sized platelet aggregates (V) was higher in patients with unstable angina (3.0+/-0.5x10(4)) than in those with stable exertional angina (1.4+/-0.3x10(4), P=.017) and chest pain syndrome (0.7+/-0.2x10(4), P=.0003). The number of small-sized platelet aggregates was higher in the cardiac events (+) group than in the cardiac events (-) group (5.5+/-0.9x10(4) vs. 1.6+/-0.4x10(4), P=.0001). A previous study elucidated that small-sized platelet aggregates ultimately developed into medium-sized and large-sized aggregates as platelet aggregation proceeds. Therefore, the production of small-sized platelet aggregates is more sensitive for hyperaggregability. Furthermore, the production of small-sized platelet aggregates increased significantly in patients with unstable angina than in those with stable exertional angina and chest pain syndrome. These findings suggest that a tendency toward thrombus formation increases markedly in patients with unstable angina and increased number of small-sized platelet aggregates on admission predicts poor prognosis during in-hospital stay in patients with unstable angina.     

Measurement of GPV released by activated platelets using a sensitive immunocapture ELISA--its use to follow platelet storage in transfusion.

Thrombin, the most potent platelet agonist, plays a central role in haemostasis and in the occurrence of thrombotic events. This agonist activates platelets by cleaving the PAR G-protein coupled receptors and by binding to glycoprotein (GP) Ib and also cleaves GPV at the platelet surface to liberate the soluble 69 kDa fragment GPVf1. Monoclonal antibodies (MoAbs) to GPV were developed as tools to study the mechanism of platelet GPV cleavage and measure release of GPV in pathological situations. Specificity of the MoAbs for GPV was confirmed by flow cytometry and immunoprecipitation of proteins from human platelets and Dami megakaryocytic cells. A sensitive immunocapture sandwich ELISA for soluble GPV was developed using two MoAbs recognizing different epitopes of GPV and purified platelet or recombinant GPV as reference protein. This ELISA was employed to determine the mean plasma concentration of GPV in 100 normal individuals (17.3 ng/ml), to demonstrate the dose-dependent release of GPVf1 from washed platelets stimulated with thrombin and to follow the progressive release of GPVf1 during storage of therapeutic platelet concentrates. The present report describes a sensitive GPV ELISA of direct application to survey the processing and storage of platelet concentrates for transfusion and of potential value to monitor platelet activation in thrombotic states.     

Properties of washed human platelets.

We have shown previously that washed human platelets resuspended in Tyrode solution containing albumin and apyrase maintain their disc shape and their ability to aggregate upon the addition of low concentration of ADP, providing fibrinogen is added to the suspending medium. We have now examined their responses to other aggregating and release-inducing agents. Collagen, arachidonate, thrombin, immune serum globulin, the ionophore A23, 187 and phytohaemagglutinin from Phaseolus vulgaris caused aggregation and release of granule contents. The response to adrenaline was variable. Serotonin caused the platelets to change shape but no aggregation or release occurred. Addition of a small amount of plasma was necessary for ristocetin-induced aggregation. Polylysine caused immediate platelet-to-platelet adherence with little or no release of granule contents. Responses to collagen or thrombin were greater in a modified medium containing magnesium but no calcium; in this medium, aggregation caused by ADP or polylysine was followed by the release of granule contents whereas these agents caused aggregation without release in a medium with both calcium and magnesium. When protein was omitted from the suspending medium, platelet aggregation in response to ADP was variable. In this medium, collagen and thrombin caused more extensive release than in the albumin-containing medium. Aggregation by polylysine was accompanied by release and extensive lysis in the protein-free medium. Thus, the composition of the final resuspending medium has a major effect on the responses of washed human platelets to aggregating agents.     

Assessment of omega-fatty-acid-supplemented human platelets for potential improvement in long-term storage

Uptake of omega (omega)-3 fatty acids can influence membrane stability and cell mobility. We investigated the effects of omega-3 and -6 fatty acids on the hemostatic efficacy of human platelets using an in vivo rabbit bleeding model. In vitro assays such as platelet aggregation, vWF bead-mediated ATP release and platelet adhesion to beads (measured by the residual platelet count [RPC] [free platelet count after reacting with the beads]/[baseline platelet count]x 100=%RPC; a high %RPC indicates reduced platelet function) were conducted on platelets treated with 1% fish oil (omega-3); 2% fish oil emulsion or 1% soy oil (omega-6). Oil treatment of platelets reduced the vWF bead-induced ATP release insignificantly. Addition of omega-3 agents reduced physical reactivity (%RPC) with the vWF beads by a factor of 1.2 (oil) and 1.9 (emulsion). The omega-6 oil enhanced reactivity by a factor of 1.7. After washing to remove excess reagent, platelet resuspension was most efficient with the omega-3 emulsion. Platelet function was higher with the omega-3-treated platelets (%RPC=52.3%, omega-3 oil; 63.3%, omega-3 emulsion vs. 85%, omega-6 oil; 82% untreated platelets). Ethyl-palmitate-treated thrombocytopenic rabbits were infused with human platelets. Survival times of the treated platelets, as monitored by flow cytometry (6.2-8.2 h) were comparable to untreated platelets (8.6 h). In the rabbit kidney injury model, blood loss after infusion of the treated platelets was similar to that of saline-infused rabbits (75.3+/-3.4 g). However, platelets washed prior to infusion reduced blood loss to a value comparable to that of fresh platelets (48.3+/-5 g). Furthermore, the presence of the infused platelets at the injury site was clearly visualized using FITC-tagged anti CD42a antibody. Thus, the omega-3-based agents protect the platelets from damage during the washing procedure as demonstrated in vitro by improved platelet resuspension, low %RPC, high stimulus-responsive ATP secretion and a reduction in blood loss in vivo.     

Deaggregation of human platelets aggregated by thrombin

Human platelets that have undergone the release reaction do not deaggregate readily. We examined conditions under which washed human platelets can be deaggregated after they have undergone an extensive release reaction induced by thrombin (1 or 5 U/ml). To make fibrinogen receptors unavailable, either CP/CPK (or apyrase) was used to remove released ADP, or PGE1 was used to increase cAMP. Chymotrypsin was used to digest proteins that might link platelets, and heparin to interact with released proteins and interfere with their binding to platelets and to each other. Individually, none of these caused deaggregation; heparin did not inhibit the effect of thrombin because no antithrombin III was present. Platelets exposed to thrombin (1 U/ml) which was neutralized at 90 sec by hirudin, could be deaggregated by combinations of CP/CPK (or apyrase) and chymotrypsin, or PGE1 and chymotrypsin. When a higher concentration of thrombin was used (5 U/ml) these combinations caused platelets to deaggregate only when heparin was added before thrombin induced the release reaction. Thus, when extensive release occurs three mechanisms may come into play to link human platelets: one that requires the fibrinogen receptor; a heparin-sensitive reaction that may involve the binding of released proteins; and a linkage that can be disrupted only by proteolysis, providing the other two mechanisms are also inhibited.     

Reaction of acetaldehyde with human platelets.

Platelet function defects observed in chronic alcoholics are not wholly explained by the inhibitory action of ethanol on platelet aggregation; they are not completely reproduced either in vivo by short-term ethanol perfusion into volunteers or in vitro by the addition of ethanol to platelet-rich plasma. As acetaldehyde (AcH) binds to many proteins and impairs cellular activities, we investigated the effect of this early degradation product of ethanol on platelets. AcH formed adducts with human platelets at neutral pH at 37 degrees C which were stable to extensive washing, trichloracetic acid hydrolysis and heating at 100 degrees C, and were not reduced by sodium borohydride. The amount of platelet adducts formed was a function of the incubation time and of the concentration of AcH in the reaction medium. At low AcH concentrations (less than 0.2 mM), platelet bound AcH was directly proportional to the concentration of AcH in the reaction medium. At higher concentrations (greater than or equal to 0.2 mM), AcH uptake by platelets tended to reach a plateau. The amount of adducts was also proportional to the number of exposures of platelets to pulses of 20 microM AcH. AcH adducts formation severely impaired platelet aggregation and shape change induced by ADP, collagen and thrombin. A positive correlation was established between platelet-bound AcH and inhibition of aggregation. SDS-PAGE analysis of AcH adducts at neutral pH demonstrated the binding of [14C]acetaldehyde to many platelet proteins. AcH adduct formation with membrane glycoproteins, cytoskeleton and enzymes might interfere with several steps of platelet activation and impair platelet aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transient receptor potential protein subunit assembly and membrane distribution in human platelets

We have previously suggested that the human homologue of the Drosophila transient receptor potential protein, TRPC1, is involved in conducting store-operated Ca2+ entry (SOCE) in human platelets since an antibody raised against the pore-forming region of TRPC1 inhibited SOCE. Here we have investigated plasma membrane expression of TRPC1 in human platelets and have probed for the presence of other TRPC proteins in these cells. Biotinylation revealed the presence of TRPC1 in the plasma membrane of resting platelets. Surface expression was not detectibly changed following Ca2+ store depletion or stimulation with thrombin. Western blotting demonstrated the presence of TRPC1, TRPC3, TRPC4, TRPC5 and TRPC6 in platelet lysates. TRPC1, TRPC4 and TRPC5 coimmunoprecipitated, as did TRPC3 and TRPC6. TRPC1, TRPC4 and TRPC5 were associated with detergent-resistant platelet membranes, from which they were partially released when the cells were cholesterol-depleted using methyl-beta-cyclodextrin. The distributions of TRPC3 and TRPC6 between soluble and membrane fractions were not affected by methyl-beta-cyclodextrin treatment. These results suggest that TRPC1, TRPC4 and TRPC5 form a heteromultimer associated with platelet lipid raft domains, whereas TRPC3 and TRPC6 associate independently of lipid rafts.     

Kinetics of merthiolate-induced aggregation of human platelets.

Incubation of human platelet-rich plasma (PRP) or washed platelets with merthiolate (MT; sodium ethylmercurithiosalicylate; an inhibitor of lysophosphatide: arachidonoyl transferase) leads to irreversible platelet aggregation which is parallelled by an increase in thromboxane A2 synthesis. MT-induced aggregation is preceded by a pronounced lag-period (0.5-10 min). Duration of the latter is inversely related to the concentration of MT ([MT]). Platelet responses to MT are similar to those triggered by arachidonate (AA) in that the relationships of the aggregation rates both to [MT] and [AA] are threshold and exhibit characteristic super-high values of the apparent Hill coefficients (h > 30). A typical MT-induced response can be subdivided in two sequential phases: i) cyclooxygenase-independent slow aggregation, and ii) indomethacin-abrogated rapid aggregation. MT-induced responses are blocked by PGE1 or ajoene (which inhibits binding of fibrinogen to its cell surface receptor, GPIIb/IIIa). The obtained data are interpreted both quantitatively and qualitatively in terms of a model assuming the existence of: i) a relationship between the rate of MT-inhibitable AA incorporation into phospholipids and the concentration of intracellular free AA, [AA]i; ii) a certain threshold value of [AA]i essential for triggering the second phase of the aggregation.     

Influence of human ceruplasmin on the aggregation of human platelets by adenosine diphosphate

The effect of a glycoprotein, human ceruloplasmin, on the aggregation of washed human platelets induced by ADP was found to be similar to that of normal fibrinogen. Periodate oxidation and neuraminidase treatment of ceruloplasmin destroyed its ability to potentiate ADP induced platelet aggregation. Since similar observations are found with fibrinogen, it can be concluded that the glycosidic moiety of those proteins plays an important role in platelet aggregation.     

Effect of metabolic inhibitors on thrombin-induced membrane potential changes in washed human platelets

Platelet stimulation by thrombin or other agonists is influenced by the metabolic status of the platelet, i.e., by the available supply of metabolic ATP. We have shown earlier that such stimulation by thrombin is accompanied by a dose-dependent depolarization of the platelet transmembrane potential and by a decrease in the transmembrane pH gradient. We have now studied the effect of metabolic inhibitors on the membrane potential changes as assessed with the fluorescent cation 3, 3′-dipropylthiodicarbocyanine. Preincubation of platelets with either 2-deoxy-D-glucose or antimycin A leads to a partial depolarization of the resting platelet's membrane. These pre-incubated platelets then exhibit a decreased membrane potential change upon α-thrombin stimulation.It is known that the platelet continuously replenishes its glycogen. The availability of such energy stores may be responsible for the restoration of approximately 20% of the thrombin response which we have observed after 3 hours of incubation with antimycin A. The intracellular glycogen does not appear to play a major role in the early platelet response to thrombin which is unimpaired 30 minutes after the completion of gel-filtration, the time at which the intracellular glycogen levels are at their lowest. These studies indicate that the thrombin-induced platelet membrane potential change, like other steps in platelet activation, depends upon maintenance of continuous metabolic function.     

Triglyceride lipase activity and human platelets.

The activity of triglyceride lipases in human postheparin plasma is significantly higher in platelet rich than platelet poor plasma. This holds for total activity, lipoprotein lipase (LPL) activity, and hepatic triglyceride lipase (H-TGL) activity. Gel filtration of platelet rich postheparin plasma on Sepharose 2 B will separate platelets from triglyceride lipase activity. The very small triglyceride lipase activity of isolated platelets is inhibited by 1.0 M NaCl, slightly inhibited by specific antibody to hepatic lipase, and not influenced by specific antibody to lipoprotein lipase.     

Surface labeling of human platelets by reductive methylation

A simple, reproducible method for the specific labeling of the surface proteins of human platelets with tritium is described. The labeled platelets were analyzed by two-dimensional gel electrophoresis (isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis) followed by fluorography and the results compared with those obtained by conventional methods. Reductive methylation gave an intense labeling of membrane glycoproteins. There was no cross-linking of the labeled membrane proteins by formaldehyde nor could clear evidence be found that inner proteins were labeled by permeation of the reagents through the plasma membrane. As well as previously described platelet membrane glycoproteins, several others could be detected that were not invariable seen using labeling techniques.     

Quantitative isolation of RNA from human platelets.

We have adapted the acid-guanidinium-phenol-chloroform extraction procedure of Chomczinsky and Sacchi to achieve efficient rapid recovery of total RNA from human platelets. Sufficient platelet RNA (20 micrograms of total RNA per 30 ml of whole blood) can be recovered from relatively small individual samples to perform Northern blot analysis on individual donors and detect the mRNAs for glycoproteins IIb1(GP IIb) and IIIa1(GP IIIa), 3.4 kb and 6.2 kb, respectively. Platelet GP IIb and GP IIIa mRNAs could also be reverse transcribed, and amplified in vitro by the polymerase chain reaction (PCR). Thus, our technique allows simultaneous Northern blotting and PCR, and therefore should be of great help to the characterization of inherited platelet disorders such as Glanzmann's thrombasthenia.     

Ionophoretic activities of phospholipids on human platelets.

The effects of phospholipids on calcium uptake by human platelets were investigated utilizing 45CaCl2. Among the phospholipids tested, cardiolipin and phosphatidic acid exerted a significant increase of calcium uptake by platelets. Calcium uptake was dependent upon cardiolipin concentration and incubation time. These phospholipids enhanced platelet aggregation induced by ADP (2 microM) or epinephrine (0.5 microgram/ml). Cardiolipin caused the significant release of serotonin from platelets in the absence of externally added calcium in the medium. These results may suggest that phospholipids activate platelets through their ionophoretic activities.