Aggregation Of Human Blood Platelets Research Articles

Aggregation and disaggregation kinetics of human blood platelets: Part II. Shear-induced platelet aggregation

A population balance equation (PBE) mathematical model for analyzing platelet aggregation kinetics was developed in Part I (Huang, P. Y., and J. D. Hellums. 1993. Biophys. J. 65: 334-343) of a set of three papers. In this paper, Part II, platelet aggregation and related reactions are studied in the uniform, known shear stress field of a rotational viscometer, and interpreted by means of the model. Experimental determinations are made of the platelet-aggregate particle size distributions as they evolve in time under the aggregating influence of shear stress. The PBE model is shown to give good agreement with experimental determinations when either a reversible (aggregation and disaggregation) or an irreversible (no disaggregation) form of the model is used. This finding suggests that for the experimental conditions studied disaggregation processes are of only secondary importance. During shear-induced platelet aggregation, only a small fraction of platelet collisions result in the binding together of the involved platelets. The modified collision efficiency is approximately zero for shear rates below 3000 s-1. It increases with shear rates above 3000 s-1 to about 0.01 for a shear rate of 8000 s-1. Addition of platelet chemical agonists yields order of magnitude increases in collision efficiency. The collision efficiency for shear-induced platelet aggregation is about an order of magnitude less at 37 degrees C than at 24 degrees C. The PBE model gives a much more accurate representation of aggregation kinetics than an earlier model based on a monodispersed particle size distribution.

Prevention of human pancreatic cancer cell-induced hepatic metastasis in nude mice by dipyridamole and its analog RA-233.

Several studies have provided evidence suggesting that platelets play a key role in tumor metastasis. A number of antiplatelet agents have been used to prevent tumor metastasis in animal models and humans. Antiplatelet agents, dipyridamole (adenosine transport inhibitor), and RA-233 (inhibitor of cAMP PDE) were used to prevent tumor-cell-platelet interactions both in in vitro and in vivo systems; however, the data were not very conclusive. Our studies used dipyridamole and RA-233 alone and in combination to investigate their effects on human pancreatic tumor cells (RWP-2)-induced platelet aggregation in human blood and on hepatic metastasis in nude mice. To examine effects of dipyridamole and RA-233 on liver metastasis, the tumor cells (RWP-2) were injected intrasplenically in nude mice grouped into control, dipyridamole (8 mg/kg), RA-233 (8 mg/kg), and dipyridamole plus RA-233 (8 mg/kg each). The agents were administered intraperitoneally 1 hour before and 24 hours after the tumor cell injection. When dipyridamole and RA-233 were used alone, only weak to moderate effects were seen on RWP-2 tumor cell-induced platelet aggregation. However, these agents, when combined, strongly inhibited the tumor cell-induced aggregation in human platelet-rich plasma. In tumor metastasis experiments, reductions of approximately 70% in hepatic nodules and 90% in surface area occupied by the tumor were seen with the combination treatment (dipyridamole plus RA-233) as compared with the control group of mice. This study suggests that the combination of dipyridamole and RA-233 provides an effective intervention for the antithrombotic approach to the treatment of cancer metastases.

Platelet Aggregation and Fibrinogen Binding in Human, Rhesus Monkey, Guinea-Pig, Hamster and Rat Blood: Activation by ADP and a Thrombin Receptor Peptide and Inhibition by Glycoprotein IIb/IIIa Antagonists

Platelet aggregation and fibrinogen binding in whole blood, induced either by ADP or by a 14 amino acid peptide mimicking an N-terminal region of the platelet thrombin receptor (TRP, thrombin receptor activating peptide), have been studied with blood from different species. Aggregation was assessed by counting the number of single platelets in blood before und after addition of the agonist with an automated cell counter. Both ADP (0.02-0.5 microM) and TRP (1-10 microM) were found to be potent agonists of platelet aggregation in human, rhesus monkey and guinea-pig blood, causing a near-maximal aggregatory response within 2 min of agonist addition. In contrast, hamster and rat platelets were much less responsive to both ADP and TRP in terms of the whole blood aggregation. Echistatin, RGDW and a synthetic glycoprotein (GP) IIb/IIIa antagonist, Ro 43-8857, inhibited fibrinogen binding to purified immobilized human GP-IIb/IIIa with IC50's of 1.6, 88 and 11.4 nM, respectively. In whole human blood, the respective IC50's (as determined by flow cytometric analysis of platelet fibrinogen binding) were 4.4, 1700 and 29.5 nM. The affinities of these three compounds for inhibiting fibrinogen binding in whole blood from rhesus monkeys and guinea-pigs were similar to the affinities for human platelets, but with rat blood echistatin, RGDW and Ro 43-8857 were all around 100-fold less potent. Ro 43-8857 was a potent inhibitor of ADP- or TRP-induced platelet aggregation in human, rhesus monkey and guinea-pig whole blood (IC50 of 69-320 nM) but was much less active in hamster blood.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypothesis for the receptors of human blood platelet aggregation and its inhibition by structure-activity relationship

We tried to clarify the size and the common charge distribution of the inhibition or stimulation of human platelet aggregation by structure-activity relationship. Numerous inhibiting and stimulating agents were able to enter the receptors. Inhibitory receptor had recess of 14 × 12.5Å in diameter. Stimulatory receptor had recess of 11 × 12Å in diameter. In the recess, there were three charges, two negative and one positive in the inhibitory receptor, and one negative and two positive in the stimulatory receptor, respectively. Charge distributions and conformation of inhibiting or stimulating agents were similar for the inhibitory agents, prostaglandin I 2 (PGI 2), PGD 2, PGE 1 adenosine and isoproterenol and conformation of the stimulating agents, thromboxane A 2 (TXA 2), platelet activating factor (PAF), adenosine diphosphate (ADP) and adrenaline. Each molecule had 3–10 inhibiting and stimulating conformations. The ratio of the number of conformations for inhibition and stimulating of platelet aggregation was highest for PGI 2 which showed the strongest inhibitory activity. TXA 2 was opposite in both respects.

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 IC 50 values for ADP (2 μM)-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 wtih ADP resulted in a dose-dependent disaggregation in platelet-rich plasma (IC 50 4.9 mU/ml), but not in whole blood. When both ATPDase (5.6–58.7 mU/ml) and ATP (0.5–10 μM) 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 consequences of these activities would be to control the growth of a platelet thrombus.

Separate induction of human blood platelet aggregation or cytotoxicity by different concentrations of PAF-acether and thrombin.

Using decreasing concentrations of PAF-acether or thrombin, it was possible to observe on human platelets, first, aggregation, classically associated to activation, then , below a threshold, cytotoxicity towards Schistosoma mansoni larvae, proposed here as stimulation. These two activities appeared as distinct and antithetic. However, their induction might be the consequence of triggering of the same receptors with different intensity, since PAF-induced, but not thrombin-induced, cytotoxicity could be inhibited with specific PAF-antagonists BN 52021 and BN 52024 also known to inhibit PAF-induced aggregation. These results give credit to the hypothesis that haemostatic and cytotoxic properties of platelets are two distinct functions of these blood elements.

Effects of pressure and gaseous anesthetics on the aggregation of human blood platelets and marine sponge cells: Similarities in responses

1. Aggregation of blood platelets and marine sponge cells was inhibited by nitrous oxide (N 2O) and helium (He) at elevated pressure but potentiated by xenon (Xe) despite the fact that Xe and N 2O are equipotent gaseous anesthetics. 2. The above aggregations are dependent on an extracellular source of calcium. 3. Platelet aggregation induced by phorbol myristate is independent of extracellular calcium and was inhibited by both N 2O and Xe and by high pressures of He. 4. The results argue against a common mechanism for Xe and N 2O and suggest that pressure may affect calcium interactions with binding site.

Triflusal vs aspirin on the inhibition of human platelet and vascular cyclooxygenase

1. 1. Triflusal is a salicylic derivative that inhibits platelet aggregation in human whole blood with a minimal inhibition of prostacyclin production. 2. 2. Aspirin inhibits platelet aggregation at concentrations that reduce vascular prostacyclin production.

5-Acyl-6-aryl-4-nitro-3(2H)pyridazinones and Related 4-Amino Compounds: Synthesis and Pharmacological Evaluation

Several 4-nitro- and 4-amino-5-acyl-6-aryl-3(2H)pyridazinones were prepared and their in vitro and ex vivo antiaggregatory properties were evaluated. 4-Nitro derivatives 3 generally showed good activity in vitro towards arachidonic acid (AA)-induced human blood platelet aggregation. The 4-amino compound 4a, which has weak in vitro activity, exhibited antiplatelet activity, particularly on adenosine dephosphate (ADP)-induced aggregation ex vivo in rabbit. Moreover, the same compound was shown to be active in platelet-activating factors (PAF)- induced rat paw hyperalgesia and to be endowed with low acute oral toxicity. The 4-amino derivatives 4a–m and the other pyridazinones 5–9 administered orally to rats were also found to be more potent antiinflammatory agents than acetyl salicylic acid (ASA). Compounds 3a and 4a, tested in vitro on lipopolysaccharide (LPS)-stimulated rat peritoneal macrophages, were seen to be active in the inhibition of prostaglandin E2 (PGE2) production and interleukin-1 activity. Structure–activity relationship studies in the series of antiaggregating pyridazinones 3 have shown the primary importance of the nitro and acetyl substituents at positions 4 and 5, respectively. Hydrophobic substituents at position 2 were also required for better activity.

Antibiotics from basidiomycetes. XXXVIII. 2-Methoxy-5-methyl-1,4-benzoquinone, a thromboxane A2 receptor antagonist from Lentinus adhaerens.

In the course of our screening for antithrombotic compounds using platelet rich plasma from bovine slaughter blood, 2-methoxy-5-methyl-1,4-benzoquinone (1) has been isolated from mycelial cultures of Lentinus adhaerens. The compound inhibits the U46619-induced aggregation of human blood platelets with an IC50 of 2.5 micrograms/ml (16.45 microM) and is a new thromboxane A2 receptor antagonist. This is the first report on an inhibitor of platelet aggregation derived from secondary metabolism of basidiomycetes.

Endothelium-dependent relaxant effect of thrombocytin, a serine proteinase from Bothrops atrox snake venom, on isolated pig coronary arteries

E. Glusa, H. Brauns and K. Stocker. Endothelium-dependent relaxant effect of thrombocytin, a serine proteinase from Bothrops atrox snake venom, on isolated pig coronary arteries. Toxicon 29, 725–732, 1991.—Since thrombin causes an endothelium-dependent relaxation of precontracted pig coronary arteries, the ability of thrombocytin, a serine proteinase from the venom of the common lancehead, Bothrops atrox, to induce endothelium-dependent changes in the vascular tone was investigated. Relaxation of pig coronary rings did not appear in vessels denuded of the endothelium. Thrombocytin (0.1–2.0 μg/ml) caused an endothelium-dependent, reversible, transient relaxation of PGF 2α-precontracted arteries which could be blocked by heparin and relatively high concentrations of α-NAPAP, a synthetic competitive thrombin inhibitor. Indomethacin and hirudin did not influence the relaxant effect. Both the thrombocytin- and bradykinin-induced relaxation were diminished by the guanylate cyclase inhibitor methylene blue and by N G-monomethyl- l-arginine. The thrombocytin-induced relaxation was absent in de-endothelialized vessels. Thrombocytin was able to induce aggregation of human blood platelets in Tyrode's solution at the same concentration range as used for the relaxation. Batroxobin neither relaxed precontracted arteries nor aggregated human blood platelets in vitro. The present studies show that the serine proteinase thrombocytin is not only able to aggregate platelets but may also release endothelium-derived relaxing factor from the vascular endothelium.

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.

Dipyridamole alone or combined with low‐dose acetylsalicylic acid inhibits platelet aggregation in human whole blood ex vivo [see comments

1. In a randomized, double-blind trial we compared the inhibition of the platelet-vessel wall interactions in whole blood ex vivo. There were four groups of 24 healthy volunteers each of whom were treated orally for 3.5 days with either 200 mg dipyridamole (sustained release preparation), 25 mg acetylsalicylic acid, both drugs combined or placebo twice daily. 2. The mean area of all platelets/aggregates was reduced by 6.2% +/- 4.2% (+/- s.e. mean) by placebo (n = 23), 19.8% +/- 6.7% by dipyridamole (n = 22), 53.7% +/- 4.9% by acetylsalicylic acid (n = 23) and 71.4% +/- 3.7% by the combination of both drugs (n = 24), when compared with total inhibition of aggregation by EGTA. Thus, low-dose acetylsalicylic acid inhibited aggregation (P less than 0.001). 3. Dipyridamole reduced the size of platelet aggregates (P less than 0.01, two-fold analysis of variance). The reduction was correlated with the individual dipyridamole plasma levels (P less than 0.05, analysis of covariance). The subgroup of large and very large thrombi being formed was also reduced by dipyridamole (P less than 0.05). 4. This ex vivo study demonstrates that dipyridamole alone inhibits formation of thrombi on subendothelial matrix and enhances the inhibitory effect of low dose acetylsalicylic acid in this model of thrombosis.

A comparison of the effects of three positive inotropic agents (amrinone, milrinone and medorinone) on platelet aggregation in human whole blood

Amrinone, milrinone and medorinone inhibit platelet aggregation in human whole blood. They are particularly potent inhibitors of arachidonic acid induced aggregation, inhibiting by 50% (IC 50) at concentrations of 1.5μM (milrinone), 7.5μM (medorinone) and 48μM (amrinone). Each drug was less potent at inhibiting ADP and collagen-induced aggregation. The rank order for inhibition of arachidonic acid - induced aggregation correlated well with the rank order of cyclic AMP phosphodiesterase inhibition for these drugs when coupared to the response of a reference cAMP phosphodiesterase inhibitor (CI-930) and a reference cAMP phosphodiesterase inhibitor (M & B 22948). Since inhibition of platelet aggregation in vitro occurred at clinically relevant concentrations, it is evident that these agents have potentially beneficial antithrombotic properties.

Synthesis of new 2-, 3-, and 4-substituted azidoquinolines: inhibitors of human blood platelet aggregation in vitro

A series of 2-, 3- and 4-substituted azidoquinoline derivatives were synthesized and tested for their ability to inhibit human platelet aggregation in vitro triggered by adenosine diphosphate (15 μM), collagen (5 μg/ml), platelet activating factor (10 μM), or the stable prostaglandin H 2 mimetic, U46619 (4 μM). The most active compounds (IC 50 2.5–68.3 μM) were the germinal 3,3-diazides ( 4f and 4g) and the 4-azido-3-nitroquinolines ( 6f and 6g).

Some novel inhibitors of platelet aggregation: Acute toxicity in mice and its relationship to in vitro efficacy and toxicity : II. Nipecotoylaminoalkane and nipecotoylpiperazine congeners

Four closely related nipecotoyl congeners are employed as molecular probes to evaluate the effects of systematic molecular changes upon lethal potency of the compounds. The in vivo toxicities, effected by changes in molecular structure, are compared to their in vitro concentrations inhibiting ADP-induced aggregation and epinephrine-induced primary aggregation of human blood platelets and their toxicities to mouse fibroblasts (L-929 cells) in culture. To assist in the selection of compounds which offer the greatest promise as therapeutic agents for further evaluation and to guide future development of optimal molecular structures, a ratio of acute ip LD50 (μmol/kg) [ T m] to concentration inhibiting 50% ADP-induced platelet aggregation (μmol/liter) [ A] is calculated for each compound. These ratios range from 2 41 to 24.92 for the four compounds included in this study.

ISOLATED HUMAN BLOOD PLATELETS DISCRIMINATE BETWEEN ANAESTHETIC AND NON-ANAESTHETIC GASES AT HIGH PRESSURES

We have compared the effects of the anaesthetic gases nitrogen and argon on adenosine diphosphate (ADP)-induced human blood platelet aggregation with the effects of the non-anaesthetic gas helium. All three gases showed dose-dependent inhibition of platelet aggregation. For nitrogen and argon there was a linear relationship between gas pressure and inhibition of aggregation over the range 15-68 atmospheres absolute (atm abs), whereas helium had a threshold for inhibition of approximately 34 atm abs. The inhibition by all gases was reversible after slow decompression. At pressures greater than 55 atm abs, nitrogen produced less inhibition than helium, indicating anaesthetic-pressure antagonism. Whereas pressure alone and the anaesthetic gases inhibited aggregation, the platelet shape change elicited by ADP was resistant to both nitrogen and helium, indicating that ADP binding and the early events in platelet activation were relatively unaffected by these conditions.

Effect of moderate fish intake on platelet aggregation and ATP release in human blood

A study to investigate the effect of a reasonable amount of dietary fish on collagen-induced platelet aggregation and ATP release in citrated whole blood is described. Two groups of twenty healthy male volunteers were given a daily dietary supplement consisting of 135 g of tinned mackerel paste (experimental group) or meat paste (control group) for a 6 week period. Compliance, calculated on the basis of the urinary excretion of lithium which was added to the supplements, was about 80%. The mackerel supplement had a weak but significant inhibitory effect on collagen-induced platelet aggregation (impedance method) and ATP release (firefly assay) in whole blood. Platelet release was significantly related to the compliance.

Relationships between chemical structure and inhibition of epinephrine-induced human blood platelet aggregation

The effect of structural features in a series of carbamoylpiperidine and nipecotoylpiperazine congeners on epinephrine-induced aggregation of human blood platelets is examined. Epinephrine-induced primary aggregation is effectively inhibited by the nipecotoylpiperazine derivatives (culminating at an I PA50 of 11.9 μM). While among nipecotoylpiperazine as well as carbamoylpiperidine congeners there are potent inhibitors of ADP-stimulated platelet function (cresting at an I A50 of 12.4 and 11.4 μM, respectively), the carbamoylpiperidine analogs are much less active (e.g., I PA50 of 298.1), or practically inactive, in impeding epinephrine-induced primary aggregation (PA).

Relationships between chemical structure and inhibition of ADP-stimulated human thrombocyte release of serotonin and platelet factor 4

The inhibitory potencies of carbamoylpiperidinoalkane and N-alkylnipecotoylpiperazine derivatives on ADP-stimulated human blood platelet aggregation, serotonin (5-HT) release and platelet factor 4 (PF-4) release were evaluated. The procedure was designed to allow concurrent determination of all three sets of values. Most compounds were more than twice as potent in blocking PF-4 (X = 91 +/- 1 (S.E., n = 7)%) compared to their inhibition of 5-HT (X = 38 +/- 1(S.E., n = 6)%) release; the one compound which failed to meet these criteria was still decidedly more powerful in impeding PF-4 than 5-HT release. Since the compounds' platelet aggregation-inhibitory values were within the same range as their 5-HT release-blocking potencies, but had a strikingly greater impact in arresting PF-4 release, it is suggested that the platelet plasma membrane and the lining enveloping the dense bodies may share certain commonalities, while the sheathing encasing the alpha-granules may differ from both in a tangible manner.