Rabbit Platelet-rich Plasma Research Articles

Visualization of the Platelet-Plasma Interface

Morphological and cytochemical studies of platelets have reported a surface coat, or glycocalyx, external to the plasma membrane (1). Biochemical analyses have likewise confirmed the highly adsorptive properties of platelets as transporters of coagulation factors (2). However, visualization of the platelet membrane by conventional EM procedures does not reflect this special relationship between the platelet and its plasma environment. By the routine method of alcohol-propylene oxide dehydration for Epon embedding, the lipid bilayer nature of the platelet membrane appears similar to other blood cells (Fig. 1). A new rapid embedding technique using dimethoxypropane (DMP) as dehydrating agent (13) has permitted ultrastructural analyses of the surface features of the platelet-plasma interface.Aliquots of human or rabbit platelet-rich plasma (PRP) were added to equal volumes of 6% glutaraldehyde in Millonig's buffer at 37° for 45 minutes, rinsed in buffer and postfixed in 1% osmium in Millonig's buffer for 45 minutes.

Inhibition and Potentiation of Platelet Function by Lysolecithin

The effects of lysolecithin (LPC) on aggregation, serotonin release, shape, and lysis of rabbit, pig, or human platelets in platelet-rich plasma (PRP) or Tyrode albumin solution were examined during prolonged incubation. LPC added to citrated or heparinized PRP from humans or rabbits at a final concentration above 100 muM caused instantaneous inhibition of platelet aggregation induced by adenosine diphosphate (ADP), epinephrine (human PRP only), collagen, or thrombin. The inhibitory effect of LPC was found to be partially reversible over a period of 60-90 min. LPC at final concentrations above 30 muM also caused inhibition of ADP-, collagen-, and thrombin-induced aggregation and collagen- and thrombin-induced release of serotonin in suspensions of rabbit, pig, or human platelets. With washed platelets, the inhibitory effect not only rapidly disappeared but was followed by transient potentiation of aggregation and serotonin release. This potentiating effect of LPC was most pronounced when thrombin was used as stimulus. Both inhibition and potentiation were observed at concentrations of LPC that did not cause a significant change in platelet shape or loss from platelets of lactic dehydrogenase. Inhibition and potentiation were also observed when platelets were added to suspending medium containing LPC, although considerably higher concentrations of LPC were required under these conditions. Potentiation was not observed when LPC was added to citrated or heparinized rabbit or human PRP or to washed rabbit platelets suspended in a medium containing 4% bovine serum albumin. It seemed likely that some or all of the observed effects of LPC on platelet function were due to structural modification of the platelet membrane insufficient to result in gross membrane damage or platelet lysis. In addition, the results of experiments using 14C-LPC seemed to indicate that the observed potentiating effect of LPC on platelet function may be related to its rapid uptake and metabolism by the platelets.

Inhibition and potentiation of platelet function by lysolecithin

The effects of lysolecithin (LPC) on aggregation, serotonin release, shape, and lysis of rabbit, pig, or human platelets in platelet-rich plasma (PRP) or Tyrode albumin solution were examined during prolonged incubation. LPC added to citrated or heparinized PRP from humans or rabbits at a final concentration above 100 muM caused instantaneous inhibition of platelet aggregation induced by adenosine diphosphate (ADP), epinephrine (human PRP only), collagen, or thrombin. The inhibitory effect of LPC was found to be partially reversible over a period of 60–90 min. LPC at final concentrations above 30 muM also caused inhibition of ADP-, collagen-, and thrombin-induced aggregation and collagen- and thrombin-induced release of serotonin in suspensions of rabbit, pig, or human platelets. With washed platelets, the inhibitory effect not only rapidly disappeared but was followed by transient potentiation of aggregation and serotonin release. This potentiating effect of LPC was most pronounced when thrombin was used as stimulus. Both inhibition and potentiation were observed at concentrations of LPC that did not cause a significant change in platelet shape or loss from platelets of lactic dehydrogenase. Inhibition and potentiation were also observed when platelets were added to suspending medium containing LPC, although considerably higher concentrations of LPC were required under these conditions. Potentiation was not observed when LPC was added to citrated or heparinized rabbit or human PRP or to washed rabbit platelets suspended in a medium containing 4% bovine serum albumin. It seemed likely that some or all of the observed effects of LPC on platelet function were due to structural modification of the platelet membrane insufficient to result in gross membrane damage or platelet lysis. In addition, the results of experiments using 14C-LPC seemed to indicate that the observed potentiating effect of LPC on platelet function may be related to its rapid uptake and metabolism by the platelets.

Competitive Inhibition by Lithium and Hydrogen lons of the Effect of Calcium on the Aggregation of Rabbit Platelets

ADP-induced platelet aggregation and shape change were monitored optically in citrated rabbit platelet-rich plasma (PRP) diluted with isotonic salt solutions. Lithium (Li) produced a concentration-dependent reduction in the rate of platelet aggregation but had no discernible effect on the shape change which precedes aggregation. When PRP was pre-incubated with Li, the inhibitory effect of the ion was independent of the duration and temperature of the treatment. The inhibitory effect of Li also was observed in heparinized PRP or when 5-HT was used as the aggregation-inducing agent. When Li was combined with aggregation inhibitors which enhance platelet cyclic AMP content either by activating adenylate cyclase or by inhibiting phosphodiesterase, only additive effects were observed. The inhibitory effect of Li was opposed by added calcium. Kinetic evaluation of the interaction between Li and Ca indicated that their antagonism was competitive. Added calcium also displayed competitive antagonism toward the aggregation inhibiting effect of increased hydrogen ion concentration in the pH range between 6 and 8.

The effect of somatostatin on platelet aggregation.

Somatostatin was infused intravenously over two hours to rabbits in a dose of 1.5 mug/kg min with an initial loading dose of 50 mug/kg. This dose inhibited platelet aggregation in response to both ADP and collagen. Lower doses of SRIF had no effect. The addition of SRIF in vitro to either human or rabbit platelet rich plasma also had no effect on collagen or ADP-induced platelet aggregation.

An endoperoxide aggregator (LASS), formed in platelets in response to thrombotic stimuli-purification, identification and unique biological significance

1. Low temperature liquid chromatography (LC) was the method of choice for preliminary isolation of “labile aggregation-stimulating substance” (LASS) and for its further purification. Activity of LASS in high resolution LC fractions appeared to be attributable to only one substance, a peroxide which could be changed non-enzymatically to authentic PGs E 2 and F 2α (identified by gas chromatography-mass spectrometry). We thus concluded that LASS had the structure of 15-hydroxy 9 α,11α-peroxidoprosta 5,13-dienoic acid. 2. Addition to saline-suspended human platelets of arachidonate, collagen or thrombin, resulted in platelet aggregation and formation of pro-aggregatory concentrations of LASS. Peak values of LASS were attained at 0.5–1 min after exposure to the stimuli (above) and LASS production was blocked by aspirin (20 μg/ml). 3. Pure LASS is more potent as a platelet aggregator than other putative thrombotic mediators such as ADP and epinephrine, being active at 20–100 ng/ml in human platelet-rich plasma (PRP). It is about 2–5 fold more potent on rabbit PRP or saline-suspended human platelets. The aggregation response is Ca ++ dependent and apparently requires a plasma factor such as fibrinogen. 4. Small concentrations of LASS induced aggregation which was reversible and not accompanied by significant release of platelet constituents. Higher concentrations of LASS induced aggregation which was irreversible and was accompanied by release of platelet-bound 14C-serotonin, although no evidence was obtained for the release of lysosomal enzymes. 5. LASS-induced aggregation and serotonin release was markedly potentiated in the presence of PGE 2 (20–1,000 ng/ml) and suppressed by PGE 1 (10–500 ng/ml). When the platelets were pretreated with aspirin (20–100 μg/ml) or adenosine (10–50 μM), there was a variable reduction in the aggregation response to LASS. In the presence of PGE 2 (0.5–1 μg/ml) no such marked inhibitory effects were produced and aspirin then potentiated the aggregation response to LASS. 6. The corresponding endoperoxide intermediate of PGE 1 biosynthesis (“PGR 1”) was also prepared in pure form and its effects compared with those of LASS. PGR 1 was lacking in pro-aggregatory effects and did not alter the ability of LASS to produce aggregation. Most isolated smooth muscle preparations investigated were caused to contract by nanogram concentrations of LASS or PGR 1. On gastro-intestinal tissues the endoperoxides were generally less potent than their stable PG end-products. In contrast, isolated aorta from the rabbit was >80–200 fold more sensitive to LASS than to PGE 2, PGF 2α or PGE 1. PGR 1 was ∼5–10 fold less active than LASS in producing contractions of the aorta. 7. It is concluded that platelet biosynthesis of LASS and PGE 2 mediates (in part or in full) irreversible platelet aggregation and the release reaction induced by collagen, thrombin, arachidonate, epinephrine or ADP. LASS probably does not play a major role in inflammatory process. Significance of these conclusions is discussed with relevance to hemostasis and the design of new anti-thrombotic therapy.

The Effect of Molecular Iodine on Platelet Aggregation in Vitro

SummaryWhen rabbit platelet-rich plasma was treated with 1-2 mM molecular iodine, marked irreversible aggregation of platelets was observed. The aggregation was characterized by inhibition by 1 mM reduced glutathione, acceleration by 0.1 mM NEM, and no interference with EDTA and adenosine which are powerful inhibitors of aggregation induced by ADP. Molecular iodine at a concentration of 0.5 mM did not induce aggregation and caused more than 60% inhibition of ADP- and collagen-induced aggregation. These findings indicated that the aggregation by molecular iodine was in quite different mechanisms from those by other aggregating agents such as ADP, and suggested that platelet aggregation by molecular iodine and ADP was greatly regulated by sulfhydryl groups.

Platelet aggregation induced by arachidonic acid is accompanied by release of potential inflammatory mediators distinct from PGE2 and PGF2.

ARACHIDONIC acid (AA) is converted into prostaglandins (PG) E2 and F2α by PG synthetase contained in mammalian tissues1,2. A material showing vasocontracting activity and called rabbit aorta contracting substance (RCS)3 has been suggested to be the cyclic endoperoxide intermediate formed during the biosynthesis of these prostaglandins4,5. The participation of RCS in inflammation has been proposed as its biosynthesis, like that of PG, is inhibited by anti-inflammatory drugs (AID)5–7. Platelets synthesize PG when reacted with collagen or thrombin8,9 or directly with AA10. Collagen-induced platelet aggregation is inhibited by AID11 and contact with collagen by damaged vessels is believed to be the initial stimulus for platelet aggregation in vivo12. No connexion has been proposed as yet between platelet aggregation, synthesis of PG and their common inhibition by AID. We have now demonstrated that AA induces platelet aggregation with generation of RCS and PG. As both responses were hindered by AID, it is suggested that PG synthetase interferes with platelet function other than through PG. An intermediate such as RCS may play the central role. Human or rabbit platelet rich plasma (hPRP or rPRP) and other blood elements were obtained as indicated in Fig. 1 and aggregation was studied turbidimetrically13 at room temperature.

Effect of sodium pentobarbital anaesthesia on platelet behavior in vitro and in vivo

Biolaser induced endothelial injury combined with rabbit ear chamber techniques offers a simple and precise method of measuring platelet reactivity in vivo in conscious animals. By using this method a consistent and significant depression in the number of platelet microemboli from sites of fresh endothelial injury was found during sodium pentobarbital anaesthesia (30 mg/kg). There was no consistent barbiturate induced alteration in microcirculatory blood flow, nor could the in vivo effect be related to changes in platelet count or acid base status. However, adenosine diphosphate (ADP) induced platelet aggregation in citrated rabbit platelet rich plasma (PRP) was significantly depressed during pentobarbital anaesthesia. Observations on platelet function, in vitro or in vivo, on animals subjected to barbiturate anaesthesia must therefore be interpreted with caution.

Alterations of Platelet Aggregation in the Chandler Tube: I. Turbidimetric Studies

Platelet aggregation in the Chandler tube was studied by continuous measurement of optical density during aggregation. Normal mean values for the duration of aggregation varied from 3.4 to 6.0 min. [rabbit platelet-rich plasma (PRP)] and 3.1 to 5.8 min. (human PRP). Mean thrombus formation time (TFT) varied from 17.1 to 20.9 min. (rabbit PRP) and from 13.5 to 16.4 min. (human PRP). Activation of Hageman factor shortened TFT and duration of aggregation, and minimized optical density changes during aggregation. Factor VIII and erythrocytes increased optical density changes during aggregation. The observations suggest that experimental and clinical states associated with an increased tendency to thrombosis may correlate with characteristic changes in optical density during aggregation in the Chandler system. Altered aggregation characteristics result from changes in plasma proteins, adenosine diphosphate release, catecholamine concentration, and the blood coagulation system.

Alterations of Platelet Aggregation in the Chandler Tube: II. Electron Microscopic Observations

Platelet aggregation in the Chandler tube was evaluated by electron microscopy. Some samples of rabbit platelet-rich plasma (PRP) had small (<5%) changes in optical density during aggregation. In those samples fibrin formation was conspicuous shortly after the onset of aggregation and was found between individual platelets in platelet thrombi at later stages of aggregation. Samples of PRP in which the optical density change exceeded 30% had larger platelet aggregates. Fibrin strands were present at the periphery of such aggregates, but generally were absent within aggregates, both in the initial stages of aggregation and in well-developed platelet thrombi. Platelets in both large and smaller aggregates showed loss of granules and hyaloplasm. Samples of PRP which had increased fibrin formation corresponded to experimental samples in which the blood coagulation system was activated experimentally and those of a small number of samples from normal humans.

Effect of prostaglandin E 1 on platelet aggregation in vitro and in hemorrhagic shock

Prostaglandin E 1 (PGE 1) was investigated for its inhibition of platelet aggregation both in vitro and in vivo. In vitro, PGE 1 inhibited ADP/5-HT-induced platelet aggregation in both rabbit platelet rich plasma and dog whole blood at concentrations as low as 2.8 × 10 −8 m (10 ng/ml). The duration of PGE 1 activity in either system was markedly influenced by the nature of the anticoagulant used. Rapid loss of PGE 1 activity occurred in the presence of heparin but not with sodium citrate alone or in combination with heparin. PGE 1 was inactivated rapidly in heparinized blood at 33°C, while no inactivation was seen in heparinized blood at 5°C over a period of 60 min. In vivo, PGE 1 was without activity when infused into a normal dog. However, the increased screen filtration pressure, representing platelet aggregation associated with hemorrhagic shock was reduced by PGE 1 infusion. An infusion of 5 μg/kg/min, which was without effect on ADP/5-HT-induced platelet aggregation in the normal animal, had a profound influence on platelet aggregation induced by hemorrhagic shock. The significance of these findings is discussed with respect to the shock process and the reported short biological half-life of PGE 1.

PLATELET AGGREGATION AND INACTIVATION AND INHIBITION OF ADENOSINE DIPHOSPHATE.

Adenosine diphosphate (ADP)-induced platelet aggregation in rabbit and rat platelet-rich plasma was used as an index of ADP activity, and as a means of detecting inhibitory activity to ADP. Inactivation of ADP occurred in rabbit and rat plasma, and ADP inhibitory activity appeared in the plasma of both species following incubation with the nucleotide. Platelets were not required for the inactivation of ADP, or for the formation of inhibitory activity, but both processes were enhanced in the presence of platelets. ADP inhibitory activity in rabbit and rat plasma did not resemble that of exogenous adenosine or AMP. The results do not support the assumption that known products of the degradation of ADP are solely responsible for the inhibitory activity. It is concluded that the inhibitory activity may be due to unknown products of the degradation of ADP, or to a factor elicited in response to the presence of ADP in plasma.

EFFECTS OF BACTERIAL ENDOTOXIN ON RABBIT PLATELETS. 3. COMPARISON OF PLATELET INJURY INDUCED BY THROMBIN AND BY ENDOTOXIN.

The platelet injury produced by bacterial endotoxin and thrombin have been compared in studies utilizing citrated rabbit platelet-rich plasma. Endotoxin-induced platelet injury is characterized by a lag period, is progressive, and does not produce gross clot formation. Thrombin-induced platelet injury is immediate, non-progressive, and is associated with clot formation. The quantity of thrombin required to produce clot formation in this citrated system is less than that required to produce release of platelet 5-hydroxytryptamine. The endotoxin-induced platelet injury required extremely large quantities of heparin for inhibition. The platelet injury induced by thrombin can be inhibited by small quantities of heparin. It is concluded that the injurious effects of endotoxin on platelets is mediated through some mechanism other than thrombin formation.

Effects of bacterial endotoxin on rabbit platelets. II. Enhancement of platelet factor 3 activity in vitro and in vivo.

Incubation of rabbit platelet-rich plasma with bacterial endotoxin results in activation of platelet factor 3, a precursor of blood thromboplastin. This platelet-endotoxin interaction is dose- and temperature-dependent, and is similar to the effects of antigen-antibody union in the presence of platelets. Injection of endotoxin intravenously is associated with a transient increase in platelet factor 3. Since platelet factor 3 can be demonstrated in platelet-poor plasma as well as platelet-rich plasma after endotoxin injection, it seems likely that an actual transfer of factor 3 from platelets to plasma occurs and that this activity is then rapidly inactivated or cleared from the blood stream.

Influence of β-phenyl isopropylhydrazine on the ability of blood platelets to retain 5-hydroxytryptamine

Platelet-rich plasma of rabbits, rats and humans was incubated under various treatments and the 5-hydroxytryptamine (5HT) content of separated platelets was analysed per ml of original plasma volume. More 5HT was found after the incubation when monoamine oxidase (MAO) inhibitor, β-phenyl isopropylhydrazine (PIH) was present in concentrations of 10 −4 or 10 −3M. Incubation in nitrogen instead of air had similar effect. At higher concentrations (10 −3 or 10 −2M, according to the species) PIH produced a marked 5HT depletion. Iproniazid, as well as isoniazid, which is not a MAO inhibitor, decreased the platelet 5HT when tested on rat platelet-rich plasma at 10 −2M. In rabbit's platelets PIH (from 10 −5 to 10 −3M) partly inhibited the release of 5HT produced by tetrabenazine. A nitrogen atmosphere had the same effect and PIH in addition to nitrogen offered no further inhibition. Isoniazid 10 −3M was ineffective. In suitable concentrations PIH also had an inhibitory action on the 5HT release from platelets of rats and humans. The inhibition of 5HT depletion is assumed to be connected with the inhibition of the platelets ability to inactivate their 5HT.