The modified Je-Ho-Tang, Korean herbal medicine, inhibits whole-blood aggregation and platelet adhesion to collagen under flow

We investigated the in vitro effect of modified Je-Ho-Tang (MJHT) hot–water extract on platelet aggregation and activation induced by agonists in human whole blood, and on platelet adhesion to a collagen-coated surface under flow shear stress conditions. MJHT extract potently inhibited the collagen-induced platelet aggregation in human whole blood in a dose-dependent manner, with a half-maximal inhibitory dose (IC₅₀) value of 526 µg/mL. We accessed several markers of platelet activation using receptor expression on platelet membranes, including GPIIb/IIIa-like expression (PAC-1) and P-selectin (CD62); we monitored the intracellular calcium mobilization response by flow cytometry in healthy subjects. A significant decrease in PAC-1 (P = 0.002), CD62 (P = 0.002), and intracellular calcium mobilization (P < 0.001) were seen in the presence of MJHT extract. In addition, we have used image analysis to study human platelet adhesion to collagen under physiologic flow conditions in a perfusion chamber. MJHT extract markedly decreased platelet adhesion to the collagen-coated surface. These results show that MJHT has potent anti–platelet activity.     

The effect of albumin concentration and storage time on the adhesion of washed porcine platelets to glass

The effect of storing platelet suspensions at room temperature before assessing platelet adhesiveness to glass was investigated. Changes found in adhesiveness depended on the albumin concentration of the suspension medium. The initial rate of platelet adhesion was inversely related to albumin concentration. Thereafter, during a 4-hr period, platelet adhesion decreased in suspensions with low (0–0.35%) albumin concentrations, whereas at higher concentrations, the adhesion was either not affected (1% albumin) or increased (2% albumin). These observations are explained by the different and opposite effects of albumin which adsorbs to glass more quickly than the platelets and improves the viability of the platelets during storage.     

Procedure for quantification of platelet adhesion to biomaterials by radioscintigraphy

Detection of adhered platelets on biomaterial surface that has blood-contacting application is an important test to assess its thrombogenicity. Usually, for measurement of platelet adhesion, after exposure to platelet-rich plasma (PRP) under standardized conditions the test surface is rinsed to remove non-adherent cells and is analyzed under scanning electron microscopy (SEM) to detect morphology of adhered cell and degree of aggregate formation. However, being a qualitative test it is unlikely to give an accurate estimate of platelets adhered to the surface. On the other hand, use of radiolabels enables quantification of platelets deposited on a material or device. Because of high gamma emission of (111)In, it can be used for radioscintigraphy, however, its short half life (2.5 days) is a major hurdle in using it for quantification of platelet adhesion. (125)I is a relatively strong radiolabel that is easily tagged to most of the proteins and has a relatively long half-life (60 days). The major objectives of this study are to standardize the labeling conditions to get good (125)I activity on platelets, while maintaining normal cell function after they are labeled. Considering all possible uncertainties, quantity of isotope and platelets to be used and the conditions of iodination reaction are established to get repeatable and reproducible labeling of platelets. Further, it is demonstrated that (125)I-platelets can be used to determine total number of cells adhered to titanium surface, which is known to be used as a blood-contacting biomaterial.     

Effect of PGI2 on platelet binding to partially denuded endothelial monolayer in vitro.

We have developed a new model for the investigation of platelet interaction with injured vascular endothelium. This involves the quantitative detection of platelet binding to a partially denuded endothelial cell monolayer in vitro. Porcine arterial endothelial monolayer, cultured on collagen gel containing fibrinogen and fibronectin, was partially denuded and the binding of ⁵¹Cr-platelets was measured. A synergistic increase in platelet binding was observed in the presence of fibrinogen and fibronectin. A distinct aggregation of platelets along the edge of the denuded area of the endothelial monolayer was seen. Prostacyclin (PGI₂) inhibited platelet aggregation, although adhesive platelets were still present at denuded sites.     

Thrombogenetic studies of bioprosthetic heart valve surfaces. I. In vitro platelet adhesion in a static system

Contact angle measurements, histological examinations and platelet adhesion tests on native and glutaraldehyde-treated porcine aortic valve leaflets, are reported. Contact angles measured with water or saline on native and treated leaflets do not differ appreciably. Values of less than 10 degrees for both the dry and wet leaflets were observed. Histological examination of the native and fixed tissue surfaces was carried out with both optical and scanning electron microscopy. Native leaflet surfaces appeared to be smoother with longer and straighter fibres beneath the surface than treated leaflets. Platelet adhesion tests were performed with fresh, non-anticoagulated human whole blood as well as with platelet suspension. Static platelet adhesion tests were carried out by depositing droplets of blood on the test surface. The blood droplets were washed off after a contact time of 2 minutes for whole blood and up to 32 minutes for platelet suspension. The adhering platelets were microscopically observed and counted. The platelet density on the native leaflets was found to be significantly higher than on the treated tissues. However, the platelet density did not vary with glutaraldehyde concentration in the range from 0.2 to 1.0% (v/v).     

Reductions in platelet contractile force correlate with duration of cardiopulmonary bypass and blood loss in patients undergoing cardiac surgery

Blood loss secondary to platelet dysfunction is known to be increased when the duration of cardiopulmonary bypass (CPB) is prolonged. The ability to correlate alterations in platelet function with the duration of bypass and early postoperative blood loss, however, has remained elusive. Platelet contractile force, a novel measure of platelet-mediated clot retraction, is known to be reduced following cardiac surgery and blockade of platelet adhesion receptors. The aim of this study was to determine if alterations in platelet contractile force (measured using whole blood) correlated with the duration of CPB and early postoperative blood loss. Thirty patients were entered into a study designed to measure platelet function before, during, and after CPB. Platelet aggregometry and surface expression of CD42b and CD61 were also measured (using whole blood) in a subset of subjects (n=10) to further characterize the intrinsic structural and functional defects induced by CPB. Reductions in platelet contractile force had a significant correlation with duration of CPB (r=0.564; P=0.002) and early blood loss (r=0.545; P=0.003). Although decreases in platelet contractile force and aggregation both correlated with CPB time in the smaller subset of patients tested, only platelet contractile force correlated with decreases in CD42b, CD61 and blood loss. The results of this study suggest that prolongation of CPB is related to increasing degrees of platelet dysfunction and that reductions in platelet contractile force are related to decreases in platelet adhesion receptors and early postoperative blood loss.     

The influence of platelet homogenates on thrombopoiesis in rabbits

The influence of platelet homogenates on thrombopoiesis in rabbits has been investigated. Before and five days after injection of the platelet homogenates platelet counts were determined. Moreover, the platelets were separated into four populations of different density. The platelet homogenates induced a significant increase of the platelet counts which was mainly due to an increase of the small light forms with a relative decrease of the large forms. It is concluded that platelets themselves contain a thrombopoiesis stimulating factor.     

The influence of glutathione and other thiols on human platelet aggregation

The platelet membrane contains sulfhydryl groups which are essential for normal platelet function. Reduced glutathione (GSH) and other thiols such as cysteine and 6-mercaptopurine were found to inhibit human platelet aggregation induced by adenosine diphosphate (ADP), collagen and arachidonic acid. The inhibition of ADP-induced aggregation by GSH (IC₅₀=0.61 ± 0.05 mM) was greater than that by cysteine (IC₅₀=13 ± 1 mM) or 6-mercaptopurine (IC₅₀=5.4 ± 0.2 mM). Two other thiols, dithiothreitol and beta-mercaptoethanol were found to cause platelet aggregation instead of inhibition. The interaction of GSH with the ADP receptor was noncompetitive in nature.     

Effects of trifluoperazine on platelet activation

Previous reports of the inhibitory effects of trifluoperazine on platelet responses to different aggregating agents have been conflicting, and the mechanism of action remains unclear. We have found that aggregation by minimum concentrations of collagen and arachidonic acid, and second phase aggregation by minimum concentrations of ADP, thrombin, epinephrine and the calcium ionophore A23187 were inhibited by 40–60μM trifluoperazine. The first phase of aggregation by a minimum concentration of epinephrine was completely inhibited by 100μM trifluoperazine, and the first phase of aggregation induced by ADP, thrombin or A23187 was decreased by 300μM trifluoperazine. The platelet shape change caused by collagen, but by no other aggregating agent examined, was inhibited by 300μM trifluoperazine. Secretion of ³H-5 hydroxytryptamine by minimum concentrations of ADP, collagen, epinephrine and arachidonic acid was completely suppressed by 50μM trifluoperazine. Secretion by thrombin and A23187 was incompletely inhibited by 300μM trifluoperazine. Thromboxane B₂ formation caused by all aggregating agents, except epinephrine, was incompletely suppressed by 50μM trifluoperazine, and 300μM trifluoperazine only caused complete inhibition of thromboxane B₂ formation by ADP, collagen and epinephrine. The phorbol ester, TPA, which mimics diacylglycerol by activating protein kinase C, caused aggregation and secretion. Aggregation, but not secretion, by low concentrations of TPA was inhibited by concentrations of trifluoperazine as low as 50μM. However, aggregation by a combination of TPA and A23187 was only inhibited by concentrations of trifluoperazine in excess of 100 μM. Secretion by TPA was inhibited by concentrations of trifluoperazine in excess of 200μM. Our findings suggest that low concentrations of trifluoperazine inhibit platelet activation by inhibiting phospholipase A₂, and that higher concentrations inhibit platelet responses by interfering with protein kinase C.     

On the role of the major platelet membrane glycoproteins in platelet adhesion to collagen

The effect of modification of intact rabbit platelets with chymotrypsin, trypsin or thrombin on platelet adhesion to collagen fibers was determined. Experiments were performed $\text{in vitro}$ with washed platelets suspended in a medium containing EGTA to prevent platelet aggregation. Platelet adhesion was unaffected by chymotrypsin or thrombin treatment but was reduced upon trypsin treatment. The latter effect could be attributed to the ADP released upon trypsin treatment. In the presence of high concentrations of AMP, adhesion was restored to control values. Release also occurred on thrombin treatment but this did not influence adhesion.The effect of the enzymes on platelet membrane glycoproteins was monitored by gel electrophoresis. Chymotrypsin caused the loss, at least, of the regions susceptible to lactoperoxidase-catalyzed iodination and to periodic acid-Schiff (PAS) staining from the major membrane glycoproteins I, II and III of rabbit platelets. Such degradation also occurred to glycoproteins I and II with trypsin, but thrombin was without detectable effect on the major membrane glycoproteins.The results indicate that degradation of the major membrane glycoproteins does not affect adhesion to collagen. Adhesion appears to be mediated either by protease-resistant parts of these molecules or by other membrane entities not readily attacked by proteases.     

Influence of extracellular calcium on single platelet activation as measured by whole blood flow cytometry

The influence of extracellular calcium concentrations ([Ca(2+)]) on single platelet activation and platelet aggregation was evaluated. Platelet fibrinogen binding and P-selectin expression were monitored by whole blood flow cytometry in the presence of EDTA or 0.125, 1.25, 2.5, 5, or 10 mM [Ca(2+)] and in the absence or presence of the thromboxane A(2) (TXA(2)) blockade. Platelet aggregation was measured in citrated and hirudinized platelet-rich plasma (PRP). Platelet fibrinogen binding was slightly increased at >/=2.5 mM [Ca(2+)] in unstimulated samples. ADP-induced platelet fibrinogen binding was, however, higher at 0.125 mM, but lower at 5 and 10 mM [Ca(2+)], as compared to 1.25 mM [Ca(2+)]. Platelet P-selectin expression was not affected by extracellular [Ca(2+)], except mild increases of ADP-induced platelet P-selectin expression in the presence of EDTA. TXA(2) blockade by ICI 192.605 influenced above flow cytometric analyses little. Using Born aggregometry, ADP induced more intense platelet aggregation in citrated PRP than in hirudinized PRP. TXA(2) blockade did not affect platelet aggregation in hirudinized PRP, but reduced aggregation in citrated PRP to approximately 85% of that in hirudinized samples. ADP also induced a more marked and sustained elevation of intracellar [Ca(2+)] in the presence of extracellular [Ca(2+)]. Thus, extracellular [Ca(2+)] has little influence on flow cytometric analysis of platelet P-selectin expression. High [Ca(2+)] enhances spontaneous platelet fibrinogen binding, but reduces ADP-induced platelet fibrinogen binding, while low [Ca(2+)] enhances ADP-induced platelet fibrinogen binding. Physiological [Ca(2+)] supports more intense platelet aggregation when effect of artificial TXA(2) synthesis is blocked.     

The importance of blood collection methods for assessment of platelet activation

Blood collection and processing techniques must be carefully standardized in order to make valid measurements of plasma concentrations of platelet secreted proteins. We therefore conducted this study to determine the optimum method for obtaining serial blood samples for platelet factor 4 (PF4) radioimmunoassay and circulating platelet aggregate ratios (CPA). Venous blood samples for PF4 and CPA were obtained from normal volunteers through either an indwelling 21 guage butterfly needle or by multiple separate venipunctures using the same type of needle and a modified “two-syringe” technique. Results demonstrated that there was significant platelet activation and secretion of PF4, but not CPA, that occurred within 15 minutes of needle insertion and continued during the duration of time that samples were obtained through the indwelling needle. Although there were lower plasma PF4 concentrations if a high fluid flow rate was maintained, there was still a significantly higher PF4 concentration when compared to samples obtained simultaneously by single venipuncture in the opposite arm. In addition, these results demonstrated that neither an indwelling intravenous needle in the opposite arm nor a previous venipuncture in the same site elevated PF4 concentrations.     

A recombinant protein and a chemically synthesized peptide containing the active peptides of the platelet collagen receptors inhibit ferric chloride-induced thrombosis in a rat model

We have previously reported that a recombinant protein (M(r) 47 kDa), which contains both active peptide of platelet receptors for types I and III collagen inhibits both types I and III collagen-induced platelet aggregation. In order to eliminate non-reactive portion of the protein, we have constructed a recombinant of rHyB (M r 6 kDa). In addition, we chemically synthesized a hybrid peptide with 30 amino acid residues (cHyB, M r 3 kDa) that contains each of the active peptide derived from platelet receptors for types I and III collagen and a linker of 12 amino acid residues. In the present investigation, we report that both rHyB and cHyB inhibit type I and type III collagen-induced platelet aggregation, and the adhesion of radiolabeled platelets onto rabbit aortic segments in a dose-dependent manner. We have used an animal model, which employs FeCl3 to induce thrombi formation to study the effectiveness of both rHyb and cHyB on preventing thrombi formation. We obtained results that show that both rHyB and cHyB can inhibit thrombi formation in a dose-dependent manner. These results suggest that either rHyB or cHyB may be a possible therapeutic agent in preventing thrombi formation.     

Dual effect of naloxone on blood platelet aggregation and cerebral blood flow in gerbils

The effect of naloxone on blood platelet aggregation and cerebral blood flow in gerbils was studied. Administration of naloxone in dose 1 mg/kg to intact gerbils resulted in a marked increase in platelet aggregability accompanied by 27% reduction in cerebral blood flow. Focal cerebral ischemic injury significantly enhanced platelet aggregatory response and treatment with naloxone was without any additional effect on platelet aggregation. Cerebral blood flow in ischemic hemisphere, however, increased following naloxone injection by 46%. In vitro naloxone in millimolar concentrations inhibited platelet aggregation in a dose-dependent way. Apparent decrease in fluorescence of platelet membranes tagged with fluorescence probe due to naloxone suggests conformational changes in platelet membrane as a primary mechanism for the antiaggregatory effect of naloxone in vitro.     

Further studies on the role of red blood cells in spontaneous platelet aggregation

The influence of red blood cells on platelet aggregation has recently been a subject of considerable interest. We have studied the effect of red cells on spontaneously formed platelet aggregates in rotating vials at 37 degrees C. Platelet aggregation was quantified by measuring the fall in number of single platelets with a whole blood platelet counter. Autologous packed red cells, platelet rich plasma and platelet free plasma were used to reconstitute aliquots of blood with constant platelet count but 0-60% haematocrit (Hct). The fall in platelet count was minimal at zero Hct, increased markedly with the Hct in the anaemic range and less markedly in the normal to polycythaemic ranges of Hct. Scanning electron microscopic observation of whole blood showed the presence of small platelet aggregates after about 3 mins rotation and very large aggregates after about 12 mins. ADP from red cells has been implicated in triggering platelet aggregation in whole blood. Whether aggregates are formed as a result of ADP leaking from the red cells or by their jostling physical action on the platelets is discussed. The marked effect of the red cells on spontaneous platelet aggregation however, justifies the manipulation of the Hct as a useful therapeutic option in the control of thrombotic and bleeding tendencies.     

Inhibition of tyrosine kinase activity prevents the adhesive and cohesive properties of platelets and the expression of procoagulant activity in response to collagen

Introduction

Platelet activation leads to signal transduction mechanisms, in which phosphotyrosine proteins play a relevant role.

Material and methods

Platelet suspensions were independently activated by collagen and thrombin in the absence and in the presence of two tyrosine kinase inhibitors, tyrphostin 47 and genistein. Samples were processed to visualize morphological changes by electron microscopy, to evaluate changes in cytoskeletal assembly, to analyze modifications in the expression of activation dependent antigens, and the procoagulant activity at the surface level by flow cytometry. Additional experiments applying flow conditions were performed to assess the effect of inhibiting tyrosine phosphorylation on primary platelet adhesion and fibrin formation.

Results

Inhibition of tyrosine phosphorylation blocked shape change and cytoskeletal assembly induced by collagen, and inhibited, though partially, those effects due to thrombin. Both activating agents induced the expression of the intraplatelet antigens CD62P and CD63 at the surface, although only collagen promoted expression of anionic phospholipids. Both tyrphostin 47 and genistein prevented those effects. The extent of platelet adhesion on both collagen-coated and subendothelial surfaces was significantly diminished by the presence of the tyrosine kinase inhibitors assayed. Fibrin formation was also significantly reduced.

Conclusions

Platelet shape change and secretion during platelet activation depends on tyrosine phosphorylation. In addition, primary adhesion of platelets induces signaling through tyrosine kinases to achieve full spreading, and results in the exposure of a procoagulant surface on platelets.     

The effects of glucosamine on platelet aggregation

The effects of glucosamine on platelet aggregation induced by a variety of agents were investigated. When compared with the effects of N-acetyl glucosamine (NAG), an acetylated derivative of glucosamine (1), it became apparent that the N-acetyl group was important in stimulating $\text{Staphylococcus aureus}$ induced platelet aggregation. The absence of the N-acetyl group from NAG changes it to glucosamine which is an inhibitor rather than a stimulator of $\text{S. aureus}$ induced platelet aggregation and release. Glucosamine also inhibits collagen, epinephrine, and ADP induced platelet aggregation and release.     

Effects of Ethanol on Platelet Responses Associated with Adhesion to Collagen

Adhesion of platelets to collagen in damaged blood vessels or ruptured atherosclerotic plaques is important in hemostasis and arterial thrombosis. Adhesion to collagen results in secretion of granule contents and formation of thromboxane A₂; thromboxane A₂ and released ADP synergistically promote aggregation around platelets adherent to collagen. Ethanol inhibits collagen-induced platelet aggregation, secretion, arachidonate mobilization, and thromboxane A₂ formation but does not inhibit platelet adhesion to de-endothelialized rabbit aortae. We investigated whether ethanol affects the initial signalling events and responses of platelets adherent to collagen, independent of the actions of secondary agonists. Suspensions of washed human platelets, labelled by incorporation of [³H]oleate into phospholipids, were used to measure platelet adhesion to collagen by a filtration method; studies were done in the presence of an ADP-removing system and blockers of receptors for thromboxane A₂, platelet-activating factor, serotonin, and fibrinogen. Ethanol (87 mM) did not affect the rate or extent of platelet adhesion to collagen or secretion of [¹⁴C]serotonin from prelabelled platelets adherent to collagen, but ethanol did inhibit thromboxane A₂ formation. Previous studies showed that ethanol does not affect platelet stimulation by arachidonate, leading to the suggestion that reduced mobilization of arachidonate, rather than inhibition of its conversion to thromboxane A₂, is responsible for inhibition by ethanol of thromboxane A₂ formation. Here, we show by a gel mobility shift assay and immunoblotting, that ethanol delays the collagen-induced increase in the phosphorylation of cytosolic phospholipase A₂, the enzyme responsible for arachidonate mobilization. However, ethanol has no effect on collagen-induced tyrosine phosphorylation of phospholipase Cγ2, determined by immunoprecipitation and immunoblotting. Thus, ethanol's effect on signal transduction in collagen-adherent platelets occurs distal to phosphorylation of phospholipase Cγ2 but proximal to phosphorylation of cytosolic phospholipase A₂.     

Inhibition of platelet aggregation and the release of P-selectin from platelets by cilostazol

To evaluate the in vitro effects of cilostazol, a phosphodiesterase III inhibitor, on platelet responses, we measured platelet aggregation and the levels of soluble P-selectin, a glycoprotein present on the -granule membrane in resting platelets, and cAMP. Platelet-rich plasma and washed platelets from healthy human volunteers were treated with cilostazol (5, 25 and 50 μM). Platelet-rich plasma was stimulated by ADP (1 and 5 μM) or collagen (5 μg/ml). Washed platelets were stimulated by thrombin (4 U/ml) in the presence or absence of 1 μM forskolin. In vehicle-treated samples, soluble P-selectin levels in response to 1 μM ADP-induced primary aggregation were similar to those of circulating levels of healthy volunteers but the levels in response to 5 μM ADP-induced secondary aggregation and collagen-induced aggregation increased markedly compared to those in response to primary aggregation. This result suggests that P-selectin is released from platelets according to the extent of platelet aggregation. Cilostazol inhibited platelet aggregation as well as P-selectin release in a concentration-dependent manner. Cilostazol inhibited completely thrombin-induced aggregation in the presence of 1 μM forskolin, when cAMP levels were two-fold higher than those in the absence of forskolin. Cilostazol, which increases intracellular cAMP in platelets, may be useful in the treatment of arterial occlusive diseases.