Platelet Electrophoretic Mobility Research Articles

Electrokinetic properties of human cryopreserved platelets.

A method for the cryopreservation of human platelets with glycerol/glucose is described which was a simplified modification of the method of Dayian and Pert (1979). The effect of cryoinjury of the platelet surface membrane was investigated by studying the surface electrokinetic properties of the platelet. A significant increase in platelet electrophoretic mobility was found after cryopreservation. The fresh platelets had a mean electrophoretic mobility of 1.04 +/- 0.05 microns s-1 V-1 cm-1 and cryopreserved platelets 1.18 +/- 0.05 microns s-1 V-1 cm-1, P < 0.05. However, the total platelet sialic acid of fresh platelets was 62.5 +/- 5.6 nmol 10(-9) platelets compared to 47.2 +/- 4.6 nmol 10(-9) platelets after cryopreservation, P < 0.001. Similarly, the neuraminidase-labile sialic acid was 26.4 +/- 4.3 nmol 10(-9) platelets for fresh platelets and 17.6 +/- 4.0 nmol 10(-9) platelets after cryopreservation, P < 0.001. Using polyacrylamide gel electrophoresis with Western blotting, we showed a reduction in the platelet glycoprotein Gp Ib after cryopreservation, this was confirmed by using crossed immunoelectrophoresis. Electron microscopy revealed a significant change in platelet morphology after the cryopreservation procedure with disruption of the platelet membrane and also platelet shape change. These features may explain the changes in platelet electrokinetic properties.

Platelet and fibrin modification by radiographic contrast media.

The effect of the radiographic contrast agents, iopamidol and diatrizoate, on fibrin assembly and structure as well as platelet surface charge was studied. Increasing the iopamidol concentration from 0 to 4.5 mM prolongs the fibrin gelation time from 20 to 105 seconds (an anticoagulant effect) and reduces the fibrin fiber mass/length ratio from 3.2 x 10(12) to 0.5 x 10(12) Da/cm (i.e., produces very thin fibrin fibers). Ultraviolet difference spectroscopy of fibrinogen showed both a 15-nm shift in the ultraviolet difference maximum for iopamidol (suggesting binding) and a perturbation of the aromatic amino acid side chain region for fibrinogen (suggesting a conformational change in fibrinogen) as the concentration of iopamidol was increased from 0 to 9 mg/ml. Binding of iopamidol to fibrinogen was also shown by affinity chromatography using a Sepharose-fibrinogen column. Electrophoretic quasi elastic light scattering was used to show platelet interaction with iopamidol as reflected in a reduction in the platelet electrophoretic mobility from 2.0 to 0.5 (microns-cm)/(V-sec) as the concentration of iopamidol was increased from 0 to 4.5 mM. In addition, the ionic radiopaque contrast agent, Renografin, was also studied and found to inhibit fibrin monomer assembly. Although iopamidol is not shown to be thrombogenic, iopamidol does appear to reduce platelet surface charge, bind fibrinogen, and modify fibrin clot structure.

Sialic Acid: Its Importance to Platelet Function in Health and Disease

Platelets are known to contain sialic acid, most of which resides on glycoprotein carbohydrate side-chains. A large proportion of the platelet sialic acid is neuraminidase-labile. The sialic acid of human platelets is of the N-acetyl neuramininic acid type and is implicated in platelet functional events, such as aggregation and adhesion as well as contributing to the platelet's electrophoretic mobility. In this respect the platelet has an overall electronegative charge which is predominately the result of the carboxyl group of the sialic acid moiety. Platelet subpopulations have been isolated with differing amounts of sialic acid and cleavage of platelet sialic acid by neuraminidase is associated with loss of platelets from the circulation. This has relevance to platelet aging in vivo where removal of platelet sialic acid may reveal 'senescence antigens' which could facilitate platelet 'uptake' by the reticuloendothelial system. The importance of platelet sialic acid status has been described in certain platelet disorders such as some thrombocytopenias as well as particular disease states including malignancies and atherosclerosis.

Plasma and platelet interactions: changes in platelet electrophoretic mobility

Plasma and platelet interactions: changes in platelet electrophoretic mobility

Neutralization of the Local Negative Charge Carried by Glycoprotein (GP)-Ib in Ristocetin-Induced Platelet Agglutination

Pretreatment of platelets with chymotrypsin dose-dependently decreased glycoprotein (GP)-Ib amounts as measured by SDS-PAGE, ristocetin-induced agglutination and platelet electrophoretic mobility (EPM). Decrease in platelet EPM in response to 0.75 mg/ml ristocetin alone were 7.0 +/- 2.3 and 6.8 +/- 4.3% (M +/- S.E., n = 6) for control and chymotrypsin-treated platelets, respectively (p greater than 0.2). Von Willebrand factor (vWF) alone had no effect on platelet EPM. However, in the presence of 0.75 mg/ml ristocetin, added vWF (2.9 micrograms/ml) caused a further 6.3 +/- 3.8% decrease in control platelet EPM, but caused no significant decrease in the enzyme-treated platelets (p less than 0.05). In the presence of 0.3 mg/ml ristocetin, added vWF (2.9-14.5 micrograms/ml) caused a small but significant decrease in control platelet EPM, but caused no significant decrease in the enzyme-treated platelets. These findings suggested that the GP-Ib carrying negative charge decreased by binding of vWF might facilitate a mutual approach of the GP-Ib molecules and bridge formation by vWF between different platelets.

Studies with a murine monoclonal antibody that abolishes ristocetin- induced binding of von Willebrand factor to platelets: additional evidence in support of GPIb as a platelet receptor for von Willebrand factor

A murine monoclonal antibody directed at or near a platelet membrane receptor for the von Willebrand factor was produced by the hybridoma technique. Purified F(ab')2 fragments and/or intact antibody completely blocked the agglutination of platelets induced by both ristocetin and bovine von Willebrand factor and the binding of von Willebrand factor antigen to platelets. The antibody also decreased platelet retention, prevented the reduction in platelet electrophoretic mobility caused by bovine von Willebrand factor, and decreased the serum prothrombin time. Radiolabeled F(ab')2 fragments bound to or approximately 2.5 X 10(4) sites on normal platelets with high affinity (KD or approximately 1.5 X 10(-8) M); there was no binding to platelets from 2 patients with the Bernard-Soulier syndrome. Immunoprecipitation and affinity chromatography studies indicated that the antibody binds to glycoprotein lb at a site contained on the externally oriented portion of the GPIb alpha chain (glycocalicin). An unidentified mol wt or approximately 20,000 molecule labeled by periodate/NaB3H4 coprecipitated and copurified with GPIb.

Studies With a Murine Monoclonal Antibody That Abolishes Ristocetin-Induced Binding of von Willebrand Factor to Platelets: Additional Evidence in Support of GPIb as a Platelet Receptor for von Willebrand Factor

A murine monoclonal antibody directed at or near a platelet membrane receptor for the von Willebrand factor was produced by the hybridoma technique. Purified F(ab')2 fragments and/or intact antibody completely blocked the agglutination of platelets induced by both ristocetin and bovine von Willebrand factor and the binding of von Willebrand factor antigen to platelets. The antibody also decreased platelet retention, prevented the reduction in platelet electrophoretic mobility caused by bovine von Willebrand factor, and decreased the serum prothrombin time. Radiolabeled F(ab')2 fragments bound to or approximately 2.5 X 10(4) sites on normal platelets with high affinity (KD or approximately 1.5 X 10(-8) M); there was no binding to platelets from 2 patients with the Bernard-Soulier syndrome. Immunoprecipitation and affinity chromatography studies indicated that the antibody binds to glycoprotein lb at a site contained on the externally oriented portion of the GPIb alpha chain (glycocalicin). An unidentified mol wt or approximately 20,000 molecule labeled by periodate/NaB3H4 coprecipitated and copurified with GPIb.

Role of Surface Negative Charge in Platelet Function Related to the Hyperreactive State in Estrogen-Treated Prostatic Carcinoma

The relationship between platelet surface negative charge and hyperfunction was examined by determining electrophoretic mobility (EPM), aggregability, and sialic acid of platelets in prostatic cancer, prostatic cancer with estrogen, prostatic cancer with estrogen and aspirin, prostatic hypertrophy, and healthy aged males. Estrogen treated prostatic cancer patients had significantly higher platelet EPM. A good linear correlation was found between sialic acid and EPM (r = 0.97, p less than 0.001). EPM was negatively correlated with primary aggregations by adrenaline and ADP but not with secondary or maximum aggregations, suggesting increased surface negative charge may inhibit primary aggregation. Estrogen and platelet population changes influenced surface negative charge. Neuraminidase removal of platelet surface sialic acid resulted in dose-dependent decreases of EPM which paralleled decreases in sialic acid. Aspirin treated patients and platelets incubated with aspirin in vitro both showed increased platelet EPM. These results suggest that platelet surface negative charge may directly affect platelet function.

Inhibition of von Willebrand Factor-Dependent Platelet Function by Increased Platelet Cyclic AMP and Its Prevention by Cytoskeleton-Disrupting Agents

AbstractPGI2, PGE1, dibutyryl cyclic AMP, and other agents known to raise platelet cyclic AMP were found to inhibit the initial slope of platelet aggregation in platelet-rich plasma induced by ristocetin and bovine von Willebrand factor. The inhibition was not due to an effect on the release reaction since aspirin treatment of platelets completely blocked release but did not significantly inhibit the initial slope of aggregation. These agents also inhibited the decrease in platelet electrophoretic mobility, which has previously been shown to be associated with the binding of von Willebrand factor to platelets, suggesting that they act by decreasing the interaction of von Willebrand factor with platelets. PGI2 and PGE1 did not inhibit the von Willebrand factor-dependent agglutination of formaldehyde-fixed platelets, indicating that they do not have a direct effect on the von Willebrand factor itself and that they require platelet metabolism and/or membrane protein mobility to express their inhibition. Preincubation of platelets with the microtubule-disrupting agents colchicine and vinblastine and microfilament-altering agents cytochalasin-D and cytochalasin-B prevented the inhibition produced by increasing platelet cyclic AMP, raising the possibility that the latter’s effect is mediated by changes in the platelet’s cytoskeleton. These studies suggest a new mechanism whereby agents that increase platelet cAMP may inhibit platelet adhesion.

Influences of prostaglandins on electrophoretic mobility and aggregation of rabbit platelets

Influences of prostaglandin(PG)s on electrophoretic mobilities and aggregation of rabbit platelets were studied. The PGs studied (PGI 2, PGE 1, PGD 2, PGE 2, PGF 2α, PGA 2 and PGA 1) had no effect on platelet electrophoretic mobility. However, both PGE 1 and PGI 2 in 0.3 and 3.0 μM inhibited ADP-induced aggregation and ADP-induced decrease in the mobility. PGD 2 in 0.3 and 3.0, and PGE 2 in 30 μM inhibited the aggregation but did not depress the ADP-induced decrease in the mobility. PGF 2α, PGA 2 and PGA 1 had no effect on the decrease in electrophoretic mobility and on the aggregation caused by ADP.

Polybrene-Induced Platelet Agglutination and Reduction in Electrophoretic Mobility: Enhancement by von Willebrand Factor and Inhibition by Vancomycin

It has recently been reported that the polycation Polybrene (hexadimethrine bromide), like ristocetin, agglutinates platelets more extensively in the presence of normal plasma than von Willebrand plasma. Since we have previously proposed that ristocetin may initiate agglutination by reducing platelet surface charge, I investigated the correlation between Polybrene's ability to induce agglutination and alter platelet electrophoretic mobility. In the absence of plasma, low concentrations of Polybrene produced small platelet aggregates and reduced the electrophoretic mobility. Higher concentrations were needed to produce small platelet aggregates in the presence of von Willebrand plasma; these same concentrations produced more rapid agglutination and much larger aggregates in the presence of normal plasma. The reductions in electrophoretic mobility were also greater in the presence of normal than von Willebrand plasma. Both agglutination and the reduction in mobility could be partially reversed with citrate. Vancomycin, an antibiotic that inhibits ristocetin-induced agglutination with some specificity, inhibited both Polybrene-induced agglutination and the reductions in platelet mobility. These data are consistent with our electrostatic model and support the view that reductions of platelet surface charge may be necessary (but perhaps not sufficient) to initiate the interaction between platelets and von Willebrand factor.

Polybrene-induced platelet agglutination and reduction in electrophoretic mobility: enhancement by von Willebrand factor and inhibition by vancomycin

It has recently been reported that the polycation Polybrene (hexadimethrine bromide), like ristocetin, agglutinates platelets more extensively in the presence of normal plasma than von Willebrand plasma. Since we have previously proposed that ristocetin may initiate agglutination by reducing platelet surface charge, I investigated the correlation between Polybrene's ability to induce agglutination and alter platelet electrophoretic mobility. In the absence of plasma, low concentrations of Polybrene produced small platelet aggregates and reduced the electrophoretic mobility. Higher concentrations were needed to produce small platelet aggregates in the presence of von Willebrand plasma; these same concentrations produced more rapid agglutination and much larger aggregates in the presence of normal plasma. The reductions in electrophoretic mobility were also greater in the presence of normal than von Willebrand plasma. Both agglutination and the reduction in mobility could be partially reversed with citrate. Vancomycin, an antibiotic that inhibits ristocetin-induced agglutination with some specificity, inhibited both Polybrene-induced agglutination and the reductions in platelet mobility. These data are consistent with our electrostatic model and support the view that reductions of platelet surface charge may be necessary (but perhaps not sufficient) to initiate the interaction between platelets and von Willebrand factor.

Inhibitory effect of tiaramide of aggregation in vivo and on electrophoresis of rabbit platelets.

Effects of tiaramide, aspirin an indomethacin were studied on rabbit platelet aggregation in vivo and on platelet electrophoretic mobility. When tiaramide (6 mg/kg), aspirin (30 mg/kg) or indomethacin (1.3 mg/kg) was injected into the ear vein of rabbit during 60 sec, tiaramide only inhibited ADP-induced aggregation, 20 min after the injection. All three drugs prevented collagen-induced aggregation 20 and 120 min after the injection. Tiaramide and aspirin prevented aggregation 24 hours later. The inhibitory effects on the aggregation of tiaramide are presumably independent of prostaglandin synthesis, because malondialdehyde (a metabolite of PGG2) production was not influenced. Tiaramide reduced cyclic AMP levels in platelets after 20 min incubation, despite the ability of this agent to inhibit platelet aggregation. Tiaramide, aspirin and indomethacin per se has no effect on platelet electrophoretic mobility, while tiaramide prevented the decrease in the mobility produced by ADP. Tiaramide and aspirin also depressed the decrease in the mobility produced by collagen.

Inhibitory Effect of Tiaramide on Aggregation in Vivo and on Electrophoresis of Rabbit Platelets

Effects of tiaramide, aspirin and indomethacin were studied on rabbit platelet aggregation in vivo and on platelet electrophoretic mobility. When tiaramide (6 mg/kg), aspirin (30 mg/kg) or indomethacin (1.3 mg/kg) was injected into the ear vein of rabbit during 60 sec, tiaramide only inhibited ADP-induced aggregation, 20 min after the injection. All three drugs prevented collagen-induced aggregation 20 and 120 min after the injection. Tiaramide and aspirin prevented aggregation 24 hours later. The inhibitory effects on the aggregation of tiaramide are presumably independent of prostaglandin synthesis, because malondialdehyde (a metabolite of PGG2) production was not influenced. Tiaramide reduced cyclic AMP levels in platelets after 20 min incubation, despite the ability of this agent to inhibit platelet aggregation. Tiaramide, aspirin and indomethacin per se has no effect on platelet electrophoretic mobility, while tiaramide prevented the decrease in the mobility produced by ADP. Tiaramide and aspirin also depressed the decrease in the mobility produced by collagen.

The Increase of the Electrophoretic Mobility of Platelets after Laparotomy

A fully automatic instrument for the determination of electrophoretic mobility of colloidal particles was applied to human platelets. A significant increase in platelet electrophoretic mobility was observed one day after a laparotomy. This suggests that a selective consumption of platelets with smaller surface negative charge may occur during postoperative hemostatic plug formation or under surgical stress. In addition, the difference in electrophoretic mobility observed between males and females suggests an effect of estrogen on platelets.

Spontaneous platelet aggregation in thrombotic disorders

Spontaneous platelet aggregation (SPA) has been recently reported to occur in patients with occulsive diseases. The clinical implication of SPA is however not clarified yet. To investigate the clinical importance and mechanism of this phenomenon, 287 patients who were referred to us for platelet aggregation study were, studied. SPA was positive in 19 patients, 5 in cerebral thrombosis, 3 in Behcet disease, 2 in angina pectoris and 5 in miscellaneous thrombotic disorders.Platelets from these patients can aggregate with extremely small amount of aggregating agents such as ADP, epinephrine and collagen. Maximum aggregation of SPA depends on platelet counts in platelet rich plasm. On several occasions, SPA became significant only after platelet rich plasma was kept at room temperature for 1-2 hours. SPA was markedly inhibited by apyrase at a concentration of 0.1mg/ml. Aspirin intake at a dose of 500mg daily for one week abolished SPA. Electrophoretic mobilities of platelets from 2 patients with Behcet disease who had SPA were significantly reduced. SPA platelets showed no changes in sialic acid levels or membrane glycoprotein profiles on SDS polyacrylamide gel disc electrophoresis. Malondialdehyde levels of SPA platelets were higher than normal platelets.Our study clearly indicates that SPA was frequently found in thrombotic disorders. SPA platelets were more sensitive to aggregating agents, and release of ADP plays a role in development of SPA. The mechanism of SPA remained unsolved, but decreased electrophoretic mobility and elevated malondialdehyde level of SPA platelets may give some clue for further study.

Electrophoretic mobility of platelets and its physiological significance

Increasing attention has been paid to the surface electrical charge of platelets in relation to their functions. However there are several criticisms in methodological respects. In the present investigation the effect of neuraminidase on platelets was examined utilizing newly developed instruments of electrophoresis. These are Free-Flow Electrophoresis model Vap-5 (Bender and Hobein, West Germany) and Laser-Zee System 3000 (Pen-Kem Inc., U. S. A.). Rabbits were catheterized in the carotid artery and blood was obtained with EDTA as an anticoagulant. Platelet rich plasma was washed twice with Tris-HCl buffered saline and platelets were finally suspended in pH 5.3 acetate buffered saline. Washed platelets added with various concentrations of neuraminidase or control saline were incubated at 37°C for an hour. After incubation platelets were washed and suspended in 3mM triethanolamine acetate or 0.001M HEPES-NaOH, 0.15M NaCl buffer for electrophoresis with Free-Flow or Laser-Zee system respectively.Results: Electrophoretic distribution of neuraminidase-treated platelets was shifted to the cathode side as compared with the control. The magnitude in shift of electrophoretic distribution depended upon neuraminidase concentration and there was a correlation between them (Fig. 1A, B). Relation between electrophoretic mobility and volume of platelets before and after the neuraminidase treatment was shown in Fig. 2A, B and 3. In general larger platelets were distributed in the anode side and smallers in the cathode side. With lower concentration of neuraminidase treatment this trend as shown by a regression line got greater. This result represents that the sensitivity of platelets to neuraminidase is various in accordance with the platelet volume and smaller platelets are more sensitive to neuraminidase. The platelet sensitivity to neuraminidase may have some role in platelet turn-over as smaller platelets have been believed as older ones.

Platelet Adhesiveness in Ischemic Heart Disease

The adhesiveness of platelets to collagen and glass was studies ex vivo in groups of patients with ischemic heart disease (IHD) and acute myocardial infarction (AMI). Adhesiveness was increased to glass and normal collagen in both groups of patients. No relation was found between reinfarction rate and adhesiveness in the acute stage of AMI. Electrophoretic mobility of platelets was the same in the AMI patients and a normal control group. It suggested that the increased 'retention' of platelets in IHD patients might be due more to platelet hyperaggregation or to hypersensitivity to platelet aggregating agents than to altered adhesiveness.

Electrophoretic mobility of platelets from normal and bleeder swine

Electrophoretic mobility of platelets from normal and bleeder swine

Demonstration of platelet electrophoretic mobility slowing factor (PSF) in supernatants of human lymphocyte cultures stimulated by phytohaemagglutinin (PHA).

It has been demonstrated that human peripheral lymphocytes, stimulated with PHA, form or release a factor that slows platelet electrophoretic mobility (PEM). The factor is chromatographed on Ultrogel AC a 44, has a molecular weight of approximately 45,000, is stable on heating at 56 degrees C for 30 min, but is inactivated by heating at 80 degrees C for 10 min. In addition, it is distinct from leukocyte inhibiting factor, which elutes in the range of BSA (mol. wt. 69,000). The authors conclude, in accordance with previous investigations, that cellular immunity is involved in thrombotic processes.