Plasma Platelet Factor Research Articles

Platelet activation caused by cardiac catheter blood collection, and its prevention

The study of platelet changes occurring across the coronary circulation is important in the investigation of the platelet's role in ischemic heart disease. It requires blood sampling through cardiac catheters. This could activate platelets and alter the results of tests of platelet activation and reactivity. This study was designed to examine this problem and to devise satisfactory methods for obtaining blood for platelet studies through long catheters. Blood collected through catheters introduced with a guide-wire had a much higher plasma heparin neutralising activity(HNA), platelet factor 4(PF4)and beta-thromboglobulin(βTG) than peripheral venous blood, and lower platelet count(PC). Blood collected through catheters introduced via a sheath, and kept filled with anticoagulant/antiplatelet solution until blood sampling, gave results similar to peripheral venous blood for the PC, platelet aggregate ratio, platelet fluorescent granule count, and for plasma HNA, PF4 and βTG. It is concluded that platelets are activated during blood collection through cardiac catheters; however, with appropriate precautions, blood which is satisfactory for platelet studies can be obtained.

Intra-Platelet Platelet Factor 4 (IP.PF4) and the Heparin-Mobilisable Pool of PF4 in Health and Atherosclerosis

Some patients with clinical evidence of atherosclerosis and others with diabetes were compared with appropriate controls. The intraplatelet level of platelet factor 4 (PF4) was significantly decreased in the arteriopaths and was lower in the diabetics when compared with controls. Patients with transient ischaemic attacks and stroke have even lower values. Intravenous heparin liberates large amounts of PF4 from an unknown reservoir, perhaps the endothelium, into the plasma. Arteriopaths liberated significantly more PF4 than the controls and the diabetics most. If a second heparin injection is given 24 hr after the first, the resultant plasma PF4 level was on average half that achieved after the first injection and again the patients had higher levels than the controls. Thus the reservoir originally "emptied" of PF4 by the heparin had been partially refilled in 24 hr and the reservoir in the atherosclerotic patients then contained more than the controls. Patients with atherosclerosis and especially diabetics differ from controls in the PF4 content of their platelets and in their response to heparin and in the rate of refill of the "heparin-mobilisable pool of PF4".

A New Method for the Conservation of Platelet Concentration During Extracorporeal Circulation

Pluronic F-68, a non-ionic polyol, was investigated for its effect on platelet conservation, platelet function, release reaction, and red blood cell hemolysis during cardiopulmonary bypass (CPB). Three groups of ten dogs each underwent CPB utilizing both normothermia and hypothermia. The control group (bubble oxygenator) did not receive any Pluronic F-68. Experimental group I (bubble oxygenator) and group II (membrane oxygenator) received 2 mg of Pluronic F-68 per milliliter of priming fluid. After 10 minutes of normothermic CPB, the platelet concentration decreased significantly by 48%. The platelet concentration in group I significantly decreased by 38%, and in group II the decrease was a non-significant 8%. There was also an associated increase in the plasma concentration thromboxane B2 and platelet factor 4 in both the control group and group I. A significant increase in free plasma hemoglobin was observed in the control group. The increase in group I and II was not significant. During hypothermia (20°C), a significant change in platelets, thromboxane B2, and platelet factor 4 was observed in all groups. After re-establishing normothermia, platelet concentration remained significantly low at 48% in the control group, whereas the platelet concentration in group I increased to 80%, a decrease of 20%, and in group II increased to 92%, a decrease of 8%. Free plasma hemoglobin, platelet factor 4 and thromboxane B2 remained significantly elevated in the control and group I. There was no significant elevation in platelet factor 4, free plasma hemoglobin, and thromboxane B2 in group III. Pluronic F-68, an emulsifying agent, has therefore been shown to be effective in platelet preservation during CPB.

Effect of heparin on the in vivo release and clearance of human platelet factor 4

Intravenous injection of heparin (100 U/kg) into normal volunteers resulted in an increase of platelet factor 4 (PF4) level in platelet- poor plasma from a mean value of 18.1 +/- 6.6 ng/ml before the injection to 257.9 +/- 68.3 ng/ml at 5 min after injection. PF4 antigen isolated from “postheparin plasma” by adsorption on heparin-agarose and elution with 2.0 M NaCl and “authentic PF4” isolated from human platelets showed identical patterns of migration as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Material released by washed human platelets was injected intravenously into rats. The clearance of PF4 followed a biphasic exponential pattern. The half-lives (T1/2) for the fast and slow components for control rats were 1.2 and 17.1 min. Heparin significantly extended the half-life of human PF4 in rat circulation. The clearance of PF4 injected together with heparin followed a single component model with a half-life of 27.6 min. Administration of heparin to rats that had been previously injected with human platelet releasate resulted in a 30-fold increase of plasma PF4 level in their circulation. The clearance of PF4 from the circulation of these rats (T1/2 = 45 min) fitted a single component model. We propose that PF4 is originally secreted by platelets into circulation and subsequently bound reversibly to vascular sites from which it can be released back into the circulation by heparin. The fast component of PF4 clearance that is abolished by heparin may reflect binding of this protein to the endothelial cells.

Effect of Heparin on the In Vivo Release and Clearance of Human Platelet Factor 4

Intravenous injection of heparin (100 U/kg) into normal volunteers resulted in an increase of platelet factor 4 (PF4) level in platelet-poor plasma from a mean value of 18.1 +/- 6.6 ng/ml before the injection to 257.9 +/- 68.3 ng/ml at 5 min after injection. PF4 antigen isolated from "postheparin plasma" by adsorption on heparin-agarose and elution with 2.0 M NaCl and "authentic PF4" isolated from human platelets showed identical patterns of migration as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Material released by washed human platelets was injected intravenously into rats. The clearance of PF4 followed a biphasic exponential pattern. The half-lives (T1/2) for the fast and slow components for control rats were 1.2 and 17.1 min. Heparin significantly extended the half-life of human PF4 in rat circulation. The clearance of PF4 injected together with heparin followed a single component model with a half-life of 27.6 min. Administration of heparin to rats that had been previously injected with human platelet releasate resulted in a 30-fold increase of plasma PF4 level in their circulation. The clearance of PF4 from the circulation of these rats (T1/2 = 45 min) fitted a single component model. We propose that PF4 is originally secreted by platelets into circulation and subsequently bound reversibly to vascular sites from which it can be released back into the circulation by heparin. The fast component of PF4 clearance that is abolished by heparin may reflect binding of this protein to the endothelial cells.

In vivo platelet activation with in vitro hyperaggregability to arachidonic acid in renal allograft recipients

Renal allograft recipients were investigated to determine the extent and possible nature of in vivo platelet activation. In 92 allografted patients stable for more than 4 months' duration, intraplatelet serotonin in circulating platelets was depleted significantly. In a further 16 patients studied serially for 12 to 16 weeks following transplantation, intraplatelet serotonin fell abruptly within 4 days from transplantation to very low levels, and remained thus for 10 weeks, rising toward normal at about 12 weeks. Although some patients showed abrupt falls in intraplatelet serotonin coincident with acute rejection episodes, there was no difference in intraplatelet serotonin in seven patients whose grafts functioned well immediately and remained stable, and seven in whom repeated rejection led to graft loss within 3 months. Thus, these tests of platelet function do not permit diagnosis of rejection or prediction of graft outcome. Plasma platelet factor 4 (PF4) concentrations, in contrast, were normal in most patients during the first 6 weeks after grafting, then rose and remained abnormal up to 13 years following the allograft in the long-term stable graft recipients. This discrepancy suggests a different mode of platelet activation in the first few weeks after grafting from subsequent months. Despite universal depletion of intraplatelet amines and alpha-granule contents only four out of 14 early allograft recipients had an abnormal bleeding time, and platelet aggregation thresholds with adenosine-5'-diphosphate and collagen were not different from controls. However, thresholds for platelet aggregation with arachidonic acid were reduced significantly (P less than 0.01) and thromboxane B2 generation was increased in vitro. There was no correlation between depletion of intraplatelet serotonin and circulating platelet-agglutinating material, but nine of 17 biopsy specimens from rejecting allografts taken during the first 3 months showed extensive glomerular localization of platelet membrane antigens and PF4.

Studies of β-thromboglobulin, platelet factor 4, and fibrinopeptide a in erythrocytosis due to cyanotic congenital heart disease

Plasma and serum assays of β-thromboglobulin (BTG) and platelet factor 4 (PF4), and plasma fibrinopeptide A (FPA) were measured in adults with cyanotic congenital heart disease to characterize further the hemostatic disorder. Artifactual elevations of plasma BTG, PF4, and FPA appeared to occur occasionally when a 21 or 22 gauge needle was used to collect blood. The high packed red cell volume was probably the cause. Use of a larger caliber needle (20 gauge) appeared to lessen the problem. Normal plasma FPA levels (20 gauge needle) in 8 of 9 patients suggest that chronic intravascular coagulation is not common in these patients. Serum BTG and PF4, used to estimate total platelet content of these proteins, were normal to slightly increased. That levels were not reduced implies that platelets do not usually circulate in a “spent” state. Therapeutic phlebotomy in 8 patients was associated with small decreases in plasma BTG and PF4 of uncertain clinical significance. Five of 14 patients had elevated plasma BTG with normal to only minimally increased plasma PF4. However, 10 of 10 patients tested were found to have reduced creatinine clearance, and therefore the relative contributions of platelet secretion and reduced BTG catabolism in the kidney to elevated plasma BTG levels are unclear.

Platelets, Coagulation and Fibrinolysis in Patients with Diabetic Retinopathy

Twenty control subjects, 12 insulin treated and 10 non-insulin treated diabetics were studied. All diabetics had retinopathy documented by fluorescein angiography and fluorophotometry. Factor VIIIR:Ag and plasma fibrinogen concentrations were elevated in both diabetic groups, but more so in the insulin treated patients. Within this latter group the plasma fibrinogen was also correlated with the degree of retinopathy. Platelets separated on linear isosmolar Percoll gradients showed an increase in intraplatelet beta TG content and concentration and a slight increase in volume of the lightest platelets in the insulin treated diabetics. Plasma platelet factor 4 and antithrombin III concentrations were normal and plasma beta TG levels were elevated only in those patients with renal insufficiency. Platelet aggregometry was performed in 18 diabetic subjects and found to be normal. It is concluded that abnormalities of coagulation and platelets in diabetes are determined by metabolic factors rather than the severity of microvascular disease per se.

Heparin induced thrombocytopenia. variable dependent platelet aggregating factor

The heparin dependent platelet aggregating factor in the serum from an individual with heparin induced thrombocytopenia exhibited reactivity with only 50% of an extensive panel of normal platelet rich plasmas, and this variability was independent of the platelet rich plasma platelet Factor 4 content. Individual sera of 3 other patients with heparin induced thrombocytopenia showed considerable variation in the lag phase and rate of platelet aggregation when tested with different normal platelet rich plasma preparations. These findings suggest that in the appropriate clinical setting the documentation of heparin dependent platelet aggregating factor should employ a panel of platelet rich plasmas if the initial screen is negative.

Enhanced in vivo platelet activation in diabetes mellitus.

Plasma levels of platelet factor 4 (PF4) determined in 58 apparently healthy subjects were found to increase with age. In 66 insulin dependent diabetics, PF4 plasma levels were increased but unrelated to the age of subject. Mean fibrinopeptide A levels were elevated in the diabetics but not correlated to PF4 levels. Activation of the coagulation system with thrombin generation thus seems to be operative in diabetes, but the enhanced platelet activation also observed in this disease is probably not thrombin mediated. No correlation was found between PF4 plasma concentration and the degree of vascular complication, HbA1C, blood glucose, 24-h urinary glucose, serum cholesterol or serum triglycerides.

The in vivo release of human platelet factor 4 by heparin

Intravenous and subcutaneous injection of heparin or the heparin analogue SSHA into normal volunteers induced release of platelet factor 4 (PF4) but not β-thromboglobulin (β-TG). At low heparin doses the amount of PF4 released was related to the plasma heparin concentration achieved. The rise in plasma PF4 was coincident with, and appeared to be a response to, the increase in plasma heparin concentration rather than to the absolute heparin level. After the primary response, the system became refractory to further challenge by the same heparin dose; the full initial magnitude of the response was not regained until 144 h. after heparin was first injected. The maximum amount of PF4 released corresponded to only about 5% of that potentially available from platelets. Moreover, heparin did not stimulate PF4 release from whole blood in vitro . We have demonstrated the presence of PF4 on the vascular endothelium, and suggest that this is the immediate source of the PF4 released by heparin, though it is probably initially derived from platelets. The effect of such binding on the antithrombotic potential of the endothelial surface is discussed.

Platelet Activation in Response to Phlebotomy

Plasma concentration of beta-thromboglobulin (BTG) and platelet factor 4 (PF4) were measured in three consecutive blood samples from 29 healthy male blood donors. The first sample was collected after 15 min of rest, the second immediately after a phlebotomy of 450 ml blood and the third after a further 15 min of rest. The mean baseline plasma BTG and PF4 values were 68 x 5 and 13.8 +/- 0.7 ng/ml, respectively. The second sample's mean plasma values of these two platelet-specific proteins were significantly higher (88 +/- ( and 24.1 +/- 4.1 ng/ml, respectively). The plasma concentrations of BTG and PF4 in the last sample, however, had returned to baseline levels. It is concluded that significant but short-lasting platelet activation and secretion take place in healthy subjects in response to an acute but comparatively mild blood loss even though the platelets do not participate in the process of hemostasis.

Enhanced platelet release reaction in insulin-dependent and insulin-independent diabetic patients.

Plasma beta thromboglobulin (BTG) and platelet factor 4 (PF4) levels were measured and correlated with plasma lipid and lipoprotein patterns in 20 insulin-independent diabetics, in 10 insulin-dependent diabetics and in 30 healthy controls matched to patients as to age, sex and weight. Increased plasma BTG and PF4 levels both in insulin-dependent and in insulin-independent diabetics indicate enhanced in vivo platelet activation and release reaction in these patients. No difference in BTG and PF4 values between these two groups of diabetic patients was observed in our study. A marked correlation between plasma PF4 values and plasma glucose levels was shown in insulin-dependent diabetics only whereas a significant positive correlation with plasma triglycerides and plasma triglyceride VLDL and a negative correlation with cholesterol-HDL was shown in insulin-independent diabetics.

慢性関節リウマチにおける血小板に関する研究

Plasma β-thromboglobulin (β-TG) concentration and platelet factor 4 (PF4) activity were examined and related to parameters of inflammation in patients with rheumatoid arthritis (RA). In 42 RA patients, the β-TG concentration assessed by radioimmuno-assay (β-TG RIA KIT Amersham) was significantly higher than that of 17 normal controls (p<0.002). The β-TG concentration correlated with the platelet count (γ=0.68, p<0.01) and erythrocyte sedimentation rate (γ=0.32, p<0.005). Plasma PF4 activity, tested by heparin neutralizing activity, was significantly higher than that of 17 normal controls (p<0.001). In the strongly positive CRP group (CRP 3(+) or more) PF4 activity was significantly higher than in the weakly positive CRP group (less than CRP 2(+)) (p<0.05). No correlation between PF4 activity and other parameters such as the platelet count, erythrocyte sedimentation rate, fibrinogen, antithrombinIII, or FDP was demonstrated. These results suggest that accerelated platelet functions were present in patients with clinically active RA.

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.

Studies of Human Platelet α-Granule Release In Vivo

AbstractThe clinical usefulness of measuring platelet factor 4 (PF4) and β-thromboglobulin (βTG) by radioimmunoassay in plasma, urine, and platelets has been evaluated in 105 normal subjects and 184 selected patients with suspected alterations in platelet reactivity. Since blood collection techniques are critical in minimizing in vitro artifact when measuring plasma PF4 and βTG, we developed a method for sampling and processing with respect to anticoagulant, inhibitory agents, temperature, centrifugation, and interval before processing. In 105 normal subjects, the levels of PF4 and βTG were 1.8 ± 1.0 (± 1 SD) and 6.0 ± 3.6 ng/ml, respectively, in plasma; 1.36 ± 0.24 and 4.29 ± 1.38 ng/fl × 10−6, respectively, in platelets; and 0.18 ± 0.16 ng PF4/mg creatinine in urine. Serum values for PF4 and βTG in these normal subjects, as well as in individuals with thrombocytopenia or thrombocytosis, correlated closely with the platelet concentration in whole blood (coefficient of correlation 0.947 and 0.826, respectively; p < 10−4), but the plasma levels of these platelet-specific proteins showed no correlation with platelet counts (p > 0.2). Patients with acute events such as cardiopulmonary bypass showed parallel depletion of PF4 and βTG from platelets, striking elevation of these proteins in plasma and clearance of PF4 into urine. Untransfused, severely thrombocytopenic patients with aplastic anemia had low levels of PF4 and βTG in their plasma, i.e., 0.3 ± 0.3 (p < 0.0001) and 4.8 ± 5.0 (p = 0.48) ng/ml, respectively, as well as in their platelets, 0.19 ± 0.17 (p < 0.0001) and 0.35 ± 0.16 (p < 0.0001) ng/fl × 10−6, respectively, compared with normal values. Despite marked increases in platelet destruction (shortened platelet survival and increased platelet turnover), patients with immune thrombocytopenic purpura (ITP) had normal levels of plasma PF4 and βTG (p = 0.81 and 0.29, respectively, compared with normal results). The two proteins were slightly elevated in the plasma of 41 hyperlipidemic subjects. However, these measurements could not differentiate patients into those with and without chronic vascular disease (p = 0.93). Measurements of these proteins in platelets or PF4 in urine were no more helpful than the plasma levels in predicting vascular disease. Levels in patients with atherosclerotic coronary artery disease showed substantial overlapping with levels in normal subjects. Patients with coronary artery disease who had survived out-of-hospital ventricular fibrillation without infarction had normal values of plasma PF4 (2.3 ± 1.5 ng/ml; p = 0.13) but significant elevations of plasma βTG (17.5 ± 11.9; p < 0.0001). In patients with documented atherosclerotic cardiovascular disease and angina pectoris, there was a small but statistically significant increase in plasma PF4 and βTG (3.1 ± 1.4 and 12.1 ± 2.9 ng/ml, p = 0.0003 and p < 0.0001 for PF4 and βTG, respectively, compared with normal values). Patients with a documented remote myocardial infarction also showed small increases in mean plasma PF4 values (3.1 ± 3.9 ng/ml; p = 0.002); treatment with aspirin did not decrease the plasma PF4 level (p > 0.1). We conclude that measurements of PF4 and βTG in plasma and platelets and of PF4 in urine do not identify individuals with stable chronic atherosclerotic vascular disease and thus do not generally provide a means for identifying patients at risk or following the effects of therapy. In contrast, marked elevation in plasma levels and depletion of these proteins from circulating platelets occur with acute in vivo platelet activation and release.

The interaction of platelets with aortic subendothelium: inhibition of adhesion and secretion by prostaglandin I2

We have studied the effect of prostaglandin I2 on platelet turnover, attachment to the subendothelium, and secretion following balloon deendothelialization of the rabbit aorta. Survival of 51Cr-labeled platelets in the balloon-injured animals remained normal. Thirty minutes after injury, there were 4.52 X 10(6) platelets/sq cm attached to the aortic surface, which was 87% covered by platelets. Although plasma platelet factor 4, as measured by radioimmunoassay, did not rise above the normal level of 6.8 +/- 2.6 ng/ml (mean +/- SEM) during the first hour after balloon injury, platelet factor 4 antigen was detected within the vessel wall by direct immunofluorescence within 30 min of injury. An infusion of 650–850 ng/kg/min prostaglandin I2 completely inhibited platelet aggregation and reduced surface coverage by 84% and platelet attachment by 63%. Animals given 50–100 ng/kg/min prostaglandin I2, which only partially inhibited platelet aggregation, had 70% of the aortic surface covered by platelets. Platelet factor 4 antigen was also detected within the aortic wall. Platelet attachment was normal in animals that had been given 850 ng/kg/min prostaglandin I2 prior to balloon injury but sacrificed after the infusion was stopped and ex vivo platelet aggregation had returned to normal.

The Interaction of Platelets With Aortic Subendothelium: Inhibition of Adhesion and Secretion by Prostaglandin I2

We have studied the effect of prostaglandin I2 on platelet turnover, attachment to the subendothelium, and secretion following balloon deendothelialization of the rabbit aorta. Survival of 51Cr-labeled platelets in the balloon-injured animals remained normal. Thirty minutes after injury, there were 4.52 X 10(6) platelets/sq cm attached to the aortic surface, which was 87% covered by platelets. Although plasma platelet factor 4, as measured by radioimmunoassay, did not rise above the normal level of 6.8 +/- 2.6 ng/ml (mean +/- SEM) during the first hour after balloon injury, platelet factor 4 antigen was detected within the vessel wall by direct immunofluorescence within 30 min of injury. An infusion of 650-850 ng/kg/min prostaglandin I2 completely inhibited platelet aggregation and reduced surface coverage by 84% and platelet attachment by 63%. Animals given 50-100 ng/kg/min prostaglandin I2, which only partially inhibited platelet aggregation, had 70% of the aortic surface covered by platelets. Platelet factor 4 antigen was also detected within the aortic wall. Platelet attachment was normal in animals that had been given 850 ng/kg/min prostaglandin I2 prior to balloon injury but sacrificed after the infusion was stopped and ex vivo platelet aggregation had returned to normal.

Plasma concentrations of platelet factor 4 in acute myocardial infarction.

The aim was to investigate whether in the acute stage of myocardial infarction (MI) platelet activation, as measured by plasma platelet factor 4 (PF-4), excluding that in ischaemic heart disease (IHD) is taking place. Over a period of 4 d the plasma levels of PF-4 were determined on 44 consecutive patients admitted to a coronary care unit with suspected MI. 21 of them had a definite acute MI (group 1), and 13 had evidence of IHD but no MI (group 2). In the remaining 10 subjects there was no evidence of either MI or IHD. On the first day the mean plasma PF-4 concentrations in group 1 and 2 patients were 11,8 +/- 1.1 and 15.0 +/- 2.3 ng/ml, respectively; the difference between means was not statistically significant. A peak mean PF-4 for group 1 patients (17.5 +/- 4.6 ng/ml) was recorded on the second day of study. The corresponding value for group 2 patients was lower, but not significantly so. In the latter subjects no peak PF-4 was recorded. During the last 2 d of study the plasma PF-4 concentrations tended to decrease, but the means for the 2 groups did not differ statistically. Thus, at no point in time was there a significant difference between the PF-4 values for MI and IHD patients present.

Plasma levels of platelet factor 4 in patients admitted to a coronary care unit.

Blood was obtained from 63 consecutive patients within 24 h period after the admission to a coronary care unit for the determination of plasma platelet factor 4 (PF-4) concentration. 28 of the subjects proved to have an acute myocardial infarction (MI), 24 had evidence of ischaemic heart disease (IHD) but no MI, and the remaining 11 patients had no signs of IHD. 40 healthy subjects served as controls. The mean PF-4 value in the MI group was 10.5 +/- 0.8 ng/ml. The corresponding values for patients with and without IHD were 8.7 +/- 0.6 and 8.3 +/- 0.6 ng/ml, respectively. The control mean (5.4 +/- 0.3 ng/ml) was significantly lower (P less than 0.001) than the means for all 3 groups of patients studied. The difference between the group of MI patients and patients with IHD as well as patients without IHD was only of borderline significance (0.10 greater than P greater than 0.05).