Blood Platelet Concentration Research Articles

Filtration technology

The leukocyte removal technology of platelet and red blood cell concentrates is now based on the only method: the filtration. In order to perfectly control this technology, it is necessary to know the different parameters that interfere in the quality of the leukocyte removal. These different parameters are studied here. They are classified in two types: the parameters that are related to the concentrate to filtrate: method used for the preparation, age, state (temperature, aggregates), concentration of red blood cells, platelets and leukocytes; the parameters that are related to the leukocyte removal technology and to the different steps of its carrying out: filter type (composition, size, geometry), prefilter, priming of the filter, flow rate during the filtration, rinsing and centrifugation after the filtration, elimination of the air inside the filter and the filtered concentrate, sterile connections, and finally good behavior in front of the difficulties and hitchs that could occur during the filtration procedure.

Fluorescence digital image analysis of thrombin and ADP induced rise in intracellular Ca 2+ concentration of single blood platelets

Cytoplasmic free Ca 2+ concentration, [Ca 2+] i, was estimated in single rabbit blood platelets by digital imaging microscopy with the use of the specific Ca 2+-indicator dye Fura-2. Uneven distribution and low level of [Ca 2+] i was found in the resting platelet even in the presence of extracellular 1 mM Ca 2+. Thrombin at 1 unit/ml immediately caused a transient increase in [Ca 2+] i, which was followed by a secondary and sustained increase in [Ca 2+] i. The distribution of increased levels of [Ca 2+] i was also shown to be uneven within the cell. The presence of 1 mM EGTA in the medium only slightly decreased the initial rise in [Ca 2+] i, but completely inhibited the latter phase, a sustained rise in [Ca 2+] i. This result shows that the initial rise of [Ca 2+] i might not be caused by Ca 2+ influx, but might be induced by mobilization of Ca 2+ from intracellular Ca 2+ storage sites. This speculation is further supported by the fact that the elevated [Ca 2+] i induced by thrombin immediately decreased to the base line value when 3 mM EGTA was applied. Thus, thrombin induced elevation of [Ca 2+] i is suggested to consist of two different processes, namely the mobilization of Ca 2+ from the intracellular storage sites and the successive Ca 2+ influx through the receptor activated Ca 2+ channels. Stimulation with ADP also caused a rapid elevation of platelet [Ca 2+] i, but this effect of ADP was different from that of thrombin. Thus, the ADP induced rise in [Ca 2+] i was accompanied by oscillation and was inhibited by extracellular EGTA. Our present experiment is the first report that clearly and directly reveals the differences between the effects of thrombin and ADP on [Ca 2+] i of platelets.

19F-NMR study of the effect of lead on intracellular free calcium in human platelets

Lead has been shown to affect calcium homeostasis. However, there is no prior evidence to indicate an effect of low concentrationsof lead in the environment (≈1 μM) on the intracellular free Ca 2+ concentration in any human tissue. We have investigated the effect of lead on the intracellular free Ca 2+ concentration of human blood platelets using 19F-NMR and a fluorinated intracellular Ca 2+ indicator. We report a basal intracellular free Ca 2+ value of 172 ± 8 nM. Treatment with 1, 5, 10 and 25 μM Pb 2+ resulted in average increases in intracellular free Ca 2+ of 39%, 91%, 135% and 172%, respectively. The percent increase in intracellular free Ca 2+ was linearly and positively correlated with the log of Pb 2+ concentration. Using atomic absorption spectroscopy, a significant increase in total calcium of approx. 10 nmol/mg protein was found in 25 μM Pb 2+ treated platelets. This indicates that influx of external Ca 2+ contributes to the observed increase in free Ca 2+. The results provide an explanation for the previously reported effects of lead on platelet function, and suggest a mechanism for low level lead-induced hypertension.

Mechanisms contributing to increased synthesis of plasminogen activator inhibitor type 1 in endothelial cells by constituents of platelets and their implications for thrombolysis.

We recently hypothesized that after pharmacologically induced coronary thrombolysis, increased activity of plasminogen activator inhibitor type 1 (PAI-1) retards recanalization, contributes to early reocclusion, or both. This hypothesis was based on the increased elaboration of PAI-1 that we observed in cultured liver cells exposed to growth factors releasable from platelets activated at sites of thrombosis in vivo. PAI-1 released locally is particularly likely to attenuate lysis of thrombi that are targets of thrombolytic drugs. Accordingly, the present study was performed to determine whether synthesis of PAI-1 by endothelial cells is augmented by products of platelets. Lysates from platelets (0.5-8.0 x 10(4)/mm3 media, i.e. less than 10% of the concentration of platelets in blood) increased synthesis and release of PAI-1 into both the extracellular matrix and conditioned media (by 2.8-fold and 3.3-fold within 6 and 24 hours, respectively). Synthesis of neither tissue-type plasminogen activator nor overall protein increased. Increased synthesis of PAI-1 was confirmed by immunoprecipitation of [35S]PAI-1 after metabolic labeling of cells. The increased elaboration of PAI-1 was consistent with increased transcription as reflected by the observed increase in PAI-1 mRNA of 2.2-fold in 4 hours. Effects of platelet lysates were simulated by transforming growth factor beta (TGF-beta), known to be present in platelet alpha-granules and released with platelet activation. Antibody to TGF-beta reduced the stimulation of PAI-1 synthesis by TGF-beta, as expected, by 82%.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct detection of activated platelets and platelet-derived microparticles in humans

Flow cytometry was used to determine whether activated platelets and platelet-derived microparticles can be detected directly in whole blood after a hemostatic insult. Two different in vivo models of platelet activation were examined: (1) a standardized bleeding time, and (2) cardiopulmonary bypass. Platelets and microplatelets were identified with a biotinylated anti-glycoprotein (GP)lb antibody and a fluorophore, phycoerythrin-streptavidin. Microparticles were distinguished from platelets by light scatter. Activated platelets were detected with three fluorescein-labeled monoclonal antibodies (MoAbs): (1) PAC1, which binds to the activated form of GPIIb-IIIa; (2) 9F9, a newly developed antibody that is specific for fibrinogen bound to the surface of activated platelets; and (3) S12, which binds to an alpha- granule membrane protein expressed on the platelet surface after granule secretion. In nine normal subjects, bleeding times ranged from 4.5 to 7.5 minutes. Over this time, there was a progressive increase in the amount of PAC1, 9F9, and S12 bound to platelets in blood emerging from the bleeding time wound. With all three antibodies, platelet activation was apparent as early as 30 seconds after the incision (P less than .03). Activation was accompanied by a progressive decrease in the concentration of platelets in blood from the wound, while the concentration of microparticles increased slightly. In nine patients undergoing open heart surgery, 1 hour of cardiopulmonary bypass caused a 2.2-fold increase in the relative proportion of microparticles in circulating blood (P less than .001). Moreover, bypass caused platelet activation as evidenced by a mean two- to threefold increase in PAC1 binding to platelets. Although this increase was significant (P less than .02), PAC1 binding exceeded the normal range for unstimulated control platelets in only 5 of 9 patients, and 9F9 and S12 binding exceeded the normal range in only two patients. Taken together, these studies demonstrate that it is now feasible using flow cytometry to evaluate the extent of platelet activation and the presence of platelet- derived microparticles in the circulation of humans.

Direct Detection of Activated Platelets and Platelet-Derived Microparticles in Humans

Flow cytometry was used to determine whether activated platelets and platelet-derived microparticles can be detected directly in whole blood after a hemostatic insult. Two different in vivo models of platelet activation were examined: (1) a standardized bleeding time, and (2) cardiopulmonary bypass. Platelets and microplatelets were identified with a biotinylated anti-glycoprotein (GP)lb antibody and a fluorophore, phycoerythrin-streptavidin. Microparticles were distinguished from platelets by light scatter. Activated platelets were detected with three fluorescein-labeled monoclonal antibodies (MoAbs): (1) PAC1, which binds to the activated form of GPIIb-IIIa; (2) 9F9, a newly developed antibody that is specific for fibrinogen bound to the surface of activated platelets; and (3) S12, which binds to an alpha-granule membrane protein expressed on the platelet surface after granule secretion. In nine normal subjects, bleeding times ranged from 4.5 to 7.5 minutes. Over this time, there was a progressive increase in the amount of PAC1, 9F9, and S12 bound to platelets in blood emerging from the bleeding time wound. With all three antibodies, platelet activation was apparent as early as 30 seconds after the incision (P less than .03). Activation was accompanied by a progressive decrease in the concentration of platelets in blood from the wound, while the concentration of microparticles increased slightly. In nine patients undergoing open heart surgery, 1 hour of cardiopulmonary bypass caused a 2.2-fold increase in the relative proportion of microparticles in circulating blood (P less than .001). Moreover, bypass caused platelet activation as evidenced by a mean two- to threefold increase in PAC1 binding to platelets. Although this increase was significant (P less than .02), PAC1 binding exceeded the normal range for unstimulated control platelets in only 5 of 9 patients, and 9F9 and S12 binding exceeded the normal range in only two patients. Taken together, these studies demonstrate that it is now feasible using flow cytometry to evaluate the extent of platelet activation and the presence of platelet-derived microparticles in the circulation of humans.

Biocompatibility of carbon-carbon materials: Blood tolerability

Carbon-carbon composites are well known in the field of aerospace technology. Such composites have been proposed to be used as biomaterials, particularly in contact with blood. To evaluate their haemocompatibility, samples were tested in vivo and in vitro, using radiotracers. In vivo study showed the accumulation of platelets on the exposed surface material with any surface morphology, whereas platelet concentration in blood remained constant. In vitro study allowed us to distinguish, among entrapped platelets, active adhering platelets from those mechanically retained and it appeared that the bulk structure of materials influenced the adhesion mechanism of platelets.

Cytoplasmic fragmentation associated with lymphoid leukemia in ruminants: interference with electronic determination of platelet concentration.

Leukocyte cytoplasmic fragmentation is associated with human leukemia, usually with leukocyte concentrations in excess of 1 O0,000/J.1.2.4,6 Increased attention has been drawn to leukocyte cytoplasmic fragments with the common use of automated hematology analyzers, and the recognition that such fragments interfere with the whole blood platelet counting capability of these instruments and by microscopy on hemocytometers. 1.2.4.6 This report documents interference of prominent leukemia-associated leukocyte fragmentation with automated electronic determination of platelet concentration in a cow and a sheep. This observation is important in veterinary hematology because it represents a morphologic feature of leukemia in animals and may result in errant enumeration of platelets by a variety of techniques. The S-Plus series of automated blood cell counters determines blood platelet concentration by mathematical analysis of both particle size distribution and concentration within a size range window encompassing approximately the lower third ofthe potential platelet volume range. This size window is about 1.3-1 3 fl on most automated cell counters modified for use in veterinary hematology. The instrument calculates platelet concentration after fitting a log normal curve to raw data in the size window (Fig. IA). In order for the curve fitting routine to function it must detect one mode between two minimum points within the size window. Furthermore, the mode must be between 2 fl and 10 fl on the modified system. If any of these criteria are not met, curve fitting is not done, an incomplete computation occurs, and platelet concentration is not printed. A cow and a sheep were evaluated hematologically by examination of Wright-Giemsa-stained blood films and an automated hemogram. Hemograms were done on an automated multi-channel analyzer modified for use in veterinary hematology by increasing the erythrocyte-platelet aperture bath current to 225 V (Coulter Counter Model S-Plus IV with histogram capability, Coulter Electronics Inc., Hialeah, FL). Platelet adequacy was estimated by blood film examination: no attempt was made to enumerate platelets by hemocytometer. The sheep was a 13-month-old black face ewe, which had been inoculated experimentally at 3 weeks of age with bovine leukemia virus (BLV) and was BLV-seropositive. Hematologic evaluation revealed severe nonregenerative anemia (hematocrit = 0.13) and extreme leukocytosis. The blood was diluted 1 : 10 to determine the leukocyte concentration. The total leukocyte concentration was 699,00O/pl, with 100°/o lymphoblasts. The sheep was killed for necropsy and was found to have multicentric lymphosarcoma consistent with that described following experimental infection with BLV.5 A 7-year-old Holstein cow suspected to have a displaced abomasum was brought to the Colorado State University. Masses were present in regions of the thoracic inlet, supra177

The role of platelets in progressive glomerulosclerosis: Mechanisms for intraglomerular platelet activation and pathogenetic consequences

Purkerson et al. have hypothesized that platelet aggregation and release reactions contribute to the progressive sclerosis of remnant nephrons. The suggested mechanism for platelet activation is disruption of endothelium by the high hydraulic pressures characteristic of remnant nephrons, with platelet exposure to basement membrane collagen. We herein postulate additional mechanisms whereby platelets might be activated in the microcirculation of remnant nephrons: (i) kinetic activation due to high plasma flow rates; (ii) close cell contact due to concentration of blood cells and platelets in the glomerulus; (iii) concentration of protein macromolecules that act as agonists for platelet release; (iv) glomerular release of saturated and monenoic fatty acids that stimulate platelet synthesis of thromboxane A2; (v) glomerular release of membrane-bound pro-coagulant factor, triggering a chain reaction that activates both platelets and the intraglomerular coagulation cascade. Platelet activation could play a pathogenetic role in many of the known derangements of structure and function in remnant nephrons. This paradigm may also be partially applicable to the glomerulosclerosis of diabetes mellitus.

Collection and transfusion of single donor platelets and their clinical response.

The success of transfusing platelet concentrates depends on a variety of factors; those factors are to a certain extent the subject of this paper. The quantity and quality of platelets transfused is discussed in relation to peripheral blood platelet concentration. The data obtained do not yet allow a final conclusion at present, so that further investigations are required.

In-vitro platelet sensitivity to collagen-induced aggregation and in-vitro platelet deterioration rate during storage, are both inversely proportional to the in-vivo blood platelet concentration in normal individuals: preliminary documentation of these theoretically predictable relationships

This preliminary study documents two new functional relationships for platelets obtained from normal subjects, whose individual blood platelet concentrations (IBPCs) are in the normal range. Firstly, platelets from subjects (or donors) with high-normal IBPCs are the least sensitive to collagen-induced aggregation (a significant inverse correlation). This may be of importance in determining the likely efficacy of fresh platelet transfusions obtained from donors with different IBPCs within the normal range. Secondly, platelets from subjects/donors with high-normal IBPCs deteriorate less rapidly during storage (a significant inverse correlation), perhaps due to differences in platelet biochemical activity between individuals. Such measurements of platelet deterioration rate were based upon the development of a new experimental technique, in which sterile test-PRP was incubated at 37°C with continuous (quarter-hourly) indirect measurements of changes in platelet morphology. This tube-based method is described in detail. The functional relationships observed do have a theoretical basis within the work of others, which is appropriately outlined. Particular discussion pivots around the possibility of using the deterioration rate relationship to predict how therapeutic platelet concentrates prepared from a given donor of know IBPC, may retain their morphological integrity during routine blood bank storage.

Sodium depletion increases platelet and plasma catecholamines in hypertensive men.

The catecholamine content in blood platelets is considerably higher than that in plasma, and platelet catecholamines must be taken up from plasma, since blood platelets lack enzymes for catecholamine synthesis. However, it is unknown whether platelets take up and store catecholamines during physiological in vivo increments in plasma catecholamines. Previously untreated 50-year-old men (n = 17) with mild to moderate essential hypertension were given a low sodium diet for 2 weeks. Urinary excretion of sodium decreased from 201 +/- 11 (SE) to 24 +/- 5 and 19 +/- 4 mmol/24 hr after 1 and 2 weeks, respectively. During the first week, the blood platelet concentration of norepinephrine increased from 27.2 +/- 2.9 to 39.6 +/- 4.7 pg/mg (p less than 0.005) and venous plasma norepinephrine increased from 3.7 +/- 0.4 to 5.6 +/- 0.5 pg/ml (p less than 0.005), and venous plasma dopamine increased from 26 +/- 4 to 41 +/- 5 pg/ml (p less than 0.05). During the second week, both plasma and platelet norepinephrine and dopamine remained elevated. Platelet epinephrine showed a small increase from baseline to the second week (p less than 0.05), but no concomitant increase in plasma epinephrine occurred. Thus, sodium depletion increases both platelet and plasma catecholamines and blood platelets may take up catecholamines in vivo. Platelet catecholamine content may be an integrated measure of plasma catecholamine concentrations during variations caused by sodium depletion.

Linoleic acid levels in white blood cells, platelets, and serum of multiple sclerosis patients.

We found a small but statistically significant reduction in the linoleic acid concentration of white blood cells and platelets in MS patients. The percent linoleic acid concentration (mean +/- S.D.) in the white blood cells if 24 MS patients was 8.8 +/- 1.8% as compared with 11.4 +/- 4.9 in 24 age and sex-matched controls (p less than 0.05). Platelet levels were 8.5 +/- 2.4% and 10.6 +/- 3.8% respectively (P less than 0.05). Serum linoleic acid levels were not significantly different in the two groups. The possible role of linoleic acid in the pathogenesis of MS has yet to be defined.

Serum α-6-l-fucosyltransferase is released from platelets during clotting of blood

Activity of serum α-6-l-fucosyltransferase is strictly correlated with the original concentration of blood platelets. The plasma enzyme activity is confined to intact platelets and appears in serum during the course of coagulation of blood. Activity of serum α-3-l-fucosyltransferase does not depend on platelets.

Suppression of maturation of megakaryocyte colony forming unit in vitro by a platelet-released glycoprotein.

The suppressive role of platelets on the growth of human marrow megakaryocyte colony forming units (CFU-M) in vitro was investigated by the use of a plasma clot assay. An inverse correlation was established between the number of megakaryocytic colonies grown and the platelet concentration of the plasma or the resultant serum used in the culture system. The suppressive effect of platelets on megakaryocyte colony formation reached a plateau at normal human blood platelet concentration and was specific for CFU-M growth, since marrow cell erythroid burst formation (BFU-E) and granulocytic-monocytic colony formation (CFU-GM) remained unaffected. The inhibitory activity was detectable in the supernatants of platelet suspensions aggregated by thrombin or ADP, and the inhibitory activity released from ADP-stimulated platelets was blocked by pretreatment of platelets with monoclonal antibody HuPl-m1. Partial purification of this activity was achieved by diethylaminoethyl (DEAE)-ion exchange and phytohemagglutinin (PHA)-E agarose affinity chromatography. This inhibitor is a glycoprotein with a molecular weight of 12-17K daltons. This platelet released glycoprotein does not affect the early proliferative phase of CFU-M in vitro but acts on a day 6-8 CFU-M-derived cell by adversely affecting its maturation into recognizable megakaryocytes. These findings demonstrate that a glycoprotein released from platelets suppresses the maturation of CFU-M into megakaryocytes.

Thrombocytopenia is an initial maternal response to fertilization in mice

There was an increase in weight of the spleens of pregnant and pseudopregnant mice in the first week after mating, but the increase occurred on Day 4 in pregnant mice and Day 2 in pseudopregnant mice. The retardation of the presumed hormonally induced increase in spleen weight during pregnancy corresponded with a significant reduction in the splenic platelet pool. This response by the spleen to early pregnancy suggested that platelets were being supplied to the vascular pool. There was a significant reduction in the platelet count by 10:30 h on the day of mating in pregnant mice and persisted until Day 7 of pregnancy, then returning to normal levels. This response did not occur in pseudopregnant mice. The decrease in platelet count was dependent upon the presence of fertilized eggs. It did not occur in mice sterilized by bilateral ligations of the oviducts and mated with fertile males. Thrombocytopenia did occur within 3 h of transfer of fertilized eggs to pseudopregnant recipients and the magnitude of the response was significantly correlated (b = -0.86) with the number of embryos present in the reproductive tract. An initial systemic response to pregnancy in mice was therefore an increased vascular demand for blood platelets, resulting in a significant reduction in the splenic and peripheral blood platelet concentration.

Continuous-flow techniques for platelet concentrate collection: a step toward standardization and yield predictability.

This study chronicles leukocyte- and erythrocyte-depleted platelet concentrate collection by a dual stage channel in which three variables: 1) donor peripheral blood platelet concentration, 2) total blood processed, and 3) collection volume were statistically correlated with platelet yield as determined by a multiple regression analysis of single variables. Platelet concentration in the final yield was related to donor precount and collection rate, and could be varied as indicated for individual applications. Total blood processed was established by procedure time, which in turn was defined by citrate-induced calcium changes in the donor. Reduction in peripheral blood platelet concentration averaged 24% for a mean platelet yield of 3.8 X 10(11). An average of 40% of transfused platelet concentrates were recovered in recipient peripheral blood 1 hr posttransfusion and were hemostatically effective, as determined by correction of bleeding time. Platelet yields and patient response were sustained during current collection procedures, verifying the principles described during the investigative period.

Tumor-cell-platelet aggregation does not correlate with metastatic potential of rat 13762NF mammary adenocarcinoma tumor cell clones.

The ability of tumor cells to induce platelet aggregation has been correlated with their capacities to colonize the lungs of experimental animals. We tested this hypothesis by studying the ability of cloned, low-passage metastatic tumor cell lines derived from rat 13762NF mammary adenocarcinoma to aggregate rat platelets in vitro and in vivo, and we then compared this activity to metastatic potential by determining the incidence of lung metastasis after subcutaneous or intravenous inoculation of the tumor cell clones into syngeneic rats. Our results failed to show a correlation between in vitro platelet-aggregating activity and metastatic potential. In this system we could not detect platelet-aggregation activity with the most metastatic tumor clone, while least metastatic clone clearly possessed high platelet-aggregating activity. In addition, by measuring changes in blood platelet and fibrinogen concentrations at various intervals following intravenous injection of tumor cell clones, we were able to confirm in vivo the observed in vitro differences in their platelet-aggregating activities. Thus, platelet aggregating activity is heterogeneously expressed among 13762NF cell clones and appears unrelated to spontaneous or experimental metastasis in this tumor system.

Sequential effects of lithium on haematopoiesis.

The administration of lithium salts to haematologically normal subjects is associated with increased blood neutrophil concentrations and marrow neutrophil production and with enhanced release of colony stimulating activity (CSA) required for growth of granulocyte-macrophage progenitor cells (CFU-GM) in vitro. To examine the haematopoietic changes associated with lithium salts, mice were given LiCl daily. Blood neutrophils and serum CSA levels increased as did blood platelet concentrations. These increments were preceded by expansion of marrow neutrophil production and increased concentrations of CFU-GM as well as progenitor cells for megakaryocytes (CFU-M) and erythrocytes (BFU-E and CFU-E). An earlier and sustained increase of transplantable pluripotential stem cells (CFU-S) was detected beginning at day 2 of lithium administration. The sustained increase of CFU-S with lithium was not associated with detectable changes of endogenous stem cells (E-CFU) suggesting that a portion of the stem cell pool is resistant to the proliferative effects of lithium. These studies indicate that lithium acts initially to directly or indirectly increase marrow CFU-S with later increments of progenitor cells. The more sustained increase of blood neutrophils with lithium administration may be the result of subsequent increments of CSA resulting in enhanced marrow granulocyte production.

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.