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We compared the in vitro effect of thrombopoietin (TPO) on platelet aggregation to other hematopoietic growth factors (HGFs): granulocyte colony stimulating factor (G- CSF), granulocyte macrophage colony stimulating factor (GM- CSF), interleukin-3 (IL-3), interleukin-6 (IL-6), and erythro poietin (EPO). The platelet aggregation response of the venous blood of eight volunteers with differing aggregation patterns was characterized by a platelet aggregation (PA) score that provided numerical assessment of an individual's platelet ag gregation tendency to adenosine diphosphate (ADP). The method for analysis of the data included constructing regres sion lines for each participant's PA score versus the HGF con centration (range of 0.5 nG/mL to 100 nG/mL) and calculating the regression coefficients for the slope and the Y axis inter cept. We demonstrated that the proposed method resulted in accurate assessment of the HGF effect or lack of it on the platelet aggregation response. Within the concentration range evaluated, the influence of IL-6 and G-CSF on platelet aggrega bility was insignificant and the effects of GM-CSF and IL-3 were almost undetectable. In contrast, the potentiating effect of TPO on platelet activation was dose dependent with significant enhancement for all responder types. The concentrations of TPO utilized in our in vitro experiments were in pathophysi ological range, indicating that its impact on platelet aggregation may have clinical relevance, and that monitoring platelet func tion in conjunction with TPO treatment might be advisable. Investigation of the possible synergistic effect of HGF combi nations is warranted.

Thrombopoietin (TPO), the ligand for the proto- oncogene c-mpl, has been cloned and expressed from both human and murine sources. Thrombopoietin increases platelet counts when given in vivo and acts on progenitor cells to in crease their proliferation and maturation into megakaryocytes. The effects of TPO on mature platelets were investigated by evaluating platelet aggregation and platelet activation- dependent antigen expression. Platelet aggregation score, a quantitative representation of aggregation, showed potentiation of response to ADP-induced aggregation but no direct agonist response to TPO alone. Soluble c-mpl blocked the effect of TPO on the platelet aggregation score. Flow cytometry showed that TPO at concentrations >250 U/ml (50 ng/ml) caused a minority population of platelets to express the activation mark ers CD62, CD63 and activated glycoprotein IIb/IIIa. While stem cell factor and interleukins-3 and -6 did not affect platelet activation antigen expression, interleukin-11 increased CD62 expression on platelets in vitro. The effects of TPO on antigenic expression and aggregability were partially inhibited in vitro by preincubation with aspirin. We conclude that high concentra tions of TPO promote platelet activation antigen expression on a proportion of platelets and potentiate platelet aggregability to ADP in vitro by a process that is partially inhibited by aspirin.

The turbidimetric method of Bom is the current approach for assessing the aggregation behavior of platelets. It has been of modest practical value, due to difficulty in standardizing laboratory techniques for plasma preparation and inadequate quantification of the aggregation process. We report a new technique of sedimented platelet rich plasma (SPRP) preparation that reduces the irregularities caused by factors associated with procuring and preparing blood samples and permits a more flexible protocol for laboratory practice. We quantified results with a platelet aggregation score, which is calculated by multiplying the ratio of the height of the initial wave of aggregation to the maximum height of the aggregation pattern by the total area under the aggregation curve and by the ratio of the whole blood platelet count and the number of platelets in the sample. Comparative analysis of platelet aggregation scores (n = 95) obtained with both plasma preparation techniques using a paired t test demonstrated no statistical differences ( t = 1.368, p = 0.174). To demonstrate the application of this modified method to evaluation of antiplatelet agents, the effects of aspirin and aspirin combined with citric acid on platelet aggregation were studied in vitro. The antiaggregatory effect of aspirin combined with citric acid was dependent on the pH and on their molar ratio, and was greater than the effect of aspirin alone. The SPRP protocol with platelet aggregation scoring methodology could be a valid alternative for measurement of the platelets' propensity to aggregate and the effect of antithrombotic treatments. Key Words: Platelet aggregation— Aspirin—Citric acid.

We sought to develop a predictive method, based on the integration of blood parameters found to be significantly associated with thrombosis and progression of atherosclerosis, that would be more accurate than primary reliance on elevation of separate risk factors. The study involved 1,034 male subjects. Lipid profiles and fibrinogen levels were determined for 123 men with documented clinical atherosclerosis and 123 apparently healthy volunteers. There were significant differences between these groups for fibrinogen, triglycerides, and HDL cholesterol. An algebraic expression was then developed which combined normalized values of fibrinogen, HDL cholesterol, and triglycerides with platelet count and aggregation data to yield a numerical value, the clinical atheroscterosis predisposition (CAP) factor. We then tested this CAP factor with a validation group of 788 men, 372 of whom were patients with documented clinical atherosclerosis; the rest were apparently healthy. A 3-year prospective evaluation was done for 72 of the 123 apparently healthy volunteers. The CAP factor was 90% indicative of patient status for the 372 men with documented complications of atherosclerosis and appeared to predict severe coronary disease in the 3-year prospective evaluation. This initial study suggests that integrated analysis of thrombotic factors in white males. provides a useful estimate of an individual's risk for subsequent development of clinical atherosclerosis. Key Words: Atherosclerosis-Prevention and control-Blood-Platelets-Cholesterol-Triglycerides--Fibrinogen.

This study had three objectives: (1) to determine the frequency of high platelet aggregators in a consecutive series of 268 apparently healthy volunteers who presented to our Center; (2) to assess the inhibitory effect of aspirin (ASA) on these high aggregators; (3) to determine, in a double-blind trial, whether or not the addition of citric acid (CTA) to ASA would increase its inhibitory effect in subjects who had a suboptimal response to aspirin alone. A platelet aggregation-scoring methodology developed for turbidimetric platelet aggregometry was used to quantify baseline aggregation and medicinal effects. We define a high aggregator as one whose unmedicated PA score is ≥30. We define the response of a high aggregator to ASA as poor if the medicated PA score stays at ≥30. We found that 58 of 268 apparently healthy unmedicated volunteers (22%) had PA scores ≥30. and that 27 of these (47%) had a poor response to 325 mg ASA, with an average drop in their PA scores from 49.5 ± 13.1 to 41.1 ± 8.6 (16%). Twenty-five of these 27 people were enrolled in the double-Mind study comparing the effect of ASA and ASA + CTA on platelet aggregability. Of these high aggregators who had a poor response to ASA, 12 of 25 (50%) had a good response to 162.5 mg of ASA plus 162.5 mg of CTA, with an average drop of their PA scores from 46.7 ± 13.2 to 22.0 ± 5.2 (53%). CTA alone had no effect on the PA score, which was similar to the control placebo. Our data suggest that a 1:1 combination of ASA and CTA may offer significantly greater protection agairtst arterial thrombotic events than ASA alone in subjects who respond poorly to ASA. Key Words: Platelet aggregation—Antithrombotic medication—Thrombosis.

We describe a new method of interpreting platelet aggregometry results and its correlation to platelet secretory protein levels. We studied 123 healthy male volunteers, 37-85 years of age. The aggregation response to 2.5 μM ADP was measured in a standardized platelet suspension (SPS) prepared from platelet-rich plasma. Turbidimetic measurements included amplitude of the primary aggregation wave (Amp1), maximum aggregation amplitude (Amax), and area under the aggregation curve (Sagg). A platelet aggregation (PA) score was calculated as ( Amp1 /Amax) ·Sagg ·(whole-blood platelet count/SPS platelet count). β-Thromboglobulin (β-TG)and . platelet factor 4 (PF4) levels were measured by radioimmunoassay. The PA score correlated significantly (p < 0.0001) with β-TG ( r = 0.86) and PF4 ( r = 0.87). It was concluded that the PA score correlates closely with individual subjects' propensities to platelet activation, as reflected by plasma β-TG and PF4 levels. Key Words: Platelets-Platelet aggregometry-Platelet secretory proteins.