In-vivo Platelet Activation and Anomalous Thrombospondin Levels in Severe Falciparum Malaria

To investigate in vivo platelet function in acute falciparum malaria plasma concentrations of β-thromboglobulin (β-TG), platelet factor 4 (PF4) and thrombospondin (TSP) were determined in 10 severely-ill Thai patients and 11 healthy volunteers. 8 patients recovered. At presentation, the platelet counts of the 10 patients were significantly lower (p < 0.025) than those of the controls, and a slight but significant increase (p < 0.05) in β-TG/PF4 ratios in the patients suggested low-grade platelet activation. Presentation plasma β-TG and PF4 concentrations did not differ from control values, probably due to the opposing effects of decreased circulating platelet mass and increased activation. By contrast, admission concentrations of TSP in the surviving patients were markedly lower (p < 0.001) than those of the controls; β-TG/PF4 ratios, but not TSP levels, returned to normal during treatment. Hepatic dysfunction and oliguric renal failure probably contributed to a sustained increase in plasma β - TG and TSP in the 2 fatally ill patients, but associated elevated PF4 levels indicated concomitant platelet activation. Our results support the suggestion that in vivo platelet activation, which appears to be rapidly controlled by treatment, occurs in patients with severe, non-fatal falciparum malaria. TSP production, apparently from non-platelet sources, was decreased and/or its consumption was increased in these patients, perhaps by factors such as cytoadherence of infected erythrocytes and consequent endothelial damage.     

In-vivo Platelet Activation and Anomalous Thrombospondin Levels in Severe Falciparum Malaria

To investigate in vivo platelet function in acute falciparum malaria plasma concentrations of β-thromboglobulin (β-TG), platelet factor 4 (PF4) and thrombospondin (TSP) were determined in 10 severely-ill Thai patients and 11 healthy volunteers. 8 patients recovered. At presentation, the platelet counts of the 10 patients were significantly lower (p < 0.025) than those of the controls, and a slight but significant increase (p < 0.05) in β-TG/PF4 ratios in the patients suggested low-grade platelet activation. Presentation plasma β-TG and PF4 concentrations did not differ from control values, probably due to the opposing effects of decreased circulating platelet mass and increased activation. By contrast, admission concentrations of TSP in the surviving patients were markedly lower (p < 0.001) than those of the controls; β-TG/PF4 ratios, but not TSP levels, returned to normal during treatment. Hepatic dysfunction and oliguric renal failure probably contributed to a sustained increase in plasma β - TG and TSP in the 2 fatally ill patients, but associated elevated PF4 levels indicated concomitant platelet activation. Our results support the suggestion that in vivo platelet activation, which appears to be rapidly controlled by treatment, occurs in patients with severe, non-fatal falciparum malaria. TSP production, apparently from non-platelet sources, was decreased and/or its consumption was increased in these patients, perhaps by factors such as cytoadherence of infected erythrocytes and consequent endothelial damage.     

Relationship of Platelet Specific Proteins and Other Factors to Atherosclerosis in Various Stages of Hypertension

There is considerable evidence from previous studies that platelets play an important role in the development and progression of atherosclerosis in hypertension, more so in relation to the stage of hypertension. Seventy one hypertensive patients (WHO stage I: 39, stage II: 23, stage III: 9) aged 19–84 (mean age: 56, normal controls in this study 59 and 62 respectively for each stage) and 37 aged 22-72 with a mean age of 52) were involved Hematocrit, beta-thromboglobulin (β-TG), platelet factor 4 (PF4), β-TG/PF4 ratio, total cholesterol (TC), low density lipoprotein-C, and triglycerides were higher in the hypertensive group while platelet count, circulating platelet aggregates, and high density lipoprotein-C were higher in the normotensive group. Among the hypertensives, stage I11 patients showed the highest β-TG, PF4, β-TG/PF4 ratio, triglycerides, and stage I with the least elevation. There were no significant differences noted in the ADP or epinephrine-induced platelet aggregation in both the normal and hypertensive patients. Other parameters such as heart rate, serum sodium, potassium, renal and liver function tests, plasma renin activity, aldosterone, fibrinogen thromboxane B and 6-Keto-PGF1jI showed no significant differences in both groups. This study clearly showed that β-TG/PF4 ratio and triglycerides are closely related to the stage of hypertension and are good indicators of in vivo platelet activation in hypertensives which may account for the acceleration of hypertensive vascular complications secondary to atherogenesis.     

Platelet factor 4 and β-thromboglobulin mRNAs in circulating microparticles of trauma patients as diagnostic markers for deep vein thrombosis

Deep vein thrombosis (DVT) is a common complication after trauma. The development of markers to predict DVT in trauma patients is needed, and circulating microparticles (MPs) and their contents are possible candidates. In this study, we aimed to identify platelet factor 4 (PF4) and β-thromboglobulin (β-TG) mRNAs in circulating MPs as potential markers for DVT diagnosis in trauma patients. Fifteen trauma patients diagnosed with DVT and fifteen matched patients without DVT were included in this study. Fifteen healthy volunteers also were included as controls. Circulating MPs were obtained from the plasma of all study subjects. Annexin V+?MPs and platelet-derived MPs (PMPs) were quantified using flow cytometry. PF4 and β-TG mRNAs in MPs were determined by qPCR, and the common logarithm of relative quantitation (RQ) was calculated using the comparative Ct method. Receiver-operating characteristic (ROC) curves were performed to analyze the diagnostic value of PF4 and β-TG mRNAs. No significant differences were found in Annexin V+?MPs and PMPs levels between trauma patients with and without DVT. However, both PF4 and β-TG mRNAs in MPs from the DVT group were significantly higher than the non-DVT group and healthy controls (P?=?0.014 for PF4, P?=?0.010 for β-TG). The ROC curve analysis showed that both the PF4 mRNA (area-under curve (AUC) 0.756, P?=?0.017) and the β-TG mRNA (AUC 0.751, P?=?0.019) had a positive predictive value for DVT. This finding indicates that the PF4 and β-TG mRNAs in MPs may be used as potential biomarkers for DVT diagnosis in trauma patients.

     

Effect of heparin on in vivo platelet factor 4 (PF4) release and platelet aggregation after aspirin administration

We studied the release in vivo of platelet specific proteins platelet factor 4 (PF4) and β-thromboglobulin (βTG), and confirmed that only PF4 is released, after heparin intravenous administration. A good correlation was found between platelet count and PF4 and this seems at variance with the idea that the source of released PF4 is vascular endothelium rather than platelets; however, such a possibility cannot be excluded. Although platelet function was blocked by aspirin injection heparin was still able to induce the release of PF4 and exerted a potentiating effect on platelet aggregation induced by ADP and collagen.     

Dipyridamole and Platelet Release of Platelet-derived Growth Factor

Platelet-derived growth factor (PDGF) and β-thromboglobulin (β-TG) are released from platelet alpha-granules during platelet activation. PDGF is a potent chemoattractant and mitogen for human vascular smooth muscle cells, and may be important in the development of late restenosis following angioplasty and in atherogenesis. In recent studies, where PDGF release into serum was evaluated indirectly by measuring (3)H-thymidine incorporation into fibroblasts, it was reported that the antiplatelet drug dipyridamole (DPM) decreased serum levels of PDGF. Such selective inhibition of the PDGF-release would have potential important implications for patients with atherosclerosis and for patients undergoing angioplasty. We therefore measured platelet content of PDGF and β-TG as well as platelet release of PDGF using a newly developed radioimmunoassay in healthy volunteers before and immediately after ingestion of DPM 100 mg t.i.d. for 3 days. We found no significant differences in platelet content of PDGF or β-TG before and after DPM. PDGF release from platelets isolated from plasma by gel filtration and stimulated with thrombin as well as platelet release of PDGF into serum was also unaffected by DPM. In conclusion, treatment with DPM does not affect platelet content of PDGF or β-TG. The treatment did not inhibit the platelet-release of PDGF as previously reported, neither via direct effects on platelets nor on inhibitory plasma components. DPM may, however, inhibit (3)H-thymidine incorporation into fibroblasts.     

Optimized flow cytometric assay for the measurement of platelet microparticles in plasma: pre-analytic and analytic considerations.

Platelet microparticles (PMP) are submicroscopic membrane vesicles released by platelets during activation. Flow cytometry is the most widely used method for quantifying PMP, but the optimization of the technical method has not yet been fully evaluated. This study was designed to assess the pre-analytical variables including blood sampling conditions, and to evaluate the analytical variations including effect of the platelet-specific antibodies and quantitative beads, precision, linearity and accuracy in comparison with beta-thromboglobulin, which is one of the platelet activation markers. Numbers of PMP collected into citrate-theophylline-adenosine-dipyridamole (CTAD) tubes were increased with time, but to a lesser extent than when collected into sodium citrate tubes. The precision of the PMP assay was relatively high. Excellent linear correlation was observed for dilution linearity. Regarding the platelet-specific antibodies used, anti-CD41a-labeled samples resulted in higher PMP levels than those labeled with anti-CD61 and anti-CD42a. There was no significant difference of PMP counts according to the quantitative beads. The PMP assay is well correlated with beta-thromboglobulin levels. Our findings suggest that blood samples for the PMP assay should be collected in a CTAD tube and delayed measurement is not allowed to avoid artefactual platelet activation. The PMP assay can be used successfully as a useful marker of the detection of in vivo platelet activation, provided that pre-analytical and technical points are optimally taken into consideration.     

Associations between arterial stiffness and platelet activation in normotensive overweight and obese young adults

Obese individuals have elevated platelet activation and arterial stiffness, but the strength and temporality of the relationship between these factors remain unclear. We aimed to determine the effect of increased arterial stiffness on circulating platelet activity in overweight/obese young adults. This analysis included 92 participants (mean age 40 years, 60 women) in the Slow Adverse Vascular Effects of excess weight (SAVE) trial, a clinical trial examining the effects of a lifestyle intervention with or without sodium restriction on vascular health in normotensive overweight/obese young adults. Carotid-femoral (cf), brachial-ankle (ba) and femoral-ankle (fa) pulse wave velocity (PWV) served as measures of arterial stiffness and were measured at baseline and 6, 12 and 24 months follow-up. Platelet activity was measured as plasma β-thromboglobulin (β-TG) at 24 months. Higher plasma β-TG was correlated with greater exposure to elevated cfPWV (p?=?0.02) and baPWV (p?=?0.04) during the preceding two years. After adjustment for serum leptin, greater exposure to elevated baPWV remained significant (p?=?0.03) and exposure to elevated cfPWV marginally significant (p?=?0.054) in predicting greater plasma β-TG. Greater arterial stiffness, particularly central arterial stiffness, predicts greater platelet activation in overweight/obese individuals. This relationship might partly explain the association between increased arterial stiffness and incident atherothrombotic events.     

Platelets are not hyperreactive in patients with ovarian cancer

Paraneoplastic thrombocytosis has been reported in different types of solid tumors, including ovarian epithelial cancer, and found to be associated with a worse outcome. Although the effect of cancer on increasing platelet counts is well documented, the effect of cancer on platelet functions is not well known. We compared in vitro aggregation response of platelets isolated from 34 patients with ovarian cancer to those of platelets from 19 patients with benign ovarian tumors. Aggregation studies were conducted in a light transmission aggregometer, using both a high and a low dose of ADP and collagen. We evaluated platelet preactivation by measuring the plasma concentration of β-thromboglobulin (β-TG) and platelet factor-4 (PF-4) as markers of platelet α granule secretion, using ELISA. We found that ovarian cancer is not associated with an enhanced aggregation response of platelets to ADP or collagen, and plasma concentration of β-TG and PF-4 is not higher in patients with ovarian cancer compared to those in patients with benign ovarian tumors.     

Soluble P-selectin assay: importance of correct anticoagulant choice

P-selectin is stored preformed in the-granules of platelets. Previous studies show-thromboglobulin, also stored in-granules can be readily released from platelets during the processing of whole blood. This artefactual release was rectified by using combination of various anti-platelet and anticoagulant compounds placed in the collecting tube. We investigated the levels of sP-selectin from 40 volunteers, comparing two anticoagulants, tri-sodium citrate, CTAD (a mixture of sodium citrate and citric acid, theophylline, adenosine and dipyridamole) plus iloprost and serum. Iloprost, a stable prostacyclin analogue, is a potent anti-platelet agent. We found significantly lower levels of sP-selectin ( P < 0.0001, paired t-test ) measured from blood collected into CTAD and iloprost compared to levels measured from either citrated plasma or serum. We suggest that plasma levels obtained from the blood collected into a CTAD tube containing iloprost are likely to more accurately reflect the true levels of circulating sP-selectin than those obtained when test-tube activation of platelets is allowed to continue in vitro .      

Significance of quantitative measurement of heparin‐induced platelet antibodies

Background: Heparin‐induced thrombocytopenia (HIT) is a prothrombotic immune‐mediated adverse drug reaction. Antigen and platelet activation assays are used for detection of antibodies. Quantitative results from platelet factor 4 (PF4)‐dependent immunoassays may lead to inter‐laboratory standardization of measurements. Objectives: The aim was to modify a PF4‐dependent immunoassay to measure PF4/heparin antibodies quantitatively. Methods: Over five consecutive years, 1070 samples from thrombocytopenic, heparin‐treated patients were analyzed by a PF4/heparin ELISA and the heparin‐induced platelet activation assay (HIPA). Results of ELISA assay were expressed as arbitrary units per liter (AU/L). Results: Precision of ELISA at the concentration of 50 AU/L was 3.6%. Of 1070 samples, 117 were positive for antibodies by ELISA and/or HIPA assay. The higher the antibody concentration was, the higher was the proportion of HIPA positive cases (>140 AU/L, 100%, n = 26; 100–140 AU/L, 55%, n = 20; 50–99 AU/L, 38%, n = 29; 30–49 AU/L, 17%, n = 36). Conclusions: The measurement of anti‐PF4/heparin antibody concentration is a new parameter that may improve the diagnosis of HIT. All samples with extremely strong antibody concentration were positive also by HIPA. For accuracy, antibody concentrations must be in the linear range of the assay and an international standard is needed.     

Acute Effects of Cigarette Smoking on Platelet Function and Plasma Catecholamines in Hypertensive and Normotensive Men

In this randomized controlled crossover study essential hypertensive men (n = 13) and matched normotensive controls (n = 18) were examined before and during cigarette and sham smoking to assess the acute effects of smoking on platelets and plasma catecholamines. Platelet activity in vivo was determined by measurements of the released α-granule constituent β-thromboglobulin (β-TG) in plasma and in urine. Urinary high molecular weight β-TG and venous plasma epinephrine increased significantly during smoking in the hypertensive group, but not among the normotensive men. Thus, cigarette smoking induces a mild platelet release reaction and also elicits a significantly higher epinephrine response in hypertensive men compared to normotensive controls.     

Altered plasma fibrin clot properties in essential thrombocythemia

Patients with increased thromboembolic risk tend to form denser fibrin clots which are relatively resistant to lysis. We sought to investigate whether essential thrombocythemia (ET) is associated with altered fibrin clot properties in plasma. Ex vivo plasma fibrin clot permeability coefficient (K_s), turbidimetry and clot lysis time (CLT) were measured in 43 consecutive patients with ET (platelet count from 245 to 991?×?10³/µL) and 50 control subjects matched for age, sex and comorbidities. Fibrinolysis proteins and inhibitors together with platelet activation markers were determined. Reduced K_s (?38%, p?p?p?=?0.01) and higher maximum absorbency of the turbidimetric curve (+6%, p?p?p?p?p?K_s inversely correlated with fibrinogen, PF4 and C-reactive protein. CLT positively correlated only with PAI-1. Patients with ET display prothrombotic plasma fibrin clot phenotype including impaired fibrinolysis, which represents a new prothrombotic mechanism in this disease.     

Elevated plasma levels of beta-thromboglobulin and platelet factor 4 in patients with rheumatic disorders and cutaneous vasculitis

The efficacy of plasma levels of β-thromboglubulin (β-TG) and platelet factor 4 (PF4) as markers of the presence and activity of vasculiditic processes in rheumatic diseases were evaluated, first by serial measurement of their levels in a patient with rheumatoid arthritis and a chronic leg ulcer in the course of treatment, and second in 11 patients with rheumatoid arthritis without cutaneous vasculitis, and in nine patients with a variety of rheumatic diseases with cutaneous vasculitis. In the former, plasma levels of β-TG and PF4 were elevated and slowly reduced in parallel with healing, raised again after relapse, and normalized after disappearance of the leg ulcer. In the latter, both plasma levels were elevated in all of the nine patients with cutaneous vascular lesions and in one of the 11 patients rheumatoid arthritis without skin lesions. Levels of β-TG and PF4 may be useful to estimate the presence of vascular lesions in rheumatic disorders. Received: 5 November 2001 / Accepted: 18 June 2002 Correspondence and offprint requests to: Dr Toshihide Yamamoto, Kishiwada Tokushukai Hospital, 4-22-38 Isonokami-cho, Kishiwada, Osaka 596-0001, Japan. Tel: 81-724-38-8781; Fax: 81-724-37-7395; E-mail: ikyoku@kishiwada.tokushukai.or.jp     

Incidence and clinical significance of anti-PF4/heparin antibodies of the IgG, IgM, and IgA class in 755 consecutive patient samples referred for diagnostic testing for heparin-induced thrombocytopenia

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is usually caused by anti-platelet factor 4 (PF4)/heparin antibodies, leading to intravascular platelet activation. These antibodies can be detected by PF4/polyanion antigen assays or platelet activation assays. While antigen assays are very sensitive and recognize immunoglobulin (Ig)G, IgA, and IgM antibodies, the role of IgM and IgA HIT-antibodies is debated. Platelet activation assays recognize IgG and are more specific for clinical HIT. METHODS: We analyzed sera from 755 consecutive patients referred for diagnostic testing for HIT using a PF4/heparin enzyme-linked immunosorbent assay (ELISA) for IgG, IgA, and IgM and by the heparin-induced platelet activation (HIPA) test. Clinical information was provided by the treating physicians. RESULTS: A total of 108 of 755 (14.3%) patients tested positive, 105 (13.9%) in the PF4/heparin IgG/A/M ELISA [28 (26.7%) only for IgM/A]; 53 (7.0%) sera were positive in the HIPA, of those 50 tested also positive in the ELISA. In 77 patients sufficient clinical information was provided. Available clinical information for 17 of the 28 patients who had only IgM and/or IgA detected showed plausible alternative (non-HIT) explanations in four of seven who had thromboembolic complications and in nine of 10 who had isolated HIT. CONCLUSION: Detection of IgG, IgM and IgA class antibodies by PF4/heparin ELISA yields a positive test result about twice as often as does a platelet activation assay, with only a minority of the additional patients detected likely having HIT. Thus, there is a potential for considerable over-diagnosis of HIT by laboratories that utilize only an ELISA for diagnostic testing.     

Aspirin responsive platelet thrombophilia in essential thrombocythemia and polycythemia vera

Essential thrombocythemia (ET) and polycythemia vera (PV) frequently present with erythromelalgia and acrocyanotic complications, migraine-like microvascular cerebral and ocular transient ischemic attacks (MIAs) and/or acute coronary disease. The spectrum of MIAs in ET range from poorly localized symptoms of transient unsteadiness, dysarthria and scintillating scotoma to focal symptoms of transient monocular blindness, transient mono- or hemiparesis or both. The attacks all have a sudden onset, occur sequentially rather than simultaneously, last for a few seconds to several minutes and are usually associated with a dull, pulsatile or migraine-like headache. Increased hematocrit and blood viscosity in PV patients aggravate the microvascular ischemic syndrome of thrombocythemia to major arterial and venous thrombotic complications. Phlebotomy to correct hematocrit to normal in PV significantly reduces major arterial and venous thrombotic complications, but fails to prevent the platelet-mediated erythromelalgia and MIAs. Complete long-term relief of the erythromelalgic microvascular disturbances, MIAs and major thrombosis in ET and PV patients can be obtained with low dose aspirin and platelet reduction to normal, but not with anticoagulation. Skin punch biopsies from the erythromelalgic area show fibromuscular intimal proliferation of arterioles complicated by occlusive platelet-rich thrombi leading to acrocyanotic ischemia. Symptomatic ET patients with erythromelalgic microvascular disturbances have shortened platelet survival, increased platelet activation markers β-thromboglobulin (β-TG), platelet factor 4 (PF4) and thrombomoduline (TM), increased urinary thromboxane B2 (TXB2) excretion, and no activation of the coagulation markers thrombin fragments F1+2 and fibrin degradation products. Inhibition of platelet cyclooxygenase (COX1) by aspirin is followed by the disappearance and no recurrence of microvascular disturbances, increase in platelet number, correction of the shortened platelet survival times to normal, and reduction of increased plasma levels of β-TG, PF4, TM and urinary TXB2 excretion to normal. These results indicate that platelet-mediated fibromuscular intimal proliferation and platelet-rich thrombi in the peripheral, cerebral and coronary end-arterial microvasculature are responsible for the erythromelalgic ischemic complications, MIAs and splanchnic vein thrombosis. Baseline platelet P-selectin levels and arachidonic acid induced COX1 mediated platelet activation showed a highly significant increase of platelet P-selectin expression (not seen in ADP and collagen stimulated platelets), which was significantly higher in JAK2^V617F mutated compared to JAK2 wild type ET.     

Adsorption of Anaphylatoxins and Platelet-Specific Proteins by Filtration of Platelet Concentrates with a Polyester Leukocyte Reduction Filter

Anaphylatoxins generated during storage of platelet concentrates (PCs) may potentially have side effects on platelet transfusion. We evaluated the anaphylatoxin-scavenging abilities of white blood cell reduction filters. Among the commercially available filters for PCs, one made with polyester fiber (PL50) dramatically adsorbed C3a and C4a anaphylatoxins to the respective mean level of 1,721–208 ng/ml and 1,240–141 ng/ml in 3-day-old PCs. C3a and C4a were measured as the native and des Arg form of each complement by radioimmunoassay. C3a and C4a anaphylatoxins in the supernatant plasma fraction from 3-day-old PC again decreased from 1,136 to 114 ng/ml and from 1,086 to 65 ng/ml, respectively. The filter also adsorbed 85% of platelet factor 4 (PF4) and 31% of β-thromboglobulin (β-TG), which had been released from platelets into the plasma during storage. The plasma levels of adhesive proteins such as fibronectin, fibrinogen, and von Willebrand factor, and plasma lactate dehydrogenase activity did not decrease after filtration. Another polyester filter (PL5A), on the other hand, significantly increased C3a and C4a levels with filtration. In addition, there was no PF4 adsorption ability during the filtration. The filters for red cells (RC50, BPF4, and R500A) had no anaphylatoxin adsorption capabilities. The observed specific adsorption of anaphylatoxins might be attributed to the electrostatic force between the positively charged anaphylatoxins with high p1 and the possibly negatively charged filter membranes. Since PF4 and β-TG have positively charged moieties in the C-terminal position, the same adsorption mechanism might operate. We have obtained a useful scavenging filter for the evaluation of side effects of anaphylatoxins on patients who received old PCs.     

VEGF, PF4 and PDGF are elevated in platelets of colorectal cancer patients

Platelets sequester angiogenesis regulatory proteins which suggests an avenue for developing biomarkers to monitor disease. We describe a comparison of angiogenesis regulatory proteins found in platelets of colorectal cancer patients and normal controls. Platelet and plasma content of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF), platelet factor 4 (PF4), thrombospondin-1 (TSP-1) and endostatin in 35 patients with colon cancer were compared with 84 age-matched healthy controls using ELISAs. We standardized the platelet preparation procedure, introduced process controls and normalized the respective protein levels to platelet numbers using an actin ELISA. Statistically significant differences were found in the median levels of VEGF, PF4 and PDGF in platelets of patients with cancer compared to healthy individuals. Platelet concentrations in cancer patients versus controls were: VEGF 1.3 versus 0.6 pg/10⁶, PF4 18.5 versus 9.4 ng/10⁶, and PDGF 34.1 versus 21.0 pg/10⁶. Multivariable logistic regression analysis indicated that PDGF, PF4 and VEGF were independent predictors of colorectal carcinoma and as a set provided statistically significant discrimination (area under the curve = 0.893, P < .0001). No significant differences were detected for bFGF, endostatin, or TSP-1. Reference Change Value analysis determined that the differences seen were not clinically significant. Plasma levels yielded no correlations.     

Radioimmunoassay of Platelet Factor 4 and β-Thromboglobulin: Development and Application to Studies of Platelet Release in Relation to Fibrinopeptide A Generation

Platelet and fibrinogen survival and turnover studies have shown that platelet activation and fibrin formation may occur to different degrees in different thrombotic disorders. More direct evidence of differential involvement of platelet activation and fibrin formation should be provided by specifically measuring the products of these reactions, i.e. released platelet proteins and fibrinopeptide A. Two platelet proteins, platelet factor 4 (PF4) and beta-thromboglobulin (betaTG), were isolated and characterized, and sensitive and specific radioimmunoassays were developed to measure them. These assays were employed, along with the radioimmunoassay for fibrinopeptide A (FPA), to study the release of PF4 and betaTG in relation to FPA cleavage. PF4 and betaTG were released by ADP and collagen with time course and concentration dependence similar to that of [14C]serotonin release. FPA was not cleaved from fibrinogen during ADP or collagen-induced platelet release. Thrombin caused release of PF4 and betaTG as well as cleavage of FPA. Cleavage of FPA occurred with concentrations of thrombin about 100 times less than did release of PF4 and betaTG, and release of [14C]serotinin required still higher thrombin concentrations. Release of [14C]serotonin and platelet proteins was similar as a function of time. Sodium citrate was found to inhibit platelet release induced by thrombin.     

Heparin-releasable platelet factor 4 in patients with coronary artery disease.

Recently, platelet factor 4 (PF4) release by heparin (heparin-releasable PF4) has been examined as a useful marker of the interaction between the substances liberated from circulating platelets and the vascular endothelium. We compared the plasma levels of PF4 and beta-thromboglobulin (beta-TG) after intravenous heparin injection in patients with coronary artery disease (CAD) and normal control subjects. We also studied the effects of low-dose aspirin (81 mg/day) on the plasma level of heparin-releasable PF4 in the CAD patients. Blood samples were obtained before and 5 min after the intravenous injection of heparin (1,000 IU) from 23 patients with CAD and 15 normal control subjects. Although the plasma beta-TG level remained unchanged after heparin injection, the plasma PF4 level markedly increased in both groups. There was a significant difference in plasma PF4 levels at 5 min after heparin injection between the CAD group (100.1 +/- 38.1) and the control group (61.0 +/- 24.0) (p less than 0.01). The PF4/beta-TG ratio after heparin injection was also higher in the CAD group than in the control group (p less than 0.01). There was a correlation between the PF4/beta-TG ratio after heparin and the Gensini CAD score, which defines the severity of coronary atherosclerosis (r = 0.489, n = 23, p less than 0.01). Low-dose aspirin was administered to 11 CAD patients for 246.0 +/- 28.8 days. Blood samples for the assay of PF4 and beta-TG were obtained as stated above, and platelet aggregation, thromboxane B2 (TxB2), and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) levels were also measured before and during aspirin administration.(ABSTRACT TRUNCATED AT 250 WORDS)