Plasma concentrations of platelet-specific proteins in young female acute myocardial infarction survivors and their age-matched female controls

The steady state plasma concentrations of the two platelet-specificproteins beta-thromboglobulin (BTG) and platelet factor 4 (PF4)were determined in two groups of females: 36 acute myocardialinfarction (AMI) survivors, and 38 age-matched control subjects.For the AMI patients the mean BTG and PF4 values were 57 ±4 and 14.1 ± 1.7 ng/ml, respectively. These two meanssignificantly exceeded the corresponding means for the controls,40 ± 2 and 10.3 ± 0.6 ng/ml, respectively. Similarresults have recently been reported by other workers who investigatedpatients with coronary artery disease; however, in no previousstudy were the values for BTG and PF4 related to those obtainedfor control subjects matched with respect to sex and age. Thepresent results therefore further support the concept that increasedplatelet activation and secretion is taking place in steadystate patients who previously have sustained an AMI.     

Human platelet factor 4: Preparation from outdated platelet concentrates and application in cerebral vascular disease

Platelet factor 4 (PF4), the platelet antiheparin protein, was isolated from both the supernatant and the cells of recently outdated platelet concentrates. Following purification by affinity chromatography, a competitive binding radioimmunoassay was developed to detect this protein in human plasma. The normal range was determined to be 9.4 ± 4.7 ng/ml (mean ± SD for 52 healthy adults). In order to determine whether individuals with transient ischemic attack (TIA) or stroke had measurable increments of PF4 in their plasma, radioimmunoassay studies were performed on 11 patients with well-documented TIA, 10 patients with well-documented stroke and on 16 age-matched controls hospitalized on a neurology service with disorders unrelated to arterial thrombosis. The 16 hospitalized controls had PF4 levels of 10.3 ± 9.1 ng/ml, a value not significantly different from the 52 normals (P > 0.50). Patients with TIA had PF4 levels of 24.6 ± 12.1 ng/ml, a value significantly higher than both the 52 normals (P < 0.001) and the 16 hospitalized control patients (P < 0.005). Patients with stroke had PF4 levels of 35.4 ± 29.2 ng/ml, a value significantly higher than both the 52 normals (P < 0.001) and the 16 hospitalized control patients (P < 0.005). Outdated platelet concentrates facilitate the development of a reproducible radioimmunoassay for PF4. The elevation of this platelet-derived protein in the plasma of patients with stroke and TIA provides evidence for recent or ongoing platelet activation in the cerebral vascular disease population.     

Patients with paroxysmal atrial fibrillation but not paroxysmal supraventricular tachycardia display evidence of platelet activation during arrhythmia

It is well known that chronic atrial fibrillation (CAF) and paroxysmal atrial fibrillation (PAF) are associated with a hypercoagulable state. However, pathological hemostatic changes during the paroxysmal supraventricular tachycardia (PSVT) have not yet been elucidated. To determine platelet activity in patients with PSVT, PAF and CAF, we examined the levels of β-thromboglobulin (BTG) and platelet factor 4 (PF4) during tachyarrhythmia attacks. We measured the levels of BTG and PF4, as an index of platelet activation in 15 patients with PAF (9 men, mean age 45 ± 11), and 14 patients with PSVT (8 men, mean age 40 ± 10). Levels were compared to 22 age and sex-matched healthy controls in sinus rhythm and with 25 patients with CAF (16 men, mean age 51 ± 12). Blood samples were taken during arrhythmia and 24 hours after conversion to sinus rhythm. Patients taking medications or have clinical conditions that may affect the BTG and PF4 levels were excluded.

In patients with PAF, BTG and PF4 levels were significantly higher than in controls (?p<0.009, and p = 0.002, respectively), and in patients with PSVT (?p<0.04, and p = 0.009, respectively), however, BTG and PF4 levels were significantly lower than CAF patients (?p = 0.002, and p = 0.02, respectively). Moreover, BTG and PF4 levels were significantly decreased 24 hours after conversion to sinus rhythm (?p<0.0001, and p = 0.004, respectively). Although BTG and PF4 levels in patients with PSVT were significantly lower than in patients with PAF (?p = 0.04, and p = 0.009, respectively) and CAF (?p = 0.0001, and p = 0.0001, respectively), BTG and PF4 levels were similar to controls and did not change significantly after recovery to sinus rhythm (?p = NS for all).

These results indicate that there was no platelet activation in patients with PSVT during tachyarrhythmia but significantly increased platelet activity in PAF and CAF patients. There was a significant decrement of the platelet activity to a level of control subjects twenty-four hours after cardioversion of PAF.     

Diagnostic Value of Fibrinopeptide A and Beta-Thromboglobulin in Acute Deep Venous Thrombosis and Pulmonary Embolism

ABSTRACT. The purpose of this study was to assess the predictive values of the assays of fibrinopeptide A (FPA), β-thromboglobulin (BTG) and their combination in patients suspected of having acute deep venous thrombosis (DVT) or pulmonary embolism (PE). In 80 controls the mean (± SD) plasma concentrations of FPA and BTG were 0.72±0.47 and 28.2±10.1 ng/ml, respectively. In 26 patients, in whom DVT was confirmed by phlebography and Doppler ultrasound, clearly raised mean FPA (5.62 ng/ml) and BTG (70.6 ng/ml) concentrations were measured compared to those in 13 patients in whom this disorder was excluded (1.00 and 33.6 ng/ml, respectively). Also in 25 patients, in whom PE was established by perfusion lung scanning, clearly increased mean FPA (6.28 ng/ml) and BTG (82.4 ng/ml) concentrations were measured compared to those in 12 patients without this disease (1.03 and 32.5 ng/ml, respectively). Raised FPA and BTG concentrations were also found in 20 patients with inflammatory disorders and in 10 with various types of malignancy. The mean FPA and BTG concentrations did not differ between patients with renal failure or diabetes mellitus and patients without these diseases. From the predictive values of these assays and their combination it can be concluded that raised FPA and BTG concentrations are not specific for thrombosis. However, when normal FPA and BTG concentrations are present, acute DVT or PE can safely be excluded in symptomatic patients. In the group with confirmed DVT/PE, anticoagulant treatment (heparin and phenprocoumon) brought down the mean FPA concentration to levels within the normal range in less than 1 hour while the mean BTG concentration remained elevated throughout the 10-day study period.     

Beta thromboglobulin and platelet factor 4 in bronchoalveolar lavage fluid of patients with systemic sclerosis

OBJECTIVE: To evaluate concentrations of the platelet activation markers beta thromboglobulin (BTG) and platelet factor 4 (PF4) in bronchoalveolar lavage fluid (BALF) from patients with systemic sclerosis with and without scleroderma interstitial lung disease (SLD). METHODS: BTG and PF-4 were measured by enzyme immunoassay in BALF from 37 patients with systemic sclerosis. Controls were 10 healthy subjects. BALF was collected during routine bronchoscopy from the right middle lobe. SLD was diagnosed by high resolution computed tomography of the lungs. RESULTS: BTG was detected in 11 of the patients with systemic sclerosis (29.7%) and PF4 was found in eight (21.6%). Mean (SD) concentrations of BTG and PF4 in BALF from patients with detectable levels of these platelet activation markers were 106.9 (69.8) and 35.2 (17.4) IU/ml, respectively. The BTG:PF4 ratio was more than 2:1, indicating in vivo release. Both markers were found exclusively in patients with SLD. SLD patients with detectable platelet activation markers had a significantly shorter disease duration than those with undetectable BTG/PF4. CONCLUSIONS: The study provides evidence that activation of blood platelets takes place within the lungs of patients with SLD and may contribute to the development of lung fibrosis.     

Plasma concentrations of platelet-specific proteins and serum thromboxane B2 production in response to treatment with dipyridamole. A pilot study of 27 post-myocardial infarction patients

In the present study 27 post-myocardial infarction patients were treated with Persantin capsules (Depot-Kapseln, sustained release form), containing 200 mg dipyridamole, b.i.d. over a period of 3 weeks. Baseline levels for plasma beta-thromboglobulin (BTG), platelet factor 4 (PF4), and serum thromboxane B2 (TXB2) were obtained on day 0 and subsequently on days 1, 3, 5, 7, 14 and 21. The baseline levels for plasma BTG and PF4 as well as for serum TXB2 significantly exceeded those for a control group consisting of healthy subjects. The plasma values for BTG and PF4 remained unchanged during the whole study period. During the first week of study the levels for serum X TXB2 were unchanged; however, on days 14 and 21 the means for TXB2 dropped significantly. There is experimental work to suggest that dipyridamole may exert an inhibitory effect on platelet thromboxane biosynthesis. The present results support this concept.     

Determinants of rebound thrombin activity after cessation of heparin in patients undergoing coronary interventions

This study was designed to characterize hemostatic activation (using fibrinopeptide A (FPA), a marker of thrombin activity, and β-thromboglobulin (BTG), a marker of platelet activation) sequentially in the coronary and peripheral circulation in patients during percutaneous coronary intervention (PCI) and several hours after PCI and discontinuation of heparin therapy. Heparin administered during PCI is known to nonuniformly suppress thrombin activity in the coronary. Persistent elevations of FPA in coronary sinus (CS) blood during PCI have been associated with subsequent ischemic events. As a related consideration, rebound thrombin activity has been demonstrated in peripheral blood samples several hours after cessation of heparin therapy in patients with acute coronary syndromes. Accordingly, we hypothesized that increased thrombin activity occurs in the coronary circulation after PCI and is induced by cessation of intravenous heparin to facilitate vascular sheath removal. Such a rebound prothrombotic effect, may contribute to suboptimal outcomes after PCI. In 21 patients undergoing PCI, heparin-bonded catheters were employed to obtain sequential CS and femoral vein (FV) blood samples for measurement in the coronary and peripheral circulation of plasma FPA, a marker of thrombin activity in vivo, and BTG released by platelets during degranulation. Following heparin administration samples were obtained immediately prior to (base) and during (start and end) PCI. Late samples were obtained several hours after PCI (284 ± 46 min, mean ± SD) following the cessation of heparin and prior to planned vascular sheath removal. Mean FPA concentration in CS blood was low at baseline (3.82 ± 2.09 ng/ml) and did not increase during PCI. Mean FPA concentration in CS blood increased significantly several hours after cessation of heparin (3.42 ± 2.36 vs. 7.82 ± 9.98, end vs. late, P < 0.001). In contrast, mean FPA concentration in FV blood was highest at baseline following vascular sheath insertion, decreased during PCI (69%, P < 0.05, base vs. end), and trended upward after PCI and cessation of heparin. Mean FPA values were higher at all times in FV compared with CS blood samples and were not concordant after PCI. Elevation of coronary circulation FPA after PCI was maximal in patients with myocardial infarction within 7 days (13.7 ± 12.4 vs. 5.6 ± 7.9 ng/ml, P = 0.08), but was not influenced by heparin treatment prior to PCI, a history of unstable angina, or coronary stent placement during PCI (9 of 21 patients). BTG values showed less variation than did FPA values, and cessation of heparin after PCI was not associated with an increase in BTG in CS or FV blood samples. An increase in thrombin activity occurs in the coronary circulation after PCI following discontinuation of heparin. The extent of increased thrombin activity was greatest in patients with recent myocardial infarction and was not exacerbated by coronary stent placement during PCI. This phenomenon may contribute to the important minority of ischemic complications early after PCI. Cathet. Cardiovasc. Diagn. 44:257–264, 1998. © 1998 Wiley-Liss, Inc.     

Effects of the platelet inhibitor ticlopidine on exercise tolerance in stable angina pectoris

Coronary blood flow might be reduced by platelet aggregatesor by vasospasm induced by platelet-produced thromboxane A2.Therefore the effects of the platelet inhibitor ticlopidine(500 mg daily) on platelet function and on exercise tolerancewere investigated in a double-blind placebo-controlled studyin 38 middle-aged men with stable incapacitating angina pectoris.Before and after 4 and 8 weeks of treatment, exercise testswere performed in warm and cold environments. The in vitro plateletreactivity to ADP was determined at rest and the plasma levelsof beta-thromboglobulin (BTG) and platelet factor 4 (PF4) weremeasured before and immediately after exercise. There were nosigns of increased platelet activity at rest or after exerciseas judged by the levels of BTG and PF4. Despite a potent inhibitionof platelet reactivity to ADP in vitro during ticlopidine treatment,the exercise tolerance was reduced in exercise tests in bothwarm and cold environments and in daily life. Therefore plateletactivity does not seem to play any significant role in exercisetolerance in the stable phase of angina pectoris.     

Plasma beta-thromboglobulin as a measure of platelet activity: Effect of risk factors and findings in ischemic heart disease and after acute myocardial infarction

The plasma concentration of beta-thromboglobulin (BTG), a platelet-specific protein released during platelet aggregation, is considered a sensitive marker of in vivo platelet activity. The mean plasma level in 133 asymptomatic individuals was 32.3 ± 1.1 ng/ml, and there was no difference between those with no risk factors (32.2 ± 1.2 ng/ml, n = 56), those who smoked (31.8 ± 1.8 ng/ml, n = 45), those with hyperlipidemia (32.8 ± 1.7 ng/ml, n = 15), and those exposed to both of these risk factors (34.1 ± 2.7 ng/ml, n = 17). The mean plasma BTG level in 104 patients with symptomatic ischemic heart disease was significantly elevated (40.9 ± 1.4 ng/ml, p < 0.01), but there was considerable overlap with normal levels. Although no difference was found between patients with no risk factors (38.1 ± 4.0 ng/ml, n = 13) and those with only 1 risk factor (37.0 ± 1.8 ng/ml, n = 44), patients with 2 or more risk factors had a significantly elevated plasma BTG level (45.2 ± 2.2 ng/ml, n = 47, p < 0.01). It is concluded that risk factors themselves do not increase platelet activity, but that patients with vascular disease have activated platelets that may contribute to the progression of the disease. Plasma BTG was also measured serially for 10 days in 29 patients after hospitalization with acute ischemic cardiac pain. Although the median plasma level was elevated above normal there were no acute changes in plasma BTG after either acute infarction (n = 22) or acute ischemia (n = 7), except in 2 patients in whom pericardial friction rubs developed. Thus, measurement of systemic plasma BTG did not detect platelet involvement in acute coronary occlusion or acute ischemia.     

Comparison of Blood Compatibility in Various Membrane Materials for Continuous Renal Replacement Therapy

Because hemofilters used for continuous renal replacement therapy contact with blood over a prolonged period during treatments, platelet activation may occur stronger. The purpose of this study is to clarify the blood compatibility in three hemofilters mostly used in Japan. We compared the blood compatibility of the two polysulfone (AEF: Asahi Kasei Medical Co., Tokyo, Japan and SHG: Toray Medical Co., Ltd., Tokyo, Japan) and one polymethylmethacrylate membranes (CH: Toray Medical Co., Ltd.). First, test blood was collected from healthy volunteers. Subsequently, the blood was circulated by a roller pump at the rate of 100 mL/min. We measured the platelet counts and platelet factor 4 (PF4). The platelet counts at 48 h in polymethylmethacrylate membrane were significantly less than that in polysulfone membranes. Levels of the PF4 after the circulation were 978.5 ± 200.0 ng/dL with AEF, 863.0 ± 233.9 ng/dL with SHG and 1780.0 ± 465.1 ng/dL with CH, respectively. Hemofilters with polysulfone membranes showed less platelet activation. It was inferred that the amount of PVP, the smoothness of the membrane surface, and the inner diameter of the hollow fiber affect the blood compatibility in the hemofilter.     

Patients with paroxysmal atrial fibrillation but not paroxysmal supraventricular tachycardia display evidence of platelet activation during arrhythmia

It is well known that chronic atrial fibrillation (CAF) and paroxysmal atrial fibrillation (PAF) are associated with hypercoagulable state. However, pathological hemostatic changes during the paroxysmal supraventricular tachycardia (PSVT) have not yet been elucidated. To determine platelet activity in patients with PSVT, PAF and CAF, we examined the levels of beta-thromboglobulin (BTG) and platelet factor 4 (PF4) during tachyarrhythmia attacks. We measured the levels of BTG and PF4, as an index of platelet activation in 15 patients with PAF (9 men, mean age 45+/-11), and 14 patients with PSVT (8 men, mean age 40+/-10). Levels were compared to 22 age and sex matched healthy controls in sinus rhythm and with 25 patients with CAF (16 men, mean age 51+/-12). Blood samples were taken during arrhythmia and 24 hours after conversion to sinus rhythm. Patients taking medications or have clinical conditions that may affect the BTG and PF4 levels were excluded. In patients with PAF, BTG and PF4 levels were significantly higher than in controls (p<0.009, and p=0.002, respectively), and in patients with PSVT (p<0.004, and p=0.009, respectively), however, BTG and PF4 levels were significantly lower than CAF patients (p=0.002, and p=0.02, respectively). Moreover, BTG and PF4 levels were significantly decreased 24 hours after conversion to sinus rhythm (p<0.0001), and p=0.004, respectively). Although BTG and PF4 levels in patients with PSVT were significantly lower than in patients with PAF (p=0.04, and p=0.009, respectively) and CAF (p=0.0001, and p=0.0001, respectively), BTG and PF4 levels were similar to controls and did not change significantly after recovery to sinus rhythm (p=NS for all). These results indicate that there was no platelet activation in patients with PSVT during tachyarrhythmia but significantly increased platelet activity in PAF and CAF patients. There was a significant decrement of the platelet activity to a level of control subjects twenty-four hours after cardioversion of PAF.     

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.     

Platelet Activity and Salt Sensitivity in the Pathogenesis of Systemic (Essential) hypertension in Black Africans

Black essential hypertensive patients with a mean arterial pressure of 125±3 mm Hg (mean ± SEM), and age and sex matched normotensive subjects with a mean arterial pressure of 89±2 mm Hg were studied under baseline conditions, after five days of salt restriction and after five days of salt loading. Salt sensitivity was defined as an increase of mean blood pressure exceeding 5% when progressing from low to high sodium intake. In vitro platelet responsiveness was assessed by aggregometry, and in vitro platelet activity by estimation of β-thromboglobulin (BTG) in plasma and thromboxane B₂ (TXB₂) excretion rate. Salt sensitivity was present in 66% of hypertensive and 55% of the normotensive subjects. An increased platelet aggregability to ADP (25%), to epinephrine (34%) and to collagen (12%) was found in parallel with an increased in vivo platelet activity (BTG increased by 55% and TXB₂ by 18%) in the hypertensives. All changes were significantly exaggerated in the salt sensitive as compared to salt resistant hypertensive patients.     

Workshop: Developing an Agenda for Anticoagulation Management Research

Background. Platelet activation after myocardial infarction and thrombolytic treatment has been documented; but its relationship with the onset of symptoms and with thrombolysis, and the influence of aspirin in this setting is not well defined. In this study we measured platelet activity in the early phase of myocardial infarction treated with either streptokinase or rt-PA and evaluated influence of aspirin in this framework. Methods. 41 patients (age 57 ± 6 years) were treated with thrombolytic therapy during myocardial infarction; 21 patients with 1,5 million units of streptokinase (Group 1) and 20 patients with 100 mg of rt-PA (Group 2); 10 randomly selected patients in each group were given 500 mg aspirin i.v. prior to infusion of thrombolytic drug and, subsequently, 325 mg aspirin a day orally. Consecutive samples of beta-thromboglobulin (BTG), a marker of platelet activity, were collected at admission and after thrombolysis for the following 48 hours. Results. At admission, BTG plasma levels averaged 125 ± 31 IU/ml in Group 1 and 134 ± 35 IU/ml in Group 2 (p = 0.81). Thrombolysis was followed by a similar increase of platelet activity with maximal values reached at the 3rd hour in both groups (196 ± 43 IU/ml in Group 1 and 192 ± 39 in Group 2: p < 001 versus baseline and p NS between the groups). Higher levels of BTG were observed in streptokinase-treated group starting from the 24th hour (p < 0.05). Patients treated with aspirin showed lower levels of BTG only from the 48th hour after thrombolysis in both groups. An inverse correlation was found between time elapsed from onset of symptoms to admission and BTG value on admission (r = −0.86 p < 0.001); in patients admitted within two hours from the beginning of symptoms, with higher levels of BTG, thrombolysis not induced a significant increase of platelet activity; who was observed in patients admitted later. Conclusions. A marked platelet activation is more evident in the first hours of myocardial infarction and is differently influenced by thrombolytic treatment in relation with the delay of patient presentation. Both streptokinase and rt-PA induce a similar increase of platelet activity which is more persistent after streptokinase; cyclooxygenase inhibition seems to influence the platelet activity only from the second day. Condensed abstract. Influence of aspirin on platelet activity during myocardial infarction treated with thrombolytic therapy is not well defined. Twenty-one patients treated with streptokinase (Group 1) and 20 patients treated with rt-PA (Group 2) were randomly selected to give 500 mg of aspirin i.v. prior thrombolysis and subsequently 325 mg a day orally. Platelet activity was evaluated through determination of beta-thromboglobulin plasma levels. Thrombolysis was followed by a similar increase of platelet activity in both groups with maximal values reached at the 3rd hour; higher levels of beta-thromboglobulin were observed in streptokinase-treated group starting from 24th hour. Treatment with aspirin reduced beta-thromboglobulin plasma levels only at 48th hour in both groups. This revised version was published online in August 2006 with corrections to the Cover Date.     

Decreased platelet activation and endothelial dysfunction after percutaneous mitral balloon valvuloplasty

OBJECTIVE: This study was conducted to assess the changes in platelet activation and endothelial dysfunction in patients with mitral stenosis (MS) and sinus rhythm (SR) following percutaneous mitral balloon valvuloplasty (PMBV). BACKGROUND: Systemic thromboembolism is a serious complication in patients with valvular heart disease, and its incidence is highest in those with mitral stenosis. A hypercoagulable state has also been reported in patients with mitral stenosis and sinus rhythm. A recent study has shown that patients with previous PMBV had a lower incidence of thromboembolism. METHODS AND RESULTS: The study was conducted in 21 patients (two men, 19 women, mean age=34+/-6 years) with mitral stenosis and sinus rhythm (SR) who underwent percutaneous mitral balloon valvuloplasty and 17 healthy control subjects (two men, 15 women, mean age=33+/-6 years). Biochemical markers of platelet activity (beta thromboglobulin, BTG, and soluble P-selectin, sPsel) and endothelial dysfunction (von Willebrand Factor, vWF) were measured in both control subjects' and patients' serum samples taken immediately before PMBV and 24 h after PMBV procedure. All patients underwent successful PMBV. Significant improvement of mitral valve area, pulmonary artery pressure, mean mitral gradients, and left atrial diameter were achieved in all patients after PMBV. Compared with control subjects, patients with MS had higher plasma levels of BTG (66+/-26 ng/ml vs. 14+/-6 ng/ml, P<0.001), vWF (177+/-67 units/dl vs. 99+/-37 units/dl, P<0.0001), sPsel (226+/-74 ng/ml vs. 155+/-66 ng/ml, P<0.001). There was a significant reduction of plasma levels of BTG (66+/-26 ng/ml vs. 48+/-20 ng/ml, P=0.002), vWF (177+/-67 units/dl vs. 134+/-60 units/dl, P=0.001) and P-selectin (226+/-74 ng/ml vs. 173+/-71 ng/ml, P=0.008,) 24 h after PMBV. CONCLUSION: We have shown that patients with severe MS and SR have increased platelet activation and endothelial dysfunction compared with control subjects and PMBV results in decreased platelet activity and improvement of endothelial injury.     

Measurement of soluble C-type lectin-like receptor 2 in human plasma

Detection of platelet activation in vivo is useful to identify patients at risk of thrombotic diseases. Platelet factor 4 (PF4) and β-thromboglobulin (β-TG) are used for this purpose; however, they are easily released upon the minimal platelet activation that occurs during sampling. Soluble forms of several platelet membrane proteins are released upon platelet activation; however, the soluble form of C-type lectin-like receptor 2 (sCLEC-2) has not yet been fully investigated. Western blotting with an anti-CLEC-2 antibody showed that sCLEC-2 was released from washed human platelets stimulated with collagen mimetics. To detect sCLEC-2 in plasma, we established a sandwich enzyme-linked immunosorbent assay (ELISA) using F(ab′)2 anti-CLEC-2 monoclonal antibodies. Although plasma mixed with citrate, adenosine, theophylline and adenosine (CTAD) is needed for the PF4 and β-TG assays, effects of anti-coagulants (EDTA, citrate and CTAD) on the sCLEC-2 ELISA were negligible. Moreover, while special techniques are required for blood sampling and sample preparation for PF4 and β-TG assay, the standard blood collections procedures used in daily clinical laboratory tests have shown to suffice for sCLEC-2 analysis. In this study, we found that two forms of sCLEC-2 are released after platelet activation: a shed fragment and a microparticle-bound full-length protein, both of which are detected by the sCLEC-2 ELISA. The average concentration of sCLEC-2 in the plasma of 10 healthy individuals was 97?±?55?pg/ml, whereas that in the plasma of 25 patients with diabetes mellitus (DM) was 149?±?260?pg/ml. A trend towards an increase in sCLEC-2 concentration in the DM patients may reflect in vivo platelet activation in the patients, suggesting that sCLEC-2 may have clinical significance as a biomarker of in vivo platelet activation.     

Platelet activation after adrenergic stimulation in hypertensive patients: effects of acebutolol

In 12 patients with arterial hypertension (stages I and II accordingto WHO), adrenergic stimulation was induced by the immersionof a hand in ice water for 2 min. Blood samples were withdrawnbefore, at the end of, and 15 min after the cold application:the experiment was repeated 2 h after the ingestion of 200 mgacebutolol, a selective betablocking agent. The following assayswere performed: serum nonesterified fatty acid (NEFA), plasmabeta-thromboglobulin (BTG) and PF4 with specific radioimmunoassays;thromboxane B₂ (TXB₂) in plasma was also estimated with radioimmunoassay,platelet sensitivity to exogenous prostacyclin; furthermore,the thrombin-induced thromboxane production before and afteracebutolol ingestion as well as serum TXB₂-levels were measured.The blood pressure and the heart rate were also monitored. After cold stimulation, a significant increase of NEFA, BTG,and plasma TXB₂ was observed, which was still discernible 15min after the application of cold. After acebutolol, the cold treatment led to a lower increaseof blood pressure with a reduction of the heart rate, as wellas to a diminished release of BTG, PF4 and TXB₂ no changes inthe reduced platelet sensitivity to prostacyclin were noticed.     

Fibrin formation and platelet aggregation in patients with acute myocardial infarction: effects of intravenous and subcutaneous low-dose heparin

Fibrinopeptide A (FPA) and beta thromboglobulin (BTG) were measured in 42 patients with acute myocardial infarction (AMI) allocated on admission to one of three groups: 14 patients received a heparin bolus injection of 5000 IU intravenously followed by a 2-hour intravenous infusion (830 IU/hr) (group 1), 14 patients received a heparin bolus of 5000 IU subcutaneously (group 2), and the remaining 14 patients received no anticoagulant treatment (group 3). In group 1 the initially elevated FPA level of 5.8 +/- 1.8 ng/ml dropped to 2.0 +/- 1.5 ng/ml 30 minutes after the intravenous heparin bolus injection of 5000 IU (p less than 0.001) and returned to normal (1.9 +/- 0.8 ng/ml) in 8 of 14 patients. The initially elevated BTG level of 64 +/- 21 ng/ml did not change significantly during intravenous heparin treatment, whereas there was a rapid but only transitory increase in platelet factor 4, (PF4) from 25 +/- 9 to 74 +/- 16 ng/ml (p less than 0.01) after the intravenous heparin bolus. In group 2 the initial FPA of 5.0 +/- 2.3 ng/ml was similarly elevated as in group 1 and dropped to 2.7 +/- 1.7 and 3.3 +/- 1.5 ng/ml 2 and 4 hours after 5000 IU subcutaneously (p less than 0.05), whereas 6 and 8 hours after subcutaneous heparin bolus the mean FPA levels were 4.2 +/- 1.7 and 5.5 +/- 2.0 ng/ml and no more significantly different from the initial FPA values. BTG and PF4 did not change significantly after the subcutaneous heparin bolus. In group 3 the initially elevated mean FPA level of 4.9 +/- 2.4 ng/ml did not change significantly during the first 8 hours after admission, whereas the FPA level 24 hours after admission was 8.4 +/- 3.9 ng/ml and higher than the initial value (p less than 0.01). We conclude that heparin may reduce the elevated FPA level in plasma found in patients with AMI; however, neither subcutaneous nor intravenous heparin in a dosage frequently used is sufficient to consistently normalize the elevated rate of fibrin formation found in these patients.     

Normal ranges of angiogenesis regulatory proteins in human platelets

Platelets sequester angiogenesis regulatory proteins early in tumor growth, which suggests a new avenue for monitoring disease. To date, there are no clinically relevant reference ranges for markers of early angiogenesis. We introduce a new ELISA‐based method for accurate and reproducible measurement of vascular endothelial growth factor (VEGF), platelet‐derived growth factor (PDGF), platelet factor 4 (PF4), thrombospondin‐1 (TSP‐1), fibroblast growth factor, basic (bFGF), and endostatin in platelets. To facilitate clinical applicability, the platelet levels in isolated samples were determined utilizing a new actin ELISA method. Platelets from healthy donors at single and repetitive time points were used for the assessment of normal ranges of these proteins. The physiological levels in platelets were: VEGF (0.74 ± 0.37 pg/10⁶ platelets); PDGF (23 ± 6 pg/10⁶); PF4 (12 ± 5 ng/10⁶); TSP‐1 (31 ± 12 ng/10⁶); bFGF (0.44 ± 0.15 pg/10⁶); and endostatin (5.6 ± 3.0 pg/10⁶). There was an excellent correlation (R² = 0.7) between the platelet levels calculated with the actin ELISA and complete blood count. The levels of the platelets were higher than those in platelet‐poor plasma by factors of: VEGF (215‐fold); PDGF (914‐fold); PF‐4 (516‐fold); TSP‐1 (813‐fold); and bFGF (17‐fold). The endostatin levels were nearly equivalent. The biovariability of the platelet proteins in eight healthy subjects over a 5‐week period was found to be minimal. We describe accurate and direct measurements of the concentrations of VEGF, bFGF, PDGF, TSP‐1, endostatin, and PF4 in platelets of healthy human subjects. In contrast to the highly variable levels in plasma and serum, the platelet‐derived measurements were accurate and reproducible with minimal biovariability. Am. J. Hematol., 2010. © 2010 Wiley‐Liss, Inc.     

Procoagulant Effect of Incompatible Platelet Transfusions in Alloimmunized Refractory Patients

The clinical effectiveness of platelet transfusion in refractory patients is still a subject of debate. We have evaluated the possible hemostatic effect of platelet transfusion in 16 alloimmunized thrombocytopenic patients whose platelet counts were less than 20,000/μl. Platelet concentrates were always obtained by apheresis procedures from incompatible donors. The posttransfusion platelet recovery was greater than 15% only in 3 cases. In the first 6 patients, measurements of bleeding time performed immediately before transfusion were in all cases longer than 30 min and did not change significantly 10 and 60 min after platelet transfusions. In all patients, ex vivo perfusion experiments with Baumgartner's platelet adhesion model, using native nonanticoagulated blood, were performed immediately before and 10 and 60 min after transfusion. No difference in platelet deposition onto the subendothelial surface was observed after platelet transfusion. Unexpectedly, the deposition of fibrin on the subendothelial surface was statistically augmented in the posttransfusion studies. Quantification of thrombin-antithrombin complexes (TAT) in plasma showed statistically significant elevations (p < 0.01) in the posttransfusion samples (31.9±12.6 vs. baseline 5.8±1.7 ng/ml), not justified by TAT levels in the transfused material (2.3±0.17 ng/ml). Transfusion of incompatible platelets to refractory patients may activate coagulation mechanisms in the absence of an increase in peripheral platelet count.