Plasma concentration of platelet-specific proteins and fibrinopeptide A in patients with artificial heart valves

beta-Thromboglobulin (beta TG), platelet factor 4 (PF4), fibrinopeptide A (FPA), lactic dehydrogenase (LDH), and platelet count were evaluated in patients with bioprostheses and prosthetic heart valves. beta TG and PF4 were significantly elevated in both patient groups (p less than 0.001), whereas FPA was normal. There was no significantly difference in plasma concentrations of beta TG and PF4 between patients with prosthetic heart valves and bioprostheses. LDH levels were significantly (p less than 0.001) higher and platelet count lower (p less than 0.001) in patients with prosthetic cardiac valves. The data indicate that bioprostheses do not cause haemolysis or activation of the coagulation system. The findings that the plasma concentration of platelet-specific proteins were elevated, support the assumption that both types of valves cause platelet damage.     

The significance of fibrinogen derivatives in plasma in human renal failure

The concentrations in plasma of fibrinogen derivatives fibrinopeptide A (FPA), beta 15-42 antigen and fragment E (FgE) antigen have been determined in patients with renal failure and compared to the concentrations of the platelet release products, beta-thromboglobulin (beta TG) and platelet factor 4 (PF4). In 'partial renal failure' (51Cr-EDTA clearance rate 4-60 ml/min) FPA, beta 15-42 antigen, FgE antigen and beta TG levels were significantly raised above a normal laboratory control group. These levels were further raised in a group of patients whose disease required regular maintenance haemodialysis (51Cr-EDTA clearance rate less than 4 ml/min). PF4 levels were not significantly raised in either group. A statistical analysis of all patient results revealed that FPA, beta 15-42 antigen and FgE antigen levels all correlated with beta TG levels but not with PF4 levels. It is known that beta TG is catabolized by the kidney but PF4 is not and that elevated beta TG levels in renal failure are caused by impaired elimination rather than increased production. These results suggest that the plasma levels of these three fibrinogen derivatives are elevated in renal disease at least in part by decreased elimination rather than by increased thrombin and plasmin activities alone.     

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.     

Bleeding and thrombosis in chronic myeloproliferative disorders: relation of platelet disorders to clinical aspects of the disease

Bleeding and thrombosis are a major cause of morbidity and mortality in myeloproliferative disorders (MPD). This study evaluates the relation between thrombohemorrhagic complications and platelet abnormalities in different subgroups of MPD. In 57 MPD patients thrombohemorrhagic complications occurred in 71% of patients with polycythemia rubra vera and 50% of patients with osteomyelofibrosis and primary thrombocythemia but in only 29% of patients with chronic myelogenous leukemia. Increased beta-thromboglobulin and platelet factor 4 plasma levels, platelet aggregation defects, and increased dispersion of the platelet volume distribution curve were most frequent in those subgroups where most serious thrombohemorrhagic complications were observed, and multiple platelet-related abnormalities were often found simultaneously. Fibrinopeptide A plasma levels were rarely elevated, however. Our results indicate that platelet abnormalities associated with bleeding and thrombosis are primarily determined by the clinical subgroup of myeloproliferative disease.     

Effect of glycaemic control, metformin and gliclazide on platelet density and aggregability in recently diagnosed type 2 (non-insulin-dependent) diabetic patients

Platelet density profiles, intraplatelet nucleotides, intraplatelet beta thromboglobulin (beta TG), plasma beta TG levels, intraplatelet cyclic AMP (cAMP) levels, platelet release reaction, platelet thromboxane (TX)B2 production and plasma fibrinogen levels were investigated in 24 newly diagnosed, non-insulin-dependent diabetic patients and 12 comparable controls. These variables were measured at diagnosis, after a 3-6 week dietary run-in period, and again after 6 months on treatment with either metformin or gliclazide therapy. With dietary restriction of refined carbohydrate and oral hypoglycaemic therapy, there was a reduction in platelet density (p less than 0.05), intraplatelet nucleotides (p less than 0.001), intraplatelet beta TG (p less than 0.001), plasma beta TG (p less than 0.001) and there was an increase in intraplatelet cAMP levels (p less than 0.05). Although these platelet variables returned towards normal, only the platelet density mean returned to within the normal range. There was no significant change in the platelet TXB2 production and plasma fibrinogen levels with treatment. Metformin and gliclazide were equally effective in the glycaemic control of non-insulin-dependent diabetes, and there was no difference between the platelet variables measured in the two groups. We would therefore suggest that improvement of glycaemic control, rather than any specific effect of the oral hypoglycaemic agent employed, is the most important factor in returning these parameters towards normality.     

Qualitative platelet defects in chronic myeloproliferative disorders: evidence for reduced ATP secretion

19 consecutive untreated patients with chronic myeloproliferative disorders and thrombocytosis were subjected to comprehensive platelet function tests including platelet aggregometry. 12 patients had essential thrombocythaemia (ET) and 7 patients had polycythaemia vera (PV). Bleeding time was normal. Arachidonic acid, collagen and ristocetin aggregation were abnormal only in a minority of patients, whereas ADP aggregation was impaired in 16 out of 19 patients. The most conspicuous findings were abolished second-wave adrenalin aggregation, increased ADP aggregation threshold, and markedly reduced ATP secretion during collagen-induced aggregation. This triad of qualitative platelet defects seems to be a good diagnostic marker of chronic myeloproliferative disease with thrombocytosis.     

Augmented oxidative stress of platelets in chronic smokers. Mechanisms of impaired platelet-derived nitric oxide bioactivity and augmented platelet aggregability.

OBJECTIVES: We investigated whether impaired platelet-derived nitric oxide (PDNO) bioactivity and augmented platelet aggregability in chronic smokers are related to the imbalance of the intraplatelet redox state through increased oxidative stress. BACKGROUND: Chronic smoking impairs PDNO release and augments platelet aggregability. However, their mechanisms are unknown. METHODS: Collagen-induced PDNO release, platelet aggregation, plasma and intraplatelet vitamin C and reduced glutathione (GSH), intraplatelet cyclic guanosine 3',5'-monophosphate (cGMP) and intraplatelet nitrotyrosine production, which is a marker of the peroxynitrite formation, were measured in 11 chronic smokers and 10 age-matched nonsmokers. RESULTS: Release of PDNO and levels of intraplatelet cGMP were lower, and platelet aggregation was greater, in smokers than in nonsmokers. Intraplatelet vitamin C and GSH levels were lower in smokers than in nonsmokers. Intraplatelet nitrotyrosine production was greater in smokers than in nonsmokers. Next, we investigated the effects of oral vitamin C administration (2 g). After vitamin C administration, intraplatelet vitamin C levels were increased and not different at 2 h between the two groups. Then, PDNO release, intraplatelet cGMP levels and platelet aggregation in smokers were restored to the levels of nonsmokers. In smokers, PDNO release and consumption of GSH during platelet aggregation were inversely correlated, and consumption was much less after vitamin C administration. Vitamin C administration decreased intraplatelet nitrotyrosine production in smokers. CONCLUSIONS: Impaired PDNO bioactivity and augmented platelet aggregability may be caused by an imbalance of the intraplatelet redox state through increased oxidative stress in smokers.     

Levels of angiogenic proteins in plasma and platelets are not different between patients with hepatitis B/C-related cirrhosis and patients with cirrhosis and hepatocellular carcinoma

Increasing evidence suggests that levels of angiogenic proteins within blood platelets change at the earliest stages of cancer development and may thus provide a promising diagnostic and prognostic tool. Patients with cirrhosis have increased risk of developing hepatocellular carcinoma (HCC). We aimed to study whether development of HCC in hepatitis-related cirrhosis results in changes in platelet levels of angiogenic proteins. We studied the intraplatelet levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), endostatin, platelet factor 4 (PF4) and thrombospondin type 1 (TSP-1) in 38 consecutive patients with hepatitis B- or C-related liver cirrhosis with or without HCC in addition to plasma levels of the same proteins. Twenty healthy volunteers were included to establish reference values for the various tests. Intraplatelet levels of VEGF, bFGF, HGF and endostatin were significantly higher in patients compared to controls. Intraplatelet levels of PDGF, PF4 and TSP-1 were comparable between patients and controls. Plasma levels of VEGF, bFGF and endostatin were comparable between patients and controls. Plasma levels of PDGF, PF4 and TSP-1 were decreased in patients, but this difference disappeared when levels were corrected for platelet count. Intraplatelet and plasma levels of all proteins assessed were comparable between patients with and without HCC. In conclusion, the intraplatelet levels of some angiogenic proteins are elevated in cirrhosis, but do not discriminate between patients with and without HCC. Thus, intraplatelet levels of angiogenic proteins do not seem useful as diagnostic or prognostic biomarker of HCC in cirrhotic patients.     

Platelet 5-hydroxytryptamine transport and storage in myeloproliferative disorders

Platelets of patients with myeloproliferative disorders (MD) such as polycythaemia vera (PV), chronic myelogenous leukaemia (CML), idiopathic myelofibrosis (IM) and essential thrombocythaemia (ET) have been found to have low 5HT levels measured both by a fluorimetric and a liquid chromatographic assay. Km and Vmax for platelet active uptake of 3H-5HT were not significantly different in controls and patients. Inhibition of 5HT reuptake by imipramine or induction of moderate release by fenfluramine were not sufficient to distinguish the group of MD platelets from controls, although some patients had less of a tendency to retain intraplatelet amine. The low platelet 5HT content found in our patients seems not to be the consequence of disturbed active transport of 5HT across platelet membrane. Although defective storage of this amine within the cell is probable, the results of the present study do not rule out the possibility that platelets from MD patients undergo in vivo activation by endogenous stimuli not inhibited by aspirin. 10 d treatment with aspirin did not result in any significant rise in intraplatelet 5HT concentration.     

Assessment of platelet activation in myeloproliferative disorders with complementary techniques.

Bleeding and thrombosis in myeloproliferative disorders (MPD) are common events, sometimes both are present in the same patient during the course of the disease. Platelet activation in patients with MPD is often suggested. The present study analyses the presence of circulating activated platelets, using simultaneously flow cytometry and aggregometric studies in MPD. We studied 28 patients: 13 with polycythaemia vera, seven with essential thrombocythaemia, and eight chronic myeloid leukaemia. We performed functional tests, aggregation and adenosine triphosphate (ATP) release and flow cytometric assays (mepacrine staining and platelet activation markers CD62, CD63 and fibrinogen binding (B-FG)). Twenty-one MPD samples (75%) had reduced aggregation and ATP release. Acquired delta-SPD was detected in 11 of 28 MPD patients (39%), and we found no association between reduced mepacrine labelling and abnormal ATP release. High levels of activation markers were obtained: CD62 in 19 of 28 patients (68%), CD63 in 13 of 28 patients (46%) and B-FG in 19 of 28 patients (68%). The most prevalent abnormality was a reduced aggregation and ATP release. The lack of association between ATP release and mepacrine labelling suggests that other mechanisms, besides the deficit of intraplatelet ATP/adenosine diphosphate, might occur. High levels of activation markers were also observed. We conclude that both tests are complementary and necessary to understand the functional status of platelets in MPD.     

Plasma fibronectin in myeloproliferative disorders and chronic granulocytic leukaemia

A significant reduction of plasma fibronectin levels was found in polycythaemia vera and myelofibrosis, the lowest levels being found in patients with marked splenomegaly. Plasma fibronectin concentration was normal in essential thrombocythaemia, and only modest reduction was seen in chronic granulocytic leukaemia in either controlled chronic phase or blast cell crisis. In a patient with myelofibrosis, the plasma fibronectin rose from less than 100 mg/l to 177 mg/l after splenectomy. Possible explanations include increased consumption of plasma fibronectin in the expanded mononuclear phagocyte system present in the liver and spleen, reduced hepatic synthesis, and the clearance of circulating immune complexes. Low plasma fibronectin concentrations may increase susceptibility to infection.     

An abnormal pattern of multiple platelet function abnormalities and increased thromboxane generation in patients with primary thrombocytosis and thrombotic complications.

Platelet aggregation (PA) induced by ADP, collagen and epinephrine, plasma levels of beta-thromboglobulin (beta TG) and thromboxane B2 (TXB2) and serum TXB2 generation were studied in 11 patients with primary thrombocytosis (7 with essential thrombocythaemia and 4 with polycythaemia vera) and compared with 16 healthy subjects. 5 patients suffered from peripheral vascular ischaemia and another 3 had venous thrombosis, but none had bleeding complications. The patients showed an abnormal pattern of platelet function and of thromboxane generation distinct from the healthy subjects in three aspects. a) Shape change was 5-26 times greater, the lag-time of collagen PA was 2.3-2.9 times longer and the extent of epinephrine PA was nil or very low. ADP- or collagen-induced PA was also reduced (p less than 0.02). b) Plasma TXB2 generation (corrected to a normal platelet concentration) stimulated by the three PA inducers was within the range of the healthy subjects in spite of the reduced extent of PA. c) Plasma beta TG level and serum TXB2 generation (both corrected to a normal platelet concentration) were 2.9-7.1 times higher (p less than 0.001) indicating enhanced in vivo platelet activation and possibly increased thrombin generation. These abnormalities were not detected in another 4 patients with secondary thrombocytosis. The abnormal pattern of platelet function and thromboxane generation can be a useful laboratory method in the evaluation of patients with primary thrombocytosis. It might also explain the thrombotic complications which occurred in 8 of the patients in a manner such that increased or normal TXB2 generation overcomes the reduced extent of PA. In this respect, the pronounced serum TXB2 synthesis might be a marker of intravascular thrombosis.     

Platelet function during the acute phase of dengue hemorrhagic fever

Platelet aggregation, plasma betathromboglobulin (BTG) and platelet factor 4 (PF4) were studied in 35 children with dengue hemorrhagic fever. The suppression of platelet aggregation was demonstrated during acute phase of DHF in both shock and non-shock patients. Simultaneous with abnormal platelet aggregation, there was increased release of BTG and PF4 from platelets into plasma during the acute phase which lasted only 3-4 days after shock or subsidence of fever. Acute phase plasma during DHF infection was also shown to have a stimulatory effect on the aggregation of autologous platelets. In this study we showed that there was an increase in platelet secretory activity of BTG and PF4 along with an impairment of the platelet aggregation during acute phase of DHF.     

Elevated plasma beta-thromboglobulin levels in multiple myeloma and in polycythaemia vera.

Plasma beta-thromboglobulin levels were determined in 22 multiple myeloma patients, in 15 patients with polycythaemia vera and in 70 healthy controls. In both multiple myeloma and polycythaemia vera significantly increased plasma concentrations of this platelet-specific protein were found.     

The Clinical Significance of Beta-Thromboglobulin and Platelet Factor-4 in Polycythaemic Patients

Simultaneous assays of platelet factor 4 (PF-4) and beta-thromboglobulin (βTG) were performed in 192 cases of myeloproliferative syndromes (polycythaemia vera and primary thrombocytosis, as defined by the Polycythaemia Vera Study Group). The results led to the following conclusions:

• (I) both assays must be combined in order to avoid a poor interpretation due to marker release in vitro;
• (II) the ‘normality’ of the values must take the platelet number into account, even in the ‘normal’ range of this parameter;
• (III) the sensitivity of the βTG assay is greater than that of PF-4 when considering the correlation of the marker values with arterial accidents;
• (IV) the predictive value of an excessive level of βTG and/or PF-4 is difficult to define, since only 13 of the cases studied had a vascular accident during the 12-month follow-up period, and the levels of the markers in these patients were not statistically different from the levels in those patients not experiencing such accidents.

     

Platelet Function Abnormalities in the Myeloproliferative Disorders

Platelet function studies were performed in 21 patients with myeloproliferative disorders. Blunted serotonin and first wave epinephrine aggregation were noted in 75 % of the patients studied. Abnormalities in platelet adhesion, aspirin bleeding time, and serotonin aggregation were found in all patients with a bleeding history. Similar abnormalities were present in certain patients without a bleeding history; this latter group of patients may have an increased bleeding risk when appropriately stressed. Abnormalities of platelet function were unrelated to the platelet count in patients with chronic myelogenous leukaemia; in contrast, subjects with polycythaemia vera showed fewer abnormalities when the platelet count was less than 500,000/μl.     

Platelets in Myeloproliferative Disorders

Certain platelet functions were evaluated in 24 patients with secondary polycythaemia (SP) and in a large number of patients suffering from myeloproliferative disorders (MD'S): 89 patients with chronic myeloid leukaemia (CML) at different stages of development, 58 with polycythaemia vera (PV), 23 with essential thrombocythaemia (ET), and 25 with agnogenic myeloid metaplasia (AMM). Bleeding time, epinephrine-induced platelet aggregation and adhesiveness agree with those generally reported in the literature; they are independent of thrombocytosis, the haemoglobin level and the leucocyte count. Macrothrombocytosis, evaluated by an electronic method, was only found in CML, mainly during acute blast crisis. An increased percentage of light platelets was a constant feature in all groups except in the SP and in 20 % of the PV. The most severe abnormalities were observed in AMM and CML in the acute stage; in the chronic phase of CML there is no correlation between the severity of platelet abnormalities and the survival of the patients.     

Thromboembolic complications in an asplenic HbE-beta-thalassaemia patient

A patient, double heterozygous for HbE-beta-thalassaemia, had recurrent thromboembolic complications following splenectomy. In addition to marginally decreased protein C and S plasma levels, laboratory studies revealed a persistent thrombocytosis and markedly elevated plasma concentrations of platelet factor 4 (PF4). PF4 neutralizes the heparin anticoagulant activity. The increased PF4 levels explained the initial heparin resistance observed during anticoagulant treatment in this patient. Subsequent heparin loading tests revealed an abnormal reaction of the PF4 plasma levels, i.e. no increase of PF4. However, upon repeated heparin injections this PF4 response normalized, which may be due to depletion of the endothelial cell associated heparin mobilizable PF4 pool. These observations may be of relevance for the treatment of similar patients.     

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.