Comparison between induced platelet aggregation and circulating platelet aggregates as platelet function tests in patients with transient ischemic attacks

Routine blood analysis, platelet counts, number of circulating platelet aggregates (CPA) and platelet aggregation in vitro against adenosine-diphosphate (ADP), epinephrine and collagene were studied in 45 healthy controls, in 10 hospitalized patients with other neurological diseases than stroke and in 12 patients with transient ischemic attacks (TIA) before and after prophylactive treatment with anticoagulants (AC) or anti-platelet drugs (APD).
Except for lower hemoglobin and hematocrit levels in women, sex, smoking, oral contraceptives or pregnancy did not significantly influence the routine blood parameter. Smoking females taking oral contraceptives had an increased number of CPA and the most easily induced aggregation in vitro .
Patients with TIA had no significant differences in blood or platelet findings versus the healthy controls (except smoking females on oral contraceptives) or the non-stroke patients, even though individual patients could have high numbers of CPA and an easily induced platelet aggregation in vitro . Treatment with AC did not influence platelet function, whereas APD therapy decreased the number of CPA and inhibited the secondary platelet aggregation in vitro .     

Platelet activation in pregnancy-induced hypertension

BACKGROUND: Although excess platelet activation, as indicated by increased plasma beta thromboglobulin (beta-TG), has been shown in pregnancy-induced hypertension (PIH), platelet adhesion, platelet morphology and a comparison of platelet and soluble (plasma) levels of the adhesion molecules P-selectin (pPsel and sPsel, respectively) have not been studied. METHODS: We conducted a cross-sectional study of 35 consecutive women with PIH (age 31+/-6 years), 31 consecutive women with normotensive pregnancies (age 29+/-5 years) and 30 normotensive non pregnant women (age 30+/-5 years). Platelet adhesion was studied in vitro by binding to fibrinogen-coated microwells, platelet morphology [mass and volume by flow cytometry], whole-platelet P-selectin (pPsel) by ELISA of the lysate of 2 x 10(8) cells, and the plasma markers soluble P-selectin (sP-sel) and beta-TG, by ELISA. RESULTS: The women with PIH had significantly raised sPsel, pPsel and (as expected) beta-TG (all p<0.05), when compared to the normotensive pregnant women and controls. However, in PIH platelet adhesion was similar to that in the normotensive pregnancy, but still higher than the normal controls (p<0.001). There was no difference among the three groups with respect to platelet mass and volume. pPsel and platelet adhesion correlated with gestational age and with systolic and diastolic blood pressure (all p<0.05). CONCLUSIONS: Increased platelet activation and adhesion develop during normal pregnancy, with some indices being further altered in PIH.     

Physiological coagulation inhibitors (protein S, protein C and antithrombin III) in severe preeclamptic states and in users of oral contraceptives

Protein C, protein S and antithrombin III were evaluated in normal pregnancy, severe preeclampsia and chronic hypertension with superimposed severe preeclampsia. The same study was performed on a group of 10 normal women using oral contraceptives. In normal pregnancy a significant decrease in the level of free and total PS was observed in the 2nd trimester of pregnancy and was sustained throughout the remaining months. No significant changes in the levels of protein C and antithrombin III were observed during normal pregnancy. In preeclamptic states a significant decrease in protein C was observed. It was more evident in severe preeclampsia when compared with the normal pregnancy group at similar gestational age. No statistically significant differences in protein S were found when the normal and pathological groups were compared. Antithrombin III decreased only slightly in the severe preeclamptic group. The decrease in protein C and antithrombin III levels in severe preeclampsia could be related with the microthrombotic state that these patients may present. However, the role played by protein S, which decreases during normal pregnancy and in preeclampsia, is not clear. A decrease in the level of total protein S was observed in the group of women using oral contraceptives. No significant changes in protein C and antithrombin III levels were observed in this group.     

Platelet behaviour in normal pregnancy, pregnancy complicated by essential hypertension and pregnancy-induced hypertension

Platelet behaviour was studied throughout pregnancy in a group of women who remained normotensive and a group with essential hypertension (EHT). Women who developed pregnancy-induced hypertension (PIH) were also studied together with a group of non-pregnant female controls. We determined the sensitivity of platelets to arachidonic acid (AA) and determined the effects of dazoxiben, a thromboxane synthetase inhibitor, on AA-induced platelet behaviour. A marked increase in platelet reactivity was evident in all three groups throughout pregnancy; platelets became more sensitive to AA and less sensitive to the inhibitory effects of dazoxiben. The change was apparent as early as 16 weeks gestation. In normotensive pregnancy and in EHT platelet behaviour had returned to normal six weeks after delivery. Platelets from women who developed PIH were more sensitive to AA than those from the other pregnant women and platelet reactivity had not returned to normal six weeks after delivery. The results indicate that alterations in platelet behaviour may contribute to the vascular complications that are known to be associated with pregnancy and with PIH in particular.     

Factor VIII levels and the risk of pre-eclampsia, HELLP syndrome, pregnancy related hypertension and severe intrauterine growth retardation

OBJECTIVE: Recently, acquired as well as genetic prothrombotic factors are associated with thrombotic events. These factors have also been related to conditions of uteroplacental insufficiency such as pre-eclampsia, HELLP syndrome and severe intrauterine growth restriction (IUGR). The aim of this study was to determine whether elevated factor VIII levels are associated with uteroplacental insufficiency, in particular pre-eclampsia, HELLP syndrome or pregnancy-induced hypertension and intrauterine growth retardation. METHODS: Plasma samples of 75 women with a history of pregnancy complicated by pre-eclampsia, HELLP syndrome, pregnancy induced hypertension or intrauterine growth restriction were tested for factor VIII:C (FVIII:C) levels at a minimum of 10 weeks post-partum. Laboratory results were compared to factor VIII:C levels found in a healthy control group of 272 women. RESULTS: Mean factor VIII:C levels were similar at 123 IU/dl in both the patient group and the controls. In a logistic regression model, after adjusting for age and blood group, no effect of factor VIII:C levels on the risk of pregnancy complications was observed, with the exception of IUGR with (OR 2.9, CI 1.0-8.7) or without hypertension (OR 2.0, CI 0.7-6.4). CONCLUSION: If the elevated level of factor VIII would be the sole factor responsible for the increased risk observed, one would expect to find an effect of blood group on risk as well (blood group being an important determinant of FVIII:C). While no such effect could be shown a causal relationship between elevated levels of factor VIII and conditions of uteroplacental insufficiency such as pre-eclampsia, HELLP syndrome, pregnancy-induced hypertension and IUGR is not very likely.     

Platelet activation and endogenous thrombin potential in pre-eclampsia

Introduction

Platelets and the coagulation system may be involved in the pathogenesis of pre-eclampsia. We investigated whether platelet and coagulation activation markers, are elevated in pre-eclampsia.

Materials/methods

Case-control study in which activated platelets, platelet-monocyte/ neutrophil aggregates, platelet microparticles (measured by flow cytometry) and four markers of thrombin generation capacity (endogenous thrombin potential (ETP), peak height, lag time and time to peak) using the Calibrated Automated Thrombogram system were assessed in pregnant women of similar gestational age with (n = 46) and without (n = 46) pre-eclampsia, and in healthy non-pregnant women (n = 42).

Results

The percentage of, CD62P+ platelets (p = 0.013), CD62P+ platelet microparticles (p = 0.029) and platelet-monocyte aggregates (p = 0.019) were significantly higher in women with pre-eclampsia than the pregnant controls. Both groups of pregnant women had significantly higher ETP and peak height (p  < 0.001) than the healthy non pregnant group and the women with pre-eclampsia had significantly higher ETP and peak height (p < 0.001) than the normotensive pregnant controls.

Conclusion

In the most comprehensive laboratory analysis to date, we found evidence of both platelet and coagulation activation in women with pre-eclampsia.     

Fibrinolytic response to venous occlusion and fibrin fragment D-dimer levels in normal and complicated pregnancy

The fibrinolytic response to venous occlusion was assessed in 29 women with normal or complicated pregnancy, by measurements of total t-PA and free t-PA with specific ELISAs. The release of t-PA from the vessel wall was 11 +/- 9 ng/ml in non-pregnant women (mean +/- SD, n = 6) but was markedly reduced throughout pregnancy. Following venous occlusion, free t-PA increased by 12 +/- 11 ng/ml in non-pregnant women but remained below the detection limit of 2 ng/ml towards the end of pregnancy. A markedly reduced t-PA release with absence of free t-PA was also observed during late pregnancy in patients with insulin-dependent diabetes mellitus, intra-uterine growth retardation and pre-eclampsia. Plasma levels of fragment D-dimer of cross-linked fibrin were measured with a specific ELISA in 79 pregnant women. D-dimer levels were 129 +/- 36 ng/ml (mean +/- SD, n = 8) in non-pregnant women and increased to 400 +/- 170 ng/ml (n = 25) and 440 +/- 220 ng/ml (n = 22) during the second and third trimester of pregnancy respectively. Significantly higher levels than observed in uncomplicated third trimester pregnancies were found in 3 out of 6 diabetic and in 2 out of 7 pre-eclamptic women. It is concluded that the t-PA release after venous occlusion is significantly reduced during pregnancy. In addition, released t-PA is rapidly inhibited. The levels of fragment D-dimer increase during pregnancy, suggesting that, notwithstanding the marked impairment of the fibrinolytic response to venous occlusion, the fibrinolytic system remains functionally active.     

Elevated fibrinopeptide A (FPA) in patients with Lesch-Nyhan syndrome.

The detection of elevated fibrinopeptide A (FPA) level in a patient with the Lesch-Nyhan syndrome complicated with cerebral infarction prompted us to examine FPA level in 3 other patients with the syndrome. FPA level significantly increased in all patients. Fibrinopeptide B beta 15-42 (FPB beta 15-42) level was increased in two, and both beta-thromboglobulin (beta TG) and platelet factor 4 (PF4) levels were elevated in one patient. These results suggest coagulation abnormalities in patients with Lesch-Nyhan syndrome.     

Variation of serum myoglobin levels in normal individuals. With reference to the use of myoglobin measurements for the detection of women carrying genes for the X-linked muscular dystrophies

The extent of variation in the serum myoglobin (Mb) levels of normal healthy females were investigated so that the value of Mb measurements for carrier detection could be properly assessed. Factors considered were sex, age, menstrual status, pregnancy, diurnal variation and daily activity. In each instance, Mb levels were compared with those for serum creatine kinase (CK). A significant difference (P less than 0.001) was found between the Mb levels of men and women but no relationship could be found between several measurements of body build and the Mb or CK levels of either sex. An apparent correlation of Mb concentrations with age was found in control groups of both sexes up to 85 years of age, but this was not significant below 60 years of age. No differences in Mb levels were found among premenarchal, menstruating, postmenopausal or hysterectomised women and neither smoking nor the use of oral contraceptives seemed to affect Mb levels. Serum CK levels were very much higher in premenarchal (P less than 0.001) and postmenarchal (P less than 0.05) teenagers than in adult controls. No difference was found between the mean Mb levels of non-pregnant and pregnant (6-40 weeks) women. The mean value for CK activity was significantly lower than normal in the pregnant women (P less than 0.001). A marked circadian rhythm of serum MB levels was found with samples taken at 5 p.m. being approximately 20% lower than those taken at 9 a.m. This unexpected decrease during the day persisted with 48 hours of bedrest suggesting that this rhythm is inherent and unrelated to muscular activity.     

Effects of pregnancy and contraception on lamotrigine disposition: new insights through analysis of lamotrigine metabolites.

OBJECTIVE: To investigate possible underlying mechanisms for alterations in lamotrigine (LTG) kinetics by gestation and use of contraceptives. METHODS: Plasma concentrations of LTG and its main metabolite lamotrigine-2-N-glucuronide (2-N-GLUC) were measured in 31 women on LTG taking combined oral contraceptives (COC), in 12 with contraceptive intrauterine devices containing levonorgestrel (CIUD), and in 20 on LTG without hormonal contraception (controls). We also measured the levels of LTG and 2-N-GLUC in plasma and urine in eight women during pregnancy, and up to three months postpartum. LTG levels in plasma were measured by high-performance liquid chromatography method (HPLC) and N-2-GLUC in urine and plasma and LTG in urine by liquid chromatography-mass spectrometry (LC/MS). RESULTS: There were no significant differences in LTG dose/concentration (D/C), or N-2-GLUC/LTG ratios between women with CIUD and controls. In contrast, compared to controls, the LTG D/C ratio was 56% higher in women taking COC (mean+/-SD, 83+/-47 versus 53.0+/-24.2; p<0.01) and N-2-GLUC/LTG ratio 82% higher in women taking COC (mean 0.477+/-0.212 SD versus 0.262+/-0.127; p<0.0003. The 2-GLUC/LTG ratios were 154% higher in plasma and 122% higher in urine in late pregnancy compared with baseline 3months postpartum. CONCLUSIONS: Our data indicate that the alterations in LTG kinetics in pregnancy as well as those induced by COC are mainly explained by enzymatic induction of the N-2-glucuronide pathway. In addition we found no evidence for an interaction between LTG and CIUD.     

Pre-eclampsia and nasal CPAP: part 1. Early intervention with nasal CPAP in pregnant women with risk-factors for pre-eclampsia: preliminary findings

BACKGROUND: Pre-eclampsia is a leading cause of maternal-fetal morbidity and mortality. Significant overlap exists between the risk factors for pre-eclampsia and sleep-disordered breathing. Nasal continuous positive airway pressure (CPAP) has been proposed as therapy for pre-eclampsia. This prospective, longitudinal study was designed to characterize sleep-related breathing patterns in pregnant women with pre-eclampsia risk factors, and to describe the effects of early nasal CPAP therapy in these patients. METHODS: Twelve pregnant women with pre-eclampsia risk factors underwent polysomnography to characterize sleep-related breathing abnormalities and baseline blood pressure determination. Patients with airflow-limitation underwent nasal CPAP titration and were treated with optimal pressures. Periodic assessments of CPAP compliance and tolerance, sleep quality, and blood pressure control were performed until delivery or pre-eclampsia onset. CPAP retitration was performed between weeks 20 and 22 of pregnancy. RESULTS: Mean respiratory disturbance index was 8.5+/-2.6 events/h of sleep, and initial nasal CPAP pressures were 5-6 cm H(2)O with an increase to 6-9 cm H2O after recalibration. All subjects with chronic hypertension maintained blood pressures below 140/90 with a mean diurnal blood pressure of 122+/-2.5 mmHg over 83+/-1.5 mmHg. Patient characteristics of obesity and prior pre-eclampsia were associated with pregnancies complicated by spontaneous abortion, premature delivery, or pre-eclampsia. CONCLUSIONS: Early application of nasal CPAP in pregnant women alleviated sleep-related breathing disturbances but was not sufficient to prevent negative pregnancy outcomes. Obesity and prior pre-eclampsia appeared to be important factors and were associated with the worst complications. However, nasal positive pressure may still be beneficial to decrease severity of outcomes, particularly if individualized to patient risk factors, more particularly hypertension at pregnancy onset.     

Effects of oral contraceptives, or lanosterol, on ADP-induced aggregation and binding of 125I-fibrinogen to rat platelets

The aggregation to ADP and the binding of 125I-fibrinogen to platelets from rats treated with oral contraceptives or normal platelets treated in vitro with lanosterol were compared to their respective controls. Both types of platelets showed a significant increase in ADP-induced aggregation and in binding of fibrinogen, indicating that the effect of oral contraceptives could be partly due to increased levels of lanosterol in platelet membrane.     

A prospective study of haemostatic tests at 28 weeks gestation as predictors of pre-eclampsia and growth retardation

A panel of haemostatic tests was performed on 400 primiparous women at 28 weeks to test whether one or more could predict the development of pregnancy complications. Fifteen women subsequently developed pre-eclampsia with significant proteinuria and 13 delivered growth retarded infants. There were no significant differences between mothers in the pre-eclampsia group and 22 randomly selected controls. A stepwise logistic discriminant analysis of the data did not produce a significant model. In the growth retarded group only beta thromboglobulin levels were significantly lower than in the controls (p less than 0.05), although in the logistic discriminant analysis the inclusion of both beta thromboglobulin and fibrin degradation products led to a borderline significant improvement in fit of the model. We conclude that the haemostatic variables studied are not significantly changed at 28 weeks nor clinically useful predictors of either pre-eclampsia or fetal growth retardation.     

In vivo platelet release in myeloproliferative disorders

The in vivo platelet release reaction in 22 patients with myeloproliferative disorders has been studied by measuring plasma concentrations of the platelet release product beta-thromboglobulin (beta TG). Mean beta TG and mean beta TG: whole blood platelet count ratio were significantly raised in the patient group taken as a whole compared to an age matched control group. No significant increases were observed in the plasma concentrations of thrombin and plasmin sensitive fibrinogen fragments fibrinopeptide A (FpA) and B beta 1-42. The patients were divided into those who had normal, increased or decreased responses to in vitro ADP-induced platelet aggregation. Mean beta TG and the mean beta TG: whole blood platelet count ratio were higher in the increased and decreased responders to ADP than in the normal aggregation group, but the differences in means were not statistically significant. Aspirin given to six patients at a dose sufficient to eliminate the secondary phase of ADP-induced platelet aggregation reduced mean beta TG and the mean beta TG: whole blood platelet count ratio but did not alter mean FpA and B beta 1-42. It is concluded that the enhanced platelet release reaction seen in myeloproliferative disorders is independent of plasma protease activity that arises when coagulation and fibrinolytic systems are activated.     

Mutation in the methylenetetrahydrofolate reductase gene might be a risk factor for cerebrovascular disease in peripartum and under oral contraceptive use

Nine women (age 22-43 years) with cerebrovascular diseases (CVD) related to pregnancy, puerperium or contraceptive use were studied. Five were pregnant, 2 were post partum and 2 were taking oral contraceptives. All under- went a complete etiological examination including assessment for the thermolabile C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene mutation. Three of the 9 patients were homozygotic for the C677T MTHFR mutation, and 3 were heterozygotic. In these 6 patients, no other etiology could be found. Mutation in the thermolabile MTHFR gene might be an important cause for CVD related to peripartum or contraceptive use.     

Analogous features of cold-promoted activation and contact system activation in human plasmas: the role of cryptic soluble surfaces in asthma, oral contraception and pregnancy

Aspects of cold-promoted activation (CPA) and contact system activation (CSA) of human plasma were assayed in 13 normal subjects, 11 asthmatics and 15 patients with history of allergy to x-ray contrast media, to see whether the phenomena were correlated. Cold activation commonly occurs in stored plasma in 60% of women taking oral contraceptives, 93% of women in 3rd trimester of pregnancy, but only 15% of normal subjects. It is assayed by measuring kallikrein. CSA also occurs at low temperatures, but is dependent on soluble negatively-charged surfaces. It is assayed using dextran SO4 or polybrene. In this study, kallikrein amidolytic activity with dextran SO4-CDA and cold-dilution CDA were positively correlated in asthmatics. The allergic patients showing elevated B-CDA values also had shortened Thrombotest times with cold dilution, while those with normal B-CDA values had normal coagulation times. The significance of these results were discussed, including the theoretical possibility that cryptic surface phenomena may be involved in the heightened coagulability of plasma in women taking oral contraceptives.     

Intracranial venous thrombosis in young women

Twenty women of child-bearing age with intracranial venous thrombosis are reported. Thirteen developed thrombosis post-partum; 6 while on oral contraceptives. In one patient no cause was found. All diagnoses were confirmed by carotid angiography and in 10 out of 14 patients laboratory data indicated a hypercoagulable state. Coagulation studies were performed in 14 normal puerperal women, matched by age, who served as controls. No statistical differences was found between thrombotic and control groups. Radiologic signs of sino-venous occlusion are reviewed. Conservative treatment with anti-edematous agents, anticonvulsants and antiplatelet aggregants is advocated.     

Increased heparin cofactor II levels in women taking oral contraceptives

Heparin cofactor II (HCII) is a thrombin inhibitor present in human plasma whose activity is enhanced by heparin. HCII exhibits important homologies with antithrombin III, the main heparin-enhanced thrombin inhibitor. Cases of recurrent thromboembolism have been recently reported in patients with HCII deficiency. Since the use of oral contraceptives (OC) is associated with an increased risk of thrombosis, the study of the plasma levels of HCII was undertaken in women taking contraceptive pills. Plasma HCII levels were found significantly higher in 62 women taking low-estrogen content OC (1.20 +/- 0.28 U/ml) than in 62 age matched women not taking OC (0.94 +/- 0.16 U/ml) or in 62 men (0.96 +/- 0.19 U/ml). Significant correlations between HCII and fibrinogen levels were reported in the three groups. From the pooled data of the two control groups (men and women not taking OC), the normal range for plasma HCII levels was defined to be between 0.60 and 1.30 U/ml (mean +/- 2 SD). Two cases of low HCII levels (less than 0.60 U/ml) were found in the control groups, but none in the group of women taking OC. It is concluded that the use of oral contraceptives is associated with a rise in HCII levels and that the screening for HCII deficiency has to be performed at distance of any OC therapy.     

Prothrombotic genotypes are not associated with pre-eclampsia and gestational hypertension: results from a large population-based study and systematic review

DNA samples collected as part of a large population-based case-control study were genotyped to examine the associations of five prothrombotic gene polymorphisms with pre-eclampsia (PE) and gestational hypertension (GH). The polymorphisms studied were: G1691A in Factor V (Factor V Leiden; FVL), prothrombin G20210A, methylenetetrahydrofolate reductase (MTHFR) C677T, plasminogen activator inhibitor-1 4G/5G and the platelet collagen receptor alpha2beta1 C807T. A group of 404 women who developed PE were retrospectively compared with 303 women with GH and 164 control women. The frequency of genotypes did not differ significantly between cases of PE or GH and controls for any of the five polymorphisms studied. We conclude that these prothrombotic genotypes are not associated with the development of PE or GH in our population. The systematic review supports our conclusion, for all but cases of severe disease. which appear to be associated with FVL and, to a lesser extent, MTHFR C677T. There is little value in antenatal screening for prothrombotic polymorphisms to predict the development of pre-eclampsia or gestational hypertension.