High prevalence of the prothrombin gene mutation in women with intrauterine growth retardation, abruptio placentae and second trimester loss.

BACKGROUND: It has been reported recently that obstetric complications are associated with thrombophilias. Our objective was to investigate the association between pregnancy complications and the guanine 20210 adenine (G20210A) mutation in prothrombin gene. METHODS: Two hundred and twenty-two women (study group) with obstetric complications were tested for the prothrombin mutation. Indications for testing were: severe preeclampsia, mild preeclampsia, intrauterine growth retardation, severe abruptio placentae, unexplained stillbirth, second trimester loss, and three or more consecutive spontaneous abortions. We also tested 156 healthy women who had at least one normal pregnancy and comprised the control group. RESULTS: Demographic data of the study and control groups were similar. Twenty-eight women of the study group (13%) were found to be heterozygous carriers of the 20210 variant of the prothrombin gene compared to five (3.2%) of the control group, p=0.001, odds ratio (OR) 2.9; 95% confidence interval (CI) 1.3-6.5. Compared to the control women, the prothrombin gene mutation was significantly more prevalent in women with IUGR, abruptio placentae, and second trimester loss but not in women with mild or severe preeclampsia, stillbirth and habitual abortion. CONCLUSIONS: Our data demonstrate that the mutation in the prothrombin gene is associated with specific pregnancy complications.

The procoagulant activity of red blood cells from patients with severe preeclampsia.

OBJECTIVE: Our purpose was to determine whether red blood cells from patients with severe preeclampsia may exhibit increased membrane exposure of procoagulant phospholipids (i.e., phosphatidylserine), which may initiate intravascular clotting and platelet activation. STUDY DESIGN: The study group comprised 28 women: 9 with severe preeclampsia in the third trimester of pregnancy, 10 normotensive with uncomplicated pregnancies, and 9 age-matched, nonpregnant, healthy women. The exposure of phosphatidylserine on the outer membrane phospholipid layer was analyzed with use of isolated, washed red blood cells that were added as a source of phospholipids to a "prothrombinase" coagulation complex. RESULTS: The resultant thrombin formed was measured by an amidolytic assay. Thrombin generation significantly increased on the addition of red blood cells from women with preeclampsia (741 +/- 132 mU/ml/min) compared with red blood cells from normotensive pregnant (422 +/- 228 mU/ml/min) and nonpregnant women (316 +/- 268 mU/ml/min, p = 0.0008). CONCLUSION: This study indicates that in patients with preeclampsia the red blood cells exhibit a significant procoagulant surface that may trigger thrombin formation, thereby playing a role in the hypercoagulable state.

Enhancement of recombinant tissue-type plasminogen activator thrombolysis with a selective factor Xa inhibitor derived from the leech Hirudo medicinalis: comparison with heparin and hirudin in a rabbit thrombosis model.

OBJECTIVE: To compare the efficacy of Yagin, a factor Xa inhibitor derived from the leech Hirudo medicinalis, with those of heparin and hirudin as adjuncts to recombinant tissue-type plasminogen activator (rTPA) thrombolysis in a rabbit thrombosis model. METHODS: Thirty-one animals were allocated randomly to three groups, all administered four boluses of 0.25 mg/kg rTPA every 10 min for 30 min, 17 mg/kg aspirin intravenously, and heparin (as a 100 IU/kg bolus followed by infusion of 50 IU/kg heparin per h), hirudin (as a 2 mg/kg bolus followed by infusion of 1 mg/kg hirudin per h), or Yagin (as an 80 micrograms/kg bolus followed by infusion of 43 micrograms/kg Yagin per h). RESULTS: Administration of Yagin was associated with a significant acceleration of the reflow time, this time being 14.5 +/- 1.2 min with Yagin, 25.8 +/- 5.2 min with heparin (P < 0.0001, versus Yagin), and 28.7 +/- 16.0 min with hirudin (P = 0.012, versus Yagin). Overall patency did not differ significantly among the three groups. CONCLUSIONS: At the indicated single doses, inhibition of factor Xa by a relatively low concentration of Yagin was found to be superior than that with either heparin or hirudin for accelerating rTPA thrombolysis.

Platelet dysfunction associated with experimental acute canine ehrlichiosis.

To determine whether platelet dysfunction occurs in canine ehrlichiosis, platelet aggregation studies in response to collagen/epinephrine, thrombin and adenosine diphosphate (ADP) were carried out by the indirect method, using sera from six dogs experimentally infected with Ehrlichia canis. Samples of serum taken before infection and four and 20 days after infection were tested by incubation with platelet-rich plasma from a seronegative healthy dog. Platelet aggregation was significantly inhibited in five of six infected dogs in response to at least one of the agonists used. A significant increase in preaggregation lag time was recorded in response to collagen/epinephrine in sera taken 20 days after infection from three of five dogs (P < 0.05). When compared with the preinfection values, a significant increase of 45 per cent in the mean preaggregation lag time was detected (P < 0.05). Maximal relative aggregation responses to ADP decreased significantly in one serum sample taken four days and one taken 20 days after infection (P < 0.01) and there was a significantly lower relative slope for one serum sample 20 days after infection (P < 0.05). Maximal relative aggregation responses to thrombin were significantly decreased together with their relative slopes in serum samples from two of four dogs four days after infection (P < 0.05). The results suggest that platelet dysfunction may occur in the acute stage of canine ehrlichiosis, and may be a contributing factor to the tendency to bleed commonly observed in this disease. Antiplatelet antibodies directed against platelet glycoproteins may play a role in the inhibition of platelet aggregation.

Detection of the Glanzmann's thrombasthenia mutations in Arab and Iraqi-Jewish patients by polymerase chain reaction and restriction analysis of blood or urine samples.

Severe Glanzmann's thrombasthenia is relatively frequent in Iraqi-Jews and Arabs residing in Israel. We have recently described the mutations responsible for the disease in Iraqi-Jews--an 11 base pair deletion in exon 12 of the glycoprotein IIIa gene, and in Arabs--a 13 base pair deletion at the AG acceptor splice site of exon 4 on the glycoprotein IIb gene. In this communication we show that the Iraqi-Jewish mutation can be identified directly by polymerase chain reaction and gel electrophoresis. With specially designed oligonucleotide primers encompassing the mutation site, an 80 base pair segment amplified in healthy controls was clearly distinguished from the 69 base pair segment produced in patients. Patients from 11 unrelated Iraqi-Jewish families had the same mutation. The Arab mutation was identified by first amplifying a DNA segment consisting of 312 base pairs in controls and of 299 base pairs in patients, and then digestion by a restriction enzyme Stu-1, which recognizes a site that is absent in the mutant gene. In controls the 312 bp segment was digested into 235 and 77 bp fragments, while in patients there was no change in the size of the amplified 299 bp segment. The mutation was found in patients from 3 out of 5 unrelated Arab families. Both Iraqi-Jewish and Arab mutations were detectable in DNA extracted from blood and urine samples. The described simple methods of identifying the mutations should be useful for detection of the numerous potential carriers among the affected kindreds and for prenatal diagnosis using DNA extracted from chorionic villi samples.

von Willebrand factor multimer patterns in pregnancy-induced hypertension.

Microangiopathy and disseminated platelet aggregation have been reported in thrombotic thrombocytopenic purpura (TTP) and pregnancy-induced hypertension (PIH). Since unusually large von Willebrand factor (vWF) multimers have been implicated in the evolvement of TTP, we analyzed factor VIII/vWF parameters in patients with PIH. Mean vWF: Ag level was significantly higher in 27 patients with PIH as compared to 20 matched healthy pregnant women (358 +/- 160 u/dl vs. 274 +/- 125 u/dl. p less than 0.05). Moreover, plasma vWF: Ag levels and the ratio of vWF: Ag to factor VIII were found to be linearly correlated to the severity of PIH. In contrast, no significant differences in mean levels of factor VIII and ristocetin cofactor were observed between these groups. Crossed immunoelectrophoresis of vWF revealed a higher incidence of a pre-peak and an increased migration index in the PIH group as compared to the control group (60% vs. 44% and 1.27 +/- 0.26 vs. 1.19 +/- 0.18, p less than 0.01 respectively). Analysis of plasma vWF multimer patterns by 1.4% agarose electrophoresis in 0.1% SDS revealed excessive amounts of large, medium and small size multimers in the PIH patients. Conceivably, the quantitative changes in vWF multimers reflect endothelial injury and may play a role in the microangiopathy observed in PIH.

Immunologic and biochemical characterization of homozygous and heterozygous Glanzmann thrombasthenia in the Iraqi-Jewish and Arab populations of Israel: comparison of techniques for carrier detection.

To define biochemically and immunologically the platelet defect in Iraqi-Jews and Arabs with Glanzmann thrombasthenia in Israel, we tested the platelets of 32 thrombasthenics and 37 obligate carriers from 19 families with affected members. Thrombasthenic platelets were devoid of glycoprotein IIb (GPIIb) as judged by polyacrylamide gel electrophoresis and devoid of the GPIIb/IIIa complex as judged by radio-electroimmunoassay. Binding of a murine monoclonal antibody directed at GPIIb and/or GPIIIa to intact thrombasthenic platelets averaged less than 2% of the control value. Evaluation of the number of molecules of antibody bound per platelet permitted discrimination between controls and obligate carriers with a high degree of accuracy (sensitivity = 91.9%, specificity = 92.3%). Obligate carriers could also be discriminated from controls by determining the ratio of GPIIb to GPIb by polyacrylamide gel electrophoresis and by quantifying the GPIIb/IIIa complex by radio-electroimmunoassay. These studies indicate that the thrombasthenics in Israel have the severe form of the disease (type I) and that the platelets of heterozygotes have significantly reduced amounts of both total and surface-exposed GPIIb and/or GPIIIa.

A relatively high frequency of severe (type III) von Willebrand's disease in Israel.

Sixty patients with von Willebrand's disease belonging to 34 unrelated families were classified into the various types of the disease by analysis of the multimer patterns of von Willebrand factor. Type I disease was observed in 62% of the families, a finding similar to the recently published British and Swedish series of patients. Type III (severe) von Willebrand's disease was observed more frequently in the Israeli group (29%) than in the other two series. Of the 16 patients with type III disease eight were Arabs living in Israel, the West Bank and the Gaza Strip (total population about 1.5 X 10(6). Thus, it appears that the frequency of type III disease is very high among Arabs (5.3/10(6]. Sibship analysis of all families affected by the type III mutant gene was compatible with an autosomal recessive mode of inheritance. An attempt was made to define carriers of type III disease and to distinguish them from type I patients and from healthy subjects. In 15 obligate carriers of type III disease mean levels of factor VIII clotting activity, of von Willebrand factor and of ristocetin cofactor were significantly higher than the corresponding mean values observed in 31 symptomatic and 12 asymptomatic type I patients, and in turn significantly lower than the corresponding mean values observed in 30 healthy subjects. Ristocetin cofactor was the best criterion for discrimination among carriers of type III disease, normal subjects and type I patients.

Factor VII in plasma of women taking oral contraceptives. Lack of cold activation under blood bank conditions.

Factor VII in plasma from about 15 percent of healthy subjects undergoes activation when samples are kept in plastic tubes at 4 degrees C. In women taking oral contraceptives, this phenomenon is observed much more frequently. If this phenomenon occurred under blood bank conditions as well, the transfusion of such plasma from donors taking oral contraceptives to patients afflicted by trauma could enhance thromboembolism. Plasma packs of 72 female donors taking oral contraceptives were separated and stored at 4 degrees C for 24 hours in the blood bank. No significant change in factor VII:C level was observed: The initial level was 110.2 +/- 6.2 U per dl (mean +/- SEM), and the 24-hour level, 97 +/- 3.3 U per dl. Among the 72 donors, 10 were identified as cold activators; their factor VII:C level increased from 85.6 +/- 2.5 U per dl (mean +/- SEM) to 222.0 +/- 7.5 U per dl when their plasma samples were kept in plastic tubes for 24 hours at 4 degrees C. In contrast, the factor VII:C level in the plasma packs kept simultaneously in the blood bank at 4 degrees C was only 101.1 +/- 9.0 U per dl at 24 hours. Thus, it appears that plasma from donors taking oral contraceptives can be used safely even when they are not frozen immediately.

PIP: Factor 7 in plasma from 15% of healthy subjects undergoes activation when samples are kept in plastic tubes at 4 degrees Celsius. In women taking oral contraceptives (OCs), this phenomenon is observed more frequently. If this phenomenon occurred under blood bank conditions as well, the transfusion of such plasma from donors taking OCs to patients afflicted by trauma could enhance thromboembolism. Plasma pavks of 72 female donors taking OCs were separated and stored at 4 degrees Celsius for 24 hours in the blood bank. No significant change in factor VII:C level was observed; the initial level was 110.2 +or- 6.2 U/dl (mean +or- SEM), and the 24 hour level was 97 +or- 3.3 U/dl. Among the 72 donors, 10 were identified as cold activators; their factor VII:C level increased from 85.6 +or- 2.5 U/dl (mean +or- SEM) to 222.0 +or- 7.5 U/dl when their plasma samples were kept in plastic tubes for 24 hours at 4 degrees Celsius. In contrast, the factor VII:C level in the plasma packs kept simultaneously in the blood bank at 4 degrees Celsius was only 101.1 +or- 9.0 U/dl at 24 hours. Thus, it appears that plasma from donors taking OCs can be used safely even when not frozen immediately.