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The protein C (PC) anticoagulant pathway plays a crucial role in the regulation of fibrin formation via proteolytic degradation of the procoagulant cofactors factor Va and VIIIa by activated PC (APC). PC circulates in plasma as a zymogen, which is activated, on the surface of endothelial cells by the thrombin-thrombomodulin complex. Another endothelial cell-specific protein, the endothelial cell PC/APC receptor (EPCR), binds PC on the endothelial cell surface and further enhances the rate of PC activation. Normal APC generation depends on the precise assemblage, on the surface of endothelial cells, of at least four proteins: thrombin, thrombomodulin (TM), PC and EPCR. Therefore, any change in the efficiency of this assemblage may cause reduced APC generation and an increase in the risk of thrombosis. In the last years, several reports have suggested the association between mutations in TM and EPCR genes and venous and arterial thrombosis. Surprisingly, no studies have been reported linking mutations with levels of circulating APC, the final product of the interaction between thrombin, TM, PC and EPCR. Here, we describe the previously reported mutations in the TM and EPCR genes, and present the design and evaluation of a new strategy to investigate TM, EPCR, PC and prothrombin gene mutations in arterial and venous thrombosis.

This study examines the evolution of the thrombotic activity in patients with myocardial infarction (MI) treated with aspirin (200 mg/day) for 2 years after MI. Plasma samples of 10 patients were collected at 7, 30, 60, 90, 120, 150, 180, 360 and 720 days. In all the samples we measured fibrinogen (Fg), high molecular weight Fg (HMW-Fg), fibrinopeptide A (FPA), prothrombin fragment 1+2 (F1+2), beta-thromboglobulin (beta-TG), von Willebrand factor (vWF), tissue factor (TF) and TF pathway inhibitor (TFPI). The plasma Fg, HMW-Fg, FPA, F1+2, beta-TG and vWF levels were significantly elevated in the patients at the beginning of the study as compared to the normal group. The 95% confidence intervals were Fg 277-333 mg/dl, HMW-Fg 200-244 mg/dl, FPA 5.3-16.5 ng/ml, F1+2 1.4-1.8 nmol/l, beta-TG 110-118 IU/ml and vWF 139-195%. At thirty days Fg and HMW-Fg returned to normal levels, whereas the increase in FPA and F1+2 levels persisted throughout the study. At 120 and 150 days, respectively, beta-TG and vWF returned to normal levels. The increase in thrombin generation and activity pointed to a persistent hypercoagulable state 2 years after MI. Plasma levels of TF and TFPI showed no statistically significant variations with respect to the normal values over the 2-year period studied. In conclusion, these results suggest a persistent generation and activity of thrombin and cellular activation in these patients after MI.

This study compares the extent of inhibition of thrombin generation and activity achieved in patients with acute myocardial infarction receiving fibrinolytic treatment (streptokinase SK, or rt-PA) and concomitant intravenous heparin treatment adjusted to the patients' weight with that achieved with the same heparin regimen but without fibrinolytic therapy. The study involved 90 patients, grouped according to their treatment: SK+heparin; rt-PA+heparin, and heparin without thrombolytic agents. Prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), fibrinopeptide A (FPA) and activated partial thromboplastin time were measured. Patients treated with SK+heparin or rt-PA+heparin and higher F1+2 plasma levels than the patients treated with heparin alone at 12, 48 and 72 h in the case of SK+heparin, and at 12, 24, 48 and 72 h in that of rt-PA+heparin. Compared to baseline, the plasma levels of FPA were decreased in the three treatment groups at 24-48 h. There were no significant changes in TAT and FPA plasma levels among the three treatment groups at the different times. After thrombolytic therapy with both SK and rt-PA, there was an increase in thrombin generation, although high-dose intravenous heparin inhibited the different increases in thrombin associated with the thrombolytic agents to the same extent.

In order to find out whether hemorheological alterations precede the atherosclerotic lesions and could constitute an atherothrombotic risk factor, we studied the hemorheological profile in 150 healthy normotensive offspring whose parents were diagnosed as having essential hypertension WHO I-II grade and in another group of 40 children with familial hypercholesterolemia (FH) but without vascular atherosclerosis lesions, whose parents also suffer from FH. In offspring of hypertensive individuals, a significant increase in the fibrinogen level with respect to the control group was found both in males and females. In addition, only the female offspring showed a higher leucocyte count. FH children showed increased erythrocyte aggregation and increased plasma viscosity with respect to the control group. The fact that the rheological alterations appear prior to the development of the vascular lesion suggests that they could play a role in the pathogenesis of the atherosclerotic process.

Eighteen patients with familial hypercholesterolemia treated with lovastatin (40 mg/day) for three months were studied to find out whether the drug induces modifications in the lipid composition of the erythrocyte membrane and whether these induce changes in the rheological behaviour of the red blood cell. Our results show that the changes observed in plasma lipids correlate with a significant decrease in the cholesterol/phospholipid ratio of the red blood cell membrane. These changes in the lipid composition of the cell are statistically related to a decrease in the erythrocyte aggregability and an improvement in blood filterability probably due to an increase in the red blood cell deformability.

The binding of unstimulated and thrombin-stimulated platelets was studied with immobilized fibrinogen and fibrin on polystyrene. The amount of fibrinogen bound to the polystyrene support was 2 micrograms/tube, which represents 2.35 micrograms/cm2. Immobilized fibrin was obtained by adding thrombin (5 nM) to immobilized fibrinogen. The number of unstimulated 111In-platelets bound to immobilized fibrinogen and fibrin was similar (3.2 +/- 0.3 x 10(6) and 3.1 +/- 0.4 x 10(6) platelets/micrograms fibrin(ogen), respectively). The platelet binding steadily increased. In the first 2 min, the binding rate was 0.23 x 10(6) platelets/micrograms fibrinogen/min. The binding rate then increased rapidly and saturation was reached at 10 min. The extent of the adhesion of resting platelets to immobilized fibrinogen is about one half that of the same platelets stimulated with thrombin. In thrombin-stimulated 111In-platelets, the binding to immobilized fibrinogen and fibrin is time dependent, and saturation is reached at 5 min. The early rate of thrombin-stimulated platelet binding to fibrinogen is about twice that of binding to fibrin (1.25 and 0.74 x 10(6) platelets/micrograms fibrin(ogen)/min, respectively). In saturation conditions, 1 microgram fibrinogen binds 5.7 +/- 0.6 x 10(6) thrombin-stimulated platelets and 1 microgram fibrin binds 4.6 +/- 0.5 x 10(6) thrombin-stimulated platelets. Our results indicate that the rate of platelet aggregation is faster than fibrin formation, and the rate of fibrinogen-platelet binding is faster than that of fibrin-platelet binding. Therefore, after thrombin stimulation, the binding of platelets to fibrin must be secondary to the binding of platelets to fibrinogen.

Patients with ischaemic heart disease have high levels of plasminogen activator inhibitor type 1 (PAI-1). These levels can constitute an independent thrombotic risk factor. In addition, PAI-1 synthesis is greater in the atherosclerotic vessels than in the normal vessels and this can also constitute an atherogenic risk factor. There is a good correlation between insulinaemia and PAI-1, but it has not been demonstrated that the circulating level of PAI-1 depends exclusively on insulinaemia. The increased levels of PAI-1 correlate with other coronary risk factors, especially hypertriglyceridaemia, but high PAI-1 levels are also found in normolipaemic patients. Vascular synthesis of PAI-1 can be promoted by high levels of circulating lipoprotein a. Finally, an exercise programme can increase the fibrinolytic activity of heart disease patients towards normal.

Several parameters of fibrinolytic and protein C pathways were evaluated in three groups of patients with high (HR), moderate (MR) and low (LR) postoperative thrombotic risk undergoing major gynaecological surgery. The HR and MR groups were subjected to low molecular weight heparin (LMW) prophylaxis. A significant increase in plasminogen activator inhibitor type 1 (PAI-1) antigen and activity levels was observed in the HR patient group in comparison with the MR and LR groups in the preoperative and early postoperative period. In all the groups studied, the maximum increase in the levels of PAI-1 was seen on day 1 after surgery. However, the D-dimeric levels reached the highest level on day 7. A significant increase in activated protein C:alpha 1 antitrypsin (APC:alpha 1AT) complex levels was observed in the HR group in comparison with the LR group, and a strong decrease in protein C inhibitor in the early postoperative period was detected in all the groups. In spite of heparin prophylaxis, 2 HR patients were diagnosed as deep vein thrombosis (DVT) during the postoperative period. Both patients showed pre-operative levels of PAI-1 antigen or activity and APC:alpha 1AT complexes above the mean + 1 SD of the pre-operative levels in the HR group. In conclusion, in HR patients a hypofibrinolytic and hypercoagulable state was detected in the pre-operative and early postoperative periods. The prophylactic LMW heparin dose used in the present report (20 mg/day x 7) was insufficient to prevent DVT in the HR group. At present our HR patients are given higher doses of LMW heparin (40 mg/day x 7).

Several fibrinolytic parameters were determined in plasma and amniotic fluid from normotensive pregnancies complicated by intrauterine fetal growth retardation (IUGR) and severe preeclamptic (PE) patients with IUGR and compared with data from normal pregnancies. A significant decrease in plasminogen activator type 2 (PAI-2) and urokinase levels in plasma and amniotic fluid was observed in IUGR groups in comparison with normal pregnancy. No significant differences were observed between the control and IUGR groups in relation to the other fibrinolytic parameters, except for plasma PAI type 1 and tissue-type plasminogen activator levels, which were significantly increased in the PE group. A significant positive correlation was observed between birth weight and PAI-2 levels in both plasma and amniotic fluid, but the plasma PAI-2 levels showed a higher correlation. In conclusion, these results suggest that the PAI-2 level measured in plasma is a more adequate marker of placental function than the PAI-2 level measured in amniotic fluid.

Plasma beta-thromboglobulin (beta TG), platelet procoagulant activity (PPA) and malondialdehyde were evaluated in a control group and in 59 patients with chronic coronary heart disease (CHD) undergoing long-term anticoagulant therapy (ACT) with acenocoumarol, and within 2 months after its termination. The patients were clinically stabilized after more than 1 year of an acute myocardial infarction. An increase in beta TG and PPA was found in the patients, both with or without ACT, when compared to the control group. In addition, PPA was found to be higher in older and hypercholesterolemic patients on ACT. After ACT suppression, PPA activity increased significantly, particularly in younger and normocholesterolemic patients. The results of the present study suggest that CHD patients on ACT have some platelet hyperactivity and that the termination of ACT induces an increase in platelet function, particularly in PPA.

Some properties of fibrinogen from 30 newborn and 30 normal adult plasmas obtained under the same conditions have been studied. The results show that the high-molecular weight fibrinogen (HMW-Fg) was decreased by nearly 25% in newborn plasma as compared to normal adult values. The kinetics of fibrin gel formation was decreased in newborn as compared with normal adult plasmas (gelation rates 2.2 +/- 1 vs. 5.6 +/- 0.5 x 10(-4) OD/s, lag time 128 +/- 25 vs. 72 +/- 3 s). The release rates of fibrinopeptide A (FPAp +/- FPA) and fibrinopeptide B (FPB) were decreased in newborn fibrinogen (FPAp + FPA 10.8 and FPB 1.2 x 10(-3) s-1) as compared to normal adult fibrinogen (FPAp + FPA 11.8 and FPB 1.7 x 10(-3) s-1). Analysis by SDS-PAGE of the reduced, highly cross-linked fibrin from the newborns showed that only 23% of the alpha-chain participates in the formation of alpha-chain polymers. The results suggest that the retarded release rate of fibrinopeptides and the decrease in the HMW fibrinogen concentration are causes of the prolonged kinetics of fibrin gel formation in newborns.

Rabbit abdominal aortas and human umbilical arteries are currently used as substrata for the study of platelet adhesion and aggregate formation under flow conditions. Using immunohistochemical and ultrastructural methods, we have analyzed both vessel surfaces. The reactivity towards platelets of the subendothelium (SE) exposed on these vessels after mechanical or enzymatic digestion (alpha-chymotrypsin) was morphometrically quantified and the nature of the interaction studied in the electron microscope. After mechanical damage, the ultrastructural study of rabbit aortas showed a clearly defined internal elastic lamina (IEL). In contrast, umbilical vessels lacked a consistent IEL and masses of amorphous material often located deeper in the media were the main constitutents of the SE. Immunohistochemical labeling of the von Willebrand factor bound to both types of vessel differed considerably. Quantification of platelet interactions after perfusion of citrated blood showed qualitative differences between mechanically damaged rabbit or human vessels. Enzymatic digestion produced a more thrombogenic surface on rabbit aortas (p less than 0.01 vs. nondigested), but decreased their reactivity towards platelets on umbilical arteries (p less than 0.01 vs. nondigested). The ultrastructural study of the interacting platelets revealed that aggregates, when present, were found on the extracellular matrix underlying endothelial cells of rabbit aortas, but interacting with fibrillar structures probably derived from cell elements of the media in the case of umbilical arteries. These findings indicate that rabbit aortas and umbilical arteries possess structural characteristics that result in different thrombogenic properties with respect to circulating platelets.

Various parameters of the haemostatic and, more importantly, of the fibrinolytic systems were examined in 5 patients with severe pre-eclampsia/eclampsia. All 5 showed signs of haemolysis, elevated liver enzymes and low platelet count (HELLP syndrome). A significant decrease in platelet count (46.4 X 10(9)/litre) and an increase in beta-thromboglobulin (137 ng/ml) were observed in the HELPP patients in comparison with normal pregnant women (267 X 10(9)/litre and 49.3 ng/ml, respectively). No significant differences in fibrinogen levels, activated partial thromboplastin time, prothrombin time and fibrin(ogen) degradation products between the HELLP patient group and the normal pregnant group were observed. However, the antigenic levels of tissue-type plasminogen activator and type 1 plasminogen activator inhibitor were significantly higher in the HELLP pre-eclamptic women than in gestational age-matched controls. In contrast, the antigen levels of type 2 plasminogen activator inhibitor were significantly lower in the patients. These results indicate that platelet activation and alterations in plasminogen activator inhibitors type 1 and type 2 may be involved in the pathophysiological pathway of this syndrome.

A report on a new case of Pelger-Huët (PH) leukocyte anomaly in the homozygous form in an 18-month-old girl is presented. Clinically, the proband shows no special symptoms dependent on the leukocyte anomaly, but polydactyly affects both hands (six fingers on one hand) and feet (six toes on both feet). Her pedigree reveals the existence of three couples in which both husband and wife are heterozygous carriers of the PH anomaly. Study of the family lineage suggests that transmission of the leukocyte anomaly is prevailingly dominant in nature with complete penetration and variable expression.

Hemostatic parameters, especially soluble fibrin monomer complexes, have been studied in 114 patients with intrauterine fetal death. The patients were classified into three groups according to the duration of fetal retention. A correlation was observed between a longer duration of fetal retention and the hemostatic alteration. Moreover, in the groups of patients in which this hemostatic disorder was more evident, an improvement in these alterations was detected after fetal evacuation. A hemorrhagic picture with consumption of coagulation factors and cross-linked fibrin oligomers was detected after uterine evacuation in only 1 case and required an emergency hysterectomy and the administration of blood and plasma. In the rest of the patients, fetal evacuation was sufficient to normalize the parameters and no hemorrhagic complications were observed. The favorable evolution and minimal hemostatic alterations in the majority of the patients with intrauterine fetal death, when early obstetric management was established, are discussed.

The incubation of human fibrinogen with plasmin gives rise to an early degradation product with a molecular weight of 63,000, calculated by polyacrylamide-gel electrophoresis sodium dodecyl sulfate (PAGE-SDS); this product is resistant to the action of the plasmin for short incubation times and represents 1 +/- 0.2% of the original quantity of fibrinogen. The fragment was isolated from the incubated mixture by gel filtration and has a no-identity reaction with fibrinogen fragment D against fibrinogen fragments D antiserum. The reduction gives rise to three polypeptidic chains with molecular weights of 36,000, 17,000 and 10,000 (calculated by PAGE-SDS). Study of the carbohydrate content of these polypeptidic chains from the reduced 63,000 MW fragment indicates that there is only one electrophoretic region which contains periodic acid-Schiff (PAS) positive material. As the 63,000 MW fragment has a reaction against fibrinogen fragment D antiserum and also contains PAS positive material, according to Pepper and co-workers, it can be taken to arise from the HOOC-terminal region of fibrinogen.

The effects of continuous intravenous perfusion of somatostatin on the incidence of ulcers and the histamine and serotonin gastric mucosal content in rats subjected to 12 h of restraint were studied. 51 sets of Sprague-Dawley male rats (102 animals) were divided at random into two groups. Those in group I were immobilized and treated with an intravenous infusion of isotonic saline solution. Animals in group II were also immobilized and given intravenous cyclic somatostatin dissolved in isotonic saline solution; the initial dose was 25 microgram and a continuous infusion of 2.5 microgram/kg/h was administered thereafter. A decrease in the ulcer index was observed in the rats perfused with somatostatin (p less than 0.01) as well as a decrease in the histamine (p less than 0.001) and serotonin (p less than 0.01) gastric mucosal contents. These effects of somatostatin on restraint-induced stress ulcer in rats are probably to be explained by microcirculatory changes in the gastric mucosa determined by the vasoactive amines (histamine and serotonin). They constitute an important factor in the pathogenesis of these lesions.

A 27-year-old pregnant woman with severe congenital hypofibrinogenemia was studied from the 18th to the 40th week of pregnancy, after which she had a normal delivery. The results of the quantitative and qualitative fibrinogen studies done on the patient made it possible to rule out associated dysfibrinogenemia. No variations in the concentration and function of fibrinogen were observed during gestation. The only treatment given was a transfusion of fresh plasma prior to delivery. The importance of fibrinogen in maintaining normal placental insertion and the obstetric management of predelivery and delivery are discussed.

The adhesiveness of platelets to collagen and glass was studies ex vivo in groups of patients with ischemic heart disease (IHD) and acute myocardial infarction (AMI). Adhesiveness was increased to glass and normal collagen in both groups of patients. No relation was found between reinfarction rate and adhesiveness in the acute stage of AMI. Electrophoretic mobility of platelets was the same in the AMI patients and a normal control group. It suggested that the increased 'retention' of platelets in IHD patients might be due more to platelet hyperaggregation or to hypersensitivity to platelet aggregating agents than to altered adhesiveness.

A method for the evaluation in vitro of platelet-collagen adhesiveness has been developed. This method is based on counting platelets both before and after their contact with an insoluble collagen (IC) suspension. The results are expressed as adhesiveness index (AI). A number of variables which could influence the test has been evaluated and standardized in a way suitable for the routinary use of the technique described.

A family with inherited factor VII deficiency is described. The propositus is a 9-year-old girl with chronic haemorrhagic history of epistaxis and bleeding after tooth extractions. Her factor VII coagulant activity was less than 3% using rabbit thromboplastin. She had a factor VII antigen level of 50%. Both parents were heterozygous for factor VII deficiency. The father had procoagulant factor VII (VII-C) of 44% and factor VII antigen (VII-Ag) of 74%, and the mother had 54% of factor VII-C and 85% of VII-Ag. Her only brother had normal levels of factor VII-C (100%). Additionally, some abnormalities in the fibrinolytic system were detected both in the propositus and her brother with shortened euglobulin lysis times and increased functional levels of plasminogen activator. To our knowledge, the clinical association of inherited factor VII deficiency and familial fibrinolytic disturbances has not been described so far.

We have studied the clinical efficacy and pharmacokinetics of cefuroxime in 3 children aged 5, 7 and 10 months who were suffering from meningitis due to Haemophilus influenzae group b (beta-lactamase producers). The MICs of chloramphenicol against these three beta-lactamase-producing H. influenzae isolates were 8, 3.1 and 16 micrograms/ml, and those of cefuroxime were 0.25, 0.5 and 0.12 microgram/ml, respectively. The dosage of cefuroxime was 100 mg/kg/day divided in four 6-hourly intravenous doses for 15 days, and 10 mg given intrathecally every 48 h. Blood and spinal fluid levels were determined by microbiological assay. All the 3 children had excellent clinical and bacteriological responses with negative spinal fluid cultures after 48 h of treatment.

Studies of the turbidity profiles of diluted (1/55, v/v) normal plasma, thrombin activity free serum plus commercial fibrinogen, and 0.15 M NaCl, pH 7.4, plus commercial fibrinogen, activated by thrombin or reptilase and measured at 350 nm, have shown that the latency time (LT) hardly varies for the fibrinogen concentration within limits of 0.03-0.15 mg/ml; however, it does vary for the thrombin concentration. The rate of gelation (RG) varies linearly with the fibrinogen (FG) concentration, conforming to the equation RG = 0.027 (FG)1.8; it hardly varies for thrombin concentrations greater than 0.50 NIH U/ml. On the other hand, RG values obtained for 0.46 NIH U/ml of thrombin or 0.92 BU/ml of reptilase show no significant differences. The variation in LT for the thrombin or reptilase concentration allows the rate of activation to be estimated, giving values of 5.9 X 10(-12) and 3.2 X 10(-12) mol/U/s, respectively, for a fibrinogen concentration in plasma of 1.1 X 10(-10) mol/ml. The mean value estimated for the ratio LT/FG in normal plasma is 35.76 +/- 18.3 and 85.62 +/- 18.3 s mg-1 ml for activation by thrombin and reptilase, respectively. We have studied in normal plasma the parameters that define the gelation of fibrin as measured by turbidity curves and their variation according to the fibrinogen concentration. This permits us to establish the kinetics of fibrin gel formation and normal range values.