A hypofibrinolytic state in overweight patients with cerebral venous thrombosis and isolated intracranial hypertension.

Evidence suggests that isolated intracranial hypertension (iIH) is often associated with cerebral venous thrombosis (CVT). In eight patients referred to our Institution for iIH who were later shown to harbor CVT we have performed a comprehensive coagulation work-up, including genetic tests for inherited predisposition to thrombophilia, to clarify the etiology of sinus venous thrombosis. All subjects were women. All but one were overweight. There were high plasma concentrations of D dimer, thrombin-anti-thrombin complexes or prothrombin fragments 1 and 2, further supporting the neuroimaging diagnosis of CVT. Importantly, seven of eight cases had a raised level of plasminogen activator inhibitor 1, a well known inhibitor of fibrinolysis related to obesity. Tissue plasminogen activator levels were elevated accordingly. Factor V gene mutation was present in one subject, and the 20,210 prothrombin gene mutation was found in another individual. Three patients had elevated plasmatic levels of homocysteine. In conclusion, the present study provides solid evidence that impaired fibrinolysis probably related to overweight, acting in concert with other prothrombotic abnormalities, is involved in the pathogenesis of CVT presenting as iIH.

Increased formation of distinct F2 isoprostanes in hypercholesterolemia.

BACKGROUND: F2 isoprostanes are stable, free radical-catalyzed products of arachidonic acid that reflect lipid peroxidation in vivo. METHODS AND RESULTS: Specific assays were developed by use of mass spectrometry for the F2 isoprostanes iPF2alpha-III and iPF2alpha-VI and arachidonic acid (AA). Urinary excretion of the 2 F2 isoprostanes was significantly increased in hypercholesterolemic patients, whereas substrate AA in urine did not differ between the groups. iPF2alpha-III (pmol/mmol creatinine) was elevated (P<0.0005) in homozygous familial hypercholesterolemic (HFH) patients (85+/-5. 5; n=38) compared with age- and sex-matched normocholesterolemic control subjects (58+/-4.2; n=38), as were levels of iPF2alpha-VI (281+/-22 versus 175+/-13; P<0.0005). Serum cholesterol correlated with urinary iPF2alpha-III (r=0.41; P<0.02) and iPF2alpha-VI (r=0. 39; P<0.03) in HFH patients. Urinary excretion of iPF2alpha-III (81+/-10 versus 59+/-4; P<0.05) and iPF2alpha-VI (195+/-18 versus 149+/-20; P<0.05) was also increased in moderately hypercholesterolemic subjects (n=24) compared with their controls. Urinary excretion of iPF2alpha-III and iPF2alpha-VI was correlated (r=0.57; P<0.0001; n=106). LDL iPF2alpha-III levels (ng/mg arachidonate) were elevated (P<0.01) in HFH patients (0.32+/-0.08) compared with controls (0.09+/-0.02). The concentrations of iPF2-III in LDL and urine were significantly correlated (r=0.42; P<0.05) in HFH patients. CONCLUSIONS: Asymptomatic patients with moderate and severe hypercholesterolemia have evidence of oxidant stress in vivo.

Plasma lipoprotein(a) levels in subjects attending a metabolic ward. Discrimination between individuals with and without a history of ischemic stroke.

In this cross-sectional study we compared the abilities of lipoprotein(a) [Lp(a)], plasminogen activator inhibitor-1 (PAI-1), and tissue plasminogen activator (TPA) to discriminate between individuals with and without a history of stroke from among subjects in a metabolic ward. A total of 210 subjects (108 men and 102 women; mean age, 63.8 years; range, 31 to 86 years) provided plasma and DNA samples for the study. Of these, 51 men and 50 women had a history of ischemic stroke. The 109 subjects without a history of stroke were compared with those with such a history for major risk factors for ischemic events. Mean plasma TPA and PAI-1 levels significantly (P < .001) discriminated among subjects younger than 70 years with a history of stroke. The mean plasma Lp(a) level of stroke subjects (21.9 mg/dL) did not differ significantly from that of control subjects (15.2 mg/dL). However, among individuals < 70 years old, Lp(a) plasma levels > 50 mg/dL were more common among stroke patients (8 with versus 1 without, P < .01 by chi 2 test). A molecular variation in the 5' flanking region of the apo(a) gene that has been related to elevated Lp(a) plasma levels (G/A-914) was not strongly correlated with circulating levels of Lp(a), nor did Lp(a) levels correlate with a polymorphism of the apo(a) gene (G/A-21), which is strongly linked (P < .001) to the G/A-914 variation. In this setting, the relation between Lp(a) and cerebral ischemia appears to be limited to individuals below 70 years with elevated (> 50 mg/dL) plasma levels of the lipoprotein.

Plasma free fatty acid metabolism during storage of platelet concentrates for transfusion.

New containers allow storage of platelet concentrates (PC) at 22 degrees C for up to 7 days, during which glycolytic and oxidative metabolism is vigorous. Recent evidence suggests that 85 percent of adenosine triphosphate regeneration is based on oxidative metabolism and that substrates other than glucose may be used. Because platelets can oxidize free fatty acids (FFA) as a possible source of energy during storage, the authors studied their availability, distribution, and turnover. Plasma FFA concentration was unchanged after 1 day of PC storage but significantly increased on Days 3, 5, and 7. Platelet-free plasma (PFP) stored under the same conditions as PC demonstrated a progressive increase in FFA, suggesting that some of the FFA accumulating in PC were derived from plasma rather than platelets. Indeed, during PC storage, plasma triglycerides decreased significantly, suggesting that they are a possible source of the increased levels of FFA found on Day 3 and thereafter. Thus, PC have a plasma FFA pool available continuously for oxidation during storage. Studies with radiolabeled palmitate suggested that FFA oxidation by platelets occurs during storage. The current findings show that plasma FFA could be a significant substrate for oxidative metabolism during storage of PC and that the oxidized FFA are replenished at least in part from plasma. These results may allow platelet storage to be improved, particularly in synthetic media.

Trial of repeated low-dose aspirin in diabetic angiopathy.

We compared the ability of aspirin to suppress platelet aggregation and thromboxane synthesis in ten normal subjects and ten patients with diabetic angiopathy and high rate of entry of new platelets into the circulation. When single doses of 100 to 1,000 mg aspirin were ingested daily for 1 month, there were time gaps between doses in which platelets from diabetics and normals aggregated and formed thromboxane ex vivo in response to the combination of arachidonic acid plus collagen. Similar gaps were also found for diabetics, but not for normals, following four daily doses (every six hours) of 25 or 100 mg. Our data show that dose schedules of aspirin which may suffice in normals are not effective in patients with diabetic angiopathy, presumably because these patients have a high rate of entry of new platelets into the circulation. We suggest that continual suppression of platelet thromboxane synthesis and aggregation by low-dose, "slow-release" preparations of aspirin would be an ideal long-term approach for the prevention of thrombosis in patients with a high rate of entry of new platelets into the circulation.

Increased fibrinogen binding to platelets from patients with familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is a disease marked by a high incidence of thrombotic episodes and hypersensitivity of the patients' platelets to naturally occurring aggregating agents. Prostaglandin/thromboxane (PG/Tx) formation, adenosine 5'-diphosphate (ADP) secretion, and fibrinogen binding to platelets are all believed to be involved in the mechanisms of platelet aggregation. Therefore, we studied the interrelated roles of these processes in the platelets of nine FH patients and 10 controls. In response to ADP, collagen, or thrombin, FH platelets bound about twice as much 125I-fibrinogen as controls. This ratio did not change after suppression of PG/Tx formation by aspirin. With or without aspirin, FH platelets always aggregated in response to significantly lower concentrations of these agents than did platelets from normal controls. After stimulation with thrombin or collagen, the hyperaggregable platelets from FH patients were shown to bind significantly more fibrinogen than control platelets even when PG/Tx formation was suppressed (aspirin) and secreted ADP was scavenged (apyrase). To determine whether the increased fibrinogen binding observed in FH platelets is due to a qualitative or quantitative abnormality of the platelet receptor, we used a monoclonal antibody (B79.7) that is specific for the receptor. The amount of B79.7 that bound to platelets from control and FH subjects was similar. In addition (as in normal individuals), the antibody inhibited aggregation and fibrinogen binding of FH platelets.

Platelet fibrinogen binding in diabetes mellitus. Differences between binding to platelets from nonretinopathic and retinopathic diabetic patients.

While it is known that platelets from diabetic patients bind more fibrinogen than do platelets from normal subjects, there has been no study comparing this phenomenon in platelets from nonretinopathic and retinopathic patients. We have made such a comparison and have found the following. In agreement with previous reports, platelets from nonretinopathic diabetic patients bind abnormally high amounts of fibrinogen. No differences in the amount of fibrinogen bound to platelets (stimulated by collagen or thrombin) were found when data from nonretinopathic and retinopathic patients were compared. However, while aspirin (an inhibitor of thromboxane synthesis) reduced the abnormally high fibrinogen binding of platelets from nonretinopathic patients to normal control levels, it did not normalize the high fibrinogen binding of platelets from retinopathic diabetic patients. The combination of aspirin plus apyrase (an ADP scavenger) almost suppressed fibrinogen binding and aggregation of platelets from normal or nonretinopathic diabetic subjects, whereas it had a somewhat lesser effect on binding and aggregation of platelets from retinopathic subjects. By using a monoclonal antibody (B59.2) to the platelet receptor for fibrinogen, we determined that this receptor was the same in platelets from normal as well as nonretinopathic diabetic subjects and that this antibody could suppress the binding of fibrinogen and the aggregation of platelets from both types of patients just as it did in platelets from normal subjects. Thus, our data indicate that, while platelets from both retinopathic and nonretinopathic patients are hyperaggregable and show abnormally high binding of fibrinogen, they differ in that these abnormalities can be normalized in platelets from nonretinopathic patients by suppressing prostaglandin/thromboxane formation and scavenging ADP, but not in those from retinopathic patients.

A myeloma paraprotein with specificity for platelet glycoprotein IIIa in a patient with a fatal bleeding disorder.

Impaired platelet aggregation, normal shape change, and agglutination and normal ATP secretion and thromboxane synthesis in response to high concentrations of thrombin or arachidonic acid were found in a patient with multiple myeloma and hemorrhagic tendency. The purified IgG1 kappa or its F(ab1)2 fragments induced similar changes when added in vitro to platelet-rich plasma from normal subjects. In addition, the paraprotein inhibited adhesion to glass microbeads, fibrin clot retraction, and binding of radiolabeled fibrinogen or von Willebrand factor to platelets exposed to thrombin or arachidonic acid without affecting intraplatelet levels of cAMP. The radiolabeled para-protein bound to an average of 35,000 sites on normal platelets but it bound to less than 2,000 sites on the platelets from a patient with Glanzmann's thrombasthenia. Immunoprecipitation studies showed that the platelet antigen identified by the paraprotein was the glycoprotein IIIa. Furthermore, binding of radiolabeled prostaglandin E1 (PGE1) to resting platelets as well as binding of von Willebrand factor to platelets stimulated with ristocetin were entirely normal in the presence of patient's inhibitor. These studies indicate that bleeding occurring in dysproteinemia may be the result of a specific interaction of monoclonal paraproteins with platelets. In addition, our data support the concept that the interaction of fibrinogen and/or von Willebrand factor with the platelet glycoprotein IIb-IIIa complex is essential for effective hemostasis.

Increased binding of fibrinogen to platelets in diabetes: the role of prostaglandins and thromboxane.

Previous studies suggested a role for prostaglandins or thromboxane A2, or both in the exposure of fibrinogen receptors on normal platelets in response to several aggregating agents. Platelets from diabetics are known to be more sensitive to aggregating agents and to produce more prostaglandins and thromboxane than platelets from normal subjects. We compared fibrinogen binding to platelets from diabetic subjects with binding to platelets from normal subjects and determined whether aspirin (which inhibits the formation of prostaglandins and thromboxane) would inhibit the binding of fibrinogen to platelets from diabetic subjects and whether this correlated with its effects on platelet aggregation. We found the following: Aspirin suppressed thromboxane formation and rendered the platelets less sensitive to the induction of aggregation by adenosine diphosphate (ADP) or collagen. The amount of U-46619 [( 15s]-hydroxy-11-alpha, 9-alpha [epoxy-methano]-prosta[5Z,13E]-dienoic acid, a stable analog of prostaglandin endoperoxide/thromboxane A2) necessary to induce aggregation, was similar in normal and diabetic subjects and was unchanged after ingestion of aspirin. Binding of 125I-fibrinogen following stimulation of platelets by ADP or collagen was greater in diabetic (because more binding sites were exposed) than in normal subjects. However, following stimulation by U-46619, binding was similar in diabetic and normal subjects. Aspirin caused a reduction in the exposure of binding sites on both platelets from diabetic and normal subjects, so that (in this respect) platelets from diabetic subjects became more like those from normal subjects. Effects of the monoclonal antibody B59.2, which is specific for the platelet glycoprotein IIb-IIIa complex (the presumed receptor for fibrinogen on the platelet surface) were also studied. The amount of this antibody that bound to platelets was the same for normal and diabetic subjects both before and after aspirin and with or without stimulation by ADP or collagen. In addition, B59.2 inhibited aggregation and fibrinogen binding in both platelets from diabetic and normal subjects. The combined data suggest that the glycoprotein IIb-IIIa complex of platelets from diabetic subjects is similar to that of platelets from normal subjects and that the increased fibrinogen binding and aggregation of platelets from diabetic subjects in response to ADP or collagen is mediated by increased formation of prostaglandin endoperoxide or thromboxane A2, or both.

Mouse antithrombotic assay: a simple method for the evaluation of antithrombotic agents in vivo. Potentiation of antithrombotic activity by ethyl alcohol.

We present a simple method for screening antithrombotic agents. When male Swiss-Webster mice (25-34 g; N = 143) were given an i.v. injection of 0.1 ml of a mixture of collagen (dose, 15 micrograms/mouse) and epinephrine (dose, 1.8 micrograms/mouse), 94% died within 5 min or remained paralyzed for more than 15 min. Examples of the use of the system for the study of antithrombotic agents are given. Solutions of agents were administered to the animals i.p. about 1 hr before the thrombotic challenge. Aspirin (20 mg/kg), OKY-1581 (30 mg/kg) and ethanol (2 gm/kg), administered as single agents in aqueous medium, protected (P less than .01, chi 2 test) 40, 50 and 35% of the animals, respectively. Heparin (150 U/kg) was ineffective. Combinations of ethanol with indomethacin or indobufen provided complete protection, whereas ethanol plus aspirin protected 84% of the animals and ethanol plus OKY-1581 protected 70% of the animals. Dipyridamole alone (3 mg/kg), dipyridamole (1.65 mg/kg) plus ethanol or dipyridamole (1.65 mg/kg) plus aspirin were ineffective. The method appears well suited for screening potential antithrombotic agents which act primarily against platelet thromboembolism.