James P. Chen

Hemorrhagic fever virus-induced changes in hemostasis and vascular biology.

Viral hemorrhagic fever (VHF) denotes a virus-induced acute febrile, hemorrhagic disease reported from wide areas of the world. Hemorrhagic fever (HF) viruses are encapsulated, single-stranded RNA viruses that are associated with insect or rodent vectors whose interaction with humans defines the mode of disease transmission. There are 14 HF viruses, which belong to four viral families: Arenaviridae, Bunyaviridae, Filoviridae and Flaviviridae. This review presents, in order, the following aspects of VHF: (1) epidemiology, (2) anomalies of platelets and coagulation factors, (3) vasculopathy, (4) animal models of VHFs, (5) pathogenic mechanisms, and (6) treatment and future studies. HF viruses produce the manifestations of VHFs either by direct effects on cellular functions or by activation of immune and inflammatory pathways. In Lassa fever, Rift Valley fever and Crimean–Congo HF, the main feature of fatal illness appears to be impaired/delayed cellular immunity, which leads to unchecked viremia. However, in HF with renal syndrome and dengue HF, the immune response plays an active role in disease pathogenesis. The interplay of hemostasis, immune response, and inflammation is very complex. Molecular biologic techniques and the use of animal models have helped to unravel some of these interactions.

Acute hypoxemia does not increase blood fibrinolytic activity in man

The influence of acute hypoxemia on blood fibrinolytic activity was investigated in 12 healthy males. Physiologically significant hypoxemia was produced by inspiration of 13% oxygen for 30 min. Six healthy males were exposed to hypoxemia at rest and 6 males to hypoxemia during exhaustive physical exercise on an ergocyclometer. During control experiments both groups of health males inspired 21% oxygen. In 5 patients with manifest respiratory insufficiency the effect of hypoxemia at rest was studied during withdrawal of oxygen treatment for up to 2.5 hours. No increase in fibrinolytic activity (measured with euglobulin clot lysis and fibrin plates) due to hypoxemia was observed either in resting healthy males or in patients. In healthy males the increase in fibrinolytic activity after physical exercise at 13% oxygen was even somewhat lower compared to 21% oxygen. No changes in other hemostatic parameters (activated partial thromboplastin time, factor VIII-related antigen, fibrinogen, plasminogen, alpha-2-antiplasmin, fibrin(ogen) degradation products) that could be attributed to hypoxemia, were observed in any group tested. It was concluded that short-term acute hypoxemia does not increase blood fibrinolytic activity in man.

Monoclonal antibodies from rats immunized with fragment D of human fibrinogen

Abstract Fischer rats were immunized with fragment D (Fg-D) of human fibrinogen (Fg) to obtain antibody specific for neoantigens unique to this molecule. Absorption of serum with whole Fg indicated that some of the antibody produced reacted preferentially with Fg-D. Hybridoma cultures were prepared by fusion of immune rat spleen cells with mouse myeloma P3-X63-Ag8. Monoclonal antibodies obtained from these cultures fell into two classes: (a) Those reacting equally well with Fg and Fg-D. (b) Those reacting preferentially but not absolutely with Fg-D. Antibody from hybridoma 104-14, a member of the first group had an affinity for Fg-D of 1.5 × 10 9 M −1 while antibodies from 106-59 and 106-71 (group 2) demonstrated much lower affinities of 1.0 × 10 7 and 4.7 × 10 6 M −1 , respectively. The cross reactivity of antibodies in the second group indicated that they react with protein conformations that are altered during production of Fg-D from Fg.

Radioimmunoassay of fibrinogen-fibrin degradation products: assay for fragment E-related neoantigen-methodological aspects.

Abstract E fragment and its antigenically-related degradation products of human fibrinogen (Fg) and/or fibrin (Fb) have been measured in both normal and pathological plasmas by a specific radioimmunoassay (RIA) for the neoantigenic expression of E(Eneo) which is engendered after the plasmic cleavage of Fg or Fb. High titer antisera were produced in rabbits against Fg-E (E derived from Fg). The anti-Fg-E sera harvested were absorbed with a cyanogen bromide-activated immunoadsorbent column coupled with intact Fg. The Fg-absorbed antisera were employed to develop a double antibody RIA which detected

Radioimmunoassay of fragment E-related neoantigen: validation studies and clinical application

Summary. Measurement of fibrinogen‐fibrin degradation products (FDP) levels in plasma may provide a direct index of plasmin action, and increased levels of FDP would indicate coagulopathy. We have established an E‐neoantigen radioimmunoassay (Eneo RIA) that can determine normal and pathological plasma levels of E‐related FDP. The assay employs rabbit antiserum produced against fragment E derived from a plasmin digest of fibrinogen and subsequently absorbed with fibrinogen. The absorbed antiserum contains antibodies which are equally reactive with fibrinogen derived E (Fg‐E) and fibrin derived E (Fb‐E) but not with fibrinogen at 1 mg/ml. The Eneo RIA was validated by assay parallelism and by recovery experiments. Plasma Eneo immunoreactivities in 14 normals were 4–22 ng/ml (mean 12.7 ng/ml). Plasma Eneo levels in 23 of 24 patients with neoplastic and haematological diseases were elevated above normal (range 27–2027 ng/ml). Unusually high Eneo values were observed with three patients whose diseases were complicated by either disseminated intravascular coagulation (DIC) or deep vein thrombosis. After heparin therapy, the Eneo level of a patient with chronic DIC declined. A pathological plasma was eluted from a Sephadex G‐200 column and Eneo immunoreactivity was determined on the eluates. The gel filtration pattern of Eneo indicates that E‐related FDP is a family of plasmic fragments derived from crosslinked fibrin.

Unique immunological cross-reactivity between fragments D and/or E of three heterologous mammalian fibrinogens

Abstract Using rabbit antisera directed against human, canine and bovine fibrinogen respectively, the antigenic relationships between human, canine and bovine fibrinogens and their D and E fragments were examined. Most of the cross-reaction between human and canine fibrinogens appears to occur through Fragment E. With respect to the antigenic relationship of human and bovine fibrinogens, the composition of cross-reacting antibodies which react through D and/or E may vary in different antisera. In addition, the human cross-reaction with bovine fibrinogen which involves fewer antigenic determinants than human and canine fibrinogens may also encompass relatively loosely folded polypeptide chains between the D and E cores.

Factors affecting the appearance of a pre-peak during crossed immunoelectrophoresis of factor VIII related antigen

Abstract Conditions were examined which elicit “pre-peak” formation during crossed immunoelectrophoresis (CIEP) of normal, human factor VIII related antigen (FVIII:RAg). Pre-peak artifacts could be induced by sample handling conditions leading to cryoprecipitate, platelets or platelet fragments in the plasma. Under conditions not promoting pre-peak artifacts, only 25% of the hemophilic samples tested showed pre-peaks. No other abnormality tested produced pre-peaks with the exception of samples from recently infused patients. Pre-peaks appear, therefore, to be of limited predictive value.

Decreased antithrombin iii level associated with failure of anticoagulation

A 67-year-old man with adenocarcinoma of the colon had recurrent pulmonary emboli. The patients level of serum antithrombin III was found to be below normal. Heparin therapy led to further reduction, resulting in failure of adequate anticoagulation. The patient subsequently died of massive pulmonary emboli despite heparin therapy. We believe the antithrombin III level should be routinely determined for patients receiving heparin therapy.

Variant von Willebrandʼs Disease and Pregnancy

The clinical course and coagulation profile of a pregnant patient with variant von Willebrands disease were followed from the second trimester through puerperium. The clinical course was characterized by a normal delivery and absence of abnormal bleeding or need for replacement therapy. The coagulation profile demonstrated an increase in factor VIII procoagulant activity, factor-VIII-related antigen, and platelet aggregation activity in response to ristocetin prior to delivery. Postpartum, these factors decreased to prepregnancy values with distinctly different patterns. Factor VIII procoagulant activity continued to rise for 5 days after delivery and then decreased with a half-life of approximately 6 days. Factor-VIII-related antigen began to decrease just prior to delivery, displaying a half-life or approximately 6 days. Ristocetin cofactor activity, however, dropped immediately postpartum and displayed a half-life of approximately 6 hr. The ristocetin cofactor activity was associated with factor-VIII-related antigen, which displayed a significantly smaller molecular weight than does normal factor-VIII-related antigen. Larger aggregates of factor-VIII-related antigen. Larger aggregates of factor-VIII-related antigen did not appear during the pregnancy, and ristocetin cofactor activity could not be demonstrated in fragments of less than 0,8 x 10(6).