Clinical and Applied Thrombosis/Hemostasis

The correct approach to the management of the asymptomatic carrier with a recognized inherited thrombophilic disorder is uncertain because reliable in formation of the risk of spontaneous (unprovoked) throm bosis in these disorders is not available. To determine the best available estimate of the annual incidence of spon taneous thrombosis in asymptomatic carriers of disorders that have been linked to familial thrombophilia, we per formed a literature review. Using Medline search from 1965 to 1992, supplemented by manual searches, we re trieved all articles that presented data on antithrombin III, protein C, protein S, dysfibrinogenemia, plasmino gen, histidine-rich glycoprotein, heparin cofactor II, and fibrinolysis in relation to thrombosis. Publications were included in the analysis if they (1) reported one or more probands with thrombotic disease and a heterozygous biochemical abnormality of the hemostatic system, (2) assessed the presence of this abnormality in family mem bers independent of the presence or absence of a history of thrombotic disease, and (3) assessed the presence of a history of thrombotic disease in all available family mem bers. The biochemical status and clinical details of all family members reported were extracted from each eligi ble article. For each abnormality the odds ratio for throm bosis was compared in family members with and without the biochemical abnormality. If applicable, thrombosis- free survival and age-specific incidences of thrombosis were calculated. The thrombotic episodes were classified as spontaneous or secondary to a recognized risk factor, and the proportion of spontaneous episodes was calcu lated. The influence of diagnostic suspicion bias in symp tomatic patients with a family history of thrombosis was reduced by recalculating the absolute incidence of throm bosis from the odds ratio after adjusting the incidence of venous thrombosis in nonaffected family members to that observed in the general population. Statistically signifi cant associations between the presence of a biochemical abnormality and a history of venous thrombosis were found for antithrombin III deficiency types 1 and 2a and 2b, protein C deficiency type 1, and protein S deficiency type I. Dysfibronogenemia was statistically significantly associated with venous as well as arterial thrombosis. Thirty-five to 67% of the events were classified as being provoked, as they occurred following exposure to a rec ognized risk factor for thrombosis. The recalculated an nual incidence of spontaneous thrombosis was 0.6 to 1.6%/year. It is concluded that this relatively low inci dence does not warrant life-long continuous use of anti coagulant prophylaxis since the reported risk of major and fatal bleeding associated with the use of oral antico agulants is 2-3 and 0.4%/year, respectively.

A method is presented using a new reagent containing propylgallate for the quantitative determina tion of lupus anticoagulant. The amount of an optimized phospholipid standard required by the clotting reaction was found to be 32-50 μg/ml at a 95% confidence level, with a mean of 41 μg/ml. This method eliminates the ef fect of heparin therapy, coumadin therapy, factor-VIII inhibitor, factor-IX inhibitor, and single-factor deficien cies that afflict presently used lupus anticoagulant screen ing and confirmatory procedures. Using this method, it should be possible to detect lupus anticoagulant in pa tients at a much lower level and follow the effect of ther apy on lupus anticoagulant.

A new in vitro system for the evaluation of platelet function, PFA-100 (currently under evaluation) is characterized. The system monitors platelet aggrega tion on a collagen-epinephrine-coated membrane as whole blood sample is aspirated under controlled flow conditions through a microscopic aperture cut into the membrane. The time required for the platelet plug to oc clude the aperture (closure time) is indicative of the plate let function in the sample. Incubation of normal blood samples with antibodies to glycoprotein (GP) Ib, GPIIb- IIIa, von Willebrand factor, or with the peptide Arg-Gly- Asp-Ser, resulted in a concentration-dependent prolonga tion in closure time. An anti-fibrinogen antibody did not impact the closure time. Closure time was also prolonged when the hematocrit or the platelet count was lowered to pathological values. The study indicates that PFA-100 could detect defects in platelet adhesion or aggregation. The simplicity of the test makes PFA-100 unique for rapid screening of a variety of platelet dysfunction.

Plasma samples from 10 healthy persons and 10 patients with acute-phase reaction were heparinized in vitro to obtain 0.00-0.70 U/ml. The activated partial thromboplastin time (aPTT) was then determined, using an optical method (Automated Coagulation Laboratory) and four reagents (actin, Cephotest, Platelin, and Throm bosil). The heparin sensitivity showed variation between individuals and was lower in acute-phase plasma than in normal plasma. There was also a marked difference in heparin sensitivity among the different reagents; actin was the least sensitive reagent, while Platelin was the most sensitive reagent in normal plasma and Thrombosil the most heparin-sensitive reagent in acute-phase plasma. Delay in testing prolonged the aPTT values in both acute- phase and normal heparinized plasma. With actin and Ce photest, a delay of 90 min at 22°C resulted in 30-50% prolongation of the aPTT. A delay of 150 min caused a prolongation of 75-110% with actin. Cephotest, Platelin, and Thrombosil were less prolonged. Ex vivo samples from heparinized plasma showed similar degrees of pro longation. Storage at 4°C resulted in less prolongation. Assuming a therapeutic range of 0.35-0.70 U/ml of hep arin, the therapeutic aPTT ratio ranges in heparinized, acute-phase plasma were 1.5-3.0 for actin and 2.5-4.5 for Cephotest, Platelin, and Thrombosil. These results un derscore certain limitations in monitoring heparin therapy with the aPTT system. Unless the assay is performed within 30 min after sampling, unduly prolonged aPTT val ues will be recorded. This may lead to underdosing of the patient.

Efegatran (LY294468), a tripeptide arginal in hibitor of the catalytic site of thrombin, is being devel oped as a parenteral anticoagulant for the treatment of acute coronary syndromes. Efegatran was studied in a canine model of coronary artery thrombosis to determine its ability to prevent thrombus formation and as an ad junctive anticoagulant to thrombolysis, in phase I clinical studies, and phase II clinical studies in unstable angina. In the preclinical in vivo studies in dogs, efegatran pro duced a dose-dependent increase in clotting times and demonstrated a selectivity for thrombin time (TT) changes. The activated partial thromboplastin time (APTT)-TT ratio (that is, based on the dose to double each clotting time) determined in dogs from ex vivo blood samples was 8: 1. This observation was similar to that obtained during the phase I studies in normal volunteers where the APTT-TT ratio was 12:1. The canine and hu man clotting systems responded similarly at doses of efe gatran where comparisons could be made (0.25-1.0 mg/ kg/h). The kinetics of the anticoagulant activity of efega tran in dogs and humans were linear and nonsaturable over the dose ranges studied. Efegatran was also found to be an effective adjunctive anticoagulant during streptoki nase-induced thrombolysis in dogs, preventing reocclu sion without increasing bleeding risk. The novel an tithrombin, efegatran, has demonstrated dose-dependent and safe anticoagulation in animal and human studies. Efegatran is presently undergoing phase II clinical studies in unstable angina and acute myocardial infarction pa tients.

Protein S can be determined by functional or immunological assays. Electroimmunodiffusion (EID) or enzyme immunoassays (enzyme-linked immunosorbent assay; ELISA) are the commonly employed techniques for measuring protein S and C4b-binding protein (C4b- BP) immunologically. Procedures for these assays are time-consuming and labor-intensive. The introduction of microlatex immunoassays (LIATEST system; Diagnos tica Stago, Asnieres-Sur-Seine, France) has provided an alternative for rapid and reliable immunological determi nation. We have placed the microlatex immunoassay for total and free protein S (TPS, FPS) and C4b-BP, using the light-scattering mode, on the Automated Coagulation Laboratory (ACL) 300 Plus (Instrumentation Laboratory, Lexington, MA, U.S.A.). We also placed a functional activity assay of protein S (STACLOT protein S; Amer ican Bioproducts, Parsippany, NJ, U.S.A.) on the ACL 300 Plus. The performance characteristics for the assays yielded a within-run coefficient of variance (CV) of 2.5- 4.6% (n = 13) for TPS, 4.0-4.8% (n = 13) for FPS, 1.9- 3.0% (n = 11) for C4b-BP, and 2.3-5.9% for protein S activity. The interrun CV was 2.1-5.7% (n = 24), 3.7- 7.0% (n = 12), 2.6-7.0% (n = 16), and 4.0-8.4% (n = 27), respectively. Analytical recovery was 94-109, 97-100, 91-103, and 99-103%, respectively. The normal ranges determined on plasmas from 30 healthy individuals were 113 ± 37 (mean ± 2 SD) for TPS, 106 ± 35 for FSP, 111 ± 22 for C4b-BP, and 107 ± 34 for protein S activity. The results for the microlatex immunoassay and either the EID or the ELISA methods showed excellent correla tions for FPS and C4b-BP; the correlations between LIATEST and either EID or ELISA for TPS were also relatively high. The functional activity of protein S cor related well with FPS. Microlatex immunoassays, using the light-scattering mode for TPS, FPS, or C4b-BP, and the functional assay of protein S can be adapted on the ACL 300 Plus system with a high accuracy and reproduc ibility and with considerable time saving.

The hepatosplenic form of schistosomiasis mansoni is associated with impairment of blood coagula tion, a result of reduced synthesis of clotting factors and inhibitors, enhanced fibrinolytic activity, or "consump tion" of plasma clotting proteins. The pathogenesis of the latter mechanism has not been elucidated. Schistosoma mansoni (Sm) has a long lifespan as an intravascular fluke, probably a consequence of its ability to evade host immunologic defense mechanisms. In order to determine if the parasite is able to disturb hemostasis in vivo, we studied 21 non-alcoholics with normal body mass index, and HBsAg, anti-HBc, and anti-HCV negative adult males, who were not using drugs. Eleven had the hepa tointestinal form of the disease, with viable Sm ova in stools, and 10 were healthy volunteers (control group). Blood specimens were drawn by a two-syringe technique for both the performance of liver tests and determination of hemostasis proteins (prothrombin, antithrombin III, protein C, protein S, high-molecular-weight kininogen, and plasminogen). The latter proteins were assayed im munologically in plasma using specific antibodies. Statis tical analysis of the results failed to demonstrate any sig nificant difference between the two groups. We may therefore conclude that there is no derangement of he patic protein synthesis in the mild form of the disease and that Sm probably escapes host defense mechanisms in vivo, and does not contribute to the consumption coagu lopathy described in the hepatosplenic form of the dis ease.

This study was designed to determine whether systemic amyloidosis is an additional risk factor for he mostatic abnormalities in hemodialysis patients and to evaluate local alterations of the hemostatic process within the patent-functional native arteriovenous fistula (AVF). Concentrations of in vivo molecular hemostatic markers, including prothrombin fragment_{1 +2} (PF 1.2), thrombin antithrombin III complex (TAT) and plasmin-α ₂ antiplasmin complex (PAP) were determined in plasma samples taken simultaneously from AVFs and contralat eral upper extremity large veins of hemodialysis patients associated with and without systemic amyloidosis. Seven amyloid (2 women, 5 men, aged 34 ± 6 years), and 13 non-amyloid patients (4 women, 9 men, aged 36 ± 7 years) on maintenance hemodialysis and 20 healthy vol unteers (8 women, 12 men, aged 36 ± 9 years) were in cluded in the study. Peripheral vein PF 1.2 and TAT lev els showed no difference between amyloid and non- amyloid patient groups, but both were significantly higher than control group. PF 1.2 and TAT levels were also found to be elevated in fistulas when compared with that of peripheral vein in both amyloid and non-amyloid pa tient groups. Determination of PAP in peripheral veins of each group revealed significantly higher levels in amyloid hemodialysis patients than in non-amyloid patients and controls. PAP levels were significantly higher in fistulas of amyloid patients than in non-amyloid patients. In con clusion, this study confirms enhanced coagulation and fibrinolysis in hemodialysis patients and excessive fibri nolysis in amyloid patients with remarkable contribu tion of AVF on enhanced coagulation and fibrinolysis.

It is usual to stop prophylaxis of most surgical patients at hospital discharge. However, there is increas ing evidence that some patients are at risk of thromboem bolism after treatment is stopped. Some evidence and several proposals from the literature are presented, but to date, the situation remains unclear.

A case of systemic lupus erythematosus (SLE) presenting as superior vena cava syndrome is pre sented. This 19-year-old woman was admitted for head ache and swelling in her face and neck. Initial evaluation revealed that she had a superior vena cava syndrome. Thoracal computed tomography (CT) and intravenous digital substraction angiography demonstrated the occlu sion in the superior vena cava. ANA and antiDNA titers were high and anticardiolipin antibodies were positive. Along with immunosuppressive treatment, she had been administered intravenous streptokinase. To our knowl edge, no SLE patient presenting with such an unusual form has been described in the English literature.