Conventional chromogenic heparin assays are influenced by patient's endogenous plasma Antithrombin levels.

OBJECTIVES: To determine, in vitro, the influence of plasma AT concentrations on heparin levels as measured by commercially available chromogenic kits. METHODS: Purified AT was added to plasma that was immune depleted of AT at the following final concentrations: 0, 0.5, 1.0, 1.5, 2.0, 2.5 u/ml. Heparin was added to each of the AT concentrations at the following final concentrations: 0, 0.2, 0.4 u/ml. Heparin levels were then determined using 5 different Anti-Xa (n=4) or Anti-IIa (n=1) commercial kits. All kits provided purified AT to be added to the assay system. RESULTS: When heparin was added to plasma, heparin concentrations measured were dependent on plasma concentrations of AT (p<0.001). When plasma concentrations of AT were less than 1.0 u/ml heparin concentrations were underestimated and when plasma concentrations of AT were greater than 1.0 u/ml actual heparin levels were over estimated. There was no significant difference in the findings between the various commercial Xa or IIa kits. In the absence of heparin, anticoagulant activity was detected when plasma AT concentrations were above 1.0 u/ml. There was a statistically significant correlation between plasma concentrations of AT and the amount of anticoagulant activity (p<0.001). CONCLUSIONS: Conventional chromogenic heparin assays are influenced by endogenous plasma AT levels.

Prothrombotic disorders in infants and children with cerebral thromboembolism.

BACKGROUND: To our knowledge, the contribution of prothrombotic conditions to cerebral thromboembolism has never been prospectively studied in a large series of pediatric patients. METHODS: The Hospital for Sick Children, Toronto, Ontario, established a program in January 1992 to diagnose and treat children (term newborn to 18 years old) with arterial ischemic stroke or sinovenous thrombosis. The routine evaluation for prothrombotic conditions included plasminogen, antithrombin, protein C, free protein S, activated protein C resistance, IgG and IgM anticardiolipin antibody, and lupus anticoagulant. We analyzed samples taken within 2 years of the event. We report results on patients seen from January 1, 1992, to January 1, 1997. RESULTS: Ninety-two patients (47 males and 45 females) entered the program during the study interval. Patients ranged from newborn to 18 years in age. Arterial ischemic stroke occurred in 78% of patients while sinovenous thrombosis occurred in 22%. All were tested for prothrombotic disorders. One or more abnormal results were present in 35 (38%) of the 92 patients. The majority (21/35) had multiple abnormal test results. The abnormal test results were anticardiolipin antibody (33%), plasminogen (9.5%), activated protein C resistance (9%), protein C (7%), antithrombin (12.5%), lupus anticoagulant (8%), and free protein S (11.5%). Male sex predicted the presence of prothrombotic abnormalities (relative risk, 1.7; 95% confidence interval, 1.2-2.5), but stroke type (relative risk, 0.8; 95% confidence interval, 0.7-1.1), age group, and presence of other risk factors did not predict abnormal testing. CONCLUSIONS: A significant proportion (38%) of children with cerebral thromboembolism had evidence of prothrombotic conditions. In particular, there was a predominance of children with anticardiolipin antibody (33%). These data support a recommendation that children with cerebral thromboembolism be evaluated for prothrombotic disorders.

Correlation of the in vivo and in vitro activities of antithymocyte sera (ATS) with the immunizing antigen dose.

Rabbits were immunized with 1, 3, 10, 30, 100, 300, or 1,000 x 10(6) murine thymocytes per kg according to the method of Levy and Medawar. Thus, 33 individual and 6 pooled antimouse antihymocyte serum (ATS) preparations were obtained and tested for in vivo immunosuppressive (graft-protective) as well as for in vitro thymocytotoxic activity. It was found that: (1) at least 3 x 10(6) thymocytes/kg were necessary for inducing ATS of appreciable immunosuppressive activity; (2) rabbits immunized with 30 x 10(6) thymocytes/kg supplied sera of the most potent immunosuppressive activity; (3) the increase of the immunizing antigen dose over 30 x 10(6) thymocytes/kg resulted in ATS preparations of decreased immunosuppressive activity; (4) the graft-protective activity of an ATS pool corresponded to the average of the activities of the individual ATS preparations from which the pool had been mixed, i.e., the process of pooling itself did not modify the immunosuppressive activity; and (5) there was a good correlation (r = 0.72, P less than 0.001) between the in vivo immunosuppressive (graft-protective) activity and the in vitro thymocytotoxic titre of ATS preparations. The theoretical and practical significance of these results if discussed.

Thrombin regulation in children differs from adults in the absence and presence of heparin.

The physiologic mechanisms that protect children from thromboembolic complications are not known. We investigated the regulation of thrombin in children because of its central importance to thrombosis. The capacity to generate thrombin in vitro (chromogenic assay) was decreased by 26% in plasmas from children (1-16 yrs; n = 102) compared to adults ([20-45 yrs; n = 20; p < 0.001]). The addition of purified prothrombin to plasmas from children increased thrombin generation to adult values. The capacity of plasmas to inhibit 125I-alpha-thrombin was increased by 21% in children compared to adults (p = 0.020), with significantly more thrombin complexed to alpha 2-macroglobulin (alpha 2M) in children. When DVT occur in children, adult guidelines for heparin therapy are used. At low heparin concentrations (0.1 and 0.2 U/ml), thrombin generation was decreased by 30% in children compared to adults (p < 0.001). At high heparin levels (0.4 U/ml), thrombin generation was negligible in all plasmas. ATIII inhibited over 95% of thrombin in all plasmas in the presence of heparin. In summary, thrombin regulation differs in children from adults and may protect children from thromboembolic complications. When DVT do occur, heparin requirements may differ in children compared to adults.

Do coagulation screening tests detect increased generation of thrombin and plasmin in sick newborn infants?

BACKGROUND: Disseminated intravascular coagulation (DIC) is usually diagnosed in sick infants who have prolonged clotting times, depletion of platelets and coagulation factors, and elevated levels of fibrin derivatives. However, the diagnostic accuracy of abnormal coagulation profiles in neonates at risk of DIC has been uncertain. Since DIC is characterized by activation of both the coagulation and fibrinolytic systems, the objective of this study was to determine whether coagulation screening tests correctly identify infants with biochemical evidence of increased thrombin and plasmin generation. METHODS: Non-surgical patients in a tertiary care nursery who were sick enough to require an indwelling arterial catheter for monitoring purposes, were enrolled in a prospective cohort study. Blood samples for thrombin/antithrombin III (TAT) complexes and the plasmin-derived fibrinopeptide B beta 1-42 were drawn 36 to 72 h after birth from a free-flowing arterial line. Platelet counts, D-Dimer levels, plasma fibrinogen concentrations and prothrombin times, expressed as International Normalized Ratios or INR, were measured at the same time. RESULTS: One hundred patients were studied. Fifty-seven infants had elevated levels of TAT (> or = 4 micrograms/l) and B beta 1-42 (> or = 4 nmol/l). The sensitivities of platelets < 150 x 10(9)/l, D-Dimer > 500 ng/ml, fibrinogen < 1.5 g/l, and INR > 1.5 were 39%, 30%, 12%, and 11%, respectively. Corresponding specificities were 88%, 91%, 98%, and 95%. CONCLUSIONS: Abnormal coagulation screens in sick newborn infants strongly support a diagnosis of DIC. However, normal screens do not exclude activation of the coagulation and fibrinolytic systems.

Circulating dermatan sulfate and heparan sulfate/heparin proteoglycans in children undergoing liver transplantation.

The liver produces dermatan sulfate (DS), heparan sulfate (HS) and heparin glycosaminoglycans (GAG) and in the presence of hepatic disease, tissue levels of the DS GAG increase dramatically. We hypothesized that in children undergoing liver transplantation plasma levels of DS would be increased. Plasma from children undergoing liver transplantation were tested preoperative, intra operative and post operative at 24-48 h, and 1-3 weeks. Fluctuating levels of DS, HS and heparin anticoagulant activity were detected at all timepoints. The anticoagulant activity was purified and gel chromatography of the material displayed a mean Mr 110,000 D. Reductive elimination decreased the mean Mr 24,000 D indicating the activity resides on a proteoglycan (PG). The purified material was subjected to further chromatography and two peaks of anticoagulant activity resolved, compatible with at least two separate PGs, one with DS GAG chains and the additional PG(s) with HS and heparin GAG chains.

Enhanced thrombin regulation during warfarin therapy in children compared to adults.

The intensity of warfarin therapy for prevention of primary and secondary thromboembolic complications in paediatric patients, is extrapolated from guidelines for adults, which may not be optimal. Therefore, we assessed thrombin regulation ex vivo and in vitro in plasmas from 40 children (1 to 18 years old with a median age of 13 years) and 27 adults receiving warfarin with an international normalized ratio of 2 to 3 (child: 2.5 +/- 0.15; adult: 2.4 +/- 0.14). Ex vivo concentrations of prothrombin fragment 1.2 were significantly lower in children (0.30 +/- 0.03 nM) compared to adults (0.45 +/- 0.04 nM; p <0.01). Thrombin generation in defibrinated plasmas (Arvin) was decreased and delayed for children compared to adults when activated by either activated partial thromboplastin time (child = 32 +/- 1.7, adult = 45 +/- 1.9 microM x s) or prothrombin time (child = 35 +/- 0.7, adult = 46 +/- 1.0 microM x s) reagents (p <0.01 for both). Although plasma concentrations of factors (F) II, FVII, FIX, F X, protein C and protein S were similar, more of the thrombin generated was complexed to alpha2 macroglobulin (alpha2M) at times close to peak thrombin activity (60 s) in plasma from children (general linear analysis of variance; p <0.03). Thus, increased alpha2M levels may enhance thrombin regulation in paediatric compared to adult patients receiving warfarin, suggesting that clinical trials in children, using less intense warfarin treatment, may be required to determine optimum therapy.

Homozygous protein C deficiency: description of a new mutation and successful treatment with low molecular weight heparin.

We present a kindred with a new mutation of the protein C gene, in which the proband had an unusual clinical presentation. The relationship between warfarin induced skin necrosis and level of anticoagulation was investigated. The pharmacokinetics of protein C concentrate was assessed to determine frequency of replacement therapy. The clinical and biochemical efficacy of therapy with low molecular weight heparin (LMWH) was assessed. The effect of long-term LMWH on bone density in the growing child was monitored using whole body densitometry. Warfarin therapy required an INR of greater than 3.5 to avoid skin necrosis. If protein C replacement was to be used, doses of 100 U/kg/day would have been required to maintain protein C levels consistently at or above 0.20 U/ml. While receiving prophylactic therapy with LMWH for almost 3 years, there were no episodes of recurrent thrombosis, no skin necrosis and no bleeding. Biochemical markers of in vivo thrombin generation were suppressed and within the normal range. Bone density continued to increase at the normal rate throughout the treatment period. LMWH is an effective form of long-term therapy for homozygous protein C deficient patients with measurable protein C levels.

MHC-specific graft-protective and delayed-type hypersensitivity (DTH) suppressive activity of a CD4+ CD8+, alpha beta T cell receptor (TCR) positive lymphoma isolated from a tolerant mouse.

Two lymphomas were found in, and isolated from A (H-2a) mice in which permanent transplantation tolerance was induced to CBA (H-2k) histocompatibility antigens by the neonatal injection of (CBAxA)F1 spleen cells. They proved to be of recipient origin and were transferable to syngeneic A mice, growing as disseminated lymphomas (L33 and L46) and killing the recipients rapidly. Analysis of the cell surface antigens disclosed that both lymphomas had an immature T cell phenotype [Thy-1+, CD5+, CD3low, TCR alpha beta low, CD4low, CD8high, heat-stable antigen (HSA) positive, and CD44-, MHC class II-, CD45R-, sIg-, Gr-1-, CD11b-]. Intraperitoneal (i.p.) injection of syngeneic A mice with viable L33 lymphoma cells resulted in a dose-dependent, significant prolongation of the mean survival times of "specific" CBA and MHC-identical B10.BR skin allografts as compared to the survival of appropriate grafts in non-lymphoma-bearing controls. The survival times of third party MHC-incompatible B10 (H-2b) and B10.D2 (H-2d) allografts were only slightly prolonged in A mice inoculated with L33 cells. The graft-protective effect was not abrogated if the proliferative capacity of the L33 cells was blocked by in vitro mitomycin C (MMC) pretreatment. Furthermore, the inoculation of L33 lymphoma into A mice significantly inhibited their DTH response to the sensitizing CBA histocompatibility antigens. In contrast, the L46 lymphoma had no effect on the survival of CBA allografts and the DTH reactivity. These data suggest that the CD4+CD8+TCR alpha beta + L33 T cell lymphoma originating from a neonatally tolerant mouse has a specific immunosuppressive effect on the in vivo reactivity of syngeneic mice to the tolerance-inducing (MHC class I) alloantigens.

Pathophysiological effects of human TNF-alpha-producing tumor xenografts in immunosuppressed mice.

Groups of CBA mice immunosuppressed with anti-thymocyte serum (ATS) treatment were xeno-transplanted with either HeLa human cervical carcinoma cells or genetically modified cells expressing the human tumor necrosis factor-alpha (TNF) gene (All cells). Both cell lines were highly resistant to the cytotoxic effects of TNF. If 3 x 10(6) tumor cells were inoculated s.c. into female mice, HeLa cells grew progressively into large tumors and killed 74% of the recipients, while TNF-expressing All cells caused fatal tumor growth only in 22% of the mice. 3 x 10(6) or 1.5 x 10(7). All cells produced progressive tumor growth and lethality in all male recipients. In sera of all the A11-cell-transplanted mice, biologically active TNF was detected shortly (4.5 h) after tumor inoculation (6 39 U/ml), decreasing to below detection level in the circulation by day 3. In recipients of 15 million A11 cells, circulating TNF reappeared and reached high levels (12-1000 U/ml) 3 to 7 weeks later, when the animals bore large tumors (14-23 mm). Generally, such mice became cachectic, severely anemic, hypothermic, and soon died. On account of calcium mobilization from bones, their serum Ca levels were high. Electron microscopy revealed severe liver damage, but there were no signs of chronic arthritis. These results suggest that ATS-treated mice xenotransplanted with TNF-gene-transfected A11 human tumor cells provide a new model for studying the pathophysiological and anti-tumor effects of TNF.