Novel inherited risk factors for venous thrombosis.
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Europe↗
Biomedical subjects
N S Key
Publications and source records attributed to N S Key.
Interferon-alpha restores beta1-integrin-dependent, collagen-mediated platelet aggregation in a patient with chronic myelogenous leukemia.
Although interferon-alpha (IFN-alpha) induces hematologic remissions in 70% to 80% of patients with chronic myelogenous leukemia (CML) and complete or near-complete cytogenetic remissions in 10% to 20% of patients, the exact mechanisms underlying these clinical results remain unclear. We have hypothesized that IFN-alpha acts at least in part through restoration of beta1-integrin function on malignant hematopoietic progenitors that can promote adhesion of malignant progenitors to the marrow microenvironment. This may then restore microenvironmental inhibition of progenitor proliferation and induce tumor dormancy. We demonstrate that IFN-alpha administration to a patient suffering from a clinically severe bleeding diathesis reversed the defective collagen-mediated aggregation of platelets expressing normal numbers of functionally inactive collagen receptors. This is the first in vivo demonstration that IFN-alpha can up-regulate the function of adhesion receptors in CML and supports the premise that IFN-alpha induces remissions by restoring normal integrin-mediated interactions between progenitors and microenvironmental components.
Collagen↗
Home treatment of mild to moderate bleeding episodes using recombinant factor VIIa (Novoseven) in haemophiliacs with inhibitors.
OBJECTIVE: To assess the safety and efficacy of a fixed dose of recombinant activated factor VII (rFVIIa; NovoSeven) in the home setting for mild to moderately severe joint, muscle; and mucocutaneous bleeding episodes in patients with haemophilia A or B with inhibitors. DESIGN: Multicentre, open-label, single arm, phase III study of one year duration. METHODS; Patients or their caregivers administered up to three doses of rFVIIa (90 microg/kg i.v.) at 3 h intervals within 8 h of the onset of a mild to moderate bleeding episode. Once the subject considered that rFVIIa had been "effective" with regard to haemostasis (after 1-3 injections), one further (maintenance) dose of rFVIIa was administered. RESULTS: Of 60 patients enrolled, 56 experienced at least one bleed, and 46 completed the one year study. 614 of 877 bleeds (70%) were evaluable according to protocol definitions. Haemostasis was rated as "effective" in 92% (566/614) of evaluable bleeds after a mean of 2.2 injections. For successfully treated episodes, the time from onset of bleeding until administration of the first injection was 1.1+/-2.0 h (mean+/-SD). Twenty-four hours after initial successful response, haemostasis was reported as having been maintained in 95% of cases. Efficacy was comparable for muscle, joint and target joint, and mucocutaneous bleeding episodes. In an intent-to-treat analysis of all 877 bleeding events, efficacy outcomes were equivalent to the evaluable bleeds, with an effective response in 88% of treated episodes. Treatment-related adverse events occurred in 32 (3% of all) bleeding episodes and consisted of re-bleeds/new bleeds in more than 50% (18/32) of these events. A single episode of superficial thrombophlebitis was the only thrombotic complication encountered, and there were no patient withdrawals due to adverse events. Development of FVII(a) antibodies could not be detected, and hypersensitivity reactions to rFVIIa were not reported. CONCLUSION: rFVIIa is effective and well tolerated when used in the home setting to treat mild to moderate bleeding episodes in patients with haemophilia A or B with inhibitors.
Adolescent↗
Platelet factor 4 binds to glycanated forms of thrombomodulin and to protein C. A potential mechanism for enhancing generation of activated protein C.
Platelet factor 4 (PF4) is an abundant platelet alpha-granule heparin-binding protein. We have previously shown that PF4 accelerates up to 25-fold the proteolytic conversion of protein C to activated protein C by the thrombin.thrombomodulin complex by increasing its affinity for protein C 30-fold. This stimulatory effect requires presence of the gamma-carboxyglutamic acid (Gla) domain in protein C and is enhanced by the presence of a chondroitin sulfate glycosaminoglycan (GAG) domain on thrombomodulin. We hypothesized that cationic PF4 binds to both protein C and thrombomodulin through these anionic domains. Qualitative SDS-polyacrylamide gel electrophoresis analysis of avidin extracts of solutions containing biotinylated PF4 and candidate ligands shows that PF4 binds to GAG+ but not GAG- forms of thrombomodulin and native but not Gla-domainless protein C. Quantitative analysis using the surface plasmon resonance-based BIAcoreTM biosensor system confirms the extremely high affinity of PF4 for heparin (KD = 4 nM) and shows that PF4 binds to GAG+ thrombomodulin with a KD of 31 nM and to protein C with a KD of 0.37 microM. In contrast, PF4 had no measurable interaction with GAG- thrombomodulin or Gla-domainless protein C. Western blot analysis of normal human plasma extracted with biotinylated PF4 demonstrates PF4 binding to protein C in a physiologic context. Thus, PF4 binds with relative specificity and high affinity to the GAG- domain of thrombomodulin and the Gla domain of protein C. These interactions may enhance the affinity of the thrombin.thrombomodulin complex for protein C and thereby promote the generation of activated protein C.
Binding Sites↗
Molecular unraveling of von Willebrand's disease: still some way to go.
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Humans↗
Simultaneous occurrence of posttransfusion purpura due to anti-HPA-1a and a delayed transfusion reaction due to anti-Jk(b)
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Aged↗
Detection of the factor VLeiden mutation. Development of a testing algorithm combining a coagulation assay and molecular diagnosis.
The Coagulation and Molecular Diagnostic laboratories at the University of Minnesota Medical School (Minneapolis) have collaborated to develop a diagnostic algorithm to identify all factor VLeiden mutation carriers without performing unnecessary and expensive genetic testing. The algorithm uses a coagulation assay for activated protein C resistance (APCR) to determine the need for genetic testing. We report the results of our experience validating this program. We compared the sensitivity, specificity, and positive and negative predictive values of two measures of APCR, the APCR ratio and the normalized ratio. We found that the normalized ratio was the more sensitive but less specific parameter to determine the need for genetic testing. By using the normalized ratio as the standard by which to refer patients to the Molecular Diagnostics Laboratory, all mutation carriers were identified. We found a large overlap in both measures of APCR between symptomatic patients with normal genotype and mutation carriers. Furthermore, we demonstrated that increased factor VIII levels with a normal genotype are associated with apparent APCR. In this article we also review other correlates of apparent APCR.
Adult↗
Molecular and biochemical approaches in the identification of heterozygotes for homocystinuria.
We compared biochemical and molecular methods for the identification of heterozygous carriers of mutations in the cystathionine beta-synthase (CBS) gene. Eleven relatives of seven unrelated patients with homocystinuria due to homozygous CBS deficiency and controls were studied with respect to total homocysteine concentrations before and after methionine loading. In addition, we determined CBS activity in cultured skin fibroblasts and tested for the presence of five known mutations by a PCR-based method in these seven patients, their relatives and controls. The results demonstrate that measurement of homocysteine after methionine loading and assay of CBS enzyme activity in cultured fibroblasts identify most but not all heterozygotes. There was significant correlation between homocysteine concentrations and CBS activities only after methionine loading (r = 0.12, 0.48, 0.48 and 0.50 at 0, 4, 6 and 8 h, respectively). Among the homozygous patients, molecular approaches identified five T833C and two G919A mutations out of 14 independent alleles, confirming the studies of others that these represent the two most prevalent mutations. In addition, we found that three of six heterozygotes with the T833 C allele had post-methionine loading homocysteine levels which overlapped with controls and of the other three, one (as well as an obligate heterozygote who did not carry any of the five mutant alleles tested) had CBS activity comparable to that of controls. These findings demonstrate that genotyping is useful as an adjunctive method for the diagnosis of the heterozygous carrier state of CBS deficiency.
Adolescent↗
Hypotensive reactions to white cell-reduced plasma in a patient undergoing angiotensin-converting enzyme inhibitor therapy.
BACKGROUND: Hypotensive reactions to platelet transfusions performed with white cell (WBC)-reduction filters with negatively charged surfaces have been reported recently in patients taking angiotensin-converting enzyme (ACE) inhibitors. Experimental studies have shown that the filter material can activate bradykinin, which may cause symptoms in patients with reduced bradykinin catabolism. Symptomatic adverse reactions after the administration of fresh-frozen plasma (FFP) through a WBC-reduction filter have not been reported in a patient on ACE Inhibitor medication. CASE REPORT: A 58-year-old man with congenital coagulation factor V deficiency and hypertension treated with an ACE inhibitor was admitted for rehabilitation after orthopedic surgery. On 3 consecutive days, he received FFP through a WBC-reduction filter; within minutes of the beginning of each infusion, he experienced a drop in blood pressure, facial erythema, abdominal pain, and anxiety. When the infusions were stopped, symptoms quickly abated without treatment. Multiple prior transfusions of unfiltered FFP and FFP filtered through a WBC-reduction filter made by a different manufacturer, as well as subsequent transfusions of unfiltered FFP, had not produced such reactions. CONCLUSION: Facial flushing, hypotension, and abdominal pain after FFP administration in a patient on ACE inhibitor medication appeared to be associated with a specific type of WBC-reduction filter. This association and other reported studies suggest that special caution is warranted when patients who are treated with ACE inhibitors receive blood components administered through WBC-reduction filters capable of generating bradykinin.
Abdominal Pain↗
Serologic evidence that factor IX inhibitor in the plasma of hemophilia B patients detects factor IX on normal red cells.
BACKGROUND: Patients with hemophilia B lack factor IX (F IX). These patients may become alloimmunized after the transfusion of F IX concentrates and may develop F IX inhibitors, which have been characterized as polyclonal IgG4 alloantibodies. Two cases in which F IX inhibitors caused difficulty in compatibility testing and antibody identification were encountered. It was hypothesized that, because F IX is present in normal plasma, it might be adsorbed by red cells in vivo and then be detected during antibody screening tests with serum containing F IX inhibitors. CASE REPORT: Sera from two African American half-brothers with hemophilia B were incompatible with all common and rare red cell phenotypes tested in the anti-human globulin test, but did not react with each other's red cells. The brothers' red cell antibodies were neutralized with both normal plasma and a commercially available F IX concentrate, which indicated that the red cell incompatibility was most probably caused by their F IX inhibitors. Red cells from an unrelated patient with hemophilia B and a very low titer of F IX inhibitor were tested against the half-brothers' sera and did not react. The compatible red cells from one of the half-brothers and the unrelated patient with hemophilia B adsorbed F IX from normal plasma or F IX concentrate after 37 degrees C incubation; this rendered them incompatible with the plasma containing F IX inhibitor from the other half-brother. CONCLUSION: F IX appears to be present on normal red cells and may be detected during compatibility and antibody identification procedures when serum or plasma containing F IX inhibitors is tested.
Adolescent↗
Toward an understanding of the pathophysiologic mechanism of thrombosis in the antiphospholipid antibody syndrome.
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Antibodies, Anticardiolipin↗
Platelet factor 4 stimulates thrombomodulin protein C-activating cofactor activity. A structure-function analysis.
Thrombomodulin (TM) is an anionic (pI approximately 4) protein cofactor that promotes thrombin (THR) cleavage of protein C to generate activated protein C (APC), a potent anticoagulant. We find that the cationic platelet alpha-granule protein platelet factor 4 (PF4) stimulates 4-25-fold the cofactor activity of rabbit TM and two differentially glycanated versions of an extracellular domain human TM polypeptide in which the glycosaminoglycan (GAG) is either present (GAG+ TM) or absent (GAG- TM) with an ED50 of 3.3-10 micrograms/ml. No such stimulation occurs in response to beta-thromboglobulin or thrombospondin, or when protein C lacking its gamma-carboxyglutamic acid (Gla) domain is the substrate. Heparin and chondroitin sulfates A and E reverse PF4 stimulation. PF4 minimally affects the Kd for THR but decreases 30-fold (from 8.3 to 0.3 microM) the Km for protein C of APC generation by GAG+ TM. PF4 also strikingly transforms the [Ca2+] dependence profile of rabbit and GAG+ TM to resemble that of GAG- TM. A potential explanation for this is that PF4, like Ca2+, induces heparin-reversible alterations in native (but not Gla-domainless) protein C conformation as assessed by autofluorescence emission analysis. We conclude that PF4 stimulates TM APC generation by interacting electrostatically with both the TM GAG and the protein C Gla domain to enhance markedly the affinity of the THR.TM complex for protein C. By this mechanism, PF4 may play a previously unsuspected role in the physiologic regulation of clotting.
Blood Platelets↗
C-reactive protein induces human peripheral blood monocytes to synthesize tissue factor.
The acute inflammatory response is frequently accompanied by serious thrombotic events. We show that C-reactive protein (CRP), an acute-phase reactant that markedly increases its serum concentration in response to inflammatory stimuli, induced monocytes to express tissue factor (TF), a potent procoagulant. Purified human CRP in concentrations commonly achieved in vivo during inflammation (10 to 100 micrograms/mL) induced a 75-fold increase in TF procoagulant activity (PCA) of human peripheral blood mononuclear cells (PBM), with a parallel increase in TF antigen levels. CRP-induced PCA was completely blocked by a monoclonal antibody against human TF but not by irrelevant murine IgG. Dot blot analysis showed a significant increase of TF mRNA after 4 hours of incubation with CRP, followed by a peak of PCA within 6 and 8 hours. Actinomycin D and cycloheximide blocked CRP-stimulated PCA, suggesting that de novo TF protein synthesis was required. Endotoxin (LPS) contamination of CRP was excluded as the mediator of TF synthesis because: (1) CRP was Limulus assay negative; (2) induction of TF PCA by CRP was not blocked by Polymyxin B, in contrast to LPS-induced PCA; (3) antihuman CRP IgG inhibited CRP-induced PCA, but not LPS-induced PCA; (4) CRP was able to stimulate TF production in LPS-pretreated PBM refractory to additional LPS stimulation; and, (5) unlike LPS, CRP was incapable of inducing TF in human umbilical vein endothelial cells. We suggest that CRP-mediated TF production in monocytes may contribute to the development of disseminated intravascular coagulation and thrombosis in inflammatory states.
Antibodies, Monoclonal↗
Eosinophil cationic granule proteins impair thrombomodulin function. A potential mechanism for thromboembolism in hypereosinophilic heart disease.
Thromboembolism is a prominent but poorly understood feature of eosinophilic, or Loeffler's endocarditis. Eosinophil (EO) specific granule proteins, in particular major basic protein (MBP), accumulate on endocardial surfaces in the course of this disease. We hypothesized that these unusually cationic proteins promote thrombosis by binding to the anionic endothelial protein thrombomodulin (TM) and impairing its anticoagulant activities. We find that MBP potently (IC50 of 1-2 microM) inhibits the capacity of endothelial cell surface TM to generate the natural anticoagulant activated protein C (APC). MBP also inhibits APC generation by purified soluble rabbit TM with an IC50 of 100 nM without altering its apparent Kd for thrombin or Km for protein C. This inhibition is reversed by polyanions such as chondroitin sulfate E and heparin. A TM polypeptide fragment comprising the extracellular domain that includes its naturally occurring anionic glycosaminoglycan (GAG) moiety (TMD-105) is strongly inhibited by MBP, whereas its counterpart lacking the GAG moiety (TMD-75) is not. MBP also curtails the capacity of TMD-105 but not TMD-75 to prolong the thrombin clotting time. Thus, EO cationic proteins potently inhibit anticoagulant activities of the glycosylated form of TM, thereby suggesting a potential mechanism for thromboembolism in hypereosinophilic heart disease.
Aged↗
Herpes simplex virus type I does not require productive infection to induce tissue factor in human umbilical vein endothelial cells.
BACKGROUND: Herpes simplex virus (HSV)-infected endothelium is a model for vascular injury and possibly the development of atherosclerosis. In vitro infection of human umbilical vein endothelial cells (HUVEC) by HSV-1 results in a number of changes including the expression of a procoagulant activity (PCA) compatible with that due to tissue factor (TF) synthesis. In this study, we have further characterized this PCA using more stringent assays for TF, and examined whether virus rendered incapable of replication retains the ability to stimulate TF synthesis in HUVEC. EXPERIMENTAL DESIGN: Confluent monolayers of HUVEC were exposed to intact or ultraviolet/heat-inactivated HSV-1. At appropriate time intervals, TF PCA was assessed by clotting assays, and TF antigen by an enzyme-linked immunosorbent assay specific for TF. The appearance of mRNA specific for TF was performed by Northern blotting. RESULTS: TF activity was demonstrated by both 1-stage and 2-stage clotting assays; the dependence of the latter on factor VIIa, and the inhibition by specific blocking antibodies to human TF support the notion that the PCA is indeed due to TF. Furthermore, cellular TF antigen levels were found to parallel TF activity, and there was a transient de novo expression of TF mRNA. Tissue factor PCA in HSV-infected HUVEC remained "encrypted"; that is, full clotting activity was not expressed in the absence of cellular disruption in a situation analogous to that seen in all normal cells thus far examined that express TF PCA. However, this response did not appear to be dependent upon replicative infection of HSV-1 within the endothelial cell since a similar (although lesser) induction of TF PCA was present in cells that had been exposed to virus previously rendered incapable of replication. CONCLUSIONS: HSV-1 induces PCA in HUVEC which is clearly TF-dependent; this response does not require viral replication. These data indicate increased complexity in HSV interactions with vascular endothelium and imply induction of some procoagulant functions by nonproductive infection.
Blood Coagulation Factors↗
Prothrombotic phenotype diversity of human aortic endothelial cells in culture.
We have previously demonstrated that human aortic endothelium exhibits morphologic heterogeneity in situ, and this heterogeneity can be reproduced in culture. In this study, we have compared prothrombotic properties of cultured endothelial cells (EC) from areas of human aorta at high risk for atherosclerosis (HP-EC) with EC from areas at low risk (LP-EC). Using paired cultures from the same donors, we have found that the expression of cell surface thrombomodulin (TM)--as measured by the ability to generate activated protein C (APC) from protein C in the presence of thrombin--is relatively reduced on HP-EC compared to LP-EC (respectively, 4.98 +/- 4.43 vs. 5.83 +/- 4.37 pM APC/min/cm2; p = .03, n = 12). Furthermore, HP-EC more efficiently assemble the prothrombinase complex on their cellular surface, resulting in an increased rate of thrombin generation from prothrombin (9.81 +/- 3.10 (HP-EC) vs. 7.96 +/- 3.20 nM thrombin/min/cm2 (LP-EC); p less than .03, n = 7). The combination of reduced TM expression and increased prothrombinase complex assembly on HP-EC suggests a prothrombotic phenotype in these cells. These findings may be important in the pathogenesis of thrombosis associated with atherosclerotic plaques.
Aorta↗
Hemolytic anemia in protoporphyria: possible precipitating role of liver failure and photic stress.
Hemolytic anemia is not a common clinical feature in protoporphyria. In this report, we describe two patients in whom we have encountered severe hemolytic anemia. Both individuals had advanced hepatic disease as a complication of their porphyria and were undergoing orthoptic liver transplantation. The onset of hemolysis appeared to be related to the development of liver disease, and in both cases, the operative procedure acutely exacerbated the red cell destructive process. We suggest that liver involvement in protoporphyria may unmask hemolytic anemia, and that red cells in these individuals are hypersensitive to photooxidative stress, such as might occur during a prolonged operative procedure.
Adolescent↗
Vascular endothelial cell proteoglycans are susceptible to cleavage by neutrophils.
Heparan sulfate proteoglycan associated with vascular endothelial cells in vivo plays an important role in a number of endothelial functions, including the inhibition of intravascular coagulation and extravasation of plasma proteins and blood cells. In this report, we demonstrate that polymorphonuclear neutrophils, as well as cell-free neutrophil supernatants, lead to a rapid and dramatic loss of proteoglycan from endothelial cells in the absence of evidence of cell lysis. This cleavage appears to be relatively (although not absolutely) selective for heparan sulfate and is mediated by neutrophil-derived serine proteases. Inhibitors of neutrophil elastase appear to be the most effective inhibitors of proteoglycan release. Furthermore, purified human neutrophil elastase also leads to cleavage of cellular proteoglycans, although not to the same extent as neutrophils or neutrophil supernatants. Proteoglycans compared with all other protein-containing macromolecules appear to be especially vulnerable to neutrophil-mediated cleavage. The results of this study may be germane to the interaction of neutrophils with endothelium during the inflammatory process, during which the loss of endothelial heparan sulfate proteoglycan may play a critical role in the vascular injury that often accompanies inflammation.
Cells, Cultured↗