Barry White

Activated protein C inhibits lipopolysaccharide-induced nuclear translocation of nuclear factor kappaB (NF-kappaB) and tumour necrosis factor alpha (TNF-alpha) production in the THP-1 monocytic cell line

Activated protein C (APC) protects against sepsis in animal models and inhibits the lipopolysacharide (LPS)‐induced elaboration of proinflammatory cytokines from monocytes. The molecular mechanism responsible for this property is unknown. We assessed the effect of APC on LPS‐induced tumour necrosis factor α (TNF‐α) production and on the activation of the central proinflammatory transcription factor nuclear factor‐κB (NF‐κB) in a THP‐1 cell line. Cells were preincubated with varying concentrations of APC (200 µg/ml, 100 µg/ml and 20 µg/ml) before addition of LPS (100 ng/ml and 10 µg/ml). APC inhibited LPS‐induced production of TNF‐α both in the presence and absence of fetal calf serum (FCS), although the effect was less marked with 10% FCS. APC also inhibited LPS‐induced activation of NF‐κB, with APC (200 µg/ml) abolishing the effect of LPS (100 ng/ml). The ability of APC to inhibit LPS‐induced translocation of NF‐κB is likely to be a significant event given the critical role of the latter in the host inflammatory response.

Use of protein-C concentrate, heparin, and haemodiafiltration in meningococcus-induced purpura fulminans

BACKGROUND Inflammatory and coagulation processes are both affected in meningococcaemia. Severe acquired protein-C deficiency in meningococcaemia is usually associated with substantial mortality: in survivors, skin grafts, amputation, and end-organ failure are not uncommon. Protein C is a natural anticoagulant and also has important anti-inflammatory activity. We assessed the effects of early replacement therapy with protein-C concentrate together with continuous veno-venous haemodiafiltration and conventional treatment in meningococcaemia. METHODS 12 patients aged between 3 months and 27 years with meningococcaemia and severe acquired protein-C deficiency (mean 0.20 IU/mL) were studied. All patients had septic shock, widespread purpura, skin necrosis, and disseminated intravascular coagulopathy. After a test dose of protein-C concentrate, patients received a continuous infusion with the dose adjusted daily to keep the plasma concentration between 0.8 and 1.2 IU/mL. 11 patients were given unfractionated intravenous heparin (10-15 IU kg-1 h-1). Nine patients had haemodiafiltration and one had peritoneal dialysis. The Glasgow meningococcal septicaemia prognostic score and the paediatric risk of mortality score predicted a minimum mortality of 80% and 57%, respectively. FINDINGS No patient died. No adverse reactions to the treatment were seen. Two patients had lower-limb amputations, one of whom had a thrombotic cerebrovascular accident; both patients had received the protein-C concentrate and heparin later than the rest of the group (60 h [16.97] vs 12 h [3.13]). One patient developed chronic renal failure despite receiving protein-C infusion 15 h after admission. INTERPRETATION The acquired severe deficiency of protein C in meningococcaemia contributes to the pathogenesis of the thrombotic necrotic lesions in the skin and other organs and probably has an important role in the inflammatory response. Protein-C therapy is merely one approach to improve the host response in this syndrome. We suggest that a double-blind, randomised, controlled multicentre trial is needed to confirm our results.

Infectious purpura fulminans: diagnosis and treatment.

Purpura fulminans is a term used to describe an acute, often lethal, syndrome of disseminated intravascular coagulation (DIC) and purpuric skin. The skin lesion is rapidly progressive, characterized by microvascular thrombosis in the dermis which ultimately results in perivascular haemorrhage and necrosis with minimal inflammation. Clinically the lesion can be distinguished from simple haemorrhage into the skin, in that the lesions of purpura fulminans are usually raised, indurated, and have a circumferential area of redness, and over time the lesions coalesce, blister and break down reflecting tissue necrosis. Although these lesions are frequently attributed to haemorrhage, skin biopsies clearly reveal that the pathophysiological lesions are in fact dermal vessel thromboses. The microvasculature of other body organs is also affected which ultimately results in necrosis and dysfunction. Inherited and acquired abnormalities of the protein C (PC) anticoagulant pathway are now believed to be responsible for the majority of patients with this clinical syndrome. The three commonest clinical situations where purpura fulminans is seen are severe bacterial infections, especially meningococcal disease, homozygous PC or protein S (PS) deficiency and autoimmune PS or PC deficiency. Rarer clinical conditions associated with purpura fulminans include warfarin-induced skin necrosis, cholestasis and antiphospholipid syndrome (Adcock & Hicks, 1990).

Low-molecular weight and unfractionated heparins induce a downregulation of inflammation: decreased levels of proinflammatory cytokines and nuclear factor-κB in LPS-stimulated human monocytes

Unfractionated heparin (UFH) and low‐molecular weight heparin (LMWH) are well defined anticoagulant agents. Recent data suggest that both LMWH and UFH may also have potent anti‐inflammatory properties; however, their mechanism of action responsible for the anti‐inflammatory effect is not yet fully elucidated. This study was designed to assess the effect of LMWH and UFH on human monocytes production of inflammatory markers and nuclear translocation of nuclear factor (NF)‐κB. Cultured monocytes were pretreated for 15 min with LMWH or UFH (10 μg and 1 μg/million cells) before stimulation with lipopolysaccharide (LPS) at a dose of 1 ng/million cells. Proinflammatory cytokines tumour necrosis factor (TNF)‐α, interleukin (IL)‐8, IL‐6 and IL‐1β release were subsequently measured by enzyme‐linked immunosorbent assay at 6 h, and nuclear translocation of the proinflammatory NF‐κB was assessed at 2 h. Treatment with pharmacological doses of LMWH and UFH significantly attenuated LPS‐induced production of TNF‐α, IL‐8, IL‐6 and IL‐1β as well as NF‐κB translocation. These results indicate equivalent and significant heparin anti‐inflammatory properties at low doses on monocyte‐mediated immune response. The inhibition of NF‐κB activation certainly represents one of the mechanisms by which heparin exerts its anti‐inflammatory effect. LMWH and UFH therefore appear as potential therapeutic inhibitors of inflammation.

Replacement therapy for invasive procedures in patients with haemophilia: literature review, European survey and recommendations.

Summary.  Although most surgical and invasive procedures can be performed safely in patients with haemophilia, the optimal level and duration of replacement therapy required to prevent bleeding complications have not been established conclusively. For providing more insight into optimal therapy during invasive procedures, a literature review of surgical procedures in patients with haemophilia was conducted. Concomitantly, current practice was surveyed in 26 European Haemophilia Comprehensive Care Centres, representing 15 different countries. The review identified 110 original papers published between 1965 and 2007. Of these, only two studies were randomized controlled trials. Target levels and the duration of replacement therapy in the published studies were as follows. For major orthopaedic surgery: preoperative targets were 80–90%; postoperative targets showed a high degree of variation, with trough levels ranging from 20% to 80%, duration 10–14 days; for liver biopsy, 70–100%, 1–7 days; tonsillectomy: 90–100%, 5–11 days; indwelling venous access device insertion: 100%, 3–10 days; circumcision: 50–60%, 2–4 days; dental surgery: 30–50%, single treatment. With the exception of dental surgery, current practice in Europe, as assessed by the survey, was largely in agreement with published data. In conclusion, this study provides both a comprehensive review and a large survey of replacement therapy in patients with haemophilia undergoing invasive procedures; these data have informed the consensus practical treatment recommendations made in this paper. This study highlights the need for better‐designed studies in order to better define minimal haemostatic levels of replacement therapy and optimal treatment duration.

Randomized comparison of unfractionated heparin with corticosteroids in severe active inflammatory bowel disease

Heparin therapy may be effective in steroid resistant inflammatory bowel disease.

Protamine sulfate down-regulates thrombin generation by inhibiting factor V activation

Protamine sulfate is a positively charged polypeptide widely used to reverse heparin-induced anticoagulation. Paradoxically, prospective randomized trials have shown that protamine administration for heparin neutralization is associated with increased bleeding, particularly after cardiothoracic surgery with cardiopulmonary bypass. The molecular mechanism(s) through which protamine mediates this anticoagulant effect has not been defined. In vivo administration of pharmacologic doses of protamine to BALB/c mice significantly reduced plasma thrombin generation and prolonged tail-bleeding time (from 120 to 199 seconds). Similarly, in pooled normal human plasma, protamine caused significant dose-dependent prolongations of both prothrombin time and activated partial thromboplastin time. Protamine also markedly attenuated tissue factor-initiated thrombin generation in human plasma, causing a significant decrease in endogenous thrombin potential (41% +/- 7%). As expected, low-dose protamine effectively reversed the anticoagulant activity of unfractionated heparin in plasma. However, elevated protamine concentrations were associated with progressive dose-dependent reduction in thrombin generation. To assess the mechanism by which protamine mediates down-regulation of thrombin generation, the effect of protamine on factor V activation was assessed. Protamine was found to significantly reduce the rate of factor V activation by both thrombin and factor Xa. Protamine mediates its anticoagulant activity in plasma by down-regulation of thrombin generation via a novel mechanism, specifically inhibition of factor V activation.

von Willebrand's disease: an important cause of dysfunctional uterine bleeding

We assessed the prevalence of von Willebrands disease (VWD) in patients with objectively confirmed dysfunctional uterine bleeding. A case–control study was designed to include 38 patients with objectively confirmed dysfunctional uterine bleeding and 38 age-matched controls with normal menstrual blood loss (MBL). Menorrhagia was defined as a mean MBL of greater than 80 ml on three consecutive menses as measured by the alkali haematin method. von Willebrand factor antigen, von Willebrand factor activity (VWF:Ac) and factor VIII:C were measured on three serial venous blood samples 1 week apart. VWD was diagnosed in five of 38 (13%) patients with menorrhagia and one of 38 (2.6%) patients with normal menstrual blood loss. The mean VWF:Ac value was significantly reduced in patients with menorrhagia (mean ± standard deviation, 84.5 ± 26.7 IU/dl versus 103.9 ± 34.5 IU/dl;P < 0.01) and this effect persisted after exclusion of patients diagnosed with VWD. Failure to investigate patients for VWD will limit the potential benefits of medical therapies such as tranexamic acid or nasal desmopressin [1-desamino-8-D-arginine vasopressin, (DDAVP)] and, in addition, will lead to an increased risk associated with surgical intervention in patients with undiagnosed VWD.

Primary thromboprophylaxis for cancer patients with central venous catheters--a reappraisal of the evidence.

Venous thromboembolism (VTE) is responsible for an estimated 25 000 deaths per annum in UK hospital practice. It is well established that many of these deaths could be prevented through the use of appropriate thromboprophylaxis. This issue is of particular relevance in oncology practice, where the risks of VTE and bleeding are both significantly higher than those observed in general medical patients. Cancer patients with in-dwelling central venous catheters (CVCs) are at particularly high risk of developing thrombotic complications. However, the literature has produced conflicting conclusions regarding the efficacy of using routine primary thromboprophylaxis in these patients. Indeed such is the level of confusion around this topic, that the most recent version of the American College of Chest Physicians (ACCP) guidelines published in 2004 actually reversed their previous recommendation (published in 2001). Nevertheless, minidose warfarin continues to be routinely used in many oncology centres in the UK. In this article, we have performed a systematic review of the published literature regarding the efficacy and the risks, associated with using thromboprophylaxis (either minidose warfarin or low-dose LMWH) in cancer patients with CVC. On the basis of this evidence, we conclude that there is no proven role for using such thromboprophylaxis. However, asymptomatic CVC-related venous thrombosis remains common, and further more highly powered studies of better design are needed in order to define whether specific subgroups of cancer patients might benefit from receiving thromboprophylaxis.

Desmopressin: therapeutic limitations in children and adults with inherited coagulation disorders.

Desmopressin (DDAVP), a synthetic analogue of vasopressin has been successfully used in the treatment of type I von Willebrands disease (VWD), mild factor VIII (FVIII) deficiency and intrinsic platelet function defects (PFDs) for almost three decades. However, there is limited published data documenting its efficacy and the reliability of circulating plasma FVIII:C as a surrogate marker of response to therapy in VWD. We report the haemostatic response to DDAVP in 133 consecutive patients (91 type I VWD, 20 mild FVIII deficiency and 22 PFDs). Minimal therapeutic response to DDAVP (0·3 µg/kg) was defined by normalization 30 min post‐ infusion of bleeding time for PFDs, factor VIII:C (FVIII:C) for mild haemophilia A, and von Willebrand factor antigen (VWF:Ag), von Willebrand factor functional activity (VWF:Ac) and FVIII:C for VWD. Nine out of 91 (10%) VWD patients failed to achieve minimal therapeutic response to DDAVP; plasma FVIII:C levels were an unreliable surrogate marker of DDAVP response as 6 out of 9 (67%) of these patients had normal post‐infusion FVIII:C levels. Five out of the 20 (25%) patients with mild FVIII deficiency and 5 out of 22 (23%) patients with PFDs failed to achieve a minimal therapeutic response to DDAVP. DDAVP is an effective therapy in the majority of patients with type I VWD, PFDs and mild FVIII deficiency. The significant failure rate associated with this therapy supports the recent recommendations that response should be assessed in all patients at the time of diagnosis. FVIII:C is an unreliable guide of response to DDAVP in patients with VWD and therefore VWF:Ag and VWF:Ac should also be assessed. Failure to demonstrate the response of VWF:Ag, VWF:Ac and FVIII:C to DDAVP in patients with VWD is likely to increase the risk of haemorrhagic complications in patients with bleeding episodes or who are undergoing surgery.