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We examined recombinant activated factor VII (rVIIa) administered by continuous infusion to eight patients with inhibitors to factor VIII, undergoing elective surgery. rVIIa was infused at a fixed rate of 16.5 microg/kg/h for a median of 13.5 days (range 1-26). There was effective haemostasis at this infusion rate in only one of two minor procedures and two of six major operations. Three patients experienced excessive bleeding despite plasma factor VII activity around 10 IU/ml. Serious bleeding occurred in two other patients caused by procedural errors unrelated to rVIIa and required re-operation. The median rVIIa clearance on day 1 was 57 ml/h/kg (range 18-100) and on day 3 was 100 ml/h/kg (range 61-200). Clearance on the final infusion day was not significantly different from day 3. The infusion did not induce pathological activation of the coagulation mechanism. The only thrombotic adverse events were two episodes of superficial thrombophlebitis of the infused vein in one subject. In conclusion, the 16.5 microg/kg/h infusion rate reliably achieves plasma factor VII activity levels of 10 IU/ml, but this level does not provide reliable haemostasis.

Intra-arterial desmopressin caused dose and time dependent increases (p <0.001 for all) in forearm blood flow (all doses) and plasma tissue plasminogen activator (t-PA) concentrations (desmopressin > or = 70 ng/min). Although plasma t-PA concentrations rose in both forearms, there was a modest local release of t-PA in the infused forearm (14 ng/100 mL of tissue/min, p <0.05). At desmopressin doses > or = 300 ng/min, plasma von Willebrand factor (vWf) and Factor VIII:C concentrations rose in both forearms (p <0.001) and correlated with the rise in interleukin-6 concentrations (r = 0.92, p <0.001: r = 0.85, p = 0.002 respectively). Neither desmopressin nor substance P caused t-PA, vWf or Factor VIII:C release in the patients, although desmopressin increased plasma interleukin-6 concentrations as in healthy volunteers. We conclude that desmopressin releases t-PA, vWf and Factor VIII:C predominantly via systemic mechanisms, possibly mediated by cytokine release. Patients with type 3 vWD appear to have a generalised failure to release t-PA acutely despite a normal interleukin-6 response to desmopressin infusion.

Twenty six patients with mild or moderate haemophilia A and inhibitors are described. The inhibitor was detected at a median age of 33 years, after a median of 5.5 bleeding episodes. This usually following intensive replacement therapy. The median presenting inhibitor titre was antihuman 11.6 BU/ml, antiporcine 1.45 BU/ml. Plasma basal factor VIII level declined from a median of 0.08 IU/ml to 0.01 IU/ml following the inhibitor development. This caused spontaneous bleeding in 22 and a bleeding pattern similar to acquired haemophilia in 17. Bleeding was often severe and caused two deaths. The inhibitor disappeared spontaneously, or following immune tolerance induction, in 16 cases after a median of 9 months (range 0.5-46), with a return to the original baseline VIIIC level and bleeding pattern accompanied inhibitor loss. The inhibitor persisted in the remainder of the cases over a median period of 99 months (range 17-433 months) of follow-up. Inhibitors are an uncommon complication of mild haemophilia which frequently persist and may be associated with severe, life-threatening, haemorrhage. Forty-one percent of treated haemophilic family members had a history of factor VIII inhibitors, suggesting a familial predisposition to develop inhibitors in these kindreds. Sixteen patients from 11 families were genotyped. Seven different missense mutations affecting the light chain were detected and two in the A2 domain. Five patients from three families had a mutation causing a substitution of Trp2229 by Cys in the C2 domain which appears to predispose to inhibitor formation since 7 of the 18 affected individuals have a history of inhibitor development.

Hepatitis G virus (HGV), a novel flavivirus, has been implicated as a cause of posttransfusion hepatitis. We have performed a longitudinal study in a cohort of haemophiliacs (n = 68) who previously received non-virus inactivated coagulation factor concentrates to assess both patterns of HGV viraemia and any associated liver disease. Hepatitis C virus (HCV) RNA was present in 58/68 and co-infection with human immunodeficiency virus (HIV) was present in 15/68. HGV RNA was detected in 17/68 (25%) samples from the mid-1980s. There was no association between either HIV infection (p = 0.74) or co-infection with a particular HCV genotype (p = 0.62). However, there was a relationship between HGV viraemia and the severity of haemophilia (p = 0.0004) with HGV RNA detected in 5/19, 9/16 and 3/32 patients with mild, moderate and severe haemophilia respectively. A longitudinal study was performed in 15/17 haemophiliacs with HGV viraemia using stored serum samples from the 1980s and 1990s. HGV viraemia persisted in 8/15 and cleared in 7/15 over a variable period of time. A Weibull model was constructed to estimate the duration of HGV viraemia in the study group. The 75th and 90th percentiles for the duration of HGV were estimated to be 8.7 years (95%, confidence interval 4.8-15.7) and 23.6 years (95% confidence interval 11.8-47.1) respectively. Laparoscopic liver inspection/biopsy was performed in 25/68. There was no association between severity of liver disease and HGV viraemia (p = 0.43). This study demonstrates considerable variation in patterns of HGV viraemia in haemophiliacs. We found little evidence to implicate HGV as a major cause of chronic liver disease in haemophiliacs.

Circulating factor VIII (fVIII) levels increase during inflammation suggesting that fVIII synthesis or secretion is stimulated during acute inflammation. To examine the mechanisms underlying this increase in circulating factor VIII, we have developed a sensitive and reliable semiquantitative assay for fVIII mRNA utilising competitive reverse transcriptase-polymerase chain reaction (RT-PCR, and used this to study two human liver cell lines, Hep-G2 and Chang Liver cells. These cells were cultured under basal conditions or following treatment with interleukin-1, -2 and 6 (IL-1, -2, and -6). Following 18h culture with IL-6 (maximum concentration 40 U/ml), these levels had risen 6 and 9 fold respectively, with no concomitant rise in control RNA levels. The dose responses for both cell types were similar, with an ED50 of 11 U/ml. The time course of this response was also similar in both cell lines with the increase in fVIII mRNA first reaching significance by 3 h, and reaching maximum levels by 12 h. IL-1 and IL-2 had no effect at any of the doses studied. This study provides the first evidence for regulated expression of fVIII in human cell lines, and suggests that increased plasma fVIII levels during the acute phase response may be due to increased expression of fVIII mRNA.

Recombinant factor VIIa was used to treat 38 patients with acquired haemophilia participating in the Novoseven compassionate-use program. 19 were male, median age 59, range 2-89 years. The median pre-treatment anti-human (H) and anti-porcine (P) inhibitor titre was H 43 BU/ml (range 1-4500) and P 4.5 BU/ml (range 0-1600). Recombinant factor VIIIa was used as first-line therapy for 14 bleeding episodes and as salvage-therapy for 60 episodes which failed to respond to blood-product therapy given for a median of four days (range 1-21 days) prior to treatment with rVIIa. Pre-rVIIa treatment was not reported for four episodes. The indications for treatment were 7 haemarthroses, 40 muscle haematomas, 20 urinary or GI haemorrhages and 3 surgical interventions. The median starting dose of rVIIa was 90.4 microg/kg (range 45-181). A median of 28 doses (range 1-541) were given per episode, over a median 3.9 days (range 0-43). Efficacy was assessed clinically 8 and 24 h after the start of rVIIa and at the end of treatment. A good response was obtained in all 14 bleeds for which rVIIIa was used as first-line therapy. The response after 24 h of rVIIa salvage-therapy for 60 bleeds was good in 75%, partial in 17% and poor in 8%. Efficacy was unreported in 4 cases. The median prothrombin time (PTT) shortened from 12 s (range 9.3-20) pre-treatment to 8.8 s (range 6-14) during treatment. The clinical response did not correlate with the dose of rVIIa used, the type of bleed or the degree of shortening of the PTT following rVIIa infusion. Three patients died from haemorrhagic complications of acquired haemophilia. This mortality of 7.9% is lower than previously reported for this condition. Although one patient developed DIC during treatment with rVIIa, this was probably attributable to hypovolaemic shock, massive transfusion and the use of PCCs. This study demonstrates that rVIIa is a safe, useful and effective treatment for bleeding in patients with acquired haemophilia.

The effects on blood flow and plasma fibrinolytic and coagulation parameters of intraarterial substance P, an endothelium dependent vasodilator, and sodium nitroprusside, a control endothelium independent vasodilator, were studied in the human forearm circulation. At subsystemic locally active doses, both substance P (2-8 pmol/min) and sodium nitroprusside (2-8 microg/min) caused dose-dependent vasodilatation (p <0.001 for both) without affecting plasma concentrations of PAI-1, von Willebrand factor antigen or factor VIII:C activity. Substance P caused local increases in t-PA antigen and activity (p <0.001) in the infused arm while sodium nitroprusside did not. At higher doses, substance P increased blood flow and t-PA concentrations in the noninfused arm. We conclude that brief, locally active and subsystemic infusions of intraarterial substance P cause a rapid and substantial local release of t-PA which appear to act via a flow and nitric oxide independent mechanism. This model should provide a useful and selective method of assessing the in vivo capacity of the forearm endothelium to release t-PA acutely.

Recent studies using assays for surrogate markers of thrombogenicity in man have demonstrated that activation of the coagulation system occurs following infusion of clinical doses of prothrombin complex concentrates (PCC) but not after the same doses of high-purity factor IX concentrates (HP-FIX) in patients with haemophilia B. Here we have investigated the mechanism of such thrombogenesis by applying assays that detect early-through to late-events in coagulation system activation in a pharmacokinetic cross-over study of 50 IU/kg PCC and a new HP-FIX product in haemophilia B patients. Satisfactory recoveries and half-lives were observed for both concentrates. HP-FIX caused no increases in thrombin-antithrombin III complex (TAT), prothrombin activation peptide fragment F1+2 (F1+2), factor X activation peptide (FXAP) or factor VIIa (FVIIa). In contrast the same dose of factor IX in the form of PCC was followed by significant increases over pre-infusion levels of TAT, F1+2 and FXAP, but not FVIIa. Elevations of FIXAP occurred after both HP-FIX and PCC but did not reach normal levels and were attributed to normalisation of the FIX concentration in those patients whose levels of FIXAP were initially low. We conclude that the thrombogenic trigger associated with PCC infusion occurs at the level of factor X activation. In the absence of any increase in FVIIa, we would attribute this to the likely presence of FIXa in the PCC.

The effects of exogenous testosterone on the haemostatic system were studied in a group of 32 healthy men undergoing a clinical trial of hormonal male contraception. The men received 200 mg testosterone oenanthate (TE) weekly i.m., and plasma samples were taken pretreatment, at defined time points up to 52 weeks of treatment, and 4 and 8 weeks after discontinuing TE. This dose of TE caused a 2-fold increase in trough plasma testosterone levels. TE caused a fall in plasma fibrinogen concentration after 16 weeks of treatment. This was sustained for the duration of TE treatment and recovered to pretreatment levels during the recovery phase. There was also a sustained fall in the level of C4b binding protein which showed a rebound to levels above pretreatment during recovery. Levels of antithrombin III and prothrombin fragment F1.2 rose initially during TE treatment, and levels of protein C, protein S (free) and plasminogen activator inhibitor fell, but the concentrations of these factors all returned to pretreatment levels during continued treatment. There was no change in the plasma concentrations of beta-thromboglobulin, tissue plasminogen activator, protein S (total), or D-dimer. There was a sustained increase in haemoglobin concentration and haematocrit, without any change in platelet count. The observed changes were consistent with mild activation of the haemostatic system during initial treatment with testosterone. After several months the raised activation markers had returned to pretreatment levels indicating that a new equilibrium had been established which did not appear to be prothrombotic.(ABSTRACT TRUNCATED AT 250 WORDS)

In a group of six normal male volunteers, infusion of DDAVP, venous occlusion and exercise were shown to increase plasma levels of factor VIII and plasminogen activator, activity and antigen, to different extents and at differing rates. Any mechanisms suggested to explain release of these proteins by various stimuli should account for such differences. All three stimuli could also increase plasma levels of prostacyclin metabolites, although this was only significant for high doses of DDAVP. Other potential endothelial markers, such as fibronectin and thrombospondin, showed no specific increase after any of the stimuli.

Eight patients on warfarin with rheumatic heart disease and prosthetic cardiac valves were selected for study on the basis of persistently elevated plasma beta-thromboglobulin (beta-tg) and platelet factor 4 (PF4) concentrations. Platelet mean lifespan and fibrinogen half life were short, and positively correlated, and both were inversely related to the plasma concentration of the platelet specific proteins. Antithrombin III (ATIII) levels were also reduced. Treatment with sulphinpyrazone resulted in lengthening of both platelet and fibrinogen survival, a rise in ATIII but no change in the beta tg or PF4 concentrations. It is concluded that patients with abnormal cardiac valves and raised plasma levels of beta tg or PF4 have, despite warfarin, a consumption coagulopathy that can be inhibited by sulphinpyrazone.

During the collection of samples for plasma beta-thromboglobulin (beta-TG) determination, it is well established that artificially high values can be observed due to in-vitro release. To estimate the reliability of a single beta-TG measurement, blood samples were collected simultaneously from both arms on two separate occasions in 56 diabetic patients selected for a clinical trial. From each arm, blood was taken into two tubes containing an anticoagulant mixture with (tube A) and without (tube B) PGE1. The overall mean value of B-TG in tube B was 1.14 times higher than in tube A (p less than 0.01). The markedly large between-arms variation accounted for the most part of within-subject variation in both tubes and was significantly greater in tube B than in tube A. Based on the difference between B-TG values from both arms, the number of subjects with artificially high B-TG values was significantly higher in tube B than in tube A on each occasion (overall rate: 28% and 14% respectively). Estimate of between-occasions variation showed that B-TG levels were relatively stable for each subject between two occasions in each tube. It is concluded that the use of PGE1 decreases falsely high B-TG levels, but a single measurement of B-TG does not provide a reliable estimate of the true B-TG value in vivo.