Search

Haemophilia published by John Wiley & Sons Ltd Introduction: Advances in genomic sequencing have facilitated the sequencing of genes associated with disorders of haemostasis. The identification of variants within genes and access to curated data incorporating structural, functional, evolutionary as well as phenotypic data has become increasingly important in order to ascribe pathogenicity. Aim: The European Association for Haemophilia and Allied Disorders (EAHAD) Coagulation Factor Variant Database Project aims to provide a single port of entry to a web-accessible resource for variants in genes involved in clinical bleeding disorders. Results: New databases have evolved from previously developed single gene variant coagulation database projects, incorporating new data, new analysis tools and a new common database architecture with new interfaces and filters. These new databases currently present information about the genotype, phenotype (laboratory and clinical) and structural and functional effects of variants described in the genes of factor (F) VII (F7), FVIII (F8), FIX (F9) and von Willebrand factor (VWF). Conclusion: The project has improved the quality and quantity of information available to the haemostasis research and clinical communities, thereby enabling accurate classification of disease severity in order to make assessments of likely pathogenicity.

Hereditary blood coagulation factor VII (FVII) deficiency is a rare autosomal recessive bleeding disorder resulting from variants in the gene encoding FVII (F7). Integration of genetic variation with functional consequences on protein function is essential for the interpretation of the pathogenicity of novel variants. Here, we describe the integration of previous locus‐specific databases for F7 into a single curated database with enhanced features. The database provides access to in silico analyses that may be useful in the prediction of variant pathogenicity as well as cross‐species sequence alignments, structural information, and functional and clinical severity described for each variant, where appropriate. The variant data is shared with the F7 Leiden Open Variation Database. The updated database now includes 221 unique variants, representing gene variants identified in 728 individuals. Single nucleotide variants are the most common type (88%) with missense representing 74% of these variants. A number of variants are found with relatively high minor allele frequencies that are not pathogenic but contribute significantly to the likely pathogenicity of coinherited variants due to their effect on FVII plasma levels. This comprehensive collection of curated information significantly aids the assessment of pathogenicity.

Introduction: Haemophilia treatment varies significantly between individuals, countries and regions and details of bleed rates, factor consumption and injection frequency are often not available. Aim: To provide an overview of the FVIII/FIX treatment practice and outcome for patients with haemophilia A (HA) or haemophilia B (HB) across Europe. Methods: Non-interventional, 12-month retrospective study where anonymized data were retrieved from haemophilia centres/registers in Belgium, France, Germany, Italy, Spain, Sweden and the United Kingdom. Male patients (all ages) receiving coagulation factor treatment 24 months prior to the study, with basal FVIII/FIX levels ≤5 IU dL-1, without inhibitors, were included. Data were summarized descriptively. Results: In total, 1346 patients with HA and 312 with HB were included in the analysis; 75% and 57% had severe disease (FVIII/FIX < 1 IU dL-1) respectively. Prophylaxis was most common for severe haemophilia, especially for children, whereas on-demand treatment was more common for moderate haemophilia in most countries. The mean (SD) prescribed prophylactic treatment ranged from 67.9 (30.4) to 108.4 (78.1) (HA) and 32.3 (10.2) to 97.7 (32.1) (HB) IU kg-1 per week, across countries. Most patients on prophylaxis were treated ≥3 times/week (HA) or two times/week (HB). The median annual bleeding rate (ABR) for patients on prophylaxis ranged from 1.0 to 4.0 for severe HA, and from 1.0 to 6.0 for severe HB, while those with moderate haemophilia generally had slightly higher ABRs. Median ABRs for on-demand-treated severe HA ranged from 4.5 to 18.0, and for HB, 1.5 to 14.0. Conclusion: Treatment practice varied greatly between centres and countries and patients treated on-demand and prophylactically both experienced bleeds, emphasizing the need for further optimization of care. (Less)

Patients with bleeding disorders previously frequently became infected with hepatitis C virus. We identified the number of patients infected in Scotland and assessed several aspects of the outcomes of HCV infection and its treatment comparing these with cohorts infected for other reasons. We calculated the number of individuals infected in Scotland (cohort A) starting with the total number of patients treated in Scottish haemophilia centres registered on the UKHCDO database between 1970 and 1989. Cases were then removed or added based on additional information from centre records. A second cohort B, consisted of 255 patients from cohort A and 47 patients HCV infected outside Scotland, but with follow-up data from Scottish centres around their HCV infection. We estimate that 455 patients with bleeding disorders became infected by coagulation factor provided by NHS Scotland. In 302 individuals with documented HCV infection, rates of natural clearance (17.4%), genotype spread (64% genotype 1) and responses to antiviral therapy (14.5% with monotherapy; 38.8% with combination therapy) were similar to those in other cohorts. Thirty-four liver biopsies were performed without adverse event and liver transplantation has been performed in 11 patients, seven for liver failure, four for hepatocellular carcinoma. Around 455 patients with bleeding disorders became HCV infected in Scotland before 1989. The natural history of HCV infection and responses to treatment are similar to those in other HCV-infected cohorts. Liver transplantation has been used successfully for the treatment of end-stage liver failure and hepatocellular carcinoma.

Gynaecological cancer is common. It is highly amenable to effective treatment, but thrombosis remains a common complication. There is controversy about whether microparticles (MPs), particularly tissue factor (TF) positive MPs, are increased in patients with malignancy and/or thrombosis. We therefore set out to investigate the relationship between MPs of different cellular origins, in patients with gynaecological malignancy. We hypothesised that patients with gynaecological malignancy have increased numbers of MPs. We measured MPs released by different cell types in these patients, and correlated the results with measures of haemostatic activation.We measured the number of platelet-derived MPs (PMPs), endothelial cell-derived MPs (EMPs), leucocyte-derived MPs (LMPs), TF+ve MPs and annexin V (AV) binding MPs in fresh plasma by flow cytometry in patients with gynaecological malignancy and a control group. We also measured D-dimers, prothrombin fragments 1 and 2 (PF12) and thrombin-antithrombin (TAT) complexes as indirect markers of haemostatic activation.The number of MPs (from all cell types) was similar in the two patient groups, with no significant differences. The number of circulating TF+ve MPs was also similar between the two groups. D-dimers (p0.001) and PF12 (p=0.009) were significantly higher in the malignant group reflecting haemostatic activation, but there was no correlation between the level of D-dimers, PF12 and TAT and MP numbers.Using fresh samples, MPs were not significantly increased in patients with gynaecological malignancy. There was, however, evidence of haemostatic activation in the patients with malignancy, but no correlation between the number of MPs and haemostatic activation.

Microparticles (MPs) are considered to be important biological effectors of several different physiological and pathological processes. There is increasing evidence of their role in haemostasis and thrombosis, and also of their importance in cancer cell survival, invasiveness and metastasis. The level of circulating MPs has been assessed in many different disease states, and there are reports that patients with malignancy and patients with thrombosis have increased levels of circulating MPs and MP-dependent thrombogenic potential. Research into the function and effect of MPs is currently hampered by a lack of standardization in the methods used to identify and quantify them. As these methods improve it is likely that MP assays will be of use both diagnostically and therapeutically in the future.

Pharmacovigilance is an essential element of any drug treatment and considering the history of adverse events due to products used to treat inherited bleeding disorders, it should be an integral component of modern haemophilia treatment. Because inherited bleeding disorders and adverse events are rare, a multicentre, preferably multinational, adverse event reporting scheme for all clotting factor products is required. EUHASS is a European, prospective, multicentre adverse event reporting scheme in the field of inherited bleeding disorders.

OBJECTIVE To examine the effects of acute insulin-induced hypoglycemia on inflammation, endothelial dysfunction, and platelet activation in adults with and without type 1 diabetes. RESEARCH DESIGN AND METHODS We studied 16 nondiabetic adults and 16 subjects with type 1 diabetes during euglycemia (blood glucose 4.5 mmol/l) and hypoglycemia (blood glucose 2.5 mmol/l). Markers of inflammation, thrombosis, and endothelial dysfunction (soluble P-selectin, interleukin-6, von Willebrand factor [vWF], tissue plasminogen activator [tPA], high-sensitivity C-reactive protein [hsCRP], and soluble CD40 ligand [sCD40L]) were measured; platelet-monocyte aggregation and CD40 expression on monocytes were determined using flow cytometry. RESULTS In nondiabetic participants, platelet activation occurred after hypoglycemia, with increments in platelet-monocyte aggregation and P-selectin (P ≤ 0.02). Inflammation was triggered with CD40 expression increasing maximally at 24 h (3.13 ± 2.3% vs. 2.06 ± 1.0%) after hypoglycemia (P = 0.009). Both sCD40L and hsCRP (P = 0.02) increased with a nonsignificant rise in vWF and tPA, indicating a possible endothelial effect. A reduction in sCD40L, tPA, and P-selectin occurred during euglycemia (P = 0.03, P ≤ 0.006, and P = 0.006, respectively). In type 1 diabetes, both CD40 expression (5.54 ± 4.4% vs. 3.65 ± 1.8%; P = 0.006) and plasma sCD40L concentrations increased during hypoglycemia (peak 3.41 ± 3.2 vs. 2.85 ± 2.8 ng/ml; P = 0.03). Platelet-monocyte aggregation also increased significantly at 24 h after hypoglycemia (P = 0.03). A decline in vWF and P-selectin occurred during euglycemia (P ≤ 0.04). CONCLUSIONS Acute hypoglycemia may provoke upregulation and release of vasoactive substances in adults with and without type 1 diabetes. This may be a putative mechanism for hypoglycemia-induced vascular injury.

Cardiovascular disease is a major cause of morbidity and mortality in patients with chronic obstructive pulmonary disease (COPD), which may in part be attributable to abnormalities of systemic vascular function. It is unclear whether such associations relate to the presence of COPD or prior smoking habit.To undertake a comprehensive assessment of vascular function in patients with COPD and healthy control subjects matched for smoking history.Eighteen men with COPD were compared with 17 healthy male control subjects matched for age and lifetime cigarette smoke exposure. Participants were free from clinically evident cardiovascular disease.Pulse wave velocity and pulse wave analysis were measured via applanation tonometry at carotid, radial, and femoral arteries. Blood flow was measured in both forearms using venous occlusion plethysmography during intrabrachial infusion of endothelium-dependent vasodilators (bradykinin, 100-1,000 pmol/min; acetylcholine, 5-20 microg/min) and endothelium-independent vasodilators (sodium nitroprusside, 2-8 microg/min; verapamil, 10-100 microg/min). Tissue plasminogen activator (t-PA) was measured in venous plasma before and during bradykinin infusions. Patients with COPD have greater arterial stiffness (pulse wave velocity, 11 +/- 2 vs. 9 +/- 2 m/s; P = 0.003; augmentation index, 27 +/- 10 vs. 21 +/- 6%; P = 0.028), but there were no differences in endothelium-dependent and -independent vasomotor function or bradykinin-induced endothelial t-PA release (P0.05 for all).COPD is associated with increased arterial stiffness independent of cigarette smoke exposure. However, this abnormality is not explained by systemic endothelial dysfunction. Increased arterial stiffness may represent the mechanistic link between COPD and the increased risk for cardiovascular disease associated with this condition.

There have been four highly probable instances of variant Creutzfeldt-Jakob disease (vCJD) transmission by non-leucocyte depleted red cell concentrates and it is now clear that the infectious agent is transmissible by blood components. To date there in no reported evidence that the infectious agent has been transmitted by fractionated plasma products, e.g. factor VIII concentrate. This review outlines current and potential risk management strategies including donor deferral criteria, the potential for donor screening, blood component processing and prion reduction filters, plasma product manufacture and the difficulties in identification and notification of those considered 'at risk of vCJD for public health purposes'.

Summary. Objective: Pregnancy is associated with marked changes in vascular physiology and an increased risk of thrombosis. The aim of the study was to assess the effect of pregnancy on the acute release of tissue plasminogen activator (t-PA) from the endothelium. Methods and results: Ten primigravida pregnant women were recruited in the third trimester of pregnancy (week 36 ± 1) and compared with 20 age-matched non-pregnant women (day 9.8 ± 0.3 of menstrual cycle). Blood flow and plasma fibrinolytic factors were measured in both forearms by venous occlusion plethysmography and blood sampling, respectively, during unilateral brachial artery infusions of bradykinin (100–1000 pmol min−1). Pregnant women had higher plasma plasminogen activator inhibitor type 1 (PAI-1) antigen concentrations (77.1 ± 12.4 vs. 21.5 ± 9.8 ng mL−1; P = 0.004) that resulted in lower basal t-PA/PAI-1 ratios (0.2 ± 0.1 vs. 0.6 ± 0.1; P = 0.02) and plasma t-PA activity concentrations (0.17 ± 0.02 vs. 0.58 ± 0.06 IU mL−1; P

Animal models suggest a vasomotor role for the B1 kinin receptor in cardiovascular disease states. In patients with heart failure treated with angiotensin-converting enzyme inhibition (ACEi), or combined B1/B2 receptor antagonism, but not B2 receptor antagonism alone, causes vasoconstriction. However, B1 agonism has no effect on vasomotor or fibrinolytic function. Findings from transgenic animals lacking the B2 receptor suggest that these conflicting data may be explained by cross-talk between B1 and B2 receptors. We hypothesized that B1 stimulation causes vasodilatation and tissue plasminogen activator release in the human forearm when B2 receptor signaling is inhibited. Forearm blood flow was measured in 16 patients with heart failure receiving ACEi. In double-blinded crossover studies, intrabrachial Lys-[Leu8]-des-Arg9-bradykinin (B1 antagonist), lys-des-Arg9-bradykinin (B1 agonist), bradykinin (B2 agonist), and sodium nitroprusside (endothelium-independent vasodilator) were infused alone or with HOE-140 (B2 antagonist). HOE-140 did not affect basal vascular tone or t-PA release, but it abolished bradykinin-induced vasodilatation and t-PA release (P < 0.0001). Blood flow and t-PA release were unaffected by B1 agonism or antagonism in the presence and absence HOE-140. Our findings do not support a role for crosstalk between the B1 and B2 kinin receptors in the human peripheral circulation.

Objectives We sought to test the hypothesis that cigarette smoking adversely alters protease-activated receptor type 1 (PAR-1)-mediated vascular effects in vivo in humans. Background Distinct from its role in the coagulation cascade, thrombin exerts its major cellular and cardiovascular actions via PAR-1. The activation of PAR-1 causes endothelium-dependent arterial vasodilation and the release of endogenous fibrinolytic factors. Methods Forearm blood flow was measured with venous occlusion plethysmography in 12 cigarette smokers and 12 age- and gender-matched nonsmokers during intrabrachial infusions of PAR-1–activating-peptide (SFLLRN; 5 to 50 nmol/min), bradykinin (100 to 1,000 pmol/min), and sodium nitroprusside (2 to 8 μg/min). Plasma tissue plasminogen activator (t-PA) and plasminogen-activator inhibitor 1 antigen and activity concentrations were measured throughout the experiment. Results All agonists caused dose-dependent increases in forearm blood flow (p Conclusions Cigarette smoking causes marked impairment of PAR-1–mediated endothelial vasomotor and fibrinolytic function. Relative arterial stasis and near abolition of t-PA release will strongly promote clot propagation and vessel occlusion. These findings suggest a major contribution of impaired endothelial PAR-1 action to the increased atherothrombotic risk of cigarette smokers.

The purpose of this study was to determine the role of the endothelium in the vascular actions of protease-activated receptor type 1 (PAR-1) activation in vivo in man.Background Thrombin is central to the pathophysiology of atherothrombosis. Its cellular actions are mediated via PAR-1. Protease-activated receptor type 1 activation causes arterial vasodilation, venoconstriction, platelet activation, and tissue-type plasminogen activator release in man.Methods Dorsal hand vein diameter was measured in 6 healthy volunteers before and after endothelial denudation. Forearm arterial blood flow, plasma fibrinolytic factors, and platelet activation were measured in 24 healthy volunteers during venous occlusion plethysmography. The effects of inhibition of prostacyclin, nitric oxide (NO), and endothelium-derived hyperpolarizing factor on PAR-1 responses were assessed during coadministration of aspirin, the "NO clamp" (L-N-G-monomethyl arginine and sodium nitroprusside), and tetraethylammonium ion, respectively.Results Endothelial denudation did not affect PAR-1-evoked venoconstriction (SFLLRN; 0.05 to 15 nmol/min). Although aspirin had no effect, SFLLRN-induced vasodilation (5 to 50 nmol/min) was attenuated by the NO clamp (p < 0.0001) and tetraethylammonium ion (p < 0.05) and abolished by their combination (p < 0.01). The NO clamp augmented SFLLRN-induced tissue-type plasminogen activator and plasminogen activator inhibitor type 1 antigen (p < 0.0001) release, but tetraethylammonium ion and aspirin had no effect. SFLLRN-induced platelet activation was unaffected by NO or prostacyclin inhibition.Conclusions Acting via PAR-1, thrombin causes contrasting effects in the human vasculature and has a major interaction with the endothelium. This highlights the critical importance of endothelial function during acute arterial injury and intravascular thrombosis, as occurs in cardiovascular events including myocardial infarction and stroke.

Since the 1970s, mortality in the hemophilia population has been dominated by human immunodeficiency virus (HIV) and few reports have described mortality in uninfected individuals. This study presents mortality in 6018 people with hemophilia A or B in the United Kingdom during 1977 to 1998 who were not infected with HIV, with follow-up until January 1, 2000. Given disease severity and factor inhibitor status, all-cause mortality did not differ significantly between hemophilia A and hemophilia B. In severe hemophilia, all-cause mortality did not change significantly during 1977 to 1999. During this period, it exceeded mortality in the general population by a factor of 2.69 (95% confidence interval [CI]: 2.37-3.05), and median life expectancy in severe hemophilia was 63 years. In moderate/mild hemophilia, all-cause mortality did not change significantly during 1985 to 1999, and median life expectancy was 75 years. Compared with mortality in the general population, mortality from bleeding and its consequences, and from liver diseases and Hodgkin disease, was increased, but for ischemic heart disease it was lower, at only 62% (95% CI: 51%-76%) of general population rates, and for 14 other specific causes it did not differ significantly from general population rates. There was no evidence of any death from variant Creutzfeldt-Jakob disease or from conditions that could be confused with it.

Objective— The endothelium-derived fibrinolytic factor tissue plasminogen activator (t-PA) is a major determinant of vessel patency after coronary plaque rupture and thrombosis. We assessed whether endothelial fibrinolytic capacity predicts atherothrombotic events in patients with coronary heart disease. Methods and Results— Plasma t-PA and plasminogen activator inhibitor (PAI)-1 concentrations were measured during intrabrachial substance P infusion in 98 patients with angiographically proven stable coronary heart disease. Forearm blood flow was measured during infusion of substance P and sodium nitroprusside. Cardiovascular events (cardiovascular death, myocardial infarction [MI], ischemic stroke [CVA], and emergency hospitalization for unstable angina) were determined during 42 months of follow-up. Patients experiencing a cardiovascular event (n=19) had similar baseline characteristics to those free of events. Substance P caused a dose-dependent increase in plasma t-PA concentrations ( P P P =0.03 for trend); patients with the lowest quartile of t-PA release had the highest rate of adverse events ( P =0.01). Conclusion— Endothelial fibrinolytic capacity, as measured by stimulated t-PA release, predicts the future risk of adverse cardiovascular events in patients with coronary heart disease. We suggest that endothelial fibrinolytic capacity is a powerful novel determinant of cardiovascular risk.

Background— Protease-activated receptor type 1 (PAR-1) has been proposed as the principal thrombin receptor in humans, although its actions in vivo have not been defined. The aim of the present study was to determine the direct vascular actions of PAR-1 agonism in humans. Methods and Results— Dorsal hand vein diameter was measured by the Aellig technique in 14 healthy volunteers during local intravenous SFLLRN (PAR-1 agonist; 0.05 to 15 nmol/min) and SLIGKV (PAR-2 agonist; 1.6 to 160 nmol/min) infusions. The venous effects of SFLLRN were further assessed in the presence or absence of norepinephrine or the glycoprotein IIb/IIIa antagonist tirofiban. Forearm blood flow was measured by venous occlusion plethysmography in 16 volunteers during infusion of SFLLRN (1 to 50 nmol/min), SLIGKV (160 to 800 nmol/min), and the endothelium-dependent vasodilator bradykinin (100 to 1000 pmol/min). Platelet-monocyte binding (a sensitive measure of platelet activation) and plasma tissue plasminogen activator (tPA), plasminogen-activator inhibitor 1, and von Willebrand factor concentrations were measured at intervals throughout the study. SFLLRN caused dose-dependent venoconstriction ( P P P P P P Conclusions— We have demonstrated that PAR-1 agonism causes platelet activation, venous constriction, arterial dilatation, and tPA release in vivo in humans. These unique and contrasting effects provide important insights into the physiological and pathophysiological role of thrombin in the human venous and arterial circulations.

Elevated plasma t-PA (tissue plasminogen activator) and serum CRP (C-reactive protein) concentrations are associated with an adverse cardiovascular risk. In the present study, we investigated whether acute local inflammation causes vascular dysfunction and influences t-PA release in patients with stable coronary heart disease. Serum CRP, plasma t-PA and PAI-1 (plasminogen activator inhibitor type 1) concentrations were determined in 95 patients with stable coronary heart disease. A representative subpopulation of 12 male patients received an intra-brachial infusion of TNF-α (tumour necrosis factor-α) and saline placebo using a randomized double-blind cross-over study design. Forearm blood flow and plasma fibrinolytic and inflammatory variables were measured. Serum CRP concentrations correlated with plasma t-PA concentrations (r=0.37, P

To investigate whether sildenafil citrate, a selective phosphodiesterase type 5 inhibitor, may improve endothelial vasomotor and fibrinolytic function in patients with coronary heart disease.Randomised double blind placebo controlled crossover study.16 male patients with coronary heart disease and eight matched healthy men received intravenous sildenafil or placebo. Bilateral forearm blood flow and fibrinolytic parameters were measured by venous occlusion plethysmography and blood sampling in response to intrabrachial infusions of acetylcholine, substance P, sodium nitroprusside, and verapamil.Forearm blood flow and acute release of tissue plasminogen activator.Mean arterial blood pressure fell during sildenafil infusion from a mean (SEM) of 92 (1) to 82 (1) mm Hg in patients and from 94 (1) to 82 (1) mm Hg in controls (p0.001 for both). Sildenafil increased endothelium independent vasodilatation with sodium nitroprusside (p0.05) but did not alter the blood flow response to acetylcholine or verapamil in patients or controls. Substance P caused a dose dependent increase in plasma tissue plasminogen activator antigen concentrations (p0.01) that was unaffected by sildenafil in either group.Sildenafil does not improve peripheral endothelium dependent vasomotor or fibrinolytic function in patients with coronary heart disease. Phosphodiesterase type 5 inhibitors are unlikely to reverse the generalised vascular dysfunction seen in patients with coronary heart disease.

Summary As a result of immunological and nucleic-acid screening of plasma donations for transfusion-transmissible viruses, and the incorporation of viral reduction processes during plasma fractionation, coagulation-factor concentrates (CFC) are now judged safe in terms of many known infectious agents, including hepatitis B and C viruses, HIV, and human T-cell lymphotropic virus. However, emerging pathogens could pose future threats, particularly those with blood-borne stages that are resistant to viral-inactivation steps in the manufacturing process, such as non-lipid-coated viruses. As outlined in this Review, better understanding of infectious diseases allows challenges from newly described agents of potential concern in the future to be anticipated, but the processes of zoonotic transmission and genetic selection or modification ensure that plasma-derived products will continue to be subject to infectious concerns. Manufacturers of plasma-derived CFC have addressed the issue of emerging infectious agents by developing recombinant products that limit the need for human plasma during production. Such recombinant products have extended the safety profile of their predecessors by ensuring that all reagents used for cell culture, purification steps, and stabilisation and storage buffers are completely independent of human plasma.

Thrombolytic therapy fails to achieve reperfusion in almost a third of patients with acute myocardial infarction. Thrombin activatable fibrinolysis inhibitor (TAFI) and soluble CD40 ligand (sCD40L) are novel endogenous fibrinolytic and atherothrombotic factors that determine clot stability. We investigated whether admission plasma thrombin activatable fibrinolysis inhibitor (TAFI) and soluble CD40 ligand (sCD40L) concentrations predicted reperfusion following thrombolytic therapy in patients with acute myocardial infarction.Prior to administration of thrombolytic therapy, venous blood was collected from 110 patients presenting with acute ST segment elevation myocardial infarction and plasma assayed for tissue plasminogen activator (t-PA) antigen and activity, plasminogen activator inhibitor type-1 antigen (PAI-1), TAFI antigen and activity, C-reactive protein (CRP) and sCD40L concentrations. Reperfusion was determined using continuous ST segment monitoring.Reperfusion occurred in 77 (70%) patients with a mean treatment to reperfusion time of 83 +/- 46 min. Peak creatine kinase was significantly lower in patients who reperfused (1578 +/- 1199 versus 2200 +/- 1744 U/L; P0.05) and correlated with time to reperfusion (r = 0.44 [95% CI: 0.23 - 0.61], P = 0.0001). There was a modest correlation between plasma TAFI antigen and activity (r = 0.3 [95% CI: 0.04 - 0.53]; P0.05). There were no significant associations between coronary reperfusion and plasma concentrations of t-PA, PAI-1, TAFI, CRP or sCD40L.Systemic plasma TAFI, sCD40L and CRP concentrations do not predict reperfusion in patients receiving thrombolytic therapy for acute ST elevation myocardial infarction.

Objective— Vascular expression of the B 1 kinin receptor is markedly upregulated with left ventricular dysfunction and angiotensin-converting enzyme (ACE) inhibition, but its function remains unclear. Inhibitors of ACE potentiate bradykinin-mediated B 2 receptor-dependent vasodilatation and tissue plasminogen activator (tissue-type plasminogen activator [t-PA]) release. We investigated the contribution of the B 1 receptor to the maintenance of vascular tone and t-PA release in patients with heart failure. Methods and Results— Eleven patients were treated with enalapril (10 mg twice daily) or losartan (50 mg twice daily) in a randomized double-blind crossover trial. During week 6 of each treatment, patients received an intrabrachial infusion of Lys-des-Arg 9 -bradykinin (B 1 agonist; 1 to 10 nmol/min), bradykinin (30 to 300 pmol/min), Lys-[Leu 8 ]-des-Arg 9 -bradykinin (B 1 antagonist; 1 to 10 nmol/min), and norepinephrine (60 to 540 pmol/min). Blood flow and t-PA release were measured using venous occlusion plethysmography and blood sampling. Bradykinin ( P 9 -bradykinin, caused vasodilatation and t-PA antigen and activity release. Norepinephrine ( P 8 ]-des-Arg 9 -bradykinin, caused vasoconstriction. Compared with losartan, enalapril augmented bradykinin-mediated vasodilatation ( P P 1 receptor-mediated responses. Conclusions— The B 1 kinin receptor does not have a major vasomotor or fibrinolytic role in patients with heart failure. Augmentation of kinin-mediated vasodilatation and t-PA release by ACE inhibition is restricted to the B 2 receptor.

Hyperhomocysteinaemia is a prothrombotic condition that may cause oxidative endothelial injury and impair endogenous fibrinolysis. Vitamin supplementation enhances endothelial function in hyperhomocysteinaemic patients, but responses in patients with co-existing coronary artery disease have been variable. It is also unknown whether hyperhomocysteinaemia is associated with reduced fibrinolytic responses in patients with coronary artery disease. The study aims were to test the hypothesis that patients with recent myocardial infarction and hyperhomocysteinaemia have impaired endothelium-dependent vasomotion and fibrinolysis that is rectified by vitamin supplementation. From a cohort of 120 patients admitted with acute myocardial infarction, 18 patients were recruited from the upper (n=9) and lower (n=9) plasma homocysteine quartiles into a randomized double-blind placebo-controlled crossover trial. Following a 4-week course of placebo or folate/cyanocobalamin/pyridoxine supplements, FBF (forearm blood flow) was measured using venous occlusion plethysmography during intra-arterial substance P (4–16 pmol/min), acetylcholine (5–20 μg/min) and sodium nitroprusside (2–8 μg/min) infusions. All vasodilators caused dose-dependent increases in infused FBF (P

Angiotensin-converting enzyme and neutral endopeptidase (EC 3.4.24.11; neprilysin) are metallopeptidases present on the endothelium that metabolize bradykinin. Inhibitors of angiotensin-converting enzyme potentiate bradykinin-mediated vasodilatation and endothelial tissue plasminogen activator release. Combined angiotensin-converting enzyme and neutral endopeptidase inhibition may have additional beneficial cardiovascular effects mediated through bradykinin potentiation. We investigated the effects of local neutral endopeptidase inhibition on the vascular actions of bradykinin in heart failure patients maintained on chronic angiotensin-converting enzyme inhibition. Ten patients received intrabrachial infusion of thiorphan (30 nmol/min), a neutral endopeptidase inhibitor, in a randomized double-blind placebo-controlled crossover trial. Thiorphan was coinfused with Lys-des-Arg 9 -bradykinin (1 to 10 nmol/min), bradykinin (30 to 300 pmol/min), atrial natriuretic peptide (10 to 100 pmol/min), and sodium nitroprusside (2 to 8 μg/min). Bradykinin, atrial natriuretic peptide, and sodium nitroprusside caused dose-dependent vasodilatation (peak blood flow 14.4±2.2, 3.6±0.6, and 8.6±1.3 mL per 100 mL/min, respectively; P P P P P

Objective— We sought to assess pharmacodynamic responses to the bradykinin antagonist B9340 and to determine the contribution of the endothelial bradykinin receptor to stimulated tissue plasminogen activator (t-PA) release in humans. Methods and Results— Bilateral forearm blood flow and plasma fibrinolytic variables were measured in 8 volunteers during 100 minutes of intrabrachial infusions of saline placebo, B9340 at 4.5 nmol/min, or B9340 at 13.5 nmol/min. On each occasion, intra-arterial bradykinin (30 to 3000 pmol/min) and substance P (4 to 16 pmol/min) were coinfused for 10 minutes at each dose. To assess the onset and offset of action, 6 additional subjects on 2 occasions received intra-arterial bradykinin (100 pmol/min) for 60 minutes with a coinfusion of either saline placebo or B9340 (13.5 nmol/min) for 12 minutes. During placebo infusion, bradykinin and substance P caused dose-dependent vasodilatation in the infused forearm ( P P P =NS). B9340 caused a reversible inhibition of bradykinin-induced vasodilatation ( P Conclusions— B9340 is a potent, reversible, and selective competitive receptor antagonist of bradykinin-induced vasodilatation and t-PA release in humans.

Objective—Inflammation contributes to the pathogenesis of cardiovascular disease, potentially through the actions of proinflammatory cytokines. We assessed the direct effects of local intra-arterial tumor necrosis factor-α (TNF-α), interleukin-6, and endotoxin on blood flow and endogenous tissue plasminogen activator (t-PA) release in vivo in humans.Methods and Results—In a double-blind, randomized, placebo-controlled trial, blood flow, plasma cytokine, and fibrinolytic parameters were measured using venous occlusion plethysmography and blood sampling. Ten subjects received intrabrachial TNF-α, interleukin-6, and endotoxin infusions, and 8 additional subjects received intrabrachial infusions of bradykinin, acetylcholine, and sodium nitroprusside after pretreatment with TNF-α. TNF-α but not interleukin-6, endotoxin, or placebo caused a gradual and sustained ≈20-fold increase in plasma t-PA concentrations (PPPPConclusions—Intra-arterial TNF-α causes an acute local vascular inflammation that is associated with impaired endothelium-dependent vasomotion as well as a sustained and substantial increase in endothelial t-PA release. TNF-α has potentially both adverse vasomotor and beneficial profibrinolytic effects on endothelial function.

After surgery in haemophilia, haemostasis is difficult to maintain in the presence of an antifactor VIII antibody. This study assessed the pharmacokinetics of recombinant activated factor VII (rFVIIa) and its efficacy in securing post-operative haemostasis in haemophiliacs with inhibitors. Continuous infusion of rFVIIa was evaluated for elective major orthopaedic surgery in nine patients with neutralizing antibodies to FVIII and at high risk of bleeding. After an initial preoperative bolus of 90 micro g/kg, rFVIIa was infused at a fixed rate of 50 micro g/kg/h for a median of 20 d (range 7-20 d). The median plasma FVII coagulant activity (FVII:C) at 24 h, 72 h and 20 d after surgery was 38 IU/ml (range 22-169 IU/ml), 45 IU/ml (range 17-88 IU/ml) and 31 IU/ml (range 27-46 IU/ml) respectively. The median plasma FVIIa:C at the same time points was 51 (range 24-211), 63 (range 22-99) and 44 (range 28-76) IU/ml respectively. Median total rFVIIa clearance remained stable during the rFVIIa continuous infusion period and was 40 (range 9-70), 34 (range 17-86) and 48 (range 32-55)ml/kg/h at the end of 24 h, 72 h and 20 d infusion respectively. Post-operatively, there were bleeds in six patients, which settled readily after a single bolus of rFVIIa (60 micro g/kg). There was a good clinical outcome for all patients. These data indicate that rFVIIa infusion at 50 micro g/kg/h achieves continuous plasma FVII procoagulant activity in excess of 30 IU/ml (12-15 nmol/l) and provides adequate haemostatic control for inhibitor patients during major orthopaedic surgery.

ObjectivesThe purpose of this study was to investigate in vivo the effects of acute systemic inflammation on the endogenous fibrinolytic capacity in men.BackgroundSystemic inflammation and endogenous fibrinolysis play a major role in the pathogenesis of coronary artery disease. Although previous studies have shown impaired endothelium-dependent vasomotor function, the effects of inflammation on the endothelial release of the fibrinolytic factor tissue plasminogen activator (t-PA) are unknown.MethodsIn a double-blind randomized placebo-controlled crossover trial, we administered a mild inflammatory stimulus, Salmonella typhivaccine, or saline placebo to eight healthy men on two separate occasions. Six hours after vaccination, blood flow and plasma fibrinolytic variables were measured in both arms during intrabrachial infusions of bradykinin (40 to 1,000 pmol/min), acetylcholine (5 to 20 μg/min), and sodium nitroprusside (2 to 8 μg/min).ResultsCompared with placebo, the S. typhivaccination caused a rise in white cell count (11.1 ± 0.5 ×109/l vs. 7.9 ± 0.8 ×109/l; p = 0.004) and plasma interleukin-6 concentrations (6.9 ± 1.4 pg/ml vs. 1.6 ± 0.4 pg/ml; p = 0.01) in addition to a significant augmentation of t-PA antigen (45 ± 9 ng/100 ml/min at peak dose vs. 24 ± 8 ng/100 ml/min at peak dose; p = 0.016, analysis of variance) and activity (104 ± 15 IU/100 ml/min vs. 54 ± 12 IU/100 ml/min; p = 0.006, analysis of variance) release during bradykinin infusion. Forearm blood flow increased in a dose-dependent manner after bradykinin, acetylcholine and sodium nitroprusside infusions (p < 0.001), but this was unaffected by vaccination.ConclusionsOur results showed that acute systemic inflammation augmented local forearm t-PA release in men, which suggests that acute inflammation may invoke a protective response by enhancing the acute endogenous fibrinolytic capacity in healthy men. Further studies are needed to clarify whether this response is impaired in patients with cardiovascular disease.

We have developed a novel assay for quantitation of human von Willebrand factor (vWF) messenger RNA (mRNA) using the ABI 7700™ quantitative PCR system (Applied Biosystems, Warrington, Cheshire, UK). To allow accurate measurement of the vWF mRNA concentration in samples, we have used 18S ribosomal RNA as an internal control. The specificity of the assay was demonstrated by polyacrylamide gel electrophoresis and ABI Prism® BigDye™ terminator cycle sequencing of the PCR product. We have also assessed the assay by using RNA from both a hepatocyte cell line which does not express vWF mRNA, and an endothelial cell line which does express vWF mRNA. By mixing varying amounts of these RNAs together, we were able to adjust the vWF quantity in known increments while keeping the 18S ribosomal RNA internal control constant. The quantitation of vWF mRNA correlated with the proportion of endothelial cell RNA (r2=0.912, p

Background: In patients with acutely symptomatic but nonruptured abdominal aortic aneurysm (AAA), emergent repair is associated with an increased mortality rate as compared with semi-elective repair. Previous results have shown that ruptured but not asymptomatic AAA repair is associated with intense thrombin generation and inhibition of systemic fibrinolysis. The purpose of this study was to determine whether circulating markers of coagulation and fibrinolysis may be used to distinguish acutely symptomatic nonruptured and ruptured AAA. Methods: A prospective study was performed of 44 patients who underwent emergency AAA repair for suspected rupture. Platelet count, fibrinogen level, prothrombin time, activated partial thromboplastin time, tissue plasminogen activator (t-PA) activity, plasminogen activator inhibitor (PAI) activity, prothrombin fragment (PF) 1+2 level, and D dimer level were measured before surgery. Results: When compared with ruptured AAAs (n = 37), acutely symptomatic nonruptured AAAs (n = 7) were associated with increased fibrinogen level ( P =.033), reduced activated partial thromboplastin time ( P =.043), increased t-PA activity ( P =.023), reduced PAI activity ( P =.005), reduced PF 1+2 level ( P =.001), and reduced D dimer level ( P =.005; all P values determined with Mann-Whitney test). The differences in t-PA activity ( P =.01), PAI activity ( P =.004), and PF 1+2 level ( P =.01) persisted in patients whose conditions were normotensive. In all patients, a PF 1+2 level of greater than or equal to 2.5 nmol/L was associated with a sensitivity, specificity, and positive and negative predictive value for rupture of 89%, 86%, 97%, and 60%, respectively. In patients whose conditions were normotensive, PAI activity of greater than or equal to 16 AU/mL was associated with a sensitivity, specificity, and positive and negative predictive value of 83%, 100%, 100%, and 88%, respectively. Conclusion: These data show that acutely symptomatic nonruptured AAA is associated with increased systemic fibrinolysis (caused by reduced fibrinolytic inhibition) and reduced thrombin generation as compared with rupture. Preoperative hemostatic markers, particularly PF 1+2 level and PAI activity, may distinguish acutely symptomatic nonruptured from ruptured AAA. (J Vasc Surg 2002;35:661-5)

The European Study on the Clinical Outcomes and Resource Utilization associated with Haemophilia Care was designed to compare various health outcomes associated with on-demand and prophylactic factor substitution methods in European haemophilia patients. While the primary objective of this research is to conduct an economic analysis, an important component of this study is to evaluate quality-of-life differences that may exist between patients who utilize these two styles of therapy. Quality-of-life research has emerged as a primary measure of health outcomes because it allows the augmentation of traditional clinical indicators of health with data gathered from the patient's perspective. A total of 1033 haemophilia patients from 16 European haemophilia treatment centres were enrolled in this study. The SF-36, a multidimensional quality-of-life instrument, was administered to all participants. This instrument measures eight health-related quality-of-life dimensions: physical functioning, physical role limitations, bodily pain, general health, vitality, social functioning, emotional role limitations, and mental health. All haemophilia subjects enrolled in the study scored significantly lower than the population normative means in the three physical dimensions and in the general health dimension. HIV-negative haemophiliac subjects differed significantly by factor substitution type in a multivariate analysis examining all eight health dimensions. Univariate analyses testing each dimension separately indicated that patients treated prophylactically reported significantly less bodily pain, better general health, and scored significantly higher in the physical functioning, mental health, and social functioning dimensions. While these results suggest that health-related quality-of-life may be better for haemophilia patients treated prophylactically, future prospective studies that gather periodic quality-of-life data over time should be conducted.

Over the past 10 years considerable use has been made of recombinant factor VIIa (rFVIIa) for the treatment of patients with inhibitors to coagulation factors. During this time, its place in the management of acute bleeds and surgery has become better defined. Although pharmacokinetic studies report the half-life of rFVIIa as 2.7 h, there is considerable intersubject variability. Moreover, rFVIIa is cleared more rapidly in children than in adults. Assays for the measurement of rFVIIa plasma levels are not readily available in clinical diagnostic laboratories, although there is evidence that plasma FVII:C levels, measured by a one-stage prothombin-based assay, reflect the plasma concentration of rFVIIa:C. The level of FVII:C required to achieve haemostasis in different clinical circumstances remains uncertain. In order to overcome the logistic difficulties of repeated frequent bolus injections, and potentially to minimise usage, administration of rFVIIa by continuous infusion has been reported. However, there is some uncertainty as to whether continuous infusion of rFVIIa has similar therapeutic efficacy to an equivalent total dose administered by bolus injections. The extensive clinical experience with rFVIIa in haemophilic patients with inhibitors has been recorded in descriptive accounts of the Compassionate Use Programme and the Emergency Use Study. On the basis of the apparent clinical efficacy and safety reported in these studies, prospective randomised trials of different dose regimens have been undertaken for the treatment of acute bleeds and surgery. These have helped to define the minimum dose needed to achieve haemostasis. There remains considerable uncertainty about the minimal effective dose and appropriate duration of therapy in different clinical circumstances. There is therefore a need for the development of evidence-based guidelines for the use of rFVIIa in bolus and continuous infusion regimens in different settings, and for the therapeutic value of measuring plasma concentrations of rFVIIa, to facilitate the optimal use of this product. Furthermore, additional randomised clinical trials will help ensure that rFVIIa is used in the most clinically and cost effective way.

We conducted a multicentre, cross- sectional study of 1042 haemophilia subjects across Europe to compare various health outcomes associated with on-demand vs. prophylactic factor-substitution therapy. Demographic, medical history, and healthcare resource utilization data were analysed along with the number of bleeding events over the past 6 months. Treatment-cost data were also examined to provide preliminary information for future economic studies. A logistic regression analysis, controlling for other statistically significant covariates, showed that patients treated on demand were 3.4 times more likely to have had a joint bleed over the previous 6 months than those treated with prophylaxis. Multiple regression analyses further confirmed these findings, because on-demand subjects had, on average, 5.15 more joint bleeds over the reporting period than patients treated with prophylaxis. Notably, these findings were even more dramatic for younger haemophilia patients when our study sample was stratified by age. Due to the high cost of factor replacement, healthcare costs were significantly higher for subjects treated prophylactically. While hospital costs for prophylaxis subjects were, on average, lower, statistically significant cost savings for prophylactic subjects were not noted. These results suggest that clinicians and health policy decision-makers should consider the advantages of prophylactic therapy for haemophilia patients in formulating treatment protocols and allocating health resources.

Background . Measurements of activated coagulation time do not correlate with plasma concentration of heparin. This study investigated the effects of a patient-specific method to manage anticoagulation and its reversal in pediatric patients undergoing cardiopulmonary bypass. Methods . Infants and children were randomly assigned to receive either a standard dose of heparin (300 IU/kg; group C, n=13) or an individualized dose, calculated by an in vitro heparin dose-response test (group HC, n=13). Protamine dose was based on a 1 mg/1 mg ratio of total administered heparin for patients in group C and of the residual heparin concentration in group HC. Results . Administered heparin was significantly higher and total protamine dose was significantly reduced in the HC group (both p ≤ 0.001). There was less thrombin generation ( p = 0.02) and fibrinolysis ( p = 0.05) in group HC. Blood loss and requirement for transfusion of blood and fresh frozen plasma were also lower in group HC (all p ≤ 0.05). Conclusions . An individualized management of anticoagulation and its reversal results in less activation of the coagulation cascade, less fibrinolysis, and reduced blood loss and need for transfusions. Further studies are warranted to better define the clinical impact of these findings.

SummaryWe 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 μg/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 μg/kg/h infusion rate reliably achieves plasma factor VII activity levels of 10 IU/ml, but this level does not provide reliable haemostasis.

We present two cases in which the occurrence of acquired haemophilia is associated with the use of depot preparations of the thioxanthenes zuclopenthixol and flupenthixol. These drugs have not previously been implicated in the aetiology of acquired haemophilia.

To estimate the resources required to manage patients with haemophilia in Scotland, we studied the demographic features, hospital admissions and causes of deaths for individuals with haemophilia A and B and von Willebrand disease, treated with blood products, during the period 1980-94 living in central Scotland. Data were obtained from 413 adults and children (93% ascertainment). The age distribution in 1980 revealed a paucity of individuals over 60 years but the number in this age group increased over the study period. Of those with haemophilia A and B, 63 and two respectively, became HIV positive. Hospital admissions rose from 103 to 168 per annum; the number of annual bed days utilized also increased, but there was marked annual fluctuation (790-1832). The rate of admission was greater for those with severe haemophilia A and this increased during the 15-year period mainly due to the clinical consequences of human immunodeficiency virus (HIV) and hepatitis C virus (HCV). The admission rate for haemophilia B was significantly lower than that for haemophilia A, and was similar for all degrees of severity of the disorder. Throughout the 15-year period the incidence of admissions for acute bleeds was constant, as was the average duration in hospital. For those with a factor VIII inhibitor, the rate of admission was about double the rate of those without an inhibitor, although the duration of hospital stay was similar for both groups. There were 61 deaths; the death rate increased during the study period principally due to HIV and HCV, and 12 patients died from haemorrhage. We conclude that: (i) the life expectancy for haemophiliacs in Scotland was generally increasing, although HIV and HCV caused increasing mortality and morbidity (as shown by the increase in hospital admissions); (ii) hospital bed usage for the treatment of acute bleeds continued to be required, but fluctuated greatly; and (iii) the clinical impression that haemophilia B is less clinically severe than haemophilia A is confirmed by objective data. The planning implications for haemophilia care in Scotland and similar countries are discussed.

Coagulation factor concentrates are known to inhibit a variety of immune reactions when assessed in vitro. This study assessed the immunomodulatory activity of a wide range of coagulation factor concentrates by measuring their inhibition of PHA-stimulated lymphocyte proliferation and reduction in IL-2 secretion. The hypothesis that TGF-β is responsible for most of these effects was tested by measuring biologically active TGF-β and immunoreactive TGF-β1 in the concentrates and comparing the levels recorded with immunosuppressive activity. In addition, the coagulation factors were compared directly with a standard preparation of TGF-β in a TGF-β-specific bioassay and in lymphocyte proliferation assays. Although there was a broad correlation between levels of total or active TGF-β and immunosuppressive activity across all of the coagulation factors tested, individual data sets showed clear discrepancies. Implying that TGF-β probably serves as a surrogate marker for other immunomodulatory contaminants and that neither TGF-β nor any other single substance could account for all of the immunosuppressive activity observed. Furthermore, there was a difference of more than 100-fold in the relative potencies of coagulation factors and pure TGF-β, when compared in immunosuppression assays, indicating that the different assays did not measure the same substance. Whereas anti-TGF-β antibody almost completely blocked the activity of coagulation factor concentrates (TGF-β-specific bioassay) and abrogated the effect of authentic TGF-β (immunosuppression assays) at high concentrations it achieved

Summary Background A newly discovered DNA virus, transfusion- transmitted virus (TTV), has been implicated as a cause of post-transfusion hepatitis. We investigated the frequency of TTV viraemia in UK blood donors, and the extent to which TTV contaminates blood products such as factor VIII and IX clotting factors. We also investigated the possible aetiological role of TTV in cryptogenic fulminant hepatic failure (FHF). Methods We extracted DNA from plasma of blood donors and patients with FHF, and from blood products (factor VIII and IX clotting-factor concentrates, immunoglobulin preparations). We detected TTV by PCR using primers from a conserved region in the TTV genome. Findings TTV viraemia was detected in 19 (1·9%) of 1000 non-remunerated regular blood donors. Infection occurred more frequently in older donors (mean age 53 years), compared with the age prolife of donors infected with hepatitis C virus and other parenterally-transmitted viruses. TTV contamination was found in ten (56%) of 18 batches of factor VIII and IX concentrate manufactured from such non- remunerated donors, and in seven (44%) of 16 batches of commercially available products. Whereas solvent or detergent treatment had little effect on the detection of TTV in factor VIII and IX by PCR, this virucidal step seemed to inactivate TTV infectivity. TTV infection was detected in four (19%) of 21 patients with FHF; in three cases, infection was detected at the onset of disease and could thus not be excluded from its aetiology. Interpretation TTV viraemia is frequent in the blood-donor population, and transmission of TTV through transfusion of blood components may have occurred extensively. Clinical assessment of infected donors and recipients of blood and blood products, and assessment of TTV's aetiological role in hepatic and extra-hepatic disease, are urgently needed.

Objective : Effective endogenous fibrinolysis requires rapid release of endothelial tissue plasminogen activator (t-PA). Using the nitric oxide synthase inhibitor, l- N G-monomethylarginine (l-NMMA), we examined the contribution of endogenous nitric oxide to substance P-induced t-PA release in vivo in man. Methods : Blood flow and plasma fibrinolytic and haemostatic factors were measured in both forearms of 8 healthy male volunteers who received unilateral brachial artery infusions of substance P (2–8 pmol/min) and l-NMMA (1–4 μg/min). Results : Substance P caused dose-dependent increases in blood flow ( P