Activation, activity and inactivation of factor VIII in factor VIII products.

INTRODUCTION: Factor VIII (FVIII) products used in haemophilia A treatment show inter-and intra-product and inter-assay differences in specific activity. The mechanistic basis of these differences remains unclear. AIM: The aim of this study was to mechanistically compare the functional properties of an in-house excipient-free full-length FVIII standard and pharmacologic recombinant products containing full-length (products A and B) or B-domainless (C and D) FVIII. METHODS: Factor VIII protein concentration was quantitated by ELISA. Product potency determinations (APTT, intrinsic tenase assays) and kinetic analyses detailing these products' activations by thrombin and FXa, their spontaneous and activated protein C (APC) catalysed inactivation and their performances in coagulation proteome reconstructions were studied +/- von Willebrand factor (VWF). Computational models were developed to facilitate interpretation of empirical data. RESULTS: Factor VIII protein content per manufacturer activity unit was highest for product C with the other three products similar to the standard. Potency estimates, done five different ways, varied 20-30% in inter- and intra-assay comparisons, with product B consistently showing lower specific activity. Kinetic analyses showed the five FVIII species to differ somewhat in maximum rate of activation, the maximum level of activity achieved, the rate of spontaneous or APC catalysed inactivation and the magnitude of the effect of VWF on these parameters. When evaluated both computationally and empirically in the context of tissue factor initiated thrombin generation, product C appears the most dissimilar. CONCLUSION: Assessments of FVIII activation/inactivation dynamics report larger differences between FVIII products than standard functional assays. However, all FVIII products promote a 'normal' thrombin generation response to TF.

Thrombin generation and bleeding in haemophilia inhibitor patients during immune tolerance induction.

BACKGROUND: Inhibitor formation complicates haemophilia treatment and requires immune tolerance induction to rid inhibitors over 5 BU. In the prospective, randomized International Immune Tolerance Study, immune tolerance induction was equally effective with high-dose (HD) (200 IU kg-1 day-1 ) and low-dose (LD) (50 IU kg-1 3× per week) factor VIII, but haemorrhages were twofold higher in the LD arm. This finding was unexpected as inhibitors neutralize FVIII activity. We hypothesized that the thrombin generation assay (TGA), a global measure of clot formation, might predict bleeding better than FVIII levels. METHODS: We evaluated TGA using relipidated tissue factor (TF) on 83 thawed, recalcified corn trypsin inhibitor/citrate plasma samples from 31 subjects (17 HD, 14 LD) who participated on the ITI study, and who had sufficient sample available and appropriate informed consent. RESULTS: There were no significant differences in peak thrombin, estimated thrombin potential, maximum rate or lag time between HD and LD arms; between pre-, during and post-ITI time points, or after FVIII spiking. In 19 subjects (12 HD, 7 LD) with anti-FVIII<1.0 BU, the prevalence of non-neutralizing antibody (NNA) and neutralizing antibody (NA) was 89.5% (17/19), and the latter strongly correlated with anti-VIII titer, r = 0.73 [95% CI: 0.55, 0.88]. CONCLUSION: In haemophilia inhibitor patients, thrombin generation is present, but does not predict bleeding risk. Following tolerance induction, NNA remains detectable in the majority.

Product-dependent anti-factor VIII antibodies.

The development of anti-factor (F)VIII antibodies in haemophilia A (HA) subjects undergoing replacement therapy has been well documented. The correlation between antibody development and the FVIII product used for replacement therapy remains a subject of discussion. The aim of this study was to evaluate the presence of anti-FVIII antibodies towards three commercial rFVIII products in 34 HA subjects' plasmas. Antibodies were quantitated by a Multiplex Fluorescence Immunoassay. All plasmas contained anti-FVIII antibodies at variable concentrations ranging from 50 nm to 570 µm. Eleven of the 20 HA subjects treated with one (r)FVIII product contained inhibitory anti-FVIII antibodies (0.8-3584 BU). The inhibitory antibody titre and the molar concentrations of total antibody were mildly correlated (r(2) = 0.6). Pronounced differences in antibody recognition with the three rFVIII products were observed. For the group treated with Product 'A', the titre towards this product was 2.4-fold higher than that observed with another full-length rFVIII-containing product (Product 'B') and almost four-fold higher than that measured with a B domain-less rFVIII product (Product 'C'). For the group of 14 HA subjects treated with FVIII other than Product 'A', only one showed higher antibody titre when measured with this product. Our data suggest that the development of anti-FVIII antibodies is biased towards the product used for treatment and that a significant fraction of antibodies bind to the B domain of FVIII.

Measuring the mechanical properties of blood clots formed via the tissue factor pathway of coagulation.

Thrombelastography (TEG) is a method that is used to conduct global assays that monitor fibrin formation and fibrinolysis and platelet aggregation in whole blood. The purpose of this study was to use a well-characterized tissue factor (Tf) reagent and contact pathway inhibitor (corn trypsin inhibitor, CTI) to develop a reproducible thrombelastography assay. In this study, blood was collected from 5 male subjects (three times). Clot formation was initiated in whole blood with 5 pM Tf in the presence of CTI, and fibrinolysis was induced by adding tissue plasminogen activator (tPA). Changes in viscoelasticity were then monitored by TEG. In quality control assays, our Tf reagent, when used at 5 pM, induced coagulation in whole blood in 3.93 ± 0.23 min and in plasma in 5.12 ± 0.23 min (n=3). In TEG assays, tPA significantly decreased clot strength (maximum amplitude, MA) in all individuals but had no effect on clot time (R time). The intraassay variability (CVa<10%) for R time, angle, and MA suggests that these parameters reliably describe the dynamics of fibrin formation and degradation in whole blood. Our Tf reagent reproducibly induces coagulation, making it an ideal tool to quantify the processes that contribute to mechanical clot strength in whole blood.

Alloantibodies to factor VIII in haemophilia.

Up to one-third of haemophilia A patients develop factor VIII (FVIII) alloantibodies (inhibitors). The Bethesda assay detects inhibitors but is relatively insensitive. Recently, a new fluorescence-based immunoassay (FLI) was developed for antibody detection. The aim of this study was to assess the prevalence of inhibitors as measured by FLI. Assays of FVIII, FVIII inhibitor by Bethesda assay with Nijmegen modification, and FVIII inhibitor by FLI were performed on adult patients with haemophilia A. Data were complete for 46 patients (median age 39), of whom 72% were severe, 7% moderate and 22% mild. The Bethesda assay was positive in only two patients (4%), while FLI was positive in 23 of 46 patients (50%), with values ranging from 0.4 to 33.7 nm (median 3.5 nm). FLI titres exceeded 7.0 nm in 19.5% of patients, all but one of whom had severe haemophilia. FLI antibody-positive patients were less likely to be HIV positive (30% vs. 70%, P = 0.02). The use of a prophylaxis regimen was associated with a lower incidence of antibody; only two of 23 patients with detectable antibody and none of those with antibody >7 nm were on a prophylaxis regimen, while nine of 23 patients without antibody were on prophylaxis, (P = 0.03). There was no difference in inhibitor presence in patients using recombinant versus plasma-derived factor. Antibodies detected by FLI are frequent in patients with haemophilia A, but are less common in those who are HIV positive or are receiving regular FVIII prophylaxis.

Potency and mass of factor VIII in FVIII products.

Several factor (F) VIII products of different origin and structure are being used for haemophilia A treatment worldwide. The assessment of FVIII concentration in these products is done using activity assays, which are dependent upon the assay and its modifications. To evaluate FVIII products for potency and for FVIII concentration and specific activity, three activity-based assays [activated partial thromboplastin time (APTT), intrinsic FXase and synthetic coagulation proteome] and two immunoassays (ELISA and western blotting) were used in this study with albumin-free full-length recombinant (r) FVIII as a standard. In all activity assays, products A and B (both contain full-length rFVIII) at 1 U mL(-1) showed potency similar to that of the 0.7 nm (1 U mL(-1)) rFVIII standard. Product E (contains truncated rFVIII) was less potent in the APTT (83% of standard) and product C (contains plasma FVIII) was less potent in FXase assays (66%). The ELISA immunoassay revealed that the specific activity of FVIII proteins in products A-C and E varied over a wide range (3900-13 200 U mg(-1)) and was higher for most lots when compared with the standard (5000 U mg(-1)), whereas the specific activity of product D (contains plasma FVIII) was lower than expected (3200-4800 U mg(-1)). (i) FVIII potency estimated in different assays gives dissimilar results; (ii) the specific activity of FVIII in various FVIII products is different and inconsistent. Thus, the administration of an equal FVIII potency in units means the administration of different amounts of FVIII protein, which may partly explain apparent discrepancies in product performance.

Blood coagulation dynamics in haemostasis.

Our studies involve computational simulations, a reconstructed plasma/platelet proteome, whole blood in vitro and blood exuding from microvascular wounds. All studies indicate that in normal haemostasis, the binding of tissue factor (TF) with plasma factor (F) VIIa (extrinsic FXase complex) results in the initiation phase of the procoagulant response. This phase is negatively regulated by tissue factor pathway inhibitor (TFPI) in combination with antithrombin (AT) and the protein C (PC) pathway. The synergy between these inhibitors provides a threshold-limited reaction in which a stimulus of sufficient magnitude must be provided for continuation of the reaction. With sufficient stimulus, the FXa produced activates some prothrombin. This initial thrombin activates the procofactors and platelets required for presentation of the intrinsic FXase (FVIIIa-FIXa) and prothrombinase (FVa-FXa) complexes which drive the subsequent propagation phase; continuous downregulation of which is provided by AT and the thrombin-thrombomodulin-PC complex. FXa generation during the propagation phase is largely (>90%) provided by the intrinsic FXase complex. TF is required for the initiation phase of the reaction but becomes non-essential once the propagation phase has been achieved. The propagation phase catalysts (FVIIIa-FIXa and FVa-FXa) continue to drive the reaction as blood is resupplied to the wound site by flow. Ultimately, the control of the reaction is governed by the pro- and anticoagulant dynamics and the supply of blood reactants to the site of a perforating injury. Our systems have been utilized to examine the qualities of hypothetical and novel antihaemorrhagic and anticoagulation agents and in epidemiologic studies of venous and arterial thrombosis and haemorrhagic pathology.

Thrombin generation in acute coronary syndrome and stable coronary artery disease: dependence on plasma factor composition.

BACKGROUND: Acute coronary syndrome (ACS) is associated with thrombin formation, triggered by ruptured or eroded coronary atheroma. We investigated whether thrombin generation based on circulating coagulation protein levels, could distinguish between acute and stable coronary artery disease (CAD). METHODS AND RESULTS: Plasma coagulation factor (F) compositions from 28 patients with ACS were obtained after onset of chest pain. Similar data were obtained from 25 age- and sex-matched patients with stable CAD. All individuals took aspirin. Patients on anticoagulant therapy were excluded. The groups were similar in demographic characteristics, comorbidities and concomitant treatment. Using each individual's coagulation protein composition, tissue factor (TF) initiated thrombin generation was assessed both computationally and empirically. TF pathway inhibitor (TFPI), antithrombin (AT), factor II (FII) and FVIII differed significantly (P < 0.01) between the groups, with levels of FII, FVIII and TFPI higher and AT lower in ACS patients. When thrombin generation profiles from individuals in each group were compared, simulated maximum thrombin levels (P < 0.01) and rates (P < 0.01) were 50% higher with ACS while the initiation phases of thrombin generation were shorter. Empirical reconstructions of the populations reproduced the thrombin generation profiles generated by the computational model. The differences between the thrombin generation profiles for each population were primarily dependent upon the collective contribution of AT, FII and FVIII. CONCLUSION: Simulations of thrombin formation based on plasma composition can discriminate between acute and stable CAD.

Effect of rosuvastatin on risk markers for venous thromboembolism in cancer.

Essentials Statins lower venous thromboembolism risk in general but have not been studied in cancer patients. We completed a randomized trial of rosuvastatin vs. placebo among cancer patients on chemotherapy. Rosuvastatin did not significantly lower prothrombotic biomarkers including D-dimer. The role of statins in venous thrombosis prevention in cancer patients remains unknown. SUMMARY: Background Statin therapy is associated with lower risk of venous thromboembolism (VTE) but has not been prospectively evaluated in patients with advanced cancer. Objectives We determined if statin administration in this high-risk population reduces the risk of VTE, based on established and emerging biomarkers. Patients/Methods This double-blind, crossover, randomized controlled trial among patients with advanced cancer receiving systemic therapy allocated participants to rosuvastatin 20 mg daily or placebo for 3-4 weeks prior to crossover to the alternative therapy, with a 3-5-week washout. D-dimer, C-reactive protein (CRP), soluble (s)P-selectin, factor VIII (FVIII), thrombin generation and exploratory biomarkers focusing on endogenous thrombin potential, including tissue factor (TF), activated factor IX (FIXa) and activated factor XI (FXIa), were measured at the start and end of both treatment periods. The primary outcome was change in D-dimer with rosuvastatin compared with placebo. Results Of 38 enrolled participants, 24 (63%) completed the study. Rosuvastatin did not cause statistically significant changes in D-dimer levels or any other biomarker. CRP levels decreased by 40%; 4.3 mg L-1 (95% confidence interval, -11.0 to +2.5 mg L-1 ) compared with placebo. In post-hoc analysis, participants who received rosuvastatin initially during their first line of treatment had a 13% decrease in D-dimer. Circulating TF, FIXa and FXIa were detected in 26%, 68% and 71% of cancer patients despite not being found in healthy individuals. Conclusions Rosuvastatin did not cause favorable changes in biomarkers of VTE risk in advanced cancer patients receiving chemotherapy. The role of statin therapy as thromboprophylaxis in the cancer population remains uncertain.

Citrate anticoagulation and the dynamics of thrombin generation.

BACKGROUND: Sodium citrate has been used as an anticoagulant to stabilize blood and blood products for over 100 years, presumably by sequestering Ca(++) ions in vitro. Anticoagulation of blood without chelation can be achieved by inhibition of the contact pathway by corn trypsin inhibitor (CTI). OBJECTIVE: To evaluate the influence of citrate anticoagulation on the performance of blood, platelet-rich and platelet-poor plasma assays. METHODS: Blood was anticoagulated in three ways: by collection into citrate, CTI and citrate with CTI. Plasma was prepared using each anticoagulation regimen. Functional analyses included calibrated automated thrombography, thromboelastography, plasma clotting, the synthetic coagulation proteome and platelet aggregation. Coagulation reactions were initiated with tissue factor-phospholipid and Ca(++) (when indicated). RESULTS: In all cases, citrate anticoagulation resulted in reaction dynamics significantly altered relative to blood or plasma stabilized with CTI alone. Subsequent experiments showed that calcium citrate itself impairs coagulation dynamics. CONCLUSION: Coagulation analyses using blood that has been exposed to citrate and recalcified do not yield reliable depictions of the natural dynamics of blood coagulation processes.

Peptidomimetic inhibitors for activated protein C: implications for hemophilia management.

BACKGROUND: Several clinical studies and experiments with transgenic mice have suggested that the severity of the bleeding phenotype in hemophilic patients is substantially reduced in association with impaired inactivation of factor (F) Va by activated protein C (APC) in the presence of the FV Leiden mutation. Experiments using a synthetic coagulation proteome model showed that the presence of FV Leiden significantly increased thrombin generation in the absence of FVIII or FIX. OBJECTIVE: To test the effect of APC inhibition on thrombin generation in hemophilia. METHODS: Prothrombinase and a synthetic coagulation proteome model of tissue factor-triggered thrombin generation were used. RESULTS: Peptide-based APC inhibitors, which mimic the P4-P4' residues surrounding the APC cleavage site at Arg306 of FVa, were synthesized. These compounds are specific and reversible inhibitors of APC, with Ki values as low as 1-2 microM; most have insignificant affinity for FXa or thrombin. The affinity for APC is dependent upon the location and character of the protecting groups. Representatives of this group of compounds inhibit FVa inactivation by APC and prolong FVa functional activity in the prothrombinase complex. When evaluated in a synthetic coagulation proteome model, one inhibitor partially compensated for the absence of FVIII. CONCLUSIONS: Synthetic APC inhibitors may be useful as adjuvants for hemophilia treatment.

Immunologic quantitation of tissue factors.

The large number of conflicting reports on the presence and concentration of circulating tissue factor (TF) in blood generates uncertainties regarding its relevance to hemostasis and association with specific diseases. We believe that the source of these controversies lies in part in the assays used for TF quantitation. We have developed a highly sensitive and specific double monoclonal antibody fluorescence-based immunoassay and integrated it into the Luminex Multi-Analyte Platform. This assay, which uses physiologically relevant standard and appropriate specificity controls, measures TF antigen in recombinant products and natural sources including placenta, plasma, cell lysates and cell membranes. Comparisons of reactivity patterns of various full-length and truncated TFs on an equimolar basis revealed quantitative differences in the immune recognition of TFs by our antibodies in the order of TF 1-263 > 1-242 > 1-218 > placental TF. Despite this differential recognition, all TF species are quantifiable at concentrations < or = 2 pM. Using a calibration curve constructed with recombinant TF 1-263 and plasma from healthy individuals (n = 91), we observed the concentration of TF antigen in plasma to be substantially lower than that generally reported in the literature: TF antigen in plasma of 72 individuals (79%) was below 2 pM (quantitative limit of our assay); TF antigen levels between 2.0 and 5.0 pM could be detected in six individuals (7%); and in 14% (13 plasmas), the non-specific signal was higher than the specific signal, and thus TF levels could not be determined. These differential recognition patterns affect TF quantitation in plasma and should be considered when evaluating plasma TF-like antigen concentrations.

Effects of an acidic environment on coagulation dynamics.

Essentials Acidosis, an outcome of traumatic injury, has been linked to impaired procoagulant efficiency. In vitro model systems were used to assess coagulation dynamics at pH 7.4 and 7.0. Clot formation dynamics are slightly enhanced at pH 7.0 in blood ex vivo. Acidosis induced decreases in antithrombin efficacy offset impairments in procoagulant activity. SUMMARY: Background Disruption of hydrogen ion homeostasis is a consequence of traumatic injury often associated with clinical coagulopathy. Mechanisms by which acidification of the blood leads to aberrant coagulation require further elucidation. Objective To examine the effects of acidified conditions on coagulation dynamics using in vitro models of increasing complexity. Methods Coagulation dynamics were assessed at pH 7.4 and 7.0 as follows: (i) tissue factor (TF)-initiated coagulation proteome mixtures (±factor [F]XI, ±fibrinogen/FXIII), with reaction progress monitored as thrombin generation or fibrin formation; (ii) enzyme/inhibitor reactions; and (iii) TF-dependent or independent clot dynamics in contact pathway-inhibited blood via viscoelastometry. Results Rate constants for antithrombin inhibition of FXa and thrombin were reduced by ~ 25-30% at pH 7.0. At pH 7.0 (+FXI), TF-initiated thrombin generation showed a 20% increase in maximum thrombin levels and diminished thrombin clearance rates. Viscoelastic analyses showed a 25% increase in clot time and a 25% reduction in maximum clot firmness (MCF). A similar MCF reduction was observed at pH 7.0 when fibrinogen/FXIII were reacted with thrombin. In contrast, in contact pathway-inhibited blood (n = 6) at pH 7.0, MCF values were elevated 6% (95% confidence interval [CI]: 1%-11%) in TF-initiated blood and 15% (95% CI: 1%- 29%) in the absence of TF. Clot times at pH 7.0 decreased 32% (95% CI: 15%-49%) in TF-initiated blood and 51% (95% CI: 35%-68%) in the absence of TF. Conclusions Despite reported decreased procoagulant catalysis at pH 7.0, clot formation dynamics are slightly enhanced in blood ex vivo and suppression of thrombin generation is not observed. A decrease in antithrombin reactivity is one potential mechanism contributing to these outcomes.

Thrombin generation profiles in deep venous thrombosis.

BACKGROUND: Reliable markers and methods to predict risk for thrombosis are essential to clinical management. OBJECTIVE: Using an integrated approach that defines an individual's comprehensive coagulation phenotype might prove valuable in identifying individuals at risk for experiencing a thrombotic event. METHODS: Using a numerical simulation model, we generated tissue factor (TF) initiated thrombin curves using coagulation factor levels from the Leiden Thrombophilia Study population and evaluated thrombotic risk, by sex, age, smoking, alcohol consumption, body mass index (BMI) and oral contraceptive (OC) use. We quantitated the initiation, propagation and termination phases of each individuals' comprehensive TF-initiated thrombin generation curve by the parameters: time to 10 nm of thrombin, maximum time, level and rate (MaxR) of thrombin generated and total thrombin. RESULTS: The greatest risk association was obtained using MaxR; with a 2.6-fold increased risk at MaxR exceeding the 90th percentile. The odds ratio (OR) for MaxR was 3.9 in men, 2.1 in women, and 2.9 in women on OCs. The association of risk with thrombin generation did not differ by age (OR:2.8 OR:2.5), BMI (OR:2.9 OR:2.3) or alcohol use. In both numerical simulations and empirical synthetic plasma, OC use created extreme shifts in thrombin generation in both control women and women with a prior thrombosis, with a larger shift in thrombin generation in control women. This suggests an interaction of OC use with underlying prothrombotic abnormalities. CONCLUSIONS: Thrombin generation based upon the individual's blood composition is associated with the risk for thrombosis and may be useful as a predictive marker for evaluating thrombosis on an individual basis.

Coagulation factor XII genetic variation, ex vivo thrombin generation, and stroke risk in the elderly: results from the Cardiovascular Health Study.

BACKGROUND: The relationships of thrombin generation (TG) with cardiovascular disease risk are underevaluated in population-based cohorts. OBJECTIVES: To evaluate the relationships of TG influenced by the contact and tissue factor coagulation pathways ex vivo with common single-nucleotide polymorphisms (SNPs) and incident cardiovascular disease and stroke. PATIENTS/METHODS: We measured peak TG (pTG) in baseline plasma samples of Cardiovascular Health Study participants (n = 5411), both with and without inhibitory anti-factor XIa antibody (pTG/FXIa(-) ). We evaluated their associations with ~ 50 000 SNPs by using the IBCv2 genotyping array, and with incident cardiovascular disease and stroke events over a median follow-up of 13.2 years. RESULTS: The minor allele for an SNP in the FXII gene (F12), rs1801020, was associated with lower pTG in European-Americans (ß = - 34.2 ± 3.5 nm; P = 3.3 × 10(-22) ; minor allele frequency [MAF] = 0.23) and African-Americans (ß = - 31.1 ± 7.9 nm; P = 9.0 × 10(-5) ; MAF = 0.42). Lower FXIa-independent pTG (pTG/FXIa(-) ) was associated with the F12 rs1801020 minor allele, and higher pTG/FXIa(-) was associated with the ABO SNP rs657152 minor allele (ß = 16.3 nm; P = 4.3 × 10(-9) ; MAF = 0.37). The risk factor-adjusted ischemic stroke hazard ratios were 1.09 (95% confidence interval CI 1.01-1.17; P = 0.03) for pTG, 1.06 (95% CI 0.98-1.15; P = 0.17) for pTG/FXIa(-) , and 1.11 (95% CI 1.02-1.21; P = 0.02) for FXIa-dependent pTG (pTG/FXIa(+) ), per one standard deviation increment (n = 834 ischemic strokes). In a multicohort candidate gene analysis, rs1801020 was not associated with incident ischemic stroke (ß = - 0.02; standard error = 0.08; P = 0.81). CONCLUSIONS: These results support the importance of contact activation pathway-dependent TG as a risk factor for ischemic stroke, and indicate the importance of F12 SNPs for TG ex vivo and in vivo.

The function of factor XI in tissue factor-initiated thrombin generation.

The influence of plasma and platelet factor (F)XI on thrombin generation initiated with 10 pm tissue factor (TF) in a synthetic coagulation model was evaluated in the presence of either 2 x 108 mL-1 platelets or the equivalent (2 microm) phospholipids. In either system, with all proteins present at physiological concentrations, FXI (30 nm) had no effect on thrombin generation. With phospholipids in the absence of FXI, an increase in vitamin K-dependent proteins (VKDP) (up to 500%) significantly prolonged the initiation phase of thrombin generation and decreased maximum thrombin levels. The inhibition was principally caused by the elevated prothrombin and FIX concentrations. When 30 nm FXI was added with elevated VKDP and phospholipids, the initiation phase was decreased and the maximum thrombin levels generated substantially increased. In experiments with platelets (with and without plasma FXI), an increase in VKDP had little effect on the initiation phase of thrombin generation. These data indicate that (i) FXI has no effect on thrombin generation at 10 pm TF and physiological concentrations of VKDP; (ii) platelets and plasma FXI are able to compensate for the inhibitory effects of elevated VKDP.

What is all that thrombin for?

The hemostatic process initiated by the exposure of tissue factor to blood is a threshold limited reaction which occurs in two distinct phases. During an initiation phase, small amounts of factor (F)Xa, FIXa and thrombin are generated. The latter activates the procofactors FV and FVIII to the activated cofactors which together with their companion serine proteases form the intrinsic FX activator (FVIIIa-FIXa) and prothrombinase (FVa-FXa) which generate the bulk of FXa and thrombin during a propagation phase. The clotting process (fibrin formation) occurs at the inception of the propagation phase when only 5-10 nM thrombin has been produced. Consequently, the vast majority (greater than 95%) of thrombin is produced after clotting during the propagation phase of thrombin generation. The blood of individuals with either hemophilia A or hemophilia B has no ability to generate the intrinsic FXase, and hence is unable to support the propagation phase of the reaction. Since clot based assays conclude before the propagation phase they are not sensitive to hemophilia A and B. The inception and magnitude of the propagation phase of thrombin generation is influenced by genetic polymorphisms associated with thrombotic and hemorrhagic disease, by the natural abundance of pro- and anticoagulants in healthy individuals and by pharmacologic interventions which influence thrombotic pathology. Therefore, it is our suspicion that the performance of the entire process of thrombin generation from initiation through propagation and termination phases of the reaction are relevant with respect to both hemorrhagic and thrombotic pathology.

Does the genotype predict the phenotype? Evaluations of the hemostatic proteome.

In this review, the complexity arising from the heterogeneity of the human hemostatic proteome is introduced and discussed with respect to impact on the diagnosis, prophylaxis and therapeutic interventions in thrombotic and hemorrhagic diseases. In the 'healthy' population, coagulation factor levels extend over a 2-4-fold range in concentration. In addition, the qualitative performance of these proteins is governed by many molecular events which are influenced both by genetic instructions which influence post-translational modification and by environmental processes that alter coagulation proteins during circulation. As a consequence, the stimulus-response coupling which follows tissue factor presentation to blood and the subsequent expression of thrombin activity is highly variable even in the 'normal' population. The consequences of this molecular heterogeneity and its potential influence on the diagnosis, prophylaxis and ultimate therapy of coagulation diseases are illustrated. It is the intention of the authors to be provocative; encouraging further investigations to understand the clinical significance of the heterogeneity of the human hemostatic proteome.

How factor VIIa works in hemophilia.

The influence of elevated platelet concentration and recombinant factor VIIa (rFVIIa) on thrombin generation at 5 pM tissue factor (TF) in a synthetic mixture corresponding to hemophilia B (SHB) and "acquired" hemophilia B blood (AHBB) produced in vitro by an antifactor IX antibody was evaluated. (a) Thrombin generation in SHB and AHBB was delayed and reduced; (b) with 10 nM rFVIIa or 5x normal platelets (10 x 10(8)/mL) SHB and AHBB showed a slight increase in thrombin generation; (c) in the absence of TF, almost no thrombin generation was detected in SHB and AHBB in the presence of 10 nM rFVIIa and 10 x 10(8)/mL activated platelets (5x normal); (d) with TF, 10 nM rFVIIa and 3-5x normal nonactivated platelets (6-10 x 10(8)/mL), thrombin levels approaching normal values were attained. FVIIa appears to function effectively and locally by the combined effect of TF expression and platelet accumulation at the site of a vascular lesion.

Factor VIIa replacement therapy in factor VII deficiency.

Factor (F)VII deficiency is an autosomal recessive disorder for which a replacement therapy is not universally available; recombinant FVIIa has been utilized as a therapeutic substitute. As FVII competes with FVIIa for binding to tissue factor in initiating the extrinsic pathway of blood coagulation, a lower dose of FVIIa replacement in cross-reacting material-negative (CRM-) individuals can achieve hemostasis. Three coagulation models (computational, synthetic and in vitro whole blood) were used to predict the FVIIa levels needed to provide apparent hemostasis in a non-bleeding state. Our whole blood results show that a 'normalized' coagulation profile for FVII-deficient individuals has an initiation phase that ends at 5.8 +/- 0.5 min (clot time) and the propagation phase of thrombin generation (thrombin-antithrombin III) yields a maximum concentration of 380 +/- 29 nmol L(-1). When CRM- FVII-deficient subjects were infused with a prophylactic dose of 23 micro g kg(-1) of recombinant FVIIa, 6-8 h postinfusion resulted in a comparable normalized whole blood profile. This FVIIa concentration (0.3-0.7 nmol L(-1)/equivalent dose: 0.8-1.8 micro g kg(-1)) is approximately 1/10 that currently used in treating FVII-deficient individuals and suggests that therapies should be altered relative to the concentration of the FVII zymogen.