Comparison of bleeding risk scores in patients with atrial fibrillation: insights from the RE-LY trial.

BACKGROUND: Oral anticoagulation is the mainstay of stroke prevention in atrial fibrillation (AF), but must be balanced against the associated bleeding risk. Several risk scores have been proposed for prediction of bleeding events in patients with AF. OBJECTIVES: To compare the performance of contemporary clinical bleeding risk scores in 18 113 patients with AF randomized to dabigatran 110 mg, 150 mg or warfarin in the RE-LY trial. METHODS: HAS-BLED, ORBIT, ATRIA and HEMORR2 HAGES bleeding risk scores were calculated based on clinical information at baseline. All major bleeding events were centrally adjudicated. RESULTS: There were 1182 (6.5%) major bleeding events during a median follow-up of 2.0 years. For all the four schemes, high-risk subgroups had higher risk of major bleeding (all P < 0.001). The ORBIT score showed the best discrimination with c-indices of 0.66, 0.66 and 0.62, respectively, for major, life-threatening and intracranial bleeding, which were significantly better than for the HAS-BLED score (difference in c-indices: 0.050, 0.053 and 0.048, respectively, all P < 0.05). The ORBIT score also showed the best calibration compared with previous data. Significant treatment interactions between the bleeding scores and the risk of major bleeding with dabigatran 150 mg BD versus warfarin were found for the ORBIT (P = 0.0019), ATRIA (P < 0.001) and HEMORR2 HAGES (P < 0.001) scores. HAS-BLED score showed a nonsignificant trend for interaction (P = 0.0607). CONCLUSIONS: Amongst the current clinical bleeding risk scores, the ORBIT score demonstrated the best discrimination and calibration. All the scores demonstrated, to a variable extent, an interaction with bleeding risk associated with dabigatran or warfarin.

Use of non-vitamin K antagonist oral anticoagulants in atrial fibrillation patients: insights from a specialist atrial fibrillation clinic.

BACKGROUND: Non-vitamin K antagonist oral anticoagulants (NOACs) are broadly preferable to vitamin K antagonists (VKAs) for stroke prevention in non-valvular atrial fibrillation (AF) given their overall net clinical benefit. We report an audit of the profile of OAC usage and adverse events in patients attending a specialist AF clinic. METHODS: Patients attending our specialist AF clinic who were commenced on NOACs for SPAF between January 2013 and August 2014 were included and electronic medical records were retrospectively reviewed between August 2014 and November 2014, to collect demographic, clinical and outcome data. Outcomes included cerebrovascular and bleeding events, death, switching between NOACs or to VKA, dose changes, cessation of NOACs and the reasons for these. To provide perspective, descriptive comparisons were made with a historical cohort of warfarin users attending the specialist AF clinic prior to the introduction of NOACs. RESULTS: We report data on 813 patients as follows: (i) 233 consecutive patients (mean (standard deviation) age 74 (10) years, 45.1% female) initiated on NOACs, with median (interquartile range) CHA2 DS2 -VASc score 3 (2-5) and HAS-BLED score 1 (1-2); and (ii) a historical cohort of 580 patients on warfarin (mean (SD) age 75 (10) years, 42.1% female) with broadly similar demographics. Overall, 54.5% (127/233) were started on rivaroxaban, 22.7% (53/233) on dabigatran and 22.7% on apixaban. Two patients experienced a transient ischaemic attack; 31 patients (13%) contributed to 37 documented bleeding events of which five bleeds (in four patients, 1.7%) were classified as major. There were seven deaths; cause of death was not available for three and the others were not related to NOACs. Eighteen (7.7%) patients switched NOACs, 2 (0.9%) patients switched to warfarin and 8 (3.4%) had their NOACs stopped. There were no ischaemic strokes in the NOAC cohort, compared with nine in the warfarin cohort, with a similar rate of major bleeding (1.7% for NOACs and 1.6% for warfarin). There were more gastrointestinal haemorrhages in the NOAC cohort (3.4% vs. 0.7% with warfarin). CONCLUSION: In this specialist AF clinic, patients prescribed NOACs had a favourable adverse event profile with good efficacy for stroke prevention, with a low rate of cessation or switch to warfarin.

Gender differences in stroke prevention in atrial fibrillation in general practice: using the GRASP-AF audit tool.

BACKGROUND: Women represent a large proportion of patients with atrial fibrillation (AF) and tend to have higher risk of stroke. AIMS: This study examines gender differences in the utilisation of oral anticoagulation (OAC) and prognosis (i.e. stroke and death) in AF patients in UK general practice. DESIGN: Retrospective observational study. METHODS: The Guidance on Risk Assessment and Stroke Prevention in Atrial Fibrillation (GRASP-AF) tool was employed to identify AF patients from 11 general practices in Darlington, England. RESULTS: Two thousand two hundred and fifty-nine AF patients (mean±SD age 76 ± 12 years; 46% female) were identified. Based on CHA2 DS2 -VASc score 95% of women and 90% of men were at moderate-high risk of stroke. Women with moderate-high risk of stroke were treated with OAC less frequently than men (47% vs. 52%, p = 0.006). Overall rates of stroke and all-cause mortality were higher among women than men (p = 0.02 and p < 0.001). However, there was no significant gender difference in these outcomes in patients receiving OAC (p = 0.52 for stroke, p = 0.18 for death). Among people not receiving OAC where indicated, female gender was associated with an increased risk of stroke before (p = 0.01), and after (p = 0.04), adjustment for stroke risk factors. Women not receiving OAC had a higher risk of death on univariate regression analysis (p = 0.002), but not after adjustment for stroke risk factors (p = 0.53). CONCLUSION: Women with AF are at higher risk of stroke than men without OAC. The gender-related differences in risk of stroke disappear if OAC is used. Despite this, women are more likely not to receive OAC.

Adjusted prognostic association of depression following myocardial infarction with mortality and cardiovascular events: individual patient data meta-analysis.

BACKGROUND: The association between depression after myocardial infarction and increased risk of mortality and cardiac morbidity may be due to cardiac disease severity. AIMS: To combine original data from studies on the association between post-infarction depression and prognosis into one database, and to investigate to what extent such depression predicts prognosis independently of disease severity. METHOD: An individual patient data meta-analysis of studies was conducted using multilevel, multivariable Cox regression analyses. RESULTS: Sixteen studies participated, creating a database of 10 175 post-infarction cases. Hazard ratios for post-infarction depression were 1.32 (95% CI 1.26-1.38, P<0.001) for all-cause mortality and 1.19 (95% CI 1.14-1.24, P<0.001) for cardiovascular events. Hazard ratios adjusted for disease severity were attenuated by 28% and 25% respectively. CONCLUSIONS: The association between depression following myocardial infarction and prognosis is attenuated after adjustment for cardiac disease severity. Still, depression remains independently associated with prognosis, with a 22% increased risk of all-cause mortality and a 13% increased risk of cardiovascular events per standard deviation in depression z-score.

Routine versus aggressive upstream rhythm control for prevention of early atrial fibrillation in heart failure: background, aims and design of the RACE 3 study.

BACKGROUND: Rhythm control for atrial fibrillation (AF) is cumbersome because of its progressive nature caused by structural remodelling. Upstream therapy refers to therapeutic interventions aiming to modify the atrial substrate, leading to prevention of AF. OBJECTIVE: The Routine versus Aggressive upstream rhythm Control for prevention of Early AF in heart failure (RACE 3) study hypothesises that aggressive upstream rhythm control increases persistence of sinus rhythm compared with conventional rhythm control in patients with early AF and mild-to-moderate early systolic or diastolic heart failure undergoing electrical cardioversion. DESIGN: RACE 3 is a prospective, randomised, open, multinational, multicenter trial. Upstream rhythm control consists of angiotensin converting enzyme inhibitors and/or angiotensin receptor blockers, mineralocorticoid receptor antagonists, statins, cardiac rehabilitation therapy, and intensive counselling on dietary restrictions, exercise maintenance, and drug adherence. Conventional rhythm control consists of routine rhythm control therapy without cardiac rehabilitation therapy and intensive counselling. In both arms, every effort is made to keep patients in the rhythm control strategy, and ion channel antiarrhythmic drugs or pulmonary vein ablation may be instituted if AF relapses. Total inclusion will be 250 patients. If upstream therapy proves to be effective in improving maintenance of sinus rhythm, it could become a new approach to rhythm control supporting conventional pharmacological and non-pharmacological rhythm control.

Stop and Go: Barriers and Facilitators to Care Home Research.

Barriers to care home research have always existed, but have been thrown into sharp relief by the COVID-19 pandemic. Existing infrastructure failed to deliver the research, or outcomes, which care home residents deserved and we need to look, again, at how these barriers can be taken down. Barriers can be categorised as procedural (encountered before research starts), system (encountered during research) or resident-specific. To tackle these, research regulatory bodies need to adopt a standardised approach to how care home research is developed and designed, reviewed and regulated, and how such approaches can enable recruitment of as wide a range of residents and their representatives as possible, including those without the mental capacity to consent for research. Establishment of local, inter-disciplinary collaborations between universities, general practices, health and social care providers and care homes is another priority. This should be based on pre-existing models such as the 'Living lab' model developed in The Netherlands and now being implemented in the UK and Austria. These changes are critical to develop a sustainable research model. If well designed this will deliver better outcomes for residents and align with the individual and organisational priorities of those who care for them.

ADAMTS-13 and von Willebrand factor: a dynamic duo.

von Willebrand factor (VWF) is a key player in hemostasis, acting as a carrier for factor VIII and capturing platelets at sites of vascular damage. To capture platelets, it must undergo conformational changes, both within its A1 domain and at the macromolecular level through A2 domain unfolding. Its size and this function are regulated by the metalloproteinase ADAMTS-13. Recently, it has been shown that ADAMTS-13 undergoes a conformational change upon interaction with VWF, and that this enhances its activity towards its substrate. This review summarizes recent work on these conformational transitions, describing how they are controlled. It points to their importance in hemostasis, bleeding disorders, and the developing field of therapeutic application of ADAMTS-13 as an antithrombotic agent in obstructive microvascular thrombosis and in cardiovascular disease.

Enhanced activity of an ADAMTS-13 variant (R568K/F592Y/R660K/Y661F/Y665F) against platelet agglutination in vitro and in a murine model of acute ischemic stroke.

Essentials ADAMTS13 requires a substrate-induced conformational change to attain full activity in vitro. The efficacy of wild type ADAMTS13 in models of thrombosis/stroke may be enhanced by pre-activation. A pre-activated ADAMTS13 variant exhibits enhanced proteolysis of platelet agglutinates. This ADAMTS13 variant is protective in a murine model of stroke at a lower dose than WT ADAMTS13. SUMMARY: Background ADAMTS-13 circulates in a closed conformation, only achieving full proteolytic activity against von Willebrand factor (VWF) following a substrate-induced conformational change. A gain-of-function (GoF) ADAMTS-13 variant (R568K/F592Y/R660K/Y661F/Y665F) is conformationally preactivated. Objectives To establish how the hyperactivity of GoF ADAMTS-13 is manifested in experimental models mimicking the occlusive arterial thrombi present in acute ischemic stroke. Methods The ability of GoF ADAMTS-13 to dissolve VWF-platelet agglutinates was examined with an assay of ristocetin-induced platelet agglutination and in parallel-flow models of arterial thrombosis. A murine model of focal ischemia was used to assess the thrombolytic potential of GoF ADAMTS-13. Results Wild-type (WT) ADAMTS-13 required conformational activation to attain full activity against VWF-mediated platelet capture under flow. In this assay, GoF ADAMTS-13 had an EC50 value more than five-fold lower than that of WT ADAMTS-13 (0.73 ± 0.21 nm and 3.81 ± 0.97 nm, respectively). The proteolytic activity of GoF ADAMTS-13 against preformed platelet agglutinates under flow was enhanced more than four-fold as compared with WT ADAMTS-13 (EC50 values of 2.5 ± 1.1 nm and 10.2 ± 5.6 nm, respectively). In a murine stroke model, GoF ADAMTS-13 restored cerebral blood flow at a lower dose than WT ADAMTS-13, and partially retained the ability to recanalize vessels when administration was delayed by 1 h. Conclusions The limited proteolytic activity of WT ADAMTS-13 in in vitro models of arterial thrombosis suggests an in vivo requirement for conformational activation. The enhanced activity of the GoF ADAMTS-13 variant translates to a more pronounced protective effect in experimental stroke.

Pleiotropic effects of antithrombin strand 1C substitution mutations.

Six different substitution mutations were identified in four different amino acid residues of antithrombin strand 1C and the polypeptide leading into strand 4B (F402S, F402C, F402L, A404T, N405K, and P407T), and are responsible for functional antithrombin deficiency in seven independently ascertained kindreds (Rosny, Torino, Maisons-Laffitte, Paris 3, La Rochelle, Budapest 5, and Oslo) affected by venous thromboembolic disease. In all seven families, variant antithrombins with heparin-binding abnormalities were detected by crossed immunoelectrophoresis, and in six of the kindreds there was a reduced antigen concentration of plasma antithrombin. Two of the variant antithrombins, Rosny and Torino, were purified by heparin-Sepharose and immunoaffinity chromatography, and shown to have greatly reduced heparin cofactor and progressive inhibitor activities in vitro. The defective interactions of these mutants with thrombin may result from proximity of s1C to the reactive site, while reduced circulating levels may be related to s1C proximity to highly conserved internal beta strands, which contain elements proposed to influence serpin turnover and intracellular degradation. In contrast, s1C is spatially distant to the positively charged surface which forms the heparin binding site of antithrombin; altered heparin binding properties of s1C variants may therefore reflect conformational linkage between the reactive site and heparin binding regions of the molecule. This work demonstrates that point mutations in and immediately adjacent to strand 1C have multiple, or pleiotropic, effects on this serpin, leading ultimately to failure of its regulatory function.

The central role of thrombin in hemostasis.

Following vascular injury, blood loss is controlled by the mechanisms of hemostasis. During this process, the serine proteinase, thrombin, is generated both locally and rapidly at sites of vessel damage. It plays a pivotal role in clot promotion and inhibition, and cell signaling, as well as additional processes that influence fibrinolysis and inflammation. These functions involve numerous cleavage reactions, which must be tightly coordinated. Failure to do so can lead to either bleeding or thrombosis. The crystal structures of thrombin, in combination with biochemical analyses of thrombin mutants, have provided insight into the ways in which thrombin functions, and how its different activities are modulated. Many of the interactions of thrombin are facilitated by exosites on its surface that bind to its substrates and/or cofactors. The use of cofactors not only extends the range of thrombin specificity, but also enhances its catalytic efficiency for different substrates. This explains a paradox (i.e. thrombin is a specific proteinase, and yet one that has multiple, and sometimes opposing, substrate reactions). In this review, we describe the context in which thrombin acts during hemostasis and explain the roles that its exosites and cofactors play in directing thrombin function. Thereafter, we develop the concept of cofactor competition as a means by which the activities of thrombin are controlled.

Further characterization of ADAMTS-13 inactivation by thrombin.

BACKGROUND: The multimeric size and platelet-tethering function of von Willebrand factor (VWF) are modulated by the plasma metalloprotease, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13). In vitro ADAMTS-13 is susceptible to proteolytic inactivation by thrombin. OBJECTIVES: In this study, we aimed to characterize the inactivation of ADAMTS-13 by thrombin and to assess its physiological significance. METHODS AND RESULTS: By N-terminal sequencing of cleavage products, and by mutagenesis, we identified the principal thrombin cleavage sites in ADAMTS-13 as R257 and R1176. Using a library of 76 thrombin mutants, we highlighted the functional importance of exosite I on thrombin in the proteolysis of ADAMTS-13. Proteolysis of ADAMTS-13 by thrombin caused an 8-fold reduction in its affinity for VWF that contributed to its loss of VWF-cleaving function. Intriguingly, thrombin-cleaved ADAMTS-13 both bound and proteolyzed a short recombinant VWF A2 domain substrate (VWF115) normally. Following activation of coagulation in normal plasma, endogenous ADAMTS-13, but not added ADAMTS-13, appeared resistant to coagulation-induced fragmentation. An estimation of the K(m) for ADAMTS-13 proteolysis by thrombin was appreciably higher than the physiological concentration of ADAMTS-13. This was corroborated by the comparatively low affinity of ADAMTS-13 for thrombin (K(D) 95 nM). CONCLUSIONS: Together, our data suggest that ADAMTS-13 is protected from rapid proteolytic inactivation by thrombin in normal plasma. Whether this remains the case under pathological situations involving elevated/sustained generation of thrombin remains unclear.

Proteolysis of the endothelial cell protein C receptor by neutrophil proteinase 3.

BACKGROUND: The endothelial cell protein C receptor (EPCR) presents protein C to the thrombin:thrombomodulin complex on the endothelium of large vessels, and enhances the generation of activated protein C (APC) and activation of protease-activated receptor-1. A previous report has demonstrated binding of soluble (s) EPCR to activated neutrophils via surface proteinase 3 (PR3). METHODS: We now report further characterization of this interaction. Activated neutrophils and purified PR3 both decrease endothelial cell (EC) surface EPCR, suggestive of its proteolysis. RESULTS: When added to purified recombinant sEPCR, PR3 produced multiple cleavages, with early products including 20 kDa N-terminal and C-terminal (after Lys(176)) fragments. The binding of active site blocked PR3 to sEPCR was studied by surface plasmon resonance. Estimates of the K(D) of 18.5-102 nM were obtained with heterogeneous binding, suggestive of more than a single interaction site. CONCLUSIONS: This work demonstrates PR3 binding to and proteolysis of EPCR and suggests a mechanism by which anticoagulant and cell protective pathways can be down-regulated during inflammation.

Ethnic differences in cancer incidence and mortality: the Birmingham Factory Screening Project.

BACKGROUND: There is little information on ethnic differences in the incidence of cancer and cancer mortality among adults in the UK, particularly concerning Afro-Caribbean people. AIM: To examine differences in the incidence of malignant cancer and cancer mortality rates among White European, Afro-Caribbean, and South-Asian people, and to examine baseline demographic predictors of cancer mortality. DESIGN: Longitudinal cohort study. METHODS: We compared ethnic differences in the incidence of malignant cancer and cancer mortality over a mean (SD) follow-up of 19.9 (4.8) years, in relation to baseline demographic characteristics and blood pressure variables, in the 2713 participants (2090 White European men and women, 428 Afro-Caribbean men and women, and 195 South Asian men) enrolled in the Birmingham Factory Screening Project whose survival status on 31 December 2003 was known. RESULTS: White European women had a significantly higher incidence of cancer compared to Afro-Caribbean women (p=0.019). In addition, South Asian men had a significantly lower incidence of cancer compared to White European men (p<0.0001) and Afro-Caribbean men (p=0.048). The incidence of cancer was similar in White European and Afro-Caribbean men (p=1.00). Overall incidence densities of cancer and death from cancer were 0.6% and 0.3% per 100 person-years of observation, respectively. Age, ethnicity, and smoking status were independent predictors of both cancer incidence and cancer mortality. DISCUSSION: The incidence of, and death from, cancer are both lower in minority ethnic groups in the UK, than in their White European counterparts.

Association of protein S p.Pro667Pro dimorphism with plasma protein S levels in normal individuals and patients with inherited protein S deficiency.

A dimorphism in PROS1 gene (c.A2,001G, p.Pro667Pro) has been associated with significantly reduced levels of both free and total protein S in carriers of the GG genotype. It is not known how the GG genotype could influence PS levels in normals, whether it could influence the levels of protein S in carriers of mutations in PROS1 gene and whether this genotype acts as an isolated or additive risk factor for venous thrombosis. With this as background, we evaluated the association of p.Pro667Pro dimorphism with free and total protein S centrally measured in a panel of 119 normal controls, 222 individuals with low protein S and 137 individuals with normal PS levels belonging to 76 families with protein S deficiency enrolled in the ProSIT study. Transient expression of recombinant wild type protein S and p.Pro667Pro protein S was performed to evaluate the role of the A to G transition at position 2001 in vitro. The p.Pro667Pro polymorphism was also expressed together with a p.Glu67Ala variant to assess a possible influence on protein S levels in protein S deficient subjects. Free and total protein S levels were significantly lower in normal women. In normal women only was the GG genotype associated with significantly lower free protein S levels in comparison to AA and AG genotypes (P=0.032). No significant influence of GG genotype was observed in patients, either with known mutations or with low protein S levels. These data were confirmed by in vitro transient expression, showing no difference in secretion levels of the p.Pro667Pro variant (even in association with the p.Glu67Ala mutation), compared to the wild type protein S. The genotype in itself was neither a significant risk factor for venous thrombosis nor a risk modifier in patients with known mutations.

Conformational quiescence of ADAMTS-13 prevents proteolytic promiscuity.

Essentials Recently, ADAMTS-13 has been shown to undergo substrate induced conformation activation. Conformational quiescence of ADAMTS-13 may serve to prevent off-target proteolysis in plasma. Conformationally active ADAMTS-13 variants are capable of proteolysing the Aα chain of fibrinogen. This should be considered as ADAMTS-13 variants are developed as potential therapeutic agents. Click to hear Dr Zheng's presentation on structure function and cofactor-dependent regulation of ADAMTS-13 SUMMARY: Background Recent work has revealed that ADAMTS-13 circulates in a 'closed' conformation, only fully interacting with von Willebrand factor (VWF) following a conformational change. We hypothesized that this conformational quiescence also maintains the substrate specificity of ADAMTS-13 and that the 'open' conformation of the protease might facilitate proteolytic promiscuity. Objectives To identify a novel substrate for a constitutively active gain of function (GoF) ADAMTS-13 variant (R568K/F592Y/R660K/Y661F/Y665F). Methods Fibrinogen proteolysis was characterized using SDS PAGE and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Fibrin formation was monitored by turbidity measurements and fibrin structure visualized by confocal microscopy. Results ADAMTS-13 exhibits proteolytic activity against the Aα chain of human fibrinogen, but this is only manifest on its conformational activation. Accordingly, the GoF ADAMTS-13 variant and truncated variants such as MDTCS exhibit this activity. The cleavage site has been determined by LC-MS/MS to be Aα chain Lys225-Met226. Proteolysis of fibrinogen by GoF ADAMTS-13 impairs fibrin formation in plasma-based assays, alters clot structure and increases clot permeability. Although GoF ADAMTS-13 does not appear to proteolyse preformed cross-linked fibrin, its proteolytic activity against fibrinogen increases the susceptibility of fibrin to tissue-type plasminogen activator (t-PA)-induced lysis by plasmin and increases the fibrin clearance rate more than 8-fold compared with wild-type (WT) ADAMTS-13 (EC50 values of 3.0 ± 1.7 nm and 25.2 ± 9.7 nm, respectively) in in vitro thrombosis models. Conclusion The 'closed' conformation of ADAMTS-13 restricts its specificity and protects against fibrinogenolysis. Induced substrate promiscuity will be important as ADAMTS-13 variants are developed as potential therapeutic agents against thrombotic thrombocytopenic purpura (TTP) and other cardiovascular diseases.

The anti-heparin properties of human low-density lipoprotein.

High-density (HDL), low-density (LDL) and very low-density lipoproteins (VLDL) have been purified from normal human plasma by a combination of ultracentrifugation in high-density salt and agarose gel filtration. The ability of these lipoproteins to inhibit different molecular weight heparin fractions has been compared, using incubation mixtures comprised of antithrombin III and factor Xa. Residual factor Xa activity was measured using the chromogenic peptide substrate Bz-Ile-Glu-Gly-Arg-pNA. LDL inhibited the high molecular weight (but not low molecular weight) heparin accelerated neutralisation of factor Xa by antithrombin III. VLDL showed a similar, though much reduced anti-heparin activity, while the addition of HDL to the factor Xa incubation mixture produced no measurable anti-heparin activity. These observations suggest that certain plasma lipoproteins may selectively modulate the inhibitory action of heparin against factor Xa.

The heparin-accelerated neutralisation of bovine alpha and beta thrombins by antithrombin III.

The heparin-accelerated neutralisation of bovine alpha and beta thrombins has been examined using a peptide substrate H-D-phenylalanyl-pipecolyl-arginine-paranitroanilide-HCl to measure thrombin amidase activity. Alpha and beta thrombins were both neutralised by antithrombin III and this neutralisation was further accelerated by the presence of small amounts of heparin. Low and high molecular weight heparin and heparins fractionated by their affinity for antithrombin III were all able to accelerate the neutralisation of alpha and beta thrombin. This work is therefore unable to confirm reports that alpha and beta thrombins have different heparin sensitivities.

Evidence for a plasma inhibitor of the heparin accelerated inhibition of factor Xa by antithrombin III.

The ability of heparin fractions of different molecular weight to potentiate the action of antithrombin III against the coagulation factors thrombin and Xa has been examined in purified reaction mixtures and in plasma. Residual thrombin and Xa have been determined by their peptidase activities against the synthetic peptide substrates H-D-Phe-Pip-Arg-pNA and Bz-Ile-Gly-Arg-pNA. High molecular weight heparin fractions were found to have higher anticoagulant activities than low molecular weight heparin when studied with both thrombin and Xa incubation mixtures in purified mixtures and in plasma. The inhibition of thrombin by heparin fractions and antithrombin III was unaffected by other plasma components. However, normal human plasma contained a component that inhibited the heparin and antithrombin III inhibition of Xa particularly when the high molecular weight heparin fraction was used. Experiments using a purified preparation of platelet factor 4 suggested that the platelet-derived heparin-neutralizing protein was not responsible for the inhibition.

Thrombin-induced fibrinopeptide B release from normal and variant fibrinogens: influence of inhibitors of fibrin polymerization.

Thrombin preferentially cleaves fibrinopeptides A (FPA) from fibrinogen resulting in the formation of desAA-fibrin from which most of the fibrinopeptides B (FPB) are then released with an enhanced rate. Kinetics of fibrinopeptide release from normal and dysfunctional fibrinogens were investigated in order to further characterize the mechanism of accelerated FPB release during desAA-fibrin polymerization. Dysfunctional fibrinogens London I and Ashford, exhibiting primary polymerization abnormalities (i.e., an abnormality present when all fibrinopeptides have been cleaved), which in the case of fibrinogen London I is believed to be caused by a defect in the D-domain, were shown to exhibit a decreased rate of FPB release compared with normal fibrinogen. While Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization, was shown to decrease the rate of FPB release from normal fibrinogen by a factor of 5, normal fragment D1, although inhibiting clot formation of normal fibrinogen, did not influence the acceleration of FPB release. On the other hand, the presence of fragment D1 did not enhance FPB release from fibrinogen London I, suggesting that interaction of D-domains in functional isolation with desAA-fibrin E-domains is not sufficient to enhance FPB release. Although clot formation was inhibited by the concentrations of fragment D1 used, the formation of small desAA-fibrin oligomers was hardly affected. Thus, small fibrin polymers, but not desAA-fibrin monomers, act as optimal substrates for the release of FPB by thrombin.