Thrombin generation and cell-dependent hypercoagulability in sickle cell disease.

Essentials Sickle cell disease is increasingly being recognized as a chronic hypercoagulable state. Thrombin generation is elevated in the whole blood, but not the plasma of sickle cell patients. Whole blood thrombin generation inversely correlates to erythrocyte phosphatidylserine exposure. Acquired protein S deficiency is likely explained by binding of protein S to sickle red cells. Click to hear Dr Hillery discuss coagulation and vascular pathologies in mouse models of sickle cell disease. SUMMARY: Introduction Sickle cell disease (SCD) is a hypercoagulable state with chronic activation of coagulation and an increased incidence of thromboembolic events. However, although plasma pre-thrombotic markers such as thrombin-anithrombin complexes and D-dimer are elevated, there is no consensus on whether global assays of thrombin generation in plasma are abnormal in patients with SCD. Based on our recent observation that normal red blood cells (RBCs) contribute to thrombin generation in whole blood, we hypothesized that the cellular components in blood (notably phosphatidylserine-expressing erythrocytes) contribute to enhanced thrombin generation in SCD. Methods Whole blood and plasma thrombin generation assays were performed on blood samples from 25 SCD patients in a non-crisis 'steady state' and 25 healthy race-matched controls. Results Whole blood thrombin generation was significantly elevated in SCD, whereas plasma thrombin generation was paradoxically reduced compared with controls. Surprisingly, whole blood and plasma thrombin generation were both negatively correlated with phosphatidylserine exposure on RBCs. Plasma thrombin generation in the presence of exogenous activated protein C or soluble thrombomodulin revealed deficiencies in the protein C/S anticoagulant pathway in SCD. These global changes were associated with significantly lower plasma protein S activity in SCD that correlated inversely with RBC phosphatidylserine exposure. Conclusion Increased RBC phosphatidylserine exposure in SCD is associated with acquired protein S deficiency. In addition, these data suggest a cellular contribution to thrombin generation in SCD (other than RBC phosphatidylserine exposure) that explains the elevated thrombin generation in whole blood.

The influence of prophylactic factor VIII in severe haemophilia A.

Haemophilia A individuals displaying a similar genetic defect have heterogeneous clinical phenotypes. Our objective was to evaluate the underlying effect of exogenous factor (f)VIII on tissue factor (Tf)-initiated blood coagulation in severe haemophilia utilizing both empirical and computational models. We investigated twenty-five clinically severe haemophilia A patients. All individuals were on fVIII prophylaxis and had not received fVIII from 0.25 to 4 days prior to phlebotomy. Coagulation was initiated by the addition of Tf to contact-pathway inhibited whole blood ± an anti-fVIII antibody. Aliquots were quenched over 20 min and analyzed for thrombin generation and fibrin formation. Coagulation factor levels were obtained and used to computationally predict thrombin generation with fVIII set to either zero or its value at the time of the draw. As a result of prophylactic fVIII, at the time of the blood draw, the individuals had fVIII levels that ranged from <1% to 22%. Thrombin generation (maximum level and rate) in both empirical and computational systems increased as the level of fVIII increased. FXIII activation rates also increased as the fVIII level increased. Upon suppression of fVIII, thrombin generation became comparable in both systems. Plasma composition analysis showed a negative correlation between bleeding history and computational thrombin generation in the absence of fVIII. Residual prophylactic fVIII directly causes an increase in thrombin generation and fibrin cross-linking in individuals with clinically severe haemophilia A. The combination of each individual's coagulation factors (outside of fVIII) determine each individual's baseline thrombin potential and may affect bleeding risk.

Activated protein C inhibitor for correction of thrombin generation in hemophilia A blood and plasma1.

BACKGROUND: Replacement therapy for hemophilic patient treatment is costly, because of the high price of pharmacologic products, and is not affordable for the majority of patients in developing countries. OBJECTIVE: To generate and evaluate low molecular weight agents that could be useful for hemophilia treatment. METHODS: Potential agents were generated by synthesizing specific inhibitors [6-(Lys-Lys-Thr-[homo]Arg)amino-2-(Lys[carbobenzoxy]-Lys[carbobenzoxy]-O-benzyl)naphthalenesulfonamide] (PNASN-1)] for activated protein C (APC) and tested in plasma and fresh blood from hemophilia A patients. RESULTS: In the activated partial thromboplastin time-based APC resistance assay, PNASN-1 partially neutralized the effect of APC. In calibrated automated thrombography, PNASN-1 neutralized the effect of APC on thrombin generation in normal and congenital factor VIII-deficient plasma (FVIII:C < 1%). The addition of PNASN-1 to tissue factor-triggered (5 pm) contact pathway-inhibited fresh blood from 15 hemophilia A patients with various degrees of FVIII deficiency (FVIII:C < 1-51%) increased the maximum level of thrombin generated from 78 to 162 nm, which approached that observed in blood from a healthy individual (201 nm). PNASN-1 also caused a 47% increase in clot weight in hemophilia A blood. CONCLUSIONS: Specific APC inhibitors compensate to a significant extent for FVIII deficiency, and could be used for hemophilia treatment.

Coagulation procofactor activation by factor XIa.

SUMMARY BACKGROUND: In the extrinsic pathway, the essential procofactors factor (F) V and FVIII are activated to FVa and FVIIIa by thrombin. In the contact pathway and its clinical diagnostic test, the activated partial thromboplastin time (APTT) assay, the sources of procofactor activation are unknown. In the APTT assay, FXII is activated on a negatively charged surface and proceeds to activate FXI, which activates FIX upon the addition of Ca(2+). FIXa feeds thrombin generation through activation of FX. FIXa is an extremely poor catalyst in the absence of its FVIIIa cofactor, which, in the intrinsic FXase complex, increases FXa generation by approximately 10(7). One potential APTT procofactor activator in this setting is FXIa. OBJECTIVE: To test the hypothesis that FXIa can activate FVIII and FV. METHODS: Recombinant FVIII and plasma FV were treated with FXIa, and the activities and integrities of each procofactor were measured using commercial clotting assays and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: Kinetic analyses of FXIa-catalyzed activation and inactivation of FV and FVIII are reported, and the the timing and sites of cleavage are defined. CONCLUSIONS: FXIa activates both procofactors at plasma protein concentrations, and computational modeling suggests that procofactor activation during the preincubation phase of the APTT assay is critical to the performance of the assay. As the APTT assay is the primary tool for the diagnosis and management of hemophilias A and B, as well as in the determination of FVIII inhibitors, these findings have potential implications in the clinical setting.

Thrombin generation and bleeding in haemophilia A.

Haemophilia A displays phenotypic heterogeneity with respect to clinical severity. The aim of this study was to determine if tissue factor (TF)-initiated thrombin generation profiles in whole blood in the presence of corn trypsin inhibitor (CTI) are predictive of bleeding risk in haemophilia A. We studied factor(F) VIII deficient individuals (11 mild, 4 moderate and 12 severe) with a well-characterized 5-year bleeding history that included haemarthrosis, soft tissue haematoma and annual FVIII concentrate usage. This clinical information was used to generate a bleeding score. The bleeding scores (range 0-32) were separated into three groups (bleeding score groupings: 0, 0 and < or = 9.6, >9.6), with the higher bleeding tendency having a higher score. Whole blood collected by phlebotomy and contact pathway suppressed by 100 microg mL(-1) CTI was stimulated to react by the addition of 5 pM TF. Reactions were quenched at 20 min by inhibitors. Thrombin generation, determined by enzyme-linked immunosorbent assay for thrombin-antithrombin was evaluated in terms of clot time (CT), maximum level (MaxL) and maximum rate (MaxR) and compared to the bleeding score. Data are shown as the mean+/-SD. MaxL was significantly different (P < 0.001) between the groups: 504 +/- 114, 315 +/- 117 and 194 +/- 91 nM; with higher thrombin concentrations in the groups with lower bleeding scores. MaxR was higher in the groups with a lower bleeding score; 97 +/- 51, 86 +/- 60 and 39 +/- 16 nM min(-1) (P = 0.09). No significant difference was detected in CT among the groups, 5.6 +/- 1.3, 4.7 +/- 0.7 and 5.6 +/- 1.3 min. Our empirical study in CTI-inhibited whole blood shows that the MaxL of thrombin generation appears to correlate with the bleeding phenotype of haemophilia A.

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.