BACKGROUND:Patients with end-stage renal disease (ESRD) who receive enoxaparin are at increased risk for adverse bleeding episodes. This phenomenon appears to occur despite judicious monitoring of antifactor Xa (aFXa) activity. Better monitoring parameters are needed to quantify the anticoagulant effects of enoxaparin in the ESRD population.OBJECTIVES:The objective of this study was to determine the utility of using thrombin generation time (TGT), platelet contractile force (PCF) and clot elastic modulus (CEM) to monitor the degree of anticoagulation in ESRD subjects, and to compare these results to aFXa activity, the current gold-standard monitoring parameter.METHODS:Eight healthy volunteers without renal dysfunction and eight ESRD subjects were enrolled into this study. Subjects received a single dose of enoxaparin 1 mg kg(-1) subcutaneously, and blood samples were obtained for the determination of aFXa activity, TGT, PCF and CEM at baseline, 4, 8, and 12 h postdose.RESULTS:Baseline, 4, 8, and 12-h aFXa activity concentrations were not different between groups. However, the corresponding TGT at 8 and 12 h was significantly prolonged in the ESRD group (P = 0.04, and P = 0.008, respectively). The 4-h peak TGT trended toward significance (P = 0.06). There were no differences in PCF or CEM across time.CONCLUSIONS:These data suggest that the parameter aFXa activity is a poor predictor of the anticoagulant effect of enoxaparin in patients with ESRD. Thrombin generation time appears to be more sensitive to the antithrombotic effects of enoxaparin in this population. Further large-scale trials are needed to corroborate these data.

Patients with renal dysfunction that receive hemodialysis (HD) are highly sensitive to low molecular weight heparin (LMWH) drugs such as Enoxaparin. There have been numerous reports of hemorrhagic effects with these drugs in this population. Although current guidelines recommend judicious monitoring of antifactor Xa activity in HD patients to prevent adverse events, this parameter is poorly correlated to efficacy and toxicity. Newer, more specific parameters such as thrombin generation time (TGT), platelet contractile force (PCF) and clot elastic modulus (CEM) may play a role in monitoring LMWH drugs in high-risk populations such as those with renal dysfunction. To determine the utility of monitoring TGT, PCF and CEM for this purpose, we conducted a prospective clinical trial in 8 non-thrombosed HD and 8 control subjects. All subjects received escalating doses of Enoxaparin 0.25, 0.50 and 1.0 mg/kg subcutaneously during this study. Blood samples were obtained four hours post-dose to capture the peak effect of Enoxaparin on antifactor Xa activity, TGT, PCF and CEM. Pearson's correlation was used to determine the relationships between antifactor Xa activity and TGT, PCF and CEM at each dose, respectively. Repeated measures analysis of covariance was used to assess for intergroup differences in the slopes of each regression line using the group status and antifactor Xa concentration as the covariates. The correlation coefficient (r), coefficient of determination (r2) and p-value for each parameter in each group are listed in the Tables. The figure illustrates the relationship between antifactor Xa and TGT in both controls and ESRD patients. These findings suggest that TGT, PCF and CEM are highly correlated to antifactor Xa activity in patients with and without renal dysfunction. There were no differences in the slopes of the regression lines between groups. Further studies are needed to determine if TGT, PCF and CEM provide more useful clinical information regarding the level of anticoagulation in high-risk patients receiving low-molecular weight heparin therapy.

Purpose: The onset of force production due to thrombin generation (TGT), the amount of force produced (PCF) and the elastic modulus (CEM) of clots have been shown to be dose dependent functions of increasing recombinant FVIIa (rFVIIa) concentrations in a variety of patient blood samples. We performed a dose dependent comparison of the effects of rFVIIa and NN1731, an enhanced activity analogue of rFVIIa, in blood from normal volunteers and from hemophilia patients with and without factor VIII inhibitors.

Antifactor Xa activity is the gold standard monitoring parameter for low molecular weight heparin (LMWH) derivatives. It is frequently measured in high-risk populations, such as patients with renal dysfunction. Despite antifactor Xa monitoring, however, bleeding in renal dysfunction patients receiving LMWH remains a problem. This study determined the relationship between antifactor Xa activity and three novel coagulation monitoring parameters: thrombin generation time (TGT), platelet contractile force (PCF) and clot elastic modulus (CEM). This study also assessed the effect of renal dysfunction on these relationships. This was an ex vivo pharmacodynamic study of the relationship between antifactor Xa activity and TGT, PCF and CEM in subjects both with and without renal dysfunction. Thirty subjects completed this study (10 controls, 10 chronic kidney disease subjects, and 10 end-stage renal disease subjects receiving hemodialysis). Blood samples obtained from participants were spiked with increasing enoxaparin concentrations (0.25, 0.5, 1.0 and 3.0 IU mL(-1)). Samples were analyzed for TGT, PCF and CEM. The relationship between antifactor Xa activity and TGT, PCF and CEM was determined by Pearson's correlation. The effect of renal dysfunction on the relationship between antifactor Xa activity and TGT, PCF and CEM was determined by analysis of covariance. There is strong correlation between antifactor Xa activity and TGT, CEM and PCF. The presence of renal dysfunction significantly prolongs the TGT, and decreases the CEM relative to controls. These results suggest that patients with renal dysfunction have a greater pharmacodynamic response to LMWH, independent of the pharmacokinetics of LMWH.

Reports of the potential clinical utility of recombinant Factor VIIa (rFVIIa, NovoSeven®, NovoNordisk) in a variety of bleeding disorders have raised the possibility that this agent may have broad spectrum hemostatic properties. We have previously demonstrated that rFVIIa produces dose dependent correction of hemostatic status in patients with Factor VIII deficiency and/or Factor VIII inhibitor. In this study we measured the dose dependent effects of rFVIIa on Platelet Contractile Force (PCF), Clot Elastic Modulus (CEM) and Thrombin Generation Time (TGT) (Hemodyne® Analyzer, Hemodyne, Inc.) in patients with factor IX deficiency. Results were compared to those seen with recombinant factor IX (BeneFIX® [GI, Wyeth]) and factor nine concentrate (Mononine® [Aventis-Behring]). Blood from three patients with factor IX deficiency was collected via aseptic venipuncture into evacuated tubes containing 3.2% sodium citrate. The samples were spiked with increasing amounts of each hemostatic agent so as to yield levels ranging from 25 to 200% of the recommended dose. At baseline, each of the factor IX deficient patients had prolonged TGT, decreased PCF and decreased CEM. Despite the fact that each patient had less than 1% of normal factor IX activity, there was significant variability between the patients in their baseline values. TGT for the three patients varied from 587 to 1200 seconds. Baseline PCF ranged from 0.9 to 1.4 Kdynes, while CEM varied from 1.6 to 5.4 Kdynes/cm2. Normal TGT, PCF and CEM values for asymptomatic controls (mean ± SD, n=25) were 392±100 seconds, 7.8±1.3 Kdynes and 18.8±5.6 Kdynes/cm2 respectively. At the 100% of recommended dose, each of the factor IX agents produced shortening of the TGT, and increases in PCF and CEM. The degree of normalization of these parameters did not appear to depend on the baseline values. The patient with the worst baseline parameters completely normalized his values at 100% doses of BeneFIX® and Mononine®. The patient with the best baseline values also normalized his parameters with BeneFIX® but not with Mononine®. The patient with intermediate baseline abnormalities improved all parameters with both factor IX products but appeared to respond better to BeneFIX® at the 100% dose level. The response to NovoSeven®, at 100% of the dose recommended for treatment of factor VIII inhibitor patients, was detectable but clearly inferior to the response to the factor IX agents. These results indicate significant variability in hemostatic status between factor IX deficient patients both at baseline and in response to factor replacement. While rFVIIa has activity in these patients, the dose previously shown to correct TGT in factor VIII deficient patients may not be appropriate for factor IX deficient patients.

Purpose: Both HCV and HIV are common in hemophiacs. Because of increased bleeding risks, little data are available on the safety of percutaneous outpatient liver biopsy (LBx) and histologic spectrum of disease in this population. Aims: To report our experience with percutaneous LBx in a cohort of hemophiliacs infected with HCV and describe the spectrum of disease and impact of HIV coinfection. Methods: A retrospective review of consecutive patients with hemophilia and HCV was performed. All patients were positive for HCV RNA. Demographic, biochemical, and histologic parameters and data regarding administration of factor concentrates given both prior and following biopsy were recorded. All LBx were performed with a 16 gauge klatskin needle after factor replacement and histology was assessed by the Knodell histologic activity index (HAI) for inflammation (0–18) and fibrosis (0–4). Mild disease was defined as a fibrosis score 0–1 and advanced fibrosis as bridging fibrosis/cirrhosis (3/4). Results: Twenty seven patients (all male, mean age 37, 22 hemophilia A, 5 hemophilia B) underwent successful percutaneous LBx without bleeding complication. HIV coinfection was present in 44% (mean CD4 382, all on HAART) and associated with higher AST, alkaline phosphatase (AP), lower platelets (PL), higher fibrosis scores, and more advanced fibrosis including all cases of cirrhosis when compared to HCV moninfection desipte similar demographic features and disease duration. Conclusions: Outpatient percutaneous LBx can be safely performed in patients with hemophila. The spectrum of liver disease included a significant proportion with advanced fibrosis which was much more common in those coinfected with HIV.Table

Background: Patients with renal dysfunction who undergo systemic anticoagulation with enoxaparin are at increased risk for bleeding. Although there is decreased renal clearance of enoxaparin in this population, the clinical utility of monitoring antifactor Xa activity is controversial because it is weakly correlated to bleeding. The goal of this study was to investigate the role of other novel anticoagulation markers, such as thrombin generation time, platelet contractile force, and clot elastic modulus, while controlling for antifactor Xa activity in patients with and without renal dysfunction. Methods: Thirty anticoagulant- and antiplatelet-naive subjects completed this trial (10 controls, 10 patients with chronic kidney disease, and 10 patients with end-stage renal disease [ESRD]). Blood samples were obtained and spiked ex vivo with increasing concentrations of enoxaparin antifactor Xa activity (0.25, 0.5, 1.0, and 3.0 IU/mL). Thrombin generation time, platelet contractile force, and clot elastic modulus were measured in each group at each antifactor Xa activity concentration. Results: Subjects with ESRD had an approximately 50% greater anticoagulant effect, determined by thrombin generation time prolongation, than controls at antifactor Xa activity concentrations of 0.5 to 3.0 IU/mL. This may explain why subjects with ESRD with seemingly therapeutic antifactor Xa levels still experience adverse bleeding. There were no intergroup differences in platelet function, determined by platelet contractile force and clot elastic modulus. Conclusion: Antifactor Xa poorly predicts the degree of anticoagulation in patients with ESRD administered low-molecular-weight heparin (LMWH). Thrombin generation time may be a clinically useful anticoagulation monitoring tool to monitor LMWH therapy, especially in patients with renal dysfunction. Additional randomized prospective studies are needed to corroborate these findings.

Prothrombin activation requires the direct interplay of activated platelets and plasma clotting factors. Once formed, thrombin causes profound, irreversible activation of platelets and reinforces the platelet plug via fibrin formation. Delayed or deficient thrombin production increases bleeding risk. Commonly employed coagulation assays, the prothrombin and partial thromboplastin times, use clot formation as a surrogate marker of thrombin generation. These assays routinely utilize platelet-poor plasma and completely miss the effects of platelets. Other markers of thrombin generation, prothrombin fragment 1 + 2 (F1 + 2) and thrombin-antithrombin complex, are typically measured after the fact. We report a simple assay, which employs the onset of platelet contractile force (PCF) as a surrogate marker of thrombin generation. PCF generation occurs concomitant with the burst of F1 + 2 release. The time between assay start and PCF onset is termed the thrombin generation time (TGT). TGT is prolonged in clotting factor deficiencies and in the presence of direct and indirect thrombin inhibitors. TGT shortens to normal with clotting factor replacement and shortens with administration of recombinant factor VIIa. TGT is short in thrombophilic states such as coronary artery disease, diabetes and thromboangiitis obliterans and prolongs toward normal with oral and intravenous anticoagulants.

While recombinant factor VIIa (rFVIIa) shows promise as a broad-spectrum hemostatic agent, questions remain regarding the most appropriate dose and the best way to monitor its effects. In this study we tested the sensitivity of a thrombin dependent platelet assay, platelet contractile force, to the effects of rFVIIa in normal, factor-deficient, and inhibitor-containing blood samples. Dose dependent effects of rFVIIa on platelet contractile force (PCF) and clot elastic modulus (CEM) were measured in all blood samples. rFVIIa minimally affected PCF and CEM in normal blood clotted with thrombin or batroxobin. While rFVIIa minimally altered PCF and CEM in factor VIII (FVIII) deficient blood clotted with thrombin, rFVIIa increased PCF and CEM and shortened the lag phase in a dose dependent manner in batroxobin-induced clots. The effects of rFVIIa in factor IX (FIX) deficient blood mirrored the effects seen in FVIII deficient samples. Whether clotted with thrombin or batroxobin, baseline PCF and CEM were abnormally low in FVIII deficient samples containing FVIII inhibitors. In such samples, rFVIIa caused dose dependent improvement of PCF, CEM, and lag phases. In one patient with a spontaneous inhibitor, rFVIIa caused dose dependent increases in PCF and CEM in blood clotted with either enzyme. rFVIIa corrects the deficient thrombin generation seen in FVIII and FIX deficiency, and in blood containing FVIII inhibitors. As a consequence, platelet function is improved and clot structure is enhanced. Platelet contractile force and clot elastic modulus measurements are sensitive to the dose dependent effects of rFVIIa.

SummaryRapid laboratory markers that correlate with patient risk would facilitate the decision making regarding admission of patients with chest pain (CP). Platelet contractile force (PCF) and clot elastic modulus (CEM) are elevated in patients undergoing coronary bypass grafting. This study assessed PCF, CEM, and platelet aggregation in patients presenting to the emergency department with chest pain (CP). Results were compared with fifty normal controls. Both the total group of CP patients (n = 100) and the subset of patients (n = 36) with documented coronary arterys disease (CAD) had significantly elevated PCF and CEM, and significantly decreased platelet aggregation relative to normal (p <0.001 for the total group, p <0.008 for patients with CAD). Patients with electrocardiographic evidence of ischemia had the highest PCF and CEM values of any patient group. Increased PCF and CEM were not due to higher platelet counts, and PCF did not differ by race.

Clot retraction is a thrombin-dependent, platelet-mediated contraction of the cellular clot mass. In this Study, the effects of delayed, deficient and inhibited thrombin generation on the development of platelet contractile force (PCF) and clot elastic modulus (CEM) were measured. When normal citrated whole blood is clotted by the addition of exogenous thrombin (1 U/ml) and calcium (10 mmol/l), PCF and CEM start to develop within the first minute and begin to level off by 1200 s. If identical samples are clotted with batroxobin (0.21 mug/ml) and calcium (10 mmol/l), both PCF and CEM development are delayed approximately 5 min. After 1200 s of clotting, however, values in the batroxobin system approach those seen with exogenous thrombin. If the added calcium concentration is held constant at 10 mmol/l, increasing the exogenous thrombin concentration from 0 to 0.5 U/ml results in increased PCF and CEM values. Above 0.5 U thrombin, the effect plateaus. At exogenous calcium of 10 mmol/l, increasing batroxobin concentrations (0-0.210 mug/ml) caused a 75% increase in PCF and a 55% increase in CEM. The increase in CEM reached a plateau above 0.05 mug batroxobin/ml. The effects of varying calcium concentrations were evaluated at constant batroxobin (0.21 mug/ml) and thrombin (1 U/ml) concentrations. With thrombin, PCF and CEM increased by > 700% as CaCl2 increased from 0 to 5 mmol/l. Above 5 mmol/l, no additional increases occurred. With batroxobin, PCF did not develop at CaCl2 concentrations less than or equal to 2.5 mmol/l. Above 2.5 mmol/l CaCl2, PCF values increased and at 10 mmol/l CaCl2 were equal to those seen with thrombin. CEM in batroxobin-mediated clots peaked at 10 mmol/l CaCl2 but were 40% less than the values found in thrombin-mediated clots. When the thrombin inhibitor P-PACK was added to the batroxobin system, dose-dependent decreases in PCF and CEM were noted. At 120 mumol/l, P-PACK totally suppressed PCE PCF in blood from a factor VIII-deficient patient varied significantly when clotted with batroxobin versus thrombin. PCF development in factor VIII-deficient blood was normal with thrombin but is delayed and depressed with batroxobin. PCF values in factor VIII-deficient blood did not reach the thrombin value after 1200 s of clotting, and CEM was significantly less. These results confirm that PCF development is thrombin dependent and that delay or reduction of PCF development results in structurally weaker clots. (C) 2002 Lippincott Williams Wilkins.

Blood loss secondary to platelet dysfunction is known to be increased when the duration of cardiopulmonary bypass (CPB) is prolonged. The ability to correlate alterations in platelet function with the duration of bypass and early postoperative blood loss, however, has remained elusive. Platelet contractile force, a novel measure of platelet-mediated clot retraction, is known to be reduced following cardiac surgery and blockade of platelet adhesion receptors. The aim of this study was to determine if alterations in platelet contractile force (measured using whole blood) correlated with the duration of CPB and early postoperative blood loss. Thirty patients were entered into a study designed to measure platelet function before, during, and after CPB. Platelet aggregometry and surface expression of CD42b and CD61 were also measured (using whole blood) in a subset of subjects (n=10) to further characterize the intrinsic structural and functional defects induced by CPB. Reductions in platelet contractile force had a significant correlation with duration of CPB (r=0.564; P=0.002) and early blood loss (r=0.545; P=0.003). Although decreases in platelet contractile force and aggregation both correlated with CPB time in the smaller subset of patients tested, only platelet contractile force correlated with decreases in CD42b, CD61 and blood loss. The results of this study suggest that prolongation of CPB is related to increasing degrees of platelet dysfunction and that reductions in platelet contractile force are related to decreases in platelet adhesion receptors and early postoperative blood loss.

Background: Aprotinin interferes with heparin binding to platelets and decreases blood loss during cardiopulmonary bypass (CPB). Heparin abolishes platelet force during CPB, and the extent of platelet force recovery after protamine administration appears to correlate with blood loss. This study assessed the effect of aprotinin on heparin suppression of platelet force. Methods: Platelet force was measured using the Hemodyne(R) Hemostasis Analyzer. Clots were formed from platelet-rich plasma (PRP) by the addition of batroxobin and 10 MM CaCl2. Clotting conditions included pH 7.4, ionic strength 0.15 M, fibrinogen level 1 mg/ml and 75,000 platelets/mul. Results: After 1200 s of clotting, force was reduced from 7110 +/- 1190 to 450 +/- 450 dyn by 0.2 U/ml of heparin. Platelet force in aprotinin [20 mug/ml (140 KIU/ml)] containing PRP was not suppressed by heparin addition (7480 +/- 2410 dyn). Aprotinin [40 mug/ml (280 KIU/ml)]. addition to previously heparinized plasma counteracted heparin force suppression. Aprotinin (40 mug/ml) increased platelet force from 5630 to 11,138 +/- 562 in PRP devoid of heparin. Aprotinin did not affect thrombin activity, fibrin structure, platelet aggregation or secretion. Conclusions: Aprotinin counteracts heparin suppression of platelet force and enhances platelet force in the absence of heparin. Aprotinin-heparin-platelet interactions may help explain aprotinin's ability to reduce blood loss during CPB. (C) 2003 Elsevier Science Ltd. All rights reserved.

Eighty percent of patients with diabetes mellitus die a thrombotic death. Seventy-five percent of these deaths is due to cardiovascular complications, and the remainder is due to cerebrovascular events and peripheral vascular complications. Vascular endothelium, the primary defense against thrombosis, is abnormal in diabetes. Endothelial abnormalities undoubtedly play a role in the enhanced activation of platelets and cloning factors seen in diabetes. Coagulation activation markers, such as prothrombin activation fragment 1+2 and thrombin-antithrombin complexes, are elevated in diabetes. The plasma levels of many clotting factors including fibrinogen, factor VII, factor VIII, factor SI. factor XII, kallikrein, and von Willebrand factor are elevated in diabetes. Conversely, the level of the anticoagulant protein C (PC) is decreased. The fibrinolytic system, the primary means of removing clots, is relatively inhibited in diabetes due to abnormal clot structures that are more resistant to degradation and an increase in plasminogen activator inhibitor type 1 (PAI-1). Increased circulating platelet aggregates, increased platelet aggregation in response to platelet agonists, increased platelet contractile Force (PCF), and the presence of higher plasma levels of platelet release products, such as beta -thromboglobulin, platelet factor 4, and thromboxane beta (2), demonstrate platelet hyperactivity in diabetes. This constellation of findings supports the clinical observation that diabetes is a hypercoagulable state. This article briefly reviews the published evidence for this conclusion and the putative roles played by hyperglycemia and hyperinsulinemia in its development. (C) 2001 Elsevier Science Inc. All rights reserved.