Frederick A. Ofosu

Thrombin structure and function: why thrombin is the primary target for antithrombotics.

Thrombin has both beneficial and harmful effects. In order of importance, at very low concentrations, alpha-thrombin firstly amplifies its own generation through the activation of factors V and VIII, which are the primary targets of antithrombotic agents. It secondly functions at the cellular level where, at low concentrations it activates platelets, and at higher concentrations, induces endothelial cell changes (e.g., shape changes, albumin transport release of plasminogen activators and other substances). It thirdly converts fibrinogen into clottable fibrin and becomes actively incorporated into the forming thrombus. In addition, it activates protein C, which in turn degrades factors V and VIII (and/or their activated forms) and causes the shutdown of thrombin generation. When compared to other serine proteinases of the blood coagulation and fibrinolytic systems, alpha-thrombin is unique in that it loses most of its proenzyme activation fragment and has developed multisite short-ranged bridge-binding interactions, which appear to explain thrombin specificity. To understand thrombin is to understand haemostasis.

The haemorrhagic and antithrombotic effects of dermatan sulphate

Summary. Heparin and dermatan sulphate are effective antithrombotic agents but the clinical use of heparin is complicated by haemorrhage. The haemorrhagic effect of dermatan sulphate is unknown. In this study we compared the antithrombotic, haemorrhagic and anticoagulant effects of heparin and dermatan sulphate in rabbits. The antithrombotic effect was measured as prevention of venous thrombus formation. The haemorrhagic effect was measured as 51Cr‐blood loss from standardized cuts in rabbit ears. The anticoagulant effect was measured as changes in the APTT, TCT and circulating anti‐factor Xa level, and the formation of 125I‐thrombin/inhibitor complexes ex vivo. The effect of heparin and dermatan sulphate on collagen‐induced platelet aggregation was measured ex vivo. Maximal antithrombotic effects of heparin and dermatan sulphate were achieved with 70 and 500 μg/kg respectively. A 20‐fold increase in heparin dose caused an 8‐fold increase in blood loss and higher doses (40‐ and 80‐fold increases) caused further dose‐related increases in blood loss (13‐ and 35‐fold increases respectively). In contrast, a 20‐ to 40‐fold increase in the antithrombotic dose of dermatan sulphate did not increase blood loss and an 80‐fold dose increase caused only a 7‐fold increase in blood loss. There was no relationship between the antithrombotic and haemorrhagic effects of either heparin or dermatan sulphate and their anticoagulant activities. In contrast, there was a relationship between the dose‐related enhancement of blood loss by these glycosaminoglycans and the inhibition of collagen‐induced platelet aggregation ex vivo. These results suggest that dermatan sulphate is less haemorrhagic than heparin at equivalent antithrombotic doses, and that the haemorrhagic effect is associated with a glycosaminoglycan‐induced platelet defect.

Preliminary study to identify cancer patients at high risk of venous thrombosis following major surgery

Summary This study investigated whether the pre‐surgical plasma levels of TAT and F1 + 2 of patients undergoing major surgery for localized tumours could identify patients at higher risk of thrombosis, and how heparin prophylaxis affected in vivo coagulation after cancer surgery. We measured the pre‐and post‐operative levels of TAT, F1+2, total factor VII (FVIIt) and zymogen FVII (FVIIz) in 117 cancer patients, with and without heparin prophylaxis. The end points of this study were DVT. ihitially detected by 125I‐fibrinogen uptake test and confirmed by ascending venography. Pre‐operative [TAT] and [Fl+2] of the cancer patients were significantly higher than those of age‐matched control subjects (n=50) (P<0.005 and P<0.05, respectively): pre‐operative [FVII] was not significantly different. One of the 83 patients receiving prophylaxis, and 8/34 not receiving prophylaxis developed post‐operative DVT. Of the parameters evaluated, only the pre‐operative [TAT] > 3. 5 ng/ml identified patients at higher risk for post‐operative DVT. Heparin reduced plasma TAT levels and FVII consumption following surgery, suggesting that heparin modulates coagulation associated with cancer surgery. The results of this study also suggest that the pre‐operative [TAT] may identify patients with higher risk for post‐operative DVT.

Anticoagulant effects of heparin in neonatal plasma.

ABSTRACT: Available data on the anticoagulant effects of heparin in neonatal plasma are scarce and conflicting: relative to adult plasma, neonatal plasma has been reported to show both resistance as well as sensitivity to heparin. We explored this apparent paradox by comparing how well heparin accelerated inhibition of exogenous thrombin and prevented thrombin generation in defibrinated neonatal and adult plasmas. Using amidolytic assays, we determined the effects of heparin on 7) the neutralization of exogenous human a-thrombin and on 2) the formation of endogenous thrombin activity after contact activation and recalcification. Neonatal plasma proved resistant to heparin (0.05 U/ mL) during inhibition of added thrombin (15 NIH U/mL). Inhibition of thrombin in heparinized neonatal plasma became as efficient as in adult plasma only after raising the AT III activity to normal adult values. However, de novo generation of thrombin activity was very susceptible to inhibition by heparin, even in neonatal plasmas with physiologically low AT HI levels. Peak thrombin activity generated in neonatal plasma in the absence of heparin was 50% or less of peak adult activity, and this already reduced ability of neonatal plasma to generate thrombin activity upon prothrombin activation was further decreased by heparin (0.05-0.2 U/mL). We conclude that due to the neonatal AT III deficiency, added thrombin is inactivated less effectively by heparin in neonatal than in normal adult plasma. Yet, the generation of thrombin activity is impaired in neonatal plasma and easily suppressed by heparin. We speculate that newborn infants may be resistant to heparin therapy during overt thrombotic disease, when neutralization of abnormal thrombin activity is the therapeutic goal. In contrast, lower plasma heparin levels may be required to prevent the formation of thrombin activity in newborn infants than in adult patients.

Differences in the clinically effective molar concentrations of four direct thrombin inhibitors explain their variable prothrombin time prolongation.

Four direct thrombin inhibitors (DTIs), lepirudin, bivalirudin, argatroban, and melagatran, differ in their ability to prolong the prothrombin time (PT). Paradoxically, the DTI in clinical use with the lowest affinity for thrombin (argatroban) causes the greatest PT prolongation. We compared the effects of these DTIs on various clotting assays and on inhibition of human and bovine factor Xa (FXa). On a mole-for-mole basis, lepirudin was most able to prolong the PT, activated partial thromboplastin time (APTT), and thrombin clotting time (TCT), whereas argatroban had the least effect. At concentrations that doubled the APTT (argatroban, 1 micromol/l; melagatran, 0.5 micromol/l; bivalirudin, 0.25 micromol/l; lepirudin, 0.06 micromol/l), the rank order for PT prolongation was: argatroban > melagatran > bivalirudin > lepirudin. Although the Kis associated with inhibition of human FXa by melagatran (1.4 micromol/l) and argatroban (3.2 micromol/l) approach their therapeutic concentrations, inhibition of FXa did not appear to be a major contributor to PT prolongation, since argatroban also prolonged the PT of bovine plasma (despite a Ki for bovine FXa of 2,600 micromol/l). Only melagatran inhibited prothrombinase-bound FXa. We conclude that the differing effects of the DTIs on PT prolongation are primarily driven by their respective molar plasma concentrations required for clinical effect. DTIs with a relatively low affinity for thrombin require high plasma concentrations to double the APTT; these higher plasma concentrations, in turn, quench more of the thrombin generated in the PT, thereby more greatly prolonging the PT.

Plasma factor VII and thrombin–antithrombin III levels indicate increased tissue factor activity in sickle cell patients

Although the mechanisms involved in the persistent clinical complications of sickle cell disease have not yet been fully delineated, previous studies suggest that sickle cell (HbSS) patients have a disposition to generate more thrombin and plasmin in vivo than normal subjects. The reasons for the impaired regulation of haemostasis in HbSS patients is poorly understood. We report studies evaluating the extent to which in vivo coagulation and fibrinolysis are altered in HbSS patients in steady state. The concentrations of total factor VII (F(VII)t), factor VII zymogen (F(VII)z), thrombin–antithrombin III (TAT), fibrinopeptide A(FPA), and fibrin D‐dimer in plasmas of 50 normal controls (HbAA) and 45 HbSS steady state patients, were measured using sensitive and specific enzyme‐linked immunoassays. The average plasma concentration of F(VII)t, in sickle cell plasma was significantly lower than that of the control subjects (0·70 ± 0·19 U/ml versus 1·16 ± 0·41 U/ml), whereas F(VII)z in the patients and controls were 0·47 ± 0·15 U/ml and 1·15 ± 0·33 U/ml respectively, P<0·001. Both measures of factor VII suggest a higher factor VII turnover in sickle cell disease. The mean concentration of TAT in the plasma of HbSS patients were significantly higher than those of HbAA controls (371 ± 44 pM versus 42 ± 2 pM) (P<0·001), a difference that is strongly indicative of higher rates of in vivo thrombin generation by HbSS patients. Plasmas of HbSS patients had significantly higher concentrations of FPA compared to those of the control subjects (12·85 ± 1·96 ng/ml versus 4·22 ± 0·37 ng/ml) (P<0·001). The D‐dimer levels were also higher in the HbSS than control plasmas (1029·6 ± 58·6 ng/ml versus 224·3 ± 27·6 g/ml) (P<0·001), with the patients’values being indicative of enhanced fibrinolysis. These results strongly suggest accelerated in vivo coagulation and fibrinolysis in HbSS patients even during steady state. They are consistent with the hypothesis that haemostasis is less tightly regulated in the HbSS patients than in HbAA controls. The altered regulation of haemostasis may contribute to the initiation of vaso‐occlusive processes associated with sickle cell painful episodes.

Respective role of antithrombin III and heparin cofactor II in the in vitro anticoagulant effect of heparin and of various sulphated polysaccharides

Summary. The in vitro anticoagulant effects of standard heparin (SH) and of seven other sulphated polysaccharides (SPS) were investigated by measuring activated partial thromboplastin time (APTT) prolongation of normal plasma and of plasmas selectively depleted of antithrombin III (AT III), of heparin cofactor II (HCII) and of both heparin cofactors. This allowed the determination of the relative contribution of each of the two heparin cofactors to the SPS anticoagulant effect. The SPS varied in their relative activities as catalysts of thrombin inhibition by purified AT III or HC II. The anticoagulant activities of heparin and dermatan sulphate were primarily attributable to their ability to enhance thrombin inhibition by AT III and HC II respectively. Heparin had an additional minor anticoagulant activity which was independent of both AT III and HC II. Pentosan polysulphate, high molecular weight dextran sulphate, heparin with low affinity for AT III and a sulphated heparin derivative had weaker anticoagulant activities in normal plasma than standard heparin. The anticoagulant activities of these last four SPS in plasma depleted of both AT III and HC II were similar to their respective activities in normal plasma. This suggests that these SPS act by directly preventing thrombin generation rather than by enhancing thrombin inhibition.

A comparative evaluation of assays for markers of activated coagulation and/or fibrinolysis : thrombin-antithrombin complex, D-dimer and fibrinogen/fibrin fragment E antigen

Measurements were made of levels of D‐dimer in plasma and serum, thrombin–antithrombin complex (TAT) in plasma and fibrinogen/fibrin fragment E antigen (FgE) in serum in a normal healthy control group and in patients with a range of disorders associated with hypercoagulability. Levels were determined in 31 normal healthy controls, 30 patients with disseminated intravascular coagulation (DIC), 21 patients with deep venous thrombosis (DVT), 27 patients with myocardial infarction (MI), 26 patients with acute leukaemia and 56 patients with liver disease. Considering all subjects, significant correlations were established between the results of all assays. Notably high correlations (r>0·9) were established between plasma and serum levels of D‐dimer, between plasma levels of D‐dimer and serum levels of FgE, and between serum levels of D‐dimer and FgE. All assays showed very high discrimination (sensitivity) between the normal control group and patients with DIC (97–100%), but there were marked differences between the assays in sensitivity for DVT and MI. In general, the FgE assay was more sensitive than the D‐dimer assay, whilst both the FgE and D‐dimer assays were more sensitive than the TAT assay. The same trends were apparent in the capability of the assays to discriminate between the normal control group and patients with acute leukaemia and liver disease: disorders with an unknown prevalence of activation of coagulation/fibrinolysis. Our results indicated that measurements of fibrinogen/fibrin degradation products (FDPs) in serum were almost unaffected by artefacts. The data further suggested that the broad‐spectrum FgE assay was better than the more specific D‐dimer assay in detecting clinical hypercoagulability. Our study showed that, in the clinical conditions examined, FDPs were more effective markers of hypercoagulability than TAT.

Hypercoagulability after trauma: hemostatic changes and relationship to venous thromboembolism.

BACKGROUND Major trauma induces a hypercoagulable state, which is frequently complicated by pathological thrombosis. However the sequential changes in coagulation markers and their relationship to clinical thrombosis have been poorly characterized. METHODS We measured several markers of in vivo coagulation and fibrinolysis and their regulation serially for 2 weeks after multi-system trauma in a prospective cohort of patients who received no anticoagulant prophylaxis. Asymptomatic deep vein thrombosis (DVT) was assessed by routine bilateral venography between day 12 and 14. Clinically suspected DVT and pulmonary embolism (PE) were investigated in a standardized manner. RESULTS Among the 135 cohort patients the overall venous thromboembolism (VTE) rate was 59%. Markers of thrombin generation were markedly increased within 24 hours of injury, remained persistently elevated for about 5 days and then decreased by day 14. No early compensatory increase in Tissue Factor Pathway Inhibitor (TFPI) or the complex of Factor Xa and TFPI (FXa-TFPI) was seen; FXa-TFPI remained depressed throughout the study. There was no inverse relationship demonstrated between markers of thrombin generation and thrombin regulation. Acquired APC resistance and hypofibrinolysis did not appear to be important contributors to hypercoagulability after trauma. None of the coagulation markers were independently predictive of VTE. Increasing age was the only significant, independent predictor of VTE. CONCLUSION Major trauma leads to significantly increased and persistent thrombin generation with disruption of its regulation. Coagulation markers do not appear to add independent predictive value in detecting VTE. Increasing age is the most important clinical predictor of VTE after trauma.

Effects of genetic fusion of factor IX to albumin on in vivo clearance in mice and rabbits.

Individuals with haemophilia B require replacement therapy with recombinant or plasma‐derived coagulation factor IX (fIX). More benefit per injected dose might be obtained if fIX clearance could be slowed. The contribution of overall size to fIX clearance was explored, using genetic fusion to albumin. Recombinant murine fIX (MIX), and three proteins with C‐terminal epitope tags were expressed in HEK 293 cells: tagged MIX (MIXT), tagged mouse serum albumin (MSAT) and MFUST, in which MIX and MSAT were fused in a single polypeptide chain. Proteins MFUST and MIXT were two‐ to threefold less active in clotting assays than MIX. In mice, the area under the clearance curve (AUC) was reduced for MFUST compared with MSAT or plasma‐derived MSA (pd‐MSA); the terminal catabolic half‐life (t0·5) did not differ amongst the three proteins. Two minutes after injection, >40% of the injected MFUST was found in the liver, compared with <10% of either MSAT or pd‐MSA. In rabbits, the AUC for MFUST was reduced compared to MIXT, MSAT, or pd‐MSA, while the t0·5 of the fusion protein fell between that of MIXT and MSAT or pd‐MSA. Similar results were obtained with non‐radioactive fused or non‐fused recombinant human fIX in fIX knockout mice. The clearance behaviour of the fusion protein thus more closely resembled that of fIX than that of albumin despite a modest increase in terminal half‐life, suggesting that fIX‐specific interactions that are important in determining clearance were maintained in spite of the increased size of the fusion protein.