Ranjit S. More

Prevention and management of bleeding complications after thrombolysis

In terms of patient numbers treated, coronary thrombolysis has now far outstripped all other forms of thrombolytic therapy. Because of the need for speed in initiating coronary thrombolysis, both the decision to administer such treatment and its supervision are usually in the hands of those trained in general medicine or cardiology rather than haematologists. In general, this policy has led to a widespread but seldom inappropriate use of thrombolysis, and to a very favourable balance between benefits and side effects. Several large controlled trials of thrombolytic therapy have provided data from which the incidence of bleeding complications can be estimated, although the absence of a standardised set of criteria makes cross-trial comparison difficult [1,2]. The most satisfactory definition of a “major” bleeding complication is one which causes death or permanent disability, or which prolongs the patient’s hospital stay. Transfusion requirements are an unsatisfactory criterion, as the indications seem to vary from country to country, and from unit to unit. There is a clear-cut

The effect of surgical preparation and in-vitro distension on the intrinsic fibrinolytic activity of human saphenous vein

The objective of this study was to assess the effect of distension on the intrinsic fibrinolytic activity of human saphenous vein during its preparation for use as a bypass conduit prior to coronary artery surgery. Fibrinolytic activity was studied using fibrin plate techniques and Chromogenic assays for extractable tPA and uPA. Samples were obtained from patients undergoing routine coronary surgery. Fibrinolytic activity was compared in control vein (untouched) vein that had been prepared for use as a bypass conduit (surgical dissection, ligation of side branches and careful but uncontrolled manual distension) and segments of dissected vein distended in vitro to pressures of 230 or 120 mmHg. Uncontrolled distension and distension to 230 mmHg impaired fibrinolytic activity as determined by areas of lysis on fibrin plates (p < 0.05), (p < 0.005), tPA activity (p < 0.005) (p < 0.005) and uPA activity (p < 0.05), (p < 0.005). Distension to 120 mmHg had no effect on the fibrinolytic activity of human saphenous vein. Impaired fibrinolytic activity caused by uncontrolled distension of saphenous vein prior to its use as a vascular conduit may contribute to early vein graft thrombosis and can be avoided using controlled distension to < 120 mmHg.

ASSESSMENT OF MYOINTIMAL CELLULAR KINETICS IN A MODEL OF ANGIOPLASTY BY MEANS OF PROLIFERATING CELL NUCLEAR ANTIGEN EXPRESSION

A detailed temporal assessment of cellular proliferation was carried out by means of immunostaining for proliferating cell nuclear antigen in a normolipemic rabbit model of balloon angioplasty to the iliac arteries. Assessment was made at 30 minutes, 2 hours, 1 day, 3 days, 7 days, 14 days, 1 month and 3 months after the procedure. Intimal hyperplasia was first noted at day 3; a prominent layer was formed by day 14. Cellular proliferation in the vessel media was observed as early as day 1 (percentage of positive-staining cells 0.5% +/- 0.2%), reaching a maximum by day 7 (16.9% +/- 5.1%) before returning to baseline levels by 1 month (0.2% +/- 0.02%); in the intima, cellular proliferation was first noted at day 7 (0.7% +/- 0.3%) and reached a maximum at day 14 (4.1% +/- 0.4%) before returning to baseline levels at 1 month (0.3% +/- 0.1%). Use of proliferating cell nuclear antigen expression in this model of angioplasty provided a simple and reproducible method of assessing cellular proliferation after vascular injury and may prove useful for monitoring the effects, in experimental models, of agents for reducing myointimal hyperplasia.

Prothrombin fragment F1+2 concentrations for monitoring anticoagulation therapy with heparin

We have compared the activated partial thromboplastin time with measurement of prothrombin fragment F1 + 2 concentrations (ELISA assay) during a 24-h period in a group of patients (n = 10) who had undergone elective coronary angioplasty and were anticoagulated post-procedure with heparin 1000 U/h. Four hours after the procedure all the patients were adequately anticoagulated according to activated partial thromboplastin time (median ratio 4.7:1) and the prothrombin fragment F1 + 2 concentration was significantly lower than pre-angioplasty values (0.5 vs 1.4 nmol/l, p = 0.04). At 24 h the median activated partial thromboplastin time ratio was still higher than the pre-procedure value (1.35 vs 0.9, p < 0.01), but the prothrombin fragment F1 + 2 concentration had risen to 2.1 nmol/l, with more variability in individual results within the patient group for the prothrombin fragment F1 + 2 concentration than for activated partial thromboplastin time (interquartile ranges (Q1, Q3) prothrombin fragment F1 + 2, 1.2-2.5; activated partial thromboplastin time, 1.2-1.5). The activated partial thromboplastin time is the standard method of monitoring the anticoagulant effect of heparin but may not fully reflect the functional coagulation status and accurately identify individual patients with less than adequate anticoagulation. Prothrombin fragment F1 + 2 concentrations may provide a more reliable indicator in individual patients of functional coagulation status in certain important situations where anticoagulation is critical such as following complicated coronary angioplasty.

Monitoring anticoagulation following intracoronary procedures: which method?

We have assessed bedside kits for monitoring the activated partial thromboplastin time and the activated clotting time by comparing them with laboratory activated partial thromboplastin time values. To determine the accuracy of anticoagulation we have concurrently measured the plasma heparin concentrations, and plasma prothrombin fragment F1 + 2 concentrations. Serial samples were taken from patients undergoing elective percutaneous transluminal coronary angioplasty (n = 14). Readings were taken pre-procedure, 30 min after administration of a heparin bolus (10,000 U) and 1, 2 and 3 h after commencement of a constant heparin infusion (15 U/kg/h) postprocedure. Activated partial thromboplastin time results obtained with the bedside kit compared reliably with laboratory values (r = 0.8), were rapidly available and were reflected by appropriate changes in prothrombin fragment F1 + 2 and heparin concentrations. However, the relationship between activated partial thromboplastin time values and activated clotting time was less precise (r = 0.59). Therefore, for routine and frequent monitoring of anticoagulation with heparin, a bedside activated partial thromboplastin time kit provides adequate control of therapy but in instances were particularly tight control of anticoagulation is required, use of prothrombin fragment F1 + 2 concentrations may be more appropriate.

Quantifying the Effect of Locally Delivered Anticoagulant Drugs: Modification of an in vivo Model of Venous Thrombosis

Increasingly, attention is focusing on the local delivery of antiplatelet and fibrinolytic therapy as a means of preventing intravascular thrombosis. A simple, reproducible model of thrombosis, based upon vascular damage is needed to test these agents in vivo. We have therefore modified a rat vena cava model of venous thrombosis based upon vascular injury and stasis. Wistar rats are anaesthetised, the inferior cava dissected free and a segment isolated by slings distally above the iliacs and proximally above the left renal vein. All other tributaries are ligated. Vascular injury is induced by externally applying soft-jaw clamps for 5 min. Agents to be tested are introduced into the isolated segment via a left renal vein cannula left in situ for 15 min and then flushed from the cava. Blood is allowed to refill the segment, all remaining slings are tied and the animal left for 30 min before being killed. The cava is then opened and thrombus removed and weighed. Scanning electron microscopy of the cava after clamping shows areas of normal endothelium interspersed with areas of denuded endothelium and exposed subendothelial connective tissue. Histological and immunohistochemical staining indicates the thrombus is composed of red cells, platelets and fibrin. The model was validated by assessing the effects of 2 different doses of locally delivered tissue-type plasminogen activator (tPA). The mean weights of thrombus were [mg (SD)]: Control (saline; n = 8) 39.0 (8.73), tPA 0.01 mg/ml (n = 6) 45.5 (10.56) and tPA 1 mg/ml (n = 8) 3.5 (3.4). Comparing 1 mg/ml tPA vs. 0.01 mg tPA vs. control, p < 0.001 (Mann-Whitney test).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of intraluminal application of tissue-type plasminogen activator on the fibrinolytic activity of experimental vein grafts

OBJECTIVE The aim was to quantify the effect of intraluminally applied tissue-type plasminogen activator (tPA) on the fibrinolytic activity of experimental vein grafts and assess the effect of pretreatment of the vein on early platelet and thrombus formation using histological techniques. METHODS A pig model of bilateral saphenous venin-carotid artery grafts was used. In each animal one side of the neck was grafted using vein distended to 230 mm Hg and pretreated with tPA (1 mg.ml-1) for a period of 15 min before grafting (treated graft). The perfused in situ for 2 h after implantation and before analysis. Changes in local fibrinolytic activity were quantified using fibrin plate techniques and specific chromogenic assays for tPA and urokinase (uPA) in tissue extract (n = 6 animals). Histological assessment was made using light and scanning microscopy (n = 4 animals). RESULTS Surgical preparation and distention significantly reduced the fibrinolytic activity of pig saphenous vein in terms of areas of lysis produced on fibrin plates (P < 0.05), tPA activity (P < 0.05), and uPA activity (P < 0.05). Pretreatment of distended vein with tPA before grafting significantly enhanced its fibrinolytic activity after 2 h perfusion compared to control (untreated) grafts, as assessed by areas of lysis on fibrin plates (P < 0.05) and specific tPA activity (P < 0.05). Treated grafts also showed qualitatively less platelet and thrombus formation on histological examination. CONCLUSIONS Pretreatment of surgically harvested vein by intraluminal application of tPA before grafting enhances its fibrinolytic activity after exposure to 2 h perfusion in vivo. This technique requires further investigation to validate its potential as a means of providing local anticoagulation to veins implanted as arterial grafts thereby reducing the incidence of early graft thrombosis.

In vitro studies of a bispecific monoclonal antibody antithrombotic conjugate [ATC(3)]

We assessed whether chemical conjugation of a monoclonal antibody (M735) against platelet glycoprotein IIb/IIIa receptors to urokinase and to a monoclonal antibody which binds to damaged endothelium (P14G11) resulted in enhanced inhibition of platelet aggregation with maintenance of localizing features (to damaged endothelium). Conjugation of M735 (an IgM monoclonal antibody) to urokinase and P14G11 (an IgG2a monoclonal antibody) was carried out using SPDP [N-Succinimidyl-3-(2-pyridyldithio)-propionate] as the cross-linking reagent. The resulting triple conjugate [ATC(3)] had fibrinolytic activity and retained platelet and damaged endothelium localizing features. Enhanced inhibition of platelet aggregation (induced by either adenosine diphosphate (ADP), collagen, or thrombin) was observed compared with both an unconjugated mixture of the three individual components and to the M735-urokinase conjugate. We conclude that it is possible to produce a bimonoclonal antibody-urokinase agent which has both potent antiplatelet aggregatory effects and additional targeting or localizing features.

Therapeutic levels of nitroglycerine do not affect the uptake and release of heparin by endothelial cells in vitro

Intravenous heparin and nitroglycerin are frequently given in combination to patients with acute coronary syndromes such as unstable angina and post myocardial infarction angina. Heparin is prescribed since it has been shown that intracoronary thrombus formation is important in the pathophysiology of these acute conditions. However, it has been demonstrated that intravenous nitroglycerin can interfere with the anticoagulant effect of heparin. The exact mechanism of the interaction is unknown but it has been suggested that there is a direct effect on plasma heparin characterised by a reduction in circulating plasma heparin levels. Heparin binds to the surface of endothelial cells in a process that is time dependent, reversible and exhibits saturation kinetics. A possible mechanism of the observed effects on the plasma heparin levels produced by nitroglycerin may be the altered handling of heparin by endothelial cells. We have investigated this further by assessing the effects of therapeutic doses of nitroglycerin on heparin uptake and release by endothelial cells, using 35S labelled heparin and human umbilical vein endothelial cell cultures.