Pharmacological profile of sulodexide.

Since its introduction, sulodexide has been used on and off for several indications. More recently this agent has become revitalized and tested in newer indications. Sulodexide is composed of glycosaminoglycan that includes a mixture of fast-moving heparin and dermatan sulfate. It exerts its anticoagulant and antithrombotic action through interactions with both AT and HCII. Sulodexide has been proven to have effects on the fibrinolytic system, platelets, endothelial cells, inflammation and more recently metalloproteases. The administration of sulodexide results in the release of lipoprotein lipase and has been shown to reduce the circulating level of lipids. It has also shown to decrease the viscosity of both whole blood and plasma. Sulodexide differs from heparin in its oral bioavailability and longer half-life. There is also less bleeding associated with sulodexide. In addition, oral administration of sulodexide does not interfere with the pharmacologic actions of commonly used agents. Similar to heparin, sulodexide releases TFPI which contributes to its antithrombotic effect and anti-inflammatory properties. Sulodexide has been proven to be effective in peripheral arterial thrombosis and venous thrombosis. It is also clinically active in the treatment of venous leg ulcers and intermittent claudication. More recent data suggest that sulodexide can be used in tinnitus and in vascular vertigo. Additional studies in these indications are required. Sulodexide was generally safe and well tolerated in the clinical trials, without any severe bleeding complications. Therefore sulodexide appears to be a good treatment for all arterial and venous diseases and for the prevention of progression of disease.

Comparative tissue factor pathway inhibitor release potential of heparins.

Tissue factor pathway inhibitor (TFPI) is released following the administration of unfractionated heparin, low-molecular-weight heparins, defibrotide and PI-88. In this study, the comparative effects of heparin, a low-molecular-weight heparin-gammaparin and a heparin-derived oligosaccharide mixture-subeparin (C3) were studied on functional and immunologic tissue factor pathway inhibitor activity levels in a non-human primate (Macaca mulatta) model. The dose-dependent effect was studied following intravenous and subcutaneous administration. Following the administration of 1 mg/kg of heparin, gammaparin, and C3, the functional levels of TFPI at 5 minutes were 2.40, 2.56, and 1.08 U/mL and the corresponding TFPI immunologic levels were 4.3-, 4.0-, and 2.1-fold, increased, respectively, over the baseline value. From these results, it can be concluded that heparin and gammaparin produced similar levels of TFPI release. Hence, gammaparin and heparin have similar TFPI release potential despite their differences in molecular weight. The influence of molecular weight, charge density, and interactions with heparin cofactor II on TFPI release are also discussed.

Successful use of recombinant hirudin and its monitoring by ecarin clotting time in patients with heparin-induced thrombocytopenia undergoing off-pump coronary artery revascularization.

Refludan (lepirudin-rDNA for injection) is the first direct thrombin inhibitor approved by the United States FDA for anticoagulation to patients with heparin-induced thrombocytopenia (HIT). It was monitored by ecarin clotting time (ECT) assay in patients with HIT. Case histories and clotting parameters for three patients undergoing off-pump coronary artery revascularization procedure are discussed. The first patient received r-hirudin at a dose of 0.2 mg/kg intravenous (IV) bolus followed by 0.15 mg/kg/hour infusion. The second patient received 0.4 mg/kg IV bolus followed by infusion of 0.15 mg/kg/hour infusion. The third patient with renal failure received 0.2 mg/kg IV bolus followed by an infusion of 0.02 mg/kg/hour. Blood samples were drawn at baseline, 5 minutes post bolus and every 15 minutes during the coronary artery revascularization procedure. ECT was performed immediately on the citrated whole blood samples using the ECT cards in conjunction with the point-of-care, the thrombolytic assessment system (TAS) Analyzer (Pharmanetics, Raleigh, NC). The plasma samples were then analyzed for APTT and liquid ECT assay performed on a kinetic centrifugal analyzer (ACL 300 Plus). The ECT by cards was ideally maintained above 600 seconds during the surgical procedure. Additional boluses of Refludan were given as and when necessary (ECT < 600 sec) in order to maintain adequate anticoagulation. The calculated circulating concentrations of Refludan, following a bolus administration, based on the ECT cards, liquid ECT and APTT were 3.20 +/- 1.3, 3.51 +/- 1.35 and 2.02 +/- 1.19 microg/mL, respectively.

Blood levels of nitric oxide, C-reactive protein, and tumor necrosis factor-alpha are upregulated in patients with malignancy-associated hypercoagulable state: pathophysiologic implications.

Endogenous generation of nitric oxide (NO) plays an important role in the regulation of cardiovascular and inflammatory responses. This mediator is synthesized by a family of enzymes collectively known as NO synthase. Several isoforms of this enzyme have been identified and can be grouped as constitutive or inducible. Increased production of NO is reported in several inflammatory disorders, such as sepsis, arthritis, thrombotic thrombocytopenic purpura (TTP), and antiphospholipid syndrome. In addition, NO upregulates cyclo-oxygenase-2 and synthesis of several other inflammatory cytokines. Inflammation and thrombotic complications are usually associated with malignancy. Earlier reports indicate the upregulation of tumor necrosis factor-alpha (TNF-alpha), C-reactive protein (CRP), and tissue factor (TF) in patients with malignancy. To determine the relationship between inflammatory cytokines and NO in cancer patients with hypercoagulable states, baseline plasma samples from 160 patients with confirmed malignancy and hypercoagulable state were analyzed for NO levels. A chemical method based on a chemiluminescent reaction between NO and ozone using a highly sensitive gas phase NO analyzer was used. CRP, TF, and TNF-alpha were measured using enzyme-linked immunosorbent assay methods. Of the 160 patients who were plasma tested, the baseline NO levels ranged from 13.7 to 98.6 microM (63.1+/-15.9 microM, mean+/-SD) in contrast to age-matched control, which ranged from 9.1 to 34.6 microM (19.8+/-6.2 microM, mean+/-SD, n=138). Cancer patients also showed marked variations in the NO levels. Eighteen of 60 cancer patients exhibited greater than 60 microM NO levels. The CRP, TNF-alpha and TF were also significantly elevated. A correlation between CRP (r(2)=0.73) and NO levels was noted in cancer patients with hypercoagulable state. These data suggest that the pathogenesis associated with malignancy/hypercoagulable state is associated with an inflammatory component. In addition, the observed hemodynamic changes in some of the cancer patients may be due to increased NO production.

Influence of different anticoagulant agents on fibrinopeptide a generation.

The purpose of this study was to determine the in vitro effects of different anticoagulant drugs on fibrinopeptide A (FPA) generation inhibition and to identify whether there is any correlation between FPA generation, Hemochron ACT, global clotting assays, and chromogenic assays. Unfractionated heparin is a conventionally used anticoagulant. New anticoagulant drugs such as low molecular weight heparins (LMWHs), pentasaccharide, and antithrombin drugs are now approved for various indications. Anti-Xa drugs are in various phases of clinical development. The influence of different anticoagulant agents has been studied on fibrinopeptide A generation, Hemochron celite ACT, global clotting assays, and chromogenic anti-Xa and anti-IIa assays. Different LMWHs (Clivarin, Dalteparin, Enoxaparin, and Tinzaparin), anti-Xa agents (Pentasaccharide, DX-9065a and unfractionated heparin), and anti-IIa agents (PEG-Hirudin, Hirudin, Efegatran and Argatroban) were studied. The blood from healthy volunteers (n=4) was drawn for each drug. Imuclone FPA enzyme-linked immunosorbent kit assay, Hemochron celite ACT assay, global clotting assays (PT, APTT, Heptest-HI, thrombin time), and Loyola chromogenic anti-Xa and anti-IIIa assays were studied. Pentasaccharide demonstrated minimal effects on the whole blood clotting time such as ACT and on inhibition of FPA generation (IC50 > 25 microg/mL). DX-9065a exhibited a significant prolongation of ACT and marked inhibition of FPA generation (IC50 = 4.12 microg/mL). Unfractionated heparin showed a marked inhibition of FPA generation (IC50 = 5.16 microg/mL). Pentasaccharide, DX-9065a and UFH showed a marked correlation between ACT and inhibition of FPA generation. LMWHs demonstrated concentration-dependent inhibition of FPA generation. LMWHs studied showed good correlation between FPA generation inhibition and ACT test. Similar correlation was seen between FPA generation inhibition and the APTT, anti Xa (heptest-HI assay) and anti-IIa activity. Anti-IIa drugs demonstrated concentration-dependent inhibition of FPA generation. Their FPA generation inhibition potency is correlated with the ACT assay. A strong correlation between Hemochron ACT and FPA generation inhibition was observed. Based on this significant correlation, the FPA generation inhibition can be predicted by point-of-care ACT assay.

Antithrombin and anti-Xa agents in the control of thrombogenesis.

Recent developments as well as future trends in the area of thrombin inhibitors and anti-Xa agents are reviewed.

Day-night variations in blood levels of nitric oxide, T-TFPI, and E-selectin.

Circadian (8/24 hours) variations in serum nitric oxide (NO), total tissue factor pathway inhibitor (T-TFPI). and E-selectin levels were studied in healthy adults and in subjects with type II diabetes. We postulated a possibility a functional relationship between them because vascular endothelium is the primary site of their synthesis and functions. NO is released by the action of eNO synthase isoform and modulates physiologic responses (e.g., vascular dilation, relaxation, increasing blood flow, inhibition of platelet and white blood cell adhesion); T-TFPI, a coagulation inhibitor, is also released from endothelial cells, and is bound to plasma lipoproteins and to glycosaminoglycans; E-selectin is expressed on endothelial cells after activation by inflammatory cytokines (interleukin-1beta and tumor necrosis factor-alpha) and elevated levels have been reported in a variety of pathologic conditions, including diabetes. We found that obese diabetic subjects had greater mean concentrations of NO and E-selectin than healthy men, 39.25 versus 12.71 microM and 81.51 versus 26.03 ng/mL, respectively. The T-TFPI levels were essentially similar in both groups of men, 47.10 versus 48.76 ng/mL. We observed that the time of peak concentrations of T-TFPI and E-selectin was similar to the timing of NO trough levels, suggesting a possible functional relationship. It may be hypothesized, therefore, that the higher concentrations of NO, unbalanced by increases in T-TFPI and E-selectin, may result in increased vascular wall uptake of lipoproteins in diabetic subjects, who are at greater risk than healthy men for developing diffuse atherosclerosis.

Tissue factor pathway inhibitor release induced by defibrotide and heparins.

We evaluated the release of tissue factor pathway inhibitor (TFPI) induced by defibrotide (DF), a single-stranded, negatively charged polydeoxyribonucleotide extracted from mammalian organ. Ten normal volunteers were injected with an intravenous bolus of 400 mg DF and 2,000 IU unfractionated heparin (UFH). In addition, three volunteers were also injected with an intravenous bolus of 2,000 anti-Xa U of two low-molecular-weight heparins (LMWHs), enoxaparin and nadroparin. UFH caused a 4-fold increase in plasma TFPI at 5 minutes, with a decrease that was parallel to the heparin level measured by the anti-Xa assay. However, at 80 minutes, although the plasma anti-Xa activity of UFH was almost undetectable, the level of TFPI remained 2-fold baseline. DF induced an increase of TFPI that was 2-fold higher than the baseline level, with a steady state achieved between 5 and 20 minutes. At 40 minutes, the TFPI levels returned to basal level. This pattern was not coincident with the clearance of DF and at 40 minutes, the concentration of DF was still one third of the levels at 5 minutes (25.4 +/- 4.04 microg/mL). Both of the LMWHs induced a similar TFPI peak level at 5 minutes (1.5-fold increase) and at 40 minutes the TFPI levels returned to the initial levels. At 5 minutes, both LMWHs showed a higher plasma anti-Xa activity than UFH, which was detectable even at 80 minutes. The current study demonstrated that one of the mechanisms of the antithrombotic activity of DF is mediated via TFPI. Furthermore, the release of TFPI by heparin is mediated by non-antithrombin III binding fragments. Thus, polyanionic electrolytes are capable of releasing TFPI irrespective of their antithrombin III effect.

Endogenous release of tissue factor pathway inhibitor by topical application of an ointment containing mucopolysaccharide polysulfate to nonhuman primates.

Several studies have shown that tissue factor pathway inhibitor (TFPI) is released after the intravenous and subcutaneous administration of heparin and heparin-related drugs. Mucopolysaccharide polysulfate (MPS) is a preparation of glycosaminoglycans (GAGS) derived from mammalian cartilage, which has several structural and functional properties similar to heparin. Previous reports have shown that MPS is capable of releasing TFPI after intravenous administration. Therefore, this investigation was performed to determine the ability of topically administered MPS to release TFPI in a nonhuman primate model. A group of four monkeys were administered 3% MPS ointment in a dosage of 0.5 g/kg corresponding to 15 mg MPS/kg; another four monkeys were administered placebo ointment at a dosage of 0.5 g/kg once a day for 5 days in a period of 10 days. No effect of MPS was observed on the coagulation assays activated partial thromboplastin time (APTT), thrombin time (TT) and Heptest or on the platelet count. However, both the total and free TFPI levels were significantly and progressively elevated over the 10-day period in comparison to the placebo control group (P<.05). It is proposed that the ability of the topically administered MPS to increase the free and total TFPI levels may be one of the modes of action that contributes to the anticoagulant and anti-inflammatory actions of this agent.

Anticoagulant and antiprotease profiles of a novel natural heparinomimetic mannopentaose phosphate sulfate (PI-88).

Heparinomimetic mannopentaose phosphate sulfate (PI-88) (Progen Industries Ltd. Brisbane, Australia), currently developed as an anticoagulant and antiproliferative agent, mainly is composed of a pentomannan. However, tetrasaccharide and disaccharide components are also present. The molecular profile and the anticoagulant potency of PI-88 are investigated in this study. Gel permeation chromatography and polyacrylamide gel electrophoresis analyses were carried out to determine the molecular profile and separation of components of PI-88, respectively. Potentiation of antithrombin III (ATIII) and heparin cofactor-II (HC-II) activity were measured using chromogenic substrate assay. In order to determine anticoagulant and antiprotease effects of PI-88, various global anticoagulant tests, such as the prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), Hep-test (Haemachem Inc., St. Louis), ecarin clotting time (ECT), activated clotting time (ACT), and thromboelastography (TEG) were used. Anti-Xa and anti-IIa activities also were measured. The effect of PI-88 on the release of tissue factor pathway inhibitor (TFPI) was performed in nonhuman primates who received PI-88 and in endothelial cell culture systems. The relative susceptibility of PI-88 to heparinase I, protamine sulfate (PS), and platelet factor 4 (PF4) also was evaluated. The high-performance liquid chromatography profiles of PI-88 showed that its average molecular weight is approximately 2300 Da. Separation and gradient electrophoretic patterns of PI-88 showed that it is composed of five different fractions. This agent activates HC-II through inhibiting the thrombin generation but not inhibiting ATIII. Although PI-88 produced a concentration-dependent prolongation of all of the clotting tests, ECT gave the best correlation in the dose-response curve (ECT, r2 = 0.94; TT, r2 = 0.84; APTT, r2 = 0.69). Heparinomimetic mannopentaose phosphate sulfate (PI-88) exhibited marked inhibition of FIIa, but not of FXa. Heparinase I failed to produce significant neutralization of PI-88 in all the assays used, whereas PS and PF4 partially neutralized the effects of this compound. Heparinomimetic mannopentaose phosphate sulfate (PI-88) produced fivefold increase in the TFPI levels at 15 minutes after intravenous (IV) injection to primates. The incubation of PI-88 in endothelial cell culture system also showed a strong effect on TFPI release. These results suggest that PI-88 exhibited strong antithrombotic and anticoagulant activity in addition to its known antiproliferative properties. Because of the molecular characteristics and the dual nature of the pharmacologic action of PI-88, this agent represents an attractive pharmacologic agent for the control of thrombotic and proliferative disorders.

The effect of a novel glycoprotein IIb/IIIa antagonist, SR 121566A, on platelet aggregation and activation in rhesus monkeys.

SR 121566A represents a peptidomimetic glycoprotein IIb/IIIa (GP IIb/IIIa) inhibitor 3-[N- 4-[4-(aminoiminomethyl)phenyl]-1,3-thiazol-2-yl ) -N-(1-carboxymethylpiperid-4-yl) amino] propionic acid, trihydrochloride. To investigate the intravenous and subcutaneous pharmacodynamics of this agent, a primate model ( Macaca mulatta) was used. The IC50 for adenosine diphosphate (ADP) (10 micromol/L)-induced platelet aggregation in this primate platelet system was found to be 45 +/- 6 nmol/L. Comparatively in the human platelet rich plasma system, SR 121566A demonstrated an IC50 of 39 +/- 4 nmol/L. Graded doses of SR 121566A in the range of 25-400 microg/kg were administered intravenously. Blood samples were drawn from individual groups of primates (n = 4-6) at varying periods of time up to 24 hours after administration of SR 121566A. The pharmacodynamic effects were measured by platelet aggregation using ADP (10 micromol/L) as an agonist. In addition, flow cytometric methods were used to measure thrombin receptor-activating peptide (TRAP) (6.25 micromol/L)-induced platelet activation. In the subcutaneous studies, 50, 100, 250, and 400 microg/kg of SR 121566A was administered with an identical blood-drawing schedule and analysis as with the intravenous studies. In the intravenous studies, all doses of SR 121566A produced > 80% inhibition of platelet aggregation 5 minutes after the administration of the drug. The duration of the inhibitory effect is proportional to the dose administered and the 50% recovery time ranged from 2 to 15 hours. By flow cytometry, TRAP-induced P-selectin expression was also blocked for a varying duration of time in a dose-dependent fashion. The subcutaneous studies showed > 90% inhibition of platelet aggregation, which was observed at 15 minutes after administration of both 50 and 100 microg/kg of the drug. The recovery time after the subcutaneously administered doses was found to be shorter than the intravenously administered doses. These studies demonstrate that SR 121566A is an effective platelet inhibitor with predictable pharmacokinetic and pharmacodynamic characteristics.

Ecarin clotting time is sensitive to heparinoids: comparison of two different techniques.

Ecarin clotting time (ECT) is currently developed for the specific monitoring of antithrombin drugs, such as hirudin, argatroban, and hirulog. Aqueous reagent and dry chemistry technology have become available for ECT monitoring of antithrombin agents. Currently, many heparinoids and heparinomimetic drugs are being developed. These agents activate heparin cofactor II (HCII) and primarily mediate their effects by inhibiting thrombin. Although the test is specific for antithrombin agents, heparin cofactor II-mediated thrombin inhibitors are capable of prolonging the ECT. In order to study the relative effects of some of these agents, ECT was measured in human plasma supplemented with Pl-88 (a sulfated pentomanose; Progen Industries Limited, Sydney, Australia), aprosulate, pentosan polysulfate, dermatan sulfate, unfractionated heparin (UFH), and recombinant hirudin (r-hirudin). All agents were supplemented to the citrated-pooled plasma prepared from 10 healthy volunteers at a graded dosage of 0 to 100 microg/ml. These techniques gave comparable results for all of the agents used (PI-88, r2 = 0.99; r-hirudin, r2 = 0.98; UFH, r2 = 0.98; dermatan sulfate, r2 = 0.95; aprosulate, r2 = 0.95; pentosan polysulfate, r2 = 0.94). The relative anticoagulant effects of various agents used on ECT varied widely, exhibiting their potency in the following order: r-hirudin = pentosan polysulfate > dermatan sulfate > PI-88 > aprosulate > UFH. The sensitivity of ECT was adjusted by varying the concentration of the ecarin reagent. The results suggest that HCII-mediated inhibition of thrombin can be detected by using ECT reagents.

Anticoagulant and antiprotease effects of a novel heparinlike compound from shrimp (Penaeus brasiliensis) and its neutralization by heparinase I.

Heparin is usually obtained from mammalian organs, such as beef lung, beef mucosa, porcine mucosa, and sheep intestinal mucosa. Because of the increased use of heparin in the production of low-molecular-weight heparin (LMWH), there is a growing shortage of the raw material needed to produce LMWHs. A previous report described the structural features of a novel LMWH from the shrimp (Penaeus brasiliensis). In order to compare anticoagulant and antiprotease effects of this heparin, global anticoagulant tests, such as the prothrombin time, activated partial thromboplastin time, thrombin time, and Heptest, were used. Amidolytic anti-Xa and anti-IIa activities were also measured. The relative susceptibility of this heparin to flavobacterial heparinase was also evaluated. The United States Pharmacopeia (USP) potency of shrimp heparin (SH) was found to be 28 U/mg. SH produced a concentration-dependent prolongation of all of the clotting tests and exhibited marked inhibition of FXa and FIIa. Heparinase treatment resulted in a marked decrease of the anticoagulant effects and neutralized the in vitro anti-IIa actions. However, the anti-Xa activities were only partially neutralized. Protamine sulfate was only partially effective in neutralizing the anticoagulant and antithrombin effects of SH. SH also produced marked prolongation of activated clotting time, which was neutralized by heparinase but not by protamine sulfate. These results suggest that SH is a strong anticoagulant with comparable properties to mammalian heparins and can be used in the development of clinically useful antithrombotic-anticoagulant drugs.

Plasma tissue factor pathway inhibitor levels as a marker for postoperative bleeding after enoxaparin use in deep vein thrombosis prophylaxis in orthopedics and general surgery.

Low-molecular-weight heparins (LMWH) are widely used as antithrombotic prophylactic pharmaceutical agents in orthopedic and general surgery. Their antithrombotic characteristics are expressed by plasma mediators such as anti-Xa. anti-IIa, and increased release of tissue factor pathway inhibitor (TFPI) from vascular endothelium. The purpose of this clinical research is to study the relation between plasma levels of these mediators and postoperative bleeding. Forty-one consecutive patients undergoing hip or knee arthroplasty (n = 36) and colectomy (n = 5) received the standard enoxaparin (a LMWH) dose preoperatively (general surgery) or immediately postoperatively (orthopedic surgery). Major bleeding was defined as a postoperative drop of > or = 5 g/dL) of hemoglobin. The authors observed that there was a linear relationship between an increase in free/total TFPI ratio levels and postoperative bleeding. When that ratio increased by >60%, the hemoglobin dropped to >5 g/dL (n = 13). This relationship between free/total TFPI ratio increase and postoperative bleeding was statistically significant (P < 0.001). Those who did not bleed (hemoglobin drop was less than 5 g/dL) (n = 28) had a ratio increase (if any) of less than 50%. However, the authors did not observe any statistical relationship between anti-Xa, anti-IIa, or prothrombin time and postoperative bleeding in patients receiving LMWH for deep vein thrombosis prophylaxis in orthopedic and general surgery patients. The authors recommend a pre- and postoperative ratio level measurement whenever major bleeding is anticipated, as adjustments of LMWH dose or frequency might be necessary.

Emerging anticoagulant and thrombolytic drugs.

Since its discovery, heparin has been used intensely as an anticoagulant for several medical and surgical indications. However, efforts are in progress to replace heparin because of its serious complications, such as intraoperative and postoperative bleeding, osteoporosis, alopecia, heparin resistance, heparin rebound, heparin-induced thrombocytopenia (HIT) and thrombosis syndrome (HITTS), and other disadvantages. Significant developments in the field of new anticoagulants have resulted in the evaluation and introduction of low molecular weight heparins (LMWHs) and heparinoids, hirudin, ancrod, synthetic peptides and peptidomimetics. However, despite significant progress in the development of these new anticoagulants, a better or an ideal anticoagulant for cardiovascular patients is not yet available and heparin still continues to amaze both basic scientists and the clinicians. To minimise the adverse effects of heparin, newer approaches to optimise its use in combination with the new anticoagulants may provide better clinical outcome. In our experience, the off-label use of argatroban at a dose of 300 microg/kg iv. bolus followed by 10 microg/kg/minute infusion in combination with aggrastat (a glycoprotein [GP] IIb/IIIa inhibitor) at a dose of 10 microg/kg iv. bolus followed by an infusion of 0.15 microg/kg/minute in patients with HIT undergoing percutaneous coronary interventions resulted in elevation of celite activated clotting time (ACT) to 300 seconds followed by a gradual decline and the ACT remained above 200 seconds even after 200 min of drug administration. A bewildering array of newer anticoagulants now exist, such as LMWHs and heparinoids, indirect or direct thrombin inhibitors, oral thrombin inhibitors, such as melagatran (AstraZeneca) and HC-977 (Mitsubishi Pharmaceuticals), Factor IXa inhibitors, indirect or direct Factor Xa inhibitors, Factor VIIa/tissue factor (TF) pathway inhibitor, newer antiplatelet agents, such as GPIIb/IIIa inhibitors, fibrin specific thrombolytic agent, such as tenecteplase and modulation of the endogenous fibrinolytic activity by thrombin activatable fibrinolytic inhibitor (TAFI), Factor XIIIa inhibitors and PAI-1 inhibitors. The quest for newer anticoagulant, antiplatelet and fibrinolytic agents will continue until ideal agents are found.

Tissue factor pathway inhibitor: an update of potential implications in the treatment of cardiovascular disorders.

Tissue factor (TF) plays a crucial role in the pathogenesis of thrombotic, vascular and inflammatory disorders. Thus, the inhibition of this membrane protein provides a unique therapeutic approach for prophylaxis and/or treatment of various diseases. Tissue factor pathway inhibitor (TFPI), the only endogenous inhibitor of the TF/Factor VIIa (FVIIa) complex, has recently been characterised biochemically and pharmacologically. Studies in patients demonstrated that both TF and TFPI may be indicators for the course and the outcome of cardiovascular and other diseases. Based on experimental and clinical data, TFPI might become an important drug for several clinical indications. TFPI is expected to inhibit the development of post-injury intimal hyperplasia and thrombotic occlusion in atherosclerotic vessels as well as to be effective in acute coronary syndromes, such as unstable angina and myocardial infarction. Of special interest is the inhibition of TF-mediated processes in sepsis and disseminated intravascular coagulation (DIC), which are associated with the activation of various inflammatory pathways as well as of the coagulation system. A Phase II trial of the efficacy of TFPI in patients with severe sepsis showed a mortality reduction in TFPI- compared to placebo-treated patients and an improvement of organ dysfunctions. TFPI can be administered exogenously in high doses to suppress TF-mediated effects, alternatively high amounts of TFPI can be released from intravascular stores by other drugs, such as heparin and low molecular weight heparins (LMWH). Using this method high concentrations of the inhibitor are provided at sites of tissue damage and ongoing thrombosis. At present, clinical studies with TFPI are rather limited so that the clinical potential of the drug cannot be assessed properly. However, TFPI and its variants are expected to undergo further development and to find indications in various clinical states.

Differential effects of unfractionated heparin and low-molecular-weight heparins on tissue thromboplastin inhibition test.

Circulating anticoagulants are endogenously produced substances that interfere with in vitro tests of coagulation like activated partial thromboplastin time (APTT), and cause prolongation of the clotting times. Evaluation of the abnormal APTT involving various factor assays and mixing studies may provide inconclusive and ambiguous results. Tissue thromboplastin inhibition test (TTIT) is one of the screening assays for detection of circulating anticoagulants. However, this test is influenced by the presence of unfractionated heparin (UFH) from concentrations 0.2 U/mL and higher. Since low-molecular-weight heparins (LMWHs) are increasingly used for the prevention of thrombotic disorders and may replace UFH in the future, in this study the authors studied the influence of LMWHs on the performance of TTIT and compared the results with UFH. UFH and LMWHs showed a prolongation of TTIT in the concentration range of 0.25-1.0 U/mL. The marked prolongation of the TTIT with UFH and different LMWHs is in decreasing order of UFH > ardeparin > tinzaparin > dalteparin > enoxaparin. Patients with circulating anticoagulants who are given LMWHs may have false-positive results of TTIT and this influence should be kept in mind during patient management.

Plasma tissue factor pathway inhibitor levels as a marker for postoperative bleeding after enoxaparin use in deep vein thrombosis prophylaxis in orthopedics and general surgery.

Low-molecular-weight heparins (LMWH) are widely used as antithrombotic prophylactic pharmaceutical agents in orthopedic and general surgery. Their antithrombotic characteristics are expressed by plasma mediators such as anti-Xa, anti-IIa, and increased release of tissue factor pathway inhibitor (TFPI) from vascular endothelium. The purpose of this clinical research is to study the relation between plasma levels of these mediators and postoperative bleeding. Forty-one consecutive patients undergoing hip or knee arthroplasty (n = 36) and colectomy (n = 5) received the standard enoxaparin (a LMWH) dose preoperatively (general surgery) or immediately postoperatively (orthopedic surgery). Major bleeding was defined as a postoperative drop of > or = 5 g/dL) of hemoglobin. The authors observed that there was a linear relationship between an increase in free/total TFPI ratio levels and postoperative bleeding. When that ratio increased by > 60%, the hemoglobin dropped to > 5 g/dL (n = 17). This relationship between free/total TFPI ratio increase and postoperative bleeding was statistically significant (P < 0.001). Those who did not bleed (hemoglobin drop was less than 5 g/dL) (n = 24) had a ratio increase (if any) of less than 50%. However, the authors did not observe any statistical relationship between anti-Xa, anti-IIa, or prothrombin time and postoperative bleeding in patients receiving LMWH for deep vein thrombosis prophylaxis in orthopedic and general surgery patients. The authors recommend a pre- and postoperative ratio level measurement whenever major bleeding is anticipated, as adjustments of LMWH dose or frequency might be necessary.

An update on heparins at the beginning of the new millennium.

Unfractionated heparin has enjoyed the sole anticoagulant status for almost half a century. Besides an effective anticoagulant, this drug has been used in several additional indications. Despite the development of newer anticoagulant drugs, unfractionated heparin has remained the drug of choice for surgical anticoagulation and interventional cardiology. In the area of hematology and transfusion medicine, unfractionated heparin has continued to play a major role as an anticoagulant drug. The development of low-molecular-weight heparins (LMWHs) represents a refinement for the use of heparin. These drugs represent a class of depolymerized heparin derivatives with a distinct pharmacologic profile that is largely determined by their composition. These drugs produce their major effects by combining with antithrombin and exerting antithrombin and anti-Xa inhibition. In addition, the LMWHs also increase non-antithrombin-dependent effects such as TFPI release, modulation of adhesion molecules, and release of profibrinolytic and antithrombotic mediators from the blood vessels. The cumulative effects of each of the different LMWHs differ and each product exhibits a distinct profile. Initially these agents were developed for the prophylaxis of postsurgical deep-vein thrombosis. However, at this time these drugs are used not only for prophylaxis, but also for the treatment of thrombotic disorders of both the venous and arterial type. To a large extent, the LMWHs have replaced unfractionated heparin in most subcutaneous indications. With the use of these refined heparins, outpatient anticoagulant management has gone through a dramatic evolution. For the first time, patients with thrombotic disorders can be treated in an outpatient setting. Thus, the introduction of LMWHs represents a major advance in improving the use of heparin. The development of the oral formulation of heparin and LMWHs also provides an important area that may impact on the use of heparin and LMWHs. The increased awareness of heparin-induced thrombocytopenia has necessitated the development of newer methods to identify patients at risk of developing this catastrophic syndrome. Furthermore, a strong interest has developed in alternate drugs or the management of patients with this syndrome. Despite the development of alternate anticoagulants that are mostly antithrombin derived (hirudins, hirulog), these agents have failed to provide similar clinical outcome as heparin in many indications. However, antithrombin drugs are useful in the anticoagulant management of heparin-compromised patients. The FDA has approved a recombinant hirudin (Refludan) and a synthetic antithrombin agent, argatroban (Novastan), for this indication. The development of synthetic heparin pentasaccharide and anti-Xa agents may have an impact on the prophylaxis of thrombotic disorders. However, these monotherapeutic agents do not mimic the polytherapeutic actions of heparin. Furthermore, these agents do not inhibit thrombin. Heparin and LMWHs are capable of inhibiting not only factor Xa and thrombin, but other serine proteases in the coagulation network. The only way the newer drugs can mimic the actions of heparin is in combination modalities (polytherapeutic approaches). It has been suggested that newer antiplatelet drugs also exhibit anticoagulant actions. While these drugs may exhibit weak effects on thrombin generation, none of the currently available antiplatelet drugs exhibit any degree of antithrombin actions. It is likely that heparins synergize or augment the effects of the new antiplatelet drugs. Currently, combination approaches are used to anticoagulate patients in these studies. The dosage of heparins has been arbitrarily reduced. This may not be an optimal procedure. Additional clinical studies are needed to study these combinations where the alterations of these drugs are compared. Such combinations will require newer monitoring approaches. The development of oral thrombin agents, GP IIb