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.

Antithrombin agents as anticoagulants and antithrombotics: implications in drug development.

The development of direct thrombin inhibitors goes back nearly four decades. Organic synthetic benzamidine derivatives were initially developed as direct antithrombin agents. Later, the structural analysis of fibrinogen, leading to the identification of thrombin cleavage sites, resulted in the recognition of specific peptide sequences where thrombin cleaved fibrinogen. These observations led to the development of synthetic peptide derivatives as inhibitors of thrombin. The leech salivary extract contained natural hirudin, the structural elucidation of which led to the development of a recombinant equivalent protein (r-hirudin). Understanding the biochemical actions of thrombin and the structure of various inhibitors prompted the development of hirulogs, a class of hybrid molecules with two sites of action. Currently, several of these thrombin inhibitors are being developed for various indications in both intravenous and subcutaneous protocols. The increased interest in thrombin inhibitors is also prompted by reports of heparin-induced thrombocytopenia (HIT) with heparin and the need to anticoagulate patients with alternate drugs. These agents produce a direct anticoagulant response by targeting thrombin. In addition, the amplification of the coagulation cascade by thrombin activation of factors V and VIII, stabilization of fibrin by activated factor XHI, and platelet activation is also inhibited by these thrombin inhibitors. Some of the synthetic thrombin inhibitors are also capable of inhibiting other enzymes in the coagulation cascade. Thrombin inhibitors therefore exert a complex effect on the coagulation network and should be carefully evaluated in clinical trials. These drugs can be used for prophylactic and therapeutic and surgical indications. However, the different thrombin inhibitors have shown distinct pharmacologic differences. There is now an interest in developing oral antithrombin inhibitors. Such issues as antagonism, laboratory monitoring, drug interactions, and long-term safety remain unresolved. Current research is focused on addressing these issues.

Usefulness of intravenous enoxaparin for percutaneous coronary intervention in stable angina pectoris.

This pilot study was designed to determine whether the low molecular weight heparin, enoxaparin, could be used for elective percutaneous coronary intervention (PCI) to provide antithrombotic effects without the full systemic anticoagulation that occurs with the use of unfractionated heparin. Sixty patients were randomized to receive intravenous enoxaparin (1 mg/kg bolus dose) or unfractionated heparin at the time of coronary intervention. Laboratory testing was performed at baseline, 5 minutes, and 4 hours after study drug to test if a single bolus dose of intravenous enoxaparin can consistently achieve therapeutic antithrombotic effect, thus eliminating the need for multiple doses of heparin and closely monitoring levels of anticoagulation during PCI. Thirty percent of patients who received unfractionated heparin required a second bolus of intravenous heparin to achieve the target-activated clotting time of 300 seconds before PCI. Enoxaparin showed antithrombotic properties comparable to that of unfractionated heparin as measured by anti-Xa levels, with less inhibition of thrombin (factor IIa) at the time points measured (p <0.0001). Angioplasty success rates, in-hospital ischemia, bleeding, and vascular complications were similar in both groups. Thus, intravenous enoxaparin has predictable and effective antithrombotic effects during elective PCI. Although the level of anticoagulation attained with enoxaparin is significantly lower than that after unfractionated heparin, no increase in ischemic complications were noted. The use of a single bolus of intravenous enoxaparin, without the need for measuring the activated clotting time or titrating heparin anticoagulation, has the potential for simplifying the performance and perhaps enhancing the safety of PCI.

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.

Global and molecular hemostatic markers in acute myeloid leukemia.

Patients with acute myeloid leukemia have multiple hemostatic and thrombotic complications, which may or may not result from disseminated intravascular coagulation. Previous studies incorporating routine coagulation analyses failed to detect any clinically useful information in most of these patients. In this study, the first comprehensive evaluation of the various aspects of the hemostatic system in a population of patients with acute myeloid leukemia was performed. Eighteen patients (23-71 years of age) were studied at either diagnosis or relapse. Hemostatic studies were performed at onset and on days 3, 7, and 30 after initiation of therapy. The bone marrow blast counts ranged from 8% to 98%; prothrombin time and activated partial thromboplastin time showed only minor prolongations in a few of these patients. However, in all patients measurement of platelet-associated markers revealed elevated platelet factor 4 and thromboxane B2 and normal 6-keto-prostaglandin F1 alpha levels. Fibrinolytic markers showed an increase in D-dimer and tissue plasminogen activator and a decrease in alpha 2-antiplasmin levels. Plasminogen, plasminogen activator inhibitor, and fibrinogen levels were normal. Coagulation markers demonstrated a decrease in protein C and antithrombin III levels and an elevation of the thrombin-antithrombin complex. The pretreatment values for all hemostatic markers studied were similar to the values obtained on days 3, 7, and 30 during treatment. This investigation demonstrated a subclinical activation of the components of the hemostatic system possibly leading to a hypercoagulable state. Although only six patients (33%) experienced hemorrhagic complications, the risk of bleeding and/or thrombosis was strongly evident in all patients. The significance of finding abnormal levels of specific molecular markers of hemostasis will be established in the future application of such markers in clinical evaluations of leukemic patients known to be at risk for coagulation disorders.

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.

Defibrotide mediated inhibition of serotonin, endothelin-I, thromboxane, and serum induced contraction of canine femoral and pulmonary arterial rings.

Defibrotide is a polydeoxyribonucleotide derived agent with a weight average of 15 to 18 kDa. By virtue of its chemical nature, this polyelectrolyte agent exerts multiple pharmacologic actions at various plasmatic and cellular sites. In recent studies, this agent has been demonstrated to exhibit cardioprotective and vasomodulatory actions. To test the effect of defibrotide on the vascular smooth muscle contractile responses, its effects were evaluated on contractile response induced by agonists in canine femoral and pulmonary arterial smooth muscle preparation. These arterial preparations were harvested from anesthetized and anticoagulated (Heparinized 3-5 U/ml) dogs. Defibrotide was administered to dogs at 10 mg/kg, i.v. and segments of canine arteries were harvested at 30 minutes after the administration of this agent. The control and defibrotide treated canine arterial ring preparations were tested against serotonin, endothelin-I, serum and control platelet rich plasma(PRP) with arachidonic acid(AA). The contractile response of arterial rings obtained for treated groups were measured using serotonin, endothelin-I, serum and PRP/AA as agonists. The contractile action of serotonin, endothelin-I, serum and PRP/AA were inhibited by pretreatment of the animal with defibrotide. The arterial ring isolated from dogs treated with defibrotide exhibited a weaker contraction. These studies support the hypothesis that defibrotide modulates endothelial function and the response to the contractile actions of various agonists may be related to this 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.

TFPI antigen levels in normal human volunteers after intravenous and subcutaneous administration of unfractionated heparin and a low molecular weight heparin.

Tissue factor pathway inhibitor (TFPI) is an important mediator of the in vivo anticoagulant/antithrombotic properties of unfractionated heparin (UFH) and low molecular weight heparin (LMWH). The vascular pool of TFPI is released into the circulation after intravenous and subcutaneous administration of both UFH and LMWH. We have administered LMWH (Ardeparin) and UFH to normal human volunteers in a dose dependent manner. Our results demonstrate that the TFPI antigen levels increase upon the intravenous and subcutaneous administration of UFH and Ardeparin. Because of the better bioavailability of LMWH by the subcutaneous route at equigravimetric dosages, Ardeparin released more TFPI than UFH. However, when given intravenously an identical release of TFPI from the vasculature has been observed. The plasma concentration of TFPI was increased 0.5-2 fold when UFH or Ardeparin was administered subcutaneously and was 3 fold higher when administered intravenously. This profound increase in TFPI antigen levels was dependent on the dosage of Ardeparin administered. This release in TFPI correlates with prolongation of the Heptest clotting assay. However, it appears from this study that TFPI release precedes the elevation of the Heptest clotting time.

Inhibitory effects of TFPI on thrombin and factor Xa generation in vitro--modulatory action of glycosaminoglycans.

The effect of tissue factor pathway inhibitor (TFPI) on thrombin and factor Xa generation was studied in an in vitro system using a prothrombin complex concentrate. It was found that TFPI, via the direct inhibition of factor Xa and the tissue factor/factor VIIa complex, inhibited both the further generation of factor Xa and the generation of thrombin in a concentration-dependent manner. The generation of thrombin (IC50 255 ng/ml) was more pronounced than that of factor Xa (IC50 684 ng/ml). The inhibitory activity of TFPI was significantly enhanced when unfractionated heparin was present in the assay system at a concentration of 10 micrograms/ml which did not show any inhibitory effects on protease generation in the same system. Furthermore, the influence of TFPI at subthreshold concentrations (100 ng/ml and 200 ng/ml, resp.) on the inhibitory action of unfractionated heparin (UFH), a low molecular weight heparin (LMWH), heparan sulfate (HS) and the synthetic heparin pentasaccharide (PS) was investigated. Whereas in the concentration range used (0.3-40 micrograms/ml) these glycosaminoglycans did not inhibit thrombin and factor Xa generation, after supplementation of the system with TFPI a concentration-dependent inhibition of the generation of the proteases up to 40-50% was seen for UFH, LMWH and HS. TFPI did not increase the activity of PS.

Laboratory monitoring of pentasaccharide in a dog model of hemodialysis.

Varying dosages of pentasaccharide (400-800 nmol/kg) were compared to a 250-U/kg single bolus dosage of unfractionated heparin (UFH) in a dog model of hemodialysis. Several laboratory assays were used to monitor the effects of pentasaccharide and UFH. The pentasaccharide did not produce any anticoagulant effects as measured by the activated partial thromboplastin time. However, in the anti-Xa chromogenic assay and the Heptest assays, there was a dose-dependent prolongation after pentasaccharide administration. In the group of dogs administered 800 nmol/kg of pentasaccharide, there was a 50% decrease in the thrombin antithrombin (TAT) complex level after 60 minutes on dialysis. In the UFH-treated dogs, wide variations in assays were observed. There was a marked elevation in the activated partial thromboplastin time and Heptest assays up to 6 hours after UFH administration. Both anti-Xa and anti-IIa activity was measured up to 4 hours. In the TAT assay, UFH was found to have a stronger effect in suppressing the formation of TAT in comparison to the pentasaccharide. These results suggest that pentasaccharide can be used as a replacement for UFH in a dog model of hemodialysis to keep the dialysis circuit patent. In addition, the anti-Xa-based assays such as the Heptest and the chromogenic anti-Xa assays can be used to monitor the effects of pentasaccharide in this model.

Efficacy of pentasaccharide in a dog model of hemodialysis.

A synthetic heparin pentasaccharide with sole anti-Xa actions has been evaluated for its antithrombotic efficacy in a dog model of hemodialysis. Various dosages of pentasaccharide, 400-800 nmol/kg, were compared with a single bolus dose of unfractionated heparin (250 U/kg). The primary endpoint in these studies was the duration of dialysis time. In addition, dialyzer filter content, venous trap protein, celite and saline ACT and hematocrit measurement. Pentasaccharide at dosages of 600 and 800 nmol/kg produced an extension of dialysis time (> 180 minutes) in contrast to unfractionated heparin at 250 U/kg which only produced antithrombotic effects for periods of up to 150 +/- 42 minutes (n = 5). At a lower dosage of 400 nmol/kg pentasaccharide produced weaker effects and the dialysis circuit was patent for periods of 122 +/- 14.8 (n = 5) minutes. The saline and celite ACT times were not extended at any dosage of pentasaccharide; however, at 250 U/kg, a strong effect was noted with unfractionated heparin (> 800 secs, 647 +/- 211 secs.), respectively. A dose dependent antithrombotic effect was also evident in the studies on the filter clots and venous trap protein content. No difference in the hematocrit was noted in any group. These results clearly suggest that despite the fact that pentasaccharide does not produce any prolongation of the coagulation times, it produces a dose dependent antithrombotic effect in this model of dog hemodialysis. Furthermore, these results also suggest that pentasaccharide at an appropriate dosage can be used as an alternate antithrombotic agent during hemodialysis.

Antithrombin agents as anticoagulants and antithrombotics. Implications in drug development.

Synthetic and recombinant thrombin inhibitors have undergone several clinical evaluations for thrombotic and cardiovascular indications. While the initial trials were focused in coronary indications, more recently, these agents are also developed for the prophylaxis and therapeutic management of thromboembolic disorders. Hirudin, PEG-hirudin and argatroban are in advanced clinical development. Recombinant hirudin has been approved in Europe as a substitute anticoagulant for the management of HIT patients. Several additional clinical trials are currently carried out to demonstrate the usefulness of these agents in thrombotic and cardiovascular indications. Despite these developments such issues as dosage optimization, laboratory monitoring, neutralization and drug interactions require additional studies for the optimal development of these drugs.

Molecular markers of hemostatic activation. Implications in the diagnosis of thrombosis, vascular, and cardiovascular disorders.

Until recently, the diagnosis of thromboembolic disorders remained difficult to establish before the occurrence of a pathologic event. Clearly, thrombosis is the result of a progressive alteration of the blood and vasculature. Various molecular markers of hemostatic alteration are found in increased or decreased amounts predisposing to thrombosis or in increased circulating amounts during the activation process.

The role of tissue factor pathway inhibitor in the mediation of the antithrombotic actions of heparin and low-molecular-weight heparin.

It is widely accepted that antithrombin III (ATIII) mediated anti-Xa and anti-IIa effects are the sole determinant of the antithrombotic actions of unfractionated heparin (UFH) and low-molecular-weight heparins (LMWHs). However, there are several unexpected observations such as the greater than 100% bioavailability of subcutaneously administered LMWH as measured by a chromogenic based anti-Xa method. The authors have proposed that, besides ATIII mediated antiprotease actions, additional endogenous factors may be responsible for the observed therapeutic and prophylactic actions of heparins. With the identification of tissue factor pathway inhibitor (TFPI) some of the unexpected effects of heparins can now be clarified. To investigate the role of heparin-releasable TFPI on LMWHs the anti-Xa and TFPI antigen levels after prophylactic and therapeutic administration of UFH and LMWHs have been studied in defined clinical trials. Regardless of the dosage designation (mg/kg or units/kg) each LMWH followed a distinct TFPI release profile. Similarly, in the intravenous studies these LMWHs produced an instantaneous increase in the TFPI antigen level. The anti-Xa effects did not always follow the same pattern as the TFPI antigen levels. These data suggest that the anti-Xa potency of a given LMWH is not the sole determinant of the antithrombotic actions of heparin and LMWH. In addition to pharmacologic agents, the effect of sequential compression devices (SCD) on the release of TFPI was also studied. A two-fold increase in TFPI antigen levels was observed in normal volunteers undergoing long leg compression for 1 h.(ABSTRACT TRUNCATED AT 250 WORDS)

A supersulfated low-molecular-weight heparin (IK-SSH) increases plasma levels of free and total tissue factor pathway inhibitor after intravenous and subcutaneous administration in humans.

Unfractionated as well as low-molecular-weight heparins (LMWH) are known to cause an increase in blood levels of tissue factor pathway inhibitor (TFPI). To study the effect of a newly developed supersulfated LMWH (IK-SSH, Iketon Farmaceutici) on TFPI concentrations in human plasma, the compound was injected into volunteers at doses of 0.14, 0.33 and 0.66 mg/kg intravenously or 0.33, 0.66 and 1.0 mg/kg subcutaneously. At certain known times blood was drawn and plasma levels of both total and free TFPI were measured using enzyme-linked immunosorbent assay methodology. Baseline plasma concentrations of TFPI were 72.2+/-3.1 ng/ml for total and 10.8+/-0.8 ng/ml for free TFPI. Intravenous or subcutaneous injection of IK-SSH led to a strong and long-lasting rise in TFPI levels which were increased more than 5-fold for total TFPI and more than 30-fold for free TFPI. Maximum TFPI levels were reached 5-10 min after intravenous and 60 min after subcutaneous administration. IK-SSH caused prolongation of ex-vivo clotting times in the APTT and Heptest assay, whereas thrombin time was not affected. Anticoagulant actions of IK-SSH showed a significant correlation to plasma concentrations of TFPI and they are thought to be based at least partially on the release of TFPI from vascular sites.

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.

Soluble P-selectin in human plasma: effect of anticoagulant matrix and its levels in patients with cardiovascular disorders.

P-Selectin represents a cell surface glycoprotein that is constitutively present in the Weibel-Palade bodies of endothelial cells and in the alpha-granules of platelets. In inflammation and thrombogenic conditions, plasmatic P-selectin levels are markedly elevated, indicating the leakage of this marker from these sites. In this study, a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) utilizing a monoclonal soluble P-selectin (sP-selectin) antibody was employed to assess this marker in blood samples collected in various anticoagulants such as heparin, hirudin, sodium citrate (3.2% and 3.8%), and ethylenediaminetetraacetic acid (EDTA). The soluble P-selectin levels ranged from 26 ng/mL to 44 ng/mL. Sodium citrate (3.8%) was used to collect platelet-poor plasma (PPP) from patients with heparin-induced thrombocytopenia (HIT), coronary angioplasty (CA), or coronary atherectomy (CAT). In comparison with the control group (approximately 30 ng/mL), all of these patient groups showed a marked elevation of sP-selectin levels (HIT = 96 ng/mL [n = 18], CA = 46 ng/mL [n = 6] and CAT = 60 ng/mL [n = 10]). In platelet-rich plasma (PRP) preparations using various anticoagulants, the sP-selectin levels were markedly higher, ranging from 87 ng/ mL to 117 ng/mL (n = 10). In patients recruited into a clinical trial (the argatroban [ARG] 911 Study), in which argatroban was used as an alternate anticoagulant in patients with HIT, a 25% to 35% decrease in sP-selectin levels was observed after 72 hours of argatroban treatment. In addition, the relative ratio between levels in PRP and PPP in these patients differed, suggesting that the anticoagulant matrix influences the sP-selectin levels. These data clearly suggest that the anticoagulant matrix and blood collection procedures may significantly influence the plasmatic P-selectin levels. Furthermore, in different clinical conditions, elevation of this marker may reflect endogenous platelet activation; however, optimal anticoagulant for blood collection is important for proper diagnostic validation.

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.

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.