Molecular aspects of thrombosis and antithrombotic drugs

There have been major advances in our understanding of thrombosis and antithrombotic drugs. This review focuses on the molecular aspects of thrombus formation and antithrombotic therapy. Molecules involved in arterial thrombosis are derived from inflammatory cells in the atherosclerotic plaque and blood platelets. These molecules work in concert to promote plaque instability and thrombogenicity. Thrombus formation on the ruptured plaque is mediated by platelet and coagulation activation. By contrast, molecules involved in venous thrombosis are derived from the activated coagulation cascade. Platelets appear to play a secondary role. The antithrombotic drugs are classified according to their targeted constituents: antiplatelet agents and anticoagulants; the latter are further divided into non-specific anticoagulants, such as vitamin K antagonists and heparin, and direct thrombin inhibitors, including hirudin and argatroban. Currently available antiplatelet agents target glycoprotein IIbIIIa (abciximab, tirofiban, eptifibatide), cyclooxygenase-1 (aspirin) or adenosine diphosphate receptor, P2Y12 (clopidogrel).     

Pharmacological assessment of the antithrombotic activity of the peptide thrombin inhibitor, D-methyl-phenylalanyl-prolyl-arginal (GYKI-14766), in a canine model of coronary artery thrombosis.

The antithrombotic activity of the tripeptide thrombin inhibitor, D-methyl-phenylalanyl-prolyl-arginal (GYKI-14766), was compared to heparin in a model of canine coronary artery thrombosis. Thrombogenesis was initiated by electrolytic injury of the intimal surface of the left circumflex coronary artery. Drug administration was started 15 min before initiation of intimal injury. Clotting times and ex vivo platelet aggregation were determined on citrated blood samples. Gingival template bleeding times were determined. Clotting times (thrombin time; activated partial thromboplastin time, APTT; prothrombin time, PT) increased in a dose-dependent manner with both anticoagulants. The two anticoagulants selectively inhibited thrombin-induced platelet aggregation. GYKI-14766 and heparin were found to delay thrombosis significantly when compared to vehicle-treated animals; minimum effective antithrombotic doses were 0.25 mg/kg/h and 80 U/kg + 30 U/kg/h, respectively. GYKI-14766 (0.25 mg/kg/h) had no effect on template bleeding time, APTT or PT. Heparin (80 U/kg + 30 U/kg/h), however, was associated with a 2.5- to 3.0-min increase in template bleeding time, a 1.8-fold and 1.7-fold increase in APTT and PT, respectively. Antithrombotic efficacy was achieved at doses of GYKI-14766 that did not affect APTT, PT or template bleeding time, whereas antithrombotic efficacy observed with heparin was associated with significant increases in APTT, PT and template bleeding time. These data demonstrate that the tripeptide thrombin inhibitor, GYKI-14766, could potentially prove to be a safer and more effective antithrombotic agent than heparin.     

Factor XI and contact activation as targets for antithrombotic therapy

The most commonly used anticoagulants produce therapeutic antithrombotic effects either by inhibiting thrombin or factor Xa (FXa) or by lowering the plasma levels of the precursors of these key enzymes, prothrombin and FX. These drugs do not distinguish between thrombin generation contributing to thrombosis from thrombin generation required for hemostasis. Thus, anticoagulants increase bleeding risk, and many patients who would benefit from therapy go untreated because of comorbidities that place them at unacceptable risk for hemorrhage. Studies in animals demonstrate that components of the plasma contact activation system contribute to experimentally induced thrombosis, despite playing little or no role in hemostasis. Attention has focused on FXII, the zymogen of a protease (FXIIa) that initiates contact activation when blood is exposed to foreign surfaces, and FXI, the zymogen of the protease FXIa, which links contact activation to the thrombin generation mechanism. In the case of FXI, epidemiologic data indicate this protein contributes to stroke and venous thromboembolism, and perhaps myocardial infarction, in humans. A phase 2 trial showing that reduction of FXI may be more effective than low molecular weight heparin at preventing venous thrombosis during knee replacement surgery provides proof of concept for the premise that an antithrombotic effect can be uncoupled from an anticoagulant effect in humans by targeting components of contact activation. Here, we review data on the role of FXI and FXII in thrombosis and results of preclinical and human trials for therapies targeting these proteins.     

Endovascular stents: a ‘break through technology’, future challenges

Coronary stents were developed to overcome the two main limitations of balloon angioplasty, acute occlusion and long term restenosis. Coronary stents can tack back intimal flaps and seal the dissected vessel wall and thereby treat acute or threatened vessel closure after unsuccessful balloon angioplasty. Following successful balloon angioplasty stents can prevent late vessel remodeling (chronic vessel recoil) by mechanically enforcing the vessel wall and resetting the vessel size resulting in a low incidence of restenosis. All currently available stents are composed of metal and the long-term effects of their implantation in the coronary arteries are still not clear. Because of the metallic surface they are also thrombogenic, therefore rigorous antiplatelet or anticoagulant therapy is theoretically required. Furthermore, they have an imperfect compromise between scaffolding properties and flexibility, resulting in an unfavourable interaction between stents and unstable or thrombus laded plaque. Finally, they still induce substantial intimal hyperplasia which may result in restenosis. Future stent can be made less thrombogenic by modifying the metallic surface, or coating it with an antithrombotic agent or a membrane eluting an antithrombotic drug. The unfavourable interaction with the unstable plaque and the thrombus burden can be overcome by covering the stent with a biological conduit such as a vein, or a biodegradable material which can be endogenous such as fibrin or exogenous such as a polymer. Finally the problem of persisting induction of intimal hyperplasia may be overcome with the use of either a radioactive stent or a stent eluting an antiproliferative drug.     

A laboratory method for parallel determination of platelet aggregation and thrombin generation

Optimal use of combinations of antiaggregating and antithrombotic drugs in vivo requires improved methods for testing of possible synergistic drug effects. Therefore, we developed an in vitro model that allows parallel evaluation of the influence of drugs on the time course of platelet aggregation inhibition and on thrombin generation inhibition. Platelet rich plasma samples were incubated with different amounts of antiaggregating and/or antithrombotic agents and the effects on thrombin generation, lag phase until the onset of platelet aggregation, and on inhibition of platelet aggregation were detected. Plasma activation was performed by addition of high (29 nM final concentration, "high coagulant challenge") or low (5 pM final concentration, "low coagulant challenge") amounts of tissue factor. Thus, platelet activation is initiated in our model by endogenously generated thrombin and not by exogenously added agonists, as usual in conventional evaluation of platelet aggregation. The combination of glycoprotein IIb/IIIa inhibitors (eptifibatide or abciximab) and anticoagulants (unfractionated heparin, low molecular weight heparin, or recombinant hirudin) used in this study exhibited an additive effect on prolongation of the lag phase until the onset of platelet aggregation. Under high but not under low coagulant challenge the combination of eptifibatide and anticoagulants had a synergistic inhibitory effect on platelet aggregation. Combination of abciximab and anticoagulants had no additive inhibitory effects on platelet aggregation under both high and low coagulant challenge. Under low coagulant challenge combination of eptifibatide but not abciximab with low molecular weight heparin significantly reduced the thrombin generation. We suggest that our laboratory method might be useful for pre-clinical testing of in vitro effects of glycoprotein IIb/IIIa inhibitors and anticoagulants.     

Nonpeptide factor Xa inhibitors III: effects of DPC423, an orally-active pyrazole antithrombotic agent,on arterial thrombosis in rabbits

DPC423 [1-[3-(aminomethyl)phenyl]-N-[3-fluoro-2'-(methylsulfonyl)[1,1'-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide] is a synthetic, competitive, and selective inhibitor of coagulation factor Xa (fXa) (K(i): 0.15 nM in humans, 0.3 nM in rabbit). The objective of this study was to compare effects of DPC423, enoxaparin (low-molecular-weight heparin), and argatroban (thrombin inhibitor) on arterial thrombosis and hemostasis in rabbit models of electrically induced carotid artery thrombosis and cuticle bleeding, respectively. Compounds were infused i.v. continuously from 60 min before artery injury or cuticle transection to the end of experiment. Carotid blood flow was used as a marker of antithrombotic effect. Antithrombotic ED(50) values were 0.4 mg/kg/h for enoxaparin (n = 6), 0.13 mg/kg/h for argatroban (n = 6), and 0.6 mg/kg/h for DPC423 (n = 12). DPC423 at the maximum antithrombotic dose increased activated partial thromboplastin time and prothrombin time (n = 6) by 1.8 +/- 0.07- and 1.8 +/- 0.13-fold, respectively, without changes in thrombin time and ex vivo thrombin activity. The antithrombotic effect of DPC423 was significantly correlated with its ex vivo anti-fXa activity (r = 0.86). DPC423 at 1, 3, and 10 mg/kg p.o. increased carotid blood flow (percent control) at 45 min to 10 +/- 4, 24 +/- 6, and 74 +/- 7, respectively (n = 6/group). Cuticle bleeding times (percent change over control) determined at the maximum antithrombotic dose were 88 +/- 12 for argatroban, 69 +/- 13 for heparin, 4 +/- 3 for enoxaparin, 5 +/- 4 for DPC423, and -3 +/- 2 for the vehicle (n = 5-6/group), suggesting dissociation of antithrombotic and bleeding time effects for DPC423 and enoxaparin. The combination of aspirin and DPC423 at ineffective antithrombotic doses produced significant antithrombotic effect. Therefore, these results suggest that DPC423 is a clinically useful oral anticoagulant for the prevention of arterial thrombosis.     

Anticoagulation with rivaroxaban: covering a broad spectrum of thromboembolic disease

Traditional anticoagulants such as heparin and vitamin K antagonists have been the mainstay of antithrombotic therapy for many years. However, these drugs have a number of well-recognized drawbacks: unfractionated heparin, low molecular weight heparins and fondaparinux are administered parenterally, and vitamin K antagonists require routine coagulation monitoring. Novel, single-target oral anticoagulants have been developed that do not need routine coagulation monitoring, including the direct factor Xa inhibitors rivaroxaban, apixaban and edoxaban, and the direct thrombin inhibitor dabigatran. Rivaroxaban is the most advanced in its clinical development. Across all ten Phase III/IV trials, rivaroxaban met the primary efficacy end points and generally had similar incidences of bleeding, with a comparable safety profile to standard-of-care.     

三联抗栓药物治疗老年不稳定型心绞痛临床疗效观察

目的观察三联抗栓药物治疗老年不稳定型心绞痛临床效果。方法选择该门诊老年不稳定型心绞痛(UAP)患者84例,在常规治疗基础上加用拜阿司匹林,氯吡格雷,低分子肝素三联抗栓药物治疗,并观察治疗前后心绞痛发作及硝酸甘油用量情况。结果患者心绞痛发作次数、心绞痛持续时间及硝酸甘油用量情况均较治疗前明显减少,治疗前后有显著性差异(P<0.01)。结论三联抗栓药物治疗UAP可进一步减少心绞痛发作,改善临床症状,且安全性良好。     

Haemostasis and antithrombotic drugs: pharmacology and novel therapeutic approaches

Considerable progress has been made during the last years with the development of new antiplatelet and antithrombotic agents. The introduction of the ADP-antagonist Clopidogrel some years ago was the first alternative to oral acetylsalicylic acid for long-term treatment. Prasugrel, another ADP-antagonist, is currently in advanced stages of clinical trials. Regarding antithrombotic drugs the interest has been focussed on small molecule direct inhibitors of thrombin and factor Xa. (Xi)Melagatran is one of the recent developments in direct thrombin inhibitors, Dabigatran may follow soon. Several factor Xa inhibitors are currently subject to clinical trials, including Rivaroxaban; others may follow. From a pharmacological point of view these compounds are likely to improve and enlarge the spectrum of available antiplatelet and antithrombotic drugs, respectively.     

Drugs for percutaneous coronary interventions

Many combinations of anticoagulants and antiplatelet drugs have been used to treat patients undergoing a percutaneous coronary intervention (PCI). Among anticoagulants, enoxaparin has some theoretical advantages over unfractionated heparin, but in clinical trials no advantage in outcome has been demonstrated in PCI. Bivalirudin, a direct thrombin inhibitor, has been associated with a lower risk of bleeding than unfractionated heparin. Among antiplatelet drugs, use of clopidogrel plus aspirin has led to a reduction in cardiovascular events; all patients undergoing PCI should receive both. Addition of a GP IIb/IIIa inhibitor may lower mortality rates further, particularly in high-risk patients such as diabetics.     

Basic fibroblast growth factor enhances the coupling of intimal hyperplasia and proliferation of vasa vasorum in injured rat arteries.

Basic fibroblast growth factor (bFGF) is mitogenic for smooth muscle cells (SMC) and angiogenic. We examined the in vivo effects of bFGF in balloon denuded carotid arteries of laboratory rats. bFGF was administered continuously from polymer-based devices at 34 ng/d into the periadventitial space of rat carotid arteries for 2 wk. Intimal hyperplasia was not observed in the absence of injury or with lipopolysaccharide induced endothelial dysfunction. Different degrees of vascular injury produced proportionally more intimal hyperplasia. bFGF increased the intimal hyperplastic response 1.3-fold with severe vascular injury, and 2.4-fold with more mild injury. Increased cell proliferation, not extracellular matrix production, accounted for these effects. Cell density was unchanged for the control and bFGF-treated groups, and the number of proliferating intimal cells at 2 wk rose to an amount equivalent to the increase in mass; 1.9- and 4.0-fold for severe and lesser injury, respectively. The relative ability of heparin to reduce SMC proliferation was not altered by the presence of bFGF.bFGF also induced profound angiogenesis within and surrounding the polymeric releasing device, and in the vasa vasorum immediately around the injured arteries. bFGF's effect on vasa was linearly related to the amount of SMC proliferation within the blood vessel. Thus, the in vivo mitogenic and angiogenic potential of bFGF are coupled, and may be similarly modulated by the products of local injury and/or factors in the vessel wall.     

Antithrombotics in atrial fibrillation and coronary disease

Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia and approximately 18–45% of AF patients have concomitant coronary artery disease (CAD). Several studies have demonstrated that oral anticoagulation is the mainstay of therapy for stroke prevention in AF. Similarly, antiplatelet therapy including aspirin and P2Y12 inhibitor is recommended in the management of acute coronary syndrome and stable CAD. Despite the high prevalence of CAD with AF, practice guidelines are scarce on the appropriate antithrombotic regimen due to lack of large-scale randomized clinical trials. The use of direct thrombin and factor Xa inhibitors for stroke prevention in AF has also complicated the possible combinations of antithrombotic therapies. This review aims to discuss the available evidence regarding aspirin as an antithrombotic strategy, the role of novel anticoagulants and the specific clinical situations where aspirin may be beneficial in patients with AF and CAD.     

Safety of pancreatic surgery with special reference to antithrombotic therapy: A systematic review of the literature

BACKGROUNDPostpancreatectomy hemorrhage (PPH) is the most severe type of complication after pancreatic surgery, although the effect of antithrombotic therapy (ATT) on PPH is largely unknown. The safety and efficacy of chemical thromboprophylaxis for venous thromboembolism (VTE) remains controversial. AIMTo elucidate the effect of ATT on PPH. METHODSPublished articles between 2013 and 2020 were searched from PubMed and Google Scholar, and after careful reviewing of all studies, studies concerning ATT and pancreatic surgery were included. Data such as study design, type of surgical procedures, type of antithrombotic drugs, and surgical outcome were extracted from the studies.RESULTSNineteen published articles with a total of 37863 patients who underwent pancreatic surgery were included in the systematic review. Fourteen were cohort studies, with only three being prospective in nature. Two studies demonstrated that in patients receiving chronic ATT, which were mostly managed by heparin bridging, the risk of PPH was higher compared with those without ATT, and one study showed that patients with direct-acting oral anticoagulants managed by heparin bridging had significantly higher postoperative bleeding rates than others. The remaining six studies reported that pancreatic surgery can be safely performed in patients receiving chronic ATT, even under preoperative aspirin continuation. Concerning chemical thromboprophylaxis for VTE, most studies have shown a potentially high risk of PPH in patients undergoing chemical thromboprophylaxis; however, its effectiveness against VTE has not been statistically demonstrated, particularly among Asian patients.CONCLUSIONPancreatic surgery in chronically ATT-received patients can be safely performed without an increase in the occurrence of PPH, although the safety and efficacy of chemical thromboprophylaxis for VTE during pancreatic surgery is still controversial. Further investigation using reliable studies with good design is required to establish definite protocols or guidelines.     

Oral antithrombotic use among myocardial infarction patients

OBJECTIVE: To examine the use of oral antithrombotics (i.e., antiplatelet agents, oral anticoagulants) after myocardial infarction (MI) in the Netherlands from 1988 to 1998. METHODS: Retrospective follow-up of 3800 patients with MI, using data from the PHARMO Record Linkage System. RESULTS: From 1988 to 1998, oral antithrombotic treatment increased significantly from 54.0% to 88.9%. In 1998, only 75.8% of patients who experienced a MI in the late 1980s received oral antithrombotic treatment compared with 94.4% of those who experienced a recent MI. CONCLUSIONS: Oral antithrombotics were considerably underused in patients with a past history of MI. Therefore, these patients should be reviewed for antithrombotic therapy to assess whether their failure to use oral antithrombotics was right or wrong, and whether treatment should be initiated if possible.     

Prior use of antithrombotic agents and neurological functional outcome at discharge in patients with ischemic stroke

Summary.  Background:  Studies in experimental animals have suggested that antithrombotic agents may have a neuroprotective effect after an ischemic injury. The aim of this study was to analyze the effect of prior use of antithrombotic agents (antiplatelets or anticoagulants) on neurological functional outcome in patients with acute ischemic stroke. Subjects and methods:  Consecutive patients included in the Perugia Stroke Registry were considered for this analysis. Neurological functional outcome was evaluated at discharge using the modified Rankin Scale (mRS ≥ 3 disabling stroke). Results:  Of the 1921 patients included in the analysis (mean age 76.3 ± 12.5 years; 53% males), 662 (34.5%) were on antithrombotic treatment (581 antiplatelets, 71 anticoagulants and 10 antiplatelets associated with anticoagulants). One hundred and twenty-two patients (6.4%) died in hospital; at discharge 712 patients (37.1%) were disabled and 1087 patients (56.6%) were non-disabled. Fifty-four (44.3%) of the deceased patients and 270 (37.9%) of disabled patients were on antithrombotic treatment, while 338 (31.1%) non-disabled patients were taking antithrombotic agents. From multivariate analysis, age and stroke severity were associated with an adverse outcome. Male gender, dyslipidemia, stroke due to small vessel disease and no history of previous stroke were associated with an improved outcome, while no correlation was found between prior use of antithrombotic agents and outcome (mortality odds ratio; OR = 1.32, 95% confidence interval; CI 0.85–2.04; P  = 0.20, mortality or disability OR = 0.95, 95% CI 0.72–1.25; P  = 0.80). Conclusion:  Prior use of antithrombotic agents does not improve the functional outcome in patients with acute ischemic stroke.     

Bleeding risk and reversal strategies for old and new anticoagulants and antiplatelet agents

Summary. The most important adverse effect of antithrombotic treatment is the occurrence of bleeding. In the case of severe bleeding in a patient who uses anticoagulant agents or when a patient on anticoagulants needs to undergo an urgent invasive procedure, it may be useful to reverse anticoagulant treatment. Conventional anticoagulants such as vitamin K antagonists may be neutralized by administration of vitamin K or prothrombin complex concentrates, whereas heparin and heparin derivatives can be counteracted by protamine sulphate. The anti‐hemostatic effect of aspirin and other antiplatelet strategies can be corrected by the administration of platelet concentrate and/or desmopressin, if needed. Recently, a new generation of anticoagulants with a greater specificity towards activated coagulation factors as well as new antiplatelet agents have been introduced and these drugs show promising results in clinical studies. A limitation of these new agents may be the lack of an appropriate strategy to reverse the effect if a bleeding event occurs, although experimental studies show hopeful results for some of these agents.     

A pharmacologic overview of current and emerging anticoagulants

For over 50 years, anticoagulant options for the treatment and prevention of thrombosis have been limited mainly to traditional agents such as unfractionated heparin and oral vitamin K antagonists such as warfarin. These traditional agents are fraught with limitations that complicate their clinical use. A variety of novel anticoagulants with improved pharmacologic and clinical profiles have recently been introduced or are in development, offering benefits over traditional therapies. Specifically, progress has been made in the development of low-molecular-weight heparins, factor Xa inhibitors, and direct thrombin inhibitors. Because of their convenience and ease of use, some of these novel compounds are competing with the traditional anticoagulants and are needed additions to the antithrombotic arsenal.     

Pharmacological interruption of acute thrombus formation with minimal hemorrhagic complications by a small molecule tissue factor/factor VIIa inhibitor: comparison to factor Xa and thrombin inhibition in a nonhuman primate thrombosis model

This study was designed to evaluate the antithrombotic efficacy and bleeding propensity of a selective, small-molecule inhibitor of tissue factor/factor VIIa (TF/VIIa) in comparison to small-molecule, selective inhibitors of factor Xa and thrombin in a nonhuman primate model of thrombosis. Acute, spontaneous thrombus formation was induced by electrolytic injury to the intimal surface of a femoral blood vessel, which results in thrombus propagation at the injured site. The TF/FVIIa inhibitor 3-amino-5-[1-[2-([4-[amino(imino)methyl]benzyl]amino)-2-oxoethyl]-3-chloro-5-(isopropylamino)-6-oxo-1,6-dihydropyrazin-2-yl]benzoic acid dihydrochloride (PHA-927F) was fully effective in prevention of thrombosis-induced vessel occlusion at a dose of 400 microg/kg/min, i.v., in the arterial vasculature (femoral artery). Neither the effective dose nor multiples up to 4.4-fold the effective arterial plasma concentration elicited any significant effect on bleeding time or blood loss from either the bleeding time site or the surgical (femoral isolation) site. Small-molecule inhibitors of factor Xa or thrombin were effective arterial antithrombotic agents; however, in contrast to the TF/FVIIa inhibitor, they both elicited substantial increases in bleeding propensity at the effective dose and at multiples of the effective plasma concentration. These data indicate that TF/VIIa inhibition effectively prevented arterial thrombosis with less impact on bleeding parameters than equivalent doses of factor Xa and thrombin inhibitors.     

Why does aspirin decrease the risk of venous thromboembolism? On old and novel antithrombotic effects of acetyl salicylic acid

It is well established that aspirin, an irreversible inhibitor of platelet cyclooxygenase activity, is effective in secondary prevention of arterial thromboembolic events. The pooled results of the recent randomized, multicenter WARFASA and ASPIRE aspirin trials showed a 32% reduction in the rate of recurrence of venous thromboembolism (VTE) in patients receiving aspirin following VTE. These clinical data support evidence that platelets contribute to the initiation and progression of venous thrombosis and aspirin inhibits thrombin formation and thrombin‐mediated coagulant reactions. In addition to the known acetylation of serine 529 residue in platelet cyclooxygenase‐1, the postulated mechanisms of aspirin‐induced antithrombotic actions also involve the acetylation of other proteins in blood coagulation, including fibrinogen, resulting in more efficient fibrinolysis. This review summarizes current knowledge on the aspirin‐induced antithrombotic effects that potentially explain clinical studies showing reduced rates of VTE events in aspirin‐treated subjects.